Detection Of Viral Pathogens In Onions And Garlic In Inner Mongolia Autonomous Region And Establishment Of A Multiplex PCR Detection System For Five Garlic Viral Pathogens

Onion(Allium cepa)and garlic(Alium sativum)are the main horticultural crops in Inner Mongolia Autonomous Region,with high edible and medicinal value.The cultivation mode has been promoted in Inner Mongolia Autonomous Region,and the planting area and annual output rank among the top.However,due to the cold climate in the main cultivation area and the prevalence of onion yellow dwarf virus(OYDV)and other viral diseases,the situation is becoming increasingly severe.In order to detect the main viral pathogens related to Allium vegetables in Inner Mongolia Autonomous Region,this study used conventional RT-PCR technology to identify and detect viral pathogens in onions,garlic,and surrounding weeds in the main cultivation area.The results showed that the detection rates of viral pathogens in onions were 22.4%(30/134)for Shallot yellow stripe virus(SYSV),25.4%(34/134)for OYDV,7.5%(10/134)for Leek yellow stripe virus(LYSV),and 9.0%(12/134)for Cucumber mosaic virus(CMV).The detection rates of viral pathogens in garlic were 80.0%(56/70)for OYDV,77.1%(50/70)for LYSV,20.0%(14/70)for Garlic latent virus(GLV),10.0%(7/70)for CMV,10.0%(7/70)for Garlic virus A(GVA),2.9%(2/70)for Garlic virus B(GVB),5.7%(4/70)for Garlic virus D(GVD),1.4%(1/70)for Garlic virus E(GVE),and 21.4%(15/70)for Garlic virus X(GVX).The detection rates of viral pathogens in surrounding weeds were 20%(2/10)for SYSV and 10%(1/10)for OYDV.Phylogenetic analysis of Inner Mongolia isolates of ten viral pathogens based on the ML tree constructed using the effective nucleotide sequences showed two main distribution patterns.CMV and SYSV were the first pattern,with different sources of cloned sequences clustered on the same branch,and the others were the second pattern,with isolates from various leagues distributed on two or more branches.The ML tree constructed based on the amino acid sequences was distributed in multiple groups(except for one cloned sequence of GVE not within the scope of analysis).The results of the consistency analysis between the effective nucleotide sequences and their corresponding reference sequences showed that the variations between the ten viral pathogen isolates and their corresponding reference sequences were large,with the highest range being 75.1%-99.2%for LYSV and the lowest being 80.4%-92.7%for SYSV.In addition,several onion plants exhibiting prominent symptoms of viral diseases were found to be negative for known viral pathogens.Therefore,leaf samples were collected from these symptomatic onion plants,pooled,and subjected to high-throughput sequencing.A nearly complete genome sequence of SYSV was obtained from the sequencing data.Subsequently,using RACE(Rapid Amplification of cDNA Ends)technology,the complete genome sequence of SYSV was obtained.Genetic diversity analysis,phylogenetic analysis,sequence alignment analysis,and recombination analysis were performed on the isolated strains of SYSV The results revealed the following:(1)SYSV evolution follows a neutral evolution model and is less influenced by environmental factors;(2)The P1 coding region exhibited a high nucleotide polymorphism coefficient and low sequence conservation,indicating a highly variable feature in this region;(3)SYSV exhibited distinct geographic variations;(4)Five recombination events were identified in the complete genome sequence of SYSVThis study designed multiple sets of primers targeting the five common viral pathogens(GLV,GVD,GVX,LYSV,OYDV)of garlic based on conventional PCR detection.Through optimization of factors such as internal reference selection,annealing temperature,extension time,and primer concentration,a multiplex PCR detection system was established,which can simultaneously detect the five major viral pathogens of garlic(GLV,GVD,GVX,LYSV,OYDV).The reaction system includes 2 X M5 Hiper Ultrafast Mix 10 μ L and different amounts of upstream and downstream primers for each virus.The sample cDNA template is 4 μL.The reaction program includes 40 cycles of denaturation at 94℃ for 30s,annealing at 56℃ for 30s,and extension at 72℃ for 1min,followed by a final extension at 72℃ for 10min.The multiplex system has been preliminarily applied to garlic samples from 15 provinces in Inner Mongolia,Yunnan,Anhui,Henan,Hebei,Shaanxi,Liaoning,Guizhou,Sichuan,Zhejiang,Shandong,Jiangsu,Hubei,and Shanxi.The results show that the multiplex PCR system has strong specificity,and although the resolution of individual virus amplification bands is low or unstable,it does not affect the accuracy of the detection results.Therefore,this system can be promoted and applied.