STUDIES ON THE BACTERIAL DIE-BACK AND CANKER DISEASE OF POPLAR

Artificial freezing caused leaf necrosis and twig die-back but did not produce cracks or cankers on living branches of three varieties of poplar. Freezing increased the damage caused by a well-established infection of the bacterial die-back and canker but checked new lesions. Freezing prior to infection has no appreciable effect on the establishment of the disease.     

Role of Extracellular Polysaccharide (EPS) Slime of Plant Pathogenic Bacteria in Protecting Cells to Reactive Oxygen Species

Pseudomonas syringae pv. phaseolicola , a phytopathogenic bacterium, seemed very sensitive in planta to the adverse action of reactive oxygen species (ROS) produced by two chemical systems. The disease symptoms in host plants were also suppressed by ROS. Several other plant pathogenic bacteria ( P. syringae pv. pisi, Erwinia amylovora, Xanthomonas campestris pv. pelargonii ) as well as P. fluorescens were also sensitive in vitro to the inhibiting or killinig action of ROS. It was shown that O₂ and H₂O₂ were produced in our two chemical systems and were involved in the killing action. OH'however was not involved in the adverse action on bacteria of the ROS. Superoxide dismutase and catalase were able to reverse the killing action of ROS. When the EPS slime around bacteria was removed by washing and centrifuging the cells, bacteria were more sensitive to ROS. However, when the cells of EPS- mutants were washed and centrifuged, their sensitivity to the killing action of ROS did not change because the lack of slime around the mutant cells.
The EPS^- Tn5 mutants of P. syringae pv. phaseolicola and the natural EPS^- mutant of E. amylovora were more sensitive to ROS than the wild type strains. These results support the idea that the EPS slime protects bacteria from ROS (O^-₂ and H₂O₂).     

BACTERIAL DISEASES OF STONE-FRUIT TREES IN BRITAIN

A bacterial shoot wilt, observed at the East Malling Research Station in 1923 and 1924, is described.
A bacterium isolated from infected shoots has been shown by inoculation experiments to be the cause of the disease.
The organism not only infects young green shoots, but experiments have shown that it can also cause leaf spots, and that it can induce gummosis and cankers on woody branches and on stems.
Cultural studies of the organism suggest that it has not previously been described, so the name Ps. prunicola is proposed for it.     

Branch sacrifice: cavitation-associated drought adaptation of riparian cottonwoods

In their native riparian zones (floodplains), Populus deltoides (prairie cottonwood) and P. fremontii (Fremont cottonwood) commonly experience substantial branch die-back. These trees occur in semi-arid areas of North America and unexpectedly given the dry regions, they are exceptionally vulnerable to xylem cavitation, drought-induced air embolism of xylem vessels. We propose that the vulnerability to cavitation and branch die-back are physiologically linked; drought-induced cavitation underlies branch die-back that reduces transpirational demand enabling the remaining shoot to maintain a favorable water balance. This proposal follows field observation along various western North American rivers as precocious branch senescence, the yellowing and death of leaves on particular branches during mid- to late summer, was common for P. deltoides and P. fremontii during hot and dry periods of low stream-flow. Branches displaying precocious senescence were subsequently dead the following year. The proposed association between cavitation, precocious senescence and branch die-back is also supported by experiments involving external pressurization of branches to about 2.5 MPa with a branch collar or through an adjacent cut-branch. The treatments induced xylem cavitation and increased leaf diffusive resistance (stomatal closure) that was followed by leaf senescence and branch death of P. deltoides. P. trichocarpa (black cottonwood) appeared to be less affected by the pressurization treatment and this species as well P. angustifolia (narrowleaf cottonwood) and P. balsamifera (balsam poplar) seldom display the patchy summer branch senescence typical of P. deltoides and P. fremontii. ’Branch sacrifice’ describes this cavitation-associated senescence and branch die-back that may provide a drought adaptation for the prairie and Fremont cottonwoods. Received: 13 May 1999 / Accepted: 4 November 1999     

