叶绿素荧光成像技术的葡萄霜霉病早期检测

葡萄霜霉病对葡萄生产构成严重威胁,尽早防治是治理霜霉病的关键.为了对该病进行早期检测,以PCR检测获取的霜霉病相对生物量作为霜霉病侵染的依据,从暗适应—光适应—暗弛豫3个光合生理状态连续变化过程中,采集80个人工接种霜霉菌叶片和80个健康对照叶片连续6 d的叶绿素荧光图像.对比健康和接种叶片叶绿素荧光动力学曲线、参数图...     

Antifungal activity of diketopiperazines extracted from Alternaria alternata against Plasmopara viticola: an ultrastructural study

Three dipeptides, belonging to the family of diketopiperazines (DKPs), were extracted from broth culture of the grapevine endophyte Alternaria alternata, and were tested against Plasmopara viticola on leaves of grapevine plants grown in greenhouse. DKPs, used at different concentrations (10(-3), 10(-4), 10(-5) and 10(-6)M) both singularly and in mixtures, demonstrated real effectiveness in inhibiting P. viticola sporulation when applied 2 or 24h after pathogen inoculation. Moreover, no necrotic lesions or other phytotoxicity symptoms were observed on DKP-treated grapevine leaf tissues. Ultrastructural analysis performed on grapevine leaf tissues revealed that the DKPs used singularly and in mixture, at above reported concentrations, did not cause leaf tissue damages. By contrast, hyphae of P. viticola exhibited marked structural changes, similar to those induced by the endophyte A. alternata. This demonstrates the involvement of these metabolites in the relationship of P. viticola and the endophyte. Further experimental trials will be carried out in the next future in order to test the effectiveness of these molecules also under field conditions, and to better understand the mechanism of action involved in the pathogen inhibition.     

Two simplified fluorescent staining techniques to observe infection structures of the oomycete Plasmopara viticola in grapevine leaf tissues

Plasmopara viticola, the causal agent of grapevine downy mildew, is an obligate biotrophic oomycete that grows in the intercellular spaces of host tissues and develops haustoria in the cells. Histological observations are the most effective methods to visualize and quantify the development of the infection structures. We chose two staining techniques leading to high resolution and contrast between parasite structures and host-plant tissues with a minimum of sample preparation: Blankophor and KOH-aniline blue fluorescent stainings. Blankophor (50 ppm in water or 15% KOH) staining was used to study the zoospore encystement on the leaf surface after release from sporangia. The aniline blue dye (0.05% in 0.067 M K(2)HPO(4), pH 9-9.5, after hot KOH whitening) was used to observe the invasive structures inside host tissues that lead to the production of sporangiophores and infectious sporangia. We tested modifications of some parameters of the procedures to determine the most appropriate for high throughput analyses adapted to our pathosystem and equipment facilities.     

Cytological modifications in maize plants infected by barley yellow dwarf virus and maize dwarf mosaic virus

Three inbred lines of maize (33-16, MO17 and B73) differing in their susceptibility to Barley yellow dwarf virus and Maize dwarf mosaic virus were studied to compare the ultrastructural modifications induced by the two viruses in leaf tissues of different age. The results demonstrate that the alterations induced by the two viruses in the different maize lines could depend on the particular line tested.     

基于高光谱成像的南瓜叶片叶绿素分布可视化研究

叶绿素浓度是植物生长的指示剂,而叶片的SPAD值则可以反映植物叶绿素含量,从而监测植物的生长状况。本文采用可见-近红外(380～1 030 nm)高光谱成像技术可以实现南瓜叶片SPAD值的可视化,同时根据叶片霜霉病疫情与叶绿素含量呈显著正相关进而可以快速诊断霜霉病疫情。通过测定健康叶片和感染不同霜霉病疫情的叶片光谱曲线,采用竞争性自适应重加权算法(CARS)进行特征波段的选择,可以得到10条特征波段,再结合偏最小二乘回归法(PLSR)进行南瓜叶片SPAD的预测。结果表明,通过对48个样本的训练,对23个样本进行预测,可以得到南瓜叶片SPAD较好的预测效果,其中R_C=0.918,RMSECV=3.932; R_CV=0.846,RMSECV=5.254; R_P=0.881,RMSEP=3.714。根据叶片光谱特征波段与SPAD之间的线性回归方程可以计算叶片各个像素点的SPAD值,最后采用图像处理技术可以得到南瓜叶片SPAD的可视化分布图,同时也反映了霜霉病的感染分布,进而判断南瓜叶片的霜霉病疫情。该研究为监测植物生长状况及判别南瓜叶片霜霉病疫情奠定了理论基础。     

