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Growth and germination of Trichoderma spp. under the influence of soil fungistasis
[ 235 ] Trans. Br. mycol. Soc. 64 (2), 235-241 (1975) Printed in Great Britain
GROWTH AND GERMINATION OF TRICHODERMA SPP. UNDER THE INFLUENCE OF SOIL FUNGISTASIS By C. P. MITCHELL*
AND
N.]. DIX
Biology Department, The University, Stirling (With
1
Text-figure)
The effect of soil fungistasis on the germination and growth of 19 Trichoderma isolates from seven species groups has been measured on four parameters. The most widespread effect was in reducing the final percentage germination but other parameters of many isolates remained unaffected. The potential amount of growth in the presence of fungistasis has been calculated and called the Theoretical Colonization Index. The value of the index, which varied considerably between isolates, was modified by fungistasis mainly through a reduction in the final percentage germination.
The competitive saprophytic ability of a fungus, or the inherent factors involved in determining the ability of a fungus to colonize a substrate in soil, were first recognized by Garrett (1950) as rapid spore germination and growth, ability to produce appropriate enzymes, ability to produce antibiotics and the tolerance of antibiotics. Park (1960) put forward the view that the ability to tolerate the antagonistic activities of competing organisms was the key to competitive success. The ability to produce antibiotics and enzymes depends in the first instance on the germination of spores and growth. The amount of growth will be determined by the degree of tolerance of the organism to the antagonistic interactions in the soil. In soil, fungistasis is a major contributory factor to the antagonistic background whether it is regarded as an inhibitory effect produced by lack of nutrients or a true antibiosis. This paper is concerned with an attempt to measure and compare the ability of 19 Trichoderma isolates from seven species groups to grow and germinate in the presence of soil fungistasis. The overall effect of soil fungistasis on these fungi has been assessed by measuring four parameters; final percentage germination, rate of germination, latent period of germination and rate of germ-tube growth. Some of this information has been used to calculate the potential amount of growth when in contact with soil by the formulation of a growth index. This index has been termed the Theoretical Colonization Index (TCI) and is considered to reflect the ability of that spore population to grow and colonize a substrate in soil. II< Present address: Department of Forestry, University of Aberdeen, Old Aberdeen, AB92UU.
Transactions British Mycological Society MATERIALS AND METHODS
The species groups of Trichoderma selected for these experiments were T. hamatum (Ben.) Bain (2 isolates), T. hareianum Rifai (3 isolates), T. koningii Oud. (I isolate), T. longibrachiatum Rifai (I isolate), T. pseudokoningii Rifai (I isolate), T. saturnispora Hammill (I isolate), T. viride Pers. ex S. F. Gray (10 isolates). Full details of their origins are given in Mitchell (1973). The sensitivity of the spores to fungistasis was measured using the method developed by Dix (1967, 1972). Sifted air-dried soil (1'5 kg) of pH 4.6 from a mixed deciduous woodland was made up to 60 % water holding capacity in a plastic seed tray and the surface smoothed. Strips of sterile Whatman number I filter paper 3 x 2 em were moistened with sterile distilled water and placed on the soil surface, making sure that there was even contact with the soil. A peptone agar block 600 p,m thick was placed on the surface of each of the pieces of paper. A drop (0'2 em") of a spore suspension was placed on the surface of each block. Controls were similarly prepared blocks placed on sterile microscope slides on the surface of the same soil, the tray was sealed with a sheet of glass and incubated at 25 DC. At set time intervals three agar blocks and a control slide were removed from the soil, stained with lactophenol and cotton blue, and examined microscopically. At least 200 spores were counted randomly on each agar block and the number germinated expressed as a percentage which was arc-sin transformed for use in further analyses. Measurements were made of 30 germ-tubes chosen randomly for each treatment. A map measurer was used to measure (in arbitrary units) the length of germ-tubes drawn with the aid of a projection microscope at x 400 magnification. Spore suspensions for use in the experiments were collected from cultures growing on potato dextrose agar after a 3-week incubation period. The spores were washed off the culture with sterile distilled water, concentrated and washed twice by centrifugation and resuspended in sterile distilled water to give a final concentration of 45 x 10 5 cm- 3• RESULTS
The percentage germination of each of the replicates at each time interval was plotted against time. A logistic curve was found to be the best fit for the data. The curves obtained are typified by the results for T. hamatum (22) spores as shown in Fig. I. The form of the curve differs from that obtained by Dix (1972) and Dix & Christie (1974). This is probably because they sampled before and after and not during the lag period of the curve. Their results will therefore relate only to the exponential and later stages of the curve. The final percentage germination was obtained from the graph and was taken as the percentage germination on the upper plateau of the logistic curve. The results for the final percentage germination are given in Table I. The percentage reduction in performance based on that of the control has been calculated so that comparisons may be made between the species and isolates (see Table 2). There was a wide variation in sensitivity to fungistasis as indicated by the
Fungistasis. C. P. Mitchell and N. J. Dix
237
100
.. ......---
80
g
•
(; o
60
<:
02
I
~
......
