Detection: A broad range of phytoplasma strains belonging to different subgroups of the phytoplasma 16S rDNA gene were detected using the assay. In details, the assay is specific for 16SrI-B (American aster yellows), 16SrI-C (Clover phyllody), 16SrII-A, (Sesame phyllody), 16SrIII-A (Green valley X), 16SrIII-B (Crepis biennis yellows), 16SrIII-H (Poinsettia branchinducing), 16SrV-A (Elm yellows), 16SrV-B (Jujube witches’ broom), 16SrV-C (Alder Yellows, Grapevine yellows), 16SrV-D (Grapevine yellows), 16SrV-E (Rubus stunt), 16SrVI (Lucerne virescence), 16SrVII (Ash yellows), 16SrIX (Pichris echioides yellows), 16SrX-A (Apple proliferation), 16SrX-B (German stone fruit yellows), 16SrXI (Flower stunting), 16SrXIIA (Bois noir, Sour cherry).
Matrix: Leaves with symptoms (leaf petioles and midveins, stems or inner bark). Although phytoplasmas can be detected in roots and bark scrapings of dormant trees, generally it is best to test for phytoplasmas at the end of summer.
Other information: kit developed and optimized by Qualiplante according to Christensen et al., 2004. A verification was performed by Qualiplante (data not published) and the performance characteristics of the kit are the same as the original publication.
Probes and primers for phytoplasma detection were based on alignments of 16S rDNA from a range of phytoplasma strains (one of each phytoplasma 16Sr group), bacteria, and mycoplasmas. Primers were designed to amplify DNA from a broad range of phytoplasma strains, excluding amplification of bacterial DNA.
No cross reactions were observed for the following bacteria: Agrobacterium radiobacter, Arthrobacter globiformis, A. oxydans, Bacillus gibsonii, B. megaterium, Clavibacter michiganense, Paenibacillus macerans, Pseudomonas putida, Ralstonia pickettii and Rhodococcus equi.
This method was evaluated by testing phytoplasmas from 18 subgroups and was found to have an analytical sensitivity equal to or up to ten times higher than conventional nested PCR, depending on the host–phytoplasma combination. A test performance study was realized during the EUPHRESCO project FruitPhytoInterlab (2011).
The real-time PCR test from Christensen et al., 2004 is recommended by the European and Mediterranean Plant Protection Organization (www.eppo.int) – PM7/133, Bulletin (2018) 48 (3), 414–424.
Taq-Man® technology, FAM fluorophore.
Included reagents: Direct Master Mix, RT-Enzyme, Negative Control, Positive Control.