Isolation, Molecular Characterization and First Report of Dothiorella Gregaria Associated with Fruit Rot of Walnuts of Jammu and Kashmir, India

Walnuts are important nutrient dense food and are regularly consumed due to the presence of numerous essential macronutrients, micronutrients and health promoting phenolic compounds (Polyphenols). The walnuts kernels purchased from market places of J&K, India were observed to be affected by Dothiorella gregaria species causing the quality detoriation and decrease in polyphenol content thus undeniably with decreased antioxidant properties. D. gregaria Infected walnut kernels were having some brown and black spots and some were having white mycelial growth and however, most samples were asymptomatic. Pathogenicity testing revealed that the pathogen was able to develop all the symptoms under experimental conditions and the reisolated pathogen was morphologically similar to D. gregaria. The samples infected with this pathogen showed considerable decrease in polyphenol content, 10.9 ± 2.66 mgGAE/g (mean ± standard deviation) thus decreased antioxidant quality as compared to the samples which showed zero incidence of this pathogen, 52.50 ± 4.27 mgGAE/g (mean ± standard deviation). Furthermore, the pathogen was studied using polyphasic approach involving morphological, molecular and phylogenetic analysis. Combined nucleotide dataset of nuclear ribosomal ITS and tef1-α revealed that Dothiorella gregaria (NY6) formed a clade with Dothiorlla iberica (MAEC33), Dothiorella sarmentorium (MAEC28) and Dothiorella iberica (CAA905) strains with 83% bootstrap support. Besides, we observed six nucleotide changes, four were insertions or deletions and two were substitutions in the 502-bp region of the ITS rRNA gene when we compared our isolate to the most equivalent sequences submitted to NCBI GenBank. This is the first report of D. gregaria affecting walnuts purchased from various markets in J&K, India, causing fruit rot in walnuts after harvest. Given that local farmers store and export walnuts, it could pose an emerging threat to their livelihood. However, creating post-harvesting interventions for D. gregaria and knowing more about the fruit rot in walnuts can be benefited from morphological and molecular identification using several gene loci, genetic variability in the ITS rRNA gene, and total phenol analyses.