不同寄主植物西花蓟马内生细菌多样性分析

为探讨不同寄主植物对西花蓟马Frankliniella occidentalis内生细菌分布的影响,应用Illumina MiSeq第二代测序技术对取食不同寄主植物的西花蓟马内生细菌的16S rDNA V5~V7区域进行测定。结果显示,Alpha多样性分析发现,西花蓟马内生细菌在取食不同寄主植物后多样性表现为西葫芦Cucurbita pepo L. >菊花Dendranthema morifolium(Ramat.) Tzvelev >曼陀罗Datura stramonium L. >玫瑰Rosa rugosa Thunb. >牵牛花Pharibitis purpurea (L.) Voigt >辣椒Capsicum annuum L.;在门水平的菌群组成上,变形菌门在各样品中所占比例最高,是西花蓟马内生菌中的优势菌门,厚壁菌门为次优势菌菌门。在属水平上内生细菌也表现出较大的差异,其中假单胞菌属Pseudomonas和欧文氏菌属Erwinia在西花蓟马取食各寄主植物时均有分布,取食辣椒、玫瑰、曼陀罗时,优势菌属均为欧文氏菌属Erwinia,取食菊花和牵牛花时,优势菌属为立克次体Rickettsia,而取食西葫芦时,则为短波毛单胞菌属Brevundimonas;利用KEGG数据库,对内生细菌进行基因功能预测分析发现,内生细菌群富集了高丰度的代谢功能基因,且主要集中于碳水化合物代谢、氨基酸代谢以及能量代谢。综上所述取食不同寄主植物时西花蓟马内生细菌多样性与群落结构具有一定的差异,寄主植物可对西花蓟马内生细菌群落组成产生重要的影响;且各寄主植物上西花蓟马内生细菌基因功能主要集中在碳水化合物代谢、氨基酸代谢和能量代谢。     

The contribution of endophytic bacteria to Albizia lebbeck-mediated phytoremediation of tannery effluent contaminated soil

Toxicity of chromium often impairs the remediation capacity of plants used in phytoremediation of polluted soils. In this study, we have identified Albizia lebbeck as a prospective chromium hyperaccumulator and examined cultivable diversity of endophytes present in chromium-treated and control saplings. High numbers (22–100%) of endophytic bacteria, isolated from root, stem, and leaf tissues, could tolerate elevated (1–3 mM) concentrations of K₂CrO₇. 16S rRNA gene sequence-based phylogenetic analysis showed that the 118 isolates obtained comprised of 17 operational taxonomic units affiliated with the proteobacterial genera Rhizobium (18%), Marinomonas (1%), Pseudomonas (16%), and Xanthomonas (7%) but also with members of Firmicutes genera, such as Bacillus (35%) and Salinococcus (3%). The novel isolates belonging to Salinococcus and Bacillus could tolerate high K₂CrO₇ concentrations (3 mM) and also showed elevated activity of chromate reductase. In addition, majority (%) of the endophytic isolates also showed production of indole-3-acetic acid. Taken together, our results indicate that the innate endophytic bacterial community assists plants in reducing heavy metal toxicity.     

Increased plant growth and copper uptake of host and non-host plants by metal-resistant and plant growth-promoting endophytic bacteria

The effects of inoculation with two metal-resistant and plant growth-promoting endophytic bacteria (Burkholderia sp. GL12 and Bacillus megaterium JL35) were evaluated on the plant growth and Cu uptake in their host Elsholtzia splendens and non-host Brassica napus plants grown in natural Cu-contaminated soil. The two strains showed a high level of ACC deaminase activities. In pot experiments, inoculation with strain GL12 significantly increased root and above-ground tissue dry weights of both plants, consequently increasing the total Cu uptake of E. splendens and Brassica napus by 132% and 48.2% respectively. Inoculation with strain JL35 was found to significantly increase not only the biomass of B. napus, consequently increasing the total Cu uptake of B. napus by 31.3%, but Cu concentration of E. splendens for above-ground tissues by 318% and roots by 69.7%, consequently increasing the total Cu uptake of E. splendens by 223%. The two strains could colonize the rhizosphere soils and root interiors of both plants. Notably, strain JL35 could colonize the shoot tissues and significantly increase the translocation factors and bioaccumulation factors of E. splendens. These results suggested that Burkholderia sp. GL12 and B. megaterium JL35 were valuable bacterial resource which had the potential in improving the efficiency of Cu phytoextraction by E. splendens and B. napus in a natural Cu-contaminated soil.     

