Research On The Detection Technology Of Phytoplasma Related To Sisal Purple Leafroll Disease And Its Dynamics In Plants

Sisal purple leaf curl is a devastating disease that has occurred on sisal（Agave sisalana）in recent years.The research of this subject shows that sisal purple leafroll disease is caused by phytoplasma.Because the phytoplasma has the characteristics of being unable to be cultivated,low abundance in the plant and uneven distribution.Therefore,the establishment of efficient molecular detection technology is very necessary for the monitoring and detection of phytoplasma diseases.In this study,a high-efficiency single-tube nested PCR detection and real-time fluorescent quantitative LAMP detection technology system for sisal purple leafroll disease-related phytoplasma was established.The former is preferably used to monitor the seasonal changes of sisal purple leafroll disease-related phytoplasma.First,a single-factor experiment was performed on the annealing temperature of the inner and outer primers of the single-tube nested PCR,and the optimal inner and outer primer annealing temperature was screened in a certain temperature range.After determining the optimal annealing temperature of the inner and outer primers,16 combinations were designed by orthogonal experiments for factors such as the concentration of inner and outer primers,the content of d NTPs,and the amount of Ex Taq enzyme in the single-tube nested PCR reaction system.The test results show that the amplification effect is best when 64°C is determined as the annealing temperature of the outer primer Sis-F1/R1 and 54°C is determined as the annealing temperature of the inner primer Sis-F2/R2.The best reaction system（25μL）finally determined through single factor experiment and orthogonal design experiment is:5μL of d NTPs at a concentration of 2.5 mmol/L,10×Ex Taq Buffer 2.5μL,Ex Taq DNA Polymerase 1μL,inner primer Sis-F2/R2 at a concentration of 15μmol/L,1μL each,at a concentration of 0.02μmol/L The outer primer Sis-F1/R1 is 1μL each,the template DNA is 1μL,and finally 11.5μL dd H₂O is added.The above reaction system can only detect bands from the DNA template of the diseased purple leafroll disease plant,which has a high degree of specificity.The minimum detection limit of the detection system is the relevant phytoplasma plasmid DNA concentration≥1 fg/μL.Secondly,through the screening of real-time fluorescent quantitative LAMP primers,the primer set LAMP-2 was selected from the two sets of LAMP primers for real-time fluorescent quantitative loop-mediated isothermal amplification.Through the orthogonal design method,an optimized combination screening experiment was carried out on the factors affecting the LAMP reaction system’s internal and external primer concentration comparison,the amount of Bst DNA polymerase,and the Mg²⁺concentration.The best reaction system（25μL）for real-time fluorescence quantitative LAMP amplification finally screened out is:10×Bst-DNA Polymerase Buffer 2.5μL;10 m mol/L d NTPs 3.5μL;5 m mol/L F3 and B3 each 1μL;20 mmol/L FIP and BIP each 1.5μL;100 m mol/L Mg²⁺1.25μL;Bst 2.0 DNA Polymerase 0.5μL;SYTO-9 green fluorescent dye 1μL;DNA template 1μL;finally add 10.25μL dd H₂O.Under the condition of 63℃,the detection system can only show an amplification curve with a clear inflection point and a clear exponential phase from the template of the purple leafroll disease diseased DNA,and the Ct value is less than 35.0;Conversely,the amplified DNA samples of other non-purple leafroll disease strains were all a straight line.That is to say,the established real-time fluorescent LAMP reaction of phytoplasma related to sisal purple leafroll disease is specific.At the same time,the detection system can specifically detect sisal purple leafroll disease-related phytoplasma plasmid DNA concentration≥1 fg/μL.Third,to explore the changes in the content of related phytoplasma in purple leaf roll disease sisal plants at different times,a single-tube nested PCR detection technology system was used to monitor the relative temporal and spatial changes of phytoplasma in purple leaf roll disease diseased plants.The results showed that the relative content distribution of phytoplasma in each part of the sisal plant was uneven.The relative content of roots was highest in March and lowest in December.The temperature change may also affect the relative content of phytoplasma.The single-tube nested PCR detection and real-time fluorescent quantitative LAMP detection technology system for sisal purple leaf roll disease-related phytoplasma established in this research can be used for subsequent disease monitoring,correlation investigation,pathogenicity,and functional verification.