The Low-Temperature-Induced Viable-But-Nonculturable State Affects the Virulence of Ralstonia solanacearum Biovar 2

ABSTRACT The physiology and virulence of Ralstonia solanacearum biovar 2 strain 1609, kept in water at 4 and 20 degrees C, were studied. At 20 degrees C, total cell and plate count (colony forming units; CFU) numbers were similar, between log 5.03 and log 5.55 CFU, and log 5.03 and log 5.51 cells per ml, at days 0 and 132, respectively. However, CFU in the cultures kept at 4 degrees C dropped from log 6.78 CFU/ml at day 0 to below detection after 84 days. The presence of catalase in the agar resulted in higher CFU, and at day 84, log 1.95 CFU/ml still was detectable. No colonies were observed at day 125. The presence of viable-but-nonculturable (VBNC) cells in the 4 degrees C cultures was confirmed using SYTO9 viability staining. Viable cell numbers were log 1.77 higher than CFU on plates with catalase. At day 84 and after 125 days, log 3.70 viable cells per ml still were present. Shifts in subpopulations differing in viability were found by flow cytometric sorting of 4 degrees C-treated cells stained with SYTO9 (healthy) and propidium iodide (PI; compromised). The SYTO9-stained cell fractions dropped from 99 to 39%, and the PI-stained fractions increased from 0.7 to 33.3% between days 0 and 125. At 20 degrees C, the SYTO9-stained fraction remained stable at 99% until day 132. SYTO9-stained cells sorted from 4 degrees C cultures at day 100 were injected into tomato plants. Upon incubation for 30 days, these plants did not show wilting. However, more than log 4.19 CFU and log 8.17 cells were recovered from these plants. Cells from colonies isolated from the nonwilted plants did not regain their virulence as demonstrated by subsequent injection into several new sets of tomato plants. Cells from 4 degrees C cultures injected at day 125 were not able to cause wilting of, or proliferate in, tomato plants. The threat posed by VBNC R. solanacearum cells upon incubation at 4 degrees C was thus ephemeral because cells lost their capacity to cause disease after 125 days.     

Effect of a Soil Amendment on the Survival of Ralstonia solanacearum in Different Soils

ABSTRACT The effect of a soil amendment (SA) composed of urea (200 kg of N per ha) and CaO (5,000 kg/ha) on the survival of Ralstonia solanacearum in four Philippine soils was investigated in a series of laboratory experiments. Within 3 weeks after application, the SA either caused an initial decrease, a final decline, or no change in the pathogen population, depending on the particular soil type. An initial decrease occurred in a soil with a basic pH and resulted in a significantly (P < 0.001) lower pathogen population immediately and at 1 week after amending the soil. This decrease was probably due to the high pH in the soil during urea hydrolysis. A final decline in the R. solanacearum population after 3 weeks occurred in two soils in which nitrite accumulated after 1 week. In these soils, no decline in bacterial levels occurred when nitrite formation was inhibited by 2-chloro-6-trichloromethylpyridine. In the soil with low pH, no nitrite accumulated and the R. solanacearum population did not decline. The suppressive effects of pH and nitrite on R. solanacearum growth were confirmed by in vitro experiments. Ammonium reduced the growth of R. solanacearum, but was not suppressive. Interactions of pH with ammonium and nitrite also occurred, whereby ammonium reduced growth of R. solanacearum only at pH 9 and nitrite was suppressive only at pH 5. Nitrate had no effect on R. solanacearum growth in vitro.     

Survival of Ralstonia solanacearum and Ralstonia pseudosolanacearum in drain water

The survival in drain water of two strains of Ralstonia solanacearum and three strains of Ralstonia pseudosolanacearum, including two strains able to cause wilt in roses, was determined. Water draining from drip‐irrigated rock wool mats on which roses were grown was supplemented with the pathogen and survival was monitored at 4, 12, 20 and 28°C for up to 112 days. All strains were able to survive for at least 112 days in drain water at 12 and 20°C, but at 4°C maximum survival was 56 days. At 28°C, the survival period was strain dependent, but was at least 56 days. Populations declined gradually in non‐sterile drain water to a low level (maximum 100 cfu mL^?1 after 112 days). In sterile drain water (autoclaved prior to addition of populations), no or only a limited decline in populations was found at 112 days, dependent on strain and temperature. Drain water that tested negative for Ralstonia in the dilution plating assay was tested for the presence of cells in a viable but non‐culturable state (VBNCs). Tomato plants were inoculated, but no symptoms developed, and plants sampled 22 days post‐inoculation were negative in a plating assay. Therefore, no indications were found that VBNCs were present.     

