一株人参内生1-氨基环丙烷-1-1羧酸(ACC)脱氨酶活性细菌的筛选、鉴定及其对宿主生长的影响

【目的】从人参内生细菌中获得具有1-氨基环丙烷1-羧酸(ACC)脱氨酶活性的菌株,并进行促生效果的验证。【方法】结合初筛和复筛的方法筛选具有ACC脱氨酶活性的人参内生菌株;采用Ashby培养基和固氮酶基因验证其固氮潜能;菌碟法及钼锑抗比色法测定其解磷能力;CAS方法检测产生铁载体能力;通过室内及田间试验测定菌株对人参生长的促进作用。通过形态学、生理生化测定及16S rRNA序列分析明确菌株的分类地位。【结果】从120株人参内生菌中获得了一株具有较高ACC脱氨酶活性的菌株JJ8-3,其酶活性为α-酮丁酸6.7μmol/(mg·h);且具有解磷特性、固氮潜能和产生铁载体能力;能明显促进人参种子及根部的生长;经鉴定菌株JJ8-3为荧光假单胞菌(Pseudomonas fluorescens)。【结论】获得了一株具有ACC脱氨酶活性的人参内生细菌,将为其在促进植物生长中的应用和研究奠定基础。     

ACC脱氨酶活性菌株ACC 30的分离、 鉴定及其促生作用

【目的】筛选具有1-氨基环丙烷-1-羧酸(简称ACC)脱氨酶活性的菌株,并探索该类菌的促生作用,有助于研发微生物肥料,实现农业增产。【方法】以ACC为唯一氮源,从土壤中富集和分离ACC脱氨酶产生菌;测定ACC脱氨酶的比活力,对酶活性最强的菌株根据形态和培养特征、生理生化特征及16S rDNA序列进行分类鉴定;分别采用菌液培养接种法与菌液浸种接种法初步研究该菌株对紫花苜蓿幼苗生长的促生作用。【结果】筛选得到6株ACC脱氨酶阳性细菌,其中菌株ACC 30酶活性最高,为0.217 U/mg,根据培养特征观察和生理生化指标测定结果,结合16S rDNA序列比对分析,确定ACC 30为产气肠杆菌(Enterobacter aerogenes)。促生试验表明,ACC 30可促进紫花苜蓿幼苗根伸长,菌液培养接种法与菌液浸种接种法两种处理方法下ACC 30分别使幼苗根相对伸长135%、136%,促生作用均明显且基本一致。但是,两种方法处理下ACC 30均抑制幼苗下胚轴伸长。【结论】筛选获得ACC脱氨酶活性菌株ACC 30,其酶活性较高且促生作用明显,有望进一步研发成为微生物肥料。     

美洲大蠊肠道内生细菌的分离及其初步抑菌活性研究

分离鉴定美洲大蠊肠道内生细菌,并初步研究了其抑菌活性。采用平板稀释法对美洲大蠊肠道内生细菌进行分离纯化,分离菌株通过形态学观察和16S rDNA基因序列BLAST比对进行种属鉴定。以金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌、肺炎克雷伯菌、大肠埃希氏菌4株细菌为对象,采用牛津杯法初步鉴定各分离菌株的抑菌活性。BLAST比对分析结果表明:从15只美洲大蠊肠道内分离鉴定出125株内生细菌,分属12科20属。初步抑菌活性显示:部分菌株对金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌、肺炎克雷伯菌、大肠埃希氏菌有明显抑菌活性。美洲大蠊肠道内生菌种类繁多,而且较多菌株具有抑菌活性。     

具ACC脱氨酶活性的碱蓬内生细菌的分离、鉴定及其生物学特性

【目的】具有1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性的盐生植物碱蓬内生细菌的分离及生物学特性的研究有助于探索内生细菌与宿主植物耐盐性的关系。【方法】采用研磨法从健康碱蓬植株的根、茎、叶中分离具有ACC脱氨酶活性的碱蓬内生细菌,根据形态特征、生理生化、API鉴定系统和16S rRNA对菌种进行鉴定,并分别测定了菌株产ACC脱氨酶、铁载体、吲哚乙酸、赤霉素、脱落酸、蛋白酶及溶磷、固氮和拮抗病原菌的特性。【结果】将分离得到的内生细菌LP11、SS12、TW1和TW2分别鉴定为栖稻假单胞菌(Pseudomonas oryzihabitans)、假单胞菌(Pseudomonas sp.)、成团泛菌(Pantoea agglomerans)和恶臭假单胞菌(Pseudomonas putida),4株菌除具有较高的ACC脱氨酶活力之外,还可不同程度地产生铁载体、吲哚乙酸、赤霉素和脱落酸,且均有溶磷作用,但无固氮能力及蛋白酶活力,唯有菌株SS12对萝卜枯萎病菌(Fusariumoxysporum f.sp.conglutinans)和黄瓜枯萎病菌(F.oxysporum f.sp.cucumerinum)具有拮抗作用。【结论】从盐生植物碱蓬中分离到的假单胞菌属和泛菌属内生细菌,具有丰富多样的生物学特性。     