可见光光谱和机器学习的温室黄瓜霜霉病严重度定量估算

温室黄瓜霜霉病严重度的准确估算是科学防治霜霉病的前提条件,对于减少农药使用量、提升温室黄瓜品质和农民经济效益具有重要意义。机器学习在植物病害诊断领域的应用越来越广泛,已经取得了丰富的研究成果,病害严重程度的估算萌发了新的思路。利用霜霉病可见光图像并结合机器学习方法,开展温室黄瓜霜霉病严重度快速准确定量估算研究。利用数码相机采集温室黄瓜霜霉病叶片图像并进行预处理,剔除病害图像的背景。以黄瓜霜霉病叶片图像为输入,构建基于卷积神经网络(CNN)的估算模型。利用可见光光谱颜色特征(CVCF)结合支持向量机进行温室黄瓜霜霉病病斑图像分割,采用SURF(speeded up robust features)特征及形态学操作对分割结果进行优化。在获取黄瓜霜霉病病斑分割图像后,提取病斑图像RGB, HSV, L~*a~*b~*, YCbCr和HSI共5个颜色空间15个颜色分量的平均值和标准差2个颜色特征,以及在此基础上结合灰度共生矩阵提取各颜色分量的对比度、相关性、熵和平稳度4个纹理特征,共计90个特征;利用Pearson相关性分析进行特征优选,采用与温室黄瓜霜霉病严重程度实际值相关性高的图像特征构建浅层机器学习估算模型,包括支持基于向量机回归(SVR)的估算模型和基于BP神经网络(BPNN)的估算模型。基于以上3种估算模型开展黄瓜霜霉病严重度定量估算,采用决定系数(R^2)和归一化均方根误差(NRMSE)对估算模型准确率进行定量评价。结果表明,模型估算的温室黄瓜霜霉病严重度与实际值之间具有良好的线性关系,其中,基于CNN的估算模型准确率最高,模型的R^2为0.919 0, NRMSE为23.33%,其次是基于BPNN的估算模型,其R^2为0.890 8, NRMSE为24.64%,基于SVR的估算模型的准确率最低,其R^2为0.8901, NRMSE为31.08%。研究结果表明,利用黄瓜霜霉病可见光图像数据,结合卷积神经网络估算模型,实现了温室黄瓜霜霉病严重度的准确估算,能够为温室黄瓜霜霉病的科学防治提供参考,提高病害防治效率,减少农药使用。     

基于多光谱荧光成像技术和SVM的葡萄霜霉病早期检测研究

葡萄霜霉病是全球危害最严重的葡萄病害,对该病进行早期检测和防治,可提高葡萄品质和产量,提出一种基于多光谱荧光成像技术（MFI）和支持向量机模型（SVM）的霜霉病早期检测方法。对人工接种霜霉病的葡萄叶片（145个）和健康对照叶片（145个）从叶背面连续6天进行多光谱荧光成像,获得试验叶片16个荧光参数（4个单独波段F440,F520,F690,F740及其相互比值）的图像。在分析不同荧光波段图像随接种天数（DPI）变化规律基础上,通过单因素方差分析和相关性分析,优选出进行霜霉病早期检测的4个波段特征F520,F690,F440/F740,F690/740,利用这4个特征构建基于SVM的霜霉病检测模型。试验发现,16个荧光参数都有早期检测霜霉病的潜力,四个单独波段中F440和F520比F690和F740对霜霉病的侵染更敏感,6DPI才显症的病斑能在F440和F520波段2DPI（接种后第二天）的荧光图像中凸显,接种叶片F440和F520波段荧光强度均随着DPI增加快速升高,在2DPI显著高于健康叶片（p<0.01）,并随着DPI增加更加显著（p<0.0001）;接种叶片F690和F740波段荧光强度均随着DPI增加逐渐减小,1DPI-3DPI与健康叶片无显著差异,从4DPI开始显著低于健康叶片（p<0.05）,并在5DPI-6DPI更加显著（p<0.01）;健康叶片荧光参数变化很小。F440极易受干扰,变异系数最大,F520最稳定。随着DPI增加,叶片被侵染程度加深,4个特征融合的SVM模型对健康和接种叶片检测准确率逐渐提高,1DPI的准确率为65.6%,3DPI检测准确率为82.2%,整个试验周期（1DPI-6DPI）的平均检测准确率达84.6%,高于单一特征中最优波段F520的阈值检测结果（1DPI的准确率为61.1%,3DPI检测准确率为78.9%,整个试验周期为80.0%）。结果表明利用MFI技术和SVM模型能实现霜霉病显症前的早期检测,为便携式葡萄霜霉病早期诊断设备的开发提供了理论依据。     

