• 中国中文核心期刊
  • 中国科学引文数据库(CSCD)核心库来源期刊
  • 中国科技论文统计源期刊(CJCR)
  • 第二届国家期刊奖提名奖
Volume 32 Issue 6
Dec.  2019
Article Contents
Turn off MathJax

Citation:

Identification and Pathogenicity of Phytophthora Species from Wild Apple Forests in Xinjiang, China

  • Corresponding author: HUAI Wen-xia, huaiwx@caf.ac.cn
  • Received Date: 2019-01-21
    Accepted Date: 2019-04-03
  • Objective To survey the diseases caused by Phytophthora in wild apple forests in Xinyuan county and Gongliu county of Xinjiang Uygur Autonomous Region aiming at isolating and identifying Phytophthora species, evaluating the pathogenicity of these species, and providing information for finding out the causes of the decline of wild apple forest. Method Phytophthora species were surveyed by the method of collecting samples of suspected Phytophthora diseases, baiting techniques from streams and soils in the wild apple forest in Xinyuan county and Gongliu county on the north slope of the Tianshan Mountains in Xinjiang. The pure isolates of Phytophthora spp. were obtained through selective medium and subculturing. Then these isolates were classified and identified by morphological observation combined with rDNA-ITS sequence analysis, and the pathogenicity of the identified Phytophthora species was determined by in vitro leaf inoculation test. Result Based on morphological characteristics and sequence analysis, a total of 88 Phytophthora isolates were identified from five species, including 65 isolates of Phytophthora lacustris Brasier, Cacciola, Nechwatal, Jung & Bakonyi, 16 isolates of Phytophthora gonapodyides (Petersen) Buisman, 3 isolates of Phytophthora plurivora T. Jung & T. I. Burgess, 2 isolates of Phytophthora gregata T. Jung, M. J. C. Stukely & T. I. Burgess and 2 isolates of Phytophthora sp. 1.The former four known Phytophthora species inoculated on the leaves of wild apples were found to produce lesions except for the control, and the most obvious symptoms were formed with the isolate of P. lacustris on the leaves. Conclusion The relatively richness of Phytophthora species were examined in Xinjing wild apple forest. And visible lesions on inoculated wild apple leaves were formed with four Phytophthora species, indicating their definite pathogenicity.
  • 加载中
  • [1]

    Cavalier-Smith T. Only six kingdoms of life[J]. Proceedings of the Royal Society B, 2004, 271:1251-1262. doi: 10.1098/rspb.2004.2705
    [2] 郑小波.疫霉菌及其研究技术[M].北京:中国农业出版社, 1997.

    [3]

    Erwin D C, Ribeiro O K. Phytophthora diseases worldwide[J]. St. Paul, MN:American Phytopathological Society, 1996, 90(6):1092.
    [4]

    Brar S, Tabima J F, Mc Dougal R L, et al. Genetic diversity of Phytophthora pluvialis, a pathogen of conifers, in New Zealand and the west coast of the United States of America[J]. Plant Pathology, 2018, 67(5):1131-1139. doi: 10.1111/ppa.12812
    [5]

    Goheen E M, Hansen E M, Kanaskie A, et al. Sudden oak death caused by Phytophthorara morum in Oregon[J]. Plant Disease, 2007, 68(4):441-441.
    [6]

    Rizzo D M, Garbelotto M, Hansen E M. Phytophthorara morum:integrative research and management of an emerging pathogen in California and Oregon forests[J]. Annual Review of Phytopathology, 2005, 43(1):309-335. doi: 10.1146/annurev.phyto.42.040803.140418
    [7]

    Rizzo D M, Garbelotto M, Davidson J M, et al. Phytophthorara morum as the cause of extensive mortality of Quercus spp. and Lithocarpus densiflorus in California[J]. Plant Disease, 2007, 86(3):205-214.
    [8]

    Reeser P, Sutton W, Hansen E. Phytophthora species in tanoak trees, canopy-drip, soil, and streams in the sudden oak death epidemic area of south-western Oregon, USA[J]. New Zealand Journal of Forestry Science, 2011, 41S:S65-S73.
    [9]

    Jung T, Burgess T I. Re-evaluation of Phytophthora citricola isolates from multiple woody hosts in Europe and North America reveals a new species Phytophthora plurivora sp. nov.[J]. Persoonia, 2009, 22(5):95-110.
    [10] 林培钧, 崔乃然.天山野果林资源-伊犁野果林综合研究[M].北京:中国林业出版社, 2000.

