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Glasshouse experiments were conducted to study infection and disease development in rockwool-grown rose plants inoculated with Ralstonia pseudosolanacearum. A R. pseudosolanacearum strain isolated from rose plants was more aggressive than strains from anthurium or curcuma. The three rose cultivars tested, Avalanche, Red Naomi, and Armando, differed in susceptibility. At 20°C, the rose strain caused hardly any symptoms over a 6-week period, whereas at 28°C typical wilt symptoms were observed within 2 weeks after stem inoculation of Armando, the most susceptible cultivar. Inoculating roots with the rose strain resulted only in weak atypical symptoms. Nevertheless, inoculating roots of cv. Armando at a relatively low inoculum dose of 104 cfu/ml led to high densities in the base of stems in one out of two experiments. R. pseudosolanacearum occasionally spread from stem inoculated plants with symptoms in rockwool slabs. This limited spread resulted in a low infection incidence, and only of plants directly adjacent to the plants with symptoms.

Ralstonia solanacearum causes a lethal bacterial wilt disease in many plants by colonizing the vascular tissues of the hosts. Upon inoculation of tomato seedlings through either leaf or root, the wilting symptoms occur first at the apical region and then proceed downward along the shoot. The systemic order of the disease initiation and progression in the host, independent of the site of pathogen inoculation, is yet to be investigated. To understand the disease progression more clearly, we have carried out a systematic study of the pathogen localization by GUS staining of inoculated tomato seedlings, at 24‐hour intervals from 0 days post‐inoculation (dpi) to 5 dpi. In both inoculation methods, pathogen colonization was observed at 1 dpi at the apical meristem as well as the cotyledon leaves, where the disease initiates. As the disease progressed, colonization by the pathogen towards the lower region of the shoot was observed. Disease consistency and pathogenicity magnitude were observed to be higher using the leaf inoculation method than the root inoculation method. Several R. solanacearum transposon‐induced mutants that were reduced in virulence by root inoculation but virulent by leaf inoculation were obtained. Using GUS staining, it was observed that these mutants were unable to localize in the shoot region when inoculated in the root. Our study indicates that the apical meristem and the cotyledon leaves are the first regions to be colonized in inoculated tomato seedlings, which might explain the disease initiation from this region.

Moko disease, caused by the bacterium Ralstonia solanacearum, is one of the most devastating diseases of Musa spp. in Colombia, where banana and plantain are major crops. The disease epidemiology is poorly understood and little is known about the diversity of the bacterial populations associated with this disease. This study assessed the diversity, phylogenetic relationship and pathogenicity of R. solanacearum strains associated with moko disease in Colombia. For this, the genetic diversity of 65 isolates obtained from four banana/plantain‐growing regions was evaluated by using multiplex PCR and analysing the partial sequences of the mutS, rplB and egl genes. These analyses revealed that all the strains belonged to the R. solanacearum phylotype II, sequevars 4 and 6. In addition, the phylogenetic analysis assorted the strains into three subgroups, which matched the region of isolation: (i) central region (i.e. Eastern plains and Andes, IIB/4); (ii) northwest (i.e. Uraba and a few strains from Magdalena, IIB/4); and (iii) north coast (Magdalena and a few strains from Uraba, IIA/6). In addition, this evolutionary pattern was associated with pathogenicity, as 63 of the 65 isolates caused wilting of banana and plantain plants under greenhouse conditions, whilst only 32, those isolated from the central region, caused such symptoms in tomato plants. In conclusion, this study shows that banana and plantain crops in Colombia foster genetically diverse strains of R. solanacearum that belong to at least three different genetic groups, which show biogeographic and host range association.

Bacterial wilt, caused by Ralstonia solanacearum, is a devastating soilborne disease in plants that limits the production of many crops worldwide. Although management of bacterial wilt has so far been unsuccessful, enhancing host resistance to the pathogen may be an effective control strategy. Recently, magnesium oxide (MgO) was found to induce defence responses against R. solanacearum in tomato plants. Here, the mechanisms underlying MgO‐induced defence responses against R. solanacearum (MgO‐i DARS) were investigated using Arabidopsis thaliana as a host plant. MgO‐i DARS was confirmed in A. thaliana mutants deficient in jasmonic acid or ethylene signalling pathways as well as in the wildtype (Col‐0) plants. In contrast, no MgO‐i DARS was found in A. thaliana mutants deficient in the salicylic acid (SA) production (sid2‐2) and signalling pathways (tga1‐1 and npr‐1). MgO treatment led to significant accumulation of SA in both roots and shoots of Col‐0. The SA biosynthesis gene isochorismate synthase 1 (ICS1) was induced in roots and shoots of A. thaliana treated with MgO. An NADPH oxidase gene respiratory burst oxidase homolog D (AtRbohD) was up‐regulated in both roots and shoots of Col‐0 treated with MgO. No MgO‐i DARS was observed in A. thaliana mutants deficient in AtRbohD. These results suggest that SA and RBOHD‐mediated ROS are pivotal for MgO‐i DARS in A. thaliana.

