Jnurnal, 1987, 3, 75-86 ‘Variation in N2...
Jnurnal, 1987, 3, 75-86
‘Variation in N2 fixation, N and P contents of
mycorrhizal Vigna unguiculata in relation to
the progressive development of extra-
radical hyphae of Glomus mosseae
M. Gueye*, H. G. Diemt & Y. R. Dommergues+
* MIl$CENKXRA,
B.P. 53, Bambey, Sertegal, and tluborrltoire BSSFT (CTFTIORSTOMJCNRS)
45 bis,
Avenue de la Belle Gabrielle, 94736 Nogent sur Mante, France
Received 10 February 1986; revised and accepted 31 October 1986
Iutroduction
Earlier studies have shown that field-grown cowpea (V@a unguiculutu) is dependent
on vesicular-arbuscular (VA) mycorrhizae for growth and phosphorus (P) uptake
(Yost & Fox 1979). ‘I’he VA mycorrhizal dependence of cowpea has been further
confirmed experimentally by Bagyaraj & Manjunath (1980) and Islam et al. (1980)
even when using non-sterile soil. It is believed that the improved growth of
mycorrhizal cowpea is related to increased absorption of some minera1 nutrients,
especially P and Zn (Bagyaraj & Manjunath 1980; Gueye 1983; Ollivier et uf. 1984).
In their experiment with Vignu zmguicdutu inoculated with different species of VA
‘<
fungi and given different forms of phosphate, Ollivier et al. (1982) showed that the
increase of P concentration in mycorrhizal plants varied significantly with the fungal
species and forms of phosphate used. Yost Bt Fox (1979) studied the P content of
cowpea leaves as affected by soil P status and showed that cowpea was dependent on
VA mycorrhixae for P uptake over a wide range of soi1 P contents,
Information is available on the beneficial effect of VA mycorrhizae on plant growth
in connection with P fertilization but very little is known about the time course of P
uptake during the growth cycle of cowpea. Furthermore, although it is well
established that the increase of P uptake in mycorrhizal plants is due mainly to the
better exploration of soil by extra-radical mycorrhiial hyphae, no work has yet been
done to assess the development of extra-radical mycorrhixal hyphae in the cowpea
rhiiosphere .
This work was initiated to determine the relation between the development on
.
‘ .
interna1 and extra-radical mycorrhizal hyphae and nodule and shoot dry weights, N
and P concentrations, shoot water content and Nz fixation (&Hz reduction activity) in
mycorrhizal cowpeas. Comparison of these characteristics with those of non-
@ Oxford University Press 1987
76 M. Gueye, H. G. Diem and Y. R. Dommerpes
mytorrhizal plants during the plant growth cycle should improve our knowledge of the
VA mycorrhizal symbiosis in cowpea.
MateriaIs and methods
Surface sterilized seeds of cowpea (cv. 58-185 with growth cycle of !XI days) were
genminated in sterile sand for two days. Each germinated seed was then transplanted
to a. pot containing 1.5 kg of autoclaved soil from the Centre National de la Recherche
Agricole in Bambey (psamment; vemacular name of soi1 Dek; see Table 1).
Table 1 Characterlstics of Bambey Dek soi1
Total C (%)
0.51
Total N (%)
0.03
Total P (mg P/kg)
74
Available P (O&n;* mg P/kg)
4
Clay (%)
6.4
Loam (%)
7.3
Sand (%)
86.3
PH @Cl)
7.0
PH (HZ~)
7.8
* Olsen et ul. (1954)
At the time of transplanting inoculations were carried out as follows. In the first
treatment (R) each seedling was inoculated with 1 ml of a Rhizobium culture, strain
0R.S 407 containing 10’ cells/ml. In the second (RM), each seedling was dually
inoculated with Rhizobium as above and 10 ml of an inoculum of Glomus mosseae
(Nicol. & Gerd.) Gerdemann & Trappe. This inoculum was prepared by blending c.
100 g (fresh weight) of infected mots, hyphae and spores from mycorrhizal and non-
nodulated cowpea in 11 sterile water. Ail control pots (R) each received 1 ml filtered
washings from the mycorrhizal inoculum in order to ensure that they also received
contaminating micro-organisms but not mycorrhiza. Each treatment was replicated 50
times. During the experiment each pot was watered when necessary and received 100
ml of Hewitt solution once a week (Hewitt 196i).
Ten successive harvests of five replicates of each treatment started five days after
planting and cottinued thereafter at five-day intervals. At each harvest nodulated
mots were assayed for nitrogenase activity using the acetylene reduction method
(Hardy et al. 1%8). Shoot water content was determined. Nodule and shoot dry
weights were obtained by drying to constant weight at 80°C. Total N and P contents of
the dry ground shoots from each treatment were estimated by the micro-Kjeldhal and
the vanadomolybdate (Jackson 1964) methods respectively. Root samples were
prepared for infection assessment by clearing and staining with Trypan Blue in
lactophenol. Frequency (percentage of infected mot pieces 0.3 cm in length) and
intensity (percentage of infected mot volume) were then assessed according to
Ollivier et af. (1982). The length of extra-radical. hyphae per cm of root was estimated
as follows. The entire mot system of each plant was gently washed from the soil by
dipping the mots in water, and randomly selected samples of mots (c. 50 cm in total
length) were then stained with Trypan Blue as described above. Al1 the extra-radical
hyplhae attached to mots were collected under a dissecting microscope and
/
Extra-radical mycorrhizal hyphae 77
,
:
homogenized in 5 ml water at high speed.The:n five 25 ~1 amounts were removed and
dispersed evenly in a thin layer of 1.5% sterile water agar in a Petri dish. When the
liquid was completely absorbed, the agar la’yer bearing the mycelium from a 25 ~1
volume was evaluated using an eyepiece micrometer and expressed as cm of hyphae
per cm of root. Means and standard errors were calculated from five replicates.
Results
The mycorrhizal infection of cowpea, the amount of extra-radical hyphae of Clornus
mosseae and the effect of mycorrhizal inoculation on shoot and nodule development
during the whole period of the experiment arle represented in Table 2. In the tropical
climate of Senegal, infection of cowpea roots by Glomus mosseae started as early as
day 10 after inoculation. Five days later, the infection frequency reached a high level
(87%:) which did not differ significantly from that obtained at the end of the
experiment. The development of Glomus mosseae within the root (infection intensity)
was also rapid and dramatically increased when plants were 15 days old. However,
increase in the development of intra-radical hyphae seemed to be progressive and
reached the maximum level only when plants were 35 days old (infection intensity:
70%) *
The growth of extraradical hyphae started only when the cowpea root system was
Table 2 Shoot weight, nodu!e weight and myconrhizal infection of
unguiculuta cv. 58-
185 inoculated with Rhizobium alone or with Rhizobium plus Glomus mosseae and cultivated in
sterile Bambey Dek soil
Shoot weight cglplant) Nodule
Extra-
Intraradical infection (%)
dry weight radical
Days Treatment Fresh
Dry
(mg/plant) development*
Frequency Intensity
-
-
5
RM
1.70 a b
0.15 a
0
n d
0
0
1.45 a
0.18 a
n d
0
_’ . .
. . . . .
10 R
3.72 a b c 0.40 a b
i
n d
ii
0
..
:RM
3.68 a b c 0.43 a b
0
n d
39 a
6 a
15 RM
7.23
6.55 cde
bal 0.92
0.89 abc
abc
5
6 a
n d
8; b
4 : b
20 R
8.38 d e
1.33 c d
6 a
0
&b
0
R M
8.23 cde
1.15 bc
35 lx
2.80 a
42 b
25
R
11.80 ef
2.13 d
15 a b
!lO
;b
0
R M
15.88 fg
2.05 d
97 d e
b
42 b
30
R
18.65 g h
3.33 ef
42 bc
nd
;b
0
R M
21.85 hi
3.03 e
121 ef
n d
53 b
35 R
17.90 gh
3.13 ef
33 b c
ii10
0
0
R M
215.78 j
3.93 fg
140 ef
b
100 b
70 c
40 R
26.15 ij
4.48 g
63 c d
nd
0
0
RM
30.08 j
5.28 h
158 f
n d
100 b
70 c
45 R
20.50 gh
3.65 ef
60 bcd
0
1;
0
R M
36.43 k
6.43 i
120 ef
6.07 b
b
75 c
50 R
27.03 j
5.75 hi
55 bed
n d
0
0
RM
40.28 k
8.05 j
121 ef
n d
100 b
75 c
R, plants inoculated with Rhizobium strain ORS407
RM, plants inoculated with Rhizobium strain ORS407 and G10mu.s mosseue
nd, not determined; *, estimated as cm of hyphae per cm of mot
Values followed by the same letter in each cohunn do not differ significantly (P=O.Ol) (Duncan 1955)
78 M. Gueye, H. G. Diem and Y. R. Dommergues
well infected but was then very rapid between clay 20 and 25 (Table 2). The amount of
exkaradical hyphae obtained at day 25 was not significantly different from that
obtained at the end of the experiment. By this time (day W), the beneficial effect of
GZomus mosseae inoculation essentially concerned the nodulation, which is shown by
the significant difference between nodule weight of mycorrhizal and non-mycorrhizd
plants. The significant increase in shoot weighlt by Glomus mosseae inoculation was
obrserved only when plants were 35-40 days old.
