Plant and Soil 140: 121-125, 1992.

© 1992 Kluwer Academic Publishers'. Printed in the Netherlands. PLSO 91/)3

Effect of the VAM fungus Glomus sp. on the growth and yield of
soybean inoculated with Bradyrhizobium japonicum

H. VEJSADOVA, D. SIBLIKOVA z, H. HRSELOVA and V. VANOURA

Institute of Microbiology, Czechoslovak Academy of Sciences, Videfiskgl 1083, CS-142 20 Prague,
Czechoslovakia and ~Research Institute of Crop Production, CS-160 O0 Prague, Czechoslovakia

Received 27 February 1991. Revised October 1991

Key words: Bradyrhizobium japonicum, dry matter yield, Glomus sp., Glycine max, nitrogen fixation,
nodule mass, root infection

Abstract

The effect of two Bradyrhizobium japonicum strains (D344 and Urbana), on the frequency and
intensity of infection by a VAM fungal Glomus sp. and the effect of VAM on biomass production by
nodulating plants were tested in soybean growing in a soil containing low levels of accessible P and N.
During the initial stage of vegetative growth, mycorrhiza frequency in roots inoculated with the two
rhizobial strains did not differ. However, during flowering it was 178% higher in roots with the strain
D344 than in the presence of the strain Ubrana. At final harvest (green pods) the VAM frequency did
not differ in the presence of either strain. VAM positively affected biomass production, foliar
concentrations of P, Zn and Cu, and number and dry matter yield of pods, but did not increase
concentrations of total N and K. In nonmycorrhizal plants total nitrogenase activity (not nodule mass)
and growth were higher with the rhizobial strain Urbana. The greatest nitrogenase activity, growth and
yield occurred in the presence of the VAM fungus, and did not differ for plants with different strains of
rhizobia.

