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An easy, rapid method has been needed to test the pathogenicity of strains of Ralstonia pseudosolanacearum, the cause of serious bacterial wilt on ginger (Zingiber officinale). When ginger leaves were clip-inoculated with race 4 strains, yellowing and V-shaped lesions developed on leaves, but no lesions formed after inoculation with race 1 strain or on mock-inoculated leaves. Tests of different bacterial concentrations as inoculum showed that the optimal concentration was greater than or equal to 106 cfu/ml. This simple, high-throughput leaf clipping inoculation method provides an efficient, easy method to evaluate pathogenicity of R. pseudosolanacearum race 4 on ginger.

Populations of the bacterial wilt pathogen, Burkholderia caryophylli, in infected plants were estimated using our newly developed carnation seedling BIO-PCR assay, which combines pre-incubation in a selective medium and polymerase chain reaction. Bacterial density in leaves and in stems from each node was examined after injured roots on plants were inoculated. B. caryophylli was consistently isolated from the lower leaves from 5 to 35 days after inoculation; thus, leaves can be assayed without sacrificing parent stock. The BIO-PCR assay has higher specificity for closely related species than a conventional assay on the selective medium, and consistently detects B. caryophylli.

We previously identified Mitsuaria sp. TWR114 and nonpathogenic Ralstonia sp. TCR112 as potential biocontrol agents to suppress tomato bacterial wilt caused by Ralstonia pseudosolanacearum. Because commercial biocontrol products require a practical cost-effective application method that maximizes their performance, we investigated whether the combined application of TWR114 and TCR112 enhances the biocontrol of bacterial wilt. In pot experiments, all the tested inoculum ratios (i.e., 1:1, 1:2, and 2:1) of the TWR114 + TCR112 treatment significantly suppressed the incidence of bacterial wilt, even at 28 days post-challenge inoculation (dpi) (13–47% wilt incidence), while 60% of plants treated with the individual isolates developed bacterial wilt within 10–12 dpi. The pathogen population in the rhizosphere and aboveground regions decreased considerably after the TWR114 + TCR112 treatment compared with that in the individual treatments. Moreover, the pathogen population in the aboveground parts of TWR114 + TCR112-treated plants had decreased to an undetectable level by 28 dpi. After inoculation with the pathogen, the expression of several tomato defense-related genes was higher in the TWR114 + TCR112-treated plants than in those treated with the individual isolates. Altogether, the results indicate that TWR114 and TCR112 applied together have a synergistic suppressive effect and that stronger defense priming might contribute to the improved biocontrol. The combination of both isolates may be a very promising approach for controlling tomato bacterial wilt in the future.

Seventy strains of Ralstonia solanacearum from solanaceous crops in Myanmar were molecularly characterized by rep-PCR DNA fingerprinting. Cluster analysis based on the fingerprints (total 33 fingerprint types) revealed seven main clusters at 80% similarity level. Strains in phylotype I showed 29 fingerprint types and were divided into six clusters, most of which were closely correlated with host plant, geographic origin, sequevar and/or biovar. In contrast, phylotype II strains showed four DNA fingerprint types within a single cluster. These results suggest that genetic diversity of the strains is endemic and that phylotype I and II may be invasive strains.

Ralstonia solanacearum, the causal agent of bacterial wilt of tomato, grows in infected plants and migrates from the roots into the soil. We investigated the effectiveness of bacterial wilt-resistant tomato rootstock in reducing the migration of R. solanacearum from susceptible scions into the soil. Rootstock stems were either 3–5 cm tall (low-grafted, LG) or ≥ 10 cm tall (high-grafted, HG). After inoculation of scions of the susceptible cultivar (SC) with R. solanacearum below the first flower, there was no difference in disease progression among LG, HG, and ungrafted SC plants, and plants had wilted by 2 weeks. However, the rate of detection of R. solanacearum in the soil of wilted plants was reduced by grafting. The size of the R. solanacearum population in the soil of fully wilted plants increased in the order of HG

