Yeasts in the Flowers of Wild Fleabane [Erigeron annus (L.) Pers.]

  • cc icon
  • ABSTRACT

    BACKGROUND

    Yeasts associated with fleabane flowers were identified using isolation methods previously applied in yeast biotechnology. A culture-based approach was required for isolation of many yeast strains associated with fleabane.

    METHODS AND RESULTS

    We spread homogenized fleabane flowers onto GPY medium containing chloramphenicol, streptomycin, Triton X-100, and L-sorbose. We isolated 79 yeast strains from the flowers of wild fleabane, and identified the yeasts via phylogenetic analysis of isolates from agar plates. The yeast species included 39 isolates of Aureobasidium pullulans, 17 of the genus Candida, 14 of the genus Rhodosporidium, 6 of the genus Cryptococcus, and 3 of the genus Rhodotorula.

    CONCLUSION

    Yeast isolates associated with fleabane flowers included A. pullulans (39 isolates) and other yeast species (40 isolates). Such yeast isolates may have biotechnological potential.


  • KEYWORD

    Fleabane , Erigeron annus (L.) Pers. , ITS gene , Wild yeast

  • Introduction

    Erigeron annus (L.) Pers. is an annual or biennial herb of the Compositae, and is a North America native pioneer species that often colonizes disturbed areas such as pastures, vacant fields, roadsides, railways, and waste areas; the herb was introduced to Korea in 1910 (Park, 2009). Flowering occurs in June and July, cephalization is white, the ligulate flower is pistillate, and the tubular flower is yellow with a long pappus (Korea National Arboretum, 2012). The leaves are edible and are commonly used in folk medicine to treat indigestion, stomach ache, diarrhea caused by enteric pathogens, and hypoglycemia (Lee, 1996; Lee, 2003; Yoo, et al., 2008). Little is known, however, about the ecological roles of plant yeasts.

    Yeasts play roles in many complex processes of various ecosystems such as plant tissues (stems, flowers, and fruits); insects; soils; aquatic environments; and extreme environments (Fonseca and Inácio, 2006; Raspor and Zupan, 2006; Botha, 2011). Industrial attributes of yeasts include the primary roles they play in many food fermentations yielding beers, ciders, wines, sake, distilled spirits, bakery products, industrial enzymes, and agricultural products (Deak, 2009; Tamang and Fleet, 2009).

    In this study, we characterized the yeast species associated with fleabane flowers. Previous studies have attempted to do the same, but their sampling strategies were casual or insufficient. Moreover, only minimal phylogenetic analyses of cultural yeast isolates have been performed in the past (Halloran et al., 2013); therefore, our results provide a basis for future studies on yeast biotechnology.

    Materials and Methods

      >  Yeast isolation

    Erigeron annus (L.) Pers. samples were aseptically collected from a residential area in Uljin with the aid of autoclaved scissors and forceps, and placed in clean plastic bags. Flower samples were stored in a cooler during transfer to the laboratory (in Uljin) and processed the same day. Each sample was washed three times with 10 mM potassium phosphate buffer and stored in an autoclaved container. Several washed flower samples were placed in tubes (Falcon Plastics, Los Angeles, CA, USA) filled to 10 mL with 10 mM potassium phosphate buffer and homogenized using an autoclavable hand homogenizer (T10 Basis; IKA, Staufen, Germany). Homogenized samples (1 mL) were placed on sterile solid media, plated using a glass spreader, and incubated at 25℃ for 2-5 days. Yeast colonies that grew on large plates (Nunc Bioassay dishes, 245 × 245 × 25 mm, Thermo Scientific, Roskilde, Denmark) were selected with autoclaved toothpicks and inoculated into 96-deep-well plates (Assay Block, 2 mL/well, 96 well square v-bottom; Costar, Cambridge, MA, USA) prior to liquid culture at 25C at 800 rpm for 48 hours (Choi et al., 2013).

    The media used for screening included (all % values are w/w): dichloran-glycerol 18% (DG18) agar (MB Cell, Seoul, Korea); dropout base (DOB) with complete amino acid supplement mixture (CSM) agar (MP Bio, Santa Ana, CA, USA); GPY agar (4% glucose, 0.5% peptone, 0.5% yeast extract, and 1.5% agar); and Sabouraud chloramphenicol gentamicin (SCG) agar (MB Cell). Antibiotics (100 mg/L of both chloramphenicol and streptomycin) were added to each medium to repress bacterial growth, and 0.1% (v/v) Triton X-100 and 0.4% (w/v) L-sorbose were added to repress fungal growth. Yeasts were cultured on DG18, DOB with CSM, GPY, and SCG agar media in square plates (245 × 245 × 25 mm). All colonies from plates yielding multiple colonies were picked and cultured separately. In total, 79 individual isolates were transferred to fresh plates three times and then processed for sequencing of the internal transcribed spacer (ITS) genes.

