Wild-Type and Nodulation-Mutant Soybean Plants'. Myeong-Je Cho and James E. Harper*. Department of Agronomy (M.-J.C.) and United States Department of ...
Received for publication January 28, 1991 Accepted April 8, 1991
Plant Physiol. (1991) 96, 1277-1282 0032-0889/91/96/1 277/06/$01 .00/0
Root Isoflavonoid Response to Grafting between Wild-Type and Nodulation-Mutant Soybean Plants' Myeong-Je Cho and James E. Harper* Department of Agronomy (M.-J.C.) and United States Department of Agriculture, Agricultural Research Service, Plant Physiology and Genetics Research Unit (J.E.H.), University of Illinois, 1201 W. Gregory, Urbana, Illinois 61801 mutants have greater nodule numbers, nodule dry weights, and nitrogenase activities than their respective wild types. In addition, nonnodulating soybean mutants have also been isolated by Carroll et al. (5) and Harper (15) from Bragg and Williams, respectively. Based on grafting studies (9-1 1), the supernodulation and hypernodulation phenotypes derived from wild-type Bragg were reported to be shoot controlled, while the nonnodulation phenotype was root controlled. It was proposed that a shootderived inhibitor(s) was involved in autoregulation of nodulation (14). Gresshoff et al. (13) reported that feeding methanol/water extracts from the inoculated Bragg wild type to nts382 supernodulated mutant plants suppressed modulation by 60 to 80%, but extracts from uninoculated wild-type or nts382 plants were not inhibitory to modulation. The isoflavones, daidzein and genistein, have been isolated and identified as major inducers of nod genes in B. japonicum (18). In addition, coumestrol and daidzein have also been shown to promote the growth of B. japonicum (8). Our recent studies (6, 7) have shown that the Williams-derived hypernodulating mutants (NOD 1-3 and NOD2-4) accumulate higher concentrations of isoflavonoid compounds (daidzein, genistein, and coumestrol) than does the parent at 9 to 12 d after inoculation with B. japonicum. There were, however, no significant differences in isoflavonoid concentrations among lines when plants were not inoculated. In this study, root isoflavonoid concentrations and nodulation characteristics from reciprocal and self grafts between the Williams parent and hypernodulating and nonnodulating mutants were compared. The objective was to determine if shoot control of hypernodulation was possibly related to root isoflavonoid levels. MATERIALS AND METHODS Plant Culture and Grafting Three soybean (Glycine max [L.] Merr.) lines were evaluated, including the parent line (Williams), a hypernodulating mutant (NOD 1-3), and a nonnodulating mutant (NN5). Both mutants were previously selected from the cv Williams (12, 15). Seeds were surface-sterilized with 5% (v/v) Clorox2 plus
ABSTRACT It was previously reported that the hypemodulating soybean (Glycine max [L.] Merr.) mutants, derived from the cultivar Williams, had higher root concentration of isoflavonoid compounds (daidzein, genistein, and coumestrol) than did Williams at 9 to 12 days after inoculation with Bradyrhizobium japonicum. These compounds are known inducers of nod genes in B. japonicum and may be involved in subsequent nodule development. The current study involving reciprocal grafts between NODI-3 (hypemodulating mutant) and Williams showed that root isoflavonoid concentration and content was more than twofold greater when the shoot genotype was NOD1-3. When grafted, NOD1-3 shoots also induced hypemodulation on roots of both Williams and NOD1-3, while Williams shoots induced normal modulation on both root genotypes. This shoot control of hypemodulation may be causally related to differential root isoflavonoid levels, which are also controlled by the shoot. In contrast, the nonnodulating characteristic of the NN5 mutant was strictly root controlled, based on reciprocal grafts. Delayed inoculation (7 days after planting) resulted in greater nodule numbers on both NOD1-3 and Williams, compared with a seed inoculation treatment. The modulation pattern of grafted plants was independent of whether the shoot portion was derived from inoculated seed or uninoculated seed, when grafted at day 7 onto seedling roots derived from inoculated seed. This observation, coupled with the fact that no difference existed in nodule number of NOD1-3 and Williams until after 9 days from seed inoculation, indicated that if isoflavonoids play a role in differential modulation of the hypemodulating mutant and the wild type, the effect is on advanced stages of nodule ontogeny, possibly related to autoregulation, rather than on initial infection stages.
