Induction of resistance in cocoa against Crinipellis perniciosa and Verticillium dahliae by acibenzolar- S-methyl (ASM)

The benzothiadiazole compound acibenzolar- S -methyl (ASM) was assessed as an inducer of resistance against Crinipellis perniciosa , agent of witches' broom, and Verticillium dahliae , agent of vascular wilt, both on cocoa. ASM induced a reduction in incidence of witches' broom of up to 84·5% when sprayed 30 days before inoculation on cocoa seedlings of cv. Catongo. ASM also induced a reduction in severity of Verticillium wilt to 55·4% on cv. Theobahia. For both pathosystems, effects of dose on disease were not clearly observed. The efficacy of the inducer increased with the interval between sprayings and the respective inoculations with the pathogens. In another experiment, the effect of ASM on the control of witches' broom on cocoa seedlings was compared with that of cuprous oxide and tebuconazole, all sprayed 15 days before inoculation. ASM reduced disease incidence by 60·1% compared with the inoculated control. ASM was superior to tebuconazole, and there was also a tendency for ASM to be better than cuprous oxide. To understand the mechanism of action of ASM as an inducer of resistance, alterations in the levels of total phenolics, polyphenol oxidases and peroxidases were evaluated 3, 15 and 30 days after spraying of seedlings of cv. Catongo. Enzyme activities from seedlings of cv. Theobahia were evaluated 30 days after spraying. On cv. Catongo, no significant differences in total phenolic content and polyphenol oxidase activity were detected after spraying. However, an increase in peroxidase activity was detected at all times of evaluation. On cv. Theobahia, significant increases in activities of peroxidase and polyphenol oxidase were detected, indicating that defence responses due to ASM were dependent on host genotype.     

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     

基质中添加西兰花残体对大丽轮枝菌GFP标记菌株在棉花体内扩展的影响

 由大丽轮枝菌引起的棉花黄萎病是一种难以防治的土传病害,西兰花残体还对其有一定的防治作用,防效为62.82%。为解析西兰花残体对棉花黄萎病的防治机制,本研究通过土壤杆菌转化的方法构建了大丽轮枝菌的绿色荧光标记菌株,采用共聚焦显微镜观察标记菌株在添加西兰花残体营养基质中和空白对照营养基质种植的棉花体内的侵染和扩展情况。结果表明构建的标记菌株gfp-wx-1与野生菌株wx-1的生物学特性无显著性差异。同时发现大丽轮枝菌在添加西兰花残体营养基质中棉花体内扩展较慢,具体表现为:在空白营养基质种植的棉花体内,大丽轮枝菌在接种第2 d时就可侵染根尖表层及根内部,第3 d到达茎部维管束,第5 d叶片可观察到少量病原菌,第7 d第一片子叶呈现大量病原菌,后期迅速扩展,叶片出现黄萎病斑;而在营养基质中添加西兰花残体种植的棉花体内,大丽轮枝菌在接种第2 d时就可侵染根尖表层,第3 d侵染根尖内部,第5 d侵染茎部维管束,直到第7 d第一片子叶才出现少量病原菌,后期扩展慢,叶片病斑较少。本研究通过荧光定量PCR方法检测了大丽轮枝菌在两个处理棉花根部定殖情况,结果表明西兰花残体能够显著降低大丽轮枝菌在棉花根部定殖的量。该研究初步明确了西兰花残体对棉花黄萎病的防治机制,为棉花黄萎病的绿色防治提供了一条新的途径。     

