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Effects of lytic enzymes and natural soil on the fine structure of conidia of Cochliobolus sativus
[ 343 ] Trans. Br. mycol. Soc. 54 (3), 343-350 (1970) Printed in Great Britain
EFFECTS OF LYTIC ENZYMES AND NATURAL SOIL ON THE FINE STRUCTURE OF CONIDIA OF COCHL/OBOLUS SAT/VUS By K. M. OLD Department of Biological Sciences, University of Dundee W. M. ROBERTSON Scottish Horticultural Research Institute, Invergowrie, Dundee AND
(With Plates 34 to 36) The effects of natural soil and lytic enzymes on two wild-type, pigmented and two hyaline isolates of Cochliobolus sativus were studied. Pigmented isolates resisted lysis by enzymes and survived for more than 2 weeks on natural soil. Hyaline isolates lysed after a few hours in enzyme solutions and after a few days on natural soil. The fine structure of pigmented and hyaline isolates was compared before and after exposure to soil and enzymes. Resistance of wild-type conidia to lysis by soil bacteria and enzymes was associated with a broad electron-dense surface layer, corresponding to the distribution of pigment in the cell wall.
The importance of a soil-borne plant pathogen may well depend on its ability to survive for extended periods in the hostile soil environment where nutrient deficiency (Lloyd & Lockwood, 1966) and the products of antagonistic micro-organisms (Mitchell & Alexander, 1963) may lead to fungal lysis. There is evidence that pigmented hyphae or other propagules tend to survive in soil longer than hyaline structures (Linderman & Tousson, 1966; Lloyd & Lockwood, 1966). This has led several workers to compare the susceptibility of hyaline and pigmented species to enzyme preparations under controlled conditions of temperature and pH. Potgeiter & Alexander (1966) showed that whereas glucanase and chitinase caused visible lysis or erosion of cell walls of intact cells of Fusarium solani and Neurospora crassa, mycelium of Rhiroctonia solani was unaffected by either enzyme. Cell-wall fractions of R. solani were lysed to some extent by a combination of both enzymes. They suggested that a melanin-like substance in the hyphae of R. solani conferred resistance to lysis. This concept was supported by later work on Aspergillus phoenicis and Sclerotium rolfsii (Bloomfield & Alexander, 1967). Conidia of A. phoenicis and sclerotia of S. rolfsii are pigmented, whereas hyphae are hyaline. Conidia and sclerotia were resistant to lysis, but could be made susceptible by mechanical abrasion. The melanin, apparently conferring resistance to enzyme attack, was located in the outer layers of the conidia and sclerotia. Similarly, pigmented walls of A. nidulans were resistant to lytic enzymes, whereas cell
344
Transactions British Mycological Society
walls of a mutant hyaline isolate were readily lysed (Kuo & Alexander, 1967). Jones & Webley (1968) studied the effects of a glucanase preparation from a streptomycete on young hyaline, and on old, pigmented walls of a sterile fungus and found that hyaline cell walls were more susceptible to lysis. Durrell (1964) studied the fine structure of conidia of several pigmented species of fungi, and showed electron micrographs depicting electron-dense material in the outer layers of the conidial wall. Histological tests indicated that this was likely to be melanin. Old (1967, 1969) showed that conidia of C. sativus were capable of surviving extended periods in soil, although after 30 days an increasing proportion of conidia became perforated and lysed. The agent responsible has not yet been isolated, although electron micrographs of ultrathin sections show bacterium-like organisms associated with the eroded conidial wall (Old & Robertson, 1970). During investigations of the effect of snail gut enzyme on conidia it was found that a mutant hyaline strain obtained from Dr R. D. Tinline was susceptible to lysis whereas wild-type pigmented conidia were not affected (Old, 1969). Chinn & Tinline (1963) in a study of the ability of several pigmented and hyaline isolates of Cochliobolus sativus to germinate on natural soil, present data indicating that spore colour is simply inherited. The investigation reported here was undertaken to compare the survival of pigmented and hyaline isolates on natural soil, and to compare the susceptibility of the isolates to several lytic enzymes. Wherever possible ultrathin sections of conidia were prepared and comparisons of the fine structure of the cell walls of the several isolates were made, before and after exposure to natural soil and enzymes. By these means it should be possible to discover any structural differences between hyaline and pigmented isolates, and relate this to the survival of the conidia in soil, and their resistance or susceptibility to enzymic erosion. MATERIALS AND METHODS
Four isolates of C. sativus were studied, two pigmented isolates from oats, designated C I and C 2 (PI. 34, fig. 2) and two hyaline isolates, T 4 and 64 (PI. 34, fig. I) (Chinn & Tinline, 1964). Pigmented isolates sporulated readily on Czapek Dox agar, but the hyaline isolates sporulated best on soil extract agar after exposure to several cycles of 12 h near u.v. irradiation followed by 12 h in the dark. Garden soil, pH 7, was collected and treated as described previously (Old, 1969). Plates containing soil and fungal inoculum were loosely wrapped in plastic sheets and incubated in metal boxes containing moist filter-paper. Soil moisture content was maintained at approximately 30 % on an oven-dry basis. Aqueous suspensions of conidia were added to the soil surface at marked locations, and recovered at intervals using cellulose acetate tape (Old, 1967) for determination of germination and lysis. When conidia were required for examination of fine structure, tapes were pressed against the surface of agar plates. Conidia adhering to the agar were picked up with a fine needle and fixed.
