应用多聚酶链式反应(PCR)技术检测和定量分析柑桔黄龙病病原

 应用多聚酶链式反应(PCR)技术对柑桔黄龙病病原进行检测和定量分析。研究结果表明:PCR技术能准确、快速地检测出田间和温室内罹病的柑桔、长春花以及带菌柑桔木虱样本中的柑桔黄龙病病原(BLO),而健康植株和柑桔木虱样本则呈阴性反应。采用的PCR引物(Primers)是根据柑桔黄龙病病原亚洲株系的DNA序列(In-2.6,基因库号码:M9491)设计的。应用竞争性定量多聚酶链式反应(Q-PCR)方法能测定病原在不同样品中的含量。该技术关键在于:病原DNA和浓度系列稀释后的竞争物DNA,在同一试管中进行DNA扩增反应时,共用相同的引物。由于彼此互相竞争反应引物的结果,病原的PCR产物和竞争物的PCR产物在数量上呈反向线性相关。因竞争物的浓度为已知,便可从线性关系中推算出病原的含量。     

柑桔木虱生物学特性及防治研究

柑桔木虱雌成虫必须选择寄主嫩梢产卵的特性与年发生代数关系密切。其嗜好寄生九里香,九里香年抽梢次数比柑桔多,在九里香上1年可能发生9—11代,在柑桔上可能发生6—7代(福州地区)。春夏秋季(旬严均气温分别为19.6℃、28℃和24℃)最长与平均成虫寿命及世代历期分别为98天、40天和52—59天;59天、32天和18—22天;65天、30天和25—30天。越冬成虫寿命长达260天。若虫和成虫的致死“温/时”分别为—3℃/小时和—10℃/小时。40％氧化乐果的800—1000倍液对木虱有特效,专性杀螨剂托尔克和普特丹对木虱无兼治作用。     

柑桔木虱(Diaphorina citri Kuwayama)与柑桔黄龙病流行关系的初步研究

 1973-1978年用从田间病树上采集的柑桔木虱成虫放饲在398株健康柑桔苗上,有32株发病。用病树上采集的柑桔木虱高龄若虫置健苗上所羽化的成虫放饲的56株,有5株发病。未接虫的110株没有发病。初步说明柑桔木虱成虫可以传病。     

柑桔木虱传递柑桔黄龙病的初步研究

柑桔木虱是柑桔黄龙病主要媒介昆虫。几年来的试验初步表明:无病植株上繁殖木虱成虫,在病树上吸毒20—30天以上就可传病。黄龙病病原在成虫体内约经20—30天循回期、带毒后木虱成虫,虫口密度大(20头/苗以上),吸食传毒1天即可发病;虫口密度小(1—5头/苗),吸食传毒7—14天也能传病。潜伏期一般2—8个月,个别株2年才发病,病梢于冬季回接,3—5个月后也表现出和虫传发病苗相同的斑驳状病症。     

柑桔黄龙病的病原在木虱体内循回期的研究

 本研究应用生物测定和电镜检验的方法,探讨黄龙病病原在其介体昆虫(柑桔木虱)体内的循回期。前者的结果表明,其循回期长短不一,短的为3天或少于3天,长的达26-27天。木虱成虫能终身携带病原。获"毒"饲育若虫新羽化的成虫也能传病。其中,羽化后1天和2-5天便能传病的单虫分别占供试虫(组)数的3%和5%。电镜检验结果,在获"毒"饲育后3天的成虫唾液腺细胞内能观察到病原体,有力支持了采用生物测定循回期的可靠性。根据由获"毒"饲育若虫新羽化的成虫就能传病的新认识,建议治虫防病的重点应放在治卵和若虫上。     

柑桔黄龙病的病原在木虱体内循回期的研究

本研究应用生物测定和电镜检验的方法,探讨黄龙病病原在其介体昆虫(柑桔木虱)体内的循回期。前者的结果表明,其循回期长短不一,短的为3天或少于3天,长的达26—27天。木虱成虫能终身携带病原。获“毒”饲育若虫新羽化的成虫也能传病。其中,羽化后1天和2—5天便能传病的单虫分别占供试虫(组)数的3%和5%。电镜检验结果,在获“毒”饲育后3天的成虫唾液腺细胞内能观察到病原体,有力支持了采用生物测定循回期的可靠性。根据由获“毒”饲育若虫新羽化的成虫就能传病的新认识,建议治虫防病的重点应放在治卵和若虫上。     

