Dennis Pitt

Production of a monoclonal antibody specific to the genus Trichoderma and closely related fungi, and its use to detect Trichoderma spp. in naturally infested composts.

Studies of the interactions between hyperparasitic fungi and their hosts are severely hampered by the absence of methods that allow the unambiguous identification of individual genera in complex environments that contain mixed populations of fungi, such as soil or compost. This study details the development of a monoclonal antibody (MF2) that allows the detection and recovery of Trichoderma spp. in naturally infested composts, and the visualization of hyperparasitic strains of Trichoderma during antagonistic interactions with their hosts. Murine monoclonal antibody MF2, of immunoglobulin class M (IgM), was raised against a protein epitope of a glycoprotein antigen(s) specific for species of the genus Trichoderma and for the closely related fungi Gliocladium viride, Hypomyces chrysospermus, Sphaerostilbella spp. and Hypocrea spp. MF2 did not react with antigens from Gliocladium catenulatum, Gliocladium roseum, Nectria ochroleuca and Clonostachys spp., nor with a range of unrelated soil- and compost-borne fungi. Extracellular production of the MF2 antigen was constitutive. Western-blotting analysis showed that MF2 bound to a ladder of proteins with apparent molecular masses in the range 35-200 kDa. Immunofluorescence studies showed that MF2 bound strongly to the cell walls of hyphae and phialides and the intercalary and terminal chlamydospores of Trichoderma spp., whereas immunogold electron microscopy revealed strong binding of MF2 to the cell walls and septa of hyphae and to the cell walls of phialoconidia. In immunofluorescence studies of dual cultures of Trichoderma and Rhizoctonia solani, only the cell walls of the hyperparasite, which coiled around the host, were stained by MF2. The specificity of MF2 enabled the development of a combined baiting-ELISA technique for the detection of Trichoderma spp. in naturally infested composts. The specificity of this technique was confirmed by phylogenetic analysis based on sequences of the ITS1-5.8S-ITS2 rRNA-encoding regions of the isolates.

Effects of dimethomorph on the morphology and ultrastructure of Phytophthora

Dimethomorph at a concentration of 1·0 μ m had profound effects on the morphology and ultrastructure of Phytophthora. The principal changes in ultrastructure were associated with extensive proliferation and aberrant deposition of cell-wall material. In actively growing regions of P. infestans, abnormal wall ingrowths (pegs) were laid down within 2 h exposure, some giving rise to false septa that occluded hyphae. The location of false septa in this species and in P. erythroseptica and P. cactorum coincided with the constrictions in hyphae that gave treated colonies a beaded morphology. Altered wall biogenesis was also manifested as thickening and in the development of more than one cell wall layer. Aberrant wall depositions contained ground and membranous cytoplasmic inclusions. Other ultrastructural changes induced by dimethomorph were the accumulation of fingerprint vacuoles, dense bodies and membrane debris (whorls). It is concluded that in the presence of dimethomorph there is loss of control of the biochemical processes involved in normal cell wall biogenesis.

Histochemical Demonstration of Certain Hydrolytic Enzymes within Cytoplasmic Particles of Botrytis cinerea Fr.

SUMMARY: Established histochemical methods were used to locate the activities of several acid and neutral hydrolases within cytoplasmic particles of Botrytis cinerea Fr. The Gomori procedure revealed acid phosphatase activity and this was used as a marker to show that these particles in fresh material were inactive until subjected to treatments which affected the permeability of lipid-protein membranes. This behaviour was interpreted as indicating that these particles may be comparable with the lysosomes of animal cells. It was shown that acid phosphatase, acid deoxyribonuclease II, β-galactosidase and several esterases were localized within these particles. Attempts to demonstrate β-D-glucuronidase activity were unsuccessful; aryl sulphatase activity was weak.

Histochemical and Ultrastructural Characterization of Vacuoles and Spherosomes as Components of the Lytic System in Hyphae of the Fungus Botrytis cinerea

An integrated approach to acid phosphatase (EC 3.1.3.2) histochemistry by the azo-dye and lead-capture (‘Gomori’) methods in phosphate-starved hyphae of the fungus Botrytis cinerea revealed strikingly different patterns of localization of activity staining. Reaction product formed with the azo-dye method was found in numerous small organelles (<;0.5 µm diameter), which also accumulated the lipophilic dye Nile Red and mislocalized the formazan indicating mitochondrial succinate dehydrogenase activity. Such small organelles were stained only weakly and sporadically with the lead-capture method; instead, lead phosphate deposits were produced mainly in large vacuoles (up to 2.5 µm diam.), similar to those accumulating the vital dye Neutral Red. Additionally, acid phosphatase activity was detected in apical secretory vesicles with the lead-capture method but not with the azo-dye method. Ultrastructural studies by transmission electron microscopy confirmed the presence of large vacuoles which showed evidence of autophagic activity, and of small moderately osmiophilic organelles. The latter are considered to be spherosomes rather than lysosomes because of their weak reaction with the lead-capture method and their high lipid content. It is suggested that their apparently strong reaction with the azo-dye method is caused partly by false localization due to the lipophilic nature of the reaction product.

Purification of a phospholipase from Botrytis and its effects on plant tissues

Abstract A phospholipase from Botrytis cinerea , grown in pure culture, was purified more than 1000-fold. Whilst it possessed no acyl hydrolase activity toward a variety of p -nitrophenyl fatty acyl derivatives, phosphatidyl choline (lecithin) acted as a substrate; the enzyme being of either type ‘A’ or ‘B’ specificity. When the purified enzyme was applied to washed beetroot discs, betacyanin leakage was induced. Loss of substances which absorb at 260 nm also occurred when washed potato tuber discs were incubated with the enzyme. Incubation with a lysosome-enriched fraction from potato sprout tissues resulted in increased acid phosphatase activity in the incubation supernatant. The phospholipase had no macerating effect on a variety of plant tissues, nor did it cause disruption of isolated protoplasts from these tissues. Studies with mitochondria from mung beans revealed no effects of the enzyme upon the respiratory activity of these organelles. The result suggested that a major site of action of the B.cinerea phospholipase was the lysosomes.

