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Resistance and susceptibility of fungal spores to lysis
Trans. Br. mycol. Soc. 58 (g), 489-497 (1972) Printed in Great Britain
RESISTANCE AND SUSCEPTIBILITY OF FUNGAL SPORES TO LYSIS By STELLA B. CHU*
AND
M. ALEXANDER
Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York (With g Text-figures) Spores of Fusarium oxysporum, Colletotrichum lagenarium, Aspergillus oryzae, Thielaviopsis basicola and Penicillium atrovenetum were lysed to varying extents in soil, but the spores of A. niger and A. phoenicis were resistant under identical conditions. A streptomycete enzyme preparation which digested F. oxysporum and C. phomoides spore walls and catalysed the release of N.acetylhexosamine and reducing sugars had some effect on walls of Helminthosporium turcicum and an albino strain of Thielaviopsis basicola and little or no effect on spores of a pigmented strain of T. basicola or of other fungi. Chitinase and p-I,g-glucanase preparations caused little lysis of F. oxysporum conidia, but the addition of a protease enhanced digestion. The release of N-acetylhexosamine and reducing sugars by chitinase and P-I,g-glucanase preparations suggests that F. oxysporum and C. lagenarium spore walls contain chitin and a P-I,g-gluean.
Soil micro-organisms are known to produce substances which bring about the lysis of fungi (Chinn & Ledingham, 1958; Lockwood, 1967), and information is available on the enzymic mechanism of heterolysis of fungal hyphae. Lysis of the hyphae of many of these organisms is the result of hydrolysis of the walls by chitinase and a fI-I,3-glucanase (Skujins, Potgieter & Alexander, 1965). The mycelium of some species and other fungal structures are resistant to lysis, and melanin appears to account for the lack of rapid destruction of the hyphae of Rhi;:;octonia solani (Potgieter & Alexander, 1966) and Aspergillus nidulans (Kuo & Alexander, 1967). In some fungi resistance to lysis is not due to melanin but apparently to a wall-localized heteropolysaccharide (Ballesta & Alexander, 1971). Many fungi whose walls are readily lysed produce spores or resting bodies not quickly digested in the soil. Spores of other fungi are probably destroyed by mechanisms and enzymes similar to those which decompose the hyphae. A streptomycete has been observed to lyse the arthrospores but to have little effect on the sporangiospores of Mucor ramannianus (Jones, Bacon, Farmer & Webley, 1968), and Verticillium hemileiae is reported to digest spores of Bemileia vastatrix (Garcia Acha, Leal & Villanueva, 1965). However, the conidia of Aspergillus phoenicis and Cochliobolus sativus seem to be quite resistant (Bloomfield & Alexander, 1967; Old & Robertson, 1970). The present study was conducted to assess the resistance and susceptibility of spores of several fungi to microbial lysis. ... Present address: Department of Anatomy, University of Michigan, Ann Arbor, Mich. 48104. 34-2
49°
Transactions British Mycological Society MATERIALS AND METHODS
Cultures were grown under a fluorescent lamp at room temperature on potato dextrose agar (Difco). The spores were harvested by scraping them from the mycelium, and they were then washed with sterile distilled water and passed through sterile cheesecloth. A liquid medium consisting of 2 % malt extract and 0'1 % yeast extract, with a pH of 5'1 after autoclaving, was used for the production of large quantities of Fusarium and Colletotrichum spores. These cultures were incubated on a rotary shaker at 30°C, and the spores were harvested by filtering the cultures through sterile cheesecloth, collecting the spores in the filtrate by centrifugation and washing them three times with sterile distilled water. The basal medium for the isolation and growth of the Streptomyces, which were obtained from soil, consisted of 0·6 g MgSO",.7H20, 4'0 g KNO a, 0'7 g K 2HPO"" 0'3 g KH 2PO"" 0'5 g CaC1 2, 10 mg yeast extract, 20 p,g FeCla, 18/kg ZnSO"" 2 p,g MnC1 2, 20 g agar and 20 g Aspergillus oryzae mycelium (wet weight) in 1'01 distilled water. The liquid basal medium modified to contain 10 % (wet weight) of A. oryzae hyphae was used for the production of lytic enzymes. The A. oryzae mycelium, a gift from Wallerstein Laboratories, was homogenized before addition to the medium. The pH of this medium was 6'5 after autodaving. The buried slide method of Chinn (1953) was used to study the lysis of spores in soil. To obtain spores killed by ultraviolet light, freshly harvested spores suspended in small amounts of water were placed under ultraviolet light at 253'7 nm on a rotary shaker at room temperature. Mter the exposure they were transferred to fresh medium for testing of viability. A suspension containing killed spores or mycelium and 1'5 % molten agar was placed on clean microscopic slides. Dead spores were used to avoid spore germination in the soil. Mter the agar had solidified, the slides were placed vertically in pots and covered with a silt loam. The soils were maintained at room temperature at 20-22 % moisture content. Slides were withdrawn at intervals. The adhering soil was dislodged with a stream ofwater and the slides were dried prior to microscopic examination. To obtain lytic preparations, cultures of Streptomyces grown for 7 days in 1'51 liquid basal medium with A. oryzae mycelium as carbon source were inoculated into 30 1 of the same medium in a 40 1 fermentor. Mter 2-4 days the cultures were harvested by centrifugation, and crystalline (NH",hSO", was gradually added to the supernatant with constant stirring to 70 % of saturation. The solution was kept at 4°C for 2 days. The precipitate which formed was collected by centrifugation and dissolved in 0'05 M phosphate buffer, pH 6'5' The solution was clarified by centrifugation. The clear supernatant then was dialysed against distilled water for 24 hat 4° and lyophilized. The enzyme preparation, spores and phosphate buffer were incubated in tubes at 37-39° on a shaker. In some instances mannitol at a final concentration of 0·8 M was included. At the end of the incubation period the enzyme was inactivated by immersing the tubes in boiling water. Lysis of spores was observed microscopically, and the optical density was deter-
Lysis offungal spores. Stella B. Chu and M. Alexander
491
