我国苹果主产区潜隐性病毒侵染特征研究
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摘要
苹果病毒病是对果树危害最大、影响最广的一种世界性病害。果树感染病毒后,一般树体生长量减少,产量降低,果实品质下降,不耐贮藏,严重时树体生长急剧衰退至枯死。在尚无有效的药剂防治和治疗措施情况下,繁殖无病毒苗木、实施果园无毒化栽培是防治苹果病毒病的最有效措施。国外诸多发达国家早在20世纪60年代已经开始发展无病毒化栽培,目前已基本普及。而我国对苹果病毒病研究起步较晚,无毒化栽培推广缓慢。正确认识病毒病的危害,准确掌握目前苹果病毒病的发生情况是制定和推广无毒化栽培政策的基础。近年来我国苹果病毒病的调查鉴定和研究工作相对薄弱,一些参考数据相对较老或者不全,因此对全国进行病毒危害现状调查对推进我国无毒化栽培有重要意义。本研究以建立高效、简化、准确率高、适合大规模样品的检测为目标对苹果病毒的RT-PCR反应体系进行优化,并通过对病毒时空分布规律的研究筛选最佳检测部位和取样时间,在此基础上对我国三个苹果主产区的病毒侵染状况做全面调查。主要结果如下:
1.苹果潜隐性病毒检测体系的优化在三种潜隐性病毒的常规两步单重RT-PCR检测体系基础上,对检测效率更高的多重RT-PCR检测体系进行了优化。
(1)对ASPV的引物进行了重新设计,筛选出了最佳的引物对组合。
(2)对两步多重RT-PCR检测体系进行了优化。优化后的RT体系中的ES Reaction Mix和EasyScript RT/RI Enzyme Mix包含了反应所需的主要试剂,只需加入适量总RNA即可。使操作步骤简化,工作效率提高,而且减少了人为出错和污染的几率,更加减少了误差。再者,PCR扩增过程使用含Taq酶的ES MIX,不仅包括了扩增过程所需要的除引物和反转录产物外的所有试剂,而且含溴酚蓝染料。PCR扩增完成后,产物直接上样电泳,节省了上样时间,使得操作步骤简化,检测效率提高。
(3)优化的一步多重RT-PCR检测体系使用新型试剂盒,该试剂已将RT和PCR反应所需的Buffer、Mg2+、dNTP、反转录酶、RNA酶抑制剂和Taq酶等按比例配成两种缓冲液Mix。反应体系配制时,只需按说明加入适量已含溴酚蓝染料Mix、总RNA模板和引物即可用于反应,扩增完成直接上样电泳。这比传统的RT-PCR方法操作步骤更简单,大大降低了污染几率和误差,同时节省了上样时间,使得检测过程更省时,工作效率更高。另外,对生产上危害较重的苹果锈果类病毒和苹果花叶病毒的RT-PCR反应体系进行了优化,建立了操作步骤更简单、效率更高的RT-PCR检测体系。
2.ASPV和ACLSV在树体内的时空分布规律采用荧光定量RT-PCR技术,对2010年4月到12月采集的枝条顶端、枝皮组织和根皮组织,用苹果肌动蛋白mRNA序列引物作为内参,以4月份枝皮组织的表达量作为标准,进行相对表达差异分析,结果显示:
(1)ASPV在枝皮组织中的含量呈先降低再升高再降低的趋势,即6月份的含量最低,8月份稍有增高,10月份达到5个采集时期的最高值,12月份略有下降,最高含量约是最低含量的5.5倍;ASPV在枝条顶端的含量呈现先降低后升高的趋势,即6月份含量最低,8月份略增,10月份继续升高,12月份达最高值,最高含量约是最低含量的3.0倍;ASPV在根皮中的分布规律为先升高再降低,即4月份含量最低,8月份含量增加,12月份略有下降,最高含量约是最低含量的2.3倍。
(2)ACLSV在树体内的时空分布规律与ASPV基本相似,仅枝条顶端的含量变化略有不同,即ACLSV在10月份的增量小于ASPV,且此时含量低于12月份。
3.苹果潜隐性病毒在我国苹果主产区的分布特征通过对我国8个苹果主产省,50个果园的2144个样品分析结果表明:
(1)采样果园均不同程度地受到苹果潜隐性病毒的侵染,果园病毒侵染率达100%,危害情况较严重。
(2)三种苹果潜隐性病毒(ASGV、ACLSV、ASPV)的整体发生率分别为:94.80%、59.33%、64.72%;总体混合侵染率为59.05%。相对而言,ASGV的侵染率最高,ACLSV的侵染率最低。
(3)三种苹果潜隐性病毒在不同品种间的侵染也有一定差异。三大主栽品种富士、红星、嘎拉的病毒侵染率都较高,ASGV的侵染率最高几乎都为100%;ASPV和ACLSV的侵染率相对最低。部分品种如金冠、乔纳金、秦冠等的病毒侵染率也较高,一些砧木品种和地方品种的染毒情况较低。
(4)在地区分布上,陕西省和河北省受三种苹果潜隐性病毒侵染较严重,复合侵染率也较高;其他地区ASGV侵染率较高,但ASPV和ACLSV的侵染相对较轻。
Virus disease is a severe worldwide disease that affects the production and quality of fruit trees. In generally, the apple trees with virus infection resulted in less growth and crop yiled, lower fruit quality, intolerance storage and sharp tree growth decline to the dead seriously. Without effective chemical control and treatment measures, breeding virus-free seedling and drug-free cultivation are the most effective measure against apple virus diseases. As early as the 1960s, many foreign countries began to develop the cultivation of virus-free and was basically popularized now. China had been the late start of studying on apple viruses and still has less development of virus-free cultivation now. The correct understanding and investigation the presence of virus diseases in China are the basic of the creation and promotion of the virus-free cultivation. So