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Some properties of a staling substance from Fusarium oxysporum
[ 54 1
]
Trans. Brit. mycol. Soc. 47 (4), 541-546 (1964) Printed in Great Britain
SOME PROPERTIES OF A STALING SUBSTANCE FROM FUSARIUM OXYSPORUM By D. PARK*
Department
ofBotany,
The University oj Manchester
A method is described for the rapid detection of a staling substance produced by F. oxysporum Schlect. ex Fr. in liquid cultures. The staling is not due to removal of substances from the medium, and is not an osmotic or pH effect. The substance is non-volatile and is labile in the presence of the fungus, but is stable after sterilizing. It is adsorbed by charcoal, cellulose and asbestos. The amount produced by the fungus is related to the tolerance of it by the fungus under any given cultural conditions, and this tolerance is affected by carbon and nitrogen nutrition. The substance was active against all of a range of fungi tested.
Park (I 96 I) described a sequence of morphogenetic events in Fusarium oxysporum and some other fungi. This sequence was attributed to increasing concentration in the medium of a staling substance produced by the fungus during growth. It was suggested that the substance acted by altering the normal pattern of growth of the young hyphal tip. One of the properties of the substance was its lability which made biossay by spore germination and other relatively lengthy methods impracticable. The present paper describes a preliminary investigation in which a method of rapid bioassay of a staling substance produced in a medium of the same nutrient composition as that used in the earlier study was tested. METHOD
The same isolate of F. oxysporum was used. It was grown in liquid medium having the composition: glucose, 0'7 g.; MgS04.7H20, 0'5 g.; KH 2P04, 0'2 g.; NH4NOa, o- I g.; distilled water to I 1., roo ml. batches in 250 ml. conical flasks being inoculated and incubated at 25° C. for I week. Most of the mycelium was removed from the liquid by centrifuging or by straining through nylon gauze. Both treatments allowed small amounts of hyphal segments and some chlamydospores and conidia to pass through with the liquid. The liquid obtained in this way is referred to in this paper as staled solution. The test fungus, Aspergillus niger van Tiegh., was grown on agar plates inoculated by streaking a spore suspension across one diameter and incubating at 25° for 3 or 4 days. The medium had the composition given above and was gelled with IO g./I. Oxoid NO.3 agar. Individual drops of staled solution were pipetted on to the margin of the test colony so that the hyphal tips were wetted by it. The reaction of those tips at the surface of the medium to the presence of the
*
Now at Department of Botany" Queen's University of Belfast, N. Ireland.
542
Transactions British Mycological Society
staled solution included total cessation of growth at the apex, slight swelling of this region, vacuolation of the usually granular cytoplasm at the site, and, later, the initiation oflateral branches some distance behind the inhibited apex. Because treatments given to staled solutions before application resulted in some differences in intensity of these reactions, the criterion finally adopted as most satisfactory indication of activity of a staled solution was complete inhibition of apical growth. A temporary check to growth, with or without swelling or attenuation of hyphal diameter, followed by re-growth of the same apex was considered a negative result indicating that the solution had lost its activity during treatment. A positive result, indicating retention of activity by the solution through the treatment, was scored only when the treated tips were permanently occluded, even though this was followed sooner or later by the development of one or more lateral branches distal to the treated apex. This complete cessation of the original growing apex is termed permanent inhibition. RESULTS
Osmotic pressure Drops of uninoculated liquid medium of the composition given and also concentrations of t, 2, 5 and 10 times, and distilled water, were individually applied as described. In no case was there more than a temporary check to growth (less than 10 min.}, the tips in all the treatments grew on and there was no stimulated development of lateral branches. Staled solution was diluted to t, 1, 1 and -{"6 concentrations. The land ! dilutions caused permanent inhibition of all tips; the 1 dilution permanently inhibited some in the treated area, but others continued growth after a temporary check; the -{"6 dilution caused no permanent alteration to apical regions. These observations show that osmotic pressure is not responsible for the effect, and that the activity is not due to removal of substances from the medium, but rather to a positive effect that can rapidly be diluted to a level having no activity, the 1 dilution being on the borderline of effectiveness. Hydrogen ion concentration The pH of uninoculated solution was 4·5; for staled solution it was 3. 1-3.2 • Uninoculated solutions and staled solutions were adjusted by NaOH and Hfll over the range pH 3 to 8 and tested. None of the adjusted uninoculated solutions caused permanent inhibition, while all the adjusted staled solutions did so. The activity therefore is not a pH effect, nor was it removed by alteration of pH within the range used. Age ofstaled culture When solutions were tested at daily intervals after inoculation there was no detectable activity after 1 and 2 days; after 3 days a permanent inhibition was detectable but branching occurred closer to the apex than subsequently. After 4 days the complete inhibitory effect as found in
Fungus staling substance. D. Park 7-day staled solution was obtainable. Activity persisted solutions up to 39 days, the maximum tested.
