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Nodulation, N2 fixation and dry matter yield of soybean [Glycine max (L.) merrill] inoculated with effective and ineffective Bradyrhizobium japonicum strains
Soil Bid Biochem. Vol.26,No. 7,pp.X83-889. 1994 Pergamon
003%0717(94)E0004-J
Copyright g; 1994Elsevier Science Ltd in Great Brilain. All rights reserved
Printed
0038-0717/94
$7.00 + 0.00
NODULATION, N, FIXATION AND DRY MATTER YIELD OF SOYBEAN [GLYCINE MAX (L.) MERRILL] INOCULATED WITH EFFECTIVE AND INEFFECTIVE BRAD YRHZZOBZUM JAPONZCUM STRAINS D.S. DARAMOLA,'S.
K.A.DANso**
and G. HARDARS~N~
‘Institute of Agricultural Research and Training, Obafemi Awolowo University, Ibadan, Nigeria, *Joint FAOjIAEA Division, International Atomic Energy Agency, P.0 Box 100, A-1400 Vienna, Austria and ‘FAO/IAEA Laboratory in Seibersdorf, Austria (Accepted
I5 December
1993)
Summary-Many soils harbour rhizobia of varying effectiveness, and this could influence the outcome of seed inoculation with selected rhizobial strains. A greenhouse study was made to compare the effect of inoculating an effective Brudyrhizobium japonicum strain onto seed (s) or into the bulk soil (b) with treatments containing an additional inoculation (with the same strain, or an ineffective B. juponicum strain) onto seed or into soil. Nodulation, N, fixation and growth of soybean were measured, using the “N isotope dilution method to quantify the N fixed. The method of inoculation had significant effects on nodule distribution along soybean roots, nodule number and fresh weight, and N, fixation. In general, soil inoculation resulted in more nodules being formed, more uniform distribution of nodules on the root, and greater nitrogen fixation. The influence of the ineffective strain (in combinations containing both strains) on nodulation and Nz fixation depended on where each or both strains were inoculated. Generally, highest N, fixation was achieved by inoculating the effective strain into soil. N, fixation by seed-inoculated effective strain was significantly depressed in the presence of the soil-inoculated ineffective strain (Es + Ib) but not when both strains were inoculated onto seed (Es + Is), or when both strains were inoculated together into soil (Eb + Ib). The trend for %Ndfa was generally similar to total nitrogen fixed. Our results therefore indicate that there can still be substantial N2 fixation in legumes by highly effective strains even in the presence of less effective strains if the effective strains are fairly well distributed in soil.
INTRODUCTION Many soils are deficient in highly effective rhizobia,
necessitating inoculation with desired strains, to ensure substantial N, fixation. Rhizobium is generally inoculated directly onto seeds, and less frequently into the bulk soil. Many soils already harbour native Rhizobium strains of different competitiveness. In addition to the intrinsic competitiveness of an inoculated strain, the method of inoculation may seriously affect nodulation success (Danso and Owiredu, 1988; Hardarson et al., 1989; Weaver and Frederick, 1974) and needs to be considered in some soils. Seed inoculation has been widely used because it results in early nodulation and forms prominent nodules clustered mostly around the crown of the root, postulated to be crucial for nitrogen fixation in the early stages of crop development (Hardarson et al., 1989). In contrast, the direct inoculation of rhizobia into soil results in a better distribution of rhizobia in the whole soil, and thus like the well distributed naturally occurring strains, are capable of forming many nodules, located on both primary and *Author for correspondence.
lateral roots. Such inoculated strains are also capable of competing better with the naturally-occurring Rhizobiutn strains (Danso and Owiredu, 1988). The importance of rhizobial distribution in soil on strain competitiveness and effective nodulation has been demonstrated by Wadisirisuk et al. (1989) who reported not only increased nodulation, but also more uniform spread of the nodules on the root system of soybean when soil was inoculated with B. japonicum. The nodules located on the lateral and younger segments of the root system (i.e. below the crown root layer) have been suggested by Danso and Bowen (1989) and Hardarson et al. (1989) to make a substantial contribution to N2 fixation during the period when the N demand of plants is greatest. Given that rhizobia migrate poorly in soil (Hamdi, 1971; Danso and Bowen, 1989), soil inoculation is an effective method for planting rhizobia where they are required for maximum effect. Towards improving inoculation success, some studies have examined the benefits of combining seed inoculation with soil inoculation (Ciafardini and Barbieri, 1987; Danso et al., 1990). Because many soils contain more than one strain of differing effectiveness (Singleton and Tavares, 1986) the interaction between strains of contrasting
D. S. DARAMOLA
884
effectiveness is receiving considerable attention (Robinson, 1969; Jones and Russell, 1972; Rolfe et al., 1980; Wadisirisuk et al., 1989; Danso et al., 1990). Singleton and Stockinger (1983) in a study using combinations of ineffective and effective strains reported significant increases in the total N of shoot as both the proportion and number of nodules formed by the effective strain increased. Singleton and Stockinger (1983) further reported that 95% of maximum N accumulation was obtained when only 75% of the nodules were effective. Rhizobium inoculation may be performed using either single, double or multi-strains. Which of these approaches is better is still a matter of controversy (Bagyaraj and Hedge, 1978; Burton, 1981). Some inoculant-producing companies, however, prefer to produce multi-strain inoculants to provide a compensatory mechanism to cope with constraints which host-strain-nvironment interactions may pose (Burton and Martinez, 1980). In contrast, singlestrain inoculants are still being produced by others to prevent dominance and antagonism by a more aggressive component strain in a mixed culture (Schwinghamer and Brockwell, 1978). Given that an effective strain on one plant may be of low effectiveness on another (Jones and Hardarson, 1979; FAO, 1984), a mixed strain inoculation could end up with effective strains competing with ineffective strains. Results of studies by Wadisirisuk et al. (1989), using single and mixed strain inoculants and different inoculation sites (on seed or into soil) showed differences in N2 fixation. More data are needed on inter-strain interactions and the effect of methods and site of inoculation on Nz fixation. Our objective was to mimic situations in which Rhizobium strains which differ widely in effectiveness have to compete for nodulation, and to evaluate how the site of initial inoculation of each or both strains will influence nodulation and N, fixation in soybean. MATERIALS
AND METHODS
The experiment was conducted in a greenhouse at the FAO/IATA Laboratory, in a Typic Eurocrepts soil (pH 7.4,0.3% organic matter) sieved to < 2 mm. The soil did not contain native bradyrhizobia (Zapata et al., 1987). 3 kg of a I : I mixture of sieved soil and washed sand were weighed into 5 1 size plastic pots with three holes at the bottom. Soil was watered by capillary rise from water contained in a saucer placed beneath each pot. The Bradyrhizobium japonicum strains used were: effective (E) strain 6 IA 124a, also referred to as J5 and ineffective (I) strain THA I, also referred to as 512, originally obtained from LiphaTech (Nitragin) Inc., Milwaukee, Madison and the Biological Nitrogen Fixation Center in Bangkok, respectively. In addition, strain J5 had been developed into a streptomycin-resistant mutant capable of growing in yeast
et
al.
