烟草根际可培养微生物多样性及防病促生菌的筛选

【背景】根际微生物在植物根部生态系统中扮演着重要角色,影响着植物的营养吸收和健康生长。【目的】了解常年不发病烟田烤烟品种K326根际可培养微生物的多样性,筛选具有防病促生功能的菌株,为烟草病害绿色防控提供资源。【方法】采用传统培养方法对烟草根际土壤中的细菌和真菌进行分离鉴定,评价菌株的促生特性及病原菌拮抗能力,并进一步验证典型菌株对盆栽烟苗的促生效果。【结果】共获得261株微生物菌株,包括160株细菌和101株真菌。经分子鉴定,细菌中以变形菌门(Proteobacteria)和厚壁菌门(Firmicutes)为主要类群;真菌中以子囊菌门(Ascomycota)和毛霉菌门(Mucoromycota)为主要类群。在属水平上,细菌以假单胞菌属(Pseudomonas)和芽孢杆菌属(Bacillus)为主,真菌以曲霉属(Aspergillus)和青霉属(Penicillium)为主。从不同种水平上进一步选择44株细菌为代表菌株,发现它们均具有不同程度的吲哚-3-乙酸(Indole-3-Acetic Acid,IAA)产生能力,9株能够溶解有机磷,16株能够溶解无机磷,13株产生铁载体,14株产...     

植物根际促生菌促生特性研究进展

根际微生物组是决定农作物健康状况的关键因素之一,也是调节农作物与生物和非生物环境相互作用的重要因素。植物根际促生菌(plant growth-promoting rhizobacteria, PGPR)为农作物宿主提供了多种有益作用,通过化学交流以复杂的方式与农作物、土壤相互作用,进而促进农作物生长。本文综述了PGPR对农作物的促生机制、PGPR与农作物的互作及其在农业实践中的应用,并展望了PGPR在农业实践中应用的发展趋势,以期为今后PGPR的应用和研究提供新的思路和理论支撑。     

植物根围促生细菌（PGPR）作用机制的研究进展

结合生防作用和促生作用2个方面综述近年植物根围促生菌（PGPR）作用机制研究进展。     

青稞根腐病防病促生细菌的筛选及其菌剂防效

【背景】由燕麦镰孢(Fusarium avenaceum)和麦根腐平脐蠕孢(Bipolaris sorokiniana)等病原真菌引起的青稞(Hordeum vulgare L. var. nudum Hook. f.)根腐病普遍发生,对藏区农业生产和发展产生了极大威胁。【目的】从青稞健康植株根际土壤中筛选具优良防病促生能力的菌株,通过研究菌株间的互作效应以筛选优良菌剂配伍,制作防效优良的防病促生菌剂,以期为青稞根腐病的防治及青稞增产提供优质的菌剂资源。【方法】采用选择性培养基筛选促生菌株,采用平板对峙法筛选拮抗菌株;采用灌根法和叶面接种法测定菌株致病性;采用乙炔还原法测定固氮能力,钼蓝比色法测定溶磷能力,采用高效液相色谱法测定分泌吲哚乙酸能力;采用16S rRNA基因序列分析鉴定菌种;田间测定菌剂防效,并采用TOPSIS综合分析法进行综合评价。【结果】共分离出具固氮、溶磷能力菌株23株,并从中筛选得到6株拮抗能力良好的菌株,其中5株溶有机磷、3株溶无机磷、3株固氮菌,而且均分泌吲哚乙酸;测定其致病性发现对青稞、燕麦、油菜、豌豆等采样区常见作物均无致病性;经鉴定分别为枯草芽孢杆菌(Ba...     

瓜菜枯萎病及生防根际微生物组研究进展

枯萎病是顽固性土传病害,称为瓜菜中的“癌症”,已成为制约我国瓜菜产业可持续健康发展的瓶颈问题。本文简要介绍瓜菜枯萎病危害,并从细胞壁降解酶、毒素、信号传导和致病基因等方面综述瓜菜枯萎病灾变机制,然后从根际微生物组自身与病原菌、土壤层面和植物层面等重点阐述了根际微生物组防治和抵御瓜菜枯萎病的机理,最后对枯萎病发生和抑制关键因子挖掘、核心微生物组构建及根际微生物组分子机制等进行了展望,期望生防微生物防治病害发生进入一个崭新且高效的阶段,为加快提升作物抗逆性机理研究提供一定思路。     

