土壤大丽轮枝菌微菌核的快速定量检测

 微菌核是大丽轮枝菌在土壤中的主要存活结构和黄萎病的初侵染来源。对土壤中大丽轮枝菌微菌核进行定量是黄萎病监测和预警的基础。本研究以大丽轮枝菌Internal Transcribed Spacer (ITS)区特异性引物对P1/P2扩增产物的重组质粒为标准品,构建SYBR Green I实时荧光定量PCR反应的标准曲线,结合土样水筛法建立了土壤大丽轮枝菌微菌核定量检测体系。同时,建立了土壤中微菌核数量与棉花黄萎病发病率的关系模型。结果表明,实时定量PCR检测灵敏度比常规PCR高10倍,检测下限为1个微菌核/克土,在5.54×10²～5.54×10⁷copies范围内,DNA拷贝数的对数值与Ct值具有良好的线性关系。建立的土壤中微菌核个数n与Ct值之间的关系为n=e^7.3-Ct/3.905。温室人工接种微菌核数量与棉花黄萎病发病率间的线性关系为y=2.710n+0.251。     

Toxicity of 1,2-dibromoethane and 1,3-dichloropropene to microsclerotia of Verticillium dahliae

The toxicity of two fumigants to microsclerotia (MS) of Verticillium dahliae in air and in soil was determined in sealed containers. 1, 2-Dibromoethane (DBE, ethylene dibromide) at 470 μ ml^?1 of air, or at 12.5 μ g^?1 of soil, killed 97% of the MS, both after incubation for 16 days. 1, 3-Dichloropropene (1, 3-D) at 20 μ ml^?1 of air, however, killed 100% of the MS after incubation for 30 h, and at 100 μ g^?1 of soil after incubation for 3 days. Higher temperatures increased the toxicity of both DBE and 1, 3-D to the MS. The toxicity of DBE increased with increase in soil moisture between 0–80% of field capacity while the toxicity of 1, 3-D increased between 0–20% of field capacity and was constant between 20–80% of field capacity.     

Formation of microsclerotia of Verticillium dahliae in petioles of infected ash trees

Under young ash trees infected withVerticillium dahliae, over 10% of the petioles of fallen leaves were colonized by the pathogen. Counts of microsclerotia in six petioles yielded an average number of 1500 per cm of petiole. Windblown leaves from infected trees very probably contribute to dissemination of the pathogen in forest stands and nurseries.     

土壤环境因素对棉花黄萎病菌微菌核存活的影响

 试验研究了土壤温度、湿度、pH值和有机质对棉花黄萎病菌微菌核存活的影响。结果表明:40、30和0℃对微菌核土壤存活影响较大,10和20℃影响较小,尤其以10℃影响最小;土壤含水量越高,对微菌核存活影响越大,在含水量低于15%时,对存活影响较小,尤其以5%的含水量影响最小,表明微菌核对土壤干旱逆境具有较强的抵抗能力;pH值低于5.5或高于8.5的土壤条件对微菌核存活影响较大,pH值为6.5~7.5影响较小;土壤有机质含量越高,微菌核存活率越低,含量低,存活率高。本研究结果表明,只要改善和控制棉花黄萎病菌土壤存活的生态条件,就可以有效地减少土壤存活菌量,达到控制病害发生与发展的目的。     

Germination of individual microsclerotia ofVerticillium dahliae

Viability of individual microsclerotia (MS) ofVerticillium dahliae of different sizes (<37 μm, 37–74 μm, 75–125 μm) was studied throughout repeated transfers in a moist chamber. Germination rate and number of germinating hyphae were positively correlated with the size of the MS. After 13 transfers, some MS of the above size ranges still germinated; some MS germinated until they lost their viability; others did not germinate for a while and then started germinating again. The length of germination time of MS on a synthetic medium was 8 days, with slight differences between MS originating from soil and those from one-year-old potato stems. Fresh MS from potato stems, however, completed their germination after 36 hours. Fewer MS (size <37 μm) were isolated from soil by the wet-sieving than by the dry-sieving technique. This finding and microscopic observations lend further support to the assumption that MS exist in soil partly in clusters.     