As a major component of the cell wall, lignin has been suggested to play an important role in plant defense response to various pathogens. However, how lignin is involved in plant pathogen interaction is still unclear. Here, we cultivated a series of transgenic tobacco lines with a range of differences in lignin content and composition. Evaluation of pathogen resistance in these plants indicated that lower total lignin content aggravated the severity of tobacco black shank and bacterial wilt diseases, while increased sinapyl lignin (S) alleviated the disease symptoms. The regression analysis indicated both lignin content and S lignin were positive correlation with the disease resistance, particularly these two factors had additive effects and they exhibited the more strong correlation with disease resistance when they combined together. Neither guaiacyl lignin (G) nor S/G ratio showed close correlation with disease resistance. The expressions of pathogenesis-related protein genes PR2 and PR3 were induced after pathogen inoculation. But the up-regulation of PR2 and PR3 did not associated to disease resistance with lignin. These data collectively suggest that both total lignin content and S lignin are main factors that contribute to basic defense response in tobacco. This article is protected by copyright. All rights reserved.

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.

Bacterial wilt, caused by Ralstonia solanacearum, is emerging as a major threat to potato production in Ethiopia, reaching epidemic proportions in the Chencha district recently, with a prevalence of 97% of potato fields in 2015. The recent disease outbreak in the district coincided with a significant introduction of seed potatoes. This research was therefore initiated to genetically characterize the pathogen so as to trace its source, identify its relationship with outbreaks in the rest of the country, and make intervention recommendations. Ralstonia solanacearum isolates were sampled both from seed and ware potato fields in Chencha and from seed potato fields in production regions suspected of being potential sources of the pathogen. Multiplex PCR and phylogenetic analysis of partial endoglucanase gene sequences identified all of the isolates as phylotype IIB sequevar 1. VNTR sequence analysis distinguished 11 different haplotypes, nine of which were unique to the Chencha district. However, one of the haplotypes was common to all seed potato producer regions of Ethiopia except for the Shashemene area. The unique and diverse VNTR haplotypes of the pathogen in Chencha indicates that it is well established in the district. When a geographical map of the VNTR haplotypes was superimposed with the main cross-regional seed potato distribution pattern of the country, it became evident that the pathogen was being disseminated via latently infected seed from the Holeta-Jeldu area in the Central Highlands of Ethiopia. Identification of largely uninfected highland districts and multiplication of high-grade seed potato exclusively in those districts should be given priority.

Ralstonia solanacearum sensu lato causes bacterial wilt in many economically important agronomic crops and tree species. Eucalyptus is one of the recorded hosts but, despite the losses to forestry, little is known regarding the disease. The aim of this study was to identify the Ralstonia species that are associated with bacterial wilt of Eucalyptus trees growing in different parts of the world. Phylotyping, sequevar and multilocus sequence analysis were used to identify and determine the genetic diversity of the available isolates. The isolates obtained from Africa and Asia grouped in phylotype I, and those isolates from South America in phylotype II. Following the recent reclassification of R. solanacearum sensu lato, these results show that there are two Ralstonia species associated with bacterial wilt of Eucalyptus, namely, R. solanacearum and R. pseudosolanacearum. The isolates from South America represented R. solanacearum, while those from Africa and Asia were R. pseudosolanacearum. Analysis of the endoglucanase (egl), transcriptional regulator (hrpB) and DNA mismatch repair (mutS) gene regions supported this finding. In addition, one previously unidentified sequevar was identified using partial endoglucanase (egl) gene sequence analysis of those isolates obtained from Colombia. This study raises questions regarding the aetiology of the two Ralstonia species found on Eucalyptus, their importance relating to quarantine, potential impact and management of the disease with which they are associated.

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.

