Isozyme analysis for the identification of Pseudomonassyringae pathovar pisi strains

Distinction between Pseudomonas syringae pathovar (pv.) pisi (Ps. syr. pisi) , responsible for bacterial blight of pea ( Pisum sativum ), and pv. syringae (Ps. syr. syringae) , still requires strain inoculation onto peas. Patterns of enzymes including esterase (EST) and superoxide dismutase (SOD) were examined for diagnostic purposes. Profiles of 59 Ps. syr . pisi strains and 53 Ps. syr . syringae strains were compared. Pseudomonas syringae pisi was characterized by one unique zymotype for SOD and two slightly different zymotypes for EST. Pseudomonas syringae syringae zymotypes were very heterogeneous with 10 different zymotypes for SOD and 32 for EST. Twenty-four percent of the Ps. syr . syringae strains shared SOD zymotype 1 of Ps. syr . pisi , thus preventing the use of this enzymatic system for identification. In contrast, the two EST zymotypes of Ps. syr. pisi strains were specific to the pathovar and could be used for its identification. The two Ps. syr. pisi EST patterns were correlated to race structure of the pathovar, zymotype 1 corresponding to races 2, 3, 4 and 6, and zymotype 2 to races 1, 5 and 7. Esterase isozyme profiling was proposed as a new identification procedure for bacterial pea blight agent.     

Evaluation of determinative tests for pathovars of Pseudomonas syringae van Hall 1902

The utility of 36 presumptive determinative tests for 32 pathovars of Pseudomonas syringae was investigated. A total of 395 strains was examined. Most strains of 12 of these pathovars ( Ps. syringae pv. cannabina, Ps. syr. delphinii, Ps. syr. glycinea, Ps. syr. helianthi, Ps. syr. lachrymans, Ps. syr. mori, Ps. syr. morsprunorum, Ps. syr. phaseolicola, Ps. syr. 'porri', Ps. syr. papulans, Ps. syr. savastanoi and Ps. syr. tabaci ) formed clusters when test data were compared by centroid analysis. Pseudomonas syr. syringae, Ps. syr. aptata, Ps. syr. atrofaciens, Ps. syr. dysoxyli and Ps. syr. japonica formed a single cluster, indicating their possible synonymy. Strains of Ps. syr. antirrhini and Ps. syr. tomato were indistinguishable, as were those of Ps. syr. garcae and Ps. syr. oryzae. Strains of Ps. syr. berberidis, Ps. syr. coronafaciens, Ps. syr. eriobotryae, Ps. syr. maculicola, Ps. syr. passiflorae, Ps. syr. pisi and Ps. syr. striafaciens and Ps. syr. tagetis did not form distinguishable clusters.
The tests which reliably differentiated pathovars are recorded in a determinative scheme.     

The development of a conductimetric assay for automated detection of metabolically active soft rot Erwinia spp. in potato tuber peel extracts

Four media were tested for their ability to detect the soft rot potato pathogens Erwinia chrysanthemi (Ech) and Erwinia carotovora ssp. atroseptica (Eca) in potato tubers by means of automated conductance measurements. The specificity of the conductimetric assays was determined by testing a set of different Erwinia spp. and potato-associated saprophytes, including the genera Pseudomonas, Bacillus, Enterobacter and Flavobacterium. All bacteria tested produced conductance responses in Special Peptone Yeast Extract, whereas in minimal medium with L-asparagine only Erwinia spp. and Pseudomonas spp. were able to generate large conductance responses. In minimal medium supplemented with glucose and trimethylamine- N -oxide only Enterobacteriaceae, Erwinia spp. included, generated conductance responses, while with pectate as sole carbon source only Erwinia spp. produced distinct conductance responses. The pectate medium proved to be particularly useful for specific automated conductimetric detection of Erwinia spp. in potato peel extracts. Within 48 h, the detection threshold of the conductimetric assay for Eca varied between 10² and 10³ cfu per ml peel extract at both incubation temperatures of 20° and 26°C. Ech was detected at concentrations of 10⁴–10⁵ or 10³–10⁴ cfu ml^-1 at 20° and 26°C, respectively. To eliminate 'false'-positive reactions in conductimetry caused by Erwinia carotovora ssp. carotovora , results of the conductance measurements have to be confirmed by other techniques, like serology or DNA assays.     

