Differentiation of lineages within “Colletotrichum gloeosporioides s.l.” associated with cassava anthracnose disease by BOX‐ and ERIC‐PCRs

Abstract

Several molecular techniques have been used to differentiate species or genetic lineages of microorganisms prior to sequencing. Among them, BOX-and ERIC-PCRs may provide specific banding patterns for different species, allowing its differentiation.Therefore, the objective of this study was to evaluate these techniques as a tool for differentiation of phylogenetic lineages belonging to the Colletotrichum gloeosporioides species complex associated with cassava anthracnose disease. Sets of BOX-and ERIC-PCR primers were used to assess the differentiation of lineages belonging to the complex with 81 C. gloeosporioides sensu lato (s.l.) isolates from different cassava producing regions. Some were identified by sequencing, such as Colletotrichum fructicola, Colletotrichum tropicale, C. gloeosporioides s.s, Colletotrichum theobromicola, Colletotrichum siamense, Colletotrichum brevisporum and Colletotrichum sichuanensis.The primers were able to amplify DNA fragments from all isolates. The ERIC-PCR presented a wider range of banding patterns in comparison to BOX-PCR, providing better differentiation of the individuals, as well as a higher correlation with the phylogenetic data was obtained by ERIC-PCR and the combined data set for "BOX-/ ERIC-PCRs," inferred by Mantel test. However, the use of concatenated data (BOX-/ ERIC-PCRs) reduced the discriminatory capacity presented by ERIC-PCR alone, probably due to the lowest resolution of BOX-PCR. Therefore, ERIC-PCR technique enabled efficient differentiation of isolates belonging to the C. gloeosporioides complex and can be used to analyse multiple isolates in a collection and also being an important tool as a guide in the decision-making process prior to sequencing. Based on this methodology, it was possible to identify two new species associated with cassava anthracnose disease, C. brevisporum and C. sichuanensis, being the first report of these two species associated with cassava anthracnose disease in Brazil. K E Y W O R D SColletotrichum spp., DNA fingerprint, Rep-PCR, species complex | 219 LOPES SILVA Et AL.

Colletotrichum species causing cassava (Manihot esculenta Crantz) anthracnose in different eco-zones within the Recôncavo Region of Bahia, Brazil

“…However, its production is strongly limited by many pests where cassava anthracnose disease (CAD), caused by Colletotrichum spp., is considered one of the most destructive cassava leaf diseases especially in main cassava producing countries worldwide (William et al 2012;Sangpueak et al 2018) CAD is characterized by cankers on stems, branches, fruits, leaf spots, and diebacks on aerial parts of diseased plants (Kunkeaw et al 2010;Silva et al 2018). Despite the recent reports of different Colletotrichum species associated with cassava antracnose in Brazil (Bragança et al 2016;Oliveira et al 2016Oliveira et al , 2018Silva et al 2019), little is known about its distribution, incidence, prevalence and disease severity across different production regions. Furthermore, there are no data regarding the relationship of CAD incidence and severity and agroecological practices and/or climatic zones in Brazilian conditions.…”

“…The study of genetic diversity of plant pathogens and species to identify and characterize causal agents of diseases is a key point for selection of adequate isolates for screening of resistance in plant breeding programs (Ramdial and Rapersad 2015). More recently, Silva et al (2019) demonstrated that enterobacterial repetitive intergenic consensuspolymerase chain reaction markers (ERIC-PCR) can be used as a complementary tool for discrimination of species within the Colletotrichum gloeosporioides complex associated with CAD in Brazil with a very high correlation with multilocus phylogenetic analysis.…”

“…A survey was conducted by Silva et al (2019) during the rainy season in 2014 (April-July), and diseased leaves and stems of cassava were collected in different production areas in the Recôncavo Region of the State of Bahia, Brazil. A total of 50 cassava fields distributed among 18 municipalities were visited and evaluated for the incidence and severity of anthracnose.…”

“…Monosporic isolates were obtained and ERIC-PCR amplifications followed the protocol described in Silva et al (2019), based on the growth of the isolates in sucrose and yeast extract broth and DNA extraction using the CTAB method. Pathogenicity was confirmed based on the in vitro detached leaf assay method proposed by Kunkeaw et al (2010) for pre-screening resistant genotypes.…”

“…A total of 82 Colletotrichum spp. gloeosporioides sensu lato isolates within 18 municipalities were identified by Silva et al (2019) associated with anthracnose symptoms of cassava. The number of isolates per municipality ranged from two to 24 for 'Muritiba' and 'Nazaré', respectively.…”