ROWB is indeed associated with phytoplasma, and if
so, of what kind, and whether local weed plants or
crops also showing phytoplasma symptoms could be
reservoirs of the same agent.
The incidence of the disease was sporadic. In 2006,
Russian olive trees at our research station in Tehran
(530 km south-east of Urmia) also showed ROWB
symptoms, and samples were collected from these
trees for phytoplasma tests, which proved positive
(ROWBp-T). In addition, during a survey in 2005,
three alfalfa (Medicago sativa) plants surrounding the
infected Russian olive trees at Urmia were found to
have witches’ broom and little-leaf symptoms
(Fig. 1b) and were collected for testing, again with
positive results (AlWBp-U); samples were also
collected from other weeds as well as from tomatoes
showing big bud symptoms and growing nearby.
Total DNA was extracted from frozen phloem
tissue of two Russian olive infected trees from each
site following the cetyl trimethyl ammonium bromide
(CTAB) procedure (Ahrens and Seemuller 1992).
PCR amplification was performed using the universal
phytoplasma primers P1/P7 (Schneider et al. 1995)
followed by primers R16F2/R16R2 (Gundersen and
Lee 1996) as nested primers after diluting the DNA
from first step. Total DNA from Tomato big bud
(TBB) phytoplasma was used as a positive control.
DNA from healthy Russian olive plants or sterilized
distilled water was used as negative controls in each
PCR. The PCR products were analyzed by electrophoresis
in agarose gels and visualized by UV
following staining with ethidium bromide.
For nucleotide sequence analysis of the ROWBp-U
isolate, the P1/P7 amplicon was used. This product
was readily visible in agarose gels, while we had to
perform a nested PCR to be able to visualize a product
in agarose gels from ROWBp-T and AlWBp-U. DNA
sequencing was performed in an Applied Biosystems