Construction Of The Co-culture System Of Candidatus Liberibacter Asiaticus

Citrus Huanglongbing(HLB) —— is the lethal disease of the citrus industry caused by phloem-limiting bacteria — Candidatus Liberibacter, only Candidatus Liberobacter asiaticum been found in China currently. The culture of the pathogen is the key to overcome Huanglongbing. Although scientists have put much manpower and many material resources in the pathogen culture still can not get the pure culture which limits the development of pathogenesis and etiology research greatly. So far, there is still have no effective methods of disease control. Overview the previous studies, the traditional pure culture method mostly been used. Pure culture method misunderstand the causes and nature of the reason of uncultured, ignoring the interactive relationship between environmental symbiotic microbes and target pathogenic bacteria. In this study, the traditional pure culture method has been altered. We use the diseased citrus plants as culture material, optimize the culture system, depend on the q PCR technique to monitor target bacteria, screening the associated bacteria which associated with a growth promoting effect on the pathogen, establish a co-culture system which simulate the original ecology environment for pathogen and associated bacteria.Samples(roots, branches, leaves and fruits) has been collected from Guangxi, Guangdong, Zhejiang, Fujian, Hunan. The optimized co-culture system include: the culture sample material use diseased citrus fruit pith; surface disinfection use 95% high concentrations ethanol ignition surface method; the co-culture medium(MCLA medium) formulations include: QS inducer(c AMP, AHL), growth factors(NADP, NAD, ATP, Rpf), anti-Oxygen toxicity(CAT, POD, sodium thioglycollate, sodium pyruvate), fungicide(cycloheximide), natural environment factor(citrus juice, citric acid, amino acid), phosphate buffer solution(p H6.4) et al.; 28 ℃, dark and microaerobic culture conditions. We use quantitative Real-time PCR(q PCR) to determine the change of pathogen population.Microaerobic and aerobic cultivation was used to separate the endophyte after surface disinfection. Associated bacteria will be added to Candidatus Liberobacter asiaticum culture system and been cultured under the facultative anaerobic cocultivition conditions. 63 strains from the host plant phloem endophytic bacteria was used as the research object, 3 strains were screened have the promotion of Huanglongbing pathogen and 5 strains have the inhibition. We has been successfully proliferated the pathogen of Huanglong(Candidatus Liberobacter asiaticum) in liquid culture conditions by use the Huanglongbing co-culture system Currently, q PCR detection confirm that the Proliferation rate of Candidatus Liberobacter asiaticum is up to 50 times. The co-culture system realized the co-cultured of Ca. L. asiaticum and associated bacteria basically, the research completed the stable and effective cocultivition system.The completion of co-culture system not only to promote the basic research of the etiology and pathogen biology of Candidatus Liberobacter asiaticum, but also help the developmen and research of antiseptic and development of early diagnosis. This research provide a new way for the prevention and control of HLB.