Distribution Characteristics Of Apple Latent Virus In Hebei Province

Apple virus diseases are widely distributed in the main producing areas of the world, affecting seriously, spreading fast, difficult to cure and so on. Apple virus Interfere the normal physiological function of the apple tree, resulting in the reduction of tree vigor, yield and the quality of fruits, causing huge economic losses. Therefore, the cultivation of virus-free apple is extremely urgent, and geting hold of of the distribution of apple virus diseases is the basis of this work and theoretical direction.So, in the research, we maked use of the apple trees of orchards in the main apple producing areas as materials, using RT-PCR technique to detect the apple latent virus. Finally, we made a full investigation of distribution characteristics of apple latent virus in the major apple production areas of Hebei, and made detection methods further improved and optimized. The main results are as follows:1.Distribution characteristics of apple latent virus in the major apple production areas of Hebei: (1).In 29 samples orchard of 7 cities of Hebei Province, we have a sampling survey, All the Orchards have been subjected to apple latent virus infection in varying degrees. The virus infection rate was 100%. (2).The overall of three kinds apple latent virus (ASGV, ASPV, ACLSV) infection rate: 91.63%, 88.48%, 81.45%, in different orchards and different varieties. Overall mixed infection rate was 68.04%. Relatively speaking, The infection rate of ASGV was the highest, Contrary, ACLSV of the lowest infection rate. (3).The infection rate of apple latent viruses was not deviations significantly, between different ages of trees. But, the data analyzed showed that the infection rates of three kinds of latent virus were rising in the New Orchard. The infection rates of saplings about 1-3 years old were generally higher than the results of other old trees. (4).The infection rate of three kinds of apple latent virus was not deviations significantly, among the different varieties. In comparison, the latent virus infection rate of Fuji, Red Delicious, Golden Delicious, Orin were generally higher than that of Railst, Gala, Jonagold. Among them, the virus infection rate of Golden Delicious was relatively the highest, three types of virus infection was averagely higher than 90%; The virus infection rate of Railst was relatively lightest, infection rate of three types virus was between 70%- 85%. (5).In the region, infection rates of three kinds of latent virus were gradually increasing from south to north, there were local specialty. Among them, infection rate of apple latent virus in Zhangjiakou was generally higher than that in other regions, infection rates of three types virus were generally above 90%.The infection rates of individual orchards was up to 100%, the mixed infection rate was up to 84.96%. the infection rate of apple latent virus in Hengshui was relatively lower than that in other regions, infection rates of three types virus were generally beween 80% -90%,and the mixed infection rate was 64.68 percent.2. Improvement and optimization of detection system for Apple latent virus in field :(1)Optimization of RNA extraction for mature tissue : Taking account of situation that field-apple organization are rich in polysaccharides, polyphenols and a large number of secondary metabolites, We have improved the conventional method of RNA extraction for apple tissue from four: In the process of RNA extraction, test containers need autoclave and high temperature baking only,without using chloroform soaked or DEPC treatment. Instead of DEPC treated water, ddH2O was used in the study; The process of RNA extraction was carried out at room temperature,and used regular high-speed centrifuge instead of high-speed freezing centrifuge; Adjust the ratio of extracts and plant material, reducing the amount of plant material for the conventional methods of1/ 20, overcoming the interference of impurities effectively; The combination of two methods separate polysaccharides: In the conditions of high concentration of Na+ (K+), using of phenol/chloroform, the quality of total RNA was improved considerably. (2).The exploration for batch system of RT-PCR technology detecting latent virus for field-apple: A uniform system will be used for each of the three apple latent virus: for reverse reaction; We have used RT-PCR technique to study its mass detection system, RT-PCR reagent composition: 10×buffer, MgCl2 (25mM), dNTPs(2.5mM), the downstream primer, TaqDNA polymerase, RNasin, PCRMarker, ultra-pure water. According to different reagents and different proportions, mixed reagent was prepared: No1, No2, No3. The above three reagents can be used separately ASGV, ASPV, ACLSV for rapid detection, in the safe storage period (four months).3. Optimization of sample collection and preservation programs: As the main test materials, the growing point of the shoot tips and young leaves were collected and put into the container box with foam insulation directly. The samples should be stored and transported at low temperature (0℃) by adding ice. Researchers must replace the melting ice continually. In this case, the longest time of storage and transport is up to five days and does not affect the extraction of total RNA of samples. If you can not meet the conditions,you can store and transport the samples at room temperature storage, the longest time of storage and transport for young leaves is up to five days.