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     

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.     

Nodule distribution on the roots of soybean and a supernodulating mutant in sand-vermiculite

The distribution of nodules of soybean (Glycine max (L.) Merr.) cultivar Bragg and the supernodulating mutant derivative nts382 was examined on the primary root relative to the first emerging lateral root, and on laterals relative to the base of the roots of plants grown in sand-vermiculite. Mutant nts382 nodulates profusely even in the presence of nitrate and appears defective in a systemic autoregulatory response that regulates nodule number in soybean. Nodules were clustered on primary roots about the first 4 cm down from the first emerging lateral root in both genotypes. Nodulation profiles showed reduced nodulation in younger and older regions of the primary root. Similarly, nodules appeared clustered close to the base of the lateral roots. Decreasing inoculum dose shifted nodule emergence to younger regions of the primary root and to lateral roots emerging in younger portions of the primary root. Our results indicate that the supernodulating mutant is able to regulate nodule number in both primary and lateral roots in the particulate matrix.     

The genetic interaction between non-nodulation and supernodulation in soybean: an example of developmental epistasis

Summary The interaction between three non-nodulation mutants (nod49, nod772 and nod139) and a supernodulation mutant (nts382) of soybean was studied by analysing the progeny from crosses between these mutants. Previously it had been shown that the non-nodulation mutants arose from single mutation events and that nod49 and nod772 are allelic, whereas nod139 represents another gene required for nodulation. Analysis of progeny from crosses between nts382 and the wild type showed that this mutant also arose from a single mutation. Complementation tests demonstrated that the mutation responsible for supernodulation in nts382 is not allelic to either of these non-nodulation characters, and that it segregates independently. Progeny were identified that were homozygous for both supernodulation and non-nodulation, and these plants were incapable of nodulation. Thus, non-nodulation is epistatic over supernodulation and this is discussed in terms of the developmental blockage in the two mutant types. The identification and confirmation of these double mutants of the supernodulation and non-nodulation mutations are described. Although the non-nodulation mutations behave as recessive characters in a wild-type background, these mutations are incompletely dominant in a genetic background homozygous for supernodulation. The significance of these results to the understanding of nodule ontogeny is discussed.     

The Auxin Transport Inhibitor N-(1-Naphthyl)phthalamic Acid Elicits Pseudonodules on Nonnodulating Mutants of White Sweetclover

The collection of symbiotic (sym) mutants of white sweetclover (Melilotus alba Desr.) provides a developmental sequence of mutants blocked early in infection or nodule organogenesis. Mutant phenotypes include non-nodulating mutants that exhibit root-hair deformations in response to Rhizobium meliloti, mutants that form ineffective nodules lacking infection threads, and mutants that form infection threads and ineffective nodules. Mutant alleles from both the sym-1 and the sym-3 loci exhibited a non-nodulating phenotype in response to R. meliloti, although one allele in the sym-1 locus formed ineffective nodules at a low frequency. Spot-inoculation experiments on a non-nodulating allele in the sym-3 locus indicated that this mutant lacked cortical cell divisions following inoculation with R. meliloti. The auxin transport inhibitor N-(1-naphthyl)phthalamic acid elicited development of pseudonodules at a high frequency on all of the sweetclover sym mutants, including the non-nodulating mutants, in which the early nodulin ENOD2 was expressed. This suggests that N-(1-naphthyl)phthalamic acid activates cortical cell divisions by circumventing a secondary signal transduction event that is lacking in the non-nodulating sweetclover mutants. The sym-3 locus and possibly the sym-1 locus appear to be essential to early host plant responses essential to nodule organogenesis.     

