Bacterial canker of stone-fruits: Infection experiments with Pseudomonas mors-prunorum and P. syringae

The effect was compared of inoculating Pseudomonas mors-prunorum and P. syringae at high inoculum concentration through wounds in plum stems and cherry branches, and through cherry-leaf scars. In the wound inoculations a North American cherry strain of P. syringae was considerably more virulent than any of three indigenous cherry strains of P. mors-prunorum, but two pear strains of P. syringae were less virulent. P. mors-prunorum showed a much greater capacity to invade through leaf scars, particularly towards the end of the leaf-fall period, when all three P. syringae strains were largely ineffective. Evidence is discussed which suggests that the greater infectivity of the P. mors-prunorum strains through leaf scars was related to their capacity to colonize the host tissues from small inocula. The P. syringae strains died out in cherry cankers earlier than P. mors-prunorum strains and they were less stable during host passage. The results of the experiments indicate that virulence in these organisms is a complex phenomenon and determined by several factors.     

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.     

The control of apple canker (Nectria galligena) in a young orchard with established infections

In a factorial experiment on canker control the efficiency of phenylmercurie nitrate (PMN) applied at leaf fall, before bud burst or both was compared with the application of dodine, dithianon, triforine, thiabendazole, benomyl or carbendazim in May and June. Of the total number of cankers which developed in unsprayed trees 76–78% resulted from infections in April to August of each year of the experiment. Infection was reduced significantly by all of the fungicides applied in summer. Of these carbendazim was outstanding, controlling both summer and autumn infections. Sporulation throughout the summer was suppressed by carbendazim and to a lesser extent benomyl, but whereas the suppressant effect of carbendazim persisted until long after leaf fall that of benomyl was evident only until August. Dodine, dithianon, triforine and thiabendazole had no significant effect on spore production. Dithianon and dodine showed highest toxicity to the germination of Nectria galligena spores of all fungicides used in summer. All of the fungicides controlled apple scab (Venturia inaequalis) although thiabendazole was the least effective. Autumn applications of PMN reduced canker incidence but their value alone was not as great as carbendazim, dithianon, benomyl or dodine applied in summer. PMN applied in spring reduced the number of cankers in trees receiving no other fungicides but tended to increase the incidence of infections, particularly in autumn, when used in conjunction with fungicides applied in May and June. PMN applied before bud burst reduced sporulation of N. galligena for a few weeks, after which production resumed and in late summer and autumn actually exceeded the controls. None of the fungicides had any direct effect on leaf fall. Infection of the crotch and basal leaf scars was more common in summer than in autumn whereas infection of leaf scars above the basal region was more common in autumn than in summer. The rootstocks of many of the trees became infected via callus tissue associated with adventitious root development and this was controlled by the carbendazim treatment.     

Evaluation of Pathogenicity and of Cultural and Biochemical Tests for Identification of Pseudomonas syringae Pathovars syringae, morsprunorum and persicae from Fruit Trees

Comparative studies were based on 216 isolates of the Pseudomonas syringae pathovars syringae, morsprunorum and persicae, mainly originating from fruit trees in several European countries, but also from USA, USSR, South Africa, New Zealand and Turkey. All the identified morsprunorum isolates were obtained from stonefruit trees, which were also a source of some syringae isolates. Fluorescent strains HR negative on tobacco leaves were regarded as saprophytic pseudomonads. Nearly all the HR positive but none of the HR negative isolates caused lesions on sweet cherry shoots and fruitlets. The largest lesions on sweet cherry shoots were caused by the morsprunorum isolates. Syringae strains always caused lesions on pear fruitlets, whereas the morsprunorum strains never did so. Some degree of specialization within the pathovar syringae seemed to exist. Cultural and biochemical tests well suited to differentiate the three pathovars were as follows: colour after growth in sucrose nutrient broth, liquefaction of gelatin, activity of β-glucosidase (arbutin) and tyrosinase, use of L-leucine, L(+)tartrate and DL-lactate as sole carbon source, fluorescent pigment production, crystaline inclusions in the nutrient agar medium, and growth rate in 0.2% yeast extract nutrient broth. Tests for longevity on nutrient agar with 5% sucrose turned out to be fairly unreliable. A tube assay gave the most consistent results when studying utilization of organic substrates. The capability for gelatin liquefaction correlated with virulence, and mucoid isolates showed a tendency for higher aggressiveness than rough colony variants. Page of protein extracts obtained from freeze-pressed bacterial cells revealed that the pathovars syringae and morsprunorum could be differentiated due to specific isoenzyme patterns for esterases and acid phosphatases. It was concluded that several pathogenicity, cultural and biochemical tests allowed an unequivocal differentiation of the Pseudomonas syringae pvs. syringae and morsprunorum. Some of the discriminating biochemical characters may, play a role during pathogenesis.     

