豆薯细菌性角斑病的病原鉴定

 在安徽滁州的豆薯叶片上发现一种由细菌侵染引起角斑症状的病害,从角斑上分离到具有致病性的非荧光的杆状细菌,菌株的表型特征、细菌学特征、LOPAT试验和生理生化试验表明该细菌与丁香假单胞菌(Pseudomonas syringae van Hall)相似,BIOLOG系统鉴定结果与丁香假单胞菌豌豆致病变种(P.syringae pv.pisi)相近,接种试验表明豆薯菌株能侵染大豆、菜豆和眉豆,但对豌豆的致病性差;在豇豆、绿豆和蚕豆上不表现症状。结果表明豆薯细菌性角斑病是一种新病害,病原菌属于丁香假单胞菌群的一个新的致病变种,命名为P.syringae pv.pachyrhizus nov.     

Comparison of Ethylene Production by Pseudomonas syringae and Ralstonia solanacearum

ABSTRACT Strains of Pseudomonas syringae pv. pisi and Ralstonia solanacearum produced ethylene at rates 20- and 200-fold lower, respectively, than strains of P. syringae pvs. cannabina, glycinea, phaseolicola, and sesami. In the current study, we investigated which ethylene biosynthetic pathways were used by P. syringae pv. pisi and R. solanacearum. Neither the activity of an ethylene-forming enzyme nor a corresponding efe gene homolog could be detected in R. solanacearum, suggesting synthesis of ethylene via 2-keto-4-methyl-thiobutyric acid. In contrast, 2-oxoglutarate-dependent ethylene formation was observed with P. syringae pv. pisi, and Southern blot hybridization revealed the presence of an efe homolog in this pathovar. The efe genes from P. syringae pvs. cannabina, glycinea, phaseolicola, pisi, and sesami were sequenced. Nucleotide sequence comparisons indicated that the efe gene in pv. pisi was not as highly conserved as it was in other P. syringae pathovars. The pv. pisi efe homolog showed numerous nucleotide substitutions and a deletion of 13 amino acids at the C-terminus of the predicted gene product. These sequence alterations might account for the lower rate of ethylene production by this pathovar. All ethylene-producing P. syringae pathovars were virulent on bush bean plants. The overlapping host range of these pathovars suggests that horizontal transfer of the efe gene may have occurred among bacteria inhabiting the same host.     

Evaluation of physiological and serological profiles of Pseudomonas syringae pv. pisi for pea blight identification

Phenotypic variability of the pea blight bacterium, Pseudomonas syringae pv. pisi, was studied on a large collection of strains isolated in France, as well as those obtained from foreign collections. Some other pseudomonads encountered on peas, particularly P.s. pv. syringae, were included in the study to evaluate differential tests for identification purposes. All the isolates that induced watersoaking on the pea cultivar Kelvedon Wonder after inoculation were considered to be P.s. pv. pisi. The other pseudomonads gave either no reaction or a hypersensitive reaction. When they corresponded to P. syringae according to the LOPAT test, they were referred to as P.s. pv. syringae.
P.s. pv. pisi did not show a single uniform phenotype. The variation of the different tests was estimated (fluorescence+ 93%; esculin-86%; dl-lactate-85%; homoserine + 75%; INA + 97%). Three O-sero-groups contained P.s. pv. pisi strains: APT-PIS (88.5%), HEL2 (11.4%) and RIB (0.1%). When the main criteria were combined, eight profiles were encountered within P.s. pv. pisi. This diversity was not linked to race structure or geographical origin of the strains. Profile PI was the most frequent (72.8%), and it was specific to the pathovar pisi . The strains belonging to the other profiles could be confused with some P.s. pv. syringae strains because of the serological heterogeneity of that pathovar. For instance, the pv. pisi strains belonging to profiles P2 and P4 resembled some of the P.s. pv. syringae found on peas and required pathogenicity tests on pea for their identification. The confusing pea isolates represented 12.8% of the total 4740 strains studied.     

