Competition between Medicago truncatula and wheat for 15N labeled soil nitrogen and influence of phosphorus

Pergamon

0038-0717(95)00114-x

Soil Biol. Biochem. Vol. 28, No. 1, pp. 8M8, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0038-0717/96 $15.00 + 0.00

COMPETITION BETWEEN MEDICAGO TRUNCATULA AND WHEAT FOR “N LABELED SOIL NITROGEN AND INFLUENCE OF PHOSPHORUS K. ELABBADI,’ M. ISMAILI’ and L. A. MATERON** ‘University Center

Moulay Ismail, for Agricultural

Faculty of Science, P.O. Box 4010, Meknes, Morocco and *International Research in the Dry Areas (ICARDA), P.O. Box 5466, Aleppo, Syria (Accepted 21 July 1995)

Summary---To determine competition for nitrogen uptake, a greenhouse experiment was conducted using annual Me&ago rruncatula cf. Jemalong (medic) and Triticum turgidurn spp. durum cv. Karim (wheat) grown alone or in mixture, over five successive crop cycles. Pots were supplied with P equivalent to 0, 50, 100 and 150 kg P,O, haa’ at the beginning of each crop cycle. The soil was labeled by growing wheat the labeled plants on soil fertilized with N enriched with 10.43 atom % ‘rN excess and by incorporating wheat as organic matter (2.08 atom % 15N excess) back into the soil. This method allowed estimation of Nz fixed by medic and N transfer to wheat. Compared to the sole medic crop, dry herbage yield and total N of medic grown in mixture, were reduced. Medic plants alone (unmixed) assimilated 80% more soil N that wheat alone at the higher rate of P. When in mixture, wheat plants took up more soil N than medic plants. The percentage N derived from synbiosis (%Ndfa), by the sole medic, averaged 60%. In mixture, %Ndfa of the medic was 80%. The total N difference method gave higher values for %Ndfa (622%) than the isotope dilution technique, depending on the P rate. This was because P rates affected the N absorption of medic and wheat differently. Dry herbage yield and total N of medic and wheat grown alone and in mixture were improved by P supply. Competition between medic and wheat was affected by P fertilization, favoring wheat which became more competitive for uptake of soil N at the higher P rates. When no P was added to the soil, mixed medic took up 60% of the soil N absorbed by sole medic; at the higher I’ rate, this was reduced to 34%. Transfer of N fixed by medic to wheat was detected only after the second crop cycle.

for both methods are similar-it must not fix any N2, and it must have the same N uptake pattern and obtain its N from the same soil volume as the Nz-fixing plant (Vose and Victoria, 1986; Weaver, 1986). Selection of the reference plant for use in the ID method is critical (Fried and Broeshart, 1975; Rennie, 1986; Vose and Victoria, 1986). A description of the ID method, its limitations and problems are discussed in several review articles (Rennie and Rennie, 1983; Witty, 1983; Vose and Victoria, 1986; Danso, 1986, 1988). The N balance of wheat and medic in cropping systems should be investigated to improve agronomic management practices. Wheat and medic are not grown in mixture but in rotation. However, during the rotation the pasture with regenerating medic is infested with volunteer wheat and the wheat field is infested with selfregenerating medic and we were interested in studying these two cases. Our objectives, were (i) to measure N2 fixed in pure and mixed stands of medic, (ii) to estimate the amounts (if any) of N transferred from medic to wheat, (iii) to determine competitive ability of medic and wheat for mineral N using i5N-label, and (iv) to determine the effects of P supply.

