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Soil-borne plant pathogens significantly threaten crop production due to lack of effective control methods. One alternative to traditional agrochemicals is microbial biocontrol, where pathogen growth is suppressed by naturally occurring bacteria that produce antimicrobial chemicals. However, it is still unclear if pathogenic bacteria can evolve tolerance to biocontrol antimicrobials and if this could constrain the long-term efficacy of biocontrol strategies. Here we used an in vitro experimental evolution approach to investigate if the phytopathogenic Ralstonia solanacearum bacterium, which causes bacterial wilt disease, can evolve tolerance to antimicrobials produced by Pseudomonas bacteria. We further asked if tolerance was specific to pairs of R. solanacearum and Pseudomonas strains and certain antimicrobial compounds produced by Pseudomonas. We found that while all R. solanacearum strains could initially be inhibited by Pseudomonas strains, this inhibition decreased following successive subculturing with or without Pseudomonas supernatants. Using separate tolerance assays, we show that the majority of R. solanacearum strains evolved increased tolerance to multiple Pseudomonas strains. Mechanistically, evolved tolerance was most likely linked to reduced susceptibility to orfamide lipopeptide antimicrobials secreted by Pseudomonas strains in our experimental conditions. Some levels of tolerance also evolved in the control treatments, which was likely correlated response due to adaptations to the culture media. Together, these results suggest that plant-pathogenic bacteria can rapidly evolve increased tolerance to bacterial antimicrobial compounds, which could reduce the long-term efficacy of microbial biocontrol.
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Antiinfecciosos , Ralstonia solanacearum , Ralstonia solanacearum/fisiología , Enfermedades de las Plantas/microbiología , Pseudomonas , PlantasRESUMEN
BACKGROUND: Ralstonia solanacearum species complex (RSSC) strains are destructive plant pathogenic bacteria and the causative agents of bacterial wilt disease, infecting over 200 plant species worldwide. In addition to chromosomal genes, their virulence is mediated by mobile genetic elements including integrated DNA of bacteriophages, i.e., prophages, which may carry fitness-associated auxiliary genes or modulate host gene expression. Although experimental studies have characterised several prophages that shape RSSC virulence, the global diversity, distribution, and wider functional gene content of RSSC prophages are unknown. In this study, prophages were identified in a diverse collection of 192 RSSC draft genome assemblies originating from six continents. RESULTS: Prophages were identified bioinformatically and their diversity investigated using genetic distance measures, gene content, GC, and total length. Prophage distributions were characterised using metadata on RSSC strain geographic origin and lineage classification (phylotypes), and their functional gene content was assessed by identifying putative prophage-encoded auxiliary genes. In total, 313 intact prophages were identified, forming ten genetically distinct clusters. These included six prophage clusters with similarity to the Inoviridae, Myoviridae, and Siphoviridae phage families, and four uncharacterised clusters, possibly representing novel, previously undescribed phages. The prophages had broad geographical distributions, being present across multiple continents. However, they were generally host phylogenetic lineage-specific, and overall, prophage diversity was proportional to the genetic diversity of their hosts. The prophages contained many auxiliary genes involved in metabolism and virulence of both phage and bacteria. CONCLUSIONS: Our results show that while RSSC prophages are highly diverse globally, they make lineage-specific contributions to the RSSC accessory genome, which could have resulted from shared coevolutionary history.
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Bacteriófagos , Ralstonia solanacearum , Bacteriófagos/genética , Humanos , Filogenia , Profagos/genética , Ralstonia solanacearum/genética , Virulencia/genéticaRESUMEN
Although plant pathogens are traditionally controlled using synthetic agrochemicals, the availability of commercial bactericides is still limited. One potential control strategy could be the use of plant growth-promoting bacteria (PGPB) to suppress pathogens via resource competition or the production of antimicrobial compounds. This study aimed to conduct in vitro and in vivo screening of eight Pseudomonas strains against Ralstonia solanacearum (the causative agent of bacterial wilt) and to investigate underlying mechanisms of potential pathogen suppression. We found that inhibitory effects were Pseudomonas strain-specific, with strain CHA0 showing the highest pathogen suppression. Genomic screening identified 2,4-diacetylphloroglucinol, pyoluteorin, and orfamides A and B secondary metabolite clusters in the genomes of the most inhibitory strains, which were investigated further. Although all these compounds suppressed R. solanacearum growth, only orfamide A was produced in the growth media based on mass spectrometry. Moreover, orfamide variants extracted from Pseudomonas cultures showed high pathogen suppression. Using the "Micro-Tom" tomato cultivar, it was found that CHA0 could reduce bacterial wilt disease incidence with one of the two tested pathogen strains. Together, these findings suggest that a better understanding of Pseudomonas-Ralstonia interactions in the rhizosphere is required to successfully translate in vitro findings into agricultural applications.
