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Made available in DSpace on 2022-04-28T19:49:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01-01 Survival niches for phytopathogenic bacteria are important for disease management. We study the survival of Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), the causal agent of bacterial wilt in common bean and bacterial tan spot in soybean, in the soil. While Cff strains from common bean already have several studies, the ecology of soybean strains needs more information. We used the strain Feij.2628A from common bean, and CffR1, Cff2R, and Cff4R from soybean. The soils were infested with bacterial suspension (107 CFU.mL−1) from each of the strains. For experiments under laboratory conditions, they were incubated in BOD incubator (biochemical oxygen demand) at 20 °C. Under natural field conditions, pots were maintained in the field without supplementary irrigation. The survival of Cff strains was evaluated every seven days until they were not detected. The identity of the bacterium was confirmed in all treatments by PCR. Under laboratory conditions, the survival period of Feij.2628A ranged from 140 to 154 days, and between 77 to 119 days for the soybean strains. Feij.2628A and Cff1R survived for a maximum of 91 days in soil with higher clay content, under natural field conditions. Cff survival was negatively influenced by high temperatures, low soil moisture, and precipitation in some treatments. These results demonstrated the potential survival of Cff strains from common bean and soybean in the soil, being potential inoculum sources for subsequent cultivations of these crops. Fallow periods ranging from three to four months may reduce the amount of inoculum in soils under these conditions. Department of Plant Protection School of Agriculture São Paulo State University (Unesp), Avenida Universitária Department of Plant Protection School of Agriculture São Paulo State University (Unesp), Avenida Universitária

Bacterial wilt caused by Ralstonia solanacearum is a major devastating soil-borne disease impeding eggplant cultivation worldwide. The present investigation was conducted to recognize and assess bacterial wilt resistant rootstocks among Solanum melongena (Haritha, Surya, SM 1, SM 2, SM 3, SM116, and SM 398), Solanum torvum (St TNAU 1 and St KAU 1), and Solanum sisymbrifolium (SS 1) for vigor, yield and qualitative traits of ‘Green Long Hybrid’ scion through grafting. The artificial inoculation method as well as the sick plot method of bacteria wilt screening were adopted. The root dip method of artificial inoculation was found to be the most reliable method as compared to media drenching and stem inoculation methods. All the rootstocks except Solanum sisymbrifolium possessed a high degree of bacterial wilt resistance (in artificial as well as sick plot conditions). Maximum plant spread, stem girth, number of primary branches, yield per plant (6.69 kg), number of fruits per plant (94.80), fruit length (22.22 cm), root length (63.65 cm), and root spread (87.05 cm) were exhibited by Green Long Hybrid scion when grafted onto Solanum melongena cv. Haritha rootstock. Taller plants with high fruit girth (10.97 cm) and average fruit weight (78.00 g) of scion were recorded on SM 116 rootstock. The highest dry matter content (11.12%) and total phenolic content (113.30 mg/100 g) of fruits were observed in scion when SM 398 and SM 3 were used as rootstocks, respectively. Overall, ‘Haritha’ cultivar was found to be the best rootstock for grafting in the current study. Thus, grafting technology can be effectively used for the control of bacterial wilt as well as for acquiring higher yield in eggplant.

Made available in DSpace on 2021-06-25T10:24:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-05-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Knowledge of the ecological survival niches of Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), the causal agent of bean bacterial wilt, is essential for the efficient disease management. Our study evaluated the survival of Cff in the phyllospheres and rhizospheres of barley, black oat, canola, common bean, forage turnip, maize, pearl millet, ryegrass, sorghum, soybean, sunflower, velvet bean, wheat and white oat planted in a greenhouse. The aerial parts of the plants were inoculated by spraying a bacterial suspension (107 CFU.mL−1) from the strain Feij. 2628A, which is resistant to rifampicin and pathogenic to common bean. The soil of the pots was infested with 200 mL of the same suspension. Cff survival was evaluated every seven days for 70 days, and the survival periods were confirmed by selecting strains from all samples and performing PCR with specific primers. Cff survived for at least seven days in the phyllosphere, and 21 days in the rhizosphere of all evaluated crops. Based on our results, barley, black oat, canola, forage turnip, maize, pearl millet, ryegrass, sorghum, soybean, sunflower, velvet bean, wheat and white oat can be potential asymptomatic hosts for Cff, and their cultivation in succession with common bean is not recommended in areas with a history of bacterial wilt occurrence. Department of Plant Protection São Paulo State University (Unesp) School of Agriculture, Avenida Universitária, 3780 Department of Plant Protection São Paulo State University (Unesp) School of Agriculture, Avenida Universitária, 3780 CAPES: 001