Sensitivity of Bacillus coagulans spores to combinations of high hydrostatic pressure, heat, acidity and nisin

We investigated the combined effects of pressure, temperature, pH, initial spore concentration and the presence of nisin on the survival of spores of Bacillus coagulans. Spores were more sensitive to pressure both at lower pH and at higher treatment temperatures. An additional 1.5-log₁₀ reduction in cfu ml^-1 was observed when pH was lowered from 7.0 to 4.0 during pressurization at 400 Mpa and 45°C. A 4-log₁₀ cfu ml^-1 reduction was observed when the temperature was increased from 25°C to 70°C during pressurization at 400 Mpa. The spores were sensitive to nisin at concentrations as low as 0.2 IU ml^-1. At least a 6-log₁₀ reduction was generally achieved with pressurization at 400 Mpa in pH 4.0 buffer at 70°C for 30 min when plated in nutrient agar containing 0.8 IU ml^-1 nisin.     

Resistance of bacterial strains to dry conditions: use of anhydrous silica gel in a desiccation model system

The Viability of 18 bacterial strains desiccated on anhydrous silica gel and stored at a temperature of 22°C for at least 3 months was determined. According to their stability in the dried state, these strains could be classified into three typical groups. Group 1, containing Gram-positive strains and Salmonella serotypes, was marked by a very slow decrease of the concentration of culturable cells from day 14 on (respectively day 21 for Salmonella thompson . The rate of decrease expressed as regression coefficient (b) ranged from —0.000389 to —0.00521 log (cfp ml^-1) per d. The Group 2 strains Enterobacter cloacae and Escherichia coli did not reach a comparable slow decrease in the dry material within the indicated time period. Regression coefficients were respectively —0.04406 and —0.03412 log (cfp ml^-1) per d. The reciprocal values —(1/b) were respectively 23 d per log (cfp ml^-1) and 29 d per log (cfp ml^-1), indicating the time periods in which a reduction of 1 log unit of culturable cells occurred. Group 3 strains Pseudomonas aeruginosa, Aeromonas hydrophila and Aer. sobria were marked by a significant susceptibility to cell damage caused during desiccation and reconstitution. A high initial decrease (ID) of the concentration of culturable organisms seems to be a characteristic property of these bacterial strains: culturable organisms could not be detected after storage for 1 d ( Aer. hydrophila, Aer. sobria ) or 7 d ( Ps. aeruginosa ). The wide range of resistance of the different bacterial strains tested indicated that the silica gel model system is a suitable tool for microbiological challenge tests to investigate the survival of micro-organisms exposed to desiccation and their stability in dry materials.     

Comparison of conductimetric and traditional plating techniques for detecting yeasts in fruit juices

Frozen fruit juice concentrates containing an average microbial population of log₁₀ 1.54 cfu ml^-1 were examined by traditional plating techniques and direct and indirect conductimetry. The initial populations in diluted (1:4) concentrates increased to an average of log₁₀ 3.82 cfu ml^-1 during incubation at 25°C for 24 h. Incubation before plating and subjecting to conductimetric tests also facilitated the resuscitation of cells that may have been freeze-injured. Yeasts were recovered in equal numbers on acidified (pH 3.5) potato dextrose agar and dichloran rose bengal chloramphenicol agar (pH 5.6). Yeasts and bacteria were recovered on orange serum agar. Detection times determined by indirect conductimetry correlated fairly well ( r = -0.73) with populations (cfu ml^-1) detected on traditional plating media. Populations in diluted concentrates which were not incubated before examination were detected conductimetrically in an average of 48.9 h, whereas detection times for diluted concentrates incubated for 24 h at 25°C before testing were reduced to an average of 14.1 h. Examination by conventional (direct) conductimetry required an additional 10–20 h to reach changes in conductance of 5 μS h^-1.     

Effect of heat treatment on Listeria monocytogenes and Gram-negative bacteria in sheep, cow and goat milks

Sheep milk, compared with cow and goat milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65°C in a test-tube method. This effect was not solely due to fat content as cow milk artificially reconstituted to 10% homologous fat was not as protective. Listeria monocytogenes in whole sheep, cow and goat milks at an inoculum level of 1 times 10⁶ cfu ml^-1 was heated at 68°C for 15 s in the plate pasteurizer and survival was only detected in whole sheep milk after heating. Whole sheep, cow and goat milks containing high levels of L. monocytogenes (1 times 10⁶ cfu ml^-1) could not survive the current HTST plate pasteurization protocol.     

