DNA PROBES AS MOLECULAR MARKERS TO MONITOR THE SEASONAL OCCURRENCE OF WALNUT WITCHES-BROOM MYCOPLASMA-LIKE ORGANISM

A rapid and sensitive method was developed to monitor the occurrence of walnut witches'-broom (WWB) mycoplasmalike organism (MLO) in infected walnut (Juglans nigra) trees. Walnut leaf tissues from both asymptomatic and symptomatic trees were collected monthly during the growing seasons of 1990 and 1991 and freeze-dried. Southern blots of CTAB-extracted DNA without endonuclease digestion were used for hybridization with P-32-labeled chromosomal and extrachromosomal WWB MLO DNA probes. The presence of WWB MLO DNA was detected in DNA extracts from a single tree at least 1 mo prior to the seasonal appearance of WWB symptoms when extrachromosomal DNA probes, but not chromosomal DNA probes, were used. Also, hybridization results and observation of symptom development indicate that WWB MLO may not be uniformly distributed within infected walnut trees. Results from two-dimensional electrophoresis experiments suggested that there were two double-stranded, circular, extrachromosomal DNA molecules, approximately 5 and 1.5 kb.