Please wait a minute...
Journal of Integrative Agriculture  2022, Vol. 21 Issue (6): 1683-1693    DOI: 10.1016/S2095-3119(21)63856-X
Plant Protection Advanced Online Publication | Current Issue | Archive | Adv Search |
A rapid multiplication system for 'Candidatus Liberibacter asiaticus' through regeneration of axillary buds in vitro
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
Citrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, P.R.China
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      

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.
Keywords:  Citrus       'Candidatus Liberibacter asiaticus'        multiplication        in vitro citrus plantlets  
Received: 23 August 2021   Accepted: 19 October 2021
Fund: This work was supported by the National Key R&D Program of China (2018YFD0201500 and 2018YFD1000300), the National Natural Science Foundation of China (31972393), and the China Agriculture Research System of MOF and MARA (CARS-26).  
About author:  LEI Tian-gang, E-mail:; Correspondence CHEN Shan-chun, Tel: +86-23-683409709, E-mail:; ZHOU Chang-yong, Tel: +86-23-68349707, E-mail:

Cite this article: 

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

Bové J M. 2006. Huanglongbing: A destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology, 88, 7–37. 
Bové J M. 2014. Huanglongbing or yellow shoot, a disease of Gondwanan origin: Will it destroy citrus worldwide? Phytoparasitica, 42, 579–583.
Bové J M, Garnier M. 2003. Phloem- and xylem-restricted plant pathogenic bacteria. Plant Science, 164, 423–438. 
Christensen N M, Nicolaisen M, Hansen M, Schulz A. 2004. Distribution of phytoplasmas in infected plants as revealed by real-time PCR and bioimaging. Molecular Plant–Microbe Interactions, 17, 1175–1184.
Coletta-Filho H D, Carlos E F, Alves K C S, Pereira M A R, Boscariol-Camargo R L, de Souza A A, Machado M A. 2010. In planta multiplication and graft transmission of ‘Candidatus Liberibacter asiaticus’ revealed by real-time PCR. European Journal of Plant Pathology, 126, 53–60. 
Davis M J, Mondal S N, Chen H, Rogers M E, Brlansky R H. 2008. Co-cultivation of ‘Candidatus Liberibacter asiaticus’ with actinobacteria from citrus with Huanglongbing. Plant Disease, 92, 1547–1550.
Deng X, Gao Y, Chen J, Pu X, Kong W, Li H. 2012. Curent situation of “Candidatus Liberibacter asiaticus” in Guangdong, China, where citrus Huanglongbing was first described. Journal of Integrative Agriculture, 11, 424–429. 
Ding F, Duan Y P, Paul C, Brlansky R H, Hartung J S. 2015. Localiztion and distribution of ‘Candidatus Liberibacter asiaticus’ in citrus and periwinkle by direct tissue blot immune assay with an anti-OmpA polyconal antibody. PLoS ONE, 10, e0123939. 
Ding F, Paul C, Brlansky R, Hartung J S. 2017. Immune tissue print and immune capture-PCR for diagnosis and detection of Candidatus Liberibacter asiaticus. Scientific Reports, 7, 46467.
Duan Y, Zhou L, Hall D G, Li W, Doddapaneni H, Lin H, Liu L, Vahling C M, Gabriel D W, Williams K P, Dickerman A, Sun Y, Gottwald T. 2009. Complete genome sequence of citrus Huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’ obtained through metagenomics. Molecular Plant-Microbe Interactions, 22, 1011–1020.
Folimonova S Y, Achor D S. 2010. Early events of citrus greening (Huanglongbing) disease development at the ultrastructural level. Phytopathology, 100, 949–958.
Fu S M, Liu H W, Liu Q H, Zhou C Y, Hartung J S. 2019. Detection of ‘Candidatus Liberibacter asiaticus’ in citrus by concurrent tissue print-based qPCR and immunoassay. Plant Pathology, 68, 796–803.
Fujiwara K, Iwanami T, Fujikawa T. 2018. Alterations of Candidates Liberibacter asiaticus-associated microbiota decrease survival of Ca. L. asiaticus in vitro assays. Frontiers in Microbiology, 9, 3089.
Gasparoto M C G, Coletta-Fiho H D, Bassanezi R B, Lopes S A, Lourenco S A, Amorin L. 2012. Influence of temperature on infection and establishment of ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ in citrus plants. Plant Pathology, 61, 658–664. 
