番茄青枯病拮抗菌的筛选

利用点接法从山东寿光和苍山不同种植年限的蔬菜大棚中分离得到的45株菌株以及实验室保存的19株菌株中,筛选到14株对番茄青枯病病原细菌有拮抗作用的菌株,然后通过牛津杯法复筛得到6株抑菌效果较好的细菌株。通过温室盆栽试验表明拮抗菌X10的防治效果最好,液体菌剂防治效果达到了81.8%,固体菌剂防治效果达到了65.4%,具有良好的应用前景。对菌株X10进行了培养特征、形态特征和生理生化特征测定,鉴定为侧胞短杆芽胞杆菌(Brevi Bacillus laterosporus)。     

青枯菌致病机理及作物抗青枯病研究进展

青枯菌(Rdstonia solancearum)是引起植物青枯病的病原细菌.青枯菌通过T3S(Ⅲ型分泌系统)、T2S(Ⅱ型分泌系统)等分泌系统将多种毒性因子输送到胞外使寄主植物致病.转基因抗病、培育抗性品种和生物防治是防治青枯病的主要途径.     

不同青枯病抗性的番茄品种内生细菌生理群数量研究

本论文对青枯病抗性不同的番茄品种其内生细菌生理群数量变化进行了研究,结果表明,番茄内生细菌生理群数量的变化随品种抗性、生育期和季节的不同而变化.在7大类生理群细菌中,氨化细菌的数量最多,且在幼苗期以后,高抗青枯病番茄品种中数量明显高于高感品种,初步认为,氨化细菌可能是影响青枯病发生的关键性微生物.番茄抗病品种在不同生育期,其内生细菌的总体数量要比感病品种多,呈交替波动变化.氨化细菌、硝化细菌、固氮细菌和反硫化细菌平均数量均表现为在夏季高于冬季,硫化细菌的数量则冬季高于夏季,厌气性细菌数量最少.     

Relationship Between Ralstonia solanacearum Diversity and Severity of Bacterial Wilt Disease in Tomato Fields in China

A field survey was conducted to determine the relationship between Ralstonia solanacearum diversity and severity of bacterial wilt disease in tomato plants grown in plastic greenhouses. Both vegetative and reproductive stages of the plants were surveyed, and the symptoms were empirically categorized into five scales: 0 (asymptomatic): 1st, 2nd, 3rd and 4th. The bacterial wilt pathogen was isolated from infected plants at each disease scale; pathogenic characteristics and population densities of the bacterial strains were assessed. Two hundred and eighty‐two isolates were identified as R. solanacearum, which were divided into three pathogenic types, virulent, avirulent and interim, using the attenuation index (AI) method and a plant inoculation bioassay. Ralstonia solanacearum was detected in all asymptomatic and symptomatic tomato plants, with population numbers, ranging from 10.5 to 86.7 × 105 cfu/g. However, asymptomatic plants harboured only avirulent or interim R. solanacearum, whereas tomato plants displaying 1st or 2nd disease degree contained interim and virulent strains. Additionally, 3rd and 4th degree plants harboured only virulent strains. The disease was more severe in vegetative‐stage plants (disease severity index (DSI) 0.20) with higher total numbers of interim and virulent R. solanacearum strains than those in reproductive‐stage plants (DSI 0.12). Three pathotypes of R. solanacearum coexisted in a competitive growth system in the tomato field, and their distribution closely correlated with the severity of tomato bacterial wilt.     

EpsR Modulates Production of Extracellular Polysaccharides in the Bacterial Wilt Pathogen Ralstonia (Pseudomonas) solanacearum

Ralstonia solanacearum is the causal agent of bacterial wilt of many agriculturally important crops. Exopolysaccharide synthesized by products of the epsI operon is the major virulence factor for R. solanacearum. Expression of epsI has been demonstrated to be under the control of several proteins, including several two-component regulators. Overexpression of EpsR was found previously to reduce the amount of synthesis specifically from the epsI promoter. Here we present data that a single chromosomal copy of epsR activates the epsI promoter, suggesting that EpsR is a concentration-dependent effector of epsI gene expression. Furthermore, the ability of EpsR to modulate epsI expression is dependent on the phosphorylation state of EpsR. Gel mobility shift assays suggest that EpsR can specifically bind the epsI promoter and that this binding requires a phosphorylated form of EpsR.     

Using the Ralstonia solanacearum Tat Secretome To Identify Bacterial Wilt Virulence Factors

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.     

