广东梅州柑橘黄龙病和病毒病的发生调查及其黄龙病菌原噬菌体多样性

doi:10.3864/j.issn.0578-1752.2020.08.007

中国农业科学 ›› 2020, Vol. 53 ›› Issue (8): 1572-1582.doi: 10.3864/j.issn.0578-1752.2020.08.007

广东梅州柑橘黄龙病和病毒病的发生调查及其黄龙病菌原噬菌体多样性

崔一平,彭埃天(),宋晓兵,程保平,凌金锋,陈霞

广东省农业科学院植物保护研究所/广东省植物保护新技术重点实验室,广州 510640

收稿日期:2019-09-16 接受日期:2019-11-18 出版日期:2020-04-16 发布日期:2020-04-29
通讯作者: 彭埃天
作者简介:崔一平,E-mail: kktracy@163.com。
基金资助:
国家重点研发计划(2018YFD0201500);国家重点研发计划(2017YFD0202000);科技创新战略专项资金(高水平农科院建设)(R2018QD-059);广东省现代农业产业技术体系创新团队建设项目(2019KJ108);广东省现代农业产业技术体系创新团队建设项目(2018LM1077);植物疫病防控省级项目(0835-190Z22803281);梅州市政府购买服务合同项目(2018063001)

Investigation on Occurrence of Citrus Huanglongbing and Virus Diseases, and Prophage Genetic Diversity of Huanglongbing Pathogen in Meizhou, Guangdong

CUI YiPing,PENG AiTian(),SONG XiaoBing,CHENG BaoPing,LING JinFeng,CHEN Xia

Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou 510640

Received:2019-09-16 Accepted:2019-11-18 Online:2020-04-16 Published:2020-04-29
Contact: AiTian PENG

摘要/Abstract

摘要：

【目的】调查柑橘黄龙病（Huanglongbing,HLB）和病毒病在广东省梅州市柑橘主产区的危害情况,分析该地区柑橘黄龙病菌（CLas）的原噬菌体多样性。【方法】以16S rDNA为模板设计引物,利用实时荧光定量PCR（qPCR)检测梅州市不同抽检地在2017—2019年3年间柑橘黄龙病的发病情况;同时分别采用已报道的柑橘衰退病毒（Citrus tristeza virus,CTV）、柑橘裂皮病毒（Citrus exocortis viroid,CEVd）、柑橘褪绿矮缩病毒（Citrus chlorotic dwarf-associated virus,CCDaV）、柑橘碎叶病毒（Citrus tatter leaf virus,CTLV）和柑橘黄脉病毒（Citrus yellow vein clearing virus,CYVCV）的特异性检测引物,提取样品的RNA,反转后以样品的cDNA为模板,通过普通PCR的方法分析梅州市抽检地区柑橘病毒病的发生情况。此外,以基于2种原噬菌体类型（SC1和SC2）对应的超变异基因区域设计的2对引物（Lap-TJ-F/Lap-TJ-R1和Lap-TJ-F/Lap-TJ-R2）,运用普通PCR方法分析该地区CLas菌株原噬菌体的遗传多样性。【结果】除2018年9月梅州大浦顺兴公司蜜柚基地的抽检样品外,梅州市其他被检地区均发现CLas。总体来说,梅州市的脐橙CLas检出率为55.3%,蜜柚为61.5%,沙田柚为61.7%。其中2017年采集自平原县大拓镇的蜜柚和沙田柚样品CLas检出率为100%,兴宁市白马镇的蜜柚为100%,沙田柚为80%;其他抽检地区的样品CLas检出率在16.7%—83.3%。虽然梅州市部分地区的检出率较高,但其病原菌的含量并不高,大部分处于柑橘黄龙病发病初、中期,属可防可控阶段;此外,梅州市柑橘病毒的总检出率不高,CTV、CTLV和CYVCV为梅州的主要病毒,CEVd和CCDaV没有检测到。CTV、CTLV和CYVCV在脐橙上的检出率依次为78.9%、7.9%和21.1%,蜜柚为15.4%、25.0%和9.6%,沙田柚为6.4%、2.1%和4.3%。基于Lap-TJ-F/Lap-TJ-R1和Lap-TJ-F/Lap-TJ-R2来研究CLas原噬菌体多样性的PCR扩增条带共有4种类型（SC1-1、SC1-2、SC2-1和SC2-2）,经过PCR电泳和测序得出,来自梅州市脐橙和沙田柚的CLas菌株以SC2-1型为主,蜜柚上以SC1-1型为主。【结论】梅州柑橘产区的柑橘黄龙病目前属于可防可控阶段,其病原菌菌株的原噬菌体在不同品种上具有其独特性;由于在抽检时发现有柑橘病毒病的存在,因此在种植过程中需加强种苗监管,同时应采取有效措施对柑橘黄龙病和柑橘木虱（Diaphorina citri）进行绿色防控。

