A Pectinase-deficient Ralstonia solanacearum Strain Induces Reduced and Delayed Structural Defences in Tomato Xylem

It has been suggested that oligogalacturonides (OGAs) released by bacterial pectinases can induce plant defence responses. To test this hypothesis, resistant tomato cultivar LS-89 and susceptible cultivar Ponderosa were inoculated with either wild-type Ralstonia solanacearum strain K60 or a pectinase-deficient triple mutant K60-509, which lacks endo-polygalacturonase PehA, exo-poly-alpha- d -galacturonosidase PehB, and pectin methylesterase Pme. K60 induced structural defence responses, including electron-dense materials (EDMs) in vessels and apposition layers (ALs) in parenchyma cells adjacent to xylem vessels colonized by bacteria in LS-89 stems. In contrast, LS-89 infected with K60-509 did not have any EDMs in vessels at 4 days after inoculation (DAI), and had them only rarely at 7 DAI. In LS-89 infected with K60-509, ALs were rarely observed in parenchyma cells adjacent to vessels at 4 DAI, and while they were present at 7 DAI, they were thinner than ALs induced by K60. The bacterial density in LS-89 stems infected with K60-509 was lower than in stems infected with K60 at 4 DAI, but the strains reached similar population sizes by 7 DAI, showing the pectinase-deficient mutant colonized resistant stems more slowly than did the wild-type strain. Vessels infected with K60-509 contained fewer EDMs at 7 DAI than were observed at either 4 or 7 DAI in vessels colonized by K60, although bacterial density in the xylem tissues containing K60-509 at 7 DAI was about the same as in the xylem tissues containing K60 at 4 DAI. Neither the wild-type strain nor the pectinase-deficient mutant induced these histopathological changes on susceptible cultivar Ponderosa. These results indicate that R. solanacearum pectinases play some role in eliciting histopathological changes in LS-89, likely by releasing OGAs that trigger plant structural defences.     

Ralstonia solanacearum genes induced during growth in tomato: an inside view of bacterial wilt

The phytopathogen Ralstonia solanacearum has over 5000 genes, many of which probably facilitate bacterial wilt disease development. Using in vivo expression technology (IVET), we screened a library of 133 200 R. solanacearum strain K60 promoter fusions and isolated approximately 900 fusions expressed during bacterial growth in tomato plants. Sequence analysis of 307 fusions revealed 153 unique in planta-expressed (ipx) genes. These genes included seven previously identified virulence genes (pehR, vsrB, vsrD, rpoS, hrcC, pme and gspK) as well as seven additional putative virulence factors. A significant number of ipx genes may reflect adaptation to the host xylem environment; 19.6%ipx genes are predicted to encode proteins with metabolic and/or transport functions, and 9.8%ipx genes encode proteins possibly involved in stress responses. Many ipx genes (18%) encode putative transmembrane proteins. A majority of ipx genes isolated encode proteins of unknown function, and 13% were unique to R. solanacearum. The ipx genes were variably induced in planta; beta-glucuronidase reporter gene expression analysis of a subset of 44 ipx fusions revealed that in planta expression levels were between two- and 37-fold higher than in culture. The expression of many ipx genes was subject to known R. solanacearum virulence regulators. Of 32 fusions tested, 28 were affected by at least one virulence regulator; several fusions were controlled by multiple regulators. Two ipx fusion strains isolated in this screen were reduced in virulence on tomato, indicating that gene(s) important for bacterial wilt pathogenesis were interrupted by the IVET insertion; mutations in other ipx genes are necessary to determine their roles in virulence and in planta growth. Collectively, this profile of ipx genes suggests that in its host, R. solanacearum confronts and overcomes a stressful and nutrient-poor environment.     