Microscopic characterization of early nodulation events in a non-nodulating soybean mutant (NN5) and lack of response to high inoculum dose

The microscopic events leading to nodulation in normally nodulatingsoybean [Glycine max (L.) Merr.] genotypes, and the effectsof Bradyrhizobium strain and inoculum dose on nodulation, wereexamined in the NN5 non-nodulating mutant derived from cv. Williams.The NN5 mutant possesses the recessive genes rj₅ and ,rj₆. BradyrhizoblumJaponicum strain USDA 110 cells attached normally to the rootsurface of NN5, many in a polar manner as in its wild-type parent,but failed to induce root hair curling and sub-epidermal celldivision in the root. Co-culturing NN5 and Williams did notmodify nodulation of either genotype. Hydroponically-grown NN5seedlings did not nodulate at a high inoculum dose (1 x 10¹⁰cells seedling^–1) of any B. japonicum strain tested (USDA110, USDA 26, USDA 136, and the tryptophan metabolic variantsB-14075 and ta 11 Nod⁺). A higher inoculum dose of 3 x 10 USDA136 cells seedling also failed to induce nodulation in NN5 andnod139 (a non-nodulating mutant of cv. Bragg). The lack of nodulationof NN5 at any inoculum dose is contrary to previous observationsof sparse nodulation of other non-nodulating mutants at highinoculum dose. Genetic control of non-nodulation in NN5 is probablysimilar to nodl39. Key words: Nodulation events, non-nodulating mutant, soybean     

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.     

Growth comparisons of a supernodulating soybean (Glycine max) mutant and its wild-type parent

The growth of a supernodulating, nitrate-tolerant soybean [ Glycine max (L.) Merr.] mutant nts 382 (nitrate-tolerant symbiosis) was compared to that of its wild-type parent, cv. Bragg, over the first 50 days after sowing. Plants were grown either inoculated in the absence of an external nitrogen source or uninoculated in the presence of 5 m M KNO₃. For both treatments, nts 382 growth up to 13 days after planting was faster than that of cv. Bragg. Thereafter, supernodulation of inoculated nts 382 occurred and growth of cv. Bragg was faster; shoot and root dry weight increments and leaf area were greater in cv. Bragg, but the N content of nts 382 was higher. Relative growth and net assimilation rates were lower in nts 382, which had faster shoot and root respiration rates. Shoot growth of uninoculated plants was similar for both mutant and wild-type but roots of nts 382 were slightly smaller than those of cv. Bragg. Total plant N content was similar in uninoculated cv. Bragg and nts 382 but the latter had a higher leaf N content. Early lateral root formation (prior to nodule emergence) was greater in nts 382 regardless of whether rhizobia or KJNO₃ were present. We conclude that nts 382 has some inherent differences from its parent but that supernodulation significantly retards plant growth.     

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.     

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.     

Responses of Rj(1) and rj(1) Soybean Isolines to Inoculation with Bradyrhizobium japonicum

We evaluated the symbiotic phenotypes of nodulation-restrictive and normal soybean isolines by inoculating Clark (genotypically Rj₁Rj₁) and mutant Clark-rj₁ (genotypically rj₁rj₁) seedlings in plastic growth pouches. Nodules first appeared on Clark seedlings inoculated with Bradyrhizobium japonicum USDA 94 after 6 days. The mean number of nodules per plant was 13.9 ± 0.8 after 24 days. In contrast, Clark-rj₁ seedlings first nodulated at 12 days, and the mean number of nodules per plant was only 1.7 ± 0.3 at 24 days. Segments from infectible zones of primary roots, i.e. near the position occupied by the root tip at the time of inoculation, were sectioned serially. Clark roots contained cortical cell divisions and a few infection threads in question mark-shaped root hairs by 2 days after inoculation. Typical nodules developed soon thereafter. Analogous serially sectioned segments from Clark-rj₁ roots lacked these responses. This prompted us to section nodules and adjacent tissues from other parts of Clark and Clark-rj₁ roots. Clark roots contained cortical cell divisions, many associated with infected root hairs. Cortical cell divisions occasionally were present in Clark-rj₁, and a few infection threads were visible in surface cells. The presence of infection threads within Clark-rj₁ nodules was confirmed by transmission electron microscopy. Thus, although B. japonicum USDA 94 fails to elicit the wild-type spectrum of responses in the infectible zones of primary roots, it can infect Clark-rj₁ via infection threads.     