mined at 625 nm. The solution was passed through a Millipore filter (0·65 fim pore size), and the N-acetylhexosamine and reducing sugar content of the filtrates was determined. N-Acetylhexosamine was estimated colorimetrically at 540 nm by the method of Tracey (1955) using an N-acetylglucosamine standard, and reducing sugar concentration was determined by the method of Somogyi (1952) with a glucose standard. The absorbancy of the filtrate at 260 nm was also measured. The glucanase activity was assessed by incubating together 1'0 ml of the enzyme preparation, 1'0 ml of a solution containing 1% (w/v) laminarin and 3'0 ml of0'05 M acetate-phosphate buffer, pH 5'7. Chitinase activity was measured in reaction mixtures containing the enzyme, acetate-phosphate buffer and 1'0 ml ofdispersed chitin (10 mg/ml). Lipase activity was tested using an olive-oil emulsion by the titrimetric method for serum lipase (Trietz, Borden & Stepleton, 1959). Protease activity was estimated with casein as substrate (Kunitz, 1947). The assays were conducted at 37°. Commercial chitinase, protease and lipase were obtained from Worthington Biochemical Corp., Freehold, N.J. Glucanase was prepared from basidiomycete QM 806 grown in a starch medium as described by Reese & Mandels (1959). This glucanase preparation was devoid of chitinase activity. RESULTS
Mter 5 days, mycelial walls of Aspergillus oryzae placed in soil showed distinct signs of breakage. Mter 20 days the growth of soil organisms was clearly discernible and the hyphae gradually disappeared. An isolate of Streptomyces sp. was obtained from this soil by using A. oryzae mycelium as a carbon source in agar plates. Under identical test conditions the spores of Aspergillus niger and A. phoenicis were resistant to lysis in soil, and only indications of discoloration of the spores were detected after 2 weeks. By contrast, Colletotrichum lagenarium spores were ruptured to a significant extent by the fifth day. Spores of Fusarium oxysporum 61765 and F. oxysporum f.sp. lycopersici were discoloured, and broken walls were detected in less than a week. Although Helminthosporium turcicum spores had lost their cytoplasmic contents during the first week the walls still appeared to be intact. Mter a 7-day incubation period a few A. oryzae spores were broken. Thielaviopsis basicola spores lost their long side walls as well as cytoplasm within 5 days, and Penicillium atrovenetum spores exhibited wall breakage by the seventh day. To study further their lysis the spores of F. oxysporum 61765 were incubated with a crude lytic enzyme preparation derived from a Streptomyces. For this purpose, o· I 25 mg of the enzyme preparation and 0'5 mg ofspores were added per ml of 0'05 M phosphate buffer (pH 6'5). Lysis was evident microscopically at 2 h as dissolution of the spore walls and the production of spherical bodies. The lysis, measured as percentage fall in optical density, was 60 for spore suspensions incubated with enzyme preparation and about 30 without enzyme, in a 16-h incubation period with no added mannitol. If mannitol was included in the reaction mixture the percentage lysis was 45 with enzyme and 20 without enzyme. However,
Transactions British Mycological Society
492
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no lysis was observed microscopically in the absence of the enzyme, although the cytoplasm in the spores appeared to have coagulated. The release of N-acetylhexosamine and ultraviolet-absorbing constituents as the spores lysed is shown in Fig. I, corrected for the release occurring in the absence of the enzymes. The yield ofN-acetylhexosamine increased as the incubation progressed, while the release of ultravioletabsorbing substances was marked only during the initial 2 h. The formation of reducing sugars was appreciable during the first 4 h. In lysis of F. oxysporum 267 spores by the crude enzyme preparation derived from the Streptomyces, the enzyme preparation and spores were suspended in o' 10 M phosphate buffer (pH 6'0), each to a final concentration of 1'0 mg/m!. With these test conditions, spherical bodies were quite evident after 8 h. As shown in Fig. 2, N-acetylhexosamine, ultravioletabsorbing materials and reducing sugars were rapidly released as the walls were digested. When 2'0 ml of a suspension of Colletotrichum phomoides spores was incubated in 5'0 ml of buffer under comparable conditions, microscopic observation revealed the formation of large numbers of wall-less spherical bodies after 8 h, by which time much of the spore wall must have been dissolved. The percentage lysis and the release of N-acetylhexosamine, ultraviolet-absorbing compounds and reducing sugars as these spores were lysed are shown in Fig. 3. No turbidity changes in suspensions ofthe spores of this fungus were noted in the absence of the enzyme. A comparison was made of the susceptibility of spores of several fungi to lysis by the Streptomyces enzyme preparation. Spores of A. niger with diminished pigmentation were obtained by growing the fungus for 5-7 days on potato dextrose agar containing o· I % dimethyl sulphite, I % dimethyl sulphoxide or 0'1 % dimethyl sulphoxide (Carley, Watson &
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Huber, 1967). Spores of A. niger and of other fungi grown on potato dextrose agar were incubated at 37° with 1'0 mg/ml of the crude enzyme preparation of Streptomyces 3 in 0'05 M phosphate buffer (pH 6,6) in the presence of 0'01 % merthiolate. Few of the spores of A. niger M 77 with diminished pigmentation released cytoplasmic material when incubated for more than 16 h with the enzyme preparation. Spores of Aspergillus awamoi, A. conicus, A. ory.<:,ae NRRL 1809, A. oryzae ATCC 9102, A. phoenicis, Cunninghamella elegans, P. atrovenetum, Moniliniafructicola, Graphium sp., Zygorf!ynchus moelleri and normal pigmented A. niger spores were resistant to the lytic enzyme preparation during 18 h incubation. On the other hand, spores of Colletotrichum lagenarium, C. phomoides and four strains of F. oxysporum were extensively lysed by the enzyme system. A small percentage of A. oryzae NRRL 1988 spores lost their outer walls or had broken walls by the end of the incubation period, and the cytoplasmic material was released. Most of the spores of an albino strain of Thielaviopsis basicola 3 had broken walls, they lost their cytoplasmic material and the residual spore walls were appreciably dissolved in the test period. Few spores of three non-albino strains of T. basicola were lysed. Helminthosporium turcicum spores released cytoplasmic material from their ends, which were destroyed by the enzyme preparation. Spores ofF. oxysporum 267 and C. lagenarium were harvested from cultures grown for 3-4 days in the liquid medium. To test their susceptibility to the streptomycete enzyme preparation, 10 mg of spores and 1'0 mg of the crude enzyme in a total volume of 10 ml were incubated for 4 h at with shaking. The preparation, in the quantity used, contained 6'25 units of protease and 1·6 I units of lipase and released 70 ,umole of glucose and 1·88 ,umole N-acetylhexosamine per hour from laminarin and chitin, respectively. As shown in Table I, the spores were digested readily, and reducing sugars and N-acetylhexosamine were liberated.
