Metabolite Formation by Fungal Pathogens of Potatoes (Solanum tuberosum L.) in the Presence of Bioprotective Agents.

The potato is a crop of global importance for the food industry. This is why effective protection against pathogens is so important. Fungi as potato pathogens are responsible for plant diseases and a significant reduction in yields, as well as for the formation of mycotoxins. This study focuses on the effect of three natural biocides, yeast Metschnikowia pulcherrima, lactic acid bacteria Lactiplantibacillus plantarum, and aqueous garlic extract, on the improvement of the physiology of planted potato tubers and the reduction in mycotoxin formation. The secondary metabolites produced by the fungal pathogens of genera Fusarium, Alternaria, Colletotrichum, Rhizoctonia, and Phoma in the presence of these biocontrol agents were compared to profiles obtained from contaminated potatoes. Analysis of liquid chromatography coupled with tandem mass spectrometry data showed the presence of 68 secondary metabolites, including the mycotoxins: alternariol, alternariol methyl ether, altertoxin-I, aurofusarin, beauvericin, diacetoxyscirpenol, enniatin B, and sterigmatocystin. The studies showed that the applied biocontrol agents had a positive effect on the physiological parameters of potatoes (including root growth, stem growth, gas exchange, and chlorophyll content index) and on the reduction in the production of mycotoxins and other secondary metabolites by Fusarium, Alternaria, and Phoma.

AaCaM is required for infection structure differentiation and secondary metabolites in pear fungal pathogen Alternaria alternata.

AIMS: Calmodulin (CaM), acts as a kind of multifunctional Ca2+ sensing protein, which is ubiquitous in fungi, is highly conserved across eukaryotes and is involved in the regulation of a range of physiological processes, including morphogenesis, reproduction and secondary metabolites biosynthesis. Our aim was to understand the characteristics and functions of AaCaM in Alternaria alternata, the causal agent of pear black spot. METHODS AND RESULTS: A 450 bp cDNA sequence of AaCaM gene of A. alternata was cloned by the PCR homology method. Sequence analysis showed that this protein encoded by AaCaM was a stable hydrophilic protein and had a high similarity to Neurospora crassa (CAA50271.1) and other fungi. RT-qPCR analysis determined that AaCaM was differentially upregulated during infection structural differentiation of A. alternata both on hydrophobic and pear wax extract-coated surface, with a 3.37-fold upregulation during the hydrophobic induced appressorium formation period (6 h) and a 1.46-fold upregulation during the infection hyphae formation period (8 h) following pear wax induction. Pharmaceutical analysis showed that the CaM-specific inhibitor, trifluoperazine (TFP), inhibited spore germination and appressorium formation, and affected toxins and melanin biosynthesis in A. alternata. CONCLUSIONS: AaCaM plays an important role in regulating infection structure differentiation and secondary metabolism of A. alternata. SIGNIFICANCE AND IMPACT OF STUDY: Our study provides a theoretical basis for further in-depth investigation of the specific role of AaCaM in the calcium signalling pathway underlying hydrophobic and pear wax-induced infection structure differentiation and pathogenicity of A. alternata.

Isolation and characterization of endophytic fungi having plant growth promotion traits that biosynthesizes bacosides and withanolides under in vitro conditions.

Endophytes are regarded with immense potentials in terms of plant growth promoting (PGP) elicitors and mimicking secondary metabolites of medicinal importance. Here in the present study, we explored Bacopa monnieri plants to isolate, identify fungal endophytes with PGP elicitation potentials, and investigate secretion of secondary metabolites such as bacoside and withanolide content under in vitro conditions. Three fungal endophytes isolated (out of 40 saponin producing isolates) from leaves of B. monnieri were examined for in vitro biosynthesis of bacosides. On morphological, biochemical, and molecular identification (ITS gene sequencing), the isolated strains SUBL33, SUBL51, and SUBL206 were identified as Nigrospora oryzae (MH071153), Alternaria alternata (MH071155), and Aspergillus terreus (MH071154) respectively. Among these strains, SUBL33 produced highest quantity of Bacoside A3 (4093 µg mL-1), Jujubogenin isomer of Bacopasaponin C (65,339 µg mL-1), and Bacopasaponin C (1325 µg mL-1) while Bacopaside II (13,030 µg mL-1) was produced by SUBL51 maximally. Moreover, these aforementioned strains also produced detectable concentration of withanolides-Withaferrin A, Withanolide A (480 µg mL-1), and Withanolide B (1024 µg mL-1) respectively. However, Withanolide A was not detected in the secondary metabolites of strain SUBL51. To best of our knowledge, the present study is first reports of Nigrospora oryzae as an endophyte in B. monnieri with potentials of biosynthesis of economically important phytomolecules under in vitro conditions.

