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IV. The Microflora of Grassland Soils
FRANCIS E. CLARK A N D ELDOR A. PAUL
chemical properties, and the type and amount of plant cover. Approaches
used by individual workers to study soil microfloras have varied widely.
Collectively, soil mycologists have placed a major emphasis on the preparation of floristic lists of the fungi in soil, while soil bacteriologists
were for many years concerned largely with the total bacterial count or
with the enumeration of physiological groups, such as the cellulolytic,
denitrifying, or nitrogen-fixing bacteria. It appears difficult or impossible
to arrange the formidable body of data collected by plate count procedures into any comprehensive picture of the microflora in grassland soil.
In the following paragraphs, discussion will be centered almost entirely
on the question of whether or not there are qualitative differences in the
bacterial and fungal floras of grassland and nongrassland soil.
A. BACTERIAA N D ACTINOMYCETES
The bacteria, the most numerous of the free-living microorganisms in
soil, vary in size from cells invisible or barely visible in the light microscope to clubbed, stalked, and branching cells and filaments many microns in length. Although some half dozen or more orders of bacteria are
recognized, a great majority of the commonly occurring soil forms are
placed in the orders Eubacteriales and Actinomycetales. In the soil microbiological literature, these two orders are usually referred to as the bacteria and the actinomycetes, respectively.
In extended study of the soil bacteria in New Zealand tussock grassland, Stout (1958,1960,1961) found that the majority of the bacteria
encountered were species of Pseudomonas. The aerobic spore-forming
bacilli were also a relatively common group. A portion of Stout’s data
is shown in Table X. Inasmuch as soil bacteriologists in other countries
Percentile Distribution in Different Genera of 4 I5 Bacterial
Isolates from New Zealand Soils”
Aerobacter spp. and
“Compiledfrom data of Stout (1960). Percentages in first vertical column based on study
of 162 isolates, and in second and third columns, on 135 and I18 isolates, respectively.
THE MICROFLORA OF GRASSLAND
had reported that the predominant bacteria in soils generally were members of the genus Arthrobacter (Jensen, 1933; Topping, 1937; Gibson,
1939; Lochhead, 1940; Clark, 1940), the possibility was raised that there
might be a different microflora dominant in grassland, or at least in New
Zealand tussock grassland. To investigate this possibility, Robinson and
MacDonald ( 1964) studied the bacterial flora of such grassland. They
concluded that the bacterial flora therein was essentially the same as that
commonly encountered in most soils. In an independent study, Loutit
and Loutit (1966) found that species of Arthrobacter, Nocardia, and
Mycobacterium formed the major portion of the bacterial flora in New
Zealand grassland. Their work, a part of which is summarized in Table
XI, confirmed observations of Robinson and MacDonald (1 964).
Bacterial Types Encountered in New Zealand Grasslando.*
“Compiled from data of Loutit and Loutit (1966).
*Values represent the frequency distribution (%); the distribution within each vertical
column is based on study of 400 random isolates picked from the plating media employed.
Taylor (1938) surveyed 90 soils selected from widely separated parts
of Canada for the occurrence of Arthrobacter globiformis. In prairie,
woodland, garden, orchard, and mixed crop soils not strongly acid, A .
globiformis was invariably present in large numbers. The pasture and
grassland soils surveyed did not group themselves apart from the other
soils in percentages of A . globiformis colonies found in total plate counts.
Vandecaveye and Katznelson ( 1938) compared the microflora of forested
and grassland soils developed from the same parent material under similar
climatic conditions in the northwestern United States. There appeared to
be no distinct association between kinds of bacteria and type of vegetation. Aerobic cellulose-destroying bacteria and anaerobic nitrogen-fixing
bacteria were sparsely encountered in both soils. Ross ( 1 958,1960),
studying the nonsymbiotic nitrogen-fixing bacteria in tussock grassland,
FRANCIS E. CLARK AND ELDOR A. PAUL
found numbers of Clostridium comparable to those reported for other
temperate soils. Similar findings for other groups of bacteria are common
in the literature. However, there is evidence that nitrifying bacteria are
less abundant in grassland than in nongrassland soils. Discussion of this
evidence will be deferred until a later section dealing with nitrogen transformations. In brief, and with possible exception for the nitrifying bacteria, the existing literature on the bacterial flora of grassland does not
show it to be greatly different from that in soils generally.
