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 ethylene-inhibitor aminoethoxyvinylglycine restores normal nodulation by Rhizobium leguminosarum biovar. viciae on Vicia sativa subsp. nigra by suppressing the ‘Thick and short roots’ phenotype

Nodulation of Vicia sativa subsp. nigra L. by Rhizobium bacteria is coupled to the development of thick and short roots (Tsr). This root phenotype as well as root-hair induction (Hai) and root-hair deformation (Had) are caused by a factor(s) produced by the bacteria in response to plant flavonoids. When very low inoculum concentrations (0.5–5 bacteria·ml^-1) were used, V. sativa plants did not develop the Tsr phenotype and became nodulated earlier than plants with Tsr roots. Furthermore, the nodules of these plants were located on the primary root in contrast to nodules on Tsr roots, which were all located at sites of lateral-root emergence. The average numbers of nodules per plant were not significantly different for these two types of nodulation. Root-growth inhibition and Hai, but not Had, could be mimicked by ethephon, and inhibited by aminoethoxyvinylglycine (AVG). Addition of AVG to co-cultures of Vicia sativa and the standard inoculum concentration of 5·10⁵ bacteria·ml^-1 suppressed the development of the Tsr phenotype and restored nodulation to the pattern that was observed with very low concentrations of bacteria (0.5–5 bacteria·ml^-1). The delay in nodulation on Tsr roots appeared to be caused by the fact that nodule meristems did not develop on the primary root, but only on the emerging laterals. The relationship between Tsr, Hai, Had, and nodulation is discussed.Abbreviations AVG aminoethoxyvinylglycine - cfu colonyforming units - Had root-hair deformation - Hai root-hair induction - NB naringenin-bacteria filtrate - Tsr Thick and short roots     

Nodulation of Glycine max by Six Bradyrhizobium japonicum Strains with Different Competitive Abilities

The root nodule locations of six Bradyrhizobium japonicum strains were examined to determine if there were any differences which might explain their varying competitiveness for nodule occupancy on Glycine max. When five strains were added to soybeans in plastic growth pouches in equal proportions with a reference strain (U.S. Department of Agriculture, strain 110), North Carolina strain 1028 and strain 110 were the most competitive for nodule occupancy, followed by U.S. Department of Agriculture strains 122, 76, and 31 and Brazil strain 587. Among all strains, nodule double occupancy was 17% at a high inoculum level (10⁷ CFU pouch⁻¹) and 2% at a low inoculum level (10⁴ CFU pouch⁻¹). The less competitive strains increased their nodule representation by an increase in the doubly occupied nodules at the high inoculum level. Among all strains, the number of taproot and lateral root nodules was inversely related at both the high and low inoculum levels (r = −0.62 and −0.69, respectively; P = 0.0001). This inverse relationship appeared to be a result of the plant host control of bacterial infection. Among each of the six strains, greater than 95% of the taproot nodules formed at the high inoculum density were located on 25% of the taproot length, the nodules centering on the position of the root tip at the time of inoculation. No differences among the six strains were observed in nodule initiation rates as measured by taproot nodule position. Taproot nodules were formed in the symbiosis before lateral root nodules. One of the poorly competitive strains (strain 76) occupied three times as many taproot nodules as lateral root nodules when competing with strain 110 (nodules were harvested from 4-week-old plants). Among these six wild-type strains of B. japonicum, competitive ability evidently is not related to nodule initiation rates.     

