Ralstonia Strains Research Articles

Genotypic and Phenotypic Analyses Show Ralstonia solanacearum Cool Virulence is a Quantitative Trait Not Restricted to "Race 3 biovar 2".

Most Ralstonia solanacearum species complex strains cause bacterial wilts in tropical or subtropical zones, but the group known as Race 3 biovar 2 (R3bv2) is cool virulent and causes potato brown rot at lower temperatures. R3bv2 has invaded potato-growing regions around the world but is not established in the United States. Phylogenetically, R3bv2 corresponds to a subset of the R. solanacearum phylotype IIB clade, but little is known about the distribution of the cool virulence phenotype within phylotype IIB. Therefore, genomes of 76 potentially cool virulent phylotype IIB strains and 30 public genomes were phylogenetically analyzed. A single clonal lineage within the sequevar 1 subclade of phylotype IIB that originated in South America has caused nearly all brown rot outbreaks worldwide. To correlate genotypes with relevant phenotypes, we quantified virulence of ten Ralstonia strains on tomato and potato at both 22°C and 28°C. Cool virulence on tomato did not predict cool virulence on potato. We found that cool virulence is a quantitative trait. Strains in the sequevar 1 pandemic clonal lineage caused the most disease, while other R3bv2 strains were only moderately cool virulent. However, some non-R3bv2 strains were highly cool virulent and aggressively colonized potato tubers. Thus, cool virulence is not consistently correlated with strains historically classified as R3bv2 group. To aid detection of sequevar 1 strains, this group was genomically delimited in the LINbase web server and a sequevar 1 diagnostic primer pair was developed and validated. We discuss implications of these results for the R3bv2 definition.

Ralstonia solanacearum species complex in Australia.

The Ralstonia solanacearum species complex (RSSC) causes vascular wilt of many crops and is considered one of the most destructive plant pathogenic bacteria worldwide. The species complex was recently resolved into a stable taxonomy of three species aligning with the previously determined phylotypes, namely R. solanacearum (phylotype II), R. pseudosolanacearum (phylotype I and III), and R. syzygii (phylotype IV). Knowing which Ralstonia species and subspecies are established in Australia is important to Australia's biosecurity and market access. The goal of this study was to analyse Australia's Ralstonia culture collections and to assign the isolates to the modern taxonomic groups. The results shed light on the identity, distribution, and pathogenicity of the Ralstonia strains in Australia. Ralstonia solanacearum, R. pseudosolanacearum phylotype I, and R. syzygii phylotype IV-11 are present in Australia but have limited geographic ranges. We identified two aberrant RSSC strains that have genetic similarity to R. syzygii based on sequevar analysis, but do not yield a phylotype IV multiplex PCR band, similar to the known aberrant strain ACH732. The aberrant strains may represent a novel species. Three new sequevars were determined, 72, 73 and 74. Several Ralstonia lineages remain undetected in Australia, providing evidence that they are absent. These include R. pseudosolanacearum phylotype III and the phylotype I mulberry infecting strains; R. solanacearum strains IIC and the Moko causing strains; and R. syzygii subsp. celebesensis, and R. syzygii subsp. syzygii. This study fulfilled Koch's postulates for the Australian strains, R. solanacearum wilted potato plants, and R. pseudosolanacearum wilted blueberry plants, the hosts from which they were initially isolated. The data supports the hypothesis that Australia has native and introduced strains of Ralstonia.

RSSC-Lineage Multiplex PCR Assay Detects and Differentiates Ralstonia solanacearum, R. pseudosolanacearum, R. syzygii, and the R3bv2 Subgroup