Behavior of Ralstonia solanacearum Race 3 Biovar 2 During Latent and Active Infection of Geranium

ABSTRACT Southern wilt of geraniums (Pelargonium hortorum), caused by the soilborne bacterium Ralstonia solanacearum race 3 biovar 2 (R3bv2), has inflicted significant economic losses when geranium cuttings latently infected with this quarantine pest were imported into the United States. Little is known about the interaction between R. solanacearum and this ornamental host. Using UW551, a virulent R3bv2 geranium isolate from a Kenyan geranium, we characterized development of Southern wilt disease and R3bv2 latent infection on geranium plants. Following soil inoculation, between 12 and 26% of plants became latently infected, carrying average bacterial populations of 4.8 x 10(8) CFU/g of crown tissue in the absence of visible symptoms. Such latently infected plants shed an average of 1.3 x 105 CFU/ml in soil run-off water, suggesting a non-destructive means of testing pools of asymptomatic plants. Similarly, symptomatic plants shed 2 x 10(6) CFU/ml of run-off water. A few hundred R. solanacearum cells introduced directly into geranium stems resulted in death of almost all inoculated plants. However, no disease transmission was detected after contact between wounded leaves. Increasing temperatures to 28 degrees C for 2 weeks did not convert all latently infected plants to active disease, although disease development was temperature dependent. Holding plants at 4 degrees C for 48 h, a routine practice during geranium cutting shipment, did not increase frequency of latent infections. R. solanacearum cells were distributed unevenly in the stems and leaves of both symptomatic and latently infected plants, meaning that random leaf sampling is an unreliable testing method. UW551 also caused potato brown rot and bacterial wilt of tomato, surpassing race 1 strain K60 in virulence on tomato at the relatively cool temperature of 24 degrees C.     

The Biological Control of Fusarium oxysporum,the Causal Agent of Potato Rot

This study was carried out in vivo and in vitro to search the biological control possibilities of soil borne dry rot causal agent Fusarium oxysporum causing yield losses in potato. In this study, 2 Pantoea agglomerans (BRTB and RK-92), 2 Bacillus pumilus (RK-103 and TV-67C), 7 Bacillus subtilis (BAB-140, TV-12H, TV-6F, EK?7, TV-17C, CP?1 and TV-125A), 3 Bacillus megaterium (TV-103B, TV-87A and TV-91C), 1 Ochrobactrum anthropi (A-16B), 1 Agrobacterium radiobacter (A-16) and 1 Bacillus thuringiensis subsp. kurstaki (BAB-410) bacterial and 2 Trichoderma harzianum (ET 4 and ET 14) fungal isolates tested efficacy in previous studies were used. In pot trial, the experiment was established with the most effective five bacterial strains (BRTB 66.22%; RK-103, 50.90%; BAB-140 50.00%; TV-103B 49.10%; TV-12H 48.65% and TV-6F 48.20%) and two fungal isolates (ET 4; 69.44%, ET 14; 66.66%). BRTB, the most effective bacterial strain, prevented completely the development of the pathogen. Based on the application time of BRTB, infection was not observed on seedlings on growing from tubers inoculated with pathogen 4?h after dipping into the bacterial solution. In storage treatments, BRTB was the most efficacy isolate when compared with others. As a result, BRTB strain of Pantoea agglomerans can be candidate in the biological control of F. oxysporum.

