Phylogeny and population structure of brown rot- and Moko disease-causing strains of Ralstonia solanacearum phylotype II

The ancient soilborne plant vascular pathogen Ralstonia solanacearum has evolved and adapted to cause severe damage in an unusually wide range of plants. In order to better describe and understand these adaptations, strains with very similar lifestyles and host specializations are grouped into ecotypes. We used comparative genomic hybridization (CGH) to investigate three particular ecotypes in the American phylotype II group: (i) brown rot strains from phylotypes IIB-1 and IIB-2, historically known as race 3 biovar 2 and clonal; (ii) new pathogenic variants from phylotype IIB-4NPB that lack pathogenicity for banana but can infect many other plant species; and (iii) Moko disease-causing strains from phylotypes IIB-3, IIB-4, and IIA-6, historically known as race 2, that cause wilt on banana, plantain, and Heliconia spp. We compared the genomes of 72 R. solanacearum strains, mainly from the three major ecotypes of phylotype II, using a newly developed pangenomic microarray to decipher their population structure and gain clues about the epidemiology of these ecotypes. Strain phylogeny and population structure were reconstructed. The results revealed a phylogeographic structure within brown rot strains, allowing us to distinguish European outbreak strains of Andean and African origins. The pangenomic CGH data also demonstrated that Moko ecotype IIB-4 is phylogenetically distinct from the emerging IIB-4NPB strains. These findings improved our understanding of the epidemiology of important ecotypes in phylotype II and will be useful for evolutionary analyses and the development of new DNA-based diagnostic tools.     

Influence of Native Microbiota on Survival of Ralstonia solanacearum Phylotype II in River Water Microcosms

Ralstonia solanacearum phylotype II biovar 2 causes bacterial wilt in solanaceous hosts, producing severe economic losses worldwide. Waterways can be major dissemination routes of this pathogen, which is able to survive for long periods in sterilized water. However, little is known about its survival in natural water when other microorganisms, such as bacteriophages, other bacteria, and protozoa, are present. This study looks into the fate of a Spanish strain of R. solanacearum inoculated in water microcosms from a Spanish river, containing different microbiota fractions, at 24°C and 14°C, for a month. At both temperatures, R. solanacearum densities remained constant at the initial levels in control microcosms of sterile river water while, by contrast, declines in the populations of the introduced strain were observed in the nonsterile microcosms. These decreases were less marked at 14°C. Lytic bacteriophages present in this river water were involved in the declines of the pathogen populations, but indigenous protozoa and bacteria also contributed to the reduced persistence in water. R. solanacearum variants displaying resistance to phage infection were observed, but only in microcosms without protozoa and native bacteria. In water microcosms, the temperature of 14°C was more favorable for the survival of this pathogen than 24°C, since biotic interactions were slower at the lower temperature. Similar trends were observed in microcosms inoculated with a Dutch strain. This is the first study demonstrating the influence of different fractions of water microorganisms on the survival of R. solanacearum phylotype II released into river water microcosms.     

Effects of ecological factors on the survival and physiology of Ralstonia solanacearum bv. 2 in irrigation water.

