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

Document Type : Research Article


1 University of Zabol

2 Agricultural and Natural Resources Center of Khorassan Razavi, Mashhad.

3 University of Yazd

4 Khorassan Razavi, with M.Sc. degree


Introduction: 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.
Materials and Methods: 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.
Results and discussion: 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.
Conclusion: 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.


1- Adaskaveg J.E., Ogawa J.M., and Buttler E.E. 1990. Morphology and ontogeny of conidia in Wilsonomyces carpophilus gen.nov. and comb.nov., causal pathogen of shot hole disease of Prunus spicies. Mycologia, 31:275-290.
2- Ahmadpour A., Ghosta Y., Javan-Nikkhah M., Fatahi Moghadam M.R., and Ghazanfari K. 2011. A study on specificity and host range of Wilsonomyces Carpophilus, the causal agent of shot hole disease of stone fruit trees and evaluation of relative resistance of some peach cultivars. Iranian Journal of Plant Protection Science (Iranian Journal of Agricultural Sciences), 42(2): 251-259. (in Persian with English Abstract)
3- Campbell J., and Skoglund L. 2011. Epidemic of Shot Hole Disease. Urban IPM Newsletter, 1(3): 1-6.
4- Cheragali V., Sarpeleh A., and Razavi M. 2012. Genetic diversity within Monosporascus cannonballus isolates in Iran. Iranian Journal of Plant Pathology, 48: 1-12. (in Persian with English abstract)
5- Evans K., Frank E., Gunnell J., Pace M., and Shao M. 2008. Coryneum or Shot hole Blight. Utah State University Extension and Plant Pest Diagnostic Laboratory. 1-3.
6- Jafar poor B., Hatami B., and Hadad Irani nejad K. 1983. Illustrated collection of important fruit trees pests and diseases in the in Khorasan province. Publications of Jihad-e Daneshgahi of the Ferdowsi University of Mashhad. (in Persian)
7- Jalali F., Safaie N. and Abbasi S. 2010 Study of the genetic diversity of Iranian isolates of Macrophomina phaseolina causal agent of soybean charcoal rot, using ISSR and REP-PCR markers. Journal of Genetic Novin, 5(3):57-68. (in Persian)
8- Jedryczka M., Rouxel T., and Balesdent M.H. 1999. Rep-PCR based genomic fingerprinting of isolates of Leptosphaeria maculans from Poland. European Journal of Plant Pathology, 105: 813–823.
9- Karimi E., Safaei N., Shamsbakhsh M. 2009. Genetic diversity and pathogenic variability of Sclerotinia sclerotiorum isolates using rep-PCR marker. Iranian Journal of Plant Pathology, 45(3): 199-212. (in Persian with English abstract)
10- Kim W.K., Mauthe W., Hausner G., and Klassen G.R. 1990. Isolation of high molecular weight DNA and doublestranded RNAs from fungi. Canadian Journal of Botany, 68: 1898-1902.
11- Klimesova M., and Prasil K. 1989. Morphological variability of the conidia of Alternaria alternata (Hyphomycetes). Novitates Botanicae Universitatis Carolinae, 5: 7-27.
12- Komijani S. Razavi M. and Aminian H. 2010. Study on the genetic diversity of Mycosphaerella graminicola cause of septoria leaf blotch of wheat in Iran using SSR, SCAR and rep-PCR markers. Iranian Journal of Plant Pathology, 45: 287-300. (in Persian)
13- Louws F.J., Fulbright D.W., Stephens, C.T., and De Bruijn F.J. 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Applied and Environmental Microbiology, 60: 2286-2295.
14- Mehta Y.R. 1998. Severe outhbreak of Stemphylium leaf blight, a new disease of cotton in Brazil. Plant Diseases, 82: 333-336.
15- Mehta A., Mehta Y.R., and Rosato Y.B. 2002. ERIC- and REP-PCR amplify non-repetitive fragments from the genome of Drechslera avenae and Stemphylium solani. FEMS Microbiology Letters, 211: 51-55.
16- Motallebi P., Javan-Nikkhah M., Okhovvat S. M. and Fotouhifar Kh. B. 2009. A study on population structure of Pyricularia grisea isolated from rice, based on rep-PCR molecular marker and Identification of vegetative compatibility groups. Iranian Journal of Plant Protection Science (Iranian Journal of Agricultural Sciences), 42(2):227-239. (in Persian)
17- Murray H.G., and Thompson W.F. 1980. Rapid isolation of high molecular weight DNA. Nucleic Acid Research, 8: 4321-4325.
18- Rafaila C., and Zaharia A. 1979. Biological and ecological feutures of Stigmina carpophila (Lev.) M. B. Ellis needed for the determination of forecasting and monitoring elements. Analel Institutului Cercetari Pentru Protection Plantelor, 14: 51-60.
19- Redondo C., Cubero J., and Melgarejo P. 2009. Characterization of Penicillium species by ribosomal DNA sequencing and BOX, ERIC and REP-PCR analysis. Mycopathologia, 168: 11–22.
20- Rogers S.O., Rehner S., Bledsoe C., Muller G.J., and Ammirati J.F. 1989. Extraction of DNA from Basidiomycetes for ribosomal DNA hybridization. Candian Journal of Botany, 67: 1235- 1243.
21- Toda T., Hyakumachi M., and Arora D. 1999. Genetic relatedness among and within different Rhizoctonia solani anastomosis groups as assessed by RAPD, ERIC and rep-PCR. Microbiological Research, 154: 247-258.
22- Van Belkum A., Scherer S., Van Alphen L., and Verbrugh H. 1998. Short sequence DNA repeats in prokaryotic genomes. Microbiology and Molecular Biology Review, 62: 275-293.
23- Versalovic J., Schneider M., De Bruijn F.J., and Lupski L.R. 1994. Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods of Molecular and Cell Biology, 5: 25–40.
24- Yousefi A., .Panjehkeh N., .HagianShahri M., .Salari M., and FalahatiRastegar M. 2010. Evaluation of Shot Hole Disease Incidence and Severity on Stone Fruit Trees in Razavi Khorasan Province .Journal of Plant Protection, 24 :218-221. (in Persian)