Study On Infection Characteristics Of Apple Latent Virus In China’s Main Producing Areas

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.