日本柑桔黄龙病菌电镜观察及PCR检测
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摘要
柑桔黄龙病是由韧皮部杆菌Candidatus Liberibacter引起的一种柑桔毁灭性病害，在多个国家与地区均有发生。该病在日本1988年于冲绳县西表岛初次发现，随后河野于1994年在冲绳本岛发现该病并确认，近年来在日本有向北扩展的趋势，最近在鹿儿岛县奄美大岛也有发现。
自1970年Bove等用电镜观察到柑桔黄龙病病原以来，电镜观察一直是作为一种重要的鉴定和诊断手段，后来基于扩增16S rDNA特异性片断的PCR检测方法成了柑桔黄龙病检测的主要方法。
由于柑桔黄龙病病原只存在于韧皮部筛管细胞中，而对柑桔叶片维管组织固定比较困难。本研究通过对材料细切，小心去掉木质部部分以便于固定。由于植物组织中都含有一定的空气，致使树脂不能顺利浸润细胞器，这就给材料包埋带来困难，这可以通过在预包埋过程中处以真空处理以抽出植物组织中的空气，以便更好包埋。本研究对日本柑桔体内的柑桔黄龙病原菌的形态结构进行了电镜观察，并与发生在其它地区的柑桔黄龙病的病原进行了比较，结果表明柑桔黄龙病病原体存在于柑桔韧皮部的筛管细胞中，有时可见寄主细胞膜内陷成小泡状，柑桔黄龙病病菌日本株系绝大多数是球形和短椭圆形，极少观察到长条形病原，直径为100-500nm，外被一层胶状物质包围，厚约10-50nm，这与其它地区发现的柑桔黄龙病病原体形态不同。本研究还对植物总DNA的提取过程和PCR扩增程序进行了优化，利用Ready-To-Go~(TM) PCR bead法可以快速、准确检测到柑桔黄龙病病原体。
Citrus Huanglongbing caused by Candidatus Liberibacter is one of the most severe citrus diseases, which has been reported in many countries and areas. In Japan, citrus Huanglongbing was observed in the island of Iriomote in Okinawa and in the main island of Okinawa in 1988 and in 1994 respectively. The disease was also observed in kagoshima island recently. It is intend to spread to the north area.
The prokaryote associated with citrus Huanglongbing was first observed in 1970 by Lafleche and Bove in the phloem of infected sweet orange leaves. The electron microscope is one of the most important direction methods to study the morphology and to detect the pathogen associated with citrus Huanglongbing. According to the sequence of 16S RNA, a fast and accurate method based on PCR has been developed to detect the pathogen and now it is the most frequently used method.
Because the pathogen of citrus huanglongbing is restricted in the sieve tubes in phloem which is surrounded by xylem tissue, it is difficult to fix the vascular tissue. In order to get good fixation, we wiped off the xylem tissue and cut the slices into 1mm cubes. Application of a few drops of Fixative No.1(2% glutaraldehyde in 0.1M cacodylate buffer at pH7.2) to the tissue before cutting into slices can prevent sticking and drying and initiate the fixing process. The air in the plant tissues prevent the resin to reach the organelles, which makes the fixation difficult. During the pre-embedding process, application of vacuum will make a rather good embedding. The pathogen of citrus huanglongbing found in Japan was detected in ultrathin section of infected citrus leaves and a comparation with the pathogen found in other areas was also carried out. It has show that the pathogen associated with Japanese citrus huanglongbing differ from others in some characters as the pathogen is mostly in round or oval forms, rarely in filamentous forms, diameter in 100-500nm with a 10-50nm envelope. A rapid and accurate detection method, with an improved DNA extraction process and Ready-To-GoTM PCR procedure was also developed.
