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.     

青霉素喷施防治柑橘黄龙病试验初报

柑橘黄龙病是世界性分布的柑橘毁灭性病害,严重制约着柑橘产业的发展．为探究青霉素对柑橘黄龙病的防治效果,本试验采用喷施的方式对柑橘黄龙病病树进行处理,同时结合实时荧光定量ＰＣＲ技术对病树内黄龙病菌量进行动态监测,确定青霉素对柑橘黄龙病的相对防效．结果表明连续喷施４次青霉素３个月和７个月后病树中黄龙病菌含量均出现不同程度的上调,相对防效分别为－１７０.１６％和－６３.９６％．以上结果表明喷施的方式进行青霉素处理黄龙病病树对柑橘黄龙病并不能取到防治效果．     

柑橘黄龙病检测方法研究进展

黄龙病是柑橘生产上的一种毁灭性病害,该病在北美、南美、西亚、东亚和南亚的一些国家和地区时有发生,造成巨大的经济损失,严重威胁着世界柑橘产业。柑橘黄龙病的传统检测方法有病害田间诊断、指示作物鉴定、病原显微镜观察、血清学、DNA-DNA杂交和PCR检测等方法。近年来,美国一些科学家把高光谱图像技术应用在柑橘黄龙病检测领域,提高了检测速度,降低了检测成本。综述了国内外关于柑橘黄龙病检测诊断方面的研究进展,介绍了该领域传统检测方法的特点和存在问题以及高光谱图像检测技术的主要特点,着重介绍了高光谱图像技术在黄龙病检测方面的最新应用技术、设备和实例。提出了柑橘黄龙病诊断领域,在实现无损检测的前提下,降低成本、提高检测实时性和诊断速度的发展趋势。     

柑桔对黄龙病病原与病毒混合侵染的反应

已知在福建省柑桔产区发生的由嫁接传染的病害有黄龙病、衰退病、裂皮病和碎叶病。由于长期以来柑桔采用无性繁殖的结果,造成这几种病害多以两种或两种以上的混合侵染形式存在。对苗期进行混合接种的试验结果表明,病状表现的严重程度与柑桔对该病原的抗性程度的密切关系,感病的病状明显,抗病的病状不明显或不表现;但各组合接种株与其过氧化物酶同酶的变化则表现出比较复杂的关系。福建省柑桔的主栽品种是芦柑、雪柑和福桔,它们对衰退病、裂皮病和碎叶病具有一定的抗性,所以这３种病原的混合侵染对本砧的柑桔生产没有造成严重的危害。     

柑桔黄龙病研究的回顾与展望

柑桔黄龙病是由一种叫做韧皮部杆菌(Candidatus Liberibacter)的病原引起的,已经确定的病原有亚洲、非洲和美洲3个种。病害通过种苗、嫁接和木虱介体等途径传播。病害可根据田间症状诊断,也可以通过血清学、电镜技术和PCR等分子生物学等技术进行鉴定。目前,柑桔黄龙病主要依靠综合防治的方法。开展病原的人工培养、抗病育种、致病机理和病害防治方法等方面的研究对揭示病原特性和病害防治等都具有十分重要的意义。     

柑桔黄龙病的为害现状及防控措施

南平市延平区柑桔生产兴起于20世纪80年代，至1996年全区柑桔种植面积达6133hm^2、总产4．06万t，当年柑桔特产税收入93．1万元，成为当地农业支柱产业。随着我国主要农产品从长期短缺到丰年有余的转变，1997年全区柑桔出现了严重“卖难”问题，1999年又遇到历史罕见的冻害，全区柑桔业受到致命的打击。2001年全区柑桔面积仅4200hm^2，比1996年锐减1933hm^2。种植的品种中，雪柑1667hm^2，中晚熟蜜柑1400hm^2，芦柑600hm^2，脐橙、雪橙及柚类面积533hm^2，质差、价低的雪柑和中晚熟蜜柑占柑桔总面积的73％。20世纪80年代至90年代末延平区柑桔业大起大落。为避免重蹈覆辙，促进当地柑桔业的健康发展，笔者结合工作，进行调查分析并提出相应对策。现将延平区柑桔的为害现状及防控措施介绍如下。     

柑橘黄龙病检测技术研究进展

冠以“柑橘（Ｃｉｔｒｕｓ ｒｅｔｉｃｕｌａｔａ Ｂｌａｎｃｏ．）癌症”之称的黄龙病暂被认为是由韧皮部难养细菌引起的对柑橘产业造成严重威胁的毁灭性病害,它给柑橘产业的健康发展造成了严重的威胁．鉴于柑橘黄龙病检测技术对柑橘黄龙病防治的重要性,文章对柑橘黄龙病及其病原、黄龙病的检测技术进行了综述,并对柑橘黄龙病的检测技术未来发展进行了展望.     

