香蕉枯萎病菌4号生理小种致病相关基因foABC1的分离

通过对香蕉枯萎病菌4号小种致病突变体B1233的进一步研究,分离了被突变的致病相关基因foABC1,同源性分析及保守结构预测该基因编码一类ABC转运蛋白,其功能可能同稻瘟病菌的ABC转运蛋白一样,负责真菌毒素的泵出,或是像其他真菌的ABC转运蛋白,在病原菌侵染寄主植物时能忍耐植物因防卫反应所释放的植保素或抗毒素类物质。     

pH值对香蕉枯萎病菌4号生理小种生长的影响

【背景】香蕉枯萎病菌4号生理小种(镰刀菌)是香蕉产业的致命威胁。已有研究表明土壤pH值越高,香蕉枯萎病发病率越低,但是现有pH值对镰刀菌影响的研究大都是用强酸强碱调节pH值,pH值没有缓冲体系保护,而且尚未检测试验终点时介质的pH值。此外,关于pH值对香蕉枯萎病菌4号生理小种(Foc4)影响的研究尚不系统,难以用于指导生产实践。【目的】为系统地了解土壤酸碱度对Foc4生长的影响。【方法】在pH 3.0-11.0之间设定9个pH值梯度,模拟酸性到碱性土壤pH值条件,于室内培养条件下系统研究pH值对Foc4生长、产孢、孢子萌发的影响及其生长过程对环境pH值的影响。【结果】弱酸性至中性环境(pH 5.0-7.0)最适宜于香蕉枯萎病菌的生长、产孢和孢子萌发。弱碱性处理(pH8.0和pH9.0)孢子平均萌发率较弱酸性环境处理(pH5.0和pH6.0)下降了73.1%。与pH 6.0酸性处理相比,pH 8.0和pH 9.0处理的产孢量分别下降了52.3%和68.1%。【结论】香蕉枯萎病菌Foc4生长和萌发过程会产酸,但是在缓冲体系液体培养基中,除了pH 9.0和pH10.0处理终点培养液pH值分别下降了0.34和0.27个单位外,其它处理起始和终点的pH值无差异。说明在缓冲体系液体培养基中的研究结果可以反映环境pH值对Foc4生长和萌发的影响。在作物可以生长的pH值范围内(pH5.0-9.0),碱性和微碱性条件(pH8.0-9.0)能明显抑制Foc4生长、产孢和孢子萌发。     

香蕉枯萎菌基因组DNA提取方法的研究

以香蕉枯萎菌菌株为试验材料,在SDS～CTAB法和高盐沉淀法等基础上加以改进,对两种提纯香蕉枯萎菌基因组DNA的方法进行了比较研究。结果表明：高盐沉淀法是适合于香蕉枯萎菌基因组DNA提取的方法。该方法提取的DNA OD260／OD280的比值为1．841,DNA产量为0．81mgDNA／g菌丝体。基因组DNA经琼脂糖凝胶电泳得到一条带型较宽且清晰的DNA谱带,基本无DNA碎带;将提取的DNA直接用于PCR扩增,得到带多而且清晰、整齐、基本无拖尾的RAPD图谱。     

尖孢镰刀菌古巴专化型MAPK FoSlt2敲除突变体的转录组分析

尖孢镰刀菌古巴专化型Fusarium oxysporum f. sp. cubense（FOC）是威胁香蕉生产的重要土传病原真菌。丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）FoSlt2信号通路在调控尖孢镰刀菌古巴专化型的生长发育、细胞壁完整性和致病性方面发挥着重要作用。为了揭示FoSlt2信号通路的致病机理和寻找农药靶标,本研究利用高通量RNA-seq技术对该病菌野生型菌株和FoSlt2敲除突变体菌株的转录组进行了比较分析,结果表明差异表达基因共有2 164个,其中上调表达基因有1 184个,下调表达基因有980个。Gene Ontology（GO）功能分析结果表明,差异表达基因主要参与在结合、催化分子功能组和代谢过程、细胞过程生物学通路中。KEGG 功能富集分析结果表明,差异基因主要参与戊糖和葡糖醛酸盐转换、氨基糖和核苷酸糖、氨基葡聚糖降解、磷酸肌醇和碳类物质代谢通路,说明这些通路与尖孢镰刀菌古巴专化型的生长发育和致病性相关。该研究为尖孢镰刀菌古巴专化型致病机制的阐明奠定了理论基础。     

农杆菌介导的香蕉枯萎病菌4号生理小种转化体系的优化

香蕉枯萎病是世界范围内香蕉种植区最为严重的病害之一,严重威胁和影响着香蕉产业的发展.本文针对香蕉枯萎病病原菌的4号生理小种,建立了农杆菌介导的转化体系,确定了影响转化效率主要因子的优化体系是:农杆菌在IM培养基诱导前农杆菌OD_(600)为0.15、农杆菌经IM液体培养基诱导的时间为7 h、乙酰丁香酮(AS)浓度为150 μmol/L、Focr4孢子浓度为1×10~6个/mL、共培养时间为48 h、培养温度为25℃、诱导培养基pH值为5.5.在此条件下,转化效率能达到700～800个转化子/10~6个香蕉枯萎病菌孢子.PCR验证表明外源的T-DNA已经成功随机地整合到该病原菌基因组中.目前,应用该转化体系已获得2 300多个转化子,为后续克隆相关致病基因打下了良好基础.     

