Host Range ofVerticillium dahliae in cultivated species in Crete

To determine the host range ofVerticillium dahliae among the cultivated species in Crete, Greece, studies were carried out during 1992–2000. Based on disease symptoms observed on 28 vegetable and forage species grown in a field naturally infected byV. dahliae, and an extensive survey of the most common cultivated species grown under natural conditions, seven hosts belonging to four botanical families not previously reported as susceptible to Verticilliun wilt, and 12 hosts belonging to seven families new for Greece, were recorded. The worldwide new hosts are: anise (Anethum graveolens), chard (Beta vulgaris ssp.cicla), chickpea (Cicer arietinum), wild sweet pea (Lathyrus ochrus), lentil (Lens culinaris), marigold (Tagetes erecta) and vetch (Vicia sativa). These species could be infected by their hosts-of-originV. dahliae isolates during pathogenicity tests. http://www.phytoparasitica.org posting Jan. 13, 2002.     

Weed hosts ofVerticillium dahliae in crete: Susceptibility, symptomatology and significance

A survey of common and uncommon weed species usually showing Verticillium wilt symptoms was carried out during 1992–2000 in Crete, Greece.Verticillium dahliae was isolated in 48 out of 182 sampled fields, in which several weed species were grown, from several locations in Oropedio, Lasithi. Altogether, 124 isolates ofV. dahliae were recovered from the vascular stem-tissue of 19 weed species, belonging to ten botanical families. Pathogenicity trials with 13 out of 19 weed species that have never been reported as hosts of the fungus, using for inoculation isolates which originated from the same weed species, resulted in infection of all of them, showing various disease symptoms. Seven weed species (Anthemis melanolepis, Cardaria draba, Convolvulus arvensis, Erodium sp.,Euphorbia helioscopia, Helminthotheca echioides andSinapis alba) are new hosts worldwide, and six additional species (Euphorbia sp.,Lactuca serriola, Raphanus raphanistrum, Sinapis arvensis, Sonchus oleraceus andTrifolium sp.) are new hosts for Greece. The most susceptible (isolation frequency: 27.9–52.8%, moderate disease severity) species were:Capsella bursa-pastoris, C. draba, Chenopodium album, Senecio vulgaris andSolanum nigrum. Less susceptible (isolation frequency: 4.8–17.8%, slight disease severity) were:Amaranthus sp.,A. melanolepis, C. arvensis, Erodium sp.,Euphorbia sp.,E. helioscopia, H. echioides, L. serriola, Malva sylvestris, R. raphanistrum, S. alba, S. arvensis, S. oleraceus andTrifolium sp. Some species —C. draba, C. album, L. serriola andS. nigrum L. — that usually showed external and vascular wilt symptoms, occasionally exhibited only reduced growth. Visible symptoms under natural field conditions in all 13 weed species that had never been reported as hosts ofV. dahliae were similar to those observed after their artificial inoculation. The fungus was not isolated fromFoeniculum vulgare ssp.piperitum, Oxalis corniculata andStellaria media, among other species. http://www.phytoparasitica.org posting Sept. 18, 2002.     

棉花黄萎病病原菌大丽轮枝菌的快速分子检测

大丽轮枝菌Verticillium dahliae是引起棉花黄萎病的土传病害病原真菌。快速及时地检测出大丽轮枝菌,对棉花黄萎病的早期预警及后期防治具有重要意义。采用聚合酶链式反应(polymerase chain reaction,PCR)检测技术对已报道大丽轮枝菌的检测引物进行验证、筛选和改进,获得1对改进的特异性PCR引物VDS-F/VDS-R。在优化的反应体系与扩增条件下,能特异性地从大丽轮枝菌基因组扩增出l条约520 bp的产物条带,检测灵敏度达到10~(-2)ng/μL;利用该引物可特异性地从含有大丽轮枝菌的土壤及棉花植株组织中检测出病原菌;采用巢氏PCR法对人工病土的检测灵敏度达到了10个孢子/g土。表明本引物的PCR检测体系可用于棉花黄萎病的早期快速检测。     

Selection and Screening of Endorhizosphere Bacteria from Solarized Soils as Biocontrol Agents Against Verticillium dahliae of Solanaceous Hosts