International audience; Bacterial wilt caused by Ralstonia solanacearum is a destructive disease for many crops. The aim of this study was to investigate the phylogenetic relationships and genetic structure of an R.solanacearum population from diverse origins in Taiwan. All 58 tested isolates belonged to phylotype I, except the two biovar 2 isolates from potato. These belonged to phylotype IIB sequevar 1 and were identical to known potato brown rot strains, which were probably introduced. Phylotype I isolates were grouped into 10 sequevars. Sequevar 15 was predominant (34 out of 56 isolates). Its distribution covered the whole island and it was largely associated with solanaceous crops such as tomato, and with tomato field soil. A total of 14 haplotypes were identified based on a partial endoglucanase gene sequence. Parsimony network analysis revealed that haplotype A was the oldest haplotype in the local population. It encompassed all but one of the sequevar 15 isolates. Large variation in virulence on tomato was observed among the 58 isolates, and seven pathotypes were identified. Significant genetic differentiation was detected among pathotypes. Moreover, genetic differentiation was detected between biovar 3 and biovar 4 subgroups and between the strains associated with solanaceous and non-solanaceous species, but none was detected between strains from different geographic origins. The results suggest that the phylotype I population in Taiwan is homogeneous, while mutation and local adaptation to specific ecological niches keep shaping the population.

Bacterial wilt caused by Ralstonia solanacearum is a serious disease of peanut (Arachis hypogaea) in China. However, the molecular basis of peanut resistance to R. solanacearum is poorly understood. Arachis duranensis, a wild diploid species of the genus Arachis, has been proven to be resistant to bacterial wilt, and thus holds valuable potential for understanding the mechanism of resistance to bacterial wilt and genetic improvement of peanut disease resistance. Here, suppression subtractive hybridization (SSH) and macroarray hybridization were employed to detect differentially expressed genes (DEGs) in the roots of A. duranensis after R. solanacearum inoculation. A total of 317 unique genes were obtained, 265 of which had homologues and functional annotations. KEGG analysis revealed that a large proportion of these unigenes are mainly involved in the biosynthesis of phytoalexins, particularly in the biosynthetic pathways of terpenoids and flavonoids. Subsequent real-time polymerase chain reaction (PCR) analysis showed that the terpenoid and flavonoid synthesis-related genes showed higher expression levels in a resistant genotype of A. duranensis than in a susceptible genotype, indicating that the terpenoids and flavonoids probably played a fundamental role in the resistance of A. duranensis to R. solanacearum. This study provides an overview of the gene expression profile in the roots of wild Arachis species in response to R. solanacearum infection. Moreover, the related candidate genes are also valuable for the further study of the molecular mechanisms of resistance to R. solanacearum.

The effect of plant age at the time of inoculation on the severity of bacterial wilt and canker disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm) was examined in six greenhouse experiments. The period during which inoculations led to wilt and death of tomato plants was defined. This period, designated ‘window of vulnerability’, ranged from transplanting to the 17- to 18-leaf stage. Plants inoculated after this period expressed disease symptoms but did not wilt or die. No significant changes in disease incidence were observed when leaves of different ages were inoculated. Yield accumulation was significantly reduced in plants inoculated within the window of vulnerability compared with those inoculated after this period. Expression of virulence genes, viz. celA, encoding a secreted cellulase, and the serine protease-encoding pat-1, chpC and ppaA, was induced during the early stages after inoculation in plants inoculated within the window of vulnerability. Differences in Cmm population between plants inoculated within and outside of this period were insignificant after the first week post-inoculation, indicating that differences in disease severity, yield loss and expression of virulence determinants are not correlated with Cmm population level. Results suggest that implementation of precautionary measures during the window of vulnerability to avoid secondary spread of Cmm will have a season-long effect on plant mortality and may minimize, or even prevent, yield losses.