Production of growth hormones inCasuarina cunninghamiana root nodules induced byFrankia strain HFPCgI4

Summary Measurements of auxin and cytokinin activities in extracts ofCasuarina root nodules were made. The nodules were induced either by pure culture ofFrankia strain CgI4 or by crushed nodule inoculum. Levels of cytokinin activity were significantly higher in root nodules induced by pureFrankia culture than in those induced by crushed nodule inoculum. However, the reasons for this are unknown. Seasonal variation in levels of cytokinin activity inCasuarina nodules has also been detected.Dedicated to the memory of Professor John G. Torrey     

Influence of the Numbers of Rhizobium supplied on the Subsequent Nodulation of the Legume Host Plant

The effect of numbers of Rhizobium supplied on the subsequentnumbers of nodules formed was tested with Phaseolus radiatusvar. aurea in water culture. Nine weeks after inoculation plants supplied with from 88,700to 887,000 bacteria per c.c. of culture solution bore significantlymore nodules than those given from 89 to 887 per c.c. An inoculumof 44,350 per c.c. produced intermediate numbers of nodules. One week after inoculation, the percentage of curled and ofinfected root-hairs on the seedlings was roughly proportionalto the logarithm of the bacterial numbers supplied, while thenumbers of nodules produced at this stage already showed a significantdifference between extreme doses of inoculum. Nine weeks after inoculation the final nodule numbers were relatedto the percentages of curled and of infected root-hairs afterone week. The final nodule numbers are related to the growth of the rootsystem since the final number of nodules per gramme of rootis independent of the initial dose of inoculum, the effect ofwhich was mainly to cause an increase in root growth.     

Early Events in the Infection of Soybean (Glycine max L. Merr) by Rhizobium japonicum: I. LOCALIZATION OF INFECTIBLE ROOT CELLS

The infectible cells of soybean roots appear to be located at any given time just above the zone of root elongation and just below the position of the smallest emergent root hairs. The location of infectible cells on the primary root at the time of inoculation was inferred from the position of subsequent nodule development, correcting for displacement of epidermal cells due to root elongation. Marks were made on the seedling growth pouches at the time of inoculation to indicate the position of the root tip and the zones of root hair development. Virtually all of the seedlings developed nodules on the primary root above the marks made at the root tips at the time of inoculation. None of the plants formed nodules on the root where mature root hairs were present at the time of inoculation. These results and profiles of nodulation frequency indicate that the location of infectible cells is developmentally restricted. When inoculations were delayed for intervals of 1 to 4 hours after marking the positions of the root tips, progressively fewer nodules were formed above the root tip marks, and the uppermost of these nodules were formed at progressively shorter distances above the marks. These results indicate that the infectibility of given host cells is a transient property that appears and then is lost within a few hours. The results also indicate that host responses leading to infection and nodulation are triggered or initiated in less than 2 hours after inoculation. The extent of nodulation above the root tip mark increased in proportion to the logarithm of the number of bacteria in the inoculum.     

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.     

Cowpea Rhizobia Producing Dark Nodules: Use in Competition Studies

During a program of screening rhizobia from West Africa, it was found that some strains produced nodules of unusually dark appearance on cowpeas, but not on peanuts, soybeans, pigeon peas, or mung beans. The dark pigmentation was in the bacteroid zone, was not correlated with nodule effectiveness, and was additional to the leghemoglobin pigment. Only rhizobial strains with a nongummy (“dry”) colony morphology produced dark nodules. Visually distinguishable pink and dark nodules formed on the same root when a mixture of pink and dark strains was applied as inoculum. The dark-nodule phenotype was therefore appraised as a marker and found to be useful for studying nodulation competition with strains of the orthodox pink-nodule type. The competitiveness of 10 pink-nodule strains was examined relative to a black-nodule strain, IRc 256; a range of competitiveness was obtained of less competitive than, equally competitive to, or more competitive than IRc 256. Patterns of primary (early) nodulation were generally the same as patterns of secondary (later) nodulation. Mixed infections by dark and pink strains produced piebald nodules, the frequency of occurrence of which was much greater among primary than among secondary nodules.     