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Transactions British Mycological Society 30
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Table
I.
En;:,ymic lYsis of spores of Fusarium oxysporum and Colletotrichum lagenarium Crude Chitinase, streptoChitinase, Chitinase, glucanase, mycete Chitinase, glucanase, glucanase, protease, preparation glucanase protease lipase lipase
Lysis (%) F. oxysporum C. lagenarium Morphological changes· F, oxysporum C, lagenarium Reducing sugar release (,umole) F. oxysporum C. lagenarium N-Acetylhexosamine release (,umole) F. oxysporum C. lagenarium
26 28
++++ ++++ 58 '2 110 0,85 2·64
13 28
32 29
17 31
32 31
+ ++
+++ ++
+ ++
++++ ++
1'5
9'9 36
3'0 36
8'9 40
44 0'49 3'30
0'5 6 3'28
0'37 2'14
0·64 3'14
• Changes observed microscopically, The symbols +, + + and + + + represent 5-25, 25-50 and 5<>--75 % of the spores showing broken walls, and + + + + represents almost complete loss of spore walls.
495
Lysis offungal spores. Stella B. Chu and M. Alexander
Table 2. Lysis tifFusarium oxysporum and Colletotrichum lagenarium spores and product release Crude extract Activities in amount of enzyme used (Jtmole/h) N-Acetylhexosamine formation from chitin Reducing sugar release from laminarin Lysis(%) F. oxysporum C. lagenarium Morphological change* F. oxysporum C. lagenarium Reducing sugar release (Jtmole/24 h) F. oxysporum C. lagenarium N-Acetylhexosamine release (Jtmole/24 h) F. oxysporum C. lagenarium * See Table
Fraction
0'58 22
0,60 40
16 38
25 43
+++ +++
+++ ++
4'9 6'7
16·6 8'1
0'28 0'9 6
I
0'29 0·86
I.
Under identical conditions the basidiomycete glucanase-eommercial chitinase combinations did not cause appreciable destruction of F. OXYsporum spores, but the mixture was effective on C. lagenarium spores, either as viewed microscopically or on the basis of reducing sugar release (Table I). The addition of the protease and of the protease and lipase permitted more extensive lysis ofF. oxysporum spores, but the effect ofthese enzymes on sugar release was modest. The aliquots of the enzymes which were employed contained 6'0 units ofprotease and I·60 units oflipase and formed 62 pmole of glucose per hour from laminarin and 1·80 pmole of Nacetylhexosamine per hour from chitin. The crude enzyme preparation (150 mg) was dissolved in 15 ml of 0'05 M tris-HCI buffer, pH 8'2, and the solution was placed on a DEAESephadex column, I I mm diam x 150 mm long, previously equilibrated with the same buffer. The column was eluted with 100 ml of the buffer before starting continuous elution with 800 ml ofbuffer containing a linear gradient of NaCI (0-0'5 M). Fractions of 5 ml each were collected, and those having ;1-1,3-glucanase and chitinase activities were pooled to give five fractions. To test their lytic activity, aliquots of the fractions were incubated with 2'5 mg of spores for 32 h at 37° with shaking in a final volume of 5 m!. The solution contained 0'05 M phosphate buffer (pH 6·6) and o· I % merthiolate to prevent microbial growth. The insoluble spore residue was removed by passage through a Millipore filter before analysis. The five fractions collected all had ;1- I ,g-glucanase and chitinase activity. However, only the first fraction caused appreciable lysis of F. oxysporum spores (Table 2). Since the ;1-I,g-glucanase and chitinase activities of this fraction were not different from those of other fractions, which caused no appreciable lysis, it seems that additional enzymes must be involved in disruption of the spores, The data also suggest that a glucan and an N-acetylhexosamine polymer are present in the spore walls since the enzymes catalyse release of the appropriate monomers.
