Diversity of endophytic fungi in Eucalyptus microcorys assessed by complementary isolation methods
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Brazil is the world’s largest producer country of eucalyptus. Although widely applied in the charcoal industry, no studies have focused on the microorganisms associated with Eucalyptus microcorys. Here, we evaluated the composition and structure of endophytic fungal communities in leaves of E. microcorys through two isolation techniques. A total of 120 fresh leaves were collected in a year-long survey at an eucalyptus plantation in the State of São Paulo (Brazil). Endophytic fungi were isolated by particle filtration (PF) and direct leaf fragment plating (LP) in two media: modified dicloran and synthetic nutrient agar, both supplemented with rose bengal and chloramphenicol. The isolates were grouped into morphospecies and identified by morphology and DNA sequencing. We recovered a total of 709 isolates, representing 59 taxa. All taxa found are reported as endophytic for the first time for E. microcorys. Castanediella eucalypticola and Neophaeomoniella eucalypti are new occurrences reported for Brazil. The LP technique recovered a higher number of taxa and isolates than the PF. However, the PF technique retrieved a higher species/isolate ratio than the LP method, 0.12 and 0.09, respectively. Fungal diversity assessed by diversity metrics did not significantly differ between isolation methods. Both techniques recovered a high number of unique taxa, demonstrating that neither method would individually represent the species richness from E. microcorys. The use of LP and PF provided a greater number of observed taxa and consequently new occurrence of species for Brazil.
KeywordsCulture-dependent methods Leaf plating Particle filtration Endophytes
The authors acknowledge “CAPES—Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” for providing a scholarship to the first author. We thank “IF—Instituto Florestal” for providing permit (# 260108-001.102/2015) to work at FEENA. We also thank Sérgio Ricardo Christofoletti for providing assistance during fieldwork at FEENA. We would like to thank “ICMBio—Instituto Chico Mendes de Conservação da Biodiversidade” for the collecting permits (# 38466-2 and #31534 issued to LTL and AR, respectively). We also thank Sérgio Kakazu for sequencing the DNA samples. We are grateful to two anonymous reviewers and the editor, Marc Stadler, for providing insightful and constructive comments to this manuscript.
- Barathikannan K, Ramasamy KP, Manohar CS, Meena RM (2017) Diversity and decolorization potential of fungi isolated from the coral reef regions off Kavaratti, India. Indian J Geo Mar Sci 46(3):497–503Google Scholar
- Carmichael JW, Kendrick WB, Conners IL, Sigler L (1980) Genera of hyphomycetes. Univ. Alberta Press: 386 ppGoogle Scholar
- Crous PW (1998) Mycosphaerella spp. and their anamorphs associated with leaf spot diseases of Eucalyptus. American Phytopathological Society (APS Press). 170 pp.Google Scholar
- Fakhrunnisa MH, Ghaffar A (2006) Seed-borne mycoflora of wheat, sorghum and barley. Pak J Bot 38(1):185–192Google Scholar
- Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41(41):95–98Google Scholar
- Hammer Ø, Harper DAT, Ryan PD (2001) PAST: paleontological statistics software package for education and data analysis. Palaeontol Electron 4(1):1–9Google Scholar
- Hyde K D, Bussaban B, Paulus B, Crous PW, Lee S, Mckenzie EH, et al (2007) Diversity of saprobic microfungi. Biodivers Conserv 16(1), 7–35. https://doi.org/10.1007/s10531-006-9119-5
- Kharwar RN, Gond SK, Kumar A, Mishra A (2010) A comparative study of endophytic and epiphytic fungal association with leaf of Eucalyptus citriodora Hook., and their antimicrobial activity. World J Microbiol Biotechnol 26(11):1941–1948. https://doi.org/10.1007/s11274-010-0374-y CrossRefGoogle Scholar
- Marsberg A, Slippers B, Wingfield MJ, Gryzenhout M (2014) Endophyte isolations from Syzygium cordatum and a Eucalyptus clone (Myrtaceae) reveal new host and geographical reports for the Mycosphaerellaceae and Teratosphaeriaceae. Australas Plant Pathol 43(5):503–512. https://doi.org/10.1007/s13313-014-0290-y CrossRefGoogle Scholar
- Möller EM, Bahnweg G, Sandermann H, Geiger HH (1992) A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi fruit bodies and infected plant tissues. Nucleic Acids Res 20(22):6115–6116. https://doi.org/10.1093/nar/20.22.6115 CrossRefPubMedPubMedCentralGoogle Scholar
- Nirenberg HI (1976) Untersuchungen uber die morphologische und biologische Differenzierung in der Fusarium-Sektion Liseola. Mitt Biol Bundesanst Land-u Forstwirtsch Berlin-Dahlem. https://doi.org/10.2307/3758963
- Oliveira C, Regasini LO, Silva GH, Pfenning LH, Young MCM, Berlinck RGS, Bolzani VS, Araujo AR (2011) Dihydroisocoumarins produced by Xylaria sp. and Penicillium sp. endophytic fungi associated with Piper aduncum and Alibertia macrophylla. Phytochem Lett 4(2):93–96. https://doi.org/10.1016/j.phytol.2010.11.003 CrossRefGoogle Scholar
- Pavlic-Zupanc D, Maleme HM, Piškur B, Wingfield BD, Wingfield MJ, Slippers B (2017) Diversity, phylogeny and pathogenicity of Botryosphaeriaceae on non-native Eucalyptus grown in an urban environment: a case study. Urban For Urban Green 26(1):139–148. https://doi.org/10.1016/j.ufug.2017.04.009 CrossRefGoogle Scholar
- Petrini O (1991) Fungal endophytes of tree leaves. In: Andrena J, Hirano SS. Microbial ecology of leaves. pp. 179–197. https://doi.org/10.1007/978-1-4612-3168-4_9
- R Core Team (2016) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/.
- Sociedade Brasileira de Sivicultura (2015) http://www.sbs.org.br. Accessed 19 Dec 2017.
- Tamura K, Peterson D, Peterson N, Steche G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739. https://doi.org/10.1093/molbev/msr121 CrossRefPubMedPubMedCentralGoogle Scholar
- Unterseher M, Schnittler M (2009) Dilution-to-extinction cultivation of leaf-inhabiting endophytic fungi in beech (Fagus sylvatica L.)—different cultivation techniques influence fungal biodiversity assessment. Mycol Res 113(5):645–654. https://doi.org/10.1016/j.mycres.2009.02.002 CrossRefPubMedGoogle Scholar
- White TJ, Burns T, Lee S, Taylor J (1990) PCR protocols: a guide to method and applications. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) Amplification and direct sequencing of fungal ribosomal rRNA genes for phylogenetics. Academic Press, Massachusetts, pp 315–352. https://doi.org/10.1016/0168-9525(90)90186-a Google Scholar
- Wulandari NF, Anun C, Hyde KD, Duong L, de Gruyter J, Meffert J, Groenewald JZ, Crous PW (2009) Phyllosticta citriasiana sp. nov., the cause of Citrus tan spot of Citrus maxima in Asia. Fungal Divers 34(1):23–39Google Scholar
- Zhang XY, Tang GL, Xu XY, Nong XH, Qi SH (2014) Insights into deep-sea sediment fungal communities from the East Indian Ocean using targeted environmental sequencing combined with traditional cultivation. PLoS One 9(10):e109118. https://doi.org/10.1371/journal.pone.0109118 CrossRefPubMedPubMedCentralGoogle Scholar