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.
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