Competitive outcome among four pasture species in sterilized and unsterilized soils

Sod Bid. Biochcm. Vol. 23. No. 9. pp. 837-843. 1991 Printed in Great Brilain. All r@hts reserved

copyrigh1(0

0038-0717/91 $3.00 + 0.00 1991 Rrgamos Press pk

COMPETITIVE OUTCOME AMONG FOUR PASTURE SPECIES IN STERILIZED AND UNSTERILIZED SOILS ROY TURKINGT~N and ELENA KLEIN Botany Department, University of British Columbia. Vancouver. British Columbia. Canada V6T 124 (Accepted

8 March 1991)

Summary-Four pasture species (Dactylis glomerata. Holcus lanatus. Lolium perenne and Trifoiiutn repens) were grown in monoculture and in all possible 2-. 3-. and Cspecies combinations in pots. One set of pots was filled with sterilized soil in which most soil microorganisms and mycorrhiza had been eliminated, a second set was unsterilized and had an added Rhi:obium inoculum. The experiment had four successive destructive harvests. For each plant species,regardlessof the identity of its competitors.percentage survival was lowest in unsterilized soils, but the mean weight of survivors was unalfected. except for T. repens which had an increased biomass. In addition, at the first harvest the microorganisms and each of the plant species had a significant effect on the relative growth rates of each of the target plant species but this effect was not continued to the final harvest. It is argued that either (a) in the unsterilized soils microorganisms inhibit germination of some seeds or adversely affect young seedlings. and that they compete with growing plants for limited resources, (b) sterilization eliminates most of the bacteria present and this along with the added Rhizohium inoculum might contribute to the higher survival in sterilized soil. or (c) the nature of sterilization growing plants.

procedure

alone increases the availability

of essential resources to

INPRODUCTlON

METtIODS

Pastures and grasslands arc dynamic and complex systems. Dcspitc their complexity. rcpcatablc patterns of population and community structure arc readily dctcctablc, patterns which may bc due to abiotic conditions. compctitivc interactions, grazing, and other factors. A largely unresolved factor in these systems is the below-ground activities of soil invcrtebrates (Seastcdt et (11.. 1988). mycorrhiza (but see Newman, 1988; Harley, 1989) and other microorganisms (Newman, 1978; Rovira, 1978). These have their own direct effects on plant growth (Gaskins et ol., 1985) but also indirectly influence plant growth by interacting with each other and by altering soil abiotic conditions. There arc a number of greenhouse and laboratory studies (Cerligione er al.. 1987; Sco et 01.. 1988) but few field studies on the effects of microorganisms on plant growth (Fitter, 1985; Anderson and Liberta, 1989). Often the field studies produce equivocal and conflicting results (Ingham and Detling. 1986; Seastedt er of., 1988). This study is one in a series that has been designed to investigate the role of microorganisms, particularly Rhizobium(Shivji and Turkington, 1988; Thompson et al.. 1990; Chanway et al., 1989) and Bacillus (Chanway er 01.. 1990) on the growth, competitive interactions, and plant morphology of some pasture species. This study was undertaken to determine the effect of soil microorganisms on competitive outcome among four pasture species. This was done by comparing the outcome of competition in sterilized soils from which most microorganisms had been eliminated, with the outcome in unsterilized soils with an added Rhkobium inoculum.

