First report of a phytoplasma associated with abnormal proliferation of cladodes in cactus pear (Opuntia ficus-indica) in Italy

While Mexico is the main cactus pear-producing country, Italy is the most important producer in the Mediterranean basin. No phytoplasma disease of cactus pear has been reported, despite previous detection of phytoplasmas in related species such as Opuntia tuna (Casper et al., 1970) and Opuntia linguiformis showing witches' broom symptoms (Cai et al., 2002). In 2003, three cactus pear plants showing abnormal growth were observed in the DISTEF collection. The plants showed severe proliferation of cladodes with lack of flowers, fruits and spine production. Viral particles were not observed by transmission electron microscopy in sap from any of the affected plants. Total DNA was extracted from the affected plants and from two symptomless cactus pears as described by Cai et al. (2002). This was used as template for phytoplasma-specific 16S rDNA PCR amplification using one of three universal primer pairs: P1/P7, R16f2/r2 (Lee et al., 2000) or fU5/rU3 (Lorenz et al., 1995). DNA preparations from phytoplasma reference strains maintained in periwinkle were used as positive controls. To produce enough amplicon for further characterization by RFLP analysis, nested primer pair R16f2/r2 was used to reamplify P1/P7-primed rDNA products. RFLP analysis was performed with restriction enzymes AluI, HhaI, HpaII, MseI and TaqI. Phytoplasma-specific PCR products were amplified from all three plants showing symptoms with P1/P7 and fU5/rU3 primers by direct PCR, and with R16f2/r2 primers by nested PCR, but symptomless plants were always negative in all three PCR assays. The RFLP patterns obtained from analysis of rDNA amplicons from the former samples were identical to the pattern obtained for reference strain faba bean phyllody phytoplasma, a member of the 16S rDNA RFLP subgroup 16SrII-C. This is the first report of a phytoplasma infecting O. ficus-indica.