Bacterial wilt strains in the Ralstonia solanacearum species complex (RSSC) pose serious threats to economically important crops worldwide. In 2014, Safni et al. proposed the reclassification of the RSSC into three genomospecies: R. solanacearum (Rsol), R. pseudosolanacearum (Rpseu), and R. syzygii (Rsyz). The revision requires the proper identification of strains for diagnostic and epidemiological studies. In response, we developed the inexpensive and user-friendly RSSC-Lineage Multiplex PCR, which effectively detects plant-pathogenic Ralstonia strains in general and also distinguishes between Rpseu, Rsol, Rsyz, and the high-security Select Agent “race 3 biovar 2” subgroup of Rsol, also known as the phylotype IIB-1 potato brown rot pandemic lineage. Genomes were retrieved from the NCBI GenBank database and screened for unique gene regions using OrthoMCL and other comparative genomic approaches. Specific primers were designed for each genomospecies, Ralstonia in general, and “race 3 biovar 2.” AT-rich flaps were added at the 5ʹ position of each primer to optimize the reaction thermodynamics. The specificity was tested in silico using the NCBI GenBank genome database and an in-house database. The in vitro specificity and accuracy of the tool was validated with 113 representative Ralstonia strains and 24 strains from other genera. The assay is highly specific, generating neither false positives nor false negatives. Primer set detection limits ranged from 10 to 100 pg. The assay also detected and differentiated strains from naturally and artificially inoculated plant hosts. This tool is highly specific, reliable, and economical for culture characterization, diagnostics, surveys, quarantine decisions, and epidemiological studies. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Genome Resource Announcement of 3 Phylotype I and 14 Phylotype II Ralstonia solanacearum Species Complex Isolates from South America

The Ralstonia solanacearum species complex is a group of gram-negative bacteria that cause lethal wilting on a broad range of plant species. Ralstonia pathogens were responsible for the Moko disease epidemic in plantains, bananas, and Heliconia sp. in Central and South America in the 1960s. To investigate the genomic diversity of Ralstonia strains isolated from Central and South America during this epidemic, we conducted Illumina whole-genome sequencing on 17 Ralstonia isolates. Our phylogenetic analysis showed that three of the isolates were phylotype I strains and 14 isolates were phylotype IIB strains. This genome announcement provides new genomic resources to investigate pathogen evolution, epidemiology, and virulence. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2023.

Ralstonia solanacearum pandemic lineage strain UW551 overcomes inhibitory xylem chemistry to break tomato bacterial wilt resistance.

Plant-pathogenic Ralstonia strains cause bacterial wilt disease by colonizing xylem vessels of many crops, including tomato. Host resistance is the best control for bacterial wilt, but resistance mechanisms of the widely used Hawaii 7996 tomato breeding line (H7996) are unknown. Using growth in exvivo xylem sap as a proxy for host xylem, we found that Ralstonia strain GMI1000 grows in sap from both healthy plants and Ralstonia-infected susceptible plants. However, sap from Ralstonia-infected H7996 plants inhibited Ralstonia growth, suggesting that in response to Ralstonia infection, resistant plants increase inhibitors in their xylem sap. Consistent with this, reciprocal grafting and defence gene expression experiments indicated that H7996 wilt resistance acts in both above- and belowground plant parts. Concerningly, H7996 resistance is broken by Ralstonia strain UW551 of the pandemic lineage that threatens highland tropical agriculture. Unlike other Ralstonia, UW551 grew well in sap from Ralstonia-infected H7996 plants. Moreover, other Ralstonia strains could grow in sap from H7996 plants previously infected by UW551. Thus, UW551 overcomes H7996 resistance in part by detoxifying inhibitors in xylem sap. Testing a panel of xylem sap compounds identified by metabolomics revealed that no single chemical differentially inhibits Ralstonia strains that cannot infect H7996. However, sap from Ralstonia-infected H7996 contained more phenolic compounds, which are known to be involved in plant antimicrobial defence. Culturing UW551 in this sap reduced total phenolic levels, indicating that the resistance-breaking Ralstonia strain degrades these chemical defences. Together, these results suggest that H7996 tomato wilt resistance depends in part on inducible phenolic compounds in xylem sap.

Validating Methods To Eradicate Plant-Pathogenic Ralstonia Strains Reveals that Growth In Planta Increases Bacterial Stress Tolerance.

