杨树枯萎病菌实时荧光定量PCR检测方法的建立及应用
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摘要
为建立荧光定量PCR检测杨树枯萎病的方法和明确前期分离的拮抗菌N6-34对杨树枯萎病致病菌尖孢镰刀菌的作用,本试验采用常规PCR法扩增尖孢镰刀菌的特异性片段,将此片段与载体连接构建质粒,提取质粒DNA在实时荧光定量PCR仪上采用两步法进行扩增并绘制实时荧光定量PCR标准曲线,同时对杨树进行不同灌根处理(T1:1×10~6 cfu/mL的尖孢镰刀菌孢子悬液和未接N6-34菌株的发酵培养液各20 mL;T2:1×10~6 cfu/mL的尖孢镰刀菌孢子悬液和灭活的N6-34菌株发酵培养液各20 mL;T3:1×10~6 cfu/mL的尖孢镰刀菌孢子悬液和N6-34菌株的发酵培养液各20 mL),并于10、20、30 d取样检测杨树根际尖孢镰刀菌数量。结果表明:本研究建立的方法能应用于土传尖孢镰刀菌导致的杨树枯萎病的检测;T1处理的尖孢镰刀菌的数量随处理时间的延长呈先下降后上升的趋势,T2和T3中的尖孢镰刀菌数量则呈下降趋势,表明N6-34能够抑制尖孢镰刀菌。本研究对指导杨树种植和病害预报具有极为重要的应用价值,能够为杨树病害管理提供科学依据。
This study was aimed to construct a real-time fluorescent quantitative PCR method for detection of poplar wilt pathogen and make clear of the effect of antagonistic bacterium N6-34 isolated earlier on Fusarium oxysporum. The specific fragment amplified by routine PCR was lingated with the vectors to constrct the plasmid, and then the plasmid DNA was extracted and amplified by two-step method on a real-time fluorescent quantitative PCR instrument. The stantard curve of real-time fluorescent quantitative PCR was also obtained. In addition, an experiment was conducted by irrigating root of poplar with different solutions. Three treatments were designed as T1(20 mL of 1×10~6 cfu/mL Fusarium oxysporum spore suspension and 20 mL of fermented culture solution with no N6-34), T2(20 mL of 1×10~6 cfu/mL Fusarium oxysporum spore suspension and 20 mL of fermented culture solution with inactivated N6-34) and T3(20 mL of 1×10~6 cfu/mL Fusarium oxysporum spore suspension and 20 mL of fermented culture solution with activated N6-34). The quantity of Fusarium oxysporum at the rhizosphere of poplar was detected after treated for 10, 20 and 30 days. The results showed that the real-time fluorescent quantitative PCR method established in this study could be used for the detection of poplar wilt caused by soil-borne Fusarium oxysporum. The quantity of Fusarium oxysporum decreased first and then increased under the T1 treatment, and decreased under the T2 and T3 treatments, which indicated that the antagonistic bacterium N6-34 could inhibit Fusarium oxysporum. This study would have very important application value for guiding poplar planting and disease prediction, and could provide scientific bases for poplar disease management.
This study was aimed to construct a real-time fluorescent quantitative PCR method for detection of poplar wilt pathogen and make clear of the effect of antagonistic bacterium N6-34 isolated earlier on Fusarium oxysporum. The specific fragment amplified by routine PCR was lingated with the vectors to constrct the plasmid, and then the plasmid DNA was extracted and amplified by two-step method on a real-time fluorescent quantitative PCR instrument. The stantard curve of real-time fluorescent quantitative PCR was also obtained. In addition, an experiment was conducted by irrigating root of poplar with different solutions. Three treatments were designed as T1(20 mL of 1×10~6 cfu/mL Fusarium oxysporum spore suspension and 20 mL of fermented culture solution with no N6-34), T2(20 mL of 1×10~6 cfu/mL Fusarium oxysporum spore suspension and 20 mL of fermented culture solution with inactivated N6-34) and T3(20 mL of 1×10~6 cfu/mL Fusarium oxysporum spore suspension and 20 mL of fermented culture solution with activated N6-34). The quantity of Fusarium oxysporum at the rhizosphere of poplar was detected after treated for 10, 20 and 30 days. The results showed that the real-time fluorescent quantitative PCR method established in this study could be used for the detection of poplar wilt caused by soil-borne Fusarium oxysporum. The quantity of Fusarium oxysporum decreased first and then increased under the T1 treatment, and decreased under the T2 and T3 treatments, which indicated that the antagonistic bacterium N6-34 could inhibit Fusarium oxysporum. This study would have very important application value for guiding poplar planting and disease prediction, and could provide scientific bases for poplar disease management.
