Characterization of Acidovorax citrulli strains isolated from solanaceous plants

Acidovorax citrulli is the causal agent of bacterial fruit blotch disease of cucurbits. Strains of this pathogen are distributed into two major groups: Group I strains have been mainly isolated from melon and other non-watermelon cucurbits, while Group II strains have been mainly recovered from watermelon. Here we report the characterization of strains T1 and EP isolated from diseased tomato and eggplant plants, respectively, and further confirmed to belong to A. citrulli species. Based on PCR, PFGE, and rep-PCR, these strains showed high similarity to the Group II strain 7a1. Sequencing and comparative analyses revealed that the genomes of T1 and EP aligned with that of the Group II model strain AAC00-1, over 97.88% and 99.22%, respectively. The virulence of T1, EP, and 7a1 determined on tomato, eggplant, and watermelon was similar and significantly higher than that of Group I strain M6. In contrast, M6 was more virulent on melon. Expression levels of seven virulence genes measured 24 hr after inoculation of tomato, eggplant, watermelon, and melon showed that the expression pattern was generally similar in strains 7a1, T1, and EP, whereas for M6 the expression was high only on melon. Overall, our results indicate that the solanaceous strains belong to Group II. To the best of our knowledge, this is the first study that reports characterization of A. citrulli strains isolated from solanaceous species. The fact that A. citrulli is able to naturally colonize and cause disease in non-cucurbit crops poses additional challenges for management of this important pathogen.     

Erwinia amylovora populations resistant to oxolinic acid in Israel: prevalence, persistence and fitness

Oxolinic acid (OA) has been the only bactericide used against fire blight in pear and quince orchards in Israel since 1998. OA-resistant Erwinia amylovora strains (Ea-OA^R) were detected in several orchards in two restricted areas in the northern Galilee region during 1999–2001. In the following years, resistant strains could not be detected in some of these locations. Documenting the fate of Ea-OA^R strains in commercial orchards at eight sites in northern Israel during 2000/03 revealed that the resistant population appeared irrespective of the number of sprays applied and the severity of the disease. The persistence of the Ea-OA^R populations varied from site to site, ranging from 4 to 20 months; these differences could be attributed to the fire blight management activities of growers. Comparative studies on the fitness of Ea-OA^R and E. amylovora strains sensitive to OA (Ea-OA^S) were conducted in vitro and in planta using two strains of each group. In four of the six comparisons, disease incidence on detached blossoms inoculated with Ea-OA^S was significantly higher than that on blossoms inoculated with Ea-OA^R. In two experiments conducted on 8-year-old pear trees grown under netting, the colonization of Ea-OA^S in blossoms, annual shoots and perennial spurs was significantly higher than that of the Ea-OA^R. In two experiments conducted on 2-year-old trees grown under netting in an experimental station, the incidence of shoots exhibiting fire blight symptoms and the rate of symptom progress within the branches were significantly higher in trees inoculated with Ea-OA^S than in those inoculated with Ea-OA^R. The results of this study suggest that OA-resistant E. amylovora strains have lower fitness than wild-type strains. These findings may have implications for fire blight management.     

Efficacy of oxolinic acid and other bactericides in suppression ofErwinia amylovora in pear orchards in Israel

