Plant and Soi1 68, 321-329 (1982)....
Plant and Soi1 68, 321-329 (1982). 0032-079X/82/0683-0323$01.35.
Ms. 5088
fi 1982 Martinus NijhoffLDr W. Junk Puhlishers, Thc Elague. Printed in the Nethdards.
Effect of inoculation with Glomus mosseae on nitrogen fixation hy
field grown soybeans
F. GANRY
ISRAJIRAT, Bamhey, Senegal
H. G. DIEM and Y. R. DOMMERGUES
CNRSJORSTOM BP. 1386, Dakar, Senegul
Key words Alginate A value Endomycorrhiza Glomus moswae Inoculant Nitrogen fixation
Rhizohium japonicum Soybean
Summary
This field study was undertaken to determine the effect of inoculation with Glomus
mosseae on N, fixation and I’ uptake by soybean. The inoculation with Glomus mosseae was achieved
using a new type of inoculant, alginate-entrapped (AE) enclomycorrhizal fungus. N, fixation was
assessed using the A value method. In P-fertilized plots, inoculation with AE Glomus mosseae
increased the harvest index based on dry weight ( t 20%) and N content of seeds (+ 17TJ, the A value (
+ 31%) and % N derived from fixation (+ 75%). Inoculation with AE Glomus mosseae decreased the
coefficient of variation for the A value and for the dry weights of the different plant parts.
Introduction
Most experiments on the effect of inoculation of soybean with vesicular-
arbuscular (VA) fungi reported up to now have been carried out under
greenhouse conditions. These experiments have clearly shown growth increases
of soybean in response to infection by effective strains of VA fungi4. These
growth increases have been attributed to a better exploitation of the labile pool of
soi1 Pio. Pot experiments 1’4,23 have also clearly indicated that VA mycorrhizae
cari greatly assist noclulation and N, fixation of soybeans inoculated with
rhizobia, a result similar to that already reported for other legumes, such as
Stylosanthes’“. This stimulation of the legume N,-fixing activity is probably at
least partly the result of the improvement of the P uptake by the plant. The few
field experiments carried out up to now on soybean in Florida2’, in North
Carolina 21 and in Bangalor, India have confirmed the beneficial effects of
inoculation by VA fungi. However, the reported positive effect on the soybean
yields were not always statistically significant 2.
The first aim of the experiment reported here was to gain further
understanding of the effect of VA infection on P uptake and N, fixation in the
C,.lA ..A-- AL- A -.,.l..- --&L..18 --.L1-L :- ..----.. !--3 ~~ -. .
322
GANRY, DIEM AND DOMMERGUES
Materials and methods
Field experiments were carried out at ’ tire ISRA (Institut Sénégalais de Recherches Agricoles)
research station ofSefa, South Senegal, in 980. The soi1 was a leached ferruginous tropical soilO(alfic
eutrustox) in which soybean had ncver be,t n grown (Table 1).
Table 1. Properties of soi1 from experimet ta1 plots
-
-
C20 Clll
2@40 cm
- -
Texture
Sand (SO-2000 um) (%)
13.3 ’
69.1
Clay (< 2 w) CYJ
12.2 /
15.3
I
pH, H,O (1/2.5)
5.9 /
5.8
Organic C (06)
0.48 1/
0.408
Organic N (%)
o.o4t/
0.041
Exchangeahle cations
S @WI00 g)
2.01
1.94
T (meq/lOO 8)
2.14
2.93
V=S/Tx 100
73
/
66
Total P, ppm
217 ’
216
Available P (Truog), ppm
5.4
2.9
S: Total exchangeable cations.
T: Total exchange capacity.
Experimental design
Split-plot experimental design was used mwith six replicates. Ah the plots were inoculated with
Rhizohiunljaponicum.
There were two mai 1 fireatments: one in which no P was added. and another in
which P was added at the rate of 22 kg P/h%. The surface of main-plot was 2X m ‘. There were two
subtreatments one involving inoculation ,with alginate-gel witbout G/omu.c moswae. the other
involving inoculation with AE Glomus rn uieat>. The surface of subplot was 25 rn’. Al1 main plots
received starter nitrogen fertihzer ( 17 kg I\\ /ha), and K as KCI (90 kg K/ha) was applied at the time of
sowing and at the time of flowering. This experimental design was part of a larger experiment,
presented elsewhere”, designed to study tlweffect of different fertilizers and Rlhizobium inoculation
on soybedn yield.
Two statistical analyses were performrd. The frst, involving the whole split-plot design (which
comprised both P-fertilized and non P-f:rtilized plots) was performed according to the methods
*- .l-~IImm , ~~~ . . . . . . 4 ..Jo.~_~.-^..-..~,p,.-l. _..- 24 l-1. --,._,_- J:..+n,.,.ra*,,,;nn ~.,,,~,\\“+j,‘,p_
EFFECT OF G. MOSSEA& ON N FIXATION OF SOY BEANS
323
The soybean cultivar used was cv. 44A/3 obtained by ISRA. The Rhizobium peat-base inoculant
tstrain USDA 138) which co,ntained 3.10” living cells per g, was applied to the seedling bed at the rate
of 220 g per 28 mZ plot.
