Genetic diversity of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> strains from China

A survey of bacterial wilt in China collected 286 strains of Ralstonia solanacearum from 17 plant species in 13 Chinese provinces to investigate genetic diversity using the biovar (bv.) and phylotype classification schemes. A phylotype-specific multiplex-PCR showed that 198 isolates belonged to phylotype I (bv. 3, 4 and 5) and 68 to phylotype II (bv. 2 and bv. 1). A phylogenetic analysis examined the partial sequence of the egl and hrpB gene of all strains and the genetic diversity of 95 representatives was reported, demonstrating that Chinese strains are partitioned into phylotype I (Asia) and II (Americas). Phylotype I strains (historically typed bv. 3, 4 and 5), had considerable phylogenetic diversity, including 10 different sequevars: seven previously described sequevars 12 to 18 and three new sequevars: 34, 44 and 48. Chinese strains Z1, Z2, Z3, Z7, Pe74 and Tm82 were not genetically distinguishable from the edible ginger reference strain ACH92 (r4-bv. 4) for sequevar 16. This is believed to be the first report of this ginger group in China. All Chinese bv. 2 strains falling into the genetically and phenotypically diverse phylotype II were placed into phylotype IIB sequevar 1 (historically the Andean race3-bv. 2 potato brown rot agent). In both the egl and hrpB sequence-based trees, strains isolated from mulberry were present in two distinct branches found in sequevars 12 and 48 (reference strains R292 and M2, respectively).     

PCR-based specific detection of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> race 4 strains

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 (165bp) from genomic DNA obtained from all mioga and curcuma and some ginger isolates; another set (21F-21R) amplified one band (125bp) 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 × 10²cfu.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB118756 and AB118757     

Analysis of genetic and biological characters of Japanese potato strains of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis>

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.     

Genetic diversity of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> strains from Mexico associated with Moko disease

The aim of this study was to develop and validate a standard area diagram set (SADs) to assess the severity of peach rust, caused by Tranzschelia discolor. The proposed SADs includes ten images of leaves with a range of severity (0.1, 0.5, 1.0, 2.0, 5.0, 10, 15, 20, 25 and 30%). The SADs was validated by 14 raters who had no experience in plant disease severity estimation. In the first step of the validation, the raters made severity estimates of 50 leaves with a range of rust severity without using SADs. In the second step, the same raters estimated severity of rust on the same 50 leaves using the SADs to aid estimation. Lin’s concordance correlation analysis showed that both precision and accuracy improved when the raters used the SADs compared to the assessments made without SADs. Accuracy, as measured by the coefficient of bias (C _b ) improved from 0.70 to 0.98, without and with SADs, respectively, and precision measured by the correlation coefficient (r) improved from 0.85 to 0.90, without and with SADs, respectively. Overall agreement, measured by Lin’s concordance correlation coefficient (ρ _c ), improved from 0.59 to 0.88 without and with SADs, respectively. Furthermore, estimates were more reliable when using SADs: the coefficient of determination (R²) was 0.60 without and 0.73 with SADs; and the intra-class correlation coefficient (ρ) was 0.72 without, and 0.86 with SADs. Thus, the use of SADs improved the precision, accuracy and reliability of visual estimates of severity of peach rust.     

Contribution of the type II secretion system in systemic infectivity of <Emphasis Type="Italic">Ralstonia </Emphasis><Emphasis Type="Italic">solanacearum</Emphasis> through xylem vessels

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<KAN-2>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.     

