de Nova Whole Genome Sequencing of Marssonina Juglandis Causing Walnut (Juglandis Regia L.) Anthracnose and Development of PCR-Based Diagnosis Using Simple Sequence Repeat (SSR) Markers

Walnut anthracnose, caused by Marssonina juglandis, is one of the economically important diseases of walnuts worldwide. The pathogen is a filamentous fungus belonging to the class Leotiomycetes. In this study, we isolated and purified the Marssonina juglandis from walnut leaves collected from the walnut orchard of SKUAST-Kashmir. The whole genome of the pathogen was sequenced using the Illumina HiSeq NGS platform. The whole genome of the pathogen was found to be 63.6354 Mb in size with 1916 scaffolds, 12086 genes, 205 total tRNAs, and 6884 SSRs containing 5861 SSRs with 150 flanking regions. A phylogenetic analysis using whole-genome alignment revealed that Marssonina juglandis is closely related to Cryphonectria parasitica and Coniella lustricola. The BlastP analysis using Uniprot, Pfam, and KOG showed 7612, 5515, and 5163 protein hits out of 12086 proteins. The total number of genes responsible for biological processes, molecular function, and cellular components were 1583, 1429, and 1843, respectively. In the Venn diagram, 3912 genes were found to be common in all three software, namely Uniprot, Pfam, and KOG including NR. In addition, we developed the SSR markers based on the whole genome and standardized them against 25 isolates of the Marssonina juglandis collected, isolated, and purified from the walnut-infected leaves in Jammu and Kashmir, India. Out of 30 randomly selected SSRs, 17 SSRs showed successful PCR amplification in 25 pathogen isolates that were found to be highly polymorphic. Further, a PCR-based detection protocol using SSR markers was also developed for early detection of the disease in the field for timely management. We found two primers that could successfully amplify the pathogen in DNA extracted directly from the infected walnut leaves.