利用PCR检测柑桔黄龙病病原研究(英文)

对引起柑桔黄龙病的类菌原体（ＢＬＯ）的ＰＣＲ检测方法进行了研究．结果表明,利用ＰＣＲ方法不但能对ＢＬＯ、ＤＮＡ进行离体扩增,而且对各种不同来源的感病植物样品也能很好地进行检测．本文首次报道了从未显症的植物上用ＰＣＲ方法检测到了ＢＬＯ,技接试验证实了该方法的可靠性．正是由于本方法可在病原侵染早期进行检测这一优点,从而可控制带病苗木的扩散．该技术也可被用于无毒苗木的筛选,从而控制病害发生．该技术对ＢＬＯ引起的黄龙病的检测不但简单、有效而且实用     

柚类果树叶片斑驳病的病原鉴定与检测

柚类是柑桔中的一类重要品种,在我国有广泛的分布、近年来,在福建等产区发现柚类病树日趋２增多、基病状为叶片斑驳、小果、植株矮化、暂称为叶片斑驳病。本有通过组织嫁接从病柚树传染到柚健苗和芦柑健苗。在电下观察,其病原功体的形态和细胞壁构造与柑桔黄龙病的病原十分要似。应用柑桔黄龙病病原ＤＮＡ的特异性引物进行ＰＣＲ试验产生的ＰＣＲ产物,其分子量与柑桔黄龙病病原ＤＮＡＰＣＲ产物的基本相同,为５６３ｂｐ;以及应     

柑桔黄龙病及其控防对策

柑桔黄龙病是柑桔生产中危害最重、威胁最大的一种毁灭性病害。文章在分别对柑桔黄龙病的病名、危害与分布、病原的认识过程、诊断技术、病原侵染来源、病原脱除等进行综述的前提下,提出严格实施检疫、重病园成片更新、应用无病毒苗木、及时挖除病株、大面积联防联治、严格防治柑桔木虱等柑桔黄龙病的防控对策。     

柑桔黄龙病PCR检测技术体系优化分析

根据柑桔黄龙病病菌亚洲种16S rDNA基因序列设计特异引物,建立了检测柑桔黄龙病的快速PCR方法,并研究了PCR体系的各主要组分对PCR反应的影响,确立了黄龙病菌的PCR检测优化体系。应用PCR检测方法对大田植株及柑桔脱毒苗进行抽样检测,结果表明:PCR检测优化体系具有快速、灵敏度高、重复性好、检测成本低等优点,适用于柑桔黄龙病的早期诊断及无病毒苗木的大量检测。     

柑桔黄龙病的防治措施

柑桔黄龙病是一种毁灭性的柑桔类病害，其病原是一种病毒性的类质菌体。此病主要通过带病的苗木、接穗、柑桔木虱等传播。该病的症状表现为：树冠顶部梢黄化或斑驳，叶脉肿胀，吉质脆硬，导致果实品味酸，无经济价植，后期烂根，1～2年后全株枯死。该病给果农带来较大的风险。柑桔黄龙病的防治可用以下措施     

应用PCR技术检测柑桔黄龙病病原的研究

应用ＰＣＲ技术检测柑桔黄龙病病原的研究邓晓玲唐伟文（华南农业大学植物保护系，广州，５１０６４２）ＴＨＥＳＴＵＤＩＥＳＯＮＤＥＴＥＣＴＩＯＮＯＦＣＩＴＲＵＳＨＵＡＮＧＬＯＮＧＢＩＮＰＡＴＨＯＧＥＮＢＹＰＯＬＹＭＥＲＡＳＥＣＨＡＩＮＲＥＡＣＴＩＯＮＤｅｎ...     

