Development Loop-mediated Isothermal Amplification (LAMP) For Detection Of Resistant Diseases In Strawberry

Strawberry anthracnose is caused by Colletotrichum gloeosporioides and Botrytis cinerea can lead to strawberry gray mold,which are the main diseases in the agricultural production of strawberry.Chemical control is the main means of preventing and curing anthracnose and gray mold diseases in strawberry production.The level of control effect will depen on the resistant of pathogenic fungi to fungicide.The traditional method for detecting resistance is isolated and purified the pathogen,then transfered to the potato dextrose agar medium containing fungicide,according to the growth inhibition effect of the agent on the pathogen to identify the resistant isolates,which takes 5 to 7 days even extended to a few weeks,and is time-consu ming and laborious.To achieve rapid detection of pathogen resistance,Loop-mediated isothermal amplification(LAMP)was established to obtain reliable detection results within 1-2 h,which will rapidly detect and monitor the pathogen resistance and the resistance development,so as to guide the medication reasonably.[Method]For gray mold disease,the rapid detection of LAMP assays for resistant-pathogen were established in this study.The specificity and mismatched LAMP primers were screened to identifiy the resistant strains.Components and conditions of LAMP reaction were optimized,then LAMP assay were used to detect field samples of trawberry gray mold and then samples were taken back to the laboratory for isolation and culture,and the traditional distinguishing dosage method was used to test the resistance,the results were compared with the LAMP assay to validate the LAMP assay for the dynamic monitoring of resistant populations and the assessment of resistance risk.For anthracnose,the rapid detection system of LAMP was established for the detection of anthracnose occurrence in strawberry seedling stage,after that,isolates were collected,isolated and detected the resistance of the pathogen by distinguishing dosage method,and the molecular mechanism of resistance was analyzed,based on the above,developing the LAMP assay to rapid detect resistance.[Purpose]Strawberry anthracnose is caused by Colletotrichum gloeosporioides and Botrytis cinerea can lead to strawberry gray mold,which are the main diseases in the agricultural production of strawberry.Benzimidazole and Qo inhibitor(QoI)fungicides are frequently used for disease management.However,there are many hosts of C.gloeosporioides and B.cinerea,at the choice pressure of the fungicide,once the resistance occurs,the level of resistance will develop rapidly for its short life cycle and prolific reproduction rate.And they have characteristics of many mutation sites and latent infection,so the rapid detection and monitoring of pathogen resistance have become particularly important to guide the rational use of fungicides.[Method]In this paper,the resistance of pathogens was detected by distinguishing dosage method,and the molecular mechanism of pathogen resistance was further explored.The rapid molecular detection system of resistance and latent infection was established:Loop-mediated isothermal amplification(LAMP)Technology to detect field samples,through the mismatched base primers specific identification of resistant strains,and the difference between the measurement method of comparison test for the dynamic monitoring of drug-resistant groups,resistance risk assessment study.[Result]1.Development a visual technique for the on-site rapid monitoring of carbendazim-resistant Botrytis cinerea using loop-mediated isothermal amplification.The LAMP mismatched primers,based on the E198A(GAG→GCG)point mutation,were designed to detect the E198A genotype specifically.HNB acted as a visual LAMP reaction indicator that turned the violet colored into a sky blue color.The detection limit of this assay was 100 copies and 10 times more than PCR.This LAMP assay could be applied to detect B.cinerea with the E198A genotype with100%accuracy compared with distinguishing dosage method.2.In this study,we established a Loop-mediated isothermal amplification(LAMP)system for the monitoring and evaluation of the risk of development of B.cinerea resistance to QoI fungicides;the method uses two LAMP assays.The first assay detects G143A mutants of B.cinerea,which are highly resistance to QoI fungicides.BCbi143/144 introns in B.cinerea are thendetected by the second assay.HNB acts as a visual LAMP reaction indicator.The optimum reaction conditions of the LAMP assays were 61°C for 50min,and the detection limit of the LAMP assays was 100×10~-44 ng/μL.We directly pre-treated the field samples by using All-DNA-Fast-Out to extract DNA within ten minutes,then performed the LAMP assay to achieve one-step rapid detection.In conclusion,we established a rapid and sensitive LAMP assay system for resistance risk assessment and for monitoring QoI-resistance of B.cinerea in the field.3.A rapid detection system for direct detection of strawberry anthracnose from plant on the plant body was developed.The field sample pretreatment was quickly carried out by LFD(lateral lateral flow test paper)and alkaline polyethylene glycol extraction for the purpose of detecting strawberry anthrax.4.The rapid detection system of LAMP was established for the specific detection of C.gloeosporioides ofthe G143A mutant genotype with the high resistance to QoI fungicides and E198A mutant genotypewith the high benzimidazole fungicides,respectively.The results are consistent with distinguishing dosage method.[Conclusion]The results of this study can achieve the purpose of rapid detection of strawberry diseases resistance in field,and it is great practical significance to adjust the strategy of strawberry production,improving the effect of drug control and reducing the dosage of fungicides.