Growth and nutrient uptake of sorghum cultivated with vesicular-arbuscular mycorrhiza isolates at varying pH

This study was conducted to determine the effects of different pH regimes on root colonization with four vesicular-arbuscular mycorrhiza (VAM) isolates, and VAM effects on host plant growth and nutrient uptake. Sorghum [Sorghum bicolor (L.) Moench] was grown at pH 4.0, 5.0, 6.0 and 7.0 (±0.1) in hydroponic sand culture with the VAM isolates Glomus etunicatum UT316 (isolate E), G. intraradices UT143 (isolate I), G. intraradices UT126 (isolate B), and an unknown Glomus isolate with no INVAM number (isolate A). Colonization of roots with the different VAM isolates varied differentially with pH. As pH increased, root colonization increased with isolates B and E, remained unchanged with isolate I, and was low at pH 4.0 and high at pH 5.0, 6.0, and 7.0 with isolate A. Isolates E and I were more effective than isolates A and B in promoting plant growth irrespective of pH. Root colonization with VAM appeared to be independent of dry matter yields or dry matter yield responsiveness (dry matter produced by VAM compared to nonmycorrhizal plants). Dry matter yield responsiveness values were higher in plants whose roots were colonized with isolates E and I than with isolates A and B. Shoot P concentrations were lower in plants colonized with isolates E and I than with isolates A and B or nonmycorrhizal plants. This was probably due to the dilution effect of the higher dry matter yields. Neither the VAM isolate nor pH had an effect on shoot Ca, Mg, Zn, Cu, and Mn concentrations, while the VAM isolate affected not only P but also S, K, and Fe concentrations. The pH x VAM interaction was significant for shoot K, Mg, and Cu concentrations.     

Ectomycorrhiza formation between Pseudotsuga menziesii seedling roots and monokaryotic and dikaryotic isolates of Laccaria bicolor

Seedling roots of Pseudotsuga menziesii were colonized with three monokaryotic isolates and one dikaryotic isolate of Laccaria bicolor to assess the effect of fungal genotype on ectomycorrhiza formation. Ectomycorrhizas resulting from colonization by the dikaryotic isolate had a multilayered mantle and a cortical Hartig net. One monokaryotic isolate (ss7) formed ectomycorrhizas comparable in anatomy to those induced by the dikaryotic isolate. Two other monokaryotic isolates (ss5, ss1) failed to form mantles or Hartig nets. Roots colonized by these isolates developed characteristics indicating an incompatible reaction.     

Colonization of Fusarium Wilt‐Resistant and Susceptible Watermelon Roots by a Green‐Fluorescent‐Protein‐tagged Isolate of Fusarium oxysporum f.sp. niveum

Interactions between watermelon and a green fluorescent protein (GFP)‐tagged isolate of Fusarium oxysporum f.sp. niveum race 1 (Fon‐1) were studied to determine the differences in infection and colonization of watermelon roots in cultivars resistant to and susceptible to Fusarium wilt. The roots of watermelon seedlings were inoculated with a conidial suspension of the GFP‐tagged isolate, and confocal laser scanning microscopy was used to visualize colonization, infection and disease development. The initial infection stages were similar in both the resistant and susceptible cultivars, but the resistant cultivar responded differentially after the pathogen had penetrated the root. The pathogen penetrated and colonized resistant watermelon roots, but further fungal advance appeared to be halted, and the fungus did not enter the taproot, suggesting that resistance is initiated postpenetration. However, the tertiary and secondary lateral roots of resistant watermelon also were colonized, although not as extensively as susceptible roots, and the hyphae had penetrated into the central cylinder of lateral roots forming a dense hyphal mat, which was followed by a subsequent collapse of the lateral roots. The initial infection zone for both the wilt‐susceptible and wilt‐resistant watermelon roots appeared to be the epidermal cells within the root hair zone, which the fungus penetrated directly after forming appressoria. Areas where secondary roots emerged and wounded root tissue also were penetrated preferentially.     