SDSC-TAB和高盐沉淀法提取香蕉枯萎病菌基因组DNA的比较

以香蕉枯萎病菌菌株为试验材料,采用SDS- CTAB法和高盐沉淀法提纯香蕉枯萎病菌基因组DNA。结果表明:高盐沉淀法是适合于香蕉枯萎病菌基因组DNA提取的方法。该方法提取的DNAOD2 60 2 80值显示产物纯度较高;经琼脂糖凝胶电泳得到一条带型较宽且清晰的DNA谱带,DNA浓度较高,基本无DNA碎带;不用RNase处理,已无RNA的干扰,无需任何纯化处理即可用于PCR扩增和RAPD分析。同时对DNA提取过程中的细节问题进行了探讨与分析。     

香蕉枯萎病菌Argonaute基因功能分析及其调控小RNA发生

尖孢镰刀菌古巴专化型Fusarium oxysporum f.sp.cubense是威胁香蕉生产的重要土传病原真菌,其中4号生理小种(Foc4)能感染几乎所有的栽培品系.Argonaute蛋白(AGO)介导的RISC复合体在RNAi干扰中起到重要作用.Foc4含有两个进化上高度保守的AGO蛋白,本研究利用同源重组技术获...     

香蕉枯萎病菌粗毒素对地衣芽胞杆菌生长和培养液上清蛋白组成的影响

本文研究了香蕉枯萎病菌4号生理小种湛江菌株(Foc 4-zj)产生的粗毒素对地衣芽胞杆菌R21菌株生长及其培养液中蛋白组成变化的影响。实验结果表明, Foc 4-zj的粗毒素能够抑制R21菌株的生长, 缩短其生长周期; 减少培养液上清蛋白含量以及改变蛋白质的种类; 低剂量的粗毒素有利于拮抗蛋白的积累, 而高剂量的粗毒素则相反。     

Investigation of the diversity of effector genes in the banana pathogen,Fusarium oxysporum f. sp. cubense,reveals evidence of horizontal gene transfer

It is hypothesized that the virulence of phytopathogenic fungi is mediated through the secretion of small effector proteins that interfere with the defence responses of the host plant. In Fusarium oxysporum, one family of effectors, the Secreted In Xylem (SIX) genes, has been identified. We sought to characterize the diversity and evolution of the SIX genes in the banana‐infecting lineages of F. oxysporum f. sp. cubense (Foc). Whole‐genome sequencing data were generated for the 23 genetic lineages of Foc, which were subsequently queried for the 14 known SIX genes (SIX1–SIX14). The sequences of the identified SIX genes were confirmed in a larger collection of Foc isolates. Genealogies were generated for each of the SIX genes identified in Foc to further investigate the evolution of the SIX genes in Foc. Within Foc, variation of the SIX gene profile, including the presence of specific SIX homologues, correlated with the pathogenic race structure of Foc. Furthermore, the topologies of the SIX gene trees were discordant with the topology of an infraspecies phylogeny inferred from EF‐1α/RPB1/RPB2 (translation elongation factor‐1α/RNA polymerase II subunit I/RNA polymerase II subunit II). A series of topological constraint models provided strong evidence for the horizontal transmission of SIX genes in Foc. The horizontal inheritance of pathogenicity genes in Foc counters previous assumptions that convergent evolution has driven the polyphyletic phylogeny of Foc. This work has significant implications for the management of Foc, including the improvement of diagnostics and breeding programmes.     

