The effects of grazing or cutting a perennial ryegrass and white clover sward on the microflora of the soil

Soil Biol. Biochem. Vol. 4, pp. 199-205. Pergamon Press 1972. Printed in Great Britain.

THE EFFECTS OF GRAZING OR CUTTING A PERENNIAL RYEGRASS A N D WHITE CLOVER SWARD ON THE MICROFLORA OF THE SOIL D. M. HALL and ERNA GROSSBARD* Grassland Research Institute, Hurley, Maidenhead, Berkshire SL6 5LR

(Accepted4 November 1971) Summary--An investigation was carried out on soils from under perennial ryegrass and white clover swards comparing the effect of grazing and cutting on the soil microflora. The dilution plate count method was used with three different media: soil extract, cellulose and plant protein agars. Over a 3-yr period changes occurred in the microflora of the surface 2" 5 cm layer of the soil; numbers of actinomycetes and bacteria generally increased under grazing whereas fungi increased under cutting. An increase in the numbers of propagules of actinomycetes and bacteria as shown by colony counts and in biological activity as revealed by the determination of carbon dioxide and mineral-nitrogen produced during incubation of the soil may have been due to increases in the amount of organic matter in the soil under grazed swards. INTRODUCTION STUDIES of the microbial populations in virgin and arable soils and in litter are numerous (Timonin, 1935; Ivarson and Sowden, 1959; Christensen, Whittingham and Novak, 1962; Witkamp, 1963). Investigations of the flora under grassland are, however, relatively few and have been reviewed by Clark and Paul (1970). This report describes some microbiological studies on a long-term grassland experiment at Hurley. In this experiment different amounts of carbon dioxide and mineral-nitrogen were produced during the incubation of soils sampled from beneath grazed and cut swards (Clement and Williams, 1962); marked differences were also shown in the yields of wheat crops grown subsequently on the same plots (Williams, Clement and Heard, 1960). In the present work an attempt was made to correlate biological activities (carbon dioxide and mineral-nitrogen production) and grassland management (grazing or cutting) with counts of microbial propagules. MATERIALS AND METHODS

Sources of materials Samples of soil were collected in 1960, 1961 and 1962 from plots which had been sown to a mixture of S.23 perennial ryegrass (Lolium perenne L.) with S.100 and S. 184 white clover (Trifolium repens L.), and then either, (i) grazed rotationally about 6 times each year--"grazed" plots, or (ii) cut 4 times each year (with removal of herbage)--"cut" plots. All plots were grazed once during the first 6 months after sowing to assist in establishing the swards; experimental treatments were then introduced. All plots received annual dressings * Present address: Agricultural Research Council, Weed Research Organization, Begbroke Hill, Yarnton, Oxford OX5 1PF. S . B . B . 4/2--F 199

200

D.M. HALL AND ERNA GROSSBARD

of nitrogen (80 kg N/ha) and sufficient phosphorus and potassium to replace amounts removed in the herbage (Grassland Research Institute, 1956). The soil samples were taken from beneath swards of three different ages (6, 30 and 42 months after sowing) in September of each year. This was made possible by sowing fresh plots to ryegrass and white clover every spring. The sampling was repeated on all plots in each of three successive weeks to provide replication. Thus the total number of samples taken each year is given by Managements of sward × ages of swards × replication in time = 2 × 3 X 3 = 18.

Field sampling and soil preparation A sample was made up from ten soil cores, 2.5 cm diam. and 7.5 cm deep taken at random from a plot. These cores were placed separately in waxed cartons sterilized by u.v. irradiation. In the laboratory the surface 2-5 cm layer was cut from each core and these portions bulked to make up the final sample. All surface organic matter and stones were removed and the bulked cores chopped up and thoroughly mixed. The final samples were stored for a maximum of 3 days in fresh sterile cartons at 5°C before plating out.

Preparation of media All media except the suspension of plant protein were sterilized by autoclaving (20 min at 1 kg/cm z) and the agar media adjusted to give a pH of 7 when solidified at room temperature. Soil extract. Soil was collected in either autumn or early spring from areas on which, during the previous season, wheat had been grown following 3 yr under a ryegrass/white clover sward; this sward had been grazed and, like the wheat which followed, had received complete fertilizer treatment. Sterile soil extract (Wallace and Lochhead, 1950) was stored below 0°C. Cellulose suspension was prepared from absorbent cotton wool (5 per cent w/v); carboxymethyl cellulose gum (0.5 g/600 ml) was added to stabilize the suspension (Skinner, 1960). This suspension (80 ml) was diluted with 1 1. of mineral-salts agar (White and Downing, 1951). Yeast extract (0.01 per cent w/v) and ferric chloride (a trace) were added immediately before pouring the plates. Plant protein suspension was prepared from fresh lucerne material (5 per cent w/v; Grossbard and Hall, 1962). This suspension (80 ml) was diluted with 1 1. of mineral-salts agar (White and Downing, 1951) from which all mineral nitrogen was omitted. Yeast extract (0-01 per cent w/v), ferric chloride (a trace) and calcium chloride (a trace) were added. The protein suspension and mineral-salts agar were combined immediately before pouring the plates. Growth inhibitors. Antibiotics were added aseptically to all media except that used for anaerobic bacteria to suppress unwanted organisms. Rose bengal (0.003 per cent w/v) and streptomycin (0-026 per cent w/v "Stryzolin", May & Baker) were used to inhibit bacteria and actinomycetes. Actidione (0.004 per cent w/v) was used to suppress fungi and to allow the growth of actinomycetes and bacteria.