Transaczions British Mycological Society DISCUSSION
The present findings demonstrate the differences among fungi in the susceptibility of their various spores to microbial lysis. These differences were evident both when the spores were exposed to the activities of the natural soil community and when incubated with a crude enzyme preparation, and suggest marked dissimilarities in the chemistry of the walls of these organisms. Indeed, differences must exist at different sites on the surface of a single structure, as indicated by the selective destruction of only portions of the spores of H. turcicum. Resistance to lysis may have a significant ecological value in the maintenance of a species in an ecosystem in which mycolytic bacteria, actinomycetes and fungi are abundant. The action of preparations containing chitinase and P-l ,3-glucanase as well as the release of N-acetylhexosamine and reducing sugars is evidence that the spore walls of F. oxysporum and C. lagenarium possess chitin and glucan components accessible to these two enzymes. The two polysaccharides have also been reported to be localized in the walls of Mucor rouxii and Neurospora crassa spores (Bartnicki-Garcia & Reyes, 1964; Mahadevan & Mahadkar, 1970), but the additional presence of melanin in the conidial walls of A. phoenicis seems to explain why these structures, though containing carbohydrates usually hydrolysed with little difficulty, are not destroyed by polysaccharide-hydrolysing enzymes excreted by mycolytic organisms (Bloomfield & Alexander, 1967). The enhanced lysis and release of reducing sugars from F. oxysporum spores observed upon the addition of protease to the chitinase-glucanase mixture may reflect the existence of a protein conferring structural rigidity on the surface structure, proteins being abundant in the spore walls of certain fungi (Horikoshi & !ida, 1964; Sturgeon, 1966). Spore walls of Physarum polycephalum contain only a single sugar, galactosamine (McCormick, Blomquist & Rusch, 1970), and the susceptibility to lysis and the persistence of these structures in natural ecosystems should prove quite interesting. This investigation was supported by U.S. Public Health Service grant UI-OOI20 from the National Center for Urban and Industrial Health. REFERENCES
BALLESTA, J.-P. G. & ALEXANDER, M. (1971). Resistance of Zygorhynchus species to lysis. Journal of Bacteriology 106, 938-945. BARTNIcKI-GARCIA, S. & REYES, E. (1964). Chemistry of spore wall differentiation in Mucor TOuxii. Archiues of Biochemistry and Biophysics 108, 125-133. BLOOMFIELD, B. J. & ALEXANDER, M. (1967). Melanins and resistance of fungi to lysis. Journal of Bacteriology 93, ]276-1280. CARLEY, H. E., WATSON, R. D. & HUBER, D. M. (1967). Inhibition of pigmentation in Aspergillus niger by dimethylsulfoxide. Canadian Journal of Botany 45, 145]-1453. CHINN, S. H. F. (1953). A slide technique for the study of fungi and actinomycetes in soil with special reference to Helminthosporium satiuum. Canadian Journal of Botany 31, 7 18-7 24. CHINN, S. H. F. & LEDiNGHAM, R.J. (1958). Application of a new laboratory method for the determination of the survival of Helminthosporium satiuum spores in soil. Canadian Journal of Botany 36, 28g-295.
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GARCIA ACHA, 1., LEAL, ]. A. & VILLANUEVA, J. R. (1965)' Lysis of uredospore germ tubes of rusts by species of Verticillium. Phytopathology 55, 40-42. HORIKOSHI, K. & IIDA, S. (1964). Studies on the spore coats of fungi. 1. Isolation and composition of the spore coats of Aspergillus oryzae. Biochimica et Biophysica Acta 83, 197-203. JONES, D., BACON,J. S. D., FARMER, V. C. & WEBLEY, D. M. (1968). Lysis of cell walls of Mucor ramannianus Moeller by a Streptomyces sp. Antonie van Leeuwenhoek Journal qf Microbiology and Serology 34, 173-182. KUNITZ, M. (1947). Crystalline soybean trypsin inhibitor. II. General properties. Journal of General Physiology 30, 29 I-310. Kuo, M.-J. & ALEXANDER, M. (1967). Inhibition of the lysis of fungi by melanins. Journal of Bacteriology 94, 624-629. LOCKWOOD, J. L. (1967). The fungal environment of soil bacteria. In The ecology of soil bacteria (ed. by T. R. G. Gray and D. Parkinson), pp. 44-65. Liverpool University Press. MCCORMICK,J.]., BLOMQUIST,]. C. & RUSCH, H. P. (1970). Isolation and characterization of a galactosamine wall from spores and spherules of Physarum polycephalum. Journal of Bacteriology 104, I II 9-I 125. MAHADEVAN, P. R. & MAHADKAR, U. R. (1970). Major constituents of the conidial wall of Neurospora crassa. Indian Journal of Experimental Biology 8, 207-210. OLD, K. M. & ROBERTSON, W. M. (1970). Effects oflytic enzymes and natural soil on the fine structure of conidia of Cochliobolus sativus. Transactions ofthe British Mycological Society 54, 343-35 0 . POTGIETER, H. J. & ALEXANDER, M. (1966). Susceptibility and resistance of several fungi to microbial lysis. Journal of Bacteriology 91, 1526-1532. REESE, E. T. & MANDELS, M. (1959). fI-D-I,3-glucanases in fungi. Canadian Journal of Microbiology 5, 173-185. SKUJINS, ].]., POTGIETER, H.J. & ALEXANDER, M. (1965). Dissolution of fungal cell walls by a streptomycete chitinase and fJ-( I -+ 3) glucanase. Archives of Biochemistry and Biophysics III, 358-364. SOMOGYI, M. (1952). Notes on sugar determination. Journal of Biological Chemistry 195, 19-2 3. STURGEON, RJ. (1966). Components of the spore wall of Pithomyces chartarum. Nature 209, 204. TRACEY, M. V. (1955). Chitin. In Modern methods ofplant analysis, vol. 2 (ed. by K. Paech and M. V. Tracey), pp. 264-274. Berlin: Springer. TRIETZ, N. W., BORDEN, T. & STEPLETON, J. D. (1959). An improved method for the determination of lipase in serum. American Journal of Clinical Pathology 31, 148-154.
