解淀粉芽孢杆菌HAB-6抑菌物质及其相关基因的分析

芽孢杆菌是重要的生防菌株,分析了解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HAB-6菌株的抑菌物质及其抑菌活性。采用特异性PCR和液质联用(LC-MS)等方法对HAB-6菌株的抑菌物质进行初步研究。通过对编码合成脂肽类抗生素的10个基因进行PCR扩增,分别得到与yndj、srf AB、itu D、fen D、itu C同源的基因5个;扩增编码合成脂肽类物质关键合成酶基因,得到与fen B、lpa-14、sfp、myc B、itu A同源的基因5个;LC-MS结果显示,HAB-6菌株能产生C14Iturin A、C17Iturin A、C13Surfactin A、C15Surfactin C等脂肽类化合物,但通过与病原真菌对峙抑菌活性检测,脂肽类物质没有抑制真菌活性。结果表明,HAB-6菌株具有脂肽类物质的合成基因和相关调控基因,代谢产生伊枯草菌素(iturin)、表面活性素(surfactin),但是酸沉淀提取得到的脂肽类物质不是HAB-6菌株抑制真菌的主要物质,其抑菌成分种类和代谢脂肽类物质的途径可能与已知的途径有所不同,有待进一步研究。

解淀粉芽胞杆菌启动基因的克隆及其对地衣杆菌α-淀粉酶基因的调控

用大肠杆菌启动基因探针质粒pHE5克隆了解淀粉芽胞杆菌的启动基因。供体菌DNA的HindⅢ片段与pHE5重组后转化大肠杆菌,获得一批带启动基因的抗四环素转化子,其中四株的抗性达到200μg/mL,从这四株高抗性转化子中提取质粒,并对其中的一个重组质粒pAE23的插入片段进行限制性图谱分析和缺失研究,获得了一个缺失了部份片段,四环素抗性仍达到200μg/mL的衍生质粒pAED23,经酶切分析证明其上的启动基因位于0.8kb的EcoR Ⅰ—HindⅢ片段上。点杂交分析证实该片段来自解淀粉芽胞杆菌的DNA。将地衣杆菌的α-淀粉酶基因亚克隆至pAED23启动基因下游的HindⅢ位点上能增强该基因在大肠杆菌中的表达。

生防菌株B9601-Y2对作物生长和产量的影响

为获知解淀粉芽孢杆菌植生性亚种(Bacillus amyloliquefaciens subsp. plantarium)菌株B9601-Y2在田间的促生长和增产应用效果,探讨其在农业生产中作为生物肥料的潜力,通过田间试验对解淀粉芽孢杆菌菌株B9601-Y2促进作物生长的效果进行验证。田间试验结果表明,接种菌株B9601-Y2能明显促进作物的生长发育,作物各项生长指标如出苗率、株高和单果质量等均有提高,更重要的是其能显著提高作物的经济学产量。黄瓜、番茄和玉米接种菌株B9601-Y2后,产量比对照分别提高9.5%～12.6%、18.9%～24.8%和7.3%～11.5%。

Screening of Degrading Bacteria for Phthalic Acid Esters Under Cadmium Stress and Its Application

In order to enrich the degrading microbial resources for phthalic acid esters(PAEs)under cadmium stress,a degrading bacterium B-7 for di(2-ethylhexyl)phthalate(DEHP)was screened from the strains stored in the laboratory by means of inorganic salt liquid medium containing DEHP and cadmium,and the characteristics of the strain were studied.Strain B-7 was Bacillus amyloliquefaciens,which had high biosafety and excellent degradability to DEHP.The optimum temperature for degradation was 25–40℃,and the optimum pH value was 6–8.Strain B-7 was cultured in inorganic salt medium(MSM)with an initial DEHP concentration of 400 mg/L and cadmium content of 10 mmol/L for 4 d,and its degradation rate of DEHP was up to 93.1%.In addition,the strain had a strong degradation ability to dimethyl phthalate(DMP),diethyl phthalate(DEP),and dibutyl phthalate(DBP).In soil contaminated by cadmium and DEHP,the synergic degradation of B-7 and indigenous microorganisms in soil significantly increased the degradation rate of DEHP,indicating that this strain had potential application value in the field of microbial remediation of soil contaminated by cadmium and PAEs.

Effects of Bacillus amyloliquefaciens Biofertilizer on Brassica juncea var. multiceps Growth and N_2O Emission from Soil

In this study, Bacil us amyloliquefaciens A3 was continual y incubated in shake fIasks contalning wastewater from sweet potato starch production as an ef-fective biofertiIizer for cuItivation of Brassica juncea var. multiceps（XueIihong）. Based on pot experiments in the greenhouse, the effects of chemical fertiIizers （CN）, biofertiIizer （BF）, inactivated broth （BI）, starch wastewater （SW） and the combination of biofertiIizer and chemical fertiIizer （BC） on the yield, NO3- content and NO2- con-tent of XueIihong, soiI physicochemical properties and N2O emission were investi-gated. The resuIts showed that the yield of XueIihong in BC and CN treatments was improved by five times compared with CK; BF and SW treatments had insignifi-cant impact on the yield of XueIihong. Compared with CN treatment, BCL treatment exhibited simiIar improving effects on the yield of XueIihong, in which NO3- content of XueIihong and soiI was reduced by 16.4%-73.6% and 22%-29%, which reduced the risk of nitrogen eIuviations in soiI; average N2O fIux （FPV30） in BCL treatment was reduced by 58.3%-73.1% compared with CN treatment. In concIusion, B. amy-loliquefaciens is a feasibIe Iow-cost biofertiIizer for sustalnabIe vegetabIe farming with a great potential for starch wastewater utiIization.

