Skip to main content
SpringerLink
Log in

Assessment of endophytic yeast diversity in rice leaves by a culture-independent approach

  • Original Paper
  • Published:
Antonie van Leeuwenhoek Aims and scope Submit manuscript

Abstract

Endophytic microorganisms inhabit internal plant tissues in the host plant without causing any symptoms or negative effects. Although the diversity of endophytes has been evaluated by both culture-dependent and culture-independent methods, less information is available on yeast communities. Therefore, in this study a culture-independent method was used to examine endophytic yeasts associated with rice leaves based on the large subunit of ribosomal DNA using a semi-nested PCR technique. Sequence analysis indicated that the colonization frequency and the relative species frequency (RF) of endophytic yeast phylotypes were 0.41 and 0.06, respectively, and the majority of the yeast phylotypes were basidiomycetous yeasts. The phylotypes were designated as five known species (Cryptococcus victoriae, Debaryomyces hansenii, Debaryomyces vindobonensis, Meyerozyma guilliermondii and Pseudozyma antarctica), together with seventeen phylotypes closest to Candida metapsilosis, Cryp. foliicola, Cryp. laurentii, Pseudozyma abaconensis, Pseudozyma aphidis and Trichosporon asahii, among which some could be novel species. The most prevalent phylotypes were those closest to Cryp. foliicola (47.5 % RF) followed by D. hansenii (22.8 % RF) and P. antarctica (16.8 % RF). The presence of the phylotypes related to species known for their potential applications as biocontrol agents and plant growth promoting hormone producers suggests that they may have valuable applications. In addition, our findings revealed the occurrence of novel phylotypes at high frequency, which should encourage extensive studies to discover novel yeast species and to understand their roles in the rice leaves.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Altschul SF, Madden TL, Schaffer JZ, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Anderson IC, Cairney JWG (2004) Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environ Microbiol 6:769–779

    Article  CAS  PubMed  Google Scholar 

  • Arenz BE, Held BW, Jurgens JA, Blanchette RA (2010) Fungal colonization of exotic substrates in Antarctica. Fungal Divers 49:13–22

    Article  Google Scholar 

  • Arnold AE, Mejia LC, Kyllo D, Rojas EI, Maynard Z, Robbins N, Herre EA (2003) Fungal endophytes limit pathogen damage in tropical trees. Proc Natl Acad Sci USA 100:15649–15654

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Bakys R, Vasaitis R, Barklund P, Ihrmark K, Stenlid J (2009) Investigations concerning the role of Chalara fraxinea in declining Fraxinus excelsior. Plant Pathol 58:284–292

    Article  Google Scholar 

  • Burdorf RJ, Laing MD, Morris CD, Jamal-Ally SE (2014) A procedure to evaluate the efficiency of surface sterilization methods in culture-independent fungal endophyte studies. Braz J Microbiol 45:977–983

    Article  Google Scholar 

  • Chae HS, Park GN, Kim SH, Jo HJ, Kim JT, Jeoung HY, An DJ, Kim NH, Shin BW, Kang YI, Chang KS (2012) Rapid direct identification of Cryptococcus neoformans from pigeon droppings by nested PCR using CNLAC1 gene. Poult Sci 91:1983–1989

    Article  CAS  PubMed  Google Scholar 

  • Chalutz E, Wilson CL (1990) Postharvest biocontrol of green and blue mold and sour rot of citrus fruit by Debaryomyces hansenii. Plant Dis 74:134–137

    Article  Google Scholar 

  • Collado J, Platas G, Gonzales I, Pelaez F (1999) Geographical and seasonal influence on the distribution of fungal endophytes in Quercus ilex. New Phytol 143:525–532

    Article  Google Scholar 

  • de Garcia V, Zalar P, Brizzio S, Gunde-Cimerman N, van Broock M (2012) Cryptococcus species (Tremellales) from glacial biomes in the southern (Patagonia) and northern (Svalbard) hemispheres. FEMS Microbiol Ecol 82:523–539

    Article  PubMed  Google Scholar 

  • Díaz C, Molina AM, Nähring J, Fischer R (2013) Characterization and dynamic behavior of wild yeast during spontaneous wine fermentation in steel tanks and amphorae. Biomed Res Int 2013:Art ID 540465. doi:10.1155/2013/540465

  • Fell JW, Boekhout T, Fonseca A, Scorezetti G, Statzell-Tallman A (2000) Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. Int J Syst Evol Microbiol 50:1351–1371

    Article  CAS  PubMed  Google Scholar 

  • Felsenstein J (1981) Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17:368–376

