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Further experimental studies of bean rhizosphere fungi
[ 45 I
]
Trans. Br. mycol. Soc. 52 (3) , 451- 457 (1969) Printed in Great Britain
FURTHER EXPERIMENTAL STUDIES OF BEAN RHIZOSPHERE FUNGI By N.J. DIX* Department ofBotany, Chelsea College ofScience and Technology Some soil fungi are isolated frequently from the rhizosphere of the French bean iPhaseoius vulgaris cv. Prince) but only very rarely from the living or decaying roots. The ability of some of these fungi to colonize substrates in competition with the bean root invading fungi Fusarium osysporum and Gliocladium roseuni acting as potential antagonists has been measured. The spores of four out of five of these fungi were able to colonize dead virgin bean roots in sterile soil, and in a non-sterile soil , large pieces of dead virgin leaf substrate, in the presence of the potential antagonists. In a sterile soil the competitiveness of most of these fungi was considerably reduced when dead bean root substrates had been precolonized 24 h previously by on e of the potential antagonists. These results are discussed in relation to the general ecology of these fungi in the soil and their behaviour in the rh izosphere of the French bean.
In a previous study the course of the colonization of the roots of the French bean (Phaseolus vulgaris cv, Prince) by fungi from the rhizosphere was determined (Dix , 1964). Subsequently the spores of a selected number of these rhizosphere fungi were subjected to tests to determine their sensitivity to soil mycostasis (Dix, 1967). Among these species sensitivity could in many cases be correlated with the ecological behaviour of the fungus. Some fungi frequently isolated from the rhizosphere failed to colonize living or decaying bean roots and many of them were found to be relatively highly sensitive to the mycostatic factor in soils. It was suggested that these species may germinate in the rhizosphere but fail to colonize the roots because they succumb to antagonisms generated by the presence of other growing fungi. In this process an important factor for some species may be their relatively high sensitivity to mycostasis which may delay their germination in the rhizosphere. In order to test these hypotheses experimentally the ability of some of these fungi to colonize substrates in the soil in the presence ofsome potential antagonists has been determined. EXPERIMENTAL
Five non-colonizers of bean roots (the test fungi) were chosen for further study. Four with spores relatively highly sensitive to mycostasis Doratomyces purpureofuscus (Fr.) Morton & Smith, Mucor plumbeus Bon., Penicillium nigricans Bain. ex. Thorn, Trichocladium asperum (Corda) Harz. and another Trichothecium roseum (Pers.) Link ex. S. F. Gray, relatively insensitive to mycostasis.
* Present address: Department of Biology, The University, Stirling.
29-2
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Transactions British Mycological Society
The two bean root colonizing fungi chosen as potential antagonists were Fusarium oxysporum Schlecht. ex Fr., and Gliocladium roseum Bain. F. osysporum is relatively insensitive to mycostasis and they both appear early in the succession of living bean root surfaces and quickly become dominant organisms. They are therefore thought likely to be antagonists of the test fungi attempting to colonize bean roots from the rhizosphere. A point of significance here is that some strains at least of Gliocladium roseum are known to be mycoparasitic (Barnett & Lilly, 1962).
Experiment I In this experiment the colonization from spores of a sterile virgin dead bean root substrate by the test fungi was studied in the presence of spores of the potential antagonists. The fungi used in these experiments had been isolated from the rhizosphere and roots of the French bean and maintained in culture on suitable nutrient agar slopes in 113.6 ml (40z) medical flat bottles. Spores were obtained for the experiment from young cultures not more than 14 days old by shaking about 15 ml of sterile water in the culture. The spore suspensions were then filtered through sterile cotton wool, to remove large debris, and collected in sterile centrifuge tubes. A spore suspension of known concentration was then prepared by centrifuging and resuspending in sterile water. The final concentration was adjusted with the aid of a Thoma counting slide. To each suspension of spores of the test fungi was then added a suspension of spores of one of the antagonists. The final mixture gave a spore concentration of 45 x IoD/ml for each fungus. Into this spore suspension were then placed the prepared bean root substrates. The roots were agitated in the mixture, withdrawn, and buried about I em deep in sterile soil. The soil was forest loam autoclaved in large crystallizing dishes (14'5 x 6·6 em high) with lids. Bean root substrates were obtained from young French bean plants germinated in sterile sand. Fleshy portions of root about 1'5 em long were selected and lengths of cotton thread tied to them. They were then placed between cotton wool pads moistened with water in a Petri dish and autoclaved at 103'4 kN/ m 2 (lslb/in2 ) for 10 min. After 5 days incubation at 20°C the roots were recovered from the soil by means of the attached threads. They were then given two preliminary washings by shaking in vials of sterile water followed by five washes in sterile water in the sterile washing apparatus used in previous experiments (Dix, 1964). After washing the roots were chopped and teased into small pieces in sterile Petri dishes (one per dish). A suitable cooled nutrient agar was then poured on top and the dishes incubated at 20°. The dishes were examined at intervals and the presence or absence of the sporulating stage of the test fungus was recorded in at least twenty-five dishes in which the antagonist was present. The twenty-five observations were made between three experiments in which different spore populations were used. The colonization of the substrates by Trichothecium roseum was also studied in similar experiments but in the absence of spores of the antagon-
Rhizospherefungi. N.J. Dix
453
ists, The via bility of the spore populations used was checked by incubating a sample at 20 0 at 100 % r.h, in a damp chamber.
