【Objective】This study aimed to establish a quintuple PCR method for rapid and simultaneous detection of Ralstonia solanacearum,Fusarium spp.,Pectobacterium spp.,Enterobacter spp.,and Pythium spp.,which provided tech...【Objective】This study aimed to establish a quintuple PCR method for rapid and simultaneous detection of Ralstonia solanacearum,Fusarium spp.,Pectobacterium spp.,Enterobacter spp.,and Pythium spp.,which provided technical support for early diagnosis of various soil-borne diseases on ginger.【Method】For five types of soil-borne pathogens causing ginger bacterial wilt and rhizome rot,specific primer combinations were designed and screened,the optimal quintuple reaction system was established by exploring optimal primer concentrations,annealing temperature,and sensitivity,and was applied to detect field plant samples to verify its utility.【Result】Specific primers pairs Rs1F/Rs1R,En1F/En1R,and Py1F/Py1R were designed according to flic gene of Ralstonia solanacearum,rpoB gene of Enterobacter spp.,and 18S rDNA of Pythium spp.,and combined with reported Fusarium spp.specific primers Fu3/Fu4 and specific primers 23SPecF/23SPecR of Pectobacterium spp.,a quintuple PCR reaction system for ginger soil-borne pathogens has been established(25.00μL):above primer dosage was 1.20,0.20,0.60,1.60,and 0.15μL respectively;2×PCR Mix 12.50μL;DNA templates of different pathogens were 1.00μL each;added ddH_(2)O to 25.00μL.Annealing temperature was optimized to 55.4℃.The specific fragments with sizes of 516,370,266,207,and 159 bp could be amplified simultaneously in the established quintuple PCR system,and the detection limit of this system for Ralstonia solanacearum,Enterobacter spp.and Pythium spp.reached 10^(-1)pg/μL,for Fusarium spp.and Pectobacterium spp.was 1 pg/μL,and for detecting five pathogens simultaneously was 10^(3)pg/μL.The multiplex PCR system established in this study could successfully detect the diseased plant samples from the field.【Conclusion】The quintuple PCR system established is able to rapid ly and accurately detect Ralstonia solanacearum,Fusarium spp.,Pectobacterium spp.,Enterobacter spp.,and Pythium spp.,which provides a useful tool for timely diagnosis and epidemic monitoring of various soil-borne diseases of ginger.展开更多
为研究留茬高度对烤烟生长发育与产质量影响,在烟株打顶前,砍掉地上部分,设置5,10,15,20 cm 4个留茬高度,分析其大田农艺性状、干物质积累、烤后烟叶化学成分与感官质量。结果表明:随着留茬高度增加烤烟株高、叶面积与干物质积累量整体...为研究留茬高度对烤烟生长发育与产质量影响,在烟株打顶前,砍掉地上部分,设置5,10,15,20 cm 4个留茬高度,分析其大田农艺性状、干物质积累、烤后烟叶化学成分与感官质量。结果表明:随着留茬高度增加烤烟株高、叶面积与干物质积累量整体呈上升趋势,茎干物质量占全株比例上升,根下降;提高留茬高度,烤烟各项经济指标有所提高,留茬20 cm处理产值为27302.20元·hm^(-2)。烤后原烟杂色烟比例较高,身份较厚,结构稍紧致,提高留茬高度烟叶成熟度、叶片结构有所提高,与正常烟株相比,留茬再生烟株总糖、还原糖含量降低,烟碱含量升高,感官评价结果较差,提高留茬高度总糖、还原糖、糖碱比呈先上升后下降趋势,留茬20 cm处理外观质量与感官质量评价结果最好。烟株打顶前,仅留地上部5~20 cm,烟株能正常生长发育,当留茬高度为20 cm时,其经济性状最佳,感官质量与外观质量最优。生产上可通过保留底部茬高20 cm,降低烟叶冰雹灾害损失。展开更多
基金National Natural Science Foundation of China(32270237)Guangxi Key Research and Development Plan Project(Guike AB21238002)Basic Scientific Research Project of Guangxi Academy of Agricultural Sciences(Guinongke 2024YP082)。
文摘【Objective】This study aimed to establish a quintuple PCR method for rapid and simultaneous detection of Ralstonia solanacearum,Fusarium spp.,Pectobacterium spp.,Enterobacter spp.,and Pythium spp.,which provided technical support for early diagnosis of various soil-borne diseases on ginger.【Method】For five types of soil-borne pathogens causing ginger bacterial wilt and rhizome rot,specific primer combinations were designed and screened,the optimal quintuple reaction system was established by exploring optimal primer concentrations,annealing temperature,and sensitivity,and was applied to detect field plant samples to verify its utility.【Result】Specific primers pairs Rs1F/Rs1R,En1F/En1R,and Py1F/Py1R were designed according to flic gene of Ralstonia solanacearum,rpoB gene of Enterobacter spp.,and 18S rDNA of Pythium spp.,and combined with reported Fusarium spp.specific primers Fu3/Fu4 and specific primers 23SPecF/23SPecR of Pectobacterium spp.,a quintuple PCR reaction system for ginger soil-borne pathogens has been established(25.00μL):above primer dosage was 1.20,0.20,0.60,1.60,and 0.15μL respectively;2×PCR Mix 12.50μL;DNA templates of different pathogens were 1.00μL each;added ddH_(2)O to 25.00μL.Annealing temperature was optimized to 55.4℃.The specific fragments with sizes of 516,370,266,207,and 159 bp could be amplified simultaneously in the established quintuple PCR system,and the detection limit of this system for Ralstonia solanacearum,Enterobacter spp.and Pythium spp.reached 10^(-1)pg/μL,for Fusarium spp.and Pectobacterium spp.was 1 pg/μL,and for detecting five pathogens simultaneously was 10^(3)pg/μL.The multiplex PCR system established in this study could successfully detect the diseased plant samples from the field.【Conclusion】The quintuple PCR system established is able to rapid ly and accurately detect Ralstonia solanacearum,Fusarium spp.,Pectobacterium spp.,Enterobacter spp.,and Pythium spp.,which provides a useful tool for timely diagnosis and epidemic monitoring of various soil-borne diseases of ginger.