INTRODUCTION

In an intercropped system, the presence of a legume increases growth
for correspondence. 83

K. Elabbadi et al.

84 MATERIALS AND METHODS

A greenhouse experiment was conducted in 20-I pots filled with sieved silty clay soil and sand in the proportion 1: 2. Soil chemical characteristics were: pH(H*O), 8.00; pH(KCI), 7.50; organic matter, 1.68%; total N, 0.12%; and, available P, 20 pg ml-‘. Prior to the study, a wheat (Triticum turgidum spp. durum cv. Karim) crop was grown in all pots containing soil fertilized with the equivalent of 85 kg ammonium sulfate enriched with 10.43 atom % lSN excess, applied in solution (200ml pot-’ containing the equivalent of 167 mg N pot-‘). Ninety days after planting the wheat, the shoots and roots were harvested, oven-dried for 48 h at 70°C and ground (0.5 mm). The resulting weighted average %N and atom %“N excess of the mixed powder of wheat root and shoot were 1.92 and 2.08, respectively. The mixed powder of wheat root and shoot was incorporated in equal amounts (45.5 g plant material pot-‘) into the same soil in each pot as an organic source of “N. This labeling is assumed to be relatively more stable, and released more steadily during plant growth, than when an inorganic “N source is used (Broadbent et al., 1982; Ismaili and Weaver, 1986). The study comprised four phosphorus treatments: PO (15 mg kg-‘) was the unfertilized control, PI (50 kg P,O, ha-’ or 400 mg P,O, pot-‘), P2 (100 kg P,O, ha-’ or 800 mg P,O, pot-‘) and P3 (150 kg P,05 ha-’ or 1200mg P,Os pot-‘). P treatments were applied at the beginning of each crop cycle. Three cropping systems were compared: two monocultures and one mixture. Monocultures were sole wheat and sole medic (Medicago truncatula cv. Jemalong). The mixture was a 50:50 mixture of wheat and medic plants. Wheat and Rhizobiuminoculated medic seeds were sown, then thinned to leave 20 plants pot-’ for the two cropping systems (pure or mixture). This operation was repeated for each successive crop cycle. There were two crop cycles per year, one from September to January and the other from January to May; from May to September, the pots were left uncropped without disturbing the soil. Before seeding the subsequent crop, the top 15 cm of the soil was carefully mixed. At physiological maturity (at 50% flowering) the above-ground biomass was harvested for both wheat and medic. The shoot samples were dried and weighed for dry matter yield determinations and ground for total N and atom % 15N excess determinations. Total N was measured by Kjeldahl analysis (Nelson and Sommers, 1973) and “N was measured with a mass spectrometer (Porter and O’Deen, 1977). Roots were extracted at the end of the fourth cycle by sieving. Root samples were dried and weighed for dry matter yield determinations and ground for total N and atom % 15Nexcess determinations. At the end of the fourth cycle the soil of each pot was well mixed and then returned to the same pot. Before retuning the soil to its corresponding pot, soil samples were

taken and sieved (< 2 mm) for atom % “N excess determination. A fifth crop cycle was then grown to investigate the effect of not displacing the roots from the pots and not mixing the soil at the end of the first 4 crop cycles on the results of the experiment. To determine legume and wheat N derived from soil and N, fixation, the ID and TND methods were used. Biological nitrogen fixation (BNF) by the legume (%Ndfa) was calculated using wheat as reference plant: %Ndfa =

l-

atom % 15N excess in legume x 100 atom % 15N excess in wheat >

The fraction of N in the legume derived from soil and labeled wheat material (%Ndfs), is given by, %Ndfs = 100 - %Ndfa Nitrogen transfer was expressed as the percentage of wheat shoot-N derived from BNF by medic (%NT) according to the following equation: %NT = l-

atom % 15N excess of wheat in mixture

x 100 > Analysis of variance was performed on data from in a completely randomized design with three replications. Differences between the means were tested by the least significant difference (LSD) test. Differences reported were significant at P < 0.05. atom % “N excess of wheat alone

