Plant and Soil 122, 259-266 (1990). © Kluwer Academic Publishers. Printed in the Netherlands.

PLSO 8126

Growth promotion of maize by legume soils A. FYSON and A. OAKS Department of Biology, McMaster University, 1280 Main St. I4I., Hamilton, Ontario, Canada L85 4K1 Received 3 February 1989. Revised August 1989

Key words:

alfalfa soil, fungicides, growth promotion, vesicular arbuscular mycorrhiza, Zea mays

Abstract

Maize (Zea mays cv W64A x W182E) was grown in a low nutrient sandy loam. Inoculation with legume soils (4.1% v/v) gave a 3 to 4 fold increase in shoot growth relative to the control after 5 to 8 weeks growth in greenhouse conditions. Plants were routinely irrigated with 1/10 Hoagland solution (with 10 mM KNO 3). With half strength Hoagland solution (10mM KNO3) there was no clear growth response. This growth response was observed with a variety of legume soils but not with any of the maize soils tested. The response to alfalfa soil was eliminated or much reduced by gamma irradiation (3.6 Mrad) or autoclaving of the inoculum. The bactericide streptomycin had no effect on the growth response whereas the fungicides benomyl and PCNB eliminated it. This suggests that fungi and not bacteria are involved in the growth promotion.

Introduction

effects on subsequent crops. To examine this aspect of crop rotation we have inoculated a low nutrient sandy loam with legume soils and have observed a significant growth response in 5 to 8 week old maize plants. In this paper we present experiments designed to characterize that growth response.

Maize (Zea mays) is a major cash crop in southern Ontario and is planted on the same land year after year. This results in yield declines (Bolton et al., 1976; Dumanski et al., 1986; Hesterman et al., 1986; Ketcheson, 1980), which may be overcome by rotation with grasses or legumes. The benefits of a legume crop in certain instances is more than can be accounted for by residual nitrogen (Bolton et al., 1976; Hesterman et al., 1986). Continuous maize cultivation may result in reduction in soil wet aggregate stability (Reid and Goss, 1980) and reduce the extent of the soil sheath encasing the root (Fyson and Oaks, 1987). This and other factors may have deleterious effects of soil water holding capacity, increase soil compaction and increase erosion which may cause significant reductions in potential yield (Battiston et al., 1987). Such trends are reversed by rotation with a legume crop. The role of legumes in increasing soil nitrogen through symbiotic nitrogen fixation is well established. The effect of the legume on other soil microbial factors whether beneficial or pathological is little known but is likely to have profound

Materials and methods

Maize cultivation Routinely, maize (cv W64A x W182E) was planted in 12 L pots in a pasteurized mixture of a sandy loam, sphagnum peat and Perlite (9:4:2/ v:v:v). However, in certain instances nonpasteurized field soils were also used. The sandy loam (Flamborough series) was obtained from the Millgrove Loam Farm, Flamborough, Ontario. An analysis of this soil (Ontario Ministry of Agriculture and Food) gave the following: - - pH 6.4; nitrate N 220 ppm; P 1 ppm; K 48 ppm; Ca 525 ppm; Mg 132 ppm; chloride 172 ppm; sulphates 800 ppm; Zn0.14ppm. In experiments with soil additions, 500 ml ofinoculum was thoroughly mixed with 12 L 259

260

Fyson and Oaks

of the standard soil prior to planting. Seedlings were thinned to one per pot after emergence. Plants were irrigated with 500ml of a 1/10 Hoagland solution which contained 10 mM KNO 3three times per week. In one set of experiments, 1/2 Hoagland solution was used to examine the effects of soil nutrient concentration on corn growth. Plants were grown in a greenhouse with supplemental high intensity mercury and sodium lamps and maintained on a 25 °C/ 20 °C, 16 h/8 h, day/night cycle. From May to September, the daytime temperature often exceeded 25 °C. Plants were harvested at 5 to 8 weeks, when a growth response was well established.

