First report of a ‘Candidatus Phytoplasma australasiaticum’ strain associated with stunting disease of opuntia in Jordan

Cultivation of prickly pear (Opuntia ficus-indica L., family Cactaceae) is of high value in dry-land agriculture in Jordan. In May 2021, symptoms including thickening and severe stunting of the cladodes and deformation of fruits were observed on prickly pear plants cultivated in southern Jordan, Madaba region (31.593565°N, 35.850111°E), with a 15% incidence across three cactus fields. To verify the occurrence of a graft-transmissible disease, wedge grafting was performed on asymptomatic opuntia rootstocks, resulting in thickened cladodes and deformed fruits within 5 weeks. Samples of cladodes from naturally infected plants were collected from 14 symp- tomatic and one asymptomatic plant. A nested polymerase chain reaction performed by using the primer pair P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995), followed by R16F2n/R16R2 (Gundersen and Lee 1996), am- plified a fragment of the expected size only from the symptomatic samples. Direct amplicon sequencing followed by BLAST comparison with both GenBank and EPPO Q-Bank databases (https://qbank.eppo.int/phytoplasmas/) allowed the identification of a ‘Candidatus Phytoplasma australasiae = australasiaticum’ strain (GenBank accession no. PQ319761, designated strain “Cact1”), with a 100% sequence identity to the reference strain (GenBank accession no. Y10097, ribosomal subgroup 16SrII-D) (Rodrigues et al. 2023; White et al. 1998). The phylogenetic analysis of strain “Cact1” with phytoplasma strains of ribosomal subgroups in group 16SrII, detected in cactus in China, Italy, and Turkey, showed that strain “Cact1” does not cluster with any of them. Indeed, comparison with all sequences in GenBank showed that the phytoplasma from Jordan clusters with those enclosed in the 16SrII group under the species ‘Ca. P. australasiae = australasiaticum’, showing about 99.00% identity to this phytoplasma and having an identity to ‘Ca. P. aurantifolia = citri’ of about 98.50%, which is below the accepted threshold for ‘Ca. Phytoplasma’ strain differentiation (Bertaccini et al. 2022). This identification was confirmed by amplifying and sequencing the leucyl transfer RNA synthetase (leuS) gene (Abeysinghe et al. 2016) (GenBank accession no. PQ349195) that showed 100% sequence identity with 100% coverage to the Parthenium hysterophorus phyllody phyto- plasma strain “PR08” from India (GenBank accession no. CP097207), identified as a strain of ‘Ca. P. australasiae = australasiaticum’ (Rodrigues et al. 2023; White et al. 1998). Other phytoplasma strains having 100% identity on the leuS gene but with 95 to 96% coverage were reported in pearl millet, soybean, and alfalfa from India (GenBank accession nos. MW020555, MW020562, and MW020559, respectively). The leuS gene sequence has been relevant to confirm the identification of phytoplasmas infecting various im- portant agricultural crops, especially in Asian countries (Tiwari et al. 2023). It is thus necessary to investigate insect vector(s) presence and search for other economically important hosts and for alternative host weeds for this phyto- plasma, considering that the disease and the associated phytoplasmas are present and spreading in other regions of Jordan.