The formation of nitrogen-fixing nodules on soybean (Glycine max [L.] Merr.) roots occurs in response to infection by Bradyrhizobiumjaponicum. After bacterial infection, the nodulation of a normal soybean plant is restricted by a plant process called autoregulation, in which the nodule formation on one part of the root systemically suppresses subsequent nodule formation in other root regions (2, 17, 21). Supernodulating and hypernodulating mutants that appear to have altered this autoregulatory control of modulation have recently been derived from Bragg and Williams parents (4, 12). These
2 Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the vendor or product by the United States Department of Agriculture, and does not imply its approval to the exclusion of other vendors or products that may also be suitable.
' Supported in part by the American Soybean Association, Research Project 88412.
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one drop of Tween 20 for 10 min and rinsed with deionized distilled water. Seeds were planted in sterilized sand in perforated trays and watered from the bottom with distilled water. Seeds were germinated and subsequently grown in growth chambers programmed for 14-h photoperiods at 650 ismol photons m-2 s-' at 29C and 10-h dark periods at 20C. At 7 d after planting, depending on experiment, seedlings were either transplanted to nutrient solution or were subjected to a grafting treatment and transplanted 4 d later. Bradyrhizobium japonicum strain USDA 110 was used to inoculate either seeds or seedling roots as indicated in the Table and Figure legends. Inoculum was grown in liquid yeast extractmannitol-gluconate medium (2) and diluted with sterilized deionized water to 108 cells/mL. An uninoculated control was included with some studies as indicated. For studies involving grafting, grafts were made on 7-d-old seedlings in the sand trays used for germination. Wedgeshaped grafts with cotyledons left on the scion were used, essentially as described by Bezdicek et al. (1). Grafts were held in place by = l-cm sections of plastic drinking straws (2.5 mm i.d.) which had been split on one side to allow for expansion. Use of the smaller straw eliminated the need for any band as described (1). Each tray of grafted plants was covered by a transparent plastic lid embedded into the moist sand to maintain high humidity around the seedlings. Grafted seedlings were grown for 2 d in the dark followed by 2 d in diurnal light/dark before transplanting. Both intact and grafted seedlings were transplanted to 18L polypropylene trays containing a modified minus N Hoagland nutrient solution as previously described (12). Seedlings were suspended through holes in Styrofoam lids placed over the trays. The solution pH was maintained at 6.5 ± 0.5 with ion exchange resin columns (16). Nutrient solutions were completely changed at 7-d intervals after transplanting.
HPLC Analysis of Isoflavonoid Compounds
Grafted plants were analyzed for isoflavonoid compounds by HPLC as previously reported (7). Samples were taken at 15 and 20 d after grafting and inoculation, with four and
Plant Physiol. Vol. 96, 1991
three replicates, respectively. Replicates consisted of two plants at 15 d and one plant at 20 d.
C2H2 Reduction Assay and Nodule Observations Twenty-five days after inoculation, plants were harvested for in vivo C2H2 reduction assay as described earlier (12), except that a single modulated root was incubated in 250-mL gas-tightjars with 10% (v/v) C2H2. Four single-plant replicates of each treatment were evaluated. The jars were sealed and 25 mL of C2H2 was injected. After the assay, nodules were removed from the roots and counted. Plant roots and nodules were then dried for dry matter determinations. The ontogeny of nodule development was followed in two separate studies: one involving grafted plants and another comparing seed inoculation with delayed inoculation. Observation of nodule development was made with a light microscope (Bausch & Lomb Inc., Rochester, NY) at 16x magnification. Statistical Analysis
Analysis of variance was performed for most experiments and treatment means were compared using Fisher's LSD when significant F tests occurred. Due to unequal sample variance when comparing nodule numbers ofhypernodulating, normal modulating, and nonnodulating lines, data are presented as means
±
SD.
