中国柑橘黑斑病相关的叶点霉属真菌种类、遗传多样性和快速诊断技术研究
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摘要
柑橘黑斑病(Citrus Black Spot, CBS),也称黑星病,病原的有性态为柑橘球座菌(Guignardia citricarpa Kiely),属子囊菌门,座囊菌目；无性态为柑橘叶点霉菌(Phyllosticta citricarpa (McAlpine) van der Aa),属半知菌类,腔孢纲,球壳孢目。病菌主要危害果实,在果皮上形成病斑导致鲜果的商品性下降,甚至无法上市鲜售。CBS病菌被欧盟列为A1类严禁入境的有害生物,也是美国严禁入境的有害生物,因此是包括我国在内有黑斑病发生国家柑橘鲜果出口的重要障碍。尽管我国是柑橘的原产地之一,栽培柑橘种类繁多,黑斑病发生普遍,但对不同类型柑橘的黑斑病菌病原种类是否一致,以及病菌的生物学特性和分子变异并无研究。本研究从我国柑橘主产区几大栽培柑橘上收集黑斑病或疑似黑斑病症状的果实和叶片,分离培养获得代表性菌株若干,在此基础上开展这些代表性菌株的种类鉴定、种内遗传多样性分析,以及种特异性引物的筛选和相关的PCR鉴定体系的建立。主要研究结果如下：
1.建立了P. citriasiana的分子鉴定技术在ITS1区域设计了针对于亚洲柑橘叶点霉菌P. citriasiana的特异性上游引物Pca8,结合下游引物ITS4,建立了准确、灵敏和快速鉴定P. citriasiana的PCR体系。利用该体系可从P. citriasiana菌株中扩增出488bp的特异性条带,而不能从柑橘叶点霉菌P. citricarpa和首都叶点霉菌P. capitalensis(-一种内生菌),以及其他柑橘常发病害病原菌中扩增出条带。该体系检测P. citriasiana DNA最低为12pg,整个过程可在3小时内完成。
2.明确了中国柑橘上叶点霉属真菌种类自2007-2011年,从我国10个省市的宽皮橘(Citrus reticulata)、柚(C. maxima)、葡萄柚(C. paradisi)、甜橙(C. sinensis)以及柠檬(C. limon)等具典型或非典型黑斑病症状的果实和叶片上分离获得496个Phyllosticta菌株。通过形态学比较,可将这些菌株分为4类。选择74个代表性菌株,克隆测定其18S rDNA和28S rDNA的部分区域、核糖体DNA的两个转录间隔区及其中的5.8S亚基(internal transcribed spacer, ITS)、肌动蛋白(Actin,ACT),延长因子(translation elongation factor-1alpha, TEF1),β微管蛋白(β-tubulin, Tub)和钙调蛋白(calmodulin, Cal)基因的部分序列,单独或合并后构建系统进化树,结果发现这74个菌株分为4个明显的分支。其一为P. citricarpa,即被欧盟和美国列为检疫对象的柑橘叶点霉菌。该病菌的寄主有宽皮橘、甜橙和柠檬,但没有从柚上发现；其二为P. citriasiana,引起柚黑斑病(也被称为棕褐斑病,‘'tan spot"),寄主为沙田柚和琯溪蜜柚,但未从其他柑橘种上发现；其三是P. capitalensis,即一种内生菌,可从本研究中的各种柑橘的有症或无症材料上获得；其四是一个有别于已有的Phyllosticta spp.,本文暂时将之定为Phyllosticta citrichinaensis X.H. Wang, K.D. Hyde&H.Y. Li。该菌可从本研究中的各种柑橘的非典型黑斑病症状的材料上获得。
在这4个Phyllosticta spp中,P. citrichinaensis的分生孢子最小,附属丝最长,短杆状孢子和子囊孢子最大；P. citrichinaensis在PDA, CMA和MEA培养基表面形成具有多个环形凹槽的菌落,在OA培养基上不产生黄色素；当以葡萄糖、蔗糖、果糖、半乳糖、甘露醇、山梨醇、麦芽糖分别作为唯一碳源时,P.citrichinaensis生长较差,菌落直径小于其他三种Phyllosticta;当以尿素、酒石酸铵、天门冬氨酸、乙酸铵、(NH4)2SO4、NH4NO3、NaNO3、胰蛋白胨、脯氨酸、甘氨酸分别作为唯一氮源时,P. citrichinaensis生长最差,菌落直径均小于其他三个Phyllosticta种,且P. citrichinaensis几乎不能利用尿素、酒石酸铵、天门冬氨酸、乙酸铵、(NH4)2SO4、NH4NO3; P. citrichinaensis生长pH范围为3.0-6.0,最适为4.0,其次为3.0。在pH为3.0-5.0的PDA培养基上, P. citrichinaensis比P.citriasiana和P. citricarpa生长快,但比P. capitalensis生长慢。
