Strains Of Ralstonia Solanacearum Research Articles

EVALUATION OF GENETIC DIVERSITY OF RALSTONIA SOLANACEARUM STRAINS USING RAPD-PCR

There were analysed 9 strains of Ralstonia solanacearum, obtained from New Zealand collection and collection of the Department of phytopathogenic bacteria of D.K. Zabolotny Institute of Microbiology and Virology on the basis of RAPD-PCR. According to the obtained results high level of genetic diversity was established and phylogenic tree was constructed. The distribution of the strains into genetic clusters shown similarity between the strains of biovar 1. No association was found between amplified sequences and determination of lipopolysaccharide chemotypes.

Endo- and exopolygalacturonases of Ralstonia solanacearum are inhibited by polygalacturonase-inhibiting protein (PGIP) activity in tomato stem extracts

Polygalacturonases (PGs) of wild-type and non-virulent phenotype conversion mutant (PC) strains of Ralstonia solanacearum were compared by investigating their activities and their inhibition by polygalacturonase-inhibiting proteins (PGIPs) from tomato stems. In cultures of wild-type strain ToUdk2, slimy (s), retarded slimy (rs) and non-slimy (ns) colonies appeared. The conversion of the 's' into the 'rs' colony form coincided with the beginning of PG production. PG activity of the PC strain increased about 5 h earlier (at 6 hpi), and was up to 35 times higher in media supplemented with two different tomato stem extracts or polygalacturonic acid, compared to the wild-type at 6 hpi, and generally 4-8 times higher across test media and time. By hydrophobic interaction chromatography (HIC), fluorophor-assisted carbohydrate-polyacrylamid-gel electrophoresis (FACE-PAGE) and mass spectrometry analyses, endo-PG PehA, exo-PGs PehB and PehC were identified. PGs of the PC mutant consisted mainly of endo-PG. The increased PG production after supplementing the medium with tomato cell wall extract was reflected by a higher activity of exo-PGs for both strains. Total PGs (endo-PG and exo-PGs) activities were inhibited by PGIPs of tomato stem extracts. PGIP activity was concentration dependent, constitutively present, and not related to resistance nor susceptibility of tomato recombinant inbred lines to R. solanacearum. The proteinaceous character of the inhibiting component was inferred from ammonium sulphate precipitation. For the first time a plant PGIP activity against a bacterial pathogen is reported. Observations support that endo- and exo-PG synthesis is governed by a sensitive regulatory network, which, in interaction with PGIP and cell wall degradation products, leads to generation or avoidance of elicitor-active oligomers, and, thus, may contribute to the development of the compatible or incompatible interaction.

First Report of Bacterial Wilt Caused by Ralstonia solanacearum on Mesona chinensis in China.

