The biological control of plant pathogens is of paramount importance nowadays, since the conventional use of chemical pesticides has been seriously questioned because of environmental and human health hazard.

In 1992, a Master's student from the University Austral of Chile (Valdivia), isolated a bacterial strain from the surface of healthy raspberry fruits that showed a high inhibition against the fungus Botrytis cinerea, an important post harvest plant pathogen of grapes, berries and other small fruits (Silva, 1992). This bacterial strain, designated as A47, also presented similar activity toward Erwinia carotovora and Ralstonia solanacearum, both potato pathogens. Using both microbiological and biochemical tests, the bacterium was classified as Bacillus subtilis, and determined to be the producer of an antibiotic responsible for the inhibition, potentially useful for biological control. This molecule was later identified as an antibiotic of the iturin group, attending to its chemical and antibiotic properties. Iturin compounds constitute a family of lipopeptides excreted from several strains of B. subtilis when grown on liquid media.

There is vast information about the selection of antagonistic microorganisms from nature, their effects, and the characterization of the metabolites involved. However, few data are available about the genetic alteration of the wild strains to improve their antagonistic effect. We believe that this is a powerful tool to produce new strains and products for biological control of plant pathogens, because the genetic modification using mutagenic agents is a simple technique and do not requires the insertion of a foreign DNA in the antagonistic microorganism.

The objectives of this research were:

1) To select by random mutagenesis of the wild type A47 Bacillus subtilis a strain with increased activity against the plant pathogens Botrytis cinerea, Ralstonia solanacerarum and Erwinia carotovora var. Carotovora.

2) To optimize culture conditions for the production of the antibiotic metabolite.

3) To isolate the metabolite with antibiotic activity

Results

Twenty colonies isolated from strain A47 were mutated with acridine orange and many mutants were obtained. From these, five mucous colonies were selected with an increased activity with respect to the wild strain. Such strains were labeled as M40, M20, M16, M12 and M4. Results from inhibition tests against B. cinerea are presented in Figures 1a and Figure b, where the stronger antagonistic effect of all mutant strains with respect to wild strain is apparent. Strain M40 was selected for further studies because it produced bigger inhibition zones on B. cinerea (Figure 1b), Erwinia caratovora and Ralstonia solanacearum.

The selected mutant, strain M40 was mucous-type and formed big, butyrous and round colonies on agar. Under the microscope, cells were short bacilli forming irregular aggregates. As the wild strain, the mutant was Gram positive.

It was later determined that mannitol (a sugar) was a better carbon source than glucose for antibiotic production and that the addition of calcium ion to the culture media, increased the antibiotic production in the mutant strain.

The antibiotic was resistant to high temperatures (thermostability), which is characteristic of iturin antibiotics.

Finally, the antibiotic was isolated from the culture media using extraction with methanol and chromatography HPLC (High performance liquid chromatography).

Discussion

Previous studies demonstrated that a strain of Bacillus sp, labeled as A47, produced a thermostable metabolite active in vitro against the phytopathogens B. cinerea, E. carotovora and R. solanacearum. The metabolite was thermostable, protease resistant, soluble in methanol and classified as an antibiotic of the iturin group attending to its chemical and antibiotic properties (Silva, 1992).

In this work, wild strain A47 was subjected to mutation with acridine orange with the purpose of improving its antagonistic capacity towards phytopathogens. After mutation of wild strain A47 with acridine orange, five mutant strains were isolated with increased activity against the target phytopathogens. From those, strain M40 was selected for further studies. This strain formed big, mucous colonies on agar, quite different from star-shaped colonies in the wild strain. Morphological differences were also appreciated under the microscope, where strain M40 were regular short bacilli as opposed to much larger bacilli in the wild strain.

We found that the synthesis of the antibiotic was increased when the mutant strain M40 was cultured on medium containing mannitol instead of glucose as carbon source. This is in agreement with Besson et al. 1987, who reported mannitol, fructose and sucrose as better carbon sources than glucose for the synthesis of iturin A in B. subtilis.

Calcium ion at a level of 0.5% had a significant effect on antibiotic production by the mutant strain M40. In 1991, Besson and Michel, 1991 proved that Ca²⁺ and Mn²⁺ promoted iturin A and bacillomycin L precipitation after excretion into the medium. In our case, the increase in antibiotic concentration in the medium can be a consequence of the increase in cell wall permeability in the mutant strain M40 promoted by Ca²⁺.

The target site for iturin A on yeast cells is in the plasma membrane. The antibiotic promotes cellular lysis and rapidly increases permeability to K⁺, which has been associated with its fungicidal activity (Besson et al. 1984). The ability of iturin antibiotics to increase membrane permeability of target microorganisms is due to the formation of ion channels on the cell membranes.

The different thermal treatments conducted with supernatants from cultures of the mutant strain M40, demonstrate the thermoresistance of the inhibitory metabolite, which is in agreement with the reported thermoresistance of iturin antibiotics produced by Bacillus.

The antibiotic produced by the mutant strain M40 was simply and efficiently purified by reverse phase HPLC.

Further research is needed to determine the chemical structure of the antibiotic produced by the mutant strain M40 within the family of iturins. More knowledge is required on the mechanisms of biocontrol of phytopathogens to develop rational strategies for the application of the antagonists and their metabolites within the agroecosystem. Once elucidated such strategies, genetic engineering can provide an efficient way of gathering desirable characteristics from different organisms in one ecologically adapted to a particular system. There is still a long way to go before a sound system is developed to protect plants from their predators without altering the ecological balance among species. The use of bioantagonists is certainly a very promising route.

BESSON, F. and MICHEL, G. Influence of divalent ions on the solubility of iturin and bacillomycin L, antifungal peptidolipids of Bacillus subtilis. Microbios, 1991, vol. 65, no. 262, p. 15-21.

BESSON, F.; CHEVANET, C. and MICHEL, G. Influence of the culture medium on the production of iturin A by Bacillus subtilis. Journal of General Microbiology, March 1987, vol. 133, no. 3, p. 767-772.

BESSON, F.; PEYPOUX, F.; QUENTIN, M.J. and MICHEL, G. Action of antifungal peptidolipids from Bacillus subtilis on the cell membrane of Sacharomyces cerevisiae. Journal of Antibiotics (Tokyo), February 1984, vol. 37, no. 2, p. 172-177.

SILVA, S. Control biológico de Botrytis cinerea Pers. Ex. Fr. en frambueso (Rubus Idaeus L.) mediante bacterias antagonistas. Tesis de Magister en Ciencias, Facultad de Ciencias Agrarias, Universidad Austral de Chile, 1992, 178 p.

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