Experiment
§____
•
•• §
8"""'-
~I
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Control
40
0 0
20
o Fig.
I.
4
8
12 Time (h)
16
20
The effect offungistasis on percentage germination of T. hamatum
22
spores.
percentage reduction in final percentage germination. When subjected to Student's t tests the values of the final percentage germination for the control and the experiment were found to be significantly different from one another in all cases. T. viride (49) showed the least sensitivity, T. viride (53) the greatest. The rate of germination to the final (maximum) percentage germination was taken as the regression coefficient of the straight line obtained from a plot oflog1 0 Nmax -N/N against time (where N is the percentage germination at any time t and N max is the maximum percentage germination). The rates of germination for the different isolates, as regression coefficients, are shown in Table 1. In order to ascertain if the regression lines, and hence the rates, calculated for the control and experiment were significantly different from one another they were subjected to a variance (F ) ratio test. In all cases, except that of T. longibrachiatum and T. viride (50) there was a reduction in the germination rate but this was significant for only six of the fungi tested (T. pseudokoningii, T. viride (14), T. viride (28), T. viride (50), T . viride (52), and T. viride (53)). T. viride (53) being the most sensitive (see Table 2). The latent period of germination as a measure of the lag phase is here defined as the time taken for 25 % of the spore population to germinate and is calculated by substituting the known values in the regression equation obtained in th e calculation of the rate of germination. The values for the latent period in the control and experiment and the percentage reduction in performance for each of the isolates are given in Tables 1 and 2. The germ-tube growth rates, for the different isolates are given as regression coefficients in Table 1. These have been calculated from the
N w
00
Table
I.
Measurements offinal percentage germination, rate of germination, latentperiod of germination and germ-tube growth rate under conditions of soilfungistasis Final % germination
Latent period (h)
Germination ra te (as regression coeff.)
Germ tube growth rate (as regression coeff.)
Control
Experiment
Control
Experiment
Control
Experiment
Control
Experiment
s....~
~15)
74'0 79'8
39'6 59'S
12,6 13'0
24'9 13'6
-0'1817 -0' 2167
-0'1443 -0'1701
0'1222 0'°970
0' 0673 0'07 05
~.
(I)
80'3 58,6 81'2 77,8
66,6 25'4 59'9 36.6
47'1 85'4 74'0
36'S 43·3 53'6
/0,8 16,6 12,6 13,6 16'3 14'1 I 1'4
9'2 <24'0 20'1 21'8 19'0 19'8 12'3
-0'2390 -o'II39 -0'2 167 -0'1270 -0'095° -0'1507 -0'1 786
-0' 22 15 -0' 1109 -0' 1864 -0'1229 -0'14 16 -0'08 17 -0'1537
0'093 8 0'°433 0'1133 0'003 6 0' 0224 0'°742 0'05 8 1
0' 08°4 0' 0 14° 0'°792 0'0102 0'0076 0' 0294 0'0467
,
A
T, hamatum 22) T, harzianum
(129~ (20 T . koningii T, longibrachiatum T. pseudokoningii T , satumispora T, viride
(2~
(14
~:~~ 4-9) (5°) (51) (52) (53) (54)
,
,
,
\
,
A
\
,
I
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...~
b:1
::!. ~. ~
~ ~
S'
()tj
50 '8 81'S 45'9 8°'9 67'7 8°'9 78'0 83 '0 5 0'7 72'2
35'2 21'7 25'2 48'1 62'3 66'3 4 1'9 24'6 7.8 5 1'0
15'3 13'9 19'2 iI '3 II'S II'O
14'6 15,6 17'6 13'4
23'S 35'1 33'6 13'1 12·8 9'6 18'7 33'1 <2!'O I '3
-0'1418 -0'1423 -0'1367 -0'1968 -0'154 1 -0'25 19 -0'1447 -0'1457 -0'1383 -0'lg86
-0'1344 -0' 0675 -0'09 86 -0'14-63 -0'17 03 -0'1442 -0'1437 -0'085 2 -0'04-53 -0'0986
0'025 1 0'0828 0'°7 11 0' 1093 0' 1069 0'1338 0' 0849 0'0612 0'07 18 0'04 63
0'0281 0'0610 0'09640'064-2 0'05 69 0' 1092 0' 0669 0'03 17 0'0166 0'050 1
~.