far, the identification and investigation of apple virus disease hazard is relatively weak and only has less comprehensive or old data. So the viruses investigation in apple orchards of the main producing areas in China is of great significance for carrying out the virus-free cultivation. The study concerned the development of the detection systems for three apple latent viruses, which have the chief characteristics of reliable, rapid, more efficient and simplified, accessible for mass-testing. The second aim was to identify the suitable tissue used for the analysis of infection. Under these conditions, the reaserch, using the samples collected respectively from different cultivars in commercial orchards located in three main apple-producing areas, will do a comprehensive infection investigation of the three main latent viruses. The main results are as follows:
1. Development and optimization of detection systems for apple latent viruses On the basis of the traditional two-step of RT-PCR systems of three latent viruses, the multiplex RT-PCR detection system, which has higher detection efficiency, were optimized.
(1) The primer pair for ASPV was redesigned and chose the best combination with two other pairs.
(2)The detection system of two-step multiplex RT-PCR was optimized. The ES Reaction Mix and EasyScript RT/RI Enzyme Mix, which contained the main reagents of the reverse transcription required, were just used with appropriate amount of total RNA. Using the Mix would be simplified the steps and lowered the risk of contamination and error. Furthermore, ES Taq Mix, which including Taq DNA polymerase and other reagents except the amplification primers and reverse transcription product, was used for the PCR amplification process. When amplification was completed, the product because of bromophenol blue dye contained, were directly used for running the agarose gels. That could save the sampling time and make detection efficiency improved.
(3) The one-step multiplex RT-PCR was performed using the commercial kit. The two master mixes typically contained all the components required for RT-PCR, by containing reaction Buffer, Mg2+, dNTP mix, reverse transcriptase, RNA inhibitor, and DNA Polymerase. The reaction mix was prepared according to the instructions, and also added total RNA and the primer pairs. The mix also contained bromophenol blue dye ,and the amplification product were quickly analyzed on agarose gels in TBE buffer. In comparison with conventional RT-PCR assay, the optimal test system have less operations, lower contamination, smaller errors and more efficiency. In addition, the two RT-PCR reaction systems of Apple scar skin viroid and Apple mosaic virus, which cause serious harms on apple production, were optimized and established the more simple and efficient detection assay.
