Identification of Bacteriophages for Biocontrol of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae is a reemerging pathogen which causes bacterial canker of kiwifruit (Actinidia sp.). Since 2008, a global outbreak of P. syringae pv. actinidiae has occurred, and in 2010 this pathogen was detected in New Zealand. The economic impact and the development of resistance in P. syringae pv. actinidiae and other pathovars against antibiotics and copper sprays have led to a search for alternative management strategies. We isolated 275 phages, 258 of which were active against P. syringae pv. actinidiae. Extensive host range testing on P. syringae pv. actinidiae, other pseudomonads, and bacteria isolated from kiwifruit orchards showed that most phages have a narrow host range. Twenty-four were analyzed by electron microscopy, pulse-field gel electrophoresis, and restriction digestion. Their suitability for biocontrol was tested by assessing stability and the absence of lysogeny and transduction. A detailed host range was performed, phage-resistant bacteria were isolated, and resistance to other phages was examined. The phages belonged to the Caudovirales and were analyzed based on morphology and genome size, which showed them to be representatives of Myoviridae, Podoviridae, and Siphoviridae. Twenty-one Myoviridae members have similar morphologies and genome sizes yet differ in restriction patterns, host range, and resistance, indicating a closely related group. Nine of these Myoviridae members were sequenced, and each was unique. The most closely related sequenced phages were a group infecting Pseudomonas aeruginosa and characterized by phages JG004 and PAK_P1. In summary, this study reports the isolation and characterization of P. syringae pv. actinidiae phages and provides a framework for the intelligent formulation of phage biocontrol agents against kiwifruit bacterial canker.     

Genomic Analysis of the Kiwifruit Pathogen Pseudomonas syringae pv. actinidiae Provides Insight into the Origins of an Emergent Plant Disease

The origins of crop diseases are linked to domestication of plants. Most crops were domesticated centuries – even millennia – ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease.     

DNA markers for identification of Pseudomonas syringae pv. actinidiae

The specific DNA fragment was screened by RAPD analysis of Pseudomonas syringae pv. actinidiae, as well as similar strains that were isolated from kiwifruits. The primer C24 detected a fragment that is specific in P. syringae pv. actinidiae. This fragment was cloned. The pathovar-specific fragment was detected from a Southern blot analysis of the genomic DNAs of P. syringae pv. actinidiae using the cloned fragment as a probe. The sequence size of the cloned fragment was determined as 675 bp. A DNA Database search suggested that the fragment was a novel one. Approximately 9 kb of a single fragment was detected only in the P. syringae pv. actinidiae by a Southern blot analysis of the genomic DNAs of P. syringae pv. actinidiae. Similar strains were also detected with the use of the cloned fragment as a probe. Since the genomic DNAs were digested with HindIII without a cleavage site, the result reveals that the cloned fragment exists on the genome of P. syringae pv. actinidiae as a single copy. A pair of primers that produced a 492 bp single fragment (only in the strains of P. syringae pv. actinidiae) were synthesized, based on the pathovar-specific sequences of the cloned fragment of P. syringae pv. actinidiae. The development of the primers and probe made it possible to diagnose the bacterial canker infection from leaves or trunks of kiwifruit trees before any symptom appeared on the tree.     

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.     

Pseudomonas syringae pv. actinidiae detection in kiwifruit plant tissue and bleeding sap

The rapid spreading of the disease during last few years highlighted the need of a quick, sensitive and reliable method for Pseudomonas syringae pv. actinidiae (Psa) detection, to find possible inoculum sources and limit the pathogen spreading. A PCR method, using new primers designed on the gene encoding a putative outer membrane protein P1, was developed to detect Psa in symptomatic and asymptomatic tissue; a nested‐PCR was also applied. Bleeding sap samples, collected in early spring from orchards with symptomatic and asymptomatic trees, were used both for PCR assays and for pathogen isolation and identification. The PCR and nested PCR methods were able to detect Psa presence at very low concentration from plant and pollen extracts; RFLP analyses with BclI on PCR and nested PCR amplicons confirmed the assay specificity, while the digestion with BfmI and AluI allowed to discriminate Psa strains isolated before 2008 from those isolated after 2008. Furthermore, the PCR and nested PCR on crude bleeding sap samples detected the presence of the pathogen in 3 and 5 of the 15 assayed samples, respectively. Direct isolation from the same samples and bacterial identification confirmed the results of molecular analysis.     

