Inhibition of Aflatoxin Production by Citrinin and Non-Enzymatic Formation of a Novel Citrinin-Kojic Acid Adduct.

Screening for microorganisms that inhibit aflatoxin production from environments showed that Penicillium citrinum inhibited aflatoxin production by Aspergillus parasiticus. The inhibitory substance in the culture medium of P. citrinum was confirmed to be citrinin (CTN). RT-PCR analyses showed that CTN did not inhibit expressions of aflatoxin biosynthetic genes (aflR, pksL1, and fas-1) of A. parasiticus, whereas feeding experiments using A. parasiticus showed that CTN inhibited the in vivo conversion of dihydrosterigmatocystin to AFB2·AFG2. These results suggest that CTN inhibits a certain post-transcriptional step in aflatoxin biosynthesis. CTN in the culture medium of A. parasiticus was found to be decreased or lost with time, suggesting that a certain metabolite produced by A. parasiticus is the cause of the CTN decrease; we then purified, characterized, and then analyzed the substance. Physico-chemical analyses confirmed that the metabolite causing a decrease in CTN fluorescence was kojic acid (KA) and the resulting product was identified as a novel substance: (1R,3S,4R)-3,4-dihydro-6,8-dihydroxy-1-(3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl)-3,4,5-trimethyl-1H-isochromene-7-carboxylic acid, which was named "CTN-KA adduct". Our examination of the metabolites' toxicities revealed that unlike CTN, the CTN-KA adduct did not inhibit aflatoxin production by A. parasiticus. These results indicate that CTN's toxicity was alleviated with KA by converting CTN to the CTN-KA adduct.

Detoxication of Citrinin with Kojic Acid by the Formation of the Citrinin-Kojic Acid Adduct, and the Enhancement of Kojic Acid Production by Citrinin via Oxidative Stress in Aspergillus parasiticus

Our previous work showed that citrinin (CTN) produced bay Penicillium citrinum inhibited the production of aflatoxin by Aspergillus parasiticus. We also reported that CTN was non-enzymatically converted to a novel CTN-KA adduct with kojic acid (KA) in aqueous condition. We herein observed that unlike CTN, the CTN-KA adduct does not show antimicrobial activity against Escherichia coli or Bacillus subtilis or any cytotoxic effect on HeLa cells, suggesting that CTN was detoxified by KA by the formation of the CTN-KA adduct. To examine the function of KA production by fungi, we isolated A. parasiticus mutants with impaired KA production. When the mutants were incubated in either liquid or agar medium supplemented with CTN, they were more susceptible to CTN than the wild KA-producing strain. The same results were obtained when we used the A. oryzae KA-producing strain RIB40 and KA-non-producing strains. When KA was added to the CTN-containing agar medium, the inhibition of growth by CTN was remarkably mitigated, suggesting that the production of KA protected the fungal growth from CTN's toxicity. We also observed that CTN enhanced the production of KA by A. parasiticus as well as A. oryzae strains. Reverse transcription-PCR showed that CTN enhanced the expression of KA biosynthetic genes (kojA, kojR, and kojT) of A. parasiticus. However, the enhancement of KA production with CTN was repressed by the addition of α-tocopherol or butylated hydroxy anisole, suggesting that KA production is enhanced by oxidative stress via the formation of reactive oxygen species caused by CTN. In contrast, α-tocopherol did not affect inhibition of AF production as well as fungal growth by CTN, suggesting that the regulation of these inhibitions with CTN might be different from that of KA production. We propose a regulation scheme of CTN for each of KA production, AF production, and fungal growth in A. parasiticus.

verA Gene is Involved in the Step to Make the Xanthone Structure of Demethylsterigmatocystin in Aflatoxin Biosynthesis.