Verticillium dahliae antagonistic endorhizosphere bacteria were selected from root tips of tomato plants grown in solarized soils. Fifty-three out of the 435 selected bacterial isolates were found to be antagonistic against V. dahliae and several other soilborne pathogens in dual cultures. Significant biocontrol activity against V. dahliae in glasshouse trials was demonstrated in three of 18 evaluated antagonistic isolates, provisionally identified as Bacillus sp. Although fluorescent pseudomonads were also isolated from root tips of tomato plants, none of the tested isolates exercised any significant antagonistic activity against V. dahliae in dual cultures. So these isolates were not tested in glasshouse trials in this study. Finally, two of the most effective bacterial isolates, designated as K-165 and 5-127, were shown to be rhizosphere colonizers, very efficient in inhibiting mycelial growth of V. dahliae in dual cultures and successfully controlling Verticillium wilt of solanaceous hosts. In glasshouse experiments, root dipping or soil drenching of eggplants with bacterial suspension of 10⁷cfu ml^–1 resulted in reduced disease severity expressed as percentage of diseased leaves (40–70%) compared to the untreated controls under high V. dahliae inoculum level (40 microsclerotia g^–1 soil). In heavily Verticillium infested potato fields, experiments with potato seeds dusted with a bacterial talc formulation (10⁸cfu g^–1 formulation), showed a significant reduction in symptom development expressed as percentage of diseased potato plants and a 25% increase in yield over the untreated controls. As for their effectiveness in increasing plant height, both bacterial isolates K-165 and 5-127 produced indolebutyric, indolepyruvic and indole propionic acids. Both antagonists are considered as plant growth promoting rhizobacteria bacteria since significantly increased the height of treated plants compared with the untreated controls. Chitinolytic activity test showed that both isolates were able to produce chitinase. Testing rhizospheric and endophytic activity of the antagonists it was shown that although the bacteria are rhizosphere inhabitants they also preferentially colonize the endorhizosphere of tomatoes and eggplants. Fatty acid analysis showed that isolate K-165 could belong to Paenibacillus alvei while 5-127 to Bacillus amiloliquefaciens.     

向日葵黄萎病病原菌轮枝菌的多重DPO-PCR检测方法

为建立可同时快速检测引起向日葵黄萎病害的2种检疫性病原菌大丽轮枝菌Verticillium dahliae Kleb.和黑白轮枝菌V.albo-atrum Reinke et Berthold的方法,根据2种病原菌的β-tubulin基因分别设计特异性DPO引物,建立多重DPO-PCR检测方法,并对其特异性和灵敏度进行评价。结果表明,所设计的DPO引物特异性强,仅大丽轮枝菌和黑白轮枝菌可分别扩增出225 bp与151 bp的特异性条带,其它向日葵病害的7种病原菌及阴性对照均无目的条带;反应体系中引物终浓度为0.2μmol/L、退火温度为60℃时,30个扩增循环的检测灵敏度均可达0.05 ng菌丝DNA量;在45~65℃退火温度范围内均可高效扩增靶基因片段,表明该方法退火温度范围宽。所建立的检测方法能够准确、高效地检测引起向日葵黄萎病的大丽轮枝菌和黑白轮枝菌,可用于向日葵种子带菌筛查及田间病害诊断检测。     

向日葵黄萎病菌突变体库的建立及其阳性转化子的鉴定

为揭示向日葵大丽轮枝菌Verticillium dahliae Kleb.的致病机理,利用农杆菌介导法将带有潮霉素抗性标记和绿色荧光蛋白报告基因的双元载体转入大丽轮枝菌的分生孢子中并获得阳性转化子,以野生型菌株为对照,对随机挑取的阳性转化子的菌落形态、菌丝生长速率、产孢量、粗毒素分泌量和致病力进行了研究。结果表明,共获得800株阳性转化子,随机选取的40株阳性转化子中有2株的菌落只产生白色气生菌丝,不能形成黑色微菌核。与对照相比,40株转化子的生长速率均有不同程度降低,其中转化子A1生长速度降低最显著,菌落直径仅为3.28 cm,比对照下降了38.92%。40株转化子中有3株的产孢量高于对照,其中转化子A9的产孢量最高,为3.50×10~7个/mL,比对照提高1.10倍;转化子A1的产孢量最低,仅为1.35×10~7个/mL,比对照下降了19.16%。40株转化子中有4株的粗毒素分泌量较对照显著升高,占测定菌株的10%,有24株较对照显著降低,占60%,其余12株与对照无显著差异。40株转化子中有3株的致病力较对照显著增强,占测定菌株的7.5%;有7株的致病力较对照显著降低,占17.5%;其余30株与对照无显著差异。     

