Monitoring And Transcriptome Analysis Of The Infection Of Sweet Potato Virus Disease (SPVD) In Sweet Potato

Sweet potato [Ipomoea batatas L.(Lam)] is one of the world's most important food,feed crops and industrial processing raw materials.China is a big producer of sweet potato,but the sweet potato production in some areas of China decreased significantly because of diseases in recent years.Sweet potato virus disease(SPVD)is a devastating disease of sweet potato,which is caused by the mixed infection of Sweet potato feathery mottle virus(SPFMV)and Sweet potato chlorotic stunt virus(SPCSV).Sweet potato infected by SPVD might show dwarf plants,leaf chlorosis,distortion,deformities and another symptoms,and the yield would be seriously affected.In order to deeply understand the occurrence,spread,control,monitoring and pathogenic mechanism of SPVD,and to prevent and control the large-scale spread of SPVD,and to reduce the loss of sweet potato production caused by SPVD,we surveyed the propagation law of SPVD,under natural infection and artificial friction infection,in two circumstances,and carried out the effect appraisal to different prevention and control measures.A fast and efficient SPVD pathogeny detection method was established,and the comparative transcriptomtic analysis of healthy and SPVD pathogeny infected sweet potato was performed.The results are as follows:1.Sweet potato varieties resistance,prevention and control methods of SPVDDuring the year of 2014 and 2015,the propagation law of SPVD were surveyed by using natural infection and artificial friction infection test in the field using sweet potato varieties(lines)Yushu No.2,Yushu No.4,Yushu No.6,Yushu No.12,Yushu No.33,Xushu No.22,Nanshu No.88,CT1,Ningzi No.1 and 0841-14.The results showed that the resistance of Yushu No.2,Xushu No.22,Ningzi No.1 and 0841-14 were relatively weak.On the contrary,the resistance of CT1,Yushu No.4 and Yushu No.12 were relatively strong.For sweet potato types,SPVD infection could be found in the starch-type sweet potato,dual-purpose sweet potato,and edible red-fleshed sweet potato and purple-fleshed sweet potato varieties(lines).The typical symptoms of SPVD generally appear on the top young leaves of plants firstly,and then the whole plant showed distorted or chlorotic dwarf symptom,and finally the plants in the vicinity of the infected plants will also be infected.During the seedling growth stage,the dispersal of SPVD was relatively serious,and there was no large-scale dispersal of SPVD in the later stage of plant growth.Artificial friction test showed the friction of the main stem and lateral branch stem could cause SPVD infection easily,while friction of the leaf could result in leaf damage and therefore reduce the infection of SPVD.The plants gengrated from the storage roots of those plants without obvious SPVD symptoms and were diagnosed as healthy plants and harvested in 2014 would also show SPVD symptoms in 2015,indicating that SPVD might be contained in the harvested storage roots.During the year of 2014 and 2015,by using sweet potato variety Xushu No.22 and setting up 8 independent blocks,we measured the effect of SPVD prevention and control methods,such as cutting off the infected leaf,pulling out the disease plants,spraying pesticide,placing yellow sticky traps,placing fly net,placing trap lamp,using of virus-free seedling.The results showed that,after two years of treatment,the largest number of infected plants were those under the control treatment.The plants treated using the methods of pulling out the disease seedlings,spraying pesticide,placing fly net and using virus-free seedling were not infected.After being treated using the method of cutting off the infected leaf,the plants were also found to be infected.There was a small amount of plants,under the trestment of placing yellow sticky traps and placing trap lamp,were also found to be infected.Our results proved that there are several measures of preventing the large-scale spread of SPVD to some extent,such as the selecting and breeding of varieties resistant to SPVD,strictly preventing the seedling-stage SPVD infection,monitoring and removeing the plants with SPVD infected top young leaves.In addition,due to the existence of SPVD latent infection,the seed storage roots harvested from SPVD infected area should not be used.The experiment proved that the elimination of the pathogen or virus media could effectively prevent and control the occurrence of SPVD.Furthermore,in the production practice,virus-free seedling,pulling out the disease plants timely,placing fly net and spraying pesticide were feasible and effective means of prevention and control of SPVD.However,the methods of cutting off the infected leaves,placing yellow sticky traps and placing trap lamp exhibited no obvious effect on SPVD prevention.2.Establishment of SPVD