In the biosynthesis of aflatoxin, verA, ver-1, ordB, and hypA genes of the aflatoxin gene cluster are involved in the pathway from versicolorin A (VA) to demethylsterigmatocystin (DMST). We herein isolated each disruptant of these four genes to determine their functions in more detail. Disruptants of ver-1, ordB, and hypA genes commonly accumulated VA in their mycelia. In contrast, the verA gene disruptant accumulated a novel yellow fluorescent substance (which we named HAMA) in the mycelia as well as culture medium. Feeding HAMA to the other disruptants commonly caused the production of aflatoxins B1 (AFB1) and G1 (AFG1). These results indicate that HAMA pigment is a novel aflatoxin precursor which is involved at a certain step after those of ver-1, ordB, and hypA genes between VA and DMST. HAMA was found to be an unstable substance to easily convert to DMST and sterigmatin. A liquid chromatography-mass spectrometry (LC-MS) analysis showed that the molecular mass of HAMA was 374, and HAMA gave two close major peaks in the LC chromatogram in some LC conditions. We suggest that these peaks correspond to the two conformers of HAMA; one of them would be selectively bound on the substrate binding site of VerA enzyme and then converted to DMST. VerA enzyme may work as a key enzyme in the creation of the xanthone structure of DMST from HAMA.

Effect of Cu on the fluorescence of the Cu-hyperaccumulator lichen Stereocaulon sorediiferum.

Stereocaulon sorediiferum is expected to be a Cu-hyperaccumulator lichen and has fluorescent substances. To clarify the relationship between the fluorescence (FL) of the lichen and its Cu concentration, we collected S. sorediiferum samples at Cu-contaminated and uncontaminated sites in Japan, determined the concentration of Cu, K, Mg, Al, Ca, Mn, Fe, Zn, chlorophyll a,b, and total carotenoids in them, analyzed lichen secondary metabolites and fluorescent substances extracted from them, and measured the FL of them and their extracts. We found that the FL intensity of S. sorediiferum samples is significantly negatively correlated with their Cu concentration. The application of its FL for Cu monitoring may allow a new nondestructive quantitative method for assessing Cu contamination. The spectroscopic and chromatographic analysis shows that the fluorescent substances negatively correlated with Cu concentration are not major lichen secondary metabolites (lobaric acid and atranorin) and remain after immersion in acetone. The correlation analysis and the comparison with the causal relationship between Cu concentration and the chlorophyll a/b ratio suggest that the reason for the decrease in FL intensity with increasing Cu concentration is a structural change of the fluorescent substances by accumulated Cu. These findings lead to a better understanding of the relationship between the FL of S. sorediiferum and its Cu concentration and provide new insights into fluorescent lichen substances.

Determination of iron species, including biomineralized jarosite, in the iron-hyperaccumulator moss Scopelophila ligulata by Mössbauer, X-ray diffraction, and elemental analyses.

Scopelophila ligulata is an Fe-hyperaccumulator moss growing in acidic environments, but the mechanism of Fe accumulation remains unknown. To understand the mechanism, we determined Fe species in S. ligulata samples. The moss samples were collected from four sites in Japan. The concentrations of Fe, P, S, Cl, and K in them were measured by induced coupled plasma mass spectrometry. Fe species in some of them were determined by Mössbauer spectroscopy and were confirmed by X-ray diffraction analysis. Fe species in S. ligulata samples were determined to be jarosite, ferritin, high-spin Fe(II) species, and akaganeite. To our knowledge, this is the first report on the biomineralization of jarosite in mosses. This result, combined with the fact that bacteria, a fungus, and a grass mineralize jarosite, suggests that its biomineralization is a common characteristic in a wide variety of living organisms. These findings indicate that the biomineralization of jarosite occurs not only in the region-specific species but in species adapted to a low-pH and metal-contaminated environment in different regions, provide a better understanding of the mechanism of Fe accumulation in the Fe-hyperaccumulator moss S. ligulata, and offer new insights into the biomineralization of jarosite.

Response of secondary metabolites to Cu in the Cu-hyperaccumulator lichen Stereocaulon japonicum.

