Study On Pathogen Identification And Detection Technique Of Garlic Virus

The garlic in HeiLongJiang was widely infected by complex viruses,through investigating.Natural infected garlic crops exhibited diversiform symptoms such as masaic,crimple,leaf roll,abnormality,etiolation,putrescence and so on.In my experiment,the viruses infecting garlics were detected and identified by serology,bioassay,electron microscope,molecular biological techniques.Through bioassay,indicator hosts were inoculated by garlic viruses,the garlic viruse pathogenys were elementarily identified.The biologic assay validated that Allium porrum,Allium cepa,Vicia faba and Gomphrena globosa was better indicator host of LYSV,OYDV,GCLV and PVX respectively.Virus particles were observed in purified sap from infected garlic or infected indicator host.Two kinds of flexous filamentous particles were observed,the one was about 300-1000nm long,the other was about 1850-2000nm long.Virus particles were observed by electron microscopy ultra-thin section.,there were two kinds of virus particles,the one species was filamentous,the other was rhabdoviruses.They exist in cytoplasm and formed various inclusion bodys.Long filamentous and rhabdoviruses were first found in garlic. The leaves were detected by DAS-ELISA,and the result showed that masaic garlic contained LYSV and PVX,etiolation garlic contained OYDV and GCLV,leaf roll garlic contained GCLV,LYSV and OYDV.Thereinto,PVX were first detected in garlic.The serological assay for 37 garlic field samples in HeiLongJiang showed that the infection rate of LYSV,GCLV,OYDV,ShLV and PVX was 95%,86%,74%,50% and 78% respectively and most of them were complex infected.With the specific primers which were designed based on the Garlic common latent virus (GCLV) coat protein conserved sequence (with optimizing the detection system),one gene fragment (300bp) was amplified by reverse transcription polymerase chain reaction (RT-PCR) using the total RNA of the plant infected by GCLV.The gene was choned and sequenced.The result was compared with the sequence of other isolates of GCLV in GenBank.The result showed that the highest homology could reach 98% of nucleic acid and 97% of amino acid.The detecting specification and sensitivity were verified.With the specific primers which were designed based on the Leek yellow stripe virus (LYSV) nucleic acid conserved sequence,one gene fragment (1032bp) was amplified by RT-PCR using the total RNA of the plant infected by LYSV.The gene was choned and sequenced,the CP sequence was included.The sequence of HLJ was compared with the sequence of the other 23 isolates of LYSV,the result showed that the highest homology could reach 88.6% of nucleic acid and 93.1% of amino acid,which proved that the virus was LYSV.The CP amino acid sequences of different isolates of LYSV were compared,the result showed:these were great differences on the amino acid sequence among the LYSV isolates,the homology rangeed from 78.5～100%.The phylogenic tree of LYSV was clustered into four groups.The isolate of HLJ was in the same group with the isolates of South Korea and Japan,they represented regionally correlation.With the specific primers which were designed based on the Potato virus X(PVX) CP,the gene fragment (714bp) was respectively amplified by RT-PCR with the same reacted condition using the total RNA of garlics and potatoes infected by PVX.The genes were choned and sequenced.The CP sequence of garlic isolate P1 was compared with the sequence of other isolates of PVX,the result showed that the highest homology could reach 97.0% of nucleic acid and 100% of amino acid,which shows that the viruse of PVX was successfully emplified in garilc.The CP nucleic acid sequences homology ranged from 75.0%～100%,the CP amino acid sequences homology ranged from 85.5%～100%.The phylogenic tree of PVX was clustered into two groups.Thereinto,the garlic isolate P1 had high affinity with the potato and rice isolates of Beijing,they represented regionally correlation.Simultaneity,The CP nucleic acid sequences homology could reach 88.3% and the amino acid sequences homology could reach 99.2% between P1 and P2,which showed that garlic was the natural host of PVX.