利用PCR检测柑桔黄龙病病原研究

对引起柑桔黄龙病的类菌原体的ＰＣＲ检测方法进行了研究。结果表明，利用ＰＣＲ方法不但能对ＢＬＯ，ＤＮＡ进行离体扩增，而且对各种不同来源的感病植物样品也能很好地进行检测。本首次报道了从未显症的植物上用ＰＣＲ方法检测到了ＢＬＯ，枝接试验证实了该方法的可靠性。     

单克隆抗体-胶体金标记技术快速检测柑桔黄龙病

应用单克隆抗体一胶体金标记技术,采用徒手切片法和斑点试验法检测柑桔黄龙病获得成 功。该方法检测速度快,操作简单,成本低,适合柑桔种苗大批量检测和田间普查。采用单抗金标技术 对田间栽培的柑桔品种、柑桔实生苗、试管微芽嫁接苗、热处理脱毒后的嫁接苗进行检测,结果表明 田间栽培的6个柑桔品种不论是否显示黄龙病症状均检测到柑桔黄龙病病菌,而消毒种子播种所得 实生苗及试管微芽嫁接苗为无病株,带病株经过30d 40℃恒温热处理后产生的新梢及其嫁接苗绝大 部分也是无病株。     

柑桔黄龙病防控技术研究与应用

根据柑桔黄龙病病原PCR检测结果,分析其病原检出率不高与检测方法、检测部位等相关,提供了"红鼻果"作为芦柑黄龙病田间诊断的依据。通过田间调查,分析总结了至2000年前永春县柑桔黄龙病长期未蔓延危害的原因在于果园管理均衡,施药次数较多,柑桔木虱没有大量生存繁衍的空间;而近年柑桔黄龙病蔓延危害的主要原因是"三农"状况发生变化,劳力转移,失管果园多,病源多、柑桔木虱大量危害,病害迅速传播。论述了近年永春县柑桔黄龙病防控技术的实践应用与取得的成效;指出柑桔规模化连片种植,分散管理,难以统一落实防控措施是黄龙病防控工作的难点。探讨了黄龙病产区发展柑桔生产的措施。     

单抗─胶体金法及PCR技术诊断柑桔黄龙病

单抗┐胶体金法及ＰＣＲ技术诊断柑桔黄龙病ＴｈｅＤｉａｇｎｏｓｉｓｏｎＨｕａｎｇｌｏｎｇＤｉｓｅａｓｅｏｆＣｉｔｒｕｓｗｉｔｈＣｏｌｏｉｄａｌＧｏｌｄＬａｂｅｌｅｄＭｏｎｏｃｌｏｎａｌＡｎｔｉｂｏｄｙＴｅｃｈｎｉｑｕｅａｎｄＰＣＲＡｍｐｌｉｆｉｃａｔｉ...     

柑橘黑点病菌(Diaporthe citri)快速分子检测技术

柑橘黑点病是一种严重危害柑橘生产的世界性真菌病害,快速及时地检测出柑橘黑点病病菌(Diaporthe citri)对柑橘黑点病早期诊断和科学防控具有重要的实践意义。本研究基于β-微管蛋白序列,设计柑橘黑点病病菌的特异引物对,基于常规PCR和SYBR Green I实时荧光定量PCR(qRT-PCR)建立了快速分子检测体系,对柑橘黑点病病菌的菌丝和典型带病叶片进行检测验证。结果发现,在常规PCR检测中,该引物可从柑橘黑点病病菌获得244 bp的特异性扩增片段,检测灵敏度达0.78 ng·μL^-1;在实时qRT-PCR中,该引物也只有在柑橘黑点病病菌中获得唯一的产物吸收峰,检测灵敏度达0.35 ng·μL^-1。利用PCR检测体系对发病黑点叶片进行检测,结果显示,常规PCR的阳性检出率为30%,qRT-PCR的阳性检出率为60%,表明qRT-PCR比常规PCR的灵敏度更高。因此,研究设计的柑橘黑点病病菌特异性引物,以及所建立的常规和实时qRT-PCR检测方法具有速度快、特异性强等特点,可以用于柑橘黑点病病菌的分子鉴定,对病害诊断具有参考价值。     