Plant-pathogenic bacteria in the Ralstonia solanacearum species complex (RSSC) cause highly destructive bacterial wilt disease of diverse crops. Wilt disease prevention and management is difficult because RSSC persists in soil, water, and plant material. Growers need practical methods to kill these pathogens in irrigation water, a common source of disease outbreaks. Additionally, the R. solanacearum race 3 biovar 2 (R3bv2) subgroup is a quarantine pest in many countries and a highly regulated select agent pathogen in the United States. Plant protection officials and researchers need validated protocols to eradicate R3bv2 for regulatory compliance. To meet these needs, we measured the survival of four R3bv2 and three phylotype I RSSC strains following treatment with hydrogen peroxide, stabilized hydrogen peroxide (Huwa-San), active chlorine, heat, UV radiation, and desiccation. No surviving RSSC cells were detected after cultured bacteria were exposed for 10 min to 400 ppm hydrogen peroxide, 50 ppm Huwa-San, 50 ppm active chlorine, or temperatures above 50°C. RSSC cells on agar plates were eradicated by 30s of UV irradiation and killed by desiccation on most biotic and all abiotic surfaces tested. RSSC bacteria did not survive the cell lysis steps of four nucleic acid extraction protocols. However, bacteria in planta were more difficult to kill. Stems of infected tomato plants contained a subpopulation of bacteria with increased tolerance of heat and UV light, but not oxidative stress. This result has significant management implications. We demonstrate the utility of these protocols for compliance with select agent research regulations and for management of a bacterial wilt outbreak in the field. IMPORTANCE Bacteria in the Ralstonia solanacearum species complex (RSSC) are globally distributed and cause destructive vascular wilt diseases of many high-value crops. These aggressive pathogens spread in diseased plant material and via contaminated soil, tools, and irrigation water. A subgroup of the RSSC, race 3 biovar 2, is a European and Canadian quarantine pathogen and a U.S. select agent subject to stringent and constantly evolving regulations intended to prevent pathogen introduction or release. We validated eradication and inactivation methods that can be used by (i) growers seeking to disinfest water and manage bacterial wilt disease outbreaks, (ii) researchers who must remain in compliance with regulations, and (iii) regulators who are expected to define containment practices. Relevant to all these stakeholders, we show that while cultured RSSC cells are sensitive to relatively low levels of oxidative chemicals, desiccation, and heat, more aggressive treatment, such as autoclaving or incineration, is required to eradicate plant-pathogenic Ralstonia growing inside plant material.

Genome-wide identification of type III effectors and other virulence factors in Ralstonia pseudosolanacearum causing bacterial wilt in ginger (Zingiber officinale).

Ralstonia pseudosolanacearum causes bacterial wilt in ginger, reducing ginger production worldwide. We sequenced the whole genome of a highly virulent phylotype I, race 4, biovar 3 Ralstonia pseudosolanacearum strain GRsMep isolated from a severely infected ginger field in India. R. pseudosolanacearum GRsMep genome is organised into two replicons: chromosome and megaplasmid with a total genome size of 5,810,605bp. This strain encodes approximately 72 effectors which include a combination of core effectors as well as highly variable, diverse repertoire of type III effectors. Comparative genome analysis with GMI1000 identified conservation in the genes involved in the general virulence mechanism. Our analysis identifiedtype III effectors, RipBJ and RipBO as present in GRsMep but absent in the reported genomes of other strains infecting Zingiberaceae family. GRsMep contains 126 unique genes when compared to the pangenome of the Ralstonia strains that infect the Zingiberaceae family. The whole-genome data of R. pseudosolanacearum strain will serve as a resource for exploring the evolutionary processes that structure and regulate the virulence determinants of the strain. Pathogenicity testing of the transposon insertional mutant library of GRsMep through virulence assay on ginger plants identified a few candidate virulence determinants specific tobacterial wilt in ginger.

Characterization of clinical Ralstonia strains and their taxonomic position

To improve understanding of the role of Ralstonia in cystic fibrosis (CF), whole genomes of 18 strains from clinical samples were sequenced using Illumina technology. Sequences were analysed by core genome Multi-Locus Sequence Typing, Average Nucleotide Identity based on BLAST (ANIb), RAST annotation, and by ResFinder. Phylogenetic analysis was performed for the 16S rRNA gene, and the OXA-22 and OXA-60 ß-lactamase families. The minimal inhibitory concentrations (MICs) were determined using broth microdilution. ANIb data for the 18 isolates and 54 strains from GenBank, supported by phylogenetic analysis, showed that 8 groups of clusters (A-H), as well as subgroups that should be considered as species or subspecies. Groups A-C contain strains previously identified as Ralstonia solanacearum and Ralstonia pseudosolanacearum. We propose that group A is a novel species. Group B and C are Ralstonia syzygii, Ralstonia solanacearum, respectively. Group D is composed of Ralstonia mannitolilytica and Group E of Ralstonia pickettii. Group F and G should be considered novel species. Group H strains belong to R. insidiosa. OXA-22 and OXA-60 family ß-lactamases were encoded by all strains. Co-trimoxazole generally showed high activity with low MICs (≤1 mg/l) as did ciprofloxacin (≤0.12 mg/l). MICs against the other antibiotics were more variable, but generally high. RAST annotation revealed limited differences between the strains, and virulence factors were not identified. The taxonomy of the genus Ralstonia is in need of revision, but sequencing additional isolates is needed. Antibiotic resistance levels are high. Annotation did not identify potential virulence factors.

