一株高效甘蔗内生固氮菌NN08200的鉴定及其对甘蔗的促生长作用

【背景】生产上过高的氮肥投入是我国农业可持续发展的重要限制因子之一。利用生物固氮是减少氮肥施用量最为有效的途径,植物内生固氮菌资源的挖掘和利用对我国农业可持续发展具有重要实践意义。【目的】筛选高效甘蔗内生固氮菌,并对其联合固氮效率及促生长功能进行评价。【方法】从广西甘蔗茎基部组织分离筛选到一株内生固氮菌株NN08200,利用乙炔还原法测定固氮酶活性,通过菌落PCR扩增nif H基因确定菌株为固氮菌;通过菌株培养性状和菌体形态观察、Biolog细菌鉴定系统和16SrRNA基因序列分析确定该菌株的分类;采用盆栽接种测定菌株对甘蔗的实际促生长作用,并利用15N同位素稀释法测定其相对固氮效率。【结果】菌株NN08200的固氮酶活性达到2445nmolC2H4/(h·m L),菌株的nif H基因长度为339bp,与甘蔗内生固氮醋酸杆菌Gluconacetobacter diazotrophicus PAL5菌株的nif H相似性达99%;根据菌株培养性状和菌体形态观察、Biolog细菌鉴定系统和16SrRNA基因序列分析结果,菌株NN08200属于泛菌属(Pantoeasp.)细菌;盆栽接种菌株NN08200能显著提高甘蔗幼苗的株高和干重,15N同位素分析结果表明接种该菌株甘蔗植株的根、茎和叶从空气中获得氮素的百分率分别为7.49%、15.02%和10.79%,其联合固氮效率显著优于甘蔗内生固氮模式菌株G. diazotrophicus PAL5,利用后者接种的甘蔗根、茎和叶从空气中获得氮的百分率分别为3.53%、9.44%和4.87%。【结论】菌株Pantoea sp. NN08200是高效甘蔗内生固氮菌,其固氮促生长效果明显高于G. diazotrophicus PAL5菌株,可望研发成为优良固氮微生物肥料生产菌种,并可进一步用于甘蔗联合固氮菌作用机理的相关研究。     

间作大豆对甘蔗根际土壤细菌及固氮菌多样性的影响

为探讨间作大豆(Glycine max)对甘蔗(Saccharum officinarum)根际土壤细菌及固氮细菌多样性的影响, 收集和开发固氮菌资源, 筛选高效甘蔗联合固氮体系, 选用3个甘蔗栽培品种‘ROC22’、‘GT21’、‘B8’与大豆品种‘Guizao 2’进行间种栽培, 采用巢式PCR特异扩增细菌16S rRNA基因片段和固氮细菌nifH基因片段, 并结合变性梯度凝胶电泳(DGGE)技术, 对间作大豆的甘蔗根际土壤细菌及固氮细菌进行系统演化和多样性分析。聚类分析结果显示, 间作大豆改变了甘蔗根际土壤细菌及固氮细菌原来的群落组成结构, 尤其对固氮菌群落组成的改变更大, 但对群落物种的优势度影响较小。Shannon-Wiener多样性指数和Simpson多样性指数分析结果表明, 甘蔗-大豆间作显著影响甘蔗根际土壤中细菌和固氮菌的多样性, 其中对固氮细菌多样性的影响较大。不同甘蔗品种的根际土壤细菌和固氮菌在间作大豆条件下表现出不同的多样性, ‘ROC22’和‘GT21’间作处理甘蔗根际土壤固氮细菌的Shannon-Wiener多样性指数显著高于单作处理, 而‘ROC22’与大豆间作处理的甘蔗根际土壤固氮菌多样性最为丰富。在大豆生长盛期, 间作处理的甘蔗根际土壤细菌多样性最为丰富, 不同处理间的差异也最大, 随后下降。总体来看, 甘蔗-大豆间作显著地影响根际土壤细菌和固氮菌的群落结构和群落多样性, 有助于对甘蔗合理间作栽培模式的认识和筛选高效甘蔗联合固氮体系。     

