猕猴桃溃疡病菌的田间分布及其传播规律
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摘要
猕猴桃溃疡病是由丁香假单胞菌猕猴桃致病变种引起的毁灭性细菌病害,风雨和伤口是溃疡病传播的主要影响因素和途径。明确溃疡病菌在田间感病植株上的分布及其侵染后的传播规律对其防治具有重要意义。基于此,研究主要依靠病菌分离和纯化富集后直接提取总DNA的方法,利用特异性引物ACT1/ACT2和PsaF1/PsaR2鉴定猕猴桃溃疡病菌的田间园区主要分布,使用扫描电子显微镜并结合不同时间点溃疡病的发生,确定溃疡病菌的初侵染源和潜伏部位、初侵染途径和传播规律。结果显示猕猴桃溃疡病菌在田间主要分布于地上部分的叶片、新稍、侧枝、主枝和主蔓中,表土和根系分布较低,只在根际土壤中分离出溃疡病菌;同时发现在叶片伤口处溃疡病病菌的数量远大于其他完好区域,是最主要的初侵染点。春季和秋季为猕猴桃溃疡病两次发病高峰,冬、夏季溃疡病菌于植物组织分布较少。研究的开展为下一步侵染机理的研究和田间防治奠定了基础。
The kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae is one of the most devastating disease. Wind, rain and wounds are considered as the main influencing factors and the ways of canker disease transmission. It was of great significance for the prevention and control of the distribution of the pathogen in the orchard and the spread of the infection. Based on this, in this study, we mainly relied on the method of direct extraction of total DNA after isolation and purification of bacteria, using the specific primers ACT1/ACT2 and PsaF1/PsaR2 to identify the main distribution of Pseudomonas syringae pv. actinidiae in kiwifruit; and determine the initial infection of ulcer pathogens and latent sites, the path of initial infection and transmission by scanning electron microscopy and combined with different occured time of Pseudomonas syringae pv. actinidiae. The results showed that the main pathogens of the canker were found in the main vines, main branches, collateral branches, new shoots and leaves of aboveground part. The distribution of the topsoil and roots was very low. At the same time, it was found that the number of canker disease in leaf wound was much larger than that of other intact regions, which was the primary infection point. Pseudomonas syringae pv. Actinidiae have two peaks which are Spring and Autumn, however, there are just a few in the plant tissues in summer and winter. The research were carried out to lay the foundation for the next step in the study of the mechanism of infection and field control.
The kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae is one of the most devastating disease. Wind, rain and wounds are considered as the main influencing factors and the ways of canker disease transmission. It was of great significance for the prevention and control of the distribution of the pathogen in the orchard and the spread of the infection. Based on this, in this study, we mainly relied on the method of direct extraction of total DNA after isolation and purification of bacteria, using the specific primers ACT1/ACT2 and PsaF1/PsaR2 to identify the main distribution of Pseudomonas syringae pv. actinidiae in kiwifruit; and determine the initial infection of ulcer pathogens and latent sites, the path of initial infection and transmission by scanning electron microscopy and combined with different occured time of Pseudomonas syringae pv. actinidiae. The results showed that the main pathogens of the canker were found in the main vines, main branches, collateral branches, new shoots and leaves of aboveground part. The distribution of the topsoil and roots was very low. At the same time, it was found that the number of canker disease in leaf wound was much larger than that of other intact regions, which was the primary infection point. Pseudomonas syringae pv. Actinidiae have two peaks which are Spring and Autumn, however, there are just a few in the plant tissues in summer and winter. The research were carried out to lay the foundation for the next step in the study of the mechanism of infection and field control.
引文
[1]赵利娜, 胡家勇, 叶振风, 等. 猕猴桃溃疡病病原菌的分子鉴定和致病力测定[J]. 华中农业大学学报, 2012, 31(5):604-608.
[2]GAO X, HUANG Q, ZHAO Z, et al. Studies on the infection, colonization, and movement of Pseudomonas syringae pv.actinidiaein kiwifruit tissues using a GFPuv-labeled strain[J]. PLoS One, 2016, 11(3): e0151169.
[3]秦虎强, 高小宁, 赵志博, 等. 陕西猕猴桃细菌性溃疡病田间发生动态和规律[J]. 植物保护学报, 2013, 40(3):225-230.