具ACC脱氨酶活性大豆根瘤内生菌的筛选、抗性及促生作用北大核心CSCD

【目的】从大豆根瘤中筛选具ACC(1-氨基环丙烷-1-羧基)脱氨酶活性的内生细菌,对活性菌株的抗盐碱性、系统分类地位以及代表菌株的促生长作用进行研究,为发掘和应用抗逆、促生优良菌种资源提供理论基础。【方法】以ACC作为唯一氮源测定菌株产ACC脱氨酶特性,采用标准曲线法测定α-丁酮酸含量,比色法定量测定ACC脱氨酶活力,固体平板筛选法对活性菌株进行抗性分析,通过菌体形态及生理生化特性测定、16S rRNA基因序列同源性分析鉴定菌株分类地位,采用盆栽试验验证代表菌株的促生作用。【结果】从河南省13个市(地区)36个点采集的大豆根瘤中筛选出8株ACC脱氨酶内生细菌,其中菌株DD132的酶活性最高(15.712 U/mg)。筛选菌株可耐受4%–6%NaCl,其中菌株DD165、DD132可耐受9%NaCl盐浓度。在pH 11时5株(DD14、DD132、DD67、DD141、DD131)生长良好,说明这些菌株有较强耐碱性。8株产ACC脱氨酶菌株分属于4属,即芽孢杆菌属(Bacillus)、肠杆菌属(Enterobacter)、寡养单胞菌属(Stenotrophomonas)和泛菌属(Pantoea)。接种试验表明内生菌DD132对小麦幼苗生长具有明显促生长作用。【结论】大豆根瘤内具ACC脱氨酶高活性菌株有较强耐盐碱性,其中菌株DD132对小麦幼苗生长有明显促生长作用。为发掘和应用抗逆、促生的优良菌种资源提供理论基础。     

中华补血草内生与根际具ACC脱氨酶活性细菌的筛选及其生物多样性

【目的】获得江苏沿海滩涂盐生药用植物中华补血草内生及根际具有1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性的细菌,研究其遗传多样性和潜在促生活性。【方法】从中华补血草和根际土壤分离筛选具有ACC脱氨酶活性的菌株,对其ACC脱氨酶活性定量检测,通过16S r RNA基因序列分析确定菌株系统发育地位。同时研究其固氮、溶磷、产植物生长素吲哚乙酸(IAA)及耐盐能力。【结果】分离筛选获得18株具有ACC脱氨酶活性的内生与根际细菌,定量检测发现其中有13株菌的ACC脱氨酶含量在20 nmolα-KA/(mg Pr·h)以上,有11株菌可以固氮,7株菌能够解磷,9株菌产生IAA。菌株的Na Cl盐耐受范围多数在0–13%之间。16S r RNA基因测序表明,活性菌株分属于7个属,多样性丰富,节杆菌属(Arthrobacter)为优势类群。其中菌株KLBMP 5180为节杆菌属的潜在新种。【结论】江苏沿海滩涂盐生药用植物中华补血草共生环境中具有丰富多样的具ACC脱氨酶活性的菌株,并存在潜在新物种资源,具有进一步研究价值。     