豆科植物根瘤内生细菌的发现及其研究进展

近年来研究报道显示,某些豆科植物与根瘤菌在形成固氮共生体的同时,其根瘤内还存在多种其他类群的内生细菌,该现象在根瘤菌研究领域越来越引起关注和重视。本文综述了根瘤内生土壤杆菌、非共生根瘤菌、其它细菌的发现、种类及其对共生关系和植物生长的影响等研究进展,同时对该研究方向提出一些初步观点和认识,旨在增加人们对根瘤微生态的了解,拓展根瘤菌研究与应用的视野。     

柑橘黄龙病株不同部位内生细菌群落结构的多样性

采用PCC(Pearson correlation coefficient)、系统聚类和多样性指数研究了柑橘黄龙病(citrus huanglongbing,HLB)植株不同部位叶片、枝条及其根部内生细菌的相互关系及其群落结构多样性.结果表明,从供试柑橘中分离得到26株内生细菌(叶片10株,枝条14株,根部2株)隶属于10个属19个种.Nested-PCR结果显示,柑橘植株不同空间叶片中,黄龙病病原的阳性检出率为58.3％.用LSD法比较显示,柑橘内生细菌在不同器官的分布量大小依次为根＞叶＞枝条.上、中、下不同部位叶片和枝条内生细菌分布量差异不显著,而东、西、南、北不同方位的叶片和枝条内生细菌分布量差异显著(P＜5％).柑橘内生细菌之间的PCC分析表明,Bacillus pumilus和Bacillus sp.与柑橘黄龙病病原菌呈显著负相关,Bacillus sp.和Bacillus pumilus与Bacillus subtilis呈显著正相关.Bacillus brevis和Escherichia hermannii与Bacillus vesiculariss存在显著正相关.对柑橘不同器官内生细菌的PCC分析表明,同种器官的内生细菌之间呈正相关,不同器官的内生细菌之间呈负相关.以内生细菌在各器官的分布量为指标分析不同器官之间的相关性,结果表明,叶片与枝条呈正相关,PCC为0.55,叶片与根部、枝条与根部均呈负相关,PCC分别为-0.19和-0.13.16S rDNA序列聚类分析表明,柑橘内生细菌可分为两大类,第Ⅰ大类为革兰氏阳性菌,第Ⅱ大类为革兰氏阴性菌.第Ⅰ大类又可以分为芽孢杆菌属和短小杆菌属两个亚类,第Ⅱ大类也可以分两个亚类.对柑橘黄龙病病原与叶片内生细菌进行聚类分析,可以分为三类,第Ⅰ类特征为内生细菌存在所有部位的叶片;第Ⅱ类特征为内生细菌与黄龙病病原菌存在负相关性;第Ⅲ类特征为内生细菌只存在特定部位叶片.当马氏距离为27.23时,可将柑橘黄龙病内生细菌群落结构聚为3类,第Ⅰ类特点是在不同部位叶片均有分布,为完全分布类型,且分布量较大,第Ⅱ类特点是在各器官中均为不完全分布类型,且分布量不均匀,第Ⅲ类特点是根部分布特性且分布量较大.分析多样性指数表明,SHANNON(H1)指数在叶片最高,根部最低.SIMPSON(D)指数在枝条最高,根部最低.Pielou指数在根部最高,枝条最低.     