A Monogenic Dominant Resistance of Tomato to Bacterial Wilt in Hawaii 7996 is Associated with Plant Colonization by Pseudomonas solanacearum

Hawaii 7996, a tomato cultivar resistant to bacterial wilt caused by P. solanacearum was crossed with Floradel, a susceptible cultivar and the F1 and F2 seeds were obtained. Inoculated plants were tested in the field for bacterial wilt resistance and colonization by P. solanacearum. The F1 did not wilt and a significant 3:1 segregation for non wilting: wilting was observed in the F2, indicating a monogenic dominant resistance in Hawaii 7996. In the F2 and in Hawaii 7996, resistance was, associated to the limitation of bacterial spread in the stem. The degree of resistance of Floradel, the F2 and Hawaï 7996 was correlated to colonization at midstem. The usefulness of plant colonization criteria for breeding programs is discussed.     

Hrp Mutants of Pseudomonas solanacearum as Potential Biocontrol Agents of Tomato Bacterial Wilt

There have been many attempts to control bacterial wilt with antagonistic bacteria or spontaneous nonpathogenic mutants of Pseudomonas solanacearum that lack the ability to colonize the host, but they have met with limited success. Since a large gene cluster (hrp) is involved in the pathogenicity of P. solanacearum, we developed a biological control strategy using genetically engineered Hrp mutants of P. solanacearum. Three pathogenic strains collected in Guadeloupe (French West Indies) were rendered nonpathogenic by insertion of an omega-Km interposon within the hrp gene cluster of each strain. The resulting Hrp mutants were tested for their ability to control bacterial wilt in challenge inoculation experiments conducted either under growth chamber conditions or under greenhouse conditions in Guadeloupe. Compared with the colonization by a pathogenic strain which spread throughout the tomato plant, colonization by the mutants was restricted to the roots and the lower part of the stems. The mutants did not reach the fruit. Moreover, the presence of the mutants did not affect fruit production. When the plants were challenge inoculated with a pathogenic strain, the presence of Hrp mutants within the plants was correlated with a reduction in disease severity, although pathogenic bacteria colonized the stem tissue at a higher density than the nonpathogenic bacteria. Challenge inoculation experiments conducted under growth chamber conditions led, in some cases, to exclusion of the pathogenic strain from the aerial part of the plant, resulting in high protection rates. Furthermore, there was evidence that one of the pathogenic strains used for the challenge inoculations produced a bacteriocin that inhibited the in vitro growth of the nonpathogenic mutants.     

Hrp- Mutants of Pseudomonas solanacearum as Potential Biocontrol Agents of Tomato Bacterial Wilt

There have been many attempts to control bacterial wilt with antagonistic bacteria or spontaneous nonpathogenic mutants of Pseudomonas solanacearum that lack the ability to colonize the host, but they have met with limited success. Since a large gene cluster (hrp) is involved in the pathogenicity of P. solanacearum, we developed a biological control strategy using genetically engineered Hrp^- mutants of P. solanacearum. Three pathogenic strains collected in Guadeloupe (French West Indies) were rendered nonpathogenic by insertion of an ω-Km interposon within the hrp gene cluster of each strain. The resulting Hrp^- mutants were tested for their ability to control bacterial wilt in challenge inoculation experiments conducted either under growth chamber conditions or under greenhouse conditions in Guadeloupe. Compared with the colonization by a pathogenic strain which spread throughout the tomato plant, colonization by the mutants was restricted to the roots and the lower part of the stems. The mutants did not reach the fruit. Moreover, the presence of the mutants did not affect fruit production. When the plants were challenge inoculated with a pathogenic strain, the presence of Hrp^- mutants within the plants was correlated with a reduction in disease severity, although pathogenic bacteria colonized the stem tissue at a higher density than the nonpathogenic bacteria. Challenge inoculation experiments conducted under growth chamber conditions led, in some cases, to exclusion of the pathogenic strain from the aerial part of the plant, resulting in high protection rates. Furthermore, there was evidence that one of the pathogenic strains used for the challenge inoculations produced a bacteriocin that inhibited the in vitro growth of the nonpathogenic mutants.     

番茄抗青枯病基因的AFLP分子标记

用番茄高抗青枯病品种“T51A”与高感青枯病品种“T9230”配制杂交组合,接种鉴定其正反交Ｆ₁代及Ｆ₂代分离群体的青枯病发生情况。结果表明,T51A对青枯病的抗性属于细胞质遗传,受1对杂合基因加性控制。用64个EcoRＩ/ＭseＩ引物组合对“T51A”、“T9230”两个亲本及其Ｆ₂代抗病和感病基因池进行AFLP分析,共扩增出约4200条可分辨的带,其中2条为稳定的差异。用“T51A”和“T9230”杂交产生的Ｆ₂代分离群体对2个特异条带与目的基因的遗传连锁性进行分析,发现特异条带AAG/CAT与暂定名为RRS-342的抗青枯病基因紧密连锁,二者之间的遗传距离为6.7 cM。将AAG/CAT片段回收、克隆和测序,成功地将其转化为SCAR标记,可以更加方便地用于对番茄青枯病基因的标记辅助选择。      

