Establishment And Application Of Loop-mediated Isotheral Amplication(LAMP) In Detection Of Rhizoctonia Solani AG1-IA And Ustilaginoidea Virens

Rice sheath blight and rice false smut are two important diseases that seriously affect rice yield and quality during the growth of rice.With the increase of fertilization levels and the diversification of planting systems,the occurrence of rice sheath blight and rice blight is becoming more and more serious.In this study,the molecular identification method was used to clarify the pathogens and anastomosis groups of rice sheath blight in major rice regions in northeast China.Loop-mediated isothermal amplification?LAMP?detection systems for Rhizoctomia solani AG1-IA and Ustilaginoidea virens were established respectively and the accurate detection of two pathogens in the field was achieved.The specific research results are as follows:Molecular identification of pathogens and anastomosis groups of rice sheath blight in three provinces of northeast China.From 2015 to 2017,214 strains of rice sheath blight were isolated from 17 main rice producing areas in Heilongjiang,Jilin and Liaoning provinces.The specific primers of different pathogens and anastomosis groups of rice sheath blight pathogen were used to identify the pathogens and anastomosis group of 214 isolates,and sequence of the internal transcription spacer of r DNA?ITS?was used to analyze the belonging of the anastomosis group of Rhizoctonia oryzae-sativae.The results showed that 214 isolates belonged to two pathogens.For Rhizoctonia solani and Rhizoctonia oryzae-sativae,the number of strains was 198 and 16,accounting for 92.52% and 7.48%,respectively.Rhizoctonia solani strains belong to two anastomosis groups,the number of AG1-IA and AG4 strains was 191 and 7 accounting for 96.46% and 3.54%,respectively.Phylogenetic analysis of ITS sequence showed that Rhizoctonia oryzae-sativae belonged to AG-Bb.In different years,the occurrence frequency and regional distribution of different populations and anastomosis groups of rice sheath blight pathogens had no significant change,but different populations and anastomosis groups of rice sheath blight pathogens in different regions had obvious differentiation characteristics.AG1-IA distributed in all rice producing areas,and it was the major anastomosis group with the highest frequency in Panjin,Liaoning province.AG-Bb of Rhizoctonia oryzae-sativae appeared most frequently in Jilin city,Tonghua city and Meihekou city of Jilin province.Development and application of a loop-mediated isothermal amplification assay for Rhizoctonia solani AG1-IA.In this study,Rhizoctomia solani AG1-IA r DNA-ITS was used as the target gene sequence.Based on the loop-mediated isothermal amplification technology,4specific LAMP primers and 1 loop primer were designed and a RS-ITS-LAMP rapid detection system based on the color changes of hydroxynaphthol blue?HNB?was established.Moreover,the reaction temperature,reaction time and 6 main factors concentration of the reaction system were optimized to determine a best reaction system.The optimal reaction conditions were determined to be constant temperature amplification at 64? for 60 min.The optimal reaction system was 10×Isothermal Amplification Buffer 2.5 ?L,MgSO₄?6 mmol/L?1.5 ?L,betaine?1.4 mol/L?3.5 ?L,d NTPs?1.8 mmol/L?4.5 ?L,outer primer RS-F3/B3?0.2?mol/L?0.5 ?L,inner primer RS-FIP/BIP?1.0 ?mol/L?2.5 ?L,loop primer RS-LB?0.4?mol/L?1 ?L,Bst DNA polymerase?0.32 U/?L?1 ?L,DNA template 1 ?L,HNB?192?mol/L?2 ?L,and dd H2 O supplemented to 25 ?L.The specificity verification results showed that DNA of Rhizoctonia solani AG1-IA strains showed a positive blue reaction after DNA amplification.However,the test strains of other soil-habitating fungi,rice common pathogenic fungi and other pathogenic pathogens showed a violet negative reaction.The results of sensitivity tests showed that the detection limit of RS-ITS-LAMP assay for Rhizoctonia solani AG1-IA is 1 pg·?L?-1? of genomic DNA,which is two orders of magnitude higher than conventional PCR technology.The results of the application of the detection system showed that the positive results of LAMP detection in the artificially inoculated plants tissue,field diseased plants tissue and field soil were positive.Development and application of a loop-mediated isothermal amplification assay for Ustilaginoidea virens.With the Translation elongation factor 1? of Ustilaginoidea virens as target gene sequence,four specific LAMP primers and two loop primers were designed.A simple,rapid and sensitive UV-tef-LAMP detection system based on SYBR Green I color visualization was established.Moreover,the reaction temperature,reaction time and 6 major factors concentration were optimized to determine the optimal reaction system.The optimal reaction conditions were determined to be constant temperature amplification at 65? for 60 min.The optimal reaction system was 10×Isothermal Amplification Buffer 2.5 ?L,MgSO₄?6mmol/L?1.5 ?L,betaine?1.2 mol/L?3 ?L,d NTPs?1.4 mmol/L?3.5 ?L,outer primer UV-F3/B3?0.2 ?mol/L?0.5 ?L,inner primer UV-FIP/BIP?1.0 ?mol/L?2.5 ?L,loop primer UV-LF/LB?0.6 ?mol/L?1.5 ?L,Bst DNA polymerase?0.32 U/?L?1 ?L,DNA template 1 ?L,and dd H2 O were added to 25 ?L.After the reaction was completed,0.25 ?L of SYBR Green I was added.The specificity verification results showed that the reaction solution after DNA amplification of Ustilaginoidea virens strains from different sources by adding SYBR Green I showed a yellow-green positive response,while DNA of other soil-habitating fungi,ricecommon pathogenic fungi and other pathogenic pathogens strains all showed orange negative reactions.In the sensitivity test,the minimum detection limit of UV-tef-LAMP technology is100 fg·?L?-1?.The application results of this detection system show that the minimum detection limit of soil samples with conidia of Ustilaginoidea virens is 10 conidia/250 mg soil.The sensitivity limit of soil samples with artificially added thick spore powder was 0.4 ?g/g.The detection of rice false smut balls in different disease forms in the field was positive,and the soil samples in the field could also detect Ustilaginoidea virens.