Roadmap to new virulence determinants in Pseudomonas syringae: insights from comparative genomics and genome organization

Systematic comparison of the current repertoire of virulence-associated genes for three Pseudomonas syringae strains with complete genome sequences, P. syringae pv. tomato DC3,000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a, is prompted by recent advances in virulence factor identification in P. syringae and other bacteria. Among these are genes linked to epiphytic fitness, plant- and insect-active toxins, secretion pathways, and virulence regulators, all reflected in the recently updated DC3,000 genome annotation. Distribution of virulence genes in relation to P. syringae genome organization was analyzed to distinguish patterns of conservation among genomes and association between genes and mobile genetic elements. Variable regions were identified on the basis of deviation in sequence composition and gaps in syntenic alignment among the three genomes. Mapping gene location relative to the genome structure revealed strong segregation of the HrpL regulon with variable genome regions (VR), divergent distribution patterns for toxin genes depending on association with plant or insect pathogenesis, and patterns of distribution for other virulence genes that highlight potential sources of strain-to-strain differences in host interaction. Distribution of VR among other sequenced bacterial genomes was analyzed and future plans for characterization of this potential reservoir of virulence genes are discussed.     

香梨内生拮抗细菌的筛选及对梨火疫病的生防潜力

该研究以健康香梨树的新鲜花器、当年生新生枝条、叶片和果实为试验材料,采用植物组织内生细菌分离法获得内生细菌菌株,培养菌落,并采用平板对峙法初筛和发酵液复筛对梨火疫病菌(Erwinia amylovora)、梢枯病菌(Pseudomonas syringae pv.syringae)和腐烂病菌(Valsa mali va...     

Pseudomonas syringae pv. actinidiae: a re-emerging, multi-faceted, pandemic pathogen

Pseudomonas syringae pv. actinidiae is the causal agent of bacterial canker of green-fleshed kiwifruit (Actinidia deliciosa) and yellow-fleshed kiwifruit (A. chinensis). A recent, sudden, re-emerging wave of this disease has occurred, almost contemporaneously, in all of the main areas of kiwifruit production in the world, suggesting that it can be considered as a pandemic disease. Recent in-depth genetic studies performed on P. syringae pv. actinidiae strains have revealed that this pathovar is composed of four genetically different populations which, to different extents, can infect crops of the genus Actinidia worldwide. Genome comparisons of these strains have revealed that this pathovar can gain and lose the phaseolotoxin gene cluster, as well as mobile genetic elements, such as plasmids and putative prophages, and that it can modify the repertoire of the effector gene arrays. In addition, the strains currently causing worldwide severe economic losses display an extensive set of genes related to the ecological fitness of the bacterium in planta, such as copper and antibiotic resistance genes, multiple siderophore genes and genes involved in the degradation of lignin derivatives and other phenolics. This pathogen can therefore easily colonize hosts throughout the year. TAXONOMY: Bacteria; Proteobacteria, gamma subdivision; Order Pseudomonadales; Family Pseudomonadaceae; Genus Pseudomonas; Pseudomonas syringae species complex, genomospecies 8; Pathovar actinidiae. MICROBIOLOGICAL PROPERTIES: Gram-negative, aerobic, motile, rod-shaped, polar flagella, oxidase-negative, arginine dihydrolase-negative, DNA 58.5-58.8 mol.% GC, elicits the hypersensitive response on tobacco leaves. HOST RANGE: Primarily studied as the causal agent of bacterial canker of green-fleshed kiwifruit (Actinidia deliciosa), it has also been isolated from yellow-fleshed kiwifruit (A. chinensis). In both species, it causes severe economic losses worldwide. It has also been isolated from wild A. arguta and A. kolomikta. DISEASE SYMPTOMS: In green-fleshed and yellow-fleshed kiwifruits, the symptoms include brown-black leaf spots often surrounded by a chlorotic margin, blossom necrosis, extensive twig die-back, reddening of the lenticels, extensive cankers along the main trunk and leader, and bleeding cankers on the trunk and the leader with a whitish to orange ooze. EPIDEMIOLOGY: Pseudomonas syringae pv. actinidiae can effectively colonize its host plants throughout the year. Bacterial exudates can disperse a large amount of inoculum within and between orchards. In the spring, temperatures ranging from 12 to 18 °C, together with humid conditions, can greatly favour the multiplication of the bacterium, allowing it to systemically move from the leaf to the young shoots. During the summer, very high temperatures can reduce the multiplication and dispersal of the bacterium. Some agronomical techniques, as well as frost, wind, rain and hail storms, can contribute to further spreading. DISEASE CONTROL: An integrated approach that takes into consideration precise scheduled spray treatments with effective and environmentally friendly bactericides and equilibrated plant nutrition, coupled with preventive measures aimed at drastically reducing the bacterial inoculum, currently seems to be the possible best solution for coexistence with the disease. The development of resistant cultivars and pollinators, effective biocontrol agents, including bacteriophages, and compounds that induce the systemic activation of plant defence mechanisms is in progress. USEFUL WEBSITES: Up-to-date information on bacterial canker research progress and on the spread of the disease in New Zealand can be found at: http://www.kvh.org.nz. Daily information on the spread of the disease and on the research being performed worldwide can be found at: http://www.freshplaza.it.     