Lichen secondary metabolites are known to be associated with heavy metal uptake and tolerance in lichens. Understanding the relationship between their secondary metabolites and heavy metals in them is important for clarifying the mechanisms of their heavy metal accumulation and tolerance. To determine the relationships between the concentrations of secondary metabolites and Cu in the Cu-hyperaccumulator lichen Stereocaulon japonicum and to clarify its response to Cu, we collected Cu-contaminated and uncontaminated samples of the lichen and determined relative concentrations of secondary metabolites and concentrations of Cu, K, glucose, and sugar alcohols in them. We found significant negative correlations between the relative concentrations of secondary metabolites-atranorin and stictic acid-and the concentration of Cu. These negative correlations can be interpreted in one of two ways: (a) S. japonicum itself reduced the relative concentrations of secondary metabolites in response to the increase of Cu concentration or (b) its carbon and energy metabolism was damaged by Cu stress, resulting in the reduction of the relative concentrations of secondary metabolites. The analysis of K, glucose, and sugar alcohols showed no effect of Cu on these concentrations, which means that the carbon and energy metabolism was not damaged by Cu stress. Therefore, the negative correlations can be interpreted that S. japonicum itself reduced the relative concentrations of secondary metabolites with the increase of Cu concentration. These findings provide a deeper understanding of the response of secondary metabolites to Cu in the lichen.

Importance of prumycin produced by Bacillus amyloliquefaciens SD-32 in biocontrol against cucumber powdery mildew disease.

BACKGROUND: Powdery mildew disease of cucurbits is caused mainly by Podosphaera fusca, which is one of the most important limiting factors in cucurbit production worldwide. Previously we reported that Bacillus amyloliquefaciens biocontrol strain SD-32 produces C17 bacillomycin D and [Ile 2002]surfactin, and that these metabolites play important roles in SD-32's biocontrol over cucumber gray mold disease. Our further investigation demonstrated that the culture broth and its supernatant suppressed cucumber powdery mildew disease in greenhouse experiments. However, the active principle(s) remained unknown. RESULTS: The active compound was isolated from the culture supernatant after anti-powdery mildew disease activity-guided purification and identified as prumycin. Prumycin significantly suppressed the disease, whereas bacillomycin D and [Ile 2002]surfactin did not. Prumycin did not induce the expression of plant defense genes (PR1a and VSP1), suggesting that it does not act via plant defense response. Light microscopic observations of prumycin-treated cucumber cotyledon suggested that prumycin inhibits the conidial germination of P. fusca. CONCLUSION: This study demonstrates that prumycin is a major factor in SD-32's suppression of cucumber powdery mildew disease. Our findings shed light for the first time on prumycin's role in biocontrol by Bacillus against this disease. © 2017 Society of Chemical Industry.

Relationship between metal and pigment concentrations in the Fe-hyperaccumulator moss Scopelophila ligulata.

Scopelophila ligulata is known to be a Fe-hyperaccumulator moss; however, its mechanism of accumulation and the effects of Fe on pigments remain unclear. To clarify the effects, we measured its metal and pigment concentrations. The Fe concentration in S. ligulata was 10-61 times higher than that in normal mosses, confirming that the moss is a Fe-hyperaccumulator. The black samples of S. ligulata had the highest Fe concentration (2.9 wt%) and the second in the order of decreasing Fe concentration (2.2 wt%), which explains their color and indicates that the excess amount of Fe is distributed through the plant body. Moreover, we observed that the concentration of Ca is negatively correlated with the concentrations of pigments and, conversely, that the concentration of K is positively correlated with the concentrations of pigments. This inverse relationship between Ca and K can be explained by the reduced uptake of K in S. ligulata in response to Ca stress, which is supported by the fact that the concentration of Ca is negatively correlated with that of K. These findings provide a better understanding of the relationships between metals and pigments in the Fe-hyperaccumulator moss S. ligulata.

Synthesis of photoreactive ivermectin B1a derivatives and their actions on Haemonchus and Bombyx glutamate-gated chloride channels.