Figure 1 shows that shoot water content in non-mycorrhizal plants dramatically
decreased at the early stage of cowpea growth to c. 5% at day 25 and remained
constant until the end of the growth cycle. Water content in mycorrhizal plants was
relatively constant during the actively growing stage of cowpea and only progressively
decreased as the plants became older.
4
5
15
25
35
45 52
Days after planting
Fig. 1 Time course of water content of shoots in mycorrhizal (Q-0) and non-mycorrhiil (M) plants
during the growth cycle of Vigna unguiculafa.
The acetylene reduction activity (ARA) per plant (Fig. 2) reflected the nodule dry
weight pattem with high activity for mycorrhizal plants and low activity of non-
mycorrhizal plants, Values of ARA per plant decreased after day 40 probably because
of nodule degeneration as suggested by low Va:lues of specific ARA (Fig. 3) found in
this period.
Variations of N and P contents in shoot tissues during the growth cycle of cowpea
are indicated in Figs 4 and 5 respectively. Surprisingly, N content was similar in
mycorrhizal plants and non-mycorrhizal plants. Time course of P content in
mycorrhizal plants was quite different from that of non-mycorrhizal plants. Figure 5
shows that shoot P content in the two treatments progressively declined up to day 20.
After this critical period, there was a rapid incrlease of P concentration in mycorrhizal
plants whereas P concentration in non-mycorrhizal plants remained constant.
Phtrsphorus concentration in mycorrhizal plants increased up to the initial value at day
25 ;and then slightly decreased again when the plants reached the second half of the
experiment. In spite of this decline, P concentration in mycorrhizal plants was still
Extra-radical mycorrhizal hyphae 79
Day!; after planting
FIg 2. Tiie course of acetylene reduction activity per plant (ARAIplant) in mycorrhizal (O-0) and nan-
mycorrhizal (M) plants during the growth cycle of Vigna unguiculata.
twice as high as that in non-mycorrhizal ornes during a long period preceding the end
of the experiment (Fig. 5).
DISCWSiOll
Mycorrhizal infection and growth of hyphlae
In many studies, mycorrhizal infection is assessed only at the end of the experiment,
generally two months or more after planting. Such late observation indicates only the
susceptibility of a given host plant to mycorrhizal infection after a long period in
contact with mycorrhizal inoculum. Many authors (Rich & Bird 1974; Yost & Fox
1979; Abbott & Robson 1981; Gam-y et al. 1985) have already emphasized the need to
assess mycorrhizal infection at the early s.tage of plant growth. Taking into account
,_
-‘_.
.
. .
,’
5
15
2.5
3 5
45
52
Days after planting
Fig. 3 ‘lïme course of aœtylcne reduction activity per g (dry weight) of nodule (specific ARA) in
mycorrhizal (0-O) and non-mycorrhizal (H) plants during the gowth cycle of Vigna unguicdata.
80 M. Gueye, H. G. Diem and Y. R. Dommergues
h 4-
aeë9> 3.
ë8
z 2.
c
1
3
15
25
35
45
52
Days after planting
Fig. 4 Tûne course of nitrogen content in mycorrhizal (O-0) and non-mycorrhizal (M) plants dmiog
the g;rowth cycle of Vigna unguicufata.
,,:
“’
:
I
’
*
-
-
-
’
*
.
-
1
0
5
15
25
3.5
45
52
Days after planting
Fig. 5 Tiie course of phosphorus content in mycorrhizal(O-0) and non-mycorrbizal (0-W’ plants ‘.
during the growth cycle of V@a unguicti.
these recommendations, we have periodically examined the course of mycorrhizal
formation from the beginning of the experiment, as well as the progressive
development of extra-radical hyphae of this fungus in the soil.
Establishment of Clornus mosseae in cowpea roots occurred c. 10 days after
inoculation. By this time 38% of observed root pieces were already infected,
confirming the observation that under tropical conditions, mycorrhizal infection of
trop legumes is rapid (Germani et al. 1980; Ganry et al. 1985). Smith & Bowen (1979)
attributed the rapid infection of legume roots by mycorrhizal fungi to the effect of high
temperature (25°C) which favoured early mycorrhizal infection, and which may be
important in the successful exploitation of VA mycorrhizae. We have also considered
early mycorrhizal infection as a prerequisite for the successful inoculation of field-
grown trop plants with introduced VA mycorrhizal fungi (Gant-y et al. 1985), and we
inferred from this experiment that uninoculated plants became mycorrhizal later than
inoculated plants because of the sparse indigen0u.s VA mycorrhizal population in the
field. Table 2 shows that infection frequency (percentages of root pieces infected)
rapidly increased to its highest values as soon as day 25. We assume that thii
parameter gives some information on the spread of mycorrhizal infection in the total
Extra-radical mycorrhizal hyphae 81
root system but is not correlated with the: effect of G1omu.s mosseae on plant
responses.
Spread of mycorrhizal infection in the root system is generally estimated by the
percentage of root length infected using the line intercept method (Ambler & Young
1977; Giovanetti & Mosse 1980). However, Kucey & Paul (1982) found that this
percentage does not give a good indication oA mycorrhizal biomass within a root; it
only indicates the proportion of the length of root system containing mycorrhizal
fungal structures. Although Sanders et al. (1977) found a direct relationship between
the weight of external mycorrhizal mycelium and the length of infected roots we
prefer to express the intensity of mycorrhizal infection by the percentage of root
volume infected, which probably reflects better the development of the fungus within
the ro’ot tissues (Ollivier et al. 1982). The variation pattern of mycorrhizal infection, as
indicated by intensity percentage, differs from that indicated by the frequency
percentage (Table 2); mycorrhizal infection intensity increased progressively and only
reached the plateau by the end of the experiment. This agrees with the findings of
Bethlenfalvay et al. (1982) which showed that the amount of intra-radical mycelium
increased throughout the life of the host plant.
Although the role of the extra-radical myt&lium is vital in exploring the soil and
increasing the rate of spread of infection (Sanders et al. 1977), few authors have
attempted to estimate the amount of extra-raIdical hyphae. The present work reports
that lengths as large as 5 cm (per cm of root) of extra-radical hyphae of Glomus
mosseae were attained rapidly when plants were well infected, as indicated by the
frequency and intensity of mycorrhizal infection at day 20 (Table 2). According to
Sanders et al. (1977), the amount of extra-radical hyphae of Glomus mosseae and
other mycorrhizal fungi was about 3.6 kg ((dry wt)/cm. If we convert our data to
biomass, assuming the hyphae are cylindrical and 1 hrn in diameter, and using a
conversion factor of 0.35 g/cm (Van Veen & :Paul 1979), the biomass of extra-radical
hyphae is c. 14.0 p,g/cm dry weight. T~US, u:nder the conditions of our experiment,
Glomus mosseae grew profusely in the rhizosphere of cowpea as plant host. As
already reported by Bethlenfalvay et al. (1982), we also found that the growth of
extra-radical hyphae stopped between day 25 and 35 while hyphae continued to
develop extensively within the roots. Growth of extra-radical hyphae of Gtomus
mosseae started to increase when mycorrhizall infection intensity was about 42% and
became stabilized when it was about 60% (Table 2). Our results are consistent with
those found previously in faba bean inoculated with the same fungus (Kucey & Paul
1982),. We do not know the reason of the early cessation of the rapid extra-radical
growth phase. Because extra-radical hyphae ,take up nutrients from the host tissues,
we hypothesize that their growth is strictly controlled by the host plant.
