Identification,characterization and optimization of phosphate solubilizing rhizobacteria (PSRB) from rice rhizosphere

Two billion people worldwide take rice (Oryza sativa L.) as a staple food. Phosphorus (P) and Nitrogen (N) are the major requirements of rice; although these are available in limited concentrations within rice growing regions. Among different types of Plant growth-promoting rhizobacteria (PGPR), Phosphate solubilizing rhizobacteria (PSRB) constitute an important class. These are known for plant growth promotion by enhancing P and N uptake. PSRB are nowadays used as biofertilizers to restore the soil health. Under the present investigation identification, characterization and optimization of phosphate solubilizing activity of these microbes at different pH, temperature and salt concentrations was carried out. Thirty-seven isolates were recovered from different regions of rice rhizosphere on Pikovskaya (PVK) agar among which 15 isolates were recovered from R.S. Pura, 12 isolates from Bishnah and 10 isolates were recovered from Akhnoor sector of Jammu, India. A prominent halo zone of clearance was developed around the colonies of 12 different isolates, indicating phosphate solubilization activity. Four distinct isolates were amplified, cloned and sequenced for taxonomic identification using 16S primers. The results indicated that PS 1, PS 2, PS 3, PS 4 were related to Pseudomonas aeruginosa, Bacillus subtilis strain 1, B. subtilis strain 2, B. subtilis strain 3, respectively. These strains when grown at a wide range of ecological factors showed maximum growth at pH between 6.8 and 8.8, temperature between 28 °C and 37 °C and salinity between 1% and 2%. Screening for phosphate solubilization activity revealed that the halo zone diameter formed by these isolates extended from 2.1 to 3.2 mm. The phosphate solubilizing efficiency (SE) ranged from 35.4 to 50.9 with highest value of 50.9 by PS4 and maximum P solubilization of 10.22 µg/ml was recorded by PS4 at 7th day. Phosphate solubilization activity of these identified PSRB strains can be utilized and explored in the rice growing belts of Jammu region which are deficient in phosphorus. MIC value for zinc sulphate heptahydrate in 12 isolates varied from 1 mg/ml to 6 mg/ml. Phosphate solubilization activity and MIC of these identified PSRB strains can be utilized and explored in the rice growing belts of Jammu region which are deficient in phosphorus.     

Biological hardening of micropropagated Picrorhiza kurrooa Royel ex Benth., an endangered species of medical importance

Three plant growth-promoting rhizobacteria viz. Bacillus megaterium, B. subtilis and Pseudomonas corrugata were used for biological hardening of micropropagated plantlets of Picrorhiza kurrooa. The bacterial isolates antagonized the fungal spp. postulated to cause death of micropropagated plants in plate-based assays and positively influenced survival and growth parameters in greenhouse investigation.     

植物根际促生菌对3种土传真菌病害病原的抑制作用

【目的】获取促生同时可防治3种土传真菌病害(Fusarium oxysporum、Sclerotinia sclerotiorum和Rhizoctonia solani)的生防菌,并明确其抑菌效果。【方法】利用前期研究获得的17株促生菌,采用平板对峙法测定其对病原真菌的拮抗作用及对菌丝生长的抑制作用。【结果】可有效拮抗立枯丝核菌的生防菌有6株,其中促生菌株FX2和LM4-3的抑制率达73.82%;拮抗尖孢镰刀菌的生防菌有7株,其中FX2的抑制率达到66.81%;拮抗油菜菌核病菌的生防菌有4株,其中菌株LHS11的抑制率高达85.71%。菌株LHS11和JM170通过次生代谢物抑制病原真菌。所有的生防菌对病原菌的菌丝生长均有一定的抑制作用。【结论】筛选得到对3种真菌病害病原具有较好生防作用的菌株LHS11和FX2。     

