Seed Transmission of Verticillum dahlia in Olive as Detected by a Highly Sensitive Nested PCR-Based Assay

Abstract

To determine whether the spread of Verticillium dahliae to new olive growing areas can be seed-borne,
fruit samples of V. dahliae-infected symptomatic and asymptomatic trees of two olive cultivars (Shimlali and Nabali)
were randomly collected in November and December 2003 from two olive-growing areas in Jordan. Seeds were excised
from the fruits and some of the seeds were sown to produce progeny seedlings. Both seeds and the seedlings
were tested for V. dahliae infection using standard plating and a nested polymerase chain reaction (PCR)-based
assay that used primers from the internal transcribed spacer (ITS) regions of nuclear ribosomal RNA (rRNA) genes.
The sensitivity of the nested PCR-based assay was investigated by amplifying the crude DNA of conidia. The incidence
of V. dahliae infection in seeds and seedlings was significantly higher with the nested PCR-based assay than
with the plating procedure in both symptomatic and asymptomatic trees of both olive cultivars. Infection rates were
significantly higher in symptomatic than in asymptomatic trees and, in general, higher for the cv. Shimlali than the
cv. Nabali. The incidence of V. dahliae infection in the seedlings was significantly higher than that in the seeds. The
expected DNA fragments were amplified from all the concentrations of V. dahliae conidial suspensions used (2 104;
2 103; 2 102; 20 and 2 conidia µl-1) indicating that the assay was highly sensitive. Olive seeds of the two cultivars
transmitted V. dahliae to the progeny seedlings in different percentages up to a maximum of 35%. Infected olive seed
contributes significantly to pathogen dissemination.