Alternaria brassicae produces a host-specific protein toxin from germinating spores on host leaves.

Spore suspensions of Alternaria brassicae, the causal agent of gray leaf spot in Brassica plants, were incubated on the leaves of cabbage (B. oleracea) and spore germination fluid (SGF) was collected after 48 h. A high molecular weight (HMW) fraction (>10 kDa) was separated from the SGF by ultrafiltration. In a detached leaf assay, the HMW fraction induced visible symptoms only on host leaves and the toxicity was lost by treatment with proteinase K or heat at 60 degrees C for 15 min, indicating the presence of host-specific protein toxin(s). A protein toxin in the HMW fraction was purified by several chromatography steps. The toxin induced water-soaked symptoms followed by chlorosis at concentrations of 0.5 to 1 microg/ml on host leaves, but not on nonhost leaves even at 50 microg/ml. The toxin also had infection-inducing activity when added to spore suspension of a nonpathogenic isolate of A. alternata, causing symptoms similar to the infection of A. brassicae only on host leaves. These results indicate that a new host-specific protein toxin named ABR-toxin is released from germinating spores of A. brassicae on host leaves. ABR-toxin migrated as a protein of 27.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of ABR-toxin was estimated to be approximately 7.0 and 21 N-terminal amino acid residues were sequenced.

Isolation of microorganisms and substances inhibitory to aflatoxin production.

Aflatoxins are toxic and carcinogenic secondary metabolites produced by Aspergillus flavus and A. parasiticus. The contamination of crops, feeds, and foods with aflatoxins can have serious effects on the health of humans and animals. Although many studies have been done to develop aflatoxin-control strategies, most are limited in their effectiveness. As part of an effort to develop control procedures, we have devised simple and safe methods that are useful for identifying microorganisms that effectively inhibit aflatoxin production by fungi. These include the microtitre agar plate assay using norsolorinic acid-accumulating mutant fungi, the ultraviolet light photography method using an instant film, the tip culture method, a convenient RNA extraction method for reverse transcription-polymerase chain reaction (RT-PCR) analysis, and other methods. Results of a recent trial have shown that Achromobacter xylosoxidans significantly inhibited aflatoxin production by A. parsiticus, and that the main inhibitory substance produced by the bacterium was cyclo(L-leucyl-L-prolyl). This result confirms that the methods described herein are useful for identifying microorganisms that inhibit aflatoxin production by fungi and could contribute to the development of methods to reduce aflatoxin contamination in commodities.

Botcinins E and F and Botcinolide from Botrytis cinerea and structural revision of botcinolides.

Botcinins E and F were isolated together with the known botcinolide. The structures of botcinins E and F were determined to be 3-O-deacetylbotcinin A (5) and 3-O-deacetyl-2-epi-botcinin A (6), respectively, by spectroscopic methods and chemical conversion. The structure of botcinolide was revised on the basis of spectroscopic data and chemical conversion. Botcinolide was originally reported as a nine-membered lactone (7), but the revised structure is the seco acid of botcinin E (13). Thus botcinolide is renamed botcinic acid, and homobotcinolide is renamed botcineric acid. Reinvestigation of the spectroscopic data reported for all botcinolide analogues indicates that 4-O-methylbotcinolide and 3-O-acetyl-2-epibotcinolide are the same as a methyl ester of botcinic acid (13a) and botcinin A (1), respectively, and that 2-epibotcinolide may be the same as botcinin E (5). Compounds 5, 6, and 13 showed weak antifungal activity against Magnaporthe grisea, a pathogen of rice blast disease.

Botcinins A, B, C, and D, metabolites produced by Botrytis cinerea, and their antifungal activity against Magnaporthe grisea, a pathogen of rice blast disease.

Four new metabolites, botcinins A-D, were isolated from the culture filtrate of a strain of Botrytis cinerea. Their structures were determined by spectroscopic methods, mainly NMR techniques, molecular modeling, and the modified Mosher's method. They exhibited antifungal activities against Magnaporthegrisea, a pathogen of rice blast disease. Botcinins B and C have a MIC of 12.5 microM, and botcinins A and D are not active below 100 microM.

A new inhibitor of 5'-hydroxyaverantin dehydrogenase, an enzyme involved in aflatoxin biosynthesis, from Trichoderma hamatum.

Screening for inhibitors of 5'-hydroxyaverantin dehydrogenase, an enzyme involved in aflatoxin biosynthesis, resulted in the isolation of a new metabolite (1) from Trichoderma hamatum. On the basis of spectroscopic data, 1 was determined to be 4, 6-dihydroxy-5-methoxy-6a-methylcyclohexa[de]indano[7, 6-e]cyclopenta[c]2H-pyran-1,9-dione.