广佛手黄龙病的鉴定

袁蒙; 潘利明; 周良云; 罗碧; 王浩; 杨全

doi:10.13422/j.cnki.syfjx.20192413

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广佛手黄龙病的鉴定

药学基础 | 更新时间：2021-02-09

- 广佛手黄龙病的鉴定
- Identification of Citrus medica var. sarcodactylis Infected with Citrus Huanglongbing
- 中国实验方剂学杂志 2020年26卷第5期页码：117-122
- 作者机构：
  
  广东药科大学　中药学院，国家中医药管理局　岭南药材生产与开发重点研究室，国家中药材产业技术体系　广州综合试验站，广东省南药规范化种植与综合开发工程技术研究中心，广州　 510006
- 作者简介：
  
  [第一作者]　袁蒙，在读硕士，从事中药资源开发与品质评价研究，Tel：020-39252353，E-mail：964485970@qq.com
  *杨全，博士，教授，从事中药资源开发与品质评价研究，Tel：020-39252353，E-mail：yangquan7208@vip.163.com
- 基金信息：
  
  国家重点研发计划项目(2017YFC1700704);广东省中医药局中药资源普查专项(20163008);2017年广东省岭南中药材保护资金专项(粤财社[2017]60号)
- DOI：10.13422/j.cnki.syfjx.20192413
  中图分类号： R289; R284.1; R22; R2-031
- 收稿日期：2019-02-22，
  
  网络出版日期：2019-09-04，
  
  纸质出版日期：2020-03-05
- 稿件说明：
移动端阅览
袁蒙, 潘利明, 周良云, 等. 广佛手黄龙病的鉴定[J]. 中国实验方剂学杂志, 2020,26(5):117-122.

Meng YUAN, Li-ming PAN, Liang-yun ZHOU, et al. Identification of Citrus medica var. sarcodactylis Infected with Citrus Huanglongbing[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(5): 117-122.
袁蒙, 潘利明, 周良云, 等. 广佛手黄龙病的鉴定[J]. 中国实验方剂学杂志, 2020,26(5):117-122. DOI： 10.13422/j.cnki.syfjx.20192413.

Meng YUAN, Li-ming PAN, Liang-yun ZHOU, et al. Identification of Citrus medica var. sarcodactylis Infected with Citrus Huanglongbing[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(5): 117-122. DOI： 10.13422/j.cnki.syfjx.20192413.

摘要

目的：

该文从多方面进行分析、比较，及时、准确地鉴别广佛手黄龙病，以便于及时确定病害、防控病情。

方法：

通过性状分析，逆转录-聚合酶链式反应(RT-PCR)，酶切反应及实时荧光定量PCR(Real-time PCR)检测多种手段相结合，对广佛手黄龙病进行鉴定。

结果：

从性状上来看，感病广佛手有典型的叶片斑驳型黄化、果相对较小，甚至畸形的特征，但并没有发现“红鼻子果”现象，这些均可以作为田间初步判别广佛手黄龙病的依据；RT-PCR检测及酶切反应结果表明，引物为OI1/OI2c时，广佛手病株有特异性条带1 160 bp，且能被Xba I酶切成520 bp和640 bp，这与其他被黄龙病侵染的柑橘属植物的检测结果一致；Real-time PCR检测结果显示，被黄龙病侵染的广佛手叶片有扩增曲线和熔解曲线，熔链温度为82 ℃，且

值在24.6～28.2，而广佛手正常株没有扩增现象出现。

结论：

性状分析可以在田间初步判别黄龙病，但是具有一定的主观性，RT-PCR及Real-time PCR检测能进一步确定广佛手黄龙病，且qPCR检测更加灵敏，还可以定量。通过性状分析以及分子鉴定的结合，能够更加及时、准确地确定广佛手黄龙病。

Abstract

Objective:

To analyze and compare different samples in many aspects to identify

Citrus medica

var.

sarcodactylis

infected with Huanglongbing(HLB) timely and accurately

in order to prevent and control the disease in time.