三种蓝莓根际细菌的分离及其对蓝莓苗生长发育的影响

【目的】从3种蓝莓根际土壤中分离细菌,探究蓝莓根际土壤细菌多样性,并筛选具有产酸、促生长、抑菌性能的菌株,为蓝莓专用微生物肥料的研究提供优质菌株资源和理论基础。【方法】选用5种培养基分离3种蓝莓根际土壤细菌,并进行16S rRNA基因测序和系统发育分析。筛选产酸、产吲哚-3-乙酸（indole-3-acetic acid,IAA）和铁载体、固氮、溶磷和抑制灰葡萄孢生长的菌株,挑选最适菌株制备菌剂进行蓝莓苗盆栽实验验证促生能力,并检测菌剂对蓝莓元素吸收和根际土壤肥力的影响。【结果】从3种蓝莓根际土壤分离得到124株细菌,挑选70株代表性菌株进行16S rRNA基因测序,分布于3个门21个属,其中芽孢杆菌属（Bacillus）、假单胞菌属（Pseudomonas）、链霉菌属（Streptomyces）和红球菌属（Rhodococcus）为优势分离菌群。代表性菌株中,21.4%的菌株能产酸,21.4%的菌株产吲哚-3-乙酸,47.1%的菌株具有固氮潜力,65.7%的菌株具有解磷能力,14.3%的菌株能产铁载体。少量菌株同时具有产酸、产IAA、固氮、解磷和抑菌等能力。选取具有产酸和多种促生特征的菌株绿针假单胞菌CSM-70和双鱼假单胞菌CSM-129进行盆栽蓝莓苗处理,发现2株菌均能显著促进蓝莓苗的生长发育并调控根际土壤pH,其中菌株CSM-70处理还显著促进了蓝莓叶片氮、磷元素的吸收,提升了土壤速效钾、碱解氮的含量。【结论】蓝莓根际细菌多样性高且蕴藏着丰富的促生长菌株,绿针假单胞菌CSM-70和双鱼假单胞菌CSM-129能够促进蓝莓苗生长、调控根际土壤pH和肥力,并促进植株养分吸收,具有蓝莓专用微生物菌剂研制与应用的潜力。     

植物根际促生菌的筛选及其对玉米的促生效应

[目的]以不同植物根及根际土壤为研究材料,进行植物根际促生菌(PGPR)的筛选,并探索其植物促生作用机制.[方法]以解磷、固氮、产氨、产IAA和拮抗3种常见病原真菌为筛选标准,测定了初筛菌株的多项促生能力,并通过对这些菌分别单独回接和多菌混接的玉米盆栽试验,测定了其对玉米的促生效应.[结果]从渭南、成阳、安康、商洛和榆林5地分离得到的158株菌中有17株茵具有上述多种植物促生作用的菌株.盆栽试验的测定结果表明:单独接种和多菌混合接种在玉米株高、根长、茎长、茎平均直径和干重方面与对照组相比较都有所增加,尤其是在多个指标上,多菌混合接种所显示出的促生效应均明显优于单菌接种.[结论]所筛选到的具有多种促生能力的菌株,可以为进一步构建植物根际促生菌(PGPR)菌群提供良好的种质资源.     

花生根际促生复合菌剂对连作花生生理生化指标和根际细菌群落的影响

【目的】利用多功能根际促生菌剂促进花生生长,缓解连作障碍对花生的生长抑制。【方法】从10年连作花生根际土中筛选根际微生物,测定其促生及拮抗能力,并经16S rRNA基因测序确定菌株分类。选取3株功能互补且相互之间无生长抑制的根际促生菌制备微生物复合菌剂,利用发芽及盆栽试验验证微生物复合菌剂的促生效应。利用高通量测序技术对花生根际土壤细菌的16S rRNA基因的V3-V4区进行测序分析。【结果】从连作花生根际中共筛选获得37株具有促生及抑制植物病原菌生长能力的根际促生菌,选取3株制备微生物复合菌剂。与空白对照相比,复合菌剂显著提升花生发芽率13.22%;与单独使用根际促生菌相比,复合菌剂显著提升花生发芽率分别为6.99%、7.51%、8.87%。施用复合菌剂对花生根系形态、根瘤数量、叶绿素含量、植株光合参数和植株抗氧化酶活均有显著促进作用,花生根系总长、根尖数、主根直径、根系体积和根系活力分别增加了43.50%、49.31%、15.11%、16.92%和112.16%;花生苗期、花针期叶片叶绿素含量和光合作用均得到显著的提高;花生根瘤数量每株增加34个。施用复合菌剂后对花生的根际细菌多样性影响并不显著,在门水平上的优势菌门变形菌门(Proteobacteria)、放线菌门(Actinobacteriota)和拟杆菌门(Bacteroidota)占70%以上。在属水平上新鞘氨醇菌属(Novosphingobium)和鞘氨醇单胞菌属(Sphingomonas)是优势菌属。【结论】本研究研制的花生根际促生复合菌剂可有效促进花生种子发芽、花生根系生长、提高叶片叶绿素含量以及促进植物光合作用,为缓解花生连作障碍及丰产增收提供有效的技术支持。     