Introduction biosis has been measured as an increased uptake

Positive effects of dual colonization of soybean
roots by vesicular-arbuscular mycorrhizal fungi
(VAM) and diazotrophic rhizobia have been in-
vestigated by a number of authors (Barea and
Azc6n-Aquilar, 1983; Gianinazzi-Pearson et al.,
1981; Pacovsky et al., 1986). The relationship
between the VAM fungus and rhizobia in terms
of effects on plant yield is influenced by differ-
ences among species, strains and cultivars of the
symbionts (Carling and Brown, 1980). Both
rhizobia and VAM fungi are active in root corti-
cal cells and, hence, it can be assumed that the
presence of one microsymbiont will affect the
activity of the other. Most studies have concen-
trated on indirect relationships between VAM
fungi and rhizobia, in which a successful sym-
of nutrient elements, P in particular, by the plant
(Ames and Bethlenfalvay, 1987) and an in-
creased N 2 fixation and nodule mass (Fredeen
and Terry, 1988), but more direct non-nutrition-
al effects of VAM fungus-rhizobia interactions
have also been reported (Bethlenfalvay et al.,
1985; Ross and Harper, 1970). However, studies
concerning the effect of rhizobial strains on the
development of a VAM fungus (Bayne and Beth-
lenfalvay, 1987; Kucey and Paul, 1982) and,
inversely of VAM fungal species on nodulation
(Bethlenfalvay et al., 1982) are relatively scarce.
In the present work, the effect of two different
strains of rhizobia on VAM frequency and inten-
sity, and the influence of a VAM fungus on dry
matter yield, number and yield of pods and foliar
concentrations of N, P, K, Zn and Cu of nodulat-
122 Vejsadov6 et al.
ing plants, has been investigated in soybean
growing in a soil containing low levels of access-
ible P and N. In addition, the activity of the
rhizobial strains was measured as total (TNA)
and specific (SNA) nitrogenase activities and
rhizobial infection was determined as nodule dry
mass.
Materials and methods
Experimental protocol
Soybean plants (Glycine max (L.) Merr., cv.
Sluna) were grown (1) inoculated only with
Bradyrhizobium japonicum strains D344 or Ur-
bana, and (2) inoculated with a Glomus sp.
isolate and with either strain D344 or Urbana.
The VAM fungal isolate is taxonomically related
to the species Glomus claroideum Schenck and
Smith and was isolated from an experimental
plot of the Research Institute of Crop Produc-
tion Prague, in Bfilice near Tfebofi. Soybean
seeds were surface sterilized for 15 min in 7%
calcium hypochlorite and left to germinate for
3-4 days at room temperature in perlite. 0.5 mL
of a suspension of rhizobia (109 cells per mL)
from the collection of the Research Institute of
Crop Production, Prague, was applied directly
onto the germinated seed at planting. At the
same time, VAM fungal inoculum in the form of
30 non-sterile spores of Glomus sp. was added
under each plant.
Growth conditions
Plants were cultivated in pots containing 750 mL
of a steamed soil-perlite mix (75 : 25%, one plant
per pot; 30 replicates per treatment). Soil
characteristics were: pH (H20) 6.0,
2 6 m g k g - I P (Olsen), 21mgkg -1 mineral N,
0.11% total N, 2 8 m g k g - ~ K , 3 6 m g k g - l M g ,
9 mg kg- J Cu, 35 mg kg-~ Zn. The substratum
was steamed for 6 hours at 75°C on three con-
secutive days. Soybean plants were grown in a
greenhouse under natural day length and light
intensity (spring-summer) and watered regularly
with distilled water. Ten plants per treatment
were harvested after 4 weeks (initial stage of
vegetative growth), 7 weeks (during flowering)
and 12 weeks (green pod formation).
Assays
Roots and shoots were dried at 105°C until con-
stant mass and weighed. N 2 fixation was mea-
sured on the basis of nitrogenase C2H 2 reducing
activity (Hardy et al., 1973). Roots were stained
with a trypan blue-lactophenol mixture (Phillips
and Hayman, 1970), and frequency of VAM
infection (percentage of infected roots) and
VAM intensity (percentage of colonized cortical
tissue) were evaluated microscopically according
to Trouvelot et al. (1986). Total nitrogen in the
leaves was determined by the automated Dumas
method. Phosphorus content in the soil and in
the plant was determined using the spec-
trophotometric method of Watanabe and Olsen
(1965). Samples were digested in a mixture of
concentrated sulfuric acid and 30% hydrogen
peroxide (1016 Digester, Tecator). Concentra-
tions of potassium, copper and zinc were de-
termined by means of atomic absorption spec-
trometry. Data were statistically analysed using
the one-way analysis of variance ANOVA on the
basis of which LSD values ( p ~<0.05 for N = 5)
for any two compared means were calculated.
Results and discussion
VAM formation, N z fixation and nodulation
The VAM Glomus sp. successfully colonized soy-
bean roots in the presence of either the rhizobial
strains D344 or Urbana (Table l). At the initial
stage of vegetative growth (after 4 weeks) mycor-
rhiza frequency in roots did not differ for the two
rhizobial strains. However, during flowering
(after 7 weeks) this was 178% higher in plants
with the strain D344 as compared to plants with
the strain Urbana (Table 1). At final harvest
after 12 weeks (green pods), the frequency of the
VAM infection did not differ in plants inoculated
with the different strains of rhizobia. The intensi-
ty of infection in roots inoculated with the strain
D344 was significantly higher (+33%) than in
the presence of the strain Urbana. No VAM
infection could be detected in roots inoculated
with the rhizobial strains alone.
The VAM fungus influenced nodule dry mass
and fixation activity of both rhizobial strains
 Effect of Glomus sp. on soybean 123

