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Ecological studies on cocco1d bacteria in a pine forest soil—III. competitive interactions between bacterial strains in soil
Soil Biol. Biochem. Vol. 5, pp. 463-472. Pergamon Press 1973. Printed in Great Britain.
ECOLOGICAL STUDIES ON COCCOID BACTERIA IN A PINE FOREST SOIL-III. COMPETITlVE INTERACTIONS BETWEEN BACTERIAL STRAINS IN SOIL W. E. LOWE* and T. R. G. GRAY Hartley Botanical Laboratories, University of Liverpool, England (Accepted 12 October 1972)
Summary--Strains of bacteria with differing degrees of overall (phenetic) similarity have been paired and grown together and separately in both acid and alkaline soils from a pine forest. A decrease in yield of an organism when grown in the presence of a second organism was taken as evidence of competition. It was shown that, contrary to expectations, pairs of organisms with different degrees of similarity might have similar competitive interactions. It is postulated that this is because one key property could result in a marked change in competitive ability while not markedly altering the taxonomic relationships of the organisms. The possibility that spatial separation of organisms can result in two or more organisms with identical functions becoming established in soil is discussed.
INTRODUCTION
have shown that organisms may have remarkably uniform reactions to changes in their environment, even if they are phenetically distinct. Soil acidity and low substrate levels seem to be of major importance in limiting the growth of organisms in the soils they examined. Nevertheless, it is possible that these organisms may have different functions in the soil, i.e. occupy separate niches. If they do, they may be spatially separated in different microenvironments or grow together in one microenvironment in which they utilize different substrate components. If they are spatially separated, they would not be expected to compete with one another; if they are not spatially separated and perform either identical or different functions, they would be expected to compete with one another to differing degrees. The occurrence of competition between two populations in a heterogeneous environment like soil might be used, therefore, as an indication that they are occupying similar microenvironments. In an attempt to detect competition between higher plants in model systems, de Wit (1960) compared growth of a single species plant population with the growth of the same population growth together with a second species population. Yield at maturity was used as a measure of growth. A decrease in yield of either species when grown together, as opposed to growth separately, was considered an indication of competition. The object of the present investigation was to see if de Wit’s methods could be applied to the detection of competition between related bacteria. In these experiments, the results of growth were expressed by determining the per cent relative yield (van den Bergh, 1968). This is obtained by measuring the yield of an organism in the presence of a second organism and expressing this as a percentage of the yield when it is grown on its own. In Fig. 1 the per cent relative yields for a hypothetical experiment with two organisms, A and B are LOWE AND GRAY (1973)
* Present address: Department of Soil Science, University of Saskatchewan, Saskatoon, Canada. 463
464
W. E. LOWE AND T. R. G. GRAY
recorded. One hundred per cent relative yield indicate that the organism is unaffected by the presence of another; 0 per cent yield indicates complete eI~mination of an organism by another. In this experiment, organisms were introduced in pure culture and in a 50:50 mixture at the start. As the population grew, it can be seen that the yield of A was unaffected and reached the same level as in pure culture. On the other hand, species B declined and was severely affected. ------.---“-----
e-m
-__-
,~qcsz l-h..__
‘-.
~-x---x
Specie**-.
~
1
I
I
I
I
I
I
I
2
3
4
5
6
Time,
doys
FIG. 1. Per cent relative yield of two organisms, A and B, following their mixture in equal amounts at time zero. Species A is unaffected while species B declines.
MATERI~S
AND ~THOD~
The most typical bacteria of the clusters determined by the numerical taxonomic survey of coccoid bacteria in soil (Lowe and Gray, 1972a) were used in the present study. These organisms were paired so that interactions between similar organisms and less similar organisms could be compared. The pairs chosen are listed in Table 1. TABLE 1. PAIRING OF ORGANISMS FOR INTERACTION EXPERIMENTS
Organisms
Cluster*
‘A Similarity (S,)*
% Similarity (.S,)*
75.2
55.3
~ta~b~vZucucc~~(A8 I) Arthrobacter (A49)
2 41
54.6
35.9
Arthrobacter Arthrobacter
(C39)
411 4i
84.3
64.4
(A49)
Micrococcus Arthrobacter
(A34) (A49)
1 43
58.9
39.1
* Lowe and Gray (1972a).
Sterile C horizon soil (pH 8.1) and Al horizon soil (amended to ptl 7 *5} were prepared as described by Lowe and Gray (1973) and placed in 10 g quantities in sterile 2.5 ml screw cap bottles. Bacteria1 suspensions were also prepared (Lowe and Gray, 1973) so that when
STUDIES
ON COCCOID
BACTERIA
IN SO&--III
465
1 mf of suspension was introduced into the soil, the soil contained approx. l-0 x lo6 bacteria/g and had a moisture content of 10% (wove. When it was required to introduce mixtures of bacteria into the soil, equal amounts of suspension were mixed and l-0 ml of the mixture added to the soil. Soils were incubated at 25°C for 7 days and samples taken at 1, 2, 3 and 7 days. Numbers of bacteria were determined using the dilution plate count technique. Because two organisms were present in the mixture, it was necessary to modify the plating technique to enable the two types to be distinguished from one another. This was achieved by surface inoculation of peptone yeast extract agar plates with 0 +1 ml suspension (~ruikshauk, 1965), followed by transfer of alt developing colonies to diagnostic media using a replica technique (Lederberg and Lederberg, 1952). Table 2 shows the diagnostic tests used to distinguish between strains. Two tests were used for each pair of organisms so that for any given pair, one member gave a positive result in one test and a negative result in the other and vice versa. The efficiency of the method was tested using organisms A81 and A49, which could also be distinguished visually by colony characteristics on the original isolation plates. Errors were of the order of 1 per cent, provided that colonies were we11 separated, i.e. below 300 per plate. 2. TESTS USED TO DIFFERENTIATE BETWEEN
TABLE
PAIR MEMBERS INTO SOIL TOGETHER
Organisms
Aesculin hydrolysis
A21 A49
Starch hydrolysis - 24h + 24 h
i_ 24 h - 24 h
WHEN
INTRODUCED
Acid from sucrw+e NA NA
A81 A49
::
- 24h + 24 h
+ 48 h - 48 h
A34 A49
NA NA
- 24h f- 24 h
-I- 48 h - 48 h
c39 A49 NA-Not
- 24h +- 24h
+ 24 h - 24 h applied.
f+f
Positive result.
