Syringae Isolates Research Articles

Genomic characterization of Pseudomonas syringae pv. syringae from Callery pear and the efficiency of associated phages in disease protection.

The Pseudomonas syringae species complex is a heterogeneous group of plant pathogenic bacteria associated with a wide distribution of plant species. Advances in genomics are revealing the complex evolutionary history of this species complex and the wide array of genetic adaptations underpinning their diverse lifestyles. Here, we genomically characterize two P. syringae isolates collected from diseased Callery pears (Pyrus calleryana) in Berkeley, California in 2019 and 2022. We also isolated a lytic bacteriophage, which we characterized and evaluated for biocontrol efficiency. Using a multilocus sequence analysis and core genome alignment, we classified the P. syringae isolates as members of phylogroup 2, related to other strains previously isolated from Pyrus and Prunus. An analysis of effector proteins demonstrated an evolutionary conservation of effectoromes across isolates classified in PG2 and yet uncovered unique effector profiles for each, including the two newly identified isolates. Whole-genome sequencing of the associated phage uncovered a novel phage genus related to Pseudomonas syringae pv. actinidiae phage PHB09 and the Flaumdravirus genus. Finally, using in planta infection assays, we demonstrate that the phage was equally useful in symptom mitigation of immature pear fruit regardless of the Pss strain tested. Overall, this study demonstrates the diversity of P. syringae and their viruses associated with ornamental pear trees, posing spill-over risks to commercial pear trees and the possibility of using phages as biocontrol agents to reduce the impact of disease.IMPORTANCEGlobal change exacerbates the spread and impact of pathogens, especially in agricultural settings. There is a clear need to better monitor the spread and diversity of plant pathogens, including in potential spillover hosts, and for the development of novel and sustainable control strategies. In this study, we characterize the first described strains of Pseudomonas syringae pv. syringae isolated from Callery pear in Berkeley, California from diseased tissues in an urban environment. We show that these strains have divergent virulence profiles from previously described strains and that they can cause disease in commercial pears. Additionally, we describe a novel bacteriophage that is associated with these strains and explore its potential to act as a biocontrol agent. Together, the data presented here demonstrate that ornamental pear trees harbor novel P. syringae pv. syringae isolates that potentially pose a risk to local fruit production, or vice versa-but also provide us with novel associated phages, effective in disease mitigation.

First Report of Bacterial Canker of Pecan Caused by Pseudomonas syringae pv. syringae in China.

Pecan (Carya illinoinensis) is a world-famous nut tree that is widely cultivated in China, especially in Jiangsu Province (Zhang et al. 2015). In April 2022, cankers on trunks were recorded in pecan (cv. Pawnee) fields located in Taizhou (32°27'58″ N, 120°0'49″ E), Jiangsu. Cankers on the trunks resulted in wilt of the plants. Usually, the color of infected bark on the trunk became darker than the healty bark. When the outer bark was peeled away, the inner tissues were water-soaked, often with reddish streaks. In the surveyed orchards, disease incidence ranged from 10 to 20% among young saplings (about 200 three-year-old trees). While no fungal mycelium or spores were found in the diseased areas by microscope, bacterial colonies were isolated by surface-sterilizing small fragments (25 mm2) of symptomatic tissue in 0.5% NaOCl, rinsing the sections twice in sterilized water, and then streaking them on Luria-Bertani (LB) plates. More than 20 bacterial isolates were obtained and all isolates induced a hypersensitive response on Nicotiana tabacum. All isolates were fluorescent on King's medium B, and were gram-negative based on lysis by KOH. Isolates were positive for levan formation, negative for oxidase and arginine dihydrolase, and did not cause soft rot on potato slices. Based on above information, the isolates thus belonged to Lelliot's LOPAT group 1, P. syringae (Lelliott and Stead 1988). The 16S rRNA sequences of five representative isolates (accession numbers OP175939-OP175943) were amplified by PCR, sequenced, and compared with the NCBI GenBank database (Weisburg et al. 1991; Sarkar and Guttman 2004), finding a 99.92% genetic similarity with a previously reported 16S rRNA sequence of a Pseudomonas syringae pv. syringae (Pss) isolate (accession numbers NW389777). Additional housekeeping genes gap1(accession numbers OP186937-OP186941), rpoD (accession numbers OP186952-OP186956), gyrB (accession numbers OP186947-OP186951), and gltA (accession numbers OP186942-OP186946) were PCR-amplified and sequenced as reported by Hwang et al. (2005), followed by multilocus sequence typing analysis (MLSA). Molecular phylogenetic trees (MEGA vesion 6.0, maximum likelihood with Jukes-Cantor model, 1,000 bootstraps) were generated based on each of these five DNA regions and revealed that all five isolates were clustered together with the strains in P. syringae genomospecies 2, and grouped these isolates with Pss in the PAMDB database (Hwang et al. 2005). As a result, these isolates were identified as Pss. Pathogenicity on pecan (cv. Pawnee) was confirmed by cutting the trunks of two-year-old pecan trees with sterilized blades dipped in cell suspensions containing 107 CFU/ml of each isolate. Plants inoculated in a similar manner with sterile water served as negative controls. The inoculated plants were incubated in a greenhouse maintained at 25°C and 80% relative humidity. After 7 to 8 days, all inoculated plants showed the symptoms of necrosis previously described for the original field plants, while the control plants did not show symptoms. The bacteria reisolated from the inoculated plants were identified as Pss using the LOPAT tests. These results and the sequence analysis of the 16S rRNA and four housekeeping genes described above, fulfilled Koch's postulates. No target bacteria were isolated from the control plants. To our knowledge, this is the first report of Pseudomonas syringae pv. syringaecausing bacterial canker of pecan worldwide. The identification of this pathogen will allow the study of strategies for managing the disease.

