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American Journal of Agricultural and Biological Sciences
Year: 2009  |  Volume: 4  |  Issue: 2  |  Page No.: 146 - 151

Biological Control of Sclerotinia sclerotiorum, Causal Agent of Sunflower Head and Stem Rot Disease, by Use of Soil borne Actinomycetes Isolates

F. Baniasadi, G.H. Shahidi Bonjar, A. Baghizadeh, A. Karimi Nik, M. Jorjandi, S. Aghighi and P. Rashid Farokhi    

Abstract: Problem statement: High level of biosafety and non adverse effects on the environment of biocontrol strategies of pest management, are priorities of tomorrow's world agriculture. Actinomycetes are active biocontrol agents due to their antagonistic properties against wide range of plant pathogens particularly fungi. Fungal pathogens are liable for a big part of damages in agriculture economy.
Approach:
In the present research antifungal bioactivity of 50 isolates of Actinomycetes collected from soils of Kerman province of Iran was investigated against Sclerotinia sclerotiorum (Lib.), the causal agent of stem rot in sunflower, through agar disc method and dual culture bioassays. The Streptomyces isolate No. 363 was propagated in submerged cultures and active crude was prepared upon which several biological characterizations performed. Greenhouse studies were achieved to confirm laboratory results.
Results:
Among the tested Streptomyces isolates, 10 isolates revealed antagonistic properties in dual culture procedure from which isolate No. 363 showed highest bioactivity. The active metabolite of Streptomyces isolate No. 363 was polar and well soluble in H2O. Using agar-disc method, progressive growth of the pathogen was highly reduced by the antagonist through exhibiting ability to constitute fungus-free zones of inhibitions. The results indicated that isolate No. 363 was a proper candidate for field biocontrol studies.
Conclusion:
Results may open a horizon for production of resistant transgenic plants having antifungal properties originated from biologically active Streptomyces spp. recognition and production of effective metabolite(s) of Streptomyces spp. which was responsible for antifungal activities will be our commercial goal due to rich reserves of soil borne Actinomycetes in Iran.

Fig. 1 and 2). Based on the screening results, in comparision to others, Streptomyces isolate No. 363 had maximum inhibition zone against S. sclerotiorum and selected for further evaluations.

Monitoring activity in shaked culture: The results of activity versus time in submerged culture are indicated in Fig. 3. In rotary submerged culture, activity of Streptomyces isolate No. 363 against S. sclerotiorum reached the maximum at 7th post inoculation day. Activity versus post seeding time in submerged media cultures is shown in Fig. 3. Since the activity reaches its maximum after 7th day of post seeding, this time was used to harvest cultures for preparation of crude extract for future investigations.

Fig. 1: Bioassay results of Streptomyces isolates against Sclerotinia. sclerotiorum. Clockwise from top: Isolate 139, isolate 347, 246 and blank agar disk (control). Center disk is S. sclerotiorum agar inoculum disk. Clear inhibition zones around the Streptomyces agar disks are representatives of lack of growth and bioactivity against the pathogen

Determination of MIC: In well diffusion-method, MIC of the crude of Streptomyces isolate No. 363 against S. sclerotiorum was determined as 1.25 mg ml-1.

Solubility of active crude in organic solvents: Solubility results are shown in Table 1. As the results show, the active principle (s) has a polar nature and well soluble in distilled water.

Shelf life or stability of active crude: Stability of the active crude in DM solvent was 75 days, assayed by using Agar diffusion-method against S. sclerotiorum.

Table 1: Bioassay results of solubility tests of the antifungal principle (s) of Streptomyces isolate No. 363 against Sclerotinia sclerotiorum in fractions of different solvents evaluated by well diffusion-method at 10 mg ml-1 of dry crude
*S: Supernatant, *P: Pellet, +: Soluble, -: Insoluble

Fig. 2: Bioassay result in Agar Disk-Method of Streptomyces isolate No. 363 against Sclerotinia sclerotiorum. Center disk is S. sclerotiorum agar inoculum disk

Fig. 3: In vitro bioassay results of Streptomyces isolate 363 against Sclerotinia sclerotiorum in rotary cultures indicative of production time versus inhibition zones

Fig. 4: In vivo greenhouse results in sunflower seedlings (P): In plants inoculated with the pathogen alone and (A+P): Plants inoculated with both pathogen and the antagonist Streptomyces isolate 363, (A): Plants inoculated with Streptomyces isolate 363 alone and (C): Untreated control plants

Fig. 5: Digital scanning electron micrograph of spore chains of Streptomyces isolate 363

Determination of TIP: Bioactivity of active isolate diminished to zero at 90°C.

