【作者】 淮稳霞；

【导师】 田国忠；

【作者基本信息】 中国林业科学研究院 ， 森林保护， 2013， 博士

【摘要】 疫霉菌（Phytophthora de Bary）是一类世界性分布的重要植物病原菌，该属的大多数种都对寄主植物具有严重的危害性和破坏性。近年来，鉴于一些疫霉入侵种（如栎树猝死病菌P. ramorum、康沃尔疫霉P. kernoviae和P. alni）对生态和经济造成的重要影响，疫霉菌也受到人们的日益关注。世界公认的疫霉菌种类从20世纪90年代中期的58个种，增加到目前的116种，其中最近10年里在全球森林生态系统中发现的疫霉属新种就达20余种。中国目前报道的疫霉菌总共有28种，其中多为农作物的病原真菌。迄今为止，我国对由疫霉菌引起的林木病害的研究报道很少，缺乏对森林疫霉病菌种类、分布、寄主范围、危害程度等的调查，更缺少对森林疫霉检测鉴定方法及防控对策的研究。自2005年至2011年，本研究利用诱捕分离技术对我国西南部分地区杜鹃-栎树林中的疫霉菌进行了监测和调查，从林间溪流和土壤中共诱捕分离得到疫霉纯菌株275株，并依据rDNA-ITS等基因序列分析和形态学特征，将全部菌株鉴定为9个种，分别是P.borealis E Hansen, Sutton&Reeser、P. cryptogea Pethybridge&Lafferty、P. gonapodyides(Petersen) Buisman）、P. gregata T. Jung, M.J.C. Stukely&T. Burgess、P. plurivora T. Jung&T.I. Burgess、 P. lacustris Brasier, Cacciola, Nechwatal, Jung&Bakonyi和P. taxonPgChlamydo以及2个新种。其中，隐地疫霉P. cryptogea和节水霉状疫霉P. gonapodyides2个为国内疫霉原有纪录种，P. borealis、P. gregata、P. plurivora、P. lacustris和P. taxonPgChlamydo为5个国内疫霉新记录种。其中，2005-2010年期间在云南迪庆地区的杜鹃-栎树林中诱捕分离得到了73株8种疫霉，分别为P. taxon PgChlamydo43株、P. lacustris9株、P. plurivora6株、P. gonapodyides4株、P. gregata3株、P. cryptogea1株以及两个新种7株；分布广泛的疫霉P. taxonPgChlamydo，为该地区的优势种。而2006-2011年，从四川甘孜和西藏林芝诱捕分离得到的疫霉菌共为202株5种，分别是P. gonapodyides为94株、P. borealis47株、P. taxonPgChlamydo33、P. plurivora17株和P. lacustris11株；其中P. gonapodyides和P. borealis来自于四川甘孜2个监测点和西藏2个监测点，是四川甘孜和西藏林芝地区的优势种。分离自云南迪庆杜鹃-栎树林溪流和土壤中的两个新种均为异宗配合，属于ITS Clade6。尽管这两个疫霉新种在形态学上同Clade6中的一些疫霉菌比较相似，但通过ITS和Cox I基因序列的系统发育分析，却与这些疫霉存在着显著的不同。根据这两种疫霉的首次发现地点，将其定名Phytophthora bitahaiensis Huai, Zhao, Tian&Hansen sp. nov.和Phytophthora nixiensis Huai, Zhao, Tian&Hansen sp. nov.，并对其分别进行了描述。在交配型配对时，疫霉P. bitahaiensis可同交配型为A2的其他疫霉菌株配对形成球形或近球形的藏卵器和围生的雄器，而P. nixiensis则表现为自身不育。这两种疫霉的孢子囊多为卵形、宽卵形，均无乳突，巢式或延伸式内层出；在PDA、CA和V8S培养基上，P.bitahaiensis的气生菌丝较少，菌落多呈花瓣状，而P. nixiensis在3种培养基上产生的气生菌丝都非常丰富；利用离体叶片接种培养法，分别用紫丁香（Syringa oblata Lindl.）、日本晚樱（Cerasus serrulata G. Don ex London var. lannesiana (Carr.) Makino）、中林46杨（Populus xeuramericana (Dode)Guiner CL.‘zhonglin-46’）、白玉兰（Magnolia denudataDesr.）、冬青（Ilex purpurea Hassk）、栓皮栎（Quercus variabilis Blume）6种植物的叶片，对P. bitahaiensis和P. nixiensis的代表菌株的致病性进行初步测定，结果表明两种疫霉对这些植物叶片均具有一定的致病性。诱捕分离自我国云南迪庆、四川甘孜和西藏林芝地区杜鹃-栎树林中的P.gonapodyides、P. taxon PgChlamydo及新种P. bitahaiensis等疫霉种内的不同菌株间都存在较多的ITS或coxI基因序列核苷酸多态性位点。这些疫霉的遗传多样性明显与其所分离地区的地理、植被、气候等环境的多样性有关。在疫霉菌快速检测技术研究中，利用疫霉属特异性引物YPh1F/Yph2R和Lyse and GoPCR Reagent，通过直接PCR法对26个疫霉菌株、17个腐霉菌株和3立枯丝核菌菌株进行了检测试验，结果显示引物YPh1F/Yph2R特异性较强，可以将疫霉菌同其他真菌区分来；该法省去了提取DNA的繁琐步骤，可以实现疫霉菌的快速准确检测和鉴定。另外，利用节水霉状疫霉（P. gonapodyides）和栗黑水疫霉（P. cambivora）菌丝可溶性蛋白作为免疫抗原分别注射家兔制备了两种多克隆抗血清，并用Western blot分析了抗血清的专一性和灵敏度及用于疫霉菌或疫霉病害的野外、口岸等现场诊断和调查的可行性。更多还原

【Abstract】 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.更多还原