Ann Microbiol (2015) 65:667–675 DOI 10.1007/s13213-014-0905-0 ORIGINAL ARTICLE The diversity of culturable yeasts in the phylloplane of rice in Thailand Savitree Limtong & Rungluk Kaewwichian Received: 2 January 2014 /Accepted: 21 April 2014 /Published online: 10 May 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract One hundred and fifty-six yeast strains were obtained Cyberlindnera fabianii, Cyberlindnera rhodanensis and by the enrichment technique from the phylloplanes of 85 rice leaf Wickerhamomyces ciferrii in Wickerhamomycetaceae; samples collected from seven provinces in Thailand. On the basis Debaryomyces nepalensis, Meyerozyma caribbica, of the D1/D2 domain of the large subunit rRNA gene sequence Meyerozyma guilliermondii, Millerozyma koratensis,and analysis, 156 strains were identified as 34 known species in 18 Yamadazyma mexicanum in Debaryomycetaceae; Pichia genera consisting of 25 species in 13 genera of the phylum kudriavzevii in Pichiaceae; and Lachancea thermotolerans in Ascomycota and nine species in five genera of the phylum Saccharomycetaceae. The species in Basidiomycota viz. Basidiomycota. The species in the phylum Ascomycota com- Cryptococcus flavescens, Cryptococcus laurentii, Cryptococcus prised 24 species in 12 genera of the order Saccharomycetales aff. laurentii and Cryptococcus rajasthanensis in the Tremellales and one species viz. Yarrowia lipolytica in Saccharomycetales lineage, Bulleromyces clade, Tremellales, Tremellomycetes, incertae sedis. The 24 species viz. Candida glabrata in the Agaricomycotina; Pseudozyma antarctica and Pseudozyma Nakaseomyces clade of Saccharomycetaceae, Candida jaroonii, aphidis in Ustilaginales, Ustilaginomycetes, Candida membranifaciens and Candida terebra in the Ustilaginomycotina; Rhodotorula taiwanensis and Yamadazyma clade of Debaryomycetaceae, Candida Sporobolomyces blumeae in Sporidiobolales, pseudolambica in the Pichia clade of Pichiaceae, Candida Microbotryomycetes, Pucciniomycotina; and Trichosporon ruelliae in the Metschnikowia clade of Metschnikowiaceae, and asahii in Trichosporonales, Tremellomycetes, three unaffiliated clade Candida species (Candida catenulata, Agaricomycotina. The most prevalent species was Candida rugosa and Candida tropicalis); Clavispora lusitaniae, R. taiwanensis with a 23 % frequency of occurrence followed Kodamaea ohmeri, Metschnikowia koreensis and by Candida tropicalis (16 %) and Cryptococcus fabianii (12 %). Metschnikowia lopburiensis in Metschnikowiaceae; Keywords Phylloplane . Rice . Yeast . Thailand . D1/D2 Electronic supplementary material The online version of this article domain (doi:10.1007/s13213-014-0905-0) contains supplementary material, which is available to authorized users. * : S. Limtong ( ) R. Kaewwichian Introduction Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand e-mail: [email protected] Rice is one of the world’s most important food crops. About 90 % of the world’s rice is cultivated in Asia. In Thailand, a S. Limtong tropical Asian country, the Office of Agricultural Economics, Center for Advanced Studies in Tropical Natural Resources, National Research University- Kasetsart University, Kasetsart University, Ministry of Agriculture and Cooperatives reported the rice Bangkok 10900, Thailand cultivation area to be approximately 13,192,400 hectares and the production of rice for the year 2012 to be 38,830,600 tons. Present Address: Rice is a type of grass belong to the family Poaceae, a group of R. Kaewwichian Bioresources Technology Unit, National Center for Genetic monocotyledonous plants. The rice species that is cultivated in Engineering and Biotechnology (BIOTEC), Pathumthani, Thailand Thailand is Asian rice, Oryza sativa. 668 Ann Microbiol (2015) 65:667–675 The phylloplane, or phyllosphere, is a term for the above strains isolated from the phylloplane of sugarcane in Thailand ground surface of plants and usually refers to the external surface (Kaewwichian et al. 2012, 2013a, 2013b; Kaewwichian and of plant leaves, a dominant aerial part of plant (Phaff and Starmer Limtong 2014). 