INVESTIGATION OF FIRE-BLIGHT OF PEAR IN ENGLAND

Outbreaks of fire-blight, caused by Erwinia amylovora , in Kentish orchards during 1957 constitute the first authenticated record of the disease in Europe. The origin of the disease is unknown, but evidence suggests that it was not present in the orchards prior to 1957.
The seasonal development and symptoms of the disease are described in detail. Fire-blight attacked trees only during the growing season; infection began in the blossoms, particularly summer blossoms, and spread into the branches forming cankers, in many of which the pathogen remained viable during the winter 1957–8. In the orchards examined there was a higher incidence of new infections by July 1958 on trees attacked the previous year than on those which were then healthy, and this is discussed in relation to overwintering sources of inoculum. The distribution of the disease in orchards during 1957 and 1958 was consistent with spread by pollinating insects rather than rain.
The outstanding susceptibility of the variety Laxton's Superb in all localities was attributed partly to its tendency to produce abundant secondary blossom. Inoculations of blossoms, fruits, shoots and branches showed it also to have less inherent resistance than the varieties Williams's Bon Chrétien and Conference, neither of which has been seriously affected in the field. The progress of symptoms following inoculation of pear and infection experiments with apple, Cotoneaster sp., Pyracantha sp., and other hosts, are described.
A comparison of climatic conditions favouring the disease in the U.S.A., i.e. high temperatures and high humidity, with meteorological records at East Malling, suggests that epidemic infection of spring blossom would be exceptional in this country.     

Periplasmic glucans of Pseudomonas syringae pv. syringae.

We report the initial characterization of glucans present in the periplasmic space of Pseudomonas syringae pv. syringae (strain R32). These compounds were found to be neutral, unsubstituted, and composed solely of glucose. Their size ranges from 6 to 13 glucose units/mol. Linkage studies and nuclear magnetic resonance analyses demonstrated that the glucans are linked by beta-1,2 and beta-1,6 glycosidic bonds. In contrast to the periplasmic glucans found in other plant pathogenic bacteria, the glucans of P. syringae pv. syringae are not cyclic but are highly branched structures. Acetolysis studies demonstrated that the backbone consists of beta-1,2-linked glucose units to which the branches are attached by beta-1,6 linkages. These periplasmic glucans were more abundant when the osmolarity of the growth medium was lower. Thus, P. syringae pv. syringae appears to synthesize periplasmic glucans in response to the osmolarity of the medium. The structural characteristics of these glucans are very similar to the membrane-derived oligosaccharides of Escherichia coli, apart from the neutral character, which contrasts with the highly anionic E. coli membrane-derived oligosaccharides.     