红外热成像与近红外光谱结合快速检测潜育期番茄花叶病

现有的番茄花叶病无损检测方法无法在潜育期内,即显症之前进行早期识别导致施药不及时或者盲目过度施药。设计与试制了红外热成像信息采集系统,主要包括：光箱、红外热成像仪、温度及升降控制器、加热板和升降载物台。该系统能够根据温度起止节点的要求,人为调节拍摄温度。在江苏大学现代农业装备与技术省部共建重点实验室Venlo型温室中进行非抗病性番茄品种辽宁农科院L-402的培育。采用叶面摩擦接种花叶病毒(Tobacccco mosaic virus, ToMV),分为轻度感染组(LI),重度感染组(SI);LI组为磷酸缓冲液稀释500倍后的病毒液接种,SI组为病毒原液接种。对照组(CG)喷施等量磷酸缓冲液。接种10 d后叶片开始出现病斑,证明接种后9 d为番茄花叶病的潜育期。使用红外热成像系统采集了三个组共计144个样本的红外热成像图,计算叶表最大温差(MTD) 以表征潜育期内连续9 d内的叶面温度变化情况。CG组叶片的MTD值差异极小,而接种后叶片MTD值随着病毒侵染时间的推进发生了显著的变化。接种6 d后MTD值差异最大可达1.63 ℃,第7 d开始差异逐步缩小,表明病毒的扩散范围增大导致病叶越来越多的区域被侵染使得整体叶温上升。光谱采集采用两种方法进行,一种是根据热像图的MTD值计算判别出温度突变区域后采集光谱,记为热像采集法(TCM);另一种是不考虑病灶位置,在叶尖、叶中、叶基三个区域分别随机选择一个点采集光谱后求平均值,记为随机采集法(RCM)。TCM确定三个光谱采集点的选择原则是：LI组接种后3,6和9 d的温度突变区域平均MTD值比CG组温度分别高出0.3,0.7和0.5 ℃。SI组接种后3,6和9 d的温度突变区域平均MTD值比CG组温度分别高出0.5,1.2,0.8 ℃。差值达到此标准的病灶位置才定为TCM的可选区域。对所有样本采用支持向量机(SVM)算法建立识别模型。采用主成分分析对2 151个波长点的光谱信息进行压缩,前6个主成分所对应的累积方差贡献率已到达99%。分别对感病3,6和9 d的样本按照2∶1的比例划分校正集和预测集,对预测集样本的病害程度进行识别。两种方法所建立的模型的总识别率分别为92.59%和99.77%。采用TCM建立的光谱识别模型中仅有接种后3 d的一个LI组样本未能识别出来,被误判成CG组样本外,其余组识别率均达到了100%。结果表明近红外光谱法识别番茄花叶病是可行的。采用红外热成像结合近红外光谱法能够建立识别率更高的番茄花叶病潜育期识别模型,克服点源采样随机性,对后续管控流程和突破作物早期精准用药的关键技术探索,建立更为精准的温室智能施药系统提供了新的思路。     

