The Role of Extracellular Melanoproteins of Venturia inaequalis in Host Susceptibility

Carder

Smith

1977

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I N T R O D U C T I O NVenturia inaequalis, the apple scab organism, has previously been shown to produce biologically-active proteinaceous pigmented metabolites in culture (Hignett & Kirkham, 1967;Kirkham & Hignett, 1973;Hignett, 1973). The material stimulated the disease when applied as a spray 2 or 3 days after inoculation of test plants; it also showed affinity for ribosomes in vivo and in vitro. Work on its origins, and some physical and chemical characteristics of both intra-and extracellular pigments are described here.The malt extract medium used in earlier work contained large amounts of polysaccharide which interfered with melanoprotein purification. To avoid this problem a more suitable medium was developed. M E T H O D SGrowth of V. inaequalis and preparation of pigments. Venturia inaequalis was grown on modified basal medium NC (Kirkham, rgy), in which the hydrolysed casein was replaced by 2.5% (w/v) apple twig extract. This was made by refluxing 330 g powdered Edward VII apple shoots (I-year-old) for 6 h in 1.5 1 water. After vacuum filtration the residue was washed with 330 ml water and the washings were pooled and concentrated at 50 "C under vacuum to 330 ml. Particulate material was removed by centrifuging at 15000 g for 10 min. After adjusting the pH to 5.8, 3 vol. cold ethanol was added and left overnight at o "C. The precipitate was removed by centrifuging as before. The supernatant was evaporated under vacuum at 37 "C to a syrup, and diluted to 200 ml with water. After extraction with 7 x 70 ml ether, theaqueous layer of neutral wood extract (NWE) was evaporated under vacuum at 37 "C to 80 ml (approximately 30 yo, w/v), readjusted to pH 5-8 and stored over chloroform at o "C. The fraction not retained during dialysis (24 h) (NWED) was used for routine cultures.Cultures (1200 ml) were grown for 14 days at 18 "C in aerated, stirred flasks, and then mycelium was filtered off, washed exhaustively with water and freeze-dried. Culture fluid was reduced in volume by 75 % at 37 "C under vacuum, followed by dialysis against six changes of water (72 h) at 5 "C. The pigmented solution was concentrated to 10 mg ml-l (determined by weighing) and mixed with I vol. ethanol pre-cooled to -I I "C. After 3 h at o "C the precipitate (N50) was separated by centrifuging, dissolved in 4 M-urea and dialysed to remove urea. Two more volumes of cold ethanol were added to the ethanolic supernatant. After 16 h at o "C the precipitate (N75) was removed, taken up in water and dialysed at 5 "C against water (six changes) for 72 h. Intracellular pigment was obtained from the freeze-dried mycelium after six extractions with cold (-I I "C) acetone. The extracted mycelium was ground with a small quantity of acid-washed sand and sufficient 0.1 M-sodium phosphate buffer, pH 7-4, to form a smooth slurry. This was filtered through cotton-wool and centrifuged for 10 min at 30000 g. The clear brown supernatant (designated IN) was dialysed against water (four changes) at 5 "C for 48 h. Particulate material was then filtered out.Nuc...

Section: Results a N D Discussionmentioning

confidence: 66%

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mentioning

confidence: 99%

Origin and Characteristics of Venturia inaequalis Melanoproteins Active in the Apple Scab Disease

Carder

Smith

1977

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“…;upport the view that the parasite elaborates substances which may interfere with the functioning of the host cell wall (and hence with solute transport) and also with host metabolisni at the site of protein synthesis. Previous work with fungal pigment and kinetin has indicated that solute transport and the hormone system of infected plants are closely associated with the scab disease, which is also affected by incident light (Hignett & Kirkham, 1967;Kirkharn & Hignett, 1973). Plant hormones have been involved with the ribosomelpolyribosome system (Evins & Varner, 1972;Trewavas, 1968), with RNA and protein synthesis (Srivastava & Arglebe, 1968;VonAbrams & Pratt, 1968), and with the light incident upon the plants (Back & Richmond, 1969;Jackson & Field, 1972).…”

Section: Discussionmentioning

confidence: 99%

“…Coloured substances made by the plant pathogen Venturia inaequalis have been shown to be closely implicated in the progress of the apple scab disease (Hignett & Kirkham, 1967;Kirkham & Hignett, 1973).…”

Section: Introductionmentioning

confidence: 99%

The Adsorption of Silver-labelled Fungal Pigment by Bacterial and Apple Plant Ribosomes