Histological examination of horse chestnut infection by Pseudomonas syringae pv. aesculi and non-destructive heat treatment to stop disease progression

Since its emergence in Northwest Europe as a pathogen that infects trunks and branches of Aesculus spp. (the horse chestnuts) approximately one decade ago, Pseudomonas syringae pv. aesculi has rapidly established itself as major threat to these trees. Infected trees exhibit extensive necrosis of phloem and cambium, which can ultimately lead to dieback. The events after host entry leading to extensive necrosis are not well documented. In this work, the histopathology of this interaction is investigated and heat-treatment is explored as method to eradicate bacteria associated with established infections. The early wound-repair responses of A. hippocastanum, both in absence and presence of P. s. pv. aesculi, included cell wall lignification by a distinct layer of phloem and cortex parenchyma cells. The same cells also deposited suberin lamellae later on, suggesting this layer functions in compartmentalizing healthy from disrupted tissues. However, monitoring bacterial ingress, its construction appeared inadequate to constrain pathogen spread. Microscopic evaluation of bacterial dispersal in situ using immunolabelling and GFP-tagging of P. s. pv. aesculi, revealed two discriminative types of bacterial colonization. The forefront of lesions was found to contain densely packed bacteria, while necrotic areas housed bacterial aggregates with scattered individuals embedded in an extracellular matrix of bacterial origin containing alginate. The endophytic localization and ability of P. s. pv aesculi to create a protective matrix render it poorly accessible for control agents. To circumvent this, a method based on selective bacterial lethality at 39 °C was conceived and successfully tested on A. hippocastanum saplings, providing proof of concept for controlling this disease by heat-treatment. This may be applicable for curing other tree cankers, caused by related phytopathogens.     

Pseudomonas syringae pv. syringae B728a hydrolyses indole-3-acetonitrile to the plant hormone indole-3-acetic acid

Nitrilase enzymes catalyse the hydrolysis of nitrile compounds to the corresponding carboxylic acid and ammonia, and have been identified in plants, bacteria and fungi. There is mounting evidence to support a role for nitrilases in plant–microbe interactions, but the activity of these enzymes in plant pathogenic bacteria remains unexplored. The genomes of the plant pathogenic bacteria Pseudomonas syringae pv. syringae B728a and Pseudomonas syringae pv. tomato DC3000 contain nitrilase genes with high similarity to characterized bacterial arylacetonitrilases. In this study, we show that the nitrilase of P. syringae pv. syringae B728a is an arylacetonitrilase, which is capable of hydrolysing indole-3-acetonitrile to the plant hormone indole-3-acetic acid, and allows P. syringae pv. syringae B728a to use indole-3-acetonitrile as a nitrogen source. This enzyme may represent an additional mechanism for indole-3-acetic acid biosynthesis by P. syringae pv. syringae B728a, or may be used to degrade and assimilate aldoximes and nitriles produced during plant secondary metabolism. Nitrilase activity was not detected in P. syringae pv. tomato DC3000, despite the presence of a homologous nitrilase gene. This raises the interesting question of why nitrilase activity has been retained in P. syringae pv. syringae B728a and not in P. syringae pv. tomato DC3000.     

Bacterial canker of plum trees, caused by Pseudomonas syringae pathovars, as a serious threat for plum production in the Netherlands

In the Netherlands, bacterial canker in plum trees (Prunus domestica) is a serious and recent problem in plum production. It is caused by Pseudomonas syringae pathovars syringae and morsprunorum. The trunks of the affected plum trees are girdled by bacterial cankers resulting in sudden death of infected trees in 3-4 years after planting. Disease incidences can be very high, and sometimes complete orchards have to be removed. Recently, plum cultivation in the Netherlands has changed from a relatively extensive into an intensive cultivation. However, due to the risks of losses of trees due to bacterial canker, growers are reluctant to plant new plum orchards. In general nurseries and fruit growers are not familiar with bacterial diseases and lack knowledge in order to prevent infections. Therefore, control strategies to manage plum decline have to be developed.     

