DNA PROBES AND PCR PRIMERS FOR THE DETECTION OF A PHYTOPLASMA ASSOCIATED WITH PEANUT WITCHES-BROOM

EcoRI restriction fragments of genomic DNA from the phytoplasma associ ated with peanut witches'-broom (PNWB) were cloned in plasmid pGEM-3Zf (+). Cloned inserts from seven PNWB-phytoplasma-specific recombinant p lasmids and two subcloned plasmids were excised with restriction enzym es, labeled with digoxigenin, and used as probes. Probe PNWB281 and it s derivative subclones PNWB281-4 and PNWB281-5 hybridized with DNA fro m PNWB-phytoplasma infected peanut and periwinkle specifically but not with DNA from healthy plants or plants infected with phytoplasmas ass ociated with sweetpotato witches'-broom (SPWB), loofah, Ipomoea obscur a, and paulownia witches'-broom, elm and aster yellows, rice yellow dw arf, and bamboo little leaf disease. Six other probes hybridized with DNA derived from PNWB and SPWB-phytoplasma-affected periwinkle but not with DNA from healthy plants or plants infected with other phytoplasm as mentioned. In Southern hybridizations, four of the nine cloned and subcloned probes could differentiate the PNWB-phytoplasma from SPWB-ph ytoplasma. Three primer pairs for PCR were synthesized according to th e partial sequences at both ends of the cloned inserts and were able t o distinguish PNWB-phytoplasma from SPWB-phytoplasma by using PCR for the first time. A minimum of 1 pg and 10 pg of total DNA from diseased periwinkle and peanut, respectively, was sufficient to amplify the sp ecific PNWB-phytoplasma PCR fragments, allowing the detection of PNWB- phytoplasma DNA from healthy-looking periwinkle plants two weeks after graft inoculation.