2. The spatial and temporal distribution patterens of ASPV and ACLSV in tree body The sample phloem+periderm+cortex parenchyma, shoot tip and root bark collected from April to December in 2010 were tested by fluorescence quantitative RT-PCR. With the sequences of domestic Malus mRNA for actin as the internal control, the study was used the expression value of the phloem material in April as standard, and analyzed the relative expression variance. The results are summaried as follows:
(1) The virus concentration of ASPV decreased at first then increased, and followed declined in phloem+periderm+cortex parenchyma throughout the year. The lowest concentriation was appeared in June, then slightly increased in August. In October , The relative concentration was still higher than in August, and was the maximum in the year. Then in December, compared with October, the concentration was reduced. The highest relative concentration was about 5.5 times higher than the lowest in phloem +periderm+ cortex parenchyma; The concentration of ASPV in the shoot tip at various times of the year performed firstly decreased then increased. The minimum ASPV relative concentration was obtained testing shoot tip collected in June. The shoot tip sampled later mostly showed a continuous increase in relative concentrations of ASPV until the highest value in December. The average ASPV relative concentration was 3 times higher in December than in June; The distribution characteristics of ASPV in the root bark was increased in August, and then gradually decreased in December. The maximum ASPV relative concentration in August was 2.3 times higher than the minimum value in April.
(2) The distribution patterns of ACLSV were mostly similar with ASPV. The slight difference is that the ACLSV relative concentration in shoot tip had less increment in Octorber. So the minimum ACLSV relative concentration was obtained in December, and the distribution characteristics of ACLSV in the shoot tip was gradually increased from June until December.
3. Distribution characteristics of apple latent virus in the major apple production areas of China Totally 2144 samples in 50 commercial orchards from 8 provinces were Collected. On the basis of the detection results presented in this paper we conclude that:
(1) All the orchards have been subjected to apple latent virus infection in varying degrees. The virus infection rate was 100%. In summary, apple viruses are widely distributed in apple growing orchards and cause serious affect.
(2) The overall of three kinds apple latent virus (ASGV, ACLSV,ASPV) infection rate: 94.80%, 59.33%, 64.72%, in different orchards and different varieties. Overall mixed infection rate was 59.05%. Relatively speaking, The infection rate of ASGV was the highest, on the contrary, ACLSV and ASPV of the lower infection rates.
(3) The infection rates of three apple latent viruses were not deviations significantly, among the different varieties. In comparison, the latent virus infection rates of Fuji, Gala, Starking were generally higher, the infection rates of Apple stem grooving virus were almost 100%, ACLSV and ASPV of the lower infection rates. Some cultivars, just like Golden Delicious, Jonagold, and Qinguan, had higher virus infection rates. Some rootstock varieties and local cultivars were of lower exposure situations.
(4) In the regions of Shaanxi Province and Hebei Province, the virus infection situations of three apple latent were most seriously, the composite infection rate were also highest; In other regions, the infection rates of ASGV were higher, but the infection rates of ASPV and ACLSV were relatively lighter.