Conidia of Cochliobolus. II. K. M. Old and W. M. Robertson 345 The effects of several enzyme preparations on conidia were studied. Three enzymes were used: snail gut enzyme (Koch Light) as a 20 % solution in phosphate buffer, pH 7, stabilized with 20 %sucrose (Bachmann & Bonner, 1959); chitinase (Calbiochem) at a concentration of 1 mg/ml in acetate buffer, pH 4.8 (Glaser & Brown, 1957); and lysozyme as used by Mach & Tatum (1963). Enzyme treatments were carried out in watch glasses with 2 ml of the reaction mixture incubated at either 27 or 35 DC on a rotary shaker. Conidia were examined by light or electron microscopy at intervals up to 24 h for evidence of lysis or germination. Conidia from soil, sporulating colonies, or enzyme treatments were fixed in 3 % glutaraldehyde, followed by 1 % osmium tetroxide. Sectioning and staining were as previously described (Old & Robertson, 1969). Sections were examined in an AEI EM 6 electron microscope. RESULTS
Fine structure of conidia During examination of the fine structure of control conidia, marked differences were observed in the appearance of the cell walls of pigmented and hyaline conidia. A certain amount of information had already been acquired concerning the multilaminate nature of the cell wall of pigmented conidia (Old & Robertson, 1969) and this was confirmed by further sectioning and examination. In addition to the electron-dense outer layer and two fibrillar layers, one denser than the other, an inner, densely fibrillar layer was found, bordering the cell lumen (Pl. 34, fig. 3). An alternative method of fixation in potassium permanganate described by Vitols, North & Linnane (196 I) gave a similar picture of the cell-wall structure (Pl. 34, fig. 5). When hyaline conidia of both isolates were examined the cell contents frequently showed details of nuclei, mitochondria, and lipid granules not visible in preparations of wild-type conidia. This suggests an increased permeability of hyaline conidia to the fixatives employed. The laminar nature of the cell wall of hyaline conidia was less well defined (Pl. 34, fig. 4). In all cells a very thin boundary layer of electron-dense material was present. The rest of the cell wall was fibrillar with an inner densely fibrillar layer detectable. This layer was usually lobed in outline when seen in transverse section (TS). In suitable preparations the protoplast had a well-defined double membrane. Longitudinal sections (LS) showed septa with a simple central pore, in common with other descriptions of ascomycete genera (Bracker, 1967). Conidia were multinucleate, as many as four nuclei were observed in a single 60 nm section. Lysis of conidia on soil Conidia of isolate C I (pigmented) and T 4 (hyaline) were placed on the surface of natural and steamed soil, and recovered at 15 clay intervals up to 45 days for determination of viability. Conidia of T 4 lysed readily on natural soil and virtually no conidia were recovered after 15 clays incubation. Pigmented conidia of isolate C I suffered no decrease in
Transactions British Mycological Society viability until 30 days incubation when 10 % showed perforations of the kind described earlier (Old & Robertson, 1969) and by 45 days, 15 % were perforated. On steamed soil perforation of conidia did not occur. These results with isolate C I were consistent with results reported previously (Old, 1969) and with results obtained with isolate C2, whereas isolate 64 (hyaline) behaved like T 4 on natural soil. It was clear that hyaline isolates had a very transient existence on soil compared to the more resistant wild-type isolates. This was confirmed in a series of experiments in which all 4 isolates were incubated for 12 days on natural and steamed soil. Observations were made on conidia recovered from the soil with adhesive tapes and stained with cotton blue in lactophenol. No estimates of viability of recovered conidia were made as it had already been shown that pigmented isolates remained viable for at least 15 days and preliminary experiments indicated that virtually all hyaline conidia which were not lysed were capable of germinating. Neither pigmented isolate lysed during 12 days on either soil, but both hyaline isolates suffered a marked degree of lysis, particularly on natural soil (Table 1). The experiment was repeated with both hyaline isolates with similar results. Table 1. Germination and lysis ofconidia oj three isolates oj Cochliobolus sativus incubated on natural soil Isolate and soil treatment C2 (Wild-type) Natural Steamed T 4 (Hyaline) Natural Steamed 64 (Hyaline) Natural Steamed
Germination on soil or agar (%) Day 1
Lysed conidia (%)
--,
,---
Day 12
Day 3
Day 6
0
0
0
0
()
0
0
0
q
0
18
0
6
17 7
64
6
3
0
12
0
4
24 5
37
4
2 6
Day
I
13
5
Observations were made on large numbers of hyaline conidia recovered from natural soil, using light microscopy. Lysis was typically accompanied by swelling and rounding of cell contents, and rupture of the cell wall (PI. 34, fig. 6). Relatively few conidia were examined with the electron microscope. However, evidence was obtained that soil bacteria aggregated around and lysed hyaline conidia in as little as 8 days (PI. 35, fig. 8). Bacteria often adhered firmly to the conidium wall (PI. 36, fig. 11), which became breached in several places (PI. 36, fig. 12). The fibrillar layers of the cell wall were more readily lysed than the narrow, electron-dense outer layer (PI. 36, fig. 13).
Effects oj lytic enrymes on conidia Of the three enzymes tested, only lysozyme had no effect on any isolate. The effect of snail gut enzyme on isolates C2, T 4 and 64 is shown in Table 2. Both hyaline isolates lysed readily, isolate C 2 was resistant to
Conidia of Cochliobolus. II. K. M. Old and W. M. Robertson
347
lysis. The experiment was repeated with both hyaline isolates and C I, giving similar results. Chitinase had an effect similar to snail gut enzyme, pigmented isolates were resistant and only hyaline conidia lysed after 3 h in chitinase at 35°. During lysis in either enzyme solution the first sign visible with the light microscope was swelling of the protoplasts. Cells then ruptured at fairly discrete areas and the contents were extruded (Pl. 34, fig. 7). Table 2. Percentages o] non-germinated (ng), germinated (g) and lysed (1) conidia oj three isolates oj Cochliobolus sativus after treatment with snail gut enryme or chitinase solutions Isolate ,-
'-~_.
