三种PCR方法检测柑橘黄龙病菌的效果比较

为了比较常规PCR、巢式PCR和实时荧光定量PCR方法在大田检测中对柑橘黄龙病(Huanglongbing, HLB)的检测效果, 首先比较了3种检测方法对柑橘黄龙病菌检测的灵敏度, 结果发现：3种检测方法的灵敏度依次为常规PCR<巢式PCR<实时荧光定量PCR。运用3种检测方法对广东5个柑橘品种上的189个黄龙病疑似病样进行检测, 结果发现：黄龙病检出率依次为常规PCR<巢式PCR<实时荧光定量PCR。研究表明：常规PCR适合以较低成本大规模检测黄龙病; 实时荧光定量PCR具有最大的检测灵敏度; 巢式PCR检测技术同时具有前两者的一些优点, 但操作较复杂, 适合技术熟练的研究者使用。     

柑橘黄龙病田间诊断与检测技术研究进展

目前防控柑橘黄龙病的措施主要包括培育无病毒苗、防治木虱、铲除病树等。而多数防控措施的开展均需灵敏、可靠的早期诊断技术配合。综述了柑橘黄龙病的田间诊断方法以及基于电镜、血清学、高光谱、淀粉显色、常规PCR、巢式PCR、LAMP PCR、定量PCR、数字PCR的检测方法 ,并阐述了各自的优缺点。其中,定量PCR是目前较成熟和具有较高灵敏度的检测技术,可配合其他低成本检测方法对未显症、假阴性等样品进行早期检测;可实现单分子DNA绝对定量分析,数字PCR检测技术在柑橘黄龙病检测中也具有较好的应用前景。     

基于近红外技术柑橘黄龙病田间快速检测方法研究

柑橘黄龙病在感病植株和健康植株之间传播速度快,因此快速检测柑橘黄龙病对其防治十分关键。本文研究了近红外技术快速检测柑橘黄龙病的方法。采用PLS-LDA建立的模型对未参与建模的样品进行了检测,结果表明,该模型检测的准确率与普通PCR检测的结果符合率达到100%,假阳性率小于1%。该技术具有检测周期短、无污染等优点,可用于田间黄龙病的快速检测。     

三种分子检测体系的比较及柑橘果园黄龙病监测

为了评价3种PCR分子检测体系对柑橘黄龙病(citrus huanglongbing,HLB)大田诊断效果,综合比较了常规PCR、巢式PCR和SYBR Green Ⅰ荧光定量PCR(SG Ⅰ-qPCR)方法对柑橘黄龙病菌检测的灵敏度、特异性和准确度等参数,并用SYBR Green Ⅰ荧光定量PCR和巢式PCR监测广西柑橘园疑似HLB样品425个,比较了2种检测体系的阳性检出率。基于CQULA04F/CQULA04R引物对的SYBR Green Ⅰ荧光定量PCR的灵敏度可达10 ag/μL;而巢式PCR灵敏度为100 ag/μL,巢式PCR较常规PCR检测灵敏度高104倍。疑似样品的HLB病原SYBR Green Ⅰ荧光定量PCR和巢式PCR检出率分别为46.6%、40.0%。各检测体系的灵敏性、特异性、符合度依次为SYBRGreen Ⅰ荧光定量PCR>巢式PCR>常规PCR。研究表明,SYBR Green Ⅰ荧光定量PCR可作为果园大规模HLB早期诊断和监测的首选,而在缺乏定量检测仪器时,巢式PCR也可用于HLB的检测,但需注意避免空气污染导致的假阳性。     

柑橘黄龙病PCR检测引物比较研究

柑橘黄龙病潜伏期长、尚无可用于大田的有效治疗药剂,快速、准确的早期检测是防控柑橘黄龙病的关键.PCR检测是目前柑橘黄龙病最常用的早期检测方法.为提高柑橘黄龙病PCR检测的准确性和检出率,本研究依据已测序的黄龙病菌全基因组序列对检测引物OI2c进行了改进(标记为OI2c-gj),将其与其他常用的7对PCR检测引物进行了特...     

