Development of PCR based SSR markers for Wilsonomyces carpophilus: A PCR based diagnosis protocol for early detection of shot hole disease infecting stone fruit crops

Abstract

Background The conidial Ascomycota fungus Wilsonomyces carpophilus causing shot hole of stone fruits is a major constrain in the production of stone fruits worldwide. Shot hole 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 current research was carried out to develop a successful detection protocol for shot hole disease of stone fruits viz., peach, plum, apricot, cherry and almond using PCR based SSR markers that were designed from the Wilsonomyces carpophilus genome using Genome-wide Microsatellite Analysing Tool package (GMATA) software to detect the pathogen at early stages. Diseased leaf samples of stone fruits were collected and the pathogen isolated on potato dextrose agar (PDA) medium and maintained on Asthana and Hawkers’ medium. For detection protocol, healthy and infected leaf samples of stone fruits were collected and DNA was extracted from the isolated pathogen cultures as well as from leaves infected shot hole disease along with the healthy control (DNA from healthy leaves of stone fruits). The successful amplification was observed in DNA extracted from all the pathogen isolates and from leaf samples with shot hole symptoms but was not observed in control (DNA from healthy leaves) thus confirming the detection of this disease from all the infected samples. Conclusion PCR based SSR makers were successfully designed for the for Wilsonomyces carpophilus causing shot hole disease in stone fruits and almond in nuts first time. PCR based detection protocol was successfully developed for the detection of pathogen directly from infected leaves of stone fruits such as peach, plum, apricot, cherry and almond among the nuts.