广西柑桔木虱北延情况及其原因的初步分析

柑桔木虱(Daiphorina citri kuway-ama)是柑桔黄龙病的传病媒介,它的发生与柑桔黄龙病的流行密切相关.1982年以来,作者在参加全区柑桔黄龙病和柑桔木虱普查后,又多次对灵川、兴安和灌阳三县进行调查,同时在兴安和灌阳县设立监测哨,定期对柑桔木虱的动态进行调查监测.1987年8～10月间,又对1982年普查时未发现柑桔木虱的兴安等7个县进行了调查.结果所调查的7个县全都有了柑桔木虱发生,柑桔木虱在广西的分布北缘已到了最北县份.作者以调查的结果绘成《广西柑桔木虱北延情况示意图》,此图可见,1980年以来,广西柑桔木虱有两次大规模和一次小规模北移;第一次大规模北移该虫的分布北缘线由1980年的北纬25度向北推移了0.5度,分布范围扩大了7个县(市);第二次是小规模北移,分布北缘线仅向北延伸了15公里.第三次大规模北移,分布北缘线由北纬25.5度又向北推移了0.5度,分布范围扩大了7个县.经过上述三次北移,该虫在广西的分布范围扩大了14个县(市),致使广西全境都有了柑桔木虱发生.作者对柑桔木虱逐年向北迁移的原因作初步分析:1、柑桔木虱大规模向北扩散的最可能途径是:随气流迁移.如上所述,在短短的几年里,此虫就作了三次北移,范围波及14个县(市).如不是借风力作迁移,是不可能达到的.2、大量的虫源是柑桔木虱北延的     

柑桔溃疡病菌实时荧光定量PCR检测与应用

根据地毯草黄单孢Xac306菌株(Xanthomonas axonopodis pv.citri,Xac306)已知全基因组中独有蛋白基因序列设计的特异性引物对和探针,建立并优化SYBR Green I(SGI)荧光染料和Taq-Man探针实时荧光定量PCR检测体系,用于柑桔溃疡病早期诊断鉴定。结果表明,建立的两种定量PCR体系均能特异地检出Xac的细胞和其基因组DNA,而对其它测试的植物病原菌和柑桔表面的腐生黄单孢菌都不能检出。SGI法和TaqMan探针法对Xac细菌悬浮液的检测灵敏度均可达到1~5个细菌/反应,对Xac靶标片段DNA的检测灵敏度可达1fg/μL。两种定量PCR检测方法比常规PCR灵敏度高2~3个数量级。对田间采集的328个柑桔显症、疑似症状和无症带菌材料富集培养样品进行了实际检测,结果表明,实时荧光PCR适合柑桔无症带菌样品的早期检测。     

快速检测单头柑橘木虱体内黄龙病病原

本研究通过采用简易的柑橘木虱制样方法,利用PCR检测技术在单头柑橘木虱体内检测到黄龙病病原,并利用XbaⅠ限制性内切酶将从柑橘木虱体内扩增的黄龙病病原16SrDNA酶切成520bp和640bp两个片段,证实了木虱体内黄龙病病原为亚洲种。本研究成功地建立了一套快速检测柑橘木虱体内黄龙病病原的方法,可为检疫工作者和相关研究人员提供重要参考。     

膠氨铜根治柑桔溃疡病

柑桔溃疡病是一种细菌性传染性检疫病害,其致病菌为甘蓝黄单胞杆菌柑桔致病变种Xanthomonas campestris pV.citri (Hasse) Dye。感病植株在叶、枝、果上产生溃疡病斑,导致落叶、落果,树势衰弱,病果商品价值大大降低。寄主遍及橙、柑、桔、柚、枳、香橼,柠檬、金柑、九里香等数十种的芸香科植物。     

木虱传播柑桔黄龙病及电镜检验报告

 本试验在福建省福州市及广东省侥平县两地进行。1978年以来,在福州市以蕉柑、福桔和新会甜橙病树为毒源树,以柑桔实生健苗为指示植物,进行木虱传试验。结果在329株指示苗中,发病40株,发病率12.2%,潜育期短的半年,长的5年。对照指示苗72株均未见发病。1982~1984年,在饶平县以蕉柑病树为毒源树,以椪柑实生健苗为指示植物,共试验70株,发病33株,发病率47.1%,潜育期短的2个月,长的8个月。对照指示苗9株未见发病。木虱传染病苗的病状与毒源树的病状相同,均表现黄梢和叶片斑驳。获"毒"木虱的唾液腺和脂肪体的超薄切片样本在电镜下均观察到原核细胞微生物(称为类立次克体或类细菌)。唾液腺内的病原体分布在外皮层细胞,内皮层细胞和腺腔内。其菌体的形状大小和膜壁结构等特征均与毒源树及木虱传染病苗的筛管细胞内的相同,木虱获得和传递柑桔黄龙病病原的方式与蚜虫传播病毒中的持久性类型十分相似。     