Effect of time of inoculation on in vitro ectomycorrhizal colonization and nodule initiation in Acacia holosericea seedlings

The complex interactions that occur in systems with more than one type of symbiosis were studied using one isolate of Bradyrhizobium sp. and the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch inoculated on to the roots of Acacia holosericea A. Cunn. ex G. Don in vitro. After a single inoculation with Bradyrhizobium sp., bacteria typically entered the roots by forming infection threads in the root hair cells via the curling point of the root hair and/ or after intercellular penetration. Sheath formation and intercellular penetration were observed on Acacia roots after a single inoculation with Pisolithus tinctorius but no radial elongation of epidermal cells. Simultaneous inoculation with both microorganisms resulted in nodules and ectomycorrhiza on the root system, occasionally on the same lateral root. On lateral roots bearing nodules and ectomycorrhiza, the nodulation site was characterized by the presence of a nodule meristem and the absence of an infection thread; sheath formation and Hartig net development occurred regularly in the region of the roots adjacent to nodules. Prior inoculation with Bradyrhizobium sp. did not inhibit ectomycorrhizal colonization in root segments adjacent to nodules in which nodule meristems and infection threads were clearly present. Conversely, in ectomycorrhizae inoculated by bacteria, the nodule meristem and the infection thread were typically absent. These results show that simultaneous inoculation with both microorganisms inhibits infection thread development, thus conferring an advantage on fungal hyphae in the competition for infection sites. This suggests that fungal hyphae can modify directly and/or indirectly the recognition factors leading to nodule meristem initiation and infection thread development.     

Can ectomycorrhizal colonization of Pinus resinosa roots affect their decomposition?

In many forest ecosystems, fine root litter comprises a large pool of organic carbon and nutrients. In temperate climates ectomycorrhizal fungi colonize the roots of many forest plant species. If ectomycorrhizal colonization influenced root decomposition, it could significantly influence carbon sequestration and nutrient cycling. Fungal tissues and fine roots may decompose at different rates and, therefore, ectomycorrhizal colonization may either hasten or retard root decomposition. Unfortunately, no comparisons of the decomposition of roots and ectomycorrhizal fungi have yet been made. Therefore, we compared decomposition of Pinus resinosa fine roots and ectomycorrhizal fungi from a Pinus resinosa plantation. We also compared the decomposition rates of nonmycorrhizal Pinus resinosa fine roots with roots colonized by nine species of ectomycorrhizal fungi. We found that the several tested isolates of ectomycorrhizal fungi decomposed far more rapidly than the fine roots and that ectomycorrhizal colonization either had no significant effect on root decomposition or significantly increased root decomposition depending on the isolate of fungus. We conclude that the composition of an ectomycorrhizal fungal community may affect carbon and nutrient cycling through its influence on root decomposition.     

Early events in the life of apple roots: variation in root growth rate is linked to mycorrhizal and nonmycorrhizal fungal colonization

Early events of mycorrhizal and nonmycorrhizal fungal colonization in newly-emerging roots of mature apple (Malus domestica Borkh) trees were characterized to determine the relationship of these events to fine root growth rate and development. New roots were traced on root windows to measure growth and then collected and stained to quantify microscopically the presence of mycorrhizal and nonmycorrhizal fungal structures. Most new roots were colonized by either mycorrhizal or nonmycorrhizal fungi but none less 25 days old were ever internally colonized by both. Compared to nonmycorrhizal colonization, mycorrhizal colonization was associated with faster growing roots and roots that grew for a longer duration, leading to longer roots. While either type of fungi was observed in roots as soon as 3 days after root emergence, intraradical colonization by mycorrhizal fungi was generally faster (peaking at 7 to 15 days) than that by nonmycorrhizal fungi and often occurred more frequently in younger roots. Only 15 to 35% of the roots had no fungal colonization by 30 days after emergence. This study provides the first detailed examination of the early daily events of mycorrhizal and nonmycorrhizal fungal colonization in newly emerging roots under field conditions. We observed marked discrimination of roots between mycorrhizal and nonmycorrhizal fungi and provide evidence that mycorrhizal fungi may select for faster growing roots and possibly influence the duration of root growth by non-nutritional means.     