3.建立巢式多重PCR鉴定柑橘上四种叶点霉属真菌的技术体系比较4种叶点霉属真菌的ITS1及18S区域序列,设计并筛选了针对P. citrichinaensis、P. citricarpa、P. capitalensis和P. citriasiana特异性上游引物Pcc1、Pc1、Pct4和Pea8(见1),并均以通用引物ITS4作为下游引物。以已知该4种菌的菌株DNA作为对照,首先使用ITS4/ITS5对供试菌株进行第一轮扩增,PCR产物稀释50-100倍后,同时加入引物对Pcc1/ITS4、Pc1/ITS4、Pct4/ITS4和Pca8/ITS4,在退火温度为60℃条件下进行第二轮PCR扩增,通过扩增条带与已知种菌株的相应扩增条带大小的比较,确定待测样品所属的叶点霉属菌的种类。该体系的灵敏度达到2ag的DNA。与传统PCR相比其灵敏度至少提高106倍。不仅可用于试验室培养的待测菌株的鉴定,还可以用于果实疑似黑斑病的病斑快速鉴定。
4.明确了中国柑橘叶点霉属真菌遗传多样性通过对影响PCR反应的主要成分模板DNA、Mg2+、dNTPs、TaqDNA聚合酶及引物等条件的优化,建立了适合柑橘叶点霉菌的ISSR-PCR反应体系。利用该体系从100条引物中筛选出11条扩增条带稳定、清晰、重复性好和多态性高的引物。利用筛选的11条引物和优化的ISSR-PCR体系,对供试的110个来自柑橘的叶点霉属菌株进行扩增,共扩增出197个条带,多态性条带为194个,多态率为98.5%,证明中国柑橘上叶点霉属真菌遗传多样性丰富。利用NTSYS-pc2.10软件对扩增的条带进行分析并构建UPGMA聚类图,显示中国柑橘叶点霉属真菌可以分为四个分支,每分支对应着P. citricarpa, P.citriasiana, P. capitalensis和P. citrichinaensis。P. citricarpa的遗传分化与寄主种类相关,而与地理来源无关。来自甜橙和柠檬的菌株与来自宽皮柑橘的菌株分布在不同的分支中,而来自砂糖橘的菌株又聚为一个分支。而P. citriasiana, P. capitalensis和P. citrichinaensis(?)中群内的遗传变异与地理距离和寄主均无相关性。
Citrus Black Spot (CBS) caused by Guignardia citricarpa (Dothideales, Loculoascomycetes in Ascomycota) Kiely. The anamorph of this pathogen is Phyllosticta citricarpa (McAlpine) van der Aa, belongs to Sphaeropsidales, Coelomycetes in Fungi Imperfecti. CBS is a common disease in China, mainly causes diverse spots on the rind of Citrus fruit, results in the fruits become unsightly and unsuitable for the fresh fruit market. The pathogen of CBS P. citricarpa is an Al quarantine pathogen for countries of the European Union, and it is also regulated as a quarantine pest in the USA. The export of citrus fruits from China and other countries where CBS is present is heavily regulated. As one of the origin places, there are many cultivated Citrus species and varieties in China, and many Citrus species are susceptible to CBS. However, there is no research to demonstrate whether the pathogen on different Citrus species is belonged to the same species. The biological and molecular characteristics of the pathogen from China are remaining unknown. In the study, Phyllosticta strains were obtained from infected fruits and leaves of the majority of the cultivated Citrus species and varieties across the main citrus producing regions of China, and identified using the methods of morphological and molecular biology. A nested-multiple PCR technology for diagnosing the4Phyllosticta species from citrus was established. Genetic diversity of4Phyllosticta species were further studied, the results were described as follows.