Greenhouse legume soils Alfalfa (Medicago sativa cv Iroquois) was planted in a 9:4:2 (v:v:v) mix of the Millgrove black loam, sphagnum peat and Perlite to which soil from an alfalfa field was added. The alfalfa soil was sub-cultured after 3--6 months by adding 10 percent of a pot culture to a pot of the soil mix described under corn cultivation above. Alfalfa soil cultures were maintained for at least 3 months prior to use in inoculation experiments. They were irrigated with water only. An analysis of the soil (Ontario Ministry of Agriculture and Food) gave the following: - - pH 7.4; nitrate N 28 ppm; P l p p m ; K14ppm; Ca105ppm; Mgl7ppm; Zn0.11 ppm. The following legumes were maintained in the greenhouse in the same conditions: - - R e d clover (Trifolium pratense), white sweet clover (Melilotus alba), vetch (Vicia sativa) and soybean (Glycine max).

Other soils The following field soils were tested as inocula to assess their effect on maize growth:--l)Elora alfalfa and maize soils. In this case, alfalfa or maize had been the only crop for 7 years (silt loam of the Conestoga series from Guelph University experiment ER); 2) Aylmer soybean soil. Soybeans were grown for two years after maize (sandy loam of the Miami series from the farm of R E Humphrey, Aylmer Ontario); 3) Troy alfalfa soil. Alfalfa was

grown for three years (from the farm of S Hunt, Troy, Ontario).

Soil treatments A bactericide (streptomycin sulphate at 500 mg L-l) and four fungicides (benomyl [Benlate] at 36mgL l, etridiazol [Truban] at 0.18 ml L- ~, metalaxyl [Ridomil] at 0.18 mg L- l, polychloronitrobenzene [PCNB-Quintozene] at 1.5 g L -l ) were tested for their effect on the growth response of maize to alfalfa soil inoculation. The formulations were thoroughly mixed with the soil prior to planting of maize. Rates were estimated as comparable to those used commercially in the field. When required the alfalfa soil was sterilized by autoclaving (1 h at 121 °C) or gamma irradiation (3.6 Mrad). A lower dosage of gamma irradiation (100krad) was also used as a selective killer of microorganisms. Gamma irradiation was carried out using a cobalt-60 source at the McMaster University Nuclear Reactor.

Estimation of vesicular arbuscular mycorrhizal (VAM) infection At harvest, the root systems were washed under the tap after shaking to remove loose soil. Roots infected with vesicular arbuscular mycorrhizal (VAM) fungi are a bright yellow colour in our system and a visual estimate of percentage yellow roots was used as a measure of the percentage of infected roots. Percentage VAM infection was also measured by clearing in 1 M KC1 at room temperature overnight and staining by the method of Brundrett et al. (1984), followed by microscope observation of the presence or absence of infection at 300--500 intersection points between roots and grid lines in a Petri dish.

Determination of shoot nitrate, phosphate and K concentrations Nitrate concentration in shoot tissues was determined colorimetrically by the method of Cataldo et al. (1975). Phosphate concentration was deter-

Growth promotion of maize by legume soils mined colorimetrically by a modified Fiske-Subarrow method (Clark and Switzer, 1977). K concentration was determined by flame photometry (Analytical Methods Manual, 1981. Environment Canada Inland Waters Directorate).

261

growth response to alfalfa soil inoculation (Fig. 2). Gamma irradiation (3.6Mrad) of the alfalfa soil prior to use as an inoculum eliminated the growth response. The growth response was also much reduced by autoclaving. This indicates that we are dealing with a microbial rather than a nutrient effect.

Results

Inoeulum size

Effect of soil nutrient status

The effect of greenhouse alfalfa soil inoculum on the growth of maize is shown in Figure 1. A significant (P < 0.05) growth response was found with 100 mL inoculum per pot (0.83 % by volume) but not with 20mL per pot (0.14 % by volume). 500 mL per pot (4.1% by volume) was chosen for most experiments as this gave a large growth response by 5 to 8 weeks from sowing. Similar results were obtained when non-pasteurized sandy loam or field soils from the Elora plots were used (data not shown). Other legume soils (red clover, white sweet clover, vetch, soybean) maintained in the greenhouse also gave a significant growth response in maize when inoculated at 500mL/pot (data not shown).