Bacillus amyloliquefaciens IUMC7 and its culture supernatant, which we previously found to suppress Fusarium wilt, were found here to have antimicrobial activity against Ralstonia solanacearum. In in vivo experiments, mushroom compost inoculated with IUMC7 significantly reduced disease severity in tomato plants over that in control soils. The R. solanacearum population decreased in soil inoculated with IUMC7. A TLC–bioautography assay showed that one of the antimicrobial substances produced by IUMC7 is likely an iturin-like lipopeptide. These results suggest that these antimicrobial compounds are responsible for disease suppression and that mushroom compost containing IUMC7 has potential as a biocontrol product.

Pythium oligandrum (PO) is a common field soil oomycete with worldwide distribution, and there is no direct evidence that PO is pathogenic to agriculturally important plants. PO has received considerable attention as a potential biocontrol agent particularly in Europe and the United States because of its ability to parasitize a wide range of fungal and oomycete plant pathogens (Takenaka 2011). To develop biological control measures with native PO isolates, we isolated PO from field soils in Hokkaido, Japan and elucidated their biocontrol mechanisms by analyzing interactions between plant pathogens and PO on host plants such as tomato, sugar beet, and potato. In addition, we developed a formulation based on PO oospores for practical use in cooperation with private companies and prefectural agricultural research centers and examined the efficacy of the formulation in controlling some agriculturally important diseases. In this paper, I will introduce the main findings of the studies. Colonization by PO in the tomato rhizosphere and its interaction with bacterial wilt pathogen, Ralstonia solanacearum

A new bacterial disease was observed on three Curcuma species, C. longa (turmeric), C. aromatica (wild turmeric) and C. zedoaria (zedoary) in Okinawa Prefecture, Japan in January 2014. At harvest, leaves suddenly wilted, curled and yellowed, and the whole plant finally died. The wilted plants had rotted rhizomes with internal discoloration. Creamy-whitish bacteria were isolated from the rhizomes of each Curcuma species. These bacteria were identified as Ralstonia solanacearum race 4, biovar 4 and phylotype I based on bacteriological characteristics, pathogenicity tests and DNA-based analyses. The names bacterial wilt of turmeric, wild turmeric and zedoary are proposed for the diseases.

Ralstonia solanacearum (Smith) (Yabuuchi et al. 1996) is the causal organism of bacterial wilt of more than 200 species representing 50 families of plants, including economically important crops such as potato, tomato, eggplant, and banana. Bacterial wilt is one of the most important diseases causing a major constraint on farmers. The disease has been widely recognized in tropical, subtropical and warm temperate regions of the world (Hayward 1991, 1994a). Isolates of R. solanacearum differ in host range, geographical distribution, pathogenicity, epidemiological characters, and physiological properties. To describe this intraspecific variability of R. solanacearum, researchers have devised many classification schemes to identify this bacterium and understand its evolution (Horita and Tsuchiya 2009). For more than 5 decades, R. solanacearum has been mainly classified according to race and biovar, based on differences in host range and biochemical properties, respectively (Denny and Hayward 2001; Hayward 1964, 1994b). Consequently, over the last 3 decades, molecular biological studies have been introduced to classify and identify R. solanacearum strains from diverse origins. In various genetic analyses, genetic variability between and within the population of R. solanacearum has been assessed. On the basis of results obtained by molecular analyses, R. solanacearum has become defined as a species complex (Gillings and Fahy 1994; Poussier et al. 2000b; Villa et al. 2005), and a new classification system has been suggested (Fegan and Prior 2005; Remenant et al. 2011). In this paper, I discuss three topics: (1) current classification schemes of R. solanacearum, (2) the genetic diversity of the pathogenic strains, in particular, those of potato and Zingiberaceae plants in relation to continuously present or newly emergent diseases in Japan, and (3) perspectives on disease control strategies.

