Distribution And Removal Of Apple Scar Skin Viroid In Apple Tree

In recent years,the diseases caused by apple viroids have brought great threats to the apple industry.Apple Scar Skin Viroid(ASSVd)can reduce the yield and quality of apple fruits.However,the detection and removal technology of ASSVd is not perfect.Therefore,the purpose of this study was to clarify the content difference of ASSVd in different tissues of apple trees in the field,and to provide guidance for selecting suitable detection tissues;at the same time,the effect of chemical reagent melatonin(MT)on the elimination of ASSVd was studied,and apple virus-free seedlings were obtained,in order to provide materials and technical support for virus-free seedling breeding practice.The main results are as follows:1.The effects of ASSVd infection on fruit quality and tree growth and development were clarified.The fruits and leaves of’Qincui’infected with ASSVd were used as materials.First,ASSVd infection caused a’flower face’phenotype on the fruit surface,and the fruit coloring area was significantly reduced by 18%compared with the control,which seriously affected the fruit quality.The infection of ASSVd in fruit quality resulted in a significant decrease in soluble sugar content by 1.78%,a significant increase in peel hardness by 28.68%,and a hardening of fruit taste.In addition,the leaf size and fresh weight of the leaves were significantly reduced by 11.22 cm~2 and 5.40 g.There was no significant difference in chlorophyll content.The activities of antioxidant enzymes CAT,POD and SOD in leaves were significantly up-regulated compared with the control.The microscopic cell structure of each tissue was observed by electron microscope.It was found that the cell structure of the peel tissue changed most obviously.The cells of the infected peel tissue shrank and became smaller,and the arrangement became irregular and irregular.In addition,it was found that the cell structure of the ovary of the flower and the cell of the phloem of the branch were also significantly smaller than the control.There was no significant effect on the cell structure of the leaves.2.The temporal and spatial distribution of ASSVd in trees during the growing season was revealed.The distribution of ASSVd in different tissues of’Qincui’trees infected with ASSVd was clarified.After the winter dormancy period,the content of ASSVd was the highest in the branches from the flowering stage to the fruit ripening,which was 8.9 times that of the leaves.The fruit development period is concentrated in the peel tissue,and the peel with the highest content in September is 66.7 times that of the leaf with the lowest content.After fruit picking,the relative content of branches was the highest,which was 20.0 times that of leaves.According to the variation of ASSVd content in different tissues in different periods,the best detection tissue in April,May and November was branches,and the best detection tissue from June to October was the peel part of fruit.This result has practical significance for guiding ASSVd detection.3.The’Qincui’tissue culture seedlings infected with ASSVd were used as experimental materials to clarify the effect of exogenous melatonin on the removal of ASSVd.Firstly,four concentration gradients of melatonin(0,10,20 and 30μM)were set for tissue culture seedlings.The results showed that the plant height and proliferation coefficient of tissue culture seedlings were significantly higher than those of other treatments under 20μM melatonin treatment.After 28 days of subculture,the content of ASSVd in tissue culture seedlings with 20μM melatonin was also the lowest.At the same time,compared with the addition of other concentrations of melatonin,the contents of endogenous hormones JA,ACC and ZT were significantly increased,and the content of ABA was significantly decreased.20μM melatonin was selected for subsequent detoxification test.In the detoxification test of melatonin combined with thermotherapy and shoot tip culture,the addition of melatonin obtained 88%shoot tip survival rate and 80%regeneration rate.The survival rate without melatonin was63%,and the regeneration rate was 60%.RT-PCR virus detection of regenerated seedlings showed that the detoxification rate of melatonin treatment was 42%,which was higher than27%of the control group.In the experiment of melatonin combined with heat treatment and shoot tip cryotherapy detoxification,the addition of melatonin also increased the survival rate,regeneration rate and detoxification rate of shoot tip.The detoxification rate of ASSVd treated with melatonin reached 63%,which was higher than 25%of the control group.