Endophytic Yeasts Colonize Roots of Ulmus parvifolia Jacq. and Quercus salicina Blume

Introduction

Yeasts are important in various industries and in traditional food fermentation (Botha, 2011; Deak, 2009; Fonseca and Inacio, 2006; Raspor and Zupan, 2006; Tamang and Fleet, 2009). In this study, we investigated the endophytic yeast taxa associated with roots of Ulmus parvifolia and Quercus salicina, and evaluated their bioethanol production. To our knowledge, only one previous study has conducted phylogenetic analysis of endophytic yeast isolates (Rosen and Kunjappu, 2013).

U. parvifolia and Q. salicina evergreen trees are mainly distributed in southern Korea and surrounding islands (Lee, 2006). U. parvifolia (U. parvifolia Jacq.) is of the Ulmaceae family and is found in deciduous broad-leaf forests in the middle-southern region of Korea and on Jeju Island. U. parvifolia is distributed from 100m to approximately 500m above sea level (asl) on Jeju Island and hill slopes in Gotjawal and Orum, where it forms evergreen broad-leaf forests (Lee, 1996; Lee, 2003). Stem bark of U. parvifolia has diuretic and expectorant properties, and leaves harvested during summer and autumn are used to treat edema and kidney/bladder stones (Lee & Park, 2011). Q. salicina Blume of the Fragaceae family is an evergreen tree species distributed on Jeju and Ulleung Islands, as well as various southern islands (Lee, 2003). It is distributed from 100 m to approximately 700m asl on hill slopes in the Gotjawal region, where it forms evergreen broad-leaf forests. It is used as a diuretic, anti-inflammatory, and to treat kidney stones (Kim et al., 2008; Leeetal.,2014).

Ethanol production by the non-conventional yeast genus Kluyveromyces has been reported (Choi et al., 2008; Goshima et al., 2013; Kang et al., 2010). Few studies have investigated the endophytic yeast communities of plant roots, which provide various habitats for microbial communities.

In this study, we evaluated the endophytic yeast taxa associated with roots of U. parvifolia and Q. salicina.

Materials and Methods

Yeast isolation from roots of U. parvifolia and Q. salicina

U. parvifolia and Q. salicina on Dongbackdongsan (latitude, 33 30 26.5'; longitude, 126 43 31.1'; 179m asl), Seonheul, Jocheon, and Jeju Islands were identified and characterized by Lee (1996). In this study, root samples were aseptically collected from Dongbaekdongsan, Jeju, and Gotjawal using clean forceps and placed in clean plastic bags. Roots were processed according to Kim et al. (2016a, 2017). Plant samples with soil were transported to the laboratory in Uljin, and processed within 1 week. The washed root samples were placed in Falcon tubes (Falcon, Los Angeles, CA) containing 10 mL of potassium phosphate buffer (10mM), and homogenized using a hand homogenizer (T10 basic; IKA, Germany). Homogenized and diluted samples (1 mL) were plated on sterile agar media using a glass spreader and incubated at 25C for 25 days. The agar media used were as follows: DG18 agar (MB Cell, Seoul), DOB with CSM agar (MP Bio, CA, USA), SCG agar (MB Cell, Seoul), and GPY agar. These media were supplemented with antibiotics (100 mg/L chloramphenicol and streptomycin), 0.1% Triton X-100, and 0.4%lsorbose (Kim et al., 2016a).

Culture of yeast isolates

Yeasts were cultured on agar media in square plates (245×245×25 mm, Nunc Bio-Assay Dish; Thermo Scientific, Roskilde, Denmark). All colonies from one or two plates were picked and cultured separately. In total, 25 isolates were transferred to fresh plates three times and then processed for sequencing of their internal transcribed spacer (ITS) regions (Kim et al., 2016a; 2017).

ITS sequencing

Phylogenetic identification was performed as reported previously (Kim et al., 2016a; Kim et al., 2017; White et al., 1990). The ITS regions of the yeast isolates were sequenced using a PRISM BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and an ABI Prism 3730XL DNA Analyzer (Applied Biosystems) by Macrogen Inc. (Seoul, Korea). The nucleotide sequences obtained were deposited in DDBJ/EMBL/GenBank under the following accession numbers: LC229700LC229712 (13 U. parvifolia isolates) and LC229713LC229724 (12 Q. salicina isolates).

