The overuse of chemical pesticides has caused serious environmental problems and thus the demand for safer pesticides is increasing. One alternative is microbial pesticides that suppress plant pathogens via their microbial activities. As microbial pesticides that are friendlier to the environment, we succeeded in using Bacillus subtilis and Alcaligenes facalis, which showed a broad suppressive spectrum against various kinds of plant pathogens not only in vivo but also in a plant test.
The B. subtilis we isolated showed suppressive activity against more than 30 kinds of plant pathogens, either bacterial or fungal. Investigation of the mechanism based on such a broad suppressive spectrum is under way by genetic and biochemical analyses. Figure 1 shows the mechanism we have clarified so far.
|
| Fig. 1. Multifunctions by Bacillus subtilis to suppress plant pathogens. |
This bacterium produces two antifungal lipopeptides, iturin A, and plipastatin, and a biosurfactant, surfactin. Surfactin is a very powerful biosurfactant. Iturin and plipastatin exhibit powerful antifungal activities. These three substances consist of amino acids and fatty acids as side chains and thus are easily biodegradable in soil in sharp contrast with persistent chemical pesticides. Genes encoding surfactin were cloned by a genome project, and our group cloned genes encoding iturin A and plipastatin.
B. subtilis produces an iron-chelating agent, 2,3-DHBG (2,3-dihydroxybenzoylglycine), which deprives iron from plant pathogens. The gene encoding 2,3- DHBG was found to be controlled in collabortaion with the genes encoding iturin and surfactin. This bacterium also produces proteases with high activity that attack the cell wall of plant pathogens. From these analytical data, we speculate that the mixed production of these substances and the cooperative function against plant pathogens are the main reasons why B. subtilis has a broad suppressive spectrum against various plant pathogens.
In order to utilize the bacterium and its metabolites as biological agents, an efficient production system is essential. We demonstrated that the solid-state fermentation (SSF) of B. subtilis using soybean curd residue as substrate is an efficient method of producing cells and their metabolities and the productivity of lipopeptides in SSF was 30 times higher than that in liquid cultivation. The products obtained by SSF can be directly applied to soil as an organic fertilizer that also functions as a biological pesticide.
The utilization of waste from food industries for the growth of B. subtilis will be helpful for the recycling of the waste. In liquid cultivation, we have established the optimum culture conditions where 10-20 g/liter of lipopeptides is produced.
Plant tests of tomato as shown in Fig. 2 prove the strong suppressive effect of B. subtilis on damping-off caused by Rhizoctonia solani.
 |  |
| Fig. 2. The result of plant test using B. subtilis. Damping off of cucumber (left) is significantly suppressed by addition of B. subtilis (right). | |
Genetic engineering is applied to produce new strains of B. subtilis with enhanced activity and multiple functions against plant diseases by introducing a gene encoding Bt-protein (insecticidal protein) or the chitinase gene, which is effective for attaking the cell wall of fungal pathogens. We have already bred B. subtilis that shows resistance to chemical pesticides, and this has enabled co-use of this bacterium with chemical pesticides. Co-use of the bacterium and the chemical pesticides reduces the amount of chemical pesticides used to 1/10 - 1/100 of that when only chemical pesticides are used.
A. faecalis suppresses plant pathogens by producing hydroxylamine by heterotrophic nitrification. Damping-off of tomato was effectively suppressed in a plant test.