Amplification of Polyketide Synthase gene fragments in ochratoxigenic and nonochratoxigenic black aspergilli in grapevine

Members of the Aspergillus section Nigri, also known as black aspergilli produce Ochratoxin A (OTA), a contaminant of wine. Despite potentially severe health effects and economic losses caused by OTA in wine, almost nothing is known about the genetics that lies behind its biosynthesis in black aspergilli in grapevine. In this work, degenerate primer sets were used to amplify 49 Polyketide Synthase (PKS) gene fragments in reference strains of A. carbonarius, A. niger and A. tubingensis. Deduced amino acid sequences were then compared with those of aolc35-12 and aoks1, two PKS genes involved in OTA biosynthesis in A. westerdijkiae. A putative homologue of aolc35-12 was found in A. carbonarius (63% amino acid identity), the main OTA producer on grapes and in an ochratoxigenic A. niger strain (58%). In A. niger this fragment corresponded to an15g07920, a PKS already annotated in the sequenced A. niger CBS 513.88 genome as putatively involved in OTA biosynthesis. No aolc35-12 candidates were found in atoxigenic A. tubingensis isolates and no putative homologues of aoks1 were found in any of the screened strains. A screening of A. niger field isolates using specific primers for an15g07920 indicated that the absence of this gene is apparently related to a failure to produce OTA. The present work gives a first insight into the genetics of OTA biosynthesis in black aspergilli in grapevine and represents a starting point for further investigation of the OTA biosynthesis pathway and the development of molecular methods to detect the producers in vineyards

Risk assessment of the occurrence of black aspergilli on grapes grown in an alpine region under a climate change scenario

Members of the Aspergillus section Nigri, also known as black aspergilli, are responsible for the ochratoxin A (OTA) and fumonisins contamination of wine. The presence of black aspergilli in vineyards has been investigated extensively in warm climates, in which the incidence of these aspergilli on grapes and levels of OTA contamination of wines are commonly high. However, a detailed description of black aspergilli populations is needed in wine-producing cool regions to establish a baseline in view of the strengthening of temperature increase and in case of summer rainfall decrease. With this in mind, we isolated and characterized black aspergilli from grapes grown in an alpine region in Northern Italy (Trentino) during a 3-year sampling. Black aspergilli were isolated from around 10% of the grape berries and most of the isolates were classified as A. niger, A. tubingensis and A. uvarum. A. carbonarius was isolated only once. OTA production was detected only in the A. carbonarius isolate and in one A. niger. Most of A. niger isolates were able to produce fumonisins. The presence of mycotoxins biosynthesis genes was assessed in A. niger isolates. An15g07920, a polyketide synthase (PKS) gene involved in OTA biosynthesis, was detected by PCR only in the single ochratoxigenic isolate. This strong correlation was not observed for anfum1, anfum6 and anfum8, three genes included in the A. niger fumonisin biosynthesis gene cluster, which were detected in different A. niger isolates not able to produce fumonisins. Projections of mean daily temperatures and monthly rainfall indicate that the presence of black aspergilli on grapes grown in vineyards of these valleys will probably increase in the futur

Gnomoniopsis castanea is the main agent of chestnut nut rot in Switzerland

Nuts of sweet chestnut have been an important food source for the alpine population in Switzerland since the Middle Ages and are still valued today for the preparation of traditional food commodities. Nut quality is reduced by insect damage and by various pathogenic fungi. In the last few years, producers and consumers perceived an increase of brown nut rot; while the nut rot agent Gnomoniopsis castanea was reported locally in southern Switzerland, its presence has not been investigated over large areas until now. This study assessed the incidence of brown nut rot and identified the causal agent present in Switzerland. Fully ripened nuts were collected from the main sweet chestnut growing areas of Switzerland. A filamentous fungus morphologically identified as G. castanea was isolated from 10 to 91% of the sampled nuts, despite only 3 to 21% of the sampled nuts showing brown rot symptoms. This fungus was isolated from symptomatic chestnuts as well as from apparently healthy chestnuts. Our results suggest a possible endophytic lifestyle in ripened nuts as well as in branches, leaves and unripe nuts as previously found. Species identity of 45 isolates was confirmed by EF-1alpha, beta-tubulin and ITS sequencing. Concatenation of β-tubulin and calmodulin sequences showed that several haplotypes were present at each sampling locality. No other nut rot pathogens could be isolated in this study, suggesting that G. castanea is the main causal agent of nut rot in Switzerland. The presence of this species is reported for the first time in a site in northern Switzerland. Further studies are needed to assess the influence of meteorological conditions and chestnut varieties on the incidence of G. castanea in order to provide prevention strategies for chestnut growers

Draft Genome Sequences of Clostridium tyrobutyricum Strains FAM22552 and FAM22553, Isolated from Swiss Semihard Red-Smear Cheese.

