Aflatoxins are potent carcinogenic and toxic substances that are produced primarily by Aspergillus flavus and Aspergillus parasiticus. We found that a bacterium remarkably inhibited production of norsolorinic acid, a precursor of aflatoxin, by A. parasiticus. This bacterium was identified as Achromobacter xylosoxidans based on its 16S ribosomal DNA sequence and was designated A. xylosoxidans NFRI-A1. A. xylosoxidans strains commonly showed similar inhibition. The inhibitory substance(s) was excreted into the medium and was stable after heat, acid, or alkaline treatment. Although the bacterium appeared to produce several inhibitory substances, we finally succeeded in purifying a major inhibitory substance from the culture medium using Diaion HP20 column chromatography, thin-layer chromatography, and high-performance liquid chromatography. The purified inhibitory substance was identified as cyclo(L-leucyl-L-prolyl) based on physicochemical methods. The 50% inhibitory concentration for aflatoxin production by A. parasiticus SYS-4 ‫؍(‬ NRRL2999) was 0.20 mg ml Aflatoxins are highly toxic, carcinogenic, and teratogenic secondary metabolites that are produced by certain strains of Aspergillus flavus and Aspergillus parasiticus (reviewed in reference 20). Recently, several strains of Aspergillus nomius, Aspergillus pseudotamarii, Aspergillus bombycis, and Aspergillus ochraceoroseus have also been reported to produce aflatoxin. These fungi are ubiquitous and grow on a variety of agricultural products under appropriate temperature and moisture conditions. Aflatoxins have been detected in numerous agricultural commodities, such as cereal grains, whole wheat, rye breads, oil seeds, cottonseed, etc. (25). The toxicity and carcinogenicity of aflatoxins have made contaminated commodities a significant health hazard all over the world. In fact, the incidence of liver cancer is high in regions with high endemic aflatoxin concentrations. Furthermore, the annual costs resulting from crop losses due to aflatoxin contamination and the costs involved in monitoring and disposal of contaminated commodities affect the agricultural economy (34).Many studies have focused on developing aflatoxin control strategies, including genetic engineering for crop resistance, biological control with competitive, nonaflatoxigenic strains of the fungus A. flavus (6, 9), and regulation of aflatoxin biosynthesis by fungicides, pesticides, inhibitory substances originating from plants, and microbial substances (15,24,27). However, most of these strategies have been shown to be limited in effectiveness. Many microorganisms have been studied to control aflatoxin production. Sweedy and Dobson (32) have reported that bacteria, yeast, molds, actinomycetes, and algae can be used to lower aflatoxin levels in foods and feeds. Ono et al. have reported that aflastatin A, which has been isolated from mycelial extracts of Streptomyces sp., effectively inhibits aflatoxin production (17, 23). Saprophytic yeasts, such as Pichia anomala, Candida krusei, and others, have...

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.

The pathway from averufin (AVR) to versiconal hemiacetal acetate (VHA) in aflatoxin biosynthesis was investigated by using cell-free enzyme systems prepared from Aspergillus parasiticus. When (1S,5S)-AVR was incubated with a cell extract of this fungus in the presence of NADPH, versicolorin A and versicolorin B (VB), as well as other aflatoxin pathway intermediates, were formed. When the same substrate was incubated with the microsome fraction and NADPH, hydroxyversicolorone (HVN) and VHA were formed. However, (1R,5R)-AVR did not serve as the substrate. In cell-free experiments performed with the cytosol fraction and NADPH, VHA, versicolorone (VONE), and versiconol acetate (VOAc) were transiently produced from HVN in the early phase, and then VB and versiconol (VOH) accumulated later. Addition of dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the same reaction mixture caused transient formation of VHA and VONE, followed by accumulation of VOAc, but neither VB nor VOH was formed. When VONE was incubated with the cytosol fraction in the presence of NADPH, VOAc and VOH were newly formed, whereas the conversion of VOAc to VOH was inhibited by dichlorvos. The purified VHA reductase, which was previously reported to catalyze the reaction from VHA to VOAc, also catalyzed conversion of HVN to VONE. Separate feeding experiments performed with A. parasiticus NIAH-26 along with HVN, VONE, and versicolorol (VOROL) demonstrated that each of these substances could serve as a precursor of aflatoxins. Remarkably, we found that VONE and VOROL had ring-opened structures. Their molecular masses were 386 and 388 Da, respectively, which were 18 Da greater than the molecular masses previously reported. These data demonstrated that two kinds of reactions are involved in the pathway from AVR to VHA in aflatoxin biosynthesis: (i) a reaction from (1S,5S)-AVR to HVN, catalyzed by the microsomal enzyme, and (ii) a new metabolic grid, catalyzed by a new cytosol monooxygenase enzyme and the previously reported VHA reductase enzyme, composed of HVN, VONE, VOAc, and VHA. A novel hydrogenation-dehydrogenation reaction between VONE and VOROL was also discovered.