Glutamate-gated chloride channels (GluCls) are inhibitory neurotransmitter receptors that are present only in invertebrates such as nematodes and insects. These channels are important targets of insecticidal, acaricidal, and anthelmintic macrolides such as avermectins, ivermectin (IVM), and milbemycins. To identify the amino acid residues that interact with IVM in GluCls, three IVM B1a derivatives with different photoreactive substitutions at C-13 were synthesized in the present study. These derivatives displayed low- or subnanomolar affinity for parasitic nematode (Haemonchus contortus) and silkworm (Bombyx mori) GluCls expressed in COS-1 cells. The derivatives also activated homomeric H. contortus GluCls expressed in Xenopus oocytes. The results indicate that synthesized photoreactive IVM B1a derivatives have superior affinity and functionality for chemically labeling the macrolide-binding site in GluCls. .

Synergistic Effects of [Ile7]Surfactin Homologues with Bacillomycin D in Suppression of Gray Mold Disease by Bacillus amyloliquefaciens Biocontrol Strain SD-32.

We previously reported that Bacillus amyloliquefaciens biocontrol strain SD-32 produces powerful antifungal lipopeptides, C17 bacillomycin D homologues. In the course of the investigation we found that the antifungal activity of the culture supernatant of this bacterium was not ascribed exclusively to bacillomycin D. We attempted to identify metabolites other than bacillomycin D to gain insight into the mechanism for the biocontrol by this bacterium. After purifying the fractions of the culture supernatant exhibiting synergistic activity with bacillomycin D, we isolated two new cyclic lipodepsipeptides, anteiso-C13 and iso-C13 [Ile(7)]surfactins, together with three known [Ile(7)]surfactins. Interestingly, [Ile(7)]surfactins showed synergistic activities with bacillomycin D to gray mold disease on cucumber leaves but not to Botrytis cinerea itself in vitro, suggesting that the synergistic effects might be on infection processes of the fungus. Actually, we observed that they did not show synergistic actions on conidial germination or mycelial growth of B. cinerea on the leaves.

Effects of Cu on the content of chlorophylls and secondary metabolites in the Cu-hyperaccumulator lichen Stereocaulon japonicum.

Understanding the relationship between Cu and Cu-hyperaccumulator lichens is important for their application in monitoring and assessing heavy metal pollution. We investigated the Cu-hyperaccumulator lichen Stereocaulon japonicum at several Cu-polluted and control sites in Japan, and found the lichen to be widely distributed. Its concentrations of Cu, chlorophylls, and secondary metabolites, chlorophyll-related indices, and absorption spectra were measured, and we observed negative effects of Cu on these concentrations and indices. For highly Cu-polluted samples (>100ppm dry weight), however, we found significant linear correlations between Cu and chlorophyll concentrations. This can be considered as the response of the photobiont in S. japonicum to Cu stress. In highly Cu-polluted samples the chlorophyll-related indices and concentration of total secondary metabolites were almost constant regardless of Cu concentration. This suggests that the increase in chlorophyll concentration with the increase in Cu concentration enhances photosynthetic productivity per unit biomass, which will allow the production of extra structure and energy for maintaining the chlorophyll-related indices under Cu stress. The relationship between the increase in chlorophyll concentration of S. japonicum and the decrease in secondary metabolite concentration of the lichen can be explained by considering the balance of carbohydrates in the lichen. We found that a spectral index A372-A394 can be a useful index of the concentrations of Cu and total secondary metabolites in S. japonicum. These findings show the adjustment of the content of chlorophylls and secondary metabolites in S. japonicum to Cu stress, and provide a better understanding of the relationship between Cu and the Cu-hyperaccumulator lichen.

Biosynthesis of integric acid isolated from the wood-decay fungus Xylaria feejeensis 2FB-PPM08M.

The biosynthesis of integric acid, a secondary metabolite of the wood-decay fungus Xylaria feejeensis strain 2FB-PPM08M, has been studied. Labeling experiments using [1-(13)C], [2-(13)C] and [1,2-(13)C2] acetate and L-methionine (methyl-(13)C) were separately performed with fungal culture. The labeling patterns of these metabolites indicated the same origin, and determined that integric acid was formed through the condensation of a sesquiterpene and a polyketide. These experiments showed that side chain of compounds would be synthesized by the polyketide pathway, while the ring carbon indicated the biosynthesis of compounds via the mevalonate pathway.