Volatility
543 III
culture
ofthe agent
Park (196I) was unable to find any evidence that the staling factor in agar cultures ofF. oxysporum was volatile. In liquid cultures in the present investigation violent forced aeration of the culture solution during the 7-day growth period had no diminution effect on the activity of the resulting staled solution. Similarly a 7 days' forced aeration under sterile conditions of sterile staled solution (which as described later retains its activity in unaerated conditions) had no detectable effect on the level of inhibitory activity of the solution. Exposing S ml. quantities of the staled solution to reduced pressure (40 em. mercury) for I hr. also did not reduce the activity. There is no evidence from these observations that the active agent is volatile.
Storage
ofstaled solution
The two methods of removing the bulk of the fungus from the culture to obtain staled solution both allow some residual inoculum to remain. During storage at laboratory temperature (about 200 C.) staled solution was tested at intervals for activity. In one such series of tests the activity was present from 0 to 17l hr.; at 18 hr. there was continued growth through the original tip of some treated tips after a temporary check, at 18l hr. most of them grew on in this way, and from 19 to 24 hr. no activity was detectable. In various repetitions of this test there were differences in the precise timing of the loss of the activity from stored staled solution but the same general effect occurred. In tests made after a longer period of storage the inhibitory activity returned concurrently with mycelial development from the residual inoculum. Autoclaved staled solution showing persistent activity on storage (see next section) was re-inoculated with a small amount of spores and mycelium from a 7-day culture. After 22l hr. incubation at 200 the solution had lost activity, but after 46l hr. activity had returned, again concurrently with visible growth of the added inoculum.
Sterilization of staled solution Heating staled solution at temperatures up to 1000 C. for periods of up to 20 min. did not remove the activity. Various methods of sterilizing staled solutions were examined. Autoclaving at ISlb./sq. in., filtration through sintered glass, and exposure to u.v, light allowed activity to remain unimpaired. Filtration through a Sterimat pad, on the other hand, completely removed activity from the solution. Autoc1aved active solution stored at laboratory temperature retained activity for up to 48 days, the maximum time tested.
Adsorption
ofthe active agent
Early results had shown that removal of the mycelium from 7-day cultures by filtration through Whatman No. I filter-paper was deleterious
544
Transactions British Mycological Society
since activity was usually completely lost after such treatment, or very much diminished. The total loss of activity after filtration through an asbestos Sterimat pad paralleled this result. The exposure to reduced pressure during this process did not account for the inactivation of the agent. Adding cellulose powder, asbestos fibre, or 'Norit OL' activated charcoal to staled solution, and removal of the solids by centrifuging, all result ed in total disappearance of activity from the solutions. Park (1961) showed that' Cellophane ' placed over the surface of staled agar cultures permitted renewal of activity in the previously inhibited fungus in the region. 'Cellophane ' was here laid over marginal hyphae of the test fungus and staled solution put on the 'Cellophane'. Some morphogenetic effects occurred in the tips underneath, but there was no permanent inhibition. Controls with distilled water on 'Cellophane' behaved no differently from those treated with water directly. Hyphae of the test fungus growing on 'Cellophane' that had been laid over the medium reacted in the usual way to staled solution by permanent inhibition. Thus' Cellophane' removes the activity of the staled solution diffusing through it, but when fungus and 'Cellophane' are both in contact with the liquid the fungus reacts before the 'Cellophane' has had any detectable effect on the activity of the staled solution. The' Cellophane' does not directly affect the sensitivity of the test fungus. When activity of staled solutions was recorded, only those hyphae at the agar surface were considered, but it was apparent that hyphae deeper in the medium were also affected. The reactions at increasing depth in the agar were similar to those recorded for the effect of increasing dilution of the staled solution. Inhibition of the apex occurred down to 0'9 mm. below the agar surface, and the effect was evident as soon as IO min. after application of the drop to the surface. Agar thus allows the rapid diffusion of the inhibitor, but appears to exert a dilution or attenuation effect on its activity. This is much less marked than that given by 'Cellophane'. Staled solution allowed to stand on agar without any fungus for 1t hr. lost its activity, whereas solutions in contact with glass over the same surface area still retained complete activity. Agar therefore absorbs or adsorbs the inhibitor from solution in contact with it. This finding raised the question of how long a contact between staled solution and the hyphae is necessary before a response can develop. Tips flooded with staled solution for 1 min. and then washed with distilled water or with uninoculated solution developed no permanent inhibition, whereas contact for 2 min. followed by washing gave the full effect. Thus the inhibitor takes 1-2 min. to attain an effective concentration at the site of action.