Predictive modeling of Pseudomonas syringae virulence on bean using gradient boosted decision trees.

Pseudomonas syringae is a genetically diverse bacterial species complex responsible for numerous agronomically important crop diseases. Individual P. syringae isolates are assigned pathovar designations based on their host of isolation and the associated disease symptoms, and these pathovar designations are often assumed to reflect host specificity although this assumption has rarely been rigorously tested. Here we developed a rapid seed infection assay to measure the virulence of 121 diverse P. syringae isolates on common bean (Phaseolus vulgaris). This collection includes P. syringae phylogroup 2 (PG2) bean isolates (pathovar syringae) that cause bacterial spot disease and P. syringae phylogroup 3 (PG3) bean isolates (pathovar phaseolicola) that cause the more serious halo blight disease. We found that bean isolates in general were significantly more virulent on bean than non-bean isolates and observed no significant virulence difference between the PG2 and PG3 bean isolates. However, when we compared virulence within PGs we found that PG3 bean isolates were significantly more virulent than PG3 non-bean isolates, while there was no significant difference in virulence between PG2 bean and non-bean isolates. These results indicate that PG3 strains have a higher level of host specificity than PG2 strains. We then used gradient boosting machine learning to predict each strain’s virulence on bean based on whole genome k-mers, type III secreted effector k-mers, and the presence/absence of type III effectors and phytotoxins. Our model performed best using whole genome data and was able to predict virulence with high accuracy (mean absolute error = 0.05). Finally, we functionally validated the model by predicting virulence for 16 strains and found that 15 (94%) had virulence levels within the bounds of estimated predictions. This study strengthens the hypothesis that P. syringae PG2 strains have evolved a different lifestyle than other P. syringae strains as reflected in their lower level of host specificity. It also acts as a proof-of-principle to demonstrate the power of machine learning for predicting host specific adaptation.

Characterization and Genetic Diversity of Pseudomonas syringae pv. syringae Isolates Associated with Rice Bacterial Leaf Spot in Heilongjiang, China.