RESULTS Nodulation and Growth Characteristics of Grafted Plants Data of Table I show the modulation characteristics of reciprocal- and self-grafted plants between the NOD1-3 hypernodulating mutant and the Williams wild type at 25 d after inoculation. NOD 1-3 shoots induced hypernodulation when grafted onto roots of Williams and NOD1-3. In contrast, the Williams wild-type shoots gave wild-type modulation patterns on roots of both soybean lines. Grafted NOD 1-3 shoots induced four times as many nodules as Williams shoots, irrespective of root genotype. Nodule mass and C2H2
Table I. Nodulation and Growth Characteristics of Reciprocal- and Self-Grafted Plants between NOD1-3 and the Williams Parent Seeds were germinated in sterilized sand and 7-d-old seedlings were grafted and then inoculated with B. japonicum. Grafted seedlings were covered by transparent plastic lids to provide high humidity and were maintained in the dark for 2 d followed by diurnal light for 2 d. Four days after grafting and inoculation, seedlings were transplanted to the 18-L trays containing a modified minus N Hoagland nutrient solution. Twenty-five days after inoculation, plants were harvested for nodule measurement and C2H2 reduction assay. Values represent means of four replicates for each grafting treatment and values in parentheses represent root dry weight without nodules. Graft
(Shoot/Root)
Williams/Williams Williams/NOD1-3 NODl -3/Williams NOD1-3/NOD1-3 LSDo.05
Nodule No. No. plant-
Nodule Dry Wt mg plant-'
Root
Shoot
C2H2Reducton moml C2H4 plant' h-'
Dry Wt
Dry Wt
196 187 772 752 165
56 47 104 108 24
6.20 5.11 11.19 11.64 2.03
0.375 (0.319) 0.333 (0.286) 0.251 (0.147) 0.236 (0.128) 0.065 (0.046)
.
g plant-
0.617 0.616 0.312 0.390 0.098
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SOYBEAN ROOT ISOFLAVONOID RESPONSE TO GRAFTING
reduction activities were also enhanced by the NOD 1-3 shoot when grafted to either NOD 1-3 or Williams roots, compared with the situation in which Williams was used as the shoot source for grafting. Williams shoots induced greater shoot and root growth than did NOD 1-3 shoots. The root genotype had no effect on growth of either root or shoot when a common shoot was grafted. Microscopic observation of soybean modulation showed that nodule numbers continued to increase throughout the 24-d growth period after inoculation with all grafting treatments (Fig. 1). Through 9 d after inoculation, no significant difference in nodule number among grafting treatments was measurable. After that time, however, grafts involving NOD1-3 shoots had significantly greater nodule numbers than those involving Williams shoots, regardless of whether the grafted root was from Williams or NOD 1-3. This difference increased with time. Greater nodule numbers were detected in all graft combinations upon microscopic observation (Fig. 1) than when physically removed and counted (Table I), due to inability to remove the very small nodules. Isoflavonoid Analysis of Root Extracts from Grafted Plants When hypernodulating mutant NOD1-3 shoots were grafted onto roots of either Williams or NOD 1-3, higher root isoflavonoid concentrations (and contents, data not shown) were observed at 15 d after inoculation, compared with grafts involving Williams shoots (Fig. 2). Self grafts ofNOD1-3 had even higher isoflavonoid concentrations than did reciprocal grafts of hypernodulating mutant NOD 1-3 shoots onto Williams roots. However, there was no significant difference in root isoflavonoid concentration between self and reciprocal grafts involving Williams shoots. A repeat evaluation (data not shown) of root isoflavonoids at 20 d after inoculation 1800 1600 CO
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provided confirmation of the pattern and level of isoflavonoids presented in Figure 2. Shoot versus Root Control of Nodulation The modulation pattern of grafted plants was independent of whether the shoot portion was derived from inoculated seed or noninoculated seed, when grafted at day 7 onto seedling roots derived from inoculated seed (Table II). This finding was true across all combinations involving Williams, NOD 1-3, and NN5. Seven-day-delayed inoculation (Table I) resulted in considerably greater numbers of nodules per plant than did seed inoculation (Table II). The shoot control of hypernodulation and normal modulation noted for seedlinginoculated plants (Table I) was confirmed with grafts of seedinoculated plants (Table II). In contrast to the hypernodulation phenotype, the inability of the NN5 nonnodulating mutant to form nodules was strictly determined by the root (Table II). Regardless of whether the grafted shoot was that of Williams or NOD 1-3, the NN5 roots failed to form nodules. Grafting nonnodulating mutant NN5 shoots onto both Williams and NOD 1-3 roots resulted in similar nodule numbers to that observed when Williams served as the shoot source.
