Characterization of Pseudomonas syringae pv. actinidiae (Psa) isolated from France and assignment of Psa biovar 4 to a de novo pathovar: Pseudomonas syringae pv. actinidifoliorum pv. nov. Since 2008, bacterial canker of kiwifruit (Actinidia deliciosa and A. chinensis) caused by Pseudomonas syringae pv. actinidiae (Psa) has resulted in severe economic losses worldwide. Four biovars of Psa can be distinguished based on their biochemical, pathogenicity and molecular characteristics. Using a range of biochemical, molecular and pathogenicity assays, strains collected in France since the beginning of the outbreak in 2010 were found to be genotypically and phenotypically diverse, and to belong to biovar 3 or biovar 4. This is the first time that strains of biovar 4 have been isolated outside New Zealand or Australia. A multilocus sequence analysis based on four housekeeping genes (gapA, gltA, gyrB and rpoD) was performed on 72 strains representative of the French outbreak. All the strains fell into two phylogenetic groups: one clonal corresponding to biovar 3, and the other corresponding to biovar 4. This second phylogenetic group was polymorphic and could be divided into four lineages. A clonal genealogy performed with a coalescent approach did not reveal any common ancestor for the 72 Psa strains. Strains of biovar 4 are substantially different from those of the other biovars: they are less aggressive and cause only leaf spots whereas Psa biovars 1, 2 and 3 also cause canker and shoot die-back. Because of these pathogenic differences, which were supported by phenotypic, genetic and phylogenetic differences, it is proposed that Psa biovar 4 be renamed Pseudomonas syringae pv. actinidifoliorum pv. nov. Strain CFBP 8039 is designated as the pathotype strain.
The first outbreaks of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae biovar 3 were detected in France in 2010. P. syringae pv. actinidiae causes leaf spots, dieback, and canker that sometimes lead to the death of the vine. P. syringae pv. actinidifoliorum, which is pathogenic on kiwi as well, causes only leaf spots. In order to conduct an epidemiological study to track the spread of the epidemics of these two pathogens in France, we developed a multilocus variable-number tandemrepeat (VNTR) analysis (MLVA). MLVA was conducted on 340 strains of P. syringae pv. actinidiae biovar 3 isolated in Chile, China, France, Italy, and New Zealand and on 39 strains of P. syringae pv. actinidifoliorum isolated in Australia, France, and New Zealand. Eleven polymorphic VNTR loci were identified in the genomes of P. syringae pv. actinidiae biovar 3 ICMP 18744 and of P. syringae pv. actinidifoliorum ICMP 18807. MLVA enabled the structuring of P. syringae pv. actinidiae biovar 3 and P. syringae pv. actinidifoliorum strains in 55 and 16 haplotypes, respectively. MLVA and discriminant analysis of principal components revealed that strains isolated in Chile, China, and New Zealand are genetically distinct from P. syringae pv. actinidiae strains isolated in France and in Italy, which appear to be closely related at the genetic level. In contrast, no structuring was observed for P. syringae pv. actinidifoliorum. We developed an MLVA scheme to explore the diversity within P. syringae pv. actinidiae biovar 3 and to trace the dispersal routes of epidemic P. syringae pv. actinidiae biovar 3 in Europe. We suggest using this MLVA scheme to trace the dispersal routes of P. syringae pv. actinidiae at a global level.A gricultural systems are continuously afflicted by emerging infectious diseases (1), which can have significant agronomic and economic consequences. A thorough knowledge of the causal agent (propagation and contamination pathways, suitable environmental conditions, host range, and pathogenicity) is essential for determining and implementing efficient disease-management measures. Pathogen genotyping yields precious information for understanding the diversity and population structure of the bacterial organisms responsible for outbreaks. It enables hypotheses about the dispersion routes of bacterial populations or clonal lineages involved in epidemics. Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) (2) is a powerful and portable genotyping method. It has been demonstrated that MLVA has a higher sensitivity and resolution than any other genotyping methods, such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence type (MLST), applied for an in-depth study of bacteria populations or epidemic outbreaks (3, 4). The aim of MLVA is to use PCR to target the tandem repeats with a motif of more than five nucleotides and to analyze the variability of their pattern in order to discriminate isolates. Generally, VNTR loci evolve according to the stepwise mutation model (SMM) by gain or l...