ACUTE DIE-BACK OF CLOVE TREES IN THE ZANZIBAR PROTECTORATE

The most widespread die-back of clove trees ( Eugenia aromatica) in the Zanzibar Protectorate is caused by Cryptosporella eugeniae sp.nov. The most noticeable symptom is the death of a branch or a portion of a branch, or, in young saplings, of the entire tree; in mature trees infection eventually leads to the semi-moribund trees now common in almost every clove plantation. The fungus invariably enters through a wound, most often one resulting from harvest damage. Pycnidia, and later perithecia, develop near the point of entry. Internally the infected wood is clearly distinguishable from the healthy by a dark red-brown stain.
Experimental infections that reproduce typical symptoms can rarely be caused in plants aged 18 to 36 months, and not at all in young seedlings. Experimental infection of older plants becomes more certain with increasing age, and with saplings of 7–10 years old failure is rare.
The prevalence of Cryptosporella die-back is almost entirely attributable to the destructive methods of harvesting now practised, combined with the presence of much infected material present in the plantations. Suggested control measures are based on the removal of old sources of infection and prevention of future damage, combined with remedial treatment where possible.     

Bacterial canker of poplars in Britain: The cause of the disease and the role of leaf-scars in infection

Bacterial canker and die-back of poplars in Britain is caused by Aplano-bacterium populi Ridé. Pseudomonas syringae (van Hall), which has been suggested as the causal agent, plays at most only a minor role in canker lesions. It may cause a shoot blight in spring, a distinct pathological condition frequently associated with bacterial canker. Marked variation in infectivity of bacterial slime between seasons and at different dates of collection within one year appears to be the main cause for situations in which varieties, resistant in trials to inoculation with slime, have proved susceptible in the field. Leaf scars afford the main avenues for infection but their infectibility declines rapidly during October. The implications of the temporal separation of availability of natural inoculum and available infection sites are discussed.     

Influence of Pseudomonas syringae culture conditions on initiation of the hypersensitive response of culture tobacco cells.

The inhibitor sensitivity and timing of the ionic response of suspension-cultured tobacco cells were used as a bioassay for the Pseudomonas syringae signal that elicits the hypersensitive response in resistant plants. The ionic response of tobacco cell suspensions inoculated with P. syringae pv. syringae 61 and P. syringae pv. pisi grown in rich media was inhibited by rifampin, tetracycline, and streptomycin during a 2- to 2.5-h induction stage. Coculturing the bacteria with tobacco cells for 3 h or more before inoculating fresh tobacco cells specifically abolished the sensitivity of the ionic response to these inhibitors and reduced the response time of the tobacco cells from 3 to 1 h. The apparent activation of the bacteria during coculture was not dependent on the plant cells and could be achieved by incubating the bacteria in a nitrogen-deficient medium containing a metabolizable carbon source. Addition of proteose peptone and Casamino Acids to this medium suppressed activation of the bacteria. The results suggest that the hypersensitive response-eliciting signal forms late in the induction stage, perhaps as a result of the derepression of some of the P. syringae genes functional in elicitation of the hypersensitive response. The nature of the activated state remains elusive but is consistent with the accumulation of protein(s) whose activity indirectly elicits the ionic response.     