The efficacy of oxolinic acid (at 200 and 300 μg a.i./l) and of several antibiotic compounds (streptomycin sulfate at 100 μg a.i./l, glycocide B at 700 μg a.i./l, kasugamycin at 80 μg a.i./l and gentamicin sulfate at 30 and 60 μg a.i./l) againstErwinia amylovora, the causal agent of fire blight in pears, was evaluated in 43 orchard experiments in 1997–2000 in Israel. In addition to the above orchard experiments, the efficacy of the bactericides was tested in live experiments with artificial inoculation. Natural fire blight symptoms were observed in 16 of the 43 experiments; in 13 of them, disease intensity and its distribution among the experimental plots provided a basis for data analysis, leading to reliable conclusions concerning the efficacy of the tested bactericides. Oxolinic acid at 300 μg a.i./l was highly effective againstE. amylovora and reduced disease severity significantly in all experiments, as compared with the untreated plots; however, a concentration of 200 μg a.i./l was not effective in some cases. Among the tested antibiotics, only gentamicin sulfate was as effective as oxolinic acid. Results of the artificial inoculation experiments corroborated those obtained in the naturally infected orchards. The pre-infection activity of oxolinic acid was determined on blossom clusters that were sprayed with the bactericide before inoculation. Control efficacy on blossom clusters sprayed 1–4 days before inoculation ranged from 68% to 80%, a level which did not differ significantly from that observed on blossom clusters sprayed on the day of inoculation (80% control). The postinfection activity of oxolinic acid was determined on blossom clusters that were sprayed with the compound after inoculation. Oxolinic acid was as effective when applied 1 or 2 days after inoculation as when it was applied on the day of inoculation; however, application of the bactericide 3 days after inoculation no longer resulted in significant disease suppression. Oxolinic acid has been used commercially in Israel since 1998 with appreciable success. Contribution No. 533-00 from the Agricultural Research Organization     

Characterization of Pectobacterium brasiliense strains from potato and vegetables in Israel

Pectobacterium brasiliense (Pbr) infects a wide range of crops worldwide, causing potato blackleg and soft rot and vegetable soft rots. This study aimed to characterize the genetic diversity and virulence variability among 68 Pbr strains isolated from either symptomless potato progeny tubers, diseased potato plants, ware potatoes wash water, or vegetables grown in Israel, as well as strains isolated from symptomless seed tubers grown in Europe, or diseased potato plants grown in France. The collection was typed using PCR and TaqMan real-time PCR analyses, dnaX sequence analysis, pulsed-field gel electrophoresis (PFGE), and pectolytic activity. dnaX phylogeny grouped almost all strains in a common genetic clade related to Pbr, which was distinct from the other Pectobacterium species. PFGE analysis identified two main clusters, including one major group of 47 strains with 95%–100% similarity. Maceration assays on two potato cultivars showed significant differences between strains but with no correlations with the source of the strains nor the status of the host (with/without symptoms). Molecular (dnaX sequences and PFGE profiles) and phenotypic analyses (tuber maceration tests) showed that the tested Pbr strains are not a homogeneous group. Analysis of the tested Pbr strains isolated from potato and vegetables grown in fields with a history of potato cultivation suggests that seed tubers imported from Europe may be the main source for Pbr in Israel. To the best of our knowledge, this is the first study that describes biodiversity and population structure of P. brasiliense isolated from potato and vegetables under hot climate conditions.     

The significance of guttation in the secondary spread of Clavibacter michiganensis subsp. michiganensis in tomato greenhouses

Bacterial canker, caused by Clavibacter michiganensis subsp. michiganensis (Cmm), can spread in commercial tomato greenhouses causing epidemics. Results of greenhouse experiments with Cmm‐contaminated tools demonstrated disease spread for only a limited distance (<4 plants) from infected plants. However, touching symptomless infected plants bearing guttation droplets prior to touching nearby plants spread the pathogen over longer distances within rows (>22 plants). The pathogen was exuded in large numbers in the guttation fluid of infected plants; its presence in the guttation fluid was not influenced by the inoculation procedures, leaf age or the volume of the guttation droplets. Population size of Cmm and the incidence of leaflets with epiphytic bacteria were significantly higher in plants placed in a guttation‐induction chamber than in those kept in a growth chamber with high humidity, suggesting exudation through guttation contributed to the formation of epiphytic populations on leaflets. This new knowledge may provide a simple and environmentally friendly means for decreasing the spread of the disease by avoiding contact with plants during periods when they bear guttation droplets.     

Characterization of theErwinia amylovora population in Israel

A collection of 205 strains ofErwinia amylovora isolated in Israel over a period of 12 years has been established. The strains were isolated from different varieties of pear, apple, loquat and quince grown in Israel, and collected from different locations in the country. They were characterized in respect to degree of virulence on several hosts and serological and molecular characters. Pathogenicity tests carried out on flowering branches of pear and apple, shoots of pears, and on trees of pear and loquat grown in containers outdoors, revealed no significant differences in the severity of blossom blight or shoot blight among the various strains. ELISA and immunofluorescence assays revealed no serotypic groups among the Israeli strains. Genomic diversity was studied by random amplified polymorphic DNA (RAPD) analysis using 24 arbitrary 10-base primers. All the strains examined (45 Israeli and 11 from Egypt, Cyprus and Greece) produced the same RAPD patterns with each of the primers used. Amplification patterns were indistinguishable from those produced by strains isolated from the neighboring countries. Results presented in this study suggest that the population ofE. amylovora in Israel is homogenous.     