Surface colonization of lodgepole pine (Pinus contorta var. latifolia [Dougl. Engelm.]) roots by Pseudomonas fluorescens and Paenibacillus polymyxa under gnotobiotic conditions

Indirect immunofluorescence techniques and confocal scanning laser microscopy were used to identify rhizobacterial strains on the root surfaces of pine seedlings, which were grown from seeds under gnotobiotic conditions. Conifer plant growth promoting rhizobacterial strains Paenibacillus polymyxa L6 and Pw-2, and the forest soil isolate Pseudomonas fluorescens M20, were inoculated onto surface-disinfested pine seeds, singly, or in dual combinations: strains L6 + M20, or strains Pw-2 + M20. Segments containing particular root microsites (root tip, root hair zone, or areas of lateral root emergence) were sampled randomly from roots 7 or 13 weeks after inoculation, and the colonization of roots by each bacterium was observed. Root segments were also sampled from individual roots at six different points along the length of the root, and the qualitative colonization of younger areas, closer to the root tip, contrasted with that of older areas, closer to the root base. The ability of strain M20 to colonize root areas adjacent to sites of lateral root emergence improves in the presence of either P. polymyxa strain, while the ability of the P. polymyxa strains to colonize these areas was not affected. More rhizobacteria were also generally observed on younger root tissues than on areas closer to the root base.     

Suppression of plant defence response by a mycorrhiza helper bacterium

The aim of the present study was to determine whether the mycorrhiza helper bacterium Streptomyces sp. AcH 505 could serve as a biocontrol agent against Heterobasidion root and butt rot. Bacterial influence on mycelial growth of Heterobasidion sp. isolates, on the colonization of wood discs and Norway spruce (Picea abies) roots was determined. The effect of AcH 505 on plant photosynthesis, peroxidase activity and gene expression, and needle infections were investigated. AcH 505 was antagonistic to 11 of 12 tested fungal Heterobasidion isolates. The antagonism resulted in a suppression of fungal colonization of Norway spruce roots and wood discs. Mycelial growth rate of the 12th strain, Heterobasidion abietinum 331 was not affected by AcH 505, and colonization of roots by this fungal strain was promoted by AcH 505. Bacterial inoculation led to decreased peroxidase activities and gene expression levels in roots. AcH 505 promotes plant root colonization by Heterobasidion strains that are tolerant to antifungal metabolites produced by the bacterium. This may result from unknown bacterial factors that suppress the plant defence response.     

The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) inhibits the stimulation of in vitro lateral root formation and the colonization of the tap-root cortex of Norway spruce (Picea abies) seedlings by the ectomycorrhizal fungus Laccaria bicolor

Norway spruce ( Picea abies (L.) Karst.) seedlings were inoculated with the ectomycorrhizal fungus Laccaria bicolor ((Marie) Orton), strain S238 N, in axenic conditions. The presence of the fungus slowed tap–root elongation by 26% during the first 15 d after inoculation and then stimulated it by 136%. In addition, it multiplied in vitro lateral root formation by 4.3, the epicotyl growth of the seedlings by 8.4 and the number of needles by 2. These effects were maintained when the fungus was separated from the roots by a cellophane membrane preventing symbiosis establishment, thus suggesting that the fungus acted by non-nutritional effects. We tested the hypothesis that IAA produced by L. bicolor S238 N would be responsible for the stimulation of fungal induced rhizogenesis. We showed in previous work that L. bicolor S238 N can synthesize IAA in pure culture. Exogenous IAA supplies (100 and 500 μ m ) reproduced the stimulating effect of the fungus on root branching but inhibited root elongation. The presence of 2,3,5-triiodobenzoic acid (TIBA) in the culture medium significantly depressed lateral root formation of inoculated seedlings. As TIBA had no significant effect on IAA released in the medium by L. bicolor S238 N, but counteracted the stimulation of lateral rhizogenesis induced by an exogenous supply of IAA, we suggest that TIBA inhibited the transport of fungal IAA in the root. Furthermore TIBA blocked the colonization of the main root cortex by L. bicolor S238 N and the formation of the Hartig net. These results specified the role of fungal IAA in the stimulation of lateral rhizogenesis and in ectomycorrhizal symbiosis establishment.     

Rapid in vitro ectomycorrhizal infection onPinus densiflora roots byTricholoma matsutake

The root systems of 11-wk-oldPinus densiflora seedlings were inoculated with a hyphal suspension ofTricholoma matsutake and aseptically incubated for 4 wk in a forest soil without supplying exogenous carbohydrates. One week following inoculation, fungal hyphae had colonized the root surface and bound soil particles together establishing a root-substrate continuum. Fungal hyphae were visible within the main root cortex following clearing bleaching and staining. In the ensuing days, fungal colonization was observed within elongating lateral roots in which Hartig net formation was confirmed 4 wk after inoculation. This is the first report of rapid ectomycorrhizal infection ofP. densiflora seedings byT. matsutake.     

