Use of light, scanning electron microscopy and bioassays to evaluate parasitism by entomopathogenic fungi of the red scale insect of palms (Phoenicococcus marlatti Ckll., 1899)

We have evaluated the parasitism of the red scale insect of the date palm (Phoenicococcus marlatti) by entomopathogenic fungi, using light microscopy (LM), scanning electron microscopy (SEM) and low temperature scanning electron microscopy (LTSEM). Beauveria bassiana, Lecanicillium dimorphum and Lecanicillium cf. psalliotae, were inoculated directly on the scale insects or on insect infested plant material. We found that L. dimorphum and L. cf. psalliotae developed on plant material and on scale insects, making infection structures. B. bassiana was a bad colonizer of date palm leaves (Phoenix dactylifera L.) and did not parasite the scale insects.     

Ultrastructure of spined conidia and hyphae of the rice false smut fungus Ustilaginoidea virens

Spined conidia and hyphae of Ustilaginoidea virens were examined by light and electron microscopy. Bright-field light microscopy showed that conidia were round to elliptical and warty on the surface with diameters approximately ranging from 3 to 5 microm. Scanning electron microscopy revealed the globose to irregularly rounded and ornamented conidia with prominent spines. The spines were pointed at the apex or irregularly curved, and approximately 200-500 nm long. Ultrastructure of spined conidia and hyphae revealed by transmission electron microscopy showed lipid globules and vacuoles in the cytoplasm enclosed by an electron-transparent cell wall. Conspicuous electron-dense spines were evident on the surface of conidia, and had obclavate or irregularly protruding shapes with varying heights along the conidial cell wall. Microfibrillar structures with stretching or branching patterns were evident in the spine matrix. Some conidia were interconnected by spines from the neighboring conidia by their extended outgrowth. Hyphae had concentric bodies that showed an electron-transparent core surrounded by an electron-dense layer. One or more intrahyphal hyphae were found in hyphal cytoplasm. The fungus is thought to form concentric bodies and intrahyphal hyphae as survival mechanisms against the water- and nutrient-deficient environments that may occur in the necrotic regions of host plants.     

Nonhost-associated proliferation of intrahyphal hyphae of citrus scab fungus Elsinoe fawcettii: refining the perception of cell-within-a-cell organization

Ultrastructural aspects of intrahyphal hyphae formation were investigated in Elsinoe fawcettii by transmission electron microscopy. Desiccated and hydrated cultures of E. fawcettii hyphae in liquid and solid media were prepared to determine the effects of water/nutrient availability and media fluidity on the formation of intrahyphal hyphae of the fungus. In all the culture conditions, intrahyphal hyphae were observed in enclosing hyphae. Electron-transparent hyphal cell walls clearly delimited intrahyphal hyphae from the cytoplasm of enclosing hyphae. Intrahyphal hyphae occupied most of the lumen of the enclosing hyphae, and showed intact hyphal cytoplasm with distinct organelles and inclusions. Intrahyphal hyphae were found to grow out of the degenerated hyphae that were almost devoid of cellular contents (simple intrahyphal hyphae). Some intrahyphal hyphae appeared to push aside a septum and passed into the adjacent hyphal cell. Besides a single intrahyphal hypha, instances were noted where enclosing hyphae contained several individual intrahyphal hyphae (multiple intrahyphal hyphae). Other enclosing hyphae contained intrahyphal hyphae, which also had intrahyphal hyphae (compound intrahyphal hyphae). The cell wall of intrahyphal hyphae showed the continuity with the cell wall of the enclosing hyphae. Concentric bodies typical of ascomycetes occurring in dry habitats were not found in all the types of hyphae. These results suggest that intrahyphal hyphae formation of E. fawcettii does not require plant defense responses. The fungus is thought to form intrahyphal hyphae during the saprophytic phase in ex planta ecological niches as well as the parasitic phase in host parts.     

Biotrophic interaction of Sporisorium scitamineum on a new host—Saccharum spontaneum