(-1
NA NA
Negative result.
Inferaction of bacteria on dilution plates. It was necessary to show that when two kinds of bacteria were growing on isolation plates together, no interaction occurred between their colonies. If it did, then it would be impossible to distinguish it from interactions occurring between bacteria in the soil. This was tested by preparing surface inoculated dilution plates from pure cultures and mixed suspensians of all the organism pairs investigated. No interactions between colonies of the different organisms used was observed (Table 3). It was concluded therefore that any interactions that did occur in the experiment would be the result of events in the soil. It was assumed also that since there was no evidence of antibiotic production by the isolates on rich media, interactions in low nutrient status soil would probabIy not be due to antibiosis.
W. E. LOWE AND T. R. G. GRAY
466
TABLE 3. INTERACTIONS ONDILUTION PLATES,JUDGED BY THE NUMBERS OF BACTERIADEVELOPINGONPLATES PREPARED FROM PIJREANDMIXEDBACTERIALSUSPENSIONS
Numbers of bacteria (X lo-‘) and standard error of the mean
No. in 0.5 ml pure culture
No. in 0.5 ml + 0.5 ml mixed culture
A21 A49
116.6 & 7.2 lOO*l + 4.1
119.6 f 5.1 96.0 & 3.3
A81 A49
44.2 & 3,l 49.2 :t 2.9
42.8 5 3.9 44.8 i 3.7
A34 A49
71.9 + 4.6 89.4 h 6.8
67.0 & 2.3 90.4 + 8.1
c39 A49
12750 & 8.4 99.4 -_I6.3
117.3 i 7-G 103.4 + 4.9
Organisms
Ex~ra~t~~~~~ba~~~~~afrom soil. After the addition of bacteria to soil, different bacterial strains might be adsorbed differentially by particles (Zvyagintsev, 1962); some might be harder to remove than others and hence be under-represented on dilution plates prepared from the samples. In order to test this possibility, a known number of cells of the bacteria to be examined were inoculated into soil and then allowed to stand for 12 h at 4°C. It was assumed that little or no growth would occur at this temperature. Dilution plates were prepared from soil samples containing the bacteria and the numbers of bacteria compared with those originally present. Table 4 records the results and shows that the efficiency of the TABLE
4.
EFFIENCIENCYOF
EXTRACTION
PROCESS
FORBACTERIA~NSOILFOR
12h
AT
4°C
B A
Organisms
No. of bacteria* (X 10e6 per ml) initial suspension
Calculated no. of bacteria* ( x 10v6 per ml) suspension after extraction
A/B x 100%
Efficiency of extraction
C horizon soil A21 A81 A34 A49 c39
208.5 137.3 167.5 126.8 43.3
* + & + &
5.6 4.3 2.4 3.6 1.1
i: 30.2 _Ir 14.8 i: 32.4 -4 11.9 i: 3.5
102.4 107-2 95,9 100.5 97.9
A21 ASI A34 A49 c39
AI horizon soil (pH amended to 7.5) 208.5 i 5.6 186.3 & 16.8 137-3 f 4.3 121.6 & 12.4 167.5 i 2.4 138.4 -i: 19.6 126-8 i 3-6 129.6 j, 15.3 43.3 f 1.1 47.3 & s.4
88.6 82.6 102.2 102.3
* Standard error of the mean included.
213.4 147.2 160-6 127.4 40.8
89.4
STUDIES ON COCCOID BACTERIA
IN SOIL-III
467
extraction process was about 100 per cent, within the limits of error of the experiment. It was concluded that differential adsorption would not seriously affect interpretation of the results, although it is possible that as populations grow some bacteria might adhere more strongly to soil particles than others. There appears to be no easy way of testing this. Efict of varying inoculum size on bacterial yield in soil. In the original experiments performed by de Wit (1960) and van den Bergh (1968), the total input of organisms into the system remained constant, i.e. total number of A + B equalled total numbers of A or total numbers of B in single species systems. Therefore, in the present study of bacteria, n cells/g were introduced into soil in pure culture and n/2 cells/g soil for each component of the mixture. Preliminary experiments were carried out to determine the effect of initial inoculum size on final yield to see whether a large inoculum resulted in large yields, a small inoculum in small yields. Experiments were conducted in which the yield of cells in A, horizon soil (pH 7 *5) and C horizon soil, both inoculated with 1 *O x lo6 cells/g and 5 x lo5 cells/g, were compared. The results of these experiments for strain A34 in C horizon soil are recorded in Fig. 2. All other strains gave similar results and are therefore omitted. It is evident that initial inoculum size cannot be related to final yield and, after 7 days incubation at 25”C, plate counts of bacteria from soils with different inocula were almost identical. Therefore, determination of per cent relative yield may be expected to give an acceptable indication of interaction occurring between organisms introduced into sterile soil together.