自1970年Bove等用电镜观察到柑桔黄龙病病原以来，电镜观察一直是作为一种重要的鉴定和诊断手段，后来基于扩增16S rDNA特异性片断的PCR检测方法成了柑桔黄龙病检测的主要方法。
由于柑桔黄龙病病原只存在于韧皮部筛管细胞中，而对柑桔叶片维管组织固定比较困难。本研究通过对材料细切，小心去掉木质部部分以便于固定。由于植物组织中都含有一定的空气，致使树脂不能顺利浸润细胞器，这就给材料包埋带来困难，这可以通过在预包埋过程中处以真空处理以抽出植物组织中的空气，以便更好包埋。本研究对日本柑桔体内的柑桔黄龙病原菌的形态结构进行了电镜观察，并与发生在其它地区的柑桔黄龙病的病原进行了比较，结果表明柑桔黄龙病病原体存在于柑桔韧皮部的筛管细胞中，有时可见寄主细胞膜内陷成小泡状，柑桔黄龙病病菌日本株系绝大多数是球形和短椭圆形，极少观察到长条形病原，直径为100-500nm，外被一层胶状物质包围，厚约10-50nm，这与其它地区发现的柑桔黄龙病病原体形态不同。本研究还对植物总DNA的提取过程和PCR扩增程序进行了优化，利用Ready-To-Go~(TM) PCR bead法可以快速、准确检测到柑桔黄龙病病原体。
Citrus Huanglongbing caused by Candidatus Liberibacter is one of the most severe citrus diseases, which has been reported in many countries and areas. In Japan, citrus Huanglongbing was observed in the island of Iriomote in Okinawa and in the main island of Okinawa in 1988 and in 1994 respectively. The disease was also observed in kagoshima island recently. It is intend to spread to the north area.
The prokaryote associated with citrus Huanglongbing was first observed in 1970 by Lafleche and Bove in the phloem of infected sweet orange leaves. The electron microscope is one of the most important direction methods to study the morphology and to detect the pathogen associated with citrus Huanglongbing. According to the sequence of 16S RNA, a fast and accurate method based on PCR has been developed to detect the pathogen and now it is the most frequently used method.
Because the pathogen of citrus huanglongbing is restricted in the sieve tubes in phloem which is surrounded by xylem tissue, it is difficult to fix the vascular tissue. In order to get good fixation, we wiped off the xylem tissue and cut the slices into 1mm cubes. Application of a few drops of Fixative No.1(2% glutaraldehyde in 0.1M cacodylate buffer at pH7.2) to the tissue before cutting into slices can prevent sticking and drying and initiate the fixing process. The air in the plant tissues prevent the resin to reach the organelles, which makes the fixation difficult. During the pre-embedding process, application of vacuum will make a rather good embedding. The pathogen of citrus huanglongbing found in Japan was detected in ultrathin section of infected citrus leaves and a comparation with the pathogen found in other areas was also carried out. It has show that the pathogen associated with Japanese citrus huanglongbing differ from others in some characters as the pathogen is mostly in round or oval forms, rarely in filamentous forms, diameter in 100-500nm with a 10-50nm envelope. A rapid and accurate detection method, with an improved DNA extraction process and Ready-To-GoTM PCR procedure was also developed.
引文
1. Garnier M, et. al.. Distribution of greening Liberobacter species in fifteen African and asian countries. Proc. (abstracts) 13th Conf. IOCV, 1995, 103.
2．林孔湘．柑桔黄梢(黄龙)病研究．植物病理学报，1956，2：1-42．
3. Tanaka, S. and Y. Doi. Studies on mycoplasma-like organisms suspected cause of citrus likubin and leaf mottling. Bull Fac. Agric. Tamagawa Univ., 1974, 14: 64-70.
4. Schneider, H.. Anatomy of greening diseased sweet orange shoots. Phytopathology, 1968, 58:1155-1160.
5. Wu, S. and H.C. Faan. Recent research on citrus yellow shoot in Guangdong Province. Proc. FAO-UNDP Greening Workshop, Lipa, Philippines, 1988, 2nd: 82-83.
6. Martinez, A.L.. Combined effects of greening and seedling yellows pathogens in citrus. Proc. Conf. Int. Org. Citrus Virol., 5th, 1972, 25-27.
7. Bhagabati, K.N., Nariani, T. K.. Interaction of greening and tristeza pathogens in Kagzi lime (Citrus anrantifolia (Christm.) Swing.) and their effect on growth and development of disease symptoms. Indian Phytopathol., 1980, 33:292-295.
8．林孔湘，朱麦拉．柑桔黄梢(黄龙)病研究，镰刀菌在发病上的作用．植物病理学报，1957．2：169-175．
9. Manicom, B., Q. and S.P. van Vuuren. Symptoms of greening disease with special imphasis on African greening. Proc.Int. Asia-Pacific Conf. Citrus Rehabil. 4th, 1990, 149-157.
10. Nariani, T.K., S.P.. Raychaudhuri, and R.B. Bhalla. Greening virus of citrus in India. Indian Phytopathol, 1967, 20:146-150.
11．陈其暴．潮柑黄龙病研究报告．新农季刊，1943，3：142-175．
12. McClean, A.P.D., Oberholzer, P. C. J.. Greening disease of sweet orange: Evidence that it is caused by a transmissible virus. S. Afr. J. Agric. Sci., 1965, 8: 253-276.
