Please wait a minute...
Journal of Integrative Agriculture  2016, Vol. 15 Issue (1): 111-119    DOI: 10.1016/S2095-3119(15)61085-1
Plant Protection Advanced Online Publication | Current Issue | Archive | Adv Search |
Evaluation of thermotherapy against Huanglongbing (citrus greening) in the greenhouse
 FAN Guo-cheng, XIA Yu-lu, LIN Xiong-jie, HU Han-qing, WANG Xian-da, RUAN Chuan-qing, LU Lian-ming, LIU Bo
1、Fruit Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350013, P.R.China
2、National Science Foundation Center for Integrated Pest Management, North Carolina State University, Raleigh NC 27606, USA
3、Agricultural Bio-Resources Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003, P.R.China
4、Zhejiang Citrus Research Institute, Taizhou 318020, P.R.China
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
摘要  Huanglongbing (HLB, or citrus greening) is the most destructive disease of citrus, which is associated with Candidatus Liberibacter asiaticus (Las). Few management options are available, aside from preventive measures such as removing infected plants, planting disease-free seedlings, and managing the insect vector. In this study, we assessed the efficacy of thermotherapy against HLB under controlled greenhouse conditions. A total of 60 two-year-old, graft-infected Citrus reticulata Blanco plants were used. The plants were randomly divided into three groups (45°C, 48°C, and untreated control), with five plants/replicate (rep) and four reps/treatment. The treated plants were placed in phytotrons for a 4-h treatment session, repeated once per week for three consecutive weeks. Disease remission was observed eight weeks post-treatment. Real-time PCR assays revealed that Las titers in HLB-affected seedlings were significantly reduced in both 45 and 48°C treatments four weeks after treatment, with the exception of eight plants. In contrast, Las titers in the untreated control plants increased significantly during the same period, with a maximum increase of 28-fold. Except for seven plants, Las titers in the new flushes of treated plants decreased more than 90% eight weeks after treatment. Las titers in mature leaves of treated plants decreased 56 and 60% in average at 45 and 48°C, respectively, eight weeks after treatment. The HLB symptoms and Las titer of seedings were markedly alleviated eight weeks after treatment in both 45 and 48°C treatments. Our results laid a good foundation for the further development of citrus free-disease seedling cultivation and Huanglongbing control in the field. The whole plants were replaced for scion or branch in previous as the research object in this study, and the expression of Huanglongbing symptoms combined with real-time polymerase chain reaction (PCR) were used to evaluate the effect of heat treatment in the greenhouse.

Abstract  Huanglongbing (HLB, or citrus greening) is the most destructive disease of citrus, which is associated with Candidatus Liberibacter asiaticus (Las). Few management options are available, aside from preventive measures such as removing infected plants, planting disease-free seedlings, and managing the insect vector. In this study, we assessed the efficacy of thermotherapy against HLB under controlled greenhouse conditions. A total of 60 two-year-old, graft-infected Citrus reticulata Blanco plants were used. The plants were randomly divided into three groups (45°C, 48°C, and untreated control), with five plants/replicate (rep) and four reps/treatment. The treated plants were placed in phytotrons for a 4-h treatment session, repeated once per week for three consecutive weeks. Disease remission was observed eight weeks post-treatment. Real-time PCR assays revealed that Las titers in HLB-affected seedlings were significantly reduced in both 45 and 48°C treatments four weeks after treatment, with the exception of eight plants. In contrast, Las titers in the untreated control plants increased significantly during the same period, with a maximum increase of 28-fold. Except for seven plants, Las titers in the new flushes of treated plants decreased more than 90% eight weeks after treatment. Las titers in mature leaves of treated plants decreased 56 and 60% in average at 45 and 48°C, respectively, eight weeks after treatment. The HLB symptoms and Las titer of seedings were markedly alleviated eight weeks after treatment in both 45 and 48°C treatments. Our results laid a good foundation for the further development of citrus free-disease seedling cultivation and Huanglongbing control in the field. The whole plants were replaced for scion or branch in previous as the research object in this study, and the expression of Huanglongbing symptoms combined with real-time polymerase chain reaction (PCR) were used to evaluate the effect of heat treatment in the greenhouse.
Keywords:  Candidatus Liberibacter asiaticus       Citrus reticulata       thermotherapy       bacterial titer  
Received: 22 December 2014   Accepted:
Fund: 

This research was funded by the United States Department of Agriculture (USDA)-APHIS-PPQ-CPHST and North Carolina State University joint project (2012-0195-01) and the Special Fund for Agro-Scientific Research in the Public Interest, China (201003067-05).

Corresponding Authors:  LIU Bo, Moblie: +86-13905917339, Fax: +86-591-87884262, E-mail: fzliubo@163.com     E-mail:  fzliubo@163.com
About author:  FAN Guo-cheng, E-mail: guochengfan@126.com;These authors contributed equally to this study.