Petroleum Hydrocarbon Bioremediation Using Native Fungal Isolates and Consortia.

Crude oil spills as a result of natural disasters or extraction and transportation operations are common nowadays. Oil spills have adverse effects on both aquatic and terrestrial ecosystems and pose a threat to human health. This study have been concerned with studying the capability of six fungal species (Curvularia brachyspora, Penicillium chrysogenum, Scopulariopsis brevicaulis, Cladosporium sphaerospermum, Alternaria alternata, and Stemphylium botryosum) and three fungal consortia (FC), FC1 (P. chrysogenum and C. brachyspora), FC2 (S. brevicaulis and S. botryosum), and FC3 (S. brevicaulis, S. botryosum, and C. sphaerospermum), to remediate petroleum hydrocarbons (PHs). Qualitative and quantitative changes in polyaromatic hydrocarbons (PAHs) and saturated hydrocarbons (SH) mixtures and the patterns of PHs degradation have been examined using HPLC and GC. Studying the GC chromatogram of C. sphaerospermum revealed severe degradation of SHs exhibited by this species, and the normal-paraffin and isoparaffin degradation percentage have been valued 97.19% and 98.88%, respectively. A. alternata has shown the highest significant (at P ˂ 0.05) PAH degradation percent reaching 72.07%; followed by P. chrysogenum, 59.51%. HPLC data have revealed that high-molecular-weight PAH percent/total PAHs decreased significantly from 98.94% in control samples to 68.78% in samples treated with A. alternata. FC1 and FC2 consortia have exhibited the highest significant PH deterioration abilities than did the individual isolates, indicating that these fungal consortia exhibited positive synergistic effects. The study supports the critical idea of the potential PAH and SH biodegradation as a more ecologically acceptable alternative to their chemical degradation.

Levels of Alternaria Toxins in Selected Food Commodities Including Green Coffee.

Alternaria toxins are emerging mycotoxins, candidates for regulation by European Authorities. Therefore, highly sensitive, confirmatory, and reliable analytical methodologies are required for their monitoring in food. In that context, an isotope dilution LC-MS/MS method was developed for the analysis of five Alternaria toxins (Altenuene, Alternariol, Alternariol monomethylether, Tentoxin, and Tenuazonic Acid) in a broad range of commodities including cereals and cereal-based products, tomato-based products, tree nuts, vegetable oils, dried fruits, cocoa, green coffee, spices, herbs, and tea. Validation data collected in two different laboratories demonstrated the robustness of the method. Underestimation of Tenuazonic Acid level in dry samples such as cereals was reported when inappropriate extraction solvent mixtures were used as currently done in several published methodologies. An investigation survey performed on 216 food items evidenced large variations of Alternaria toxins levels, in line with data reported in the last EFSA safety assessment. The analysis of 78 green coffee samples collected from 21 producing countries demonstrated that coffee is a negligible source of exposure to Alternaria toxins. Its wide scope of application, adequate sample throughput, and high sensitivity make this method fit for purpose for the regular monitoring of Alternaria toxins in foods.

Two Phytotoxins Isolated from the Pathogenic Fungus of the Invasive Weed Xanthium italicum.

