Lack of mycorrhizal autoregulation and phytohormonal changes in the supernodulating soybean mutant <Emphasis Type="Italic">nts1007</Emphasis>

Autoregulatory mechanisms have been reported in the rhizobial and the mycorrhizal symbiosis. Autoregulation means that already existing nodules or an existing root colonization by an arbuscular mycorrhizal fungus systemically suppress subsequent nodule formation/root colonization in other parts of the root system. Mutants of some legumes lost their ability to autoregulate the nodule number and thus display a supernodulating phenotype. On studying the effect of pre-inoculation of one side of a split-root system with an arbuscular mycorrhizal fungus on subsequent mycorrhization in the second side of the split-root system of a wild-type soybean (Glycine max L.) cv. Bragg and its supernodulating mutant nts1007, we observed a clear suppressional effect in the wild-type, whereas further root colonization in the split-root system of the mutant nts1007 was not suppressed. These data strongly indicate that the mechanisms involved in supernodulation also affect mycorrhization and support the hypothesis that the autoregulation in the rhizobial and the mycorrhizal symbiosis is controlled in a similar manner. The accumulation patterns of the plant hormones IAA, ABA and Jasmonic acid (JA) in non-inoculated control plants and split-root systems of inoculated plants with one mycorrhizal side of the split-root system and one non-mycorrhizal side, indicate an involvement of IAA in the autoregulation of mycorrhization. Mycorrhizal colonization of soybeans also resulted in a strong induction of ABA and JA levels, but on the basis of our data the role of these two phytohormones in mycorrhizal autoregulation is questionable.     

Autoregulation and Nitrate Inhibition of Nodule Formation in Soybean cv. Enrei and its Nodulation Mutants

The regulation and nitrate inhibition of nodule formation insoybean, Glycine max (L.) Merr., was further examined usingthe nodulation mutants of cv. Enrei. The non-nodulating mutantsEn115, Enl282, and En1314 produced extremely few markedly-curledroot hairs which were all devoid of infection threads, and invariablyfailed to initiate sub-epidermal cell divisions (SCDs) in theroot cortex. A considerable number of arrested SCDs was foundbefore nodule emergence in Enrei, but not in En6500 which hadsignificantly more SCDs that progressively increased at moreadvanced stages of nodule ontogeny. These observations indicatethat autoregulation acts by blocking the developmental stagebefore nodule emergence. In both Enrei and En65OO, the maturationof emerged nodules was restricted by a late-acting nodulationcontrol mechanism that is apparently unrelated to autoregulation.Reciprocal wedge-grafts of plants inoculated at sowing showedthat the control of the supernodulating phenotype resides inthe shoot, while the non-nodulating phenotype is strictly root-controlled.The nodulation phenotype of the current non-nodulating mutantsresults not from an alteration of the autoregulatory mechanism,but from mutation that exerts a root-localized effect that blocksSCDs which trigger the autoregulatory mechanism. Reciprocalgrafting experiments on Enrei and En6500 seedlings grown undervarious nitrate levels suggest that nitrate inhibition of nodulation,like autoregulation, is shoot-controlled. Since these two processesare invariably expressed together, they are probably causallyrelated, acting synergistically to regulate nodule formationin soybean. These results indicate that the regulation and nitrateinhibition of nodulation in the nodulation mutants of cv. Enreiare similar to those of cv. Bragg nodulation mutants. Key words: Autoregulation, nitrate-tolerant symbosis, non-nodulating mutants, soybean, supernodulating mutant     

Suppression of arbuscular mycorrhizal colonization and nodulation in split-root systems of alfalfa after pre-inoculation and treatment with Nod factors