(Accepted for publication 5 December 197 I )
RESISTANCE AND SUSCEPTIBILITY OF FUNGAL SPORES TO LYSIS By STELLA B. CHU*
AND
M. ALEXANDER
Laboratory of Soil Microbiology, Department of Agronomy, Cornell University, Ithaca, New York (With g Text-figures) Spores of Fusarium oxysporum, Colletotrichum lagenarium, Aspergillus oryzae, Thielaviopsis basicola and Penicillium atrovenetum were lysed to varying extents in soil, but the spores of A. niger and A. phoenicis were resistant under identical conditions. A streptomycete enzyme preparation which digested F. oxysporum and C. phomoides spore walls and catalysed the release of N.acetylhexosamine and reducing sugars had some effect on walls of Helminthosporium turcicum and an albino strain of Thielaviopsis basicola and little or no effect on spores of a pigmented strain of T. basicola or of other fungi. Chitinase and p-I,g-glucanase preparations caused little lysis of F. oxysporum conidia, but the addition of a protease enhanced digestion. The release of N-acetylhexosamine and reducing sugars by chitinase and P-I,g-glucanase preparations suggests that F. oxysporum and C. lagenarium spore walls contain chitin and a P-I,g-gluean.
Soil micro-organisms are known to produce substances which bring about the lysis of fungi (Chinn & Ledingham, 1958; Lockwood, 1967), and information is available on the enzymic mechanism of heterolysis of fungal hyphae. Lysis of the hyphae of many of these organisms is the result of hydrolysis of the walls by chitinase and a fI-I,3-glucanase (Skujins, Potgieter & Alexander, 1965). The mycelium of some species and other fungal structures are resistant to lysis, and melanin appears to account for the lack of rapid destruction of the hyphae of Rhi;:;octonia solani (Potgieter & Alexander, 1966) and Aspergillus nidulans (Kuo & Alexander, 1967). In some fungi resistance to lysis is not due to melanin but apparently to a wall-localized heteropolysaccharide (Ballesta & Alexander, 1971). Many fungi whose walls are readily lysed produce spores or resting bodies not quickly digested in the soil. Spores of other fungi are probably destroyed by mechanisms and enzymes similar to those which decompose the hyphae. A streptomycete has been observed to lyse the arthrospores but to have little effect on the sporangiospores of Mucor ramannianus (Jones, Bacon, Farmer & Webley, 1968), and Verticillium hemileiae is reported to digest spores of Bemileia vastatrix (Garcia Acha, Leal & Villanueva, 1965). However, the conidia of Aspergillus phoenicis and Cochliobolus sativus seem to be quite resistant (Bloomfield & Alexander, 1967; Old & Robertson, 1970). The present study was conducted to assess the resistance and susceptibility of spores of several fungi to microbial lysis. ... Present address: Department of Anatomy, University of Michigan, Ann Arbor, Mich. 48104. 34-2
49°
Transactions British Mycological Society MATERIALS AND METHODS
Cultures were grown under a fluorescent lamp at room temperature on potato dextrose agar (Difco). The spores were harvested by scraping them from the mycelium, and they were then washed with sterile distilled water and passed through sterile cheesecloth. A liquid medium consisting of 2 % malt extract and 0'1 % yeast extract, with a pH of 5'1 after autoclaving, was used for the production of large quantities of Fusarium and Colletotrichum spores. These cultures were incubated on a rotary shaker at 30°C, and the spores were harvested by filtering the cultures through sterile cheesecloth, collecting the spores in the filtrate by centrifugation and washing them three times with sterile distilled water. The basal medium for the isolation and growth of the Streptomyces, which were obtained from soil, consisted of 0·6 g MgSO",.7H20, 4'0 g KNO a, 0'7 g K 2HPO"" 0'3 g KH 2PO"" 0'5 g CaC1 2, 10 mg yeast extract, 20 p,g FeCla, 18/kg ZnSO"" 2 p,g MnC1 2, 20 g agar and 20 g Aspergillus oryzae mycelium (wet weight) in 1'01 distilled water. The liquid basal medium modified to contain 10 % (wet weight) of A. oryzae hyphae was used for the production of lytic enzymes. The A. oryzae mycelium, a gift from Wallerstein Laboratories, was homogenized before addition to the medium. The pH of this medium was 6'5 after autodaving. The buried slide method of Chinn (1953) was used to study the lysis of spores in soil. To obtain spores killed by ultraviolet light, freshly harvested spores suspended in small amounts of water were placed under ultraviolet light at 253'7 nm on a rotary shaker at room temperature. Mter the exposure they were transferred to fresh medium for testing of viability. A suspension containing killed spores or mycelium and 1'5 % molten agar was placed on clean microscopic slides. Dead spores were used to avoid spore germination in the soil. Mter the agar had solidified, the slides were placed vertically in pots and covered with a silt loam. The soils were maintained at room temperature at 20-22 % moisture content. Slides were withdrawn at intervals. The adhering soil was dislodged with a stream ofwater and the slides were dried prior to microscopic examination. To obtain lytic preparations, cultures of Streptomyces grown for 7 days in 1'51 liquid basal medium with A. oryzae mycelium as carbon source were inoculated into 30 1 of the same medium in a 40 1 fermentor. Mter 2-4 days the cultures were harvested by centrifugation, and crystalline (NH",hSO", was gradually added to the supernatant with constant stirring to 70 % of saturation. The solution was kept at 4°C for 2 days. The precipitate which formed was collected by centrifugation and dissolved in 0'05 M phosphate buffer, pH 6'5' The solution was clarified by centrifugation. The clear supernatant then was dialysed against distilled water for 24 hat 4° and lyophilized. The enzyme preparation, spores and phosphate buffer were incubated in tubes at 37-39° on a shaker. In some instances mannitol at a final concentration of 0·8 M was included. At the end of the incubation period the enzyme was inactivated by immersing the tubes in boiling water. Lysis of spores was observed microscopically, and the optical density was deter-
Lysis offungal spores. Stella B. Chu and M. Alexander
491
