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American Journal of Agricultural and Biological Sciences
Year: 2009  |  Volume: 4  |  Issue: 1  |  Page No.: 72 - 78

Biocontrol of Botrytis allii Munn the Causal Agent of Neck Rot, the Post Harvest Disease in Onion, by Use of a New Iranian Isolate of Streptomyces

M. Jorjandi, G.H. Shahidi Bonjar, A. Baghizadeh, G.R. Sharifi Sirchi, H. Massumi, F. Baniasadi, S. Aghighi and P. Rashid Farokhi    

Abstract: Problem statement: Soil Actinomycetes particularly Streptomyces spp. have showed antagonistic activity against wide range of plant pathogens. In the recent decades they have attracted high interests as biocontrol agents. Onion neck rot or gray mold caused by Botrytis allii have imposed economic post harvest damages to onion bulbs and decreased its storage durability and market value.
Approach:
To investigate for biocontrol means against the pathogen, antagonistic activity of 50 isolates of soil actinomycetes were assayed through agar disk method and dual culture bioassays. Active isolates were exposed to chloroform for detection of antibiotic. Minimum Inhibitory Concentration (MIC) value and solubility of active crude extract in organic solvents were determined for Streptomyces isolate No. 347 which showed a unique and stable property of inhibiting Botrytis allii. To investigate the antagonistic effect of Streptomyces isolate No 347 on control of onion gray mold, four different treatments were tested by means of Tukey HSD test.
Results:
From the tested isolates, 13 showed anti gray mold activities. Exposure of active isolates to chloroform revealed that Streptomyces isolates No 347, 263 and 350 retained their antifungal activities. The active metabolite(s) of Streptomyces isolate No 347 was polar, soluble in H2O but insoluble in chloroform and methanol. MIC of the crude was determined as 0.05 mg mL-1 against B. allii. Stability of the active crude in distilled water at room temperature (12-30°C) was about 6 months. Statistical studies indicated that Streptomyces isolates No 347 can decrease losses of neck rot with significant level (p<0.05).
Conclusion:
The future goals include investigation of the antifungal genes in active isolates as candidates for genetic engineering of onion for increased tolerance against B. allii.

Fig. 1.

Chloroform assay: Among 13 effective Streptomyces isolates, 3 isolates of 347, 263 and 350, retained their antifungal activities after exposure to chloroform.

Scanning electron microscope studies: Scanning electron micrograph of mycelia of Streptomyces isolate No. 347 is showed in Fig. 2. Streptomyces isolate No 263 has been previously identified as Streptomyces sindeneusis[10].

Fig. 1: Test result of screening using dual culture bioassay. Center, agar plug of Botrytis allii with radial growth; left, plain agar plug as control; right, Streptomyces isolate No. 347; top, Streptomyces isolate No. 263 and bottom, Streptomyces isolate No. 350 showing inhibitory effect against mycelial growth and sporulation of B. allii

Fig. 2: Scanning electron micrograph of mycelia of Streptomyces isolate No. 347

Antifungal activity of submerged cultures: Activity versus post seeding time in submerged media cultures of Streptomyces isolate No 347 is showed in Fig. 3.

Since the activity reached its maximum after 4 days of post seeding, this time was used to harvest cultures for preparation of crude extract for further studies. Test result of bioactivity of aqueous culture medium of Streptomyces isolate No 347 in well diffusion method on lawn culture of Botrytis allii at the peak day is showed in Fig. 4. Crude extract was prepared by separating spores and mycelia via passing the culture media through two layers of cheese cloth and Whatman® filter paper and drying the filtrate to dryness using cold blower overnight. Prepared dried crudes were kept refrigerated before use.

Fig. 3: Activity versus post seeding time in submerged cultures of Streptomyces isolate No. 347 monitored by well diffusion-method against Botrytis allii

Fig. 4: Test result of bioactivity of aqueous culture medium of Streptomyces isolate No. 347 in well diffusion method on lawn culture of Botrytis allii; top, uninoculated culture medium as control and bottom, Streptomyces isolate No. 347 inoculated culture medium, 4th day after seeding of submerged medium showing inhibition of mycelial growth of B. allii

Solubility of active crude in organic solvents: Solubility results of crude extract of Streptomyces isolate No 347 are showed in Table 1. As the results show, apparently the active principle (s) has a polar nature since activity is recoverable only in H2O supernatants and pellets of tested organic solvent.

