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.

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

In June 2010, young plants of kiwifruit growing in the French regions of Rhone-Alpes (Actinidia deliciosa 'Summer') and Aquitaine (A. chinensis 'Jintao') showed small, angular, necrotic leaf spots and cankers on some canes that was sometimes associated with production of a red exudate. Most of the affected canes died, and in a few cases after a few months, the entire plant died. Symptoms were consistent with Pseudomonas syringae pv. actinidiae, a bacterium that affects several species of Actinidia (A. deliciosa and A. chinensis, the two most important commercial species of kiwifruit). A recent outbreak of this disease is devastating the Italian kiwifruit industry. Bacterial colonies were isolated on King's medium B (KB) from leaf spots and infected canes. Three isolates from Aquitaine and 10 from Rhone-Alpes were retained for further characterization. The 13 isolates were gram negative, induced a hypersensitive reaction when infiltrated in tobacco plants, did not have a cytochrome c oxidase, an arginine dehydrolase or urease activity, did not hydrolyze esculin, starch, or gelatine, and did not induce ice nucleation. When plated on KB, these strains did not show strong fluorescence usually associated with strains of P. syringae. Complete lack of fluorescence reported for the pathotype strain ICMP 9817 has not been observed for those strains. They showed the same weak fluorescence as the strains of P. syringae pv. actinidiae recently isolated from Italy. Those characteristics match those of strains of P. syringae pv. actinidiae (3). Using total DNA of the 13 strains, the pathotype strain, and primers PsaF1/R2 (2), a 280-bp fragment was amplified by PCR, supporting the strains as being P. syringae pv. actinidiae. The amplicon from 6 of the 13 strains was sequenced and found to be 100% similar to the corresponding DNA fragment of the pathotype strain ICMP 9617 (GenBank AY342165). Partial sequences of 1,381 bp of the 16S rDNA gene of four of the six isolates, three strains isolated from Rhone-Alpes and one strain isolated from Aquitaine, were obtained by amplification with primers 27f and 1492r (1). Except for the sequence of strain 181, which was isolated from Aquitaine and had a 1 bp difference (GenBank JF323026), the other sequences were 100% identical to each other (GenBank JF323027 to JF323029). These four sequences were 99% identical to the 16SrDNA sequences of ICMP 9617, the pathotype strain of P. syringae pv. actinidiae (GenBank AB001431). These four strains and the pathotype strain were sprayed (1 × 109 CFU/ml) on leaves of four 6- to 8-week-old seedlings of A. chinensis each. After 4 days, small, necrotic, angular spots were found on all plants inoculated with those four strains and the pathotype strain. No symptoms were found on plants treated with water only. From those leaf spots, bacteria that had all the characteristics of P. syringae pv. actinidiae (as previously described) were isolated. Recently, two different haplotypes for the housekeeping gene cts were described for P. syringae pv. actinidiae (4), the strains isolated from France belong to the haplotype I. This is the same haplotype to which strains isolated from the recent Italian outbreak also belong. To our knowledge, this is the first report of bacterial canker of kiwifruit in France. References: (1) V. Gurtler and V. A. Stanisich. Microbiology 142:3 1996. (2) J. Rees-George et al. Plant Pathol. 59:453, 2010. (3) Y. Takikawa et al. Ann. Phytopathol. Soc. Jpn. 55:437, 1989. (4) J. L. Vanneste et al. N.Z. Plant Prot. 63:7, 2010.

First Report of Tomato chlorotic dwarf viroid in Tomato in France.

