| Tomato spotted wilt virus（TSWV）is the representative member of the Tospoviridae in Bunyavirales,it is listed as one of the ten species of plant viruses that are the most harmful in the world.Pepper is an important vegetable in China,Chongqing is one of the main pepper producing and consumption areas in China,thus,pepper planting becomes one of the key industries in Chongqing.TSWV was detected on pepper in Chongqing area in 2015,and it has been widespread in Chongqing.It is clear that the resistance of peper varieties to TSWV in Chongqing area provides better evidence for breeders,and is of great significance for TSWV controlling in Chongqing pepper.In this study,TSWV was inoculated by artificial friction on the main varieties of pepper planted Chongqing.Resistance analysis of main pepper varieties by Dot enzyme linked immunosorbent assay（Dot-ELISA）,Reverse transcription polymerase chain reaction（RT-PCR）and real-time fluorescence quantitative PCR（Real-time quantitative PCR）,further analyzed the virus accumulation of TSWV in different main cultivars of peper,to determine the resistance of the main varieties of Chongqing peper.Finally,the genetic variation of TSWV on different resistant peper was analyzed.Through friction inoculation,8 varieties of pepper,which called Yu La NO.10,YuJiao NO.12,YuJiao NO.5,Yan Jiao 425,ZaoChun,Yan Jiao 11,Yan Jiao 417and ChaoTianJiao were inoculated with TSWV.The incidence of different pepper varieties was detected by ELISA and RT-PCR at 20 dpi;Symptom observation after inoculation,combined with the classification standard of tomato Verticillium virus disease,the disease index of different varieties of pepper was analyzed.The results showed that the detection results of ELISA were in accordance with the results of RT-PCR;the incidence and disease index of Yan Jiao 417 were the highest（51.4%and 61.11）,Yan Jiao 417 is the most sensitive variety to TSWV;the second was the Yan Jiao 425,Yu La NO.10、YuJiao NO.12 and ChaoTianJiao are more sensitive to TSWV;the resistance of ZaoChun and YuJiao NO.5 to TSWV was relatively strong;the resistance of Yan Jiao 11 to TSWV was the strongest,and the incidence and disease index were 17.1%and 25.In order to further clarify the resistance of 8 pepper varieties to TSWV.Real time fluorescence quantitative PCR Technology was used for the detection of virus accumulation in different resistant pepper.Furthermore,the resistance of the main cultivars to tomato Verticillium wilt virus was further confirmed.The result showed that among the 8 different pepper varieties,the accumulation of TSWV virus in Yan Jiao417 was the highest;followed by ChaoTianJiao,Yan Jiao 425,Yu La NO.10,YuJiao NO.12,ZaoChun and YuJiao NO.5;the accumulation of TSWV virus in Yan Jiao 11was the least.The results further confirmed that Yan Jiao 417 was a susceptible variety,and Yan Jiao 11 was a resistant variety.The accumulation level of TSWV in different pepper varieties varies in different periods,the accumulation of TSWV was measured at 15 dpi、30 dpi、60 dpi、80 dpi、100 dpi、150 dpi,.After inoculation,the accumulation of TSWV was increased first and then decreased.TSWV replication and accumulation in Yan Jiao 417、Yu La NO.10、Yan Jiao 425、ZaoChun and ChaoTianJiao reached the peak at 60 dpi;TSWV replication and accumulation in Yan Jiao 11、YuJiao NO.12 and YuJiao NO.5 reached the peak of at 85 dpi.Yan Jiao 417 and Yan Jiao 11 are Chongqing pepper varieties,but the resistance varies greatly.To clarify the level of variation of TSWV in these two pepper varieties.In this study,TSWV was inoculated with pepper cultivar Yan Jiao 417 and Yan Jiao 11with different resistance.Amplification gene of non-structural proteins of TSWV in inoculated plants.The construction of the early（50 dpi）and late（150 dpi）varieties of Yan Jiao 417 and the early（50 dpi）and late（150 dpi）populations of Yan Jiao 11 were studied,analys the genetic structure and variation of the four populations.The results of the analysis showed that the genetic diversity of the TSWV NSs genome population was different in different resistance peppers.The haplotype diversity and nucleotide diversity of TSWV NSs genomic population in Yan Jiao 417 were:0.284±0.102 and0.00068±0.00042.The haplotype diversity and nucleotide diversity of TSWV NSs genomic population in Yan Jiao 11 were:0.649±0.086 and 0.00088±0.00022.TSWV NSs gene did not detect recombination on these two types of pepper.The genetic structure of the four populations is consistent with the"quasispecies"characteristic of the virus.The percentage of mutated clones in the early population of Yan Jiao 11,the late Yan Jiao 11 period,the early Yan Jiao 417,and the late Yan Jiao 417 population were:42.10%、38.89%、6.67%、16.67%,and each mutation frequency are:5.30×10-4、4.43×10-4、1.32×10-4、1.48×10-4.It can be seen that the genetic variation of the NSs genome of TSWV in Yan Jiao 11 is higher than that of Yan Jiao 417.The comparison of the main sequences of the four populations revealed that the main sequence homology of the early and late of Yan Jiao 11 was 100%,and that of Yan Jiao 417 was also 100%.However,compare the alignment of the main sequence of Yanjiao 11,the main sequence of Yanjiao 417,and the sequence of the NSs gene on the inoculated Nicotiana tabacum,a A→G mutation in the NSs gene of TSWV was found on the 766nucleotide sequence of the 1500 nucleotide sequence of Yan Jiao 11.However,there was no mutation in the NSs gene of Yanjiao 417 and the inoculated N.benthamiana,but the mutation in this site resulted in the change of glutamic acid to glycine.In summary,this study identified the resistance of 8 varieties of peppers planted in Chongqing to TSWV.Among them,Yanjiao 417 was the most sensitive to infection by TSWV,and Yanjiao 11 showed the strongest resistance;The analysis of the genetic variation in pepper 417 and Yanjiao 11 showed that the genetic variation of TSWV in Yanjiao 11 was even more abundant.This study has certain reference value against the breeding of TSWV pepper varieties and the effective prevention and control of TSWV induced diseases. |
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