内生枯草芽孢杆菌B-001菌株内生定殖研究及生物学特性

用浸种、浸根、淋根、伤茎、伤叶和喷雾等接种方法测试枯草芽孢杆菌B-001菌株入侵烟草植株的途径,发现该菌可通过自然孔口和伤口入侵寄主植物;取接种植株的茎部组织切片在电镜下观察,发现该菌株在烟草的微管束和细胞内定殖。生长特性研究结果表明该菌株最适生长pH为7.5,最适生长温度为30℃,48～96h内处于稳定生长期。     

N~+注入诱变芽孢杆菌选育高产抗菌物质菌株

本研究通过N+注入芽孢杆菌选育高产抗菌物质菌株。注入N+的能量为25keV,剂量为50×2.6×1013、80×2.6×1013、100×2.6×1013、120×2.6×1013和150×2.6×1013N+/m2。结果表明最佳的注入剂量为50×2.6×1013N+/m2,选育出了1株高产菌株(Bacillus subtilisfmbJ224),其抗菌物质产量是出发菌株的1.96倍,经传代培养,其高产性能稳定。同时通过对该菌株的发酵特性研究,发现其产抗菌物质的模式为延滞合成型。     

球形芽胞杆菌S-腺苷甲硫氨酸合成酶基因的克隆和表达

根据S-腺苷甲硫氨酸合成酶(SAMS)基因核苷酸序列的保守性,利用苏云金芽胞杆菌(Bacillus thuringiensis)基因组中SAMS序列信息设计引物。通过PCR扩增出球形芽胞杆菌(Bacillus sphaericus)SAMS基因,插入大肠杆菌(Escherichia coli)表达载体pGEX-6P-1,获得重组质粒pGEX-SAM。序列分析表明,来自球形芽胞杆菌的SAMS与大肠杆菌(K02129)、枯草芽胞杆菌(U52812)、酿酒酵母(U17246)、小鼠(J05571)和人(BC018359)的SAMS基因,分别有63.9%、75.4%、58.8%、59.1%和55.7%的同源性。由于一级结构存在明显的差异,将该重组的质粒转入大肠杆菌DH5α并成功地表达了SAMS。通过亲和层析纯化出球形芽胞杆菌的SAMS,SDS-PAGE分析显示蛋白分子量为43kD。HPLC对SAMS的活性分析表明,该酶可催化ATP和蛋氨酸形成S-腺苷甲硫氨酸(SAM)。     

尼古丁降解菌L1的分离鉴定与降解特性分析

通过对分离于烟草叶片的154株具有降解尼古丁细菌菌株筛选，获得能高效降解尼古丁的L1菌株。通过形态观察，16S rDNA序列分析和生理生化测定将其鉴定为Bacillus simplex。菌落生长密度测定和高压液相色谱分析表明， L1菌株最适生长尼古丁浓度为1g/L，随着菌落密度增加，尼古丁降解效率逐渐升高，36 h最高降解率达到75.0%。而低尼古丁含量不利于L1菌株的生长，过高尼古丁含量对L1菌株的生长和降解效率具有反馈抑制作用。菌株L1降解尼古丁过程中无色素产生，说明其尼古丁代谢途径有别于节杆菌。     

Effects of biocontrol bacteria and earthworms on Aphanomyces euteiches root-rot and growth of peas (Pisum sativum) studied in a pot experiment

ABSTRACT

The role of below-ground interactions between microbial biocontrol agents and soil fauna for combatting soil-borne plant diseases have not been studied sufficiently. This study tested the hypothesis that the beneficial bacterium Bacillus velezensis UCMB5113 and the anecic earthworm Lumbricus terrestris positively influence health and growth of peas (Pisum sativum L.) infested with the pathogen Aphanomyces euteiches causing root-rot disease. A greenhouse fully factorial experiment studied the effects of A. euteches, B. velezensis and L. terrestris on the emergence, growth and health of pea plants. The factors B. velezensis and L. terrestris resulted in taller plants (p = .003 and p = .030). B. velezensis treatment resulted in a higher biomass of shoots and roots (p ≤ .001 and p = .005). The effects increased with the presence of both factors (p = .036). Earthworms reduced the disease symptoms significantly (p = .032). The decreased disease symptoms caused by the earthworms might be due to the consumption of A. euteiches (direct effect) as well as soil disturbance (indirect effect). Interactions between the microorganisms added and the earthworms were shown. B. velezensis and L. terrestris can be useful for enhancement of plant growth and for biological control of root-rot in peas.     

Yield response of wheat and barley to inoculation of plant growth promoting rhizobacteria at various levels of nitrogen fertilization

The yield response of a wheat (Kirik) and a barley (Tokak 157/37) cultivar to inoculation with Azospirillum brasilense Sp246 and Bacillus sp. OSU‐142 was studied in relation to three levels of N fertilization (0, 40, and 80 kg ha^–1) under field conditions in Erzurum, Turkey, in 1999 and 2000. Seed inoculation with A. brasilense Sp246 significantly affected yield and yield components, both in wheat and barley. On average of years and N doses, inoculation with A. brasilense Sp246 increased spike number per m², grain number per spike, grain yield, and crude protein content by 7.2, 5.9, 14.7, and 4.1 % in wheat and by 6.6, 8.1, 17.5, and 5.1 % in barley, respectively, as compared to control. Inoculation with Bacillus sp. OSU‐142 significantly increased kernel number per spike in wheat, but no significant effect was determined in the other characteristics. Grain yields and yield components were also higher at all levels of nitrogen fertilizer in the inoculated plots as compared to the control. However, these increases diminished at high fertilizer levels. These results suggest that application of the growth promoting bacteria A. brasilense Sp246 may have the potential to be used as a biofertilizer for spring wheat and barley cultivation in organic and low‐N input agriculture.     