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Day.,s after Grafting and Inoculation Figure 1. Changes in modulation of reciprocal- and self-grafted plants between NOD1 -3 and the Williams parent. Nodules were counted with a light microscope (x16) at 3-d intervals through a 24-d growth period after grafting and inoculation. Other details as in Table I legend. Values represent means of three replicates for each grafting treatment within a sampling time and vertical bars are LSDo.05; n.s. denotes nonsignificant differences among treatments at the 0.05 level.
Delayed Inoculation Effect on Nodulation
Seven-day-delayed inoculation markedly increased nodule numbers of both NOD 1-3 and Williams, compared with the seed-inoculation treatment (Fig. 3A). The 7-d-delayed inoculation treatment induced modulation mainly (about 90% of total nodules) on the branched lateral roots of both soybean lines (Fig. 3B). In contrast, seed-inoculated plants formed nodules primarily on the tap root at the early observation times but the percentage of nodules on the tap root decreased
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Table II. Nodulation Characteristics of Reciprocal- and Self-Grafted Plants among Williams, NOD1-3, and NN5 with Inoculation Treatments Seeds were inoculated with B. japonicum prior to planting, with the exception of those seeds giving rise to shoot portions designated below as "noninoculated." Germination, grafting, and subsequent growth conditions were as described in Table I. Plants were harvested for nodule measurement at 26 d after inoculation and germination. Values expressed are as means + SD (n = 3 or 4). Graft (Shoot/Root)
Inoculated/inoculated Williams/Williams Williams/NOD1-3 Williams/NN5 NOD1-3/Williams NOD1-3/NOD1-3 NOD1-3/NN5 NN5/Williams NN5/NOD1-3 NN5/NN5 Noninoculated/inoculated Williams/Williams Williams/NOD1-3 Williams/NN5 NOD1-3/Williams
NOD1-3/NOD1-3 NOD1-3/NN5 NN5/Williams NN5/NOD1-3 NN5/NN5
No.ule
Nodule
No. plant-'
mg plant-'
33 ± 13 36 ± 6 0±0 242 ± 30 203 ± 18 0±0 34 ± 10 53 ± 9 0±0
38 ± 7 28 ± 4 0±0 63 ± 10 75 ± 3 0±0 33 ± 5 34 ± 2 0±0
53 ± 10 37 ± 3 0±0 255 ± 29 194 ± 25 0±0 37 ± 5 42 ± 12 0±0
34 ± 11 29 ± 6 0±0 94 ± 17 69 ± 22 0±0 29 ± 3 24 ± 4 0±0
zein, genistein, and coumestrol) than did the Williams parent when inoculated with B. japonicum. The current study extended this work by determining root isoflavonoid concentrations and contents from reciprocal and self grafts between NOD 1-3 and the Williams parent. Isoflavonoid analyses from root extracts of grafted plants showed that NOD1-3 shoots had markedly higher root isoflavonoid concentrations (Fig. 2) and contents (data not shown) in roots of both lines compared with Williams shoots. These results are consistent with the hypothesis that root isoflavonoid levels may be related to differential modulation expression by a mechanism that is unrelated to the previously reported involvement of isoflavonoids on nod gene induction (18). Measuring isoflavonoid levels in root exudates did not appear pertinent to this study, because the responses noted appear to be postinfection. This conclusion is consistent with the observation that Bragg and its supernodulating nts382 mutant were similar in terms of 2400