Effect of carbon and nitrogen nutrition The effect of varying the concentration of the C and N sources on the production of the inhibitor by F. oxysporum was tested first in liquid medium. For N, in addition to the basal medium, the same but with 0, 0'3, I, 3 and 10 g.jl.NH4NOs were used. After supporting growth
Fungus staling substance. D. Park
545
of the fungu s for I week staled solutions from th e first three of these caused permanent inhibition of A. niger, while the last three did not. Thus in a medium containing th e standard amount of glucose, increased N lowered the amount of the inhibitor produced. For C, media containing 0,0'2,0'7,2,7 and 20 g. glucose/I ., respectively, were used ; staled solutions from the first of these gave no permanent inhibition while the remaining five did. However, at 0'2 g./I. activity was no longer evident at dilution of the staled solution to 1; at 0'7 g./I. activity was evident at this dilution but not at i, whereas in the last three media dilution to i still had full effect. Thus, increasing the amount of glucose in a medium containing the standard amount of N permitted the development of greater conc entrations of the inhibitor in the stal ed solution. The effect of similar variation in composition of the medium supporting growth of A. niger upon th e sensitivity of that fungus was examined using media gelled with agar. In all media permanent inhibition of the tips developed after treatment with standard stal ed solution. There were, however, marked and interesting differences in the morphological responses of the hyphal tips in the few minutes before the walls had completely set, and in th e form of branching after subsequent growth further back. These morphological effects still require detail ed analysis but they do give an indication that with increasing conc entration of glucose in the medium on which th e test fungus is grown, it becomes less sensitive, while with increasing N it is more sensitive. This indication was confirmed by preparing serial dilutions from I to l6 of standard staled solution and applying them to the test fungu s on the different media. The results (T able I) show clearly that high glucose level confers tolerance and high N increases sensitivity . Table I. Effect of dilutions ofstandard staled solution on Aspergillus niger growing on media with diJferent C andN concentrations NH.NO s g.I ).
Glucose g,/I.
A
Dilution [
t t 1 --le_
A
0
0' 1
0 '3
1 '0
3
10
0
0 '2
0 '7
+
+ +
+ +
+ + +
+ + +
+ + + +
+ + + +
+ + +
+ + +
i.
