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

Abstract

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

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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URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2020.08.007

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Figures/Tables 6

Table 1

Table 2

Detection of CLas in main citrus producing areas in Meizhou City"

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

Table 2

Table 3

Table 4

Fig. 1

Fig. 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-	-

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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