Isolation, Identification And Rapid Detection Techniques Of Phvtophthora Species From Rhododendron-oak Forests In Southwest China

Phytophthora species are plant pathogens with world-wide distribution, and the vastmajority of them are notorious pathogens. With the increased attention given to the genusPhytophthora in the last decade in response to the ecological and economic impact of severalinvasive species (such as P. ramorum, P. kernoviae, and P. alni), there has been a significantincrease in the number of described species. There are now approximately116species ofPhytophthora described in the literature of which58were recognized by Erwin and Ribeiro1996. In the last decade, about20new Phytophthora species or taxa were recovered fromforests and natural ecosystems. In China, there are currently28described species ofPhytophthora, most of which are pathogens of agricultural crops or ornamental plants. So farthere are few reports of Phytophthora diseases of trees or of Phytophthora surveys in theforests in China. Therefore, the objectives of this study were to isolate and identifyPhytophthora species by baiting techniques from streams and soil in the oak forests, toevaluate the genetic diversity of Phytophthora populations using a combination ofmorphological and molecular tools and to detect Phytophthora rapidly by serological anddirect PCR methods.In the surveys from2005to2011, a total of275isolates of Phytophthora spp. wererecovered from baited leaves and soils in rhododendron-oak forests of south-eastern China.Nine Phytophthora species were identified by observation of morphological features andITS1-5.8S-ITS2rDNA sequence analysis. The eight taxa included two originally reportedspecies in China, P. cryptogea Pethybridge&Lafferty and P. gonapodyides (Petersen) Buisman,first reports in China of five recently described species, including P. borealis E Hansen, Sutton&Reeser, P. gregata T. Jung, M.J.C. Stukely&T. Burgess, P. plurivora T. Jung&T.I. Burgess,P. lacustris Brasier, Cacciola, Nechwatal, Jung&Bakonyi and P. taxon PgChlamydo, and twopreviously unrecognized species, Phytophthora sp.1and P. sp.2. Phytophthora species were surveyed by collecting soil samples and placing bait leaves inselected streams during June to October in the years2005,2006and2010at three sites in oakforests in Diqing Tibetan Autonomous Prefecture of NW Yunnan province, China. EightPhytophthora species were identified, which included43P. taxon PgChlamydo isolates, nine P.lacustris isolates, six P. plurivora isolates, four P. gonapodyides isolates, three P. gregataisolates, one P. cryptogea isolate and seven isolates of two previously unrecognized species.The most numerous species, P. taxon PgChlamydo and the second most abundant species, P.lacustris, were recovered at all three sites.In the surveys from2006to2011,202isolates of Phytophthora spp. were recovered frombaited leaves at six sites in rhododendron-oak forests in Nyingchi, Tibet Autonomous Regionand Ganzi, Sichuan Province. Only five Phytophthora species were identified, including47P.borealis isolates,94P. gonapodyides isolates,33P. taxon PgChlamydo isolates,17P. plurivoraisolates and11P. lacustris isolates. The most numerous species, P. gonapodyides and thesecond most abundant species, P. borealis, were both recovered at four sites in Tibet andSichuan Province.Two novel heterothallic species from Phytophthora ITS Clade6were isolated from baitedleaves in Phytophthora surveys of forest streams and soils in Diqing of NW Yunnan province.They are similar in morphology to other Clade6species, however they are phylogeneticallydistinctly different from other clade6species based on the sequence analysis of ITS and Cox Igene. According to their first recovered sites, the two new species were described asPhytophthora bitahaiensis Huai, Zhao, Tian&Hansen sp. nov. and Phytophthora nixiensisHuai, Zhao, Tian&Hansen sp. nov. P. bitahaiensis formed globose or subglobose oogonia andamphigynous antheridia when paired with A2mating type testers. P. nixiensis was sexuallysterile, with no oogonia formed in single-strain culture or when paired with mating type testersof heterothallic species. Sporangia of these two species were mostly nonpapillate, ovoid tobroadly ovoid, internal nested or extended proliferation. All three P. bitahaiensis isolatesformed petaloid colonies with sparse to limited aerial mycelium, but two isolates of P. nixiensis both formed fluffy aerial mycelium on PDA, CA and V8S. Preliminary pathogenicity testsusing detached leaves of six plant species, Cerasus serrulata G. Don ex London var.lannesiana (Carr.) Makino, Ilex purpurea Hassk, Magnolia denudata Desr., Syringa oblataLindl., Populus×euramericana (Dode) Guiner CL.‘zhonglin-46’ and Quercus variabilisBlume were conducted, with representative isolates of P. bitahaiensis and P. nixiensis asinocula. Visible lesions on inoculated leaves were formed with all isolates, indicating theirdefinite pathogenicity.Genetic diversity in the species based on the nucleotide polymorphism of the ITS or coxIgene was examined in the collected populations of P. gonapodyides, P. taxon PgChlamydo, P.bitahaiensis etc., and interpreted in light of the various environments (geographic regions,vegetation and climate etc.) from which they were isolated.Using primers YPh1F/Yph2R specific to Phytophthora spp and Lyse and Go PCR Reagent,the pathogen detection tests were performed with26isolates of Phytophthora,17isolates ofPythium and3isolates of Rhizoctonia solani. The results showed that only Phytophthorastrains could be detected by this method so as to distinguish them from other fungi, simplifythe DNA extraction procedure and realize rapid and accurate detection and identification. Thetwo sera against a soluble protein fraction of P. gonapodyides and P. cambivora were producedby immunizing the rabbits. Western blot analysis was conducted to dertemine the specificityand sensitivity for detecting Phytophthora spp and for diagnosing the related diseases.