乙氧氟草醚对脐橙的杀梢效果及对柑橘木虱栖息分布的影响

柑橘木虱偏好产卵于柑橘嫩梢新芽上，在统一抹芽放梢后及时施药防治木虱，成为防控柑橘黄龙病的重要措施。近年通过杀梢剂处理杀灭新发嫩梢代替人工抹梢受到果农的关注和使用，并已成为防治黄龙病的重要技术手段。本研究以纽荷尔脐橙为供试树种，喷施不同质量浓度的乙氧氟草醚药液，探究其杀梢效果及对柑橘木虱栖息分布的影响，并建立了嫩梢萎蔫的分级标准和木虱栖息分布的研究方法。结果表明:乙氧氟草醚对脐橙树苗和定植树有相似的杀梢效果，质量浓度为10 mg/L时即有一定的杀梢作用，40~55 mg/L时杀梢效果最佳，速效性优异，对柑橘叶片和成熟期果实无药害。采用40和55 mg/L的乙氧氟草醚药液对树苗喷雾，3 d后，嫩梢长度抑制率分别为86.1%和124.7%，嫩梢直径抑制率分别为83.8%和94.8%；7 d后，校正杀梢率分别为93.7%和84.1%，木虱校正死亡率分别为7.8%和21.4%，木虱栖息抑制率分别为81.0%和84.4%。采用40、55 mg/L的乙氧氟草醚药液对定植树喷雾，3 d后嫩梢长度抑制率分别为85.9%和118.8%，嫩梢直径抑制率分别为83.9%和104.1%；7 d后校正杀梢率分别为96.6%和82.4%。嫩梢萎蔫可使嫩梢上的木虱虫卵和若虫死亡，对成虫死亡率影响小，但栖息环境发生迁移。因此，乙氧氟草醚施用于脐橙可快速杀梢，减少木虱的食物来源，有效控制木虱种群数量。     

Detection by PCR of Candidatus Liberibacter asiaticus, the bacterium causing citrus huanglongbing in vector psyllids: application to the study of vector–pathogen relationships

Citrus huanglongbing (HLB), previously called greening, is a serious citrus disease in Asia, eastern and southern Africa. It is caused by Candidatus Liberibacter asiaticus (Las), a phloem-limited, nonculturable bacterium transmitted by the Asian citrus psyllid ( Diaphorina citri ) in Asia. A PCR-based assay was developed for monitoring Las in vector psyllids using a rapid DNA extraction from psyllid bodies and PCR amplification. The entire procedure for Las detection in psyllids can be completed within 5 h. Using this method, Las can be accurately detected in psyllid adults as well as nymphs in different instar stages. The assay is sensitive enough for Las detection in single-psyllid extract from adult, fifth, fourth and third instars. In a transovarial transmission experiment, Las was not detected in eggs or in offspring produced by Las-carrying psyllid females. In a retention test, the Las-carrying psyllids remained Las-positive for 12 weeks after they were moved to common jasmine orange, a Las-immune plant. From these experimental results it was concluded that Las persists in the Asian citrus psyllid vector, but is not transovarially transmitted by the vector. These data help in understanding epidemiological characteristics of Las and psyllids in citrus HLB.     

Different symptoms in carrots caused by male and female carrot psyllid feeding and infection by ‘Candidatus Liberibacter solanacearum’

Carrot psyllid Trioza apicalis was recently found to carry the plant pathogenic bacterium ‘Candidatus Liberibacter solanacearum’ (CLs). To confirm the transmission of bacteria by the psyllids and to dissect the symptoms caused in carrot plants by psyllid feeding and CLs infection, a greenhouse experiment with single psyllids feeding on separate plants was performed. A positive correlation was found between the amount of CLs bacteria in the psyllids and in the corresponding plants exposed to feeding, indicating CLs transmission. The female psyllid feeding caused more severe damage than male feeding, and resulted in a substantial decrease in the root weight. Female psyllid feeding also significantly reduced the carrot leaf weight and increased the number of curled leaves. The number of curled leaves was also increased by the nymphs when their number exceeded 10 per plant. A high titre of CLs bacteria significantly reduced root weight, while not affecting the weight or number of the leaves. However, the amount of CLs correlated with the number of leaves showing discolouration symptoms. Microscopy of infected carrot plants revealed that the phloem tubes throughout the whole plant, from leaf veins to the root tip, were colonized by bacteria. The bacterial cells appeared to be long and thin flexible rods with tapering ends and a transversally undulated surface. Microscopy also revealed collapsed phloem cells in the infected carrots. Damage in the phloem vessels is likely to reduce the sucrose transport from source leaves to the root, explaining the observed leaf discolouration and reduction in root weight.     