C2 ,-----"-------.,
ng
Snail gut enzyme Phosphate buffer Chitinase Acetate buffer
IUO
33 100 100
T4 ,-------------A-_----,
g
I
ng
0 67 0 0
0 0 0
27 8 25
0
100
g
92
0 0 0
64 ,----~
I
ng
g
I
73
19 16
2 84
79
0
75
33
0
0
100
0
0 67 0
Ultrathin sections were prepared from conidia immersed in snail gut enzyme for 3 and 6 h and from conidia immersed in chitinase for 3 h. When treated with snail gut enzyme the outline of hyaline conidia became indented. The outer electron-dense layer was breached in one or more places (Pl. 35, fig. 10). In several conidia cell walls had a macerated appearance with fibres separated from one another, although it is possible that this effect was enhanced by shearing forces during sectioning. As the outer layer was eroded further, the conidia sometimes assumed bulging, distorted shapes. As in conidia treated with snail gut enzyme, lysis by chitinase appeared to be initiated at fairly discrete areas of the cell wall. The region that lysed most rapidly was situated just within the outer electron-dense layer (Pi. 36, fig. 14), corresponding to the region most susceptible to bacterial lysis when conidia were incubated in natural soil (Pl. 36, fig. 13). Sections through conidia in an advanced state of lysis showed general erosion of the fibrillar layers suggesting that chitin is the main microfibrillar element. Conidia were incubated in chitinase in sodium acetate buffer at 35°, pH 4'8. The cytoplasm of both enzyme treated and control conidia in buffer solution was disorganized and vacuolated (Pi. 35, fig. 9). Subsequently it was found that conidia failed to germinate after incubation in buffer solution for 4 h at 35°. It could therefore be considered that the chitinase was acting upon dead or dying conidia. This criticism could not be levelled at the snail gut enzyme treatment which was conducted at 27°. Control conidia in phosphate buffer germinated normally, and in conidia undergoing lysis, regions of the cytoplasm that were not adjacent to severely eroded cell wall contained intact nuclei and mitochondria.
Transactions British Mycological Society DISCUSSION
The most obvious difference in the fine structure of wild-type and hyaline isolates is the presence of a broad electron-dense layer in the outer layer of wild-type conidia. This was visible in specimens fixed in glutaraldehyde and osmic acid or in potassium permanganate. The layer was similar to that shown by Durrell (1964) for Stemphyllium, Coccosporium and Humicola. Durrell states that spore walls of Helminthosporium satiuum (C. satiousi had bands of electron-dense material rather than a continuous layer. This may be a reflexion of differences in isolates, or variation in fixation and staining methods. The electron density of the outer layer is associated with the presence of the pigment. Durrell identified melaninlike substances in C. satious and several other fungi by histochemical methods. Light microscopy studies of thick sections of Araldite-embedded conidia have confirmed the location of a brown pigment in the outer layer of the conidium wall. However, extraction and analysis of the pigment as done with several other fungi (Bartnicki-Garcia & Reyes, 1964; Lingappa, Sussmann & Bernstein, 1963; Sussmann, Lingappa & Bernstein, 1963) would be needed to prove its melanin-like nature. Of the four isolates studied here, the two pigmented ones were resistant to lytic enzymes, and the two hyaline isolates were readily lysed. This is consistent with previous work (Potgeiter & Alexander, 1966; Bloomfield & Alexander, 1967; Kuo & Alexander, 1967;]ones & Webley, 1968) and supports the concept that pigments, particularly if located in the surface layers of fungal propagules, can confer resistance to lysis. We have also shown that hyaline conidia rapidly lyse on natural soil whereas pigmented conidia survive for much longer periods. The role of lytic enzymes of microbial origin in the survival of fungal propagules in soil is not clearly established (Baker, 1968). It seems reasonable that the resistance to lytic enzymes shown by pigmented conidia contributes to their longevity on soil. Studies by Old & Robertson (1969, 1970) on aged conidia of C. sativus on soil show that the outer layer of the conidial wall is the region most resistant to decomposition in the soil. These studies, together with observations on bacteria aggregated on hyaline conidia and apparently causing lysis of the cell wall (PI. 35, fig. 8), are direct evidence that enzymes of exogenous microbial origin may lyse fungal conidia in soil. Particularly as this lysis bore a striking resemblance to that caused in vitro by chitinase and snail gut enzyme. The fact that the pigmentation of the conidium wall is due to a single gene (Tinline & Dickson, 1958) suggests strongly that resistance to lytic enzymes in vitro, and survival for long periods in soil in the presence of a diverse microflora, are attributable to the presence of the pigment, rather than to several, unrelated properties of the wild-type conidia. How then could the pigment in surface layers protect the conidium from enzyme attack? Wild-type conidia of C. saiiuus seem to be less permeable to fixatives than hyaline conidia, as shown by less favourable structural detail of cytoplasm in electron micrographs. It may be that pigmented walls are less permeable to enzymes than are hyaline walls. Another possibility suggested by Kuo & Alexander (1967) is that pigments
Conidia of Cochliobolus. II. K. M. Old and W. M. Robertson 349 (melanins) inhibit enzyme activity. It has been shown that release of pigments in plant tissue may inhibit enzymes of phytopathogenic fungi and limit lesion size (Deverall & Wood, r96r). Perhaps a similar mechanism could render pigmented conidia resistant to lysis by soil bacteria. Whatever the mechanism involved, our observations provide evidence that soil bacteria lyse hyaline conidia in soil, and that the resistance of pigmented conidia to lytic enzymes is related to their longevity on soil. We thank Professor D. A. T. Dick, Anatomy Department, Dundee University for the use of the electron microscope, and Mrs E. Lloyd-Davis for her kind co-operation. Thanks are also due to Dr C. E. Taylor for reading the manuscript and to Messrs S. Malecky and J. Sutherland for photographic work. REFERENCES