Ralstonia solanacearum is the causal organism of bacterial wilt of more than 200 species representing 50 families of plants in tropical, subtropical, and warm temperate regions in the world. Traditionally classified into five races based on differences in host range, R. solanacearum has also been grouped into six biovars on the basis of biochemical properties. With recent developments in molecular biology, various DNA-based analyses have been introduced and used to confirm that this binary system does not completely represent the diversity within R. solanacearum strains. Therefore, a new hierarchical classification scheme has been suggested, which defines R. solanacearum as a species complex and reorganized the concept of the species as a monophyletic cluster according to a phylogenetic analysis based on genomic sequence data. Here we discuss the current bacterial wilt situation and genetic relationships based on the recent classification system of Japanese R. solanacearum strains as well as worldwide strains. We also review the genetic, biochemical, and pathological characteristics of R. solanacearum strains, in particular, those affecting potato and Zingiberaceae plants as distinctly important pathogens in relation to continuously problematic and recent emergent diseases in Japan.

In August 2010, bacterial soft rot was found on root chicory (Cichorium intybus var. sativum) in Hokkaido, Japan. Severely infected plants in fields were discolored, had wilted foliage, and black necrosis of petioles near the crown. Wilted leaves subsequently collapsed and died, forming a dry, brown or black rosette. The root and crown became partially or wholly soft-rotted. Slimy masses on infected areas of roots, turned dark brown or black. Gram-negative, rod-shaped, peritrichously flagellated, facultatively anaerobic bacteria were exclusively isolated from rotted roots, and typical symptoms were reproduced after inoculation with the strains. The bacteria were identified as Dickeya dianthicola, Pectobacterium carotovorum subsp. carotovorum, and Pectobacterium carotovorum subsp. odoriferum based on further bacteriological characterization and the sequence analysis of the malate dehydrogenase gene and 16S rRNA gene. These bacteria should be included with the previously reported Dickeya (=Erwinia) chrysanthemi in Saitama Prefecture, Japan, as causal pathogens of bacterial wilt of chicory.

Ralstonia solanacearum is responsible for bacterial wilt disease. Specific and accurate identification of this pathogen is essential for protection of susceptible crops as well as breeding resistant varieties. Historically, R. solanacearum has been classified into biovars based on the use of sugar and alcohol as carbon sources, into races based on its ability to infect different hosts, more recently into phylotypes based on the intergenic transcribed sequence of the ribosomal RNA genes 16S and 23S and into sequevars based on the endoglucanase gene (egl) sequence. Race 3 biovar 2 (R3Bv2) is widespread in South and Central America, and in Brazil it is present in all potato-producing regions as the most prevalent strain. In this study, we classified 53 Brazilian R. solanacearum biovar 2 (Bv2) strains by traditional and molecular methods. PCR with specific primers confirmed all 53 bacterial strains as belonging to the R. solanacearum species complex, and all were classified as biovar 2A or 2T based on acidification of sugars and alcohols. Multiplex phylotype PCR assigned all strains to phylotype II. Phylogenetic analysis of egl sequences showed that most Bv2 strains from Brazil analyzed in this study did not cluster with known sequevars and are less clonal than the R3Bv2 strains reported for other countries. This is the first study to address the diversity of a collection of Brazilian R. solanacearum strains using the phylotype and sequevar classification scheme.

An organic hydroponic system that we developed has potential to control root diseases including bacterial wilt of hydroponically grown tomato. In inoculation tests with Ralstonia solanacearum during tomato plant cultivation in conventional inorganic hydroponics and in our organic system, many of the tomato seedlings in the conventional system wilted and died, but none of the seedlings in the organic hydroponics wilted or developed any symptoms, suggesting that the organic system can suppress this bacterial wilt disease. Interestingly, a rhizosphere biofilm, formed only on roots in the organic hydroponic system, may be responsible for the suppression of the bacterial wilt.