Fable I. VAM frequency and intensity in roots of soybean after 7 weeks' plant growth (Table 2). During
plants inoculated with Glomus sp. and different Bradyr- flowering, total nitrogenase activity (TNA) was
hizobium strains significantly higher in roots with the strain Ur-
Plant age Treatment Frequency Intensity bana than with the strain D344. At the same
(weeks) (%) (%) time, the presence of the VAM fungus stimulated
4 D" 0.00 0.00 TNA and nodule mass significantly in both rhizo-
D + VAM ~ 6.60 0.79 bial strains. On the other hand, values for
U~ 0.00 0.00
specific nitrogenase activity (SNA) were higher
U + V A M '~ 5.52 3.00
L S D (0.05) 4.81 3.01
at 7 weeks in the strain D344 than in the strain
Urbana. Mycorrhizal infection substantially in-
D 0.00 0.00 creased SNA at 4 weeks of vegetative growth
D + VAM 18.40 17.00 then reduced it during the flowering stage (7
U 0.00 0.00
weeks).
U + VAM 6.62 4.21)
L S D ({).05) 4.72 3.85
Dr)' mass production
12 D 0.00 0.00
D + VAM 61.20 38.60 The strain Urbana alone significantly improved
U 0.00 0.00
dry mass production of shoots during flowering
U + VAM 59.20 29.80
L S D (0.05) 2.89 6.00
as compared to strain D344 (Table 3). However,
the dry mass of shoots of VAM plants was only
" Roots inoculated with strain D344.
Roots inoculated with strain D344 and Glomus sp.
significantly higher than non-VAM plants inocu-
Roots inoculated with strain Urbana. lated with the strain D344 during this period.
" Roots inoculated with strain Urbana and Glomus sp, VAM infection in the presence of either rhizobial
strain positively influenced biomass production
during later phases of growth, giving dry mass
increases of shoots and roots at the final harvest.

Table 2. T o t a l ( T N A ) and specific (SNA) nitrogenase activi-

ty and rhizobial infection as nodule dry mass of soybean
plants inoculated with Glornus sp. and different Bradyr- Table 3. N u m b e r of pods and dry mass of roots, shoots, pods
hizobium strains of soybean plants inoculated with Glomus sp. and different
Bradyrhizobium strains
Plant age Treatment TNA ~ SNA ¢ Nodule
(weeks) dry mass (g) Plant age Treatment Pods D r y mass (g)
(weeks) (number
4 D" 1.41 43.21 (I.036
per plant) Roots Shoots Pods
D + VAM" 4.57 268.37 0.037
U' 2.61 51.06 0.044 1") a (I.27 tl.64 -
U + VAM a 4.81 247.25 0.030 I) + VAM ~' 0.25 0.66 -
L S D (0.05) 4.04 42.78 11.{/36 U" 0,27 {).73 -
U + V A M ~L - 0.25 0.60 -
D 1 1.79 203.99 (1.058 L S D (0.05) {), 11 {I.32 -
D + VAM 19.16 58.92 {).318
U 17.85 149.14 0.136 D - 0.32 1.71 -
U + VAM 22.75 107.27 0.191 D + VAM b - 0.63 3.37 -
L S D (0.05) 4.01 37.29 0.149 U - 0.53 289 -
U + VAM d - {I.65 3.47 -.
12 D 1.11 3.(13 0.363 I,SD (0.05) - 0.13 0,64 -
D + VAM 2,05 4.16 (t.494
U 1,77 4.53 {t.433 12 D 16 tl.79 9.77 5.22
U + VAM 2,71 4.53 {/.624 D + VAM 21) 1.11 12.36 6.31
L S D (0.05) 1.37 2.96 0.154 U 14 0.96 10.01 4.78
U + VAM d 19 1.25 12.46 5.411
""'"'" A s in Table 1.
L S D (0.05) 2 (I.29 2.06 0.26
" txmol C2H ~ h ~ plant ~.
' txmol C : H a h ' (g nodule dry mass) ' ;,,",~,d AS in T a b l e 1.
124 Vejsadov6 et al.

