Identification of Pseudomonas syringae pv. syringae causing bacterial leaf blight of Miscanthus sinensis

Journal of Plant Diseases and Protection - In the late summer of 2015, severe leaf blight occurred on Miscanthus sinensis grown on natural riverside lands of the Han River in Seoul, South Korea....     

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

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

Bacterial leaf spot and blight of crucifer plants (Brassicaceae) caused by Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis

Bacterial leaf spot and blight diseases caused by Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pcal) are becoming a significant concern for producers of crucifer crops worldwide. Since Psm was first described in 1911, many have reported on its diverse phenotypic, genetic and pathogenic characteristics. Japanese isolates of Psm are also heterogeneous and differ in their host preferences. Pcal was first described in 2002 and has quickly spread globally. Recent work demonstrated that some isolates that had been identified as Psm are actually Pcal. Pcal was also shown to be split into two groups, A and B, based on bacteriological properties, genetic traits and pathogenicity. Group A of Pcal consists mostly of isolates from Japanese radish and radish, isolated before 1990s, that are more aggressive on radish leaves but less aggressive on other Brassica plants compared with group B. Group B of Pcal consists of recent isolates from various crucifer plants including the pathotype of Pcal. In this review, we suggest that group A of Pcal may have existed since the 1950s and survived as a relatively minor pathogen on radish or Japanese radish, whereas group B emerged in the late 1990s, causing global epidemics because of its stronger virulence on various Brassica crops. We also suggest that emergence of a new group of a pathogenic bacterium may cause a re-emergence or new epidemics of a disease that previously was of minor importance.     

Pseudomonas syringae pv. syringae on pepper seedlings in Italy

Pseudomonas syringae pv. syringae causing leaf spot on pepper seedlings grown in a plant bed is reported in Italy for the first time. The pathogen was identified by means of biochemical, physiological and pathogenicity tests as well as by SDS-polyacrylamide gel electrophoresis of whole-cell proteins. The bacterial isolates showed positive for ice nucleation and biocide production.     

Bacterial brown spot on Avena storigosa Schereb. caused by Pseudomonas syringae pv. alisalensis

Avena storigosa Schereb. (bristle oat) is used as a green manure in crop rotations and as an antagonist of nematodes in Nagano Prefecture, Japan. In 2011, necrotic, brown, water-soaked lesions were observed on young bristle oat plants. A pathogenic bacterium was isolated from symptomatic leaves of infected plants and produced the same symptoms after inoculation. Bacteriological properties of the bacterial isolates from bristle oat matched those of Pseudomonas syringae pathovars. The host range of the bristle oat isolates was identical to that of P. syringae pv. alisalensis. This is the first report of bristle oat disease caused by P. syringae pv. alisalensis.     

Comparison of ethylene-producing Pseudomonas syringae strains isolated from kudzu (Pueraria lobata) with Pseudomonas syringae pv. phaseolicola and Pseudomonas syringae pv. glycinea

The relationships among strains of Pseudomonas syringae pv. glycinea (Psg) and Pseudomonas syringae pv. phaseolicola (Psp) isolated from kudzu ( Pueraria lobata) and bean ( Phaseolus vulgaris) were investigated. All strains tested showed a close phenotypic similarity, with the exception of the utilization of inositol and mannitol as well as the production of toxins. On this basis the strains could be divided into three groups. Group 1 consists of all strains of pathovar glycinea, group 2 includes all Psp strains isolated from kudzu, and all Psp strains isolated from bean belong to group 3. This grouping was also reflected in the genetic fingerprints using the polymerase chain reaction (PCR) with primers that anneal to dispersed repetitive bacterial sequences (rep-PCR). The rep-PCR generated fingerprints were unique for each of the three groups. The strains of group 2, Psp strains isolated from kudzu, possess certain characteristics of group 1 (ethylene production) and group 2 (phaseolotoxin production). The Psp strains from kudzu can be clearly differentiated from Psp strains isolated from bean. They utilize mannitol, produce ethylene, and are strongly pathogenic to kudzu, bean, and soybean. The results obtained show that the Psp strains from kudzu should be separated from the pathovar phaseolicola and should represent their own pathovar.     