RESULTS

Medic and wheat dry wt and total N accumulation was higher in the first harvest (Table 1). The decrease was most severe after the first cycle. This may have been the result of(i) planting two crops per year, (ii) “N-labeled plant material was only added at the first crop, and (iii) seeding was repeated in the same pot without disturbing the root systems (there was no evidence of poor soil aeration). Medic responded well to P fertilizer especially in the first crop, with a 2.24-fold increase in dry wt between PO and P3, compared to 1.59-fold for wheat. In the mixture, medic response was only 1.61-fold, compared to 1.91-fold for wheat. The effect of added P on wheat dry wt and total N increase was evident in all crop cycles. In general, the short dry wt/plant of medic in the mixture was lower than in the monoculture at the P2 and P3 rates (Table 1). In contrast, wheat grown with medic yielded more shoot dry wt/plant than wheat grown in pure stand (Table 1). This difference was maintained over time and was accentuated by increasing the P rate. In the second, third and fourth mixed crop cycles, the P3 rate induced a decrease in dry wt and total N production of medic (Table 1). In the fifth crop, P3 rate gave a slight increase in mixed medic growth probably because the soil was removed from the pots, aerated and then returned to the pots. Overall plant

Competition Table I. Harvested

above-ground

between

dry wt (DW, g plant-‘) in mixture over

medic and wheat for N uptake

85

and total N (TN, mg plant-‘) production by wheat and medic grown solely and five

crops

successive

in

the greenhouse

Crop cycle 2

I

4

3

5

DW

TN

DW

TN

DW

TN

DW

TN

DW

TN

I .28 1.44 I .77 2.03

14.39 18.36 22.53 25.65

0.66 0.73 0.9 I 1.04

13.15 Il.69 il.71 24.79

0.65 0.69 0.87 0.84

7.57 8.36 IO.18 II.27

0.33 0.54 0.68 0.73

4.10 9.18 10.58 II.36

0.56 0.61 0.71 0.75

8.19 8.32 7.77 8.69

Mixed wheat PO PI P2 P3 LSD’

I .73 2.21 2.66 3.31 0.19

22.12 27.81 34.33 42.37 2.16

I .06 1.17 I .30 I .43 0.10

25.30 26.79 21.44 30.46 I .47

0.73 0.97 I .28 I .38 0.10

10.92 12.55 14.73 17.05 I.12

0.64

0.62 0.83 I.13 0.07

10.20 10.23 II.20 14.54 0.66

0.67 0.88 I .03 I.31 0.12

II.83 13.22 15.71 22.04 I.81

Sole medic PO PI P2 P3

1.69 2.32 2.79 3.19

14.39 18.36 22.53 25.65

0.57 0.77 0.91 I.10

13.15 17.69 17.71 24.79

0.47 0.60 0.67 0.7 I

7.57 8.36 10.18 I I .27

0.41 0.47 0.53 0.53

4.10 9.18 10.58 II.36

0.39 0.61 0.75 0.78

8.19 8.32 7.77 8.69

Mixed medic PO PI P2 P3 LSD*

I .66 1.84 2.36 2.67 0.19

50.96 63.7 97.34 105.10 9.54

0.68 0.84 0.85 0.64 0.13

21.39 19.78 19.53 15.14 2.1 I

0.40 0.63 0.68 0.54 0.13

13.17 16.06 15.69 14.59 2.34

0.47 0.63 0.62 0.44 0.07

10.37 15.62 15.59 II.91 0.46

0.48 0.59 0.71 0.82 0.05

12.54 14.06 16.19 19.18 3.71

CroD

Sole wheat PO PI P2 P3

*LSD (0.05) is for the intetaction

P x cropping

system (sole or mixed) PO, PI, P2 and P3 are respectively

dry weights from all pots were much lower than those of the first cropping cycle. The percentage of N in medic and wheat derived from soil in the fimt crop is shown in Table 2. In the sole-cropping system, N uptake from soil by medic was more than that by wheat; this difference was most pronounced at P3. The N uptake from soil by medic was, however, substantially reduced in the mixture, and P appeared to have no influence on the percentage contribution of soil N to the medic plants when mixed (Table 2). The total N derived from the soil by wheat grown in mixture with medic was higher than that of wheat grown alone (Table 2). In the mixture, wheat took up much more of the soil N than medic. Phosphorus treatments increased uptake of soil N for both species. At PO, soil N uptake by medic in mixture was only about 60% of that in sole medic, while at P3 medic in mixture took up only 34% of the soil N absorbed by the corresponding sole medic.