Ralstonia solanacearum Pectin Methylesterase Is Required for Growth on Methylated Pectin but Not for Bacterial Wilt Virulence

Ralstonia (Pseudomonas) solanacearum causes bacterial wilt, a serious disease of many crop plants. The pathogen produces several extracellular plant cell wall-degrading enzymes, including polygalacturonases (PGs) and pectin methylesterase (Pme). Pme removes methyl groups from pectin, thereby facilitating subsequent breakdown of this cell wall component by PGs, which are known bacterial wilt virulence factors. R. solanacearum PGs could not degrade 93% methylated pectin unless the substrate was first demethylated by Pme, but as the degree of methylation of the pectin substrate decreased, PG activity increased. Primers derived from a published pme sequence generated an 800-bp DNA probe fragment, which identified Pme-encoding plasmids from a R. solanacearum genomic library. A pme chromosomal mutant had no detectable Pme activity in vitro and no longer grew on 93% methylated pectin as a carbon source. Curiously, the pme mutant, which had no detectable PG activity on highly methylated pectin, was just as virulent as the wild-type strain on tomato, eggplant (aubergine), and tobacco. Since PG activity is required for full virulence, this result suggests that the pectin in these particular hosts may not be highly methylated, or that the breakdown of highly methylated pectin is not a significant factor in the disease process in general. A positive response regulator of PG production called PehR was not required for wild-type Pme production. However, a mutant strain lacking PhcA, which is a global regulator of several virulence genes, produced no detectable Pme activity. Thus, pme expression is directly or indirectly regulated by PhcA but not by PehR.     

Invasiveness of Avirulent Strains of Pseudomonas solanacearum in Tomato Cultivars, Resistant or Susceptible to Bacterial Wilt

Tomato plant colonization by avirulent strains of Pseudomonas solanacearum was studied. The strain 8217R a spontaneous rough mutant was able to penetrate into roots by nature opennings and colonize plants at high levels. The strain 8173, a hrp^- engineered fluidal mutant, colonized the plants to a lesser extent than 8217R. There was no difference in colonization observed at taproots and hypocotyls between resistant and susceptible cultivars with the avirulent strains 8217R and 8173, contrary to the plant-virulent strain interaction. Results indicated that the bacterial spread was limited in the two cultivars regardless of their resistance or susceptibility and suggested the existence of a host defence mechanism, limiting the bacterial spread.     

Direct Delivery of Inoculum to Shoot Tissue Interferes with Genotypic Resistance to Ralstonia solanacearum in Tomato Seedlings

Employing known susceptible and resistant genotypes and pure bacterial inoculum (0.1 OD; 10⁸ CFU/ml^?1), five different inoculation methods were tried to assess the response of tomato genotypes to Ralstonia solanacearum. This included seed‐soaking inoculation, seed‐sowing followed by inoculum drenching, or at 2‐week stage through petiole‐excision inoculation, soaking of planting medium with inoculum either directly or after imparting seedling root‐injury. Seed‐based inoculations or mere inoculum drenching at 2 weeks did not induce much disease in seedlings. Petiole inoculation induced 90–100% mortality in susceptible checks but also 50–60% mortality in normally resistant genotypes within 7–10 days. Root‐injury inoculation at 2‐week seedling stage appeared the best for early and clearer distinction between resistant and susceptible lines. The observations suggest a role played by the root system in governing genotypic resistance to the pathogen. Direct shoot inoculation is to be adopted only for selecting highly resistant lines or to thin down segregating populations during resistance breeding.     

Field Performance of Resistant Potato Genotypes Transformed with the EFR Receptor from Arabidopsis thaliana in the Absence of Bacterial Wilt (Ralstonia solanacearum)

Bacterial wilt caused by the pathogen Ralstonia solanacearum is a devastating disease of potato crops. Harmonizing immunity to pathogens and crop yield is a balance between productive, economic, and environmental interests. In this work, the agronomic performance of two events of potato cultivar INIA Iporá expressing the Arabidopsis thaliana EFR gene (Iporá EFR 3 and Iporá EFR 12) previously selected for their high resistance to bacterial wilt was evaluated under pathogen-free conditions. During two cultivation cycles, the evaluated phenotypic characteristics were emergence, beginning of flowering, vigor, growth, leaf morphology, yield, number and size of tubers, analyzed under biosecurity standards. The phenotypic characteristics evaluated did not show differences, except in the morphology of the leaf with a more globose appearance and a shortening of the rachis in the transformation events with respect to untransformed Iporá. The Iporá EFR 3 genotype showed a ~40% yield decrease in reference to untransformed Iporá in the two trials, while Iporá EFR 12 did not differ statistically from untransformed Iporá. Iporá EFR 12 shows performance stability in the absence of the pathogen, compared to the untransformed control, positioning it as an interesting candidate for regions where the presence of the pathogen is endemic and bacterial wilt has a high economic impact.     

