Solanacearum Strains Research Articles

Relationship Between Avirulence Gene (avrA) Diversity in Ralstonia solanacearum and Bacterial Wilt Incidence

Bacterial wilt, caused by Ralstonia solanacearum, is a serious disease of tobacco in North and South Carolina. In contrast, the disease rarely occurs on tobacco in Georgia and Florida, although bacterial wilt is a common problem on tomato. We investigated whether this difference in disease incidence could be explained by qualitative characteristics of avirulence gene avrA in the R. solanacearum population in the southeastern United States. Sequence analysis established that wild-type avrA has a 792-bp open reading frame. Polymerase chain reaction (PCR) amplification of avrA from 139 R. solanacearum strains generated either 792-bp or approximately 960-bp DNA fragments. Strains that elicited a hypersensitive reaction (HR) on tobacco contained the 792-bp allele, and were pathogenic on tomato and avirulent on tobacco. All HR-negative strains generated a approximately 960-bp DNA fragment, and wilted both tomato and tobacco. The DNA sequence of avrA in six HR-negative strains revealed the presence of one of two putative miniature inverted-repeat transposable elements (MITEs): a 152-bp MITE between nucleotides 542 and 543, or a 170-bp MITE between nucleotides 461 and 462 or 574 and 575. Southern analysis suggested that the 170-bp MITE is unique to strains from the southeastern United States and the Caribbean. Mutated avrA alleles were present in strains from 96 and 75% of North and South Carolina sites, respectively, and only in 13 and 0% of the sites in Georgia and Florida, respectively. Introduction of the wildtype allele on a plasmid into four HR-negative strains reduced their virulence on both tobacco and tomato. Inactivation of avrA in an HR-positive, avirulent strain, resulted in a mutant that was weakly virulent on tobacco. Thus, the incidence of bacterial wilt of tobacco in the southeastern United States is partially explained by which avrA allele dominates the local R. solanacearum population.

DNA Analysis of Ralstonia solanacearum and Related Bacteria Based on 282-bp PCR-Amplified Fragment

Strains of Ralstonia solanacearum, Pseudomonas syzygii, and the blood disease bacterium (BDB) from different countries were tested for polymerase chain reaction amplification of the 282-bp fragment using the primer pair 759 and 760. These 282-bp fragments from 49 strains of R. solanacearum, six strains of P. syzygii, and two strains of BDB were sequenced. A phylogenetic tree was generated based on the sequence alignment. The R. solanacearum strains were divided into three groups. Group I was composed of strains belonging to biovars 3, 4, 5, and biovar N2 from Japan. Most of the strains from this group were of Asian origin except for two strains from Australia and Guyana (GMI 1000), the type strain. Group II was composed of strains belonging to biovars 1 and 2 and biovar N2 from Brazil. Group III was composed of strains belonging to biovar N2 from Japan and the Philippines. All strains of P. syzygii and BDB clustered in group III. Based on nucleotide differences of the 282-bp fragments, restriction enzyme NlaIII was capable of differentiating R. solanacearum strains into the three groups. Restriction analysis of 165 R. solanacearum isolates from the Philippines using NlaIII showed that all biovar 3 and 4 (group 1) strains had restriction fragments of 116 and 166 bp, strains belonging to biovars 1 and 2 (group 2) showed no restriction, and one strain belonging to biovar 2 (group 3) showed restriction fragments of 54 and 228 bp in size. Thus, NlaIII could be used for rapid differentiation of R. solanacearum strains. Additionally, other restriction enzymes, such as McrI, BsiEI, and MnlI could be used to differentiate R. solanacearum strains from P. syzygii strains.