Alternariol and altenuisol were isolated as the major phytotoxins produced by an Alternaria sp. pathogenic fungus of the invasive weed Xanthium italicum. Altenuisol exhibited stronger phytotoxic effect compared with alternariol. At 10 µg/mL, alternariol and altenuisol promoted root growth of the monocot plant Pennisetum alopecuroides by 11.1 % and 75.2 %, respectively, however, inhibitory activity was triggered by the increase of concentration, with root elongation being suppressed by 35.5 % and 52.0 % with alternariol and altenuisol at 1000 µg/mL, respectively. Alternariol slightly inhibited root length of the dicot plant Medicago sativa at 10-1000 µg/mL, whereas altenuisol stimulated root growth by 51.0 % at 10 µg/mL and inhibited root length by 43.4 % at 200 µg/mL. Alternariol and altenuisol did not exert strong regulatory activity on another dicot plant, Amaranthus retroflexus, when tested concentration was low, however, when the concentration reached 1000 µg/mL, they reduced root length by 68.1 % and 51.0 %, respectively. Alternariol and altenuisol exerted similar effect on shoot growth of three tested plants but to a lesser extent. It is noteworthy to mention that this is the first report on the phytotoxicity of altenuisol.

Metabolomics Reveals the Response of the Phenylpropanoid Biosynthesis Pathway to Starvation Treatment in the Grape Endophyte Alternaria sp. MG1.

Phenylpropanoid (PPPN) compounds are widely used in agriculture, medical, food, and cosmetic industries because of their multiple bioactivities. Alternaria sp. MG1, an endophytic fungus isolated from grape, is a new natural source of PPPNs. However, the PPPN biosynthesis pathway in MG1 tends to be suppressed under normal growth conditions. Starvation has been reported to stimulate the PPPN pathway in plants, but this phenomenon has not been well studied in endophytic fungi. Here, metabolomics analysis was used to examine the profile of PPPN compounds, and quantitative reverse transcription-polymerase chain reaction was used to detect the expression of key genes in the PPPN biosynthesis pathway under starvation conditions. Starvation treatment significantly increased the accumulation of shikimate and PPPN compounds and upregulated the expression of key genes in their biosynthesis pathways. In addition to previously reported PPPNs, sinapate, 4-hydroxystyrene, piceatannol, and taxifolin were also detected under starvation treatment. These findings suggest that starvation treatment provides an effective way to optimize the production of PPPN compounds and may permit the investigation of compounds that are undetectable under normal conditions. Moreover, the diversity of its PPPNs makes strain MG1 a rich repository of valuable compounds and an extensive genetic resource for future studies.

Effects of ultraviolet-c treatment on growth and mycotoxin production by Alternaria strains isolated from tomato fruits.

Large amounts of tomato fruits and derived products are produced in China and may be contaminated by Alternaria mycotoxins, which may have the potential risks for human health. There is thus an increasing interest in reducing the mycotoxins. In the present study, 26 Alternaria strains isolated from tomato black rots were identified according to morphological and molecular grounds, and their mycotoxigenic abilities for alternariol (AOH), alternariol monomethyl-ether (AME) and tenuazonic acid (TeA) were also investigated. The results showed that A. alternate was the predominant species with incidence values of 65.4% (17/26), followed by A. brassicae (7/26) and A. tenuissima (2/26). A. alternate isolates showed the highest capacity for AOH, AME and TeA production among the studied isolates either in vitro or in vivo, suggested that A. alternata may be the most important mycotoxin-producing species in tomato fruits. Thus, UV-C irradiation was used to reduce the mycotoxin produced by A. alternata in our study. The results showed that low dose of UV-C irradiation (0.25 kJ/m2) could effectively inhibit mycotoxins production and penetration in tomatoes. Upon treatment with UV-C, there was 79.6, 76.4 and 51.4% of reduction in AOH, AME and TeA penetration when compared to untreated fruits. This may be associated with the enhanced phenolics by UV-C irradiation. In fact, the induced phenolics were including p-coumaric, ferulic and pyrocatechuic acids, of which p-coumaric acid (1.0 mM) displayed the highest reduction of TeA with 60.2%, whereas ferulic acid (1.0 mM) showed strong inhibitory effects on the AOH and AME production by 59.4 and 79.1%, respectively. Therefore, the application of UV-C irradiation seems to be a promising method for reducing the potential risk of Alternaria mycotoxins in fruits and also for enhancing phenolics of processing products.

Purification, crystallization and anticancer activity evaluation of the compound alternariol methyl ether from endophytic fungi Alternaria alternata.

