Phaeomoniella chlamydospora and Phaeoacremonium spp. in grapevines from Uruguay

Symptoms corresponding to esca and Petri diseases have been described in Uruguay as being associated with Phaeomoniella chlamydospora and Phaeoacremonium spp. Isolates of Phaeoacremonium spp. recovered from diseased grapevines were characterized and identified. Additionally, specific primers developed for Pa. chlamydospora and Phaeoacremonium spp. were evaluated for direct detection of these fungi in asymptomatic grapevine tissues. The bark was removed, and the trunk underneath was surface-disinfected. Chips from symptomatic grapevines were plated on potato dextrose agar and incubated at 25 degrees C. Isolates were identified morphologically and phylogenetically. Sequences of ITS rDNA, beta-tubulin and actin genes made it possible to identify 52 isolates as Pa. chlamydospora, 33 as Pm. aleophilum, and one as Pm. australiense. The Pm. aleophilum isolates were divided into three groups by their growth pattern and their colony shape at 37 C: a) white colonies with yellow or brown reverse; b) brown colonies with clear margin and a dark center on the reverse; c) brown colonies with dark-brown diffusible pigments and brown reverse. Further studies are required to explain these differences. Primers Pac1f/Pac2r specific for Phaeoacremonium spp., Pal1N/Pal2 specific for Pm. aleophilum, F2bt/R1bt specific for Pm. aleophilum and ten other Phaeoacremonium species, and Pch1/Pch2 and Pmo1f/Pmo2r, both specific for Pa. chlamydospora were evaluated on the DNA of target fungi and some of other fungi frequently isolated from diseased vine tissues. F2bt/R1bt and Pmo1f/Pmo2r were selected, and were used in a nested PCR to detect Phaeoacremonium spp. and Pa. chlamydospora in asymptomatic canes of nursery mother grapevines. Nine out of ten sampled grapevines tested positive for one of the fungi. Molecular diagnosis is potentially a useful method to assess the health of mother grapevines.