Cách Trồng Dưa Hấu Vỏ Xanh Ruột Vàng Lạ Mắt Khiến Chị Em Phát Cuồng Săn Lùng

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  • Cách Trồng Dưa Hấu Vỏ Xanh Ruột Vàng Lạ Mắt Khiến Chị Em Phát Cuồng Săn Lùng Dưa Hấu Vỏ Xanh Ruột Vàng đang được nhiều chị em ưa chuộng dù có giá cao gấp 3 – 5 lần dưa hấu đỏ. Loại dưa này mê hoặc người dùng bởi màu sắc lạ và vị ngọt dịu. Đây là giống cây cho trái quanh năm, có thể trồng kinh tế hoặc trồng chậu tại nhà.

    Cách Trồng Dưa Hấu Vỏ Xanh Ruột Vàng Lạ Mắt Khiến Chị Em Phát Cuồng Săn Lùng

    TÔI YÊU NÔNG NGHIỆP CHIA SẺ THÔNG TIN VỀ DƯA HẤU VỎ XANH RUỘT VÀNG VỚI CÁC BẠN

    Dưa hấu ruột vàng có nguồn gốc từ châu Phi, tên khoa học Citrullus Lanatus, thuộc họ Bầu bí (Cucurbitaceae). Loại trái cây này vỏ mỏng, cứng, ruột vàng ươm nhiều nước, thường được lựa chọn để giải nhiệt.

    Hiện nay, trên thị trường giá dưa hấu ruột vàng có giá cao hơn dưa hấu đỏ, khoảng 20.000 – 30.000 đồng/ ký nhưng vẫn được các bà nội trợ ưa chuộng.

    Dưa hấu ruột vàng cho trái quanh năm

    Ngày Tết Việt Nam thì dưa hấu là thức quả không thể thiếu, vài năm gần đây dưa hấu ruột vàng đang được ưa chuộng chưng Tết. Màu vàng của ruột tượng trưng cho tài lộc, may mắn. Màu xanh của vỏ tượng trưng cho hy vọng, niềm vui năm mới.

    Vì nhu cầu thị trường cao nên giống dưa hấu này đã được nhân rộng, trồng kinh tế ở nhiều tỉnh thành như các tỉnh miền Tây Nam Bộ, Hòa Bình, Hải Dương,…

    Dưa hấu vỏ xanh ruột vàng trồng chậu rất cói giá dịp tết

    Dưa hấu vỏ xanh ruột vàng là loại cây chịu nhiệt cực tốt, khả năng kháng sâu bệnh cao, dễ trồng, trồng quanh năm, dù ở thời điểm nào cây cũng phát triển, sinh trưởng tốt, có thể dễ dàng đậu quả ngay cả trong mùa mưa. Loại dưa hấu này là cây sinh trưởng ngắn ngày, chỉ sau chỉ sau 60 – 80 ngày gieo trồng bạn đã có thể thu hoạch được những quả dưa chín ngọt mát.

    Dưa hấu vỏ xanh ruột vàng cho năng suất cao

    Đây là giống cây trồng có hiệu quả kinh tế cao vì vốn đầu tư thấp nhưng năng suất lại cao, sản lượng trung bình đạt 20 – 25 tấn/ ha , chính vì thế đang trở thành giống cây trồng mới được nhiều nhà nông lựa chọn. Mỗi quả dưa hấu vỏ xanh ruột vàng nặng khoảng 3 – 4 ký, có quả nặng đến 6 ký. Mỗi vụ thu hoạch, hộ trồng dưa hấu ruột vàng thu lãi khoảng 15 – 20 triệu đồng/ ha.

    Dưa hấu vỏ xanh ruột vàng có vỏ mỏng nhưng dai và cứng, màu xanh sáng, rất tiện lợi cho việc vận chuyển đi xa và bảo quản. Thịt dưa hấu vàng tươi, thơm nhẹ, ngọt dịu, ít hạt, cắn vào thấy ngập nước nhưng lại khô ráo, không chảy nước như các loại dưa hấu đỏ thông thường. Chính vì những ưu điểm trên mà dưa hấu ruột vàng đang có tiềm năng kinh tế lớn, có giá trị xuất khẩu cao.

    Bên cạnh đó, dưa hấu vỏ xanh ruột vàng cũng rất dễ dàng trồng tại nhà . Cây sai trái ngay cả khi trồng chậu, trồng thùng xốp. Bạn cần chọn chậu hoặc thùng xốp có kích thước lớn để đủ không gian cho cây phát triển. Bạn cũng có thể chọn những chậu sứ đẹp để trồng dưa hấu ruột vàng chưng Tết.

    Dưa hấu vỏ xanh ruột vàng dễ trồng tại nhà

    GỬI THÔNG TIN VÀO MẪU BÊN DƯỚI ĐỂ ĐẶT HÀNG

    Gần đây có rất nhiều thông tin về dưa hấu tiêm thuốc gây hoang mang dư luận, tại sao bạn không tự trồng tại nhà để cả gia đình yên tâm thưởng thức quả ngọt giải nhiệt? Ngoài có trái ngon, sạch lại còn có cây đẹp trang trí nhà cửa. Liên hệ với Tôi Yêu Nông Nghiệp để sở hữu ngay gói hạt giống dưa hấu vỏ xanh ruột vàng siêu hấp dẫn. Shop cam kết hạt F1 đúng giống, tỷ lệ nảy mầm cao.

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    LỢI ÍCH CỦA TRÁI DƯA HẤU RUỘT VÀNG CÓ THỂ BẠN CHƯA BIẾT

    Dưa hấu chứa rất nhiều vitamin

    Uống nước ép dưa hấu mỗi ngày ngăn ngừa ung thư

    Mẹo chọn dưa hấu ngon ngọt cho các chị em: Dưa hấu càng nặng tay càng mọng nước

    GỬI THÔNG TIN VÀO MẪU BÊN DƯỚI ĐỂ ĐẶT HÀNG

    Quả dưa phải nặng tay, vỗ kêu bốp bốp. Quả dưa càng nặng hơn kích thước bao nhiêu thì càng mọng nước và ngọt. Nếu dưa nhẹ so với kích thước thì có thể sẽ bị xốp.

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    HƯỚNG DẪN CHI TIẾT CÁCH TRỒNG VÀ CHĂM SÓC HẠT GIỐNG DƯA HẤU VỎ XANH RUỘT VÀNG

    Thời vụ: Dưa hấu ruột vàng có thể trồng được quanh năm thời điểm nào cũng sinh trưởng tốt, tuy nhiên năng suất cao nhất và nhanh thu hoạch nhất là vụ xuân và vụ hè. Nếu bạn trồng dưa hấu để bán dịp Tết hoặc trồng cảnh chưng Tết thì nên bắt đầu gieo hạt từ cuối tháng 10 âm lịch.

    Dưa hấu ruột vàng trồng được quanh năm

    Đất trồng: không kén đất nhưng nên chọn đất đất thịt nhẹ, đất pha cát. Bạn có thể trồng luân canh dưa hấu với các loại cây thuộc họ bầu bí.

    Khi cây con được 1 tuần là có thể mang đi trồng

    Chăm sóc

    Mật độ: Nếu trồng kinh tế, bạn có thể trồng với mật độ 833 – 909 cây/ ha (50 – 60 gram hạt giống), lên liếp khi trồng, mỗi cây cách nhau 50-60cm, hàng cách hàng 5 m.

    Ngưng tưới trước khi thu hoạch để dưa ngọt hơn

    Dưa hấu ruột vàng có thể ra trái kể cả mùa mưa

    Vì là cây ít sâu bệnh, sinh trưởng khỏe nên bạn không cần chăm sóc quá cầu kỳ.

    Mẹo: để tăng dộ ngọt của dưa hấu ruột vàng, trước khi thu hoạch bạn nên ngưng tưới nước cho cây. Bạn yên tâm là cây sẽ không chết vì giống dưa này có khả năng chịu hạn cực tốt. Sau thu hoạch xong bạn phải tưới điều độ trở lại để đảm bảo lượng trái mùa 2.

    Giàn chữ X trồng kinh tế

    Thu hoạch: chỉ hơn 1 tháng kể từ lúc gieo bạn đã có thể thu hoạch được dưa hấu ruột vàng ngọt mát, giải nhiệt rất tốt.

    Cách trồng dưa hấu leo giàn

    Giàn trụ trồng chậu làm cảnh

    Khi trồng chậu, bạn nên chọn kiểu

    giàn hình trụ cho cây dễ leo, với kiểu giàn này, trái sẽ lủng lẳng bên ngoài rất đẹp mắt

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  • Hiện nay, thay vì trồng ớt ngọt theo phương pháp canh tác truyền thống thì trồng Hạt giống ớt ngọt thủy canh được người nông dân ưa chuộng bởi những ưu điểm như năng suất cao, dễ dàng kiểm soát sâu bệnh, hạn chế rủi ro về thời tiết và có thể thâm canh quanh năm. Cách trồng Ớt ngọt rất đơn giản nên bạn có thể tự trồng ngay tại nhà.

    CÂY TRỒNG HẠT GIỐNG CHIA SẺ THÔNG TIN VÈ GIỐNG ỚT NGỌT VỚI BẠN

    Ớt ngọt (Sweet peppers) hay ớt chuông có tên khoa học là Capsicum annuum Group. Ớt chuông có nguồn gốc ở Mexico, Trung Mỹ, và phía Bắc Nam Mỹ. Quả ớt chuông có rất nhiều màu sắc như đỏ, vàng, cam, xanh lục, sô-cô-la/nâu,…

    Khác với các giống ớt thông khác, ớt ngọt không hề cay, mùi vị thanh ngọt, thịt dày và vỏ mỏng. Trái to, tròn, bóng đẹp nên rất được ưa chuộng trồng chậu. Cây sinh trưởng khỏe, ít sâu bệnh, khả năng phân nhánh tốt.

    Quả ớt chuông rất giàu dinh dưỡng, chứa nhiều chất chống oxi hóa và vitamin C, carotene, lycopene rất cao. Ớt ngọt nhanh cho thu hoạch, chỉ sau 75-80 ngày bạn đã thu hoạch được những quả ớt bóng đẹp nặng 250 -350 g và chế biến các món ăn rất ngon cho gia đình. Phù hợp với các món nướng, nhồi nhân, hoặc xào…

    Nếu trồng để làm kinh tế thì ớt ngọt được nhiều người tiêu dùng tò mò, giá của loại ớt này cũng khá cao. Nếu trồng ở nhà thì có thể gieo trồng trong chậu, giống như trồng cây cảnh. Ớt ngọt có thể trồng thủy canh, không những thế, ớt ngọt còn tượng trưng cho may mắn. Trồng một chậu ớt ngọt thủy canh trong nhà sẽ mang đến tài vận cho cả gia đình.

    PHƯƠNG PHÁP THỦY CANH GIÚP ỚT ỚT NGỌT SAI TRÁI TRĨU QUẢ

    Bột thủy canh TC-Mobi là sản phẩm dinh dưỡng thủy canh thế hệ mới với thành phẩm dạng bột độc đáo mang lại nhiều tiện ích và chất lượng cao cho khách hàng khi sử dụng. TC-MOBI tạo ra một môi trường dinh dưỡng tuyệt vời và tối ưu cho cây sinh trưởng để cho ra các sản phẩm rau, quả tươi ngon và sạch 100%.

    Thành phần chính: N-P2O5-K2O: 15-4-18%. Vi lượng: 250ppm B, 250ppm Mn, 28ppm Zn, 12ppm Cu, 7ppm Mo, 120ppm Fe.

    Với 100gr được pha 200 lít nước, với mỗi 20 lít nước / chậu cách 30 ngày thêm 10gr bột thủy canh cho cây. Ví dụ: Chậu cây 20 lít cách 30 ngày thêm 10gr vào chậu. Liều lượng sử dụng:

    Bột thủy canh TC-Mobi là sản phẩm dinh dưỡng thủy canh thế hệ mới nhất

    Để Nguồn nước: Phải đảm bảo nguồn nước trồng hạt giống ớt cu tý thủy canh là nước sạch, điều này giúp cây trồng không bị nhiễm vi khuẩn độc hại. Rọ trồng thủy canh: Nên chọn những loại rọ có kích thước phù hợp, không quá to, không quá nhỏ, phải đảm bảo đủ chỗ cho cây phát triển Giá thể: Tốt hơn hết nên dùng xơ dừa hoặc trấu, bởi những loại giá thể này khả năng giữ ẩm và thoát nước cực kỳ tốt. Dung dịch thủy canh: Có thể chọn mua những loại dung dịch dinh dưỡng chuyên dụng. Bút đo PH, bút đo nồng độ PPM

    Ớt Cu Tý được sinh trưởng và phát triển tốt theo phương pháp thủy canh thì quy trình trồng và chăm sóc phải đúng kỹ thuật. Và theo đó, khâu chuẩn bị dụng cụ cũng như giống cây là vô cùng quan trọng.

    hoặc Chắc chắn Giống Ớt Ngọt Thủy Canh này sẽ khiến bạn không thất vọng khi sở hữu chúng. Liên hệ ngay vớicaytronghatgiong.com để lại thông tin bên dưới để sở hữu cho mình Giống Ớt Ngọt Thủy Canh ngay nào!

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    HƯỚNG DẪN TRỒNG THỦY CANH HẠT GIỐNG ỚT NGỌT

    Kỹ thuật trồng hạt giống ớt cu tý thủy canh:

    Bạn có thể tận dụng bao nion, bao xi măng, chậu, khay, thùng xốp có sẵn trong nhà hoặc mảnh đất trống trong vườn để trồng hạt giống ớt ngọt. Dưới đáy khay đục lỗ để thoát nước

    Khi cây con được khoảng 30 – 35 ngày thì đem ra cấy với khoảng cách 60cm x 30cm x 35cm. Khi cấy xong chuyển vào nơi ít ánh nắng hoặc che chắn khoảng 4 – 5 ngày để cây mới cây nhanh chóng bén rễ và không bị cháy nắng. Tưới nước ngày 2 lần vào lúc sáng sớm vào chiều tối.

    Ngâm hạt giống Ớt bằng nước ấm (khoảng 30 độ C) trong vòng 6 – 10 tiếng thì đem gieo hạt xuống đất đã chuẩn bị sẵn. Tưới nước bằng vòi phun nhẹ.

    Chăm sóc:

    Ớt Ngọt ưa phát triển ở đất màu mỡ, cát pha hoặc thịt nhẹ và có độ pH từ 5,5 – 7. Nên bón lót với vôi rồi phơi ải từ 7 – 10 ngày trước khi gieo trồng hạt giống để xử lý các mầm bệnh có trong đất. Bạn có thể mua đất sẵn hoặc tiến hành trộn đất với phân bò hoai mục, phân gà, phân trùn quế, vỏ trấu, xơ dừa, than bùn, mùn hữu cơ…

    Sau khi trồng phải tưới cho ớt hàng ngày cho đến khi cây hồi xanh, ở giai đoạn sinh trưởng sau nên thường xuyên tới giữ ẩm cho cây trong suốt thời gian sinh trưởng.

    Dung dịch thủy canh mua về được pha với nước theo tỷ lệ ghi sẵn trên vỏ hộp nên chẳng cần phải có nhiều kinh nghiệm cũng trồng được cây theo phương pháp này. Liều lượng sử dụng: Với 100gr được pha 200 lít nước

    Bón bột thủy canh: cứ sau 7-10 ngày thì thêm bột thủy canh theo tỷ lệ trên bao bì hướng dẫn để cây phát triển tốt. Vơi gói bột thủy canh kèm theo bộ sản phẩm thì có thể sử dụng cho 200 lít nước.

    Vô cùng đơn giản cho quy trình trồng ớt ngọt bằng phương pháp thủy canh mà bạn không nên bỏ qua

    THÔNG TIN KỸ THUẬT VỀ HẠT GIỐNG ỚT NGỌT DÀNH CHO CHUYÊN GIA

    – chuyên cung cấp các loại CÂY TRỒNG HẠT GIỐNG hạt giống Cherry Nhiệt Đới F1, shop chuyên hạt giống hoa và chuyên bán các loại hạt giống cây ăn trái, cây mini, hạt giống sen mini Nhật Bản và rất nhiều loại hạt giống mini khác.

    Tag: Hạt giống ớt ngọt, ớt chuông

    MIỄN PHÍ TRÊN TOÀN QUỐC Giao hàng

    GỬI THÔNG TIN VÀO MẪU BÊN DƯỚI ĐỂ ĐẶT HÀNG

    Công ty với 12 năm trong ngành Cam Kết Hạt Giống Đúng Chất Lượng Đúng Giống như trên

    --- Bài cũ hơn ---

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  • – Hạt giống: Bạn có thể mua hạt giống quả tại các cửa hàng. Tuy nhiên nên lựa chọn hạt giống chất lượng, tỉ lệ nảy mầm cao, không sâu bệnh, như vậy năng suất gieo trồng sẽ cao hơn.

    Những hạt giống dưa hấu mini được trồng thử nghiệm sau 30 ngày đã chuẩn bị bắt đầu bắc giàn và cho ra hoa

    Đất trồng: Dưa hấu mini thích hợp với mọi loại đất nhưng tốt nhất nên lựa chọn đất nhiều mùn, như vậy cây sẽ phát triển tốt và cho nhiều quả. Bạn có thể mua đất tribat để trồng tại các cửa hàng hoặc trộn trấu tươi để tăng thêm độ tơi xốp của đất.

    – Chậu trồng: Lựa chọn chậu bằng nhựa hoặc thùng xốp để trồng.

    – Gieo hạt: bỏ đất vào chậu, gieo hạt trực tiếp lên đất. Giữ ẩm khoảng 4 – 9 ngày hạt sẽ nảy mầm và phát triển thành cây con. Đến khi ra bốn lá thì có thể đưa vào trồng trong đất.

    – Làm giàn: Dưa hấu mini cần làm giàn khi cây bắt đầu leo. Giàn dưa hấu mini không khác gì với cách trồng dưa leo, vì vậy bạn có thể dùng dây thép hoặc cọc nhỏ làm giàn cho dưa. Còn đối với chậu treo hay thùng xốp thì các bạn có thể tận dụng trồng cạnh các hàng rào, cây sẽ bám vào và leo rất nhanh. Nên làm giàn trước khi xuống cây giống.

    Tưới nước: Bạn duy trì độ ẩm cho bộ rễ phát triển. Chú ý không nên tưới nước quá nhiều dễ làm cây ngập úng, chú ý không để chậu trồng bị ngập hay thùng xốp bị đọng nước, cây không chết nhưng chậm phát triển, tốt nhất nên giữ ẩm vừa phải để cây phát triển tốt nhất.

    – Phòng trừ sâu bệnh: Dưa hấu mini có khả năng chịu hạn tốt nên không cần lo lắng về sâu bệnh. Bạn cũng có thể phòng tránh bằng cách vệ sinh sạch sẽ đất và chậu trồng ngày từ khâu đầu tiên. Nếu trồng bằng chậu treo thì giảm rất nhiều thiệt hại do dế và ốc sên.

    – Cây cho hoa từ 45 – 60 ngày. Cây cho trái quanh năm, cứ 3 – 4 ngày bạn thu hoạc 1 đợt. Cây phát triển tốt trong môi trường khí hậu Việt Nam, nắng và mưa nhiều sẽ cho năng suất cao hơn.

    Theo chúng tôi

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  • Cây ” Dưa hấu tí hon” có tên khoa học là Melothria scabra, các tên gọi khác như dưa hấu chuột, dưa hấu Mexico, Cucamelon, Pepquino. Cây thuộc họ dây leo có trái ăn được. Trước đây, Pepquino là loại cây dại ở Nam Mỹ, sau đó những người nông dân Hà Lan đem giống dưa này về trồng trong nhà kính. Từ đó, dưa hấu tí hon trở thành đặc sản và được bán khắp châu Âu. Dưa hấu Pepquinno được dùng để ăn trong các bữa ăn nhẹ hoặc làm món khai vị. Người Anh thì quan niệm quả pepquino này đem lại may mắn cho họ vì nó hiếm và độc đáo.

    Cây này có nhiều tính năng như quả dùng để ăn, cây để làm cảnh. Trồng dưa hấu tí hon sẽ cho rất nhiều trái, vừa đẹp vừa thích mắt vì nó có hoa vàng li ti.

    + Chậu trồng: Trồng trong chậu hoặc thùng xốp. Mỗi thùng có kích thước 50×70 bạn có thể trồng được 4 cây với khoảng cách là 40cm.

    + Đất trồng: Cây thích hợp mọi loại đất nhưng nếu trồng trên loại đất có nhiều mùn là phù hợp nhất vì cây phát triển tốt nhất và cho quả nhiều nhất. Nếu có trấu tươi bạn nên thêm vào để tăng độ tơi xốp của đất.

    + Phân bón: Phân hữu cơ là lựa chọn tốt nhất cho loại cây này.

    + Hạt và cây giống: Cây được trồng từ hạt. Hạt được ươm khoảng 4 đến 9 ngày thì bắt đầu nẩy mầm, khi cây được 4 lá thì chúng ta bắt đầu đưa vào trồng.

    + Giữ ấm hạt trong các giá thể (có thể là tro trấu, mạt dừa v.v…) khoảng 4 đến 9 ngày và tưới một ít nước (khoảng 24oC) hàng ngày. Sau 4-9 ngày, khi hạt bắt đầu nẩy mầm thì tiếp tục tưới nước ở nhiệt độ 18oC – 21oC, đến khi ra bốn lá thì có thể đưa vào trồng trong đất.

    – Dưa hấu chuột không khó trồng và chăm sóc. Trồng dưa hấu tí hon không khác so với cách trồng dưa leo, thậm chí, dưa hấu chuột chịu hạn và chịu lạnh tốt hơn rất nhiều. Bạn không cần phải lo lắng về sâu bệnh, khi mùa đông khắc nghiệt, cũng như phải am hiểu các kĩ thuật cắt tỉa phức tạp.

    + Cây sẽ mất 15 ngày để phát triển bộ rễ, trong thời gian này chú ý nhiều đến độ ẩm của đất, cây chịu được hạn nhưng không chịu được ngập úng, chú ý không để chậu trồng bị ngập hay thùng xốp bị đọng nước, cây không chết nhưng chậm phát triển, tốt nhất nên giữ ẩm vừa phải để cây phát triển tốt nhất.

    + Bạn chỉ tưới nước vừa đủ để giữ ẩm đất, cây có thể chịu được nắng và có thể bạn không tưới 2 đến 3 ngày mà cây không chết.

    + Cây bắt đầu có hoa từ ngày thứ 45 đến 60. Theo tài liệu thì cây thu hoạch trái quanh năm, 3 đến 4 ngày thu hoạch 1 đợt. Theo nguồn tổng hợp từ hội làm vườn các nước, mỗi cây trồng trong chậu cảnh treo sẽ cho 4kg trái/ năm. Nếu chăm sóc tốt cây sẽ cho quả nhiều hơn. Hiện tại cây phát triển tốt trong môi trường khí hậu Việt Nam, nắng và mưa nhiều.

    + Cây ít bệnh, không bị sâu hại nhưng cây non cần chú ý bọ cánh cứng và ốc sên gây hại (cắn đứt ngọn non), cây cũng cần phòng các loại dế cắn đứt rễ. Nếu trồng bằng chậu treo thì giảm rất nhiều thiệt hại do dế và ốc sên.

    Quả dưa hấu nhỏ xinh Pepquino đang làm mê mẩn những người yêu trái cây ở Mỹ và Anh có làm bạn hào hứng không? Nếu câu trả lời là “có” thì hãy bắt tay vào trồng luôn nào. Bạn có thể mua giống hạt dưa hấu tí hon ở các cửa hàng bán giống rau sạch online, giá một hạt giống dưa hấu loại này từ 20.000 đồng, một cây non từ 100.000 đồng.

    --- Bài cũ hơn ---

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  • 1. Chuẩn bị:

    – Hạt giống: Bạn có thể tìm mua hạt giống dưa chuột mini nhập khẩu từ Mỹ tại các cửa hàng chuyên phân phối và kinh doanh hạt giống uy tín để đảm bảo chất lượng quả. Giá tham khảo khoảng 65.000 đồng/ gói (10 hạt).

    – Đất trồng: Loại đất thích hợp để trồng giống cây này là đất mùn hữu cơ, đất thịt tơi xốp hoặc đất xơ dừa. Lưu ý không dùng đất bị vón cục và san bằng bề mặt đất trồng cho bằng phẳng.

    – Nơi gieo trồng: Sử dụng các loại chậu cảnh hoặc tận dụng khay nhựa, cốc nhựa, thùng xốp, vỏ hộp,… để ươm hạt giống.

    – Ánh sáng, nước: Dưa chuột ưa ánh sáng nên bạn cần chọn vị trí nhiều ánh nắng mặt trời để cây phát triển tốt. Chuẩn bị loại bình xịt phun sương để tưới ẩm cho cây.

    – Đầu tiên, chúng ta tiến hành ngâm hạt giống với nước ấm (tỉ lệ 2 sôi 3 lạnh) trong vòng khoảng từ 2 – 4 tiếng đồng hồ. Sau đó cho hạt vào bông gòn/ khăn ướt hoặc miếng vải ẩm và ủ vài ngày cho đến khi hạt nứt mầm thì đem gieo.

    – Cho đất đã chuẩn bị vào chậu/ khay/ hộp xốp,… Tạo trên bề mặt đất những lỗ nhỏ sâu chừng 1cm và gieo hạt trực tiếp vào các lỗ này.

    – Dùng bịt xịt phun sương phun đều nước lên bề mặt khay để tạo độ ẩm và đặt khay ra ngoài nơi có nắng tốt.

    – Sau khoảng 1 – 2 tuần, các hạt được gieo sẽ bắt đầu nảy mầm. Khi cây con đạt chiều cao khoảng 5cm với hai lá mầm to và chồi lá ở giữa đang chuẩn bị nhú thì bạn đem tách ra và trồng vào chậu lớn hơn.

    – Sau khoảng 3 tuần, cây con sẽ đạt chiều cao khoảng 10 – 15cm, lá chồi sẽ mọc và phát triển nhanh.

    – Dưa chuột nói chung và dưa chuột mini nói riêng đều ưa sáng, vì thế bạn nên lưu ý trồng cây ở nơi có nhiều ánh nắng mặt trời.

    – Thường xuyên vun xới đất cho tơi xốp, làm sạch cỏ dại xung quanh, nếu cần thiết có thể bón thêm phân dạng loãng pha với nước rồi phun đều.

    – Khoảng cách trồng cây con phải cách nhau tầm 40 – 50cm. Đây là khoảng cách thích hợp để các cây không cạnh tranh nhau về ánh sáng cũng như nguồn đất.

    – Vào mùa nắng, bạn tưới nước 2 lần mỗi ngày vào buổi sáng và chiều. Khi cây càng lớn, nhất là thời kỳ ra hoa trái rộ, thì càng tăng cường lượng nước tưới cũng như khu vực xung quanh gốc. Vào mùa mưa nên chú ý vấn đề thoát nước để tránh tình trạng cây bị ngập úng.

    Sau khoảng 50 – 60 ngày là chúng ta đã có thể thu hoạch dưa chuột mini. Kích thước quả trưởng thành khoảng từ 4 – 6 cm, quả màu trắng hoặc vàng nhạt lạ mắt, ăn có độ giòn, vị thơm mát ngọt dịu. Vì thời gian thu hoạch nhanh chóng cũng như ra quả hầu như quanh năm nên các bà nội trợ có thể tha hồ tận hưởng thành quả của mình.

    Cách trồng cà chua tại nhà

    Bước 1: Chuẩn bị hạt giống hoặc cây giống

    Mua cây cà chua giống ngoài chợ hoặc các cửa hàng cây giống. Nếu muốn trồng từ hạt, có thể gieo hạt vào bất kỳ vật dụng nào từ cốc, bát, hộp nhựa…, để chúng trong nhà ở gần cửa sổ hoặc những nơi có nắng.

    Bước 2: Chuẩn bị chỗ trồng

    Vị trí trồng lý tưởng nhất là nơi có đầy đủ ánh nắng mặt trời và cây đòi hỏi đất trồng phải thật giàu chất dinh dưỡng hữu cơ. Nếu bạn không thể tự ủ phân xanh hữu cơ tại nhà thì có thể mua sẵn ở các cửa hàng.

    Cây non phải được trồng sâu xuống đất từ 50 – 75% chiều cao thân, đặc biệt là những cây thân cao, mảnh dẻ. Và tưới nước ướt hết đất dưới gốc để cây không bị sốc với môi trường mới.

    Tưới nước đều đặn cho cây 1 hoặc 2 lần/ ngày, để đảm bảo cây có đủ nước để phát triển tốt. Lượng nước cho mỗi lần tưới ít hay nhiều phù thuộc vào cây nhỏ hay lớn.

    (Chú ý: Giữ ẩm cho đất không phải là tưới nước liên tục. Nếu đất bị sũng nước sẽ giết chết bộ rễ và tạo điều kiện để nấm phát triển, nhất là khi trời thực sự ấm áp hoặc nóng.)

    Bước 5: Thêm giàn, cọc hoặc lồng…

    Xem xét làm thêm giàn leo, cọc hoặc lồng để hỗ trợ cây cà chua leo lên cao khoảng 14 ngày sau khi trồng. Một chiếc lồng cần có chiều cao tối thiểu là 1.2 mét, thậm chí là cao hơn nếu cây phát triển tốt.

    Hãy tìm kiếm loại phân hữu cơ tốt để kích thích cây ra quả. Đất càng giàu hữu cơ thì chất lượng quả cà chua sẽ càng tốt hơn. (Chi tiết có thể nhờ chủ cửa hàng tư vấn).

    Bước 7: Chăm sóc cây khi ra quả

    Trung bình từ 45 – 90 ngày, phổ biến nhất là 60 ngày sau khi trồng sẽ xuất hiện quả. Quả ban đầu thường nhỏ và có màu xanh lá cây.Khi đã đạt kích thước hoàn chỉnh thì quả sẽ chuyển dần sang màu đỏ đậm hơn. Điều này có nghĩa quả đã bắt đầu chín và sẵn sàng để thu hoạch.

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  • Cách Trồng Dưa Chuột Bao Tử
  • Mách Chị Em Hai Cách Trồng Dưa Chuột Trong Thùng Xốp Cho Quả Sai Lúc Lỉu
  • Cách Trồng Cây Dưa Chuột Cho Ra Nhiều Quả
  • Hướng Dẫn Trồng Và Chăm Sóc Dưa Leo Tại Nhà
  • Dưa chuột là loại quả yêu thích của rất nhiều người, đặc biệt là chị em phụ nữ. Không chỉ thanh mát, dịu nhẹ, loại quả này còn dễ dàng được trồng trong nhà phố bởi ra quả gần như quanh năm. Ngoài những loại dưa chuột giống to quen thuộc hay loại dưa chuột mini da xanh, trên thị trường hiện nay còn xuất hiện loại hạt giống dưa chuột mini màu trắng.

    – Hạt giống: Bạn có thể tìm mua hạt giống dưa chuột mini nhập khẩu từ Mỹ tại các cửa hàng chuyên phân phối và kinh doanh hạt giống uy tín để đảm bảo chất lượng quả. Giá tham khảo khoảng 65.000 đồng/ gói (10 hạt).

    – Đất trồng: Loại đất thích hợp để trồng giống cây này là đất mùn hữu cơ, đất thịt tơi xốp hoặc đất xơ dừa. Lưu ý không dùng đất bị vón cục và san bằng bề mặt đất trồng cho bằng phẳng.

    – Nơi gieo trồng: Sử dụng các loại chậu cảnh hoặc tận dụng khay nhựa, cốc nhựa, thùng xốp, vỏ hộp,… để ươm hạt giống.

    – Ánh sáng, nước: ưa ánh sáng nên bạn cần chọn vị trí nhiều ánh nắng mặt trời để cây phát triển tốt. Chuẩn bị loại bình xịt phun sương để tưới ẩm cho cây.

    – Đầu tiên, chúng ta tiến hành ngâm hạt giống với nước ấm (tỉ lệ 2 sôi 3 lạnh) trong vòng khoảng từ 2 – 4 tiếng đồng hồ. Sau đó cho hạt vào bông gòn/ khăn ướt hoặc miếng vải ẩm và ủ vài ngày cho đến khi hạt nứt mầm thì đem gieo.

    – Cho đất đã chuẩn bị vào chậu/ khay/ hộp xốp,… Tạo trên bề mặt đất những lỗ nhỏ sâu chừng 1cm và gieo hạt trực tiếp vào các lỗ này.

    – Dùng bịt xịt phun sương phun đều nước lên bề mặt khay để tạo độ ẩm và đặt khay ra ngoài nơi có nắng tốt.

    – Sau khoảng 1 – 2 tuần, các hạt được gieo sẽ bắt đầu nảy mầm. Khi cây con đạt chiều cao khoảng 5cm với hai lá mầm to và chồi lá ở giữa đang chuẩn bị nhú thì bạn đem tách ra và trồng vào chậu lớn hơn.

    – Sau khoảng 3 tuần, cây con sẽ đạt chiều cao khoảng 10 – 15cm, lá chồi sẽ mọc và phát triển nhanh.

    – Dưa chuột nói chung và dưa chuột mini nói riêng đều ưa sáng, vì thế bạn nên lưu ý trồng cây ở nơi có nhiều ánh nắng mặt trời.

    – Thường xuyên vun xới đất cho tơi xốp, làm sạch cỏ dại xung quanh, nếu cần thiết có thể bón thêm phân dạng loãng pha với nước rồi phun đều.

    – Khoảng cách trồng cây con phải cách nhau tầm 40 – 50cm. Đây là khoảng cách thích hợp để các cây không cạnh tranh nhau về ánh sáng cũng như nguồn đất.

    – Vào mùa nắng, bạn tưới nước 2 lần mỗi ngày vào buổi sáng và chiều. Khi cây càng lớn, nhất là thời kỳ ra hoa trái rộ, thì càng tăng cường lượng nước tưới cũng như khu vực xung quanh gốc. Vào mùa mưa nên chú ý vấn đề thoát nước để tránh tình trạng cây bị ngập úng.

    Sau khoảng 50 – 60 ngày là chúng ta đã có thể thu hoạch dưa chuột mini. Kích thước quả trưởng thành khoảng từ 4 – 6 cm, quả màu trắng hoặc vàng nhạt lạ mắt, ăn có độ giòn, vị thơm mát ngọt dịu. Vì thời gian thu hoạch nhanh chóng cũng như ra quả hầu như quanh năm nên các bà nội trợ có thể tha hồ tận hưởng thành quả của mình.

    Các món ăn hấp dẫn như salad, gỏi, dưa chuột cuộn tôm thịt,… hay đơn giản là ngâm dưa leo trong nước để tạo nên loại thức uống mát lành, giúp thanh lọc cơ thể là Detox Water đang rất thịnh hành tại Mỹ.

    --- Bài cũ hơn ---

  • Cách Trồng Dưa Chuột Thái
  • Trồng Ngay Giống Dưa Chuột Chùm Siêu Trái, Ăn Rất Giòn
  • Hướng Dẫn Trồng Dưa Leo Nhật Bản
  • Cách Trồng Dưa Leo Cho Quả “sai Chĩu Giàn” Quanh Năm
  • Cách Trồng Dưa Leo Thủy Canh Sai Quả, Không Bị Đắng
  • Hướng Dẫn Trồng Lan Mini Cattleya

    --- Bài mới hơn ---

  • Các Loài Hoa Lan Ngộ Nghĩnh
  • Phòng Trừ Kiến Trên Cây Lan
  • Tại Sao Địa Lan Hay Rụng Lá?
  • Lá Lan Hồ Điệp Mềm Và Nhăn Nheo?
  • Hiện Tượng Lá Lan Hồ Điệp Bị Héo Và Nhăn Nheo
  • Năm 1992, khi về hưu vợ chồng tôi quyết định trở lại California, miền đất nắng ấm quanh năm, nơi mà chúng tôi có đã nhiều kỷ niệm ban đầu với miền đất mới đã mở rộng vòng tay đón nhận những kẻ tỵ nạn khốn khổ, vội vàng bỏ nước ra đi vào năm 1975.

    Rời Peoria, Illinois, nơi vì sinh kế và vì số phận long đong chúng tôi đã chịu suốt 16 mùa đông lạnh lẽo có khi xuống tới -25°F. Trong mớ hành trang trở về thủ đô của người tỵ nạn, ngoài quần áo còn có vài thùng chứa những cây lan chúng tôi đã nuôi từ năm 1976. Trong số cây lan này có cây Lc. Little Pink Snow do ông bạn già George Hotchkiss người Mỹ gốc Pháp tặng cho khi chúng tôi nói chuyện với nhau về loài hoa muôn hương, muôn sắc này.

    Thời kỳ đó, giống lan Mini Cattleya rất hiếm, giá đắt như vàng, cây chỉ có 3 nhánh nhỏ, thân cây cao chừng 10 cm, lá dài 12-16 cm, ngang 3 cm, hoa chiều rộng chừng 5 cm mầu trắng như tuyết, chiếc lưỡi mầu tím hồng tuyệt đẹp.

    Đến nay, khóm lan này tính ra đã sống trên 33 năm, bằng tuổi với đứa cháu gái nội của chúng tôi. Đây không còn là cây lan tầm thường nữa mà là một một kỷ vật thân thương cho nên nhiều bạn bè muốn xin một vài nhánh, chúng tôi không nỡ lòng nào chia cắt cho người khác. 

    Xuất hiện sâu bệnh  trên cây Cattleya mini vì thiếu chăm sóc:

    Nhưng mùa hè năm nay cây đã quá cằn cỗi, lớp thân cây ông cha già nua ốm yếu choán gần hết chỗ cư trú cho đám con cháu sau này. Mảnh vỏ cây cho lan bám vào đã bị mục nát, lại thêm mấy năm trời bị bỏ lăn bỏ lóc vì thương nhớ người bạn đời đã trên 55 năm chung sống bỏ tôi về bên kia thế giới. Khi nhìn đến thì ôi thôi! lũ rệp sáp (Boiduvale scales) đã thừa cơ phá hoại, thân cây, lá cây bị đốm đen, đốm vàng lỗ chỗ, mầm non bị còi cọc không lớn lên được. Thêm vào đó lũ sâu bi (Isopod, Pillbug, Sow Bug) dùng đó làm hang ổ, ăn hết đầu rễ non làm cho khi đến mùa hoa chỉ còn 1-2 chiếc không còn xum xuê như trước. Nếu không cứu vãn tình thế, bảo vật này có lẽ cũng theo người vợ yêu quý ra đi, cho nên đành phải ra tay chỉnh đốn lại mặc dầu vết thương khi mổ mật chưa lành.

    Cách xử lý khi cây cattleya mini bị sâu bệnh:

    Cắt bỏ những thân cây già cỗi, không còn mắt non, không thể sinh con đẻ cái, những chiếc lá bị rệp đốt nhăn nheo và rể đã bị mục thối. Khi cắt nên quan sát cho kỹ, cần phải để lại 3-5 nhánh nuôi các mầm non. Ngăn ngừa vi khuẩn xâm nhập cây sau khi cắt: Dùng Physan 20 pha với 2 thìa cà phê cho 4 lít nước, phun toàn thân cây lá và rễ đề phòng vi trùng xâm nhập theo các vết cắt. Pha thuốc trừ rệp sáp : pha một chai cồn 70% khoảng 16 oz (473 ml) và một chai nước cùng một dung tích, 1/2 thìa cà phê xà phòng rửa bát, 1/2 thìa cà phê dầu ăn loại thực vật. Cho tất cả vào chiếc bình 2 lít lắc cho thật đều, rồi phun cả mặt trên mặt dưới lá, thân cây. Dùng bàn chải đánh răng mềm chà xát những chỗ rệp bám để diệt tận gốc rễ và trứng rệp.

    Chuẩn bị chậu trồng Cattleya mini

    Chọn một miếng vỏ cây làm giá thể trồng (cork bark, Quercus suber). Đây là một giống cây mọc ở Portugal, Algeria, Spain, Morocco, France, Italy and Tunisia, vỏ cây không bị dễ bị mục. Đặt một lớp sơ dừa dầy 2-3 cm xuống dưới rồi bỏ một nắm vỏ cây nhỏ vào. Những nhánh lan nhỏ đã bị cắt, nếu cần sẽ buộc lại với nhau cho cây khỏi nghiêng ngả.

    Cuối cùng dùng giây cột chặt toàn bộ vào với nhau. Trồng thêm vài cây cóc mẳn cho đẹp mắt. 

    Tưới nước có pha B1 pha 2 thìa súp với 4 lít nước và để cây vào chổ rợp mát trong vòng 1 tháng mới đưa ra chỗ có nắng.

