Open Access

Characterization of a novel yeast species Metschnikowia persimmonesis KCTC 12991BP (KIOM G15050 type strain) isolated from a medicinal plant, Korean persimmon calyx (Diospyros kaki Thumb)

AMB Express20177:199

https://doi.org/10.1186/s13568-017-0503-1

Received: 7 August 2017

Accepted: 1 November 2017

Published: 10 November 2017

Abstract

The yeast strain Metschnikowia persimmonesis Kang and Choi et al., sp. nov. [type strain KIOM_G15050 = Korean Collection for Type Cultures (KCTC) 12991BP] was isolated from the stalk of native persimmon cultivars (Diospyros kaki Thumb) obtained from different regions of South Korea and was characterized phenotypically, genetically, and physiologically. The isolate grew between 4 and 40 °C (optimum temperature: 24–28 °C), pH 3–8 (pH optimum = 6.0), and in 0–4% NaCl solution (with optimal growth in absence of NaCl). It also exhibited strong antibiotic and antimicrobial activities. Morphologically, cells were characterized by the presence of long, needle-shaped ascospores. Based on 18S ribosomal DNA gene sequence analysis, the new species was found to belong to the genus Metschnikowia as a sister clade of Metschnikowia fructicola. We therefore conclude that this yeast isolate from D. kaki is a new member of the genus Metschnikowia and propose the name M. persimmonesis sp. nov. This strain has been deposited in the KCTC for future reference. This discovery provides a basis for future research on M. persimmonesis sp. nov., including its possible contribution to the medicinal properties of the host persimmon plant.

Keywords

Diospyros kaki ThumbPersimmonKorean herbal medicineYeast Metschnikowia persimmonesis sp. nov

Introduction

The genus Metschnikowia of the family Metschnikowiaceae (Order Saccharromycetales) comprises single-celled species that reproduce via budding of vegetative cells and is characterized by the presence of one or two needle-shaped ascospores on elongated asci (Mendonca-Hagler et al. 1993; Pretorius 2000; Marinoni et al. 2006; Kuan et al. 2016). A total of 35 Metschnikowia species have been described to date in a wide range of hosts, including flowers, fruits, flower-pollinating insects, and lacewings (Mendonca-Hagler et al. 1993; Lachance et al. 2005; Guzmán et al. 2013; De Vega et al. 2014; Kuan et al. 2016; Álvarez-Pérez et al. 2016). Yeasts provide a variety of unique bioactive metabolites that have medicinal importance (Mager and Winderickx 2005; VanderMolen et al. 2013). They are also used as biocontrol agents along with bacteria in agriculture, especially for the prevention of post-harvest diseases in fruits (Janisiewicz et al. 2001; Kurtzman and Droby 2002; Spadaro et al. 2008; Csutak et al. 2013; Türkel et al. 2014) such as Metschnikowia fructicola isolated from grapes (Kurtzman and Droby 2002). Persimmon (Diospyros kaki Thumb.); a Korean medicinal plant (Mallavadhani et al. 1998; Kotani et al. 2000; Heo 2001; Giordani et al. 2011; Tsubaki et al. 2012) is one of the many plants that are inhabited by a number of fungal species, including the microfungus Fusicladium levieri (Scholler 2003); Metschnikowia reukaufii and M. gruessii which are mainly found in the nectar (Canto et al. 2015). In a study, it was observed that D. kaki fruit stalk is inhibited by a number of microorganisms including bacteria and fungi (Choi et al. 2017). However, despite the fact that a number of studies have attempted to isolate yeast inhabiting different parts of persimmon over the years, no study is known to have been conducted to isolate yeast species from the fruit stalk of D. kaki. In this study therefore, we describe a new yeast species Metschnikowia persimmonesis Kang and Choi et al., sp. nov. (type strain KIOM_G15050 = KCTC 12991BP) isolated from the stalk of native Korean D. kaki cultivars obtained from different regions of South Korea. M. persimmonesis was identified as a novel species based on pyrosequencing of its 18S rDNA gene. We characterized this species morphologically, genetically, and physiologically, and evaluated its growth in the presence of various nutrients and environmental stimuli. We found that M. persimmonesis produces needle-shaped ascospores which is a basic characteristic of members of the genus Metschnikowia. Its taxonomic membership was confirmed by a phylogenetic analysis that revealed M. fructiola as the closest sister species. This novel yeast strain has been given Korean Patent Submission Number (10-2016-0137873) and international Patent Submission Number (PCT-KR2017-010681) and deposited in the Korean Collection for Type Cultures (KCTC) for future reference and research.

Materials and methods

Sample preparation and specimen isolation

The origin of the yeast strain used in this study was the fruit stalk and calyx of D. kaki obtained in South Korea and processed by suspending the cleaned dried and pulverized samples in 50 ml Luria-Bertani (LB) or potato dextrose (PD) medium (Fig. 1). A 15-ml volume of each sample from each of the medium was transferred to a 50-ml tube and incubated at 25 °C with shaking at 100 rpm for 48 h. LB and PD cultures were streaked onto agar-containing LB and PD medium respectively and a single colony was isolated by sub-culturing four times via streaking on fresh plates. Single colonies were stored at − 20 °C in a 1.5-ml Eppendorf tube.
Fig. 1

