First Report of a ‘ <i>Candidatus</i> 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 five weeks. Samples of cladodes from naturally infected plants were collected from fourteen symptomatic and one asymptomatic plant. A nested polymerase chain reaction (PCR) performed by using primer pair P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995), followed by R16F2n/R16R2 (Gundersen and Lee 1996), amplified a fragment of the expected size only from the symptomatic samples. Direct amplicon sequencing followed by blast comparison to both, GenBank and EPPO-QBank 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) (White et al., 1998; Rodrigues et al., 2023). The phylogenetic analysis of strain ´Cact1´ with phytoplasma strains of ribosomal subgroups in group 16SrII and detected in cactus in China, Italy and Turkey showed that strain ´Cact1´ is not clustering with any of them (Figure 1). Indeed, comparison with all sequences in GenBank, show 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%, that is below the accepted threshold for ‘Ca. Phytoplasma’ strains 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 phytoplasma strain ´PR08´ from India (GenBank accession no. CP097207), identified as a strain of ‘Ca. P. australasiae=australasiaticum’ (White et al., 1998; Rodrigues et al., 2023). Other phytoplasma strains having 100% identity on the leuS gene but with 95-96% coverage were reported in pearl millet, soybean and alfalfa from India (GenBank accession no. MW020555, MW020562 and MW020559 respectively). The leuS gene sequence has been relevant to confirm the identification of phytoplasmas infecting various agricultural important 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 phytoplasma considering that the disease and the associated phytoplasmas are present and spreading in other regions of Jordan.