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...     

柑橘黄龙病病情消长动态与立体式防效探讨

通过对涌泉镇2004-2012年全镇柑橘黄龙病全面普查,结果表明,无核蜜橘柑橘黄龙病病菌感染至显症潜伏期可达1年以上,其年度扩散消长呈单峰型曲线变化规律。坚持每年"一挖两治"(挖除病树阻断菌源,加强种苗接穗管治及柑橘木虱防治阻断菌链)和"三防五关"(春季清园和严防从病区采入接穗高接换种,夏季进行夏梢柑橘木虱防治,秋冬季进行秋梢柑橘木虱防治和彻底查挖病树,狠抓种苗检疫关、治虫防病关、疫情普查关、病树挖除关和健身控病关)立体式防控,其防控效果可达87%~93%,从而保障柑橘产业健康持续发展。     

湘南地区柑橘黄龙病的PCR检测

针对近年湘南部分柑橘产区出现叶片黄化、果实畸形及全株矮化、橘树枯死等柑橘黄龙病疑似症状,应用多聚酶链式反应(PCR)技术对湘南8个柑橘产区的柑橘进行了检测,结果发现6个柑橘产区有柑橘黄龙病为害.同时建立了一套快速、准确的柑橘黄龙病PCR检测技术体系.     

基于光谱信息的柑橘黄龙病无损检测及分级模型构建

柑橘黄龙病(Huanglongbing,HLB)是世界柑橘生产上最具毁灭性的病害,给果农和相关产业造成了巨大的损失.以柑橘叶片为载体,利用高光谱图像技术采集柑橘叶片表面的高光谱图像,用ENVI4.7进行图像处理,提取感兴趣区域(Region of Intest,ROI),统计感兴趣区域平均光谱数据,并进行相关植被植物的运算,最后通过PLS-DA(Partial Least Squares Discrimination Analysis)判别法进行鉴别并分类.结果表明:基于平均光谱值和植被指数的PLS-DA判别模型都能对健康、缺锌和HLB叶片进行鉴别.其中基于平均光谱值的PLS-DA模型鉴别健康柑橘叶片样品的灵敏度为100％,特异度为100％,准确度为100％;鉴别缺锌柑橘叶片样品的灵敏度为80.6％,特异度为91.7％,准确度为88.9％;鉴别HLB叶片的灵敏度为89.3％,特异度为88.3％,准确度为88.9％.基于植被指数的PLS-DA判别模型鉴别健康柑橘叶片样品的灵敏度为100％,特异度为100％,准确度为100％;鉴别缺锌柑橘叶片样品灵敏度为92.5％,特异度为89.3％,准确度为90.1％;鉴别HLB叶片的灵敏度为86.4％,特异度为95.3％,准确度为90.1％.识别正确率较高,说明利用高光谱进行柑橘黄龙病病情分类是可行的.     

一种快速鉴定柑橘黄龙病耐性种质材料的方法

【目的】研发一种快速鉴定柑橘黄龙病耐性种质材料的方法,为加快柑橘黄龙病耐性育种进程和提高育种效率打下基础。【方法】以收集的36份疑似对柑橘黄龙病具有耐性的柑橘种质为材料,采用直接高接于感染黄龙病菌柑橘树上的方法(高接染毒鉴定法),通过田间症状观察结合定量PCR检测对试验材料的耐病性进行鉴定评价。【结果】春季高接供试材料1个月后接芽萌发,3个月后(2018年6月5日)首次在KH-14上出现典型的叶片斑驳型黄化症状,4个月后(2018年7月5日)观察有23份种质材料的叶片出现斑驳型黄化症状,另外11份种质材料未表现症状;6个月后(2018年9月4日)观察发现KH-18、KH-12、KHY-4、KHY-5和KHY-6等5份种质材料的生长虽然较正常,但叶片已出现黄化症状,只有KH-21的枝梢生长良好,无黄梢和斑驳型黄化叶,初步判定为对黄龙病具有耐性的种质材料。对6份种质材料的定性PCR检测结果表明,KH-21为阴性,其他5份材料为阳性;实时荧光定量PCR检测结果,KH-21的平均黄龙病菌含量为1870.0个细胞/μg DNA,对照材料平均黄龙病菌含量为372285.5个细胞/μg DNA,表明KH-21对柑橘黄龙病具有耐性。【结论】高接染毒鉴定法鉴定周期在6个月左右,具有通量大、时间短、效率高、成本低及结果准确等优点,能达到快速鉴定柑橘品种对柑橘黄龙病耐性的目的,可在柑橘黄龙病耐性鉴定中推广使用。     