Roots of legumes establish symbiosis with arbuscular mycorrhizal fungi (AMF) and nodule-inducing rhizobia. The existing nodules systemically suppress subsequent nodule formation in other parts of the root, a phenomenon termed autoregulation. Similarly, mycorrhizal roots reduce further AMF colonization on other parts of the root system. In this work, split- root systems of alfalfa (Medicago sativa) were used to study the autoregulation of symbiosis with Sinorhizobium meliloti and the mycorrhizal fungus Glomus mosseae. It is shown that nodulation systemically influences AMF root colonization and vice versa. Nodules on one half of the split-root system suppressed subsequent AMF colonization on the other half. Conversely, root systems pre-colonized on one side by AMF exhibited reduced nodule formation on the other side. An inhibition effect was also observed with Nod factors (lipo-chito-oligosaccharides). NodSm-IV(C16:2, S) purified from S. meliloti systemically suppressed both nodule formation and AMF colonization. The application of Nod factors, however, did not influence the allocation of (14)C within the split-root system, excluding competition for carbohydrates as the regulatory mechanism. These results indicate a systemic regulatory mechanism in the rhizobial and the arbuscular mycorrhizal association, which is similar in both symbioses.     

Charcoal and shrubs modify soil processes in ponderosa pine forests of western Montana

In split-root systems of alfalfa (Medicago sativa L.), already existing nodules or arbuscular mycorrhizal roots suppress further establishment of symbiosis in other root parts, a phenomenon named autoregulation. Roots treated with rhizobial nodulation signals (Nod factors) induce a similar systemic suppression of symbiosis.In order to test the hypothesis that flavonoids play a role in this systemic suppression, split-root systems of alfalfa plants were inoculated on one side of the split-root system with Sinorhizobium meliloti or Glomus mosseae or were treated with Nod factor. HPLC-analysis of alfalfa root extracts from both sides of the split-root system revealed a persistent local and systemic accumulation pattern of some flavonoids associated with the different treatments. The two flavonoids, formononetin and ononin, could be identified to be similarily altered after rhizobial or mycorrhizal inoculation or when treated with Nod factor.Exogenous application of formononetin and ononin partially restored nodulation and mycorrhization pointing towards the involvement of these two secondary compounds in the autoregulation of both symbioses.     

Interaction between supernodulating or non-nodulating mutants of soybean and two arbuscular mycorrhizal fungi

 Twelve nodulation mutants (seven non-nodulating and five supernodulating) of soybean [Glycine max (L.) Mirr.] were screened for arbuscular mycorrhizal colonization in the presence of either Glomus etunicatum Becker and Gerdemann or Gigaspora margarita Becker and Hall. The cultivars showed variation in colonization parameters. The two supernodulating mutants En6500 and NOD1–3 had higher frequencies of colonization with 2.5–4.5 times higher arbuscular abundance than the respective wild types. The enhanced mycorrhization resulted in significant enhancement of P uptake by En6500. The non-nodulating mutants showed decreases in mycorrhizal parameters. Mutants En1282 and Harosoy^–exhibited aborted infection after formation of typical appressorium-like structures at some sites. However, none of these had the non-mycorrhizal phenotype. Growth and nutrient-uptake parameters should be considered while studying plant mutants for mycorrhization. Accepted: 7 July 2000     

Relationship between autoregulation and nitrate inhibition of nodulation in soybeans

Ten of 11 supernodulating mutants of soybean [ Glycine max (L.) Merr.] cv. Bragg, in which nodulation was far in excess of that in the wild type, showed pronounced tolerance of nodulation to applied nitrate. Mutant nts (nitrate-tolerant symbiosis) 1116 had an intermediate nodulation response and also showed some inhibition by nitrate. Mutant 1029, a revertant of nts382 (an extreme supernodulator), showed a wild-type nodulation pattern and was equally sensitive to nitrate as cv. Bragg. Grafting experiments with cv. Bragg and nts382 indicated that both supernodulation and tolerance of nodulation to nitrate were dependent on shoot factors. Total leaf nitrate reductase (EC 1.6.6.1 and EC 1.6.6.2) activity of the supernodulating mutants was similar to that in cv. Bragg. We conclude from these results that the inhibitory effect of nitrate on nodule initiation and development in soybean depends on an interaction between nitrate and the autoregulation singal. In the supernodulating mutants, the autoregulation signal is either altered or absent and cosequently nodulation in these mutants is not sensitive to nitrate.     

