MOLECULAR HYBRIDIZATION AND PCR AMPLIFICATION OF NON-RIBOSOMAL DNA TO DETECT AND DIFFERENTIATE STOLBUR PHYTOPLASMA ISOLATES FROM ITALY

Abstract

Amplification of ribosomal phytoplasma DNA by direct or nested PCR and stolbur-specific primers, followed by restriction length polymorphism analysis were used to detect and identify stolbur phytoplasmas in symptomatic alfalfa, celery, field bindweed, grapevine, olive and tomato plant samples from different regions of Italy. Alfalfa appears to be a new host for this phytoplasma. All isolates showed RFLP patterns indistinguishable from one another and from the one of the Serbian reference isolate from pepper. In Southern hybridization experiments, EcoRI and HindIII restricted DNAs from stolbur-infected herbaceous hosts hybridized with two riboprobes from random-cloned chromosomal DNA fragments of a Sardinian stolbur isolate from tomato, yielding more or less complex patterns. None of the probes hybridized with DNAs from stolbur-unrelated phytoplasma infected or healthy plants or olives known to harbour stolbur phytoplasma. With one of the probes, two types of molecular hybridization profiles were detected among stolbur-infected samples. Based on the nucleotide sequence of this probe, a new non-ribosomal primer pair was designed. These primers amplified a stolbur-specific fragment of 720 bp from stolbur- infected samples, but not from symptomatic olives, healthy plants or plants infected with unrelated phytoplasmas. To verify whether absence of amplified fragments in gels was due to lack of priming or to undetectably low amplification of stolbur DNA, negative PCR products were tested in hybridization assays with the primers parental probe. Positive hybridization signals were obtained with such samples but not with products from healthy DNAs or negative PCR controls. The same probe was also used to detect stolbur DNA on membranes printed with tissues from field-infected tomato plants or from glasshouse-maintaned stolbur-infected periwinkles or young asymptomatic tomato seedlings grafted with infected field material.