    Tôi hy vọng rằng mùa xuân năm tới cây lan thân thương sẽ hoàn toàn hồi phục và cho nhiều hoa như những năm về trước và yên trí không phải vất vả cứ vài năm một lần thay chậu nữa.

    Placentia mùa hè 2008

    BÙI XUÂN ĐÁNG

    --- Bài cũ hơn ---

  • Những Đóm Trắng Tròn Xuất Hiện Trong Chậu Lan Hồ Điệp?
  • Chăm Sóc Và Thuần Dưỡng Lan Rừng Thế Nào?
  • Tham Khảo Cách Đặt Tên Cho Lan Rừng
  • Giới Thiệu Tổng Quát Về Hoa Lan
  • 10 Lý Do Khiến Lan Cháy Đầu Rễ Và Chậm Ra Rễ
  • Fungi And Food Spoilage, 3Rd Ed

    --- Bài mới hơn ---

  • Kinh Nghiệm Chọn Mua Phân Bón Lá Npk Nhập Khẩu
  • Tổng Hợp 5 Loại Phân Bón Dưỡng Quả Cam Sành Tăng Độ Ngọt
  • Quy Trình Bón Phân Cho Sầu Riêng Vùng Tây Nguyên
  • Cơ Hội Đến Từ Cổ Phiếu Dầu Khí, Thép Và Phân Bón
  • Bán Buôn, Bán Lẻ Axit Fulvic 90% (Fulvic Acid) Tan Trong Nước
  • Fungi and Food Spoilage

    “This page left intentionally blank.”

    John I. Pitt

    l

    Ailsa D. Hocking

    Fungi and Food Spoilage

    13

    John I. Pitt Honorary Research Fellow CSIRO Food and Nutritional Sciences North Ryde, NSW 2113 Australia

    ISBN 978-0-387-92206-5 e-ISBN 978-0-387-92207-2 DOI 10.1007/978-0-387-92207-2 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2009920217 # Springer ScienceþBusiness Media, LLC 2009 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer ScienceþBusiness Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expssion of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer ScienceþBusiness Media (www.springer.com)

    Preface to the Third Edition

    In contrast to the second edition, the third edition of ”Fungi and Food Spoilage” is evolutionary rather than revolutionary. The second edition was intended to cover almost all of the species likely to be encountered in mainstream food supplies, and only a few additional species have been included in this new edition. The third edition repsents primarily an updating – of taxonomy, physiology, mycotoxin production and ecology. Changes in taxonomy reflect the impact that molecular methods have had on our understanding of classification but, it must be said, have not radically altered the overall picture. The improvements in the understanding of the physiology of food spoilage fungi have been relatively small, reflecting perhaps the lack of emphasis on physiology in modern microbiological science. Much remains to be understood about the specificity of particular fungi for particular substrates, of the influence of water activity on the growth of many of the species treated, and even on such basic parameters as cardinal temperatures for growth and the influence of pH and pservatives. Since 1997, a great deal has been learnt about the specificity of mycotoxin production and in which commodities and products-specific mycotoxins are likely to occur. Changes in our understanding of the ecology of the included species are also in most cases evolutionary. A great number of papers have been published on the ecology of foodborne fungi in the past few years, but with few exceptions the basic ecology of the included species remains. Recent changes in our understanding of foodborne fungi include the realisation that Aspergillus carbonarius is a major source of ochratoxin A in the world food supply, that A. westerdijkiae and not A. ochraceus is the other common Aspergillus species making this toxin and that these species are responsible for ochratoxin A in foods outside the cool temperate regions, where Penicillium verrucosum is the important species. In recent years a number of new species have been found to be capable of producing aflatoxin, but the fact remains that most aflatoxin in the global food supply is produced by A. flavus and A. parasiticus. The taxonomy of Fusarium species is still undergoing major revision. However, the renaming of Fusarium moniliforme as F. verticillioides is the only change of importance here. Recent publications have improved our understanding of species – mycotoxin relationships within Fusarium.

    v

    vi

    Among the colleagues who helped us to ppare this edition, we wish to particularly thank Dr Anne-Laure Markovina, now of the University of Sydney, who assisted in literature searches and some cultural and photographic work, and Mr N.J. Charley who has continued his excellent work of curating the FRR culture collection, on which so much of the descriptive work in this book is based.

    Preface to the Third Edition

    Preface to the First Edition

    vii

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    Preface to the Second Edition

    In planning for the second edition of ”Fungi and Food Spoilage”, we decided that the book would benefit from a larger format, which would permit improved illustrations, and from some expansion of the text, in both numbers of species treated and overall scope. These aims have been realised. The Crown Quarto size has allowed us to include substantially larger, clearer illustrations. Many new photographs and photomicrographs have been added, the latter taken using a Zeiss Axioscop microscope fitted with Nomarski differential interference contrast optics. We have taken the opportunity to include more than 40 additional species descriptions, to add a new section on mycotoxin production for each species and to update and upgrade all of the text. Since the first edition, changes in the climate for stabilising fungal nomenclature have resulted in development of a list of ”Names in Current Use” for some important genera, including Aspergillus and Penicillium. Names of species used in the second edition are taken from that list, which was given special status by the International Botanical Congress, Tokyo, 1994. Names used in this edition have priority over any other names for a particular species. Publication of a list of ”Authors of Fungal Names” (P.M. Kirk and A.E. Ansell, Index of Fungi, Supplement: 1-95, 1992) has also stabilised names of authorities for all fungal species. Abbreviations of authors’ names used in this edition conform to those recommended by Kirk and Ansell. Some progress in standardisation of methods and media has also been made, primarily through the efforts of the International Commission on Food Mycology. The first edition included some 400 references. When we began revisionary work, we felt that the number of references in the area of food mycology had probably doubled or increased by perhaps 150% during the intervening years. In fact, this second edition includes over 1900 references, almost a five-fold increase over the 1985 edition! This provides a clear indication that interest in, and study of, food mycology has greatly increased in recent years. Modern referencing systems have enabled us to expand information from tropical sources, especially in Asia and Africa, but we are conscious of the fact that treatment of fungi found in foods on a worldwide basis remains rather incomplete. We gratefully acknowledge support and assistance from colleagues who have contributed to this new edition. Ms J.C. Eyles formatted and printed the camera

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    x

    ready copy, Ms C. Heenan collated, arranged and formatted the illustrations and Mr N.J. Charley looked after the culture collection, culture growth and colony photography. Without this level of support, the book would not have been completed.

    Preface to the Second Edition

    Contents 1

    Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    1

    2

    The Ecology of Fungal Food Spoilage . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Water Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Hydrogen Ion Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Gas Tension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Consistency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Nutrient Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Specific Solute Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8 Preservatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.9 Conclusions: Food Preservation . . . . . . . . . . . . . . . . . . . . . . . .

    3 3 4 5 7 8 8 8 9 9

    3

    Naming and Classifying Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Taxonomy and Nomenclature: Biosystematics . . . . . . . . . . . . . 3.2 Hierarchical Naming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Zygomycotina. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Ascomycotina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Basidiomycotina . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 The Ascomycete – Conidial Fungus Connection . . . . . . . . . . . . 3.7 Dual Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8 Practical Classification of Fungi . . . . . . . . . . . . . . . . . . . . . . . .

    11 11 12 12 13 15 15 15 16

    4

    Methods for Isolation, Enumeration and Identification . . . . . . . . . . . . 4.1 Sampling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Enumeration Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1 Direct Plating. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2 Dilution Plating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.3 Incubation Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Sampling Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Air Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Isolation Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.1 Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.2 Moulds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.3 Short Term Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Choosing a Suitable Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6.1 General Purpose Enumeration Media. . . . . . . . . . . . . . . 4.6.2 Selective Isolation Media. . . . . . . . . . . . . . . . . . . . . . . . . 4.6.3 Techniques for Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6.4 Techniques for Heat-Resistant Fungi . . . . . . . . . . . . . . . 4.6.5 Other Plating Techniques . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Estimation of Fungal Biomass . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.1 Chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.2 Ergosterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.3 Impedimetry and Conductimetry . . . . . . . . . . . . . . . . . .

    19 19 19 20 21 22 22 23 23 23 24 24 25 26 27 30 32 33 34 34 35 37 xi

    xii

    5

    Contents

    4.7.4 Adenosine Triphosphate (ATP) . . . . . . . . . . . . . . . . . 4.7.5 Fungal Volatiles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7.6 Immunological Techniques . . . . . . . . . . . . . . . . . . . . . 4.7.7 Molecular Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8 Identification Media and Methods. . . . . . . . . . . . . . . . . . . . . . 4.8.1 Standard Methodology . . . . . . . . . . . . . . . . . . . . . . . . 4.8.2 Plating Regimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8.3 Inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8.4 Additional Media and Methods . . . . . . . . . . . . . . . . . 4.8.5 Identification of Fusarium Species. . . . . . . . . . . . . . . . 4.8.6 Yeasts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9 Examination of Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.1 Colony Diameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.2 Colony Characters. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.3 Preparation of Wet Mounts for Microscopy. . . . . . . . 4.9.4 Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.9.5 Microscopes and Microscopy . . . . . . . . . . . . . . . . . . . 4.10 Preservation of Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.10.1 Lyophilisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.10.2 Other Storage Techniques . . . . . . . . . . . . . . . . . . . . . . 4.11 Housekeeping in the Mycological Laboratory. . . . . . . . . . . . . 4.11.1 Culture Mites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.11.2 Problem Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.11.3 Pathogens and Laboratory Safety . . . . . . . . . . . . . . . .

    37 37 38 40 41 41 41 41 42 43 44 45 45 45 46 46 47 48 48 49 50 50 51 51

    Primary Keys and Miscellaneous Fungi . . . . . . . . . . . . . . . . . . . . . . . . 5.1 The General Key . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1 Notes on the General Key . . . . . . . . . . . . . . . . . . . . . . 5.2 Miscellaneous Fungi. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 Genus Acremonium Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Genus Alternaria Nees: Fr.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Genus Arthrinium Kunze . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Genus Aureobasidium Viala and G. Boyer . . . . . . . . . . . . . . . . 5.7 Genus Bipolaris Shoemaker . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8 Genus Botrytis P. Micheli: Fr. . . . . . . . . . . . . . . . . . . . . . . . . . 5.9 Genus Chaetomium Kunze . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.10 Genus Chrysonilia Arx . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.11 Genus Cladosporium Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.12 Genus Colletotrichum Corda . . . . . . . . . . . . . . . . . . . . . . . . . . 5.13 Genus Curvularia Boedijn. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.14 Genus Drechslera S. Ito . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.15 Genus Endomyces Reess . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.16 Genus Epicoccum Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.17 Genus Fusarium Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.18 Genus Geotrichum Link: Fr.. . . . . . . . . . . . . . . . . . . . . . . . . . . 5.19 Genus Hyphopichia Arx and van der Walt. . . . . . . . . . . . . . . . 5.20 Genus Lasiodiplodia Ellis and Everh. . . . . . . . . . . . . . . . . . . . . 5.21 Genus Monascus Tiegh. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.22 Genus Moniliella Stolk and Dakin . . . . . . . . . . . . . . . . . . . . . . 5.23 Genus Nigrospora Zimm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    53 54 55 56 58 60 64 65 67 68 70 73 75 81 82 85 86 88 89 122 124 125 127 129 131

    Contents

    xiii

    5.24 5.25 5.26 5.27 5.28 5.29 5.30

    Genus Pestalotiopsis Steyaert . . . . . . . . . . . . . . . . . . . . . . . . . . Genus Phoma Sacc. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus Stemphylium Wallr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus Trichoconiella B.L. Jain. . . . . . . . . . . . . . . . . . . . . . . . . Genus Trichoderma Pers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus Trichothecium Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genus Ulocladium Preuss . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    133 134 136 137 139 140 142

    6

    Zygomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Genus Absidia Tiegh. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Genus Cunninghamella Matr. . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Genus Mucor P. Micheli: Fr. . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Genus Rhizomucor (Lucet and Costantin) Vuill. . . . . . . . . . . . 6.5 Genus Rhizopus Ehrenb.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 Genus Syncephalastrum J. Schrot. ¨ …………………. 6.7 Genus Thamnidium Link . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    145 148 149 151 157 158 165 167

    7

    Penicillium and Related Genera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1 Genus Byssochlamys Westling . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 Genus Eupenicillium F. Ludw. . . . . . . . . . . . . . . . . . . . . . . . . . 7.3 Genus Geosmithia Pitt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4 Genus Paecilomyces Bainier . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Genus Scopulariopsis Bainier . . . . . . . . . . . . . . . . . . . . . . . . . . 7.6 Genus Talaromyces C.R. Benj.. . . . . . . . . . . . . . . . . . . . . . . . . 7.7 Genus Penicillium Link. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.7.1 Penicillium subgenus Aspergilloides Dierckx . . . . . . . . . 7.7.2 Penicillium subgenus Furcatum Pitt. . . . . . . . . . . . . . . . 7.7.3 Penicillium subgenus Penicillium . . . . . . . . . . . . . . . . . . 7.7.4 Penicillium subgenus Biverticillium Dierckx . . . . . . . . .

    169 170 175 182 183 187 188 194 196 207 223 263

    8

    Aspergillus and Related Teleomorphs. . . . . . . . . . . . . . . . . . . . . . . . . . 8.1 Genus Emericella Berk. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Genus Eurotium Link: Fr. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3 Genus Neosartorya Malloch and Cain . . . . . . . . . . . . . . . . . . . 8.4 Genus Aspergillus Fr.: Fr. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    275 279 281 292 295

    9

    Xerophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1 Genus Basipetospora G.T. Cole and W.B. Kendr. . . . . . . . . . . 9.2 Genus Chrysosporium Corda . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Genus Eremascus Eidam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Genus Polypaecilum G. Sm. . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5 Genus Wallemia Johan-Olsen. . . . . . . . . . . . . . . . . . . . . . . . . . 9.6 Genus Xeromyces L.R. Fraser . . . . . . . . . . . . . . . . . . . . . . . . .

    339 340 342 347 348 350 353

    10

    Yeasts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    357

    11

    Fresh and Perishable Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Spoilage of Living, Fresh Foods . . . . . . . . . . . . . . . . . . . . . . . 11.2 Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.1 Citrus Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.2 Pome Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.3 Stone Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    383 383 383 384 385 386

    xiv

    Contents

    11.3

    11.4 11.5 11.6

    12

    11.2.4 Tomatoes and other Solanaceous Fruit. . . . . . . . . . . 11.2.5 Melons and other Cucurbits . . . . . . . . . . . . . . . . . . . 11.2.6 Grapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.7 Berries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.8 Figs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.9 Tropical Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.1 Peas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.2 Beans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.3 Onions and Garlic . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.4 Potatoes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.5 Roots and Tubers . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.6 Yams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.7 Cassava . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.8 Leafy and other Green Vegetables . . . . . . . . . . . . . . Dairy Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Meats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cereals, Nuts and Oilseeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6.1 Wheat, Barley and Oats. . . . . . . . . . . . . . . . . . . . . . . 11.6.2 Rice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6.3 Maize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6.4 Soybeans and Mung Beans . . . . . . . . . . . . . . . . . . . . 11.6.5 Other Beans and Pulses . . . . . . . . . . . . . . . . . . . . . . . 11.6.6 Sunflower Seed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6.7 Sorghum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6.8 Peanuts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6.9 Cashews and Brazil Nuts . . . . . . . . . . . . . . . . . . . . . . 11.6.10 Almonds, Hazelnuts, Walnuts and Pecans . . . . . . . . 11.6.11 Pistachios . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6.12 Copra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    Spoilage of Stored, Processed and Preserved Foods . . . . . . . . . . . . . . . 12.1 Low Water Activity Foods: Dried Foods . . . . . . . . . . . . . . . . 12.1.1 Cereals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.2 Flour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.3 Pasta. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.4 Bakery Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.5 Maize . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.6 Soybeans, Mung Beans, other Beans and Chickpeas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.7 Nuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.8 Peanuts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.9 Hazelnuts, Walnuts, Pecans and Almonds . . . . . . . . 12.1.10 Pistachio Nuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.11 Other Nuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.12 Coconut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.13 Spices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.14 Coffee Beans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.15 Cocoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1.16 Dried Meat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    387 387 388 389 389 390 391 391 391 391 392 392 392 392 393 393 394 395 395 396 396 397 398 398 398 398 399 399 399 400 401 401 402 403 403 403 404 405 406 406 407 408 409 409 410 410 411 412

    Contents

    xv

    12.2

    Low Water Activity Foods: Concentrated Foods . . . . . . . . . . 12.2.1 Jams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.2 Dried Fruit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.3 Fruit Cakes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.4 Confectionery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.5 Fruit Concentrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.6 Honey and Syrups . . . . . . . . . . . . . . . . . . . . . . . . . . . . Low Water Activity Foods: Salt Foods . . . . . . . . . . . . . . . . . . Intermediate Moisture Foods: Processed Meats . . . . . . . . . . . Heat Processed Acid Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . Preserved Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    412 412 413 414 415 415 416 416 417 418 418 419

    Media Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    423

    Glossary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    427

    References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    431

    Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    503

    12.3 12.4 12.5 12.6 12.7

    Chapter 1

    Introduction

    From the time when primitive man began to cultivate crops and store food, spoilage fungi have demanded their tithe. Fuzzes, powders and slimes of white or black, green, orange, red and brown have silently invaded – acidifying, fermenting, discolouring and disintegrating, rendering nutritious commodities unpalatable or unsafe. Until recently, fungi have generally been regarded as causing only unaesthetic spoilage of food, despite the fact that Claviceps purpurea was linked to human disease more than 200 years ago, and the acute toxicity of macrofungi has long been known. Japanese scientists recognised the toxic nature of yellow rice 100 years ago, but 70 years elapsed before its fungal cause was confirmed. Alimentary toxic aleukia killed many thousands of people in the USSR in 1944-1947; although fungal toxicity was suspected by 1950, the causal agent, T-2 toxin, was not clearly recognised for another 25 years. Forgacs and Carll (1952), in a prophetic article, warned of the danger from common spoilage fungi, but it was not until 1960, when the famous “Turkey X” disease killed 100,000 turkey poults in Great Britain, and various other disasters followed in rapid succession, that the Western world became aware that common spoilage moulds could produce significant toxins. Since 1960 a seemingly endless stream of toxigenic fungi and potentially toxic compounds has been discovered. On these grounds alone, the statement ”It’s only a mould” is no longer acceptable to food microbiologist, health inspector or consumer. The demand for accurate identification and characterisation of food spoilage fungi has become urgent. In the flurry of research into mycotoxins, however, it must not be forgotten that food spoilage as such

    J.I. Pitt, A.D. Hocking, Fungi and Food Spoilage, DOI 10.1007/978-0-387-92207-2_1, Ó Springer ScienceþBusiness Media, LLC 2009

    1

    2

    the process applied. As examples we can take Polypaecilum pisce on salt fish, Xeromyces bisporus on fruit cake, Cladosporium herbarum on refrigerated meat, Zygosaccharomyces bailii in pserved juices, Z. rouxii in jams and fruit concentrates, Aspergillus flavus on peanuts, Eurotium chevalieri on hazel nuts, Penicillium roqueforti on cheeses, Byssochlamys fulva in acid canned foods . . . the list of quite specific food – fungus associations is extensive. The study of such associations is one of the more important branches of the young discipline, food mycology. This book sets out to document current knowledge on the interaction of foods and fungi, in the context of spoilage and toxicity, not food production or biotechnology. Four aspects are examined. First, ecology: what factors in foods select for particular kinds of fungi? A chapter is devoted to the physical and chemical parameters which influence the growth of fungi in foods. Second, methodology: how do we isolated fungi from foods? What are the best media to use? How do we go about identifying food spoilage fungi? Third, the commodity: what fungi are usually associated with a particular food? Here ecological factors interact to produce a more or less specific habitat. Major classes of foods and their associated spoilage fungi are described. Finally, the fungus: what fungus is that? In a series of chapters, the main food spoilage moulds and yeasts are described and keyed, together with others commonly associated with food but not noted for spoilage. Where possible, further information is

    1

    Introduction

    given on known habitats and sources, physiology, heat resistance, etc., together with a selective bibliography. Accurate information on mycotoxin production is also included. As far as possible, the pcise terminology for fungal structures used by the pure mycologist and indeed most necessary for him has been avoided in these chapters. Some concepts and terms are of course essential: these have been introduced as needed and are listed in a glossary. The taxonomic sections of this book are designed to facilitate identification of food spoilage and common food contaminant fungi. A standardised plating regimen is used, originally developed for the identification of Penicillium species (Pitt, 1979b) and extended here to other genera relevant to the food industry. Under this regimen, cultures are incubated for 1 week at 5, 25 and 378C on a single standard medium and at 258C on two others. In conjunction with the appropriate keys, this system will enable identification of most foodborne fungi to species level in just 7 days. For a few kinds of fungi, notably yeasts and xerophiles, subsequent growth under other more specialised conditions will be necessary. Finally, this book is dedicated to the general food microbiologist. May it help to restore equilibrium and assist in continued employment, when the quality assurance manager demands: ”What is it?” . . . ”How did it get in?” . . . ”What does it do?” . . . ”How do we get rid of it?” . . . and, worst of all . . . ”Is it toxic?”

    Chapter 2

    The Ecology of Fungal Food Spoilage

    Food is not commonly regarded as an ecosystem, perhaps on the basis that it is not a ”natural” system. Nevertheless an ecosystem it is and an important one, because food plants and the fungi that colonise their fruiting parts (seeds and fruit) have been co-evolving for millennia. The seed and nut caches of rodents have provided a niche for the development of storage fungi. Fallen fruit, as they go through the cycle of decay and desiccation, have provided substrate for a range of fungi. Humans have aided and abetted the development of food spoilage fungi through their vast and varied food stores. It can be argued, indeed, some rapidly evolving organisms, such as haploid asexual fungi, are moving into niches created by man’s exploitation of certain plants as food. Food by its very nature is expected to be nutritious: therefore, food is a rich habitat for microorganisms, in contrast with the great natural systems, soil, water and plants. Given the right physico-chemical conditions, only the most fastidious microorganisms are incapable of growth in foods, so that factors other than nutrients usually select for particular types of microbial populations. Perhaps the most important of these factors relates to the biological state of the food. Living foods, particularly fresh fruits, vegetables, and also grains and nuts before harvest, possess powerful defence mechanisms against microbial invasion. The study of the spoilage of such fresh foods is more properly a branch of plant pathology than food microbiology. The overriding factor determining spoilage of a fresh, living food is the ability of specific microorganisms to overcome defence mechanisms. Generally speaking, then, spoilage of

    fresh foods is limited to particular species. Such specific relationships between fresh food and fungus are discussed in Chapter 11 and under particular species. Other kinds of foods are moribund, dormant or nonliving, and the factors which govern spoilage are physical and chemical. There are eight principal factors: (l) water activity; (2) hydrogen ion concentration; (3) temperature – of both processing and storage; (4) gas tension, specifically of oxygen and carbon dioxide; (5) consistency; (6) nutrient status; (7) specific solute effects; and (8) pservatives. Each will be discussed in turn below.

    2.1 Water Activity Water availability in foods is most readily measured as water activity. Water activity (aw), is a physicochemical concept, introduced to microbiologists by Scott (1957), who showed that aw effectively quantified the relationship between moisture in foods and the ability of microorganisms to grow on them. Water activity is defined as a ratio: aw ¼ p=po ; where p is the partial pssure of water vapour in the test material and po is the saturation vapour pssure of pure water under the same conditions.

    J.I. Pitt, A.D. Hocking, Fungi and Food Spoilage, DOI 10.1007/978-0-387-92207-2_2, Ó Springer ScienceþBusiness Media, LLC 2009

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    Water activity is numerically equal to equilibrium relative humidity (ERH) expssed as a decimal. If a sample of food is held at constant temperature in a sealed enclosure until the water in the sample equilibrates with the water vapour in the enclosed air space (Fig. 2.1a), then aw ðfoodÞ ¼ ERH ðairÞ=100: Conversely, if the ERH of the air is controlled in a suitable way, as by a saturated salt solution, at equilibrium the aw of the food will be numerically equal to the generated ERH (Fig. 2.1b). In this way, aw can be experimentally controlled, and the relation of aw to moisture (the sorption isotherm) can be studied. For further information on water activity, its measurement and significance in foods see Duckworth (1975); Pitt (1975); Troller and Christian (1978); Rockland and Beuchat (1987). In many practical situations, aw is the dominant environmental factor governing food stability or spoilage. A knowledge of fungal water relations will then enable pdiction both of the shelf life of foods and of potential spoilage fungi. Although the water relations of many fungi will be considered inpidually in later chapters, it is pertinent here to provide an overview. Like all other organisms, fungi are profoundly affected by the availability of water. On the aw scale, life as we know it exists over the range 0.9999þ to 0.60 (Table 2.1). Growth of animals is virtually confined to 1.0-0.99 aw; the permanent wilt point of mesophytic plants is near 0.98 aw; and most microorganisms cannot

    Fig. 2.1 The concept of water activity (aw) (a) the relationship between aw and equilibrium relative humidity (ERH); (b) one method of controlling aw by means of a saturated salt solution, which generates a specific ERH at a specific constant temperature

    The Ecology of Fungal Food Spoilage

    grow below 0.95 aw. A few halophilic algae and bacteria can grow in saturated sodium chloride (0.75 aw), but are confined to salty environments. Ascomycetous fungi and conidial fungi of ascomycetous origin comprise most of the organisms capable of growth below 0.9 aw. Fungi capable of growth at low aw, in the psence of extraordinarily high solute concentrations both inside and out, must be ranked as among the most highly evolved organisms on earth. Even among the fungi, this evolutionary path must have been of the utmost complexity: the ability to grow at low aw is confined to only a handful of genera (Pitt, 1975). The degree of tolerance to low aw is most simply expssed in terms of the minimum aw at which germination and growth can occur. Fungi able to grow at low aw are termed xerophiles: one widely used definition is that a xerophile is a fungus able to grow below 0.85 aw under at least one set of environmental conditions (Pitt, 1975). Xerophilic fungi will be discussed in detail in Chapter 9. Information about the water relations of many fungi remains fragmentary, but where it is known it has been included in later chapters.

    2.2 Hydrogen Ion Concentration At high water activities, fungi compete with bacteria as food spoilers. Here pH plays the decisive role. Bacteria flourish near neutral pH and fungi cannot compete unless some other factor, such as low water

    2.3 Temperature

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    Table 2.1 Water activity and microbial water relations in perspectivea aw

    Perspective

    Foods

    1.00

    Blood, plant wilt point, seawater

    0.95

    Most bacteria

    Vegetables meat, milk fruit Bread

    0.90 0.85 0.80

    Staphylococcus aureus

    Ham Dry salami

    0.75

    Salt lake Halophiles

    0.70

    Jams Salt fish Fruit cake Confectionery Dried fruit Dry grains

    Moulds

    Yeasts

    Basidiomycetes Most soil fungi Mucorales Fusarium Rhizopus, Cladosporium Aspergillus flavus Xerophilic Penicillia Xerophilic Aspergilli Wallemia Eurotium Chrysosporium Eurotium halophilicum

    Basidiomycetes Most ascomycetes Zygosaccharomyces rouxii (salt) Zygosaccharomyces bailii Debaryomyces hansenii

    0.65 Xeromyces bisporus Zygosaccharomyces rouxii (sugar) 0.60 DNA disordered a Modified from data of J.I. Pitt as reported by Brown (1974). Water activities shown for microorganisms approximate minima for growth reported in the literature.

    activity or a pservative, renders the environment hostile to the bacteria. As pH is reduced below about 5, growth of bacteria becomes progressively less likely. Lactic acid bacteria are exceptional, as they remain competitive with fungi in some foods down to about pH 3.5. Most fungi are little affected by pH over a broad range, commonly 3-8 (Wheeler et al., 1991). Some conidial fungi are capable of growth down to pH 2, and yeasts down to pH 1.5. However, as pH moves away from the optimum, usually about pH 5, the effect of other growth limiting factors may become apparent when superimposed on pH. Figure 2.2 is an impssion of the combined influence of pH and aw on microbial growth: few accurate data points exist and the diagram is schematic. For heat-processed foods, pH 4.5 is of course critical: heat processing to destroy the spores of Clostridium botulinum also destroys all fungal spores. In acid packs, below pH 4.5, less severe processes may permit survival of heat-resistant fungal spores (Section 2.3).

    2.3 Temperature The influence of temperature in food pservation and spoilage has two separate facets: temperatures during processing and those existing during storage.

    As noted above, heat-resistant fungal spores may survive pasteurising processes given to acid foods. Apart from a few important species, little information exists on the heat resistance of fungi. Much of the information that does exist must be interpted with care, as heating menstrua and conditions can vary markedly, and these may profoundly affect heat resistance. High levels of sugars are generally protective (Beuchat and Toledo, 1977). Low pH and pservatives increase the effect of heat (Beuchat, 1981a, b; Rajashekhara et al., 2000) and also hinder resuscitation of damaged cells (Beuchat and Jones, 1978). Ascospores of filamentous fungi are more heat resistant than conidia (Pitt and Christian, 1970; Table 2.2). Although not strictly comparable, data of Put et al. (1976) indicate that the heat resistance of yeast ascospores and vegetative cells is of the same order as that of fungal conidia. Among the ascomycetous fungi, Byssochlamys species are notorious for spoiling heat processed fruit products (Olliver and Rendle, 1934; Richardson, 1965). The heat resistance of B. fulva ascospores varies markedly with isolate and heating conditions (Beuchat and Rice, 1979): a D value between 1 and 12 min at 908C (Bayne and Michener, 1979) and a z value of 6-7C8 (King et al., 1969) are practical working ps. The heat resistance of

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    The Ecology of Fungal Food Spoilage

    Fig. 2.2 A schematic diagram showing the combined influence of water activity and pH on microbial growth

    B. nivea ascospores is marginally lower (Beuchat and Rice, 1979; Kotzekidou, 1997a). Ascospores of Neosartorya fischeri have a similar heat resistance to those of Byssochlamys fulva, but have been reported less frequently as a cause of food spoilage. Heat resistant fungi are discussed further in Chapter 4. Food products may be stored at ambient temperatures, in which case pvention of spoilage relies on other parameters, or under refrigeration, where temperature is expected to play a pservative

    role. Food frozen to -108C or below appears to be microbiologically stable, despite some reports of fungal growth at lower temperatures. The lowest temperatures for fungal growth are in the range -7 to 08C, for species of Fusarium, Cladosporium, Penicillium and Thamnidium (Pitt and Hocking, 1997). Nonsterile food stored at ca. 58C in domestic refrigerators, where conditions of high humidity pvail, will eventually be spoiled by fungi of these genera. At high aw and neutral pH, psychrophilic bacteria may also be important (mostly Pseudomonas species).

    Table 2.2 Comparative heat resistance of ascospores and conidiaa Survivors (%) Fungus

    Spore type

    Initial viable count/ml

    Ascospores 5.0 102 Conidia 7.3 102 Eurotium chevalieri Ascospores 1.0 103 Conidia 8.9 102 Xeromyces bisporus Ascospores 1.0 103 Aspergillus candidus Conidia 3.8 102 Wallemia sebi Conidia 7.1 102 a Heated at temperatures shown for 10 min. Data from Pitt and Christian (1970). Eurotium amstelodami

    508C

    608C

    708C

    93 107 103 128 93 102 42

    85 0.3 62 0.1 30 0 0

    3 0 21 0 0.3 0 0

    2.4 Gas Tension

    Thermophilic fungi, i.e. those which grow only at high temperatures, are rarely of significance in food spoilage. If overheating of commodities occurs, however, in situations such as damp grain, thermophiles can be a very serious problem. Thermotolerant fungi, i.e. species able to grow at both moderate and high temperatures, are of much greater significance. Aspergillus flavus and A. niger, able to grow between ca. 8 and 458C, are among the most destructive moulds known.

    2.4 Gas Tension Food spoilage moulds, like almost all other filamentous fungi, have an absolute requirement for oxygen. However, many species appear to be efficient oxygen scavengers, so that the total amount of oxygen available, rather than the oxygen tension, determines growth. The concentration of oxygen dissolved in the substrate has a much greater influence on fungal growth than atmospheric oxygen tension (Miller and Golding, 1949). For example, Penicillium expansum grows virtually normally in 2.1% oxygen over its entire temperature range (Golding, 1945), and many other common food spoilage fungi are inhibited only slightly when grown in nitrogen atmospheres containing approximately 1.0% oxygen (Hocking, 1990). Paecilomyces variotii produced normal colonies at 258C under 650 mm of vacuum (Pitt, unpublished). Most food spoilage moulds appear to be sensitive to high levels of carbon dioxide, although there are notable exceptions. When maintained in an atmosphere of 80% carbon dioxide and 4.2% oxygen, Penicillium roqueforti still grew at 30% of the rate in air (Golding, 1945), provided that the temperature was above 208C. In 40% CO2 and 1% O2, P. roqueforti grew at almost 90% of the rate in air (Taniwaki et al., 2001a). Xeromyces bisporus has been reported to grow in similar levels of carbon dioxide (Dallyn and Everton, 1969). Byssochlamys species appear to be particularly tolerant of conditions of reduced oxygen and/or elevated carbon dioxide. Growth of Byssochlamys nivea was little affected by replacement of nitrogen in air by carbon dioxide, and growth in carbon dioxide-air mixtures was proportional only to

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    oxygen concentration, at least up to 90% carbon dioxide (Yates et al., 1967). Both Byssochlamys nivea and B. fulva were capable of growth in atmospheres containing 20, 40 or 60% carbon dioxide with less than 0.5% oxygen, but inhibition increased with increasing carbon dioxide concentration (Taniwaki et al., 2001a). Byssochlamys fulva is capable of growth in 0.27% oxygen, but not in its total absence (King et al., 1969). It is also capable of fermentation in fruit products, but psumably only if some oxygen is psent. At least some species of Mucor, Rhizopus and Fusarium are able to grow and ferment in bottled liquid products and sometimes cause fermentative spoilage. Growth under these conditions may be yeast-like. Species of Mucor, Rhizopus and Amylomyces used as starter cultures in Asian fermented foods can grow under anaerobic conditions, demonstrated by growth in an anaerobe jar with a hydrogen and carbon dioxide generator (Hesseltine et al., 1985). Other authors have reported growth under anaerobic conditions of such fungi as Mucor species, Absidia spinosa, Geotrichum candidum, Fusarium oxysporum and F. solani (Stotzky and Goos, 1965; Curtis, 1969; Taniwaki, 1995). The yeast-like fungus Moniliella acetoabutans can cause fermentative spoilage under totally anaerobic conditions (Stolk and Dakin, 1966). As a generalisation, however, it is still correct to state that most food spoilage problems due to filamentous fungi occur under aerobic conditions, or at least where oxygen tension is appciable, due to leakage or diffusion through packaging. In contrast, Saccharomyces species, Zygosaccharomyces species and other fermentative yeasts are capable of growth in the complete absence of oxygen. Indeed, S. cerevisiae and Z. bailii can continue fermentation under several atmospheres pssure of carbon dioxide. This property of S. cerevisiae has been harnessed by mankind for his own purposes, in the manufacture of bread and many kinds of fermented beverages. Z. bailii, on the other hand, is notorious for its ability to continue fermenting at reduced water activities in the psence of high levels of pservatives. Fermentation of juices and fruit concentrates may continue until carbon dioxide pssure causes container distortion or explosion. The closely related species Zygosaccharomyces rouxii is a xerophile and causes

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    spoilage of low-moisture liquid or packaged products such as fruit concentrates, jams and dried fruit. The difference in oxygen requirements between moulds and fermentative yeasts is one of the main factors determining the kind of spoilage a particular commodity will undergo.

    2.6 Nutrient Status As noted in the pamble to this chapter, the nutrient status of most foods is adequate for the growth of any spoilage microorganism. Generally speaking, however, it appears that fungal metabolism is best suited to substrates high in carbohydrates, whereas bacteria are more likely to spoil proteinaceous foods. Lactobacilli are an exception. Most common mould species appear to be able to assimilate any food-derived carbon source with the exception of hydrocarbons and highly condensed polymers such as cellulose and lignin. Most moulds are equally indifferent to nitrogen source, using nitrate, ammonium ions or organic nitrogen sources with equal ease. Some species achieve only limited growth if amino acids or proteins must provide both carbon and nitrogen. A few isolates classified in Penicillium subgen. Biverticillium are unable to utilise nitrate (Pitt, 1979b).

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    The Ecology of Fungal Food Spoilage

    Some xerophilic fungi are known to be more demanding. Ormerod (1967) showed that growth of Wallemia sebi was strongly stimulated by proline. Xerophilic Chrysosporium species and Xeromyces bisporus also require complex nutrients, but the factors involved have not been defined (Pitt, 1975). Yeasts are often fastidious. Many are unable to assimilate nitrate or complex carbohydrates; a few, Zygosaccharomyces bailii being an example, cannot grow with sucrose as a sole source of carbon. Some require vitamins. These factors limit to some extent the kinds of foods susceptible to spoilage by yeasts. A further point on nutrients in foods is worth making here. Certain foods (or nonfoods) lack nutrients essential for the growth of spoilage fungi. Addition of nutrient, for whatever reason, can turn a safe product into a costly failure. Two cases from our own experience illustrate this point, both involving spoilage by the pservativeresistant yeast Zygosaccharomyces bailii. In the first, a highly acceptable (and nutritious) carbonated beverage containing 25% fruit juice was eventually forced from the Australian market because it was impractical to ppare it free of occasional Z. bailii cells. Effective levels of pservative could not be added legally and pasteurisation damaged its flavour. Substitution of the fruit juice with artificial flavour and colour removed the nitrogen source for the yeast. A spoilage free product resulted, at the cost of any nutritional value and a great reduction in consumer acceptance. The other case concerned a popular water-ice confection, designed for home freezing. This confection contained sucrose as a sweetener and a pservative effective against yeasts utilising sucrose. One production season the manufacturer decided, for consumer appeal, to add glucose to the formulation. The glucose provided a carbon source for Zygosaccharomyces bailii, and as a result several months production, valued at hundreds of thousands of dollars, was lost due to fermentative spoilage.

    2.7 Specific Solute Effects As stated earlier, microbial growth under conditions of reduced water availability is most satisfactorily described in terms of aw. However,

    2.9 Conclusions: Food Preservation

    the particular solutes psent in foods can exert additional effects on the growth of fungi. Scott (1957) reported that Eurotium (Aspergillus) amstelodami grew 50% faster at its optimal aw (0.96) when aw was controlled by glucose rather than magnesium chloride, sodium chloride or glycerol. Pitt and Hocking (1977) showed a similar effect for Eurotium chevalieri and reported that the extreme xerophiles Chrysosporium fastidium and Xeromyces bisporus grew poorly if at all in media containing sodium chloride as the major solute. In contrast Pitt and Hocking (1977) and Hocking and Pitt (1979) showed that germination and growth of several species of Aspergillus and Penicillium was little affected when medium aw was controlled with glucose-fructose, glycerol or sodium chloride. Zygosaccharomyces rouxii, the second most xerophilic organism known, has been reported to grow down to 0.62 aw in fructose (von Schelhorn, 1950). Its minimum aw for growth in sodium chloride is reportedly much higher, 0.85 aw (Onishi, 1963). Some fungi are halophilic, being well adapted to salty environments such as salted fish. Basipetospora halophila and Polypaecilum pisce grow more rapidly in media containing NaCl as controlling solute (Andrews and Pitt, 1987; Wheeler et al., 1988c). Such fungi have been called halophilic xerophiles to distinguish them from obligately halophilic bacteria.

    2.8 Preservatives Obviously, pservatives for use in foods must be safe for human consumption. Under this constraint, food technologists in most countries are limited to the use of weak acid pservatives: benzoic, sorbic, nitrous, sulphurous, acetic and propionic acids – or, less commonly, their esters. In the concentrations permitted by most food laws, these acids are useful only at pH levels up to their pKa plus one pH unit, because to be effective they must be psent as the undissociated acid. For studies of the mechanism of action of weak acid pservatives see Warth (1977, 1991); Brul and Coote (1999); Stratford and Anslow (1998) and Stratford and Lambert (1999).

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    The use of chemical pservatives in foods is limited by law in most countries to relatively low levels and to specific foods. A few fungal species possess mechanisms of resistance to weak acid pservatives, the most notable being Zygosaccharomyces bailii. This yeast is capable of growth and fermentation in fruit-based cordials of pH 2.9-3, of 458C Brix and containing 800 mg/L of benzoic acid (Pitt and Hocking, 1997). The yeast-like fungus Moniliella acetoabutans can grow in the psence of 4% acetic acid and survive in 10% (Pitt and Hocking, 1997). Of the filamentous fungi, Penicillium roqueforti appears to be especially resistant to weak acid pservatives and this property has been suggested as a useful aid to isolation and identification (Engel and Teuber, 1978).