200
160
.p .F :: 120 .a ';; P 80
40
i
I
1
I
I
I
I
I
2
3
4
5
6
Time,
I ?
days
FIG. 2. Growth of Micrococcus A34 in C horizon soil from different inoculum sizes. Initial inoculum size: (O-O) 1 .O x 10 per ml; (0-O) 5.0 x lo5 per ml. Numbers of bacteria expressed as millions per gramme oven dry soil. Vertical lines represent 95 per cent confidence limits.
468
W. E. LOWE AND T. R. G. GRAY
Interactions between organisms in soil
Experiments on the growth of organisms in pairs as outlined earlier were carried out in triplicate in soil incubated at 25°C for 7 days. The initial inoculum sizes are given in Table 5 and were measured by sampling soil immediately after mixing the inoculum with the soil. Inoculum sizes were standardized as closely as possible but there are some slight discrepancies between numbers of certain pair members. These were considered to be unimportant. TABLE
5.
INOCULUM SIZES USED IN THE INVESTIGATION OF INTERACTIONS BETWEEN BACTERIAL POPULATIONS Inoculum
Al Pairs of organisms
horizon
size (No.
bacteria
soil + CaC03
Pure
(pH
x 1O-6 per g oven dry soil)*
7.5)
Mixture
C horizon soil (pH 8.1) Pure
Mixture
A21 A49
23.650.6 25.7 5 0.5
12.2 f 1.0 9.6 * 0.2
22.7 10.8 20.2 f 0.9
11.8 5 1.0 9.6 f 1.0
A81 A49
8.5 & 0.7 9.6 f 0.2
5.3 + 0.2 4.5 * 0.3
8.9 rir 0.9 9.8 h 0.2
5.2 & 0.2 4.5 i 0.5
A34 A49
12.0 jy 1.0 17.6 & 0.4
6.7 i 1.1 9.3 i 0.8
14.1 ho.6 12.0 * 1.5
6.8 10.2 9.0 * 0.4
c39 A49
24.5 f 1.0 19.8 & 2.4
10.7 & 2.4 11.3 f 0.8
14.0 & 3.2 18.2 f 1.8
10.3 & 1.8 10.2 f 0.8
* Standard error of the means also included.
Growth curves obtained from pure cultures of organisms were similar to those already reported by Lowe and Gray (1973) and are not repeated here. However, the per cent relative yields for all pairs of organisms are plotted in Figs. 3-6. The results obtained varied considerably. In the mixture of A34 (Micrococcus) and A49 (ArthrobacterJ both organisms were unaffected by the presence of the other and produced high yields in both A, and C horizon soils (Fig. 6). These organisms had comparatively low percentage similarity coefficients of 58 -9 per cent S, and 39.1 per cent S, (Lowe and Gray, 1972) and it is possible that they occupy separate niches within the soil since they did not compete. When A21 (Arthrobacter,) was mixed with A49, only A49 was affected while A21 still gave a high yield, at least in the A, horizon soil (Fig. 3). This suggests that these two organisms are not spatially separated in the soil but that one (A21) was better fitted to grow in this environment (Garrett, 1970) than the other. These organisms had relatively high percentage similarity coefficients of 75.2 per cent S, and 58 *3 % S,. In the remaining cases, when A81 (Staphylococcus) and C39 (ArthrobacterJ were mixed with A49 (Figs 4 and 5 respectively), both organisms showed reduced yields although the similarity between C39 and A49 was high (84.3 per cent S,, 64.4 per cent Sj) while between A81 and A49 it was low (54.6 per cent S, and 35.9 per cent S,). This suggests that it is not possible to correlate the degree of spatial separation with the degree of overall similarity of the organisms involved and as suggested earlier it is probable that some organisms inhabiting the same niche may utilize different components of it. Thus A81 is biochemically active in sugar fermentation while A49 is not (Lowe and Gray, 1972); a fermentative ability suggests the ability of the organism to grow anaerobically.
STUDIES ON COCCOID BACTERIA
60
IN SOIL-III
469
-
A49
40
A21 20
t
I
I
I 2
3
Time,
1 4
I 5
I 6
1 7
days
FIG. 3. Per cent relative yields of organisms A21 and A49 when added to A1 horizon soil (pH 7.5) and C horizon soil (pH 8.1). (O-0) A1 horizon soil (pH 7.5); (e-0) C horizon soil (pH 8-l), organism A49, (A-A) AI horizon soil (pH 7.5); (A-----& C horizon soil (pH 8 - l), organism A21. GENERAL
DISCUSSION
It has been suggested that if a group of organisms is shown to differ from a second group of organisms in a large sample of randomly chosen properties, then a further sampling of randomly chosen properties should confirm the difference between the groups (Sokal and Sneath, 1963). In the present investigation, it has been shown that groups of coccoid bacteria can be distinguished by examination of a large number of morphological and biochemical properties (Lowe and Gray, 1972). A second sample of properties has not been investigated but the ability of organisms to compete with one another for soil substrates has been assessed. Garrett (1950) coined the phrase competitive saprop~ytic ability which he defined as ‘the summation of physiological characteristics that make for success in competitive colonization of dead organic material’. Later Garrett (1970) equated this term with the general fitness of a microbe to exist in one or more ecological niches. It could be argued, therefore, that if organisms are shown to have different levels of fitness, they are different from one another in a large set of unknown characteristics and are therefore both ecologically and phenetically distinct. The assessment of competitive interactions made here is not broad enough to test properly the validity of this idea. In particular, interactions between very much less similar organisms would have been useful, e.g. pseudomonads and micrococci. However, some tentative conclusions can be reached. Firstly, the correlation between ecological and phenetic similarity
.
