嫁接西瓜对西瓜枯萎病菌侵染的防御机制研究

为了揭示嫁接提高西瓜抗枯萎病的机制,该研究以嫁接西瓜为材料,采用扫描电镜观察了枯萎病菌侵染下寄主的组织结构变化,荧光定量分析了相关防卫基因的表达,比较了嫁接西瓜对枯萎病菌侵染的抗感反应。结果显示:(1)枯萎病菌侵染后,与自根西瓜相比,嫁接西瓜的根部木质部导管通过快速形成膜状物、侵填体及细胞壁增厚阻塞菌丝入侵;自根西瓜防御反应较嫁接西瓜晚,严重侵染时薄壁细胞降解,导管组织脱落导致维管系统空洞,从而使植株呈现萎蔫症状,该现象在嫁接西瓜中没有发现。(2)枯萎病菌侵染后,嫁接西瓜比自根西瓜具有较高的防卫基因表达水平,其中:嫁接西瓜中,CHI、APX和PPO基因的表达随枯萎病菌侵染时间的延长而升高,而PAL呈现先升高后降低的表达趋势,但仍高于本底表达;自根西瓜中,仅PPO基因在枯萎病菌侵染后表达上调,而其他基因的表达则是先升高后降低,与嫁接西瓜中的PAL基因表达一致。研究表明,嫁接植株一方面通过快速的组织结构响应,另一方面从转录水平提高了相关防卫基因的表达,最终使植株具有抗病性;推测防御基因在嫁接植株与枯萎病菌互作中的强烈诱导响应可能是嫁接植株抗病的分子机制之一。     

香蕉胁迫相关蛋白基因的克隆及其表达分析

通过随机克隆测序的方法从香蕉根系cDNA文库中获得胁迫相关蛋白基因,命名为MaSAP1(GenBank登录号为AGH14257.1)。扩增获得的cDNA序列与质粒OZ092的目的片段序列一致,表明MaSAP1是香蕉SAP基因编码框全长cDNA,包含一个510bp的最大开放阅读框,编码一个长169个氨基酸的蛋白质。蛋白质序列同源比对发现其含有完整的A20和AN1基序结构。系统进化树比对分析表明,MaSAP1与水稻和獐茅的亲缘关系较近。组织特异性研究表明,MaSAP1基因在香蕉根和果实中的表达量较高,在茎中的表达量最低。实时荧光定量PCR分析表明MaSAP1响应激素的处理,同时也响应干旱、低温、高盐和枯萎病菌的侵染等胁迫。可见,MaSAP1基因在植物生长发育和植物响应逆境中具有重要作用。     

海岛棉GbGSTU7的克隆及其对枯萎病菌侵染的应答

该研究基于前期转录组测序数据分析,采用RT-PCR方法从海岛棉06-146中克隆得到参与海岛棉抗枯萎病调控的候选基因谷胱甘肽转移酶基因(GbGSTU7),对其序列进行生物信息学分析,并对GbGSTU7基因在病菌侵染下不同组织的表达特性进行分析,以探讨GbGSTU7基因与棉花抗枯萎病的关系。结果显示:(1)GbGSTU7基因CDS全长711 bp,编码236个氨基酸,属于GST家族tau类,二级结构由5个β折叠和9个α螺旋构成,三级结构呈球状。(2)枯萎病菌侵染后,GbGSTU7基因表达量在抗枯萎病品种06-146的根中呈下降趋势,48 h达到最低,茎和叶中呈先升高再降低的趋势,在茎中12 h达到最高,在叶中24 h达到最高;在感枯萎病品种‘新海14’的根中呈先下降再上升趋势,且在8 h达到最低,在茎和叶中的趋势无明显规律,但在茎中8 h达到最高,在叶中4 h达到最高;且两个材料中均表现为GbGSTU7基因在根中的表达量最低时,茎的表达量升到最高。研究表明,GbGSTU7基因在海岛棉抗、感病材料的各组织中均可响应枯萎病菌的诱导表达,且均具有明显的组织特异性;推测GbGSTU7基因可能与棉花抗枯萎病有着密切的关系,可能在棉花抵御病菌侵染的过程中发挥重要的调控作用。     