Competition in Kenyan soils between Rhizobium leguminosarum biovar phaseoli strain Kim5 and R. tropici strain CIAT899 using the gusA marker gene

The contribution of appropriate inoculum strains to more efficient nitrogen fixation by legumes has been difficult to assess due to the laborious nature of the assays involved in assessing establishment of inoculum strains in the field. The use of marker genes, in particular the GUS system, changes this, making it possible to assess occupancy by the inoculum strain in large numbers of nodules on whole root systems. Here we used the GUS system to evaluate the competitive ability of two rhizobial strains, Rhizobium leguminosarum bv. phaseoli strain Kim5 and R. tropici strain CIAT899 in two soil types from Kenya. The results confirm that Kim5 is a highly competitive strain, forming 86% of the nodules in a near-neutral pH soil. Although the competitiveness of CIAT899 is enhanced in an acid (pH 4.5) soil it still only formed 35% of the nodules. There were no differences between inoculum strains in their efficiency of nitrogen fixation in either soil type, and virtually no N2-fixation occurred in the acid soil due to the lack of tolerance of the Phaseolus genotype to soil acidity.     

Correlation between infection by Rhizobium leguminosarum and lectin on the surface of Pisum sativum L. roots

The lectin on the surface of 4- and 5-dold pea roots was located by the use of indirect immunofluorescence. Specific antibodies raised in rabbits against pea seed isolectin 2, which crossreact with root lectins, were used as primary immunoglobulins and were visualized with fluorescein- or tetramethylrhodamine-isothiocyanate-labeled goat antirabbit immunoglobulin G. Lectin was observed on the tips of newly formed, growing root hairs and on epidermal cells located just below the young hairs. On both types of cells, lectin was concentrated in dense small patches rather than uniformly distributed. Lectin-positive young hairs were grouped opposite the (proto)xylematic poles. Older but still-elongating root hairs presented only traces of lectin or none at all. A similar pattern of distribution was found in different pea cultivars, as well as in a supernodulating and a non-nodulating pea mutant. Growth in a nitrate concentration which inhibits nodulation did not affect lectin distribution on the surface of pea roots of this age. We tested whether or not the root zones where lectin was observed were susceptible to infection by Rhizobium leguminosarum. When low inoculum doses (consisting of less than 10⁶ bacteria·ml^-1) were placed next to lectin-positive epidermal cells and on newly formed root hairs, nodules on the primary roots were formed in 73% and 90% of the plants, respectively. Only a few plants showed primary root nodulation when the inoculum was placed on the root zone where lectin was scarce or absent. These results show that lectin is present at those sites on the pea root that are susceptible to infection by the bacterial symbiont.Abbreviations FITC fluorescein isothiocyanate - TRIC tetramethylrhodamine isothiocyanate     

Nodulation gene regulation and quorum sensing control density-dependent suppression and restriction of nodulation in the Bradyrhizobium japonicum-soybean symbiosis

The nodulation of Glycine max cv. Lambert and the nodulation-restricting plant introduction (PI) genotype PI 417566 by wild-type Bradyrhizobium japonicum USDA110 is regulated in a population-density-dependent manner. Nodulation on both plant genotypes was suppressed (inhibited) when plants received a high-density inoculum (10(9) cells/ml) of strain USDA110 grown in complex medium, and more nodules were produced on plants receiving a low-cell-density inoculum (10(5) cells/ml). Since cell-free supernatants from strain USDA110 grown to high cell density in complex medium decreased the expression of an nodY-lacZ fusion, this phenomenon was attributed to bradyoxetin-induced repression of nod gene expression. Inoculation of either the permissive soybean genotype (cv. Lambert) or PI 417566 with 10(9) cells/ml of the nodD2, nolA, nodW, and nwsB mutants of USDA110 enhanced nodulation (up to 24%) relative to that seen with inoculations done with 10(5) cells/ml of the mutants or the wild-type strain, indicating that these genes are involved in population-density-dependent nodulation of soybeans. In contrast, the number of nodules produced by an nodD1 mutant on either soybean genotype was less than those seen with the wild-type strain inoculated at a low inoculum density. The nodD2 mutant outcompeted B. japonicum strain USDA123 for nodulation of G. max cv. Lambert at a high or low inoculum density, and the results of root-tip-marking and time-to-nodulate studies indicated that the nolA and nodD2 mutants nodulated this soybean genotype faster than wild-type USDA110. Taken together, the results from these studies indicate that the nodD2 mutant of B. japonicum may be useful to enhance soybean nodulation at high inoculum densities and that NodD2 is a key repressor influencing host-controlled restriction of nodulation, density-dependent suppression of nodulation, perception of bradyoxetin, and competitiveness in the soybean-B. japonicum symbiosis.     