     

Potato bacterial wilt in India caused by strains of phylotype I, II and IV of Ralstonia solanacearum

Bacterial wilt or brown rot is one of the most devastating diseases of potato caused by a bacterium Ralstonia solanacearum (Smith 1986) Yabuuchi et al. (Microbiol Immunol 39:897–904 1995). Traditionally, R. solanacearum is classified into five races (r) on the basis of differences in host range and six biovars (bvs) on the basis of biochemical properties. Recently using molecular methods, R.?solanacearum has been classified 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. In the present study, 75 bacterial strains, isolated from wilt infected potatoes from various potato growing regions of India, were classified by traditional and molecular methods. The identity of all the strains was confirmed as R. solanacearum as expected single 280-bp fragment resulted in all the strains following PCR amplification using R. solanacearum specific universal primer pair 759/760. Biovar (bv) analysis, based on utilization of disaccharide sugars and hexose alcohols, categorised the 75 strains into bv2 (78.7 %), 2 T (5.3 %), 3 (5.3 %) and 4 (10.7 %). The phylotype specific multiplex PCR assigned 78.7 % strains to phylotype II, 16.0 % to phylotype I and 5.3 % to phylotype IV. Phylogenetic analysis of egl gene sequences clustered all fifty nine phylotype II (bv2) strains with reference strain IPO1609 (IIB-1), all four phylotype IV (bv2T) strains with reference strain MAFF301558 (IV-8), three phylotype I (bv3) strains with reference strain MAFF211479 (I-30) and all eight phylotype I (bv4) and one phylotype I (bv3) strain with reference strain CIP365 (I-45). The study concluded that the Indian potato strains of R. solanacearum belong to three out of four phylotypes namely: the Asian phylotype I, the American phylotype II, and the Indonesian phylotype IV. This is the first study to address the diversity of R. solanacearum from potato in India using phylotype and sequevar scheme. We also report here for the first time the occurrence of phylotype IV sequevar 8 (bv2T) strain of R. solanacearum causing potato bacterial wilt in mid hills of Meghalaya in India.     

Überleben von Ralstonia solanacearum Biovar 2 (Rasse 3), dem Erreger der Schleimkrankheit der Kartoffel, in Boden und auf verschiedenen Materialien (Mikrokosmos)

Microcosm studies were carried out to test the survival of Ralstonia solanacearum biovar 2 (race 3) in soil at the permanent wilting point (wp) water content and at field capacity (fc) water content and on various material. Soils were placed at permanent ?5°C, 4°C, 15°C and 20°C and weekly fluctuating ?10/0/+10°C and the material at 5, 15 °C, 20°C with relative humidity (rh) uncontrolled or at constant 10% or 90%. In soil, survival was clearly dependent on temperature independent of water content. At 20°C Ralstonia solanacearum could be reisolated up to 364 days, at 15°C up to 290 days, at 4°C up to 209 days and at fluctuating temperatures (?10/0/+10°C) only up to 18 days. The lower the temperature, the more the population declined. At 15°C and 20°C appr. 10⁷ cfu/g soil were detected after 100 days, whereas at ?5°C only 10² cfu/g soil were detected after only 18 days. The pathogen was longer detectable in sandy-clay loam than in lighter sandy soil. It could be longer reisolated at wilting point and the populations did not decline as rapidly as at field capacity. Ralstonia solanacearum could best survive on material surfaces like rubber, plastic and varnished metal with maximum survival of 40 days at 5°C and 10% rh. In general there is a low risk of Ralstonia solanacearum overwintering under European climatic conditions when the fields are cleared of plant debris and the soil is frozen. Contamined material surfaces pose the risk of pathogen transmission to healthy tubers.     

Ralstonia (Pseudomonas) solanacearum Race 3 (Biovar 2) in Surface Water and Natural Weed Hosts: First Report on Stinging Nettle (Urtica dioica)

The population dynamics of the brown rot bacterium Ralstonia (Pseudomonas) solanacearum in surface water of two selected water-areas were monitored over a two-year period. In some cases during summer, high bacterial numbers (up to 10⁶ cfu l^–1) were observed. In a host plant survey a few plants of stinging nettle (Urtica dioica) were found to be a natural host of the bacterium when plants were growing with their roots in contaminated water. The significance of U. dioica in the epidemiology of the brown rot bacterium is not yet known and subject to further investigation. Pathogenicity of R. solanacearum to stinging nettle (U. dioica) and bittersweet (Solanum dulcamara) was demonstrated in a greenhouse experiment.     