Steps in preparing D. kaki samples

Substrate culture and strain characterization

Colonies growing on potato dextrose agar (PDA) and LB agar medium were inoculated in 5 ml LB liquid medium or potato dextrose broth (PDB) in sterilized test tubes and cultured at 28 °C. To obtain growth curves, 100 ml LB liquid medium or PDB were incubated at 28 °C with shaking at 150 rpm in a 500-ml round flask. The inoculum was combined with a 3% volume of culture medium containing preculture (with 595-nm absorbance values of 0.379 and 0.377, respectively), and cultured for 0, 6, 12, 18, 24, 36, 48, 72, 96, 120, and 168 h. Absorbance readings at 595 nm were obtained for tenfold dilutions of sample and used to generate a standard curve; this was used to determine the number of viable cells, which is expressed as colony-forming units (cfu) per μl. The number of cells in the culture was calculated by counting the viable cells as follows: 1 ml of culture was transferred to a test tube containing 9 ml sterilized water and serially diluted. Three replicates of 100 cells each were prepared on separate plates that were incubated at 28 °C for 2 days. The number of colonies on each plate was counted and is expressed as an average value (cfu per ml). M. persimmonesis was also sub-cultured for 24 h in tryptic soy agar (TSA), nutrient agar (NA), R2A, Bennett’s agar, and ISP2.

Microscopic examination of M. persimmonesis

The yeast strain was spread on PDA plates using sterile bent streaking rod. Plates were then incubated at 28 °C for 48 h. Suspected colonies of M. persimmonesis yeast were re-streaked on sterile PDA plates and incubated at 28 °C for 48 h. This procedure was repeated three times to obtain pure colonies of M. persimmonesis which was then examined by microscopy. A wet mount slide was prepared for examination in which a drop of water was placed on a microscope slide and a small amount of yeast cells were transferred to the drop with a sterile streaking stick and stirred. The drop was covered with a coverslip and viewed under the microscope. Cell morphology was evaluated by phase-contrast microscopy (Eclipse 80i; Nikon, Tokyo, Japan).

Mating compatibility of the isolated colonies was performed by culturing on yeast carbon base (Difco) supplemented with 1.5% agar and 0.01% ammonium sulfate at 28 °C then periodically examined by phase contrast microscopy (Eclipse 80i; Nikon, Tokyo, Japan).

M. persimmonesis DNA extraction and quantification

Single colonies from the 13 samples cultured in LB and PD were labeled as L and P, respectively; 100 mg of each sample were pulverized and combined with 200 μl distilled water. The mixture was heated at 100 °C for 10 min. Genomic DNA was extracted using a DNeasy Plant Mini kit (Qiagen, Hilden, Germany). The amount and quality of genomic DNA were verified by electrophoresis on a 0.8% agarose gel, and the concentration was determined at 260 nm using a Nano Drop 1000 spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA).

Polymerase chain reaction (PCR) amplification and identification of M. persimmonesis based on 18S rDNA sequence

Polymerase chain reaction amplification of 18S rDNA was performed in a final reaction volume of 30 μl using 30 pmol of the universal solvent-based primers ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′; sequence ID: 3) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′; sequence ID: 4), 20 ng genomic DNA, and Solg 2× Taq PCR Pre-Mix (Solgent, Daejeon, Korea). The reaction was carried out in a 9700 thermal cycler (Applied Biosystems, Singapore) under the following conditions: 95 °C for 15 min; 35 cycles of 50 °C for 40 s, 50 °C for 40 s, and 72 °C for 90 s; and 72 °C for 5 min. A 10-μl volume of the amplified 18S rDNA product was visualized by 1.5% agarose gel electrophoresis and was observed as a 600-bp band under ultraviolet light. The DNA fragment was purified using a kit (Solgent) and inserted into the pGEM-Teasy vector (Promega, Madison, WI, USA), which was transformed into XL1-Blue MRF competent cells (Stratagene, La Jolla, CA, USA). Single colonies were selected for insert verification. T7 and SP6 primer sites were analyzed with an ABI3730 automatic DNA sequencer (Applied Biosystems, Foster City, CA, USA) and compared with sequences in the National Center for Biotechnology Information (NCBI) Genbank database (http://www.ncbi.nlm.nih.gov). The GenBank Accession Number for the 18s rDNA and 26s rDNA of the yeast M. persimmonesis was MF446617 and MF446618 respectively.

DNA extraction, PCR, and pyrosequencing

Pyrosequencing methods have been described in detail elsewhere (Buée et al. 2009; Serkebaeva et al. 2013). Colonies on LB and PD agar plates were collected using sterile streaking sticks and transferred to sterile plastic conical tubes for identification by pyrosequencing. Samples were ground using a Precellys Grinder (Bertin Technologies, Montigny-le-Bretonneux, France), centrifuged, and washed twice with 1× phosphate-buffered saline (Biowhittaker, Walkersville, MD, USA). Pellets were stored at − 20 °C in 1.5-ml Eppendorf tubes before DNA isolation.

Genomic DNA was extracted from colonies of 13 samples using the MagListo 5M Genomic DNA Extraction kit (Bioneer, Daejeon, Korea) according to the manufacturer’s instructions. The DNA concentration was determined at 220 nm on a NanoDrop 1000 spectrophotometer and sample quality was confirmed on a 1% agarose gel. The 600-bp 18S DNA gene was amplified using the universal fungal 18S DNA primers ITS1 and ITS4. PCR amplification was carried out in a 2720 thermal cycler (Applied Biosystems, Foster City, CA, USA) under the following conditions: 95 °C for 15 min; 35 cycles of 95 °C for 20 s, 53 °C for 40 s, and 72 °C for 90 s; and 72 °C for 5 min.