One hundred and eighty 21 cm dia pots were filled with a growth mixture containing equal volumes of peat, soil, pcrlite and washed sand. An additional 180 new and clean pots were filled with a similar soil mix in which the soil and peat had been steam-sterilimd at I kg/cm’ for 90 min. This eliminated or significantly reduced the microorganism population of the soil mix. The unsterilized pots had 5 ml of Rhizobiurn Ieguminosarum biovar rri/olii inoculum (trade name Nitragin) mixed into the upper 2cm of the soil. !Seeds of the grasses Dacrylis glornerara (Orchardgrass), Holcus lanurus (Velvet grass), tolium p4renne (perennial ryegrass) and Trifolium repens (white clover), from a commercial supplier, were divided into two groups. Those to be sown in the sterilized soils were surface-sterilized by enclosing in a cloth bag and dipping in ethanol (80%) for 45s. in 1.2% sodium hypochlorite for IS min, followed by five 3 min washes in sterile distilled water; the other group of seeds was not surface-sterilized. In mid-June, seeds were added to each pair of sterilized and unsterilized pots in either monoculture, all 2-. 3-, or 4-species combinations. In all pots the sowing density was 2.5 seeds per cm’. The experiment was done outdoors at the University of British Columbia field station. Pots would have been contaminated continuously by airborne microorganisms, by rain-splash etc., but to reduce the expected contamination of sterilized treatments, all pots were placed on I cm high plywood blocks which were resting on sheets of plastic. All pots were randomized and the treatments replicated three times. The first harvest was taken after 5 weeks and the remaining three harvests at every 4 weeks until mid-October. Each harvest was destructive and used the plants from 45 sterilized and 45 unsterilized pots. At each harvest, the number of individual plants (grasses and T. repew) was counted. Plant shoots were separated by species, dried at IOBT for 4 days, and weighed. At the final harvest all measurements were made on a 25% subsample of each pot. By the end of the experiment, although the

831

ROY TURKINGTON and ELENA

838

sterilized treatments had undoubtedly been contaminated sad nodules were observed on the roots of T. reppnr (a problem also reported by de Wit cr al.. 1966) a very clear distinction in plant growth still remained between sterilized and unsterilized soils. Analysis of variance was performed on log,, transformed data using PRDC GLM in the SAS statistical package

(Freund and Littell. 1981). Two-sample f-tests were performed using the Midas statistical package (Fox and Guire. 1976).

RESULfS

The indices of growth are percentage survival of sown individuals and the average dry weight of surviving individuals. From these. the relative growth rate (RGR) and the relative yield total (RYT) between successive harvest can be calculated. For clarity of presentation, percentage survival and mean dry weight will be presented for the final harvest only (Table I; Figs I and 2) and RGR (Table 2; Fig. 3) for the four successive harvests.

Doclylis

Surkal

KLEIN and growth

Many combinations of plant species in both sterilized and unsterilized soils had significant effects on the survival and growth of the four species at harvests one and two (data not shown). Most of these were the effects of a single species on a target species. On average, H. lunotus is the weaker competitor with the other species showing no significant differences in their effects on each of the target species. Other significant effects in the early harvests were (a) interactions involving a single-species and the presence or absence of microorganisms, or (b) plant x plant effects which nearly all included 1: repens in the mixture. The presence of the microorganisms had a significant (P c 0.001) influence on the growth of D. glomerata. L. perenne and T. repens throughout the first three harvests (P -c 0.03 for H. lono~). All effects had diminished by the fourth harvest and significant effects were almost exclusively microorganism effects or the effects of a single species on a target species (Table I).

glomsrolo

Holcus lonolus

100 go-

80

80-

yj

70 -

et 1.

70

60 -

,’

60

&

50

5o-

II

r-l

0 = al 2 a”

40 JO

20 10

MHLTHHLH LTTL

LTTL

T

T

B

a0

5

*,?I

70

*?

iz

70

3 w

60

2

60

& 0 E

50

50

40

& 0 5

40

;

30

e

30

2

20

cc

20

10

60

10 liDilL

DDHD H L L

H L

Fig. I. The percentage survival to the fourth harvest of D. glomeruru (D). H. hurus (H). L. per-e (L) and T. reppns (T) growing in monoculture (M) and with various mixtures of species, in sterilized (solid bars) and unsterilized (open bars) soil.

