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Survival of endophytic diazotrophic bacteria in soil under different moisture levels
Oliveira, André L.M.;Canuto, Erineudo L.;Silva, Edmilson E.;Reis, Veronica M.;Baldani, José I.;
Brazilian Journal of Microbiology , 2004, DOI: 10.1590/S1517-83822004000300005
Abstract: the effects of soil moisture on the survival of three diazotrophic bacteria species (azospirillum amazonense, gluconacetobacter diazotrophicus and azospirillum brasilense) were tested. soil moisture had little influence on the survival of a. brasilense, which is considered a free-living species. on the other hand, increased soil moisture extended the survival of the endophytes a. amazonense and g. diazotrophicus. these results indicate that nitrogen-fixing endophytic species are more affected by soil moisture than associative nitrogen-fixing species.
Transformation and expression of specific insecticide gene Bt cry3A in resident endogenetic bacteria isolated fromApriona germari (Hope) larvae intestines
特异性杀虫基因Bt cry3A在桑粒肩天牛幼虫两种肠道常驻内生菌中的转化和表达

Zhongkang Wang,Wei He,Guoxiong Peng,Yuxian Xi,Qiang Li,Youping Yin,

微生物学报 , 2008,
Abstract: Objective] Transforming the specific insecticidal gene Bt cry3A into the dominant resident endogenetic bacteria in intestines of Apriona germari (Hope) larvae to construct transgenic bacteria that can colonize and express the insecticidal gene Bt cry3A perfectly in intestines of Apriona germari (Hope) larvae. Method] We isolated and identified the dominant resident endogenetic bacteria by traditional methods and molecular method based of 16S rDNA analysis. Two Escherichia coli - Bacillus thuringiensis shuttle plasmid pHT305a and pHT7911 which contained specific insecticidal gene Bt cry3A were transformed into two resident endogenetic bacteria Brevibacillus brevis Ag12 and Bacillus thuringiensis Ag13 isolated from A. germari larvae intestines respectively by electro-transformation. Results] Eighteen species of bacteria have isolated and identified from Apriona germari larvae intestines and two of them (Brevibacillus brevis Ag12 and Bacillus thuringiensis Ag13) were selected as starting bacteria to recieve the Bt cry3A. The 4 transgenic engineering strains Ag12-7911, Ag12-305a, Ag13-7911 and Ag13-305a were obtained successfully and validated by testing the plasmid stability in recombinants, transformants vegetal properties, crystal poisonous protein observation, expressional protein SDS-PAGE. The Bt cry3A gene had been transformed into Brevibacillus brevis and Bacillus thuringiensis. Both bioassay and examination of the engineering strains in intestines after feeding them to larvae showed that all these trans-formant strains (Brevibacillus brevis Ag12-305a, Bacillus thurigiensis Ag13-305a, Brevibacillus brevis Ag12-7911 and Bacillus thurigiensis Ag13-7911) could colonize and express 65 kDa protoxin in intestines of A. germari larvae and had insecticidal activity. Conclusion] We obtained four transgenic bacteria that can colonize and express the target insecti-cide gene Bt cry3A in A. germari larvae. They may be developed as a new insecticide.
Response of micropropagated sugarcane varieties to inoculation with endophytic diazotrophic bacteria
Oliveira, André Luiz Martinez de;Canuto, Erineudo de Lima;Reis, Ver?nica Massena;Baldani, José Ivo;
Brazilian Journal of Microbiology , 2003, DOI: 10.1590/S1517-83822003000500020
Abstract: previous studies estimated that sugarcane could obtain up to 60% of total nitrogen accumulated from bnf. here a mixture of five endophytic diazotrophic strains was tested in a field trial, inoculated in two micropropagated sugarcane varieties and three locals, to determine the effects on commercial crop conditions. the sugarcane plantlets were inoculated in vitro, and after 17 months of growing in the field, the productivity and bnf contribution showed to be influenced by the plant genotype and soil type. the highest bnf contributions was observed in the poorest soil for both varieties. smaller increases in productivity were observed for sp 701143 variety grown in soil with low or medium fertility. in contrast, a decrease in the stem productivity was observed in the sp 813250 variety grown in the three localities.
Characterization of Bacillus thuringiensis sigK disruption mutant and its influence on activation of cry3A promoter
苏云金芽胞杆菌sigK 基因插入失活突变体的特点及其对cry3A基因启动子活性的影响

