几种尖孢镰刀菌专化型中SIX1、SIX4、SIX6、SIX8同源基因分析

尖孢镰刀菌在与寄主的相互作用中分泌几个特定的富含半胱氨酸的小分子量蛋白进入木质部中启动致病力,被称为SIX(secreted in xylem)蛋白,为明确其在不同寄主中的作用,本研究比较分析了几种尖孢镰刀菌专化型中SIX1、SIX4、SIX6、SIX8同源基因序列。根据已完成的尖孢镰刀菌古巴专化型1号(Foc1)与4号生理小种(Foc4)全基因组测序序列信息及相关SIX基因序列设计引物,应用PCR方法扩增分析56株尖孢镰刀菌古巴专化型与18株其它专化型及非致病型尖孢镰刀菌菌与其它种或属共21株菌株中的SIX1、SIX4、SIX6、SIX8基因。结果表明:设计的SIX1、SIX4、SIX6、SIX8基因引物均不能从非致病性尖孢镰刀菌与其它镰刀属种或其它属的菌株DNA中扩增出目的条带;SIX1基因的2个引物均能从供试的Foc菌株DNA中扩增出目的条带,同时可从部分其它专化型菌株DNA中扩增出目的条带;SIX4基因引物仅能从供试的尖孢镰刀菌番茄专化型与部分甘蓝专化型菌株DNA中扩增出目的条带;SIX6引物仅能从供试Foc1、Foc2、Foc4菌株DNA中扩增出目的条带;SIX8基因引物能从所有供试的致病尖孢镰刀菌中DNA扩增出目的条带。研究发现的SIX6基因序列提供了快速鉴定尖孢镰刀菌古巴专化型的检测方法,同时为深入研究尖孢镰刀菌各个专化型中SIX基因的功能奠定基础。     

湖北省蔬菜根际土壤中的镰孢菌分布特征

为明确湖北省蔬菜产区镰孢菌(Fusarium spp.)的种类及分布特征,本研究从湖北省13个地级市蔬菜产区采集根际土壤样品55份,对土样中的镰孢菌进行分离、纯化和鉴定,获得271株镰孢菌;通过镰孢菌特异性引物扩增、测序等分子生物学技术,鉴定出尖孢镰孢菌(F. oxysporum)118株,占分离菌株43.6%;木贼孢菌(F. equiseti)46株,占17.0%;共享镰孢菌(F.commune)45株,占16.6%;茄病镰孢菌(F. solani)31株,占11.4%;藤仓镰孢菌(F. fujikuroi)11株,占4.1%;镰孢霉菌(F.delphinoides)6株,占2.2%;轮纹镰孢菌(F. concentricum)5株,占1.8%;三线镰孢菌(F. tricinctum)3株,占1.1%;镰孢菌属(Fusarium spp.)6株,占2.2%。通过与致病菌尖孢镰孢菌番茄专化型(F. oxysporum f. sp. lycopersici)菌株进行对峙培养,筛选出拮抗镰孢菌16株,包括尖孢镰孢菌(F. oxysporum)4株,茄病镰孢菌(F. solani)4株,轮纹镰孢菌(F. concentricum)3株,三线镰孢菌(F.tricinctum)3株,木贼孢菌(F. equiseti)和藤仓镰孢菌(F. fujikuroi)各1株。本研究明确了湖北省蔬菜产区的镰孢菌属真菌的种类和多样性特征,为蔬菜镰孢菌病害的防治提供科学依据。     

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。     

滩涂盐碱地根际镰孢菌种类、非生物胁迫抗性及致病性

镰孢菌Fusarium spp.通常引起多种作物病害,但其中某些种类可作为植物内生菌从而提高植物的生长和抗逆性。因此,研究非农田生态系统镰孢菌物种多样性和生态功能具有重要的科学意义。本研究对分离自滩涂盐碱地盐生植物根际的23株镰孢菌进行了种类鉴定、非生物胁迫抗性及毒性评价研究。结果表明：经形态学和多基因联合建树分析(ITS+rpb2+tef1-α),这些菌株属于7个系统发育种,多数菌株属于尖孢镰孢菌F.oxysporum、恶臭镰孢菌F.foetens和藤仓镰孢菌F.fujikuroi。在7种非生物胁迫条件下[H2O2、亚硫酸氢钠甲萘醌(menadionesodiumbisulfite,MSB)、KCl、山梨醇(sorbitol)、荧光增白剂(calcofluorwhite,CFW)、刚果红(congored)、NaCl]测定菌株抗性,上述3种镰孢菌与Fusariumsp.1表现出相似的抗性能力;Fusariumsp.2和Fusarium sp.3尤其在离子胁迫和渗透胁迫方面表现出较强的适应性;芳香镰孢菌F. ambrosium则对多种胁迫均比较敏感。PCR检测表明大多数菌株含有恩镰孢菌...     