Classification and identification of M. persimmonesis

Purified PCR products were used to construct gene libraries and determine nucleotide sequences using a GS Junior Titanium Sequencing kit (Roche Diagnostics, Indianapolis, IN, USA). Sequence reads obtained by pyrosequencing were analyzed as previously described (Kim et al. 2012; Diouf et al. 2015). Sequences obtained with GS Junior were compared with the 18S rDNA gene sequences from the non-redundant sequence database using the NCBI Basic Local Alignment Search Tool (BLAST; v.2.2.30+) for taxonomic identification and analysis of microbial diversity. Each sequence was then classified taxonomically using NCBI Metagenome Analyzer (MEGAN; v.5.10.1), and the results were used for taxonomic classification. Isolates were classified according to the hierarchy of super-kingdom, kingdom, phylum, class, order, family, genus, and species. A rooted phylogenetic tree was generated by assigning all sequences to each node. Each group of identified microorganisms is presented according to the Integrated Taxonomic Information System (ITIS) taxonomic counting heat maps. Microsoft Excel (Redmond, WA, USA) was used for data analysis.

Growth characteristics of M. persimmonesis at various pH

Solutions of sterilized tryptic soy broth (TSB) medium (20 ml) with various pH were prepared and sterilized with a 0.2-μm pore filter. Culture broth was collected in TSA medium and resuspended in 3 ml TSB. A 100-μl volume of each suspension was inoculated and incubated at 28 °C for 2 days with shaking at 150 rpm. Cell viability was determined by measuring the turbidity at 595 nm with a spectrophotometer.

Growth characteristics of M. persimmonesis at various NaCl concentrations

The growth characteristics of isolates were evaluated in nutrient broth (NB; 20 ml) containing NaCl concentrations of 0, 0.1, 0.3, 0.5, 1, 2, 3, 4, 5, 7, 10, 12, 15, 18, 20, and 24% (with Ml). Cultures were grown on nutrient agar (NA) and then resuspended in 3 ml NB; 100 μl of culture were then incubated at 28 °C with shaking at 150 rpm for 2 days. Yeast growth was then determined by measuring the turbidity of cells at 595 nm with a spectrophotometer.

Growth characteristics of M. persimmonesis at various temperatures

Isolates were subcultured in TSA; cultures were transferred to 125-ml Erlenmeyer flasks containing 30 ml of TSB and incubated at temperatures of 4, 10, 15, 18, 20, 24, 28, 30, 35, 37, 40, 45, and 50 °C on a shaker at 160 rpm. Growth in TSB medium was evaluated by measuring turbidity at 640 nm at predetermined time intervals over 48 h at the above temperatures, as previously described (Janisiewicz et al. 2001).

Saccharide test

API 20 C AUX and API 5 CH (both from bioMérieux, Marcy l’Étoile, France) were used to identify strains for the sugar (carbon) availability test according to the manufacturer’s protocols. These tests include 19 carbohydrate assimilation test strains along with a negative control and are based on turbidity measurements. Isolates were cultured on PDA and TSA plates. Colonies were collected and re-suspended in 0.85% NaCl to obtain a MacFarland turbidity of 2. A 100-μl aliquot of the turbid solution was thoroughly mixed with 7 ml API 20 C medium ampoule 2, and the resultant mixture was transferred to API 20 C AUX and API 5 CH cups using a sterile pipette. Strips were read after incubation for 48 h at 30 °C.

Antimicrobial activity (inhibition) test

The disc-diffusion method (Balouiri et al. 2016) was used to evaluate antimicrobial activity of the isolates. Agar plates were inoculated with a standardized inoculum of Botrytis cinerea as the test microorganism. Filter paper discs containing predetermined concentrations of M. persimmonesis were placed on the agar surface. The plates were then incubated at 26 °C for 72 h, after which the diameter of growth inhibition zones was measured. The procedure was repeated using Fusarium oxysporum as the test organism instead of B. cinerea.

Antibiotic test

Antibiotic susceptibility was evaluated using 6-mm BBL Sensi-Discs (BD Biosciences, Franklin Lakes, NJ, USA) containing nystatin (50 μg), nystatin (30 μg), salinomycin (30 μg), amphotericin (2 μg), or gramicidin S (30 μg) and other antibiotics including teicoplanin, amikacin, ampicillin, lincomycin, nalidixic acid, kanamycin, gentamicin, streptomycin, neomycin, penicillin, vancomycin, erythromycin, oleandomycin, amoxicillin, spiramycin, rifampicin, polymixin B, bacitracin, tetracycline, cycloheximide, roxithromycin, sphingomyelin, apramycin, hygromycin B, capreomycin, sisomycin, phosphomycin, chloramphenicol, and streptomycin.