    [11] 于少帅, 赵文霞, 姚艳霞, 等.新疆野苹果枯枝症状级别与水杨酸含量、胸径关系研究[J].林业科学研究, 2019, 32(2):111-116.

    [12] 阎国荣, 许正.天山野生果树主要病害及其分布[J].干旱区研究, 2001, 18(2):47-49.

    [13] 刘爱华, 张新平, 温俊宝, 等.天山野苹果林苹果小吉丁与苹果腐烂病复合危害研究[J].新疆农业科学, 2014, 51(12):2240-2244.

    [14] 牛程旺, 王静茹, 朱小琼, 等.新疆野果林褐腐病菌的种类[J].菌物学报, 2016, 35(12):1514-1525.

    [15] 王智勇, 张彦龙, 杨忠岐, 等.苹小吉丁(鞘翅目:吉丁甲科)幼虫龄数的测定[J].林业科学研究, 2013, 26(6):786-789.

    [16] 孔婷婷, 刘爱华, 岳朝阳, 等.天山野苹果林苹果小吉丁虫生真菌调查[J].北方园艺, 2017(1):138-141.

    [17] 薛煜, 刘雪峰, 项存悌.林病研究方法[M].哈尔滨:东北林业大学出版社, 2013.

    [18]

    Nechwatal J, Bakonyi J, Cacciola S O, et al. The morphology, behaviour and molecular phylogeny of Phytophthora taxon Salixsoil and its redesignation as Phytophthora lacustris sp. nov.[J]. Plant Pathology, 2012, 62(2):355-369.
    [19]

    Brasier C M, Hamm P B, Hansen E M. Cultural characters, protein patterns and unusual mating behaviour of Phytophthora gonapodyides isolates from Britain and North America[J]. Mycological Research, 1993, 97(11):1287-1298. doi: 10.1016/S0953-7562(09)80160-3
    [20]

    Jung T, Stukely M J, Hardy G E, et al. Multiple new Phytophthora species from its clade 6 associated with natural ecosystems in Australia:evolutionary and ecological implications[J]. Persoonia, 2011, 26(1):13-39. doi: 10.3767/003158511X557577
    [21] 淮稳霞.中国西南地区杜鹃-栎树林中疫霉菌的分离鉴定及快速检测技术研究[D].北京, 中国林业科学研究院博士学位论文, 2013.

    [22]

    White T J, Bruns T D, Lee S B, et al. Amplification and direct sequencing of fungal ribosomal RNA Genes for phylogenetics//PCR-Protocols and Applications-A Laboratory Manual[M]. San Diego, CA: Academic Press, 1990.
    [23]

    Altschul S F. Basic local alignment search tool[J]. Journal of Molecular Biology, 1990, 215(3):403-410.
    [24]

    Katoh K, Rozewicki J, Yamada K D. MAFFT online service:multiple sequence alignment, interactive sequence choice and visualization[J]. Briefings in Bioinformatics, 2017, (4):1-7.
    [25]

    Tamura K, Peterson D, Peterson N, et al. MEGA5:molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[J]. Molecular Biology and Evolution, 2011, 28(10):2731-2739. doi: 10.1093/molbev/msr121
    [26]

    Maddison W P, Maddison D R. Mesquite: a modular system for evolutionary analysis[CP]. Version 3.5.1, 2018. http://www.mesquiteproject.org.
    [27]

    De D I, Vercauteren A, Speybroeck N, et al. Effect of host factors on the susceptibility of Rhododendron to Phytophthorara morum[J]. Plant Pathology, 2010, 59(2):301-312. doi: 10.1111/j.1365-3059.2009.02212.x
    [28]

    Huai W X, Tian G, Hansen E M, et al. Identification of Phytophthora species baited and isolated from soil and streams in northwestern Yunnan province, China[J]. Forest Pathology, 2013, 43(2):87-103. doi: 10.1111/efp.12015
    [29] 李雯雯.中国典型生境中森林疫霉的调查和分类研究[D].北京, 中国林业科学研究院硕士学位论文, 2018.