Simple SummaryIn Northeast China, rice bacterial brown leaf spot caused by Pseudomonas syringae pv. syringae is among the most damaging rice diseases. This disease results in significant yield losses. This study focuses on a comprehensive analysis of the pathogen, population structure, and genetic diversity within the species based on biochemical tests and genetic characterizations. Our results indicate high genetic heterogeneity in Pseudomonas syringae pv. syringae isolates, and clustering of testing isolates and reference strains are related with the genomospecies 1. This work contributes to the physiological classification of the P. s. pv. syringae isolated from Heilongjiang Province, China and the results present new data concerning the phylogeny and genetic diversity. Such studies have not been reported about P. s. pv. syringae from this region yet.In China, rice is one of the most important cereal crops. Rice bacterial brown leaf spot caused by P. s. pv. syringae is among the most damaging rice diseases in the Heilongjiang Province of China and results in substantial yield losses. In this study, a comprehensive analysis of the pathogen, population structure, and genetic diversity within the species was performed. For this purpose, 176 bacterial isolates of P. s. pv. syringae collected from 15 locations were characterized by using biochemical tests such as the LOPAT test, and genetic characterizations such as multilocus sequence analysis (MLSA) and repetitive PCR, using BOX, REP and ERIC primers. Biochemical testing and detection of syrB genes confirm the presence of P. s. pv. syringae, genetic characterization by MLSA and genetic fingerprinting by repetitive PCR confirmed that high genetic heterogeneity exists in the P. s. pv. syringae isolates, and clustering of the tested isolates and reference strains are related with the same genomospecies 1. This work contributes to the physiological classification of the P. s. pv. syringae isolated from Heilongjiang Province, China, and the results present new data concerning the phylogeny and genetic diversity. This type of study about P. s. pv. syringae has been not reported from this region until now.

Identification of Host Resistance to Bacterial Canker Disease on Apricot, Plum and Peach Grown in Punjab and KPK, Pakistan

Bacterial canker disease caused by the pathogen Pseudomonas syringae pv. syrinagae (Pss) has been spread widely in Pakistan, in Punjab and KPK provinces specially in strone fruits like apricot, Peach and Plum. During the 2014–2015 growing period, diseased samples were obtained from peach, plum and apricot trees showing bacterial canker symptoms in Punjab and KPK provinces and 48 Pseudomonas syringae isolates were obtained. The main aim of this research study was to find out host resistance in the cultivars of Apricot, Peach and Plum against bacterial canker caused by Pss in Peach, Plum and Apricot cultivars grown in Punjab and KPK province of Pakistan. In a pathogenicity test, three Pss isolates (PS3, PS9 and PS17) were found to be highly virulent in peach, plum and apricot and were tested for cultivar resistance to Pss. Leaves and shoots of five varieties of Peach Early grand, Florida king, 4 ½, 5 ½, 6 ½, Four varieties of Plum Red beauty, Fazal-e- manani, Stanley, Producer and Two varieties of Apricot Chinese apricot and golden amber were foliar sprayed with a mixed culture of Pss having concentration of 10-8 cfu ml−1 . Sprayed cultivars were covered with plastic bags for 3 days for retaining moisture and kept in glass house and were keenly observed for appearance of symptoms. 5 ½ of peach, Fazal-e-manani of plum and Chinese apricot of apricot were found resistant while 6 ½ of peach, Stanley of plum were found susceptible and Golden amber of apricot was found moderately susceptible to Pss. This is the first report of host resistance response of apricot, peach and plum cultivars grown in Pakistan to Pss.

Characterization of Pseudomonas syringae Isolated from Systemic Infection of Zucchini in Australia.

Zucchini plants with symptoms including twisted petioles, necrotic leaves, crown rot, and internal fruit rot were found in Bundaberg, Australia, at a commercial field for the first time during late autumn 2016, resulting in direct yield losses of 70 to 80%. Three Pseudomonas syringae strains isolated from symptomatic leaf (KL004-k1), crown (77-4C), and fruit (KFR003-1) were characterized and their pathogenicity evaluated on pumpkin, rockmelon, squash, and zucchini. Biochemical assays showed typical results for P. syringae. The three isolates differed, however, in that two produced fluorescent pigment (KFR003-1 and 77-4C) whereas the third, KL004-k1, was nonfluorescent. Multilocus sequence analysis classified the isolates to phylogroup 2b. The single-nucleotide polymorphism analysis of core genome from the Australian and closely related international isolates of P. syringae showed two separate clusters. The Australian isolates were clustered based on fluorescent phenotype. Pathogenicity tests demonstrated that all three isolates moved systemically within the inoculated plants and induced necrotic leaf symptoms in zucchini plants. Their identities were confirmed with specific PCR assays for P. syringae and phylogroup 2. Pathogenicity experiments also showed that the Eva variety of zucchini was more susceptible than the Rosa variety for all three isolates. Isolate KL004-k1 was more virulent than 77-4C on pumpkin, rockmelon, squash, and zucchini. This study expands the knowledge of P. syringae isolates that infect cucurbits and provides useful information for growers about the relative susceptibility of a range of cucurbit species.

