湖南省柑橘茎陷点型衰退病为害情况调查及其弱毒系筛选研究
摘要
湖南作为中国柑橘栽培大省,柑橘产业近几年来得到了快速的发展,面积与产量均已跃居全国第一。长期以来,由于抗柑橘衰退病(CTV)枳(Poncitrustrifoliate)砧的大量使用,使得CTV并未对湖南省的柑橘生产造成太大影响,但近年来随着茎陷点型的CTV强毒系的入侵,该病害对我省的柑橘产业所带来的危害越来越严重,并且由于该病可被蚜虫传播,无毒树也很容易被再感染,所以防治更为困难。因此,本研究旨在摸清湖南省目前柑橘衰退病为害情况的同时,通过RT-PCR结合测序的方法初步筛选出CTV的弱毒株系,为交叉保护试验在生产实践中的应用奠定基础。
本研究试验进程及结果如下:
1.对湖南省各柑橘产业大县的主栽品种进行了茎陷点型CTV发生为害的初步调查,调查发现部分果园的强毒系病株已经完全失去生产价值,树势极其衰弱,枝叶稀疏黄化严重,茎陷点明显,甚至有完全无果的现象,并且其受害程度随时问的推移而加重。
2.通过RT-PCR结合测序的方法对其毒系进行分型研究,结果发现各地区的衰退株系并没有出现非常专一的地区特异性,这可能与目前各县市的品种交流开始频繁起来有关,而衰退株系本身具有特异性,优良但却未经过脱毒的品种通过嫁接繁殖将本地的衰退株系带到别地,成为强毒系。
3.初步筛选出CTV弱毒株系。引用了Sambade设计的引物,通过RT-PCR法进行CTV株系鉴定,对经RT-PCR法鉴定为弱毒的样品进行序列的测定,之后再对其进行系统发育树状图进行分析,同时对测得序列相应氨基酸序列的分析。并同时结合酶切的方法,利用ScaⅠ酶及TaqⅠ酶对各株系进行酶切反应进行弱毒系的初步筛选,为CTV弱毒株系的筛选提供进一步的依据。
4.交叉保护试验。利用3种常用的CTV指示植物进行了交叉保护(Mild StrainCross Protection,MSCP)试验,观察其筛选出的弱毒系的交叉保护作用能力。
Hunan is one of the most important citrus producers in China, which citrus industry by rapid development in recent years, area and production have jumped first in the country. Due to widely utilization of CTV-resistant trifoliate rootstock, up to now CTV has not caused severe damages to the citrus industry. However, as the strong CTV strain's infection, some economic losses in citrus have been demonstrated. CTV can be carry with aphides, lead to the tree of innocuity be easy to infected, so it's hard to control. Present study aims at the find out of damage situation in Hunan, use the RT-PCR technique and the analysis of DNA sequences, selected mild CTV strains forPractice MSCP in Hunan.
1. Preliminary investigation on stem pitting of CTV in citrus varieties of theHunan citrus industry county found that some of the highly virulent orchard plants has been completely lost production value, trees extremely recession, branches Sparse yellow serious depression stems obvious point, even no fruit, and the extent of their victims with the passage of time.
2. By RT-PCR method of sequencing the toxicity of its type conducted research and found that all regions of the recession lines and did not appear very specific areas of specificity, this may be the current county and the city of species frequent exchanges began with , But it has not been good virus-free varieties by grafting to the local recession breeding lines to other places, and the recession itself with specific lines, the local department may be attenuated to other places will become of the severe isolate.
3. Preliminary screening to mild strain of CTV. Quoted Sambade design primer, through RT-PCR method to identify the virus samples of the sequence, At the same time, measured the corresponding sequence of amino acid sequence analysis, CTV mild strain of the basis for further screening . At the same time use the method of ScaI and TaqI enzyme digestion screening to mild strain of CTV.
4. Mild Strain Cross Protection experiment. Use of three kinds of commonly used instructions of the CTV to research Mild Strain Cross Protection experiment.
本研究试验进程及结果如下:
1.对湖南省各柑橘产业大县的主栽品种进行了茎陷点型CTV发生为害的初步调查,调查发现部分果园的强毒系病株已经完全失去生产价值,树势极其衰弱,枝叶稀疏黄化严重,茎陷点明显,甚至有完全无果的现象,并且其受害程度随时问的推移而加重。
2.通过RT-PCR结合测序的方法对其毒系进行分型研究,结果发现各地区的衰退株系并没有出现非常专一的地区特异性,这可能与目前各县市的品种交流开始频繁起来有关,而衰退株系本身具有特异性,优良但却未经过脱毒的品种通过嫁接繁殖将本地的衰退株系带到别地,成为强毒系。
3.初步筛选出CTV弱毒株系。引用了Sambade设计的引物,通过RT-PCR法进行CTV株系鉴定,对经RT-PCR法鉴定为弱毒的样品进行序列的测定,之后再对其进行系统发育树状图进行分析,同时对测得序列相应氨基酸序列的分析。并同时结合酶切的方法,利用ScaⅠ酶及TaqⅠ酶对各株系进行酶切反应进行弱毒系的初步筛选,为CTV弱毒株系的筛选提供进一步的依据。
4.交叉保护试验。利用3种常用的CTV指示植物进行了交叉保护(Mild StrainCross Protection,MSCP)试验,观察其筛选出的弱毒系的交叉保护作用能力。
Hunan is one of the most important citrus producers in China, which citrus industry by rapid development in recent years, area and production have jumped first in the country. Due to widely utilization of CTV-resistant trifoliate rootstock, up to now CTV has not caused severe damages to the citrus industry. However, as the strong CTV strain's infection, some economic losses in citrus have been demonstrated. CTV can be carry with aphides, lead to the tree of innocuity be easy to infected, so it's hard to control. Present study aims at the find out of damage situation in Hunan, use the RT-PCR technique and the analysis of DNA sequences, selected mild CTV strains forPractice MSCP in Hunan.
1. Preliminary investigation on stem pitting of CTV in citrus varieties of theHunan citrus industry county found that some of the highly virulent orchard plants has been completely lost production value, trees extremely recession, branches Sparse yellow serious depression stems obvious point, even no fruit, and the extent of their victims with the passage of time.
2. By RT-PCR method of sequencing the toxicity of its type conducted research and found that all regions of the recession lines and did not appear very specific areas of specificity, this may be the current county and the city of species frequent exchanges began with , But it has not been good virus-free varieties by grafting to the local recession breeding lines to other places, and the recession itself with specific lines, the local department may be attenuated to other places will become of the severe isolate.
3. Preliminary screening to mild strain of CTV. Quoted Sambade design primer, through RT-PCR method to identify the virus samples of the sequence, At the same time, measured the corresponding sequence of amino acid sequence analysis, CTV mild strain of the basis for further screening . At the same time use the method of ScaI and TaqI enzyme digestion screening to mild strain of CTV.
4. Mild Strain Cross Protection experiment. Use of three kinds of commonly used instructions of the CTV to research Mild Strain Cross Protection experiment.
引文
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