+:l .....
~
~. Q
Fungistasis. C. P. Mitchell and N. J. Dix Table
2.
Percentage reduction in performance due to soilfungistasis % reduction in final % germination
T, hamatum (IS) (22) T, harzianum (I) ( 129) (20) T, kongingii T, longibrachiatum T, pseudokoningii T, satumispora T, viride (2) (14) (28) (48) (49) (so) (SI) (S2) (S3) (54)
239
% reduction in latent period
% reduction in
germination rate
% reduction in germ-tube growth rate
46'5*** 2S'4***
49'4 4'4
20'6 NS 21'S NS
44'9* 27'3 NS
17'0*** S6'8*** 26'2*** S2'9*** 22'7*** 49'3** 27'6**
0'0 100'0 37'3 37'6 14'2 28,8 7'3
7'3 NS 2,6 NS 14'0 NS 3'2 NS 0'0 NS 4S'8*** 13'9 NS
14'3 NS 67'7** 3°'1 NS 0'0 NS 66'1* 6°'4* 19'6 NS
3°'7*** 73'4** 45"1* 40'6*** 8'0* 18'1** 46'3*** 7°'4*** 84'6*** 29'4***
34'9 6°'4 42'8 13,8 10'2 0'0 21'9 S2'9 100'0 17,8
S'2 NS 52'6** 27'9*** 2S'7 NS 0'0 NS 42'8* 0'7 NS 42'9*** 67'3*** 28'9 NS
0'0 NS 26'3 NS 0'0 NS 41'3 NS 46'8* 18'4 NS 21'2 NS {8'2 NS 76'9* 0'0 NS
* Significant: P < 0'05, ** Significant: P < 0'01. *** Significant: P < 0'001. NS, Not significant: P > o-og.
Table 3, The theoretical colonization index andpercentage reduction T, hamatum (IS) (22) T, harzianum (I) (129) (20) T. koningii T, longibrachiatum T, pseudokoningii T, saturnispora T, viride (2) (14) (28) ({8) t49) (So) (SI) (S2) (53) (S4)
16
Control
Experiment
Reduction t %)
6011'3 6278'4
S24'8 1626,S
9 1'3 74'1
8 184'6 103° ' 1 4723'1 1089'2 765'0 9498'6 277S'o
4°9 2'4 123'6 12S5"1 201'1 2so'8 422'8 117S'4
5°'0 88'0 73'4 81'S 67'2 9S'6 S7'6
54°'3 S890'6 2632'8 8125'7 6442'6 2862'6 S021'8 S839'6 4495'2 4983'8
230'6 367'2 21S'4 1686,6 ISIS,6 IISI'O 471'9 273'4 45'2 2188'3
S7'3 93'8 9 1,8 79'2 76'S S9'8 90'6 9S'3 99'0 so'o
Myc:64
240
Transactions British Mycological Society
average of the common log values of the germ-tube lengths at each time. The variance (F) ratio was used to test for significant differences between the regression lines. Only six of the isolates (T. hamatum (15), T. harzianum (129), T. longibrachiatum, T. pseudokoningii, T. viride (49) and T. viride (53)) had germ-tube growth rates on soil significantly different from those in the control (see Table 2). The TCI was calculated by taking the product of the percentage germination at any time and the mean germ-tube length at the same time. The TCI for each of the isolates after 24 h incubation is shown in Table 3 where for comparative purposes the TCI for each has also been expressed as a percentage reduction based on that of the same population in the control. The use of the TCI enables predictions to be made of the ability of different spore populations to germinate in the presence of a simple substrate in soil and colonize it. The advantage of this index is that it is a measure not only of the effect of antagonism on one area of germination but of the overall effect on the ability of the spores to colonize a substrate. There was a large variation in the TCI of the different isolates, but in most cases fairly substantial growth was made. T. viride (53) with a TCI of only 45'2 shows the least growth whereas T. harzianum (I) with an index of 4092'4 has the highest growth under antagonistic conditions. DISCUSSION
In general many Trichoderma spp. appear to be relatively insensitive to soil fungistasis. Soil fungistasis had its greatest effect, among the species tested, on the final percentage germination, since in many cases, once germination had begun, growth and the rate of germination were not significantly affected. However, there was considerable variation in the value of the TCI, even among different isolates of the same species, suggesting that there might be considerable variation in the amount of growth different strains might make in the soil. All, however, were capable of growth indicating that it would theoretically be possible for all of them to exert an antagonistic effect upon competing organisms. The percentage reduction in the TCI has a higher positive correlation with the percentage reduction in the final percentage germination (r = 0'7759, P < 0'001), than the germ-tube growth rate (r = 0'4774, P < 0'05)' This suggests that in