+,
2
7
20
+ +
+
+
permanent inhibition; - , lack of permanent inhibition , DIS CUSSION
The results describ ed here provide clear evidence of an inhibitor not present in the original medium but produced by th e growth ofF. oxy sporum. The effect is not a negative one resulting from th e removal of some factor from the medium since it can itself be removed by adsorption and can be diluted out. The effect is not one of pH or osmotic pressure. The factor causes effects closely similar to th e staling effects described by Park (196 I) in agar cultures of F. oxysporum. The first detectable activity after more than 2 days coincides with th e time of development of
546
Transactions British Mycological Society
staling in the agar medium; in plates seeded with F. oxysporum staling effects were first seen after 2 days, and in centrally inoculated spreading colonies an area reached maximum staling after slightly more than 2 days from its inception at the growing margin. The disappearance of the inhibitor during storage of the staled solution agrees with the lability of the staling factor in the agar medium, while its persistence in autoclaved solution points to a biological removal by the fungus that produces it. The subsequent return of the inhibitor with re-growth of the fungus from residual or added inoculum also coincides with the dynamic pattern of autolysis balanced by growth described earlier from the agar cultures. It was suggested by Park (1961) that the inhibitor in a staled region became reduced by natural lability to a level permitting some growth, which in turn led to formation of more inhibitor increasing again to a fungistatic level. In the present study as in the earlier one there was no evidence that volatility could account for the disappearance from stored media. The removal of the effect by 'Cellophane' also parallels the finding in the earlier study. The production of greater concentrations of the inhibitor by F. oxysporum in media with high C and low N agrees with the greater tolerance of the test fungus, A. niger, under these same conditions if it is assumed that this is a common factor and that nutrition affects its production by the two organisms in the same way. From the earlier study it would be expected that under any set of conditions production would be limited by tolerance under those conditions so that when a fungistatic level is reached further growth, and hence further production of the inhibitor, is prevented until the concentration again falls below that level. Staled solution has been applied to tips of Absidia glauca Hagem., Achlya ambisexualis J. R. Raper, Alternaria tenuis auct., Aspergillus nidulans (Eidam) Wint., Botrytis cinerea Pers. ex Fr., Epicoccum nigrum Link, Fusarium culmorum (W. G. Smith) Sacc., Mucor plumbeus Bon., Neurospora crassa Shear & Dodge, Penicillium claviforme Bain., Phycomyces blakesleeanus Burgeff, Phytophthora erythroseptica Pethybr., Polyporus betulinus (Bull.) Fr., Pythium debaryanum Hesse, P. proliferum de Bary, Rhizopusstolonifer (Ehrenb. ex Fr.) Lind., Saprolegnia sp., Sordariafimicola (Rob.) Ces, & de Not., Trichothecium roseum Link ex Fr., and Zygorhynchus moelleri Yuill. growing on the agar medium, and while there were differences in the specific reactions to the solution all developed permanent inhibition. No fungus has been tested that has not shown this reaction to staled solution from F. oxysporum. The factor active in this test seems therefore to cause an effect like that investigated earlier in not being restricted in its action to the species producing it. REFERENCE
PARK, D. (1961). Morphogenesis, fungistasis and cultural staling in Fusarium oxysporum Snyder & Hansen. Trans. Brit. mycol. Soc. 44, 377-390.
(Accepted for publication 12 April 1964)
]
Trans. Brit. mycol. Soc. 47 (4), 541-546 (1964) Printed in Great Britain
SOME PROPERTIES OF A STALING SUBSTANCE FROM FUSARIUM OXYSPORUM By D. PARK*
Department
ofBotany,
The University oj Manchester
A method is described for the rapid detection of a staling substance produced by F. oxysporum Schlect. ex Fr. in liquid cultures. The staling is not due to removal of substances from the medium, and is not an osmotic or pH effect. The substance is non-volatile and is labile in the presence of the fungus, but is stable after sterilizing. It is adsorbed by charcoal, cellulose and asbestos. The amount produced by the fungus is related to the tolerance of it by the fungus under any given cultural conditions, and this tolerance is affected by carbon and nitrogen nutrition. The substance was active against all of a range of fungi tested.
Park (I 96 I) described a sequence of morphogenetic events in Fusarium oxysporum and some other fungi. This sequence was attributed to increasing concentration in the medium of a staling substance produced by the fungus during growth. It was suggested that the substance acted by altering the normal pattern of growth of the young hyphal tip. One of the properties of the substance was its lability which made biossay by spore germination and other relatively lengthy methods impracticable. The present paper describes a preliminary investigation in which a method of rapid bioassay of a staling substance produced in a medium of the same nutrient composition as that used in the earlier study was tested. METHOD
The same isolate of F. oxysporum was used. It was grown in liquid medium having the composition: glucose, 0'7 g.; MgS04.7H20, 0'5 g.; KH 2P04, 0'2 g.; NH4NOa, o- I g.; distilled water to I 1., roo ml. batches in 250 ml. conical flasks being inoculated and incubated at 25° C. for I week. Most of the mycelium was removed from the liquid by centrifuging or by straining through nylon gauze. Both treatments allowed small amounts of hyphal segments and some chlamydospores and conidia to pass through with the liquid. The liquid obtained in this way is referred to in this paper as staled solution. The test fungus, Aspergillus niger van Tiegh., was grown on agar plates inoculated by streaking a spore suspension across one diameter and incubating at 25° for 3 or 4 days. The medium had the composition given above and was gelled with IO g./I. Oxoid NO.3 agar. Individual drops of staled solution were pipetted on to the margin of the test colony so that the hyphal tips were wetted by it. The reaction of those tips at the surface of the medium to the presence of the
*
Now at Department of Botany" Queen's University of Belfast, N. Ireland.
