scholarly journals Towards the Identification of Type III Effectors Associated with Ralstonia solanacearum Virulence on Tomato and Eggplant

2015 ◽  
Vol 105 (12) ◽  
pp. 1529-1544 ◽  
Author(s):  
Flora Pensec ◽  
Aurore Lebeau ◽  
M. C. Daunay ◽  
Frédéric Chiroleu ◽  
Alice Guidot ◽  
...  
Keyword(s):  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Evolutionary Dynamics ◽  
Allelic Diversity ◽  
Chain Reaction ◽  
Field Collection ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Dna Microarray Data ◽  
Polymerase Chain

For the development of pathogen-informed breeding strategies, identifying the microbial genes involved in interactions with the plant is a critical step. To identify type III effector (T3E) repertoires associated with virulence of the bacterial wilt pathogen Ralstonia solanacearum on Solanaceous crops, we used an original association genetics approach combining DNA microarray data and pathogenicity data on resistant eggplant, pepper, and tomato accessions. From this first screen, 25 T3Es were further full-length polymerase chain reaction-amplified within a 35-strain field collection, to assess their distribution and allelic diversity. Six T3E repertoire groups were identified, within which 11 representative strains were chosen to challenge the bacterial wilt-resistant eggplants ‘Dingras multiple Purple’ and ‘AG91-25’, and tomato Hawaii7996. The virulence or avirulence phenotypes could not be explained by specific T3E repertoires, but rather by individual T3E genes. We identified seven highly avirulence-associated genes, among which ripP2, primarily referenced as conferring avirulence to Arabidopsis thaliana. Interestingly, no T3E was associated with avirulence to both eggplants. Highly virulence-associated genes were also identified: ripA5_2, ripU, and ripV2. This study should be regarded as a first step toward investigating both avirulence and virulence function of the highlighted genes, but also their evolutionary dynamics in natural R. solanacearum populations.

scholarly journals Ralstonia solanacearum Race 3, Biovar 2, the Causal Agent of Brown Rot of Potato, Identified in Geraniums in Pennsylvania, Delaware, and Connecticut

Plant Disease ◽  
2003 ◽  
Vol 87 (4) ◽  
pp. 450-450 ◽  
Author(s):  
S. H. Kim ◽  
T. N. Olson ◽  
N. W. Schaad ◽  
G. W. Moorman
Keyword(s):  
United States ◽  
Polymerase Chain Reaction ◽  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
The United States ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chain Reaction ◽  
Carbohydrate Utilization ◽  
Tetrazolium Chloride ◽  
Polymerase Chain

