First Report of Colletotrichum karstii Causing Anthracnose on Annona muricata in Brazil

Annona muricata L. (Annonaceae), commonly known as soursop, is an important crop for the food industry and possesses a wide spectrum of biological activities (Moghadamtousi et al. 2015). Colletotrichum species are major plant pathogens causing anthracnose on Annona spp. (Kamei et al. 2014). This is an important disease in tropical regions, causing severe soursop production losses every year (McMillan 1986). In May 2017, anthracnose-like symptoms, consisting of typical dieback, were observed on A. muricata saplings in a forest nursery in the city of Viçosa (Minas Gerais state, Brazil). The necrotic lesions started with brown to black streaks, becoming white in color with large necrotic borders containing black acervuli. A single-conidium culture was obtained on potato dextrose agar (PDA). Leaf fragments were washed in running water and then surface disinfested by consecutive immersion in 70% ethanol for 1 min, in 1% sodium hypochlorite for 3 min, and in sterile distilled water for 1 min. The culture was deposited at the Otávio de Almeida Drumond Culture Collection (DOA 1430 COAD 2391). Colonies on PDA were gray to pale salmon, with abundant conidiomata (acervuli) and aerial mycelium in dispersed tufts. Acervuli were circular to elliptical, arranged irregularly, disrupting the outer epidermal cell wall of the host. Conidiogenous cells were cylindrical and hyaline; conidia cylindrical shaped, one-celled, smooth-walled, (9.18 to) 10.13 to 15.46 × 4.56 to 7.99 μm (x- = 12.42 ± 1.39 × 5.49 ± 0.65, n = 30). Appressoria were dark brown to black, circular to clavate, 6.14 to 10.83 × (4.87 to) 5.13 to 7.42 μm (x- = 8.51 ± 1.19 × 5.99 ± 0.64, n = 30). Morphological characteristics were consistent with those described in the literature for C. karstii (Yang et al. 2011). DNA was extracted, and the primers pairs ACT-512F/ACT-783R, CAL-228F/CAL2Rd, CHS-79F/CHS-354R, and ITS1/ITS4, were used to amplify the actin, calmodulin, chitin synthase 1, and internal transcribed spacer genes, respectively. The sequences were deposited in GenBank under accession numbers MH138304, MH138305, MH138306, and MH138307. Results from Bayesian inference and maximum likelihood estimation analyses using consensus sequences placed the Colletotrichum isolate reported here in a same clade with other C. boninense specimens (study ID 22342 deposited in TreeBASE). The results suggest that C. karstii clade is well-supported (100/1.00) and forms a sister clade to the C. novae-zelandiae clade (86/1.00). The isolate under study, COAD 2391, clustered closely together with the C. karstii reference isolate CBS 127552 with a bootstrap support of 98% and a Bayesian posterior probability value of 0.88. To confirm pathogenicity, mycelial plugs were placed onto 25 wounds in each of six A. muricata saplings. On control treatment, only PDA medium plugs without fungal culture were placed on the wounded leaves. Plants were kept for 48 h in a mist irrigation chamber at 25°C and subsequently maintained in a growth chamber (25 ± 2°C, 80 ± 5% humidity, 12-h photoperiod). The symptoms included dark brown necrotic lesions, irregularly round in shape. After 10 days, the pathogen was successfully reisolated from inoculated plant tissues, and the cultures were identical to the original strains, confirming Koch’s postulates. In Brazil, C. karstii has been previously reported on other crops, such as araticum fruit (Annona crassiflora), Malabar chestnut (Pachira aquatica), papaya (Carica papaya), Brazilian cherry (Eugenia uniflora), mango (Mangifera indica), apple (Malus domestica), and southern highbush blueberry (Vaccinium corymbosum) (Damm et al. 2012; Farr and Rossman 2018). However, this is the first report of C. karstii causing anthracnose on A. muricata leaves in Brazil.