盐穗木根际产ACC脱氨酶耐盐菌株的筛选及鉴定

从新疆五家渠103团盐碱地盐穗木根际的3份土样中筛选出10株产ACC脱氨酶的菌株,通过对这10株菌株的ACC脱氨酶活性和耐盐能力的测定,结果显示其中有4株菌株ACC脱氨酶活性较高,且能在盐(NaCl)浓度12%生长。根据ACC脱氨酶活性以及耐盐性能的测定结果,选择W5、W8、W9这3株菌株进行鉴定,通过生理生化实验、Biolog鉴定、16S rDNA测序分析后,鉴定结果显示W5、W8为乳酪短杆菌(Brevibacterium casei),W9为普沙根瘤菌(Rhizobium pusense)。     

含ACC脱氨酶活性的复合菌株提高苜蓿抗盐碱能力研究

为探究含ACC脱氨酶活性的复合菌株对盐碱胁迫下紫花苜蓿的促生效果,以3株含ACC脱氨酶活性的植物根际促生菌拜式不动杆菌Y-2(Acinetobacter beijerinckii)、粘质沙雷氏菌BC-3(Serratia marcescens)和产气肠杆菌CE-1(Enterobacter aerogenes)为供试菌株,通过菌株拮抗反应测试,筛选可以混合使用的菌株,测定单菌株和复合菌株的耐盐碱特性和促生特性,并进行盐碱土盆栽试验,评价其对紫花苜蓿的促生效果。结果表明,复合菌株Y-2+CE-1和BC-3+CE-1间无拮抗反应,可以混合使用,在p H值为10和盐浓度为7%的条件下生长良好。单菌株和复合菌株均具有ACC脱氨酶活性、分泌植物生长素(IAA)能力和溶磷能力,且复合菌株的促生特性优于单菌株。此外,接种单菌株和复合菌株均能不同程度地促进紫花苜蓿的生长,其中复合菌株Y-2+CE-1和BC-3+CE-1促生效果显著,与对照相比,株高、根长、地上干重、根干重分别提高20.3%和20%、51.4%和45.9%、47%和36.4%、68.8%和52%,且促生效果优于单菌株。可见,含ACC脱氨酶活性的复合菌株能缓解盐碱胁迫,对紫花苜蓿具有明显的促生效果。本研究结果为研制复合菌肥,治理和利用盐碱地奠定了理论基础。     

一株百合内生细菌 Burkholderia sp. FJb-2的分离鉴定及其体外抑菌促生效应

从北京市房山区百合资源圃采集红芯百合(Fangio)植株样品,通过组织块分离法分离内生细菌;进一步以百合灰霉病菌—灰葡萄孢菌( Botrytis cinerea)ACCC36423、百合叶尖干枯病菌—葡萄座腔菌( Botryosphaeria dothidea)ACCC37263和百合枯萎病菌—尖孢镰刀菌( Fusarium oxysporum)F01139等几种主要百合病原真菌为指示菌,采用平板对峙法筛选具有拮抗活性的拮抗菌株;并对拮抗菌株进行产 1-氨基环丙烷 -1-羧酸(ACC)脱氨酶活性、产生长素(IAA)、溶磷、产铁载体等多种植物促生特性测定;最后通过形态观察及 16S rRNA基因序列分析,初步鉴定其种属。结果从百合茎部分离获得一株内生细菌菌株 FJb-2;该菌株对灰葡萄孢菌、葡萄座腔菌和尖孢镰刀菌均有较高拮抗活性,对病原真菌生长的抑制率范围为 60%～ 81%;该菌株产 ACC脱氨酶活性为     

