Study Isolates of Fusarium Stem and Root Rot Disease of Greenhouse Cucumber Using Pathogenicity Tests, Vegetative Compatibility Groups and Molecular Marker

Document Type : Research Article

Authors

1 South Kerman Agricultural and Natural Resources Research and Education Center, AREEO, Jiroft

2 Sciences and Research Branch, Islamic Azad University, Tehran

3 Islamic Azad University

Abstract

Introduction: Crops Production under greenhouse condition has been increased during the last decade in Iran. Two formae speciales of Fusarium oxysporum including F. oxysporum f.sp. radicis-cucumerinum (Forc), causing stem and root rot and F. oxysporum f.sp. cucumerinum (Foc) causing wilt in cucumber are the most important diseases of cucumber worldwide. Root and stem rot disease of cucumber caused by Forc is a very important disease of cucumber, recorded for the first time in Greece over 1989. Root and stem rot have been recorded on cucumber in Canada in 1994, in France in 1998, in China in 1999, and in Spain in 2000, causing significant yield losses. Using resistant varieties is the best and durable control method of this disease. Having knowledge on the variability and genetic diversity among the population of pathogen is necessary for screening cucumber cultivars to find resistant varieties. The objective of this research was to characterize the isolates of Forc causing stem and root rot of greenhouse cucumber in Jiroft region using pathogenicity test, vegetative compatibility groups and molecular marker assay.
Material and Methods: To study the genetic diversity among isolates of Fusarium on cucumber, many greenhouses were investigated for wilt, stem and root rot disease in Jiroft and Kahnuj, Kerman, Iran. Fusarium isolates were recovered from symptomatic cucumber plants during 2009-2011 growing season. The pathogenicity test, vegetative compatibility groups (VCGs) and RAPD marker were used to study the genetic diversity among isolates. Isolated fungi was inoculated on seedlings of commercial susceptible variety of cucumber (Negin) at four leaves stage and two different temperatures under greenhouse condition for separating two different mentioned Fusarium formae speciales. The Forc and Foc are, respectively, pathogenic at 17-21 oC and 25-30 oC. One to two weeks after inoculation, the results of pathogenicity tests were reported. Different plant species such as tomato, pepper, watermelon, melon, and cantaloupe were used to confirmed forma specialis of cucumber. To determine vegetative compatibility groups (VCGs), Fusarium Nit mutants were produced and complementation tests were carried out using Puhalla (1985) and Correll et al (1987) methods. RAPD primers series SBS was employed to study genetic diversity at molecular level.
Results and Discussion: Overall, 45 Fusarium isolates were recovered from infected cucumber. 42 isolates were identified as F. oxysporum and three isolates as Fusarium sp. Based on pathogenicity test, cultural and morphological characters, symptoms expression, experimental host range on some other plant species, 36 isolates were identified as F. oxysporum f. sp. radicis-cucumerinum and six isolates as F. oxysporum f. sp. cucumerinum. These cultural and morphological characteristics were similar to those of F. oxysporum. f.sp. radicis-cucumerinum described by Vakalounakis (1996) and later in other works (Punja& Parker, 2000; Cercauskas et al., 2001; Vatchev, 2007). Most of isolates showed high degree of disease severity index. Fusarium stem and root rot disease of greenhouse cucumber in Jiroft occuring mainly at bearing and harvesting time. A stripe shape stem lesion started from soil level and progress toward aerial parts. A whitish to pink/orange color due to mycelia mat and spore formation of Fusarium oxysporum on surface of stem were also observed. The causal fungal pathogen was easily isolated from infected roots, crown and stem. In total, 288 nit mutants were recovered from 36 isolates. Among these, 53.4% belonged to nit1, 25.2% to nit3, and 21.4% to NitM. Three VCGs groups were identified and arbitrary designated as VCG-A, VCG-B and VCG-C. PCR reactions were conducted using RAPD primers. High polymorphism among fungal isolates was found. The size of amplified bands ranged from 250 to 2500 bp. Cluster analysis of RAPD data using UPGMA method and Dice’s coefficient distinguished two main groups at 84% similarity level.
Conclusions: Our results showed that the F. oxysporum f. sp. radicis-cucumerinum is more prevalent forma specialis in greenhouse cucumber at Jiroft region. Due to difference among isolates of Forc in terms of aggressiveness and genetic diversity, it is proposed to use multigene resistant cultivars to achieve better control management. This work is the first attempt to assess genetic diversity among Fusarium isolates causing cucumber root and stem rot based on pathogenicity test, VCG and RAPD molecular markers in Kerman province, Iran.

