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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。
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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。
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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%。
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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。
图 1 基于最大似然法构建的疫霉菌clade 6系统发育树
Figure 1. Maximum likelihood phylogenetic tree established for Phytophthora species in clade 6
图 2 基于最大似然法构建的疫霉菌clade 2系统发育树
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)。
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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。
图 3 4种疫霉菌在胡萝卜琼脂培养基上生长的菌落形态
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 4a,4b)。
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 4c,4d)。
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。
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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。
表 1 野苹果林疫霉菌分布信息表
Table 1. Distribution of Phytophthora species in wild apple forests
种类
Species时间
Time地点
Site方法
Method数量
Quantity湖沼疫霉
P. lacustris2016 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. gonapodyides2016 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. plurivora2016 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 -
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。
新疆野苹果林中疫霉菌种类鉴定及致病性研究
Identification and Pathogenicity of Phytophthora Species from Wild Apple Forests in Xinjiang, China
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摘要:
目的 通过研究新源县和巩留县野苹果林中疫霉菌的种类和致病性差异,为探讨新疆野苹果林衰亡原因提供基本资料,为野苹果林病害防治提供科学依据。 方法 采用疑似疫霉病害样品采集、土壤诱捕、林间溪流诱捕等方法对新疆野苹果林中的疫霉菌进行了调查采样和监测诱捕,并利用选择性培养基分离获得疫霉纯菌株。通过形态学观察和rDNA-ITS序列分析相结合的方法对所获得的疫霉菌株进行分类鉴定,并通过离体叶片接种试验对所鉴定的疫霉菌进行致病性测定。 结果 依据形态学特征和序列分析,鉴定得到5个种共计88株疫霉,分别为65株湖沼疫霉Phytophthora lacustris Brasier,Cacciola,Nechwatal,Jung & Bakonyi、16株节水霉状疫霉Phytophthora gonapodyides(Petersen)Buisman、3株多寄主疫霉Phytophthora plurivora T. Jung & T. I. Burgess、2株聚疫霉Phytophthora gregata T. Jung,M. J. C. Stukely & T. I. Burgess和2株Phytophthora sp. 1。将前4种疫霉接种离体野苹果叶片后,发现它们均产生病斑,其中湖沼疫霉在离体叶片上产生的病症最为明显。 结论 新疆野苹果林中具有多种疫霉菌,且所鉴定的4种疫霉菌均对野苹果叶片有一定的致病性。 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. -
Key words:
- wild apple forests
- / Phytophthora species
- / identification
- / pathogenicity
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表 1 野苹果林疫霉菌分布信息表
Table 1. Distribution of Phytophthora species in wild apple forests
种类
Species时间
Time地点
Site方法
Method数量
Quantity湖沼疫霉
P. lacustris2016 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. gonapodyides2016 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. plurivora2016 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 -
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