Patterns of interaction between isolates of three pathovars of Pseudomonas syringae and accessions of a range of host and nonhost legume species

Isolates of three pathovars of Pseudomonas syringae were tested against 10 legume species. Some isolates of all pathovars showed cultivar-specific interactions with at least one legume species outside the expected host range. Lablab purpureus and Phaseolus lunatus were found to be hosts to isolates of both P. syringae pv. glycinea and P. syringae pv. phaseolicola, while Lathyrus latifolius was host to isolates of P. syringae pv. pisi and P. syringae pv. glycinea . Lens culinaris showed patterns of interaction with isolates of all three pathovars. Gene models based on mathematical estimates of minimum gene numbers agreed with those previously published for the interactions of P. syringae pv. pisi with Pisum sativum and P. syringae pv. phaseolicola with Phaseolus vulgaris. Two different gene-for-gene models based on five resistance/avirulence gene pairs were proposed to explain observed interactions between Glycine max and P. syringae pv . glycinea . Pathogen isolates which contained no known avirulences defined on their respective host species were found to carry cryptic avirulences recognized by other plant species. Estimates of minimum gene numbers required to explain the interactions of a plant species with all pathogen isolates or to explain the interactions of the isolates of one pathovar with all plant accessions were consistently lower than the sum of the minimum gene numbers required to explain the interactions of each individual component.     

Specificity of polyclonal antibodies to different antigenic preparations of Pseudomonas syringae pv. pisi strain UQM551 and Pseudomonas syringae pv. syringae strain L

Polyclonal antibodies were produced against sonicated and heat-killed cells of Pseudomonas syringae pv. pisi strain UQM551 and Pseudomonas syringae pv. syringae strain L, and their specificities were compared. Evidence is presented that the serological specificity between these two pathovars lies in surface antigens. Of the surface antigens purified and tested, only flagella and lipopolysaccharide from the cell wall showed no cross-reactivity with heterologous antisera. Antisera to glutaraldehyde-fixed flagella of the two strains showed a high level of specificity. At a species or genus level, antisera prepared from heat-killed cells of P. syringae distinguished this species from all other bacterial species and genera tested, including strains of Pseudomonas fluorescens, Escherichia coli, Agrobacterium and Rhizobium.     

Production of syringomycins and syringopeptins by Pseudomonas syringae pv. atrofaciens

All virulent strains of Pseudomonas syringae pv. atrofaciens produce in vitro substances with syringomycin-like features. All strains inhibited the growth of Geotrichum candidum in the plate assay although the extent of their growth inhibition was variable.
Purification of bioactive culture extracts of a highly virulent strain by ion exchange chromatography (Whatman CM52) yielded a main fraction which inhibited the growth of Rhodotorula pilimanae and Bacillus megaterium , and was phytotoxic to tobacco and wheat plants. In particular, the injection of this fraction in the culm of wheat plants caused phytotoxic symptoms on leaves similar to those caused in nature by the pathogen. The further purification by HPLC of the above fraction gave rise to four main bioactive substances which have been identified by spectroscopic methods (FAB-MS) and amino acid analysis as syringomycin E, syringomycin G, syringopeptin 25A and syringopeptin 25B, toxic lipodepsipeptides thus far recognized to be produced by most strains of P. syringae pv. syringae . The injection of both syringomycin E and syringopeptin 25A in wheat leaves caused necrotic symptoms; however, syringopeptin 25A was at least six times more active than syringomycin E. The possible role of the toxins in the disease development on cereals and the need for a careful examination of pathogenetic and biochemical features of P. syringae pv. atrofaciens to establish the relationships of the two pathovars in the 'syringae group' are discussed.     