柑桔黄龙病亚洲种病原(Candidatus Liberobacter asiaticus)16S rDNA 的PCR-SSCP分析

应用PCR—SSCP技术对我国柑桔主要产区、泰国和法国留尼湾等地收集到的柑桔黄龙病亚洲种病原（Candidatus Liberobacter asiaticus）16S rDNA进行了分析。PCR结果表明,不同的地区的柑桔黄龙病亚洲种16S rDNA在长度上没有明显区别。进一步的SSCP结果显示,不同地区柑桔黄龙病亚洲种16S rDNA序列在组成上也没有差异。显示来自不同地区、不同寄主品种的柑桔黄龙病亚洲种种内没有存在明显差异。     

中国柑橘黄龙病研究30年

综述了我国自1978年以来有关柑橘黄龙病的研究概况,包括柑橘黄龙病在我国的分布与危害、黄龙病寄主、病原研究、病害诊断与检测、传播途径与病害流行、防控技术等.     

Visible-near infrared spectroscopy for detection of Huanglongbing in citrus orchards

This paper evaluates the feasibility of applying visible-near infrared spectroscopy for in-field detection of Huanglongbing (HLB) in citrus orchards. Spectral reflectance data from the wavelength range of 350-2500 nm with 989 spectral features were collected from 100 healthy and 93 HLB-infected citrus trees using a visible-near infrared spectroradiometer. During data preprocessing, the spectral data were normalized and averaged every 25 nm to reduce the spectral features from 989 to 86. Three datasets were generated from the preprocessed raw data: first derivatives, second derivatives, and a combined dataset (generated by integrating preprocessed raw data, first derivatives and second derivatives). The preprocessed datasets were analyzed using principal component analysis (PCA) to further reduce the number of features used as inputs in the classification algorithm. The dataset consisting of principal components were randomized and separated into training and testing datasets such that 75% of the dataset was used for training; while 25% of the dataset was used for testing the classification algorithms. The number of samples in the training and testing datasets was 145 and 48, respectively. The classification algorithms tested were: linear discriminant analysis, quadratic discriminant analysis (QDA), k-nearest neighbor, and soft independent modeling of classification analogies (SIMCA). The reported classification accuracies of the algorithms are an average of three runs. When the second derivatives dataset were analyzed, the QDA-based classification algorithm yielded the highest overall average classification accuracies of about 95%, with HLB-class classification accuracies of about 98%. In the combined dataset, SIMCA-based algorithms resulted in high overall classification accuracies of about 92% with low false negatives (less than 3%).     

柑桔温室栽培研究

1996－2000年试验结果表明，在加温设施栽培条件下，早熟温州蜜柑物候期明显提早，果实可在7、8月成熟，果形端正，果皮较薄，甜酸适口。可溶性固形物含量达9.0％，花期长30d左右，座果率18.2％，产量为29.445t/hm^2。并对病虫害发生动态与相关管理技术作了探讨。     

无病容器大苗在柑桔黄龙病综合防控中的应用价值

柑桔细菌性病害黄龙病是全球柑桔业的致命威胁。采用无病苗、砍除罹病树、统防统治柑桔木虱等综合防控措施虽然有效,但在幼年园中落实难。调查表明,导致此现象的主要原因是传统使用的露地小苗一般需要3年才能长大投产,许多果农在此期间不得不外出打工以减轻经济压力。针对此种情况,提出了采用无病容器大苗建园,辅以促生栽培管理措施,使之在定植1年后就投产的栽培技术路线,藉以减低幼树期黄龙病防控压力并减慢发病速度。4年多的实践表明,容器大苗技术非常适合在黄龙病疫区使用,可大幅度提高疫区柑桔种植效益。简介了无病容器大苗技术及其原理,分析了该技术的优势,并对其推广应用前景进行展望。     