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Ralstonia solanacearum , Solanum lycopersicum , Antibacterianos/farmacología , Antibiosis , Solanum lycopersicum/microbiología , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/prevención & control , Pseudomonas/genéticaRESUMEN
BACKGROUND: Bacterial blotch is a group of economically important diseases affecting the cultivation of common button mushroom, Agaricus bisporus. Despite being studied for more than a century, the identity and nomenclature of blotch-causing Pseudomonas species is still unclear. This study aims to molecularly characterize the phylogenetic and phenotypic diversity of blotch pathogens in Western Europe. METHODS: In this study, blotched mushrooms were sampled from farms across the Netherlands, United Kingdom and Belgium. Bacteria were isolated from symptomatic cap tissue and tested in pathogenicity assays on fresh caps and in pots. Whole genome sequences of pathogenic and non-pathogenic isolates were used to establish phylogeny via multi-locus sequence alignment (MLSA), average nucleotide identity (ANI) and in-silico DNA:DNA hybridization (DDH) analyses. RESULTS: The known pathogens "Pseudomonas gingeri", P. tolaasii, "P. reactans" and P. costantinii were recovered from blotched mushroom caps. Seven novel pathogens were also identified, namely, P. yamanorum, P. edaphica, P. salomonii and strains that clustered with Pseudomonas sp. NC02 in one genomic species, and three non-pseudomonads, i.e. Serratia liquefaciens, S. proteamaculans and a Pantoea sp. Insights on the pathogenicity and symptom severity of these blotch pathogens were also generated. CONCLUSION: A detailed overview of genetic and regional diversity and the virulence of blotch pathogens in Western Europe, was obtained via the phylogenetic and phenotypic analyses. This information has implications in the study of symptomatic disease expression, development of diagnostic tools and design of localized strategies for disease management.
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Agaricus , Agaricus/genética , Bélgica , Europa (Continente) , Filogenia , Pseudomonas/genética , Reino UnidoRESUMEN
Bacterial blotch is a group of economically important diseases of the common button mushroom (Agaricus bisporus). Once the pathogens are introduced to a farm, mesophilic growing conditions (that are optimum for mushroom production) result in severe and widespread secondary infections. Efficient, timely and quantitative detection of the pathogens is hence critical for the design of localized control strategies and prediction of disease risk. This study describes the development of real-time TaqManTM assays that allow molecular diagnosis of three currently prevalent bacterial blotch pathogens: "Pseudomonas gingeri," Pseudomonas tolaasii and (as yet uncharacterized) Pseudomonas strains (belonging to Pseudomonas salomonii and Pseudomonas edaphica). For each pathogen, assays targeting specific DNA markers on two different loci, were developed for primary detection and secondary verification. All six developed assays showed high diagnostic specificity and sensitivity when tested against a panel of 63 Pseudomonas strains and 40 other plant pathogenic bacteria. The assays demonstrated good analytical performance indicated by linearity across calibration curve (>0.95), amplification efficiency (>90%) and magnitude of amplification signal (>2.1). The limits of detection were optimized for efficient quantification in bacterial cultures, symptomatic tissue, infected casing soil and water samples from mushroom farms. Each target assay was multiplexed with two additional assays. Xanthomonas campestris was detected as an extraction control, to account for loss of DNA during sample processing. And the total Pseudomonas population was detected, to quantify the proportion of pathogenic to beneficial Pseudomonas in the soil. This ratio is speculated to be an indicator for blotch outbreaks. The multiplexed assays were successfully validated and applied by routine testing of diseased mushrooms, peat sources, casing soils, and water from commercial production units.