Identification of the Chinese Box Orange (Severinia Buxifolia) as an Alternative Host of the Bacterium Causing Citrus Huanglongbing

The Chinese box orange (Severinia buxifolia) was shown by graft-inoculation and psyllid-transmission tests to be an alternative host of the bacterium causing citrus Huanglongbing (HLB). A PCR-based assay for detection of the HLB bacterium (HLBB) was used to monitor HLBB. In graft-inoculation tests, the Chinese box orange (CBO) grafted with HLBB-infected scions of Luchen sweet orange (LSO) were positive for HLBB, 2–3 months after grafting. The back-grafting test demonstrated that HLBB-infected CBO scions could transmit HLBB back to LSO hosts via grafting. In psyllid-transmission tests, psyllids (insect vectors) transmitted HLBB to CBO plants, in which HLBB could be detected 3–4 months after inoculation. Acquisition-access tests of psyllids revealed that HLBB-free psyllids can acquire HLBB from diseased CBO hosts and can transmit HLBB back to the LSO plants. A field survey verified the presence of HLBB-infected CBO plants in the vicinity of citrus orchards. In this paper, CBO is shown to be a susceptible host plant in which HLBB can exist and replicate. It is also a donor plant from which HLBB can be transmitted to citrus hosts by grafting or by psyllid vectors.     

柑桔溃疡病菌PCR快速检验检疫技术研究

 柑桔溃疡病是严重影响全世界柑桔生产的重大检疫性病害,根据柑桔溃疡病菌(Xanthomonas axonopodis pv. citri)新近公布的全基因组中独有的保守蛋白基因序列,设计筛选出一对种特异性引物(JYF5/JYR5),能专一地扩增检出柑桔组织表面所带溃疡病菌的DNA靶带(413 bp)。而柑桔叶面附生的非致病性黄单胞菌、野油菜黄单胞菌近缘种以及健康柑桔样品都不能扩增;靶细菌DNA检测下限1.56 pg/μL,靶细菌悬浮液检测下限10 cfu/μL;在不同PCR仪及各种控温方式下都能稳定地扩增出特征性靶带。这一特异、准确的柑桔溃疡病菌PCR检验技术和研制的预包被固相化PCR检测试剂盒已开始用于我国非疫生产区建设中柑桔苗木、果实的病害检疫检验。     

Quantification and ecological study of ‘Candidatus Liberibacter asiaticus’ in citrus hosts,rootstocks and the Asian citrus psyllid

The use of proper management strategies for citrus huanglongbing (HLB), caused by ‘Candidatus Liberibacter asiaticus’ (Las) and transmitted by Asian citrus psyllid (ACP) (Diaphorina citri), is a priority issue. HLB control is based on healthy seedlings, tolerant rootstock cultivars and reduction of ACP populations. Here, dynamic populations of Las in different citrus hosts and each instar of ACP were studied, together with the seasonal growth and distribution of Las in different tissues, using conventional and TaqMan real‐time PCR. Different levels of susceptibility/tolerance to HLB were seen, resulting in different degrees of symptom severity and growth effects on hosts or rootstocks. Troyer citrange, Swingle citrumelo and wood apple were highly tolerant among 11 rootstock cultivars. Regarding distribution and seasonal analysis of Las, mature and old leaves contained high concentrations in cool temperatures in autumn and spring. Las was detected earlier through psyllid transmission than through graft inoculation, and the amounts of Las (AOL) varied in different hosts. Thus, different AOL (10⁴–10⁷ copy numbers μL^?1) and Las‐carrying percentages (LCP; 40–53.3%) were observed in each citrus cultivar and on psyllids, respectively. Furthermore, both AOL and LCP were lower in nymphs than in adult psyllids, whereas the LCP of psyllids were not affected by increasing the acquisition‐access time. The present study has significant implications for disease ecology. The combination of early detection, use of suitable rootstocks and constraint of psyllid populations could achieve better management of HLB disease.     

LncRNA在柑橘木虱与黄龙病病原菌互作中的差异表达分析及验证

为明确柑橘黄龙病的唯一自然传播媒介——柑橘木虱Diaphorina citri的长链非编码RNA(long non-coding RNA,lncRNA)是否参与调控黄龙病病原菌Candidatus Liberibacter asiaticus(CLas)的侵染及复制,采用生物信息学预测及PCR扩增方法进行lncRNA的预测、特征分析、验证及差异表达分析。结果显示,柑橘木虱的13个转录组RNA-Seq数据中共有10 192个lncRNA基因,对应15 747条lncRNA转录本;与蛋白质编码基因相比,柑橘木虱lncRNA具有更少的外显子数量和更短的转录本长度;随机选取的10条lncRNA基因中,有7条lncRNA基因在无菌柑橘木虱广州品系或赣州品系中有表达,其中1条lncRNA基因TCONS_00034665在无菌广州品系和无菌赣州品系中存在差异表达;带菌和无菌柑橘木虱成虫中预测获得2个差异表达的lncRNA基因TCONS_00096118和TCONS_00234564,实时荧光定量PCR验证发现TCONS_00234564与预测结果一致,在带菌柑橘木虱成虫中高表达。表明lncRNA参与了黄龙病病原菌与寄主柑橘木虱的互作。