基质中添加西兰花残体对大丽轮枝菌GFP标记菌株在棉花体内扩展的影响

 由大丽轮枝菌引起的棉花黄萎病是一种难以防治的土传病害,西兰花残体还对其有一定的防治作用,防效为62.82%。为解析西兰花残体对棉花黄萎病的防治机制,本研究通过土壤杆菌转化的方法构建了大丽轮枝菌的绿色荧光标记菌株,采用共聚焦显微镜观察标记菌株在添加西兰花残体营养基质中和空白对照营养基质种植的棉花体内的侵染和扩展情况。结果表明构建的标记菌株gfp-wx-1与野生菌株wx-1的生物学特性无显著性差异。同时发现大丽轮枝菌在添加西兰花残体营养基质中棉花体内扩展较慢,具体表现为:在空白营养基质种植的棉花体内,大丽轮枝菌在接种第2 d时就可侵染根尖表层及根内部,第3 d到达茎部维管束,第5 d叶片可观察到少量病原菌,第7 d第一片子叶呈现大量病原菌,后期迅速扩展,叶片出现黄萎病斑;而在营养基质中添加西兰花残体种植的棉花体内,大丽轮枝菌在接种第2 d时就可侵染根尖表层,第3 d侵染根尖内部,第5 d侵染茎部维管束,直到第7 d第一片子叶才出现少量病原菌,后期扩展慢,叶片病斑较少。本研究通过荧光定量PCR方法检测了大丽轮枝菌在两个处理棉花根部定殖情况,结果表明西兰花残体能够显著降低大丽轮枝菌在棉花根部定殖的量。该研究初步明确了西兰花残体对棉花黄萎病的防治机制,为棉花黄萎病的绿色防治提供了一条新的途径。     

Characterization of resistance against the olive‐defoliating Verticillium dahliae pathotype in selected clones of wild olive

Verticillium wilt of olive is best managed by resistant cultivars, but those currently available show incomplete resistance to the defoliating (D) Verticillium dahliae pathotype. Moreover, these cultivars do not satisfy consumers' demand for high yields and oil quality. Highly resistant rootstocks would be of paramount importance for production of agronomically adapted and commercially desirable olive cultivars in D V. dahliae‐infested soils. In this work, resistance to D V. dahliae in wild olive clones Ac‐13, Ac‐18, OutVert and StopVert was assessed by quantifying the fungal DNA along the stem using a highly sensitive real‐time quantitative polymerase chain reaction (qPCR) protocol and a stem colonization index (SCI) based on isolation of V. dahliae following artificial inoculations under conditions highly conducive for verticillium wilt. Ac‐13, Ac‐18, OutVert and StopVert showed a symptomless reaction to D V. dahliae. The mean amount of D V. dahliaeDNA quantified in stems of the four clones ranged from 3.64 to 28.89 pg/100 ng olive DNA, which was 249 to 1537 times lower than that in susceptible Picual olive. The reduction in the quantitative stem colonization of wild olive clones by D V. dahliae was also indicated by a sharp decrease in the SCI. Overall, there was a pattern of decreasing SCI in acropetal progression along the plant axis, as well as correlation between positive reisolation and quantification of pathogen DNA. The results of this research show that wild olive clones Ac‐13, Ac‐18, OutVert and StopVert have a valuable potential as rootstocks for the management of verticillium wilt in olive.     

Colonization in cotton plants by a green fluorescent protein labelled strain of Verticillium dahliae

Verticillium wilt of cotton (Gossypium hirsutum) is a widespread and destructive disease caused by the soil-borne fungal pathogen Verticillium dahliae. In this study, a green fluorescent protein (GFP) labelled V. dahliae strain (TV7) was obtained by transforming gfp into defoliating strain V991. Strain TV7 was used to study infection and colonization of wilt resistant cotton cultivar Zhongzhimian KV1 and susceptible cultivar 861 with the aid of confocal laser scanning microscopy. The results showed that initial infection and colonization of V. dahliae in Zhongzhimian KV1 and 861 were similar. Conidia and hyphal colonies formed and penetrated in the root meristematic and elongation zones and in the conjunction of the lateral and main roots. The invaded conidia started to germinate by 2 hpi (hours post-inoculation), penetrated into the root cortex and vascular bundles, eventually colonized in the stem xylem vessels and grew restrictedly in the individual tracheae of both resistant and susceptible cultivars. Moreover, pathogen DNA could be detected by qPCR in roots and stems of both cultivars, but its content in the wilt susceptible cultivar 861 was much higher than that in the wilt resistant cultivar Zhongzhimian KV1. The results indicated that the resistant cultivar has ability to suppress V. dahliae reproduction.     