烟草青枯病菌拮抗细菌的筛选、鉴定和抑菌机制研究

青枯雷尔氏菌Ralstonia solanacearum造成的烟草青枯病是烟草主要毁灭性病害之一.本研究采用牛津杯法从运城盐湖湖岸土壤中筛选获得一株对烟草青枯病菌具有较好拮抗效果的菌株FY-C;并进一步分析了菌株FY-C的抑菌谱、对青枯病菌的潜在生防效果.结果 显示,经菌株FY-C无菌滤液处理24 h后的青枯病菌细胞壁...     

Immunofluorescence Colony-staining (IFC) for Detection and Quantification of Ralstonia (Pseudomonas) solanacearum Biovar 2 (Race 3) in Soil and Verification of Positive Results by PCR and Dilution Plating

A procedure was developed for specific and sensitive quantitative detection of Ralstonia (Pseudomonas) solanacearum biovar 2 (race 3) in soil. It is based on immunofluorescence colony-staining (IFC) followed by confirmation of the identity of fluorescent colonies by PCR-amplification or dilution plating on a semi-selective medium, SMSA. Addition of sucrose and the antibiotics cycloheximide and crystal violet to the non-selective trypticase soy broth agar resulted in increased colony size and staining intensity of R. solanacearum in IFC. Verification of IFC-results by picking cells from IFC-positive colonies followed by dilution plating of the suspended cells on SMSA was highly efficient. The success rate was 92% and 96% with spiked and naturally contaminated soils respectively. Several other bacterial species which cross-reacted with polyclonal antibodies in IFC also grew on SMSA and were difficult to distinguish from R. solanacearum, thereby necessitating confirmation of the results. Rapid verification of IFC-positive results directly by PCR-amplification with primers D2/B specific to division 2 of R. solanacearum had a success rate of 86% and 96% with spiked and naturally contaminated soil samples, respectively. Primers D2/B reacted with all R. solanacearum division 2 strains, and strains of R. syzygii and the banana blood disease bacterium, but not with saprophytic bacteria cross-reacting in IFC with R. solanacearum antibodies. In comparative tests, IFC was able to detect consistently ca. 100 cfu g^–1 of soil, a detection level similar to that found with direct plating on SMSA, but less laboriously, whereas detection level with a bioassay on tomato plants was only 10⁴–10⁵ cfu g^–1 of soil.     

Current classification of Ralstonia solanacearum and genetic diversity of the strains in Japan

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.     

Phylotype and sequevar variability of Ralstonia solanacearum in Brazil,an ancient centre of diversity of the pathogen

The β‐proteobacterium Ralstonia solanacearum causes bacterial wilt of many plant species. Knowledge of phylotype and sequevar variability in populations of this microorganism is useful for implementing control measures, particularly host resistance. To this end, 301 isolates of R. solanacearum were collected from different geographic regions and hosts in Brazil. Their phylotype and sequevar characterization was used to determine the amount and distribution of phenetic and phylogenetic variability. Isolates were classified into phylotypes I (n = 48), clade 1; and phylotype II, clades 2–5. Phylotype II was divided into subclusters IIA (n = 112) and IIB (n = 141). Phylotype II was widely distributed, whereas phylotype I isolates were found in Central, Northern, and Northeastern regions of Brazil. There were 108 haplotypes identified among endoglucanase (egl) gene sequences from 301 isolates and 32 haplotypes among DNA repair (mutS) gene regions from 176 isolates. The egl and mutS sequence analyses identified eight known (1, 4, 7, 18, 27, 28, 41 and 50) and four new (54, 55, 56 and 57) sequevars. Phylotype IIB showed high diversity in sequevars and host range. Multiplex PCR, using primers specific to the Moko ecotype, characterized banana and long pepper isolates as sequevar 4 and 4/NPB, respectively. This constitutes the first report of the emergent ecotype IIB/4NPB in a new host, long pepper. The majority of sequevars were associated with geographic regions. This high variability of R. solanacearum in Brazil suggests use of host resistance to control bacterial wilt should be mainly focused by region.     