棉秆生物炭对大丽轮枝菌生长及毒素作用的影响

棉花黄萎病难以防治的根源在于大丽轮枝菌Verticillium dahliae Kleb在土壤中形成的微菌核能抵抗不良环境,并在土壤中长期存活,将棉秆加工成生物炭施入棉田,可克服棉秆直接还田的缺点,从源头上防止黄萎病菌进入土壤。为探索棉秆生物炭还田对棉花黄萎病原菌的影响机理,采用液态摇床培养及平皿固态培养法研究生物炭对大丽轮枝菌菌丝生长及微菌核萌发的影响;通过平皿内棉花和甜瓜种子发芽试验研究生物炭对大丽轮枝菌毒素的减毒作用。结果表明:液态培养条件下,棉秆生物炭对大丽轮枝菌菌丝生长抑制作用不明显,在固态培养下对菌丝体生长有一定促进作用;生物炭能减慢微菌核的萌发速率,但对最终萌发率无影响;液态培养中加入棉秆生物碳对大丽轮枝菌毒素有较强的减毒作用。在摇床培养中期,加入2.0 g·L-1生物炭处理与对照棉种在培养12天时的发芽率分别为53.3%与33.3%(P0.05);前期、中期及后期加入0.5 g·L-1生物炭处理的棉花幼苗总长度分别为对照的4.9、2.1倍及3.2倍;加入棉秆生物碳处理甜瓜种子发芽率、胚根长度、胚轴长度及总苗长较对照均有不同程度增加。表明棉杆生物炭对棉花黄萎病菌大丽轮枝菌菌丝生长、微菌核萌发的抑制作用较弱,对大丽轮枝菌毒素危害有较强的减毒作用。     

检疫性轮枝菌及其近似种的鉴定

 大丽轮枝菌(Verticillium dahliae)和黑白轮枝菌（V. albo-atrum）在世界范围内引起多种作物的黄萎病,属于我国重要进境植物检疫对象。本研究对采自我国部分地区和CBS保存的多种植物病原性轮枝菌,包括黑白轮枝菌、大丽轮枝菌及其变种大丽轮枝菌长孢变种(V. dahliae var. longisporum)、三体轮枝菌(V. tricorpus)、变黑轮枝菌(V. nigrescens)和云状轮枝菌(V. nubilum),采用生物学特性观察,结合rDNA-ITS序列分析的方法,进行了比较和分析。结果表明：不同种类轮枝菌在休眠结构形态上具有一定差异,部分菌株不产生任何休眠结构。各供试菌株在15～25℃范围内均可生长,但黑白轮枝菌在30℃下生长受到强烈抑制,而其他菌株受影响较小。对供试菌株rDNA-ITS序列分析结果表明植物病原性轮枝菌可聚为9个分支,包括三体轮枝菌、变黑轮枝菌、云状轮枝菌、V. theobromae、大丽轮枝菌、大丽轮枝菌长孢变种和3个不同的黑白轮枝菌分支,黑白轮枝菌、大丽轮枝菌及其长孢变种亲缘关系较近。采用生物学性状结合rDNA-ITS序列分析能够更加有效地将两种检疫性轮枝菌从其他植物病原性轮枝菌中区分出来。     

Genetic variability and virulence among Verticillium albo-atrum isolates from hop