Identification of a novel heavy metal resistant Ralstonia strain and its growth response to cadmium exposure

A novel Ralstonia Bcul-1 strain was isolated from soil samples that was closest to Ralstonia pickettii. Broad-spectrum resistance was identified to a group of heavy metal ions and tolerance to concentrations of Cd2+ up to 400 mg L-1. Low concentrations of heavy metal ions did not have distinctive impact on heavy metal resistance genes and appeared to induce greater expression. Under exposure to Cd2+, cell wall components were significantly enhanced, and some proteins were also simultaneously expressed allowing the bacteria to adapt to the high Cd2+ living environment. The maximum removal rate of Cd2+ by the Ralstonia Bcul-1 strain was 78.97% in the culture medium supplemented with 100 mg L-1 Cd2+. Ralstonia Bcul-1 was able to survive and grow in a low nutrient and cadmium contaminated (0.42 mg kg-1) vegetable soil, and the cadmium removal rate was up to 65.76% in 9th growth. Ralstonia Bcul-1 mixed with biochar could maintain sustainable growth of this strain in the soil up to 75 d and the adsorption efficiency of cadmium increased by 16.23–40.80% as compared to biochar application alone. Results from this work suggests that Ralstonia Bcul-1 is an ideal candidate for bioremediation of nutrient deficient heavy metal contaminated soil.

Rapid Loop-Mediated Isothermal Amplification for Detection of the Ralstonia solanacearum Species Complex Bacteria in Symptomatic Potato Tubers and Plants.

The Ralstonia solanacearum species complex (RSSC) is composed of several Ralstonia species and strains that are little related and show varied host range and distinct geographic distributions. The RSSC causes wilt disease, and can thus have severe economic consequences for many important crops and ornamental plants. One such is potato (Solanum tuberosum), where infection causes brown rot of the tubers. It is important that symptomatic tubers and plants can be rapidly and easily tested, as exclusion of infected material is a cornerstone of management of bacterial diseases. A suitable method is loop-mediated isothermal amplification, a rapid, DNA-based method that can be used for specific detection of plant pathogens in infected materials. The combination of this loop-mediated isothermal amplification assay for the RSSC with a simple sample preparation method is fit for purpose for identification of this devastating disease in symptomatic tubers and plants. This methodology is rapid and cost efficient, and can be carried out outside of conventional laboratory facilities.

Threat of Brown Rot of Potato and Existing Resistance

Brown rot of potatoes caused by Ralstonia solanacearum is common in developing countries. Historically, brown rot losses in the United States have been restricted to southern states. This trend will most likely change with new Ralstonia introductions and temperature variability. A cold tolerant Ralstonia strain was used in this study to compare resistance between 18 wild accessions from eight species, nine accessions from the International Potato Center (CIP), and seven common US cultivars. Testing was done at both 18 °C and 30 °C. Of the wild potato species tested, S. commersonii and S. microdontum were the most resistant. CIP accessions varied from moderately resistant to very resistant. Four of the US cultivars were also moderately resistant. With certain accessions temperature played a role in resistance with high temperatures usually increasing severity of disease and cool temperatures promoting the occurrence of asymptomatic plants. The resistance observed appears to be partial, multigenic resistance.

The Immune Receptor Roq1 Confers Resistance to the Bacterial Pathogens Xanthomonas, Pseudomonas syringae, and Ralstonia in Tomato.