In vivo greenhouse studies: Symptoms as stem rot and wilt appeared 4-6 days after inoculation in seedlings inoculated with S. sclerotiorum, while other treatments did not develop signs of the disease. The results of this experiment which indicates promising biological control of Streptomyces isolate No. 363 against S. sclerotiorum the causal agent of head and stem rot in sunflower in greenhouse are shown in Fig. 4.

Scanning electron microscope studies: Scanning electron micrograph of spore chains of Streptomyces isolate 363 is shown in Fig. 5.

DISCUSSION

Overdose usage of chemicals as pesticides and fertilizers is common in farming in most parts of the world which threatens food safety of individuals. Some pesticides can be hardly cleaned from nature and have a potential capability to have adverse effect or destroy useful microorganisms which have positive effects in fertility of soil and growth of plants. To lower or avoid side effects, biological control is an alternative and proper choice in pest management. In ideal biological control measures, proper microorganisms are those having well adaptation in soil and rizosphere exerting effective antagonistic activity against soil pathogens persistently. In the in vitro study to fight sunflower stem rot, a natural agent (Actinomycete) was used that had effective antagonistic characteristics against S. sclerotiorum. In green house study, similar to in vitro tests, Streptomyces isolate 363 suppressed fungal diseases in inoculated plant without any contamination in the soil and plant. It also increased growth of plants. A commercial product containing Streptomyces isolate 363 or its effective metabolite is suggested to avoid head and stem rot in sunflower. Identification the effective metabolite (s) and search genes responsible for its function can be the topic for future and perfecting researches which also consider Actinomycetes (like Streptomyces isolate 363) as both biological control agents and biological fertilizers. Several workers have reported that In vitro studies have documented satisfactory results in use of Streptomyces against some pathogens[8]. Some other workers have reported biological control of S. sclerotiorum by Streptomyces spp. Tahtamouni reported In vitro antifungal activity of some of Streptomyces isolates against S. sclerotiorum which confirmed earlier findings by El-Tarabily et al.[3] who had reported such activity against S. minor in vitro, as well as those of Gupta et al. against several phytopathogenic fungi and Saadoun et al.[11,12] against several food-associated fungi and molds. Findings confirm importance of the Streptomyces isolates as biocontrol agents and also emphasize the importance of indigenous Streptomyces spp. as biocontrol agents against wide range of fungal phytopathogens[1,17] with the future perspective of replacement with chemical control measures. Genetic engineering provides an opportunity to protect plants from fungal diseases and to reduce the use of synthetic fungicides. The genes for antifungal metabolites can be engineered into plants to increase the resistance of crop plants to fungal attack, decreasing the use of environmentally unfriendly fungicides. The major factor limiting the application of this technology is the identification and isolation of useful genes that code for antifungal metabolites[1]. Performing genetic modifications of plants to resist phytopathogens is of high importance that related specialists do pay attention to. Translation of genes from an antagonist of the pathogen to plant, without making new damages or problems for plant and environment, may be a choice by which we can control diseases of plants from the early stages of growing to the end of plant life. After recognition of biocontrol agents, projects should be designed to find their effective principle(s) and gene(s) encoding related traits and at the end, translate gene(s) to the target plant. Thus it may be assumed that the antifungal-metabolite gene from Streptomyces isolate 363 may be a useful candidate for genetic engineering of agriculturally important crop plants such as sunflower for increased tolerance against S. sclerotiorum.

CONCLUSION

Results may open a perspective for production of resistant transgenic plants having antifungal properties originated from biologically active Streptomyces spp.

ACKNOWLEDGEMENT

Thanks to Horticultural Research Institute of Shahid Bahonar University of Kerman for financial support. This research is part of MSc thesis of the first author performed at the Department of Agricultural Biotechnology, in collaboration with Horticultural Research Institute and Department of Plant Pathology, College of Agriculture, Shahid Bahonar University of Kerman, Iran. This research is dedicated to Mr. Afzalipour and Mrs. Fakhereh Saba, the founders of Universities in Kerman.

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