mined at 625 nm. The solution was passed through a Millipore filter (0·65 fim pore size), and the N-acetylhexosamine and reducing sugar content of the filtrates was determined. N-Acetylhexosamine was estimated colorimetrically at 540 nm by the method of Tracey (1955) using an N-acetylglucosamine standard, and reducing sugar concentration was determined by the method of Somogyi (1952) with a glucose standard. The absorbancy of the filtrate at 260 nm was also measured. The glucanase activity was assessed by incubating together 1'0 ml of the enzyme preparation, 1'0 ml of a solution containing 1% (w/v) laminarin and 3'0 ml of0'05 M acetate-phosphate buffer, pH 5'7. Chitinase activity was measured in reaction mixtures containing the enzyme, acetate-phosphate buffer and 1'0 ml ofdispersed chitin (10 mg/ml). Lipase activity was tested using an olive-oil emulsion by the titrimetric method for serum lipase (Trietz, Borden & Stepleton, 1959). Protease activity was estimated with casein as substrate (Kunitz, 1947). The assays were conducted at 37°. Commercial chitinase, protease and lipase were obtained from Worthington Biochemical Corp., Freehold, N.J. Glucanase was prepared from basidiomycete QM 806 grown in a starch medium as described by Reese & Mandels (1959). This glucanase preparation was devoid of chitinase activity. RESULTS
Mter 5 days, mycelial walls of Aspergillus oryzae placed in soil showed distinct signs of breakage. Mter 20 days the growth of soil organisms was clearly discernible and the hyphae gradually disappeared. An isolate of Streptomyces sp. was obtained from this soil by using A. oryzae mycelium as a carbon source in agar plates. Under identical test conditions the spores of Aspergillus niger and A. phoenicis were resistant to lysis in soil, and only indications of discoloration of the spores were detected after 2 weeks. By contrast, Colletotrichum lagenarium spores were ruptured to a significant extent by the fifth day. Spores of Fusarium oxysporum 61765 and F. oxysporum f.sp. lycopersici were discoloured, and broken walls were detected in less than a week. Although Helminthosporium turcicum spores had lost their cytoplasmic contents during the first week the walls still appeared to be intact. Mter a 7-day incubation period a few A. oryzae spores were broken. Thielaviopsis basicola spores lost their long side walls as well as cytoplasm within 5 days, and Penicillium atrovenetum spores exhibited wall breakage by the seventh day. To study further their lysis the spores of F. oxysporum 61765 were incubated with a crude lytic enzyme preparation derived from a Streptomyces. For this purpose, o· I 25 mg of the enzyme preparation and 0'5 mg ofspores were added per ml of 0'05 M phosphate buffer (pH 6'5). Lysis was evident microscopically at 2 h as dissolution of the spore walls and the production of spherical bodies. The lysis, measured as percentage fall in optical density, was 60 for spore suspensions incubated with enzyme preparation and about 30 without enzyme, in a 16-h incubation period with no added mannitol. If mannitol was included in the reaction mixture the percentage lysis was 45 with enzyme and 20 without enzyme. However,
Transactions British Mycological Society
492
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no lysis was observed microscopically in the absence of the enzyme, although the cytoplasm in the spores appeared to have coagulated. The release of N-acetylhexosamine and ultraviolet-absorbing constituents as the spores lysed is shown in Fig. I, corrected for the release occurring in the absence of the enzymes. The yield ofN-acetylhexosamine increased as the incubation progressed, while the release of ultravioletabsorbing substances was marked only during the initial 2 h. The formation of reducing sugars was appreciable during the first 4 h. In lysis of F. oxysporum 267 spores by the crude enzyme preparation derived from the Streptomyces, the enzyme preparation and spores were suspended in o' 10 M phosphate buffer (pH 6'0), each to a final concentration of 1'0 mg/m!. With these test conditions, spherical bodies were quite evident after 8 h. As shown in Fig. 2, N-acetylhexosamine, ultravioletabsorbing materials and reducing sugars were rapidly released as the walls were digested. When 2'0 ml of a suspension of Colletotrichum phomoides spores was incubated in 5'0 ml of buffer under comparable conditions, microscopic observation revealed the formation of large numbers of wall-less spherical bodies after 8 h, by which time much of the spore wall must have been dissolved. The percentage lysis and the release of N-acetylhexosamine, ultraviolet-absorbing compounds and reducing sugars as these spores were lysed are shown in Fig. 3. No turbidity changes in suspensions ofthe spores of this fungus were noted in the absence of the enzyme. A comparison was made of the susceptibility of spores of several fungi to lysis by the Streptomyces enzyme preparation. Spores of A. niger with diminished pigmentation were obtained by growing the fungus for 5-7 days on potato dextrose agar containing o· I % dimethyl sulphite, I % dimethyl sulphoxide or 0'1 % dimethyl sulphoxide (Carley, Watson &
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Huber, 1967). Spores of A. niger and of other fungi grown on potato dextrose agar were incubated at 37° with 1'0 mg/ml of the crude enzyme preparation of Streptomyces 3 in 0'05 M phosphate buffer (pH 6,6) in the presence of 0'01 % merthiolate. Few of the spores of A. niger M 77 with diminished pigmentation released cytoplasmic material when incubated for more than 16 h with the enzyme preparation. Spores of Aspergillus awamoi, A. conicus, A. ory.<:,ae NRRL 1809, A. oryzae ATCC 9102, A. phoenicis, Cunninghamella elegans, P. atrovenetum, Moniliniafructicola, Graphium sp., Zygorf!ynchus moelleri and normal pigmented A. niger spores were resistant to the lytic enzyme preparation during 18 h incubation. On the other hand, spores of Colletotrichum lagenarium, C. phomoides and four strains of F. oxysporum were extensively lysed by the enzyme system. A small percentage of A. oryzae NRRL 1988 spores lost their outer walls or had broken walls by the end of the incubation period, and the cytoplasmic material was released. Most of the spores of an albino strain of Thielaviopsis basicola 3 had broken walls, they lost their cytoplasmic material and the residual spore walls were appreciably dissolved in the test period. Few spores of three non-albino strains of T. basicola were lysed. Helminthosporium turcicum spores released cytoplasmic material from their ends, which were destroyed by the enzyme preparation. Spores ofF. oxysporum 267 and C. lagenarium were harvested from cultures grown for 3-4 days in the liquid medium. To test their susceptibility to the streptomycete enzyme preparation, 10 mg of spores and 1'0 mg of the crude enzyme in a total volume of 10 ml were incubated for 4 h at with shaking. The preparation, in the quantity used, contained 6'25 units of protease and 1·6 I units of lipase and released 70 ,umole of glucose and 1·88 ,umole N-acetylhexosamine per hour from laminarin and chitin, respectively. As shown in Table I, the spores were digested readily, and reducing sugars and N-acetylhexosamine were liberated.