1. Establishment of identification system of P. citriasiana By comparing the differences of the internal transcribed spacer region of Phyllosticta spp., specific upstream primer Pca8for P. citriasiana was designed. By combining the downstream primer ITS4, an accurate, sensitive and fast identification system of P. citriasiana was established. By using the established PCR identification system, diagnostic fragments of488bp were consistently amplified from P. citriasiana, but were never amplified from P. citricarpa and endophytic P. capitalensis, nor from the common pathogens for citrus. This method is highly sensitive (12pg of genomic DNA) and rapid (with in3 hour) for detection of P. citriasiana.
2. Clarification of Phyllosticta species associated with citrus diseases in China Samples of citrus fruits and leaves with black spot or black spot-like symptoms were collected from cultivated Citrus species including mandarins, oranges, pomeloes and lemons in the ten citrus-producing provinces of China during2007to2011. Four hundred and ninety six Phyllosticta strains were isolated from these samples. These isolates were divided into four morphological groups, and74representative strains of these four groups were selected for phylogenetic analysis. Analyses inferred from the sequences of the internal transcribed spacer region of nuclear ribosomal DNA (ITS), partial actin gene (ACT), partial translation elongation factor1-alpha (TEF1),β-tubulin (Tub) and Calmodulin (Cal) showed these representative Phyllosticta isolates clustered in four distinct clades corresponding to three known, and an undescribed species. Phylogenetic trees based on individual gene and combined genes resolved the same results. Clade-I corresponded to P. citricarpa, the pathogen of black spot (the species regulated by EU and USA). It was found on oranges, mandarins and lemons, but was not found on pomeloes; Clade-Ⅱ corresponded to P. citriasiana, the pathogen of pomeloe black spot (also named as tan spot), was only found from pomeloes, but not from the other Citrus; Clade-Ⅲ corresponded to P. capitalensis, the endophytic fungus with a wide host range, was found on all citrus fruits and leaves with black spot or black spot-like symptoms in this study; Clade-IV corresponded to Phyllosticta citrichinaensis X. H. Wang, K. D. Hyde&H.Y. Li, sp. nov., a novel species described in this study, obtained from all four Citrus species with atypical citrus black spot symptoms.
Among four Phyllosticta species, P. citrichinaensis has the smallest conidia, longest mucoid apical appendage, largest spermatia and ascospores. P. citrichinaensis could be distinguished from P. citricarpa, P. citriasiana and P. capitalensis by producing colonies with multiple annular ridges on PDA, cornmeal agar (CMA), and malt extract agar (MEA), and by not producing yellow pigment in OA. On media using sucrose, fructose, galactose, mannitol, sorbitol, glucose or maltose (1%w/v) as a sole carbon source, P. citrichinaensis grew slower than the other three Phyllosticta species from Citrus. On media using urea, tartaric acid ammonium, aspartic acid, CH3COONH4,(NH4)2SO4, NH4NO3, NaNO3, tryptone, proline or glycine as a sole nitrogen source, P. citrichinaensis grew slowest than the other three Phyllosticta species from Citrus. P. citrichinaensis almost could not grow on media using urea, tartaric acid ammonium, aspartic acid, CH3COONH4,(NH4)2SO4or NH4NO3as sole nitrogen source. P. citrichinaensis could grow at pH value of3.0-6.0, but grew the best at pH4.0, followed by3.0. At pH value of3.0-5.0, P. citrichinaensis grew faster than P. citricarpa and P. citriasiana, but slower than P. capitalensis.