13. McClean, A.P.D., Oberholzer, P. C. J.. Citrus psylla, a vector of the greening disease of sweet orange. S. Afr. J. Agric. Sci., 1965, 8: 297-298.
14. Garnier, M., et al.. Genomic characterization of a liberibacter present in an ornamental rutaceous tree, Calodendrum capense, in the Western Cape Province of South Africa. Proposal of'Candidatus Liberibacter africanus subsp, capensis'. Int J Syst Evol Microbiol, 2000, 50 Pt 6: 2119-2125.
15. Reinking, A.. Diseases of economic plants in southern China. Philipp. Agric., 1919, 8: 109-135.
16．何畏冷．广东果树病害志之三．岭南学报，1937，3(1)：1-54．
17. McClean, A.P.D. and R.E. Schwarz. Greening of blotchy-mottle disease of citrus.
Phytophylactica, 1970, 2: 177-194.
18. Oberholzer, P., C, J.. The present status of citrus nutrition in South Africa. So.Africa Dept.Agr.Citrus N utr. Bul., 1947, 271:14
19. van der Merwe, A.J., Andersen, F. G.. Chromium and manganese toxicity. Is it important in Transvaal citrus greening? Farming S. Afr., 1947, 12: 439-440.
20. Oberholzer, P., C, J., D. von Staden, F,A,, and W. Basson, J.. Greening disease of sweet orange in South Africa. Proc. Conf. Int. Org. Citrus Virol., 3rd, 1965, 213-19.
21. Oberholzer, P., C,J and J.D.J.Hofmeyer. The nature and control of clonal senility in commercial varieties of citrus in South Africa. Bul. Faculty Agr.,Univ.Pretoria, Pretoria,South Africa. 1955, 46
22. Schwarz, R.E.. An insect-transmissible virus trapped on sweet orange seedlings in orchards where greening disease is common. So.Africa Jour.Agri.Sci., 1964, 7: 885-90.
23. Lafleche, D., Bove, J. M.. Mycoplasmes dans les agrumes atteints de "greening", de stubborn, on des maladies similaires. Fruits, 1970, 25: 455-65.
24. Cheema, G.S. and S.S.Bhatt. The dieback disease of citrus trees and its relation to the soils of western India. Dept.Agr.Bot., 1928, 155: 1-48.
25. Fraser, L.R. and D.Singh. Greening virus: a new threat to citrus industry. Punjba Hort. Jour., 1966, 6: 104-107.
26. Capoor, S.P., D.G.Rao., and S.M. Viswanath. A vector of the greening disease of citrus in India. Indian Jour.Agr. Sci., 1967, 37: 572-576.
27. Saglio, P., Lafleche, D., Bonissol, C., Bove, J. M.. Isolement, culture et observations an microscope electronique des structures de type mycoplasme associes a la maladie du stubborn des agrumes et leur comparison avec les structures observees dans le cas de la maladie Greening des agrumes. Physiol. Veg., 1971,9: 569-582.
28. Bove, J.M., Calavan, E. C., Capoor, S. P., Cortez, R. E., Schwarz, R. E.. Influence of temperature on symptoms of California stubborn, South African greening, Indian citrus decline and Philippines leaf mottling diseases. Proc. Conf. Int. Org. Citrus Virol., 6th, 1974, 12-15.
29. Jagoueix, S., J. Bove, M., and M. Garnier. The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria. Int J Syst Bacteriol, 1994, 44(3): 379-86.
30. Chen, M.-H., Miyakawa, T., Matsui, C.. Citrus likubin pathogens in the salivary glands of Diaphorina citri. Phytopathology, 1973, 63: 194-195.
31. Moll, J.N. and M.M. Martin. Electron microscope evidence that citrus psylla (Trioza erytreae) is a vector of greening disease in South Africa. Phytophylactica, 1973, 5: 41-44.
32. Gamier, M., Latrille, J., Bove, J. M.. Spiroplasma citri and the organism associated with likubin: comparison of their envelope systems. Proc. Conf. Int. Org. Citrus Virol., 7th, 1976, 13-17.
33. Moll, J.N. and M.M. Martin. Comparison of the organism causing greening disease with several plant pathogenic gram-negative bacteria, rickettsia-like organisms and mycoplasma-like organisms. Coll. INSERM, 1974, 33: 89-96.