Susceptibility of European pear cultivars to Pseudomonas syringae pv. syringae using immature fruit and detached leaf assays

The susceptibility of thirty-three pear cultivars and two pear rootstocks to four virulent strains of Pseudomonas syringae pv. syringae was evaluated by inoculating detached immature fruits and young leaves. The four strains were similarly virulent and did not show cultivar specificity although they were isolated from different pear cultivars and exhibited different biochemical profiles. The most frequently planted pear cultivars, Conference, Abate Fetel, General Leclerc, Williams, D. Comice, El Dorado, Alexandrine, B. Anjou, Passe Crassane and the rootstock OHxF 333 were susceptible to P. syringae pv. syringae. Maximal severity values were obtained on 'Preguystar' leaves (about 90%). The rootstock Winter Nelis was less susceptible. Results with immature fruit and detached leaf assays agreed with field observations on cultivar susceptibility to bacterial blast. However, the detached leaf test gave a more accurate prediction and has the advantages that symptoms develop quickly (48 h), and leaves are available for a longer period of time than fruits. This method is proposed as a rapid and reproducible screening system of cultivar susceptibility to bacterial blast of pear.     

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

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

Similarity between Copper Resistance Genes from Pseudomonas syringae pv. actinidiae and P. syringae pv. tomato

Twenty-eight strains of Pseudomonas syringae pv. actinidiae isolated in 1984, 1987 and 1988 from kiwifruit orchards in Japan were tested for their resistance to copper sulfate. All strains isolated in 1984 were copper sensitive with a minimum inhibitory concentration (MIC) of cupric sulfate of 0.75 mM. However, some strains isolated in 1987 and 1988 were resistant, with the MIC ranging from 2.25 to 3.0 mM. All copper-resistant strains contained at least one of two plasmids, pPaCul (about 70.5 kb) or pPaCu2 (about 280 kb), or both. In a copper-resistant strain Pa429, the location of the copper-resistance gene(s) was examined by insertional inactivation with Tn5. The MIC of copper sulfate in the copper-sensitive mutant obtained by Tn5 tagging decreased from 2.75 to 0.75 mM. The 14.5 kb BamHI fragment, designated pPaCuB14, containing the same locus mutagenized with Tn5 was cloned from pPaCu1. However, pPaCuB14 did not confer copper resistance in the transformant of copper-sensitive strain Pa21R, suggesting that this clone did not contain a full set of copper-resistance gene(s). Then a cosmid library of pPaCu1 was constructed and six cosmid clones hybridized with pPaCuB14 were selected. One of the six cosmids, designated pPaCuC1, conferred a near wild-type level of copper resistance in the transformant of the copper-sensitive strain. pPaCuC1 had a homologous region that hybridized with all of the PCR-amplifled fragments of copA, copB, copR, and copS genes of P. syringae pv. tomato. DNA sequence analysis of the homologous region revealed the existence of four open reading frames (ORF A, B, R and S) oriented in the same direction. The predicted amino acid sequences of ORF A, B, R and S had 80, 70, 97 and 95% identity with CopA, B, R and S of P. syringae pv. tomato, respectively. Received 5 July 2001/ Accepted in revised form 27 September 2001     

Induction of pear blossom blast caused by Pseudomonas syringae pv. syringae

Conditions were established for inducing pear blossom blast caused by Pseudomonas syringae pv. syringae on both attached and detached shoots. The incidence of blossom blast was proportional to the logarithm of the P.s. pv. syringae population under optimal temperature, moisture, and bloom developmental stage. Highest incidence of blossom infection followed occurrence of a major exotherm (an increase in temperature caused by the heat of fusion from ice formation within blossom tissue) in the presence of P. s. pv. syringae. The exotherm was detected inside ovary tissue at temperatures ranging from –1.8 to –3.5 C. Wetness duration following the thawing process was less important than wetness during and immediately after the freeze event. Blossoms inoculated, then air-dried or removed from low-temperature treatment prior to occurrence of an exotherm, had a low incidence of infection, The full bloom stage of blossom development was more susceptible to blossom blast than either the open cluster or tight cluster stages of development.     

