| Viroid is an important class of pathogens that can cause many serious plant diseases.It is also the simplest self-replicating infectious agent currently known in nature.The genome of the viroid is small circ?Lar RNA with the size is generally in the range of 200-400 nt and does not encode proteins.Viroids are divided into two families,Pospiviroidae and Avsunviroidae according to replication strategy.To date,ICTV classifies 44 types of known viroids into 7 genera.In this study,we tested seven representative viroids: Citrus exocortis viroid(CSVd),Iresine viroid 1(IrVd-1),Potato spindle tuber viroid(PSTVd),Apple scar skin viroid(ASSVd),Avocado Sunblotch Viroid(ASBVd),Hop stunt viroid(HSVd),Peach Latent Mosaic Viroid(PLMVd).for their ability to replicate in filamentous plant pathogenic fungi,Cryphonectria parasitica as well as genetic mutation of viroid in fungi.In addition,the effects of viroid infection on the growth,development and pathogenicity of the host fungi were observed.This topic provides new research ideas and scientific basis for understanding the host range,pathogenesis and potential biological control use of viroids.By constructing infectious clones of seven viroids,in-vitro transcripts of viroid were first inoc?Lated into tobacco and infected yeast strains to detect their activities and then the viroids were artificially introduced into C.parasitica protoplasts by protoplast transfection.After the cells wereregenerated,the transfectants were cultured on the PDA medium and the fungal single-stranded RNA wasextracted and used for the RT-PCR detection of viroids infection.The effects of stable infection of viroids on the growth,development and pathogenicity of host fungi were also observed.The results are listed as follows:1.Construction of seven types of viroid infectious cDNA clones: Plasmids of seven kinds of viroids,ASBVd,HSVd,IRVd,PLMVd,ASSVd,IRVd and CSVd were prepared by oligonucleotide synthesis method.After obtaining in-vitro transcripts,they were tested in yeast strain AH109 for the infectivity.2.Investigation of the ability of seven kinds of viroids to infect filamentous fungi:After introducing seven type of viroids into C.parasitica protoplasts,RT-PCR showed that ASBVd,HSVd,and IRVd co?Ld replicate in C.parasitica.The replication of ASBVd was more stable than the other two.Total RNA of ASBVd-infected fungal strains were extracted after subc?Lture and the results show that the replication of ASBVd did not significantly decrease,while the replication of the other two viroids gradually decreased.3.Analysis of the effect of ASBVd infection on C.parasitica: Infection of ASBVdinhibited the growth of C.parasitica on PDA medium and the laboratory test for fungal pathogenicity found that ASBVd also has an effect on C.parasitica pathogenicity.4.Analysis of the spread of ASBVd in fungal hosts: Similar to fungal viruses,viroid co?Ld be vertically and horizontally transmitted.ASBVd was transmitted by host hyphal fusion as confirmed by RT-PCR.C.parasitica infected with ASBVd was cultured under light.After spor?Lation,the spores were isolated,cultured,and the single-stranded RNA of the fungus was extracted and used for RT-PCR detection,The result showed that viroid was vertically transmitted through spores.5.Analysis of the genetic variation of ASBVd in the fungal host: After the viroids infects the filamentous fungus,the infected strain was subcultured and the RNA of the 8th generation strain was extracted for RT-PCR detection.Sequence analysis showed that viroid sequence become mutated after subculture.6.Comparison of ASBVd wild-type and mutant strains and their effects on host fungi:After the ASBVd mutant strain infected the fungus,the amount of fungal aerial hyphae increased although the growth rate did not change significantly.Viroid RNA detection showed that the replication of ASBVd decreased compared to that of wild type. |
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