猕猴桃细菌性溃疡病流行预测初探

对猕猴桃溃疡病流行分析表明,影响该病发生严重程度y的生态因子是3月中下旬降水x1和1月份均温x2,其模型是y=2.1359 0.0107x1-0.6061x2;猕猴桃溃疡病发生流行的主导因子为冬季及初春旬均温和降水量的相对变差,并且由此得到病害流行的回归方程为：y=-8.127 22.739x-13.254x^2,经检验,该方程达极显著水平。     

Molecular and Physiological Characterization of Pseudomonas syringae pv. tomato and Pseudomonas syringae pv. maculicola Strains That Produce the Phytotoxin Coronatine

The chlorosis-inducing phytotoxin coronatine is produced by several Pseudomonas syringae pathovars, including glycinea, morsprunorum, atropurpurea, and the closely related tomato and maculicola. To date, all coronatine-producing pv. glycinea, morsprunorum, and atropurpurea strains that have been examined carry the gene cluster that controls toxin production on a large plasmid. In the present study the genomic location of the coronatine gene cluster was determined for coronatine-producing strains of the pv. tomato-maculicola group by subjecting their genomic DNA to pulsed-field electrophoresis and Southern blot analysis with a hybridization probe from the coronatine gene cluster. The cluster was chromosomally borne in 10 of the 22 strains screened. These 10 strains infected both crucifers and tomatoes but could not use sorbitol as a sole source of carbon. The remaining 12 coronatine-producing strains had plasmid-borne toxin gene clusters and used sorbitol as a carbon source. Only one of these strains was pathogenic on both crucifers and tomatoes; the remainder infected just tomatoes. Restriction fragment length polymorphism analysis of the pv. tomato-maculicola coronatine gene clusters was performed with probes from P. syringae pv. tomato DC3000, a tomato and crucifer pathogen. Although the coronatine cluster appeared, in general, to be highly conserved across the pv. tomato-maculicola group, there were significant differences between plasmid-borne and chromosomally borne genes. The extensively studied coronatine cluster of pv. glycinea 4180 closely resembled the plasmid-borne clusters of the pv. tomato-maculicola group.     

Studies on the Infection,Colonization, and Movement of Pseudomonas syringae pv. actinidiae in Kiwifruit Tissues Using a GFPuv-Labeled Strain

Kiwifruit bacterial canker, an economically important disease caused by Pseudomonas syringae pv. actinidiae (Psa), has caused severe losses in all major areas of kiwifruit cultivation. Using a GFPuv-labeled strain of Psa, we monitored the invasion, colonization, and movement of the pathogen in kiwifruit twigs, leaves and veins. The pathogen can invade twigs through both wounds and natural openings; the highest number of Psa is obtained in cut tissues. We determined that, following spray inoculation, Psa-GFPuv could infect leaves and cause lesions in the presence and absence of wounds. Light and transmission electron microscopic observations showed that bacterial cells colonize both phloem and xylem vessels. Bacterial infection resulted in marked alterations of host tissues including the disintegration of organelles and degeneration of protoplasts and cell walls. Furthermore, low temperature was conducive to colonization and movement of Psa-GFPuv in kiwifruit tissues. Indeed, the pathogen migrated faster at 4°C than at 16°C or 25°C in twigs. However, the optimum temperature for colonization and movement of Psa in leaf veins was 16°C. Our results, revealing a better understanding of the Psa infection process, might contribute to develop more efficacious disease management strategies.     

