Introduction

The external surface of plant leaves, which is usually referred to as the phylloplane or phyllosphere, has been recognized as an important habitat for epiphytic microorganisms (Fonseca and Inacio 2006; Phaff and Starmer 1987). In the phylloplane, the growth of microorganisms is dependent on nutrients from plant metabolites that are secreted to the phylloplane or on compounds in materials from external sources that drop on the plant surface. The plant metabolites are organic substances, mostly simple sugars e.g. glucose, fructose and sucrose, while the materials from external sources are inorganic nutrients (Xin et al. 2009). While bacteria are the most abundant phylloplane microorganisms, yeasts and yeast-like fungi such as Aureobasidium pullulans are also active phylloplane colonizers (Andrews and Harris 2000). The phylloplane of diverse temperate, tropical and Mediterranean plants have been found to be colonized by both basidiomycetous and ascomycetous yeasts (Fonseca and Inacio 2006; Glushakova and Chernov 2010; Glushakova et al. 2007; Inácio et al. 2005; Landell et al. 2010; Nakase et al. 2001; Slavikova et al. 2009). Although the common phylloplane yeasts were basidiomycetous species belonging to the genera Bullerra, Cryptococcus, Dioszegia, Rhodotorula, Sporobolomyces, Tilletiopsis and Trichosporon such as Cryptococcus laurentii, Rhodotorula mucilaginosa, Rhodotorula glutinis and Sporobolomyces roseus (de Azeredo et al. 1998; Fonseca and Inacio 2006; Glushakova et al. 2007; Nakase et al. 2001; Sharma et al. 2009; Slavikova et al. 2009), some ascomycetous species have also been found such as Debaryomyces hansenii, Hanseniaspora uvarum, Kazachstania barnetii, Metschnikowia pulcherima, Metschnikowia reukaufii, Pichia membranifaciens, Saccharomyces cerevisiae and various species of Candida (Glushakova and Chernov 2010; Glushakova et al. 2007; Koowadjanakul et al. 2011; Landell et al. 2010; Sharma et al. 2009; Slavikova et al. 2009). Yeasts colonizing the phylloplane were studied intensively; however, limited investigation on diversity of yeasts in phylloplane in tropical area was conducted so far. For example Nakase et al. (2001) reported ballistoconidium-froming yeasts found in the phylloplane of Thailand.

Indole-3-acetic acid (IAA) is the major member of plant growth promoter in the auxin class that is known to stimulate both rapid and long-term responses in plants by regulation of various developmental and physiological processes (Cleland 1990). It is produced by plants and microorganisms including bacteria (Xinxian et al. 2011), actinomycetes (Khamna et al. 2010), yeasts (El-Tarabily 2004; Nakamura et al. 1991; Nassar et al. 2005), and filamentous fungi (Ruanpanun et al. 2010). IAA-producing microorganisms are receiving attention as good sources of biofertilizer (Sasikala and Ramana 1998). Applications of IAA producing yeasts, such as S. roseus, Candida valida, R. glutinis and Trichosporon asahii, Lindera (Williopsis) saturnus and R. mucilaginosa, to promoting growth of plants have been reported (El-Tarabily 2004; Nassar et al. 2005; Perondi et al. 1996; Xin et al. 2009).

There is little information on IAA-producing yeasts, especially those of the phylloplane. Therefore, the objectives of this study were isolation and identification of yeasts isolated from phylloplane in Thailand, followed by assessment of their capacity to produce indole-3-acetic acid in vitro.

Materials and methods

Sample collection

Green and undamaged plant leaves were collected and placed in plastic bags, sealed and transferred in ice-box to laboratory. The samples were kept at 8 °C until subjected for yeast isolation.

Yeast isolation

Yeast was isolated by an enrichment technique using yeast extract malt extract (YM) broth (3 g/L yeast extract, 3 g/L malt extract, 5 g/L peptone and 10 g/L glucose) supplemented with 0.025 % sodium propionate and 0.02 % chloramphenicol (Limtong et al. 2007). Three grams of cut leaves, derived from cutting few leaves to the size that can be put into a 250 mL Erlenmeyer, was inoculated into 50 mL enrichment broth in the flask and incubated on a rotary shaker at 30 ± 3 °C for 2 days. A loopful of the enriched culture was streaked on YM agar supplemented with 25 mg/L sodium propionate and 20 mg/L chloramphenicol. Yeast colonies of different morphologies were picked and purified by cross streaking on YM agar. Purified yeast strains were suspended in YM broth supplemented with 10 % v/v glycerol and maintained at −80 °C.

Yeasts identification and phylogenetic analysis

Yeasts were identified by molecular taxonomy based on the analysis of the D1/D2 domain of the large subunit (LSU) rRNA gene sequences similarities according to a guideline of Kurtzman and Robnett (1998) that yeast strains with 0–3 nucleotide differences are conspecific or sister species and yeast strains showing nucleotide substitutions greater than 6 are usually different species. Therefore, a strain showing nucleotide substitutions greater than six from the type strain of the closest species could be designed as the novel species. Undescribed species are the species that the D1/D2 domain of the LSU rRNA gene sequences was deposited in the GenBank without description.