Control of root architecture and nodulation by the LATD/NIP transporter

The Medicago truncatula LATD/NIP gene is essential for the development of lateral and primary root and nitrogen-fixing nodule meristems as well as for rhizobial invasion of nodules. LATD/NIP encodes a member of the NRT1(PTR1) nitrate and di-and tri-peptide transporter family, suggesting that its function is to transport one of these or another compound(s). Because latd/nip mutants can have their lateral and primary root defects rescued by ABA, ABA is a potential substrate for transport. LATD/NIP expression in the root meristem was demonstrated to be regulated by auxin, cytokinin and abscisic acid, but not by nitrate. LATD/NIP''s potential function and its role in coordinating root architecture and nodule formation are discussed.Key words: nodule development, lateral root development, root architecture, symbiotic nitrogen fixation, Medicago truncatula, NRT1(PTR) gene familyUnlike most other plants, legumes form two kinds of lateral root organs: lateral roots and nitrogen-fixing root nodules that form in conjunction with compatible symbiotic rhizobium bacteria. Although the morphology and function of these two root organs is distinct, both require the function of the LATD/NIP gene, indicating shared genetic components for these two developmental processes and providing support for a model in which legume nodules evolved from a lateral root blueprint. Both lateral roots and nodules initiate in previously differentiated root cells in response to environmental and developmental cues mediated by hormones. Interestingly, regulation of nodules and lateral roots by hormones is often opposite, allowing formation of one organ or another depending on the conditions.     

Spontaneous nodules induce feedback suppression of nodulation in alfalfa

A small subpopulation of alfalfa (Medicago saliva L.) plants grown without fixed nitrogen can develop root nodules in the absence of Rhizobium. Cytological studies showed that these nodules were organized structures with no inter- or intracellular bacteria but with the histological characteristics of a normal indeterminate nodule. Few if any viable bacteria were recovered from the nodules after surface sterilization, and when the nodular content was used to inoculate alfalfa roots no nodulation was observed. These spontaneous nodules were formed mainly on the primary roots in the region susceptible to Rhizobium infection between 4 and 6 d after seed imbibition. Spontaneous nodules appeared as early as 10 d after germination and emerged at a rate comparable to normal nodules. The formation of spontaneous nodules on the primary root suppressed nodulation in lateral roots after inoculation with R. meliloti RCR2011. Excision of spontaneous nodules at inoculation eliminated the suppressive response. Our results indicate that the presence of Rhizobium is not required for nodule organogenesis and the elicitation of feedback regulation of nodule formation in alfalfa.Abbreviation RT root tip This work was supported by an endowment to the Racheff Chair of Excellence of the University of Tennessee, and the Soybean Promotion Board, Haskinsville, Tenn., USA. We are indebted to Noel Gerahty for performing the acetylene-reduction assays, and Dr. E.T. Graham for allowing the use of microscope facilities.     

The Influence of Temperature on Meloidogyne incognita on Soybean

The effects of temperature and initial inoculum density of Meloidogyne incognita on soybean growth and nematode reproduction were investigated in greenhouse temperature tanks and in controlled-growth chambers. The interactions of initial inoculum density (P_i) and soil temperature in effects on shoot growth were adequately described by multiple-regression models. At the highest temperatures (30 or 32/28 C), moderate to high inoculum killed many plants. A P_i of 27,000 eggs/15-cm-diam pot retarded shoot growth at 26 C. Only the greatest P_i (81,000 eggs/15-cm pot) suppressed shoot growth at 18, 22, or 20/16 C. Inoculation with 3,000 or 9,000 eggs/plant resulted in heavier root systems at all temperatures except 30 C. At that temperature, 9,000 eggs suppressed root growth. At 18 and 26 C, a Pi of 81,000 eggs was required to retard root growth. Nematode reproduction was related directly to temperature and P_i except at a density of 81,000 eggs/15-cm pot.     

