عنوان مقاله [English]
Introduction: Rhizoctonia solani is one of the major pathogens causing root rot in the main bean-growing regions in Zanjan province. Under favorable conditions, yield losses in commercial bean fields due to Rhizoctonia root rot have exceeded 50 percent reduction in pod and seed numbers per plants. In 2012 most isolates of the pathogen from severely infected bean fields in Zanjan were assessed to AG-4. R. solani AG-4 can attack other commercial crops such as potato, alfalfa, barley, tomato, cabbage, etc. which are grown in rotation with bean in the area. Thus, the disease is unlikely to be controlled by crop rotation. Moreover, there is no registered resistant bean cultivar against the disease in Iran. Although soil treatment with fungicides is the only effective control method in the region, according to environmental and side effects of fungicides, alternative approaches such as biocontrol method using Trichoderma species is considered.
Materials and Methods: The study was conducted using five isolates of Trichoderma (T12-0, T12-N, T19, T6, T95) received from the Department of Plant Pathology, Ferdowsi University of Mashhad (Dr. H. Rouhani) and six isolates of Trichoderma (T36,T125, T131, T93, T89, T25) collected in 2011 from rhizosphere of bean plants in the commercial bean fields of Zanjan province (Table 1). Trichoderma isolates were evaluated for their potential to antagonize in vitro the plant pathogenic fungus R. solani using three different tests. In the first test, each isolate of Trichoderma was grown in a dual culture with R. solani AG-4 strain Rh7 on PDA and incubated at 25˚C. Radii of colony of R. solani were measured after 72 h. In the second test the ability of Trichoderma isolates to produce volatile inhibitors was measured. This experiment was conducted in two conditions involving the same time culturing of Trichoderma and Rhizoctonia and isolating 72 h early growing Trichoderma. For both tests the percentage of inhibition was measured by dividing the difference between the radial growth of control and antagonized cultures of the pathogen by the radial growth of the control and multiplied by 100. All experiments were replicated three times. The third test was conducted with growing of Trichoderma and Rhizoctonia on microscopic slides covered with PDA and the interaction of hypha was studied using a microscope. For assessment of in vivo antagonistic activity, a greenhouse experiment was designed in 11 treatments with 3 replicates. Inoculum of R. solani AG-4 was prepared by inoculation of sterilized wheat grains with the fungus and the soil was inoculated with infected wheat grains. Bean seeds (red bean, cultivar Naz) were treated with conidial suspension of Trichoderma (107 ml-1 conidia) and planted in pots. Disease severity was recorded using a scale (1 to 5) according to Nelson and coworkers (1996) 30 days after sowing. Shoot length, root length, shoot weight and root weight also were measured. For all data the analysis of variance (ANOVA) was performed using SPSS Version 18.
Table 1- Trichoderma isolates used for their antagonistic activity against Rhizoctonia solani
Results and Discussion: The results indicated that the mycoparasitism was the main mechanism accounting for antagonistic activity of Trichoderma isolates. In a competitive saprophytic ability test in a dual culture on PDA, T25, T93, T12-N and T12-0 performed more effective than the other isolates, as T25 covered the whole colony surface of R. solani, grew and sproulated on the pathogen mycelia after five days. Volatile metabolites of all Trichoderma isolates inhibited mycelial growth, whereas T12-N with % 32/73 at the same time culture and T6 with %56/08 at 72 h early culture of Trichoderma isolates had the highest effect on mycelial growth of R. solani. Microscopic studies also showed that Trichoderma isolates could inhibit hyphal growth of R. solani and parasitize the pathogen by hyphal contact, penetration, hyphal coiling around the pathogen hypha which finally resulted in hyphal fragmentation and caused death in the pathogen. These effects were typically caused by all successful Trichoderma isolates specifically T12-0 and T93. Although all Trichoderma isolates used in this study, had significant reduction in disease severity of bean root rot, caused by R. solani under greenhouse conditions (P = 0.01), but the most effective disease control occurred when the seeds treated with one of the following Trichoderma isolates: T12-0, T131، T95، T12-N and T93, among them T12-0 belonging to T. virens, was the most effective isolate and the plants treated with this isolate, showed no disease symptoms. T93 also is belonged to T. virens. Two other species of Trichoderma were also observed among the successful isolates: T. harzianum (T12-N, T95) and T. viride (T131). There are several publications on the biocontrol of R. solani by these species. Out of five successful isolates in greenhouse experiment, two isolates (T131, T93) were collected from the rhizosphere of bean in the studied area (indigenous isolates), thus could have priority for usage in future field assays. Seed treatment with T6, T36, T93, T12-N and T12-0 resulted in significantly increasing of root length, shoot weight and shoot length (P =0.01). Significant increasing of plant growth parameters caused by Trichoderma isolates has been reported by many other researchers on bean and other plants.
Conclusion: According to our results, several Trichoderma isolates not only antagonize in vitro the pathogen, but also controlled the disease under greenhouse conditions, thus they possibly use the same biocontrol mechanisms in the soil as in vitro conditions. Although field conditions are more complex and the many biotic and abiotic environmental factors will have an influence on the biocontrol potential of Trichoderma species, but it is essential to use successful indigenous and non-indigenous isolates under field conditions in the studied area to assess their biocontrol potential against the disease in fields for future commercial usages.