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

作　　者：崔一平[1] 彭埃天[1] 宋晓兵[1] 程保平[1] 凌金锋[1] 陈霞[1] 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)

机构地区：[1]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

出　　处：《中国农业科学》2020年第8期1572-1582,共11页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2018YFD0201500,2017YFD0202000);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(2019KJ108,2018LM1077);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)。

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,CCDa V)、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c DNA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CCDa V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�【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 16 S 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

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 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]