Tomato chlorotic dwarf viroid (TCDVd) is a pospiviroid found naturally infecting tomato (Solanum lycopersicum L.) (3) and several ornamentals such as Brugmansia, petunia (1), and trailing verbena (4). Initially identified in North America (3), it has been reported from India, Europe (the Netherlands and United Kingdom), and Japan. At the end of 2007, 20 to 25% of tomato plants within a group of greenhouses in the Brittany Region of France were observed with top bunching, leaf curling, and epinasty symptoms. Reverse transcription (RT)-PCR with a primer pair specific for several pospiviroids (5'GGGGAAACCTGGAGCGA3' and 5'GGGGATCCCTGAAGCGC3') amplified the correctly sized fragment (approximately 360 bp) from total nucleic acid extracts from three symptomatic plants. The sequence of the uncloned amplification product (GenBank Accession No. EU729744) was determined, together with that of five cloned cDNAs. All sequences were highly related with a total of three mutations in these six sequences and they showed 96.9% (GQ169709 and AY372399) to 99.4% (AF162131) identity with TCDVd sequences present in GenBank. Identification of TCDVd was confirmed from the same plant samples by molecular hybridization with a Potato spindle tuber viroid (PSTVd)-specific probe (which cross-hybridizes with TCDVd to a certain extent) and by PCR with the PSTVd/TCDVd-specific 2A-1S primer pair (3) and sequencing of the amplified fragment. The French isolate is most closely related to the original tomato isolate from Canada (GenBank Accession No. AF162131). In a grow-out test involving 2,500 seeds from the original seed lot from which the symptomatic plants were derived, 2 of the 250 pools of 10 plants tested positive for TCDVd infection with the 3H1-2H1 primer pair (2). The sequence of the amplified product proved identical to the isolate detected in the original greenhouse plants, indicating a low level of seed transmission. As with other pospiviroids, which appear to be more and more frequently reported in greenhouse tomatoes, possible sources of infection include contaminated seeds, as seem to be the case in this first outbreak, and also transfer to tomatoes from infected ornamental hosts. This is, to the best of our knowledge, the first report of TCDVd in tomato in France. References: (1) T. James et al. Plant Pathol. 57:400, 2008. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) R. P. Singh et al. J. Gen. Virol. 80:2823, 1999. 4) R. P. Singh et al. Plant Dis. 90:1457, 2006.

Occurrence of a Strain of Potato Yellow Mosaic Geminivirus Infecting Tomato in the Eastern Caribbean.

Viruslike symptoms were observed in epidemic proportions in tomato, Lycopersicon esculentum Mill., in Guadeloupe and Martinique each year since 1992 and 1993, respectively, and in Puerto Rico since 1994 (1,3). Many tomato fields in Guadeloupe and Martinique had more than 70% of plants expressing symptoms of chlorotic mottling, leaf distortion, leaf rolling, and stunting in 1995 and 1996. The B biotype of Bemisia tabaci (aka B. argentifolii) was associated with all these epidemics. Ninety-three samples of tomato were collected from multiple locations from each island (65 samples from Guadeloupe, 11 from Martinique, and 17 from Puerto Rico) and assayed for the presence of geminivirus by polymerase chain reaction (PCR) with broad spectrum primers, PAL1v1978 and PAR1c496 for the A component, and PBL1v2040 and PCRc154 for the B component (4). Most samples tested positive for geminivirus (98% from Guadeloupe, 100% from Martinique, and 82% from Puerto Rico). Restriction analyses of amplified A component fragment with SacI, EcoRI, and AluI, and amplified B component fragment with EcoRI, AluI, VspI, PstI, and HaeIII were conducted on 34 samples (25 from Guadeloupe, six from Martinique, and three from Puerto Rico). All samples produced very similar restriction patterns, suggesting that they were infected by the same virus. One to three clones of amplified fragments of A and B components, obtained from one plant sample from each location, were at least 98.7% identical in sequence to each other and were 89.6% and 75.2 to 75.7% identical to equivalent regions in potato yellow mosaic virus (PYMV) A and B DNA, respectively (GenBank accession nos. D00940 and D00941). This isolate of PYMV was collected from potato in Venezuela in 1985 (2). Infectious full-length genomic clones of A and B component DNA from Guadeloupe were derived from the same tissue as the PCR-generated clones, and nucleotide sequences were found to be 99.1 and 99% identical to the PCR-generated fragments of A and B DNA, respectively. Nucleotide sequences of these full-length clones were 92.6 and 89.4% identical to full-length sequences of PYMV A and B DNA, respectively. The DNA sequence identity of the common regions of the A and B components between the Guadeloupe virus and PYMV was 95.1 and 91.6%, respectively. There was a nucleotide identity of 93% in the first 125 nucleotides of the coat protein gene. The virus found in Guadeloupe, Martinique, and Puerto Rico appears to be a strain of PYMV reported from Venezuela. This strain of PYMV is at least partially responsible for the epidemics in tomato in Guadeloupe, Martinique, and Puerto Rico. The similarity among geminivirus sequences at distant locations (Puerto Rico to Martinique is approximately 600 km) is unexpected and could be due to recent introductions. References: (1) J. K. Brown et al. Plant Dis. 79:1250, 1995. (2) R. H. A. Coutts et al. J. Gen. Virol. 72:1515, 1991. (3) B. Hostachy et al. Phytoma 456:24, 1993. (4) M. R. Rojas et al. Plant Dis. 77:340, 1993.