Identification Of Dominant Viruses Of Pseudostellaria Heterophylla In Fujian Province And Its Genome Sequence Analysis Of TuMV, BBWV2 And CMV

Pseudostellaria heterophylla is a traditional Chinese medicinal material,which is used as medicine with tuber root.It has the functions of replenishing the vital-qi,reinforcing the spleen and improving the weakness after illness.It is mainly produced in Fujian,Anhui and Guizhou Provinces in China and is the important economic characteristic crops of the local areas.Zherong in Fujian Province has the name of "the home of P.heterophylla in China",accounting for 60% of the national output value of P.heterophylla.P.heterophylla primarily propagates through root tubers,and often plant consecutively,causes the viral diseases of P.heterophylla occurred seriously,all these made the yield reduced and quality declined of P.heterophylla.Now virus diseases of P.heterophylla have become an important factor limiting the development of P.heterophylla industry.In this study,a total of 443 P.heterophylla root tubers and plants were collected from seven planting fields of P.heterophylla in Fujian during 2020 and 2022.The detection rate was 95.7% through RT-PCR detection,among which,BBWV2,TuMV and CMV were detected in 312,376 and 294 samples,respectively,with the detection rates of 70.4%,85.9%and 66.4%,respectively.However,TMV was undetectable in this samples.The results showed that BBWV2,TuMV and CMV were the main harmful viruse species of P.heterophylla in Fujian Province.Moreover,the combination infection rate of P.heterophylla virus was 81.3%,which indicates that combination infection in the field is widespread.In addition,to clarify source of the virus disease of P.heterophylla in field.RT-PCR was performed on 16 kinds of weeds collected from the planting area of P.heterophylla,and virus was undetected,and aphids was not observed.It was speculated that the main source of the virus might be the root of P.heterophylla.The whole genome of BBWV2,TuMV and CMV of P.heterophylla in Fujian Province were obtained by RT-PCR and 5’/3’-RACE,and then the genomic organization,sequence analysis,phylogenetic analysis and recombination analysis were carried out.The results showed that,the genome full-length of BBWV2-TFJ RNA1 and RNA2 were 5983 nt and3597nt,respectively,and it is similar to other BBWV2 isolates genomic organization,and the nucleotides of RNA1 and RNA2 from other BBWV2 isolates were 79.4%～86.5% and 72.2%～91.3%,respectively.The phylogenetic analysis of RNA1 and RNA2 nucleotide sequences showed that two large groups and two subgroups were formed in RNA1,among which BBWV2-TFJ RNA1 belonged to subgroup II-e,and it was the closest relative to the Chinese isolate DZ.In RNA2,three large groups and three subgroups were formed,among which BBWV2-TFJ RNA2 belonged to group I,and it was most closely related to Chinese isolates DZ,XC1 and ZR-GX2.RDP4 recombination analysis showed that BBWV2 of P.heterophylla had potential recombination events.The whole genome of TuMV-TFJ was 9833 nt,and it was similar to other TuMV isolates genomic organization,and the nucleotide consistency with other TuMV isolates was 80.1%～95.9%.Phylogenetic analysis based on TuMV nucleotide sequence showed that TuMV isolates formed two large groups and six subgroups.TuMV-TFJ belonged to subgroup world-B3,and it was closely related to AHBL isolated from Anhui Provinces in China.RDP4 recombination analysis showed that TuMV of P.heterophylla had potential recombination events.The whole genome of CMV isolates CMV-TFJ RNA1,RNA2 and RNA3 were 3419 nt,3068nt and 2194 nt,respectively,and it was similar to other CMV isolates genomic organization,and the nucleotides of RNA1,RNA2 and RNA3 from other CMV isolates were 78.3%～97.2%,72.3%～97.9% and 75.3%～97.4%,respectively.Based on the whole nucleotide sequence of CMV,phylogenetic analysis of CMV RNA1,RNA2 and RNA3 showed that,The CMV isolates formed two large subgroups,and subgroup I was divided into subgroup IA and subgroup IB.In RNA1,CMV-TFJ RNA1 belonged to subgroup II,and it was most closely related to the Chinese isolate SL.In RNA2,CMV-TFJ RNA2 belonged to subgroup II,and it had the closest relationship with the Chinese isolate SL.In RNA3,CMV-TFJ RNA3 belonged to subgroup II,and it was closely related to the Chinese isolate SL and the Australian isolate SW-11.RDP4 recombination analysis showed that CMV of P.heterophylla had potential recombination events.In this study,we clear and definite that BBWV2,TuMV and CMV is main pathogens,which causes the occurrence of virus diseases of P.heterophylla in Fujian Province.Molecular cloning and full-length sequence analysis of sequence of BBWV2,TuMV and CMV of P.heterophylla,providing theoretical basis for the prevention and control of virus diseases of P.heterophylla in field.