Nitrogen Partitioning During Early Development of Supernodulating Soybean (Glycine max [L.] Merrill) Mutants and their Wild-Type Parent

The relative importance of fixed N², cotyledonary N, and nitratefor growth of seedlings of soybean cv. Bragg and two of itsnitrate tolerant supernodulating (nts) mutants (intermediatents 1116 and extreme nts1007) was investigated during symbioticdevelopment in the presence of nitrate (3.0 mol m^–3) using¹⁵N techniques. Newly-fixed N₂ and nitrate were both major sourcesof N for nodule development and nitrate principally supportedearly shoot and root growth in Bragg. In the nts mutants, however,all plant parts and nodules in particular, relied more on Nstored in the cotyledons. This resulted in later nodule maturityand a period of prolonged N-starvation for the seedlings ofthe extreme supernodulator, and could be responsible for theirsubsequently lowered biomass accumulation compared to the parentcultivar. Key words: Nodules, N partitioning, supernodulating soybeans     

Regulation of the soybean-Rhizobium nodule symbiosis by shoot and root factors

The availability of soybean mutants with altered symbiotic properties allowed an investigation of the shoot or root control of the relevant phenotype. By means of grafts between these mutants and wild-type plants (cultivar Bragg and Williams), we demonstrated that supernodulation as well as hypernodulation (nitrate tolerance in nodulation and lack of autoregulation) is shoot controlled in two mutants (nts382 and nts1116) belonging most likely to two separate complementation groups. The supernodulation phenotype was expressed on roots of the parent cultivar Bragg as well as the roots of cultivar Williams. Likewise it was shown that non-nodulation (resistance to Bradyrhizobium) is root controlled in mutant nod49. The shoot control of nodule initiation is epistatically suppressed by the non-nodulation, root-expressed mutation. These findings suggest that different plant organs can influence the expression of the nodulation phenotype.     

Soybean Seedling Extracts Inhibit Supernodulation

When soybean (Glycine max ) nodulation mutant nts 382 was inoculated with Bradyrhizobium japonicum, these plants nodulated significantly more than the parental type Bragg. Nts 382 seedlings displayed wild-type nodulation pattern when aqueous extracts of young Bragg shoots were applied to the cultural medium together with nutrient solution. Application of young nts 382 shoot extracts to Bragg seedlings did not result in any apparent increase in nodule number. In graft experiments, young shoots from mutant nts 382 induced supernodulation on Bragg root stocks, while no supernodulation was observed when Bragg seedlings were used as scion and grafted onto nts 382 root stocks. Further, the effectiveness of Bragg plant extracts to suppress supernodulation on nts 382 seedlings was found to depend on the age of the plant material used, being very ineffective with extracts from 60-day-old plants. The age effect was not observed in graft experiments. These findings suggest that soybean supernodulation phenomenon may be controlled by one or a few unknown chemicals or plant hormones.     

Nitrogenase activity and ureide levels in a supernodulating soybean mutant: Effects of inoculum dose and nitrate treatment

The effects of nitrate on nitrogenase (EC 1.18.2.1) activity of soybean ( Glycine max [L.] Merr) cv. Bragg and its supernodulating mutant derivative, nts382, were compared. A short-term nitrate treatment was used to allow effects on nitrogenase activity to be studied in the absence of effects on nodule growth and a low inoculum dose, which prevented supernodulation of nts382, was employed to test for any interaction between supernodulation and the magnitude of the effect of nitrate on nitrogenase activity. At the usual inoculum dose, nitrogenase activity, per g nocule, of nts382 was lower than that of Bragg and was proportionally less affected by nitrate. Decreasing the inoculum dose increased nitrogenase activity of nts382 and also the proportional decline in response to nitrate. The decline in the ureide conentration in xylem exudate in response to nitrate was proportionally similar to the decline in nitrogenase activity per plant. However, although nitrogenase activity per plant of nts382 was several-fold less than that of Bragg, the ureide flux rate (ureide concentration x xylem sap exudation rate), was not different. At the usual inodulum dose, the ureide content of the nocules, stems plus petioles and leaves of nts382 was greater than that of Bragg. Decreasing the inoculum dose reduced the ureide content of the nodules of nts382 but not of Bragg. Ureide degradative capacity of the leaves was the same for Bragg and nts382. Low activities of 5-phosphoribosyl pyrophosphate amidotransferase (EC 2.4.2.14) and glutamine synthetase (EC 6.3.1.2) in the nodules reflected the low nitrogenase activity of nts382.     