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.

Both ‘Atago’ and ‘Shinkou’ are late-season japanese pear (Pyrus pyrifolia Nakai) cultivars with russet-brown fruit, and the progenies of crosses made between ‘Nijisseiki’ and unknown cultivars, but they display different growth habit, fruit size, and fruit quality. To clarify the difference in fruit development between the two cultivars, the levels of endogenous gibberellins (GA1, GA3, and GA4) and abscisic acid (ABA) in fruit were identified and quantified by gas chromatography/mass spectrometry, and a histological study of fruit was carried out. The results showed that cell number rather than cell size of mesocarp is responsible for the difference in fruit size between the two cultivars. Furthermore, analysis of endogenous bioactive gibberellins revealed that ‘Atago’ fruit has much higher levels of GA1, GA3, and GA4 than ‘Shinkou’ fruit during fruit development, particularly during the early period of fruit growth. However, ‘Shinkou’ has a much higher ABA level than ‘Atago’ during the early period of fruit development. Abscisic acid concentration remained at an extremely low level after the first production peak after anthesis in ‘Atago’ compared with ‘Shinkou’.

In the aflatoxin biosynthetic pathway, 5-oxoaverantin (OAVN) cyclase, the cytosolic enzyme, catalyzes the reaction from OAVN to (2S,5S)-averufin (AVR) (E. Sakuno, K. Yabe, and H. Nakajima, Appl. Environ. Microbiol. 69:6418-6426, 2003). Interestingly, the N-terminal 25-amino-acid sequence of OAVN cyclase completely matched an internal sequence of the versiconal (VHOH) cyclase that was deduced from its gene (vbs). The purified OAVN cyclase also catalyzed the reaction from VHOH to versicolorin B (VB). In a competition experiment using the cytosol fraction of Aspergillus parasiticus, a high concentration of VHOH inhibited the enzyme reaction from OAVN to AVR, and instead VB was newly formed. The recombinant Vbs protein, which was expressed in Pichia pastoris, showed OAVN cyclase activity, as well as VHOH cyclase activity. A mutant of A. parasiticus SYS-4 ‫؍(‬ NRRL 2999) with vbs deleted accumulated large amounts of OAVN, 5-hydroxyaverantin, averantin, AVR, and averufanin in the mycelium. These results indicated that the cyclase encoded by the vbs gene is also involved in the reaction from OAVN to AVR in aflatoxin biosynthesis. Small amounts of VHOH, VB, and aflatoxins also accumulated in the same mutant, and this accumulation may have been due to an unknown enzyme(s) not involved in aflatoxin biosynthesis. This is the first report of one enzyme catalyzing two different reactions in a pathway of secondary metabolism.Aflatoxins are toxic, carcinogenic, and mutagenic secondary metabolites mainly produced by certain strains of Aspergillus flavus and Aspergillus parasiticus. Contamination by aflatoxins in food and feed is a serious problem in many areas of the world. The biosynthetic pathway of aflatoxin has been studied extensively, and most of the steps have been clarified (reviewed in references 12, 16, 18, 19, and 29). Many enzymes and the genes encoding the enzymes have been isolated, and most of the enzyme genes were found to comprise a huge cluster over 70 kb long in the fungal genome (16,25,27,29).We recently reported that two enzymes are involved in the pathway from 5Ј-hydroxyaverantin (HAVN) to averufin (AVR); HAVN dehydrogenase catalyzes the conversion of HAVN to 5Ј-oxoaverantin (OAVN), and OAVN cyclase catalyzes the next reaction from OAVN to AVR (13). These enzymes have been purified and characterized. The identity of HAVN dehydrogenase with the gene product of adhA (3) was confirmed by tryptic digestion and matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis (13). The molecular masses of natural OAVN cyclase and denatured OAVN cyclase are 158 kDa and 79 kDa, respectively.In this study, we determined the amino acid sequence of the enzyme to find the gene encoding the OAVN cyclase. Surprisingly, the N-terminal sequence of OAVN cyclase was the same as a stretch of the versiconal (VHOH) cyclase sequence that was deduced from the reported vbs gene (15). VHOH cyclase has been purified independently by Lin and Anderson (10) and by McGuire et al. (11). VHOH cyclase catalyzes the co...

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.

The methanol extracts of 168 plant species from 68 families were evaluated for their inhibitory activity against lettuce seedling elongation. Among the plant species tested, 12 species had EC₅₀ values for radicle growth inhibition ranging from 0.01 to 5.00 mg fresh weight equivalent mL^-1. Enterolobium contortisiliquum, a traditionally used herbal medicine, exhibited the strongest inhibitory activity (estimated EC₅₀: 0.28 fresh weight equivalent mL^-1). Among the 12 species, Pachysandra terminalis, Tamarindus indica, and Albizia guachapele required investigation, because only little has been reported about their chemical constituents to date. The data in the present study would be useful in finding new lead compounds for natural herbicides.