Distinguishing pathovars of Pseudomonas syringae on peas: nutritional, pathogenicity and serological tests

Of the published methods to distinguish Pseudomonas syringae pv. syringae and Pseudomonas syringae pv. pisi , inoculation of susceptible cultivars was the most reliable. Results were confirmed by inoculation of lemon fruit.
A much more rapid and convenient serological method was developed to distinguish the two pathovars. Antisera against glutaraldehyde-fixed cells had a high level of specificity in Ouchterlony gel double-diffusion tests and, after cross-absorption with heterologous antigens, in indirect ELISA. Antiserum to P. syringae pv. pisi has considerable potential to detect pea seed infected with this pathogen.     

Genetic relationship between races of Pseudomonas syringae pv.pisi and cultivars of Pisum sativum

Isolates of Pseudomonas syringae pv. pisi from the UK and overseas were categorized into six races on the basis of their reactions to a range of differential pea (Pisum sativum) cultivars. Race 2 was predominant among the isolates examined and this probably reflects its relative international importance. A previously uncharacterized race (race 6) was virulent on all cultivars tested. Resistance to races 1-5 was widespread in commercial cultivars and breeding lines with more than 75% showing resistance to one or more races. A preliminary study of the inheritance of resistance indicated that for races 1, 2 and 3, resistance was controlled by different dominant genes. The genetic basis for the relationship between races of P. syringae pv. pisi and pea cultivars was explained in terms of a gene-for-gene relationship involving five matching gene pairs. With further clarification of the genetics of resistance this host-pathogen association will meet most of the requirements of a model system for the study of the genetic and molecular basis of pathogenicity and host specificity.     

Comparative dynamics of adherent and nonadherent bacterial populations on maize leaves

ABSTRACT The dynamics of the adherent and nonadherent populations of three bacterial species on maize leaves were examined to identify the extent to which bacteria adhere to leaves and the importance of this adhesion to leaf colonization. Pantoea agglomerans strain BRT98, Clavibacter michiganensis subsp. nebraskensis strain GH2390, and Pseudomonas syringae pv. syringae strain HS191R all rapidly adhered to maize leaves following inoculation, but differed in the percentage of cells that adhered to the leaves. Immediately following inoculation, the percentage of adherent cells was highest for the saprophyte P. agglomerans (8 to 10%) and was much lower for the pathogens C. michiganensis subsp. nebras-kensis and P. syringae pv. syringae (2 to 3 and <1%, respectively), although the results for P. syringae pv. syringae HS191R were based on only one experiment. In the 4 days following inoculation, the percentage of the P. agglomerans populations that adhered to the leaves increased to approximately 70%. Similarly, the percentage of C. michiganensis subsp. nebraskensis and P. syringae pv. syringae cells that resisted removal steadily increased in the days following inoculation, although these increases probably reflected both adherence and localization to endophytic sites. Based on differences in the percentage of cells adhering to several cuticular wax mutants of maize, the rapid adherence of C. michiganensis subsp. nebraskensis cells to maize leaves was influenced by the cuticular wax properties, while the rapid adherence of P. agglomerans was not. Finally, bacterial adherence to leaves was advantageous to P. agglomerans survival and growth on leaves based on the finding that the nonadherent populations of the P. agglomerans strain decreased significantly more than did the adherent populations in the 24 h following inoculation, and increased much less than did the adherent populations over the next 3 days. Similar results with the C. michiganensis subsp. nebraskensis and P. syringae pv. syringae strains indicate that bacterial adherence to leaves, bacterial movement to endophytic sites, or both were advantageous to the survival and growth of these strains on leaves.     

Plasmids in Pseudomonas syringae pv. pisi carry genes for pathogenicity

All strains tested which are pathogenic to peas and which react with antiserum to Pseudomonas syringae pv. pisi contain two to four plasmids; non-pathogens contain none. Two plasmids from a pathogenic strain were transferred individually to a non-pathogenic recipient strain of Pseudomonas syringae: both plasmids converted the recipient to a pathogen on peas and from hypersensitivity negative to hypersensitivity positive on tobacco. Neither plasmid encoded homoserine catabolism.     