It has frequently been emphasized that the conditions often existing
in grassland, namely, low soil moisture content, warm temperature, a
neutral or alkaline reaction, and a good supply of organic matter, such as
that provided by grass roots, preferentially stimulate the growth of the
actinomycetes over that of the bacteria (Alexander, I961 ; Kuster, 1967;
Kutzner, 1956; Mishustin, 1956) and that consequently the actinomycetes constitute a greater proportion of the total count in grassland than
in nongrassland soil. Orpurt and Curtis ( 1 957) and Vernon ( I958), working in Wisconsin prairie and New Zealand tussock sites, respectively,
observed that actinomycetes accounted for half the total plate count.
In contrast, there are also numerous reports that grassland soils contain
no higher percentages of actinomycetes than do adjacent cultivated or
forested soils (Vandecaveye and Katznelson, 1938; Timonin, 1935; Sandon, 1928). Data of Robinson and MacDonald (1964) given in Table XI1
show the percentage incidence of actinomycetes to be lower in undisturbed grassland than in nearby cultivated soil. In view of the conflicting
literature, it does not yet appear possible to state whether actinomycetes
are proportionately more or less numerous in grassland than in nongrassland soil.
Knowledge about fungi in grassland soils is limited and parallels that
for fungi in soil generally. Warcup ( 1967) has stated that although long
floristic lists of soil fungi have been compiled, one is still unable to give
an adequate picture of the fungal flora in a soil. While the importance of
specific substrates for fungal growth has become recognized, there remain
major problems in mycological studies, such as determining what organisms are present on a substrate in soil, differentiating between dormant
and active portions of a fungus or of different fungi, and with measuring,
in some sense, the activity of fungi in natural substrates.
During the early years of the present century, descriptive studies of
fungi took precedence over eclogical investigations concerning their
distribution in soil. The early literature on soil fungi has been adequately
Actinomycete Populations Encountered in New Zealand Soil as Shown by Three Different Plating Media“
Fortified soil extract agar
Soil extract agar
Cultivated and limed
Cultivated and limed
Cultivated and limed
“Compiled from data of Robinson and MacDonald (1964).
*Each value given represents mean of two sampling dates: November 3 and December 14.
FRANCIS E. CLARK A N D ELDOR A. PAUL
reviewed by Waksman ( 1916). The literature was sufficiently fragmentary to cause Waksman (1917) to ask whether there was a specific fungus flora of the soil, or whether the species present therein were only
occasional invaders. After study of soils from several widely separated
geographical areas, he concluded there was a distinct soil fungus flora,
with the species occurring in any particular soil dependent on a number
of soil and climatic conditions. His conclusions were generally supported
by other soil mycologists of the era (Werkenthin, 1916; Brown, 1917;
Brierly, 1923; LeClerg and Smith, 1928; Jensen, 193 1). Although some
of these workers included an occasional meadow or pasture soil in their
studies, for the most part they were concerned with nongrassland soils,
and it remained for a later group of workers to ask whether there was a
specific fungus flora for grassland soil.
Paine (1927) observed that Mucor spp. were not as abundant in grassland as in forest areas, whereas Hormodendron and Cladosporioides and
Aspergillus fumigatus were more numerous in pasture. In a study of
virgin and cultivated profiles in Manitoba, Bisby and co-workers (1933,
1935) found Aspergillus spp. relatively rare, and Penicillium spp. relatively common. Trichoderma, although present in both grassland and
forested soils, was more numerous in the latter. Fusarium was especially
common in prairie soils, and F . oxysporum was the species most frequently encountered, usually in the A horizon. They observed that Monotospora daleae (Mycogone nigra) was isolated frequently from grassland,
but never from forested soil. As this species was also encountered in
wheat fields, they concluded that it was commonly associated with the
In a study of five English grasslands, Warcup (1 95 1) found that such
soil factors as acidity and temperature were of overriding importance in
determining species distribution. He concluded that different fungal populations occur in different grassland soils. England and Rice (1957) compared the soil fungi of a tall grass prairie with that of an adjacent abandoned field in central Oklahoma. With soil sampling conducted throughout
the year, at no time did species common to both plots exceed 50%; the
average was 35.2%. There were 9 species in both plots at all sampling
periods; namely, Aspergillus fumigatus, Pencillium notaturn, Mucor
globosus, M . sphaerosporus, Monotospora brevis, Fusarium decemcellulare, F. lateritium, F. nivale, and F. orthoceras.
Orpurt and Curtis ( 1957) determined the soil microfungi for 25 prairie
sites in Wisconsin. Whereas Fusarium spp. were encountered only infrequently in forested soils, they were among the most prominent and
characteristic members of the prairie soil flora. Likewise, Aspergillus
THE MICROFLORA OF GRASSLAND
spp. were common in prairie soils, particularly in the drier sites. Penicillium spp. tended to do better in mesic prairie, and the Mucorales in the
wetter sites. The genus Emericellopsis was restricted to wet sites.
