Mycological Progress

, Volume 17, Issue 6, pp 719–727 | Cite as

Diversity of endophytic fungi in Eucalyptus microcorys assessed by complementary isolation methods

  • Lorena T. Lacerda
  • Luís F. P. Gusmão
  • Andre Rodrigues
Original Article
  • 111 Downloads

Abstract

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.

Keywords

Culture-dependent methods Leaf plating Particle filtration Endophytes 

Notes

Acknowledgements

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.

Supplementary material

11557_2018_1385_MOESM1_ESM.docx (22 kb)
ESM 1 (DOCX 22.3 kb)

References

  1. Aguín O, Sainz MJ, Ares A, Otero L, Mansilla JP (2013) Incidence, severity and causal fungal species of Mycosphaerella and Teratosphaeria diseases in Eucalyptus stands in Galicia (NW Spain). For Ecol Manag 302:379–389.  https://doi.org/10.1016/j.foreco.2013.03.021 CrossRefGoogle Scholar
  2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402.  https://doi.org/10.1093/nar/25.17.3389 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 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
  4. Barber PA, Burgess TJ, Hardy GESJ, Slippers B, Keane PJ, Wingfield MJ (2005) Botryosphaeria species from Eucalyptus in Australia are pleoanamorphic, producing Dichomera synanamorphs in culture. Mycol Res 109(12):1347–1363.  https://doi.org/10.1017/s0953756205003989 CrossRefPubMedGoogle Scholar
  5. Barradas C, Phillips AJ, Correia A, Diogo E, Bragança H, Alves A (2016) Diversity and potential impact of Botryosphaeriaceae species associated with Eucalyptus globulus plantations in Portugal. Eur J Plant Pathol 146(2):245–257.  https://doi.org/10.1007/s10658-016-0910-1 CrossRefGoogle Scholar
  6. Bernreiter A, Teijeiro RG, Garrido P, Ramos L (2016) Mycosphaerella and Teratosphaeria leaf spot diseases of Eucalyptus globulus in Ecuador. Aust Plant Dis Notes 11(1):18.  https://doi.org/10.1007/s13314-016-0204-7 CrossRefGoogle Scholar
  7. Bills GF, Polishook JD (1994) Abundance and diversity of microfungi in leaf litter of a lowland rain forest in Costa Rica. Mycologia 86(2):187–198.  https://doi.org/10.2307/3760635 CrossRefGoogle Scholar
  8. Carmichael JW, Kendrick WB, Conners IL, Sigler L (1980) Genera of hyphomycetes. Univ. Alberta Press: 386 ppGoogle Scholar
  9. Cheewangkoon R, Crous PW, Hyde KD, Groenewald JZ, To-Anan C (2008) Species of Mycosphaerella and related anamorphs on Eucalyptus leaves from Thailand. Persoonia Mol Phylogeny Evol Fungi 21(1):77–91.  https://doi.org/10.3767/003158508x370857 CrossRefGoogle Scholar
  10. Cheewangkoon R, Groenewald JZ, Summerell BA, Hyde KD, To-Anun C, Crous PW (2009) Myrtaceae, a cache of fungal biodiversity. Persoonia Mol Phylogeny Evol Fungi 23(1):55–85.  https://doi.org/10.3767/003158509X474752 CrossRefGoogle Scholar
  11. Collado J, Platas G, Paulus B, Bills GF (2007) High-throughput culturing of fungi from plant litter by a dilution-to-extinction technique. FEMS Microbiol Ecol 60(3):521–533.  https://doi.org/10.1111/j.1574-6941.2007.00294.x CrossRefPubMedGoogle Scholar
  12. Costa LC, Peixoto PEC, Gusmão LPF (2015) Effects of storage conditions and culture media on the saprobic fungi diversity in tropical leaf litter. Mycosphere 6(1):43–52.  https://doi.org/10.5943/mycosphere/6/1/5 CrossRefGoogle Scholar
  13. Crous PW (1998) Mycosphaerella spp. and their anamorphs associated with leaf spot diseases of Eucalyptus. American Phytopathological Society (APS Press). 170 pp.Google Scholar
  14. Crous PW, Wingfield MJ (1997) Colletogloeopsis, a new coelomycete genus to accommodate anamorphs of two species of Mycosphaerella on Eucalyptus. Can J Bot 75:667–674.  https://doi.org/10.1139/b97-074 CrossRefGoogle Scholar
  15. Crous PW, Kendrick WB, Alfenas AC (1997) New species of hyphomycetes associated with Eucalyptus. S Afr J Bot 63(5):286–290.  https://doi.org/10.1016/s0254-6299(15)30769-9 CrossRefGoogle Scholar