Molecular Diversity of Ralstonia solanacearum Isolated from Ginger in Hawaii

ABSTRACT The genetic diversity of Ralstonia solanacearum strains isolated from ginger (Zingiber officinale) growing on the island of Hawaii was determined by analysis of amplified fragment length polymorphisms (AFLPs). Initially 28 strains of R. solanacearum collected from five host plant species worldwide were analyzed by AFLP. A second analysis was conducted on 55 R. solanacearum strains collected from three ginger farms along the Hamakua Coast of Hawaii, the principle area of ginger cultivation in the state. From the initial analysis, R. solanacearum strains from ginger in Hawaii showed a high degree of similarity at 0.853. In contrast, the average genetic similarity between R. solanacearum strains from heliconia and ginger was only 0.165, and strains from ginger showed little similarity with strains from all other hosts. The second analysis of 55 strains from ginger on different Hawaiian farms confirmed that they were distinct from race 1 strains from tomato. Strains from ginger also showed greater diversity among themselves in the second analysis, and the greatest diversity occurred among strains from a farm where ginger is frequently imported and maintained. Our results provide evidence that R. solanacearum strains from ginger in Hawaii are genetically distinct from local strains from tomato (race 1) and heliconia (race 2).

A new and sensitive Co-operational polymerase chain reaction for rapid detection of Ralstonia solanacearum in water

Three primers from 16S rRNA were successfully assayed simultaneously in one reaction for sensitive detection of Ralstonia solanacearum in watercourses. The protocol is a modification of the Co-operational polymerase chain reaction (Co-PCR), which allows the simultaneous and co-operational action of the primers. It specifically amplified R. solanacearum strains belonging to biovars 1, 2 and 4. No products were obtained from any of the 162 unidentified isolates from river water. The sensitivity of the assay was <1 cfu/ml as determined by analysis of heat-treated water samples spiked with R. solanacearum, also containing indigenous microbiota up to 10 5 cfu/ml. The developed Co-PCR assay was more sensitive than other standard PCR assays in the analysis of 51 Spanish environmental water samples. Namely 31.3% of the samples were positive using the newly developed assay, whereas 13.7% or less positive samples were found with the other protocols. The Co-PCR improves the detection sensitivity of R. solanacearum and provides an important tool for its routine detection from environmental water samples and for epidemiological studies.

Simple sequence repeats and compositional bias in the bipartite Ralstonia solanacearum GMI1000 genome.

BackgroundRalstonia solanacearum is an important plant pathogen. The genome of R. solananearum GMI1000 is organised into two replicons (a 3.7-Mb chromosome and a 2.1-Mb megaplasmid) and this bipartite genome structure is characteristic for most R. solanacearum strains. To determine whether the megaplasmid was acquired via recent horizontal gene transfer or is part of an ancestral single chromosome, we compared the abundance, distribution and compositon of simple sequence repeats (SSRs) between both replicons and also compared the respective compositional biases.ResultsOur data show that both replicons are very similar in respect to distribution and composition of SSRs and presence of compositional biases. Minor variations in SSR and compositional biases observed may be attributable to minor differences in gene expression and regulation of gene expression or can be attributed to the small sample numbers observed.ConclusionsThe observed similarities indicate that both replicons have shared a similar evolutionary history and thus suggest that the megaplasmid was not recently acquired from other organisms by lateral gene transfer but is a part of an ancestral R. solanacearum chromosome.

Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence.

As reported previously for Ralstonia solanacearum strain GMI1000, wild-type strains AW1 and K60 were shown to produce Hrp pili. AW1 and K60 mutants lacking Hrp pili still exhibited twitching motility, which requires type 4 pili (Tfp), and electron microscopy revealed that they still made flexuous polar pili. Twitching-positive cells had an extracellular 17 kDa protein that was associated with piliation, and an internal 43-amino-acid sequence of this protein was typical of type 4 pilins. This amino acid sequence is encoded by an open reading frame, designated pilA, in the genomic sequence of GMI1000. PilA is 46% identical to a Pseudomonas aeruginosa type 4 pilin over its entire length and has all the conserved residues and motifs characteristic of type 4 group A pilins. pilA mutants did not make the 17 kDa PilA protein and did not exhibit twitching motility. When compared with its parent, an AW1 pilA mutant was reduced in virulence on tomato plants and in autoaggregation and biofilm formation in broth culture. Unlike AW1, a pilA mutant did not exhibit polar attachment to tobacco suspension culture cells or to tomato roots; it was also not naturally competent for transformation. We reported previously that twitching motility ceases in maturing AW1 colonies and that inactivation of PhcA, a global transcriptional regulator, results in colonies that continue to exhibit twitching motility. Similarly, in broth culture, expression of a pilA::lacZ fusion in AW1 decreased 10-fold at high cell density, but expression remained high in a phcA mutant. In addition, pilA::lacZ expression was positively regulated 10-fold by PehR, a response regulator that is known to be repressed by PhcA. This signal cascade is sufficient to explain why pilA expression, and thus twitching motility, decreases at high cell densities.