Detection and eradication of endophytic bacteria from micropropagated mint plants

Summary Liquid medium and an enriched agar were used to detect endophytic bacteria in micropropagated mints (Mentha spp.) within 2 to 6 d of inoculation. Bacteria isolated from the cultures were screened on several antibiotic concentrations to determine bactericidal doses. Antibiotics were also tested for phytotoxic effects. Shoot tips from infected plants were treated by immersion in liquid MS medium containing antibiotics either singly or in combination. Streptomycin applied at 1000µg/ml for a period of 10 d was effective and less phytotoxic in a larger number of cases than gentamicin (50µg/ml), neomycin (500µg/ml), or rifampicin (30µg/ml). Mint cultures that tested negative for bacteria after antibiotic treatment were multiplied, retested, and cold-stored for 1 yr or longer. Upon regrowth after storage, 25 of 30 treated cultures (83%) tested negative for bacteria. Of the 25, 8 were successfully treated with streptomycin, 1 with gentamicin, 2 with neomycin, 1 with rifampicin, and 1 with streptomycin and gentamicin; 12 required more than one treatment. An early detection system, initial trial treatment with streptomycin for infected plants, and monitoring of treated cultures successfully reduced the spread of bacterial contamination. Antibiotic treatment in liquid MS medium at pH 6.9 resulted in enhanced bactericidal activity over that seen at pH 5.5.     

元阳梯田地方水稻品种根部内生菌及根际微生物的分离与鉴定

采用组织分离法和土壤稀释涂板法,对元阳哈尼梯田2个地方品种‘月亮谷’和‘红脚老粳’的根部内生细菌及根际土壤细菌进行了分离,研究元阳梯田传统水稻品种特殊的内生菌组成.结果表明: 试验共得到399个菌株.经形态特征及生理生化鉴定,月亮谷根部和其根际土壤分别分离到8和5个属,其中5个属是共有的;红脚老粳的根部和其根际土壤中分别分离到10和7个属,其中6个属是共有的.经分子生物学鉴定,月亮谷根部分离到11个种和5个属,根际土壤分离到8个种和4个属,其中5个种和4个属是共有的;红脚老粳根部分离到9个种和5个属,根际土壤分离到10个种和3个属,其中4个种和2个属是共有的.通过分子生物学鉴定,大部分菌株都可以鉴定到种,而通过形态及生理生化特性只能初步鉴定到属,但两种方法在属层次上的鉴定结果基本一致.元阳地方水稻根部内生细菌及根际土壤细菌具有一定的种属同源性与特异性.     

Characterization of endophytic and symbiotic bacteria within plants of the endemic association Centaureetum horridae Mol

Abstract

We investigated the internal association of bacteria with Astragalus terraccianoi and Centaurea horrida, two endemic plants of the Mediterranean islands, forming the phytosociological association Centaureetum horridae, typical of windswept cliffs on the rocky shores of Asinara (Sardinia, Italy) and other limited locations. Sampling occurred in the protected natural park of the Asinara island. Roots and stems of the two plants and the root nodules of A. terraccianoi were surface sterilized in order to remove external and rhizospheric microbiota and to subsequently isolate the culturable bacterial communities. Plate counts revealed densities of endophytes between 3.7 × 10² and 2.8 × 10⁴ colony forming units per gram of fresh weight. 16S rDNA sequencing revealed the occurrence of bacteria displaying high similarity with Actinobacterium sp., Paenibacillus sp., Rhizobium sp., Methylobacterium sp., Pedobacter panaciterrae, Aerococcus viridans, Stenotrophomonas rhizophila, Bacillus sporothermodurans, Bacillus pumilus, Bacillus simplex, Bacillus flexus, Streptomyces ciscaucasicus and Dyella sp. The putative nitrogen-fixing rhizobium symbiont of A. terraccianoi was identified for the first time. It turned out to belong to the slow-growing Bradyrhizobium genus and to share a 97% similarity with Bradyrhizobium canariense. It was found to be nonculturable and to coexist in nodules with a number of different endophytes.     