542
Transactions British Mycological Society
staled solution included total cessation of growth at the apex, slight swelling of this region, vacuolation of the usually granular cytoplasm at the site, and, later, the initiation oflateral branches some distance behind the inhibited apex. Because treatments given to staled solutions before application resulted in some differences in intensity of these reactions, the criterion finally adopted as most satisfactory indication of activity of a staled solution was complete inhibition of apical growth. A temporary check to growth, with or without swelling or attenuation of hyphal diameter, followed by re-growth of the same apex was considered a negative result indicating that the solution had lost its activity during treatment. A positive result, indicating retention of activity by the solution through the treatment, was scored only when the treated tips were permanently occluded, even though this was followed sooner or later by the development of one or more lateral branches distal to the treated apex. This complete cessation of the original growing apex is termed permanent inhibition. RESULTS
Osmotic pressure Drops of uninoculated liquid medium of the composition given and also concentrations of t, 2, 5 and 10 times, and distilled water, were individually applied as described. In no case was there more than a temporary check to growth (less than 10 min.}, the tips in all the treatments grew on and there was no stimulated development of lateral branches. Staled solution was diluted to t, 1, 1 and -{"6 concentrations. The land ! dilutions caused permanent inhibition of all tips; the 1 dilution permanently inhibited some in the treated area, but others continued growth after a temporary check; the -{"6 dilution caused no permanent alteration to apical regions. These observations show that osmotic pressure is not responsible for the effect, and that the activity is not due to removal of substances from the medium, but rather to a positive effect that can rapidly be diluted to a level having no activity, the 1 dilution being on the borderline of effectiveness. Hydrogen ion concentration The pH of uninoculated solution was 4·5; for staled solution it was 3. 1-3.2 • Uninoculated solutions and staled solutions were adjusted by NaOH and Hfll over the range pH 3 to 8 and tested. None of the adjusted uninoculated solutions caused permanent inhibition, while all the adjusted staled solutions did so. The activity therefore is not a pH effect, nor was it removed by alteration of pH within the range used. Age ofstaled culture When solutions were tested at daily intervals after inoculation there was no detectable activity after 1 and 2 days; after 3 days a permanent inhibition was detectable but branching occurred closer to the apex than subsequently. After 4 days the complete inhibitory effect as found in
Fungus staling substance. D. Park 7-day staled solution was obtainable. Activity persisted solutions up to 39 days, the maximum tested.
Volatility
543 III
culture
ofthe agent
Park (196I) was unable to find any evidence that the staling factor in agar cultures ofF. oxysporum was volatile. In liquid cultures in the present investigation violent forced aeration of the culture solution during the 7-day growth period had no diminution effect on the activity of the resulting staled solution. Similarly a 7 days' forced aeration under sterile conditions of sterile staled solution (which as described later retains its activity in unaerated conditions) had no detectable effect on the level of inhibitory activity of the solution. Exposing S ml. quantities of the staled solution to reduced pressure (40 em. mercury) for I hr. also did not reduce the activity. There is no evidence from these observations that the active agent is volatile.
Storage
ofstaled solution
The two methods of removing the bulk of the fungus from the culture to obtain staled solution both allow some residual inoculum to remain. During storage at laboratory temperature (about 200 C.) staled solution was tested at intervals for activity. In one such series of tests the activity was present from 0 to 17l hr.; at 18 hr. there was continued growth through the original tip of some treated tips after a temporary check, at 18l hr. most of them grew on in this way, and from 19 to 24 hr. no activity was detectable. In various repetitions of this test there were differences in the precise timing of the loss of the activity from stored staled solution but the same general effect occurred. In tests made after a longer period of storage the inhibitory activity returned concurrently with mycelial development from the residual inoculum. Autoclaved staled solution showing persistent activity on storage (see next section) was re-inoculated with a small amount of spores and mycelium from a 7-day culture. After 22l hr. incubation at 200 the solution had lost activity, but after 46l hr. activity had returned, again concurrently with visible growth of the added inoculum.