  16. Crous PW, Wingfield MJ, Mansilla JP, Alfenas AC, Groenewald JZ (2006) Phylogenetic reassessment of Mycosphaerella spp. and their anamorphs occurring on Eucalyptus. II. Stud Mycol 55:99–131.  https://doi.org/10.3114/sim.55.1.99 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Crous PW, Schoch CL, Hyde KD, Wood R, Gueidan C, Hoog GS, Groenewald JZ (2009) Phylogenetic lineages in the Capnodiales. Stud Mycol 64(1):17–47.  https://doi.org/10.3114/sim.2009.64.02 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Crous PW, Wingfield MJ, Richardson DM, Le Roux JJ, Strasberg D, Edwards J et al (2016) Fungal Planet description sheets: 400–468. Persoonia Mol Phylogeny Evol Fungi 36(1):316–468.  https://doi.org/10.3767/003158516X692185 CrossRefGoogle Scholar
  19. Damare S, Raghukumar C, Raghukumar S (2006) Fungi in deep-sea sediments of the Central Indian Basin. Deep-Sea Res I Oceanogr Res Pap 53(1):14–27.  https://doi.org/10.1016/j.dsr.2005.09.005 CrossRefGoogle Scholar
  20. De Abreu LM, Almeida AR, Salgado M, Pfenning LH (2010) Fungal endophytes associated with the mistletoe Phoradendron perrottettii and its host tree Tapirira guianensis. Mycol Prog 9(4):559–566.  https://doi.org/10.1007/s11557-010-0663-8 CrossRefGoogle Scholar
  21. Fakhrunnisa MH, Ghaffar A (2006) Seed-borne mycoflora of wheat, sorghum and barley. Pak J Bot 38(1):185–192Google Scholar
  22. González-Teuber M (2016) The defensive role of foliar endophytic fungi for a South American tree. AoB Plants 8:50.  https://doi.org/10.1093/aobpla/plw050 CrossRefGoogle Scholar
  23. 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
  24. Hammer Ø, Harper DAT, Ryan PD (2001) PAST: paleontological statistics software package for education and data analysis. Palaeontol Electron 4(1):1–9Google Scholar
  25. Hunter G, Wingfield BD, Crous PW, Wingfield MJ (2006) A multi-gene phylogeny for species of Mycosphaerella occurring on Eucalyptus leaves. Stud Mycol 55:147–161.  https://doi.org/10.3114/sim.55.1.147 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 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
  27. Jonker JGG, Van Der Hilst F, Junginger HM, Cavalett O, Chagas MF, Faaij APC (2015) Outlook for ethanol production costs in Brazil up to 2030, for different biomass crops and industrial technologies. Appl Energy 147(1):593–610.  https://doi.org/10.1016/j.apenergy.2015.01.090 CrossRefGoogle Scholar
  28. 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
  29. Lima FDS, Soares ACF, Sousa CDS (2013) Occurrence and activity arbuscular mycorrhizal fungi in eucalypt (Eucalyptus sp.) plantations in the northern coast of Bahia, Brazil. Rev Árvore 37(2):245–255.  https://doi.org/10.1590/S0100-67622013000200006 CrossRefGoogle Scholar
  30. Linnakoski R, Puhakka-Tarvainen H, Pappinen A (2012) Endophytic fungi isolated from Khaya anthotheca in Ghana. Fungal Ecol 5(3):298–308.  https://doi.org/10.1016/j.funeco.2011.08.006 CrossRefGoogle Scholar
  31. Lombard L, Zhou XD, Crous PW, Wingfield BD, Wingfield MJ (2010) Calonectria species associated with cutting rot of Eucalyptus. Persoonia Mol Phylogeny Evol Fungi 24(1):1–11.  https://doi.org/10.3767/003158510X486568 CrossRefGoogle Scholar
  32. Lupo S, Tiscornia S, Bettucci L (2001) Endophytic fungi from flowers, capsules and seeds of Eucalyptus globulus. Rev Iberoam Micol 18(1):38–41PubMedGoogle Scholar
  33. 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
  34. Massenssini AM, Tótola MR, Borges AC, Costa MD (2016) Isolation and characterization of phosphate solubilizing bacteria from Eucalyptus sp. rhizosphere. Rev Árvore 40(1):125–134.  https://doi.org/10.1590/0100-67622016000100014 CrossRefGoogle Scholar
