Abstract
Oak decline in non-calcareous soils in south-western Spain has been associated with Phytophthora cinnamomi for decades. However, other Phytophthora species such as P. quercina and P. psychrophila have been associated with Quercus decline in the eastern part of Spain where calcareous soils are predominant. With the aim of investigating the involvement of Phytophthora spp. in oak decline in eastern Spain, two forests in different geographical areas (Alcoi and Vallivana) were selected as sampling sites. Both forests are similar in altitude, soil and vegetation composition and are located 230 km apart. Soil samples were analyzed in parallel by isolation using baiting methods and by amplicon massive sequencing. Results showed that one of the most frequent species detected in both sampling sites was P. quercina, although cultures were only obtained from Alcois holm oak forest. Pyrosequencing showed a very similar Phytophthora species composition in both areas. Furthermore, an uncultured Phytophthora taxa (named provisionally Phytophthora taxon ballota) was the dominant species, followed by P. quercina. Considering the difficulty in the isolation of Phytophthora taxon ballota, new Real-Time specific assays based in the ITS1 region were developed for the detection of this new taxa and P. quercina in environmental samples from oak declined areas. Taqman assays were tested on soil samples and on Phytophthora pure cultures. Results revealed the coexistence of both species in most of the samples, with the predominance of P. taxon ballota in terms of the amount of DNA available. Quantitation assays were high congruent with pyrosequencing results (relative number of reads per species). In order to evaluate the implication of different Phytophthora spp. in oak decline in eastern-Spain a new Real-Time specific detection protocol is proposed.