1987; Fonseca and Inacio 2006). The phylloplane is known to be Although yeasts inhabiting the phylloplane have been stud- colonized by a large number of microorganisms including bac- ied intensively, the diversity of yeasts in the phylloplane of teria, yeasts and fungi (Andrews and Harris 2000;Lindowand rice has received little attention, and only a few articles have Brandl 2003). The growth of microorganisms in the phylloplane been published so far. Among them, one article reported the is dependent on nutrients from plant metabolites that are secreted use of the ballistoconidium-fall isolation method to study to the phylloplane or materials from external sources that drop on yeasts in rice phylloplane in Thailand (Nakase et al. 2001). the plant surface. The plant metabolites are organic substances, Therefore, this study aimed to investigate the diversity of mostly simple sugars, e.g., glucose, fructose and sucrose, while yeasts in the phylloplane of rice in Thailand by using the amino acids, organic acids and sugar alcohols are also present enrichment isolation technique. (Fiala et al. 1990;Xinetal.2009). On the other hand, nutrients in the materials from external sources are inorganic nutrients (Xin et al. 2009). In addition to having relatively limited nitrogen Materials and methods sources, the microbial inhabitants of the phylloplane encounter strong ultraviolet radiation, low or fluctuating water activity, Sample collection fluctuating temperature, and antimicrobial compounds that are produced by plants or other microorganisms; as a consequence, Green and healthy leaves of rice (Oryza sativa)wererandom the microorganisms on the phylloplane are often irregularly collected from rice fields. Leaf samples were put in plastic distributed (Inácio et al. 2005; Fonseca and Inacio 2006; bags, sealed and brought in an ice-box to the laboratory. The Remus-Emsermann et al. 2012; Vorholt 2012). samples were kept at 8 °C until subjected to yeast isolation. Bacteria are the most abundant phylloplane microorganisms; however, yeasts and yeast-like fungi are also active phylloplane Yeast isolation colonizers (Andrews and Harris 2000; Lindow and Brandl 2003). The phylloplanes of both dicotyledonous and monocoty- Yeasts were isolated by an enrichment technique using yeast ledonous plants are colonized by both basidiomycete and asco- extract malt extract (YM) broth (3 g/L yeast extract, 3 g/L malt mycete yeasts (Nakase and Suzuki 1985; de Azeredo et al. 1998; extract, 5 g/L peptone and 10 g/L glucose) supplemented with Nakase et al. 2001;Inácioetal.2005; Fonseca and Inacio 2006; 250 mg/L sodium propionate and 200 mg/L chloramphenicol Glushakova et al. 2007; Slavikova et al. 2007, 2009; Glushakova (Limtong et al. 2007). Three grams of cut leaves, derived from and Chernov 2010; Landell et al. 2010). Fewer studies on yeasts cutting a few leaves to the size that could be put into a 250 ml that colonize the phylloplanes of monocotyledonous plants have Erlenmeyer, were inoculated into 50 ml enrichment broth in been reported. The ballistoconidium-fall method was used to the flask and incubated on a rotary shaker at room temperature isolate ballistoconidium-forming yeasts from dead leaves and (30±2 °C) for 2 days. A loopful of the enriched culture was stems of rice in Japan and from rice and various plants in directly streaked on YM agar supplemented with 250 mg/L Thailand (Nakase and Suzuki 1985;Nakaseetal.2001). The sodium propionate and 200 mg/L chloramphenicol and incu- prevalent yeast species associated with leaves, stems and rhizo- bated at room temperature (30±2 °C) for 2–7daysoruntil spheres of sugarcane in Brazil were found to be Cryptococcus yeast colonies appeared. Yeast colonies of different morphol- laurentii, Cryptococcus albidus, Rhodotorula mucilaginosa and ogies were picked and purified by cross streaking on YM agar. Debaryomyces hansenii (de Azeredo et al. 1998). In a recent Purified yeast strains were suspended in YM broth supple- investigation, Pseudozyma graminicola was proposed from two mented with 10 % v/v glycerol and maintained at −80 °C. strains isolated from the leaves of pasture plants in Russia (Golubev et al. 2007). Candida aechemae and Candida vrieseae Yeasts identification were proposed as two novel yeast species isolated from phyllophanes of bromolids in southernBrazil(Landelletal. Yeasts were identified by analysis of the D1/D2 domain of the 2010). Bensingtonia rectispora, a ballistoconidium-forming large subunit (LSU rRNA) gene sequence similarities (Kurtzman yeast, was a new species proposed from strains isolated from and Robnett 1998). The sequence of the D1/D2 region of the bamboo (Wang et al. 2012). Metschnikowia lopburiensis,was LSU rRNA gene was determined from PCR products amplified proposed as a new species from one strain isolated from the from genomic DNA. The D1/D2 region of the LSU rRNA gene surface of green rice leaves in Thailand (Kaewwichian et al. was amplified and sequenced with primers, NL1 and NL4 2012). Four novel yeast species viz. Metschnikowia (Kurtzman and Robnett 1998). Methods for DNA extraction saccharicola, Yamadazyma siamensis, Wickerhamomyces and amplification of the D1/D2 region of the LSU rRNA gene siamensis and Nakazawaea siamensis were proposed from and ITS region were as described previously (Limtong et al. Ann Microbiol (2015) 65:667–675 669 2007). The PCR products were checked by agarose gel electro-