Experiment 2 In this experiment the colonization of large pi eces of sterile virgin substrate from the spores of the test fungi was studied in an unsterile soil in the pres ence of spores of the antagonists. The general experimental procedure was the same as in Expt. I and differed only in the following respects. The substrate used was Giyceria maxima leaf. Young living leaves were gathered, cut into portions about I x I em and autoclaved at 103'4 kN/m 2 for 10 min. They were then shaken in a mixed spore suspension containing 45 x lOS spores of each of the antagonist and a test fungus. The substrate was then removed and incubated in the unsterilized forest loam for 5 days. After washing and plating out the presence or absence of the sporulating stage of the test fungus was recorded in at least twenty-five dishes in which the antagonist was present. The twenty-five observations were made between four experiments. Leaves often became invaded by nematodes during the course of the experiment; these leaves were disregarded. Experiment 3 In this experiment the a bility of the test fungi to colonize substrates which had been precolonized by one of the antagonists was studied in a sterile soil. For comparison with the test fungi, two late colonizers of bean roots Volutella ciliata (Alb . & Schwein.) Fr. and Chaetomium globosum Kunze ex. Fr., both with spores relatively highly sensitive to mycostasis, were included. In addition, Gliocladium roseum, which is more sensitive to mycostasis than Fusarium oxysporum, was tested against this fungus. The bean roots were prepared as in Expt. I. The roots were carefully arranged on the surface ofa colony of-one ofthe a n tagonists growing on a potato dextrose agar plate. After incubation at 20 0 for 24 h the roots were removed from the plates and shaken in a suspension of one of the test fungi at a concentration of 45 x IOs/ml. The roots were then removed and incubated at 20 0 in sterile forest loam soil for 5 days. Thereafter they were treated as in Expt. I. The presence or absence of the sporulating stage of the test fungus was recorded in at least twenty-five dishes in which the antagonist was present spread over three different experiments. RESULTS
The number of positive observations of the test fungi in these three experime nts appear in Table I expressed as percentages. Under the conditions of Expt, I the competitive ability of most of the t est fungi was high and they successfully colonized the bean root substrate in competition with the antagonists. In the absence of any background a ntagonism in the soil, any antagonisms which might have been generated in the experiment by the germination and growth of the fungi were clearly
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Transactions British Mycological Society
insufficient to influence the course of colonization of the substrate. The exception was Trichothecium roseum which was only able to colonize the substrate in the absence of the antagonists. Similar results were obtained in Expt, 2 with the unsterile soil and the large leaf substrate. In Expt. 3, however, when the substrate had b een precolonized by one of the antagonists the ability of most of the test fungi to colonize the substrate was now severely limited by one or both of the antagonists. The one exception to this was Mucorplumbeus. It is significant perhaps that the fungi Chaetomium globosum and Gliocladium roseum which do colonize bean roots were able to colonize under these conditions although a late bean root colonizer Volutella ciliata was not. Table
I.