Method:

HLB was identified through character analysis

reverse transcription-polymerase chain reaction(RT-PCR)

enzyme digestion reaction and Real-time PCR.

Result:

In terms of characters

there were typically variegated yellow leaves and relatively small fruit

even with deformity but without " red nose fruit" among

medica

var.

sarcodactylis

infected with HLB. All of these can be used as the basis for the preliminary identification of HLB in the fields. According to the RT-PCR test results and enzyme digestion reaction

when the primer was OI1/OI2c

there was specific band of 1 160 bp

which could be cut into 520 bp and 640 bp by Xba I enzyme. These results were consistent with the characters of other citrus plants infected with HLB. According to the Real-time PCR detection results

medica

var.

sarcodactylis

infected with HLB had amplification curves and dissolved peaks

with the melting temperature was 82 ℃ and

between 24.6 to 28.2

while the normal plants were not amplified.

Conclusion:

Character analysis can be used to roughly distinguish HLB in the fields

but with a certain subjectivity. RT-PCR or Real-time PCR can be used to identify

medica

var.

sarcodactylis

infected with HLB in a timely and accurate manner

and qPCR detection is more sensitive and quantitative. Through the combination of character analysis and molecular identification

medica

var.

sarcodactylis

infected with HLB can be determined more timely and accurately.

关键词

Keywords

references

国家药典委员会．中华人民共国药典：一部 [M]．北京：中国医药科技出版社， 2015 ： 178 .

金雯芳，沈丽红，任加惠，等．佛手精油及相关产品开发的研究进展 [J]．中草药， 2016 ， 47 ( 5 ): 857 - 861 .

郭晓强，颜军，邬晓勇，等．低糖佛手果脯加工工艺研究 [J]．食品科学， 2006 ， 27 ( 12 ): 953 - 955 .

陈跃文，顾振宇，刘辉，等．佛手酥加工工艺研究 [J]. 食品研究与开发， 2015 ， 36 ( 21 ): 109 - 113 .

黄有霖，郭素华，陈达炜．佛手茶的保健功效及其保健开发前景 [J]．海峡药学， 2007 ， 92 ( 9 ): 71 - 72 .

杨国伟，马越，王晓杰，等．佛手发酵酒生产工艺研究 [J]. 酿酒科技， 2009 ， 176 ( 2 ): 84 - 86 .

刘春娜，姜竹茂．低热量佛手酸奶的研制 [J]．中国乳业， 2010 ， 102 ( 6 ): 58 - 61 .

祁龙凯，林励，汪金玉，等．广东英德引种广佛手与道地产地广佛手质量的比较研究 [J]．中药新药与临床药理， 2014 ， 25 ( 1 ): 67 - 70 .

张爵玉，蒋林，王琴．广佛手的研究现状及进展 [J]．中国调味品， 2008 ， 349 ( 3 ): 20 - 23 .

岳玲，程轩轩，杨全．广佛手的资源调查研究 [J]．广东化工， 2018 ， 45 ( 11 ): 34 - 36 .

J A QURESHI , P A STANSLY ． Exclusion techniques reveal significant biotic mortality suffered by Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus [J]． Biological Control ， 2009 ， 50 ( 2 ): 129 - 136 .

T R GOTTWALD ． Current epidemiological understanding of citrus Huanglongbing [J]． Annual Review Phytopathol ， 2010 ， 48 : 119 - 139 .

苏华楠．中国柑橘黄龙病病原调查、种群遗传分化及其原噬菌体溶菌酶蛋白原核表达 [D]．重庆：西南大学， 2013 .

许美容，陈燕玲，邓晓玲．柑橘黄龙病症状与“ Candidatus Liberibacter asiaticus”PCR检测结果的相关性分析 [J]．植物病理学报， 2016 ， 46 ( 3 ): 367 - 373 .

S JAGOUEIX , J M BOVÉ , M GARNIER . PCR detection of the two 'candidatus' liberobacter species associated with greening disease of citrus [J]． Molecular and Cellular Probes ， 1996 ， 10 ( 1 ): 43 - 50 .