BACHMANN, B. J. & BONNER, D. M. (1959). Protoplasts from Neurospora crassa. J. Bact. 78, 550-556. BAKER, R. (1968). Mechanisms of biological control of soil-borne pathogens. A. Rev. Phytopath. 6, 263-294. BARTNICKI-GARCIA, S. & REYES, E. (1964). Chemistry of spore wall differentiation in Mucor rouxii. Archs Biochem. Biophys. 108, 125-133. BLOOMFIELD, B. J. & ALEXANDER, M. (1967)' Melanins and resistance of fungi to lysis. J. Bact. 93, 1276-1280. BRACKER, C. E. (1967). Ultrastructure offungi. A. Rev. Phytopath. 5, 343-374. CHINN, S. H. F. & TINLINE, R. D. (1963)' Spore germinability as an inherent character ofCochliobolus sativus. Phytopathology 53, 1109-1 I 12. CHINN, S. H. F. & TINLINE, R. D. (1964)' Inherent germinability and survival of spores of Cochliobolus sativus. Phytopathology 54, 349-352. DEVERALL, B.J. & WOOD, R. K. S. (1961). Chocolate spot of beans (Viciafaba L.)interactions between phenolase of host and pectic enzymes of the pathogen. Ann. appl. Biol. 49, 473-487. DURRELL, L. VV. (1964). The composition and structure of walls of dark fungus spores. Mycopath. Mycol, appl. 23, 339-343. GLASER, L. & BROWN, D. H. (1957)' The synthesis of chitin in cell free extracts of Neurospora crassa.]. biol. Chern. 228, 729-742. JONES, D. & WEBLEY, D. M. (1968). A new enrichment technique for studying lysis of fungal cell walls in soil. Pl. Soil. 28, 147-157. Kuo, M.J. & ALEXANDER, M. (1967)' Inhibition of the lysis of fungi by melanins. ]. Bact. 94, 624-629. LINDERMAN, R. G. & TOUSSON, T. A. (1966). Behaviour of albino chlamydospores of Thielaviopsis basicola. Phytopathology 56,887. LINGAPPA, Y., SUSSMANN, A. S. & BERNSTEIN, I. A. (1963). Effectoflight and media upon growth and melanin formation in Aureobasidium pullulans (De Bary) Am. (Pullularia pullulans). Mycopath. Mycol. appl. 20, 109-128. LLOYD, A. B. & LOCKWOOD,J. L. (1966). Lysis offungal hyphae in soil and its possible relation to autolysis. Phytopathology 56, 595-602. MACH, B. & TATUM, E. L. (1963). Ribonucleic acid synthesis in protoplasts of Escherichia coli: Inhibition by Actinomycin D. Science, N.r. 139, 1051-1052. MITCHELL, R. & ALEXANDER, M. (1963). Lysis of soil fungi by bacteria. Can.J. Microbiol. 9, 169-177. OLD, K. M. (1967). Effects of natural soil on survival of Cochliobolus sativus. Trans. Br. mycol. Soc. 50, 615-624. OLD, K. M. (1969)' Perforation of conidia of Cochliobolus sativus in natural soils. Trans. Br. mycol. Soc. 53, 207-216. OLD, K. M. & ROBERTSON, W. M. (1969). Examination of conidia ofCochliobolus sativus recovered from natural soil using transmission and scanning electron microscopy. Trans. Br, mycol. Soc. 53, 2 I 7-22 I.
35°
Transactions British Mycological Soaety
OLD, K. M. & ROBERTSON, W. M. (1970). Growth of bacteria within lysing fungal conidia in soil. Trans. Br. mycol. Soc. 54, 337-341. POTGEITER, H.J. & ALEXANDER, M. (1966). Susceptibility and resistance of several fungi to microbial lysis. ]. Bact. 91, 1526-1532. SUSSMANN, A. S., LINGAPPA, Y. & BERNSTEIN, 1. A. (1963). Effect oflight and media upon growth and melanin formation in Cladosporium mansoni, Mycopath, Mycol. appl. 20, 3°7-3 14. TINLINE, R. D. & DICKSON, J. G. (1958). Cochliobolus satiuus. I. Perithecial development and the inheritance of spore colour and rna ting type. Mycologia 50, 697-706. VITOLS, E., NORTH, R.J. & LINNANE, A. W. (1961). Studies on the oxidative metabolism of Saccharomyces cerevisiae. I. Observations on the fine structure of the yeast cell. ]. biophys. biochem. Cytol. 9, 689-699.
EXPLANATION OF PLATES
34-36
PLATE 34 Photomicrographs (PM) and electron micrographs (EM) of Cochliobolus sativus Fig. I. Hyaline conidia (PM). x 450. Fig. 2. Pigmented conidia (PM). x 550. Fig. 3. Fine structure of the cell wall of a pigmented conidium (EM) showing the electron-dense outer layer (OL), densely fibrillar layer (F I), more diffusely fibrillar layer (F2), and densely fibrillar layer lining the cell lumen (F3). x 22,500. (Note. F 2 is better shown in Fig. 5 due to some folding in this section.) Fig. 4. Fine structure of the cell wall of a hyaline conidium (EM) showing the narrow outer electron-dense layer, and the three fibrillar layers of the cell wall. X 22,500. Fig. 5. Fine structure of the cell wall ofa pigmented conidium (EM) fixed with KI\InO" showing the same laminar structure illustrated in Fig. 3. x 34,000. Fig. 6. Hyaline conidia (PM) recovered from soil and stained, showing lysis of cell walls, and adhering soil particles. x 230. Fig. 7. Hyaline conidia (PM) treated with snail gut enzyme, showing cell walls lysing and releasing pro top lasts. x 450. PLATE 35 Electron micrographs of ultrathin sections of Cochliobolus satious Fig. 8. Bacteria aggregated on a conidium recovered from soil, apparently causing lysis of cell wall fibrils (L). The dark fold (arrowed) is produced during sectioning. x 3,400. Fig. 9. Chitinase-treated conidium showing lysis of the region of the cell wall adjacent to the narrow outer electron-dense layer (arrowed). Note the vacuolated appearance of the cytoplasm related to the death of the conidium during incubation with the enzyme at 35°. x 2,550. Fig. 10. Snail gut enzyme-treated conidium showing erosion of outer electron-dense layer in two places (E), and lysis of cell-wall fibrils, particularly in the area immediately within the electrondense outer layer (arrowed). x 4,000. PLATE 36 Electron micrographs of ultrathin sections of Cochliobolus satiuus Fig. I I. Bacteria adhering to the cell wall of a hyaline conidium recovered from soil. x 35,000. Fig. 12. Soil bacteria adhering to the cell wall of a hyaline conidium, and appearing to cause perforation (P) of the electron-dense outer layer and lysis of microfibrils. x 24,000. Fig. 13. Cell wall of lysing hyaline conidium recovered from soil, showing lysis (L) of microfibrils. X 27,5°0. Fig. 14. Cell wall of lysing conidium treated with chitinase, showing lysis of microfibrils (L) in the region immediately within the electron-dense outer layer. x 24,000.
