NoPv1: a synthetic antimicrobial peptide aptamer targeting the causal agents of grapevine downy mildew and potato late blight.

Grapevine (Vitis vinifera L.) is a crop of major economic importance. However, grapevine yield is guaranteed by the massive use of pesticides to counteract pathogen infections. Under temperate-humid climate conditions, downy mildew is a primary threat for viticulture. Downy mildew is caused by the biotrophic oomycete Plasmopara viticola Berl. & de Toni, which can attack grapevine green tissues. In lack of treatments and with favourable weather conditions, downy mildew can devastate up to 75% of grape cultivation in one season and weaken newly born shoots, causing serious economic losses. Nevertheless, the repeated and massive use of some fungicides can lead to environmental pollution, negative impact on non-targeted organisms, development of resistance, residual toxicity and can foster human health concerns. In this manuscript, we provide an innovative approach to obtain specific pathogen protection for plants. By using the yeast two-hybrid approach and the P. viticola cellulose synthase 2 (PvCesA2), as target enzyme, we screened a combinatorial 8 amino acid peptide library with the aim to identify interacting peptides, potentially able to inhibit PvCesa2. Here, we demonstrate that the NoPv1 peptide aptamer prevents P. viticola germ tube formation and grapevine leaf infection without affecting the growth of non-target organisms and without being toxic for human cells. Furthermore, NoPv1 is also able to counteract Phytophthora infestans growth, the causal agent of late blight in potato and tomato, possibly as a consequence of the high amino acid sequence similarity between P. viticola and P. infestans cellulose synthase enzymes.

In vitro assessment of the antimicrobial efficacy of chitosan nanoparticles against major fish pathogens and their cytotoxicity to fish cell lines.

Nanotechnology is an emerging avenue employed in disease prevention and treatment. This study evaluated the antimicrobial efficacy of chitosan nanoparticles (CSNPs) against major bacterial and oomycete fish pathogens in comparison with chitosan suspension. Initially, the minimum inhibitory concentrations (MIC, MIC90 ) were determined and the per cent inhibition of bacterial growth was calculated. Subsequently, the minimum bactericidal concentrations (MBCs) were determined. The time-dependent disruptions of CSNP-treated pathogens were observed via transmission electron microscopy (TEM), and the effect of CSNPs on the viability of two fish cell lines was assessed. No antimicrobial effect was observed with chitosan, while CSNPs (105 nm) exhibited a dose-dependent and species-specific antimicrobial properties. They were bactericidal against seven bacterial isolates recording MBC values from 1 to 7 mg/ml, bacteriostatic against four further isolates recording MIC values from 0.125 to 5 mg/ml and fungistatic against oomycetes recording MIC90 values of 3 and 4 mg/ml. TEM micrographs showed the attachment of CSNPs to the pathogenic cell membranes disrupting their integrity. No significant cytotoxicity was observed using 1 mg/ml CSNPs, while low dose-dependent cytotoxicity was elicited by the higher doses. Therefore, it is anticipated that CSNPs are able to compete and reduce using antibiotics in aquaculture.

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples.

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM. Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells. The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Attachment, penetration and early host defense mechanisms during the infection of filamentous brown algae by Eurychasma dicksonii.

Eurychasma dicksonii is one of the most common and widespread marine pathogens and attacks a broad spectrum of more than 45 brown algal species. The present study focuses on the mechanism used by the pathogen to attach on the host cell wall and force its way into algal cells. Ultrastructural examination revealed a needle-like structure which develops within the attached spore and extends along its main axis. Particular cell wall modifications are present at the basal part of the spore (adhesorium pad) and guide the needle-like tool to penetrate perpendicularly the host cell wall. The unique injection mechanism is shared with Haptoglossa species which suggests that this is an important characteristic of early diverging oomycetes. Furthermore, the encystment and adhesion mechanism of E. dicksonii shows significant similarities with other oomycetes, some of which are plant pathogens. Staining and immunolabelling techniques showed the deposition of ß-1,3-glucans on the host cell wall at the pathogen penetration site, a strategy similar to physical responses previously described only in infected plant cells. It is assumed that the host defense in terms of callose-like deposition is an ancient response to infection.