烟草可培养内生细菌的分离及多样性分析

采用稀释平板法, 从健康烟草的根、茎、叶组织中分离到267株内生细菌。利用细菌菌落表征性状和16S rRNA序列对这些分离物进行了多样性分析。通过数值分析比较了8个菌落形态表征性状, 以类平均连锁聚类法的方式进行聚类分析, 在2.15的水平上可分成5个聚类群和56个亚群。16S rDNA序列系统发育分析表明267株分离物可分为21个类群。研究表明同一菌落形态类型的菌株在系统发育树中不一定聚为一类, 菌落形态分类与分子生物学方法分类结果不完全一致。经克隆测序分析表明, 这267株分离物分别与GenBank中6类细菌中的21个已知种相似性达到98%?99%。其中芽孢杆菌属(Bacillus)细菌是烟草可培养内生细菌的优势种群。     

Use of plant growth-promoting bacteria to enhance salinity stress in soybean (Glycine max L.) plants

The effects of three rhizobacterial isolates namely Pseudomonas fluorescens (M1), Pseudomonas putida (M2) and Bacillus subtilis (M3) were examined to enhance growth and chemical components such as chlorophyll and proline of three cultivars of soybean (Glycine max L.) under two levels of salinity stress (S1 = 200 mM and S2 = 400 mM of NaCl salt). Several morphological and physiological parameters were investigated. The highest mean values of final germination percent (FGP) were registered in cultivar Crawford (95%) followed by Giza111 cultivar (93%) in the presence of P. fluorescens, while, FGP of Clark was 85%. Mean germination time was decreased by the application of P. fluorescens or P. putida in both salt stressed and unstressed traits. All growth parameters were significantly decreased by salinity treatments, particularly at S2. A significant increase in stem length and shoot fresh weight was recorded in plants treated with P. fluorescens. This enhancing trend was followed by the application of P. putida then B. subtilis. Chlorophyll contents and plant soluble proteins were decreased, while proline content was increased as compared with control treatment. Results showed that the salt tolerant cultivar, Crawford, may have a better tolerance strategy against oxidative damages by increasing antioxidant enzymes activities under high salinity stress. These results suggest that salt induced oxidative stress in soybean is generally counteracted by enzymatic defense systems stimulated under harsh conditions. Our results showed that inoculation with plant growth-promoting rhizobacterial (PGPR) alleviated the harmful effects of salinity stress on soybean cultivars. The diversity in the phylogenetic relationship and in the level of genetic among cultivars was assessed by SDS-PAGE and RAPD markers. Among the polymorphism bands, only few were found to be useful as positive or negative markers associated with salt stress. The maximum number of bands (17) was recorded in Crawford, while the minimum number of bands (11) was recorded in Clark. Therefore, the ISSR can be used to identify alleles associated with the salt stress in soybean germplasm.     

辽宁盘锦三角洲翅碱蓬根系及内生细菌群落多样性

【目的】翅碱蓬(Suaeda heteroptera)是一种典型的盐碱指示物,对重金属和石油污染的盐碱土壤有一定的修复作用,但是关于翅碱蓬根系与根系内生微生物之间的关系、微生物的多样性以及根系微生物在生物修复中所起作用的研究较少。本文以盘锦"红海滩"的翅碱蓬为例,研究翅碱蓬根系及根系内生细菌菌群种类和结构。【方法】通过传统的培养方法和非培养的高通量测序方法对翅碱蓬根系土壤样品、根系附着物样品以及根系匀浆样品中微生物群落多样性进行分析。【结果】传统方法共分离得到67株细菌,选择代表菌28株,根据其形态特征和生理生化特征,结合16S r RNA基因序列比对进行鉴定,它们分别属于盐单胞菌属(Halomonas)、海细菌属(Marinobacterium)、芽孢杆菌属(Bacillus)、副球菌属(Paracoccus)、假单胞菌属(Pseudomonas)、游动球菌属(Planococlus)、沙雷氏菌属(Serratia)、刘志恒菌属(Zhihengliuella)等。利用高通量测序技术对样品进行多样性分析,其中根系附着物样品的菌群丰度和多样性最高,依次分别为根系土壤样品和根系匀浆样品。3个样品中有效序列群落结构可分为12个门,包括酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、绿菌门(Chlorobi)、绿弯菌门(Chloroflexi)、蓝菌门(Cyanobacteria)、厚壁菌门(Firmicutes)、芽单胞菌门(Gemmatimonadetes)、浮霉菌门(Planctomycetes)、变形菌门(Proteobacteria)、螺旋体门(Spirochaetes)和疣微菌门(Verrucomicrobia)。根系匀浆样品中蓝菌门为优势门类,占整个菌群的42%,变形菌门为次优势类群,占33%。变形菌门在根系附着物样品中为优势门类,占46%,拟杆菌门为次优势门类,占16%。根系土壤样品中拟杆菌门为优势门类,占整个菌群的37%,次优势类群为变形菌门,占20%。【结论】翅碱蓬根系和内生菌具有丰富的多样性,其根系微生物可能会在重金属和石油污染土壤的生物修复中起一定的修复作用。     