Gottwald T R. 2010. Current epidemiological understanding of citrus Huanglongbing. Annual Review of Phytopathology, 48, 119–139. 
Ha P T, He R, Killing N, Brown J K, Omsland A, Gang D R, Beyenal H. 2019. Host-free biofilm culture of “Candidatus Liberibacter asiaticus,” the bacterium associated with Huanglongbing. Biofilm, 1, 100005.
He Y, Chen S, Peng A, Zou X, Xu L, Lei T, Liu X, Yao L. 2011. Production and evaluation of transgenic sweet orange (Citrus sinensis Osbeck) containing bivalent antibacterial peptide genes (Shiva A and Cecropin B) via a novel Agrobacterium-mediated transformation of mature axillary buds. Scientia Horticulturae, 128, 99–107.
Hijaz F, Killiny N. 2014. Collection and chemical composition of phloem sap from Citrus sinensis L. Osbeck (sweet orange). PLoS ONE, 9, e101830.
Huber D M, Haneklaus S. 2007. Managing nutrition to control plant disease. Landbauforschung Völkenrode, 57, 313–322. 
Jagoueix S, Bové J M, Garnier M. 1994. The phloem-limited bacterium of greening disease of citrus is a member of the α subdivision of the Proteobacteria. International Journal of Systematic Bacteriology, 44, 379–386.
Jiang H, Wei W, Saiki T, Kawakita H, Watanabe K, Sato M. 2004. Distribution patterns of mulberry dwarf phytoplasma in reproductive organs, winter buds, and roots of mulberry trees. Journal of General Plant Pathology, 70, 168–173.
Killiny N. 2016. Generous hosts: What makes Madagascar periwinkle (Catharanthus roseus) the perfect experimental host plant for fastidious bacteria? Plant Physiology and Biochemistry, 109, 28–35.
Killiny N. 2017. Metabolite signature of the phloem sap of fourteen citrus varieties with different degrees of tolerance to Candidatus Liberibacter asiaticus. Physiological and Molecular Plant Pathology, 97, 20–29.
Killiny N, Hijza F, El-Shesheny I, Alfaress S, Jones S E, Rogers M E. 2017. Metabolomic analyses of the haemolymph of the Asian citrus psyllid Diaphorina citri, the vector of Huanglongbing. Physiological Entomology, 42, 134–145.
Krystel J, Shi Q, Shaw J, Gupta G, Hall D, Stover E. 2019. An in vitro protocol for rapidly assessing the effects of antimicrobial compounds on the unculturable bacterial plant pathogen, Candidatus Liberibacter asiaticus. Plant Methods, 15, 85–96.
Li T, Zhang L, Deng Y, Deng X, Zheng Z. 2021. Establishment of a Cuscuta campestris-mediated enrichment system for genomic and transcriptomic analyses of ‘Candidatus Liberibacter asiaticus’. Microbial Biotechnology, 14, 737–751. 
Li W, Levy L, Hartung J S. 2009. Quantitative distribution of ‘Candidatus Liberibacter asiaticus’ in citrus plants with citrus Huanglongbing. Phytopathology, 99, 139–144.
Li Y, Xu M, Dai Z, Deng X. 2018. Distribution pattern and titer of Candidatus Liberibacter asiaticus in periwinkle (Catharanthus roseus). Journal of Integrative Agriculture, 17, 2501–2508.
Lin H, Coletta-Filho H D, Han C S, Lou B, Civerolo E L, Machado M A, Gupta G. 2013. Draft genome sequence of “Candidatus Liberibacter americanus” bacterium associated with citrus Huanglongbing in Brazil. Genome Announcements, 1, e00275–e00288. 
Lin H, Pietersen G, Han C, Read D A, Lou B, Gupta G, Civerolo E L. 2015. Complete genome sequence of “Candidatus Liberibacter africanus,” a bacterium associated with citrus Huanglongbing. Genome Announcements, 3, e00733–e00748. 
Liu X, Zheng Y, Wang-pruski G, Gan Y, Zhang B, Hu Q, Du Y, Zhao J, Liu L. 2019. Transcriptome profiling of periwinkle infected with Huanglongbing (‘Candidatus Liberibacter asiaticus’). European Journal of Plant Pathology, 153, 891–906. 
Lopes S A , Bertolini E, Frare G F, Martins E C, Wulff N A, Teixeira D C, Fernandes N G, Cambra M. 2009. Graft transmission efficiencies and multiplication of ‘Candidatus Liberibacter americanus’ and ‘Ca. Liberibacter asiaticus’ in citrus plants. Phytopathology, 99, 301–306.
Louzada E S, Vazquez O E, Braswell W E, Yanev G, Devanaboina M, Kunta M. 2016. Distribution of ‘Candidatus liberibacter asiaticus’ above and below ground in Texas citrus. Phytopathology, 106, 702–709.