Interaction between silicon amendment,bacterial wilt development and phenotype of Ralstonia solanacearum in tomato genotypes

Silicon amendment significantly reduced bacterial wilt incidence expressed as area under disease progress curve for tomato genotypes L390 (susceptible) by 26.8% and King Kong2 (moderately resistant) by 56.1% compared to non-treated plants grown in hydroponic culture. However, wilt incidence in silicon-treated plants of genotype L390 reached 100% at 13 days post-inoculation (dpi), while in genotype King Kong2, plant death was retarded by 6 days, with 20% reduction of final wilt incidence. Bacterial numbers were significantly lower in silicon-treated compared to non-treated plants in King Kong2 at 2 dpi in midstems and in all organs at 5 dpi, and in Hawaii 7998 (resistant) in all organs at 2 dpi. Differences between genotypes were obvious on midstem level (5 dpi), where bacterial populations were generally significantly lower compared to roots. Increased tolerance was observed in genotypes L390 and King Kong2 with silicon treatment.Silicon accumulated in roots and was low in stems and leaves. Inoculation with Ralstonia solanacearum did not significantly affect silicon uptake and distribution. Negative correlations between root silicon content and bacterial numbers of midstems in genotypes Hawaii 7998 and King Kong2 suggested an induced resistance. Indications for an influence of host genotype and silicon treatment on the phenotypic conversion of R. solanacearum strain To-udk2-sb from fluidal to non-fluidal colonies in planta were observed.This is the first report on the effect of silicon on a bacterial disease and in a silicon-non-accumulator plant.     

Legislative measures to prevent the introduction and spread of Ralstonia solanacearum in the European Union1

Since the outbreaks of potato brown rot (caused by Ralstonia solanacearum) in EU Member States in the mid 1990s, EU measures against this pest have been strengthened. Factors in the spread of R. solanacearum are reviewed: planting material, Solanaceous weeds, water, soil, waste, machinery and equipment. The Control Directive of 1998 aims to prevent spread and ensure eradication. It provides for the establishment of a demarcated zone whenever the pest is found, for measures to prevent spread from this zone and for measures to eliminate the pest within the zone.     

Identification and use of a wild plant with antimicrobial activity against Ralstonia solanacearum, the cause of bacterial wilt of potato

Fresh aerial tissue and roots of 14 wild plants in Okinawa prefecture were investigated for their antimicrobial activity against Ralstonia solanacearum , which causes bacterial wilt of potato. A 70% aqueous ethanol extract of fresh aerial tissue of Geranium carolinianum L. showed strong antimicrobial activity against R. solanacearum . This extract also showed antimicrobial activity against the pathogens causing common scab of potato and soil rot of sweet potato. The antimicrobial substance could be extracted with hot water, and was effective against R. solanacearum in soil. In the field test, a treatment combining incorporation of dried aerial tissue into the soil and solarization was highly effective for control of bacterial wilt of potato. These findings suggest that G. carolinianum L. could be used as a biological agent for the control of bacterial wilt of potato.     

番茄青枯病病原菌拮抗菌株的筛选及其田间防控作用研究

从番茄青枯病发病严重田块的健康植株根际土壤中分离筛选得到2株高效拮抗菌株,命名为W12和W118,经16SrDNA基因鉴定均属芽胞杆菌属;用PCR扩增的方法扩增脂肽类抗生素合成基因,结果表明W12和W118含有合成bacillomycin、iturin和fengycin三种抗生素的基因;将2株拮抗菌用于田间试验,结果表明混合菌株防控效果最好,3次灌菌后防控效果达到62.3%,单独施用菌株W118较单独施用W12防控效果好,3次灌菌后防控效果达到56.7%。     