(Acceptedfor publication
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EFFECTS OF LYTIC ENZYMES AND NATURAL SOIL ON THE FINE STRUCTURE OF CONIDIA OF COCHL/OBOLUS SAT/VUS By K. M. OLD Department of Biological Sciences, University of Dundee W. M. ROBERTSON Scottish Horticultural Research Institute, Invergowrie, Dundee AND
(With Plates 34 to 36) The effects of natural soil and lytic enzymes on two wild-type, pigmented and two hyaline isolates of Cochliobolus sativus were studied. Pigmented isolates resisted lysis by enzymes and survived for more than 2 weeks on natural soil. Hyaline isolates lysed after a few hours in enzyme solutions and after a few days on natural soil. The fine structure of pigmented and hyaline isolates was compared before and after exposure to soil and enzymes. Resistance of wild-type conidia to lysis by soil bacteria and enzymes was associated with a broad electron-dense surface layer, corresponding to the distribution of pigment in the cell wall.
The importance of a soil-borne plant pathogen may well depend on its ability to survive for extended periods in the hostile soil environment where nutrient deficiency (Lloyd & Lockwood, 1966) and the products of antagonistic micro-organisms (Mitchell & Alexander, 1963) may lead to fungal lysis. There is evidence that pigmented hyphae or other propagules tend to survive in soil longer than hyaline structures (Linderman & Tousson, 1966; Lloyd & Lockwood, 1966). This has led several workers to compare the susceptibility of hyaline and pigmented species to enzyme preparations under controlled conditions of temperature and pH. Potgeiter & Alexander (1966) showed that whereas glucanase and chitinase caused visible lysis or erosion of cell walls of intact cells of Fusarium solani and Neurospora crassa, mycelium of Rhiroctonia solani was unaffected by either enzyme. Cell-wall fractions of R. solani were lysed to some extent by a combination of both enzymes. They suggested that a melanin-like substance in the hyphae of R. solani conferred resistance to lysis. This concept was supported by later work on Aspergillus phoenicis and Sclerotium rolfsii (Bloomfield & Alexander, 1967). Conidia of A. phoenicis and sclerotia of S. rolfsii are pigmented, whereas hyphae are hyaline. Conidia and sclerotia were resistant to lysis, but could be made susceptible by mechanical abrasion. The melanin, apparently conferring resistance to enzyme attack, was located in the outer layers of the conidia and sclerotia. Similarly, pigmented walls of A. nidulans were resistant to lytic enzymes, whereas cell
344
Transactions British Mycological Society
walls of a mutant hyaline isolate were readily lysed (Kuo & Alexander, 1967). Jones & Webley (1968) studied the effects of a glucanase preparation from a streptomycete on young hyaline, and on old, pigmented walls of a sterile fungus and found that hyaline cell walls were more susceptible to lysis. Durrell (1964) studied the fine structure of conidia of several pigmented species of fungi, and showed electron micrographs depicting electron-dense material in the outer layers of the conidial wall. Histological tests indicated that this was likely to be melanin. Old (1967, 1969) showed that conidia of C. sativus were capable of surviving extended periods in soil, although after 30 days an increasing proportion of conidia became perforated and lysed. The agent responsible has not yet been isolated, although electron micrographs of ultrathin sections show bacterium-like organisms associated with the eroded conidial wall (Old & Robertson, 1970). During investigations of the effect of snail gut enzyme on conidia it was found that a mutant hyaline strain obtained from Dr R. D. Tinline was susceptible to lysis whereas wild-type pigmented conidia were not affected (Old, 1969). Chinn & Tinline (1963) in a study of the ability of several pigmented and hyaline isolates of Cochliobolus sativus to germinate on natural soil, present data indicating that spore colour is simply inherited. The investigation reported here was undertaken to compare the survival of pigmented and hyaline isolates on natural soil, and to compare the susceptibility of the isolates to several lytic enzymes. Wherever possible ultrathin sections of conidia were prepared and comparisons of the fine structure of the cell walls of the several isolates were made, before and after exposure to natural soil and enzymes. By these means it should be possible to discover any structural differences between hyaline and pigmented isolates, and relate this to the survival of the conidia in soil, and their resistance or susceptibility to enzymic erosion. MATERIALS AND METHODS
Four isolates of C. sativus were studied, two pigmented isolates from oats, designated C I and C 2 (PI. 34, fig. 2) and two hyaline isolates, T 4 and 64 (PI. 34, fig. I) (Chinn & Tinline, 1964). Pigmented isolates sporulated readily on Czapek Dox agar, but the hyaline isolates sporulated best on soil extract agar after exposure to several cycles of 12 h near u.v. irradiation followed by 12 h in the dark. Garden soil, pH 7, was collected and treated as described previously (Old, 1969). Plates containing soil and fungal inoculum were loosely wrapped in plastic sheets and incubated in metal boxes containing moist filter-paper. Soil moisture content was maintained at approximately 30 % on an oven-dry basis. Aqueous suspensions of conidia were added to the soil surface at marked locations, and recovered at intervals using cellulose acetate tape (Old, 1967) for determination of germination and lysis. When conidia were required for examination of fine structure, tapes were pressed against the surface of agar plates. Conidia adhering to the agar were picked up with a fine needle and fixed.