Interstrain Competition between Representatives of Indigenous Serotypes of Rhizobium trifolii

The symbiotic characteristics of Rhizobium trifolii strains 1-01 and 2-01 were evaluated both individually and in various combinations on two cultivars (Mt. Barker and Woogenellup) of subterranean clover (Trifolium subterraneum L.). Nodules were observed on day 8 independent of cultivar or strain. Cultivar differences were measured in nodulating efficiency by 1-01 since 54% of the primary nodules were formed on cv. Mt. Barker and only 15% were formed on cv. Woogenellup in the zone above, or 1 cm below, the root tip location at the time of inoculation. The percentage of nodules formed in this zone by 2-01 was similar on both cultivars (31 to 32%). When mixtures of strains 1-01 and 2-01 (230:1 and 1:20) were used to inoculate plants, >90% of the nodules on both cultivars were occupied by the more abundant strain in the inoculum regardless of sampling date (4 or 8 weeks). In contrast, large percentages of nodules on 4-week-old plants of both cultivars exposed to a 5:1 inoculum mixture were doubly occupied (64 and 74%). By week 8 these values had decreased significantly (P ≤ 0.01) and were accompanied by large increases in the percentage of nodules occupied by either strain 1-01 alone (1 to 65%) on cv. Mt. Barker or 2-01 alone (4 to 49%) on cv. Woogenellup. The superior (cv. Mt. Barker) and inferior (cv. Woogenellup) symbiotic performance of plants inoculated with the 5:1 mixture correlated more closely with the 8-week than the 4-week nodule occupancy data. Primary nodule occupancy by 1-01 and 2-01 was significantly influenced by changes in the inoculum ratios of 1-01/2-01 from 5.7:1 to 0.67:1 on cv. Mt. Barker and from 1.9:1 to 0.67:1 on cv. Woogenellup. Despite evidence for extensive proliferation of the inoculant strains on the rhizoplanes, no evidence was obtained for either interstrain antagonism or selective proliferation as a valid reason to explain the outcome of primary nodulation.     

Competition studies with Rhizobium trifolii in a field experiment

M c L oughlin , T.J. B ordeleau L.M. & D unican , L.K. 1984. Competition studies with Rhizobium trifolii in a field experiment. Journal of Applied Bacteriology 56 , 131–135.
Competition studies were carried out in a field experiment by comparing the ability of antibiotic marked Rhizobium strains inoculated at three inoculum levels in forming nodules against an effective indigenous Rhizobium population of 1.5 times 10⁵/g. Three inoculum strains were assessed. It was seen that by increasing the level of inoculum there was a corresponding increase in the number of nodules formed by the introduced strains. G1067 formed a high proportion (85%) at the highest level, whereas the other two inoculum strains occupied less than 50% of the nodules at the same level. Persistence of the inoculum strain was measured into the second season; G1032 could not be detected, G1006 (another 'foreign' strain) formed 43% and G1067 formed 97% of the nodules sampled. The indigenous Rhizobium population isolated from nodules was shown to be heterogenous, and 15 different intrinsic antibiotic resistance patterns were found. Fifty per cent of the isolates fell in the same group which showed a similar pattern to that of the inoculum strain G1067.     

A comparison of the fluorescent ELISA and antibiotic resistance identification techniques for use in ecological experiments with Rhizobium trifolii

The fluorescent ELISA technique for the identification of bacteria was compared with antibiotic resistant mutants as marker systems for use with Rhizobium trifolii in root nodules and in soil. With an effective(CP3B) and an ineffective (R4) strain as a mixed 1:1 inoculum, there was a highly significant correlation ( P < 0.001) between the two techniques when the plants were grown at pH 5.5 when the majority of nodules were inhabited by the effective strain. At pH 6.5, where the ineffective strain predominated in the nodules, there was no correlation. The reason was that 85% of R4 nodules had volumes less than 0.1 mm₃ with bacterial numbers obviously below the necessary threshold for detection using the serological method. Both methods were efficient at enumerating rhizobia from soils although the recovery rate from a brown earth soil was significantly higher than from a peat soil. Fluorescent ELISA was able to detect rhizobia at 8.0 times 10₅ cells/ml soil suspension (1 g soil to 10 ml water) in the brown earth soil and at 2.0 times 10₅ cells/ml in the peat soil. The results are discussed in terms of the limitations of both techniques in ecological studies.     