A specific and rapid diagnostic tool has been developed to detect Xanthomonas campestris pv. musacearum, the causal agent of bacterial wilt of banana. PCR primers were developed from intergenic regions of X. campestris pv. musacearum following its partial sequence. A total of 48 primers were tested for specificity to X. campestris pv. musacearum strains collected from various regions in Uganda. These were also tested for specificity against related Xanthomonas species from the vasicola group, Xanthomonas species pathogenic to other crops, and against those existing saprophytically on banana plants. Seven primer sets (Xcm12, Xcm35, Xcm36, Xcm38, Xcm44, Xcm47 and Xcm48) were found to be very specific to X. campestris pv. musacearum. These primer sets directed the amplification of the expected product for all 52 strains of X. campestris pv. musacearum collected from different locations in Uganda. No amplification products were obtained with unrelated phytopathogenic bacteria or endophytic/epiphytic bacteria from banana. A detection limit of 103 CFU mL−1 corresponding to about four cells per PCR reaction was observed when X. campestris pv. musacearum cells were used for all the seven primer sets. The DNA samples from symptomless plant tissues also tested positive with primer set Xcm38. The specific PCR method described here is a valuable diagnostic tool which can be used to detect the pathogen at early stages of infection and monitor disease.

Bacterial wilt of forage grasses, caused by the pathogen Xanthomonas translucens pv. graminis (Xtg), is a major disease of forage grasses such as Italian ryegrass (Lolium multiflorum). The plant genotype-bacterial isolate interaction was analysed to elucidate the existence of race-specific responses and to assist the identification of plant disease resistance genes. In a greenhouse experiment, 62 selected plant genotypes were artificially inoculated with six different bacterial isolates. Significant differences in resistance were observed among L. multiflorum genotypes (P 0·05) could be observed using linear regression modelling. However, additive main effects and multiplicative interaction effects (ammi) analysis revealed five genotypes which did not cluster close to the origin of the biplot, indicating specific interactions between these genotypes and some bacterial isolates. Simple sequence repeat (SSR) markers were used to identify marker-resistance associations using the same plant genotypes and bacterial isolates. The SSR marker NFA027 located on linkage group (LG) 5 was significantly associated with bacterial wilt resistance across all six bacterial isolates and explained up to 37·4% of the total variance of AUDPC values. Neither the inoculation experiment nor the SSR analyses revealed major host genotype-pathogen isolate interactions, thus suggesting that Xtg resistance, observed so far, is effective across a broad range of different bacterial isolates and plant genotypes.

The detection of Ralstonia solanacearum (biovar 2A) in stems of symptomless plants before harvest of the potato crop, instead of tubers, would not only save highly valued planting material but would be less time-consuming and would also enhance farmers’ market decisions. Although pathogen detection in stems has been proven efficient for ring rot, this has never been investigated for bacterial wilt (BW). Therefore the possibility of detecting BW latent infection in stem pieces about three weeks before harvest was assessed in 57 fields of the Andean highlands of Peru. Two sensitive, specific and user-friendly serological methods were used to detect the pathogen in latently infected tubers and stems: double-antibody sandwich (DAS)-ELISA and indirect ELISA on nitrocellulose membrane (NCM) after enrichment of the plant extracts in a semi-specific broth. Optimum sample sizes of stems and tubers were evaluated for 37 potato crops showing between 0 and 0·1% BW incidence using a binomial distribution model to calculate the detection probabilities. Although results of detection using the two serological techniques had 100% concordance, detection probabilities were higher using DAS-ELISA, whatever the plant part tested. BW detection probabilities were higher for tubers than for stems; a 99% detection probability was obtained by analysing 400 stems sections or 250 tubers using DAS-ELISA. Detection of BW infection in symptomless plants 20 days before harvest using post-enrichment DAS-ELISA is a reliable and user-friendly technique that can easily be used by national plant protection services and seed programmes in developing countries.

A new selective medium (APCA medium) was developed for the isolation of Burkholderia caryophylli, the causal agent of carnation bacterial wilt, from both plants and soil. The optimal concentration and combination of antibiotics was investigated to determine the most selective condition for growing B. caryophylli. The resultant composition of the medium per litre was: 0·79 g (NH4)2SO4, 1·0 g KH2PO4, 0·5 g MgSO4 · 7H2O, 0·2 g KCl, 2·0 g D-arabinose, 5 mg crystal violet, 50 mg cycloheximide, 50 mg polymyxin B sulphate, 50 mg ampicillin sodium, 10 mg chloramphenicol, 25 mg blue tetrazolium, and 15 g agar. Plating efficiency ranged from 119 to 174% with an average of 141% compared to that of nutrient agar. The bacterium was successfully isolated from contaminated soil and plant tissues with this medium. Moreover, the medium almost completely inhibited the growth of other plant pathogenic bacteria and soil saprophytes. This selectivity was high enough to detect B. caryophylli in contaminated soil.