Early experiments (data not shown) established that a good growth response of maize to alfalfa soil inoculation was produced with addition of either 1 mM or 10mM KNO3 to the 1/10 strength Hoagland's solution. 10mM KNO 3 was chosen for subsequent work because at this concentration nitrogen was not limiting growth either in the presence or absence of the alfalfa soil. Experiments were carried out to assess the effect of increasing nutrient supply (1/2 strength Hoagland solution with 10 mM KNO3 instead of usual 1/10 Hoagland with 10 mM KNO3) on the response of maize to alfalfa soil inoculation. The results of two such experiments are shown in Figure 3. In both cases (and other experiments), feeding with the higher nutrient regime (1/2 Hoagland solution) resulted in greater shoot dry weight at time of harvest. In neither experiment (Fig. 3a, f) was there a significant (P ~< 0.05) growth response to greenhouse alfalfa soil inoculation when the higher nutrient regime was used. The nitrate, phosphate and K concentrations of shoot tissues from the two experiments were deter-

Effect of autoclaving or gamma irradiation of &oculum A growth response to 500mL of greenhouse alfalfa soil is usually first observed at the five leaf stage (about three weeks from sowing under our conditions). In the example shown the plants were harvested at 6 weeks. They showed a 5-fold shoot 5

12 =

10

0

8

~ 6

41

3' X

in0

c

0

20 100 500 1000

INOCULUM ADDED (ml)

Fig. 1. Influence of greenhouse alfalfa soil inoculum volume on maize shoot growth. Mean + S.E. for 5 plants.

tn O C

1

2

3

Fig. 2. Effect of gamma irradiation and autoclaving of greenhouse alfalfa soil inoculum on its effect on maize shoot growth. C, No inoculum added; 1,500 mL untreated greenhouse alfalfa soil added; 2,500mL gamma irradiated (3.6 Mrad) greenhouse alfalfa soil added; 3,500 mL autoclaved greenhouse alfalfa soil added. Mean + S.E. for 5 plants.

262

Fyson and Oaks

mined (Fig. 3). In one experiment, nitrate per g fresh weight was the same for all treatments (Fig. 3d) whereas in the other plants inoculated with greenhouse alfalfa soil with 1/10 Hoaglands and both treatments with 1/2 Hoaglands had higher shoot tissue nitrate concentrations than the 1/10 Hoaglands control (Fig. 3i). Inoculation increased tissue phosphate in both experiments at both concentrations of Hoagland solution. In none of the treatments was there a clear effect on shoot K concentration. Levels of VAM infection were measured in both experiments. No infected roots were found in control plants. With alfalfa soil inoculation, the concentration of Hoagland solution applied had no apparent effect on the percentage of roots infected by VAM fungi although the root systems were larger with 1/2 Hoagland solution. VAM infection

--

5

;:

4

O

levels were much lower in experiment 1 (Fig 3b) than in experiment 2 (Fig. 3g).

Effect of various soil inocula Figure 4 shows the results of six experiments testing the effects of various soil inocula on maize growth in our system. Figure 4a and 4b show a similar growth response with the Troy alfalfa soil as with the greenhouse alfalfa soil. The experiment shown in Figure 4c and 4d showed that a soybean soil also gave a substantial growth response. Elora maize soil (Fig. 4c, e) and Elora alfalfa soil failed to give a significant growth response in our system. Two rapeseed soils and a pasture soil gave a significant (P ~< 0.05) growth response but much less than our regular alfalfa soil (Fig. 4t"). 5' f 3,

4

cI

cn

I

0

0

b

30

ilO t

g

2O

v"

o

I-I

I-I

O

0~ ~"" 6 0 "

60

d

tnnn

~

i

40-

~ 20

20

o

O

'7

•~O.OJ °"°t HMH C A C

,

.

,

1/10 H

i

A

.

