Origin of the Outbreak in France of Pseudomonas syringae pv. actinidiae Biovar 3, the Causal Agent of Bacterial Canker of Kiwifruit, Revealed by a Multilocus Variable-Number Tandem-Repeat Analysis.

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 tandem-repeat (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.

First Report of Pseudomonas syringae pv. actinidiae, the Causal Agent of Bacterial Canker of Kiwifruit in Slovenia.

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.