The effect of soil pH on the ability of ectomycorrhizal fungi to increase the growth of Eucalyptus globulus Labill.

We examined the effect of two levels of soil pH (5 and 6) on the ability (effectiveness) of ectomycorrhizal fungi to increase the growth of Eucalyptus globulus Labill. at a deficient supply of P. Plants were inoculated with one of six fungal isolates [Laccaria laccata (Scop. ex Fr.) Berk. and Br. (isolates A and B), Pisolithus tinctorius (Pers.) Coker and Couch (isolates A and B), Descolea maculata Bough. and Mal. and Setchelliogaster sp. nov.] and were grown in a P-deficient sand, in pots, in a temperature-controlled glasshouse. Seedlings were harvested 89 days after planting and were assessed for dry matter production, tissue P concentrations, ectomycorrhizal colonization of roots and hyphal development in soil.Uninoculated plants had less than 5% of their fine root length colonized by ectomycorrhizal fungi. In contrast, inoculated plants had 30% or greater of their fine root length ectomycorrhizal. Inoculation increased the uptake of P and growth of plants for all isolates and at both levels of soil pH, although growth responses to inoculation were greater at pH 6, particularly for the two L. laccata isolates. Isolates which colonized roots most extensively increased plant growth to the greatest extent. D. maculata was the most effective fungal isolate at pH 5, and both D. maculata and L. laccata A were most effective at pH 6. The effects of soil pH on plant growth were also related to some extent to the effects of soil pH on colonized root length. Growth responses to inoculation were related less well to hyphal development in soil. The L. laccata isolates formed more hyphae in soil (on a per pot, per m of fine root, and per m of colonized fine root basis) than other fungal isolates, but were not always more effective in increasing plant grown.     

Evaluation of a Native and Introduced Isolate of Pochonia chlamydosporia against Meloidogyne javanica

Growth chamber and plastic tunnel experiments were conducted to compare the ability of a native and introduced isolate of Pochonia chlamydosporia to colonize the rhizosphere of selected plant species and survive in soil. Effects of the isolates on population density of Meloidogyne javanica and yield of tomato after single or multiple fungal applications were also determined. In growth chamber experiments, both isolates showed a similar ability to colonise the rhizosphere of selected vegetables, except for the introduced isolate, which produced more colony forming units cm^-2 of root surface on tomato and cabbage than the native one. In the tunnel house, both isolates parasitized eggs of M. javanica, and the native but not the introduced isolate increased parasitism after multiple applications. The native isolate was recovered more frequently from soil, and was a better colonizer of tomato roots than the introduced one irrespective of the number of fungal applications. Multiple fungal applications of either isolate reduced the nematode gall rating, and the native isolate also reduced the final egg population in roots. Neither isolates reduced final nematode densities in soil or affected tomato yield when compared to untreated plots.     

Manganese nutrition and accumulation of phenolics and lignin as related to differential resistance of wheat genotypes to the take-all fungus

Differential resistance of four Triticum aestivum L. genotypes to isolates of take-all fungus (Gaeuman-nomyces graminis var. ritici Walker) was tested in a complete factorial experiment set up in a growth chamber using Mn-deficient Wangary sand amended with four rates of Mn. Mn-efficient cultivars produced more dry matter at low supply of Mn. Fertilization with Mn significantly increased its accumulation in roots and shoots. The most sensitive measure of take-all infection was the total length of root stellar lesions; these lesions were reduced by Mn fertilization and were shorter in Mn-efficient genotypes. The resistance-enhancing effect of Mn was the most obvious in the Mn-inefficient genotype (Bayonet) and the least obvious in the Mn-efficient one (C8MM). Phenolics biosynthesis in roots was clicited by fungal infection, especially in the case of the highly virulent isolate. The weakly virulent isolate increased phenolics concentration in roots much more if no Mn was added, indicating that the resistance-enhancing effect of Mn may not be directly exerted through the effects on phenolics biosynthesis. Lignin concentration in roots decreased due to Mn fertilization, while no effect of take-all infection was noted. It appears that biosynthesis of phenolics and lignin in wheat roots has a low Mn requirement which can be satisfied at environmental Mn concentrations below those necessary for optimum plant growth. ei]Section editor: A C Borstlap ei]Section editor: H Lambers     

