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First Report of Colletotrichum theobromicola and C. siamense Causing Anthracnose on Cultivated and Wild Cassava Species in Brazil
- Oliveira, S. A. S., Silva, L. L., Diamantino, M. S. A. S., Ferreira, C. F.
- Plant disease 2018 v.102 no.4 pp. 819
- Bayesian theory, Colletotrichum, DNA, agar, anthracnose, cassava, cetyltrimethylammonium bromide, conidia, culture media, ethanol, fungi, genes, genetic databases, genetic variation, germplasm, hosts, leaves, nucleotide sequences, pathogenicity, photoperiod, phylogeny, polymerase chain reaction, protocols, sodium hypochlorite, staple crops, surveys, tissues, topology, tropical and subtropical fruits, Brazil
- Cassava is an important staple food crop in Brazil. Surveys were conducted during three seasons (2014 to 2016) to collect symptomatic stem and leaf samples of cultivated cassava in the Recôncavo Region of Bahia, northeastern Brazil. Additionally, samples from 10 germplasm accessions of wild cassava species were also collected. Symptomatic tissues were disinfested with 70% ethanol (30 s), 0.5% sodium hypochlorite, and then washed with sterilized distilled water three times, plated on potato dextrose agar (PDA), and incubated at 24°C for 7 days with a 12-h photoperiod. A total of 94 isolates of Colletotrichum sp. were obtained from cultivated cassava and 15 from wild species. The genetic diversity of the isolates was accessed by rep-PCR (Mahuku and Riascos 2004), with the formation of three groups: group 1 with 101 isolates closely related with C. gloeosporioides and C. fructicola, and groups 2 and 3 with no correspondence with previous reported species, with five and three isolates, respectively. Four isolates (PPAM 08, PPAM 15, Ant 38CX, and Ant 44C19) were randomly selected for further characterization, being two of each group (2 and 3). Pathogenicity was assessed using detached cassava leaves inoculated with a 5-mm plug of PDA with sporulating fungal colonies. Both wounded (sterilized needle) and nonwounded tissues were inoculated. Ten leaves were inoculated by each method and 10 noninoculated served as controls. The leaves were incubated in a moist chamber at 25°C. Symptoms appeared on foliar tissues after 3 to 5 days. To confirm the species identity, DNA was extracted by the CTAB protocol (Murray and Thompson 1980) and sequences of GAPDH, CHS-1, and CAL genes were generated (Diao et al. 2017). The DNA sequences blasted against the GenBank database were 96 to 100% similar to C. siamense (PPAM 08 and PPAM 15), and to C. theobromicola (Ant 38CX and Ant 44C19). Phylogenetic analysis was further conducted based on Bayesian inference for each gene and the concatenated alignment and the same topology and clades were found. Generated sequences for isolates PPAM 08 (GAPDH: KP763688, CAL: KP763674, and CHS-1: KP763681) and PPAM 15 (GAPDH: KP763691, CAL: KP763677, and CHS-1: KP763683) were more than 98% similar to the ex-type strain CMM 4083 (GAPDH: KC517194 and CAL: KC517209) previously described as C. dianesei (Lima et al. 2013), but reclassified as C. siamense by Liu et al. (2016). PPAM08 and PPAM15 strains were isolated from wild cassava species. The conidia (n = 50) morphology on synthetic nutrient poor agar (SNA) was similar to C. siamense, with 11.3 to 19.2 (avg. 14.8) × 4.4 to 6.8 (avg. 5.7) µm. In contrast, Ant 38CX (GAPDH: MF465790 and CAL: MF465788) and Ant 44C19 (GAPDH: MF465789, CAL: MF465787, and CHS-1: MF465791) strains were more than 98% similar to the ex-type strain ICMP:17895 (GAPDH: JX010057, CAL: JX009600, and CHS-1: JX009828), being classified as C. theobromicola. Ant 38CX and Ant 44C19 strains were isolated from anthracnose disease in cultivated cassava. On SNA, conidia (n = 50) were 11.8 to 15.2 (avg. 13.5) × 3.7 to 5.8 (avg. 4.8) μm. The species C. theobromicola was described associated with anthracnose lesions in cacao (Rojas et al. 2010), and C. siamense with anthracnose in different hosts and tropical fruits (Lima et al. 2013; Liu et al. 2016). To our knowledge, this is the first report of C. siamense causing anthracnose in two species of wild cassava (M. carthaginesis and M. tomentosa), and of C. theobromicola on cultivated cassava (M. esculenta) in Brazil.