    [30]

    Balci Y, Halmschlager E. Incidence of Phytophthora species in oak forests in Austria and their possible involvement in oak decline[J]. Forest Pathology, 2003, 33(3):157-174. doi: 10.1046/j.1439-0329.2003.00318.x
    [31]

    Cooke D E L, Drenth A, Duncan J M, et al. A molecular phylogeny of Phytophthora and related oomycetes[J]. Fungal Genetics and Biology, 2000, 30(1):17-32. doi: 10.1006/fgbi.2000.1202
    [32]

    Blair J E, Coffey M D, Park S Y, et al. A multi-locus phylogeny for Phytophthora utilizing markers derived from complete genome sequences[J]. Fungal Genetics and Biology, 2008, 45(3):266-277. doi: 10.1016/j.fgb.2007.10.010
    [33]

    Martin F N, Blair J E, Coffey M D. A combined mitochondrial and nuclear multilocus phylogeny of the genus Phytophthora[J]. Fungal Genetics and Biology, 2014, 66(3):19-32.
    [34]

    Yang X, Tyler B M, Hong C. An expanded phylogeny for the genus Phytophthora[J]. IMA Fungus, 2017, 8(2):355-384. doi: 10.5598/imafungus.2017.08.02.09
    [35]

    Liu A H, Shang J, Zhang J W, et al. Canker and fine-root loss of Malus sieversii(Ldb.)Roem. caused by Phytophthora plurivora in Xinjiang Province in China[J]. Forest Pathology, 2018, e12462. https://doi.org/10.1111/efp.1246
    [36]

    Burgess T I, Simamora A V, White D, et al. New species from Phytophthora Clade 6a:evidence for recent radiation[J]. Persoonia, 2018, 41:1-17. doi: 10.3767/persoonia.2018.41.01
    [37]

    Brasier C M, Cooke D E L, Duncan J M, et al. Multiple new phenotypic taxa from trees and riparian ecosystems in Phytophthoragona podyides-P. megasperma ITS Clade 6, which tend to be high-temperature tolerant and either inbreeding or sterile[J]. Mycological Research, 2003, 107(3):277-290. doi: 10.1017/S095375620300738X
    [38]

    Reeser P W, Sutton W, Hansen E M, et al. Phytophthora species in forest streams in Oregon and Alaska[J]. Mycologia, 2011, 103(1):22-35. doi: 10.3852/10-013
    [39]

    Cacciola S O, Williams N A, Cooke D E L, et al. Molecular identification and detection of Phytophthora species on some important Mediterranean plants including sweet chestnut[J]. Forest Snow and Landscape Research, 2001, 76(3):351-356.
    [40]

    Orlikowski L B, Ptaszek M, Rodziewicz A, et al. Phytophthora root and collar rot of mature fraxinus excelsior in forest stands in Poland and Denmark[J]. Forest Pathology, 2011, 41(6):510-519. doi: 10.1111/j.1439-0329.2011.00714.x
    [41]

    Nechwatal J, Mendgen K. Widespread detection of Phytophthora taxon Salixsoil in the littoral zone of lake constance, Germany[J]. European Journal of Plant Pathology, 2006, 114(3):261-264. doi: 10.1007/s10658-005-5593-y
    [42]

    Jung T, Nechwatal J. Phytophthora gallica sp. nov. a new species from rhizosphere soil of declining oak and reed stands in France and Germany[J]. Mycological Research, 2008, 112(10):1195-1205. doi: 10.1016/j.mycres.2008.04.007
    [43]

    Corcobado T, Cubera E, Pérezsierra A, et al. First report of Phytophthora gonapodyides involved in the decline of Quercus ilex in xeric conditions in Spain[J]. New Disease Report, 2010, 22:33. doi: 10.5197/j.2044-0588.2010.022.033
    [44]

    Robideau G P, Cock D A W A M, Coffey M D, et al. DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer[J]. Molecular Ecology Resources, 2011, 11(6):1002-1011. doi: 10.1111/j.1755-0998.2011.03041.x
  • 加载中
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Figures(6) / Tables(1)

Article views(4874) PDF downloads(60) Cited by()

Proportional views

Identification and Pathogenicity of Phytophthora Species from Wild Apple Forests in Xinjiang, China

    Corresponding author: HUAI Wen-xia, huaiwx@caf.ac.cn
  • 1. The Key Laboratory of National Forestry and Grassland Administration on Forest Protection, Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing 100091, China
  • 2. The Bureau of Natural Resources of Emin County, Xinjiang Uygur Autonomous Region, Emin 834600, China