AM真菌对青枯菌和根际细菌群落结构的影响

利用传统的平板培养与DGGE相结合的技术手段,研究了接种AM真菌对番茄根际土壤中的青枯菌和细菌群落结构的影响。结果表明,菌根根际土壤中的细菌总量和总DNA量都高于非菌根根际土壤,其中前者的青枯菌种群数量比后者低60倍;DGGE图谱也证实了AM真菌对青枯菌的抑制效应,还揭示出接种AM真菌对根际土壤中细菌群落结构所产生的复杂的影响。文章对AM真菌抑制青枯菌的机制进行了探讨。     

一株番茄青枯病菌拮抗细菌的筛选、发酵条件优化及田间小区防效

[背景]番茄青枯病是由青枯劳尔氏菌(Ralstonia solanacearum)引起的一种土传细菌性病害,该病原菌严重影响番茄的生产。[目的]筛选番茄青枯病的生防细菌,并将其用于病害防治。[方法]采用抑菌圈法、琼脂扩散法从湖南衡阳青枯病发病田的健康番茄根际土壤筛选对青枯劳尔氏菌具有较强拮抗能力的菌株,通过形态学观察、生理生化试验、16S rRNA基因和gyrA基因测序分析确定其分类地位;以单因素试验和正交试验对发酵条件进行优化;通过田间小区试验初探其防效。[结果]筛选的菌株TR-1被初步鉴定为贝莱斯芽孢杆菌(Bacillus velezensislezensis);菌株TR-1最佳培养基配方(g/L):可溶性淀粉20.0,大豆蛋白胨10.0,磷酸氢二钾5.0;最佳发酵条件:pH6.0-7.0,温度30-33℃,摇床转速160 r/min,发酵时长48 h,优化后TR-1无菌发酵上清液对青枯菌抑菌圈直径达2.95 cm,约为优化前的2倍;其田间小区防效为60.30%。[结论]通过对菌株TR-1发酵条件进行优化可大大提升其发酵液抑菌效果,而且菌株TR-1在田间小区试验中对番茄青枯病防效优...     

一株番茄青枯病生防菌的鉴定与防病、定殖能力初探

摘要：【目的】采用根系分泌物培养基筛选到一株番茄根际优势细菌YPP-9。本文分析测定该菌株对植物青枯病菌茄科雷尔氏菌的拮抗作用和控病能力,及其在番茄根际的定殖能力,并系统分析该菌株的分类学地位。【方法】以平板双重培养法和温室盆栽试验分别测定菌株对病原菌的拮抗能力和对番茄青枯病的控病能力;利用变性梯度凝胶电泳技术分析菌株在番茄根际的定殖能力;以形态学和生理生化特性以及16S rRNA基因序列分析确定菌株的分类地位。【结果】菌株YPP-9对茄科雷尔氏菌SSF-4的平板抑菌带宽为5 mm,其盆栽控制番茄青枯病的效果达63.7%。菌株YPP-9在番茄根际具有较好的定殖能力。该菌株培养24 h后菌落呈奶酪色,革兰氏染色阳性,菌体杆状、大小1.8-4.1 μm×0.9-1.1 μm,形成芽孢,芽孢中生或偏端生且为近似柱形,孢囊不膨大,无伴孢晶体,侧生鞭毛。菌株生长pH范围为pH 5.5-8.5且最适生长pH为6.0,生长温度范围为20℃-45℃且最适生长温度为30℃。The BIOLOG GP2结果显示该菌为芽孢杆菌属。16S rRNA基因序列分析显示该菌株与Bacillus fumarioli的亲缘关系最近且序列相似性为97%,且其序列号为FJ231500。该菌株的G+C含量为41.9%,甲基萘醌主要类型为MK-7,细胞壁脂肪酸的主要种类为C14:0 iso、C15:0 iso 和C16:0 iso以及C16 : 1ω7c alcohol且含量分别为28.27%、19.59%、12.93%和10.88%。【结论】菌株YPP-9对茄科雷尔氏菌具有良好的拮抗作用和盆栽控病能力,且能良好的定殖于番茄根际。分类学上,该菌株归入芽胞杆菌属（Bacillus）,并可能是一个新的种。     

Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants

Ralstonia solanacearum, which causes bacterial wilt of diverse plants, produces copious extracellular polysaccharide (EPS), a major virulence factor. The function of EPS in wilt disease is uncertain. Leading hypotheses are that EPS physically obstructs plant water transport, or that EPS cloaks the bacterium from host plant recognition and subsequent defense. Tomato plants infected with R. solanacearum race 3 biovar 2 strain UW551 and tropical strain GMI1000 upregulated genes in both the ethylene (ET) and salicylic acid (SA) defense signal transduction pathways. The horizontally wilt-resistant tomato line Hawaii7996 activated expression of these defense genes faster and to a greater degree in response to R. solanacearum infection than did susceptible cultivar Bonny Best. However, EPS played different roles in resistant and susceptible host responses to R. solanacearum. In susceptible plants the wild-type and eps(-) mutant strains induced generally similar defense responses. But in resistant Hawaii7996 tomato plants, the wild-type pathogens induced significantly greater defense responses than the eps(-) mutants, suggesting that the resistant host recognizes R. solanacearum EPS. Consistent with this idea, purified EPS triggered significant SA pathway defense gene expression in resistant, but not in susceptible, tomato plants. In addition, the eps(-) mutant triggered noticeably less production of defense-associated reactive oxygen species in resistant tomato stems and leaves, despite attaining similar cell densities in planta. Collectively, these data suggest that bacterial wilt-resistant plants can specifically recognize EPS from R. solanacearum.     

Development of ELISA for the detection of Ralstonia solanacearum in tomato: its application in seed health testing

Bacterial wilt caused by Ralstonia (formerly Pseudomonas) solanacearum is worldwide in distribution. It is one of the most destructive bacterial diseases of economically important crops. The serological assays so far developed for the detection of R. solanacearum were able to provide information as to the presence or absence of the pathogen in soil and plant materials. However, they could not discriminate between virulent and avirulent strains of the pathogen and were not specific to strains and races. In the present investigation, virulent bacterial cells (encapsulated with mucin) from tomato seeds were used as antigen and polyclonal antisera were developed in rabbit using a classical immunization protocol. Antisera thus developed were examined for the antibody titre, sensitivity, specificity, rapidity and the efficacy of the antibody in identifying the virulent and avirulent strains of the pathogen and the potential for application of this assay to the screening of infected plant materials and seeds. Our results indicate that the anti-tomato R. solanacearum: (i) has a good antibody titre of 1:10,000; (ii) can detect as few as 100 bacterial cells/ml; (iii) is tomato-specific (it reacted with tomato R. solanacearum, and not with isolates from chilli or eggplant); (iv) is reactive to all isolates of R. solanacearum from tomato; (v) is not cross-reactive with non-pseudomonads; (vi) is virulent strain-specific as it recognizes the virulent exopolysaccharide component as an antigenic determinant; (vii) reactivity could be correlated well with the degree of infection in tomato seeds and plant materials. The enzyme linked immunosorbent assay developed is sensitive, specific and rapid, therefore suitable for the detection of R. solanacearum isolates from tomato seeds during routine assays.     

Quantitative immunofluorescence of regulated eps gene expression in single cells of Ralstonia solanacearum.

Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10(7) cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of beta-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined.     

Transposon mutagenesis reveals differential pathogenesis of Ralstonia solanacearum on tomato and Arabidopsis

Ralstonia solanacearum causes a deadly wilting disease on a wide range of crops. To elucidate pathogenesis of this bacterium in different host plants, we set out to identify R. solanacearum genes involved in pathogenesis by screening random transposon insertion mutants of a highly virulent strain, Pss190, on tomato and Arabidopsis thaliana. Mutants exhibiting various decreased virulence levels on these two hosts were identified. Sequence analysis showed that most, but not all, of the identified pathogenesis genes are conserved among distinct R. solanacearum strains. A few of the disrupted loci were not reported previously as being involved in R. solanacearum pathogenesis. Notably, a group of mutants exhibited differential pathogenesis on tomato and Arabidopsis. These results were confirmed by characterizing allelic mutants in one other R. solanacearum strain of the same phylotype. The significantly decreased mutants' colonization in Arabidopsis was found to be correlated with differential pathogenesis on these two plants. Differential requirement of virulence genes suggests adaptation of this bacterium in different host environments. Together, this study reveals commonalities and differences of R. solanacearum pathogenesis on single solanaceous and nonsolanaceous hosts, and provides important new insights into interactions between R. solanacearum and different host plants.     

A polyphasic approach for studying the interaction between Ralstonia solanacearum and potential control agents in the tomato phytosphere.

