蓝莓枝干溃疡病病原鉴定及品种抗病性研究
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摘要
为明确引起蓝莓枝干溃疡病的病原菌,为该病害的田间诊断及综合防治提供参考,采用组织分离法和单孢分离法对引起蓝莓枝干溃疡病的病原菌进行了分离纯化,利用柯赫氏法则、菌落形态特征及核糖体DNA内转录间隔区序列(rDNA-ITS)对菌株进行了分类鉴定,采用活体接种病原菌和病斑长度测量的方法检测了5种蓝莓品种的抗病性。从病枝分离得到疑似病原菌菌株15株(JA1～JA8、JB1～JB7),菌株JA4和JB2均可导致蓝莓枝干溃疡病。rDNA-ITS序列分析和菌落形态学鉴定发现,JA4为拟茎点霉菌(Phomopsis sp.),JB2为葡萄座腔菌(Botryosphaeria dothidea)。接种试验表明,布里吉塔、蓝丰和蓝美人对枝干溃疡病的抗性没有显著差异。拟茎点霉菌(Phomopsis sp.)和葡萄座腔菌(Botryosphaeria dothidea)均可以引起蓝莓枝干溃疡病,拟茎点霉菌在春天条件适宜情况下可由花芽侵入,而葡萄座腔菌必须从伤口侵染,两种病原菌也可以产生复合侵染。蓝莓品种布里吉塔、蓝美人和蓝丰等品种抗性强;夏普蓝易受病原菌侵染,抗病性差。
To verify the pathogens that lead to stem canker on blueberry, and provide a theoretical basis for field diagnosis and integrated control of the disease, the pathogens causing stem canker of blueberry were isolated and purified by tissue separation and single spore separation. Identification of pathogen were carried out using Koch's postulates, morphological characteristics and inter transcriptional spacer of ribosome DNA(rDNA-ITS). In vivo inoculation of pathogen and spot length measurement were performed to detect the resistance of five blueberry cultivars to stem canker. The test results showed that both strains JA4 and JB2 could cause stem canker on blueberries. ITS sequences analyses and colony identification revealed that strains JA4 and JB2 were Phomopsis sp. and Botryosphaeria dothidea, respectively. The inoculation results indicated that there was no significant difference in the resistance to stem canker among Brigitta, Bluecrop and Bluebelle. Both Phomopsis sp. and Botryosphaeria dothidea could cause stem canker on blueberry. Phomopsis sp. could be invaded by flower buds, and Botryosphaeria dothidea could be infected by the wound. The blueberry cultivars Brigitta, Bluecrop and Bluebelle showed higher resistance than Sharpblue, which was susceptible to infection by the pathogen and showed weaker resistance than other four cultivars.
To verify the pathogens that lead to stem canker on blueberry, and provide a theoretical basis for field diagnosis and integrated control of the disease, the pathogens causing stem canker of blueberry were isolated and purified by tissue separation and single spore separation. Identification of pathogen were carried out using Koch's postulates, morphological characteristics and inter transcriptional spacer of ribosome DNA(rDNA-ITS). In vivo inoculation of pathogen and spot length measurement were performed to detect the resistance of five blueberry cultivars to stem canker. The test results showed that both strains JA4 and JB2 could cause stem canker on blueberries. ITS sequences analyses and colony identification revealed that strains JA4 and JB2 were Phomopsis sp. and Botryosphaeria dothidea, respectively. The inoculation results indicated that there was no significant difference in the resistance to stem canker among Brigitta, Bluecrop and Bluebelle. Both Phomopsis sp. and Botryosphaeria dothidea could cause stem canker on blueberry. Phomopsis sp. could be invaded by flower buds, and Botryosphaeria dothidea could be infected by the wound. The blueberry cultivars Brigitta, Bluecrop and Bluebelle showed higher resistance than Sharpblue, which was susceptible to infection by the pathogen and showed weaker resistance than other four cultivars.
引文
[1] 徐成楠, 周宗山, 迟福梅,等. 越橘葡萄座腔菌枝枯病的病原菌鉴定[J]. 园艺学报, 2013, 40(2): 231-236.XU C N, ZHOU Z S, CHI F M, et al. Research on the pathogen causing Botryosphaeria stem blight on blueberry[J]. Acta Horticulturae Sinica, 2013, 40(2): 231-236. (in Chinese with English abstract)
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[3] XU C N, ZHANG H J, ZHOU Z S, et al. Identification and distribution of Botryosphaeriaceae species associated with blueberry stem blight in China[J]. European Journal of Plant Pathology, 2015, 143(4): 737-752.