Sulphur accumulation after Verticillium dahliae infection of two pepper cultivars differing in degree of resistance

Elemental sulphur levels, sulphur localization in stems, and levels of sulphate, glutathione and cysteine were studied in pepper ( Capsicum annuum ) cvs Yolo Wonder (higher resistance) and Luesia (lower resistance) after inoculation with Verticillium dahliae , the cause of vascular wilt. Accumulation of elemental sulphur (S⁰) was first detected 10 days after inoculation in Yolo Wonder (mean S⁰ level 7·3  µ g g⁻¹ DW), and 15 days after inoculation in Luesia (mean S⁰ level 3·3  µ g g⁻¹ DW). The maximum level was reached 21 days after inoculation in Yolo Wonder (14·1  µ g g⁻¹ DW). In control plants, elemental sulphur was not detected. SEM-EDX (scanning electron microscopy-energy dispersive X-ray microanalysis) indicated that the sulphur was not restricted to a specific location, but was dispersed throughout the vascular tissue. Sulphate levels showed a decline at the end of the experiment in inoculated plants, possibly related to the increase in sulphur levels seen in the two cultivars. The differences in sulphate levels between the two cultivars may be due to faster sulphate breakdown in cv. Yolo Wonder.     

Interaction of spatially separated Pratylenchus penetrans and Verticillium dahliae on potato measured by impaired photosynthesis

Experiments were conducted under growth-chamber conditions to determine if Pratylenchus penetrans systemically alters light use efficiency (LUE) of Russet Burbank potato infected by Verticillium dahliae. Pathogen separation was achieved by inoculating potato roots with the nematode prior to injecting fungal conidia into the stem vasculature. Treatments were P. penetrans alone, V. dahliae alone, nematode and fungus together, and a no-pathogen control. Gas exchange was repeatedly and nondestructively measured on the fifth-youngest leaf with a Li-Cor LI-6200 portable photosynthesis system. By 16 and 20 days after stem injection with the fungus, LUE was synergistically impaired in jointly infected plants. Transpiration in plants infected with both pathogens was significantly reduced. However, the combined effect of nematode and fungus was synergistic in one experiment and additive in the other. Stems were destructively harvested when LUE was synergistically impaired. Coinfected potato plants contained more colony-forming units (CFU) of V. dahliae in stem sap than those infected by the fungus alone in one experiment. Evidence is provided that infection of Russet Burbank roots by P. penetrans systemically affects disease physiology associated with stem vascular infection by V. dahliae . The findings indicate that the role of the nematode in the fungus/host interaction is more than simply to facilitate extravascular and/or vascular entry of the fungus into potato roots.     

Water relations and ethylene production as related to symptom expression in cocoa seedlings infected with defoliating and non-defoliating isolates of Verticillium dahliae

Some isolates of Verticillium dahliae can induce severe defoliation on cocoa plants and others lead to wilting then desiccation of the leaves, without defoliation. The underlying mechanisms that precede the occurrence of these two distinct responses were studied in root inoculated seedlings under glasshouse conditions.
Rapid decreases in total transpiration, stomatal conductance and midday leaf water potential were closely associated with the onset of foliar symptoms, indicating that water stress is a major cause of symptom development. Water stress was most pronounced when plants were inoculated with a'non-defoliating'isolate. In contrast, a'defoliating'isolate induced accumulation of ethylene in newly developed leaves, where the first symptoms generally appeared. This hormone appeared to be responsible for the accelerated senescence and defoliation, as demonstrated by reversal with the application of the ethylene inhibitor silver thiosulphate.
Increased symptoms, water stress and ethylene production occurred in upper leaves and coincided with more intensive colonization of this part of the plant by the pathogen. This unusual pattern reflects the vascular anatomy of the host.     

