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.     

Looking inside phytoplasma-infected sieve elements: A combined microscopy approach using Arabidopsis thaliana as a model plant

Phytoplasmas are phloem-inhabiting plant pathogens that affect over one thousand plant species, representing a severe threat to agriculture. The absence of an effective curative strategy and the economic importance of many affected crops make a priority of studying how plants respond to phytoplasma infection. Nevertheless, the study of phytoplasmas has been hindered by the extreme difficulty of culturing them in vitro and by impediments to natural host plant surveys such as low phytoplasma titre, long plant life cycle and poor knowledge of natural host-plant biology. Stating correspondence between macroscopic symptoms of phytoplasma infected Arabidopsis thaliana and those observed in natural host plants, over the last decade some authors have started to use this plant as a model for studying phytoplasma-plant interactions. Nevertheless, the morphological and ultrastructural modifications occurring in A. thaliana tissues following phytoplasma infection have never been described in detail. In this work, we adopted a combined-microscopy approach to verify if A. thaliana can be considered a reliable model for the study of phytoplasma-plant interactions at the microscopical level.The consistent presence of phytoplasma in infected phloem allowed detailed study of the infection process and the relationship established by phytoplasmas with different components of the sieve elements. In infected A. thaliana, phytoplasmas induced strong disturbances of host plant development that were mainly due to phloem disorganization and impairment. Light microscopy showed collapse, necrosis and hyperplasia of phloem cells. TEM observations of sieve elements identified two common plant-responses to phytoplasma infection: phloem protein agglutination and callose deposition.     

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.     

Cellular localisation of calcium ions during potato hypersensitive response to Potato virus Y

Ca(2+) is one of the most universal and versatile signalling molecules and is involved in almost every aspect of cellular processes. Accumulating evidence suggests that Ca(2+) serves as a messenger in many growth and developmental processes and in plant responses to biotic and abiotic stresses. Numerous signals have been shown to induce transient elevation of cytoplasmic [Ca(2+)](cyt) in plants. The calcium free ions were detected cytochemically in Solanum tuberosum cv. Rywal tissues as a hypersensitive response (HR) from 10h to 5 days after a Potato virus Y (PVY) infection. Calcium was detected in vivo by its reaction with Alizarin S Red, producing an intense red staining in contact with calcium free ions. Calcium was found in the necrotic area of the epidermal and mesophyll cells 3 days after the PVY infection (when morphological symptoms on potato leaves appeared). Calcium ions were detected cytochemically in HR also by its reaction with potassium pyroantimonate. Inoculation with PVY(NTN) and also PVY(N) Wi induced a rapid hypersensitive response during which highly localised increased accumulation of electron-dense deposits of calcium pyroantimonate were detected. Calcium deposition was observed in necrotic and non-necrotic areas, starting from 10h after PVY infection. The deposits were present along ER cisternae, chloroplasts and mitochondria envelopes connected with PVY particles. The precipitates of calcium antimonate were detected near the nuclear envelope, inside karyolymph and along tracheary elements, especially when virus particles were present inside.     

Ultrastructural analysis of Vitis vinifera leaf tissues showing atypical symptoms of Plasmopara viticola

In an abandoned farm in Tuscany a year by year regression of downy mildew disease on grapevines was observed and a decrease in virulence as well as vigor and fertility of the causal fungus, Plasmopara viticola. Anomalous spots of the fungus (i.e. atypical coloration of leaves or mosaic) on leaf tissues of a sensitive Vitis vinifera grapevine were observed. The anomalous symptoms were often associated with the typical 'oil spots' and were present under environmental conditions favourable for a normal development of the disease. An ultrastructural study was carried out on leaf tissues of grapevine plants aimed at clarifying the cause of this phenomenon and detecting whether there were alterations in P. viticola mycelium and endophytes present. ELISA was also performed to check the presence of grapevine viruses in the plants. TEM results demonstrated that characteristic P. viticola was present in leaf samples showing oil spots, while, both the fungus and the host tissues showed cytological alterations in leaves with mosaic symptoms. Finally, hyphae were absent in leaf tissues without downy mildew spots, but showing severe ultrastructural modifications. Several plant virus infections were found in these grapevines. Literature reports that the development and sporulation of some phytopathogenic fungi inside their hosts can be limited by virus infections. Further experimental approaches are required to determine if resistance to P. viticola can be induced by viral infections in grapevines.     