Genetically marked Rhizobium identifiable as inoculum strain in nodules of soybean plants grown in fields populated with Rhizobium japonicum.

The fate of an inoculum strain of Rhizobium japonicum was studied using a genetically marked strain I-11O subline carrying resistance markers for azide, rifampin, and streptomycin (I-110 ARS). At the time of planting into a field populated with R. japonicum, seeds of soybean cultivars Kent and Peking were inoculated with varying cell densities of strain I-110 ARS. At various times during the growing season, surface-sterilized root nodules were examined for the presence of the inoculum strain by plating onto selective media. The recovery of the inoculum strain was unambiguous, varying, in the case of Kent cultivar, from about 5% with plants (sampled at 51 days) that had been inoculated with 3 X 10(8) cells per cm of row to about 20% with plants (sampled at 90 days) that had been inoculated with 3 X 10(9) cells per cm. The symbiotically incompatible interaction of Peking and strain 110 in Rhizobium-populated field soil was confirmed by the finding that at 60 days after planting, only one nodule in 360 sampled contained strain I-110 ARS. The use of genetically marked Rhizobium bacteria was found to provide for precise identification of the inoculum strain in nodules of field-grown soybeans.     

Genetically marked Rhizobium identifiable as inoculum strain in nodules of soybean plants grown in fields populated with Rhizobium japonicum

The fate of an inoculum strain of Rhizobium japonicum was studied using a genetically marked strain I-11O subline carrying resistance markers for azide, rifampin, and streptomycin (I-110 ARS). At the time of planting into a field populated with R. japonicum, seeds of soybean cultivars Kent and Peking were inoculated with varying cell densities of strain I-110 ARS. At various times during the growing season, surface-sterilized root nodules were examined for the presence of the inoculum strain by plating onto selective media. The recovery of the inoculum strain was unambiguous, varying, in the case of Kent cultivar, from about 5% with plants (sampled at 51 days) that had been inoculated with 3 X 10(8) cells per cm of row to about 20% with plants (sampled at 90 days) that had been inoculated with 3 X 10(9) cells per cm. The symbiotically incompatible interaction of Peking and strain 110 in Rhizobium-populated field soil was confirmed by the finding that at 60 days after planting, only one nodule in 360 sampled contained strain I-110 ARS. The use of genetically marked Rhizobium bacteria was found to provide for precise identification of the inoculum strain in nodules of field-grown soybeans.     

Regulation of nodulation in Alnus incana-Frankia symbiosis

We have studied regulation of nodulation in Alnus incana (L.) Moench using double inoculations in plastic pouches and a slide technique to observe root hair deformation. Initially, the distribution of nodules between main and lateral roots appeared quite constant, independent of the concentration of inoculum (1 to 250 μg of crushed nodules plant⁻¹). Susceptibility to infection after the second inoculation was restricted to lateral roots after the initial infections developed. When pre-existing nodules were excised before the second inoculation, subsequent nodules appeared to arise where infections had arrested at stages earlier than actual nodule emergence. We observed that root hairs formed postinoculation were very crowded and short with a pronounced deformation. No nodules were found later on this region of the root, suggesting a loss of susceptibility in this region. Split-root experiments with delays between inoculation of the first and second side of the root system showed irreversible, systemic inhibition of nodulation on the second side starting between 3 and 6 days after the inoculation of the first side. Only when compatible, infective strains were used in the first inoculation, was nodule formation inhibited after the second inoculation. We conclude that autoregulation of nodulation operates in Alnus incana and on a time scale similar to what is found in some legumes.