Trichoderma spp. soil fungistasis modifies the TCI most significantly through a reduction in germination rather than through a reduction in the growth rate of germ-tubes. Differences in the numbers of spores germinating may therefore be the most important factor contributing to differences in the size of the TCI in Trichoderma spp. The percentage reduction in the TCI is also correlated positively with percentage reduction in the rate of germination (r = 0'5168, P < 0'05), and the latent period of germination (r = 0,6586, P < 0'01). If the TCI is a valid measure of growth and reflects the competitive saprophytic ability of a fungus under antagonistic conditions then it would appear that the numbers of spores germinating in a Trichoderma population may be the most significant factor determining whether colonization of a substrate proceeds or not. Dix (1972) posed the question on
Fungistasis. C. P. Mitchell and N. J. Dix what aspect of germination does soil fungistasis have its most important effect? He suggested that provided some germination takes place, the most significant effect was likely to be on the growth rate of germ-tubes. These results would indicate that this concept may underestimate the significance of the contribution to differences in total growth made by the number of spores germinating where the differences between the growth rates of different species in the presence of soil fungistasis are slight compared with differences in the numbers of spores germinating. However, where competition for colonization is between germinating single spores differences in the amounts of growth produced will be due mainly to differences in the growth rate of the germ-tubes. Once again the question arises as to whether colonization in soil is most often effected by the germination of isolated single spores or by a number of spores germinating in locally dense populations. A number of interesting relationships exist between the effects of soil fungistasis on the different parameters of germination. The reduction in the latent period was correlated positively with both the reduction in the final percentage germination and the germ-tube growth rate (r = 0,8882, P < o-oor and r = 0'5430, P < 0'05 respectively). Dix (r972), for Penicillium spp., found a correlation only between reduction in the latent period and the final percentage germination. The latent period in the control was correlated positively with the reduction in the final percentage germination (r = 0'9055, P < o·oor). A similar result was obtained by Steiner & Lockwood (r969). Analysis of the results ofDix (r972) shows the same correlation. Thus the present results and those of Dix (r 972) support the hypothesis of Steiner & Lockwood (r969) that the time of germination can have a significant effect upon the response of the spore to fungistasis. This work is part of a thesis presented for the degree of Ph.D. in the University of Stirling. One of us (C. P. Mitchell) is indebted to the Science Research Council for financial support. REFERENCES
DIX, N.]. (1967). Mycostasis and root exudation: factors influencing the colonization of bean roots by fungi. Transactions of the British Mycological Society 50, 23-3 I. DIX, N.]. (1972). Effect of soil fungistasis on spore germination and germ tube growth in Penicillium species. Transactions of the British Mycological Society 58, 59-66. DIX, N.]. & CHRISTIE, P. (1974). Changing sensitivity to soil fungistasis with age in Drechslera rostrata spores and associated permeability changes. Transactions of the British Mycological Society 62, 527-535. GARRETT, S. D. (1950). Ecology of the root inhabiting fungi. Biological Reviews 1115, 220-254. MITCHELL, C. P. (1973). Antagonistic interactions involving Trichoderma species. Ph.D. Thesis, University of Stirling. PARK, D. (1960). Antagonism - the background to soil fungi. The Ecology of Soil Fungi (ed. D. Parkinson &]. S. Waid), pp. 148-159. Liverpool: University Press. STEINER, G. W. & LOCKWOOD, ]. L. (1969). Soil fungistasis: sensitivity of spores in relation to germination time and size. Phytopathology 59, 1084-1092.