Sporisorium scitamineum is a biotrophic smut fungus harbored inside the smut gall on the top internodal region of Saccharum spontaneum, a wild relative of sugarcane (Saccharum officinarum). The interactions of spined conidia of S. scitamineum with S. spontaneum were examined during the different stages of plant growth starting from the bud stage to the decaying stage. The spores in the soil from the polyetic inocula grew into confined epidermal cells of the buds and finally sporulated in the topmost internodal region. Hyphae invasion of the plant tissues were restricted to the point of infection. Culms of infected plants in late October sporulated, notably; hyphal sporulation produced shorter hyphal stolons. Remarkably, the nodal regions of infected plants had no spores and fragmented hyphae. On the basis of microscopic analyses, hyphae and spores were absent in all internodes above the ground till the topmost smut gall region. This result indicated that, S. scitamineum undergoes tissue-confined invasion of S. spontaneum. By associating culture medium method with polymerase chain reaction (PCR) on plant portions void of smut gall, S. scitamineum was not detected, indicating that colonization was not systemic. It was observed that the biotrophic interaction resulted in structural reorganization in the restricted region of infection forming erect cylindrical structure, in which the fungus was sandwiched between the central stalk and sheath, and possibly played a key role in preventing inflorescence. Comparatively, a significant difference in the rate of teliospores germination between reference Ustilago esculenta (26.6%, P < 0.05) and S. scitamineum (62.9%, P < 0.05) at 20° C was observed. This study also provides insights on the effect of different temperature regimes on the germination of S. scitamineum teliospores in vitro.     

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.     

Accumulation and localization of extensin protein in apoplast of pea root nodule under aluminum stress

Cell wall components such as hydroxyproline-rich glycoproteins (HRGPs, extensins) have been proposed to be involved in aluminum (Al) resistance mechanisms in plants. We have characterized the distribution of extensin in pea (Pisum sativum L.) root nodules apoplast under short (for 2 and 24 h) Al stress. Monoclonal antibodie LM1 have been used to locate extensin protein epitope by immunofluorescence and immunogold labeling.The nodules were shown to respond to Al stress by thickening of plant and infection thread (IT) walls and disturbances in threads growth and bacteria endocytosis. Immunoblot results indicated the presence of a 17-kDa band specific for LM1. Irrespective of the time of Al stress, extensin content increased in root nodules. Further observation utilizing fluorescence and transmission electron microscope showed that LM1 epitope was localized in walls and intercellular spaces of nodule cortex tissues and in the infection threads matrix. Al stress in nodules appears to be associated with higher extensin accumulation in matrix of enlarged thick-walled ITs. In addition to ITs, thickened walls and intercellular spaces of nodule cortex were also associated with intense extensin accumulation.These data suggest that Al-induced extensin accumulation in plant cell walls and ITs matrix may have influence on the process of IT growth and tissue and cell colonization by Rhizobium bacteria.     

THE ELECTRON-BEAM IRRADIATION ON THE HIGH TEMPERATURE SUPERCONDUCTING THIN FILMS: A NUMERICAL SIMULATION OF THE LOW TEMPERATURE SCANNING ELECTRON MICROSCOPY

Low-temperature scanning electron microscopy (LTSEM) is a promising measuring technique for probing the spatial distribution of the superconducting properties of the high-temperature superconducting (HTS) thin films. A theoretical analysis of the electron-beam irradiation on the HTS thin films in the LTSEM has been carried out. An inhomogeneously distributed grain array model has been applied in the analysis, and some numerical sim-ulations have been carried out on the electron-beam induced voltage (EIV) signals in the LTSEM experiments. The comparisons of our numerical results with the LTSEM experi-mental data indicate that it is quite reasonable to use a two-dimensional Josephson junction array for stimulating the inhomogeneous HTS thin film sample. Our numerical results also show that the EIV signals are influenced by the electron-beam power used in the LTSEM, and a reduction of the electron-beam power is suggested in order to eliminate the errors in estimating the local values of critical temperature T_c and critical current I_c by the sample temperature and the bias current at which the first EIV signal occurs.     

Correlative light and scanning electron microscopy of the same sections gives new insights into the effects of pectin lyase on bordered pit membranes in Pinus radiata wood

Bordered pits are structures in the cell walls of softwood tracheids which permit the movement of water between adjacent cells. These structures contain a central pit membrane composed of an outer porous ring (margo) and an inner dense and pectin-rich disc (torus). The membrane is overarched on each side by pit borders. Pits may be aspirated, a condition where the torus seals against the pit border, effectively blocking the pathway between cells. In living trees this maintains overall continuity of water conduction in xylem by sealing off tracheids containing air. Drying of timber results in further pit aspiration, which reduces wood permeability to liquid treatment agents such as antifungal chemicals. One possible way to increase permeability is by treating wood with pectin lyase to modify or remove the torus. The effectiveness of this treatment was initially evaluated using light microscopy (LM) of toluidine blue stained wood. Pectic material is coloured pink-magenta with this stain, and loss of this colour after treatment has been interpreted as indicating destruction of the torus. However, correlative light (LM) and scanning electron (SEM) microscopic observations of identical areas of toluidine blue stained sections revealed that many unstained pits had intact but modified tori when viewed with SEM. These observations indicate that LM alone is not sufficient to evaluate the effects of pectin lyase on pit membranes in wood. Combining LM and SEM gives more complete information.     