关键词: 柑橘黄龙病, 韧皮部杆菌亚洲种, 柑橘病毒病, 原噬菌体, 遗传多样性

Abstract:

【Objective】The objective of this study is to investigate the impacts of citrus Huanglongbing (HLB) and virus diseases on citrus production in Meizhou City of Guangdong Province, and to reveal the local ‘Candidatus Liberibacter asiaticus’ (CLas, the pathogen of HLB in China) strains genetic diversity based on the prophage region.【Method】The primers were designed with 16S rDNA as the template, and the incidence of HLB in different sampling sites in Meizhou City in the 3 years from 2017 to 2019 was detected by qPCR. After RNA extraction, RT-PCR followed by ordinary PCR were applied to detect Citrus tristeza virus (CTV), Citrus exocortis viroid (CEVd), Citrus chlorotic dwarf-associated virus (CCDaV), Citrus tatter leaf virus (CTLV) and Citrus yellow vein clearing virus (CYVCV) using their own reported primers, separately. Two pairs of primers (Lap-TJ-F/Lap-TJ-R1 and Lap-TJ-F/Lap-TJ-R2) were designed based on the supervariant gene regions corresponding to two prophage types (SC1 and SC2), and the genetic diversity of CLas prophage regions was analyzed using ordinary PCR method.【Result】The results of CLas detection showed that only the sample from one orchard (Honey pummelo base in Shunxing Company in Dapu County) in September, 2018 was free, other inspected areas in Meizhou all had CLas. Overall, the CLas detection rate of navel orange in Meizhou was 55.3%, the honey pummelo was 61.5%, and the Shatian pummelo was 61.7%. Among them, the CLas detection rate of honey pummelo and Shatian pummelo collected from Datuo Town, Pingyuan County in 2017 was 100%, the detection rate of honey pummelo and Shatian pummelo in Baima Town, Xingning City was 100% and 80%, respectively. The detection rate of samples in other sampling areas was 16.7%-83.3%. Although the detection rate in some areas of Meizhou was high, the content of pathogenic bacteria was not high, most of them were at the early and middle stages of HLB, which belonged to a preventable and controllable stage. In the same region, the total detection rate of citrus viruses in Meizhou City was not high. CTV, CTLV and CYVCV were the main viruses in Meizhou, CEVd and CCDaV were not detected. The detection rate of CTV, CTLV and CYVCV on navel orange was 78.9%, 7.9% and 21.1%, on honey pummelo was 15.4%, 25.0%, and 9.6%, on Shatian pummelo was 6.4%, 2.1%, and 4.3%, respectively. Through prophage genetic diversity analysis of CLas strains in Meizhou City, four CLas prophage amplicon types (SC1-1, SC1-2, SC2-1 and SC2-2) were observed in the CLas from all the tested samples, SC2-1 represented the predominant CLas type in navel orange and Shatian pummelo in Meizhou City, and SC1-1 was predominant in honey pummelo.【Conclusion】The citrus HLB in Meizhou citrus producing area is currently at a controllable stage. The prophage of its pathogenic CLas strain is unique in different varieties. Due to the existence of citrus virus diseases during spot inspection, seedling supervision needs to be strengthened during the planting process and effective measures should be taken to prevent and control HLB and Diaphorina citri.

Key words: citrus Huanglongbing, ‘Candidatus Liberibacter asiaticus’ (CLas), citrus virus disease, prophage, genetic diversity

崔一平,彭埃天,宋晓兵,程保平,凌金锋,陈霞. 广东梅州柑橘黄龙病和病毒病的发生调查及其黄龙病菌原噬菌体多样性[J]. 中国农业科学, 2020, 53(8): 1572-1582.

CUI YiPing,PENG AiTian,SONG XiaoBing,CHENG BaoPing,LING JinFeng,CHEN Xia. Investigation on Occurrence of Citrus Huanglongbing and Virus Diseases, and Prophage Genetic Diversity of Huanglongbing Pathogen in Meizhou, Guangdong[J]. Scientia Agricultura Sinica, 2020, 53(8): 1572-1582.