Heterotrophic bacterial populations in the mineral waters of thermal springs in Spain

The microbiological quality and heterotrophic bacterial populations of 26 thermal mineral water springs in Spain were studied. In most of the springs the number of viable aerobes was less than 10³ cfu ml^-1 and the number of sporulated bacteria less than 10² cfu ml^-1. No significant differences were foundin the counts obtained with Plate Count Agar (PCA) and PCA diluted 1 : 10 and incubated at 22°, 37° and 45°C. Total coliforms were found in 14 springs, faecal streptococci in three, spores of sulphite-reducing Clostridium and Pseudomonas aeruginosa in seven. Neither Escherichia coli nor Staphylococcus aureus were found. A total of 665 strains were isolated and 85·4% of these identified; 329 were Gram-positive and 239 were Gram-negative. The genera most prevalent present in the springs were Pseudomonas (in 92.3%), Bacillus (65.4%), Enterobacter, Micrococcus and Staphylococcus (50%), Acinetobacter (42.3%), Arthrobacter (38.4%), Clostridium (27%) and Xanthomonas (23%). Gram-negative bacteria predominated in the mesothermal springs and Gram-positive bacteria in the hyper- and hypothermal springs. The most common Gram-negative rod species isolated were Ps. fluorescens, Ps. aeruginosa, Ps. putida, Ent. agglomerans, Ent. sakazakii, Ac. calcoaceticus and Ent. amnigenus.     

Relatedness of Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. antirrhini

The relationships among strains of Pseudomonas syringae pv. tomato, Ps. syr. antirrhini, Ps. syr. maculicola, Ps. syr. apii and a strain isolated from squash were examined by restriction fragment length polymorphism (RFLP) patterns, nutritional characteristics, host of origin and host ranges. All strains tested except for Ps. syr. maculicola 4326 isolated from radish ( Raphanus sativus L.) constitute a closely related group. No polymorphism was seen among strains probed with the 5.7 and 2.3 kb Eco RI fragments which lie adjacent to the hrp cluster of Ps. syr. tomato and the 8.6 kb Eco RI insert of pBG2, a plasmid carrying the β-glucosidase gene(s). All strains tested had overlapping host ranges. In contrast to this, comparison of strains by RFLP patterns of sequences homologous to the 4.5 kb Hind III fragment of pRut2 and nutritional properties distinguished four groups. Group 1, consisting of strains of pathovars maculicola, tomato and apii , had similar RFLP patterns and used homoserine but not sorbitol as carbon sources. Group 2, consisting of strains of pathovars maculicola and tomato , differed from Group 1 in RFLP patterns and did not use either homoserine or sorbitol. Group 3 was similar to Group 2 in RFLP patterns but utilized homoserine and sorbitol. This group included strains of the pathovars tomato and antirrhini , and a strain isolated from squash. Group 4, a single strain of Ps. syr. maculicola isolated from radish, had unique RFLP patterns and resembled Group 3 nutritionally. The evolutionary relationships of these strains are discussed.     

Methods for recovery and immunodetection of Xanthomonas campestris pv. phaseoli in navy bean seed

Non-selective enrichment procedures were evaluated for recovery of Xanthomonas campestris pv. phaseoli (fuscans strain) from artificially inoculated navy bean seed. A marked increase in recovery of the pathogen was obtained when the mixtures (bacterium plus bean seed) were suspended in Pseudomonas Agar F medium at 28°C for 48 h. Detection of this pathogen by indirect immunofluorescence microscopy (IF) and indirect enzyme-linked immunosorbent assay (ELISA) with a specific monoclonal antibody was compared. The IF system was not only more sensitive but also more reliable than ELISA for detection of the pathogen. The method is particularly useful for evaluation of the common bacterial blight status of seedlots before planting out.     

Measuring the spermosphere colonizing capacity (spermosphere competence) of bacterial inoculants

Spermosphere establishment by bacteria which were coated onto seeds was studied using soybean seeds treated with four bacterial strains at levels of log10 1 to 4 colony-forming units (cfu) per seed planted in a field soil mix, and incubated 48 h. Each strain at every inoculum level developed spermosphere population densities of log10 4 to 8 cfu/seed, demonstrating an average multiplication of log10 3 cfu/seed. An alternative method was developed to differentially rank bacteria for spermosphere colonizing capacity, based upon incorporation of bacteria into a soil and monitoring the resulting spermosphere population densities around noninoculated seeds after 4 days at 14 degrees C. Fifty-seven bacterial strains which were isolated from soybean roots or from water samples, including Pseudomonas putida, P. putida biovar B, P. fluorescens, Serratia liquefaciens, Enterobacter aerogenes, and Bacillus spp. were tested in the spermosphere colonization assay. Average spermosphere population densities for the 57 strains ranged from 0 to log10 7.0 cfu/seed. Strains of a given taxon demonstrated marked diversity with ranges from 0 to log10 6.0 cfu/seed for Bacillus spp. and from log10 1.4 to 7.0 cfu/seed for Pseudomonas putida. The relative ranking of representative strains was consistent in repeating experiments. The potential usefulness of the assay for efforts to develop competitive bacterial inoculants for crop seeds is discussed.     

