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length from cut to the 3rd bud and the length of 1st plus 2nd internodes were registered,
as was the length of the necroses. Re-isolations were made for all replicates and for all
experimental and control treatments. Each shoot was divided in different segments,
depending on the length of the necrosis, and re-isolation was made from each segment
by taking five pieces of symptomatic wood tissue and placing them onto PDA plates, with
250mg L -1 chloramphenicol.
Conclusions were drawn based on the percentage of re-isolation, as these gave a more
precise idea of the efficacy of the fungicides. For cv. Syrah all treatments revealed a level
of re-isolation lower or similar to the reference carbendazim+flusilazol, except for the
BUC 74700F F and BUC 40300 F treatments (8 and 6, respectively). However, when the
prevention and slowing down of the disease progression were considered, the best
results were obtained for treatments 3 (BUC 31300 F) and 7 (BUC 61700 F). In these
cases Pa. chlamydospora was not reisolated from the segment 4. For cv. Touriga
Nacional only treatment 7 (BUC 61700 F) was able to provide a lower or similar level of
fungal re-isolation than carbendazim+flusilazol. In this case, however, when prevention
or slowing down the disease progression was considered, the best treatment was 7 (BUC
61700 F). This was the only treatment from which Pa. chlamydospora was not re-
isolated.
To complement the classical method of re-isolation, a molecular approach based on
nested-PCR method was used, in order to confirm the presence of Pa. chlamydospora in
the grapevine wood tissues. This method involves two sets of primers, used in two
successive runs of polymerase chain reaction. The first set (ITS4 and ITSF1) was used to
amplify a fragment of the ITS region of fungi, and the second set (Pch1 and Pch2) to
amplify, within the first run product, a specific fragment of 360 pb from the ITS region of
Pa. chlamydospora. The results obtained by nested-PCR were not always in agreement
with those obtained from the re-isolation method, but in general the PCR-based method
was more sensitive.
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