棉花抗枯萎病相关转录因子基因的克隆与表达

以枯萎病菌诱导棉花基因表达谱中获得的差异表达bZIP作为探针,采用电子克隆结合RT-PCR方法从棉花抗枯萎病品种‘中棉所12’中克隆了1个TGA转录因子基因,命名为GhTGA2.2。序列分析表明,该基因的cDNA全长1 356bp,编码451个氨基酸,预测分子量为50.04kD,等电点为5.85,含有保守的bZIP结构域。系统进化树分析表明,GhTGA2.2属于bZIP亚家族的TGA转录因子,与拟南芥AtTGA2、烟草NtTGA2.2亲缘关系最近。qRT-PCR分析表明,经枯萎病菌诱导后,GhTGA2.2基因在抗病品种中呈上调表达,随处理后时间的推移,其相对表达量呈先升高后降低的趋势,并于处理后24h表达量达到最大;水杨酸诱导后1h,GhTGA2.2基因相对表达量迅速增加;茉莉酸和乙烯诱导后GhTGA2.2基因的相对表达量明显降低,呈下调表达。研究推测,GhTGA2.2基因可能通过水杨酸信号传导途径参与对枯萎病菌的防御反应。     

转GhB301基因烟草的防御酶活性及抗病相关基因表达分析

为了明确棉花ERF-B3亚族转录因子基因GhB301在烟草异位表达后(抗枯萎病中)的功能,该研究以过表达GhB301基因烟草和野生型烟草为材料,采用枯萎病菌孢子悬浮液接菌方法,分析病原菌侵染前后防御酶活性变化以及防卫相关基因的表达变化与抗病性的关系。结果显示:(1)棉花枯萎病菌处理15d后,2个转基因株系烟草叶片黄化程度与野生型相比较轻。(2)棉花枯萎病菌处理后,过表达GhB301转基因烟草和野生型烟草叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)的活性较未接菌对照显著提高,并且酶活峰值出现均早于野生型材料;转基因材料叶片的POD、PAL、PPO活性均在处理3d后达到峰值,而野生型材料叶片的POD、PAL活性在处理5d后才达到峰值。(3)接种棉花枯萎病菌后活性氧相关基因、乙烯(ET)/茉莉酸(JA)途径相关基因、病程相关基因的表达量在转基因株系OE1和OE2中均受到明显影响。研究推测,GhB301在烟草中的异位表达激活了防卫相关基因的表达,提高了防御酶的活性,从而增强了烟草对枯萎病菌的抗性。     

喷施油菜素内酯对棉花类固醇5α~-还原酶基因(GhDET2)在干旱胁迫下表达影响的分析

为了解植物类固醇5α~-还原酶基因(DET2)作为油菜素内酯(BR)生物合成途径中限速酶的作用。利用RACE技术克隆获得棉花的Gh DET2基因,并进行生物信息学分析,然后对棉花‘新陆早17号’幼苗使用PEG6000模拟干旱胁迫24 h,进行不同浓度油菜素内酯(brassinolide,BR)的喷施处理,分析Gh DET2基因在棉花幼苗根、茎、叶中的表达情况。序列分析表明Gh DET2基因的开放阅读框有774 bp,编码257个氨基酸。荧光定量分析结果显示,用BR处理棉苗3 h和6 h后Gh DET2基因表达量在叶中明显提高,而在根和茎中只略微提高,12 h和24 h时Gh DET2基因表达量较前期还有所降低。喷施BR在短时间内促进Gh DET2基因表达量的升高,推测BR喷施能够短期高效地增强棉花的抗旱能力。     

芒果SEPALAATA基因的克隆与表达分析

以芒果品种‘吕宋’(Mangifera indica L.Carabao)为试材,利用同源克隆和RACE技术从花序中获得了1个芒果SEPALAATA(SEP)基因cDNA全长,命名为MSEP1(GenBank中登录号为KP702299)。MSEP1基因的cDNA全长为921bp,包含一个长度为726bp开放阅读框,编码241个氨基酸,蛋白质相对分子质量为27.7kD,理论等电点为5.79。序列比对和系统进化树分析表明,MSEP1具有保守的MADS-box及半保守的K区,属于MADS-box家族的SEP亚家族。器官特异性表达分析表明,MSEP1基因在芒果根、茎中表达量较低,在叶片、花芽中表达量较高,而在花序中表达量最高。研究推测,MSEP1基因可能在芒果生殖生长中发挥重要作用。     

