First Report of Colletotrichum plurivorum Causing Anthracnose on Cucumis sativus in Brazil

Abstract

Cucumbers have great economic and social importance. Annual worldwide production is approximately 80 million tons (FAOSTAT, 2019), 184 thousand tons of which are produced in Brazil (IBGE, 2020). Leaves with symptoms of anthracnose (necrotic brown or angular spots) were observed on cucumber plants grown in organic systems in September 2021, Pernambuco, Brazil (8°7’45’’S, 35°16’167’’W). About 40% of the plants fields were infected. Samples were collected and fragments were cut from the margins of the symptomatic tissue. The fragments were superficially disinfected with 70% ethanol (30 s) and 2% sodium hypochlorite (2 min), then washed three times with sterile distilled H2O and dried on sterile filter paper. The fragments were placed on potato dextrose agar (PDA) containing chloramphenicol (50 mg/L) and incubated at 28 ± 2 °C for 3 days. From the fungal isolates obtained, a representative specimen of Colletotrichum spp. was isolated, purified by subculturing from emergent hyphae tips and used for morphological characterization, phylogenetic analysis, and pathogenicity testing. The fungus isolated on PDA formed gray to grayish-black colonies with white aerial mycelia after 7 days. Ascomata were globose to subglobose, 120–200 × 100–150 μm in size (n = 10). Setae formed directly on the hyphae. Asci were 50–70 × 10–12 μm in size, 8‐spored, unitunicate, thin-walled, and clavate. Ascospores were 14–22 × 4–5 μm in size (n = 30), hyaline, slightly curved to curved with obtuse to slightly rounded ends. Conidia were hyaline, smooth-walled, aseptate, straight, cylindrical, the apex and base rounded, and 12–15 × 5 μm in size, (n = 30). For molecular identification, the nuclear ribosomal internal transcribed spacers (nrITS), actin (ACT), beta-tubulin (TUB), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were sequenced (Damm et al. 2019). The sequences obtained were deposited in GenBank (nrITS: OP720945, ACT: OP723523, TUB: OP723525, and GAPDH: OP723524). The sequences from the nrITS region, ACT, TUB2, and GAPDH were highly similar to those from C. plurivorum: nrITS - CBS 125474 (539/539 - 100%; NR_160828); ACT - CBS 125474 (270/271 - 99%; MG600925), TUB2 - CBS 125474 (517/518 - 99%; MG600985); and GAPDH - CBS 125474 (197/197 - 100%; MG600781), respectively. Multilocus phylogenetic analysis was performed using Bayesian inference, which showed that the isolate C. plurivorum FPO04 clustered in the same clade as the ex-type of C. plurivorum (CBS 125474). In the pathogenicity test, leaves of five healthy cucumber plants, previously injured in the middle region with sterile needles, were inoculated with 50 µl of a conidial suspension (1 × 106 spores mL -1) prepared from 7-day-old of colonies of C. plurivorum. Sterile distilled water was used as negative controls. The inoculated plants were maintained in a humid greenhouse chamber for 24 hours. After 7 days, the same anthracnose symptoms seen in the field were observed on the inoculated plants. Control plants remained healthy. Colletotrichum plurivorum was reisolated from symptomatic leaves, fulfilling Koch's postulates. This species has been reported from several crops, including Abelmoschus esculentus (okra) (Damm et al. 2019) and Glycine max (soybeans) (Zaw et al. 2019). To our knowledge, this is the first report of C. plurivorum causing anthracnose on cucumber leaves in Brazil. This report lays the groundwork for future studies to determine management practices for control of this disease in C. sativus.