Bacterial wilt caused by Ralstonia solanacearum, is one of the serious soil-borne diseases in flue-cured tobacco production areas in China. However, it was found that the occurrence of bacterial wilt not only varied with different cultivars but also varied with various sites even under the condition of planting the same variety. It suggests that the occurrence of bacterial wilt could be controlled by regulating soil microorganism and the growth environment. Therefore, the relationship between microorganism’s diversity in the rhizosphere and actual incidences were evaluated on the number of symptomatic plants and the functional diversity of soil microorganisms in field trials on two sites. The results showed that the incidence of bacterial wilt in the same cultivar was varied between the two experimental sites. However, the same trend was found in the number of rhizospheric microbes among the three varieties except for fungi. Also, a good relationship was found between the number of pathogens, microbial functional diversity in the rhizosphere, and actual incidences. The utilization intensity of carbohydrates, amino acids, carboxylic acids, polymers, amines, and carbon utilization of rhizospheric soil microbial community was negatively related with the occurrence of bacterial wilt while it was on the opposite for the utilization intensity of phenolic acids. The results revealed that the mechanism for resistance in flue-cured tobacco was directly associated with the number of pathogenic microorganisms in rhizosphere and the diversity of rhizospheric microbes. By improving the diversity of rhizospheric microbes, the incidence of tobacco bacterial wilt disease can be reduced.

Made available in DSpace on 2021-06-25T10:49:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) A crop rotation system can reduce phytopathogen inoculum, and consequently reduce the incidence and severity of disease. Bean bacterial wilt is an important disease and several crops can act as inoculum sources for Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff). The aim was to evaluate the influence of crop rotation systems on 10 common bean cultivars against bacterial wilt. Experiments were located in two different areas in Brazil (2012), cultivated under a no-tillage system and with a history of the disease. The evaluated rotation systems were black oat, wheat and fallow followed by common bean. After harvest, 10 dry bean cultivars were sown, together with a fallow area. Disease incidence and severity were assessed at 45, 60 and 80 days after sowing. Also evaluated were four agronomical parameters: pod numbers, grain numbers, total weight of grains per plant and weight of 100 grains. Epiphytic and endophytic colonization by Cff was evaluated in black oat and wheat, and in crop debris. The rotation systems significantly influenced the incidence and severity of bacterial wilt in common bean cultivars in both areas and negatively affected the four agronomic parameters. The cultivars BRS Campeiro, BRS Estilo, IPR Tuiuiú, IPR Tangará and IPR Campos Gerais presented low severity and incidence of the disease and higher productivity. Cff strains were recovered from black oat, wheat and crop debris. Our results reinforce the importance of planting non-Cff host crops in rotation systems with common bean, as well as planting cultivars with a level of resistance to bacterial wilt. Departamento de Proteção Vegetal Faculdade de Ciências Agronômicas (FCA) Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Avenida Universitária, 3780 Departamento de Proteção Vegetal Faculdade de Ciências Agronômicas (FCA) Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Avenida Universitária, 3780

Bacterial wilt (BW), caused by Ralstonia solanacearum species complex (RSSC), is a major disease affecting tomato and other solanaceous crops produced in Louisiana and worldwide. The most effective and economic management strategy for BW is the use of crop genetic resistance. However, regional genetic variation among RSSC strains has made stable and durable resistance elusive. A collection of tomato, eggplant and pepper (TEP) accessions that responds to globally diverse strains in the RSSC was screened for resistance to six RSSC strains, belonging to phylotypes I and II, from Louisiana, United States (US). Wilt severity was quantified from six days up to 25 days post-inoculation (DPI). Wilting increased most rapidly to 10 DPI and was generally more severe with Phylotype II strains. However, wilt response profiles depended on the accession-strain combination. Differences in aggressiveness, as measured by the area under the disease progress stairs, were observed among the phylotype I and II strains on TEP. Wilting responses were the most severe with the tomato accessions. Hawaii 7996, TML46, CLN1463 and R3034 were highly resistant to all phylotype I strains, but Hawaii 7996 displayed the best resistance to phylotype II strains. Remarkably, eggplant accession Ceylan SM 164 was resistant to all six strains. Eggplant accessions SM6, Surya, and AG91–25, as well as pepper accessions CA8 and MC4, were moderately to highly resistant to all six strains. Although resistance spectrum and levels of these tomato, eggplant and pepper accessions need to be confirmed by further experiments, our results already indicate that these accessions are potential BW-resistant rootstock candidates for grafting to susceptible market-driven tomato, eggplant and pepper produced in this US state.

