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.     

Effect of cold-water irrigation on bacterial wilt pathogen of tomato

Bacterial wilt caused by Ralstonia solanacearum is one of the most devastating bacterial diseases of plants worldwide. Management of bacterial wilt in tomato and other crops has been difficult, and so novel but easily implemented control methods are being sought. To evaluate the effect of cold-water irrigation on bacterial wilt of tomato, four treatments were used in which CF (chemically fertilized) soil and CF + FYM (chemical fertilizer + farmyard manure [FYM]) soil were inoculated with a bacterial suspension (R. solanacearum strain YU1Rif43) at 10⁶ colony forming units (CFU) g^?1 soil. Tomato seedlings were grown in Agri-pots in a plant growth chamber. The soil was irrigated with water that was kept at the same temperature in each treatment: 4, 10, 20, or 30°C. Incidence and severity of wilt, counting of the colonies of the culturable population of pathogen, and dry-mass and height of the plants were examined. After 45 days and in both kinds of soil, most of the plants had wilted in soil irrigated at 30°C. Wilt incidence was substantially reduced when transplanted seedlings were irrigated at lower temperatures (4–20°C). Survival of R. solanacearum was also reduced after being irrigated with water at lower temperatures, indicating that the reduced incidence of wilt was linked to reduced survival of the pathogen. Dry-mass and plant height were slightly higher under control conditions than in soils irrigated at lower temperatures. This study suggests that cold-water irrigation could significantly reduce bacterial wilt of tomato and have an adverse effect on survival of the wilt pathogen.     

内生菌B47的定殖能力及其对番茄青枯病的防治作用

从番茄茎分离的内生枯草芽孢杆菌菌株B47对番茄青枯病有较好的防治作用,利用该菌株的抗链霉素突变菌株,研究其在土壤和番茄植株根、茎中的定殖能力及其对番茄青枯病的防治作用。结果表明,枯草芽孢杆菌菌株B47可在土壤和番茄植株中定殖。B47施到土壤中后的15~45天,其数量逐步增加,45天后,其数量逐步下降。B47在土壤中的定殖能力随土壤的种类和土壤的处理情况而异。施入菜地土后的第45天,B47在非灭菌土中的数量是9.91×105cfu/g土壤干重,而在灭菌土中的数量是9.84×107cfu/g土壤干重。接种后,番茄植株根和茎中的B47数量,从苗期到结果期逐渐增加,但到了成熟期呈下降趋势。B47和番茄青枯病菌混合施入土壤后,随B47的数量增加番茄青枯病菌的数量显著降低。当番茄植株根和茎中B47的含量分别为1.17×104cfu/g鲜重和3.33×104cfu/g鲜重时,接种番茄青枯病菌后的第20天,对番茄青枯病的防治效果达79.79%。     

Bacterial wilt-resistant tomato rootstock suppresses migration of <Emphasis Type="Italic">ralstonia solanacearum</Emphasis> into soil

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?<?LG?<?SC. These results show that grafting onto resistant rootstock strongly suppressed the migration of R. solanacearum into the soil by the time of full wilting, and the effect was stronger with a longer rootstock. Migration of R. solanacearum into soil increased with increasing disease severity in SC, LG and HG. These facts suggest that early uprooting of slightly infected plants could control the spread of the bacteria into the soil.     

Development of a specific molecular tool for the detection of epidemiologically active mulberry causing-disease strains of Ralstonia solanacearum phylotype I (historically race 5-biovar 5) in China

Ralstonia solanacearum causes bacterial wilt disease in many plant species, including mulberry. Here, we used a suppression subtractive hybridization (SSH) approach to identify specific DNA fragments in R. solanacearum race 5-biovar 5. The genome of the R. solanacearum M7 strain was subtracted from that of the GMI1000 strain, resulting in the identification of 85 subtracted fragments. The primer set MG67-F/R for identification of Ralstonia solanacearum race 5-biovar 5 strains was designed on the basis of the clone MG67 sequence. Furthermore, a multiplex PCR was developed by using the primer set MG67-F/MG67R in combination with the species-specific primer pair 759/760. A 156 bp r5-bv5-specific fragment, together with a 282 bp species-specific fragment, was amplified from all tested R. solanacearum r5-bv5 strains. The sensitivity of the multiplex PCR made it possible to detect concentrations as low as 10² CFU ml^?1 of pure culture. Moreover, the r5-bv5-specific multiplex PCR was successfully applied to detect Ralstonia solanacearum race 5-biovar 5 strains in diseased mulberry samples. Therefore, the multiplex PCR assay can be used as a reliable diagnostic technique to enable researchers to rapidly identify isolates of R. solanacearum race 5-biovar 5.     