Percentage occurrence of the testfungi in the three experiments Antagonist
Fusarium osysporum-,
, - - - --A
Expt,
Doratomyces purpureofuscus Penicillium nigricans Mu corplumbeus Trichocladium asperum Trichothecium roseum
Gliocladium roseum Volutella ciliata Chaetomium globosum
I
93 95
2
3 100
100
16
63
20
100
47
100
10 0 90
100
100
85 a
83
17 a 93 25 95
None
~
100
10 0
a
Gliocladium roseum 3
75 o
II
60
o 100
DIS CUSSION
A simple explanation of why the test fungi did not colonize bean roots from the rhizosphere could be that they failed to germinate in the presence of the root. Ifroot exudates never reach a sufficiently high level to overcome the effects of soil mycostasis, then the spores of some fungi, which may be more sensitive to mycostasis than some others, may not be able to germinate. This may well be so in the case of Mucor plumbeus which in Expt. 3 was able to successfully compete for substrates precolonized by antagonists but which, although common in the rhizosphere, is known to have only a 2 % frequency of isolation from French bean roots (Dix, 1964). This fungus is known also to make nutritional demands upon the environment before the spores can germinate. These nutrients appear to be potentially available in the soil and were therefore considered unlikely to be generally limiting (Dix, 1967). It is possibl e, however, that there may be local nutrient deficiencies. If the spores of the test fungi germinate in the rhizosphere of the French bean, the experimental evidence is that they may do so later than the spores of either Fusarium oxysporum or Gliocladium roseum. In Expt. I, in the absence of any background antagonism in a sterile soil, it is reasonable to assume the spores of the fungi would have germinated as rapidly as their inherent charact eristics would allow. The results were that most of the best fungi successfully colonized the bean root substrates. The inference from this is that if the spores of all the bean rhizosphere fungi could germinate as
Rhizosphere fungi. N. J. Dix
455
successfully in the rhizosphere, then they would all colonize bean roots at least in the later stages of their decomposition. The fact that they do not suggests that they may not all germinate rapidly in the rhizosphere, and Expt. 3 demonstrates the possible consequences. In this experiment in the absence of any background antagonism, the inability of the test fungi to colonize the substrate appears to be entirely due to the occupation a short time previously of the substrate by one of the antagonists. The antagonisms so generated by the growth of earlier colonizers on the substrate appear to be sufficient in themselves to prevent further colonization by other species. Differences exist between one of the antagonists and the test fungi in their inherent growth characteristics. Compared with Fusarium oxysporum the average growth rates of the test fungi are considerably less and the spores of some species germinate more slowly. The average rate of growth (rnmjday) and the average latent period for spore germination in hours at 20 0 of the antagonists and the test fungi are as follows (growth rates first): Doratomyces purpureofuscus, 4.0, 12; Penicillium nigricans, 2'3, 13; Mucor plumbeus, 4'5, 7; Trichocladium asperum, 5'2, 7; Trichothecium roseum, 5'7, 7; Gliocladium roseum, 4'0, 12; Fusarium oxysporum, r o-5, 8. The growth rates were calculated from four replicate experiments on tap water agar inoculated with standard disks cut from tap water agar cultures. The latent period is the average of three experiments performed on Czapek-Dox agar. The spore population was said to have germinated, and the latent period ended, when about 50 % of the spores had germ tubes at least equal to the spore width. These differences, however, did not limit the ability of any species, save possibly one, to colonize the substrates in Expt. I. This suggests that the prior occupation of a substrate by a fungal species in nature, in some cases at least, cannot be due to any inherent differences which may exist between one species and another in the speed with which spores germinate or the rate at which mycelium grows. Other mechanisms must operate and one of these could be soil mycostasis which could delay the germination of the spores of some species. Mycostasis is known to have an unequal effect on the germination of spores of different species (Jackson, 1958; Dix, 1967). This differential effect may operate in the soil under certain conditions, e.g. low nutrient levels or high competition for nutrients. These experimental results would suggest that the key to the behaviour of the test fungi in the French bean rhizosphere lies in their relatively high sensitivity to soil mycostasis and would seem to support the view put forward by Dix (1967) that the germination of these spores is delayed in the rhizosphere until root exudation builds up at special sites by which time the adjacent root surface is already well colonized by more rapidly germinating species, i.e. those with spores relatively less sensitive to mycostasis such as Fusarium osysporum and Gliocladium roseum. The former species being unable to compete are thus prevented from colonizing the decaying root. The rate of spread of one of the early colonizers, Fusarium oxysporum, over tap water agar medium indicates that it has great potential for rapid spread over and occupation of substrates as they become available (see above). This could be an important secondary characteristic of this fungus which allied with its relatively low sensitivity to mycostasis enables it to dominate root substrates. The ability of a fungus when in