During the last decade, a new bacterial disease has impaired the yield of vegetable sweet potato (30–80%) in Taiwan. Infected plants developed stunting, root and stem rot, vascular discoloration and wilting. Ten bacterial isolates that caused the same symptoms in sweet potatoes after inoculation were reisolated and classified as Ralstonia solanacearum phylotype I biovar 4 based on physical and molecular analyses. Moreover, these isolates also caused wilting in convolvulaceous, solanaceaous and cruciferous plants. This report is the first of bacterial wilt of sweet potato caused by R. solanacearum in Taiwan.

New rapid diagnostic methods are urgently needed to discriminate the quarantine pathogen Ralstonia solanacearum (Rs) race 3 biovar 2 (R3B2) from other populations of Rs that lack the adaptation to cause bacterial wilt disease in temperate regions. We used an in silico bioinformatic approach to identify several genome sequences potentially specific to R3B2 strains. Primer sets were designed to PCR-amplify sequences in these regions, and four sets were ultimately shown to be >99% accurate for detection of R3B2 strains. On the basis of these results, several primers were designed to enable development of a loop-mediated isothermal amplification assay that was rapid, technologically simple, and essentially 100% accurate for identification of R3B2 when applied to a comprehensive collection of geographically diverse Rs strains. We fortuitously found that a sequence in one of the “R3B2-specific” regions has ~90% identity to a sequence present in strains of the blood disease bacterium (BDB), a member of the Rs species complex that infects banana. Alignments of these sequences allowed design of a second PCR primer set that proved 100% accurate for identification of BDB strains when tested on the 22 BDB strains available to us. These results demonstrate the power of in silico genomic subtraction for rapid identification of population-specific DNA sequences and for the development of simple, reliable detection methods for Rs subpopulations.

We assessed the geographic distribution, biovar, phylotype, DNA fingerprints (rep-PCR), and/or endoglucanase sequence of potato bacterial wilt pathogen, Ralstonia solanacearum (Rs), in Japan. Rs has been isolated from potato fields in southwestern, warm, temperate regions. Of the 188 isolates, 74 belonged to biovar N2 (39%), 44 to biovar 3 (24%), and 70 to biovar 4 (37%). Biovars N2 and 4 strains were widely distributed, from northern (Hokkaido) to southern (Okinawa) Japan. Based on the results of multiplex-PCR analysis, every potato strains belonged to either phylotype I or IV. Phylotype I comprised both biovars 3 and 4 strains. On the other hand, phylotype IV included biovar N2 strains. None of the strains belonged to phylotype II or III or biovar 1 or 2. Phylogenetic analysis based on DNA fingerprints and endoglucanase gene sequences clarified the genetic diversity of the Japanese potato strains and the close genetic relationship between the Japanese strains and the Asian strains in phylotypes I and IV.

The viable but nonculturable (VBNC) state is induced in the bacterial wilt pathogen Ralstonia solanacearum under prolonged environmental stress. These VBNC cells lose their ability to grow on standard media such as CPG agar, but some of the cells can recover this ability on media supplemented with sodium pyruvate (SP), that degrades hydrogen peroxide. Recently, we suggested that some of the cells in the low-temperature-induced SP-recoverable VBNC state regained their ability to grow on CPG agar after exposure to moderate temperature. These revived cells also retained their virulence on tomato. Although R. solanacearum is detectable on semiselective media, VBNC cells are not detectable on any known semiselective media for the pathogen. To create a suitable medium to detect VBNC cells, we therefore added various compounds that can either degrade hydrogen peroxide or serve an antioxidant function in a semiselective medium, modified SMSA. SP at 5 g/l most improved the sensitivity of R. solanacearum detection. Furthermore, counts on modified SMSA plates for R. solanacearum that had been added to field soil also increased after the addition of 5 g/l SP. SP thus improved the medium’s sensitivity for the detection of R. solanacearum by rescuing a portion of the VBNC cells.