北部湾徐闻海域红树内生细菌物种多样性及其杀线虫活性研究

为发掘红树植物内生细菌资源和寻找新型微生物杀虫剂,该研究从北部湾徐闻海域采集7种红树植物共16份样品,设计10种分离培养基,使用稀释涂布法分析红树植物内生细菌的分布特征,通过16S rRNA分子生物学方法对内生细菌进行多样性分析,并利用秀丽隐杆线虫模型,通过杀线虫活性实验测试内生细菌乙酸乙酯提取物的杀线虫活性。结果表明:(1)从16份红树植物各组织器官中获得33株内生细菌,分布于19个科23个属。其中,芽孢杆菌属(Bacillus)为优势菌属,并发现了10株潜在的新种或新属。(2)筛选到具有显著杀线虫活性的菌株IMDGX 4725和IMDGX 4744,半数致死浓度(LC₅₀)分别为61.58、100.89mg·mL^-1。研究结果证实了徐闻海域红树具有多样性丰富的内生细菌,同时部分细菌具有较强的杀线虫活性,具有发现新型微生物杀虫剂的潜力。     

Chemical characterization of root exudates from rice (Oryza sativa) and their effects on the chemotactic response of endophytic bacteria

Root exudates represent an important source of nutrients for microorganisms in the rhizosphere and seem to participate in early colonization inducing chemotactic responses of rhizospheric bacteria. We characterized the root exudates collected from rice plantlets cultured under hydroponic conditions and assessed their effects on the chemotaxis of two strains of endophytic bacteria, Corynebacterium flavescens and Bacillus pumilus, collected from the rice rhizosphere. We compared these chemotactic effects on endophytic bacteria with those on two strains of plant-growth-promoting bacteria, Azospirillum brasilense (isolated from the corn rhizosphere) and Bacillus sp. (from the rice rhizosphere). The root exudates were collected at different time intervals. The highest concentration and diversity of amino acids and carbohydrates were found during the first 2 weeks after seeding. Histidine, proline, valine, alanine, and glycine were the main amino acid residues identified during the 4 weeks of culture. The main carbohydrates identified were glucose, arabinose, mannose, galactose, and glucuronic acid. The chemotactic responses of the analyzed endophytic bacteria to root exudates were 3.9 to 5.1 times higher than those of A. brasilense and 2.2 to 2.8 times higher than Bacillus sp. Our results indicate that rice exudates may induce a higher chemotactic response for endophytic bacteria than for other bacterial strains present in the rice rhizosphere.     

根瘤内生细菌

在研究根瘤菌与豆科植物共生关系的过程中,人们发现根瘤中同时定居着许多与根瘤菌不同的内生菌,这些非共生细菌生活在根瘤中,但不引起植物产生明显的病害,为根瘤内生细菌.根瘤内生细菌不仅将植物作为其栖息场所,而且对宿主植物有促生、防病、内生联合固氮等广泛的生物学作用.     

马衔山中国沙棘根瘤内共生细菌多样性研究

采集甘肃省兰州市马衔山中国沙棘(Hippophae rhamnoidoes)根瘤样品,采用高通量测序和纯培养方法,分析中国沙棘根瘤内定殖的内共生细菌的组成、相对丰度和物种多样性,并比较两种方法研究结果的差异。在不同的微生物分类单元,高通量测序检测到中国沙棘根瘤内共生细菌24门50纲90目167科215属;而纯培养方法仅检测到3门5纲7目8科8属。进一步分析沙棘根瘤内共生细菌主要类群的相对丰度,发现高通量测序与纯培养方法的结果有明显差异,在科和属的分类单元上差异尤其显著。两种方法都表明中国沙棘根瘤内共生细菌具有丰富的多样性,但高通量测序能够较为全面的反映中国沙棘根瘤内共生细菌的群落组成,而纯培养方法仅能够分离到部分可培养的中国沙棘根瘤内共生细菌,在很大程度上极可能低估中国沙棘根瘤内共生细菌的物种组成并高估其丰度。     

