Identification of QTLs for Ralstonia solanacearum race 3-phylotype II resistance in tomato.

Resistance against a Ralstonia solanacearum race 3-phylotype II strain JT516 was assessed in a F(2:3) and a population of inbred lines (RIL), both derived from a cross between L. esculentum cv. Hawaii 7996 (partially resistant) and L. pimpinellifolium WVa700 (susceptible). Resistance criteria used were the percentage of wilted plants to calculate the AUDPC value, and bacterial colonization scores in roots and stem (hypocotyl and epicotyl) assessed in two independent greenhouse experiments conducted during the cool and hot seasons in Réunion Island, France. Symptoms were more severe during the cool season trials. Heritability estimates in individual seasons ranged from 0.82 to 0.88, depending on resistance criterion. A set of 76 molecular markers was used for quantitative trait loci (QTL) mapping using the single- and composite- interval mapping methods, as well as ANOVA. Four QTLs, named Bwr- followed by a number indicating their map location, were identified. They explained from 3.2 to 29.8% of the phenotypic variation, depending on the resistance criterion and the season. A major QTL, Bwr-6, and a minor one, Bwr-3, were detected in each season for all resistance criteria. Both QTLs showed stronger effects in the hot season than in the cool one. Their role in resistance to R. solanacearum race 3-phylotype II was subsequently confirmed in the RIL population derived from the same cross. Two other QTLs, Bwr-4 and Bwr-8, with intermediate and minor effects, respectively, were only detected in the hot season, demonstrating that environmental factors may strongly influence the expression of resistance against the race 3-phylotype II strain JT516. These QTLs were compared with those detected in the RIL population against race 1-phylotype I strain JT519 as well as those detected in other previous studies in the same genetic background against other race 1-phylotype I and II strains. This comparison revealed the possible occurrence of some phylotype-specific resistance QTLs in Hawaii 7996.

Partial sequencing of the hrpB and endoglucanase genes confirms and expands the known diversity within the Ralstonia solanacearum species complex.

We determined partial hrpB and endoglucanase genes sequences for 30 strains of Ralstonia solanacearum and one strain of the blood disease bacterium (BDB), a close relative of Ralstonia solanacearum. Sequence comparisons showed high levels of variability within these two regions of the genome involved in pathogenicity. Phylogenetic analysis based upon sequence comparisons of these two regions revealed three major clusters comprising all Ralstonia solanacearum isolates, the BDB strain constituted a phylogenetically distinct entity. Cluster 1 and cluster 2 corresponded to the previously defined divisions 1 and 2 of Ralstonia solanacearum. Moreover, two subclusters could be identified within cluster 2. The last cluster, designated cluster 3 in this study, included biovar 1 and N2 strains originating from Africa. This recently described group of strains was confirmed to be clearly different from the other strains suggesting a separate evolution from those of both divisions 1 and 2.

Resistance to bacterial wilt in somatic hybrids between Solanum tuberosum and Solanum phureja.

Somatic hybrid plants were produced after protoplast electrofusion between a dihaploid potato, cv. BF15, and a wild tuber-bearing relative, Solanum phureja, with a view to transferring bacterial wilt resistance into potato lines. A total of ten putative hybrids were selected. DNA analysis using flow cytometry revealed that six were tetraploids, two mixoploids, one amphiploid and one octoploid. In the greenhouse, the putative hybrids exhibited strong vigor and were morphologically intermediate, including leaf form, flowers and tuber characteristics. The hybrid nature of the ten selected plants was confirmed by examining isoenzyme patterns for esterases and peroxidases, and analysis of RAPD and SSR markers. Analysis of chloroplast genome revealed that eight hybrids possessed chloroplast (ct) DNA of the wild species, S. phureja, and only two contained Solanum tuberosum ct type. Six hybrid clones, including five tetraploids and one amphiploid, were evaluated for resistance to bacterial wilt by using race 1 and race 3 strains of Ralstonia solanacearum, originating from Reunion Island. Inoculations were performed by an in vitro root dipping method. The cultivated potato was susceptible to both bacterial strains tested. All somatic hybrids except two were tolerant to race 1 strain, and susceptible to race 3 strain. Interestingly, the amphiploid hybrid clone showed a good tolerance to both strains.

Genetic diversity of african and worldwide strains of ralstonia solanacearum as determined by PCR-restriction fragment length polymorphism analysis of the hrp gene region

The genetic diversity among a worldwide collection of 120 strains of Ralstonia solanacearum was assessed by restriction fragment length polymorphism (RFLP) analysis of amplified fragments from the hrp gene region. Five amplified fragments appeared to be specific to R. solanacearum. Fifteen different profiles were identified among the 120 bacterial strains, and a hierarchical cluster analysis distributed them into eight clusters. Each cluster included strains belonging to a single biovar, except for strains of biovars 3 and 4, which could not be separated. However, the biovar 1 strains showed rather extensive diversity since they were distributed into five clusters whereas the biovar 2 and the biovar 3 and 4 strains were gathered into one and two clusters, respectively. PCR-RFLP analysis of the hrp gene region confirmed the results of previous studies which split the species into an "Americanum" division including biovar 1 and 2 strains and an "Asiaticum" division including biovar 3 and 4 strains. However, the present study showed that most of the biovar 1 strains, originating from African countries (Reunion Island, Madagascar, Zimbabwe, and Angola) and being included in a separate cluster, belong to the "Asiaticum" rather than to the "Americanum" division. These African strains could thus have evolved separately from other biovar 1 strains originating from the Americas.