Sequence Analysis and Detection of Ralstonia solanacearum by Multiplex PCR Amplification of 16S–23S Ribosomal Intergenic Spacer Region with Internal Positive Control

Polymerase chain reaction (PCR) methods for detection and differentiation of Ralstonia solanacearum strains were compared. The 16S–23S rRNA gene ITS sequence data revealed the two main sequence clusters (divisions I and II) of R. solanacearum and further subclusters of division II. Based on this sequence data, primers were designed which differentiated divisions I and II. Furthermore, to improve reliability of the PCR assay for routine detection of R. solanacearum in host plants, a novel multiplex PCR assay was developed in which the pathogen-specific sequences are coamplified with host plant DNA as an internal PCR control (IPC). The assay was validated during routine testing of potato samples submitted in official surveys. Of 4300 samples from 143 cultivars, 13 tested positive in both multiplex PCR and immunofluorescence (IF) assays and could be confirmed by bioassay in tomato seedlings and reisolation of the pathogen. The IPC was successfully amplified from all samples tested. A further 12 samples gave positive IF results which were not confirmed by either the multiplex PCR or tomato bioassay, indicating a greater specificity of the latter two assays.

Twitching motility of Ralstonia solanacearum requires a type IV pilus system.

Twitching motility is a form of bacterial translocation over firm surfaces that requires retractile type IV pili. Microscopic colonies of Ralstonia solanacearum strains AW1, K60 and GMI1000 growing on the surface of a rich medium solidified with 1.6% agar appeared to exhibit twitching motility, because early on they divided into motile 'rafts' of cells and later developed protruding 'spearheads' at their margins. Individual motile bacteria were observed only when they were embedded within masses of other cells. Varying degrees of motility were observed for 33 of 35 strains of R. solanacearum in a selected, diverse collection. Timing was more important than culture conditions for observing motility, because by the time wild-type colonies were easily visible by eye (about 48 h) this activity ceased and the spearheads were obscured by continued bacterial multiplication. In contrast, inactivation of PhcA, a transcriptional regulator that is essential for R. solanacearum to cause plant disease, resulted in colonies that continued to expand for at least several additional days. Multiple strains with mutations in regulatory genes important for virulence were tested, but all exhibited wild-type motility. Many of the genes required for production of functional type IV pili, and hence for twitching motility, are conserved among unrelated bacteria, and pilD, pilQ and pilT orthologues were identified in R. solanacearum. Colonies of R. solanacearum pilQ and pilT mutants did not develop spearheads or rafts, confirming that the movement of cells that had been observed was due to twitching motility. Compared to the wild-type parents, both pilQ and pilT mutants caused slower and less severe wilting on susceptible tomato plants. This is the first report of twitching motility by a phytopathogenic bacterium, and the first example where type IV pili appear to contribute significantly to plant pathogenesis.

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

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

Bacteriophage P4282, a parasite of Ralstonia solanacearum, encodes a bacteriolytic protein important for lytic infection of its host.