1/2 H

f

C AJ ,C 1/10 H

A

1/2 H

Fig. 3. Influence oi' Hoagland concentration on response of maize to greenhouse alfalfa soil inoculation. Fig. 3a--e Experiment 1. a) Shoot growth with 1/10 or 1/2 Hoagland solution (10 mM KNO3) with (A) or without (C) greenhouse alfalfa soil. b) Percentage roots infected by VAM fungi, c) Phosphate concentration in shoots, d) Nitrate concentration in shoots, e) K concentration in shoots. Mean _.+ S.E. for 5 plants. Fig. 3f--j Experiment 2. f) Shoot growth with 1/10 or 1/2 Hoagland solution (10 mM KNO3) with (A) or without (C) greenhouse alfalfa soil. g) Percentage roots infected by VAM fungi, h) P concentration in shoots, i) Nitrate concentration in shoots, j) K concentration in shoots. Mean + S.E. for 5 plants.

Growth promotion of maize by legume soils

2121 ,12S HH ,

a 12 10

uJ

0H C

C

2

Q

1

ilc n o.n n

2

C

4

5

lb

IIIIII0 000

11

10

8

8

4 2 0

C

1

5

6

263

0 C

SOIL

4

7

C

la

lc

8

9

10

INOCULUM ADDED

Fig. 4. Influence of various soil inocula on maize shoot growth. Results of 6 experiments (Fig. 4a,t) are shown. Soil inocula were as follows: C, no inoculum added; 1, greenhouse alfalfa soil; 2, Troy alfalfa soil; 3, 12 week dry stored greenhouse alfalfa soil; 4, Elora maize soil; 5, Aylmer soybean soil; 6, gamma irradiated (100 Krad) greenhouse alfalfa soil; 7, Elora alfalfa soil; 8, winter rape soil (Elora); 9, spring rape soil; 10, pasture soil; la, 14 month old greenhouse alfalfa soil; lb, 4 month old greenhouse alfalfa soil; lc, 3 month old greenhouse alfalfa soil. Mean __+ S.E. for 5 plants.

The experiment shown in Figure 4b tested the growth response to the regular inoculum (freshly shaken from alfalfa roots) and to greenhouse alfalfa soil air dried and stored at room temperature for 12 weeks. In each case the inocula elicited a significant growth response (P < 0.05). Inoculum in which alfalfa had been grown for 4 or 14 months gave a significant (P ~< 0.05) growth response (Fig. 4, soils la and lb). There was no obvious growth response when alfalfa plants had been grown for 3 months (Fig. 4, lc) or less. Four months was chosen as the minimum age for routine experiments.

eliminated the growth response to alfalfa soil inoculation. Etridiazol and metalaxyl had no significant effect on the growth response at the concentrations used. Low level gamma irradiation (100 krad) eliminated the growth response. In these experiments (benomyl experiments excepted), the percentage of roots infected b y VAM was estimated by visual assessment of yellow root pigmentation (Fig. 5). Treatments which reduced (metalaxyl) or destroyed the growth response (PCNB, gamma irradiation) substantially reduced or eliminated VAM infection. Etridiazol and streptomycin treatment had no clear effect on VAM infection.

Effect of a bactericide, fungicides and low level gamma irradiation of inoculum

Discussion

The effect of streptomycin, four fungicides and low level gamma irradiation of the alfalfa soil inoculum on the growth response of maize to alfalfa soil inoculation are shown in Figure 5. Streptomycin at 500mgL-' killed most bacteria (Plant counts of < 10Zml soil -I when plated on nutrient agar) but had no effect on the growth response of maize. Higher concentrations of streptomycin were deleterious to plant growth in the presence or absence of alfalfa soil inoculum. Two non selective fungicides, benomyl and PCNB

In our conditions, addition of alfalfa soil consistently gave a substantial growth response in maize shoots after 5 to 8 weeks. Elimination or reduction of this effect by gamma irradiation or autoclaving the inoculum and the low nutrient status of the alfalfa soil (see materials and methods) suggests that microbial factors in the alfalfa soil are responsible for this effect. The influence of inoculum size on the growth response suggests that the number of propagules of the active microorganism(s) may be a limiting factor. The growth response with legume

264

Fyson and Oaks a)streptomycin

.t b) benornyl (36mg.I "1 ) c) PCNS | 1.59.1 "1 ) 10 s u l p h a t e ( ~ O m g . I "l) 5

"~10 I"~ 8

;e

~

~4 o o T

32

2

I

O

0

6' 4'