Results

Morphology

After 3 days of culture at 28 °C on PDA, cells were globose (3.0−7.0 × 4.0–9.0 µm) or ovoidal (6.0–9.0 µm) and grew as single cells or in small clusters (Fig. 2a). Budding cells and chlamydospores were common (Fig. 2a, b). Colonies were round, convex, smooth, and red-pigmented white.
Fig. 2

Metschnikowia persimmonesis KIOM_G15050 (KCTC 12991BP)

Growth curves characteristics of M. persimmonesis in PDB and LB medium

Metschnikowia persimmonesis that grew in PDA and TSA media (Fig. 3) was used for growth curve generation and strain characterization.
Fig. 3

Growth of M. persimmonesis on different media

Metschnikowia persimmonesis grew rapidly from 12 to 24 h after induction in PDB (Fig. 4). The growth rate subsequently slowed between 36 and 168 h and then increased slightly to reach a maximal value at 168 h. A similar trend was observed in LB medium, except that growth was maximal at 48 h and then decreased up until the end of the culture period at 168 h.
Fig. 4

Growth according to medium of Metschnikowia persimmonesis KIOM G15050 strain

Standard growth curve for M. persimmonesis

Samples cultured for 2 days were diluted to predetermined ratios and the number of cells was counted for samples with an absorbance of 1.0 or less to prepare a standard curve (Fig. 5). The equation of the line for the standard curve was determined as follows.
$${\text{Y }} = { 5}. 2 7 { } + { 5}. 1 8 {\text{x }}({\text{x:}}{\text{ absorbance}},{\text{ y:}}{\text{ Log no}}.{\text{ {of cells}}})$$
Fig. 5

Bacterium amount standard curve of Metschnikowia persimmonesis KIOM G15050 strain

Growth characteristics of M. persimmonesis at different pH

The growth of M. persimmonesis in TSB varied from pH 2.6 to 10.6. The growth rate increased with increases in pH up to pH 6.0 before declining (Table 1 and Fig. 6).
Table 1

Summary of growth tests and other test characteristics of M. persimmonesis

Tests

Results

Tests

Results

Tests

Results

Tests

Results

Tests

Results

Assimilation at 96 h of:

Oxidation at 96 h of:

Antimicrobial susceptibility test

API 2 °C AUX test

API 50 CH test

Water

Water

Teicoplanin

*

Glucose

+

Control

Fumaric acid

v

Acetic acid

v

Amikacin

*

Glycerol

+

Glycerol

+

l-Malic acid

v

Formic acid

Ampicillin

*

2-Keto-d-gluconate

+

Erythritol

Succinic acid mono-methyl ester

Propionic acid

Lincomycin

*

l-Arabinose

d-Arabinose

Bromo-succinic acid

Succinic acid

+

Nalidixic acid

*

d-Xylose

+

l-Arabinose

l-Glutamic acid

v

Succinic acid mono-methyl ester

v

Kanamycin

*

Adonitol

+

d-Ribose

γ-Amino-butyric acid

l-Aspartic acid

Gentamicin

*

Xylitol

+

d-Xylose

+

α-Keto-glutaric acid

l-Glutamic acid

v

Streptomycin

*

d-Galactose

+

l-Xylose

2-Keto-d-gluconic acid

v

l-Proline

v

Neomycin

*

Inositol

d-Adonitol

+

d-Gluconic acid

v

d-Gluconic acid

+

Penicillin

*

d-Sorbitol

+

Methyl-β-d-xylopyranoside

Dextrin

Dextrin

Vancomycin

*

α-Methyl-d-glucoside

+

d-Galactose

+

Inulin

Inulin

Erythromycin

*

N-Acetyl-d-glucosamine

+

Glucose

+

d-Cellobiose

+

Cellobiose

+

Oleandomycin

*

d-Cellobiose

+

d-Fructose

+

Gentiobiose

v

Genntiobiose

+

Amoxicillin

*

d-Lactose(bovine origin)

d-Mannose

+

Maltose

Maltose

+

Spiramycin

*

d-Maltose

+

l-Sorbose

+

Maltotriose

Maltotriose

+

Rifampicin

*

d-Saccharose (Sucrose)

+

l-Rhamnose

d-Melezitose

v

d-Melezitose

+

Polymixin B

*

d-Trehalose

Dulcitol

d-Melibiose

d-Melibiose

Nystatin

*+

d-Melezitose

+

Inositol

Palatinose

Palatinose

+

Bacitracin

*

d-Raffinose

d-Manntol

+

d-Raffinose

d-Raffinose

Tetracycline

*

  

d-Sorbitol

+

Stachyose

Stachyose

Cycloheximide

*+

  

Methyl-α-d-mannopyranoside

Sucrose

Sucrose

+

Roxithromycin

*

  

Methyl-α-d-glucoopyranoside

+

d-Trehalose

v

d-Trehalose

Sphingomyelin

*

  

N-Aceylglucosamine

+

Turanose

v

Turanose

+

Apramycin

*

  

Amygdalin

+

N-Acetyl-d-glucosamine

N-Acetyl-d-glucosamine

+

Salinomycin

*+

  

Arbutin

+

d-Glucosamine

v

α-d-Glucose

+

Hygromycin B

*

  

Esculin

+

α-d-Glucose

+

d-Galactose

v

Capreomycin

*

  

Salicin

+

d-Galactose

v

d-Psicose

Sisomycin

*

  

d-Celoibiose

+

d-Psicose

l-Sorbose

+

Amphotericin

*+

  

d-Maltose

+

l-Rhamnose

Salicin

v

Gramicidin S

*+

  

d-Lactose (bovine origin)

l-Sorbose

d-Mannitol

+

Phosphomycin

*

  

d-Melibiose

α-Methyl-d-glucoside

v

d-Sorbitol

+

Chloramphenicol

*

  

d-Saccharose (sucrose)

+

β-Methyl-d-glucoside

v

d-Arabitol

Streptomycin

*

  

d-Trehalose

+

Amygdalin

Xylitol

v

    