Number and mass of pods were significantly
higher in VAM plants than in non-VAM plants,
and pod production in the presence of the rhizo-
bial strain D344 was always significantly greater
than with the strain Urbana.
These results correspond well to the develop-
ment of VAM which was consistently higher at 7
and 12 weeks in roots inoculated with the strain
D344. Furthermore, mycorrhizal plants pro-
duced a 25% greater biomass as compared with
non-VAM plants, irrespective of the rhizobial
strain used.
N, P, K, Zn and Cu concentrations in leaves
Concentrations of N and K in VAM plants did
not significantly differ from that in non-VAM
plants at any growth stage (Table 4). After 4
weeks' plant growth, P concentrations in leaves
of mycorrhizal plants increased with respect to
those of non-mycorrhizal plants in the presence
of either strain of rhizobia. During flowering,
these differences were no longer significant and
at final harvest phosphorus concentrations were
only higher in leaves of plants inoculated with
the combination VAM + D344. VAM infection
stimulated Zn uptake by plants in the presence
of both rhizobial strains and Zn concentrations
in leaves of mycorrhizal plants was greater at all
harvests. At the final harvest (12 weeks) its
concentration in mycorrhizal plants was 113%
and 77% higher than in non-VAM plants, in the
presence of the strain D344 and Urbana respec-
tively. Concentrations of Cu were significantly
higher in VAM plants than in non-VAM plants at
the initial stage of growth but not at flowering
and increased later (at final harvest) in plants
inoculated with the strain D344 (Table 4).
In our experiments, the mutual relationship
between a VAM fungus, Bradyrhizobium and
soybean were strongly dependent on the growth
stage of the plants, as previously reported by
Bethlenfalvay et al. (1985). The development of
mycorrhizal infection during the whole growth
period was influenced by the rhizobial strain
used (Table 1). Plants, when inoculated with the
strain D344, showed a more rapid development
of mycorrhizal infection than those inoculated
with the strain Urbana. However, the final fre-
quency of VAM infection did not differ signifi-
cantly between roots inoculated with either rh-
izobial strains although infection intensity re-
mained higher with D344. Results concerning N
nutrition seem conflicting. Although nitrogenase
activities and nodulation tended to be greater in
nodulated VAM plants at 4 and 12 weeks, the

Table 4. Concentrations of phosphorus, nitrogen, potassium, zinc and copper in leaves of soybean plants inoculated with Glomus
sp. and different Bradyrhizobium strains
Plant Treatment P N K Zn Cu
age (mg g 1 DW) (tzgg 1 DW)
(weeks)
Da 3.65 49.80 35.86 54.28 11.06
D + VAM b 4.62 46.40 49.86 62.60 14.04
Uc 4.68 48.40 50.78 50.66 10.34
U + VAM a 5.71 46.40 51.96 96.32 13.94
LSD (0.05) 0.95 10.21 11.70 7.91 2.09

D 3.43 31.80 33.36 50.50 8.74

D + VAM 3.86 31.40 38.22 93.54 7.38
U 3.29 36.20 33.32 49.92 9.08
U + VAM 3.14 31.80 31.38 97.92 6.68
LSD (0.05) 1.29 9.62 4.51 18.36 1.37

12 D 1.26 30.00 24.24 62.26 6.56

D + VAM 2.28 31.60 26.08 132.64 10.64
U 1.55 31.80 24.10 73.12 6.76
U + VAM 1.59 30.40 23.90 129.22 2.56
LSD (0.05) 0.50 9.50 5.78 15.11 2.18
a,b,c,d As in Table 1.

nitrogen concentrations of VAM and non VAM
plants inoculated with strains D344 and Urbana
did not differ, indicating an equal nitrogen sup-
ply to plants in all treatments (Table 4) . This
could be due to a recycling of N in plants during
the different growth phases .
VAM clearly stimulated the uptake of P, Zn
and Cu by soybean plants under conditions of
low reserves of P and N in the soil, irrespective
of the different concentrations of individual ele-
ments in leaves and for both strains of rhizobia .
The present results indicate that in spite of dif-
ferences in rhizobial activity, the presence of the
VAM fungus was of overriding significance . The
low level of N reserves in the test soil was within
the concentration range when both rhizobia are
active (Skrdleta et al ., 1984) .
Acknowledgements
The authors thank Dr J Dedina, Institute of
Physiology, Prague, for providing AAS analyses
and Ing D Kovafova, for the nitrogen determina-
tion and Dr V Gianinazzi-Pearson for critical
suggestions .
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