Régine Samson

Erwinia carotovora subsp. odorifera subsp. nov., associated with odorous soft rot of chicory (Cichorium intybus L.).

Eleven strains of Erwinia carotovora that were isolated mainly, but not exclusively, from slimy rot of witloof chicory and were previously designated “atypical” E. carotovora subsp. atroseptica strains were characterized and compared with strains of E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi (including the type strains). The 11 atypical E. carotovora subsp. atroseptica strains produced a typical bananalike odor when they were inoculated onto witloof chicory leaves. DNA-DNA homology experiments, biochemical tests, tests to determine carbon utilization patterns, and tests to identify the volatile metabolites produced from rotting witloofs were performed. The volatile end products of witloof decay were analyzed by gas chromatography. Alcohols, methylketones, and ethylacetate were produced by all of the Erwinia strains which we studied, whereas propyl acetate, isobutyl acetate, isoamyl acetate, and 2-actamyl acetate were produced only by the flavoring witloof soft-rot strains. A DNA relatedness study was performed by hybridizing DNAs with a tritium-labeled DNA and estimating the δTm values (δTm is the difference between the thermal denaturation midpoint of a homoduplex and the thermal denaturation midpoint of a heteroduplex). The 11 flavoring strains constituted a tight DNA hybridization group (79 to 91% related to type strain CFBP 1878 isolated from witloof). Strains of E. carotovora subsp. carotovora were 59 to 88% related to strain CFBP 1878T (T = type strain) (δTm range, 3 to 4.5°C), indicating that they belonged to the same species but another subspecies. E. carotovora subsp. atroseptica and E. carotovora subsp. betavasculorum appeared to be less closely related to strain CFBP 1878T than E. carotovora subsp. carotovora was, exhibiting 53% homology (δTm, 7°C) and 48 to 51% homology (δTm, 8.5°C), respectively, with strain CFBP 1878T. Therefore, we propose that the 11 flavoring strains are members of a new subspecies, Erwinia carotovora subsp. odorifera. We examined 95 biochemical characteristics, API strip tests, and assimilation tests in Biotype galleries and identified nine tests which can be used for phenotypic differentiation of the new subspecies.

The Relationship of Host Range, Physiology, and Genotype to Virulence on Cantaloupe in Pseudomonas syringae from Cantaloupe Blight Epidemics in France.

ABSTRACT In 1993, a bacterial blight caused important losses of cantaloupe (Cucumis melo var. cantalupensis) in southwestern France and has now been reported in all cantaloupe-growing regions of France. The causal agent of this blight is Pseudomonas syringae, although on a worldwide basis this bacterium has not been a major pathogen of melon for over 50 years. To identify the pathovar of the cantaloupe pathogen, we employed biochemical tests, plasmid and chromosomal profiling, and host range studies for 23 strains from cantaloupe and 47 reference strains of 14 pathovars of P. syringae. Numerical analysis of 119 traits, serological typing, syringomycin production, and BOX-polymerase chain reaction profiles did not allow us to differentiate among pathovars related to P. syringae pv. syringae. Host range studies of cantaloupe and references strains on 18 plant species showed that virulence to sugar beet was a common feature of strains virulent on cantaloupe, but was not common to strains avirulent on cantaloupe. Virulence to other species of plants varied among strains, but the overall extent of the host range was proportional to aggressiveness to cantaloupe. We propose that the strains attacking cantaloupe in France be considered P. syringae pv. aptata and that adequate host range testing may reveal that this pathovar is the cause of cantaloupe blight reported in other parts of the world.

Multiphasic approach for the identification of the different classification levels of Pseudomonas savastanoi pv. phaseolicola

