Establishment Of LAMP Rapid Detection System For Apple Diseases In Yantai

As of 2018,more than 100 apple diseases have been reported.These diseases can be divided into leaf diseases,stem diseases,fruit diseases and root diseases according to the location of occurrence.Apple from Yantai has been selected as the first brand of fruit industry in China for 10 consecutive years.However,the apple diseases has brought huge economic losses to the apple industry.At present,the control methods of Apple disease are mainly prophylaxis and early treatment,so it is very important to identify and detect the pathogen at the right time.Due to the similarity of the disease,and the separation and identification requires very long cultivation time,the traditional morphological separation and identification methods can no longer meet the needs of today.And the common molecular biology detection methods have strict requirements for experimental operations and test instruments,so it is not suitable for routine testing in the field.Therefore,the establishment of a fast,accurate and efficient detection system is of great significance for the timely and effective control of various diseases of apples.Loop-mediated isothemal amplification（LAMP）is widely used in medicine,food,health and other fields.This technology has also been used in the detection of plant pathogens too.It provides a new idea for the early detection of plant pathogens in the future.In this study,Lamp fast detection method of five common fungal diseases（Apple mould core,Apple canker,Apple bitter rot,Apple spot leaf drop and apple red spot disease）of apple in Yantai area was established.At the same time,the detection conditions were optimized and verified in the field.The follows are the research results:1.LAMP and PCR specific primers were designed for the internal reference genes and specific genes of four pathogenic fungi.2.Five methods of genomic DNA extraction were screened,and three methods of DNA extraction suitable for the four pathogenic fungi in this study were obtained.They are All-DNA-Fast-out,KOH-PEG and Kit method for DNA extraction of plant genome.All of these three methods can successfully extract the mycelial DNA of four pathogenic bacteria.But in the field sample detection experiment of lamp detection system,it is found that All-DNA-Fast-out is only suitable for extracting the DNA of pathogenic bacteria in leaves,and the Kit method for DNA extraction of plant genome is suitable for extracting the DNA of pathogenic bacteria in leaves,branches and fruits.3.Lamp fast detection method of Apple mould core,Apple canker,Apple bitter rot,Apple spot leaf drop and apple red spot disease were established successfully.This method has very high interspecific specificity.It can testing Colletotrichum gloeosporioides 10^-9ng/μL in 75 min,testing Trichothecium roseum 10^-99 ng/μL in 90 min,testing Alternaria Alternaria 10^-66 ng/μL in 90 min,and testing Valsa mali 10^-99 ng/μL in 90 minutes.4.The LAMP detection system was optimized in this study.It was found that the best reaction time and temperature of four pathogenic fungi were 60 min and 65℃.The optimum final concentration of reaction of Apple canker’s FIP/BIP was 0.6μmol/L,and the optimum final concentration of reaction of other diseases’s FIP/BIP were 0.4μmol/L.5.In order to determine the feasibility of the LAMP detection method,the LAMP detection system established in this experiment was used to specific detecte five kinds of samples collected in the experimental field of Yantai Academy of Agricultural Sciences,including twenty healthy samples and twenty suspected infected samples.The results are compared with PCR detection and traditional isolation and identification detection methods,we found that the PCR and LAMP detection rates of the four pathogenic fungi were found to be higher than those of traditional separation and detection methods,while the detection rates of LAMP detection were higher than PCR detection.