高光谱影像的苹果花叶病叶片花青素定量反演

花叶病是苹果叶片常见的病毒性病害,患病叶片的花青素含量出现异常。以叶片花青素含量作为病害严重程度的定量化指标,使用高光谱成像技术获取感染花叶病的苹果叶片的高光谱图像,分析叶片的光谱特征,通过任意两个波段的反射率的不同数学组合,构建并筛选对染病叶片花青素含量高度敏感的最优光谱指数,进而建立苹果叶片花青素含量的高光谱估算模型,最终实现苹果叶片花青素含量分布状况的可视化表达。结果表明,随着病害严重程度的增大,苹果叶片的花青素含量升高;叶片染病区域的光谱反射率在整个可见光区域明显增加,而且出现了红边蓝移现象。通过两两波段组合构建的三种光谱指数(NDSI_(770,722),RSI_(717,770),DSI_(581,520))与苹果叶片花青素含量的相关系数绝对值均达到0.8以上。在构建的四种苹果叶片花青素含量估算模型中,选用三个光谱指数为参数、并使用偏最小二乘回归方法建立的Anth-PLSR模型精度最高(R2=0.823, RMSE=0.056)。采用Anth-PLSR模型对患病叶片的高光谱图像进行逐像元解算,得到苹果花青素含量分布图。进一步通过叶片花青素含量分布图计算苹果叶片整叶的花青素含量平均值,作为苹果叶片健康程度的定量化指标。此外,通过提取整叶光谱均值、使用同样模型可简洁有效地估算苹果整叶花青素含量平均值。为苹果叶片花叶病病害监测提供了一种直观、快速的技术手段。     

基于叶绿素荧光光谱分析的黄瓜霜霉病害预测模型

为了实现对黄瓜病害的快速无损准确预测,基于激光诱导叶绿素荧光光谱分析技术,建立了温室黄瓜霜霉病害的预测模型.通过测定健康叶片、病菌接种3d叶片和接种6d叶片的光谱曲线,采用一阶导数光谱预处理方法,结合主成分分析数据降维方法对三组光谱数据进行特征信息提取后,建立主成分得分散点图,依据累积贡献率选取10个主成分代替导数光谱...     

MALDI-based intact spore mass spectrometry of downy and powdery mildews

Fast and easy identification of fungal phytopathogens is of great importance in agriculture. In this context, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a powerful tool for analyzing microorganisms. This study deals with a methodology for MALDI-TOF MS-based identification of downy and powdery mildews representing obligate biotrophic parasites of crop plants. Experimental approaches for the MS analyses were optimized using Bremia lactucae, cause of lettuce downy mildew, and Oidium neolycopersici, cause of tomato powdery mildew. This involved determining a suitable concentration of spores in the sample, selection of a proper MALDI matrix, looking for the optimal solvent composition, and evaluation of different sample preparation methods. Furthermore, using different MALDI target materials and surfaces (stainless steel vs polymer-based) and applying various conditions for sample exposure to the acidic MALDI matrix system were investigated. The dried droplet method involving solvent evaporation at room temperature was found to be the most suitable for the deposition of spores and MALDI matrix on the target and the subsequent crystallization. The concentration of spore suspension was optimal between 2 and 5?×?10(9) spores per ml. The best peptide/protein profiles (in terms of signal-to-noise ratio and number of peaks) were obtained by combining ferulic and sinapinic acids as a mixed MALDI matrix. A pretreatment of the spore cell wall with hydrolases was successfully introduced prior to MS measurements to obtain more pronounced signals. Finally, a novel procedure was developed for direct mass spectra acquisition from infected plant leaves. Copyright ? 2012 John Wiley & Sons, Ltd.     