The Plant Disease Diagnostic Laboratory of the Pennsylvania Department of Agriculture received diseased geranium (Pelargonium × hortorum) samples from several Pennsylvania (PA) greenhouses in 1999 and 2000 and from one Delaware (DE) greenhouse in 1999. Originating from Guatemala, plants exhibited yellowing, wilting, stunting, and bacterial oozing from the vascular tissues. Isolations on yeast dextrose-CaCO3 (YDC) and triphenyl-tetrazolium-chloride (TTC) agars resulted in off-white mucoid colonies and white, fluidal colonies with pink centers, respectively. Such colonies are typical of Ralstonia solanacearum (1). The disease was similar to a bacterial wilt of geranium caused by an unidentified biovar of R. solanacearum (3). Preliminary tests using Biolog MicroLog 3 (Hayward, Ca; 4.01A) and enzyme-linked immunosorbent assay (ELISA) (Agdia Inc., Elkhart, IN; BRA 33900/0500) identified the organism as R. solanacearum. For pathogenicity tests, a 10-μl droplet of water suspension containing 1 × 106 CFU of each of five geranium strains (PDA 22056-99, 81849-99, 81862-99, 51032-00, and 64054-00) per milliliter was placed on a stem wound made by cutting off the terminal growth of each of 4 6-leaf stage plants of geranium ‘Orbit Scarlet’, tomato ‘Rutgers’, potato ‘Russet Norkotah’, and eggplant ‘Black Beauty’ in a growth chamber at 28°C, 86% relative humidity, and 12 h light/dark cycle. Water was included as a control. The five strains caused severe yellowing and wilting within 10 days. Colonies typical of R. solanacearum were reisolated from symptomatic tissue on YDC and TTC. To determine the specific biovar, 20 pathogenic geranium strains from PA and DE plus a strain of R. solanacearum originally isolated from a geranium plant of Guatemalan origin received from Connecticut in 1995 were grown up to 28 days on Ayers mineral medium supplemented with a 1% final concentration of D-cellobiose, dextrose, meso-inositol, lactose, maltose, D-ribose, trehalose, mannitol, sorbitol, or dulcitol (1). Acid was produced by 21 test strains from the first five carbohydrates only. Such carbohydrate utilization is typical of bv 2 (1). Bv 2 identification was confirmed by real-time polymerase chain reaction using bv 2-specific primers and probes (N. Schaad, unpublished) designed from a bv 2-specific DNA fragment (2). All tested strains were positive using ELISA. In contrast, strains of bv 2 from geraniums in Wisconsin and South Dakota were reported to be negative using ELISA (4). From our results, it appears that bv 2 was introduced into the United States on geraniums from Guatemala in 1995 and 1999. This cool climate bv 2, a regulated agent by the Agricultural Bioterrorism Protection Act of 2002, has caused extensive crop loss in potatoes in Europe, but has not been found in potatoes in the United States. References: (1) T. P. Denny and A. C. Hayward. Ralstonia solanacearum. Pages 151–174 in: Lab Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad et al. eds. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001. (2) M. Fagen et al. Development of a diagnostic test based on the polymerase chain reaction (PCR) to identify strains of R. solanacearum exhibiting the Biovar 2 genotype. Pages 34–43 in: Bacterial Wilt Disease: Molecular and Ecological Aspects. P. H. Prior et al. eds. Springer-Verlag, Berlin, 1998. (3) D. L. Strider et al. Plant Dis. 65:52, 1981. (4) L. Williamson et al. (Abstr.) Phytopathology 91 (Suppl.):S95, 2001.

scholarly journals Genetic Diversity and Pathogenicity of Ralstonia solanacearum Causing Tobacco Bacterial Wilt in China

Plant Disease ◽  
2016 ◽  
Vol 100 (7) ◽  
pp. 1288-1296 ◽  
Author(s):  
Yanyan Li ◽  
Ji Feng ◽  
Hailong Liu ◽  
Lin Wang ◽  
Tom Hsiang ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Gene Diversity ◽  
Integrated Management ◽  
Chain Reaction ◽  
Soilborne Disease ◽  
Progress Curve ◽  
Polymerase Chain ◽  
Different Levels

Bacterial wilt caused by Ralstonia solanacearum is the most serious soilborne disease of tobacco (Nicotiana tabacum) in China. In this study, 89 strains were collected in 2012 to 2014 from across the four major tobacco-growing areas in China. The strains were identified as phylotype I by multiplex polymerase chain reaction and further divided into seven sequevars based on polymorphisms in the endoglucanase (egl) gene. Among the seven sequevars, four (15, 17, 34, and 44) have been previously described as pathogens of tobacco and two (13 and 14), which are reported here on tobacco, were previously found only on other plants. In addition, a new sequevar named 54 was identified. Strains from tobacco from different regions showed different levels of genetic diversity based on partial egl gene sequences. The farther north the distribution, the lower the gene diversity found. Pathogenicity of 27 representative strains was assessed by inoculation onto three tobacco cultivars of varying susceptibility. Through cluster analysis of area under the disease progress curve values, the 27 strains were classified into different pathotypes based on virulence; however, no obvious associations were found between sequevar and pathotype. These results will assist in determining geographical distribution of strains, and provide the foundation for breeding and integrated management programs in China.

scholarly journals Genome context influences evolutionary flexibility of nearly identical type III effectors in two phytopathogenic Pseudomonads

2021 ◽  
Author(s):  
David A Baltrus ◽  
Qian Feng ◽  
Brian H Kvitko
Keyword(s):  
Pseudomonas Syringae ◽  
Evolutionary Dynamics ◽  
Effector Proteins ◽  
In Planta ◽  
Genomic Context ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Genome Context ◽  
The Impact ◽  
Multiple Type