To enhance bacterial wilt resistance in tobacco expressing a foreign protein, we isolated the bacteriolytic gene from a bacteriophage that infects Ralstonia solanacearum. The bacteriolytic protein of phage P4282 isolated in Tochigi Prefecture was purified from a lysate of R. solanacearum M4S cells infected with the phage, and its bacteriolytic activity was assayed by following the decrease in the turbidity of suspensions of R. solancacearum M4S cells. The molecular weight of the bacteriolytic protein was approximately 71 kDa, and the sequence of the N-terminal 13 amino acids was determined. We used oligonucleotide probes based on this amino acid sequence to isolate the bacteriolytic gene from phage P4282 DNA. This gene of 2061 bp encodes a product of 687 amino acids, whose calaculated molecular weight was 70.12 kDa. The bacteriolytic gene was placed under the control of an inducible promoter. and the plasmid was transformed into Escherichia coli NM522. The soluble proteins extracted from E.coli NM522 cells harboring the plasmid with the bacteriolytic gene showed obvious bacteriolytic activities against several strains of R. solanacearum isolated in various districts in Japan. DNA fragments from five phages, isolated in Niigata, Aomori, Okinawa, Fukushima and Yamaguchi Prefectures, hybridized to the bacteriolytic gene of phage P4282. These observations indicate that the bacteriolytic protein shows nonspecific activity against R. solanacearum strains, and a sequence similar to that of the bacteriolytic gene is conserved in the DNA of other bacteriophages. These results indicate that the generation of transgenic (tobacco) plants expressing the bacteriolytic gene of phage P4282 might result in enhanced resistance to bacterial wilt in tobacco.

Evidence that Ralstonia eutropha (Alcaligenes eutrophus) contains a functional homologue of the Ralstonia solanacearum Phc cell density sensing system.

In the phytopathogen Ralstonia (Pseudomonas) solanacearum, control of many virulence genes is partly mediated by the Phc cell density sensing system. Phc uses a novel self-produced signal molecule [3-hydroxypalmitic acid methyl ester (3-OH PAME)], an atypical two-component system (PhcS/PhcR), and a LysR-type activator (PhcA) to regulate a reversible switching between two different physiological states. While Phc is present in most R. solanacearum strains, it is apparently absent from other pseudomonad plant pathogens and prokaryotic genomes that have been sequenced. Here, we report discovery of a phcA orthologue in the non-pathogenic, facultative chemolithoautotroph Ralstonia eutropha (Alcaligenes eutrophus) that fully complements R. solanacearum phcA mutants. We also demonstrate that some R. eutropha produce an extracellular factor that complements R. solanacearum mutants deficient in production of the 3-OH PAME signal molecule that controls phcA. Additionally, Southern blot hybridization analysis suggested that R. eutropha harbours other Phc components, such as PhcB (a biosynthetic enzyme for 3-OH PAME) and PhcS (a 3-OH PAME-responsive sensor kinase). Analysis of a phcA-null mutant of R. eutropha showed that phcA (and probably Phc) positively activates motility, in contrast to R. solanacearum where it represses motility. Similarly, the R. eutropha phcA mutant was unaffected in siderophore production, whereas inactivation of phcA in R. solanacearum increases siderophore production. Although our data strongly suggest that R. eutropha has a functional Phc-like system and support the phylogeny of Ralstonia, it implies that Phc may have a different physiological and ecological function in R. eutropha.

Microbiological factors affecting the colonization of tomato roots by Ralstonia solanacearum YU1Rif43lux

We investigated the effect of prior colonization of sterile soils by a range of soil microbes on the growth of Ralstonia solanacearum YUIRif43 in an earlier report (Soil Sci. Plant Nutr., 46, 449–459, 2000). Here, we report the effect of prior colonization of tomato roots, the host plant of the pathogen, by soil microbes on succeeding colonization by the pathogen. When the soil microbial community was introduced onto tomato roots, subsequent colonization by YUIRif431ux, a lux-marked mutant of YUIRif43, was remarkably suppressed compared with that in the absence of microorganisms which had previously colonized roots (hereafter referred to as priorcolonists). Soil bacteria, rather than fungi, were mainly responsible for the suppression. Next, the effect of individual microbial strains on the colonization by YUIRif43lux was examined. Some strains did not show any significant suppressive effect on subsequent colonization by YUIRif43Iux, unlike others. All the R. solanacearum strains used in this study, especially R. solanacearum NPSI (a weakly virulent mutant of YUl), were effective prior colonists against colonization by YUIRif431ux, that is, they markedly suppressed subsequent colonization by YUIRif431ux. Most of the strains that were effective prior colonists in soil in the earlier report were also effective at suppressing the colonization of tomato roots by YUIRif43Iux. YUIRif43lux competed on tomato roots with the strains that did not show previously an effect against colonization by YUIRif431ux, while root colonization by YUlRif43lux was reduced by co-inoculation with the strains that previously showed a colonization effect. These results suggested that soil bacteria were likely to be responsible for the suppression of colonization by YUIRif43lux of tomato roots, and that strains belonging to R. solanacearum among individual microbial strains might be the most effective antagonists against colonization by YUIRif431ux. Using Tn5-induced derivatives of a strain, Pseudomonas fluorescens MelRC2, one of the most effective priorcolonists, the contribution of motility, antibiosis, rhizosphere competence to the suppressiveness of P. fluorescens MelRC2 against colonization by YUIRif43lux were examined.