Jellywort (Mesona chinensis Benth) is an herbaceous plant in the Lamiaceae (mint) family. The plant is referred to as 'Xiancao' (weed from angels) in Chinese and primarily used to make grass jelly, a popular refreshing drink. Currently, Xiancao cultivation is a fast-growing industry with a high profit margin in southern China. An estimated 7,000 ha is grown with a value of more than $50 million USD. In June, 2009, a wilting disease of Xiancao was observed in Guangdong and the neighboring Guangxi and Fujian provinces with incidence up to 50% in the severest case. Affected plants initially show withering symptoms on apical leaves during the daytime with recovery at night. As the disease develops, withering leaves spread downward, eventually encompassing the whole plant. Leaves lose vigor but remain green. After 3 to 4 days, wilting becomes irreversible. Roots and basal stem tissues blacken and rot, leading to plant death. Longitudinal sectioning of the basal stem shows browning of vascular tissues with whitish ooze visible when compressed. To investigate the disease etiology, 12 Xiancao plants from three cultivars showing typical wilting symptoms were collected from a production field in Zengcheng City of Guangdong Province in June 2010. A total of 27 bacterial isolates showing large, elevated, and fluidal colonies with a pale red center were isolated from vascular tissue on tripheny tetrazolium chloride medium (3) after incubation at 30° for 2 days. Fifteen 45-day-old Xiancao plants (cv. Zhengcheng 1) were inoculated by injection of 20 μl of bacterial suspension (1 × 108 CFU/ml) into the middle stem. Sterile water was used as a negative control. After 4 to 6 days of incubation in a greenhouse (28 to 30°), all (15 of 15) inoculated plants developed wilting symptoms as described above. The same bacterium was reisolated from inoculated plants. The five negative control plants did not show any wilting symptoms. With the same artificial inoculation procedure, this bacterium also caused similar wilting disease in tobacco, potato, tomato, pepper, and eggplant. An inoculation test with a tomato strain of Ralstonia solanacearum resulted in similar symptoms. On the basis of symptomatology and bacterial culture characteristics, R. solanacearum (formerly Pseudomonas solanacearum) was suspected as the causal agent. For confirmation, the universal bacterial 16S rDNA primer set E8F/E1115R (1) was used to amplify DNA from pure culture. A 1,027-bp DNA sequence was obtained and deposited in GenBank with Accession No. HQ159392. BLAST search against the current version of GenBank database showed 100% similarity with the 16S rDNA sequences of 26 R. solanacearum strains. Furthermore, primer set 759/760 (4) amplified a specific 280-bp fragment. Along with the result from multiplex PCR (2), the bacterium was identified as R. solanacearum Phylotype I. To our knowledge, this is the first report of a disease caused by R. solanacearum on M. chinensis.

Genetic diversity of Ralstonia solanacearum strains from China assessed by PCR-based fingerprints to unravel host plant- and site-dependent distribution patterns

Bacterial wilt caused by Ralstonia solanacearum is a serious threat to crop production in China. A collection of 319 R. solanacearum strains isolated from 14 different diseased host plants collected in 15 Chinese provinces was investigated by BOX fingerprints in order to test the influence of the site and the host plant on their genetic diversity. Phylotype, fliC-RFLP patterns and biovar were determined for all strains and the sequevar for 39 representative strains. The majority of strains belonged to the Asian phylotype I, shared identical fliC-RFLP patterns and were assigned to four biovars (bv3:123; bv4:162; bv5:3; and bv6:11). Twenty strains were phylotype II, assigned to biovar 2, and had distinct fliC-RFLP patterns. BOX-PCR fingerprints generated from the genomic DNA of each strain revealed a high diversity of the phylotype I strains, where 28 types of BOX fingerprints could be distinguished. While many BOX clusters comprised isolates from different provinces and several host plants, some groups contained isolates that were plant or site specific. All phylotype II isolates originating from 10 provinces belonged to sequevar 1 and displayed identical BOX patterns as the potato brown rot strains from various regions of the world.

Extracts from Ralstonia solanacearum induce effective resistance to the pathogen in both Arabidopsis and solanaceous plants

Plants defend themselves against microbial invasions by detecting conserved molecules, collectively called pathogen-associated molecular patterns (PAMPs). PAMPs-triggered basal resistance is the first inducible layer of plant defense. Here we found that Ralstonia solanacearum strain RS1002 can efficiently grow and cause disease in ecotype Columbia-0 of the model plant Arabidopsis thaliana in a manner dependent on the Hrp type III secretion system (T3SS). The extent of disease symptoms caused by R. solanacearum was reduced in plants pretreated with ΔhrpY mutant deficient in the functional Hrp T3SS. Pretreatment with a boiled extract (BE) from R. solanacearum had a similar inhibitory effect on disease development or bacterial multiplication in both Arabidopsis and several solanaceous plants. Simultaneous inoculations with BE and R. solanacearum did not induce BE-mediated resistance, nor did a BE treatment with proteinases. These results indicate that host plants recognize an unknown proteinaceous PAMP in the BE to induce disease resistance and that the Hrp T3SS of R. solanacearum can suppress it. From an analysis using Arabidopsis mutants lacking PAMP receptors, the elongation factor Tu of R. solanacearum was shown to partially contribute to BE-mediated basal resistance in Arabidopsis plants.

