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

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

Identification of open reading frames unique to a select agent: Ralstonia solanacearum race 3 biovar 2

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.     

Detection and functional characterization of a large genomic deletion resulting in decreased pathogenicity in Ralstonia solanacearum race 3 biovar 2 strains

Bacterial wilt (brown rot) disease of potato caused by Ralstonia solanacearum is one of the most important bacterial diseases and a major constraint on potato production worldwide. Through a comparative genomic analysis between R.?solanacearum'race 3 biovar 2' (R3bv2) strains, we identified a 77 kb region in strain UW551 which is specifically absent in the hypoaggressive strain IPO1609. We proved that IPO1609 indeed carries a 77 kb genomic deletion and provide genetic evidence that occurrence of this deletion is responsible for almost complete loss of pathogenicity of this strain. We carried out a functional analysis of this 77 kb region in strain UW551 using a combination of gene deletion and functional complementation approaches which identified the methionine biosynthesis genes metER as having a major contribution to IPO1609 pathogenesis. Deletion of the metER genes significantly impacts pathogenicity of R3bv2 strains but does not lead to methionine auxotrophy nor reduced ability to multiply in planta. In addition, this study indicated that three type III secretion system effectors or a type VI secretion system present within the 77 kb region have no or very minor contribution to pathogenicity.     

Influence of native microbiota on survival of Ralstonia solanacearum phylotype II in river water microcosms

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

Degradation of starch in poplar cells at low temperatures

In Vitro Cellular & Developmental Biology - Plant - Carbon is essential for plant growth. Starch, the main storage carbohydrate, plays an important role in the plant life cycle. When poplar...     

Tropical Strains of Ralstonia solanacearum Outcompete Race 3 Biovar 2 Strains at Lowland Tropical Temperatures

Bacterial wilt, caused by members of the heterogenous Ralstonia solanacearum species complex, is an economically important vascular disease affecting many crops. Human activity has widely disseminated R. solanacearum strains, increasing their global agricultural impact. However, tropical highland race 3 biovar 2 (R3bv2) strains do not cause disease in tropical lowlands, even though they are virulent at warm temperatures. We tested the hypothesis that differences in temperature adaptation and competitive fitness explain the uneven geographic distribution of R. solanacearum strains. Using three phylogenetically and ecologically distinct strains, we measured competitive fitness at two temperatures following paired-strain inoculations of their shared host, tomato. Lowland tropical strain GMI1000 was only weakly virulent on tomato under temperate conditions (24°C for day and 19°C for night [24/19°C]), but highland tropical R3bv2 strain UW551 and U.S. warm temperate strain K60 were highly virulent at both 24/19°C and 28°C. Strain K60 was significantly more competitive than both GMI1000 and UW551 in tomato rhizospheres and stems at 28°C, and GMI1000 also outcompeted UW551 at 28°C. The results were reversed at cooler temperatures, at which highland strain UW551 generally outcompeted GMI1000 and K60 in planta. The superior competitive index of UW551 at 24/19°C suggests that adaptation to cool temperatures could explain why only R3bv2 strains threaten highland agriculture. Strains K60 and GMI1000 each produced different bacteriocins that inhibited growth of UW551 in culture. Such interstrain inhibition could explain why R3bv2 strains do not cause disease in tropical lowlands.     

Seasonal variation in the lateral distribution of mayfly nymphs in a boreal river

Seasonal variations in the lateral distribution of mayfly nymphs were investigated in the North Swedish river Vindelälven; a river with large seasonal differences in water level, current velocity and ice conditions, A total of 22 mayfly species about equally divided between summer and winter species were found along a single transect. At low water level in late summer and autumn most species were distributed over the whole transect. In winter the populations occupying the littoral moved into deeper water, so avoiding the ice. Only very small mortalities were detected in the ice. Prior to the spring flood most characteristically still water species moved to the shallow uppermost littoral while lotic species and the burrowing Ephemera vulgata L. did not show the same aggregation near the shore. It is suggested that these lateral movements allow the nymphs to avoid adverse environmental conditions and to exploit food resources over the whole transect.     

Quantitative immunofluorescence of regulated eps gene expression in single cells of Ralstonia solanacearum.

Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10(7) cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of beta-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined.     