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。     

海南省柑橘主产区黄龙病和病毒病的发生危害情况调研初报

柑橘是我国南方重要的水果之一,柑橘黄龙病和柑橘病毒病已经对我国柑橘的安全生产造成了严重威胁。为了解柑橘黄龙病和病毒病对海南柑橘产业的危害情况,本研究通过荧光定量PCR(Quantitative PCR)和反转录PCR(RT-PCR)分别对随机采集自海南柑橘主产区澄迈县3个福橙种植园和琼中县4个绿橙种植园共计109个样品进行了柑橘黄龙病和柑橘5种主要病毒病的检测。结果表明:澄迈县福橙标准化种植示范基地和琼中县营根镇绿橙种植园2个新植橘园的柑橘黄龙病发病较轻,检出率仅为8%和11%,而其他5个橘园的柑橘黄龙病检出率均在75%以上,严重的达100%。除此之外,7个橘园均未检测到柑橘褪绿矮缩病毒;澄迈县和琼中县的衰退病毒病检出率分别在52.9%和77.8%以上,黄脉病毒病在83.3%和90%以上。澄迈县还检测到碎叶病毒病的存在,检出率在38.1%以上,除红湖农场外还存在裂皮病毒病,检出率在11.8%以上。琼中县的绿橙种植园3还存在裂皮病毒病,检出率为43.8%。综上所述,柑橘黄龙病和病毒病已经严重威胁到海南省福橙和绿橙主产区的柑橘生产安全。加强对海南柑橘苗木安全生产的同时,亟需采取有效的田间综合防控措施,以促进海南柑橘产业的健康发展。     

柑橘黄龙病菌核酸检测技术研究进展

候选韧皮部杆菌（Candidatus Liberibacter spp.）是一类通过虫媒传播，寄生于韧皮部的革兰氏阴性细菌，引起柑橘上的毁灭性病害——黄龙病（Huanglongbing,HLB）。柑橘感染黄龙病后在3～5年内便会衰亡或丧失结果能力，目前尚无有效防治药剂，果园若不及时在感染初期铲除病树，病害会在柑橘木虱的活动下迅速蔓延至整个果园，乃至摧毁整个地区的柑橘产业，造成不可估量的损失。培育无毒苗木、尽早发现和彻底铲除病树是防控柑橘黄龙病的关键，因此建立快速、精准的检测方法是防控黄龙病的基础。本文主要综述了柑橘黄龙病各种核酸检测方法及其优缺点，以期对建立更加灵敏、准确、高效的检测技术提供参考。     

柑橘黄龙病症状与“Candidatus Liberibacter asiaticus”PCR检测结果的相关性分析

柑橘黄龙病症状较为复杂,且因寄主品种、生长期、病程等因素而异。利用PCR检测其病原菌"Candidatus Liberibacter spp."是目前柑橘黄龙病诊断的可靠方法之一。分析柑橘黄龙病症状与PCR检测结果的相关性有助于提高黄龙病的田间诊断准确率。本研究结果表明,与PCR检测相比,根据症状诊断柑橘黄龙病具有较高的假阳性率(8.20%)和假阴性率(50%);通过分析1 839个样品的症状与病原PCR检测结果发现,表现为斑驳型黄化、均匀黄化和"绿岛"这3种叶部症状以及"红鼻子果"和畸形果这2种果实症状的PCR病原检出率高;具有斑驳和黄化、黄化和"绿岛"、"绿岛"和花叶等复合症状样品的PCR检测"Ca.L.asiaticus"的阳性率最高;直径小于1 cm的幼果中的"Ca.L.asiaticus"检测稳定性低。这些结果为更准确地通过症状诊断柑橘黄龙病提供了科学依据。     

柑橘花器和种子中黄龙病菌的定量分布及应用

为准确、快速测定感病柑橘花器和种子中黄龙病菌（Candidatus Liberibacter asiaticus,Las）的含量,比较了SYBR Green Ⅰ实时荧光定量PCR（SGI-qPCR）和单管双引物对TaqMan探针qPCR（STDP-qPCR）的检测灵敏度,并用STDP-qPCR法定量检测了感病沙田柚花器和种子中的Las。结果显示,STDP-qPCR检测灵敏度为1×10⁰拷贝/μL,比SGI-qPCR高100倍;花器中的雄蕊、花瓣、雌蕊和花粉等组织,以及种子的种皮和胚乳组织中均可检测到Las,但含量差异较大,其中种皮组织中Las含量最高,达到109 842个细胞/μg DNA,花粉中的Las含量最低,为308个细胞/μg DNA,所有种壳中均未检测到Las;基于雄蕊组织的黄龙病分子诊断准确率达93.8%。表明Las在感病柑橘花器和种子中呈不均匀分布,基于雄蕊组织的黄龙病诊断方法可辅助用于该病害的高通量检测。     