Cite this article: 

FAN Guo-cheng, XIA Yu-lu, LIN Xiong-jie, HU Han-qing, WANG Xian-da, RUAN Chuan-qing, LU Lian-ming, LIU Bo. 2016. Evaluation of thermotherapy against Huanglongbing (citrus greening) in the greenhouse. Journal of Integrative Agriculture, 15(1): 111-119.

Ark P A, Gardner M W. 1944. Carrot bacterial blight as it affectsthe roots. Phytopathology, 34, 415-420

Bakai-Golan R. 1973. Postharvest heat treatment to controlAlternaria tenuis Auct. rot in tomato. PhytopathologiaMediterranea, 12, 108-111

Bové J M. 2006. Huanglongbing: a destructive, newly-emerging,century-old disease of citrus. Journal of Plant Pathology,88, 7-37

Bové J M, Bonnet P, Garnier M, Aubert B. 1980. Penicillinand tetracycline treatments of greening disease-affectedcitrus plants in the glasshouse, and the bacterial natureof the procaryote associated with greening. In: CalavanE C, Garnsey S M, Timmer L W, eds., Proceedings of the8th Conference of the International Organization of CitrusVirologists. University of California, Riverside, USA. pp.97-102

Bové J M, Calavan E C, Capoor S P, Cortez R E, Schwarz RE, Weathers L G, Cohen M. 1974. Influence of temperatureon symptom of California stubborn, South African greening,Indian citrus decline and Philippines leaf mottling disease.In: Weathers L G, Cohen M, eds., Proceedings of the 6thConference of the International Organization of CitrusVirologists. University of California, Berkeley, USA. pp.12-15

Campbell A I. 1961. The effect of rubbery wood virus on thestoolbed production of two clonal apple rootstocks. Journalof Horticultural Science, 36, 268-273

Chen N R, Dai Y M, Wu T X, Xu T. 1981. Control ofyellow shoot disease of citrus by trunk injection oftetracycline hydrochloride and some other medicines. ActaPhytophylacica Sinica, 8, 163-169 (in Chinese)

Devash Y, Okon Y, Henis Y. 1980. Survival of Pseudomonastomato in soil and seeds. Phytopathology, 99, 175-185

Fan G C, Liu B, Wu R J, Li T, Cai Z J, Ke C. 2009. Thirty yearsof research on citrus huanglongbing in China. Fujian Journalof Agricultural Sciences, 24, 183-190 (in Chinese)

Gao R X, Zheng H M. 1981. On control of yellow shoot diseaseof citrus. Acta Phytophylacica Sinica, 8, 53-58 (in Chinese)

Grondeau C, Ladonne F, Fourmond A, Poutier F, Samson R.1992. Attempt to eradicate Pseudomonas syringae pv. pisifrom pea seeds with heat treatments. Seed Science andTechnology, 20, 515-525

Grondeau C, Samson R, Sands D C. 1994. A review ofthermotherapy to free plant materials from pathogens,especially seeds from bacteria. Critical Reviews in PlantSciences, 13, 57-75

GHRG (Guangxi Huanglongbing Research Group). 1976.The pathogen of huanglongbing and the study on usingacheomycin and terramycin to control huanglongbing.Citrus Scientific and Technical News Report, 2, 28-31(in Chinese)

Hoffman M T, Doud M S, Williams L, Zhang M Q, Ding F, StoverE, Duan Y P. 2013. Heat treatment eliminates ‘CandidatusLiberibacter asiaticus’ from infected citrus trees undercontrolled conditions. Phytopathology, 103, 15-22

Jindal K K, Thind B S, Soni P S. 1989. Physical and chemicalagents for the control of Xanthomonas campestrispv. vignicola from cowpea seeds. Seed Science andTechnology, 17, 371-382

Josephson K L, Gerba C P, Pepper I L. 1993. Polymerasechain reaction detection of nonviable bacterial pathogens.Applied and Environmental Microbiology, 59, 3513-3515

Ke C, Wang Z S. 1988. Study on the use of antibiotics tocontrol citrus huanglongbing. Fujian Journal of AgriculturalSciences, 3, 1-10 (in Chinese)

Kelly L, Smith D L, Snary E L, Johnson J A, Harris A D,Wooldridge M, Morris J G. 2004. Animal growth promoters:To ban or not to ban? A risk assessment approach.International Journal of Antimicrobial Agents, 24, 205-212

Li W B, Hartung J S, Levy L. 2007. Evaluation of DNAamplification methods for improved detection of ‘CandidatusLiberibacter species’ associated with citrus huanglongbing.Plant Disease, 91, 51-58

Li W B, Levy L, Hartung J S. 2009. Quantitative distribution of‘Candidatus Liberibacter asiaticus’ in citrus plants with citrushuanglongbing. Phytopathology, 99, 139-144

