Molecular Detection Of Viral Diseases In Chewing Cane From Guangdong And Effect Evaluation Of Virus-free Seedlings

Chewing cane is the main economic crops in southern China.The chewing cane variety is mainly’Badila’,which is single variety,and it is asexual reproduction crops.It is easy to be infected by virus and cause many kinds of virus diseases,which can reduce the yield and quality of chewing cane.The production of virus-free chewing cane seedlings is the main way to control the virus disease.In this study,molecular detection of virus diseases in chewing cane growing areas in Guang dong province was carried out to identify the main types and occurrence of virus in chewing cane areas,so as to provide target virus for virus-free of seedlings.On the other hand,the virus removal rate,production performance and physiological and biochemical characteristics of virus-free seedlings were evaluated,which provided scientific basis for the development of virus-free chewing cane seedlings.The main findings are as follows:（1）In this study,we developed a visualized,rapid,simple,accurate and sensitive reverse transcription loop-mediated isothermal amplification（RT-LAMP）assay for the detection of Sugarcane streak mosaic virus（SCSMV）and Sugarcane mosaic virus（SCMV）in sugarcane.Screen out the specific primer set of SCSMV（P2-F3:5’-CCAGCGACAAC ATCAAGCAT-3’;P2-B3:5’-CGCCCAATC GTTGAACTGT-3’;P2-FIP:5’-GGCGGAACGTACAGGGATTTCACAACGAGTCAAGC TGGTAGT-3’;P2-BIP:5’-CGCTCAAAACAGAAGCCAAGGCGGCGGC TGATGAAGAGATG-3’）and SCMV specific primer set（C1-F3:5’-CTTTC TGTTAACATACAAACCG-3’;C1-B3:5’-CATTGT CCAACTTCCGTTTA-3’;C1-FIP:5’-ATGGC TTCATACCATCTATCAAAC TCAAC AAC AAGAC ATATCAAACAC-3’;C1-BIP:5’-GATGACACACAAATGAC AGTTGTCGTGAG CAACCGTTC TCAA-3’）.It can be used for specific detection of target virus.The technology can be useful for sugarcane quarantine and production of virus-free chewing cane seedlings.This is the first report of a RT-LAMP based assay for the detection of SCSMV in sugarcane.（2）The virus species and disease occurrence in major chewing cane producing areas in Guang dong were investigated.One hundred and six samples of chewing cane with suspected symptoms of mosaic disease were collected.The molecular detection results showed that the positive detection rates of SrMV,SCMV and SCYLV were 97.2%,98.1% and 95.3%,respectively.The co-infection rates of SrMV and SCMV;SrMV and SCYLV;SCMV and SCYLV;and SrMV,SCMV,and SCYLV were 95.3%,92.5%,93.4%,and 90.6%,respectively.No positive samples of SCSMV,JGMV,MDMV,SCBV were detected in any of the samples.Sugarcane mosaic disease and sugarcane yellow leaf disease usually occurred in chewing cane planted in Guang dong.These findings will be helpful for the development of virus-free chewing cane seedlings in the future.（3）To survey the virus elimination effect of virus-free chewing cane seedlings,we collected 83 samples of virus-free seedling from heeling-in nursery,first nursery,secondary nursery and production field to detect virus.The molecular detection results showed that the positive detection rate of SCMV was 3.61%.The positive sample of SrMV was not found.The positive detection rate of SCYLV was 93.97%.In this study,the virus-free seedlings developed by techniques of shoot apical meristem culture can effectively remove SCMV and SrMV in the chewing cane seedlings,but the removal effect of SCYLV needs further study.（4）The production performance,physiological and biochemical characteristics of virus-free chewing cane seedlings were evaluated.The results show:（1）Compared with infected seedlings,the stem yield of virus-free seedlings was increased by 88.41%-98.11%,plant height increased by 48.66-57.49cm,the internode length increased by 2.16-2.68cm,the single stem weight increased by 67.80%-168.09%,the reducing sugar increased by 0.91-1.15 percentage points,the sucrose content increased by-0.06-1.33 percentage points,and the bending resistance increased by 106.49%-189.73%.Virus-free seedlings significantly increase the yield,quality and lodging resistance of chewing cane.（2）The chlorophyll content,photosynthetic index（Pn,Gs and Tr）,activity and relative gene expression of photosynthetic key enzymes（PEPC and Rubisco）of the virus-free seedling were higher than the infected seedlings during the growth,indicating that the virus-free seedlings can increase the chlorophyll content,photosynthetic key enzyme activity and gene expression,thereby improving photosynthesis efficiency and promoting photosynthesis.（3）The content of O^2-and MDA of the virus-free seedlings was lower than the infected seedlings during the growth.The activity of antioxidant defense enzymes SOD,POD and CAT of the virus-free seedlings was gradually higher than that of infected seedlings with the development of the growth period.It indicated that the active oxygen had less damage to the virus-free seedlings,and the degree of membrane lipid peroxidation was low.The virus-free chewing cane seedlings had higher antioxidant enzyme activity,and it is concluded that the virus-free seedlings had improved the resistance to stress.