菠菜内生固氮菌的分离及其功能特性研究

内生固氮菌具有促生、病原菌的防治及生物固氮等作用,并且占据着植物组织内有利于营养供应和微环境适宜的生态位,是重要的微生物菌种资源。从菠菜根内分离筛选优势内生固氮菌7株,其中假单胞菌属(Pseudomonas)5株,红球菌属(Rhodococcus)和黄杆菌属(Flavobacterium)各1株。菌株Pseudomonas sp.BCE6和Pseudomonas sp.BC-E8固氮酶活性较高,分别为(13.19±0.32)和(12.11±0.96)C2H4nmol/(mg protein·h),与圆褐固氮菌(ACCC11103)的固氮酶活性具有极显著性差异(P0.01);菌株Pseudomonas sp.BC-E7具有固氮酶活性[(8.42±0.03)C2H4nmol/(mg protein·h)]、产IAA能力[(59.58±4.15)μg/m L]、ACC脱氨酶活性[(5.067±0.376)μmol/(mg protein·h)],拮抗立枯丝核菌ACCC36124(Rhizoctonia solani)、麦类赤霉病ACCC36272(Gibberella zeae)等4种常见土传病害。菠菜内生固氮菌Pseudomonas sp.BC-E7是一株多功能内生菌,具有较好的生产应用前景。     

水稻根际促生细菌促生生理活性评价

为了考察水稻特异性PGPR菌株各生理活性对水稻幼苗初生根数、次生根数、茎干重和根干重的影响,筛选优良PGPR菌株,采用改良的CAS检测法、Salkowski比色法、钼蓝比色法和1-氨基环丙烷-1-羧酸(ACC)脱氨酶检测法分别测定菌株产铁载体、产吲哚乙酸(IAA)、溶磷能力和ACC脱氨酶活性,利用Hoagland半固体培养基和土壤盆栽法培养水稻幼苗。结果表明,IAA产量低的菌株对水稻幼苗初生根数、次生根数和茎干重的增长有明显的促进效果;根干重明显随铁载体产量增加而增加;溶磷活性高的菌株对水稻幼苗茎干重和根干重的增加均有明显的促进作用;低ACC脱氨酶活性的菌株促进水稻幼苗初生根的生长,活性高的菌株促进次生根数的增加。根据半固体试验结果,选择7株具有明显促生效果的菌株进行盆栽试验,结果证实,这些菌株均具有显著的促生效应。     

不同盐分条件下植物促生菌对燕麦和黑麦草幼苗的促生效应

从盐生植物根际土中分离得到4株含1-氨基环丙烷-1-羧酸(ACC)脱氨酶的植物促生菌(PGPR),通过无菌育种袋栽培试验,考查其在不同盐分条件下对燕麦和黑麦草幼苗的促生效应。结果表明,4株菌对5 g/kg或10 g/kg NaCl盐分胁迫下的燕麦和黑麦草幼苗均表现出显著地缓解促生效应,其中假单胞菌属S1最显著,10 g/kg NaCl比无NaCl时促生作用更大。4株PGPR的ACC脱氨酶活性与植物生长参数(根长和下胚轴长)之间具有极显著的正相关性(Pearson相关系数>0.81)。     

三株产ACC脱氨酶的植物根际促生菌产酶条件优化及促生作用研究

为提高植物根际促生菌（PGPR）菌株产1-氨基环丙烷-1-羧酸（ACC）脱氨酶的能力,对3株PGPR菌株进行了最佳产酶条件优化及玉米促生试验。在单因素试验的基础上,研究了p H、温度、ACC底物浓度、Na Cl浓度、摇床转速对ACC脱氨酶酶比活力的影响,以p H、温度、ACC底物浓度为主要因素,进行响应面最佳产酶工艺优化。最终得到W5菌株的产酶条件为p H 8、温度32℃、ACC脱氨酶底物浓度6.0 mmol/L;BS2-2菌株的产酶条件为p H 10、温度30℃、ACC脱氨酶底物浓度6.0 mmol/L;5#菌株产酶条件为pH 9、温度30℃、ACC脱氨酶底物浓度6.0 mmol/L;较未优化前酶比活力分别提高了6.6、2、4倍。在盆栽试验中,W5菌株促生效果最为显著。利用高产ACC脱氨酶的PGPR能够促进玉米生长,可为制作为微生物复合菌剂奠定基础。     