Verticillium wilt, caused by Verticillium albo-atrum or V. dahliae, is an important disease of many worldwide crop species. In Europe, V. albo-atrum isolates infecting hop express different levels of virulence, inducing mild or lethal disease syndromes, and it is therefore an attractive model for studying the virulence of this pathogen. In this work, eleven amplified fragment length polymorphism (AFLP) primer combinations were used to analyze genetic variability among 55 V. albo-atrum hop isolates from four European hop growing regions, as well as isolates from other hosts and V. dahliae isolates. Cluster analysis divided V. albo-atrum and V. dahliae isolates into two well-separated groups. Within the V. dahliae cluster, isolates were separated without host specific grouping, although no host adapted isolates were included. In V. albo-atrum, the alfalfa isolates were distinct from isolates of other hosts, where a high association with virulence was observed in hop and tomato isolates. All lethal hop isolates were genetically different from mild hop isolates. The lethal hop isolates from England and Slovenia expressed the same virulence phenotype, although they showed a different AFLP pattern. The mild hop isolates formed two subgroups, to which isolates clustered irrespective of geographical location. These data suggest multiple origins of V. albo-atrum hop isolates, and the possible appearance of new virulent isolates in the future in other hop growing regions.     

Possibilities of biological and chemical control of Verticillium wilt in pepper

In pathogen populations in Serbia, the incidence, pathogenic and morphological characters ofVerticillium spp. were studied. Biological and chemical control ofVerticillium was investigated in pepper plants (Capsicum annuum L. cv. ‘Soroksari’) with the biofungicide Polyversum^® (Pythium oligandrum) and the conventional fungicides benomyl and propamocarb-hydrochloride. On the basis of macroscopic and microscopic characters of the isolates originating from eight localities in Serbia, it was established that they apparently belong to the speciesVerticillum dahliae. The isolates differed in their pathogenic characters. However, all of them caused marked wilting symptoms on pepper plants 40 days after inoculation, conducted when there were more than nine fully developed leaves on the primary stem. The fungicides were applied either before or after inoculation. Benomyl was the most efficient fungicide in wilt control (88.2% when applied after inoculation and 94.6% when applied before inoculation). Polyversum proved more efficient (66.6%) when applied before rather than after inoculation. Propamocarb-hydrochloride provided sufficient Verticillium wilt control; its efficacy and that of Polyversum were similar, and less efficient than benomyl, but still significantly different from the disease control.     

Tolerance to a non-host isolate of Verticillium dahliae in tomato

Tolerance to Verticillium spp. is a condition in which a host plant develops few symptoms despite substantial colonization by the pathogen. In the present paper we have shown that Craigella tomatoes are tolerant to a non-host isolate of V. dahliae, Dvd E6. Symptom expression was used to quantify disease and quantitative PCR to assess the amount of fungus in the stems. The classical incompatible and compatible interactions between Craigella resistant or Craigella susceptible near isolines and V. dahliae, race 1 were used for comparative purposes. Additional experiments using cytological assessment and quantitative PCR showed that in the tolerant interactions one plant defence response, vascular coating, was deployed as effectively as in resistant plants, limiting pathogen distribution. However, a second defence response, which causes the cyclical elimination of fungus from the stem in the classical interactions either does not occur or is substantially delayed in tolerant plants. Thus, the Verticillium population remains stable and substantial throughout the studied time course.     

棉花内生真菌CEF-373菌株对棉花黄萎病的防效及其作用机理

为明确棉花内生真菌CEF-373菌株对棉花黄萎病的防效及其作用机理,利用圆盘滤膜法和平板对扣培养法测定菌株CEF-373对大丽轮枝菌Verticillium dahliae菌丝生长的抑制作用,测定其对棉花黄萎病的温室和田间防效,并通过活性氧含量及防御基因表达情况来分析其作用机理。结果表明,菌株CEF-373的挥发性代谢产物和非挥发性代谢产物对大丽轮枝菌菌丝的生长均有显著抑制作用,抑制率最高分别可达37.75%和100.00%。用1×107CFU/mL的菌株CEF-373分生孢子悬浮液灌根后,对棉花黄萎病的温室防效可达71.12%,用质量比为3%的菌株CEF-373固体菌剂拌土栽培后,对棉花黄萎病的温室防效可达62.74%,防治作用显著。菌株CEF-373的发酵液滴灌和微生物肥料处理40 d后,对棉花黄萎病的田间防效达到最大,分别为36.23%和27.71%,而在后期有所降低。菌株CEF-373可以诱导棉花叶片中细胞活性氧的爆发;且菌株CEF-373成功激活了苯丙氨酸解氨酶、过氧化物酶、多酚氧化酶、几丁质酶和病程相关蛋白基因PR10的表达,对大丽轮枝菌的侵染具有抵御作用。表明棉花内生真菌CEF-373菌株通过抑制大丽轮枝菌生长以及诱导寄主系统抗病性来有效防治棉花黄萎病,具有较好的生物防治应用前景。     