[4]SERIZAWA S, ICHIKAWA T, TAKIKAWA Y, et al. Occurrence of bacterial canker of kiwifruit in Japan: description of symptoms, isolation of the pathogen and screening of bactericides[J]. Japanese Journal of Phytopathology, 1989, 55(4):427-436.
[5]KOH Y J, LEE D H. Canker of kiwifruit by Pseudomonas syringae pv. Morsprunorum[J]. Korean Journal of Plant Pathology, 1992: 119-122.
[6]MAZAREI M, MOSTOFIPOUR P. First report of bacterial canker of kiwifruit in Iran[J].Plant Patholothy, 1994, 43(6):1055-1056.
[7]SCONICHINI M. Occurrence of Pseudomonas syringae pv. actinidiae on kiwifruit in Italy[J]. Plant Pathology, 1994, 43(6):1035-1038.
[8]方炎祖, 朱晓湘, 王宇道. 湖南猕猴桃病害调查研究初报[J]. 四川果树科技, 1990(1):28-29.
[9]VANNESTE J L, POLIAKOFF F, AUDSSEAU C, et al. First report of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit in France[J]. Plant Disease , 2011, 95:1311.
[10]BALESTRA G M, RENZI M, MAZZAGLIA A. First report of bacterial canker of Actinidia deliciosa caused by Pseudomonas syringae pv. actinidiae in Portugal[J]. New Disease Reports, 2010, 22(10):2044-0588.
[11]KIM G H, KIM K H, SON K I, et al. Outbreak and spread of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae biovar 3 in Korea [J]. Plant Pathology Journal, 2016, 32(6):545-551.
[12]承河元, 李瑶, 万嗣, 等.安徽省猕猴桃溃疡病菌鉴定[J]. 安徽农业大学学报, 1995, 22(3):219-228.
[13]刘绍基, 唐显富, 王忠肃.四川省苍溪猕猴桃溃疡病的发生规律[J]. 中国果树, 1996, 12(1) :25-26.
[14]梁英梅, 张星耀, 田呈明, 等.陕西省猕猴桃枝干溃疡病病原菌鉴定[J].西北林学院学报, 2000, 15(1):37-39.
[15]林尤剑, 高日霞.福建猕猴桃病害调查与鉴定[J].福建农业大学学报, 1995, 24(1):49-50.
[16]SAWADA H, TAKEUCHI T, MATAUDA I. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyl transferase gene (argK) and 16S-23S rRNA intergenic spacer sequences[J]. Applied and Environmental Microbiology, 1997, 63(1): 282-288.
[17]SAWADA H, SSUZUKI F, MATSUDA I, et al. Phylogenetic analysis of Pseudomonas syringae pathovars suggests the horizontal gene transfer of argK and the evolutionary stability of hrp gene cluster[J]. Journal of Molecular Evolution, 1999, 49(5):627-644.
[18]HAN H S, KOH Y J, HUR J S, et al. Identification and characterization of coronatine-producing Pseudomonas syringae pv. Actinidiae[J]. Journal of Microbiology and Biotechnology, 2003, 68(11): 6423-6230.
[19]VANNESTE J L. Pseudomonas syringae pv. actinidiae (Psa): a threat to the New Zealand and global kiwifruit industry [J]. New Zealand Journal of Crop & Horticultural Science, 2012, 40(4):265-267.
[20]SCORTICHINI M, MARCELLETTI S, FERRANTE P, et al. Pseudomonas syringae pv. actinidiae: a re-emerging, multi-faceted, pandemic pathogen[J]. Molecular Plant Pathology , 2012, 13:631-640.
[21]CUNTY A, POLIAKOFF F, RIVOAL C, et al. Characterization of Pseudomonas syringae pv. actinidiae (Psa) isolated from France and assignment of Psa biovar 4 to a de novo pathovar: Pseudomonas syringae pv. actinidifoliorum pv. nov.[J]. Plant Pathology, 2015, 64(3):582-596.
[22]FUJIKAWA T, SAWADA H. Genome analysis of the kiwifruit canker pathogen Pseudomonas syringae pv. actinidiae biovar 5[J]. Scientific Reports, 2016, 6:21399.