An improved enrichment broth for the sensitive detection of Ralstonia solanacearum (biovars 1 and 2A) in soil using DAS–ELISA

A reliable, sensitive, low-cost and easy-to-use technique is described for the detection of Ralstonia solanacearum (the causal organism of bacterial wilt, BW) in soil. A total of 273 potato isolates belonging to five different biovars (Bv), originating from 33 countries worldwide, were tested and successfully detected by antibodies produced at the International Potato Center (CIP). Isolates of R. solanacearum belonging to Bv1 and Bv2A were successfully detected by double antibody sandwich–enzyme-linked immunosorbent assay (DAS–ELISA) at low population levels after incubation of soil suspensions for 48 h at 30°C in a new semiselective broth containing a potato tuber infusion. Detection thresholds of 20 and 200 CFU g⁻¹ inoculated soil were obtained for Bv1 and Bv2A, respectively. Sensitivity of detection of Bv2A was similar or even higher in five different inoculated soil types. No cross-reactions were obtained in DAS–ELISA after enrichment of soil suspensions (i) prepared from 23 different soils sampled in BW-free areas in six departments of Peru; and (ii) inoculated with 10 identified bacteria and 136 unknown isolates of soil microbiota isolated from eight different locations. Only the blood disease bacterium gave a low-level reaction after enrichment. In naturally infested soils, average sensitivities of 97·6 (SE 14·8) and 100·9 (SE 22·6) CFU g⁻¹ were obtained for biovars 1 and 2A, respectively. By making serial dilutions of the soil suspension before enrichment, densities of R. solanacearum could be determined in a semiquantitative way. Results also showed that composite samples of five soils could be analysed to assess field soil populations without reducing detection sensitivity.     

Rapid field detection of Ralstonia solanacearum in infected tomato and potato plants using the Staphylococcus aureus slide agglutination test

Ralstonia solanacearum is the major cause of bacterial wilt, which affects over 200 species of plants, many of economic importance. Current methods for detection and identification of the pathogen rely on isolation on semi-selective media followed by a combination of serological and molecular techniques and plant inoculation. Such methods are time-consuming, and require extensive laboratory facilities and skilled personnel. A reliable and rapid screening technique, which could be applied in the field, would reduce the number of samples submitted for laboratory testing and provide a vital component in disease control measures. A programme for the control of R. solanacearum has been introduced in Portugal and a Staphylococcus aureus slide agglutination test was used directly on tomato and potato plants in the field and on bacterial cultures under laboratory conditions. The results obtained show that this technique can play a major role in control programmes by providing a sensitive tool for the rapid detection of the pathogen.     

Structural changes of homogalacturonan,rhamnogalacturonan I and arabinogalactan protein in xylem cell walls of tomato genotypes in reaction to Ralstonia solanacearum

Healthy and Ralstonia solanacearum-inoculated tomato genotypes susceptible or resistant to bacterial wilt including recombinant inbred lines (RILs) deriving from a cross between the resistant genotype Hawaii7996 and the susceptible Wva700 were compared for symptom and bacterial population development, and for the composition and structure of pectic polysaccharides and arabinogalactan proteins (AGPs) of xylem cell walls by immunological staining of tissue prints. Constitutive differences were observed between resistant and susceptible RILs, with a higher degree of methyl-esterification of homogalacturonan (HG) detected by antibody JIM7 in the resistant plants. After inoculation, decreased methyl-esterification of HG indicated by stronger labeling with antibody JIM5 was observed in all susceptible genotypes and in five of eleven resistant genotypes, with a clear increase in the non-blockwise de-esterification pattern of HG (LM7) only in the susceptible lines, indicating the mode of action of the pectinmethylesterase of R. solanacearum. In the susceptible lines infection generally leads to increased branching of rhamnogalacturonan I indicated by the detection of arabinan (LM6) and galactan (LM5) side chains, and of arabinogalactan protein (LM2), while only few of the resistant genotypes reacted with changes in these epitopes. All the resistant, symptomless genotypes contained relatively high pathogen populations in stems. A clear relation between cell wall composition and degree of latent infection of resistant genotypes was not found.