Antibacterial activity of geraniin from sugar maple leaves: an ultrastructural study with the phytopathogen Xanthomonas campestris pv. vitians.

Effect of geraniin extracted from sugar maple (Acer saccharum) leaves on the viability of the phytopathogen Xanthomonas campestris pv. vitians was evaluated with the SYTOX Green nucleic acid stain, penetrating only compromised membranes, and plate counts. In parallel, structural changes of treated bacteria were examined in transmission electron microscopy (TEM). Based on SYTOX Green and plate counts, geraniin at the minimum bactericidal concentration (3.125 mg/ml) increased mortality after 45 min by 37% and 62%, respectively, when compared with controls. According to observations in TEM, geraniin caused morphological alterations of these rod-shaped bacteria, including degradation of their envelopes, as also suggested by the incorporation of SYTOX. These alterations were often accompanied by cytoplasm leakage and the formation of more pronounced whitish areas in the cytoplasm similar to vacuolization. Moreover, multi-membranous and/or -wall systems were at times formed in the treated bacteria. The presence of some extracellular electron-dense material was frequently noted around the treated bacteria. The matrix surrounding control bacteria tended to disappear after geraniin treatment. This study highlights for the first time the effect of geraniin on bacterial ultrastructure, thus contributing to a better understanding of the mechanism by which this molecule exerts antibacterial activity.

First Extensive Microscopic Study of Butternut Defense Mechanisms Following Inoculation with the Canker Pathogen Ophiognomonia clavigignenti-juglandacearum Reveals Compartmentalization of Tissue Damage.

Ophiognomonia clavigignenti-juglandacearum endangers the survival of butternut (Juglans cinerea) throughout its native range. While screening for disease resistance, we found that artificial inoculations of 48 butternut seedlings with O. clavigignenti-juglandacearum induced the expression of external symptoms, but only after a period of dormancy. Before dormancy, compartmentalized tissues such as necrophylactic periderms (NPs) and xylem reaction zones (RZs) contributed to limiting pathogen invasion. Phenols were regularly detected in RZs, often in continuity with NPs during wound closure, and confocal microscopy revealed their presence in parenchyma cells, vessel plugs and cell walls. Vessels were blocked with tyloses and gels, particularly those present in RZs. Suberin was also detected in cells formed over the affected xylem by the callus at the inoculation point, in a few tylosis walls, and in longitudinal tubes that formed near NPs. Following dormancy, in all inoculated seedlings but one, defensive barriers were breached by O. clavigignenti-juglandacearum and then additional ones were produced in response to this new invasion. The results of this histopathological study indicate that trees inoculated in selection programs to test butternut canker resistance should go through at least one period of dormancy and that asymptomatic individuals should be dissected to better assess how they defend themselves against O. clavigignenti-juglandacearum.

Factors Influencing the Regional Dynamics of Butternut Canker.

Butternut (Juglans cinerea) is an important component of native biodiversity in eastern North America. Of urgent concern is the survival of butternut, whose populations are declining rapidly, in large part due to an exotic pathogen, Ophiognomonia clavigignenti-juglandacearum, that causes butternut canker. The disease presently occurs throughout the range of butternut in North America, causing branch and stem cankers, dieback, and tree mortality. Despite the existential threat posed by O. clavigignenti-juglandacearum to butternut, a detailed understanding of the factors that drive cross-scale disease patterns is lacking. Therefore, we investigated the association of a range of factors, including tree attributes, topography, and weather, with butternut canker spatial dynamics at different scales using data collected in the province of Quebec, Canada. Trunk canker damage and dieback showed distinct geographic patterns. Bark phenotype was not significantly associated with trunk canker damage. Results suggest that open or dominant trees may show less dieback than intermediate or suppressed trees. Probability of the presence of trunk canker and percent dieback were proportional to the tree diameter at breast height. Temperature was positively associated with disease severity at a 1-km2 scale. Our results provide strong evidence that multiple factors, notably weather, influence butternut canker epidemiology.

An Overview of Canadian Research Activities on Diseases Caused by Phytophthora ramorum: Results, Progress, and Challenges.