1.苹果潜隐性病毒检测体系的优化在三种潜隐性病毒的常规两步单重RT-PCR检测体系基础上,对检测效率更高的多重RT-PCR检测体系进行了优化。
(1)对ASPV的引物进行了重新设计,筛选出了最佳的引物对组合。
(2)对两步多重RT-PCR检测体系进行了优化。优化后的RT体系中的ES Reaction Mix和EasyScript RT/RI Enzyme Mix包含了反应所需的主要试剂,只需加入适量总RNA即可。使操作步骤简化,工作效率提高,而且减少了人为出错和污染的几率,更加减少了误差。再者,PCR扩增过程使用含Taq酶的ES MIX,不仅包括了扩增过程所需要的除引物和反转录产物外的所有试剂,而且含溴酚蓝染料。PCR扩增完成后,产物直接上样电泳,节省了上样时间,使得操作步骤简化,检测效率提高。
(3)优化的一步多重RT-PCR检测体系使用新型试剂盒,该试剂已将RT和PCR反应所需的Buffer、Mg2+、dNTP、反转录酶、RNA酶抑制剂和Taq酶等按比例配成两种缓冲液Mix。反应体系配制时,只需按说明加入适量已含溴酚蓝染料Mix、总RNA模板和引物即可用于反应,扩增完成直接上样电泳。这比传统的RT-PCR方法操作步骤更简单,大大降低了污染几率和误差,同时节省了上样时间,使得检测过程更省时,工作效率更高。另外,对生产上危害较重的苹果锈果类病毒和苹果花叶病毒的RT-PCR反应体系进行了优化,建立了操作步骤更简单、效率更高的RT-PCR检测体系。
2.ASPV和ACLSV在树体内的时空分布规律采用荧光定量RT-PCR技术,对2010年4月到12月采集的枝条顶端、枝皮组织和根皮组织,用苹果肌动蛋白mRNA序列引物作为内参,以4月份枝皮组织的表达量作为标准,进行相对表达差异分析,结果显示:
(1)ASPV在枝皮组织中的含量呈先降低再升高再降低的趋势,即6月份的含量最低,8月份稍有增高,10月份达到5个采集时期的最高值,12月份略有下降,最高含量约是最低含量的5.5倍;ASPV在枝条顶端的含量呈现先降低后升高的趋势,即6月份含量最低,8月份略增,10月份继续升高,12月份达最高值,最高含量约是最低含量的3.0倍;ASPV在根皮中的分布规律为先升高再降低,即4月份含量最低,8月份含量增加,12月份略有下降,最高含量约是最低含量的2.3倍。
(2)ACLSV在树体内的时空分布规律与ASPV基本相似,仅枝条顶端的含量变化略有不同,即ACLSV在10月份的增量小于ASPV,且此时含量低于12月份。
3.苹果潜隐性病毒在我国苹果主产区的分布特征通过对我国8个苹果主产省,50个果园的2144个样品分析结果表明:
(1)采样果园均不同程度地受到苹果潜隐性病毒的侵染,果园病毒侵染率达100%,危害情况较严重。
(2)三种苹果潜隐性病毒(ASGV、ACLSV、ASPV)的整体发生率分别为:94.80%、59.33%、64.72%;总体混合侵染率为59.05%。相对而言,ASGV的侵染率最高,ACLSV的侵染率最低。
(3)三种苹果潜隐性病毒在不同品种间的侵染也有一定差异。三大主栽品种富士、红星、嘎拉的病毒侵染率都较高,ASGV的侵染率最高几乎都为100%;ASPV和ACLSV的侵染率相对最低。部分品种如金冠、乔纳金、秦冠等的病毒侵染率也较高,一些砧木品种和地方品种的染毒情况较低。
(4)在地区分布上,陕西省和河北省受三种苹果潜隐性病毒侵染较严重,复合侵染率也较高;其他地区ASGV侵染率较高,但ASPV和ACLSV的侵染相对较轻。
Virus disease is a severe worldwide disease that affects the production and quality of fruit trees. In generally, the apple trees with virus infection resulted in less growth and crop yiled, lower fruit quality, intolerance storage and sharp tree growth decline to the dead seriously. Without effective chemical control and treatment measures, breeding virus-free seedling and drug-free cultivation are the most effective measure against apple virus diseases. As early as the 1960s, many foreign countries began to develop the cultivation of virus-free and was basically popularized now. China had been the late start of studying on apple viruses and still has less development of virus-free cultivation now. The correct understanding and investigation the presence of virus diseases in China are the basic of the creation and promotion of the virus-free cultivation. So far, the identification and investigation of apple virus disease hazard is relatively weak and only has less comprehensive or old data. So the viruses investigation in apple orchards of the main producing areas in China is of great significance for carrying out the virus-free cultivation. The study concerned the development of the detection systems for three apple latent viruses, which have the chief characteristics of reliable, rapid, more efficient and simplified, accessible for mass-testing. The second aim was to identify the suitable tissue used for the analysis of infection. Under these conditions, the reaserch, using the samples collected respectively from different cultivars in commercial orchards located in three main apple-producing areas, will do a comprehensive infection investigation of the three main latent viruses. The main results are as follows:
1. Development and optimization of detection systems for apple latent viruses On the basis of the traditional two-step of RT-PCR systems of three latent viruses, the multiplex RT-PCR detection system, which has higher detection efficiency, were optimized.
(1) The primer pair for ASPV was redesigned and chose the best combination with two other pairs.