Uptake and movement of the fungicide imazalil following trunk injection into apple and plum trees by a novel, rapid technique

A hand-held apparatus, produced by modifying a veterinary multiple dosing gun, was used to pressure-inject aqueous solutions of imazalil into the trunks of plum or apple trees after harvest, before bud burst or during flowering. Bioassay showed that post harvest injection led to movement of imazalil up and down the trunk and into branches of both apple and plum trees. Movement in the trunk after injection before bud burst also occurred in plums but was limited in apples and fungicide was detected in branches only in apples and only at the end of the season. Injection at flowering time resulted in movement of imazalil into branches of apple, but not plum, trees. Imazalil was detected in twigs of apple, but not plum, trees but only after flowering time injections. The technique could be useful for control of tree diseases provided due attention is paid to dose, to the number and siting of injection holes and to the linking of injection time with the epidemiology of the target disease.     

Phylogenetic Analysis of Pseudomonas syringae Pathovars Suggests the Horizontal Gene Transfer of argK and the Evolutionary Stability of hrp Gene Cluster

Pseudomonas syringae are differentiated into approximately 50 pathovars with different plant pathogenicities and host specificities. To understand its pathogenicity differentiation and the evolutionary mechanisms of pathogenicity-related genes, phylogenetic analyses were conducted using 56 strains belonging to 19 pathovars. gyrB and rpoD were adopted as the index genes to determine the course of bacterial genome evolution, and hrpL and hrpS were selected as the representatives of the pathogenicity-related genes located on the genome (chromosome). Based on these data, NJ, MP, and ML phylogenetic trees were constructed, and thus 3 trees for each gene and 12 gene trees in total were obtained, all of which showed three distinct monophyletic groups: Groups 1, 2 and 3. The observation that the same set of OTUs constitute each group in all four genes suggests that these genes had not experienced any intergroup horizontal gene transfer within P. syringae but have been stable on and evolved along with the P. syringae genome. These four index genes were then compared with another pathogenicity-related gene, argK (the phaseolotoxin-resistant ornithine carbamoyltransferase gene, which exists within the argK–tox gene cluster). All 13 strains of pv. phaseolicola and pv. actinidiae used had been confirmed to produce phaseolotoxin and to have argK, whose sequences were completely identical, without a single synonymous substitution among the strains used (Sawada et al. 1997a). On the other hand, argK were not present on the genomes of the other 43 strains used other than pv. actinidiae and pv. phaseolicola. Thus, the productivity of phaseolotoxin and the possession of the argK gene were shown at only two points on the phylogenetic tree: Group 1 (pv. actinidiae) and Group 3 (pv. phaseolicola). A t test between these two pathovars for the synonymous distances of argK and the tandemly combined sequence of the four index genes showed a high significance, suggesting that the argK gene (or argK–tox gene cluster) experienced horizontal gene transfer and expanded its distribution over two pathovars after the pathovars had separated, thus showing a base substitution pattern extremely different from that of the noncluster region of the genome. Received: 18 January 1999 / Accepted: 25 May 1999     

Identification of viruses isolated from plum trees affected by decline, line-pattern and ringspot diseases

Viruses obtained from plum trees infected with either decline, line-pattern or ringspot diseases were characterized and identified. All the viruses were serologically related to Prunus necrotic ringspot virus (NRSV); those from trees with decline or ringspot were serologically indistinguishable from cherry strains of NRSV but differed in pathogenicity, whereas the virus from trees with line pattern was closely related to apple mosaic virus. When returned from herbaceous hosts to Prunus, line-pattern and ringspot isolates reproduced symptoms characteristic of the diseased sources. One virus isolate from trees with decline diminished the vigour of young plum trees. Comparison with other investigations shows that at least two unrelated viruses cause plum line-pattern disease in America and Europe.     

