162 B. Bir´o et al. Applied Soil Ecology 15 2000 159–168
analysis of variance two-way ANOVA. Comparison of means was made by using the least significant dif-
ferences LSD=p0.05. Regression analysis of the nodulation data and the ARA measurements used the
Statgraphics 5.0 program.
3. Results
3.1. Selection of the rhizobium inoculum Table 2 shows the result of a preliminary exper-
iment, where six candidate R. meliloti strains were used to inoculate sterile alfalfa seedlings, grown in
a Thornton agar with six replicates Vincent, 1970. After 8 weeks of growth, the number of root nodules
and the ARA were estimated Hardy et al., 1973. The number of root nodules on the alfalfa seedlings
varied between 4.5 and 12. The data mostly positively correlating with that of ARA 10.7–35.6 ppm ethy-
lene h
− 1
per plant. Correlation coefficient r
2
=0.998 at p=0.0017 level. The equation of the correlation
curve was y=log 1.293x
1.137
. With strain Bk 51, some small and white nodules developed. This was not
significantly related to performance the ARA mea- surements. The strains Lu 41, K 41, S 57 showed
the highest efficiency. These strains were selected as candidates for further inoculations as a mixed
population.
Table 2 A sterile seedling experiment for estimating the efficiency ARA
and nodulating ability of some R. meliloti strains, as potential candidates for the inoculation of alfalfa in pot experiments n=6
a
Laboratory code of rhizobia
Root nodules number per plant
ARA ppm Uninoculated control
Bk 51 12±2.0
28.7±6.2 Lu 41
b
8 .6±2.5
35 .6±4.1
K 4 1
6 .3±1.0
25 .8±4.7
S 5 7
5 .7±1.2
20 .9±3.2
Kt 12 5.0±2.9
18.5±3.6 Kt 41
4.5±1.5 10.7±1.8
a
Strains originated from the Commercial Inocula Collection of the Research Institute for Soil Science and Agricultural Chemistry
RISSAC-MTA TAKI of the Hungarian Academy of Sciences, Budapest.
b
Strains set as italics were selected for the pot experiment, as 1:1:1 v:v:v mixed inoculum approximately 10
6
CFU ml
− 1
.
3.2. Effect of mono- or multilevel inoculations on the dry matter accumulation
All diazotrophs as dual- MR, MS or tripar- tite MRS combinations with the AMF M in-
creased dry matter production in the sterilised G and in the re-suspended GB treatments, where the
other competitive AMF populations were excluded Table 3. G. fasciculatum M 107, as single inoculum
also significantly enhanced growth. This was further increased in the RM dual-inoculated tripartite sys-
tems. The R. meliloti R strain-mixture, which was selected for great effectivity also resulted in the same
beneficial effect on the dry matter accumulation in the non-sterilised soil. A much higher increase was
found, however, in the multilevel co-inoculation treat- ments, where the AMF was accompanied both by
the associative and the symbiotic Azospirillum and Rhizobium
nitrogen-fixers synergistic effect of the MRS treatments.
Re-inoculation of the gamma-sterilised soils with the AM-free soil extract GB reconstructed the orig-
inal C microflora in the rhizosphere. The same dry matter yield was found in the controls in comparison
with the non-treated C soil.
In the original non-sterilised soil C a uniform ben- eficial effect was found for the plant growth as a re-
sult of the single inoculation of microsymbionts, both by the diazotrophs R, S and by the AMF M. The
tripartite and the multilevel systems with the Azospir- illum
and the Rhizobium diazotrophs MR, MS and MRS treatments, however, have resulted in the same
dry matter accumulation, as the non-inoculated con- trols Table 3.
3.3. Nutrient uptake of the inoculated plants among the various soil conditions
Both the macro- and microelement content was affected by the microsymbiont co-inoculations, and
influenced by the soil substrates C, G, GB used. The most positive effect of the single- or multilevel
microbial treatments occurred with the sterilised sub- strates, where the indigenous AMF were excluded.
The inoculated strain of the mycorrhizal fungi G. fasciculatum
M 107 was effective in increasing, both the N-, P- and the K-content of the alfalfa shoot, in
the G, GB treatments.