[4] 余磊, 唐旭兵, 赵建荣,等. 云南蓝莓枝干溃疡病病原菌鉴定及rDNA-ITS序列分析[J]. 云南农业大学学报, 2014, 29(1): 27-31.YU L, TANG X B, ZHAO J R, et al. Identification and the internal transcribed spacer regions analysis of the pathogen causing stem canker on blueberry in Yunman[J]. Journal of Yunnan Agricultural University, 2014, 29(1):27-31. (in Chinese with English abstract)
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[6] ESPINOZA J G, BRICE?O E X, CHáVEZ E R,et al. Neofusicoccum spp. associated with stem canker and dieback of blueberry in Chile[J]. Plant Disease, 2009, 93(11): 1187-1194.
[7] 方中达. 植病研究方法[M]. 3版. 北京: 中国农业出版社,2007.
[8] 凌丹燕, 李永强, 路梅, 等. 短柄樱桃干腐病病原菌的鉴定及其生物学特性[J]. 浙江师范大学学报(自然科学版), 2016, 39(2): 200-206.LING D Y, LI Y Q, LU M, et al. Identification and biological characteristic researches of rot pathogen isolated from Chinese cherry(Prunus pseudocerasus)[J].Journal of Zhejiang Normal University (Natural Sciences), 2016, 39(2): 200-206. (in Chinese with English abstract)
[9] KUMAR S, STECHER G, TAMURA K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[J]. Molecular Biology and Evolution, 2016, 33(7): 1870-1874.
[10] SLIPPERS B, CROUS P W, DENMAN S,et al. Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea[J]. Mycologia, 2004, 96(1): 83-101.
[11] ZHAO X, ZHANG G, LI B, et al. Seasonal dynamics of Botryosphaeria dothidea infections and symptom development on apple fruits and shoots in China[J]. European Journal of Plant Pathology, 2016, 146(3): 507-518.
[12] WANG F, ZHAO L, LI G,et al. Identification and characterization of Botryosphaeria spp. causing gummosis of peach trees in Hubei Province in central China[J]. Plant Disease, 2011, 95(11): 1378-1384.
[13] 马瑜, 柯杨, 王琴, 等. 核桃溃疡病症状及其病原菌鉴定[J]. 果树学报, 2014, 31(3): 443-447.MA Y, KE Y, WANG Q, et al. A stem canker disease of walnut (Juglans regia)caused by Botryosphaeria dothidea[J].Journal of Fruit Science, 2014, 31(3): 443-447. (in Chinese with English abstract)
[14] 李文英, 李夏, 解开治, 等. 葡萄座腔菌科真菌的系统学和多样性探讨[J]. 生物多样性, 2017, 25(8): 874-885. LI W Y, LI X, XIE K Z, et al. Systematics and species diversity of Botryosphaeriaceous fungi[J].Biodiversity Science, 2017, 25(8): 874-885. (in Chinese with English abstract)
[15] DAYKIN M E, MILHOLAND R D. Histopathology of blueberry twig blight caused by Phomopsis vaccinii[J]. Phytopathology, 1990, 80(8): 736-740.
[16] WRIGHT A F, HARMON P F. Identification of species in the Botryosphaeriaceae family causing stem blight on southern highbush blueberry in Florida[J]. Plant Disease, 2010, 94(8): 966-971.
[17] PHILLIPS A J, OUDEMANS P V, CORREIA A, et al. Characterisation and epitypification of Botryosphaeria cortices, the cause of blueberry cane canker[J]. Fungal Diversity, 2006, 21(4): 141-155.
[18] TENNAKOON K, RIDGWAY H J, JASPERS M V, et al. Botryosphaeriaceae species associated with blueberry dieback and sources of primary inoculum in propagation nurseries in New Zealand[J]. European Journal of Plant Pathology, 2017, 150(2): 363-374.
[19] KOIKE S T, ROONEY-L S, WRIGHT A F. First report of stem blight of blueberry in California caused by Neofusicoccum parvum[J]. Plant Disease, 2014, 98(9): 1280.
[20] SLIPPERS B, WINGFIELD M J. Botryosphaeriaceae as endophytes and latent pathogens of woody plants: diversity ecology and impact[J]. Fungal Biology Reviews, 2007, 21(2): 90-106.
[21] SLIPPERS B, SMIT W A, CROUS P W, et al. Taxonomy phylogeny and identification of Botryosphaeriaceae associated with pome and stone fruit trees in South Africa and other regions of the world[J]. Plant Pathology, 2007, 56(1): 128-139.
[22] 徐成楠, 张红军, 迟福梅,等. 蓝莓枝枯病菌Botryosphaeria dothidea的寄主范围研究[J]. 中国果树, 2015(2): 43-45.XU C N, ZHANG H J, CHI F M,et al. Study on host range of blueberry stem canker pathogen Botryosphaeria dothidea[J]. China Fruits, 2015(2): 43-45. (in Chinese)
[23] FARR D F, CASTLEBURY L A, ROSSMAN A Y, et al. A new species of Phomopsis causing twig dieback of Vaccinium vitis-idaea(lingonberry)[J]. Mycological Research, 2002, 106(6): 745-752.