839

Compcrition among plants in stcriiizcd soils i.e. unsterilizedn skrikd soils(summedover all plant species annbinatioaa). singk and growth of four target speciesat barvest4. Values a F-probabilities

Table 1. The inf!uenceof microor~s, and titura

spozies,

of specks, on the wrvivd

Target succis

Rtcat

Mean dry

SUIViVJ

Microo~nirmr Dactyli. Hokw L.olinm T?ifdiUWl Dactylis,H&w Dactylit, Lo&m Dactyl&. Tri/diwn lfolcw, L.&m Holcus. TriJiilium Ldium. Tri/oliwn Dactylic. Holcus. Lolium Dactylis. Holctu. Tri/oium Dactyl&. Lolium. Trifolium Holeus. Ldium. Trifolium

Ldim

H&u

DWtylit

NS

0.0001 O.CQOl 0.0001 0.0232 0.0054 NS

0.0276 0.0001 0.0005 0.0126 NS NS

-

0.0050

Dactylis

SUtiVd

wi&I

0.0003 -

Mean dry

PcrunI NS 0.0003 0.0001 0.0002 NS NS -

swvivrl

weight

NS 0.0142

0.0026 O.OWl 0.0001 0.0001 NS -

0.0023 O.WOi NS

NS NS -

Ns -

o.wO4 0.0414 NS NS

0.0247

NS

-

-

NS -

0.0374

Ttifdth

Mcandty

weight

-

-

Pcramt

NS -

glomerola

Penzen SUWiVd

0.0001 o.wi4 0.0192 O.Mw)I NS NS 0.0079 -

0.0001 NS

NS -

Man dry Weigh1 0.0001 0.0431 NS 0.0076 NS NS IG -

-

-

-

-

NS

NS

NS -

NS -

-

-

Holcur

lonotur

2 s r 0 J 0 .E: 0 .c, 0 9 k a. E 0 ‘5 3

1.75 1.5 1.25 1 0.75 0.5 0.25 0

tl

tl

I.

LTT

H

L T

IllIL MDLTD

Trifollum

MDHTDDH

II

T

T

MDHLDD

L

D T

I

:DL T

repens

H

L

Fig. 2. The average weight of 100 individuals, at the fourth harvest, of D. glomcrora (D). H. /attutus (H). L. perennc (L) and T. repens (T) growing in monoculture (M) and with various mixtures of spccics, in sterilized (solid bars) and unsterilized (open bars) soil.

840

ROY TURKINGYGN

and ELENAKLEIN For all target species, regardless of the identity of its competitor percentage survival was reduced in unsterilized soils (Fig. 1). Nearly all peaks for the without-microorganisms treatment (solid bars on Fig. I) involved T. repens; in contrast, none of the peaks for the with-microorganisms treatment (open bars on Fig. 1) involved T. repens, except when T. repens was the target species. While all target species had fewer survivors in unsterilized soils, the pattern did not hold for dry weight of survivors. Microorganisms retarded growth of L. perenne, had little effect on D. glomeruta and H. lanatus, and significantly increased dry weight of T. repens in all treatments (Table I; Fig. 2). Relative growth rate (RGR)

The mean relative growth rate, R. is a measure of the average rate, during a given period, at which a plant produces new material, and is calculated by: K,_, = log W, - lo& W, IT2 - T, where W, and W, are the dry weights at times T, and T, respectively. R is measured in units of wt wt-’ t-‘, and in this study was mg g-’ week-’ (Fig. 3). Few of the significant effects were species interaction effects so only the etIccts of single species on a target species have been tabulated (Table 2) and graphed (Fig. 3). Each of the species, and the microorganisms, had a significant influcncc on the mean relative growth rate of target spccics D. glomerara. If. lanatus and L. pmmne during the first growth interval (Tr, to r,), but only microorganisms and L. perenne had a significant cfI’cct on T. repens during this period. In only a few instances did a significant cffcct on mean relative growth rate continue through the second growth period, and ncvcr to the fourth period (Table 2). For the three grasses thcrc was a general pattern of a decreasing R during the four growth periods, although there is often a tendency for the rate to be slowest for the third, rather than the fourth, period. Where significant differences (Table 2) do exist, higher values of K for the grasses occur in sterilized soils, and for T. repes in the unsterilized soils. Relative yield total (R YT)

