In summer 2012, carrot (Daucus carota L.) plants displaying symptoms of leaf yellowing, stunting and proliferation of dwarfed shoots with bushy tops, and a dense hairy growth of secondary roots were observed. Symptomatic carrots were collected from three fields used for seed production and located in Region Centre of France near Orléans. The presence of psyllids (Psyllidae) in one of the fields was reported but they were not clearly identified. Fifty percent of the field was infected. Due to a large amount of plant debris, the harvested seeds were difficult to separate and the germination rate was low (from 10 to 77%), rendering them unmarketable. The symptoms observed were similar to those described for carrots infected by Aster yellows phytoplasma and ‘Candidatus Liberibacter solanacearum’ in Europe (3). Total DNA was extracted from petiole and root tissue of 16 symptomatic and 6 asymptomatic carrots (cv. Amsterdam, CAC3075), 2 samples of black nightshade leaves (Solanum nigrum) collected from the same fields, and 2 samples of carrot plants (cv Berlicum) grown in a high containment greenhouse, using a cetyl trimethyl ammonium bromide (CTAB) buffer extraction method. All DNA extracts were tested for phytoplasmas (1) and for ‘Ca. L. solanacearum’ by real-time PCR (2). DNA extracts were also tested for ‘Ca. L. solanacearum’ by PCR using primer pairs OA2/OI2c and CL514F/R to amplify a portion of 16S rDNA and rpIJ/rpIL ribosomal protein genes, respectively (4). DNA from greenhouse carrot plants yielded no amplicon with all PCR. Phytoplasma was not detected in any of the tested samples. However, amplification was observed with the real-time PCR assay for ‘Ca. L. solanacearum’ (2) for all DNA samples extracted from symptomatic and asymptomatic field carrots (cycle threshold [ct] values between 16.75 and 30.59), and from S. nigrum (ct between 31.62 and 33.25). For field carrot DNA, a 1,168-bp 16S rDNA fragment and a 669-bp rpIJ/rpIL fragment were amplified whereas DNA from S. nigrum yielded no amplicon. Four amplicons obtained from these PCR assays with both primer pairs from symptomatic carrot samples were sequenced directly (Beckmann Coulter Genomics, Grenoble, France). BLAST analysis of the 16S rDNA sequences (KF357911) showed 99% nucleotide identity to those of ‘Ca. L. solanacearum’ amplified from carrot in Finland (GU373049). The rpIJ/rpIL nucleotide sequences (KF357912) were 99% identical to sequences of the analogous rpIJ/rpIL ‘Ca. L. solanacearum’ ribosomal protein gene from carrot in Spain (JX308305). These results confirmed the presence of ‘Ca. L. solanacearum’ in all symptomatic and asymptomatic carrot sampled in Region Centre, France. To our knowledge, this is the first report of this pathogen in carrot in France. These results, in addition to those previously obtained (4), suggest a wider distribution of ‘Ca. L. solanacearum’ than previously reported in Europe and should lead plant health managers to consider this pathogen as an emerging threat. References: (1) N. M. Christensen et al. Mol. Plant Microbe Interact. 17:1175, 2004. (2) W. Li et al. J. Microbiol. Methods 78:59, 2009. (3) J. E. Munyaneza et al. Plant Dis. 94:639, 2010. (4) J. E. Munyaneza et al. Plant Dis. 96:453, 2012.

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.

‘Candidatus Liberibacter solanacearum’ (Lso) is an emerging phytopathogenic bacterium that causes significant crop losses worldwide. This bacterium has been identified in association with diseases of several solanaceous crops in the United States and New Zealand, and with carrot and celery crops in several European countries. Five Lso haplotypes (LsoA, LsoB, LsoC, LsoD, and LsoE) have now been described worldwide. In France, symptoms of Lso were observed on plants of the Apiaceae family in several regions. One hundred and ninety-two samples of apiaceous plants were collected from 2012 to 2016 in different geographical regions and were tested for the occurrence of Lso by real-time PCR assay. In addition to carrot and celery, Lso was detected in four other apiaceous crops: chervil, fennel, parsley, and parsnip. These new findings suggest that Lso has a wider natural host range within the Apiaceae family than expected. To identify the Lso haplotypes present in France, we sequenced and analyzed the 16S rRNA gene and the 50S ribosomal protein rpIJ-rpIL gene region from a representative bacterial collection of 44 Lso-positive samples. Our SNP analysis revealed the occurrence of two distinct bacterial lineages that correspond to haplotypes D and E. Then, we assessed the phylogenetic relationships between strains isolated from France and a worldwide collection of Lso isolates by using the rpIJ-rpIL gene region sequences. The neighbor-joining tree constructed delineated five clusters corresponding to the five Lso haplotypes, with LsoD and LsoE being closely related phylogenetically. Altogether, the data presented here constitute a first step toward a better understanding of the genetic diversity among Lso haplotypes in France, and provide new insights into the host range of this emerging bacterial species.

‘Candidatus Liberibacter solanacearum’ is a bacterium associated with several vegetative disorders on solanaceous and apiaceous crops. Following the recent detection of the bacterium in carrots in Europe, and particularly carrot plants used for seed production in France, two independent laboratories conducted experiments on the transmission of this pathogen by seed and had discordant results: one study showed no bacterial transmission to plants, and the other showed transmission to carrot seedlings starting from the fourth month of culture. To test the hypothesis that growing conditions affect seed transmission efficiencies, trials were renewed in 2015 on four lots of 500 carrot seeds naturally contaminated with ‘Ca. L. solanacearum’ and two lots of 100 healthy seeds. The plants were grown for 6 months in an insect-proof NS2 greenhouse. Sets of 108 plants from the contaminated lots and 24 plants from the healthy lots were individually analyzed each month using real-time PCR to detect the bacterium. The detection tests on seeds and plants from healthy lots were always negative. During the 6 months of the trial, no plants from the contaminated seed lots tested positive for the bacterium or showed any infection symptoms. These results indicate that transmission of ‘Ca. L. solanacearum’ by carrot seed is rare and difficult to reproduce.

A working group established in the framework of the EUPHRESCO European collaborative project aimed to compare and validate diagnostic protocols for the detection of “Flavescence dorée” (FD) phytoplasma in grapevines. Seven molecular protocols were compared in an interlaboratory test performance study where each laboratory had to analyze the same panel of samples consisting of DNA extracts prepared by the organizing laboratory. The tested molecular methods consisted of universal and group-specific real-time and end-point nested PCR tests. Different statistical approaches were applied to this collaborative study. Firstly, there was the standard statistical approach consisting in analyzing samples which are known to be positive and samples which are known to be negative and reporting the proportion of false-positive and false-negative results to respectively calculate diagnostic specificity and sensitivity. This approach was supplemented by the calculation of repeatability and reproducibility for qualitative methods based on the notions of accordance and concordance. Other new approaches were also implemented, based, on the one hand, on the probability of detection model, and, on the other hand, on Bayes’ theorem. These various statistical approaches are complementary and give consistent results. Their combination, and in particular, the introduction of new statistical approaches give overall information on the performance and limitations of the different methods, and are particularly useful for selecting the most appropriate detection scheme with regards to the prevalence of the pathogen. Three real-time PCR protocols (methods M4, M5 and M6 respectively developed by Hren (2007), Pelletier (2009) and under patent oligonucleotides) achieved the highest levels of performance for FD phytoplasma detection. This paper also addresses the issue of indeterminate results and the identification of outlier results. The statistical tools presented in this paper and their combination can be applied to many other studies concerning plant pathogens and other disciplines that use qualitative detection methods.

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