Studies on red light-induced resistance of broad bean to Botrytis cinerea: I. Possible production of suppressor and elicitor by germinating spores of pathogen

When detached broad bean leaves were preinoculated with virulent strain B304 of Botrytis cinerea 24 h before a challenge inoculation with strain B304, lesion formation by B304 was significantly inhibited in red light but not in the dark. In leaves that were preinoculated with avirulent strain 021 and then challenged by B304, however, lesion formation was not inhibited even under red light. Such differences in lesion formation after the challenge inoculation with an avirulent strain were also observed with lesions caused by Alternaria alternata, a nonpathogen of broad bean and by avirulent strain 021R in the presence of germination fluid from spores of strains B304 and 021R. These results suggest the possibility that virulent B. cinerea produced a suppressor involved in induced susceptibility and an elicitor involved in resistance induced by red light during spore germination.     

A study of populations of Pseudomonas syringae pv. syringae on stonefruits in Victoria

In a survey of the major stonefruit nurseries in Victoria during winter 1978 and 1979, Pseudomonas syringae pv. syringae , the causal organism of bacterial canker, was found to be present on most of the stonefruit material in all nurseries but was detected most frequently on apricot.
The epiphytic populations of P.s. pv. syringae on leaves, buds and shoots of apricot and cherry were assessed periodically between 1979 and 1983 by determining the proportion of trees bearing the bacterium or by counting numbers of bacteria. Populations consistently reached peak levels during spring and late autumn, with highest levels in spring. Populations were lowest during mid- to late summer. High proportions of tree contamination and high populations coincided with periods when maximum temperatures ranged from 19° to 25°C, and when rainfall was moderately high. The significance of these findings in the light of information from other studies on the seasonal variability of host susceptibility, and in relation to chemical control, is discussed.
There was no evidence of occurrence of P.s. pv. morsprunorum in Victoria.     

Influence of Ring Nematode Infestation and Calcium, Nitrogen, and Indoleacetic Acid Applications on Peach Susceptibility to Pseudomonas syringae pv. syringae

ABSTRACT Two field experiments were conducted to study the effects of added nitrogen, calcium, and indoleacetic acid, in the presence or absence of ring nematodes (Mesocriconema xenoplax), on susceptibility of peach to bacterial canker. When noninfested soil was inoculated with ring nematodes, peach tree susceptibility to bacterial canker infection caused by Pseudomonas syringae pv. syringae was dramatically increased after a period of 2 years. However, no evidence was found that ring nematode infestation increased tree water stress or, in turn, altered plant calcium uptake. Soil fumigation with methyl bromide prior to planting in a commercial orchard significantly reduced both nematode populations and peach tree susceptibility to bacterial canker infection when compared with nonfumigated treatments. In both experiments, tree susceptibility, as measured by canker length following inoculation of stems with P. syringae pv. syringae, was negatively correlated with plant tissue nitrogen content and positively correlated with tissue calcium content. A principal components analysis showed that tissue nitrogen and calcium levels were negatively correlated, and that high-nitrogen, low-calcium tissues were less susceptible to bacterial canker than low-nitrogen, high-calcium tissues. These results indicate that the increased susceptibility of peach to P. syringae pv. syringae under nematode infestation conditions is mediated by both nutritional effects (primarily nitrogen) and nutritional-independent effects, but do not support previous reports of beneficial effects of calcium for reducing bacterial canker.     