双光子成像观测拟南芥细胞骨架及根尖生长过程

细胞骨架在植物细胞及其运动、发育变化等过程中起着重要作用。本文利用MS培养基在无菌条件下培养转染GFP(绿色荧光蛋白)的拟南芥,使其经过从种子萌发、植株生长直至开花结果的完整生命周期;在此过程中,利用适合对较大较厚样品进行活体四维成像的双光子激光扫描显微术,观测活的拟南芥种子、根尖、导管和根毛等器官内的细胞骨架形态,以及拟南芥根尖生长发育的动态过程,量化计算出根尖的生长速度。用低浓度(10-10mol.L-1)吲哚-3-乙酸刺激拟南芥,观测到根尖生长速度比正常情况下提高约3.8倍。     

A gamma-tubulin antibody against a plant peptide sequence localises to cell division-specific microtubule arrays and organelles in plants

Gamma tubulin (gamma-tubulin) is involved in microtubule initiation in the eukaryotes. In animal cells it is localised to centrosomes and to other, non-centrosomal sites of microtubule initiation. In addition, cytoplasmic complexes containing gamma-tubulin (gamma-TuRCs; gamma-somes) have been described, which are multiprotein complexes involved in microtubule initiation. Most localisations of gamma-tubulin in plants have previously been achieved using an antibody directed towards a conserved peptide sequence found in animal cells, showing co-localisation with all microtubule arrays throughout the cell cycle. Because different antibodies may give various patterns of subcellular localisation, in the present study we raised a polyclonal antibody ('Hayley') to the plant peptide sequence EDFATQGGDRKDVFFY (bold letters indicate plant-specific amino acids) to further investigate the subcellular distribution in plants. Immunoblotting using wheat root tip protein extracts revealed a 58 kDagamma-tubulin-like peptide as has been described before. Immunofluorescence microscopy of wheat root-tip cells, however, revealed localisation of gamma-tubulin to a subset of mitotic microtubule arrays and the cytokinetic phragmoplast, but not to interphase cortical arrays or the preprophase band of microtubules. This lack of labelling may be caused by a restriction of antibody access during interphase, but more likely by a cell division-specific conformational change in the gamma-tubulin molecule. Our antibody also gave an organelle-like labelling, not described before, which may represent storage forms or precursors of gamma-tubulin, perhaps related to plastid-based microtubule initiation in hepatics and hornworts.     

Subcellular impact of sonoporation on plant cells: Issues to be addressed in ultrasound-mediated gene transfer

Sonoporation (membrane perforation via ultrasonic cavitation) is known to be realizable in plant cells on a reversible basis. However, cell viability may concomitantly be affected over the process, and limited knowledge is now available on how such cytotoxic impact comes about. This work has investigated how sonoporation may affect plant cells at a subcellular level and in turn activate programmed cell death (PCD). Tobacco BY-2 cells were used as the plant model, and sonoporation was applied through a microbubble-mediated approach with 100:1 cell-to-bubble ratio, free-field peak rarefaction pressure of either 0.4 or 0.9 MPa, and 1 MHz ultrasound frequency (administered in pulsed standing-wave mode at 10% duty cycle, 1 kHz pulse repetition frequency, and 1 min duration). Fluoroscopy results showed that sonoporated tobacco cells may undergo plasma membrane depolarization and reactive oxygen species elevation (two cellular disruption events closely connected to PCD). It was also found that the mitochondria of sonoporated tobacco cells may lose their outer membrane potential over time (observed using confocal microscopy) and consequently release stores of cytochrome-c proteins (determined by Western Blotting) into the cytoplasm to activate PCD. These findings provide insight into the underlying mechanisms responsible for sonoporation-induced cytotoxicity in plant cells. They should be taken into account when using this membrane perforation approach for gene transfection applications in plant biotechnology.     

Ultrastructure of Tuta absoluta parasitized eggs and the reproductive potential of females after parasitism by Metarhizium anisopliae

Among the microorganisms used in biological control, the muscadine fungus Metarhizium anisopliae (Metsch.) Sorok. is produced and formulated world wide aiming to control pests from several agricultural crops. This work evaluated effects of M. anisopliae isolate UFRPE-6 on the fecundity and mortality of Tuta absoluta (Lepidoptera: Gelechiidae) females and the mechanism of infection on eggs. The infection of the females by the fungus did not affect their oviposition and fecundity; however it affected the survival with total and confirmed mortality of 54.2% and 37.14%, respectively. The eggs were treated with suspension at concentration of 10(6)conidia/mL. The analysis under scanning electron microscopy showed that conidia germination and penetration processes in the eggs of T. absoluta started within the period of 6h after the inoculation. Several hyphal bodies were observed from 12h and an intense extrusion of the mycelium covering all the external surface of the eggs 72h after inoculation. Despite its moderate activity in adults, the isolate URPE-6 of M. anisopliae showed promising in the control of T. absoluta due to its pathogenicity and virulence to eggs from this pest.     