Simultaneous quantification of sporangia and zoospores in a biotrophic oomycete with an automatic particle analyzer: disentangling dispersal and infection potentials.

Quantitative pathogenicity traits drive the fitness and dynamics of pathogens in agricultural ecosystems and are key determinants of the correct management of crop production over time. However, traits relating to infection potential (i.e. zoospore production) have been less thoroughly investigated in oomycetes than traits relating to dispersal (i.e. sporangium production). We simultaneously quantified sporangium and zoospore production in a biotrophic oomycete, for the joint assessment of life-cycle traits relating to dispersal and infection potentials. We used an automatic particle analyzer to count and size the sporangia and/or zoospores produced at t = 0 min (no zoospore release) and t = 100 min (zoospore release) in 43 Plasmopara viticola isolates growing on the susceptible Vitis vinifera cv. Cabernet Sauvignon. We were able to differentiate and quantify three types of propagules from different stages of the pathogen life cycle: full sporangia, empty sporangia and zoospores. The method was validated by comparing the sporangium and zoospore counts obtained with an automatic particle analyzer and under a stereomicroscope (manual counting). Each isolate produced a mean of 5.8 ± 1.9 (SD) zoospores per sporangium. Significant relationships were found between sporangium production and sporangium size (negative) and between sporangium size and the number of zoospores produced per sporangium (positive). However, there was a significant positive correlation between total sporangium production and total zoospore production. This procedure can provide a valid quantification of the production of both sporangia and zoospores by oomycetes in large numbers of samples, facilitating joint estimation of the dispersal and infection potentials of plant pathogens in various agro-ecological contexts.

Communication between filamentous pathogens and plants at the biotrophic interface.

Fungi and oomycetes that colonize living plant tissue form extensive interfaces with plant cells in which the cytoplasm of the microorganism is closely aligned with the host cytoplasm for an extended distance. In all cases, specialized biotrophic hyphae function to hijack host cellular processes across an interfacial zone consisting of a hyphal plasma membrane, a specialized interfacial matrix, and a plant-derived membrane. The interface is the site of active secretion by both players. This cross talk at the interface determines the winner in adversarial relationships and establishes the partnership in mutualistic relationships. Fungi and oomycetes secrete many specialized effector proteins for controlling the host, and they can stimulate remarkable cellular reorganization even in distant plant cells. Breakthroughs in live-cell imaging of fungal and oomycete encounter sites, including live-cell imaging of pathogens secreting fluorescently labeled effector proteins, have led to recent progress in understanding communication across the interface.

Confocal microscopy in plant-pathogen interactions.

The development of confocal microscopy and its application to studies of plant-pathogen interactions have revolutionised research into the role of selected molecules and cell components in pathogen infection strategies and plant defence responses. Confocal microscopy allows high-resolution visualisation of a variety of fluorescent and fluorescently tagged molecules in both fixed and living cells, not only in single cells but also in intact tissues. Confocal microscopes greatly improve image quality by reducing interference by out-of-focus light and can capture high-resolution serial optical sections through samples in the z-axis. In combination with a range of computational image analysis techniques, confocal microscopy provides a powerful tool by which molecules, molecular interactions, and cell components can be localised and studied.

Immunolocalization of pathogen effectors.

The use of polyclonal antibodies enables the detection of proteins on a cellular and even subcellular level. Immunolocalization can be used on all pathosystems even if one or both partners of the interaction are unamenable to molecular tools like transformation. This chapter provides detailed information about how to obtain high quality antibodies, how to prepare samples, and finally how to detect the proteins. Methods for light and electron microscopy are presented.

Evidence for the importance of enzymatic digestion of epidermal walls during subepidermal sporulation and pustule opening in white blister rusts (Albuginaceae).

