多菌灵、三环唑对大丽轮枝菌微菌核、黑色素形成及致病力的影响

 在离体和活体条件下测定了多菌灵、三环唑对大丽轮枝菌的微菌核黑色素形成的影响,以及经2种杀菌剂处理产生的形态变异菌株对棉苗的致病力。结果表明:多菌灵在培养基内含量超过0.1μg/ml时,即可抑制大丽轮枝菌微菌核的形成,随着培养基内多菌灵浓度的增加,微菌核形成时间逐渐延长,形成量逐渐减少;三环唑浓度为0.5μg/ml时,可抑制微菌核的黑色素形成,微菌核从黑色变为浅红至红褐色。三环唑对微菌核黑色素形成呈可逆抑制,变色的微菌核菌落移入不含药的培养基后,大多可恢复形成黑色素。培养基内三环唑浓度的提高,也可抑制大丽轮枝菌微菌核的形成;因2种杀菌剂的抑制而丧失形成微菌核的白色菌丝体移入不含药的培养基,微菌核形成能力也不能恢复。多菌灵和三环唑经棉株吸收后均能抑制植株内大丽轮枝菌微菌核的形成,但对微菌核色素的形成未见有明显影响。2种杀菌剂处理产生的形态变异菌株的致病力与野生型菌株致病力差异不显著。     

落叶型大丽轮枝菌T-DNA插入突变体库的构建

采用ATMT技术建立大丽轮枝菌落叶型菌株XJ2008菌株的T-DNA插入突变体文库,共获得6 043个突变体。从中随机挑选104个突变体,以野生型XJ2008菌株为参照,评价其致病性、菌落生长速率、分生孢子及微菌核的产生能力等。结果表明,有12.5%的突变体丧失产孢能力,4.8%的突变体的生长速率显著减慢,8.7%的突变体的生长速率显著加快,12.5%的突变体丧失产生微菌核的能力,47.1%的突变体的致病性显著低于野生型菌株XJ2008,且突变体2-736、2-740、2-745的病情指数分别约为野生型菌株XJ2008的0.184、0.168和0.197倍。该突变体库突变体遗传稳定性好,性状多样性丰富。     

A rapid diagnostic assay for detection and quantification of the causal agent of strawberry wilt from field samples

Verticillium dahliae Kleb, the cause of Verticillium wilt disease, is a destructive pathogen that leads to severe yield losses in strawberry fields and thus considerable economic damages. Although rapid identification and detection methods are becoming available more, pathogen quantification remains one of the main challenges in the disease management. In this study, a real-time polymerase chain reaction (rtPCR) assay was developed to quantitatively assess V. dahliae abundance directly from affected roots and soil collected from different areas in Estonia. A specific primer pair based on the ribosomal DNA (rDNA) internally transcribed spacer was designed for SYBR Green-based assay. Strawberry plant and soil samples were randomly collected from different areas in Estonia and analyzed for V. dahliae by soil plating technique and rtPCR assay. The assay was specific for V. dahliae so that the minimum detection limit was 0.93?pg?µl^?1 of pathogen DNA and the lowest amount of V. dahliae detected in soil was 10.48?pg?µl^?1 of target DNA corresponding to one microsclerotia per gram of soil. This technique allowed rapid detection and quantification of the pathogen DNA at the picogram level in soils and even in symptomless plants, facilitating the screening of the pathogen in diverse areas. This is the first study about the rtPCR technique being used successfully to assess populations of V. dahliae with high specificity and sensitivity in Estonia strawberry fields. Results of this research can be useful for growers and agricultural organizations to improve available disease management strategies against Verticillium wilt.     

Effects of no tillage and genetic resistance on sunflower wilt by Verticillium dahliae

From 2001 to 2006 crop seasons three field experiments were run to compare the effect of no tillage (NT) vs. conventional tillage (CT) on Verticillium wilt of sunflower. One experiment had sunflower monocropping (SM) and the others the sequence wheat–sunflower (WS) with 6 years of fescue pasture or seven WS cycles as previous crops. All experimental fields have history of the disease. One cultivar with low resistance was used in SM and two genotype-resistance levels (high and low; characterized by six and eight cultivars each, respectively) in the WS sequences. Leaf mottle severity in all environments, Verticillium dahliae colony forming units (CFU)/g of soil in 2005 or 2006 trials, density of microsclerotia in the stem pith at 0.5 m above the soil line in WS, and grain yield and oil content in WS with fescue as previous crop were recorded. Every year, disease severity was higher in CT than in NT in all trials. In SM, disease severity increased during the 3 years in CT from 58% to 88%, while in NT disease severity remained around the initial level (49%). The V. dahliae-CFU/g of soil after 3 years in SM or three cycles of WS was approximately three times higher in CT than in NT. In WS, density of microsclerotia in stem pith were higher in CT than in NT. In WS with fescue pasture as previous crop, grain yield and oil content tend to increase with NT in relation to CT. The combination of NT and high-resistant genotypes reduced the disease and the production of microsclerotia in stem pith to very low values. NT + high resistance should be viewed as a preventative, not a curative disease management option because the microsclerotia persist in soil for a long time. Therefore, NT + high resistance programs should be initiated early, before inoculum builds up to high levels in the soil. Thus, the combination of NT and high-resistant cultivars promises to be an interesting tool to manage V. dahliae and Verticillium wilt in sunflower and would have potential in other crops like alfalfa, cotton or strawberry.     