Glomus mossrac was multiplied on roots of Mgmt unguicnlam which had been inoculated with roots
fragments heavily infected with the VA fungus. After two month’s growth the roots of I’lgna
unyuiculatn were harvested, washed on a sieve to remove extra soi1 and the mixture of infected roots,
spores, extramatrical hyphae was then thoroughly homogenised in a Waring blender for 3 seconds
three times (100 g in 1000 ml of water). The suspension thus obtained was entrapped in calcium
alginateaccording themethod already describedfor Rhizobium’” The resulting product consisted of
wet beads of alginate-entrapped (AE) Glornus mosseue which were stored in a cold room. Each bead
contained ca 12 mg (fresh weight) of infected roots, spores and hyphae, inoculation was performed by
introducing 10--l 5 beads 3-4 cm deep into the soi1 around each seedling when they were 15 days old.
The total rainfall before sowing (June to July 19) was 153 mm and 504 mm during the growth cycle
(July 19 ta October 15). The rainfall distribution was fairly even; however. a dry period did occur from
September 10 to October 1 at the time of pod flling.
Amount cflnitrogenfixed hJ> soq’heun
This was evaluated according to the A value methods which involves simultaneous determination
of the A values by uninoculated soybean (Au) and by inoculated soybean (Ai) using “N-labelled
nitrogen fertilizer.
For the sake of clarity. we give hereafter the definition in short of the ‘A’ value method used for the
determination of hxed-N, clearly by Fried and Broeshart *:
1 - The available amount of N in a source is designated by ‘A’, which is a cancrpt.
2 - The ‘A’ value is expressed in equivalent units of kg N/ha as nitrogen fertilizer applied
(ammonium sulfate in our experiment).
3 - Symbiotic N, fixation by legume trop is confronted with 3 sources ofN: a, Soi1 N; b, Fertilizer
N jammonium sulfate in our experiment); c, N supplied by N, hxing mechanism in nodules.
4 - We need a non nodulating (non nod.) trop with the same growth-period for the determinalion
of A ‘Soil’ value.
5 - From nodulating trop, we cari determine (using labelled fertilizer) the A ‘Soi1 + fixation’
value.
6 .- In our experiment. we are in the situation where nodulating trop and non nodulatingcrop ‘sec’
the same available amount of soil N but received ditferent amount of fertilizer N.
7 - A ‘Fix.’ = A ‘Soi1 + fix.’ - A ‘Soif
If:
A ‘fertilizer’ = rate of fertilizer applied (17 N)
y,, Ndff = “;) of N derived from fertilizer
9; Ndf fixation = “;, of N derived from fixation
T.;, Ndf soi1 = 9, of N derived from soi1
We cari Write:
y;Ndff
1; Ndf fixation
T,< Ndf Soi1
-.. =: ~-.
A fertilizer
A fixation
A Soi1
8 -.- Fixed-N (kgiha) = 00 Ndf fixation :< total N (kg N/ha).
In n,,v rvn&m~nt th Pn_n -,,,A _ -_-..,,. . . . ..-.. _^^.. 1-.-J ----L-1- --’
1 .<
If fertilization and inoculation with Glomus mossrn~~ on plant dry weight, total N and P contents of field-grown soybeans
Plant dry wt (kg;ha)
Total N (kg/ha)
Total P (kg/ha)
-
IUS
Seeds
Straw
Seeds
Seeds
Straw
Seeds
Seeds
Straw
Seeds
me
+ straw
+ straw
+ straw
~.
1486(33) 3098(27) 4584(28)
95
23
119
5 . 8
1.0
6 . 9
1381(30) 3391(14) 4772(18)
8 7
2 5
112
5 . 4
1.1
6 . 5
1546(12) 4128(15) 5674(11)
1 0 0
43
1 4 3
7 . 7
2 . 9
1 0 . 6
1848( 4 ) 4164( 8 ) 6012( 6 )
116
3 8
154
9.2
2 . 5
1 1 . 7
(21)
(2.3
(20)
(22)
(30)
(22)
(24)
(37)
(22)
:“-
osseae
(15)
( 8)
( 9)
(15)
(20)
(15)
(19)
(35)
(21)
Q
ient of variation (“A). Data related to straw, including husk. The only significant effect was the main effect of P fertilization (F test, blocks with split-
5
7
0
E
z
:
i
E
c
E
EFFECT OF G. MOSSEAE ON N FIXATION OF SOYBEANS
325
The amount of fixed N,, expressed in kg N/ha, was ohtained by multiplying the difference Ai-Au by
the percent utilization of the fertilizer by the soybeans.
Plants were carefully harvested avoiding contamination with soi1 N. The samples were dried at 65%
70°C for 24 h, weighed, ground into a 40 mesh powder, analysed for P content according to the usual
vanadomolybdophosphate method and for total nitrogen content according to the Kjeldahl method.
‘“N analyses were carried out at the Seibersdorf Laboratory (IAEA) using Dumas’ method (the
combustion performed in this technique converts total N directly to N,) and emission spectrometry.
Assessment ofmycorrhizal ir$ection
The roots were stained according to themethod of Phillips and Hayman’s, tut into 5 mm segments
and observed under a dissecting microscope to determine the percentage ofinfected roots (frequency).
Results and discussion
lntrrpretution of data related to the split-plot design
This interpretation indicated that the only significant treatments were P
fertilization, which increased the weight, total P and N content of soybean (Table
2), and inoculation with AE Glomus mosseae, which significantly decreased the
amount of N fertilizer taken up by the plant (Table 3). The beneficial effect of P
fertilization on soybean yield was to be expected but the result with inoculation
with AE Glomus mosseae is worth underlining since this is the first time that the
effect of inoculation with AE Glomus mosseae on N fertilizer uptake is
demonstrated in situ using the A value method. The decrease in N fertilizer
uptake observed occurred concurrently with an increase in N, fixatïon, which
confirms that by improving P and water uptake during the dry period
inoculation with AE Glomus mosseae cari enhance N, fixation (Table 3). One
striking effect of P fertilization was that it decreased the plant heterogeneity
Table 3. Sources of N and A value
Treatments
Fixed N,
Soi1 N
Fertilizer N
A value
_ .--_ ~-- -.~- ~.