Effects of Bacillus amyloliquefaciens Biofertilizer on Tea Yield and Quality

[Objective] This study was aimed to explore the effects of Bacil us amy-loliquefaciens biofertilizer on tea yield and quality. [Method] The field plot experiment was conducted with the biofertilizer treatments and control to investigate 100-bud weight and main biochemical components. [Result] The treatments by Bacil us amy-loliquefaciens biofertilizer, which was fermented using sweet potato starch wastewa-ter （SPSW） as culture medium, improved 100-bud weight and tea quality significant-ly under the concentration of 0.8×108, 1.6×108 and 3.2×108 cfu/ml with the dose of 1 L/m2 for 4 times. At the optimum concentration of 1.6 ×108 cfu/ml, the biofertilizer treatment increased the 100-bud weight by 22.3%, water extracting materials by 21.9%, amino acids content by 8.83%, tea polyphenol content by 9.76%, and de-creased theine content by 8.32%, respectively. Compared with the control, there was no significant difference between the SPSW treatment and the control. [Con-clusion] The production of the B. amyloliquefaciens biofertilizer could consume SP-SW, and the application of the biofertilizer could improved the tea yield and quality, which provided references for the development of ecological agriculture.

Bacillus amyloliquefaciens Strain W19 can Promote Growth and Yield and Suppress Fusarium Wilt in Banana Under Greenhouse and Field Conditions

Plant growth-promoting rhizobacteria （PGPR） are considered to be the most promising agents for cash crop production via increasing crop yields and decreasing disease occurrence. The Bacillus amyloliquefaciens strain W19 can produce secondary metabolites （iturin and bacillomycin D） effectively against Fusarium oxysporum f. sp. cubense （FOC）. In this study, the ability of a bio-organic fertilizer （BIO） containing strain W19 to promote plant growth and suppress the Fusarium wilt of banana was evaluated in both pot and field experiments. The results showed that application of BIO significantly promoted the growth and fruit yield of banana while suppressing the banana Fusariurn wilt disease. To further determine the beneficial mechanisms of the strain, the colonization of green fluorescent protein-tagged strain W19 on banana roots was observed using confocal laser scanning microscopy and scanning electron microscopy. The effect of banana root exudates on the formation of biofilm of strain W19 indicated that the banana root exudates may enhance colonization. In addition, the strain W19 was able to produce indole-3-acetic acid （IAA）, a plant growth-promoting hormone. The results of these experiments revealed that the application of strain W19-enriched BIO improved the banana root colonization of strain W19 and growth of banana and suppressed the Fusarium wilt. The PGPR strain W19 can be a useful biocontrol agent for the production of banana under field conditions.

Suppression of Fusarium Wilt of Banana with Application of Bio-Organic Fertilizers

Fusarium wilt is one of the most serious diseases of banana plants caused by soil-borne pathogen Fusarium oxysporum f.sp. cubense(FOC). In this study a pot experiment was conducted to evaluate the effects of different bio-organic fertilizers(BIOs) on Fusarium wilt of banana, including the investigations of disease incidence, chitinase and β-1,3-glucanase activities of banana plants, and FOC populations as well as soil rhizosphere microbial community. Five fertilization treatments were considered, including chemical fertilizer containing the same N, P and K concentrations as the BIO(control), and matured compost mixed with antagonists Paenibacillus polymyxa SQR-21 and Trichoderma harzianum T37(BIO1), Bacillus amyloliquefaciens N6(BIO2), Bacillus subtilis N11(BIO3), and the combination of N6 and N11(BIO4). The results indicated that the application of BIOs significantly decreased the incidence rate of Fusarium wilt by up to 80% compared with the control. BIOs also significantly promoted plant growth, and increased chitinase andβ-1,3-glucanase activities by 55%–65% and 17.3%–120.1%, respectively, in the banana roots. The population of FOC in the rhizosphere soil was decreased significantly to about 104 colony forming units g-1with treatment of BIOs. Serial dilution plating and denaturing gradient gel electrophoresis analysis revealed that the application of BIOs increased the densities of bacteria and actinomycetes but decreased the number of fungi in the rhizosphere soil. In general, the application of BIOs revealed a great potential for the control of Fusarium wilt disease of banana plants.

Menaquinone-7 production from maize meal hydrolysate by Bacillus isolates with diphenylamine and analogue resistance

A menaquinone-7（MK-7） high-producing strain needs to be isolated to increase MK-7 production, in order to meet a requirement of MK-7 given the low MK-7 content in food products. This article focuses on developing MK-7 high-producing strains via screening and mutagenesis by an atmospheric and room temperature plasma（ARTP） mutation breeding system. We isolated an MK-7-producing strain Y-2 and identified it as Bacillus amyloliquefaciens, which produced（7.1±0.5） mg/L of MK-7 with maize meal hydrolysate as carbon source. Then, an MK-7 highproducing strain B. amyloliquefaciens H.β.D.R.-5 with resistance to 1-hydroxy-2-naphthoic acid, β-2-thienylalanine, and diphenylamine was obtained from the mutation of the strain Y-2 using an ARTP mutation breeding system. Using strain H.β.D.R.-5, efficient production of MK-7 was achieved（（30.2±2.7） mg/L）. In addition, the effects of nitrogen sources, prenyl alcohols, and MgSO_4 on MK-7 production were investigated, suggesting that soymeal extract combined with yeast extract, isopentenol, and MgSO4 was beneficial. Under the optimized condition, the MK-7 production and biomass-specific yield reached（61.3±5.2） mg/L and 2.59 mg/L per OD600 unit respectively in a 7-L fermenter. These results demonstrated that strain H.β.D.R.-5 has the capacity to produce MK-7 from maize meal hydrolysate, which could reduce the substrate cost.