Bacterial wilt of potato caused by Ralstonia solanacearum is a severe threat to potato cultivation in Kerman province, located in southeastern Iran and the third largest potato producer in the country. The present study intends to determine the precise phylogenetic position, sequevar, and genetic diversity of R. solanacearum strains associated with the disease. To this end, the major potato-growing regions of the province (Jiroft and Laleh Zaar) were surveyed to collect tuber samples from affected potato plants showing typical wilt symptoms. Twenty-three strains were collectively purified and subjected to phenotypic and genotypic characterization. Biochemical tests showed that all the strains belonged to R. solanacearum biovar 2A. Furthermore, phylotype-specific multiplex polymerase chain reaction results indicated that the strains belonged to phylotype II. For sequevar typing as well as the determination of the phylotype subcluster (IIA/IIB), polymerase chain reaction amplicons of the endoglucanase precursor gene of 11 representative strains were sequenced. In phylogenetic analysis, the strains were grouped with the reference strains of phylotype IIB-1 retrieved from the GenBank. Then, to assess genetic diversity, 19 selected strains were subjected to genomic fingerprinting, i.e., amplified fragment length polymorphism (AFLP) using seven primer pair combinations. The AFLP data showed heterogeneity among the strains of R. solanacearum and were grouped into four clusters. With our knowledge, this is the first report of the phylotype, and sequevar determination (i.e., IIB-1) in addition to AFLP-based genomic fingerprinting of R. solanaceraum strains causing potato wilt in southeastern Iran.

In vitro experiments showed that six isolates, belonging to different biovars, were equally susceptible to mustard essential oil (MEO) vapor. The proportion of cell mortality and colony growth inhibition were directly related to the dose of MEO. A 24 h exposure to 1 or 2 μL of AITC per liter of box volume resulted in mortality of 89% and 92% of cells, respectively, and exposure to 10 μL L−1 resulted in 100% cell mortality. The growth of developing colonies was reduced by 89% at 2 or 4 μL L−1 and by 96% at 6 or 10 μL L−1. The leakage of intracellular materials increased with increasing doses of MEO vapor. The decline in soil inoculum density by fumigation was directly related to the MEO dose. The initial inoculum density of approximately 3 × 105 colony-forming units per gram of soil declined by 67, 91 and 96% at fumigation doses of 25, 50 and 75 μL of allyl isothiocyanate (AITC) per liter of soil, respectively. The pathogen was not detected at a fumigation dose of 100 μL L−1. Disease control was also related to the fumigation dose. After 48 days, the bacterial wilt symptoms did not develop on the tomatoes when the soil was fumigated at 100 μL L−1 or higher.

Bacterial wilt (BW) caused by Ralstonia solanacearum (Ras) is a very destructive disease in solanaceous crops. The molecular mechanism underlying BW resistance in solanaceous plants is still unclear. Using RNA sequencing, we investigated the transcriptional response in the seedling roots of a BW-resistant cultivar D101 and a BW-susceptible cultivar Changbohuang (CBH) of tobacco at 3 h after inoculation with Ras. In total, 158 and 835 differentially expressed genes (DEGs) were detected in D101 and CBH, respectively. Only 41 DEGs were in common between the two cultivars. The number of DEGs in D101 was much smaller than that in CBH, suggesting that D101 was less affected by the inoculation than CBH because of its higher resistance, which could be expressed at the early stages of Ras infection. Transcriptome analysis revealed that in D101 two sets of upregulated genes were significantly enriched in two corresponding groups of gene ontology terms regarding glutathione and flavonoids metabolisms, respectively, suggesting that glutathione and flavonoids are probably two types of main substances conferring the early resistance against Ras infection in tobacco root. Our findings provide a valuable clue to deeply investigate the molecular mechanism of BW resistance in tobacco and solanaceous plants.

Bacterial wilt (BW) incited by Ralstonia pseudosolanacearum (Rps), is one among the most economically important and devastating disease prevalent in all the ginger growing countries. Several strategies encompassing cultural, physical and chemical means have been reported to manage bacterial wilt but with limited success. In the present study, a technology integrating physical (soil solarization), chemical (soil amelioration with calcium chloride) and biological (ginger apoplastic bacterium - Bacillus licheniformis) methods has been developed to manage BW efficiently, economically and eco-friendly. The results indicated that, CaCl2 (2 to 4%) is inhibitory to R. pseudosolanacearum under in vitro conditions. In planta evaluation under challenge inoculation showed 71%, 98% and 100% reduction in BW with B. licheniformis, 3% and 4% CaCl2, respectively. Subsequent field evaluation involving soil solarization followed by soil amelioration with CaCl2 or with B. licheniformis resulted in significant reduction in the population of R. pseudosolanacearum from 108 to 103. Further field evaluation in farmer’s plot in BW endemic regions also resulted in 100% disease suppression adopting the technology. The results emanated from the present study indicated that the technology developed which includes soil solarization along with soil amelioration with either CaCl2 3% or B. licheniformis would serve as a viable and effective integrated strategy for the management of BW in ginger.