沙打旺黄萎病菌的分离鉴定及其生物学特性和遗传多样性

为明确沙打旺Astragalus adsurgens黄萎病病原菌的种类及其生物学特性和遗传多样性,结合形态学特征和分子生物学方法对分离自疑似感染黄萎病沙打旺植株的病原菌进行鉴定,并依照柯赫氏法则进行致病性验证,同时在室内条件下对病原菌的生物学特性进行测定,分别利用大丽轮枝菌Verticillium dahliae生理小种和交配型特异性引物对病原菌的生理小种和交配型进行PCR分子鉴定。结果表明,共分离到13株病原菌菌株,其形态学特征和培养性状基本相似,经鉴定引起沙打旺黄萎病的病原菌为大丽轮枝菌。该菌具有较宽的温度和酸碱度适应范围,最适生长和产孢的温度为25℃,最适生长和产孢的pH范围分别为pH 7～9和pH 6～7;该病原菌生长和产孢的最适碳源为可溶性淀粉和D-甘露醇,最适生长氮源为牛肉膏和胰蛋白胨,最适产孢氮源为甘氨酸。分离获得的13株大丽轮枝菌菌株均属于2号生理小种和MAT1-2-1交配型,表明无性繁殖可能是大丽轮枝菌的主要繁殖形式。     

Incidence of Verticillium wilt of artichoke in eastern Spain and role of inoculum sources on crop infection

Surveys of 94 artichoke fields throughout the artichoke production areas of Comunidad Valenciana (eastern Spain) were conducted from 1999 to 2002 to determine the incidence and distribution of Verticillium wilt.Verticillium dahliae was isolated from 80.9% of the sampled fields, and detected in all artichoke-growing areas, with a mean disease incidence of 53.8% infected plants. The disease was found to cause severe damage to cv. ‘Blanca de Tudela’, which is the most important artichoke cultivar grown in Spain, and was also observed on the seed-propagated cv. ‘Imperial Star’. In field trials to study the role of infected planting material and soil inoculum on infection of artichoke plants during the cropping season,V. dahliae was transmitted from infected stumps to the plants, confirming that the use of infected stumps could have greatly contributed to the dissemination of the pathogen. Inoculum density ofV. dahliae in soil had an effect on crop infection, in that a higher number of microsclerotia per gram of soil resulted in a higher percentage of infected plants. In addition, yield of cv. Blanca de Tudela was significantly affected byV. dahliae infection, showing that a higher percentage of infection corresponded with lower yield. http://www.phytoparasitica.org posting July 21, 2005.     

Retrotransposon-like elements in the genome of Verticillium dahliae may be used as DNA markers for fungal species and pathotypes

Using differential hybridization, two DNA fragments, VDf35 and VDf90, specific to Verticillium dahliae, were isolated. These fragments contained truncated open reading frames (ORFs) homologous to the gypsy-type retrotransposon. The ORFs of VDf35 and VDf90 were pol and gag homologs, respectively. In addition, VDf90 had a pol homolog without an ORF sequence. The pol homologs in VDf35 and VDf90 were similar to each other, and these two DNA fragments had completely identical sequences. Genomic Southern analysis revealed that numerous copies of these homologs existed in V. dahliae, suggesting that V. dahliae carries a gypsy-like retroelement. Genomic Southern and polymerase chain reaction (PCR) analysis also indicated that a large number of these homologs exist in V. longisporum as well as in V. dahliae, but only a few were present in V. albo-atrum. No homolog was found in either V. nigrescens or V. tricorpus. The uneven distribution of these homologs of the retroposon-like elements among Verticillium species suggested a close genetic kinship between V. dahliae and V. longisporum. PCR primers designed from VDf35 showed species- or pathotype-specific amplification. Therefore, this sequence may be useful as a DNA marker to identify species and pathotypes of V. dahliae. This is the first report on a retrotransposon-like sequence in the genome of phytopathogenic Verticillium species.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB095264 (VDf90) and AB095265 (VDf35)     