The genetic characterization of Pseudomonas syringae pv. tagetis based on the 16S–23S rDNA intergenic spacer regions

Pseudomonas syringae pv. tagetis, a plant pathogen being considered as a biological control agent of Canada thistle (Cirsium arvense), produces tagetitoxin, an inhibitor of RNA polymerase which results in chlorosis of developing shoot tissues. Although the bacterium is known to affect several plant species in the Asteraceae and has been reported in several countries, little is known of its genetic diversity. The genetic relatedness of 24 strains of P. syringae pv. tagetis with respect to each other and to other P. syringae and Pseudomonas savastanoi pathovars was examined using 16S–23S rDNA intergenic spacer (ITS) sequence analysis. The size of the 16S–23S rDNA ITS regions ranged from 508 to 548 bp in length for all 17 P. syringae and P. savastanoi pathovars examined. The size of the 16S–23S rDNA ITS regions for all the P. syringae pv. helianthi and all the P. syringae pv. tagetis strains examined were 526 bp in length. Furthermore, the 16S–23S rDNA ITS regions of both P. syringae pv. tagetis and P. syringae pv. helianthi had DNA signatures at specific nucleotides that distinguished them from the 15 other P. syringae and P. savastanoi pathovars examined. These results provide strong evidence that P. syringae pv. helianthi is a nontoxigenic form of P. syringae pv. tagetis. The results also demonstrated that there is little genetic diversity among the known strains of P. syringae pv. tagetis. The genetic differences that do exist were not correlated with differences in host plant, geographical origin, or the ability to produce toxin.     

Evaluation of the Potential of five Housekeeping Genes for Identification of Quarantine Pseudomonas syringae

The Pseudomonas syringae species, containing approximately 60 pathovars, causes bacterial plant diseases on numerous crops and results in great economic losses. It is difficult to rapidly and accurately identify and detect P. syringae due to its complicated classification system. It has also been shown that housekeeping genes have great potential in phylogenetic analysis and classification of bacteria. In this study, phylogenetic analyses of five housekeeping genes, including 16S rRNA, rpoD, gapA, gltA and gyrB, were performed for 100 Pseudomonas strains. Our results showed that two housekeeping genes (rpoD and gapA) had the maximum ability in distinguishing the majority of Pseudomonas pathovars, whereas rpoD exhibited the best for precise and efficient detection of five P. syringae strains, which is of quarantine significance to China.     

Pseudomonas syringae colonizes distant tissues in Nicotiana benthamiana through xylem vessels

The ability to move from the primary infection site and colonize distant tissue in the leaf is an important property of bacterial plant pathogens, yet this aspect has hardly been investigated for model pathogens. Here we show that GFP‐expressing Pseudomonas syringae pv. syringae DC3000 that lacks the HopQ1‐1 effector (PtoDC3000ΔhQ) has a strong capacity to colonize distant leaf tissue from wound‐inoculated sites in N. benthamiana. Distant colonization occurs within 1 week after toothpick inoculation and is characterized by distant colonies in the apoplast along the vasculature. Distant colonization is blocked by the non‐host resistance response triggered by HopQ1‐1 in an SGT1‐dependent manner and is associated with an explosive growth of the bacterial population, and displays robust growth differences between compatible and incompatible interactions. Scanning electron microscopy revealed that PtoDC3000ΔhQ bacteria are present in xylem vessels, indicating that they use the xylem to move through the leaf blade. Distant colonization does not require flagellin‐mediated motility, and is common for P. syringae pathovars that represent different phylogroups.     

Phage biocontrol to combat Pseudomonas syringae pathogens causing disease in cherry

Bacterial canker is a major disease of Prunus species, such as cherry (Prunus avium). It is caused by Pseudomonas syringae pathovars, including P. syringae pv. syringae (Pss) and P. syringae pv. morsprunorum race 1 (Psm1) and race 2 (Psm2). Concerns over the environmental impact of, and the development of bacterial resistance to, traditional copper controls calls for new approaches to disease management. Bacteriophage-based biocontrol could provide a sustainable and natural alternative approach to combat bacterial pathogens. Therefore, seventy phages were isolated from soil, leaf and bark of cherry trees in six locations in the south east of England. Subsequently, their host range was assessed against strains of Pss, Psm1 and Psm2. While these phages lysed different Pss, Psm and some other P. syringae pathovar isolates, they did not infect beneficial bacteria such as Pseudomonas fluorescens. A subset of thirteen phages were further characterized by genome sequencing, revealing five distinct clades in which the phages could be clustered. No known toxins or lysogeny-associated genes could be identified. Using bioassays, selected phages could effectively reduce disease progression in vivo, both individually and in cocktails, reinforcing their potential as biocontrol agents in agriculture.     

Comparative Genome Analysis Provides Insights into the Evolution and Adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum

A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree.     

