arbuscular mycorrhizal formation in undisturbed soil counteracts compacted soil stress for pigeon...
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Arbuscular mycorrhizal formation in undisturbed soil
counteracts compacted soil stress for pigeon pea
K. Yano*, A. Yamauchi, M. Iijima, Y. Kono
School of Agricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
Accepted 7 January 1998
Abstract
Plant growth is sometimes restricted with soil compaction under no-tillage conditions, although undisturbed soils are favorable
to arbuscular mycorrhizal (AM) fungi of a symbiont. We examined growth responses of the pigeon pea (Cajanus cajan (L.)
Millsp.) to soil disturbance and inoculation with an AM fungus (Gigaspora margarita Becker & Hall) in a pot experiment. The
AM fungal inoculum was added to the soil and wheat was grown. After 6 months the shoot of wheat was removed and the soil
was either disturbed or remained undisturbed. Subsequently, pigeon pea was grown and harvested after 3 months. The
colonization and spore density of Gigaspora were signi®cantly greater in undisturbed soil than in disturbed soil. Undisturbed
soil showed higher penetrometer resistance and resulted in impaired shoot growth of the pigeon pea with lower shoot-to-root
(S/R) ratio than disturbed soil. However, inoculation with the AM fungus reduced the stress impact of undisturbed soil on the
pigeon pea without affecting the soil resistance and S/R ratio. A possible reason for reducing the stress impact was increase in
speci®c root length, rather than P in¯ow with the AM formation. It is a novel ®nding that AM formation in undisturbed soil
could promote root elongation despite the fact that soil was seriously compacted. # 1998 Elsevier Science B.V.
Keywords: Arbuscular mycorrhiza; Cajanus cajan (L.) Millsp.; Gigaspora margarita Becker & Hall; Soil disturbance; Soil compaction; Tillage
1. Introduction
Reduced tillage systems are important not only for
economic but also for environmental reasons, namely
the saving of energy input and soil erosion. However,
it is pointed out that reduced tillage may cause
increased soil compaction which is detrimental to
crop yields (e.g., Vyn and Raimbault, 1993). In gen-
eral, development of plant root systems is restricted in
compacted growth media (Iijima and Kono, 1991;
Iijima et al., 1991). Consequently, it is desirable to
restore and to enhance the functions of root systems
such as water and nutrient absorption, when the soil is
compacted under reduced tillage.
Arbuscular mycorrhizal (AM) fungi have been
found to form functional mycorrhizas enhancing some
nutrient uptake, especially phosphorus (P), in many
plant species (Harley, 1991), while reduced tillage is
likely to be favorable to AM fungi. Mulligan et al.
(1985), for example, found that excessive tillage with
accompanying soil disturbance reduced the coloniza-
tion of roots by AM fungi. It is generally recognized
that soil disturbance can reduce AM colonization, at
least in young plants (Evans and Miller, 1988; Fair-
child and Miller, 1988; Jasper et al., 1989a, b). A
Applied Soil Ecology 10 (1998) 95±102
*Corresponding author. Tel.: +81-52-789-4024; fax: +81-52-
789-4012; e-mail: [email protected]
0929-1393/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved.
P I I S 0 9 2 9 - 1 3 9 3 ( 9 8 ) 0 0 0 3 4 - 1
plausible mechanism for increased AM formation
without soil disturbance is that the hyphal network
structure is well-preserved in undisturbed soils, which,
therefore, can serve as an inoculum of higher infec-
tivity (e.g., Evans and Miller, 1990; Jasper et al.,
1989a, b).
If AM formation becomes intensive in undisturbed
soil conditions it might compensate for the restricted
functions of root systems, particularly nutrient absorp-
tion. However, little is known about whether such
intensive AM formation can reduce the stress impact
of undisturbed soil on host plants, because stress
factors of undisturbed soils were almost negligible
in those studies that investigated the relationship
between soil disturbance and AM colonization.
Therefore, we conducted a pot experiment to exam-
ine whether inoculation with an AM fungus (Giga-
spora margarita Becker & Hall) to a preceding crop
(winter wheat) can reduce the adverse effects of
undisturbed soil on the growth of subsequently planted
pigeon pea (Cajanus cajan (L.) Millsp.), which is
known to be susceptible to physical properties of
the soil (Okada et al., 1991). Furthermore, we assumed
that if AM formation can reduce the stress impact of
non-disturbed soil, improvement in plant growth
would take place in two ways: by increasing P absorp-
tion per unit root length, as indicated by Nadian et al.