3. Establishment of nested-multiple PCR identification system of four Phyllosticta species By comparing the sequence differences of the first internal transcribed spacer region and18s region of four Phyllosticta species associated with citrus, species-specific upstream primers Pccl, Pcl, Pct4and Pca8(above1) for P. citrichinaensis, P. citricarpa, P. capitalensis and P. citriasiana were designed. By combination with ITS4as downstream primer, an individual PCR diagnosis of each species was established and optimized. For diagnosis of a suspect Phyllosticta culture or a lesion that was suspected to be related to Phyllostica, the DNA of the culture or lesion was used as template, ITS4/ITS5was used to the first round amplification, the amplified product (diluted by50-100fold) was then used as template, and using mixture of primers Pccl/ITS4, Pcl/ITS4, Pct4/ITS4and Pca8/ITS4was used for the second round PCR amplification at annealing temperature of60℃. Meanwhile, the DNA of standard isolate belonging to P. citrichinaensis, P. citricarpa, P. capitalensis and P. citriasiana respectively was used as positive control. The exact Phyllosticta species was identified by comparison the diagnosis bands with standard isolate of each species after electrophoresis. The nested-multiple PCR could detect the fungal DNA as low as2ag and increased the detection sensitivity at least106-fold compared to the traditional PCR method. The established nested-multiple PCR system in the study could not only identify Phyllosticta spp. culture, but also could identify suspect Phyllosticta spp.-associated lesion on citrus rind.
4. Genetic diversity of Phyllosticta associated with citrus By optimization the concentration of DNA templates, Mg2+, dNTPs, TaqDNA polymerase and primers, a suitable ISSR-PCR system for Phyllosticta was established.11primers amplified stable, clear, repeatable, high polymorphism productions were screened from100primers. Eleven primers and the optimized ISSR-PCR system were used to investigate the genetic diversity of110isolates of Phyllosticta, and197bands were amplified, of which194polymorphism bands. The polymorphism rate was98.5%, showing rich genetic diversity of Phyllosticta. NTSYS-pc2.10was used to build UPGMA dendrogram. It showed that the isolates of Phyllosticta were divided into four clades, corresponded to P. citricarpa, Pcitriasiana, P. capitalensis and P. citrichinaensis. Genetic differentiation of P. citricarpa was host-related, not geographical distance-related. Isolates collected from sweet oranges and lemons were distinct from isolates from mandarins, they gathered in different clades. Isolates collected from Citrus reticulata cv. Shatangju were different from isolates from other mandarins, and those isolates gathered in another clade. The genetic variation of P. citriasiana, P. capitalensis and P. citrichinaensis was not related to host and geographical distance.
1.建立了P. citriasiana的分子鉴定技术在ITS1区域设计了针对于亚洲柑橘叶点霉菌P. citriasiana的特异性上游引物Pca8,结合下游引物ITS4,建立了准确、灵敏和快速鉴定P. citriasiana的PCR体系。利用该体系可从P. citriasiana菌株中扩增出488bp的特异性条带,而不能从柑橘叶点霉菌P. citricarpa和首都叶点霉菌P. capitalensis(-一种内生菌),以及其他柑橘常发病害病原菌中扩增出条带。该体系检测P. citriasiana DNA最低为12pg,整个过程可在3小时内完成。