Moderate temperature fluctuations rapidly reduce the viability of Ralstonia solanacearum race 3, biovar 2, in infected geranium, tomato, and potato plants.

Most Ralstonia solanacearum strains are tropical plant pathogens, but race 3, biovar 2 (R3bv2), strains can cause bacterial wilt in temperate zones or tropical highlands where other strains cannot. R3bv2 is a quarantine pathogen in North America and Europe because of its potential to damage the potato industry in cooler climates. However, R3bv2 will not become established if it cannot survive temperate winters. Previous experiments showed that in water at 4°C, R3bv2 does not survive as long as native U.S. strains, but R3bv2 remains viable longer than U.S. strains in potato tubers at 4°C. To further investigate the effects of temperature on this high-concern pathogen, we assessed the ability of R3bv2 and a native U.S. strain to survive typical temperate winter temperature cycles of 2 days at 5°C followed by 2 days at -10°C. We measured pathogen survival in infected tomato and geranium plants, in infected potato tubers, and in sterile water. The population sizes of both strains declined rapidly under these conditions in all three plant hosts and in sterile water, and no culturable R. solanacearum cells were detected after five to seven temperature cycles in plant tissue. The fluctuations played a critical role in loss of bacterial viability, since at a constant temperature of -20°C, both strains could survive in infected geranium tissue for at least 6 months. These results suggest that even when sheltered in infected plant tissue, R3bv2 is unlikely to survive the temperature fluctuations typical of a northern temperate winter.

Growth and pathogenicity characteristics of Ralstonia solanacearum strain RS1100 in long-term stationary phase culture

Nonsporulating gram-negative bacteria remain metabolically active and also develop increased resistance to a variety of environmental stresses after exponential growth has stopped and cells enter stationary phase. Ralstonia solanacearum is a nonsporulating gram-negative soil-borne pathogen that causes lethal wilt diseases of many plants around the world. Its growth and pathogenicity characteristics for entering longterm stationary phase were investigated by a prolonged 20- day laboratory culture. The data obtained from viability trial revealed that ≈99% of R. solanacearum strain RS1100 (race 1) died at day 14 in SPA medium and then survivors maintained at ≈105 cfu ml–1 until the 20th day. Binary fission was observed and two modes of death procedures were assumed for growth of R. solanacearum under transmission electronic microscope, nucleoid condensation or nucleoid fragmentation and cytolysis. The latent period for R. solanacearum causing plant wilt prolonged while the cell density reduced gradually. The pathogen retained its aggressiveness at low density as 1.13 × 105 cfu ml–1 at 20th day. Both mucoid and nonmucoid forms of R. solanacearum were coexistent throughout the experiment with varying ratio. The results here confirm that upon entering long-term stationary phase R. solanacearum can survive the stress conditions of nutrient starvation during prolonged stationary phase.

Plant ferredoxin-like protein (PFLP) outside chloroplast in Arabidopsis enhances disease resistance against bacterial pathogens