      >  Sequencing and phylogenetic analysis

    The detailed methodology of sequencing and phylogenetic analysis has been previously published(Choi et al., 2013). The primer set used to amplify ITS genes from the yeast strains were the previously described ITS1 (5′-TCCGTAGGTGAACCTGCG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers (White et al., 1990). For polymerase chain reaction (PCR), DNA was extracted from yeast colonies growing on agar plates using the Instagene Matrix method, according to the manufacturer’s protocol (Bio-Rad Laboratories, Hercules, CA, USA).

    PCR was performed with 20 ng genomic DNA as template in a 30-μL reaction volume containing EF-Taq DNA polymerase (Solgent, Daejeon, Korea). The PCR program included the following steps: 95℃ for 5 minutes; followed by 35 cycles of 95℃ for 2 minutes, 55℃ for 60 s, and 72℃ for 60 seconds; and a final extension step for 10 min at 72℃. Amplification products were purified using a multiscreen filter plate (Millipore Corp., Bedford, MA, USA). Sequencing was performed using a PRISM BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Hi-Di formamide (Applied Biosystems) was added to DNA samples containing the extension products. The mixtures were incubated at 95℃ for 5 minutes followed by 5 minutes on ice, and analyzed on an ABI Prism 3730XL DNA Analyzer (Applied Biosystems). DNA sequencing was performed by Macrogen Inc. (Seoul, Korea). Nucleotide sequences for the 79 isolates reported in this paper were deposited in DDBJ(DNA Data Bank of Japan)/GenBank under the following accession numbers: LC018741-LC018819.

      >  Phylogenetic analysis of Aureobasidium pullulans interspecies

    The nucleotide sequences of the ITS genes were aligned using the ClustalW2 program of the EMBL-EBI website. A BLAST search was used to identify the GenBank sequences most closely related to those of the yeast isolates. Phylogenetic trees were constructed using the neighbor-joining method, using MEGA5 for Windows (Tamura et al., 2011), and featured bootstrap analyses of 1,000 samples. Evolutionary distances were calculated using the Kimura two-parameter method (Saitou and Nei, 1987).

    Results and Discussion

    Here, yeast isolates colonizing fleabane flowers were isolated and phylogenetically analyzed. Seventy-nine strains from whole yeast isolates were identified from a single plate of homogenized flower samples. The strains included, A. pullulans (39 isolates), Candida (17 isolates), Rhodosporidium (14 isolates), Cryptococcus (6 isolates), and Rhodotorula (3 isolates) (Fig. 1). Shown in Fig. 1 is a summary phylogram of the yeast isolates. A. pullulans, Candida spp., and Rhodosporidium spp. dominated the yeast composition of the plant flower, representing 49%, 21%, and 18% of the isolates, respectively. The least abundant yeasts were Cryptococcus spp. (8%) and Rhodotorula spp. (4%). Among the isolates were several species, including C. tropicalis (11 isolates), C. parapsilosis (6 isolates), Rhodosporidium azoricum (13 isolates), and R. fluvial (1 isolate) for which there was little prior knowledge regarding their habitat and function (Käppeli and Fiechter, 1977). Karatay and Dönmez (2010) showed that C. tropicalis and R. mucilaginosa produce biodiesel in media containing molasses, while Rhodosporidium isolates were also shown to produce biodiesel in a snow crab study (unpublished data). As shown in Fig. 1, this is the first yeast composition to be defined in fleabane flowers. Specially, five genera colonized the flowers, and future studies should explore community structure. In addition, A. pullulans, Candida, and Rhodosporidium were major colonizers, suggesting that yeast strains may be plant-specific.