    2.9 Conclusions: Food Preservation It is evident from the above discussion that the growth of fungi in a particular food is governed largely by a series of physical and chemical parameters, and definition of these can assist greatly in assessing the food’s stability. The situation in practice is made more complex by the fact that such factors frequently do not act independently, but synergistically. If two or more of the factors outlined above act simultaneously, the food may be safer than expected. This has been described by Leistner and Rodel (1976) as the ”hurdle concept”. ¨ This concept has been evaluated carefully for some commodities such as fermented sausages and is now widely exploited in the production of shelf stable bakery goods and acid sauces. For most fungi, knowledge remains meagre about the influence of the eight parameters discussed here on germination and growth. However, sufficient information is now available that some rationale for spoilage of specific commodities by certain fungi can be attempted, especially where one or two parameters are of overriding importance. This topic is considered in later chapters devoted to particular commodities.

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    Chapter 3

    Naming and Classifying Fungi

    As with other living organisms, the name applied to any fungus is a binomial, a capitalised genus name followed by a lower case species name, both written in italics or underlined. The classification of organisms in genera and species was a concept introduced by Linneaus in 1753 and it is the keystone of biological science. It is as fundamental to the biologist as Arabic decimal numeration is to the mathematician. Here the analogy ends: the concept of ”base 10” is rigorous; the concept of a species, fundamental as it is, is subjective and dependent on the knowledge and concepts of the biologist who described it.

    3.1 Taxonomy and Nomenclature: Biosystematics Once biologists began to describe species and to assemble them into genera, questions about their relationships began to arise: is species x described by Jones in 1883 the same as species y described by Smith in 1942? Does species z, clearly distinct from x and y in some characters, belong to the same genus? The study of these relationships is termed taxonomy. Modern taxonomy is based on sound scientific principles, but still involves subjective judgment. When the decision is made that species x and species y are the same, however, the taxonomist must follow clearly established procedures in deciding which name must be used (”has priority”). The application of these procedures is termed nomenclature and, for fungi, plants and algae, is governed by the International Code of Botanical Nomenclature (ICBN). The ICBN is a relatively complex document of about 70 Articles dealing with all aspects of

    correctly naming plants, algae and fungi. It is amended every 6 years by special sessions at each International Botanical Congress and is republished thereafter. The 17th version of the ICBN (the Vienna code) is the most recently published (McNeill et al., 2006). The ICBN impinges only indirectly on the work of the practicing mycologist or microbiologist. It is nevertheless of vital importance to the orderly naming of all plant life; to ignore the ICBN is to invite chaos. Where confusion arises over the correct name for a botanical species – a constant source of irritation to the nontaxonomist – it stems usually from one of three causes: indecision by, or disagreement among, taxonomists on what constitutes a particular species; incorrect application of the provisions of the ICBN; or ignorance of earlier literature. To return to our example, when species x and species y are seen to be the same, x has priority because it was published earlier; y becomes a synonym of x. Important synonyms are often listed after a name to aid the user of a taxonomy, and this procedure has been followed here. Through ignorance, the same species name may be used more than once, for example, Penicillium thomii Maire 1915 and P. thomii K.M. Zalessky 1927. The name P. thomii has been given to two quite different fungi. Clearly P. thomii Maire has priority; the later name is not valid. To avoid ambiguity, correct practice in scientific publication is to cite the author of a species at first mention, and before any formal description. The ICBN provides rules to govern change of genus name also. In our example, if species z is transferred to the genus to which species x and y belong, it retains its species name but takes the new

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    genus name. The original author of the name z is placed in brackets after the species name, followed by the name of the author who transferred it to the correct genus. For example, Citromyces glaber Wehmer 1893 became Penicillium glabrum (Wehmer) Westling 1893 on transfer to Penicillium by Westling in 1911. Note the use of Latinised names: glaber (masculine) became glabrum (neuter) to agree with the gender of the genus to which it was transferred. Further points on the use of the ICBN arise from this example. P. glabrum retains its date of original publication, and therefore takes priority over P. frequentans Westling 1911 if the two species are combined. When Raper and Thom (1949) combined the two species, a taxonomically correct decision, they retained the name P. frequentans, which was nomenclaturally incorrect, causing confusion when Subramanian (1971) and Pitt (1979b) took up the correct name. It is worth pointing out that the confusion in this and similar situations arose from Raper and Thom’s action in ignoring the provisions of the ICBN, not from that of later taxonomists who correctly interpted it.

    connection between the Ascomycetes, the fungi that produce sexual spores in sacks, and the Deuteromycetes, where spores are always asexual, has been known for a long time. However, molecular taxonomy has provided the fundamental assurance needed to make this change. From the point of view of the food mycologist, this is a mixed blessing. The demands of the molecular systematists may yet make the taxonomy of foodborne fungi even more complicated. The taxonomic system used here is believed to be both practical and in line with the current ”best practice” of the nomenclaturalists. The hierarchical subpisions in Kingdom Fungi of interest in the psent context are shown below, using as examples three genera and species important in food spoilage:

    3.2 Hierarchical Naming

    Note that names of genera, species and varieties are italicised or underlined, while higher taxonomic ranks are not. Three subkingdoms of the kingdom Fungi include genera of significance in food spoilage. As indicated in the examples above, these are Zygomycotina, Ascomycotina and (much less commonly) Basidiomycotina. Fungi from each of these subkingdoms have quite distinct properties, shared with other genera and species from the same subkingdom. Unlike other texts, this book will not rely on initial recognition of a correct subkingdom before identification of genus and species can be undertaken. Nevertheless, identification of the subkingdom can provide valuable information about a fungus, so the principal properties of these three subkingdoms are described below.

    A given biological entity, or taxon in modern terminology, can be given a hierarchy of names: a cluster of related species is grouped in a genus, of related genera in families, of families in orders, orders in classes, and classes in subkingdoms. Similarly a species can be pided into smaller entities: subspecies, varieties and formae speciales (a term usually reserved for plant pathogens). In most modern classifications, the fungi are ranked, like plants and animals, as a separate kingdom. Traditionally, fungi have been pided into several subkingdoms, based on spore type and some environmental considerations. Modern molecular methods have revolutionised this. Fungi have been shown to be more closely related to animals than plants, where traditional taxonomy has always placed them. Some of the so-called ”lower fungi” have been shown not to be fungi of all (though mycologists will no doubt continue to study them). The most important change from the point of view of the food mycologist is the demise of the subkingdom Deuteromycotina, and its absorption (almost entirely) into the Ascomycotina. The

    Kingdom Subkingdom Class Order Family Genus Species Variety

    Fungi Zygomycotina Zygomycetes Mucorales Mucoraceae Rhizopus stolonifer

    Fungi Ascomycotina Plectomycetes Eurotiales Trichocomaceae Eurotium chevalieri intermedius

    Fungi Basidiomycotina Wallemiomycetes Wallemiales Wallemiaceae Wallemia sebi

    3.3 Zygomycotina Most fungi within the subkingdom Zygomycotina belong to the class Zygomycetes. Fungi in this class possess three distinctive properties:

    3.4 Ascomycotina

    1. Rapid growth. Most isolates grow very rapidly, often filling a Petri dish of malt extract agar with loose mycelium in 2-4 days. 2. Nonseptate mycelium. Actively growing mycelia are without septa (cross walls) and are essentially unobstructed. This allows rapid movement of cell contents, termed ”protoplasmic streaming”, which can be seen readily by transmitted light under the binocular microscope. In wet mounts the absence of septa is usually obvious (Fig. 3.1a). 3. Reproduction by sporangiospores. The reproductive structure characteristic of Zygomycetes is the sporangiospore, an asexually produced spore which in genera of interest here is usually produced inside a sac, the sporangium, on the end of a long specialised hypha. Sporangiospores are produced very rapidly. From the food spoilage point of view, the outstanding properties of Zygomycetes are very rapid growth, especially in fresh foods of high water activity; inability to grow at low water activities (no Zygomycetes are xerophiles); and lack of resistance to heat and chemical treatments. From the food safety point of view, Zygomycetes have rarely been reported to produce mycotoxins.

    3.4 Ascomycotina The subkingdom Ascomycotina is distinguished from Zygomycotina by a number of fundamental characters, the most conspicuous being the production of septate mycelium (Fig. 3.1b). Consequent on

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    this, growth of fungi in this subkingdom is usually slower than that of Zygomycetes, although there are some exceptions. Fungi in the subkingdom Ascomycotina, loosely called ”ascomycetes”, characteristically produce their reproductive structures, ascospores, within a sac called the ascus (plural, asci, Fig. 3.2a, b). In most fungi, nuclei normally exist in the haploid state. At one point in the ascomycete life cycle, diploid nuclei are produced by nuclear fusion, which may or may not be pceded by fusion of two mycelia. These nuclei undergo meiosis within the ascus, followed by a single mitotic pision and then differentiation into eight haploid ascospores. In most genera relevant to this work, asci can be recognised in stained wet mounts by their shape, which is spherical to ellipsoidal and smoothly rounded; size, which is generally 8-15 mm in diameter; and the psence when maturity approaches of eight ascospores tightly packed within their walls. At maturity asci often rupture to release the ascospores, which are thick walled, highly refractile, and often strikingly ornamented (Fig. 3.2c, d). Two other characteristics of asci are significant: generally they mature slowly, after incubation for 10 days or more at 258C, and they are usually borne within a larger, macroscopic body, the general term for which is ascocarp. Genera of interest here usually produce asci and ascospores within a spherical, smooth-walled body, the cleistothecium (Fig. 3.3a), or a body with hyphal walls, the gymnothecium (Fig. 3.3b). Ascospores are highly condensed, refractile spores, which are often resistant to heat, pssure

    Fig. 3.1 (a) Nonseptate mycelium of Syncephalastrum racemosum; (b) septate mycelium of Fusarium equiseti

    14

    3

    Naming and Classifying Fungi

    Fig. 3.2 Asci and ascocarps: (a) asci of Talaromyces species; (b) asci of Byssochlamys fulva; (c) ascospores of Eupenicillium alutaceum; (d) ascospores of Neosartorya quadricincta. Bars ¼ 5 mm

    and chemicals. Almost all xerophilic fungi are ascomycetes. Besides their sexual spores, ascospores, ascomycetes commonly produce asexual spores. Formed after mitotic nuclear pision, these spores are borne singly or in chains, in most genera of interest here from more or less specialised hyphal structures. The general term for this type of spore is conidium (plural, conidia), but other more specialised terms

    exist for specific kinds of conidia. Along the evolutionary process, some Ascomycetes with welldeveloped asexual stages lost the ability to produce ascospores, and rely entirely on conidia for dispersal. Conidia, and the specialised hyphae from which they are borne, are astonishingly perse in appearance. The size, shape and ornamentation of conidia and the complexity of the structures producing them

    Fig. 3.3 (a) Cleistothecia of Eupenicillium; (b) gymnothecia of Talaromyces. SEM. Bars = 50 mm

    3.7 Dual Nomenclature

    provide the basis for classification of Ascomycetes that no longer produce the ascosporic (sexual) stage. Lacking ascospores, conidial fungi are not usually heat resistant, but conidia may be quite resistant to chemicals. Some conidial fungi are xerophilic.

    3.5 Basidiomycotina The Subkingdom Basidiomycotina includes mushrooms, puffballs and the plant pathogenic rusts and smuts. Until recently it was not considered of any interest to the food mycologist. However, molecular studies indicate that the small brown species Wallemia sebi, long a curiosity because of its lack of resemblance to any other fungus, is a basidiomycete. It has no obvious phylogenetic affinity with any other genus and has now been classified in its own order, Wallemiales (Zalar et al., 2005). Only one other species of foodborne fungi, Trichosporonoides nigrescens Hocking and Pitt (1981), has a known affinity with this subkingdom.

    3.6 The Ascomycete – Conidial Fungus Connection It was established more than a century ago that many fungal species carry the genetic information to produce both ascospores and conidia. These two kinds of spores are produced by different mechanisms and have different functions, so they are not always formed simultaneously. Not surprisingly, mycologists sometimes have given different generic and species names to a single fungus producing both an ascosporic and a conidial state. The usage of these names under the ICBN depends on the circumstances under which they were originally given. Some of these circumstances are discussed briefly below. The ascomycete state, now usually referred to as the teleomorph, is regarded by nomenclaturalists as the more important reproductive state, and the name applied to the teleomorph should be used when the ascomycete state is psent. If the conidial state is also in evidence, the fungus is now a holomorph and is still correctly known by the teleomorph name. If the conidial state, known as the

    15

    anamorph, has a separate name, this strictly speaking applies to the conidial state. It should be used only when the ascomycete state is absent, or to refer specifically to the conidial state if the ascomycete is psent. However, the reader is warned that some anamorphic names are, and will continue to be, in common use for holomorphic fungi. Under the Articles of the ICBN, a generic name originally given to an anamorphic or conidial fungus cannot be used for a teleomorphic or ascomycetous fungus. For example, the name Penicillium, originally given to an anamorphic fungus with no known teleomorph, cannot be used for the teleomorphs later found to be produced by other Penicillium species. Such teleomorphs are classified in the genera Eupenicillium or Talaromyces, depending on whether ascospores are produced in cleistothecia or gymnothecia. Correct species names for the ascomycetous and conidial states of a single holomorphic fungus may or may not be the same, depending both on the circumstance in which the names were originally given, and on later synonymy. For example, Eupenicillium ochrosalmoneum Scott and Stolk and Penicillium ochrosalmoneum Udagawa refer to the teleomorph and anamorph of a single fungus. Udagawa (1959) described the anamorph; the teleomorph was later found, in the same isolate, by Scott and Stolk (1967). On the other hand, the anamorph of Eupenicillium cinnamopurpureum Scott and Stolk (1967) is Penicillium phoeniceum van Beyma (1933), with P. cinnamopurpureum Abe ex Udagawa (1959) as a synonym. Scott and Stolk (1967) found a teleomorph in Udagawa’s P. cinnamopurpureum; Pitt (1979b) later showed that this species was a synonym of the earlier P. phoeniceum. E. cinnamopurpureum, the first name applied to the teleomorph, is unaffected by this change in the anamorph name. In passing, note that ”Abe ex Udagawa” indicates invalid (incomplete) publication of this species by Abe, with validation later by Udagawa. The species dates from the year of validation.

    3.7 Dual Nomenclature An important point here is that some isolates of Penicillium phoeniceum regularly produce the teleomorphic state Eupenicillium cinnamopurpureum, while

    16

    others, taxonomically indistinguishable, fail to produce a teleomorph at all. Because of this, it is essential to have a separate name for teleomorph and anamorph. The system of two names for a single fungus, known as dual nomenclature, has a place in the classification of fungi despite its apparent complexity. In the descriptions in later chapters, fungi for which both teleomorphs and anamorphs are known have both names listed. As noted above, if both states are found in a particular isolate, the teleomorph name is the more appropriate: to use that given to the anamorph is not incorrect, but this name is more sensibly applied to the conidial state only. Dual nomenclature would be relatively simple if the relationship between anamorph and teleomorph was always one to one. This is not the case. As has already been mentioned, species classified in Penicillium may produce teleomorphs in two genera, Eupenicillium and Talaromyces. On the other hand Talaromyces produces anamorphs in two genera, Penicillium and Paecilomyces. Aspergillus is the anamorph of eight or ten teleomorphic genera. Most teleomorph-anamorph relationships encountered in food mycology belong to the genera mentioned here. These relationships will be described where necessary under these particular genera.

    3.8 Practical Classification of Fungi Fungi are classified in a vast array of orders, families, genera and species. Among natural organisms, the numbers of taxa of fungi are rivalled only by those of the flowering plants and insects. Estimates of fungal species range as high as 1.5 million; only 5% of this number have so far been described (Hawksworth, 1991). Many fungi are highly specialised. Some will grow only in particular environments such as soil or water; many are obligate parasites and require a specific host, such as a particular plant species, and will not grow in artificial culture; many grow only in association with plant roots. From the point of view of the food microbiologist, these kinds of fungi are irrelevant. In one sense, most fungi which spoil foods are also highly specialised, their speciality being the ability to obtain nutrients from, and hence grow on, dead, dormant or moribund plant material more or less regardless of source. The

    3

    Naming and Classifying Fungi

    principal factors influencing food spoilage by fungi are physico-chemical and have already been outlined in Chapter 2. The point being made here is that food spoilage fungi are classified in just a few orders and a relative handful of genera. For this reason there is much to be said for food mycologists avoiding the use of a traditional, hierarchical classification as outlined above and employing a less formal approach to the identification of the fungi of interest to them. In the psent work, this pragmatic approach has been followed as far as possible:

    The use of specialised terms has been kept to a

    minimum, while being cognisant of the need for clarity of expssion. Hierarchical classification has been avoided as far as possible, consistent with retaining a logical approach to the psentation of fungi which are related or of similar appearance. Identification procedures used have been designed to be simple and comphensible, avoiding the use of specialised equipment or procedures unavailable in the routine laboratory. To this end, identification of nearly all species included in this work is based entirely on inoculation of a single series of Petri dishes, incubation under carefully standardised conditions and examination by traditional light microscopy. A standard plating regimen has been used for the initial examination of all isolates (except yeasts), so that identification procedures can be carried out without foreknowledge of genus or even subkingdom. Cultural characters, which can be broadly defined as the application of microbiological techniques to mycology, have been used throughout.

    The use of cultural characters has long been implicit in the study of fungi in pure culture on artificial substrates, especially in such genera as Aspergillus and Penicillium, genera of paramount importance in food spoilage. In Penicillium, cultural characters have been used as taxonomic criteria since the turn of the 20th century, but have assumed greater importance through the work of Pitt (1973, 1979b), who used the measurement of colony diameters, following incubation under standardised conditions, as a taxonomic criterion. The use of

    3.8 Practical Classification of Fungi

    pure culture techniques and growth data in fungal taxonomy is now widespad. Food microbiologists, the primary audience for this book, are familiar with cultural techniques and the use of a wide range of media and varied incubation conditions, so the authors make no apology for the taxonomic approach used in the psent work. This approach is a logical extension of the system used by the first author in Penicillium taxonomy and which has been found to have a much broader applicability. In the field of mycology, different genera have been studied by many different people of varied backgrounds and for different reasons. Consequently, keys and descriptions have been based on a wide variety of media, often traditional formulations incorporating all sorts of natural products. This heterogeneity makes comparisons difficult

    17

    and adds unnecessary complexity to the task of the nonspecialist confronted with a range of fungal genera. The approach used here has been to examine every isolate (excluding yeasts) by a single system: inoculation onto a standard set of Petri dishes and examination of them culturally and microscopically after 7 days incubation. Most of the genera and species included in this book can be identified immediately, at that point. Only in exceptional cases has it been found necessary to reinoculate isolates onto a further set of media in order to complete identification. The exceptional fungi are first the xerophiles, many of which grow poorly if at all on the standard media, and, second, genera such as Fusarium and Trichoderma, in which some species cannot readily be differentiated on the standard regimen. Details of the techniques used are given in Chapter 4.

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    Chapter 4

    Methods for Isolation, Enumeration and Identification

    This chapter describes techniques and media suitable for the enumeration, isolation and identification of fungi from foods. Some techniques are similar to those used in food bacteriology; others have been developed to meet the particular needs of food mycology. Most of the media have been specifically formulated for foodborne fungi. The approach taken here is designed to provide a systematic basis for the study of food mycology. In 1984 a group of about 30 of the world’s foremost scientists in food mycology met in Boston, Massachusetts, USA, to hear and discuss a wide range of psentations that explored many aspects of methodology in food mycology. Agreement was reached on broad issues and areas requiring further work pinpointed. The proceedings were published as ”Methods for the Mycological Examination of Food” (King et al., 1986). At a second workshop, held in Baarn, the Netherlands, in 1990, results of a number of collaborative studies on media and methods were psented and some standardised protocols developed. The proceedings, published as ”Modern Methods in Food Mycology” (Samson et al., 1992), provided a comphensive overview of current thinking in this field. The working group which organised those two workshops was then formalised as the International Commission on Food Mycology (ICFM), a commission under the auspices of the Mycology Division of the International Union of Microbiological Societies (IUMS). ICFM is dedicated to international standardisation of methods in food mycology. Subsequent ICFM workshops were held in Copenhagen, Denmark (1994), Uppsala, Sweden (1998), Samsø, Denmark (2003) and Key West,

    Florida (2007). Papers from the third and fourth workshops were published in the International Journal of Food Microbiology and the proceedings of the fifth (Samsø) workshop were published as ”Advances in Food Mycology” (Hocking et al., 2006a). The methodology described below is based on recommendations from ICFM and repsents current thinking within the food mycology community. However, no formal endorsement from ICFM is implied.

    4.1 Sampling It must be emphasised at the outset that results from mycological assays of foods are only as good as the samples used. However, sampling is beyond the scope of this text. Excellent treatises on sampling plans for food bacteriological purposes have been produced by the International Commission on Microbiological Specifications for Foods (ICMSF, 1986, 2002) and are generally applicable to food mycology.

    4.2 Enumeration Techniques Quantification of the growth of filamentous fungi is more difficult than for bacteria or yeasts. Vegetative growth consists of hyphae, which are not readily detached from the substrate and which survive blending poorly. When sporulation occurs,

    J.I. Pitt, A.D. Hocking, Fungi and Food Spoilage, DOI 10.1007/978-0-387-92207-2_4, Springer ScienceþBusiness Media, LLC 2009

    19

    20

    very high numbers of spores may be produced, causing sharp rises in viable counts, often without any great increase in biomass. The estimation of fungal growth or biomass is not easy, because no primary standard exists (such as cell numbers used for yeasts and bacteria). Although techniques for quantifying biomass have improved in recent years, most food laboratories continue to rely on viable counting (dilution plating) for detecting and quantifying fungal growth in foods. As well as dilution plating, a second standard method, known as direct plating, has been developed for estimating fungal numbers and growth in foods. Both methods are described in detail below. Techniques for biomass estimation will be discussed later.

    4.2.1 Direct Plating Contributions at the international workshops mentioned above emphasised the use of direct plating as the pferred method for detecting, enumerating and isolating fungi from particulate foods such as grains and nuts. In direct plating, food particles are placed directly on solidified agar media. In most situations, particles should be surface disinfected before plating, as this removes the inevitable surface contamination arising from dust and other sources and permits recovery of the fungi actually growing in the particles. This process provides an effective measure of inherent mycological quality and permits assessment of the potential psence of mycotoxins as well. Surface disinfection should be omitted only where surface contaminants become part of the downstream mycoflora, for example, in grain intended for flour manufacture. Even here, surface disinfection before direct plating provides the most realistic appraisal of actual grain quality. Results from direct plating analyses are expssed as percentage infection of particles. The technique provides no direct indication of the extent of fungal invasion in inpidual particles. However, it is reasonable to assume that a high percentage infection is correlated with extensive invasion in the particles and a higher risk of mycotoxin occurrence.

    4 Methods for Isolation, Enumeration and Identification

    The standard protocol recommended by the ICFM (Hocking et al., 2006a, p. 344) is given below, with amplification where necessary. Surface disinfection. Surface disinfection is carried out by immersing particles in a chlorine solution. Household chlorine bleach, nominally 4-5% active chlorine, is effective. Dilute the chlorine 1 to 10 with water before use, to provide an approximately 0.4% solution. Immerse particles for 2 min, stirring occasionally, then drain the chlorine. Chlorine solutions are rapidly denatured by organic matter, so it is important to use a surplus of chlorine solution (10 times the volume of the particles) and to use the solution only once. This process is conveniently carried out in 250-500 ml beakers. Place 50 or more particles in the beaker and add chlorine. To dislodge air bubbles, immediately stir with a pair of forceps, leaving them in the solution, and pferably cover the beaker with a watch glass. The watch glass simplifies decanting of the chlorine, and the forceps, disinfected by the chlorine, may be used to plate the particles. Studies in our laboratory have shown that the treatment outlined here may be inadequate under some conditions. In commodities such as peanuts or maize where high levels of Aspergillus flavus or Penicillium species may be psent, surface disinfection may be difficult. Here 2 min immersion in 70% ethanol followed by 2 min in 0.4% chlorine is recommended. Rinsing. After the chlorine is poured off, particles may be rinsed once with sterile water. Use a 1 min treatment, with stirring, then pour the water off. Again a watch glass should cover the beaker during this period, and the sterile forceps should be used for stirring. It is not clear whether rinsing fulfils any essential function. Early direct plating regimens used agents such as mercuric chloride for disinfection, and rinsing was essential to remove such toxic materials before they penetrated the particles too deeply. However, chlorine is effectively denatured by the particles and is believed to penetrate very little. In our opinion, the rinsing step can be omitted without loss of efficacy of the treatment, with savings in time and materials, and reduced risk of recontamination from the air. Plating. After disinfection and the optional rinse, particles should be plated onto solidified agar, at the rate of 6-20 particles per plate, depending on

    4.2 Enumeration Techniques

    particle size. Use the disinfected forceps. Plating should be carried out immediately: keep the watch glass on the beaker if this is not possible. Incubation. Incubate plates upright, under normal circumstances for 5 days at 258C. See more detailed notes below. Examination. After incubation, examine plates visually, count the numbers of infected particles and expss results as a percentage. Differential counting of various genera is often possible. Correct choice of media, a stereomicroscope and experience will all assist in this process.

    4.2.2 Dilution Plating Dilution plating is the appropriate method for mycological analysis of liquid or powdered foods. It is also suitable for grains intended for flour manufacture and other situations where total fungal contamination is relevant. Sample pparation. The two most common methods of sample pparation for dilution plating are stomaching and blending: stomaching is recommended by ICFM (King et al., 1986). The Colworth Stomacher (Sharpe and Jackson, 1972), or equivalent equipment (e.g. BagMixer1, Interscience, Saint Nom La Brete`che, France), is a very effective device for dispersing and separating fungi from finely pided materials such as flour and spices, and soft foods such as cheeses and meats, and its use is strongly recommended. Treatment time in the stomacher should be 2 min. Harder or particulate foods such as grains, nuts or dried foods, like dried vegetables, should be soaked before stomaching. Soaking times from 30 to 60 min are generally sufficient, but for extremely hard particles such as dried legumes, soaking for up to 3 h may be required. Comminution in a Waring Blender or similar machine is a suitable alternative for these types of samples and may give a more satisfactory homogenate. Blending times should not exceed 60 sec, as longer treatments may fragment mycelium into lengths too short to be viable or overheat the homogenate. The sample size used should be as large as possible. If a stomacher or BagMixer 400 is used, a sample size of 10-40 g is suitable.

    21

    Diluents. The recommended diluent is aqueous 0.1% peptone (Hocking et al., 2006a, p. 344), suitable for both filamentous fungi and yeasts. Saline solutions, phosphate buffer or distilled water are no longer recommended by ICFM as they may be deleterious to yeasts (Mian et al., 1997). The addition of a wetting agent such as polysorbate 80 (Tween 80) may be desirable for some products, but the natural wetting ability of the peptone is usually adequate. Special diluents may be necessary in some circumstances. If yeasts are to be enumerated from dried products or juice concentrates, the diluent should also contain 20-30% sucrose, glucose or glycerol, as the cells may be injured or be susceptible to osmotic shock. Dilution. Serial dilutions of fungi are carried out by the same procedures as those used in bacteriology, and the recommended dilution rate is 1:10 (=1+9). Fungal spores sediment more quickly than bacteria, so it is important to draw aliquots for dilution or plating as soon as possible, pferably within 1 min (Beuchat, 1992). Plating. Spad (surface) plating is recommended. When pour plates are used, fungi develop more slowly from beneath the agar surface and may be obscured by faster growing colonies from surface spores. Hence spad plating allows more uniform colony development, improves the accuracy of enumeration of the colonies and makes subsequent isolation of pure cultures easier. The optimum inoculum for surface plating is 0.1 ml. Best results will be obtained if plates are dried slightly before use. After adding the inoculum, spad it evenly over the agar surface with a sterile bent glass rod (”hockey stick”). Sterilise the rod by flaming it with ethanol before use. A plate spinner is a useful accessory. It is usually possible to enumerate plates with up to 150 colonies, but if a high proportion of rapidly growing fungi are psent, the maximum number which can be distinguished with any accuracy will be lower. Because of this restriction on maximum numbers, it may be necessary on occasion to accept counts from plates with as few as 10-15 colonies. Clearly, such limitations on numbers per plate and the overgrowth of slow colonies will result in higher counting errors than are usually achieved with bacteria or yeasts. Enumerating yeasts is less difficult. In the absence of filamentous fungi, from 30 to 300 colonies per

    22

    plate can be counted and errors will be comparable with those to be expected in bacterial enumeration. Incubation. The standard incubation conditions are 258C for 5 days. However, other conditions may be more suitable in some circumstances (see notes below). Reporting results. As in bacteriology, results from dilution plating are expssed as viable counts per gram of sample. Note that such results are not directly comparable with those obtained from direct plating and may not offer a direct indication of the extent of fungal growth.

    4.2.3 Incubation Conditions As noted above, the standard incubation conditions specified by ICFM are 258C for 5 days (Hocking et al., 2006a). Undoubtedly, 258C is the most suitable temperature for routine work in temperate to subtropical environments. Few if any common fungi are sensitive to this temperature, even those which grow readily under refrigeration. Higher temperatures are unacceptable in the temperate zone: 308C is close to the upper limit for growth of some important Penicillium species. In tropical regions, incubation at 308C is recommended as a more realistic temperature for enumerating fungi from commodities stored at ambient temperatures. In cool temperate regions such as Europe, 228C may be a more suitable incubation temperature. When used for fungi, Petri dishes should be stored upright. The principal reason is that some common fungi can shed large numbers of spores during handling, which in an inverted dish will be transferred to the lid. Reinversion of the Petri dish for inspection or removal of the lid may liberate spores into the air or onto benches and cause serious contamination problems.

    4.3 Sampling Surfaces Methods are outlined below for directly sampling the mycoflora of surfaces of commodities such as fruits, meats, cheeses, salamis and dried fish and also packaging materials, machinery and walls.

    4 Methods for Isolation, Enumeration and Identification

    The techniques are based on those described by Langvad (1980) for studying the fungal flora of leaves. If samples are particulate, or can be cut up, sterile forceps can be used to pss pieces of a suitable size (up to about 10 mm2) onto a suitable medium in a Petri dish. The sample is then removed, leaving an impssion, and any spores or mycelium transferred will form colonies within a few days. This technique is known as pss plating. For packaging materials such as cardboard, an alternative method is to cut a piece which will fit in a standard Petri dish. The dish is ppared by adding a sterile filter paper moistened with 10% glycerol and then placing a bent glass rod on it as a separator. After adding the sample, a thin layer of an appropriate agar medium is poured over its surface. To reduce evaporation, the dish should be sealed with Parafilm or a similar material or placed in a polyethylene bag before incubation at 258C for a few days. If contamination levels are not too heavy, the number and types of moulds psent can be effectively estimated by this method. Colonies may be subcultured for identification. For sampling walls or other surfaces, or for nondestructive sampling, impssions may be taken using adhesive tape. Carefully handled, tape coming from the roll will be virtually sterile. Press a short length of tape firmly onto the surface to be sampled, adhesive side down, then transfer it, still with the same side down, onto a suitable growth medium. After 1-2 days incubation at 258C, the tape may be removed to allow development and sporulation of colonies. Surface sampling techniques for assessment of sanitation in food production and processing areas are discussed by Evancho et al. (2001). Surfaces may be sampled by using sterile swabs or by agar contact methods. The swab method involves rubbing a moistened sterile cotton swab over the test surface and placing the swab in a dilution bottle to be subsequently diluted and plated on appropriate media. Agar contact plates, also known as RODAC (replicate organism direct agar contact) plates, are restricted to use on smooth or semi-smooth flat surfaces. An alternative to agar contact plates is the agar slice technique, where agar is filled into a syringe-like apparatus or into an artificial sausage casing. The solidified agar can be pushed out onto

    4.5 Isolation Techniques

    the surface to be sampled, then the portion making contact sliced off with a sterile scalpel or wire and placed in a Petri dish for incubation.

    4.4 Air Sampling Air sampling in the food processing environment is discussed by Evancho et al. (2001). The simplest method of air sampling is by sedimentation or settle plates. A Petri dish containing an appropriate agar medium is exposed to the atmosphere for a fixed period of time (usually 15-60 min), then closed and incubated at 258C (Samson et al., 2004a). This method can be useful in food production areas, as it gives a direct indication of the number and types of fungi likely to come into contact with exposed product. However, the settle plate technique lacks pcision, and volumetric air sampling is a much more reliable indicator of air quality. A number of air sampling devices are commercially available. Of these, the Anderson sampler (either the two-stage or six-stage model; Anderson Instruments Inc, Atlanta, GA, USA) is probably the best, giving accurate and consistent results (Buttner and Stetzenbach, 1993). However, the Anderson sampler is expensive and requires mains power or a large battery for operation. In factory situations, the dry cell battery-operated Biotest

    23

    Hycon RCS and RCS High Flow centrifugal air samplers (Biotest, Solihull, UK) and the MAS-100 air sampler (Merck, Darmstadt, Germany) are more convenient, as they are small and readily portable. The Biotest RCS Plus sampler was reported to give comparable results to larger and more sophisticated machines, but its sampling efficiency gradually decreased for particle sizes below 4 mm (Benbough et al., 1993).

    4.5 Isolation Techniques The term ”isolation” is used here in its strict sense: the pparation of a pure culture, free from any contamination and ready for identification.

    4.5.1 Yeasts Streaking techniques commonly used for bacterial purification are equally suitable for the isolation of yeasts (Fig. 4.1). A method widely used by yeast specialists is to disperse a portion of a colony in 2- 3 ml of sterile water, then streak a single loop of this suspension over the whole surface of a plate, moving the loop slowly down from top to bottom while simultaneously moving it rapidly across the plate

    Fig. 4.1 Petri dishes of yeasts growing on malt extract agar, showing a streaking method suitable for producing isolated colonies

    24

    from side to side. After suitable incubation, wellseparated single colonies should appear in the lower half of the plate (Fig. 4.1). If all of these single colonies appear to be of similar size and appearance (taking into account the effect of crowding), the culture may be judged to be pure. Microscopic checks of some single colonies are also desirable. Disperse a needle point of cells from a colony in a drop of water, add a cover slip and examine by bright field illumination at about 400. Cell outlines will be clearly visible. Note that, unlike those of bacteria, yeast cell sizes often vary considerably in a pure pparation. Purity is indicated not so much by uniformity of cell size within a pparation as by similarity in microscopic cell appearance from colony to colony. When a culture is considered to be pure, streak it onto an appropriate slant.

    4.5.2 Moulds Streaking techniques are ineffective for filamentous fungi and are not recommended. Isolation depends on picking a small sample of hyphae or spores – judged to be pure by eye, by hand lens or pferably under the stereomicroscope – and placing this sample on a fresh plate as a point inoculum. Purity is subsequently judged by uniformity in appearance of the colony which forms after incubation. The appearance of a mixed culture depends on the growth rates of the fungi psent. If rates are perse, a mixed culture is often indicated by a clump of dense hyphae at the inoculum point, surrounded by looser wefts of spading hyphae. With fungi of approximately equal growth rates, mixtures are often indicated by colonies with sectoring growth: sectors will show differences in mycelial, spore or reverse colours or in radial growth rates. The simplest starting place for isolating fungi is an enumeration plate with well-separated colonies. Use a needle of platinum or nichrome, pferably cut to a chisel point with a pair of pliers, or a steel sewing needle. Sterilise it by heating, then plunge the tip into cold agar and leave until cool – with nichrome or steel this will require several seconds. With the tip of the cold, wet needle pick off a few spores or a tuft of mycelium – just enough to be

    4 Methods for Isolation, Enumeration and Identification

    4.6 Choosing a Suitable Medium

    25

    require free access to oxygen for typical growth and sporulation. Oxygen starvation during growth will at best lead to retarded sporulation or at worst death of the culture. Long-term pservation of fungi is dealt with later in this chapter.

    4.6 Choosing a Suitable Medium Food laboratories often rely on a single all-purpose medium to produce a ”yeast and mould” count in everything from raw material to finished product. But just as the food bacteriologist uses selective media for particular purposes, so too food mycologists are developing a range of media suited to specific applications. It is plainly unrealistic to expect a single medium to answer all questions about mould and yeast contamination in all foods. The fungi that spoil meats or fresh vegetables are not the same as those that grow on dried fish. Although often used for this purpose, very dilute media such as potato dextrose agar are of little or no value for enumerating fungi from dried foods.

    The most important pision in types of enumeration media lies between those suitable for high water activity foods, such as eggs, meat, vegetables and dairy products, and those suited to the enumeration of fungi in dried foods. The most suitable media for dried foods depend on the type of food, the major categories being foods low in soluble solids such as cereals, high sugar foods such as confectionery and dried fruit, and salt foods. A second consideration lies in whether the primary interest is in moulds, or yeasts, or both; and a third concerns the psence or absence of pservatives. Finally, media are available for specific mycotoxigenic fungi, notably Aspergillus flavus and related species and Penicillium verrucosum plus P. viridicatum. An overview of media considered most suitable for particular purposes is given in Table 4.1. The table is derived from Pitt and Hocking (1997), together with recent recommendations from ICFM (Samson et al., 1992; Hocking et al., 2006a).

    Table 4.1 Recommended media for fungal detection, enumeration and isolationa Type of food

    Selecting for

    Medium

    Remarks

    Fresh foods: milk and milk products, fruit, cheese, sea foods

    Moulds Yeasts General

    DRBC TGY, MEA, OGY DRBC

    Blend (where necessary) and dilution plate

    Freshly harvested grains, nuts

    General Dematiaceous Hyphomycetes Fusarium Yeasts

    DRBC DRBC, CZID CZID TGY, MEA, OGY

    Direct plate Direct plate Direct plate Dilution plate

    Fruit juices, fresh

    Yeasts

    TGY, MEA, OGY

    Dilution plate

    Fruit juices, pserved

    Preservative resistant yeasts

    TGYA, malt acetic agar

    Dilution plate

    Fruit juices, to be pasteurised, or pasteurised products

    Heat resistant moulds

    PDA, MEA

    Special protocol

    Fruit juice concentrates

    Xerophilic yeasts

    MY50G

    Special diluents

    Dried foods in general

    General

    DG18

    Direct plate

    Stored cereals, nuts

    General Dematiaceous Hyphomycetes Fusarium

    DG18 DRBC, CZID CZID

    Direct plate Direct plate Direct plate

    26

    4 Methods for Isolation, Enumeration and Identification

    Table 4.1 Recommended media for fungal detection, enumeration and isolationa (continued) Type of food

    Selecting for

    Medium

    Remarks

    Grain for milling into flour

    General

    DG18

    Stomach or blend and dilution plate

    Dried fruit, confectionery, chocolate, etc.

    Xerophilic moulds and yeasts

    MY50G

    Direct plate

    Fastidious xerophiles – in psence of Eurotium spp.

    MY50G MY70GF

    Direct plate Direct plate

    General

    DG18

    Halophilic xerophiles

    MY5-12, MY10-12

    Direct plate or pss plate Direct plate or pss plate

    Fungi producing aflatoxins

    AFPA

    Direct or dilution plate

    DRYS

    Direct or dilution plate

    Salt foods, e.g. salt fish

    General

    General Fungi producing ochratoxins a For medium acronyms, see Section 4.6.

    4.6.1 General Purpose Enumeration Media To be effective, a general purpose enumeration medium must fulfil several requirements (Pitt, 1986). As these are sometimes overlooked, they are listed here:

    to inhibit bacterial growth completely, without

    affecting growth of foodborne fungi (filamentous or yeasts); to be nutritionally adequate and support the growth of fastidious fungi; to suppss the growth of rapidly spading fungi, especially the Mucorales, but not to pvent their growth entirely, so they too can be enumerated; to slow radial growth of all fungi, to permit counting of a reasonable number of colonies per plate, without inhibiting spore germination; to promote growth of relevant fungi; and to suppss growth of soil fungi or others generally irrelevant in food spoilage.