Abstract:  Objective To survey the diseases caused by Phytophthora in wild apple forests in Xinyuan county and Gongliu county of Xinjiang Uygur Autonomous Region aiming at isolating and identifying Phytophthora species, evaluating the pathogenicity of these species, and providing information for finding out the causes of the decline of wild apple forest. Method Phytophthora species were surveyed by the method of collecting samples of suspected Phytophthora diseases, baiting techniques from streams and soils in the wild apple forest in Xinyuan county and Gongliu county on the north slope of the Tianshan Mountains in Xinjiang. The pure isolates of Phytophthora spp. were obtained through selective medium and subculturing. Then these isolates were classified and identified by morphological observation combined with rDNA-ITS sequence analysis, and the pathogenicity of the identified Phytophthora species was determined by in vitro leaf inoculation test. Result Based on morphological characteristics and sequence analysis, a total of 88 Phytophthora isolates were identified from five species, including 65 isolates of Phytophthora lacustris Brasier, Cacciola, Nechwatal, Jung & Bakonyi, 16 isolates of Phytophthora gonapodyides (Petersen) Buisman, 3 isolates of Phytophthora plurivora T. Jung & T. I. Burgess, 2 isolates of Phytophthora gregata T. Jung, M. J. C. Stukely & T. I. Burgess and 2 isolates of Phytophthora sp. 1.The former four known Phytophthora species inoculated on the leaves of wild apples were found to produce lesions except for the control, and the most obvious symptoms were formed with the isolate of P. lacustris on the leaves. Conclusion The relatively richness of Phytophthora species were examined in Xinjing wild apple forest. And visible lesions on inoculated wild apple leaves were formed with four Phytophthora species, indicating their definite pathogenicity.

  • 疫霉菌(Phytophthora de Bary)是一类广泛分布于自然界中的重要植物病原菌,可寄生于果树、林木、农作物等多种植物,引起枝枯、溃疡、腐烂、猝倒,甚至是植株死亡[1-3]。在全球目前已被正式描述的150余种疫霉菌中,有些可危及重要林木和观赏植物,对森林健康构成巨大威胁,严重影响森林景观[4]。例如,樟疫霉(Phytophthora cinnamomi Rands)严重威胁澳大利亚桉树森林生态系统;多枝疫霉(Phytophthorara ramorum Werres, De Cock & E. M. Hansen)已被包括我国在内的多个国家列入了进境植物检疫性有害生物名单,该病菌造成美国西海岸栎类等多种树木大量枯萎死亡[5-7];多寄主疫霉(Phytophthorara plurivora T. Jung & T.I. Burgess)可以侵染欧洲冷杉(Abies alba Mill.)、欧洲赤杨(Alnus glutinosa(L.)Gaertn.)、灰桤木(Alnus incana(L.) Moench)等多种植物,常寄生于这些植物根部或茎基部,引起根腐或茎基溃疡,造成植物衰退、枯萎甚至死亡[8-9]

    新疆野苹果(Malus sieversii (Ledeb.) Roem.)是天山野果林的主要组成部分,是中国濒危二级重点保护植物,同时也是现代栽培苹果(Malus domestica (Borkh.))的祖先,主要分布在新源县和巩留县[10-11]。近些年来,由于过度放牧等人类活动和苹小吉丁、野苹果腐烂病等病虫危害多种因素,造成新疆野苹果林出现大面积的衰退和死亡,致使野苹果林面积减少50%左右[10-15]。通过前期调查,我们发现受害野苹果树除了苹小吉丁和腐烂病菌典型危害症状外,有些还出现顶稍枯萎、枝干溃疡、茎基腐烂等症状,其受害状与疫霉菌危害症状较为相似。因此,为了明确新疆野苹果林中疫霉菌的种类,探讨其与野苹果树衰退和死亡的关系,本研究自2016年起对我国新疆伊犁地区新源县和巩留县野苹果林中的疑似疫霉病害进行调查,通过rDNA-ITS基因序列分析和形态学观察对从病害症状组织分离、土壤诱捕和林间溪流诱捕获得的疫霉菌进行分类鉴定,并利用离体叶片对所鉴定的疫霉菌进行致病性初步测定,以期为后续研究其对新疆野苹果林衰退的影响提供理论基础。

1.   材料与方法
  • 采样地点位于新疆伊犁哈萨克自治州的新源县阿勒玛勒乡野果林改良场(43°22′N, 83°36′E)和巩留县库尔德宁镇交勒赛(43°13′N, 82°46′E),属于野苹果林集中分布区。新源阿勒玛勒乡无霜期为145~165 d,年均降水量500~580 mm, 年均气温7.7℃,1月平均气温-8.1℃,7月平均气温20.4℃,冬季逆温现象明显;巩留县库尔德宁镇交勒赛无霜期为150 d左右,年均降水量200~780 mm,年均气温7.4℃,属山地高寒气候[15-16]