Merfa M V, Pérez-López E, Naranjo E, Jain M, Gabriel D W, De La Fuente L. 2019. Progress and Obstacles in Culturing ‘Candidatus Liberibacter asiaticus’, the bacterium associated with Huanglongbing. Phytopathology, 109, 1092–1101.
Nation J E. 2015. Insect Physiology and Biochemistry. CRC  Press, New York.
Parker J K, Wisotsky S R, Johnson E G, Hijaz F M, Killiny N, Hilf M E, De La Fuente L. 2014. Viability of ‘Candidatus Liberibacter asiaticus’ prolonged by addition of citrus juice to culture medium. Phytopathology, 104, 15–26. 
Peng A, Zhang J, Zou X, He Y, Xu L, Lei T, Yao L, Li Q, Chen S. 2021. Pyramiding the antimicrobial PR1aCB and AATCB genes in ‘Tarocco’ blood orange (Citrus sinensis Osbeck) to enhance citrus canker resistance. Transgenic Research, 30, 635–647. 
Sechler A, Schuenzel E L, Cooke P, Donnua S, Thaveechai N, Postnikova E, Stone A L, Schneider W L, Damsteegt V D, Schaad N W. 2009. Cultivation of ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’ associated with Huanglongbing. Phytopathology, 99, 480–486.
Stewart E J. 2012. Growing unculturable bacteria. Journal of Bacteriology, 194, 4151–4160.
Tatineni S, Sagaram U S, Gowda S, Robertson C J, Dawson W O, Iwanami T, Wang N. 2008. In planta distribution of ‘Candidatus Liberibacter asiaticus’ as revealed by polymerase chain reaction (PCR). Phytopathology, 98, 592–599. 
Teixeira D C, Saillard C, Eveillard S, Danet J L, Costa P I, Ayre A J, Bové J M. 2005. ‘Candidatus Liberibacter americanus’, associated with citrus Huanglongbing (greening disease) in São Paulo state, Brazil. International Journal of Systematic and Evolutionary Microbiology, 55, 1857–1862.
Teixeira D C, Saillard C, Couture C, Martins E C, Wulff N A, Jagoueix S, Yamamoto P T, Ayres A J, Bové J M. 2008. Distribution and quantification of Candidatus Liberibacter americanus, agent of Huanglongbing disease of citrus in São Paulo State, Brasil, in leaves of an affected sweet orange tree as determined by PCR. Molecular and Cellular Probes, 22, 139–150. 
Walter A J, Duan Y, Hall D G. 2012. Titers of ‘Ca. Liberibacter asiaticus’ in Murraya paniculata and Murraya-reared Diaphorina citri are much Lower than in citrus and citrus-reared psyllids. HortScience, 47, 1449–1452.
Wang N, Pierson E A, Setubal J C, Xu J, Levy J G, Zhang Y, LI J, Rangel L T, Martins J. 2017. The Candidatus Liberibacter–host interface: Insights into pathogenesis mechanisms and disease control. Annual Review of Phytopathology, 55, 20.1–20.32. 
Wu F, Huang J, Xu M, Fox E G, Beattie G A C, Holford P, Deng X. 2018. Host and environmental factors influencing ‘Candidatus Liberibacter asiaticus’ acquisition in Diaphorina citri. Pest Manage Science, 74, 2738–2746. 
Ukuda-Hosokawa R, Sadoyama Y, Kishaba M, Kuriwada T, Anbutsu H, Fukatsu T. 2015. Infection density dynamics of the citrus greening bacterium “Candidatus Liberibacter asiaticus” in field populations of the psyllid Diaphorina citri and its relevance to the efficiency of pathogen transmission to citrus plants. Applied and Environmental Microbiology, 81, 3728–3736. 
Wulff N A, Zhang S, Setubal J C, Almeida N F, Martins E C, Harakava R, Kumar D, Rangel L T, Foissac X, Bové J M, Gabriel D W. 2014. The complete genome sequence of ‘Candidatus Liberibacter americanus’, associated with citrus Huanglongbing. Molecular Plant–Microbe Interactions, 27, 163–176. 
Zheng Z, Xu M, Bao M, Wu F, Chen J, Deng X. 2016. Unusual five copies and dual forms of nrdB in “Candidatus Liberibacter asiaticus”: Biological implications and PCR detection application. Scientific Reports, 6, 39020. 
Zhao X Y. 2017. Review of Studies on Prevention and Control of Citrus Huanglongbing. 1st ed. China Agricultural Press, Beijing. (in Chinese)
Zhou C. 2020. The status of citrus Huanglongbing in China. Tropical Plant Pathology, 45, 279–284. 
Zou X, Jiang X, Xu L, Lei T, Peng A, He Y, Yao L, Chen S. 2017. Transgenic citrus expressing synthesized cecropin B genes in the phloem exhibits decreased susceptibility to Huanglongbing. Plant Molecular Biology, 93, 341–353. 

No related articles found!
No Suggested Reading articles found!