Li W B, Hartung J S, Levy L. 2006. Quantitative real-time PCRfor detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. Journal ofMicrobiological Methods, 66, 104-115

Lin C Y. 1981. Studies on black rot of cruciferous crops causedby Xanthomonas campestris pv. campestris in Taiwan. PlantProtection Bulletin (Taiwan), 23, 157-167

Lin K H. 1963. Further studies on citrus yellow shoot. ActaPhytophylacica Sinica, 2, 243-251 (in Chinese)

Lin K H, Lo H H. 1965. A preliminary study on thermotherapy ofyellow shoot disease of citrus. Acta Phytophylacica Sinica,4, 169-174 (in Chinese)

Lin K H, Zheng R Y. 1964. A preliminary study on citrus yellowshoot disease virus and resistance of the diseased citrusbudwood to heat. Acta Phytopathologica Sinica, 7, 61-65(in Chinese)

Lister R M, Bancroft J B, Nadakavukaren M J. 1965. Somesap-transmissible viruses from apple. Phytopathology, 55,859-870

Lo X H, Luo Z D, Tang W W. 1981. Studies on the thermotherapyof citrus yellow shoot disease. Acta Phytophylacica Sinica,8, 47-52 (in Chinese)

Luo Z D. 1991. Preliminary report on the efficacy of heatedtetracycline hydrochloride solution in treating citrusyellow shoot disease. Journal of South China AgriculturalUniversity, 12, 38-42 (in Chinese)

Martinez A L, Nora D M, Armedilla A L. 1970. Suppressionof symptoms of citrus greening disease in the Philippinesby treatment with tetracycline antiobiotics. Plant DiseaseReproter, 54, 1007-1009

McClean A P D, Oberholzer P C J. 1965. Citrus psylla, a vectorof the greening disease of sweet orange. South AfricanJournal of Agricultural Science, 8, 297-298

Murray M G, Thompson W F. 1980. Rapid isolation of highmolecular weight plant DNA. Nucleic Acids Research, 8,4321-4326

Nariani T K, Raychaudhuri S P, Viswanath S M. 1971. Responseof greening pathogen of citrus to certain tetracyclineantibiotics. Current Science, 40, 552.Naumann K, Karl H. 1988. Possibilities of disinfectingbean seeds infected with Pseudomonas syringae pv.phaseolicola. Nachrichtenblatt fur den Pflanzenschutz inder DDR, 42, 204-208 (in German)

Rao Y P, Durgapal J C. 1966. Seed transmission of bacterialblight disease of Sesamum (Sesamum orientale L.) anderadication of seed infection. Indian Phytopathology, 19,402-403

Sands D C, Fourest E, Rehms L D. 1989. Dry heat seedtreatment for Xanthomonas campestris pv. translucens.In: Klement Z, ed., 7th International Conference onPlant Pathogenic Bacteria. June 11-16, 1989 BudapestAkademiaiKiado, Budapest. p. 51.

Schwarz R E, Van Vuuren S P V. 1971. Decrease in fruitgreening of sweet orange by trunk injection of tetracyclines.Plant Disease Reporter, 55, 747-750

Shekhawat G S, Srivastava D N. 1971. Control of bacterial leafstreakof rice (Oryza sativa L.). Indian Journal of AgriculturalSciences, 41, 1098-1101

Shekhawat P S, Jain M L, Chkravarti B P. 1982. Detectionand seed transmission of Xanthomonas campestris pv.campestris causing black rot of cabbage and cauliflowerand its control by seed treatment. Indian Phytopathology,35, 442-447

Smith D L, Dushoff J, Morris J G. 2005. Agricultural antibioticsand human health. PLoS Medicine, 2, 0731-0735

Smith D L, Johnson J A, Harris A D, Furuno J P, PerencevichE N, Morris J G. 2003. Assessing risks for a pre-emergentpathogen: Virginiamycin use and the emergence ofstreptogramin resistance in Enterococcus faecium. TheLancet Infectious Diseases, 3, 241-249

Tamietti G. 1982. Seed treatment trials to control halo blightof bean. Informatore Fitopatologico, 32, 47-50 (in Italian)

Tatineni S, Sagaram U S, Gowda S, Robertson C J, DawsonW O, Iwanami T, Wang N. 2008. In planta distributionof ‘Candidatus Liberibacter asiaticus’ as revealed bypolymerase chain reaction (PCR) and real-time PCR.Phytopathology, 98, 592-599

Xu M R, Liang M D, Chen J C, Xia Y L, Zheng Z, Zhu Q, DengX L. 2013. Preliminary research on soil conditioner mediatedcitrus Huanglongbing mitigation in the field in Guangdong,China. European Journal of Plant Pathology, 137, 283-293

Zandbergen M. 1965. Hot water treatment for bulbs. In: RoyalHorticultural Society Daffodil-Tulip Yearbook. London theRoyal Horticultural Society, UK. p. 187.