根际ACC脱氨酶活性细菌的分离及其促生作用研究

从不同来源的植物根际土壤中分离出能以1-氨基环丙烷-1-羧酸(ACC)为唯一氮源生长的细菌4株,经测定均具有较高的ACC脱氨酶活性,并进一步研究了菌株对植物的促生长作用。首先进行了种子根长试验,利用菌株菌悬液处理玉米和番茄种子,结果表明,经过细菌1101、2101、4201和GXGD002处理,种子的生根长度较对照组均有明显增长,玉米种子根长相对伸长率依次为78.73%、61.72%、47.37%、47.08%;番茄种子根长相对伸长率依次为61.19%、55.84%、54.34%、51.19%。其次进行了番茄穴盘栽培试验,分别在播种后40 d和55 d收苗,测定植株的株高、茎粗、根长等生长指标,结果表明,菌株4201和GXGD002具有显著促进生长作用,其处理组植株在株高、根长、根系活力等方面较对照组均有明显增强。     

产ACC脱氨酶植物根际促生菌的筛选及其对修复植物高羊茅生长的影响

以1-氨基环丙烷-1-羧酸(ACC)为唯一氮源,从石油污染的土壤中分离得到一株具有ACC脱氨酶活性的菌株D5A.该菌在以ACC为唯一氮源条件下,其ACC脱氨酶比活力为0.084 U/mg.另外,D5A还具有产生吲哚乙酸(IAA)、耐盐以及溶磷的特性.在液体培养条件下该菌株产IAA高达112 mg/L,在90 g/kg盐含量的培养基中仍能够正常生长且具有较强的溶解矿物磷能力.同时该菌对酸碱具有良好的适应性,在初始pH4～10的LB培养基中生长良好.种子发芽试验表明在3 g/kg和6 g/kg浓度NaC1的逆境条件下,该菌株能显著提高高羊茅种子的发芽率和芽长.最后,通过对其进行生理生化特性和16S rDNA序列分析,该菌株初步鉴定为克雷伯氏菌(Klebsiellasp.).     

马铃薯内生固氮菌的分离及其促生特性研究

内生固氮菌是一类重要的植物促生菌,占据着植物组织内有利于营养供应和微环境适宜的生态位,能更好的发挥促生功能,促生机制包括固氮、解磷、产植物生长素、产ACC脱氨酶、产铁载体及拮抗病害等。本文从连作3年马铃薯根筛选获得8株内生固氮菌,其中短芽孢杆菌属(Brevibacterium)3株,芽孢杆菌属(Bacillus)3株和泛菌属(Pantoea)2株。促生特性研究发现:菌株Brevibacterium sp.GWR4具有较高固氮酶活性[16.206nmol C2H4/(mg蛋白质·h)],与圆褐固氮菌的固氮酶活性具有极显著性差异(P0.01);菌株Pantoea spp.GWR2、GWR3具有极高的产IAA能力,分别为186.07、162.21μg/m L,GWR2兼具产ACC脱氨酶活性[3.74μmol/(mg蛋白质·h)],GWR3兼具溶解无机磷的能力;菌株Brevibacterium sp.GWR5可以拮抗尖孢镰刀菌MLSOF(Fusarium xysporum)和茄病镰刀菌MLS-QB(Fusarium solani),兼具溶解有机磷和产IAA能力;Bacillus spp.GWR7、GWR8兼具固氮和产IAA能力。盆栽试验显示,分别接种Pantoea sp.GWR3,Brevibacterium spp.GWR4、GWR5和Bacillus spp.GWR7、GWR8后小白菜鲜重显著高于未接菌对照处理。可见,这5株菌兼具多种促生特性且对小白菜具有较好的促生效果,有望进一步研究开发成为微生物肥料生产菌种。     