Made available in DSpace on 2019-10-06T15:27:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-06-15 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Bacterial wilt caused by Curtobacterium flaccumfaciens pv. flaccumfaciens is among the diseases that affect Phaseolus vulgaris L. This disease has been frequently detected in bean fields and causes severe production losses in Brazil. The aim of this research was to examine the genetic diversity existing among twenty-four isolates of C. flaccumfaciens collected from their native and alternative host, and a collection of sixty strains belonging to four phytopathogenic pathovars preserved at the French Collection for Plant-associated Bacteria (CIRM-CFBP) by multilocus sequence analysis (MLSA) based on six housekeeping genes (atpD, dnaK, gyrB, ppK, recA and rpoB). A phylogenetic tree with the concatenated sequences of six genes showed high genetic diversity among the strains. For instance, strains belonging to C. f. pv. flaccumfaciens do not cluster together within the species. Similar results were obtained with a minimal MLSA scheme using gyrB and recA, which we propose for reliable identification at the species level of Curtobacterium isolates. No correlation was identified between phylogeny and pathogenicity in the Curtobacterium flaccumfaciens strains analyzed in this work. The specific primers CffFOR2 and CffREV4 designed by Tegli et al. (Letters in Applied Microbiology, 35(4), 331–337, 2002) to detect C. f. pv. flaccumfaciens in naturally infected bean seeds proved to be efficient for the detection of bean-pathogenic strains. Instituto Federal de Educação Ciência e Tecnologia de Minas Gerais, Campus Santa Luzia, Rua Érico Veríssimo, 317 Departamento de Agronomia Universidade Estadual de Londrina (UEL), Rod PR 445, Km 380, C.P. 10.011 Faculdade de Ciências Agronômicas Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Rod Alcides Soares, Km 3 IRHS INRA AGROCAMPUS-Ouest Université d’Angers SFR 4207 QUASAV CIRM-CFBP, 42 Rue Georges Morel Faculdade de Ciências Agronômicas Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Rod Alcides Soares, Km 3

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

Cucurbit bacterial wilt, caused by Erwinia tracheiphila, is a devastating disease of cucurbit crops in the Midwest and Northeast U.S. Current management of bacterial wilt relies primarily on insecticide applications to control striped and spotted cucumber beetles (Acalymma vittatum and Diabrotica undecimpunctata howardi, respectively), which vector E. tracheiphila. Development of alternative management strategies is constrained by a lack of understanding of bacterial wilt etiology. The impact of host age on rate on symptom development and extent of bacterial movement in the xylem of muskmelon (Cucumis melo cv. Athena) was evaluated following wound inoculation of 2- to 8-week-old plants in growth chamber experiments. Wilting occurred more rapidly in plants after inoculating E. tracheiphila into 2- or 4-week-old plants than 6- or 8-week-old plants. Recovery of viable cells from stem segments revealed that vascular spread of E. tracheiphila was more extensive below than above the inoculation point. These findings provide experimental evidence that host age impacts the rate of symptom development in cucurbit bacterial wilt and that movement of the xylem-inhabiting pathogen E. tracheiphila within muskmelon plants occurs primarily in the downward direction.

High incidence of wilt disease (35–45%) of tomato (Solanum lycopersicum L.) was recently observed in two different locations of Tamil Nadu state of India. Infected plants showed typical wilt symptoms which include sudden green wilting of foliage followed by drooping of leaves downwards. The characteristic white milky ooze observed on the cut end of infected stem indicated bacterial etiology. Upon isolation, bacterium produced white fluidal irregular colonies with characteristic pink center on 2, 3, 5 triphenyl tetrazolium chloride amended nutrient medium. The bacterium caused typical green wilting 4–5 days post inoculation (dpi) on one month old tomato plants. The identity of bacterium was confirmed as Ralstonia solanacearum using 16S rRNA gene sequences as well as by R. solanacearum specific PCR. Further studies confirmed that the bacterium belonged to race 1 and biovar 3. In depth genetic characterization using multilocus sequence typing (MLST) revealed that the wilt outbreaks in Tamil Nadu were caused by genetically distinct race 1 strains of R. solanacearum. While the isolate, TRsR found clustered with R. solanacearum isolates previously reported from Andaman and Nicobar Islands-India, TRsP is clustered with isolates reported from Kerala, India. The results of our MLST based genetic analysis clearly indicated horizontal spread of bacterial wilt disease of tomato as a consequence of pathogen migration with the expansion of cultivation in India.