Genetic diversity of Ralstonia solanacearum as assessed by PCR-RFLP of the hrp gene region, AFLP and 16S rRNA sequence analysis, and identification of an African subdivision.

The genetic diversity among strains in a worldwide collection of Ralstonia solanacearum, causal agent of bacterial wilt, was assessed by using three different molecular methods. PCR-RFLP analysis of the hrp gene region was extended from previous studies to include additional strains and showed that five amplicons were produced not only with all R. solanacearum strains but also with strains of the closely related bacteria Pseudomonas syzygii and the blood disease bacterium (BDB). However, the three bacterial taxa could be discriminated by specific restriction profiles. The PCR-RFLP clustering, which agreed with the biovar classification and the geographical origin of strains, was confirmed by AFLP. Moreover, AFLP permitted very fine discrimination between different isolates and was able to differentiate strains that were not distinguishable by PCR-RFLP. AFLP and PCR-RFLP analyses confirmed the results of previous investigations which split the species into two divisions, but revealed a further subdivision. This observation was further supported by 16S rRNA sequence data, which grouped biovar 1 strains originating from the southern part of Africa.

Specific detection of biovars of Ralstonia solanacearum in plant tissues by Nested-PCR-RFLP

A sensitive and specific assay, based on a Nested-PCR-RFLP protocol, was developed for the detection of biovars of Ralstonia solanacearum, the causal agent of bacterial wilt. Oligonucleotide primer pairs were selected within the hrp gene region. Specific amplification of the hrp fragments was obtained for all R. solanacearum strains and also for two closely related species, Pseudomonas syzygii and the blood disease bacterium. No amplification was observed for a wide range of other bacterial species, including R. pickettii and Burkholderia cepacia. Digestion with HindII provided four distinct restriction profiles specific to biovars or groups of biovars of R. solanacearum: one for biovar 1 strains originating from the Southern part of Africa, one for American biovar 1 and biovars 2 and N2 strains, one for biovars 3 and 4 strains, and one for biovar 5 strains. When applied to either pure culture or infected plant tissues, Nested-PCR allowed detection as low as 103 cfu ml−1, which corresponds to 1 cfu per reaction. Amplification was partially or completely inhibited by compounds contained in plant extracts (potato plant and potato tuber, tomato, tobacco, eggplant, pepper and Pelargonium asperum). A combined PVPP/BSA treatment prior to amplification permitted reliable Nested-PCR detection of R. solanacearum strains in plant samples. Nested-PCR-RFLP, assessed with isolates from Reunion Island but also applicable to any R. solanacearum strain, provides a wide range of possible uses for identification, detection and epidemiological investigations.

A Single Locus Leads to Resistance of Arabidopsis thaliana to Bacterial Wilt Caused by Ralstonia solanacearum Through a Hypersensitive-like Response