盐胁迫油菜根际中含假单胞菌（Pseudomonas spp.1）的氨基环丙烷-1-羧酸 (ACC) 脱氨基酶特征研究

When exposed to biotic or abiotic stress conditions,plants produce ethylene from its immediate precursor 1-aminocyclopropane-1-carboxylate(ACC),leading to retarded root growth and senescence.Many plant growth-promoting rhizobacteria contain the enzyme ACC deaminase and this enzyme can cleave ACC to form α-ketobutyrate and ammonium,thereby lowering levels of ethylene.The aim of this study was to isolate and characterize ACC deaminase-producing bacteria from the rhizosphere of salt-stressed canola(Brassica napus L.).Out of 105 random bacterial isolates,15 were able to utilize ACC as the sole source of nitrogen.These 15 isolates were also positive for indole acetic acid(IAA) production.Phylogenetic analysis based on partial 16 S rDNA sequences showed that all isolates belonged to fluorescent Pseudomonas spp.In the canola rhizosphere investigated in this study,Pseudomonas fluorescens was the dominant ACC deaminase-producing species.Cluster analysis based on BOX-A1R-based repetitive extragenic palindromic-polymerase chain reaction(BOX-PCR) patterns suggested a high degree of genetic variability in ACC deaminase-producing P.fluorescens strains.The presence of indigenous ACC-degrading bacteria in the rhizosphere of canola grown in saline soils indicates that these bacteria may contribute to salinity tolerance.     

Drought-Tolerant Pseudomonas spp. Improve the Growth Performance of Finger Millet (Eleusine coracana (L.) Gaertn.) Under Non-Stressed and Drought-Stressed Conditions

Application of plant growth-promoting bacteria(PGPB)is an environmentally sustainable option to reduce the effects of abiotic and biotic stresses on plant growth and productivity.Three 1-aminocyclopropane-1-carboxylic acid(ACC)deaminase-producing drought-tolerant bacteria were isolated from a rain-fed agriculture field in the Central Himalaya of Kumaun region,Uttarakhand,India and evaluated for their efficiency in improving finger millet(Eleusine coracana(L.)Gaertn.)plant growth under non-stressed and drought-stressed conditions.These bacteria withstood a substrate metric potential of -1.0 MPa(30% polyethylene glycol 8000)and therefore were considered drought-tolerant.These strains were identified as Pseudomonas spp.by fatty acid methyl ester analysis and 16S rRNA gene sequencing.The ACC deaminase activity of these strains was characterized at the biochemical level,and the presence of acd S gene,the structural gene for ACC deaminase,was confirmed by the polymerase chain reaction.Two sets of pot trials in glass house were set up,one for normal(non-stressed)and the other for drought-stressed conditions.After 5 weeks,one set of plants was subjected to drought stress for 5 d,while the other set continued to be watered.The same growth parameters were recorded for both sets of plants after 40 d of plant growth.The results of pot trials showed that treatments inoculated with ACC deaminase-producing bacterial strains significantly improved the growth performance of finger millet plants and foliar nutrient content as compared to uninoculated treatments under both non-stressed and drought-stressed conditions.In addition,a significant increase in antioxidant activity was observed,wherein bacterial stain inoculation improved plant fitness by protecting it from oxidative damage induced by drought.     

Characterization of phosphate solubilizing fluorescent pseudomonads from the rhizosphere of Aloe vera (L.)