20
-
A49
-0
2 h .-f z z a
I
I
I
I
4
5
1
I
100 ---_-_-_-_-_-_-_--_ A
I
2
3
Time,, FIG.
I
6
7
days
4. Per cent relative yields of organisms A81 and A?9 when added to A1 horizon soil (pH 7.5) and C horizon soil (pH 8 -1). For key see Fig. 3. 100--_--_-*-_--__-_____* a0 A49
Time,
days
FIG. 5. Per cent relative yieIds of organisms C39 and A49 when added to A1 horizon soil (pH
7.5) and C horizon soil (pH 8.1). For key see Fig. 3.
STUDIES ON COCCOID BACTERIA
471
IN SOIL-III
40
A49
s t
A34
‘“;, , , , , , 1
2
Time, Eirc.6. Per
3
4
5
6
1
7
days
cent relative yields of organisms A34 and A49 when added to AI horizon soil (pH 7 -5) and C horizon soil (pH 8.1). For key see Fig. 3.
is not very precise. The quite different degrees of similarity of the pair members A81/A49 and C39JA49 which had similar competitive responses is indicative of this. The probable reason for this is that while it is the summation of properties of an organism that finally determine its fitness or competitive ability, the individual properties do not contribute equally to this summation. One particular property might result in the exclusion of an organism from a particular niche, e.g. obligate aerobes could not grow in anaerobic niches. In this respect, the concept of taxonomic overall similarity differs from that of ecological overall similarity, for alteration of a single property in a taxonomic scheme would not result in the exclusion of a microorganism from a polythetic taxonomic group. Secondly, it is unhkely that competitive interactions of this type are of overriding importance in the establishment of bacteria in particular microenvironments. The soil is so heterogeneous and the bacterial populations so widely spread out within it (Gray, 1967) that the establishment of bacteria on a soil particle is more likely to be due to coincidence rather than the outcome of competition between strains arriving simultaneously on the same particle. This is not to say that competition studies tell us nothing about bacteria in a soil, for an inability to compete under favourable conditions will suggest spatial separation of zones of activity. Such considerations affect the applicability of Gause’s concept of competition to soil organisms. Gause (1934) supposed that if two different organisms occupied the same niche, one would sooner or later eliminate the other. However, if organisms such S.B.B. 5/d--F
472
W. E. LOWE AND T. R. G. GRAY
as bacteria arrive in microenvironments by chance and are so thinly spread out in the soil that the chances of two different strains arriving in the same environment simultaneously with any regularity are small, then it is probable that more than one organism wili be able to exploit one and the same type of niche. For competition to occur, it is also necessary that bacteria be growing actively in soi1. There is a large body of evidence (Gray and Williams, 1971) to suggest that the growth rates of microorganisms in soil are slow and that organisms remain dormant for long periods of time. Some of this evidence concerns the lack of available energy to support rapid growth of the comparatively large microbial populations present in soil, and one might ask, therefore, where the energy came from to support microbial growth in the present experiments. It is known that sterilization increases the availability of nutrients in soil (Warcup, 1957; Eno and Popenoe, 1964) and Jenkinson (1966) has shown that quite large respiratory flushes occur following the inoculation of microbes into sterile soil, due to the release of nutrients from the killed natural microflora. A similar situation probably accounts for the pattern of results in these experiments. Acknowledgements-We wish to thank Professor A. D. BRADSHAWfor his helpful advice in designing these experiments. One of us (W.E.L.) wishes to thank the Science Research Council for a grant enabling him to carry out this work. REFERENCES CRUIKSHANKR. (1956) Me&c& Microbiology. 11th Ed. Livingstone, Edinburgh. DE Wrr C. T. (1960) On competition, Versl. Landbouwsk. Onderz. R~ksla~dbProe~ Stn., 66. ENO C. F. and POPENOEH. (1964) Gamma radiation compared with steam and methyl bromide as a soil sterilizing agent. Proc. Soil Sci. Sot. Am. 28,533-535.. GARR~X~S. D. (1950) Biology of root infecting fungi. Biof. Rev. l&220-2.54. GARRETSS. D. (1970) Pathogenic Roo~“I~fec~i~gFungi. p. 114. University Press, Cambridge. GAUSE F. G. (1934) The Struggle for Existence. Williams & Wilkins, Baltimore. GRAYT. R. G. (1967) Stereoscan electron microscopy of soil microorganisms. Science, N. Y. 155,1&S-1670. GRAY T. R. G. and WILLIAMSS. T. (1971) Soil Micro-organisms. Oliver 8z Boyd, Edinburgh. JENKINSONS. D. (1966) Studies on decomposition of plant material in soil-II. Partial soil sterilization and the soil biomass. J. Soil Sci. 17, 280-302. LEDERBERGJ. and LEDERBERGE. M. (1952) Replica plating and indirect selection of bacterial mutants. J. Bact. 63, 399-406. LOWE W. E. and GRAY T. R. G. (1972) Ecological studies on coccoid bacteria in a pine forest soil-I. Classification. Soil Biol. Biochem. 4, 459-468. LOWE W. E. and GRAY T. R. G. (1973) Ecological studies on coccoid bacteria in a pine forest soil-II. Growth of bacteria introduced into soil. Soil Biol. Biochem. 5,449%462. SOKAN R. R. and SNEATHP. H. A. (1963) Principles of ~arner~ca~Taxonomy. Freeman, San Fransisco. WARCUPJ. H. (1957) Chemical and biological aspects of soil sterilization. Soils Fertil. 20 l-5. VAN DEN BERGHJ. P. (1968) An analysis of yields of grasses in mixed and pure stands. VersE. ~aadboa~sk. Onderz. R~kslandbProe~ Stn., 7 14. ZVYAGINTSEV D. G. (1962) Adsorption of micro-organisms by soil particles. Soviet Soif Sci. 140-144.