植物根际促生菌的筛选及鉴定

【目的】植物根际促生菌(PGPR)和植物的互作关系往往不稳定,PGPR菌群有可能提高菌株对野外环境的适应性。为此,本文根据PGPR促生机制的多样性,从不同植物根际土壤进行了PGPR的筛选及鉴定。【方法】首先,按照固氮、解磷、解钾、拮抗6种常见病原真菌,同时能在植物根际定殖为基本初筛标准,然后在实验室条件下测定初筛菌株的多项促生能力(PGP),最后通过生理生化试验和16SrRNA基因序列分析对所筛菌株进行鉴定。【结果】从江苏扬州、盐城等地土壤样品筛选出14株PGPR,具有体外抑菌、产NH3、产IAA、产HCN、产嗜铁素、解磷、溶钾、固氮以及产抗生素等促生能力。分类鉴定结果显示:7株属于假单胞菌属(Pseudomonas)、3株属于类芽孢杆菌属(Paenibacillus)、2株为芽孢杆菌属(Bacillus)、1株为布克霍尔德氏菌属(Burkholderia)、1株为欧文氏菌属(Erwinia)。【结论】所筛细菌具有多种促生能力,且能在根际定殖,为进一步构建多功能PGPR广适菌群提供菌株资源。     

落地生根内生固氮菌多样性和促生特性

【背景】内生固氮菌可以定殖于植物体内为植物提供营养物质,还能通过代谢促进植物生长,目前对于落地生根内生菌的研究鲜见报道。【目的】研究落地生根中内生固氮菌多样性。【方法】从表面消毒的植物组织中分离纯化内生菌,并通过乙炔还原法测定菌株的固氮酶活性。采用SDS-PAGE全细胞蛋白电泳和IS指纹图谱对菌株聚类,各类群代表菌株进行16S rRNA基因系统发育分析和生理生化鉴定。测定菌株固氮、分泌生长素和ACC脱氨酶、产铁载体、溶磷和解钾等促生特性。【结果】从落地生根中分离纯化出26株内生固氮菌,聚为5个类群,隶属于4个属的5个菌种,且各类群代表菌株具有多种促生功能。【结论】从落地生根中分离获得的内生菌具有丰富的遗传多样性和促生特性,并且存在新的微生物资源,有待开发利用。     

宁夏枸杞内生细菌的多样性及其抑菌活性研究

【目的】对宁夏枸杞各药用部位内生细菌的分布特征、遗传多样性和抑菌活性进行分析。【方法】采用菌落计数和16S rRNA基因序列分析法研究枸杞内生细菌的分布特征、遗传多样性,采用琼脂扩散法测定其抑菌活性。【结果】从各药用组织器官中分离出内生细菌34株,隶属于7科11属,内生细菌的数量和群落组成存在明显的组织特异性,其数量表现为根皮>叶>花>果实,而多样性则表现为花>根皮>叶>果实。芽孢杆菌属为枸杞优势内生菌群,分布于所有组织中;抑菌实验结果表明有76.5%的内生菌对一种或多种病原菌的生长有抑制作用,芽孢杆菌属菌株R2、R7、L3和短波单胞菌属的R3拮抗番茄炭疽杆菌和玉米大斑病菌的能力较强,而多数菌株对大肠杆菌和金黄色葡萄球菌的抑制能力较弱。【结论】枸杞可培养内生细菌遗传多样性丰富,对植物病原菌有较强的抑制活性。     

Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars

We have isolated 576 endophytic bacteria from the leaves, stems, and roots of 10 rice cultivars and identified 12 of them as diazotrophic bacteria using a specific primer set of nif gene. Through 16S rDNA sequence analysis, nifH genes were confirmed in the two species of Penibacillus, three species of Microbacterium, three Bacillus species, and four species of Klebsiella. Rice seeds treated with these plant growth-promoting bacteria (PGPB) showed improved plant growth, increased height and dry weight and antagonistic effects against fungal pathogens. In addition, auxin and siderophore producing ability, and phosphate solubilizing activity were studied for the possible mechanisms of plant growth promotion. Among 12 isolates tested, 10 strains have shown higher auxin producing activity, 6 isolates were confirmed as strains with high siderophore producing activity while 4 isolates turned out to have high phosphate-solubilizing activity. These results strongly suggest that the endophytic diazotrophic bacteria characterized in this study could be successfully used to promote plant growth and inducing fungal resistance in plants.     

荧光假单胞菌抗生性代谢产物合成相关基因的研究现状

荧光假单胞菌(Pseudomonas fluorescens)是一种重要的植物根际促生菌,它能够产生藤黄绿脓菌素、2,4-二乙酰基藤黄酚、硝吡咯菌素、吩嗪-1-羧酸等抗生性次级代谢产物,可抑制多种病原物,在农作物土传病害的生物防治研究中具有重要意义.总结了荧光假单胞菌中已确立的抗生性次级代谢产物的合成机制,重点阐述了相关基因的结构、功能,以及利用生物工程技术对荧光假单胞菌进行遗传操作的最新进展,同时对荧光假单胞菌在生物防治中的应用和其作为生防菌剂的前景进行了展望.     

川蔓藻内生及根际细菌多样性与抑菌活性研究

该研究采用稀释涂布法结合形态观察、16S rRNA基因序列分析,对广西北海川蔓藻(Ruppia maritima)内生及根际细菌的物种多样性进行了研究,并采用琼脂扩散法和光度计法分析了其粗提物抑制马尔尼菲青霉菌活性。结果表明:从川蔓藻中分离到可培养内生细菌26株,根际可培养细菌31株。分别将内生细菌归属为10科12属13种,根际分离出细菌归属为9科14属19种,其中5株根际细菌可能为潜在新种。获得8株细菌对马尔尼菲青霉菌有抑制活性,总阳性率为25.0%。其中,菌株BGMRC 2015、BGMRC 2059、BGMRC 2043的粗提物表现出较强的抑制马尔尼菲青霉菌效果,其MIC分别为(1.800±0.045)、(1.881±0.061)、(1.604±0.021)mg·m L~(-1)。川蔓藻中可培养细菌具有较高的物种多样性,蕴藏着丰富的新物种资源,且富含抑菌活性良好的菌株。     

桐花树内生和根际细菌多样性及抗血栓活性研究

红树植物内生菌在红树共生体的物质循环、能量传递和健康维护等方面起着重要作用。为探究红树植物内生菌的多样性,进一步揭示内生菌在红树共生体的功能多样性提供菌种资源,该研究选择6种分离培养基和采用传统稀释涂布法对从广西北海滩涂上采集的桐花树组织和根际土壤样品进行分离,对获得的可培养细菌进行多样性分析,并通过体外溶栓实验筛选出具有抗血栓活性的菌株。结果表明:(1)基于16S rRNA基因序列系统进化分析,从桐花树组织和根际土壤中共获得125株细菌;分布于变形菌门(Proteobacteria)、放线菌门(Actinobacteria)和厚壁菌门(Firmicutes) 3个门27个科39个属74个种中,芽孢杆菌属为优势菌属,菌株数量占13.5%。(2)抗血栓活性实验表明,初筛获得18株具有抗血栓活性细菌,总阳性率为24.32%;将初筛有活性的菌株进行复筛和重复验证实验,进一步验证其活性,结果复筛出3株细菌B1850、B1989和B2632具有很强抗血栓活性。综上所述,广西北海滩涂上红树植物桐花树中存在丰富的可培养细菌资源,具有从中挖掘新的纤溶酶和开发溶血栓药物的潜力。     