Inulin

Arbutin

v

Glycerol

    

d-Melezitose

+

Salicin

v

Tween 80

    

d-Rafinose

Maltitol

v

      

Amidon (starch)

d-Mannitol

v

      

Glycogen

d-Sorbitol

      

Xylitol

+

Adonitol

v

      

Gentiobiose

+

d-Arabitol

      

d-Turanose

+

Xylitol

v

      

d-Lyxose

i-Erythritol

      

d-Tagatose

Glycerol

v

      

d-Fucose

Tween 80

      

l-Fucose

l-Arabinose

      

d-Arabitol

+

d-Arabinose

      

l-Arabitol

d-Ribose

      

Potassium glucoaate

+

d-Xylose

+

      

Potassium 2-ketogluconate

+

Succinic acid mono-methyl ester plus d-xylose

v

      

Potassium 5-ketogluconate

N-Acetyl-l-glutamic acid plus d-xylose

v

        

Quinic acid plus d-xylose

+

        

d-Glucuronic acid plus d-xylose

v

        

Dextrin plus d-xylose

v

        

α-d-Lactose plus d-xylose

v

        

d-Melibiose plus d-xylose

v

        

d-Galactose plus d-xylose

+

        

m-Inositol plus d-xylose

v

        

1,2- Propanediol plus d-xylose

v

        

Acetoin plus d-xylose

v

        

V , variable; −, negative; +, positive; *+, sensitive or susceptible to the antibiotic; *−, resistant to the antibiotic

Fig. 6

Growth of M. persimmonesis in different pH range

Growth characteristics of M. persimmonesis at different NaCl concentrations

Metschnikowia persimmonesis was cultured in nutrient broth with different NaCl concentrations. M. persimmonesis exhibited maximal growth in the absence of NaCl; the growth rate was inversely related to NaCl concentration, with the lowest rate recorded at 24% NaCl (Fig. 7).
Fig. 7

Growth of M. persimmonesis in different sodium chloride concentration

Growth temperature

Metschnikowia persimmonesis was inoculated on TSA and cultured at temperatures ranging from 4 to 50 °C. Growth was inhibited at higher temperatures (45 and 50 °C) and the optimum growth temperature was 25 °C.

Physiological tests

After incubation for 24–48 h, M. persimmonesis cultured on PDA and TSA was tested for carbon source availability using API 20 C AUX and API 5 CH. M. persimmonesis was found to metabolize glycerol, d-xylose, d-adonitol, d-galactose, glucose, d-fructose, d-mannose, mannose, l-sorbose, d-mannitol, d-sorbitol, methyl-α-d-glucopyranoside, glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, esculin, salicin, d-cellobiose, d-maltose, d-saccharose (sucrose), d-trehalose, d-melezitose, xylitol, and zenose. Carbohydrates added as a carbon source included gentiobiose, d-turanose, d-arabitol, potassium gluconate, and potassium 2-ketogluconate. However, erythritol, d-arabinose, l-arabinose, d-ribose, l-xylose, d-xylopyranoside, l-rhamnose, dulcitol, inositol, d-methyl-α-d-mannopyranoside, d-lactose (bovine origin), d-melibiose, insulin, d-raffinose (d-lactose, amidon (starch), glycogen, d-lyxose, d-tagatose, d-fucose), l-fucose, l-arabitol, and potassium 5-ketogluconate carbohydrates were not used as carbon sources. We also found that d-sorbitol, α-methyl-d-glucoside, d-maltose, d-saccharose (sucrose), and d-melezitose were used as a carbon source and induced pigment production (Table 1).

Discussion

Metschnikowia represents one of the most phylogenetically diverse genera of ascomycetous yeast comprising of 35 known species in a wide range of habitats (Kuan et al. 2016), and yet are difficult to distinguish due to similarities in assimilation reactions among different species (Kurtzman and Droby 2002). Both M. persimmonesis and M. fructicola show very similar assimilation reactions, apart from the fact that they exhibit negative and positive reactions, respectively, to l-sorbose, sucrose, and maltose (Table 1). A microscopic examination revealed that M. persimmonesis has unique morphology, including a thick wall and elongated asci (Fig. 2). But since these characteristics are also shared by M. fructicola (Kurtzman and Droby 2002), the growth test and morphological features alone cannot be used to distinguish M. persimmonesis from M. fructicola. The use of phylogenetic analysis of conserved DNA and protein sequences is vital in the taxonomical classification (Cummings et al. 2013). In fact, recent study showed that pyrosequencing makes it possible to simultaneously profile large numbers of samples (Chun et al. 2010) using sequence repeats of the chromosomal rDNA array. Indeed, rDNA repeats within a genome have identical sequences due to sequence homogenization, and can therefore be used for species identification and barcoding (Sipiczki et al. 2013). The compared sequences generated by GS Junior to the NCBI nucleotide sequence database using BLAST gave a resultant file that was saved in xml format, and taxonomic classification was performed using NCBI taxonomic information and MEGAN v.5.10.6. More than 660,000 taxon types were returned by the search, with individual species classified in a hierarchical manner (super kingdom, kingdom, phylum, class, order, family, genus, and species). MEGAN provided a lowest common ancestor assignment algorithm that was used to assign the lowest taxon to each sequence. All sequences were assigned to each node to complete the rooted phylogenetic tree. The generated IT IS taxonomic counting heat maps were used to complete the identification of M. persimmonesis and the entries in Genbank placed this strain of yeast as a sister clade of M. fructicola (Fig. 8).
Fig. 8

Phylogenic tree indicating position of M. persimmonesis in genus Metschnikowia

The maximum growth rate of M. persimmonesis in TSB was recorded at pH 6.0 (Table 1 and Fig. 7). This is within the same pH range for other species within this genus such as M. andauensis, which showed maximal growth at pH 6.5 (Manso et al. 2014) and M. pulcherrima, which showed maximum growth rate at optimum pH ranging from 5.0 to 7.5 (Spadaro et al. 2010). These results therefore indicate that the optimal pH for growth of Metschnikowia species is between pH 5–7.5.