Integrative Conjugative Elements (ICEs) are replicons that can insert and excise from chromosomal locations in a site specific manner, can conjugate across strains, and which often carry a variety of genes useful for bacterial growth and survival under specific conditions. Although ICEs have been identified and vetted within certain clades of the agricultural pathogen Pseudomonas syringae, the impact of ICE carriage and transfer across the entire P. syringae species complex remains underexplored. Here we identify and vet an ICE (PmaICE-DQ) from P. syringae pv. maculicola ES4326, a strain commonly used for laboratory virulence experiments, demonstrate that this element can excise and conjugate across strains, and contains loci encoding multiple type III effector proteins. Moreover, genome context suggests that another ICE (PmaICE-AOAB) is highly similar in comparison with and found immediately adjacent to PmaICE-DQ within the chromosome of strain ES4326, and also contains multiple type III effectors. Lastly, we present passage data from in planta experiments that suggests that genomic plasticity associated with ICEs may enable strains to more rapidly lose type III effectors that trigger R-gene mediated resistance in comparison to strains where nearly isogenic effectors are not present in ICEs. Taken together, our study sheds light on a set of ICE elements from P. syringae pv. maculicola ES4326 and highlights how genomic context may lead to different evolutionary dynamics for shared virulence genes between strains.

scholarly journals Broad Diversity of Ralstonia solanacearum Strains in Cameroon

Plant Disease ◽  
2009 ◽  
Vol 93 (11) ◽  
pp. 1123-1130 ◽  
Author(s):  
Gabriel Mahbou Somo Toukam ◽  
Gilles Cellier ◽  
Emmanuel Wicker ◽  
Caroline Guilbaud ◽  
Rémi Kahane ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Ralstonia Solanacearum ◽  
Central Africa ◽  
Sequence Analyses ◽  
Chain Reaction ◽  
Specific Pcr ◽  
Economic Threat ◽  
The Indian Ocean ◽  
Polymerase Chain ◽  
Reference Strains

In 2005, an extensive survey of bacterial wilt in Cameroon collected 110 strains of Ralstonia solanacearum from wilting tomato, potato, pepper, huckleberry (Solanum scabrum), sesame, and amaranth. The genetic diversity and phylogeny of selected strains from Cameroon were assessed by multiplex–polymerase chain reaction (PCR), race 3/biovar 2–specific PCR, and sequence analyses of the mutS and egl genes. These data were compared with those from 33 reference strains covering the known diversity within the R. solanacearum species complex. Strains isolated in Cameroon clustered into three of the four known phylotypes: I (Asian), II (American), and III (African). Lowland tomato strains belonged to phylotype I and were quite homogeneous. The strains belonging to phylotype II were genetically diverse, and partitioned into subclusters IIA and IIB (sequevar 1, race 3/biovar 2). Cameroon strains in the African phylotype III were distinct from reference strains from Zimbabwe or the Indian Ocean, highlighting the genetic diversity present within this phylotype. Strains from potatoes growing in the highlands of West Cameroon fell into both phylotypes II (race 3/biovar 2) and III. These phylotype II and III highland strains attacked both potato and tomato and could therefore pose an economic threat to potato and tomato crops throughout Central Africa. This is the first comprehensive report on the genetic diversity of R. solanacearum strains in Cameroon.

scholarly journals Genetic Diversity and Aggressiveness of Ralstonia solanacearum Strains Causing Bacterial Wilt of Potato in Uruguay

Plant Disease ◽  
2011 ◽  
Vol 95 (10) ◽  
pp. 1292-1301 ◽  
Author(s):  
M. I. Siri ◽  
A. Sanabria ◽  
M. J. Pianzzola
Keyword(s):  
Genetic Diversity ◽  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Potato Tubers ◽  
Bacterial Strains ◽  
Potato Plants ◽  
Polymerase Chain ◽  
First Time ◽  
Different Levels