Determination of MIC: In well diffusion-method, MIC of the crude was determined as 0.05 mg mL-1 against B. allii.

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 B. allii.

Table 1: Bioassay results of solubility tests of the antifungal principle (s) of Streptomyces isolate No. 347 against Botrytis allii in fractions of different solvents indicated by well diffusion-method at 20 mg mL-1 of dry crude
a: Supernatant; b: Pellet; +: Soluble; -: Insoluble

Fig. 5: Antifungal activity of Streptomyces sp. isolate No. 347 in control of Botrytis allii at 24°C. Top left: control bulb, received no treatment; top right, bulb received only Streptomyces sp. isolate No. 347; bottom left: bulb received both Streptomyces sp. isolate No. 347 and B. allii and bottom right: bulb received treatment with B. allii

In vivo studies: The results of biological control of onion Botrytis gray mold in four tested treatments are shown in Fig. 5. Treatment of onion bulbs with pathogen resulted in development of typical gray mold symptoms which was progressive throughout the experiment. No visible symptoms developed in control or treatment of Streptomyces isolate No. 347 alone however, mild symptoms developed in treatment of B. allii plus Streptomyces isolate No. 347 which percentage of symptoms onsets recorded and used in statistical data analysis. Figure 6 shows antifungal antagonistic activity of Streptomyces sp. isolate No. 347 in control of Botrytis allii in neck rot of onion bulbs.

Statistical data analysis: Statistical analysis of data with multiple comparisons of Tukey test indicated which there was no significant difference between Streptomyces isolate No. 347 and control (untreated).

Fig. 6: Biocontrol of Botrytis gray mold by use of Streptomyces isolate No. 347. (a): Botrytis allii, (b): Botrytis allii plus Streptomyces isolate No. 347, (c): Control (untreated) and (d): Streptomyces isolate No. 347

Table 2: Multiple comparisons of four treatments in biocontrol of onion gray mold by Streptomyces isolate No. 347 by means of Tukey HSD test
*: The significant level was set at p<0.05.

However, there was significant difference between bulbs treated with B. allii plus Streptomyces isolate No. 347 and bulbs treated with B. allii at 0.05 level indicative of high level of antagonistic effect of Streptomyces isolate No 347 against Botrytis allii as shown in Table 2.

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[10]. The pioneering work of Waksman showed that Actinomycetes are capable of producing medically useful antibiotics[12]. The purpose of this research was reducing gray mold damages by antifungal activity of Streptomyces isolate No 347. The results may also be considered for further studies of Actinomycetes microflora in native Iranian soils with the goal to find new agents in biocontrol of storage diseases of products. Post harvest diseases caused by phytopathogenic fungi such as Botrytis allii are of major problems in storage worldwide. Genetic engineering provides an opportunity to protect plants from fungal diseases and to reduce the use of chemical fungicides. The genes for antifungal metabolites from Streptomyces isolate No 347 can be engineered into onion plants to increase the resistance of the bulbs to fungal attack, decreasing the use of environmentally unfriendly fungicides in the field. The major factor limiting the application of this technology is the identification and isolation of useful genes that code for antifungal metabolites in Streptomyces isolate No 347. Further research of the project would be focused on field and long-term storage evaluation of the antagonist against the pathogen in the cosmopolitan malady of onion neck rot. As reported by Purvis et al.[4], there are about 5 million units (50 pound bags) of low pungency, sweet onions produced in the Vidalia onion growing area at Georgia, USA, annually. Approximately half of them, 2.4 million units, are put in Controlled Atmosphere (CA) storage to extend the marketing period. Onions coming out of CA storage command a higher price, usually $20-24 per unit compared to $10-14 (or less) per unit on the fresh market. Losses during storage range from 10-20% in good years to 50-70% in poor years. The primary cause of storage losses is rots from infection by the fungus, Botrytis allii.

CONCLUSION

Annual losses caused by Botrytis allii in storage can cost only in Vidalia onion growers from $5-25 million. Genetically engineered onions bearing resistant genes against the pathogen would save the world money and produce, however, public concern on genetically engineered crops yet to receive special concern.

ACKNOWLEDGMENT

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 and is dedicated to Mr. Afzalipour and Mrs. Fakhereh Saba, the founders of Universities in Kerman.

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