B. Bir´o et al. Applied Soil Ecology 15 2000 159–168 163
Table 3 Shoot dry weight Sh, root dry weight Rt and the total dry matter production Sh+Rt of alfalfa
a
M. sativa L. Strains
b
Substrates and dry matter yield g per pot C
G GB
Sh Rt
Sh+Rt Sh
Rt Sh+Rt
Sh Rt
Sh+Rt Control
1.56a 1.72a
3.28a 0.97a
1.04a 2.35a
1.66a 1.80a
3.46a M
1.72b 2.51b
4.23b 2.33b
2.84b 5.17b
2.49c 2.33b
4.82b MR
1.60a 1.59a
3.19a 2.07b
3.93b 5.25c
2.27b 2.94b
5.21c MS
1.71b 2.10a
3.81a 2.01b
2.71b 4.72b
2.10b 2.76b
4.86b MRS
1.59a 2.38a
3.97a 2.50b
3.38b 5.87c
2.04b 3.29c
5.33c R
1.73b 2.53b
4.30b n.d.
c
n.d. n.d.
n.d. n.d.
n.d. S
1.73b 2.50b
4.22b n.d.
n.d. n.d.
n.d. n.d.
n.d. RS
1.74b 2.64b
4.37b n.d.
n.d. n.d.
n.d. n.d.
n.d.
a
Plants were grown for 3 months in either the original- C, gamma-sterilised- G or AM-free-resuspended GB chernozem soil with single-, dual- or multilevel microbial treatments. Dry matter was collected at the 6th, 9th and 12th weeks and is summarised here g DW
per pot. Each value is the mean of three pots. n.d.: not determined.
b
M: mycorrhiza G. fasciculatum M 107, R: rhizobium R. meliloti S 57+Lu-41+K 41, S: spirillum A. brasilense Km5 or their combinations, respectively.
c
Not determined.
In relation to nitrogen uptake among the original non-sterilised soil conditions C, positive effects of
rhizobium inoculation were found in all of the combi- nations R, RS or MRS. In case of the Azospirillum
diazotroph bacteria an enhanced N content was found in the MS treatment; the mycorrhizal inoculation re-
sulted in an additive positive effect.
Uptake of P and K were also increased by the in- oculated R. meliloti strain-mixture as single- R, or
Table 4 The quantity of the main nutrients N, P, K in the shoot biomass of alfalfa in the pot experiment
a
Strain combinations Substrates and macro-elements
b
C G
GB N
P K
N P
K N
P K
Control 39.8a
2.6b 27.7a
18.1a 2.2a
17.8a 37.1a
2.5a 29.7a
M 38.2a
2.0a 24.9a
73.2c 4.4b
35.0b 65.5c
4.1b 36.8b
MR 39.1a
2.5ab 28.7a
56.2b 4.0b
32.8b 55.2b
3.8b 33.4ab
MS 49.6b
2.4ab 27.5a
51.3b 4.0b
30.5b 61.6c
4.2b 34.4ab
MRS 50.7b
2.2ab 26.2a
71.4c 5.0c
37.0b 64.2c
4.2b 34.4ab
R 56.7b
3.6c 32.7b
n.d. n.d.
n.d. n.d.
n.d. n.d.
S 42.4a
2.5ab 27.8ab
n.d. n.d.
n.d. n.d.
n.d. n.d.
RS 48.4b
3.2c 31.6ab
n.d. n.d.
n.d. n.d.
n.d. n.d.
a
Plants were grown in a non-sterilised calcareous chernosem soil C: all microbes, in a gamma-sterilised soil G: no microbes and in a sterilised substrate, to which a mycorrhiza-free soil extract was added GB: no mycorrhiza. The effect of the single- M, R, S, dual-
MR, MS, RS and multilevel MRS inoculations were assessed by using bacterial and fungal microsymbionts. M: G. fasciculatum M 107, R: R. meliloti S 57+Lu 41+K 41, S: A. brasilense Km 5. n.d.: not determined.
b
Values denoted by the same letter are not significantly different at p
5
level. Significant differences are given in italics.
dual systems with the other nitrogen-fixing bacteria A. brasilense Km 5-RS. With the mycorrhizal M,
G. fasciculatum M 107 partner Table 4 the effect
was smaller. Difference between the mono- and multilevel co-
inoculations were not observed for the concentrations of elements such as the Ca, Mg, Fe, Zn, Cu and S in the
shoot biomass. In the non-mycorrhizated G, GB con- trol substrates, however, an increased element-content
164 B. Bir´o et al. Applied Soil Ecology 15 2000 159–168
was found, as a result of the gamma-sterilisation. The effect of the additional AM-free rhizosphere mi-
croflora depended on the microelement, studied. Any microbial re-inoculation GB decreased the Mg, Zn
and Cu and S content of the alfalfa shoot-biomass, compared to the gamma-sterilised substrate G. The
concentration of Fe was increased by the re-suspension of the AM-free soil extract in the control substrates.
In the original normal soil C, the content of some elements was unaffected by the inoculated microsym-
bionts. With Ca slightly increased, but not significant, uniform uptake occurred with the single- or dual di-
azotroph treatments C+R, C+S and C+RS, respec- tively Table 5.