Pseudomonas syringae pv. actinidiae from Recent Outbreaks of Kiwifruit Bacterial Canker Belong to Different Clones That Originated in China

A recently emerged plant disease, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis), is caused by Pseudomonas syringae pv. actinidiae (PSA). The disease was first reported in China and Japan in the 1980s. A severe outbreak of PSA began in Italy in 2008 and has spread to other European countries. PSA was found in both New Zealand and Chile in 2010. To study the evolution of the pathogen and analyse the transmission of PSA between countries, genomes of strains from China and Japan (where the genus Actinidia is endemic), Italy, New Zealand and Chile were sequenced. The genomes of PSA strains are very similar. However, all strains from New Zealand share several single nucleotide polymorphisms (SNPs) that distinguish them from all other PSA strains. Similarly, all the PSA strains from the 2008 Italian outbreak form a distinct clonal group and those from Chile form a third group. In addition to the rare SNPs present in the core genomes, there is abundant genetic diversity in a genomic island that is part of the accessory genome. The island from several Chinese strains is almost identical to the island present in the New Zealand strains. The island from a different Chinese strain is identical to the island present in the strains from the recent Italian outbreak. The Chilean strains of PSA carry a third variant of this island. These genomic islands are integrative conjugative elements (ICEs). Sequencing of these ICEs provides evidence of three recent horizontal transmissions of ICE from other strains of Pseudomonas syringae to PSA. The analyses of the core genome SNPs and the ICEs, combined with disease history, all support the hypothesis of an independent Chinese origin for both the Italian and the New Zealand outbreaks and suggest the Chilean strains also originate from China.     

Effector loss drives adaptation of Pseudomonas syringae pv. actinidiae biovar 3 to Actinidia arguta

A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia chinensis. In contrast, A. arguta (kiwiberry) is not a host of Psa3. Resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have been isolated and analyzed, revealing a 51 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors–AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a –suggesting a resistance response in A. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes in Actinidia for breeding durable resistance into future kiwifruit cultivars.     

Bactericidal Compounds Controlling Growth of the Plant Pathogen Pseudomonas syringae pv. actinidiae,Which Forms Biofilms Composed of a Novel Exopolysaccharide

Pseudomonas syringae pv. actinidiae is the major cause of bacterial canker and is a severe threat to kiwifruit production worldwide. Many aspects of the disease caused by P. syringae pv. actinidiae, such as the pathogenicity-relevant formation of a biofilm composed of extracellular polymeric substances (EPSs), are still unknown. Here, a highly virulent strain of P. syringae pv. actinidiae, NZ V-13, was studied with respect to biofilm formation and architecture using a flow cell system combined with confocal laser scanning microscopy. The biofilm formed by P. syringae pv. actinidiae NZ V-13 was heterogeneous, consisting of a thin cellular base layer 5 μm thick and microcolonies with irregular structures. The major component of the EPSs produced by P. syringae pv. actinidiae NZ V-13 bacteria was isolated and identified to be an exopolysaccharide. Extensive compositional and structural analysis showed that rhamnose, fucose, and glucose were the major constituents, present at a ratio of 5:1.5:2. Experimental evidence that P. syringae pv. actinidiae NZ V-13 produces two polysaccharides, a branched α-d-rhamnan with side chains of terminal α-d-Fucf and an α-d-1,4-linked glucan, was obtained. The susceptibility of the cells in biofilms to kasugamycin and chlorine dioxide was assessed. About 64 and 73% of P. syringae pv. actinidiae NZ V-13 cells in biofilms were killed when kasugamycin and chlorine dioxide were used at 5 and 10 ppm, respectively. Kasugamycin inhibited the attachment of P. syringae pv. actinidiae NZ V-13 to solid surfaces at concentrations of 80 and 100 ppm. Kasugamycin was bacteriostatic against P. syringae pv. actinidiae NZ V-13 growth in the planktonic mode, with the MIC being 40 to 60 ppm and a bactericidal effect being found at 100 ppm. Here we studied the formation, architecture, and composition of P. syringae pv. actinidiae biofilms as well as used the biofilm as a model to assess the efficacies of bactericidal compounds.     