缺了氮磷钾,叶子发黄变枯又卷曲

正柑橘品种丰富并且各有特点,味道甘甜,营养丰富,是深受人们喜爱的一种水果。柑橘对土壤的适应范围较广,pH值4.5-8均可生长,以pH值5.5-6.5为最适宜。但近些年,随着人们对柑橘的需求量越来越多,柑橘的种植面积也在猛增。随之出现的柑橘缺素导致的病害也越来越严重。柑橘黄化现象常有出现,叶片发黄,光合作用降低,创造的有机物减少,树势变弱,抗病能力差,产量低。有的果农看见柑橘叶片黄了,认为是黄龙病,很慌恐,怕传播到健康树上,立刻砍树。其实柑橘叶片变黄并不一定是黄龙病,大部分很有可能是缺素引起的黄化。     

柑橘黄龙病综合防控技术

<正>柑橘黄龙病是柑橘生产上的毁灭性病害,可以为害包括橘类、橙类、柚类等柑橘栽培品种,病害传播蔓延迅速,对柑橘生产造成严重的危害。柑橘黄龙病的防控必须采取得力措施,从传播媒介控制、健康种苗繁育、染病植株砍除等多个环节入手,做到综合防控、标本兼治、持续治理。重点在"苗、虫、铲、检"四个字上下功夫。苗:就是推广健康种苗,从源     

湛江农垦红江橙主产区2个果园黄龙病的巢式PCR检测分析

柑橘黄龙病(HLB)是柑橘生产上的一种毁灭性病害,对柑橘生产为害较重。为了明确柑橘黄龙病在红江橙果园中的分布及危害情况,进一步为柑橘黄龙病的综合防控提供有价值的参考,采用Nested PCR分子检测技术,于2016～2018年对湛江农垦红江橙主产区具有黄化、斑驳等症状的305个红江橙样本进行检测。结果表明,305个红江橙叶片样本中,HLB的阳性检出率为53.1%;其中红江农场38队红江橙果园黄龙病的阳性检出率为55.6%,广垦(湛江)红江橙农业科技有限公司横江红江橙果园黄龙病的阳性检出率为48.6%。柑橘黄龙病已在湛江农垦红江橙果园普遍发生,生产上应引起重视。     

湖南省柑橘黄龙病菌的PCR检测及序列分析

从湖南省8个市(县)采集柑橘黄龙病疑似植株的叶片,以特异引物进行PCR检测。结果表明:8个市(县)所采样品均检测到了柑橘黄龙病病原菌,其中新宁、武冈、隆回、洞口和通道县为首次发现,各地病原菌序列与柑橘黄龙病亚洲种(M94319)的同源性高达99.1%～100%;不同地区的柑橘黄龙病病原菌存在微小差异,其中新宁与通道县病株差异稍大。这说明由特异性引物建立的PCR检测技术具有特异性强、准确率高、可重复性好等特点,可用于柑橘黄龙病的快速检测。     