Delivery methods for introducing endophytic bacteria into maize

The effectiveness of fivemethods for delivery of ten endophyticbacteria into maize stem and root tissues wasstudied in greenhouse conditions at EmbrapaMilho Sorgo, Sete Lagoas, MG, Brazil. Thedelivery methods included seed inoculation,soil drench, foliar spray, pruned-root dip andseed inoculation + soil drench. The bacterialendophytes were previously isolated from maizeplants, and reinoculated and recovered aftertreatments from maize, cv BR201. Each of thefive methods led to establishment and recoveryof bacterial endophytes in root tissues, butonly four isolates were recovered by the seedtreatment method. All 10 isolates wererecovered by pruned-root dip and seed treatment+ soil drench. No isolates were recovered instem tissues by the seed treatment method, andin the root by foliar spray method. However,all isolates were recovered in stem tissue bypruned-root dip method. The pruned-root dip wasthe most efficient method to deliver bacterialendophytes into maize. The isolate, BR201, wasrecovered by almost all methods in root andstem tissues. The results demonstrate thatendophytic bacteria can be recovered from maizetissues following inoculation by the differentmethods described, but the delivery depends onthe methods used and the endophytic bacterialisolate.     

柠条根瘤内生细菌的抗逆性及遗传多样性

采用16S rDNA PCR-RFLP和序列分析方法对分离自柠条根瘤的40株内生细菌的遗传多样性及系统发育进行分析,并对菌株的耐盐性、耐酸碱性和生长温度范围进行测定.结果表明:40株供试菌株共产生9种遗传图谱类型;对各类型代表菌株进行16S rDNA序列测定,结合形态特征和生理生化检测结果,表明供试菌株分别归属于芽孢杆菌属(Bacillus)、Inguilinus属、申氏杆菌属(Shinella)和不动杆菌属(Acinetobacter),遗传多样性较为丰富;57.5％的菌株可耐受4％的NaCl,75％的菌株可在pH 11.0的条件下生长,85％的菌株经60℃热激处理后仍能继续生长,显示柠条根瘤内生细菌具有较强的抗逆性,菌株LWEN 07和LWEN 15抗逆能力最为显著.     

Occurrence,physiological and molecular analysis of endophytic diazotrophic bacteria in gramineous energy plants

Endophytic diazotrophic bacteria could be isolated from the energy plants Pennisetum purpureum, Miscanthus sinensis, Miscanthus sacchariflorus and Spartina pectinata using semisolid nitrogen free media. Higher levels of diazotrophic bacteria were found if no nitrogen fertilizer was applied. The bacteria were characterized on the basis of typical morphology, physiological tests, and the use of phylogenetic oligonucleotide probes. They belong partially to the species Azospirillum lipoferum and Herbaspirillum seropedicae while others supposedly represent a new species of Herbaspirillum. Using PCR-fingerprinting techniques a limited genetic diversity of these isolates was found which may indicate an adaptation to the specific conditions of the interior of these plants.     