Effect on plant growth, nodulation and N&xation
The effect of inoculation with Gtomus mosseace on nodulation was particularly marked
from day 20. This seems to be related to the onset of growth of the extra-radical
hyphae (Table 2). Since they are probably stimulated by P supply from actively
growing extra-radical hyphae, mycorrhizal plants produced six times more nodule dry
matter than non-mycorrhizal plants. Stimulation of nodulation at day 20 may be
important in field situations, because annual plants cari derive maximum benefit from
N2 fixation at this growth stage. Even thouglh the difference in nodulation between
mycorrhizal and non-mycorrhizal plants decreased at the end of the experiment, the
82 M. Gueye, H. G. Diem and Y. R. Dommergues
former still produced twice as much nodule dry weight as the latter plants (Table 2).
NT-living activity, (ARA/plant) (Fig. 2), was clearly higher in mycorrhizal than in
non-mycorrhizal plants. The increase of Nz-fixing activity was relatively more rapid in
mycorrhizal than in non-mycorrhizal plants between day 15 and 25 since during this
period increase in nodule weight of mycorrhizal plants (6-97 mg) was also much more
rapid than that of non-mycorrhizal plants (5-15 mg). According to these data,.it is
clear that high Nz-fixing activity occurred as soon as plants were infected by VA fungi.
The influence of early high N@ring activity due to mycorrhizal inoculation on grain
yield of soybean has already been found and discussed by Ganry et al. (1985).
Noldulation as well as Nz-fixing activity seemed not to be correlated with the
development of intra-radical hyphae (Table 2). By contra&, nodule dry weight (Table
2) and ARA per plant (Fig. 2) dramatically increased when extra-radical hyphae
started to develop (Table 2). This was concomitant with the enhancement of P inflow
which occurred by this time (Fig. 5).
In our study, the stimulating effect of Glonzus mosseae on plant growth was
significant only 45 days after inoculation.The increase of shoot dry weight was about
77% at this time. The delay in plant growth response to mycorrhizal infection suggests
that large amounts of P taken up by Glomus moss.eae were first needed for nodulation
rather than for plant gowth.
Effet on shoot water content
It is generally recognized that mycorrhizae protect plants against long periods of water
stress by enhancing P absorption from SO~I (Levy & Krikun 1980; Safir et al. 1972;
Allen & Boosalis 1983; Sieverding 1984). The rapid decrease of water content in non:
mycorrhizal plants between day 5 and 25 (Fig. 1) suggests that they were greatly
deficient in P during their active growth phase whereas in mycorrhizal plants water
content remained steady during the same period. The water content of the shoots was
higher in mycorrhizal than in non-mycorrhizal plants, from day 20 to 35, a period.
when extra-radical hyphae developed profusely (Table 2), thus enhancing P infIow.
into the plant as suggested by higher P content in mycorrhizal plants (Fig. 5). It has
been suggested that drought resistance of mycorrhizal plants was due to a decreased
resistance of plant tissues to water flow and therefore an enhanced water transport
throughout the plant. Such an enhanced water transport was found to be mediated by
a better P nutrition in mycorrhizal plants (Safir er’ al. 1972). By contrast, P-deficient
plants are more susceptible to drought (Atkinson & Davison 1973).
Effect on N and P contents
Surprisingly, variation patterns of N content in non-mycorrhizal and mycorrhizal
plants were similar throughout the experiment (Fig. 4), but there were differences
between time courses of P content in mycorrhizal and non-mycorrhizal plants (Fig. 5).
During the early growth stage of mycorrhizal plants (up to day 20, Fig. 5) the marked
decrease of P content was due to the utilization of significant amounts of available P
for growth and nodulation and probably also to the competition for P by the fungal
endophyte, thus creating a transitory sink for P. This would explain the significant
lower P content in mycorrhizal plants compared to that in non-mycorrhizal plants at
day 20, (Fig. 5). Bethlenfalvay et al. (1982) also found that shoot P content in the
controlls was significantly higher than in mycorrhizal plants during the first six weeks of
growth of soybean. However, Fig. 5 shows that the supposed sink for P in mycorrhiil
<. -2’
.::
: . 2, . ,
.
.
1 * ”
Extra-radical mycorrhizal hyphae 83
plants was subsequently and rapidly compensated by enhanced P uptake resulting
from the development of extra-radical hyphae. This suggestion is supported by the
marked increase of P content in mycorrhizal plants after day 20 (Fig. 5). During the
following growth phase of cowpea, P content in mycorrhizal plants was always
significantly higher than that in non-mycorrhizal plants, which constantly remained
low from day 25. Such P content pattems in mycorrhizal or non-mycorrhizal plants
were obtained mathematically in a simulation study by Sanders & Sheikh (1983).
In conclusion, the time course of P content in cowpeas consisted of three phases: (1) a
decaeasing critical phase prior to the devellopment of extra-radical hyphae between
day 5 and 20; (2) a P enrichment phase following the development of extra-radical
hyphae hetween day 20 and 25; (3) a slightly decreasing phase which was then
siabilized as the plant matured. Such a three-phase pattem of P percentage in
mycorrhizal plants has already been reported by Snellgrove et al. (1982) in the case of
Allium porrum.
Acknowledgement
We thank Moussa Niang for valuable technical assistance.
References
ABWIT, L. K. & ROBSON, A. D. 1981 Infectîvity and effectiveness of vesicular-arbuscular
my&rbizal fungkeffect of inoculum type. Australian Journal of Agricultural Research 32,
631-639.
ALLEN, M. F. & Boosa~~s, M. G. 1983 Effects of two species of VA mycorrhizal fungi on
drought tolerance of winter wheat. New Ph~~tologist 93, 67-76.
AM%ER, J. R. B YOUNG, J. L. 1977 Techniques for determining mot length infected by
vesicular-arbuscular mycorrbizae. Soil Science Society of America Journal 41,551-556.
ATKINSON, D. B DAMSON, A. W. 1973 Tbe effects of phosphorus deficiency on water content
and response to drought. New Phytologkt 72, 307-313.
BAGYARM, D. J. L MANJUNATH, A. 1980 Response of trop plants to VA mycorrhizd inoculation
in an unsterile Indian soil. New Phytologist !85, 33-36.
BEWLENFALVAY, G. J., PA~~VSKY, R. S. B :BROWN, M. S. 1982 Parasitic and mutualistic
associations between a mycorrhizal fungus and soybean: development of the endophyte.
Phytopathology 72,854-897.
DUNCAN, D. B. 1955 Multiple range and multiple F tests. Biomezrks 11, l-42.
GANRY, F., DIEM, H. G., WEY, J. & DOMMERGUES, Y. 1985 Inoculation with Glomus mosseae
improves Nz fixation by field-grown soybea~ Biology and Fertiliry of Soils 1, 15-23.
GEXMANI, G,, DIPM, H. G. a D~MM~RCUES, Y. 1980 Muenœ of 1,2dibromo-3-chlore-propane
(DBCP) on mycorrhizal infection of field-grown groundnut. In Tropical Mycorrhiza
Research, ed. Mikola, P. pp. 245-246. Oxford: Clarendon Press.
GIOVANEITI, M. a Moss~, B. 1980 An evaluation of techniques for measuring vesicular
arbuscular mycorrbizal infection in roots. New Phytologist 84,489-500.
GUEYE, M. 1983 Vigna unguiculata en symbiose avec Rhizobium et Glomus mosseae. Th&e
Doctorat 3& cycle, p. 150, ORSTOM, Paris.
HARDY, R. W. F., HOLSTEN, R. D., JACKSON, E. K. a BURNS, R. C. 1968 Tbe aœtylene assay
for Nz fixation: laboratory and field evaluation. Pfant Physiology 43, 1185-1207.
Hmvrrr, E. J. 1966 Sand and Watcr Culture Methods used in the Study of Pkmt Nutrition.
Technical communication No. 22 Second edn. London: Commonwealth Agricultural Bureau.
~SIAM, R., A~MABA, A. ct !~ANDE~S, F. E. 1980 Response of cowpea (Vigna unguiculata) to
inoculation with VA mycorrbizal fungi ,and to rock phosphate fertilization in some
unsteriliid Nigerian soils. Planf and Soi1 $8, 107-117.
JACKSON, M. L. 1964 Soil Chcmical Analyslr, Englewood Cliffs, N.J.: Prentiœ Hall.
KUCEY, R. M. N. & PAUL, E. A. 1982 Biomass of mycorrhizal fur@ associated with bean mots.
Soil Biology and Biochetitry 14,413-414.
84 M. Gueye, H. G. Diem and Y. R. Dommergues
LEW, 1. 6r KRIKUN, J. 1980 Effect of vesicular-arbuscular mycorrhiza in Citrus jumbhiri water
relations. New Phytologist 85, 25-32.