(Accepted for publication r5 August r974) 16'2
GROWTH AND GERMINATION OF TRICHODERMA SPP. UNDER THE INFLUENCE OF SOIL FUNGISTASIS By C. P. MITCHELL*
AND
N.]. DIX
Biology Department, The University, Stirling (With
1
Text-figure)
The effect of soil fungistasis on the germination and growth of 19 Trichoderma isolates from seven species groups has been measured on four parameters. The most widespread effect was in reducing the final percentage germination but other parameters of many isolates remained unaffected. The potential amount of growth in the presence of fungistasis has been calculated and called the Theoretical Colonization Index. The value of the index, which varied considerably between isolates, was modified by fungistasis mainly through a reduction in the final percentage germination.
The competitive saprophytic ability of a fungus, or the inherent factors involved in determining the ability of a fungus to colonize a substrate in soil, were first recognized by Garrett (1950) as rapid spore germination and growth, ability to produce appropriate enzymes, ability to produce antibiotics and the tolerance of antibiotics. Park (1960) put forward the view that the ability to tolerate the antagonistic activities of competing organisms was the key to competitive success. The ability to produce antibiotics and enzymes depends in the first instance on the germination of spores and growth. The amount of growth will be determined by the degree of tolerance of the organism to the antagonistic interactions in the soil. In soil, fungistasis is a major contributory factor to the antagonistic background whether it is regarded as an inhibitory effect produced by lack of nutrients or a true antibiosis. This paper is concerned with an attempt to measure and compare the ability of 19 Trichoderma isolates from seven species groups to grow and germinate in the presence of soil fungistasis. The overall effect of soil fungistasis on these fungi has been assessed by measuring four parameters; final percentage germination, rate of germination, latent period of germination and rate of germ-tube growth. Some of this information has been used to calculate the potential amount of growth when in contact with soil by the formulation of a growth index. This index has been termed the Theoretical Colonization Index (TCI) and is considered to reflect the ability of that spore population to grow and colonize a substrate in soil. II< Present address: Department of Forestry, University of Aberdeen, Old Aberdeen, AB92UU.
Transactions British Mycological Society MATERIALS AND METHODS
The species groups of Trichoderma selected for these experiments were T. hamatum (Ben.) Bain (2 isolates), T. hareianum Rifai (3 isolates), T. koningii Oud. (I isolate), T. longibrachiatum Rifai (I isolate), T. pseudokoningii Rifai (I isolate), T. saturnispora Hammill (I isolate), T. viride Pers. ex S. F. Gray (10 isolates). Full details of their origins are given in Mitchell (1973). The sensitivity of the spores to fungistasis was measured using the method developed by Dix (1967, 1972). Sifted air-dried soil (1'5 kg) of pH 4.6 from a mixed deciduous woodland was made up to 60 % water holding capacity in a plastic seed tray and the surface smoothed. Strips of sterile Whatman number I filter paper 3 x 2 em were moistened with sterile distilled water and placed on the soil surface, making sure that there was even contact with the soil. A peptone agar block 600 p,m thick was placed on the surface of each of the pieces of paper. A drop (0'2 em") of a spore suspension was placed on the surface of each block. Controls were similarly prepared blocks placed on sterile microscope slides on the surface of the same soil, the tray was sealed with a sheet of glass and incubated at 25 DC. At set time intervals three agar blocks and a control slide were removed from the soil, stained with lactophenol and cotton blue, and examined microscopically. At least 200 spores were counted randomly on each agar block and the number germinated expressed as a percentage which was arc-sin transformed for use in further analyses. Measurements were made of 30 germ-tubes chosen randomly for each treatment. A map measurer was used to measure (in arbitrary units) the length of germ-tubes drawn with the aid of a projection microscope at x 400 magnification. Spore suspensions for use in the experiments were collected from cultures growing on potato dextrose agar after a 3-week incubation period. The spores were washed off the culture with sterile distilled water, concentrated and washed twice by centrifugation and resuspended in sterile distilled water to give a final concentration of 45 x 10 5 cm- 3• RESULTS
The percentage germination of each of the replicates at each time interval was plotted against time. A logistic curve was found to be the best fit for the data. The curves obtained are typified by the results for T. hamatum (22) spores as shown in Fig. I. The form of the curve differs from that obtained by Dix (1972) and Dix & Christie (1974). This is probably because they sampled before and after and not during the lag period of the curve. Their results will therefore relate only to the exponential and later stages of the curve. The final percentage germination was obtained from the graph and was taken as the percentage germination on the upper plateau of the logistic curve. The results for the final percentage germination are given in Table I. The percentage reduction in performance based on that of the control has been calculated so that comparisons may be made between the species and isolates (see Table 2). There was a wide variation in sensitivity to fungistasis as indicated by the
Fungistasis. C. P. Mitchell and N. J. Dix
237
100
.. ......---
80
g
•
(; o
60
<:
02
I
~
......