Biocontrol of Fusarium oxysporum f.sp. cubense tropical race 4 by formulated cells and cell-free extracts of Streptomyces griseus in sterile soil environment

The biocontrol activities of cells and cell-free extracts of Streptomyces griseus was tested against Fusarium oxysporum f.sp. cubense tropical race 4 (FOC race 4) in a sterile soil environment. They were first formulated in sodium alginate, kaolin clay and in alginate–kaolin combination, prior to introducing into sterile soil inoculated with 6 log₁₀ cfu FOC race 4 g^?1 soil. Results revealed that bioformulated cells of S. griseus, irrespective of the materials used, were generally more effective in inhibiting growth of FOC race 4 when compared to non-formulated cells of S. griseus. Kaolin was the most suitable inert material as formulation of S. griseus with kaolin effectively suppressed FOC race 4, with only 5.40 log₁₀ cfu g^?1 of FOC race 4 recovered after 20 days. Kaolin formulations also allowed good cell recovery post-formulation. Alginate was less desirable as poorer control was demonstrated, with 6.12 and 6.16 log₁₀ cfu g^?1 of FOC race 4 recovered from soils treated with alginate only and alginate–kaolin formulated S. griseus, respectively. Bioformulations did not benefit cell-free extracts at all. Our study suggests formulation of cells of S. griseus is more beneficial than cell-free extracts and kaolin is the preferred material for formulation.     

香蕉枯萎病菌Fow1基因的克隆及序列分析

为了解Fow1基因在尖镰刀菌古巴专化型侵染香蕉过程中的作用,及其与尖镰刀菌古巴专化型生理小种1号和生理小种4号之间的致病力差异的关系,采用PCR和RT-PCR方法扩增了2个生理小种的Fow1基因,并对扩增产物进行了克隆测序及相似序列搜索和比对,还对基因编码的蛋白进行了结构预测和功能分析。研究结果表明2个生理小种Fow1基因开放阅读框均为957bp,编码318个氨基酸,基因序列和氨基酸序列差异小,而且两个生理小种Fow1基因所编码的蛋白均具有酵母线粒体载体蛋白典型的结构特征,推测Fow1基因可能为香蕉枯萎病菌在香蕉组织中定殖所必需。从Fow1基因序列及其编码蛋白的氨基酸序列看,2个生理小种致病力的差异与Fow1基因并无明显对应关系,这为进一步研究Fow1基因功能奠定了基础。     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

基于RAPD、ISSR和AFLP对西瓜枯萎病菌遗传多样性的评价

利用RAPD、ISSR和AFLP分子标记技术对50个西瓜枯萎病菌株进行了分析。结果表明,21个RAPD引物、21个ISSR引物和21对AFLP引物分别对供试菌株扩增出113、134和389条带,三种分子标记的遗传相似系数比较一致,均可揭示西瓜枯萎病菌的遗传变异特点。三种分子标记产生的聚类分析结果存在一定差异,其中RAPD类群与生理小种和地理来源之间均不存在明显关系;而AFLP和ISSR类群与生理小种之间存在一定相关性,与菌株的地理来源关系不明显。     

应用PCR-RFLP和巢式PCR检测黄瓜尖镰孢菌

以3株黄瓜尖镰孢菌(Fusarium oxysporum f.sp.cucumarinum)、23株镰孢菌属(Fusariumspp.)真菌和分离自土壤的20株真菌、6株细菌和7株放线菌为材料,采用化学裂解法提取总DNA,进行PCR-RFLP和巢式PCR检测,试验证明PCR-RFLP程序不能完全区分Fusarium属内不同种,而巢式PCR对黄瓜尖镰孢菌具有特异性.运用优化的PCR-RFLP和巢式PCR检测程序对染病黄瓜组织进行了检测,结果表明,两种方法均可在接种发病早期(未显症时)检测出黄瓜枯萎病菌,PCR-RFLP在感病品种接种后3d即可检测到病原菌,而巢式PCR在接种后5d才能检测到病原菌.     

基于多基因序列分析对尖孢镰孢菌古巴专化型（香蕉枯萎病菌）生理小种的鉴定

由尖孢镰孢菌古巴专化型Fusarium oxysporum f. sp. cubense, Foc引起的香蕉枯萎病是香蕉生产上的毁灭性病害,自1996年以来已对我国华南地区香蕉生产造成了严重危害。传统上香蕉枯萎病菌生理小种的鉴定主要采用人工接种鉴别寄主尔后测定病菌致病性的方法,但实验周期长,且受季节影响。以来自澳大利亚的香蕉枯萎病菌生理小种1号（BW1）、2号（Race 2）、3号（Race 3）以及亚热带4号（BW4）为对照,对分离自我国华南地区主要香蕉产区（广东、广西、海南、福建等省区）的14株香蕉枯萎病菌的单孢菌株进行致病性测定,并结合热带4号小种（TR4）和亚热带4号小种（ST4）的分子特异检测方法,确定其生理小种类型;同时,利用ITS、TEF-1α、IGS、histone H3、β-tubulin等 5个主要用于镰孢菌系统发育学研究的基因,研究不同地区不同来源的Foc菌株之间的亲缘关系及其与非病原尖孢镰孢菌的关系,并评价这5个基因在香蕉枯萎病菌生理小种鉴定上的应用价值。研究结果表明：（1）来源于我国华南地区的4号小种主要为热带4号小种;（2）TEF-1α、IGS、histone H3等3个基因片段能够将Foc中不同生理小种的菌株划分成不同的系统发育谱系,与致病性测定的结果具有对应关系,也能较好地反映尖孢镰孢菌种内菌株的亲缘关系,可用于香蕉枯萎病菌生理小种鉴定;（3）我国Foc 1号生理小种的遗传多样性高于4号生理小种,Foc 1号生理小种的菌系与来自香蕉果实上的非病原尖孢镰孢菌的亲缘关系比其与Foc 4号生理小种的菌系的亲缘关系更近。     