[23]LIU P, XUE S Z, HE R, et al. Pseudomonas syringae pv. actinidiae isolated from non-kiwifruit plant species in China[J]. European Journal of Plant Pathology, 2016, 145:743 754.
[24]PRENCIPE S, NARI L, VITTONE G, et al. Effect of bacterial canker caused by Pseudomonas syringae pv. actinidiae on postharvest quality and rots of kiwifruit Hayward′[J]. Postharvest Biology & Technology, 2016, 113:119-124.
[25]STEFANI E, GIOVANARDI D. Dissemination of Pseudomonas syringae pv. actinidiae through pollen and its epiphytic life on leaves and fruits[J]. Phytopathologia Mediterranea, 2011, 50(3):489-496.
[26]KOH Y, NOU I. DNA markers for identification of Pseudomonas syringae pv. Actinidiae[J]. Molecules and Cells, 2002, 13:309-314.
[27]REES-GEORGE J, VANNESTE J L, CORNISH D A, et al. Detection of Pseudomonas syringae pv. actinidiae using polymerase chain reaction (PCR) primers based on the 16S-23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions[J]. Plant Pathology, 2010, 59(3):453-464.
[28]LIU P, FANG J, CHEN K, et al. Phenylethanol promotes adhesion and biofilm formation of the antagonistic yeast Kloeckera apiculata for the control of blue mold on citrus[J]. FEMS Yeast Research, 2014, 14(4): 536-546.
[29]黄其玲, 高小宁, 赵志博, 等. GFPuv标记猕猴桃溃疡病菌的生物学特性及其在土壤、根系中的定殖[J].中国农业科学, 2013, 46(2):282-291.
[30]VANNESTE J L, GIOVANARDI D, YU J, et al. Detection of Pseudomonas syringae pv. actinidiae in kiwifruit pollen samples[J]. New Zealand Plant Protection, 2011, 64:246-251.
[2]GAO X, HUANG Q, ZHAO Z, et al. Studies on the infection, colonization, and movement of Pseudomonas syringae pv.actinidiaein kiwifruit tissues using a GFPuv-labeled strain[J]. PLoS One, 2016, 11(3): e0151169.
[3]秦虎强, 高小宁, 赵志博, 等. 陕西猕猴桃细菌性溃疡病田间发生动态和规律[J]. 植物保护学报, 2013, 40(3):225-230.
[4]SERIZAWA S, ICHIKAWA T, TAKIKAWA Y, et al. Occurrence of bacterial canker of kiwifruit in Japan: description of symptoms, isolation of the pathogen and screening of bactericides[J]. Japanese Journal of Phytopathology, 1989, 55(4):427-436.
[5]KOH Y J, LEE D H. Canker of kiwifruit by Pseudomonas syringae pv. Morsprunorum[J]. Korean Journal of Plant Pathology, 1992: 119-122.
[6]MAZAREI M, MOSTOFIPOUR P. First report of bacterial canker of kiwifruit in Iran[J].Plant Patholothy, 1994, 43(6):1055-1056.
[7]SCONICHINI M. Occurrence of Pseudomonas syringae pv. actinidiae on kiwifruit in Italy[J]. Plant Pathology, 1994, 43(6):1035-1038.
[8]方炎祖, 朱晓湘, 王宇道. 湖南猕猴桃病害调查研究初报[J]. 四川果树科技, 1990(1):28-29.
[9]VANNESTE J L, POLIAKOFF F, AUDSSEAU C, et al. First report of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit in France[J]. Plant Disease , 2011, 95:1311.
[10]BALESTRA G M, RENZI M, MAZZAGLIA A. First report of bacterial canker of Actinidia deliciosa caused by Pseudomonas syringae pv. actinidiae in Portugal[J]. New Disease Reports, 2010, 22(10):2044-0588.
[11]KIM G H, KIM K H, SON K I, et al. Outbreak and spread of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae biovar 3 in Korea [J]. Plant Pathology Journal, 2016, 32(6):545-551.
[12]承河元, 李瑶, 万嗣, 等.安徽省猕猴桃溃疡病菌鉴定[J]. 安徽农业大学学报, 1995, 22(3):219-228.
