A protocol for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in carrot seeds using real‐time PCR was developed. The bacterium was detected in 23 out of 54 carrot seed lots from 2010 to 2014, including seeds collected from diseased mother plants. The average total number of ‘Ca. L. solanacearum’ cells in individual seeds ranged from 4·8 ± 3·3 to 210 ± 6·7 cells per seed from three seed lots, but using propidium monoazide to target live cells, 95% of the cells in one seed lot were found to be dead. Liberibacter‐like cells were observed in the phloem sieve tubes of the seed coat and in the phloem of carrot leaf midrib from seedlings. The bacterium was detected as early as 30 days post‐germination, but more consistently after 90 days, in seedlings grown from PCR positive seed lots in an insect‐proof P2 level containment greenhouse. Between 12% and 42% of the seedlings from positive seed lots tested positive for ‘Ca. L. solanacearum’. After 150 days, symptoms of proliferation were observed in 12% of seedlings of cv. Maestro. ‘Candidatus Liberibacter solanacearum’ haplotype E was identified in the seeds and seedlings of cv. Maestro. No phytoplasmas were detected in seedlings with symptoms using a real‐time assay for universal detection of phytoplasmas. The results show that to prevent the entry and establishment of the bacterium in new areas and its potential spread to other crops, control of ‘Ca. L. solanacearum’ in seed lots is required.
Double-stranded RNAs purified from the V2356 ('Successa') sour cherry source of the Shirofugen stunt disease (SSD) were sequenced using a 454 pyrosequencing multiplex approach. The 15,646 reads obtained were assembled into 279 contigs, 5 of which, totaling almost 16.9 kbp and 5,332 reads (34% of sample reads), showed high Blast scores and homology to Little cherry virus 1 (LChV1). The five contigs were further assembled manually into three supercontigs spanning the full LChV1 genome with only two small gaps (17 and 55 bases). Completion of the sequencing of the viral genome was performed using targeted polymerase chain reaction and primers designed from the contigs. No evidence for the presence of other viral agents in the V2356 source could be obtained in the remaining contigs or singletons. The V2356 LChV1 isolate is only ≈76% identical with the reference complete LChV1 sequences and, in particular, with the ITMAR isolate associated with the Kwanzan stunting syndrome. However, it is highly homologous (97 to 100% identity) in two short genome regions with divergent LChV1 from North America, providing the first complete sequence for such divergent isolates. Although not providing a definite proof, the failure to detect any other viral agent in the V2356 SSD source and the identification of LChV1 in a second, independent, source of the disease suggests that LChV1 isolates could be responsible for the SSD syndrome.
High‐throughput sequencing (HTS) technologies have revolutionized plant pest research and are now raising interest for plant pest diagnostics, with plant virus diagnostics at the forefront of development. However, the application of HTS in plant pest diagnostics raises important challenges that plant health regulators will have to address. Adapted infrastructures, technical guidelines and training are pivotal for further use and adoption of the HTS technologies in the phytosanitary framework.
A polyvalent nested reverse transcription-polymerase chain reaction (RT-PCR) test using degenerate primers containing inosine (polyvalent degenerate oligonucleotides [PDO]) was developed for filamentous fruit tree viruses belonging to the genera Trichovirus, Capillovirus, and Foveavirus. The 362-bp product was amplified from nucleic acid extracts obtained from Prunus and Malus leaf samples. All the viruses targeted were detected, demonstrating the polyvalence of the test. The variability of a collection of Apple chlorotic leaf spot virus isolates was analyzed using the sequence of the PDO RT-PCR amplified cDNAs. The technique was also used to screen stone fruit materials infected with known agents or with virus-like graft-transmissible diseases of unknown etiology. The results obtained further validated the broad specificity of the assay, with positive amplification obtained for uncharacterized or partially characterized viruses associated with cherry and peach disorders. Sequencing the amplified PCR products either directly or after cloning allowed the identification of variants of known agents and the tentative identification of two new agents, a Trichovirus and a Foveavirus. In addition, sequence comparisons demonstrated that the sequence of the targeted region is phylogenetically informative and of predictive taxonomic value.
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