Long-distance transport of signals during symbiosis: Are nodule formation and mycorrhization autoregulated in a similar way?

Legumes enter nodule symbioses with nitrogen-fixing bacteria (rhizobia), whereas most flowering plants establish symbiotic associations with arbuscular mycorrhizal (AM) fungi. Once first steps of symbiosis are initiated, nodule formation and mycorrhization in legumes is negatively controlled by a shoot-derived inhibitor (SDI), a phenomenon termed autoregulation. According to current views, autoregulation of nodulation and mycorrhization in legumes is regulated in a similar way. CLE peptides induced in response to rhizobial nodulation signals (Nod factors) have been proposed to represent the ascending long-distance signals to the shoot. Although not proven yet, these CLE peptides are likely perceived by leucine-rich repeat (LRR) autoregulation receptor kinases in the shoot. Autoregulation of mycorrhization in non-legumes is reminiscent to the phenomenon of “systemic acquired resistance” in plant-pathogen interactions.Key words: arbuscular mycorrhiza, autoregulation, CLE peptides, mutant, nodulation, split-root systemUnder natural conditions, growth of plants is often limited by the availability of nutrients such as nitrogen and phosphorous. Plants have therefore developed strategies to acquire nutrients with the help of soil microorganisms. Most land plants enter mutualistic root symbioses with arbuscular mycorrhizal (AM) fungi, whereas legumes form special root nodules containing nitrogen-fixing bacteria, so-called rhizobia.^1–4 Establishment and maintenance of symbiosis requires plant resources, such as photosynthetically assimilated carbon. To minimize these costs, host plants are able to control the degree of their symbiotic interactions. Above a critical threshold level further establishment of symbiosis is restricted—a feedback phenomenon termed autoregulation of symbiosis. Autoregulation can be experimentally demonstrated in split-root systems. When legume roots are already infected by rhizobia on one side of a split-root, further nodule development is “systemically” inhibited on the other side. Similarly, prior colonization of split-roots by AM fungi on one half suppresses later fungal root colonization on the other half. Hence, important elements of the symbiotic autoregulation circuit are not only localized in roots, but also in aerial parts of the plant, implicating transport of signals in vascular bundles (Fig. 1). Whereas autoregulation of nodulation in legumes has been studied for many decades,^5–9 the first publications clearly stating a shoot-controlled autoregulation of mycorrhization in split-root systems appeared in 2000 for the non-legume barley (Hordeum vulgare) and thereafter for alfalfa (Medicago sativa) and soybean (Glycine max).^10–13 The data from these split-root experiments are supported by the findings that supernodulating (or hypernodulating) loss-of-autoregulation mutants displayed either an increased degree of AM colonization and/or a higher abundance of arbuscules.^14–16Open in a separate windowFigure 1Proposed model of shoot-controlled autoregulation of symbiosis in a split-root system. Prior infection of root A by rhizobia or AM fungi systemically suppresses later establishment of symbiosis in root B. Expression of specific CLE peptides (and/or other peptide hormones) is induced in response to rhizobial nodulation signals (Nod factors) and perhaps also in response to colonization by AM fungi (stage 1). The CLE peptides (and/or other signals) are then presumed to be transported in the xylem to the shoot, where they are perceived by leucine-rich repeat (LRR) autoregulation receptor kinases (stage 2). As a result of autoregulation signaling in the shoot, an unknown shoot-derived inhibitor (SDI) is produced (stage 3) and transported as a phloem-mobile signal to the root. Perception and action of the SDI signal in roots would then inhibit nodulation and root colonization by AM fungi (stage 4).     