    Fulfilling the above requirements necessitates the use of potent inhibitory compounds, and there is sometimes a fine line between inhibition of undesirable microorganisms and suppssion of growth of those being sought. Modern fungal enumeration media rely on the use of antibiotics at neutral pH

    for the inhibition of bacteria. Such media allow better recovery of moribund and sensitive fungi than the acidified media commonly used in the past. For many years rose bengal has been added to media to slow colony spad, while more recently 2,6-dichloro-4-nitroaniline (dichloran) has been added to inhibit rapidly spading moulds. Many common spoilage fungi, Aspergillus and Penicillium species in particular, develop better on media with adequate nutrients. Low nutrient media of very high aw, such as potato dextrose agar, have lost favour because they are selective against some species in these genera. The media described below are considered to be the most satisfactory general purpose enumeration media available at this time (Hocking et al., 2006a). Formulations are given in the Media Appendix. Dichloran rose bengal chloramphenicol (DRBC) agar. DRBC (King et al., 1979, Pitt and Hocking, 1997) is recommended for both moulds and yeasts. It is particularly suited to fresh and high aw foods (Hocking et al., 1992). This medium contains both rose bengal (25 mg/kg) and dichloran (2 mg/kg), which restrict colony spading without affecting spore germination unduly. Compact colonies allow crowded plates to be counted more accurately. This combination of inhibitors also effectively restricts the rampant growth of most of the common mucoraceous fungi such as Rhizopus and

    4.6 Choosing a Suitable Medium

    27

    Fig. 4.2 Petri dishes of (a) DRBC and (b) RBC showing effective control of Rhizopus growth by rose bengal and dichloran in DRBC

    Mucor (Fig. 4.2), although it does not completely control some other troublesome genera such as Trichoderma. In routine use, it is recommended that DRBC plates be incubated away from light at 258C for 5 days. Dichloran 18% glycerol (DG18) agar. Hocking and Pitt (1980) developed DG18 to be selective for xerophilic fungi from low moisture foods such as stored grains, nuts, flour and spices. DG18 was designed for enumeration of a range of nonfastidious xerophilic fungi and yeasts. However, it has been shown since that it supports growth of the common Aspergillus, Penicillium and Fusarium species, as well as most yeasts, and many other common foodborne fungi. DG18 can now be described as a general purpose medium with emphasis on enumeration of fungi from dried foods. DG18 is also recognised to be a very useful medium for enumeration of airborne fungi (Wu et al., 2000; Samson et al., 2004a). It is an effective inhibitor of Mucoraceous fungi, and bacteria are totally suppssed. However, growth of Eurotium species (pviously known as ”the Aspergillus glaucus group”) is still somewhat too rapid, and colonies may have diffuse margins. The addition of detergent to DG18 has been reported to be an improvement in this respect (Beuchat and de Daza, 1992). Although DG18 is a satisfactory isolation medium for Eurotium species, it is not suitable for their

    identification. Eurotium species are usually identified on Czapek yeast extract agar with 20% sucrose (CY20S), described later in this chapter. Other general purpose media. Under circumstances where rapidly spading moulds do not cause problems, two alternative general purpose enumeration media are satisfactory. These are rose bengal chloramphenicol agar (RBC; Jarvis, 1973), from which DRBC was developed, and oxytetracycline glucose yeast extract agar (OGY; Mossel et al., 1970). OGY has been found to be very suitable for yeasts in the absence of moulds (Andrews, 1992a). Most of the media discussed above are available in ready to use dehydrated form from media suppliers such as Oxoid, Difco, BBL, etc.

    4.6.2 Selective Isolation Media Although considerable progress has been made in the past 20 years, the formulation of selective media for foodborne fungi still requires a great deal of research. The availability of effective media can greatly simplify the isolation and identification of significant food spoilage and mycotoxigenic fungi. Most attention has been paid so far to the requirements of xerophilic fungi because of their failure to develop on standard high aw media. For mycotoxigenic fungi, satisfactory media exist only for

    28

    4 Methods for Isolation, Enumeration and Identification

    Coconut Cream agar (CCA) to detect aflatoxin production in Aspergillus flavus and A. parasiticus. CCA can be made using any brand of commercial canned coconut cream (available from Asian food stores in many places). Dilute 50:50 with water, add agar (1.5%) and autoclave. Inoculate solidified plates with up to four colonies (picked with a wet needle) and incubate at 308C for 5-7 days. Examine, reverse upmost, under long wave length UV light. Colonies producing aflatoxins will fluoresce bluish white or white, especially in the centres. Ignore fluorescence from the Petri dish itself. Use an uninoculated coconut cream agar plate as a control (Dyer and McCammon, 1994). Plates inoculated with known nontoxigenic and toxigenic strains are also useful controls. A. parasiticus isolates almost always produce aflatoxins. Media for fungi producing ochratoxin A. Although ochratoxin A was first described from Aspergillus ochraceus, recent molecular studies indicate that A. westerdijkiae is the major ochratoxin A producing species in Aspergillus Section Circumdati (Frisvad et al., 2004). Aspergillus carbonarius is also an important source of ochratoxin A, particularly in grape products (Abarca et al., 2004; Leong et al., 2006a and references therein). However, there are no selective or indicative media for these fungi. When DG18 is used as the isolation medium, selection of colonies with light brown (ochre) or black sporulation is a good starting point for detecting A. westerdjikiae or A. carbonarius respectively. Coconut cream agar (Dyer and McCammon, 1994) can also be used to screen for ochratoxin production in A. carbonarius and species in Aspergillus Section Circumdati (Heenan et al., 1998). Plates are best incubated at 258C rather than 308C for potentially ochratoxigenic species of Aspergillus. Penicillium verrucosum is the most important ochratoxin a producer in the genus Penicillium (Pitt, 1987), although P. nordicum can also produce this mycotoxin (Frisvad and Samson, 2004). Frisvad (1983) developed Dichloran rose bengal yeast extract sucrose agar (DRYS) for selective enumeration of P. verrucosum. P. nordicum, P. viridicatum and P. aurantiogriseum are also selected by DRYS. P. verrucosum and P. nordicum produce ochratoxin A, P. verrucosum also produces citrinin, while the latter two species produce xanthomegnin and viomellein. According to Frisvad (1983), P. verrucosum colonies on DRYS have a violet brown reverse, and

    4.6 Choosing a Suitable Medium

    the latter two species produce yellow colonies with a yellow reverse. The incubation regimen recommended by Frisvad (1983) is 7-8 days at 208C. Subsequently, Frisvad et al. (1992) developed dichloran yeast extract sucrose 18% glycerol agar (DYSG) on which P. verrucosum produces a red brown reverse. Because of its lower aw, DYSG inhibits rapidly growing fungi such as Rhizopus and Mucor more effectively than DRYES. In a study of 76 samples of wheat, rye and barley, Lund and Frisvad (2003) found that DYSG was more effective than DRYES for screening grain samples for the psence of P. verrucosum and potential ochratoxin A contamination. Media for Fusarium species. Dichloran chloramphenicol peptone agar (DCPA; Andrews and Pitt, 1986) can be used to isolate Fusarium species from grains and other substrates. The medium was developed from Nash-Snyder medium (Nash and Snyder, 1962) a medium for enumeration of Fusarium species from soils. DCPA uses a low level of dichloran as a substitute for the high level of pentachloronitrobenzene (PCNB), and chloramphenicol rather than the antibiotic mixture used by Nash and Snyder (1962). When Fusarium species are dominant, DCPA is effective for their isolation from grains, animal feeds and soil. However, DCPA has been found to be less effective in mixed populations. DCPA is a very useful medium for the identification of Fusarium species because it often induces the abundant formation of macroconidia (Hocking and Andrews, 1987). A much more stringent medium, effective for isolation of most Fusarium species occurring in foods, is Czapek iprodione dichloran agar (CZID; Abildgren et al., 1987). As well as dichloran, this medium contains the fungicide iprodione. CZID is highly selective for Fusarium species, and is probably the best medium for Fusarium enumeration and isolation. CZID is suitable for isolation of Fusarium species by direct plating of surface disinfected grains and other commodities, or dilution plating of homogeneous samples such as flour. Some questions remain concerning whether CZID may be too selective and not support growth of all foodborne Fusarium species. However, the common species all grow well. Castella´ et al. (1997) developed a Fusarium selective medium (MGA 2.5) using 2.5 mg/L malachite green as the selective agent. They reported that MGA 2.5 was more selective than Nash-Snyder medium as it did not allow development of colonies of other fungal genera.

    29

    Bragulat et al. (2004) compared the efficacy of MGA 2.5 medium with DCPA, CZID and several other Fusarium media, using pure cultures of twelve Fusarium species commonly found in foods as well as naturally contaminated samples. They reported that there was no statistically significant difference in colony counts of the Fusarium spp. tested, but that colonies on MGA 2.5 were smaller than the other media. MGA 2.5 did not allow growth of other fungi such as Zygomycetes and yeasts from naturally contaminated samples, thus providing better selectivity than the other media. Media for dematiaceous Hyphomycetes. Although designed primarily for Fusarium isolation, CZID has been found in our laboratory to be very useful for isolating dematiaceous Hyphomycetes, provided that iprodione is added at half the usual concentration, as full strength iprodione tends to restrict colony diameters too severely. Alternaria, Bipolaris, Curvularia, Stemphylium and Ulocladium species will grow and sporulate well when incubated for 5 days at 258C with a 12 h photoperiod. Drechslera species will grow but will not sporulate on this medium. DRBC is also of value for isolating these fungi, but some species do not sporulate readily on it. DCPA, originally developed for isolation of Fusarium, may also be used as an isolation medium for the dematiaceous Hyphomycete genera mentioned above. Alternaria, Curvularia and related genera usually grow rapidly and sporulate well on DCPA. However, fungi should not be maintained or stored on DCPA for more than two weeks, as ammonia is produced by aging cultures. Media for xerophilic fungi. Xerophilic fungi are of great importance in food spoilage, and hence media and techniques for their enumeration and isolation have received much attention. Xerophiles range from those which grow readily on normal media and which are only marginally xerophilic, such as many Aspergillus and Penicillium species, to those, such as Xeromyces bisporus, which will not grow at all on normal (high aw) media. It is not surprising that no single medium is suitable for quantitative estimation of all xerophilic fungi found causing food spoilage. As noted earlier, DG18 was developed as a general medium for xerophiles, and remains the medium of choice for this purpose. DG18 should be used in any general examination of the mycoflora of dried foods. Media for extreme xerophiles. Fungi discussed here include Xeromyces bisporus, xerophilic

    30

    4 Methods for Isolation, Enumeration and Identification

    The most satisfactory medium is malt extract yeast extract 70% glucose fructose agar (MY70GF). MY70GF is of similar composition to MY50G, except that it contains equal parts of glucose and fructose to pvent crystallisation of the medium at the concentration used (70% w/w). It is made in a similar manner to MY50G. Growth of even the extreme xerophiles on MY70GF is extremely slow, and plates should be incubated for at least 4 weeks at 258C. Once growth is apparent, pick off small portions of colonies and transfer them to MY50G, to allow more rapid growth and sporulation. Media for halophilic xerophiles. Some xerophilic fungi from salted foods such as salt fish grow more rapidly on media containing NaCl and hence are correctly termed halophilic xerophiles. Malt extract yeast extract 5% salt 12% glucose agar (MY5-12) and malt extract yeast extract 10% salt 12% glucose agar (MY10-12) are suitable for these fungi. Techniques for enumeration and isolation are similar to those described above for other extreme xerophiles.

    4.6.3 Techniques for Yeasts The simplest enumeration and growth medium for most food spoilage yeasts is malt extract agar (MEA). Although originally introduced as a growth medium for moulds, its rich nutritional status makes it very suitable for yeasts, and its relatively low pH (usually near 5.0) reduces the possibility of bacterial contamination. More recently, tryptone glucose yeast extract agar (TGY) has been recommended for enumeration of yeasts (Hocking et al., 1992, 2006a). Due to a higher glucose concentration (10%) and higher pH (5.5-6.0), this medium is more effective in recovery of stressed yeast cells, and colony development is usually faster than on MEA. However, its higher pH means that an antibiotic should be incorporated for enumeration of yeasts from food samples which may contain bacteria. Recommended antibiotics are chloramphenicol or oxytetracycline at the concentrations used in DRBC and OGY. Both MEA and TGY are suitable for enumeration of yeasts in products such as fruit juices, fruit purees and yoghurt, where moulds are usually psent only in low numbers.

    4.6 Choosing a Suitable Medium

    31

    and replace it with sterile water. Leave the cap loose, incubate at room temperature or 308C and watch for evidence of fermentation. Shaking the container daily will help to detect gases resulting from fermentation. Classical enrichment techniques used in bacteriology can also be used for yeasts. TGY broth has been used very successfully in our laboratory for enrichment of low numbers of yeasts in liquid products. Add 10 ml of product to 90 ml TGY broth and incubate at 258C for 3-4 days, or 308C for 2-3 days. Look for signs of fermentation and streak out onto TGY agar. Detection of pservative resistant yeasts. A few species of yeasts are able to grow in products containing pservatives such as sorbic, benzoic and acetic acids or sulphur dioxide. The most important of these is Zygosaccharomyces bailii. The simplest and most effective way to screen for pservative resistant yeasts is to spad or streak product onto plates of malt acetic agar (MAA), which is MEA with 0.5% acetic acid added (Pitt and Richardson, 1973) or TGY with 0.5% acetic acid added (TGYA, Hocking, 1996). MAA and TGYA are made by adding glacial (16 N) acetic acid to melted and tempered basal medium to give a final concentration of 0.5%. Mix and pour immediately. These media cannot be held molten for long periods or remelted because of their low pH (approximately 3.2 and 3.8 for MAA and TGYA, respectively). The acetic acid does not need sterilisation before use. MAA and TGYA are suitable media for monitoring raw materials, process lines and products containing pservatives for resistant yeasts. They are also effective for testing pviously isolated yeasts for pservative resistance. Erickson (1993) developed a selective medium for Zygosaccharomyces bailii. Zygosaccharomyces bailii medium (ZBM) is based on Sabouraud dextrose agar amended with fructose, NaCl, tryptone, yeast extract and trypan blue dye, then acetic acid (0.5%) and potassium sorbate (0.01%) are added to make the medium selective. It is designed as a plating medium for detection of Z. bailii in acidified ingredients in conjunction with hydrophobic grid membrane filtration (Erickson, 1993). When compared with MAA and TGYA in an interlaboratory study, ZBM was found to be highly selective for Zygosaccharomyces bailii, to the exclusion of other important pservative resistant yeasts such as Schizosaccharomyces pombe and Pichia

    32

    membranaefaciens. In addition, recovery of Z. bailii cells sublethally injured by lyophilisation was significantly lower on ZBM than on TGYA or MAA (Hocking, 1996). Its high selectivity and complex formulation make ZBM unsuitable for routine laboratory use as a medium for detection of pservative resistant yeasts. Enrichment of pservative resistant yeasts. A technique capable of detecting yeast numbers as low as 1 cfu/ml within 4 days has been developed in our laboratory (Hocking et al., 1996). This method is particularly suitable for the detection of Zygosaccharomyces bailii in raw materials or finished product, but can also be used for the detection of Schizosaccharomyces pombe, Pichia membranaefaciens and other species of pservative resistant yeasts. The method involves a 2to 3-day enrichment step followed by a plating step with a further 2 days of incubation. Triplicate 20 ml tryptone glucose yeast extract (TGY) broths containing 0.5% acetic acid (TGYA) are each inoculated with 1 g or 1 ml of product and the broths incubated at 308C. After incubation for 48 and 72 h, 0.1 ml from each broth is spad plated onto TGY agar containing 0.5% acetic acid and the plates incubated at 308C. The detection time of the method is shortened by incubating broths and plates at 308C rather than the traditional temperature of 258C, as the optimum growth temperature for Zygosaccharomyces bailii is 30-328C (Jermini and Schmidt-Lorenz, 1987b). The sensitivity of the method is greatly increased by using triplicate broths instead of single or duplicate broths and by spad plating 0.1 ml from each broth instead of streaking a loopful onto TGYA agar. This method has been used to detect low numbers of cells of Zygosaccharomyces bailii in experimentally inoculated cordial syrup, mayonnaise, salad dressing and barbecue sauce and other pservative-resistant yeasts such as Schizosaccharomyces pombe, Pichia membranaefaciens and some pservative resistant strains of Saccharomyces cerevisiae. Yeasts of intermediate pservative resistance (e.g. Debaryomyces hansenii, Candida krusei and Torulaspora delbrueckii) can also be detected by this method. Better recoveries were obtained using TGY than a malt extract agar and broth system, possibly due to the fact that the pH of TGY broth + 0.5% acetic acid is 3.8, compared with pH 3.2 for MEA + 0.5% acetic acid, and there is a lower concentration of glucose (2%) in the ME

    4 Methods for Isolation, Enumeration and Identification

    system compared with 10% in TGY. Yeasts which are unable to grow in the psence of acetic acid or other weak acid pservatives (sorbic or benzoic acids and their salts) are not detected by this method.

    4.6.4 Techniques for Heat-Resistant Fungi Heat resistant spoilage fungi, such as Byssochlamys, Talaromyces, Neosartorya and Eupenicillium species can be selectively isolated from fruit juices, pulps and concentrates by laboratory pasteurisation using various methods (Beuchat and Rice, 1979; Hocking and Pitt, 1984; Beuchat and Pitt, 2001; Houbraken and Samson, 2006). Three methods are described here: a plating method based on that of Murdock and Hatcher (1978), a direct incubation method and a filtration method for liquid samples such as liquid sugar. Plating method. If the sample to be tested is more concentrated than 358Brix, it should first be diluted 1:1 with 0.1% peptone or similar diluent. For very acid juices such as passionfruit, normally about pH 2.0, the pH should be adjusted to 3.5-4.0. Two 50 ml samples are taken for examination. Erlenmeyer flasks (250 ml) or polyethylene Stomacher bags may be used as heat penetration into these containers will be rapid. If using Stomacher bags, the tops should be heat sealed. If a heat sealer is not available, the tops may be folded over and secured with a clip and should not be fully immersed. The two samples are heated in a closed water bath at 808C for 30 min, then rapidly cooled. Each 50 ml sample is then mixed with an equal volume of double strength MEA distributed over four 150 mm Petri dishes. The Petri dishes are loosely sealed in a plastic bag to pvent drying and incubated at 308C for up to 30 days. Plates are examined weekly for growth. Most moulds will produce visible colonies within 10 days, but incubation for up to 30 days will allow for the possible psence of badly heat damaged spores, which may germinate very slowly. This long incubation time also allows most moulds to mature and sporulate, aiding their identification. The main problem associated with this technique is the possibility of aerial contamination of the plates with common mould spores, which will give false positive results. The growth of green Penicillium

    4.6 Choosing a Suitable Medium

    colonies, or colonies of common Aspergillus species such as A. flavus and A. niger, is a clear indication of contamination as these fungi are not heat resistant. To minimise this problem, plates should be poured in clean, still air or a Class 2 biohazard cabinet if possible. If a product contains large numbers of heat resistant bacterial spores (e.g. Bacillus species), antibiotics can be added to the agar. The addition of chloramphenicol (100 mg/l of medium) will pvent the growth of these bacteria. Direct incubation method. A more direct method used for screening fruit pulps and other semisolid materials avoids the problems of aerial contamination. Place approximately 30 ml of pulp in each of three or more flat bottles such as 100 ml medicine flats. Heat the bottles in the upright position for 30 min at 808C and cool, as described pviously. The bottles of pulp can then be incubated directly, without opening and without the addition of agar. They should be incubated flat, allowing as large a surface area as possible, for up to 30 days at 308C. Any mould colonies which develop will need to be subcultured onto a suitable medium for identification. If containers such as Roux bottles are available, larger samples can be examined by this technique, but heating times must be increased. Bottle contents should reach at least 758C for 20 min when checked by a thermometer suspended near the centre of the pulp. For further details of the above methods see Hocking and Pitt (1984) or Beuchat and Pitt (2001). Filtration method for liquid sugars. This method permits the detection of very low numbers of cells in clear liquids such as liquid sugar. Sample size should be at least 100 g, taken after vigorously shaking the container from which the sample is drawn. Add 100 ml diluent (0.1% aqueous peptone) to 2 50 g samples and mix well to dissolve. Filter both samples sequentially through the same sterile 0.45 mm membrane filter. After both samples have passed through the filter, rinse the interior of the funnel with 3 20- 30 ml volumes of sterile diluent. Remove the filter from the filter holder using sterile forceps and place it in a sterile bottle or Stomacher bag. Add 10 ml diluent to the bottle or bag containing the filter and place in a water bath at 758C for 30 min. Ensure the sample is submerged in the water bath (weigh down if necessary). Cool rapidly to room temperature, shake well, then pide the 10 ml of diluent between three Petri dishes. Add a generous portion of MEA with

    33

    antibiotics to each plate, mix the agar and sample well, then let the plates solidify. Incubate at 308C for up to 30 days, examining weekly. Count colonies and report count per 100 g. This method was developed by BCN Research Laboratories, Rockford, Tennessee, USA.

    4.6.5 Other Plating Techniques Three other techniques which were developed for counting bacteria have been applied to fungal enumeration. Spiral plate count. Zipkes et al. (1981) evaluated the application of the spiral plate procedure to the enumeration of yeasts and moulds. They compared this procedure with the traditional pour plate and streak plate methods and found that spiral plating gave a higher overall recovery and lower replicate plating errors than the other two methods. The medium they used was potato dextrose agar, but the technique should be no less efficient using the media recommended here. Automation of spiral plate counting was studied by Manninen et al. (1991). They compared counts of pure bacterial, yeast and mould cultures using standard plating methods and spiral plate counts determined both manually and with a laser colony detector. They concluded that counts were not significantly different except where large colonies (10-15 mm diameter) of Rhizopus oligosporus were enumerated. Other Rhizopus and Mucor species are also likely to interfere with this method unless a suitable plating medium (such as DRBC) is used. Alonso-Calleja et al. (2002) found that the spiral plate technique compared well with standard plate counting on OGYE agar for enumeration of yeasts and moulds in goat’s milk cheeses; however, Garcı´ a-Armesto et al. (2002) found the method unsuitable for enumeration of yeasts in raw ewe’s milk. Hydrophobic grid membrane filters. Membrane filters overprinted with a square hydrophobic grid have been developed for rapid enumeration of bacteria. The hydrophobic grid membrane filter TM (HGMF) system is marketed as ISO-Grid by Neogen (Lansing, MI, USA). The HGMF ”count” is determined by a most probable number (MPN) calculation. Brodsky et al. (1982) applied the HGMF technique to counting yeasts and moulds in foods. They compared it with spad plating on

    34

    4 Methods for Isolation, Enumeration and Identification

    4.7 Estimation of Fungal Biomass A deficiency in all of the enumeration techniques which rely on culturing fungi is that the result is at best poorly correlated with growth or biomass. Biomass is usually regarded as the fundamental measure of fungal growth in biotechnology, but it is not easy to quantify under the conditions existing in foods. Mycelial dry weight is most commonly used as a biomass estimate, but its relationship to mycelial wet weight and to metabolism varies widely in foods, due to the great influence of aw on both of the latter parameters. Fungi growing at reduced aw can be expected to be more dense than at high aw due to increased concentrations of internal solutes, though this is exceptionally difficult to measure experimentally. The question of a satisfactory fundamental measure of fungal biomass remains unanswered. Despite these basic problems, several chemical and biochemical techniques are available to estimate the extent of fungal growth in commodities. These techniques rely either on some unique component of the fungus that is not found in other microorganisms or foods or on immunological or molecular techniques. Some are still in the developmental phase: the most important ones are described briefly here.

    4.7.1 Chitin Chitin is a polymer of N-acetyl-D-glucosamine and is a major constituent of the walls of fungal spores and mycelium. It also occurs in the exoskeleton of insects but is not psent in bacteria or in foods. Hence the chitin content of a food or raw material can provide an estimate of fungal contamination. Chitin is most effectively assayed by the method of Ride and Drysdale (1972). Alkaline hydrolysis of the food sample at 1308C causes partial depolymerisation of chitin to produce chitosan. Treatment with nitrous acid then causes partial solubilisation and deamination of glucosamine residues to produce 2,5-anhydromannose, which is estimated colorimetrically using 3-methyl-2-benzothiazolone hydrazone hydrochloride as the principal reagent. Alkaline hydrolysis is more readily accomplished at

    4.7 Estimation of Fungal Biomass

    1218C in an autoclave (Jarvis, 1977). Improved assay sensitivity was achieved by derivatisation of glucosamine and other products with o-phthalaldehyde, separation by high performance liquid chromatography and detection of fluorescent compounds with a spectrofluorimeter (Lin and Cousin, 1985). Ekblad and Nasholm (1996) also described an HPLC method which measured fluorescence of a 9-fluorenylmethylchloroformate derivative of glucosamine. The chitin assay remains rather complex and slow, usually requiring about 5 h. A number of studies have indicated that the chitin assay is a valuable technique for estimating the extent of fungal invasion in foods such as maize and soybeans (Donald and Mirocha, 1977), wheat (Nandi, 1978) and barley (Whipps and Lewis, 1980) to estimate mycorrhizal fungi and fungal pathogens in plant material and soil (Ekblad and Nasholm, 1996; Ekblad et al., 1998; Penman et al., 2000; Singh, 2005) and measure wood rotting fungi (Nilsson and Bjurman, 1998). Particular attention has been paid to the possibility of developing the chitin assay as a replacement for the Howard mould count for tomato products (Jarvis, 1977; Bishop et al., 1982; Cousin et al., 1984). The chitin assay has some shortcomings and has been severely criticised by some authors (e.g. Sharma et al., 1977). The relationship between dry weight and chitin content varies at least twofold for different food spoilage fungi (Cousin et al., 1984; Lin and Cousin, 1985; Cousin, 1996). Some foods contain naturally occurring amino sugars such as glucosamine and galactosamine, which should be removed by acetone extraction prior to hydrolysis (Whipps and Lewis, 1980). Products from rot-free tomatoes give positive glucosamine assays even after acetone extraction (Cousin et al., 1984) and chitin content does not increase proportionally with fungal growth (Sharma et al., 1977). Insect contamination of grain samples has been reported to produce grossly misleading results (Sharma et al., 1977), but the psence of fruit flies in tomatobased products was less serious (Lin and Cousin, 1985). Materials such as stored grains frequently contain insect fragments and need to be checked before chitin assays are attempted. Because of these difficulties, the use of chitin as a chemical assay for fungi in foods has largely been superseded by the ergosterol assay.

    35

    36

    mycelial mass, and varied with substrate, aeration and growth phase. The ergosterol content was low during the rapid growth phase but tended to increase, at times sharply, as growth slowed. Taniwaki (1995) demonstrated that in fungi growing in atmospheres low in O2 and with elevated CO2 levels, the ergosterol content of hyphae was significantly reduced. In atmospheres containing 60% CO2, ergosterol content per unit of hyphal length was up to six times less than in air. Growth medium also affected ergosterol concentrations: on average, seven foodborne fungal species grown on PDA produced more than twice as much ergosterol per unit hyphal length as when grown on CYA (Taniwaki, 1995; Taniwaki et al., 2006). However, there was a reasonable correlation between ergosterol and mycelium dry weight for seven of the eight species tested. Eurotium chevalieri was the exception: this species produced little ergosterol and appeared to produce several other sterols (Taniwaki et al., 2006). Marı´ n et al. (2005) examined 16 species of food spoilage fungi and concluded that ergosterol content and colony diameters were better correlated to fungal biomass than fungal counts were. Marı´ n et al. (2008) showed that, for 14 common food spoilage fungi, correlation coefficients between ergosterol and colony diameters were sufficiently significant over a range of aw values (0.95-0.85), pHs (5-7) and potassium sorbate concentrations (0.5-1.5%) for both parameters to be useful in growth modelling. Quantifying ergosterol production in foods has proved more difficult. Seitz et al. (1977) showed a good correlation between damage in rice grains and their ergosterol content, between ergosterol in wheat and rainfall during the growing season and between ergosterol content and fungal invasion in several sorghum hybrids. Matcham et al. (1985) reported good correlations between linear extension of Agaricus bisporus grown on rice grains and chitin, ergosterol and laccase production. Ergosterol content correlated with colony counts of fungi on wheat grains at 0.95 aw but not at 0.85 aw (Tothill et al., 1992). Using a stereomicroscope for visual examination, they concluded that sound grain contained up to 6 mg/g ergosterol, microscopically mouldy grain 7.5-10 mg/g and visibly mouldy grain more than 10 mg/g ergosterol. From studies on ergosterol levels, colony counts and mould growth in a variety of grain samples, Schnu¨rer and Jonsson (1992) concluded that ergosterol correlated with colony counts

    4 Methods for Isolation, Enumeration and Identification

    better on DG18 (r = 0.77) than on MEA (r = 0.69). Ergosterol levels of food grade wheat ranged from 2.4 to 2.8 mg/g dry weight, samples from field trials (of unspecified quality) from 3.0 to 5.6 mg/g and feed grains from 8 to 15 mg/g dry weight. After an extensive survey of ergosterol levels in Danish crops, Hansen and Pedersen (1991) concluded that the normal levels of ergosterol in barley were 7.6 – 2.8, wheat for bread making 5.0 – 1.5, rye for bread making 6.8 – 2.2, peas 2.2 – 2.7 and rapeseed 2.4 – 1.3 mg/g dry weight. Ochratoxin A in barley correlated well with ergosterol content and reached significant levels when ergosterol increased to 25 mg/g dry weight. However, aflatoxin B1 became detectable in cottonseed meal when ergosterol reached only 4 mg/g. ”Burned” rapeseed, a measure of quality, became significant when ergosterol reached 1.4 mg/g dry weight. Lamper et al. (2000) found that ergosterol content correlated well (r = 0.87) with deoxynivalenol levels in wheat inoculated with Fusarium graminearum or F. culmorum. Moraes et al. (2003) found that there was good correlation between mould counts and ergosterol content of Brazilian maize (r = 0.94) but a poor correlation (r = 0.4) between ergosterol and aflatoxin content. Pietri et al. (2004) found significant correlation between ergosterol content of Italian maize and the major mycotoxins, fumonisin B1 (1995 crop) or zearalenone and deoxynivalenol (1996 crop). Ergosterol content correlated strongly with fat acidity values and germination ability of stored canola (Pronyk et al., 2006). These authors also noted that Penicillium and Aspergillus species contributed more to ergosterol than Eurotium species. Ergosterol levels in sound canola were between 1.46 and 1.67 mg/g, whereas levels above 2 mg/g indicated significant levels of spoilage (Pronyk et al., 2006). Karaca and Nas (2006) examined ergosterol content of dried figs and found good correlation (r = 0.92) between aflatoxin and ergosterol in reject figs which were fluorescent, but no significant correlation with patulin content. Kadakal et al. (2005) found good correlation (r = 0.98) between ergosterol and patulin in apple juice and that both patulin (r = 0.99) and ergosterol (r = 0.99) were linearly related to the proportion of decayed apples used to make the juice. Ergosterol has been used to assess mould growth in cheese with variable results (Pecchini, 1997; Taniwaki et al., 2001a).

    4.7 Estimation of Fungal Biomass

    Ergosterol content has also been investigated as an indicator of the mycological status of tomato products. Battilani et al. (1996) found a significant correlation between ergosterol, Howard mould count (HMC) and fungal growth, but with a high level of uncertainty. Kadakal et al. (2004) found a linear relationship between degree of decay in tomato pulp and HMC (r = 0.97) and ergosterol (r = 0.96) and concluded that ergosterol has the potential to be used in quality assessment of tomatoes. Sio et al. (2000) described an improved method for extraction of ergosterol from tomato products. Other applications of ergosterol as a measurement of fungal biomass include estimation of mould spores in indoor air and aerosols (Miller and Young, 1997; Robine et al., 2005; Lau et al., 2006), estimation of wood decay by fungi (Eikenes et al., 2005) and estimation of fungi in soil and wetlands (e.g. Headley et al., 2002; Zhao et al., 2005). The ergosterol assay is reported to have a high sensitivity and, in contrast to the chitin assay, requires only 1 h for completion (Seitz et al., 1979). Despite its limitations, it appears to be a useful indicator of fungal invasion of foods and to hold promise as a routine technique for quality control purposes.

    4.7.3 Impedimetry and Conductimetry Metabolites produced by growth of microorganisms in liquid media alter the medium’s impedance and conductance. The use of changes in these properties as a measure of bacterial growth was suggested by Hadley and Senyk (1975) and was first applied to yeasts by Evans (1982) and to moulds by Jarvis et al. (1983). Most of the subsequent work on fungi has been carried out with yeasts, but the methodology is often applicable to moulds also. During the 1980s there were a number of studies aimed at optimising media for inducing detectable and reproducible changes in either conductance or capacitance during fungal growth (see Pitt and Hocking, 1997). Watson-Craik et al. (1989, 1990) studied 27 mould species on a wide range of both commercially available and specially ppared media and concluded that conductance and capacitance were both medium and species specific. The use of media high in ammonium ions and glucose, with added yeast extract and peptone, decreased the

    37

    influence of product variability and induced higher conductance changes (Owens et al., 1992). An impedimetric method for detection of heat resistant fungi in fruit juices was described by Nielsen (1992). The detection limit in artificially contaminated juices was one Neosartorya ascospore per millilitre, detectable in 100 h. Huang et al. (2003) reported an impedance method for detection of bacteria and fungi in bottled water which shortened the detection time from 5 days to 27.1 h for fungi and from 48 to 11.3 h for bacteria. Although impedimetry and conductimetry promised to be effective rapid methods when used under well-defined conditions for a specific purpose with a particular kind of food, the methodology does not appear to have been broadly taken up for food mycology applications.

    4.7.4 Adenosine Triphosphate (ATP) ATP has also been suggested as a measure of microbial biomass because bioluminescence techniques provide a very sensitive assay (Jarvis et al., 1983). Provided that background levels of ATP in plant or other cells are very low, or that microorganisms can be effectively separated from such other materials, the method has some potential as a microbial assay. A good correlation was shown between ATP production and viable counts of six species of psychrotrophic yeasts grown in pure culture (Patel and Williams, 1985). The effective detection of low levels of yeasts in carbonated beverages by ATP has also been reported (LaRocco et al., 1985). However, living plant cells contain high levels of ATP and fungi are often very difficult to separate from food materials. Moreover, extraction of molecules from fungal cells is notoriously difficult, so this potential may be difficult to realise in food mycology. The most widespad application of ATP bioluminescence in the food industry is for monitoring hygiene of surfaces in food production facilities (Easter, 2007).

    4.7.5 Fungal Volatiles Methods for detection and characterisation of fungal volatiles are finding increasing applications in

    38

    4 Methods for Isolation, Enumeration and Identification

    of volatile compound production as an indicator of mould deterioration in grains has been extensively assessed and reviewed (Kaminski and Wasowicz, 1991; Schnu¨rer et al., 1999; Magan and Evans, 2000; Paolesse et al., 2006; Balasubramanian et al., 2007). Fungal volatiles can be used to detect potential mycotoxin contamination, to discriminate between fungal species (Sunesson et al., 1995; Keshri et al., 1998) and even between toxigenic and non toxigenic strains of particular fungi (Sahgal et al., 2007). Karlshøj et al. (2007a) used an electronic nose to differentiate between closely related Penicillium species (P. camemberti, P. nordicum, P. paneum, P. carneum, P. roqueforti and P. expansum) from cheese. Volatile profiles can be used to pdict mould spoilage in bakery products (Vinaixa et al., 2004; Marı´ n et al., 2007a) and to detect and differentiate between toxigenic and non toxigenic P. verrucosum strains in bakery products (Needham and Magan, 2003). Volatile profiles have also been used to differentiate between toxigenic and non toxigenic Fusarium strains (Keshri and Magan, 2000; Demyttenaere et al., 2004), to identify mycotoxins (aflatoxins, ochratoxin A and deoxynivalenol) in durum wheat (Tognon et al., 2005) and to detect and quantify ochratoxin A and deoxynivalenol in barley (Olsson et al., 2002). This technology has also been applied to pdict the psence of P. expansum and patulin in apple products (Karlshøj et al., 2007b) and to detect and discriminate diseases of potato tubers (Kushalappa et al., 2002) and stemend rot and anthracnose in mangoes (Moalemiyan et al., 2006). Electronic nose technology has also been used for early detection of moulds in libraries and archives (Pinzari et al., 2004).

    4.7 Estimation of Fungal Biomass

    Enzyme-linked immunosorbent assay (ELISA). The pparation of antigens from three common foodborne fungi (Penicillium aurantiogriseum, Mucor racemosus and Fusarium oxysporum) was described by Notermans and Heuvelman (1985). Preparation of immunoglobulin antibodies against these antigens was followed by development of an ELISA assay. Fungi were detected in both unheated and heat processed foods by this method. Antigens were relatively genus specific: the M. racemosus antigens reacted with other Mucor and Rhizopus species, and the Penicillium antigen reacted with the Aspergillus species tested. It was subsequently shown that the Penicillium antigen reacted with 43 of 45 Penicillium species tested and that antigen production correlated with mycelial weight and was unaffected by culture conditions, medium, temperature and aw (Notermans et al., 1986). The Penicillium antigen also reacted with Aspergillus flavus and the level of antigen correlated with aflatoxin production (Notermans et al., 1986). ELISA techniques have also been studied as a potential replacement for the Howard mould count. Antigens from tomato moulds (Alternaria alternata, Geotrichum candidum and Rhizopus stolonifer) were used to produce an ELISA test sensitive to 1 mg/g of mould in tomato. A correlation was observed between antigen formation and mould added to tomato puree, while background interference was very low (Lin et al., 1986). The method was tested against a broader range of foods, with encouraging results (Lin and Cousin, 1987). Robertson and Patel (1989) improved the sensitivity of the method for tomato paste by using a polyclonal antibody against Botrytis cinerea, Mucor piriformis and Fusarium solani in addition to the three species used by Lin et al. (1986). ELISA based methodology has been reported for the detection of Fusarium species in corn (Meirelles et al., 2006), cornmeal (Iyer and Cousin, 2003) and grain (Rohde and Rabenstein, 2005). Correlations with other measures of fungal growth (ergosterol, colony count, mycotoxin levels) were variable. Aspergillus species have also been examined as targets for immunological detection because of their importance in mycotoxin contamination. ELISA detection of A. ochraceus in wheat (Lu et al., 1995), coffee powder, chilli powders and poultry feed (Anand and Rati, 2006) has been investigated, with reasonable correction with other parameters

    39

    40

    Schwabe et al. (1992) compared the latex agglutination assay with ergosterol production for detection of Penicillium, Aspergillus and Fusarium species in pure culture. They concluded that the two methods were comparable for Penicillium and Aspergillus but that ergosterol was more sensitive for Fusarium. In food samples, both the latex agglutination test and ergosterol were effective means of detecting mould growth, but no clear correlation existed in values obtained by the two methods. Kesari et al. (2004) used a latex agglutination test to detect teliospores of Karnal bunt (Tilletia indica) in single grains of wheat and were able to detect a few as 750 teliospores, which they reported as suitable for single seed analysis. Fluorescent antibody techniques. Fluorescent antibody techniques have also been used directly for the detection of mould in foods. Warnock (1971) detected Penicillium aurantiogriseum in barley by this method, while Robertson et al. (1988) used antisera from five fungi to visualise moulds and simplify their detection in the Howard mould count technique.

    4 Methods for Isolation, Enumeration and Identification

    4.8 Identification Media and Methods

    Sequencing of the ITS region along with ”housekeeping genes” such as calmodulin, b-tubulin and elongation factor 1-a is now commonly applied for purposes of identification and phylogenetic analysis of important food spoilage and mycotoxigenic fungi in the genera Aspergillus (Varga, 2006; Geiser et al., 2007; Peterson, 2008), Penicillium (Peterson, 2004, 2006; Samson et al., 2004b; Wang and Zhuang, 2007; Serra et al., 2008) and Fusarium (O’Donnell et al., 2004; Scott and Chakraborty, 2006; Leslie et al., 2007). Despite the apparent power of molecular techniques, they need to be applied with some caution, particularly when comparing DNA sequences with those in the publicly available databases to identify yeast or mould isolates. Correct identification relies on the database sequences having the correct name attached to them by the depositor, which is not always the case. If the identification makes sense, the percent homology is 98% or greater and the number of base pairs on which the homology is scored is high, then the answer is probably correct.

    4.8 Identification Media and Methods 4.8.1 Standard Methodology The identification keys in this book are based primarily on the standardised procedure described for the identification of Penicillium species by Pitt (1979b). Cultures are grown for 7 days on three standard media at 258C, and on one of these at 5 and 378C also. The three Media are Czapek yeast extract agar (CYA; Pitt, 1973) used at all three temperatures; malt extract agar (MEA; Raper and Thom, 1949) and 25% glycerol nitrate agar (G25N; Pitt, 1973). Their formulae are given in the Media Appendix. Preparation time of CYA and G25N is reduced by the use of Czapek concentrate (Pitt, 1973), which is added to the media at the rate of 1% of the aqueous portion. As media ingredients have become more purified in recent years, difficulties with extent and colour of sporulation on CYA have been encountered, especially with some Penicillium species. To overcome this problem, Czapek concentrate has been reformulated (Pitt, 2000) by the inclusion of traces of zinc and copper (Smith, 1949) (see Media Appendix).

