scholarly journals A Novel, Sensitive Method to Evaluate Potato Germplasm for Bacterial Wilt Resistance Using a Luminescent Ralstonia solanacearum Reporter Strain

2014 ◽  
Vol 27 (3) ◽  
pp. 277-285 ◽  
Author(s):  
Andrea Paola Zuluaga Cruz ◽  
Virginia Ferreira ◽  
María Julia Pianzzola ◽  
María Inés Siri ◽  
Núria S. Coll ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Accurate Method ◽  
Quantitative Detection ◽  
Screening Methods ◽  
Reporter Strain ◽  
In Planta ◽  
Breeding Programs ◽  
Potato Germplasm ◽  
Pathogen Colonization

Several breeding programs are under way to introduce resistance to bacterial wilt caused by Ralstonia solanacearum in solanaceous crops. The lack of screening methods allowing easy measurement of pathogen colonization and the inability to detect latent (i.e., symptomless) infections are major limitations when evaluating resistance to this disease in plant germplasm. We describe a new method to study the interaction between R. solanacearum and potato germplasm that overcomes these restrictions. The R. solanacearum UY031 was genetically modified to constitutively generate light from a synthetic luxCDABE operon stably inserted in its chromosome. Colonization of this reporter strain on different potato accessions was followed using life imaging. Bacterial detection in planta by this nondisruptive system correlated with the development of wilting symptoms. In addition, we demonstrated that quantitative detection of the recombinant strain using a luminometer can identify latent infections on symptomless potato plants. We have developed a novel, unsophisticated, and accurate method for high-throughput evaluation of pathogen colonization in plant populations. We applied this method to compare the behavior of potato accessions with contrasting resistance to R. solanacearum. This new system will be especially useful to detect latency in symptomless parental lines before their inclusion in long-term breeding programs for disease resistance.

scholarly journals Ralstonia solanacearum Needs Motility for Invasive Virulence on Tomato

2001 ◽  
Vol 183 (12) ◽  
pp. 3597-3605 ◽  
Author(s):  
Julie Tans-Kersten ◽  
Huayu Huang ◽  
Caitilyn Allen
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Soft Agar ◽  
Cell Protein ◽  
In Planta ◽  
Tomato Plants ◽  
Bacterial Wilt Disease ◽  
Virulence Assay ◽  
Flagellar Filament

ABSTRACT Ralstonia solanacearum, a widely distributed and economically important plant pathogen, invades the roots of diverse plant hosts from the soil and aggressively colonizes the xylem vessels, causing a lethal wilting known as bacterial wilt disease. By examining bacteria from the xylem vessels of infected plants, we found thatR. solanacearum is essentially nonmotile in planta, although it can be highly motile in culture. To determine the role of pathogen motility in this disease, we cloned, characterized, and mutated two genes in the R. solanacearum flagellar biosynthetic pathway. The genes for flagellin, the subunit of the flagellar filament (fliC), and for the flagellar motor switch protein (fliM) were isolated based on their resemblance to these proteins in other bacteria. As is typical for flagellins, the predicted FliC protein had well-conserved N- and C-terminal regions, separated by a divergent central domain. The predicted R. solanacearum FliM closely resembled motor switch proteins from other proteobacteria. Chromosomal mutants lackingfliC or fliM were created by replacing the genes with marked interrupted constructs. Since fliM is embedded in the fliLMNOPQR operon, the aphAcassette was used to make a nonpolar fliM mutation. Both mutants were completely nonmotile on soft agar plates, in minimal broth, and in tomato plants. The fliC mutant lacked flagella altogether; moreover, sheared-cell protein preparations from the fliC mutant lacked a 30-kDa band corresponding to flagellin. The fliM mutant was usually aflagellate, but about 10% of cells had abnormal truncated flagella. In a biologically representative soil-soak inoculation virulence assay, both nonmotile mutants were significantly reduced in the ability to cause disease on tomato plants. However, the fliC mutant had wild-type virulence when it was inoculated directly onto cut tomato petioles, an inoculation method that did not require bacteria to enter the intact host from the soil. These results suggest that swimming motility makes its most important contribution to bacterial wilt virulence in the early stages of host plant invasion and colonization.

scholarly journals Evaluation of Resistance Sources of Tomato (Solanum lycopersicum L.) to Phylotype I Strains of Ralstonia solanacearum Species Complex in Benin

Agronomy ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1513
Author(s):  
Herbaud Zohoungbogbo ◽  
Adonis Quenum ◽  
Judith Honfoga ◽  
Jaw-Rong Chen ◽  
Enoch Achigan-Dako ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Species Complex ◽  
Block Design ◽  
Agricultural System ◽  
Sources Of Resistance ◽  
Breeding Programs ◽  
Solanum Lycopersicum L ◽  
The University ◽  
Phylotype I