Effect of calcium nutrition on resistance of tomato against bacterial wilt induced by Ralstonia solanacearum

This study investigated the effect of calcium nutrition on tomato bacterial wilt caused by Ralstonia solanacearum and the regulation of resistance mechanisms. Plants cultured in nutrient solution with calcium concentrations of 0.5, 5.0, and 25.0 mM, were inoculated with R. solanacearum by the root dip method. Severity of disease development, Ca concentration in tomato root and shoot tissues, hydrogen peroxide (H₂O₂) concentration, peroxidase (POD, EC 1.11.1.7) and polyphenol oxidase (PPO, EC 1.10.3.2) in tomato leaves were analyzed. Disease severities of low, medium and high Ca treatments were 100 %, 77.1 % and 56.8 % respectively. Plant growth in high Ca treatment was significantly better than those in low Ca treatment in height, stem diameter and biomass. Tomato plants absorbed significantly more Ca in roots and shoots as the level of Ca in the nutrient solution increased. In addition, H₂O₂ level in high Ca treatment rose faster and reached a higher peak with 10.86 μM gFW^?1(31.32 % greater than medium Ca plants). The activities of POD and PPO also have a greater increase in high Ca treatment with 99.09 U gFW^?1 and 107.24 U gFW^?1 compared to 40.70 U gFW^?1 and 77.45 U gFW^?1 in low Ca treatment. A negative correlation was found between Ca concentration, level of H₂O₂, POD, PPO in tomato, and disease severity, indicating that they played an important role in resistance of tomato to this disease. These results suggested that Ca was involved in the regulation of H₂O₂ concentration, and activity of POD and PPO in tomato.     

Fusarium solani f.sp.cucurbitae andF. oxysporum f.sp.niveum inoculum densities in tunisian soils and their effect on watermelon seedlings

Populations ofFusarium solani f.sp.cucurbitae (Fsc) andFusarium oxysporum f.sp.niveum (Fon) in naturally infested soil of watermelon fields were counted by the soil dilution method with subsequent pathogenicity tests. Inoculum density varied within the same region from one field to another, ranging between 9 and 1600 CFU g^?1 soil forFsc and from 0 to 200 CFU g^?1 soil forFon. Fusarium crown- and root-rot-diseased seedlings were observed in most soils (93%); however, Fusarium wilt was observed in only 34% of soil samples. The disease incidence on cv. ‘Giza’ (Y) increased significantly with inoculum density in the soil (X) (P<0.001). ForFsc, the relationship between inoculum density and disease incidence was characterized by the equation Y=0.0005X+0.165 (R²=0.67). ForFon, the equation was Y=0.003X?0.0014 (R²=0.88). Based on these equations, the estimated inoculum densities required to cause 50% disease incidence (DI₅₀) on cv. Giza plants was 670 and 171 CFU g^?1 soil forFsc andFon, respectively.     

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.     

Risks for infection of strawberry plants with an aerosolized inoculum of <Emphasis Type="Italic">Xanthomonas fragariae</Emphasis>

Xanthomonas fragariae is the causative agent of angular leaf spot of strawberry, a quarantine organism in plant propagation material in the European Union. Field experiments were conducted to assess the risks for infection of strawberry plants through dispersal of an aerosolized inoculum. In practice, pathogen aerosols can be formed during mowing of an infected crop or by water splashing on symptomatic plants during overhead irrigation or rain. In our experiments, aerosols were generated by spraying suspensions of X. fragariae with a density of 10⁸ cfu ml^?1 or water under pressure vertically up into the air. In strawberry plants (cv Elsanta) placed at 1.3, 5 and 10 m distance downwind from the spray boom, infections were found, as evidenced with a combination of dilution–plating and molecular techniques, but more frequently in plants wetted prior to inoculation than in plants kept dry. A logarithmic decrease in infection incidence was found with the distance to the inoculum source. Symptomatic plants were found up to 5 m distance from the inoculum source. No infected plants were found in plants placed 4 m upwind or treated with water. In glasshouse studies, it was shown that under conditions favorable for disease development, spray-inoculation of strawberry plants with estimated densities of X. fragariae as low as 2000 cfu per plant were able to cause symptoms both in cv Elsanta and cv Sonata. Results indicate that there is a considerable risk on infections of strawberry plants exposed to aerosolized inoculum.     