Cotyledons are the main source of secondary spread of Acidovorax citrulli in melon nurseries

Acidovorax citrulli (Ac) is the causal agent of bacterial fruit blotch (BFB) disease resulting in substantial economic damage to cucurbit crops worldwide. The plant parts and cultural practices (irrigation methods and bactericidal sprays) that affect the secondary spread of Ac in melon nurseries were investigated in this study. Overhead irrigation dispersed the pathogen from infected seedlings to 95% of the neighbouring healthy seedlings, with 80% of them displaying high disease severity. In contrast, when sub‐irrigation by floating was employed, the neighbouring plants of the infected ones did not display disease symptoms and were not colonized by Ac. Foliar treatment with Kocide after cotyledon emergence reduced disease incidence to 40%, with 37% of the plants displaying low disease severity. Assessment of Ac populations in different parts of the seedlings revealed that cotyledons were the most colonized part of the plant. Images of fluorescent binocular and confocal laser‐scanning microscopy of seedlings infected with a GFP‐labelled Ac strain showed that the pathogen forms abundant aggregates on the surface of cotyledons, colonizes the intercellular spaces of the parenchymatic tissues extensively, and moves through the vascular system of the hypocotyls, leading to infection of emerging leaves. Results of this study indicate that preventing secondary spread of Ac in melon nurseries by sub‐irrigation combined with a bactericidal spray at the cotyledon stage may provide an effective means for BFB control.     

Characterization of Dickeya strains isolated from potato grown under hot‐climate conditions

Dickeya strains isolated in Israel in 2006–2010 were characterized by dnaX sequence analysis, pulsed‐field gel electrophoresis (PFGE), biochemical assays and pectolytic activity, and found to be homogeneous: most of them could be classified as ‘Dickeya solani’. Of the 34 strains isolated from imported seed tubers or potato plants grown from imported seed, 32 were typed as ‘D. solani’ and only two were characterized as Dickeya dianthicola. Biovar typing indicated that all ‘D. solani’ strains were biovar 3. ‘Dickeya solani’ strains were most closely related to Dickeya dadantii subsp. dieffenbachiae according to PFGE and dnaX analyses and both species exhibited high pectolytic activity. Expression levels of two putative virulence genes, pelL (encoding a pectic enzyme) and dspE (encoding a type III effector) were significantly induced in ‘D. solani’ strains isolated from potato plants or tubers grown in hot climates such as the Negev region in Israel, compared to those isolated from seed tubers imported from the Netherlands, France or Germany. Results of this study support the hypothesis that ‘D. solani’ strains isolated in Israel are also clonal; however, they appear to be more virulent than strains isolated in Europe.     

Molecular diagnostic procedures for production of pathogen-free propagation material

Production of disease-free propagation material is a major means of controlling most bacterial diseases of plants, particularly when neither resistant clones nor effective chemical treatments are available. For this purpose sensitive, specific and rapid detection methods are required. The advent of molecular biology and, in particular, the polymerase chain reaction (PCR) has opened new ways for the characterization and identification of plant pathogens and the development of disease-management strategies. PCR-based detection methods rely on the development of primers for the specific detection of the pathogen. The use of pathogenicity genes as targets for primer design is the preferred procedure for obtaining specific primers but other procedures may also be useful for this purpose. In the present review we describe four examples of procedures for detecting four important bacterial pathogens in Israel: Erwinia herbicola pv gypsophilae in gypsophila, Xanthomonas campestris pv pelargonii in geranium, Agrobacterium tumefaciens in asters and roses, and Xanthomonas campestris pv campestris in crucifers. Procedures for constructing specific PCR primers for each bacterium are illustrated and discussed as well as the combination of PCR with other methods.