Finding sources of resistance to bacterial wilt (BW) caused by Ralstonia solanacearum species complex is a crucial step toward the development of improved bacterial wilt-resistant tomato varieties. Here, we evaluated new sources of bacterial wilt-tolerant/resistant tomato lines and identified associated phylotype/sequevar of R. solanacearum strains in Benin. Eighteen F5 lines and five checks were evaluated in two hotspots: the experimental site of the World Vegetable Center, Cotonou Benin, and the Laboratory of Genetics, Biotechnology and Seed Science of the University of Abomey-Calavi. Experiments were laid out in a randomized complete block design with four replicates. Data were collected on bacterial wilt incidence, horticultural and fruit traits and yield components. Across the two experiments, the F5 lines showed no wilting, while the local variety ‘Tounvi’ used as susceptible check showed 57.64% wilting. The wilting was due to BW and was associated with sequevars I-14, I-18 and I-31 of phylotype I. AVTO1803, AVTO1955-6 and H7996 were the highest yielding lines with 20.29 t·ha−1, 17.66 t·ha−1 and 17.07 t/ha, respectively. The sources of resistance to BW can be recommended to national agricultural system for dissemination or used in tomato breeding programs.

scholarly journals Leaf-to-Whole Plant Spread Bioassay for Pepper and Ralstonia solanacearum Interaction Determines Inheritance of Resistance to Bacterial Wilt for Further Breeding

2021 ◽  
Vol 22 (5) ◽  
pp. 2279
Author(s):  
Ji-Su Kwon ◽  
Jae-Young Nam ◽  
Seon-In Yeom ◽  
Won-Hee Kang
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Evaluation Method ◽  
Inheritance Of Resistance ◽  
Breeding Programs ◽  
Whole Plant ◽  
Resistance Trait ◽  
Breeding Efficiency ◽  
Serious Disease ◽  
Chili Peppers

Bacterial wilt (BW) disease from Ralstonia solanacearum is a serious disease and causes severe yield losses in chili peppers worldwide. Resistant cultivar breeding is the most effective in controlling BW. Thus, a simple and reliable evaluation method is required to assess disease severity and to investigate the inheritance of resistance for further breeding programs. Here, we developed a reliable leaf-to-whole plant spread bioassay for evaluating BW disease and then, using this, determined the inheritance of resistance to R. solanacearum in peppers. Capsicum annuum ‘MC4′ displayed a completely resistant response with fewer disease symptoms, a low level of bacterial cell growth, and significant up-regulations of defense genes in infected leaves compared to those in susceptible ‘Subicho’. We also observed the spreading of wilt symptoms from the leaves to the whole susceptible plant, which denotes the normal BW wilt symptoms, similar to the drenching method. Through this, we optimized the evaluation method of the resistance to BW. Additionally, we performed genetic analysis for resistance inheritance. The parents, F1 and 90 F2 progenies, were evaluated, and the two major complementary genes involved in the BW resistance trait were confirmed. These could provide an accurate evaluation to improve resistant pepper breeding efficiency against BW.

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

2007 ◽  
Vol 73 (12) ◽  
pp. 3779-3786 ◽  
Author(s):  
Enid T. Gonz�lez ◽  
Darby G. Brown ◽  
Jill K. Swanson ◽  
Caitilyn Allen
Keyword(s):  
Virulence Factors ◽  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Acid Tolerance ◽  
Bioinformatic Analysis ◽  
Wild Type ◽  
In Planta ◽  
Tomato Plants ◽  
Tat System

ABSTRACT 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.

scholarly journals Viability, Stability and Biocontrol Activity in Planta of Specific Ralstonia Solanacearum Bacteriophages After Their Conservation Prior to Commercialization and Use

2021 ◽  
Author(s):  
Belén Álvarez ◽  
Laura Gadea-Pallás ◽  
Alejandro Rodríguez ◽  
Begonya Vicedo ◽  
Àngela Figàs-Segura ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Reference Strain ◽  
Specific Activity ◽  
Experimental Period ◽  
In Planta ◽  
Tomato Plants ◽  
Preservation Method ◽  
Biocontrol Activity ◽  
Susceptible Tomato