Dilutions, plating and incubation Soil (50 g) was shaken on an end-over-end shaker for 30 rain with NaC1 solution (0.75 per cent w/v, 450 ml). Serial dilutions were prepared using this saline solution; each dilution was shaken for 1 min before withdrawing suspension for further dilution. Plates were inoculated with 1 ml of soil suspension in 15 ml of molten agar medium. F o r anaerobic cultures, 1 ml of the suspension was added to 20 ml of molten agar medium in a McCartney

EFFECT OF GRASS MANAGEMENT ON SOIL MICROFLORA

201

bottle; when the agar had set, a layer (3 mm) of sterilized liquid paraffin was poured on top and the cap screwed tight. TABLE1.

DILUTIONS OF SOIL SUSPENSIONS AND PERIODS OF INCUBATION USED FOR THE VARIOUS ORGANISMS AND MEDIA

Organism

Medium

Dilution of s o i l suspension

Replication Period of incubation (days)

Aerobic bacteria and actinomycetes

Soil extract Plant protein Cellulose

1 × 10 6 25 x 104 25 x 104

4 4 4

12 15 6

Anaerobic bacteria

Soil extract

1 x 106 or 25 x 104

5

28

Fungi

Soil extract Plant protein Cellulose

1 x 104 or 5 x 10a 5 x 10a 5 x 10a

4 4 4

6 6 6

Dilutions plated, treatment replications and times of incubation in the dark at 25°C are given in Table 1. Moisture contents were determined by drying to constant weight at 100°C. The pH of the soil was determined in suspension in water (40 per cent w/v) using an electrode.

Recording of colonies After incubation (Table 1) the plates and McCartney bottles were examined without being opened, using a × 12 lens and transmitted light. All colonies growing on the soilextract medium and all colonies producing cleared zones in cellulose and plant protein agar were counted. Actinomycetes (mostly streptomycetes) were distinguished from bacteria when aerial mycelium had developed. RESULTS AND DISCUSSION The mean numbers of colonies which developed on the three media from the microbial propagules in soils beneath swards of different ages and under different managements are listed in Table 2. It must be stressed that the numbers refer to colonies arising from propagules, i.e. active cells, hyphal elements and spores, and it is accepted that a high but unknown proportion of the colonies of actinomycetes and fungi on the plates can be expected to arise from spores; such colonies are indistinguishable from those originating from hyphal elements. This work was carried out in the belief that the numbers of propagules formed or surviving in the soil as well as normal vegetative growth will be affected by environmental conditions. A statistical analysis of the data summarized in Table 2 indicates the two following trends.

Influence of management Actinomycetes and bacteria were more numerous in soils beneath grazed swards than beneath cut swards whereas relatively more fungi were found under cut swards.

Influence of age of swards Beneath the grazed swards the numbers of actinomycetes and cellulolytic aerobic bacteria increased during the period from 6- to 30-months from sowing but there was no further

6 30 42 M

6 30 42 M

6 30 42 M

Aerobic bacteria

Anaerobic bacteria

Fungi

0-63 0.67 0.65

25.5 18-4 22-0

333 337 335

10"9 14"4 12"7

Grazed

0.80

21"5

235

8" 1

0"83 1 "04 0"94

19.0 15-5 17"3

225 218 222

7"9 9"0 8-5

Cut

Sward management

4- 0.052++(c)

:~ 0.117 (a) ± 0.1143 (b)

± 1-66"(c)

::k 4"04 (a) :k 4.20 (b)

:k29" 26Hc)

~ 4 9 . 7 2 (a) :L53.84 (b)

:k 1-06~(c)

~ 2" 18*(a) :k 2.30 (b)

Standard errors of means

Soil extract agar

0"17 0" 15 0-16

14"8 12"4 13" 6

7.4 6.4 6"9

Grazed

0.20

10.9

3.2

0-21 0"24 0.23

7"5 10"4 9" 0

4"5 3-3 3"9

Cut

Sward management

4-0-028"(c)