    Based on BLAST searches and phylogenetic analyses, we found several interspecies of A. pullulans (Fig. 2). A. pullulans is known as black yeast because it produces melanin. Previous data showed that this yeast is the dominant species on the flower, leaf and stem of tiger lily, covering 97%, 35%, and 42% of each of these surfaces, respectively (Kim and Kim, 2015). To identify species and/or interspecies, deep-clade phylogenetic analyses of the A. pullulans isolates was performed, revealing that the yeast strains were most closely related to Group I (37 isolates) and Group II (2 isolates), while none belonged to Groups III or IV. In Group I, isolate F-97 was separated from the rest of the group. Isolates F-3 through F-99 and the remaining 25 isolates were not related to Group I. Generally, A. pullulans produces polysaccharides, including pullulan and β-glucan, which find industrial and medical applications (Yurlova and de Hoog, 1997; Cheng et al., 2011; Muramatsu et al., 2012). Recently, A. pullulans has been shown to produce (poly)malic acid (Nagata et al., 1993), lipase (Leathers et al., 2013), laccase (Rich et al., 2011), mannitol oils (Price et al., 2013), biocontrol agents (Mari et al., 2012), biosurfactants (Kim et al., 2015), valuable lipids (Turk et al., 2004), and siderophores (Ma et al., 2012). A. pullulans from plant flowers have also been found to produce several biosurfactants, depending on their phylogenetic class (unpublished data).

    The present study identified the main yeast isolates from fleabane, including a phylogenetic assessment of the A. pullulans isolates. However, the roles played by yeasts (including A. pullulans) in host plants still require further exploration. Future studies should address the distribution of yeast communities using an extensive sampling strategy for more robust group assessments.