ABSTRACT Strains of Ralstonia solanacearum have been shown to cause bacterial wilt in some, but not all, ecotypes of Arabidopsis thaliana. We demonstrate here that after inoculation of the leaves of resistant ecotype S96 with R. solanacearum strain Ps95 necrosis around the inoculation site rapidly appeared and no further symptoms developed in the plant. Leaves of susceptible ecotype N913 completely wilted 7 days after inoculation with Ps95, and symptoms spread systemically throughout the whole plant within 2 weeks after inoculation. These results suggest that the resistance of Arabidopsis S96 to R. solanacearum is due to a response similar to the hypersensitive response (HR) observed in other plant diseases. Northern blot analysis of the expression of defense-related genes, known to be differentially induced during the HR in Arabidopsis, indicated that pathogenesis-related protein PR-1, glutathione S-transferase (GST1), and Cu, Zn superoxide dismutase (SOD) mRNAs increased significantly in S96 leaves between 3 to 12 h after infiltration with Ps95. The induction of these genes in susceptible ecotype N913 by Ps95 was clearly delayed. Genetic analysis of crosses between resistant ecotype S96 and susceptible ecotype N913 indicated that resistance to Ps95 is due to a single dominant locus.

During Infection of Its Host, the Plant Pathogen Ralstonia solanacearum Naturally Develops a State of Competence and Exchanges Genetic Material

In this work we investigated the ability of the plant pathogen Ralstonia solanacearum to develop a state of competence in planta and to be genetically transformed during the infection process. Tomato plants infected with R. solanacearum GMI1000 were inoculated with plasmid DNA. R. solanacearum clones expressing the marker gene were selected only during the period of time that bacteria were actively multiplying inside the plant vessels. Moreover, experiments in which R. solanacearum strains harboring different marker genes were co-inoculated into the plant demonstrated that infecting bacterial strains exchange genetic information in planta by a mechanism that is likely to be transformation. To our knowledge, these results constitute the first demonstration of soil microorganisms developing a state of competence in planta. The biotechnological implications of this result will be discussed.

Highly specific PCR-diagnosis to determine Pseudomonas solanacearum strains of different geographical origins

Using a PCR-based assay with highly specific primers, we were able to clearly identify all of 28 different Pseudomonas solanacearum strains, whereas none of the other bacteria tested gave a cross reaction. The PCR sensitivity in standard dilution experiments of pure strains was in the range of 10 to 100 cells. The assay was also investigated for its suitability in routine diagnosis of potato tubers and tomato plants inoculated with various amounts of P. solanacearum; it reached a sensitivity of 103 cells per specimen. The region between primers PS96H and PS96I was sequenced for the first time and aligned. A total of 17 P. solanacearum strains have been sequenced, resulting in six different sequence groups. When the variable sequence was analyzed, a high correlation between point mutations and geographical origin of the P. solanacearum strains was revealed. The PCR assay described in this study combined with automatical sequencing of the amplificated region provides a powerful tool for the epidemiology of P. solanacearum.

Distribution and Multiplication of Ralstonia solanacearum (Synonym Pseudomonas solanacearum) in Tomato Plants of Resistant Rootstock Cultivar LS-89 and Susceptible Ponderosa.

We examined the distribution and multiplication of Ralstonia solanacearum (Synonym Pseudomonas solanacearum) in roots and stems of tomato plants of resistant rootstock cultivar LS-89 and susceptible cultivar Ponderosa. In a growth chamber at 30°C, 30-day-old seedlings were inoculated with R. solanacearum strain 8107S after trimming lateral roots and immersing the root ball in a bacterial suspension of 107 cfu/ml. In inoculated Ponderosa plants, bacteria were recovered from all taproot and stem samples tested. Pathogen density increased in root and stem tissues to 108 or 109 cfu/g fresh matter in 10 days. Inoculated LS-89 plants were latently infected with the pathogen. Recovery of the pathogen in these LS-89 plants at 14 days after inoculation was about 90% in taproots and the upper hypocotyls, and about 80% in the first internodes, but the percentage decreased higher in the stem (30% recovery at the fifth internode). Pathogen density increased to 106 cfu/g fresh matter in taproots, upper hypocotyls and the first internodes in the first 4 days, but it was steady afterwards. Transverse sections of the upper hypocotyls of infected plants showed bacterial masses in the primary and secondary xylem tissues in Ponderosa, but only in a portion of the primary xylem tissues in LS-89. These results indicate that the bacteria are localized in the primary xylem tissues and their movement is limited in resistant LS-89 plants.