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Bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC) is a serious threat to potato production in Uganda. However, little is known about the extent of the disease and the type of the pathogen strains involved. A nationwide survey was conducted to study BW prevalence and incidence in potato, and potato tuber and stem samples of potential alternative hosts were collected for pathogen isolation. DNA was extracted from pure cultures for genetic diversity studies. The pathogen was phylotyped by multiplex PCR; then, a subset of isolates was typed at sequevar level. Isolates of the same sequevar were then haplotyped using multilocus tandem repeat sequence typing (TRST) schemes. BW prevalence and incidence in potato farms were 81.4 and 1.7%, respectively. Three RSSC phylotypes were identified, with the majority of the strains belonging to Phylotype II (80%) followed by Phylotype I (18.5%) and III (1.5%). Phylotype I strains belonged to Sequevar 31, and Phylotype II strains belonged to Sequevar 1. Potato-associated Phylotype II Sequevar 1 strains were more diverse (27 TRST haplotypes) than nonpotato Phylotype I (5 TRST haplotypes). Mapping of TRST haplotypes revealed that three TRST haplotypes of Phylotype II Sequevar 1 strains play an important epidemiological role in BW of potato in Uganda being disseminated via latently infected seed.[Formula: see text]Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
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Epidemiología Molecular , Ralstonia solanacearum , Solanum tuberosum , Tipificación Molecular , Filogenia , Enfermedades de las Plantas/microbiología , Ralstonia solanacearum/clasificación , Ralstonia solanacearum/genética , Solanum tuberosum/microbiología , UgandaRESUMEN
Blackleg and soft rot of potato, caused by Pectobacterium and Dickeya spp., are major production constraints in many potato-growing regions of the world. Despite advances in our understanding of the causative organisms, disease epidemiology, and control, blackleg remains the principal cause of down-grading and rejection of potato seed in classification schemes across Northern Europe and many other parts of the world. Although symptom recognition is relatively straightforward and is applied universally in seed classification schemes, attributing disease to a specific organism is problematic and can only be achieved through the use of diagnostics. Similarly as disease spread is largely through the movement of asymptomatically infected seed tubers and, possibly in the case of Dickeya spp., irrigation waters, accurate and sensitive diagnostics are a prerequisite for detection. This chapter describes the diagnostic pathway that can be applied to identify the principal potato pathogens within the genera Pectobacterium and Dickeya.
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ADN Bacteriano/análisis , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Pectobacterium/genética , Pectobacterium/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Solanum tuberosum/microbiología , ADN Bacteriano/genética , Enterobacteriaceae/patogenicidad , Pectobacterium/patogenicidad , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Tubérculos de la Planta/química , Tubérculos de la Planta/microbiología , Especificidad de la EspecieRESUMEN
Pectinolytic Gram-negative bacteria were isolated from different waterways in the UK and Finland. Three strains (174/2(T), 181/2 and Dw054) had the same 16S rRNA gene sequences which shared 99% sequence similarity to species of the genus Dickeya, and a phylogeny of related genera confirmed attribution to this genus. Fatty acid profile analysis of all three strains found a high proportion of C16 : 1ω7c/C16 : 1ω7c and C16 : 0 fatty acids, and library profile searches found closest matches to Dickeya chrysanthemi. Production of a concatenated phylogeny using six loci, recA, gapA, atpD, gyrB, infB and rpoB, provided a high-resolution phylogeny which placed strains 174/2(T) and 181/2 as a distinct clade, separated from the other species of the genus Dickeya by a relatively long branch-length. DNA-DNA hybridization analysis with a limited number of reference species also supported the distinctiveness of strains 174/2(T) and 181/2 within the genus Dickeya. All three strains could be phenotypically distinguished from other species of the genus by fermentation of melibiose and raffinose but not D-arabinose or mannitol. The name Dickeya aquatica sp. nov. is proposed for the new taxon; the type strain is 174/2(T) (â= NCPPB 4580(T)â= LMG 27354(T)).