34. Gamier, M., Bove, J. M.. Structure trilamellaire des deux membranes qui entourent les organismes procaryotes associes a la maladie du greening des agrumes. Fruits, 1977, 32: 749-752.
35. Bove, J., M., Bonnet, P., Gamier, M., Aubert, B.. Penicillin and tetracycline treatment of greening disease affected citrus plants in the glasshouse, and the bacterial nature of the procaryote associated with greening, proc. Conf. Int. Org. Citrus Virol. 8th, 1980, 91-102.
36. Aubert, B. and J. Bove, M.. Effect of penicillin or tetracycline injections of citrus trees affected by greening disease under field conditions in Reunion Island. Proc. Conf. Int. Org. Citrus Virol., 8th, 1980, 103-108.
37. Gamier, M., Danel, N., Bove, J. M.. The greening organism is a gram negative bacterium. Proc. Conf. Int. Org. Citrus Virolo 9th, 1984, 115-124.
38. Gamier, M. and J. Bove, M.. Transmission of the organism associated with citrus greening disease from sweet orange to periwinkle by dodder. Phytopathology, 1983, 73: 1358-1363.
39. Ghosh, S.K., Raychaudhuri, S. P., Varma, A., Nariani, T. K.. Isolation and culture of mycoplasma associated with citrus greening disease. Curr. Sci., 1971,40: 299-300.
40. Nariani, T.K., Ghosh, S. K., Kumar, D., Raychaudhuri, S. P.. Viswanath, S. M., Detection and possibilities of therapeutic control of the greening disease of citrus caused by mycoplasmas. Proc. Indian Nat. Sci. Acad. Ser. B, 1975, 41: 334-339.
41. Sodhi, S.S., Dhillon, S. S., Jeyarajam, R., Cheema, S. S.. Mycoplasmalike organism associated with citrus greening. Proc. Indian Natl. Acad. Sci. Ser. B, 1973, 41: 386-89.
42. Garnett, H.M.. Isolation and characterization of the greening organism. Citrus Subtrop. Fruit J., 1984, 611: 4-6.
43. Duncan, F., Garnett, H. M.. Serological studies of the greening organism. Citrus Subtrop. Fruit J., 1984,611: 9-10.
44. Ariovich, D. and H.M. Garnett. The use of immuno-gold staining techniques for detection of/a bacterium associated with greening diseased citrus. Phytopathology, 1989,79: 382-384.
45. Garnett, H.M., Mochaba, F., Civerolo, E. L., Lee, R. F., Brlansky, R. H., Bock, D..
Serological comparison of putative greening isolates. Proc. Conf. Int. Org. Citrus Virol., 11th, 1991.
46. Graca, J.V.d.. CITRUS GREENING DISEASE. Annu. Rev. Phytopathol., 1991,29: 109-136.
47. Labuschagne, N., Botha, A. D., Kotze, J. M.. Study of greening symptoms on inoculated plants. Proc. Symp. Citrus Greening, Nelspruit, S. Afr., 1984, 28-33.
48. Gardner, J.M., Feldman, A. W., Zablotowicz, R. M.. Identity and behaviour of xylem-residing bacteria in rough lemon roots of Florida citrus trees. Appl. Environ. Microbiol., 1982,43: 1331-42.
49. Gamier, M., Martin-Gros, G., Bove, J. M.. Monoclonal antibodies against the bacterial-like organism associated with citrus greening disease. Ann. Inst. PasteudMicrobiol., 1987,138: 639-650.
50. Villechanoux, S., et al.. Detection of Several Strains of the Bacterium-Like Organism of Citrus Greening Disease by DNA Probes. Curt Microbiol, 1992,24: 89-95.
51. Villechanoux, S., et al.. The genome of the non-cultured, bacterial-like organism associated with citrus greening disease contains the nusG-rplKAJL-rpoBC gene cluster and the gene for a bacteriophage type DNA polymerase. Curr Microbiol, 1993,26(3): 161-166.
52. Murray, R.G.E. and K.H. Schleifer. Taxonomic Notes: A Proposal for Recording the Properties of Putative Taxa of Procaryotes. Int J Syst Bacteriol, 1994.44: 174-176.
53. Murray, R.G.E. and E. Stackebrandt. Taxonomic Notes: Implementation of the Provisional Status Candidatus for Incompletely Described Procaryotes. Int J Syst Bactedol, 1995, 45:186-187.
54. Truper, H., G.. How to name a prokaryote Etymological considerations, proposals and practical advice in prokaryote nomenclature. FEMS Microbiology Reviews, 1999, 23: 231-249.