Ralstonia solanacearum is a pathogen that causes bacterial wilt producing severe damage in staple solanaceous crops. Traditional control has low efficacy and/or environmental impact. Recently, the bases of a new biotechnological method by lytic bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 with specific activity against R. solanacearum were established. However, some aspects remain unknown, such as the survival and maintenance of the lytic activity after submission to a preservation method as the lyophilization. To this end, viability and stability of lyophilized vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 and their capacity for bacterial wilt biocontrol have been determined against one pathogenic Spanish reference strain of R. solanacearum in susceptible tomato plants in different conditions and making use of various cryoprotectants. The assays carried out have shown satisfactory results with respect to the viability and stability of the bacteriophages after the lyophilization process, maintaining high titres throughout the experimental period, also with respect to the capacity of the bacteriophages for the biological control of bacterial wilt, controlling this disease in more than 50% of the plants. The results offer good prospects for the use of lyophilization as a conservation method for the lytic bacteriophages of R. solanacearum in view of their commercialization as biocontrol agents.

scholarly journals The RSc0454-Encoded FAD-Linked Oxidase Is Indispensable for Pathogenicity in Ralstonia solanacearum GMI1000

2019 ◽  
Vol 32 (6) ◽  
pp. 697-707
Author(s):  
Xun Hu ◽  
Zhiwen Zhao ◽  
Tao Zhuo ◽  
Xiaojing Fan ◽  
Huasong Zou
Keyword(s):  
Lactate Dehydrogenase ◽  
Succinate Dehydrogenase ◽  
Gene Transcription ◽  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Cell Aggregation ◽  
Wilt Disease ◽  
In Planta ◽  
Bacterial Wilt Disease ◽  
Dehydrogenase Gene

Ralstonia solanacearum is the causal agent of bacterial wilt disease. Here, we report that a large FAD-linked oxidase encoded by RSc0454 in GMI1000 is required for pathogenicity. The FAD-linked oxidase encoded by RSc0454 is composed of 1,345 amino acids, including DUF3683, lactate dehydrogenase (LDH), and succinate dehydrogenase (SDH) domains. The RSc0454 protein showed both LDH and SDH activities. To investigate its role in pathogenicity, a deletion mutant of the RSc0454 gene was constructed in GMI1000, which was impaired in its ability to cause bacterial wilt disease in tomato. A single DUF3683, LDH, or SDH domain was insufficient to restore bacterial pathogenicity. Mutagenesis of the RSc0454 gene did not affect growth rate but caused cell aggregation at the bottom of the liquid nutrient medium, which was reversed by exogenous applications of lactate, fumarate, pyruvate, and succinate. qRT-PCR and promoter LacZ fusion experiments demonstrated that RSc0454 gene transcription was induced by lactate and fumarate (both substrates of LDH). Compared with the downregulation of the succinate dehydrogenase gene sdhBADC and the lactate dehydrogenase gene ldh, RSc0454 gene transcription was enhanced in planta. This suggests that the oxidase encoded by RSc0454 was involved in a redox balance, which is in line with the different living conditions of R. solanacearum.

scholarly journals Hydroxycinnamic Acid Degradation, a Broadly Conserved Trait, Protects Ralstonia solanacearum from Chemical Plant Defenses and Contributes to Root Colonization and Virulence

2015 ◽  
Vol 28 (3) ◽  
pp. 286-297 ◽  
Author(s):  
Tiffany M. Lowe ◽  
Florent Ailloud ◽  
Caitilyn Allen
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Root Colonization ◽  
Xylem Sap ◽  
Hydroxycinnamic Acid ◽  
Plant Colonization ◽  
In Planta ◽  
Acetyl Bromide ◽  
Defense Compounds

Plants produce hydroxycinnamic acid (HCA) defense compounds to combat pathogens, such as the bacterium Ralstonia solanacearum. We showed that an HCA degradation pathway is genetically and functionally conserved across diverse R. solanacearum strains. Further, a feruloyl-CoA synthetase (Δfcs) mutant that cannot degrade HCA was less virulent on tomato plants. To understand the role of HCA degradation in bacterial wilt disease, we tested the following hypotheses: HCA degradation helps the pathogen i) grow, as a carbon source; ii) spread, by reducing HCA-derived physical barriers; and iii) survive plant antimicrobial compounds. Although HCA degradation enabled R. solanacearum growth on HCA in vitro, HCA degradation was dispensable for growth in xylem sap and root exudate, suggesting that HCA are not significant carbon sources in planta. Acetyl-bromide quantification of lignin demonstrated that R. solanacearum infections did not affect the gross quantity or distribution of stem lignin. However, the Δfcs mutant was significantly more susceptible to inhibition by two HCA, namely, caffeate and p-coumarate. Finally, plant colonization assays suggested that HCA degradation facilitates early stages of infection and root colonization. Together, these results indicated that ability to degrade HCA contributes to bacterial wilt virulence by facilitating root entry and by protecting the pathogen from HCA toxicity.