In Europe, the meadow spittlebug Philaenus spumarius is the main known vector of the quarantine bacterium Xylella fastidiosa. So far detection and identification of X. fastidiosa has more often been performed from plant matrices than insects, mainly using a real‐time PCR and multilocus sequence typing (MLST) approach. Detection of X. fastidiosa in its insect vectors would enhance knowledge of the epidemiologic situation in France, specifically in the already infected Corsica and Provence‐Alpes‐Côte d’Azur (PACA) regions. The aim of this study was to validate a methodological approach to detect X. fastidiosa in P. spumarius, analysed individually or in groups of 10, using real‐time PCR and MLST, and to apply the approach to more than 4,000 individuals collected between 2015 and 2019 from infected areas. The corresponding results expanded our knowledge of the epidemiology of X. fastidiosa in France: (a) X. fastidiosa subsp. multiplex including the sequence types ST6 and ST7 were identified in the insect vector; (b) the rate of positive insects per infected area was as high as 33.3% in Corsica or 50% in the PACA region; (c) positive adults were found during winter; and (d) the bacterial load in P. spumarius (droplet digital PCR) usually ranged from 103 to 104 cells per insect, but could be as high as 105 or 106 cells per insect for some individuals (13%). The subspecies and sequence types detected in P. spumarius corresponded to the situation officially reported for plants in the same areas.
Xylella fastidiosa is a xylem-limited bacterium native to America and classified as a priority pest for EU regulation. Since 2013, X. fastidiosa has been identified in European countries with a Mediterranean climate, such as Italy, France, Spain and Portugal, with different subspecies and sequence types (ST) detected. Since 2015 X. fastidiosa subsp. multiplex ST6 and/or ST7 has been detected in Corsica and the Provence-Alpes-Côte d’Azur in almost 70 plant species, whereas X. fastidiosa subsp. pauca ST53 has been found in only two host plants. In this study, we report two new variants, recently detected in two separated areas of the PACA region, genetically related to the subspecies multiplex and assigned to (i) ST88 detected on Polygala myrtifolia, Hebe sp., Osteospermum ecklonis, Lavandula x intermedia, Coronilla glauca and Euryops chrysanthemoides and (ii) ST89: detected on Myoporum sp. and Viburnum tinus. Both variant strains were isolated in vitro. Moreover, we report here the identification of X. fastidiosa subsp. multiplex ST6 in a new region of the South of France, Occitanie (Aude), in plants from natural and urban settings and from a nursery.
In May 2013, 20 plants in a production orchard of kiwifruit (Actinidia deliciosa) cv. Hayward in the seaside area of Primorska showed small, angular, coalescing necrotic leaf spots and cankers on green shoots. In the following 2 weeks, disease progressed to wilting and shoot dieback with exudates. Symptoms were consistent with Pseudomonas syringae pv. actinidiae. Circular, flat, granulated colonies with entire margins were isolated from leaf spots on King's medium B (KB) and on sucrose nutrient agar with boric acid, cephalexine, and cycloheximide. Strains were purified on KB and showed weak fluorescence upon a prolonged incubation (>10 days) and belonged to P. syringae LOPAT group Ia (+---+). DNA was extracted from strains and plant extracts with Chelex 100 resin and Bio-Nobile QuickPick Plant Kit (Turku, Finland), respectively. PCR products of expected sizes were generated by PCR assays (2,4) from all strains and plant extract, supporting the strains as being P. syringae pv. actinidiae. Two strains (NIB Z 1870 and 1871) were further identified by cytochrome C oxidase (negative), glucose metabolism (oxidative), aesculine (negative), and nitrate (negative). Their partial rpoD gene sequences (GenBank Accession Nos. KJ724117 and KJ724118) (3) were identical to the sequence of the P. syringae pv. actinidiae pathotype strain NCPPB 3739 (FN433222, 100% coverage) and to the sequence of P. syringae pv. theae at 96% coverage (FN433271). BOX-PCR fingerprinting and multilocus sequence analysis (MLSA) based on four housekeeping