---.--...-- -
-- - - -- ---
P fertilization Glomus
(kg Ww
mossea4~
70
kg/ha
%
kg@
%
k/ha
Wha
0
0
37
44
58
69
4.0
4.8
422 (32)
0
+
38
42
58
65
3.9
4.3
43 1(32)
22
0
24
34
73
104
3.8
5.4
439(16)
22
+
41
63
56
86
2.9
4.4
576( 6)
Main effect of P fertilization
(15)
(24)
!
326
/
GANRY, DIEM AND DOMMERGUES
expressed by the coefficient of variation (CV). Thus the CV for the dry weight of
seeds which was 30-35oi, in plots Nlthout P was only 4-12’:<, in plots with P (Table
2). The application rate of P wa: 11ow (22 kg P/ha) but sufficient to decrease the
heterogeneity of the distribution of available P in the soil.
The effect of either treatments, (IP application or inoculation with AE Glomus
mosseue on mycorrhizal infectior of the roots was not significant (Table 4),
probably because the roots were :;ampled when the plant were too old (pod filling
stage), which allowed plenty of ti,me for the native VA fungi to invade the roots.
Table 4. Root infection of soyhean by VI hungi
i
--I-
Treatments
/
.~~~
/
Infection
P fertilization
Inoculation with
frequency (“0)
(kg WW
Glomus mosseae
0
0
38 (27)
0
+
46 (22)
22
0
36(28)
22
f
42 (24)
No significant differences.
In brackets, coefficient of variation (li,).
,
/
It was not possible to carry cld a statistical analysis of the harvest index.
However, table 5 shows that in thte plots with no P fertilization the effect of
inoculation with AE Glomus moweae on the harvest index* expressed as dry
weight, total N or total P was negafiive whereas it was positive in the plots with P
fertilization. In other words the teneficial effect of inoculation with AE Glomus
mosseae on the harvest index occt rred only when a small amount of P (22 kg/ha)
was applied to the soil, which conftrned the suggestion that P application may be
necessary for the expression of t.?e: beneficial effect of VA inoculation17. The
differences observed between plan& with or without AE Glomus mosseae was
probably accentuated by thc drot.ght period which occurred during pod-filling.
Inoculation with AE Glomus mtwseae descreased the coefficient of variation of
the different parameters studied including the A value. For this, latter parameter
the coefficient decreased from 16 to 6% in the P treated plots.
The high variability of contra1 plots (without AE Glomus mosseae) was
attributed to the patchy distributi XII of VA fungi reflected by a high coefficient of
variation of infection. 1noculati)tr with AE Glomus mosseue improved the
EFFECT OF G. MOSSEAI: ON N FIXATION OF SOYBEANS
321
Table 5. Estimation of the harvest index resulting from inoculation with Glomus mosseae (G.m.)
P. fertilizer
G.m.
D.W.
N
P
0
0
0.32
0.80
0.84
+
0.29
0.78
0.83
0,”
- 9.1
-2.5
-- 1.2
+
0
0.21
0.70
0.73
+
0.31
0.75
0.79
ut0
+ 14.8
+7.1
-+ 8.2
2, = relative increase or decrease in harvest index.
Harvest index = weight, total N or P content of seeds as a proportion of weight, total N or P
content of trop.
following VA inoculation has recently been found by Morandi et al.’ 5 using
raspberry. It is interesting to note that during their growth VA inoculated
soybeans were visually better developed and greener than uninoculated plants.
lnterpretation of data concerning P-fertilized plots alone
Table 6 shows that inoculation with AE Glomus mosseae signitîcantly
increased dry weight (+ 20x), total N content of seeds (+ 17x), the A value (
+ 3 1%) and PJ, N derived from fixation ( + 75%). The increase in total N was not
surprising since the beneficial effect of VA fungi on the N content of seeds has
already been reported 21,22 Increase in the A value is probably related to the
.
stimulation of N, fixation, which is itself a consequence of endomycorrhizal
increase of ion uptake3. ‘This has been clearly shown in glass-house conditions
and, in a few instances, in the field using indirect methods of assessments, such as
nodule number or nodule weight and acetylene reduction activity
measurements’*4*22.
Table 6. Significant effects of inoculation with C/ornus mosseae (study restricted to the P-fertilized
plots)
Inoculation
Dry ,weight
Total N
A value
Fixed N,
with
of seeds
of seeds
(kg/ha)
(io)
Glornus mosseuc
(k/ha)
Uw’ha)
0
1546(100)
99.6(100)
439(100)
23.7 (100)
+
1848(120)
116.2(117)
576(131)
41.4(175)
/
328
/
GANRY, DIEM AND DOMMERGUES
Conclusion
The present field study on soybeans confirms most of the experimental data
obtained under glass-house candi ;icms on the effect of inoculation with VA fungi,
Le. increased dry weight, total b and N. There is a strong indication that
inoculation with Glomus mosseac imcreases the harvest index, which is a most
valuable result. Moreover, by reiucing uptake of fertilizer N and soi1 N and
simultaneously promoting N, fira,iion, VA mycorrhizae conserve the stock of
soi1 N and thus contribute to improving the N balance which is often negative in
grain legume crops 25. However, eren under the best conditions (i.e. in P-fertilized
plots inoculated with AE Glomu~ mosseae) the total amount of N, fixed is low
(63 kg/ha) and only 41% of trop N is derived from N, fixation whereas higher
figures have reported”.