Understanding Reaction of Potato (Solanum tuberosum) to Ralstonia solanacearum and Relationship of Wilt Incidence to Latent Infection

Bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases affecting more than 200 plant species, including solanaceous crops. The pathogen is known to cause complicated symptoms ranging from visible to latent ones. Understanding crop's reaction to the pathogen and the underlying relatedness of latent infection to wilt incidence is of paramount importance. Thus, a number of potato cultivars including improved and otherwise were evaluated under greenhouse and field conditions. Accordingly, twenty‐eight of the cultivars tested under greenhouse conditions were resistant to the pathogen with scores ranging from 0.77 to 1.17 of 5. Nonetheless, under field conditions, only 2 of 28 cultivars found to be ‘resistant’ under greenhouse conditions, showed adequate resistance to the pathogen, indicating the significant impact of environment on the activity of the pathogen and reaction of the crop. Percentage wilt incidence and latent infection showed significant (P < 0.05) positive correlation, with r = 0.9438. Thus, evaluation of crop's performance based on the combination of the parameters like field wilt incidence and proportion of latent infection gave us better picture of the overall crop feat, than using wilt incidence as a sole parameter of evaluation as has been the case in most studies. Moreover, the established correlation of latent infection with field wilt incidence will also help us understand the disease epidemiology and design effective management measures, accordingly.     

Grafting Tomato Cultivars Resistant or Susceptible to Bacterial Wilt: Analysis of Resistance Mechanisms

Grafting experiments were carried out in order to understand tomato resistance mechanisms to Pseudomonas solanacearum . Resistant scions grafted on susceptible root-stocks wilted, indicating that vascular tissues of resistant cultivars were not tolerant to higher bacterial populations than susceptible ones. Colonization frequencies and bacterial densities observed in plant grafted on resistant or susceptiblle root-stocks showed that resistance was correlated to the limitation of bacterial spread in the lower part of the stem.     

Two Host-Induced Ralstonia solanacearum Genes, acrA and dinF, Encode Multidrug Efflux Pumps and Contribute to Bacterial Wilt Virulence

Multidrug efflux pumps (MDRs) are hypothesized to protect pathogenic bacteria from toxic host defense compounds. We created mutations in the Ralstonia solanacearum acrA and dinF genes, which encode putative MDRs in the broad-host-range plant pathogen. Both mutations reduced the ability of R. solanacearum to grow in the presence of various toxic compounds, including antibiotics, phytoalexins, and detergents. Both acrAB and dinF mutants were significantly less virulent on the tomato plant than the wild-type strain. Complementation restored near-wild-type levels of virulence to both mutants. Addition of either dinF or acrAB to Escherichia coli MDR mutants KAM3 and KAM32 restored the resistance of these strains to several toxins, demonstrating that the R. solanacearum genes can function heterologously to complement known MDR mutations. Toxic and DNA-damaging compounds induced expression of acrA and dinF, as did growth in both susceptible and resistant tomato plants. Carbon limitation also increased expression of acrA and dinF, while the stress-related sigma factor RpoS was required at a high cell density (>10⁷ CFU/ml) to obtain wild-type levels of acrA expression both in minimal medium and in planta. The type III secretion system regulator HrpB negatively regulated dinF expression in culture at high cell densities. Together, these results show that acrAB and dinF encode MDRs in R. solanacearum and that they contribute to the overall aggressiveness of this phytopathogen, probably by protecting the bacterium from the toxic effects of host antimicrobial compounds.     

Role of Phenylalanine Ammonia Lyase and Polyphenol Oxidase in Host Resistance to Bacterial Wilt of Tomato

Plants respond to bacterial pathogen attack by activating various defence responses, which are associated with the accumulation of several factors like defence-related enzymes and inhibitors which serve to prevent pathogen infection. The present study focused on the role of the defence-related enzymes phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) in imparting resistance to tomato against bacterial wilt pathogen Ralstonia solanacearum . The temporal pattern of induction of these enzymes showed maximum activity at 12 h and 15 h for PAL and PPO, respectively, after the pathogen inoculation (hpi) in resistant cultivars. Twenty different tomato cultivars were analyzed for PAL, PPO and total phenol content following pathogen inoculation. The enzyme activities and total phenol content increased significantly (P < 0.05) in resistant cultivars upon pathogen inoculation. The increase in enzyme activities and total phenol content were not significant in susceptible and highly susceptible cultivars. The role of PAL and PPO in imparting resistance to tomato against bacterial wilt disease is discussed.