基于可见光光谱分析的黄瓜白粉病识别研究

白粉病是黄瓜常见病害之一,传播速度极快,严重时可造成黄瓜大量减产,对其进行快速准确识别,对黄瓜白粉病诊断和防治具有重要意义,应用可见光谱技术,结合主成分分析和支持向量机算法,实现对黄瓜白粉病的快速识别。配制白粉病菌孢子悬浮液,并人工接种于科研温室内的黄瓜叶片上,以诱发黄瓜白粉病,待白粉病有一定面积暴发后,利用海洋光学USB2000+型便携式光谱仪对黄瓜叶片光谱信息进行采集,利用五点取样法采集样本,在5个检查点,每点选取2株黄瓜进行调查,每株选取4枚感病叶片,每枚叶片随机选取5个感病区域进行光谱采集,共计采集200个感病叶片光谱样本,同样采集200个健康叶片样本作为对照。通过Ocean Optics Spectra-Suite软件采集漫反射标准白板信息和光谱仪暗电流实现光谱仪校正,调节积分时间、扫描次数以及平滑度等参数来实现光谱曲线平滑处理,以有效抑制光谱噪声,对光谱特征进行分类识别,去掉首尾噪声较大的波段,保留光谱的可见光波段进行研究,最终选取450~780 nm波段范围作为研究对象。利用主成分分析对所研究波段范围内的高维光谱数据（947维）进行降维处理,根据主成分的累计贡献率,选取前5个主成分作为分类模型的输入,以白粉病和健康叶片的判别结果作为输出,利用支持向量机算法,通过对样本的分类学习训练构建黄瓜白粉病和健康叶片的分类识别模型,随机选取120个样本作为训练集用于分类模型构建,其余80个样本作为测试集用于模型检验,并通过选取不同的核函数来获得最优模型。利用混淆矩阵对分类识别模型的准确率进行评价,当选取径向基核函数时,分类识别模型对黄瓜健康叶片和白粉病叶片的识别准确率最高,分别为100%和96.25%,总准确率为98.125%,具有较高的准确率。结果表明,利用可见光光谱信息并结合主成分分析和支持向量机算法,可以实现对黄瓜白粉病的快速准确识别,为黄瓜病害诊断提供了方法和参考依据。     

Fourier Transform Mid-Infrared Photoacoustic Spectroscopy for Presymptomatic Detection of Powdery Mildew Infection in Rubus corchorifolius L.

A powdery mildew fungi-induced disease appearing on leaves of Rubus corchorifolius L. has been observed in China, which resulted in seriously influencing the yield and quality of the fruit, and a presymptomatic detection of powdery mildew infection is needed to guarantee the yield and quality through removing the fungi in an early stage. Depth-profiling Fourier transform mid-infrared photoacoustic spectroscopy was applied to characterize both the healthy leaves and powdery mildew-infected leaves of Rubus corchorifolius L. The profiled surface could be divided into out layer (depth of about 1.32 µm) and deep layer (depth of about 1.87 µm). There were numerous differences in the total spectral range (500–4000 cm^?1) between healthy leaves and infected leaves, especially the intensity of absorption bands of 2800–3000 cm^?1 (aliphatic C–H vibration) and 2250–2350 cm^?1 (CO₂) significantly decreased when the leaf was infected by powdery mildew. For the out layer the standard spectral variance between healthy leaf and infected leaf was 7.33 × 10², whereas it was 1.86 × 10⁴ for the deep layer; the standard spectral variance between out layer and deep layer for healthy leaf was 3.38 × 10³, whereas it was 1.84 × 10⁴ for infected leaf, which implied that both out layer and deep layer responded to powdery mildew infection. Combining spectral differences between healthy leaf and infected leaf and variances between out layer and deep layer, a presymptomatic detection of powdery mildew infection was successfully made, which provided an alternative option and noninvasive method for the fast diagnosis of powdery mildew infection on Rubus corchorifolius L.     