Insights into the role of ethylene perception in tomato resistance to vascular infection by Verticillium dahliae

A Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system was employed to investigate the role of the tomato ethylene receptor ETR4. By comparing wilting symptoms of verticillium wilt in wild-type, ethylene-insensitive Never ripe ( Nr ) mutant tomato plants and ETR4 -silenced plants, it was demonstrated that disease severity in the Nr and ETR4 -silenced plants was statistically reduced compared to wild-type plants. Disease incidence and severity were reduced by 11 and 20%, respectively, in the Nr plants compared to the wild-type plants, at 33 days post-inoculation (d.p.i.). In the ETR4 -silenced plants, disease incidence and severity were reduced by 14 and 15%, respectively, compared to the TRV -only-inoculated plants, at 37 d.p.i. Quantification of Verticillium dahliae by qPCR revealed that the reduction in symptom severity in the Nr plants was associated with significant reduction of growth of the pathogen in the vascular tissues of the Nr plants compared to that in the wild-type plants, suggesting that impaired perception of ethylene via the Never-ripe receptor results in increased disease resistance. Fungal reduction was evident at each sampling day in the Nr plants, ranging from 1·5 to 1·75 times less than that in the wild-type plants. Fungal quantification in the ETR4- silenced and TRV -only-inoculated plants showed similar levels of fungal biomass.     

Response of strawberry genotypes to inoculation with isolates of Verticillium dahliae differing in host origin

Eight strawberry genotypes known to differ in susceptibility to verticillium wilt were inoculated with eight isolates of Verticillium dahliae originally obtained from six different host crops: strawberry, potato, watermelon, mint, eggplant (aubergine) and cauliflower. Inoculation experiments were conducted in replicated field trials during two successive years. Known susceptible genotypes developed typical symptoms of verticillium wilt in both years. Although isolates manifested a wide range of aggressiveness, differences were significant only on the most susceptible strawberry genotype. Two isolates originating from strawberry were among the most aggressive of the eight tested, whereas the least aggressive isolate was obtained from cauliflower. Six strawberry genotypes that were regarded as resistant to verticillium wilt based on previous tests were also resistant in the present study, regardless of the isolate used. Overall, strawberry genotypes represented the largest source of variation in these experiments, with variance components approximately 10-fold greater than those associated with either isolate or the isolate × genotype interaction. The results suggest it should be possible to develop resistance to verticillium wilt in strawberry that is broadly effective against isolates of diverse host origin.     

喷施外源激素对棉株棉花黄萎病发病程度的影响

通过温室人工接菌 ,研究了喷施 4种外源激素 3-吲哚乙酸、赤霉素、脱落酸和反 -玉米素对棉苗棉花黄萎病发病程度的影响。结果表明赤霉素在5.0～80.0μg/ml浓度范围内 ,提高了棉苗抗病性 ,但棉苗表现徒长、叶片畸形 ;反 -玉米素25.0～80.0μg/ml明显促进了病情发展 ;3-吲哚乙酸10μg/ml,脱落酸0.5μg/ml,反 -玉米素10 μg/ml的处理对降低棉苗的发病率和发病程度作用较明显。     

Reliable detection of unevenly distributed Verticillium dahliae in diseased olive trees

Verticillium wilt caused by Verticillium dahliae is one of the most threatening diseases of olive worldwide. For pre‐planting and post‐planting control of verticillium wilt in olive trees, availability of a rapid, reliable and non‐destructive method for detection of V. dahliae is essential. For such a method, suitable and easily performed sampling and efficient processing of samples for extraction of DNA are necessary. In this study, the suitability of young twig and leaf samples of olive trees, which are easy to collect and extract DNA from, were assessed for the detection of V. dahliae in routine procedures. The lower (about 50 cm from the tip) and top parts (about 5 cm from the tip) of twigs, as well as leaves from infected olive trees were screened for V. dahliae infection and distribution using real‐time PCR. The biomass of V. dahliae detected in individual twigs was highly variable, but there was no significant difference between mean quantities of V. dahliae DNA detected in top and lower parts of twigs. Furthermore, it was demonstrated that analysis of combined samples containing DNA extracted from five twigs of an infected tree accurately detected the presence of the pathogen. Similarly, testing combined samples of 5–10 leaves enabled reliable detection of the pathogen in an infected tree. The development of this assay enables reliable detection of V. dahliae in infected olive trees that can aid in management decisions for the implementation of integrated disease management.     