International trade and travel are the driving forces behind the spread of invasive plant pathogens around the world, and human-mediated movement of plants and plant products is now generally accepted as the primary mode of their introduction, resulting in huge disturbance to ecosystems and severe socio-economic impact. These problems are exacerbated under the present conditions of rapid climatic change. We report an overview of the Canadian research activities on Phytophthora ramorum. Since the first discovery and subsequent eradication of P. ramorum on infected ornamentals in nurseries in Vancouver, British Columbia, in 2003, a research team of Canadian government scientists representing the Canadian Forest Service, Canadian Food Inspection Agency, and Agriculture and Agri-Food Canada worked together over a 10-year period and have significantly contributed to many aspects of research and risk assessment on this pathogen. The overall objectives of the Canadian research efforts were to gain a better understanding of the molecular diagnostics of P. ramorum, its biology, host-pathogen interactions, and management options. With this information, it was possible to develop pest risk assessments and evaluate the environmental and economic impact and future research needs and challenges relevant to P. ramorum and other emerging forest Phytophthora spp.

Variation in stem morphology and movement of amyloplasts in white spruce grown in the weightless environment of the International Space Station.

One-year-old white spruce (Picea glauca) seedlings were studied in microgravity conditions in the International Space Station (ISS) and compared with seedlings grown on Earth. Leaf growth was clearly stimulated in space whereas data suggest a similar trend for the shoots. Needles on the current shoots of ground-based seedlings were more inclined towards the stem base than those of seedlings grown in the ISS. Amyloplasts sedimented in specialized cells of shoots and roots in seedlings grown on Earth while they were distributed at random in similar cells of seedlings tested in the ISS. In shoots, such amyloplasts were found in starch sheath cells located between leaf traces and cortical cells whereas in roots they were constituents of columella cells of the cap. Nuclei were regularly observed just above the sedimented amyloplasts in both organs. It was also frequent to detect vacuoles with phenolic compounds and endoplasmic reticulum (ER) close to the sedimented amyloplasts. The ER was mainly observed just under these amyloplasts. Thus, when amyloplasts sediment, the pressure exerted on the ER, the organelle that can for instance secrete proteins destined for the plasma membrane, might influence their functioning and play a role in signaling pathways involved in gravity-sensing white spruce cells.

Fungal colonization and host defense reactions in Ulmus americana callus cultures inoculated with Ophiostoma novo-ulmi.

The host-pathogen interaction leading to Dutch elm disease was analyzed using histo- and cyto-chemical tests in an in vitro system. Friable and hard susceptible Ulmus americana callus cultures were inoculated with the highly aggressive pathogen Ophiostoma novo-ulmi. Inoculated callus tissues were compared with water-treated callus tissues and studied with light microscopy (LM), transmission-electron microscopy (TEM), and scanning-electron microscopy (SEM). New aspects of this interaction are described. These include the histological observation, for the first time in plant callus cultures, of suberin with its typical lamellar structure in TEM and the intracellular presence of O. novo-ulmi. Expression of the phenylalanine ammonia lyase gene, monitored by real-time quantitative polymerase chain reaction, was correlated with the accumulation of suberin, phenols, and lignin in infected callus cultures. This study validates the potential use of the in vitro system for genomic analyses aimed at identifying genes expressed during the interaction in the Dutch elm disease pathosystem.

The vegetative life-cycle of the clover pathogen Cymadothea trifolii as revealed by transmission electron microscopy.

The vegetative life-cycle of Cymadothea trifolii (anamorph Polythrincium trifolii), causing sooty blotch of clover, is described using chemically as well as cryofixed and freeze-substituted samples. The pathogen enters the leaf through stomata and proliferates intercellularly. Nutrients are assumedly obtained via an interaction apparatus produced within the pathogen's hyphae, opposite to which the host cell is triggered to invaginate its plasmalemma. Rare attempts of 'self-parasitism' were also seen. Entering the conidial stage, stromata are laid down under the lower epidermis. The dying tissue above may explain the necrotic spots observed on infected leaflets. Foot cells in the conidial stromata produce thick-walled conidiophores, which grow sympodially. New conidiophores may grow into empty shells of old ones. Conidia are detached after pores between them and conidiophores have become plugged by organelles resembling Woronin bodies. Conidia are usually two-celled and their walls contain chitin and beta-1,3-glucans as indicated by labelling with gold-conjugated wheat germ agglutinin and anti-beta-1,3-glucan antibodies. Both conidiophores and conidia contain a structure which we regard as a new organelle with as yet unknown function.