高寒草地珠芽蓼内生拮抗固氮菌Z19的鉴定及其固氮功能

【目的】从东祁连山高寒草地珠芽蓼内生细菌中筛选和鉴定具有固氮能力和拮抗能力的内生细菌。【方法】采用16S rRNA基因序列同源性分析和生理生化指标测定方法对该菌株进行鉴定。【结果】从珠芽蓼中分离获得的21株内生细菌中6株内生菌具有固氮能力,76.2%具有抑菌能力,其中5株内生细菌对5种以上的病原菌有抑制作用;菌株Z19具有较强固氮能力和分解纤维素的能力,其纤维素溶解圈直径与菌落直径比达3.33,产生的纤维素酶活性为0.31 U,且对辣椒立枯丝核病菌(Rhizoctonia solani)、油菜菌核病菌(Sclerotinia sclerotiorum)、番茄灰霉病菌(Botrytis cinerea)、小麦根腐病菌(Bipolaris sorokiniana)和番茄早疫病菌(Alternaria solani)具有较强拮抗能力;经PCR扩增和测序,获得了菌株Z19的固氮基因(nifH)序列和16S rRNA基因序列,在GenBank中的登录号分别为EU693340和EU236746;菌株Z19呈革兰氏阳性,杆状,产芽孢。【结论】结合生理生化特征及16S rRNA基因序列同源性比较,鉴定其为枯草芽孢杆菌(Bacillus subtilis)。     

多功能固氮菌筛选及其在土壤生态修复中的应用

以固氮酶和ACC(1-aminocyclopropane-1-carboxylate,1-氨基环丙烷-1-羧酸)脱氨酶活性为指标筛选功能菌株,研制菌剂,用于修复生荒地土壤生态。采用乙炔还原法测定菌株固氮酶活性;比色法定量测定ACC脱氨酶活性;PCR扩增得到菌株16S r RNA,通过序列分析、比对研究菌株的系统发育地位;通过形态、生理生化特征和16S r DNA序列比对鉴定菌种。结果显示,筛选到固氮酶活性大于9 nmol/(h·mg)的菌株共计47株,其中43株的固氮酶活性大于阳性对照固氮菌剂生产菌株圆褐固氮菌ACCC11103。筛选到产生ACC脱氨酶的菌株20株,ACC脱氨酶活性为0.326-21.980μmol/(h·mg)。将筛选到的功能菌株接种小白菜测试促生效应,其中47株使普通白菜(Brassica campestris)鲜重增加。16S r RNA测序鉴定显示筛选到的51株功能菌株系统发育地位上属于农杆菌、节杆菌、芽孢杆菌、伯克氏菌、鞘氨醇杆菌、屈挠杆菌、剑菌、肠杆菌、溶杆菌、微球菌、类芽孢杆菌、叶杆菌、假单胞菌、根瘤菌、中华根瘤菌、芽孢乳杆菌等16属31种。选取高效菌株7012、7134、7144和7164作为核心菌株制备了固氮多功能菌剂,应用于生荒地,试验地土壤微生物数量显著提高,土壤蔗糖酶、过氧化氢酶、脲酶、磷酸酶、纤维素酶、木聚糖酶活性极显著高于未施用菌剂的对照处理。多功能固氮菌制剂对于提高生荒地土壤生物活性、改善土壤生态环境具有重要作用和应用价值。     

贵州喀斯特地区具ACC脱氨酶活性细菌的分离和鉴定

1-氨基环丙烷-1-羧酸(ACC)脱氨酶能够降解乙烯前体,从而有助于植物生长。具有ACC脱氨酶活性细菌在旱胁迫下具有植物促生作用。本研究从贵州地区选取典型喀斯特地貌区域159份土壤样品中分离并鉴定出具有ACC脱氨酶活性的细菌188株。利用16S r DNA测序分析将这些菌株归为14属63种,优势菌属为假单胞菌属和伯克氏菌属,分别有ACC脱氨酶活性的细菌18种和17种。对63种菌株的ACC脱氨酶活性定量检测,酶活最高的菌株是AL30ADF120(Cupriavidus oxalaticus),为3.639 U/mg。据我们所知,这是第一个有关喀斯特地区土壤中的具有ACC脱氨酶活性细菌名录,为将来研究ACC脱氨酶活性细菌的植物促生作用奠定了基础。     