Growth promotion and yield enhancement of peanut (Arachis hypogaea L.) by application of plant growth-promoting rhizobacteria

Although plant growth-promoting rhizobacteria (PGPR) have been reported to influence plant growth, yield and nutrient uptake by an array of mechanisms, the specific traits by which PGPR promote plant growth, yield and nutrient uptake were limited to the expression of one or more of the traits expressed at a given environment of plant–microbe interaction. We selected nine different isolates of PGPR from a pool of 233 rhizobacterial isolates obtained from the peanut rhizosphere on the basis of ACC-deaminase activity. The nine isolates were selected, initially, on the basis of germinating seed bioassay in which the root length of the seedling was enhanced significantly over the untreated control. All the nine isolates were identified as Pseudomonas spp. Four of these isolates, viz. PGPR1, PGPR2, PGPR4 and PGPR7 (all fluorescent pseudomonads), were the best in producing siderophore and indole acetic acid (IAA). In addition to IAA and siderophore-producing attributes, Pseudomonas fluorescens PGPR1 also possessed the characters like tri-calcium phosphate solubilization, ammonification and inhibited Aspergillus niger and A. flavus in vitro. P. fluorescens PGPR2 differed from PGPR1 in the sense that it did not show ammonification. In addition to the traits exhibited by PGPR1, PGPR4 showed strong in vitro inhibition to Sclerotium rolfsii. The performances of these selected plant growth-promoting rhizobacterial isolates were repeatedly evaluated for 3 years in pot and field trials. Seed inoculation of these three isolates, viz. PGPR1, PGPR2 and PGPR4, resulted in a significantly higher pod yield than the control, in pots, during rainy and post-rainy seasons. The contents of nitrogen and phosphorus in soil, shoot and kernel were also enhanced significantly in treatments inoculated with these rhizobacterial isolates in pots during both the seasons. In the field trials, however, there was wide variation in the performance of the PGPR isolates in enhancing the growth and yield of peanut in different years. Plant growth-promoting fluorescent pseudomonad isolates, viz. PGPR1, PGPR2 and PGPR4, significantly enhanced pod yield (23–26%, 24–28% and 18–24%, respectively), haulm yield and nodule dry weight over the control in 3 years. Other attributes like root length, pod number, 100-kernel mass, shelling out-turn and nodule number were also enhanced. Seed bacterization with plant growth-promoting P. fluorescens isolates, viz. PGPR1, PGPR2 and PGPR4, suppressed the soil-borne fungal diseases like collar rot of peanut caused by A. niger and PGPR4 also suppressed stem rot caused by S. rolfsii. Studies on the growth patterns of PGPR isolates utilizing the seed leachate as the sole source of C and N indicated that PGPR4 isolate was the best in utilizing the seed leachate of peanut, cultivar JL24. Studies on the rhizosphere competence of the PGPR isolates, evaluated on the basis of spontaneous rifampicin resistance, indicated that PGPR7 was the best rhizoplane colonizer and PGPR1 was the best rhizosphere colonizer. Although the presence of growth-promoting traits in vitro does not guarantee that an isolate will be plant growth promoting in nature, results suggested that besides ACC-deaminase activity of the PGPR isolates, expression of one or more of the traits like suppression of phytopathogens, solubilization of tri-calcium phosphate, production of siderophore and/or nodulation promotion might have contributed to the enhancement of growth, yield and nutrient uptake of peanut.     