1973

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S U M M A R YExtracellular material produced by Venturia inaequalis (the apple scab disease organism) formed a tightly bound complex with silver. The silver complex (in aqueous solution) was taken up by whole detached apple leaves (cultivar MM 109) and was observed in the petiole cytoplasm as ribosome-like particles. Diffuse deposits of silver were observed in the cell walls.The silver complex was also taken up by a proportion of the particles present in ribosome preparations made from apple-leaf tissues and from bacterial cultures. Such complexed ribosomes were apparently swollen by internal adsorption of the substance, some showing (in the electron microscope) a faint halo round the central core; the cleft between the sub-units was visible. Comparatively few ribosomes from Pseudomonas rnors-prunorurn showed strong affinity for the silver complex. The ribosomal particles were similar in most respects to the particles seen in treated leaf petioles. Silver-complexed particles prepared from isolated ribosomes were degraded by 'Pronase' to ragged skeletal remnants which were further digested by pancreatic ribonuclease. The latter enzyme did not attack intact particles.The results support evidence from earlier work that the progress of the disease is partly controlled by the effects of fungal pigments on host solute transport (possibly mediated via the cell-wall membrane system) and on hormone-controlled plant metabolism and photosynthesis (via the protein-synthesizing system).

“…Production of extracellular phenoloxidase by V. inaequalis would tend to enhance this effect, thereby inhibiting its growth. The fungus would also be deprived of phenolic substrates, which would hinder its capacity to synthesize melanoprotein which is active in the apple scab disease (Hignett & Kirkham, 1967 ;Hignett, 1973). In view of the generally observed changes in virulence of isolates of I/.…”

Section: R E S U L T S a N D Discussionmentioning

confidence: 99%

The Properties of Extracellular Enzymes of Venturia inaequalis and Their Association with Loss of Virulence of the Fungus in Culture

Roberts

Carder

1979

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Ri bonuclease, deoxyribonuclease, acid phosphatase and phenoloxidase were detected in preparations of extracellular melanoprotein isolated from cultures of Venturia inaequalis. The high initial levels of activity produced per g fungus declined within a few days of inoculation, to an approximately constant level. About six times as much activity was released by conidia germinating in medium enriched with wood extract than was released in basal medium. After 5 d incubation, the rate of production of enzymically active melanoprotein reflected the rate of growth of the fungus. Isolated melanoprotein stored at 4 "C for 3 months showed up to 190% more hydrolase activity than was measured originally, but longer storage caused a subsequent decrease in activity. Only phenoloxidase decreased continuously during storage. The apparent stability of the bond between melanin and protein under dissociating conditions contrasted with the reversible formation of complexes between the fungal product and polyelectrolytes.A set of re-isolates of different ages produced randomly variable levels of hydrolase activity when cultured repeatedly over a period of 16 months. On each occasion, however, the specific and total activity of each hydrolase was (with one marginal exception) lower in cultures of the oldest re-isolate than in those of the later re-isolates. In contrast, the specific activity of phenoloxidase was highest in cultures of the oldest re-isolate. These changes were associated with the decline in virulence observed in stored re-isolates of clone E, of V. inaequalis. I N T R O D U C T I O NExtracellular enzymes associated with melanoprotein produced by cultures of Venturia inaequalis were studied with a view to elucidating the biological stability of the pathogen. An attempt was also made to correlate the various levels of enzyme activity produced in culture by different clones with the corresponding levels of virulence observed on test plants. METHODS Growth of V. inaequalis andpreparation of extracellulav melanoproteins.Stock cultures of each isolate were maintained under paraffin oil at 4 "C (Kirkham, 1957). Using the methods of Hignett et al. (1977), extracellular melanoprotein was isolated from 40 ml shake cultures of V. inaequalis and fractionally precipitated with ethyl alcohol giving two fractions (N50 and N75). The cultures were usually grown on basal medium augmented with diffusible neutral wood extract (NWED) CHignett et a [., 1977). For cultures grown on basal medium without NWED, 0.05% (w/v) asparagine was added as an alternative nitrogen source. Growth curves were obtained by taking duplicate 40 ml shake cultures at intervals up to 18 d after inoculation. The mycelium was filtered off, washed exhaustively and dried to constant weight over phosphorus pentoxide. Non-diffusible material in the culture fluid was isolated by dialysis at 4 "C against five changes of 1 1 water