The Ralstonia solanacearum species complex is one of the most destructive bacterial plant pathogens for many crops. Cavendish banana is an important staple food which is severely impacted by the bacterial wilt Moko disease, caused by R. solanacearum race 2. In Mexico, banana plantations comprise 75,000 ha, supporting the economy of more than 100,000 families. Moko disease is present in the states of Chiapas and Tabasco, which are responsible for >60% of the total banana production. Each year, thousands of diseased plants are eradicated there because of Moko disease. The goal of this study was to genotype and characterizes Mexican Moko strains. Most of the strains were pathogenic to banana (PB), but two strains were found to be unable to develop wilt symptoms (NPB). A phylogenetic tree based on egl placed all Mexican strains in phylotype IIA, sequevar 6. Mexican Moko strains share identical egl sequence with strains from Central America and Caribbean countries, supporting the Caribbean origin proposed for sequevar 6 by some authors. The Mexican Seq6 nonpathogenic to banana (6NPB) and pathogenic (6 PB) strains were clustered apart in sister clades, in hrpB and pga phylogenetic trees, suggesting that this lineage is under divergence in Mexico. This is the first report worldwide of race 2 NPB strains genotyped in sequevar 6, and extends the currently known phenotypic diversity of R. solanacearum.

Bacterial wilt (causal organism-Ralstonia solanacearum) is one of the most important soil-borne diseases of eggplant (Solanum melongena) worldwide. The best way to control it economically is to develop cultivars resistant to this soil-borne pathogen. Resistance gene (R-gene) cloning and sequencing to obtain resistance gene analogs (RGAs) is one of the most recent approaches for obtaining disease resistant cultivars. Three different types of degenerative PCR based primers were used for isolating RGAs from resistant cultivated/wild species through amplification of the target region of nucleotide binding site-leucine rich repeat (NBS-LRR). Genetic diversity was observed in the sequences isolated, and six sequences showing specific conserved motifs were shortlisted as Solanum melongena RGAs (Sm7RGA1, Sm7RGA2, Sm7RGA4, Sm7RGA8 and Sm7RGA10) and Solanum viarum RGA (SvRGA2). The RGAs isolated in this study belong to both toll interleukin-1 receptors (TIR)-NBS-LRR and non-TIR-NBS-LRR type R-genes and show similarity with other plant resistance genes. This study also confirmed the hypothesis that dicots have both TIR and non-TIR resistance genes. The present study on eggplant RGAs will help develop RGA based markers linked to bacterial wilt in eggplant and other plant species. Further, it will provide information and pave the way for elucidation at the molecular level of wild and cultivated species’ mechanism of resistance to bacterial wilt. This is the first report of NBS-LRR class resistance genes/RGA in resistant eggplant and its wild relatives against bacterial wilt (BW).

Bacterial wilt, caused by Ralstonia solanacearum (Smith, US Department of Agriculture, 12, 1–28, 1986) Yabuuchi et al. (Microbiology and Immunology, 39, 897–904, 1995) (formerly Pseudomonas solanacearum), causes 35–90 % yield loss in tomato production in hot-humid tropical regions. Commonly used strategies for minimizing incidence of bacterial wilt include choosing resistant varieties, cocopith based growing media, chemical methods, grafting on resistant rootstocks, application of biocontrol agents and field management practices. The cultivation of resistant varieties is the most appropriate and eco-friendly way to manage the soil borne diseases in crops but the same is not fitting well with bacterial wilt in tomato. In this review, a systematic analysis has been made on reaction of 526 genotypes of tomato to R. solanacearum pathogen tested over a 30 year period in Andaman Islands and identified 37 genotypes as highly resistant (HR), 58 as resistant (R) and 151 as moderate resistant (MR). None of the claimed resistant genotypes exhibited stable and durable resistance against this pathogen and no single genotype reported in HR category (after 2002) except only two genotypes in R category. The period for a genotype to shift from resistance to susceptible is very short because of pathogen evolution hence, it is necessary to constantly develop new resistant varieties in tomato against R. solanacearum. The review also suggests breeding strategies for rapid development of bacterial wilt resistant varieties in tomato in order to minimize the yield losses especially in the tropical conditions.

Bacterial wilt is one of the important constraints in the cultivation of solanaceous vegetables in India. The disease is caused by Ralstonia solanacearum, a soil bacterium. We have collected 232 isolates of R. solanacearum infecting solanaceous vegetables (eggplant, tomato and chilli) and other crops from different parts of India. Pathogenicity of the isolates was tested on eggplant, tomato and chilli and the pathogen was confirmed by PCR. Multiplex PCR and biochemical tests indicated that all the isolates were phylotype I and biovar 3. Ninety-five representative isolates selected based on geographical region, host range and pathogenicity were subjected to further phylogenetic and diversity analysis. Sequence analysis of egl, pga and hrpB genes of 95 isolates and genetic diversity of 50 representative isolates was reported and discussed. Indian isolates within the Phylotype I did not group based on the host or geographical location, except clustering of isolates from the Andaman Islands. Indian isolates clustered into two sub groups based on egl and pga trees indicating the presence of two major population groups. Sub group 1 is the dominant group in the data set and consists of unknown/newer sequevars, and sub group 2 consist of mainly the isolates which are designated with sequevar numbers based on egl sequences. In the hrpB based tree, the sub group 2 is the dominant group in the data set and it is the same for the sub group 1 of the egl tree. Indian phylotpe I R. solanacearum strains are phenotypically diverse including the previously described sequevars 14, 17, 44, 47 and 48. Our studies indicated the existence of R. solanacearum isolates with unknown/newer sequevars; the diversity existing among the phylotype I isolates might be due to a continuous evolutionary process. To our knowledge this is the first detailed report on the diversity of phylotype I R. solanacearum strains infecting solanaceous vegetables and the existence of unknown/newer sequevars in India.