Thornton (1958, 1960, 1965), investigating the fungal flora of New
Zealand grassland, observed greater numbers of mycelia from soils under
pasture than under forest, and confirmed Orpurt and Curtis (1957) that
Fusarium was absent from forested soil but common in grassland, F.
oxysporum being one of the dominant species. Other fungi dominant in
tussock grassland were Trichoderma viride, Rhizoctonia spp., Cladosporium herbarum, Mucor hiemalia, Mortierella spp., and Penicillium
spp. The observation that the soil fungal patterns for five pastures were
very similar led Thornton to believe in the existence of a similar environment in those soils for fungi. Grass roots appeared to be the dominant
factor in the development of a uniform environment. Fungal species recovered from the surfaces of grass roots were mainly the same species as
those recovered from soil apart from roots; also they were primarily the
same species as Waid ( 1957) had observed as responsible for the decomposition of ryegrass roots in soil.
Mishra ( 1 965, 1966), in studies on the seasonal distribution and variation in the fungal flora of grasslands of Varanasi, India, compiled extended observations on the frequency of occurrence of individual species.
The commonest species throughout the year were Thielaviu terricolu,
Chaetomium globosum, Aspergillus niger, A . terreus, and Paecilomyces
fusisporus. He concluded that though a limited number of fungal species
showed specificity for different localities, no general principles could be
laid down concerning the specificity of fungal floras in relation to grass
consociations. Ray and Dwivedi (1962) and Dwivedi (1966) also made
mycological studies on Indian grassland. The majority of their isolates
belonged to Aspergillus, Trichoderma and Cladosporium. In Fusarium,
only F. nivule was commonly encountered. Penicillium spp. were sparsely
encountered; only one species, P. funiculosum, was common to all the
grassland sites investigated.
Table XI11 shows the frequency of occurrence of species of fungi at
differing profile depths on the Matador grassland site in Saskatchewan.
These data emphasize that which has so often been noted by workers
elsewhere, namely, the dominance of Fusarium spp. in grassland. They
also show that the relative frequencies of occurrence of different genera
are not constant at differing profile depths.
A fungal phenomenon long known as common in grassland is the fairy
ring (Shantz and Piemeisel, 1917; Bayliss-Elliot, 1926). In fairy rings
there is dominant development of one or few basidiomycetes, such as
F U N C I S E. CLARK A N D ELDOR A. PAUL
Marasrnius oreades and Clitocybe gigantea. Their dense mycelial zone
contains a restricted population of microfungi, with fewer species being
present as compared with uninvaded soil (Warcup, 1951).
Frequency of Occurrence of Species of Fungi in Platings
of Matador Grassland Soil, April-October Inclusive"
Sterile dark forms
D. Parkinson (personal communication).
"Numbers represent percentages of washed particles yielding the fungi.
C. ALGAEA N D LICHENS
Although algae are found in soils everywhere, they have not been
widely studied by soil microbiologists. The most intensive investigations
have been conducted on soils in Russia (Shtina, 1957, 1963) and on the
waterlogged and underwater soils of rice fields in India and Japan (Lewin,
1962). Other studies worthy of mention have been made by Bristol Roach
(1926, 1927) and Lund (1945, 1967) in England, Tchan (1953, 1959) in
Australia, and Durrell (1959) and Shields and Durrell (1 964) in America.
The algae, being photosynthetic, mainly inhabit the soil surface and the
undersurfaces of transparent or translucent rocks. Their numbers usually
are directly proportional to the availability of light at the ground surface,
or inversely proportional to the completeness of ground cover by higher
plants. Cameron and Fuller (1960) found extensive crusts of soil algae in
desert regions sparsely covered by plants. Piercy ( 1917) found that Chlorohormidiurnflaccidurn grew abundantly in wet periods after drought had
killed the grass cover and before a new sward appeared. Small ponds that
occur in grassland, particularly if they serve as watering holes for grazing
animals and thus become enriched in mineral nutrients from animal droppings, frequently exhibit heavy blooms or surface growths of algae.
Possibly greater light intensity at the soil surface accounts for the reportedly richer algal flora found in grassland than in forested soil (Peter-
THE MICROFLORA O F GRASSLAND
sen, 1935; John, 1942). The latter found an average of 15 species in seven
forested soils and of 27 species in six grassland soils.
Other workers have emphasized the uniformity of algal types in soil.