[24] LOMBARO L, VAN LEEUWEN G C M, GUARNACCIA V, et al. Diaporthe species associated with Vaccinium with specific reference to Europe[J]. Phytopathologia Mediterranea, 2014, 53(2): 287-299.
[25] 岳清华, 赵洪海, 梁晨, 等. 蓝莓拟茎点枝枯病的病原[J]. 菌物学报, 2013, 32(6): 959-966.YUE Q H, ZHAO H H, LIANG C, et al. The pathogen causing Phomopsis twig blight of blueberry[J]. Mycosystema, 2013, 32(6): 959-966. (in Chinese with English abstract)
[26] 李媛, 石凌波, 费诺亚, 等. 蓝莓茎溃疡病病原菌鉴定及品种抗病性研究[J]. 植物病理学报, 2017,47(6):721-729.LI Y, SHI L B, FEI N Y, et al. Identification of causative agent of blueberry stem canker and screening of the disease resistant cultivars[J]. Acta Phytopathologica Sinica, 2017,47(6):721-729. (in Chinese with English abstract)
[27] 罗利娟, 习平根, 姜子德, 等. 纯培养下拟茎点霉分生孢子的形成及意义[J]. 菌物学报, 2004, 23(3): 375-380. LUO L J, XI P G, JIANG Z D, et al. Taxonomic significance of conidial formation of Phomopsis in pure culture[J]. Mycosystema, 2004, 23(3): 375-380. (in Chinese with English abstract)
[2] 吴林. 中国蓝莓35年:科学研究与产业发展[J]. 吉林农业大学学报, 2016, 38(1): 1-11.WU L. Thirty-five years of research and industry development of blueberry in China[J]. Journal of Jilin Agricutural University, 2016, 38(1):1-11. (in Chinese with English abstract)
[3] XU C N, ZHANG H J, ZHOU Z S, et al. Identification and distribution of Botryosphaeriaceae species associated with blueberry stem blight in China[J]. European Journal of Plant Pathology, 2015, 143(4): 737-752.
[4] 余磊, 唐旭兵, 赵建荣,等. 云南蓝莓枝干溃疡病病原菌鉴定及rDNA-ITS序列分析[J]. 云南农业大学学报, 2014, 29(1): 27-31.YU L, TANG X B, ZHAO J R, et al. Identification and the internal transcribed spacer regions analysis of the pathogen causing stem canker on blueberry in Yunman[J]. Journal of Yunnan Agricultural University, 2014, 29(1):27-31. (in Chinese with English abstract)
[5] 徐成楠, 周宗山, 张红军, 等. 蓝莓枝干溃疡病病原鉴定[J]. 植物病理学报, 2012, 42(5): 532-535.XU C N, ZHOU Z S, ZHANG H J, et al. Identification of the pathogen causing stem canker of blueberry[J]. Acta Phytopathologica Sinica, 2012, 42(5): 532-535. (in Chinese with English abstract)
[6] ESPINOZA J G, BRICE?O E X, CHáVEZ E R,et al. Neofusicoccum spp. associated with stem canker and dieback of blueberry in Chile[J]. Plant Disease, 2009, 93(11): 1187-1194.
[7] 方中达. 植病研究方法[M]. 3版. 北京: 中国农业出版社,2007.
[8] 凌丹燕, 李永强, 路梅, 等. 短柄樱桃干腐病病原菌的鉴定及其生物学特性[J]. 浙江师范大学学报(自然科学版), 2016, 39(2): 200-206.LING D Y, LI Y Q, LU M, et al. Identification and biological characteristic researches of rot pathogen isolated from Chinese cherry(Prunus pseudocerasus)[J].Journal of Zhejiang Normal University (Natural Sciences), 2016, 39(2): 200-206. (in Chinese with English abstract)
[9] KUMAR S, STECHER G, TAMURA K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[J]. Molecular Biology and Evolution, 2016, 33(7): 1870-1874.
[10] SLIPPERS B, CROUS P W, DENMAN S,et al. Combined multiple gene genealogies and phenotypic characters differentiate several species previously identified as Botryosphaeria dothidea[J]. Mycologia, 2004, 96(1): 83-101.
[11] ZHAO X, ZHANG G, LI B, et al. Seasonal dynamics of Botryosphaeria dothidea infections and symptom development on apple fruits and shoots in China[J]. European Journal of Plant Pathology, 2016, 146(3): 507-518.