extract mannitol (YEM) medium supplemented with 500 kg streptomycin ml- ’ (Danso and Alexander, 1974). The strains were grown separately in YEM broth for 7 days, the cells were harvested by centrifugation in a refrigerated centrifuge at 5000~ for 20 min, washed twice with sterile distilled water and re-suspended in sterile water to give a cell density of 3 x lo8 cells ml-’ for J5 and 6 x lo8 cells ml-’ for 512, as measured by a dilution plate count (Somasegaran and Hoben, 1985). Peat-based inoculants were prepared by mixing 4.5 ml of J5 with 15 g peat to give ca 5.2 x 10’ cells g-’ peat inoculant (dilution plate count). For 512, a 4.5 ml-suspension was inoculated into 15 g peat, and the plate count indicated a population of 5.5 x IO’ cells gg’ peat. A mixed-strain peat inoculant containing J5 and 512 was formulated by combining 2.0 ml of J5 with 1.0 ml of 512, and adding water to bring up the volume to 5 ml. The suspension was then incorporated into 15 g peat to give ca 5.8 x 10’ cells g-’ moist peat. For liquid inoculation, 10 ml of J5 was diluted in 100 ml water for soil inoculation. In order to have approximately equal cell concentrations of J5 and 512 per unit of soil in the mixed strain liquid inoculant, 5 ml of J5 and 2.5 ml of 512 were mixed and made up to a 100 ml cell suspension. The experiment consisted of a total of eight strains and site of inoculation treatments, plus an inoculated control and a reference non-nodulating Clay soybean isoline as shown in Table 1. Soybean [Glycine max (L.) Merrill] seeds (cv. Clay) were inoculated by coating surface-sterilized seeds (Vincent, 1970) with one strain or a mixture of both strains immediately before sowing. For strain incorporation into the bulk soil, a 100 ml cell suspension of either or both strains (obtained as described above) was mixed into the soil at planting. Uninoculated soils received 100 ml of sterile distilled water. Each pot was planted with four inoculated or uninoculated seeds and thinned to two seedlings 7 days after planting (DAP), followed immediately with the addition of a 50 ml solution of (NH,),SO, enriched with 10% “N atom excess to each pot, to give the equivalent of 1Opg N g-‘. Each treatment, harvested at flowering (52 DAP) and at physiological
I.
Table
inoculation
Inoculation On seed (S) E’
treatments and their designation the text
treatment Into
soil
(b)
Treatment
Nil
Es
Nil E I
E
I
Es+ lb
E
IsfEb
Nil
E+I Nil
E+I Nil
Nil
Nil
‘E
and I
denote respeclively.
designation
Eb Es+Eb
: I
I
in
Is + Ib
Es+
Is
Eb + Ib Uninoculated Non-nodulating
effective
and
soybean
inefkctive
strains.
Nodulation by effective and ineffective strains maturity (67 DAP), was replicated four times, in a randomized complete block design. Plants were harvested 2cm above soil level, chopped into 2cm pieces and dried in the oven at 70°C for 2 days. Roots were carefully removed from each pot and any adhering soil washed with tap water, before sectioning into O-5, 5-10, IO-15 and > 15 cm soil depth segments for nodule counts. Nodules from the different segments were later bulked for fresh weight determination. Nodule-forming Bradyrhizobium strains were identified at the 57 DAP harvest from 10 randomly sampled nodules per plant. For this, each nodule was crushed in sterile distilled water in a Petri-dish, and the suspension streaked on YEM with or without 500 ng streptomycin ml-’ (Somasegaran and Hoben, 1985). In the case of dual-strain inoculation, the proportion of nodules formed by the streptomycin labelled strains (J5 or E) was estimated as % of positive growth on YEM containing streptomycin. Nitrogen in plants was determined by Kjeldhal digestion (Eastin, 1978) and atom % “N excess by mass spectrometry (Fiedler and Proksch, 1975). Nitrogen fixation was calculated using the 15N isotope dilution equation (Fried and Middelboe, 1977), using uninoculated and non-nodulating soybean (cv. Clay) as reference plants. Statistical analysis was by ANOVA, and treatment comparisons were based on the Least Significant Difference (LSD) among treatments.