Experiment
§____
•
•• §
8"""'-
~I
O s<: 0
Control
40
0 0
20
o Fig.
I.
4
8
12 Time (h)
16
20
The effect offungistasis on percentage germination of T. hamatum
22
spores.
percentage reduction in final percentage germination. When subjected to Student's t tests the values of the final percentage germination for the control and the experiment were found to be significantly different from one another in all cases. T. viride (49) showed the least sensitivity, T. viride (53) the greatest. The rate of germination to the final (maximum) percentage germination was taken as the regression coefficient of the straight line obtained from a plot oflog1 0 Nmax -N/N against time (where N is the percentage germination at any time t and N max is the maximum percentage germination). The rates of germination for the different isolates, as regression coefficients, are shown in Table 1. In order to ascertain if the regression lines, and hence the rates, calculated for the control and experiment were significantly different from one another they were subjected to a variance (F ) ratio test. In all cases, except that of T. longibrachiatum and T. viride (50) there was a reduction in the germination rate but this was significant for only six of the fungi tested (T. pseudokoningii, T. viride (14), T. viride (28), T. viride (50), T . viride (52), and T. viride (53)). T. viride (53) being the most sensitive (see Table 2). The latent period of germination as a measure of the lag phase is here defined as the time taken for 25 % of the spore population to germinate and is calculated by substituting the known values in the regression equation obtained in th e calculation of the rate of germination. The values for the latent period in the control and experiment and the percentage reduction in performance for each of the isolates are given in Tables 1 and 2. The germ-tube growth rates, for the different isolates are given as regression coefficients in Table 1. These have been calculated from the
N w
00
Table
I.
Measurements offinal percentage germination, rate of germination, latentperiod of germination and germ-tube growth rate under conditions of soilfungistasis Final % germination
Latent period (h)
Germination ra te (as regression coeff.)
Germ tube growth rate (as regression coeff.)
Control
Experiment
Control
Experiment
Control
Experiment
Control
Experiment
s....~
~15)
74'0 79'8
39'6 59'S
12,6 13'0
24'9 13'6
-0'1817 -0' 2167
-0'1443 -0'1701
0'1222 0'°970
0' 0673 0'07 05
~.
(I)
80'3 58,6 81'2 77,8
66,6 25'4 59'9 36.6
47'1 85'4 74'0
36'S 43·3 53'6
/0,8 16,6 12,6 13,6 16'3 14'1 I 1'4
9'2 <24'0 20'1 21'8 19'0 19'8 12'3
-0'2390 -o'II39 -0'2 167 -0'1270 -0'095° -0'1507 -0'1 786
-0' 22 15 -0' 1109 -0' 1864 -0'1229 -0'14 16 -0'08 17 -0'1537
0'093 8 0'°433 0'1133 0'003 6 0' 0224 0'°742 0'05 8 1
0' 08°4 0' 0 14° 0'°792 0'0102 0'0076 0' 0294 0'0467
,
A
T, hamatum 22) T, harzianum
(129~ (20 T . koningii T, longibrachiatum T. pseudokoningii T , satumispora T, viride
(2~
(14
~:~~ 4-9) (5°) (51) (52) (53) (54)
,
,
,
\
,
A
\
,
I
\
...~
b:1
::!. ~. ~
~ ~
S'
()tj
50 '8 81'S 45'9 8°'9 67'7 8°'9 78'0 83 '0 5 0'7 72'2
35'2 21'7 25'2 48'1 62'3 66'3 4 1'9 24'6 7.8 5 1'0
15'3 13'9 19'2 iI '3 II'S II'O
14'6 15,6 17'6 13'4
23'S 35'1 33'6 13'1 12·8 9'6 18'7 33'1 <2!'O I '3
-0'1418 -0'1423 -0'1367 -0'1968 -0'154 1 -0'25 19 -0'1447 -0'1457 -0'1383 -0'lg86
-0'1344 -0' 0675 -0'09 86 -0'14-63 -0'17 03 -0'1442 -0'1437 -0'085 2 -0'04-53 -0'0986
0'025 1 0'0828 0'°7 11 0' 1093 0' 1069 0'1338 0' 0849 0'0612 0'07 18 0'04 63
0'0281 0'0610 0'09640'064-2 0'05 69 0' 1092 0' 0669 0'03 17 0'0166 0'050 1
~.