禾本科植物联合固氮研究及其应用现状展望

综述了近年来从禾本科植物体内和根际发现的内生固氮菌和根际固氮菌的种类、特征及对宿主的促生机理,以及固氮菌接种剂在农业生产中的应用现状和存在的问题,指出影响联合固氮菌接种效果的主要因素有土著微生物的竞争;植物基因型差异和环境条件的变化,如结合态氮(氨、亚硝酸盐、硝酸盐等)对固氮酶的合成阻遏和较高的氧分压对联合固氮菌的固氮效率影响．提出了发掘和利用禾本科植物的生物固氮潜力的努力方向：从自然界分离筛选获得广谱高效固氮菌株;应用基因工程构建耐铵、泌铵型联合固氮菌;诱导禾本科植物形成固氮根瘤;充分发挥植物内生固氮菌的优势．     

4株茶树根际促生菌菌株的鉴定及促生作用

【背景】根际促生菌可以促进植物生长、提高植物抗性。茶树根际具有特殊的根土微生物生境,可以获得具促生作用的有益微生物。【目的】探究4株茶树根际促生菌菌株的分类地位及促生作用,筛选优良的根际促生菌菌株。【方法】通过形态、生理生化特征、16S rRNA基因序列同源性比对鉴定4株茶树根际促生菌,采用钼锑抗比色法测定溶磷量,通过比色法测定ACC脱氨酶活性、CAS法测定产铁载体能力、Salkowski法测定产IAA (Indoleacetic acid)的能力进行促生作用研究,通过盆栽实验测试白菜、空心菜、苋菜及水稻的株高及鲜重以分析促生效应。【结果】鉴定KKS-6-N1为放射型土壤杆菌(Agrobacteriumradiobacter), KKS-7-N7为铜绿假单胞菌(Pseudomonas aeruginosa),GD3为Pseudomonashunanensis,GD12为弯曲芽孢杆菌(Bacillusflexus)。固氮菌株KKS-6-N1可产铁载体;固氮菌株KKS-7-N7具有解磷及产铁载体能力,分泌的IAA含量高达101.29mg/L;解钾菌株GD3具溶磷能力,分泌的ACC脱氨酶酶活为8.09μmol/(mg·h),相对铁载体含量为0.31;具固氮解钾性能的菌株GD12分泌的ACC脱氨酶活性为14.46μmol/(mg·h)。盆栽试验表明,4个菌株对白菜、空心菜、苋菜的株高和鲜重均有明显促进作用,尤以GD3效果更甚。【结论】茶树根际促生菌菌株Pseudomonas hunanensis GD3促生作用显著,具有开发成微生物菌肥的潜力。     

三株固氮类芽孢杆菌的特点及其对中国青菜的产量和土壤酶活性的影响（英文）

【目的】本研究分析三株固氮菌PGPR性状特征及其对中国青菜产量和土壤酶活的影响。【方法】氮(N)-修复(固氮)细菌被认为是一种能够促进植物生长和增产的施氮方式。在本研究中,我们用无氮培养基分离出了30株根际固氮细菌:11株来自小麦根际,16株来自中国青菜根际和3株来自莲花根际。基于16S r DNA序列分析,对小麦、中国青菜和莲花等植物根际中属于类芽孢杆菌属的主要固氮细菌进行研究。【结果】本研究从这30株固氮菌中筛选出三株属于类芽孢杆菌属(Paenibacillus)的细菌,分别命名为P-4、W-7和L-3,它们的固氮酶活性不但高于对照组(圆褐固氮菌),而且可以有效抑制两种或三种植物病原菌的生长,即核盘菌(Sclerotinia sclerotiorum)、玉蜀黍赤霉(Gibberella zeae)和棉花黄萎病菌(Verticillium dahliae)。菌株W-7还具有溶解难溶磷的能力,中国青菜在接种菌株W-7和L-3后,其鲜重显著增加,同时改变了田间土壤蔗糖酶、磷酸酶和过氧化氢酶的活性;而接种了菌株P-4对植物的生长和酶活性没有显著的影响。【结论】土壤蔗糖酶、磷酸酶和过氧化氢酶活性与中国青菜的生物量呈正相关。同时,菌株W-7和L-3具有促进植物产量和提高土壤质量的良好潜力。     