RESULTS AND DISCUSSION
Nodulation Significant differences (P < 0.05) in nodule distribution (Fig. I), nodule number and fresh weight (Table 2) occurred among treatments. Nobules were more dispersed on the roots of treatments in which the effective strain was soil-applied (Eb), whether alone or in combination with the ineffective strain. For example, nodule distribution on roots in the Eb and Eb + Ib treatments was better than the respective corresponding treatments, Es and Es + Is. Kamicker and Brill (1987) similarly reported that nodule distribution on roots can be affected by the method of inoculation. The treatment with the sole ineffective strain (Is + Ib) was overall the poorest in nodulation. Among the three inoculation treatments with only the effective strain, Es (which we consider as our control treatment formed the lowest number of nodules, with those for Eb and Es + Eb being similar at both harvests. However, nodule weight differences were not statistically significant (P < 0.05) across treatments. The formation of abundant nodules therefore resulted in individual nodules being smaller than for those treatments that formed fewer nodules. A similar observation was reported for a supernodulating soybean mutant (Gunawardena et al., 1993).
885
Inoculation with a mixture of the ineffective and effective strains resulted in variable effects on nodulation and nodule weight. Wadisirisuk et al. (1989) reported that the method of inoculation can influence the competitiveness of strains. Except for where both strains were inoculated onto seed (Es + Is), in which case the effective strain formed fewer (43%) nodules, % nodulation due to the effective strain was always higher (mean, 64%), indicating that the effective strain was more competitive than the ineffective strain. By inoculating the effective strain onto the seed, a second inoculation with the ineffective strain onto the seed (whether on the seed or into soil) had no effect on total number of nodules formed at the 67 DAP harvest; the same trend was observed for nodule fresh weights. In both dual-strain inoculation treatments where the effective strain had been inoculated into soil (Is + Eb and Es + Eb), nodule numbers were enhanced relative to Es. Similar results were obtained for nodule fresh weights when either Eb or a mixture of E + I was soil-applied compared to Es. Interestingly, the Is + Eb inoculation gave the highest nodulation at 67 DAP, and would suggest a stimulatory effect on the effective strain by the seed-inoculated ineffective strain. A similar observation was reported by Wadisirisuk et al. (1989), while Rolfe et al. (1980) reported that the interaction between a non-nodulating mutant of R. trifolii and an ineffective mutant resulted in the formation of effective (N,-fixing) nodules. In another study, Ohlendorf and Martensson (1990) reported an interaction between two R. leguminosarum strains that resulted in some nodulation by a strain that when inoculated alone would not form any nodules on the strain-specific Afgh. I pea cultivar. These results point to the complex and variable outcome of the interaction between rhizobia of different effectiveness and competitiveness. Our results suggest that in soils containing ineffective strains, it should still be possible to obtain significant benefits by inoculating with a competitive, effective strain. It appears that the dominant factor in the increased nodulation was the Eb inoculation. In earlier studies, Hardarson et a/. (1989) and Wadisirisuk et al. (1989) had made similar observations. The better nodulation where strains were soil-applied could be attributed to the fact that being more homogeneously distributed in soil, they are more likely to encounter roots than when the rhizobia are seed-applied and thus localized near the vicinity of the emerging roots (Weaver and Frederick, 1974; Danso and Bowen, 1989). We may also infer from the nodulation data that it is better to inoculate an effective strain (whether single or mixed) into soil than localizing it on seed, an approach that characteristically results in nodulation mainly around the crown (Ciafardini and Barbieri, 1987; Hardarson et al., 1989). However, the inoculation of rhizobia into soil may be more laborious than the conventional seed inoculation method, and may also require
D. S. DARAMOLA et
886
al.
80 52 DAP
i
I=.:
seed bulk soil
E
I
E
I
I
I
1
E
E
E+I E+I
E
67 DAP
I=.:
seed bulk soil
E
I
E
1
I
I
I
E
E
E+I E
_ E+I
Fig. 1. Nodule numbers of soybean at 52 and 67 DAP when effective (E) and ineffective (1) strains were inoculated on to the seed (S) or into bulk soil (B).
mechanization. Given the potential benefit of soil inoculation, it is worth thinking seriously about devising practical methods for doing this in the field. Soil inoculation will be very useful, for example, for replacing or diluting the population of native rhizobia which are often not as effective as selected inoculant strains (Bergersen et al., 1971), or when conditions are unfavourable for seed inoculation, such as when seeds are dusted with toxic fungicides (Brockwell et al., 1980).
Nitrogen jixation
Very little N,-fixing activity was detected at 52 DAP in plants inoculated with only the ineffective strain (Ib + Ic) (Table 3). The treatment that gave the highest fixation was where the effective and ineffective strain were inoculated together into soil (Eb + Ib). Most other treatments gave statistically similar (P < 0.05) %Ndfa. The same trend was observed at 67 DAP. As with %Ndfa, total N fixed was lowest in the Is + Ib treatment, highest were the Es + Eb and
Nodulation by effective and ineffective strains Table
2. Nodulation
of soybean
as affected
by varying
887
methods
and
site of
inoculation Inoculation
Nodule
seed
soil
Nodule
number?
plant -’ Days 52
fresh weight
plant after
’
planting
67
52
67
E
Nil
36.2 b*
37.4 c
I.1 ab
I .4 ab
Nil
E
86.6 a
89.0
I.2 a
1.4 ab
E
E
79.6 a
90.0 ab
1.2 a
1.6 a
I
I
10.5 c
9.6 d
0.4 c
0.4 c
b
E
1
18.3 c
27.0 c
0.8 b
1.2 b
I
E
80.8 a
104.2 a
I.2 a
1.7 a
E+I
Nil
34.6 b
34.8 d
1.2 a
1.3 ab
Nil
E+I
74.0 a
87.9 b
I.1 ab
I.6 ab
LSD
(0.05)
%CV *Numbers
within
different tMean
a column
14.8
14.8
0.3
0.4
19.0
17.0
21.0
21.0
followed
of three
letter
are not statistically
replicates.