Diversity, pathogenicity and biocontrol efficacy of Pseudomonas syringae isolated from plants in northern Jordan

Diversity, pathogenicity, and biocontrol efficacy of Pseudomonas syringae strains isolated from a wide range of habitats in Jordan were investigated. Thirty-five infected plant samples (vegetable leaves and woody plant twig) were randomly collected. King's B agar medium was implemented and 124 colonies of P. syringae candidates were selected. Phenotypic, biochemical and pathogenicity tests were further carried out to confirm the identity of P. syringae isolates. The frequency of fluorescent isolates varied between 11- 50 %. Even though all isolates were shown to induce chlorosis of tobacco leaves, only two were able to macerate potato slices causing their rotting. This may be due to variation in secretion of cellulase and pectinase enzymes involve in pathogenicity as clearly discussed in this study. Interestingly, 55% of isolates were found to clearly inhibit growth of E. coli. Exceptional inhibition was noticed in P. syringae isolated from tomato and a possible effect of syringomycin was suggested. Therefore, P. syringae obtained in this study was recommended to have a pivotal role in the biological control system. Results obtained from the experiment of ice nucleation activity revealed that this phenotypic feature dominates woody plants.

Resistance to Potassium Phosphite in Phytophthora Species Causing Citrus Brown Rot and Integrated Practices for Management of Resistant Isolates.

Phytophthora citrophthora, P. syringae, P. nicotianae, and less commonly P. hibernalis are causal agents of brown rot of citrus fruit in California. The chronic disease occurs during the winter season, requires annual management, and has limited California citrus exports because of quarantines in some markets. Potassium phosphite (KPO3) is registered as a pre- and postharvest fungicide in the United States to manage Phytophthora brown rot. We evaluated the in vitro toxicity of KPO3 to 65, 60, and 38 isolates of P. citrophthora, P. syringae, and P. nicotianae, respectively, that were obtained from major growing regions of California. Frequency distributions of effective concentrations to inhibit mycelial growth by 50% (EC50 values) were not normally distributed, with skewness values of 1.84, 1.60, and -0.51 for each species, respectively. Isolates considered sensitive (EC50 values <25 µg/ml), moderately resistant (EC50 values from 25 to 75 µg/ml), or resistant (EC50 values >75 µg/ml) were identified for each species. The majority of P. citrophthora (83.1%) and P. syringae (78.3%) isolates were sensitive, whereas most P. nicotianae isolates (86.8%) were moderately resistant or resistant. Resistance factors were calculated as 65, 19, and 10 for the three species, respectively. In preharvest field trials, KPO3 (2,280 g/ha) applications were not effective in reducing citrus brown rot incidence when orange fruit were inoculated with a resistant (EC50 = 161.9 µg/ml) isolate of P. citrophthora, demonstrating the potential for field resistance. Oxathiapiprolin (32.6 g/ha), however, was highly effective, indicating the absence of multidrug resistance. Postharvest treatments with KPO3 were only effective in reducing brown rot caused by the resistant isolate of P. citrophthora to a low incidence when high rates (8,000 µg/ml) were used in heated (54°C) applications. The sensitive and moderately resistant isolates were managed using rates of 4,000 µg/ml, but heated treatments at this rate were needed to reduce brown rot to commercially acceptable levels when decay was caused by a moderately resistant isolate.