Purification and physiological properties of two lipolytic enzymes of Solanum tuberosum

Abstract Two lipolytic enzymes have been separated and partially purified from potato tubers. One enzyme of higher isoelectric value, possessed acyl hydrolase activity toward a number of p -nitrophenyl fatty acyl derivatives, the relative activity depending on the fatty acyl chain length. There was also some activity towards phosphatidyl choline. The other enzyme possessed phospholipase and galactolipase activity, but showed a low acyl hydrolase activity towards p -nitrophenyl fatty acyl derivatives. When applied to plant tissues, the enzyme with the greater acyl hydrolase activity caused rapid ion efflux from discs of potato tuber and beetroot, foflowed by reabsorption of ions by the tissues. The purified phospholipase did not produce this effect but induced acid phosphatase leakage from lysosome-enriched fractions of potato sprout tissue. No maceration of tissue or protoplast disruption was observed when either of the two enzymes were incubated with a variety of plant preparations.

Purification, characterization and exit routes of two acid phosphatases secreted by Botrytis cinerea

The two forms of acid phosphatase secreted by Botrytis cinerea were purified, characterized and found to possess fundamentally different properties. The phosphate-repressible acid phosphatase was a monomer of clearly defined molecular weight (55–56 kDa), with a polypeptide component of 43 kDa. The enzyme was secreted through the hyphal, apex and accumulated in the culture fluid. Its high affinity for a wide range of substrates rendered it suitable for a putative role as an extracellular phosphate-scavenging enzyme. Secretion of a constitutive acid phosphatase occurred at low levels in phosphate-enriched culture; at phosphate starvation, the activity of this enzyme was enhanced ten-fold in the culture fluid and two-fold in the cell wall. The constitutive enzyme had strikingly different properties from those of the repressible form. It consisted of a 93 kDa polypeptide which was heavily glycosylated such that the secreted protein displayed size heterogeneity of 140–200 kDa by SDS-polyacrylamide gel electrophoresis and 320–450 kDa by native Sephacryl S-300 chromatography. Irrespective of the phosphate status, most of the secreted constitutive enzyme activity was retained by the cell wall. These results were consistent with ultrastructural histochemical observations which suggested that this enzyme form was secreted by a novel pathway via the fusion of vacuoles with the plasma membrane in mature hyphal segments.

Acid phosphatase secretion by Botrytis cinerea

Botrytis cinerea secreted low levels of a constitutive acid phosphatase into liquid culture when grown in the condition of phosphate starvation or in the presence of high concentrations (4 MM) of organic phosphate esters or inorganic phosphate. During phosphate starvation the overall acid phosphatase activity secreted into the culture fluid was increased up to 80-fold due to the appearance of a second (phosphate-repressible) enzyme form. On native polyacrylamide electrophoresis gels, the constitutive enzyme displayed size heterogeneity, whereas the repressible enzyme was resolved as a discrete band. Both forms of the enzyme reacted with p -nitrophenylphosphate under such conditions, but only the constitutive form was found to hydrolyse β-glycerophosphate. Histochemical staining with these two substrates with the Gomori lead-capture method revealed significant staining of secretory vesicles in the hyphal tip region only in phosphate-starved mycelium with p -nitrophenylphosphate as the substrate. By contrast, vacuolar structures were stained with both substrates under conditions of phosphate starvation as well as abundance of inorganic phosphate. The implications of these observations for the likely exit route of the two acid phosphatase forms are discussed.

The antifungal properties of Minimedusa polyspora

Colonies of Fusarium oxysporum f. sp. narcissi in dual culture with Minimedusa polyspora were overgrown, with the contents of the hyphae showing coagulation and vacuolation followed by cell death. Lysis ensued after development of numerous small holes in the hyphae followed by rapid disintegration of the walls. Minimedusa also directly entwined hyphae of F. oxysporum by means of short side branches which developed appressorium-like attachment structures and wall penetration pegs. Whilst hyphae of Minimedusa were not seen to grow intracellularly they were observed within the remains of dead hyphae of Fusarium. Neutral red staining indicated that Fusarium hyphae were apparently killed prior to contact between the two fungi. Bioautography and NMR studies on culture filtrates of Minimedusa revealed five antifungal triene compounds, of which one was a polyacetylene component, thereby providing further evidence for antibiosis as the primary mechanism of antagonism.

Histochemical and ultrastructural characterization of fungal mitochondria

This report demonstrates the striking variability of mitochondria within and between different species of fungi from all major taxa, i.e., Oomycetes ( Phytophthora erythroseptica ), Zygomycotina ( Basidiobolus ranarum, Mucor mucedo ), Ascomycotina ( Sordaria fimicola ), Deuteromycotina ( Botrytis cinerea, Fusarium culmorum ) and Basidiomycotina ( Schizophyllum commune ). For light microscopy (LM), a simple procedure was used to highlight mitochondria in living hyphae. They appeared filamentous, sometimes exceeding 25 m in length, and were occasionally branched. Ultrastructural features were investigated by transmission electron microscopy (TEM) which revealed that mitochondria of P. erythroseptica and B. ranarum had tubular cristae whereas the cristae in all other species were lamellate. The value of mitochondrial ultrastructure as a tool for fungal phylogenetic analysis is discussed.