Plant Science

Specific scopeThis Standard describes a national regulatory control system for Bactericera cockerelli and the bacterial pathogen ‘Candidatus Liberibacter solanacearum’ the cause of zebra chip disease in potato. The scope is as follows: Exclusion from the EPPO region of B. cockerelli an efficient vector of ‘Ca. L. solanacearum’ within solanaceous crops (e.g. potato, tomato) Eradication of incursions of B. cockerelli Exclusion from the EPPO region of ‘Ca. L. solanacearum’ haplotypes A and B. Although reference will only be made to haplotypes A and B, the Standard would also apply to new non‐European haplotypes of ‘Ca. L. solanacearum’ which may have different host ranges, or which may be vectored more efficiently by psyllids which are widespread in the region. The reduction of the risk of spreading ‘Ca. L. solanacearum’ haplotypes C, D and E to potato production systems and potatoes being moved within the EPPO region may be recommended in future when more information is available but is not covered in this Standard.Specific approvalFirst approved in 2017‐09.

‘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.

The use of proper management strategies for citrus huanglongbing ( HLB ), caused by ‘ Candidatus Liberibacter asiaticus’ (Las) and transmitted by Asian citrus psyllid ( ACP ) ( Diaphorina citri ), is a priority issue. HLB control is based on healthy seedlings, tolerant rootstock cultivars and reduction of ACP populations. Here, dynamic populations of Las in different citrus hosts and each instar of ACP were studied, together with the seasonal growth and distribution of Las in different tissues, using conventional and TaqMan real‐time PCR . Different levels of susceptibility/tolerance to HLB were seen, resulting in different degrees of symptom severity and growth effects on hosts or rootstocks. Troyer citrange, Swingle citrumelo and wood apple were highly tolerant among 11 rootstock cultivars. Regarding distribution and seasonal analysis of Las, mature and old leaves contained high concentrations in cool temperatures in autumn and spring. Las was detected earlier through psyllid transmission than through graft inoculation, and the amounts of Las ( AOL ) varied in different hosts. Thus, different AOL (10 4 –10 7 copy numbers μL −1 ) and Las‐carrying percentages ( LCP ; 40–53.3%) were observed in each citrus cultivar and on psyllids, respectively. Furthermore, both AOL and LCP were lower in nymphs than in adult psyllids, whereas the LCP of psyllids were not affected by increasing the acquisition‐access time. The present study has significant implications for disease ecology. The combination of early detection, use of suitable rootstocks and constraint of psyllid populations could achieve better management of HLB disease.

AbstractIsothermal recombinase polymerase amplification (RPA) assays for the specific detection of “Candidatus Phytoplasma mali (Ca. P. mali),” the causal agent of apple proliferation, were developed. The assays amplify a fragment of the imp gene and amplimers were detected either by fluorescence in real‐time mode (TwistAmp®exo assay) using a fluorophore‐labelled probe or by direct visualization employing a lateral flow device (TwistAmp®nfo assay/Milenia®HybriDetect). The RPA assays specifically amplified DNA from “Ca. P. mali” strains, and cross‐reactivity with other phytoplasmas or plant DNA was not observed. The limit of detection was determined with a cloned imp standard, and positive results were obtained down to 10 copies with both RPA assay formats. In comparison with a TaqMan real‐time PCR assay based on the same target gene, the RPA assays were equally sensitive, but results were obtained faster. Simplified nucleic acid extraction procedures from plant tissue with Tris‐ and CTAB‐based buffers revealed that crude Tris–DNA extracts were a suitable source for RPA tests while larger concentrations of CTAB were inhibitory. This is the first report of RPA‐based assays for the detection of “Ca. P. mali”. The assays are suitable for high‐throughput screening of plant material and point‐of‐care diagnostic and can be potentially combined with a simplified DNA extraction procedure.