AIMS: Medicinal plant-associated endophytic fungi are important sources of precious bioactive compounds, contributing more than 80% of the natural drugs for various ailments. The present study was aimed at evaluating the anticancer activity of the crystallized compound alternariol methyl ether (AME) against hepatocellular carcinoma (HCC) both in vitro and in vivo from an endophytic fungus residing in the medicinal plant Vitex negundo. METHODS AND RESULTS: The secondary metabolites from the endophytic fungus Alternaria alternata MGTMMP031 were isolated. Purification and characterization of the compound was performed and the potential compound was identified as AME. The crystal structure of AME was unambiguously confirmed by X-ray analysis. AME has been checked for its antibacterial and anticancer properties which showed its effectiveness against various bacteria and demonstrated marked anti-proliferative activity against the human HCC cells (HUH-7) both in vitro and in vivo. Mode of actions included cell cycle arrest, reducing the level of markers enzymes of liver cancer and preventing tumour growth. CONCLUSIONS: Alternariol methyl ether acts as a potential therapeutic target against HCC. The compound was isolated and the crystal structure was obtained for the first time from the endophytic fungus A. alternata MGTMMP031. In the present study, the crystallized structure of AME was obtained by slow evaporation technique. It can be concluded that AME acts as a potential therapeutic target against HCC. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic fungi residing in the medicinal plants have strong biological significance and bioactive compounds from these fungi provide better therapeutic targets against diseases.

Isolation of taxol producing endophytic fungus Alternaria brassicicola from non-Taxus medicinal plant Terminalia arjuna.

In the present study, an endophytic fungal strain was isolated from its non-Taxus host plant Terminalia arjuna and identified as Alternaria brassicicola based on its morphological characteristics and internal transcribed spacer sequence analysis. This fungus was grown in potato dextrose broth and analyzed for the presence of taxol by using chromatographic and spectrometric techniques. The ethyl acetate extract of A.brassicicola was subjected to column chromatography. Among the different fractions, the fraction 7 showed positive to taxol, which was further confirmed by UV absorption, HPLC, FTIR spectra and LC-ESI-MS by comparing with the authentic taxol (Paclitaxel). The peaks of fraction 7 obtained by UV spectroscopy, FTIR and HPLC analysis were quite similar to that of standard taxol confirming the presence of taxol. A parent ion peak of m/z 854.95 was observed in the LC-ESI-MS spectrum which was similar to paclitaxel with reported m/z of 854 [M+H]+ ion. A. brassicicola produced about 140.8 µg/l taxol as quantified through HPLC. Present study results suggest that the endophytic fungus A.brassicicola serves as a potential source for the production of taxol isolated from non-Taxus plant.

Secondary metabolite profiles of small-spored Alternaria support the new phylogenetic organization of the genus.

The group of the small-spored Alternaria species is particularly relevant in foods due to its high frequency and wide distribution in different crops. These species are responsible for the accumulation of mycotoxins and bioactive secondary metabolites in food. The taxonomy of the genus has been recently revised with particular attention on them; several morphospecies within this group cannot be segregated by phylogenetic methods, and the most recent classifications proposed to elevate several phylogenetic species-groups to the taxonomic status of section. The purpose of the present study was to compare the new taxonomic revisions in Alternaria with secondary metabolite profiles with special focus on sections Alternaria and Infectoriae and food safety. A total of 360 small-spored Alternaria isolates from Argentinean food crops (tomato fruit, pepper fruit, blueberry, apple, wheat grain, walnut, pear, and plum) was morphologically identified to species-group according to Simmons (2007), and their secondary metabolite profile was determined. The isolates belonged to A. infectoria sp.-grp. (19), A. tenuissima sp.-grp. (262), A. arborescens sp.-grp. (40), and A. alternata sp.-grp. (7); 32 isolates, presenting characteristics overlapping between the last three groups, were classified as Alternaria sp. A high chemical diversity was observed; 78 different metabolites were detected, 31 of them of known chemical structure. The isolates from A. infectoria sp.-grp. (=Alternaria section Infectoriae) presented a specific secondary metabolite profile, different from the other species-groups. Infectopyrones, novae-zelandins and phomapyrones were the most frequent metabolites produced by section Infectoriae. Altertoxin-I and alterperylenol were the only compounds that these isolates produced in common with members of section Alternaria. None of the well-known Alternaria toxins, considered relevant in foods, namely alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), tentoxin (TEN) or altenuene (ALT), were produced by isolates of this section. On the other hand, strains from section Alternaria (A. tenuissima, A. arborescens, and A. alternata sp.-grps.) shared a common metabolite profile, indistinguishable from each other. AOH, AME, ALT, TEN, and TeA were the most frequently mycotoxins produced, together with pyrenochaetic acid A and altechromone A. Alternaria section Alternaria represents a substantial risk in food, since their members in all types of crops are able to produce the toxic metabolites.