The decrease in growth of M. persimmonesis with increase in Sodium chloride concentration is related to previous studies on some members of genus Metschnikowia in which the growth of M. arizonensis and M. dekortorum on yeast mold agar with 5% NaCl was weakly positive and was inhibited at a just a concentration of 10% (Lachance and Bowles 2002). Indeed, yeast growth decreases with increasing NaCl concentration (Speakman et al. 1928). Thus, growth inhibition of M. persimmonesis at 24% NaCl concentration (Fig. 8) further proves that low tolerance to NaCl may be a general characteristic of Metschnikowia species.

The optimum growth temperature (25 °C) for M. persimmonesis is within the same optimum growth temperature range for some of the members of genus Metschnikowia. For instance, an acid protease from the marine yeast M. reukaufii W6b exhibited maximal activity at 25 °C (Li et al. 2010), which was the temperature that promoted optimum growth of M. pulcherrima (Csutak et al. 2007). In fact, most yeasts have optimal growth temperatures between 20 and 25 °C (Kurtzman and Fell 1998).

Metschnikowia persimmonesis exhibited strong antibacterial activity against B. cinerea and F. oxysporum (Fig. 9) and was resistant to both antibiotics and streptomycin.
Fig. 9

Inhibition test of M. persimmonesis on Fusarium oxysporum and Botrytis cineria

Thus, the susceptibility of PDA was lower than that of amphotericin and gramicidin S. Studies have showed that a number of members of genus Metschnikowia are used in post-harvest control of diseases. M. fructicola has been used to manage post-harvest fruit rot (Kurtzman and Droby 2002), whereas M. pulcherrima has been used against blue mold in apple (Janisiewicz et al. 2001). As shown by size of zone of inhibition (Fig. 10), the high antimicrobial activity of M. persimmonesis may also be explored for biocontrol of post-harvest diseases.
Fig. 10

Diameter of growth inhibition zones by M. persimmonesis on Fusarium oxysporum at 4 days

Abbreviations

KCTC: 

Korean Collection for Type Cultures

KIOM: 

Korea institute of oriental medicine

LB: 

Luria-Bertani

PD: 

potato dextrose

PDA: 

potato dextrose agar

PDB: 

potato dextrose broth

TSA: 

tryptic soy agar

PCR: 

polymerase chain reaction

NCBI: 

National Center for Biotechnology Information

TSB: 

tryptic soy broth

BLAST: 

Basic Local Alignment Search Tool

Declarations

Authors’ contributions

YK and JC isolated the new novel strain for the very first time. All authors contributed during experimental work and scientific writing of this manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

Data has been deposited at a publicly available repository at Korean Collection for Type Cultures and the genomic information submitted to National Center for Biotechnology Information.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Funding

This work was supported by grants from Development of Anti-fungicide using Korean Herbal Medicine-Associated New Yeast Strain (KIOM G15050) from the Diospyros Kaki Calyx (K17690) and Development of Foundational Techniques for the Domestic Production of Authentic Herbal Medicines based on the Establishment of Molecular Authentication System (K17403) at Korea Institute of Oriental Medicine through the Ministry of Science, ICT & Future Planning, Republic of Korea.

Publisher’s Note

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
Korea Institute of Oriental Medicine (KIOM)
(2)
University of Science & Technology (UST), Korean Medicine Life Science
(3)
Natural Chemotherapeutics Research Institute (NCRI), Ministry of Health
(4)
Bioresources Industrialization Research Division, Nakdonggang National Institute of Biological Resources
(5)
Department of Food Science, Institute of Fusion Biotechnology, Gyeongnam National University of Science and Technology
(6)
Forest Research Department, Gyeongsangnam-do Forest Environment Research Institute
(7)
AtoGen Co., Ltd.
(8)
Korea Research Institute of Bioscience and Biotechnology (KRIBB)
(9)
Department of Food Science and Biotechnology, Chungbuk National University

