First Report of Fusarium flocciferum Causing Wilt Disease of Chili (Capsicum annuum) in Northern Himalayas

Abstract

In August 2020 chili (Capsicum annuum L.) showing wilt symptoms were collected from different districts of the Kashmir: Pulwama, Srinagar, Baramulla, and Anantnag. From each district one location was selected for sample collection and a total of 23 chili isolates were isolated. The tissue bit technique was used to isolate fungus from the infected samples on potato dextrose agar (PDA) medium, purified using the single spore technique, maintained at 25⁰±1℃ and then stored at 4° C (Ferniah et al. 2014) . Initially cultural characteristics appeared as white colonies which gradually turned to pale white colored and attained a growth of 90 mm in 18 days of incubation at 25 ± 1°C. Microscopic observations revealed that mycelium was branched and cylindrical, 3.53–4.98 µm in width. Microconidia were ellipsoidal, hyaline, 0-1 septa werepresent, and 6-7 x 3-4 µm in size. Macroconidia were cylindrical, hyaline, 2-6 septa, measuring 20-60 x 40-45 µm in size. Molecular identification of the pathogens with ITS, TEF, and RPB2 was successfully carried out and the fungi was confirmed as Fusarium flocciferum infecting chili. Amplified PCR products were sequenced and were successfully submitted and accessioned in GenBank with accession number OM189458, OM441199, OR484037 for ITS, TEF, and RPB2 gene. To confirm Koch’s postulates pathogenicity test was carried out using rhizosphere inoculation technique (Najar et al. 2011, Parihar et al. 2022). In total 7 replications for sand maize meal medium (potting mixture) was prepared by autoclaving 90 g of sand and 10 g of maize meal in 250 ml of erlenmeyer flask comprising 40 ml of distilled water. The spore suspension at 100 µl per pot was inoculated and was mixed with the sterilized potting mixture in a ratio of (2:1) and up to seven days pathogen was allowed to infect the soil (Davey and Papavizas 1962; Hami et al. 2021). Then chili seeds (cv. Kashmir long-1) were sown in infected potting mixture and grown for three weeks to allow the pathogen to infect the host plants. F. flocciferum took six weeks for appearance of symptoms in the infected potted plants. Control mock inoculation of the potting mixture was carried out using water droplets instead of spore suspension at 100 µl per pot. Seven replications were kept for both inoculated and un-inoculated / control mock pots. The plants developed initial symptoms from light green to yellowish discoloration of leaves followed by the drooping, shriveling, and ultimately leading to death. The collar region of the plant was cut vertically and observed that vascular bundles showed brownish spots and discoloration, indicating wilt as the cause of death. The pathogens were re-isolated and inoculated from all infected plants, then compared with their original pure culture inoculated first, which completely resembled based on morphological, cultural, and pathogenic characteristics. No symptoms were observed on control plants. A phylogenetic analysis was also carried out using ClustalW software that grouped the species identified by different genes into different clades. F. flocciferum has been reported earlier in pea, faba bean and bamboo (Kainthola et al. 2022; Šišić et al. 2020) . In solanaceous crops, this species have been explored as wilt pathogens for the first time from India, indicating diversifying nature of Fusarium flocciferum across various hosts including solanaceous crops.