Characterization and screening of plant probiotic traits of bacteria isolated from rice seeds cultivated in Argentina

Many seeds carry endophytes, which ensure good chances of seedling colonization. In this work, we have studied the seed-borne bacterial flora of rice varieties cultivated in the northeast of Argentina. Surface-sterilized husked seeds of the rice cultivars CT6919, El Paso 144, CAMBA, and IRGA 417 contained an average of 5×10⁶ CFU/g of mesophilic and copiotrophic bacteria. Microbiological, physiological, and molecular characterization of a set of 39 fast-growing isolates from the CT6919 seeds revealed an important diversity of seed-borne mesophiles and potential plant probiotic activities, including diazotrophy and antagonism of fungal pathogens. In fact, the seed-borne bacterial flora protected the rice seedlings against Curvularia sp. infection. The root colonization pattern of 2 Pantoea isolates from the seeds was studied by fluorescence microscopy of the inoculated axenic rice seedlings. Both isolates strongly colonized the site of emergence of the lateral roots and lenticels, which may represent the entry sites for endophytic spreading. These findings suggest that rice plants allow grain colonization by bacterial species that may act as natural biofertilizers and bioprotectives early from seed germination.     

Investigation into the ability of roots of the poikilohydric plant Craterostigma plantagineum to survive dehydration stress

The ability of the root system of the poikilohydric plant Craterostigma plantagineum to survive dehydration was investigated. The data presented here reveal that the root system is capable of surviving dehydration, but shortly after rehydration the root system senesces. Two weeks after rehydration the growth of a complete new root system is initiated. During dehydration sucrose accumulates from 36 to a maximum of 111 micromol g-1 DW in the roots. It is suggested that the accumulation of sucrose protects the root system during dehydration. There are major stores of stachyose in the roots of Craterostigma (making up over 40% of the dry weight of the tissue) and during dehydration these stores are metabolized. It is suggested that these stachyose stores act as carbohydrate reserves for the synthesis of sucrose. However, over 350 micromol g-1 DW stachyose is metabolized in the roots, which is well in excess of that required for the accumulation of sucrose observed. It is likely that the stachyose reserves in the root system are translocated to other regions of the plant to support carbohydrate metabolism during dehydration of the tissue. During rehydration, the stachyose reserves return to their original level within 96 h. There is no change in the elevated sucrose content of the roots over this period. Thus the roots maintain the protective properties of sucrose much longer than they are needed. The maintenance of high sucrose contents in rehydrating roots is discussed as a possible survival strategy against recurrent desiccation events.     

An image analysis method for determination of spatial colonization patterns of bacteria in plant rhizosphere

A method that allows the rapid visualization of bacterial spatial colonization patterns on roots for the determination of general colonization trends was developed. This method, which analyzes images of roots, and bioluminescence-enhanced images of bacterial colonization patterns on these roots, was used to study the colonization patterns of seed-applied Enterobacter cloacae strain E6 on 3-day-old cucumber plants. Conventional dilution-plating methods indicated that E6 colonized cucumber tap roots in high populations and that these populations significantly decreased as the distance from the seed increased. In addition to confirming these observations, image analysis indicated that colonization by E6 significantly decreased on lateral roots as the distance increased horizontally away from the tap root, and that this bacterium did not evenly cover the most densely colonized regions of the cucumber root system. Results from these experiments indicate that the majority of E6 populations on cucumber roots after seed application are limited to the upper regions of the tap root and that E6 does not effectively colonize other regions of the root system. Received: 15 June 1988 / Received revision: 19 November 1998 / Accepted: 29 November 1998     

Localized alteration in lateral root development in roots colonized by an arbuscular mycorrhizal fungus

