Development Of The Loop-mediated Isothermal Amplification Assay For Detecting Tobaccomosaic Virus In Soil And Analysis Of Thetransmitted Efficiency Of The Soil

Tobacco mosaic virus(TMV) is a very important pathogen which causes serious diseases on crops, such as tobacco and tomato, around the world. In Chinese major tobacco-growing areas, the disease even leads the 5%-20% loss. Previous studies proved that the soil contaminated with TMV play a important role in the occurrence and prevalence of TMV. However, the propagation efficiency of TMV in soil has not yet been reported.In this study, based on the development of Loop-mediated isothermal amplification(LAMP) assay for detecting TMV, we detected the TMV in the soil of tobacco rhizosphere and the tabacco leaves that cellected from field in Guiyang, Hunan Province. We also cloned and sequenced the coat protein sequence of TMV detected from the soil of tobacco rhizosphere and the tabacco leaves, and anaylzed the relationship to confirm that the TMV in the tabacco leaves were from the the soil of tobacco rhizosphere. Furher more, experiments were also carried out in the green house to evaluate the the propagation efficiency of TMV in soil. Our results are as follows:1. The total RNA was extracted from the tobacco rhizosphere soil collected from field in Guiyang, Hunan Province in 2014 using the soil EZNA TM Soil RNA Kit,respectively. Wiht the specific primer pairs FIP \ BIP and F3 \ B3, we developed the LAMP for detecting TMV. The reaction conditions of LAMP, including the concentrations of d NTPs, Mg2+, reaction temperature and the reaction times were optimized.The specificity and sensitivity of LAMP were testified. The optimum RT-LAMP can be carried out under the concentrations of 4 mmol·L-1 Mg2+ and 6mmol·L-1 d NTPs. LAMP reaction was carried out at 61℃ for 40 min. Sensitivity of the RT-LAMP assay was 100-folder than that of a standard reverse-transcription polymerase chain reaction(RT-PCR)2. In the field planted tobacco, we used nylon mesh to cover the tobacco for preventing the insect. 35 leaves and the soils of tobacco rhizosphere were collected from the covered tobacco every 15 days for three times, and were used as samples fordetceting TMV. The RNA of all collected samples was extracted and used as templated for reverse transcription, respectively. After reverse transcription, LAMP method was used to detect TMV. Tobaccoes infeceted by TMV and its rhizosphere soil contaminated with TMV accounted for the total number of infected rhizosphere soil percentage from the initial 12% increase to 96%, the tobacco infected rate of 8.57% initially, increased to94.28%, a larger increase; in laboratory greenhouse, the experimental group are 6tobaccos, there are 5 infected TMV, the incidence of 83.33%. The control 1 and control2 have 2 and 1 infected TMVrespectively, the incidence are 33.33% and 16.67%. The study prove that soil has a high spread efficiency of TMV with exact data.3. The analysis of CP sequences of TMV from soil samples GYS1, GYS3,GYS6 and tobacco samples GYL1, GYL2, GYL3, GYL5, GYL6, GYL7 in Guiyang showed that the nucleotide homology between tobacco and soil samples is high,, which are 96.3%-99.4%. In the phylogenetic tree, the strain KJ406323.1 from Russia,LN651235.1, AM412008.1 from Vietnam, HQ896025.2 from Henan, China and the strains of our samples are all in the same branch.There are highly similarity among them.Nucleotide sequence homology with the HQ896025.2 strain are 97.1%-98.1%.In summary, our results showed that the LAMP is high specific, sensitive and economical, which makes it a quick method for detecting TMV from the soil. The soil contaminated with TMV play a important role in the occurrence and prevalence of TMV.The analysis of TMV CP sequence homologyshowed that TMV detected in the soil and the leaves was the same strain. Our results provided a basis for the future control of TMV caused diseases.