茶树根内生细菌的分离及其茶多酚耐受性的初步研究

本实验研究了茶树根内生细菌的分离方法及对茶多酚的耐受性,并对内生细菌进行了初步的鉴定。结果表明,分离茶树内生细菌时采用75%的酒精2min和2%次氯酸钠20min表面消毒处理,消毒彻底;含茶多酚的细菌营养平板可作为茶树根内生细菌的选择性分离平板。茶树内生细菌对茶多酚的耐受浓度,最高不高于0.5g/L;经鉴定主要为芽孢杆菌属(Bacillus sp.)。     

抗茶树冰核细菌内生菌的筛选及鉴定

从茶树内生菌中进行了冰核细菌拮抗菌的筛选,得到菌株Y1,通过对菌株Y1进行形态学观察、生理生化指标测定及16βS rDNA序列测定和序列同源性分析,将菌株Y1鉴定为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）。本研究获得了茶树内生拮抗菌株,明确了菌株Y1的种属,有利于冰核细菌生物防治的开展。     

茶树内生细菌的分布规律初探

以铁观音、黄旦、毛蟹等9种茶树品种一年生有性系苗和扦插苗为试验材料,分别对茶树不同器官的内生细菌数量进行测定分析,结果表明：茶树的根、茎、叶中均存在大量的内生细菌。内生细菌数量在茶树不同器官中的分布规律为：侧根〉主根〉茎部〉叶片。器官间内生细菌数量的差异达到显著水平,茶树有性系后代和无性系品种器官间内生细菌数量的变异系数分别为70.8%和31.9%。茶树有性系后代的侧根和主根的内生细菌数量多于无性系茶树品种;而无性系茶树品种的茎部和叶片内生细菌数多于茶树有性系后代。     

茶树根系耐铝促生内生细菌的分离鉴定及其特性研究

茶树喜酸耐铝,且低浓度的铝促进茶树生长,然而其调控机理并不清晰。从耐铝促生菌的角度,探究其可能的原因。以铝处理的茶树根系为材料,经分离鉴定,得到可培养的内生细菌38株,其中厚壁菌门27株,放线菌门11株。从利用1-氨基环丙烷-1-羧酸（ACC）能力、溶磷能力、产铁载体能力和分泌吲哚乙酸（Indole-3-acetic acid,IAA）能力对38株内生细菌进行了探究,结果表明,38株内生细菌都有一种以上的促生能力,其中厚壁菌FBA、FPC以及放线菌AMM、ACP032155等菌株的综合促生能力较好;38株内生细菌在1 mmol·L^-1 Al³⁺浓度下均能存活,其中放线菌AME2耐铝能力最强,在8 mmol·L^-1 Al³⁺浓度下仍能存活,说明铝能促进茶树耐铝促生菌的生长,从而间接促进茶树的生长,为选育具有显著耐铝促生能力的茶树内生细菌用于茶树的栽培育种奠定基础。     

拮抗炭疽病的茶树内生菌筛选、鉴定及培养条件优化

为筛选高效拮抗茶树炭疽病的内生细菌,以茶树健康叶片为材料,采用平板对峙拮抗法进行筛选,并对筛选到的菌株进行鉴定、抑菌效果评价及培养条件优化。从分离的162株内生细菌中筛选到1株对茶树胶孢炭疽菌有较好抑制效果的拮抗细菌X13。形态学、生理生化鉴定及16 S rDNA系统进化发育分析显示,分离的菌株X13为枯草芽孢杆菌（Bacillus subtilis）。菌株X13对病原菌菌丝生长抑制率为61.6%。生长曲线表明,菌株X13对数生长期为接种后2～14 h。响应面优化的培养条件为4.0%（质量百分浓度）玉米粉,1.0%（质量百分浓度）的硝酸钠,接种量3.5%（体积分数）。本研究结果可为茶树炭疽病防治及生防菌剂的开发提供重要参考。     

一株野生大豆内生细菌YDX14菌株的分离、鉴定及促生效应研究

为筛选具有促生效应的内生细菌资源,从野生大豆叶片中分离筛选出1株内生细菌菌株YDX14,采用形态学观察和分子生物学分析方法对该菌株进行鉴定;通过测定菌株溶磷能力、产IAA能力、产ACC脱氨酶能力等研究菌株的促生特性;通过水培促生试验测定菌株对小麦幼苗的促生作用,并采用单因素和正交试验确定菌株的最佳发酵条件.结果表明:Y...     

