Danny Rioux

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

ABSTRACT 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.

Suberized tyloses in trees : an ultrastructural and cytochemical study

The nature of the wall layers observed in suberized tyloses was studied in Populus basalmifera L., Ulmus americana L. and Quercus rubra L. As the suberin layers were present only in tyloses that had completed their expansion, most of the results concern mature tyloses. The cyto- and immunocytochemical tests were conducted, respectively, with an exoglucanase having a binding affinity for β(1→4)-D-glucans, the subunits of cellulose, and with two monoclonal antibodies specific for un-esterified and esterified pectic molecules. In the three species, labelling for pectic compounds was intense over the external layer of tyloses but usually more dispersed or nearly absent over the layer corresponding to a primary wall that was, however, intensely labelled for β(1→4)-D-glucans. The outer wall layer, comparable to a middle lamella in mature tyloses, was continuous with similar material that appeared to be secreted by the tylosis. This material was particularly abundant in pit chambers, in void spaces between the tylosis and the vessel wall, particularly at the junction of the vessel and two adjacent cells, and close to the rim of vessel perforation plates. In P. balsamifera, a single suberized layer or occasionally a succession of suberized and cellulose-containing layers was observed internal to the tylosis primary wall. In U. americana, the wall of tylosis was similar to that of P. balsamifera except that, at times, a secondary-wall-like layer was formed and only a single suberized layer was observed. In Q. rubra, the suberized layer was always observed internal to the tylosis secondary wall. Simple pits were also constantly noted in Q. rubra tyloses. The occasional occurrence of a cellulosic layer internally to the suberized layer was observed in the three species. Histochemical tests revealed that lignin was also an important component of the tylosis wall. The tyloses frequently contained phenolic compounds in close association with the suberized layers. The significance of the formation of suberized tyloses in trees is discussed.

Anatomical and Physiological Aspects of Resistance to Dutch Elm Disease

Most elm species (Ulmus spp.), considered by many to be the most beautiful urban trees, are well adapted to the numerous stressful conditions in cities. Their major shortcoming, however, is their susceptibility to Dutch elm disease (DED). Although millions of elms have died since the first observation of DED about 70 years ago in Europe, they are not verging on extinction because they have a great capacity for regeneration, which explains why large elms affected by DED in woodland areas are gradually replaced by younger and smaller elm trees.

Ultrastructural and cytochemical studies of host and pathogens in some fungal wilt diseases: retro- and introspection towards a better understanding of DED

This report presents a survey of previous and more recent ultrastructural and cytochemical investigations of disease development in elm, caused by Ophiostoma novo-ulmi, with results of some comparative studies of other wilt diseases caused by f.spp., of Fusarium oxysporum and of Verticillium sp. For cytochemical studies, probes complexed to colloidal gold to detect cellulose, pectin, chitin, and DNA were used. Thus, the formation of tyloses, pit membrane alterations, and the disease effect on parenchyma cells in mature as well as in young tissue were characterized. Vessel lumina in these plants, in diverse situations, contained heterogeneous matter, among which occurred masses of opaque matter and in certain instances pectin fibrils secreted by parenchyma cells and tyloses. Numerous globoid, opaque bodies of regular sizes, which have been mostly overlooked previously, abounded in vessel elements of U. americana and U. glabra, including trees injected with glucose solutions prior to inoculation. Coarser fibrillar material was also noted in vessel lumina, but mostly shortly after inoculation. Other peculiar forms of O. novo-ulmi are also described. The possible role of the components under study is discussed, and a model for DED is proposed in which hindrance to vessel invasion, including downward spread of the pathogen, and reactions of parenchyma cells are complementary and considered to be conducive to defence mechanisms, including compartmentalization of the invaded xylem.

Formation of Ligno-Suberized Tissues in Jack Pine Resistant to the European Race of Gremmeniella abietina.

ABSTRACT A histological study was conducted to provide insights into the defense mechanisms of Pinus banksiana resistant to the European (EU) race of Gremmeniella abietina in naturally infected sites. At the time of sampling, the only apparent symptom was a blight induced at the tip of the shoots. The identity of G. abietina during microscopic examinations was confirmed by an immunogold labeling method. Once the fungus had succeeded in penetrating the bracts through stomata, it invaded the stem cortex and the phloem cells and attained the vascular cambium. The progression of the pathogen to the pith was possible principally through intense colonization of needle traces but also by the invasion of the rays. Ligno-suberized tissues confining the pathogen within the necrotic area were revealed by histochemical tests. Well-defined boundaries were initiated at the base of healthy needles and at the vascular cambium level. They regularly formed one continuous suberized barrier completely crossing the shoot from one needle to the other. A nonlamellar form of suberin was observed in transmission electron microscopy. Restoration of cambial activities and tissue regeneration following necrophylactic periderm formation were suggested as essential factors in the defense system of P. banksiana against the EU race of G. abietina. To our knowledge, this is the first demonstration of an anatomical defense mechanism of a conifer against Scleroderris canker.

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.