Table 2. Uptake

of mineral N from soil by medic and wheat during the first croo Medic Sole

Wheat Sole

Mixed

TNdfs

%Ndfs

TNdfs

%Ndfs

TNdfs

TNdfs

PO PI

16.2 21.2

40.0 33.9

9.6 12.0

I9 I9

14.5 18.0

20.4 27.0

P2 P3 LSD

39.3 46.0 3.3

42.7 33.4 N’S

17.5 15.7 0.1

I8 I5 NS

22.5 25.5 1.6

34.0 42.0 0.3

P

Mixed

TNdfs (mg plant -‘) and1 %Ndfs are total N and %N of medic and wheat plant shoots ‘derived from soil sources. (PO = 0; PI = 50; P2 = 100: P3 = ISOIng P,O, ha-‘).

0, 50, 100 and I50 kg P,O, ha-‘.

Comparisons between 15N concentration of wheat grown alone or in mixture (Table 3) indicated that there was a dilution, independently of P rates: the 15N enrichment of the mixed wheat in the first, second and third cycles was lower than sole wheat. In the fourth cycle, this dilution was more important in the Table 3. Effect of phosphorus and cropping system on “N enrichment of medic and wheat during five cropping seasons, Sl, S2, S3, S4 and S5 Atom % “N excess s4 Crop

Sl Shoot

s2 Shoot

s3 Shoot

Shoot

Root

Soil

s5 Shoot

Sole medic PO PI P2 P3 SE

0.389 0.313 0.389 0.308 0.012

0.412 0.380 0.387 0.348 0.007

0.239 0.197 0.230 0.227 0.006

0280 0.266 0.234 0.237 0.006

0.207 0.191 0.172 0.183 0.004

0.035 0.025 0.033 0.044 0.003

0.248 0.208 0.154 0.138 0.013

Mix. medic PO PI P2 P3 SE

0.184 0.185 0.170 0.142 0.008

0.365 0.308 0.296 0.345 0.010

0.056 0.096 0.156 0.080 0.01 I

0.169 0.218 0.227 0.263 0.01 I

0.153 0.156 0.142 0.145 0.003

0.037 0.054 0.049 0.045 0.005

0.243 0.091 0.108 0.113 0.018

Sole wheat PO PI P2 P3 SE

0.972 0.924 0.912 0.920 0.010

0.556 0.580 0.561 0.545 0.004

0.358 0.322 0.288 0.338 0.008

0.311 0.290 0.325 0.304 0.004

0.255 0.250 0.245 0.244 0.008

0.069 0.074 0.074 0.077 0.004

0.342 0.330 0.328 0.317 0.003

Mix. wheat PO PI P2 P3 SE

0.903 0.938 0.964 0.958 0.01 I

0.488 0.529 0.546 0.465 0.010

0.361 0.305 0.298 0.323 0.006

0.306 0.295 0.289 0.306 0.003

0.173 0.197 0.204 0.206 0.005

0.037 0.054 0.049 0.045 0.005

0.301 0.297 0.206 0.170 0.017

PO, PI, P2, and P3 are respectively SE is the standard error.

~

0, 50, 100. and I50 kg P,O, ha

I.

K. Elabbadi er al.

86

Table 4. Nitrogen fixation in mixed and sole medic using the isotope dilution (ID) and total N difference (TND) methods Mixed medic

Sole medic %Ndfa

P PO PI P2 P3 LSD

N fixed (mg plant - ‘)

%Ndfa N fixed (mg plant ‘)

ID

TND

LSD

ID

TND

LSD

ID

TND

60.0 66.1 57.3 66.5 NS

64.0 70.9 75.5 81.4 3.9

NS 4.5 3.7 I.1

24.3 41.3 52.7 91.5 3.6

25.5 44.5 70.0 112.0 9.5

NS NS 13.1 6.4

79.4 79.8 82.3 85. I NS

40.5 51.0 79.8 89.4 8.0

roots than in the shoots. In the fifth crop, however, a significant (P < 0.05) shoot 15N dilution was observed, especially at P3. The absorption of enriched 15Nby medic was in all cases lower than that observed for wheat (Table 3) due to N, fixation by medic during the five successive crop cycles. Enrichment of medic shoots was not affected by P treatment. The atom % “N excess of the mixed medic was lower than sole medic due to a greater proportion of legume N derived from air (%Ndfa) when grown in the mixture than when grown in pure stand (Table 4). Values for N, fixation by medic in the first crop cycle calculated by the TND method were higher than those measured by the ID method, especially when higher P rates were applied (Table 4).