Xanthomonas species, Pseudomonas syringae and Ralstonia species are bacterial plant pathogens that cause significant yield loss in many crop species. Generating disease-resistant crop varieties can provide a more sustainable solution to control yield loss compared to chemical methods. Plant immune receptors encoded by nucleotide−binding, leucine−rich repeat (NLR) genes typically confer resistance to pathogens that produce a cognate elicitor, often an effector protein secreted by the pathogen to promote virulence. The diverse sequence and presence/absence variation of pathogen effector proteins within and between pathogen species usually limits the utility of a single NLR gene to protecting a plant from a single pathogen species or particular strains. The NLR protein Recognition of XopQ 1 (Roq1) was recently identified from the plant Nicotiana benthamiana and mediates perception of the effector proteins XopQ and HopQ1 from Xanthomonas and P. syringae respectively. Unlike most recognized effectors, alleles of XopQ/HopQ1 are highly conserved and present in most plant pathogenic strains of Xanthomonas and P. syringae. A homolog of XopQ/HopQ1, named RipB, is present in most Ralstonia strains. We found that Roq1 confers immunity to Xanthomonas, P. syringae, and Ralstonia when expressed in tomato. Strong resistance to Xanthomonas perforans was observed in three seasons of field trials with both natural and artificial inoculation. The Roq1 gene can therefore be used to provide safe, economical, and effective control of these pathogens in tomato and other crop species and reduce or eliminate the need for traditional chemical controls.

Complete Genome Sequence of 3-Chlorobenzoate-Degrading Bacterium Cupriavidus necator NH9 and Reclassification of the Strains of the Genera Cupriavidus and Ralstonia Based on Phylogenetic and Whole-Genome Sequence Analyses.

Cupriavidus necator NH9, a 3-chlorobenzoate (3-CB)-degrading bacterium, was isolated from soil in Japan. In this study, the complete genome sequence of NH9 was obtained via PacBio long-read sequencing to better understand the genetic components contributing to the strain's ability to degrade aromatic compounds, including 3-CB. The genome of NH9 comprised two circular chromosomes (4.3 and 3.4 Mb) and two circular plasmids (427 and 77 kb) containing 7,290 coding sequences, 15 rRNA and 68 tRNA genes. Kyoto Encyclopedia of Genes and Genomes pathway analysis of the protein-coding sequences in NH9 revealed a capacity to completely degrade benzoate, 2-, 3-, or 4-hydroxybenzoate, 2,3-, 2,5-, or 3,4-dihydroxybenzoate, benzoylformate, and benzonitrile. To validate the identification of NH9, phylogenetic analyses (16S rRNA sequence-based tree and multilocus sequence analysis) and whole-genome sequence analyses (average nucleotide identity, percentage of conserved proteins, and tetra-nucleotide analyses) were performed, confirming that NH9 is a C. necator strain. Over the course of our investigation, we noticed inconsistencies in the classification of several strains that were supposed to belong to the two closely-related genera Cupriavidus and Ralstonia. As a result of whole-genome sequence analysis of 46 Cupriavidus strains and 104 Ralstonia strains, we propose that the taxonomic classification of 41 of the 150 strains should be changed. Our results provide a clear delineation of the two genera based on genome sequences, thus allowing taxonomic identification of strains belonging to these two genera.

Characterization of Ralstonia strains infecting tomato plants in South Africa

Ralstonia spp., the causal agents of bacterial wilt, cause severe yield losses of Solanaceous crops, including tomato. The disease is difficult to control due to the pathogen's ability to survive in soil and to cause latent infections. Therefore, characterizing Ralstonia strains is important in developing effective strategies for diagnoses, quarantine and selection of biocontrol agents. In this study, 50 Ralstonia strains previously isolated from wilted tomato plants in different locations in South Africa were obtained from the culture collection of the Agricultural Research Council, Pretoria, and private seed companies. A phylogenetic analysis of the endoglucanase gene sequences revealed that 49 strains were R. pseudosolanacearum and only one was R. solanacearum. R. pseudosolanacearum strains grouped into two sequevars, 18 and 31, while the single R. solanacearum strain grouped into the cold-adapted sequevar 1, also known as race 3 biovar 2. The pathogenicity results revealed that the selected strains were pathogenic on tomato seedlings. This study thus revealed that bacterial wilt of tomato is caused primarily by R. pseudosolanacearum, but R. solanacearum IIB-1 is also present in South Africa. This information is relevant in terms of the implementation of quarantine measures, notably to put into place measures that will prevent the introduction of strain IIB-1 into other provinces of South Africa.