牡丹根部内生细菌的分离鉴定及脂肽类物质的拮抗活性研究

【目的】从牡丹(Paeonia suffruticosa Andr.)根部组织中分离鉴定内生细菌,测定拮抗菌株脂肽类活性物质的体外抑菌活性。【方法】采用平板对峙法筛选出对牡丹灰霉病菌(Botrytis paeoniae Oadem)、牡丹炭疽病菌(Gloeosporium sp.)、牡丹黑斑病菌(Altenaria sp.)、牡丹黄斑病菌(Phyllosticta commonsii)有拮抗作用的内生细菌。基于形态特征、生理生化特性和16S rRNA基因序列同源性鉴定拮抗菌株。根据脂肽类抗菌物质合成相关基因序列对拮抗菌株进行基因扩增检测,采用酸沉淀法提取拮抗菌株的脂肽类物质,平板对峙法测定脂肽类物质的体外抑菌活性。【结果】从牡丹根部组织中共分离获得62株内生细菌,其中菌株Md31和Md33对4种病原菌均有较明显的抑制作用。Md31和Md33被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。通过对菌株Md31和Md33进行5个脂肽类合成功能基因bmyB、fenD、ituC、srfAA和srfAB的检测,序列同源性分析,表明两个菌株具有合成脂肽类物质的能力。菌株Md31和Md33的脂肽类粗提物对所测试的牡丹病原真菌均具有不同程度的抑制作用。【结论】获得了2株对牡丹病原菌有良好抑制效果的解淀粉芽孢杆菌Md31和Md33,两个菌株的脂肽类粗提物也具有较强的体外抑菌活性,该研究为牡丹内生细菌的进一步开发应用奠定了基础。     

银沙槐内生放线菌抗菌活性及其与内生细菌的拮抗关系

从荒漠濒危植物银沙槐根部分离到内生放线菌12株和内生细菌31株.以大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌为指示菌对内生放线菌进行了抗菌活性筛选.结果表明：3株放线菌对3种指示菌均有抑制作用,2株完全无抑菌作用.采用菌块抑制法分析内生放线菌对来自同一植物内生细菌的抑制效果,44.9%的组合出现抑制现象,最大抑菌圈直径为28.5mm,最小者仅8.25 mm,主要抑制对象是革兰氏阴性菌和芽孢菌;55.1%的组合无抑制现象.说明内生菌的相互作用具有多样性.     

浑源黄芪内生细菌的菌群组成及其功能

【目的】分离、鉴定浑源黄芪内生细菌,筛选潜在促生菌,并研究绿叶挥发物对其生长的影响。【方法】以山西浑源7年生传统采收期黄芪为材料,采用平板培养法分离内生菌,16S rRNA基因序列进行菌株鉴定;通过培养基中添加1-氨基环丙烷-1-羧酸(1-aminocyclopropane-1-carboxylate,ACC)、色氨酸及缺氮素培养的方式进行含ACC脱氨酶、吲哚乙酸产生及固氮菌初筛;通过培养基中添加Ca_3(PO_4)_2、钾长石的方式进行解磷、解钾菌初筛;电感耦合等离子体质谱等方法进行定量;通过液体培养基中添加绿叶挥发物的方式,研究其对含ACC脱氨酶菌株的影响;利用顶空气相色谱-质谱法测定黄芪绿叶挥发物含量。【结果】从浑源黄芪根中分离得85株代表性内生菌株,分别属于变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)的13个属,其中假单胞菌属(Pseudomonas)、泛菌属(Pantoea)和葡萄球菌属(Staphylococcus)菌株数量较高,占分离菌株总数的80.00%。筛选获得的促生菌中,具有吲哚乙酸合成能力的菌株所占比例最高(69.41%),其次为含ACC脱氨酶和具有固氮活性的菌株,分别占分离菌株的40.00%和31.76%,而解磷、解钾菌株所占比例较低(分别为14.12%、7.06%);双重促生效应菌株中,兼具吲哚乙酸合成与含ACC脱氨酶菌株所占比例最高(37.65%),其次为兼具吲哚乙酸合成和固氮活性、兼具含ACC脱氨酶和固氮活性的菌株,分别占分离菌株的28.24%和24.71%,兼具含ACC脱氨酶和解钾活性的菌株占比最低(1.18%)。2–50μmol/L正己醛和Z-3-己烯醛、5–125μmol/L正己醇具有促进部分含ACC脱氨酶内生菌株生长的作用。【结论】吲哚乙酸产生和含ACC脱氨酶的内生菌株占比高、绿叶挥发物促生菌的存在很可能是浑源黄芪内生菌群适应栖息地独特生境的产物,绿叶挥发物很可能是影响浑源黄芪内生菌群组成与功能的重要因素之一。