Durrell (1959), in an investigation of the algae in 223 soil samples representing widely dissimilar types of Colorado soils, found 85 species of
algae representing 40 genera. In buffalo grass sod, species of Phormidium,
Chlorococcum, Nostoc, Schizothrix, Protococcus and Stichococcus were
present. Particularly common were Phormidium tenue and Chlorococcum
humicola. These same species were also present under saltgrass, in white
alkali spots, in soil from an old dried stream bed, in mud from the edge of
pools, and also in each of three successive seasonal samplings from ten
marked stations across a cultivated grass area. Durrell concluded that
there was a great similarity of algal species not only for diverse Colorado
soils but also between his isolates and those reported from other countries. Flint (1958) also noted a marked resemblance in the occurrence of
the major groups of algae of three tussock grasslands in New Zealand to
that of algae reported for soils in other countries. More recently, Balloni
and Materassi (1968) failed to find meaningful differences in the algal
floras of virgin and cultivated soils in Venezuela.
Lichens are symbiotic associations of algae and fungi in which one or
both members of the association undergo considerable change in morphology. It is for this reason that the lichens are considered as taxonomic
entities. Lichens are able to colonize extremely inhospitable land surfaces, such as exposed rock. The lichens are not a dominant part of the
microflora in mature grassland insofar as biomass and decomposer activity are concerned. Their importance in ecosystem function lies in their
influences on soil water regimes, their possible contribution to nitrogen
fixation, and their release of nutrients through soil or rock weathering.
V. Biomass a n d Bioactivity Measurements
OF THE BIOMASS
Estimates by direct microscopy of the number of bacterial cells in
cultivated soils are usually of the order of several billions per gram of
soil. Strugger ( 1948) using fluorescent microscopy observed from 2-9
billion viable cells per gram. Taylor (1936) using the dilution ratio method
for direct counting reported soil bacterial populations of approximately
three billion per gram. Clark (1967) concluded that two billion cells per
gram could be taken as a representative value for the bacterial population
of a productive agricultural soil. On the common generalization that one
trillion bacterial cells weigh 1 g, a bacterial biomass of two billion cells
FRANCIS E. CLARK A N D ELDOR A. PAUL
per gram of soil amounts to 450 g live weight per m2 to a depth of 15 cm.
Taking the water content of the bacterial cell as 80% (Camp, 1963), the
live-weight estimate can be restated on a dry weight basis as 90 g/m2to a
depth of 15 cm.
Individual soils of course vary greatly in their microbial content. Published estimates of live-weight bacterial biomass in soil are usually expressed as a range of values. Alexander ( 196 1) has stated this range to be
33-330 g/m2/15 cm; Jensen (1963), 100-1OOOg; Russell (1950), 170-390
g; Krasil’nikov ( 1 944),67-720 g; Latter and Cragg ( 1967), 2 1 - 135 g; and
Stockli (1956), 160-380 g. Averaging the above estimates gives a value
of 270 g live weight, or 54 g dry weight, for the bacterial biomass per m2 to
a depth of 15 cm.
In the older literature concerning estimates of the fungal biomass in
soil, the fungi were commonly stated to possess a biomass ranging from
equivalence to twice that of the soil bacteria. Recent measurements
(Stockli, 1956; Jackson, 1965, Latter et al., 1967) of hyphal lengths in
soil by direct microscopy range from 100 to 2000 mlg. The mean value
obtained by Jackson (1965) for seven soil samples was very close to 120
m of hyphal length per gram of soil. Transposing this measurement to
biomass, using an average hyphal diameter of 3 p , gives a value of 38 gl
m2/15 cm, or somewhat less than the 54 g estimate derived above for the
bacterial biomass. It is interesting to note that Latter and Cragg ( 1 967)
found the biomass of fungi in a Juncus community to be approximately
two-fifths that of the bacterial biomass.
Data collected by Babiuk and Paul (1970) and Parkinson (personal
communication) in their studies on the magnitude of the microbial biomass in grassland at the Matador site in Saskatchewan are shown in
Table XIV. Their data were calculated on the basis that the average size
of the bacteria and actinomycetes was 0.6 by 1 p, and the average diameter of the fungi, 2.5 p. The bacterial diameter of 0.6 p, determined by
microscopic examination using fluorescein isothiocyanate, may be somewhat low, but even the use of 0.8 p as the diameter would still indicate
that the fungal biomass was roughly twice that of the bacteria, if viewed
on a mean seasonal basis. The data indicate that the ratio between biomass estimates of the bacteria plus actinomycetes and that of the fungi
at the 0-10 cm depth is similar to the ratio between the two groups at the
0-30 cm depth. There did not appear to be any positive correlation between the changes in biomass of bacteria and fungi during the season.
Whereas the bacteria approached their highest value in May, the fungi
were at their lowest. The biomass values tabulated are perhaps the best
current estimates for bacteria and fungi in grassland soil, and accordingly