The fate of Ralstonia solanacearum bv. 2, the causative agent of brown rot in potato, in aquatic habitats of temperate climate regions is still poorly understood. In this study, the population dynamics and the physiological response of R. solanacearum bv. 2 were tested in sterile pure water and in agricultural drainage water obtained from waterways near potato cropping fields in The Netherlands. The behaviour of five different biovar 2 isolates in drainage water at 20 degrees C was very similar among strains. One typical isolate with consistent virulence (strain 1609) was selected for further studies. The effects of temperature, light, canal sediment, seawater salts, and the presence of competing microorganisms on the survival of strain 1609 were assessed. Moreover, the impacts of the physiological state of the inoculum and the inoculum density were analyzed. The population dynamics of strain 1609 in sterile pure water were also characterized. In sterile pure water, the fate of R. solanacearum 1609 cells depended strongly on temperature, irrespective of inoculum density or physiological state. At 4 degrees C and 44 degrees C, strain 1609 CFU numbers showed declines, whereas the strain was able to undergo several cell divisions at 12 degrees C, 20 degrees C, and 28 degrees C. At 20 degrees C and 28 degrees C, repeated growth took place when the organism was serially transferred, at low inoculum density, from grown water cultures into fresh water devoid of nutrients. Both at low and high cell densities and regardless of physiological state, R. solanacearum 1609 cells persisted as culturable cells for limited periods of time in drainage water. A major effect of temperature was found, with survival being maximal at 12 degrees C, 20 degrees C, and 28 degrees C. Temperatures of 4 degrees C, 36 degrees C, or 44 degrees C induced accelerated declines of the culturable cell numbers. The drainage water biota had a strong effect on survival at 12 degrees C, 20 degrees C, and 28 degrees C, as the persistence of strain 1609 was significantly enhanced in sterile drainage water systems. Furthermore, there was a negative effect of incident light, in a light:dark regime, on the survival of R. solanacearum 1609 in natural drainage water. Also, levels of seawater salts realistic for drainage water in coastal areas were detrimental to strain survival. Ralstonia solanacearum 1609 showed considerable persistence in canal sediment saturated with drainage water, but died out quickly when this sediment was subjected to drying. Evidence was obtained for the conversion of R. solanacearum 1609 cells to nonculturable cells in water microcosms kept at 4 degrees C, but not in those kept at 20 degrees C. A substantial fraction of the cells found to be nonculturable were still viable, as evidenced by the direct viable count and by staining with the redox dye 5-cyano-2,3-ditolyl tetrazolium chloride. The potential occurrence of viable-but-nonculturable cells in natural waters poses a problem for the detection of R. solanacearum by cultivation-based methods.     

Pig slurry reduces the survival of Ralstonia solanacearum biovar 2 in soil

The effect of added pig slurry and solarization on the survival of Ralstonia solanacearum biovar 2 strain 1609 in soil was analysed in soil microcosms and field plots. In addition, the invasion of potato plants by R. solanacearum and the development of disease symptoms were determined, as measures of induced disease suppressiveness. In untreated soil, R. solanacearum showed slow population declines in both microcosms and the field from, initially, 10(6-)10(7) to 10(3)-10(4) CFU.(g dry soil)(-1) in about 9 weeks. The suppressiveness assays of these untreated soils after this period revealed that most of the plants that were used developed wilting symptoms and (or) contained the pathogen in their lower stem parts, as shown by immunofluorescence colony staining and PCR. The addition of pig slurry resulted in a significantly lower population size of R. solanacearum as well as reduced numbers of infected and (or) diseased plants in the soil suppressiveness tests. On the other hand, solarization of soil also decreased R. solanacearum survival but did not enhance soil suppressiveness as measured by development of disease symptoms and (or) plant invasion after 9 weeks. Combined soil solarization and pig slurry addition showed an additive effect of both treatments. Healthy-looking plants, primarily from soils treated with pig slurry and solarization, incidentally revealed the latent presence of R. solanacearum in the lower stem parts. The mechanism behind the enhanced population declines and disease suppressiveness induced by pig slurry is unclear but shifts in community profiles were clearly discernible by PCR - denaturing gradient gel electrophoresis 9 weeks after pig slurry addition in the field experiment, indicating induced changes in the bacterial community structure.     

Monitoring the survival of fish-pathogenic Francisella in water microcosms

In this report, the survival behaviour of fish pathogenic Francisella in water microcosms was investigated under laboratory conditions. Two isolates of Francisella noatunensis (NCIMB14265(T) and PQ 1106), from fish held in seawater and freshwater, were inoculated into natural (nonsterile) and sterile sea- and freshwater microcosms, respectively, and monitored under different temperature conditions (4, 8 and 12 °C) over a period of 60 days. The culturability of the strains was inversely related to the water temperature. Strain NCIMB14265(T) was found to survive longer in seawater than PQ 1106 held in freshwater at equivalent temperatures. The survival of both strains was higher in sterile than in nonsterile microcosms. These results were confirmed by quantitative PCR analysis targeting the succinate dehydrogenase (sdhA) gene. A cell viability assay coupled with FISH analyses showed that F. noatunensis cells enter a viable but not culturable (VBNC) state after a period in water. However, although metabolically active, the VBNC cells were not pathogenic to cod (Gadhus morhua) following an intraperitoneal challenge, under the conditions tested. The data presented contribute to a better understanding of the behaviour of F. noatunensis in natural seawater and freshwater environments, and show the need for further investigation of the role of VBNC cells in the environmental transmission of this pathogen.     