大豆内生细菌的筛选和鉴定

从黑龙江省不同地点采集大豆,从不同组织中分离内生细菌,并用分离得到的内生细菌对病原真菌做皿内拮抗试验,经过两次筛选有生物拮抗活性的内生菌菌株。结果表明,从大豆各种组织中分离得到137株内生细菌,其中2株对多种植物病原真菌具有拮抗作用,经初步鉴定2菌株均为芽孢杆菌(Bacillus sp.)。     

茶树内生真菌的分离和鉴定研究进展

植物内生真菌是指那些在其生活史的某一时期或全部阶段内生活在植物体内的、但对寄生植物组织并不引起明显的病害症状的真菌。目前,有关植物内生真菌生物学、生理生态学、次生产物化学的研究日益增多,显示了内生真菌具有重要的研究意义与广泛的应用前景,但茶树内生真菌的分离与鉴定研究尚未见系统报道。要开发茶树内生真菌,首先是从茶树体内分离得到微生物菌株,其次是对这些微生物菌株进行鉴定,本文就茶树内生真菌的分离与鉴定研究方法作了总结,以期为进一步研究和开发利用茶树内生真菌奠定基础。     

小粒野生稻内生细菌的分离鉴定和促生功能分析

小粒野生稻（Oryza minuta）是具有良好生物和非生物胁迫抗性的野生稻资源，挖掘小粒野生稻促生相关内生细菌可为其微生态研究和菌肥开发提供参考信息。本研究对小粒野生稻的根、茎和叶进行内生细菌分离，共获得85个菌株。通过促生功能分析发现，43株内生细菌具有溶磷能力，19株具有固氮作用，29株产铁载体，13株产吲哚乙酸。分离自根部的OMR2-3菌株和叶部的OML3-4菌株具有较高吲哚乙酸分泌能力，分别为17.22 mg/L和16.59 mg/L。形态学和分子鉴定显示，OMR2-3菌株和OML3-4菌株分别为阴沟肠杆菌（Enterobacter cloacae）和路德维希肠杆菌（Enterobacter ludwigii）。通过温室促生效果测定发现，2个菌株对多年生稻粳型品种PR23和籼型品种云大107具有显著促生效果，可以不同程度促进多年生稻幼苗的株高、根长、鲜质量、叶绿素和氮素含量。以上结果说明，小粒野生稻蕴含丰富的促生相关内生细菌资源，从中筛选到的内生阴沟肠杆菌OMR2-3菌株和路德维希肠杆菌OML3-4菌株具有开发成为多年生稻生物微生物菌肥的潜力，有助于多年生稻绿色轻简化的生产模...     

盐碱地大豆根内促生拮抗细菌DCX的分离及鉴定

根系内生细菌资源的开发与利用是绿色农业发展的研究重点，而盐碱地大豆根内生细菌的分离与鉴定鲜有报道。本文以大庆盐碱地大豆根系为材料，分离纯化大豆内生细菌，采用平板对峙法和比色法测定内生细菌的抑菌能力和分泌IAA能力，选取抑菌谱广且分泌IAA能力强的株菌，通过检测形态学特征、生理生化性质、结构基因和功能基因序列的系统发育分析，明确细菌的分类地位。结果表明从大庆盐碱地大豆根内分离获得的一株内生细菌DCX，对10种植物病原真菌均具有拮抗作用，其中，对玉米新月弯孢菌、大豆菌核病菌、稻瘟病菌、禾谷镰刀菌的抑菌率分别为50.00%、78.12%、56.25%、43.75%，分泌IAA的能力为2.56±0.41μg·mL-1（n=6）。经形态学、生理生化试验及系统进化分析鉴定，DCX菌株属于解淀粉芽孢杆菌（Bacillus amyloliquefaciens），该菌株在pH11、NaCl浓度 10%的NA培养基中生长良好，可为盐碱地的作物病害防治提供资源。     