ECOLOGICAL STUDIES ON COCCOID BACTERIA IN A PINE FOREST SOIL-III. COMPETITlVE INTERACTIONS BETWEEN BACTERIAL STRAINS IN SOIL W. E. LOWE* and T. R. G. GRAY Hartley Botanical Laboratories, University of Liverpool, England (Accepted 12 October 1972)
Summary--Strains of bacteria with differing degrees of overall (phenetic) similarity have been paired and grown together and separately in both acid and alkaline soils from a pine forest. A decrease in yield of an organism when grown in the presence of a second organism was taken as evidence of competition. It was shown that, contrary to expectations, pairs of organisms with different degrees of similarity might have similar competitive interactions. It is postulated that this is because one key property could result in a marked change in competitive ability while not markedly altering the taxonomic relationships of the organisms. The possibility that spatial separation of organisms can result in two or more organisms with identical functions becoming established in soil is discussed.
INTRODUCTION
have shown that organisms may have remarkably uniform reactions to changes in their environment, even if they are phenetically distinct. Soil acidity and low substrate levels seem to be of major importance in limiting the growth of organisms in the soils they examined. Nevertheless, it is possible that these organisms may have different functions in the soil, i.e. occupy separate niches. If they do, they may be spatially separated in different microenvironments or grow together in one microenvironment in which they utilize different substrate components. If they are spatially separated, they would not be expected to compete with one another; if they are not spatially separated and perform either identical or different functions, they would be expected to compete with one another to differing degrees. The occurrence of competition between two populations in a heterogeneous environment like soil might be used, therefore, as an indication that they are occupying similar microenvironments. In an attempt to detect competition between higher plants in model systems, de Wit (1960) compared growth of a single species plant population with the growth of the same population growth together with a second species population. Yield at maturity was used as a measure of growth. A decrease in yield of either species when grown together, as opposed to growth separately, was considered an indication of competition. The object of the present investigation was to see if de Wit’s methods could be applied to the detection of competition between related bacteria. In these experiments, the results of growth were expressed by determining the per cent relative yield (van den Bergh, 1968). This is obtained by measuring the yield of an organism in the presence of a second organism and expressing this as a percentage of the yield when it is grown on its own. In Fig. 1 the per cent relative yields for a hypothetical experiment with two organisms, A and B are LOWE AND GRAY (1973)
* Present address: Department of Soil Science, University of Saskatchewan, Saskatoon, Canada. 463
464
W. E. LOWE AND T. R. G. GRAY
recorded. One hundred per cent relative yield indicate that the organism is unaffected by the presence of another; 0 per cent yield indicates complete eI~mination of an organism by another. In this experiment, organisms were introduced in pure culture and in a 50:50 mixture at the start. As the population grew, it can be seen that the yield of A was unaffected and reached the same level as in pure culture. On the other hand, species B declined and was severely affected. ------.---“-----
e-m
-__-
,~qcsz l-h..__
‘-.
~-x---x
Specie**-.
~
1
I
I
I
I
I
I
I
2
3
4
5
6
Time,
doys
FIG. 1. Per cent relative yield of two organisms, A and B, following their mixture in equal amounts at time zero. Species A is unaffected while species B declines.
MATERI~S
AND ~THOD~
The most typical bacteria of the clusters determined by the numerical taxonomic survey of coccoid bacteria in soil (Lowe and Gray, 1972a) were used in the present study. These organisms were paired so that interactions between similar organisms and less similar organisms could be compared. The pairs chosen are listed in Table 1. TABLE 1. PAIRING OF ORGANISMS FOR INTERACTION EXPERIMENTS
Organisms
Cluster*
‘A Similarity (S,)*
% Similarity (.S,)*
75.2
55.3
~ta~b~vZucucc~~(A8 I) Arthrobacter (A49)
2 41
54.6
35.9
Arthrobacter Arthrobacter
(C39)
411 4i
84.3
64.4
(A49)
Micrococcus Arthrobacter
(A34) (A49)
1 43
58.9
39.1
* Lowe and Gray (1972a).
Sterile C horizon soil (pH 8.1) and Al horizon soil (amended to ptl 7 *5} were prepared as described by Lowe and Gray (1973) and placed in 10 g quantities in sterile 2.5 ml screw cap bottles. Bacteria1 suspensions were also prepared (Lowe and Gray, 1973) so that when
STUDIES
ON COCCOID
BACTERIA
IN SO&--III
465
1 mf of suspension was introduced into the soil, the soil contained approx. l-0 x lo6 bacteria/g and had a moisture content of 10% (wove. When it was required to introduce mixtures of bacteria into the soil, equal amounts of suspension were mixed and l-0 ml of the mixture added to the soil. Soils were incubated at 25°C for 7 days and samples taken at 1, 2, 3 and 7 days. Numbers of bacteria were determined using the dilution plate count technique. Because two organisms were present in the mixture, it was necessary to modify the plating technique to enable the two types to be distinguished from one another. This was achieved by surface inoculation of peptone yeast extract agar plates with 0 +1 ml suspension (~ruikshauk, 1965), followed by transfer of alt developing colonies to diagnostic media using a replica technique (Lederberg and Lederberg, 1952). Table 2 shows the diagnostic tests used to distinguish between strains. Two tests were used for each pair of organisms so that for any given pair, one member gave a positive result in one test and a negative result in the other and vice versa. The efficiency of the method was tested using organisms A81 and A49, which could also be distinguished visually by colony characteristics on the original isolation plates. Errors were of the order of 1 per cent, provided that colonies were we11 separated, i.e. below 300 per plate. 2. TESTS USED TO DIFFERENTIATE BETWEEN
TABLE
PAIR MEMBERS INTO SOIL TOGETHER
Organisms
Aesculin hydrolysis
A21 A49
Starch hydrolysis - 24h + 24 h
i_ 24 h - 24 h
WHEN
INTRODUCED
Acid from sucrw+e NA NA
A81 A49
::
- 24h + 24 h
+ 48 h - 48 h
A34 A49
NA NA
- 24h f- 24 h
-I- 48 h - 48 h
c39 A49 NA-Not
- 24h +- 24h
+ 24 h - 24 h applied.
f+f
Positive result.