RYT describes the mutual relationship of mixtures of species that may, or may not, be making demands on the same resources. The relative yield (RY) of a species is calculated by: RY of species I =

Yield of I in mixture Yield of I in pure stand’

The RYT is the sum of the RYs for all of the species in a mixture. In only three cases (D. gfomerata-T. repens, P = 0.002; L. perenne-T. repens, P = 0.038; D. glomerata-H. lanatus-T. repens, P = 0.007) were there significant differences between

the sterilized and unsterilized treatments and each of these occurred in the final growth period and each had T. repens as a component of the mixture. Considering all species mixtures there are no general trends due to either the presence or absence of microorganisms; there are almost an equal number of cases of RYT > I .O and RYT < 1.0 for both sterilized and unsterilized treatments. Values of RYT > 1.0 tend to

I

. .-

800-j Holcus lonotus

72

glomerofo

T3

74

700-

600 -

repens

perenne

800 _ Trifolium

800 ,LoIium

-

;

E

:

f

_aX

400

700 600 500

8oo

-300

Tl

Tl

! T3

72

T3

. 0 with D. gtomeratr , 0 with L pcrenoc A A with T. repeos

T2

0 with ft. Imates 0 with L percnoc b A wilh T. repens

l l

T4

T4

I

9) Two species

0

Trifollum

u Tl

ropens

74

74

A with L. pererme

A A with T. repcor

72

-300w

coo,

-300

-200

-100

100

200

300

400

500

600

700

600

mix

~. ---

- -

.

__

-._-_.

- -_-

_ --.---.-

-

. . -.

__---

_-

-_--.-

Fig. 3. The relative growth rate of D. glomerara. H. hna~u~. L. prennc and T. repent over four successive time intervals when grown in (A) monoculture and in @I 2-s@= mixtures. in sterilii (open symbols, dotkd tines) and unsterilized (solid symbols. solid tint) soil.

c

11

600-j

:

CJBsoo-

700-

f

;

g~~,Ooctylis

_?

A) Monoculture

ROYTURKINGIVN and ELP~AKLEIN

842

occur in species mixtures which include F. repens; there are 28 values of RYT > 1.23 and all include T. repens (data not shown). DISCCSSION

Under natural conditions plants continuously interact with other organisms, herbivores, neighbouring plants, and a broad array of microorganisms both above ground and below ground. Association with soil microorganisms can influence plant growth and, therefore, the nature of interactions with other neighbouring plants and ultimately have an effect on population and community structure (see reviews by Newman, 1978; Rovira. 1978; Gaskins er al., 1985; Turkington er al., 1988). Often the association is beneficial to the plant if the microorganisms solubilize minerals, fix Nr, produce growth-promoting hormones, or suppress pathogens. In contrast, soil microorganisms can adversely affect plants by immobilizing essential nutrients (Barber, 1978). by competing with plants for available nutrients, and by secreting toxins (Hoagland and Williams, 1985). Any of these mechanisms can be speculatively applied to the descriptive plant responses observed in our study. For each of the four species, pcrccntagc survival to the fourth harvest was reduced in the unsterilized soils a result consistent with survivorship of .Schirachyrium scopuriunt (littlc blucstcm grass) in unfumigatcd soils (Anderson and Libcrta. 1989). Surprisingly, the reduced numbers wcrc not compcnsated for by an incrcascd weight of individual survivors, except for T. repens, nor did it change the pattern of rclativc compctitivc ability. Thus, the total plant dry weight per pot for each of the three grasses was greater in sterilized soils bccausc thcrc were more survivors. It is tempting to argue that microorganisms in the unsterilized soils were exerting a negative influence, perhaps pathogenic, at the germination or early seedling stages of all species. but the influence did not apply to the growing plant. However, because total plant dry weight per pot was always significantly lower in unsterilized grass pots, this suggests that microorganisms may be competing with the grasses for essential resources. Seastedt ef 01. (1988) came to a similar conclusion with studies on a tallgrass prairie. They added sugar and inorganic N to plots and recorded changes in population densities of micro- and macro-arthropods, nematodes and microbes. Bacteriovorous nematodes responded signilicantly by doubling their densities in sugar-plots, presumably because of increased bacterial densities. Plant biomass also increased in the sugar-plots. The parallel response by plants and fauna suggests that they both compete for the same resources. Howcvcr, enhanced growth in sterilized soils may simply bc related to the sterilization process which has been shown to increase the availability of inorganic nutrients (Jakobsen and Andersen. 1982). and to alIect inorganic nutrient competition effects among mycorrhiza and soil microorganisms (Ridge, 1976). The data do not permit us to distinguish between these options. The combined effects of the sterilization procedure possibly increasing the availability of inorganic nutri-