Influence of Pseudomonas syringae culture conditions on initiation of the hypersensitive response of culture tobacco cells

The inhibitor sensitivity and timing of the ionic response of suspension-cultured tobacco cells were used as a bioassay for the Pseudomonas syringae signal that elicits the hypersensitive response in resistant plants. The ionic response of tobacco cell suspensions inoculated with P. syringae pv. syringae 61 and P. syringae pv. pisi grown in rich media was inhibited by rifampin, tetracycline, and streptomycin during a 2- to 2.5-h induction stage. Coculturing the bacteria with tobacco cells for 3 h or more before inoculating fresh tobacco cells specifically abolished the sensitivity of the ionic response to these inhibitors and reduced the response time of the tobacco cells from 3 to 1 h. The apparent activation of the bacteria during coculture was not dependent on the plant cells and could be achieved by incubating the bacteria in a nitrogen-deficient medium containing a metabolizable carbon source. Addition of proteose peptone and Casamino Acids to this medium suppressed activation of the bacteria. The results suggest that the hypersensitive response-eliciting signal forms late in the induction stage, perhaps as a result of the derepression of some of the P. syringae genes functional in elicitation of the hypersensitive response. The nature of the activated state remains elusive but is consistent with the accumulation of protein(s) whose activity indirectly elicits the ionic response.     

Microscopic examination of natural sessile bacterial populations from an alpine stream.

Natural populations of bacteria assoiciated with the slime on submerged surfaces in a mountain stream were examined by phase-contrast and electron microscopy. The slime contained large numbers of bacteria which were predominantly gram-negative as determined by their cell wall structure. Examination of the in situ distribution of cells revealed that they were enmeshed in an extensive fibrous matrix whose component fibrils were stained with ruthenium red. The arrangement of slime fibrils immediately around individual bacterial cells suggested that this material was produced by these bacteria. This slime facilitated microcolony development and also anchored the bacteria to a particular surface. It is proposed that these slime-enmeshed microcolonies constitute functional communities within which most sessile bacteria live.     

Relationship of total viable and culturable cells in epiphytic populations of Pseudomonas syringae.

The direct viable count method, used to detect viable but nonculturable bacteria in aquatic systems, was modified to examine epiphytic populations of Pseudomonas syringae. Viable-population sizes determined from the number of cells that elongated when incubated with yeast extract and nalidixic acid were compared with those determined by the conventional plate count method. The plate count method accurately determined the number of viable cells in epiphytic P. syringae populations in a state of active growth under conditions of high relative humidity. The plate count method also accurately determined the number of viable cells in P. syringae inoculum, or a growing P. syringae population, subject to desiccation stress under conditions of low relative humidity. In epiphytic populations of P. syringae older than 80 h, however, the plate count underestimated the viable-population size by about two- to fourfold, suggesting that up to 75% of the P. syringae population was nonculturable. These nonculturable cells may have entered a starvation-survival state, induced by low nutrient availability in the phyllosphere environment. Epiphytic P. syringae populations undergoing rapid size changes due to growth and death under fluctuating environmental conditions in the field should be accurately enumerated by the plate count method. However, the possible underestimation of viable-population size under some circumstances should be considered in epidemiological studies of phytopathogenic bacteria and when genetically engineered microorganisms in terrestrial ecosystems are monitored.     

Competitive Exclusion of Epiphytic Bacteria by IcePseudomonas syringae Mutants

The growth of ice nucleation-active and near-isogenic ice nucleation-deficient (Ice) Pseudomonas syringae strains coexisting on leaf surfaces was examined to determine whether competition was sufficient to account for antagonism of phylloplane bacteria. The ice nucleation frequency spectra of 46 IceP. syringae mutants, obtained after mutagenesis with ethyl methanesulfonate, differed both quantitatively and qualitatively, but the mutants could be grouped into four distinct phenotypic classes. The numbers of ice nucleation-active bacteria and ice nuclei active at -5 degrees C were reduced on plants colonized with IceP. syringae mutant strains before challenge inoculations with an IceP. syringae wild-type strain. Frost injury to plants pretreated with IceP. syringae strains was also reduced significantly compared with that to control plants and was correlated with the population size of the IceP. syringae strain and with the numbers of ice nuclei active at -5 degrees C. An IceP. syringae strain colonized leaves, flowers, and young fruit of pears in field experiments and significantly reduced the colonization of these tissues by IceP. syringae strains and Erwinia amylovora as compared with untreated trees.