Characterization of cultivated fungi isolated from grape marc wastes through the use of amplified rDNA restriction analysis and sequencing

Abstract

Microbial assessment of grape marc wastes, the residual solid by-product of the wine-industry, was performed by identifying phylogenetically the fungal culturable diversity in order to evaluate environmental and disposal safety issues and to discuss ecological considerations of applications on agricultural land. Fungal spores in grape marc were estimated to 4.7 x 10(6) per g dry weight. Fifty six fungal isolates were classified into eight operational taxonomic units (OTUs) following amplified ribosomal DNA restriction analysis (ARDRA) and colony morphology. Based on 18S rRNA gene and 5.8S rRNA gene-ITS sequencing, the isolates representing OTUs #1, #2, #3, and #4, which comprised 44.6%, 26.8%, 12.5%, and 5.3%, respectively, of the number of the total isolates, were identified as Aspergillus fumigatus, Bionectria ochroleuca, Haematonectria haematococca, and Trichosporon mycotoxinivorans. The isolates of OTU#5 demonstrated high phylogenetic affinity with Penicillium spp., while members of OTUs #6 and #7 were closer linked with Geotrichum candidum var. citri-aurantii and Mycocladus corymbifer, respectively (95.4 and 97.9% similarities in respect to their 5.8S rRNA gene-ITS sequences). The OTU#8 with a single isolate was related with Aspergillus strains. It appears that most of the fungal isolates are associated with the initial raw material. Despite the fact that some of the species identified may potentially act as pathogens, measures such as the avoidance of maintaining large and unprocessed quantities of grape marc wastes in premises without adequate aeration, together with its suitable biological treatment (e.g., composting) prior to any agriculture-related application, could eliminate any pertinent health risks.

Dynamics of Panax ginseng Rhizospheric Soil Microbial Community and Their Metabolic Function

“…Aliquot (100 μ L) of each transformation was spread on LB/ampicillin/IPTG/X-gal plates and incubated at 37°C for 16 h. For each sample, 192 white colonies were picked out, which were amplified by 27f and 1492r primer that the positive clones were confirmed through. Then, 10 μ L target insert fragments (about 1,500 bp) were digested by 3 U restriction endonuclease Hinf I (TaKaRa) [ 13 , 14 ] and Pst I (TaKaRa) [ 15 ] at 37°C for 2 h. Clones having the same restriction patterns were defined as an operational taxonomic unit (OTU).…”

Cultivation of Trichosporon mycotoxinivorans in sugarcane bagasse hemicellulosic hydrolyzate

“…Abe et al (2010) have described the presence of T. mycotoxinivorans in fresh shavings of palm tissue that were damaged by beetle larvae (Coleoptera: Dryophthoridae) in Japan. It occurrence was also described in grape marc in southern Peloponnese, Greece, by Ntougias et al (2010), which reports it as closely related to an unidentified member of Trichosporon clade, isolated from the xylophagous beetle Phrenapetes bennetti (Tenebrionidae), collected in Panama by Suh et al (2005). Hickey et al (2009) have found T. mycotoxinivorans to be a novel respiratory pathogen in patients with cystic fibrosis.…”