    Article  CAS  PubMed  Google Scholar 

  • Gai CS, Lacava PT, Maccheroni W Jr, Glienke C, Araújo WL, Miller TA, Azevedo JL (2009) Diversity of endophytic yeasts from sweet orange and their localization by scanning electron microscopy. J Basic Microbiol 49:441–451

    Article  PubMed  Google Scholar 

  • Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118

    Article  CAS  PubMed  Google Scholar 

  • Ghosh S, Santra T, Chakarvarty A (2013) Study of antagonistic yeasts isolated from some natural sources of West Bengal. Agric Biol J N Am 4:33–40

    Article  Google Scholar 

  • Glushakova AM, Kachalkin AV, Chernov IY (2014) Yeasts in the flowers of entomophilic plants of Moscow region. Microbiology 83:125–134

    Article  CAS  Google Scholar 

  • Guillamón JM, Sabaté J, Barrio E, Cano J, Querol A (1998) Rapid identification of wine yeast species based on RFLP analysis of the ribosomal internal transcribes spacer (ITS) region. Arch Microbiol 169:387–392

    Article  PubMed  Google Scholar 

  • Gusmão DS, Santos AV, Marini DC, Bacci M Jr, Berbert-Molina MA, Lemos FJA (2010) Culture-dependent and culture-independent characterization of microorganisms associated with Ades aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut. Acta Trop 115:275–281

    Article  PubMed  Google Scholar 

  • Harrison E, Muir A, Stratford M, Wheals A (2011) Species-specific PCR primers for the rapid identification of yeasts of the genus Zygosaccharomyces. FEMS Yeast Res 11:356–365

    Article  CAS  PubMed  Google Scholar 

  • Hernández-Montiel LG, Ochoa JL, Troyo-Diéguez E, Larralde-Corona CP (2010) Biocontrol of postharvest blue mold (Penicillium italicum Wehmer) on Mexican lime by marine and citrus Debaryomyces hansenii isolates. Postharvest Biol Technol 56:181–187

    Article  Google Scholar 

  • Higginbotham SJ, Arnold AE, Ibañez A, Spadafora C, Coley PD, Kursa TA (2013) Bioactivity of fungal endophytes as a function of endophyte taxonomy and the taxonomy and distribution of their host plants. PLoS ONE 8:e73192. doi:10.1371/journal.pone.0073192

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Holland SM (2003) Analytic Rarefaction 1.3. http://strata.uga.edu/software/. Accessed 3 Oct 2014

  • Hou Y, Ma Z, Dong S, Chen YH, Yu X (2013) Analysis of yeast-like symbiote diversity in the brown planthopper (BPH), Nilaparvata lugans Stål, using a novel nested PCR-DGGE protocol. Curr Microbiol 67:263–270

    Article  CAS  PubMed  Google Scholar 

  • Ibeas JI, Lozano I, Perdigones F, Jimenez J (1996) Detection of Dekkera-Brettanomyces strains in sherry by a nested PCR method. Appl Environ Microbiol 62:998–1003

    CAS  PubMed Central  PubMed  Google Scholar 

  • Jones MDM, Richards TA (2011) Environmental DNA analysis and the expansion of the fungal tree of life. In: Pöggeler S, Wöstemeyer J (eds) Evolution of fungi and fungal-like organisms: the mycota XIV. Springer-Verlag, Berlin, pp 37–54

    Chapter  Google Scholar 

  • Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Khunnamwong P, Surussawadee J, Jindamorakot S, Limtong S (2014) Wickerhamiella siamensis f.a., sp. nov., a novel endophytic and epiphytic yeast species isolated from sugarcane leaf in Thailand. Int J Syst Evol Microbiol 64:3849–3855

    Article  PubMed  Google Scholar 

  • Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120

    Article  CAS  PubMed  Google Scholar 

  • Kurtzman CP, Robnett CJ (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leewenhoek 73:331–371

    Article  CAS  Google Scholar 

  • Larran S, Monaco C, Alippi HE (2001) Endophytic fungi in leaves of Lycopersicon esculentum mill. World J Microbiol Biotechnol 17:181–184

    Article  Google Scholar 

  • Limtong S, Kaewwichian R (2015) The diversity of culturable yeasts in the phylloplane of rice in Thailand. Ann Microbiol 65:667–675

    Article  CAS  Google Scholar 

  • Limtong S, Kaewwichian R, Yongmanitchai W, Kawasaki H (2014) Diversity of culturable yeasts in phylloplane of sugarcane in Thailand and their capability to produce indole-3-acetic acid. World J Microbiol Biotechnol 30:1785–1796