55. Schwarz, R.E.. Indexing of greening and exocortis through fluorescent marker substances. Proc. Conf. Int. Org. Citrus Virol., 4th, 1968, 118-124.
56. Wu, S.P.. Direct fluorescence detection for diagnosing citrus yellow shoot disease. Proc. Workshop Citrus Greening Disease, Fuzhou, China. 3pp, 1987.
57. Garnier, M., G. Martin-Gros, and J. Bove, M.. Monoclonal antibodies against the bacterial-like organism associated with citrus greening disease. Ann Inst Pasteur Microbiol, 1987, 138(6): 639-650.
58. Garnier, M., M.L. Iskra, and J.M. Bove. Towards monoclonal antibodies against the apple proliferation mycoplasma-like organism. Isr J Med Sci, 1987, 23(6): 691-695.
59. Hocquellet, A., J. Bove, M., and M. Garnier. Production and evaluation of
non-radioactive probes for the detection of the two 'Candidatus Liberobacter' species associated with citrus huanglongbing (greening). Mol Cell Probes, 1997, 11(6): 433-438.
60. Jagoueix, S., J. Bove, M., and M. Gamier. PCR detection of the two 'Candidatus' Liberobacter species associated with greening disease of citrus. Mol Cell Probes, 1996, 10(1): 43-50.
61. Hocquellet, A., et al.. Detection and identification of the two Candidatus Liberobacter species associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of the beta operon. Mol Cell Probes, 1999, 13(5): 373-379.
62．林孔湘．柑桔黄梢(黄龙)病的进一步研究．植物保护学报. 1963.2: 242-251.
63. Martinez, A.L., Nora, D. M., Armedilla, A. L.. Suppression of symptoms of citrus greening disease in the Philippines with tetracycline antibiotics. Plant Dis. Reptr., 1970, 54: 1007-1009.
64. Cheema, S.S., Kapur, S. P., Sharma, 0. P.. Chemotherapeutic control of greening disease of citrus through bud dip treatment. Indian J. Virol., 1986, 2: 104-107.
65. Schwarz, R.E. and S.P. van Vuuren. Decreases in fruit greening of sweet orange by trunk injections with tetracyclines. PlantDis. Reptr, 1970, 55: 747-750.
66. Capoor, S.P. and M.J. Thimmalachar. Cure of greening affected plants by chemotherapeutic agents. Plant Dis. Reptro, 1973, 57: 160-163.
67. Aubert, B. and S. Quilici. Biological control of the African and Asian citrus psyllids (Homoptera:Psylloidea), through eulophid and encyrtid parasites (Hymenoptera:Chalcidoidea) in Reunion Island. Proc. Conf. Int. Org. Citrus Virol. 9th, 1984, 100-108.
68．林传光．关于柑桔黄龙病几个问题的讨论．植物保护学报，1963，2：237-241．
69．林孔湘．评“关于柑桔黄龙病几个问题的讨论”．植物保护学报，1964，3：165-171．
70．邓晓玲，唐伟文．应用PCR技术检测柑桔黄龙病病原的研究．华南农业大学学报，1996，17：119-120．
71．田亚南，柯穗，柯冲．柑桔黄龙病诊断法的研究进展．福建农业学报，1998，13：27-35
72. Kim, K.S., C.K.Pallaghy(1996, modified 1997), Purification of plasmid DNA (miniprep) with high yields using diatomaceous earth. U.S.Dept. Commerce/NOANNMFS/ NWFSC/Molecular Biology Protocols: http://www.nwfs.noaa.gov/protocols/dna-prep.html
2．林孔湘．柑桔黄梢(黄龙)病研究．植物病理学报，1956，2：1-42．
3. Tanaka, S. and Y. Doi. Studies on mycoplasma-like organisms suspected cause of citrus likubin and leaf mottling. Bull Fac. Agric. Tamagawa Univ., 1974, 14: 64-70.
4. Schneider, H.. Anatomy of greening diseased sweet orange shoots. Phytopathology, 1968, 58:1155-1160.
5. Wu, S. and H.C. Faan. Recent research on citrus yellow shoot in Guangdong Province. Proc. FAO-UNDP Greening Workshop, Lipa, Philippines, 1988, 2nd: 82-83.
6. Martinez, A.L.. Combined effects of greening and seedling yellows pathogens in citrus. Proc. Conf. Int. Org. Citrus Virol., 5th, 1972, 25-27.