Studies on the biosynthesis of ralfuranones in Ralstonia solanacearum

Ralfuranones, aryl-furanone secondary metabolites, are involved in the virulence of Ralstonia solanacearum in solanaceous plants. Ralfuranone I (6) has been suggested as a biosynthetic precursor for other ralfuranones; however, this conversion has not been confirmed. We herein investigate the biosynthesis of ralfuranones using feeding experiments with ralfuranone I (6) and its putative metabolite, ralfuranone B (2). The results obtained demonstrated that the biosynthesis of ralfuranones proceeded in enzymatic and non-enzymatic manners.     

青枯菌hrp基因的研究进展

青枯菌的hrp基因可诱发植物的超敏反应.对其基因组全序列测定表明:hrp基因簇位于基因组的大质粒上,共有20多个基因组成.从青枯菌中分离得到的可直接诱发植物超敏反应的效应蛋白主要为pop基因编码,它由hrp基因编码的类型Ⅲ蛋白分泌通道释放.目前的研究表明:(1)在hrp基因簇中,hrpY、hrpX及hrpV与分泌通道的一种纤毛的组装有关;(2)hrpB是整个类型Ⅲ蛋白分泌通道基因的转录激活子并作用于基因组中的其它效应基因;(3)hrpG是植物信号对hrp,基因的表达进行级联调控的组分之一.     

Identification of QTLs for Ralstonia solanacearum race 3-phylotype II resistance in tomato

Resistance against a Ralstonia solanacearum race 3-phylotype II strain JT516 was assessed in a F_2:3 and a population of inbred lines (RIL), both derived from a cross between L. esculentum cv. Hawaii 7996 (partially resistant) and L. pimpinellifolium WVa700 (susceptible). Resistance criteria used were the percentage of wilted plants to calculate the AUDPC value, and bacterial colonization scores in roots and stem (hypocotyl and epicotyl) assessed in two independent greenhouse experiments conducted during the cool and hot seasons in Réunion Island, France. Symptoms were more severe during the cool season trials. Heritability estimates in individual seasons ranged from 0.82 to 0.88, depending on resistance criterion. A set of 76 molecular markers was used for quantitative trait loci (QTL) mapping using the single- and composite- interval mapping methods, as well as ANOVA. Four QTLs, named Bwr- followed by a number indicating their map location, were identified. They explained from 3.2 to 29.8% of the phenotypic variation, depending on the resistance criterion and the season. A major QTL, Bwr-6, and a minor one, Bwr-3, were detected in each season for all resistance criteria. Both QTLs showed stronger effects in the hot season than in the cool one. Their role in resistance to R. solanacearum race 3-phylotype II was subsequently confirmed in the RIL population derived from the same cross. Two other QTLs, Bwr-4 and Bwr-8, with intermediate and minor effects, respectively, were only detected in the hot season, demonstrating that environmental factors may strongly influence the expression of resistance against the race 3-phylotype II strain JT516. These QTLs were compared with those detected in the RIL population against race 1-phylotype I strain JT519 as well as those detected in other previous studies in the same genetic background against other race 1-phylotype I and II strains. This comparison revealed the possible occurrence of some phylotype-specific resistance QTLs in Hawaii 7996.     

Influence of light and natural microbiota of the Butrón river on E. coli survival

The survival of an E. coli strain in water samples from the Butrón river has been studied. The input of E. coli cells in the aquatic system breaks down the established balance among the components of the natural microbiota: E. coli becomes the object of the active protozoal predation whereas the autochtonous heterotrophic community become alternative preys. As a result of this new situation, the natural microbiota increases but returns to the initial values once the E. coli cells have been removed from the system. The effect of the temperature of incubation on the survival is exerted through the effect of this parameter on the predatory activity of the protozoa. Light has a lethal and direct action on the E. coli cells, the effect of this parameter is even superior to that of predation.     

