Plant development in a mycorrhizal field-grown mixture

0038-0717/91 53.00 + 0.00 Copyright 0 1991 Pcrgamon Rcss plc

Soil Biol. B&hem. Vol. 23, No. 1, pp. 661-665, 1991 Printed in Great Britain. All rights reserved

INTERSPECIFIC N-TRANSFER AND PLANT DEVELOPMENT IN A MYCORRHIZAL FIELD-GROWN MIXTURE CHANTALHAMEL* and DONALD L. SMITH Plant Science Department, Macdonald College of McGill University, 21 I1 I Lakeshore Road, Ste-Anne de Bellevue, Quibec, Canada H9X 1CO (Accepted 20 January 1991) Summary-In the field, a mycorrhiil mixture of corn and soybean was compared to non-mycorrhixal and to P-compensated plant mixtures. The extent of “N-transfer from soybean to corn was assess&. Plant development and the competitive relationship between the components of the mixtures were also examined. After having labelled selected soybean plants with isotopic NH,NO, by feeding roots induced on their stems, a greater amount of i5N-transfer to corn was measured in mycorrhixa inoculated plots than in control plots. The growth of both corn and soybean plants was greatly enhanced when inoculated with Glomur intraradix, and the effect of the fungus could not be replicated by fertilization. Inoculation and P fertilization had similar effects on P, K and Mg uptake by plants, but their effects differed regarding Ca absorption. Inoculation with the mycorrhixal fungus favoured the grass component of the mixture over the legume. Even if more N appeared to be transferred from soybean to corn when plants were mycorrhixal, the nutrient status of the plants suggests that the growth increase can be attributed mainly

to a better P uptake by mycorrhizal plants, and that the significance of interspecific mycorrhixae-mediated N-transfer may be limited.

INTRODUClION

In intercrops and pastures, N-transfer from legumes to grasses can partly substitute for N fertilizers. Values of N-transfer ranging from 2 to 17% of legume N were reported by Agboola and Fayemi (1972), Ledgard et al. (1985) and Brophy et al. (1987), and Willey (1979) estimated the contribution of an intercropped legume to corn at as much as 40kgNha-‘. Accepted mechanisms of N transfer are: (a) by direct root excretion and leaching from leaves and (b) decomposition of fallen leaves (Burton et al., 1983). Recently, results from some experiments indicated enhancement of interspecific N-transfer between plants colonized by a common mycelium of vesicular-arbuscular mycorrhizal fungi. Transfer of lSN from clover to ryegrass (Haystead et al., 1988) and from soybean to corn (Van Kessel et al., 1985) was significantly increased when the plants were mycorrhizal. Francis et al. (1986) grew Festuca ovino and Plantago lanceolata in a split root system. A nutrient gradient was created between the donor and the receiver plant and therefore, a source-to-sink relationship was established between the associated plants. The receiver plant grew better and had a higher N content when mycorrhixal. In the field, a similar N source-to-sink relationship may also be found between legumes and grasses growing together in soil of low fertility. Some field studies have investigated the occurrence of mycorrhizae-mediated N-transfer from legume to

grass grown in association (Barea et al., 1987; 1989a; 1989b). However, for the study of mycorrhixae in grass-legume associations, the validity of the “Ndilution method used by Barea et al. for assessment of N-transfer had been questioned (Hamel et al., 1991). The role of VA mycorrhizae in increasing N-transfer between legumes and associated grasses under field conditions needs to be assessed. We have examined the effect of mycorrhixal colonization on transfer of 15N from 15N-labelled soybean to corn growing in mixture in the field.