NH n C

3°t 12o

CP

A

AP

" 1n 2

onn

>10

~ o C CP A AP d)et rldlazol (0.18 ml.I "1 )

C CP A AP ,e ) met alaxyl (0.18mg.I " 1 ) f ) Y i r radlation(lOOkrad)

25

15 o 10

~

0

h nnll

4

01n

00 0O

.1o, C

CP

A

AP

C

CP

A

AP

n

1o C

A

IA

SOIL T R E A T M E N T

Fig. 5. Effect of a bactericide, fungicides and low level gamma irradiation on the response of maize to greenhouse alfalfa soil inoculation. ' Shoot dry weight and % roots infected by VAM fungi: C, control; A, alfalfa soil added (500 mL/pot); CP, control + pesticide; AP, + alfalfa soil (500mL/pot), + pesticide; IA, gamma irradiated (100krad) alfalfa soil added (500mL/pot). Mean + S.E. for 5 plants.

soils from the field and the smaller yet still significant response with rapeseed and pasture soils suggests that this is a general phenomenon. Absence of a response with the Elora soils indicates that the microbial factors are absent or much reduced in some field soils. The increase in size of the growth response with time of cultivation of alfalfa in the inoculum soil suggests that populations of the active microorganism(s) increase with time in our greenhouse pot cultures. The failure of streptomycin to eliminate or reduce the growth response to alfalfa soil inoculation and the elimination of the growth response by the non-selective fungicides benomyl and PCNB implicates fungi rather than bacteria as the growth promoting factor(s). These two fungicides also eliminated VAM infection as has been reported in other studies (summarised in Menge, 1982; Trappe et al., 1984). Benomyl also eliminated a growth response to inoculation with the VAM fungus Glomus deserticola in our system (data not shown). Metalaxyl and etridiazol which are used to control

phycomycetous plant pathogens (including Phytophthora and Pythium) had no effect on the growth response to alfalfa soil inoculation at the concentrations used. These two fungicides also had no clear effect on levels of VAM infection. Experiments using Glomus versiforme in our greenhouse maize growth assay established that metalaxyl (0.18 mg L- l ) had little effect on VAM infection or the growth response. Groth and Martinson (1983) found that metalaxyl increased VAM infection in maize and soybeans but had no effect on plant growth. In contrast, Jabaji-Hare and Kendrick (1987) found a reduction in VAM infection and growth in leek (Allium porrum) with the same concentration of the fungicide as was used in our experiments. Our results with metalaxyl and benomyl are compatible with an involvement of VAM fungi in the alfalfa soil growth response but do not rule out a role for other beneficial fungal species. Low level gamma irradiation (100krad) of the alfalfa soil inoculum knocked out the growth response in three of four experiments (e.g. Fig. 5t). In

Growth promotion of maize by legume soils the other (Fig. 2d), a small but significant (P ~< 0.05) growth response was observed after inoculation. In all four cases, no V A M infection was f o u n d in roots o f plants inoculated with the treated soil. Jacobsen (1985) reported that 100 krad o f g a m m a irradiation eliminated mycorrhizal infection by one o f two fungal species examined. O u r results are compatible with an i m p o r t a n t role for V A M in the g r o w t h response o f maize but do not rule out the possibility that other fungal species m a y be involved. A l t h o u g h our greenhouse soil mix is not p h o s p h o r u s deficient in terms o f reco m m e n d e d levels, maize plants not inoculated with alfalfa soil often show signs o f P deficiency when the 1/10 strength H o a g l a n d solution was used. It is well established that V A M increases the ability o f plants to extract P f r o m soil (Harley and Smith, 1983) and it is possible that in our experiments, the improved P nutrition and growth o f inoculated plants is due to V A M . However, other soil microorganisms have been shown to be important in P mobilization. F o r example, Penicillium bilaji in the wheat rhizosphere increases P mobilization and increases crop yields (Kucey, 1987; 1988). Increasing the nutrient supply (including P) in our experiments eliminated the growth response but percentage o f roots infected by V A M fungi was little changed. Inoculation increased shoot phosphate concentration with both levels o f nutrient supply. It is possible that in these conditions, P supply is no longer limiting growth. A l t h o u g h V A M infection is associated with the g r o w t h response to alfalfa soil inoculation, we c a n n o t say unequivocally that V A M fungi are responsible for the growth response. Experiments currently being carried out are designed to identify the microorganisms involved in the growth response in our system.

