The use of polymerase chain reaction and molecular hybridization for detection of phytoplasmas in different plant species in Mexico

Abstract

The oligonucleotide primer pairs MMF/MMR, R16F2/R16R2, P1/Tint, P1/Bltvaint, P1/Ayint, P1/P7 and P3/P7 were used in polymerase chain reaction (PCR) for detection of phytoplasmas in coconut palms (Cocos nucifera), marigold (Tagetes erecta), potato (Solanum tuberosum), periwinkle (Catharanthus roseus), and cassava (Manihot esculenta). There were differences in specificity in the amplification of DNA sequences depending of the primer pair set used and plant species tested. Primer pair P1/Tint amplified single DNA fragments from all above plant species, including symptomless coconut palms collected from lethal yellowing (LY) affected areas. The primer set MMF/MMR yielded consistent results in the detection of phytoplasmas from coconut palms, marigold, and periwinkle phyllody. DNA fragments amplified with primer pairs R16F2/R16R2 and P1/Tint from the above plant species mentioned, were digested with restriction endonucleases EcoR1 or AluI. Marigold and periwinkle showed an identical pattern and was similar in some extent to that of coconut palm. DNA fragments amplified from coconut palm (LY269 bp) and periwinkle (PW1.2 bk), were cloned into vector pGEM-T and used to transform cells of Escherichia coli strain; then, they were used as probes in DNA-DNA hybridization assays. These probes are targeted to anneal to the operon of the ribosomal DNA of the 16S gen of phytoplasmas. Probe PW1.2 hybridized with DNA samples from all plant species tested, including jipi palm (Caludovica palmata). Probe LY269 hybridized only with DNA extracted from coconut and jipi palms, and periwinkle, but not with DNA of marigold, potato and cassava. Both probes hybridized with DNA extracted from symptomless coconut palms from coconut lethal yellowing affected areas, whereas, no hybridization was detected with DNA extracted from healthy plants of all species tested. This is the first report of molecular detection in Mexico of phytoplasmas infecting cassava