Non-destructive methods of detecting Curtobacterium flaccumfaciens pv. flaccumfaciens in mungbean seeds

An indirect immunofluorescent assay (IF) with specific monoclonal antibodies (MAbs) and a semi-selective agar medium for Curtobacterium flaccumfaciens pv. flaccumfaciens , developed in this study, were compared with foliar symptoms and microscopic bacterial ooze for detection of this pathogen in mungbean seed 6 d and 28 d after germination. The IF method detected more infected seedlings than the other three methods at both samplings. Symptomless carriers of C. flaccumfaciens pv. flaccumfaciens in mungbean were detectable only by the IF method and, less frequently, by plating out on media. Poor agreement between the IF and other methods was found. The IF method gave the best agreement in the detection of the pathogen between early and late samplings of individual germinated seed. Currently, the IF technique with a specific MAb is being used for selecting clean seedlings for production of disease-free seed.     

Effect of pH on isoamylase production by Pseudomonas amyloderamosa WU 5315

The isoamylase activity of Pseudomonas amyloderamosa WU 5315 was stable over the pH range from 5.5 to 6.25 while only about 30% of the activity remained at pH 6.5. Low isoamylase activity (418 U ml^-1) was produced by the cells grown at high pH. Activity reached almost 3000 U ml^-1 when pH was kept below 6.0 during the fermentation. With 1% glucose plus 2% maltose instead of 3% maltose as carbon source, however, no pH control was required and the isoamylase activity of Ps. amyloderamosa WU 5315 increased to 3400 U ml^-1.     

Anaerobic degradation of pectin by mixed consortia and optimization of fermentation parameters for higher pectinase activity

Samples from biogas digesters, sewage ponds, animal house effluents and food processing wastes were used in enrichment systems seeking anaerobic bacteria producing pectinases. Among the 46 anaerobic consortia developed from various samples, four showed high pectinase activity under static anaerobic conditions. Investigation of fermentation variables showed the optimum conditions for pectinase activity were pH 7.0, 45°C and 72 h of growth with 0.5% pectin in the cultivation medium. A 1.4- to 1.6-fold increase in the pectinase activity was achieved under these conditions. The maximum yield of enzymes (62.72 U ml-1 of pectinase, 4.74 U ml^-1 of polygalacturonase, 113.30 U ml^-1 of pectin lyase, 2.10 U ml^-1 of pectinesterase, 0.75 U ml^-1 of total cellulase and 9.27 U ml^-1 of xylanase) was recorded with the consortia C-S2 developed from decomposed plant samples collected from a pond.     

Efflux of ions and ATP depletion induced by pediocin PA-1 are concomitant with cell death in Listeria monocytogenes Scott A

Domination of Carnobacterium divergens LV13 by a bacteriocin-producing (bac⁺) organism Carnobacterium piscicola LV17 was dependent on the level of inoculum of the producer strain and its bacteriocin production. When C. piscicola LV17 was grown in APT broth from an initial inoculum of α-10⁴ cfu ml^-1, bacteriocin was not produced (bac^-) although maximum population was reached. The culture remained bac^- during subsequent inoculation at 10²-10⁷ cfu ml^-1 unless it was first grown on solid medium or if heat-treated supernatant fluids from a bac⁺ culture of C. piscicola LV17, LV17A or LV17B were added to the culture prior to the stationary phase of growth. Use of purified carnobacteriocins from C. piscicola LV17A and LV17B confirmed their role in regulation of the bac⁺ phenotype. The need for induction might account in part for differences in bacteriocin production by cultures in liquid and on solid growth media.     

Comparison of the Vitek, API 20E and Gene-trak® systems for the identification of Yersinia enterocolitica

The current improvements in nucleic acid hybridization technology provide new techniques for the identification of micro-organisms. One such technique is the Gene-trak® DNA hybridization system (Framingham, MA, USA), which was introduced in 1983. The objective for this study was to evaluate the new Gene-trak® Yersinia enterocolitica kit in comparison with the API 20E and Vitek systems. A total of 101 strains including 18 reference non- Yersinia strains from the authors' stock culture collection and 83 suspected positive isolates from CIN agar were tested. Of these 83 isolates, 40 were identified as Y. enterocolitica after incubation at 37°C for 24 with the API 20E system; 37 strains were identified at 30°C for 48 h. The Gene-trak® method gave positive results with 39 strains. The Vitek system gave positive results with 27 strains.
With the Gene-trak® method, Y. enterocolitica was detectable in mixed cultures provided that the numbers of cfu ml^-1 were equal to or above 10⁶ Y. enterocolitica ml^-1. Although enrichment procedures are still needed, the system provides a quick detection of these food-borne pathogens.     