Eb + Ib inoculations, with the values for most of the remaining treatments being similar. Our data indicate that nodule weight more closely reflected the nitrogen-fixing abilities of the inoculation treatments than nodule numbers. The corresponding regression coefficients (r) were, 0.70 (P < 0.01) and 0.65 (P < 0.05), respectively. That at 67 DAP, plants inoculated with only ineffective rhizobia contained 22 %Ndfa suggests that this strain was not completely ineffective. The soil used is known to be free of indigenous bradyrhizobia (Zapata et al., 1987) and this was confirmed in the present study; the uninoculated reference plants remained nodule-free throughout the study. It is, however, interesting that except for one of the mixed strain inoculation treatments (Es + Ib), the ineffective strain generally did not decrease nitrogen fixed relative to where the sole effective strain was inoculated onto seed. However, inoculating the ineffective strain into soil prior to planting seeds inoculated with the effective strain resulted in reduced N, fixation. Thus, one factor common with the two best N,-fixing inoculation treatments (Eb + Eb and Eb + Is) is, that both had the effective strain in the soil, in support of the suggested general superiority of soil inoculation over Table
by an identical
(P > 0.05).
3. Effects
of inoculation
seed inoculation (Hardarson et al., 1989). Singleton and Stockinger (1983) also reported that a deleterious effect on N, fixation was observed in their study only when infection by the ineffective brain was great enough to reduce nodule mass. Dry matter and N yielcls
The lowest dry matter and N yields were recorded on plants inoculated with only the ineffective strain, uninoculated soybean and non-nodulating soybean (Table 4). The indications therefore are: (i) that soil N was insufficient for optimum plant growth, (ii) that the ineffective strain did not fix much N, and (iii) inoculation with the effective strain resulted in greater N accumulation because of the substantial input from N, fixation, which consequently enhanced dry matter yields. However, once inoculated with the effective strain (irrespective of whether together with the ineffective strain or not), dry matter and N yields were little affected, compared to the control treatment, Es. These results are therefore in contrast to those of nodulation and N, fixation (Table 3). The most likely explanation is, that the N fixed by each of these inoculation treatments must have satisfied the
alternatives
on nitrogen
fixation
in soybeans
Inoculation seed
Soil
Ndfa
% Ndfa Days 52
after
67
(mg
plant-‘)
planting 52
67
E
Nil
75.1 b’
67.8
19.0 b
19.5 b
Nil
E
77.9 b
15.3 ab
23.9 ab
19.5 b
E
E
77.3 b
65.4
23.0 ab
25.4 a
I
I
7.5 d
21.9 d
0.8 c
2.5 d
E
I
76.5 b
53.8 c
18.8 b
14.0 c 19.4 b
b bc
I
E
80.8 ab
70.1
b
21.0 b
Efl
Nil
65.0 c
66.0
b
20.5 b
17.7 b
Nil
E+I
88.6
82.2 a
26.6 a
23.7 a
LSD
( < 0.05)
% cv *Numbers different
within
a column
(P > 0.05).
8.4
I I.9
5.3
3.3
8.4
13.0
19.0
13.0
followed
by an identical
letter
are not statistically
D. S.
888
DARAMOLA et al.
Table 4. Drv matter and nitroeen Inoculation Seed
Soil
vield of sovbean
Dry matter yield (g plant ‘)
inoculated
differentlv
N yield (mg plant
‘)
Days after planting 52 E Nil E E E+I Nil Uninocculated Non-nodulating LSD < 0.05 %CV
Nil E E I I E Nil E+I
1.9 2.3 2.4 1.4 I.7 2.1 2.4 2.2 I.1 I.1
52
61 bc* a a dc cd abc a ab c c
0.4 13.9
‘Numbers within a column followed different (P > 0.05).
minimum required for optimum growth under the prevailing conditions. Greater differences in dry matter and N yields occurred at 52 DAP than at 67 DAP.
Also, those plants that fixed less nitrogen used up more soil N for growth (data not presented). Despite the lack of significant differences among most inoculation treatments, it is interesting that for both N yield and dry matter, the Es + Eb treatment still gave the highest values, followed by Eb + Ib, at 67 DAP (Table 4). The treatment, Es + Ib, that fixed the lowest amount of N among inoculation treatments containing E (Table 2), similarly gave the poorest dry matter and N yields. Acknowledgements-We
are grateful to MS H. Axmann and the analytical group of the IAEA Laboratory in Seibersdorf for the total nitrogen and nitrogen ratios analyses, and to MS Marie-And&e Abliischer for typing the manuscript. The senior author thanks the International Atomic Energy Agency for the fellowship awarded to conduct this research. REFERENCES Bagyaraj D. J. and Hedge S. V. (1978) Response of cowpea (Vigna unguiculala L. Walp) to Rhozobium seed inoculation. Current Science 13, 548-549. Bergersen F. J., Brockwell J., Gibson A. H. and Schwinghamer E. A. (1971) Studies of natural populations and mutants of Rhizobium in the improvement of legume inoculants. Planr and Soil, Special‘Volume, 3-16. Brockwell J.. Gault R. R.. Chase D. L.. Helv F. W.. Zorin M. and Corbin E. J. (1980) An appraisal of practical alternatives to legume seed inoculation. Field experiments on seed bed inoculation with solid and liquid inoculants. Australian Journal of Agricuhural Research 31, 4760.