Phylogenetic analysis of yeast isolates

Multiple alignments of ITS sequences were performed using the Clustal Omega software on the EMBL website (http://www.ebi.ac.uk/tools/msa/clustalo/). A BLAST sequence similarity search was performed to identify the GenBank sequence of the yeast strain most closely related to each isolate. Phylogenetic trees were constructed by the neighbor-joining method using MEGA5 software for Windows (Tamura et al., 2011), including bootstrap analyses based on 1000 samples, and evolutionary distances were calculated by the Kimura two-parameter method (Saitou and Nei, 1987).

Bioethanol and biosurfactant production

Bioethanol production was measured after anaerobic culture of three ethanol-producing isolates for 72 h. Bioethanol was measured by NICEM (National Instrumentation Center for Environmental Management, Seoul, Korea) by high-performance liquid chromatography (Hcolumn, Dionex Ultimate3000, USA), using a refractive index detector (ERC, RefractoMAX520, Japan) and ultraviolet light at 210nm. Surface tension of yeast cultures was measured using a Du Noy ring tensiometer (Sigma Model 700, KSV Instruments Ltd., Helsinki, Finland).

Results and Discussion

Endophytic yeasts were isolated from homogenized roots of U. parvifolia and Q. salicina, and subjected to phylogenetic analysis using their ITS sequences. In total, 25 yeast isolates were obtained. The genera Cyberlindnera (12 isolates) and Rhodotorula (8 isolates) predominated (Fig. 2a). This is to our knowledge the first study to identify yeasts associated with U. parvifolia and Q. salicina roots.

In a previous study, the dominant yeast genus from Mankyua chejuense roots was Cyberlindnera (140 isolates), followed by Candida (11 isolates) and Kluyveromyces (1isolate). Dendropanax morbifera roots harbored Vanderwaltozyma (49.3%, 40 isolates), Cryptococcus (49.3%, 40 isolates), and Kluyveromyces (1.2%, 1 isolate). Moreover, the Kluyveromyces isolate exhibited a high level of bioethanol production (Kim et al., 2017).

C. saturnus is distributed worldwide in freshwater, soil, and leaf litter (Kurtzman 2011). Rhodotorula mucilaginosa is a ubiquitous basidiomycetous yeast species found in plants, soil, and aquatic environments, as well as extreme environments such as uranium leachate (Sampaio 2011). Trichosporon porosum is present in soil, trees, and bat guano, but its utility in biotechnology, food, agriculture, and medicine is unknown (Sugita2011).

The Kluyveromyces isolates in this study were similar to K. hubeiensis, colonies of which on GPY medium are wheat-colored, have a rough texture, a round undulated margin, and umbonate elevation. K. hubeiensis ferments ethanol by the simultaneous saccharification and fermentation process (Choi et al., 2008).

U. parvifolia and Q. salicina are found in subtropical regions, the vegetation of which comprises mainly broad-leaved evergreen trees. Such trees predominate in the Seonheul-ri forest area of Korea. We investigated bioethanol and biosurfactant production by the Cyberlindnera and Rhodotorula (Fig. 2) isolates. As shown in Table 1, Kluyveromyces showed a high level of bioethanol production. However, none of the isolates produced detectable levels of biosurfactants. In contrast, Aureobasidium pullulans strains from wild flowers produce several types of biosurfactant (Kim et al., 2016b).

In conclusion, we isolated yeasts from roots of U. parvifolia and Q. salicina growing at Gotjawal, in the east of Jeju Island. The genera Cyberlindnera and Rhodotorula predominated, and Kluyveromyces produced a detectable level of bioethanol. These yeast isolates may have biotechnological applications as biosurfactant and bioethanol producers.

ACKNOWLEDGEMENT

Research has been supported by a grant from the National Research Foundation of Korea (NRF), funded by Korean government MSIP (Ministry of Science, ICT and Future Planning) (No. NRF-2014R1A2A1A11052888).

Tables & Figures

Fig. 1a. Photographs of U. parvifolia habitatin Seonhel Gotjawal (A), flower (B), fruits (C), and roots (D).

Fig. 1b. Photographs of Q. salicina habitat in Seonhel Gotjawal (A), flower (B), fruits (C), and roots (D).

Fig. 2. Phylogenetic tree based on the ITS region sequences of yeast isolates from U. parvifolia (A) and Q. salicina (B) roots. Strains isolated in this study are indicated in bold. Numbers are confidence levels determined from 1,000 replicate bootstrap samplings.

Table 1. Bioethanol productivity of yeasts associated with roots of U. parvifolia and Q. salicina

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