Alleviation of salt stress by plant growth regulators and IAA producing bacteria in wheat

The action of phytohormone producing bacteria and plant growth regulators on germination and seedling growth of wheat under saline conditions were studied. Seed dormancy enforced by salinity (100 mM NaCl) was substantially alleviated and the germination was promoted by gibberellin, auxin, zeatin, and ethephon from 54 to 97%. The IAA producing bacterial strains Pseudomonas aureantiaca TSAU22, Pseudomonas extremorientalis TSAU6 and Pseudomonas extremorientalis TSAU20 significantly increased seedling root growth up to 25% in non-salinated conditions and up to 52% at 100 mM NaCl, compared to control plants. It is concluded that growth regulators considerably alleviated salinity-induced dormancy of wheat seeds. The facts mentioned above make it possible to recommend root colonizing bacteria that produce phytohormone to alleviate salt stress of wheat grown under conditions of soil salinity.     

无花果内生真菌的分离及其鉴定

从无花果(Ficus carica)的根、茎、叶中分离得到87株内生真菌,经显微形态特征观察鉴定为14个属,其中根部35株涉及8个属,茎部37株涉及10个属,叶部15株涉及3个属。结果表明,无花果的不同部位内生真菌的数量、分布和种群存有差异。     

8种贵州药用植物内生放线菌的分离及多样性研究

【背景】药用植物内生菌能产生多种活性较好的次级代谢产物,是新抗生素发现的重要来源。目前,药用植物内生菌的研究主要集中在内生真菌,内生放线菌研究相对较少,是一个尚未充分开发的新领域。【目的】研究贵州药用植物内生放线菌多样性,以期发现新种放线菌或产新活性物质的放线菌,为微生物药物研究奠定基础。【方法】药用植物样品表面消毒后用粉碎机粉碎,采用9种不同分离培养基分离放线菌;通过提取放线菌基因组DNA,PCR扩增16S rRNA基因序列,测定序列并提交EzBioCloud数据库进行序列比对,构建系统进化树,进行内生放线菌多样性分析。【结果】通过形态特征初步排重,从8种药用植物样品中共分离得到内生菌62株,测定其16S rRNA基因序列,序列比对结果表明其中的57株菌分布于放线菌域的5目8科16属,分别是短小杆菌属(Curtobacterium)、链霉菌属(Streptomyces)、微杆菌属(Microbacterium)、纤维单胞菌属(Cellulomonas)、植物杆菌属(Plantibacter)、原小单孢菌属(Promicromonospora)、动球菌属(Kineococcus)、壤球菌属(Agrococcus)、沼杆菌属(Patulibacter)、气微菌属(Aeromicrobium)、拉贝达氏菌属(Labedella)、叶居菌属(Frondihabitans)、冷杆菌属(Frigoribacterium)、节杆菌属(Arthrobacter)、动孢菌属(Kineosporia)、Amnibacterium属,其中短小杆菌属(Curtobacterium)为优势菌属,菌株M8JJ-5和M2KJ-4的16S rRNA基因序列分别与有效发表菌株AmnibacteriumkyonggienseKSL51201-037T(FJ527819)和Aeromicrobiumfastidiosum DSM10552T (Z78209)的相似性最高,相似性分别为97.29%和98.95%,可能为潜在新物种。【结论】贵州药用植物中蕴藏着多样性丰富的放线菌且具有从中发现新放线菌的潜力,能为新抗生素的发现提供菌种储备,也为今后对该地区放线菌资源的认识和开发利用提供理论依据。     

灯台树内生放线菌多样性及抗菌活性评价

从云南西双版纳采集7份灯台树样品,经过表面消毒,用4种分离培养基分离得到105株内生放线菌。经16S rRNA基因序列分析鉴定,分布于7个科9个属。利用5种植物病原真菌指示菌对所有菌株的发酵液进行抗菌活性检测。结果显示,有12.4%、14.3%、11.4%、12.4%、8.6%的菌株分别对镰刀霉、疫霉、赤星霉、苹果炭疽、白色念珠菌有抗性。对3株具有广谱抗菌活性的菌株进行再次发酵和抗菌活性复筛,结果显示这3株的抗菌活性稳定,并可能含有生物碱类化合物。