Conidia of Cochliobolus. II. K. M. Old and W. M. Robertson 345 The effects of several enzyme preparations on conidia were studied. Three enzymes were used: snail gut enzyme (Koch Light) as a 20 % solution in phosphate buffer, pH 7, stabilized with 20 %sucrose (Bachmann & Bonner, 1959); chitinase (Calbiochem) at a concentration of 1 mg/ml in acetate buffer, pH 4.8 (Glaser & Brown, 1957); and lysozyme as used by Mach & Tatum (1963). Enzyme treatments were carried out in watch glasses with 2 ml of the reaction mixture incubated at either 27 or 35 DC on a rotary shaker. Conidia were examined by light or electron microscopy at intervals up to 24 h for evidence of lysis or germination. Conidia from soil, sporulating colonies, or enzyme treatments were fixed in 3 % glutaraldehyde, followed by 1 % osmium tetroxide. Sectioning and staining were as previously described (Old & Robertson, 1969). Sections were examined in an AEI EM 6 electron microscope. RESULTS
Fine structure of conidia During examination of the fine structure of control conidia, marked differences were observed in the appearance of the cell walls of pigmented and hyaline conidia. A certain amount of information had already been acquired concerning the multilaminate nature of the cell wall of pigmented conidia (Old & Robertson, 1969) and this was confirmed by further sectioning and examination. In addition to the electron-dense outer layer and two fibrillar layers, one denser than the other, an inner, densely fibrillar layer was found, bordering the cell lumen (Pl. 34, fig. 3). An alternative method of fixation in potassium permanganate described by Vitols, North & Linnane (196 I) gave a similar picture of the cell-wall structure (Pl. 34, fig. 5). When hyaline conidia of both isolates were examined the cell contents frequently showed details of nuclei, mitochondria, and lipid granules not visible in preparations of wild-type conidia. This suggests an increased permeability of hyaline conidia to the fixatives employed. The laminar nature of the cell wall of hyaline conidia was less well defined (Pl. 34, fig. 4). In all cells a very thin boundary layer of electron-dense material was present. The rest of the cell wall was fibrillar with an inner densely fibrillar layer detectable. This layer was usually lobed in outline when seen in transverse section (TS). In suitable preparations the protoplast had a well-defined double membrane. Longitudinal sections (LS) showed septa with a simple central pore, in common with other descriptions of ascomycete genera (Bracker, 1967). Conidia were multinucleate, as many as four nuclei were observed in a single 60 nm section. Lysis of conidia on soil Conidia of isolate C I (pigmented) and T 4 (hyaline) were placed on the surface of natural and steamed soil, and recovered at 15 clay intervals up to 45 days for determination of viability. Conidia of T 4 lysed readily on natural soil and virtually no conidia were recovered after 15 clays incubation. Pigmented conidia of isolate C I suffered no decrease in
Transactions British Mycological Society viability until 30 days incubation when 10 % showed perforations of the kind described earlier (Old & Robertson, 1969) and by 45 days, 15 % were perforated. On steamed soil perforation of conidia did not occur. These results with isolate C I were consistent with results reported previously (Old, 1969) and with results obtained with isolate C2, whereas isolate 64 (hyaline) behaved like T 4 on natural soil. It was clear that hyaline isolates had a very transient existence on soil compared to the more resistant wild-type isolates. This was confirmed in a series of experiments in which all 4 isolates were incubated for 12 days on natural and steamed soil. Observations were made on conidia recovered from the soil with adhesive tapes and stained with cotton blue in lactophenol. No estimates of viability of recovered conidia were made as it had already been shown that pigmented isolates remained viable for at least 15 days and preliminary experiments indicated that virtually all hyaline conidia which were not lysed were capable of germinating. Neither pigmented isolate lysed during 12 days on either soil, but both hyaline isolates suffered a marked degree of lysis, particularly on natural soil (Table 1). The experiment was repeated with both hyaline isolates with similar results. Table 1. Germination and lysis ofconidia oj three isolates oj Cochliobolus sativus incubated on natural soil Isolate and soil treatment C2 (Wild-type) Natural Steamed T 4 (Hyaline) Natural Steamed 64 (Hyaline) Natural Steamed
Germination on soil or agar (%) Day 1
Lysed conidia (%)
--,
,---
Day 12
Day 3
Day 6
0
0
0
0
()
0
0
0
q
0
18
0
6
17 7
64
6
3
0
12
0
4
24 5
37
4
2 6
Day
I
13
5
Observations were made on large numbers of hyaline conidia recovered from natural soil, using light microscopy. Lysis was typically accompanied by swelling and rounding of cell contents, and rupture of the cell wall (PI. 34, fig. 6). Relatively few conidia were examined with the electron microscope. However, evidence was obtained that soil bacteria aggregated around and lysed hyaline conidia in as little as 8 days (PI. 35, fig. 8). Bacteria often adhered firmly to the conidium wall (PI. 36, fig. 11), which became breached in several places (PI. 36, fig. 12). The fibrillar layers of the cell wall were more readily lysed than the narrow, electron-dense outer layer (PI. 36, fig. 13).
Effects oj lytic enrymes on conidia Of the three enzymes tested, only lysozyme had no effect on any isolate. The effect of snail gut enzyme on isolates C2, T 4 and 64 is shown in Table 2. Both hyaline isolates lysed readily, isolate C 2 was resistant to
Conidia of Cochliobolus. II. K. M. Old and W. M. Robertson
347
lysis. The experiment was repeated with both hyaline isolates and C I, giving similar results. Chitinase had an effect similar to snail gut enzyme, pigmented isolates were resistant and only hyaline conidia lysed after 3 h in chitinase at 35°. During lysis in either enzyme solution the first sign visible with the light microscope was swelling of the protoplasts. Cells then ruptured at fairly discrete areas and the contents were extruded (Pl. 34, fig. 7). Table 2. Percentages o] non-germinated (ng), germinated (g) and lysed (1) conidia oj three isolates oj Cochliobolus sativus after treatment with snail gut enryme or chitinase solutions Isolate ,-
'-~_.