Differential sensitivity of nodulation to ethylene in soybean cv. Bragg and a supernodulating mutant

We previously found that the ethylene inhibitor Ag⁺ could overcome the inhibitory effect of nitrate on nodulation of soybean ( Glycine max ) cv. Bragg. The same treatment increased nodulation quantitatively under non-inhibitory conditions, strongly suggesting involvement of ethylene in the control of nodulation in this species. Supernodulation mutants that lack internal autoregulation of nodulation, however, had biosynthesis capacity similar to the wild type. In the present work, the effects of ethylene on nodulation of 'Bragg' and two separate, but allelic, supernodulating mutants ( nts382 and nts1007 ) were compared. The nodulation process appeared much more sensitive than plant growth and development to ethylene, which reduced the number of nodules per plant, but nearly twofold more in the wild type than in the supernodulation mutants. The cause–effect relationship is established by the counteracting effect of Ag⁺ and the fact that the stronger the inhibition by ethylene, the higher the recovery of nodulation ability with the ethylene antagonist. This higher tolerance of or lower sensitivity to ethylene in nts382 persists even under low inoculum dose, where nodule number and mass could be decreased to wild-type levels. Differences between the mutant and the wild type in the triple response test do not appear to support differences in ethylene perception on a whole-plant basis. The results suggest that sensitivity of nodulation to ethylene might have been affected in supernodulation mutants.     

Rhizobial Nodulation Factors Stimulate Mycorrhizal Colonization of Nodulating and Nonnodulating Soybeans

Legumes form tripartite symbiotic associations with noduleinducing rhizobia and vesicular-arbuscular mycorrhizal fungi. Co-inoculation of soybean (Glycine max [L.] Merr.) roots with Bradyrhizobium japonicum 61-A-101 considerably enhanced colonization by the mycorrhizal fungus Glomus mosseae. A similar stimulatory effect on mycorrhizal colonization was also observed in nonnodulating soybean mutants when inoculated with Bradyrhizobium japonicum and in wild-type soybean plants when inoculated with ineffective rhizobial strains, indicating that a functional rhizobial symbiosis is not necessary for enhanced mycorrhiza formation. Inoculation with the mutant Rhizobium sp. NGR[delta]nodABC, unable to produce nodulation (Nod) factors, did not show any effect on mycorrhiza. Highly purified Nod factors also increased the degree of mycorrhizal colonization. Nod factors from Rhizobium sp. NGR234 differed in their potential to promote fungal colonization. The acetylated factor NodNGR-V (MeFuc, Ac), added at concentrations as low as 10-9 M, was active, whereas the sulfated factor, NodNGR-V (MeFuc, S), was inactive. Several soybean flavonoids known to accumulate in response to the acetylated Nod factor showed a similar promoting effect on mycorrhiza. These results suggest that plant flavonoids mediate the Nod factor-induced stimulation of mycorrhizal colonization in soybean roots.     

Efficiency of Nodule Initiation and Autoregulatory Responses in a Supernodulating Soybean Mutant

We compared the formation of nodules on the primary roots of a soybean cultivar (Glycine max (L.) Merr. cv. Bragg) and a supernodulating mutant derivative, nts382. Inoculation with Bradyrhizobium japonicum USDA 110 at different times after seed imbibition showed that the roots acquired full susceptibility to infection only between 3 and 4 days postgermination. When the plants were inoculated with serial dilutions of a bacterial suspension, the number of nodules formed in the initially susceptible region of the roots was linearly dependent on the logarithm of the inoculum dose until an optimum dose was reached. At least 10-fold-lower doses were required to induce half-maximal nodulation responses on nts382 than on the wild type. However, at optimal doses, about six times as many nodules formed in the initially susceptible region of the roots in nts382. Since there was no appreciable difference in the apparent rates of nodule emergence, the increased efficiency of nodule initiation in the supernodulating mutant could have resulted from a lower threshold of response to bacterial symbiotic signals. Two inoculations (24 h apart) of G. max cv. Bragg revealed that there was a host-mediated regulatory response that suppressed nodulation in younger portions of the primary roots, as reported previously for other soybean cultivar-Bradyrhizobium combinations. Similar experiments with nts382 revealed a comparable suppressive response, but this response was not as pronounced as it was in the wild type. This and other results suggest that there are additional control mechanisms for nodulation that are different from the systemic autoregulatory control of nodulation altered in supernodulating mutants.     