The diversity of 40 strains of Ralstonia solanacearum causing bacterial wilt of potato in the major potato-growing areas of Iran was assessed. Based on rep-PCR genomic fingerprinting, strains fell into two distinct groups. The first group contained 37 of the 40 strains and the second consisted of three strains from a narrow tropical region in Iran. The three strains from the narrow tropical region were found to be phenotypically and genotypically most similar to R. solanacearum biovar 2T strains, whereas all other strains were phenotypically and genotypically identified as being R. solanacearum biovar 2/race 3. Phylogenetic analysis of endoglucanase gene sequence information of two of the strains from the tropical region revealed that they belonged to phylotype II of the R. solanacearum species complex and had 100% sequence similarity to a biovar 2T strain from potato in Peru. This is the first report of the presence of R. solanacearum phylotype II/biovar 2T in Iran and the first report of the existence of this group of R. solanacearum outside South America.

Ralstonia solanacearum strain OE1-1 causes bacterial wilt on tobacco plants. The popA-mutant 31b, derived from OE1-1 by insertion of transposon Tn4431, did not cause wilt on tobacco plants inoculated through the roots. However, when 31b was directly inoculated into xylem vessels, the tobacco plants wilted, similarly to those inoculated with OE1-1. 31b retained its exopolysaccharide productivity and its type-III secretion function. Furthermore, 31b grew in intercellular spaces and systemically infected tobacco plants, similarly to OE1-1. popA consists of an operon with popB and popC, and suppression of popB and popC expression resulting from polar mutation by transposon insertion did not affect the virulence of 31b. The mutated popA (popA31b) was composed of 960 nucleotides, including 39 derived from Tn4431. A recombinant mutant from OE1-1, where popA31b was introduced by marker exchange, showed the same phenotype as 31b. PopA31b protein was extracellularly secreted by 31b co-cultured with Arabidopsis thaliana. These results suggest that PopA31b extracellularly secreted by 31b in intercellular spaces may be implicated in suppression of disease development, leading to inability of the bacteria to induce wilt on plants. Taken together, interactions between host plants and R. solanacearum existing in intercellular spaces immediately after invasion may be involved in disease development.

When the biocontrol agent Pythium oligandrum (PO) colonizes the rhizosphere, it suppresses bacterial wilt disease in tomato (Solanum lycopersicum cv. Micro-Tom) caused by Ralstonia solanacearum, and a homogenate of its mycelia exhibits elicitor activity, inducing an ethylene (ET)-dependent defence response in Micro-Tom. Since salicylic acid (SA) and jasmonic acid (JA) play an important role in plant defence responses to pathogens, the involvement of SA- and JA-dependent signal transduction pathways in resistance to R. solanacearum was investigated in tomato roots treated with a mycelial homogenate of PO. Bacterial wilt disease was also suppressed in tomato cv. Moneymaker treated with the PO homogenate. However, the SA-inducible PR-1(P6) gene was not up-regulated in either Micro-Tom or Moneymaker. SA did not accumulate in homogenate-treated roots in comparison with distilled water-treated controls, even 24 h after inoculation. Induced resistance against R. solanacearum was not compromised in SA-non-accumulating NahG transgenic plants treated with the PO homogenate. On the other hand, the expression of the JA-responsive gene for the basic PR-6 protein was induced in both tomato cultivars treated with the PO homogenate. Furthermore, quantitative disease assays showed that the induced resistance against R. solanacearum was compromized in PO homogenate-treated jai1-1 mutant plants defective in JA signalling. These results indicated that the JA-dependent signalling pathway is required for PO-induced resistance against R. solanacearum in tomato.

Fifty-nine Ralstonia solanacearum isolates from diverse crops and regions were collected and characterized to determine the distribution and diversity of this soilborne pathogen in Guatemala. Three distinct types were present: a phylotype I, sequevar 14 strain, probably originating from Asia, infecting tomatoes and aubergines at moderate elevations; a phylotype II, sequevar 6 strain of American origin causing Moko disease in lowland banana plantations; and a phylotype II, sequevar 1 (race 3 biovar 2) strain causing brown rot on potatoes, Southern wilt of Pelargonium spp. and bacterial wilt of greenhouse tomatoes at high elevations. These data on strain diversity will inform effective regional efforts to breed for wilt resistance. A sensitive enrichment method did not detect the pathogen in fruits from naturally infected commercial tomato plants in Guatemalan fields and greenhouses, although it was detected in 6% of fruits from a wilt-resistant hybrid. Low numbers of R. solanacearum cells were also infrequently detected in fruits from plants artificially inoculated in the growth chamber with either race 3 biovar 2 or a phylotype II tomato strain.

