JIA-2021-1450 一种基于腋芽离体再生技术的柑橘黄龙病菌快速增殖系统的构建

doi:10.1016/S2095-3119(21)63856-X

摘要/Abstract

摘要：

本研究以感染黄龙病菌亚洲种（‘Candidatus Liberibacter asiaticus’, CLas）的长叶橙（Citrus sinensis Osbeck）为材料，建立CLas侵染长叶橙的茎段培养方法，利用PCR和荧光定量PCR检测分析腋芽离体再生嫩梢中CLas的增殖情况。在此基础上，取CLas侵染长叶橙枝条进行表面消毒处理，切取腋芽嫁接于柑橘试管砧木，进行试管培养，利用PCR和荧光定量PCR检测腋芽再生嫩梢中CLas的增殖规律；采用直接组织印迹免疫法（Direct tissue blot immune assay，DTBIA）对试管苗中CLas的分布进行检测。培养基中添加适合浓度的激素可促进感病柑橘茎段离体培养的萌芽，萌芽率最高的培养基为MS+6-BA 1.0 mg·L^-1+GA 0.2 mg·L^-1+IAA 0.2 mg·L^-1。茎段萌芽30 d时，嫩梢病原PCR检测的阳性率达75%，其CLas浓度平均为温室条件下培养茎段原叶片中脉的28.2倍，最高为484.2倍。试管嫁接苗萌芽10、15、20、25、30和40 d时，嫩梢CLas PCR检测的阳性率分别为10%、15%、15%、20%、55%和70%，CLas浓度分别为7.5×10⁴ 、2.2×10⁶ 、1.4×10⁷、2.2×10⁷ 、1.2×10⁸和1.4×10⁸ cells μg^-1 DNA；萌芽30和40 d的试管苗中CLas浓度超过10⁸的比例分别达30%和40%。DTBIA检测结果显示，试管苗中黄龙病菌的分布比温室保存的感病柑橘中均匀。CLas侵染柑橘试管苗的主要症状为嫩梢枯死、停止生长、叶黄和落叶。感病试管苗的死亡率在萌芽40 d后会急剧升高，60 d时死亡率达82.0%。CLas在通过腋芽嫁接的柑橘试管苗中能快速增殖，该方法的建立为深入开展柑橘黄龙病病原生物学、病原-寄主互作及抗菌药物快速筛选等研究奠定一定基础。本研究建立了一种快速增殖和高浓度富集柑橘黄龙病菌的培养方法。

Abstract: ‘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). Firstly, 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), and it 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 7.5×10⁴ to 1.4×10⁸ cells μg^-1 of citrus DNA during the first 40 DAG. The percentages of shoots with >1×10⁸ CLas cells μg^-1 DNA were 30% and 40% at 30 and 40 DAG, respectively. Direct tissue blot immune assay (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 in vitro culture. This method will be useful for studying plant–HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.

Key words: Citrus , 'Candidatus Liberibacter asiaticus' , multiplication , in vitro citrus plantlets

. JIA-2021-1450 一种基于腋芽离体再生技术的柑橘黄龙病菌快速增殖系统的构建[J]. Journal of Integrative Agriculture, 2022, 21(6): 1683-1693.

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. A rapid multiplication system for 'Candidatus Liberibacter asiaticus' through regeneration of axillary buds in vitro[J]. Journal of Integrative Agriculture, 2022, 21(6): 1683-1693.

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