(1996); and increasing the root length, as indicated by
Yano et al. (1996). The former case allows root
development to remain restricted, while the latter case
provides greater penetration capability to overcome
high mechanical resistance.
2. Materials and methods
2.1. Plant growth
A commercial nutrient-poor substrate (Akatamat-
suchiTM; subsoil of an Andosol aggregated arti®cially
into 5 mm aggregations) was used so that excess soil
nutrient could not inhibit colonization of inoculated
AM fungus. This soil contained only a trace of Truog-
P and no spores of Gigaspora. Plastic pots (16 cm
diameter, 30 cm height) were ®lled with the substrate.
Fifteen gram of AM fungus inoculum (Cerakin-
kongTM, Central Glass, Japan) per pot, containing
approximately 1500 spores of Gigaspora margarita
Becker & Hall, were applied as the inoculation treat-
ment, while the same amount of inoculum sterilized
with an autoclave were applied as non-inoculation
treatment. Each inoculum was added into the soil
layer 10 cm below the surface.
Surface-sterilized seeds of wheat (Triticum aesti-
vum L., cv. Norin 61) were sown in each pot on 18
December 1993, and thinned to one plant per pot three
weeks later. Approximately 250 ml of Hoagland solu-
tion, in which the P concentration was adjusted to one-
third strength, was applied weekly to each pot. The
wheat plants were harvested on 22 June 1994. The
shoots were removed but the roots remained in the
pots. There were no apparent differences in shoot and
root growth of wheat between inoculated and non-
inoculated treatments. Then, to disturb the soil, it was
taken out and shuf¯ed thoroughly using a shovel in a
container to cut it into 5 mm particle size, while in
another pot the soil was not disturbed. Consequently,
four soil treatments were prepared: non-inoculated
with the AM fungus and undisturbed soil (NI-UD);
inoculated and undisturbed soil (I-UD); non-inocu-
lated and disturbed soil (NI-D); and inoculated and
disturbed soil (I-D). Four replicate pots were prepared
for each treatment.
On 4 July 1994, surface-sterilized seeds of pigeon
pea (Snow Brand, Japan) were sown in each pot and
thinned to one plant per pot 2 weeks later. Fertilizer
was not applied to the pigeon pea. Water was supplied
into each pot every three days until excess water
drained from the pot.
2.2. Determination of the shoot dry matter and P
content
At 90 days after planting, shoots were harvested and
oven dried at 708C for 48 h. The dried samples were
weighed and ground. The P concentration in the shoots
was determined colorimetrically by following the
phosphovanado±molybdate method after the plant
materials had been digested with nitric acid and per-
chloric acid (Hanson, 1950).
2.3. Measurement of soil penetration resistance and
spore density
After harvesting the shoots, soil penetration resis-
tance in each pot was measured using a penetrometer
96 K. Yano et al. / Applied Soil Ecology 10 (1998) 95±102
(DIK-5520, Daiki rika kogyo, Japan). To ensure the
soil was at its maximum water holding capacity, water
was supplied into each pot until the excess water
drained from the pot 1 day before the measurement.
The root system was then carefully removed from the
pot soil, washed with tap water to completely remove
any adhered soil. Washed root system was weighed
freshly after removing excess water with a paper
towel, and then preserved in FAA (formalin 1: acetic
acid 1: 70% ethyl alcohol 18 by volume). The remain-
ing soil was thoroughly mixed, and then the fungus
spore density was determined. The fungus spores were
collected from 100 ml volume of pot soil using the
method of sucrose density gradient centrifugation
after wet sieving (Daniels and Skipper, 1982), and
were counted under a stereoscope.
2.4. Measurement of root length and AM
colonization
The total root length was measured with a root
length scanner (Commonwealth Aircraft, Australia).
After measurement of the length, roots were further
cut into about 1 cm segments. The root segments were
randomly collected and cleared in 10% KOH (Phillips
and Hayman, 1970). These segments were then
bleached by 1/10 diluted H2O2 to ensure clearing
and then stained with trypan blue in lactoglycerol.
Percentages of the total root length colonized were
estimated using the grid intersect method (Giovanneti
and Mosse, 1980).
2.5. Statistical analysis
The data were analyzed by two-way analysis of
variance (ANOVA), in which the variation sources
consisted of the inoculation (I or NI) and soil dis-
turbance (D or UD) treatments. To improve normality,
log and arcsine square root transformations were
performed for data on the percentage colonized root
length and the spore density respectively. Duncan's
multiple range tests were performed to evaluate the
signi®cance of differences among the four treatment
combinations.