银杏EPSPS基因克隆及表达分析

利用RACE技术,克隆到银杏EPSPS合酶基因(GbEPSPS)的cDNA序列并对其进行生物信息学分析.结果表明:银杏GbEPSPS cDNA长1 403 bp(GenBank登录号GU084139),其包含一个1 035 bp的ORF框,编码344个氨基酸;预测该基因编码蛋白质分子量为36.87 kD,其等电点为5.75.系统进化树分析表明,银杏EPSPS蛋白质序列与其他物种的EPSPS同源性较高.半定量RT-PCR分析结果显示,EPSPS基因在银杏的叶和果实中表达量最高,其次为茎,根中表达水平最低.草甘膦处理能显著诱导银杏EPSPS基因表达量升高;紫外光对银杏EPSPS基因的表达具有诱导作用;ABA诱导GbEPSPS表达量先升后降;GbEPSPS转录水平受到42℃高温显著诱导.     

苎麻转录因子基因BnMYB3的克隆及表达分析

该研究采用RACE技术,从苎麻中克隆到1个MYB转录因子基因(BnMYB3)的全长cDNA序列(GenBank登录号为MF741320.1)。生物信息学分析表明,BnMYB3基因cDNA全长为1 216bp,包括900bp编码区序列,编码含有299个氨基酸的蛋白,其分子量约为33.63kD,理论等电点为9.16;该蛋白质含有2个典型的MYB结构域,属于R2R3-MYB。从苎麻基因组中克隆了BnMYB3基因1 681bp启动子序列,该序列包含ABRE、GARE-motif、CGTCA-motif和TGACG-motif等多个逆境相关的顺式作用元件。实时荧光定量PCR分析表明,BnMYB3为组成型表达基因,在茎和叶中的表达量显著高于根;BnMYB3基因能够响应镉胁迫,且表达量随镉胁迫处理时间和处理浓度的增加而显著上升。     

木霉诱导下山新杨PodaPIN9基因的组织表达调控

PIN蛋白具有多个跨膜结构域,影响着高等植物生长素的外向运输和众多生长发育过程。木霉菌是能促进植物生长、提高其对多种病害防御作用的生防因子。研究木霉菌对木本植物山新杨生长素的极性分布的影响有重要意义。克隆了山新杨PodaPIN9基因,对其核酸和蛋白序列进行分析;同时,构建的进化树显示PodaPIN9与6个物种的9条PIN基因具有高度一致性（>80%）。qRT-PCR分析表明,PodaPIN9在山新杨茎尖、成熟叶和根中均有表达。在根中表达量极低;在茎尖和叶中的表达量极高,分别是根中的503和346倍。该基因受木霉影响在茎尖和叶中的表达量均显著下调;而根中的表达量显著上调,达到对照的32.01倍。并发现根际接种木霉48 h会使杨树茎尖、叶和根中生长素含量降低。说明木霉菌能影响杨树茎尖、叶和根中IAA水平及PodaPIN9的表达量。并且Pearson相关性分析表明在茎尖、叶和根中,PodaPIN9表达量与IAA水平具有不同的相关性。     

Screening of <i>Bacillus subtilis</i> HAAS01 and Its Biocontrol Effect on <i>Fusarium</i> wilt in Sweet Potato

Fusarium wilt, a disease caused by Fusarium oxysporum f.sp batatas (Fob) is an important disease in sweet potato production. Using endophytic bacteria for biological control of sweet potato diseases is one of the important ways. A Bacillus subtilis with antagonistic effect on Fusarium wilt of sweet potato was isolated from soil by confrontation culture. According to the biological characteristics, 16S rDNA sequence analysis, and physiological and biochemical analysis, the Bacillus subtilis HAAS01 was named. A pot experiment was conducted for the biological control experiment of strain HAAS01, and the endogenous hormone content, antioxidant enzyme activity, soluble protein content, and related gene expressions of sweet potato plants were detected. The results showed that the HAAS01 strain could promote the production of endogenous hormones and resist the infection of plant diseases together with defensive enzymes and upregulation of related gene expressions. In summary, Bacillus subtilis HAAS01 was effective in controlling Fusarium wilt of sweet potato and has potential for application and development.     