新疆木碱蓬(Suaeda dendroides)根际耐盐促生细菌的筛选及鉴定

【背景】新疆是全国最大的盐渍化土壤分布区,土壤盐渍化严重影响作物生长,耐盐促生菌可以有效改善土壤肥力,提高作物抗逆性,促进植物生长,提高土壤利用率。【目的】分离筛选获得木碱蓬(Suaeda dendroides)根际土壤中耐盐促生菌菌株,对优良促生作用的菌株进行分子鉴定,挖掘微生物资源,为微生物菌剂的研制奠定基础。【方法】采用传统的分离方法筛选获得木碱蓬根际耐盐微生物,采用"三级筛选体系"筛选获得耐盐促生菌菌株,用CTAB法提取菌株DNA,对菌株16S rRNA基因测序进行系统发育分析,确定耐盐促生菌菌株的分类地位。【结果】从木碱蓬根际共分离获得耐盐微生物58株,8株具有固氮活性,解磷活性菌株12株,具有解钾活性菌株15株,产IAA活性的菌株3株,具有较强产氨活性的菌株2株。经过促生平板筛选,菌株GTZW50-5和MH-F促进了拟南芥生长,表现出较好的促生效果,通过小麦盆栽试验,菌株GTZW50-5对小麦的根长以及株高具有显著的促生作用,在一定程度上提高了植物体内的叶绿素含量。MH-F菌株对小麦的根系具有较显著的促生作用,且对小麦的叶绿素及脯氨酸含量在不同盐浓度下都有所提高。经过系统发育分析,GTZW50-5与Bacillus vallismortis (AY603658)序列相似度达到99.43%,鉴定属于Bacillus vallismortis,MH-F与Enterobacterludwigii(JTLO01000001)序列相似度为98.34%,鉴定属于Enterobacter属。【结论】菌株GTZW50-5与MH-F均具有较好的促生效果,这为耐盐微生物资源的开发和利用提供了理论依据。     

樱桃根际促生细菌

微生物肥料通过其中所含微生物的生命活动,产生有益于植物生长的物质,可使土壤中无效营养有效化,可将植物无法利用的土壤、有机肥中的物质分解为可供作物生长所利用的营养成分,这样不仅使有机肥的优势得以充分发挥,还能提高化肥的利用率,减少化肥使用量。接种微生物肥料,被普遍认为是一项环境友好、经济有效、提高农林产品产量和品质的技术措施。果树、蔬菜、中草药、苗木花卉等经济作物以及部分大田作物普遍存在严重的连作障碍问题,植物根系发育不良、土传病害加重等,利用化肥、农药等化学措施不能从根本上解决这些问题。研究与实践表明,利用植物根际促生细菌(Plant growth promoting rhizobacteria,PGPR)解决作物的连作障碍问题是一条行之有效的途径。本刊2012年第12期刊登了陈波、杜秉海等的文章"樱桃根际促生细菌的筛选与鉴定"[1]。作者从樱桃根际土壤中筛选具有良好应用潜力的细菌分离物。利用盆栽试验,验证其促生效果。对促生效果较好的细菌分离物,进行形态学观察、生理生化测定、16S rRNA基因序列及系统发育树分析以确定其分类地位。筛选出4株(AI5、AI21、PII17、PI7)具有良好应用潜力的细菌分离物,盆栽试验结果表明对樱桃生长有明显的促进作用。该研究对于开发樱桃专用PGPR制剂,促进樱桃根系发育和养分吸收,提高樱桃抗逆性等具有重要实践意义;同时为进一步研究相关菌株在樱桃根际定殖规律以及与樱桃的互作机制奠定了基础。在生物肥料的生产中具有广阔的应用前景。近年来该研究团队先后开展了利用GFP标记菌株,研究其在樱桃根际的定殖动态[2],菌株发酵条件优化[3]、吸附载体筛选及储存试验[4]、田间示范试验等[5-8]。发表相关论文6篇,获得职务发明专利授权2项、实用新型专利1项,获得农业部微生物肥料登记证1个,以该研究为主要内容的"经济林木新型生物肥料研究与开发"课题于2013年通过山东省科技厅组织的成果鉴定。但这并不意味着樱桃生物肥料开发成功。不同的环境条件,诸如气象、土壤性质或其他土著微生物活力等均会对细菌生长造成影响,成功的实验室试验并不一定在田间原位能成功,希望将来能针对不同作物筛选和培育高效优良微生物功能菌株,扩大菌种资源库,确定多菌株组合,改进复合菌剂生产工艺,尽早产业化。     