柑橘黄化花叶病毒侵染性克隆构建及应用

【目的】构建柑橘黄化花叶病毒（citrus yellow mosaic virus,CYMV）侵染性克隆,为深入研究其分子特性及致病机理打下基础。【方法】利用In-Fusion同源重组技术将分段扩增的CYMV基因组序列与三元表达载体pCY重组连接,构建该病毒1.4倍基因组全长DNA克隆并开展序列分析。将所获克隆通过农杆菌介导接种尤力克柠檬实生苗,通过分子检测、症状和病毒粒子观察及再嫁接实验鉴定其侵染性。利用所获侵染性克隆接种不同的柑橘品种及草本植物,通过RT-PCR检测确定侵染率,观察不同柑橘品种受侵染后的症状差异。基于侵染性克隆构建ORFⅠ和ORFⅡ分别替换为绿色荧光蛋白基因（green fluorescent protein,gfp）的突变体,分析突变对病毒侵染性的影响。【结果】利用In-Fusion同源重组技术获得CYMV 1.4倍基因组全长DNA克隆7个。其中,CYMV-3与已登录GenBank的9个CYMV分离株基因组相应核苷酸序列一致性为90%—100%,与分离株CYMV-SO(AF347695)同源性最高并在遗传进化树上聚为一簇。侵染性鉴定结果表明,CYMV-3接种的14...     

Detection of Huanglongbing (citrus greening) based on hyperspectral image analysis and PCR

Huanglongbing (HLB, citrus greening) is one of the most serious quarantine diseases of citrus worldwide. To monitor in real-time, recognize diseased trees, and efficiently prevent and control HLB disease in citrus, it is necessary to develop a rapid diagnostic method to detect HLB infected plants without symptoms. This study used Newhall navel orange plants as the research subject, and collected normal color leaf samples and chlorotic leaf samples from a healthy orchard and an HLB-infected orchard, respectively. First, hyperspectral data of the upper and lower leaf surfaces were obtained, and then the polymerase chain reaction (PCR) was used to detect the HLB bacterium in each leaf. The PCR test results showed that all samples from the healthy orchard were negative, and a portion of the samples from the infected orchard were positive. According to these results, the leaf samples from the orchards were divided into disease-free leaves and HLB-positive leaves, and the least squares support vector machine recognition model was established based on the leaf hyperspectral reflectance. The effect on the model of the spectra obtained from the upper and lower leaf surfaces was investigated and different pretreatment methods were compared and analyzed. It was observed that the HLB recognition rate values of the calibration and validation sets based on upper leaf surface spectra under 9-point smoothing pretreatment were 100% and 92.5%, respectively. The recognition rate values based on lower leaf surface spectra under the second-order derivative pretreatment were also 100% and 92.5%, respectively. Both upper and lower leaf surface spectra were available for recognition of HLB-infected leaves, and the HLB PCR-positive leaves could be distinguished from the healthy by the hyperspectral modeling analysis. The results of this study show that early and nondestructive detection of HLB-infected leaves without symptoms is possible, which provides a basis for the hyperspectral diagnosis of citrus with HLB.     

‘Extended spectral angle mapping (ESAM)’ for citrus greening disease detection using airborne hyperspectral imaging

Hyperspectral (HS) imaging is becoming more important for agricultural applications. Due to its high spectral resolution, it exhibits excellent performance in disease identification of different crops. In this study, a novel method termed ‘extended spectral angle mapping (ESAM)’ was proposed to detect citrus greening disease (Huanglongbing or HLB), which is a very destructive disease of citrus. Firstly, the Savitzky–Golay smoothing filter was used to remove spectral noise within the data. A mask for tree canopy was built using support vector machine, to separate the tree canopies from the background. Pure endmembers of the masked dataset for healthy and HLB infected tree canopies were extracted using vertex component analysis. By utilizing the derived pure endmembers, spectral angle mapping was applied to differentiate between healthy and citrus greening disease infected areas in the image. Finally, most false positive detections were filtered out using red-edge position. An experiment was carried out using an HS image acquired by an airborne HS imaging system, and a multispectral image acquired by the WorldView-2 satellite, from the Citrus Research and Education Center, Lake Alfred, FL, USA. Ground reflectance measurement and coordinates for diseased trees were recorded. The experimental results were compared with another supervised method, Mahalanobis distance, and an unsupervised method, K-means, both of which showed a 63.6 % accuracy. The proposed ESAM performed better with a detection accuracy of 86 % than those two methods. These results demonstrated that the detection accuracy using HS image could be enhanced by focusing on the pure endmember extraction and the use of red-edge position, suggesting that there is a great potential of citrus greening disease detection using an HS image.     