Determining the elemental composition of Calotropis procera using X‐ray Analytical Microscopy

Caloptropis procera (Oshar) is a desert plant that did not receive much attention from the science community. The objective of this study was to investigate the elemental composition of the different parts of the plant using an X‐ray analytical microscope, to identify the elements naturally present in the plant and in the future detect the presence of any contaminants absorbed by the plants from the surrounding environment. Stalks, leaves and flowers from three Oshar plants were qualitatively and quantitatively analyzed. Leaves were scanned to establish the elemental spatial distribution within individual leaves. Subsequently, parts of the plants were dried, crushed and pulverized, then analyzed to determine elemental concentrations. The major elements present throughout the plant were Cl, K and Ca with varied concentrations. Other elements (Mg, Si, P, Fe, Sr, Mn and Br) are present in the leaf with various low concentrations of <5%. Major, minor and trace elements present in the various plant parts were determined. The outcome of this study will be used as a pilot for further investigations pertaining to the utilization of the Oshar plant for environmental cleaning purposes. Copyright © 2012 John Wiley & Sons, Ltd.     

Localisation of hydrogen peroxide accumulation during Solanum tuberosum cv. Rywal hypersensitive response to Potato virus Y

The reactive oxygen species hydrogen peroxide (H₂O₂) was detected cytochemically in Solanum tuberosum cv. Rywal tissues as a hypersensitive response (HR) 24 and 48 h after a Potato virus Y (PVY) infection.Hydrogen peroxide was detected in vivo by its reaction with 3.3-diaminobenzidine, producing a reddish-brown staining in contact with H₂O₂. Hydrogen peroxide was detected in the necrotic area of the epidermal and mesophyll cells 24 and 48 h after the PVY infection. Highly localised accumulations of H₂O₂ were found within xylem tracheary elements, and this was much more intensive than in non-infected leaves. Hydrogen peroxide was detected cytochemically in HR also by its reaction with cerium chloride, producing electron-dense deposits of cerium perhydroxides.Inoculation with PVY^NTN and also PVY^N Wi induced a rapid hypersensitive response during which highly localised accumulations of H₂O₂ was detected in plant cell walls. The most intensive accumulation was present in the bordering cell walls of necrotic mesophyll cells and the adjacent non-necrotic mesophyll cells. Intensive electron-dense deposits of cerium perhydroxide were found along ER cistrenae and chloroplast envelopes connected with PVY particles. The precipitates of hydrogen peroxide were detected in the nuclear envelope and along tracheary elements, especially when virus particles were present inside. The intensive accumulation of H₂O₂ at the early stages of potato–PVY interaction is consistent with its role as an antimicrobial agent and for this reason it has been regarded as a signalling molecule.     

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.     

Electron microscopy studies on luteovirid transmission by aphids

Transmission electron microscopy (TEM) observations have been extensively applied to follow the route of luteovirids in their vectors. Luteovirids are icosahedral plant viruses which are phloem-limited and strictly transmitted in a circulative manner by aphids. Virus particles, acquired by aphids while feeding on an infected plant, circulate in the aphid's body without replication and are internalized during this process in two different cell types (intestinal and accessory salivary gland cells). The endocytosis mechanism at the gut level seems to rely on a clathrin-mediated entry process and virions are observed in the aphid's gut cells in various vesicular structures. After exocytosis from intestinal cells, virions are released in the aphid's body cavity where they are thought to bind to symbionin, an endosymbiotic protein. Transcytosis of the accessory salivary gland cells occurs similarly as at the gut level but in the reverse direction. Using engineered virus mutants, viral proteins required for transmission and involved in virus retention in the hemocoel have been identified. Virus mutants poorly or non aphid-transmitted have also been localized in the aphid's body by TEM. These observations reveal the crucial role of the minor capsid protein in gut internalization. While not strictly required, this protein seems to play an important role in the efficiency of this process by interacting with putative virus receptors localized on the gut apical membrane. More recently, some aphid proteins have also been shown to exhibit in vitro virus binding capacity and could potentially be components of the endocytotic apparatus.     

Functional Magnetic Resonance Imaging in Intact Plants—Quantitative Observation of Flow in Plant Vessels

Quantitative magnetic resonance (MR) images of flow velocities in intact corn plants were acquired using magnetization-prepared MR microscopy. A phase contrast flow imaging technique was used to quantitate water flow velocities and total volume flow rates in small xylem vessels. The simultaneous measurement of the transpiration of the whole plant was achieved by using a closed climate chamber within the MR magnet. The total volume flow rate and the transpiration values were in close correlation. Functional magnetic resonance imaging in intact plants was performed by light stimulation of the transpiration inside of the magnet. The change in the flow velocities in the xylem vessels of single vascular bundles was in correlation with the changes in the transpiration. Significant differences were observed between the xylem vessels in different vascular bundles. Furthermore, flow velocity measurements were performed on excised plant stems and visualized by the uptake of the MR contrast agent, gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA). A comparison between the phase contrast flow imaging and the contrast media uptake showed to be in good agreement with each other.     