毒死蜱降解菌株Bacillus latersprorus DSP的降解特性及其功能定位

从土壤中分离到一株能有效降解毒死蜱的细菌DSP，该分离株鉴定为侧芽孢杆菌（Bacillus latersprorus）。在纯培养条件下测定了分离株DSP对毒死蜱的降解性能。在接种量为菌浓度OD415=0．2，pH7．0、25℃条件下，测得1、10mg L^-1毒死蜱的降解符合一级动力学特征，其降解半衰期分别为1．48d、5．00d，100mg L^-1毒死蜱对DSP菌有明显的抑制作用；分离株DSP在不同pH及温度下对毒死蜱的降解作用为pH7．0〉pH5．0〉pH9．0，35℃〉25℃〉15℃。该菌株含有一个20kb左右的质粒，通过吖啶橙与升温法对质粒消除实验证实，随着质粒的丢失，菌株利用毒死蜱的能力也丧失，用热击法和CaCl2法将菌株质粒转人大肠杆菌JM109和质粒消除处理的DSP^-菌中，随着质粒的获得，这些转化子获得了降解毒死蜱的能力。研究结果表明，Bacillus latersprorus DSP降解毒死蜱的功能和质粒有关。     

生防细菌NCD-2中抑菌功能相关基因的定位及克隆

枯草芽孢杆菌NCD-2菌株是一株有效防治棉花黄萎病的细菌,它通过产生抑菌物质达到对大丽轮枝菌的抑制作用。以该菌株作为有效成分的微生物农药已通过国家农药登记。通过原生质体转化法将含有转座子mini-Tn10的质粒pHV1249转入枯草芽孢杆菌NCD-2中,获得转化子。对转化子通过高温诱导转座子转座,获得4 000个NCD-2菌株的突变子。对这些突变子进行对大丽轮枝菌的抑菌作用测定,筛选到2株抑菌作用增强的突变子和4株抑菌作用降低的突变子。采用染色体步移技术对此6个突变子中转座子插入位点基因的侧翼序列进行克隆和测序,结合枯草芽孢杆菌168菌株的全基因组序列对测序结果进行序列同源性和基因定位分析,结果表明:抑菌作用增强的2个突变子中,转座子插入到一个功能未知的基因内部,此基因与枯草芽孢杆菌168菌株中的yvoA基因的同源性为98%;抑菌作用降低的4个突变子中,转座子插入位点对应于枯草芽孢杆菌168菌株中的phoP基因内部,插入位点的侧翼序列与枯草芽孢杆菌168菌株中的phoP基因的同源性达到98%。     

ELISA在苏云金杆菌杀虫蛋白毒素检测中的应用及新进展

苏云金杆菌是目前应用较广泛的生物杀虫细菌之一，其产生的杀虫蛋白毒素是主要的杀虫成分，目前对苏云金杆菌的毒力和杀虫蛋白毒素的检测主要采用生物测定、SDS-PAGE等方法，但这些方法的灵敏度较差。本文综述了酶联免疫吸附分析法在苏云金杆菌蛋白毒素检测中的应用，以及在检测中采用的ELISA的类型和基本原理，并对其应用前景进行了展望。     

芽孢桿菌對百合抗灰霉病之抗性誘導

Lily leaf blight,caused by Botrytis elliptica,is an important fungal disease in Taiwan.In order to decrease the disease incidence in Lilium formosanum,the efficacy of application of rhizobacteria for induced systemic resistance(ISR)was examined in this study.Over 300 rhizobacteria were isolated from the rhizosphere of L.formosanum healthy plants and 63 were identified by the analysis of fatty acid profiles.The ability of disease suppression of 13 strains was demonstrated by soil drench application of bacterial suspensions to the rhizosphere of L.formosanum seedlings.Biocontrol experiments were carried out with Bacillus cereus and Pseudomonas putida strains on Lilium Oriental hybrid cultivars Acapulco and Star Gazer besides L.formosanum in the greenhouse and field conditions.The assay with a B.cereus strain good for disease suppression showed that the protection against B.elliptica on L.formosanum could last for at least ten days,consistent with the good association of B.cereus with lily roots.Analysis of the expression of LfGRP1 and LsGRP1,encoding glycine-rich protein of L.formosanum and cv.Star Gazer,respectively,reveals a difference between that in response to the treatment of B.cereus and that singly induced by B.elliptica,suggesting that plant defense responses driven by B.cereus follows a signaling pathway different from that triggered by B.elliptica.According to the results of biocontrol assay and LfGRP1/LsGRP1 gene expression analysis with the culture filtrates of B.cereus strains,we presume that more than one eliciting factor of ISR are generated by B.cereus and some of them exhibit thermostable and autoclave-tolerant traits.This is the first report about ISR-eliciting rhizobacteria and determinants effective for foliar disease suppression on lily.