迟熟蕉柑茎尖嫁接砧木对比试验及病毒类病害鉴定

分别以枳和阿尔及利亚夏橙作为砧木,以迟熟蕉柑芽作为接穗进行茎尖嫁接,结果枳砧嫁接成活率为9．1％,阿尔及利亚夏橙砧嫁接成活率为54．5％,将两种砧木嫁接苗再嫁接在枳砧上,其3年生幼树均生长正常,表明阿尔及利亚夏橙是茎尖嫁接迟熟蕉柑的优良砧木。应用指示植物鉴定方法和PER检测技术鉴定7株茎尖嫁接苗的柑橘碎叶病、衰退病、裂皮病和黄龙病,结果1、3．4、5、6号迟熟蕉柑单株不带碎叶病、衰退病、裂皮病和黄龙病,可作为繁殖无病苗木的备选材料;2、7号迟熟蕉柑单株不带碎叶病、裂皮病和黄龙病,但带有衰退病,推测衰退病毒源可能来自取芽的田间植株。     

Preliminary Studies on Species and Distribution of Citrus Viroids in China

Citrus viroids are the small but economically important RNA pathogens. For investigating their occurrence and distribution in China, 65 viroid samples collected from 8 major citrus cultivated regions were evaluated using one-step or multiplex onestep RT-PCR and biological indexing for specifically detection of Citrus exocortis viroid （CEVd）, Citrus bent leaf viroid （CBLVd）, Hop stunt viroid （HSVd）, Citrus viroid-Ⅲ （CVd-Ⅲ） and Citrus viroid-Ⅳ （CVd-Ⅳ）. The results showed that there were at least 4 kinds of citrus viroids （CEVd, CBLVd, HSVd, and CVd-Ⅲ） on citrus trees in China. Most of the infected citrus plants harbored more than one viroid species, and two plants were infected with up to 4 citrus viroids. Sweet orange was more frequently infected by viroids than other citrus varieties. It is the preliminary report on the species and distribution of citrus viroids in China.     

Distribution pattern and titer of Candidatus Liberibacter asiaticus in periwinkle (Catharanthus roseus)

Candidatus Liberibacter asiaticus (CaLas), an uncultured Gram-negative alphaproteobacterium, is the causal agent of Huanglongbing (HLB) in citrus. CaLas resides in phloem sieve tubes and has been shown to be unequally distributed in different tissues. Although HLB is a disease of citrus plants, it has been demonstrated that periwinkle can serve as an experimental host of CaLas, which can be transmitted from citrus to periwinkle via the parasitic plant dodder (Cuscuta spp.). To investigate the distribution of CaLas in various periwinkle tissues, the bacteria were transmitted from an infected periwinkle plant to healthy periwinkles by top-grafting. The movement of the inoculum and associated titer changes were observed over time in various tissues. CaLas could be detected in the leaves, main stems, and roots of infected periwinkle by conventional PCR, and in all three tissues a clear time-dependent change in CaLas titer was observed, with titer increasing soon after inoculation and then decreasing as disease symptoms became severe. The highest titer was found at 25, 35 and 35 days after inoculation in leaves, main stems and roots, respectively. The titer in leaves was much higher than in the main stems and roots at the same time point, and the spatial distribution of CaLas in the leaves, main stems and roots of infected periwinkle was uneven, similar to what has been shown in citrus. The results provide guidance for selecting the proper periwinkle tissues and sampling times for early detection of CaLas.