Phenotypic and genotypic analysis of Pseudomonas syringae recovered from symptomatic beans and associated weeds in Northern Spain

Thirty isolates of Pseudomonas syringae other than pathovar (pv.) phaseolicola, recovered from beans (18) and weeds (12) in a Northern Spanish region, were studied by a polyphasic approach, including phenotypic tests, detection of levansucrase and toxin (syringomycin and syringopeptin) genes, typing by RAPD (Random Amplified Polymorphic DNA) and PFGE (Pulsed-Field Gel Electrophoresis). The isolates were phenotypically heterogeneous, and both RAPD and PFGE, probed to be highly discriminative, as they were almost able to differentiate at the level of isolate. Phylogenetic analysis based on conserved genes of the core genome (rpoD, gltA and gyrB) distributed the isolates in three different clades (2A, 2B and 2C) belonging to phylogroup 2 of the P. syringae complex. According to toxin production, 18 of them, within clade 2B, were assigned to P. syringae pv. syringae and five of these caused symptoms after experimental inoculation on bean pods. Four of the five pathogenic isolates were obtained from weeds, supporting the role of weeds associated with bean crops, particularly Cyperus rotundus, Malva sylvestris, and Polygonum lapathifolium, as a reservoir of the pathogen. In conclusion, results of this study suggest that apart from P. syringae pv. phaseolicola, P. syringae isolates circulating in our region are mostly epiphytes, and support the role of weeds associated with bean crops as reservoir of pathogenic strains of P. syringae pv. syringae.

Susceptibility of Serbian plum cultivars to indigenous bacterial and Monilinia laxa isolates

The susceptibility of Serbian plum cultivars to pathogens originating from their phyllosphere was evaluated by inoculating detached young leaves and mature fruits. The virulence of indigenous isolates of the bacteria Pseudomonas syringae, Pseudomonas congelans, Erwinia persicina, Clavibacter michiganensis and Rhizobium nepotum was tested on detached leaves of four Serbian plum cultivars (Ranka, Pozegaca, Cacanska lepotica and Cacanska rodna). The Pseudomonas syringae isolates formed intense symptoms within 48 hours on all tested cultivars with severity index values in the range of 41 - 47%. The other isolates had significantly lower severity values or no symptoms were developed. This study demonstrates for the first time pathogenicity of Pseudomonas congelans on plum, with symptom intensity not significantly different from P. syringae after 96 h of incubation. Virulence of Monilinia laxa isolates was tested on mature fruit of the Cacanska rodna and Pozegaca cultivars and was detected in both of them. Higher susceptibility to M. laxa was recorded for the Pozegaca cultivar, with a fruit infection rate between 43 and 66%. In the case of the Rodna cultivar, no statistically significant difference in the fruit infection rate was detected between the four tested M. laxa isolates. These data indicate significant susceptibility of Serbian plum cultivars to indigenous P. syringae and M. laxa isolates.

A comparative evaluation of freezing criteria and molecular characterization of epiphytic ice-nucleating (Ice+) and non-ice-nucleating (Ice−) Pseudomonas syringae and Pseudomonas fluorescens

The ice-nucleation-active bacteria that produce ice-nucleating proteins (INPs), especially Pseudomonas species, may play a critical potential role in frost injury. The ice-minus variant of bacteria is a mutant, lacking the genes responsible for the production of INPs. The aim of the project was the isolation of relevant bacteria. Pseudomonas-like bacteria were screened from 66 leaves samples and were identified by biochemical as well as molecular methods with rpoD, 16S rDNA-flu and hrpl primers. The ice nucleation activity (INA) of isolates was performed using the drop-freezing method. The existence of the ina gene in isolates was investigated by specific inaZ and inaW primers. The effect of various temperatures, salt concentration and pH on growth and INA were investigated. Finally, the pathogenicity and freezing of INA+ and INA− bacteria on tomatoes and oranges were estimated. In this study, 57 isolates were identified as Pseudomonas including five P. syringae strains and seven P. fluorescens strains. The freezing profiles revealed that P. fluorescens strain 22A as INA− bacteria and three strains of P. syringae 18A, 52A and 52B are also recommended as strong INA+ strains. Most of the P. syringae isolates were active at −2 to −8 °C (type I and II), whereas P. fluorescens strains had an efficient activity at −8 to −12 °C (type III). Unlike the selected P. syringae bacteria, P. fluorescens strains did not show signs of frost injury on tomatoes and oranges.

