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American Journal of Applied Sciences
Year: 2009  |  Volume: 6  |  Issue: 1  |  Page No.: 194 - 199

Biological Control of Rice Blast (Magnaporthe oryzae) by use of Streptomyces sindeneusis isolate 263 in Greenhouse

M. Ebrahimi Zarandi, G.H. Shahidi Bonjar, F. Padasht Dehkaei, S.A. Ayatollahi Moosavi, P. Rashid Farokhi and S. Aghighi    

Abstract: Soil Actinomycetes particularly Streptomyces spp. have antagonistic activity against wide range of plant pathogens. In the recent decades they have attracted high interests as biocontrol agents. In search for finding such principles, in vitro suppression of Magnaporthe oryzae the causal agent of rice blast disease was studied by use of Streptomyces sindeneusis isolate 263 in greenhouse. Spray of rice seedling-leaves with of mixed spore suspension of the pathogen and S. sindeneusis isolate 263 resulted in strong inhibition of the pathogen and suppression of leaf symptoms. Propagation of the antagonist crude sap was performed in aqueous cultures and bioactivity was monitored in shaked cultures. Ongoing goals of this research include isolation, characterization and identification of the active metabolites and future goals include identification of active genes for use in development of recombinant DNAs in transgenic rice varieties bearing elevated resistance to infections by M. oryzae.

Figure 1 shows two antagonists and the suppressive reaction of M. oryzae. Figure 2 shows bioassay results of S. sindeneusis isolate 263 against M. oryzae measured in agar disk-method. In both methods, S. sindeneusis isolate 263 show antifungal inhibitory effects on the pathogen.


Fig. 1: Suppressive reaction of two Streptomyces isolates on Magnaporthe oryzae. Left: Streptomyces isolate 339 and right: Streptomyces isolate 328. Center disk is Magnaporthe oryzae agar disk which its growth towards the antagonists is inhibited clearly

Fig. 2: Bioassay results of Streptomyces isolates against Magnaporthe oryzae. Clockwise from top: Streptomyces sindeneusis isolate 263, blank agar disk (control), the Streptomyces isolate 202. Center disk is Magnaporthe oryzae agar disk

Table 1: Morphological and physiological characterization of Streptomyces sindeneusis isolate 263
F: Flexuous; Sm: Smooth; 1: Positive; 0: Negative;-: No utilization; +: Utilization

Fig. 3: In vitro bioassay results of Streptomyces sindeneusis isolate 263 against Magnaporthe oryzae in rotary cultures indicative of production time versus inhibition zones

Monitoring activity and growth curve: Activity reached maximum after 5 days in rotary cultures. In shaked cultures, this interval was used to harvest cultures to prepare crude extract for use in further investigations. Activity versus post seeding time in rotary cultures is presented in Fig. 3.

Taxonomy of streptomyces isolate 263: Isolate 263 was identified as Streptomyces sindeneusis isolate 263 which is a new record from Iran. Table 1 shows the complete identification of this isolate based on morphological and biochemical characterization. Figure 4 shows scanning electron micrograph of spore chains of S. sindeneusis isolate 263.

Determination of MIC: In well diffusion-method, MIC of the crude was determined as 3.125 mg mL-1 against M. oryzae.

Solubility active crude in organic solvents: Solubility results are indicated in Table 2. As the results show, apparently there is more than one active principle involved since activity is traceable both in polar and nonpolar solvents of H2O, methanol and chloroform.

Shelf life or stability of active crude: Stability of the active crude in distilled water at room temperature (12-30°C) was about six months, assayed by using Agar diffusion-method against M. oryzae.


Table 2: Bioassay results of solubility tests of the antifungal principle(s) of Streptomyces sindeneusis isolate 263 against Magnaporthe oryzae in fractions of different solvents indicated by well diffusion-method at 20 mg mL-1 of dry crude
*S: Supernatant, P: Pellet

Fig. 4: Scanning electron micrograph of spore chains of Streptomyces sindeneusis isolate 263

Determination of TIP: Bioactivity of active isolate diminished to zero at 170°C.

Fungicidal and/or fungistatic activity: Transfer of blocks from inhibition zones to fresh PDA plates revealed afterward growth of the pathogen which was indicative of fungistatic activity of S. sindeneusis.

In vivo greenhouse studies: The results of biological control of Streptomyces sindeneusis isolate 263 against M. oryzae the causal agent of rice blast are indicated in Fig. 5. Treatment of plants with pathogen only resulted typical blast symptoms and percent of diseased leaf area evaluated 8% and in treatment of plants with pathogen plus Streptomyces evaluated 0.5% according to the method developed by IRRI. That is indicative of significant reduction in the number of lesions in pots which received the antagonist.


Fig. 5: In vivo greenhouse results in rice plants indicative blast disease (A) in plants inoculated with the pathogen alone and (B) plants inoculated with the Streptomyces sindeneusis isolate 263 alone, (C) plants inoculated with both pathogen and the antagonist, S. sindeneusis isolate 263 and (D) untreated control plants

Fig. 6: Comparative result of Biological activity of Streptomyces sindeneusis isolate 263 against Magnaporthe oryzae on rice seedlings. (A, B): indicate leaf symptoms resulted from infections by M. oryzae and (C-F): show the effect of mixed spore sprays of leaves with both S. sindeneusis isolate 263 and the pathogen which indicates strong inhibition in blast development

Treatment of rice plants with antagonist in B and C groups had no significant effect on dry weight of rice plants. Figure 6 indicates comparative result of biological activity of S. sindeneusis isolate 263 against M. oryzae on rice seedlings. A and B indicate leaf symptoms resulted from infections by M. oryzae and C-F show the effect of mixed spore sprays of leaves with both S. sindeneusis isolate 263 and the pathogen which indicates high degree of inhibition in blast development.

DISCUSSION

In sustainable agriculture natural biofungicides are safe and pro environment. Since most of synthetic fungicides do harm the ecosystem to some extent, their usage should be banned and switched to safer strategies as biological control techniques. Our findings represent the presence of potential antifungal metabolite (s) in S. sindeneusis isolate 263 against M. oryzae. Antifungal activity of the isolate found in this study highlights its importance as a candidate for further investigation in biological control of the world-wide destructive rice blast disease. The genes encoding many antifungal characteristics are currently being used by agribusiness to create genetically modified plants that have increased fungal resistance in the field. We believe that the results of these findings can form the avenue for production of resistant transgenic-plants with recombinant DNA having antifungal genes cloned from biologically active Streptomyces isolates which would lead to environmentally safer measures in plant-pest management.

ACKNOWLEDGMENTS

This research is dedicated to Mr. A. Afzalipour and Mrs. Fakhereh Saba the founders of Universities in Kerman. This paper is part of M. Ebrahimi Zarandi dissertation fulfilled for degree of Master of Science in Dept. of Plant Pathology, College of Agriculture, Kerman University of Shahid Bahonar, Kerman, Iran.

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