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https://www.chinaagrisci.com/CN/Y2020/V53/I8/1572

图/表 6

表1

表2

梅州市柑橘主产区黄龙病菌的检测"

采样时间Sampling time	采集地 Location (Village/Town/County)	品种 Variety	样品数（个）Number	检出率 Detection rate (%)	含菌量 HLB content (×10³ copies/g)	Ct范围 Ct range
2019-06	平远县仁居镇飞龙村Feilong/Renju/Pingyuan	脐橙 Navel orange	11	63.6	0.069-735	21.18-35.27
	平远县仁居镇六吉村 Liuji/Renju/Pingyuan	脐橙 Navel orange	10	30.0	0.078-0.123	34.39-35.08
	梅县区石扇镇西南村 Xi’nan/Shishan/Meixian	蜜柚 Honey pummelo	10	70.0	0.14-393	22.13-34.19
	梅县区人民广场森林公园 People’s Square Forest Park in Meixian District	蜜柚 Honey pummelo	12	66.7	0.073-277	22.66-35.17
	梅县区石扇镇西南村 Xi’nan/Shishan/Meixian	沙田柚 Shatian pummelo	10	60.0	0.073-576	21.55-35.18
	梅县区城东镇玉水村 Yushui/Chengdong/Meixian	沙田柚 Shatian pummelo	10	80.0	0.072-856	20.95-35.20
	大浦县大东镇东光村 Dongguang/Dadong/Dapu		15	60.0	0.069-1.02	31.18-35.27
2018-01	平远县仁居镇李氏家庭农场 Family Farm of LI/Renju Town/Pingyuan County	脐橙^×Navel orange	11	54.5	0.072-49.7	25.27-35.19
	平远县仁居镇李氏家庭农场 Family Farm of LI/Renju Town/Pingyuan County	脐橙^*Navel orange	6	83.3	0.071-0.76	31.62-35.22
	梅县区雁洋镇南福村 Nanfu/ Yanyang/Meixian	蜜柚 Honey pummelo	6	16.7	0.059	35.50
	大埔县湖寮镇双髻山 Shuangji/Huliao/Dapu		5	80.0	0.042-0.31	32.98-36.02
	大埔县百侯镇新乐村 Xinle/Baihou/Dapu		5	60.0	0.135-1.7	30.43-34.24
2018-09	大埔顺兴公司蜜柚基地 Honey pummelo base in Shunxing Company in Dapu County		5	0	0	0
2018-09		沙田柚Shatian pummelo	5	0	0	0
2017-07	兴宁市白马镇 Baima/Xingning	蜜柚Honey pummelo	6	100.0	0.199-1036	20.66-32.26
2017-07	兴宁市白马镇 Baima/Xingning	沙田柚Shatianpummelo	5	80.0	0.052-0.683	31.78-35.68
2017-08	平远县大拓镇 Datuo/Pingyuan	蜜柚Honey pummelo	3	100.0	0.232-1106	20.56-33.42
2017-08	平远县大拓镇 Datuo/Pingyuan	沙田柚Shatian pummelo	2	100.0	0.18-1742	19.87-33.81
2017-2019	梅州市 Meizhou City	脐橙Navel orange	38	55.3	0.069-735	21.18-35.27
		蜜柚Honey pummelo	52	61.5	0.042-1106	20.56-36.02
		沙田柚Shatian pummelo	47	61.7	0.052-1742	19.87-35.68

表2

表3

表4

图1

图2

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采集地 Location		品种 Variety	种植年限 Crop years (a)	面积 Area (hm²)
平远县 Pingyuan County	仁居镇飞龙村Feilong Village, Renju Town	脐橙Navel orange	>2	100
平远县 Pingyuan County	仁居镇六吉村Liuji Village, Renju Town	脐橙Navel orange	>1	100
梅县区 Meixian District	石扇镇西南村Xi’nan Village, Shishan Town	蜜柚Honey pummelo	>20	100
	人民广场森林公园People’s Square Forest Park	蜜柚Honey pummelo	>5	>10
	石扇镇西南村Xi’nan Village, Shishan Town	沙田柚Shatian pummelo	>2	300
	城东镇玉水村Yushui Village, Chengdong Town		>25	>10
大埔县Dapu County	大东镇东光村Dongguang Village, Dadong Town		>9	1000