Extracts from <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> induce effective resistance to the pathogen in both <Emphasis Type="Italic">Arabidopsis</Emphasis> and solanaceous plants

Plants defend themselves against microbial invasions by detecting conserved molecules, collectively called pathogen-associated molecular patterns (PAMPs). PAMPs-triggered basal resistance is the first inducible layer of plant defense. Here we found that Ralstonia solanacearum strain RS1002 can efficiently grow and cause disease in ecotype Columbia-0 of the model plant Arabidopsis thaliana in a manner dependent on the Hrp type III secretion system (T3SS). The extent of disease symptoms caused by R. solanacearum was reduced in plants pretreated with ΔhrpY mutant deficient in the functional Hrp T3SS. Pretreatment with a boiled extract (BE) from R. solanacearum had a similar inhibitory effect on disease development or bacterial multiplication in both Arabidopsis and several solanaceous plants. Simultaneous inoculations with BE and R. solanacearum did not induce BE-mediated resistance, nor did a BE treatment with proteinases. These results indicate that host plants recognize an unknown proteinaceous PAMP in the BE to induce disease resistance and that the Hrp T3SS of R. solanacearum can suppress it. From an analysis using Arabidopsis mutants lacking PAMP receptors, the elongation factor Tu of R. solanacearum was shown to partially contribute to BE-mediated basal resistance in Arabidopsis plants.     

Visualization of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> cells during biocontrol of bacterial wilt disease in tomato with <Emphasis Type="Italic">Pythium oligandrum</Emphasis>

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.     

Genetic diversity of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> strains causing bacterial wilt of solanaceous crops in Myanmar

In 2013 and 2014, an extensive survey of bacterial wilt in Myanmar was performed, and 70 strains of Ralstonia solanacearum (Rs) were collected from wilting plants of tomato, potato, chili and eggplant. Myanmar Rs strains were characterized by traditional and molecular methods. Polymerase chain reaction (PCR) test using Rs-specific primer set amplified one specific band (281-bp) from template DNA of all strains. Pathogenicity tests on the four solanaceous plants differentiated the strains into six pathogenic groups. Biovar determination tests showed that biovar 3 strains predominated (63%) among all Rs strains. Biovar 4 strains (7%) were obtained from both tomato and chili strains, whereas biovar 2 (30%) strains were isolated only from potato. Multiplex-PCR analysis indicated that tomato, eggplant and chili strains belonged to phylotype I, whereas potato strains comprised phylotype I and phylotype II. Strains in phylotype I, which was suggested to have originated from Asia, were the most prevalent in all surveyed areas. Phylogenetic analysis based on the endoglucanase (egl) gene sequences revealed that Myanmar strains partitioned into two major clusters that corresponded to phylotype I and II. Strains in phylotype I were further divided into seven subclusters, each corresponding to a distinct sequevar (15, 17, 46, 47, 48, unknown 1 or unknown 2). All strains in phylotype II belonged to sequevar 1. This is the first comprehensive report of the presence of diverse Rs strains in Myanmar.     

Application of matrix-assisted laser desorption ionization time-of-flight mass spectrometry for rapid and accurate identification of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> and <Emphasis Type="Italic">Ralstonia pseudosolanacearum</Emphasis>

Ralstonia solanacearum “species complex” (RSSC) represents soil-borne plant pathogenic bacteria, consisting of diverse and widespread strains that cause bacterial wilt on a wide range of host plants. A recent polyphasic taxonomic study has divided the RSSC into three bacterial species; Ralstonia pseudosolanacearum (phylotypes I and III), Ralstonia solanacearum (phylotype II) and Ralstonia syzygii (phylotype IV). Currently, standard identification of RSSC in plant health laboratories mainly relies on performance of two tests that are based on a different principle. However, these tests are inadequate to precisely discriminate among the three bacterial species in the RSSC. The accurate identification of each of the three bacterial species in the RSSC requires additional molecular tests, including a phylotype determination. These methodologies are labor-intensive, time consuming and rather impractical for routine identification purposes in a plant health laboratory. We explored the potential for an accurate identification of R. pseudosolanacearum (phylotypes I and III) and R. solanacearum (phylotype II) in RSSC, upon implementation of the MALDI-TOF MS tool, and after the creation and validation of an in-house database supplementing the commercial database and covering the entire known genetic diversity in RSSC. MALDI-TOF MS is an emerging approach for identification of bacterial plant pathogens and has been shown to be robust and reproducible. Additionally, when compared to the conventional microbial identification methods it is shown to be less laborious and less expensive. Validation data demonstrated that our in-house database (Mass Spectra Profiles, MSPs) was very specific resulting in the rapid and accurate identification of Ralstonia solanacearum (phylotype II), and Ralstonia pseudosolanacearum (phylotypes I and III). Additionally, no false positive results were obtained with our in-house database for other related Ralstonia sp., such as the R. picketii isolate PD 3286, or for the Pseudomonas syringae and Pseudomonas spp. isolates.     