MATERIALSAND METHODS Site The experiment was conducted in the summer of 1988, at the E. Lods Research Center of McGill University, on a Chicot fine sandy-loam containing 87.5 mg available P kg-’ of soil (Bray 2) and 70 mg K, 350 mg Mg and 1950 mg Ca kg-’ of soil (ammonium acetate extract). The initial pH of the soil was 7.6. After fumigation with methyl bromide (59 g m2 under a plastic sheet for 3 days), available N was measured. Soil N content was low, probably due to immobilization following incorporation of straw (4 t ha-‘) in the fall of the year preceding the experiment. There was 4.5 mg NH, and 4.6 mg NO, kg-’ of soil. The soil was fertilized with K only. KC1 was incorporated in the spring at a rate of 78 kg ha-‘. Experimental design

Corn (Zeu muys L. cv. pioneer 3809) and soybean (Glycine mux (L.) Merrill cv. Maple Arrow) were

*Present address: Institut de Recherche en Biologie Vigetale, 4101 Sherbrooke Est, Montreal, Quebec, Canada HlX 2B2.

sown in the same row in 3 m x 3 m plots, at densities 661

CEIAN-~ALHAMEL~~~DONALDL.SMITH

662

equivalent to 40,000 and 250,000 plants ha-’ respectively. There were 75 cm between the rows. The experiment consisted of four treatments randomized into each of four complete blocks. Plants were either mycorrhixal and did not receive P fertilizer, or were not mycorrhixal and received either 0, 53 or 106 kgP ha-‘. These rates of P fertilization correspond to 0, 0.66 time and 1.32 times the rate recommended for that soil (Anonymous, 1987). Increasing rates of P fertilizer were applied to the control plots in an attempt to compensate for the lack of fungusassisted P uptake and to produce a range of plant growth responses. Seeding Leek (Aflium porum) root pieces (40 g m-’ of row) colonized by Glomus intraradix Schenck & Smith were spread in 7-cm deep hand-made furrows and covered with 5 cm of soil. The non-mycorrhizal plots received autoclaved (126°C for 20 min) leek roots and 12.5 ml m-t of a solution of filtered (Whatman no 1) inocuhun washing. Seeds of corn and soybean were then placed in the furrow and Bradyrhizobium japonicum inoculum (Nitragin Co.) was broadcast over them before closing the furrows. After emergence, plant density was carefully adjusted to 3 corn and 19 soybean plants m-r of row. N-15 labelling Root growth was induced on selected soybean stems by air-layering, 29 days after emergence. Moist sand : Turface (1: 1, v/v) filled plastic pouches were placed around the stem of two soybean plants adjacent to one randomly selected corn plant in each plot. Some IBA powder (Chipman Inc.) was applied to two scars made on soybean stems, to improve root induction. Seventeen days later, 31 mg of “NH,NO, (99% “N) in 3 ml of water was injected into each pouch. Again, 7 and 14 days later, 63.5 mg and 80 mg “NH,N03 respectively, were similarly applied into each pouch. Sampling and harvest Plants were sampled for tissue analysis 35 days after emergence. Corn plants were then at the

2000

seedling stage and soybean plants had reached growth stage RI (Fehr et al., 1971). The first two expanded leaves of corn and soybean were collected, dried at 70°C for 2 days, ground (~2 mm) and assayed for N by a standard Kjeldahl method, and for P by the method of Murphy and Riley (1962) after wet digestion of the plant material (Thomas et al., 1967). At mid-season, a fence of metallic wire mesh was installed around the plots to keep soybean leaves on site as the plants senesced. Fallen leaves were collected regularly and dried. After 4 months, 1.3 m of the two middle rows of each plot were harvested. Plant tops were dried, ground, and assayed for P and N content, as described above, and for K, Ca, Mg, Zn and Mn by atomic absorption spectrometry. The percentages of “N in labelled soybean plants and their adjacent corn plant was also determined by emission spectrometry (Fiedler and Proksch, 1974) after drying of their Kjeldahl ammonium solutions under vacuum. Plants from a “N free area were used for determination of rsN natural abundance. The roots were sampled to determine the extent of root colonization by the mycorrhixal fungus. Percentage of root colonization was determined by the grid intersect method (Giovanetti and MOW, 1980) after clearing and staining the roots with acid fuchsin (Brundrett et al., 1984). Plant root density was also evaluated (Tennant, 1975) from soil samples taken to a depth of 15 cm, at 15 cm intervals from three randomly selected corn plants, using 10 cm (dia) cores. An analysis of variance (Steel and Tot-tie, 1980) was conducted on all measured variables using PC-SAS (Ray, 1982).