Although the present experiment was not specially designed to check the
performance of the inoculum con ;ir;ting of a polymer-entrapped VA fungus, the
results reported here suggest that ..hiis new type of inoculum could be successfully
used in the field. It would be interestiing to compare it with other methods used to
introduce VA fungi into soil, such as the soi1 pellet method described by Hall “.
Acknowledgements
We thank Dr. V. /sianinazzi-Pearson for reviewing the manuscript and
Youssouph N’Diaye and Moussa Niang fo/r tlrchnical assistance. The investigation was funded in part
by IAEA (joint FAOIIAEA coordinated r,s::arch programme).
1
Received 4 May 1982. Revised August lYri2
References
1
Asimi S, Gianinazzi-Pearson V and C ianinazzi S 1980 Influence of increasing soi1 phosphorus
levels on interactions between vesicula,:-arbuscular mycorrhizae and Rhizobium in soybeans.
Can. J. Bot. 58,2200-2205.
2
Bagyaraj D J, Manjunath A and Pa .il R B 1979 Interaction between a vesicular-arbuscular
mycorrhiza and Rhizobium and theil eiRècts on soybean in the field. New Phytol. 82, 141-146.
3
Bowen G D 1980 Mycorrhizal rôles ‘n,tropical plants and ecosystems. In Tropical M ycorrhiza
Research. Ed. P Mikola. Clarendon ‘ress, Oxford, pp 165-190.
4 Carling D E and Brown M F 1980 Relative effect of vesicular-arbuscular mycorrhizal fungi on
the growth and yield of soybeans. So’l. Sci. Soc. Am. J. 44, 528-532.
5 Carling D E, Riehle W G, Brown M F and Johnson D R 1978 Effects of a vesicular-arbuscular
nodulating soybeans. Phytopatholog
6
Ithaca. 434 p.
EFFECT OF G. MOSSEAE ON N FIXATION OF SOYBEANS
329
9 Ganry F and Wey J 1981 Inoculation par Rhizohiumjaponicum et fumure phosphatée du soja
au Sénégal Report on the FAO/IAEA Coordinated Research Programme on the use of Isotopes
in Studies on Biological Dinitrogen Fixation. Contract n’ Dl-SEN 2375, IAEA, Vienna (in
press).
10 Gianinazzi-Pearson V, Fardeau J C, Asimi S and Gianinazzi S 1981 Source of additional
phosphorus absorbed from soi1 by vesicular-arbuscular mycorrhizal soybeans. Physiol. Veg. 19,
33-44.
1 1
Hall 1 R 1979 Soil pellets to introduce vesicular-arbuscular mycorrhizal fungi into soil. Soi1
Biol. Biochem. 11,85-86.
12
Herridge D F 1981 A whole-system approach to quantifying biological nitrogen fixation by
legumes and associated gains and losses of nitrogen in agricultural systems. In Proc. Workshop
on Biological Nitrogen Fixation Technology for Tropical Agriculture. Cali Colombia March 9-
13 (in press).
13 Jung G, Mugnier J, Diem H G and Dommergues Y R 1982 Polymer-entrapped Rhizobium as
an inoculant for legumes. Plant and Soi1 65,219-231.
14
Lecompt M 1965 L’expérimentation et les engrais. SPIEA, Paris. 91 p.
15
Morandi D, Gianinazzi S and Gianinazzi-Pearson V 1979 Intérêt de l’endomycorhization
dans la reprise et la croissance des framboisiers issus de multiplication végétative in vitro. Ann.
Amél. Plantes 29,23-30.
16
Mosse B 1977 Plant growth responses to vesicular-arbuscular mycorrhiza. Responses of
Stylosanthes and maïze to inoculation in unsterile soils. New Phytol. 78,277-288.
17
Mosse B and Hayman D S 1980 Mycorrhiza in agricultural plants. In Tropical Mycorrhiza
Research. Ed. P Mikola. Clarendon Press, Oxford. pp 213-230.
18
Phillips J M and Hayman D S 1970 Improved procedures for clearing roots and staining
parasitic and vesicuiar-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans.
Br. Mycol. Soc. 55, 158--l 61.
19
Quidet P et Masmejean T 1962 Organisation, conduite et interprétation des essais de fumure.
Services Agronomiques de la Société Commerciale des Potasses d’Alsace. SEDA, Paris, 108 p.
20
Ross J P 1971 Effect of phosphate fertilization on yield of mycorrhizal and non mycorrhizdl
soybeans. Phytopathology 61,140&1403.
21
Ross J P and Harper J A 1970 Effect of Endogone mycorrhiza on soybean yields.
Phytopathology 60, 1552-1556.
22 Schenck N C and Hinson K 1973 Responses of nodulating and non-nodulating soybeans to a
species of Endogone mycorrhiza. Agron. J. 65,849-850.
23 Skipper H D and Smith G W 1979 Influence of soi1 pH on the soybean-endomycorrhiza
symbiosis. Plant and Soi1 53,559-563.
24 Snedecor G W and Cochran W C 1971 Statistical methods. Sixth Edition. The Iowa State
University Press, Ames Iowa, USA.
25
Wetselaar R and Ganry F 1982 Nitrogen balance in tropical agrosystems. In Microbiology of
Tropiçdi Soils. Eds. Y R Dommergues and H G Diem. Martinus Nijhoff, The Hague (1n press).
