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

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 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.
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.
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.