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

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

Bacterial inflorescence rot of grapevine caused by Pseudomonas syringae pv. syringae

Molecular sequencing (rpoB) and standard pathological and microbiological methods identified Pseudomonas syringae pv. syringae (Pss) as the causal agent of bacterial inflorescence rot of grapevines (Vitis vinifera) in three vineyards in Tumbarumba, NSW, Australia in 2006 and 2007. Pss strains from shrivelled berries and necrotic inflorescences of diseased grapevines were used to inoculate leaves and inflorescences of potted cv. Semillon grapevines. Pss caused disease symptoms similar to those experienced in the field, including angular leaf lesions, longitudinal lesions in shoot tissues and rotting of inflorescences from before flowering until shortly after fruit set. High humidity promoted symptom severity. The necrotic bunch stem and leaf lesions were susceptible to the development of Botrytis cinerea infections. Cryo‐scanning electron microscopy (cryoSEM) indicated that Pss entered leaves and inflorescence tissues via distorted, open, raised stomata surrounded by folds of tissue that appeared as ‘star‐shaped’ callose‐rich complexes when viewed by UV light microscopy. In necrotic tissues, cryoSEM revealed Pss within petiole parenchyma cells and air‐filled rachis xylem vessels. This is the first report of inflorescence and hence fruit loss caused by Pss in grapevines. The disease is described as ‘bacterial inflorescence rot’ and regarded as one that expands the previously reported pathology of grapevines caused by P. syringae. This study also indicated that infection by Pss might promote destructive B. cinerea infections when the fungus is already present but latent, although further experimentation is needed to prove such an interaction.     

Inheritance of resistance to angular leaf spot (Pseudomonas syringae pv. lachrymans) in cucumber and identification of molecular markers linked to resistance

Angular leaf spot is a common disease of cucumber ( Cucumis sativus ) caused by Pseudomonas syringae pv. lachrymans . Genetics of resistance to this disease was investigated using two sets of parameters: (i) disease severity, i.e. the number and size of necrotic and chlorotic lesions on the infected leaves, and (ii) presence or absence of a chlorotic halo around the necrotic spots on the infected leaves. Disease severity appears to be controlled by multiple genes and the heritability of the resistance was estimated to be 53%. The presence or absence of the chlorotic halo was determined to be governed by a single gene, with the presence of the halo (the susceptible phenotype) being a dominant character. A RAPD marker linked to the gene conferring the chlorotic halo was identified. Genetic distance between this marker, OP-AO07, a polymorphic 420 bp amplicon in the DNA of the susceptible plants, and the locus encoding the chlorotic halo was estimated to be 13 cM.     

Highly specific assays to detect isolates of Pseudomonas syringae pv. actinidiae biovar 3 and Pseudomonas syringae pv. actinidifoliorum directly from plant material

Pseudomonas syringae pv. actinidiae (Psa) is responsible for bacterial canker of kiwifruit. Biovar 3 of Psa (Psa3) has been causing widespread damage to yellow‐ and green‐fleshed kiwifruit (Actinidia spp.) cultivars in all the major kiwifruit‐producing countries in the world. In some areas, including New Zealand, P. syringae pv. actinidifoliorum (Pfm), another bacterial pathogen of kiwifruit, was initially classified as a low virulence biovar of Psa. Ability to rapidly distinguish between these pathovars is vital to the management of bacterial canker. Whole genome sequencing (WGS) data were used to develop PCR assays to specifically detect Psa3 and Pfm from field‐collected material without the need to culture bacteria. Genomic data from 36 strains of Psa, Pfm or related isolates enabled identification of areas of genomic variation suitable for primer design. The developed assays were tested on 147 non‐target bacterial species including strains likely to be found in kiwifruit orchards. A number of assays did not proceed because although they were able to discriminate between the different Psa biovars and Pfm, they also produced amplicons from other unrelated bacteria. This could have resulted in false positives from environmental samples, and demonstrates the care that is required when applying assays devised for pure cultures to field‐collected samples. The strategy described here for developing assays for distinguishing strains of closely related pathogens could be applied to other diseases with characteristics similar to Psa.     

Occurrence of Pseudomonas syringae pv. actinidiae on kiwifruit in Italy

Pseudomonas syringae pv. actinidiae has been isolated from kiwifruit plants for the first time in Italy. Biochemical tests were consistent with those characterizing the type-strain; pathogenicity tests yielded severe blights in the inoculated kiwifruit plants and no symptoms on lilac, pear and peach. Nutritional tests as well as whole-cell protein profiles revealed slight differences between the strains isolated in Japan and those of the present study. The main symptoms observed in the field are a red-rusty exudation covering the bark of twigs and trunks, blight of young canes and plants, angular leaf spots surrounded by chlorotic haloes and tiny cankers along the twigs.