OLLMBR, B., BERTHEAU, Y. DIEM, H. G. dr GIANINAZZI-PEARSON, V. 1982 Influence
de la variété de Vigna unguiculatu L. Walp dans l’expression de trois associations
endomycorhiziennes à vesicules et arbuscules. Canadian Journal of Botany 61, 354-358.
OLLM~R, B., DIEM, H. G., PINTA, M. d DOMMERGUE-S, Y. R. 1984 Effect of endomycorrhizae
on the concentration of P and Zn in Vigna unguiculata shoots. Agrochimica 28, 341-352.
OLSEN, S. R., &LE, L. V., WATANABE, F. S. dr DEAN, L. A. 1954 Estimation of available
phosphorus in soils by extraction with sodium bicarbonate. Circular of Vnited s’tates
Department of Agriculture No. 939.
Rtcrr, .J. R. dr BIRD, G. W. 1974 Association of early season vesicular mycorrhizae with
increased growth and development of cotton. Phytopathology 64, 1421-1425.
SAFIR, G. R., BOYER, J. S. dr GERDEMANN, J. W. 11972 Nutrient status and mycorrhii
enhancement of water transport in soybeans. Plant physiology 49,700-703.
SANDEKS, F. E. & SHEIKH, N. A. 1983 The development of vesicular-arbuscular mycorrhizal
infection in plant root systems. Plant and Soi1 71, 223-246.
SANDERS, F. E., TINKER, P. B., BLACK, R. L. B. 6r PALI~ERLY, S. M. 1977 The development of
mycorrhizal root systems: 1. Spread of infection and growth promoting effects with four
species of vesiculararbuscular endophyte. New Phytologist 78, 257-268.
SIEVERDING, E. 1984 Influence of soi1 water regimes on VA mycorrhiza. 3. Comparison of 3
mycorrhizal fungi and their influence on transpiration. Zeitschrift fiir Acker und Pflanzenbau
153,52-61.
SMKH, S. E. dr BOWEN G. D. 1979 Soi1 temperature, mycorrhizal infection and nodulation of
Medicago truncatula and Trifolium subterraneum. Soi1 Biology and Biochemistry 11, 469-
473.
SNELLCIROVE,
R. C., SPLITETOESSER, W. E. STRIBLE~, D. P. a TINKER, P. B. 1982 The
distribution of carbon and the demand of the fungal symbiont in leek plants with vesicular-
arbuscular mycorrhizas. New Phytologist 92, 75-87.
VAN VEEN, J. A. 6r PAUL, E. A. 1979 Conversion of biovolume measurements of soil organisms,
grown under various moisture tensions, to biomass and their nutrient content. Applied ami
Environmental Microbiology 37, 686-692.
Yosr, R. S. dr Fox, R. L. 1979 Contribution of mywrrhizae to P nutrition of crops growing on
an oxisol. Agronomy Journal 71,903~908.
Summary
Vigne unguiculata cv. 58-185 grown in a sterile Dek soi1 was inoculated with Rhizobium sp. or
Rhizobium sp. plus Glomus mosseae. Response of the host plant to the treatments was
estimated by periodic measurements of shoot and nodule dry weights, Nz fixation (%Hz
reduction activity) and N and P contents up to the 50th day of the growth cycle. It was only 45
days after planting that shoot dry weight of dually inoculated plants differed significantly from
that of plants inoculated with Rhizobium sp. alone. Nodule dry weight and Nz fixation of dually
inoculated plants were significantly higher than those of plants inoculated with Rhizobium sp.
alone from day 20 after planting, but there was no significant difference in N content (%).
During the first 20 days, shoot P content (%) of both sets of plants decreased progressively, P
content of dually inoculated plants being lower than that of the others. Later, P content of
dually inoculated plants increased rapidly whereas P content of the other plants remained
constant. Increase in nodule dry weight, Nz fixation and P content of dually inoculated plants
wrresponded to the onset of the development of the extra-radical hyphae of Glomus mosseue in
the rhizosphere.
Résumé
Variation dans fa fixation de Nz, les teneurs en N et P chez Vigna unguic.ulata
mycorhizé en relation avec le développement progressif des hyphes extraradica!es de
Glornus mosseae
On a inoculé V. unguiculata poussant dans un sol Dek stérile avec Rhizobium et Rhizobium
plus Glomus mosseae. On a recherché la réponse de la plante-hôte à ces deux traitements en
Extra-radical mycorrhizal hyphae 85
ic
j,a*nbhiri w a t e r
estimant périodiquement les poids des nodules et des parties a&iennes de la plante, la fixation
d’azote (activite réductrice de &Hz), les teneurs en N et P jusqu’au 50” jour du cycle de
MN.. V. 1982 Influence
végetation. C’est seulement au 45’jour aprés la plantation que le poids sec des parties adriennes
)n cle trois associations
des plantes inoculées avec deux symbiotes (plantes doublement inoculées) diffère significative-
f Botany 61., 354-3.58.
ment de celui des plantes inoculées avec Rhizobium seul. Le poids sec des nodules et la fixation
ffect of endomycorrhizae
NZ des plantes doublement inoculées sont significativement plus éleves que ceux des plantes
rochimica 23, 341-352.
inoculées avec Rhizobium seul au 20” jour aprts la plantation mais il n’y a pas de différence
1 Estimation of available
significative pour la teneur en N (%). Pendant les 20 premiers jours, la teneur en P (X) des
ïrcular of United States
parties aériennes des deux catégories de plantes décroit progressivement; la teneur en P des
plantes doublement inoculees est plus faible que celle des plantes inoculees seulement avec
jicular mycorrhizae with
Rhizobium. Plus tard, la teneur en P des plantes doublement inoculees augmente rapidement
1421-1425.
tandis que celle des autres plantes reste constante. L’accroissement du poids sec des nodules, de
status and mycorrhizal
la fixation d’azote et de la teneur en P observe chez les plantes doublement inoculées
700-703.
correspond au démarrage du developpement des hyphes extra-radicales de Clornus mosseae
ir-arbuscular mycorrhizal
dans la rhizos.phére.
1977 The development of
Resumen
moting effects with four
Variacibn en la jijacih de N y en lus contenidos de ,Y y P de Vigna unguiculata
l-.268.
niza. 3. Comparison of 3
micorrizada en relacidn con el desarrrollo progresivo de las hifas extra radiculares de
ir Acker und PfIanzenbau
Glomus mosseae
Se cultiv6 V@a unguiculata cv. 58-185 en un suelo ester-il tipo Dek, se inocul6 con Rhizobium
Bction and nodulation of
sp. o con Rhizobium sp. mas Glomus mosseae. La respuesta de la planta huésped a 10s
MI Biochemistry 11, 469-
tratamientos se estudi6 midiendo periodicamente el peso s~cco de la parte aerea y de 10s
nodules, la fijaci6n de N (actividad reductora de CzHz) y 10s contenidos de N y P hasta el50“ dfa
‘INKER, P. H. 1982 The
del ciclo de crecimiento. La diferencia entre el peso seco de 1.a parte aerea de las plantas con
X:L~ plants with vesicular-
doble inoculaci6n y aquellas inoculadas con Rhizobium sp. unicamente, no fue significativa
hasta 45 dias desptiés de la siembra. A 10s 20 dias de la siembra tanto el peso seco de 10s nodules
ements of soi1 organisms,
como la fijaci6n de nitr6geno de las plantas con doble inoculation cran significativamente
ent content. Applied and
superiores a 10s valores obtenidos para las plantas con solo Rhizobium SP., aunque no se
observaron diferencias en el contenido en N (%). Durante 110s primeros 20 dfas del ciclo el
ition of crops growing on
contenido en P (%) de ambos grupos de plantas disminuy6 progresivamente, siendo 10s valores
obtenidos por las plantas con doble inoculaci6n inferiores a 10s de las dem& M& tarde el
contenido en P de las plantas con doble inoculation aument rapidamente manteniendose
constante el dle las demas. El incremento en el peso seco de 10s nodules, en la fijaci6n de N y en
rd with Rhitobium sp. or
el contenido en P de las plantas con doble inoculation se correspondi6 con el inicio del
t to the treatments was
desarrollo de las hifas extraradiculares de Glomus mosseae.
ights, Nz fixation (C?H,
nvth cycle. It was only 45
fiffered significantly from
t and Nz fixation of dually
lated with Rhizobium sp.
rente in N content (%).
ecreased progressively, P
rers. Later, P content of
e other plants remained
1 ldually inoculated plants
hae of Glomus mosseae in
ICZ Vigna unguiculata
vphts extruradicales de
R.hirobium et Rhizobium
i ces deux traitements en
‘Variation in N2 fixation, N and P contents of
mycorrhizal Vigna unguiculata in relation to
the progressive development of extra-
radical hyphae of Glomus mosseae
M. Gueye*, H. G. Diemt & Y. R. Dommergues+
* MIl$CENKXRA,
B.P. 53, Bambey, Sertegal, and tluborrltoire BSSFT (CTFTIORSTOMJCNRS)
45 bis,
Avenue de la Belle Gabrielle, 94736 Nogent sur Mante, France
Received 10 February 1986; revised and accepted 31 October 1986
Iutroduction
Earlier studies have shown that field-grown cowpea (V@a unguiculutu) is dependent
on vesicular-arbuscular (VA) mycorrhizae for growth and phosphorus (P) uptake
(Yost & Fox 1979). ‘I’he VA mycorrhizal dependence of cowpea has been further
confirmed experimentally by Bagyaraj & Manjunath (1980) and Islam et al. (1980)
even when using non-sterile soil. It is believed that the improved growth of
mycorrhizal cowpea is related to increased absorption of some minera1 nutrients,
especially P and Zn (Bagyaraj & Manjunath 1980; Gueye 1983; Ollivier et uf. 1984).