瓜类枯萎病菌遗传多样性和亲缘关系的SRAP分析

尖孢镰刀菌可造成不同瓜类的枯萎病.为明确不同寄主、不同地区的瓜类枯萎病菌菌株间的遗传多样性及亲缘关系,采用相关序列扩增多态性(SRAP)分子标记技术,对来源于不同地区、不同寄主的95株尖孢镰刀菌的基因组DNA进行多态性扩增.以筛选出的19对引物共扩增出238条带,多态性比率为100%,平均每对引物扩增出12.5个位点和12.5个多态性位点;尖孢镰刀菌苦瓜专化型共扩增出166条带,其中145条为多态性条带,多态性比率为87.4%,平均每对引物扩增出8.7个位点和7.7个多态性位点,说明尖孢镰刀菌的遗传变异较为广泛.瓜类枯萎病菌株间的遗传相似系数范围为0.68～0.99,样品间的平均Nei遗传多样性指数和Shannon指数分别为0.2390和0.3718.在遗传相似系数为0.74时,可将供试的95株尖孢镰刀菌划分为苦瓜、黄瓜、西瓜、甜瓜4个专化群.在SRAP聚类树中,同一寄主的尖孢镰刀菌聚在一个分支上,其中尖孢镰刀菌苦瓜专化型菌株间的遗传相似系数范围为0.78～0.99,Nei遗传多样性指数为0.1811,平均Shannon指数为0.2750,表明尖孢镰刀菌苦瓜专化型的遗传变异较大,且菌株的聚群与地理来源存在相关性.     

香蕉上的镰孢菌种类及其系统发育关系(英文)

镰孢菌属真菌是香蕉上的重要病原菌,主要引起香蕉枯萎病以及香蕉冠腐病,在我国已明确引起香蕉枯萎病的病原为尖孢镰孢古巴专化型 Fusarium oxysporum f. sp. cubense(FOC)1号和4号生理小种,但是引起香蕉冠腐病的镰孢菌种类还未明确。为了解香蕉上镰孢菌在种间及种内水平上的多样性,2008–2011 年间作者从华南地区不同的水果市场及香蕉果园采集香蕉样品90份,分离得到143株镰孢菌。通过形态学观察及基于 EF-1α基因的系统进化分析鉴定出10种镰孢菌,即F. oxysporum、F. solani、F. camptoceras、F. pallidoroseum、F. stiloides、F. chlamydosporum、F.verticillioides、F. proliferatum、F. concentricum、F. sacchari,以及藤仓赤霉复合种(Gibberella fujikuroi species complex,GFC)中 3 个未定名的类群。轮纹镰孢 F. concentricum 及甘蔗镰孢 F.sacchari 是香蕉果实中最常见种,前菌为我国首次报道,后菌是首次报道与香蕉有关。对从香蕉上分离的藤仓赤霉复合种(GFC)及尖孢镰孢复合种(FOSC)的EF-1α序列进行了系统发育分析,其GFC中的27个菌株组成的单系群可分为7个不同的亚群,分别为 F.verticillioides、F. proliferatum、F. concentricum、F. sacchari 以及3个没有描述过的菌系 Fusarium sp. 1、Fusarium sp.2和 Fusarium sp.3;FOSC中的50个菌株形成2大分枝共12个谱系,分离自我国华南地区的21株尖孢镰孢形成7个谱系,其中 13株已知的香蕉枯萎病病原菌分布在3个谱系中,我国大陆的香蕉枯萎病病原菌菌株与来源于台湾地区及东南亚的菌株亲缘关系较近,FOC1号生理小种的遗传分化大于4号生理小种,FOC 1号生理小种与分离自香蕉果实上的尖孢镰孢菌的亲缘关系比与FOC 4号生理小种的亲缘关系更近。研究结果表明,我国香蕉上存在着丰富的镰孢菌种类,而且种内遗传多样性丰富。     