Selection of antagonistic bacteria isolated from the Physalis peruviana rhizosphere against Fusarium oxysporum

Aims: Cape gooseberries (Physalis peruviana) have become increasingly important in Colombia for both domestic consumption and the international export market. Vascular wilting caused by Fusarium oxysporum is the most damaging disease to P. peruviana crops in Colombia. The control of this pathogen is mainly carried out by chemical and cultural practices, increasing production costs and generating resistance. Therefore, the objectives of this study were to test rhizobacteria isolates from P. peruviana rhizosphere against F. oxysporum under in vitro and in vivo conditions. Methods and Results: Over 120 strains were isolated, and five were selected for their high inhibition of F. oxysporum growth and conidia production under in vitro conditions. These strains inhibited growth by 41–58% and reduced three‐ to fivefold conidia production. In the in vivo assays, all the tested isolates significantly reduced fungal pathogenicity in terms of virulence. Isolate B‐3·4 was the most efficient in delaying the onset of the first symptoms. All isolates were identified as belonging to the genus Pseudomonas except for A‐19 (Bacillus sp.). Conclusions: Our results confirmed that there are prospective rhizobacteria strains that can be used as biological control agents; some of them being able to inhibit in vitro F. oxysporum growth and sporulation. Significance and Impact of the Study: Incorporating these bacteria into biological control strategies for the disease that causes high economical losses in the second most exported fruit from Colombia would result in a reduced impact on environment and economy.     

Stability of transformed antagonistic Fusarium oxysporum strains in vitro and in soil microcosms

The stability of a genetically modified strain of Fusarium oxysporum used as antagonist against phytopathogenic formae speciales of F. oxysporum was evaluated both in vitro and in microcosm assays. The Escherichia coli hygromycin B phosphotransferase gene (hph), conferring hygromycin B resistance, was introduced by genetic transformation into a recipient strain marked by benomyl resistance and a dark red pigmentation. Hybridization with the complete plasmid suggested that the integration had generally occurred in a multiple-tandem array at multiple sites. Among nine independent transformants tested, only three of them were mitotically stable after four rounds of vegetative growth with no selective pressure, while six showed various changes in the integration pattern. One transformant had lost the ability to grow in the presence of hygromycin B. In soil microcosms all the transformants maintained the hygromycin B resistant phenotype, but six of them showed rearrangement of transforming DNA. Only one strain (coded T26.40) underwent no obvious rearrangement both after in vitro growth and after recovery from the soil microcosm. The nine transformants were used in three biological control experiments against Fusarium wilt of carnation in comparison to two untransformed reference strains and to the recipient mutant. A high degree of variability in the biocontrol activity was observed throughout the experiments and only transformant T26.40 consistently controlled the incidence of disease. The results are discussed in relation to risk assessment of the release of transgenic antagonistic fungi.     