Albugo candida, A. ipomoeae-panduratae, Pustula tragopogonis, Wilsoniana bliti and W. portulacae are widespread obligate biotrophic plant pathogens causing white blister diseases on a variety of flowering plants. Their subepidermal mode of sporulation is unique amongst Oomycetes and leads to blister-like structures on their hosts similar to those produced by true rusts (Uredinales). Unlike in true rusts, sporangia are colourless and produced in chains; the first formed, primary sporangium, differing in size and morphology from subsequent secondary sporangia. According to current interpretations of pustule development the rising pressure of the growing chains of sporangia tear off the epidermal layer from the mesophyll and, in the end, ruptures the epidermis to release the sporangia. This is not convincing considering the rigidity of the epidermal layer and the fact that thin-walled mesophyll cells show no signs of pressure endurance. Our detailed light-, scanning electron-, and transmission electron microscopic observations provide evidence that pustule development and opening are regulated and delicate processes that involve directed enzymatic dissection of host tissue cell walls. The process starts when intercellular hyphae separate the epidermal layer from the parenchyma, forming a cavity in which sporulation takes place. Then thick-walled sporogenous hyphae with club-shaped but thin-walled tips develop and produce sporangia in basipetal succession from the apices of the sporogenous hyphae. The short-living primary sporangia attach tightly to the inner cell walls of the epidermal layer and undergo dramatic cytological changes during pustule maturation, including vacuolisation and development of numerous electron-dense vesicles that might deliver cell wall degrading enzymes. In ripe pustules, the disintegration of areas of epidermal cells leads to the opening of the pustules and to the release of the secondary sporangia. Also the comparison of samples prepared for scanning electron microscopy with fresh pustules, as well as the comparison of the inner epidermal layers detached by the pathogens and detached by force supports our conclusion that delicate enzymatic activity and not force are involved in pustule development and opening by these highly sophisticated pathogens.

Different pathotypes of the sunflower downy mildew pathogen Plasmopara halstedii all contain isometric virionsdagger.

Eight pathotypes of Plasmopara halstedii were screened to investigate the occurrence of virions and the potential viral influence on the pathogenicity of the sunflower downy mildew pathogen. In 23 of 26 P. halstedii isolates derived from eight countries in Europe, North America and South America, virions were detected by transmission electron microscopy. By contrast, there were no ultrastructural indications of virus-like particles in eight other related Oomycetes. The virions of representative P. halstedii isolates were morphologically and biochemically characterized and compared among each other. Regardless of their host's pathotypes, the geographical origin of the isolate and the sensitivity towards the fungicide metalaxyl, the viral characters obtained were uniform. The virions were isometric and measured approximately 37 nm in diameter. One polypeptide of c. 36 kDa and two segments of single-stranded RNA (3.0 and 1.6 kb) were detected. Both viral RNA segments were detected by capillary electrophoresis in the three remaining P. halstedii isolates where virions were undetectable by transmission electron microscopy. Virus-specific primers for the 1.6 kb-segment were synthesized and used to determine and compare a partial sequence of the viral coat protein among virions of different P. halstedii pathotypes. In all tested isolates, fragments of 0.7 kb were amplified which were directly sequenced. Sequence variation was insignificant. As both less aggressive and more aggressive P. halstedii isolates contained virions, the presence or absence of virions could not explain the diverse aggressiveness of the downy mildew pathogen towards sunflower. Moreover, the results indicated that pathogenicity of P. halstedii was not related to variation in morphological or biochemical characters of the virions.

Combresomyces cornifer gen. sp. nov., an endophytic peronosporomycete in Lepidodendron from the Carboniferous of central France.

Structurally preserved periderm of the lycophyte Lepidodendron rhodumnense from the Visean (Mississippian) of central France contains a peronosporomycete (Combresomyces cornifer gen. sp. nov.) that occurs in the form of pyriform to subglobose terminal oogonia. On the surface is a conspicuous ornamentation, which may have formed through condensation of a mucilaginous extra-oogonial wall secretion. Some oogonia contain thin-walled spherules, which may represent (walled) oospheres or spores of an endoparasitic fungus (?chytrid), whereas single, large spheres in the interior are interpreted as oospores. Antheridia adpressed to several of the specimens are clavate and paragynous. This discovery sheds light on the morphology and biology of peronosporomycetes in a terrestrial ecosystem some 330My ago. Although the organism occurs exclusively in the periderm of L. rhodumnense, it is not known whether it represents a symptomless endophyte, pathogen, or saprotroph.

