Caracterização de espécies de Colletotrichum em vegetação de Mata Atlântica no Estado do Rio de Janeiro, Brasil

Abstract

Colletotrichum sp. are responsible for anthracnose in several crops of agronomic interest, causing considerable economic loss in the pre and post-harvest, being among the 10 most important groups of phytopathogenic fungi, however, its occurrence in wild hosts has been less studied. The objective of this work was the morphological and molecular characterization of Colletotrichum species from Atlantic Forest vegetation, collected mainly in the Municipal Natural Park of Curió (PNMC) from Paracambi and other municipalities in the State of Rio de Janeiro, with symptoms of anthracnose. The samples were taken to the laboratory and observed under stereoscopic microscope to identify acervuli of Colletotrichum sp., where thin handmade sections of the plant material were made or by using cryomicrotome, in order to assemble the slides for study, staining the sample with cotton blue and covering it with a coverslip to observe the structures of the fungus in optical microscopes. The measurements of the fungal structures were taken through a micrometric eyepiece. The fungal samples were isolated in BDA (Potato-dextrose-agar) medium for the cultural characterization, and appressoria culture was performed in a cover slip to observe its formation. The botanical specimens were identified, and the samples of the vegetative material were herborized and incorporated into the “Verlande Duarte Silveira” Phytopathological Herbarium (UFRJ) located in the Department of Entomology and Plant Pathology (DENF), Institute of Biological Sciences and Health (ICBS) at the Federal Rural University of Rio de Janeiro (UFRRJ). The fungal isolates were transferred to test tubes with BDA medium, inserted in the mycological collection of the DENF and preserved by the Castellani method. The 16 isolates identified in this study were labeled with the codes CC201801 to CC201816. Molecular techniques were used for DNA extraction, PCR amplification of the ITS1-5.8S-ITS2 (Internal Transcribed Spacer) region of rDNA using the primers ITS1 and ITS4 and sequencing in the 3500 Applied Biosystems. The contigs obtained were assembled in the BioNumerics 7.0 program (Applied Maths - bioMérieux) and the phylogenetic analysis was conducted in the MEGA X program. Phylogenetic analysis was performed using the maximum likelihood method based on the Tamura-Nei model, using the ITS sequences obtained in GenBank through the access code described in previous works. The morphological characterization identified that of the 16 samples studied, eleven occurrences are reported for the first time in the world (CC201804, CC201805, CC201806, CC201807, CC201808, CC201810, CC201812, CC201813, CC201814, CC201815, CC201816); three for the first time in Brazil (CC201802, CC201803, CC201811) and two (CC201801, CC201809) for the first time in RJ, in their hosts. The molecular characterization indicated that seven isolates are grouped into the gloeosporioides species complex (CC201801, CC201802, CC201803, CC201806, CC201811, CC201812, CC201815), seven in the boninense complex (CC201804, CC201805, CC201807, CC201808, CC201809, CC201814, CC201816) and the isolate CC201813 diverges from all of them by the peculiar morphological characteristic of the conidium and the distance in the phylogenetic tree, suggesting to be a new species within the genus Colletotrichum.