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Transactions British Mycological Society
occupation of a substrate to prevent other fungus species from colonizing does not appear to be uncommon, neither is it confined to specific ecological groups (Barton, 1961; Bruehl & Lai, 1966; Lai & Bruehl, 1968). The activity of Fusarium oxysporum and Gliocladium roseum against some of the test fungi here therefore appears to be part of a general antagonistic phenomenon but which from these experiments appears to be more active against some species than others. No experiments have been carried out to determine the nature of the antagonistic mechanism but it appears that specific antibiotic substances are not necessarily involved. Many of the fungi acting as antagonists in this way are not known antibiotic producers. Competition for nutrients or space or the production of staling substances by the precolonizers could be involved. In special instances the mycoparasitic activity of certain species such as Gliocladium roseum could be important. Barton (1960) concluded that the production of staling substances was responsible and this appears from his experiments to be the most satisfactory general explanation. Park (1963) has demonstrated the widespread production of staling substances by fungi and their widespread action affecting the growth of fungi, and recently Dwivedi & Garrett (1968) demonstrated that an important factor determining the ability of a soil fungus to colonize nutrient agar plates was a tolerance of fungistatic substances produced during the earlier growth of other fungi on the medium. The fungus Trichothecium roseum from these experiments appears to be a particularly weak competitor and these results are in agreement with some observations made by Park (1955) on the behaviour of this fungus. He found no colonization of substrates on the surface or beneath an unsterile soil and also the slow colonization of substrates under sterile conditions. The weak competitiveness of the fungus seems to be entirely consistent with the observation that although it appears to be relatively insensitive to soil mycostasis it is unable to colonize French bean roots. The results of Expt. 2 indicates the kind of substrate which fungi relatively highly sensitive to mycostasis could compete for and successfully colonize in nature. Large portions of dead or senescent plant material falling on to the soil may quickly release considerable quantities of organic exudates. These could have the effect of causing the rapid germination of the fungal species immediately adjacent to the substrate irrespective of their sensitivity to soil mycostasis. Whilst such substrates would not be virginal (Webster, 1956, 1957), they might be sparsely populated by fungal species. They would provide a larger surface area for colonization than the root, which may be an important feature for species attempting to colonize. The root surface and tissues may therefore be a rather special habitat for saprophytic fungi. My thanks are due to Mrs]. Poole for valuable technical assistance.
Rhizospherefungi. N.J. Dix
457
REFERENCES
BARNEl1, H. L. & LILLY, V. G. (1962). A destructive mycoparasite Gliocladium roseum. Mycologia 54. 72-77· BARTON, R. (1960). Antagonism amongst some sugar fungi. In The ecology of soil fungi (cd. by D. Parkinson and]. S. Waid). Liverpool University Press . BARTON, R. (1961). Saprophytic activity of Pythium mamillatum in soils. II. Factors restricting Pythium mamillatum to pioneer colonization of substrates. Trans. Br, mycol. Soc. 44, 105-118. BRUEHL, G. W. & LAI, P. (1966). Prior-colonization as a factor in the saprophytic survival of several fungi in wheat straw. Phytopathology 56,766-768. DIX, N.]. (1964). Colonization and decay of bean roots. Trans. Br, mycol. Soc. 47, 28529 2. DIX, N.]. (1967).~Mycostasisand root exudation: Factors influencing the colonization of bean roots by fungi. Trans. Br. mycol. Soc. 50, 23-31. DWIVEDI, R. S. & GARRETT, S. D. (1968). Fungal competition in agar plate colonization from soil inocula. Trans. Br. mycol. Soc. 51, 95-101. ]ACKSON, R. M. (1958). Some aspects of soil fungistasis. ]. gen. Microbiol. 19, 390-401. LAI, P. & BRUEHL, G. W. (1968). Antagonism among Cephalosporium gramineum, Trichoderma spp., and Fusarium culmorum. Phytopathology 58, 562-566. PARK, D. (1955). Experimental studies on the ecology of fungi in soil. Trans. Br. mycol. Soc. 38, 13°-142. PARK, D. (1963). Evidence for a common fungal growth regulator. Trans. Br. mycol, Soc. 46, 541-548. WEBSTER,]. (1956). Succession of fungi on decaying cocksfoot culms. I. ]. &01. 44, 5 17-544. WEBSTER,]. (1957). Succession offungi on decaying cocksfoot culms. II.]. &01. 45, I-gO.