First Report of Bacterial Wilt Disease Caused by Ralstonia solanacearum on Blueberries (Vaccinium corymbosum) in Florida

In the fall of 2016 and spring of 2017 Ralstonia solanacearum was isolated from blueberry plants (Vaccinium corymbosum) at seven farms in six counties in Florida. Symptoms were bronzing of leaves, marginal leaf necrosis, and bacterial streaming. The symptomatic plants resembled blueberry plants infected with Xylella fastidiosa. Symptomatic tissue samples were removed, surface sterilized, cut into distilled water, and streaked onto triphenyltetrazolium chloride medium (Kelman, 1954). Mucoid colonies with red pigmented, egg-shaped pattern characteristic of R. solanacearum formed within 48 hr at 28°C. In the fall of 2016, all bacterial strains were isolated from the Southern highbush blueberry cultivar ′Arcadia′, but as we widened our search via extension communications with growers, strains were isolated from other highbush cultivars. Ralstonia strains were characterized into phylotypes and sequevars by sequencing both the Internal Transcribed Spacer (ITS) region of the 16S rRNA gene (AMB013: CATCCACCGCTTGT...

Mineral Phosphate Solubilizing Bacteria Isolated from Various Plant Rhizosphere under Different Aluminum Content

The objectives of this study was to isolate and characterize the mineral phosphate solubilizing bacteriafrom rhizosphere and evaluate their potential as plant growth promoting bacteria in Al-toxic soils. The halozone formation method was used to isolate PSB using the media containing insoluble phosphates (Ca-P or Al-P)as a source of phosphate. Eight of acid and Al-tolerant PSB isolates that were able to solubilize Ca-P wereobtained from rhizosphere of clover, wheat, corn, and sunflower grown in Al-toxic soil. Identification of theisolates based on the 16S rRNA gene sequence analysis demonstrated that the isolates were strains of Burkholderia(5 strains), Pseudomonas (1 strain), Ralstonia (1 strain), and unidentified bacterium (1 strains). All PSB isolatesshowed the capability to dissolve Ca-P, and only 1 strain (Ralstonia strain) was able to dissolve Al-P in agar platemedium. The P-solubilization by these isolates was correlated with pH of medium. Inoculation of the bacterialstrains on clover on Al-toxic medium showed that all isolates increased the plant dry weight compared withuninoculated treatment. Our results showed that those PSB isolates have potential to be developed as a biofertilizerto increase the efficiency of P-inorganic fertilizer used in Al-toxic soils.

Biocontrol of potato wilt by selective rhizospheric and endophytic bacteria associated with potato plant.

Ralstonia solanacearum is the causative agent of wilt disease in plants, which constitutes a severe problem to agricultural crops, particularly for potato production in Madagascar. The present study focuses on the isolation, in vitro and in vivo assays of potential rhizospheric and endophytic bacteria associated with healthy potato plant, capable to inhibit the growth of Ralstonia solanacearum for controlling potato bacterial wilt. A total of 77 bacteria strains were isolated from six soil rhizospheric samples and six vegetal material samples of healthy potatoes in the district of Antsirabe II. Forty of them were telluric actinomycetes, 25 were endophytic actinomycetes and 12 were fluorescent Pseudomonas spp. An additional 30 phytopathogenic isolates were obtained from six rhizopsheric soil samples of diseased potatoes. Morphological, cultural, biochemical characterization and molecular identification with the Ralstonia solanacearum specific primers 759/760 revealed that 24 of the pathogenic isolates belong to the Ralstonia solanacearum species, biovar two; the causal agent of potato bacterial wilt. Isolates from healthy plants were, then, examined in vitro and in vivo for their antagonistic activity against Ralstonia solanacearum strain for their potential to improve potato plant growth. In vitro antagonism of actinomycete and Pseudomonas isolates against Ralstonia solanacearum development was performed using agar diffusion technique, while in vivo tests were conducted under greenhouse conditions. Ten antagonistic strains including two Pseudomonas, four telluric actinomycetes, and four endophytic actinomycetes inhibited the tested Ralstonia strain. Four strains, E7, E13 (endophytic actinomycete from root potatoes), S25 (telluric actinomycetes) and P7 (fluorescent Pseudomonas), showed high antagonistic activity against the pathogen with zones of inhibition from 23 to 40 mm. Of the fours strains tested in greenhouse, E7 significantly reduced (p < 0.05) the percentage of Ralstonia solanacearum that infected plants by 72.04%. The isolates E13 and S25 have also been demonstrated to improve plant growth by increase of plant height to 44.63% and 44.84%, fresh weight to 68.75% and 75.85% and dry weight to 86.17% and 115.42%, respectively compared with non-treated control. Morphological and cultural characterization of these three active isolates showed that they belong to the genus Streptomyces. The antagonism of these isolates against Ralstonia solanacearum according to in vitro and in vivo tests results, along with their high efficiency as regards the improvement of plant development, suggests that these three actinomycete strains E7, E13 and S25 could be useful for biocontrol of potato bacterial wilt.