Clostridium tyrobutyricum is the main microorganism responsible for late blowing defect in cheeses. Here, we present the draft genome sequences of two C. tyrobutyricum strains isolated from a Swiss semihard red-smear cheese. The two draft genomes comprise 3.05 and 3.08 Mbp and contain 3,030 and 3,089 putative coding sequences, respectively

Genomic approach to studying nutritional requirements of Clostridium tyrobutyricum and other Clostridia causing late blowing defects.

Clostridium tyrobutyricum is the main microorganism responsible for the late blowing defect in hard and semi-hard cheeses, causing considerable economic losses to the cheese industry. Deeper knowledge of the metabolic requirements of this microorganism can lead to the development of more effective control approaches. In this work, the amino acids and B vitamins essential for sustaining the growth of C. tyrobutyricum were investigated using a genomic approach. As the first step, the genomes of four C. tyrobutyricum strains were analyzed for the presence of genes putatively involved in the biosynthesis of amino acids and B vitamins. Metabolic pathways could be reconstructed for all amino acids and B vitamins with the exception of biotin (vitamin B7) and folate (vitamin B9). The biotin pathway was missing the enzyme amino-7-oxononanoate synthase that catalyzes the condensation of pimeloyl-ACP and l-alanine to 8-amino-7-oxononanoate. In the folate pathway, the missing genes were those coding for para-aminobenzoate synthase and aminodeoxychorismate lyase enzymes. These enzymes are responsible for the conversion of chorismate into para-aminobenzoate (PABA). Two C. tyrobutyircum strains whose genome was analyzed in silico as well as other 10 strains isolated from cheese were tested in liquid media to confirm these observations. 11 strains showed growth in a defined liquid medium containing biotin and PABA after 6-8 days of incubation. No strain showed growth when only one or none of these compounds were added, confirming the observations obtained in silico. Furthermore, the genome analysis was extended to genomes of single strains of other Clostridium species potentially causing late blowing, namely Clostridium beijerinckii, Clostridium sporogenes and Clostridium butyricum. Only the biotin biosynthesis pathway was incomplete for C. butyricum and C. beijerincki. In contrast, C. sporogenes showed missing enzymes in biosynthesis pathways of several amino acids as well as biotin, folate, and cobalamin (vitamin B12). These observations agree with the results of growth experiments of these species in liquid media reported in the literature. The results of this study suggest that biotin and folate are potential targets for reducing late blowing in cheese and highlight the usefulness of genomic analysis for identifying essential nutrients in bacteria

Assessment of the ochratoxin A production ability of Aspergillus tubingensis

Aspergillus tubingensis is a black Aspergillus frequently isolated from different agricultural products, including grapes. Conflicting results have been published in recent years about its ability to produce ochratoxin A (OTA), a potent nephrotoxic and carcinogenic mycotoxin. This study re-examined six A. tubingensis strains deposited in international culture collections for OTA production. OTA could not be detected in any A. tubingensis extract using HPLC coupled with a fluorescence detector (FLD), whereas it was easily detected in ochratoxigenic A. niger extracts used as positive control. The same outcome was obtained using LC-MS. The presence of other metabolites with retention times similar to the OTA signal in the A. tubingensis extracts or background noise of the growth media may be reasons for the misinterpretation of the chromatograms obtained by HPLC-FLD