Development ofBradyrhizobium infections in supernodulating and non-nodulating mutants of soybean (Glycine max [L.] Merrill)

Summary The early events in the development of nodules induced byBradyrhizobium japonicum were studied in serial sections of a wild type (cv. Bragg), a supernodulating mutant (nts 382) and four non-nodulating mutants (nod49, nod139, nod772, andrj ₁) of soybean (Glycine max [L.] Merrill). Cultivar Bragg responded to inoculation in a similar manner to that described previously for cv. Williams; centres of sub-epidermal cell divisions were observed both with and without associated infection threads and most infection events were blocked before the formation of a nodule meristem. The non-nodulating mutants (nod49, nod772, andrj ₁) had, at most, a few centres of sub-epidermal cell divisions. In general, these were devoid of infection threads and did not develop beyond the very early stages of nodule ontogeny. Sub-epidermal cell divisions or infection threads were never observed on mutant nodl39. This mutant is not allelic to the other non-nodulating mutants and represents a defect in a separate complementation group or gene that is required for nodulation. The supernodulating mutant nts382, which is defective in autoregulation of nodulation, had a similar number of sub-epidermal cell divisions as the wild-type Bragg, but a much greater proportion of these developed to an advanced stage of nodule ontogeny. Mutant nts382, like Bragg, possessed other infection events that were arrested at an early stage of development. The results are discussed in the context of the progression of events in nodule formation and autoregulation of nodulation in soybean.Abbreviations nts nitrate tolerant symbiosis - RT root tip (i.e., position of the tap root tip at the time of inoculation) - SERH shortest emerging root hair (i.e., position of the shortest emerging root hair on the tap root at the time of inoculation) - SCD subepidermal cell divisions     

Shoot apex removal does not alter autoregulation of Modulation in soybean

The suppression of new nodule development in soybean (Glycine max (L.) Merr.) has been previously demonstrated to involve the shoot through reciprocal grafts between the wild-type cultivar Bragg and its supernodulating mutant nts382. Using the same grafting technique, but modified through the excision of the shoot apex region and emerging lateral shoots, we show here that autoregulation of nodule number still existed despite apex removal. This radical treatment lowered total nodule number per plant as well as root, shoot and nodule dry weight. Bragg shoots grafted onto nts382 roots gave wild-type nodulation (26 nodules, 15mg total nodule mass) as compared to nts382 shoots grafted onto Bragg roots (340 nodules, 277 mg total nodule mass). Specific nodule mass differed between supernodulating (about 0·5-1·0mg per nodule) and wild-type nodulating (2·3 mg per nodule) plants. In contrast to other growth characteristics, apex removal did not affect specific nodule size, except in plants with wild-type shoots and nts382 (supernodulation) roots. Apex removal only slightly affected the percentage of nodule weight per total root weight in nts382, but had a severe effect in wild type. Growth reductions varied between the normal and supernodulating plants. The fact that autoregulation of nodulation still functions in plants devoid of functional shoot apices suggests that the autoregulation signal may not be derived from the apex regions and that the leaf may be a likely source.     

Characteristics of certain symbiotic pea mutants by traits related to hormonal status

Association between traits for hormonal status and nodulation in the mutants of pea Pisum sativum L. with abnormal nodulation and original forms was analyzed. The sensitivity of plant tissues to exogenous phytohormones and changes in the concentration of the major auxin, indolyl-3-acetic acid, in plant roots during interaction with rhizobia were examined. Association between Nod(++)-phenotype and auxin balance was revealed: the supernodulating mutants were more sensitive to auxin treatment than the parental cultivars. Mutations in the sym8 gene, in contrast to those in the sym5 gene, had no effect on plant hormonal status. The level of indolyl-3-acetic acid during interaction with rhizobia depended on the time after inoculation and plant genotype. The mutations affecting nodulation were suggested to change auxin level in roots.     