(2)The detection system of two-step multiplex RT-PCR was optimized. The ES Reaction Mix and EasyScript RT/RI Enzyme Mix, which contained the main reagents of the reverse transcription required, were just used with appropriate amount of total RNA. Using the Mix would be simplified the steps and lowered the risk of contamination and error. Furthermore, ES Taq Mix, which including Taq DNA polymerase and other reagents except the amplification primers and reverse transcription product, was used for the PCR amplification process. When amplification was completed, the product because of bromophenol blue dye contained, were directly used for running the agarose gels. That could save the sampling time and make detection efficiency improved.
(3) The one-step multiplex RT-PCR was performed using the commercial kit. The two master mixes typically contained all the components required for RT-PCR, by containing reaction Buffer, Mg2+, dNTP mix, reverse transcriptase, RNA inhibitor, and DNA Polymerase. The reaction mix was prepared according to the instructions, and also added total RNA and the primer pairs. The mix also contained bromophenol blue dye ,and the amplification product were quickly analyzed on agarose gels in TBE buffer. In comparison with conventional RT-PCR assay, the optimal test system have less operations, lower contamination, smaller errors and more efficiency. In addition, the two RT-PCR reaction systems of Apple scar skin viroid and Apple mosaic virus, which cause serious harms on apple production, were optimized and established the more simple and efficient detection assay.
2. The spatial and temporal distribution patterens of ASPV and ACLSV in tree body The sample phloem+periderm+cortex parenchyma, shoot tip and root bark collected from April to December in 2010 were tested by fluorescence quantitative RT-PCR. With the sequences of domestic Malus mRNA for actin as the internal control, the study was used the expression value of the phloem material in April as standard, and analyzed the relative expression variance. The results are summaried as follows:
(1) The virus concentration of ASPV decreased at first then increased, and followed declined in phloem+periderm+cortex parenchyma throughout the year. The lowest concentriation was appeared in June, then slightly increased in August. In October , The relative concentration was still higher than in August, and was the maximum in the year. Then in December, compared with October, the concentration was reduced. The highest relative concentration was about 5.5 times higher than the lowest in phloem +periderm+ cortex parenchyma; The concentration of ASPV in the shoot tip at various times of the year performed firstly decreased then increased. The minimum ASPV relative concentration was obtained testing shoot tip collected in June. The shoot tip sampled later mostly showed a continuous increase in relative concentrations of ASPV until the highest value in December. The average ASPV relative concentration was 3 times higher in December than in June; The distribution characteristics of ASPV in the root bark was increased in August, and then gradually decreased in December. The maximum ASPV relative concentration in August was 2.3 times higher than the minimum value in April.
(2) The distribution patterns of ACLSV were mostly similar with ASPV. The slight difference is that the ACLSV relative concentration in shoot tip had less increment in Octorber. So the minimum ACLSV relative concentration was obtained in December, and the distribution characteristics of ACLSV in the shoot tip was gradually increased from June until December.
3. Distribution characteristics of apple latent virus in the major apple production areas of China Totally 2144 samples in 50 commercial orchards from 8 provinces were Collected. On the basis of the detection results presented in this paper we conclude that:
(1) All the orchards have been subjected to apple latent virus infection in varying degrees. The virus infection rate was 100%. In summary, apple viruses are widely distributed in apple growing orchards and cause serious affect.
(2) The overall of three kinds apple latent virus (ASGV, ACLSV,ASPV) infection rate: 94.80%, 59.33%, 64.72%, in different orchards and different varieties. Overall mixed infection rate was 59.05%. Relatively speaking, The infection rate of ASGV was the highest, on the contrary, ACLSV and ASPV of the lower infection rates.
(3) The infection rates of three apple latent viruses were not deviations significantly, among the different varieties. In comparison, the latent virus infection rates of Fuji, Gala, Starking were generally higher, the infection rates of Apple stem grooving virus were almost 100%, ACLSV and ASPV of the lower infection rates. Some cultivars, just like Golden Delicious, Jonagold, and Qinguan, had higher virus infection rates. Some rootstock varieties and local cultivars were of lower exposure situations.
(4) In the regions of Shaanxi Province and Hebei Province, the virus infection situations of three apple latent were most seriously, the composite infection rate were also highest; In other regions, the infection rates of ASGV were higher, but the infection rates of ASPV and ACLSV were relatively lighter.
引文
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