Sterilization of staled solution Heating staled solution at temperatures up to 1000 C. for periods of up to 20 min. did not remove the activity. Various methods of sterilizing staled solutions were examined. Autoclaving at ISlb./sq. in., filtration through sintered glass, and exposure to u.v, light allowed activity to remain unimpaired. Filtration through a Sterimat pad, on the other hand, completely removed activity from the solution. Autoc1aved active solution stored at laboratory temperature retained activity for up to 48 days, the maximum time tested.
Adsorption
ofthe active agent
Early results had shown that removal of the mycelium from 7-day cultures by filtration through Whatman No. I filter-paper was deleterious
544
Transactions British Mycological Society
since activity was usually completely lost after such treatment, or very much diminished. The total loss of activity after filtration through an asbestos Sterimat pad paralleled this result. The exposure to reduced pressure during this process did not account for the inactivation of the agent. Adding cellulose powder, asbestos fibre, or 'Norit OL' activated charcoal to staled solution, and removal of the solids by centrifuging, all result ed in total disappearance of activity from the solutions. Park (1961) showed that' Cellophane ' placed over the surface of staled agar cultures permitted renewal of activity in the previously inhibited fungus in the region. 'Cellophane ' was here laid over marginal hyphae of the test fungus and staled solution put on the 'Cellophane'. Some morphogenetic effects occurred in the tips underneath, but there was no permanent inhibition. Controls with distilled water on 'Cellophane' behaved no differently from those treated with water directly. Hyphae of the test fungus growing on 'Cellophane' that had been laid over the medium reacted in the usual way to staled solution by permanent inhibition. Thus' Cellophane' removes the activity of the staled solution diffusing through it, but when fungus and 'Cellophane' are both in contact with the liquid the fungus reacts before the 'Cellophane' has had any detectable effect on the activity of the staled solution. The' Cellophane' does not directly affect the sensitivity of the test fungus. When activity of staled solutions was recorded, only those hyphae at the agar surface were considered, but it was apparent that hyphae deeper in the medium were also affected. The reactions at increasing depth in the agar were similar to those recorded for the effect of increasing dilution of the staled solution. Inhibition of the apex occurred down to 0'9 mm. below the agar surface, and the effect was evident as soon as IO min. after application of the drop to the surface. Agar thus allows the rapid diffusion of the inhibitor, but appears to exert a dilution or attenuation effect on its activity. This is much less marked than that given by 'Cellophane'. Staled solution allowed to stand on agar without any fungus for 1t hr. lost its activity, whereas solutions in contact with glass over the same surface area still retained complete activity. Agar therefore absorbs or adsorbs the inhibitor from solution in contact with it. This finding raised the question of how long a contact between staled solution and the hyphae is necessary before a response can develop. Tips flooded with staled solution for 1 min. and then washed with distilled water or with uninoculated solution developed no permanent inhibition, whereas contact for 2 min. followed by washing gave the full effect. Thus the inhibitor takes 1-2 min. to attain an effective concentration at the site of action.
Effect of carbon and nitrogen nutrition The effect of varying the concentration of the C and N sources on the production of the inhibitor by F. oxysporum was tested first in liquid medium. For N, in addition to the basal medium, the same but with 0, 0'3, I, 3 and 10 g.jl.NH4NOs were used. After supporting growth
Fungus staling substance. D. Park
545
of the fungu s for I week staled solutions from th e first three of these caused permanent inhibition of A. niger, while the last three did not. Thus in a medium containing th e standard amount of glucose, increased N lowered the amount of the inhibitor produced. For C, media containing 0,0'2,0'7,2,7 and 20 g. glucose/I ., respectively, were used ; staled solutions from the first of these gave no permanent inhibition while the remaining five did. However, at 0'2 g./I. activity was no longer evident at dilution of the staled solution to 1; at 0'7 g./I. activity was evident at this dilution but not at i, whereas in the last three media dilution to i still had full effect. Thus, increasing the amount of glucose in a medium containing the standard amount of N permitted the development of greater conc entrations of the inhibitor in the stal ed solution. The effect of similar variation in composition of the medium supporting growth of A. niger upon th e sensitivity of that fungus was examined using media gelled with agar. In all media permanent inhibition of the tips developed after treatment with standard stal ed solution. There were, however, marked and interesting differences in the morphological responses of the hyphal tips in the few minutes before the walls had completely set, and in th e form of branching after subsequent growth further back. These morphological effects still require detail ed analysis but they do give an indication that with increasing conc entration of glucose in the medium on which th e test fungus is grown, it becomes less sensitive, while with increasing N it is more sensitive. This indication was confirmed by preparing serial dilutions from I to l6 of standard staled solution and applying them to the test fungu s on the different media. The results (T able I) show clearly that high glucose level confers tolerance and high N increases sensitivity . Table I. Effect of dilutions ofstandard staled solution on Aspergillus niger growing on media with diJferent C andN concentrations NH.NO s g.I ).