Keywords


1- Ahn I. P., Chung H. S., and Lee Y. H. 1998. Vegetative compatibility groups and pathogenicity among isolates of Fusarium oxysporun f. sp. cucumerinum. Plant Disease, 82: 244-246.
2- Alves Santos F. M., Martinez B. D., Rodriguez M. M. C., and Diez J. J. 2007. Cultural characteristics, pathogenicity and genetic diversity of Fusarium oxysporum isolates from tobacco fields in Spain. Physiological and molecular Plant Pathology, 71: 26-32.
3- Armstrong G. M., and Armstrong J. K. 1978. Formae specialis and races of Fusarium oxysporum causing wilt of Cucurbitaceae. Phytopathology, 68: 19- 28.
4- Bouhot D. 1981. Some aspects of the pathogenic potential in formae specialis and races of Fusarium oxysporum on cucurbitaceae. Pages 318-326 in: Fusarium: Disease, Biology, and Taxonomy. P. E. Nelson T. A. Toussoun and R. J. Cook eds. Pennsylvania State University Press, University Park.
5- Cerkauskas R. F., Brown J., and Ferguson G. 2001. First report of Fusarium stem and root rot of greenhouse cucumber caused by Fusarium oxysporum f. sp. radicis-cucumerinum in Ontario. Plant Disease, 85(9): 1028-1028
6- Correll J. C., Klittich C. J. R., and Leslie J. F. 1987. Nitrate nonutilizing mutants of Fusamm oxysporum and their use in vegetative compatibility tests. Phytopathology, 77: 1640- 1646.
7- Gerlagh M., and Blok W. J. 1988. Fusarium oxysporum f. sp. cucurbitacearum n. f. embracing all formae specialis of F. oxysporum attacking cucurbitaceous crops. Netherlan Journal of Plant Pathology, 94: 17-31.
8- Karaca G., and Kahveci E. 2010. First report of Fusarium oxysporum f. sp. radicis-cucumerinum on cucumbers in Turkey. Plant Pathology, 59(6): 1173-1174
9- Mehmet T. F., and Kurt S. 2010. Pathogenicity, vegetative compatibility and amplified fragment length polymorphism (AFLP) analysis of Fusarium oxysporum f. sp. radicis-cucumerinum isolates from Turkish greenhouses. Phytoparasitica, 83(3): 253-260.
10- Moreno A., Alferez A., Aviles M., Dianez F., Blanco R., Santos M., and Tello J. C. 2001. First report of Fusarium oxysporum f. sp. radicis-cucumerinum on cucumber in Spain. Plant Disease, 85(11): 1206-1206.
11- Najafiniya M., and Shahabi I. 2013. Genetic diversity among Iranian isolates of Fusarium oxysporum f. sp. radicis-cucumerinum using vegetative compatibility groups and RAPD molecular marker. Asian Mycological Congress 2013 and the 13th International Marine and Freshwater Mycology symposium. China National Convention Center (CNCC), Beijing, China, August 19-23, 2013, p56.
12- Najafinia M., and Sharma P. 2009. Cross pathogenicity among isolates of Fusarium oxysporum causing wilt in cucumber and muskmelon. Indian phytopathology, 62(1): 9-13.
13- Najafinia M., and Sharma P. 2011. Characterization of Indian isolates of Fusarium oxysporum f. sp. cucumerinum using vegetative compatibility groups (VCGs) and RAPD assay. Indian phytopathology, 64(1): 12-18.
14- Nelson P. E., Toussoum T. A., and Marasas W. F. O. 1983. Fusarium species: A Illustrated manual for identification. The Pennsylvania State University Press, 226 pp.
15- Puhalla J. E. 1985. Classification of strains of Fusarium oxysporum on the basis of Vegetative Compatibility. Canadian Journal of Botany, 63: 179-183.
16- Punja Z. K., and Parker M. 2000. Development of Fusarium root and stem rot, a new disease on greenhouse cucumber in British Columbia, caused by Fusarium oxysporum f.sp. radicis-cucumerinum. Canadian Journal of Plant Pathology, 22: 349–63.
17- Shahriari D., and Zare R. 2006. Fusarium stem and root rot of greenhouse cucumber. Proceedings of the 17th Iranian Plant Protection Congress, Karaj, Iran (In Persian).
18- Shahriari D., Molavi E., Aminian H., and Etebarian H. R. 2011. Histopathological response of resistant and susceptible cultivars of cucumber to Fusarium oxysporum f.sp. radicis-cucumerinum, the causal agent of Fusarium stem and root rot. Seed and Plan Production Journal 27(1): 375-391. (In Persian with English summary)
19- Vakalounakis D. J. 1996. Root and stem rot of cucumber caused by Fusarium oxysporum f. sp. radicis-cueumerinum f. sp. nov. Plant Disease, 80: 313-316.
20- Vakalvunakis D. J., and Fragkiadakis G. A. 1999. Genetic diversity of Fusarium oxysporum isolates from cucumber differentiation by pathogenicity, vegetative compatibility and RAPD fingerprinting. Phytopathology, 89: 161-168.
21- Vakalounakis D. J., Doulis A. G., and Klironomou E. 2005. Characterization of Fusarium oxysporum f. sp. radicis-cucumerinum attacking melon under natural conditions in Greece. Plant Pathology, 54: 339–346.
22- Vakalounakis DJ., Wang Z., Fragkiadakis GA., Skaracis GN., and Li D-B. 2004. Characterization of Fusarium oxysporum isolates obtained from cumber in China by pathogenicity, VCGs and RAPD. Plant Disease, 88: 645–9.
23- Vatchev T. D. 2007. First report of Fusarium root and stem rot of greenhouse cucumber caused by Fusarium oxysporum f.sp. radicis-cucumerinum in Bulgaria. Bulgarian Journal of Agricultural Science, 13: 151-152.
24- Vatchev T. D. 2015. Fusarium root and stem rot of greenhouse cucumber: aerial distribution of inoculum. Bulgarian Journal of Agricultural Science, 21: 650–654.
25- Woltz SS., Jones JP., and Scott JW. 1992. Sodium chloride, nitrogen source, and lime influence Fusarium crown rot severity in tomato. HortScience, 27: 1087-1088.