The intercellular biotrophic leaf pathogen Cymadothea trifolii locally degrades pectins, but not cellulose or xyloglucan in cell walls of Trifolium repens.

The intercellular ascomycetous pathogen Cymadothea trifolii, causing sooty blotch of clover, proliferates within leaves of Trifolium spp. and produces a complex structure called interaction apparatus (IA) in its own hyphae. Opposite the IA the plant plasmalemma invaginates to form a bubble. Both structures are connected by a tube with an electron-dense sheath. Using immunocytochemistry on high-pressure frozen and freeze-substituted samples, we examined several plant and fungal cell wall components, including those in new host wall appositions at the interaction site, as well as a fungal polygalacturonase. Within the tube linking IA and host bubble, labelling was obtained for cellulose and xyloglucan but not for rhamnogalacturonan-I and homogalacturonans. The IA labelled for chitin and beta-1,3-glucans, and for a fungal polygalacturonase. Plant wall appositions reacted with antibodies against callose, xyloglucans and rhamnogalacturonan-I. Cymadothea trifolii partly degrades the host cell wall. Structural elements remain intact, but the pectin matrix is dissolved. A fungal polygalacturonase detected in the IA is probably a key factor in this process. Owing to the presence of chitin and beta-1,3-glucans, the IA itself is considered an apoplastic compartment.

Ultrastructural alterations of Erwinia carotovora subsp. atroseptica caused by treatment with aluminum chloride and sodium metabisulfite.

Aluminum and bisulfite salts inhibit the growth of several fungi and bacteria, and their application effectively controls potato soft rot caused by Erwinia carotovora. In an effort to understand their inhibitory action, ultrastructural changes in Erwinia carotovora subsp. atroseptica after exposure (0 to 20 min) to different concentrations (0.05, 0.1, and 0.2 M) of these salts were examined by using transmission electron microscopy. Plasma membrane integrity was evaluated by using the SYTOX Green fluorochrome that penetrates only cells with altered membranes. Bacteria exposed to all aluminum chloride concentrations, especially 0.2 M, exhibited loosening of the cell walls, cell wall rupture, cytoplasmic aggregation, and an absence of extracellular vesicles. Sodium metabisulfite caused mainly a retraction of plasma membrane and cellular voids which were more pronounced with increasing concentration. Bacterial mortality was closely associated with SYTOX stain absorption when bacteria were exposed to either a high concentration (0.2 M) of aluminum chloride or prolonged exposure (20 min) to 0.05 M aluminum chloride or to a pH of 2.5. Bacteria exposed to lower concentrations of aluminum chloride (0.05 and 0.1 M) for 10 min or less, or to metabisulfite at all concentrations, did not exhibit significant stain absorption, suggesting that no membrane damage occurred or it was too weak to allow the penetration of the stain into the cell. While mortality caused by aluminum chloride involves membrane damage and subsequent cytoplasmic aggregation, sulfite exerts its effect intracellularly; it is transported across the membrane by free diffusion of molecular SO2 with little damage to the cellular membrane.

Ultrastructure of the infection process of potato tuber by Helminthosporium solani, causal agent of potato silver scurf.

Silver scurf is an important postharvest disease affecting potato tubers worldwide, caused by Helminthosporium solani. In the present study, key steps of infection of potato tubers (cv. 'Dark Red Norland') by H. solani were described using transmission (TEM) and scanning electron microscopy (SEM). The fungus entered potato tubers mainly via hyphae, although germ tubes were also able to directly penetrate the tubers. An extracellular sheath was observed around hyphae growing over the surface of tubers and the host cell wall appeared lyzed at the point of penetration. Observations suggested that both mechanical and enzymatic processes are involved in periderm penetration. Hyphae of H. solani, 9 h after tuber inoculation, were present intracellularly mostly in the periderm and in some cortical cells. Two days after inoculation, host cells were invaded and both infected and neighbouring host cells showed signs of necrosis (disrupted cytoplasm, absence of typical organelles or endomembrane systems, collapsed peridermal cells) that were not observed in healthy control tubers. Four days after inoculation, completing the infection cycle, conidiophores emerged from peridermal cells directly by erupting through the host cell walls.