7. Bhagabati, K.N., Nariani, T. K.. Interaction of greening and tristeza pathogens in Kagzi lime (Citrus anrantifolia (Christm.) Swing.) and their effect on growth and development of disease symptoms. Indian Phytopathol., 1980, 33:292-295.
8．林孔湘，朱麦拉．柑桔黄梢(黄龙)病研究，镰刀菌在发病上的作用．植物病理学报，1957．2：169-175．
9. Manicom, B., Q. and S.P. van Vuuren. Symptoms of greening disease with special imphasis on African greening. Proc.Int. Asia-Pacific Conf. Citrus Rehabil. 4th, 1990, 149-157.
10. Nariani, T.K., S.P.. Raychaudhuri, and R.B. Bhalla. Greening virus of citrus in India. Indian Phytopathol, 1967, 20:146-150.
11．陈其暴．潮柑黄龙病研究报告．新农季刊，1943，3：142-175．
12. McClean, A.P.D., Oberholzer, P. C. J.. Greening disease of sweet orange: Evidence that it is caused by a transmissible virus. S. Afr. J. Agric. Sci., 1965, 8: 253-276.
13. McClean, A.P.D., Oberholzer, P. C. J.. Citrus psylla, a vector of the greening disease of sweet orange. S. Afr. J. Agric. Sci., 1965, 8: 297-298.
14. Garnier, M., et al.. Genomic characterization of a liberibacter present in an ornamental rutaceous tree, Calodendrum capense, in the Western Cape Province of South Africa. Proposal of'Candidatus Liberibacter africanus subsp, capensis'. Int J Syst Evol Microbiol, 2000, 50 Pt 6: 2119-2125.
15. Reinking, A.. Diseases of economic plants in southern China. Philipp. Agric., 1919, 8: 109-135.
16．何畏冷．广东果树病害志之三．岭南学报，1937，3(1)：1-54．
17. McClean, A.P.D. and R.E. Schwarz. Greening of blotchy-mottle disease of citrus.
Phytophylactica, 1970, 2: 177-194.
18. Oberholzer, P., C, J.. The present status of citrus nutrition in South Africa. So.Africa Dept.Agr.Citrus N utr. Bul., 1947, 271:14
19. van der Merwe, A.J., Andersen, F. G.. Chromium and manganese toxicity. Is it important in Transvaal citrus greening? Farming S. Afr., 1947, 12: 439-440.
20. Oberholzer, P., C, J., D. von Staden, F,A,, and W. Basson, J.. Greening disease of sweet orange in South Africa. Proc. Conf. Int. Org. Citrus Virol., 3rd, 1965, 213-19.
21. Oberholzer, P., C,J and J.D.J.Hofmeyer. The nature and control of clonal senility in commercial varieties of citrus in South Africa. Bul. Faculty Agr.,Univ.Pretoria, Pretoria,South Africa. 1955, 46
22. Schwarz, R.E.. An insect-transmissible virus trapped on sweet orange seedlings in orchards where greening disease is common. So.Africa Jour.Agri.Sci., 1964, 7: 885-90.
23. Lafleche, D., Bove, J. M.. Mycoplasmes dans les agrumes atteints de "greening", de stubborn, on des maladies similaires. Fruits, 1970, 25: 455-65.
24. Cheema, G.S. and S.S.Bhatt. The dieback disease of citrus trees and its relation to the soils of western India. Dept.Agr.Bot., 1928, 155: 1-48.
25. Fraser, L.R. and D.Singh. Greening virus: a new threat to citrus industry. Punjba Hort. Jour., 1966, 6: 104-107.
26. Capoor, S.P., D.G.Rao., and S.M. Viswanath. A vector of the greening disease of citrus in India. Indian Jour.Agr. Sci., 1967, 37: 572-576.
27. Saglio, P., Lafleche, D., Bonissol, C., Bove, J. M.. Isolement, culture et observations an microscope electronique des structures de type mycoplasme associes a la maladie du stubborn des agrumes et leur comparison avec les structures observees dans le cas de la maladie Greening des agrumes. Physiol. Veg., 1971,9: 569-582.
28. Bove, J.M., Calavan, E. C., Capoor, S. P., Cortez, R. E., Schwarz, R. E.. Influence of temperature on symptoms of California stubborn, South African greening, Indian citrus decline and Philippines leaf mottling diseases. Proc. Conf. Int. Org. Citrus Virol., 6th, 1974, 12-15.
29. Jagoueix, S., J. Bove, M., and M. Garnier. The phloem-limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria. Int J Syst Bacteriol, 1994, 44(3): 379-86.