Comprehensive characterization of an aspen (&amp;lt;i&amp;gt;Populus tremuloides&amp;lt;/i&amp;gt;) leaf litter sample that maintained ice nucleation activity for 48 years

Abstract. Decaying vegetation was determined to be a potentially important source of atmospheric ice nucleation particles (INPs) in the early 1970s. The bacterium Pseudomonas syringae was the first microorganism with ice nucleation activity (INA) isolated from decaying leaf litter in 1974. However, the ice nucleation characteristics of P. syringae are not compatible with the characteristics of leaf litter-derived INPs since the latter were found to be sub-micron in size, while INA of P. syringae depends on much larger intact bacterial cells. Here we determined the cumulative ice nucleation spectrum and microbial community composition of the historic leaf litter sample 70-S-14 collected in 1970 that conserved INA for 48 years. The majority of the leaf litter-derived INPs were confirmed to be sub-micron in size and to be sensitive to boiling. Culture-independent microbial community analysis only identified Pseudomonas as potential INA. Culture-dependent analysis identified one P. syringae isolate, two isolates of the bacterial species Pantoea ananatis, and one fungal isolate of Mortierella alpina as having INA among 1170 bacterial colonies and 277 fungal isolates, respectively. Both Pa. ananatis and M. alpina are organisms that produce heat-sensitive sub-micron INPs. They are thus both likely sources of the INPs present in sample 70-S-14 and may represent important terrestrial sources of atmospheric INPs, a conclusion that is in line with other recent results obtained in regard to INPs from soil, precipitation, and the atmosphere.

Biocontrol of Pseudomonas syringae pv. syringae affecting citrus orchards in Tunisia by using indigenous Bacillus spp. and garlic extract

Citrus blast and black pit that became increasingly important bacterial diseases are caused by Pseudomonas syringae pv. syringae. This study aimed to evaluate the antibacterial potential of Bacillus species strains and garlic extracts against two P. syringae isolates (BAT13 and DAPP-PG115). The Bacillus species strains were isolated from symptomless citrus leaves. Under in vitro conditions, 21 Bacillus species strains and garlic extract displayed antibacterial activity against the pathogen. Under greenhouse conditions, antagonistic bacteria, garlic extract, and copper sulfate confirmed their antimicrobial effect on P. syringae and reduced significantly the extend of stem necrosis 10 weeks after inoculation by BAT13 up to 60.55, 56.11, and 45.83%, and by DAPP-PG115 up to 70.83, 62.5, and 46.52%, in respect to relevant treatments. Garlic extract was the most effective treatment in our hands, and it suggests that Allium sativum extract could be used to control and prevent infection by the pathogen.

Characterisation of Pseudomonas syringae isolates from apricot orchards in north-eastern Italy

A set of 31 isolates were obtained from apricot (Prunus armeniaca L.) orchards showing symptoms of blast and gummosis in orchards located in north-eastern Italy, mostly during three subsequent growing seasons (2014–2016). These isolates were identified by means of their colony morphology, pathogenicity (hypersensitive reaction in tobacco leaves and the detection of both syringomycin and coronatine coding genes), genetic features (BLASTn analysis of the rpoD gene) and physiological tests (GATTa) as Pseudomonas syringae pv. syringae and P. syringae pv. morsprunorum race 1 and race 2 strains, respectively. Phylogenetic analysis based on the rpoD gene sequence showed that the two pathovars and two races are well separated and highlighted high intrapathovar/race variation for both P. syringae pv. syringae and P. syringae pv. morsprunorum race 2. Pseudomonas syringae pv. morsprunorum race 1 formed a unique clade that had high sequence homology. Repetitive element-based polymerase chain reaction fingerprinting analysis using the ERIC, REP and BOX primers again showed both high intra-pathovar variation within the P. syringae pv. syringae isolates, which grouped into six distinct clusters, and notable intra-race genetic diversity within both P. syringae pv. morsprunorum races. All 31 strains showed tolerance to copper at 200 ppm in vitro. Among these isolates, 13 were highly resistant (up to 500 ppm) and revealed the presence of the cusCBA gene cluster. All P. syringae pv. syringae strains showed ice nucleation activity, and nine were markedly active (up −3 °C) and characterised by the presence of the INAz gene. Finally, this study is the first report of the isolation of P. syringae pv. morsprunorum race 2 strains from apricot in Italy.