Methods for DNA isolation and amplification of the D1/D2 domain of the LSU rRNA gene were as described previously by Limtong et al. (2007). The PCR product was checked by agarose gel electrophoresis and purified by using the QIA quick purification kit (Qiagen, Hilden, Germany). The purified product was sequenced commercially by Macrogen Inc. (Seoul, Korea) with primers, NL1 and NL4. The sequences were compared pairwise using a BLAST search (Altschul et al. 1997).

A phylogenetic tree was constructed from the evolutionary distance data with Kimura’s two-parameter correction (Kimura 1980), using the neighbor-joining method (Saitou and Nei 1987). Confidence levels of the clades were estimated from bootstrap analysis (1,000 replicates; Felsenstein 1985).

Determination of indole-3-acetic acid production

Production of indole-3-acetic acid (IAA) by the phylloplane yeasts was investigated by the method of Xin et al. (2009). A yeast culture cultivated for 1–2 days on YM agar at 25 °C was inoculated in 5 mL of yeast extract peptone dextrose (YPD) broth (10 g/L yeast extract, 2 g/L peptone and 2 g/L dextrose) supplemented with 1 g/L l-tryptophan in a test tube and incubated on a shaker at 30 ± 2 °C and 150 rpm for 7 days. An aliquot of 1.5 mL of the culture broth was centrifuged at 8,000 rpm for 5 min and the supernatant was collected for determination of IAA concentration. One mL of supernatant was mixed with 1 mL of Salkowski reagent (12 g/l FeCl3 and 7.9 M H2SO4; Glickmann and Dessaux 1994), and the intensity of pink color developing in the mixture after 30 min was quantified with a spectrophotometer (UV-1700, Shimadzu, Japan) at a wavelength of 530 nm. Calibration curve using pure IAA was established for calculation of IAA concentration. Growth was determined as dry weight by drying cells after centrifugation at 100 °C until constant weight was obtained.

Results

Sample collection and yeast isolation

Yeasts were isolated from the phylloplane of 76 leaf samples of 45 plant species and 21 samples of unknown plants which had been collected from 19 locations in seven provinces in the eastern, central, north–eastern and peninsular regions of Thailand during April and May 2009 (Table 1).

Table 1 Leaf from diverse plant species and yeasts isolated from phylloplane with their accession numbers of the D1/D2 domain of the large subunit rRNA gene
Full size table

A total of 114 yeast strains and 10 strains of yeast-like fungus were obtained from 91 samples representing 93.8 % of the samples investigated (Table 1). Among these 91 samples three samples contained only the yeast-like fungus.

Yeast identification

On the basic of the D1/D2 domain of the LSU rRNA gene sequence similarity and the generally accepted criteria of Kurtzman and Robnett (1998), 96 yeast strains were identified to be 36 species in 18 genera (Tables 1 and 2). Thirty-four species were in 16 genera of Phylum Ascomycota (15 genera) viz. Candida (15 species), Clavispora (1 species), Cyberlindnera (1 species), Debaryomyces (1 species), Hanseniaspora (3 species), Hyphopichia (1 species), Kazachstania (1 species), Kluyveromyces (1 species), Kodamaea (1 species), Lachancea (1 species), Metschnikowia (1 species), Meyerozyma (1 species), Pichia (2 species), Starmerella (1 species), Torulaspora (2 species) and Wickerhamomyces (1 species), and two species were in Phylum Basidiomycota (2 genera) viz. Sporidiobolus (1 species), and Trichosporon (1 species). Four strains were similar to three undescribed species in Ascomycota and nine strains require further analysis for identification. Three strains were found to represent two novels Candida species which were previously proposed to be C. sirachaensis and C. sakaeoensis. The 10 strains of yeast-like fungus were identified to be A. pullulans (Table 1, 2). Phylogenetic positions of all yeast species obtained in this study were shown in Fig. 1. Most Candida species were distributed in five phylogenetically distinct clades including Kodamaea, Lodderomyces-Spathospora, Nakaseozyma, Starmerella and Yamadazyma clades while Candida rugosa was not placed in any clade.

Table 2 Frequency of isolation of phylloplane yeasts obtained from diverse plant species in Thailand
Full size table
Fig. 1
figure 1

Phylogeny of yeasts isolated from phylloplanes based on the D1/D2 domains of the LSU rRNA gene

Full size image

The results indicated that 98.0 % of the strains isolated by the enrichment technique were ascomycetous yeasts, and only 2.0 % represented basidiomycetous species. The dominant species was Candida tropicalis, although only 14 strains of this species were isolated from 14 samples, represented a 14.3 % frequency of isolation (Table 2). Ten strains of A. pullulans, the yeast-like fungus, were isolated from 10 samples, giving a frequency of isolation of 7.8 %.