Lixin Du,Juan Wei,Lili Han,Zhen Chen,Jie Zhang,Fuping Song,Dafang Huang,

微生物学报 , 2011,
Abstract: 摘要: 【目的】构建苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt) sigK 基因插入失活突变体,分析突变体特点并明确其对cry3A 基因启动子的影响。【方法】采用同源重组技术在苏云金芽胞杆菌HD-73 菌株sigK 基因中插入卡那霉素抗性基因,构建了sigK 基因插入失活突变体。通过生长曲线测定、扫描电子显微镜观察晶体、芽胞形成情况和芽胞计数及SDS-PAGE 等方法分析了突变体的特点; 构建了遗传恢复菌株对上述性状进行了功能验证; 利用启动子融合lacZ 技术检测了cry3A 基因启动子的转录活性。【结果】获得了苏云金芽胞杆菌HD-73 菌株sigK 基因突变体,生长曲线测定表明,突变体较出发菌株在稳定期后期生长较慢; 扫描电子显微镜观察和芽胞计数分析显示,突变体丧失了形成芽胞和晶体的能力; SDS-PAGE 分析表明突变体中伴胞晶体蛋白的表达量明显低于出发菌株和恢复菌株。利用载体pHT315 携带sigK 基因及其启动子在突变株中表达,所获得的遗传恢复菌株恢复了突变株产生芽胞和晶体的能力; sigK 基因的突变可以提高cry3A 基因启动子在产胞后期的转录活性,对cry3A 启动子指导的Cry 蛋白表达量没有显著影响。【结论】本研究证明sigK 基因为苏云金芽胞杆菌芽胞形成所必需,并影响伴胞晶体蛋白的产量; sigK 基因功能的丧失有利于cry3A 基因启动子在产胞后期的转录。
Endophytic colonization of rice (Oryza sativa L.) by the diazotrophic bacterium Burkholderia kururiensis and its ability to enhance plant growth
Mattos, Katherine A.;Pádua, Vania L.M.;Romeiro, Alexandre;Hallack, Leticia F.;Neves, Bianca C.;Ulisses, Tecia M.U.;Barros, Claudia F.;Todeschini, Adriane R.;Previato, José O.;Mendon?a-Previato, Lucia;
Anais da Academia Brasileira de Ciências , 2008, DOI: 10.1590/S0001-37652008000300009
Abstract: burkholderia kururiensis is a diazotrophic bacterium originally isolated from a polluted aquifer environment and presents a high level of similarity with the rice endophyte "b. brasilensis" species. this work assessed the ability of b. kururiensis to endophytically colonize rice plantlets by monitoring different tissues of root-inoculated plants for the presence of bacterial growth in different media, electron microscopy and by 16s rdna analysis. observations of roots, stems and leaves of inoculated rice plantlets by electron microscopy revealed b. kururiensis colonization predominantly on root hair zones, demonstrating endophytic colonization primarily through the endodermis, followed by spreading into xylem vessels, a possible pathway leading to aerial parts. although indifferent for the bacterial growth itself, addition of a nitrogen source was a limiting factor for endophytic colonization. as endophytic colonization was directly associated to an enhanced plant development, production of phytohormone auxin/indole-3-acetic acid by b. kururiensis was assayed with transgenic rice plantlets containing an auxin-responsive reporter (dr5-gus). our findings suggest the ability of auxin production by plant-associated b. kururiensis which may have a stimulatory effect on plant development, as evidenced by activation of dr5-gus. we hereby demonstrate, for the first time, the ability of b. kururiensis to endophytically colonize rice, promoting both plant growth and rice grain yield.
Structural interaction between GFP-labeled diazotrophic endophytic bacterium Herbaspirillum seropedicae RAM10 and pineapple plantlets 'Vitória'
Baldotto, Lílian Estrela Borges;Olivares, Fábio Lopes;Bressan-Smith, Ricardo;
Brazilian Journal of Microbiology , 2011, DOI: 10.1590/S1517-83822011000100015
Abstract: the events involved in the structural interaction between the diazotrophic endophytic bacterium herbaspirillum seropedicae, strain ram10, labeled with green fluorescent protein, and pineapple plantlets 'vitória' were evaluated by means of bright-field and fluorescence microscopy, combined with scanning electron microscopy for 28 days after inoculation. after 6 hours of inoculation, h. seropedicae was already adhered to the roots, colonizing mainly root hair surface and bases, followed by epidermal cell wall junctions. bacteria adherence in the initial periods occurred mainly in the form of solitary cells and small aggregates with pleomorphic cells. bacteria infection of root tissue occurred through the cavities caused by the disruption of epidermal cells during the emergence of lateral roots and the endophytic establishment by the colonization of intercellular spaces of the cortical parenchyma. moreover, within 1 day after inoculation the bacteria were colonizing the shoots. in this region, the preferred sites of epiphytic colonization were epidermal cell wall junctions, peltate scutiform trichomes and non-glandular trichomes. subsequently, the bacteria occupied the outer periclinal walls of epidermal cells and stomata. the penetration into the shoot occurred passively through stoma aperture followed by the endophytic establishment on the substomatal chambers and spread to the intercellular spaces of spongy chlorenchyma. after 21 days of inoculation, bacterial biofilm were seen at the root hair base and on epidermal cell wall surface of root and leaf, also confirming the epiphytic nature of h. seropedicae.