采集地 Location	品种 Variety	样品 Sample	Ct-value	CLas		扩增类型 Amplified type
采集地 Location	品种 Variety	样品 Sample	Ct-value	qPCR	Ordinary PCR	扩增类型 Amplified type
广州市白云区钟落潭镇 Zhongluotan Town, Baiyun District, Guangzhou City	砂糖橘Sugar orange	Z1	22.08	A+	A+	SC2-1 (2)
	默克特Murcott	Z2	25.89	A+	A+	SC2-1(2)
	蜜柚Honey pummelo	Z3	22.08	A+	A+	SC2-1
平远县仁居镇 Renju Town, Pingyuan County	脐橙Navel orange	R1	21.18	A+	A+	SC2-1
		R2	35.20	A+	A-	SC2-1
		R3	34.80	A+	A-	SC2-1
		R4	N	A-	A-	-
梅县区石扇镇 Shishan Town, Meixian District	蜜柚Honey pummelo	SM1	22.13	A+	A+	SC1-1
	蜜柚Honey pummelo	SM2	32.69	A+	A-	SC1-1
	沙田柚Shatian pummelo	S1	21.55	A+	A+	SC2-1
	沙田柚Shatian pummelo	S2	33.96	A+	A-	SC2-1
梅县区城东镇 Chengdong Town, Meixian District	沙田柚Shatian pummelo	C1	31.68	A+	A-	SC2-1
梅县区城东镇 Chengdong Town, Meixian District	沙田柚Shatian pummelo	C2	34.06	A+	A-	SC1-2
梅县区人民广场森林公园 People’s Square Forest Park in Meixian District	蜜柚Honey pummelo	G1	22.66	A+	A+	SC2-1
梅县区人民广场森林公园 People’s Square Forest Park in Meixian District	蜜柚Honey pummelo	G2	34.60	A+	A-	SC2-1
大埔县大东镇 Dadong Town, Dapu County	蜜柚Honey pummelo	D1	31.18	A+	A-	SC2-1
大埔县大东镇 Dadong Town, Dapu County	蜜柚Honey pummelo	D2	34.92	A+	A-	-

广东梅州柑橘黄龙病和病毒病的发生调查及其黄龙病菌原噬菌体多样性

Investigation on Occurrence of Citrus Huanglongbing and Virus Diseases, and Prophage Genetic Diversity of Huanglongbing Pathogen in Meizhou, Guangdong

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采集地 Location (Village/Town/County)	品种 Variety	样品数（个） Number	检出率Detection rate (%)
采集地 Location (Village/Town/County)	品种 Variety	样品数（个） Number	CTV	CEVd	CCDaV	CTLV	CYVCV
平远县仁居镇飞龙村 Feilong/Renju/Pingyuan	脐橙 Navel orange	11	100.0	0	0	0	0
平远县仁居镇六吉村 Liuji/Renju/Pingyuan	脐橙 Navel orange	10	50.0	0	0	0	0
梅县区石扇镇西南村 Xi’nan/Shishan/Meixian	蜜柚 Honey pummelo	10	50.0	0	0	0	0
梅县区人民广场森林公园 People’s Square Forest Park in Meixian County	蜜柚 Honey pummelo	10	0	0	0	0	0
梅县区石扇镇西南村 Xi’nan/Shishan/Meixian	沙田柚 Shatian pummelo	12	0	0	0	0	0
梅县区城东镇玉水村 Yushui/Chengdong/Meixian		10	0	0	0	0	0
大埔县大东镇东光村 Dongguang/Dadong/Dapu		15	0	0	0	0	0
平远县仁居镇李氏家庭农场 Family Farm of LI, Renju Town, Pingyuan County	脐橙^×Navel orange	11	81.8	0	0	9.1	72.7
平远县仁居镇李氏家庭农场 Family Farm of LI, Renju Town, Pingyuan County	脐橙^*Navel orange	6	83.3	0	0	33.3	0
梅县区雁洋镇南福村 Nanfu/Yanyang/Meixian	蜜柚 Honey pummelo	6	0	0	0	50	0
大埔县湖寮镇双髻山 Shuangji/Huliao/Dapu		5	0	0	0	100	0
大埔县百侯镇新乐村 Xinle/Baihou/Dapu		5	0	0	0	60	0
大浦顺兴公司蜜柚基地 Honey pummelo base in Shunxing Company in Dapu County		5	0	0	0	20	40
	沙田柚Shatian pummelo	5	0	0	0	0	0
平远县大拓镇 Datuo/Pingyuan	蜜柚Honey pummelo	3	100.0	0	0	0	100.0
平远县大拓镇 Datuo/Pingyuan	沙田柚Shatian pummelo	2	100.0	0	0	50.0	100.0
兴宁市白马镇 Baima/Xingning	蜜柚Honey pummelo	6	0	0	0	20.0	0
兴宁市白马镇 Baima/Xingning	沙田柚Shatian pummelo	5	10.0	0	0	0	0
梅州市 Meizhou City	脐橙Navel orange	38	78.9	0	0	7.9	21.1
	蜜柚Honey pummelo	52	15.4	0	0	25.0	9.6
	沙田柚Shatian pummelo	47	6.4	0	0	2.1	4.3

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