三株固氮类芽孢杆菌的特点及其对中国青菜的产量和土壤酶活性的影响

【目的】本研究分析三株固氮菌PGPR性状特征及其对中国青菜产量和土壤酶活的影响。【方法】氮(N)-修复(固氮)细菌被认为是一种能够促进植物生长和增产的施氮方式。在本研究中,我们用无氮培养基分离出了30株根际固氮细菌:11株来自小麦根际,16株来自中国青菜根际和3株来自莲花根际。基于16S r DNA序列分析,对小麦、中国青菜和莲花等植物根际中属于类芽孢杆菌属的主要固氮细菌进行研究。【结果】本研究从这30株固氮菌中筛选出三株属于类芽孢杆菌属(Paenibacillus)的细菌,分别命名为P-4、W-7和L-3,它们的固氮酶活性不但高于对照组(圆褐固氮菌),而且可以有效抑制两种或三种植物病原菌的生长,即核盘菌(Sclerotinia sclerotiorum)、玉蜀黍赤霉(Gibberella zeae)和棉花黄萎病菌(Verticillium dahliae)。菌株W-7还具有溶解难溶磷的能力,中国青菜在接种菌株W-7和L-3后,其鲜重显著增加,同时改变了田间土壤蔗糖酶、磷酸酶和过氧化氢酶的活性;而接种了菌株P-4对植物的生长和酶活性没有显著的影响。【结论】土壤蔗糖酶、磷酸酶和过氧化氢酶活性与中国青菜的生物量呈正相关。同时,菌株W-7和L-3具有促进植物产量和提高土壤质量的良好潜力。     

甘蔗联合固氮的回顾与展望

在巴西,某些甘蔗品种能通过生物固氮获得生长所需氮素。对甘蔗根际固氮菌和内生固氮菌的研究曾引领了非豆科植物联合固氮的研究。内生固氮菌Gluconacetobacter diazotrophicus表现出很多特性,与Herbaspirillum seropedicae、H.rubrisubalbicans、Nitrospirillum amazonense和Paraburkholderia tropica组成的固氮菌剂能联合甘蔗固氮并促进甘蔗生长。近年来的研究发现分类上属于Bradyrhizobium和Rhizobium等属的根瘤菌在与甘蔗联合的核心固氮菌群之中,以不结瘤的方式在甘蔗体内活跃地表达固氮酶基因。综述了这些甘蔗联合固氮菌的特色研究并探讨优化甘蔗联合固氮的策略。     

辣椒根际促生菌的分离筛选及抗病促生特性研究

获得辣椒根际促生菌(Plant growth promoting rhizobacteria,PGPR)并探究其抗病促生特性。采用固氮、无机磷和有机磷培养基从江苏省徐州市采集的辣椒根际土壤中分离筛选根际促生菌株(PGPR),通过形态特征及16S rDNA序列分析进行菌株鉴定,对菌株的固氮、解磷、分泌3-吲哚乙酸(IAA)能力及对4种辣椒病害病原菌抗病能力进行探究。得到13株辣椒PGPR菌株,经鉴定分别属于Bacillus、Pseudomonas、Lelliottia、Siccibacter、Achromobacter、Microbacterium和Paenibacillus;13株PGPR菌株均有固氮功能;其中7株可解有机磷,分别属于Lelliottia、Bacillus、Siccibacter、Microbacterium、Paenibacillus;5株可解无机磷,分别属于Lelliottia、Bacillus、Siccibacter、Pseudomonas;3株具有分泌IAA能力,分别属于Lelliottia、Siccibacter、Bacillus;5株具有抗病能力,分别属于Bacillus、Lelliottia、Siccibacter。辣椒根际土壤含有在农业生产上具有潜在的应用价值的多功能根际促生菌。     