Bacterial wilt, caused by Ralstonia solanacearum, is a major disease affecting potato (Solanum tuberosum) production worldwide. Although local reports suggest that the disease is widespread in Uruguay, characterization of prevalent R. solanacearum strains in that country has not been done. In all, 28 strains of R. solanacearum isolated from major potato-growing areas in Uruguay were evaluated, including 26 strains isolated from potato tubers and 2 from soil samples. All strains belonged to phylotype IIB, sequevar 1 (race 3, biovar 2). Genetic diversity of strains was assessed by repetitive-sequence polymerase chain reaction, which showed that the Uruguayan strains constituted a homogeneous group. In contrast, inoculation of the strains on tomato and potato plants showed, for the first time, different levels of aggressiveness among R. solanacearum strains belonging to phylotype IIB, sequevar 1. Aggressiveness assays were also performed on accessions of S. commersonii, a wild species native to Uruguay that is a source of resistance for potato breeding. No significant interactions were found between bacterial strains and potato and S. commersonii genotypes, and differences in aggressiveness among R. solanacearum strains were consistent with previously identified groups based on tomato and potato inoculations. Moreover, variation in responses to R. solanacearum was observed among the S. commersonii accessions tested.

scholarly journals The awr Gene Family Encodes a Novel Class of Ralstonia solanacearum Type III Effectors Displaying Virulence and Avirulence Activities

2012 ◽  
Vol 25 (7) ◽  
pp. 941-953 ◽  
Author(s):  
Montserrat Solé ◽  
Crina Popa ◽  
Oriane Mith ◽  
Kee Hoon Sohn ◽  
Jonathan D. G. Jones ◽  
...  
Keyword(s):  
Gene Family ◽  
Pseudomonas Syringae ◽  
Ralstonia Solanacearum ◽  
Tomato Dc3000 ◽  
Type Iii Effectors ◽  
Type Iii ◽  
New Gene ◽  
Transient Overexpression ◽  
Effector Recognition

We present here the characterization of a new gene family, awr, found in all sequenced Ralstonia solanacearum strains and in other bacterial pathogens. We demonstrate that the five paralogues in strain GMI1000 encode type III-secreted effectors and that deletion of all awr genes severely impairs its capacity to multiply in natural host plants. Complementation studies show that the AWR (alanine-tryptophan-arginine tryad) effectors display some functional redundancy, although AWR2 is the major contributor to virulence. In contrast, the strain devoid of all awr genes (Δawr1-5) exhibits enhanced pathogenicity on Arabidopsis plants. A gain-of-function approach expressing AWR in Pseudomonas syringae pv. tomato DC3000 proves that this is likely due to effector recognition, because AWR5 and AWR4 restrict growth of this bacterium in Arabidopsis. Transient overexpression of AWR in nonhost tobacco species caused macroscopic cell death to varying extents, which, in the case of AWR5, shows characteristics of a typical hypersensitive response. Our work demonstrates that AWR, which show no similarity to any protein with known function, can specify either virulence or avirulence in the interaction of R. solanacearum with its plant hosts.

scholarly journals Ralstonia solanacearum Virulence Increased Following Large Interstrain Gene Transfers by Natural Transformation

2011 ◽  
Vol 24 (4) ◽  
pp. 497-505 ◽  
Author(s):  
Bénédicte Coupat-Goutaland ◽  
Dominique Bernillon ◽  
Alice Guidot ◽  
Philippe Prior ◽  
Xavier Nesme ◽  
...  
Keyword(s):  
Ralstonia Solanacearum ◽  
Natural Transformation ◽  
Comparative Genomic ◽  
Type Iii Effectors ◽  
New Genes ◽  
Tomato Isolate ◽  
Polymerase Chain ◽  
Genomic Regions ◽  
The Impact ◽  
Reaction Sequencing

Horizontal gene transfer (HGT) is a major driving force of evolution and is also likely to play an important role in the threatening emergence of novel pathogens, especially if it involves distantly related strains with substantially different pathogenicity. In this study, the impact of natural transformation on pathogenicity in six strains belonging to the four phylotypes of the plant-pathogenic bacterium Ralstonia solanacearum was investigated. The study focused on genomic regions that vary between donor and recipient strains and that carry genes involved in pathogenicity such as type III effectors. First, strains from R. solanacearum species complex were naturally transformed with heterologous genomic DNA. Transferred DNA regions were then determined by comparative genomic hybridization and polymerase chain reaction sequencing. We identified three transformant strains that acquired large DNA regions of up to 80 kb. In one case, strain Psi07 (phylotype IV tomato isolate) acquired 39.4 kb from GMI1000 (phylotype I tomato isolate). Investigations revealed that i) 24.4 kb of the acquired region contained 20 new genes, ii) an allelic exchange of 12 genes occurred, and iii) 27 genes (33.4 kb) formerly present in Psi07 were lost. Virulence tests with the three transformants revealed a significant increase in the aggressiveness of BCG20 over its Psi07 parent on tomato. These findings demonstrate the potential importance of HGT in the pathogenic evolution of R. solanacearum strains and open new avenues for studying pathogen emergence.