    2016和2017年6—9月,在上述两样地内开展调查和采样,将样地内出现叶片和枝梢枯萎、枝干溃疡等疑似疫霉危害症状的野苹果叶片、枝条和树皮等组织装入无菌袋带回实验室。同时,随机选取衰弱的野苹果树,挖取20~30 cm深处的根际土壤200 g置于无菌密封袋,加入少量无菌水,浸泡2~3片健康的野苹果叶片或白蜡树叶片作为诱饵来诱捕土壤中的疫霉菌。此外,在两个样地中各选择一溪流平缓处设置诱捕监测点(分别记作XY和GL),用野苹果或白蜡树的健康叶片做诱饵浸泡在林间溪水中,每隔5~7 d取出更换一次叶片。原叶片观察是否产生病斑。具有病斑的叶片直接进行分离培养;若未产生病斑,则将其放入无菌密封袋18℃保湿培养数日,待诱饵叶片产生病斑后,再进行分离培养。

  • 将采集的病样以及诱饵叶片使用组织分离法[17]分离后,放置于VARP+选择培养基上20℃培养3~5 d,待长出菌落后,将培养皿放置在显微镜下观察,并挑取疑似疫霉和腐霉的菌丝转入VARP选择培养基上纯化,最后转入玉米培养基(CMA)观察形态和保存。

  • 将分离获得的疫霉纯菌株转入胡萝卜汁培养基(CA)平板上,在20℃条件下培养5~15 d后,观察菌落形态,并在显微镜下观察和拍照。菌株在V8汁固体培养基(V8S)培养4~7 d后,用打孔器在菌落边缘打出菌饼3~5块放入培养皿中,并加入适量无菌水,于20℃、正常日光条件下培养24 h后,置于显微镜下观察其游动孢子囊、藏卵器、雄器和卵孢子等形态结构特征并拍照[9, 18-21]。单株培养不产生藏卵器、雄器和卵孢子的疫霉菌,可通过直接配对法用参考菌株栗疫霉黑水病菌(Phytophthora cambivora (Pe-tri) Buisman)A1型和A2型(美国俄勒冈州立大学Everett Hansen教授实验室提供)来诱导其有性器官的形成。

  • 所获得的疫霉菌株于马铃薯葡萄糖琼脂(PDA)培养基上20℃培养10 d后,刮取少量菌丝,采用CTAB法进行总DNA的提取。随后利用引物ITS4和ITS6对rDNA-ITS序列进行PCR扩增[22]。扩增产物经琼脂糖凝胶电泳检测后,送北京美吉桑格生物医药科技有限公司进行双向测序。测序结果利用Staden Package 1.6.0 (MRC, Cambridge, England)进行序列拼接。将来自NCBI网站[23]的参考序列与已测菌株序列基于最大似然法构建系统发育树[24-26]

  • 通过离体叶片培养法对所鉴定的疫霉菌进行致病性测定[26],每个菌株设3个重复,以接种无菌琼脂块作为对照。于25℃培养7 d后,采用十字交叉法测量病斑大小并拍照。随后用VARP培养基分离病健交界处,鉴定是否为所接种疫霉菌。

2.   结果与分析
  • 2016年,从新源和巩留两个样地共采集叶片及枝梢枯萎、枝干溃疡等疑似疫霉危害症状的野苹果树叶片、枝条、树皮和果实等组织样品90份,但均未分离到疫霉菌;采集土壤样品123份,经过诱捕分离获得6株疫霉菌;采集溪流诱捕样品10份,分离纯化后得到4株疫霉菌。2017年,采集土壤样品63份,获得2株疫霉菌;采集溪流诱捕样品40份,得到76株疫霉菌。

  • 所获得的88株疫霉菌的完整ITS序列长度范围为761 bp至820 bp,这些序列均已提交至GenBank(登录号:MK027402-MK027461)。序列比对结果显示,3个疫霉菌株的ITS序列与多寄主疫霉参考菌株的ITS序列(FJ665225)完全相同,一致性为100%;65个疫霉菌株的ITS序列与参考菌株湖沼疫霉(Phytophthora lacustris Brasier, Cacciola, Nechw., T. Jung & Bakonyi)的序列(HQ012956)一致性为99.76~%100%;16个疫霉菌株的ITS序列与节水霉状疫霉(Phytophthora gonapodyides (H.E. Petersen) Buisman(JN547620))序列一致性为99.63%~99.76%;2个疫霉菌株的ITS序列与聚疫霉Phytophthora gregata T. Jung, Stukely & T.I. Burgess参考菌株的ITS序列(HQ012941)最为相近,一致性为99.51%;另外,有2株疫霉(CYP31和CYP74)可能属于同一个新种,它们的ITS序列与疫霉Phytophthora sp. BR333(HQ643355)最为相近,一致性为99.76%~99.88%。