植物根际促生枯草芽孢杆菌JC01筛选

【目的】筛选高效植物根际促生细菌,阐明产挥发性有机化合物(VOC)菌株JC01的促生机制。【方法】选取从植物根际中分离得到的838株细菌,以固氮、解(溶)磷以及分泌嗜铁素、吲哚乙酸(IAA)活性为指标,对其促生能力进行赋值评估,将赋值在3分以上的107株细菌进行指纹图谱分析,挑选其中不同簇的20株促生潜力细菌进行温室实验,以评价赋值体系与温室促生效果之间的关系,进一步探究具有较好促生效果菌株JC01的作用机理。【结果】共筛选出了来源于指纹图谱中不同簇的4株具有较好促生效果的菌株,细菌的平板活性赋值与促生效果之间的相关系数r大于0.6。其中,菌株JC01分泌的具有促生作用的VOC能够增强番茄植株IAA信号通路关键基因的表达,减弱脱落酸(ABA)、乙烯(ETH)信号通路关键基因的表达。JC01经16S r RNA基因鉴定为枯草芽孢杆菌。【结论】细菌的平板活性赋值与促生效果之间存在较高的正相关性,枯草芽孢杆菌JC01可能通过产生VOC对番茄生长进行调控。     

花生根际多功能固氮菌的分离及其耐盐碱特性研究

旨在获得具有固氮、溶磷及分泌IAA的多功能耐盐碱根际促生菌(PGPR),提高盐碱地花生产量。采用平板涂布法对盐碱地花生根际固氮菌进行了分离,结合16S rDNA序列鉴定和乙炔还原法测定固氮酶活。共获得22个固氮菌株,其固氮酶活性为125.82-346.32 nmol C_2H_4/(h·mL)。所有菌株均具有产ACC脱氨酶及溶解难溶性磷源活性。其ACC脱氨酶活性为0.12-1.26U/mg;溶解磷酸三钙量为1.45-53.58 mg/L,溶解磷酸铁量为1.45-8.27 mg/L,溶解磷酸铝量为1.1-22.82 mg/L。其中,10株具有分泌IAA的能力,分泌量在1.36-17.80μg/mL,占供试菌株的55%。对这10株促生菌进行NaCl、pH耐受性测定,结果显示最大pH耐受力为9,最大NaCl耐受力为1.5 mol/L。这10菌株具有较强的耐盐碱性,具有在盐碱地中应用的潜力。     

Isolation,partial characterization,and the effect of plant growth-promoting bacteria (PGPB) on micro-propagated sugarcane in vitro

We report the isolation of nitrogen fixing, phytohormone producing bacteria from sugarcane and their beneficial effects on the growth of micropropagated sugarcane plantlets. Detection of the nitrogen fixing bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) method indicated the presence of up to 10⁶ bacteria per gram dry weight of stem and 10⁷ bacteria per gram dry weight of root of field-grown sugarcane. Two nitrogen fixing bacterial isolates were obtained from stem (SC11, SC20) and two from the roots (SR12, SR13) of field-grown plants. These isolates were identified as Enterobacter sp. strains on the basis of their morphological characteristics and biochemical tests. The isolate SC20 was further characterized by 16S rRNA sequence analysis, which showed high sequence similarity to the sequence of Enterobacter cloacae and Klebsiella oxytoca. All the isolates produced the phytohormone indoleacetic acid (IAA) in pure culture and this IAA production was enhanced in growth medium containing tryptophan. The bacterial isolates were used to inoculate micro-propagated sugarcane in vitro where maximum increase in the root and shoot weight over control was observed in the plantlets inoculated with strain SC20. By using the¹⁵N isotope dilution technique, maximum nitrogen fixation contribution (28% of total plant nitrogen) was detected in plantlets inoculated with isolate SC20.     