Verticillium longisporum and Fusarium solani: two new species in the complex of internal discoloration of horseradish roots

This study was conducted to determine the causal agent(s) of internal discoloration of horseradish roots. In 1999, 133 roots from 31 fields, and in 2000, 108 roots from nine fields, were assayed to determine the incidence and severity of internal discoloration of horseradish roots as well as the pathogen(s) associated with discoloured tissue. Verticillium dahliae , Verticillium longisporum and Fusarium solani were isolated from 14, 16 and 23% of roots in 1999, and from 24, 20 and 19% of roots in 2000, respectively. Verticillium longisporum on horseradish was identified for the first time. Pathogenicity tests of isolated microorganisms were conducted on horseradish in the glasshouse. In one experiment on the susceptible cultivar 1573, roots (sets) were inoculated by dipping the sets in a suspension of either V. dahliae microsclerotia, V. longisporum microsclerotia, or F. solani conidia and then grown in a soil mix over 5 months. Plants inoculated with any of the three species developed root discoloration similar to that observed in commercial fields. Internal root discoloration symptoms developed over a period of 5 months. For all three pathogens, severity of root discoloration was significantly higher after 5 months compared with 2 months from inoculation. In another experiment on cultivar 1590, tissue culture-generated seedlings and sets were planted in an infested soil mix with V. dahliae or V. longisporum and grown in the glasshouse. Plants developed root discoloration, as observed in the field. The pathogens were reisolated from inoculated plants in both experiments. No pathogen was isolated from the control plants in the experiments. The results of this study suggest that internal discoloration of horseradish roots is a disease complex caused by at least three fungal species.     

陆地棉对黄萎病抗性的分子标记研究

 利用陆地棉标准系TM-1和常抗棉2个陆地棉品种杂交并自交,获得109个F₂单株及F_2:3家系为作图群体,以SSR、RAPD和SRAP 3种分子标记进行抗黄萎病性状的分子标记筛选。结果从1611对(条)引物中仅筛选到70对(条)多态性引物,获得75个多态性位点并进行标记间的连锁性分析。75个标记构建了一个包括15个连锁群,全长535 cM的陆地棉品种间分子标记遗传连锁图,标记间平均距离为11.15 cM,有27个标记不能进入任何连锁群。连锁群的标记数最少2个,最多6个;长度从1.0 cM到92.7 cM不等。对其F_2:3家系的成株期抗黄萎病性状即平均病情指数的分布进行分析,显示其呈正态分布,进一步说明陆地棉对黄萎病的抗性为数量遗传;单标记分析及复合区间作图,检测出与抗黄萎病性相关的3个QTL,分别位于第3、5、6连锁群上,贡献率分别为14.15%、3.45%和18.78%。另外,对该群体生长过程中黄萎病不同发病高峰期的病情也进行了分析。     

Verticillium dahliae Modifies the Concentrations of Proline, Soluble Sugars, Starch, Soluble Protein and Abscisic Acid in Pepper Plants

The objective of this research was to study the levels of some organic solutes, such as total protein, total soluble sugars, starch and proline in leaves, as well as abscisic acid concentration in xylem of pepper plants inoculated with Verticillium dahliae Kleb. Healthy and inoculated plants were always kept well watered. Measurements were made at time intervals after inoculation. Leaf water potential rapidly decreased as a consequence of fungal infection. However, relative water content in leaves only changed significantly from day 20 after inoculation, and such decreases coincided with a sharp build up of proline and total soluble sugars in leaves. Starch and protein levels, as well as abscisic acid concentration in xylem, declined in healthy and inoculated peppers as they became older. However, such decreases were more pronounced in infected plants, especially soon after inoculation. Results suggest that proline and total soluble sugars accumulation could be sensors of the damage caused by the fungal infection.     