Ralstonia solanacearum biovar 2, the causative agent of brown rot in potato, has been responsible for large crop losses in Northwest Europe during the last decade. Knowledge on the ecological behaviour of R. solanacearum and its antagonists is required to develop sound procedures for its control and eradication in infested fields.A polyphasic approach was used to study the invasion of plants by a selected R. solanacearum biovar 2 strain, denoted 1609, either or not in combination with the antagonistic strains Pseudomonas corrugata IDV1 and P. fluorescens UA5-40. Thus, this study combined plating (spread and drop plate methods), reporter gene technology (gfp mutants) and serological (imunofluorescence colony staining [IFC]) and molecular techniques (fluorescent in situ hybridization [FISH], PCR with R. solanacearum specific primers and PCR-DGGE on plant DNA extracts). The behaviour of R. solanacearum 1609 and the two control strains was studied in bulk and (tomato) rhizosphere soil and the rhizoplane and stems of tomato plants.The results showed that an interaction between the pathogen and the control strains at the root surface was likely. In particular, R. solanacearum 1609 CFU numbers were significantly reduced on tomato roots treated with P. corrugata IDV1(chr:gfp1) cells as compared to those on untreated roots. Concomitant with the presence of P. corrugata IDV1(chr:gfp1), plant invasion by the pathogen was hampered, but not abolished.PCR-DGGE analyses of the tomato rhizoplane supported the evidence for antagonistic activity against the pathogen; as only weak R. solanacearum 1609 specific bands were detected in profiles derived from mixed systems versus strong bands in profiles from systems containing only the pathogen. Using FISH, a difference in root colonization was demonstrated between the pathogen and one of the two antagonists, i.e. P. corrugata IDV1(chr:gfp1); R. solanacearum strain 1609 was clearly detected in the vascular cylinder of tomato plants, whereas strain IDV1 was absent.R. solanacearum 1609 cells were also detected in stems of plants that had developed in soils treated with this strain, even in cases in which disease symptoms were absent, indicating the occurrence of symptomless infection. In contrast, strain 1609 cells were not found in stems of several plants treated with either one of the two antagonists.The polyphasic analysis is valuable for testing antagonistic strains for approval as biocontrol agents in agricultural practice.     

The effect of the substance exin on development of microbial infections and isolation of ethylene in plants

Effects of Exin on infection of tomato, potato, and cabbage plants with Pseudomonas solanacearum and Erwinia carotovora and a fungus Sclerotium rolfsii were studied. The treatment of infected plants with Exin caused no significant effect on the development of the disease. Treatment with streptomycin as a standard for comparison completely inhibited the growth of these microorganisms. Pretreatment with Exin one to eight days before infecting inhibited the development of diseases. The numbers of tomato and potato plants damaged among those infected with P. solanacearum were lower by 10 and 35% respectively. In field experiments (350 plants per variant), treatment with Exin decreased the development of wilt caused by S. rolfsii and P. solanacearum and rot caused by E. carotovora. Treatment with Exin activated the release of ethylene for not less than 30 days. Possible mechanisms of the effects of Exin are discussed.     

Ralstonia solanacearum strains from Martinique (French West Indies) exhibiting a new pathogenic potential

We investigated a destructive pathogenic variant of the plant pathogen Ralstonia solanacearum that was consistently isolated in Martinique (French West Indies). Since the 1960s, bacterial wilt of solanaceous crops in Martinique has been caused primarily by strains of R. solanacearum that belong to either phylotype I or phylotype II. Since 1999, anthurium shade houses have been dramatically affected by uncharacterized phylotype II strains that also affected a wide range of species, such as Heliconia caribea, cucurbitaceous crops, and weeds. From 1989 to 2003, a total of 224 R. solanacearum isolates were collected and compared to 6 strains isolated in Martinique in the 1980s. The genetic diversity and phylogenetic position of selected strains from Martinique were assessed (multiplex PCRs, mutS and egl DNA sequence analysis) and compared to the genetic diversity and phylogenetic position of 32 reference strains covering the known diversity within the R. solanacearum species complex. Twenty-four representative isolates were tested for pathogenicity to Musa species (banana) and tomato, eggplant, and sweet pepper. Based upon both PCR and sequence analysis, 119 Martinique isolates from anthurium, members of the family Cucurbitaceae, Heliconia, and tomato, were determined to belong to a group termed phylotype II/sequevar 4 (II/4). While these strains cluster with the Moko disease-causing strains, they were not pathogenic to banana (NPB). The strains belonging to phylotype II/4NPB were highly pathogenic to tomato, eggplant, and pepper, were able to wilt the resistant tomato variety Hawaii7996, and may latently infect cooking banana. Phylotype II/4NPB constitutes a new pathogenic variant of R. solanacearum that has recently appeared in Martinique and may be latently prevalent throughout Caribbean and Central/South America.     

