ISHS Acta Horticulturae 234: VII International Symposium on Virus Diseases of Ornamental Plants
CLONED NUCLEIC ACID HYBRIDIZATION PROBES IN DETECTION AND CLASSIFICATION OF MYCOPLASMALIKE ORGANISMS (MLOS) |
| Authors: | Robert E. Davis, I. Lee, Ellen L. Dally, N. Dewitt, Sharon M. Douglas |
| Abstract:
MLO DNA suitable for molecular cloning was obtained by a method developed during this work. The method, which involves a procedure for isolation of sieve cells from infected plants (Lee, I.M., and R. E. Davis. 1983. Phytopathology 73:1540 – 1543.), should be applicable to a broad range of phloem-inhabiting pathogens. DNA fragments from 4 (four) different MLOs (aster yellows, AY; tomato big bud, BB; elm yellows, EY; and an unknown, ORCH1 MLO) were cloned in plasmid vectors pSP64 and pSP65. Recombinant plasmids or cloned inserts were labeled with [32P] or with biotin and employed as probes in dot-hybridizations with nucleic acid extracted from MLO-infected plants of periwinkle (Catharanthus roseus). Patterns of hybridization indicated that the AY, ORCH1, BB, and blueberry stunt disease MLOs belong to a cluster of strains that share greater nucleotide sequence homology with one another than with other MLOs tested. Results from use of [32P] -labeled single-stranded RNA probes, transcribed in vitro from linearized recombinant plasmids, confirmed the concept of an AY-related MLO strain cluster, distinguished between strains in this cluster, and indicated a close genetic relatedness between eastern AY and a strain of western AY MLO. Biotinylated DNA probes were successfully applied in dot-hybridizations for the detection of various MLOs in C. roseus and other host plant species and of AY MLO in the insect vector Macrosteles fascifrons. MLOs detected included those of alfalfa witches broom in alfalfa (Medicago sativa); clover yellow edge, X-disease, clover phyllody, and eastern AY in clover (Trifolium repens); eastern and western AY, and clover phyllody in aster (Callistephus chinensis); and AY in celery (Apium graveolens var. dulce) and M. fascifrons. | |
Download Adobe Acrobat Reader (free software to read PDF files) | |