Factors that Affect Spread of Pseudomonas syringae in the Phyllosphere

ABSTRACT Successful spread of an organism to a new habitat requires both immigration to and growth on that habitat. Field experiments were conducted to determine the relative roles of dispersal (i.e., immigration) and bacterial multiplication in spread of Pseudomonas syringae pv. syringae in the phyllosphere. To study spread, individual plots consisted of three nested concentric squares with the inner 6 m(2) planted to snap beans serving as the sink. Each sink, in turn, was surrounded by a barrier zone, usually 6 m wide, which was surrounded by a 6-m-wide source area. The source areas were planted with snap bean seeds inoculated with doubly marked strains derived from wild-type P. syringae pv. syringae B728a. The treatments were designed to test the effects of the nature and width of the barrier zone and suitability of the habitat in the sinks on spread of P. syringae pv. syringae. The marked strains introduced into the source areas at the time of planting were consistently detected in sink areas within a day or two after emergence of bean seedlings in the sources as assessed by leaf imprinting and dilution plating. The amounts of spread (population sizes of the marked strain in sinks) across barrier zones planted to snap bean (a suitable habitat for growth of P. syringae pv. syringae), soybean (not a favorable habitat for P. syringae pv. syringae), and bare ground were not significantly different. Thus, the nature of the barrier had no measurable effect on spread. Similarly, spread across bare-ground barriers 20 m wide was not significantly different from that across barriers 6 m wide, indicating that distance on this scale was not a major factor in determining the amount of spread. The suitability of the sink for colonization by P. syringae pv. syringae had a measurable effect on spread. Spread to sinks planted to clean seed was greater than that to sinks planted with bean seeds inoculated with a slurry of pulverized brown spot diseased bean leaves, sinks planted 3 weeks before sources, and sinks planted to a snap bean cultivar that does not support large numbers of P. syringae pv. syringae. Based of these results, we conclude that the small amount of dispersal that occurred on the scale studied was sufficient to support extensive spread, and suitability of the habitat for multiplication of P. syringae pv. syringae strongly influenced the amount of spread.     

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.     

Serological detection and identification of bacteria from plants by the conjugated Staphylococcus aureus slide agglutination test

A rapid slide agglutination test using polyclonal antisera conjugated to protein A-rich whole-cell Staphylococcus aureus was developed for the detection and identification of bacteria from plants. The specificity and sensitivity of the technique was evaluated in 18 antibody/antigen combinations, representing six bacterial genera ( Erwinia, Lactobacillus, Pseudomonas, Rhizobium, Rhodococcus and Xanthomonas ). For two pathovars of Pseudomonas syringae the specificity of the technique was increased by the use of antisera prepared to somatic extracts.
The advantages of the Staphylococcus aureus agglutination technique include speed, simplicity and the ability to identify organisms directly from infected plant tissues. It was applied to the detection of Pseudomonas syringae pv. phaseolicola and pv. pisi in lesions on bean and pea, respectively, to P. gladioli pv. alliicola and Lactobacillus sp. from rotted onion bulbs and specific strains of Rhizobium phaseoli in bean root nodules.     

Rapid Diagnosis of Pseudomonas syringae pv. papulans, the Causal Agent of Blister Spot of Apple, by Polymerase Chain Reaction Using Specifically Designed hrpL Gene Primers

ABSTRACT The identification and detection of Pseudomonas syringae pv. papulans, the causal agent of blister spot of apple, on apple leaves and fruit was achieved by polymerase chain reaction amplification of a specific DNA fragment of the hrpL sequence. The consensus primers hrpL(1) and hrpL(2) were designed based on the alignment of pseudomonad hrpL gene sequences available in nucleic acid data banks. This primer set produced a 631-bp amplicon from 37 of the 57 pseudomonads strains tested. These strains belonged to genomospecies 1 and 2, as described by Gardan et al. (8). The amplicon obtained from 30 of these strains was digested with eight restriction enzymes. Three different restriction patterns were produced from strains belonging to genomospecies 1, resulting in A1 and A2 patterns, while strains belonging to genomospecies 2 were characterized by a B pattern. Patterns A1 and A2 differed at only two sites, a Bsp 143I site located at nucleotide 360 and a MseI site located at nucleotides 22-24. Group A2 consisted solely of P. syringae pv. papulans strains. The hrpL gene in P. syringae pv. papulans strain CFBP3323 was sequenced. Two primer sets, Pap1/Pap2 and Pap1/Pap3, were designed and tested for specificity to P. syringae pv. papulans. These primers amplified expected fragments of 242 and 303 bp, respectively. Pap1/Pap2 amplified a fragment only with P. syringae pv. papulans DNA, while Pap1/Pap3 amplified all tested strains belonging to genomospecies 1. A diagnostic procedure using the Pap1/Pap2 primer set was successful for the detection of P. syringae pv. papulans in diseased fruit and artificially inoculated leaves.     