尖孢镰孢一新硝酸盐营养突变类型与营养体亲和性

从73个尖孢镰孢(Fusarium oxysporum)不同专化型菌株上获得684个硝酸盐营养突变株(nit mutant)。作相关氮源利用试验及亚硝酸反应后,鉴定出一新硝酸盐营养突变类型:亚硝酸盐还原酶结构基因类型,命名为nit8,占总突变株的6.7%。同时被鉴别的还有nit1、nit3和Nit M三种突变类型,它们分别占突变株总数的81.0%,3.8%和8.5%。此外,首次引入一种亚硝酸反应在这类研究中的应用,还提出了互补指数概念与公式来表示nit突变株营养体之间亲和的能力。     

棘孢木霉挥发性次级代谢产物检测及抑菌活性分析

木霉是一类具有重要生防价值的丝状真菌。文中首先对分离自浙江省绍兴市和广东省佛山市共12株棘孢木霉Trichoderma asperellum进行平板拮抗评价,然后采用顶空固相微萃取气质联用法(HS-SPME-GC-MS)检测拮抗性较好的两株菌的挥发性次级代谢产物。结果表明,棘孢木霉ZJSX5003和GDFS1009菌丝生长迅速,对尖孢镰孢菌Fusariumoxysporum抑制率分别达73%和74%。挥发性次级代谢产物主要是醇类和酮类,其中包含异丁醇、异戊醇、3-甲基-3-丁烯-1-醇、3-羟基-2-丁酮、2,3-丁二醇和6-正戊基-2H-吡喃-2-酮(6-PAP)。进一步通过体外抑菌试验,证实6-PAP具有较好的抑制尖孢镰孢菌的效果,为开发以木霉菌代谢产物如6-PAP为主要成分的生防制剂提供指导。     

尖孢镰孢一新硝酸盐营养突变类型与营养体亲和性

从７３个尖孢镰孢不同专化型菌株上获得６８４个硝酸盐营养突变株。作相关氮源利用试验及亚硝酸反应后,鉴定出一新硝酸盐营养突变类型,亚硝酸盐还原酶结构基因类型,命名为ｎｉｔ８,占总突变株的６．７％,同时被鉴别的还有ｎｉｔ１,ｎｉｔ３和Ｎｉｔ Ｍ三种突变类型,它们分别占突变株总数的８１．０％,３．８％和８．５％。此外,首次引入一种亚硝酸反应在这类研究中的应用,还提出了互补指数概念与公式来表示ｎｉｔ突变株营     

抗油桐枯萎病木霉菌的分离鉴定及抑菌作用研究

油桐是我国重要的木本油料植物,其生产的桐油作为天然的优良干性油在工业上具有广泛用途。但由油桐专化型尖孢镰孢菌Fusarium oxysporum f. sp. fordiis（Fof-1）侵染引起的枯萎病给油桐产业造成了毁灭性灾害,目前尚无有效防治手段。众多实践证明,利用生防菌可以有效防治土传枯萎病。本研究发现,在抗病油桐根围土壤中木霉菌的相对丰度较高,并从中分离获得了16株木霉菌;通过形态学鉴定和ITS-TEF1双基因联合构建系统进化树,鉴定出4种木霉菌：拟康宁木霉Trichoderma koningiopsis（TkonT1）、螺旋木霉T. spirale（TspiT2）、深绿木霉T. atroviride（TatrT3）和哈茨木霉T. harzianum（TharT4）;通过对峙培养试验,发现木霉菌株TkonT1、TharT4和TspiT2具有较好的抑菌效果;进一步显微观察发现菌株TkonT1和TatrT3可缠绕在尖孢镰孢菌菌丝体上或穿入菌丝体内营寄生生长,吸收病菌菌丝体养分进而导致病菌菌丝体破裂和细胞原生质消解。结果表明,从抗病油桐根围土壤中获得的拮抗木霉菌株可用于油桐枯萎病的生物防治。     

Characterization of the formae speciales of Fusarium oxysporum causing wilts of cucurbits by DNA fingerprinting with nuclear repetitive DNA sequences.