 The morphological responses of root systems to localized colonization by endophytes is not well understood. We examined the responses of lateral roots to the arbuscular mycorrhizal (AM) fungus Gigaspora margarita Becker & Hall inoculated locally into the soil. Peanut (Arachis hypogaea L.) and pigeon pea (Cajanus cajan (L.) Millsp.) were examined. Root boxes filled with nutrient-poor soil in were inoculated in one half with the fungus and in the other half with a sterilized inoculum. Responses were apparent after 30 days but not after 20 days. Overall, lateral root development was more advanced in inoculated soil. This was clearly observed for 2nd- and 3rd-order lateral roots, but less clear for 1st-order lateral roots in both species, although percentage of colonized root length was higher in 1st-order lateral roots. Whilst in peanut the responses were clearly evident at the level of lateral roots initiated on more proximal parts of the tap root axis, they occurred on more distal parts in pigeon pea. We conclude that plants under nutrient-poor conditions give priority to mycorrhizal roots when partitioning assimilation products within the root system. Thus, AM formation may induce local morphological alteration of root systems. Accepted: 29 August 1996     

Phosphorus effect on phosphatase activity in endomycorrhizal maize

Success of a mycorrhizal symbiosis is influenced by the availability of phosphorus (P) in the soil. Maize ( Zea mays L. cv. Great Lakes 586) plants were grown under five different levels of soil P, either in the presence or absence of formononetin or the vesicular‐arbuscular mycorrhizal (VAM) fungus Glomus intraradices Schenck and Smith. We detected physiological differences in mycorrhizal roots very early in the development of symbiosis, before the onset of nutrient‐dependent responses. Under low P levels, VAM roots accumulated a greater shoot dry weight (13%), root P concentration (15%) and protein concentration (30%) than non-VAM roots, although root growth was not statistically significantly different. At higher P levels, mycorrhizal roots weighed less than non-VAM roots (10%) without a concomitant host alteration of growth or root P concentration. Mycorrhizal colonization decreased as soil P increased. Formononetin-treatment enhanced colonization of the root by G. intraradices and partially overcame inhibition of VAM colonization by high soil P concentrations. This is the first report that formononetin improves root colonization under high levels of soil P. Acid phosphatase (ACP) and alkaline phosphatase (ALP) activities were closely related to the level of fungal colonization in corn roots. ACP activity in corn roots responded more to soil P availability than did ALP activity (38% more). These results suggest that ACP was involved in the increased uptake of P from the soil, while ALP may be linked to active phosphate assimilation or transport in mycorrhizal roots. Thus, soil P directly affected a number of enzymes essential in host-endophyte interplay, while formononetin enhanced fungal colonization.     

Colonization of wheat by Azospirillum brasilense Cd is impaired by saline stress

The effect of saline stress on the colonization of wheat was analyzed by using Azospirillum brasilense Cd carrying the fusion of the reporter gene lacZ (β-galactosidase) with the N₂ fixation gene promoter nifA. Colonization was also studied by inducing para-nodules on wheat roots using 2,4-D, establishing that these structures acted as bacterium protected niches. Bacteria grown under standard conditions were distributed along the whole root system, except the elongation zone, and colonized the para-nodules. Bacteria experiencing saline stress were mainly localized at the root tips and the lateral roots. In 2,4-D treated plants, most of the bacteria were present around the basal surface of the modified lateral root structures. Using the MPN method, there were not statistical differences between the numbers of control and stressed bacteria. As this method estimates endophytic colonization in contrast with the one using X-gal, which emphasizes colonization on the root surface, both procedures demonstrated to be necessary, concluding that salt treatment reduced surface colonization (X-gal) but not colonization inside the root. The bacterial counts made on inoculated wheat roots indicated higher numbers of both control and stressed bacteria in roots treated with 2,4-D compared with untreated roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Cotyledonary Inhibitor of Root Nodulation in Pisum sativum

Root nodule formation was studied in 11-day-old seedlings of Pisum sativum L. cv. Alaska, which were inoculated with Rhizobium leguminosarum at the time of planting. On intact plants an identical nodulation pattern was observed in the lateral roots attached to the two xylem strands connected to the cotyledons. When one cotyledon was removed before germination, however, a highly significant increase in nodultion was observed on the lateral roots attached to the xylem strand which no longer was connected to a cotyledon. Excision of one cotyledon also caused an alteration in the radial location of root nodules on the primary root. Under these conditions there was a distinct promotion of nodular proliferations in the root cortex opposite the primary phloem poles. The fact that removal of one cotyledon increased nodulation on the lateral roots but had no effect on the rhizobial infection of lateral root hairs suggested that a cotyledonary inhibitor acts at a step between the infection process and the appearance of a macroscopic nodule. The data were interpreted in terms of an inhibitor of cortical cell division which is translocated from the cotyledons to the developing root via the phloem.