大连枣疯病病原鉴定
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摘要
植原体是一类没有细胞壁、存在于植物韧皮部筛管细胞中的植物病原微生物。它可引起植物黄化、丛枝、花变叶、矮缩或花器退化等症状,导致粮食、果树、蔬菜、花卉等几百种植物致病。它所引起的病害已遍布世界各地,我国发生也很严重,其危害程度已上升为仅次于真菌、病毒病害的第三大类植物病害。
本研究根据植原体病害的症状学特点,对我国部分省区果树植原体病害进行了病害调查并采样,以基于植原体,16S rRNA,基因设计的通用引物、运用巢式PCR,对其采集到的病样进行了分子生物学检测,以查明该类病害在果树上的发生现状。对采样中检测出来的大连市枣疯病植原体,进行了基因序列分析及计算机模拟的RFLP分析,填补了辽宁省大连地区枣疯病病原鉴定的空白。主要研究结果如下:
1、以基于植原体,16S rRNA,基因设计的通用引物(R16mF2/R16mR1,及R16F2/R16R2)、运用直接及巢式,PCR,对从辽宁、河南、湖北等果树产区采集到的、100,多个疑似植原体病害的样本,进行了分子生物学检测,仅在辽宁省大连市的枣疯病样本中,出现了植原体特异的扩增条带。
2、对大连市发病较严重的营城子区,进行了枣疯病发病率和病情指数的调查。该区枣疯病平均发病率为,22%、病情指数,8.67。电镜观察感病叶片的病组织超薄切片,在其韧皮部筛管细胞中发现了典型的植原体细胞。
3、对大连枣疯株系(JWB-DL)的16S rDNA基因进行了序列测定,该序列(GenBank登录号为EF661582)为1851个核苷酸。将JWB-DL与NCBI中登录的序列进行Blast,序列比对,结果与属于16SrV,的榆树黄化植原体(GenBank登录号为AF189214)具有较高的同源性(98%),推断JWB-DL应属于同一个组即榆树黄化组(16SrV)。计算机模拟RFLP分析也表明它属于榆树黄化组(16SrV)。将JWB-DL与所属植原体组中的各亚组代表株系进行16S rDNA序列比对,通过构建的系统发育树的聚类分析,表明JWB-DL,与CLY,,植原体同属于16SrV-B亚组。JWB-DL与其它种JWB相比,部分rRNA操纵子序列有所不同,表明JWB-DL是一个独特种。JWB-DL核糖体蛋白基因rplV-rpsC基因的核酸序列与麻纤维丛枝植原体(GenBank号:EF029093)一致,而不同于其它JWB植原体,它代表了一种新的、截然不同的Candidatus Phytoplasma ziziphi植原体。
Phytoplasmas are phytopathogenic prokaryotes which lacking cell walls and inhibit phloem sieve elements in infected plants. Phytoplasma have been associated with diseases in several hundred plant species, and it may cause the plant exhibiting yellows, witches broom, phyllody and others symptoms. In recent years, this disease has the tendency of causing more loss.
According to the symptoms of phytoplasmas, we investigated diseases of phytoplasma and collected samples around the country. Suspicious samples of phytoplasma were detected and identified by nested-PCR assay, in which the universal primers for 16S rRNA of phytoplasma were used. The jujube witches’broom phytoplasma after identified were analyzed by sequencing and computer-simulated RFLP. It is the first time to identify and classify phytoplasma of JWB in Dalian, Liaoning Province. Main results and conclusion of this research are as follows:
1.Suspicious samples of phytoplasma being collected from Liaoning, Henan, Hubei and other provinces were detected and identified by nested-PCR method, in which universal primers(R16mF2/R16mR1and R16F2/R16R2) of phytoplasma were used to amplifying 16S rRNA gene. As a result, only in DNA samples from Dalian of Liaoning Province, specific bands were amplified by PCR method, and which was identified as jujube witches’-broom phytoplasma disease subsequently.
2.The incidence and disease index of JWB in Yingchengzi district in Dalian were investigated. Ultra structure of young tissues affected by phytoplasma were observed using ultra-thin section of electronic microscopy technique, the typical phytoplasmas bodies were observed in the phloem of diseased plant of jujube witches’broom.
3.The PCR-amplified DNA segment of JWB-DL was sequenced. Results show that, the length of JWB-DL is 1851bp, G+C% is 44%, GenBank Accession No. is EF661582. Homologic analysis of nucleotide is 98% between JWB-DL and elm yellows phytoplasma (GenBank Accession No.AF189214). Therefore, the jujube trees should be infected by a type of phytoplasma which is belonging to the elm yellows group (16SrV). Analyzing sequence variations of 16S rDNA among JWB-DL and other JWB strains, partial rRNA operon in JWB-DL was different from others, indicating that JWB-DL is a distinct strain. The nucleotide sequence of the JWB-DL phytoplasma rplV-rpsC (GenBank Accession No. EF661581) is more identical to that of hemp fiber witches’broom phytoplasma (GenBank Accession No.EF029093) than those in JWB phytoplasma strains described previously. To our knowledge, this is the first report of a JWB disease in northeastern China, and JWB-DL represents a new, distinct‘Candidatus Phytoplasma ziziphi’-related strain.