C2 ,-----"-------.,
ng
Snail gut enzyme Phosphate buffer Chitinase Acetate buffer
IUO
33 100 100
T4 ,-------------A-_----,
g
I
ng
0 67 0 0
0 0 0
27 8 25
0
100
g
92
0 0 0
64 ,----~
I
ng
g
I
73
19 16
2 84
79
0
75
33
0
0
100
0
0 67 0
Ultrathin sections were prepared from conidia immersed in snail gut enzyme for 3 and 6 h and from conidia immersed in chitinase for 3 h. When treated with snail gut enzyme the outline of hyaline conidia became indented. The outer electron-dense layer was breached in one or more places (Pl. 35, fig. 10). In several conidia cell walls had a macerated appearance with fibres separated from one another, although it is possible that this effect was enhanced by shearing forces during sectioning. As the outer layer was eroded further, the conidia sometimes assumed bulging, distorted shapes. As in conidia treated with snail gut enzyme, lysis by chitinase appeared to be initiated at fairly discrete areas of the cell wall. The region that lysed most rapidly was situated just within the outer electron-dense layer (Pi. 36, fig. 14), corresponding to the region most susceptible to bacterial lysis when conidia were incubated in natural soil (Pl. 36, fig. 13). Sections through conidia in an advanced state of lysis showed general erosion of the fibrillar layers suggesting that chitin is the main microfibrillar element. Conidia were incubated in chitinase in sodium acetate buffer at 35°, pH 4'8. The cytoplasm of both enzyme treated and control conidia in buffer solution was disorganized and vacuolated (Pi. 35, fig. 9). Subsequently it was found that conidia failed to germinate after incubation in buffer solution for 4 h at 35°. It could therefore be considered that the chitinase was acting upon dead or dying conidia. This criticism could not be levelled at the snail gut enzyme treatment which was conducted at 27°. Control conidia in phosphate buffer germinated normally, and in conidia undergoing lysis, regions of the cytoplasm that were not adjacent to severely eroded cell wall contained intact nuclei and mitochondria.
Transactions British Mycological Society DISCUSSION
The most obvious difference in the fine structure of wild-type and hyaline isolates is the presence of a broad electron-dense layer in the outer layer of wild-type conidia. This was visible in specimens fixed in glutaraldehyde and osmic acid or in potassium permanganate. The layer was similar to that shown by Durrell (1964) for Stemphyllium, Coccosporium and Humicola. Durrell states that spore walls of Helminthosporium satiuum (C. satiousi had bands of electron-dense material rather than a continuous layer. This may be a reflexion of differences in isolates, or variation in fixation and staining methods. The electron density of the outer layer is associated with the presence of the pigment. Durrell identified melaninlike substances in C. satious and several other fungi by histochemical methods. Light microscopy studies of thick sections of Araldite-embedded conidia have confirmed the location of a brown pigment in the outer layer of the conidium wall. However, extraction and analysis of the pigment as done with several other fungi (Bartnicki-Garcia & Reyes, 1964; Lingappa, Sussmann & Bernstein, 1963; Sussmann, Lingappa & Bernstein, 1963) would be needed to prove its melanin-like nature. Of the four isolates studied here, the two pigmented ones were resistant to lytic enzymes, and the two hyaline isolates were readily lysed. This is consistent with previous work (Potgeiter & Alexander, 1966; Bloomfield & Alexander, 1967; Kuo & Alexander, 1967;]ones & Webley, 1968) and supports the concept that pigments, particularly if located in the surface layers of fungal propagules, can confer resistance to lysis. We have also shown that hyaline conidia rapidly lyse on natural soil whereas pigmented conidia survive for much longer periods. The role of lytic enzymes of microbial origin in the survival of fungal propagules in soil is not clearly established (Baker, 1968). It seems reasonable that the resistance to lytic enzymes shown by pigmented conidia contributes to their longevity on soil. Studies by Old & Robertson (1969, 1970) on aged conidia of C. sativus on soil show that the outer layer of the conidial wall is the region most resistant to decomposition in the soil. These studies, together with observations on bacteria aggregated on hyaline conidia and apparently causing lysis of the cell wall (PI. 35, fig. 8), are direct evidence that enzymes of exogenous microbial origin may lyse fungal conidia in soil. Particularly as this lysis bore a striking resemblance to that caused in vitro by chitinase and snail gut enzyme. The fact that the pigmentation of the conidium wall is due to a single gene (Tinline & Dickson, 1958) suggests strongly that resistance to lytic enzymes in vitro, and survival for long periods in soil in the presence of a diverse microflora, are attributable to the presence of the pigment, rather than to several, unrelated properties of the wild-type conidia. How then could the pigment in surface layers protect the conidium from enzyme attack? Wild-type conidia of C. saiiuus seem to be less permeable to fixatives than hyaline conidia, as shown by less favourable structural detail of cytoplasm in electron micrographs. It may be that pigmented walls are less permeable to enzymes than are hyaline walls. Another possibility suggested by Kuo & Alexander (1967) is that pigments
Conidia of Cochliobolus. II. K. M. Old and W. M. Robertson 349 (melanins) inhibit enzyme activity. It has been shown that release of pigments in plant tissue may inhibit enzymes of phytopathogenic fungi and limit lesion size (Deverall & Wood, r96r). Perhaps a similar mechanism could render pigmented conidia resistant to lysis by soil bacteria. Whatever the mechanism involved, our observations provide evidence that soil bacteria lyse hyaline conidia in soil, and that the resistance of pigmented conidia to lytic enzymes is related to their longevity on soil. We thank Professor D. A. T. Dick, Anatomy Department, Dundee University for the use of the electron microscope, and Mrs E. Lloyd-Davis for her kind co-operation. Thanks are also due to Dr C. E. Taylor for reading the manuscript and to Messrs S. Malecky and J. Sutherland for photographic work. REFERENCES