香蕉枯萎病菌果胶甲基酯酶基因的克隆与生物信息学分析

旨在了解香蕉枯萎病菌(Fusarium oxysporumf.sp.cubense)4号生理小种(FOC4)PME基因序列特征,根据同源物种PME相关序列设计引物,利用PCR和RT-PCR技术,克隆FOC4序列基因和开放阅读框,命名为Foc4Pme。结果表明,所获得的PME基因均含有2个内含子和3个外显子,990 bp的片段,编码329个氨基酸。预测编码蛋白有信号肽,具有1个功能位点,其分子质量和等电点分为34.894 8 kD和9.17,该蛋白为稳定存在的蛋白。该蛋白疏水性最大值为2.022,最小值为-2.156,大部分区域为亲水区。该基因编码的蛋白具有一定保守性,进化上与镰刀菌亲缘关系最近。     

Colonization of Fusarium Wilt‐Resistant and Susceptible Watermelon Roots by a Green‐Fluorescent‐Protein‐tagged Isolate of Fusarium oxysporum f.sp. niveum

Interactions between watermelon and a green fluorescent protein (GFP)‐tagged isolate of Fusarium oxysporum f.sp. niveum race 1 (Fon‐1) were studied to determine the differences in infection and colonization of watermelon roots in cultivars resistant to and susceptible to Fusarium wilt. The roots of watermelon seedlings were inoculated with a conidial suspension of the GFP‐tagged isolate, and confocal laser scanning microscopy was used to visualize colonization, infection and disease development. The initial infection stages were similar in both the resistant and susceptible cultivars, but the resistant cultivar responded differentially after the pathogen had penetrated the root. The pathogen penetrated and colonized resistant watermelon roots, but further fungal advance appeared to be halted, and the fungus did not enter the taproot, suggesting that resistance is initiated postpenetration. However, the tertiary and secondary lateral roots of resistant watermelon also were colonized, although not as extensively as susceptible roots, and the hyphae had penetrated into the central cylinder of lateral roots forming a dense hyphal mat, which was followed by a subsequent collapse of the lateral roots. The initial infection zone for both the wilt‐susceptible and wilt‐resistant watermelon roots appeared to be the epidermal cells within the root hair zone, which the fungus penetrated directly after forming appressoria. Areas where secondary roots emerged and wounded root tissue also were penetrated preferentially.     

Purification and characterization of an exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici

Abstract An exopolygalacturonase produced by Fusarium oxysporum f. sp. radicis lycopersici , a fungus that produces root rot, was purified by gel filtration and ion exchange chromatography. It had a M _r 68 K, a pH optimum of 5.6 and an optimum temperature of 60°C. This polygalacturonase was inhibited by calcium ions and had a K _m of 0.64 mM using sodium polypectate as substrate. The exo mode of action of this enzyme was revealed by thin-layer chromatography of hydrolysed substrate.     

Detection and Quantification of Fusarium oxysporum f. sp. niveum Race 1 in Plants and Soil by Real-time PCR

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is the most serious soil-borne disease in the world and has become the main limiting factor of watermelon production. Reliable and quick detection and quantification of Fon are essential in the early stages of infection for control of watermelon Fusarium wilt. Traditional detection and identification tests are laborious and cannot efficiently quantify Fon isolates. In this work, a real-time polymerase chain reaction (PCR) assay has been described to accurately identify and quantify Fon in watermelon plants and soil. The FONRT-18 specific primer set which was designed based on identified specific sequence amplified a specific 172 bp band from Fon and no amplification from the other formae speciales of Fusarium oxysporum tested. The detection limits with primers were 1.26 pg/μl genomic DNA of Fon, 0.2 pg/ng total plant DNA in inoculated plant, and 50 conidia/g soil. The PCR assay could also evaluate the relationships between the disease index and Fon DNA quantity in watermelon plants and soil. The assay was further used to estimate the Fon content in soil after disinfection with CaCN₂. The real-time PCR method is rapid, accurate and reliable for monitoring and quantification analysis of Fon in watermelon plants and soil. It can be applied to the study of disease diagnosis, plant-pathogen interactions, and effective management.