D C TEIXEIRA , C SAILLARD , S JAGOUEIX EVEILLARD ， et al . A new liberibacter species，Candidatus Liberibacter americanus，is associated with Huanglungbing in Sao Paulo State，Brazil. [J]． Int J Syst Evolut Microbiol ， 2005 ， 55 ( 5 ): 1857 - 1862 .

R HARAKAVA ， J L MARAIS ， J OCHASAN . Improved sensitivity in the detection and differentiation of citrus huanglongbing bacteria from South Africa and the Philippines [C]// Proceedings of 14th Conference of the IOCV. Riverside: Proceedings of 14th Conference of the IOCV ， 2000 .

W LI , J S HARTUNG , L LEVY ． Quantitative Real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus Huanglongbing [J]． J Microbiological Methods ， 2006 ， 66 ( 1 ): 104 - 115 .

S SUBANDIYAH , T IWANAMI , S TSUYUMU ， et al . Comparison of 16S rDNA and 16S/23S intergenic region sequences among Citrus greening organisms in Asia [J]． Plant Disease ， 2000 ， 84 ( 1 ): 15 - 18 .

D C TEIXEIRA , C SAILLARD , C COUTURE . Distribution and quantification of Candidatus Liberibacter americanus，agent of huanglongbing disease of citrus in Saõ Paulo State，Brasil，in leaves of an affected sweet orange tree as determined by PCR [J]． Mol Cellular Probes ， 2008 ， 22 ( 3 ): 139 - 150 .

O A REINKING . Diseases of economic plants in southern China [J]． Phillip Agric ， 1919 ， 8 ( 4 ): 109 - 135 .

林孔湘 . 柑桔黄梢(黄龙)病研究Ⅱ-关于病原的探讨 [J]．植物病理学报， 1956 ， 2 ( 1 ): 13-42，104 .

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 [J]. 植物病理学报， 1957 ， 3 ( 2 ): 169 - 176 .

M GARNIER , N DANEL , J M BOVÉ . The greening organism is a gram negative bacterium [J]． Int Organ Citrus Virologists Conference Proceedings ， 1984 : 115 - 124 .

X WANG , H SU , L HUANG ， et al . Identification of a novel 1033-nucleotide deletion polymorphism in the prophage region of ‘candidatus liberibacter asiaticus’： potential applications for bacterial epidemiology [J]． J Phytopathol ， 2015 ， 163 ( 7/8 ): 681 - 685 .

罗志达，叶自行，许建楷，等．柑桔黄龙病的田间诊断方法 [J]．广东农业科学， 2009 ， 228 ( 3 ): 91 - 93 .

S VILLECHANOUX , M GARNIER , F LAIGRET ， 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 [J]． Curr Microbiol ， 1993 ， 26 ( 3 ): 161 - 166 .

邓晓玲，梁志慧，唐伟文．快速检测柑桔黄龙病病原的研究 [J]．华南农业大学学报， 1999 ， 20 ( 1 ): 4 - 7 .

田亚南，柯穗，李韬，等．应用电镜与PCR技术检测琯溪蜜柚黄龙病病原 [J]．植物病理学报， 2000 ， 30 ( 1 ): 76 - 81 .

Y IFTIKHAR , S RAUF , U SHAHZAD ， et al . Huanglongbing: pathogen detection system for integrated disease management-a review [J]． J Saudi Soc Agr Sci ， 2016 ， 15 ( 1 ): 1 - 11 .

Z WANG , Y YIN , H HU ， et al . Development and application of molecular-based diagnosis for Candidatus liberibacter asiaticus，the causal pathogen of citrus Huanglongbing [J]． Plant Pathol ， 2006 ， 55 ( 5 ): 630 - 638 .

J K MORGAN , L ZHOU , W LI ， et al . Improved Real-time PCR detection of ' Candidatus Liberibacter Asiaticus' from Citrus and psyllid hosts by targeting the intragenic Tandem-repeats of its prophage genes [J]． Mol Cell Probes ， 2012 ， 26 ( 2 ): 90 - 98 .

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