Molecular Detection Techniques Of Kiwifruit Crown Gall Pathogens

Kiwifruit crown gall is a root disease of Kiwifruit caused by bacteria,which can cause root tissue swelling and malformation of kiwifruit,affecting nutrition absorption and normal physiological function of Kiwifruit,in severe cases,it can lead to plant death.In this study,kiwifruit root cancer was studied.The pathogenic bacteria of kiwifruit crown gall were isolated and purified by tissue separation,using morphological and molecular biological methods to identify pathogen,to clarify the pathogen species of Kiwifruit crown gall.By using genome-specific genes to establish kiwifruit crown gall,the rapid PCR detection system and loop-mediated isothermal amplification（LAMP）technology.The rapid detection methods provide technical support basis for the early diagnosis of Kiwifruit crown gall.The main research results are as follows:（1）Through morphological observation,physiological and biochemical reactions,and molecular biological techniques,combined with 16S r DNA and Rec A-gyr B-atp D-rpl B polygene sequence analysis,the pathogenic bacteria of kiwifruit crown gall in Guizhou Province were identified as Agrobacterium fabacearum.The biological characteristics showed that the optimal carbon source for culture of A.fabacearum.pathogen was arabinose,nitrogen source was（NH₄）₂HPO₄,inorganic salt was K₂HPO₄,and 28℃was the optimum temperature,p H value was8.0,the inoculation amount was 5%.（2）The PCR rapid detection Aar F/R and Afa F/R were screened and determined as the best primers to establish a PCR rapid detection technique for kiwifruit root cancer pathogen.The results showed that no specific amplification for the near-source species of the strains and Agrobacterium spp.Isolated in the healthy soil of kiwifruit crown gall,and the pathogen of A.fabacearum amplified specific bands.The optimum annealing temperature Aar F/R primers were 53℃,the number of cycles was 35 times,and the detection limit of DNA optimized Aar F/R primers was 5×10^-2ng/μL.The optimal annealing temperature and cycles of Afa F/R primers was 55.9°C and 40 cycles,and the minimum detection concentration was 5×10^-3 ng/μL.Two pairs of Aar F/R and Afa F/R primers were used to detect the field samples.The results showed that the genome of the diseased soil and the diseased tissue DNA amplified specific bands,and there was no specific amplification in the healthy tissue and the healthy soil,which indicated that Aar F/R and Afa F/R primers had strong specificity and were suitable for the early diagnosis of kiwifruit root cancer.（3）The LAMP primer design and screening showed that the primer group 48-Lamp and 9-Lamp had high specificity,and both pairs of primer groups could amplify A.fabacearum,but no amplification effect on other strains.The sensitivity of48-Lamp and-Lamp and 9-Lamp primer groups were detected by fluorescence visual and agarose gel electrophoresis.The results showed that the primer group of 9-Lamp had high sensitivity,and the minimum detection limit of genome DNA was5×10^-8 ng/μL.The primer group 48-Lamp and 9-Lamp detection system was applied to the genomic DNA of kiwifruit tissue and soil in the field suspected samples of kiwifruit crown gall.The diseased soil and tissue could be amplified by LAMP,and the fluorescence visual test results were positive,while the healthy soil and tissue had no LAMP amplification reaction.In this study,the pathogens of kiwifruit crown gall were separated and identified,combined with comparative genomes to establish a specific and efficient PCR detection technology and a convenience,specific,and sensitive LAMP detection technology to achieve molecular detection of kiwifruit crown gall.Establishing a molecular detection system for kiwifruit crown gall provides technical support for the early diagnosis of the disease,which is beneficial to the timely,scientific and effective prevention and control of kiwifruit crown gall and reduces economic losses,which has important guiding significance.