[13]刘绍基, 唐显富, 王忠肃.四川省苍溪猕猴桃溃疡病的发生规律[J]. 中国果树, 1996, 12(1) :25-26.
[14]梁英梅, 张星耀, 田呈明, 等.陕西省猕猴桃枝干溃疡病病原菌鉴定[J].西北林学院学报, 2000, 15(1):37-39.
[15]林尤剑, 高日霞.福建猕猴桃病害调查与鉴定[J].福建农业大学学报, 1995, 24(1):49-50.
[16]SAWADA H, TAKEUCHI T, MATAUDA I. Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyl transferase gene (argK) and 16S-23S rRNA intergenic spacer sequences[J]. Applied and Environmental Microbiology, 1997, 63(1): 282-288.
[17]SAWADA H, SSUZUKI F, MATSUDA I, et al. Phylogenetic analysis of Pseudomonas syringae pathovars suggests the horizontal gene transfer of argK and the evolutionary stability of hrp gene cluster[J]. Journal of Molecular Evolution, 1999, 49(5):627-644.
[18]HAN H S, KOH Y J, HUR J S, et al. Identification and characterization of coronatine-producing Pseudomonas syringae pv. Actinidiae[J]. Journal of Microbiology and Biotechnology, 2003, 68(11): 6423-6230.
[19]VANNESTE J L. Pseudomonas syringae pv. actinidiae (Psa): a threat to the New Zealand and global kiwifruit industry [J]. New Zealand Journal of Crop & Horticultural Science, 2012, 40(4):265-267.
[20]SCORTICHINI M, MARCELLETTI S, FERRANTE P, et al. Pseudomonas syringae pv. actinidiae: a re-emerging, multi-faceted, pandemic pathogen[J]. Molecular Plant Pathology , 2012, 13:631-640.
[21]CUNTY A, POLIAKOFF F, RIVOAL C, et al. Characterization of Pseudomonas syringae pv. actinidiae (Psa) isolated from France and assignment of Psa biovar 4 to a de novo pathovar: Pseudomonas syringae pv. actinidifoliorum pv. nov.[J]. Plant Pathology, 2015, 64(3):582-596.
[22]FUJIKAWA T, SAWADA H. Genome analysis of the kiwifruit canker pathogen Pseudomonas syringae pv. actinidiae biovar 5[J]. Scientific Reports, 2016, 6:21399.
[23]LIU P, XUE S Z, HE R, et al. Pseudomonas syringae pv. actinidiae isolated from non-kiwifruit plant species in China[J]. European Journal of Plant Pathology, 2016, 145:743 754.
[24]PRENCIPE S, NARI L, VITTONE G, et al. Effect of bacterial canker caused by Pseudomonas syringae pv. actinidiae on postharvest quality and rots of kiwifruit Hayward′[J]. Postharvest Biology & Technology, 2016, 113:119-124.
[25]STEFANI E, GIOVANARDI D. Dissemination of Pseudomonas syringae pv. actinidiae through pollen and its epiphytic life on leaves and fruits[J]. Phytopathologia Mediterranea, 2011, 50(3):489-496.
[26]KOH Y, NOU I. DNA markers for identification of Pseudomonas syringae pv. Actinidiae[J]. Molecules and Cells, 2002, 13:309-314.
[27]REES-GEORGE J, VANNESTE J L, CORNISH D A, et al. Detection of Pseudomonas syringae pv. actinidiae using polymerase chain reaction (PCR) primers based on the 16S-23S rDNA intertranscribed spacer region and comparison with PCR primers based on other gene regions[J]. Plant Pathology, 2010, 59(3):453-464.
[28]LIU P, FANG J, CHEN K, et al. Phenylethanol promotes adhesion and biofilm formation of the antagonistic yeast Kloeckera apiculata for the control of blue mold on citrus[J]. FEMS Yeast Research, 2014, 14(4): 536-546.
[29]黄其玲, 高小宁, 赵志博, 等. GFPuv标记猕猴桃溃疡病菌的生物学特性及其在土壤、根系中的定殖[J].中国农业科学, 2013, 46(2):282-291.
[30]VANNESTE J L, GIOVANARDI D, YU J, et al. Detection of Pseudomonas syringae pv. actinidiae in kiwifruit pollen samples[J]. New Zealand Plant Protection, 2011, 64:246-251.