    41

    4.8.3 Inoculation As shown in Fig. 4.3, Petri dishes of CYA and MEA for incubation at 258C are inoculated with a single culture at three points, equidistant from the centre and the edge of the plate and from each other. Plates of the other media are inoculated with two points per culture, as illustrated. With some fungi, especially Penicillium and Aspergillus, it is important to minimise colonies from stray spores. The most satisfactory technique is to inoculate plates with spores suspended in semisolid agar (Pitt, 1979b). Dispense 0.2-0.4 ml of melted agar (0.2%) and detergent (0.05%), such as polysorbitan 80 (Tween 80), in small vials and sterilise. To use, add a needle point of spores and mycelium to a vial and mix slightly. Then, before flaming the needle, use it to stab inoculate the 58C plate;

    42

    4 Methods for Isolation, Enumeration and Identification

    Fig. 4.3 Schematic of regimen used for culturing fungal isolates for identification

    residual spores on the needle make a good inoculum. Next, take a sterile loop, mix the vial contents thoroughly and inoculate the standard plates. Used vials can be sterilised by steaming and reused several times before being washed or discarded.

    4.8.4 Additional Media and Methods The plating regimen outlined above can be used to identify most of the fungi described in subsequent chapters of this book. Some exceptions exist, because certain genera either grow poorly or fail to sporulate on the standard media. As noted earlier in this chapter, fastidious xerophiles are identified on MY50G agar. Eurotium species, traditionally identified on Czapek agar with 20% sucrose, are identified here on Czapek yeast extract agar with 20% sucrose (CY20S; see Appendix). Trichoderma species are best identified on potato dextrose agar (PDA) after a relatively short incubation time (3-4 days), as the structures tend to autolyse as cultures mature. Penicillium subgenus Penicillium. Many species classified in subgenus Penicillium are morphologically similar, and identification using traditional morphological techniques remains difficult. These

    Penicillia are very common in foods, and many produce mycotoxins, so correct identification is often critical. Species within subgenus Penicillium fall into two groups: those with an affinity for proteinaceous foods and those which grow more vigorously in foods high in carbohydrate. Frisvad (1981, 1985) introduced creatine sucrose agar (CREA) to permit differentiation between these two groups. Creatine (as the sole nitrogen source) permitted growth of the former group while inhibiting the latter. Incorporation of bromocresol purple enabled visualisation of pH changes, either acid or alkaline, depending on the creatine or sucrose metabolism of a particular species. However, discrimination between positive and negative responses was not always clear, and the main tabulation of species reactions to CREA (Table 1 in Frisvad, 1985) was difficult to interpt. Frisvad (1993) subsequently produced a number of variations of CREA, including acid and neutral pH formulations and substitution of sucrose with fructose or lactose. Although the merits of each formulation were discussed, no firm recommendation resulted from this exercise. Pitt (1993a) modified Frisvad’s strongly alkaline CREA medium by studying several sucrose and creatine concentrations over a wide pH range. The result was neutral creatine sucrose agar (CSN), a medium producing eight different reactions among

    4.8 Identification Media and Methods

    the 20 Penicillium subgenus Penicillium species tested. When included in the normal plating regimen for identification of Penicillium cultures from the subgenus Penicillium, CSN provides a very useful aid to distinguishing between these difficult and closely related species. See Chapter 7 for details of use of CSN, its reactions and interptation. Its formulation is given in the Media Appendix. Dematiaceous Hyphomycetes. The natural habitats for many dematiaceous Hyphomycetes are plants or plant material, so finding suitable laboratory media and conditions to induce typical sporulation can be difficult. Alternaria, Curvularia, Stemphylium and Ulocladium species are best identified from dichloran chloramphenicol malt extract agar (DCMA; Andrews, 1992b) plates incubated for 7 days at 258C under lights. Conidial characteristics of Bipolaris species vary with type of medium. Species described here are best identified from tap water agar (TWA) containing a natural substrate such as sterilised wheat or millet seeds, or wheat straw. Drechslera species will grow well on DCMA but sporulation is poor. The best sporulation is achieved with V-8 juice (V-8 J) agar or TWA with one of the natural substrates mentioned above, incubated at 258C for 7-10 days under lights with a 12 h photoperiod. Light/dark periodicity is important as Drechslera cultures require light to produce conidiophores and darkness to produce conidia, possibly due to light inactivation of flavine necessary for conidial formation (Knan, 1971; Platt et al., 1977). For the identification of Trichoderma and Fusarium species, potato dextrose agar (PDA) is used. Fusarium species also require additional methods and media as outlined below.

    4.8.5 Identification of Fusarium Species Fusarium isolates exhibit unusually high variability in colony morphology and also may deteriorate rapidly in culture. Thus, they should be identified as soon as possible after initial isolation with a minimum of subculturing to avoid deterioration. It is common practice to ppare cultures of Fusaria from single spores for growth on identification media, as this reduces both of these problems.

    43

    Single sporing. The technique for pparing single spore cultures is as follows (Nelson et al., 1983; Leslie and Summerell, 2006). Pour about 10 ml of 2% water agar into unscratched glass or plastic Petri dishes and allow to dry, either by holding the plates at room temperature for several days or by placing them inverted in an oven at 37-458C for about 30 min. Prepare a suspension of conidia in a 10 ml sterile water blank so that it contains 1-10 spores per low-power (10) microscope field when a drop from a 3 mm loop is examined on a slide. With experience, this concentration can be gauged simply by observing the turbidity of the suspension. Pour the suspension of spores onto a dried water agar plate, drain off the excess liquid and incubate in an inclined position at 20-258C for 18-20 h. After incubation, open the Petri dish, shake off any accumulated moisture droplets and examine under a stereomicroscope using transmitted light. The germinating conidia should be visible under 25 magnification. A dissecting needle with a flattened end and sharpened edges is used to cut out small squares of agar containing single, germinating conidia. These single conidia are then transferred on the agar blocks to the desired growth medium. If the original culture is contaminated with bacteria, a drop of 25% lactic acid may be added to the water blank. Allow this acidified spore suspension to stand for 10 min before pouring onto a water agar plate. Germination of acid-treated Fusarium conidia may be delayed by 24 h or more. Media. Two media have been used in this book for the identification of Fusarium isolates: potato dextrose agar (PDA) for colony characteristics and colour and dichloran chloramphenicol peptone agar (DCPA) for the development of diagnostic macro-, micro- and chlamydoconidia. A third medium, carnation leaf agar (CLA), is recommended by some Fusarium specialists for Fusarium cultivation and identification (Nelson et al., 1983; Leslie and Summerell, 2006). CLA is an excellent medium, on which most Fusarium species readily produce their diagnostic macroconidia. Production of macroconidia on DCPA is usually comparable with that on CLA, but microconidia and chlamydoconidia are often more plentiful on CLA due to greater production of aerial hyphae. DCPA is used in the psent work rather than CLA,

    44

    however, because dried, gamma-irradiated carnation leaves are difficult to obtain in many localities. Inoculation and incubation. For identification by the methods used in this book, single spore cultures of Fusarium isolates should be ppared on agar blocks as outlined above, inoculated, one per plate, onto two plates each of PDA and DCPA and incubated at 258C for 7 days. Inpidual plates may be used for each medium, or alternatively pided plates may be used, with one medium on each half of the plate. Illumination during incubation is essential for the production of macroconidia. The light source may be diffuse daylight (not direct sunlight) or light from a bank of fluorescent tubes. A photoperiod of 12 h per day is normally used. Alternating temperatures of 20 and 258C have been recommended (Nelson et al., 1983; Leslie and Summerell, 2006) but are not essential. A simple light bank may be constructed from a standard 40 watt fluorescent fixture with two cool white tubes, suspended 0.5-l m above the laboratory bench or shelf supporting the cultures. The addition of a black light tube (e.g. Philips TL 40 W/80 RS F40BLB) is also desirable and in some cases essential to induce macroconidial or chlamydoconidial production.

    4.8.6 Yeasts Yeast identification systems. Identification of foodborne yeasts remains a difficult task, as colony characteristics and microscopic morphology are of limited value. Generally it has been necessary to use biochemical and physiological tests such as fermentation of carbohydrates, assimilation patterns for a range of carbon and nitrogen sources and growth at various temperatures. Details of these methods and media may be found in Kurtzman and Fell (1998), Kurtzman et al. (2003) or Barnett et al. (2000). Molecular methods (see below) are being used increasingly in yeast identification, replacing time consuming biochemical testing. Identification using systems based on biochemical and physiological testing is complex and time consuming. However, a number of attempts have been made to assist those who wish to persevere with yeast identification. Several simplified systems

    4 Methods for Isolation, Enumeration and Identification

    have been published in the literature, and both automated and manual yeast identification systems are now commercially available. Dea´k and Beuchat (1987) published a simplified identification key (SIK) which included 215 species of foodborne yeasts. They subsequently modified their system, restricting it to the 76 species most frequently occurring in foods (SIM), and reported that it was much more successful than the API 20C system (BioMe´rieux, Marcy-l’Etoile, France) for identification (Dea´k, 1992). The SIM uses only two Petri dishes and three test tubes to examine each strain for ability to assimilate 10 carbon sources, fermentation of glucose, assimilation of nitrate and splitting of urea. These biochemical tests are supplemented by morphological observations. SIM separates the yeasts into six groups by a dichotomous key utilising the results of five key tests. Further tests are used to differentiate the yeasts in these six groups using secondary dichotomous keys. A dichotomous key to 25 common species of foodborne yeasts was published by Smith and Yarrow (1995), who used 17 biochemical and physiological tests to distinguish the species. Of the commercial systems available, the most widely used for foodborne yeasts are the Biolog, which is an automated system, and the BioMe´rieux ID32C yeast identification strips which can be read manually or automatically using the ATB system. BioMe´rieux also markets the fully automated VITEK 2 system, based on a card containing 64 tests. However, the database comprises only 46 clinically important species (Aubertine et al., 2006) and so has limited application to foodborne yeasts. The Biolog (Biolog Inc., Hayward, CA, USA; http://www.biolog.com/microID.html) utilises a yeast identification test panel (YT MicroPlateTM) consisting of a matrix of 8 12 wells. The first three rows contain 35 carbon source oxidation tests using tetrazolium violet as an indicator of oxidation. The next five rows contain carbon assimilation tests which are scored turbidimetrically against a negative control panel containing only water. The last row contains two carbon sources and tests for the co-utilisation of various carbon sources with D-xylose. The hardware (Biolog MicroStation Reader) consists of an automated plate reader coupled with a computer, which interpts the

    4.9 Examination of Cultures

    results and compares them with the resident database which currently includes 267 species. Manual interptation of the Biolog plates is not recommended. This system has been designed with the food industry in mind, and the database contains all the common foodborne yeasts, unlike other systems which are usually aimed at the clinical market. The ATB ID32C system (BioMe´rieux, Marcyl’Etoile, France) is an automated system utilising the BioMe´rieux ID32C yeast identification strips. These strips contain 30 assimilation tests plus a positive (glucose) and a negative control well, all of which are inoculated with a yeast suspension of specified density. The strips are incubated at 308C for 48-72 h. As with the Biolog, the ATB automated system consists of a plate reader attached to a computer. The database associated with the ATB system contains 63 yeast species. The BioMe´rieux ID32C strips can be read manually, and the results enable identification of yeasts using published keys or computer identification programmes such as that of Barnett et al. (1996). This system may be used with reasonable success, particularly when the test results from the ID32C strips are supplemented with extra tests (glucose fermentation, urease production, nitrate utilisation, growth in 0.5 and 1% acetic acid and in 50 and 60% glucose, growth at 378C, production of pseudohyphae, ascospore formation and morphological observations) to give a more comphensive base for the identification. Growth in 0.5 and 1% acetic acid indicates pservative resistance, and growth in 50 and 60% glucose gives an indication of ability to grow at reduced aw. Both these parameters are important in determining a yeast’s ability to cause spoilage in particular products. Identification using DNA sequencing is increasingly becoming the method of choice, as extensive databases such as GenBank are freely available for identification purposes. The 600- 650 nucleotide D1/D2 region of the large subunit (26S) ribosomal DNA is the most widely targeted section of the genome, and sometimes the ITS region may also be used (Kurtzman et al., 2003). Sequencing of the D1/D2 region, along with some supplementary physiological and biochemical tests, is the identification method currently used in our laboratory.

    45

    Even using the available systems, identification of yeasts still requires some specialist knowledge and interptation and remains time consuming. Our experience indicates that no more than 12 species of spoilage yeasts are of real concern in foods. It is possible to differentiate these few species by relatively simple techniques, i.e. colony and microscopic morphology, growth on the standard media used for filamentous fungi, growth on other media which test for pservative resistance, ability to use nitrate as a nitrogen source and adaptation to high NaCl concentrations. Details of these techniques are given in Chapter 10.

    4.9 Examination of Cultures As noted above, all cultures for identification should first be grown on the standard regimen described earlier. After 7 days incubation, the following examination should be carried out and then the general key to fungi in Chapter 5 should be used. That key will be of assistance even in the event of cultures failing to grow under one or more of the standard conditions.

    4.9.1 Colony Diameters Measure the diameters of macroscopic colonies in millimetres from the reverse side (Fig. 4.4). Microscopic growth or germination at 58C is assessed by low power microscopy (60-100), by putting the 58C Petri dish on the microscope stage and examining by bright field transmitted light. Growth at 378C is assessed macroscopically only; germination of spores at 378C is an unreliable character.

    4.9.2 Colony Characters Colony appearance can be judged by eye or with a hand lens, but examination is more effective if a stereomicroscope is used. Magnifications in the range of 5-25 are the most useful. Characters such as type and location of sporing structures and

    46

    4 Methods for Isolation, Enumeration and Identification

    Fig. 4.4 Technique for measuring colony diameters by transmitted light

    extent of sporulation are best gauged with the stereomicroscope. Reflected light is usually more effective than transmitted light. To determine colony colours, examine colonies by daylight or by daylight-type fluorescent light. In some genera, reference to a colour dictionary is helpful. The Methuen ”Handbook of Colour” (Kornerup and Wanscher, 1978) has been used in this work and is highly recommended.

    4.9.3 Preparation of Wet Mounts for Microscopy Fungi should always be examined microscopically as wet mounts rather than fixed and stained like bacteria. To ppare a wet mount, use an inoculating needle to cut out a small portion of the colony which includes sporing structures. Examination with the stereomicroscope can be an invaluable aid here. For freely sporing fungi with little mycelium, cut a piece of colony near the edge, where fruiting structures are young and spore numbers not excessive. Take structures which may enclose spores, i.e. cleistothecia, from near colony centres, where the probability of mature spores is highest. If the only differentiated parts of the colony appear to be

    buried in the agar, e.g. pycnidia, take a sample of these with a small piece of the agar. Float the cut colony sample from the needle onto a slide with the aid of a drop of 70% ethanol. It may be necessary to tease out the specimen with the needle and the corner of a cover slip (square cover slips are best). Fungal specimens may be highly hydrophobic: the ethanol helps to wet the pparation, minimising the amount of entrapped air. When most of the ethanol has evaporated, add a drop of lactic acid (for phase or interference contrast optics) or lactofuchsin stain (see below) for bright field. Add a cover slip; if necessary remove excess liquid from the pparation by gently blotting with facial tissue or similar absorbent paper. The pparation is now ready for examination.

    4.9.4 Staining A wide variety of stains are in use for mycological work. However, most are time consuming to ppare, or to use, or are slow to act, because fungal walls and spores are highly resistant to stains. By far the most effective stain for use in food mycology is lactofuchsin (Carmichael, 1955), which suffers from none of these faults. It consists of 0.1% acid fuchsin

    4.9 Examination of Cultures

    dissolved in lactic acid of 85% or higher purity. Young actively growing fungal structures are pferentially coloured bright pink, so sporing structures can usually be readily distinguished against a background of older mycelium. Cleistothecial initials, developing asci and maturing ascospores are also seen more readily in pparations stained with lactofuchsin. Like most other mycological stains, lactofuchsin is corrosive. Take care to clean it off microscope parts or skin! Be especially careful of the objective faces, because lactic acid will slowly corrode the relatively soft glass used in lenses.

    47

    equipped with an eyepiece micrometer, which is essential for measuring dimensions of spores and sporogenous structures. In the examination of fungal mounts, it is stressed that it is most important to use low power optics before succumbing to the temptation to use oil immersion. The principal reason is that fungal pparations usually remain as small clumps and do not disperse as bacteria do. Only under low power is the search for the optimal area of the slide for the observation of fruiting structures likely to be rewarded. Once a suitable area is located under the 10 or 16 objective, move to the 40. This should be the lens most used; microscope optics are such that only the finest details of ornamentation can be observed more effectively under oil immersion than at this magnification. Aligning the microscope. Correct alignment of the microscope is essential, so that its resolution is as high as possible and it can be used for long periods without discomfort. An incorrectly aligned microscope will lead to poor observation, discomfort, fatigue, headache and eye strain. A person of normal visual acuity should be able to use a correctly aligned instrument throughout a whole working day without discomfort. The steps to correctly align a microscope are given below. They should be read in conjunction with the microscope manufacturer’s instructions. 1. Mount a slide on the stage and bring it into approximate focus. If a ppared slide is not available, a slide marked with a marking pen or ink line is a satisfactory substitute. 2. Close the microscope’s field diaphragm (the one at the microscope base nearer the light source). The image of the diaphragm opening should now be visible in the microscope field. If it is, first focus it with the condenser focusing knob and then centre it in the field with the condenser centring screws. If the diaphragm opening cannot be seen, first rack the condenser up and down and watch to see if the opening becomes visible; if it does not, rack the condenser to its highest position and then slowly open the field diaphragm until the opening comes into view. Centre the diaphragm approximately and proceed as above. 3. For bright field optics, the condenser diaphragm should be adjusted each time the objective power is changed. Remove one ocular; close the

    48

    condenser diaphragm so that the field seen down the open tube is about two-thirds its maximum size. With phase contrast and interference contrast systems, this adjustment is less critical. The pceding steps align the microscope itself and should be checked frequently. If optimal illumination is desired, each step should be carried out for each new slide and each objective change. As a routine habit, the whole process should take only a few seconds. If the available microscope is not equipped with a built-in light source, a field diaphragm and a fully centring condenser, it is unlikely to be satisfactory for the identification of small spored fungi such as Penicillium species. The following steps are designed to align the observer with the microscope, compensating for inpidual differences in sight. Provided settings on the microscope are remembered, these steps need be carried out only occasionally, to check that visual acuity has not altered. Different settings will be needed for an inpidual with and without spectacles or contact lenses. 4. Assuming the microscope is binocular, pull the oculars out to their greatest distance apart and then, while watching a focused field, move them gradually together until a single circular field is seen without strain or head movement. Note the distance on the scale between the oculars; this is the inpidual’s interpupillary distance. Repeat this operation two or three times until satisfied that the correct distance has been found. This distance should always remain the same and be similar on any microscope. 5. Under the 40 or 100 objective, locate a tiny, readily recognised point on the slide and focus on it. Take a piece of white card and place it between the focusing ocular and the corresponding eye. Leave the eye open. Now focus the tiny point with the other eye, carefully, with the microscope fine focus. Next, transfer the white card to the other ocular and, using the focusing collar beneath the ocular, refocus the tiny point. Remove the card and note the setting on the scale. Repeat until satisfied the correct setting has been found. 6. On some microscopes, the eyepiece micrometer can be focused independently. Use the focusing system on the ocular itself to focus the micrometer. Note the setting on the scale on the side of the ocular.

    4 Methods for Isolation, Enumeration and Identification

    Always check the settings on the microscope before use and after making measurements with the micrometer. It is very easy to upset the ocular alignment when measuring.

    4.10.1 Lyophilisation For unstable cultures, and indeed for the long term storage of any food spoilage fungi, freeze drying or lyophilisation is probably the best method of pservation. Many commercial systems are now available for carrying out this process. A satisfactory menstruum for lyophilisation of most fungi is l.5 normal strength reconstituted nonfat milk powder (15% in distilled water). For fungi with hydrophobic conidia, such as Aspergillus and Penicillium, a small amount of detergent (0.05%) such as polysorbitan 80 (Tween 80) should be added to the milk. Dispense the milk in 10 ml lots in small tubes or 12.5 ml (0.5 oz) McCartney bottles, and sterilise by steaming for 20 min on three successive days (the Tyndallisation process). The milk must be stored at 208C or above between steamings, to permit bacterial spores to germinate.

    4.10 Preservation of Fungi

    4.10.2 Other Storage Techniques A variety of systems other than lyophilisation have been proposed for long term storage of fungi. Of these, liquid nitrogen storage has found most acceptance with major culture collections. This type of storage appears to be superior to any other for plant pathogens and fungi which will not sporulate in pure culture. However, liquid nitrogen systems are expensive to establish and maintain and are only suitable for large collections. Freezer units which run at very low temperatures (-808C or below) are available and are well suited to the needs of culture collections. In our laboratory we routinely store cultures at -808C, using glycerol (60-80%) as a cryoprotectant. Spore suspensions are ppared by taking conidia or ascospores from a freely sporulating sector of the colony, dispersing them in the glycerol then freezing in screw capped

    49

    cryovials. An 80% solution of glycerol remains viscous at -808C, which enables cultures to be removed from the freezer for subculture without the need for defrosting. For smaller laboratories that do not have access to lyophilisation, liquid nitrogen or ultra-low temperature storage, some simple techniques exist that can be used to maintain fungal cultures over relatively long periods (e.g. 1-10 years) without the need for subculturing. Water storage. A simple and inexpensive method of fungal culture pservation is storage of agar blocks in water (Smith and Onions, 1994). Small agar blocks (7-10 mm2) are cut from the growing margin of a young fungal colony and placed in sterile water in a bottle such as a Bijou bottle (6.25 ml or 0.25 oz McCartney bottle). The rubber lined cap is screwed down and the bottles stored in a cool room (1-108C). Cultures may be revived by removal of a block and placing it on a suitable growth medium. Using this method, cultures have been reported to remain viable and retain their characteristics for up to 7 years (Boeswinkel, 1976; Smith and Onions, 1994). Some yeasts may be maintained by storing as suspensions in water (Kirsop, 1984). Growth from a late logarithmic slant culture is suspended in sterile distilled water and transferred to a sterile container so that 90% of the volume is filled with the suspension. Containers are stored at room temperature. Survival of some Candida, Saccharomyces, Cryptococcus, Rhodotorula and Schizosaccharomyces species for up to 4 years has been reported (Kirsop, 1984). Silica gel storage. Many fungal cultures may be maintained for long periods (often more than 10 years) by drying spore suspensions onto silica gel (Smith and Onions, 1983; Smith, 1984). This method is not suitable for mycelial cultures, but can be used with some success for yeasts (Kirsop, 1984). As silica gel liberates heat when moistened, the technique depends on keeping the cultures cool enough to avoid damaging the spores during pparation. Medium grain plain (non-indicating) silica gel of 6-22 mesh is placed in suitable glass bottles (Bijou or McCartney bottles) to a depth of about 1 cm and sterilised, either by dry heat at 1808C for 2-3 h or by autoclaving at 1218C for 15 min. Autoclaved silica gel must be thoroughly

    50

    dried in an oven before use. Bottles are pcooled by placing in a tray of ice or refrigerating for 24 h, then transferring to an ice tray for inoculation. Suspensions of fungal spores or yeasts are ppared in sterile skimmed milk (as for lyophilisation, above) and the suspension added to the cooled silica gel to wet three-quarters of it. The bottles and gel are allowed to dry at room temperature for 10-14 days with caps slightly loosened. Caps are then screwed down and the bottles stored at 48C (storage at room temperature is also satisfactory) in air-tight containers over indicating silica gel to absorb any moisture. Cultures are revived by shaking a few crystals of silica gel onto a suitable growth medium (broth culture may be better for yeasts). Survival varies according to species or even strain, but survival of yeasts for up to 5 years (Kirsop, 1984) and fungi for more than 10 years (Smith and Onions, 1983) has been reported.

    4.11 Housekeeping in the Mycological Laboratory Like any other microbiological laboratory, a mycological laboratory should be kept in a clean condition. Discard unwanted cultures regularly and dispose of them by steaming or autoclaving. Wipe bench tops regularly with ethanol (70-95%). Floors should be wet mopped or polished only with machines equipped with efficient vacuum cleaners and dust filters. Where possible store food and plant materials away from the laboratory. Open Petri dishes carefully. Use small inocula on wet needles. Transport Petri dishes to the stereomicroscope stage before removing lids. Do not bump cultures during transport. Contrary to popular belief, a well-run mycological laboratory is not a source of contamination to bacteriological laboratories. The air in a mycological laboratory should not carry a significant population of fungal spores. The reverse problem can occur, however, because bacteria multiply more rapidly than do fungi. Bacterial spores are often psent in food laboratories, readily infect fungal plates and can rapidly outgrow and inhibit fungal mycelia, especially at 378C.

    4 Methods for Isolation, Enumeration and Identification

    If for any reason fungal spore concentrations do build up in a laboratory and cause an unacceptable level of contamination, the air should be purified. The simplest technique is to spray the air throughout the laboratory with an aerosol before it is closed in the evening. Any aerosol spray, such as a room deodoriser or air freshener, is effective. Aerosol droplets entrain fungal spores very efficiently and carry them to the floor. A more drastic and effective treatment in cases of severe contamination is to spray a solution of 2% thymol in ethanol around the room and close it for a weekend. The spray is rather pungent, and while not harmful to humans, it effectively kills fungal spores and mites (see below). Do not leave cultures on benches before fumigation.

    4.11.1 Culture Mites A major hazard in growing and maintaining fungal cultures is the culture mite. Many species of mites live on fungal hyphae as their main or sole diet in nature and find culture collections an idyllic environment. Mites crawl from culture to culture, contaminating them with fungi and bacteria as they go or, given long enough, may eat them out entirely. Mites are very small (0.05-0.15 mm long), usually just visible to the observant naked eye. They are arachnoids, related to spiders, and hermaphroditic. Each mite leaves a trail of eggs about half adult size as it goes. Eggs hatch within 24 h and reach adulthood within 2 or 3 days. The damage an unchecked mite plague can do to a fungal culture collection has to be experienced to be believed. The most common sources of mites are plant material, soil, contaminated fungal cultures and mouldy foodstuffs. Mites can also be carried on large dust particles. Building work near a laboratory almost always induces a mite infestation. The avoidance of losses due to mites requires constant vigilance. Always watch for telltale signs, such as contaminants growing around the edges of a Petri dish, a ”moth-eaten” appearance to colonies or ”tracks” of bacterial colonies across agar. Examination of suspect material or cultures under the stereomicroscope will readily reveal the psence of mites and mite eggs.

    4.11 Housekeeping in the Mycological Laboratory

    Adult mites are rapidly killed by freezing, and mite eggs will only survive 48-72 h at -208C. Cultures contaminated by mites can often be recovered by freezing for 48 h, then subculturing from uninfected portions of the culture with the aid of the stereomicroscope. Suspect food and other samples being brought into the laboratory should also be frozen for 24 h to destroy mites before enumeration or subculturing is carried out. Infestation by mites can be minimised by good housekeeping, i.e. by avoiding accumulation of dust or old cultures in the laboratory. It is also good practice to handle and store food and plant samples well away from areas where fungi are inoculated and incubated. To control a mite infestation, remove all contaminated material, including cultures. Freeze Petri dishes and culture tubes which must be recovered; autoclave, steam or add alcohol to all others. Clean benches thoroughly with sodium hypochlorite (household bleach) or 70% ethanol. Incubators can be disinfested with aerosol insecticides.

    4.11.2 Problem Fungi There are three fungal invaders which should be watched for carefully in a food mycology laboratory: Aspergillus fumigatus, Rhizopus stolonifer and Chrysonilia sitophila. The first is a human pathogen; the others can cause a contamination chain which is difficult to break. Aspergillus fumigatus may cause invasive aspergillosis in the lungs or serious allergenic responses in some inpiduals. It is sound practice to immediately kill and discard cultures of this fungus as soon as it is recognised. On no account should it be used for experimental studies in food spoilage or biodeterioration without pcautions to pvent dissemination of spores. The morphology of A. fumigatus is described in detail in Chapter 8, but it is readily recognisable in the unopened Petri dish:

    colonies are low, dull blue and broadly spading, with a velvety texture;

    growth is very rapid at 378C, covering a Petri dish in 2 days;

    long columns of blue conidia are readily seen under the stereomicroscope.

    51

    4.11.3 Pathogens and Laboratory Safety While it must be said that any fungus which is capable of growth at 378C is a potential mammalian pathogen, the physiology of the healthy human is highly resistant to nearly all of the fungi encountered in the food laboratory. Nevertheless, fungi which can grow at 378C should be treated with caution. In particular, the habit of sniffing cultures is to be avoided wherever possible. It is true that odours produced by fungi have been used quite frequently as taxonomic criteria, especially in older

    52

    publications, but their subjective and ephemeral nature makes them of little value for this purpose, and the risks involved are serious. Some laboratories regard fungal volatiles as such a serious risk that cultures such as Penicillium and Aspergillus species are handled in a biohazard cabinet. Many types of fungal spores are allergenic or carry mycotoxins. Inhalation of fungal spores should be avoided as far as possible. Of the fungi described in this book, only the Aspergilli normally pose any direct threat to health. Aspergillus fumigatus has already been mentioned, and care should also be taken when handling cultures of Neosartorya species, which are closely related to

    4 Methods for Isolation, Enumeration and Identification

    A. fumigatus and also grow prolifically at 378C. Other Aspergillus species, particularly A. flavus, A. niger and A. terreus, have been isolated from human specimens from time to time. For more details, see de Hoog et al. (2000). These species appear to be mainly opportunists and pose little threat to healthy people. Careful handling and good housekeeping are all that are required. However, immunocompromised inpiduals are in a different category. It is increasingly evident that such people have little resistance to fungal infection. Persons suspected to be immunocompromised, regardless of the cause, should not work in a mycology laboratory nor indeed be permitted knowingly to enter one.

    Chapter 5

    Primary Keys and Miscellaneous Fungi

    Principles underlying fungal classification have been outlined in Chapter 3, including a brief overview of the relevant pisions of the Kingdom Fungi and their principal methods of reproduction. Some further detailed information is necessary in this chapter to assist in the use of the keys which follow. Ascomycetes. As discussed in Chapter 3, Ascomycetes produce ascospores in asci (Fig. 3.2). One genus, Byssochlamys, produces asci which are unenclosed; all other genera of relevance here produce asci in some kind of fruiting body, or ascocarp. The two kinds of ascocarp commonly seen in food spoilage fungi, the cleistothecium and the gymnothecium, have been described and illustrated in Chapter 3 (Fig. 3.3). Both types of ascocarp are usually pale or brightly coloured, not dark, and release ascospores by rupturing irregularly. Of genera relevant here, cleistothecia are produced by Emericella, Eurotium, Eupenicillium, Monascus and Neosartorya, and gymnothecia by Talaromyces. A third class of ascocarp, less commonly encountered in foodborne fungi, is the perithecium. Perithecia have cellular walls like cleistothecia, but are distinguished by the psence of an apical pore or ostiole through which asci or ascospores are liberated; also asci are long and clavate with ascospores arranged linearly within them. In the one perithecial genus of interest here, Chaetomium, the perithecia are black and have stout hyphae attached to the walls (Fig. 5.10). Conidial fungi. The strictly conidial fungi, also known as Fungi imperfecti or Deuteromycetes or anamorphic fungi, possess an amazing variety of ways of producing conidia. Terminology for structures bearing conidia and for conidia themselves has

    become astonishingly complex in recent years; fortunately most of it is not essential for the recognition of the genera discussed in this text. Terms which are important in the keys which follow are described below. A fundamental pision within the asexual fungi separates genera which form conidia aerially, grouped in the class Hyphomycetes, from those in which conidia are borne in some sort of enveloping body, the class Coelomycetes. Hyphomycetes. Fungi have developed seemingly endless ways of extruding or cutting off conidia, solitarily or in chains, from fertile cells which themselves may be borne solitarily or aggregated into more or less ordered structures. Hyphomycete taxonomy attempts to thread a way through this maze. In general, type and degree of aggregation of the fertile cells, and type of conidium, provides the basis for generic classification, while details of these characters and of spore size, shape and ornamentation are more commonly used to distinguish species. Features of conidia used in the keys in this work are length, septation, ornamentation and colour, particularly whether walls are light or dark. The method of conidium formation (ontogeny) is seldom emphasised here, because terminology is complex and distinctions may not be obvious. The principal point to note is the disposition of conidia: they may be borne solitarily, i.e. just one conidium per point of production; singly, i.e. successively from a single point, but unattached to each other; or in chains. Solitary conidia are borne on a relatively broad base and usually adhere to the fertile cell. Conidia formed in chains are usually extruded from a small cell of determinate length, often a

    J.I. Pitt, A.D. Hocking, Fungi and Food Spoilage, DOI 10.1007/978-0-387-92207-2_5, Ó Springer ScienceþBusiness Media, LLC 2009

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    54

    phialide, which in most genera narrows to a distinct neck. Conidia borne singly may be extruded in this same manner, or be borne by extrusion from a pore in a hypha or fertile cell, or be cut off by hyphal fragmentation. Phialidic Hyphomycetes. Hyphomycetes may produce phialides solitarily (the genus Acremonium) or in loosely ordered structures (Trichoderma) or highly ordered structures (Aspergillus, Penicillium and related genera). Genera of interest here with less ordered phialidic structures can mostly be differentiated by macroscopic characters, e.g. colony diameters and colours. However, differentiating genera with highly ordered phialidic structures will necessitate careful microscopic examination. Phialides in Aspergillus, Penicillium and related genera are sometimes borne directly on a stalk or stipe which arises from a hypha; in other species, however, the phialides are borne from supporting cells, termed metulae (sing. metula) and in some species the metulae may in turn be supported by other cells, termed branches or rami (sing. ramus). The whole structure, including the stipe, is called a conidiophore. In Aspergillus, stipes are always robust, with thick walls and usually without septa; the stipe terminates in a more or less spherical swelling, the vesicle, which bears phialides, or metulae and phialides, over most of its surface. In Aspergillus, phialides (and metulae) are always produced simultaneously, and this feature can readily be recognised by examining young developing conidiophores (Fig. 8.1a). Similar structures, though smaller, are produced by some Penicillium species: these are clearly distinguished from Aspergillus species by stipes which are septate and by phialides which are produced over a period of time (successively; Fig. 8.2b). Most Penicillium species, and those of related genera, do not produce phialides on vesicles, but in a cluster directly on a stipe, or on metulae and/or rami. The fruiting structure in Penicillium and related genera is termed a penicillus, while that in Aspergillus (for want of a better term) is called a head (or more recently, an aspergillum, Klich, 2002). Coelomycetes. As noted earlier, Coelomycetes produce conidia within an enveloping body, termed a conidioma (pl. conidiomata). In Petri dish culture, conidiomata are produced on or just under the agar surface and are macroscopically visible, usually being 100-500 mm in diameter. Two kinds of conidioma are important here: the pycnidium, a more

    5 Primary Keys and Miscellaneous Fungi

    or less spherical body with one or more pores (ostioles) through which conidia are released, and the acervulus, a flat body from which conidia are released by lifting or rupturing of a lid. The majority of Coelomycetes are pathogens on plants and many have not been studied in pure culture. In consequence, their taxonomy is difficult and genera and species are often poorly delimited. For a complete account of Coelomycete taxonomy see Sutton (1980). Yeasts. Yeasts are fungi which have developed the ability to reproduce by forming single vegetative cells by budding or, in a few species, by fission, in a manner similar to bacteria. Like bacteria, and unlike fungal spores, such cells are metabolically active and may in turn reproduce by budding (or fission). Yeast cells may survive for long periods both in culture and in nature. In consequence many yeasts produce true spores rarely or not at all. Yeasts are readily distinguished from filamentous fungi on the agar plate by their soft-textured colonies and limited growth. They are usually also readily distinguished from bacteria by their raised and often hemispherical colonies, white or pink colours and lack of ”bacterial” odour. If in doubt, make a simple wet mount of a colony in water or lactofuchsin, add a cover slip and examine with the oil immersion lens. Yeast cells are larger than bacteria, measuring at least 3 2 mm and are nonuniform in size. If the culture is not too old, some cells will usually show developing buds. Yeasts cannot be classified solely by morphological features or growth on the standard media, and so are considered in a separate chapter (Chapter 10).

    5.1 The General Key The taxonomic terms discussed above will enable the use of the general and miscellaneous keys which follow, although some other taxonomic terms may be introduced in discussions of particular genera. It is emphasised that these keys are designed for use on isolates which have been incubated for 7 days on the standard plating regimen outlined in Chapter 4. Colony diameters should be measured in millimetres from the reverse side by transmitted light. The general key has been designed to be as simple as possible and suitable for routine use, but it should be read in conjunction with the notes below it.

    5.1 The General Key

    55

    General key to food spoilage fungi 1

    No growth on any standard medium in 7 days Growth on one or more standard media

    Chapter 9 – ”Xerophilic fungi” 2

    2 (1)

    Colonies yeasts, either recognisably so on isolation or in culture, i.e. colonies soft, not exceeding 10 mm diam on any standard medium Growth filamentous, exceeding 10 mm diam on one or more standard media

    Chapter 10 – ”Yeasts” 3

    3 (2)

    Growth on CYA and/or MEA faster than on G25N Growth on G25N faster than on CYA and MEA

    4 (3)

    Hyphae frequently and conspicuously septate Hyphae lacking septa or septa rare

    5 (4)

    No mature spores psent in 7 days Mature spores psent in 7 days

    6 9

    6 (5)

    Immature fruiting structures of some kind psent No fruiting structures (or spores) detectable by lowpower microscopy or wet mounts from CYA or MEA

    7

    5 Chapter 6 – ”Zygomycetes”

    7 (6)

    Colonies and fruiting structures white or brightly coloured Colonies or fruiting structures dark

    8 (7)

    Colonies and fruiting structures white Colonies or fruiting structures brightly coloured

    9 (5)

    Spores (conidia) less than 10 mm long, borne in chains on clustered fertile cells (phialides), on well-defined stipes Spores (conidia) of various sizes, borne singly or solitarily, or if borne in chains, then chains not in aggregates

    10 (9)

    11 (10)

    Conidia blue or green, phialides produced successively on vesicles, vesicles less than 10 mm diam, stipes usually septate Conidia variously coloured, phialides and/or metulae produced simultaneously on vesicles, vesicles larger than 10 mm diam, stipes nonseptate

    Couplet 1. No growth on any standard medium indicates an extreme xerophile, i.e. Xeromyces bisporus or a Chrysosporium species, or a nonviable culture. Next inoculate culture onto MY50G for 7 days at 258C. If growth occurs, enter the key in Chapter 9, ”Xerophilic Fungi”; no growth on MY50G indicates a nonviable culture. Chrysosporium and Xeromyces

    See section on ”Miscellaneous fungi” below 8 Continue incubation; when spores mature, refer to section on ”Miscellaneous fungi” below Chapter 8 – ”Aspergillus and its teleomorphs” Chapter 7 – ”Penicillium and related genera”

    Phialides or metulae and phialides borne on more or less spherical swellings on the stipe apices Phialides borne on penicilli, i.e. on unswollen stipes with or without intervening metulae and rami

    5.1.1 Notes on the General Key

    4 Chapter 9 – ”Xerophilic fungi”

    10 See section on ”Miscellaneous fungi” below

    11 Chapter 7 – ”Penicillium and related genera”

    Chapter 7 – ”Penicillium and related genera”

    Chapter 8 – ”Aspergillus and its teleomorphs”

    isolates are usually white or rarely golden brown. If the original culture used as inoculum is coloured other than pure white or golden brown, it is probably nonviable. Couplet 2. Yeasts are usually readily distinguished by slow growth, soft, easily sampled colonies, small spherical to ellipsoidal cells, often of variable size and shape and by reproduction by budding. See Chapter 10, ”Yeasts” for identification procedures.

    56

    5 Primary Keys and Miscellaneous Fungi

    Couplet 3. The ability to grow more rapidly on G25N than on CYA or MEA indicates a xerophile (at least for keying purposes here). Check the key in Chapter 9, ”Xerophilic Fungi”. Some isolates can be identified from the standard plates, while others will require growth on CY20S or MY50G for identification. Couplet 4. The absence of septa in young, growing hyphae indicates an isolate belongs to subkingdom Zygomycotina, discussed here in Chapter 6 ”Zygomycetes”. Couplets 5, 6. Some isolates from a variety of fungal genera will not produce spores on the standard media in 7 days. Continue to incubate such cultures, pferably in diffuse daylight such as a laboratory window sill, at temperatures near 258C. Also inoculate such cultures onto two or three plates of DCMA and incubate these at 258C or thereabouts in darkness and in diffuse daylight or, if possible, under fluorescent illumination (see Chapter 4). After 1-2 weeks, check again for spores or fruiting bodies. If such structures are not seen, the isolate is unlikely to be significant in foods. Apparently asporogenous cultures should also be checked with a stereomicroscope while scraping up a sector of the colony with a needle. Fruiting bodies submerged in the agar will sometimes become visible with this technique. Couplet 7. Some isolates which produce white or brightly coloured fruiting bodies also produce very sparse aerial conidial structures which are easily overlooked. Check such cultures carefully with the stereomicroscope; if conidial structures are found, make a wet mount and reenter the key at Couplet 5.