RESULTS Mycorrhizal colonization At harvest, mycorrhizal fungi had colonized on average, 80% of the root length of plants sampled in the inoculated plots, while less than 1% of root length were mycorrhizally infected in the control plots (data not shown).

(4 G

r

3.5r 3.0 -

i!i

1500

g

8

i E .2 m

2.5

-

(‘I

a a ab

r

1000

g

1.5-

500

M

PO

Pl

P2

Fig. 1. (A) Dry biomass of corn and soybean and (B) corn :soybean dry mass ratios at harvest. Plants were inoculated with Gfomus fnrraradtx (M), or non-inoculated and given no phosphate (PO),0.66 time (PI) or 1.32 times (P2) the P recommended rate. Bars height represent the mean of four replicates and are not significantly different (P < 0.05, ANOVA protected LSD test) when lahelled with the same letter.

Mycorrhiza involvement in plant mixtures Table 3. “N enriclunent of corn as affected by inoculation with G/emus turarudix (M). or given no phosphate (PO), 0.66 (PI) or 1.32 times (P2) the phosphate rate recommended

Table 1. N and P concentration 35 days after emergence in soybean and corn inoculated with Glomus introradix (M), or non-inoculated and given no phosphate (PO), 0.66 (PI) or 1.32 times (P2) the phosphate rate recommended Concentration (mg g-l) Soybean Treatment

M

44.2 43.1 40.6 45.3

PO

Pi P2

a a a a

2.65 2.06 2.35 2.64

N a b b a

Treatment

a a a a

2.29 ab 1.73 b 2.28 ab 2.58 a

156.2 a 19.8 b 15.6b 11.3b

0.6075 0.2789 0.1099 0.0855

a b b b

Numbers are means of four replicates and are not significantly ditTerent (P
Numbers are means of four replicates and are not significantly different (P < 0.05, ANOVA protected LSD test) when followed by the same letter within a column.

Plant biomass production of up to 1.32 times the P rate recommended for the soil, mycorrhizal

Despite the provision fertilizer

(9)

M PO PI P2

P

35.5 34.1 37.1 36.3

“N atom excess in corn 0)

‘JN in corn

Corn P

N

663

plants grew much larger than any of the P-compensated controls Fig. l(a)]. The difference between mycorrhizal and control plant biomass was especially large for corn plants, where the biomass of the mycorrhizal plants was increased 1.75 fold over that of controls fertilized with 1.32 times the recommended P rate. Mycorrhizal soybean plants produced 1.27 fold more biomass than the control plants fertilized with the highest amount of P fertilizer. The corn: soybean dry mass ratio was almost doubled in the mycorrhizal plots as compared to the non-fertilized controls [Fig. l(b)]. P fertilization, up to 0.66 time the recommended P rate, also increased the corn : soybean ratio. However, above this rate, the ratio decreased as corn plants did not respond to the highest P fertilization level.

the highest tissue P content, followed by controls fertilized with 1.32 times the recommended P rate, then, by the ones receiving the lower P rate, and finally, by the non-fertilized controls (Table 2). Inoculated plants and P-fertilized controls differed also in that P fertilization increased the Ca content in soybean tissues and had no significant effect on corn Ca content while, in contrast, mycorrhizal coloniration decreased the Ca content in corn and had no significant effect on soybean Ca level. In addition, soybean Zn content was decreased by P fertilization and not affected by inoculation. Nevertheless, inoculation and P fertilization had similar effects on plant K and Mg contents, as both treatments increased plant K content and reduced Mg content. Neither treatment affected corn Zn content. Although plant N content tended to be higher in the presence of the fungus, inoculation did not significantly increase corn N concentration (Table 2). Transfer of “N

