The phyllosphere is colonized by complex microbial communities, which are adapted to the harsh habitat. Although the role and ecology of nonpathogenic microorganisms of the phyllosphere are only partially understood, leaf microbiota could have a beneficial role in plant growth and health. Pesticides and biocontrol agents are frequently applied to grapevines, but the impact on nontarget microorganisms of the phyllosphere has been marginally considered. In this study, we investigated the effect of a chemical fungicide (penconazole) and a biological control agent (Lysobacter capsici AZ78) on the leaf microbiota of the grapevine at three locations. Amplicons of the 16S rRNA gene and of the internal transcribed spacer were sequenced for bacterial and fungal identification, respectively. Pyrosequencing analysis revealed that the richness and diversity of bacterial and fungal populations were only minimally affected by the chemical and biological treatments tested, and they mainly differed according to grapevine locations. Indigenous microbial communities of the phyllosphere are adapted to environmental and biotic factors in the areas where the grapevines are grown, and they are resilient to the treatments tested. The biocontrol properties of phyllosphere communities against downy mildew differed among grapevine locations and were not affected by treatments, suggesting that biocontrol communities could be improved with agronomic practices to enrich beneficial populations in vineyards. Plants support a complex micro-ecosystem, and they host distinct bacterial communities on and inside various plant organs (1). The aerial part of plants (phyllosphere) is normally colonized by a variety of bacteria, filamentous fungi and yeasts (2). Microbial phyllosphere communities are complex and composed by many uncultured microorganisms (2, 3), which are adapted to the harsh environmental conditions (4, 5). In particular, microbial epiphytes of the phyllosphere are exposed to the atmosphere and must deal with direct UV radiation, wide fluctuations in temperature, low water availability, and limited access to nutrients (2,6). Therefore, the composition of phyllosphere communities could be affected by environmental factors, such as UV radiation, air pollution, and nitrogen fertilization, as well as by biotic factors, such as plant species and invading microorganisms (6-8). Moreover, the phyllosphere is an open system and microbes can invade plant leaves by migration from the atmosphere, soil, other plants, insects, and animals (9).The phyllosphere has been less intensively studied than the rhizosphere and has received considerable attention in recent years (1). The interest in phyllosphere microbiology was initially driven by investigations into plant pathogens, but most phyllosphere-colonizing microorganisms live as commensals and/or mutualistic symbionts on their host plants (2,4,6). Phyllosphere communities are involved in functional processes as large in scale as the carbon cycle, nitrogen fixation, and degradation of organic pollu...

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

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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.

Ten polymorphic microsatellite loci were isolated and characterized from the rice- and maize-infecting Basidiomycete fungus Rhizoctonia solani anastomosis group AG-1 IA. All loci were polymorphic in two populations from Louisiana in USA and Venezuela. The total number of alleles per locus ranged from four to eight. All 10 loci were also useful for genotyping soybean-infecting R. solani AG-1 isolates from Brazil and USA. One locus, TC06, amplified across two other AG groups representing different species, showing species-specific repeat length polymorphism. This marker suite will be used to determine the global population structure of this important pathogenic fungus.

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 future.

Aims: To develop two assays based on the loop-mediated isothermal amplification (LAMP) of DNA for the quick and specific identification of Aspergillus carbonarius and ochratoxigenic strains of the Aspergillus niger clade isolated from grapes. Methods and Results: Two sets of primers were designed based on the polyketide synthase genes involved or putatively involved in ochratoxin A (OTA) biosynthesis in A. carbonarius and A. niger clade. Hydroxynaphthol blue was used as indirect method to indicate DNA amplification. The limit of detection of both assays was comparable to that of a PCR reaction. Specificities of the reactions were tested using DNA from different black aspergilli isolated from grapes. The two LAMP assays were then used to identify A. carbonarius and ochratoxigenic A. niger and A. awamori grown in pure cultures without a prior DNA extraction. Conclusions: The two LAMP assays permitted to quickly and specifically identify DNA from OTA-producing black aspergilli, as well as isolates grown in pure culture. Significance and Impact of the Study: Monitoring vineyards for the presence of OTA-producing strains is part of the measures to minimize the occurrence of OTA in grape products. The two LAMP assays developed here could be potentially used to speed the screening process of vineyards for the presence of OTA-producing black aspergilli.

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.