引文
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[2] B?hlenius H, ?vergaard R. Impact of seedling type on early growth of poplar plantations on forest and agricultural land[J]. Scandinavian Journal of Forest Research, 2016, 31(8):733-741.
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[4] 任嘉红,班虎栋,叶建仁,等. 吡咯伯克霍尔德氏菌JK-SH007的发酵条件及其对杨树溃疡病的防治效果[J]. 中国生物防治, 2010, 26(3): 300-306.
[5] 任嘉红,王艳芳,叶建仁. 杨树腐烂病拮抗细菌的筛选及其定殖研究[J]. 西部林业科学,2009, 38(2): 6-11.
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[8] 谢宝多,马白菡,胡湘芝,等. 欧美杨枯萎病的研究[J]. 中南林学院学报, 1993, 13(1): 25-33.
[9] 黄建强,廖树青. 欧美杨枯萎病症状和病原初探[J]. 湖南林业科技, 2001, 28(4): 52-53.
[10] 刘彩云,季洪亮. 园林树木真菌性枯萎病研究进展及其防治策略[J]. 山东林业科技,2014,44(6): 100-103,128.
[11] 洒荣波. 杨树内生拮抗菌N6-34抗菌活性物质与防病机制[D]. 泰安:山东农业大学, 2018.
[12] 郑亚明,骆勇,周益林,等. 实时荧光定量PCR在植物病害流行学中的应用[J]. 植物保护,2011, 37(4): 18-22.
[13] 陈璐,谢学文,石延霞,等. 实时荧光定量PCR检测蔬菜病原细菌技术[J]. 中国蔬菜,2013(4): 1-5.
[2] B?hlenius H, ?vergaard R. Impact of seedling type on early growth of poplar plantations on forest and agricultural land[J]. Scandinavian Journal of Forest Research, 2016, 31(8):733-741.
[3] 张楠. 杨树病虫害防治技术[J]. 吉林农业, 2016(22):97-99.
[4] 任嘉红,班虎栋,叶建仁,等. 吡咯伯克霍尔德氏菌JK-SH007的发酵条件及其对杨树溃疡病的防治效果[J]. 中国生物防治, 2010, 26(3): 300-306.
[5] 任嘉红,王艳芳,叶建仁. 杨树腐烂病拮抗细菌的筛选及其定殖研究[J]. 西部林业科学,2009, 38(2): 6-11.
[6] 项存悌,高克祥,何秉章,等. 杨树烂皮病生物防治的研究[J]. 东北林业大学学报,1991,19(6): 15-25.
[7] 李伶俐,韩正敏,吕明亮,等. 杨树枯萎病菌茄类镰刀菌的生物学特性[J]. 林业科技开发, 2009, 23(4): 51-54.
[8] 谢宝多,马白菡,胡湘芝,等. 欧美杨枯萎病的研究[J]. 中南林学院学报, 1993, 13(1): 25-33.
[9] 黄建强,廖树青. 欧美杨枯萎病症状和病原初探[J]. 湖南林业科技, 2001, 28(4): 52-53.
[10] 刘彩云,季洪亮. 园林树木真菌性枯萎病研究进展及其防治策略[J]. 山东林业科技,2014,44(6): 100-103,128.
[11] 洒荣波. 杨树内生拮抗菌N6-34抗菌活性物质与防病机制[D]. 泰安:山东农业大学, 2018.
[12] 郑亚明,骆勇,周益林,等. 实时荧光定量PCR在植物病害流行学中的应用[J]. 植物保护,2011, 37(4): 18-22.
[13] 陈璐,谢学文,石延霞,等. 实时荧光定量PCR检测蔬菜病原细菌技术[J]. 中国蔬菜,2013(4): 1-5.