3.4. Abundance of the inoculated microbes in the rhizosphere of the alfalfa
For the AMF, the infection frequency F, in the targeted roots was high all near to the 100 and was
not significantly changed for in the different microbial treatments data not shown. The arbuscular content
of the infected parts a, , however, was a more vari- able as a function of the single-, dual- or multilevel
co-inoculations Fig. 1. Addition of the AM-free soil suspension the normal rhizosphere constituents to
the gamma-sterilised substrates G, GB increased the arbusculum content of the alfalfa root system. A syn-
Fig. 1. Nodule-number I, arbusculum content II and abundance of the associative diazotrophs III on the alfalfa roots after 3 months of growth in a calcareous loamy chernozem soil C: all microbes, in the same gamma-sterilised soil G: no microbes and in the sterilised
substrate, resuspended with mycorrhiza-free soil extract GB: no mycorrhiza. The effect of the single- M, R, S, dual- MR, MS and multilevel MRS coinoculations were assessed after 3 months of growth in a pot experiment among controlled conditions. M: vesicular
AMF G. fasciculatum M 107, R: R. meliloti S 57+K 41+Lu 41 mixture, S: A. brasilense Km 5 and their combinations, respectively. Table 5
The amount of Ca, Mg, Fe, Zn, Cu and S in the shoot biomass of the alfalfa in a pot experiment
a
Treatments Element content
b
mg per pot Ca
Mg Fe
Zn Cu
S C
2.23a 3127a
124.7a 22.5a
7.23a 4026.7a
G 3.30c
4330c 139.4a
39.4c 10.91b
7483.3b GB
3.34c 3543b
237.5b 28.6b
7.98a 4656.7a
C+M 2.20a
3086a 92.4a
22.6a 6.62a
3670.0a C+MR
2.19a 2856a
97.9a 22.7a
6.48a 3596.7a
C+MS 2.25a
2880a 113.5a
22.7a 6.95a
4374.3a C+MRS
2.01a 3063a
91.4a 23.8a
6.97a 3390.0a
C+R 2.42b
3120a 129.0a
26.7a 6.78a
4073.3a C+S
2.37b 2943a
120.4a 20.9a
6.92a 3847.7a
C+RS 2.44b
2890a 100.4a
24.8a 7.37a
3390.0a
a
Plants were grown in a non-sterilised calcareous chernosem soil C: all microbes, re-inoculated in a gamma-sterilised soil
G: no microbes and in the sterilised substrate, which was re-suspended with a mycorrhiza-free soil extract GB: no mycor-
rhiza. The effect of the single- M, R, S, dual- MR, MS, RS and the multilevel MRS co-inoculations were assessed by using
bacterial and fungal microsymbionts. M: G. fasciculatum M 107, R: R. meliloti S 57+Lu 41+K 41, S: A. brasilense Km 5.
b
Values sharing the same letter in a column do not differ significantly at p0.05. Significant differences are given in italics.
ergistic effect was achieved, when all of the inoculated microbes were present MRS. The non-sterilised
original soil showed significantly, the lowest colonisa- tion arbusculum content=14.6 by the mycorrhiza
B. Bir´o et al. Applied Soil Ecology 15 2000 159–168 165
G. fasciculatum M 107 and Azospirillum A. brasilense
Km 5 co-inoculations C+MS. For nodule-number, after the 3 months of plant
growth, a beneficial effect was found for all mi- crobial inoculations in the sterile or AM-free soils
Fig. 1. A further enhancement was developed with the multilevel co-inoculations MRS. In the origi-
nal soil C a significantly increased nodule-number was also recorded with the mycorrhizal G. fascicu-
latum
M 107 inoculation. Dual treatments with the obligate- and the associative diazotrophs Rhizobium+
Azospirillum bacteria, RS also resulted in a synergis-
tically increased nodule-number nn=27 per pot, data not shown. This was the highest rate in the control
soil. The most probable number method MPN for cal-
culating the abundance of the associative nitrogen- fixing bacteria was also carried out for the rhizo-
sphere of the alfalfa host. These data showed that the cell-number of the Azospirillum diazotrophs increased
due to the mono- or multilevel inoculations. The high- est rates were found in case of the single- or dual my-
corrhizal co-inoculations M, MS, MRS especially in the sterile or in the AM-free soils G, GB. In the
original substrate C the lowest cell numbers were recorded in the Azospirillum-non-amended combina-
tions, such as the M, R and MR treatments. All of the mycorrhizal co-inoculations increased the abundance
of the associative diazotrophs. In the GB substrate, only the dual- or multilevel co-inoculations of the MS
or MRS treatments were effective.
4. Discussion and conclusions