30. Chen, M.-H., Miyakawa, T., Matsui, C.. Citrus likubin pathogens in the salivary glands of Diaphorina citri. Phytopathology, 1973, 63: 194-195.
31. Moll, J.N. and M.M. Martin. Electron microscope evidence that citrus psylla (Trioza erytreae) is a vector of greening disease in South Africa. Phytophylactica, 1973, 5: 41-44.
32. Gamier, M., Latrille, J., Bove, J. M.. Spiroplasma citri and the organism associated with likubin: comparison of their envelope systems. Proc. Conf. Int. Org. Citrus Virol., 7th, 1976, 13-17.
33. Moll, J.N. and M.M. Martin. Comparison of the organism causing greening disease with several plant pathogenic gram-negative bacteria, rickettsia-like organisms and mycoplasma-like organisms. Coll. INSERM, 1974, 33: 89-96.
34. Gamier, M., Bove, J. M.. Structure trilamellaire des deux membranes qui entourent les organismes procaryotes associes a la maladie du greening des agrumes. Fruits, 1977, 32: 749-752.
35. Bove, J., M., Bonnet, P., Gamier, M., Aubert, B.. Penicillin and tetracycline treatment of greening disease affected citrus plants in the glasshouse, and the bacterial nature of the procaryote associated with greening, proc. Conf. Int. Org. Citrus Virol. 8th, 1980, 91-102.
36. Aubert, B. and J. Bove, M.. Effect of penicillin or tetracycline injections of citrus trees affected by greening disease under field conditions in Reunion Island. Proc. Conf. Int. Org. Citrus Virol., 8th, 1980, 103-108.
37. Gamier, M., Danel, N., Bove, J. M.. The greening organism is a gram negative bacterium. Proc. Conf. Int. Org. Citrus Virolo 9th, 1984, 115-124.
38. Gamier, M. and J. Bove, M.. Transmission of the organism associated with citrus greening disease from sweet orange to periwinkle by dodder. Phytopathology, 1983, 73: 1358-1363.
39. Ghosh, S.K., Raychaudhuri, S. P., Varma, A., Nariani, T. K.. Isolation and culture of mycoplasma associated with citrus greening disease. Curr. Sci., 1971,40: 299-300.
40. Nariani, T.K., Ghosh, S. K., Kumar, D., Raychaudhuri, S. P.. Viswanath, S. M., Detection and possibilities of therapeutic control of the greening disease of citrus caused by mycoplasmas. Proc. Indian Nat. Sci. Acad. Ser. B, 1975, 41: 334-339.
41. Sodhi, S.S., Dhillon, S. S., Jeyarajam, R., Cheema, S. S.. Mycoplasmalike organism associated with citrus greening. Proc. Indian Natl. Acad. Sci. Ser. B, 1973, 41: 386-89.
42. Garnett, H.M.. Isolation and characterization of the greening organism. Citrus Subtrop. Fruit J., 1984, 611: 4-6.
43. Duncan, F., Garnett, H. M.. Serological studies of the greening organism. Citrus Subtrop. Fruit J., 1984,611: 9-10.
44. Ariovich, D. and H.M. Garnett. The use of immuno-gold staining techniques for detection of/a bacterium associated with greening diseased citrus. Phytopathology, 1989,79: 382-384.
45. Garnett, H.M., Mochaba, F., Civerolo, E. L., Lee, R. F., Brlansky, R. H., Bock, D..
Serological comparison of putative greening isolates. Proc. Conf. Int. Org. Citrus Virol., 11th, 1991.
46. Graca, J.V.d.. CITRUS GREENING DISEASE. Annu. Rev. Phytopathol., 1991,29: 109-136.
47. Labuschagne, N., Botha, A. D., Kotze, J. M.. Study of greening symptoms on inoculated plants. Proc. Symp. Citrus Greening, Nelspruit, S. Afr., 1984, 28-33.
48. Gardner, J.M., Feldman, A. W., Zablotowicz, R. M.. Identity and behaviour of xylem-residing bacteria in rough lemon roots of Florida citrus trees. Appl. Environ. Microbiol., 1982,43: 1331-42.
49. Gamier, M., Martin-Gros, G., Bove, J. M.. Monoclonal antibodies against the bacterial-like organism associated with citrus greening disease. Ann. Inst. PasteudMicrobiol., 1987,138: 639-650.
50. Villechanoux, S., et al.. Detection of Several Strains of the Bacterium-Like Organism of Citrus Greening Disease by DNA Probes. Curt Microbiol, 1992,24: 89-95.