YueChaoyin,LiuZiduo ZengXiaohui ShaoZongze YuZiniu,

微生物学报 , 2000,
Abstract: 将对鞘翅目昆虫有特异毒性的苏云金芽孢杆菌cry3A基因电转化到只对鳞翅目昆虫有毒性的苏云金芽孢杆菌野生型菌株YBT8031中,获得转化了BMBY001。SDSPAGE分析及镜检结果表明,cry3A基因可在该菌株中高效表达,但出发菌株中原有的cry1Ab、cry1Ac及cry2的表达则受到不同程度的影响。生物测定结果显示,转化子BMBY001对柳蓝叶甲(鞘翅目)具有较高毒力,LC50为0413μL/mL(浸叶法),对小菜蛾(鳞翅目)的毒力比野生受体菌YBT8031有所降低,LC50值为3319μL/mL。
Robson Cavalcante de Lima,Dora Inés Kozusny-Andreani,Roberto Andreani Junior,Lucas da Fonseca
Revista Facultad Nacional de Agronomía, Medellín , 2011,
Abstract: A utiliza o de inoculantes contendo microrganismos diazotróficos na cana-de-a úcar pode contribuir para a promo o do crescimento destas plantas e promover ganhos significativos na produtividade. Objetivou-se no presente trabalho caracterizar bactérias diazotróficas isoladas de colmos cana-de-a úcar. Foram utilizados os meios de cultivo semi-sólidos NFb e JNFb sem adi o de nitrogênio. Foram isoladas cinco estirpes nativas de bactérias diazotróficas as quais foram avaliadas microscopicamente pela colora o de Gram e em rela o à resistência intrínseca aos antibióticos; fungicida furadan (i.a. carbofuran) e inseticida regente (i.a. fipronil), e à capacidade de fixar o nitrogênio em condi es de casa-de-vegeta o e campo. Os resultados obtidos permitiram observar que todos os isolados apresentaram características de bacilos Gram-negativos, e ampla resistência aos antibióticos. A estirpe nativa UCCBc5 apresentou resistência ao fungicida furadan e ao inseticida regente. As bactérias isoladas UCCBc1 e UCCBc5 apresentam capacidade e eficiência de fixar o nitrogênio atmosférico em condi es de casa-de-vegeta o. Verificou-se que a estirpe UCCBc5, resistente ao furadan e ao regente apresentou capacidade fixadora do nitrogênio atmosférico, em condi es de campo. Na avalia o de doses de inoculante observou-se que existe rela o dose de inoculante/ eficiência de fixa o de nitrogênio. Estes resultados permitiram afirmar que as bactérias diazotróficas endofíticas podem ser utilizadas, em alguns casos, como substituto da aduba o nitrogenada, na cultura de cana-de- a úcar. The use of inoculants containing diazotrophic microorganism in sugarcane can contribute to promoting the growth of these plants and tp promote significant gains in productivity. The objective of this study was to characterize diazotrophic bacteria isolated from stalks of sugarcane. We used the means of a semi-solid NFB and JNFb without added nitrogen. Five strains were isolated native diazotrophs which were evaluated microscopically by Gram stain, the intrinsic resistance to antibiotics, fungicide Furadan (i.a. carbofuran) and insecticide Regent (i.a. fipronil ) and ability to fix nitrogen in greenhouse and field conditions. All isolates were characteristic of Gram-negative, and for the intrinsic resistance to antibiotics showed widespread resistance. A native UCCBc5 strain showed resistance to the fungicide Furadan and the insecticide Regente. Bacteria isolated UCCBc1 and UCCBc5 present capacity and efficiency of fixing atmospheric nitrogen under conditions of greenhouse. It was found that the
Construction of high-level expression vector for Bacillus thuringiensis