Intracellular papillae of Actinocephalus (Eriocaulaceae-Poales) roots and their interaction with fungi: a light and transmission electron microscopy study

The genus Actinocephalus comprises 25 species and is restricted to Brazil, occurring mainly in the Espinhaço Mountains of Minas Gerais and Bahia States. Previous anatomical studies have reported the occurrence of intracellular papillae in the Actinocephalus roots, without dealing with their ultrastructure and function. The purpose of this paper is to investigate the structure, the composition and the probable function of the intracellular papillae of Actinocephalus roots, based on light microscopy, transmission electron microscopy and histochemical tests. The intracellular papillae occurred in all root tissues, from the rhizodermis to the vascular cylinder; they presented different forms and sizes and, ultrastructurally, they corresponded to material deposited between the cell wall and the plasma membrane. The histochemical tests carried out were positive for cellulose, pectin and callose. The intracellular papillae are responses of the plant cells to the interaction with fungi. They work as a physical barrier restricting fungal penetration, and they may also favor the supply of water and nutrients to the plant, since they increase root absorption surface. This might explain why the species of Actinocephalus are among the tallest Eriocaulaceae despite their reduced radicular system and the nutritional deficiency of the soil in which they grow.     

Ecological and agricultural applications of synchrotron IR microscopy

The diffraction-limited spot size of synchrotron-based IR microscopes provides cell-specific, spectrochemical imaging of cleared leaf, stem and root tissues of the model genetic organism Arabidopsis thaliana, and mutant plants created either by T-DNA insertional inactivation or chemical mutagenesis. Spectra in the wavelength region from 6 to 12 μm provide chemical and physical information on the cell wall polysaccharides of mutants lacking particular biosynthetic enzymes (“Cellulose synthase-like” genes). In parallel experiments, synchrotron IR microscopy delineates the role of Arabidopsis cell wall enzymes as susceptibility factors to the fungus Erysiphe cichoracearum, a causative agent of powdery mildew disease. Three genes, pmr4, pmr5, and pmr6 have been characterized by these methods, and biochemical relations between two of the genes suggested by IR spectroscopy and multivariate statistical techniques could not have been inferred through classical molecular biology. In ecological experiments, live plants can also be imaged in small microcosms with mid-IR transmitting ZnSe windows. Small exudate molecules may be spatially mapped in relation to root architecture at diffraction-limited resolution, and the effect of microbial symbioses on the quantity and quality of exudates inferred. Synchrotron IR microscopy provides a useful adjunct to molecular biological methods and underground observatories in the ongoing assessment of the role of root–soil–microbe communication.     

Investigation of growth responses in saprophytic fungi to charred biomass

We present the results of a study testing the response of two saprophytic white-rot fungi species, Pleurotus pulmonarius and Coriolus versicolor, to charred biomass (charcoal) as a growth substrate. We used a combination of optical microscopy, scanning electron microscopy, elemental abundance measurements, and isotope ratio mass spectrometry (¹³C and ¹⁵N) to investigate fungal colonisation of control and incubated samples of Scots Pine (Pinus sylvestris) wood, and charcoal from the same species produced at 300 °C and 400 °C. Both species of fungi colonise the surface and interior of wood and charcoals over time periods of less than 70 days; however, distinctly different growth forms are evident between the exterior and interior of the charcoal substrate, with hyphal penetration concentrated along lines of structural weakness. Although the fungi were able to degrade and metabolise the pine wood, charcoal does not form a readily available source of fungal nutrients at least for these species under the conditions used in this study.     

Preparation of epidermal growth factor (EGF) conjugated iron oxide nanoparticles and their internalization into colon cancer cells

Epidermal growth factor (EGF) was conjugated with carboxymethyldextran (CMDx) coated iron oxide magnetic nanoparticles using carbodiimide chemistry to obtain magnetic nanoparticles that target the epidermal growth factor receptor (EGFR). Epidermal growth factor modified magnetic nanoparticles were colloidally stable when suspended in biological buffers such as PBS and cell culture media. Both targeted and non-targeted nanoparticles were incubated with CaCo-2 cancer cells, known to overexpress EGFR. Nanoparticle localization within the cell was visualized by confocal laser scanning microscopy and light microscopy using Prussian blue stain. Results showed that targeted magnetic nanoparticles were rapidly accumulated in both flask-shaped small vesicles and large circular endocytic structures. Internalization patterns suggest that both clathrin-dependent and clathrin-independent receptors mediated endocytosis mechanisms are responsible for nanoparticle internalization.     