Protection of crops against bacterial disease is an important issue in agricultural production. One of the strategies to lead plants become resistant against bacterial pathogens is employing a transgene, like plant ferredoxin-like protein (PFLP). PFLP is a photosynthetic type ferredoxin isolated from sweet pepper and contains a signal peptide for targeting towards chloroplasts. Our previous reports indicated that transgenic plants with this protein are more resistant against bacterial pathogens. However, this heterologous protein was visualized not only inside the chloroplasts, but also in the cytoplasm. In this article, we moved to study its heterologous expression in Arabidopsis by expressing the protein in chloroplast, apoplast and cytoplasm. This work was achieved by engineering a chloroplast target (CPF), an apoplast target (ESF), and cytoplasm target (DF) plants. The expression and subcellular localization of PFLP were analyzed by Western blot and immuno-staining by confocal microscopy, respectively. We tested the ability of the transgenic Arabidopsis for resistance to two Ralstonia solanacearum strains and their ability to increase the hypersensitive response (HR) triggered by harpin (HrpZ) from Pseudomonas syringae. The DF and ESF plants conferred resistance against bacterial wilt strains and increased HR by harpin, but no resistance found in the CPF plants. In addition, we determined the level of reduced ascorbate in all transgenic plants and further analyzed the expression of two NADPH-oxidase genes ( AtrbohD and AtrbohF) in ESF plant. Among the transgenic Arabidopsis plants, ESF plants confer the highest resistance to bacterial pathogens and followed by DF plants. We concluded that PFLP enhances disease resistance in Arabidopsis when expressed in the apoplast or in cytoplasm but not when targeted into the chloroplast. This study provides a strategy for molecular breeding to improve resistance of crops against bacterial pathogens.

PopW of Ralstonia solanacearum, a new two‐domain harpin targeting the plant cell wall

Harpins are extracellular glycine-rich proteins eliciting a hypersensitive response (HR). In this study, we identified a new harpin, PopW, from Ralstonia solanacearum strain ZJ3721. This 380-amino-acid protein is acidic, rich in glycine and serine, and lacks cysteine. When infiltrated into the leaves of tobacco (non-host), PopW induced a rapid tissue collapse via a heat-stable but protease-sensitive HR-eliciting activity. PopW has an N-terminal harpin domain (residues 1-159) and a C-terminal pectate lyase (PL) domain (residues 160-366); its HR-eliciting activity depends on its N-terminal domain. Analyses of subcellular localization and plasmolysis demonstrated that PopW targeted the onion cell wall. This was further confirmed by its ability to specifically bind to calcium pectate, a major component of the plant cell wall. However, PopW had no detectable PL activity. Western blotting revealed that PopW was secreted by the type III secretion system in an hrpB-dependent manner. Gene sequencing indicated that popW is conserved among 20 diverse strains of R. solanacearum. A popW-deficient mutant retained the ability of wild-type strain ZJ3721 to elicit HR in tobacco and to cause wilt disease in tomato (a host). We conclude that PopW is a new cell wall-associated, hrpB-dependent, two-domain harpin that is conserved across the R. solanacearum species complex.

Analysis of genetic and biological characters of Japanese potato strains of Ralstonia solanacearum

We assessed the geographic distribution, biovar, phylotype, DNA fingerprints (rep-PCR), and/or endoglucanase sequence of potato bacterial wilt pathogen, Ralstonia solanacearum (Rs), in Japan. Rs has been isolated from potato fields in southwestern, warm, temperate regions. Of the 188 isolates, 74 belonged to biovar N2 (39%), 44 to biovar 3 (24%), and 70 to biovar 4 (37%). Biovars N2 and 4 strains were widely distributed, from northern (Hokkaido) to southern (Okinawa) Japan. Based on the results of multiplex-PCR analysis, every potato strains belonged to either phylotype I or IV. Phylotype I comprised both biovars 3 and 4 strains. On the other hand, phylotype IV included biovar N2 strains. None of the strains belonged to phylotype II or III or biovar 1 or 2. Phylogenetic analysis based on DNA fingerprints and endoglucanase gene sequences clarified the genetic diversity of the Japanese potato strains and the close genetic relationship between the Japanese strains and the Asian strains in phylotypes I and IV.