scholarly journals The In Planta Transcriptome of Ralstonia solanacearum: Conserved Physiological and Virulence Strategies during Bacterial Wilt of Tomato

mBio ◽  
2012 ◽  
Vol 3 (4) ◽  
Author(s):  
Jonathan M. Jacobs ◽  
Lavanya Babujee ◽  
Fanhong Meng ◽  
Annett Milling ◽  
Caitilyn Allen
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
High Cell ◽  
In Planta ◽  
Bacterial Gene ◽  
Content Type ◽  
Xylem Fluid ◽  
Bacterial Gene Expression ◽  
Weed Host ◽  
Cell Densities

ABSTRACTPlant xylem fluid is considered a nutrient-poor environment, but the bacterial wilt pathogenRalstonia solanacearumis well adapted to it, growing to 108to 109 CFU/g tomato stem. To better understand howR. solanacearumsucceeds in this habitat, we analyzed the transcriptomes of two phylogenetically distinctR. solanacearumstrains that both wilt tomato, strains UW551 (phylotype II) and GMI1000 (phylotype I). We profiled bacterial gene expression at ~6 × 108 CFU/ml in culture or in plant xylem during early tomato bacterial wilt pathogenesis. Despite phylogenetic differences, these two strains expressed their 3,477 common orthologous genes in generally similar patterns, with about 12% of their transcriptomes significantly alteredin plantaversus in rich medium. Several primary metabolic pathways were highly expressed during pathogenesis. These pathways included sucrose uptake and catabolism, and components of these pathways were encoded by genes in thescrABYcluster. A UW551scrAmutant was significantly reduced in virulence on resistant and susceptible tomato as well as on potato and the epidemiologically important weed hostSolanum dulcamara. FunctionalscrAcontributed to pathogen competitive fitness during colonization of tomato xylem, which contained ~300 µM sucrose.scrAexpression was induced by sucrose, but to a much greater degree by growthin planta. Unexpectedly, 45% of the genes directly regulated by HrpB, the transcriptional activator of the type 3 secretion system (T3SS), were upregulatedin plantaat high cell densities. This result modifies a regulatory model based on bacterial behavior in culture, where this key virulence factor is repressed at high cell densities. The active transcription of these genes in wilting plants suggests that T3SS has a biological role throughout the disease cycle.IMPORTANCERalstonia solanacearumis a widespread plant pathogen that causes bacterial wilt disease. It inflicts serious crop losses on tropical farmers, with major economic and human consequences. It is also a model for the many destructive microbes that colonize the water-conducting plant xylem tissue, which is low in nutrients and oxygen. We extracted bacteria from infected tomato plants and globally identified the biological functions thatR. solanacearumexpresses during plant pathogenesis. This revealed the unexpected presence of sucrose in tomato xylem fluid and the pathogen’s dependence on host sucrose for virulence on tomato, potato, and the common weed bittersweet nightshade. Further,R. solanacearumwas highly responsive to the plant environment, expressing several metabolic and virulence functions quite differently in the plant than in pure culture. These results reinforce the utility of studying pathogens in interaction with hosts and suggest that selecting for reduced sucrose levels could generate wilt-resistant crops.

scholarly journals Leaf-to-whole plant spread bioassay for pepper and Ralstonia solanacearum interaction determines inheritance of resistance to bacterial wilt for further breeding

2021 ◽  
Author(s):  
Ji-Su Kwon ◽  
Jae-Young Nam ◽  
Seon-In Yeom ◽  
Won-Hee Kang
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Evaluation Method ◽  
Inheritance Of Resistance ◽  
Breeding Programs ◽  
Whole Plant ◽  
Resistance Trait ◽  
Breeding Efficiency ◽  
Serious Disease ◽  
Chili Peppers

AbstractBacterial wilt (BW) disease by Ralstonia solanacearum is a serious disease and causes severe yield losses in chili peppers worldwide. Resistant cultivar breeding is the most effective in controlling BW. Thus, a simple and reliable evaluation method is required to assess disease severity and to investigate the inheritance of resistance for further breeding programs. Here, we developed a reliable leaf-to-whole plant spread bioassay for evaluating BW disease and then, using this, determined the inheritance of resistance to R. solanacearum in peppers. Capsicum annuum ‘MC4’ displayed a completely resistant response with fewer disease symptoms, a low level of bacterial cell growth, and significant up-regulations of defense genes in infected leaves compared to those in susceptible ‘Subicho’. We also observed the spreading of wilt symptoms from the leaves to the whole susceptible plant, which denotes the normal BW wilt symptoms, similar to the drenching method. Through this, we optimized the evaluation method of the resistance to BW. Additionally, we performed genetic analysis for resistance inheritance. The parents, F1 and 90 F2 progenies, were evaluated, and the two major complementary genes involved in the BW resistance trait were confirmed. These could provide an accurate evaluation to improve resistant pepper breeding efficiency against BW.

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