Host specificity,but not high-temperature tolerance,is associated with recent outbreaks of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> in chrysanthemum in the Netherlands

Two hypotheses which might explain a recent increase in the incidence of verticillium wilt of chrysanthemums in glasshouses in the Netherlands were investigated, viz whether selection for increased resistance to elevated temperatures has occurred due to frequent steaming of soils in the glasshouses, or whether the strains of Verticillium dahliae occurring in chrysanthemum glasshouses are particularly virulent towards this host. Following artificial inoculation, five isolates of V. dahliae from chrysanthemum were pathogenic on chrysanthemum but five isolates from potato were non-pathogenic for this host. When inoculated onto potato plants, all isolates caused early senescence with no significant difference between the two groups of isolates. In amplified fragment length polymorphism analysis, the potato isolates formed a cluster distinct from all other isolates. As a group the chrysanthemum isolates were no more diverse than the potato isolates but did not form a cluster distinct from 12 other isolates tested. This suggests that high pathogenicity to chrysanthemum has developed on several occasions but that the group of potato isolates were possibly monophyletic. Microsclerotia produced in vitro from the chrysanthemum isolates had significantly lower average lethal temperature tolerance than those from the five potato isolates suggesting that being able to resist the effects of soil sterilisation by steam is not a factor in wilt of chrysanthemums in the Netherlands.     

Lignin is involved in the reduction of Verticillium dahliae var. longisporum inoculum in soil by crop residue incorporation

Verticillium wilt is an increasing problem in European cauliflower production. In this study, several crop residues were screened for their ability to reduce the viability of microsclerotia when incorporated into soil. In addition, the role of fungitoxic volatiles and lignin in the crop residue-mediated reduction in microsclerotia viability was studied.Broccoli (Brassica oleracea var. italica), cauliflower (B. oleracea var. botrytis), Indian mustard (Brassica juncea), ryegrass (Lolium perenne) and corn (Zea mays) were incorporated in naturally infested soil samples collected from two cauliflower fields in Belgium, labelled Is1 and S3. The effectiveness in reducing the viability of microsclerotia depended on the soil sample and on the type of residue. In the Is1 soil, broccoli, cauliflower and ryegrass incorporation significantly reduced the inoculum level by more than 90%, while Indian mustard significantly reduced numbers of viable microsclerotia by 50%. In the S3 soil, broccoli, cauliflower and Indian mustard were not effective, whereas ryegrass and corn incorporation reduced the microsclerotia level by 50% or more. In conclusion, incorporation of ryegrass and corn was more effective than incorporation of crucifer residues.In the conditions tested, fungicidal volatile compounds did not play an important role in Verticillium microsclerotia reduction in soil. Volatiles from broccoli and cauliflower did not affect microsclerotia viability in an in vitro bioassay, whereas the volatiles from Indian mustard killed the microsclerotia. Indian mustard incorporation in soil, however, only had a minor effect on microsclerotia viability.In the S3 soil, 1% (w/w) Kraft pine lignin, a waste product of the paper industry, had to be added to observe a significant reduction on the viability of microsclerotia, whereas in the Is1 soil, a significant effect was observed when 0.1% (w/w) Kraft pine lignin was added. Acid-insoluble lignin was extracted from all crop residues previously tested. Crop residues with high lignin content seemed to be more effective than crop residues with low lignin content. The reduction of Verticillium microsclerotia viability depended on lignin type and on crop structure, since lignin extracted from cauliflower leaves was more effective than lignin extracted from cauliflower stems and corn leaves were more effective than corn roots. Microsclerotia reduction was higher after fresh residue incorporation than after incorporation of their extracted acid-insoluble lignin, indicating that the effect of crop residue incorporation on microsclerotia viability cannot be explained solely by the effects of lignin.Incorporation of lignin-rich substrates in soil may open up new perspectives for integrated control of Verticillium.     

pH及盐分对大丽轮枝菌微菌核形成的影响

微菌核是棉花黄萎病原大丽轮枝菌(Verticillium dahliae Kleb.)在土壤中的主要存活形式及该病的侵染源,研究土壤pH及盐分对大丽轮枝菌微菌核形成的影响对明确黄萎病发生具有重要意义。采用菌丝生长速率法研究培养基pH及盐分质量浓度与种类对病原菌生长及微菌核形成的影响。供试大丽轮枝菌菌丝生长最适pH为7.0,偏酸或偏碱会抑制菌丝生长,但培养基偏碱可显著促进微菌核形成。当pH为8.0时,大丽轮枝菌菌丝生长受抑制较小,同时微菌核区面积较pH为7.0时增加22.6%。盐分质量浓度影响大丽轮枝菌菌丝生长及微菌核形成。随培养基NaCl质量浓度增加,供试大丽轮枝菌生长受到抑制,菌落面积和菌丝面积均逐渐减小,但微菌核形成量却显著增加;当NaCl质量浓度为10g·L~(-1)时,微菌核区面积较无NaCl时增加40.7%。盐分种类影响供试大丽轮枝菌生长。随盐分质量浓度增加,氯化物(NaCl和KCl)和硫酸盐(Na_2SO_4和MgSO_4)均可促进大丽轮枝菌微菌核形成,而CaCl_2则显著促进菌丝生长,并在质量浓度大于7g·L~(-1)时抑制微菌核形成。在培养环境偏碱性或氯化物和硫酸盐含盐量较高时,均可促进棉花黄萎病原大丽轮枝菌微菌核形成量增加。