Induction of secondary dormancy in sunflower seeds by high temperature. Possible involvement of ethylene biosynthesis

High temperature (45°C) inhibits seed germinition and seedling sunflower ( Helianthus annuus L. cv. Mirasol). Treatment of imbibed seeds at 45°C for more than 48 h induces a secondary dormancy, which is associated with progressive decrease of germination ability at optimal temperature (25°C) as well as with abnormal seedling growth. Ethylene (55μl l⁻¹) and 2-chloroethylphosphonic acid (ethephon) (2.5 m M ) improve germination of thermodormant seeds at 25°C. but the abnormal growth of the seedlings remains. O₂-enriched atmosphere and dry storage improve germination and normal seedling growth. The induction of thermodormancy in sunflower seeds seems associated with loss of their ability to convert 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. Possible effects of high temperature on membranes and ethylene forming enzyme (EFE) are discussed.     

Abscisic acid levels in seeds of the gibberellin-deficient mutant lh-2 of pea (Pisum sativum)

The lh-2 mutation in garden pea ( Pisum sativum L.) blocks an early step in the gibberellin (GA) biosynthesis pathway, the three-step oxidation of ent -kaurene to ent -kaurenoic acid. As a result, only low levels of GAs, including the bioactive GA₁, are found in shoots and seeds of lh-2 plants. Mutant plants are dwarf in stature, and show increased seed abortion and decreased seed weight, compared with seeds of the tall wild-type (WT) progenitor (cv. Torsdag). The aberrant seed development of lh-2 plants is associated with reduced levels of GA₁ and GA₃, and with an accumulation of abscisic acid (ABA) in young seeds (pre-contact point). This ABA accumulation is typically 3- to 4-fold, and can be up to 6-fold, compared with control plants. To investigate whether the accumulation of ABA is partly responsible for causing the observed seed abortion in lh-2 plants, we constructed a double mutant between the lh-2 allele and wil . The wil mutation blocks ABA biosynthesis, and reduces ABA levels in young seeds by 10-fold. Introduction of the wil mutation reduces the endogenous ABA levels in young lh-2 seeds, but fails to rescue the seeds from abortion. This indicates that the effects of lh-2 on seed development are not mediated through increased ABA levels, and is consistent with previous evidence that GAs are the controlling factor underlying the lh-2 seed phenotype in pea.     

Phytotoxin production by Pseudomonas syringae pv. syringae: Syringopeptin production by syr mutants defective in biosynthesis or secretion of syringomycin

Abstract Syringomycin and syringopeptin are lipodepsipeptide phytotoxins produced by Pseudomonas syringae pv. syringae . Four syr genes were identified previously and hypothesized to be involved in the regulation ( syrA ), biosynthesis ( syrB and syrC ), or export ( syrD ) of syringomycin. This study determines the influence of syr mutations on the composition of phytotoxic metabolites produced by P. syringae pv. syringae strain B301D-R. Levels of syringomycin and syringopeptin produced in liquid cultures were estimated by reverse phase HPLC analyses and differential antimicrobial assays. Significant quantities of syringopeptin were produced by both syrB and syrC mutants despite their inability to produce syringomycin. Only trace quantities of both lipodepsipeptides were produced by syrA and syrD mutants of P. syringae pv. syringae . These results indicate that syringomycin and syringopeptin are synthesized by separate pathways, but may share common mechanisms for secretion and regulation.     

Functional homologs of the Arabidopsis RPM1 disease resistance gene in bean and pea.

We showed that a bacterial avirulence (avr) gene function, avrPpiA1, from the pea pathogen Pseudomonas syringae pv pisi, is recognized by some, but not all, genotypes of Arabidopsis. Thus, an avr gene functionally defined on a crop species is also an avr gene on Arabidopsis. The activity of avrPpiA1 on a series of Arabidopsis genotypes is identical to that of the avrRpm1 gene from P.s. pv maculicola previously defined using Arabidopsis. The two avr genes are homologous and encode nearly identical predicted products. Moreover, this conserved avr function is also recognized by some bean and pea cultivars in what has been shown to be a gene-for-gene manner. We further demonstrated that the Arabidopsis disease resistance locus, RPM1, conditioning resistance to avrRpm1, also conditions resistance to bacterial strains carrying avrPpiA1. Therefore, bean, pea, and conceivably other crop species contain functional and potentially molecular homologs of RPM1.