ShARGA B. Мicroorganisms of some species from Bacillus genus and their antagonism to plant pathogenic bacteria and fungi.

Українська версія

Thesis for the degree of Candidate of Sciences (CSc)

State registration number

0402U002157

Thesis Registration Form

0402U002157.pdf

Applicant for

Specialization

  • 03.00.07 - Мікробіологія

19-06-2002

Specialized Academic Board

Д 26.233.01

D.K. Zabolotny Institute of Microbiology and Virology of the NASU

Essay

The thesis describes results of selection of antagonists among isolates of Bacillus genus and study of their properties, particularly, antibiosis against phytopathogenic Botrytis cinerea, Botrytis fabae , Erwinia spp. and some other plant pathogenic bacteria and fungi. The protective effect of these antagonists was evaluated on in vitro grown plants and on plant material from the greenhouse and field. Biological control with application of bacteria antagonistic to Botrytis and Erwinia may provide a useful alternative to chemicals. Of 270 Bacillus isolates tested, 54 (20%) were found to cause necrosis and/or chlorosis when placed on leaves of Vicia faba L. Only 14 strains (6.5%) were able to prevent chocolate spot symptoms developing in vivo and all of these antagonised B. cinerea and B. fabae in vitro through antibiosis. B. macerans BS 153 and B. subtilis BS 2924 were selected for biocontrol experiments in both the greenhouse and field in which plants were sprayed with bacterial suspensions. Detached leaves were inoculated with Botrytis at intervals after bacilli application. The beneficial effect of the biocontrol agent persisted much longer in glasshouse grown plants. ED 50 estimated for B. macerans BS 153 and B. subtilis BS 2924 at the same time were 1.3 and 1.27 times less concentrated in the greenhouse than in the field spray. 20 Among selected antagonists only B. subtilis BS 934 and its antimicrobial metabolite were able to suppress fire blight symptoms on detached flowers. The B. subtilis BS 934 was used in both the greenhouse and the field experiments in which flowers of 'Conference' pear were sprayed with bacterial suspension. The median effective dose, ED 50 of B. subtilis BS 934 suspension was about 10 times less in the greenhouse than in the field spray. From the high number of bacteria required and the rate of decline of introduced Bacillus populations in field grown plants it is concluded that improved formulations and spray application techniques are needed for better survival and protectiveeffect of the antagonists. Antimicrobic substances were produced by B. subtilis cultures in a defined medium and isolated from culture filtrate by precipitation at pH 2.5. Active fractions were extracted in ethyl-acetate, acetone and 80 % ethanol. The extracts were developed in thin-layer chromatography with ethanol : water (2 :1, v/v). Three bands were indicated under UV light (l = 350 nm) for each of extracts with Rf 0.67, 0.69 and 0.75 for B. subtilis BS 107; 0.69, 0.72 and 0.8 for B. subtilis BS 934; 0.61, 0.67 and 0.76 for B. subtilis BS 2924; 0.65, 0.69 and 0.79 for B. subtilis BS 2931. Only plaques with Rf 0.75, 0. 76, 0.79 and 0.8 were nynhidrine negative and in each case they formed an inhibitory zone, when thin layer chromatography plates contacted with agar seeded by Erwinia test cultures. The antimicrobial substances release into fermentation medium was observed from early hours of B. subtilis BS 107, BS 934, BS 2924 and BS 2931 cultivation. At that time pelleted cells contained higher amountsof antimicrobial substances than concentrated to the volume of pellet supernatant from these cultures. The higher antimicrobial activity of concentrated filtrates was observed in old cultures. The antimicrobial substances were resistant to the action of nucleases, proteases and lipase. The culture filtrate concentrats remained biologically active, when autoclaved 2 times for 35 minutes at 2 atmosphere gauge pressure in acidic, neutral and alcaline solutions. They exhibited no loss of antimicrobial activity at pH 1-14 within a month of observation and when stored at 4°C for over 1 year. B. subtilis BS 107, BS 934 or BS 2931 showed in vitro and in vivo antagonism to the potato black leg and soft rot agents. The application of antagonist or its antimicrobial substance to the cut potato tissues prevented or reduced symptoms of the diseases. In experiments B. subtilis BS 107 and B. subtilis BS 934 suppressed significantly potato rots in storage. The antimicrobial substances were active in vitro against a broad spectrum of cells. The antimicrobial products of selected antagonists have properties, which differ them from the antimicrobial metabolites described earlier for other bacilli. The substances showed the repellent properties on Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica and Erwinia chryzanthemi cells in 21 chemotaxis and suppressesed the penetration and movement of the pathogens within plant vessels. Strain B. subtilis BS 2924 produced the antimicrobial gaseous metabolite with broad spectrum of action. The addition of D(+)glucose to nutrient agar led to most higher metabolite production. The chemical tests on hydrogen cyanide, indole and ammonia were negative. Gas-liquide chromatography, IR-spectrometry and mass-spectrometry headspace analysis of B. subtilis BS 2924 culture revealed the high CO2 content and presence of toxic compounds with C=O, COOH, CH3, C2H5 and ОН-groups. The methane, ethane and ethylene were not produced by the culture.

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