2.明确了中国柑橘上叶点霉属真菌种类自2007-2011年,从我国10个省市的宽皮橘(Citrus reticulata)、柚(C. maxima)、葡萄柚(C. paradisi)、甜橙(C. sinensis)以及柠檬(C. limon)等具典型或非典型黑斑病症状的果实和叶片上分离获得496个Phyllosticta菌株。通过形态学比较,可将这些菌株分为4类。选择74个代表性菌株,克隆测定其18S rDNA和28S rDNA的部分区域、核糖体DNA的两个转录间隔区及其中的5.8S亚基(internal transcribed spacer, ITS)、肌动蛋白(Actin,ACT),延长因子(translation elongation factor-1alpha, TEF1),β微管蛋白(β-tubulin, Tub)和钙调蛋白(calmodulin, Cal)基因的部分序列,单独或合并后构建系统进化树,结果发现这74个菌株分为4个明显的分支。其一为P. citricarpa,即被欧盟和美国列为检疫对象的柑橘叶点霉菌。该病菌的寄主有宽皮橘、甜橙和柠檬,但没有从柚上发现；其二为P. citriasiana,引起柚黑斑病(也被称为棕褐斑病,‘'tan spot"),寄主为沙田柚和琯溪蜜柚,但未从其他柑橘种上发现；其三是P. capitalensis,即一种内生菌,可从本研究中的各种柑橘的有症或无症材料上获得；其四是一个有别于已有的Phyllosticta spp.,本文暂时将之定为Phyllosticta citrichinaensis X.H. Wang, K.D. Hyde&H.Y. Li。该菌可从本研究中的各种柑橘的非典型黑斑病症状的材料上获得。
在这4个Phyllosticta spp中,P. citrichinaensis的分生孢子最小,附属丝最长,短杆状孢子和子囊孢子最大；P. citrichinaensis在PDA, CMA和MEA培养基表面形成具有多个环形凹槽的菌落,在OA培养基上不产生黄色素；当以葡萄糖、蔗糖、果糖、半乳糖、甘露醇、山梨醇、麦芽糖分别作为唯一碳源时,P.citrichinaensis生长较差,菌落直径小于其他三种Phyllosticta;当以尿素、酒石酸铵、天门冬氨酸、乙酸铵、(NH4)2SO4、NH4NO3、NaNO3、胰蛋白胨、脯氨酸、甘氨酸分别作为唯一氮源时,P. citrichinaensis生长最差,菌落直径均小于其他三个Phyllosticta种,且P. citrichinaensis几乎不能利用尿素、酒石酸铵、天门冬氨酸、乙酸铵、(NH4)2SO4、NH4NO3; P. citrichinaensis生长pH范围为3.0-6.0,最适为4.0,其次为3.0。在pH为3.0-5.0的PDA培养基上, P. citrichinaensis比P.citriasiana和P. citricarpa生长快,但比P. capitalensis生长慢。
3.建立巢式多重PCR鉴定柑橘上四种叶点霉属真菌的技术体系比较4种叶点霉属真菌的ITS1及18S区域序列,设计并筛选了针对P. citrichinaensis、P. citricarpa、P. capitalensis和P. citriasiana特异性上游引物Pcc1、Pc1、Pct4和Pea8(见1),并均以通用引物ITS4作为下游引物。以已知该4种菌的菌株DNA作为对照,首先使用ITS4/ITS5对供试菌株进行第一轮扩增,PCR产物稀释50-100倍后,同时加入引物对Pcc1/ITS4、Pc1/ITS4、Pct4/ITS4和Pca8/ITS4,在退火温度为60℃条件下进行第二轮PCR扩增,通过扩增条带与已知种菌株的相应扩增条带大小的比较,确定待测样品所属的叶点霉属菌的种类。该体系的灵敏度达到2ag的DNA。与传统PCR相比其灵敏度至少提高106倍。不仅可用于试验室培养的待测菌株的鉴定,还可以用于果实疑似黑斑病的病斑快速鉴定。
4.明确了中国柑橘叶点霉属真菌遗传多样性通过对影响PCR反应的主要成分模板DNA、Mg2+、dNTPs、TaqDNA聚合酶及引物等条件的优化,建立了适合柑橘叶点霉菌的ISSR-PCR反应体系。利用该体系从100条引物中筛选出11条扩增条带稳定、清晰、重复性好和多态性高的引物。利用筛选的11条引物和优化的ISSR-PCR体系,对供试的110个来自柑橘的叶点霉属菌株进行扩增,共扩增出197个条带,多态性条带为194个,多态率为98.5%,证明中国柑橘上叶点霉属真菌遗传多样性丰富。利用NTSYS-pc2.10软件对扩增的条带进行分析并构建UPGMA聚类图,显示中国柑橘叶点霉属真菌可以分为四个分支,每分支对应着P. citricarpa, P.citriasiana, P. capitalensis和P. citrichinaensis。P. citricarpa的遗传分化与寄主种类相关,而与地理来源无关。来自甜橙和柠檬的菌株与来自宽皮柑橘的菌株分布在不同的分支中,而来自砂糖橘的菌株又聚为一个分支。而P. citriasiana, P. capitalensis和P. citrichinaensis(?)中群内的遗传变异与地理距离和寄主均无相关性。
Citrus Black Spot (CBS) caused by Guignardia citricarpa (Dothideales, Loculoascomycetes in Ascomycota) Kiely. The anamorph of this pathogen is Phyllosticta citricarpa (McAlpine) van der Aa, belongs to Sphaeropsidales, Coelomycetes in Fungi Imperfecti. CBS is a common disease in China, mainly causes diverse spots on the rind of Citrus fruit, results in the fruits become unsightly and unsuitable for the fresh fruit market. The pathogen of CBS P. citricarpa is an Al quarantine pathogen for countries of the European Union, and it is also regulated as a quarantine pest in the USA. The export of citrus fruits from China and other countries where CBS is present is heavily regulated. As one of the origin places, there are many cultivated Citrus species and varieties in China, and many Citrus species are susceptible to CBS. However, there is no research