Jacques Luisetti

Characterization of phenotypically distinct strains of Xanthomonas axonopodis pv. citri from Southwest Asia

Strains of Xanthomonas axonopodis pv. citri were isolated from Mexican lime (Citrus aurantifolia) trees in several countries in southwest Asia. These strains produced typical erumpent bacterial canker lesions on Mexican lime but not on grapefruit (C. paradisi). Lesions on grapefruit were watersoaked and blister-like in contrast to the typical erumpent lesions seen after artificial inoculation with all described pathotypes of X. axonopodis pv. citri. This group of strains hydrolysed gelatin and casein and grew in the presence of 3% NaCl as is typical of X. axonopodis pv. citri pathotype A. RFLP analyses and DNA probe hybridization assays also gave results consistent with X. axonopodis pv. citri pathotype A. Metabolic fingerprints prepared with the Biolog® system showed similarities as well as differences to X. axonopodis pv. citri pathotype A. In spite of the physiological and genetic similarities to pathotype A of X. axonopodis pv. citri, these strains had no or very little affinity for polyclonal antiserum prepared against any of the reference strains of X. axonopodis pv. citri and also did not react with monoclonal antibody A1, an antibody that detects all strains of pathotype A of X. axonopodis pv. citri. These strains were also insensitive to bacteriophage Cp3 like X. axonopodis pv. citri pathotype A and unlike X. axonopodis pv. citri pathotype B. We conclude that these strains, designated Xcc-A*, represent a variant of X. axonopodis pv. citri pathotype-A with pathogenicity limited to C. aurantifolia. The existence of extensive genotypic and phenotypic variation within pathotype A of X. axonopodis pv. citri was unexpected and further complicates the systematics of this species.

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 F2:3 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.

Evaluation of procedures for reliable PCR detection of Ralstonia solanacearum in common natural substrates.

Several procedures were compared for reliable PCR detection of Ralstonia solanacearum in common substrates (plant, seed, water and soil). In order to prevent the inhibition of PCR by substances contained in crude extracts, numerous DNA extraction procedures as well as additives to buffers or PCR mixtures were checked. Our results showed that the efficiency of these methods or compounds depended greatly upon the nature of the sample. Consequently, preparation of samples prior to PCR depended upon sample origin. Simple methods such as a combined PVPP/BSA treatment or the association of filtration and centrifugation for detecting the bacterium in plant or water samples were very powerful. DNA capture also efficiently overcame PCR inhibition problems and ensured the detection of R. solanacearum in environmental samples. However, the commercial DNA extraction QIAamp kit appeared to be the most effective tool to guarantee the accurate PCR detection of the pathogen whatever the origin of the sample; this was particularly true for soil samples where the commonly used methods for the detection of R. solanacearum were inefficient. This study demonstrates that using an appropriate procedure, PCR is a useful and powerful tool for detecting low levels of R. solanacearum populations in their natural habitats.

Specific detection of biovars of Ralstonia solanacearum in plant tissues by Nested-PCR-RFLP

A sensitive and specific assay, based on a Nested-PCR-RFLP protocol, was developed for the detection of biovars of Ralstonia solanacearum, the causal agent of bacterial wilt. Oligonucleotide primer pairs were selected within the hrp gene region. Specific amplification of the hrp fragments was obtained for all R. solanacearum strains and also for two closely related species, Pseudomonas syzygii and the blood disease bacterium. No amplification was observed for a wide range of other bacterial species, including R. pickettii and Burkholderia cepacia. Digestion with HindII provided four distinct restriction profiles specific to biovars or groups of biovars of R. solanacearum: one for biovar 1 strains originating from the Southern part of Africa, one for American biovar 1 and biovars 2 and N2 strains, one for biovars 3 and 4 strains, and one for biovar 5 strains. When applied to either pure culture or infected plant tissues, Nested-PCR allowed detection as low as 103 cfu ml−1, which corresponds to 1 cfu per reaction. Amplification was partially or completely inhibited by compounds contained in plant extracts (potato plant and potato tuber, tomato, tobacco, eggplant, pepper and Pelargonium asperum). A combined PVPP/BSA treatment prior to amplification permitted reliable Nested-PCR detection of R. solanacearum strains in plant samples. Nested-PCR-RFLP, assessed with isolates from Reunion Island but also applicable to any R. solanacearum strain, provides a wide range of possible uses for identification, detection and epidemiological investigations.

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.

Bacterial Canker of Wild Cherry Tree in France Caused by a new Pathovar of Pseudomonas syringae pv. avii (pv. nov.)

This paper described a novel bacterial pathogen that causes canker on wild cherry tree. This bacterium belongs to the Pseudomonas syringae group (LOPAT Ia). Based on pathogenicity tests, phenotypic traits and molecular characteristics, it is proposed as a new P. syringae pathovar, pv. avii.

A Tentative Explanation of the Distribution, on Reunion Island, of Bacterial Wilt Caused by Either Biovar 2 or Biovar 3 of Ralstonia solanacearum

In the Reunion Island, the distribution of race 1 and race 3 strains of Ralstonia solanacearum as determined by the wilting of susceptible plants, is clearly connected with the altitude of cropped areas, so that the temperature could be considered as the main discriminant factor. Experiments were performed in a controlled environment chamber in order to test the hypothesis of a discriminant effect of the temperature on the multiplication in tomato seedlings of two representative strains and on the issued wilting rate. Results indicate that temperature could be responsible for the lack of biovar 3 strains in cool areas but not for the very low frequency of bacterial wilt caused by biovar 2 strains recorded in warm areas. Co-inoculations of both strains point out a rather weak competitiveness of the biovar 2 strain that could be the starting point of a more complicated explanation.

Spatial and Temporal Variations in Size and Phenotypic Structure of Populations of Pseudomonas Syringae on Fruit Trees

Most pathovars of Pseudomonas syringae (P. syr.) are known to be epiphytes on their host plants. According to Leben (1965), this means that these phytopathogenic bacteria are able to survive on the aerial parts of the plants when the conditions are not favourable (mainly high temperature and low humidity), and to multiply significantly when they become favourable again (moisture after rain or dew). Quantitative variations have been reported for the epiphytic populations of P. syr. pathovars occurring on fruit trees (Crosse, 1959; English and Davis, 1960; Panagopoulos and Crosse, 1964; Panagopoulos, 1966; Ercolani, 1969; Gardan et al., 1972; Luisetti and Paulin, 1972; Latorre and Jones, 1979; Burr and Katz, 1984; Roos and Hatting, 1986; Bordjiba and Prunier, 1991) depending mainly on the stage of vegetative development but also on the plant genotype or on the climatic conditions (Latorre et al., 1985). As an example, Figure 1 shows the variations of the epiphytic populations of P. syr. pv. persicae, the causal agent of a severe die-back of peaches: a high level in spring followed by a strong decrease during summer and a significant increase during leaf fall.