Identification and grouping of mycoplasmalike organisms associated with grapevine yellows and clover phyllody diseases based on immunological and molecular analyses

Abstract

Immunofluorescent staining, dot blot hybridization, PCR, random amplified polymorphic DNA (RAPD) markers, and restriction fragment length polymorphism were used to study the genetic relatedness among mycoplasmalike organisms (MLOs) associated with several geographically diverse grapevine yellows diseases (CA1, CH1, SA1, and SA2 from Bologna, Italy; GYU from Udine, Italy; GYR from Rome, Italy; and GYG from Germany). The relationship between these and MLOs associated with clover phyllody diseases in Italy (CPhB and CPhC) and Canada (CPhCa) was also examined. Two monoclonal antibodies reacted with MLOs of GYU-, CPhB-, and CPhC-infected periwinkles. Dot blot hybridization with two cloned GYU DNA fragments, GYD-1 and GYD-2 inserts, showed that both hybridized with DNAs of GYU-, CPhB-, and CPhC-infected periwinkles but not with those of GYR and CPhCa. In addition, GYD-1 insert hybridized with DNAs of CA1, CH1, SA1, SA2, and GYG. Three primer pairs were developed in PCR experiments for this study. By using primer set GYD2P1F and GYD2P1R, a 600- bp DNA fragment was amplified only when DNAs from GYU-, CPhB-, and CPhC- infected plants were used as templates. With the primer pair GYD2P1F and GYD2P2R, a 550-bp DNA fragment was amplified from GYU, CPhB, CPhC, and GYG. The primer pair GYD1P1F and GYD1P2R, on the other hand, could amplify all isolates, although the patterns of PCR products were not identical for all isolates. Six different primers used in RAPD analysis have resulted in 13 RAPD markers which were either specific for one isolate or shared by two, three, or four isolates and thus could be used for the identification of MLO isolates. On the basis of these results, all isolates studied except GYR and CPhCa could be distinguished from one another, and they could be broadly classified into five subgroups: subgroup I, CA1 and CH1; subgroup II, SA1 and SA2; subgroup III, GYU, CPhB, and CPhC; subgroup IV, GYG; and subgroup V, GYR and CPhCa. Restriction fragment length polymorphism analyses with cloned GYD- 1 insert were in good agreement with results obtained from other approaches used in this study.