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with time. Nodules on the tap roots of seedling-inoculated plants occurred on the area that exhibited young root hairs at the time of inoculation (data not shown). With the 7-d-delay inoculation treatment, no significant difference in nodule numbers between soybean lines was detected until 9 d after inoculation, whereas with the seed-inoculation treatment, difference between lines was not detected until 18 d using the pooled analyses (Fig. 3A). Difference between lines was detectable by day 12 for the seed-inoculated treatment if analyzed as a separate experiment, due to much smaller variability in nodule number for seed inoculation than for delayed inoculation treatments (analyses not shown). As time proceeded, the difference in nodule numbers between NOD1-3 and the Williams parent became greater.
DISCUSSION The current grafting study showed that shoots from the Williams wild type induced wild-type modulation, while shoots from the NOD1-3 hypernodulating mutant induced hypernodulation on roots of both lines (Table I, II, Fig. 1). This finding confirms the conclusion made in previous grafting studies (9-1 1) that the supernodulation and hypernodulation mutants derived from Bragg are controlled through the action of the shoot. Our recent studies (6, 7) demonstrated that the hypernodulating mutants from Williams accumulated higher concentrations of isoflavonoid compounds (daid-
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Days after Inoculation Figure 3. Effect of delayed inoculation on (A) nodule numbers and (B) percentage of nodules on the tap roots of NOD11-3 and the Williams parent. Either seeds or 7-d-old seedlings were inoculated with B. japonicum. After 7 d growth in sand, plants were transplanted to 18-L trays containing nutrient solution. Seedling-inoculated plants were transplanted 1 h after inoculation. Plant roots were harvested and separated into tap roots and lateral roots. Nodules were counted with a light microscope (x16) at 3-d intervals through a 21-d growth period after inoculation. Values represent means of three replicates for each treatment within a sampling time and vertical bars are LSDo.05; n.s. denotes nonsignificant differences among treatments at the 0.05 level.
SOYBEAN ROOT ISOFLAVONOID RESPONSE TO GRAFTING
number of infection events but differed in number of infection events that subsequently progressed to form a nodule ( 19). In contrast to shoot control of hypernodulation, the nonnodulating mutant NN5 roots failed to nodulate, regardless ofwhether the shoot used in grafting was from NN5, Williams, or NOD 1-3 (Table II). Thus, the inability of the NN5 mutant to form nodules is strictly controlled by the root, not the shoot. This also confirms the conclusion of Delves et al. ( 11) that nonnodulation is root controlled in mutant nod49, regardless of whether the grafted shoot was that of nts382 or the Bragg parent. It is known from analysis for nod gene inducibility in B. japonicum that seedling extracts or exudates from nonnodulating, supernodulating, and the parent (Bragg) lines have similar competence in nod induction (20, 22). Nonnodulating mutants are either insensitive to the recognition of the plant signal compounds or unable to convert the cell division stimulus into an actual infection after response to the signal (14). Our previous result (6) also showed that, even though the inoculated NN5 nonnodulating mutant had higher isoflavonoid concentration than did the Williams parent, it did not develop any nodules, nor did it respond to the addition of isoflavonoids to the growth medium. Thus, the nonnodulation characteristic is not related to isoflavonoids (6). Shoot control of autoregulation of modulation may involve the presence of shoot-derived inhibitors) as well as activator(s). It was reported that