A useful method for preparing microsclerotial inoculum of Verticillium dahliae

Various methods of inoculating plants with Verticillium dahliae have been described; however, so far, there are no effective preparations of its microsclerotia, a major source of infection under natural field conditions. In this study, we developed a simple and effective method to prepare microsclerotial inoculum of V. dahliae using a cellophane medium that effectively promoted microsclerotia formation. In addition, a sieving process allowed the preparation of inoculum exclusively composed of microsclerotia. Our novel method provides a reproducible and stable inoculum for disease induction by V. dahliae under conditions that approximate natural infections.     

Disruption of Cerevisin via Agrobacterium tumefaciens‐mediated transformation affects microsclerotia formation and virulence of Verticillium dahliae

Agrobacterium tumefaciens‐mediated transformation (ATMT) was used to obtain a large number of Verticillium dahliae (Vd991) T‐DNA insertion mutants that were randomly integrated. Insertion mutants that produced significantly fewer microsclerotia were chosen for further analysis. Mutant T0065 was identified as having the Cerevisin gene interrupted by T‐DNA, and it was named cerevisin. The cerevisin protein showed a high amino acid sequence similarity with the vacuolar protease B. The mutant strain cerevisin displayed decreased production of microsclerotia and conidia, significantly reduced growth rate and reduction in virulence compared to the wild type. Moreover, the composition of secreted proteins differed between the cerevisin mutant and the wild type. Loss of function of Cerevisin decreased secretion of proteins of low molecular weight (14–25 kDa). Upon treatment with the secreted proteins of the mutant, the degree of leaf wilting decreased, indicating that Cerevisin is implicated in production of these proteins, which are putative pathogenicity factors of V. dahliae. The results suggest that Cerevisin is involved in controlling multiple processes of development and metabolism, plays an important role in vegetative growth and microsclerotia formation and affects virulence of V. dahliae.     

棉花抗黄萎病品种选育方法探讨

 在棉花抗病育种中,通过病圃或重病地选择抗病单株是常规的选育方法,但其在抗黄萎病育种中的效果并不理想。如何提高抗黄萎病育种效率,促进育种进程是目前急需解决的问题之一。为此,19982002年我们在北京人工病圃,对不同抗病杂交组合和选育方法的选育效果进行了研究。结果表明,在双亲本均为抗(耐)品种的杂交后代群体中,以9月中旬是抗病还是感病并没有多少关联,对黄萎病抗性的选育应选群体抗病性强的株系或组合,以其丰产性为主要选育目标;只有抗与抗(或高耐)的组合中才能选出黄萎病抗性更高的株系;在抗(耐)品种中,后期无论是抗或感的单株其后代的抗病性差异不明显     

Quantification of microsclerotia ofVerticillium dahliae in plant material by image analysis

A procedure for the quantification of microsclerotia ofVerticillium dahliae with an image analysis system was compared with counting by eye. Colonised potato plant material was used from plants grown in pathogen-free soil in a greenhouse and from twelve crops (including four potato cultivars) grown outdoors in pots filled with pathogen-free soil under natural conditions. The values obtained from the potato material from the greenhouse were comparable for both methods. Variation in the results mainly resulted from sampling errors. The numbers of microsclerotia in plants grown outdoors were overestimated by image analysis for most crops. The source of the error was related to the presence of plant and soil particles that did not discolour during boiling of the samples in sodium hydroxide. Image analysis was a suitable and reliable method for assessing the number of microsclerotia only in potato haulm samples from plants grown in pathogen-free soil in the greenhouse.Abbreviations cv coefficient of variation - IA image analysis - MS microsclerotia, microsclerotium     

北疆棉花黄萎病发病率调查及土壤中黄萎病菌微菌核数量与种群类型分析

为了解北疆棉花黄萎病的发生情况及发病与土壤中黄萎病菌微菌核数量的关系和病原菌种群类型, 2021年对北疆石河子?奎屯?博乐等8市(县)棉田棉花黄萎病发病率?土壤中黄萎病菌的微菌核数量?菌株种群类型进行了抽样调查?结果表明, 北疆未发生棉花黄萎病的棉田占49.2%, 0%<发病率<5.0%的棉田占32.7%, 发病率≥5.0%的棉田占18%?与2013年?2015年相比, 2021年无病田率分别增加17.7和12.7百分点, 发病率≥5%的棉田分别减少15.7和21.6百分点?从棉田黄萎病发病率与土壤中微菌核数量的关系来看, 整体上北疆棉田棉花黄萎病发病率与微菌核数量相关性不显著(r=0.119 1); 分区域看, 石河子-沙湾片区?奎屯-乌苏片区?精河-博乐片区棉田黄萎病发病率与微菌核数量的相关系数分别为0.033 2?0.007 6?0.062 3, 均无显著相关性; 而呼图壁-玛纳斯片区棉田黄萎病发病率与微菌核数量的相关系数为0.635 7, 呈中度正相关?土壤中的黄萎病菌菌株以菌核型为主, 占57.9%, 菌丝型占23.2%, 中间型占18.9%?用特异性引物进行PCR检测表明, 土壤中黄萎病菌落叶型菌株占97.6%, 占绝对优势?本研究将为北疆棉区棉花黄萎病的综合防控提供理论依据?     