Challenging the larvae of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> and assessing the immune responses to nematode-bacterium complex

Helicoverpa armigera is a strong insecticidal resistance developed insect pest. The understanding of its innate immune responses to emerging biocontrol agent entomopathogenic nematode-bacterial complex can provide an opportunity to control this insect in an environmentally benign manner. Study was focused on role of hemocytes changes and PO activity in Steinernema abbasi-Xenorhabdus indica challenged larvae of H. armigera over the time. Total cell count changed effectively from 10.2?±?1.81?×?10⁵ to 15.5?±?3.3?×?10⁵ cells/mm³ upto 9 h and reduced distinctly up to 8.0?±?2.49?×?10⁵ cells/ mm³ in 24 h. PO activity inclined significantly and was recorded highest at 9 h (24.67?±?1.08?×?10² units) and lowest at 24 h (14.34?±?0.74?×?10² units) in total hemolymph with a similar pattern in plasma and the cellular fraction. Phenoloxidase activity in total and cellular component of hemolymph was positively correlated with prohemocytes, granulocytes and oenocytoids. Study showed the hemocytes and PO accounted as active immune responses against nematode infection. The results provide the first insight to understand the hemolytic activity, quick immunosuppression responses of S. abbasi-X. indica and vulnerability of H. armigera.     

Factors affecting the virulence of Ralstonia solanacearum and its colonization on tobacco roots

Tobacco bacterial wilt caused by Ralstonia solanacearum is a serious disease affecting tobacco cultivation in southwest China. The response surface methodology was employed to evaluate the optimal conditions of tobacco bacterial wilt, and green fluorescent protein gene (gfp) labelling was applied to monitor the location and survival dynamics of R. solanacearum (Rs::gfp) on tobacco roots and in soil under these optimal conditions. The results showed that the highest wilt incidence was 91.13%, which occurred when the population reached 6.6 × 10⁶ CFU/g soil, the temperature was 30.55 °C, and the humidity was >81.42%. The Rs::gfp densely colonized the root tips and root hairs, and cells of Rs::gfp were observed intermittently in the elongation zone or at the point of the emerging lateral roots. The Rs::gfp number in the rhizosphere soil was 10.75‐, 73.13‐ and 74.86‐times higher than that in the bulk soil at 10, 15 and 20 days after transplantation, respectively. Increased colonization by Rs::gfp was related to the population of the pathogen, the environmental temperature and the humidity in the soil. These three conditions determined whether R. solanacearum would induce tobacco wilt. This is the first study to investigate factors affecting the virulence of a tobacco wilt bacterial pathogen, which is important for conducting field diagnosis and biocontrol of tobacco bacterial wilt.     

Recovery of Ralstonia solanacearum from canal water in traditional potato-growing areas of Egypt but not from designated Pest-Free Areas (PFAs)

Surveys over three seasons of irrigation, drainage and artesian well water throughout the major potato-growing areas of Egypt indicated that Ralstonia solanacearum bv. 2 race 3 (phylotype II sequevar 1), cause of potato brown rot, was limited to the canals of the traditional potato-growing areas in the Nile Delta region, with positive findings more commonly associated with the network of smaller irrigation canals flowing through potato-growing areas. Pathogen populations in the canals of the Delta (~100–200 cfu l^?1) were generally variable throughout the year with presence linked to potato cultivation in the immediate area. The pathogen was not detected in irrigation or drainage water associated with potato cultivation in the newly reclaimed desert areas (designated as Pest-Free Areas, PFAs) or in the main branches of the Nile upstream from these areas. In vitro studies showed that temperature and microbial activity were the main factors affecting survival of the pathogen in canal water. In experiments at temperatures of 4, 15, 28 and 35°C, survival was longest at 15°C and shortest at 35°C. Survival at 4 and 28°C tended to be intermediate between these extremes as was survival when the bacterium was grown at fluctuating temperatures. Aeration, solarisation and pH variation between 4 and 9 appeared to have little effect on survival. Survival in autoclaved or filter-sterilised canal water was longer than in untreated water irrespective of other factors with survival times exceeding 300 days at 15°C in some experiments. Evidence is presented indicating that survival in water-saturated sediment may be longer than in the overlying water suggesting that sediment may provide a protective niche for the pathogen in some circumstances. The maximum survival time in non-sterile Egyptian canal water at high inoculum pressure was estimated to be up to 300 days at optimum temperature for survival (15–30°C) suggesting the potential for long-distance spread in Egyptian surface waters from sources of contamination.     