+:l .....
~
~. Q
Fungistasis. C. P. Mitchell and N. J. Dix Table
2.
Percentage reduction in performance due to soilfungistasis % reduction in final % germination
T, hamatum (IS) (22) T, harzianum (I) ( 129) (20) T, kongingii T, longibrachiatum T, pseudokoningii T, satumispora T, viride (2) (14) (28) (48) (49) (so) (SI) (S2) (S3) (54)
239
% reduction in latent period
% reduction in
germination rate
% reduction in germ-tube growth rate
46'5*** 2S'4***
49'4 4'4
20'6 NS 21'S NS
44'9* 27'3 NS
17'0*** S6'8*** 26'2*** S2'9*** 22'7*** 49'3** 27'6**
0'0 100'0 37'3 37'6 14'2 28,8 7'3
7'3 NS 2,6 NS 14'0 NS 3'2 NS 0'0 NS 4S'8*** 13'9 NS
14'3 NS 67'7** 3°'1 NS 0'0 NS 66'1* 6°'4* 19'6 NS
3°'7*** 73'4** 45"1* 40'6*** 8'0* 18'1** 46'3*** 7°'4*** 84'6*** 29'4***
34'9 6°'4 42'8 13,8 10'2 0'0 21'9 S2'9 100'0 17,8
S'2 NS 52'6** 27'9*** 2S'7 NS 0'0 NS 42'8* 0'7 NS 42'9*** 67'3*** 28'9 NS
0'0 NS 26'3 NS 0'0 NS 41'3 NS 46'8* 18'4 NS 21'2 NS {8'2 NS 76'9* 0'0 NS
* Significant: P < 0'05, ** Significant: P < 0'01. *** Significant: P < 0'001. NS, Not significant: P > o-og.
Table 3, The theoretical colonization index andpercentage reduction T, hamatum (IS) (22) T, harzianum (I) (129) (20) T. koningii T, longibrachiatum T, pseudokoningii T, saturnispora T, viride (2) (14) (28) ({8) t49) (So) (SI) (S2) (53) (S4)
16
Control
Experiment
Reduction t %)
6011'3 6278'4
S24'8 1626,S
9 1'3 74'1
8 184'6 103° ' 1 4723'1 1089'2 765'0 9498'6 277S'o
4°9 2'4 123'6 12S5"1 201'1 2so'8 422'8 117S'4
5°'0 88'0 73'4 81'S 67'2 9S'6 S7'6
54°'3 S890'6 2632'8 8125'7 6442'6 2862'6 S021'8 S839'6 4495'2 4983'8
230'6 367'2 21S'4 1686,6 ISIS,6 IISI'O 471'9 273'4 45'2 2188'3
S7'3 93'8 9 1,8 79'2 76'S S9'8 90'6 9S'3 99'0 so'o
Myc:64
240
Transactions British Mycological Society
average of the common log values of the germ-tube lengths at each time. The variance (F) ratio was used to test for significant differences between the regression lines. Only six of the isolates (T. hamatum (15), T. harzianum (129), T. longibrachiatum, T. pseudokoningii, T. viride (49) and T. viride (53)) had germ-tube growth rates on soil significantly different from those in the control (see Table 2). The TCI was calculated by taking the product of the percentage germination at any time and the mean germ-tube length at the same time. The TCI for each of the isolates after 24 h incubation is shown in Table 3 where for comparative purposes the TCI for each has also been expressed as a percentage reduction based on that of the same population in the control. The use of the TCI enables predictions to be made of the ability of different spore populations to germinate in the presence of a simple substrate in soil and colonize it. The advantage of this index is that it is a measure not only of the effect of antagonism on one area of germination but of the overall effect on the ability of the spores to colonize a substrate. There was a large variation in the TCI of the different isolates, but in most cases fairly substantial growth was made. T. viride (53) with a TCI of only 45'2 shows the least growth whereas T. harzianum (I) with an index of 4092'4 has the highest growth under antagonistic conditions. DISCUSSION
In general many Trichoderma spp. appear to be relatively insensitive to soil fungistasis. Soil fungistasis had its greatest effect, among the species tested, on the final percentage germination, since in many cases, once germination had begun, growth and the rate of germination were not significantly affected. However, there was considerable variation in the value of the TCI, even among different isolates of the same species, suggesting that there might be considerable variation in the amount of growth different strains might make in the soil. All, however, were capable of growth indicating that it would theoretically be possible for all of them to exert an antagonistic effect upon competing organisms. The percentage reduction in the TCI has a higher positive correlation with the percentage reduction in the final percentage germination (r = 0'7759, P < 0'001), than the germ-tube growth rate (r = 0'4774, P < 0'05)' This suggests that in Trichoderma spp. soil