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2.6 2.4 2.1 1.5 2.1 2.6 2.1 2.6 1.4 I.3
ab ab a c ab ab b ab c c
0.5 16.0 by an identical
23.3 25.3 29.8 10.7 24.3 25.9 31.4 30.0 9.0 8.2
61 b b ab bc b ab a ab c c
6.1 19.2
28.5 25.9 29.2 II.4 24.3 27.1 26.1 28.3 II.4 10.7
ab ab ab bc ab ab ab ab c c
4.3 13.3
letter are not statistically
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003%0717(94)E0004-J
Copyright g; 1994Elsevier Science Ltd in Great Brilain. All rights reserved
Printed
0038-0717/94
$7.00 + 0.00
NODULATION, N, FIXATION AND DRY MATTER YIELD OF SOYBEAN [GLYCINE MAX (L.) MERRILL] INOCULATED WITH EFFECTIVE AND INEFFECTIVE BRAD YRHZZOBZUM JAPONZCUM STRAINS D.S. DARAMOLA,'S.
K.A.DANso**
and G. HARDARS~N~
‘Institute of Agricultural Research and Training, Obafemi Awolowo University, Ibadan, Nigeria, *Joint FAOjIAEA Division, International Atomic Energy Agency, P.0 Box 100, A-1400 Vienna, Austria and ‘FAO/IAEA Laboratory in Seibersdorf, Austria (Accepted
I5 December
1993)
Summary-Many soils harbour rhizobia of varying effectiveness, and this could influence the outcome of seed inoculation with selected rhizobial strains. A greenhouse study was made to compare the effect of inoculating an effective Brudyrhizobium japonicum strain onto seed (s) or into the bulk soil (b) with treatments containing an additional inoculation (with the same strain, or an ineffective B. juponicum strain) onto seed or into soil. Nodulation, N, fixation and growth of soybean were measured, using the “N isotope dilution method to quantify the N fixed. The method of inoculation had significant effects on nodule distribution along soybean roots, nodule number and fresh weight, and N, fixation. In general, soil inoculation resulted in more nodules being formed, more uniform distribution of nodules on the root, and greater nitrogen fixation. The influence of the ineffective strain (in combinations containing both strains) on nodulation and Nz fixation depended on where each or both strains were inoculated. Generally, highest N, fixation was achieved by inoculating the effective strain into soil. N, fixation by seed-inoculated effective strain was significantly depressed in the presence of the soil-inoculated ineffective strain (Es + Ib) but not when both strains were inoculated onto seed (Es + Is), or when both strains were inoculated together into soil (Eb + Ib). The trend for %Ndfa was generally similar to total nitrogen fixed. Our results therefore indicate that there can still be substantial N2 fixation in legumes by highly effective strains even in the presence of less effective strains if the effective strains are fairly well distributed in soil.
INTRODUCTION Many soils are deficient in highly effective rhizobia,
necessitating inoculation with desired strains, to ensure substantial N, fixation. Rhizobium is generally inoculated directly onto seeds, and less frequently into the bulk soil. Many soils already harbour native Rhizobium strains of different competitiveness. In addition to the intrinsic competitiveness of an inoculated strain, the method of inoculation may seriously affect nodulation success (Danso and Owiredu, 1988; Hardarson et al., 1989; Weaver and Frederick, 1974) and needs to be considered in some soils. Seed inoculation has been widely used because it results in early nodulation and forms prominent nodules clustered mostly around the crown of the root, postulated to be crucial for nitrogen fixation in the early stages of crop development (Hardarson et al., 1989). In contrast, the direct inoculation of rhizobia into soil results in a better distribution of rhizobia in the whole soil, and thus like the well distributed naturally occurring strains, are capable of forming many nodules, located on both primary and *Author for correspondence.
lateral roots. Such inoculated strains are also capable of competing better with the naturally-occurring Rhizobiutn strains (Danso and Owiredu, 1988). The importance of rhizobial distribution in soil on strain competitiveness and effective nodulation has been demonstrated by Wadisirisuk et al. (1989) who reported not only increased nodulation, but also more uniform spread of the nodules on the root system of soybean when soil was inoculated with B. japonicum. The nodules located on the lateral and younger segments of the root system (i.e. below the crown root layer) have been suggested by Danso and Bowen (1989) and Hardarson et al. (1989) to make a substantial contribution to N2 fixation during the period when the N demand of plants is greatest. Given that rhizobia migrate poorly in soil (Hamdi, 1971; Danso and Bowen, 1989), soil inoculation is an effective method for planting rhizobia where they are required for maximum effect. Towards improving inoculation success, some studies have examined the benefits of combining seed inoculation with soil inoculation (Ciafardini and Barbieri, 1987; Danso et al., 1990). Because many soils contain more than one strain of differing effectiveness (Singleton and Tavares, 1986) the interaction between strains of contrasting
D. S. DARAMOLA
884
effectiveness is receiving considerable attention (Robinson, 1969; Jones and Russell, 1972; Rolfe et al., 1980; Wadisirisuk et al., 1989; Danso et al., 1990). Singleton and Stockinger (1983) in a study using combinations of ineffective and effective strains reported significant increases in the total N of shoot as both the proportion and number of nodules formed by the effective strain increased. Singleton and Stockinger (1983) further reported that 95% of maximum N accumulation was obtained when only 75% of the nodules were effective. Rhizobium inoculation may be performed using either single, double or multi-strains. Which of these approaches is better is still a matter of controversy (Bagyaraj and Hedge, 1978; Burton, 1981). Some inoculant-producing companies, however, prefer to produce multi-strain inoculants to provide a compensatory mechanism to cope with constraints which host-strain-nvironment interactions may pose (Burton and Martinez, 1980). In contrast, singlestrain inoculants are still being produced by others to prevent dominance and antagonism by a more aggressive component strain in a mixed culture (Schwinghamer and Brockwell, 1978). Given that an effective strain on one plant may be of low effectiveness on another (Jones and Hardarson, 1979; FAO, 1984), a mixed strain inoculation could end up with effective strains competing with ineffective strains. Results of studies by Wadisirisuk et al. (1989), using single and mixed strain inoculants and different inoculation sites (on seed or into soil) showed differences in N2 fixation. More data are needed on inter-strain interactions and the effect of methods and site of inoculation on Nz fixation. Our objective was to mimic situations in which Rhizobium strains which differ widely in effectiveness have to compete for nodulation, and to evaluate how the site of initial inoculation of each or both strains will influence nodulation and N, fixation in soybean. MATERIALS
AND METHODS
The experiment was conducted in a greenhouse at the FAO/IATA Laboratory, in a Typic Eurocrepts soil (pH 7.4,0.3% organic matter) sieved to < 2 mm. The soil did not contain native bradyrhizobia (Zapata et al., 1987). 3 kg of a I : I mixture of sieved soil and washed sand were weighed into 5 1 size plastic pots with three holes at the bottom. Soil was watered by capillary rise from water contained in a saucer placed beneath each pot. The Bradyrhizobium japonicum strains used were: effective (E) strain 6 IA 124a, also referred to as J5 and ineffective (I) strain THA I, also referred to as 512, originally obtained from LiphaTech (Nitragin) Inc., Milwaukee, Madison and the Biological Nitrogen Fixation Center in Bangkok, respectively. In addition, strain J5 had been developed into a streptomycin-resistant mutant capable of growing in yeast
et
al.