一株高效甘蔗内生固氮细菌GXS16的鉴定及其对甘蔗的促生长作用

[背景] 我国甘蔗生产中氮肥过量施用严重,导致生产成本居高不下,充分发挥甘蔗与内生固氮菌的联合固氮作用,减少氮肥施用量,对促进我国甘蔗产业可持续发展具有重要意义。[目的] 筛选优势甘蔗内生固氮菌,对其基本特性、联合固氮效率及促生长功能进行评价。[方法] 从甘蔗根系分离到一株内生固氮菌GXS16,利用乙炔还原法测定固氮酶活性,通过PCR扩增nifH基因确定菌株为固氮菌;通过形态观察、Biolog检测和16S rRNA基因序列分析等对菌株进行分类;通过接种盆栽甘蔗检测菌株的促生长作用,采用¹⁵N同位素稀释法检测菌株相对固氮效率。[结果] 菌株GXS16固氮酶活性为2.42μmol-C₂H₄/（h·mL）,根据菌株培养性状和菌体形态观察、Biolog检测、16S rRNA、nifH、acdS基因序列分析结果,菌株GXS16属于伯克氏菌属（Burkholderia）;菌株GXS16还具有1-氨基环丙烷-1-羧酸脱氨酶（1-Aminocyclopropane-1-Carboxylate Deaminase,ACC）活性及合成生长素吲哚乙酸（Indoleacetic Acid,IAA）、降解无机磷的功能;接种GXS16处理甘蔗植株的株高比对照增长15%以上,干重增长20%以上,¹⁵N同位素测定显示甘蔗根、茎、叶从空气中获得氮的百分比分别为7.69%、15.64%和8.72%,效率显著优于模式菌株G.diazotrophicus PAL5。[结论] Burkholderia sp.GXS16是一株高效甘蔗内生固氮菌,具有良好应用前景。     

Contiguous organization of nitrogenase genes in a heterocystous cyanobacterium

The organization of the three structural nitrogen fixation (nif) genes that encode nitrogenase (nif K and nif D) and nitrogenase reductase (nif H) have been examined in a number of cyanobacteria. Hybridization of Anabaena 7120 nif gene probes to restriction endonuclease-digested genomic DNA has shown (a) that cyanobacteria incapable of N₂ fixation have no regions of DNA with significant homology to the three nif probes, (b) that Pseudanabaena sp., a nonheterocystous cyanobacterium, has a contiguous nif KDH gene cluster, and (c) that in contrast with other heterocystous cyanobacteria, Fischerella sp. has a contiguous nif KDH gene cluster.     

Isolation, molecular characterization and growth-promoting activities of endophytic sugarcane diazotroph Klebsiella sp. GR9

A group of endophytic diazotrophs were isolated from surface-sterilized roots and stems of different sugarcane varieties in the Tamilnadu region of India. From these, four isolates were selected, based on the highest acetylene reduction activity. Gene-specific PCR amplification confirmed the presence of nif-D genes in those isolates. The 16S rRNA sequence of isolates GR4 and GR7 had a 99.5% sequence similarity to the Pseudomonas sp. pDL01 (AF125317) and 16S rDNA sequence of isolate GR3 had a 100% similarity to that of Burkholderia vietnamiensis (AY973820). The 16S rDNA sequence of isolate GR9 was 99.79% similar to that of the Klebsiella pneumoniae type strain (KPY17657). Colonization by the isolates was confirmed using micropropagated sugarcane and sterile rice seedlings. Isolate GR9, identified as Klebsiella pneumoniae, was consistently more active in reducing acetylene as compared with the other isolates. The effects of GR9 and the sugarcane diazotroph Gluconacetobacter diazotrophicus were compared in inoculated micropropagated sugarcane plantlets. The effects of K. pneumoniae GR9, and four other diazotrophs, G. diazotrophicus, Herbaspirillum seropedicae, Azospirillum lipoferum 4B, and Burkholderia vietnamiensis in inoculated rice seedlings were compared. GR9 alone or in combination with the other diazotrophs performed best under pot conditions. The combined effects of nitrogen fixation and endophytic colonization of this diazotroph may be useful for the development of bio-inoculants.     

