Assessment of epiphytic yeast diversity in rice (Oryza sativa) phyllosphere in Thailand by a culture-independent approach.

Abstract

The epiphytic yeast diversity in rice phyllosphere in Thailand was investigated by a culture-independent technique based on the RFLP pattern and the sequence of the D1/D2 domain of the large subunit rRNA gene. Forty-four samples of rice leaf were collected randomly from six provinces. The DNA was extracted from leaf washing samples and the D1/D2 domain was amplified using PCR technique. The PCR products were cloned and then screened by colony PCR. Of total 1121 clones, 451 clones (40.2%) revealed the D1/D2 domain sequences closely related to sequences of yeasts in GenBank, and they were clustered into 45 operational taxonomic units (OTUs) at 99% homology. Of total yeast related clones, 329 clones (72.9%) were identified as nine known yeast species, which consisted of 314 clones (8 OTUs) in the phylum Basidiomycota including Bullera japonica, Pseudozyma antarctica, Pseudozyma aphidis, Sporobolomyces blumeae, Sporobolomyces carnicolor and Sporobolomyces oryzicola and 15 clones (6 OTUs) in the phylum Ascomycota including Metschnikowia koreensis, Meyerozyma guilliermondii and Wickerhamomyces anomalus. The D1/D2 sequences (122 clones) that could not be identified as known yeast species were closest to 3 and 14 species in Ascomycota and Basidiomycota, respectively, some of which may be new yeast species. The most predominant species detected was P. antarctica (42.6%) followed by B. japonica (25.9%) with 63.6 and 22.7% frequency of occurrence, respectively. The results of OTU richness of each sampling location revealed that climate condition and sampling location could affect epiphytic yeast diversity in rice phyllosphere.