Molecular Detection And Identification Of Phytoplasmas And Viruses Associated With Brassica Napus

Rapeseed(Brassica napus L.)is an important cruciferous crop worldwide.It is often threatened by many diseases.There is few research about phytoplama diseases on rapeseed and no report of them in China.In this study,we carried out disease identification of rapeseed samples with suspected diseases from Wuhan and Ezhou,Hubei Province,E'erguna,Neimenggu Province and Mianyang,Sichuan Province,China and found that some samples were infected by phytoplasmas while the others harboured various viruses.Phytoplasmas were successfully determined with Nested-PCR amplification using phytoplasma-specific primer pairs in samples from Wuhan and Ezhou,Hubei Province and E'erguna,Neimenggu Province.Phylogenetic analysis was carried out and these discovered phytoplasmas were grouped with online tool,iPhyClassifier.As a result,phytoplasmas of EZ and NMG with witches'-broom,and WH3 with dwarf belonged to subgroup 16SrI-B,while phytoplasmas of WH1 with fasciation and WH2 with peduncle clustering were members of subgroup 16SrV-H.This is the first report of phytoplasmas infecting rapeseed in China and of group 16 SrV infecting rapeseed.The host range of groups 16 SrI and 16 SrV was further investigated,and group 16 SrI was found from 307 plant species that belong to 230 genera of 83 families distributing in 56 countries/regions,while group 16 SrV from 65 species in 47 genera of 30 families,most of which are woody plants and only 9 are herbaceous plants.Phytoplasmas of subgroups 16SrI-A and 16SrI-B were found to infect crucifer,and phytoplasmas infecting rapeseeds mainly distributed in Canada,China,Croatia,Czech Republic,France,Greece,Iran,Poland,and USA,while subgroups 16 SrVB and 16SrV-H were identified from crucifer.The presence of phytoplasmas of group 16 SrV suggests the possible host expansion and host jumping and the potential risk of phytoplasmas on rapeseed.Rapeseed samples with witches'-broom and dwarf collected from Mianyang,Sichuan Province and samples with deformed,narrow and long leaves and chlorosis spot,and even with dwarf and/or witches'-broom collected from Wuhan,Hubei Province were hypothesized to be infected by viruses or other pathogens since phytoplasmas couldn't detected from them.In this case,total RNA was extracted and used for RNA-Seq to exhibit the virome.Various viruses were dected in the RNA samples,most of which are +ssRNA viruses.In samples from Mianyang,Brassica yellows virus(BrYV)and 2 kinds of Brassica yellows virus associated RNAs were identified with high FPKM values,their FPKM values were 10500.94,142424.44 and 99514.26,respectively.Many contigs were found to share high identity with 17 kinds of mycoviruses including Mitovirus,Ourmia-like virus,Narnavirus,Endornavirus,Totivirus and-ssRNA viruses,because their FPKM values were much lower than these of BrYV and its associated RNAs,suggested these viruses might from fungi living on diseased rapeseed.There were less virus-related contigs in RNA samples of Wuhan.A partial genome RNA of BrYV with a FPKM of 674515.44 was detected,suggested that Wuhan diseased samples might also be infected by BrYV.Interestingly,a virus,named Brassica napus associated narnavirus 2(BrNANV 2),had been identified in Wuhan diseased rapeseed,BrNANV 2 was similar to Phytophthora infestans RNA virus 4,the amino acid identiy and similarity of RNA replicase between the two were 79% and 87%.The FPKM of BrNANV 2 was 334215.53,about half of that of BrYV,suggesting that BrNANV 2 might infect an oomycete which lived on diseased rapeseed or might directly infect rapeseed.In general,phytoplasmas and viruses have been identified from rapeseed with different symptoms in this stusy.This is the first report of phytoplasmas infecting rapeseed in China and of group 16 SrV infecting rapeseed.BrYV and its associated RNAs,and viruses that might infect fungi/oomycete have been identifited.These findings might help to protect rapeseed from diseases and enrich our knowledge on the diversity of viruses.