Comparison and characterization of tomato big bud- and sweet potato littleleaf-group phytoplasmas

EcoRI or HindIII-restricted DNA from phytoplasmas associated with tomato bi g bud (TBB) and sweet potato little leaf (SPLL-V4) was cloned into pBluescr ipt and the recombinant plasmids were screened by reciprocal DNA hybridizat ion with TBB- and SPLL-V4 DNA. Most of the recombinant plasmids hybridized to DNA of both phytoplasmas. Southern hybridization analysis of EcoRI- or H indIII-restricted TBB- and SPLL-V4 DNA with two TBB probes (pTBB32 and pTBB 87) gave identical restriction patterns. Southern hybridizations of HindIII - or EcoRI digested DNA from 10 phytoplasma-positive field samples revealed a different restriction fragment pattern in two phytoplasmas from sweet po tato. Three TBB probes which did not hybridize to SPLL-V4 were selected for Southern blot analysis of samples. The pTBB42 and pTBB78 hybridized to nin e of the 10 TBB-symptomatic held samples producing identical patterns. pTBB 88 contained extrachromosomal DNA and hybridized with two phytoplasmas from diseased sweet potato and perennial phlox;. The SPLL-V4-specific probe pH 21 hybridized to five field samples generating different restriction patter ns. About 80 held samples were subjected to the polymerase chain reaction u sing primers deduced from pH21, pTBB32 and pTBB88, These phytoplasmas had b een previously characterized and grouped by their 16S/23S spacer region res triction fragment pattern but this classification was in most cases differe nt to the results obtained with these primers.