Inoculation and nitrate alter phytohormone levels in soybean roots: differences between a supernodulating mutant and the wild type

 The levels of different cytokinins, indole-3-acetic acid (IAA) and abscisic acid (ABA) in roots of Glycine max [L.] Merr. cv. Bragg and its supernodulating mutant nts382 were compared for the first time. Forty-eight hours after inoculation with Bradyrhizobium, quantitative and qualitative differences were found in the root's endogenous hormone status between cultivar Bragg and the mutant nts382. The six quantified cytokinins, ranking similarly in each genotype, were present at higher concentrations (30–196% on average for isopentenyl adenosine and dihydrozeatin riboside, respectively) in mutant roots. By contrast, the ABA content was 2-fold higher in Bragg, while the basal levels of IAA [0.53 μmol (g DW)⁻¹, on average] were similar in both genotypes. In 1 mM NO₃ ⁻-fed Bragg roots 48 h post-inoculation, IAA, ABA and the cytokinins isopentenyl adenine, and isopentenyl adenosine quantitatively increased with respect to uninoculated controls. However, only the two cytokinins increased in the mutant. High NO₃ ⁻ (8 mM) markedly reduced root auxin concentration, and neither genotypic differences nor the inoculation-induced increase in auxin concentration in Bragg was observed under these conditions. Cytokinins and ABA, on the other hand, were little affected by 8 mM NO₃ ⁻. Root IAA/cytokinin and ABA/cytokinin ratios were always higher in Bragg relative to the mutant, and responded to inoculation (mainly in Bragg) and nitrate (both genotypes). The overall results are consistent with the auxin-burst-control hypothesis for the explanation of autoregulation and supernodulation in soybean. However, they are still inconclusive with respect to the inhibitory effect of NO₃ ⁻. Received: 16 April 1999 / Accepted: 13 December 1999     

Ultrastructure of bacteroid-containing tissue of Pisum sativum L. peas having various regulatory mechanisms of nodulation

The infected root nodule cells of Pisum sativum cvs. Torsdag, Rondo and its supernodulating mutant nod3 have been investigated by transmission electron microscopy and morphometrically. Torsdag and nod3 developed effective nodules, when grown with or without nitrates in the growth medium. The nodules developed by Rondo were ineffective in the presence of nitrates, and otherwise effective. An obvious similarity in the fine structure of bacteroid tissue of root nodules has been observed in Torgsdag (Nod5) and the supernodulating mutant nod3, both forms being nitrate-tolerant, but nodulation being controlled by different genetic systems. The statistical processing results showed significant differences in the respective morphometric parameters of nodule cells between the plants grown according to either scheme: with and without nitrates. Combined nitrogen is likely to affect the ratio of symbionts in the infected nodule cells of cultivars with nitrate-tolerant nodulation.     

Nodulation inhibition by Rhizobium leguminosarum multicopy nodABC genes and analysis of early stages of plant infection.

During analysis of early events in the infection and nodulation of Vicia hirsuta roots inoculated with normal and mutant strains of Rhizobium leguminosarum and strains containing cloned nodulation (nod) genes, a number of novel observations were made. (i) Alternating zones of curled and straight root hairs were seen on roots of V. hirsuta inoculated with the wild-type strain of R. leguminosarum. This phasing of root hair curling was not seen if plants were grown under continuous light or continuous dark conditions. (ii) Reduced nodulation and delayed nodule initiation was observed with a strain carrying a Tn5 mutation in the nodE gene. In addition the phased root hair curling was absent, and root hair curling was observed along the length of the root. (iii) The nodABC genes cloned on a multicopy plasmid in a wild-type strain inhibited nodulation but induced a continuous root hair curling response. Those few nodules that eventually formed were found to contain bacteria which had lost the plasmid carrying the nodABC genes. (iv) With a strain of Rhizobium cured of its indigenous symbiotic plasmid, but containing the cloned nodABCDEF genes, continuous root hair curling on V. hirsuta was observed. However, no infection threads were observed, and surprisingly, it did appear that initial stages of nodule development occurred. Observations of thin sections of these early developing nodules indicated that early nodule meristematic divisions may have occurred but that no bacteria were found within the nodules and no infection threads were observed either within the nodule bumps or within any of the root hairs. It was concluded that for normal infections to occur, precise regulation of the nod genes is required and that overexpression of the root hair curling genes inhibits the normal infection process.     

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.