Biocontrol efficacy of Trichoderma spp. against sesame wilt caused by Fusarium oxysporum f. sp. sesami

Sesame (Sesamum indicum L.) is one of the most important oilseed crops in Egypt and worldwide. It is being infected with many pathogens, among these pathogens Fusarium oxysporum f.sp. sesami (Zap.) Cast is causing severe economic losses on sesame. In this study, antagonistic capability of 24 isolates of Trichoderma spp. was assessed in vitro against F. oxysporum f.sp. sesami. Two strains; T. harzianum (T9) and T. viride (T21) were revealed to have high antagonistic effect against F. oxysporum f.sp. sesami in vitro with inhibition percentage about 70 and 67%, respectively. These two isolates proved to have high ability to control Fusarium wilt disease under greenhouse conditions. The highest reduction in disease severity was achieved with T. viride followed by T. harzianum with reduction in disease severity about 77 and 74%, respectively. This study revealed that the time of application of bioagents is a decisive factor in determining the efficacy of Trichoderma isolates to control Fusarium wilt of sesame. It was revealed that the highest reduction in the disease severity was achieved when either Trichoderma viride or T. harzianum were applied 7 days before challenging with the F. oxysporum f.sp. sesami.     

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。     

Endophytic bacteria from Datura metel for plant growth promotion and bioprotection against Fusarium wilt in tomato

Nine non-pathogenic bacterial isolates, recovered from Datura metel organs and able to colonise the internal stem tissues of tomato cultivar Rio Grande, were screened for their ability to suppress tomato Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici (FOL), and to enhance plant growth. S33 and S85 isolates tested were found to be the most effective in decreasing Fusarium wilt severity by 94–95% compared to FOL-inoculated and untreated control. A significant enhancement of growth parameters was recorded on tomato plants inoculated or not with FOL. Both isolates were characterised and identified using 16S rDNA sequencing genes as Stenotrophomonas sp. str. S33 (KR818084) and Pseudomonas sp. str. S85 (KR818087). Screened in vitro for their antifungal activity towards FOL, these isolates led to 38.7% and 22.5% decrease in pathogen radial growth and to the formation of an inhibition zone of 12.75 and 8.37?mm respectively. Stenotrophomonas sp. str. S33 and Pseudomonas sp. str. S85 were found to be chitinase-, protease- and pectinase-producing strains but unable to produce hydrogen cyanide. Production of indole-3-acetic acid-like compounds, phosphate solubilising ability and pectinase activity were investigated for elucidating their plant growth-promoting traits and their endophytic colonisation ability.     

Thermodynamics of partitioning of chitinase and laminarinase in a soya lecithin liposome system and their antifungal effect against Fusarium oxysporum

Abstract

The goal of the present work was to compare the partitioning behavior of chitinase and laminarinase (from Trichoderma spp.) in soya lecithin liposomes at different temperatures and examine the activity of the resulting microencapsulated enzymes against Fusarium oxysporum. In both cases the partition coefficients (K_o/w) were greater than 1, indicating affinity of the enzymes for microencapsulation in liposomes. Enthalpy calculations indicated that the process was endothermic in the case of laminarinase and exothermic in the case of chitinase. Soya lecithin liposomes were stable for more than 20 days. The stability of the immobilized enzyme was increased in the case of chitinase, but was not changed in the case of laminarinase. Although the antifungal effects of individual immobilized preparations decreased after microencapsulation compared with non-immobilized enzymes, they were increased by the synergistic effect of both encapsulated enzymes. The application of free or immobilized enzymes was also shown to enhance the inhibitory effect of the chemical fungicide, thiabendazole, on F. oxysporum.     

百合枯萎病拮抗细菌的筛选、鉴定及其抑菌物质研究

【目的】筛选对百合枯萎病具有抑菌活性的拮抗细菌,对其抑菌活性物质进行初步分离纯化分析。【方法】以强致病力的百合尖孢镰刀菌(Fusarium oxysporum)为靶菌,采用系列稀释法和平板对峙法初筛拮抗细菌,并通过产铁载体能力、水解酶活性、土壤定殖力等多种生防特性指标进行复筛,结合形态学特征、生理生化指标和16S rRNA基因序列比对鉴定其分类地位;利用百合尖孢镰刀菌作为靶菌进行活性追踪,结合酸沉淀、快速柱色谱、HPLC等分离纯化手段,对菌发酵液中的抑菌活性物质进行纯化分析。【结果】在64株百合根际细菌和386株海洋细菌中进行初筛,得到9株对百合镰刀菌具有较强拮抗活性的菌株,最后筛选了1株拮抗活性较强且产铁载体能力和水解酶活性、土壤定殖能力较高的菌株11B91,鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其抑菌活性物质初步推测可能为iturin和fengycin脂肽类化合物。【结论】菌株11B91在百合枯萎病的生物防治中具有潜在的应用价值,证实海洋来源的微生物也具有防治陆地植物病原菌的潜力,为植物病害的防治拓宽了思路。     

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.