Soil phosphorous (P) deficiency is a major constraint to plant production which is overcome by adding inorganic-phosphate as chemical fertilizers. Fluorescent pseudomonads are the diverse group of bacteria able to mobilize sparingly soluble phosphate form. Total three hundred seven fluorescent Pseudomonas isolates were obtained from the Aloe barbadensis (Miller) rhizosphere. These Pseudomonas strains were further evaluated in vitro for their ability to solubilize phosphate and to produce indole acetic acid (IAA), hydrogen cyanide (HCN), siderophore and 1-aminocyclopropane 1- carboxylate (ACC) deaminase. Fifty three (36.8%) isolates produced IAA and 52 (36.1%) isolates produced siderophores whereas 36 (25.0%) and 31 (21.5%) isolates produced HCN and ACC deaminase, respectively. A positive correlation existed between siderophore and ACC deaminase producers. Cluster analysis showed rhizosphere as the major factor influencing the ecological distribution and physiological characterization of phosphate solubilizing bacteria (PSB). Based on partial 16S rRNA gene sequencing PSB were identified as Pseudomonas putida, Pseudomonas sp. and Pseudomonas plecoglossicida with highest phosphate solubilization ability. In conclusion, these phosphate solubilizing fluorescent pseudomonads would help in understanding their role in phosphorus solubilization and identification of potent phosphorus solubilizers from the rhizosphere of commercially grown A. barbadensis.     

Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian mustard (Brassica juncea L. Czern.)

Eleven cadmium-tolerant bacterial strains were isolated from the root zone of Indian mustard (Brassica juncea L. Czern.) seedlings grown in Cd-supplemented soils as well as sewage sludge and mining waste highly contaminated with Cd. The bacteria also showed increased tolerance to other metals including Zn, Cu, Ni and Co. The isolated strains included Variovorax paradoxus, Rhodococcus sp. and Flavobacterium sp., and were capable of stimulating root elongation of B. juncea seedlings either in the presence or absence of toxic Cd concentrations. Some of the strains produced indoles or siderophores, but none possessed C₂H₂-reduction activity. All the strains, except Flavobacterium sp. strain 5P-3, contained the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which hydrolyses ACC (the immediate precursor of plant hormone ethylene) to NH₃ and α-ketobutyrate. V. paradoxus utilized ACC as a sole source of N or energy. A positive correlation between the in vitro ACC deaminase activity of the bacteria and their stimulating effect on root elongation suggested that utilization of ACC is an important bacterial trait determining root growth promotion. The isolated bacteria offer promise as inoculants to improve growth of the metal accumulating plant B. juncea in the presence of toxic Cd concentrations and for the development of plant-inoculant systems useful for phytoremediation of polluted soils.     

Screening of Multi-Trait Rhizobacteria for Improving the Growth,Enzyme Activities,and Nutrient Uptake of Tea (Camellia sinensis)

The use of plant growth-promoting rhizobacteria (PGPR) as agricultural inputs for increasing crop production needs the selection of efficient bacteria with plant growth-promoting (PGP) attributes. Therefore, the purpose of this study was to evaluate the effects of 20 multi-traits bacteria on tea growth, nutrient uptake, chlorophyll contents, and enzyme activities under field conditions for over 3 years. These isolates were screened in vitro for their PGP traits such as the production of indole acetic acid (IAA), nitrogenase activity, phosphorus (P) solubilization, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Screening of rhizobacteria that show multiple PGP traits suggests that they stimulated overall plant growth, including shoot development and leaf yield, improving macro- and micro-nutrient uptake, chlorophyll contents, and activities of enzymes of tea plant. Use of strains with multiple PGP traits could be a more effective approach and have great potential for the environmentally-friendly tea production.     

Role of methylotrophic bacteria in managing abiotic stresses for enhancing agricultural production

Abiotic stresses are a significant factor that considerably limits plant growth and productivity. Methylotrophs are an essential group of bacteria that utilize volatile carbon compounds, are prolific colonizers of different plant parts, and play a vital role in plant growth promotion(PGP) under stress conditions.Numerous rhizospheric and phyllosphere methylotrophs have been reported to exhibit PGP activities with superior stress-tolerating capacity against drought,heavy metal, salinity, high and...     