实时荧光定量PCR检测柑橘黄龙病田间试验药剂防治效果

[目的]评价柑橘黄龙病田间试验药剂橘叶青的防控效果。[方法]采用实时荧光定量PCR检测喷施橘叶青后的柑橘叶片和果皮中黄龙病病原菌的含量。[结果]喷施橘叶青后的叶片和果皮组织含菌量明显降低,叶片组织含菌量降低了近35倍,果皮组织含菌量降低了约7倍。[结论]防治药剂橘叶青对柑橘黄龙病具有较好的防控效果,可以有效控制病原菌在柑橘组织中发展。     

柑橘黄龙病的为害传播与综合防控措施

总结柑橘黄龙病的主要危害特征、传播方式和发病条件,提出严格检疫、脱毒育苗、隔离建园、病虫防治、清除病树、加强宣传等综合防治措施。     

柑橘黄龙病药物防治策略

柑橘黄龙病(HLB)是世界范围内柑橘生产上的毁灭性病害,已在全球范围造成了巨大的经济损失。黄龙病由韧皮部限制性寄生菌Candidatus liberibacters spp.引起,主要通过柑橘木虱Diaphorina citri(Kuwayama)在田间传播。目前,黄龙病的主要防治方法有:一是传统的"三板斧"防治策略,即种植无病苗木、严防木虱(大面积连片统防统治)和挖净病树;二是药物防治,主要包括抗生素化学药物、窄谱靶向化学药物、免疫诱导物、纳米制剂等在内的多种抗菌剂。本文对近年来国内外柑橘黄龙病药物防治的相关研究进行了系统论述,以期为柑橘黄龙病菌新型防治药剂研发提供理论依据和参考。     

基于激光诱导击穿光谱的柑橘叶片黄龙病检测

采用激光诱导击穿光谱(LIBS)结合化学计量学的方法对柑橘叶片黄龙病进行定性检测.试验结果显示:柑橘叶片中营养元素P(Ⅱ)、Mn(Ⅰ)、Si(Ⅰ)和Fe(Ⅰ)的LIBS信号强度与柑橘叶片的健康程度有直接关系,其中健康、中度感染黄龙病和重度感染黄龙病的柑橘叶片中P(Ⅱ)、Mn(Ⅰ)、Si(Ⅰ)和Fe(Ⅰ)的特征光谱强度呈...     

中国柑橘黄龙病研究30年

\t\t\t\t\t目的\t\t\t\t\t柑橘黄龙病(Citrus Huanglongbing,HLB)是由韧皮部杆菌(Candidatus Liberibacter spp.)引发的重要柑橘病害。本试验利用生物信息学方法分析韧皮部杆菌全基因组,推测CLIBASIA_03230基因(命名为Clsp11)编码分泌蛋白,克隆该基因并构建其原核表达载体,表达、纯化Clsp11蛋白,以用于制备特异抗体。\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t方法\t\t\t\t\t利用CTAB法从感染柑橘黄龙病病原的长春花中提取总DNA,以此为模板经PCR扩增Clsp11,并克隆至pMD18-T载体,挑取阳性克隆测序验证;利用无缝克隆技术将Clsp11克隆至原核表达载体pET30a,通过PCR和酶切鉴定筛选阳性克隆,命名为pET30a-Clsp11;转化大肠杆菌BL21,经IPTG诱导,表达N-端融合6×His标签的Clsp11重组蛋白,SDS-PAGE电泳检测,分析目的蛋白的表达水平,并利用镍柱进行纯化。\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t\t结果\t\t\t\t\tClsp11蛋白成功表达,IPTG诱导5 h表达量显著提高,主要以包涵体形式存在于宿主菌中。\t\t\t\t\t\t\t\t\t\t\t\t\t结论\t\t\t\t\t本研究克隆了柑橘黄龙病病原Clsp11基因,构建了原核表达载体pET30a-Clsp11,并成功表达、纯化了Clsp11重组蛋白,为进一步制备特异性抗体和研究Clsp11生物学功能奠定了基础。\t\t\t\t\t\t\t\t\t     

柑橘黄龙病对柑橘产量和品质的影响

通过对不同发病程度黄龙病的柑橘进行单株产量、含糖量(可溶性固形物)和单果果重的测定,并结合柑橘外观的观测,结果表明,柑橘黄龙病不同病情级别均降低柑橘的产量、单果果重、品质和经济价值,但对柑橘的含糖量影响不大。