In their experiment with Vignu zmguicdutu inoculated with different species of VA
‘<
fungi and given different forms of phosphate, Ollivier et al. (1982) showed that the
increase of P concentration in mycorrhizal plants varied significantly with the fungal
species and forms of phosphate used. Yost Bt Fox (1979) studied the P content of
cowpea leaves as affected by soil P status and showed that cowpea was dependent on
VA mycorrhixae for P uptake over a wide range of soi1 P contents,
Information is available on the beneficial effect of VA mycorrhizae on plant growth
in connection with P fertilization but very little is known about the time course of P
uptake during the growth cycle of cowpea. Furthermore, although it is well
established that the increase of P uptake in mycorrhizal plants is due mainly to the
better exploration of soil by extra-radical mycorrhiial hyphae, no work has yet been
done to assess the development of extra-radical mycorrhixal hyphae in the cowpea
rhiiosphere .
This work was initiated to determine the relation between the development on
.
‘ .
interna1 and extra-radical mycorrhizal hyphae and nodule and shoot dry weights, N
and P concentrations, shoot water content and Nz fixation (&Hz reduction activity) in
mycorrhizal cowpeas. Comparison of these characteristics with those of non-
@ Oxford University Press 1987
76 M. Gueye, H. G. Diem and Y. R. Dommerpes
mytorrhizal plants during the plant growth cycle should improve our knowledge of the
VA mycorrhizal symbiosis in cowpea.
MateriaIs and methods
Surface sterilized seeds of cowpea (cv. 58-185 with growth cycle of !XI days) were
genminated in sterile sand for two days. Each germinated seed was then transplanted
to a. pot containing 1.5 kg of autoclaved soil from the Centre National de la Recherche
Agricole in Bambey (psamment; vemacular name of soi1 Dek; see Table 1).
Table 1 Characterlstics of Bambey Dek soi1
Total C (%)
0.51
Total N (%)
0.03
Total P (mg P/kg)
74
Available P (O&n;* mg P/kg)
4
Clay (%)
6.4
Loam (%)
7.3
Sand (%)
86.3
PH @Cl)
7.0
PH (HZ~)
7.8
* Olsen et ul. (1954)
At the time of transplanting inoculations were carried out as follows. In the first
treatment (R) each seedling was inoculated with 1 ml of a Rhizobium culture, strain
0R.S 407 containing 10’ cells/ml. In the second (RM), each seedling was dually
inoculated with Rhizobium as above and 10 ml of an inoculum of Glomus mosseae
(Nicol. & Gerd.) Gerdemann & Trappe. This inoculum was prepared by blending c.
100 g (fresh weight) of infected mots, hyphae and spores from mycorrhizal and non-
nodulated cowpea in 11 sterile water. Ail control pots (R) each received 1 ml filtered
washings from the mycorrhizal inoculum in order to ensure that they also received
contaminating micro-organisms but not mycorrhiza. Each treatment was replicated 50
times. During the experiment each pot was watered when necessary and received 100
ml of Hewitt solution once a week (Hewitt 196i).
Ten successive harvests of five replicates of each treatment started five days after
planting and cottinued thereafter at five-day intervals. At each harvest nodulated
mots were assayed for nitrogenase activity using the acetylene reduction method
(Hardy et al. 1%8). Shoot water content was determined. Nodule and shoot dry
weights were obtained by drying to constant weight at 80°C. Total N and P contents of
the dry ground shoots from each treatment were estimated by the micro-Kjeldhal and
the vanadomolybdate (Jackson 1964) methods respectively. Root samples were
prepared for infection assessment by clearing and staining with Trypan Blue in
lactophenol. Frequency (percentage of infected mot pieces 0.3 cm in length) and
intensity (percentage of infected mot volume) were then assessed according to
Ollivier et af. (1982). The length of extra-radical. hyphae per cm of root was estimated
as follows. The entire mot system of each plant was gently washed from the soil by
dipping the mots in water, and randomly selected samples of mots (c. 50 cm in total
length) were then stained with Trypan Blue as described above. Al1 the extra-radical
hyplhae attached to mots were collected under a dissecting microscope and
/
Extra-radical mycorrhizal hyphae 77
,
:
homogenized in 5 ml water at high speed.The:n five 25 ~1 amounts were removed and
dispersed evenly in a thin layer of 1.5% sterile water agar in a Petri dish. When the
liquid was completely absorbed, the agar la’yer bearing the mycelium from a 25 ~1
volume was evaluated using an eyepiece micrometer and expressed as cm of hyphae
per cm of root. Means and standard errors were calculated from five replicates.
Results
The mycorrhizal infection of cowpea, the amount of extra-radical hyphae of Clornus
mosseae and the effect of mycorrhizal inoculation on shoot and nodule development
during the whole period of the experiment arle represented in Table 2. In the tropical
climate of Senegal, infection of cowpea roots by Glomus mosseae started as early as
day 10 after inoculation. Five days later, the infection frequency reached a high level
(87%:) which did not differ significantly from that obtained at the end of the
experiment. The development of Glomus mosseae within the root (infection intensity)
was also rapid and dramatically increased when plants were 15 days old. However,
increase in the development of intra-radical hyphae seemed to be progressive and
reached the maximum level only when plants were 35 days old (infection intensity:
70%) *
The growth of extraradical hyphae started only when the cowpea root system was
Table 2 Shoot weight, nodu!e weight and myconrhizal infection of
unguiculuta cv. 58-
185 inoculated with Rhizobium alone or with Rhizobium plus Glomus mosseae and cultivated in
sterile Bambey Dek soil
Shoot weight cglplant) Nodule
Extra-
Intraradical infection (%)
dry weight radical
Days Treatment Fresh
Dry
(mg/plant) development*
Frequency Intensity
-
-
5
RM
1.70 a b
0.15 a
0
n d
0
0
1.45 a
0.18 a
n d
0
_’ . .
. . . . .
10 R
3.72 a b c 0.40 a b
i
n d
ii
0
..
:RM
3.68 a b c 0.43 a b
0
n d
39 a
6 a
15 RM
7.23
6.55 cde
bal 0.92
0.89 abc
abc
5
6 a
n d
8; b
4 : b
20 R
8.38 d e
1.33 c d
6 a
0
&b
0
R M
8.23 cde
1.15 bc
35 lx
2.80 a
42 b
25
R
11.80 ef
2.13 d
15 a b
!lO
;b
0
R M
15.88 fg
2.05 d
97 d e
b
42 b
30
R
18.65 g h
3.33 ef
42 bc
nd
;b
0
R M
21.85 hi
3.03 e
121 ef
n d
53 b
35 R
17.90 gh
3.13 ef
33 b c
ii10
0
0
R M
215.78 j
3.93 fg
140 ef
b
100 b
70 c
40 R
26.15 ij
4.48 g
63 c d
nd
0
0
RM
30.08 j
5.28 h
158 f
n d
100 b
70 c
45 R
20.50 gh
3.65 ef
60 bcd
0
1;
0
R M
36.43 k
6.43 i
120 ef
6.07 b
b
75 c
50 R
27.03 j
5.75 hi
55 bed
n d
0
0
RM
40.28 k
8.05 j
121 ef
n d
100 b
75 c
R, plants inoculated with Rhizobium strain ORS407
RM, plants inoculated with Rhizobium strain ORS407 and G10mu.s mosseue
nd, not determined; *, estimated as cm of hyphae per cm of mot
Values followed by the same letter in each cohunn do not differ significantly (P=O.Ol) (Duncan 1955)
78 M. Gueye, H. G. Diem and Y. R. Dommergues
well infected but was then very rapid between clay 20 and 25 (Table 2). The amount of
exkaradical hyphae obtained at day 25 was not significantly different from that
obtained at the end of the experiment. By this time (day W), the beneficial effect of
GZomus mosseae inoculation essentially concerned the nodulation, which is shown by
the significant difference between nodule weight of mycorrhizal and non-mycorrhizd
plants. The significant increase in shoot weighlt by Glomus mosseae inoculation was
obrserved only when plants were 35-40 days old.