The biocontrol agent Pythium oligandrum (PO) can suppress bacterial wilt caused by Ralstonia solanacearum (RS) in tomato. To understand the primary biocontrol mechanisms of bacterial wilt by PO, we pretreated tomato plants with sterile distilled water or preinoculated them with PO, followed by inoculation with RS, then observed PO and RS in fixed sections of tomato tissues using a confocal laser-scanning microscope and fluorescence labeling until 14 days after the inoculation with RS. Horizontal and vertical movement of RS bacteria was frequently observed in the xylem vessels of roots and stems of tomato plants (cv. Micro-Tom) that had not been inoculated with PO. In plants that were preinoculated with PO, the movement of RS was suppressed, and bacteria appeared to be restricted to the pit of vessels, a reaction similar to that observed in resistant rootstocks. PO colonization was mainly observed at the surfaces of taproots, the junctions between taproots and lateral roots, and the middle sections of the lateral roots. PO was not observed near wound sites or root tips where RS tended to colonize. However, RS colonization was significantly repressed at these sites in PO preinoculated plants. These observations suggest that the induction of plant defense reactions is the main mechanism for the control of tomato bacterial wilt by PO, not direct competition for infection sites.

Approximately 30,000 fluorescent bacterial strains isolated from tomato, lettuce, eggplant, Chinese cabbage, and Japanese pepper plants at seven different locations in Hyogo Prefecture, were screened for plant-growth-promoting (PGP) activity to induce disease resistance against bacterial wilt in tomato. The 37 strains that had higher PGP activity were subjected to molecular phylogenetic analyses using the sequences of the 16S rRNA, gyrB and rpoD genes. Most of the strains were identified as Pseudomonas fluorescens or its close relative, P. putida, while a few strains were grouped with more distantly related bacterial species such as Enterobacter and Stenotrophomonas. The phylogenetic relationships among tomato and lettuce isolates mostly coincided with the source locality and host plants, with a few exceptions. In contrast, isolates from Japanese pepper plants did not form their own cluster but represented several different bacterial species.

Ralstonia solanacearum strain OE1-1 (OE1-1) systemically invades tobacco plants and causes bacterial wilt. A type II secretion system (T2SS)-deficient mutant of OE1-1, derived from EZ::TN transposon-insertion, retained the ability of the parent strain to produce exopolysaccharide in vitro and grow in intercellular spaces immediately after invasion of host plants, but lost the ability to systemically infect the host. With transmission electron microscopy, the mutant was not observed in xylem vessels. These findings suggest that the T2SS contributes to systemic infection by enabling the bacteria to invade xylem vessels.

To study the behavior and mutation of Ralstonia solanacearum in Solanum toxicarium, which is resistant to bacterial wilt, S. toxicarium was grown in aseptic culture and inoculated with R. solanacearum. Although 60%–80% of the inoculated plants were wilting after 2 to 3 days, most wilted plants had recovered by 20 days after inoculation. The pathogen was reisolated from over 98% of inoculated plant stems, but the percentage of recovery decreased the closer the isolation sites were toward the upper stem sections. Three colony types, characterized as fluidal white, nonfluidal red, and a mixture of fluidal white and nonfluidal red, were reisolated from the stems. Nonfluidal red colonies were less virulent on tomato plants than fluidal white colonies.

Biological soil disinfestation (BSD), or reductive soil disinfestation, achieved by amendment with organic materials such as wheat bran followed by flooding and covering the soil surface, has been used to control some soilborne diseases including Fusarium wilt and bacterial wilt of tomato. During a BSD treatment, accumulation of acetic acid and/or butyric acid was detected with high-performance liquid chromatography. Survival of Fusarium oxysporum f. sp. lycopersici or Ralstonia solanacearum was suppressed by these organic acids. Amendment of these organic acids into soil suppressed the survival of R. solanacearum at lower concentrations than the maximum detected in BSD treatment, indicating that production of these organic acids is one of the mechanisms of control. However, F. oxysporum f. sp. lycopersici in soil survived with the maximum concentrations of these organic acids achieved by BSD; thus, involvement of factors other than organic acids may be involved.

