Agronomy and Crop Science

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.

‘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.

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.

Abstract The association of phytoplasma was investigated in symptomatic tomato (Solanum lycopersicum L.), eggplant (Solanum melongen L.), mallow (Malva spp.) and Bermuda grass (Cynodon dactylon L.) plants exhibiting witches’ broom and white leaf diseases, respectively. Total DNA was extracted from tomato (n=3), eggplant (n=2), mallow (n=2) and Bermuda grass (n=8) samples. Direct polymerase chain reaction (PCR) was performed using P1/P7 primer set, then PCR products were sequenced. Sequences obtained from tomato, eggplant and mallow shared 99% maximum nucleotide identity with phytoplasma belonging to subgroup 16SrII-D, and resulted therefore ‘Candidatus Phytoplasma australasia’-related. Sequences obtained from Bermuda grass showed 100% maximum nucleotide identity to 16SrXIV-A subgroup and were ‘Ca. P. cynodontis’-related. The study presents the first molecular confirmation and sequence data of presence of ‘Ca. P. australasia’ and ‘Ca. P. cynodontis’ in Iraq.