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.

First report of powdery mildew caused by Phyllactinia pyri-serotinae Sawada on pear (Pyrus communis L.) from India.

In August 2020 powdery mildew was observed on pear cv. Fertility at the University research field in Shalimar, Srinagar (J&K), India (34° 08' 30.5'' N and 74° 51' 42.0'' E) with a disease incidence up to 30% (100 leaves observed from ten trees). White irregularly shaped fungal colonies were observed on the abaxial leaf surface which latter covered the whole leaf surface and developed black chasmothecia. The affected leaves appeared brittle, slightly curved upwards and dropped prematurely. Mycelium was hypophyllous, septate and measured 2.0 to 5.0 µm in width. Appressoria were nipple shaped, solitary or present in opposite pairs. Conidiophores were erect, up to 440.0 µm long (n=50), mostly centrally on upper surface of mother cells. Conidiophore foot-cells were filiform, followed by 1 to 3 shorter cells, producing single conidia at the tip. Conidia were hyaline, lanceolate, with a non-papillate rounded apex, measuring55.5 to 81.4 × 14.8 to 22.5 µm (n=50) and devoid of any conspicuous fibrosin bodies. Germ tube was, filiform, twisted, arose basally and measured 2.0 to 5.0 µm in width. Chasmothecia were hypophyllous, black, scattered, globose and measured 195.0 to 255.0 µm in diameter (n=50) having 8 to 12 equatorial, acicular, up to 270.0 µm length appendages with 25.9 to 44.4 µm diameter bulbous base (n=50) and obtuse or subacute apex. Asci in a chasmothecium were clavate to saccate, 62.9 to 81.4 × 18.5 to 22.2 µm (n=50), stalked, and two- spored. Ascospores were 33.3 to 40.7 × 12.9 to 18.5 µm (n=50), pale yellowish or golden brown in color. All morphological features were consistent with Phyllactinia pyri-serotinae (Braun and Cook 2012). To confirm the fungus identity at molecular level, DNA of two isolates was extracted from chasmothecia. The internal transcribed spacer (ITS) sequence of ribosomal DNA was amplified with the primers ITS1 and ITS4 (White et al. 1990) and sequenced. The ITS sequences submitted to NCBI GenBank under Accession No. MZ505441 and MZ505442 have 97 (416/427) & 96 (424/440) per cent and 99 (424/430) & 98 (428/438) per cent base pair matching, with that of P. pyri-serotinae isolates from Japan (AB080521 and AB985507), respectively. Thus, the pathogen was identified as Phyllactinia pyri-serotinae Sawada based on morphological and molecular sequence analyses. The pathogenicity tests of both the isolates were carried out on one year old pear saplings (cv. Fertility) and repeated twice. The inoculum was prepared by collecting P. pyri-serotinae conidia in sterile distilled water from infected pear leaves. Three saplings were inoculated by spraying (15ml per sapling) the inoculum (3 x 105 spores ml-1) on leaf surfaces, while same number of saplings sprayed with sterile distilled water served as non-inoculated controls. After 15 days of incubation at 25oC in a green house, similar symptoms as observed on naturally infected plants were observed on inoculated plants and uninoculated plants remained symptomless. The pathogen of interest observed on inoculated plants was morphologically characterized and found to be similar to P. pyri-serotinae. The voucher specimen was deposited in the Herbarium Crytogamae Indiae Orientalis (HCIO), IARI, New Delhi under accession number 52213. Pear is the third most important temperate fruit grown in India (Chattopadhyay 2009) and our study reveal P. pyri-serotinae as the new causal agent of powdery mildew in addition to P. guttata (Dhar and Shah 1982) under Indian conditions.