Figure 1 shows that shoot water content in non-mycorrhizal plants dramatically
decreased at the early stage of cowpea growth to c. 5% at day 25 and remained
constant until the end of the growth cycle. Water content in mycorrhizal plants was
relatively constant during the actively growing stage of cowpea and only progressively
decreased as the plants became older.
4
5
15
25
35
45 52
Days after planting
Fig. 1 Time course of water content of shoots in mycorrhizal (Q-0) and non-mycorrhiil (M) plants
during the growth cycle of Vigna unguiculafa.
The acetylene reduction activity (ARA) per plant (Fig. 2) reflected the nodule dry
weight pattem with high activity for mycorrhizal plants and low activity of non-
mycorrhizal plants, Values of ARA per plant decreased after day 40 probably because
of nodule degeneration as suggested by low Va:lues of specific ARA (Fig. 3) found in
this period.
Variations of N and P contents in shoot tissues during the growth cycle of cowpea
are indicated in Figs 4 and 5 respectively. Surprisingly, N content was similar in
mycorrhizal plants and non-mycorrhizal plants. Time course of P content in
mycorrhizal plants was quite different from that of non-mycorrhizal plants. Figure 5
shows that shoot P content in the two treatments progressively declined up to day 20.
After this critical period, there was a rapid incrlease of P concentration in mycorrhizal
plants whereas P concentration in non-mycorrhizal plants remained constant.
Phtrsphorus concentration in mycorrhizal plants increased up to the initial value at day
25 ;and then slightly decreased again when the plants reached the second half of the
experiment. In spite of this decline, P concentration in mycorrhizal plants was still
Extra-radical mycorrhizal hyphae 79
Day!; after planting
FIg 2. Tiie course of acetylene reduction activity per plant (ARAIplant) in mycorrhizal (O-0) and nan-
mycorrhizal (M) plants during the growth cycle of Vigna unguiculata.
twice as high as that in non-mycorrhizal ornes during a long period preceding the end
of the experiment (Fig. 5).
DISCWSiOll
Mycorrhizal infection and growth of hyphlae
In many studies, mycorrhizal infection is assessed only at the end of the experiment,
generally two months or more after planting. Such late observation indicates only the
susceptibility of a given host plant to mycorrhizal infection after a long period in
contact with mycorrhizal inoculum. Many authors (Rich & Bird 1974; Yost & Fox
1979; Abbott & Robson 1981; Gam-y et al. 1985) have already emphasized the need to
assess mycorrhizal infection at the early s.tage of plant growth. Taking into account
,_
-‘_.
.
. .
,’
5
15
2.5
3 5
45
52
Days after planting
Fig. 3 ‘lïme course of aœtylcne reduction activity per g (dry weight) of nodule (specific ARA) in
mycorrhizal (0-O) and non-mycorrhizal (H) plants during the gowth cycle of Vigna unguicdata.
80 M. Gueye, H. G. Diem and Y. R. Dommergues
h 4-
aeë9> 3.
ë8
z 2.
c
1
3
15
25
35
45
52
Days after planting
Fig. 4 Tûne course of nitrogen content in mycorrhizal (O-0) and non-mycorrhizal (M) plants dmiog
the g;rowth cycle of Vigna unguicufata.
,,:
“’
:
I
’
*
-
-
-
’
*
.
-
1
0
5
15
25
3.5
45
52
Days after planting
Fig. 5 Tiie course of phosphorus content in mycorrhizal(O-0) and non-mycorrbizal (0-W’ plants ‘.
during the growth cycle of V@a unguicti.
these recommendations, we have periodically examined the course of mycorrhizal
formation from the beginning of the experiment, as well as the progressive
development of extra-radical hyphae of this fungus in the soil.
Establishment of Clornus mosseae in cowpea roots occurred c. 10 days after
inoculation. By this time 38% of observed root pieces were already infected,
confirming the observation that under tropical conditions, mycorrhizal infection of
trop legumes is rapid (Germani et al. 1980; Ganry et al. 1985). Smith & Bowen (1979)
attributed the rapid infection of legume roots by mycorrhizal fungi to the effect of high
temperature (25°C) which favoured early mycorrhizal infection, and which may be
important in the successful exploitation of VA mycorrhizae. We have also considered
early mycorrhizal infection as a prerequisite for the successful inoculation of field-
grown trop plants with introduced VA mycorrhizal fungi (Gant-y et al. 1985), and we
inferred from this experiment that uninoculated plants became mycorrhizal later than
inoculated plants because of the sparse indigen0u.s VA mycorrhizal population in the
field. Table 2 shows that infection frequency (percentages of root pieces infected)
rapidly increased to its highest values as soon as day 25. We assume that thii
parameter gives some information on the spread of mycorrhizal infection in the total
Extra-radical mycorrhizal hyphae 81
root system but is not correlated with the: effect of G1omu.s mosseae on plant
responses.
Spread of mycorrhizal infection in the root system is generally estimated by the
percentage of root length infected using the line intercept method (Ambler & Young
1977; Giovanetti & Mosse 1980). However, Kucey & Paul (1982) found that this
percentage does not give a good indication oA mycorrhizal biomass within a root; it
only indicates the proportion of the length of root system containing mycorrhizal
fungal structures. Although Sanders et al. (1977) found a direct relationship between
the weight of external mycorrhizal mycelium and the length of infected roots we
prefer to express the intensity of mycorrhizal infection by the percentage of root
volume infected, which probably reflects better the development of the fungus within
the ro’ot tissues (Ollivier et al. 1982). The variation pattern of mycorrhizal infection, as
indicated by intensity percentage, differs from that indicated by the frequency
percentage (Table 2); mycorrhizal infection intensity increased progressively and only
reached the plateau by the end of the experiment. This agrees with the findings of
Bethlenfalvay et al. (1982) which showed that the amount of intra-radical mycelium
increased throughout the life of the host plant.
Although the role of the extra-radical myt&lium is vital in exploring the soil and
increasing the rate of spread of infection (Sanders et al. 1977), few authors have
attempted to estimate the amount of extra-raIdical hyphae. The present work reports
that lengths as large as 5 cm (per cm of root) of extra-radical hyphae of Glomus
mosseae were attained rapidly when plants were well infected, as indicated by the
frequency and intensity of mycorrhizal infection at day 20 (Table 2). According to
Sanders et al. (1977), the amount of extra-radical hyphae of Glomus mosseae and
other mycorrhizal fungi was about 3.6 kg ((dry wt)/cm. If we convert our data to
biomass, assuming the hyphae are cylindrical and 1 hrn in diameter, and using a
conversion factor of 0.35 g/cm (Van Veen & :Paul 1979), the biomass of extra-radical
hyphae is c. 14.0 p,g/cm dry weight. T~US, u:nder the conditions of our experiment,
Glomus mosseae grew profusely in the rhizosphere of cowpea as plant host. As
already reported by Bethlenfalvay et al. (1982), we also found that the growth of
extra-radical hyphae stopped between day 25 and 35 while hyphae continued to
develop extensively within the roots. Growth of extra-radical hyphae of Gtomus
mosseae started to increase when mycorrhizall infection intensity was about 42% and
became stabilized when it was about 60% (Table 2). Our results are consistent with
those found previously in faba bean inoculated with the same fungus (Kucey & Paul
1982),. We do not know the reason of the early cessation of the rapid extra-radical
growth phase. Because extra-radical hyphae ,take up nutrients from the host tissues,
we hypothesize that their growth is strictly controlled by the host plant.