大丽轮枝菌蛋白激发子PevD1诱导棉花抗病性及作用机理

 PevD1是一种大丽轮枝菌(Verticillium dahliae)分泌蛋白,具有激发烟草过敏反应(HR)和系统获得性抗病(SAR)的功能。为明确蛋白PevD1诱导棉花抗病性及其作用机制,本文利用大肠杆菌表达、纯化的PevD1诱导棉苗植株,检测棉苗对大丽轮枝菌的抗性及免疫应答反应。结果表明,大肠杆菌表达的PevD1重组蛋白不是棉花品种“新陆早42号”的致萎因子,叶片注射8 μg/mL PevD1蛋白诱导3 d后根部接种大丽轮枝菌,15 d后PevD1处理组病害减轻率达35.04%。PevD1能诱导棉花叶片抗性早期信号分子H₂O₂产生和NO积累,维管束细胞壁加厚、木质素和酚类物质的积累。另外,PevD1处理能提高防御酶PAL、POD和PPO活性,提高棉花抗性基因和木质素合成相关基因PAL、C4H1、4CL的转录水平。说明PevD1通过激发棉花免疫系统而提高抗病性,该研究不仅为利用PevD1蛋白激发子控制棉花黄萎病提供了科学依据,同时也为阐明棉花与大丽轮枝菌互作机理提供了理论基础。     

Vegetative compatibility and pathogenicity of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> isolates from chrysanthemum in Turkey

A collection of 30 strains of Verticillium dahliae, recovered during 2004–2006 from 12 cultivars of chrysanthemum (Chrysanthemum morifolium) in five districts of İzmir province in Turkey, was assigned to vegetative compatibility groups (VCGs) based on pairings of complementary nitrate-nonutilizing (nit) mutants induced on a chlorate-containing medium. Of these strains, nine were assigned to VCG1, seven to VCG2A, 11 toVCG2B and one to VCG4B. The remaining two strains could not be tested for vegetative compatibility because of their inability to yield nit mutants. Pathogenicity tests conducted by the root-dip method, demonstrated that wilt of chrysanthemum in Turkey is caused by V. dahliae, and most strains in VCG1 were significantly more aggressive to chrysanthemum than those in VCGs 2 and 4B. This is the first known study in the world of the VCGs of V. dahliae isolates from chrysanthemum.     

新疆棉花黄萎病的发生现状及其病原菌的分子鉴定与ISSR分析

为掌握新疆主要植棉区棉花黄萎病的发生现状及其病原菌大丽轮枝菌Verticillium dahliae的落叶型菌系分布以及遗传变异情况,于2015年对26个新疆主要植棉区棉花黄萎病的发生情况进行了随机调查,统计新疆大丽轮枝菌的培养性状,利用大丽轮枝菌落叶型特异引物D1/D2、INTD2F/INTD2R与非落叶型特异性引物ND1/ND2、INTNDF/INTNDR对新疆大丽轮枝菌菌系进行互补鉴定,并对部分菌系的遗传变异进行简单序列重复区间(inter simple sequence repeat,ISSR)分析。结果表明:2015年新疆棉花黄萎病发病田比例为54.0%,其中病情指数在10.0以上的发病田与2013年持平,而病情指数在20.0以上的严重发病田比例为10.8%,比2013年增加3.8个百分点;新疆大丽轮枝菌的培养性状以菌核型为主,比例为70.1%,菌丝型与中间型比例分别为13.4%和16.5%;新疆大丽轮枝菌落叶型菌系比例为53.2%,26株菌株的来源地全部检出落叶型菌系;聚类分析结果显示,当遗传相似系数为0.66时,新疆大丽轮枝菌落叶型与非落叶型菌系聚为2个谱系,菌系地理来源、培养性状与大丽轮枝菌的遗传分化无明显相关性。