3. Results
3.1. Shoot growth of pigeon pea
Table 1 shows the dry weight, phosphorus (P)
concentration and P content of the harvested shoots
at 90 days after planting (DAP). All plants in the four
treatments bloomed at this stage. The shoot dry matter
decreased in the order I-D>NI-D>I-UD>NI-UD.
Although the P concentration was not signi®cantly
different among treatments in any shoot parts, it
tended to be higher in non-disturbed treatments
Table 1
Effects of inoculation with the arbuscular mycorrhizal fungus Gigaspora margarita and soil disturbance on the dry weight (DW) and
phosphorus (P) content in the shoot of pigeon pea at 90 days after planting (DAP)
Treatments DW (g plantÿ1) P conc. (mg gÿ1 DW) P content (mg plantÿ1)
Leaves Stems Total Leaves Stems Total Leaves Stems Total
NI-UD 0.69c 1.89d 2.58d 1.85a 1.16a 1.35a 1.34c 2.14c 3.49c
I-UD 1.09c 3.98c 5.07c 1.95a 1.21a 1.38a 2.12bc 4.84b 6.96b
NI-D 2.15b 5.82b 7.97b 1.63a 1.07a 1.22a 3.36ab 6.20ab 9.56ab
I-D 2.70a 7.61a 10.31a 1.75a 1.00a 1.19a 4.51a 7.42a 11.93a
ANOVA
Source Probability
Inoculation (I) 0.2066 0.0196 0.0411 0.3428 0.9135 0.9705 0.0522 0.0080 0.0108
Disturbance (D) 0.0011 <0.001 <0.001 0.0786 0.0616 0.0761 <0.001 <0.001 <0.001
I�D 0.8290 0.8396 0.9482 0.9419 0.3947 0.7631 0.6867 0.2538 0.5788
NI-UD, non-inoculated and undisturbed soil; I-UD, inoculated and undisturbed soil; NI-D, non-inoculated and disturbed soil; I-D, inoculated
and disturbed soil.
Means followed by the same letters within a column are not significantly different (p<0.05) from each other by Duncan's multiple range test.
K. Yano et al. / Applied Soil Ecology 10 (1998) 95±102 97
(NI-UD and I-UD) compared with disturbed ones (NI-
D and I-D). The P content differed between the
treatments in the same order as the shoot dry weight,
but was not signi®cantly different between I-UD and
NI-D, and between NI-D and I-D, while the P content
of NI-UD was signi®cantly lower than in the other
treatments. The shoot dry weight and the P content in
the I-ND treatment were approximately twice those in
the NI-UD treatment. ANOVA detected no signi®cant
interaction effects between inoculation and soil dis-
turbance for the dry weight and the P content.
3.2. Soil hardness, spore density and colonization by
AM fungus
Fig. 1 shows the soil penetration resistance as an
index of the soil hardness in each treatment just after
the harvest. Resistance was clearly higher in undis-
turbed soil than in disturbed soil. However, there was
no signi®cant difference between non-inoculated and
inoculated soils within the same soil disturbance
treatment.
Table 2 shows the differences among treatments in
the number of mycorrhizal spores and the percentage of root length colonized at 90 DAP. The number of
Gigaspora spores in I-UD was more than twice that in
I-D, while no spores were found in NI-UD or NI-D.
Looking at the percentage of the root length colonized,
the I-UD treatment was highest and the I-D treatment
next. Some colonization was observed in the NI-D and
NI-UD treatments but with much lower levels, due to
indigenous fungi. ANOVA results for the spore density
indicated that the interaction effect and both main
effects were signi®cant. For the percentage of root
length colonized, interaction and inoculation effects
were signi®cant but no signi®cant effect was detected
due to soil disturbance.
3.3. Development and function of root system
Fig. 2 shows the appearance of the harvested root
system in each treatment at 90 DAP. Root system
development in the NI-UD treatment was clearly
poorer compared to the other three treatments. Com-
paring the root system of NI-UD and I-UD, it was
clear that the root development in undisturbed soil was
distinctly improved by inoculation.
Table 3 shows the root fresh weight, root length,
speci®c root length, shoot-to-root (S/R) ratio and
Fig. 1. Changes in penetration resistance with soil depth in the NI-
UD (&), I-UD (&), NI-D (*) and I-D (*) treatments. Values are
shown in means�S.E. of four replicates. NI-UD, non-inoculated
and undisturbed soil; I-UD, inoculated and undisturbed soil; NI-D,
non-inoculated and disturbed soil; I-D, inoculated and disturbed
soil.