(-1
NA NA
Negative result.
Inferaction of bacteria on dilution plates. It was necessary to show that when two kinds of bacteria were growing on isolation plates together, no interaction occurred between their colonies. If it did, then it would be impossible to distinguish it from interactions occurring between bacteria in the soil. This was tested by preparing surface inoculated dilution plates from pure cultures and mixed suspensians of all the organism pairs investigated. No interactions between colonies of the different organisms used was observed (Table 3). It was concluded therefore that any interactions that did occur in the experiment would be the result of events in the soil. It was assumed also that since there was no evidence of antibiotic production by the isolates on rich media, interactions in low nutrient status soil would probabIy not be due to antibiosis.
W. E. LOWE AND T. R. G. GRAY
466
TABLE 3. INTERACTIONS ONDILUTION PLATES,JUDGED BY THE NUMBERS OF BACTERIADEVELOPINGONPLATES PREPARED FROM PIJREANDMIXEDBACTERIALSUSPENSIONS
Numbers of bacteria (X lo-‘) and standard error of the mean
No. in 0.5 ml pure culture
No. in 0.5 ml + 0.5 ml mixed culture
A21 A49
116.6 & 7.2 lOO*l + 4.1
119.6 f 5.1 96.0 & 3.3
A81 A49
44.2 & 3,l 49.2 :t 2.9
42.8 5 3.9 44.8 i 3.7
A34 A49
71.9 + 4.6 89.4 h 6.8
67.0 & 2.3 90.4 + 8.1
c39 A49
12750 & 8.4 99.4 -_I6.3
117.3 i 7-G 103.4 + 4.9
Organisms
Ex~ra~t~~~~~ba~~~~~afrom soil. After the addition of bacteria to soil, different bacterial strains might be adsorbed differentially by particles (Zvyagintsev, 1962); some might be harder to remove than others and hence be under-represented on dilution plates prepared from the samples. In order to test this possibility, a known number of cells of the bacteria to be examined were inoculated into soil and then allowed to stand for 12 h at 4°C. It was assumed that little or no growth would occur at this temperature. Dilution plates were prepared from soil samples containing the bacteria and the numbers of bacteria compared with those originally present. Table 4 records the results and shows that the efficiency of the TABLE
4.
EFFIENCIENCYOF
EXTRACTION
PROCESS
FORBACTERIA~NSOILFOR
12h
AT
4°C
B A
Organisms
No. of bacteria* (X 10e6 per ml) initial suspension
Calculated no. of bacteria* ( x 10v6 per ml) suspension after extraction
A/B x 100%
Efficiency of extraction
C horizon soil A21 A81 A34 A49 c39
208.5 137.3 167.5 126.8 43.3
* + & + &
5.6 4.3 2.4 3.6 1.1
i: 30.2 _Ir 14.8 i: 32.4 -4 11.9 i: 3.5
102.4 107-2 95,9 100.5 97.9
A21 ASI A34 A49 c39
AI horizon soil (pH amended to 7.5) 208.5 i 5.6 186.3 & 16.8 137-3 f 4.3 121.6 & 12.4 167.5 i 2.4 138.4 -i: 19.6 126-8 i 3-6 129.6 j, 15.3 43.3 f 1.1 47.3 & s.4
88.6 82.6 102.2 102.3
* Standard error of the mean included.
213.4 147.2 160-6 127.4 40.8
89.4
STUDIES ON COCCOID BACTERIA
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467
extraction process was about 100 per cent, within the limits of error of the experiment. It was concluded that differential adsorption would not seriously affect interpretation of the results, although it is possible that as populations grow some bacteria might adhere more strongly to soil particles than others. There appears to be no easy way of testing this. Efict of varying inoculum size on bacterial yield in soil. In the original experiments performed by de Wit (1960) and van den Bergh (1968), the total input of organisms into the system remained constant, i.e. total number of A + B equalled total numbers of A or total numbers of B in single species systems. Therefore, in the present study of bacteria, n cells/g were introduced into soil in pure culture and n/2 cells/g soil for each component of the mixture. Preliminary experiments were carried out to determine the effect of initial inoculum size on final yield to see whether a large inoculum resulted in large yields, a small inoculum in small yields. Experiments were conducted in which the yield of cells in A, horizon soil (pH 7 *5) and C horizon soil, both inoculated with 1 *O x lo6 cells/g and 5 x lo5 cells/g, were compared. The results of these experiments for strain A34 in C horizon soil are recorded in Fig. 2. All other strains gave similar results and are therefore omitted. It is evident that initial inoculum size cannot be related to final yield and, after 7 days incubation at 25”C, plate counts of bacteria from soils with different inocula were almost identical. Therefore, determination of per cent relative yield may be expected to give an acceptable indication of interaction occurring between organisms introduced into sterile soil together.