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494
Transactions British Mycological Society 30
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Table
I.
En;:,ymic lYsis of spores of Fusarium oxysporum and Colletotrichum lagenarium Crude Chitinase, streptoChitinase, Chitinase, glucanase, mycete Chitinase, glucanase, glucanase, protease, preparation glucanase protease lipase lipase
Lysis (%) F. oxysporum C. lagenarium Morphological changes· F, oxysporum C, lagenarium Reducing sugar release (,umole) F. oxysporum C. lagenarium N-Acetylhexosamine release (,umole) F. oxysporum C. lagenarium
26 28
++++ ++++ 58 '2 110 0,85 2·64
13 28
32 29
17 31
32 31
+ ++
+++ ++
+ ++
++++ ++
1'5
9'9 36
3'0 36
8'9 40
44 0'49 3'30
0'5 6 3'28
0'37 2'14
0·64 3'14
• Changes observed microscopically, The symbols +, + + and + + + represent 5-25, 25-50 and 5<>--75 % of the spores showing broken walls, and + + + + represents almost complete loss of spore walls.
495
Lysis offungal spores. Stella B. Chu and M. Alexander
Table 2. Lysis tifFusarium oxysporum and Colletotrichum lagenarium spores and product release Crude extract Activities in amount of enzyme used (Jtmole/h) N-Acetylhexosamine formation from chitin Reducing sugar release from laminarin Lysis(%) F. oxysporum C. lagenarium Morphological change* F. oxysporum C. lagenarium Reducing sugar release (Jtmole/24 h) F. oxysporum C. lagenarium N-Acetylhexosamine release (Jtmole/24 h) F. oxysporum C. lagenarium * See Table
Fraction
0'58 22
0,60 40
16 38
25 43
+++ +++
+++ ++
4'9 6'7
16·6 8'1
0'28 0'9 6
I
0'29 0·86
I.
Under identical conditions the basidiomycete glucanase-eommercial chitinase combinations did not cause appreciable destruction of F. OXYsporum spores, but the mixture was effective on C. lagenarium spores, either as viewed microscopically or on the basis of reducing sugar release (Table I). The addition of the protease and of the protease and lipase permitted more extensive lysis ofF. oxysporum spores, but the effect ofthese enzymes on sugar release was modest. The aliquots of the enzymes which were employed contained 6'0 units ofprotease and I·60 units oflipase and formed 62 pmole of glucose per hour from laminarin and 1·80 pmole of Nacetylhexosamine per hour from chitin. The crude enzyme preparation (150 mg) was dissolved in 15 ml of 0'05 M tris-HCI buffer, pH 8'2, and the solution was placed on a DEAESephadex column, I I mm diam x 150 mm long, previously equilibrated with the same buffer. The column was eluted with 100 ml of the buffer before starting continuous elution with 800 ml ofbuffer containing a linear gradient of NaCI (0-0'5 M). Fractions of 5 ml each were collected, and those having ;1-1,3-glucanase and chitinase activities were pooled to give five fractions. To test their lytic activity, aliquots of the fractions were incubated with 2'5 mg of spores for 32 h at 37° with shaking in a final volume of 5 m!. The solution contained 0'05 M phosphate buffer (pH 6·6) and o· I % merthiolate to prevent microbial growth. The insoluble spore residue was removed by passage through a Millipore filter before analysis. The five fractions collected all had ;1- I ,g-glucanase and chitinase activity. However, only the first fraction caused appreciable lysis of F. oxysporum spores (Table 2). Since the ;1-I,g-glucanase and chitinase activities of this fraction were not different from those of other fractions, which caused no appreciable lysis, it seems that additional enzymes must be involved in disruption of the spores, The data also suggest that a glucan and an N-acetylhexosamine polymer are present in the spore walls since the enzymes catalyse release of the appropriate monomers.