The relationships between strains of Pseudomonas savastanoi pv. phaseolicola (P. sav. phaseolicola), P. syringae pv. tabaci (P. syr. tabaci) and P. syr. syringae which all cause disease on bean; the related species P. sav. glycinea and P. syr. actinidiae, and reference bacteria, were evaluated by studying the phenotypic and genetic diversity of a collection of 62 strains. All the P. sav. phaseolicola strains tested produced characteristic watersoaked lesions on bean pods. Other pathovars produced varying combinations of symptoms including necrotic lesions, with or without watersoaked centres and sunken tissue collapse of the lesion (P. syr. tabaci) and necrotic lesions with or without sunken collapse (P. syr. syringae). At the genomospecies level, all the strains of P. sav. phaseolicola, P. sav. glycinea and P. syr. tabaci, belonging to genomospecies 2, could be separated from P. syr. syringae strains (genomospecies 1) and P. syr. actinidiae strains (unknown genomospecies) by BOX-PCR and DNA/DNA hybridisation. To distinguish P. sav. phaseolicola, within genomospecies 2, from P. sav. glycinea and P. syr. tabaci, it was necessary to perform nutritional characterisations myo-inositol negative and p-hydroxy benzoate positive for P. sav. phaseolicola strains), PCR with specific primers designed from the tox region (positive for all of the P. sav. phaseolicola strains) and serotyping, as 71% of the P. sav. phaseolicola strains reacted as O-serogroup PHA1. Important intrapathovar variation was seen by genomic fingerprinting with REP and ERIC primers, as well as with RAPD primers (AE7 and AE10) and esterase profilings. While RAPD fingerprinting detected variability correlated with two race-associated evolutionary lines, REP, ERIC and esterase profiles revealed intrapathovar variation linked to some host origins, that separated the kudzu isolates, and the mungbean isolates, from the other P. sav. phaseolicola strains.

A Review of Thermotherapy to Free Plant Materials from Pathogens, Especially Seeds from Bacteria

Abstract Treating planting materials with heat is a one-century-old method of disease control that has proved to be efficient against various pathogenic microorganisms. Thermotherapy, simple in principle, consists in heat treatment of plant parts at temperature/time regimes that kill the conserved pathogen and that are only slightly injurious to the host. Heat is applied mainly by water, air, or vapor. A large variety of plant parts can be heat treated: Whole tree, scions, vitroplants, seedlings, stalks, cuttings, sprouts, cut flowers, seeds, bulbs, tubers, corms, or fruit and vegetables in storage. The target pathogenic microorganisms are mainly fungi, viruses, and bacteria. Many studies show the success of reducing diseases by heat. Thermotherapy also is the main means, associated with meristem and tip culture, of producing virus-free explants from infected mother-plants. Most bacterial diseases of annual plants are seed-borne. Seed-transmission provides numerous foci of primary infection in the field a...

BOX-PCR-based identification of bacterial species belonging to Pseudomonas syringae: P. viridiflava group

The phenotypic characteristics and genetic fingerprints of a collection of 120 bacterial strains, belonging to Pseudomonas syringae sensu lato group, P. viridiflava and reference bacteria were evaluated, with the aim of species identification. The numerical analysis of 119 nutritional characteristics did not show patterns that would help with identification. Regarding the genetic fingerprinting, the results of the present study supported the observation that BOX-PCR seems to be able to identify bacterial strains at species level. After numerical analyses of the bar-codes, all pathovars belonging to each one of the nine described genomospecies were clustered together at a distance of 0.72, and could be separated at genomic species level. Two P. syringae strains of unknown pathovars (CFBP 3650 and CFBP 3662) and the three P. syringae pv. actinidiae strains were grouped in two extra clusters and might eventually constitute two new species. This genomic species clustering was particularly evident for genomospecies 4, which gathered P. syringae pvs. atropurpurea, coronafaciens, garcae, oryzae, porri, striafaciens, and zizaniae at a noticeably low distance.

Description of the bacterium causing blight of leek as Pseudomonas syringae pv. porri (pv. nov.).

ABSTRACT Forty bacterial strains isolated from leek blight (Allium porrum) in France and other countries were studied by conventional biochemical methods, serological reactions, numerical taxonomy, DNA-DNA hybridization, and ice nucleation activity, as well as by pathogenicity on leek and other host plants. They were compared with reference strains of Pseudomonas, mainly pathotype strains of P. syringae pathovars and strains of P. syringae pv. syringae isolated from various host plants including onions. Leek strains sorted with P. syringae species (sensu lato) by LOPAT tests (production of levan-sucrase, oxidase, pectinase, arginine dihydrolase, and hypersensitive reaction on tobacco). Leek strains were pathogenic to leek and produced symptoms identical to those observed in the field. They were the only strains in our study that could cause blight of leek. Thus, our results justify the creation of a new pathovar. Leek strains constituted a highly homogeneous DNA group and a discrete phenon by numerical taxonomy, and they belonged to O-serogroup POR. The name of P. syringae pv. porri is proposed for the bacterium causing leek blight. Criteria for routine identification are presented and taxonomic status is discussed.

