Effect of Long-Term Fungicide Applications on Virulence and Diversity of spp. Associated to Olive Anthracnose.

In this study, the presence and variability of spp. was evaluated by comparing fungal isolates obtained from olive trees under long-time phytosanitary treatments with trees without any phytosanitary treatments (treated and untreated, respectively). Olive fruits of trees of the highly susceptible 'Galega vulgar' cultivar growing in the Alentejo region were used as samples. From the 210 olive trees sampled (half from treated and half from untreated orchards), 125 (59.5%) presented spp., with a significant lower number of infected trees in treated (39) when compared to untreated orchards (86). The alignment and analysis of beta-tubulin (tub2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), chitin synthase (CHS-1) and histone H3 (HIS-3) gene sequences allowed the identification of all 125 isolates as belonging to the complex. The vast majority of the isolates (124) were identified as and one isolate, from an untreated tree, was identified as . Isolates were divided into five different groups: Group A: 39 isolates from treated trees matched in 100% with sequences from the database; Group B: 76 isolates from untreated trees matched in 100% with sequences from the database; Group C: one isolate from untreated trees presenting a single nucleotidic difference in the HIS-3 sequence; Group D: eight isolates from untreated trees presenting differences in two nucleotides in the tub2 sequences that changed the protein structure, together with differences in two specific nucleotides of the GAPDH sequences; Group E: one isolate, from untreated olive trees, matched 100% with sequences from the database in all genes. Considering the similarities of the sampled areas, our results show that the long-time application of fungicides may have caused a reduction in the number of olive trees infected with spp. but an increase in the number of fruits positive to spp. within each tree, which may suggest different degrees of virulence of isolates from trees growing different management regimes. It is imperative that the fungicides described as causing resistance are applied at appropriate times and intervals, since their efficiency decreases when applied incorrectly and new and more virulent species may arise.