LI Chao-Rui,DU Li-Xin,PENG Qi,LIANG Ying-Ping,GAO Ji-Guo,ZHANG Jie,SONG Fu-Ping,

微生物学通报 , 2013,
Abstract: 【目的】通过比较cry1A、cry3A、cry4A和cry8E四个基因的启动子转录活性, 筛选出一个强启动子, 利用强启动子构建一个苏云金芽胞杆菌(Bacillus thuringiensis, 简称Bt)高效表达载体。【方法】利用启动子融合lacZ技术检测了4种启动子的转录活性。通过扫描电子显微镜观察晶体、SDS-PAGE、蛋白定量和生物活性测定等方法对新建高效表达载体进行功能验证。【结果】构建了Pcry1A、Pcry3A、Pcry4A和Pcry8E 4个启动子融合报告基因lacZ的表达载体, 经β-半乳糖苷酶活性分析得知, 启动子活性从高到低依次为Pcry8E>Pcry1A>Pcry4A>Pcry3A。选取cry8E启动子, 以pHT315作为基础载体构建苏云金芽胞杆菌高效表达载体pHT315-8E21b, 将cry1Ac基因连接到pHT315-8E21b和广泛应用的cry3A启动子指导的pSXY-422b上, 分别转入无晶体突变株HD-73?, 获得菌株HD-8E1Ac和HD-422-1Ac。扫描电子显微镜观察显示, HD-8E1Ac菌株可以形成菱形晶体, 说明正确表达了cry1Ac基因。SDS-PAGE分析结合蛋白定量实验表明pHT315-8E21b表达效率高于pSXY-422b。对小菜蛾(Plutella xylostella)的生物活性测定表明HD-8E1Ac菌株对小菜蛾有生物活性, 且菌株活性高于HD-422-1Ac。【结论】利用强启动子Pcry8E构建了一个能在Bt中高效表达的穿梭载体pHT315-8E21b, 该载体可正确表达cry1Ac基因, 其表达效率高于被广泛应用的pSXY422b。
EFIKASI KOMBINASI Bacillus thuringiensis israelensis DAN Mesocyclops aspericornis SEBAGAI PENGENDALI HAYATI Aedes aegypti DI GENTONG AIR  [cached]
R. A. Yuniarti,Damar T. B.
Bulletin of Health Research , 2012,
Abstract: EFIKASI KOMBINASI Bacillus thuringiensis israelensis DAN Mesocyclops aspericornis SEBAGAI PENGENDALI HAYATI Aedes aegypti DI GENTONG AIR
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