  • 将供试菌株的ITS序列与各个Clade中的69株疫霉参考序列一起基于最大似然法构建系统发育树,其中支持率≥0.70的值被标注在树上,外类群为弗吉尼亚疫霉(Phytophthora virginiana Xiao Yang & C.X. Hong(KC295544))。结果显示:3个菌株位于Clade 2,85个菌株位于Clade 6。在ITS Clade 2中(图 1),有3株菌株与多寄主疫霉聚为一支,支持率为0.93。而在ITS Clade 6中(图 2),有65株菌株与湖沼疫霉聚为一支,支持率为0.84;16株与节水霉状疫霉聚为一支,支持率为0.95;2株与聚疫霉聚在一支,支持率为0.94,其也与P. taxon raspberry聚为一支,但目前聚疫霉和P. taxon raspberry被认为属于同一个种。这些结果支持将所有菌株鉴定为5种疫霉。

    Figure 1.  Maximum likelihood phylogenetic tree established for Phytophthora species in clade 6

    Figure 2.  Maximum likelihood phylogenetic tree established for Phytophthora species in clade 2

    此外,ITS序列分析结果显示Clade 6中疑有1个未描述的疫霉新种Phytophthora sp.。该种的两个菌株(CYP31和CYP74)与未描述疫霉种Phytophthora sp. BR333(HQ643355)聚为一支,支持率为0.79,且在CoxI基因序列上存在20~24个碱基差异(未发表)。该疑似新种与密西西比疫霉(Phytophthora mississippiae X. Yang, W. E. Copes, and C. X. Hong.)在ITS序列上存在5~6个碱基差异,而在CoxI基因片段序列上存在39~41个碱基差异(相关数据未展示)。

  • 经ITS序列分析鉴定的疫霉菌株,通过菌落形态和游动孢子囊等形态学特征观察,与参考文献中描述的疫霉种类进行了比对和确认。所鉴定的4种疫霉P. lacustris(菌株号CYP4)、P. gonapodyides(菌株号CYP52)、P. plurivora(菌株号CYPXYS77.1)和P. gregata(菌株号CYP9)的代表菌株在CA培养基上生长15 d,菌落形态如图 3所示。

    Figure 3.  Colony morphology of four isolates of Phytophthora specieson Carrot Agarmedia

    湖沼疫霉培养在CA培养基上呈花瓣状,少量气生菌丝分布于菌落中央,而边缘菌丝生长在培养基内部(图 3a)。该疫霉通常在蒸馏水中产生大量游动孢子囊;孢子囊多呈卵形或倒梨形,无乳突,不脱落,大小为27.1~57.5 μm ×14.8~41.1 μm,平均大小40.0 μm ×25.7 μm,长宽比为1.40~1.83(平均长宽比1.56),延伸式或巢式内层出;异宗配合,供试菌株与栗疫霉黑水病菌的A1型、A2型菌株进行配对时并未产生有性生殖器官,表现为自身不育(图 4a4b)。

    Figure 4.  Morphological structures of four strains of Phytophthora

    节水霉状疫霉于CA培养基呈玫瑰花状(图 3b);孢子囊为卵形或倒梨形,无乳突,不脱落,大小为39.8~80.9 μm× 25.8~48.0 μm,平均大小58.8 μm×36.9 μm,长宽比为1.54~1.69(平均长宽比1.59),内层出;异宗配合,代表菌株表现为自身不育,其与栗疫霉黑水病菌的A1型、A2型菌株进行配对并未产生有性生殖器官(图 4c4d)。

    多寄主疫霉在CA培养基上呈放射状(图 3c);游动孢子囊着生于不分枝的孢囊梗顶端,偶尔也会间生或侧生,有时在节点处生成近球形菌丝膨大体;孢子囊呈椭圆形、卵形、柠檬形或不规则形状。半乳突,不脱落,偶见双或多乳突,大小为38.2~79.7 μm× 29.6~55.5 μm,平均大小56.0 μm ×38.3 μm,长宽比为1.29~1.44(平均1.46);同宗配合,藏卵器为球形,壁表面光滑,雄器侧生,大小为20.4~36.0 μm,平均大小26.1 μm(图 4e~4g)。