Four genes of Medicago truncatula controlling components of a nod factor transduction pathway

Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting several key developmental responses in the roots of legume hosts. Using nodulation-defective mutants of Medicago truncatula, we have started to dissect the genetic control of Nod factor transduction. Mutants in four genes (DMI1, DMI2, DMI3, and NSP) were pleiotropically affected in Nod factor responses, indicating that these genes are required for a Nod factor-activated signal transduction pathway that leads to symbiotic responses such as root hair deformations, expressions of nodulin genes, and cortical cell divisions. Mutant analysis also provides evidence that Nod factors have a dual effect on the growth of root hair: inhibition of endogenous (plant) tip growth, and elicitation of a novel tip growth dependent on (bacterial) Nod factors. dmi1, dmi2, and dmi3 mutants are also unable to establish a symbiotic association with endomycorrhizal fungi, indicating that there are at least three common steps to nodulation and endomycorrhization in M. truncatula and providing further evidence for a common signaling pathway between nodulation and mycorrhization.     

Lack of Systemic Suppression of Nodulation in Split Root Systems of Supernodulating Soybean (Glycine max [L.] Merr.) Mutants

Wild-type soybean (Glycine max [L] Merr. cv Bragg) and a nitrate-tolerant supernodulating mutant (nts382) were grown in split root systems to investigate the involvement of the autoregulation response and the effect of timing of inoculation on nodule suppression. In Bragg, nodulation of the root portion receiving the delayed inoculation was suppressed nearly 100% by a 7-day prior inoculation of the other root portion with Bradyrhizobium japonicum strain USDA 110. Significant suppression was also observed after a 24-hour delay in inoculation. Mutant nts382 in the presence of a low nitrate level (0.5 millimolar) showed little, if any, systemic suppression. Root fresh weights of individual root portions were similar for both wild type and nts382 mutant. When nts382 was grown in the absence of nitrate, a 7-day delay in inoculation resulted in only 30% suppression of nodulation and a significant difference in root fresh weight between the two sides, with the delayed inoculated side always being smaller. Nodulation tests on split roots of nts382, nts1116, and wild-type cultivars Bragg, Williams 82, and Clark demonstrated a difference in their systemic suppression ability. These observations indicate that (a) autoregulation deficiencies in mutant nts382 result in a reduction of systemic suppression of nodulation, (b) some suppression is detectable after 24 hours with a delayed inoculation, (c) the presence of low nitrate affects the degree of suppression and the root growth, and (d) soybean genotypes differ in their ability to express this systemic suppression.     

Influence of genes determining supernodulation on root colonization by the mycorrhizal fungus Glomus mosseae in Pisum sativum and Medicago truncatula mutants

Pisum sativum (pea) mutants of the wild type cv. Frisson and six supernodulating Medicago truncatula mutants of the wild-type cv. Jemalong line J5 for their ability to form endomycorrhizas. The six mutants of M. truncatula were shown to be allelic mutants of the same gene Mtsym12, whereas distinct genes (sym28 and sym29) are known to determine the supernodulation character of the P64 and P88 pea mutants, respectively. Mutant P88 of pea and the majority of the M. truncatula mutants were significantly more colonized by the mycorrhizal fungus Glomus mosseae than their corresponding wild types, 4 weeks and 30 days after inoculation, respectively. These differences were expressed essentially in transversal intensity rather than in length intensity of root colonization and appeared to correspond to an increase in arbuscule formation. Results are discussed in relation to the mutated genes and, in particular, whether the observed effects are due indirectly to plant physiological modifications or are a direct result of possible common factors of regulation of nodulation and mycorrhizal development. Accepted: 9 February 2000     