可见光光谱和支持向量机的温室黄瓜霜霉病图像分割

针对温室现场环境下采集的黄瓜霜霉病叶片图像中存在光照不均匀和背景复杂的问题,提出了一种基于可见光光谱和支持向量机的温室黄瓜霜霉病图像分割方法。首先,提出了一种基于可见光谱的颜色特征CVCF(combination of three visible color features)及其检测方法,该颜色特征将超红特征(excess red,ExR)、H分量和b*分量三种颜色特征结合,通过设置ExR参数,降低光照条件对ExR的影响,克服了光照不均匀对病斑分割的影响。在CVCF的基础上,结合基于径向基核函数的支持向量机分类器,通过优化分类器参数构建病斑分割模型,获得了温室黄瓜霜霉病图像初始分割结果。在初始分割结果基础上,采用SURF(speeded up robust features)特征及形态学操作,对分割结果进一步优化,消除背景噪声对分割结果的影响,从而获得最终病斑分割结果。为进一步验证方法的有效性,选择了OTSU算法、K均值聚类算法和决策树算法,作对比研究。结果表明,OTSU+H*0.2,K-means+H+b*,DT+H+b*和该研究算法的错分率分别为：19.44%,40.19%,16.27%和7.37%,该算法对温室现场环境下采集的黄瓜霜霉病图像的分割效果明显优于其他对比方法。该方法能够充分克服光照不均匀和复杂背景的影响准确地提取病斑,为病害识别提供了良好的数据来源。     

病斑、病斑附近烟叶和正常烟叶的FTIR研究

用傅里叶变换红外光谱法研究了赤星病、角斑病和气候型斑点病3组病斑、病斑附近烟叶和正常烟叶,发现3组烟叶样品红外光谱的吸光度比A1631/A1025呈现相同的变化趋势,有正常烟叶病斑附近烟叶病斑的规律。对3组烟叶样品整个光谱区的原始光谱和二阶导数光谱进行距离分析,结果显示,病斑附近烟叶与正常烟叶的Pearson相关系数大于病斑与正常烟叶的Pearson相关系数,即与病斑相比,病斑附近烟叶与正常烟叶具有更紧密的关系。吸光度比A1631/A1025和Pearson相关系数的这种变化趋势表明病斑附近烟叶的化学成分在逐渐改变,即病斑附近烟叶处于正常和病变的过渡状态。研究结果表明,傅里叶变换红外光谱有望成为诊断烟草潜育期病害的方法。     

Formation of two-dimensional crystals of icosahedral RNA viruses

The formation of 2D arrays of three small icosahedral RNA viruses with known 3D structures (tomato bushy stunt virus, turnip yellow mosaic virus and bromegrass mosaic virus) has been investigated to determine the role of each component of a negative staining solution containing ammonium molybdate and polyethylene glycol. Virion association was monitored by dynamic light scattering (DLS) and virus array formation was visualised by conventional transmission electron microscopy and cryo-electron microscopy after negative staining. The structural properties of viral arrays prepared in vitro were compared to those of microcrystals found in the leaves of infected plants. A novel form of macroscopic 3D crystals of turnip yellow mosaic virus has been grown in the negative staining solution. On the basis of the experimental results, the hypothesis is advanced that microscopic arrays might be planar crystallisation nuclei. The formation of 2D crystals and the enhancing effect of polyethylene glycol on the self-organisation of virions at the air/water interface are discussed. SYNOPSIS: The formation of 2D arrays of icosahedral viruses was investigated by spectroscopic and transmission electron microscopic methods.     

The evidence of Tobacco rattle virus impact on host plant organelles ultrastructure

Tobraviruses, like other (+) stranded RNA viruses of plants, replicate their genome in cytoplasm and use such usual membranous structures like endoplasmic reticulum. Based on the ultrastructural examination of Tobacco rattle virus (TRV)-infected potato and tobacco leaf tissues, in this work we provide evidence of the participation of not only the membranous and vesicular ER structures but also other cell organelles during the viral infection cycle. Non-capsidated TRV PSG particles (potato isolate from the Netherlands) (long and short forms) were observed inside the nucleus while the presence of TRV capsid protein (CP) was detected in the nucleus caryolymph and within the nucleolus area. Both capsidated and non-capsidated viral particles were localized inside the strongly disorganized chloroplasts and mitochondria. The electron-dense TRV particles were connected with vesicular structures of mitochondria as well as with chloroplasts in both potato and tobacco tissues. At 15–30 days after infection, vesicles filled with TRV short particles were visible in mitochondria revealing the expanded cristae structures. Immunodetection analysis revealed the TRV PSG CP epitope inside chloroplast with disorganized thylakoids structure as well as in mitochondria of different tobacco and potato tissues. The ultrastructural analysis demonstrated high dynamics of the main cell organelles during the TRV PSG–Solanaceous plants interactions. Moreover, our results suggest a relationship between organelle changes and different stages of virus infection cycle and/or particle formation.     