    聚疫霉在CA培养基上呈稀疏绒毛状,气生菌丝较少(图 3d);孢子囊呈长卵形、柠檬形、梨形或倒梨形,不脱落,内层出或外层出,大小为29.9~74.8 μm ×22.5~46.1 μm,平均大小56.1 μm× 34.2 μm,长宽比为1.33~1.62(平均长宽比1.52),孢囊梗在节点处形成近椭圆形的菌丝膨大体,并且在黑暗条件下产生大量链式菌丝膨大体(图 4h~4l);供试两菌株(CYP6和CYP9)与栗疫霉黑水病菌的A1型、A2型菌株进行配对时并未产生有性生殖器官,表现为自身不育。

  • 本研究共鉴定出5种疫霉菌,其中湖沼疫霉为优势种。几种疫霉菌的分布信息见表 1

    种类
    Species
    时间
    Time
    地点
    Site
    方法
    Method
    数量
    Quantity
    湖沼疫霉
    P. lacustris
    2016 巩留 土壤诱捕 2
    2017 新源 溪流诱捕 23
    巩留 溪流诱捕 40
    节水霉状疫霉
    P. gonapodyides
    2016 新源 土壤诱捕 3
    2017 新源 溪流诱捕 3
    巩留 溪流诱捕 10
    多寄主疫霉
    P. plurivora
    2016 新源 土壤诱捕 1
    2017 新源 土壤诱捕 2
    聚疫霉P. gregata 2016 巩留 溪流诱捕 2
    Phytophthora sp. 2016 新源 溪流诱捕 1
    巩留 溪流诱捕 1

    Table 1.  Distribution of Phytophthora species in wild apple forests

  • 采用离体叶片培养法,用野苹果叶片对湖沼疫霉、节水霉状疫霉、多寄主疫霉和聚疫霉4种疫霉的代表菌株(湖沼疫霉:CYP4;节水霉状疫霉:CYP52;多寄主疫霉:CYPXYS77.1;聚疫霉:CYP9)进行了致病性测定。结果显示:接种7 d后,上述疫霉菌均侵染野苹果叶片并产生明显深褐色病斑,而所有健康对照的野苹果叶片上均无病斑形成(图 5)。利用十字交叉法测量病斑大小(见图 6),发现湖沼疫霉的菌株对野苹果离体叶片的致病力最强,病斑大小为2.3 cm;其次是节水霉状疫霉和多寄主疫霉,而聚疫霉致病力最弱,病斑大小仅为0.5 cm。从表现症状的离体叶片病斑上均能再次分离得到所接种的疫霉菌。

    Figure 5.  Symptoms on detached leaves of wild apples after inoculation with four isolates of Phytophthora species

    Figure 6.  The average size of lesions on infected leaves of wild apples 7 d after inoculation with four isolates of Phytophthora species

3.   讨论
  • 本研究在两个野苹果林样地的土壤和溪流中共分离到5种疫霉菌,分别是湖沼疫霉、节水霉状疫霉、多寄主疫霉和聚疫霉以及疑似新种Phytophthora sp.。其中前4种疫霉为广布种,在我国西南地区、河北雾灵山以及欧洲、美洲及澳洲均有发现[9, 20, 28-30]。以前的学者依据ITS、COX等基因序列将疫霉菌划分为10个Clades[31-34]。在本研究分离鉴定出的5种疫霉菌中,只有多寄主疫霉属于Clade 2,它是2009年新描述的疫霉种。这种疫霉可侵染北美红栎(Quercus rubra L.)欧洲桤木(Alnus glutinosa(L.)Gaertn.)和挪威槭(Acer platanoides L.)等植物,引起多种森林植物衰退和枯萎;该疫霉在欧洲、北美、澳洲均有发现报道,分布相当广泛[8, 20]。刘爱华等[35]从新疆野苹果树发病树皮组织和根际土壤样品中也分离获得到多寄主疫霉,并通过接种野苹果离体茎段和根部对该疫霉进行致病性测定,证明该疫霉菌可侵染野苹果的根部,使其根部坏死,但并未发现其引起野苹果茎干溃疡。本研究中所获得的两株多寄主疫霉在致病性测定中对野苹果的离体叶片也表现出较强的致病性,说明多寄主疫霉可能与新疆野苹果林的衰退和枯萎有关,但其对野外野苹果树的致病性强弱尚待更深入的研究。