(Acceptedfor publication 17 January 1969)
]
Trans. Br. mycol. Soc. 52 (3) , 451- 457 (1969) Printed in Great Britain
FURTHER EXPERIMENTAL STUDIES OF BEAN RHIZOSPHERE FUNGI By N.J. DIX* Department ofBotany, Chelsea College ofScience and Technology Some soil fungi are isolated frequently from the rhizosphere of the French bean iPhaseoius vulgaris cv. Prince) but only very rarely from the living or decaying roots. The ability of some of these fungi to colonize substrates in competition with the bean root invading fungi Fusarium osysporum and Gliocladium roseuni acting as potential antagonists has been measured. The spores of four out of five of these fungi were able to colonize dead virgin bean roots in sterile soil, and in a non-sterile soil , large pieces of dead virgin leaf substrate, in the presence of the potential antagonists. In a sterile soil the competitiveness of most of these fungi was considerably reduced when dead bean root substrates had been precolonized 24 h previously by on e of the potential antagonists. These results are discussed in relation to the general ecology of these fungi in the soil and their behaviour in the rh izosphere of the French bean.
In a previous study the course of the colonization of the roots of the French bean (Phaseolus vulgaris cv, Prince) by fungi from the rhizosphere was determined (Dix , 1964). Subsequently the spores of a selected number of these rhizosphere fungi were subjected to tests to determine their sensitivity to soil mycostasis (Dix, 1967). Among these species sensitivity could in many cases be correlated with the ecological behaviour of the fungus. Some fungi frequently isolated from the rhizosphere failed to colonize living or decaying bean roots and many of them were found to be relatively highly sensitive to the mycostatic factor in soils. It was suggested that these species may germinate in the rhizosphere but fail to colonize the roots because they succumb to antagonisms generated by the presence of other growing fungi. In this process an important factor for some species may be their relatively high sensitivity to mycostasis which may delay their germination in the rhizosphere. In order to test these hypotheses experimentally the ability of some of these fungi to colonize substrates in the soil in the presence ofsome potential antagonists has been determined. EXPERIMENTAL
Five non-colonizers of bean roots (the test fungi) were chosen for further study. Four with spores relatively highly sensitive to mycostasis Doratomyces purpureofuscus (Fr.) Morton & Smith, Mucor plumbeus Bon., Penicillium nigricans Bain. ex. Thorn, Trichocladium asperum (Corda) Harz. and another Trichothecium roseum (Pers.) Link ex. S. F. Gray, relatively insensitive to mycostasis.
* Present address: Department of Biology, The University, Stirling.
29-2
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Transactions British Mycological Society
The two bean root colonizing fungi chosen as potential antagonists were Fusarium oxysporum Schlecht. ex Fr., and Gliocladium roseum Bain. F. osysporum is relatively insensitive to mycostasis and they both appear early in the succession of living bean root surfaces and quickly become dominant organisms. They are therefore thought likely to be antagonists of the test fungi attempting to colonize bean roots from the rhizosphere. A point of significance here is that some strains at least of Gliocladium roseum are known to be mycoparasitic (Barnett & Lilly, 1962).
Experiment I In this experiment the colonization from spores of a sterile virgin dead bean root substrate by the test fungi was studied in the presence of spores of the potential antagonists. The fungi used in these experiments had been isolated from the rhizosphere and roots of the French bean and maintained in culture on suitable nutrient agar slopes in 113.6 ml (40z) medical flat bottles. Spores were obtained for the experiment from young cultures not more than 14 days old by shaking about 15 ml of sterile water in the culture. The spore suspensions were then filtered through sterile cotton wool, to remove large debris, and collected in sterile centrifuge tubes. A spore suspension of known concentration was then prepared by centrifuging and resuspending in sterile water. The final concentration was adjusted with the aid of a Thoma counting slide. To each suspension of spores of the test fungi was then added a suspension of spores of one of the antagonists. The final mixture gave a spore concentration of 45 x IoD/ml for each fungus. Into this spore suspension were then placed the prepared bean root substrates. The roots were agitated in the mixture, withdrawn, and buried about I em deep in sterile soil. The soil was forest loam autoclaved in large crystallizing dishes (14'5 x 6·6 em high) with lids. Bean root substrates were obtained from young French bean plants germinated in sterile sand. Fleshy portions of root about 1'5 em long were selected and lengths of cotton thread tied to them. They were then placed between cotton wool pads moistened with water in a Petri dish and autoclaved at 103'4 kN/ m 2 (lslb/in2 ) for 10 min. After 5 days incubation at 20°C the roots were recovered from the soil by means of the attached threads. They were then given two preliminary washings by shaking in vials of sterile water followed by five washes in sterile water in the sterile washing apparatus used in previous experiments (Dix, 1964). After washing the roots were chopped and teased into small pieces in sterile Petri dishes (one per dish). A suitable cooled nutrient agar was then poured on top and the dishes incubated at 20°. The dishes were examined at intervals and the presence or absence of the sporulating stage of the test fungus was recorded in at least twenty-five dishes in which the antagonist was present. The twenty-five observations were made between three experiments in which different spore populations were used. The colonization of the substrates by Trichothecium roseum was also studied in similar experiments but in the absence of spores of the antagon-
Rhizospherefungi. N.J. Dix
453
ists, The via bility of the spore populations used was checked by incubating a sample at 20 0 at 100 % r.h, in a damp chamber.