The effects of Calcium carbonate (CaCO3) on the survival of Ralstonia solanacearum in the laboratory and on the control of bacterial wilt in the field were investigated. In the laboratory, the influences of pH and Ca2+ on bacteria were studied as well. The laboratory results showed that soils treated with CaCO3 particles (0 ~ 1 mm) significantly inhibited the survival of R. solanacearum and increased the pH by approximately 1.5 units as compared to the control. For the growth of R. solanacearum, the suitable pH range was 6.0 ~ 7.0 with pH = 6.5 being the optimal. The results of CaCl2 and NaCl showed that the growth of R. solanacearum was significantly inhibited by Ca2+ instead of Cl−. With increase of Ca2+ concentration, the pectinase activity decreased pronouncedly, while there were no obvious effects on the production of extracellular polysaccharide. The field results indicated that CaCO3 reduced disease incidence. Treatment with organic fertilizer and CaCO3 increased soil pH and lowered the R. solanacearum population nearly 100 times and increased the Ca2+ content in tobacco significantly compared to treatments without CaCO3. It is suggested that CaCO3 could serve as a potential soil amendment for the control of bacterial wilt caused by R. solanacearum.

Ralstonia solanacearum, which consists of five races/biovars, is considered a “species-complex” and is an important phytopathogen that causes wilt disease in more than 200 plant species. R. solanacearum race 1 biovar 4 (R1bv4) has caused yield losses of 30–80 % in the vegetable sweet potato (VSP) in the last decade in Taiwan. To identify the source of the initial inoculum of R1bv4 in VSP fields, soil and cuttings from these fields were examined from 2009 to 2010. The results of the investigation indicated that the population of R1bv4 was generally distributed throughout the natural soil of VSP fields at a density ranging from 1.3 × 102 to 9.5 × 105 cfu/g soil; however, the incidence of bacterial wilt was not significantly associated with the density of the R1bv4 population in soils (R2 = 0.084). In contrast, densities of R1bv4 ranging from 2.3 × 103 to 5.9 × 105 cfu/g tissue were detected in the vine tissue of asymptomatic plants in the fields. Additional experiments demonstrated that R1bv4-free VSP cuttings without visible symptoms planted in infested soils in the greenhouse setting could carry approximately 3.1 × 105 R1bv4 cfu/g tissue, which suggests the existence of a latent period for R1bv4 in VSP plants. The results of a BIO-PCR analysis showed that R1bv4 was detected in 2.0 to 98.0 % of the VSP cuttings used for propagation in fields; in addition, the percentage of VSP cuttings carrying R1bv4 and the incidence of bacterial wilt in fields were positively correlated (R2 = 0.909). The inoculation experiments conducted in greenhouses and in fields showed that the cutting inoculum (CI) contributed more to the incidence of bacterial wilt in VSP plants than the soil inoculum (SI). In the field experiments conducted in 2010, an incidence of disease of 27.1 to 38.5 % was detected in healthy field cuttings 8 months after transplantation; in contrast, the incidence of disease in field cuttings carrying R1bv4 was 49.0 to 68.8 %. The incidence of disease was significantly lower in healthy cuttings than in cuttings carrying R1bv4 (p = 0.05).

Alfalfa (Medicago sativa), the world’s most influential forage crop, is infected by many diseases such as alfalfa bacterial wilt disease. The causal agent of bacterial crown and root rot and wilt disease is Pseudomonas viridiflava, which is a substantial pathogen of alfalfa worldwide. This pathogen spreads through the xylem and under field conditions, plants show growth stunting, chlorosis and wilting symptoms not previously reported. In this study- the first on Pseudomonas viridiflava on alfalfa from Iran, we have investigated the pathogenicity and genetic diversity of Pseudomonas viridiflava in some parts of Iran. To survey the causal agent of the disease, symptomatic plants were collected from the main alfalfa growing area. Pathogenicity of the collected strains was confirmed on alfalfa plants under green-house conditions using a completely randomized design. Determination of bacterial strains was done based on standard bacteriological methods and PCR assay using specific primers. Effects of bacterial strains on wet weight, dry weight, stem length and root length of infected plants were measured and the data were analyzed by SAS software and Duncan’s assessment. The diversity of liquid cellular proteins of bacterial strains was examined on Polyacrylamide gel. To delineate of genetic diversity the total DNA was drawn out. Fourteen random primers were used in a RAPD test. To sketch the dendrogram, RAPD fragments were used to calculate genetic diversity with NTSYS software. This data showed pathogenicity and genetic diversity of Pseudomonas viridiflava in Iran.