Host Range Specificity in Verticillium dahliae

ABSTRACT Verticillium dahliae isolates from artichoke, bell pepper, cabbage, cauliflower, chili pepper, cotton, eggplant, lettuce, mint, potato, strawberry, tomato, and watermelon and V. albo-atrum from alfalfa were evaluated for their pathogenicity on all 14 hosts. One-month-old seedlings were inoculated with a spore suspension of about 10(7) conidia per ml using a root-dip technique and incubated in the greenhouse. Disease incidence and severity, plant height, and root and shoot dry weights were recorded 6 weeks after inoculation. Bell pepper, cabbage, cauliflower, cotton, eggplant, and mint isolates exhibited host specificity and differential pathogenicity on other hosts, whereas isolates from artichoke, lettuce, potato, strawberry, tomato, and watermelon did not. Bell pepper was resistant to all Verticillium isolates except isolates from bell pepper and eggplant. Thus, host specificity exists in some isolates of V. dahliae. The same isolates were characterized for vegetative compatibility groups (VCGs) through complementation of nitrate nonutilizing (nit) mutants. Cabbage and cauliflower isolates did not produce nit mutants. The isolate from cotton belonged to VCG 1; isolates from bell pepper, eggplant, potato, and tomato, to VCG 4; and the remaining isolates, to VCG 2. These isolates were also analyzed using the random amplified polymorphic DNA (RAPD) method. Forty random primers were screened, and eighteen of them amplified DNA from Verticillium. Based on RAPD banding patterns, cabbage and cauliflower isolates formed a unique group, distinct from other V. dahliae and V. albo-atrum groups. Minor genetic variations were observed among V. dahliae isolates from other hosts, regardless of whether they were host specific or not. There was no correlation among pathogenicity, VCGs, and RAPD banding patterns. Even though the isolates belonged to different VCGs, they shared similar RAPD profiles. These results suggest that management of Verticillium wilt in some crops through crop rotation is a distinct possibility.     

棉花黄萎病防治策略

黄萎病是棉花生产中的最主要病害之一,广泛分布于世界各产棉国.由于该病是土壤传播的维管束病害,防治难度较大,至今尚无特效的防治药剂,只能依靠种植抗病品种为主的综合防治措施.但是,我国目前棉花品种的抗病性只能达到抗至耐病水平,致使该病在环境条件适宜时连续流行为害.控制该病的猖獗为害,已成为棉花生产可持续发展的主要问题之一.     

Sources of Verticillium dahliae Affecting Lettuce

ABSTRACT Since 1995, lettuce in coastal California, where more than half of the crop in North America is grown, has consistently suffered from severe outbreaks of Verticillium wilt. The disease is confined to this region, although the pathogen (Verticillium dahliae) and the host are present in other crop production regions in California. Migration of the pathogen with infested spinach seed was previously documented, but the geographic sources of the pathogen, as well as the impact of lettuce seed sparsely infested with V. dahliae produced outside coastal California on the pathogen population in coastal California remain unclear. Population analyses of V. dahliae were completed using 16 microsatellite markers on isolates from lettuce plants in coastal California, infested lettuce seed produced in the neighboring Santa Clara Valley of California, and spinach seed produced in four major spinach seed production regions: Chile, Denmark, the Netherlands, and the United States (Washington State). California produces 80% of spinach in the United States and all seed planted with the majority infested by V. dahliae comes from the above four sources. Three globally distributed genetic populations were identified, indicating sustained migration among these distinct geographic regions with multiple spinach crops produced each year and repeated every year in coastal California. The population structure of V. dahliae from coastal California lettuce plants was heavily influenced by migration from spinach seed imported from Denmark and Washington. Conversely, the sparsely infested lettuce seed had limited or no contribution to the Verticillium wilt epidemic in coastal California. The global trade in plant and seed material is likely contributing to sustained shifts in the population structure of V. dahliae, affecting the equilibrium of native populations, and likely affecting disease epidemiology.     