Characterization of Phenolic Compounds from Early and Late Ripening Sweet Cherries and Their Antioxidant and Antifungal Activities.

Early and late ripening sweet cherries were characterized for phenolic acids, and also their antioxidant capacity and potential antifungal effects were investigated. Free, conjugated, and bound phenolics were identified and quantified using ultra performance liquid chromatography-tandem mass spectrometry. Our results indicated that the early ripening cultivars contained higher free phenolic acids, which was positively related to remarkable antioxidant properties and the inhibition effects on Alternaria alternata and tenuazonic acid (TeA) accumulation. However, conjugated phenolics of the late ripening cultivars, mainly including caffeic, 2,3,4-trihydroxybenzoic, p-coumaric, and pyrocatechuic acids, achieved the highest antifungal effects and almost completely inhibited the A. alternata and TeA production. 2,2-Diphenyl-1-picrylhydrazyl testing and ferric ion reducing antioxidant power assay showed strong positive correlation with total phenolics and specific phenolics such as free epicatechin and conjugated 2,3,4-trihydroxybenzoic acids and also with antifungal activity. Results from this study provide further insights into the health-promoting phenolic compounds in sweet cherries.

Two New Metabolites from the Endophytic Fungus Alternaria sp. A744 Derived from Morinda officinalis.

Two new compounds isobenzofuranone A (1) and indandione B (2), together with eleven known compounds (3-13) were isolated from liquid cultures of an endophytic fungus Alternaria sp., which was obtained from the medicinal plant Morinda officinalis. Among them, the indandione (2) showed a rarely occurring indanone skeleton in natural products. Their structures were elucidated mainly on the basis of extensive spectroscopic data analysis. All of the compounds were evaluated with cytotoxic and α-glucosidase inhibitory activity assays. Compounds 11 and 12 showed significant inhibitory activities against four tumor cell lines; MCF-7, HepG-2, NCI-H460 and SF-268, with IC50 values in the range of 1.91-9.67 µM, and compounds 4, 5, 9, 10, 12 and 13 showed excellent inhibitory activities against α-glucosidase with IC50 values in the range of 12.05-166.13 µM.

A new Secondary metabolites of the crinoid (Comanthina schlegeli) associated fungus Alternaria brassicae 93.

Fungus Alternaria brassicae 93 isolated from crinoid (Comanthina schlegeli), which was collected from the South China Sea. Six compounds were isolated from A. brassicae 93, including one new compound (1), along with five known compounds, ochratoxin A methyl ester (2), cis-4-hydroxym-ellein (3), (R)-7-hydroxymellein (4), trans-2-anhydromevalonic (5) and protocatechuic acid (6). Their structures were determined by spectroscopic methods and comparison with reported data. Cytotoxicity against two human cancer cell lines and antibacterial activity against twelve aquatic bacteria of compound 1 were also tested.

Secondary metabolites from a strain of Alternaria tenuissima Isolated from Northern Manitoba soil.

Deoxyphomalone (1), dimethyl 4-methyl-2,6-pyridinedicarboxylate (2), stemphyperylenol (3), and N-methyl-2-pyrrolidone (4) were isolated from the fermentation broth of a strain of the fungus, Alternaria tenuissima. This fungus was isolated from the soil underlying the lichen, Peltigera didactyla, which had been collected from Wapusk National Park in Northern Manitoba. The structures of the compounds were determined by comprehensive analysis of their spectroscopic data including FT-IR, 1D and 2D NMR spectroscopy and mass spectrometry; and their bioactivities were tested against E.coli cells. The taxonomic identity of the fungus was confirmed by ITS sequencing of its ribosomal DNA.