Zhang M Q, Duan Y P, Powell C A. 2010. Bactericidal activitiesof antimicrobial molecules against huanglongbingassociated‘Candidatus Liberibacter asiaticus’ in thediseased periwinkle. Phytopathology, 100, S145-S145.Zhang M Q, Duan Y P, Zhou L J, Turechek W W, Stover E,Powell C A. 2009. Screening molecules for control of citrushuanglongbing using an optimized regeneration systemfor ‘Candidatus Liberibacter asiaticus’ infected periwinkle(Catharanthus roseus) cuttings. Phytopathology, 100,239-245

Zhang M Q, Powell C A, Zhou L J, He Z L, Stover E, DuanY P. 2011. Chemical compounds effective against thecitrus huanglongbing bacterium ‘Candidatus Liberibacterasiaticus’ in planta. Phytopathology, 101, 1097-1103

Zhao X Y, Jiang Y H, Qiu Z S, Su W F, Li C Y. 1982a. Atechnique of graft transmission of citrus yellow shootdisease (Huanglongbing). Acta Phytopathologica Sinica,12, 53-55 (in Chinese)

Zhao X Y, Qiu Z S, Su W F, Jiang Y H. 1981. The reaction ofpenicillin and tetracycline on citrus Huanglongbing. SouthChina Fruits, 4, 17. (in Chinese)

Zhao X Y, Qiu Z S, Su W F, Jiang Y H. 1982b. Continued effectof tetracycline on the elimination of citrus yellow shoot bybudwood immersion. Acta Phytophylacica Sinica, 9, 67-68(in Chinese)
[1] XUAN Zhi-you, ZHANG Song, LI Ping, YANG Fang-yun, CHEN Hong-ming, LIU Ke-hong, ZHOU Yan, LI Zhong-an, ZHOU Chang-yong, CAO Meng-ji. Apple stem grooving virus is associated with leaf yellow mottle mosaic disease on Citrus grandis cv. Huangjinmiyou in China[J]. >Journal of Integrative Agriculture, 2022, 21(7): 2031-2041.
[2] LIU Cong, LI De-xiong, HUANG Xian-biao, Zhang Fu-qiong, Xie Zong-zhou, Zhang Hong-yan, Liu Ji-hong. Manual thinning increases fruit size and sugar content of Citrus reticulata Blanco and affects hormone synthesis and sugar transporter activity[J]. >Journal of Integrative Agriculture, 2022, 21(3): 725-735.
[3] HU Guo-jun, DONG Ya-feng, ZHANG Zun-ping, FAN Xu-dong, REN Fan. Elimination of grapevine fleck virus and grapevine rupestris stem pitting-associated virus from Vitis vinifera 87-1 by ribavirin combined with thermotherapy[J]. >Journal of Integrative Agriculture, 2021, 20(9): 2463-2470.
[4] CUI Xue-jin, ZENG Chun-hua, LIU Ke-hong, TENG Cai-ling, ZHOU Chang-yong, WANG Xue-feng. Decreasing detection frequency of MITE (MCLas-A) in the population of ‘Candidatus Liberibacter asiaticus’ recently collected in southern China[J]. >Journal of Integrative Agriculture, 2020, 19(10): 2597-2601.
[5] LI Ya, XU Mei-rong, DAI Ze-han, DENG Xiao-ling. Distribution pattern and titer of Candidatus Liberibacter asiaticus in periwinkle (Catharanthus roseus)[J]. >Journal of Integrative Agriculture, 2018, 17(11): 2501-2508.
[6] HU Guo-jun, DONG Ya-feng, ZHANG Zun-ping, FAN Xu-dong, REN Fang, LI Zheng-nan. Effect of pre-culture on virus elimination from in vitro apple by thermotherapy coupled with shoot tip culture[J]. >Journal of Integrative Agriculture, 2018, 17(09): 2015-2023.
[7] WANG Xue-feng, CHEN Jiao-yue, TAN Jin , DUAN Suo, DENG Xiao-ling, CHEN Jian-chi, ZHOU Chang-yong. High genetic variation and recombination events in the vicinity of non-autonomous transposable elements from ‘Candidatus Liberibacter asiaticus’[J]. >Journal of Integrative Agriculture, 2015, 14(10): 2002-2010.
[8] DENG Xiao-ling, CHEN Jian-chi, KONG Wei-wen, LI Hua-ping. Curent Situation of “Candidatus Liberibacter asiaticus” in Guangdong, China, Where Citrus Huanglongbing Was First Described[J]. >Journal of Integrative Agriculture, 2012, 12(3): 424-429.
No Suggested Reading articles found!