! 0 - 0 3 2 (a) 4-0-034 (b)

4-2.67 (c)

~z2-52 (a) i 3 - 1 5 (b)

+0.77"~(c)

±1.49"(a) ± 1 - 5 8 (b)

Standard errors of means

Plant protein agar

a) Applies to the difference between the 6-month mean and a single 30- or 42-month mean. b) Applies to the difference between any 30-month mean and any 42-month mean. (c) Applies to the difference between the grazed and cut swards averaged over 30 and 42 months. *P < 0-05; "~P < 0"01; ++P < 0-001. M Mean of counts from the 30- and 42-month swards; 3 yr, 2 plots/yr, 3 replicates/plot, 4 plates/replicate 6-month Mean 3 yr, 2 plots/yr, 3 replicates/plot, 4 plates/replicate. 30-month Mean ~ Each 3 yr, 1 plot/yr, 3 replicates/plot, 4 plates/replicate. 42-month Mean . / For Anaerobic bacteria instead of 4 plates/replicate read 5 bottles/replicate.

l

6 30 42 M

Age of sward (months)

0.25 0"25 0.25

9.5 10.5 10" 0

6-9 8.3 7"6

Grazed

0.30

2.1

4.0

0.29 0.35 0.32

4.9 5.2 5" 1

4.2 4.2 4"2

Cut

Sward management

i o . o 3 o (c)

t 0 " 0 4 1 (a) ~0"046 (b)

~:0- 83+.(c)

+1.48~(a) ± 1 " 5 9 (b)

~0-76}(c)

i l " 27 (b)

11.15t(a)

Standard errors of means

Cellulose agar

M E A N NUMBERS OF COLONIES ( 1 0 6 / g OVEN-DRIED SOIL) ON SOIL EXTRACT AGAR, PLANT PROTEIN AGAR AND CELLULOSE AGAR OF SOIL MICROORGANISMS FROM GRAZED AND CUT SWARDS OF DIFFERENT AGES

Actinomycetes

Organism

TABLE 2.

0

Obo

EFFECT OF GRASS MANAGEMENT ON SOIL MICROFLORA

203

increase during the subsequent 30- to 42-month period. No similar trend was observed in the populations from beneath the cut swards. There was no evidence for changes in the populations of fungi and anaerobic bacteria under either type of sward during the period from 6 to 42 months after sowing. These results show the response of the soil microflora to two forms of utilization of the grass sward, namely cutting and grazing. This response is shown by the quantitative changes in microbial populations isolated on soil extract agar as well as by the specific physiological groups developing on the plant protein and cellulose agars. The main feature of these results is that the grazing treatment as compared with cutting seems to encourage the development of a larger number of propagules of actinomycetes and bacteria whilst formation of fungal propagules is favoured by cutting. These results may be compared with the data in Table 3 for carbon dioxide evolved and nitrogen mineralized during incubation of soil samples (Dr C. R. Clement, personal communication) from the same plots and sampling periods as we investigated although not TABLE 3. CARBON DIOXIDE AND MINERAL-NITROGEN OBTAINED AFTER INCUBATION OF SOIL SAMPLES FOR 20 DAYS AT 3 0 ° C

Year

Age of sward (months)

COz evolved (carbon part/106 of oven-dried soil) Grazed Cut

Mineral N (part/10p of oven-dried soil) Grazed Cut

1960

6 42

30.5 39.6

22.4 26.0

587 767

518 760

1961

6 42

23-3 53-9

27.7 45.8

504 818

460 568

1962

6 42

27"0 71.0

31.6 25"0

510 625

499 558

Data kindly furnished by Dr C. R. Clement of the Soils and Plant Nutrition Division, Grassland Research Institute. actually on the same samples and using the methods of Clement and Williams (1962). These data are available from 6- and 42-month old swards. Correlations with the microbiological data were calculated after combining data from both managements (Table 4). Significant correlations were obtained in 5 instances. Replication appears to have been too small to establish significance when managements were analysed separately. Data on biological activities in some instances follow the pattern of the microbial populations. Actinomycete numbers on all media showed correlations with carbon dioxide evolved on incubation. Also mineral-nitrogen formed during incubation was correlated positively with numbers of actinomycetes and aerobic bacteria counted on cellulose agar. This suggests that grazing encouraged the growth and activity of actinomycetes and bacteria. A higher root weight was found under grazed than cut swards (Grassland Research Institute, 1958) and over a 3-yr period greater increases were found in both organic-carbon and mineralized-nitrogen in the soils of the grazed plots compared with those of the cut plots (Clement, 1958). These increases occurred mainly within the surface 2.5 cm layer (which we examined) and, together with possible changes in composition of the organic matter, they may well have accounted for the increased numbers of the actinomycetes and bacteria in the grazed swards. The observations at Hurley are consistent with the work of