Success evaluation of the biological control of Fusarium wilts of cucumber,banana, and tomato since 2000 and future research strategies

The Fusarium wilt caused by Fusarium oxysporum strains is the most devastating disease of cucumber, banana, and tomato. The biological control of this disease has become an attractive alternative to the chemical fungicides and other conventional control methods. In this review, the research trends and biological control efficiencies (BCE) of different microbial strains since 2000 are reviewed in detail, considering types of microbial genera, inoculum application methods, plant growth medium and conditions, inoculum application with amendments, and co-inoculation of different microbial strains and how those affect the BCE of Fusarium wilt. The data evaluation showed that the BCE of biocontrol agents was higher against the Fusarium wilt of cucumber compared to the Fusarium wilts of banana and tomato. Several biocontrol agents mainly Bacillus, Trichoderma, Pseudomonas, nonpathogenic Fusarium, and Penicillium strains were evaluated to control Fusarium wilt, but still this lethal disease could not be controlled completely. We have discussed different reasons of inconsistent results and recommendations for the betterment of BCE in the future. This review provides knowledge of the biotechnology of biological control of Fusarium wilt of cucumber, banana, and tomato in a nut shell that will provide researchers a beginning line to start and to organize and plan research for the future studies.     

生物有机肥对土壤微生物群落结构变化及西瓜枯萎病的调控

【目的】研究施用生物有机肥处理土壤后对西瓜枯萎病发生的影响,明确可能与西瓜枯萎病发病密切相关的土壤微生物群落结构。【方法】设置2组不同年份材料对比,依次为施用生物有机肥后2016年发病期(CK2016、T12016)和2017年发病期(CK2017、T12017)。基于16S r RNA序列测定,利用Illumina Miseq高通量测序平台对施用生物有机肥后不同年份的土壤微生物群落组成和差异进行测序分析。【结果】试验结果发现,不同年份的土壤微生物群落中alpha多样性指数并无明显的差异,但是施用生物有机肥后的土壤相比对照土壤中细菌群落多样性略有增高。不同年份土壤细菌群落结构在门水平上差异不显著,其中变形菌门和硬壁菌门是构成这两个年份土壤细菌的重要组成部分,同时也是比较稳定的微生物类群。在属水平上分析发现主要动态变化菌属为芽孢杆菌属、肠球菌属、乳球菌属及水恒杆菌属。通过Spearman分析发现它们与西瓜枯萎病发生率的关系均为负相关。【结论】施用生物有机肥可帮助西瓜连作土壤的生态修复,对枯萎病防治起到一定的作用。通过对施用生物有机肥的不同年份土壤微生物种群结构动态变化及对枯萎病发生率呈正负相关的微生物菌属的分析研究,为如何利用调节土壤微生物群落结构来防治西瓜枯萎病提供了新的思路。     

Genetic diversity studies and identification of SSR markers associated with Fusarium wilt (Fusarium udum) resistance in cultivated pigeonpea (Cajanus cajan)

Genetic diversity and identification of simple sequence repeat markers correlated with Fusarium wilt resistance was performed in a set of 36 elite cultivated pigeonpea genotypes differing in levels of resistance to Fusarium wilt. Twenty-four polymorphic sequence repeat markers were screened across these genotypes, and amplified a total of 59 alleles with an average high polymorphic information content value of 0.52. Cluster analysis, done by UPGMA and PCA, grouped the 36 pigeonpea genotypes into two main clusters according to their Fusarium wilt reaction. Based on the Kruskal–Wallis ANOVA and simple regression analysis, six simple sequence repeat markers were found to be significantly associated with Fusarium wilt resistance. The phenotypic variation explained by these markers ranged from 23.7 to 56.4%. The present study helps in finding out feasibility of prescreened SSR markers to be used in genetic diversity analysis and their potential association with disease resistance.     

Effect of the combination of bio-organic fertiliser with Bacillus amyloliquefaciens NJN-6 on the control of banana Fusarium wilt disease,crop production and banana rhizosphere culturable microflora

Soil amended with organic amendments has been suggested to be a strategy for managing the Fusarium wilt disease which severely hindered the banana production. The effects of four fertilisation regimes, including chemical fertiliser, manure composts and bio-organic fertiliser (BIO) containing Bacillus amyloliquefaciens NJN-6 for 2-year continuous application on the banana Fusarium wilt disease incidence, crop yield and rhizosphere culturable microbial community were investigated. To explore the soil microflora, plate counting method, in vitro screening method for antagonism, eco-physiological index and culture-dependent denaturing gradient gel electrophoresis method (CD DGGE) were used. The highest banana yield, culturable bacteria, actinobacteria and Bacillus populations, culturable bacteria to fungi (B/F) value, antagonistic Bacillus ratio and lowest Fusarium wilt disease incidence were observed in the BIO treatment. Based on CD DGGE results, the BIO application significantly altered the soil bacteria structure and showed highest richness and diversity. The phylogenetic analysis of the selected bands showed that the most abundant phyla were Proteobacteria and Bacteroidetes and BIO application enriched the genera Comamonas, Chitinophaga, the species Bacillus flexus and uncultured Bacillus. All the results showed that 2-year continuous application of BIO containing B. amyloliquefaciens NJN-6 more effectively controlled Fusarium wilt disease and improved fruit yields under field conditions and modulated banana rhizosphere microflora.     