ents (Jakobsen and Andersen. 1982), random contamination of the sterilized soils by N@xing Rhizobium, and presumably by other microorganisms, and the addition of the RhL-obium inoculum, probably all contribute to the observed patterns of growth of the grasses with T. repens. Each of the grasses had their greatest growth when in mixture with T. repens in sterilized soils. The survival and growth data are reflected in the values of R. Relative growth rates are influenced by two major factors, ontogenetic drift and environmental variables. Ontogenetic drift is due to develop ments which occur within the plant and high relative growth rates early in the life cycle, and declining with time, are typical of many species (Hunt, 1978). It is difficult to disentangle ontogenetic effects from the background of a changing environment, primarily all those factors associated with changing plant density as the plants grow. However, in comparing values of a in sterilized and unsterilized soils, most of the shifts may be assumed to be due to the microorganisms. Again, the microorganisms are inhibitory for the grasses and may bc competing with the plants for essential nutrients, or perhaps are releasing toxins. The nutrient-limitation hypothesis seems more plausible considering that the effects arc rcvcrscd for contaminated “stcrilizcd” pots containing T. repens. This hypothesis is also supported in a gcncral way with the RYT data whcrc mixtures containing T, repens tends to have the highest values. Our rcsuhs show that soil microorganisms have a large inhibitory clTcct on the early lift stages of thcsc four pnsturc species. particularly on survival, but have littlc cffcct on the subscqucnt growth of seedlings to adult plants, or on their rclativc compctitivc abilities. However, sterilization clearly incrcascs the carrying capacity of the pots thus, cithcr the microorganisms in unstcrilizcd pots are competing with’thc plants for limiting resources, or the stcrilization procedure increases the availability of essential nutrients. Ackno~l~~/~c,nlenf~-Thiswork was funded by an operating grant from the Natural Sciences and Engineering Research Council of Canada. WC thank L. Mehrhoff for help with statistical analyses. REFERENCES Anderson R. C. and Liberta A. (1989) Growth of little blucstem (Shizuchyrium scoparium) (Poaceae) in fumigated and nonfumigated soils under various inorganic nutrient conditions. Americun Journal of Boruny 76, 95-104. Barber D. A. (1978) Nutrient uptake. In Interuciions Berween Non-khogenic Soil Mick.wrganisms and Plants (Y. R. Dommeraues and S. V. Krupa, Eds),. pp. . . 131-162. Elsevicr. New York. Cerligione L. J., Libcrta A. E. and Anderson R. C. (1987) EITccts of soil moisture and soil sterilization on vaiculararbuscular mycorrhiz al colonization and growth of little bluestcm (Schizuchyrium scopurium). Canudiun Journul of Botuny 66, 757-761. Chanway C. P.. Holl F. B. and Turkington R. (1989) Effect of Rhizobium Ieguminosorum biovar trifolii genotype on specificity between Trtyolium repens and L,olium perennc. Journal of &co/ogy 77. I 1% I 160. Chanway C. P.. Hall F. 8. and Turkington R. (1990)

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