Ms. 5088
fi 1982 Martinus NijhoffLDr W. Junk Puhlishers, Thc Elague. Printed in the Nethdards.
Effect of inoculation with Glomus mosseae on nitrogen fixation hy
field grown soybeans
F. GANRY
ISRAJIRAT, Bamhey, Senegal
H. G. DIEM and Y. R. DOMMERGUES
CNRSJORSTOM BP. 1386, Dakar, Senegul
Key words Alginate A value Endomycorrhiza Glomus moswae Inoculant Nitrogen fixation
Rhizohium japonicum Soybean
Summary
This field study was undertaken to determine the effect of inoculation with Glomus
mosseae on N, fixation and I’ uptake by soybean. The inoculation with Glomus mosseae was achieved
using a new type of inoculant, alginate-entrapped (AE) enclomycorrhizal fungus. N, fixation was
assessed using the A value method. In P-fertilized plots, inoculation with AE Glomus mosseae
increased the harvest index based on dry weight ( t 20%) and N content of seeds (+ 17TJ, the A value (
+ 31%) and % N derived from fixation (+ 75%). Inoculation with AE Glomus mosseae decreased the
coefficient of variation for the A value and for the dry weights of the different plant parts.
Introduction
Most experiments on the effect of inoculation of soybean with vesicular-
arbuscular (VA) fungi reported up to now have been carried out under
greenhouse conditions. These experiments have clearly shown growth increases
of soybean in response to infection by effective strains of VA fungi4. These
growth increases have been attributed to a better exploitation of the labile pool of
soi1 Pio. Pot experiments 1’4,23 have also clearly indicated that VA mycorrhizae
cari greatly assist noclulation and N, fixation of soybeans inoculated with
rhizobia, a result similar to that already reported for other legumes, such as
Stylosanthes’“. This stimulation of the legume N,-fixing activity is probably at
least partly the result of the improvement of the P uptake by the plant. The few
field experiments carried out up to now on soybean in Florida2’, in North
Carolina 21 and in Bangalor, India have confirmed the beneficial effects of
inoculation by VA fungi. However, the reported positive effect on the soybean
yields were not always statistically significant 2.
The first aim of the experiment reported here was to gain further
understanding of the effect of VA infection on P uptake and N, fixation in the
C,.lA ..A-- AL- A -.,.l..- --&L..18 --.L1-L :- ..----.. !--3 ~~ -. .
322
GANRY, DIEM AND DOMMERGUES
Materials and methods
Field experiments were carried out at ’ tire ISRA (Institut Sénégalais de Recherches Agricoles)
research station ofSefa, South Senegal, in 980. The soi1 was a leached ferruginous tropical soilO(alfic
eutrustox) in which soybean had ncver be,t n grown (Table 1).
Table 1. Properties of soi1 from experimet ta1 plots
-
-
C20 Clll
2@40 cm
- -
Texture
Sand (SO-2000 um) (%)
13.3 ’
69.1
Clay (< 2 w) CYJ
12.2 /
15.3
I
pH, H,O (1/2.5)
5.9 /
5.8
Organic C (06)
0.48 1/
0.408
Organic N (%)
o.o4t/
0.041
Exchangeahle cations
S @WI00 g)
2.01
1.94
T (meq/lOO 8)
2.14
2.93
V=S/Tx 100
73
/
66
Total P, ppm
217 ’
216
Available P (Truog), ppm
5.4
2.9
S: Total exchangeable cations.
T: Total exchange capacity.
Experimental design
Split-plot experimental design was used mwith six replicates. Ah the plots were inoculated with
Rhizohiunljaponicum.
There were two mai 1 fireatments: one in which no P was added. and another in
which P was added at the rate of 22 kg P/h%. The surface of main-plot was 2X m ‘. There were two
subtreatments one involving inoculation ,with alginate-gel witbout G/omu.c moswae. the other
involving inoculation with AE Glomus rn uieat>. The surface of subplot was 25 rn’. Al1 main plots
received starter nitrogen fertihzer ( 17 kg I\\ /ha), and K as KCI (90 kg K/ha) was applied at the time of
sowing and at the time of flowering. This experimental design was part of a larger experiment,
presented elsewhere”, designed to study tlweffect of different fertilizers and Rlhizobium inoculation
on soybedn yield.
Two statistical analyses were performrd. The frst, involving the whole split-plot design (which
comprised both P-fertilized and non P-f:rtilized plots) was performed according to the methods
*- .l-~IImm , ~~~ . . . . . . 4 ..Jo.~_~.-^..-..~,p,.-l. _..- 24 l-1. --,._,_- J:..+n,.,.ra*,,,;nn ~.,,,~,\\“+j,‘,p_
EFFECT OF G. MOSSEA& ON N FIXATION OF SOY BEANS
323
The soybean cultivar used was cv. 44A/3 obtained by ISRA. The Rhizobium peat-base inoculant
tstrain USDA 138) which co,ntained 3.10” living cells per g, was applied to the seedling bed at the rate
of 220 g per 28 mZ plot.
Glomus mossrac was multiplied on roots of Mgmt unguicnlam which had been inoculated with roots
fragments heavily infected with the VA fungus. After two month’s growth the roots of I’lgna
unyuiculatn were harvested, washed on a sieve to remove extra soi1 and the mixture of infected roots,
spores, extramatrical hyphae was then thoroughly homogenised in a Waring blender for 3 seconds
three times (100 g in 1000 ml of water). The suspension thus obtained was entrapped in calcium
alginateaccording themethod already describedfor Rhizobium’” The resulting product consisted of
wet beads of alginate-entrapped (AE) Glornus mosseue which were stored in a cold room. Each bead
contained ca 12 mg (fresh weight) of infected roots, spores and hyphae, inoculation was performed by
introducing 10--l 5 beads 3-4 cm deep into the soi1 around each seedling when they were 15 days old.