Necessity of OxyR for the hydrogen peroxide stress response and full virulence in Ralstonia solanacearum

The plant pathogen Ralstonia solanacearum, which causes bacterial wilt disease, is exposed to reactive oxygen species (ROS) during tomato infection and expresses diverse oxidative stress response (OSR) genes during midstage disease on tomato. The R. solanacearum genome predicts that the bacterium produces multiple and redundant ROS-scavenging enzymes but only one known oxidative stress response regulator, OxyR. An R. solanacearum oxyR mutant had no detectable catalase activity, did not grow in the presence of 250 μM hydrogen peroxide, and grew poorly in the oxidative environment of solid rich media. This phenotype was rescued by the addition of exogenous catalase, suggesting that oxyR is essential for the hydrogen peroxide stress response. Unexpectedly, the oxyR mutant strain grew better than the wild type in the presence of the superoxide generator paraquat. Gene expression studies indicated that katE, kaG, ahpC1, grxC, and oxyR itself were each differentially expressed in the oxyR mutant background and in response to hydrogen peroxide, suggesting that oxyR is necessary for hydrogen peroxide-inducible gene expression. Additional OSR genes were differentially regulated in response to hydrogen peroxide alone. The virulence of the oxyR mutant strain was significantly reduced in both tomato and tobacco host plants, demonstrating that R. solanacearum is exposed to inhibitory concentrations of ROS in planta and that OxyR-mediated responses to ROS during plant pathogenesis are important for R. solanacearum host adaptation and virulence.     

Highly virulent strains of Pseudomonas solanacearum that are defective in extracellular-polysaccharide production

Extracellular polysaccharide (EPS) has long been regarded as one of the most important factors involved in wilting of plants by Pseudomonas solanacearum. By means of transposon Tn5 mutagenesis, we have isolated a class of mutants that have an afluidal colony morphology but retain the ability to cause severe wilting and death of tobacco plants. One such mutant, KD700, was studied in detail. By marker exchange mutagenesis, the altered colony morphology was shown to be the result of a single Tn5 insertion in a 14.3-kilobase EcoRI fragment. This defect could be corrected by introducing a homologous clone from a cosmid library of the wild-type, parental strain K60. The Tn5-containing fragment was introduced into other P. solanacearum wild-type strains by marker exchange, and these altered strains had the same afluidal phenotype as KD700. N-Acetylgalactosamine (GalNac), the major constituent of EPS of all wild-type strains of P. solanacearum, was not detected by gas chromatography-mass spectrometry analysis of vascular fluids from wilting plants infected by KD700. In contrast, GalNac was readily detected in similar fluids of plants infected by K60. Polysaccharides extracted from culture filtrates of KD700 contained approximately one-fifth of the GalNac present in polysaccharides from K60. No differences in growth rates in culture or in planta between the mutant and the parental strains were observed. Since strains that are deficient in EPS production can remain highly virulent to tobacco, we conclude that EPS, or at least its GalNac-containing component, may not be required for disease development by P. solanacearum.     

Static suppression of tomato bacterial wilt by bacterial coagulation using a new functional polymer that coagulates bacterial cells and is highly biodegradable

Tomato bacterial wilt by Ralstonia solanacearum was suppressed by coagulation of bacterial cells without disinfection using a copolymer of methyl methacrylate with N-benzyl-4-vinylpyridinium chloride in a molar ratio of 3:1 (PMMA-co-BVP) as a polymeric coagulant for bacterial cells. When 10 mg/kg of PMMA-co-BVP was added to soil before transplanting of tomato seedlings, and 2 mg/kg was supplemented once a week after transplanting, a 51% reduction of appearance and a 54% reduction of index of symptoms were observed. PMMA-co-BVP did not exhibit bactericidal activity against R. solanacearum, and coagulation of the bacterial cells appeared to reduce the opportunity for infectious contact of roots of tomato with cells of R. solanacearum, and resulted in disease suppression. PMMA-co-BVP was shown to be highly biodegradable, and the half-life was 5.1 d when treated with activated sludge in soil.     