FTIR spectroscopic evaluation of changes in the cellular biochemical composition of the phytopathogenic fungus Alternaria alternata induced by extracts of some Greek medicinal and aromatic plants.

In this study, the biological activity of aquatic extracts of selected Greek medicinal and aromatic plants to the phytopathogenic fungus Alternaria alternata was investigated. Lamiaceae species (Hyssopus officinalis L., Melissa officinalis L., Origanum dictamnus L., Origanum vulgare L. and Salvia officinalis L.) were found to enhance significantly the mycelium growth whereas Crocus sativus appears to inhibit it slightly. M. officinalis and S. officinalis caused the highest stimulation in mycelium growth (+97%) and conidia production (+65%) respectively. In order to further investigate the bioactivity of plant extracts to A. alternata, we employed Fourier Transform Infrared Spectroscopy (FTIR). Differences of original spectra were assigned mainly to amides of proteins. The second derivative transformation of spectra revealed changes in spectral regions corresponding to absorptions of the major cellular constituents such as cell membrane and proteins. Principal component analysis of the second derivative transformed spectra confirmed that fatty acids of the cell membranes, amides of proteins and polysaccharides of the cell wall had the major contribution to data variation. FTIR band area ratios were found to correlate with fungal mycelium growth.

Biosynthesis and safety evaluation of ZnO nanoparticles.

The secrets gleaned from nature have led to the development of biomimetic approaches for the growth of advanced nanomaterials. Biological methods for nanoparticle synthesis using microorganisms, enzymes, and plants or plant extracts have been suggested as possible ecofriendly alternatives to chemical and physical methods. Here, we report extracellular mycosynthesis of ZnO-NPs by Alternaria alternata (Fr.) Keissl (1912). On treating zinc sulfate solution with fungal culture filtrate, rapid reduction of ZnSO4 was observed leading to the formation of highly stable ZnO-NPs in the solution and up-to-date literature survey showed this was the first report of biosynthesis of ZnO-NPs using this fungus. The particles thereby obtained were characterized by different analytical techniques. EDX-spectrum revealed the presence of zinc and oxygen in the nanoparticles. FTIR spectroscopy confirmed the presence of a protein shell outside the nanoparticles which in turn also support their stabilization. DLS and TEM analysis of the ZnO-NPs indicated that they ranged in size from 45 to 150 nm with average size of 75 ± 5 nm. But potential negative impacts of nanomaterials are sometimes overlooked during the discovery phase of research. Therefore, in the present study, bio-safety of mycosynthesized ZnO-NPs were evaluated by using cytotoxicity and genotoxicity assays in human lymphocyte cells, in vitro. Cytotoxicity studied as function of membrane integrity and mitochondrial dehydrogenase activity revealed significant (P < 0.05) toxicity at treatment concentration of 500 µg/ml and above. Additionally, DNA damaging potential was also studied using comet assay. The results revealed significant genotoxicity at the highest concentration (1,000 µg/ml).

Molecular characterization and detection of mutations associated with resistance to succinate dehydrogenase-inhibiting fungicides in Alternaria solani.