沙棘根瘤内生细菌中抑菌促生菌株的筛选和鉴定

【背景】植物内生细菌可产生具有抑菌和促生活性的物质,既能抑制植物病原菌对寄主植物的侵染,也能促进植物的生长。沙棘根瘤内生细菌是根瘤内除共生固氮的弗兰克氏菌外,与沙棘共生的一大类微生物。研究具有抑菌和促生活性的植物内生菌,可为微生物菌肥的研究提供理论基础。【目的】筛选具有优良抑菌和促生活性的沙棘根瘤内生细菌,初步研究其抑菌和促生活性,并对菌株进行鉴定。【方法】采用双层琼脂法、琼脂扩散法、双层平板对峙法、牛津杯法进行沙棘根瘤拮抗性内生细菌的筛选。选取抑菌活性较高的内生细菌,分别采用Salkowski比色法、ChromeAzurolS(CAS)平板检测法和钼锑抗比色法进行产吲哚乙酸、铁载体及溶磷能力的测定。采用发酵液灌根法测定沙棘根瘤内生细菌SR308对黄瓜促生作用的盆栽效果。通过形态和培养特征、生理生化试验及16S rRNA基因序列分析法对菌株TT201和SR308进行鉴定。【结果】从131株沙棘根瘤内生细菌中筛选出9株具有较强抑菌活性的内生细菌,其中菌株TT201抑菌性最佳、抑菌谱广;菌株SR308的促生活性最好,其发酵液对黄瓜的生长具有较强的促进作用。对具有较强抑菌和促生活性的菌株TT201和SR308进行鉴定的结果表明,菌株TT201为侧孢短芽孢杆菌(Brevibacilluslaterosporus),菌株SR308为蕈状芽孢杆菌(Bacillusmycoides)。【结论】获得2株具有优良抑菌和促生活性的沙棘根瘤内生细菌,为进一步开发微生物农药及菌肥提供了资源。     

Bacterial Biosynthesis of 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase and Indole-3-Acetic Acid (IAA) as Endophytic Preferential Selection Traits by Rice Plant Seedlings

In this study, bacteria were isolated from the rhizosphere and inside the roots and nodules of berseem clover plants grown in the field in Iran. Two hundred isolates were obtained from the rhizosphere (120 isolates), interior roots (57 isolates), and nodules (23 isolates) of clover plants grown in rotation with rice plants. Production of chitinase, pectinase, cellulase, siderophore, salicylic acid, hydrogen cyanide, indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylate (ACC) deaminase, solubilization of phosphate, antifungal activity against various rice plant pathogen fungi, N₂ fixation, and colonization assay on rice seedlings by these strains was evaluated and compared (endophytic isolates vs. rhizosphere bacteria). The results showed both the number and the ability of plant growth-promoting (PGP) traits were different between endophytic and rhizosphere isolates. A higher percentage of endophytic isolates were positive for production of IAA, ACC deaminase, and siderophore than rhizosphere isolates. Therefore, it is suggested that clover plant may shape its own associated microbial community and act as filters for endophyte communities, and rhizosphere isolates with different (PGP) traits. We also studied the PGP effect of the most promising endophytic and rhizosphere isolates on rice seedlings. A significant relationship among IAA and ACC deaminase production, the size of root colonization, and plant growth (root elongation) in comparison with siderophore production and phosphate solubilization for the isolates was observed. The best bacterial isolates (one endophytic isolate and one rhizosphere isolate), based on their ability to promote rice growth and colonize rice roots, were identified. Based on 16S rDNA sequence analysis, the endophytic isolate CEN7 and the rhizosphere isolate CEN8 were closely related to Pseudomonas putida and Pseudomonas fluorescens, respectively. It seems that PGP trait production (such as IAA, ACC deaminase) may be required for endophytic and rhizosphere competence as compared to other PGP traits in rice seedlings under constant flooded conditions. The study also shows that the presence of diverse rhizobacteria with effective growth-promoting traits associated with clover plants may be used for sustainable crop management under field conditions.     

Native bacterial endophytes promote host growth in a species-specific manner; phytohormone manipulations do not result in common growth responses

Background

All plants in nature harbor a diverse community of endophytic bacteria which can positively affect host plant growth. Changes in plant growth frequently reflect alterations in phytohormone homoeostasis by plant-growth-promoting (PGP) rhizobacteria which can decrease ethylene (ET) levels enzymatically by 1-aminocyclopropane-1-carboxylate (ACC) deaminase or produce indole acetic acid (IAA). Whether these common PGP mechanisms work similarly for different plant species has not been rigorously tested.

Methodology/ Principal Findings

We isolated bacterial endophytes from field-grown Solanum nigrum; characterized PGP traits (ACC deaminase activity, IAA production, phosphate solubilization and seedling colonization); and determined their effects on their host, S. nigrum, as well as on another Solanaceous native plant, Nicotiana attenuata. In S. nigrum, a majority of isolates that promoted root growth were associated with ACC deaminase activity and IAA production. However, in N. attenuata, IAA but not ACC deaminase activity was associated with root growth. Inoculating N. attenuata and S. nigrum with known PGP bacteria from a culture collection (DSMZ) reinforced the conclusion that the PGP effects are not highly conserved.