  • 1. Botha A. 2011 The importance and ecology of yeasts in soil [Soil Biology and Biochemistry] Vol.43 P.1-8 google doi
  • 2. Cheng K. C., Demirei A., Catchmark J. M. 2011 Pullulan: biosynthesis, production, and applications [Applied Microbiology and Biotechnology] Vol.92 P.29-44 google doi
  • 3. Choi S. C., Kim M. U., Kim J. S. 2013 Selective isolation and phylogeny of the yeast species associated with Aloe vera and Aloe saponaria [Korean Journal of Environmental Agriculture] Vol.32 P.240-243 google doi
  • 4. Deak T., Satyanarayana G., Kunze G. 2009 Ecology and biodiversity of yeasts with potential value in biotechnology. Yeast biotechnology: diversity and applications P.151-168 google
  • 5. Fonseca A., Inacio J., Rosa C. A., Peter G. 2006 Phylloplane yeasts. Biodiversity and ecophysiology of yeast P.263-301 google
  • 6. Halloran S. T., Mauck K. E., Fleischer S. F., Tumlinson J. H. 2013 Volatiles from intact and Lygus-damaged Erigeron annuus (L.) Pers. are highly attractive to ovipositing Lygus and its parasitoid Peristenus relictus Ruthe [Journal of Chemical Ecology] Vol.39 P.1115-1128 google doi
  • 7. Kappeli O., Fiechter A. 1977 Component from the cell surface of the hydrocarbon-utilizing yeast Candida tropicalis with possible relation to hydrocarbon transport [Journal of Bacteriology] Vol.131 P.917-921 google
  • 8. Karatay S. E., Donmez G. 2010 Improving the lipid accumulation properties of the yeast cells for biodiesel production using molasses [Bioresource Technology] Vol.101 P.7988-7990 google doi
  • 9. Kim J. S., Kim D. S. 2015 Phylogeny of the yeast species isolated from wild tiger lily (Lilium lancifolium Thunb.) [Korean Journal of Environmental Agriculture] Vol.34 P.149-154 google doi
  • 10. Kim J. S., Lee I. K., Yun B. S. 2015 A novel biosurfactant production by Aureobasidium pullulans L3-GPY from a tiger lily wild flower, Lilium lancifolium Thunb [PLoS One] Vol.10 P.e0122917 google doi
  • 11. 2012 Field Guide Naturalized Plants of Korea P.239 google
  • 12. Leathers T. D., Rich J. O., Anderson A. M., Manitchotpisit P. 2013 Lipase production by diverse phylogenetic clades of Aureobasidium pullulans [Biotechnology Letters] Vol.35 P.1701-1706 google doi
  • 13. Lee T. B. 2014 Coloured Flora of Korea P.327 google
  • 14. Lee W. T. 1996 Coloured Standard Illustrations of Korean Plants P.360 google
  • 15. Ma Z. C., Chi Z., Geng Q., Zhang F., Chi Z. M. 2012 Disruption of the pullulan synthetase gene in siderophore-producing Aureobasidium pullulans enhances siderophore production and simplifies siderophore extraction [Process Biochemistry] Vol.47 P.1807-1812 google doi
  • 16. Mari M., Martini C., Spadoni A., Rouissi W., Bertolini P. 2012 Biocontrol of apple postharvest decay by Aureobasidium pullulans [Postharvest Biology and Technology] Vol.73 P.56-62 google doi
  • 17. Muramatsu D., Iwai A., Aoki S., Uchiyama H., Kawata K., Nakayama Y., Nikawa Y., Kusanbo K., Okabe M., Miyazaki T. 2012 β-Glucan derived from Aureobasidium pullulans is effective for the prevention of influenza in mice [PLoS One] Vol.7 P.e41399 google doi
  • 18. Nagata N., Nakahara T., Tabuchi T. 1993 Fermentation production of poly(β-L-malic acid), a polyelectrolytic biopolyester, by Aureobasidium sp [Bioscience, Biotechnology, and Biochemistry] Vol.57 P.638-642 google doi
  • 19. Park S. H. 2009 New illustration and Photographs of Naturalized Plants of Korea P.124 google
  • 20. Price N. P. J., Manitchotpisit P., Vermillion K. E., Bowman M. J., Leathers T. D. 2013 Structural characterization of novel extracellular liamocins (mannitol oils) produced by Aureobasidium pullulans strain NRRL 50380 [Carbohydrate Research] Vol.370 P.24-32 google doi
  • 21. Raspor P., Zupan J., Rosa C. A., Peter G. 2006 Yeast in extreme environments. Biodiversity and ecophysiology of yeasts P.370-417 google
  • 22. Rich J. O., Manitchotpisit P., Peterson S. W., Leathers T. D. 2011 Laccase production by diverse phylogenetic clades of Aureobasidium pullulans [Rangsit Journal of Arts and Sciences] Vol.1 P.41-47 google
  • 23. Saitou N., Nei M. 1987 The neighbor-joining method: a new method for reconstructing phylogenetic trees [Molecular Biology and Evolution] Vol.4 P.406-425 google
  • 24. Tamang J. P., Fleet G. H., Satyanarayana G., Kunze G. 2009 Yeasts diversity in fermented foods and beverages. Yeast biotechnology: diversity and applications P.169-198 google
  • 25. Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. 2011 MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods [Molecular Biology and Evolution] Vol.28 P.2731-2739 google doi
  • 26. Turk M., Mejanelle L., ?entjurc M., Grimalt J. O., Gunde-Cimerman N., Plemenita? A. 2004 Saltinduced changes in lipid composition and membrane fluidity of halophilic yeast-like melanized fungi [Extremophiles] Vol.8 P.53-61 google doi
  • 27. White T. J., Bruns T., Lee S., Taylor J. W., Innis M. A., Gelfand D. H., Sninsky J. J., White T. J. 1990 Amplicationand direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications P.315-322 google
  • 28. Yoo N. H., Jang D. S., Yoo J. L., Lee Y. M., Kim Y. S., Cho J. H., Kim J. S. 2008 Erigeroflavanone, a flavanone derivative from the flowers of Erigeron annuus with protein glycation and aldose reductase inhibitory activity [Journal of Natural Products] Vol.71 P.713-715 google doi
  • 29. Yurlova N. A., de Hoog G. S. 1997 A new variety of Aureobasidium pullulans characterized by exopolysaccharide structure, nutritional physiology and molecular features [Antonie Van Leeuwenhoek] Vol.72 P.141-147 google doi
  • [Fig. 1.] (A) Pi chart, and (B) a phylogenetic tree of yeast isolates from flowers of the wild fleabane Erigeron annus (L.) Pers., based on internal transcribed spacer sequences. Strains described in the present study are in bold font. The numerals are the confidence levels derived from 1,000 replicate bootstrap samplings.
    (A) Pi chart, and (B) a phylogenetic tree of yeast isolates from flowers of the wild fleabane Erigeron annus (L.) Pers., based on internal transcribed spacer sequences. Strains described in the present study are in bold font. The numerals are the confidence levels derived from 1,000 replicate bootstrap samplings.
  • [Fig. 2.] Aureobasidium pullulans interspecies. A neighbor-joining tree of A. pullulans isolates from flowers of the wild fleabane Erigeron annus (L.) Pers., based on internal transcribed spacer sequences. Strains described in the present study are in bold font. The numerals are the confidence levels derived from 1,000 replicate bootstrap samplings.
    Aureobasidium pullulans interspecies. A neighbor-joining tree of A. pullulans isolates from flowers of the wild fleabane Erigeron annus (L.) Pers., based on internal transcribed spacer sequences. Strains described in the present study are in bold font. The numerals are the confidence levels derived from 1,000 replicate bootstrap samplings.