煤污病胁迫下3个薄壳山核桃品种的光合特性

为探究煤污病对薄壳山核桃光合特性的影响,以Stuart、Wichita、Pawnee 3种受煤污病危害程度不同的品种为对象,测定染病叶片及健康叶片的光合参数、光响应曲线以及叶绿素荧光参数.结果表明:煤污病可显著影响薄壳山核桃叶片的光合气体交换,3个品种染病叶片光合气体交换参数[净光合速率(Pn)、蒸腾速率(Tr)、胞间CO2浓度(Ci)及气孔导度(Gs)]较健康叶片均出现显著下降(P<0.05),其程度从大到小依次为Stuart>Wichita>Pawnee.就Pn而言,Stuart染病叶片下降61.07％,Wichita染病叶片下降51.26％,Pawnee染病叶片下降40.62％.光响应曲线显示,3个品种染病叶片的最大净光合速率(Pnmax)均显著低于健康叶片,暗呼吸速率(Rd)与健康叶片无显著差异,表观量子效率(AQY)均显著低于健康叶片,光补偿点(LCP)均显著高于健康叶片.3个品种染病叶片的叶绿素荧光参数(Fv/Fm、ΦPSⅡ、ETR、qP)相比于健康叶片,整体而言均出现了下降,但未达到显著水平.说明煤污病可造成薄壳山核桃叶片光合速率下降,净光合速率下降程度为40.62％～61.07％.染病叶片利用弱光的能力下降,合成的光合产物减少,而实际光合效率并未显著降低,光合机构仍较为稳定.     

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.     

Spectral difference analysis and airborne imaging classification for citrus greening infected trees

Citrus greening, also called Huanglongbing (HLB), became a devastating disease spread through citrus groves in Florida, since it was first found in 2005. Multispectral (MS) and hyperspectral (HS) airborne images of citrus groves in Florida were acquired to detect citrus greening infected trees in 2007 and 2010. Ground truthing including field and indoor spectral measurement, infection status along with GPS coordinates was conducted for both healthy and infected trees. Ground spectral measurements showed that healthy canopy had higher reflectance in the visible range, and lower reflectance in the near-infrared (NIR) range than HLB infected canopy. Red edge position (REP) also showed notable difference between healthy and HLB canopy. But the difference in the NIR range and REP were comparably more sensitive to the environment or the background noise. Accuracy for separating HLB and healthy samples reached more than 90% when a simple REP threshold method was implemented in the ground reflectance datasets, regardless of field or indoor measurement; but it did not work well with the HS images because of its low spatial resolution. Support vector machine (SVM) was able to provide a fast, easy and adoptable way to build a mask for tree canopy. High positioning error of the ground truth in the 2007 HS image led to validation accuracy of less than 50% for most of classification methods. In the 2010 image from Southern Gardens (SG) grove, with better ground truth records, higher classification accuracies (about 90% in training sets, more than 60% in validation sets for most of the methods) were achieved. Disease density maps were also generated from the classification results of each method; most of them were able to identify the severely infected areas. Simpler classification methods such as minimum distance (MinDist) and Mahalanobis distance (MahaDist) showed more stable and balanced detection accuracy between the training and validation sets in the 2010 images. Their similar infection trend with ground scouted maps showed a promising future to manage HLB disease with airborne spectral imaging.     

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.     

Citrus disease recognition based on weighted scalable vocabulary tree

Citrus Huanglongbing (HLB) is a destructive disease in citrus production that causes huge economic damage to citrus producers and related industries in the world. Early and accurate detection of HLB is a critical management step to control the spread of this disease. However, existing HLB detection methods cannot be widely adopted in citrus production due to long-time and high-cost detection period in specific laboratory environments. In view of this, a fast-response and low-cost computer vision technique is investigated for diagnosing HLB in citrus leaves. Specifically, the Gaussian mixture density (GMD) is performed to extract the leaf object from the citrus image, followed by the feature extraction and recognition of the existence of HLB in the leaf based on scalable vocabulary tree. A citrus leaf image dataset is constructed, and the experimental results show that the proposed HLB recognition method with GMD object extraction performs 95–100 % accuracy within 1 s.     

‘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.     