DISCUSSION

The competitive ability of wheat over medic (especially in the high P status soil) was greater than that of medic plants. Similar results are reported with different conditions of plant growth by other workers (e.g. Dahman and Graham, 1981; Patra et al., 1986; Danso et al., 1988). In all the crop cycles, growth of medic was improved at Pl and P2. It was depressed at P3 rate, except in the first crop cycle when soil was more favorable for plant growth. The total N of individual shoots of wheat was higher when grown in mixture than when grown in pure culture (Table 1). This indicates an obvious benefit of neighboring medic in favor of wheat; however, this could be due merely to the better competition mentioned above, so that the grass interplanted with medic would be less N-limited than when sole planted. These results do not, in themselves, support the hypothesis of transfer of N from the medic in medic-grass mixture. In the sole crop, medic took up more soil N than the wheat. This increased N uptake by sole medic compared to sole wheat was also found by Ismaili and Weaver (1986) for siratro and kleingrass. At P3, sole medic took up 46 mg plant-’ compared with 25.5 mg plant-’ taken up by sole wheat (Table 2). The %N of medic was variable depending on the leaf-to-stem ratio. By sampling time, some defoliation may have occurred

or the %N of medic in pure stand was lower in the first crop or higher in mixture. It is most likely that sole wheat obtained all its N from the soil. Compared to wheat, medic was not competing effectively for soil N and was thus forced to rely more and more on biological N, fixation. In the mixture, P treatments favored more growth of wheat than medic, increasing further the competition for soil N between the species. This is important for the wheat crop when medic is considered as a weed to be eliminated by herbicides in the rotation system. Many workers have observed similar increase in soil-derived N in grasses when they are mixed with legumes (Vallis et al., 1967; Haystead and Mariott, 1979; Morris and Weaver, 1987; Ta and Faris, 1987) and considered it due to the inability of the fixing plant to compete equally with grass for soil N. This has been described as the “N sparing” effect, whereby more soil N becomes available to a grass because of reduced uptake by a nodulated legume (Butler and Ladd, 1985). It is possible that, in addition to such a competitive advantage of the grass for soil N, some N was transferred from the legume to the grass. Evidence of such N-transfer can be obtained by comparing the atomic composition of the grass in both cropping systems (Table 3). In the first, second and third crops, wheat obtained some N transfer from the legume (Table 3) with irregular variations, independent of P concentration. In the fourth crop, mean values of root atom %“N excess of the wheat grown with medic compared with that of the wheat root N when grown alone showed a considerably greater isotope dilution (P < 0.05) in the mixture compared with the monoculture. This suggests a considerable transfer of fixed N to the wheat. However, this result could be due to fine medic roots contaminating the wheat roots during sampling rather than a genuine transfer of medic-fixed N. Less indication of N transfer was shown for the shoot. In the fourth crop cycle, some important transfer was expected from N released from legume tissues after they had been recycled through microbial decomposition as suggested by Henzell and Vallis (1977) and by Broadbent et al. (1982). This happened effectively at the fifth crop cycle where there was evidence of uptake of fixed N, obtained from the medic, possibly through underground indirect transfer involving mineralization of dead roots and sloughed nodules which would be expected to be highest in the latest crop cycle. In fact at the end of the fourth cycle, the plant roots were extracted by sieving the soil and returned to the corresponding soil in each pot; this reduced compaction and increased soil aeration at the beginning of the fifth crop cycle. The largest proportion or amount of wheat N that could have been transferred from medic in the last cycle was obtained at the P3 level where 46% (10 mg N plant-‘) of mixed wheat N were derived from fixed-N transfer. This supports the view that the