枯草芽孢菌株TL2在茶树体内的内生定殖

枯草芽孢菌株TL2接种茶树后,可以从茶树不同组织分离到细菌,其细菌种群数量随着时间逐渐减少,其细菌多样性系数也随着时间有所降低。其菌体主要分布在根部厚壁组织的细胞间隙,茎部厚角组织的细胞间隙、维管束等组织的细胞间隙、叶片的气孔器附近、上下表皮细胞间隙、厚角组织细胞间隙以及内皮层组织细胞间隙等。     

枯草芽孢菌株TL2对茶轮斑病的防病机制

枯草芽孢菌株（Bacillus subtilis）TL2能产生多种外分泌抗菌蛋白,抑制茶轮斑病菌（Pestallozzia theae）的菌丝生长及其分生孢子的形成和萌发。另外,菌株TL2通过改变茶树体内活性氧代谢相关酶系如SOD等的活性,以调节茶树受轮斑病菌侵染后活性氧的代谢平衡,同时诱导茶树产生抗性酶系如PAL和β-1,3-葡聚糖酶,以限制茶树轮斑病菌的扩展。     

桐花树内生细菌动态及拮抗植物病原菌菌株筛选鉴定

以荧光显微计数法和平板分离法分别对桐花树根茎叶内生细菌总量和可培养细菌数量进行周年动态初步测定,并对拮抗植物病原菌的可培养细菌菌株进行筛选鉴定。结果表明:桐花树根茎叶内均含有大量的内生细菌,其中根部内生细菌总量为0.88×107~49.67×107ind./g FW,7月份最大,叶部细菌总量为1.07×107~65.07×107ind./g FW,也以7月份最大,茎部细菌总量为1.1×107~19.73×107ind./g FW,以5月份最大;而根茎叶可培养细菌含量分别为1.02×103~13.18×103、0.13×103~11.24×103、0.31×103~10.36×103CFU/g FW,均以3月份数量最大,周年不同月份之间细菌总量和可培养细菌数量均差异显著。用平板对峙生长法,从86株可培养内生细菌中筛选获得1株对香蕉叶鞘腐败病菌、香蕉枯萎病菌、马拉巴栗茎腐病等植物病原菌均有较强拮抗作用的Aec23菌株,经形态、生理生化测试及16S rDNA序列比较分析,Aec23菌株初步鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。     

Insecticidal effect of recombinant endophytic bacterium containing Pinellia ternata agglutinin against white backed planthopper, Sogatella furcifera

White backed planthopper (WBPH; Sogota furcifera Horvath) has become the major threat to rice crops throughout Asia, damaging plants both through its feeding behavior and by acting as a virus vector. Here, we developed a novel method for biologically controlling WBPH by using endophytic bacterium to express anti-pest plant lectins. Strain SJ-10 of an endophytic bacterium, characterized as Enterobacter cloacae by morphological, physiological, biochemical and 16s rDNA characteristics, was isolated from rice seedlings. The Pinellia ternate agglutinin (PTA) gene was cloned into SJ-10 for expression. The positive transformant, selected by antibiotic resistance, was evaluated using PCR, SDS-PAGE and Western blot assay. After inoculation, rSJ-10 could colonize rice plants so that they expressed PTA, and then the rice was shown to have insecticidal activity against WBPH. The results showed that rSJ-10 could significantly decrease the survival and fecundity of WBPH fed on rice seedlings (p < 0.01). At day 19, the fecundity of WBPH inoculated with rSJ-10, or with wild-type SJ-10 was decreased by 86.1%, and 25.6%, respectively. At day 22, numbers of WBPH on rice in the control were 19.4 times greater than on rice inoculated with rSJ-10. At day 26, the rice seedlings all died in the control group, but the seedlings inoculated with rSJ-10 grew well. The results showed that the rice seedlings inoculated with rSJ-10 expressing PTA protein were endowed with the anti-pest activity against WBPH. Further work is needed to investigate whether the rice plants expressing rPTA are toxic to mammals. This research highlights a way to biologically control planthoppers by recombinant endophytic bacteria expressing plant lectins.     