The total rainfall before sowing (June to July 19) was 153 mm and 504 mm during the growth cycle
(July 19 ta October 15). The rainfall distribution was fairly even; however. a dry period did occur from
September 10 to October 1 at the time of pod flling.
Amount cflnitrogenfixed hJ> soq’heun
This was evaluated according to the A value methods which involves simultaneous determination
of the A values by uninoculated soybean (Au) and by inoculated soybean (Ai) using “N-labelled
nitrogen fertilizer.
For the sake of clarity. we give hereafter the definition in short of the ‘A’ value method used for the
determination of hxed-N, clearly by Fried and Broeshart *:
1 - The available amount of N in a source is designated by ‘A’, which is a cancrpt.
2 - The ‘A’ value is expressed in equivalent units of kg N/ha as nitrogen fertilizer applied
(ammonium sulfate in our experiment).
3 - Symbiotic N, fixation by legume trop is confronted with 3 sources ofN: a, Soi1 N; b, Fertilizer
N jammonium sulfate in our experiment); c, N supplied by N, hxing mechanism in nodules.
4 - We need a non nodulating (non nod.) trop with the same growth-period for the determinalion
of A ‘Soil’ value.
5 - From nodulating trop, we cari determine (using labelled fertilizer) the A ‘Soi1 + fixation’
value.
6 .- In our experiment. we are in the situation where nodulating trop and non nodulatingcrop ‘sec’
the same available amount of soil N but received ditferent amount of fertilizer N.
7 - A ‘Fix.’ = A ‘Soi1 + fix.’ - A ‘Soif
If:
A ‘fertilizer’ = rate of fertilizer applied (17 N)
y,, Ndff = “;) of N derived from fertilizer
9; Ndf fixation = “;, of N derived from fixation
T.;, Ndf soi1 = 9, of N derived from soi1
We cari Write:
y;Ndff
1; Ndf fixation
T,< Ndf Soi1
-.. =: ~-.
A fertilizer
A fixation
A Soi1
8 -.- Fixed-N (kgiha) = 00 Ndf fixation :< total N (kg N/ha).
In n,,v rvn&m~nt th Pn_n -,,,A _ -_-..,,. . . . ..-.. _^^.. 1-.-J ----L-1- --’
1 .<
If fertilization and inoculation with Glomus mossrn~~ on plant dry weight, total N and P contents of field-grown soybeans
Plant dry wt (kg;ha)
Total N (kg/ha)
Total P (kg/ha)
-
IUS
Seeds
Straw
Seeds
Seeds
Straw
Seeds
Seeds
Straw
Seeds
me
+ straw
+ straw
+ straw
~.
1486(33) 3098(27) 4584(28)
95
23
119
5 . 8
1.0
6 . 9
1381(30) 3391(14) 4772(18)
8 7
2 5
112
5 . 4
1.1
6 . 5
1546(12) 4128(15) 5674(11)
1 0 0
43
1 4 3
7 . 7
2 . 9
1 0 . 6
1848( 4 ) 4164( 8 ) 6012( 6 )
116
3 8
154
9.2
2 . 5
1 1 . 7
(21)
(2.3
(20)
(22)
(30)
(22)
(24)
(37)
(22)
:“-
osseae
(15)
( 8)
( 9)
(15)
(20)
(15)
(19)
(35)
(21)
Q
ient of variation (“A). Data related to straw, including husk. The only significant effect was the main effect of P fertilization (F test, blocks with split-
5
7
0
E
z
:
i
E
c
E
EFFECT OF G. MOSSEAE ON N FIXATION OF SOYBEANS
325
The amount of fixed N,, expressed in kg N/ha, was ohtained by multiplying the difference Ai-Au by
the percent utilization of the fertilizer by the soybeans.
Plants were carefully harvested avoiding contamination with soi1 N. The samples were dried at 65%
70°C for 24 h, weighed, ground into a 40 mesh powder, analysed for P content according to the usual
vanadomolybdophosphate method and for total nitrogen content according to the Kjeldahl method.
‘“N analyses were carried out at the Seibersdorf Laboratory (IAEA) using Dumas’ method (the
combustion performed in this technique converts total N directly to N,) and emission spectrometry.
Assessment ofmycorrhizal ir$ection
The roots were stained according to themethod of Phillips and Hayman’s, tut into 5 mm segments
and observed under a dissecting microscope to determine the percentage ofinfected roots (frequency).
Results and discussion
lntrrpretution of data related to the split-plot design
This interpretation indicated that the only significant treatments were P
fertilization, which increased the weight, total P and N content of soybean (Table
2), and inoculation with AE Glomus mosseae, which significantly decreased the
amount of N fertilizer taken up by the plant (Table 3). The beneficial effect of P
fertilization on soybean yield was to be expected but the result with inoculation
with AE Glomus mosseae is worth underlining since this is the first time that the
effect of inoculation with AE Glomus mosseae on N fertilizer uptake is
demonstrated in situ using the A value method. The decrease in N fertilizer
uptake observed occurred concurrently with an increase in N, fixatïon, which
confirms that by improving P and water uptake during the dry period
inoculation with AE Glomus mosseae cari enhance N, fixation (Table 3). One
striking effect of P fertilization was that it decreased the plant heterogeneity
Table 3. Sources of N and A value
Treatments
Fixed N,
Soi1 N
Fertilizer N
A value
_ .--_ ~-- -.~- ~.
