Development of PCR based SSR markers for Wilsonomyces carpophilus and a PCR based diagnosis protocol for the early detection of shot hole disease in stone fruit crops

Background: The conidial Ascomycota fungus Wilsonomyces carpophilus causing shot hole in stone fruits is a major constraint in the production of stone fruits worldwide. Shothole disease symptoms appear on leaves, fruits, and twigs. Successful isolation of the pathogen from different hosts on synthetic culture medium is a time consuming and tedious procedure for identification of the pathogen based on morpho-cultural characterization. Methods and results: The present research was carried out to develop a successful PCR based early detection protocol for the shot hole disease of stone fruits, viz., peach, plum, apricot, cherry, and almond using the pathogen specific SSR markers developed from the Wilsonomyces carpophilus genome using Genome-wide Microsatellite Analysing Tool package (GMATA) software. Diseased leaf samples of different stone fruits were collected from the SKUAST-K orchard and the pathogen was isolated on potato dextrose agar (PDA) medium and maintained on Asthana and Hawkers’ medium with a total of 50 pathogen isolates comprised of 10 isolates each from peach, plum, apricot, cherry and almond. The DNA was extracted from both healthy and infected leaf samples of different stone fruits. The DNA was also extracted from the isolated pathogen cultures (50 isolates). Out of 2851 SSR markers developed, 30 SSRs were used for the successful amplification of DNA extracted from all the 50 pathogen isolates. These SSRs were used for the amplification DNA from shot hole infected leaf samples of different stone fruits, but the amplification was not observed in the control samples (DNA from healthy leaves), thus confirming the detection of this disease directly from the shot hole infected samples using PCR based SSR markers. To our knowledge, this forms the first report of SSR development for the Wilsonomyces carpophilus and their validation for the detection of shot hole disease directly from infected leaves. Conclusion: PCR based SSR makers were successfully developed and used for the detection of Wilsonomyces carpophilus causing shot hole disease in stone fruits including almond in nuts for the first time. These SSR markers could successfully detect the pathogen directly from the infected leaves of stone fruits namely peach, plum, apricot and cherry including almond from the nuts.