extract mannitol (YEM) medium supplemented with 500 kg streptomycin ml- ’ (Danso and Alexander, 1974). The strains were grown separately in YEM broth for 7 days, the cells were harvested by centrifugation in a refrigerated centrifuge at 5000~ for 20 min, washed twice with sterile distilled water and re-suspended in sterile water to give a cell density of 3 x lo8 cells ml-’ for J5 and 6 x lo8 cells ml-’ for 512, as measured by a dilution plate count (Somasegaran and Hoben, 1985). Peat-based inoculants were prepared by mixing 4.5 ml of J5 with 15 g peat to give ca 5.2 x 10’ cells g-’ peat inoculant (dilution plate count). For 512, a 4.5 ml-suspension was inoculated into 15 g peat, and the plate count indicated a population of 5.5 x IO’ cells gg’ peat. A mixed-strain peat inoculant containing J5 and 512 was formulated by combining 2.0 ml of J5 with 1.0 ml of 512, and adding water to bring up the volume to 5 ml. The suspension was then incorporated into 15 g peat to give ca 5.8 x 10’ cells g-’ moist peat. For liquid inoculation, 10 ml of J5 was diluted in 100 ml water for soil inoculation. In order to have approximately equal cell concentrations of J5 and 512 per unit of soil in the mixed strain liquid inoculant, 5 ml of J5 and 2.5 ml of 512 were mixed and made up to a 100 ml cell suspension. The experiment consisted of a total of eight strains and site of inoculation treatments, plus an inoculated control and a reference non-nodulating Clay soybean isoline as shown in Table 1. Soybean [Glycine max (L.) Merrill] seeds (cv. Clay) were inoculated by coating surface-sterilized seeds (Vincent, 1970) with one strain or a mixture of both strains immediately before sowing. For strain incorporation into the bulk soil, a 100 ml cell suspension of either or both strains (obtained as described above) was mixed into the soil at planting. Uninoculated soils received 100 ml of sterile distilled water. Each pot was planted with four inoculated or uninoculated seeds and thinned to two seedlings 7 days after planting (DAP), followed immediately with the addition of a 50 ml solution of (NH,),SO, enriched with 10% “N atom excess to each pot, to give the equivalent of 1Opg N g-‘. Each treatment, harvested at flowering (52 DAP) and at physiological
I.
Table
inoculation
Inoculation On seed (S) E’
treatments and their designation the text
treatment Into
soil
(b)
Treatment
Nil
Es
Nil E I
E
I
Es+ lb
E
IsfEb
Nil
E+I Nil
E+I Nil
Nil
Nil
‘E
and I
denote respeclively.
designation
Eb Es+Eb
: I
I
in
Is + Ib
Es+
Is
Eb + Ib Uninoculated Non-nodulating
effective
and
soybean
inefkctive
strains.
Nodulation by effective and ineffective strains maturity (67 DAP), was replicated four times, in a randomized complete block design. Plants were harvested 2cm above soil level, chopped into 2cm pieces and dried in the oven at 70°C for 2 days. Roots were carefully removed from each pot and any adhering soil washed with tap water, before sectioning into O-5, 5-10, IO-15 and > 15 cm soil depth segments for nodule counts. Nodules from the different segments were later bulked for fresh weight determination. Nodule-forming Bradyrhizobium strains were identified at the 57 DAP harvest from 10 randomly sampled nodules per plant. For this, each nodule was crushed in sterile distilled water in a Petri-dish, and the suspension streaked on YEM with or without 500 ng streptomycin ml-’ (Somasegaran and Hoben, 1985). In the case of dual-strain inoculation, the proportion of nodules formed by the streptomycin labelled strains (J5 or E) was estimated as % of positive growth on YEM containing streptomycin. Nitrogen in plants was determined by Kjeldhal digestion (Eastin, 1978) and atom % “N excess by mass spectrometry (Fiedler and Proksch, 1975). Nitrogen fixation was calculated using the 15N isotope dilution equation (Fried and Middelboe, 1977), using uninoculated and non-nodulating soybean (cv. Clay) as reference plants. Statistical analysis was by ANOVA, and treatment comparisons were based on the Least Significant Difference (LSD) among treatments.