to demonstrate whether the pathogen on different Citrus species is belonged to the same species. The biological and molecular characteristics of the pathogen from China are remaining unknown. In the study, Phyllosticta strains were obtained from infected fruits and leaves of the majority of the cultivated Citrus species and varieties across the main citrus producing regions of China, and identified using the methods of morphological and molecular biology. A nested-multiple PCR technology for diagnosing the4Phyllosticta species from citrus was established. Genetic diversity of4Phyllosticta species were further studied, the results were described as follows.
1. Establishment of identification system of P. citriasiana By comparing the differences of the internal transcribed spacer region of Phyllosticta spp., specific upstream primer Pca8for P. citriasiana was designed. By combining the downstream primer ITS4, an accurate, sensitive and fast identification system of P. citriasiana was established. By using the established PCR identification system, diagnostic fragments of488bp were consistently amplified from P. citriasiana, but were never amplified from P. citricarpa and endophytic P. capitalensis, nor from the common pathogens for citrus. This method is highly sensitive (12pg of genomic DNA) and rapid (with in3 hour) for detection of P. citriasiana.
2. Clarification of Phyllosticta species associated with citrus diseases in China Samples of citrus fruits and leaves with black spot or black spot-like symptoms were collected from cultivated Citrus species including mandarins, oranges, pomeloes and lemons in the ten citrus-producing provinces of China during2007to2011. Four hundred and ninety six Phyllosticta strains were isolated from these samples. These isolates were divided into four morphological groups, and74representative strains of these four groups were selected for phylogenetic analysis. Analyses inferred from the sequences of the internal transcribed spacer region of nuclear ribosomal DNA (ITS), partial actin gene (ACT), partial translation elongation factor1-alpha (TEF1),β-tubulin (Tub) and Calmodulin (Cal) showed these representative Phyllosticta isolates clustered in four distinct clades corresponding to three known, and an undescribed species. Phylogenetic trees based on individual gene and combined genes resolved the same results. Clade-I corresponded to P. citricarpa, the pathogen of black spot (the species regulated by EU and USA). It was found on oranges, mandarins and lemons, but was not found on pomeloes; Clade-Ⅱ corresponded to P. citriasiana, the pathogen of pomeloe black spot (also named as tan spot), was only found from pomeloes, but not from the other Citrus; Clade-Ⅲ corresponded to P. capitalensis, the endophytic fungus with a wide host range, was found on all citrus fruits and leaves with black spot or black spot-like symptoms in this study; Clade-IV corresponded to Phyllosticta citrichinaensis X. H. Wang, K. D. Hyde&H.Y. Li, sp. nov., a novel species described in this study, obtained from all four Citrus species with atypical citrus black spot symptoms.