Experiment 2 In this experiment the colonization of large pi eces of sterile virgin substrate from the spores of the test fungi was studied in an unsterile soil in the pres ence of spores of the antagonists. The general experimental procedure was the same as in Expt. I and differed only in the following respects. The substrate used was Giyceria maxima leaf. Young living leaves were gathered, cut into portions about I x I em and autoclaved at 103'4 kN/m 2 for 10 min. They were then shaken in a mixed spore suspension containing 45 x lOS spores of each of the antagonist and a test fungus. The substrate was then removed and incubated in the unsterilized forest loam for 5 days. After washing and plating out the presence or absence of the sporulating stage of the test fungus was recorded in at least twenty-five dishes in which the antagonist was present. The twenty-five observations were made between four experiments. Leaves often became invaded by nematodes during the course of the experiment; these leaves were disregarded. Experiment 3 In this experiment the a bility of the test fungi to colonize substrates which had been precolonized by one of the antagonists was studied in a sterile soil. For comparison with the test fungi, two late colonizers of bean roots Volutella ciliata (Alb . & Schwein.) Fr. and Chaetomium globosum Kunze ex. Fr., both with spores relatively highly sensitive to mycostasis, were included. In addition, Gliocladium roseum, which is more sensitive to mycostasis than Fusarium oxysporum, was tested against this fungus. The bean roots were prepared as in Expt. I. The roots were carefully arranged on the surface ofa colony of-one ofthe a n tagonists growing on a potato dextrose agar plate. After incubation at 20 0 for 24 h the roots were removed from the plates and shaken in a suspension of one of the test fungi at a concentration of 45 x IOs/ml. The roots were then removed and incubated at 20 0 in sterile forest loam soil for 5 days. Thereafter they were treated as in Expt. I. The presence or absence of the sporulating stage of the test fungus was recorded in at least twenty-five dishes in which the antagonist was present spread over three different experiments. RESULTS
The number of positive observations of the test fungi in these three experime nts appear in Table I expressed as percentages. Under the conditions of Expt, I the competitive ability of most of the t est fungi was high and they successfully colonized the bean root substrate in competition with the antagonists. In the absence of any background a ntagonism in the soil, any antagonisms which might have been generated in the experiment by the germination and growth of the fungi were clearly
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Transactions British Mycological Society
insufficient to influence the course of colonization of the substrate. The exception was Trichothecium roseum which was only able to colonize the substrate in the absence of the antagonists. Similar results were obtained in Expt, 2 with the unsterile soil and the large leaf substrate. In Expt. 3, however, when the substrate had b een precolonized by one of the antagonists the ability of most of the test fungi to colonize the substrate was now severely limited by one or both of the antagonists. The one exception to this was Mucorplumbeus. It is significant perhaps that the fungi Chaetomium globosum and Gliocladium roseum which do colonize bean roots were able to colonize under these conditions although a late bean root colonizer Volutella ciliata was not. Table
I.