Identification and Functional Analysis of Type III Effector Proteins in Mesorhizobium loti

Mesorhizobium loti MAFF303099, a microsymbiont of the model legume Lotus japonicus, possesses a cluster of genes (tts) that encode a type III secretion system (T3SS). In the presence of heterologous nodD from Rhizobium leguminosarum and a flavonoid naringenin, we observed elevated expression of the tts genes and secretion of several proteins into the culture medium. Inoculation experiments with wild-type and T3SS mutant strains revealed that the presence of the T3SS affected nodulation at a species level within the Lotus genus either positively (L. corniculatus subsp. frondosus and L. filicaulis) or negatively (L. halophilus and two other species). By inoculating L. halophilus with mutants of various type III effector candidate genes, we identified open reading frame mlr6361 as a major determinant of the nodulation restriction observed for L. halophilus. The predicted gene product of mlr6361 is a protein of 3,056 amino acids containing 15 repetitions of a sequence motif of 40 to 45 residues and a shikimate kinase-like domain at its carboxyl terminus. Homologues with similar repeat sequences are present in the hypersensitive-response and pathogenicity regions of several plant pathogens, including strains of Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas species. These results suggest that L. halophilus recognizes Mlr6361 as potentially pathogen derived and subsequently halts the infection process.

Identification of a Bifunctional UDP-4-keto-pentose/UDP-xylose Synthase in the Plant Pathogenic Bacterium Ralstonia solanacearum Strain GMI1000, a Distinct Member of the 4,6-Dehydratase and Decarboxylase Family

The UDP-sugar interconverting enzymes involved in UDP-GlcA metabolism are well described in eukaryotes but less is known in prokaryotes. Here we identify and characterize a gene (RsU4kpxs) from Ralstonia solanacearum str. GMI1000, which encodes a dual function enzyme not previously described. One activity is to decarboxylate UDP-glucuronic acid to UDP-beta-l-threo-pentopyranosyl-4''-ulose in the presence of NAD(+). The second activity converts UDP-beta-l-threo-pentopyranosyl-4''-ulose and NADH to UDP-xylose and NAD(+), albeit at a lower rate. Our data also suggest that following decarboxylation, there is stereospecific protonation at the C5 pro-R position. The identification of the R. solanacearum enzyme enables us to propose that the ancestral enzyme of UDP-xylose synthase and UDP-apiose/UDP-xylose synthase was diverged to two distinct enzymatic activities in early bacteria. This separation gave rise to the current UDP-xylose synthase in animal, fungus, and plant as well as to the plant Uaxs and bacterial ArnA and U4kpxs homologs.

Pepper Accession LS2341 Is Highly Resistant to Ralstonia solanacearum Strains from Japan

Bacterial wilt caused by Ralstonia solanacearum is one of the most serious diseases in pepper (Capsicum annuum) crops in warm-temperate, subtropical, and tropical areas, including Japan. Resistant lines are a prerequisite for breeding resistant cultivars but are not well studied. Eight pepper accessions previously described as resistant to the pathogen were selected and inoculated with a highly virulent strain, KP9547. Among them, Malaysian accession LS2341 exhibited the highest resistance. Accession LS2341 was then challenged with 14 virulent strains collected from various areas in Japan. The strains cover biovars 2T, 3, and 4 and include isolates from the host plants of tomato, eggplant, and pepper. Results indicated that accession LS2341 had the highest level of resistance to all the strains of R. solanacearum examined. Therefore, accession LS2341 was confirmed as an appropriate source for the breeding of resistant cultivars of pepper in Japan and is a candidate for a potential source of resistance in other areas.

Broad Diversity of Ralstonia solanacearum Strains in Cameroon.

In 2005, an extensive survey of bacterial wilt in Cameroon collected 110 strains of Ralstonia solanacearum from wilting tomato, potato, pepper, huckleberry (Solanum scabrum), sesame, and amaranth. The genetic diversity and phylogeny of selected strains from Cameroon were assessed by multiplex-polymerase chain reaction (PCR), race 3/biovar 2-specific PCR, and sequence analyses of the mutS and egl genes. These data were compared with those from 33 reference strains covering the known diversity within the R. solanacearum species complex. Strains isolated in Cameroon clustered into three of the four known phylotypes: I (Asian), II (American), and III (African). Lowland tomato strains belonged to phylotype I and were quite homogeneous. The strains belonging to phylotype II were genetically diverse, and partitioned into subclusters IIA and IIB (sequevar 1, race 3/biovar 2). Cameroon strains in the African phylotype III were distinct from reference strains from Zimbabwe or the Indian Ocean, highlighting the genetic diversity present within this phylotype. Strains from potatoes growing in the highlands of West Cameroon fell into both phylotypes II (race 3/biovar 2) and III. These phylotype II and III highland strains attacked both potato and tomato and could therefore pose an economic threat to potato and tomato crops throughout Central Africa. This is the first comprehensive report on the genetic diversity of R. solanacearum strains in Cameroon.