    A finely drawn glass needle will sometimes be of assistance in making mounts from delicate conidial structures on the colony. Nearly all dark fruiting structures encountered will mature at 258C within 2 weeks. Light does not usually influence this process. When mature spores are formed, refer to the following section.

    5.2 Miscellaneous Fungi In this section are considered the genera which do not logically fit into some larger grouping considered elsewhere. Some are important in specific food spoilage problems, others are found in particular habitats such as cereals, while still others repsent the aerially dispersed fungal flora found as ubiquitous contaminants or saprophytes. As will be seen, they are a very heterogeneous collection. Most fungi significant in food spoilage or food contamination and not treated in other chapters are included here. It is inevitable, though, that occasional isolates from foods will not belong to the genera considered in this section. The key has not been designed to take account of this, as it would be a practical impossibility. So when an isolate appears to key out satisfactorily, it must be checked against the description to confirm the identification. Some isolates will of course belong to a recognisable genus, but not the species described; in that case the references indicated will provide further information. The miscellaneous fungal genera are considered in alphabetical order following the key.

    Key to miscellaneous genera 1

    Colonies on CYA and MEA not exceeding 60 mm diam in 7 days Colonies on CYA or MEA exceeding 60 mm diam in 7 days

    2 12

    2 (1)

    Conidia borne within a fruiting body on or beneath the agar surface Conidia borne from aerial or surface hyphae

    3 (2)

    Mycelium and conidia hyaline or brightly coloured Mycelium and/or conidia dark coloured

    4 11

    4 (3)

    Conidia with a single lateral septum Conidia nonseptate or with more than one septum

    Trichothecium 5

    5 (4)

    Conidia borne from gradually tapering fertile cells (phialides) Conidia borne directly on hyphae or by budding or hyphal fragmentation

    Phoma 3

    6 7

    5.2 Miscellaneous Fungi

    6 (5)

    Colonies exceeding 50 mm diam on CYA Colonies not exceeding 50 mm diam on CYA

    7 (5)

    Colonies exceeding 45 mm diam on MEA; conidia borne solely by the breakup of hyphae to form arthroconidia Colonies not exceeding 40 mm diam on MEA; conidia not exclusively arthroconidia

    8 (7)

    9 (8)

    10 (9)

    Budding hyphal fragments (10-50 mm long) psent Hyphal fragments absent; or if psent, not budding

    11 (3)

    Colonies low, mucoid and yeast-like, becoming grey to black in both obverse and reverse Colonies dry and velutinous, obverse green, reverse olive or deep blue black

    57

    Fusarium Acremonium

    Geotrichum 8

    Monascus 9

    Endomyces 10 Hyphopichia Moniliella

    Aureobasidium Cladosporium

    12 (1)

    Spores borne within an enclosed fruiting body on or under the agar surface Spores borne from aerial or surface hyphae

    13 17

    13 (12)

    Spores consistently less than 15 mm long The larger or all spores more than 15 mm long

    14 15

    14 (13)

    Fruiting bodies perithecia with stout, black hyphae attached to the walls Fruiting bodies pycnidia, without attached hyphae

    15 (13)

    Fruiting bodies roughly spherical (pycnidia) Fruiting bodies flat (acervuli)

    16 (15)

    Conidia hyaline or brightly coloured, nonseptate, without terminal appendages Conidia dark, with three or four septa and with spike-like, sometimes branched, terminal appendages

    Chaetomium Phoma Lasiodiplodia 16 Colletotrichum Pestalotiopsis

    17 (12)

    Colonies and conidia hyaline or brightly coloured Colonies and/or conidia dark coloured

    18 22

    18 (17)

    Colonies with grey or green areas Colonies white, orange, pink or purple

    19 20

    19 (18)

    Colonies green Colonies grey

    20 (18)

    Colonies low and persistently white Colonies floccose, white or becoming brightly coloured

    Geotrichum 21

    21 (20)

    Colonies pdominantly orange, orange conidia shed profusely around the Petri dish rim Colonies white, pink or purple, sporulation on MEA weak or absent, better on DCPA under lights

    Chrysonilia

    22 (17)

    Conidia consistently less than 15 mm long Conidia frequently exceeding 15 mm long

    Trichoderma Botrytis

    Fusarium 23 25

    58

    23 (22)

    5 Primary Keys and Miscellaneous Fungi

    Conidiophores long, branched, apically swollen, bearing closely packed pale brown conidia Conidiophores short or ill-defined, dark brown or black conidia borne irregularly

    Botrytis 24

    24 (23)

    Conidia dark brown, often with a lighter coloured band around the periphery Conidia uniformly jet black

    Arthrinium Nigrospora

    25 (22)

    Conidia approximately spherical Conidia elongate

    Epicoccum 26

    26 (25)

    Conidia with transverse septa (or thick walls between cells) only Larger conidia with both transverse and longitudinal septa or irregularly septate

    27 30

    27 (26)

    Conidia clavate (club shaped), often with long hyphal appendages at the apices; found almost exclusively on rice Conidia cylindroidal, ellipsoidal or an elongate ”D” shape; source more general

    Trichoconiella 28

    28 (27)

    Conidia cylindroidal, with parallel sides except at the terminal cells Conidia fusoid, narrowing from the central cells to the terminal cells, often bent or ”D” shaped

    Drechslera

    29 (28)

    Conidia not exceeding 40 mm long Conidia usually exceeding 40 mm long

    Curvularia Bipolaris

    30 (26)

    Conidia clavate (club shaped) Conidia spherical to roughly ellipsoidal or short cylindrical

    Alternaria 31

    31 (30)

    Conidia often tapering towards the base and sometimes pointed, i.e. pyriform or apiculate Conidia spherical to short cylindrical, not tapering from base to apex, with rounded ends

    5.3 Genus Acremonium Link Commonly referred to as Cephalosporium Corda in p-1970 literature, Acremonium is a large and varied genus characterised by the production of small, hyaline, single-celled conidia borne singly, i.e. successively but not connected to each other, from solitary phialides. A variety of species have been recorded from foods from time to time. One species, A. strictum, described here, is of relatively common occurrence. In this species, the phialides gradually taper to the apices without basal thickening or formation of a distinct neck, and conidia aggregate in balls of slime. Under the stereomicroscope, the slime balls look like large single spores, but their true nature becomes evident in wet mounts. A. strictum appears mainly in the earlier food literature under the name C. acremonium. As noted by Domsch et al. (1980), this name has been used for a variety of species, so that reports on physiology and occurrence are unreliable (Fig. 5.1).

    Acremonium strictum W. Gams

    29

    Ulocladium Stemphylium

    Fig. 5.1

    Cephalosporium acremonium (name of uncertain application; no valid authority).

    Colonies on CYA 20-30 mm diam, white or orange to pink, dense to floccose or funiculose; reverse pale or with orange to pink tones. Colonies on MEA 13-20 mm diam, similar to those on CYA or of slimy texture. Colonies on G25N 1 mg/kg, but can occur at lower levels sometimes. In ppubertal gilts, clinical signs include vulval swelling, uterine enlargement and mammary development. Mature sows can develop ovarian atrophy, constant oestrus and pseudopgnancy (Hagler et al., 2001). Prepubertal male pigs can undergo a feminising effect with mammary development, decreased testicular size and loss of libido, but mature boars are resistant (Hagler et al., 2001). It was once claimed that humans had shown similar signs in Puerto Rico, but an FDA investigation failed to confirm this (Goodman et al., 1987). T-2 toxin. T-2 toxin is the cause of alimentary toxic aleukia (ATA) a devastating disease which occurred in the USSR during and after Second World War, in times of extreme food shortage resulting in consumption of overwintered cereals (Joffe, 1978; Beardall and Miller, 1994). Many people, probably hundreds of thousands, died as a result (Marasas et al., 1984). ATA was characterised by leucopoenia, bleeding from nose, throat and gums, haemorrhagic rash, exhaustion of the bone marrow and fever. Vomiting, nausea, diarrhoea and abdominal pain also usually occurred. Decrease in immunological functions led to susceptibility to bacterial and viral diseases, and often death (Joffe, 1978; Beardall and Miller, 1994). Haemorrhagic syndrome in cattle, pigs and poultry in the United States in the 1960s was also probably due to T-2 toxin (Desjardins, 2006). T-2 toxin is produced by F. sporotrichioides and, less commonly, F. poae (Desjardins, 2006). It appears to be produced only under cold conditions, and fortunately is now uncommon. The occurrence, toxicity and biology of T-2 toxin were examined in detail by JECFA (2001). They established that T-2 (and its metabolite HT-2) were immunotoxic and haemotoxic compounds in several animal species after short-term intake. Long-term effects could not be evaluated. T-2 was at most weakly genotoxic. In the absence of long-term studies, T-2 was not classifiable as to carcinogenicity (JECFA, 2001). Fumonisins. Fumonisins are produced by F. verticillioides (formerly known as F. moniliforme), by the closely related species F. proliferatum, uncommonly by F. subglutinans and F. oxysporum,

    92

    5 Primary Keys and Miscellaneous Fungi

    with neural tube defects such as spinal bifida in a population along the Texas-Mexican border (Desjardins, 2006). For the human population, a provisional maximum tolerable daily intake (PMTDI) of 2 mg/kg body weight per day was established by JECFA (2001). Determining which particular Fusarium species produces which mycotoxins has not been easy. Unstable taxonomy and misidentification have combined to cloud the picture. In a major contribution, Marasas et al. (1984) examined several hundred Fusarium isolates, identifying them according to Nelson et al. (1983) and then assessing mycotoxin production. Sections on mycotoxins in the species descriptions which follow are based on Marasas et al. (1984), Desjardins (2006) and Leslie and Summerell (2006). Claims concerning mycotoxin production by particular species in more recent references should be treated with some caution. Cultural instability. Many Fusarium species are notorious for their instability in culture. Isolates of some species will degenerate quickly, often after only one or two transfers. For this reason, it is important to identify Fusaria as soon as possible after primary isolation. Pure cultures for identification are traditionally started from a single germinated spore, as the mass transfer of Fusaria appears to increase the rate of deterioration of strains in culture. Identification procedures. Identification of Fusarium isolates is often difficult, but the task can be made easier by observing a few basic rules:

    identify cultures as soon as possible after primary isolation;

    always grow cultures for identification from single germinated conidia; standardised media and incubation conditions; use a light bank (Chapter 4) whenever possible.

    use

    Diagnostic features. The main characters used to distinguish species of Fusarium are (1) the size and shape of the macroconidia; (2) the psence or absence of microconidia; (3) the manner in which microconidia are produced; (4) the type of phialide on which microconidia are produced; (5) the psence or absence of chlamydoconidia; and (6) the colours and morphology of colonies on PDA.

    5.17 Genus Fusarium

    93

    The morphology of macroconidia is a principal diagnostic feature for Fusarium species. Macroconidia generally have at least three septa, with a differentiated apical cell which may be pointed, rounded, hooked or filamentous, and a basal cell which may be foot-shaped, with a distinct heel, or just slightly notched. Fusarium macroconidia generally exhibit some degree of curvature, the convex and concave sides being referred to as the dorsal and ventral sides, respectively. Although some macroconidia are usually produced in the aerial mycelium, the shape and size of those in sporodochia are more regular and are used for identification purposes where possible. Microconidia are usually produced in the aerial mycelium and their shape can be very important in Fusarium identification. Most species which produce microconidia form only a single type, the most common shape being ellipsoidal to clavate. However, F. poae produces spherical to apiculate microconidia and F. sporotrichioides produces a variety of shapes: ellipsoidal, pyriform and spherical. The method of production of microconidia and the types of phialides on which they are borne are also useful diagnostic criteria. F. verticillioides produces its microconidia in long, delicate, dry chains, which are best observed by using the 10 objective of the

    Key to Fusarium species included here 1

    Microconidia abundant Microconidia rare or absent

    2 9

    2 (1)

    Colonies on PDA with mycelium and/or reverse coloured greyish rose or burgundy Colonies on PDA in shades of cream, pale salmon or violet

    3 5

    3 (2)

    4 (3)

    5 (2)

    6 (5)

    F. poae 4

    F. chlamydosporum F. sporotrichioides

    6 7 F. verticillioides F. proliferatum

    94

    5 Primary Keys and Miscellaneous Fungi

    7 (5)

    Colonies cream or bluish, sporodochia cream Colonies pale salmon or violet, sporodochia salmon

    8 (7)

    Microconidia borne on short, stout monophialides; chlamydoconidia usually produced Microconidia borne on polyphialides and slender monophialides; chlamydoconidia not produced

    9 (1)

    Colonies cream, pale salmon or brown Colonies greyish rose to burgundy

    10 (9)

    Macroconidia cigar- or spindle-shaped, produced in the aerial mycelium Macroconidia obviously curved, produced in sporodochia

    11 (9)

    Macroconidia robust, ventral side straight; aerial mycelium tan to brown Macroconidia delicate and slender, slightly or definitely curved; aerial mycelium white or pinkish

    12 (11)

    Macroconidia longer and narrower, maximum width 5.5 mm 13 (11)

    Macroconidia with elongated basal cell and long, whip-like apical cell Macroconidia with basal and apical cells not obviously elongated

    14 (13)

    Macroconidia delicate and needle-like, with sides almost parallel Macroconidia with slight to definite curvature

    Telemorph: Gibberella acuminata Wollenw. Colonies on CYA 40-50 mm diam, of dense, felty mycelium, white to greyish rose or greyish magenta; reverse uniformly pale or with areas of greyish rose. Colonies on MEA 45-65 mm diam, yellow brown centrally, greyish rose at the margins; reverse deep brownish yellow to brownish orange, occasionally pale. Colonies on G25N 9-15 mm diam. At 58C, colonies 7-12 mm diam. No growth at 378C. On PDA, colonies usually covering the whole Petri dish, of dense to floccose white to pale salmon mycelium, sometimes greyish rose at the margins; reverse dark ruby centrally, greyish ruby at the margins. On DCPA, colonies sparse, of floccose to funiculose white to pale salmon mycelium; reverse pale or with brownish red annular rings. Macroconidia relatively slender, usually with five septa, but three and four septa not uncommon, with a long, tapering apical cell and foot-shaped basal cell, distinctly but not highly curved, with

    F. oxysporum F. subglutinans 10 11

    Macroconidia short and stout, up to 7 mm wide

    Fusarium acuminatum Ellis & Everh. Fig. 5.22

    F. solani 8

    F. semitectum F. equiseti 12 13 F. culmorum (See F. graminearum) F. graminearum F. longipes 14 F. avenaceum F. acuminatum

    the widest point often one third of the distance from the base, giving a ”bottom-heavy” appearance; microconidia produced sparsely by some isolates; chlamydoconidia produced, relatively slowly. Distinctive features. Ruby to dark ruby reverse colours on PDA, and relatively slender, slightly curved macroconidia, usually with five septa, are the distinctive features of Fusarium acuminatum. However, unless chlamydoconidia are psent, this species can be confused with F. avenaceum (see below). F. armeniacum is very closely related (Burgess and Summerell, 2000). Taxonomy. Perithecia of Gibberella acuminata Wollenw., the teleomorph of F. acuminatum, are formed in the laboratory when opposite mating types are inoculated onto sterile wheat straws (Booth, 1971). Isolates of F. acuminatum show considerable variability in culture, and this variability, correlated with secondary metabolite production (Logrieco et al., 1992), has been shown to be due to the fact that two species were included within F. acuminatum. F. armeniacum (Forbes et al.) L.W. Burgess and Summerell, pviously described as F. acuminatum var. armeniacum G.A.

    5.17 Genus Fusarium

    95

    Fig. 5.22 Fusarium acuminatum (a) colonies on PDA and DCPA, 7 d, 258C; (b, c) macroconidia, bar = 10 mm

    Forbes et al. has been elevated to species level (Burgess and Summerell, 2000) being distinguished on morphological grounds, isoenzyme patterns, molecular markers and mycotoxin profiles. Macroconidia of F. armeniacum are produced in distinct apricot coloured sporodochia. Physiology. Some isolates of Fusarium acuminatum have antioxidant enzyme activity (Kayali and Tarhan, 2005). Mycotoxins. Most of the mycotoxin production reported from Fusarium acuminatum, particularly the production of trichothecene toxins, is probably more correctly due to F. armeniacum (Desjardins, 2006; Leslie and Summerell, 2006). Isolates producing type A trichothecenes including T-2 toxin, HT-2 toxin and diacetoxyscirpenol (see Pitt and Hocking, 1997) are most likely to be F. armeniacum. In a survey of 25 isolates of F. acuminatum from different sources and geographic locations,

    Logrieco et al. (1992) pided the isolates into three categories, (a) enniatin B and/or moniliformin producers (probably F. acuminatum sensu stricto), (b) T-2, HT-2 and/or neosolaniol producers (probably F. armeniacum) and (c) nontoxigenic. F. acuminatum sensu stricto produces moniliformin (Chelkowski et al., 1990; Logrieco et al., 1992) and enniatins (Logrieco et al., 1992; Kononeko et al., 1993; Desjardins, 2006; Leslie and Summerell, 2006) as well as some other minor toxins (Desjardins, 2006). Ecology. Fusarium acuminatum has been isolated from a wide variety of plants throughout the world. Although some isolates may cause severe root rot in particular legume species (Leslie and Summerell, 2006), F. acuminatum is generally regarded as a saprophyte. It has been reported to cause rot in pumpkins (Elmer, 1996), is one cause of rot in stored potatoes (Theron and Holz, 1990) and is weakly pathogenic in

    96

    5 Primary Keys and Miscellaneous Fungi

    bananas (Jime´nez et al., 1993). It is quite common in poor quality wheat from cool temperate zones (Mills and Wallace, 1979; Abramson et al., 1987). It has been isolated from developing peanut pods (Barnes, 1971), barley (Abdel-Kader et al., 1979) and, in our laboratory, from rain damaged sorghum and soybeans. The incidence of F. acuminatum in tropical commodities was low (Pitt et al., 1993, 1994). References. Domsch et al. (1980), under G. acuminata; Nelson et al. (1983); Desjardins (2006); Leslie and Summerell (2006).

    Fusarium avenaceum (Fr.) Sacc.

    Fig. 5.23

    Teleomorph: Gibberella avenacea R.J. Cooke Colonies on CYA covering the whole Petri dish, moderately deep to deep, of open, floccose

    mycelium coloured white, very pale rose or deeper greyish rose; reverse varying from pale to pale yellow or with areas of greyish rose or sometimes uniformly deep burgundy. Colonies on MEA 45- 55 mm diam, low to moderately deep, of open floccose to funiculose mycelium, coloured white, pale rose or greyish rose, sometimes brown centrally; reverse brownish orange, sometimes paler centrally or at the margins. Colonies on G25N 9- 15 mm diam. At 58C, colonies 10-12 mm diam. No growth at 378C. On PDA, colonies moderately deep to deep, of dense mycelium coloured white, pale salmon or sometimes dark brownish red, with central masses of reddish orange sporodochia, sometimes surrounded by an outer ring of paler sporodochia; reverse greyish red, with darker annular rings, paler towards the margins. On DCPA, colonies deep, of moderately dense white to pale salmon mycelium with a central mass of orange to

    Fig. 5.23 Fusarium avenaceum (a) colonies on PDA and DCPA, 7 d, 258C; (b, c) macroconidia, bar = 10 mm

    5.17 Genus Fusarium

    salmon sporodochia, often surrounded by concentric rings of sporodochia; reverse pale. Macroconidia long, slender, with four to seven septa, thin-walled, straight or slightly curved, with a tapering apical cell and a notched or foot-shaped basal cell; microconidia produced sparsely by some isolates; chlamydoconidia absent. Distinctive features. Fusarium avenaceum is distinguished by thin walled, needle-like macroconidia and by the absence of chlamydoconidia. Despite the fact that F. avenaceum and F. acuminatum are not considered by Fusarium taxonomists to be closely related, these two species can be difficult to distinguish, as isolates with macroconidia of intermediate form are not uncommon. Colony diameters on PDA at 308C after 3 days can be a useful differentiating feature: under these conditions colonies of F. avenaceum are usually 8- 15 mm diam, whereas those of F. acuminatum are 15-28 mm diam (Burgess et al., 1994). Isolates of F. avenaceum show an unusually broad range of colours on PDA and also have a very broad host range. However, extensive genetic analysis has shown no bases from splitting the species, and pathogenicity tests on single strains have confirmed the broad host range (Nalim, 2004). Taxonomy. The teleomorph of Fusarium avenaceum is Gibberella avenacea R.J. Cook. It is not usually seen in culture on the media used here. Physiology. The optimum growth temperature for Fusarium avenaceum is 358C, the minimum near -38C and the maximum 318C (Domsch et al., 1980). The minimum aw for growth is approximately 0.90 at 258C (Magan and Lacey, 1984c), and the pH optimum ranges between 5.4 and 6.7 (Domsch et al., 1980). Mycotoxins. This species has been reported to produce a variety of trichothecene and other mycotoxins. However, Nelson et al. (1983) regarded only reports of moniliformin production as accurate. Later reports have confirmed this (Chelkowski et al., 1990; Abbas et al., 1991; Bosch and Mirocha, 1992). Reports of production of fusarin C (Farber and Sanders, 1986; Thrane, 1988; Leonov et al., 1993) and enniatins (Blais et al., 1992; Kononeko et al., 1993) also appear to be reliable (Desjardins, 2006; Leslie and Summerell, 2006). Production of any trichothecene toxin has not been confirmed, and Fusarium avenaceum does not carry the tri5 gene which is essential for trichothecene production (Tan and Niessen, 2003).

    97

    Ecology. Fusarium avenaceum has a worldwide distribution wherever crops are grown, but is relatively uncommon in food commodities. It is a major component of Fusarium head blight in cereals in Europe, the US Pacific Northwest region and Canada (Desjardins, 2006). Logrieco et al. (2002) identified F. avenaceum as a component of Fusarium ear rot of maize in Europe. It has been reported from barley (Flannigan, 1969; Petters et al., 1988; Stenwig and Liven, 1988) where it may inhibit germination of malting grains (Hudec, 2007), but is of minor importance in gushing of beer (Niessen et al., 1992). Other reported sources are sorghum (Onyike and Nelson, 1992), peanuts (Joffe, 1969), pigeon peas (Maximay et al., 1992) and, in our laboratory, triticale. F. avenaceum has been reported to cause spoilage of cool-stored broccoli (Mercier et al., 1991), dry rot of stored carrots in Italy (Marziano et al., 1992) and dry rot of rutabaga (swede turnip) in Canada (Peters et al., 2007). It has occasionally caused spoilage of apples, pears, asparagus, tomatoes, eggplant and potatoes (Snowdon, 1990, 1991) and has been reported as a postharvest pathogen of stonefruit in New Zealand (Hartill and Broadhurst, 1989). References. Domsch et al. (1980), as G. avenacea; Nelson et al. (1983); Leslie and Summerell (2006).

    Fusarium chlamydosporum Wollenw. & Reinking Fig. 5.24 Fusarium fusarioides (Gonz. Frag. & Cif.) Booth

    Colonies on CYA covering the whole Petri dish, of low to moderately deep floccose mycelium, coloured white to pale rosy pink, often with surface appearing powdery due to production of microconidia; reverse pale to greyish rose or brownish red. Colonies on MEA 55-70 mm diam, of low, moderately dense mycelium in shades of yellow brown, or greyish rose to greyish ruby, paler at the margins; reverse deep yellow brown to orange brown. Colonies on G25N 15-20 mm diam. At 58C, colonies 1-2 mm diam. At 378C, colonies 5-15 mm diam. On PDA, colonies of felty mycelium, coloured pale salmon, sometimes browner, or with patches of greyish red, often with a powdery appearance from profuse microconidial production; reverse deep violet brown to dark ruby, paler at the margins. On DCPA, colonies of sparse, floccose, pale salmon mycelium, often powdery with microconidia, showing poorly defined annulations; macroconidia

    98

    5 Primary Keys and Miscellaneous Fungi

    Fig. 5.24 Fusarium chlamydosporum (a) colonies on PDA and DCPA, 7 d, 258C; (b, c) polyphialides, bar = 10 mm; (d) macroconidia, bar = 10 mm; (e) microconidia, bar = 10 mm

    occasionally produced near the colony centres in salmon sporodochia; reverse pale. Macroconidia often rare, relatively short and stout, usually with three to five septa, slightly curved; microconidia produced abundantly from polyphialides in the aerial mycelium, with zero to two septa, fusiform to slightly clavate. Chlamydoconidia usually abundant in older cultures, produced singly, in pairs or in clumps. Distinctive features. The psence of abundant fusiform microconidia borne on polyphialides is the most outstanding feature of Fusarium chlamydosporum. Also colonies on PDA have dark violet brown to dark ruby reverse colours.

    Taxonomy. Fusarium chlamydosporum has priority over F. fusarioides as the correct name for this species (Domsch et al., 1980; Nelson et al., 1983; Leslie and Summerell, 2006). Physiology. This species has minimum, optimum and maximum temperatures for growth of 5, 27 and 378C (Seemu¨ller, 1968). Mycotoxins. Production of type A trichothecenes (including T-2 toxin, HT-2 toxin, monoacetoxyscirpenol, neosolaniol and iso-neosolaniol) by Fusarium chlamydosporum was reported by Park and Chu (1993); however, subsequent studies have found no evidence of trichothecene production in this species (Desjardins, 2006). Moniliformin is the

    5.17 Genus Fusarium

    major mycotoxin produced by F. chlamydosporum (Marasas et al., 1984; Desjardins, 2006). Ecology. Fusarium chlamydosporum is mainly an inhabitant of soils in warmer climates (Domsch et al., 1980; Leslie and Summerell, 2006), and is not regarded as a plant pathogen or spoilage fungus. However, it is commonly isolated from grains in drier areas, particularly in the Middle East, southern Europe, central Asia and Australia (Leslie and Summerell, 2006), and has also been isolated from pearl millet (Wilson et al., 1993; Jurjevic et al., 2007), pecans (Huang and Hanlin, 1975) and sorghum (Rabie et al., 1975; Onyike and Nelson, 1992). A low incidence of F. chlamydosporum was found in peanuts from both Indonesia and the Philippines (Pitt et al., 1998a) and from mung beans and sorghum in Thailand (Pitt et al., 1994). Involvement in dry rot of potatoes has also been reported (Somani, 2004; Esfahani, 2006). References. Domsch et al. (1980); Nelson et al. (1983); Leslie and Summerell (2006).

    99

    Fusarium culmorum (W.G. Smith) Sacc. Fig. 5.25 Colonies on CYA covering the whole Petri dish, of dense felty mycelium, often with a floccose overlay, sometimes reaching the Petri dish rim, pale red to pastel red; reverse pastel red to deep red. Colonies on MEA 60 mm or more diam, floccose, in age often reaching the Petri dish lid, pale red to pastel red, commonly with a greyish orange to yellowish brown overlay; reverse brown to reddish brown. Colonies on G25N usually 5-10 mm diam, mycelium orange white, reverse yellow to orange. At 58C, germination. No growth at 378C. On PDA, colonies covering the whole Petri dish, of dense to floccose mycelium, pale red and pale yellow brown; reverse red to deep red. On DCPA, colonies 50-65 mm diam, of sparse mycelium, orange to pinkish white, bearing abundant macroconidia in orange sporodochia; reverse dull orange brown.

    Fig. 5.25 Fusarium culmorum (a) colonies on PDA and DCPA, 7 d, 258C; (b, c) macroconidia, bar = 10 mm

    100

    Macroconidia relatively short, wide and only slightly curved, with four to five septa, 30-45 mm long, with rounded or sometimes papillate apical cells; basal cells with a slight to definite notch, sometimes papillate. Microconidia not produced. Chlamydoconidia sometimes formed, in conidia, or intercalary in the hyphae, singly or in chains, 9-14 mm diam, smooth walled. Distinctive features. Short, stout macroconidia are the prime feature distinguishing Fusarium culmorum from most other species. F. culmorum may be confused with F. crookwellense L.W. Burgess et al., but macroconidia of the latter species have a distinctly foot-shaped basal cell, whereas those of F. culmorum are shorter and stouter, and the basal cell is not distinctly foot-shaped. Taxonomy. No teleomorph is known for this species. Physiology. Fusarium culmorum has been reported to be psychrotrophic, growing down to 08C, with an optimum at 218C and a maximum of only 318C (Arsvoll, 1975); however, Magan and Lacey (1984c) reported growth at 358C. The minimum aw for growth is 0.87 at 20-258C and pH 6.5: at pH 4.0, growth did not occur below 0.90 aw (Magan and Lacey, 1984a). A strain of F. culmorum produced deoxynivalenol optimally at 258C, but only between 0.995 and 0.97-0.96 aw (Hope and Magan, 2003; Hope et al., 2005). At 158C, deoxynivalenol was produced in lower concentrations later in the growth cycle, but over a slightly greater aw range (0.995 to 0.95-0.94 aw). The dynamics of nivalenol production for this strain was similar (Hope and Magan, 2003). Zearalenone production by F. culmorum was reported to be optimal above 258C (Bottalico et al., 1982). F. culmorum is very tolerant of low O2 tensions (Magan and Lacey, 1984b). Radiation resistance of F. culmorum was relatively high: up to 0.8 kGy were needed for a tenfold reduction in spore numbers on grain, and up to 1.39 kGy on media (O’Neill et al., 1991). Mycotoxins. Fusarium culmorum produces a variety of mycotoxins: indeed the list in our files is of more than 40 compounds. However, the most important toxins confirmed to be produced by this species (Nelson et al., 1983; Marasas et al., 1984; Desjardins, 2006; Leslie and Summerell, 2006) are deoxynivalenol, nivalenol and their derivatives (Abramson et al., 2001; Hestbjerg et al., 2002;

    5 Primary Keys and Miscellaneous Fungi

    Chandler et al., 2003; Jennings et al., 2004) and zearalenone (Bakan et al., 2001; Hestbjerg et al., 2002; Llorens et al., 2004a; Brinkmeyer et al., 2005). Moniliformin production was reported by Scott et al. (1987) but was not detected in 42 isolates of F. culmorum from Canada by Abramson et al. (2001). Reports of production of type A trichothecenes (T-2 toxin, HT-2 toxin) have not been substantiated (Leslie and Summerell, 2006). The existence of two chemotypes of Fusarium culmorum, those that produce deoxynivalenol and those that produce nivalenol (Miller et al., 1991), has been confirmed by molecular studies. Within the trichothecene gene cluster, isolates possessing the Tri7 and Tri13 genes produce nivalenol and related compounds, whereas sequences in the Tri3, Tri5 and Tri6 genes are associated with deoxynivalenol production (Chandler et al., 2003; Jennings et al., 2004; Quarta et al., 2005, 2006). Of 55 European isolates examined by Quarta et al. (2005), 11 were the nivalenol chemotype, and the remainder were deoxynivalenol producers. Jennings et al. (2004) examined 153 isolates from England and Wales, and found that the DON chemotype was dominant over the NIV chemotype (59% vs 41%, respectively). Lauren et al. (1992) examined 45 isolates of F. culmorum from New Zealand soil and pasture and found none produced deoxynivalenol or its monoacetyl isomers. Two chemotypes were identified, one producing diacetylnivalenol with culmorin as the major metabolite accounted for 95% of isolates, while the other chemotype produced diacetoxyscirpenol. Ecology. This species has a worldwide distribution in soil and as a pathogen of cereals and other hosts, with a higher incidence in temperate climates (Domsch et al., 1980; Nelson et al., 1983; Leslie and Summerell, 2006). It is an important component of the cohort of Fusarium species that cause head blight of wheat and associated cereal crops in Europe, Canada, China and other areas with cool weather during the growing season (Desjardins, 2006). In wheat it causes extensive internal damage to the grain, and reductions in flour yield and baking quality (Meyer et al., 1986). F. culmorum was reported as the dominant Fusarium species on barley in Europe (see Pitt and Hocking, 1997). It also occurs in triticale (Perkowski et al., 1988). F. culmorum has been identified as a component of Fusarium ear rot of maize in Europe (Logrieco

    5.17 Genus Fusarium

    101

    et al., 2002). F. culmorum was one cause of crown rot in bananas (Wade et al., 1993), and is a minor cause of spoilage of apples and pears (Snowdon, 1990). References. Domsch et al. (1980); Nelson et al. (1983); Desjardins (2006); Leslie and Summerell (2006).

    Fusarium equiseti (Corda) Sacc.

    Fig. 5.26

    Teleomorph: Gibberella intricans Wollenw. Colonies on CYA filling the whole Petri dish, often to the lid, of dense to floccose white mycelium; reverse pale or pale salmon. Colonies on MEA

    covering the whole Petri dish, of open, floccose white to pale brown mycelium; reverse pale, or sometimes showing areas of pale greyish red. Colonies on G25N 12-20 mm diam. At 58C, colonies of 1- 4 mm diam produced. At 378C, usually no growth, although in isolates from the tropics, colonies up to 35 diam Produced. On PDA, colonies of dense to floccose mycelium, white to pale salmon, becoming brown with age, with a central mass of orange to brown sporodochia, sometimes surrounded by poorly defined sporodochial rings; reverse pale salmon, often with a brown central area and brown flecks. On DCPA, colony appearance usually dominated by salmon, orange or brownish

    Fig. 5.26 Fusarium equiseti (a) colonies on PDA and DCPA, 7 d, 258C; (b, c) macroconidia, bar ¼ 10 mm; (d) chlamydoconidia, bar ¼ 10 mm

    102

    421

    The factors enabling fungi to cause spoilage in cheese are the ability to grow at refrigeration temperatures, to grow in low oxygen concentrations, lipolytic activity, resistance to the pservative action of free fatty acids and growth at reduced aw. Penicillium roqueforti and P. commune meet all these criteria and are thus the most successful spoilage moulds on cheese. Toxin production (roquefortine and PR toxin from P. roqueforti and cyclopiazonic acid from P. commune) is a definite, though probably small, hazard. PR-imine was detected in 50 of 60 samples of blue-vein cheese, but PR toxin was not (Siemens and Zawistowski, 1993). Roquefortine was detected in 1 of 10 samples of Valdeon cheese, a blue´ mould ripened Spanish variety (Lopez-Dias et al., 1996), in all 11 samples of European blue-mould cheeses purchased in Finland (Kokkonen et al., 2005a) and in all 30 samples of European bluemould cheese examined in Italy (Finoli et al., 2001). The levels of roquefortine detected by Kokkonen et al. (2005b) ranged from 0.8 to 12 mg/kg, whereas levels reported by Finoli et al. (2001) were lower (0.08-1.47 mg/kg). Mycophenolic acid was detected in 4 of 12 samples of mouldy Spanish Manchego ´ cheese (Lopez-Dias et al., 1996) and 1 of 11 samples of blue-mould cheese in Finland (Kokkonen et al., 2005a). Ochratoxin A has recently been reported by Dall’Asta et al. (2008) in blue-mould cheeses from Italy (23 of 54 samples) and France (7 of 14 samples) at levels from 0.25 to 3.0 mg/kg. Cyclopiazonic acid detected in six samples of Italian Taleggio, a soft, smear-ripened cheese, was confined mainly to the rind (Finoli et al., 1999). Sterigmatocystin produced by Aspergillus versicolor was detected in the surface layer of hard cheeses in the Netherlands (Northolt et al., 1980). Mycotoxin levels reported in cheese are not usually considered to be of public health significance. Mouldy cheese is unsuitable for sale and for manufacturing purposes. Protection from the Penicillia relies on clean production conditions, low temperature storage, low oxygen atmospheres, integrity of packaging materials, intact rinds, pservative impgnated wrappers and rapid turnover of stock.

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    Media Appendix

    Aspergillus flavus and parasiticus agar (AFPA) Peptone, bacteriological Yeast extract Ferric ammonium citrate Chloramphenicol Agar Dichloran (0.2% in ethanol, 1.0 ml) Water, distilled

    10 g 20 g 0.5 g 100 mg 15 g 2 mg 1l

    After addition of all ingredients, sterilise by autoclaving at 1218C for 15 min. The final pH of this medium is 6.0-6.5. Creatine sucrose neutral agar (CSN) CS concentrate Sucrose Creatine KH2PO4 Bromocresol purple Agar Water, distilled to

    10 ml 10 g 5.0 g 1.0 g 0.05 g 15 g 1l

    Creatine sucrose (CS) concentrate KCl MgSO4 7H2O FeSO4 7H2O ZnSO4 7H2O CuSO4 5H2O Water, distilled to

    5g 5g 0.1 g 0.1 g 0.05 g 100 ml

    Sterilise by autoclaving at 1218C for 15 min. Final unadjusted pH is approximately 6.8. A pH between 5.5 and 6.8 is satisfactory. For identification of Penicillium subgenus Penicillium species. Czapek concentrate NaNO3 KCl

    30 g 5g

    MgSO4 7H2O FeSO4 7H2O Water, distilled

    5g 0.1 g 100 ml

    Czapek concentrate will keep indefinitely without sterilisation. The pcipitate of Fe(OH)3 which forms in time can be resuspended by shaking before use. Czapek iprodione dichloran agar (CZID) Sucrose Yeast extract Chloramphenicol Dichloran (0.2% in ethanol, 1 ml) Czapek concentrate Trace metal solution Agar Water, distilled Iprodione (suspension)

    30 g 5g 100 mg 2 mg 10 ml 1 ml 15 g 1l 1 ml

    Add iprodione suspension . Kvashnina, E.S. 1976. (Physiological and ecological characteristics of Fusarium species Sect. Sporotrichiella). Mikol. Fitopatol. 10: 275-281. Kwasna, H., Ward, E. and Bateman, G.L. 2006. Phylogenetic relationships among Zygomycetes from soil based on ITS1/2 rDNA sequences. Mycol. Res. 110: 501-510. La Guerche, S., Dauphin, B., Pons, M., Blancard, D. and Darriet, P. 2006. Characterization of some mushroom and earthy off-odors microbially induced by the development of rot on grapes. J. Agric. Food Chem. 54: 9193-9200. Labuda, R. and Tancˇinova´, D. 2003. Eupenicillium ochrosalmoneum, a rare species isolated from a feed mixture in Slovakia. Biologia, Bratislava 58: 1123-1126. Lacey, J. 1980. Colonization of damp organic substrates and spontaneous heating. In Microbial Growth and Survival in Extreme Environments, eds G.W. Gould and J.E.L. Corry. London: Academic Press. pp. 53-70. Lahali, R., Serrhini, M.N. and Jijakli, M.H. 2004. Efficacy assessment of Candida oleophila (strain O) and Pichia anomala (strain K) against major postharvest diseases of citrus fruits in Morocco. Commun. Appl. Biol. Sci. Ghent Univ. 69: 601-609. Lahali, R., Serrhini, M.N. and Jijakli, M.H. 2005. Studying and modelling the combined effect of temperature and water activity on the growth rate of P. expansum. Int. J. Food Microbiol. 103: 315-322. Lahlali, R., Serrhini, M.N., Friel, D. and Jijakli, M.H. 2007. Predictive modelling of temperature and water activity (solutes) on the in vitro radial growth of Botrytis cinerea Pers. Int. J. Food Microbiol. 114: 1-9. Laich, F., Fierro, F., Cardoza, R.E. and Martin, J.F. 1999. Organization of the gene cluster for biosynthesis of penicillin in Penicillium nalgiovense and antibiotic production in cured dry sausages. Appl. Environ. Microbiol., 65: 1236-1240. Lamper, C., Teren, J., Bartok, T., Komoroczy, R., Mesterhazy, A. and Sagi, F. 2000. Predicting DON contamination in Fusarium-infected wheat grains via determination of the ergosterol content. Cereal Res. Commun. 28: 337-344. Lanciotti, R., Sinigaglia, M., Gardini, F. and Guerzoni, M.E. 1998. Hansenula anomala as spoilage agent of cream-filled cakes. Microbiol. Res. 153: 145-148.

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    Index

    Note: Bold face denotes fungal species description A Absidia corymbifera, 148 in tree nuts, 408 oxygen requirements, 7 ramosa, 149 Acetic acid pserves, spoilage of, 419 Candida krusei, 363 Moniliella acetoabutans, 130 Pichia membranaefaciens, 371 Acremonium, 57, 58 strictum, 58 Acute cardiac beriberi, 197 Aflatoxins, 307-309 from Aspergillus flavus, 307 from Aspergillus nomius, 311 from Aspergillus parasiticus, 322 coconut cream agar for detection of, 28 from milk, 308 Air sampling, 23 Alimentary toxic aleukia (ATA), 1, 91, 109, 117 Altenuene, 62 Alternaria, 58, 60, 142 alternata, 60, 63 in cantaloupes, 387 in cereals, 395, 396 in citrus, 385 in figs, 389 in mangoes, 390 mycotoxins, 61 in pome fruits, 386 in rice, 396 in sorghum, 398 in soybeans, 397 in tomatoes, 387 in wheat, 395 citri, 60 in citrus, 384, 385 infectoria, 60, 62 in sorghum, 398 in wheat, 396

    padwickiii, 137, 138 passiflorae, in passionfruit, 390 tenuis, 60, 62 tenuissima, 60, 62 Alternariol, 62 Alternariol monomethyl ether, 62 Altertoxin, 62 Anaerobic growth of fungi, 7 Anamorph, 15 Apples, spoilage of, 385, 386 Botrytis cinerea, 69 Epicoccum nigrum, 90 Penicillium brevicompactum, 233 Penicillium expansum, 245 Penicillium funiculosum, 264 Penicillium solitum, 258 Arthrinium, 58, 64, 132 phaeospermum, 64 state of Apiospora montagnei, 65 Ascomycete-conidial fungus connections, 15 Ascomycetes, 53 Ascomycotina, 13-15 Aspergillus, 277, 278, 279, 295 aculeatus, 277, 295 in onions, 391 alliaceus, 320 alutaceus, 317, 321 amstelodami, 282 awamori, 313 candidus, 277, 279, 297, 317 in cereals, 402 in cheese, 421 in flour and pasta, 403 heat resistance, 6 in maize, 402 in peanuts, 406 in rice, 403 in soybeans, 405 carbonarius, 277, 279, 299 in coffee beans, 410, 411 in dried vine fruit, 413

    503

    504 Aspergillus (cont.) in grapes, 388 ochratoxin A, 388, 411 chevalieri, 285 classification, 277 clavatus, 277, 279, 302 colony diameters, 278 fischeri, 293 fischerianus, 293 flavipes, 277, 279, 303 flavus, 277, 279, 305, 315, 322, 329 in Brazil nuts, 399, 409 in cashews, 399, 409 in coffee beans, 411 in copra, 400 in figs, 414 in maize, 397, 406 in peanuts, 398, 406, 407 in pistachios, 400, 409 in rice, 38, 396, 402, 403 in salt fish, 416, 417 in sorghum, 398 in soybeans, 397, 405 in spices, 410 in sunflower seed, 398 in tree nuts, 399, 401, 408, 409 fonsecaeus, 299 fumigatus, 51, 277, 279, 311 in maize products, 405 in rice, 402, 403 in tree nuts, 399, 408, 409 glaucus, 291 japonicus, 277, 279, 297 key to common species, 277 manginii, 291 nidulans, 279 niger, 277, 279, 313 in chickpeas, 406 in cocoa, 411, 412 in coffee beans, 410, 411 in dried vine fruit, 413 in figs, 389, 414 in grapes, 388 in maize, 404, 405 in onions, 391 in peanuts, 398, 406, 407 in pistachios, 400, 408, 409 in rice, 402, 403 in salt fish, 416, 417 in spices, 410 in tree nuts, 399, 407, 408 in yams, 392 niveus, 278, 279, 298, 315 nomius, 277, 279, 311 ochraceus, 278, 279, 317 in chickpeas, 406 in coffee beans, 410, 411 in dried fruit, 413 in figs, 414 in pistachios, 408 oryzae, 277, 279, 306, 310

    Index parasiticus, 277, 279, 306, 321 in peanuts, 407 in tree nuts, 408 penicillioides, 278, 279, 323, 325 in cereals, 402 in confectionery, 415 in flour, 403 in fruit cakes, 414 in salt fish, 416 in processed meats, 417 pseudoglaucus, 287 pulchellus, 299 repens, 287 reptans, 287 restrictus, 278, 279, 324, 325 in cereals, 403 in fruit cakes, 414 in jams, 412 in maize, 404 in nuts, 409 in soybeans, 405 in rice, 403 ruber, 289 rubrobrunneus, 289 sejunctus, 289 sojae, 306, 322 steynii, 278, 279, 320 sydowii, 278, 279, 329 in cashews, 399, 409 in rice, 396 in salt fish, 416 in spices, 410 tamarii, 278, 279, 328 in Brazil nuts, 409 in cashews, 399, 409 in cocoa, 411, 412 in coffee beans, 411 in maize, 404 in peanuts, 406, 407 teleomorphs, 276 terreus, 278, 279, 330 tubingensis, 313, 314 ustus, 277, 279, 332 versicolor, 278, 279, 327, 333 vitis, 282 wentii, 278, 279, 336 westerdijkiae, 278, 279, 317, 320 in coffee beans, 411 ochratoxin, 411 Aspergillus flavus and parasiticus agar (AFPA), 28, 307, 319, 322, 329, 423 ATP, estimation of, 37 Aureobasidium, 57, 65 pullulans, 65 in meat, 394 Aurofusarin, 104, 117

    B Baked goods, mycoflora of, 403-404 Eurotium chevalieri, 287

    Index Balkan endemic nephropathy, 261 Bananas, spoilage of, 390 Alternaria alternata, 62 Colletotrichum gloeosporioides, 82 Fusarium oxysporum, 108 Fusarium semitectum, 113, 114 Fusarium subglutinans, 120 Fusarium verticillioides, 121, 122 Lasiodiplodia theobromae, 127 Nigrospora spherica, 133 Pestalotiopsis guepini, 134 Phoma sorghina, 135 Barley, mycoflora of, 395 Alternaria alternata, 62, 64 Arthrinium, 65 Aspergillus clavatus, 302 Chaetomium, 73 Cladosporium, 76, 79, 81 Fusarium culmorum, 100 Fusarium graminearum, 104 Fusarium sporotrichioides, 118 Geotrichum candidum, 124 Penicillium hordei, 249 Penicillium verrucosum, 261 Basidiomycotina, 15 Basipetospora, 340 halophila, 341, 350 in salt fish, 416 Beans, mycoflora of, 391, 397, 405 Rhizopus stolonifer, 160 Beauvericin, 102, 108, 110, 112, 117, 119 Beer, spoilage of, Brettanomyces bruxellensis, 358, 362 Berries, spoilage of, 389 Bettsia, 340, 343 alvei, 343 Biltong, mycoflora of, 412 Biomass, estimation of fungal, 34 ATP estimation, 37 chitin assay, 34 conductimetry, 37 ELISA, 39 ergosterol assay, 35 fluorescent antibodies, 40 immunological techniques, 38 impedimetry, 37 latex agglutination, 39 molecular methods, 40 PCR, 40 volatiles, 37 Biosystematics, 11-17 Bipolaris, 58, 67, 85 australiensis, 67 bicolor, 67 maydis, 67 oryzae, 67 setariae, 67 spicifera, 67 Botryodiplodia theobromae, 125

    505 Botryosphaera rhodina, 125, 126 ribis, 386 Botrytis, 58, 68 allii, 69 in garlic, 391 byssoides, 69 cinerea, 68 in beans, 391 in berries, 389 in carrots, 392 in grapes, 388 in kiwifruit, 389 in peas, 391 in pome fruits, 385 in stone fruit, 386 state of Sclerotinia porri, 69 Bread, spoilage of, 403 Chrysonilia sitophila, 73, 74 Endomyces fibuliger, 88 Hyphopichia burtonii, 125 Moniliella suaveolens, 131 Penicillium roqueforti, 256 Pichia membranaefaciens, 371 Brettanomyces bruxellensis, 360, 361 intermedius, 360 Brines for olives, pickles and cheese, spoilage of, 358, 364, 365, 366, 371, 372 Butenolide, 102, 104 Butter, spoilage of, 394 Candida parapsilosis, 364 Penicillium thomii, 207 Rhodotorula mucilaginosa, 373 Byssochlamys, 169, 170 in cream cheeses, 393 fulva, 2, 5, 170, 171, 184 in heat processed acid foods, 418 heat resistance, 5, 171, 173 nivea, 6, 170, 173 oxygen requirements, 7, 171 spectabilis, 170, 184

    C Cakes, spoilage of, 404 Eurotium rubrum, 291 Penicillium crustosum, 241 Xeromyces bisporus, 355 Calonectria, 90 Candida, 360, 361 chodatii, 124 famata, 364 holmii, 366 krusei, 360, 361, 362 mogii, 379 parapsilosis, 360, 361, 363 in cheese, 420 in pserved foods, 419 in yoghurt, 393

    506 Candida (cont.) pelliculosa, 369 valida, 370 Capsicums, spoilage of, 387 Alternaria alternata, 62 Rhizopus stolonifer, 160 Carbon dioxide, tolerance of fungi to, 7 Carnation leaf agar (CLA), 43 Carpenteles, 175 Carrots, spoilage of, 392 Rhizopus stolonifer, 160 Cashews, mycoflora of, 399, 409 Aspergillus flavus, 309 Cassava, mycoflora of, 392, 393 Penicillium oxalicum, 218 Cephalosporium acremonium, 56 Cereals, mycoflora of Alternaria alternate, 60, 62 Aspergillus versicolor, 335 Chaetomium globosum, 73 Cladosporium, 78 Curvularia lunata, 84 Drechslera, 85 Endomyces fibuliger, 88 Fusarium avenaceum, 97 Fusarium culmorum, 100 Fusarium equiseti, 102 Fusarium graminearum, 104 Fusarium oxysporum, 108 Fusarium poae, 110 Fusarium sporotrichioides, 117, 118 Fusarium spp, 91, 92 Nigrospora oryzae, 132 Penicillium aurantiogriseum, 231 Penicillium chrysogenum, 237 Penicillium verrucosum, 261 Pestalotiopsis, 133 Phoma, 135, 136 Trichothecium roseum, 142 Cereals, spoilage of, 395-397, 402-406 Chaetoglobosin, 73 Chaetomium, 57, 70 brasiliense, 70 funicola, 70, 71 globosum, 70, 72 in cashews, 399, 409 in rice, 396 in soybeans, 397 in spices, 410 Cheese, manufacture, 419-421 Debaryomyces hansenii, 366 Penicillium camemberti, 235 Penicillium commune, 238 Penicillium roqueforti, 256 Cheese, mycoflora and spoilage of, 419-421 Aspergillus versicolor, 335 Candida parapsilosis, 364 Cladosporium, 75, 77, 78, 81 Debaryomyces hansenii, 366 Eurotium amstelodami, 284 Eurotium herbariorum, 292

    Index Eurotium repens, 289 Fusarium oxysporum, 108 Geotrichum candidum, 124 Moniliella suaveolans, 131 Penicillium commune, 238 Penicillium crustosum, 241 Penicillium glabrum, 200 Penicillium roqueforti, 256 Pichia membranaefaciens, 371 Rhodotorula mucilaginosa, 373 Chickpeas, mycoflora of, 398, 405, 406 Chitin assay, 34-35 Chloroanisole formation in foods, 233, 411 Aspergillus versicolor, 336 Eurotium repens, 289 Paecilomyces variotii, 185 Penicillium chrysogenum, 236 Chocolates, spoilage of, 415 Chrysosporium inops, 347 Chrysosporium xerophilum, 347 Xeromyces bisporus, 355 Zygosaccharomyces rouxii, 382 Chrysonilia, 57, 73 sitophila, 51, 73, 74 Chrysosporium, 340, 342 farinicola, 343 in chocolate, 415 in coconut, 409 fastidium, 9, 344, 346 inops, 343, 345 in confectionery, 415 isolation techniques, 30 key to xerophilic species, 343 in prunes, 413 xerophilum, 343, 347 in coconut, 409 Citreoviridin, 197 from Eupenicillium ochrosalmoneum, 182 from Penicillium citreonigrum, 197 Citrinin, 129 from Monascus ruber, 129 from Penicillium citrinum, 208 from Penicillium verrucosum, 261 Citrus, spoilage of, 384-385 Alternaria, 61, 62 Fusarium oxysporum, 108 Geotrichum candidum, 123 Penicillium digitatum, 242 Penicillium italicum, 251 Penicillium ulaiense, 251 Cladosporium, 57, 75 butyri, 131 chodati, 124 cladosporioides, 75, 78, 80, 81 in cheese, 419 in chilled meat, 394 in coconut, 409 in low salt margarine, 394 in rice, 396 in salt fish, 416 in soybeans, 397, 405

    Index in tree nuts, 399, 407 in wheat, 395, 396 in grapes, 389 herbarum, 75, 76, 77, 78 in cheese, 419, 420 in chickpeas, 398 in margarine, 394 in meat, 394 in tomatoes, 387 in tree nuts, 408 key to included species, 75 macrocarpum, 75, 78 sphaerospermum, 75, 76, 80 in citrus, 385 in maize products, 405 in strawberries, 389 suaveolens, 131, 394 Classification of fungi, 11, 16-17 Claviceps purpurea, 1 Cochliobolus lunatus, 83 Cocoa beans, mycoflora of, 411-412 Aspergillus fumigatus, 312 Candida krusei, 363 Kloeckera apiculata, 368 Coconut cream agar, 28 Coconut, spoilage of, 400, 409 Aspergillus niger, 315 Chrysosporium farinicola, 344 Chrysosporium xerophilum, 347 Coelomycetes, 54 Coffee beans, mycoflora of, 410-411 Aspergillus ochraceus and related species, 320 Ochratoxin in, 410, 411 Coffee, off flavours, 411 Aspergillus versicolor, 336 Colletotrichum, 57, 81 acutatum, 81, 82 in berries, 389 circans, 81 coccodes, 81 dermatium, 82 gloeosporioides, 81 in avocados and mangoes, 390 higginsianum, 81 lindemuthianum, 81 musae, 81, 82 in bananas, 390 Colony characters, 45 diameters, 45 Commodities, tropical, mycoflora of, 396-398 Chaetomium brasiliense, 70 Chaetomium funicola, 72 Cladosporium cladosporioides, 77 Concentrated foods, spoilage of, 412-416 Conductimetry, 37 Confectionery, spoilage of, 415 Chrysosporium inops, 347 Xeromyces bisporus, 355 Zygosaccharomyces rouxii, 382 Consistency, effect on growth, 8

    507 Copra, mycoflora of, 400 Cream cheese, spoilage of, 393, 419, 421 Creatine sucrose agar (CREA), 42, 224 Creatine sucrose neutral agar (CSN), 224, 423 Cucumbers, spoilage of, 388 Penicillium oxalicum, 218 Culmorin, 100, 104 Culture mites, 50 Cultures, examination, 45 Cunninghamella, 148, 149 berttholletiae, 149 echinulata, 149 elegans, 149 Curvularia, 58, 67, 82 lunata, 83 lunata var. aeria, 83 pallescens, 83, 8

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    Trong thiên nhiên, Phalaenopsis phát triển trong tất cả các vùng nhiệt đới châu Á, Cây phát triển trong tự nhiên ở nhiệt độ ngày là 28-35°C, đêm: 20-24°C và độ ẩm tương đối cao.

    Phalaenopsis ưa bóng râm, cây có khả năng hấp thụ chất dinh dưỡng qua rễ và lá. Rễ cũng đóng vai trò neo giữ cây.

    Lan Hồ Điệp có chừng 44 loại nguyên giống, mọc trên dãy Himalaya đến suốt châu Á và sang cả Úc châu. Việt Nam có chừng 7-8 giống. Vào năm 1750 G.E. Rumphius đã tìm ra cây Hồ Điệp nhưng lầm tưởng là một loại Angraecum và sau này Carl Blume mới tìm ra cây Phalaenopsis amabilis vào năm 1825. Theo tiếng La tinh chữ Phaluna có nghĩa là con bướm (moth) và opsis có nghĩa là giống như. Lan Hồ điệp là một loại lan thân đơn, ngắn, lá to và cứng, rễ dài. Những cây nguyên giống thường nở hoa vào mùa đông xuân, các cây lai giống hoa nở quanh năm. Nếu nuôi đúng cách lan hồ điệp có thể sống rất lâu. Có cây sống được trên 18 năm sau đó ra hoa ở ngọn rồi mới chết.

    Để có chậu lan Hồ Điệp nở hoa đúng vào những ngày Tết lại là điều không hề dễ dàn g. Bài viết này sẽ hướng dẫn bạn cách chăm sóc để lan .

    Chăm sóc lan hồ điệp ra hoa đúng tết

    Để năm nào lan hồ điệp cũng ra nhiều hoa và nở hoa vào đúng dịp Tết Nguyên Đán cổ truyền, người trồng lan hồ điệp và chơi lan hồ điệp cần nắm bắt được đặc tính của cây lan hồ điệp là chịu ẩm nhưng không chịu ướt.Ở đây có một số mẹo nhỏ giúp bạn trồng lan hồ điệp tốt hơn: Mẹo chăm sóc lan hồ điệp trước khi tiến hành việc kích thích thế nào để hồ điệp nở hoa tết.

    Thời gian thực hiện việc kích thích lan hồ điệp ra hoa đúng tết:

    Lan hồ điệp cần thời gian 2 tháng từ lúc nhú nhánh hoa đến lúc các bông hoa nở. Đồng thời, do lan hồ điệp rất lâu tàn nên ta có thể cho nó trổ bông trước mùng 1 Tết khoảng 15-20 ngày để hoa có thể nở hết và Tết đến ta có nhánh Hồ điệp thật rực rỡ.

    Do đó, việc chăm sóc, bón phân để lan Hồ Điệp nở đúng vào dịp Tết Nguyên Đán cần được tiến hành ngay từ 15-20 tháng 9 Âm Lịch cây phải nhú nụ rồi thì mới có khả năng nở hoa đúng Tết (cũng tùy theo năm, chủ yếu bạn nhớ thời gian để lan trổ hoa là 2 tháng, rồi tùy từng năm mà bạn chọn thời điểm kích hoa)

    Thời gian nở hoa của lan hồ điệp

    Cách lựa chọn cây lan hồ điệp để kích hoa tết:

    Ta chọn những cây lan đã trưởng thành, là những cây đã ra hết lá non, có ít nhất khoảng 3-4 cặp lá và đưa một khu vực riêng.

    Cách bón phân để lan hồ điệp ra hoa ngay tết:

    Ta dùng phân bón lá có tỷ lệ NPK 10-30-20 (hàm lượng Photpho, Kali cao), liều lượng từ 1/2-1g cho 4lit nước để kích thích cây ra hoa, cách 1 tuần phun 1 lần. Thực hiện trong 3 – 4 tuần thì bạn sẽ thấy cây bắt đầu cho ra vòi hoa (lớn cỡ hạt lúa)

    3 – 4 tuần thì bạn sẽ thấy cây bắt đầu cho ra vòi hoa

    Đến khi vòi hoa có độ dài khoảng 2 – 3 cm, chúng ta sẽ sử dụng phân bón lá NPK 15-20-30 (hàm lượng Kali cao). Cách 6 – 7 ngày thì phun cho cây một lần để kích thích cho vòi hoa phát triển nhanh hơn. Đồng thời, loại phân bón này cũng sẽ giúp cho màu sắc của hoa sau khi nở được thắm hơn, lâu tàn hơn và tránh được nguy cơ thối hoa.

    Sau khoảng 45 – 50 ngày, tức là khoảng vào tháng 12 âm lịch, cành hoa bắt đầu nở bông đầu tiên, và sau 2 tháng thì cây lan sẽ nở hoa rộ (ngay Tết Nguyên Đán).

    Chú ý: Thường thì cứ sau mỗi đợt ra lá, hồ điệp lại nghỉ ra lá một thời gian (trên cây không còn lá non), nếu giai đoạn này xuất hiện quá sớm (trước thời điểm xử lý phun phân “kích” cho cây lan ra bông khá lâu) thì có thể giữ cho cây luôn ở tình trạng không có lá non bằng cách hàng tuần dùng phân bón lá NPK 20-20-20 phun cho tới thời điểm xử lý hoa.