Phenotypic diversity of Xanthomonas sp. mangiferaeindicae.

Carbohydrate utilization profiles by means of the API (Appareils et Procédés d'Identification) system and sensitivity to antibiotics and heavy metal salts of 68 Xanthomonas sp. mangiferaeindicae strains isolated in nine countries from mango (Mangifera indica L.) and other genera of the Anacardiaceae were examined to assess the variability of the taxon. The strains could be separated into 10 groups according to Ward clustering. Apigmented strains isolated from the pepper tree [syn. Brazilian pepper] (Schinus terebenthifolius Raddi) could not be clearly differentiated from most apigmented strains isolated from mango. Yellow-pigmented strains isolated from mango in Brazil and Reunion Island, apigmented strains isolated from mango in Brazil and from ambarella in the French West Indies, clustered in distinct groups. The results are consistent with those of other studies, based on isozyme analysis of esterase, phosphoglucomutase and superoxide dismutase, and hrp-RFLP analysis; they indicate the need for a comprehensive taxonomic evaluation of xanthomonads associated with Anacardiaceae.

Evaluation of the Biolog Substrate Utilization System To Identify and Assess Metabolic Variation among Strains of Xanthomonas campestris pv. Citri.

Metabolic fingerprints of 148 strains of Xanthomonas campestris pv. citri originating from 24 countries and associated with various forms of citrus bacterial canker disease (CBCD) were obtained by using the Biolog substrate utilization system. Metabolic profiles were used to attempt strain identification. Only 6.8% of the studied strains were correctly identified when the commercial Microlog 2N data base was used alone. When the data base was supplemented with data from 54 strains of X. campestris pv. citri (40 CBCD-A strains, 8 CBCD-B strains, and 6 CBCD-C strains) and data from 43 strains of X. campestris associated with citrus bacterial spot disease, the percentage of correct identifications was 70%. Thus, it is recommended that users supplement the commercial data base with additional data prior to using the program for identification purposes. The utilization of Tween 40 in conjunction with other tests can help to differentiate strains associated with CBCD and citrus bacterial spot disease. These results confirmed the separation of X. campestris pv. citri into different subgroups (strains associated with Asiatic citrus canker [CBCD-A], cancrosis B [CBCD-B], and Mexican lime canker [CBCD-C]). The utilization of l-fucose, d-galactose, and alaninamide can be used as markers to differentiate strains associated with these groups. A single strain associated with bacteriosis of Mexican lime in Mexico (CBCD-D) was closely similar to CBCD-B strains.

Lateral and transversal diffusion and phase transitions in erythrocyte membranes. An excimer fluorescence study.

The lateral diffusion of the excimer-forming probe pyrene decanoic acid has been determined in erythrocyte membranes and in vesicles of the lipid extracts. The random walk of the probe molecules is characterized by their jump frequency, nu j, within the lipid matrix. At T = 35 degrees C a value of nu i = 1.6 . 10(8) s-1 is found in erythrocyte membranes. A somewhat slower mobility is determined in vesicles prepared from lipid extracts of the erythrocyte membrane. Depending on structure and change of the lipids we obtain jump frequencies between 0.8 . 10(8) s-1 and 1.5 . 10(8) s-1 at T - 35 degrees C. The results are compared with jump frequencies yielded in model membranes. The mobility of molecules perpendicular to the membrane surface (transversal diffusion) is investigated. Erythrocyte ghosts doped with pyrene phosphatidylcholine were mixed with undoped ghosts in order to study the exchange kinetics of the probe molecule. A fast transfer between the outer layers of the ghost cells tau 1/2 = 1.6 min at T = 37 degrees C) is found. The exchange process between the inner and the outer layer of one erythrocyte ghost (flip-flop process) following this fast transfer occurs with a half-life time value of t 1/2 = 100 min at T = 37 C. The application of excimer-forming probes presumes a fluid state of the membrane. Therefore we investigated the phase transition behaviour using the excimer technique. Beside a thermotropic phase transition at T = 38 degrees C and T = 33 degrees C we observed an additional fluidity change at T = 38 degrees C in erythrocyte ghost. This transition is attached to a separation of the boundary lipid layer from the intrinsic proteins. No lipid phase transition is observed in liposomes from isolated extracts of the erythrocyte membrane with our methods.

Monitoring the location profile of fluorophores in phosphatidylcholine bilayers by the use or paramagnetic quenching.

Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short range interaction we developed a spectroscopic method to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase. This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.