pathogeny detection method through fluorescence quantitative RT-PCRThe leaves of sweet potato cultivars/lines with typical SPVD symptoms were collected,and the partial genes encoding coat protein(CP)of SPFMV and heat shock protein70(HSP70)of SPCSV were cloned from the diseased leaves.Fluorescence quantitative PCR(q RT-PCR)was used to detectSPFMV CP and SPCSV HSP70 by employing primers designed according to the conserved nucleotide sequences.The rapid and accurate protocol for SPFMV and SPCSV detection was established by comparison with the typical SPVD symptoms and virus detection results by NCM-ELISA,and the detection results were also compared among leaves collected from different sweet potato cultivars/lines,among different plants collected from the same cultivar/line,and among top young leaves and mature leaves in the middle collected from the same plant.Cluster analysis showed that there were at least two types of SPFMV strains(EA and RC)and one type of SPCSV strain(WA)existing in the tested sweet potato leaves.Among the 15 tested cultivars/lines,10 cultivars/lines were detected as co-infected by SPFMV and SPCSV by employing NCM-ELISA detection and q RT-PCR methods.The results obtained from q RT-PCR detection corresponded to that from NCM-ELISA testing,indicating that the q RT-PCR detection method developed in this study could accurately reflect the co-infection degree of SPFMV and SPCSV in the sweet potato plants.Differential transcriptional levels of SPFMV CP and SPCSV HSP70 were detected in diseased leaves from different cultivars/lines or different plants of the same cultivar/line,and even in top young leaves and mature leaves in the middle from the same plant by using q RT-PCR.The detected transcriptional levels of SPFMV CP in the tested diseased leaves were higher than and were 3-556 times of that of SPCSV HSP70.In addition,q RT-PCR showed a transcriptional level of SPCSV HSP70 in two of four cultivars/lines which were detected to be only infected by SPFMV but not infected by SPCSV through NCM-ELISA detection,indicating the present q RT-PCR detection method was more sensitive and accurate when compared to the NCM-ELISA method.The q RT-PCR detection method of SPVD was established by detecting the total RNA of plant leaves.The transcription level of the genes encoding coat protein(CP)of SPFMV and heat shock protein70(HSP70)of SPCSV could indirectly reflect virus copy numbers,which makes this method more convenient and high efficient.This method could provide a useful tool for the monitoring and early warning of SPVD.3.Comparative transcriptome analysis of healthy and SPVD pathogeny-infected sweet potatoThe sweet potato cultivar 0841-14,which was susceptible to SPVD,was selected,and transcriptome sequencing used the top young leaves of both healthy plants and SPVD-infected plants that near the same plants infected by SPVD.A total of 11120 differentially expressed unigene were obtained,among which 6032 were up-regulated and 5088 were down-regulated.GO function enrichment and KEGG pathway enrichment analysis showed that the differentially expressed genes were mainly involved in the biological process of the process of metabolism,cellular process and response stimulation.Furthermore,those genes mainly involved in molecular functions such as binding,catalytic activity,transport,transcription factor activity,etc.,were up-regulated or down-regulated.SPVD infection of sweet potato might affect secondary compounds metabolism,translation process,carbohydrate metabolism,energy metabolism,and many another pathways such as ketone and terpenoids metabolism in the sweet potato plants.SPVD pathogeny could bring about several consequences to sweet potato plants.The photosynthetic efficiency decreased,and the metabolic pathway of phenylalanine severely damaged.The expression of genes encoding the key enzymes of generating flavonoids,lignin and phytoalexin were inhibited,parts of plant hormones content and resistance gene expression changed significantly.The data obtained in this study provided valuable information for the study of sweet potato genome,and provided a transcriptome clue for revealing the mechanism of SPVD pathogeny infection.In this study,the occurrence and propagation and the varieties resistance of SPVD were investigated,and the current commonly used viral disease prevention and control methods were tested,and the effective methods for SPVD prevention and control were found out.A rapid detection method of SPVD pathogeny using fluorescence quantitative RT-PCR was established,and a preliminary understanding of the response to SPVD pathogeny infection in sweet potato was obtained.Our study will useful in revealing the pathogenic mechanism of SPVD and the molecular mechanism of disease resistance in sweet potato,and will provide important information for molecular breeding of sweet potato.