204

D.M. HALL AND ERNA GROSSBARD

TABLE 4. CORRELATION COEFFICIENTS BETWEEN CARBON DIOXIDE, MINERAL-NITROGEN AND MEAN NUMBERS OF ACTINOMYCETES, AEROBIC AND ANAEROBIC BACTERIA, AND FUNGI (DATA FROM CUT AND GRAZED PLOTS COMBINED)

Medium (number of colonies)

Carbon dioxide evolved r (d.f. 10)

Mineral-nitrogen r (d.f. 10)

Actinomycetes

Soil extract Plant protein Cellulose

+0- 802t +0. 784t +0" 795t

+0" 114 +0" 360 + 0" 781t

Aerobic bacteria

Soil extract Plant protein Cellulose

+0" 559 +0- 363 +0" 379

+0" 051 - 0 - 201 +0" 896++

Anaerobic bacteria

Soil extract

-0" 339

+0" 069

Fungi

Soil extract Plant protein Cellulose

--0- 451 --0.455 -- 0.400

--0" 138 --0" 197 -- 0" 138

Organism

Least significant values of r for 10 degrees of freedom = 0.576 (5 ~o)* 0-708 (1%)t 0.823 (0" 1%)++ Eggleton (1934) which showed that populations of micro-organisms under cut grass swards were higher than under arable due to the greater quantity of organic matter derived from the root m a t and herbage debris. The present results also agree with the reported cellulolytic (Ktister, 1967) and proteolytic (Waksman and Starkey, 1932; Waksman, 1959) properties of actinomycetes. The pattern of fungal counts contrasts with those of the actinomycetes and bacteria. The lack of a positive correlation between carbon dioxide evolution and fungal counts seems somewhat unexpected in view of the known efficiency of fungi in decomposing organic materials. However, these results confirm those of Jensen (1936).

Changes between years of sampling Although soil p H did vary between samples (ranging from 6.4 to 7.4 on the grazed plots and from 5.9 to 7.2 on the cut plots) this variation was not related to the year in which sampling was carried out. Other environmental records (rainfall, soil moisture, incident solar radiation and temperature) for the various sampling periods showed that the weather varied from year to year and probably accounted for the differences in number of colonies counted. Following the work of Rovira (1959) and Rouatt and Katnelson (1960) who observed decreases in bacterial counts with decreases in light intensity under relatively constant conditions of temperature and moisture, correlation coefficients were calculated between energy of incident radiation which was measured with a Megatron (Megatron, London) and number of colonies. The energy of this radiation was calculated for periods of 3, 4, 5 and 6 days immediately before sampling and also for the 3-day period covering 4-6 days before sampling. The results showed negative correlations between number of colonies and the energy of incident radiation which contrasts with the findings of the authors cited; it should be emphasized however that the present observations were made on field soils subject to normal fluctuations in temperature and moisture. The greatest number of significant correlations was obtained and these correlations reached their highest levels of significance for

EFFECT OF GRASS MANAGEMENT ON SOIL MICROFLORA

205

the 3-day p e r i o d (days 1-3) a n d decreased b o t h in n u m b e r a n d in level o f significance as the p e r i o d lengthened to 6 days. T h e y were also lower for the 3-day p e r i o d (4-6 days before sampling) t h a n for the p e r i o d 0 - 3 days before sampling. Thus the n u m b e r s o f colonies o b s e r v e d a n d therefore p r e s u m a b l y some aspects o f m i c r o b i a l activity a p p e a r e d to be related m o s t closely to the energy o f the incident r a d i a t i o n received b y the plots d u r i n g the three days i m m e d i a t e l y before sampling. U n f o r t u n a t e l y the d a t a available d i d n o t allow this study to be extended to include p e r i o d s o f 1 a n d 2 days i m m e d i a t e l y before sampling. There are several ways in which, either singly o r in c o m b i n a t i o n energy o f incident radiation m i g h t influence the microftora in the soil. There c o u l d be, for example, a direct effect o f i n d u c e d t e m p e r a t u r e on m i c r o b i a l activities; an indirect effect t h r o u g h the a m o u n t o f moisture present in the soil, o r an indirect effect t h r o u g h p h o t o s y n t h e s i s in the plants o f the s w a r d a n d later excretion o f organic m a t e r i a l s f r o m their r o o t s ( R o v i r a , 1959) or even w i t h d r a w a l o f nutrients b y actively-growing energy-rich roots. Acknowledgements--We are indebted to the late DR WILLIAMDAVIES,former Director of the Grassland

Research Institute, for his interest and encouragement in this work. We thank the Biometrics Department for assistance and members of the Soils and Plant Nutrition Division for discussions and criticism. REFERENCES

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