Compost enhances plant resistance against the bacterial wilt pathogen Ralstonia solanacearum via up-regulation of ascorbate-glutathione redox cycle

The interactions between the pathogen Ralstonia solanacearum and potato Solanum tuberosum plants were studied to investigate the reactive oxygen species metabolic system and ascorbate (ASC)-glutathione (GSH) redox cycle in response to compost application. Single potato eyepieces were germinated and grown in pots containing sandy soil with or without compost at a rate of 7.5 g kg^?1 soil. Non-compost- and compost-treated plants (CTP) were inoculated with R. solanacearum 25 days after planting and then analyzed after 10 days, unless otherwise stated. The present results revealed that pathogen infection caused a remarkable decrease in plant growth related parameters and productivity and an increase in disease incidence. However, under these conditions compost had substantially improved plant growth and decreased disease incidence and bacterial population. R. solanacearum resulted in significant enhancement in the activities of NADPH oxidase, lipoxygenase, the production rate of superoxide and hydroxyl radicals, levels of hydrogen peroxide, membrane lipid peroxidation, and protein oxidation indicating the induction of oxidative stress in potato roots. However, the pathogen-mediated enhancement in indices of oxidative stress was considerably decreased by compost application, which enhanced the activities of ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) in infected potato plants, implying a better ROS-scavenging activity. Data also indicated that there were general increases in ASC and GSH content in infected compost treated plants, but non-compost treated ones significantly had lower levels of such redox metabolites. In addition, significantly higher ratios of ASC/DHA (dehydroascorbate) and GSH/GSSG (glutathione disulphide) were generally found in CTP than in non-compost treated-ones. The obtained results suggest that compost provides effective protection against the Ralstonia bacterial pathogen via up-regulation of the capacity of the ASC-GSH cycle and modulation of the cellular redox status, thereby eliminating ROS damage and sustaining membrane stability.     

Specific Detection of Biovars of Ralstonia solanacearum in Plant Tissues by Nested-PCR-RFLP

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

A novel strategy to reduce overwintering inoculum of Monilinia laxa

Brown rot on cherry and plum, caused by Monilinia laxa, is an important disease, for which overwintered mummified fruit is a significant inoculum source for infection of flowers and fruit in the spring. Experiments were conducted to assess the potential of applying plant protection products in winter and/or early spring to suppress sporulation on mummified fruit. Products tested included Indar 5 EW (a commercial fungicide, a.i. fenbuconazole), Aureobasidium pullulans Y126 [a candidate biocontrol agent (BCA)]), Bacillus sp. B91 (a candidate BCA) and Serenade (a commercially formulated BCA of Bacillus subtilis strain QST 713) in addition to control treated with tap water. Indar and A. pullulans Y126 significantly reduced sporulation when applied once in winter. Overall, a single treatment in early spring (February) was slightly more effective than single treatment in winter (November). Application of all products in both winter and early spring led to significant reduction in sporulation. Indar had the highest efficacy, reducing the number of spores from 9?×?10⁵ to 5?×?10³ per mummified plum when applied twice. Of the three BCAs applied on both occasions, A. pullulans Y126 had the highest efficacy, reducing the number of spores from 9?×?10⁵ to 5?×?10⁴ per mummified plum. There were no synergistic but additive effects between the two applications in winter and early spring based on the Bliss independence test. These results suggest that reducing overwintering inoculum in dormant season is effective and may be part of an integrated management strategy for brown rot on stone fruit.     