The viable but nonculturable state of Ralstonia solanacearum may be involved in long-term survival and plant infection

The role of the dormant-like viable but nonculturable (VBNC) condition in the etiology of bacterial infection was examined using a plant system. The plant-pathogenic bacterium Ralstonia solanacearum was first shown to enter into the VBNC state both in response to cupric sulfate when in a saline solution and when placed in autoclaved soil. To determine if the VBNC condition is related to pathogenesis, the physiological status of bacteria recovered from different regions of inoculated tomato plants was determined at different stages of infection. The fraction of in planta bacteria that were VBNC increased during infection and became greater than 99% by the late stage of disease. The possibility that soil-dwelling VBNC bacteria may resuscitate and infect plants was also examined. When tomato seeds were germinated in sterile soil that contained VBNC but no detectable culturable forms of R. solanacearum cells, resuscitation was observed to occur in soil adjacent to plant roots; these resuscitated bacteria were able to infect plants. This is the first report of R. solanacearum entering the VBNC state and of resuscitation of any VBNC plant-pathogenic bacteria and provides evidence that the VBNC state may be involved in explaining the persistent nature of some infections.     

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.     

培养条件对青枯雷尔氏菌脂肪酸组成的影响

应用气相色谱技术测定不同温度、培养时间、pH值等培养条件下青枯雷尔氏菌(Ralstonia solanacearum)脂肪酸的结果表明: 青枯雷尔氏菌强致病力菌株Rs-J.1.4-010704-01v的脂肪酸种类有14~34种, 主要特征脂肪酸为C16:1ω7c/C15:0 ISO 2OH(10.644 min), C16:0(10.950 min), C18:1ω7c(14.177 min), 所占总百分比含量为总脂肪酸的55.66%~75.69%; 该菌脂肪酸的种类与含量随着培养条件的改变而发生变化,     

Characteristics of Ralstonia solanacearum Biovar N2 Strains in Asia

Ralstonia solanacearum biovar N2 strains isolated in Asia were compared by biochemical tests with biovar N2 strains from South America and biovar 2 (race 3) strains from Africa, America, Asia and Europe. Distinct differences were found between Asian and South American strains of biovar N2, and between Asian biovar N2 and biovar 2 strains with respect to their ability to utilize several carbon sources. Using cluster analysis based on repetitive sequence‐based polymerase chain reaction (rep‐PCR) genomic fingerprints, the Asian biovar N2 strains were divided into two groups, group 1 containing Japanese strains and group 2 containing Indonesian and Philippine strains. The fingerprints showed the genetic diversity of biovar N2 strains in Asia.     

Characterization of CRISPR-Cas systems in the Ralstonia solanacearum species complex

Clustered regularly interspaced short palindromic repeats (CRISPRs) are composed of an array of short DNA repeat sequences separated by unique spacer sequences that are flanked by associated (Cas) genes. CRISPR-Cas systems are found in the genomes of several microbes and can act as an adaptive immune mechanism against invading foreign nucleic acids, such as phage genomes. Here, we studied the CRISPR-Cas systems in plant-pathogenic bacteria of the Ralstonia solanacearum species complex (RSSC). A CRISPR-Cas system was found in 31% of RSSC genomes present in public databases. Specifically, CRISPR-Cas types I-E and II-C were found, with I-E being the most common. The presence of the same CRISPR-Cas types in distinct Ralstonia phylotypes and species suggests the acquisition of the system by a common ancestor before Ralstonia species segregation. In addition, a Cas1 phylogeny (I-E type) showed a perfect geographical segregation of phylotypes, supporting an ancient acquisition. Ralstoniasolanacearum strains CFBP2957 and K60^T were challenged with a virulent phage, and the CRISPR arrays of bacteriophage-insensitive mutants (BIMs) were analysed. No new spacer acquisition was detected in the analysed BIMs. The functionality of the CRISPR-Cas interference step was also tested in R. solanacearum CFBP2957 using a spacer-protospacer adjacent motif (PAM) delivery system, and no resistance was observed against phage phiAP1. Our results show that the CRISPR-Cas system in R. solanacearum CFBP2957 is not its primary antiviral strategy.     