Diversity of Culturable Plant Growth-Promoting Bacterial Endophytes Associated with Sugarcane Roots and Their Effect of Growth by Co-Inoculation of Diazotrophs and Actinomycetes

Application of environmentally friendly agents to reduce the use of chemicals and to enhance growth of plants is an ultimate goal of sustainable agriculture. The use of plant growth-promoting endophytes has become of great interest as a way to enhance plant growth and additionally protect plants from phytopathogens. In this study, 135 isolates of endophytic bacteria including actinomycetes were isolated from roots of commercial sugarcane plants cultivated in Thailand and were characterized for plant growth-promoting (PGP) traits. Based on morphological and 16S rRNA sequence analysis, the endophytes were distributed into 14 genera of which the most dominant species belong to Bacillus, Enterobacter, Microbispora, and Streptomyces. Two strains of endophytic diazotrophs, Bacillus sp. EN-24 and Enterobacter sp. EN-21; and two strains of actinomycetes, Microbispora sp. GKU 823 and Streptomyces sp. GKU 895, were selected based on their PGP traits including 1-aminocyclopropane-1-decarboxylate deaminase, indole-3-acetic acid, nitrogen fixation, phosphate solubilization, and siderophore production for evaluation of sugarcane growth enhancement by individual and co-inoculation. Sixty days after co-inoculation by endophytic diazotrophs and actinomycetes, the growth parameters of sugarcane plants were significantly greater than that of individual and un-inoculated plants. The results indicated that these endophytes have high potential as PGP agents that could be applied to promote sugarcane growth and could be developed as active added value biofertilizers in the future.     

In silico characterization and transcriptomic analysis of nif family genes from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.     

The Mo- and Fe-nitrogenases of the endophyte Kosakonia sp. UYSO10 are necessary for growth promotion of sugarcane

Sugarcane is a multipurpose crop primarily used to produce sugar, energy and bioethanol. It requires high amounts of N-fertilization for optimal growth, which increases production costs and environmental degradation. The contribution of biological nitrogen fixation to Uruguayan commercial sugarcane cultivars was demonstrated previously, and diazotrophic bacteria that were isolated from the stems were characterized and identified. From this collection, the isolate UYSO10 related to the Kosakonia genus (formerly Enterobacter) was described as a plant growth-promoting endophyte of sugarcane plants. To evaluate the effect of the inoculation of wild-type and nitrogenase-deficient strains of Kosakonia sp. UYSO10 on sugarcane growth promotion under non-sterile conditions. Kosakonia sp. UYSO10 was inoculated onto sugarcane setts for plant growth promotion greenhouse experiments. Single and double mutants resulting to the nitrogenase-encoding genes (nifH, anfH) were constructed, and the phenotypes were evaluated in vitro and in vivo. Kosakonia sp. UYSO10 is able to promote sugarcane growth under non-sterile conditions, that strain UYSO10 harbors two functional nitrogenases and the inactivation of both nitrogenase-encoding genes diminish its capacity of promoting growth on sugarcane. All together, the results obtained showed that the biological nitrogen fixation ability of Kosakonia sp. UYSO10 is required for sugarcane growth promotion.     

Phenotypic and molecular characterization of native Azospirillum strains from rice fields to improve crop productivity

Beneficial microorganisms have been considered as an important tool for crop improvement. Native isolates of Azospirillum spp. were obtained from the rhizospheres of different rice fields. Phenotypic, biochemical and molecular characterizations of these isolates led to the identification of six efficient strain of Azospirillum. PCR amplification of the nif genes (nifH, nifD and nifK) and protein profile of Azospirillum strains revealed inter-generic and inter-specific diversity among the strains. In vitro nitrogen fixation performance and the plant growth promotion activities, viz. siderophore, HCN, salicylic acid, IAA, GA, zeatin, ABA, NH₃, phosphorus metabolism, ACC deaminase and iron tolerance were found to vary among the Azospirillum strains. The effect of Azospirillum formulations on growth of rice var. Khandagiri under field condition was evaluated, which revealed that the native formulation of Azospirillum of CRRI field (As6) was most effective to elevate endogenous nutrient content, and improved growth and better yield are the result. The 16S rRNA sequence revealed novelty of native Azospirillum lipoferum (As6) (JQ796078) in the NCBI database.