Magnesium oxide nanoparticles induce systemic resistance in tomato against bacterial wilt disease

Bacterial wilt is a serious problem affecting many important food crops. Recent studies have indicated that treatment with biotic or abiotic stress factors may increase the resistance of plants to bacterial infection. This study investigated the effects of magnesium oxide nanoparticles (MgO NP) on disease resistance in tomato plants against Ralstonia solanacearum, as well as its antibacterial activity. The roots of tomato seedlings were inoculated with R. solanacearum and then immediately treated with MgO NP; the treated plants showed very little inhibition of bacterial wilt. In contrast, when roots were drenched with a MgO NP suspension prior to inoculation with the pathogen, the incidence of disease was significantly reduced. Rapid generation of reactive oxygen species such as O₂^?˙ radicals was observed in tomato roots treated with MgO NP. Further O₂^?˙ was rapidly generated when tomato plant extracts or polyphenols were added to the MgO NP suspension, suggesting that the generation of O₂^?˙ in tomato roots might be due to a reaction between MgO NP and polyphenols present in the roots. Salicylic acid‐inducible PR1, jasmonic acid‐inducible LoxA, ethylene‐inducible Osm, and systemic resistance‐related GluA were up‐regulated in both the roots and hypocotyls of tomato plants after treatment of the plant roots with MgO NP. Histochemical analyses showed that β‐1,3‐glucanase and tyloses accumulated in the xylem and apoplast of pith tissues of the hypocotyls after MgO NP treatment. These results indicate that MgO NP induces systemic resistance in tomato plants against R. solanacearum.     

Prevention of pine wilt disease induced by Bursaphelenchus xylophilus and Monochamus galloprovincialis by trunk injection of emamectin benzoate

The pine wood nematode Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle is the causal agent of pine wilt disease. We evaluated the efficacy of emamectin benzoate (EB) for preventing wilt disease in the field and its effect on the vector Monochamus galloprovincialis (Olivier) (Coleoptera: Cerambycidae). Four experimental plots were delimited in a maritime pine (Pinus pinaster Aiton) forest in Portugal. Trunk-injection trials with EB included three dose-rates: 0.032 g a.i. cm^?1 diameter at breast height—DBH, n?=?75 trees; 0.064 g a.i. cm^?1 DBH, n?=?75 trees; and 0.128 g a.i. cm^?1 DBH, n?=?50 trees; along with an untreated control plot (n?=?75 trees). EB was successfully injected and translocated in pines at an effective concentration. None of the treated trees died after a period of 26 months, contrasting with a 33% mortality of non-treated pines. Analysis of residues successfully detected EB in branches of treated pines, with the quantity increasing relative to the injection dose rate, and was found to have a clear effect on the longevity and feeding activity of adult M. galloprovincialis feeding on branches. EB was efficient in preventing wilt disease and bark beetle attacks in the terrain, and its application by trunk injection is a new option for wilt disease management programs in Portugal and in Europe.     

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.     

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.     

Molecular epidemiology of Ralstonia solanacearum strains from plants and environmental sources in Portugal

Several outbreaks of bacterial wilt disease caused by the quarantine bacterium Ralstonia solanacearum were identified in Portugal. Intensive surveys recognized the bacterium as endemic in the main irrigated agricultural ecosystems. Between 1999 and 2006 all isolates of R. solanacearum were characterized as belonging to biovar 2A. In 2007, biovar 1 strains were recorded in potato fields under a confined area. A panel of 101 Portuguese isolates obtained from biotic and environmental samples was studied. Following a polyphasic approach, these isolates were analysed by SDS-PAGE of the whole cell proteins, MSP-PCR (csM13), rep-PCR (BOXA1R and ERIC-2) and FAFLP (EcoRI?+?0/MseI?+?C). A 750?bp sequence of endoglucanase (egl) gene was studied for 17 representative isolates and 95 accessions retrieved from the GeneBank. Numerical analysis of protein profiles correlated quite well with biovar subphenotype, producing a unique megacluster (r?=?71.1%). MSP-PCR was more discriminative (r?=?62%). Rep-PCR approaches displayed higher polymorphism levels with ERIC 2 primer producing high diversity indexes (D and J′). FAFLP was the most reproducible method (95%) displaying 229 polymorphic characters and the highest evenness (J′). For all the methods small clusters disclosed a clonal origin for isolates with a common geographical origin/matrix. FAFLP identified an adaptative microevolution phenomenon for surface water strains. Polyphasic approach congruence highlighted the inability of individual methods to explain the whole diversity. Mr. Bayes egl-based phylogenetic tree allocated the 17 Portuguese isolates into the sub-clusters of narrow (nhr) and broad host range (bhr) of Phylotype II unveiling the epidemiological story of R. solanacearum in Portugal and identified different populations coexisting in the same habitats. This is the first report of the presence of R. solanacearum Phylotype II, bhr strains in Western Europe.