    本研究中所鉴定的另外4种疫霉菌,即湖沼疫霉、节水霉状疫霉、聚疫霉和疑似新种Phytophthora sp.均属于Clade 6内的第二个subclade。自2000年以来,Clade 6中新增疫霉菌达20种,从而使其成为疫霉属中最大的Clade[36]。其中,湖沼疫霉于1972年首次从英国洪涝后的旱柳(Salix matsudana Koidz.)病根上分离得到,Brasier等[19]依据形态学特征将其鉴定为节水霉状疫霉,随后又发现其不同于节水霉状疫霉而将其描述为P. taxon Salixsoil[37]。该疫霉广泛存在与洪涝环境中[8, 38],不仅曾在意大利洪涝后的桃树(Amygdalus persica L.)根部病斑上分离获得过[39],还曾从欧洲白蜡树(Fraxinus excelsior L.)的根茎腐烂部分离获得过[40]。Nechwatal等[18]对湖沼疫霉进行致病性测定的结果显示,该疫霉对桤木属(Alnus Mill.)、李属(Prunus L.)和柳属(Salix L.)的一些树种具有致病性。疫霉学者们普遍认为,湖沼疫霉在潮湿的环境中是一种重要的根部致病菌[40-42]。尽管本研究中仅用野苹果离体叶片对该疫霉菌的致病性进行了初步测定,但结果也显示该疫霉菌的致病性较强。另外,湖沼疫霉是我们调查中从土壤和林间溪流中诱捕分离所获菌株数量最多的一种疫霉菌。

    与湖沼疫霉同属疫霉Clade 6b中的另外两种疫霉菌——节水霉状疫霉和聚疫霉,被认为属于弱致病菌。其中,节水霉状疫霉能引起植物的根部或果实腐烂,其寄主涉及11个科13种植物,但一般认为该疫霉所引起的病害属于危害较小的次要病害[3, 43]。而聚疫霉是2011年描述的疫霉种,分离自澳大利亚西部濒死植物的根际土,最初被鉴定为P. taxon Raspberry和P. sp. 7[20]。本研究所鉴定的几种疫霉菌对植物均有不同程度的致病性,暗示这些疫霉可能与新疆野苹果林的衰退有关。但这几种疫霉菌能否在野外直接引起野苹果树发病,或者其在野苹果林衰退中所起的作用大小还有待于进一步的调查和研究。

    目前,在疫霉属Clade 6中仅正式描述的种就有28个,另外还有很多未命名的种和杂交种[33]。本研究所发现的疑似新种Phytophthora sp.的两个菌株(CYP31和CYP74)在ITS序列上与Robideau等[44]未描述疫霉种P. sp. BR333(HQ643355)仅存在1或2个碱基差异,CoxI基因片段序列上与P. sp. BR333存在20~24个碱基差异;而与密西西比疫霉(KF112850)存在5~6个ITS序列的碱基差异和39~41个CoxI基因片段的碱基差异(相关数据未展示)。另外,通过形态学的初步观察,我们发现该疫霉的孢子囊形态也主要为卵圆形、椭圆形和倒梨形,内层出,但未观察到卵孢子、藏卵器等生殖结构。将来,可以通过诱捕获得该疑似新种的更多菌株,对这个新种进行详细鉴定和描述。

    本研究利用了疑似疫霉病害症状组织直接分离法、林间溪流诱捕法和土壤诱捕法3种方法,对新疆野苹果林中的疫霉菌进行了调查,结果显示,林间溪流诱捕法的效果最佳,获得的菌株占总菌株数的92.05%。除了本研究中所用的3种方法外,森林疫霉菌的诱捕方法还有很多种,例如刘爱华等[35]利用未成熟的青苹果从新疆野苹果树发病树皮组织和根际土壤样品中诱捕获得了多寄主疫霉。采用多种分离诱捕方法,选用不同诱饵,在不同季节重复采样等都能分离获得更多的疫霉菌株或种类,而使研究结果更具说服力。因此,在今后的调查监测中还将尝试使用林冠穿透水诱捕法、溪流水过滤法等多种方法,对新疆野苹果林中的疫霉菌开展更加系统和全面的调查和研究。

4.   结论
  • 本研究首次对新疆新源和巩留两样地野苹果林中的疫霉菌种类进行调查和研究,经ITS基因序列分析和形态学观察发现新疆野苹果林中具有多种疫霉菌,并且所鉴定出的4种疫霉菌对野苹果叶片具有不同程度的致病性,为研究疫霉菌对新疆野苹果林衰退的影响提供了理论基础。

Reference (44)

Catalog

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return