    Article  CAS  PubMed  Google Scholar 

  • Lou QZ, Lu M, Sun JH (2014) Yeast diversity associated with invasive Dendroctonus valens killing Pinus tabuliformis in China using culturing and molecular methods. Microb Ecol 68:397–415

    Article  PubMed  Google Scholar 

  • Lutz MC, Lopes CA, Rodriguez ME, Sosa MC, Sangorrín MP (2013) Efficacy and putative mode of action of native and commercial antagonistic yeasts against postharvest pathogens of pear. Intl J Food Microbiol 164:166–172

    Article  CAS  Google Scholar 

  • Martin KJ, Rygiewicz PT (2005) Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiol 5:28–38

    Article  PubMed Central  PubMed  Google Scholar 

  • Middelhoven WJ (1997) Identity and biodegradative abilities of yeasts isolated from plants growing in an arid climate. Antonie Van Leeuwenhoek 72:81–89

    Article  CAS  PubMed  Google Scholar 

  • Mittelbach M, Yurkov AM, Nocentini D, Nepi M, Weigend M, Begerow D (2015) Nectar sugars and bird visitation define a floral niche for basidiomycetous yeast on the Canary Islands. BMC Ecol 15:2–14

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Naik BS, Shashikala J, Krishnamurthy YL (2009) Study on the diversity of endophytic communities from rice (Oryza sativa L.) and their antagonistic activities in vitro. Microbiol Res 164:290–296

    Article  CAS  PubMed  Google Scholar 

  • Nakano Y, Nagahama T, Hatada Y, Nunoura T, Yakami H, Miyazaki J, Takai K, Horikoshi K (2010) Fungal diversity in deep-sea sediments—the presence of novel fungal groups. Fungal Ecol 3:316–325

    Article  Google Scholar 

  • Nasanit R, Krataithong K, Tantirungkij M, Limtong S (2015) Assessment of epiphytic yeast diversity in rice (Oryza sativa) phyllosphere in Thailand by culture-independent approach. Antonie Van Leeuwenhoek 107:1475–1490

    Article  CAS  PubMed  Google Scholar 

  • Nassar AH, El-Tarabily KA, Sivasithamparam K (2005) Promotion of plant growth by an auxin-producing isolate of the yeast Williopsis saturnus endophytic in maize (Zea mays L.) roots. Biol Fertil Soils 42:97–108

    Article  CAS  Google Scholar 

  • Nielsen DS, Teniola OD, Ban-Koffi L, Owusu M, Andersson TS, Holzapfel WH (2007) The microbiology of Ghanaian cocoa fermentations analysed using culture-dependent and culture-independent methods. Int J Food Microbiol 114:168–186

    Article  CAS  PubMed  Google Scholar 

  • Nutaratat P, Srisuk N, Arunrattiyakorn P, Limtong S (2014) Plant growth-promoting traits of epiphytic and endophytic yeasts isolated from rice and sugar cane leaves in Thailand. Fungal Biol 118:683–694

    Article  CAS  PubMed  Google Scholar 

  • Oros-Sichler M, Gomes NCM, Neuber G, Smalla K (2006) A new semi-nested PCR protocol to amplify large 18S rRNA gene fragments for PCR-DGGE analysis of soil fungal communities. J Microbiol Meth 65:63–75

    Article  CAS  Google Scholar 

  • Petrini O (1991) Fungal endophytes of tree leaves. In: Andrews JH, Hirano SS (eds) Microbial ecology of the leaves. Springer-Verlag, New York, pp 179–197

    Chapter  Google Scholar 

  • Petrini O, Sieber TN, Toti L, Viret O (1992) Ecology, metabolite production, and substrate utilization in endophytic fungi. Natl Toxins 1:185–196

    Article  CAS  Google Scholar 

  • Porter TM, Golding GB (2012) Factors that affect large subunit ribosomal DNA amplicon sequencing studies of fungal communities: classification method, primer choice, and errors. PLoS ONE 7:e35749. doi:10.1371/journal.pone.0035749

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Russo G, Libkind D, Sampio J, van Broock MR (2008) Yeast diversity in the acidic Rio Agrio-Lake Caviahue volcanic environment (Patagonia, Argentina). FEMS Microbiol Ecol 65:415–5424

    Article  CAS  PubMed  Google Scholar 

  • Singh P, Raghukumar C, Verma P, Shouche Y (2011) Fungal community analysis in the deep-sea sediments of the Central Indian Basin by culture-independent approach. Microb Ecol 61:507–517

    Article  CAS  PubMed  Google Scholar 

  • Singh P, Raghukumar C, Verma P, Shouche Y (2012) Assessment of fungal diversity in deep-sea sediments by multiple primer approach. World J Microbiol Biotechnol 28:659–667