Identification and characterization of bacteria associated with decline of ironwood (Casuarina equisetifolia) in Guam

Ironwood (Casuarina equisetifolia subsp. equisetifolia) is a nitrogen-fixing tree of considerable social, economic and environmental importance that commonly occurs in tropical/subtropical zones of Asia, the Pacific, Africa, and Central America. Ironwood decline was first noticed on Guam in 2002 and is now affecting thousands of trees and impacting the ecosystem. In 2012, a survey showed that Ralstonia solanacearum and Klebsiella spp. were associated with wetwood symptoms of declining trees. R. solanacearum strains isolated from diseased ironwood in Guam were similar to R. solanacearum strain GMI1000, having similar BOX-PCR profiles and belonging to phylotype I and biovar 3. Two Klebsiella species (K. variicola and K. oxytoca) were recovered, with K. variicola being the more prevalent species. Pathogenicity tests revealed that R. solanacearum caused wilt in tomato and ironwood seedlings, whereas neither Klebsiella spp. produced symptoms. There were no differences in virulence between Guam R. solanacearum and control strains following inoculation into tomato and ironwood from Hawaii. Additionally, no observable differences in ironwood susceptibility to Ralstonia strains from Guam or Hawaii, were observed, suggesting that the association of Guam R. solanacearum with Guam ironwood is not specific. Co-inoculation studies with both R. solanacearum and Klebsiella variicola and K. oxytoca revealed that Klebsiella sp. did not affect symptoms produced by R. solanacearum alone. In planta studies were feasible only on seedlings and young trees in Hawaii; thus, possible interactions between R. solanacearum and Klebsiealla sp. in adult trees remain to be investigated. A new in-field survey of declining ironwood is needed to better understand the role of Klebsiella and Ralstonia in ironwood tree decline in Guam.

Cupriavidus and Burkholderia species associated with agricultural plants that grow in alkaline soils

The presence of Burkholderia, Cupriavidus, and Ralstonia species in northeastern Mexico was investigated. An analysis of the root surrounding soil from different agricultural plants led to the isolation of Burkholderia and Cupriavidus species but no Ralstonia strains. Most Cupriavidus species were unknown and grouped into two clusters according to ARDRA profiles. The 16S rRNA sequence analysis showed that the Cupriavidus isolates were highly related among them and with different Cupriavidus species with validated names. However, SDS-PAGE profiles were distinct among the different ARDRA profiles and to other Cupriavidus species examined, suggesting new species in the genus. This shows that Cupriavidus is more widely associated with plants than previously appreciated. The BCC isolate was 99% similar to B. cenocepacia by recA sequence analysis. Additionally, most Cupriavidus strains from the two largest groups grew on media containing up to 0.1 mg/ml of copper, 10.0 mg/ml arsenic and 1.0 mg/ml zinc. Burkholderia strains grew on media containing up to 10.0 mg/ml zinc, 5.0 mg/ml arsenic and 0.1 mg/ml copper.

Biodegradation kinetics of 4-fluorocinnamic acid by a consortium of Arthrobacter and Ralstonia strains

Arthrobacter sp. strain G1 is able to grow on 4-fluorocinnamic acid (4-FCA) as sole carbon source. The organism converts 4-FCA into 4-fluorobenzoic acid (4-FBA) and utilizes the two-carbon side-chain for growth with some formation of 4-fluoroacetophenone as a dead-end side product. We also have isolated Ralstonia sp. strain H1, an organism that degrades 4-FBA. A consortium of strains G1 and H1 degraded 4-FCA with Monod kinetics during growth in batch and continuous cultures. Specific growth rates of strain G1 and specific degradation rates of 4-FCA were observed to follow substrate inhibition kinetics, which could be modeled using the kinetic models of Haldane–Andrew and Luong–Levenspiel. The mixed culture showed complete mineralization of 4-FCA with quantitative release of fluoride, both in batch and continuous cultures. Steady-state chemostat cultures that were exposed to shock loadings of substrate responded with rapid degradation and returned to steady-state in 10–15 h, indicating that the mixed culture provided a robust system for continuous 4-FCA degradation.