In planta distribution of 'Candidatus Liberibacter asiaticus' as revealed by polymerase chain reaction (PCR) and real-time PCR

Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic alpha-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of 'Candidatus Liberibacter asiaticus,' respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that 'Ca. Liberibacter asiaticus' was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/mug of total DNA in different tissues. A relatively high concentration of 'Ca. Liberibacter asiaticus' was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that 'Ca. Liberibacter asiaticus' was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.     

Quantification of viable <Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

Citrus Huanglongbing (HLB) is a devastating disease of citrus known to be associated with a fastidious, phloem-limited Gram-negative, yet to be cultured bacterium in the genus Candidatus Liberibacter. In the present study we have developed a method to quantify viable Candidatus Liberibacter asiaticus (Las) with the aid of ethidium monoazide (EMA) which can differentiate live from dead cells. First, calibration curves were developed with the aid of quantitative real-time PCR (QPCR) by using a plasmid template consisting of a 703 bp DNA fragment of rplKAJL-rpoBC (β-operon) region. Standard equations were then developed to quantify Las genome equivalents in citrus, periwinkle, and Asian citrus psyllid, Diaphorina citri. To overcome the limitation of quantitative PCR in discriminating between live and dead bacterial cells, EMA was used to inhibit the amplification of DNA from the dead cells of Las in plant samples. By using the standard equations and EMA-QPCR methods developed in this study, we found that the proportion of viable cells in citrus and periwinkle ranged from 17–31% and 16–28%, respectively. It was determined that a minimum bacterial concentration is required for HLB symptom development by quantifying the population of Las in symptomatic and asymptomatic leaves. The EMA-QPCR methodology developed in the present study should provide an accurate assessment of viable HLB pathogen, providing a tool to investigate disease epidemiology and thus act as a crucial component for disease assessment and management. The authors P. Trivedi and U. S. Sagaram contributed equally to this work.     

柑橘黄龙病nested-PCR检测技术在柑橘苗木生产中的应用

柑橘黄龙病(huanglongbing, HLB)是柑橘生产上的一种毁灭性病害。本研究以柑橘叶脉为材料,采用nested-PCR技术对广西部分柑橘苗圃苗木进行了黄龙病检测。结果表明,nested-PCR技术不仅可以检测已经表现症状的苗木样品,还可以检测出带病但不显症的苗木样品。2007年共检测柑橘苗木样品1 950个,2008年共检测样品1 480个,黄龙病苗木检出率分别为10.31％和6.22％。本研究对及早控制带病苗木的传播,繁育柑橘无病毒苗木具有重要的意义。     

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.     

柑橘黄龙病病原研究进展

本文对柑橘黄龙病(HLB)的发现、认识过程及病原菌的分类、检测鉴定技术进行了综述。HLB是一种十分严重的柑橘病害,主要通过木虱和嫁接进行传播,在不同的地区和国家具有不同的名字,已统一命名为HLB。对引起HLB的原因探索经历了一个漫长的过程,从水害、缺素、镰刀菌、病毒到类菌原体,最后确定为细菌。该病原属于α-亚纲,韧皮部杆菌属,分为亚洲种、非洲种和美洲种3个种,可以通过PCR技术进行检测鉴定。同时,对韧皮杆菌的人工培养以及与黄龙病相关植原体的最新研究进展进行了阐述。     

A graft-based chemotherapy method for screening effective molecules and rescuing huanglongbing-affected citrus plants