Competitive antagonism of insect GABA receptors by 4-substituted 5-(4-piperidyl)-3-isothiazolols.

γ-Aminobutyric acid (GABA) receptors are important targets of parasiticides/insecticides. Several 4-substituted analogs of the partial GABAA receptor agonist 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) were synthesized and examined for their antagonism of insect GABA receptors expressed in Drosophila S2 cells or Xenopus oocytes. Thio-4-PIOL showed weak antagonism of three insect GABA receptors. The antagonistic activity of Thio-4-PIOL was enhanced by introducing bicyclic aromatic substituents into the 4-position of the isothiazole ring. The 2-naphthyl and the 3-biphenylyl analogs displayed antagonist potencies with half maximal inhibitory concentrations in the low micromolar range. The 2-naphthyl analog induced a parallel rightward shift of the GABA concentration-response curve, suggesting competitive antagonism by these analogs. Both compounds exhibited weak insecticidal activities against houseflies. Thus, the orthosteric site of insect GABA receptors might be a potential target site of insecticides.

New series of avenanthramides in oat seed.

Avenanthramides are characteristic constituents of oat seeds. We analyzed the methanol extract of oat seeds by HPLC and detected three compounds 1, 2, and 3 eluted at retention times similar to avenanthramides. The three compounds were purified by column chromatography and HPLC. Spectroscopic analyses of 1, 2, and 3 suggested that they are amides of 4,5-dihydroxyanthranilic acid with caffeic, p-coumaric, and ferulic acids, respectively. Their identities were confirmed by comparing spectra and chromatographic behavior with compounds synthesized from 4,5-dihydroxyanthranilic acid and N-hyrdroxysuccinimide esters of hydroxycinnamic acids. LC-MS/MS analysis with multiple reaction monitoring showed that the amounts of 1, 2, and 3 were 16.5-26.9% of corresponding avenanthamides with 5-hydroxyanthranilic acid. Compounds 1, 2, and 3 showed stronger 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity than the corresponding avenanthramides with 5-hydroxyanthranilic acid, indicating the involvement of 4,5-dihydroxyanthranilic acid moiety in the scavenging of DPPH radicals.

Isolation of anteiso-C17, iso-C17, iso-C16, and iso-C15 bacillomycin D from Bacillus amyloliquefaciens SD-32 and their antifungal activities against plant pathogens.

Two new cyclic lipopeptides (3 and 4) were isolated from the culture filtrate of Bacillus amyloliquefaciens strain SD-32, together with two known metabolites, iso-C15 and iso-C16 bacillomycin D (1 and 2). Spectroscopic and chemical analyses identified the structures of the new compounds 3 and 4 as anteiso-C17 bacillomycin D, cyclic (l-Asn-d-Tyr-d-Asn-l-Pro-l-Glu-d-Ser-l-Thr-3-amino-14-methylhexadecanoic acid) and iso-C17 bacillomycin D, cyclic (l-Asn-d-Tyr-d-Asn-l-Pro-l-Glu-d-Ser-l-Thr-3-amino-15-methylhexadecanoic acid), respectively. The absolute configuration of C-3 in the ß-amino fatty acid was determined to be R on the basis of the CD spectrum of its dinitrophenyl-p-methoxyaniline derivative. The activities of compounds 1-4 were evaluated against 13 plant pathogens: the activities of anteiso- and iso-C17 bacillomycin D (3 and 4) were almost the same and stronger than those of iso-C15 and iso-C16 bacillomycin D (1 and 2); iso-C15 bacillomycin D (1) was weakest. Compounds 2-4 inhibited the growth of all fungi tested; however, Pythium aphanidermatum was not inhibited at all by any of the compounds. Furthermore, compounds 1-4 at concentrations of 80, 40, 30, and 30 µM, respectively, inhibited completely the Botrytis cinerea infection in cucumber leaf.

