Establishment And Application Of PCR And Near Infrared Spectroscopy Fast Assay For The Detection Of Citrus Huanglongbing

Citrus Huanglongbing(HLB) is one of the most devastating diseases of citrus industry and the quarantine disease of citrus industry around the world.HLB takes place in many of citrus-producing areas,and the development of citrus industry is badly restricted.HLB can lead to a series of typical symptoms of the infected trees that tree vigor declined,yellow shoots with blotchy mottling of leaves,uniformly yellowing,or lopsided or color inversion fruits (so-called‘red nose fruits’).The pathogen of Citrus Huanglongbing is one of Gram-negative bacteria,which belong to Candidatus Liberibacter.The pathogen can be divided into Candidatus Liberibacter asiaticus,Candidatus Liberibacter africanus and Candidatus Liberibacter americanus according to the sequence of 16 S rDNA,region resource and the thermo sensitivity of pathogenic bacteria.Because the Koch’s postulates of disease have not been completely fulfilled and the difficulty of pure culture of the pathogen,the study about Citrus Huanglongbing in depth is facing the serious challenge.We have a horrible problem related to the prevention and control measures about Citrus Huanglongbing,and the reasons are as follows: firstly,almost the plant of Citrus of Rutaceae family and Diaphorina citri Kuwayama can be infected by the pathogen;in addition,the pathogen can be spread by infected-scion in a long distance propagation or infectedcitrus psylla in a short range spread;what’s more,no curative chemicals and resistant varieties or cultivars can be taken.The prevention and control measure of HLB mainly focus on clearing away diseased tree,cultivating virus-free seedling of citrus and killing ACP.Research on detection of HLB,not only is the guarantee of the breeding of virus-free seedling of citrus,and the technical support of research on species of HLB and distribution of disease,but also is the important diseases prevention and control measures of containing the spread of disease.In this study,the following four aspects have been focused: 1.In order to providing the technical support of DNA extraction for pathogens from insects quickly or accurately,research on three methods of DNA extraction for Candidatus Liberibacter asiaticus from individual adult asian citrus psyllid was carried out.2.Genome of suspicious leaf samples were extracted by four DNA extraction methods of CTAB-Trion method,magnetic bead method,cracking method and soaking extracting method,and detected by LAMP assay for Citrus Huanglongbing.One DNA extraction method that can be applied to a great amount of samples for Cirtrus Huanglongbing detection has been filtrated.3.Establishment and application of fast detection of Citrus Huanglongbing by near infrared spectroscopy can provide a new method of HLB detection in field.The main results obtained are as follows.1.Three methods were taken to extracting DNA of individual adult Asian citrus psyllid,which was cultivated on sweet orange with Candidatus Liberibacter asiaticus (CLas) infected in greenhouse.The efficiency of different DNA extraction methods were evaluated by comparing the concentration,purity,PCR products of DNA,respectively.The results showed that threemethods can extract DNA of CLas and citrus psyllid.Cracking method was simple,rapid and the concentration of DNA sample was higher,while the purity was low.The method of chloroformisoamylol was complex and the concentration of DNA sample was low,but the purity was high.Animal Tissue Direct PCR Kit was simplest,the concentration was high,while the costs was high.Three methods have advantages and shortcoming,respectively.2.Several DNA extraction methods apply to a great amount of citrus samples for Citrus Huanglongbing LAMP assay were compared.Providing technical support for the prevention and control of Citrus Huanglongbing.DNA genome of citrus leaves with suspicious Huanglongbing symptoms was prepared,using CTAB-Trion method,magnetic bead method,cracking method,soaking extracting method,and amplified by LAMP and PCR,respectively.Optimal method will evaluate based on preparing procedure,operation program,the quality of DNA,prepare time,spend costs and environment-friendly.The results showed the DNA prepared by CTAB-Trion method and magnetic bead method was the best,the range of OD260/OD280 was 1.8-2.0,and the DNA can be detected by LAMP,but preparing procedure is tedious,need long preparing time and spend more costs,what’s more,CTAB-Trion method need phenol and chloroform reagents.The DNA prepared by cracking method was useful to detection although DNA quality was not as good as CTAB-Trion and magnetic bead method,furthermore preparing procedure easy,environment-friendly,short prepare time and less costs.The DNA extracted by soaking extracting method wasn’t suitable for LAMP assay because of low Purity and concentration.Cracking method was superior to other methods for LAMP assay.It would be an easy and simple to handle,eco-friendly,the results can be visualized,economical,time saving,needn’t expensive and special instrument and can apply to a great amount of samples for Citrus Huanglongbing detection with LAMP assay.3.Research on near infrared spectroscopy fast assay for the detection of citrus Huanglongbing shows that near infrared spectroscopy can apply to the detection of citrus Huanglongbing according to specific chromatogram,the optimal discriminating analysis method is PLS-LDA,and the ideal Pre-processing method of Spectral signals is MSC.The coincidence rate of the detection result of the near infrared spectroscopy fast assay for the detection of citrus Huanglongbing and PCR detection is 91% applying to 720 samples of navel orange leaves from farm.