Characterization of Burkholderia sp. XTB‐5 for Phenol Degradation and Plant Growth Promotion and Its Application in Bioremediation of Contaminated Soil

A key issue when researching land degradation is the pollution of soils. For bioremediation of contaminated soil, Burkholderia sp. XTB‐5 cells were obtained from soil and grown on mineral salt medium with initial phenol concentrations of 650 mg L⁻¹ and 850 mg L⁻¹, which were found to degrade more than 98% of phenol content in less than 4 days. About 90% of phenol content (with initial concentration of 250 mg kg⁻¹ soil) was removed from soil inoculated with XTB‐5 cells in 6 days. More than 90% of phenol content was removed within 20 days after co‐introduction of XTB‐5 cells and plants to sterilized soil in a greenhouse or to natural soil in field trials. But under the same conditions, individual introduction of plants to sterilized soil in the greenhouse reduced phenol content by about 50% and introduction to natural soil in field trials reduced phenol content by about 38%, suggesting that phytoremediation of phenol is often inefficient and microorganisms can efficiently degrade this pollutant. In addition, strain XTB‐5 was found to solubilize phosphate and produce 1‐Aminocyclopropane‐1‐Carboxylate (ACC) deaminase and siderophore. Strain XTB‐5 promoted plant growth in both phenol‐absent and phenol‐spiked soil under greenhouse and field conditions. Considering that ACC deaminase is beneficial to plant growth under adverse environmental conditions, plant growth promotion by XTB‐5 in phenol‐contaminated soil is not only due to XTB‐5 cell‐degradation of phenol and reduced phytotoxicity but also to production of ACC deaminase. Hence, Burkholderia sp. XTB‐5 presents an attractive microorganism for phytoremediation of contaminated soil and agronomic application. Copyright © 2016 John Wiley & Sons, Ltd.     

多功能植物根际促生菌 DD3 的功能特性及对大蒜幼苗的促生效果

【目的】植物根际促生菌 (plant growth promoting rhizobacteria, PGPR) 能够促进植物生长、防治病害、增加作物产量,具有较好的环境兼容性、不易引起抗性、效果持久稳定等优势。筛选高效多功能菌,探索抗病促生机理,对开发防控大蒜土传病害的微生物技术,发展“高产、优质、高效、生态、安全”的大蒜产业具有十分重要的理论和现实意义。 【方法】以实验室前期从山东省金乡县连作大蒜根部分离的 70 株细菌为供试菌株,筛选对大蒜根腐病病原真菌拟枝孢镰刀菌 (Fusarium sporotrichioides) ACCC37402 和大蒜叶枯病病原真菌链格孢属菌株 (Alternaria sp.) D14011-1 具有较好拮抗效果的菌株;采用 16S rDNA 基因序列同源性分析,对其进行初步鉴定;通过测定菌株对抗生素的敏感性、水解蛋白能力、产铁载体能力、产 HCN 能力等,研究菌株的拮抗特性;通过测定菌株的固氮能力、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;通过温室盆栽试验,进行多功能菌菌悬液及其无菌发酵液对大蒜幼苗生长的影响以及抗病效果研究。 【结果】菌株 DD3 对大蒜根腐病病原真菌 ACCC37402 和大蒜叶枯病病原真菌 D14011-1 具有较好拮抗效果,抑菌率均为 45%;经 16S rDNA 基因序列同源性分析,初步确定为芽孢杆菌属 (Bacillus) 菌株。其它功能特性研究结果显示:菌株 Bacillus sp. DD3 具有水解蛋白能力、产 HCN 能力、产铁载体能力、固氮能力、溶解有机磷和无机磷的能力;对氨苄青霉素钠、硫酸卡那霉素等 11 种抗生素无耐受性,不具有分泌吲哚乙酸 IAA 能力和产 ACC 脱氨酶能力。盆栽试验显示,接种 Bacillus sp. DD3 菌悬液对大蒜根腐病的防治效果较好,防控率达 80%。接种 Bacillus sp. DD3 及其无菌发酵液后能够促进大蒜幼苗的生长,其中接种菌悬液效果更佳。 【结论】菌株 Bacillus sp. DD3 兼具多种功能特性,对大蒜根腐病具有较好的拮抗效果,同时可以显著促进大蒜幼苗生长,有望进一步研究开发成为微生物肥料生产菌种。