    Article  CAS  PubMed  Google Scholar 

  • Solis MJL, Yurkov A, dela Cruz TE, Unterseher M (2015) Leaf-inhabiting endophytic yeasts are abundant but unevenly distributed in three Ficus species from botanical garden greenhouses in Germany. Mycol Progress 14:1019–1028

    Article  Google Scholar 

  • Suzuki M, Prasad GS, Kurtzman CP (2011) Debaryomyces Lodder & Kreger-van Rij (1952). In: Kurtzman CP, Fell JW, Boekhout T (eds) The yeasts, a taxonomic study. Elsevier, San Diego, pp 361–372

    Chapter  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Taylor MW, Tsai P, Anfang N, Ross HA, Goddard R (2014) Pyrosequencing reveals regional differences in fruit-associated fungal communities. Environ Microbiol 16:2848–5858

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Tian XL, Cao LX, Tan HM, Zeng QG, Jia YY, Han WQ, Zhou SN (2004) Study on the communities of endophytic fungi and endophytic actinomycetes from rice and their antipathogenic acitivities in vitro. World J Microbiol Biotechnol 20:303–309

    Article  Google Scholar 

  • Toju H, Tanabe AS, Yamamoto S, Sato H (2012) High-coverage ITS primers for the DNA-based identification of ascomycetes and basidiomycetes in environmental samples. PLoS ONE 7:e40863

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Trindade RC, Resende MA, Silva CM, Rosa CA (2002) Yeast associated with fresh and frozen pulps of Brazillian tropical fruits. Syst Appl Microbiol 25:294–300

    Article  CAS  PubMed  Google Scholar 

  • Vega FE, Dowd PF (2005) The role of yeasts as insect endosymbionts. In: Vega FE, Blackwell M (eds) Insect-fungal associations ecology and evolution. Oxford University Press, Oxford, pp 211–243

    Google Scholar 

  • Wang QM, Boekhout T, Bai FY (2011) Cryptococcus foliicola sp. nov. and Cryptococcus taibaiensis sp. nov., novel basidiomycetous yeast species from plant leaves. J Gen Appl Microbiol 57:285–291

    Article  CAS  PubMed  Google Scholar 

  • White TJ, Bruns TD, Lee S, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis M, Gefand D, Sninsky J, White T (eds) PCR protocols: a guide to methods and applications. Academic Press, London, pp 315–322

    Google Scholar 

  • Xin G, Glawe D, Doty SL (2009) Characterization of three endophytic, indole-3-acetic acid-producing yeasts occurring in Populus trees. Mycol Res 113:973–980

    Article  CAS  PubMed  Google Scholar 

  • Yang CH, Crowley DE, Borneman J, Keen NT (2001) Microbial phyllophere populations are more complex than previously realized. Proc Natl Acad Sci USA 98:3889–3894

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yuan ZL, Zhang CL, Lin FC, Kubicek C (2010) Identity, diversity and molecular phylogeny of the endophytic mycobiota in the roots of rare wild rice (Oryza granulate) from a nature reserve in Yunnan, China. Appl Environ Microbiol 76:1642–1652

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Thailand Research Fund through a Thailand Research Fund/TRF Research-Team Promotion Grant (RTA54800009) and the Center for Advanced Studies in Tropical Natural Resources, National Research University-Kasetsart University.

Author information

Authors and Affiliations

  1. Central Laboratory and Greenhouse Complex, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom, 73140, Thailand

    Manee Tantirungkij

  2. Department of Biotechnology, Faculty of Engineering and Industrial Technology, Silpakorn University, Sanamchandra Palace Campus, Nakhon Pathom, 73000, Thailand

    Rujikan Nasanit

  3. Department of Microbiology, Faculty of Sciences, Kasetsart University, Bangkok, 10900, Thailand

    Savitree Limtong

  4. Center for Advanced Studies in Tropical Natural Resources, National Research University-Kasetsart University, Bangkok, 10900, Thailand

    Savitree Limtong

Authors
  1. Manee Tantirungkij
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rujikan Nasanit
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Savitree Limtong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Savitree Limtong.

Ethics declarations

Conflict of interest

The authors declare that there are no conflicts of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tantirungkij, M., Nasanit, R. & Limtong, S. Assessment of endophytic yeast diversity in rice leaves by a culture-independent approach. Antonie van Leeuwenhoek 108, 633–647 (2015). https://doi.org/10.1007/s10482-015-0519-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10482-015-0519-y

Keywords

Search

Navigation