References

  1. Álvarez-Pérez S, de Vega C, Pozo IM, Lenaerts M, Assche VA, Herrera MC, Lievens B (2016) Nectar yeasts of the Metschnikowia clade are highly susceptible to azole antifungals widely used in medicine and agriculture. FEMS Yeast Res 16:fov115. https://doi.org/10.1093/femsyr/fov115 View ArticlePubMedGoogle Scholar
  2. Balouiri M, Sadiki M, Ibnsouda SK (2016) Methods for in vitro evaluating antimicrobial activity: a review. JPA 6:71–79. https://doi.org/10.1016/j.jpha.2015.11.005 Google Scholar
  3. Buée M, Reich M, Murat C, Morin E, Nilsson R, Uroz SH, Martin F (2009) 454 pyrosequencing analyses of forest soils reveal an unexpectedly high fungal diversity. New Phytol 184(2):449–456. https://doi.org/10.1111/j.1469-8137.2009.03003.x (Epub 2009 Aug 22) View ArticlePubMedGoogle Scholar
  4. Canto A, Herrera CM, García IM, García M, Bazaga P (2015) Comparative effects of two species of floricolous Metschnikowia yeasts on nectar. Anales Jard Bot Madr 72(1):019. https://doi.org/10.3989/ajbm.2396 Google Scholar
  5. Choi JE, Choi HS, Lee CK, Kang MS, Kim IJ, Choi SM, Kim GH, Seo TW, Lee YK, Moon BC, Yang MY (2017) Analysis of microbial communities in local cultivars of astringent persimmon (Diospyros kaki) fruits grown in Gyeongnam province of Korea. J Environ Biol. https://doi.org/10.22438Google Scholar
  6. Chun J, Kim YK, Lee HJ, Choi Y (2010) The analysis of oral microbial communities of wild-type and toll-like receptor 2-deficient mice using a 454 GS FLX Titanium pyrosequencer. BMC Microbiol 10:101. https://doi.org/10.1186/1471-2180-10-101 View ArticlePubMedPubMed CentralGoogle Scholar
  7. Csutak O, Vassu T, Cornea P, Grebenisan I (2007) Genetic characterization of two new Metschnikowia strains with antifungal activity. Romanian Biotech Lett 12(2):3175–3182Google Scholar
  8. Csutak O, Vassu T, Sarbu I, Stoica I, Cornea P (2013) Antagonistic activity of three newly isolated yeast strains from the surface of fruits. Food Technol Biotechnol 51(1):70–77Google Scholar
  9. Cummings PJ, Ahmed R, Durocher JA, Jessen A, Vardi T, Obom MK (2013) Pyrosequencing for microbial identification and characterization. J Vis Exp 78:e50405. https://doi.org/10.3791/50405 Google Scholar
  10. De Vega C, Guzman B, Steenhuisen LS, Johnson DS, Herrera MC, Lachance M (2014) Metschnikowia drakensbergensis sp. nov. and Metschnikowia caudata sp. nov., endemic yeasts associated with Protea flowers in South Africa. Int J Syst Evol Microbiol 64:3724–3732. https://doi.org/10.1099/ijs.0.068445-0 View ArticlePubMedGoogle Scholar
  11. Diouf M, Roy V, Mora P, Frechault S, Lefebvre T, Hervé V, Lefèvre RC, Miambi E (2015) Profiling the succession of bacterial communities throughout the life stages of a higher termite Nasutitermes arborum (Termitidae, Nasutitermitinae) using 16S rRNA gene pyrosequencing. PLoS ONE. https://doi.org/10.1371/journal.pone.0140014 Google Scholar
  12. Giordani E, Doumett S, Nin S, Bubba MD (2011) Selected primary and secondary metabolites in fresh persimmon (Diospyros kaki Thunb.): a review of analytical methods and current knowledge of fruit composition and health benefits. Food Res Int 44:1752–1767. https://doi.org/10.1016/j.foodres.2011.01.036 View ArticleGoogle Scholar
  13. Guzmán B, Lachance M, Herrera MC (2013) Phylogenetic analysis of the angiosperm-floricolous insect–yeast association: have yeast and angiosperm lineages co-diversified? Mol Phylogenet Evol 68:161–175. https://doi.org/10.1016/j.ympev.2013.04.003 View ArticlePubMedGoogle Scholar
  14. Heo J (2001) The precious mirror of oriental medicine [Dong-uibogam]. Namsandang Press, SoulGoogle Scholar
  15. Janisiewicz WJ, Tworkoski TJ, Kurtzman CP (2001) Biocontrol potential of Metcnikowia pulcherrima strains against blue mold of apple. Phytopathology 91(11):1098–1108. https://doi.org/10.1094/PHYTO.2001.91.11.1098 View ArticlePubMedGoogle Scholar
  16. Kim OS, Cho JY, Lee K, Yoon HS, Kim M, Na H, Park CS, Jeon SY, Lee HJ, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721. https://doi.org/10.1099/ijs.0.038075-0 View ArticlePubMedGoogle Scholar
  17. Kotani M, Matsumoto M, Fujita A, Higa S, Wang W, Suemura M, Kishimoto T, Tanaka T (2000) Persimmon leaf extract and astragalin inhibit development of dermatitis and IgE elevation in NC/Nga mice. J Allergy Clin Immunol 106(1):159–166. https://doi.org/10.1067/mai.2000.107194 View ArticlePubMedGoogle Scholar
  18. Kuan PCS, Ismail R, Kwan Z, Yew SM, Yeo SK, Chan CL, Toh FY, Na LS, Lee WK, Hoh CC, Yee YW, Ng PK (2016) Isolation and characterization of an Atypical Metschnikowia sp. strain from the skin scraping of a dermatitis. PLOS ONE. https://doi.org/10.1371/journal.pone.0156119 Google Scholar