Occurrence and ultrastructure of Albugo candida on a new host, Arabis alpina in Saudi Arabia.

A population of Arabis alpina (Brassicaceae) growing in Saudi Arabia was observed to be infected for the first time by the Oomycete, Albugo candida. Both conventional chemical fixation and high pressure freezing followed by freeze substitution (HPF/FS) were used to prepare zoosporangia, intercellular hyphae, haustoria, invading host cells and host-parasite interface of A. candida for study with both scanning and transmission electron microscopy. Both fixations gave good preservation of ultrastructural details and data from the two sample types were highly complementary. Scanning electron microscopic observation revealed that mature zoosporangia of A. candida are spherical or ellipsoidal in shape and characterized by a smooth surface and faint terminal secession scar at each end. Transmission electron microscopic observation indicated that coenocytic intercellular hyphae are located in intercellular spaces of host leaf tissue forming haustoria in host mesophyll cell. Each haustorium is connected to intercellular hyphae by a narrow, slender neck which enclosed by a collar as a response of host cell to infection. The cytoplasm of the haustorium contains different organelles characteristic of the Oomycetes. No obvious responses are observed in host cell organelles following infection which may be due to the presence of a compatibility between the host and the Oomycete. Modifications of the host plasma membrane around the haustorium are detected. Many tubular elements were found to be continuous with the extrahaustorial membrane. This appears to be the first report of the presence of these tubular elements in case of A. candida haustoria. These tubular elements may increase membrane surface area and consequently increase the efficacy of nutrients uptake by haustoria from host cell.

Taxonomy, molecular phylogeny, and ultrastructural morphology of Olpidiopsis porphyrae sp. nov. (Oomycetes, straminipiles), a unicellular obligate endoparasite of Bangia and Porphyra spp. (Bangiales, Rhodophyta).

Olpidiopsis porphyrae sp. nov., a marine oomycete endoparasite that infects the commercially cultivated red alga Porphyra yezoensis, is described and its phylogenetic position based on molecular data and ultrastructural morphology is discussed. O. porphyrae infects the host Porphyra by means of encysted zoospores. Spherical-shaped holocarpic thalli develop within the cytoplasm of its algal host, which produce monoplanetic, subapically biflagellate zoospores. The characteristic features of this isolate are the ellipsoidal, unicellular thallus and simple holocarpic zoosporangial development, which show morphological similarity with the genus Olpidiopsis. Laboratory infection experiments with a wide range of green, brown, and red algae revealed that O. porphyrae infects several stages of the bangialean red algae (the genera Bangia and Porphyra). Molecular phylogenetic analyses inferred from both SSU rRNA and cox2 genes showed O. porphyrae branched before the main saprolegnian and peronosporalean lineages within the monophyletic oomycete clade, indicating its phylogenetic separation from them. A single or double K-body-like organelle, which contains tubular inclusions, is found located to one side of the zoospore nucleus and shows similarities to homologous organelles previously described in O. saprolegniae. The ultrastructural morphology of O. porphyrae with zoospore initials containing K-bodies and tubular mitochondrial cristae is characteristic of oomycetes. Group I intron-like multiple insertions were found in the SSU rRNA gene of O. porphyrae. This is the first report of SSU group I introns in the class Oomycetes.

The development, ultrastructural cytology, and molecular phylogeny of the basal oomycete Eurychasma dicksonii, infecting the filamentous phaeophyte algae Ectocarpus siliculosus and Pylaiella littoralis.