本研究根据植原体病害的症状学特点,对我国部分省区果树植原体病害进行了病害调查并采样,以基于植原体,16S rRNA,基因设计的通用引物、运用巢式PCR,对其采集到的病样进行了分子生物学检测,以查明该类病害在果树上的发生现状。对采样中检测出来的大连市枣疯病植原体,进行了基因序列分析及计算机模拟的RFLP分析,填补了辽宁省大连地区枣疯病病原鉴定的空白。主要研究结果如下:
1、以基于植原体,16S rRNA,基因设计的通用引物(R16mF2/R16mR1,及R16F2/R16R2)、运用直接及巢式,PCR,对从辽宁、河南、湖北等果树产区采集到的、100,多个疑似植原体病害的样本,进行了分子生物学检测,仅在辽宁省大连市的枣疯病样本中,出现了植原体特异的扩增条带。
2、对大连市发病较严重的营城子区,进行了枣疯病发病率和病情指数的调查。该区枣疯病平均发病率为,22%、病情指数,8.67。电镜观察感病叶片的病组织超薄切片,在其韧皮部筛管细胞中发现了典型的植原体细胞。
3、对大连枣疯株系(JWB-DL)的16S rDNA基因进行了序列测定,该序列(GenBank登录号为EF661582)为1851个核苷酸。将JWB-DL与NCBI中登录的序列进行Blast,序列比对,结果与属于16SrV,的榆树黄化植原体(GenBank登录号为AF189214)具有较高的同源性(98%),推断JWB-DL应属于同一个组即榆树黄化组(16SrV)。计算机模拟RFLP分析也表明它属于榆树黄化组(16SrV)。将JWB-DL与所属植原体组中的各亚组代表株系进行16S rDNA序列比对,通过构建的系统发育树的聚类分析,表明JWB-DL,与CLY,,植原体同属于16SrV-B亚组。JWB-DL与其它种JWB相比,部分rRNA操纵子序列有所不同,表明JWB-DL是一个独特种。JWB-DL核糖体蛋白基因rplV-rpsC基因的核酸序列与麻纤维丛枝植原体(GenBank号:EF029093)一致,而不同于其它JWB植原体,它代表了一种新的、截然不同的Candidatus Phytoplasma ziziphi植原体。
Phytoplasmas are phytopathogenic prokaryotes which lacking cell walls and inhibit phloem sieve elements in infected plants. Phytoplasma have been associated with diseases in several hundred plant species, and it may cause the plant exhibiting yellows, witches broom, phyllody and others symptoms. In recent years, this disease has the tendency of causing more loss.
According to the symptoms of phytoplasmas, we investigated diseases of phytoplasma and collected samples around the country. Suspicious samples of phytoplasma were detected and identified by nested-PCR assay, in which the universal primers for 16S rRNA of phytoplasma were used. The jujube witches’broom phytoplasma after identified were analyzed by sequencing and computer-simulated RFLP. It is the first time to identify and classify phytoplasma of JWB in Dalian, Liaoning Province. Main results and conclusion of this research are as follows:
1.Suspicious samples of phytoplasma being collected from Liaoning, Henan, Hubei and other provinces were detected and identified by nested-PCR method, in which universal primers(R16mF2/R16mR1and R16F2/R16R2) of phytoplasma were used to amplifying 16S rRNA gene. As a result, only in DNA samples from Dalian of Liaoning Province, specific bands were amplified by PCR method, and which was identified as jujube witches’-broom phytoplasma disease subsequently.
2.The incidence and disease index of JWB in Yingchengzi district in Dalian were investigated. Ultra structure of young tissues affected by phytoplasma were observed using ultra-thin section of electronic microscopy technique, the typical phytoplasmas bodies were observed in the phloem of diseased plant of jujube witches’broom.
3.The PCR-amplified DNA segment of JWB-DL was sequenced. Results show that, the length of JWB-DL is 1851bp, G+C% is 44%, GenBank Accession No. is EF661582. Homologic analysis of nucleotide is 98% between JWB-DL and elm yellows phytoplasma (GenBank Accession No.AF189214). Therefore, the jujube trees should be infected by a type of phytoplasma which is belonging to the elm yellows group (16SrV). Analyzing sequence variations of 16S rDNA among JWB-DL and other JWB strains, partial rRNA operon in JWB-DL was different from others, indicating that JWB-DL is a distinct strain. The nucleotide sequence of the JWB-DL phytoplasma rplV-rpsC (GenBank Accession No. EF661581) is more identical to that of hemp fiber witches’broom phytoplasma (GenBank Accession No.EF029093) than those in JWB phytoplasma strains described previously. To our knowledge, this is the first report of a JWB disease in northeastern China, and JWB-DL represents a new, distinct‘Candidatus Phytoplasma ziziphi’-related strain.
引文
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