Glucose g,/I.
A
Dilution [
t t 1 --le_
A
0
0' 1
0 '3
1 '0
3
10
0
0 '2
0 '7
+
+ +
+ +
+ + +
+ + +
+ + + +
+ + + +
+ + +
+ + +
i.
+,
2
7
20
+ +
+
+
permanent inhibition; - , lack of permanent inhibition , DIS CUSSION
The results describ ed here provide clear evidence of an inhibitor not present in the original medium but produced by th e growth ofF. oxy sporum. The effect is not a negative one resulting from th e removal of some factor from the medium since it can itself be removed by adsorption and can be diluted out. The effect is not one of pH or osmotic pressure. The factor causes effects closely similar to th e staling effects described by Park (196 I) in agar cultures of F. oxysporum. The first detectable activity after more than 2 days coincides with th e time of development of
546
Transactions British Mycological Society
staling in the agar medium; in plates seeded with F. oxysporum staling effects were first seen after 2 days, and in centrally inoculated spreading colonies an area reached maximum staling after slightly more than 2 days from its inception at the growing margin. The disappearance of the inhibitor during storage of the staled solution agrees with the lability of the staling factor in the agar medium, while its persistence in autoclaved solution points to a biological removal by the fungus that produces it. The subsequent return of the inhibitor with re-growth of the fungus from residual or added inoculum also coincides with the dynamic pattern of autolysis balanced by growth described earlier from the agar cultures. It was suggested by Park (1961) that the inhibitor in a staled region became reduced by natural lability to a level permitting some growth, which in turn led to formation of more inhibitor increasing again to a fungistatic level. In the present study as in the earlier one there was no evidence that volatility could account for the disappearance from stored media. The removal of the effect by 'Cellophane' also parallels the finding in the earlier study. The production of greater concentrations of the inhibitor by F. oxysporum in media with high C and low N agrees with the greater tolerance of the test fungus, A. niger, under these same conditions if it is assumed that this is a common factor and that nutrition affects its production by the two organisms in the same way. From the earlier study it would be expected that under any set of conditions production would be limited by tolerance under those conditions so that when a fungistatic level is reached further growth, and hence further production of the inhibitor, is prevented until the concentration again falls below that level. Staled solution has been applied to tips of Absidia glauca Hagem., Achlya ambisexualis J. R. Raper, Alternaria tenuis auct., Aspergillus nidulans (Eidam) Wint., Botrytis cinerea Pers. ex Fr., Epicoccum nigrum Link, Fusarium culmorum (W. G. Smith) Sacc., Mucor plumbeus Bon., Neurospora crassa Shear & Dodge, Penicillium claviforme Bain., Phycomyces blakesleeanus Burgeff, Phytophthora erythroseptica Pethybr., Polyporus betulinus (Bull.) Fr., Pythium debaryanum Hesse, P. proliferum de Bary, Rhizopusstolonifer (Ehrenb. ex Fr.) Lind., Saprolegnia sp., Sordariafimicola (Rob.) Ces, & de Not., Trichothecium roseum Link ex Fr., and Zygorhynchus moelleri Yuill. growing on the agar medium, and while there were differences in the specific reactions to the solution all developed permanent inhibition. No fungus has been tested that has not shown this reaction to staled solution from F. oxysporum. The factor active in this test seems therefore to cause an effect like that investigated earlier in not being restricted in its action to the species producing it. REFERENCE
PARK, D. (1961). Morphogenesis, fungistasis and cultural staling in Fusarium oxysporum Snyder & Hansen. Trans. Brit. mycol. Soc. 44, 377-390.
(Accepted for publication 12 April 1964)