Table 2
Effects of inoculatation with the arbuscular mycorrhizal fungus
Gigaspora margarita and soil disturbance on the percentage of
colonized root length to the total root length of pigeon pea and the
spore density in the soil at 90 days after planting (DAP)
Treatments Colonized
root length (%)
Spore density
(no. per 100 ml soil)
NI-UD 15.5 c 0.0 c
I-UD 56.3 a 23.0 a
NI-D 17.3 c 0.0 c
I-D 41.5 b 9.6 b
ANOVA
Source Probability
Inoculation (I) <0.001 <0.001
Disturbance (D) 0.1066 0.0149
I�D 0.0495 0.0149
NI-UD, non-inoculated and undisturbed soil; I-UD, inoculated and
undisturbed soil; NI-D, non-inoculated and disturbed soil; I-D,
inoculated and disturbed soil.
Means followed by the same letters within a column are not
significantly different (p<0.05) from each other by Duncan's
multiple range test.
98 K. Yano et al. / Applied Soil Ecology 10 (1998) 95±102
the shoot P absorption per unit root length in each
treatment. The fresh weight decreased in the order
I-D�NI-D�I-UD>NI-UD, as with the shoot growth.
Although the tap root length did not differ among
treatments, signi®cant differences were found in the
total length of all lateral roots. ANOVA results
indicated that, for the root fresh weight, the
disturbance effect was signi®cant. For the total
Fig. 2. Root system appearence of pigeon pea in the pot of NI-UD (a), I-UD (b), NI-D (c) and I-D (d) treatments at 90 DAP. Bar indicates
10 cm length. NI-UD, non-inoculated and undisturbed soil; I-UD, inoculated and undisturbed soil; NI-D, non-inoculated and disturbed soil;
I-D, inoculated and disturbed soil.
Table 3
Effects of inoculation with the arbuscular mycorrhizal fungus Gigaspora margarita and soil disturbance on the root system development of
pigeon pea at 90 DAP
Treatments Fresh weight
(g plantÿ1)
Root length (m plantÿ1) Specific root length
(m gÿ1 FW)
S/R ratio
(g DW gÿ1FW)
P absorption
(mg P mÿ1 root length)
Tap root Lateral root
NI-UD 20.0 c 0.569 a 73.85 c 3.92 c 0.134 c 0.048 a
I-UD 34.0 b 0.598 a 192.00 b 5.69 b 0.149 c 0.036 c
NI-D 36.2 ab 0.526 a 206.03 b 5.78 b 0.221 b 0.047 ab
I-D 40.9 a 0.479 a 291.68 a 7.39 a 0.253 a 0.040 bc
ANOVA
Source Probability
Inoculation (I) 0.0518 0.9168 <0.001 0.0037 0.2306 0.1120
Disturbance (D) 0.0199 0.3379 <0.001 0.0027 <0.001 0.8045
I�D 0.3028 0.6683 0.3414 0.8667 0.6557 0.5742
NI-UD, non-inoculated and undisturbed soil; I-UD, inoculated and undisturbed soil; NI-D, non-inoculated and disturbed soil; I-D, inoculated
and disturbed soil.
Means followed by the same letters within a colum are not significantly different (p<0.05) from each other by Duncan's multiple range test.
K. Yano et al. / Applied Soil Ecology 10 (1998) 95±102 99
root length of all lateral roots, both inoculation
and disturbance effects were signi®cant at high
probabilities (p<0.001).
The speci®c root length and S/R ratio were calcu-
lated using the root fresh weight. The speci®c root
length showed differences between treatments similar
to the differences observed in the lateral root length.
ANOVA results indicated that the interaction effect
was not signi®cant while the two main effects were
signi®cant (p<0.01). The S/R ratio showed signi®-
cantly lower values in undisturbed treatments than
in disturbed ones. Plants in the I-D treatment had a
signi®cantly higher S/R ratio than plants in the NI-D
treatment while no signi®cant difference was found
between plants in the I-UD and NI-UD treatments.
ANOVA results indicated that only the soil distur-
bance effect was highly signi®cant (p<0.001). The
speci®c P absorption was obtained by dividing the
shoot P content by the total root length in each plant.
The P absorption was found to be in the order NI-
UD�NI-D�I-D�I-UD, but ANOVA could not detect
any signi®cant effects.