Variation of pathotypes and races and their correlations with clonal lineages in Verticillium dahliae

Understanding pathogenic variation in plant pathogen populations is key for the development and use of host resistance for managing verticillium wilt diseases. A highly virulent defoliating (D) pathotype in Verticillium dahliae has previously been shown to occur only in one clonal lineage (lineage 1A). By contrast, no clear association has yet been shown for race 1 with clonal lineages. Race 1 carries the effector gene Ave1 and is avirulent on hosts that carry resistance gene Ve1 or its homologues. The hypothesis tested was that race 1 arose once in a single clonal lineage, which might be expected if V. dahliae acquired Ave1 by horizontal gene transfer from plants, as hypothesized previously. In a diverse sample of 195 V. dahliae isolates from nine clonal lineages, all race 1 isolates were present only in lineage 2A. Conversely, all lineage 2A isolates displayed the race 1 phenotype. Moreover, 900‐bp nucleotide sequences from Ave1 were identical among 27 lineage 2A isolates and identical to sequences from other V. dahliae race 1 isolates in GenBank. The finding of race 1 in a single clonal lineage, with identical Ave1 sequences, is consistent with the hypothesis that race 1 arose once in V. dahliae. Molecular markers and virulence assays also confirmed the well‐established finding that the D pathotype is found only in lineage 1A. Pathogenicity assays indicated that cotton and olive isolates of the D pathotype (lineage 1A) were highly virulent on cotton and olive, but had low virulence on tomato.     

大丽轮枝菌及激素处理后棉花酵母双杂交文库的构建

大丽轮枝菌是引起棉花、马铃薯等重要作物黄萎病的土传病原真菌,其分泌的胞外蛋白是侵染寄主的重要毒力因子,因此研究胞外蛋白与寄主的相互作用具有重要意义.本研究旨在构建高效的棉花酵母双杂交cDNA文库,并通过已知大丽轮枝菌效应子VCR1互作蛋白的筛选来评价构建文库的质量.分别采用大丽轮枝菌及茉莉酸甲酯、水杨酸和乙烯处理海岛棉...     

Race 2 of Verticillium dahliae infecting tomato in Japan can be split into two races with differential pathogenicity on resistant rootstocks

Verticillium dahliae infecting tomato can be differentiated into races 1 and 2 based on differential pathogenicity on tomato cultivars carrying resistance gene Ve1. Although no commercial cultivars resistant to race 2 are available, race 2‐resistant rootstock cultivars Aibou and Ganbarune‐Karis have been bred in Japan. Nevertheless, the resistance of these rootstocks appears to be unstable in commercial tomato fields. Pathogenicity assays conducted under controlled conditions revealed that these rootstock cultivars are resistant to some isolates of race 2; this resistance is controlled by a single dominant locus, denoted by V2, based on segregation of resistance in F₂ populations from selfed rootstock cultivars. However, some other isolates of race 2 can overcome this resistance. Therefore it is proposed that the current race 2 of V. dahliae should be divided into two races, i.e. ‘race 2’ (nonpathogenic on Aibou) and ‘race 3’ (pathogenic on Aibou). The distribution of these races was surveyed in 70 commercial tomato fields in Hida, Gifu Prefecture, Japan. Race 3 was found in 45 fields, indicating that race 3 had already spread throughout the region. On the other hand, 25 fields had only race 2, and thus race 2‐resistant rootstocks would be effective for disease management in these fields. Races 2 and 3 could not be identified by genomic Southern hybridization probed with a telomere sequence, nor with previously reported race‐specific PCR assays. Elucidation of race‐determining mechanisms and development of methods for quick race identification should be made in future studies.     

防治棉花黄萎病的生防细菌NCD-2的田间效果评价及其鉴定

 生防细菌NCD-2是自棉花根围土壤分离到的一株细菌菌株,田间小区试验表明:NCD-2菌株对棉花黄萎病的防治效果2年平均为85.4%,3年大区示范防病效果为51.2%~60.6%,并且该生防细菌对棉花产量和产量构成因子(单铃重、衣分、衣指、籽指等)及纤维品质(绒长)有所提高和改善。同时,本文测定了NCD-2对主要大田作物的安全性,证明其对小麦、玉米、棉花、马铃薯、黄瓜、茄子和大豆7种作物没有致病性,并且对这些作物的出苗、生长和发育没有不良影响。应用API50CH和API20 E细菌鉴定试剂盒,将NCD-2鉴定为枯草芽孢杆菌(Bacillus subtilis Cohn.)。