Pulvinus functional traits in relation to leaf movements: a light and transmission electron microscopy study of the vascular system

Previous studies on legume pulvini suggest that the vascular system plays an important role in the redistribution of ions and transmission of stimuli during leaf's movements. However, the number of anatomical and ultrastructural studies is limited to few species. The aim of this paper is to investigate the structure and cellular features of the pulvinus vascular system of nine legume species from Brazilian cerrado, looking for structural traits pointing to its participation in the leaf's movements. Samples were excised from the medial region of opened pulvinus of Bauhinia rufa, Copaifera langsdorffii, Senna rugosa (Caesalpinioideae), Andira humilis, Dalbergia miscolobium, Zornia diphylla (Faboideae), Mimosa rixosa, Mimosa flexuosa and Stryphnodendron polyphyllum (Mimosoideae), and were prepared following light microscopy, transmission electron microscopy and histochemical standard techniques. The vascular system occupies a central position, comprises phloem and xylem and is delimited by a living sheath of septate fibers in all the species studied. This living cells sheath connects the cortex to the vascular tissues via numerous plasmodesmata. The absence of fibers and sclereids, the presence of phenolic idioblasts and the abundance and diversity of protein inclusions in the sieve tube members are remarkable features of the phloem. Pitted vessel elements, parenchyma cells with abundant cytoplasm and living fibriform elements characterize the xylem. The lack of lignified tissues and extensive symplastic continuity by plasmodesmata are remarkable features of the vascular system of pulvini of the all studied species.     

Iron nanoparticle growth induced by Kr–F excimer laser photolysis of Fe(CO)5

We report the uptake by wheat, lupin and Arabidopsis of mesoporous silica nanoparticles functionalised with amine cross-linked fluorescein isothiocyanate (MSN-APTES-FITC). The preparation of these particles at room temperature enabled the synthesis of 20 nm particles that contained a network of interconnected pores around 2 nm in diameter. The uptake and distribution of these nanoparticles were examined during seed germination, in roots of plants grown in a hydroponic system and in whole leaves and roots of plants via vacuum infiltration. The nanoparticles did not affect seed germination in lupin and there was no phytotoxicity. Following germination of wheat and lupin grown in a nutrient solution containing nanoparticles, they were found within cells and cell walls of the emerging root and in the vascular transport elements, the xylem, and in other associated cells. In leaves and roots of Arabidopsis the nanoparticles were found, following vacuum infiltration of whole seedlings, to be taken up by the entire leaf and they were principally found in the intercellular spaces of the mesophyll but also throughout much of the root system. We propose that MSNs could be used as a novel delivery system for small molecules in plants.     

Chiral nematic phase of suspensions of rodlike viruses: left-handed phase helicity from a right-handed molecular helix

We report a study on charged, filamentous virus called M13, whose suspensions in water exhibit a chiral nematic (cholesteric) phase. In spite of the right-handed helicity of the virus, a left-handed phase helicity is found, with a cholesteric pitch which increases with temperature and ionic strength. Several sources of chirality can be devised in the system, ranging from the subnanometer to the micrometer length scale. Here an explanation is proposed for the microscopic origin of the cholesteric organization, which arises from the helical arrangement of coat proteins on the virus surface. The phase organization is explained as the result of the competition between contributions of opposite handedness, deriving from best packing of viral particles and electrostatic interparticle repulsions. This hypothesis is supported by calculations based on a coarse-grained representation of the virus.     

Plant delta(15)N associated with arbuscular mycorrhization, drought and nitrogen deficiency

It has long been evident that plant (15)N chiefly reflects the processes which fractionate (15)N/(14)N rather than the (15)N of plant N source(s). It has emerged recently that one of the most important fractionating processes contributing to the whole plant (15)N is the presence/absence, type or species of mycorrhiza, especially when interacting with nutrient deficiency. Ecto- and ericoid mycorrhizas are frequently associated with (15)N-depleted foliar (15)N, commonly as low as -12 per thousand. As shown by the present study, plants having no mycorrhiza, or those infected with various species of arbuscular mycorrhiza (AM)-forming fungi, interact with varying concentrations of soil nitrogen [N] and moisture to enrich plant (15)N by as much as 3.5 per thousand. Hence the lack of a mycorrhiza, or variation in the species of AM-forming fungal associations, can account for about 25% of the usually reported variations of foliar (15)N found in field situations and do so by (15)N enrichment rather than depletion. Copyright 1999 John Wiley & Sons, Ltd.     