Mycorrhiza‐mediated interference between cover crop and weed in organic winter cereal agroecosystems: The mycorrhizal colonization intensity indicator

The mycorrhizal fungi are symbiotic organisms able to provide many benefits to crop production by supplying a set of ecosystem functions. A recent ecological approach based on the ability of the fungi community to influence plant–plant interactions by extraradical mycelium development may be applied to diversified, herbaceous agroecosystems. Our hypothesis is that the introduction of a winter cereal cover crop (CC) as arbuscular mycorrhizal fungi (AMF)–host plant in an organic rotation can boosts the AMF colonization of the other plants, influencing crop–weed interference. In a 4‐years organic rotation, the effect of two winter cereal CC, rye and spelt, on weed density and AMF colonization was evaluated. The AMF extraradical mycelium on CC and weeds roots was observed by scanning electron microscopy analysis. By joining data of plant density and mycorrhization, we built the mycorrhizal colonization intensity of the Agroecosystem indicator (MA%). Both the CC were colonized by soil AMF, being the mycorrhizal colonization intensity (M%) affected by environmental conditions. Under CC, the weed density was reduced, due to the increase of the reciprocal competition in favor of CC, which benefited from mycorrhizal colonization and promoted the development of AMF extraradical mycelium. Even though non‐host plants, some weed species showed an increased mycorrhizal colonization in presence of CC respect to the control. Under intense rainfall, the MA% was less sensitive to the CC introduction. On the opposite, under highly competitive conditions, both the CC boosted significantly the mycorrhization of coexistent plants in the agroecosystem. The proposed indicator measured the agroecological service provided by the considered CCs in promoting or inhibiting the overall AMF colonization of the studied agroecosystems, as affected by weed selection and growth: It informs about agroecosystem resilience and may be profitably applied to indicate the extent of the linkage of specific crop traits to agroecosystem services, contributing to further develop the functional biodiversity theory.     

Primary Analysis of Components in Different Soybean Nodulating Mutants and their Effects on Nitrate Reductase Activity and Nodulation

The water extracts of leaves and roots from supernodulating soybean (Glycine max (L.) Merr. ) nts 382 and nonnodulating soybean Nod 49 have been chromatographed using filtering method through the column (25 cm × 2 cm) Sephadex G25 and 4 fractions, namly, nts 382 (Nod 49) F1, nts 382 (Nod 49) F2, nts 382 (Nod 49) F3, and nts 382 (Nod 49) F4 could be distinguished according to nitrate reductase (NR) activities inhibited by the eluate. The inhibition of NR activity by the noninoculated nts 382 F2 and the nts 382 F4 in vitro were much stronger than that by the inoculated nts 382 F2 and nts 382 F4. On the contrary, the obvious inhibition of NR activity in vitro by the noninoculated Nod 49 F2 and Nod 49 F4 were substantialy strengthed again by the innoculated Nod 49 F2 and Nod 49 F4. The facts indicated that the quantity of NR inhibitors in the leaf cells of soybean nts 382 reduced after the inoculation but was that in the inoculated Nod 49 leaf cells further more accumulated. Both nodulations assays, the nodulation of soybean "Bragg " injected with inoculated nts 382 Fl, nts 382 F2, nts 382 F3 and nts 382 F4 from leaves and roots and the nodulation of soybean nts 382 injected with inoculated Nod 49 F2, Nod 49 F3 and Nod 49 F4 from leaves only showed that nts 382 Fl and nts 382 F2 increased nodules of soybean "Bragg" by 1 to 3 times but nts 382 F3 and nts 382 F4 did not. Inhibition of soybeannts 382 nodulation by inoculated Nod 49 F2 Nod 49 F3 and Nod 49 F4 expressed that the Nod 49 F4 only inhibited the nodulation strongly by one time in the experiments with nts 382 plants with leaves, and by 15 times in the experiments with nts 382 plants without leaves at 10 d of inoculation and injection and this inhibition was nonreversible even after stopping injection from the 11th day to the 15th day after inoculation.