Huanglongbing (HLB) is the most devastating disease of citrus. The global citrus industry is in urgent need of effective chemical treatments for HLB control because of its rapid spreading worldwide. Due to the fastidious nature of the pathogens, and the poor permissibility of citrus leaf surfaces, effective screening of chemicals for the HLB control can be challenging. In this study, we developed a graft-based chemotherapy method to rapidly screen potential HLB-controlling chemical compounds. In addition, we improved transmission efficiency by using the best HLB-affected scion-rootstock combination, and demonstrated the HLB bacterial titer was the critical factor in transmission. The HLB-affected lemon scions had a high titer of HLB bacterium, survival rate (83.3%), and pathogen transmission rate (59.9%). Trifoliate, a widely used commercial rootstock, had the highest survival rate (>70.0%) compared with grapefruit (52.6%) and sour orange (50.4%). Using this method, we confirmed a mixture of penicillin and streptomycin was the most effective compounds in eliminating the HLB bacterium from the HLB-affected scions, and in successfully rescuing severely HLB-affected citrus germplasms. These findings are useful not only for chemical treatments but also for graft-based transmission studies in HLB and other Liberibacter diseases.     

A phytoplasma closely related to the pigeon pea witches'-broom phytoplasma (16Sr IX) is associated with citrus huanglongbing symptoms in the state of São Paulo, Brazil

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of S?o Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fD1/rP1 was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and 'Candidatus Liberibacter asiaticus'. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known.     

Sequence and evolutionary analysis of ribosomal DNA from Huanglongbing (HLB) isolates of Western India

Citrus greening (Huanglongbing, HLB) is a widespread and economically important citrus disease all over the world. The disease is caused by a phloem-limited fastidious gram negative bacterium, “Candidatus Liberibacter spp.” which belongs to the alpha-proteobacteria group classified on the basis of its 16SrDNA sequence. Although the pathogen has been classified under three distinct groups, viz. Asian, African and American isolates, nothing is known about the status and the molecular variabilities among the Indian HLB isolates collected from different citrus cultivars grown in India. Five different HLB isolates showing variable symptoms based on their severity of infection on different citrus, viz. Mosambi, Rangpur lime, Cleopatra mandarin, acid lime and rough lemon, were studied by PCR amplification, sequence and evolutionary analysis of their 16S and 16S/23S rDNA intergenic regions. Analysis of the 16S/23S rDNA intergenic region separated all five Indian isolates from existing African isolates but failed to differentiate among Asian, American and Indian isolates. However, further analysis of complete 16S rDNA clearly indicated that Indian isolates fall within the Asian HLB group. Overall, our results suggest that all the five Indian HLB isolates taken for the current analysis belong to the Candidatus Liberibacter asiaticus strain, which showed distinct sequence variabilities and produced noticeable symptoms on the citrus trees. These results provide a robust framework for understanding how differences in pathogenicity among various HLB isolates may be related to evolutionary history.     

Widespread occurrence of “Candidatus liberibacter africanus subspecies capensis” in Calodendrum capense in South Africa

Recent studies in citrus orchards confirmed that Citrus Greening, a heat sensitive citrus disease, similar to Huanglongbing (HLB), is associated with the presence of ??Candidatus Liberibacter africanus?? (Laf) in South Africa. Neither ??Candidatus Liberibacter asiaticus?? (Las), associated with HLB, ??Candidatus Liberibacter americanus??, nor ??Candidatus Liberibacter africanus ssp. capensis?? (LafC), previously detected in the Western Cape, South Africa on an indigenous Rutaceous species, Calodendrum capense (L. f.) Thunb. (Cape Chestnut), were detected in citrus. The current study aims to determine the potential role of C. capense in the epidemiology of Citrus Greening in South Africa and whether LafC poses a risk to citriculture. A total of 278?C. capense samples were collected throughout South Africa and tested for Liberibacters using real-time PCR. While LafC was found in 100 samples, distributed from all areas where collected, no evidence of Laf infection in any sample was found . The identity of the LafC present was confirmed by sequencing the amplicon derived from conventional PCR of the ?-operon of the ribosomal protein gene region of the first 17 infected trees found and of a representative sample from each district. The Liberibacter status of 44?C. capense and 272 citrus (Midnight Valencia) trees growing in close proximity to each other for over 15?years was determined. Out of 44?C. capense specimens, 43 were infected with LafC, but none of the citrus trees were infected with LafC. Based on the results of this it appears that natural spread of LafC to citrus does not occur.