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.     

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 (>10(7) 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.     

Biological and Chemical Induction of Resistance to the Globodera tabacum solanacearum in Oriental and Flue-Cured Tobacco (Nicotiana tabacum L.)

The effects of acibenzolar-S-methyl (ASM) and four combinations of plant growth-promoting rhizobacteria (PGPR) on the reproduction of a tobacco cyst nematode, Globodera tabacum solanacearum, and growth of Nicotiana tabacum (cv. K326 and Xanthi) were tested under greenhouse and field conditions. The PGPR included combinations of Bacillus subtilis A13 with B. pumilis INR7, B. pumilis SE34, B. licheniformis IN937b, or B. amyloliquefaciens IN937a, respectively. Among the four rhizobacterial combinations, IN937a + A13 exhibited the most consistent reduction in G. t. solanacearum cysts under greenhouse and field conditions. No undesirable effects of IN937a + A13 were observed on tobacco growth under greenhouse and field conditions. Use of INR7 + A13 reduced G. t. solanacearum reproduction on flue-cured tobacco cv. K326 but not on oriental tobacco cv. Xanthi. Application of ASM reduced final numbers of G. t. solanacearum cysts, but also resulted in phytotoxicity mainly under the greenhouse conditions. When oriental tobacco seedlings were pre-grown in a IN937a + A13-treated soil-less medium, a single application of ASM at 200 mg/L one week after transplanting significantly reduced G. t. solanacearum reproduction in the field.     

An exo-poly-alpha-D-galacturonosidase, PehB, is required for wild-type virulence of Ralstonia solanacearum.

Ralstonia solanacearum, which causes bacterial wilt disease of many plant species, produces several extracellular plant cell wall-degrading enzymes that are suspected virulence factors. These include a previously described endopolygalacturonase (PG), PehA, and two exo-PGs. A gene encoding one of the exo-PGs, pehB, was cloned from R. solanacearum K60. The DNA fragment specifying PehB contained a 2,103-bp open reading frame that encodes a protein of 74.2 kDa with a typical N-terminal signal sequence. The cloned pehB gene product cleaves polygalacturonic acid into digalacturonic acid units. The amino acid sequence of pehB resembles that of pehX, an exo-PG gene from Erwinia chrysanthemi, with 47.2% identity at the amino acid level. PehB also has limited similarity to plant exo-PGs from Zea mays and Arabidopsis thaliana. The chromosomal pehB genes in R. solanacearum wild-type strain K60 and in an endo-PG PehA- strain were replaced with an insertionally inactivated copy of pehB. The resulting mutants were deficient in the production of PehB and of both PehA and PehB, respectively. The pehB mutant was significantly less virulent than the wild-type strain in eggplant virulence assays using a soil inoculation method. However, the pehA mutant was even less virulent, and the pehA pehB double mutant was the least virulent of all. These results suggest that PehB is required for a wild-type level of virulence in R. solanacearum although its individual role in wilt disease development may be minor. Together with endo-PG PehA, however, PehB contributes substantially to the virulence of R. solanacearum.     

PCR-based specific detection of Ralstonia solanacearum by amplification of cytochrome c1 signal peptide sequences

A polymerase chain reaction (PCR)-based method was developed to detect the DNA of Ralstonia solanacearum, the causal agent of bacterial wilt in various crop plants. One pair of primers (RALSF and RALSR), designed using cytochrome c1 signal peptide sequences specific to R. solanacearum, produced a PCR product of 932 bp from 13 isolates of R. solanacearum from several countries. The primer specificity was then tested using DNA from 21 isolates of Ralstonia, Pseudomonas, Burkholderia, Xanthomonas, and Fusarium oxysporum f. sp. dianthi. The specificity of the cytochrome c1 signal peptide sequences in R. solanacearum was further confirmed by a DNA-dot blot analysis. Moreover, the primer pair was able to detect the pathogen in artificially inoculated soil and tomato plants. Therefore, the present results indicate that the primer pair can be effectively used for the detection of R. solanacearum in soil and host plants.