Identification of two serological flagellar types (H1 and H2) inPseudomonas syringae pathovars

Flagellar antigen specificity was studied for the speciesPseudomonas syringae, P. viridiflava andP. cichorii. After checking their motility, bacteria were reacted against six polyclonal antisera containing anti-O (LPS) and anti-H (flagellar) antibodies by indirect immunofluorescent staining. Two distinct flagellar serotypes (H1 and H2) were described. The distribution of H1 and H2 serotypes was then determined for a collection of 88 phytopathogenicPseudomonas strains. Serotype H1 was possessed byP. syringae pv.aptata (12 strains),P. s. pv.helianthi (2),P. s. pv.pisi (11), andP. s. pv.syringae (13). Serotype H2 was possessed byP. cichorii (2),P. s. pv.delphinii (1),P. s. pv.glycinea (4),P. s. pv.lacrymans (1),P. s. pv.mori (1),P. s. pv.morsprunorum (10),P. s. pv.persicae (1),P. s. pv.phaseolicola (8),P. s. pv.tabaci (10) andP. s. pv.tomato (1).P. viridiflava (5) revealed HI, H2 and untyped flagella. The following isolates were untypable by the H1/H2 system:P. corrugata (3),P. fluorescens (2),P. tolaasii (1). H1/H2 serotypes distribution is not linked toP. syringae O-serogroups. On the other hand, H1/H2 distribution seems remarkably linked to the new genospecies of theP. syringae group.

Epiphytic life is the main characteristic of the life cycle ofPseudomonas syringae pv.pisi, pea bacterial blight agent

Pseudomonas syringae pv.pisi, pea bacterial blight agent, is seed-transmitted. Some aspects of its life cycle and its biology were investigated. The colonization of pea plants obtained from naturally infected seeds was studied in natural conditions while high populations of bacteria developed on plants showing no symptoms. Two streptomycin-resistant mutants were used to study the epiphytic life of the pathogen. Populations were monitored in different host-parasite compatibilities. When race 2 or race 6 of the pathogen was surface-inoculated on susceptible cultivars, a decrease of population size was observed during the following one to three days but was followed by an increase to levels 1000 times greater than the initial number detected, without symptoms for most of the plants. When race 2 was surface-inoculated on resistant genotypes or race 6 on non-host plants, bacteria did not multiply but population levels slightly decreased.Pseudomonas syringae pv.pisi shows a resident phase and its development is race-specific. Weeds collected in naturally contaminated pea fields, diseased or not, often harboured the pathogen but with levels smaller than those observed on peas. Pea crop debris and volunteers kept high levels of bacteria for at least eight months after the harvest of a diseased crop. As long as two pea crops are not grown one after the other in the same field, it is unlikely that debris and volunteers will act as an important inoculum source. The development of this pathogen during the growing season is considered as an important parameter to take into account for controlling the disease through seed health testing.

Serological and Molecular Size Characterization of Flagellins of Pseudomonas syringae Pathovars and Related Bacteria

Flagella from a total of 118 strains representing mostly pathovars of the phytopathogenic group Pseudomonas syringae, but also P. chlororaphis, P. cichorii, P. corrugata, P. fluorescens, P. fuscovaginae, P. stutzeri, P. viridiflava, as well as related phytopathogenic genera (Burkholderia cepacia and Ralstonia solanacearum) were characterized by immuno-fluorescent staining, SDS-PAGE, and immunoblotting. Eighty-six strains of the P. syringae group pathovars, P. cichorii and P. viridiflava were shown to possess flagella of serotypes H1 or H2, composed of a unique flagellin, whose molecular size varied between 31 and 31.5 kDa. Similarities between the P. syringae flagellin and a 31 kDa surface protein involved in pathogenicity are pointed out. The distribution of H1 and H2 antigens in the nine recently described genomospecies of P. syringae-P. viridiflava group suggested that flagellin would represent a phylogenetic marker within the arginin-dihydrolase-negative fluorescent pseudomonads. The characterization of flagellin was proposed as an identification tool at a level situated between genus and species.

Pseudomonas Leek Blight: Study of Seed Transmission

In order to monitor transmission of Pseudomonas syringae pv. porri from seed to seedlings, bacterial populations were evaluated on seed and on transplants of the same seed lots. For this work, a new semi-selective medium was defined.