injection of methanol/water extracts from the inoculated Bragg wild type (but not uninoculated wild type or either uninoculated or inoculated nts382) suppressed modulation of the nts382 supernodulating mutant (13). Gresshoff et al. (14) proposed that the suppression activity of modulation is correlated with an alteration of ABA concentration in the shoot. ABA remained at a preinoculated level in both inoculated supernodulating mutants and uninoculated wild-type plants, but it increased in inoculated wildtype plants in the shoot after 2 to 3 d, reaching a plateau by day 7 or 8 (14). The current study evaluated the grafting effect of shoots from seed-inoculated or uninoculated plants, which might have different levels of shoot inhibitorss, on modulation of seed-inoculated root stocks. The lack of any difference in modulation pattern of seed-inoculated roots when grafted on day 7 to shoots derived from seedlings from inoculated and uninoculated seed (Table II) indicates that inoculation may not significantly alter the levels of possible shoot inhibitors) through day 7. In addition, no significant difference in nodule numbers between NOD 1-3 and Williams was detected until after 9 d following seed inoculation (Fig. 3A). These results indirectly support the previous conclusions (3, 19) that subsequent nodule formation is suppressed primarily at the stage of nodule emergence rather than at the stage of initial
infection. Inoculation of 7-d-old grafted plants resulted in a marked increase of modulation in all grafting treatments of both NOD 1-3 and Williams compared with grafting of seed-inoculated plants (Tables I and II). This was likely due to more potential infection sites becoming simultaneously infected on 7-d-old seedlings as opposed to stronger autoregulatory control exercised in seed-inoculated plants. The same observation was made on ungrafted plants (Fig. 3A). This may be causally related to higher root isoflavonoid concentrations in the roots of the older plants during the period of nodule initiation and
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nodule development. Our previous results (6) showed that isoflavonoid levels increased with age of the roots of uninoculated soybean plants as well as of inoculated plants. Delayed inoculation induced nodule formation mostly on the branched lateral roots, whereas seed inoculation induced nodulation initially on the tap roots with increasing nodule numbers on the lateral roots and in the developing root zone of the tap roots with time (Fig. 3B). This again relates to the potential infectable sites at the time of inoculation. Nodules of delayed-inoculated plants were almost completely eliminated in the upper zone of the tap roots (data not shown). This is consistent with the conclusion of Bhuvaneswari et al. (2) that, when inoculations were delayed for intervals of 1 to 4 h after marking the positions of the root tips, the modulation above the root tip marks was progressively decreased. In conclusion, the results shown here support the previous conclusion (6) that isoflavonoids may be involved in differential modulation expression between hypernodulated and wild-type soybean lines at the advanced stage of nodule ontogeny rather than at the stage of initial infection. The similarity of response to grafting for the hypernodulating and nonnodulating mutants of Williams reported here and for mutants of Bragg reported previously ( 11) may indicate that the selected mutants involve similar genetic defects.
1. 2. 3.
4. 5.
6. 7.
8.
9. 10. 11.