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Enterobacteriaceae/clasificación , Filogenia , Microbiología del Agua , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Ácidos Grasos/química , Finlandia , Genes Bacterianos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Reino UnidoRESUMEN
Pectinolytic bacteria have been recently isolated from diseased potato plants exhibiting blackleg and slow wilt symptoms found in a number of European countries and Israel. These Gram-reaction-negative, motile, rods were identified as belonging to the genus Dickeya, previously the Pectobacterium chrysanthemi complex (Erwinia chrysanthemi), on the basis of production of a PCR product with the pelADE primers, 16S rRNA gene sequence analysis, fatty acid methyl esterase analysis, the production of phosphatases and the ability to produce indole and acids from α-methylglucoside. Differential physiological assays used previously to differentiate between strains of E. chrysanthemi, showed that these isolates belonged to biovar 3. Eight of the isolates, seven from potato and one from hyacinth, were analysed together with 21 reference strains representing all currently recognized taxa within the genus Dickeya. The novel isolates formed a distinct genetic clade in multilocus sequence analysis (MLSA) using concatenated sequences of the intergenic spacer (IGS), as well as dnaX, recA, dnaN, fusA, gapA, purA, rplB, rpoS and gyrA. Characterization by whole-cell MALDI-TOF mass spectrometry, pulsed field gel electrophoresis after digestion of whole-genome DNA with rare-cutting restriction enzymes, average nucleotide identity analysis and DNA-DNA hybridization studies, showed that although related to Dickeya dadantii, these isolates represent a novel species within the genus Dickeya, for which the name Dickeya solani sp. nov. (type strain IPO 2222(T)â=âLMG25993(T)â=âNCPPB4479(T)) is proposed.
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Enterobacteriaceae/clasificación , Pectinas/metabolismo , Filogenia , Solanum tuberosum/microbiología , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Europa (Continente) , Ácidos Grasos/química , Genes Bacterianos , Indoles/metabolismo , Israel , Datos de Secuencia Molecular , Tipificación de Secuencias Multilocus , Hibridación de Ácido Nucleico , Enfermedades de las Plantas/microbiología , ARN Ribosómico 16S/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Dickeya (formerly Erwinia chrysanthemi) species cause diseases on a wide range of crops and ornamental plants worldwide. Here we present the draft sequences of 17 Dickeya isolates spanning four Dickeya species, including five isolates that are currently unassigned to a species.
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Dickeya dianthicola and "Dickeya solani" are currently the dominant bacterial pathogens of potatoes in Europe. Here, we present the draft genome sequences of four strains of each pathogen.
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Variable-number tandem-repeat (VNTR) analysis was used for high-resolution discrimination among Ralstonia solanacearum phylotype IIB sequevar 1 (PIIB-1) isolates and further evaluated for use in source tracing. Five tandem-repeat-containing loci (comprising six tandem repeats) discriminated 17 different VNTR profiles among 75 isolates from potato, geranium, bittersweet (Solanum dulcamara), tomato, and the environment. R. solanacearum isolates from crops at three unrelated outbreak sites where river water had been used for irrigation had distinct VNTR profiles that were shared with PIIB-1 isolates from infected bittersweet growing upriver of each site. The VNTR profiling results supported the implication that the source of R. solanacearum at each outbreak was contaminated river water. Analysis of 51 isolates from bittersweet growing in river water at different locations provided a means to evaluate the technique for studying the epidemiology of the pathogen in the environment. Ten different VNTR profiles were identified among bittersweet PIIB-1 isolates from the River Thames. Repeated findings of contiguous river stretches that produced isolates that shared single VNTR profiles supported the hypothesis that the pathogen had disseminated from infected bittersweet plants located upriver. VNTR profiles shared between bittersweet isolates from two widely separated Thames tributaries (River Ray and River Colne) suggested they were independently contaminated with the same clonal type. Some bittersweet isolates had VNTR profiles that were shared with potato isolates collected outside the United Kingdom. It was concluded that VNTR profiling could contribute to further understanding of R. solanacearum epidemiology and assist in control of future disease outbreaks.
