| Trifolium pretense L. cv. is one of the medicinally important pastures widely grow in Gansu province. It was famous for higher isoflavone and protein content. As a good resources for extract isoflavone. Trifolium pretense L. cv. was widely grow in Minxian. The cultivated area had increased to 6666 hm2 in 2015. Chemical fertilizers and pesticides were used as a traditional way to increase the yield of Trifolium pretense L. cv. But lead to a series of questions such as: soil degradation, soil fertility and grass yield decline, soil microflora diversity of natural pasture damage. The application of bio-control manure in other crops providing a new way to solve this question. The bio-control manure produced by growth-promoting bacteria and bio-control bacteria can not only effectively promote the growth and quality of Trifolium pretense L. cv., but also replace partial chemical fertilizers and pesticides, reduce production cost, prevention soil degradation, protect the soil ecological environment. So Far, it has not been reported in literature. The aim of our work was to isolated strain resources, which had the ability of nitrogen fixation, dissolved phosphorus, secrete IAA, and antagonize pathogenic fungus. The biological characteristics, species identification, the mechanism of dissolved phosphorus were investigated in this work. A field plot experiment was conducted to investigate the effects of application the bio-control manure reducing partial chemical fertilizers on growth and quality of Trifolium pretense L. cv. The results showed that:1. Great differences of bacterial properties can be found in growth-promoting bacteria isolated from rhizosphere of Trifolium pretense L. cv.Twelve rhizobia strains were screened from rhizosphere of Trifolium pretense L. cv. which had a low growth speed on the YMA medium. The trogenase activity of rhizobia strains distributed in the range of 0.42~4.88 μmo L C2H4/m L?h. Trogenase activity of five rhizobia strains was higher than 2.00 μmo L C2H4/m L?h. The trogenase activity of MHSG8 was the highest, and higher than other rhizobia strains.Five potential plant growth promoting rhizobacteria were screened from twenty strains based on their properties of 3-indoleacetic acid(IAA) secreting, which had a well growth on King medium. IAA secreting ability distributed in the range of 9.35~12.01 μg/m L. The IAA secreting ability of MHS19 was the highest, and higher than other strains.Twenty-six phosphorus solubilizing bacteria were screened. The PKOC2 medium, which excluding the yeast extract and FeSO4?7H2O was more suitable for screening rhizosphere phosphorus solubilizing bacteria than PKO and PKOC1 medium. Glucose, MgCl2?6H2O, and(NH4)2SO4 were key factors for phosphate solubilization capacity of P-dissolving bacteria. The suitable addition amount were 18.19 g, 3.21 g, and 0.08 g, respectively. Phosphate solubilization capacity of six strains distributed in the range of 50.34~269.50 mg/L. The Phosphate solubilization capacity of MHS30 was the highest, and significantly higher than other strains(P<0.05).2. The growth-promoting bacteria isolated from rhizosphere of Trifolium pretense L. cv. had well potential for producing bio-bacterial manure.Based on the properties of nitrogen fixation, phosphate dissolving, 3-indoleacetic acid(IAA) secreting, antagonism of pathogens. Eight potential plant growth promoting rhizobacteria and one rhizobia strains were screened. The results showed that trogenase activity of rhizobia strains(MHSG8) was 4.88 μmo L C2H4/mL?h. Phosphate solubilization capacity of MHS7, MHS27, MHS30, and MHS49 distributed in the range of 153.00~269.50 mg/L. Secreting IAA ability of MHS19, MHS13 distributed in the range of 11.65~12.01 μg/m L. MHS7, MHS27, MHS31, and MHS48 had a strong ability for antagonizing pathogen(Fusarium oxysporium f. sp. Cucumerinum, Rhizoctonia solani, Fusarium oxysporum f. niveum). These strains mentioned above with well property of growth-promoting and biocontrol, which can be used to produce bio-bacterial manure.3. Several growth-promoting bacteria from rhizosphere of Trifolium pretense L. cv. were identified.Seven growth-promoting bacteria from rhizosphere of Trifolium pretense L. cv. were identified by 16 S rDNA. They were Bacillus subtilis(three strains) 、 Pseudomonas fluorescens(one strain)、Klebsiella oxytoca(one strain)、Bacillus cereus(one strain)、Bacillus pumilus(one strain)。4. The pathway of bacteria dissolved phosphorus are diverse.The citric acid and succinic acid, which secreted by MHS49 during the culture period, played a important role in additon the acidity of culture environment. This was the dissolved phosphorus mechnism of MHS49. The citric acid, tartaric acid, and formic acid which secreted by MHS30 during the culture period, played a important role in additon the acidity of culture environment. This was the dissolved phosphorus mechnism of MHS30. The dissolved phosphorus mechnism of MHS7 and MHS27 was different with MHS30 and MHS49.5. The bio-control strain resources isolated from rhizosphere of Trifolium pretense L. cv. han an effectively antagonism for pathogenic bacteria.Four bio-control strain resources were isolated from rhizosphere of Trifolium pretense L. cv. They were MHS7, MHS27, MHS31, and MHS48. MHS7, MHS27, MHS31 belonged to Bacillus subtilis, Bacillus thuringiensis, Pseudomona fluorescens. Fusarium oxysporium f. sp. Cucumerinum was effectively inhibited by MHS7 and MHS48. Inhibition rates were 73.8%, 41.8%, respectively. Rhizoctonia solani was effectively inhibited by MHS27 and MHS31. Inhibition rates were 64.3%, 71.2%, respectively. Fusarium oxysporum f. niveum was effectively inhibited by MHS27 and MHS31. Inhibition rates were 81.7%, 64.5%, respectively. These strains can produce siderophore, secrete siderophores amount of order was: MHS27> MHS31> MHS7> MHS48.6. The bio-control manure produced by growth-promoting bacteria and bio-control bacteria can effectively promote the growth and quality of Trifolium pretense L. cv.Screening of growth-promoting bacteria and bio-control bacteria can be successful colonization at rhizosphere of Trifolium pretense L. cv. The quality of the bio-control manure complied with the standard of microbiological fertilizer of China(NY227-94). By applying the bio-control manure to combine with 75% of chemical fertilizers, the total hay yield of clover was increased to 4.76% compared with control. The content of crude protein, crude ash, calcium, phosphorus, was also improved, and the content of neutral detergent fiber(NDF) and acid detergent fiber(ADF) was declined. Furthermore, the inoculant treatment(inoculant + 50% fertilizer) may not increase the total hay yield, the quality, and isoflavone content of clover, but reducing the chemical fertilizer application for 50%. The way of bio-control manure combined with chemical fertilizer providing an efficacious approach to obtain a higher hay yield for Trifolium pretense L. cv. Minshan. For reducing a large consumption of non-renewable energy, protecting the agricultural ecological environment, improving the quality of agricultural products, food security and credibility. This mode of production has a positive effect to guarantee the sustainable development of agriculture and animal husbandry. |
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