scholarly journals GENETIC DIVERSITY OF BACTERIAL WILT CAUSED BY RALSTONIA SOLANACEARUM AS ASSESSED BY PCR

2017 ◽  
Vol 29 (1) ◽  
pp. 69
Author(s):  
Muhammad Mansoor ◽  
Muhammad W. Abbas ◽  
J. D V. Elsas ◽  
Muhammad R. Bashir ◽  
Muhammad Atiq
Keyword(s):  
Genetic Diversity ◽  
Molecular Biology ◽  
Bacterial Wilt ◽  
Ralstonia Solanacearum ◽  
Wilt Disease ◽  
Multi Locus Sequence Typing ◽  
Chain Reaction ◽  
Bacterial Wilt Disease ◽  
Different Strains ◽  
Modern Tool

The current research was conducted to investigate genetic diversity of Ralstonia solanacearum for comparison of different strains that were collected mainly from Netherlands as well as from Bangladesh, Brazil, Kenya, Egypt, Pakistan and Palma. Forty six strains were included in contemporary studies whereas main biovars for these strains included biovar-2 except GMI1000 that belonged to biovar 3. Genetic diversity of bacterial wilt disease caused by R. solancearum was assessed by focusing mainly on three genes i.e. mutL, cbhA and dps. All the genes seem to be conserved but in case of mutL some strains showed divergence. Multi Locus Sequence Typing (MLST) scheme was used in this contemporary research. It was concluded that polymerized chain reaction (PCR) is the most imperative and appropriate modern tool of molecular biology to find genetic diversity in Ralstonia solanacearum causing bacterial wilt.     

Isolation of Ralstonia solanacearum hrpB constitutive mutants and secretion analysis of hrpB-regulated gene products that share homology with known type III effectors and enzymes

Microbiology ◽  
2005 ◽  
Vol 151 (9) ◽  
pp. 2873-2884 ◽  
Author(s):  
Naoyuki Tamura ◽  
Yukio Murata ◽  
Takafumi Mukaihara
Keyword(s):  
Pseudomonas Syringae ◽  
Ralstonia Solanacearum ◽  
Type Iii Secretion ◽  
Effector Proteins ◽  
Gene Products ◽  
Nudix Hydrolase ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Extracellular Secretion ◽  
Culture Supernatants

The Hrp type III secretion system (TTSS) is essential for the pathogenicity of the Gram-negative plant pathogen Ralstonia solanacearum. To examine the secretion of type III effector proteins via the Hrp TTSS, a screen was done of mutants constitutively expressing the hrpB gene, which encodes an AraC-type transcriptional activator for the hrp regulon. A mutant was isolated that in an hrp-inducing medium expresses several hrpB-regulated genes 4·9–83-fold higher than the wild-type. R. solanacearum Hrp-secreted outer proteins PopA and PopC were secreted at high levels into the culture supernatants of the hrpB constitutive (hrpB c) mutant. Using hrpB c mutants, the extracellular secretion of several hrpB-regulated (hpx) gene products that share homology with known type III effectors and enzymes was examined. Hpx23, Hpx24 and Hpx25, which are similar in sequence to Pseudomonas syringae pv. tomato effector proteins HopPtoA1, HolPtoR and HopPtoD1, are also secreted via the Hrp TTSS in R. solanacearum. The secretion of two hpx gene products that share homology with known enzymes, glyoxalase I (Hpx19) and Nudix hydrolase (Hpx26), was also examined. Hpx19 is accumulated inside the cell, but interestingly, Hpx26 is secreted outside the cell as an Hrp-secreted outer protein, suggesting that Hpx19 functions intracellularly but Hpx26 is a novel effector protein of R. solanacearum.

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