Flagellin is not a major defense elicitor in Ralstonia solanacearum cells or extracts applied to Arabidopsis thaliana

The phytopathogenic bacterium Ralstonia solanacearum requires motility for full virulence, and its flagellin is a candidate pathogen-associated molecular pattern that may elicit plant defenses. Boiled extracts from R. solanacearum contained a strong elicitor of defense-associated responses. However, R. solanacearum flagellin is not this elicitor, because extracts from wild-type bacteria and fliC or flhDC mutants defective in flagellin production all elicited similar plant responses. Equally important, live R. solanacearum caused similar disease on Arabidopsis ecotype Col-0, regardless of the presence of flagellin in the bacterium or the FLS2-mediated flagellin recognition system in the plant. Unlike the previously studied flg22 flagellin peptide, a peptide based on the corresponding conserved N-terminal segment of R. solanacearum, flagellin did not elicit any response from Arabidopsis seedlings. Thus recognition of flagellin plays no readily apparent role in this pathosystem. Flagellin also was not the primary elicitor of responses in tobacco. The primary eliciting activity in boiled R. solanacearum extracts applied to Arabidopsis was attributable to one or more proteins other than flagellin, including species purifying at approximately 5 to 10 kDa and also at larger molecular masses, possibly due to aggregation. Production of this eliciting activity did not require hrpB (positive regulator of type III secretion), pehR (positive regulator of polygalacturonase production and motility), gspM (general secretion pathway), or phcA (LysR-type global virulence regulator). Wild-type R. solanacearum was virulent on Arabidopsis despite the presence of this elicitor in pathogen extracts.     

Quantitative visible spectroscopy at low temperatures: a systematic examination

A systematic study of the enhancement of optical absorption of solutions upon freezing is presented. The enhancement factor, the ratio of absorbance of the frozen solution under given conditions to that of the solution at room temperature, is shown to increase with the optical pathlength of the sample, lower temperatures, and decreasing optical density of the solution. The enhancement factor is only weakly dependent upon wavelength under the defined conditions. This study makes possible a clearer understanding of the factors involved in low-temperature spectroscopy. Also presented are measurements of the relative contributions of the two hemes of cytochrome oxidase to the optical spectrum at ?140°C, as an example of quantitative studies at low temperatures.     

Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato

AIMS: The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation of this target molecule is found upon cell death. METHODS AND RESULTS: It was demonstrated by RNase treatment of extracted nucleic acids from R. solanacearum cell suspensions that NASBA exclusively detected RNA and not DNA. The ability of NASBA to assess viability was demonstrated in two sets of experiments. In the first experiment, viable and chlorine-killed cells of R. solanacearum were added to a potato tuber extract and tested in NASBA and PCR. In NASBA, only extracts spiked with viable cells resulted in a specific signal after Northern blot analysis, whereas in PCR, targeting 16S rDNA sequences, both extracts with viable and killed cells resulted in specific signals. In the second experiment, the survival of R. solanacearum on metal strips was studied using NASBA, PCR-amplification and dilution plating on the semiselective medium SMSA. A positive correlation was found between NASBA and dilution plating detecting culturable cells, whereas PCR-amplification resulted in positive reactions also long after cells were dead. The detection level of NASBA for R. solanacearum added to potato tuber extracts was determined at 104 cfu per ml of extract, equivalent to 100 cfu per reaction. With purified RNA a detection level of 104 rRNA molecules was found. This corresponds with less than one bacterial cell, assuming that a metabolically active cell contains ca 105 copies of rRNA. Preliminary experiments demonstrated the potential of NASBA to detect R. solanacearum in naturally infected potato tuber extracts. CONCLUSIONS: NASBA specifically amplifies RNA from viable cells of R. solanacearum even present in complex substrates at a level of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel NASBA assay will be particularly valuable for detection of R. solanacearum in ecological studies in which specifically viable cells should be determined.     

Genetic mapping of a major dominant gene for resistance to Ralstonia solanacearum in eggplant

Resistance of eggplant against Ralstonia solanacearum phylotype I strains was assessed in a F₆ population of recombinant inbred lines (RILs) derived from a intra-specific cross between S. melongena MM738 (susceptible) and AG91-25 (resistant). Resistance traits were determined as disease score, percentage of wilted plants, and stem-based bacterial colonization index, as assessed in greenhouse experiments conducted in Réunion Island, France. The AG91-25 resistance was highly efficient toward strains CMR134, PSS366 and GMI1000, but only partial toward the highly virulent strain PSS4. The partial resistance found against PSS4 was overcome under high inoculation pressure, with heritability estimates from 0.28 to 0.53, depending on the traits and season. A genetic map was built with 119 AFLP, SSR and SRAP markers positioned on 18 linkage groups (LG), for a total length of 884 cM, and used for quantitative trait loci (QTL) analysis. A major dominant gene, named ERs1, controlled the resistance to strains CMR134, PSS366, and GMI1000. Against strain PSS4, this gene was not detected, but a significant QTL involved in delay of disease progress was detected on another LG. The possible use of the major resistance gene ERs1 in marker-assisted selection and the prospects offered for academic studies of a possible gene for gene system controlling resistance to bacterial wilt in solanaceous plants are discussed.