Mineral con tent

Thirty five days after emergence, the effect of mycorrhizal inoculation on uptake of minerals by soybean could already be seen (Table 1). Mycorrhizal soybean plants and controls receiving 1.32 times the recommended P fertilizer rate had similar P tissue contents, while the less and the non-fertilized controls had lower P contents. In contrast, inoculation effects on corn P uptake were not yet apparent. At harvest, the effect of the treatments on plant P content was more marked. Mycorrhizal plants had

The percentage of N as 15N above natural abundance in corn plants was higher in inoculated than non-inoculated plots, while there was no significant difference in the i5N enrichment of control treatments (Table 3). When expressed as the actual amount of i5N in corn in excess of the natural abundance of the isotope, the difference between the mycorrhizal and the control treatments became dramatic (‘fable 3). The expression of the data on the basis of the amount of “N transferred per unit of root density gives a

Tabk 2. Nutrient concentration at harvest in soybean and corn inoculated with Glomw intrarodix (M), or non-inoculated and given no phosphate (PO), 0.66 time (PI) or 1.32 times (P2) the phosphate rate recommended

N

P

K

Ma

Ca

(meg-‘)

Treatment

Zn

Mn

(CBB-‘)

Soybean El PI P2 Corn

M

;: P3

21.4 25.9 25.4 26.6

a ab b ab

IS.6 a 14.6 a IS.1 a 14.1 a

2.9 a 1.6d l.9c 2.1 b 2.2 a I.1 c l.2c 1.6b

14.9 b 13.3c 16.1 a 16.3 a 7.4 a 5.2 b 6.0 ab 6.4 b

4.6 c 5.3 a 5.0 b 4.9 b

10.6 b 10.6 b 10.9 b 11.6a

40.5 a 39.4 a 33.3 b 31.3b

84.7 98.3 85.8 77.0

ab a ab b

1.7b 2.5 a 1.8b 1.7b

1.2b 2.4 a 2.0 a 2.0 a

18.5a 17.8 a 17.0a 16.5 a

18.0 ab 21.8ab 26.5 a 14.3 b

Numbers ara means of four replicates and are not significantly ditTerent (P ~0.05. ANOVA protected LSD test) when followed by the same letter within a column and a crop.

664

CNANTAL

HAMEL and

DONALD

L. Smrn

measure of the efficiency of the roots at transferring r5N (Fig. 2). Mycorrhizal roots appeared more efficient at transferring “N than non-mycorrhizal roots, while the roots of the non-fertilized control plants were the least efficient.