Among four Phyllosticta species, P. citrichinaensis has the smallest conidia, longest mucoid apical appendage, largest spermatia and ascospores. P. citrichinaensis could be distinguished from P. citricarpa, P. citriasiana and P. capitalensis by producing colonies with multiple annular ridges on PDA, cornmeal agar (CMA), and malt extract agar (MEA), and by not producing yellow pigment in OA. On media using sucrose, fructose, galactose, mannitol, sorbitol, glucose or maltose (1%w/v) as a sole carbon source, P. citrichinaensis grew slower than the other three Phyllosticta species from Citrus. On media using urea, tartaric acid ammonium, aspartic acid, CH3COONH4,(NH4)2SO4, NH4NO3, NaNO3, tryptone, proline or glycine as a sole nitrogen source, P. citrichinaensis grew slowest than the other three Phyllosticta species from Citrus. P. citrichinaensis almost could not grow on media using urea, tartaric acid ammonium, aspartic acid, CH3COONH4,(NH4)2SO4or NH4NO3as sole nitrogen source. P. citrichinaensis could grow at pH value of3.0-6.0, but grew the best at pH4.0, followed by3.0. At pH value of3.0-5.0, P. citrichinaensis grew faster than P. citricarpa and P. citriasiana, but slower than P. capitalensis.
3. Establishment of nested-multiple PCR identification system of four Phyllosticta species By comparing the sequence differences of the first internal transcribed spacer region and18s region of four Phyllosticta species associated with citrus, species-specific upstream primers Pccl, Pcl, Pct4and Pca8(above1) for P. citrichinaensis, P. citricarpa, P. capitalensis and P. citriasiana were designed. By combination with ITS4as downstream primer, an individual PCR diagnosis of each species was established and optimized. For diagnosis of a suspect Phyllosticta culture or a lesion that was suspected to be related to Phyllostica, the DNA of the culture or lesion was used as template, ITS4/ITS5was used to the first round amplification, the amplified product (diluted by50-100fold) was then used as template, and using mixture of primers Pccl/ITS4, Pcl/ITS4, Pct4/ITS4and Pca8/ITS4was used for the second round PCR amplification at annealing temperature of60℃. Meanwhile, the DNA of standard isolate belonging to P. citrichinaensis, P. citricarpa, P. capitalensis and P. citriasiana respectively was used as positive control. The exact Phyllosticta species was identified by comparison the diagnosis bands with standard isolate of each species after electrophoresis. The nested-multiple PCR could detect the fungal DNA as low as2ag and increased the detection sensitivity at least106-fold compared to the traditional PCR method. The established nested-multiple PCR system in the study could not only identify Phyllosticta spp. culture, but also could identify suspect Phyllosticta spp.-associated lesion on citrus rind.
4. Genetic diversity of Phyllosticta associated with citrus By optimization the concentration of DNA templates, Mg2+, dNTPs, TaqDNA polymerase and primers, a suitable ISSR-PCR system for Phyllosticta was established.11primers amplified stable, clear, repeatable, high polymorphism productions were screened from100primers. Eleven primers and the optimized ISSR-PCR system were used to investigate the genetic diversity of110isolates of Phyllosticta, and197bands were amplified, of which194polymorphism bands. The polymorphism rate was98.5%, showing rich genetic diversity of Phyllosticta. NTSYS-pc2.10was used to build UPGMA dendrogram. It showed that the isolates of Phyllosticta were divided into four clades, corresponded to P. citricarpa, Pcitriasiana, P. capitalensis and P. citrichinaensis. Genetic differentiation of P. citricarpa was host-related, not geographical distance-related. Isolates collected from sweet oranges and lemons were distinct from isolates from mandarins, they gathered in different clades. Isolates collected from Citrus reticulata cv. Shatangju were different from isolates from other mandarins, and those isolates gathered in another clade. The genetic variation of P. citriasiana, P. capitalensis and P. citrichinaensis was not related to host and geographical distance.
引文
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