A rapid multiplication system for'Candidatus Liberibacter asiaticus'through regeneration of axillary buds in vitro  

作　　者：LEI Tian-gang HE Yong-rui ZOU Xiu-ping WANG Xue-feng FU Shi-min PENG Ai-hong XU Lan-zhen YAO Li-xiao CHEN Shan-chun ZHOU Chang-yong 

机构地区：[1]Citrus Research Institute,Southwest University/National Citrus Engineering Research Center,Chongqing 400712,P.R.China

出　　处：《Journal of Integrative Agriculture》2022年第6期1683-1693,共11页农业科学学报（英文版）

基　　金：supported by the National Key R&D Program of China (2018YFD0201500 and 2018YFD1000300);the National Natural Science Foundation of China (31972393);the China Agriculture Research System of MOF and MARA (CARS-26)

摘　　要：‘Candidatus Liberibacter asiaticus(CLas)’,which causes citrus Huanglongbing(HLB)disease,has not been successfully cultured in vitro to date.Here,a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected‘Changyecheng’sweet orange(Citrus sinensis Osbeck).Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds.A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination(DAG).This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse.To minimize contamination during in vitro regeneration,CLas-infected axillary buds were micrografted onto seedlings of‘Changyecheng’sweet orange and cultured in a liquid medium.In this culture,the titers of CLas in regenerated shoots rapidly increased from 7.5×10^(4)to 1.4×10^(8)cellsμg^(-1)of citrus DNA during the first 40 DAG.The percentages of shoots with>1×10^(8)CLas cellsμg^(-1)DNA were 30 and 40%at 30 and 40 DAG,respectively.Direct tissue blot immunoassay(DTBIA)indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse.The disease symptoms in the plantlets were die-back,stunted growth,leaf necrosis/yellowing,and defoliation.The death rate of the plantlets was 82.0%at 60 DAG.Our results show that CLas can effectively multiply in citrus plantlests in vitro.This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.

关 键 词：CITRUS ‘Candidatus Liberibacter asiaticus’ MULTIPLICATION in vitro citrus plantlets 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]