First report of anthracnose caused by colletotrichum truncatum on bell pepper (Capsicum annuum) in Pakistan

Anthracnose fruit rot of bell pepper (Capsicum annuum L. var. Grossum Sendt) is an important field and postharvest disease in Pakistan causing a considerable reduction in yield. From June 2014 to July 2015, symptoms were observed on approximately 40 to 72% of the fruits growing in three fields, which totaled 15 ha in the Chakwal district (32°51′40″ N, 72°45′36″ E) of Pakistan. Circular to angular, necrotic sunken lesions with concentric rings were observed on fruit with light orange spore masses produced in black acervuli. Lesions from 48 symptomatic fruit were cut into small pieces (5 to 10 mm²) and immersed in 1% NaOCl solution for 2 minutes, rinsed three times in sterilized distilled water, and dried on sterilized filter paper. The tissue was then plated on potato dextrose agar (PDA) and incubated for 4 days at 26 ± 2°C. The colonies were initially light gray before turning dark gray after 8 to 10 days (Diao et al. 2014) with abundant black structures similar to acervuli with mucilaginous, light orange spore masses. The conidia were falcate, aseptate hyaline measuring 22 to 28 × 2.2 to 2.5 μm (mean = 25 × 2.4 μm), and originating from an acervular conidiomata surrounded with thick walled setae that were septate, straight, and dark brown measuring 82 to 130 × 3.5 to 6 μm (mean = 104 × 4.6 μm). Appressoria were light brown, irregular in shape, and 12.5 to 15 × 5 to 7.5 μm (mean = 13.8 × 6.4 μm). Morphological characteristics of 36 isolates were consistent with those of Colletotrichum truncatum (Damm et al. 2009). For molecular identification, DNA from two representative isolates, ACT1 and ACT3, was extracted, purified by GeneJet PCR purification kit, and sequences of the internal transcribed spacer (ITS1/ITS4), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Templeton et al. 1992) regions were obtained and submitted in GenBank. The accession numbers (KU051395, KX348142, and KX348141) for isolate ACT1 and (KU051397, KX276650, and KX378681) for ACT3 were assigned to ITS, GAPDH, and ACT regions, respectively. The sequences for all three genes exhibited 100% identity with C. truncatum, GenBank accession no. KX197404 (ITS), KP145568 (GAPDH), and KC110826 (ACT) for both isolates, when compared with Q-bank fungi database. Pathogenicity testing was conducted on symptomless, detached bell pepper fruit. Three unwounded fruit per isolate were inoculated with drop of 20 µl conidial suspension (10⁶ conidia/ml) harvested from 2-week-old cultures, grown on PDA. Fruits inoculated with the same volume of sterile distilled water served as the negative control. All inoculated and control fruit were kept in moist chambers and incubated at 25°C. Five days after inoculation, sunken necrotic lesions having light gray mycelia with abundant acervuli were observed on the inoculated fruit surface similar to the original symptoms observed in the field. However, control fruit remained asymptomatic. The fungal colonies reisolated from the lesions were morphologically similar to the original isolates on PDA, confirming Koch’s postulates. This pathogen was previously reported causing disease on bell pepper in Trinidad (Ramdial and Rampersad 2015) and has been reported on chili pepper in many countries of the world (Katoch et al. 2016). To the best of our knowledge, this is the first report of C. truncatum causing anthracnose on bell pepper in Pakistan.