Effect on plant growth, nodulation and N&xation
The effect of inoculation with Gtomus mosseace on nodulation was particularly marked
from day 20. This seems to be related to the onset of growth of the extra-radical
hyphae (Table 2). Since they are probably stimulated by P supply from actively
growing extra-radical hyphae, mycorrhizal plants produced six times more nodule dry
matter than non-mycorrhizal plants. Stimulation of nodulation at day 20 may be
important in field situations, because annual plants cari derive maximum benefit from
N2 fixation at this growth stage. Even thouglh the difference in nodulation between
mycorrhizal and non-mycorrhizal plants decreased at the end of the experiment, the
82 M. Gueye, H. G. Diem and Y. R. Dommergues
former still produced twice as much nodule dry weight as the latter plants (Table 2).
NT-living activity, (ARA/plant) (Fig. 2), was clearly higher in mycorrhizal than in
non-mycorrhizal plants. The increase of Nz-fixing activity was relatively more rapid in
mycorrhizal than in non-mycorrhizal plants between day 15 and 25 since during this
period increase in nodule weight of mycorrhizal plants (6-97 mg) was also much more
rapid than that of non-mycorrhizal plants (5-15 mg). According to these data,.it is
clear that high Nz-fixing activity occurred as soon as plants were infected by VA fungi.
The influence of early high N@ring activity due to mycorrhizal inoculation on grain
yield of soybean has already been found and discussed by Ganry et al. (1985).
Noldulation as well as Nz-fixing activity seemed not to be correlated with the
development of intra-radical hyphae (Table 2). By contra&, nodule dry weight (Table
2) and ARA per plant (Fig. 2) dramatically increased when extra-radical hyphae
started to develop (Table 2). This was concomitant with the enhancement of P inflow
which occurred by this time (Fig. 5).
In our study, the stimulating effect of Glonzus mosseae on plant growth was
significant only 45 days after inoculation.The increase of shoot dry weight was about
77% at this time. The delay in plant growth response to mycorrhizal infection suggests
that large amounts of P taken up by Glomus moss.eae were first needed for nodulation
rather than for plant gowth.
Effet on shoot water content
It is generally recognized that mycorrhizae protect plants against long periods of water
stress by enhancing P absorption from SO~I (Levy & Krikun 1980; Safir et al. 1972;
Allen & Boosalis 1983; Sieverding 1984). The rapid decrease of water content in non:
mycorrhizal plants between day 5 and 25 (Fig. 1) suggests that they were greatly
deficient in P during their active growth phase whereas in mycorrhizal plants water
content remained steady during the same period. The water content of the shoots was
higher in mycorrhizal than in non-mycorrhizal plants, from day 20 to 35, a period.
when extra-radical hyphae developed profusely (Table 2), thus enhancing P infIow.
into the plant as suggested by higher P content in mycorrhizal plants (Fig. 5). It has
been suggested that drought resistance of mycorrhizal plants was due to a decreased
resistance of plant tissues to water flow and therefore an enhanced water transport
throughout the plant. Such an enhanced water transport was found to be mediated by
a better P nutrition in mycorrhizal plants (Safir er’ al. 1972). By contrast, P-deficient
plants are more susceptible to drought (Atkinson & Davison 1973).
Effect on N and P contents
Surprisingly, variation patterns of N content in non-mycorrhizal and mycorrhizal
plants were similar throughout the experiment (Fig. 4), but there were differences
between time courses of P content in mycorrhizal and non-mycorrhizal plants (Fig. 5).
During the early growth stage of mycorrhizal plants (up to day 20, Fig. 5) the marked
decrease of P content was due to the utilization of significant amounts of available P
for growth and nodulation and probably also to the competition for P by the fungal
endophyte, thus creating a transitory sink for P. This would explain the significant
lower P content in mycorrhizal plants compared to that in non-mycorrhizal plants at
day 20, (Fig. 5). Bethlenfalvay et al. (1982) also found that shoot P content in the
controlls was significantly higher than in mycorrhizal plants during the first six weeks of
growth of soybean. However, Fig. 5 shows that the supposed sink for P in mycorrhiil
<. -2’
.::
: . 2, . ,
.
.
1 * ”
Extra-radical mycorrhizal hyphae 83
plants was subsequently and rapidly compensated by enhanced P uptake resulting
from the development of extra-radical hyphae. This suggestion is supported by the
marked increase of P content in mycorrhizal plants after day 20 (Fig. 5). During the
following growth phase of cowpea, P content in mycorrhizal plants was always
significantly higher than that in non-mycorrhizal plants, which constantly remained
low from day 25. Such P content pattems in mycorrhizal or non-mycorrhizal plants
were obtained mathematically in a simulation study by Sanders & Sheikh (1983).
In conclusion, the time course of P content in cowpeas consisted of three phases: (1) a
decaeasing critical phase prior to the devellopment of extra-radical hyphae between
day 5 and 20; (2) a P enrichment phase following the development of extra-radical
hyphae hetween day 20 and 25; (3) a slightly decreasing phase which was then
siabilized as the plant matured. Such a three-phase pattem of P percentage in
mycorrhizal plants has already been reported by Snellgrove et al. (1982) in the case of
Allium porrum.
Acknowledgement
We thank Moussa Niang for valuable technical assistance.
References
ABWIT, L. K. & ROBSON, A. D. 1981 Infectîvity and effectiveness of vesicular-arbuscular
my&rbizal fungkeffect of inoculum type. Australian Journal of Agricultural Research 32,
631-639.
ALLEN, M. F. & Boosa~~s, M. G. 1983 Effects of two species of VA mycorrhizal fungi on
drought tolerance of winter wheat. New Ph~~tologist 93, 67-76.
AM%ER, J. R. B YOUNG, J. L. 1977 Techniques for determining mot length infected by
vesicular-arbuscular mycorrbizae. Soil Science Society of America Journal 41,551-556.
ATKINSON, D. B DAMSON, A. W. 1973 Tbe effects of phosphorus deficiency on water content
and response to drought. New Phytologkt 72, 307-313.
BAGYARM, D. J. L MANJUNATH, A. 1980 Response of trop plants to VA mycorrhizd inoculation
in an unsterile Indian soil. New Phytologist !85, 33-36.
BEWLENFALVAY, G. J., PA~~VSKY, R. S. B :BROWN, M. S. 1982 Parasitic and mutualistic
associations between a mycorrhizal fungus and soybean: development of the endophyte.
Phytopathology 72,854-897.
DUNCAN, D. B. 1955 Multiple range and multiple F tests. Biomezrks 11, l-42.
GANRY, F., DIEM, H. G., WEY, J. & DOMMERGUES, Y. 1985 Inoculation with Glomus mosseae
improves Nz fixation by field-grown soybea~ Biology and Fertiliry of Soils 1, 15-23.
GEXMANI, G,, DIPM, H. G. a D~MM~RCUES, Y. 1980 Muenœ of 1,2dibromo-3-chlore-propane
(DBCP) on mycorrhizal infection of field-grown groundnut. In Tropical Mycorrhiza
Research, ed. Mikola, P. pp. 245-246. Oxford: Clarendon Press.
GIOVANEITI, M. a Moss~, B. 1980 An evaluation of techniques for measuring vesicular
arbuscular mycorrbizal infection in roots. New Phytologist 84,489-500.
GUEYE, M. 1983 Vigna unguiculata en symbiose avec Rhizobium et Glomus mosseae. Th&e
Doctorat 3& cycle, p. 150, ORSTOM, Paris.
HARDY, R. W. F., HOLSTEN, R. D., JACKSON, E. K. a BURNS, R. C. 1968 Tbe aœtylene assay
for Nz fixation: laboratory and field evaluation. Pfant Physiology 43, 1185-1207.
Hmvrrr, E. J. 1966 Sand and Watcr Culture Methods used in the Study of Pkmt Nutrition.
Technical communication No. 22 Second edn. London: Commonwealth Agricultural Bureau.
~SIAM, R., A~MABA, A. ct !~ANDE~S, F. E. 1980 Response of cowpea (Vigna unguiculata) to
inoculation with VA mycorrbizal fungi ,and to rock phosphate fertilization in some
unsteriliid Nigerian soils. Planf and Soi1 $8, 107-117.
JACKSON, M. L. 1964 Soil Chcmical Analyslr, Englewood Cliffs, N.J.: Prentiœ Hall.
KUCEY, R. M. N. & PAUL, E. A. 1982 Biomass of mycorrhizal fur@ associated with bean mots.
Soil Biology and Biochetitry 14,413-414.
84 M. Gueye, H. G. Diem and Y. R. Dommergues
LEW, 1. 6r KRIKUN, J. 1980 Effect of vesicular-arbuscular mycorrhiza in Citrus jumbhiri water
relations. New Phytologist 85, 25-32.