Early blight, caused by Alternaria solani, is an economically important foliar disease of potato in several production areas of the United States. Few potato cultivars possess resistance to early blight; therefore, the application of fungicides is the primary means of achieving disease control. Previous work in our laboratory reported resistance to the succinate dehydrogenase-inhibiting (SDHI) fungicide boscalid in this plant pathogen with a concomitant loss of disease control. Two phenotypes were detected, one in which A. solani isolates were moderately resistant to boscalid, the other in which isolates were highly resistant to the fungicide. Resistance in other fungal plant pathogens to SDHI fungicides is known to occur due to amino acid exchanges in the soluble subunit succinate dehydrogenase B (SdhB), C (SdhC), and D (SdhD) proteins. In this study, the AsSdhB, AsSdhC, and AsSdhD genes were analyzed and compared in sensitive (50% effective concentration [EC50] < 5 µg ml(-1)), moderately resistant (EC50 = 5.1 to 20 µg ml(-1)), highly resistant (EC50 = 20.1 to 100 µg ml(-1)), and very highly resistant (EC50 > 100 µg ml(-1)) A. solani isolates. In total, five mutations were detected, two in each of the AsSdhB and AsSdhD genes and one in the AsSdhC gene. The sequencing of AsSdhB elucidated point mutations cytosine (C) to thymine (T) at nucleotide 990 and adenine (A) to guanine (G) at nucleotide 991, leading to an exchange from histidine to tyrosine (H278Y) or arginine (H278R), respectively, at codon 278. The H278R exchange was detected in 4 of 10 A. solani isolates moderately resistant to boscalid, exhibiting EC50 values of 6 to 8 µg ml(-1). Further genetic analysis also confirmed this mutation in isolates with high and very high EC50 values for boscalid of 28 to 500 µg ml(-1). Subsequent sequencing of AsSdhC and AsSdhD genes confirmed the presence of additional mutations from A to G at nucleotide position 490 in AsSdhC and at nucleotide position 398 in the AsSdhD, conferring H134R and H133R exchanges in AsSdhC and AsSdhD, respectively. The H134R exchange in AsSdhC was observed in A. solani isolates with sensitive, moderate, highly resistant, and very highly resistant boscalid phenotypes, and the AsSdhD H133R exchange was observed in isolates with both moderate and very high EC50 value boscalid phenotypes. Detection and differentiation of point mutations in AsSdhB resulting in H278R and H278Y exchanges in the AsSdhB subunit were facilitated by the development of a mismatch amplification mutation assay. Detection of these two mutations in boscalid-resistant isolates, in addition to mutations in AsSdhC and AsSdhD resulting in an H134R and H133R exchange, respectively, was achieved by the development of a multiplex polymerase chain reaction to detect and differentiate the sensitive and resistant isolates based on the single-nucleotide polymorphisms present in all three genes. A single A. solani isolate with resistance to boscalid did not contain any of the above-mentioned exchanges but did contain a substitution of aspartate to glutamic acid at amino acid position 123 (D123E) in the AsSdhD subunit. Among A. solani isolates possessing resistance to boscalid, point mutations in AsSdhB were more frequently detected than mutations in genes coding for any other subunit.

Microbial transformations of isophorone by Alternaria alternata and Neurospora crassa.

Isophorone (3,5,5-trimethyl-2-cyclohexen-1-one), a monoterpene, and the structurally related 1,8-cineole and camphor, have demonstrated a protective effect against cancer, biological activity against a variety of microorganisms, and anti-oxidant properties. The derivatization of isophorone is, therefore, an important field of xenobiochemistry, pharmacology and toxicology. The aim of this study was to obtain derivatives of isophorone through microbial biotransformation and evaluate the biotransformation metabolites as potential antimicrobial agents. Incubation of isophorone with the fungi Alternaria alternata and Neurospora crassa afforded 4a-hydroxy- and 7-hydroxy-isophorone as transformation metabolites. The antimicrobial activities of isophorone and the metabolites were evaluated in vitro both by using agar dilution and microdilution methods. However, no significant antibacterial activity was observed when compared with those of standard substances.

Endophytic fungi from Miquelia dentata Bedd., produce the anti-cancer alkaloid, camptothecine.

Camptothecine (Campothecin, CPT), a quinoline alkaloid, is a potent inhibitor of eukaryotic topoisomerase I. Several semi-synthetic derivatives of CPT are in clinical use against ovarian, small lung and refractory ovarian cancers. While CPT is produced by several plant species belonging to the Asterid clade, in recent years, efforts have been made to isolate endophytic fungi from some of these plants as possible alternative sources of CPT. In this study we report the isolation of three endophytic fungi from fruit and seed regions of Miquelia dentata (Icacinaceae), that produce CPT, 9-methoxy CPT (9-MeO-CPT) and 10-hydroxy CPT (10-OH-CPT). All the three fungi identified as, Fomitopsis sp. P. Karst (MTCC 10177), Alternaria alternata (Fr.) Keissl (MTCC 5477) and Phomposis sp. (Sacc.) produced CPT, 9-MeO-CPT and 10-OH-CPT in mycelial mats in shake flasks containing potato dextrose broth. Methanolic and ethyl acetate extracts of these fungal species were cytotoxic to colon and breast cancer cell lines. We discuss these results in the context of the recent interest in endophytic fungi as possible alternative sources of plant secondary metabolites.