Investigation of Genetic Diversity of Wilsonomyces carpophilus in Khorasan Razavi Using rep-PCR Marker

Shot hole disease of stone fruit trees resulted from Wilsonomyces carpophilus can weaken the trees and reduce the quantity and quality of the crops worldwide particularly in semi-arid regions. Coryneum blight or shot hole disease infects all the stone fruit trees including peach, nectarine, apricot, sour cherry, plum, cherry, and almond. One of the most important strategies to manage any plant disease is to use resistant cultivars. In this way, it is very important to have knowledge about the status of genetic diversity and to determine the relationship between isolates of the causal agent fungus. The main objective of the present research was to study the genetic diversity of W. carpophilus in Khorasan Razavi province using the rep-PCR molecular fingerprinting method.
Sampling was performed from peach, nectarine, plum, apricot and cherry orchards of Quchan, Torqabeh, Shandiz, Chenaran, Neishabur, Kalat, Torbat Heidarieh and Mashhad during spring and summer of 2012 and 2013. Mono-conidial isolates were recovered from infected leaves, fruits, and twigs of different parts of orchards. Infected collected leaves, twigs, and fruits were transferred to the laboratory. By using techniques of Klimesova and Prasil (1989) and Mehta (1998) from the cut parts between infected and healthy tissues of each isolate, cuts of 2-3 mm from leaf, fruit and twig were prepared by the scalpel. These pieces were surface sterilized with 1% sodium hypochlorite liquid about 1 to 3 minutes based on the thickness of tissue. Then, the samples were cultured on PDA, MEA, and WA media and incubated at 18, 20, and 25 °C. The isolated fungi were purified and identified. The research was performed on 20 fungal isolates collected from different stone fruit trees. Genomic DNA was amplified using BOX A1R, ERIC2, ERIC1R, REP2-I, and REP1R-I primers. Thirty-eight of 39 fragments amplified were polymorphic for 100 to 5000 base pairs. Similarity matrix between isolates was calculated based on Jacquard Coefficient and cluster analysis and construction of dendrogram were done based on UPGM using NTYSIS.PC 2.0 software.
From 39 amplified bands, 38 bands (97.5%) showed polymorphism. The molecular weight of amplified DNA fragments was between 100 to 5000 bp. Based on analysis of banding pattern of REP primer set, isolates of W. carpophilus were categorized into 12 groups at the 69% similarity level. The most genetic similarity of isolates (94%) was between AK (apricot of Kalat) and PK (peach of Kalat) and the least genetic similarity of W. carpophilus isolates was between AQ (apricot of Quchan), AN (apricot of Neishabur), AC (apricot of Chenaran), PlM (plum of Mashhad), PM2 (peach of Mashhad number 2), and CC (cherry of Chenaran) with the other isolates. Based on the results of this study rep-PCR could separate isolates of W. carpophilus very well and also could separate similar isolates and hosts which have the close genetic relationship. Similar results were obtained by Edel et al. (1995) and Jedryczka et al. (1999). Edel et al. (1995) compared three different molecular methods for characterization of Fusarium oxysporum strains. The marker also separated isolates of Kalat and Chenaran geographically and to some extent isolates of plum in terms of hosting from the other isolates. Toda et al. (1999) in their study, about the investigation of genetic correlation among and within different isolates of Rhizoctonia solani by rep-PCR divided the 41 isolates into 7 groups which indicate considerable genetic diversity among isolates. Also, Karimi et al. (2010) in their study about the investigation of genetic diversity of Sclerotinia sclerotiorum at 64% similarity level, divided the isolates into 7 groups and separated most of the isolates geographically.
The results obtained in this study indicated that rep-PCR is a practical, rapid, and accurate technique for separation of W. carpophilus isolates. Considering the high genetic diversity observed in the population of this fungus, making attempts to plant cultivars with high resistance and resistant genes can largely prevent the outbreaks and intensity of the pathogen. Therefore, further researches in this area can be placed in breeding, production and reproduction of cultivars with particular resistance programs against shot hole disease. The present research study is a prelude to solving problems related to this important disease.