A semiselective medium was developed for isolating Xanthomonas campestris pv. musacearum (Xcm) from infected banana plants, soil and insect vectors. The new medium was named cellobiose-cephalexin agar (CCA) and it contained (L−1): 1 g yeast extract, 1 g glucose, 1 g peptone, 1 g NH4Cl, 1 g MgSO4·7H2O, 3 g K2HPO4, 1 g beef extract, 10 g cellobiose, 14 g agar, 40 mg cephalexin, 10 mg 5-fluorouracil and 120 mg cycloheximide. The medium was evaluated for selectivity using 21 bacterial isolates and for plating efficiency using Xcm. The bacterial isolates included a soilborne Xanthomonas species and three pathogenic Xanthomonas strains that infect cassava, cabbage and beans. Although the plating efficiency of Xcm on CCA was lower (59%) than on non-selective yeast extract peptone glucose agar (YPGA), its selectivity was significantly higher, averaging 60 and 82%, when isolating from banana fruits and soil, respectively. CCA was also superior when isolating Xcm from insect vectors, with selectivity of 48–75%, compared with 8–17% on YPGA. Xanthomonas campestris pv. phaseoli did not grow on CCA, while X. campestris pv. campestris and X. axonopodis pv. manihotis grew, but their colonies were smaller than those of Xcm. Twenty-nine out of 33 suspected Xcm strains isolated from plants, soil and insects using CCA were pathogenic when inoculated onto banana plants, indicating that CCA can be a reliable tool in isolating Xcm populations. The medium should prove useful in studies on ecology, epidemiology and management of the banana bacterial wilt pathogen that is currently ravaging bananas in East and Central Africa.

Pythium oligandrum (PO) is a mycoparasite on a wide range of fungi and suppresses diseases caused by fungal pathogens when colonizing the rhizosphere. PO and its cell wall proteins (CWPs) have elicitor activity that induces defence responses in plants. The potential of a mycelial homogenate of PO to suppress bacterial diseases was studied in roots of tomato (Lycopersicon esculentum cv. Micro-Tom) inoculated with Ralstonia solanacearum. PO-treated plants showed enhanced resistance to R. solanacearum and reduction in severity of wilt symptoms. As ethylene often acts as one of the signal molecules for induced resistance, its production following treatment of tomato roots with the mycelial homogenate or CWP of PO was measured. The level of ethylene in PO- and CWP-treated plants was transiently elevated six- to 11-fold at 4–8 h after treatment, followed by high expression of three basic ethylene-inducible defence-related genes (PR-2b, PR-3b and PR-5b). Analysis of PR-5b gene expression in the leaves of PO- and CWP-treated plants suggested that PR gene expression was induced systemically. The expression of LeERF2 and LeETR4, which confer an ethylene-dependent signalling pathway, was also significantly accelerated by such treatments. These results indicate that PO has the potential to control bacterial wilt disease and that CWP may play an important role in the induction of resistance to R. solanacearum accompanying the activation of the ethylene-dependent signalling pathway.

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.

Several seed extraction procedures, used for detection of Clavibacter michiganensis ssp. michiganensis (Cmm) in naturally infected and artificially infested tomato seed lots were evaluated. Extraction methods that included grinding the seeds were significantly better at detecting the pathogen in three different seed lots than methods that used only soaking. The detection threshold of Cmm in relation to seed sample size was determined by adding naturally infected seeds into samples of three different sizes. Cmm was detected by agar plating assay, on three media (CNS, mSCM, D2ANX), and by direct PCR from seeds and Bio-PCR (bacteria cultured on agar media prior to PCR). In samples of 10 000 seeds containing one infected seed, Cmm could be detected only by Bio-PCR and in only one replicate out of five. In samples containing five or 10 infected seeds per 10 000 seeds, three of five and five of five replicates, respectively, were detected by the three detection methods. In samples of 5000 seeds, one infected seed could be detected in all five replicates only after adding a concentration step. A high correlation (R2 = 0·9448) between artificially infested seeds and the disease incidence was found. Seed lots infested with less than 58 colony-forming units (CFU) per g did not cause disease under glasshouse conditions, whereas lots with about 1000 CFU g−1 caused disease in 78 plants out of 2000.