肇庆、云浮地区砂糖橘黄龙病PCR 快速检测技术的建立

以砂糖橘叶脉为材料,采用微量提取法提取DNA,利用柑橘黄龙病病原的特异引物对其16S rDNA 片段进行PCR 扩增,将其扩增产物纯化、回收,筛选含有黄龙病病原16S rDNA 片段的阳性克隆进行序列测定,初步建立了适用于肇庆、云浮地区砂糖橘黄龙病PCR 检测技术,并通过该技术对肇庆、云浮地区砂糖橘黄龙病的发生情况进行了初步了解。结果表明：应用16S rDNA 检测砂糖橘黄龙病在肇庆、云浮地区是可行的,肇庆、云浮地区各县砂糖橘产区均检测到感染黄龙病植株。     

亚洲韧皮杆菌在单株脐橙病树的分布情况

【目的】研究亚洲韧皮杆菌(CLas)在纽荷尔脐橙病树的分布情况,为柑橘黄龙病(HLB)的防控提供参考依据。【方法】以1~3个枝条表现斑驳黄化叶片症状的纽荷尔脐橙病树为研究对象,对主干发出的5个主枝(A、B、C为斑驳黄化枝条,D、E为无症状枝条)的叶片分别取样,运用PCR和实时荧光定量PCR(qPCR)分别检测,用2-ΔΔCt法分析树体各部分黄龙病菌含量。对于果树上表现典型HLB病果但叶片无症状的结果枝,分别检测典型病果和相应叶片的感病情况。【结果】PCR对A、B、C、D、E的阳性检出率分别为88.57%、61.11%、54.17%、6.67%和0,qPCR对A、B、C的阳性检出率均为100.00%,对D、E的阳性检出率分别为40.00%和13.33%;2-ΔΔCt法分析发现,斑驳黄化叶片中病原菌含量更高,病果的阳性检出率高于相应的无症状叶片样品。【结论】CLas在表现典型斑驳黄化症状的叶片和果实中的含量及阳性检出率均较高,病原菌在病树中分布不均匀。在进行HLB检测时,应选取典型或疑似症状样品,并运用qPCR进行检测,一旦发现树体局部发病,应当整株销毁。     

柑橘黄龙病菌对沙田柚田间性状和果实品质的影响

【目的】明确柑橘黄龙病菌对沙田柚Citrus maxima (Burm.) Merr. cv. Shatian Yu田间性状和果实品质的影响。【方法】结合田间调查和室内试验系统研究沙田柚感染黄龙病菌后叶片症状、果实内外品质和风味感官品质的变化,及病菌浓度和树龄对病程发展的影响。【结果】沙田柚嫁接病芽后,黄龙病菌浓度保持在较低水平(Ct28)时,植株叶片的黄龙病症状表现不明显,老叶轻微斑驳黄化,新叶轻微均匀黄化,抽梢正常。与健康植株(Ct35)相比,低黄龙病菌浓度(28Ct32)的植株,叶片症状、果实产量和各品质指标均无显著变化;随着发病程度的加深,高黄龙病菌浓度(Ct26)的柚树叶片出现典型斑驳黄化症状,单株柚果总产量和总结果数显著降低,果实变小变轻,着色不均匀,可食率、出汁率、可溶性固形物含量、甜味度、果肉饱满度和综合风味显著下降,酸味度和异味度反而显著提高,失去食用价值。【结论】沙田柚黄龙病病程进展较慢,但随着发病程度加深,其经济价值受到严重影响。本研究可为评价柑橘黄龙病对沙田柚品质的影响提供科学依据,为解析柚类植物的黄龙病耐性机理提供理论支撑。     

Development of a sensitive and reliable droplet digital PCR assay for the detection of ‘Candidatus Liberibacter asiaticus'

Citrus Huanglongbing(HLB, yellow shoot disease) is one of the most serious citrus diseases worldwide. To better improve the detection sensitivity, a droplet digital PCR(ddPCR) assay was developed for the rapid detection of ‘Candidatus Liberibacter asiaticus'(Las), the putative causal agent of HLB. The detection of sensitivity comparison using positive plasmid indicated that dd PCR was superior to quantitative PCR(qPCR) for detecting and quantifying Las at low concentrations. The Las detection of 40 field samples also showed that six of 13 asymptomatic samples(46.15%) with high Ct value(35) were positive by dd PCR. This methodology showed great potential for early HLB infection diagnosis.