Bacterial wilt of sweet potato caused by <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in Taiwan

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.     

Detection of race 1 strains of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in field samples in Taiwan using a BIO-PCR method

Bacterial wilt caused by race 1 strains of Ralstonia solanacearum is endemic on tomato produced in diverse agro-ecosystems in Taiwan. Using a new BIO-PCR protocol developed in this study, R. solanacearum was detected in soil, weed, and water samples collected from eight fields with different disease histories and cropping systems located in major tomato production areas. The sensitivity of the BIO-PCR was 1.9 CFU ml⁻¹ and 17 CFU g⁻¹ of soil for pure suspension and infested soil, respectively. The positive detection frequency of the BIO-PCR method was 66.6, 39.6, 23.1, and 31.8% for all tested samples of soil, weed rhizosphere soil, weed root, and water, respectively, and was higher than plating on MSM-1 medium. Detection of R. solanacearum from field soil indicated that spatial distribution of the pathogen in the field was not even regardless of the presence or absence of the disease and the different agro-ecosystems where the sampled fields were located, and the degree of unevenness was higher when tomato was absent from the field. Weed rhizosphere soils could be good sampling targets to monitor the pathogen in the field, because a higher positive detection proportion and population of R. solanacearum were found in the rhizosphere rather than the root of the collected weed samples. Symptomless weeds and contaminated irrigation, standing, or drainage waters were found to be important for the over-season survival and dissemination of R. solanacearum.     

Functional analysis of the melanin biosynthesis genes <Emphasis Type="Italic">ALM1</Emphasis> and <Emphasis Type="Italic">BRM2</Emphasis>-<Emphasis Type="Italic">1</Emphasis> in the tomato pathotype of <Emphasis Type="Italic">Alternaria alternata</Emphasis>

The tomato pathotype of Alternaria alternata (A. arborescens) produces the dark brown to black pigment melanin, which accumulates in the cell walls of hyphae and conidia. Melanin has been implicated as a pathogenicity factor in some phytopathogenic fungi. Here, two genes of the tomato pathotype for melanin biosynthesis, ALM1 and BRM2-1, which encode a polyketide synthetase and a 1,3,8-trihydroxynaphthalene (THN) reductase, respectively, have been cloned and disrupted in the pathogen. The gene-disrupted mutants, alm1 and brm2-1, had albino and brown phenotypes, respectively. The wild-type and the mutants caused the same necrotic lesions on the leaves after inoculation with spores. These results suggest that melanin is unlikely to play a direct role in pathogenicity in the tomato pathotype A. alternata. Scanning electron microscopy revealed that the conidia of both mutants have much smoother surfaces in comparison to the wild-type. The conidia of those mutants were more sensitive to UV light than those of the wild-type, demonstrating that melanin confers UV tolerance.     

Characterization of <Emphasis Type="Italic">Inago1</Emphasis> and <Emphasis Type="Italic">Inago2</Emphasis> retrotransposons in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.     

Distribution and multiplication of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in tomato plants with resistance derived from different origins

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.     

Pyruvate-amended modified SMSA medium: improved sensitivity for detection of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis>

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.     