Pseudomonas fluorescens strain PfG32R actively suppresses the occurrence of bacterial wilt disease of tomato caused by Ralstonia solanacearum. To determine the characteristics affected by spontaneous mutations in the gacS gene, three spontaneous mutants NR1, NR9, and ASW6 were tested for production of several enzymes, antimicrobial activity, and biocontrol activity against bacterial wilt disease of tomato in a greenhouse. Production of biosurfactant and of enzymes such as Tween-80 lipase, gelatin protease, and lecithinase; swarming activity; and antifungal activities against Fusarium oxysporum f. sp. radicis-lycopersici and F. graminearum were inactivated by mutations in the gacS gene, indicating that they are under the regulation of the GacS/GacA two-component regulatory system. Levan production, swimming, antifungal activity against Magnaporthe grisea, and antibacterial activity against R. solanacearum and Erwinia carotovora ssp. carotovora were not controlled by the GacS/GacA system. Biocontrol activities of the three spontaneous mutants varied from strain to strain, indicating that the biocontrol activities of PfG32R are influenced not only by the GacS/GacA system but also by other complex factors, which may not be regulated by the system.

Two primer sets were designed based on the sequence of polymorphic bands that were derived from repetitive sequence-based polymerase chain reaction (rep-PCR) fingerprinting and specifically detected in Ralstonia solanacearum race 4 strains (ginger, mioga, and curcuma isolates). One primer set (AKIF-AKIR) amplified a single band (165 bp) from genomic DNA obtained from all mioga and curcuma and some ginger isolates; another set (21F-21R) amplified one band (125 bp) from the other ginger isolates. These primer sets did not amplify the bands from genomic DNA of other R. solanacearum strains or of other related bacteria. PCR detection limit for the pathogen was 2 × 102 cfu.

The distribution and multiplication of Ralstonia solanacearum in tomato plants of 11 resistant cultivars derived from different genetic sources and susceptible cultivar Ponderosa were examined. Bacterial multiplication in stems of resistant tomato plants was suppressed owing to the limitation of pathogen movement from the protoxylem or the primary xylem to other xylem tissues. The limitation was most conspicuous in Hawaii 7996. Grafting experiments indicated that the percentage of wilting of Ponderosa scions was less on Hawaii 7996 rootstocks than that on the most resistant rootstock (LS-89) used in Japan. Hawaii 7996 could be an alternative genetic source for breeding for resistance to bacterial wilt.

Commercially available tomato cultivars were hydroponically cultured for inoculation, with Ralstonia solanacearum (K-101), which causes bacterial wilt, by pouring an inoculum suspension into the nutrient solution. Cultivar susceptibility to the bacteria was evaluated, based on the highest percentage of wilting. Because the length of time for wilt appearance varied among cultivars, some cultivars appeared to be suppressive to the translocation and/or multiplication of the invading pathogen. Thus, this hydroponic inoculation system is effective for examining levels of susceptibility in tomato cultivars to bacterial wilt.

FPT9601, a plant growth-promoting rhizobacterium (PGPR) isolated from tomato rhizosphere, can protect tomato (Lycopersicon esculentum Mill) from bacterial wilt disease caused by Ralstonia solanacearum. This strain produces antibiotics 2,4-diacetylphloroglucinol (2,4-DAPG) and hydrogen cyanide (HCN). It also produces proteases and uncharacterized siderophores (Sid). A mutant strain SM2214, obtained by Tn5 insertion, did not produce 2,4-DAPG, HCN or proteases, but overproduced Sid. Marker-exchange mutagenesis confirmed that a single transposon insertion caused the multiple phenotypic changes of this mutant. Complementation of the mutant with a 1.3-kb DNA fragment that was amplified from genomic DNA of the wild-type P. fluorescens strain by PCR could restore the lost functions of the mutant strain. Nucleotide sequencing revealed that the fragment contained a 642-bp open reading frame (ORF) highly homologous to the regulator responser gene gacA. The in vitro anti-bacterium test and plant protection experiment under greenhouse conditions indicated that the gacA gene played an important role in the suppression of tomato bacterial wilt disease.