Competition between medic and wheat for N uptake

release of N from breakdown and decomposition of dead legume tissues is the main process in N transfer from legume to associated grass (Vasilas and Ham, 1985; Goodman, 11988).This increase in turnover of N via nodule and root senescence with time has been shown by others (Haystead and Marriott, 1978; Mallarino et al., 1990). The 15N enrichment of the soil N at the end of the fourth season was lower under sole medic than under sole wheat showi-ng that fixed N, was transferred from medic to the soil through mineralization of nodules and roots during the previous crop cycle. The wheat which had higher % ‘*N excess kept the soil with higher labeled N. Values of the % 15N atom excess in soil under mixed culture were situated between those in soil under sole medic and sole wheat (Table 3) showin that the soil recovered N from medic and wheat :roots, as a result, the soil had less % “N than sole wheat and more % “N than sole medic. The sole medic crop derived an average of 60% of its N from fixation (Table 4). Despite this high %Ndfa, the amount of N taken up from soil by sole medic was higher than that of sole wheat, which depended entirely on soil N. The inclusion of wheat with medic resulted in a considerable reduction in the atom % “N excess of the legume (Table 3), indicating greater reliance on fixed N in the mixture (Table 4). Wahua and Miller (1978) observed a 3.65-fold increase in total IV, fixed in soybean intercropped with sorghum compared with soybean alone. A similar result was fou.nd by Palmason et al. (1992) in lupin-ryegrass mixture. A likely reason for grass stimulation of legume N,-fixation is depletion of soil N by the grass, which would reduce nitrate inhibition of nodulation and module function and increase the reliance of the legume on N,-fixation. In the mixture, the amount of N derived from atmospheric N,, increased significantly by P treatments indicating that under high P rate, wheat took up more soil N and was more competitive for mineral N than medic. In turn, medic fixed more atmospheric Nz to compensate for less mineral N. The %Ndfa was also increased, but not significantly (Table 4). This resulted from the greater competitiveness of wheat for soil N as the mixture was fertilized with P (Table 2). Consequently, the mixed medic increased its dependance on N, fixation more and more. The highest %Ndfa corresponded to the lowest ratio between N, fixation by mixed medic to that by sole medic (Table 4). Nitrogen fixation measured by the TND method was higher than that measured by the ID method, especially when P was added to the soil (Table 4). The main reason for this overestimation was that P had different effects on the uptake of soil N by medic and wheat (Table 2). Using wheat as the reference plant, medic was shown to take up more soil N than wheat with increasing P rates. Therefore, the basic assumption of the difference method, namely that fixing and

87

reference crops take up equal amounts of N from soil, was not proved with increasing P rate. It is reported that the TND had less precision than the ID method (Rennie, 1979) and cannot be used with confidence to estimate N, fixation (Rennie, 1984). It is also noted that the TND method generally underestimates N, fixation (Talbott et al., 1982; Papastylianou, 1987; Guffy et al., 1989) since the legume uses less soil N than the reference plant (Bole and Rennie, 1983). However, many other workers have found a similarity in measurements of biologically-fixed N using the TND method and the ID method (Legg and Sloger, 1975; Talbott et al., 1982; Materon and Danso, 1991). We conclude that the results of this experiment demonstrated that medic and wheat are not compatible in mixture because wheat dominated medic. Mixed cropping favored the growth of grass over that of the legume because of differences in their competitive ability, particularly at high P rates. The medic can fix its own N and leave soil N for the grass which in turn, can compete for other factors such as light and space. In sole-cropped system, medic absorbed more soil N than wheat, especially at the higher P rates. Phosphorus increased competition by wheat and reduced growth of medic intercropped with wheat. The isotope dilution method was more reliable for estimating N, fixation than the total N difference method. Transfer of N from nodules and root decay from medic to wheat was significant after the first crop cycle and was favored by P fertilization. The need to destroy self-regenerating medic in the cereal phase may be avoided through better management of the crop rotation system. Acknowledgements-We thank the Joint FAOIIAEA Division fir financing this study. Our special thanks to R. W. Weaver and M. P. Salema for the 15N analvsis. criticisms and suggestions.

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88

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