The morphological development, ultrastructural cytology, and molecular phylogeny of Eurychasma dicksonii, a holocarpic oomycete endoparasite of phaeophyte algae, were investigated in laboratory cultures. Infection of the host algae by E. dicksonii is initiated by an adhesorium-like infection apparatus. First non-walled, the parasite cell developed a cell wall and numerous large vacuoles once it had almost completely filled the infected host cell (foamy stage). Large-scale cytoplasmic changes led to the differentiation of a sporangium with peripheral primary cysts. Secondary zoospores appeared to be liberated from the primary cysts in the internal space left after the peripheral spores differentiated. These zoospores contained two phases of peripheral vesicles, most likely homologous to the dorsal encystment vesicles and K-bodies observed in other oomycetes. Following zoospore liberation the walls of the empty cyst were left behind, forming the so-called net sporangium, a distinctive morphological feature of this genus. The morphological and ultrastructural features of Eurychasma were discussed in relation to similarities with other oomycetes. Both SSU rRNA and COII trees pointed to a basal position of Eurychasma among the Oomycetes. The cox2 sequences also revealed that the UGA codon encoded tryptophan, constituting the first report of stop codon reassignment in an oomycete mitochondrion.

A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death.

Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.

A revision of Plasmopara penniseti, with implications for the host range of the downy mildews with pyriform haustoria.

Plasmopara penniseti is the sole member of the genus Plasmopara parasitic to Poaceae, after the genus Viennotia had been described to accommodate Plasmopara oplismeni. Morphological, ultrastructural, and molecular phylogenetic data indicate that Plasmopara penniseti is not closely related to the generic type, and it is, therefore, transferred to the newly described genus Poakatesthia. The view that the genera of downy mildews with pyriform to vesicular haustoria (Basidiophora, Benua, Bremia, Paraperonospora, Plasmopara, Plasmoverna, and Protobremia) include species parasitic to Poaceae has to be discarded. All of these genera are apparently restricted to dicotyledonous hosts.

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.

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.

Structural and developmental studies of Chlamydomyzium oviparasiticum from Rhabditis nematodes and in culture.

The oomycete (Peronosporomycete) Chlamydomyzium oviparasiticum, previously recorded as a parasite of rotifer eggs, was found infecting Rhabditis nematodes in a sample of rotting garden compost. For the first time C. oviparasiticum was cultured in liquid media, which enabled more detailed studies of zoospore behaviour and facilitated the use of confocal microscopy. Rhabditis nematodes were successfully re-infected from liquid-cultured inoculum. Light (including video) microscopy and transmission electron microscopy were used to document details of thallus development, zoospore release and resting spore morphology to enable comparison with other oomycete species. This species showed several significant saprolegnialian characters such as the 'achlyoid' pattern of spore formation, centrifugal cleavage and structured encystment vesicles. In contrast, spore release into a transient vesicle was a peronosporalean characteristic. The thick-walled resting spores showed relatively poor cytoplasmic preservation and had a thick multi-layered wall. It was still not possible to unequivocably decide whether these were chlamydospores or parthenogenically formed oospores. The phylogenetic significance of these observations is discussed.

A revision of Bremia graminicola.

Bremia graminicola (Chromista, Peronosporales) is a common downy mildew pathogen of Arthraxon spp. (Poaceae) in Central to East Asia and the only species of Bremia parasitic on grasses. Despite its widespread occurrence and apparent differences in host range and morphology compared with other species of the genus, its placement in Bremia has not been challenged for the past 90y. Its current taxonomic position is revised based on sporangiophore morphology and ultrastructure, haustorium morphology, and nu-rDNA sequence analysis. Haustorium morphology and sporangiophore ultrastructure indicate that B. graminicola is not a member of the genus Bremia, which shows affinities to Plasmopara and Paraperonospora. Based on haustorium morphology, B. graminicola appears to be more closely related to Viennotia oplismeni, although the sporangiophore morphology is strikingly different between these two taxa. This is supported by molecular analyses based on a near-representative sample of nuLSU rDNA sequences of downy mildew genera, whereby B. graminicola is revealed as the sister taxon of V. oplismeni with 100 % BS support under all phylogenetic optimality criteria applied. Relationships of this clade to other groups are less clear. However, network and reduced-consensus analyses show that this lack of resolution is mainly due to the ambiguous molecular affinities of Sclerospora graminicola. Omitting this highly divergent taxon results in considerable support for a clade comprised of taxa with globose to pyriform haustoria, including B. lactucae, and for the sister-group relationship of B. graminicola and V. oplismeni with Hyaloperonospora. Consequently, a new genus, Graminivora, is described to accommodate B. graminicola.