基于高光谱成像和判别分析的黄瓜病害识别

利用光谱成像技术(400～720 nm)识别黄瓜白粉病、角斑病、霜霉病、褐斑病和无病区域。构建高光谱图像采集系统进行样本图像的采集,预处理和光谱信息的提取。由于获得的原始光谱数据量很大,为了减少后续运算量,提高准确率,采用逐步判别分析和典型判别分析两种方法进行降维。逐步判别从55个波段中选择12个波段,典型判别从55个波段中提取2个典型变量。利用选择的光谱特征参数建立病害识别模型。逐步判别构建的模型对训练样本和测试样本的判别准确率分别为100%和94%,典型判别构建的模型对训练样本和测试样本的判别准确率均为100%。说明利用高光谱成像技术可以进行黄瓜病害的快速、准确识别,并为实现可见光谱范围内黄瓜病害的田间实时在线检测提供了可能。     

Cytopathological potato virus Y structures during Solanaceous plants infection

The ultrastructural analysis of tobacco, potato and pepper tissues during infection with necrotic strains and the ordinary Potato virus Y strain of revealed the presence of virus inclusions not only in the epidermis and mesophyll but also in the vascular tissues. For the first time cytoplasmic inclusions were documented in companion cells and phloem parenchyma as well as in xylem tracheary elements. The ultrastructural features studied in this work consisted of mostly laminated inclusions (in the traverse and longitudinal section), which were frequently connected with enlarged cisternae of endoplasmic reticulum (ER) located in the direct vicinity of the cell wall attached to virus particles opposite to plasmodesmata. It was noticed that ER participates in synthesis and condensation of the PVY inclusions. During compatible interaction of tobacco and potato plants with PVY, amorphous and nuclear inclusions were observed. Such forms were not found in pepper tissues and potato revealing the hypersensitivity reaction to the infection with PVY necrotic strains. It was stated that the forms of cytoplasmic inclusions cannot serve as a cytological criterion to distinguish the potato virus Y strains and do not depend on host resistance level. Only in compatible interaction in Solanaceous plants tissues cytoplasmic inclusions were observed from the moment the morphological symptoms appeared. In the reaction of hypersensitivity, the inclusions were found on the 24th day following the infection with the PVY necrotic strains, whereas the symptoms were observed 3 days after the PVY infection.     

Colonization of cashew plants by Lasiodiplodia theobromae: microscopical features

Lasiodiplodia theobromae is a phytopathogenic fungus causing gummosis, a threatening disease for cashew plants in Brazil. In an attempt to investigate the ultrastructural features of the pathogen colonization and its response to immunofluorescence labeling, light, confocal and electron microscope studies were conducted on different severity scale patterns of diseased plants. Lasiodiplodia-antisera was checked for cross reactivity against common cashew plants fungi. Optical microscopy analysis revealed a longitudinally sectioned hyphae located within the xylem vessels, showing an extensive hyphal development in the secondary xylem tissue. SEM images demonstrated that the fungus was found in some asymptomatic samples, particularly within the xylem vessels as confirmed by the optical images. Symptomatic sample images showed an extensive distribution of the fungus along the secondary xylem, within the vessels, infecting xylem parenchyma. A closer look in the secondary xylem parenchyma reveals a heavy and profuse invasion of the cells with a distinguishable cell wall disintegration and fully hyphae dispersal. There was no reactivity of Lasiodiplodia-antisera against mycelial extracts of Colletotrichum gloeosporioides, Phomopsis anardii and Pestalotiopsis guepinii. Following incubation of sections with the polyclonal antisera, the hyphae were intensely and regularly labeled. Rays, vessels and parenchyma cells were the preferred pathway for L. theobromae colonization. Artificial infection provides the information that the vascular cylinder is undoubtedly employed and used by the fungus for hyphae distribution. Immunofluorescence assay employed in situ was applied and the polyclonal antisera produced was able to recognize the fungus and proved to be a sensitive technique to detect it.