The effect of the operating parameters of heat recovery steam generators on combined cycle/sea-water desalination plant performance

Combined gas/steam turbine cycle plants have been proposed for cogeneration of electricity and process steam. Examples are combined-cycle power plants coupled with sea-water desalination, district heating plants, chemical industries, etc. In combined heat and power plants, the gas turbine exhaust heat is utilized through the use of heat recovery steam generators (HRSG's). As a result, these waste heat generators (boilers), whether fired or unfired, control the performance of the combined plant lower side (bottoming cycle). Moreover, any changes made in the HRSG operating parameters (i.e. the pinch point, approach temperature, first and second stage pressures, and mass ratios) can greatly affect the HRSG performance and will eventually affect the overall combined plant performance. This paper presents a method to predict the performance of the heat recovery steam generators (HRSG)/steam bottoming cycle combined with sea-water desalination plant at various steam and exhaust gas conditions.     

Nanoparticles of: Synthesis and superparamagnetic properties

By using oil in water micelles, cobalt ferrite particles having an average diameter around 3 nm were synthetised. These nanoparticles are characterized by the presence of cation vacancies and no Fe(II) is observed, as it has been described in literature previously. Chemical interfacial treatment allows to coat the particles with citrate derivatives. The magnetic properties of uncoated and coated particles strongly diluted in a polymer substrate are compared by magnetization measurements and ⁵⁷Fe M?ssbauer spectroscopy. The anisotropy constant is shown to be independent of coating, whereas the magnetization is found to be larger in the uncoated particles. Received 3 February 1998     

安徽琅琊山铜矿植物和土壤重元素同步辐射X射线荧光分析

利用同步辐射 X射线荧光分析方法研究了生长在金属矿山上的植物体内重元素的组成与含量 ,以及植物对重元素的富集特性。对采自安徽琅琊山铜矿的梓木草 (Lithospermum Erythrorhizon)和山慈菇(Tulipa Edulis) 2种植物的重元素分析结果表明 ,在锌木草各营养器官之间 ,各种重元素的分布极不均匀 ,Mn、Fe、Cu、Zn等主要富集在多年生根中。山慈菇叶片中的重元素的含量 ,比其他地方的同种植物较高。与一般植物体内重元素检测方法相比 (如火焰原子吸收光谱法 ) ,该法的灵敏度高 ,样品处理简便。     

Chromium localization in plant tissues of Lycopersicum esculentum Mill using ICP-MS and ion microscopy (SIMS)

High-resolution imaging secondary ion mass spectrometry (HRI-SIMS) in combination with inductively coupled plasma mass spectrometry (ICP-MS) were utilised to determine specific sites of chromium concentration in tomato plant tissues (roots, stems and leaves). The tissues were obtained from plants grown for 2 months in hydroponic conditions with Cr added in a form chromium salt (CrCl₃·6H₂O) to concentrations of 25 and 50 mg/L. The chemical fixation procedure used permit to localize only insoluble or strongly bound Cr components in tomato plant tissue. In this work no quantitative SIMS analysis was made. HRI-SIMS analysis revealed that the transport of chromium is restricted to the vascular system of roots, stems and leaves. No Cr was detected in epidermis, palisade parenchyma and spongy parenchyma cells of the leaves. The SIMS-300 spectra obtained from the tissues confirm the HRI-SIMS observations. The roots, and especially walls of xylem vessels, were determined as the principal site of chromium accumulation in tomato plants.     

Hydrodynamic cavitation efficiently inactivates potato virus Y in water

Waterborne plant viruses can destroy entire crops, leading not only to high financial losses but also to food shortages. Potato virus Y (PVY) is the most important potato viral pathogen that can also affect other valuable crops. Recently, it has been confirmed that this virus is capable of infecting host plants via water, emphasizing the relevance of using proper strategies to treat recycled water in order to prevent the spread of the infectious agents. Emerging environmentally friendly methods such as hydrodynamic cavitation (HC) provide a great alternative for treating recycled water used for irrigation. In the experiments conducted in this study, laboratory HC based on Venturi constriction with a sample volume of 1 L was used to treat water samples spiked with purified PVY virions. The ability of the virus to infect plants was abolished after 500 HC passes, corresponding to 50 min of treatment under pressure difference of 7 bar. In some cases, shorter treatments of 125 or 250 passes were also sufficient for virus inactivation. The HC treatment disrupted the integrity of viral particles, which also led to a minor damage of viral RNA. Reactive species, including singlet oxygen, hydroxyl radicals, and hydrogen peroxide, were not primarily responsible for PVY inactivation during HC treatment, suggesting that mechanical effects are likely the driving force of virus inactivation. This pioneering study, the first to investigate eukaryotic virus inactivation by HC, will inspire additional research in this field enabling further improvement of HC as a water decontamination technology.