fungistasis modifies the TCI most significantly through a reduction in germination rather than through a reduction in the growth rate of germ-tubes. Differences in the numbers of spores germinating may therefore be the most important factor contributing to differences in the size of the TCI in Trichoderma spp. The percentage reduction in the TCI is also correlated positively with percentage reduction in the rate of germination (r = 0'5168, P < 0'05), and the latent period of germination (r = 0,6586, P < 0'01). If the TCI is a valid measure of growth and reflects the competitive saprophytic ability of a fungus under antagonistic conditions then it would appear that the numbers of spores germinating in a Trichoderma population may be the most significant factor determining whether colonization of a substrate proceeds or not. Dix (1972) posed the question on
Fungistasis. C. P. Mitchell and N. J. Dix what aspect of germination does soil fungistasis have its most important effect? He suggested that provided some germination takes place, the most significant effect was likely to be on the growth rate of germ-tubes. These results would indicate that this concept may underestimate the significance of the contribution to differences in total growth made by the number of spores germinating where the differences between the growth rates of different species in the presence of soil fungistasis are slight compared with differences in the numbers of spores germinating. However, where competition for colonization is between germinating single spores differences in the amounts of growth produced will be due mainly to differences in the growth rate of the germ-tubes. Once again the question arises as to whether colonization in soil is most often effected by the germination of isolated single spores or by a number of spores germinating in locally dense populations. A number of interesting relationships exist between the effects of soil fungistasis on the different parameters of germination. The reduction in the latent period was correlated positively with both the reduction in the final percentage germination and the germ-tube growth rate (r = 0,8882, P < o-oor and r = 0'5430, P < 0'05 respectively). Dix (r972), for Penicillium spp., found a correlation only between reduction in the latent period and the final percentage germination. The latent period in the control was correlated positively with the reduction in the final percentage germination (r = 0'9055, P < o·oor). A similar result was obtained by Steiner & Lockwood (r969). Analysis of the results ofDix (r972) shows the same correlation. Thus the present results and those of Dix (r 972) support the hypothesis of Steiner & Lockwood (r969) that the time of germination can have a significant effect upon the response of the spore to fungistasis. This work is part of a thesis presented for the degree of Ph.D. in the University of Stirling. One of us (C. P. Mitchell) is indebted to the Science Research Council for financial support. REFERENCES
DIX, N.]. (1967). Mycostasis and root exudation: factors influencing the colonization of bean roots by fungi. Transactions of the British Mycological Society 50, 23-3 I. DIX, N.]. (1972). Effect of soil fungistasis on spore germination and germ tube growth in Penicillium species. Transactions of the British Mycological Society 58, 59-66. DIX, N.]. & CHRISTIE, P. (1974). Changing sensitivity to soil fungistasis with age in Drechslera rostrata spores and associated permeability changes. Transactions of the British Mycological Society 62, 527-535. GARRETT, S. D. (1950). Ecology of the root inhabiting fungi. Biological Reviews 1115, 220-254. MITCHELL, C. P. (1973). Antagonistic interactions involving Trichoderma species. Ph.D. Thesis, University of Stirling. PARK, D. (1960). Antagonism - the background to soil fungi. The Ecology of Soil Fungi (ed. D. Parkinson &]. S. Waid), pp. 148-159. Liverpool: University Press. STEINER, G. W. & LOCKWOOD, ]. L. (1969). Soil fungistasis: sensitivity of spores in relation to germination time and size. Phytopathology 59, 1084-1092.
(Accepted for publication r5 August r974) 16'2