  19. Kurtzman CP, Droby S (2002) Metschnikowia fructicola, a new ascosporic yeast with potential for biocontrol of postharvest fruit rots. Syst Appl Microbiol 24:395–399. https://doi.org/10.1078/0723-2020-00045 View ArticleGoogle Scholar
  20. Kurtzman C, Fell JW (1998) The yeasts, a taxonomic study. Elsevier-Science, AmsterdamGoogle Scholar
  21. Lachance MA, Bowles JM (2002) Metschnikowia arizonensis and Metschnikowia dekortorum, two new large-spored yeast species associated with floricolous beetles. FEMS Yeast Res 2:81. https://doi.org/10.1111/j.1567-1364.2002.tb00072.x PubMedGoogle Scholar
  22. Lachance MA, Ewing CP, Bowles JM, Starmer WT (2005) Metschnikowia hamakuensis sp. nov., Metschnikowia kamakouana sp. nov and Metschnikowia mauinuiana sp. nov., three endemic yeasts from Hawaiian nitidulid beetles. Int J Syst Evol Microbiol 55:1369–1377. https://doi.org/10.1099/ijs.0.63615-0 View ArticlePubMedGoogle Scholar
  23. Li J, Peng Y, Wang X, Chi Z (2010) Optimum production and characterization of an acid protease from marine yeast Metschnikowia reukaufii W6b. JOUC 9(4):359–364. https://doi.org/10.1007/s11802-010-1765-2 Google Scholar
  24. Mager WH, Winderickx J (2005) Yeast as a model for medical and medicinal research. Trends Pharmacol Sci 26(5):265–273. https://doi.org/10.1016/j.tips.2005.03.004 View ArticlePubMedGoogle Scholar
  25. Mallavadhani UV, Panda KA, Rao RY (1998) Review article number 134 pharmacology and chemotaxonomy of diospyros. Phytochemistry 49(4):901–951. https://doi.org/10.1016/S0031-9422(97)01020-0 View ArticlePubMedGoogle Scholar
  26. Manso T, Nunes C, Lima-Costa M (2014) Growth kinetics of the biocontrol agent Metschnikowia andauensis PBC-2 in submerged batch cultures. IJAFST 2(4):1–14Google Scholar
  27. Marinoni G, Piškur J, Lachance MA (2006) Ascospores of large-spored Metschnikowia species are genuine meiotic products of these yeasts. FEMS Yeast Res 3(1):85–90Google Scholar
  28. Mendonca-Hagler LC, Hagler AN, Kurtzman CP (1993) Phylogeny of Metschnikowia species estimated from partial rRNA sequences. Int J Syst Bacteriol 43(2):368–373. https://doi.org/10.1099/00207713-43-2-368 View ArticlePubMedGoogle Scholar
  29. Pretorius IS (2000) Review-tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking. Yeast 16:675–729. https://doi.org/10.1002/1097-0061(20000615)16:8<675:AID-YEA585>3.0.CO;2-B View ArticlePubMedGoogle Scholar
  30. Scholler M (2003) Fusicladium levieri, a fungal parasite of persimmon, found in Indiana. Proc Indiana Acad Sci 112(2):132–134Google Scholar
  31. Serkebaeva YM, Kim Y, Liesack W, Dedysh SN (2013) Pyrosequencing-based assessment of the bacteria diversity in surface and subsurface peat layers of a northern wetland, with focus on poorly studied phyla and candidate divisions. PLoS ONE 8(5):e63994. https://doi.org/10.1371/journal.pone.0063994 View ArticlePubMedPubMed CentralGoogle Scholar
  32. Sipiczki W, Pfliegler WP, Holb IJ (2013) Metschnikowia species share a pool of diverse rRNA genes differing in regions that determine hairpin-loop structures and evolve by reticulation. PLoS ONE 8(6):e67384. https://doi.org/10.1371/journal.pone.0067384 View ArticlePubMedPubMed CentralGoogle Scholar
  33. Spadaro D, Sabettab W, Acquadroc A, Portisc E, Garibaldib A, Gullinob ML (2008) Use of AFLP for differentiation of Metschnikowia pulcherrima strains for postharvest disease biological control. Microbiol Res 163:523–530. https://doi.org/10.1016/j.micres.2007.01.004 View ArticlePubMedGoogle Scholar
  34. Spadaro D, Ciavorella A, Dianpeng Z, Garibaldi A, Gullino ML (2010) Effect of culture media and pH on the biomass production and biocontrol efficacy of a Metschnikowia pulcherrima strain to be used as a biofungicide for postharvest disease control. Can J Microbiol 56(2):128–137. https://doi.org/10.1139/w09-117 View ArticlePubMedGoogle Scholar
  35. Speakman HB, Gee AH, Luck JM (1928) The influence of sodium chloride on the growth and metabolism of yeast. J Bacteriol 15(5):319–340PubMedPubMed CentralGoogle Scholar
  36. Tsubaki S, Ozaki Y, Yonemori K, Azuma J (2012) Mechanical properties of fruit-cuticular membranes isolated from 27 cultivars of Diospyros kaki Thunb. Food Chem 132:2135–2139. https://doi.org/10.1016/j.foodchem.2011.12.039 View ArticleGoogle Scholar
  37. Türkel S, Korukluoğlu M, Yavuz M (2014) Biocontrol activity of the local strain of Metschnikowia pulcherrima on different postharvest pathogens. Biotechnol Res Int 2014:6. https://doi.org/10.1155/2014/397167 View ArticleGoogle Scholar
  38. VanderMolen KM, Raja HA, El-Elimat T, Oberlies NH (2013) Evaluation of culture media for the production of secondary metabolites in a natural products screening program. AMB Express 3:71. https://doi.org/10.1186/2191-0855-3-71 View ArticlePubMedPubMed CentralGoogle Scholar

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© The Author(s) 2017