The genetic relatedness of five formae speciales of Fusarium oxysporum causing wilts of cucurbit plants was determined by DNA fingerprinting with the moderately repetitive DNA sequences FOLR1 to FOLR4. The four FOLR clones were chosen from a genomic library made from F. oxysporum f. sp. lagenariae 03-05118. Total DNAs from 50 strains representing five cucurbit-infecting formae speciales, cucumerinum, melonis, lagenariae, niveum, and momordicae, and 6 strains of formae speciales pathogenic to other plants were digested with EcoRV and hybridized with 32P-labeled FOLR probes. The strains were clearly distinguishable at the formae specialis level on the basis of FOLR DNA fingerprints. Fifty-two fingerprint types were detected among the 56 strains by using all FOLR probes. These probes were used to infer phylogenetic relationships among the DNA fingerprint types by the unweighted pair group method using averages and parsimony analysis. The fingerprint types detected in each of the formae speciales cucumerinum, lagenariae, niveum, and momordicae were grouped into a single cluster. However, two different genetic groups occurred in the formae specialis melonis. The two groups also differed in pathogenicity: one group caused wilts of muskmelon and oriental melon, while the second was pathogenic only to muskmelon. The fingerprint types of different formae speciales pathogenic to plants other than cucurbits were distinguishable from one another and from the fingerprints of the cucurbit-infecting strains. These results suggest that the cucurbit-infecting formae speciales are intraspecific variants distinguishable at the DNA level and in their host range.     

A robust identification and detection assay to discriminate the cucumber pathogens Fusarium oxysporum f. sp. cucumerinum and f. sp. radicis-cucumerinum

The fungal species Fusarium oxysporum is a ubiquitous inhabitant of soils worldwide that includes pathogenic as well as non-pathogenic or even beneficial strains. Pathogenic strains are characterized by a high degree of host specificity and strains that infect the same host range are organized in so-called formae speciales. Strains for which no host plant has been identified are believed to be non-pathogenic strains. Therefore, identification below the species level is highly desired. However, the genetic basis of host specificity and virulence in F. oxysporum is so far unknown. In this study, a robust random-amplified polymorphic DNA (RAPD) marker-based assay was developed to specifically detect and identify the economically important cucumber pathogens F. oxysporum f. sp. cucumerinum and F. oxysporum f. sp. radicis-cucumerinum. While the F. oxysporum radicis-cucumerinum strains were found to cluster in a separate clade based on elongation factor-1alpha phylogeny, strains belonging to F. oxysporum f. sp. cucumerinum were found to be genetically more diverse. This is reflected in the observation that specificity testing of the identified markers using a broad collection of F. oxysporum strains with all known vegetative compatibility groups of the target formae speciales, as well as representative strains belonging to other formae speciales, resulted in two cross-reactions for the F. oxysporum f. sp. cucumerimum marker. However, no cross-reactions were observed for the F. oxysporum f. sp. radicis-cucumerimum marker. This F. oxysporum f. sp. radicis-cucumerimum marker shows homology to Folyt1, a transposable element identified in the tomato pathogen F. oxysporum f. sp. lycopersici and may possibly play a role in host-range specificity in the target forma specialis. The markers were implemented in a DNA array that enabled parallel and sensitive detection and identification of the pathogens in complex samples from diverse origins.     

Identification of F. o. cucumerinum and F. o. luffae by RAPD-generated DNA probes

AIMS: To create a fast, sensitive and specific method for identifying Fusarium oxysporum f. sp. cucumerinum and F. o. luffae. METHODS AND RESULTS: Specific DNA bands were selected as probes from RAPD profiles of 13 formae speciales of F. oxysporum. The forma specialis-specific probe OPC18300c and OPC18520f could be used to identify F. o. cucumerinum and F. o. luffae by RAPD-PCR followed dot blot hybridization, respectively. CONCLUSIONS: A specific method for identifying F. o. cucumerinum and F. o. luffae was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: F. oxysporum formae speciales identification with a DNA probe can be relatively rapid and provides a method to identify the pathogen without host inoculation tests.     

尖孢镰刀菌四个专化型细胞核的初步研究

本文对尖孢镰刀菌(Fusarium oxysporum)的4个专化型12株菌的孢子萌发、核分裂时间及核DNA含量进行了比较,结果表明:不同专化型菌株孢子萌发速度基本一致,而萌发过程中发生第一次核分裂的时间不同。黄瓜、西瓜、荸荠、大豆各专化型第一次核分裂时间分别为:5.67hr、5.45hr、7.35hr、7.82hr,其核DNA含量分别为:0.321pg、o.306pg、0.177pg 0.174pg。测定结果显示出不同专化型菌株的孢子核DNA含量可能存在倍数关系,并推测F.oxysporum的黄瓜专化型与西瓜专化型为同一类型,而荸荠专化型与大豆专化型为同一类型。     

Effector gene screening allows unambiguous identification of Fusarium oxysporum f. sp. lycopersici races and discrimination from other formae speciales