Gnomoniopsis castanea is the main agent of chestnut nut rot in Switzerland

Nuts of sweet chestnut have been an important food source for the alpine population in Switzerland since the Middle Ages and are still valued today for the preparation of traditional food commodities. Nut quality is reduced by insect damage and by various pathogenic fungi. In the last few years, producers and consumers perceived an increase of brown nut rot; while the nut rot agent Gnomoniopsis castanea was reported locally in southern Switzerland, its presence has not been investigated over large areas until now. This study assessed the incidence of brown nut rot and identified the causal agent present in Switzerland. Fully ripened nuts were collected from the main sweet chestnut growing areas of Switzerland. A filamentous fungus morphologically identified as G. castanea was isolated from 10 to 91% of the sampled nuts, despite only 3 to 21% of the sampled nuts showing brown rot symptoms. This fungus was isolated from symptomatic chestnuts as well as from apparently healthy chestnuts. Our results suggest a possible endophytic lifestyle in ripened nuts as well as in branches, leaves and unripe nuts as previously found. Species identity of 45 isolates was confirmed by EF-1alpha, beta-tubulin and ITS sequencing. Concatenation of β-tubulin and calmodulin sequences showed that several haplotypes were present at each sampling locality. No other nut rot pathogens could be isolated in this study, suggesting that G. castanea is the main causal agent of nut rot in Switzerland. The presence of this species is reported for the first time in a site in northern Switzerland. Further studies are needed to assess the influence of meteorological conditions and chestnut varieties on the incidence of G. castanea in order to provide prevention strategies for chestnut growers.ISSN:0031-9465ISSN:1593-209

Draft Genome Sequences of Clostridium tyrobutyricum Strains FAM22552 and FAM22553, Isolated from Swiss Semihard Red-Smear Cheese

Clostridium tyrobutyricum is the main microorganism responsible for late blowing defect in cheeses. Here, we present the draft genome sequences of two C. tyrobutyricum strains isolated from a Swiss semihard red-smear cheese. The two draft genomes comprise 3.05 and 3.08 Mbp and contain 3,030 and 3,089 putative coding sequences, respectively

Isolation of mycotoxins producing black aspergilli in herbal teas available on the Swiss market

Black aspergilli are among the predominant fungal contaminants in herbal teas. Despite the ability of some species in this group to produce the mycotoxins ochratoxin A (OTA) and fumonisins no study had been done to investigate in detail their presence in this commodity. In the present work conventional herbal teas available on the Swiss market were investigated for black aspergilli contamination. Black aspergilli were found in 16 of 22 samples, ranging from 10 to 3500 colony forming units per gram of herbal tea. Recovered isolates were identified to the species level by calmodulin sequencing. The most frequently isolated species were Aspergillus niger, Aspergillus acidus, Aspergillus awamori and Aspergillus tubingensis. Aspergillus carbonarius, the most important OTA-producing black Aspergillus, could not be recovered. A. niger and A. awamori isolates were tested for their ability to produce fumonisins and OTA in vitro. Fumonisins were produced by 76% of A. niger and 37% of A. awamori isolates. 7% of A. niger and none A. awamori isolates could produce OTA. In total, 12 of 22 samples were found to be contaminated with black aspergilli able to produce major mycotoxins. Our results indicate that mycotoxins producing black aspergilli are widespread fungal contaminants of herbal teas. Therefore, their presence as well as the occurrence of their mycotoxins should be further investigated to assess health risks linked with the consumption of this commodity

Genetic Structure of Populations of the Rice-Infecting Pathogen Rhizoctonia solani AG-1 IA from China

Sheath blight disease (SBD) on rice, caused by Rhizoctonia solani AG-1 IA, is one of the most devastating rice diseases on a global basis, including China (in Eastern Asia), the world's largest rice-growing country. We analyzed the population genetics of nine rice-infecting populations from China using nine microsatellite loci. One allopatric population from India (Southern Asia) was included in the analyses. In total, 300 different multilocus genotypes were found among 572 fungal isolates. Clonal fractions within rice fields were 16 to 95%, suggesting that sclerotia were a major source of primary inoculum in some fields. Global Phi(ST) statistics (Phi(ST) = 42.49; P <= 0.001) were consistent with a relatively high level of differentiation among populations overall; however, pairwise comparisons gave nonsignificant R(ST) values, consistent with contemporary gene flow among five of the populations. Four of these populations were located along the Yangtze River tributary network. Gene flow followed an isolation-by-distance model consistent with restricted long-distance migration. Historical migration rates were reconstructed and yielded values that explained the current levels of population subdivision. Except for one population which appeared to be strictly clonal, all populations showed evidence of a mixed reproductive mode, including both asexual and sexual reproduction. One population had a strictly recombining structure (all loci were in Hardy-Weinberg equilibrium) but the remaining populations from China and the one from India exhibited varying degrees of sexual reproduction. Six populations showed significant F(IS) values consistent with inbreeding