In this study, colonization and disease development of axenically-grown tomato plants by Clavibacter michiganensis subsp. michiganensis (Cmm), the causative agent of bacterial wilt and canker, was investigated. For this, a spontaneous rifampicin resistant strain of Cmm was tagged with a marker that expressed a green fluorescent protein (GFP) in a stable way and which possessed a similar virulence to the parental strain. In vitro plants were drop-inoculated at the stem base and the population dynamics was determined by dilution pour-plating in a selective medium. At 3 h after inoculation, Cmm was already present in low densities in roots, stems and leaves. At 16 dpi, Cmm was found throughout the entire plant in high densities of ca. 1010 cfu g−1. Symptoms developed in the in vitro plants typical for Cmm, such as canker, wilting and growth reduction. The presence of Cmm in vascular and parenchymatic tissue of in vitro tomato plants was confirmed by epifluorescence stereo- and confocal laser scanning microscopy. This study showed that in vitro tomato plants can be effectively used for detailed studies on interactions between Cmm and its host, in particular if a GFP-tagged strain of the pathogen is used.

International audience; 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 10(2) 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.

Solanum commersonii is a wild species related to the cultivated potato. Some S. commersonii genotypes have been proven to be resistant to the pathogenic bacteria Ralstonia solanacearum, which causes damage in potato and other economically important crops. Here an expression analysis of the response of a resistant S. commersonii genotype against R. solanacearum was performed using microarrays. The aims of this work were to elucidate the molecular processes involved in the interaction, establish the timing of the response, and contribute to identify genes related to the resistance. The response to the treatment was already initiated at 6 h post-inoculation (hpi) and was established at 24 hpi; during this period, a high number of genes was differentially expressed and several candidate genes for the resistance of S. commersonii to R. solanacearum were identified. At an early stage, the photosynthetic process was highly repressed and several genes encoding proteins related to reactive oxygen species (ROS) production were differentially expressed. The induction of ERF and ACC-oxidase genes related to the ethylene pathway and PR1 related to the salicylic acid pathway suggested the induction of both pathways, and back up the previously reported hemibiotrophic nature of the pathogen. Five genes related to plant defence and observed to be differentially expressed at the first two time points were validated by real time PCR. This work gives a glimpse to the molecular processes involved in S. commersonii resistance and identifies the species as a valuable genetic source for potato breeding against bacterial wilt.

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.

In this study, we investigated the ability of DL-3-aminobutyric acid (BABA) to protect tomato against bacterial wilt caused by Ralstonia solanacearum. This was combined with studies of accumulation of total phenolic compounds, free and total salicylic acid (SA), and activity of enzymes related to plant defence, i.e., polyphenol oxidase (PPO) and catalase (CAT). Under greenhouse conditions, tomato plants pre-treated by soil drenching with BABA profoundly reduced disease severity of bacterial wilt compared to plants receiving a soil drench with water. Thus, BABA reduced leaf wilting index by 75.3 % and vascular browning index by 69.9 %, without any in vitro inhibitory activity on the pathogen. BABA treatment significantly reduced the population of R. solanacearum in stems of tomato plants and additionally also significantly increased both fresh and dry weight of roots and shoots of tomato plants compared with the inoculated control. Application of BABA resulted in a high increase in PPO activity both in plants with and without inoculation. Compared to water-treated plants, treatment with BABA also induced a significant increase of total phenolic compounds as well as of free and total SA in leaves of both inoculated and non-inoculated tomato plants at all sampling times. CAT activity decreased in tomato plants treated with BABA in comparison with the water-treated control plants and the decrease in activity correlated with an increasing total SA accumulation. These findings suggest that BABA treatment resulted in induction of resistance to bacterial wilt in tomato.

Bacterial wilt in cardamom (Elettaria cardamomum Maton) was observed in Kerala state of India. Infected plants showed wilting wherein all leaves roll or curl upward towards the midrib centre, turn yellow, and the whole plant finally dies; the collar region shows water-soaked lesions initially and turns dark brown eventually; copious quantity of bacterial exudate is observed on the cut end of the pseudostem. The bacterium was identified as Ralstonia solanacearum based on a panel of phenotypic characters such as fluidal white colony on Kelman’s medium, biovar assay and biolog assay (BiologGN), and genotypic characters such as Multiplex-PCR based phylotyping, sequences of 16S rDNA, 16-23S intergenic region, and recN gene. Collectively these tests revealed that the R. solanacearum infecting cardamom belong to biovar 3 and phylotype 1 confirming its Asian origin. Upon soil inoculation, the bacterium caused typical wilting of the cardamom plants in three weeks and ginger plantlets in two weeks. Cross transmissibility of the bacterium was observed in cardamom and ginger wherein the plants succumbed to wilt when R. solanacearum from either of the host was inoculated. BOX-PCR fingerprinting revealed that the strain is identical (100%) to a ginger strain of R. solanacearum, which is widely prevalent in the Indian sub-continent. Furthermore, Multilocus Sequence Typing (MLST) based strain comparison confirmed that cardamom and ginger strain were identical to each other at 11 loci. Apart from striking phenotypic and genotypic (allelic) similarities, geographical origin, and cross transmissibility of the cardamom strain of R. solanacearum strongly suggest that the new occurrence of wilt of cardamom in India could have an origin in bacterial wilt of ginger. Perusal of records on Ralstonia-induced bacterial wilt in crop plants, particularly among the Zingiberaceae family, reveals that this is a new report of bacterial wilt disease in small cardamom.

