| powdery mildew fungus caused by Ucinula necator is one of the most serious diseases in grape production worldwide.It is also the focus of control and monitoring research in wine grape production in the eastern foot of Helan Mountain in Ningxia.The current methods for early diagnosis and detection of powdery mildew fungus are time-consuming,laborious and lack of accuracy,which is difficult to meet the monitoring needs of overwintering and latent bacterial sources.Therefore,a simple,rapid and accurate detection method for powdery mildew fungus is urgently needed in production.Therefore,LAMP rapid detection method for powdery mildew is establish in this study to provide technical support for detection of grape samples in the field.Quantitative detection of latent bacteria counts is the key to early monitoring and early warning of the disease.However,there is still a lack of systematic studies on the relationship between latent bacteria counts of Powdery mildew and the occurrence of field diseases.Therefore,in this study,real-time Quantitative Polymerase Chain Reaction.Establishment of real-time PCR detection system,Molecular-detected disease Index(MDI)and Field disease Index(DI)The incubation period of wine grape powdery mildew is systematically study by using Arc GIS technology and other methods,in order to provide theoretical basis for the early warning and prevention of wine grape powdery mildew in the eastern foothill of Helan Mountain.The results are as follows:1.The samples of infected grape powdery mildew leaves were collected from 7 different grape planting areas in the eastern foot of Helan,Ningxia.15 strains were isolated by single spot.The primer TUB2 was designed according to the sequence of TUB2_Fw/TUB2_Rev and IGS_FW/NS1R,213 bp and 295 bp fragments were obtained respectively,and the CAPS analysis showed that the powdery mildew of wine grape in this area was genotype B;Primers Uc592/Uc946 were designed according to the sequence of CYP51 gene,and a 379 bp fragment was amplified SSCP analysis showed that powdery mildew of wine grape had two subtypes(races)in this area2.LAMP primers were designed according to the full length gene sequence of CYP51 of U.necator,and the optimal reaction conditions of LAMP were UNITF3-1/UNITB3-1 as external primers and UNITFIP-1/UNITBIP-1 as internal primers.The concentration of Bst DNA polymerase was 0.32 U·μL-1,betaine was 0.8 mol·L-1,dNTPs was 2.6 mmol·L-1,Mg2+was 4.8 mmol·L-1,the ratio of internal and external primers was 6:1,and the reaction time was 60 min at 62℃.A minimum of 1 pg·μL-1 of powdery mildew was detected.The established system is suitable for the detection of grape samples in the field,the reaction i stable and easy to operate.3.The results showed that the lowest detection limits of primer F-g-6/R-g-6 and F-P450-Un/R-Un for grape leaf DNA and U.necator DNA by gradient dilution were 1.0×10-2 ng·μL-1 and 1.0×10-5 ng·μL-1,which were 100 and 1000 times higher than those of conventional PCR.The standard curves of grape leaf DNA and U.necator DNA were established,which were y1=26.62-3.18x(R2=0.99637)and y2=17.44-3.03x(R2=0.99564).There was a good linear relationship between the cycle threshold of the standard curve and the template concentration.Using the established real-time PCR detection method to detect 30 field samples,a total of 22 samples were detected to contain U.necator.There was a very significant correlation between MDI and DI,and the correlation coefficient was 0.916.4.Three wine grape plots in three different ecological areas in the eastern foot of Helan were sampled and investigated.Using the established real-time PCR detection system of grape and grape powdery mildew,the correlation between MDI and field investigation DI was analyzed.The results showed that there was a very significant correlation between MDI and DI in the three plots,MDI of the three sample plots had the highest fitting with di after 18 days of sampling(R12=0.943,R22=0.956,R32=0.966).ArcGIS was used to simulate the amount of bacteria in the incubation period and the actual occurrence in the field of grape powdery mildew from the perspective of space.The results showed that the amount of bacteria in the incubation period and the occurrence in the field of the three experimental plots showed different spatial structures.The semi variogram was exponential model,Gaussian model and spherical model,and the spatial pattern of the field population was aggregation distribution.The prediction model[y=3.521x+0.684(R2=0.810)]was constructed by using MDI and DI to predict the field DI after 18 days.The predicted DI of the sample plot was basically consistent with the actual DI,and the coincidence rate was 96.6%.It shows that MDI can effectively predict the epidemic degree and occurrence trend of wine grape powdery mildew in the field,and realize visualization through Arc GIS technology. |
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