Genetic Characterization of Pseudomonas syringae pv. syringae Strains from Stone Fruits in California

Strains of Pseudomonas syringae pv. syringae were isolated from healthy and diseased stone fruit tissues sampled from 43 orchard sites in California in 1995 and 1996. These strains, together with P. syringae strains from other hosts and pathovars, were tested for pathogenicity and the presence of the syrB and syrC genes and were genetically characterized by using enterobacterial repetitive intergenic consensus (ERIC) primers and PCR. All 89 strains of P. syringae pv. syringae tested were moderately to highly pathogenic on Lovell peach seedlings regardless of the host of origin, while strains of other pathovars exhibited low or no pathogenicity. The 19 strains of P. syringae pv. syringae examined by restriction fragment length polymorphism analysis contained the syrB and syrC genes, whereas no hybridization occurred with 4 strains of other P. syringae pathovars. The P. syringae pv. syringae strains from stone fruit, except for a strain from New Zealand, generated ERIC genomic fingerprints which shared four fragments of similar mobility. Of the P. syringae pv. syringae strains tested from other hosts, only strains from rose, kiwi, and pear generated genomic fingerprints that had the same four fragments as the stone fruit strains. Analysis of the ERIC fingerprints from P. syringae pv. syringae strains showed that the strains isolated from stone fruits formed a distinct cluster separate from most of the strains isolated from other hosts. These results provide evidence of host specialization within the diverse pathovar P. syringae pv. syringae.     

Antimicrobial activity of essential oils of Thymus vulgaris and Origanum vulgare on phytopathogenic strains isolated from soybean

The aim of this work was to study the antimicrobial activity of essential oils obtained from Thymus vulgaris (thyme) and Origanum vulgare (oregano) on phytopathogenic Pseudomonas species isolated from soybean. Strains with characteristics of P. syringae were isolated from leaves of soybean plants with blight symptoms. Ten of these could be identified in Group Ia of LOPAT as P. syringae. Six of these were confirmed as P. syringae using 16S rRNA, indicating the presence of these phytopathogenic bacteria in east and central Argentina. All the phytopathogenic bacteria were re‐isolated and identified from the infected plants. MIC values for thyme were 11.5 and 5.7 mg·ml^?1 on P. syringae strains, while oregano showed variability in the inhibitory activity. Both essential oils inhibited all P. syringae strains, with better inhibitory activity than the antibiotic streptomycin. The oils were not bactericidal for all pseudomonads. Both oils contained high carvacrol (29.5% and 19.7%, respectively) and low thymol (1.5%). Natural products obtained from aromatic plants represent potential sources of molecules with biological activity that could be used as new alternatives for the treatment of phytopathogenic bacteria infections.     

The Occurrence of Bacterial Red Streak of Sugarcane Caused by Pseudomonas syringae pv. syringae in Iran

A bacterial leaf streak disease characterized by reddish, narrow (1–2 mm wide) streaks of variable size, and occasionally with bleached centers, was found in sugarcane (Saccharum, interspecific hybrid) fields in northern Iran. The incitant bacterium was identified as Pseudomonas syringae pv. syringae (P. s. syringae). The disease is similar in aetiology to the sugarcane ‘red streak’ disease reported recently from Japan. Cultivardependent variations in symptoms were noted., Difference in pathogenicity as well as in electrophoretic profile of cell proteins between strains of P.s. syringae causing red streak in sugarcane and those causing canker on stone fruit trees, were observed.     

Occurrence of Phytophthora Root and Stem Rot of Kiwifruit in Turkey

Vine decline of kiwifruit was observed in an orchard in Bart?n province of Turkey. Affected vines exhibited poor terminal growth, leaf discoloration and various degrees of dieback, including complete vine death. Symptoms were observed in the field on roots, crowns and stems. Two Phytophthora species were isolated from decayed cortical roots and lower stems of kiwifruits. They were identified as Phytophthora cryptogea and Phytophthora megasperma by their morphological characteristics and the analysis of sequences of the internal transcribed spacer (ITS) region of the rDNA. Pathogenicity of the isolates was tested by stem inoculation on kiwifruit seedlings. After 4 weeks, cankers developed in the plants inoculated with P. cryptogea, while no cankers formed in those inoculated with P. megasperma and in control plants. This is the first report of P. cryptogea causing root and stem rot of kiwifruit in Turkey.     