Bacterial canker of cherry trees, Prunus avium, in South Africa

In the 1980’s the causal agents of bacterial canker of cherry trees in South Africa was reported to be Pseudomonas syringae pv. syringae and Pseudomonas syringae pv. morsprunorum. Subsequently, no further studies were undertaken on the disease or causal agents. The aim of the current study was to conduct field surveys to determine the current situation pertaining to bacterial canker in the major cherry producing areas of South Africa. Following isolations from infected trees, strains were characterized using biochemical as well as multilocus sequence analyses (MLSA). Pathogenicity tests were undertaken with immature cherry fruit as well as three different cherry cultivars. Although symptoms of bacterial canker were present in all areas surveyed, P. syringae isolates were only isolated from three sites in the Western Cape Province. The isolates collected in this study showed a hypersensitive response on tobacco leaves and were pathogenic on immature cherry fruit and cherry trees. The phenotypic tests and MLSA using four genes (cts, gapA, gyrB and rpoD) showed phenotypic and genetic identity with Pseudomonas syringae pv. syringae. Selected strains induced a hypersensitive response on tobacco leaves and were pathogenic on immature cherry fruit and green cherry tree shoots. The current study shows that P. syringae pv. syringae is responsible for bacterial canker in the Western Cape Province, South Africa.

Occurrence of copper-resistant Pseudomonas syringae pv. syringae strains isolated from rain and kiwifruit orchards also infected by P. s. pv. actinidiae

During spring 2014–2015, leaves showing extensive leaf spotting were collected from Actinidia deliciosa and A. chinensis orchards located in northern, central and southern Italy where a copper-based strategy used to control bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), was ineffective. In addition, bacterial isolates were recovered from wind-driven raindrops. Psa was isolated solely from raindrops, whereas P. s. pv. syringae was consistently isolated from both symptomatic leaves and rain. The P. s. pv. syringae isolates were molecularly typed using repetitive-sequence PCR and assessed in vitro for their resistance to copper sulphate and for the presence of the cop ABCDRS gene cluster. An ad hoc quantitative real-time PCR was performed to confirm the P. s. pv. syringae capability for Actinidia spp. leaf infection using a low dose of inoculum and co-inoculation with Psa. A copper-resistant P. s. pv. syringae strain isolated from rain effectively colonized Actinidia spp. leaves from a low dose of inoculum. When such a strain was co-inoculated with a reference Psa strain, they reached a higher density than in inoculations performed singly. Copper-resistant P. s. pv. syringae strains infecting the leaves of Actinidia spp. appear to be widespread in Italy and, in some circumstances, they could have replaced Psa in leaf colonization and disease development. Populations of this phytopathogen showing resistance to copper would appear to be dispersed in the environment by the rain. This raises questions concerning the reliability of long-term strategies to control kiwifruit bacterial canker caused by Psa solely with copper compounds.

Characterization of Pseudomonas syringae pv. syringae, Causal Agent of Citrus Blast of Mandarin in Montenegro.

Citrus blast caused by bacterium Pseudomonas syringae is a very important disease of citrus occuring in many areas of the world, but with few data about genetic structure of the pathogen involved. Considering the above fact, this study reports genetic characterization of 43 P. syringae isolates obtained from plant tissue displaying citrus blast symptoms on mandarin (Citrus reticulata) in Montenegro, using multilocus sequence analysis of gyrB, rpoD, and gap1 gene sequences. Gene sequences from a collection of 54 reference pathotype strains of P. syringae from the Plant Associated and Environmental Microbes Database (PAMDB) was used to establish a genetic relationship with our isolates obtained from mandarin. Phylogenetic analyses of gyrB, rpoD, and gap1 gene sequences showed that P. syringae pv. syringae causes citrus blast in mandarin in Montenegro, and belongs to genomospecies 1. Genetic homogeneity of isolates suggested that the Montenegrian population might be clonal which indicates a possible common source of infection. These findings may assist in further epidemiological studies of this pathogen and for determining mandarin breeding strategies for P. syringae control.