Effect of simultaneous and sequential inoculations of <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>, <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> and <Emphasis Type="Italic">Phomopsis vexans</Emphasis> on eggplant in sand mix and fly ash mix soil

Effects simultaneous and sequential inoculations of Meloidogyne incognita, Ralstonia solanacearum and Phomopsis vexans were studied on the growth, chlorophyll and carotenoid contents of eggplants grown in 25% fly ash and 25% sand mix soil. Plants grown in 25% fly ash mix soil had lesser plant growth than grown in 25% sand ash mix soil. Inoculation of M. incognita / R. solanacearum or P. vexans caused reduction in plant growth, chlorophyll and carotenoid contents in both types of soils but these pathogens in combination caused a greater reduction in than individual inoculation. Inoculation of M. incognita 20 days prior to R. solanacearum caused a greater reduction in plant growth than inoculation of M. incognita prior to P. vexans. Inoculation of P. vexans prior to R. solanacearum caused a lesser reduction in plant growth, chlorophyll and carotenoid contents than inoculation of P. vexans prior to M. incognita. Inoculation of R. solanacearum 20 days prior to M. incognita caused a greater reduction in plant growth, chlorophyll and carotenoid contents than inoculation of R. solanacearum prior to P. vexans. Galling and multiplication of M. incognita was higher in plants grown in 25% sand amended soil than with 25% fly ash soil. R. solanacearum and P. vexans had adverse effects on galling and nematode multiplication. Wilt and blight indices caused by R. solanacearum and P. vexans were 3 respectively. Wilt and blight indices were 4 when two pathogens were inoculated together.     

Genetic diversity of <Emphasis Type="Italic">Rhizobium vitis</Emphasis> strains in Japan based on multilocus sequence analysis of <Emphasis Type="Italic">pyrG</Emphasis>, <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">rpoD</Emphasis>

Forty-one strains of Rhizobium vitis, either tumorigenic (Ti) or nonpathogenic, were characterized using multilocus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD. The strains separated into seven clades. Rhizobium vitis (Ti) strains isolated from Japan were divided into five genetic groups (A to E), and nonpathogenic R. vitis strains were divided into two genetic groups (F and G). This result suggests that there are new genetic groups of R. vitis in Japan. Among these groups, members of A and B groups are widely distributed throughout Japan.     

Survival of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in infected tissues of <Emphasis Type="Italic">Capsicum annuum</Emphasis> and in soils of the state of Pernambuco,Brazil

The survival of Ralstonia solanacearum A1-9^Rif race 1 phylotype I was studied in ten different soil types in the absence of the host plant as well as in infected tissues of the stem and root of bell peppers buried in the soil at 0, 5, and 15 cm. The survival time of R. solanacearum A1-9^Rif in the ten soil types ranged from 42 up to 77 days. Among the chemical and physical characteristics of the soil, clay content, residual moisture, and available water were positively correlated, and pH was negatively correlated, with survival time, population size at 42 days, and area under the population curve. The pathogen survival differed significantly in relation to the plant tissues, but not with respect to the incorporation depth of the infected tissues. The root tissue of bell pepper supported a larger bacterial population at 7 and 21 days (5 × 10⁴ and 3.1 × 10⁴ CFU g⁻¹ tissue, respectively) compared with the stem tissue (0.35 × 10⁴ and 0.48 × 10⁴ CFU g⁻¹ tissue, respectively) and also had a larger area under the population curve. On the other hand, the stem tissues presented a greater decomposition rate and pH compared with the roots. In conclusion, the different types of studied soils as well as the infected bell pepper tissues were considered potential primary sources of R. solanacearum inocula, but only for a short period.     

Bioassay-guided isolation of a novel chemoattractant for <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in tomato root exudates

Tomato root exudates were analyzed using a bioassay to detect the chemoattractant for Ralstonia solanacearum. An activated charcoal-adsorbed fraction of root exudates from tomato cultivar Oogata-fukuju had chemoattractant activity for R. solanacearum strain MAFF 730138. The active component, purified using a Sep-Pak C18 cartridge, an activated charcoal column, diol-modified silica gel, and NH₂-modified silica gel, is a new hydrophobic attractant. The final purified fraction produced a single peak in a diol-modified silica gel HPLC analysis.