Effect of metal stress on photosynthetic pigments in the Cu-hyperaccumulating lichens Cladonia humilis and Stereocaulon japonicum growing in Cu-polluted sites in Japan.

To understand the ecology and physiology of metal-accumulating lichens growing in Cu-polluted sites, we investigated lichens near temple and shrine buildings with Cu roofs in Japan and found that Stereocaulon japonicum Th. Fr. and Cladonia humilis (With.) J. R. Laundon grow in Cu-polluted sites. Metal concentrations in the lichen samples collected at some of these sites were determined by inductively coupled plasma mass spectroscopy (ICP-MS). UV-vis absorption spectra of pigments extracted from the lichen samples were measured, and the pigment concentrations were estimated from the spectral data using equations from the literature. Secondary metabolites extracted from the lichen samples were analyzed by high-performance liquid chromatography (HPLC) with a photodiode array detector. We found that S. japonicum and C. humilis are Cu-hyperaccumulating lichens. Differences in pigment concentrations and their absorption spectra were observed between the Cu-polluted and control samples of the 2 lichens. However, no correlation was found between Cu and pigment concentrations. We observed a positive correlation between Al and Fe concentrations and unexpectedly found high negative correlations between Al and pigment concentrations. This suggests that Al stress reduces pigment concentrations. The concentrations of secondary metabolites in C. humilis growing in the Cu-polluted sites agreed with those in C. humilis growing in the control sites. This indicates that the metabolite concentrations are independent of Cu stress.

New 3-O-Alkyl-4a,10a-dihydrofusarubins produced by Fusarium sp. Mj-2.

Five new 3-O-alkyl-4a,10a-dihydrofusarubins (2-6) were isolated from the culture filtrate of a strain of Fusarium sp. (Mj-2), together with the known metabolite, anhydrofusarubin (1). The structures of the new metabolites were elucidated by spectroscopic analyses to be 3-O-butyl, 3-O-3'-methylbutyl, 3-O-2'-methylbutyl and 3-O-2'-phenylethyl-4a,10a-dihydrofusarubin A, and an isomer of 3-O-2'-phenylethyl-4a,10a-dihydrofusarubin A. Their antifungal and antibacterial activities were evaluated together with a 3-O-methyl derivative (7) prepared from 3-O-butyl-4a,10a-dihydrofusarubin A (2), indicating that the size of the O-substituent at C-3 in the 4a,10a-dihydrofusarubins negatively affected the metabolites' antimicrobial activity.

Effects of aliphatic aldehydes on the growth and patulin production of Penicillium expansum in apple juice.

The effects of 16 aliphatic aldehydes with 3-10 carbons on the growth and patulin production of Penicillium expansum were examined. When P. expansum spores were inoculated into apple juice broth, some alkenals, including 2-propenal, (E)-2-butenal, (E)-2-pentenal, and (E)-2-hexenal, inhibited fungal growth and patulin production. Their minimal inhibitory concentrations were 5, 50, 80, and 80 µg/mL respectively. Vital staining indicated that these alkenals killed mycelia within 4 h. Treatment of the spores with these aldehydes also resulted in rapid loss of germination ability, within 0.5-2 d. On the other hand, aliphatic aldehydes with 8-10 carbons significantly enhanced patulin production without affecting fungal growth: 300 µg/mL of octanal and 100 µg/mL of (E)-2-octenal increased the patulin concentrations in the culture broth by as much as 8.6- and 7.8-fold as compared to that of the control culture respectively. The expression of the genes involved in patulin biosynthesis in P. expansum was investigated in mycelia cultured in apple juice broth containing 300 µg/mL of octanal for 3.5, 5, and 7 d. Transcription of the msas gene, encoding 6-methylsalicylic acid synthase, which catalyzed the first step in the patulin biosynthetic pathway was remarkably high in the 3.5-d and 5-d-old cultures as compared with the control. However, octanal did not any increase the transcription of the msas in the 7-d-old culture or that of the other two genes, IDH and the peab1, in culture. Thus the enhanced patulin accumulation with supplementation with these aldehydes is attributable to the increased amount of the msas transcript.