BACHMANN, B. J. & BONNER, D. M. (1959). Protoplasts from Neurospora crassa. J. Bact. 78, 550-556. BAKER, R. (1968). Mechanisms of biological control of soil-borne pathogens. A. Rev. Phytopath. 6, 263-294. BARTNICKI-GARCIA, S. & REYES, E. (1964). Chemistry of spore wall differentiation in Mucor rouxii. Archs Biochem. Biophys. 108, 125-133. BLOOMFIELD, B. J. & ALEXANDER, M. (1967)' Melanins and resistance of fungi to lysis. J. Bact. 93, 1276-1280. BRACKER, C. E. (1967). Ultrastructure offungi. A. Rev. Phytopath. 5, 343-374. CHINN, S. H. F. & TINLINE, R. D. (1963)' Spore germinability as an inherent character ofCochliobolus sativus. Phytopathology 53, 1109-1 I 12. CHINN, S. H. F. & TINLINE, R. D. (1964)' Inherent germinability and survival of spores of Cochliobolus sativus. Phytopathology 54, 349-352. DEVERALL, B.J. & WOOD, R. K. S. (1961). Chocolate spot of beans (Viciafaba L.)interactions between phenolase of host and pectic enzymes of the pathogen. Ann. appl. Biol. 49, 473-487. DURRELL, L. VV. (1964). The composition and structure of walls of dark fungus spores. Mycopath. Mycol, appl. 23, 339-343. GLASER, L. & BROWN, D. H. (1957)' The synthesis of chitin in cell free extracts of Neurospora crassa.]. biol. Chern. 228, 729-742. JONES, D. & WEBLEY, D. M. (1968). A new enrichment technique for studying lysis of fungal cell walls in soil. Pl. Soil. 28, 147-157. Kuo, M.J. & ALEXANDER, M. (1967)' Inhibition of the lysis of fungi by melanins. ]. Bact. 94, 624-629. LINDERMAN, R. G. & TOUSSON, T. A. (1966). Behaviour of albino chlamydospores of Thielaviopsis basicola. Phytopathology 56,887. LINGAPPA, Y., SUSSMANN, A. S. & BERNSTEIN, I. A. (1963). Effectoflight and media upon growth and melanin formation in Aureobasidium pullulans (De Bary) Am. (Pullularia pullulans). Mycopath. Mycol. appl. 20, 109-128. LLOYD, A. B. & LOCKWOOD,J. L. (1966). Lysis offungal hyphae in soil and its possible relation to autolysis. Phytopathology 56, 595-602. MACH, B. & TATUM, E. L. (1963). Ribonucleic acid synthesis in protoplasts of Escherichia coli: Inhibition by Actinomycin D. Science, N.r. 139, 1051-1052. MITCHELL, R. & ALEXANDER, M. (1963). Lysis of soil fungi by bacteria. Can.J. Microbiol. 9, 169-177. OLD, K. M. (1967). Effects of natural soil on survival of Cochliobolus sativus. Trans. Br. mycol. Soc. 50, 615-624. OLD, K. M. (1969)' Perforation of conidia of Cochliobolus sativus in natural soils. Trans. Br. mycol. Soc. 53, 207-216. OLD, K. M. & ROBERTSON, W. M. (1969). Examination of conidia ofCochliobolus sativus recovered from natural soil using transmission and scanning electron microscopy. Trans. Br, mycol. Soc. 53, 2 I 7-22 I.
35°
Transactions British Mycological Soaety
OLD, K. M. & ROBERTSON, W. M. (1970). Growth of bacteria within lysing fungal conidia in soil. Trans. Br. mycol. Soc. 54, 337-341. POTGEITER, H.J. & ALEXANDER, M. (1966). Susceptibility and resistance of several fungi to microbial lysis. ]. Bact. 91, 1526-1532. SUSSMANN, A. S., LINGAPPA, Y. & BERNSTEIN, 1. A. (1963). Effect oflight and media upon growth and melanin formation in Cladosporium mansoni, Mycopath, Mycol. appl. 20, 3°7-3 14. TINLINE, R. D. & DICKSON, J. G. (1958). Cochliobolus satiuus. I. Perithecial development and the inheritance of spore colour and rna ting type. Mycologia 50, 697-706. VITOLS, E., NORTH, R.J. & LINNANE, A. W. (1961). Studies on the oxidative metabolism of Saccharomyces cerevisiae. I. Observations on the fine structure of the yeast cell. ]. biophys. biochem. Cytol. 9, 689-699.
EXPLANATION OF PLATES
34-36
PLATE 34 Photomicrographs (PM) and electron micrographs (EM) of Cochliobolus sativus Fig. I. Hyaline conidia (PM). x 450. Fig. 2. Pigmented conidia (PM). x 550. Fig. 3. Fine structure of the cell wall of a pigmented conidium (EM) showing the electron-dense outer layer (OL), densely fibrillar layer (F I), more diffusely fibrillar layer (F2), and densely fibrillar layer lining the cell lumen (F3). x 22,500. (Note. F 2 is better shown in Fig. 5 due to some folding in this section.) Fig. 4. Fine structure of the cell wall of a hyaline conidium (EM) showing the narrow outer electron-dense layer, and the three fibrillar layers of the cell wall. X 22,500. Fig. 5. Fine structure of the cell wall ofa pigmented conidium (EM) fixed with KI\InO" showing the same laminar structure illustrated in Fig. 3. x 34,000. Fig. 6. Hyaline conidia (PM) recovered from soil and stained, showing lysis of cell walls, and adhering soil particles. x 230. Fig. 7. Hyaline conidia (PM) treated with snail gut enzyme, showing cell walls lysing and releasing pro top lasts. x 450. PLATE 35 Electron micrographs of ultrathin sections of Cochliobolus satious Fig. 8. Bacteria aggregated on a conidium recovered from soil, apparently causing lysis of cell wall fibrils (L). The dark fold (arrowed) is produced during sectioning. x 3,400. Fig. 9. Chitinase-treated conidium showing lysis of the region of the cell wall adjacent to the narrow outer electron-dense layer (arrowed). Note the vacuolated appearance of the cytoplasm related to the death of the conidium during incubation with the enzyme at 35°. x 2,550. Fig. 10. Snail gut enzyme-treated conidium showing erosion of outer electron-dense layer in two places (E), and lysis of cell-wall fibrils, particularly in the area immediately within the electrondense outer layer (arrowed). x 4,000. PLATE 36 Electron micrographs of ultrathin sections of Cochliobolus satiuus Fig. I I. Bacteria adhering to the cell wall of a hyaline conidium recovered from soil. x 35,000. Fig. 12. Soil bacteria adhering to the cell wall of a hyaline conidium, and appearing to cause perforation (P) of the electron-dense outer layer and lysis of microfibrils. x 24,000. Fig. 13. Cell wall of lysing hyaline conidium recovered from soil, showing lysis (L) of microfibrils. X 27,5°0. Fig. 14. Cell wall of lysing conidium treated with chitinase, showing lysis of microfibrils (L) in the region immediately within the electron-dense outer layer. x 24,000.
(Acceptedfor publication
10
December 1969)
Trans. Br. mycol. Soc.
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