4. Discussion
Our main objective was to examine whether inocu-
lation with the AM fungus Gigaspora margarita of a
preceding wheat can reduce the stress impact of non-
tilled (non-disturbed) soil conditions on the growth of
a succeeding pigeon pea. Undisturbed soil condition,
as measured by higher penetration resistance (Fig. 1),
clearly imposed the pigeon pea a stress due to soil
compaction (Table 1). Comparing I-UD with NI-UD,
however, the stress impact was reduced in inoculated
soil without changing the resistance.
A number of studies have shown that undisturbed
soil conditions causes intensive AM formation (Evans
and Miller, 1988, 1990; Fairchild and Miller, 1988,
1990; Jasper et al., 1989a, b, 1991; McGonigle et al.,
1990). Those studies examined young plants approxi-
mately up to 1-month old, while McGonigle and
Miller (1993) found that early intensive AM coloniza-
tion under no-tillage and ridge tillage conditions
compared to the tilled case almost disappeared from
50 DAP. Contrarily, intensive AM colonization in
undisturbed soil was still found at 90 DAP in the
present experiment (Table 2).
Despite intensive AM colonization, shoot growth at
harvest was primarily determined by soil disturbance
(i.e. disturbed>undisturbed) and secondarily deter-
mined by inoculation (i.e. inoculated>non-inoculated)
(Table 1). Adverse effects of undisturbed soils were
not emphasized in previous reports that investigated
the relationship between soil disturbance and AM
colonization. Probably because of the relatively short
experimental duration, the stress impact on the plants
would be negligible in those studies. Indeed, we could
not ®nd signi®cant differences in shoot growth
between NI-UD and NI-D at 30 DAP (data are not
shown).
The stress impact of the undisturbed soil was also
re¯ected in the S/R ratio. In general, plants in stressful
environments seem to put more of their resources into
root production (Fitter and Hay, 1981). Although the
S/R ratio was not signi®cantly affected by inoculation,
it was signi®cantly lower in both undisturbed soils,
compared to disturbed soils (Table 3). This implies
that both NI-UD and I-UD plants allocated more
amount of the photosynthate into the root system
relative to the shoot than NI-D and I-D plants, prob-
ably to adapt to the stressful environment.
It is known that roots in compacted media become
thicker (i.e., lower speci®c root length) compared to
roots in non-compacted media (Iijima et al., 1991;
Russell, 1977). While both NI-UD and I-UD plants
showed a similarly low S/R ratio, I-UD plants had a
remarkably greater speci®c root length than NI-UD
plants (Table 3). Thus, although the plants in undis-
turbed substrate gave priority to the root system as
regards the photosynthate allocation, the below-
ground use of the allocated photosynthate was quite
different. A greater speci®c root length was found not
only in I-UD but also in I-D plants (Table 3), so that
this morphological modi®cation of the root system can
be attributed to the AM fungus inoculated.
AM colonization of a root system often increases
nutrient absorption, particularly P (Harley, 1991). For
increasing P absorption by AM formation, two expla-
nations should be considered: higher absorption abil-
ity per unit root length; and extended root length. In
this study we have assumed that the former case would
allow the root system development to remain
restricted, while the latter would allow the root system
to develop with greater penetrative force to overcome
the higher mechanical resistance. Nadian et al. (1996)
100 K. Yano et al. / Applied Soil Ecology 10 (1998) 95±102
found an increase in P absorption per unit root length
in AM roots of 7-week old clover when low-P soil was
compacted. The present results, however, support the
latter case, since the improvement in root system
development in mycorrhizal plants was remarkable
(Fig. 2), particularly the lateral roots, while no
increase in P absorption per unit root length was found
(Table 3).
It is a novel ®nding that AM formation in undis-
turbed soil could promote root elongation despite the
fact that the soil was seriously compacted. According
to a sensitivity analysis using mathematical model-
ling, the rate of root elongation was more sensitive
than the root radius for P uptake (Barber, 1984).
However, most studies have assumed that the mycor-
rhizal bene®ts in nutrient uptake are due to solely the
increase in the soil volume that is exploited by the
external hyphae, extending the rhizosphere radially.
To the contrary, we pointed out elsewhere that the
bene®ts of AM formation should be taken to include
also the axially expanded rhizosphere (Yano et al.,
1996). The present results strongly imply the impor-
tance of root extension for AM bene®ts.
Acknowledgements
We are grateful S. Ban (Central Glass, Japan) for
providing AM fungus inoculum. This work was sup-
ported by a Grant-in-Aid for Scienti®c Research from
the Ministry of Education, Science and Culture, Japan
(No. 08406002).
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