genes gapA (KJ733923 and KJ733924), gltA (KJ733925 and KJ733926), gyrB (KJ733927 and KJ733928), and rpoD identified both strains as biovar 3, a highly virulent biovar of P. syringae pv. actinidiae (5). The pathogenicity of the two strains was confirmed on four plants of A. deliciosa ‘Hayward’ for each strain. Six-month-old plants were sprayed on the abaxial sides of leaves with 30 ml cell suspension prepared from a 72-h-old culture of the appropriate strain (~8 × 106 CFU/ml in 0.01 M MgSO4), covered with plastic bags for 24 h, and incubated under high relative humidity (80%) with 14 h daylight and 24/21°C day/night temperature. Three positive and three negative control plants were inoculated with the Italian P. syringae pv. actinidiae virulent strain K9 (kindly provided by Dr. Gian Luca Bianchi of the Plant Health Service of Friuli Venezia Giulia region) and 0.01 M MgSO4, respectively. After 7 days, water-soaked brown spots with pale green halos were observed on all plants inoculated with bacteria. Re-isolated bacteria were identical to the original strains in their morphology, PCR products, and rpoD sequences. Negative control plants did not develop symptoms, and no growth was observed on media. This is the first laboratory confirmation of bacterial canker of kiwifruit in Slovenia. Visual inspections carried out by the plant health authorities in 2013 and laboratory analysis confirmed additional infection with P. syringae pv. actinidiae in a single, nearby orchard. The pest status of P. syringae pv. actinidiae in Slovenia is officially declared as present, subject to official control (1). References: (1) EPPO Reporting Service. Online publication: http://archives.eppo.int/EPPOReporting/2014/Rse-1402.pdf . No. 02 2014/026, 2014. (2) A. Gallelli et al. J. Plant Pathol 93:425, 2011. (3) N. Parkinson et al. Plant Pathol. 60:338, 2011. (4) J. Rees-George et al. Plant Pathol. 59:453, 2010. (5) J. L. Vanneste et al. Plant Dis. 97:708, 2013.
The aim of this study was to characterise the performance of new molecular methods for the detection and identification of Pseudomonas syringae pv. actinidiae (Psa) and to provide validation data in comparison to the assays mentioned in official diagnostic protocols and being currently used. Eleven molecular tests for the Psa detection were compared in an interlaboratory comparison where each laboratory had to analyse the same panel of samples consisting of thirteen Psa-spiked kiwifruit wood extracts. Laboratories had to perform also isolation from the wood extracts. Data from this interlaboratory test performance study (TPS) was statistically analysed to assess the performance of each method. In order to provide complete validation data, both for detection and identification, this TPS was supplemented by a further study of identification from pure culture of phylogenetically closely related Pseudomonas spp., Psa, and bacterial strains associated with kiwifruit. The results of both these studies showed that simplex-PCRs gave good results, whereas duplex-PCR and realtime PCR were the most reliable tools for detection and identification of Psa. Nested and multiplex-PCR gave
Of American origin, a wide diversity of Xylella fastidiosa strains belonging to different subspecies have been reported in Europe since 2013 and its discovery in Italian olive groves. Strains from the subspecies multiplex (ST6 and ST7) were first identified in France in 2015 in urban and natural areas. To trace back the most probable scenario of introduction in France, the molecular evolution rate of this subspecies was estimated at 3.2165 × 10-7 substitutions per site per year, based on heterochronous genome sequences collected worldwide. This rate allowed the dating of the divergence between French and American strains in 1987 for ST6 and in 1971 for ST7. The development of a new VNTR-13 scheme allowed tracing the spread of the bacterium in France, hypothesizing an American origin. Our results suggest that both sequence types were initially introduced and spread in Provence-Alpes-Côte d’Azur (PACA); then they were introduced in Corsica in two waves from the PACA bridgehead populations.
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