RESULTS AND DISCUSSION
Nodulation Significant differences (P < 0.05) in nodule distribution (Fig. I), nodule number and fresh weight (Table 2) occurred among treatments. Nobules were more dispersed on the roots of treatments in which the effective strain was soil-applied (Eb), whether alone or in combination with the ineffective strain. For example, nodule distribution on roots in the Eb and Eb + Ib treatments was better than the respective corresponding treatments, Es and Es + Is. Kamicker and Brill (1987) similarly reported that nodule distribution on roots can be affected by the method of inoculation. The treatment with the sole ineffective strain (Is + Ib) was overall the poorest in nodulation. Among the three inoculation treatments with only the effective strain, Es (which we consider as our control treatment formed the lowest number of nodules, with those for Eb and Es + Eb being similar at both harvests. However, nodule weight differences were not statistically significant (P < 0.05) across treatments. The formation of abundant nodules therefore resulted in individual nodules being smaller than for those treatments that formed fewer nodules. A similar observation was reported for a supernodulating soybean mutant (Gunawardena et al., 1993).
885
Inoculation with a mixture of the ineffective and effective strains resulted in variable effects on nodulation and nodule weight. Wadisirisuk et al. (1989) reported that the method of inoculation can influence the competitiveness of strains. Except for where both strains were inoculated onto seed (Es + Is), in which case the effective strain formed fewer (43%) nodules, % nodulation due to the effective strain was always higher (mean, 64%), indicating that the effective strain was more competitive than the ineffective strain. By inoculating the effective strain onto the seed, a second inoculation with the ineffective strain onto the seed (whether on the seed or into soil) had no effect on total number of nodules formed at the 67 DAP harvest; the same trend was observed for nodule fresh weights. In both dual-strain inoculation treatments where the effective strain had been inoculated into soil (Is + Eb and Es + Eb), nodule numbers were enhanced relative to Es. Similar results were obtained for nodule fresh weights when either Eb or a mixture of E + I was soil-applied compared to Es. Interestingly, the Is + Eb inoculation gave the highest nodulation at 67 DAP, and would suggest a stimulatory effect on the effective strain by the seed-inoculated ineffective strain. A similar observation was reported by Wadisirisuk et al. (1989), while Rolfe et al. (1980) reported that the interaction between a non-nodulating mutant of R. trifolii and an ineffective mutant resulted in the formation of effective (N,-fixing) nodules. In another study, Ohlendorf and Martensson (1990) reported an interaction between two R. leguminosarum strains that resulted in some nodulation by a strain that when inoculated alone would not form any nodules on the strain-specific Afgh. I pea cultivar. These results point to the complex and variable outcome of the interaction between rhizobia of different effectiveness and competitiveness. Our results suggest that in soils containing ineffective strains, it should still be possible to obtain significant benefits by inoculating with a competitive, effective strain. It appears that the dominant factor in the increased nodulation was the Eb inoculation. In earlier studies, Hardarson et a/. (1989) and Wadisirisuk et al. (1989) had made similar observations. The better nodulation where strains were soil-applied could be attributed to the fact that being more homogeneously distributed in soil, they are more likely to encounter roots than when the rhizobia are seed-applied and thus localized near the vicinity of the emerging roots (Weaver and Frederick, 1974; Danso and Bowen, 1989). We may also infer from the nodulation data that it is better to inoculate an effective strain (whether single or mixed) into soil than localizing it on seed, an approach that characteristically results in nodulation mainly around the crown (Ciafardini and Barbieri, 1987; Hardarson et al., 1989). However, the inoculation of rhizobia into soil may be more laborious than the conventional seed inoculation method, and may also require
D. S. DARAMOLA et
886
al.
80 52 DAP
i
I=.:
seed bulk soil
E
I
E
I
I
I
1
E
E
E+I E+I
E
67 DAP
I=.:
seed bulk soil
E
I
E
1
I
I
I
E
E
E+I E
_ E+I
Fig. 1. Nodule numbers of soybean at 52 and 67 DAP when effective (E) and ineffective (1) strains were inoculated on to the seed (S) or into bulk soil (B).
mechanization. Given the potential benefit of soil inoculation, it is worth thinking seriously about devising practical methods for doing this in the field. Soil inoculation will be very useful, for example, for replacing or diluting the population of native rhizobia which are often not as effective as selected inoculant strains (Bergersen et al., 1971), or when conditions are unfavourable for seed inoculation, such as when seeds are dusted with toxic fungicides (Brockwell et al., 1980).
Nitrogen jixation
Very little N,-fixing activity was detected at 52 DAP in plants inoculated with only the ineffective strain (Ib + Ic) (Table 3). The treatment that gave the highest fixation was where the effective and ineffective strain were inoculated together into soil (Eb + Ib). Most other treatments gave statistically similar (P < 0.05) %Ndfa. The same trend was observed at 67 DAP. As with %Ndfa, total N fixed was lowest in the Is + Ib treatment, highest were the Es + Eb and
Nodulation by effective and ineffective strains Table
2. Nodulation
of soybean
as affected
by varying
887
methods
and
site of
inoculation Inoculation
Nodule
seed
soil
Nodule
number?
plant -’ Days 52
fresh weight
plant after
’
planting
67
52
67
E
Nil
36.2 b*
37.4 c
I.1 ab
I .4 ab
Nil
E
86.6 a
89.0
I.2 a
1.4 ab
E
E
79.6 a
90.0 ab
1.2 a
1.6 a
I
I
10.5 c
9.6 d
0.4 c
0.4 c
b
E
1
18.3 c
27.0 c
0.8 b
1.2 b
I
E
80.8 a
104.2 a
I.2 a
1.7 a
E+I
Nil
34.6 b
34.8 d
1.2 a
1.3 ab
Nil
E+I
74.0 a
87.9 b
I.1 ab
I.6 ab
LSD
(0.05)
%CV *Numbers
within
different tMean
a column
14.8
14.8
0.3
0.4
19.0
17.0
21.0
21.0
followed
of three
letter
are not statistically
replicates.