Iodole-3-acetic acid production

Among the 114 strains of yeast, 39 strains in 20 species, one strain of an undescribed species, one strain of a novel species, and two unidentified strains showed the capability of producing IAA when cultivated in YPD broth supplemented with 0.1 % l-tryptophan (Table 3). The other 71 strains grew in this medium, but no IAA was produced. All five strains of C. maltosa produced relatively high concentrations (121.4–234.1 mg/L) of IAA. This result indicated IAA production was strain-dependent; some strains of some species were able to produce IAA while others were not. All 10 strains of A. pullulans produced IAA; however, the concentrations were relatively low.

Table 3 Indole-3-acetic acid productions in YPD broth supplemented with 1 mg/L l-tryptophan in a test tube after 7 days by shake cultivation at 150 rpm and 30 ± 3 °C
Full size table

Discussion

The two findings of the present study were, first, that phylloplane yeasts are present on diverse plant species in Thailand and C. tropicalis was frequently found and second, that approximately 37.7 % of the phylloplane yeasts found were capable of in vitro IAA biosynthesis. The study found that by enrichment isolation at 30 ± 3 °C most yeasts obtained from phylloplane in Thailand were in the phylum Ascomycota (98.0 %); this is in contrast with the other investigations, which report the dominance of basidiomycetous yeasts on the phylloplane in the other regions (de Azeredo et al. 1998; Fonseca and Inacio 2006; Glushakova et al. 2007; Nakase et al. 2001; Sharma et al. 2009). This difference may have resulted from the different technique and incubation temperature employed for isolation. While in most investigations leaf washing followed by dilution plating technique was used, Nakase et al. (2001) used the ballistoconidium fall method with YM agar without any antibacterial and antifungal agents, and they found more yeast species when the incubation temperature was at 23 °C than at 30 °C. This may due to the fact that most of basidiomycete yeasts grow rather slow and they have fairly low maximum growth temperature in comparison with ascomycete yeasts and the enrichment isolation at high temperature (30 ± 3 °C) selects rapid growing yeasts. Therefore, more ascomycete yeasts were obtained.

Among yeast species found on phylloplane in this study only S. ruineniae has been stated that its primary habitat is possible to be the phylloplane (Sampaio 2011). Strains of many yeast species found in this study were reported to isolate from insects viz. Candida amphixiae, C. apicola, C. etchellsii, C. glabrata, C. trypodendroni, Debaryomyces nepalensis, Hanseniaspora guilliermondii, Hyphopichia burtonii, Kluyveromyces marxianus, Kodamae ohmeri, Lachances thermotolerans and Starmerella meliponinorum (Cadez and Smith 2011; Kurtzman 2011a, d; Lachance 2011b; Lachance and Kurtzman 2011a, b; Rosa et al. 2003) and from plants including flowers, fruits and tree parks viz. Candida jaroonii, C. nivariensis, C. potacharoeniae, C. stigmatis, Clavispora lusitaniae, Hanseniaspora opuntiae, H. thailandica, Metschnikowia koreensis, Meyerozyma guilliermondii and Pichia kudriavzevii (Cadez and Smith 2011; Imanishi et al. 2008; Kurtzman 2011b, c; Lachance 2011a, c; Lachance et al. 2011; Nakase et al. 2010; Jindamorakot et al. 2009; Sipiczki 2010). The present of these yeast species on the phylloplanes in this study may resulted from visiting of insects that carried these yeasts to the phylloplane. Strains of some species obtained in this study were previously reported to be the novel species discovered in Thailand isolated from the other sources viz. C. jaroonii (Imanishi et al. 2008), C. potacharoeniae (Nakase et al. 2010), H. thailandica (Jindamorakot et al. 2009), Kazachstania siamensis (Limtong et al. 2007) and Wickerhamomyces edaphicus (Limtong et al. 2009). Moreover, A. pullulans, the yeast-like fungus, which was found to occur regularly on the leaves of fruit trees in the Czech Republic (Slavikova et al. 2009), was also frequently found in the present study.

Investigation of the IAA production capability of the phylloplane yeasts in this study revealed that about 37.7 % of investigated yeast strains possessed this ability. It was found that among the species of one genus, both IAA producing species and non-producing species were detected. Moreover, not all strains within the same species had the ability to produce IAA. It therefore seems that IAA production capability is strain-dependent in these phylloplane yeasts. Variation in IAA biosynthesis among strains within the same species in the other microorganisms has also been reported by the other investigators (Ruanpanun et al. 2010; Tsavkelova et al. 2006).

IAA production by C. maltosa LM114 (314.3 mg/L) seemed to be higher than that reported for the other yeasts such as Lindera (Williopsis) saturnus (22.5 mg/L; Nassar et al. 2005); fungi such as Aspergillus niger (132.7 μg/mL; Bilkay et al. 2010); some actinomycetes such as Streptomyces sp. CMU-H009 (143.95 μg/mL; Khamna et al. 2010); and bacteria such as Rubrivivax benzoatilyticus JA2 (58.1 mg/L; Mujahid et al. 2001) and Klebsiellas sp. SN1 (291 mg/L; Xinxian et al. 2011). However, the concentration of IAA produced by C. maltosa LM114 needs to be confirmed with more specific method for IAA determination such as high-performance liquid chromatography or gas chromatography-mass spectrometry.