Acknowledgements We wish to thank Dr. R L Peterson, D e p a r t m e n t o f Botany, University o f Guelph for a pot culture o f Glomus versiforme; Drs. M H Miller, G Fairchild and T McGonigle o f the D e p a r t m e n t o f L a n d Resources, University o f Guelph for helpful discus sions and Helena Taivainen and Valerie Goodfellow o f the D e p a r t m e n t o f Biology, M c M a s t e r University for help in analysing and weighing the plant materials. Financial support for this w o r k was pro-

265

vided by the N a t u r a l Science and Engineering Research Council o f C a n a d a (Strategic G r a n t p r o g r a m StrN 040).

References Battison L A, Miller M H and Shelton I J 1987 Soil erosion and corn yield in Ontario. I. Field evaluation. Can. J. Soil Sci. 67, 731-745. Bolton E F, Dirks V A and Aylesworth J W 1976 Some effects of alfalfa, fertilizer and lime on corn yields on clay soil during a range of seasonal soil moisture conditions. Can. J. Soil Sci. 56, 21-25. Brundrett M C, Piche Y and Peterson R L 1984 A new method for observing the morphology of vesicular-arbuscular mycorrhizae. Can. J. Bot. 62, 2128-2134. Cataldo D A, Haroon M, Schrader L E and Youngs V L 1975 Rapid colormetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun. Soil Sci. Plant Anal. 6, 71-80. Clark J M and Switzer R L 1977 Experimental Biochemistry, 2nd Edition. W H Freeman and Co., San Francisco, 335 p. Dumanski J, Bootsma A and Kirkwood V 1986 A geographic analysis of grain corn yield trends in Ontario using a computerized land information base. Can J. Soil Sci. 66, 481--497. Fyson A and Oaks A 1987 Physical factors involved in the formation of soil sheaths on corn seedling roots. Can. J. Soil Sci. 67, 591-600. Groth D E and Martinson C S 1983 Increased mycorrhizal infection of maize and soybean after soil treatment with metalaxyl. Plant Dis. 657, 1377-1378. Harley J L and Smith S E 1983 Mycorrhizal Symbiosis. Academic Press, London, 483 p. Hesterman O B, Sheaffer E C, Burns D K, Leuschen W E and Ford J H 1986 Alfalfa dry matter and nitrogen production and fertilizer nitrogen response in legume-corn rotations. Agron. J. 78, 19-23. Jabaji-Hare S H and Kendrick W B 1987 Response of an endomycorrhizal fungus in Alliumporrum L. to different concentrations of the systemic fungicides, Metalaxyl (Ridomil) and Fosetyl-al (Aliette). Soil Biol. Biochem. 19, 95-99. Jacobsen I 1984 Mycorrhizal infectivity of soils eliminated by low doses of ionizing radiation. Soil Biol. Biochem. 16, 281282. Ketcheson J W 1980 Long-range effects of intensive cultivation and monoculture on the quality of southern Ontario soils. Can. J. Soil Sci. 60, 403-410. Kucey R M N 1987 Increased phosphorus uptake by wheat and field beans inoculated with a phosphorns-solubilising Penicillium bilaji strain and with vesicular-arbuscular mycorrhizal fungi. Environ. Microbiol. 53, 2699-2703. Kucey R M N 1988 Effect of Penicilliumbilajion the solubility and uptake of P and micro nutrients from soil by wheat. Can. J. Soil Sci. 68, 261-270. Menge J A 1982 Effects of soil fumigants and fungicides on vesicular-arbuscular fungi. Phytopathology 72, 1125-1132. Reid J B and Goss M J 1981 Effect of living roots of different

266

Growthpromotion of maize by legume soils

plant species on the aggregate stability of two arable soils. J. Soil Sci. 32, 521-541. Trappe J M, Molina R and Castellano M 1984 Reactions of

mycorrhizal fungi and mycorrhizal formation to pesticides. Annu. Rev. Phytopathol. 22, 331-359.