DlSCUsSlON

E#ect of mycorrhizal inoculation and P fertilization on plant development Plant response to P fertilization was weak and the lack of mycorrhizal infection could not be compensated for by P fertilization up to 1.32 times the rate of P fertilizer recommended for the soil. However, the possibility that P availability did not limit plant growth cannot be ruled out in this study as growth enhancement by mycorrhizal inoculation was accompanied by increased P concentration in plant tissue. It is known that vesicular-arbuscular mycorrhizal fungi can increase P uptake by plants through their phosphatase activity (Dodd et al., 1987), through a better exploration of the soil by hyphae (Bolan et al., 1987). and through the uptake of tixed soil P, which is unavailable to plant roots (Young et al., 1986). Our results emphasize the dependence of corn and soybean plants on mycorrhizal colonization for P absorption. Mycorrhizal colonization also affected the content of other elements in plant tissues. The mycorrhizal condition in corn favoured the uptake of K relative to Mg and Ca, while in soybean, both mycorrhizal colonization and P fertilization favoured K uptake. P fertilization also favoured Ca uptake by soybean. It is interesting to note that although P fertilization and mycorrhizal colonization had a similar effect on the content of Mg and K in plants, the effect of mycorrhizae and P fertilizer were different for Ca uptake and changed between soybean and corn. This suggests that the P and the mycorrhizal effect are not entirely equivalent and may also differ with plant species. Despite the great differences at harvest between biomass and tissue P content of mycorrhizal and non-mycorrhizal plants, 35 days after emergence, P-compensated and mycorrhizal corn and soybean plants had similar P contents in their tissues. This result suggests that the effect of mycorrhizal inoculation on plant P uptake was relatively slow to develop. The importance of high soil inoculum potential and early development of the symbiosis is recognized for mycotrophic annual plants (Fairchild and Miller, 1988; Read and Birch, 1988). Mycorrhizal colonization and, to a lesser extent, P fertilization, increased the proportion of corn : soybean in the mixture. It is generally accepted that mycorrhizal colonization in grass-legume associations confers an advantage to the legume (Haynes, 1980). In fact, it seems that mycorrhizal fungi will give more advantage to the more mycotrophic plant species (Hetrick et al., 1989) which is usually the legume component of the mixture. Because grasses possess a fine and fibrous root system with many root hairs, they generally benefit less from the mycorrhizal symbiosis than legumes which have a less extensive root system (Sparhng and Tinker, 1975). Corn, a

idl PO P’l P-2 Fig. 2. Amount of 15Ntransferred per unit of root density measured in plots under the different treatments. Notation as in Fig. I. Bars height represent the mean of four replicates and are not significantly different (P < 0.05. ANOVA protected LSD test) when label14 with the same kttcr.

grass species which is fairly dependent on mycorrhizae (Plenchette et al., 1983), appears to be an exception to the general rule, perhaps because it has a much coarser root system than most grasses. E#ect of mycorrhizal inoculation on “N-transfer from soybean to corn The greater amount of lSN in excess by concentration and the higher mass of ‘rN in mycorrhiil corn suggests a role for G. intraradix in 15N-transfer from soybean to corn, and confirms results obtained from pot experiments (Haystead et al., 1988; Van Kessel et al., 1985). However, the ‘rN excess in mycorrhizal plants and that of stunted P-compensated plants cannot fairly be compared. Bigger corn and soybean plants are likely to have more root contact, and therefore, to accumulate more ‘W than smaller plants. The different size of mycorrhizal and non-mycorrhizal plants confounds the effect of the fungus. To overcome this difficulty, the i5N data were expressed in terms of the amount of 15N transferred per unit of root density, a measure of root efficiency. Calculated on a root density basis, mycorrhizal inoculation increased plant root efficiency for the transfer of “N, by 2.3 fold. Mycorrhizal inoculation greatly increased lSNtransfer to corn but did not significantly alter its N concentration. These results seem to indicate that mycorrhizae-mediated interspecific N-transfer is not entirely due to improved corn root absorption when mycorrhizal, but that mycorrhizal links between soy bean and corn plants may also play a role in the enhancement of N-transfer. The relative importance of mycorrhizal links and of the improved efficiency of the corn root system for the uptake of N excreted by soybean roots, could be better assessed in pot experiments. It is difficult to estimate the importance of mycorrhizae-mediated N-transfer from this study. Although the presence of the mycorrhizal fungus increased corn N yield, the fungus did not improve significantly the N concentration in corn plant tissues, suggesting that mycorrhizae-mediated Ntransfer may have little significance at the crop production level.

Mycorrhii

involvement in plant mixtures

Acknowledgements-This research was supported by a grant of the Conseil de Recherches en P&he-et en AgroAhmcntaire du Quebec, and a grant of the Natural Sciences and Enaineerina Research Council of Canada, held by D. L. Smith. c Hamel gratefully acknowledges support by the Natural Sciences and Engineering Research Council of Canada Post Graduate Scholarship Program. REFERENCES

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