During infection of tomato, the fungus Fusarium oxysporum f. sp. lycopersici secretes several unique proteins, called 'secreted in xylem' (Six) proteins, into the xylem sap. At least some of these proteins promote virulence towards tomato and among them, all predicted avirulence proteins that can trigger disease resistance in tomato have been found. In this study, a large, worldwide collection of F. oxysporum isolates was screened for the presence of seven SIX genes ( SIX1 – SIX7 ). The results convincingly show that identification of F. oxysporum formae speciales and races based on host-specific virulence genes can be very robust. SIX1, SIX2, SIX3 and SIX5 can be used for unambiguous identification of the forma specialis lycopersici . In addition, SIX4 can be used for the identification of race 1 strains, while polymorphisms in SIX3 can be exploited to differentiate race 2 from race 3 strains. For SIX6 and SIX7 , close homologs were found in a few other formae speciales , suggesting that these genes may play a more general role in pathogenicity. Host specificity may be determined by the unique SIX genes, possibly in combination with the absence of genes that trigger resistance in the host.     

The presence of a virulence locus discriminates Fusarium oxysporum isolates causing tomato wilt from other isolates

Fusarium oxysporum is an asexual fungus that inhabits soils throughout the world. As a species, F. oxysporum can infect a very broad range of plants and cause wilt or root rot disease. Single isolates of F. oxysporum, however, usually infect one or a few plant species only. They have therefore been grouped into formae speciales (f.sp.) based on host specificity. Isolates able to cause tomato wilt (f.sp. lycopersici) do not have a single common ancestor within the F. oxysporum species complex. Here we show that, despite their polyphyletic origin, isolates belonging to f.sp. lycopersici all contain an identical genomic region of at least 8 kb that is absent in other formae speciales and non-pathogenic isolates, and comprises the genes SIX1, SIX2 and SHH1. In addition, SIX3, which lies elsewhere on the same chromosome, is also unique for f.sp. lycopersici. SIX1 encodes a virulence factor towards tomato, and the Six1, Six2 and Six3 proteins are secreted in xylem during colonization of tomato plants. We speculate that these genes may be part of a larger, dispensable region of the genome that confers the ability to cause tomato wilt and has spread among clonal lines of F. oxysporum through horizontal gene transfer. Our findings also have practical implications for the detection and identification of f.sp. lycopersici.     

Isozyme Variation and Genetic Distances of Erysiphe graminis DC. Formae Speciales

Abstract Isozymes of ten different enzymes and unspecific stained proteins were used as biochemical genetic markers to study genetic variation within and between E. graminis ff. sp. hordei, avenae, secalis and tritici. In addition, grainproteins of the corresponding host species were examined. In each forma specialis, one genotype proved to be predominant. 131 distinct isozyme and 93 protein bands were distinguishable in these genotypes. However, divergent banding patterns differed only in 8 bands from the predominant banding patterns found within the formae speciales avena, secalis and tritici. The genetic relationships between powdery mildew formae speciales and host species were computed by cluster analysis from similarity (F) and dissimilarity (D) coefficients and illustrated by phylogenetic trees. Marked correspondence was found between E. graminis ff. sp. secalis and tritici (F: 82–90%). Lower homologies were obtained from the comparison ofthese formae speciales respectively with E. graminis ff. sp. hordei (F: 28–34%) and avenae (F: 24–32%). All phylogenetic trees constructed revealed the same arrangement classification of the formae speciales with similar graduation. The comparison of the host species revealed the highest similarity between S. cereale and T. aestivum (F: 74%). Regression analysis confirmed significant correlation between the genetic relationships within host species and powdery mildew formae speciales (r²= 0.81).     

Species-specific primers for Fusarium redolens and a PCR-RFLP technique to distinguish among three clades of Fusarium oxysporum

The currently available morphological and molecular diagnostic techniques for Fusarium redolens and the three phylogenetic clades of Fusarium oxysporum are problematic. Aligned translation elongation factor 1 alpha (TEF-1 alpha) gene sequences from these species and their close relatives were used to design F. redolens-specific primers, and to identify restriction sites that discriminate among the three clades of F. oxysporum. The F. redolens-specific primers distinguished this species from all others included in the study. There were three TEF-1 alpha-RFLP patterns among formae speciales of F. oxysporum. These PCR-RFLP patterns corresponded with the three clades. These techniques provide simple and inexpensive diagnostic methods for the identification of F. redolens and members of the three clades of F. oxysporum.     

The cell wall of Fusarium oxysporum.

Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50-60% of the total mass of the wall. X-ray diffraction studies showed the presence of alpha-1, 3-glucan in the alkali-soluble cell wall fraction and of beta-1, 3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan.