Many thanks to all who responded to my inquiry last week about what additives people are currently using in media for isolating basidiomycete fungi, in view of the reduced availability of benomyl. There is a lot to think about in your replies which will be very helpful as we work out where we are headed in our own lab.

 

A number of those who replied requested that I circulate the email feedback for everyone’s benefit, so I have collated all the relevant segments and pasted them below in rough categories. I have also attached and referenced several files that two people sent. If anyone else responds separately I will circulate the email directly, if it is not already distributed on the ForPath mailing list.  Regards, Ian H.

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Original email query:

“With the reduced availability of Benlate fungicide our lab is seeking other options for a selective medium for isolating basidiomycete cultures (unlike many other fungal groups, basidiomycetes are able to grow in the presence of the added active ingredient, benomyl).

 

“I would be pleased to hear what media other laboratories may be currently using to isolate basidiomycte fungi and what people may think of their effectiveness”. – Ian H (30 Jan.).

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Still using benomyl:

 

·         “Have tried 3 other selective media, but none work well. Still using benomyl streptomycin malt agar. Please let me know what you find”.  – Dr. James (Jim) T. Blodgett, USDA Forest Service, Rocky Mountain Region, Forest Health Protection, Rapid City, South Dakota.

 

·         “We’re still using benomyl here.  It seems one small canister will last us over 100 years!” – Dr. Jim Worrall, USDA Forest Service, Rocky Mountain Region, Forest Health Protection, Gunnison, Colorado. [see also below].

 

·         Since I left WV (in 2008) I have not had to isolate for basidios.  And back in WV I had a 1lb bag of the stuff squirreled away. Out in CA I’m more often isolating Leptographium on cyclohex.  So I’m not much use.   However, can I ask that when you accumulate the answers would you be so kind as to send a synthesis to all who respond. – Martin MacKenzie, Forest Pathologist, South Sierra Service Area, Stanislaus National Forest, Sonora, California.

 

·         “That is an interesting question. I still have one bottle of benomyl which I keep safe and still did not need to use alternatives. In past we used poor and acidified media which also to an extent help in selecting basidiomycetes but not as efficiently as benomyl. I guess similar situation is with cycloheximide (actidion)which one could not buy anywhere so I had to rely on my stock and this compound is  so successfully used in selective media to isolate Ophiostomatoid fungi. I hope you will write a small review of answers and tips you get from other researchers related to your questions and share it with this group. I would be very interested to hear it”. – Adnan Uznovic, Durability and Sustainability, Advanced Building Systems, Vancouver, British Columbia.

 

·         Please inform me of the media you do end up using, as I might want to change someday.  I do have quite a bit of Benlate in the lab still, but I am always looking for good selective media. – Michelle Cram, US Forest Service, Forest Health Protection, Athens, Georgia. [see also below].

 

 

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Alternatives:

 

PCNB etc.:

 

·         “This paper lists some alternative media and their relative performance.  It’s old but I’m not aware of any major developments since then: Worrall, J.J. 1991. Media for selective isolation of hymenomycetes. Mycologia 83(3):296-302. http://www.forestpathology.org/pdfs/worrall1991media.pdf.” – Dr. Jim Worrall, USDA Forest Service, Rocky Mountain Region, Forest Health Protection, Gunnison, Colorado.

 

·         “I did a test of several media [see attached: SWFDWcram14.docx].  The PP media developed by (Kuhlman and Hendrix 1962) was very good.  I did find adding the 4 ppm of Benlate to this media (PPB media) was good at controlling the Trichoderma, but if I did not have Benlate, I would still use the PP media by Kuhlman and Hendrix.

“PP media, Kuhlman, EG, Hendrix, FF, Jr. 1962. A selective medium for the isolation of Fomes annosus. Phytopathology 52: 1310-1312.

5 g Bacto peptone, 20 g agar, 0.25g MgSO4•7H20, 0.5 g KH2PO4, 0.19 g Pentachloronitrobenzene (or 0.254 g Terrachor), 0.1 g streptomycin sulfate, into 1 liter water; autoclaved and cooled before adding 2 ml lactic acid (50%), and 20 ml ethyl alcohol (95%).

“*Peptone slows the growth of fungi that require a carbohydrate for growth.  Spore suspensions of basidiospores and conidia were tested (no data offered though) and germination was slowed down and it took 10-14 days for colonies to develop.  Trichoderma is slowed down, but still grows relatively well on the media”. – Michelle Cram, US Forest Service, Forest Health Protection, Athens, Georgia.

 

·         “You might experiment a bit with Kuhlman’s medium, which US Forest Service scientist George Kuhlman developed many years ago for culture of Heterobasidion irregulare (then considered H. annosum).  I do not have the recipe at my fingertips, but a www search should turn up the citation”. – Professor Glen R. Stanosz, Department of Forest and Wildlife Ecology, University of Wisconsin, Madison, Wisconsin.

 

·         “We still use PCNB…” - Professor Steve Woodward, Department of Plant and Soil Science, Institute of Biological and Environmental Sciences, University of Aberdeen.

 

Other azoles etc.:

 

·         “Just a thought on the question:  I suggest testing several azoles such as propiconazole (Alamo, Banner Maxx) or thiabendazole (Arbotect 20S).  Both are used as injectants for Dutch elm disease.  Also, I believe that several other azoles are used against dermatophytes (athlete's foot, tinea cruris).  I also suggest that you contact Dr. Jessie Glaeser, USFS Forest Products Lab., Madison, WI, who may provide much greater insights.  Her doctoral research was published in Mycotaxon, and she did some highly significant work on differentiating certain fungal taxa via selective media.  Good luck, for I feel sure the effective agent is out there!”  –Jay Stipes, Prof. Emeritus, Virginia Tech., Blacksburg, Virginia. 

 

·         “I would look into thiophanate methyl. Thiophanate methyl is a breakdown product of benomyl and is not effective against basidiomycetes. Ever since benomyl was phased out, rust pathologists have routinely added this to trays of young growing cereal seedlings representing resistance gene isolines, prior to inoculation with rust, in the process of identifying rust “races” ”.– Dr. Paul Zambino, US Forest Service, Region 1 Forest Health Protection, Coeur d'Alene, Idaho.

 

·         I agree.  Thiophanate would be a good candidate.  There is a good spate of azoles, and this would be an excellent research project.  – Jay Stipes.

 

·         “Here is a “fossil” formula, perhaps still working. But nowadays we use malt agar with thiabendazole (after Guillaumin, see attachment [Thiabendazolagar Rp Holdenrieder et al 19942DSCN4218.jpeg] with formula 8with very good results! only the thiabendazole need several weeks to dissolve in the lactic acid. But you can keep the stock solution forever). I think we cited this many years ago in a conference contribution: Holdenrieder, O., Schmid-Haas, P., Baumann, E. (1994): Isolation of decay fungi from increment cores: Frustrating experience from Switzerland. Proc. 8th IUFRO Int. Root and Butt Rot Conf., Uppsala and Porvoo., Aug. 8-16, 1993.: 57-581. But I cannot locate the original currently). Best regards.”  – Prof. Dr. Ottmar Holdenrieder, Forest Pathology and Dendrology Section, Dept. of Environmental Sciences, Federal Institute of Technology, Zurich. [attachment: Zhang Holdenrieder 1983 Selective Media.pdf].

 

And…:

 

·         “Instead of Benomyl (Benlate) we use to add to normal PDA or MA: chloramphenicol 0,1 g/l (Sigma-Aldrich) together with streptomicin 0,05 g/l (Sigma-Aldrich). For us it is Ok, even though I would prefer the old Benomyl, but...”. – Nicola La Porta, IASMA Research and Innovation Centre, Fondazione Edmund Mach, Sustainable Agro-ecosystems and Bioresources Dept., San Michele all’Adige, Trento, Italy.

 

____________________________

 

Ian Hood

Forest Protection Scientist

Scion (NZ Forest Research Inst. Ltd.)

49 Sala Street, Private Bag 3020, Rotorua 3046, New Zealand

DDI +64 7 343 5538
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