Conclusions/ Significance

We conclude that natural endophytic bacteria with PGP traits do not have general and predictable effects on the growth and fitness of all host plants, although the underlying mechanisms are conserved.     

Detection and Characterization of ACC Deaminase in Plant Growth Promoting Rhizobacteria

The enzyme 1-aminocyclopropane-1-carboxylate deaminase converts ACC, the precursor of the plant hormone ethylene to α-ketobutyrate and ammonium. The enzyme has been identified in few soil bacteria, and is proposed to play a key role in plant growth promotion. In this study, the isolates of plant growth promoting rhizobacteria were screened for ACC deaminase activity based on their ability to grow on ACC as a sole nitrogen source. The selected isolates showed the presence of other plant growth promoting characteristics such as IAA production, phosphate solubilization and siderophore production. The role of ACC deaminase in lowering ethylene production under cadmium stress condition was also studied by measuring in vitro ethylene evolution by wheat seedlings treated with ACC deaminase positive isolates. Nucleic acid hybridization confirmed the presence of ACC deaminase gene (acdS) in the bacterial isolates.     

A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase

Aims: 1‐Aminocyclopropane‐1‐carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. Methods and Results: A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat‐resistant polypropylene chimney‐top 96‐well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC‐utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC‐utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Conclusions: Determination of bacterial ACC consumption by the PCR‐plate ninhydrin–ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. Significance and Impact of the Study: The PCR‐plate ninhydrin–ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates.     

焦化厂地肤根内解芘细菌的筛选及促生潜力

从多环芳烃耐受植物根内分离具多环芳烃降解功能的内生细菌并研究其促生特性,为内生菌协同宿主植物修复多环芳烃污染土壤提供基础.以长期受多环芳烃污染的焦化厂区生长的地肤为材料,从其根内分离出以芘和1-氨基环丙烷-1-羧酸(ACC)为唯一碳源和氮源的内生细菌8株.通过芘降解试验,筛选得到3株高效芘降解内生细菌KSE4、KSE7和KSE8,经鉴定分别为芽孢杆菌属、假单胞菌属和鞘氨醇菌属.通过液体培养试验,研究了3株菌在芘胁迫下产ACC脱氨酶的能力和对地肤种子萌发的影响.结果表明: 随着芘浓度(0~15 mg·L^-1)的升高,ACC脱氨酶活性降低,其中KSE7的效果最好,在芘浓度为15 mg·L^-1时,地肤发芽率和芽长分别比对照提高了44.8%和61.1%,在地肤-微生物修复焦化厂污染土壤的修复中具有一定的应用潜力.     

旱地小麦根际细菌中产生1-氨基环丙烷-1-羧酸(ACC)脱氨酶菌株的分离和鉴定

采用富集定向筛选法,从旱地小麦的根际土壤中分离到2株产生1-氨基环丙烷-1-羧酸(ACC)脱氨酶的菌株AS和CS。经测定菌株AS和CS的ACC脱氨酶的比活力分别为0.018 6 U/mg和0.016 7 U/mg蛋白。根据培养特征观察和生理生化指标测定结果,结合16S rDNA碱基序列测定和系统发育同源性分析,确定菌株AS和菌株CS分别属于霍氏肠杆菌(Enterobacter hormaechei)和变形斑沙雷氏菌(Serratia proteamaculans)。     

秸秆发酵中乳酸菌复合系SFC-2对杂菌的抑制作用

目的]为探明秸秆发酵用乳酸菌复合系SFC-2对杂菌的抑制作用.[方法]从秸秆自然发酵体系中.利用平板划线法、纸片扩散法和变性梯度凝胶电泳(DGGE)筛选出13株菌作为研究该乳酸菌复合系抑菌作用的对象.[结果](1)通过16S rDNA序列分析显示13株分离菌中9株菌的近缘种为Enterobactercloacae,Klebsiella oxytoca,Bacillus cereus,Klebsiella pneumoniae,Citrobacter freundii,Klebsiellaterrigena、Citrobacter sp.,这些均为常见的致病菌或条件致病菌.(2)将筛选出的致病菌作为被测试菌株,用SFC-2的发酵上清液处理表明:SFC-2复合系的抑菌作用高于复合系分离的单一菌株和单一菌株人工组合的抑制作用.(3)SFC-2培养的14～48 h内动态检测结果表明:发酵上清液的有机酸含量有显著差异,但抑菌活性差异不显著;发酵上清液经热处理后抑菌活性不发生变化,蛋白酶K处理后抑菌活性部分丧失.     

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。