First report on detection of ‘Candidatus Phytoplasma aurantifolia’ (group 16SrII) affecting sapodilla in eastern Cuba

Sapodilla (Manilkara zapota) has become an important fruit crop in eastern Cuba due to its fresh consumption and derivates used in local commerce. Sapodilla has been identified as a potential export crop, and its pilot cultivation is being exploited in the eastern province of Las Tunas for such purposes. Symptoms of leaf yellowing and witches’ broom have been recently observed in sapodilla fields in the region of Vazquez, Las Tunas (Fig. 1). Although no data are available on the distribution of the disease throughout the province, there was patchy distribution in Vazquez sapodilla fields, with approximately 20% of plants displaying obvious symptoms, this being the first record of such symptoms in this crop in Cuba.

Leaf samples from three symptomatic and two asymptomatic plants were collected and total DNA extracted and used as template in a nested PCR with universal phytoplasma 16S rDNA primers, R16mF2/R1 and fU5/rU3. PCR amplicons (880 bp) were produced in two out of three symptomatic samples, but not from the asymptomatic ones. The two amplicons were purified, cloned (pGEMT-Easy Vector, Promega), and sequenced in both directions with M13 forward and reverse sequencing primers. Restriction profiles after digestion of PCR amplicons with Sau3AI, HpaII and HaeIII enzymes were all identical to those of ‘Candidatus Phytoplasma aurantifolia’ (group 16SrII). The 16S rDNA sequences of the phytoplasma detected in the two symptomatic sapodilla samples shared 100% identity with each other, and 99% with that of the Cleome witches'-broom phytoplasma (GenBank Accession No. EU513212), a member of group 16SrII, when compared using BLAST searches. The 16S rDNA sequence of the sapodilla phytoplasma was deposited in GenBank (EU869220). ‘Ca. Phytoplasma aurantifolia’ group phytoplasmas have been previously recorded from Cuba in papaya, particularly in the eastern region (Arocha et al., 2007), which reinforces the prevalence of this group in this part of the country. No previous record of the presence of phytoplasmas has been found in sapodilla from other countries in the region, or worldwide, and to our knowledge, this is the first record of a phytoplasma pathogen affecting this crop.