Plant Growth Promotion And Control Of Plant Disease With Bacillus Spp. And Lipopeptide

The biological control of pathogens has been studied for many years, and the introduction of plant growth-promoting rhizobacteria (PGPR) into soil or the rhizosphere has been proposed for the biological control of fungal, bacterial, and viral origin. The biological control that results from PGPR is caused by several mechanisms such as competition, antibiosis, and induced resistance. In addition, the mechanisms for PGPR mediated plant growth promotion include the bacterial synthesis of plant hormones and increased uptake of available of mineral, nitrogen and phosphorus in the soil. Bacillus spp, one kind of the PGPR, can promote plant growth and control of plant disease. In this dissertation, the mechanisms of Bacillus spp. control of plant disease and promote plant growth, fermentation optimization in lipopeptide productivity of Bacillus spp. and control of plant disease with biopesticides of lipopeptide were studied. The results obtained as below:1. The results of greenhouse experiments demonstrated that the Bacillus spp. treatments significantly enhanced tobacco height and fresh weight and obviously lowered the disease severity rating of Tobacco mosaic virus (TMV) at 28 days postinoculation (dpi). TMV accumulation in young non-inoculated leaves was remarkably less for all plants treated with Bacillus spp. RT-PCR analysis of signaling regulatory genes Coil and NPR1, and defense genes PR-1a and PR-1b in tobacco treated with Bacillus spp. elucidates its association with enhancing systemic resistance of tobacco to TMV. The further analysis of two expansion genes, which regulate plant cell growth, NtEXP2 and NtEXP6, was verified to be concomitant with growth promotion in roots and leaves of tobacco responding to Bacillus spp. In addition, reactive oxygen intermediate burst and the expression of hypersensitive cell death marker genes hsr203 and hinl were Induced in tobacco treated with Bacillus spp.2. HpaGxooc, from rice pathogenic bacterium Xanthomonas oryzae pv. oryzicola, is a member of the harpin group of proteins, eliciting hypersensitive cell death in non-host plants, inducing disease and insect resistance in plants, and enhancing plant growth. Bacillus spp. is well known for their biocontrol activity against several plant pathogens and plant growth-promoting effects. In order to further improve the growth rate and antagonistic ability of the strain and obtain high-efficiency strains, HpaGXOOc encoding gene was transport into OKB105 using genetic engineering methods. Here we address whether genetically engineered Bacillus strain OKBHF that can express and secret the HpaGxooc protein had better biocontrol function and growth promotion than the parent strain Bacillus subtilis OKB105. The results of greenhouse experiments demonstrated that OKBHF treatments had significantly greater height, fresh weight, flower and fruit numbers, and obviously lower disease severity rating of Cucumber mosaic virus (CMV) than that of plants treated with OKB 105 and other Bacillus spp. at 28 days postinoculation (dpi) with CMV. CMV accumulation in young non-inoculated leaves was remarkably less for plants treated with OKBHF than that treated with OKB 105 and other Bacillus spp. at 14,21 and 28 dpi. The expression of three homologues of the expansin genes, which regulate plant cell growth, and the Pti4 and Pti6 genes encoding tomato transcription factor, which activate the expression of a wide array of PR genes, and defense gene PR-1a, which involved in defense pathway under modulation by the regulator NPR1, that were induced in leaves of tomato plants following treatment with OKBHF or OKB 105. Higher levels of gene expression were induced by the application of OKBHF than that of OKB 105. Our results suggest that combinative effects of OKB 105 and HapGxooc in disease resistance and growth promotion might have a great potential in agricultural use.3. Effects of different fermentation conditions and cultures on lipopeptide productivity of B. subtilis G1 were investigated. Results showed that the best culture condition for lipopeptide productivity is 3%initial inoculant at pH 7.0 under 30℃for 38 h, and the optimal liquid culture is Landy broth. During the course of lipopeptide extraction, lipopeptide compounds were almost entirely precipitated at pH 4.0. Under the optimized conditions, the amount of lipopeptide crude extraction could reach 6.5 g/L and the amount of purified lipopeptide compounds could reach 2.4 g/L analysised by HPLC.4. The cheapest ferment condition of B. subtilis G1 were investigated by 30L and 1000L ferment vessel based on ferment dynamic and HPLC. The active B. subtilis G1 was inoculated into LB, cultured for about 24 h under 37%as first grade seed, then inoculated into 200 ml ferment cultural base, cultured (30℃) over night as the second grade seed. The seed was transited into the 30 L ferment vessel with 3%inoculant, containing 16 L corn medium at pH7.0. The pO2 was set as 30%, the agitation was controlled by pO2 fermented for 38 hours (30℃). Results showed that the amount of lipopeptide crude extraction could reach 5.4 g/L and the amount of purified lipopeptide compounds could reach 1.35 g/L analysised by HPLC.5. Lipopeptides produced by Bacillus subtilis is natural anti-bacterial substances, including surfactin, iturin and fengycin. Surfactin not only highly active surfactant, but also a wide range of bioactive substances, demonstrating anti-viral, anti-tumor and anti-mycoplasma activity. In addition, iturin and fengycin can antagonize a broad spectrum of fungal pathogens. Biopesticides of lipopeptide was developed by our laboratory, and obtained a patent. The results of greenhouse experiments demonstrated that biopesticides of lipopeptide significantly enhanced pepper and cucumber growth and lowered the disease severity rating. In addition, the results of field trails indicated that biopesticides of lipopeptide significantly enhanced rape growth and lowered the disease severity rating of Sclerotinia sclerotinorium.