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Repeticiones de Minisatélite , Tipificación Molecular/métodos , Ralstonia solanacearum/clasificación , Ralstonia solanacearum/genética , Genotipo , Enfermedades de las Plantas/microbiología , Ralstonia solanacearum/aislamiento & purificación , Ríos/microbiología , Reino UnidoRESUMEN
Using sequences from the recA locus, we have produced a phylogeny of 188 Dickeya strains from culture collections and identified species relatedness and subspecies clade structure within the genus. Of the six recognized species, Dickeya paradisiaca, D. chrysanthemi and D. zeae were discriminated with long branch lengths. The clade containing the D. paradisiaca type strain included just one additional strain, isolated from banana in Colombia. Strains isolated from Chrysanthemum and Parthenium species made up most of the clade containing the D. chrysanthemi type strain, and the host range of this species was extended to include potato. The D. zeae clade had the largest number of sequevars and branched into two major sister clades that contained all of the Zea mays isolates, and were identified as phylotypes PI and PII. The host range was increased from six to 13 species, including potato. The recA sequence of an Australian sugar-cane strain was sufficiently distinct to rank as a new species-level branch. In contrast to these species, Dickeya dadantii, D. dianthicola and D. dieffenbachiae were distinguished with shorter branch lengths, indicating relatively closer relatedness. The recA sequence for the type strain of D. dadantii clustered separately from other strains of the species. However, sequence comparison of three additional loci revealed that the D. dadantii type strain grouped together with the six other D. dadantii strains that were sequenced. Analysis of all four loci indicated that the D. dadantii strains were most closely related to D. dieffenbachiae. Three further branches (DUC-1, -2 and -3) were associated with these three species, which all diverged from a common origin and can be considered as a species complex. The large clade containing the D. dianthicola type strain comprised 58 strains and had little sequence diversity. One sequevar accounted for the majority of these strains, which were isolated nearly exclusively from eight hosts from Europe. Isolation of this sequevar on multiple occasions from Dianthus and (more recently) potato demonstrates that this lineage has become established in these species. The D. dadantii clade comprised 11 sequevars, and the known host range of the species was extended from eight to 19 species. New hosts included several ornamental species and potato. The clade DUC-1 was made up exclusively of potato strains originating from Europe, which had identical sequences, whilst DUC-2 strains were isolated mostly from a variety of monocotyledonous species. A single strain from Aglaonema sp. made up DUC-3. A single sequevar constituted the D. dieffenbachiae clade. The phylogenetic method described will provide a simple means for identification to the species and intraspecies level, which will support efforts to control these pathogens based on monitoring and surveillance.
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Proteínas Bacterianas/genética , Enterobacteriaceae/clasificación , Enterobacteriaceae/genética , Rec A Recombinasas/genética , Enterobacteriaceae/aislamiento & purificación , Evolución Molecular , Datos de Secuencia Molecular , Filogenia , Plantas/microbiologíaRESUMEN
In 2003 and 2004, 15 isolates of Ralstonia solanacearum were obtained from wilting plants of field-grown pepper (Capsicum annuum) in south Florida and from pot-grown hydrangea (Hydrangea paniculata and H. macrophylla) and geranium (Pelargonium × hortorum) in commercial nurseries and retention ponds in north Florida. Diagnostic immunoassays and polymerase chain reaction (PCR) analyses identified all the isolates as R. solanacearum but not race 3 biovar 2. Pathogenicity studies on tomato, pepper, and tobacco revealed that all 15 strains had similar high virulence on tomato and all caused wilting of tobacco, although there were significant differences among the strains in aggressiveness on tobacco. An indigenous Florida tomato strain, race 1 biovar 1 (Rs5), caused no disease on tobacco and little or none on pepper. The three pepper strains were more aggressive than Rs5 or two hydrangea strains on all three pepper cultivars studied. Phylogenetic analysis based on an endoglucanase gene sequence indicated that these strains had three distinct origins. The three pepper strains belonged to phylotype I biovar 3 and clustered with strains from diverse hosts in Asia belonging to sequevar 13. The six geranium strains and four of the hydrangea strains were closely related to strains in sequevar 5, a distinct subcluster of phylotype II biovar 1 strains isolated from the French West Indies and Brazil. Two other biovar 1 strains from hydrangea and strains K60, AW, and Rs5 belonged to sequevar 7 in phylotype II and probably are native to North America. None of the Florida isolates belong to the highly regulated Select Agent race 3 biovar 2 subgroup, according to both the DNA sequence analysis and the biovar phenotypic test results. However, the race 3 biovar 2-specific B2 primers weakly amplified a product from some race 1 biovar 1 strains in real-time PCR, indicating that this assay may give false positives under some conditions. Given the high cost of a misdiagnosis, it seems advisable to use at least two independent diagnostic methods to confirm that a suspect isolate is R. solanacearum R3B2. This is the first report of the presence of R. solanacearum race 1 biovar 3 or phylotype I strains in North America, and the first report confirming R. solanacearum causing natural infection of hydrangea in Florida. Thus, R. solanacearum strains that are quite distinct from presumably indigenous strains are present and can infect diverse hosts in Florida.
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An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.