[12] WANG F, ZHAO L, LI G,et al. Identification and characterization of Botryosphaeria spp. causing gummosis of peach trees in Hubei Province in central China[J]. Plant Disease, 2011, 95(11): 1378-1384.
[13] 马瑜, 柯杨, 王琴, 等. 核桃溃疡病症状及其病原菌鉴定[J]. 果树学报, 2014, 31(3): 443-447.MA Y, KE Y, WANG Q, et al. A stem canker disease of walnut (Juglans regia)caused by Botryosphaeria dothidea[J].Journal of Fruit Science, 2014, 31(3): 443-447. (in Chinese with English abstract)
[14] 李文英, 李夏, 解开治, 等. 葡萄座腔菌科真菌的系统学和多样性探讨[J]. 生物多样性, 2017, 25(8): 874-885. LI W Y, LI X, XIE K Z, et al. Systematics and species diversity of Botryosphaeriaceous fungi[J].Biodiversity Science, 2017, 25(8): 874-885. (in Chinese with English abstract)
[15] DAYKIN M E, MILHOLAND R D. Histopathology of blueberry twig blight caused by Phomopsis vaccinii[J]. Phytopathology, 1990, 80(8): 736-740.
[16] WRIGHT A F, HARMON P F. Identification of species in the Botryosphaeriaceae family causing stem blight on southern highbush blueberry in Florida[J]. Plant Disease, 2010, 94(8): 966-971.
[17] PHILLIPS A J, OUDEMANS P V, CORREIA A, et al. Characterisation and epitypification of Botryosphaeria cortices, the cause of blueberry cane canker[J]. Fungal Diversity, 2006, 21(4): 141-155.
[18] TENNAKOON K, RIDGWAY H J, JASPERS M V, et al. Botryosphaeriaceae species associated with blueberry dieback and sources of primary inoculum in propagation nurseries in New Zealand[J]. European Journal of Plant Pathology, 2017, 150(2): 363-374.
[19] KOIKE S T, ROONEY-L S, WRIGHT A F. First report of stem blight of blueberry in California caused by Neofusicoccum parvum[J]. Plant Disease, 2014, 98(9): 1280.
[20] SLIPPERS B, WINGFIELD M J. Botryosphaeriaceae as endophytes and latent pathogens of woody plants: diversity ecology and impact[J]. Fungal Biology Reviews, 2007, 21(2): 90-106.
[21] SLIPPERS B, SMIT W A, CROUS P W, et al. Taxonomy phylogeny and identification of Botryosphaeriaceae associated with pome and stone fruit trees in South Africa and other regions of the world[J]. Plant Pathology, 2007, 56(1): 128-139.
[22] 徐成楠, 张红军, 迟福梅,等. 蓝莓枝枯病菌Botryosphaeria dothidea的寄主范围研究[J]. 中国果树, 2015(2): 43-45.XU C N, ZHANG H J, CHI F M,et al. Study on host range of blueberry stem canker pathogen Botryosphaeria dothidea[J]. China Fruits, 2015(2): 43-45. (in Chinese)
[23] FARR D F, CASTLEBURY L A, ROSSMAN A Y, et al. A new species of Phomopsis causing twig dieback of Vaccinium vitis-idaea(lingonberry)[J]. Mycological Research, 2002, 106(6): 745-752.
[24] LOMBARO L, VAN LEEUWEN G C M, GUARNACCIA V, et al. Diaporthe species associated with Vaccinium with specific reference to Europe[J]. Phytopathologia Mediterranea, 2014, 53(2): 287-299.
[25] 岳清华, 赵洪海, 梁晨, 等. 蓝莓拟茎点枝枯病的病原[J]. 菌物学报, 2013, 32(6): 959-966.YUE Q H, ZHAO H H, LIANG C, et al. The pathogen causing Phomopsis twig blight of blueberry[J]. Mycosystema, 2013, 32(6): 959-966. (in Chinese with English abstract)
[26] 李媛, 石凌波, 费诺亚, 等. 蓝莓茎溃疡病病原菌鉴定及品种抗病性研究[J]. 植物病理学报, 2017,47(6):721-729.LI Y, SHI L B, FEI N Y, et al. Identification of causative agent of blueberry stem canker and screening of the disease resistant cultivars[J]. Acta Phytopathologica Sinica, 2017,47(6):721-729. (in Chinese with English abstract)
[27] 罗利娟, 习平根, 姜子德, 等. 纯培养下拟茎点霉分生孢子的形成及意义[J]. 菌物学报, 2004, 23(3): 375-380. LUO L J, XI P G, JIANG Z D, et al. Taxonomic significance of conidial formation of Phomopsis in pure culture[J]. Mycosystema, 2004, 23(3): 375-380. (in Chinese with English abstract)