香蕉植株根区土壤的真菌多样性分析

尖孢镰孢菌古巴专化型(Fusarium oxysporum f.sp.cubense)是香蕉枯萎病的病原菌,该菌是一种土壤习居菌,了解香蕉根区土壤中真菌多样性及镰孢菌属(Fusarium)真菌所占比例,对如何减少土壤中的病原菌、预防香蕉枯萎病的发生有重要的指导意义。该文通过采集不同宿根年限的香蕉健康植株和枯萎病植株的根区土壤,利用高通量测序技术测定土壤样品中的真菌种群。结果表明:(1)同一宿根年限的香蕉植株中,健康植株根区土壤中所获的reads及OTUs数量均高于枯萎病植株,说明健康植株根区土壤的真菌多样性丰富于枯萎病植株。(2)除了一年生香蕉枯萎病植株以担子菌门(Basidiomycota)为主外,其他土壤样品中均以子囊菌门(Ascomycota)为主,其中的丛赤壳科最高相对丰度来自三年生健康植株的根区土壤(26.02%),其次是五年生的枯萎病植株根区土壤(15.56%)。(3)在丛赤壳科中,镰孢菌属在三年生健康植株土壤中的相对丰度最高(2.54%),在其他样品中的相对丰度在0.1%～0.65%之间;在镰孢菌属中,腐皮镰孢菌(Fusarium solani)的相对丰度(0～1.59%...     

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。     

Control of Fusarium wilt of tomato caused by Fusarium oxysporum f. sp. lycopersici using leaf extract of Piper betle L.: a preliminary study

The main objective of this study was to evaluate the effectiveness of crude chloroform extract of Piper betle L. (PbC) in controlling Fusarium wilt of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f. sp. lycopersici. It was observed that 1% (w/w) amendment of the PbC in soil was more efficient in reducing the Fusarium population in soil than carbendazim and the combined amendment of carbendazim and PbC. Fusarium wilt control studies were carried out in a greenhouse. Variation in different parameters like shoot growth, root growth and mean fresh weights of tomato seedlings in all the treatments were recorded. Accumulation of total phenolics was also studied from the root tissues of tomato. Higher accumulation of total phenolics was observed in the Fusarium-infested plants as compared to that of healthy control and PbC-treated plants. Moreover, it was observed that the extract could reduce the symptoms and disease development. Electron microscopy studies were also done to observe the Fusarium infestation in the vascular bundles and to show the accumulation of total phenolics in the vacuoles of root tissue.     

Biological Control of Fusarium spp. in Cotton, Wheat and Muskmelon by Trichoderma harzianum

A new isolate of Trichoderma harzianum (T-35) was isolated from the rhizosphere of cotton plants from a field infested with Fusarium. Under glasshouse conditions, the antagonist was applied to soil growing in a bran/peat mixture (1:1, v/v) or as a conidial suspension or used as a seed coating. When T. harzianum was tested against Fusarium oxysporum f. sp. vasinfectum, F. oxysporum f. sp. melonis or F. roseum‘Culmorum”, a significant disease reduction, was obtained in cotton, melon and wheat, respectively. Biological control of Fusarium wilt of cotton was achieved when tested at two inoculum levels of the pathogen (2 × 10⁷ and 2 × 10⁸ microconidia/kg soil), decreasing the Fusarium spp. soil population. The long term effect of T. harzianum on Fusarium wilt of cotton was studied using successive plantings. The antagonist persisted in soil throughout three consecutive plantings, reducing the Fusarium, wilt incidence in each growth cycle. At the first planting the largest amount of preparation was found superior, whereas at the third planting, no significant difference could be observed between the four rates of Trichoderma preparation. T. harzianum (T-35) controlled Fusarium wilt in cotton and muskmelon when applied in both naturally or artificially infested alluvial vertisol and sandy-loam soils, respectively. Soil or seed treatments with the antagonist provided a similar disease control of F. roseum‘Culmorum’ and of F. oxysporum f. sp. melonis.