  35. 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
  36. 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
  37. 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
  38. Oliveira FN, Fortes GA, Paula JR, Ferri PH, Santos SC (2014) Seasonal influence on the essential oil of Eucalyptus microcorys. Nat Prod Commun 9(4):575–580PubMedGoogle Scholar
  39. Paulus BC, Gadek P, Hyde KD (2003) Estimation of microfungi diversity in tropical rainforest leaf litter using particle filtration: the effects of leaf storage and surface treatment. Mycol Res 107(6):748–756.  https://doi.org/10.1017/S0953756203007913 CrossRefPubMedGoogle Scholar
  40. 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
  41. 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
  42. Polishook JD, Bills GF, Lodge DJ (1996) Microfungi from decaying leaves of two rain forest trees in Puerto Rico. J Ind Microbiol 17(3):284–294.  https://doi.org/10.1007/BF01574703 Google Scholar
  43. 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/.
  44. Rajulu G, Suryanarayanan TS, Tangjang S (2016) Endophytic fungi of orchids of Arunachal Pradesh, North Eastern India. Curr Res Environ Appl Mycol 6(4):293–299.  https://doi.org/10.5943/cream/6/4/7 CrossRefGoogle Scholar
  45. Schulz B, Wanke U, Draeger S, Aust HJ (1993) Endophytes from herbaceous plants and shrubs-effectiveness of surface sterilization methods. Mycol Res 97(12):1447–1450.  https://doi.org/10.1016/S0953-7562(09)80215-3 CrossRefGoogle Scholar
  46. Singh P, Raghukumar C, Verma P, Shouche Y (2010) Phylogenetic diversity of culturable fungi from the deep-sea sediments of the Central Indian Basin and their growth characteristics. Fungal Divers 40(1):89–102.  https://doi.org/10.1007/s13225-009-0009-5 CrossRefGoogle Scholar
  47. Smith H, Wingfield MJ, Petrini O (1996) Botryosphaeria dothidea endophytic in Eucalyptus grandis and Eucalyptus nitens in South Africa. For Ecol Manag 89(1–3):189–195.  https://doi.org/10.1016/S0378-1127(96)03847-9 CrossRefGoogle Scholar
  48. Sociedade Brasileira de Sivicultura (2015) http://www.sbs.org.br. Accessed 19 Dec 2017.
  49. Souza ELD, Antoniolli ZI, Machado RG, Pazzini DE, Dahmer SF, Redin M, Ramires MF (2017) Ectomycorrhizal isolates in the seedling production of Eucalyptus grandis W. Hill ex. Maiden in quartzarenic neosols. Cienc Florest 27(2):471–484.  https://doi.org/10.5902/1980509827729 CrossRefGoogle Scholar
  50. 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
  51. Thambugala KM, Ariyawansa HA, Li YM, Boonmee S, Hongsanan S, Tian Q, Singtripop C, Bhat DJ, Camporesi E, Jayawardena R, Liu Z, Xu J, Chukeatirote E (2014) Dothideales. Fungal Divers 68(1):105–158.  https://doi.org/10.1007/s13225-014-0303-8 CrossRefGoogle Scholar
  52. 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
  53. Wang X, Chen G, Huang F, Zhang J, Hyde KD, Li H (2012) Phyllosticta species associated with citrus diseases in China. Fungal Divers 52(1):209–224.  https://doi.org/10.1007/s13225-011-0140-y CrossRefGoogle Scholar
  54. 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
  55. Wikee S, Lombard L, Crous PW, Nakashima C, Motohashi K, Chukeatirote E, Hyde KD (2013) Phyllosticta capitalensis, a widespread endophyte of plants. Fungal Divers 60(1):91–105.  https://doi.org/10.1007/s13225-013-0235-8 CrossRefGoogle Scholar
  56. 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
  57. 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

Copyright information

© German Mycological Society and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Departamento de Bioquímica e MicrobiologiaUniversidade Estadual Paulista (UNESP)Rio ClaroBrazil
  2. 2.Departamento de Ciências BiológicasUniversidade Estadual de Feira de Santana (UEFS)Feira de SantanaBrazil

Personalised recommendations