Seasonal variation of Ralstonia solanacearum biovar 2 populations in a Spanish river: recovery of stressed cells at low temperatures

The presence of Ralstonia solanacearum biovar 2 in the watercourses of European countries is increasing, but little is known about its ecology in aquatic habitats. The detection of this pathogen in 2000 in one Spanish river led us to study its population density at different locations on the river over a period of 3 years. During 2000 and 2001, the pathogen was recovered at low densities (10 to 80 CFU/ml) by direct plating on modified SMSA agar from water samples at 14 degrees C or higher, but its isolation was usually unsuccessful at temperatures below 9 degrees C. To monitor the pathogen's abundance in winter, we used two liquid selective media for enrichment (at 29 and 35 degrees C) and compared them by using spiked river water samples: modified Wilbrink broth (MWB) was more efficient than modified SMSA broth for double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) detection of R. solanacearum. Enrichment in MWB at both temperatures allowed us to recover R. solanacearum cells that were nonculturable on solid media up to 25 days after their entry into the viable but nonculturable state. When we applied this technique to water samples during the cold months of 2001 and 2002, we obtained the best detection results by the most-probable-number method after enrichment at 35 degrees C with MWB. The enrichment protocol was combined with DASI-ELISA and validated by Co-PCR to detect both naturally and artificially starved and cold-stressed cells in water, which were still infective. Overall, the data from this study demonstrate the effects of temperature variation on the population and culturability of R. solanacearum cells on solid media and their survival at low temperatures.     

A Duplex PCR Assay for the Detection of Ralstonia solanacearum Phylotype II Strains in Musa spp.

Banana wilt outbreaks that are attributable to Moko disease-causing strains of the pathogen Ralstonia solanacearum (Rs) remain a social and economic burden for both multinational corporations and subsistence farmers. All known Moko strains belong to the phylotype II lineage, which has been previously recognized for its broad genetic basis. Moko strains are paraphyletic and are distributed among seven related but distinct phylogenetic clusters (sequevars) that are potentially major threats to Musaceae, Solanaceae, and ornamental crops in many countries. Although clustered within the Moko IIB-4 sequevar, strains of the epidemiologically variant IIB-4NPB do not cause wilt on Cavendish or plantain bananas; instead, they establish a latent infection in the vascular tissues of plantains and demonstrate an expanded host range and high aggressiveness toward Solanaceae and Cucurbitaceae. Although most molecular diagnostic methods focus on strains that wilt Solanaceae (particularly potato), no relevant protocol has been described that universally detects strains of the Musaceae-infecting Rs phylotype II. Thus, a duplex PCR assay targeting Moko and IIB-4NPB variant strains was developed, and its performance was assessed using an extensive collection of 111 strains representing the known diversity of Rs Moko-related strains and IIB-4NPB variant strains along with certain related strains and families. The proposed diagnostic protocol demonstrated both high accuracy (inclusivity and exclusivity) and high repeatability, detected targets on either pure culture or spiked plant extracts. Although they did not belong to the Moko clusters described at the time of the study, recently discovered banana-infecting strains from Brazil were also detected. According to our comprehensive evaluation, this duplex PCR assay appears suitable for both research and diagnostic laboratories and provides reliable detection of phylotype II Rs strains that infect Musaceae.     

Influence of aquatic microbiota on the survival in water of the human and eel pathogen Vibrio vulnificus serovar E

The eel and human pathogen Vibrio vulnificus serovar E (biotype 2) is seldom isolated from natural waters, although it can survive in sterilized artificial seawater microcosms for years. The main objective of the present study was to investigate whether aquatic microbiota can limit its survival and recovery from water samples. A set of preliminary experiments of survival in microcosms containing natural seawater and water from eel farms showed that the persistence of this pathogen was mainly controlled by grazing, and secondarily by bacterial competition. The bacterial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E (VSE) strains and potential competitors. Competitors included V. vulnificus biotype 1 isolates and strains of selected species that can grow on the selective media designed for V. vulnificus isolation from water samples. Evidences of bacterial competition that was detrimental for VSE recovery were recorded. Thus, some species produced a deleterious effect on VSE strains under starvation, and others were able to use the resources more efficiently under nutrient input. These results suggest that an overgrowth of more efficient competitor bacteria in conventional media used for isolation of V. vulnificus could mask the recovery of VSE strains and explain the scarcity of reports on the isolation of this human and eel pathogen from natural waters.     