200
160
.p .F :: 120 .a ';; P 80
40
i
I
1
I
I
I
I
I
2
3
4
5
6
Time,
I ?
days
FIG. 2. Growth of Micrococcus A34 in C horizon soil from different inoculum sizes. Initial inoculum size: (O-O) 1 .O x 10 per ml; (0-O) 5.0 x lo5 per ml. Numbers of bacteria expressed as millions per gramme oven dry soil. Vertical lines represent 95 per cent confidence limits.
468
W. E. LOWE AND T. R. G. GRAY
Interactions between organisms in soil
Experiments on the growth of organisms in pairs as outlined earlier were carried out in triplicate in soil incubated at 25°C for 7 days. The initial inoculum sizes are given in Table 5 and were measured by sampling soil immediately after mixing the inoculum with the soil. Inoculum sizes were standardized as closely as possible but there are some slight discrepancies between numbers of certain pair members. These were considered to be unimportant. TABLE
5.
INOCULUM SIZES USED IN THE INVESTIGATION OF INTERACTIONS BETWEEN BACTERIAL POPULATIONS Inoculum
Al Pairs of organisms
horizon
size (No.
bacteria
soil + CaC03
Pure
(pH
x 1O-6 per g oven dry soil)*
7.5)
Mixture
C horizon soil (pH 8.1) Pure
Mixture
A21 A49
23.650.6 25.7 5 0.5
12.2 f 1.0 9.6 * 0.2
22.7 10.8 20.2 f 0.9
11.8 5 1.0 9.6 f 1.0
A81 A49
8.5 & 0.7 9.6 f 0.2
5.3 + 0.2 4.5 * 0.3
8.9 rir 0.9 9.8 h 0.2
5.2 & 0.2 4.5 i 0.5
A34 A49
12.0 jy 1.0 17.6 & 0.4
6.7 i 1.1 9.3 i 0.8
14.1 ho.6 12.0 * 1.5
6.8 10.2 9.0 * 0.4
c39 A49
24.5 f 1.0 19.8 & 2.4
10.7 & 2.4 11.3 f 0.8
14.0 & 3.2 18.2 f 1.8
10.3 & 1.8 10.2 f 0.8
* Standard error of the means also included.
Growth curves obtained from pure cultures of organisms were similar to those already reported by Lowe and Gray (1973) and are not repeated here. However, the per cent relative yields for all pairs of organisms are plotted in Figs. 3-6. The results obtained varied considerably. In the mixture of A34 (Micrococcus) and A49 (ArthrobacterJ both organisms were unaffected by the presence of the other and produced high yields in both A, and C horizon soils (Fig. 6). These organisms had comparatively low percentage similarity coefficients of 58 -9 per cent S, and 39.1 per cent S, (Lowe and Gray, 1972) and it is possible that they occupy separate niches within the soil since they did not compete. When A21 (Arthrobacter,) was mixed with A49, only A49 was affected while A21 still gave a high yield, at least in the A, horizon soil (Fig. 3). This suggests that these two organisms are not spatially separated in the soil but that one (A21) was better fitted to grow in this environment (Garrett, 1970) than the other. These organisms had relatively high percentage similarity coefficients of 75.2 per cent S, and 58 *3 % S,. In the remaining cases, when A81 (Staphylococcus) and C39 (ArthrobacterJ were mixed with A49 (Figs 4 and 5 respectively), both organisms showed reduced yields although the similarity between C39 and A49 was high (84.3 per cent S,, 64.4 per cent Sj) while between A81 and A49 it was low (54.6 per cent S, and 35.9 per cent S,). This suggests that it is not possible to correlate the degree of spatial separation with the degree of overall similarity of the organisms involved and as suggested earlier it is probable that some organisms inhabiting the same niche may utilize different components of it. Thus A81 is biochemically active in sugar fermentation while A49 is not (Lowe and Gray, 1972); a fermentative ability suggests the ability of the organism to grow anaerobically.
STUDIES ON COCCOID BACTERIA
60
IN SOIL-III
469
-
A49
40
A21 20
t
I
I
I 2
3
Time,
1 4
I 5
I 6
1 7
days
FIG. 3. Per cent relative yields of organisms A21 and A49 when added to A1 horizon soil (pH 7.5) and C horizon soil (pH 8.1). (O-0) A1 horizon soil (pH 7.5); (e-0) C horizon soil (pH 8-l), organism A49, (A-A) AI horizon soil (pH 7.5); (A-----& C horizon soil (pH 8 - l), organism A21. GENERAL
DISCUSSION
It has been suggested that if a group of organisms is shown to differ from a second group of organisms in a large sample of randomly chosen properties, then a further sampling of randomly chosen properties should confirm the difference between the groups (Sokal and Sneath, 1963). In the present investigation, it has been shown that groups of coccoid bacteria can be distinguished by examination of a large number of morphological and biochemical properties (Lowe and Gray, 1972). A second sample of properties has not been investigated but the ability of organisms to compete with one another for soil substrates has been assessed. Garrett (1950) coined the phrase competitive saprop~ytic ability which he defined as ‘the summation of physiological characteristics that make for success in competitive colonization of dead organic material’. Later Garrett (1970) equated this term with the general fitness of a microbe to exist in one or more ecological niches. It could be argued, therefore, that if organisms are shown to have different levels of fitness, they are different from one another in a large set of unknown characteristics and are therefore both ecologically and phenetically distinct. The assessment of competitive interactions made here is not broad enough to test properly the validity of this idea. In particular, interactions between very much less similar organisms would have been useful, e.g. pseudomonads and micrococci. However, some tentative conclusions can be reached. Firstly, the correlation between ecological and phenetic similarity
.
20
-
A49
-0
2 h .-f z z a
I
I
I
I
4
5
1
I
100 ---_-_-_-_-_-_-_--_ A
I
2
3
Time,, FIG.