---.--...-- -
-- - - -- ---
P fertilization Glomus
(kg Ww
mossea4~
70
kg/ha
%
kg@
%
k/ha
Wha
0
0
37
44
58
69
4.0
4.8
422 (32)
0
+
38
42
58
65
3.9
4.3
43 1(32)
22
0
24
34
73
104
3.8
5.4
439(16)
22
+
41
63
56
86
2.9
4.4
576( 6)
Main effect of P fertilization
(15)
(24)
!
326
/
GANRY, DIEM AND DOMMERGUES
expressed by the coefficient of variation (CV). Thus the CV for the dry weight of
seeds which was 30-35oi, in plots Nlthout P was only 4-12’:<, in plots with P (Table
2). The application rate of P wa: 11ow (22 kg P/ha) but sufficient to decrease the
heterogeneity of the distribution of available P in the soil.
The effect of either treatments, (IP application or inoculation with AE Glomus
mosseue on mycorrhizal infectior of the roots was not significant (Table 4),
probably because the roots were :;ampled when the plant were too old (pod filling
stage), which allowed plenty of ti,me for the native VA fungi to invade the roots.
Table 4. Root infection of soyhean by VI hungi
i
--I-
Treatments
/
.~~~
/
Infection
P fertilization
Inoculation with
frequency (“0)
(kg WW
Glomus mosseae
0
0
38 (27)
0
+
46 (22)
22
0
36(28)
22
f
42 (24)
No significant differences.
In brackets, coefficient of variation (li,).
,
/
It was not possible to carry cld a statistical analysis of the harvest index.
However, table 5 shows that in thte plots with no P fertilization the effect of
inoculation with AE Glomus moweae on the harvest index* expressed as dry
weight, total N or total P was negafiive whereas it was positive in the plots with P
fertilization. In other words the teneficial effect of inoculation with AE Glomus
mosseae on the harvest index occt rred only when a small amount of P (22 kg/ha)
was applied to the soil, which conftrned the suggestion that P application may be
necessary for the expression of t.?e: beneficial effect of VA inoculation17. The
differences observed between plan& with or without AE Glomus mosseae was
probably accentuated by thc drot.ght period which occurred during pod-filling.
Inoculation with AE Glomus mtwseae descreased the coefficient of variation of
the different parameters studied including the A value. For this, latter parameter
the coefficient decreased from 16 to 6% in the P treated plots.
The high variability of contra1 plots (without AE Glomus mosseae) was
attributed to the patchy distributi XII of VA fungi reflected by a high coefficient of
variation of infection. 1noculati)tr with AE Glomus mosseue improved the
EFFECT OF G. MOSSEAI: ON N FIXATION OF SOYBEANS
321
Table 5. Estimation of the harvest index resulting from inoculation with Glomus mosseae (G.m.)
P. fertilizer
G.m.
D.W.
N
P
0
0
0.32
0.80
0.84
+
0.29
0.78
0.83
0,”
- 9.1
-2.5
-- 1.2
+
0
0.21
0.70
0.73
+
0.31
0.75
0.79
ut0
+ 14.8
+7.1
-+ 8.2
2, = relative increase or decrease in harvest index.
Harvest index = weight, total N or P content of seeds as a proportion of weight, total N or P
content of trop.
following VA inoculation has recently been found by Morandi et al.’ 5 using
raspberry. It is interesting to note that during their growth VA inoculated
soybeans were visually better developed and greener than uninoculated plants.
lnterpretation of data concerning P-fertilized plots alone
Table 6 shows that inoculation with AE Glomus mosseae signitîcantly
increased dry weight (+ 20x), total N content of seeds (+ 17x), the A value (
+ 3 1%) and PJ, N derived from fixation ( + 75%). The increase in total N was not
surprising since the beneficial effect of VA fungi on the N content of seeds has
already been reported 21,22 Increase in the A value is probably related to the
.
stimulation of N, fixation, which is itself a consequence of endomycorrhizal
increase of ion uptake3. ‘This has been clearly shown in glass-house conditions
and, in a few instances, in the field using indirect methods of assessments, such as
nodule number or nodule weight and acetylene reduction activity
measurements’*4*22.
Table 6. Significant effects of inoculation with C/ornus mosseae (study restricted to the P-fertilized
plots)
Inoculation
Dry ,weight
Total N
A value
Fixed N,
with
of seeds
of seeds
(kg/ha)
(io)
Glornus mosseuc
(k/ha)
Uw’ha)
0
1546(100)
99.6(100)
439(100)
23.7 (100)
+
1848(120)
116.2(117)
576(131)
41.4(175)
/
328
/
GANRY, DIEM AND DOMMERGUES
Conclusion
The present field study on soybeans confirms most of the experimental data
obtained under glass-house candi ;icms on the effect of inoculation with VA fungi,
Le. increased dry weight, total b and N. There is a strong indication that
inoculation with Glomus mosseac imcreases the harvest index, which is a most
valuable result. Moreover, by reiucing uptake of fertilizer N and soi1 N and
simultaneously promoting N, fira,iion, VA mycorrhizae conserve the stock of
soi1 N and thus contribute to improving the N balance which is often negative in
grain legume crops 25. However, eren under the best conditions (i.e. in P-fertilized
plots inoculated with AE Glomu~ mosseae) the total amount of N, fixed is low
(63 kg/ha) and only 41% of trop N is derived from N, fixation whereas higher
figures have reported”.