Measurement of Horizontal and Vertical Movement of Ralstonia solanacearum in Soil

Two model systems were constructed to measure horizontal and vertical movement of bacteria in soil. These systems were applied to measuring movement of Ralstonia solanacearum (race 1, biovar 3), a causal agent of bacterial wilt of tomato, in andosol and sand at 28°C. The first system was used to measure horizontal movement of the bacteria in soil packed in a narrow horizontal frame. Suspension of the pathogen was applied to soil at one end of the frame, and bacterial number per gram of soil was measured over distance from the inoculation point after 4 days. Horizontal movement of R. solanacearum in supersaturated soil, but without flow, was possibly due to diffusion and the front advanced at 2.2 cm/day in andosol, and at 8.1 cm/day in sand. Using the same experimental system, but applying water inflow to one end of the frame only, the bacterium was detected at the front of water in andosol and sand. The front of the distribution advanced at 20.4 cm/h in andosol and 66.3 cm/h in sand. In the second experimental system, a cylinder of soil packed in a short tube was soaked with water, and soil at the top of the tube was inoculated with bacterial suspension. Immediately, soil cylinders were turned upward, and the bacterial number per gram of soil was measured along vertical distance from the inoculation point after 7 days. Using the system with andosol, the capillary water front rose to 32.5 cm over 7 days after inoculation, and R. solanacearum reached to 18.8 cm height. In sand, capillary water rose to 20.0 cm and the bacteria reached to 16.3 cm height.     

Contribution of Folate Biosynthesis to Ralstonia solanacearum Proliferation in Intercellular Spaces

The vigorous proliferation of Ralstonia solanacearum OE1-1 in host intercellular spaces after the invasion of host plants is necessary for the virulence of this bacterium. A folate auxotroph, RM, in which a mini-Tn5 transposon was inserted into pabB encoding para-aminobenzoate synthase component I, lost its ability to vigorously proliferate in intercellular spaces along with its systemic infectivity and virulence after inoculation into roots and infiltration into leaves of tobacco plants. Complementation of RM with the pabB gene allowed the mutant to multiply in intercellular spaces and to cause disease. In tobacco plants that were pretreated with folate, RM was able to vigorously proliferate in the intercellular spaces and cause disease. Interestingly, when it was inoculated through cut stems, the mutant multiplied in the plants and was virulent. Moreover, the mutant multiplied well in stem fluids but not in intercellular fluids, suggesting that the folate concentration within intercellular spaces may be a limiting factor for bacterial proliferation. Therefore, folate biosynthesis contributes to the vigorous proliferation of bacteria in intercellular spaces and leads to systemic infectivity resulting in virulence.     

Possible Mechanisms Limiting Movement of Ralstonia solanacearum in Resistant Tomato Tissues

The distribution and appearance of Ralstonia solanacearum in the upper hypocotyl tissues of root‐inoculated tomato seedlings of resistant rootstock cultivar LS‐89 (a selection from Hawaii 7998) and susceptible cultivar Ponderosa were compared to clarify the mechanism that limits the movement of the bacterial pathogen in resistant tomato tissues. In stems of wilted Ponderosa plants, bacteria colonized both the primary and the secondary xylem tissues. Bacteria were abundant in vessels, of which the pit membranes were often degenerated. All parenchyma cells adjacent to vessels with bacteria were necrotic and some of them were colonized with bacteria. In stems of LS‐89 plants showing no discernible wilting symptoms, bacteria were observed in the primary xylem tissues but not in the secondary xylem tissues. Necrosis of parenchyma cells adjacent to vessels with bacteria was observed occasionally. The pit membranes were often thicker with high electron density. The inner electron‐dense layer of cell wall of parenchyma cells and vessels was thicker and more conspicuous in xylem tissues of infected LS‐89 than in xylem of infected Ponderosa or mock‐inoculated plants. Electron‐dense materials accumulated in or around pit cavities in parenchyma cells next to vessels with bacteria, and in vessels with bacteria. Many bacterial cells appeared normal in vessels, except for those in contact with the pit membranes. These results indicate that R. solanacearum moves from vessel to vessel in infected tissues through degenerated pit membranes and that restricted movement in xylem tissues was the characteristic feature in LS‐89. The limitation in bacterial movement may be related to the thickening of the pit membranes and/or the accumulations of electron‐dense materials in vessels and parenchyma cells.