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LITERATURE CITED Bezdicek DF, Magee BH, Schillinger JA (1972) Improved reciprocal grafting technique for soybeans (Glycine max L.). Agron J 64: 558 Bhuvaneswari TV, Turgeon BG, Bauer WD (1980) Early events in the infection of soybean (Glycine max L. Merr.) by Rhizobium japonicum. Plant Physiol 66: 1027-1031 Calvert HE, Pence MK, Pierce M, Malik NSA, Bauer WD (1984) Anatomical analysis of the development and distribution of Rhizobium infections in soybean roots. Can J Bot 62: 2375-2384 Carroll BJ, McNeil DL, Gresshoff PM (1985) A supernodulation and nitrate-tolerant symbiotic (nts) soybean mutant. Plant Physiol 78: 34-40 Carroll BJ, McNeil DL, Gresshoff PM (1986) Mutagenesis of soybean (Glycine max (L.) Merr.) and the isolation of nonnodulating mutants. Plant Sci 47: 109-114 Cho M-J, Harper JE (1991) Effect of inoculation and nitrogen on isoflavonoid concentration in wild-type and nodulationmutant soybean roots. Plant Physiol 95: 435-442 Cho M-J, Harper JE (1991) Effect of localized nitrate application on isoflavonoid concentration and modulation in split-root systems of wild-type and nodulation-mutant soybean plants. Plant Physiol 95: 1106-1112 d'Arcy Lameta A (1987) Study of soybean and lentil root exudates. III. Influence of soybean isoflavonoids on the growth of rhizobia and some rhizospheric microorganisms. Plant Soil 101: 267-272 Delves AC, Higgins AV, Gresshoff PM (1987) Shoot control of supernodulation in a number of mutant soybeans, Glycine max (L.) Merr. Aust J Plant Physiol 14: 689-694 Delves AC, Higgins AV, Gresshoff PM (1987) Supernodulation in interspecific grafts between Glycine max (soybean) and Glycine soja. J Plant Physiol 128: 473-478 Delves AC, Mathews A, Day DA, Carter AS, Carroll BJ, Gresshoff PM (1986) Regulation ofthe soybean-Rhizobium nodule symbiosis by shoot and root factors. Plant Physiol 82: 588590 Gremaud MF, Harper JE (1989) Selection and initial characterization of partially nitrate tolerant modulation mutants of soybean. Plant Physiol 89: 169-173
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13. Gresshoff PM, Krotzky A, Mathews A, Day DA, Schuller KA, Olsson J, Delves AC, 'Carroll BJ (1988) Suppression of the symbiotic supernodulation symptoms of soybean. J Plant Physiol 132: 417-423 14. Gresshoff PM, Mathews A, Krotzky A, Olsson JE, Carroll BJ, Delves AC, Kosslak R, Appelbaum ER, Day DA (1988) Supernodulation and non-nodulation mutants of soybean. In R Palacios, DPS Verma, eds, Molecular Genetics of Plant-Microbe Interactions 1988. APS Press, St. Paul, pp 364-369 15. Harper JE (1989) Nitrogen metabolism mutants of soybean. In AJ Pascale, ed, World Soybean Research Conference IV, Vol IV. Asociacion Argentina De La Soja, Buenos Aires, pp 212216 16. Harper JE, Nicholas JC (1976) Control of nutrient solution pH with an ion exchange system: effect on soybean modulation. Physiol Plant 38: 24-28 17. Kosslak RM, Bohlool BB (1984) Suppression of nodule development of one side of a split-root system of soybeans caused by prior inoculation of the other side. Plant Physiol 75: 125130
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18. Kosslak RM, Bookland R, Barkei J, Paaren HE, Appelbaum ER (1987) Induction of Bradyrhizobiumjaponicum common nod genes by isoflavones isolated from Glycine max. Proc Natl Acad Sci USA 84: 7428-7432 19. Mathews A, Carroll BJ, Gresshoff PM (1989) Development of Bradyrhizobium infections in supernodulating and non-nodulating mutants of soybean (Glycine max [L.] Merrill). Protoplasma 150: 40-47 20. Mathews A, Kosslak RM, Sengupta-Gopalan C, Appelbaum ER, Carroll BJ, Gresshoff PM (1989) Biological characterization of root exudates and extracts from nonnodulating and supernodulating soybean mutants. Mol Plant-Microbe Interact 2: 283-290 21. Pierce M, Bauer WD (1983) A rapid regulatory response governing modulation in soybean. Plant Physiol 73: 286-290 22. Sutherland TD, Bassam BJ, Schuller LJ, Gresshoff PM (1990) Early modulation signals of the wild type and symbiotic mutants of soybean (Glycine max). Mol Plant-Microbe Interact 3: 122-128