Mulberry wilt disease (MWD) was recently identified in Hangzhou, Zhejiang province, China. Typical symptoms of the disease are browning of vascular tissues, leaf wilt, defoliation, and tree decline. Unlike the symptoms of bacterial wilt disease caused by Ralstonia solanacearum, symptoms of MWD generally started from the bottom of the plants and moved upward. In inoculation experiments, four selected MWD strains caused mulberry shoot leaf wilt, discoloration, and defoliation. They also induced whole plant leaf wilt, defoliation and dark brown discoloration of vascular tissue. Based on Biolog metabolic profiles, fatty acid methyl ester analysis (FAME) and sequence analysis of the partial 16S rDNA and rpoB genes four MWD strains were identified as members of the genus Enterobacter. The 16S rDNA and rpoB gene sequences revealed a close relationship among two isolates, R2-2 and R6-2, and the E. asburiae type strain JCM6051. The isolates showed >98% similarity to E. asburiae JCM6051 in their rpoB gene. These results indicated that isolates R2-2 and R6-2 belonged to E. asburiae. No similarity in 16S rDNA sequences above 97% was found between either of the remaining isolates, R11-2 or R18-2, and any recognized Enterobacter species, suggesting that the two isolates may represent novel Enterobacter species. rpoB gene similarity values between the isolates and Enterobacter spp. type strains were

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.

A total of 252 wild Solanum accessions and one population of 49 introgression lines of LA716 were screened for resistance to a race 1/biovar 4/phylotype I strain Pss186 of Ralstonia solanacearum. Most wild tomato accessions were highly susceptible. However, five accessions of S. pennellii, i.e. LA1943, LA716, LA1656, LA1732 and TL01845 were resistant to strain Pss186. These accessions were then challenged against two other race 1/phylotpye I strains Pss4 and Pss190, which were more aggressive. All the five S. pennellii accessions were susceptible to Pss4, but displayed high to moderate resistance to Pss190 with a percentage of wilted plants ranging from 0% to 60%. Pss190 is an aggressive strain that made a resistant tomato line Hawaii 7996 susceptible. Thus, the results found in this study provide evidence of the presence of strain-specific resistance. LA3501, which has an introgression segment on chromosome 6, was found to be resistant to Pss186 among the screened introgression lines. This confirms the importance of resistance trait loci on chromosome 6 that have been identified by other studies. This is the first report of S. pennellii being resistant to bacterial wilt. These new resistant sources will provide breeders with more resources to breed for stable resistance to bacterial wilt of tomato.

The ability of selected strains of fluorescent Pseudomonas spp. to cause induced systemic resistance (ISR) in Eucalyptus urophylla against bacterial wilt caused by Ralstonia solanacearum was investigated. Four of the five strains used can produce salicylic acid (SA) in vitro and, therefore, chemical SA, that is known to induce resistance in many plant species, was used as a reference treatment. Whereas a soil drench with SA did induce systemic resistance in E. urophylla, infiltration of SA into leaves did not. None of the fluorescent Pseudomonas spp. strains caused ISR against bacterial wilt when applied to the soil, but two strains, P. putida WCS358r and P. fluorescens WCS374r triggered ISR when infiltrated into two lower leaves 3–7 days before challenge inoculation. A mutant of strain WCS358r defective in the biosynthesis of the fluorescent siderophore pseudobactin, did not cause ISR, while the purified siderophore of WCS358r did, suggesting that pseudobactin358 is the ISR determinant of WCS358. A siderophore-minus mutant of WCS374r induced the same level of disease resistance as its parental strain, but the purified siderophore induced resistance as well, indicating that both the siderophore and another, unknown, inducing determinant(s) of WCS374r can trigger ISR in Eucalyptus. A possible role of WCS374r-produced SA remains uncertain. Transformation of a siderophore-minus mutant of WCS358 with the SA biosynthetic gene cluster from WCS374 did not enable this transformant to cause ISR in E. urophylla.