Multiplication ofPseudomonas solanacearum in resistant potato plants and the establishment of latent infections

When 1-mo-old plants of a wilt-resistant clone ofSolanum phureja (1386.15) were stem-inoculated with three strains ofPseudomonas solanacearum (K60, S123, and S206), the bacteria multiplied rapidly at the point of inoculation and then moved in the vascular system to other parts of the stem. Resistant plants showed a remarkable ability to support relatively high populations of the bacterium in the absence of disease symptoms. Although multiplication in this resistant clone was substantially less than in susceptible Russet Burbank potato plants, large numbers of bacteria (up to 624 × 10⁴ cells of K60 per 5-cm stem segment) reached the base of the stem of plants maintained at high temperature (28°C) for 20 days after stem inoculation. From the base of the stem, the bacteria moved rapidly into the roots and tubers. Strains ofP. solanacearum differed in their ability to cause latent tuber infection in different resistant potato clones. When 11S.phureja ×S. tuberosum hybrids were stem-inoculated, maintained at 28°C for 3 wk and then grown to maturity at 20°C., most of the clones yielded tubers infected by one or more strains. The race 1 strain (K60) was the most infectious; 53.8% of all tubers harvested from all plants inoculated with this isolate carried latent infections. Because one clone (BR 53.1) never yielded infected tubers, there appear to be genetic factors which may be useful in breeding programs aimed at eliminating latent tuber infection.     

接种PRSV番木瓜种苗胞质酵母双杂交cDNA文库的构建与分析

利用Trizol法提取接种PRsv 4 d的番木瓜种苗总RNA,分离纯化mRNA,反转录合成双链cDNA,双链cDNA末端经Pfu-DNA聚合酶补平,与EcoR I接头连接,XhoI酶切消化产生粘端.用Sepharose CL-2B柱分离纯化去除小分子cDNA片段,再与pMyr酵母表达载体连接,转化受体菌XL10-Gold,构建了接种PRSV番木瓜种苗胞质酵母双杂交cDNA文库.所获得的原始文库的克隆总数为1.8×106cfu,重组率为100%,文库滴度为2.6×109cfu/mL.对随机选取的34个克隆进行PCR鉴定,结果表明插入片段长度均大于O.5 kb且集中在1 kb左右.文库质量鉴定结果表明,该文库具有较好的库容量、较高的重组率以及较大的插入片段.     

Resistance to potato virus Y inSolanum tuberosum spp.andigena

Tests were made on lines ofSolanum tuberosum spp.andigena to determine 1) whether resistance to potato virus Y exists in the Andigena germplasm, 2) the nature of its inheritance, and 3) the type of resistance. Results of the isolation and identification studies indicated that the pathogen involved was a common strain of potato virus Y. Under field conditions susceptible plants frequently escaped infection. However, field exposures over two seasons resulted in the same ratios as tests in which the same progenies were mechanically inoculated. Mechanical inoculation at the seedling stage proved to be a reliable means of transmission and resulted in accurate screening for resistance. Of 641 tub × tub clones tested, all were susceptible to the virus. Of 366 tub × adg clones tested, 170 were resistant and 196 were susceptible. This fits a 13:15 ratio, assuming random chromatid segregation and a single dominant gene conferring resistance. Plants that were resistant following mechanical transmission were also resistant when inoculated by aphids, indicating the reliability of mechanical transmission as a means of screening for resistance. To determine the type of resistance, top-graft and approach-graft tests were made. Failure to recover the virus from resistant plants inoculated by either grafting method suggests that immunity is the type of resistance involved.     

不同产区青稞根部可培养内生固氮菌的多样性研究

为认识青稞内生固氮菌群落组成,丰富内生固氮菌资源,明确产地对青稞内生固氮菌多样性的影响,本研究利用纯培养技术分别对在拉萨、林芝、日喀则栽培的藏青2000根部内生固氮菌进行分离、纯化,并测定各菌株的固氮酶活性及高固氮酶活性菌株的溶磷、溶钾及产IAA特性,最后,通过16SrDNA基因测序和构建系统发育树研究了高固氮酶活性菌株的分类学地位。结果显示,通过Ashby培养基分离、纯化得到内生固氮菌37株,其中,具有高固氮酶活性的11株,占总分离菌株27%,有6株菌株具有分泌IAA的能力,2株具有溶钾能力,1株具有溶磷能力。16S rDNA测序结果表明,11株高固氮酶活性的菌中,有5株菌属于Bacillus,为优势菌属,其余菌株分别属于Gemmatimonas、Sphingomonas、Devosia、Agrobacterium、Mesorhizobium和Burkholderia。