Genome Sequence of the Plant Pathogen Pseudomonas syringae pv. panici LMG 2367

Pseudomonas syringae pv. panici is a phytopathogenic bacterium causing brown stripe disease in economically important crops worldwide. Here, we announce the draft genome sequence of Pseudomonas syringae pv. panici LMG2367 to provide further valuable insights for comparison of the pathovars among species Pseudomonas syringae.     

Two plant NLR proteins confer strain-specific resistance conditioned by an effector from Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae(Psa) causes bacterial canker, a devastating disease threatening the Actinidia fruit industry. In a search for non-host resistance genes against Psa, we find that the nucleotidebinding leucine-rich repeat receptor(NLR) protein ZAR1 from both Arabidopsis and Nicotiana benthamiana(Nb) recognizes Hop Z5 and triggers cell death. The recognition requires ZED1 in Arabidopsis and JIM2 in Nb plants, which are members of the ZRK pseudokinases and known components of the...     

Identification of strA-strB Genes in Streptomycin-Resistant Pseudomonas syringae pv. actinidiae Biovar 2 Strains Isolated in Korea

Bacterial canker is a devastating disease of kiwifruit caused by the bacterium Pseudomonas syringe pv. actinidiae. Canker disease of kiwifruit in Korea has been controlled using streptomycin for more than two decades. Four streptomycin-resistant strains, belonging to biovar 2, which are found only in Korea, were collected between 2013 and 2014 from different orchards located in Jeju, Korea. The genetic background for streptomycin resistance among P. syringe pv. actinidiae strains were determined by examining the presence of strA-strB or aadA, which are genes frequently found in streptomycin-resistant bacteria, and a point mutation at codon 43 in the rpsL gene. All four streptomycin-resistant strains of P. syringe pv. actinidiae investigated in this study contained strA-strB as a resistant determinant. The presence of the aadA gene and a mutation in codon 43 of the rpsL gene was not identified.     

The polysaccharide glycocalyx of Pseudomonas syringae pv. atrofaciens

It was shown by the method of electron microscopy that cells of virulent strain Pseudomonas syringae rv. atrofaciens 4394 have extracellular, probably, polysaccharide glycocalix. It consists of acid components giving positive cytochemical reaction with ruthenium red, a specific reagent to polyanions.     

AvrE1 and HopR1 from Pseudomonas syringae pv. actinidiae are additively required for full virulence on kiwifruit

Pseudomonas syringae pv. actinidiae ICMP 18884 biovar 3 (Psa3) produces necrotic lesions during infection of its kiwifruit host. Bacterial growth in planta and lesion formation are dependent upon a functional type III secretion system (T3S), which translocates multiple effector proteins into host cells. Associated with the T3S locus is the conserved effector locus (CEL), which has been characterized and shown to be essential for the full virulence in other P. syringae pathovars. Two effectors at the CEL, hopM1 and avrE1, as well as an avrE1-related non-CEL effector, hopR1, have been shown to be redundant in the model pathogen P. syringae pv. tomato DC3000 (Pto), a close relative of Psa. However, it is not known whether CEL-related effectors are required for Psa pathogenicity. The Psa3 allele of hopM1, and its associated chaperone, shcM, have diverged significantly from their orthologs in Pto. Furthermore, the CEL effector hopAA1-1, as well as a related non-CEL effector, hopAA1-2, have both been pseudogenized. We have shown that HopM1 does not contribute to Psa3 virulence due to a truncation in shcM, a truncation conserved in the Psa lineage, probably due to the need to evade HopM1-triggered immunity in kiwifruit. We characterized the virulence contribution of CEL and related effectors in Psa3 and found that only avrE1 and hopR1, additively, are required for in planta growth and lesion production. This is unlike the redundancy described for these effectors in Pto and indicates that these two Psa3 genes are key determinants essential for kiwifruit bacterial canker disease.