Although the present experiment was not specially designed to check the
performance of the inoculum con ;ir;ting of a polymer-entrapped VA fungus, the
results reported here suggest that ..hiis new type of inoculum could be successfully
used in the field. It would be interestiing to compare it with other methods used to
introduce VA fungi into soil, such as the soi1 pellet method described by Hall “.
Acknowledgements
We thank Dr. V. /sianinazzi-Pearson for reviewing the manuscript and
Youssouph N’Diaye and Moussa Niang fo/r tlrchnical assistance. The investigation was funded in part
by IAEA (joint FAOIIAEA coordinated r,s::arch programme).
1
Received 4 May 1982. Revised August lYri2
References
1
Asimi S, Gianinazzi-Pearson V and C ianinazzi S 1980 Influence of increasing soi1 phosphorus
levels on interactions between vesicula,:-arbuscular mycorrhizae and Rhizobium in soybeans.
Can. J. Bot. 58,2200-2205.
2
Bagyaraj D J, Manjunath A and Pa .il R B 1979 Interaction between a vesicular-arbuscular
mycorrhiza and Rhizobium and theil eiRècts on soybean in the field. New Phytol. 82, 141-146.
3
Bowen G D 1980 Mycorrhizal rôles ‘n,tropical plants and ecosystems. In Tropical M ycorrhiza
Research. Ed. P Mikola. Clarendon ‘ress, Oxford, pp 165-190.
4 Carling D E and Brown M F 1980 Relative effect of vesicular-arbuscular mycorrhizal fungi on
the growth and yield of soybeans. So’l. Sci. Soc. Am. J. 44, 528-532.
5 Carling D E, Riehle W G, Brown M F and Johnson D R 1978 Effects of a vesicular-arbuscular
nodulating soybeans. Phytopatholog
6
Ithaca. 434 p.
EFFECT OF G. MOSSEAE ON N FIXATION OF SOYBEANS
329
9 Ganry F and Wey J 1981 Inoculation par Rhizohiumjaponicum et fumure phosphatée du soja
au Sénégal Report on the FAO/IAEA Coordinated Research Programme on the use of Isotopes
in Studies on Biological Dinitrogen Fixation. Contract n’ Dl-SEN 2375, IAEA, Vienna (in
press).
10 Gianinazzi-Pearson V, Fardeau J C, Asimi S and Gianinazzi S 1981 Source of additional
phosphorus absorbed from soi1 by vesicular-arbuscular mycorrhizal soybeans. Physiol. Veg. 19,
33-44.
1 1
Hall 1 R 1979 Soil pellets to introduce vesicular-arbuscular mycorrhizal fungi into soil. Soi1
Biol. Biochem. 11,85-86.
12
Herridge D F 1981 A whole-system approach to quantifying biological nitrogen fixation by
legumes and associated gains and losses of nitrogen in agricultural systems. In Proc. Workshop
on Biological Nitrogen Fixation Technology for Tropical Agriculture. Cali Colombia March 9-
13 (in press).
13 Jung G, Mugnier J, Diem H G and Dommergues Y R 1982 Polymer-entrapped Rhizobium as
an inoculant for legumes. Plant and Soi1 65,219-231.
14
Lecompt M 1965 L’expérimentation et les engrais. SPIEA, Paris. 91 p.
15
Morandi D, Gianinazzi S and Gianinazzi-Pearson V 1979 Intérêt de l’endomycorhization
dans la reprise et la croissance des framboisiers issus de multiplication végétative in vitro. Ann.
Amél. Plantes 29,23-30.
16
Mosse B 1977 Plant growth responses to vesicular-arbuscular mycorrhiza. Responses of
Stylosanthes and maïze to inoculation in unsterile soils. New Phytol. 78,277-288.
17
Mosse B and Hayman D S 1980 Mycorrhiza in agricultural plants. In Tropical Mycorrhiza
Research. Ed. P Mikola. Clarendon Press, Oxford. pp 213-230.
18
Phillips J M and Hayman D S 1970 Improved procedures for clearing roots and staining
parasitic and vesicuiar-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans.
Br. Mycol. Soc. 55, 158--l 61.
19
Quidet P et Masmejean T 1962 Organisation, conduite et interprétation des essais de fumure.
Services Agronomiques de la Société Commerciale des Potasses d’Alsace. SEDA, Paris, 108 p.
20
Ross J P 1971 Effect of phosphate fertilization on yield of mycorrhizal and non mycorrhizdl
soybeans. Phytopathology 61,140&1403.
21
Ross J P and Harper J A 1970 Effect of Endogone mycorrhiza on soybean yields.
Phytopathology 60, 1552-1556.
22 Schenck N C and Hinson K 1973 Responses of nodulating and non-nodulating soybeans to a
species of Endogone mycorrhiza. Agron. J. 65,849-850.
23 Skipper H D and Smith G W 1979 Influence of soi1 pH on the soybean-endomycorrhiza
symbiosis. Plant and Soi1 53,559-563.
24 Snedecor G W and Cochran W C 1971 Statistical methods. Sixth Edition. The Iowa State
University Press, Ames Iowa, USA.
25
Wetselaar R and Ganry F 1982 Nitrogen balance in tropical agrosystems. In Microbiology of
Tropiçdi Soils. Eds. Y R Dommergues and H G Diem. Martinus Nijhoff, The Hague (1n press).