Interactions with Hosts at Cool Temperatures, Not Cold Tolerance, Explain the Unique Epidemiology of Ralstonia solanacearum Race 3 Biovar 2

Most strains of the bacterial wilt pathogen Ralstonia solanacearum are tropical, but race 3 biovar 2 (R3bv2) strains can attack plants in temperate zones and tropical highlands. The basis of this distinctive ecological trait is not understood. We compared the survival of tropical, R3bv2, and warm-temperate North American strains of R. solanacearum under different conditions. In water at 4 degrees C, North American strains remained culturable the longest (up to 90 days), whereas tropical strains remained culturable for the shortest time (approximately 40 days). However, live/dead staining indicated that cells of representative strains remained viable for >160 days. In contrast, inside potato tubers, R3bv2 strain UW551 survived >4 months at 4 degrees C, whereas North American strain K60 and tropical strain GMI1000 were undetectable after <70 days in tubers. GMI1000 and UW551 grew similarly in minimal medium at 20 and 28 degrees C and, although both strains wilted tomato plants rapidly at 28 degrees C, UW551 was much more virulent at 20 degrees C, killing all inoculated plants under conditions where GMI100 killed just over half. Thus, differences among the strains in the absence of a plant host were not predictive of their behavior in planta at cooler temperatures. These data indicate that interaction with plants is required for expression of the temperate epidemiological trait of R3bv2.

Molecular typing of Japanese strains of Ralstonia solanacearum in relation to the ability to induce a hypersensitive reaction in tobacco

The genetic diversity of 120 Ralstonia solanacearum strains isolated from a variety of host plants across Japan was assessed on the basis of hypersensitive response (HR) in tobacco leaves and phylogenetic analyses of endoglucanase gene egl, hrpB, and gyrB. Phylogenetic analysis of egl revealed that only three strains belonged to phylotype IV, and 117 strains belonged to phylotype I. Partial sequences of HrpB were identical among phylotype I strains except for one strain. Analyses using the partial nucleotide sequences of the gyrB and egl gene fragments grouped phylotype I strains into 11 gyrB and 8 egl types, respectively, whereas analyses using the partial amino acid sequences of GyrB and Egl grouped phylotype I strains into 4 GyrB and 5 Egl types, respectively. Using multilocus sequence typing of GyrB and Egl, we identified 10 unique sequence types within the Japanese phylotype I strains. Strains belonging to the GyrB42 or GyrB66 type caused wilt in tobacco, and strains belonging to GyrB2 or GyrB9 type elicited HR, demonstrating that HR induction in tobacco is genetically differentiated in the Japanese strains of R. solanacearum.

Distribution of avirulence genes avrA and popP1 in 22 Japanese phylotype I strains of Ralstonia solanacearum

Sequence analysis of hrp loci and effector genes in the flanking regions showed significantly high similarities between two phylotype I strains of Ralstonia solanacearum, GMI1000 and Japanese strain OE1-1. Further sequence analysis of the distribution of avrA and popP1, known as determinants of a hypersensitive response (HR) induction on Nicotiana tabacum (tobacco), in 22 Japanese phylotype I strains revealed that all strains had one of the two distinct avrA alleles and that 10 strains had an identical popP1 but the other 12 did not. After infiltration of tobacco leaves, more than half of these 22 strains elicited HR. In combination with the ability to induce HR, avrA and popP1 are thus not likely to be the sole determinants of HR in Japanese phylotype I strains.

