Cytology of irregular growth forms of Ophiostoma ulmi and Ophiostoma novo-ulmi growing through millipore filter membranes and sterilized elm wood sections

Abstract

When Ophiostoma ulmi or Ophiostoma novo-ulmi are grown on either 0.22- or 0.45-μm millipore filter membranes placed on impoverished agar medium, the fungus grows through these membranes and takes on various irregular pleomorphic growth forms (P-forms). Links of continuity between these forms and the more regular ones have been shown using light, confocal, and transmission electron microscopy. Tests with labelled probes, such as gold-complexed wheat germ agglutinin for chitin and β-exoglucanase for cellulosic β-1,4-glucans, have indicated that in P-forms deposition of chitin is much altered but is less so in the case of cellulosic glucan. The cytology of these forms compared with the regular fungal ones is also very different, particularly with reference to mitochondria and nuclei. Also, numerous vesiculate structures were noted in the rarely septate P-forms. Similar irregular forms with opaque contents were produced by these fungi when they were grown on sterilized elm wood sections. When these latter samples were fixed by high-pressure freezing, the following main features were noted: fungal cells with a very thin wall, slightly labelled for chitin but more intensely for cellulosic glucans; well-preserved structures, such as plasmalemma and endoplasmic reticulum; and a slightly opaque, fibril-containing extracellular sheath. Differences in labelling for galactose, whether of wall layers or cell contents, were also observed in regular and P-forms. Electron opaque bodies that labelled strongly for galactose were also numerous in P-forms in some samples.Key words: transmission electron microscopy, high-pressure freezing, gold labelling, extracellular sheaths, wall constituents.

Histopathology of Fusarium wilt of staghorn sumac (Rhus typhina) caused by Fusarium oxysporum f. sp. callistephi race 3. II. Characteristics of opaque matter associated with extensive host cell and cell wall alterations

“…The present observations reinforce the hypothesis, as already expressed for other systems and supported by similar observations (Ouellette and Baayen 2000;Ouellette and Rioux 1993;Ouellette et al 1995Ouellette et al , 1999aOuellette et al , 2004aOuellette et al , 2004bOuellette et al , 2004cOuellette et al , 2004d, that the OM occurrence primarily relates to the pathogen, as: 1) OM, including that of the VWLM, was also connected to fungal cells, and often by means of microfilaments (see also Atstatt 2003, showing some endophyte-like fungal cells with their walls pervaded by similar filamenatous structures); and 2) close similarities noted between OM present in host walls or in periplasmic areas, that of the VWLM, and content of some fungal cells and elements (compare Figs. 6d,7d,8c and element in Fig.…”

“…Similar structures often pervaded fungal walls and extracellular matter, and then seemed to stretch freely over void spaces or into the host cell walls. A similar association was equally observed in other fungal wilt pathogens (Nicole et al 1994;Ouellette et al 1995Ouellette et al , 1999bOuellette et al , 2002.…”

“…These ranged from peculiar breakdown of pit membranes of bordered pits, degradation of parenchyma cell walls, pervasion and alterations of vessel secondary walls by matter extending from their lining material and so on. These observations paralleled some of those made regarding other wilt diseases, in particular of elms infected by Ophiostoma novo-ulmi Brasier (Ouellette 1978;Ouellette andRioux 1992, 1993;Ouellette et al 1995Ouellette et al , 1999aOuellette et al , 2004a and of eggplant infected by Verticillium dahliae Kleb. (unpublished observations).…”

Tissue invasion and alteration in eggplant infected with Verticillium dahliae: A light and transmission electron microscopy study

“…Tests to detect cellulose and chitin were carried out in 1992 using the 1972 embedded samples and using colloidal gold-complexed probes as described in Ouellette et al (1995). Briefly, in tests for ␤-1,4-glucans, ultrathin sections were first floated on a drop of phosphate buffer saline (PBS) containing 0.02% polyethylene glycol (PEG) 20 000 (pH 6.5) for 5 min, then for 30 min on a drop of a gold-complexed exoglucanase (EC 3.2.1.91), rinsed with PBS and distilled water, and stained as mentioned above.…”

“…We have already described particular modes of pathogen development, of tissue alterations, of host cell reactions and related aspects concerning Dutch elm disease and Fusarium wilts of carnation, tomato and staghorn sumac (Charest et al 2004;Ouellette 1978b;Ouellette et al 1995Ouellette et al , 1999Ouellette et al , 2004aOuellette et al , 2004bOuellette et al , 2004cOuellette et al , 2004dOuellette et al , 2004e, 2005aOuellette et al , 2005b. These works present peculiar characteristics of pathogen structures, of host cell wall and content alterations related to extensive tissue invasion by opaque matter, not ascribable to a host origin, of various host cell reactions and so on, and more than circumstantial evidence was presented to show that most of the material lining vessel walls did not always correspond to the notions of coating as supported by others.…”