Effect of copper stress on cup lichens Cladonia humilis and C. subconistea growing on copper-hyperaccumulating moss Scopelophila cataractae at copper-polluted sites in Japan.

We investigated lichen species in the habitats of the copper (Cu)-hyperaccumulating moss Scopelophila cataractae and found that the cup lichens Cladonia subconistea and C. humilis grow on this moss. We performed X-ray fluorescence and inductively coupled plasma mass (ICP-MS) analysis of lichen samples and measured the visible absorption spectra of the pigments extracted from the samples to assess the effect of Cu stress on the cup lichens. The chlorophyll a/b ratio and degradation of chlorophyll a to pheophytin a were calculated from the spectral data. X-ray fluorescence analysis indicated that Cu concentrations in cup lichens growing on S. cataractae were much higher than those in control samples growing on non-polluted soil. Moreover, Cu microanalysis showed that Cu concentrations in parts of podetia of C. subconistea growing on S. cataractae increased as the substrate (S. cataractae) was approached, whereas those of C. humilis growing on S. cataractae decreased as the substrate was approached. This reflects the difference in the route of Cu ions from the source to the podetia. Furthermore, ICP-MS analysis confirmed that C. subconistea growing on S. cataractae was heavily contaminated with Cu, indicating that this lichen is Cu tolerant. We found a significant difference between the visible absorption spectra of pigments extracted from the Cu-contaminated and control samples. Hence, the spectra could be used to determine whether a cup lichen is contaminated with Cu. Chlorophyll analysis showed that cup lichens growing on S. cataractae were affected by Cu stress. However, it also suggested that the areas of dead moss under cup lichens were a suitable substrate for the growth of the lichen. Moreover, it suggested that cup lichens had allolepathic effects on S. cataractae; it is likely that secondary metabolites produced by cup lichens inhibited moss growth.

Production of M-/GM-group aflatoxins catalyzed by the OrdA enzyme in aflatoxin biosynthesis.

Aspergillus parasiticus produces the minor aflatoxins M(1) (AFM(1)), M(2) (AFM(2)), GM(1) (AFGM(1)), and GM(2) (AFGM(2)), as well as the major aflatoxins B(1) (AFB(1)), B(2) (AFB(2)), G(1) (AFG(1)), and G(2) (AFG(2)). Feeding of A. parasiticus with aspertoxin (12c-hydroxyOMST) caused AFM(1) and AFGM(1), and cell-free experiments using the microsomal fraction of A. parasiticus and aspertoxin caused production of AFM(1), indicating that aspertoxin is a precursor of AFM(1) and AFGM(1). Feeding of the same fungus with O-methylsterigmatocystin (OMST) caused AFM(1) and AFGM(1) together with AFB(1) and AFG(1); feeding with dihydroOMST (DHOMST) caused AFM(2) and AFGM(2) together with AFB(2) and AFG(2). Incubation of either the microsomal fraction or OrdA enzyme-expressing yeast with OMST caused production of aspertoxin together with AFM(1) and AFB(1). These results demonstrated that the OrdA enzyme catalyzes both 12c-hydroxylation reaction from OMST to aspertoxin and the successive reaction from aspertoxin to AFM(1). In contrast, feeding of the fungus with AFB(1) did not produce any AFM(1), demonstrating that M-/GM-aflatoxins are not produced from B-/G-aflatoxins. Furthermore, AFM(1) together with AFB(1) and AFG(1) was also produced from 11-hydroxyOMST (HOMST) in feeding experiment of A. parasiticus, whereas no aflatoxins were produced when used the ordA deletion mutant. These results demonstrated that OrdA enzyme can also catalyze 12c-hydroxylation of HOMST to produce 11-hydroxyaspertoxin, which serves as a precursor for the production of AFM(1) and AFGM(1). The same pathway may work for the production of AFM(2) and AFGM(2) from DHOMST and dihydroHOMST through the formation of dihydroaspertoxin and dihydro-11-hydroxyaspertoxin, respectively.