Genetic diversity of Ralstonia solanacearum strains from China

A survey of bacterial wilt in China collected 286 strains of Ralstonia solanacearum from 17 plant species in 13 Chinese provinces to investigate genetic diversity using the biovar (bv.) and phylotype classification schemes. A phylotype-specific multiplex-PCR showed that 198 isolates belonged to phylotype I (bv. 3, 4 and 5) and 68 to phylotype II (bv. 2 and bv. 1). A phylogenetic analysis examined the partial sequence of the egl and hrpB gene of all strains and the genetic diversity of 95 representatives was reported, demonstrating that Chinese strains are partitioned into phylotype I (Asia) and II (Americas). Phylotype I strains (historically typed bv. 3, 4 and 5), had considerable phylogenetic diversity, including 10 different sequevars: seven previously described sequevars 12 to 18 and three new sequevars: 34, 44 and 48. Chinese strains Z1, Z2, Z3, Z7, Pe74 and Tm82 were not genetically distinguishable from the edible ginger reference strain ACH92 (r4-bv. 4) for sequevar 16. This is believed to be the first report of this ginger group in China. All Chinese bv. 2 strains falling into the genetically and phenotypically diverse phylotype II were placed into phylotype IIB sequevar 1 (historically the Andean race3-bv. 2 potato brown rot agent). In both the egl and hrpB sequence-based trees, strains isolated from mulberry were present in two distinct branches found in sequevars 12 and 48 (reference strains R292 and M2, respectively).

Susceptibility of Geranium Cultivars to Ralstonia solanacearum

Sixty-one cultivars of geraniums, including zonal, regal, ivy, and scented, were tested for susceptibility to three strains of Ralstonia solanacearum: a race 1, biovar 1 (R1B1) strain P597 isolated from tomato in Florida, a R1B1 strain P673 obtained from pothos originated from Costa Rica, and a race 3, biovar 2 (R3B2) strain UW551 isolated from geranium imported from Kenya. These three strains represent populations of R. solanacearum found in the United States or imported with infected plant propagative material. A genomic comparison of the geranium cultivars was also done using amplified fragment length polymorphisms. Both R1B1 strains were more virulent than the R3B2 strain, producing wilt symptoms on most cultivars of zonal, regal, and ivy types. Variation in susceptibility of geranium cultivars to the two R1B1 strains was observed. The R3B2 strain UW551 had a much more restricted host range and was not able to infect most regal geranium cultivars when applied as a soil drench. Many of the scented cultivars were found to be resistant to all three strains of R. solanacearum when tested using the drench inoculation method. However, most scented cultivars were found to be susceptible when plants were wound-inoculated. The greatest variation in type of resistance was observed between the scented geranium cultivars and specific strains of R. solanacearum.

Study on intraspecific diversity of Ralstonia solanacearum strains in West Malaysia using whole cell fatty acid analysis

Surveys were conducted between the years of 2005 and 2006 at several locations in the northern, central and southern parts of West Malaysia to study the polymorphism of Ralstonia solanacearum strains. These sites included vegetables and farms with known hosts of the pathogen, such as banana, tomato, eggplant, chili and tobacco. Samples were collected from the suspected wilted plants and weeds, including soil and water samples, in selected areas. The bacterium was isolated in all samples using semi-selective tetrazolium chloride medium (TZC). The bacteria strains were detected by using the BIOLOG identification system and were confirmed by nested-PCR. Fatty Acid Methyl Esters (FAME) profiling was performed to determine polymorphism among 58 bacterial isolates. The results showed that the fatty acid composition varied for all R. solanacearum isolates. Grouping of R. solanacearum isolates by fatty acid composition suggested that the existence of distinct groups that were significantly related to host of bacteria but low correlation between fatty acid profiles and biovar or sampling site was detected. A unique FAME profile was found among the strains that have been isolated from banana.