Ultrastructural Alterations in Fusarium sambucinum and Heterobasidion annosum Treated with Aluminum Chloride and Sodium Metabisulfite

Aluminum chloride (AlCl(3)) and sodium metabisulfite (Na(2)S(2)O(5)) have received increasing attention as antifungal agents for the control of plant diseases. In an effort to understand their toxic action on fungi, ultrastructural changes and membrane damage in Fusarium sambucinum (Ascomycota) and Heterobasidion annosum (Basidiomycota) in response to salt exposure was investigated using transmission electron microscopy. Conidial membrane damage was quantified using SYTOX Green stain, which only enters altered membranes. The results showed that mortality of the conidia was generally closely associated with SYTOX stain absorption in F. sambucinum treated with Na(2)S(2)O(5) and in H. annosum treated with AlCl(3) or Na(2)S(2)O(5), suggesting that these salts cause membrane alterations. For both fungi, ultrastructural alterations in conidia treated with AlCl(3) and Na(2)S(2)O(5) included membrane retraction, undulation, and invagination. At higher concentrations or exposure periods to the salts, loss of membrane integrity, cytoplasmic leakage, and cell rupture were observed. Ultrastructural alterations and increased SYTOX stain absorption in salt-treated conidia appear consistent with a mode of action where AlCl(3) and Na(2)S(2)O(5) alter membrane integrity and permeability.

Alterations of Vessel Elements and Reactions of Surrounding Tissues in the DED Syndrome

A summary of the principal observations accumulated by the senior author and colleagues since 1970, concerning the histopathology of DED, is presented. An overview of events occurring in disease development are exposed such as: invasion of secondary vessel elements and accumulation therein of extraneous material (including the alveolar network), attack of vessel walls (in association with masses of electron opaque material) and surrounding tissues up to the cambial area, pathogen action on proto- and metaxylem tissues, and similar observations of differentiating tissues, where cell malformation is often pronounced. Various types of cytoplasmic reactions in the axial parenchyma of secondary xylem, are mentioned. Some of these, considered detrimental, range from a high vacuolization of the cytoplasm, of pronounced organelle alterations up to complete cytoplasmic disintegration; other reactions may be considered of a defense type: increased opacity and density of the cytoplasm, accumulation of large amounts of opaque or more translucent matter in large vacuoles, and so on. These are ordinarily accompanied by wall modifications, such as an increased density and opacity of the protective layer. Intermediate situations exist. Wall modifications in parenchyma cells and fibers in normal wood or in barrier zones are thought to be associated with trees showing some degree of resistance or having partially recovered from acute symptoms. As a corollary, susceptibility can be associated with a lack of such reactions, particularly in shoot and twigs where tissue disturbances seem to be most prominent. It is stressed that all the disturbances observed are associated with the presence of masses or strands of opaque matter. These observations point to the necessity of characterizing further these opaque constituents, or at least recognizing that a problem exists that calls for the formulation of new concepts.

A suberized perimedullary reaction zone in Populus balsamifera novel for compartmentalization in trees

Abstract Following artificial inoculation of nonhost Populus balsamifera with Ophiostoma ulmi, structural defensive tissues were formed in the xylem. Among these tissues there was a perimedullary sheath of cells, located adjacent to the invaded xylem, that originated from the dedifferentiation of perimedullary and xylem parenchyma cells. Histochemical tests revealed that this sheath was intensively suberized. A band of lignified cells was frequently detected on both sides of this suberized tissue. The formation of such a tissue at the pith margin represents a new type of anatomical barrier in relation to compartmentalization processes described for trees. Ultrastructural examination showed that the wall of cells forming this zone was generally composed of a compound middle lamella, a suberized secondary wall and a tertiary wall layer. Using colloidal gold conjugated to monoclonal antibodies against pectin and to an exoglucanase for cellulose, only limited labelling was obtained for pectin whereas labelling for cellulose was abundant in the compound middle lamella and the tertiary wall layer. In a few fibres close to this suberized zone, the latter probe also made it possible to distinguish the occasional presence of several alternating wall layers mainly composed of either suberin or cellulose. In Salix sp., another tree species belonging to the Salicaceae, this type of suberized reaction zone was also observed. The new reaction zone is similar in structure and location to a suberized barrier formed in nonwoody carnation (Dianthus caryophyllus) plants in the defense against vascular fungi.

Ultrastructure of the alveolar network and its relation to coating on vessel walls in elms infected by Ophiostoma novo-ulmi and in other plants affected with similar wilt diseases

En olmos afectados por la grafiosis, la red alveolar, demarcada por bandas filamentosas, y confluente con acumulaciones de la misma sustancia (cubricion) presentes en las paredes de los vasos, aparece regularmente en los elementos conductores. Sustancias similares tapizan las paredes de los vasos en secciones de madera delgada de olmo inoculada y esterilizada, y posteriormente criofijadas a altas presiones. Se observo que la cubricion se conecta con las celulas del micelio y que ocasionalmente contenia pequenas particulas opacas del tamano de los ribosomas, estructuras membranosas y vesiculares, asi como que, tras la incubacion de astillas lenosas cogidas de muestras enfermas incubadas en agar, aun se presentaba una sustancia similar. La cubricion y las bandas alveolares aumentaron su espesor en la confluencia con otras bandas o estructuras membranosas. Estructuras y sustancias similares aparecieron tambien en otras plantas afectadas por enfermedades similares originadas por hongos que producen marchitamiento. En todos los sistemas, la cubricion compacta no pudo ser marcada como quitina, celulosa ni pectina. En zumaque (Rhus typhina), la sonda de ADN se pego a la cubricion. En resumen, a la vista de estos datos, parece ser que la cubricion y la red alveolar no estan formados por componentes inertes, un hecho que indica su probable origen patogenico. Se sugiere que esos elementos podrian ser importantes no solo en las fases iniciales de la infeccion, sino tambien, en infecciones mas desarrolladas o recurrentes, en el momento en que los mecanismos de resistencia del hospedante no son efectivos.