Percentage occurrence of the testfungi in the three experiments Antagonist
Fusarium osysporum-,
, - - - --A
Expt,
Doratomyces purpureofuscus Penicillium nigricans Mu corplumbeus Trichocladium asperum Trichothecium roseum
Gliocladium roseum Volutella ciliata Chaetomium globosum
I
93 95
2
3 100
100
16
63
20
100
47
100
10 0 90
100
100
85 a
83
17 a 93 25 95
None
~
100
10 0
a
Gliocladium roseum 3
75 o
II
60
o 100
DIS CUSSION
A simple explanation of why the test fungi did not colonize bean roots from the rhizosphere could be that they failed to germinate in the presence of the root. Ifroot exudates never reach a sufficiently high level to overcome the effects of soil mycostasis, then the spores of some fungi, which may be more sensitive to mycostasis than some others, may not be able to germinate. This may well be so in the case of Mucor plumbeus which in Expt. 3 was able to successfully compete for substrates precolonized by antagonists but which, although common in the rhizosphere, is known to have only a 2 % frequency of isolation from French bean roots (Dix, 1964). This fungus is known also to make nutritional demands upon the environment before the spores can germinate. These nutrients appear to be potentially available in the soil and were therefore considered unlikely to be generally limiting (Dix, 1967). It is possibl e, however, that there may be local nutrient deficiencies. If the spores of the test fungi germinate in the rhizosphere of the French bean, the experimental evidence is that they may do so later than the spores of either Fusarium oxysporum or Gliocladium roseum. In Expt. I, in the absence of any background antagonism in a sterile soil, it is reasonable to assume the spores of the fungi would have germinated as rapidly as their inherent charact eristics would allow. The results were that most of the best fungi successfully colonized the bean root substrates. The inference from this is that if the spores of all the bean rhizosphere fungi could germinate as
Rhizosphere fungi. N. J. Dix
455
successfully in the rhizosphere, then they would all colonize bean roots at least in the later stages of their decomposition. The fact that they do not suggests that they may not all germinate rapidly in the rhizosphere, and Expt. 3 demonstrates the possible consequences. In this experiment in the absence of any background antagonism, the inability of the test fungi to colonize the substrate appears to be entirely due to the occupation a short time previously of the substrate by one of the antagonists. The antagonisms so generated by the growth of earlier colonizers on the substrate appear to be sufficient in themselves to prevent further colonization by other species. Differences exist between one of the antagonists and the test fungi in their inherent growth characteristics. Compared with Fusarium oxysporum the average growth rates of the test fungi are considerably less and the spores of some species germinate more slowly. The average rate of growth (rnmjday) and the average latent period for spore germination in hours at 20 0 of the antagonists and the test fungi are as follows (growth rates first): Doratomyces purpureofuscus, 4.0, 12; Penicillium nigricans, 2'3, 13; Mucor plumbeus, 4'5, 7; Trichocladium asperum, 5'2, 7; Trichothecium roseum, 5'7, 7; Gliocladium roseum, 4'0, 12; Fusarium oxysporum, r o-5, 8. The growth rates were calculated from four replicate experiments on tap water agar inoculated with standard disks cut from tap water agar cultures. The latent period is the average of three experiments performed on Czapek-Dox agar. The spore population was said to have germinated, and the latent period ended, when about 50 % of the spores had germ tubes at least equal to the spore width. These differences, however, did not limit the ability of any species, save possibly one, to colonize the substrates in Expt. I. This suggests that the prior occupation of a substrate by a fungal species in nature, in some cases at least, cannot be due to any inherent differences which may exist between one species and another in the speed with which spores germinate or the rate at which mycelium grows. Other mechanisms must operate and one of these could be soil mycostasis which could delay the germination of the spores of some species. Mycostasis is known to have an unequal effect on the germination of spores of different species (Jackson, 1958; Dix, 1967). This differential effect may operate in the soil under certain conditions, e.g. low nutrient levels or high competition for nutrients. These experimental results would suggest that the key to the behaviour of the test fungi in the French bean rhizosphere lies in their relatively high sensitivity to soil mycostasis and would seem to support the view put forward by Dix (1967) that the germination of these spores is delayed in the rhizosphere until root exudation builds up at special sites by which time the adjacent root surface is already well colonized by more rapidly germinating species, i.e. those with spores relatively less sensitive to mycostasis such as Fusarium osysporum and Gliocladium roseum. The former species being unable to compete are thus prevented from colonizing the decaying root. The rate of spread of one of the early colonizers, Fusarium oxysporum, over tap water agar medium indicates that it has great potential for rapid spread over and occupation of substrates as they become available (see above). This could be an important secondary characteristic of this fungus which allied with its relatively low sensitivity to mycostasis enables it to dominate root substrates. The ability of a fungus when in
