First Report of Colletotrichum siamense Causing Leaf Anthracnose on Cotton in India

Abstract

During 2017 and 2018, distinct leaf anthracnose symptoms were observed on cotton (Gossypium hirsutum L.) from Wardha district, Maharashtra state, India. The appearance of symptoms coincided with greater humidity and intermittent rains during the months of August and September. Among 500 Bollgard-II cotton hybrids plants, nearly 20 to 30% were found affected, which showed 35 to 40% disease severity. Symptoms initially appeared as small, circular or irregular, sunken, chlorotic spots, which later turned brown with a dark border. As the disease progressed, lesions expanded and merged, causing necrosis of the whole leaf. For pathogen isolation, 15 randomly sampled symptomatic plants were brought to the laboratory. Diseased leaf sections (5 mm2) were surface disinfested with 1% NaClO for 2 min, rinsed three times in sterile water, dried with sterilized blotter paper, and then plated on potato dextrose agar. The plates were incubated at 25 ? 2?C for 5 days under a 12-h photoperiod. Three isolates (CS1, CS2, and CS3) were recovered from infected leaves. Purified colonies were initially white, turned gray with time, and had salmon-colored conidial masses embedded in the medium in a concentric ring pattern and black acervuli aggregated mainly in the center of the culture plate. Conidia were single celled, hyaline, cylindrical having rounded ends, smooth walls, guttulate, 9.6 to 16.9 ?m in length, and 3.5 to 6.0 ?m in width (n = 100). These morphological characteristics were consistent with the species description of Colletotrichum gloeosporioides complex (Weir et al. 2012). For molecular identification, the internal transcribed spacer (ITS) region of rDNA and a partial sequence of the actin (ACT) and beta-tubulin (TUB2) genes of three isolates were amplified with ITS1/ITS4, ACT-512F/ACT-783R, and BT2Fd/BT4R primers, respectively (Damm et al. 2009). The ITS sequence (MH478170, 558 bp), ACT gene (MH494078, 283 bp), and TUB2 gene (MH494079, 321 bp) of representative isolate CS1 showed 99 to 100% identity with sequences (JX010171, FJ907423, and JX010404, respectively) obtained from the holotype isolate (BPD-I2) of Colletotrichum siamense Prihastuti, L. Cai & K.D. Hyde (Prihastuti et al. 2009; Weir et al. 2012). Pathogenicity was tested on 45-day-old Bollgard-II cotton hybrid plants by spore spray inoculation. Five plants for each isolate were inoculated with a conidial suspension (106 conidia/ml) (and control plants with sterilized water) and kept separately in a glass house at 25?C, >85% relative humidity, and a 12-h photoperiod. Plants were sprayed lightly with water in the morning and evening to maintain humidity and thus enhance the infection. The experiment was repeated. Anthracnose symptoms were observed 7 days after inoculation, whereas the control plants remained symptomless. The pathogen was reisolated from infected leaves and its identity as C. siamense confirmed based on morphological characteristics as well as on sequences of ITS, ACT, and TUB2 genes. To our knowledge, this is the first report of leaf anthracnose caused by C. siamense on cotton in India or globally. This report will be useful to plan for species-specific disease management strategies.