I
6
7
days
4. Per cent relative yields of organisms A81 and A?9 when added to A1 horizon soil (pH 7.5) and C horizon soil (pH 8 -1). For key see Fig. 3. 100--_--_-*-_--__-_____* a0 A49
Time,
days
FIG. 5. Per cent relative yieIds of organisms C39 and A49 when added to A1 horizon soil (pH
7.5) and C horizon soil (pH 8.1). For key see Fig. 3.
STUDIES ON COCCOID BACTERIA
471
IN SOIL-III
40
A49
s t
A34
‘“;, , , , , , 1
2
Time, Eirc.6. Per
3
4
5
6
1
7
days
cent relative yields of organisms A34 and A49 when added to AI horizon soil (pH 7 -5) and C horizon soil (pH 8.1). For key see Fig. 3.
is not very precise. The quite different degrees of similarity of the pair members A81/A49 and C39JA49 which had similar competitive responses is indicative of this. The probable reason for this is that while it is the summation of properties of an organism that finally determine its fitness or competitive ability, the individual properties do not contribute equally to this summation. One particular property might result in the exclusion of an organism from a particular niche, e.g. obligate aerobes could not grow in anaerobic niches. In this respect, the concept of taxonomic overall similarity differs from that of ecological overall similarity, for alteration of a single property in a taxonomic scheme would not result in the exclusion of a microorganism from a polythetic taxonomic group. Secondly, it is unhkely that competitive interactions of this type are of overriding importance in the establishment of bacteria in particular microenvironments. The soil is so heterogeneous and the bacterial populations so widely spread out within it (Gray, 1967) that the establishment of bacteria on a soil particle is more likely to be due to coincidence rather than the outcome of competition between strains arriving simultaneously on the same particle. This is not to say that competition studies tell us nothing about bacteria in a soil, for an inability to compete under favourable conditions will suggest spatial separation of zones of activity. Such considerations affect the applicability of Gause’s concept of competition to soil organisms. Gause (1934) supposed that if two different organisms occupied the same niche, one would sooner or later eliminate the other. However, if organisms such S.B.B. 5/d--F
472
W. E. LOWE AND T. R. G. GRAY
as bacteria arrive in microenvironments by chance and are so thinly spread out in the soil that the chances of two different strains arriving in the same environment simultaneously with any regularity are small, then it is probable that more than one organism wili be able to exploit one and the same type of niche. For competition to occur, it is also necessary that bacteria be growing actively in soi1. There is a large body of evidence (Gray and Williams, 1971) to suggest that the growth rates of microorganisms in soil are slow and that organisms remain dormant for long periods of time. Some of this evidence concerns the lack of available energy to support rapid growth of the comparatively large microbial populations present in soil, and one might ask, therefore, where the energy came from to support microbial growth in the present experiments. It is known that sterilization increases the availability of nutrients in soil (Warcup, 1957; Eno and Popenoe, 1964) and Jenkinson (1966) has shown that quite large respiratory flushes occur following the inoculation of microbes into sterile soil, due to the release of nutrients from the killed natural microflora. A similar situation probably accounts for the pattern of results in these experiments. Acknowledgements-We wish to thank Professor A. D. BRADSHAWfor his helpful advice in designing these experiments. One of us (W.E.L.) wishes to thank the Science Research Council for a grant enabling him to carry out this work. REFERENCES CRUIKSHANKR. (1956) Me&c& Microbiology. 11th Ed. Livingstone, Edinburgh. DE Wrr C. T. (1960) On competition, Versl. Landbouwsk. Onderz. R~ksla~dbProe~ Stn., 66. ENO C. F. and POPENOEH. (1964) Gamma radiation compared with steam and methyl bromide as a soil sterilizing agent. Proc. Soil Sci. Sot. Am. 28,533-535.. GARR~X~S. D. (1950) Biology of root infecting fungi. Biof. Rev. l&220-2.54. GARRETSS. D. (1970) Pathogenic Roo~“I~fec~i~gFungi. p. 114. University Press, Cambridge. GAUSE F. G. (1934) The Struggle for Existence. Williams & Wilkins, Baltimore. GRAYT. R. G. (1967) Stereoscan electron microscopy of soil microorganisms. Science, N. Y. 155,1&S-1670. GRAY T. R. G. and WILLIAMSS. T. (1971) Soil Micro-organisms. Oliver 8z Boyd, Edinburgh. JENKINSONS. D. (1966) Studies on decomposition of plant material in soil-II. Partial soil sterilization and the soil biomass. J. Soil Sci. 17, 280-302. LEDERBERGJ. and LEDERBERGE. M. (1952) Replica plating and indirect selection of bacterial mutants. J. Bact. 63, 399-406. LOWE W. E. and GRAY T. R. G. (1972) Ecological studies on coccoid bacteria in a pine forest soil-I. Classification. Soil Biol. Biochem. 4, 459-468. LOWE W. E. and GRAY T. R. G. (1973) Ecological studies on coccoid bacteria in a pine forest soil-II. Growth of bacteria introduced into soil. Soil Biol. Biochem. 5,449%462. SOKAN R. R. and SNEATHP. H. A. (1963) Principles of ~arner~ca~Taxonomy. Freeman, San Fransisco. WARCUPJ. H. (1957) Chemical and biological aspects of soil sterilization. Soils Fertil. 20 l-5. VAN DEN BERGHJ. P. (1968) An analysis of yields of grasses in mixed and pure stands. VersE. ~aadboa~sk. Onderz. R~kslandbProe~ Stn., 7 14. ZVYAGINTSEV D. G. (1962) Adsorption of micro-organisms by soil particles. Soviet Soif Sci. 140-144.