    Hoa Lan Hồ Điệp Cụm từ tìm kiếm — 106 hoa lan p 2 q phú nhuận tp hcm 106 hoa lan phường 2 q phú nhuận tphcm 119 hoa lan phường 2 q phú nhuận tphcm 126 hoa lan 142 hoàng văn thụ hoa lan sài gòn 160 hoa lan p phú nhuận tphcm 199 hoa lan quận phú nhuận 76 hoa lan p2 phú nhuận 86.55 au co lan rung com airwick crystal air dạng gel hương hoa lan hcm an ho diep an hoa lan ho diep ân hoa lan o lai thieu anh hoa lan ảnh hoa lan quân tử anh lan ho diep baảg gniá hoa lan đồ điệp bài thơ về hoa lan bán bình hoa lan tphcm bán các giống hoa phong lan tại thành phố hồ chí minh ban cay dia lan dep bán cay giống hoa lan tphcm bán cây giống lan hồ điệp tphcm bán cây hoa lan ban cay hoa lan gia sy ban cay hoa lan hcm ban cay hoa lan hue tphcm ban cay hoa lan kieng o tphcm bán cây hoa lan o sài gòn bán cay hoa lan quan tu bán cây hoa lan tphcm bán cây lan con sai gon bán cây lan giống hcm 2021 bán cây mô hoa lan ban cay phong lan ho diep con o hcm bán chậu cao đựng hoa lan rẻ nhất tphcm bán chậu hoa lan bán chậu hoa lan can đẹp tp hcm ban chau hoa lan dep o quan 1 bán chậu hoa lan hồ điệp sài gòn ban chau hoa lan mung khai truong hoc mon bán chậu hoa lan tím tphcm ban chau hoa lan tp hcm bán chậu hoa tulip tphcm bán chậu lan hồ điệp bán chậu trồng lan bán củ hoa lan huệ bán củ hoa lan huệ tphcm bán củ hoa tiên ông tphcm bán địa lan giống bán địa lan tết 2021 tại tphcm ban giong cay lan ho diep tphcm ban giong cay phong lan bán giống hoa hồng đen ban giong hoa lan bán giống hoa lan hồ điệp bán giống hoa lan o tphcm bán giống hoa lan tphcm ban giong lan ho diep bán giống lan hồ điệp bán giống lan hồ điệp ở tphcm bán hao lan hồ chí minh ban hchau hoa lan ho diep gia ban ho diep nhap khau tphcm ban hoa canh o ho chi minh ban hoa dia lan tai sai gon bán hoa địa lan trắng bán hoa giả giá rẻ tphcm bán hoa giả saigon ban hoa ho diep ban hoa lab bán hoa lab ban hoa lan bán hoa lan bán hoa lan bạch dương tphcm bán hoa lan các loại bán hoa lan cac loai truc tuyen gia re ban hoa lan cat canh nhap khau thailank bán hoa lan cattleya ban hoa lan cay canh tai ho chi minh ban hoa lan cay con o thanh pho ho chi minh bán hoa lan cây giống ở tp hcm bán hoa lan cấy mô bán hoa lan chậu bán hoa lan chậu chơi tết hcm ban hoa lan chau gia re tai tphcm bán hoa lan chậu treo tphcm ban hoa lan chung tet bán hoa lan con giá rẻ hcm ban hoa lan dang nụ hcm ban hoa lan dao ở tphcm bán hoa lan dạo tại tphcm ban hoa lan dendro bán hoa lan dendro bình chánh bán hoa lan denro ban hoa lan dep bán hoa lan đẹp bán hoa lan đẹp gò vấp bán hoa lan đẹp ở sài gòn bán hoa lan đẹp saigon ban hoa lan dep tai tphcm ban hoa lan duong hoang van thu bán hoa lan đường hoàng văn thụ bán hoa lan đường nguyễn hữu cảnh tại tp hcm bán hoa lan giả bán hoa lan giả đẹp bán hoa lan giả giá rẻ tphcm bán hoa lan giả giá sỉ bán hoa lan giả hcm bán hoa lan giả ỏ yp hcm bán hoa lan giá rẻ ban hoa lan gia re hcm bán hoa lan giá rẻ hcm bán hoa lan giá rẻ tại tphcm bán hoa lan giá rẻ tết 2021 tp hcm bán hoa lan giá rẻ tphcm bán hoa lan giả sai gòn ban hoa lan gia si ban hoa lan gia si tai saigon bán hoa lan giống bán hoa lan giống giá rẻ bán hoa lan giống giá rẻ tạp tphcm bán hoa lan giống giá rẻ tphcm bán hoa lan giống hcm bán hoa lan giống ho diep tphcm bán hoa lan giống ở hcm bán hoa lan giống ở quận 2 tphcm bán hoa lan giống tại hcm bán hoa lan giống tphcm ban hoa lan hac vy bán hoa lan hàn quốc tại hcm ban hoa lan hcm bán hoa lan hcm ban hoa lan hồ chí minh bán hoa lan hồ chí minh ban hoa lan ho diep bán hoa lan hồ điệp bán hoa lan hồ điệp để bàn ban hoa lan ho diep gia re bán hoa lan hồ điệp gia re hcm ban hoa lan ho diep giong tphcm ban hoa lan ho diep hcm bán hoa lan hồ điệp hcm bán hoa lan hồ điệp ở tphcm bán hoa lan hồ điệp rẻ nhất tphcm bán hoa lan hồ điệp tết 2021 giá rẻ hcm bán hoa lan hồ điệp tết 2021 tại hcm bán hoa lan hồ điệp tphcm bán hoa lan mini tại hồ chí minh ban hoa lan mokara hcm bán hoa lan mokara tại tphcm bán hoa lan mua têt bán hoa lan ngày tết tphcm ban hoa lan ngoc diem tphcm bán hoa lan ngọc điểm tphcm ban hoa lan o ben cat binh duong gia re ban hoa lan o go vap ban hoa lan o hcm bán hoa lan ở hoc môn bán hoa lan o phú nhuan bán hoa lan ở quận phú nhuận bán hoa lan ở sài gòn ban hoa lan o thanh pho ban hoa lan o thanh pho ho chi minh ban hoa lan o tphcm bán hoa lan ở tphcm bán hoa lan online bán hoa lan online tphcm ban hoa lan q 12 bán hoa lan q6 ban hoa lan quan 1 bán hoa lan quận 12 bán hoa lan quận 3 ở đâu bán hoa lan quan 7 bán hoa lan quận 7 bán hoa lan quận 7 hcm ban hoa lan quan go vap bán hoa lan quân tử ban hoa lan quan tu o tphcm ban hoa lan quan tu tai tphcm bán hoa lan quân tử tại tphcm bán hoa lan quân tử tphcm ban hoa lan re bán hoa lan re nhat ban hoa lan re nhat sai gon ban hoa lan rung bán hoa lan rừng giá rẻ ban hoa lan rung giong o tphcm bán hoa lan rừng hcm ban hoa lan rung o tp hcm ban hoa lan rung o tphcm ban hoa lan rung tai hcm bán hoa lan sac loai tp hcm bán hoa lan sài gòn ban hoa lan tai go vap bán hoa lan tại quận gò vấp bán hoa lan tại sài gòn ban hoa lan tai tp hcm bán hoa lan tại tphcm bán hoa lan tân thành ban hoa lan tet gia re tai tphcm ban hoa lan tet tphcm bán hoa lan thái tại tp hcm ban hoa lan thien nga du mau ban hoa lan thu dau mot bán hoa lan to hcm bán hoa lan tp hcm bán hoa lan tp hồ chí minh ban hoa lan tphcm bán hoa lan tphcm bán hoa lan trên hcm ban hoa lan tren mang ban hoa lan trong chau ban hoa lan trong chau tang qua tai tan phu bán hoa lan tử quan hcm ban hoa lan vai tphcm bán hoa lan vàng thơm gò vấp hcm bán hoa lan vĩ dạ tại tp hcm bán hoa lan vũ nữ vàng tại thành phố thái nguyên bán hoa lan xanh bán hoa loan đẹp nhất sài gòn bán hoa pha lê ban hoa phong lan bán hoa phong lan đỏ tphcm bán hoa phong lan tại vườn tại tp hcm ban hoa tet 2021 tp hcm ban hoc lan ho diep ban lan canh tai tphcm bán lan cấy mô hcm bán lan cấy mô tại tphcm ban lan cay mo tai tphcm 2021 ban lan chậu tp hcm quân 7 bán lan con cấy mô tphcm ban lan con gia re bán lan con hcm ban lan dia o dau tphcm bán lan điện hoa giao tận nơi ban lan giong bán lan giống bán lan giống giá rẻ bán lan giống hồ điệp ở tân phú hcm bán lan giống ho diep ở tphcm bán lan giống o duong au co hcm bán lan giống ở tphcm bán lan giống tại tphcm bán lan giống tphcm bán lan hai tại tphcm bán lan hài tphcm ban lan ho diep bán lan ho diep bán lan hồ điệp bán lan ho diep cây lẻ bán lan hồ điệp cấy mô bán lan hồ điệp cấy mô tphcm bán lan hồ điệp con bán lan hồ điệp con tphcm bán lan hồ điệp da cat hoa giá rẻ tphcm bán lan hồ điệp đang hoa bán lan hồ điệp giá rẻ ban lan ho diep gia re tp hcm bán lan hồ điệp giá rẻ tphcm bán lan hồ điệp giá rẻ tphcm 2021 ban lan ho diep giong bán lan hồ điệp giống bán lan hồ điệp giống giá rẻ tp hcm 2021 bán lan hồ điệp giống hcm bán lan hồ điệp giong tại hcm bán lan hồ điệp giống tp hcm ban lan ho diep giong tphcm bán lan hồ điệp giống tphcm ban lan hô điệp hcm bán lan hồ điệp hcm bán lan hồ điệp loại lớn tại hcm bán lan hồ điệp mini giống hcm ban lan ho diep nhap khau tphcm bán lan hồ điệp nuôi cấy mô bán lan hồ điệp ở hồ chí minh ban lan ho diep quan 1 pasteur ban lan ho diep quan 2 bán lan hồ điệp số lượng lớn ban lan ho diep tai sai gon ban lan ho diep tet bán lan hồ điệp tết 2021 ban lan ho diep thanh thai bán lan hồ điệp tphcm ban lan ho diep trong chai tai hcm bán lan ngọc điểm cấy mô bán lan ngọc điểm con tphcm ban lan ngoc diem giong bán lan ngọc điểm giống tp ho chi minh bán lan phi điệp tím tphcm ban lan quan tu ban lan rung bán lan rừng ban lan rung gia re ban lan rung gia si bán lan rừng ở hồ chí minh ban lan rung o tphcm bán lan rừng tại hcm bán lan rừng tại tphcm ban lan rung theo kg bán lan rừng theo kg bán lan rừng tphcm ban lan tai tp hcm bán lan thảo xanh lưỡi bán lan tp hcm bán lan vanda giống sai gon bán mô lan vanda tphcm bán nguyên liệu làm hoa đá tại tphcm bán nguyên liệu làm hoa pha lê tphcm ban nha 43 hoa lan bán phân kích thích hoa lan gò vấp bán phong lan cấy mô bán phong lan đẹp giá rẻ bán phong lan giả hạc tphcm ban phong lan gia re tai tphcm ban phong lan gia re tphcm bán phong lan giá rẻ tphcm ban phong lan gia re tphcm 2021 bán phong lan giống tại tphcm ban phong lan sai gon bán phong lan sỉ tphcm bán phong lan tại tphcm bán si cây giống lan hồ điệp bán sỉ hoa lan bán sỉ hoa lan giả tp hồ chí minh bán sỉ hoa lan tết hcm bán sỉ hoa lan tphcm ban si hoa laqn rung o tphcm bán sỉ lan hồ điệp bán thùng đựng gạo hoa lan tphcm bang gia lan ho diep bang gia lan nuoi cay mo tai ho chi minh bang gia phong lan ho diep banla ho diep o tphcm bảo an hoa lan bao gia hoa lan dep bệnh hoa lan ngọc điểm benh phong lan ho diep big c thủ dầu một hoa lan bình hoa đá pha lê bán ở tphcm binh hoa lan bình hoa lan 1 triệu binh hoa lan ban giay lon binh hoa lan da dep bình hoa lan đẹp binh hoa lan gia dep binh hoa lan ho diep bình hoa lan hồ điệp binh hoa lan ho diep gia binh hoa lan ho diep gia lon cao 1m hcm bình xịt hoa lan bó hoa lan đẹp bó hoa lan vang bo si hoa lan bỏ sỉ hoa lan hồ điệp bo si lan ho diep bộ sưu tập lan hồ điệp bông hồ điệp trắng mua o đua hcm bong hoa lan ho diep bong lan ho diep bún đậu hoa lan buon ban hoa lan buon ban hoa lan tai tphcm ca diem ban hoa lan giong o tphcm caách thức bán hoa lan các bệnh của hoa lan các bệnh thường gặp trên hoa lan các bệnh trên hoa lan cac cho ban hoa lan o tphcm các chợ hoa lan tại tp hcm cac cua hang hoa lan ho diep tai tp hcm cac dai li hoa lan gong o thanh pho ho chi minh các đại lý hoa lan tại tphcm các địa điểm hoa lan ở tphcm cac diem ban hoa lan tai tphcm cac điểm bán hoa lan thành phố hồ chí minh cac diem ban lan ho diep tai tp hcm cac giong hoa lan các giống hoa lan cắt cành các giống hoa lan đẹp các giống hoa lan hồ điệp các giống lan hồ điệp cac giong lan trong chiu thoi tiet nong cac hoa lan các kiểu cắm hoa lan hồ điệp cac lai hoa lan thuong ban o thanh pho các loại địa lan cac loai hao lan dep cac loai hoa lan các loại hoa lan cac loai hoa lan cattleya hoa tra mi các loại hoa lan chịu nắng các loại hoa lan đẹp các loài hoa lan đẹp nhất cac loai hoa lan gia tri tren thi truong viet nam các loại hoa lan ho diep các loại hoa lan hồ điệp các loại hoa lan hợp khí hậu tphcm các loại hoa lan nở vào dịp tết cac loai hoa lan o viet nam các loại hoa lan phổ biến ở việt nam các loại hoa lan rừng quý các loại hoa lan rừng việt nam các loại hoa lan thông dụng các loại hoa lan trong nam cac loai lan các loại lan hồ điệp các loại lan hồ điệp rừng các loài lan ở việt nam các loại lan quý cac loai lan rung de trong o tp hcm các loại lan rừng quý hiếm cac loai phan bon cham soc lan ho diep các mẫu cắm hoa lan đẹp các mẫu hoa lan bằng vải voan đẹp các nhà vườn bán lan giống ở miền bắc cac nha vuon hoa lan ngoc diem tai thu dau mot cac phan biet hoa lan cac shop hoa lan o tphcm các tiệm bán hoa lan nổi tiếng ở tp hcm cac trung tam hat giong hoa lan o tp hcm các vựa lan ho diep o tp hcm cac vuon ban hoa lan tet o sai gon cac vuon hoa lan o binh chanh các vườn hoa lan ở thành phố cac vuon hoa lan o tphcm các vườn hoa lan tại tp hcm các vườn hoa lan tại tphcm các vườn lan lớn ở thành phố hồ chí minh các vườn lan ở bình chánh các vườn lan ở củ chi cac vuon lan o ho chi minh ban si các vườn lan ở thành phố hồ chí minh các vườn lan ở tphcm cac vuon trong hoa lan can hcm cách bó hoa cưới bằng hoa lan cách bon phan cho hoa lan cách bón phân cho hoa lan cach bon phan cho lan ho diep cách cắm chậu hoa lan hồ điệp cách cắm chậu hoa lan hồ điệp bằng vải lụa cach cam hoa lan cách cắm hoa lan cách cắm hoa lan đẹp cách cắm hoa lan hồ điệp cach cam hoa lan ho diep gia cách cắm hoa lan hồ điệp giả cach cap hoa lan ho diep dep cách cấy mô hoa lan cach cham hoa ho diep cách chăm hoa lan cách chăm lan hồ điệp cách chăm sóc cây hoa lan cách chăm sóc cây hoa phong lan hồ điệp cách chăm sóc để lan hồ điệp ra hoa cach cham soc hoa lan cách chăm sóc hoa lan cách chăm sóc hoa lan hồ điệp cach cham soc hoa lan ho diep trang bao an cách chăm sóc hoa lan rừng cách chăm sóc hoa lan tại nhà cách chăm sóc hoa lan vẩy rồng cách chăm sóc hoa phong lan cách chăm sóc lan cach cham soc lan ho diep cách chăm sóc lan hồ điệp cách chăm sóc lan hồ điệp đang ra hoa cách chăm sóc lan hồ điệp ra hoa cách chăm sóc lan hồ điệp sau tết cách chăm sóc lan rừng cach chiec canh hoa lan cach chiet trong hoa lan cách chọn lan hồ điệp cách giữ hoa lan hồ điệp tươi lâu cách gói hoa lan vào thùng xop cách làm cho hoa lan ra hoa cach lam hoa ho diep bang pha le cach lam hoa lan bang pha le cách làm hoa lan bằng vải voan cách làm hoa lan hồ điệp bằng pha lê cách làm hoa lan hồ điệp bằng vải voan cách làm hoa lan hồ điệp tím bằng vải voan cách làm hoa lan ho diep vải voan cách làm hoa pha lê cách làm hoa pha lê ở tphcm cach mua chau hoa lan ho diep cach phong ngua benh cho hoa lan cach thưc câm hoa lan hồ điệp châu lớn cach trang tri hoa lan tren ban tho phat cách trị bệnh cho hoa lan cach tri sau long cho hoa lan cách trồng chăm sóc hoa lan cach trong hat phog lan ho diep cach trong hoa lan cách trồng hoa lan cách trồng hoa lan hiệu quả cach trong hoa lan ho diep cách trồng hoa lan hồ điệp cách trồng hoa lan rừng cách trồng hoa lan trên gỗ cách trồng hoa lan trong chậu cach trong lam cach trong lan cách trồng lan cach trong lan ho diep cách trồng lan ho điệp cách trồng lan hồ điệp cách trồng lan hồ điệp con cách trồng lan ho diep giong tphcm cách trồng lan hồ điệp ra hoa cách trồng lan hồ điệp sau tết cach trong lan ho diep tai hcm cach trong lan ho diep tai nha cách trồng lan hồ điệp vào chậu cách trồng lan sóc ta cách trồng lan trong chậu cách trồng phong lan sai gon cach trong va cham soc hoa lan cách trồng và chăm sóc hoa lan cach trong va cham soc lan ho diep cách trồng và chăm sóc lan hồ điệp cach tuoi hoa lan cách tưới nước cho lan ho diep cách ươm giống hoa lan cach uom lan ho diep cách ươm lan hồ điệp cachtronglanhodiep cam hoa lan cắm hoa lan cam hoa lan da cắm hoa lan để bàn cắm hoa lan đẹp cắm hoa lan hồ điệp cam hoa lan ngay tet cần bán hoa lan vanda cần mua hoa địa lan o sai gòn 2021 can mua hoa lan cân mua hoa lan cắt cành tại tphcm can mua hoa lan choi tet tai tp hcm can mua hoa lan gia re tai vuon tphcm cần mua hoa lan hồ điệp khu vực quận 1 can mua hoa phong lan giong o tphcm canh hoa lan vũ nữ rẻ tphcm cap hoa lan cát lan cat lan ho diep cau lac bo hoa lan tphcm câu lạc bộ hoa lan tphcm cay canh hoa lan quan 6 ho chi minh cây con lan hồ điệp cay giong hoa la hcm cay giong lan ho diep cây giống lan hồ điệp cây giống lan hồ điệp hồ chí mình cây giống phong lan hồ điệp ở tphcm cay ho diep cay hoa lan cay hoa lan bi vang la cay hoa lan binh chanh cây hoa lan giá bao nhiêu cay hoa lan hoang duong cay lan cây lan con cây lan dendro để bàn bán ở dĩ an cay lan ho diep cây lan hồ điệp cây lan hồ điệp giống hồ chí minh cay lan ho diep mini trang cay lan ho diep mini trng cây lan ngoc diem đắt nhất việt nam cây lan quân tử cây lan quân tử mua ở đâu tphcm cây lan ý mua ở đâu sai gon cc loại hoa lan cha lan hồ chí minh chăm sóc hồ điệp cham soc hoa lan chăm sóc hoa lan cham soc hoa lan ho diep cham soc hoa lan tai nha cham soc lan ho diep chăm sóc lan hồ điệp cham soc lan ho diep canh chăm sóc lan hồ điệp ra hoa đúng tết chăm sóc lan hồ điệp sau khi ra hoa chậu 3 cành lan hồ điêp hồ thị kỷ chau cam lan ho diep dep tai hcm chau ho diep chậu hồ điệp chậu hoa bằng gỗ chau hoa dai ho diep chau hoa ho diep chau hoa lan châu hoa lan chậu hoa lan chậu hoa lan ban tet chậu hoa lan bao nhiêu tiền chậu hoa lan can chậu hoa lan can giá rẻ chau hoa lan can tai tphcm chậu hoa lan can tphcm chau hoa lan chúc mừng sinh nhật đẹp tphcm chau hoa lan dep chậu hoa lan đẹp chau hoa lan gia chau hoa lan gia bao nhieu chau hoa lan giao hang tan noi chau hoa lan ho diep chậu hoa lan hồ điệp chau hoa lan ho diep 1 canh chậu hoa lan hồ điệp 1 mét chậu hoa lan hồ điệp đẹp chậu hoa lan hồ điệp giả chau hoa lan ho diep gia nhieu chau hoa lan ho diep nho chau hoa lan ho diep quan1 5 chậu hoa lan kiểng chau hoa lan mac nhat chậu hoa lan nhỏ bao nhiêu tiền chau hoa lan phong khach chau hoa lan quan tan phu chậu hoa lan tại quận 10 chậu hoa lan tết đang bán chạy ở hcm trên đường thành thái chậu hoa lan tp hcm chau hoa lan tphcm chau hoa phong lan dep chau hoalan ho diep chau lan chậu lan ảnh sài gòn chau lan dep chậu lan đẹp chậu lan đẹp hcm chau lan gia gia si o tp hcm chau lan ho diep chậu lan hồ điệp chau lan ho diep 12 canh co y nghia gi chau lan ho diep 3 canh chậu lan hồ điệp 5 cành chau lan ho diep bang lua cao cap chậu lan hồ điệp đẹp chau lan ho diep gia chau lan ho diep giá chậu lan hồ điệp giá chậu lan hồ điệp giả chậu lan hồ điệp ở sai gon goa hang tan noi chậu lan hồ điệp tại hồ chí minh chau lan ho diep tai sai gon chau loan ho diep nho de ban chau nhua trong lan cay mo chậu phong lan chậu phong lan giá rẻ hcm chậu trồng hồ điệp chậu trồng hoa lan chau trong hoa lan tphcm chậu trồng lan chau trong lan ho diep chauvhoa lan gia tphcm chi phi trong lan ho diep chieu cao cua gong hoa ho diep chjau hoa lan cho ban giong lan tphcm cho ban hao lan o binh chanh cho ban hat hoa ho diep tphcm cho ban hoa gia o tphcm cho ban hoa lan cho ban hoa lan ho diep tai tp hcm cho ban hoa lan hoc mon ba diem cho ban hoa lan o quan 8 cho ban hoa lan o tphcm cho ban hoa lan quan 10 cho ban hoa lan re o tphcm cho ban lan ho diep o tphcm cho ban lan ho diep quan 5 cho bán lan ho diep trang o tan phu chổ bán lan nuôi cấy mô tại tphcm cho dau moi hoa lan o dau sai gon cho dau moi hoa lan sai gon chợ đầu mối hoa lan tphcm cho hoa ho thi ky chợ hoa lan cho hoa lan đẹp nhất sai gon chợ hoa lan giá rẻ hcm chợ hoa lan ở sài gòn cho hoa lan rung chợ hoa lan rừng thành phố hcm cho hoa lan sai gon chợ hoa lan sài gòn chợ hoa lan saigon chợ hoa lan sỉ ở tphcm chợ hoa lan tp hcm chợ hoa lan tphcm cho hoa tet saigon cho lan rung sai gon cho mua hoa ho diep sai gon chổ nào bán hoa lan o tphcm cho nao o tphcm ban hoa lan cho phien hoa lan o tphcm cho thuê hoa lan cho tot mua con giong hoa lan cho xem hoa lan cattleya cho xem hoa lan cattleya choi hoa lan chồi hoa lan hồ điệp chong bong lan ho diep gia chong hoa lan chong hoa lan tai nha be quan 8 chua lan hoa ho diep re nhat sai gon chuyen ban hoa lan chuyen ban hoa lan chau tphcm quan 1 chuyen ban hoa lan ho diep chuyên bán hoa lan ở sài gòn chuyen ban hoa lan pham ngoc thach chuyên bán hoa lan sỉ lẻ hcm chuyen ban hoa lan si le phu nhuan chuyên bán hoa lan sỉ lẻ tphcm chuyên bán hoa lan tại hcm chuyên bán hoa lan tại tphcm chuyên bán hoa lan tp hcm chuyen ban hoa lan tuoi chuyen ban hoa lan tuoi gia re hcm chuyen ban lan quan tu chuyện cung cấp giống lan tphcm chuyen cung cap hoa gia si chuyên cung cấp hoa lan chuyen cung cap hoa lan ho diep chuyên cung cấp hoa lan tại tphcm chuyên cung cấp lan rừng tphcm chuyên giao lan hồ điệp giá rẻ chuyên hoa lan hcm chuyên hoanlan quận 11 chuyen lan ho diep hoc mon chuyen lan ho diep tphcm clb trang trại hoa lan tp hcm co bao nhieu loai hoa lan co nen mo shop hoa lan co so ban hoa co so ban hoa lan dendro tai ho chi minh co so ban hoa lan sai gon cơ sở bán hoa lan tại sài gòn co so ban lan ho diep hcm cơ sở hoa lan tâm trí ở thành phố hồ chí minh co so hoa lan tphcm co so hoa lan trong thanh pho co so hoa pha le o sai gon cơ sở mua bán hoa lan tại tphcm cơ sở trồng hoa lan o hcm con dương bán hoa lan nhieu ở hcm cong ty cung cap hoa lan ho diep dep tphcm công ty hoa lan hồ điệp ở sài gòn cong ty hoa lan mai huy cong ty hoa lan sai gon công ty tnhh thoa hoa lan hồ chí minh việt nam ct hoa lan dau tieng cty dich vu hoa lan q12 cửa h2ng hoa lan giả tphcm cua hang ban hat giong hoa lan cửa hàng bán hoa giả ở tphcm cửa hàng bán hoa giả tại tphcm cua hang ban hoa lan cửa hàng bán hoa lan của hàng bán hoa lan cây ơ ho chi minh cua hang ban hoa lan dat set o tphcm cua hang ban hoa lan ho diep cửa hàng bán hoa lan hồ điệp cửa hàng bán hoa lan hồ điệp 2 ba trung cua hang ban hoa lan ho diep tai tp hcm cửa hàng bán hoa lan hồ điệp tphcm cua hang ban hoa lan lon nhat tp ho chi minh cửa hàng bán hoa lan ở tp hồ chí minh cua hang ban hoa lan o tphcm cuâ hang ban hoa lan tai go vap cửa hàng bán hoa lan tại quận 11 cua hang ban hoa lan tai quan 7 cua hang ban hoa lan tai tphcm cửa hàng bán hoa lan tp hcm cửa hàng bán hoa lang ở tphcm cua hang ban hoa ln o tphcm cua hang ban lan ho diep re nhat tp hcm cua hang hoa ho diep sai gon cua hang hoa ho diep tphcm cửa hàng hoa hồ thị kỷ cua hang hoa lan cửa hàng hoa lan cua hang hoa lan 112 pham viet chanh quan 1 cua hang hoa lan anh cua hang hoa lan chau tai tp hcm cua hang hoa lan duong luong dinh cua q2 sai gon cua hang hoa lan gia re tphcm cửa hàng hoa lan hcm cua hang hoa lan ho chi minh cửa hàng hoa lan hồ chí minh cua hang hoa lan ho diep o an lac cua hang hoa lan ho diep o quan 1 cua hang hoa lan ho diep phuong hiep binh chanh cửa hàng hoa lan hồ điệp quận 2 cua hang hoa lan ho diep quan 3 cua hang hoa lan ho diep re tphcm cua hang hoa lan hồ diệp saigon cửa hàng hoa lan hồ điệp tại tphcm cửa hàng hoa lan hồ điệp tp hcm cua hang hoa lan hoang giap cửa hàng hoa lan hoàng hòa cửa hàng hoa lan hoàng văn thụ cửa hàng hoa lan hóc môn cua hang hoa lan o âu cơ quận tân phú cua hang hoa lan o hcm cửa hàng hoa lan ở phú nhuận cua hang hoa lan o sai gon cửa hàng hoa lan ở sài gòn cua hang hoa lan o tphcm cửa hàng hoa lan quận 1 cua hang hoa lan sai gon cửa hàng hoa lan sài gòn cua hang hoa lan tai nguyen chi thanh quan 10 cửa hàng hoa lan tại tp hcm cua hang hoa lan tai tphcm cửa hàng hoa lan tại tphcm cua hang hoa lan thai binh quan 1 cửa hàng hoa lan thành phố hồ chí minh cua hang hoa lan tp hcm cua hang hoa lan tphcm cửa hàng hoa tươi quận 6 cua hang lan ho dep phong lan quan tan binh cua hang lan ho diep cua hang lan ho diep quan 1 cua hang lan ho diep quan 3 cua hang lan ho diep tphcm cua hang lan ho thi ky cua hang phan bon hoa lan cua kinh hoa phong lan o sai gon cung cao hoa lan cung cap cay giong hoa lan hcm cung cấp chậu lan hồ điệp ở hồ chí minh cung cấp giống cây hồ điệp cung cap giong hoa lan cung cấp giống lan hồ điệp cung cap hoa lam chậu gia tot cung cấp hoa lan bán cành cung cấp hoa lan chậu hồ chí minh cung cap hoa lan gia si cung cấp hoa lan giá sỉ bình chánh cung cấp hoa lan giong cung cấp hoa lan hồ chí minh cung cấp hoa lan tết tphcm cung cấp lan dendro cấy mô cung cấp lan dendro giống cung cấp lan đẻno tai tphcm cung cap lan giong tphcm cung cap lan ho diep gia si cung cấp lan hồ điệp giá sỉ cung cap lan ho diep gia si tphcm cung cấp lan hồ điệp giong giá sỉ tai hcm cung cap lan ho diep o tp hcm cung cap lan ho diep tai tphcm cung cap si hoa lan ho diep tp hcm cuua hang hoa lan chuc mung sinh nhat tphcm đại hồ điệp dai l hoa lan go vap đại lí thu mua hoa lan dai ly ban giong lan ho diep hcm dai ly ban hoa lan o sai gon đại lý ban hoa lan tai tphcm dai ly ban lan ho diep o sai gon dai ly cung cap hoa lan o hcm dai ly hoa lan ho diep tphcm dai ly hoa lan o cu chi đai lý hoa lan tai thanh pho ho chi minh đại lý lan hồ điệp dai ly lan ho diep tphcm dai ly lan rung tphcm đại lý phân bón hoa lan cây cảnh hvp danh sách các loại hoa phong lan danh sach vuon lan cu chi dat bong lan ho diep gia re tphcm dặt chau hoa lan o long thanh đặt chậu lan ở đâu dat hoa lan đặt hoa lan dat hoa lan chau dat hoa lan cuoi cam tay o dau dat hoa lan ho diep dat hoa lan ho diep đặt hoa lan hồ điệp đặt hoa lan hồ điệp online tp hcm đặt hoa lan hồ điệp online tphcm dat hoa lan ho diep quan 5 dat hoa lan ho diep truc tuyen tphcm dat hoa lan ho diep uy tinh tphcm đặt hoa lan khai trương hồ chí minh đặt hoa lan sinh nhật quận 1 đặt hoa lan tại tphcm dat hoa lan tang sinh nhat q 2 đặt hoa lan tp hcm dat hoa lan tphcm dat hoa online tai tphcm q6 hao lan ho diep dat hoa phong lan ho diep hcm dat hoa phu nhuan lan ho diep đặt hoa sinh nhật hcm dat mua hoa lan cat canh tai thanh pho ho chi minh đặt mua hoa lan ở hcm dặt mua hoa lan o tphcm đặt mua hoa lan tại tphcm dat mua lan ho diep đất trồng lan hồ điệp dau hap toc orchid hoa lan dau hoa lan tai tai quan tanphu day choi hoa lan dđặt hoa lan tphcm ddịađiểm bán hoa lan tại thành phố hồ chí minh dđiểm bán hoa lan hồ điệp tphcm dendro lai ho diep di chi ban lan ho diep o tphcm dia ban giong cay hoa lan con đia chi 61 duong hoa lan pdhu nhuan hcm dia chi ban dung cu trong chau hoa lan tphcm địa chỉ bán giống hoa lan con ở tphcm dia chi ban ho lan trung buom địa chỉ bán hoa giả đẹp ở tphcm dia chi ban hoa lan dia chi ban hoa lan dat set o tphcm dia chi ban hoa lan dep o sai gon dia chi ban hoa lan dep tai tphcm địa chỉ bán hoa lan giả đẹp ở tphcm địa chỉ bán hoa lan giả ở tphcm địa chỉ bán hoa lan giả ởthanh phố hồ chí minh địa chỉ bán hoa lan giống tại tphcm dia chi ban hoa lan ho diep con gia re tai tphcm dia chi ban hoa lan ho diep gia re hcm dia chi ban hoa lan ho diep o hcm dia chi ban hoa lan ho diep o sai gon dia chi ban hoa lan ho diep si o tphcm dia chi ban hoa lan nhua o thanh pho hcm địa chỉ bán hoa lan ở gò vấp dia chi ban hoa lan o lai thieu binh duong dia chi ban hoa lan o quan 9 dia chi ban hoa lan o tphcm địa chỉ bán hoa lan quân tử tai tphcm dia chi ban hoa lan re dep dia chi ban hoa lan tai thanh pho hcm dia chi ban hoa lan tai tphcm địa chỉ bán hoa lan tại tphcm dia chi ban hoa lan tet 2021 tphcm dia chi ban hoa lan tphcm địa chỉ bán hoa lan uy tphcm địa chỉ bán hoa phong lan ở sài gòn địa chỉ bán lan cấy mô địa chỉ bán lan cấy mô ở hcm địa chỉ bán lan cấy mô tại tphcm địa chỉ bán lan cấy mô tphcm địa chỉ bán lan giống dia chi ban lan giong o tphcm địa chỉ bán lan giống ở tphcm địa chỉ bán lan giống tại tphcm địa chỉ bán lan hồ điệp địa chỉ bán lan hồ điệp con tại tphcm địa chỉ bản lan hồ điệp đẹp tại tphcm dịa chỉ bán lan ho diep giá re nhat tphcm dia chi ban lan ho diep o sai gon dia chi ban lan ho diep re tai tp hcm dia chi bán lan ho diep tai huyen binh chanh tp hcm dia chi ban lan ho diep tai sai gon địa chỉ bán lan hồ điệp tại sài gòn dia chi ban lan ho diep tai thu dau mot dia chi ban lan o tphcm dia chi ban lan vanda o hcm dia chi ban phan trung chi dung cho hoa lan hcm dia chi ban phan trung chi dung cho hoa lan tan phu hcm dia chi bo hoa lan o tp ho chi minh địa chỉ các vườn lan ở tphcm địa chỉ 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chậu 500k hoa lan chau ho diep hoa lan chậu mua ở đâu tphcm hoa lan chau o tphcm hoa lan chậu quan 2 hoa lan chậu rẻ nhất tphcm hoa lan chau sai gon hoa lan chậu sứ hoa lan châu tphcm hoa lan chậu treo đẹp hoa lan chiec hai hoa lan chợ bưởi hoa lan chợ long thành hoa lan cho van phong hoa lan chung tet hoa lan chưng tết hoa lan chung tet 2021 hoa lan chưng tet gia re tai phu nhuan hoa lan chuông bán ở đâu hoa lan chuong co ban o dau tai quan 8 hoa lan co bao nhieu mau hoa lan con gia re tphcm hoa lan công nghiệp hoa lan công tử hoa lan cu chi hoa lan cu3 hoa lan đá hoa lan đá ở hcm hoa lan đá pha lê hoa lan da pha le de ban hoa lan đại anh sai gòn hoa lan đai châu hoa lan đai châu giống mua o tphcm hoa lan đại học nông nghiệp hoa lan dambri hoa lan dangi hoa lan đất hoa lan dat gia nhat hoa lan đất sét hoa lan đất sét ở đâu tphcm hoa lan đắt tiền nhất hoa lan day hoa lan day leo hoa lan de hoa lan để bàn hoa lan delro hoa lan đen hoa lan den ro xuan tp hcm hoa lan dendro hoa lan dendro 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thung co hu k hoa lan dua sac hoa lan duc hoa hoa lan duoi chon gia bao nhieu hoa lan đuôi công hoa lan đường hoàng văn thụ hoa lan đường lê văn hưu hoa lan đường lương định của q2 hoa lan đường mai chí thọ quận 7 hoa lan duong nguyen thi thap quan7 hoa lan duong so 6 quan 9 hoa lan duong thich quang duc hoa lan đường trường sơn hoa lan duong vinh vien quan 10 hoa lan enzolife hoa lan epi trang gia bao nhieu hoa lan flower box hoa lan gan ben xe mien dong hoa lan gần sân bay hoa lan ghep go hoa lan gia hoa lan giả hoa lan giả bán tại sài gòn hoa lan gia bao nhieu hoa lan giả bao nhieu 1 canh hoa lan giả cao cấp hoa lan giả cao cấp hcm hoa lan giả cao cấp tp hcm hoa lan giả đẹp hoa lan giả giá rẻ hoa lan gia gia re tphcm hoa lan giả gò vấp hoa lan giả hcm hoa lan gia huy hoa lan giả ở sài gòn hoa lan giả o tphcm hoa lan giả ở tphcm hoa lan gia re hoa lan giá rẻ hoa lan gia re cho tot hoa lan gia re hcm hoa lan giá rẻ hcm hoa lan giá rẻ hồ chí minh hoa lan gia re o hoang van thu hoa lan giá rẻ ở sài gòn hoa lan gia re ở tphcm hoa lan gia re quan 2 hoa lan gia re sai gon hoa lan giá rẻ tại hồ chí minh hoa lan gia re tai tphcm hoa lan gia re tan phu hoa lan gia re thu duc hoa lan gia re tp hcm hoa lan gia re tphcm hoa lan giá rẻ tphcm hoa lan giả sài gòn hoa lan gia si hoa lan giá sỉ hoa lan gia si o sai gon hoa hoa lan giá sỉ ở tphcm hoa lan giá sỉ sài gòn hoa lan gia si tp hcm hoa lan gia si tphcm hoa lan gia tai tphcm hoa lan giả tặng khai trương hoa lan gia tphcm hoa lan giả tphcm hoa lan giả trang trí phòng khách thành phố hồ chí minh hoa lan gia treo chau hoa lan gia treo tuong hoa lan giao tan nha hoa lan giao tân nhà hoa lan giao tận nhà hoa lan giao tận nơi ở hcm hoa lan giay nhun hoa lan giay o tphcm hoa lan giong hoa lan giong bac hai tphcm hoa lan giống giá rẻ hcm hoa lan giống nhà bè hoa lan giống sài gòn hoa lan giống tại sài gòn hoa lan giống tp hcm hoa lan giong tphcm hoa lan giống tphcm hoa lan go vap hoa lan gò vấp hoa lan hac dinh hoa lan hạt dưa q5 hoa lan hat vy ban o dau tai tphcm hoa lan hcm hoa lan he hoa lan hinh anh gia tp hcm hoa lan ho chi minh hoa lan hồ chí minh hoa lan ho dep hoa lan ho diep hoa lan ho điệp hoa lan hô điêp hoa lan hồ điep hoa lan hồ điệp hoa lan hồ điệp 4 hồ chí minh việt nam hoa lan hồ điệp 4 ho chi minh vietnam hoa lan ho diep an binh duong hoa lan hồ điệp apollo hoa lan ho diep ba to quan 8 hoa lan hồ điệp bán ở đâu hoa lan ho diep ban o quan 12 hoa lan hồ điệp bằng vải voan hoa lan ho diep bao nhieu hoa lan hồ điệp bao nhiêu 1 chậu hoa lan hồ điệp bình thạnh hoa lan ho điệp cần những điều gì hoa lan hồ điệp cao su hoa lan ho diep chau hoa lan hồ điệp chậu hoa lan ho diep co y nghia gi trong cuoc song hoa lan ho diep dep hoa lan hồ điệp đẹp hoa lan ho diep dep quan 3 hoa lan hồ điệp đẹp thành phố hồ chí minh hoa lan hồ điệp dĩ an hoa lan hồ điệp đường nguyên đình chiểu tp hcm hoa lan ho diep đường phạm ngọc thạch hoa lan hồ điệp đường phạm văn hai tphcm hoa lan hồ điệp gần quận bình tân hoa lan hồ điệp gần quang trung gò vấp hoa lan ho diep gia hoa lan ho diep giả hoa lan hồ điệp giả hoa lan ho diep gia bao nhieu hoa lan ho diep gia nhu that hoa lan ho diep gia re hoa lan hồ điệp giá rẻ hoa lan ho diep gia re o go vap hoa lan ho diep gia re quan go vap hoa lan ho diep gia re sai gon hoa lan ho diep gia re tp hcm hoa lan hồ điệp giá rẻ tphcm hoa lan ho diep gia si hoa lan ho diep gia si o tphcm hoa lan ho diep gia si tai sai gon hoa lan hồ diệp giả tại hcm hoa lan hồ điệp giả tại tp hcm hoa lan ho diep gia tai tphcm hoa lan hồ điệp giả tan binh hoa lan ho diep gia tien canh bao nhieu hoa lan ho diep giong hoa lan ho diep hcm hoa lan hồ điệp hcm hoa lan ho diep ho chi minh hoa lan hồ điệp ho chi minh hoa lan ho diep le hoa lan ho diep lon cam hoa lan hồ điệp lương đình của quận 2 hoa lan ho diep mau tim vo chong hoa lan ho diep mau vang hoa lan ho diep mini hoa lan hồ điệp mua ở đâu hoa lan ho diep nguyen van troi hoa lan hồ điệp nhánh lớn tp hcm hoa lan ho diep nhỏ thấp hoa lan hồ điệp ở lê trọng tấn bình tân hoa lan hồ điệp ở sài gòn hoa lan ho diep o saigon hoa lan ho diep o thanh thai hoa lan ho diep ở tp hcm hoa lan ho điep o tphcm hoa lan ho diep phường 9 quận 3 hồ chí minh việt nam hoa lan ho diep q 4 hoa lan ho diep quan 2 hoa lan ho diep quan 3 hoa lan ho diep quan 4 hoa lan ho diep quan 6 hoa lan hồ điệp quận 7 hoa lan ho diep rung hoa lan ho diep rung hoa lan hồ điệp rừng hoa lan ho diep sai gon hoa lan hồ điệp sài gòn hoa lan hồ điệp sài gòn hồ chí minh hoa lan hồ điệp sài gòn ho chi minh city ho chi minh hoa lan hồ điệp sài gòn hồ chí minh việt nam hoa lan ho điệp sai gòn nở hoa quanh năm ko hoa lan ho diep sinh nhat hoa lan ho diep tai hcm hoa lan ho diep tại hồ chí minh hoa lan ho diep tai quan 3 hoa lan hồ điệp tại sai gon hoa lan hồ điệp tại tp hcm hoa lan hồ điệp tại tp thủ dầu một hoa lan ho diep tai tphcm hoa lan hồ điệp tại tphcm hoa lan ho diep tai vuon hcm hoa lan ho diep tang ban gai hoa lan ho diep tang sinh nhat hoa lan ho diep tang sinh nhat tai tphcm hoa lan ho diep tet hoa lan ho diep thai lan hcm hoa lan ho diep tiep hoa lan ho diep tim hoa lan hồ điệp tím hoa lan hồ điệp tím nhạt hcm hoa lan hồ điệp tím sai gon hoa lan ho diep tp hcm hoa lan ho diep tphcm hoa lan hồ điệp tphcm hoa lan ho diep trang hoa lan hồ điệp trắng hoa lan hồ điệp trắng 5 cành hoa lan hồ điệp trong sài gòn hoa lan ho diep vai nhap hoa lan hồ điệp vai o dau ban hoa lan ho diep vai voan hoa lan ho diep vang hoa lan hồ điệp vàng hoa lan ho diep vang o go vap hoa lan ho diep voan hoa lan hoalancaycanh com hoa lan hoàng dương tp hcm hoa lan hoang duong tphcm hoa lan hoang hau hoa lan hoang hau bang pha le hoa lan hoang hau xanh hoa lan hoang lan quan 1 hoa lan hoc mon hoa lan hóc môn hoa lan hodiep hoa lan hồng hóc môn hoa lan hoồ điệp hcm hoa lan huệ hoa lan inc hoa lan khu dan cu him lam p tan hung hoa lan kieng hcm hoa lan kiểng hồ chí minh hoa lan kieu ban o tphcm hoa lan kieu tim hoa lan kim điệp xuân hoa lan kim ly hô chi minh hoa lan kim tuyen hoa lan king hoc mon hoa lan là gì hoa lan lai thieu hoa lan len ro o thanh pho ho chi minh hoa lan leo gia hoa lan liberty hoa lan lida hoa lan linh tinh hoa lan lồng đèn hoa lan ly xa hoa lan mạc đỉnh chi tphcm hoa lan mai huy hoa lan man thiên hong ban dấu sai gon hoa lan mau do hoa lan mau vang hoa lan màu xanh hoa lan màu xanh mua tại tphcm hoa lan mini hoa lan mini gia re sai gon hoa lan mini quan 1 hoa lan mini siêu nhỏ hoa lan mini tphcm hoa lan moc lan hoa lan mokara hoa lan mokara o thu dau 1 hoa lan molara hoa lan một bông hoa lan một lá hoa lan mua o dau hoa lan mùa tết 2026 như thế nào hoa lan mun hoa lan mung dam cuoi hoa lan mừng khai trương hoa lan mừng sinh nhật hoa lan mưng sn hoa lan my hoa lan nào đắt nhất hoa lan nao de trong hoa lan nào dễ trồng nhất hoa lan nào đẹp nhất hoa lan nao lau tan nhat hoa lan nào lâu tàn nhất hoa lan nao nhieu tien hoa lan nao no mua tet hoa lan ngay 20 10 hoa lan ngay tet 2021 quan 7 hoa lan nghi hoa lan nghi saigon hoa lan ngoc diem hoa lan ngoc diem hcm hoa lan ngoc diem rung hoa lan ngọc điểm trong trong chau hoa lan ngọc điển hoa lan ngoc ha quan 2 hoa lan ngọc hoàng hoa lan nguyen bỉnh khiêm quận 1 hoa lan nguyen van troi hoa lan nhà vườn thành phố hồ chí minh hoa lan nha vuon tphcm hoa lan nhân duyên hoa lan nhập khẩu hoa lan nhựa hoa lan nhua tai hcm hoa lan nhua thành phố ho chí minh hoa lan no thang 1 hoa lan no trang long hoa lan nuôi trồng và kinh doanh hoa lan o ba diem hoa lan ở bình chánh hoa lan ở bình tân hoa lan ở chợ hồ thị kỷ hoa lan o cu chi hoa lan o go vap hoa lan o hcm hoa lan ở hcm hoa lan o sai gon hoa lan ở saigon pearl hoa lan o tan binh hoa lan o thanh pho ho chi minh hoa lan o tphcm hoa lan ở tphcm hoa lan ở trường đại học nông nghiệp thành phố hồ chí minh hoa lan o2 hoa lan og tung o binh chang tphcm hoa lan online hoa lan online hcm hoa lan online tp hcm hoa lan orchids hoa lan pasdora hoa lan phan dang luu hoa lan phat ngoc hoa lan phát tài hoa lan phi điệp tim duoc ban o dau hoa lan phong lan hoa lan phu lam hoa lan phu nhuan hoa lan phuc binh an duc hoa long an hoa lan phuc loc tho ba diem hoa lan phuong hoa lan quận 1 hoa lan quân 1 giá rẻ hoa lan quan 10 hoa lan quận 10 hoa lan quan 12 hoa lan quận 3 hoa lan quận 4 hoa lan quan 6 hoa lan quan 7 hoa lan quận 7 hoa lan quận 9 hoa lan quận gò vấp hoa lan quận tân phú hoa lan quan tu hoa lan quân tử hoa lan quân tử bán ở đâu o hcm hoa lan quan tu ban o tphcm hoa lan quân tử giá bao nhiêu hoa lan quân tử hồ chí minh hoa lan quan tu mua o dau hoa lan quân tử mua ở đâu hoa lan quân tử mua ở đâu tai tphcm hoa lan quân tử mua ở đâu tphcm hoa lan quan tu mua o hcm hoa lan quân tử ở phú mỹ tân thành mua ở đâu hoa lan quan tu o sai gon hoa lan quân tử ở tp hcm hoa lan quan tu tai tphcm ban o dau hoa lan quang ngai hoa lan quy hoa lan quý hoa lan quý hiếm hoa lan re dap o dau tp hcm hoa lan rẻ đẹp sài gòn hoa lan re hcm hoa lan re nhat o tp hcm hoa lan re o sai gon hoa lan rẻ ở tp hcm hoa lan rẻ tphcm hoa lan ren rét hoa lan renret hoa lan rủ hoa lan rung hoa lan rừng hoa lan rung com hoa lan rung dep hoa lan rừng đẹp nhất hoa lan rung gia re o quan 12 hoa lan rừng giá rẻ sài gòn hoa lan rung hcm hoa lan rung o ho chi minh hoa lan rừng ở sài gòn hoa lan rung si tai ho chi minh hoa lan rung toc tien hoa lan rung trong tu nhien hcm hoa lan sa dec hoa lan sai go

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    --- Bài cũ hơn ---

  • Cách Trồng Hoa Lan Vũ Nữ Đẹp Và Đơn Giản Nhất
  • Chia Sẻ Kinh Nghiệm Cách Trồng Hoa Lan Rừng Việt Nam
  • Kỹ Thuật Trồng Chăm Sóc Lan Thủy Tiên / Lan Kiều
  • Giới Thiệu Lan Cattleya Đại Tân
  • Giới Thiệu Lan Cattleya – Cao Đại Hùng
  • Một Số Loài Lan Rừng Đơn Thân

    --- Bài mới hơn ---

  • Ngọc Điểm – Rhynchostylis (Rink-Oh-Sty-Lis) Viết Tắt Rhy.
  • Hoa Lan Huyết Nhung Việt Nam
  • Tổng Hợp Dendrobium Rừng Việt Nam
  • Người Việt Và Hoa Lan Việt
  • Lan Hài Hằng (Paphiopedilum Hangianum)
  • Mỗi loài hoa đều có vẻ đẹp riêng. Hoa lan chiếm lĩnh thế giới thực vật bởi sự kỳ diệu, phong phú và tính đa dạng của chúng.

    Tuy không rực rỡ sắc màu như các giống lan lai ngoại nhập nhưng lan rừng có vẻ đẹp tự nhiên, thanh thoát và phần lớn có hương thơm nên hấp dẫn giới hâm mộ. Lan rừng Việt Nam có khoảng trên dưới 800 loài.

    Một Số Loài Lan Rừng Đơn Thân

    Dựa vào hình thái, cấu trúc thân cây, hoa lan phân thành 2 nhóm (theo Pfitzer):

    – Nhóm đa thân (Symbodial)

    – Nhóm đơn thân (monopodial)

    Ngoài ra còn có một nhóm trung gian giữa 2 nhóm trên nhưng gồm rất ít giống.

    Nhóm lan đơn thân gồm những cây lan tăng trưởng theo chiều cao và thân ngày càng dài ra, có thể phân nhánh trên thân, thường có nhiều rễ gió mọc dọc theo thân. Lan đơn thân rừng thường được nhân giống bằng cách tách nhánh, một số loài hiện đã được nhân giống bằng phương pháp cấy mô. Kiểu trồng tốt nhất là nên trồng ghép trên khúc gỗ, thân cây sống hoặc chết, trong giỏ gỗ với giá thể thông thoáng, thoát nước tốt.

    Sau đây là một số cây lan rừng tự nhiên Việt nam thuộc nhóm đơn thân được trồng phổ biến:

    1- CÂY LAN HỎA HOÀNG

    – Tên khác: Hoàng Yến(không phổ biến)

    – Tên khoa học: Ascocentrum miniatumLindley 1913

    a- Đặc điểm hình thái, sinh vật học:Cây thân nhỏ, lá dày, mọc hai dãy đều nhau, dày cứng màu xanh bóng. Phát hoa đứng thẳng, mọc ra từ nách lá, chùm hoa mang nhiều hoa nhỏ xếp dày đặc; đài và cánh hoa giống nhau, nở xòe rộng màu vàng cam rất đẹp. Môi gắn chặt vào gốc trụ, phân ba thùy, thùy bên nhỏ, đứng, thùy giữa to dạng cái lưỡi, có túi cựa. Nở hoa vào khoảng tháng 3-4, đặc biệt rất lâu tàn (1-2 tuần).

    b- Yêu cầu về điều kiện sinh thái:

    – Nhiệt độ: 20-25°C

    – Ánh sáng: 70-80%.

    – Ẩm độ: 40-70%.

    c- Một số cây lan rừng tự nhiên Việt Nam khác thuộc chi Ascocentrum:

    – Ascocentrum christentionianum (Tử hoàng): Hoa màu tím.

    – Ascocentrum pusillum Averyanov

    * Cây Tử Hoàng (Ascocentrum christensonianum Haager 1993):

    (Photo courtesy of Jay Pfahl)

    Lá hình dãi, màu đỏ xanh. Hoa màu tím, phát hoa dài 10-15 cm, mọc thẳng đứng từ nách lá, hoa nở vào mùa xuân.

    (* Tài liệu để tham khảo: Found in Vietnam in semi-deciduous and deciduous dry lowland forests at elevations of sealevel to 700 meters on the branches of forest trees as a hot to warm growing, monopodial epiphyte that has reddish green strap shaped leaves and blooms in the spring and fall with an erect, axillary, 4 to 6″ [10 to 15 cm] long inflorescence.)

    2- CÂY LAN CÙ LAO MINH

    – Tên Khác: Uyên ương(không phổ biến và có lẽ không phù hợp).

    – Tên khoa học: Christensionia vietnamica

    a- Đặc điểm hình thái, sinh vật học:

    Cây đơn thân, lá màu xanh lục trãi ra, chốp lá phân thùy. Có 2 dạng cây: cây lá ngắn và cây lá dài, đều cho hoa giống nhau. Hoa lớn, phát hoa thường mang 4 hoa màu xanh lá cây sáng, môi có họng cùng màu cánh hoa, bờ môi có màu trắng. Hoa nở vào khoảng tháng 6-7, hoa lâu tàn.

    b- Đặc điểm sinh thái học:

    – Phân bổ sinh thái tự nhiên: Đây là giống lan đặc hữu của Việt Nam. Trong tự nhiên, mọc dọc theo suối Ea Dong tỉnh Khánh Hòa và các khe suối ở đèo Mang Yang tỉnh Gia Lai.

    – Một số yêu cầu về điều kiện sinh thái: Nhiệt độ: 20-25°C, ánh sáng: 50%, ẩm độ: 40-70%.

    Cây lá ngắn Cây lá dài

    3- CÂY LAN NGỌC ĐIỂM

    – Tên khác: Nghinh Xuân, Tai Trâu, Đai Châu, Lan Me.

    – Tên khoa học: Rhynchostylis gigantea

    a- Đặc điểm hình thái, sinh vật học:Cây thuộc nhóm đơn thân, tăng trưởng theo chiều thẳng đứng, có rất nhiều rễ gió mọc thẳng từ thân. Lá dày, hẹp, tận cùng có hai thùy, có nhiều sọc nhạt màu chạy dọc theo chiều dài của lá. Phát hoa thòng hay cong, dài khoảng bằng chiều dài của lá, mang nhiều hoa màu trắng có điểm tím, môi có 3 thùy. Hoa có mùi thơm dễ chịu. nở hoa vào khoảng tháng 12 đến tháng 1-2, trùng vào dịp tết Nguyên Đán nên rất được ưa chuộng.

    (Theo dõi cho thấy: có hai dạng cây, cây có lá màu xanh hơi vàng, sọc trên lá rất rõ, dạng này trên cánh hoa có nhiều điểm tím hồng rất đẹp; dạng lá màu xanh lục, sọc trên lá rất nhạt hay không có sọc, dạng này trên cánh hoa có những vệt tím ít hơn, màu trắng nhiều hơn, không đẹp bằng dạng lá sọc?)

    b- Yêu cầu về điều kiện sinh thái:

    – Nhiệt độ: 25-30°C.

    – Ánh sáng: 60%.

    – Ẩm độ: 40-70%.

    c- Một số cây Ngọc Điểm lai nhập từ Thái lan:

    4- CÂY LAN HẢI YẾN

    – Tên khác: Ngọc Bích, Hải Âu.

    – Tên khoa học: Rhynchostylis coelestis

    a- Đặc điểm hình thái, sinh vật học:Cũng giống như Ngọc Điểm nhưng thân lá nhỏ hơn, màu xanh mướt, lá xếp theo rãnh, giữa cong quằn xuống, hai thùy lá không đều nhau. Phát hoa đứng thẳng, hoa màu trắng , ở phần chót có màu xanh lam, môi màu xanh hoa cà. Hương thơm đài các. Cây nở hoa vào khoảng tháng 3 – tháng 5.

    b- Yêu cầu về điều kiện sinh thái:

    – Nhiệt độ: 20-25°C.

    – Ánh sáng: 60%.

    – Ẩm độ: 40-70%.

    5-CÂY LAN BẠCH VĨ HỒ

    – Tên khác: Đuôi chồn, Đuôi sóc, Sóc lào. Ở miền Nam gọi là Đuôi chồn nhưng thường không phổ biến vì rất hiếm, miền Bắc gọi là Đuôi sóc, Sóc lào. Tên Bạch vĩ hồ có người cho rằng dùng để gọi cây Rhynchostylis retusavar alba màu trắng (xem ảnh dưới).

    – Tên khoa học: Rhynchostylis retusa

    a- Đặc điểm hình thái, sinh vật học:Lá dài, hơi mỏng và cong quặn, chốp lá phân thùy. Phát hoa dài, rũ thòng, mang nhiều hoa màu trắng hồng điểm tím, hoa có mùi thơm khá gắt, nở vào khoảng tháng 5-7.

    b- Yêu cầu về điều kiện sinh thái:

    – Nhiệt độ: 20-25°C.

    -Ánh sáng: 60%.

    -Ẩm độ: 40-70%.

    * Cây Bạch vĩ hồ đột biến màu trắng:

    6- CÂY LAN HỒNG NGỌC

    – Tên khác: Đuôi Cáo, Giáng Hương Nhiều Hoa. Ở miền Nam thường gọi là Đuôi cáo và gần như trở thành tên gọi dân gian phổ biến.

    – Tên khoa học: Aerides multiflora

    a- Đặc điểm hình thái, sinh vật học:Cây thân thẳng đứng. Lá dày, xếp thành hai dãy đối xứng, màu xanh hơi vàng và hơi nhăn nheo, có hai thùy không đều nhau. Phát hoa cong hay thòng, hoa màu trắng hồng với nhiều đốm đỏ hồng hay tím hồng,môi hầu như không có cựa, có ba thùy, thùy giữa hình tam giác màu tím đậm, hoa thơm, lâu tàn, nở vào mùa hè đến đầu mùa thu.

    b- Yêu cầu về điều kiện sinh thái:

    – Nhiệt độ: 20-25°C.

    – Ánh sáng: 50 %.

    – Ẩm độ: 40-70%.

    7- CÂY LAN BÒ CẠP TÍA

    – Tên khác: Lan nhện

    – Tên khoa học: Arachnis annamensis(Rolfe.) J. J. Smith

    a- Đặc điểm hình thái, sinh vật học:Thân khá dài, leo bò và phân nhánh. Lá tròn, dài, đầu lá có 2 thùy, mọc cách. Phát hoa dài, đứng. Hoa to màu khảm, có nhiều vết rằn ri màu nâu tía. Lá đài và cánh hoa bằng nhau, hẹp dài, trãi ra, hơi rộng ở đỉnh, đỉnh lệch cong về phía bên dưới. Hai lá đài bên cong như hai càng của con bò cạp nên được gọi là lan Bò Cạp. Môi gắn vào trụ thành một khối rất ngắn, mập, có cựa rất ngắn, môi phân làm 3 thùy, thùy giữa dài và nhọn. Ra hoa vào mùa hè, khoảng tháng 3-5, hoa lâu tàn, có mùi thơm.

    b- Đặc điểm sinh thái học:

    – Lan Bò cạp rất dễ trống, trồng ghép với gốc cây hoặc trồng như lan cắt cành, có nơi trồng làm hàng rào. Loài này cần nhiều ánh sáng, có thể trồng ngoài nắng trực tiếp.

    – Một số yêu cầu về điều kiện sinh thái: Nhiệt độ: 25-30°C; Ánh sáng: 70-100 %; Ẩm độ: 40-70%.

    --- Bài cũ hơn ---

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