FePt nanoparticles as heterogeneous Fenton-like catalysts for hydrogen peroxide decomposition and the decolorization of methylene blue

The potential effects of oxidized multi-walled carbon nanotubes (o-MWCNTs) with a length ranging from 50 to 630?nm on the development and physiology of wheat plants were evaluated by examining their effects on seed germination, root elongation, stem length, and vegetative biomass at a concentration ranging from 10 to 160???g/mL in the plant. Results indicated that after 7?days of exposure to the o-MWCNTs medium, faster root growth and higher vegetative biomass were observed, but seed germination and stem length did not show any difference as compared with controls. Moreover, a physiological study was conducted at cellular level using a traditional physiological approach to evidence the possible alterations in morphology, the cell length of root zone, and the dehydrogenase activity of seedlings. Transmission electron microscopy images revealed that o-MWCNTs could penetrate the cell wall and enter the cytoplasm after being taken up by roots. The cell length of root zone for the seedlings germinated and grown in the o-MWCNTs (80???g/mL) medium increased by 1.4-fold and a significant concentration-dependent increase in the dehydrogenase activity for the o-MWCNT-treated wheat seedlings was detected. These findings suggest that o-MWCNTs can significantly promote cell elongation in the root system and increase the dehydrogenase activity, resulting in faster root growth and higher biomass production.     

Formation of surface membranes in developing mammalian hair fibres

Mammalian hair fibres result from complex mechanisms involving synthesis, assembly and stabilisation of keratin proteins in the follicle. The developing hair shaft consists of outer cuticle cells surrounding cortical and medullary (optional) cell types. Presumptive fibre cuticle (FC) is contained by the inner root sheath (IRS) consisting of IRS cuticle, Huxley and Henle cells which are in turn enclosed in an outer root sheath (ORS) of epidermal-like cells. In the current structural studies we have used energy filtered transmission electron microscopy (Zeiss 902A) on Merino sheep skin biopsies to examine the fine sequence of morphological changes involved in forming the fibre surface membrane and the associated underlying structural bands comprising the a-layer and exocuticle. Prior to the development of the exocuticle, FC cells demonstrate a typical plasma-membrane apposed to IRS cuticle plasma-membranes separated by an intercellular space. The formation of exocuticular lamellae is followed by degradation of the residual FC surface membrane and the appearance of intercellular laminae demonstrating a stained central band. As maturation continues cleavage between IRS cuticle and FC occurs along this central band liberating hair into the pilary canal. The mature surface consists of keratinized cells containing a well developed exocuticle and a-layer coated with paired lamina (presumably two lipid containing bilayers) of material approximately 10–12 nm thick derived from the intercellular laminae. The current observations show FC surface formation is similar to process occurring in epidermal stratum corneum and that the cuticle surface membrane of mammalian fibres is not derived from a modified plasma-membrane as previously documented.     

DNA Vector Polyethyleneimine Affects Cell pH and Membrane Potential: A Time-Resolved Fluorescence Microscopy Study

Polyethyleneimine (PEI) is one of the very efficient nonviral vectors being developed and tested for artificial gene transfer into target cells. One of its serious limitations is the significant cytotoxicity of the large amounts of free PEI in the mixtures of DNA and PEI used for transfection. To further investigate the cellular effects of free PEI, we have analyzed the PEI-induced alterations of various cell parameters such as membrane heterogeneity and fluidity, cytoplasmic pH, and plasma membrane potential in a variety of cells such as Swiss 3T3 fibroblast, Chinese hamster ovary, insect cells SF9, plant cell line BY2, and Saccharomyces cerevisae. Fluorescence probes such as Nile red, SNARF-1, and cyanine dye DiSC₂(3), coupled with the technique of picosecond time-resolved fluorescence microscopy, were used in estimating the above-mentioned cell parameters. It was found that the cell membranes were largely unperturbed by PEI. However, the cytoplasmic pH showed an increase of 0.1–0.4 units when the cells were treated with PEI. The plasma membrane potential was found to be depolarized in S. cerevisae and Swiss 3T3 cells. These results suggest that the cytotoxic effects of PEI may partly originate from inhibition of regulation of cytoplasmic pH and plasma membrane potential. Further, it is proposed that the resultant cell alterations favors the transfection process.     

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.