51. Villechanoux, S., et al.. The genome of the non-cultured, bacterial-like organism associated with citrus greening disease contains the nusG-rplKAJL-rpoBC gene cluster and the gene for a bacteriophage type DNA polymerase. Curr Microbiol, 1993,26(3): 161-166.
52. Murray, R.G.E. and K.H. Schleifer. Taxonomic Notes: A Proposal for Recording the Properties of Putative Taxa of Procaryotes. Int J Syst Bacteriol, 1994.44: 174-176.
53. Murray, R.G.E. and E. Stackebrandt. Taxonomic Notes: Implementation of the Provisional Status Candidatus for Incompletely Described Procaryotes. Int J Syst Bactedol, 1995, 45:186-187.
54. Truper, H., G.. How to name a prokaryote Etymological considerations, proposals and practical advice in prokaryote nomenclature. FEMS Microbiology Reviews, 1999, 23: 231-249.
55. Schwarz, R.E.. Indexing of greening and exocortis through fluorescent marker substances. Proc. Conf. Int. Org. Citrus Virol., 4th, 1968, 118-124.
56. Wu, S.P.. Direct fluorescence detection for diagnosing citrus yellow shoot disease. Proc. Workshop Citrus Greening Disease, Fuzhou, China. 3pp, 1987.
57. Garnier, M., G. Martin-Gros, and J. Bove, M.. Monoclonal antibodies against the bacterial-like organism associated with citrus greening disease. Ann Inst Pasteur Microbiol, 1987, 138(6): 639-650.
58. Garnier, M., M.L. Iskra, and J.M. Bove. Towards monoclonal antibodies against the apple proliferation mycoplasma-like organism. Isr J Med Sci, 1987, 23(6): 691-695.
59. Hocquellet, A., J. Bove, M., and M. Garnier. Production and evaluation of
non-radioactive probes for the detection of the two 'Candidatus Liberobacter' species associated with citrus huanglongbing (greening). Mol Cell Probes, 1997, 11(6): 433-438.
60. Jagoueix, S., J. Bove, M., and M. Gamier. PCR detection of the two 'Candidatus' Liberobacter species associated with greening disease of citrus. Mol Cell Probes, 1996, 10(1): 43-50.
61. Hocquellet, A., et al.. Detection and identification of the two Candidatus Liberobacter species associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of the beta operon. Mol Cell Probes, 1999, 13(5): 373-379.
62．林孔湘．柑桔黄梢(黄龙)病的进一步研究．植物保护学报. 1963.2: 242-251.
63. Martinez, A.L., Nora, D. M., Armedilla, A. L.. Suppression of symptoms of citrus greening disease in the Philippines with tetracycline antibiotics. Plant Dis. Reptr., 1970, 54: 1007-1009.
64. Cheema, S.S., Kapur, S. P., Sharma, 0. P.. Chemotherapeutic control of greening disease of citrus through bud dip treatment. Indian J. Virol., 1986, 2: 104-107.
65. Schwarz, R.E. and S.P. van Vuuren. Decreases in fruit greening of sweet orange by trunk injections with tetracyclines. PlantDis. Reptr, 1970, 55: 747-750.
66. Capoor, S.P. and M.J. Thimmalachar. Cure of greening affected plants by chemotherapeutic agents. Plant Dis. Reptro, 1973, 57: 160-163.
67. Aubert, B. and S. Quilici. Biological control of the African and Asian citrus psyllids (Homoptera:Psylloidea), through eulophid and encyrtid parasites (Hymenoptera:Chalcidoidea) in Reunion Island. Proc. Conf. Int. Org. Citrus Virol. 9th, 1984, 100-108.
68．林传光．关于柑桔黄龙病几个问题的讨论．植物保护学报，1963，2：237-241．
69．林孔湘．评“关于柑桔黄龙病几个问题的讨论”．植物保护学报，1964，3：165-171．
70．邓晓玲，唐伟文．应用PCR技术检测柑桔黄龙病病原的研究．华南农业大学学报，1996，17：119-120．
71．田亚南，柯穗，柯冲．柑桔黄龙病诊断法的研究进展．福建农业学报，1998，13：27-35
72. Kim, K.S., C.K.Pallaghy(1996, modified 1997), Purification of plasmid DNA (miniprep) with high yields using diatomaceous earth. U.S.Dept. Commerce/NOANNMFS/ NWFSC/Molecular Biology Protocols: http://www.nwfs.noaa.gov/protocols/dna-prep.html