Multilocus sequence typing of Pseudomonas syringae sensu lato confirms previously described genomospecies and permits rapid identification of P. syringae pv. coriandricola and P. syringae pv. apii causing bacterial leaf spot on parsley

Since 2002, severe leaf spotting on parsley (Petroselinum crispum) has occurred in Monterey County, CA. Either of two different pathovars of Pseudomonas syringae sensu lato were isolated from diseased leaves from eight distinct outbreaks and once from the same outbreak. Fragment analysis of DNA amplified between repetitive sequence polymerase chain reaction; 16S rDNA sequence analysis; and biochemical, physiological, and host range tests identified the pathogens as Pseudomonas syringae pv. apii and P. syringae pv. coriandricola. Koch's postulates were completed for the isolates from parsley, and host range tests with parsley isolates and pathotype strains demonstrated that P. syringae pv. apii and P. syringae pv. coriandricola cause leaf spot diseases on parsley, celery, and coriander or cilantro. In a multilocus sequence typing (MLST) approach, four housekeeping gene fragments were sequenced from 10 strains isolated from parsley and 56 pathotype strains of P. syringae. Allele sequences were uploaded to the Plant-Associated Microbes Database and a phylogenetic tree was built based on concatenated sequences. Tree topology directly corresponded to P. syringae genomospecies and P. syringae pv. apii was allocated appropriately to genomospecies 3. This is the first demonstration that MLST can accurately allocate new pathogens directly to P. syringae sensu lato genomospecies. According to MLST, P. syringae pv. coriandricola is a member of genomospecies 9, P. cannabina. In a blind test, both P. syringae pv. coriandricola and P. syringae pv. apii isolates from parsley were correctly identified to pathovar. In both cases, MLST described diversity within each pathovar that was previously unknown.     

Biological control of bacterial speck of tomato under field conditions at several locations in north america

ABSTRACT Bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, continues to be a problem for tomato growers worldwide. A collection of nonpathogenic bacteria from tomato leaves plus P. syringae strains TLP2 and Cit7, P. fluorescens strain A506, and P. syringae pv. tomato DC3000 hrp mutants were examined in a greenhouse bioassay for the ability to reduce foliar bacterial speck disease severity. While several of these strains significantly reduced disease severity, P. syringae Cit7 was the most effective, providing a mean level of disease reduction of 78% under greenhouse conditions. The P. syringae pv. tomato DC3000 hrpA, hrpH, and hrpS mutants also significantly reduced speck severity under greenhouse conditions. The strains with the greatest efficacy under greenhouse conditions were tested for the ability to reduce bacterial speck under field conditions at locations in Alabama, Florida, and Ontario, Canada. P. syringae Cit7 was the most effective strain, providing a mean level of disease reduction of 28% over 10 different field experiments. P. fluorescens A506, which is commercially available as Blight-Ban A506, provided a mean level of disease reduction of 18% over nine different field experiments. While neither P. syringae Cit7 nor P. fluorescens A506 can be integrated with copper bactericides due to their copper sensitivity, there exist some potential for integrating these biological control agents with "plant activators", including Actigard. Of the P. syringae pv. tomato DC3000 hrp mutants tested, only the hrpS mutant reduced speck severity significantly under field conditions.