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Transactions British Mycological Society
occupation of a substrate to prevent other fungus species from colonizing does not appear to be uncommon, neither is it confined to specific ecological groups (Barton, 1961; Bruehl & Lai, 1966; Lai & Bruehl, 1968). The activity of Fusarium oxysporum and Gliocladium roseum against some of the test fungi here therefore appears to be part of a general antagonistic phenomenon but which from these experiments appears to be more active against some species than others. No experiments have been carried out to determine the nature of the antagonistic mechanism but it appears that specific antibiotic substances are not necessarily involved. Many of the fungi acting as antagonists in this way are not known antibiotic producers. Competition for nutrients or space or the production of staling substances by the precolonizers could be involved. In special instances the mycoparasitic activity of certain species such as Gliocladium roseum could be important. Barton (1960) concluded that the production of staling substances was responsible and this appears from his experiments to be the most satisfactory general explanation. Park (1963) has demonstrated the widespread production of staling substances by fungi and their widespread action affecting the growth of fungi, and recently Dwivedi & Garrett (1968) demonstrated that an important factor determining the ability of a soil fungus to colonize nutrient agar plates was a tolerance of fungistatic substances produced during the earlier growth of other fungi on the medium. The fungus Trichothecium roseum from these experiments appears to be a particularly weak competitor and these results are in agreement with some observations made by Park (1955) on the behaviour of this fungus. He found no colonization of substrates on the surface or beneath an unsterile soil and also the slow colonization of substrates under sterile conditions. The weak competitiveness of the fungus seems to be entirely consistent with the observation that although it appears to be relatively insensitive to soil mycostasis it is unable to colonize French bean roots. The results of Expt. 2 indicates the kind of substrate which fungi relatively highly sensitive to mycostasis could compete for and successfully colonize in nature. Large portions of dead or senescent plant material falling on to the soil may quickly release considerable quantities of organic exudates. These could have the effect of causing the rapid germination of the fungal species immediately adjacent to the substrate irrespective of their sensitivity to soil mycostasis. Whilst such substrates would not be virginal (Webster, 1956, 1957), they might be sparsely populated by fungal species. They would provide a larger surface area for colonization than the root, which may be an important feature for species attempting to colonize. The root surface and tissues may therefore be a rather special habitat for saprophytic fungi. My thanks are due to Mrs]. Poole for valuable technical assistance.
Rhizospherefungi. N.J. Dix
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REFERENCES
BARNEl1, H. L. & LILLY, V. G. (1962). A destructive mycoparasite Gliocladium roseum. Mycologia 54. 72-77· BARTON, R. (1960). Antagonism amongst some sugar fungi. In The ecology of soil fungi (cd. by D. Parkinson and]. S. Waid). Liverpool University Press . BARTON, R. (1961). Saprophytic activity of Pythium mamillatum in soils. II. Factors restricting Pythium mamillatum to pioneer colonization of substrates. Trans. Br, mycol. Soc. 44, 105-118. BRUEHL, G. W. & LAI, P. (1966). Prior-colonization as a factor in the saprophytic survival of several fungi in wheat straw. Phytopathology 56,766-768. DIX, N.]. (1964). Colonization and decay of bean roots. Trans. Br, mycol. Soc. 47, 28529 2. DIX, N.]. (1967).~Mycostasisand root exudation: Factors influencing the colonization of bean roots by fungi. Trans. Br. mycol. Soc. 50, 23-31. DWIVEDI, R. S. & GARRETT, S. D. (1968). Fungal competition in agar plate colonization from soil inocula. Trans. Br. mycol. Soc. 51, 95-101. ]ACKSON, R. M. (1958). Some aspects of soil fungistasis. ]. gen. Microbiol. 19, 390-401. LAI, P. & BRUEHL, G. W. (1968). Antagonism among Cephalosporium gramineum, Trichoderma spp., and Fusarium culmorum. Phytopathology 58, 562-566. PARK, D. (1955). Experimental studies on the ecology of fungi in soil. Trans. Br. mycol. Soc. 38, 13°-142. PARK, D. (1963). Evidence for a common fungal growth regulator. Trans. Br. mycol, Soc. 46, 541-548. WEBSTER,]. (1956). Succession of fungi on decaying cocksfoot culms. I. ]. &01. 44, 5 17-544. WEBSTER,]. (1957). Succession offungi on decaying cocksfoot culms. II.]. &01. 45, I-gO.
(Acceptedfor publication 17 January 1969)