Transaczions British Mycological Society DISCUSSION
The present findings demonstrate the differences among fungi in the susceptibility of their various spores to microbial lysis. These differences were evident both when the spores were exposed to the activities of the natural soil community and when incubated with a crude enzyme preparation, and suggest marked dissimilarities in the chemistry of the walls of these organisms. Indeed, differences must exist at different sites on the surface of a single structure, as indicated by the selective destruction of only portions of the spores of H. turcicum. Resistance to lysis may have a significant ecological value in the maintenance of a species in an ecosystem in which mycolytic bacteria, actinomycetes and fungi are abundant. The action of preparations containing chitinase and P-l ,3-glucanase as well as the release of N-acetylhexosamine and reducing sugars is evidence that the spore walls of F. oxysporum and C. lagenarium possess chitin and glucan components accessible to these two enzymes. The two polysaccharides have also been reported to be localized in the walls of Mucor rouxii and Neurospora crassa spores (Bartnicki-Garcia & Reyes, 1964; Mahadevan & Mahadkar, 1970), but the additional presence of melanin in the conidial walls of A. phoenicis seems to explain why these structures, though containing carbohydrates usually hydrolysed with little difficulty, are not destroyed by polysaccharide-hydrolysing enzymes excreted by mycolytic organisms (Bloomfield & Alexander, 1967). The enhanced lysis and release of reducing sugars from F. oxysporum spores observed upon the addition of protease to the chitinase-glucanase mixture may reflect the existence of a protein conferring structural rigidity on the surface structure, proteins being abundant in the spore walls of certain fungi (Horikoshi & !ida, 1964; Sturgeon, 1966). Spore walls of Physarum polycephalum contain only a single sugar, galactosamine (McCormick, Blomquist & Rusch, 1970), and the susceptibility to lysis and the persistence of these structures in natural ecosystems should prove quite interesting. This investigation was supported by U.S. Public Health Service grant UI-OOI20 from the National Center for Urban and Industrial Health. REFERENCES
BALLESTA, J.-P. G. & ALEXANDER, M. (1971). Resistance of Zygorhynchus species to lysis. Journal of Bacteriology 106, 938-945. BARTNIcKI-GARCIA, S. & REYES, E. (1964). Chemistry of spore wall differentiation in Mucor TOuxii. Archiues of Biochemistry and Biophysics 108, 125-133. BLOOMFIELD, B. J. & ALEXANDER, M. (1967). Melanins and resistance of fungi to lysis. Journal of Bacteriology 93, ]276-1280. CARLEY, H. E., WATSON, R. D. & HUBER, D. M. (1967). Inhibition of pigmentation in Aspergillus niger by dimethylsulfoxide. Canadian Journal of Botany 45, 145]-1453. CHINN, S. H. F. (1953). A slide technique for the study of fungi and actinomycetes in soil with special reference to Helminthosporium satiuum. Canadian Journal of Botany 31, 7 18-7 24. CHINN, S. H. F. & LEDiNGHAM, R.J. (1958). Application of a new laboratory method for the determination of the survival of Helminthosporium satiuum spores in soil. Canadian Journal of Botany 36, 28g-295.
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GARCIA ACHA, 1., LEAL, ]. A. & VILLANUEVA, J. R. (1965)' Lysis of uredospore germ tubes of rusts by species of Verticillium. Phytopathology 55, 40-42. HORIKOSHI, K. & IIDA, S. (1964). Studies on the spore coats of fungi. 1. Isolation and composition of the spore coats of Aspergillus oryzae. Biochimica et Biophysica Acta 83, 197-203. JONES, D., BACON,J. S. D., FARMER, V. C. & WEBLEY, D. M. (1968). Lysis of cell walls of Mucor ramannianus Moeller by a Streptomyces sp. Antonie van Leeuwenhoek Journal qf Microbiology and Serology 34, 173-182. KUNITZ, M. (1947). Crystalline soybean trypsin inhibitor. II. General properties. Journal of General Physiology 30, 29 I-310. Kuo, M.-J. & ALEXANDER, M. (1967). Inhibition of the lysis of fungi by melanins. Journal of Bacteriology 94, 624-629. LOCKWOOD, J. L. (1967). The fungal environment of soil bacteria. In The ecology of soil bacteria (ed. by T. R. G. Gray and D. Parkinson), pp. 44-65. Liverpool University Press. MCCORMICK,J.]., BLOMQUIST,]. C. & RUSCH, H. P. (1970). Isolation and characterization of a galactosamine wall from spores and spherules of Physarum polycephalum. Journal of Bacteriology 104, I II 9-I 125. MAHADEVAN, P. R. & MAHADKAR, U. R. (1970). Major constituents of the conidial wall of Neurospora crassa. Indian Journal of Experimental Biology 8, 207-210. OLD, K. M. & ROBERTSON, W. M. (1970). Effects oflytic enzymes and natural soil on the fine structure of conidia of Cochliobolus sativus. Transactions ofthe British Mycological Society 54, 343-35 0 . POTGIETER, H. J. & ALEXANDER, M. (1966). Susceptibility and resistance of several fungi to microbial lysis. Journal of Bacteriology 91, 1526-1532. REESE, E. T. & MANDELS, M. (1959). fI-D-I,3-glucanases in fungi. Canadian Journal of Microbiology 5, 173-185. SKUJINS, ].]., POTGIETER, H.J. & ALEXANDER, M. (1965). Dissolution of fungal cell walls by a streptomycete chitinase and fJ-( I -+ 3) glucanase. Archives of Biochemistry and Biophysics III, 358-364. SOMOGYI, M. (1952). Notes on sugar determination. Journal of Biological Chemistry 195, 19-2 3. STURGEON, RJ. (1966). Components of the spore wall of Pithomyces chartarum. Nature 209, 204. TRACEY, M. V. (1955). Chitin. In Modern methods ofplant analysis, vol. 2 (ed. by K. Paech and M. V. Tracey), pp. 264-274. Berlin: Springer. TRIETZ, N. W., BORDEN, T. & STEPLETON, J. D. (1959). An improved method for the determination of lipase in serum. American Journal of Clinical Pathology 31, 148-154.
(Accepted for publication 5 December 197 I )