Bacterial Canker of Peach: Effect of Tree Winter Water Content on the Spread of Infection Through Frost-related Water Soaking in Stems

Several experiments were conducted to study the influence of water content in trees on dye diffusion or controlled Pseudomonas syringae pv. persicae infection spread induced in winter by water soaking after freezing and thawing. These were carried out in a cold cabinet in excised dormant hardy 1-year-old slightly dehydrated or rehydrated peach shoots, and in whole variably hydrated peach trees cultivated outdoors in 3001 containers. In shoots, dye diffusion was more substantial and infection more widespread when water content was higher. In potted peach trees, the spread of individual bacterial cankers which was induced by inoculating different shoots over two successive winters, was more restricted when tree water content was lower. In both years, a high positive correlation coefficient was observed between individual tree water content and mean canker length. This study revealed that stem water content in peach trees during winter and frosts is a fundamental factor in infection because it can drastically affect bacteria diffusion in cortical tissues during frost- and thaw-related water soaking. The effect of water content may have important epidemiological consequences firstly due to the influence of several environmental and agronomic factors on tree water content, and then to the prevalence of frost-related water soaking in many perennial and herbaceous plants.     

Phytophthora collar rot of apple: seasonal effects on infection and disease development

One-to three-year-old trees of the apple variety Cox's Orange Pippin were highly resistant to infection by Phytophthora cactorum except during the spring from March to May. A rapid increase in resistance occurred after this time from the commencement of shoot growth. The period of susceptibility to infection by P. syringae was from October to December, when trees were dormant. Inoculations with either fungus during the respective susceptible periods caused rapidly extending lesions which girdled and killed the trees; established lesions continued to enlarge in months when trees were resistant to infection. Similar seasonal fluctuations in resistance to infection by P. cactorum also occurred in mature (35-year-old) Cox trees but the susceptible period was longer. Lesions resulting from inoculations at the optimum time (the ‘mouseear’ to ‘pink-bud’ stages of development) extended at similar rates in both young and old trees. The infrequent incidence of collar rot in young trees is probably related to factors other than inherent resistance. In resistance and pathogenicity studies young trees gave reliable and consistent results, provided that inoculations were correctly timed in relation to bud development.     

Quick Decline of Macadamia Trees: Association with Phytophthora capsici

Some macadamia trees in commercial orchards in Hawaii showing quick decline syndrome had bleeding from the trunk. Phytophthora capsici was isolated from c. 67% of such sites, and was shown to kill branches of artificially-inoculated healthy macadamia trees. The pathogen was detected in both diseased and apparently healthy bark and isolated from wood 80 mm away from the bark. Results suggest that trunk infection by P. capsici may lead to girdling and rapid decline, and attract insects which then cause some bleeding by making wounds at the sites of recent infection.     

A Phylogenomic Study of the OCTase Genes in Pseudomonas syringae Pathovars: The Horizontal Transfer of the argK–tox Cluster and the Evolutionary History of OCTase Genes on Their Genomes

Phytopathogenic Pseudomonas syringae is subdivided into about 50 pathovars due to their conspicuous differentiation with regard to pathogenicity. Based on the results of a phylogenetic analysis of four genes (gyrB, rpoD, hrpL, and hrpS), Sawada et al. (1999) showed that the ancestor of P. syringae had diverged into at least three monophyletic groups during its evolution. Physical maps of the genomes of representative strains of these three groups were constructed, which revealed that each strain had five rrn operons which existed on one circular genome. The fact that the structure and size of genomes vary greatly depending on the pathovar shows that P. syringae genomes are quite rich in plasticity and that they have undergone large-scale genomic rearrangements. Analyses of the codon usage and the GC content at the codon third position, in conjunction with phylogenomic analyses, showed that the gene cluster involved in phaseolotoxin synthesis (argK–tox cluster) expanded its distribution by conducting horizontal transfer onto the genomes of two P. syringae pathovars (pv. actinidiae and pv. phaseolicola) from bacterial species distantly related to P. syringae and that its acquisition was quite recent (i.e., after the ancestor of P. syringae diverged into the respective pathovars). Furthermore, the results of a detailed analysis of argK [an anabolic ornithine carbamoyltransferase (anabolic OCTase) gene], which is present within the argK–tox cluster, revealed the plausible process of generation of an unusual composition of the OCTase genes on the genomes of these two phaseolotoxin-producing pathovars: a catabolic OCTase gene (equivalent to the orthologue of arcB of P. aeruginosa) and an anabolic OCTase gene (argF), which must have been formed by gene duplication, have first been present on the genome of the ancestor of P. syringae; the catabolic OCTase gene has been deleted; the ancestor has diverged into the respective pathovars; the foreign-originated argK–tox cluster has horizontally transferred onto the genomes of pv. actinidiae and pv. phaseolicola; and hence two copies of only the anabolic OCTase genes (argK and argF) came to exist on the genomes of these two pathovars. Thus, the horizontal gene transfer and the genomic rearrangement were proven to have played an important role in the pathogenic differentiation and diversification of P. syringae. Received: 22 May 2001 / Accepted: 26 September 2001