Eb + Ib inoculations, with the values for most of the remaining treatments being similar. Our data indicate that nodule weight more closely reflected the nitrogen-fixing abilities of the inoculation treatments than nodule numbers. The corresponding regression coefficients (r) were, 0.70 (P < 0.01) and 0.65 (P < 0.05), respectively. That at 67 DAP, plants inoculated with only ineffective rhizobia contained 22 %Ndfa suggests that this strain was not completely ineffective. The soil used is known to be free of indigenous bradyrhizobia (Zapata et al., 1987) and this was confirmed in the present study; the uninoculated reference plants remained nodule-free throughout the study. It is, however, interesting that except for one of the mixed strain inoculation treatments (Es + Ib), the ineffective strain generally did not decrease nitrogen fixed relative to where the sole effective strain was inoculated onto seed. However, inoculating the ineffective strain into soil prior to planting seeds inoculated with the effective strain resulted in reduced N, fixation. Thus, one factor common with the two best N,-fixing inoculation treatments (Eb + Eb and Eb + Is) is, that both had the effective strain in the soil, in support of the suggested general superiority of soil inoculation over Table
by an identical
(P > 0.05).
3. Effects
of inoculation
seed inoculation (Hardarson et al., 1989). Singleton and Stockinger (1983) also reported that a deleterious effect on N, fixation was observed in their study only when infection by the ineffective brain was great enough to reduce nodule mass. Dry matter and N yielcls
The lowest dry matter and N yields were recorded on plants inoculated with only the ineffective strain, uninoculated soybean and non-nodulating soybean (Table 4). The indications therefore are: (i) that soil N was insufficient for optimum plant growth, (ii) that the ineffective strain did not fix much N, and (iii) inoculation with the effective strain resulted in greater N accumulation because of the substantial input from N, fixation, which consequently enhanced dry matter yields. However, once inoculated with the effective strain (irrespective of whether together with the ineffective strain or not), dry matter and N yields were little affected, compared to the control treatment, Es. These results are therefore in contrast to those of nodulation and N, fixation (Table 3). The most likely explanation is, that the N fixed by each of these inoculation treatments must have satisfied the
alternatives
on nitrogen
fixation
in soybeans
Inoculation seed
Soil
Ndfa
% Ndfa Days 52
after
67
(mg
plant-‘)
planting 52
67
E
Nil
75.1 b’
67.8
19.0 b
19.5 b
Nil
E
77.9 b
15.3 ab
23.9 ab
19.5 b
E
E
77.3 b
65.4
23.0 ab
25.4 a
I
I
7.5 d
21.9 d
0.8 c
2.5 d
E
I
76.5 b
53.8 c
18.8 b
14.0 c 19.4 b
b bc
I
E
80.8 ab
70.1
b
21.0 b
Efl
Nil
65.0 c
66.0
b
20.5 b
17.7 b
Nil
E+I
88.6
82.2 a
26.6 a
23.7 a
LSD
( < 0.05)
% cv *Numbers different
within
a column
(P > 0.05).
8.4
I I.9
5.3
3.3
8.4
13.0
19.0
13.0
followed
by an identical
letter
are not statistically
D. S.
888
DARAMOLA et al.
Table 4. Drv matter and nitroeen Inoculation Seed
Soil
vield of sovbean
Dry matter yield (g plant ‘)
inoculated
differentlv
N yield (mg plant
‘)
Days after planting 52 E Nil E E E+I Nil Uninocculated Non-nodulating LSD < 0.05 %CV
Nil E E I I E Nil E+I
1.9 2.3 2.4 1.4 I.7 2.1 2.4 2.2 I.1 I.1
52
61 bc* a a dc cd abc a ab c c
0.4 13.9
‘Numbers within a column followed different (P > 0.05).
minimum required for optimum growth under the prevailing conditions. Greater differences in dry matter and N yields occurred at 52 DAP than at 67 DAP.
Also, those plants that fixed less nitrogen used up more soil N for growth (data not presented). Despite the lack of significant differences among most inoculation treatments, it is interesting that for both N yield and dry matter, the Es + Eb treatment still gave the highest values, followed by Eb + Ib, at 67 DAP (Table 4). The treatment, Es + Ib, that fixed the lowest amount of N among inoculation treatments containing E (Table 2), similarly gave the poorest dry matter and N yields. Acknowledgements-We
are grateful to MS H. Axmann and the analytical group of the IAEA Laboratory in Seibersdorf for the total nitrogen and nitrogen ratios analyses, and to MS Marie-And&e Abliischer for typing the manuscript. The senior author thanks the International Atomic Energy Agency for the fellowship awarded to conduct this research. REFERENCES Bagyaraj D. J. and Hedge S. V. (1978) Response of cowpea (Vigna unguiculala L. Walp) to Rhozobium seed inoculation. Current Science 13, 548-549. Bergersen F. J., Brockwell J., Gibson A. H. and Schwinghamer E. A. (1971) Studies of natural populations and mutants of Rhizobium in the improvement of legume inoculants. Planr and Soil, Special‘Volume, 3-16. Brockwell J.. Gault R. R.. Chase D. L.. Helv F. W.. Zorin M. and Corbin E. J. (1980) An appraisal of practical alternatives to legume seed inoculation. Field experiments on seed bed inoculation with solid and liquid inoculants. Australian Journal of Agricuhural Research 31, 4760.
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2.6 2.4 2.1 1.5 2.1 2.6 2.1 2.6 1.4 I.3
ab ab a c ab ab b ab c c
0.5 16.0 by an identical
23.3 25.3 29.8 10.7 24.3 25.9 31.4 30.0 9.0 8.2
61 b b ab bc b ab a ab c c
6.1 19.2
28.5 25.9 29.2 II.4 24.3 27.1 26.1 28.3 II.4 10.7
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4.3 13.3
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