A set of PCRs for rapid identification and characterization of Pseudomonas syringae phylogroups.

Aims The aim of this study was to develop a rapid PCR-based method for the specific detection of individual phylogroups of the Pseudomonas syringae complex. Methods and Results Seven primer pairs were developed by analysing whole genomes of 54 Ps. syringae strains. The specificity and sensitivity of these primer pairs were assessed on 236 strains from a large and comprehensive Ps. syringae collection. The method was also validated by characterizing the phylogenetic diversity of 174 putative Ps. syringae isolates from kiwifruit and apricot orchards of southeastern France. Conclusion Our PCR-based method allows for the detection and characterization of nine of the 13 Ps. syringae phylogroups (phylogroups 1, 2, 3, 4, 7, 8, 9, 10 and 13). Significance and Impact of the Study To date, phylogenetic affiliation within the Ps. syringae complex was only possible by sequencing housekeeping genes. Here, we propose a rapid PCR-based method for the detection of specific phylogroups of the Ps. syringae complex. Furthermore, for the first time we reveal the presence of Ps. syringae strains belonging to phylogroups 10 and 13 as epiphytes on plants, whereas they had previously only been observed in aquatic habitats.

Characterization of Pseudomonas syringae pathovars from different sweet cherry cultivars by RAPD analysis

Pseudomonas syringae pvs., isolated from sweet cherry grown on different localities in Serbia, were genetically characterized using RAPD analysis. Four out of eleven tested primers (SPH1, DJP 17, DJ 15, and DJ 16) were selected on the basis of the differences between isolates within two pathovars - syringae and morsprunorum race 1. Cumulative RAPD analysis indicated heterogeneity within the population of both groups of tested isolates, revealing four different patterns in each group. RAPD analysis showed up to 24% differences among pv. syringae isolates, as well as 41% in comparison with the reference strain KFB0103 (pv. syringae), while differences of 15% among isolates pv. morsprunorum 1 race and 36% compared to the reference strain CFBP2119 (pv. morsprunorum 1) were observed. Isolates from locality Selenca exhibited three different genotypic patterns of pv. morsprunorum race 1 and one pattern of pv. syringae. Isolates of pv. morsprunorum collected in the same year from two plant organs (branches and leaves) of the cv. Vanda yielded two different patterns. The pv. morsprunorum on cv. Kordia and pv. syringae on cv. Regina were detected at Mikicevo locality. The same patterns were observed for isolates of pv. syringae from Kanjiza and Selenca, as well as from Gornji Tavankut in two years of isolation. Differences were noted between isolates from the same pathovar originating from Ljutovo and Mikicevo, as well as with respect to all other isolates of same pathovar.

Phylogenetic Relationships of Pseudomonas syringae pv. syringae Isolates Associated with Bacterial Inflorescence Rot in Grapevine.

Pseudomonas syringae pv. syringae causes extensive yield losses in wine-grape production in some Australian cool-climate vineyards. Putative P. syringae pv. syringae isolates from infected grapevines within a range of vineyards were genotyped using RNA polymerase β-subunit (rpoB) and multilocus sequence typing (MLST) using primers for glyceraldehyde-3-phosphate dehydrogenase (gapA), citrate synthase (gltA), DNA gyrase B (gyrB), and σ factor 70 (rpoD). The isolates were also evaluated for pathogenicity by inoculation of detached grapevine leaves. The isolates were grouped by MLST data into two well-supported clades, each containing a mixture of pathogenic and nonpathogenic grapevine isolates, indicating that P. syringae pv. syringae in Australian vineyards is genetically diverse. Each clade also contained P. syringae pv. syringae from nongrape hosts pathogenic to grapevine, demonstrating a lack of host specificity and possible potential for cross-infection of grape and other horticultural crops. Furthermore, the isolation of pathogenic P. syringae pv. syringae isolates from grapevine sucker shoots suggests that sucker shoots may allow overwintering of the pathogen. The vineyard quarantine status of P. syringae pv. syringae may need to be reconsidered, due to its easy dispersal through pruning equipment.