OLLMBR, B., BERTHEAU, Y. DIEM, H. G. dr GIANINAZZI-PEARSON, V. 1982 Influence
de la variété de Vigna unguiculatu L. Walp dans l’expression de trois associations
endomycorhiziennes à vesicules et arbuscules. Canadian Journal of Botany 61, 354-358.
OLLM~R, B., DIEM, H. G., PINTA, M. d DOMMERGUE-S, Y. R. 1984 Effect of endomycorrhizae
on the concentration of P and Zn in Vigna unguiculata shoots. Agrochimica 28, 341-352.
OLSEN, S. R., &LE, L. V., WATANABE, F. S. dr DEAN, L. A. 1954 Estimation of available
phosphorus in soils by extraction with sodium bicarbonate. Circular of Vnited s’tates
Department of Agriculture No. 939.
Rtcrr, .J. R. dr BIRD, G. W. 1974 Association of early season vesicular mycorrhizae with
increased growth and development of cotton. Phytopathology 64, 1421-1425.
SAFIR, G. R., BOYER, J. S. dr GERDEMANN, J. W. 11972 Nutrient status and mycorrhii
enhancement of water transport in soybeans. Plant physiology 49,700-703.
SANDEKS, F. E. & SHEIKH, N. A. 1983 The development of vesicular-arbuscular mycorrhizal
infection in plant root systems. Plant and Soi1 71, 223-246.
SANDERS, F. E., TINKER, P. B., BLACK, R. L. B. 6r PALI~ERLY, S. M. 1977 The development of
mycorrhizal root systems: 1. Spread of infection and growth promoting effects with four
species of vesiculararbuscular endophyte. New Phytologist 78, 257-268.
SIEVERDING, E. 1984 Influence of soi1 water regimes on VA mycorrhiza. 3. Comparison of 3
mycorrhizal fungi and their influence on transpiration. Zeitschrift fiir Acker und Pflanzenbau
153,52-61.
SMKH, S. E. dr BOWEN G. D. 1979 Soi1 temperature, mycorrhizal infection and nodulation of
Medicago truncatula and Trifolium subterraneum. Soi1 Biology and Biochemistry 11, 469-
473.
SNELLCIROVE,
R. C., SPLITETOESSER, W. E. STRIBLE~, D. P. a TINKER, P. B. 1982 The
distribution of carbon and the demand of the fungal symbiont in leek plants with vesicular-
arbuscular mycorrhizas. New Phytologist 92, 75-87.
VAN VEEN, J. A. 6r PAUL, E. A. 1979 Conversion of biovolume measurements of soil organisms,
grown under various moisture tensions, to biomass and their nutrient content. Applied ami
Environmental Microbiology 37, 686-692.
Yosr, R. S. dr Fox, R. L. 1979 Contribution of mywrrhizae to P nutrition of crops growing on
an oxisol. Agronomy Journal 71,903~908.
Summary
Vigne unguiculata cv. 58-185 grown in a sterile Dek soi1 was inoculated with Rhizobium sp. or
Rhizobium sp. plus Glomus mosseae. Response of the host plant to the treatments was
estimated by periodic measurements of shoot and nodule dry weights, Nz fixation (%Hz
reduction activity) and N and P contents up to the 50th day of the growth cycle. It was only 45
days after planting that shoot dry weight of dually inoculated plants differed significantly from
that of plants inoculated with Rhizobium sp. alone. Nodule dry weight and Nz fixation of dually
inoculated plants were significantly higher than those of plants inoculated with Rhizobium sp.
alone from day 20 after planting, but there was no significant difference in N content (%).
During the first 20 days, shoot P content (%) of both sets of plants decreased progressively, P
content of dually inoculated plants being lower than that of the others. Later, P content of
dually inoculated plants increased rapidly whereas P content of the other plants remained
constant. Increase in nodule dry weight, Nz fixation and P content of dually inoculated plants
wrresponded to the onset of the development of the extra-radical hyphae of Glomus mosseue in
the rhizosphere.
Résumé
Variation dans fa fixation de Nz, les teneurs en N et P chez Vigna unguic.ulata
mycorhizé en relation avec le développement progressif des hyphes extraradica!es de
Glornus mosseae
On a inoculé V. unguiculata poussant dans un sol Dek stérile avec Rhizobium et Rhizobium
plus Glomus mosseae. On a recherché la réponse de la plante-hôte à ces deux traitements en
Extra-radical mycorrhizal hyphae 85
ic
j,a*nbhiri w a t e r
estimant périodiquement les poids des nodules et des parties a&iennes de la plante, la fixation
d’azote (activite réductrice de &Hz), les teneurs en N et P jusqu’au 50” jour du cycle de
MN.. V. 1982 Influence
végetation. C’est seulement au 45’jour aprés la plantation que le poids sec des parties adriennes
)n cle trois associations
des plantes inoculées avec deux symbiotes (plantes doublement inoculées) diffère significative-
f Botany 61., 354-3.58.
ment de celui des plantes inoculées avec Rhizobium seul. Le poids sec des nodules et la fixation
ffect of endomycorrhizae
NZ des plantes doublement inoculées sont significativement plus éleves que ceux des plantes
rochimica 23, 341-352.
inoculées avec Rhizobium seul au 20” jour aprts la plantation mais il n’y a pas de différence
1 Estimation of available
significative pour la teneur en N (%). Pendant les 20 premiers jours, la teneur en P (X) des
ïrcular of United States
parties aériennes des deux catégories de plantes décroit progressivement; la teneur en P des
plantes doublement inoculees est plus faible que celle des plantes inoculees seulement avec
jicular mycorrhizae with
Rhizobium. Plus tard, la teneur en P des plantes doublement inoculees augmente rapidement
1421-1425.
tandis que celle des autres plantes reste constante. L’accroissement du poids sec des nodules, de
status and mycorrhizal
la fixation d’azote et de la teneur en P observe chez les plantes doublement inoculées
700-703.
correspond au démarrage du developpement des hyphes extra-radicales de Clornus mosseae
ir-arbuscular mycorrhizal
dans la rhizos.phére.
1977 The development of
Resumen
moting effects with four
Variacibn en la jijacih de N y en lus contenidos de ,Y y P de Vigna unguiculata
l-.268.
niza. 3. Comparison of 3
micorrizada en relacidn con el desarrrollo progresivo de las hifas extra radiculares de
ir Acker und PfIanzenbau
Glomus mosseae
Se cultiv6 V@a unguiculata cv. 58-185 en un suelo ester-il tipo Dek, se inocul6 con Rhizobium
Bction and nodulation of
sp. o con Rhizobium sp. mas Glomus mosseae. La respuesta de la planta huésped a 10s
MI Biochemistry 11, 469-
tratamientos se estudi6 midiendo periodicamente el peso s~cco de la parte aerea y de 10s
nodules, la fijaci6n de N (actividad reductora de CzHz) y 10s contenidos de N y P hasta el50“ dfa
‘INKER, P. H. 1982 The
del ciclo de crecimiento. La diferencia entre el peso seco de 1.a parte aerea de las plantas con
X:L~ plants with vesicular-
doble inoculaci6n y aquellas inoculadas con Rhizobium sp. unicamente, no fue significativa
hasta 45 dias desptiés de la siembra. A 10s 20 dias de la siembra tanto el peso seco de 10s nodules
ements of soi1 organisms,
como la fijaci6n de nitr6geno de las plantas con doble inoculation cran significativamente
ent content. Applied and
superiores a 10s valores obtenidos para las plantas con solo Rhizobium SP., aunque no se
observaron diferencias en el contenido en N (%). Durante 110s primeros 20 dfas del ciclo el
ition of crops growing on
contenido en P (%) de ambos grupos de plantas disminuy6 progresivamente, siendo 10s valores
obtenidos por las plantas con doble inoculaci6n inferiores a 10s de las dem& M& tarde el
contenido en P de las plantas con doble inoculation aument rapidamente manteniendose
constante el dle las demas. El incremento en el peso seco de 10s nodules, en la fijaci6n de N y en
rd with Rhitobium sp. or
el contenido en P de las plantas con doble inoculation se correspondi6 con el inicio del
t to the treatments was
desarrollo de las hifas extraradiculares de Glomus mosseae.
ights, Nz fixation (C?H,
nvth cycle. It was only 45
fiffered significantly from
t and Nz fixation of dually
lated with Rhizobium sp.
rente in N content (%).
ecreased progressively, P
rers. Later, P content of
e other plants remained
1 ldually inoculated plants
hae of Glomus mosseae in
ICZ Vigna unguiculata
vphts extruradicales de
R.hirobium et Rhizobium
i ces deux traitements en