Vegetative-compatibility groups in Verticillium dahliae from Israel

Nitrate-nonutilizing (nit) mutants were used to determine vegetative compatibility among 34 isolates of Verticillium dahliae from cotton, potato, olive, eggplant, chrysanthemum and tomato from 12 sites in Israel. Based on the formation of complementary heterokaryons, 33 isolates were assigned to two vegetative- compatibility groups (VCGs): one VCG contained 15 isolates from cotton, eggplant, chrysanthemum and olive; and the other VCG contained 18 isolates from potato, olive and cotton. The status of an additional isolate from tomato, which was compatible with both VCGs, remained unclear. In a limited pathogenicity test with 10 isolates, two (from tomato and eggplant) were pathogenic on tomato, eggplant and cotton; most isolates from cotton were pathogenic on cotton and eggplant only; and one from cotton was non-pathogenic. Fewer isolates were pathogenic on tomato than on cotton or eggplant. The diversity of vegetative compatibility found in our V. dahliae collection is comparable to that found in studies of American populations.     

Evaluation of Olive Cultivars for Resistance to Verticillium dahliae

Resistance of 23 important olive cultivars to Verticillium dahliae has been evaluated in four experiments under controlled conditions. Nine-month-old nursery olive plants were inoculated with a cotton non-defoliating (ND) (V4) or a cotton defoliating (D) (V117) isolate of V. dahliae. Resistance was evaluated by assessing symptom severity using a 0–4 rating scale and estimating the area under disease progress curves. The percentage of plants killed and of those which recovered from the disease were used as additional parameters for including a particular cultivar into a defined category. Most of the evaluated cultivars were susceptible, although at different levels, to both isolates of V. dahliae. All cultivars were more susceptible to the D pathotype than to the ND one. A group of 11 cultivars, including several important Spanish cultivars, were susceptible or extremely susceptible to both pathotypes of V. dahliae. A second group showed differences of resistance depending on the pathotype used. They were susceptible or extremely susceptible to the D pathotype but resistant or moderately susceptible to the ND one. Finally, 'Frantoio', 'Oblonga' and 'Empeltre' were moderately susceptible to the D isolate of V. dahliae and resistant to the ND one. The resistance of 'Empeltre' was evident by the plant ability to recover from infection with either isolates. 'Empeltre' is considered to be a valuable cultivar for inclusion in breeding programmes for resistance to Verticillium wilt.     

Effect of plant roots on the germination of microsclerotia ofVerticillum dahliae

Root observation boxes were used to study the effects of hosts and non-hosts on the germination of microsclerotia ofV. dahliae. The effects of roots on microsclerotia were examined within a radius of 1 mm around the root tip. Host plants such as potato and field bean induced a higher percentage of germination of the microsclerotia than a non-host such as barley. A susceptible potato cultivar stimulated germination more than a resistant cultivar. The germination percentage and the number of hyphae per microsclerotium decreased with distance from the root surface regardless of the plant species or cultivar.     

Effect of plant roots on the germination of microsclerotia ofVerticillium dahliae

Induction of germination of microsclerotia by exudates from plant roots may be important for the control ofV. dahliae. Laboratory experiments with root observation boxes were carried out to assess the influence of root tips of seven crop species and cultivars on the germination of microsclerotia ofVerticillium dahliae in soil under controlled conditions. The root density of crops was measured in a field experiment. The results of the laboratory experiments and the field experiment were combined to estimate the total effect of crops on the population of microsclerotia in the field. Germination of microsclerotia was stimulated by all crops compared to a control without a crop. Among crops, roots of potato cvs Element and Astarte had a larger stimulation effect on microsclerotia than that of potato Ostara, pea, flax, sugar beet or onion. The number of hyphae per microsclerotium decreased with distance from the root surface regardless of the crop species or cultivar. Differences in root densities, in the affected root zones and in the stimulation effect on germination of microsclerotia caused large differences among crops in the effect on the population of microsclerotia in the soil. However, growing a rop with the special purpose to reduce the level ofV. dahliae inoculum in the soil is an inefficient control measure, because only a small part of the total soil volume is affected by roots and the number of hyphae per microscleroium affected is too low.Abbreviations MS microsclerotia, microsclerotium