Variable Reaction of Tomato Lines to Bacterial Wilt Evaluated at Several Locations in Southeast Asia

Bacterial wilt (BW), caused by Pseudomonas solanacearum E.F. Smith, is one of the most destructive disease of tomato (Lycopersicon esculentum Mill.) in the tropics. Twenty tomato lines/accessions previously identified as BW-resistant were evaluated for BW reaction in fields providing high disease pressure at Subang, Indonesia; Los Baños, Philippines; Malaysian Agricultural Research and Development Institute (MARDI), Kuala Lumpur; Asian Vegetable Research and Development Center (AVRDC), Taiwan; and Taiwan Seed Improvement and Propagation Service (TSIPS). Entries also were tested in the greenhouse at the AVRDC with a P. solanacearum strain from Taiwan (Pss4) using a drench inoculation method. Objectives of the study were to identify stable sources of BW resistance for southeast Asian tomato breeding programs, and to determine the correlation between field and greenhouse reactions. Mean entry survival was 21.6% at Subang, 31.9% at Los Baños, 76.7% at the AVRDC, 93.6% at Malaysia, and 93.3% at TSIPS, indicating that most entries were resistant at MARDI and the Taiwan locations but susceptible at Subang and Los Baños. L285 (mean survival = 83.8%) and CRA 84-58-1 (mean survival = 79.4%) were the most resistant entries in the field trials. Mean survival (70.1%) of CRA 66-derived entries was significantly better than the mean of entries with resistance derived from UPCA 1169 or UPCA 1169 plus `Venus' or `Saturn'. Mean survival of AVRDC entries bred in the 1980s (59.4%) was significantly greater than mean survival of AVRDC lines bred in the 1970s (45.7%). The correlation between entry BW percent survival averaged over the five field trials and entry means from drench inoculation in the greenhouse was highly significant (r = 0.70), suggesting that the drench inoculation method is effective in selection for BW resistance.

Analysis of the Phylogenetic Relationships of Strains of Burkholderia solanacearum, Pseudomonas syzygii, and the Blood Disease Bacterium of Banana Based on 16S rRNA Gene Sequences

We determined nearly complete 16S rRNA gene sequences for 19 isolates of Burkholderia solanacearum, three isolates of the blood disease bacterium of bananas, and two isolates of Pseudomonas syzygii, the cause of Sumatra disease of cloves. The dendrogram produced by comparing all of these sequences revealed that there were two divisions, which corresponded to the results obtained previously in a restriction fragment length polymorphism analysis (D. Cook, E. Barlow, and L. Sequeira, Mol. Plant Microbe Interact. 2:113-121, 1989) and a total 16S ribosomal DNA (rDNA) sequence analysis of four isolates representing four biovars of B. solanacearum (X. Li, M. Dorsch, T. Del Dot, L. I. Sly, E. Stackebrandt, and A. C. Hayward, J. Appl. Bacteriol. 74:324-329, 1993). Division 1 comprised biovars 3, 4, and 5 and an aberrant biovar 2 isolate (strain ACH0732), and division 2 included biovars 1, 2, and N2, the blood disease bacterium, and P. syzygii. Specific nucleotides at positions 458 to 460 (UUC) and 474 (A) characterized division 2, whereas in division 1 the nucleotides at these positions were ACU and U, respectively. However, strain ACH0732 had a U at position 458, as did division 2 isolates, and G instead of U at position 474. Division 2 consisted of two subdivisions; one subdivision contained two B. solanacearum isolates that originated from Indonesia, P. syzygii strains, and blood disease bacterium strains, and the other subdivision contained all of the other division 2 isolates. Within division 1, the level of 16S rDNA sequence similarity ranged from 99.8 to 100%, and within division 2, the levels of 16S rDNA sequence similarity ranged from 99.1 to 100%. The division 1 isolates exhibited an average level of 16S rDNA sequence similarity to division 2 isolates of 99.3% (range, 99.1 to 99.5%). The occurrence of consistent polymorphisms in the 16S rDNA sequences of B. solanacearum strains, in particular unique 16S rDNA sequence differences in aberrant biovar 2 isolate ACH0732, and the occurrence of the Indonesian subdivision of division 2 suggest that this group is a rapidly evolving (tachytelic) group.