Pea-root exudates and their effect upon root-nodule bacteria

The main purpose of this investigation was to study the exudation (mechanism, sites) of various compounds by roots of pea seedlings in relation to the growth of Rhizobium leguminosarum.Chapter 1 gives a survey of the literature pertaining to plant-root exudates and their influence upon soil microorganisms. In chapter 2, material and methods used in the investigation are described.Chapter 3 deals with experiments to localize the sites of exudation of young pea roots. In agreement with the generally accepted view, the tips of the main root as well as of the lateral roots were found to be important sites of exudation (fig. 3.1). Furthermore, a considerable release of ninhydrin-positive compounds occurred during the formation of lateral roots, a process by which the main root is severely damaged (figures 3.2 and 3.3).In chapter 4, experiments are recorded on the effect of root exudates, obtained from pea seedlings, on the growth of Rhizobium leguminosarum in a mineral salts' medium. Furthermore, the behaviour of Rhizobium leguminosarum on the root surface (before and shortly after the emergence of the first lateral roots) and in the direct vicinity of roots of pea seedlings is described.Exudates from growing roots were found to enable the growth of Rhizobium leguminosarum. Addition of a carbon source to the medium considerably increased the stimulatory effect of the exudates, indicating that excessive amounts of nitrogenous compounds were present in the root exudates during the early stages of growth, whereas assimilable carbohydrates were present in limiting amounts (table 4.1).Roots of pea seedlings without lateral roots, inoculated with Rhizobium leguminosarum, were unable to support growth of the bacteria on the root surface. Even a considerable drop of bacterial numbers occurred, suggesting the release of growth-inhibiting compounds by the roots (table 4.2). After the formation of lateral roots a strong increase of bacterial numbers on the root surface was observed. This increase was restricted to that part of the main root where lateral roots were present (table 4.4).When pea seedlings were grown in Petri dishes with their roots on agar (carbon source present), mixed with a suspension of Rhizobium leguminasarum, no zone of stimulated bacterial growth was observed in the vicinity of the root (fig. 4.5). This was in contrast with seedlings of a number of other legumes which under the same experimental conditions gave a distinct zone of bacterial growth near the roots (fig. 4.4). Only where lateral roots emerged from the main pea root was a rather weak stimulation of the rhizobia observed after some time (fig. 4.6). Removal of the cotyledons and stems of the pea seedlings growing on the agar resulted in an enormous growth of Rhizobium leguminosarum around the entire root system (fig. 4.7).Lateral roots of pea seedlings growing in sterile agar along the bottom of the Petri dish upon inoculation with R. leguminosarum gave a zone of bacterial growth. This zone started at some distance from the root, apparently owing to the presence of a zone of growth inhibition surrounding the roots more closely (fig. 4.9).When R. leguminosarum was grown in tubes in an agar medium supplemented with yeast extract or glutarnic acid, growth was confined to the upper part of the agar with a maximum at some distance below the surface of the agar. However, when seedlings of various legumes were present with their roots in the agar, rhizobial growth occurred along the entire root system down to the bottom of the tube as a result of transport of oxygen through the plant and excretion by the roots (fig. 4.11). In the case of pea seedlings, the bacterial growth was present at some distance from the roots, apparently owing to a zone of inhibition surrounding the pea root (fig. 4.12).When no glutamic acid or yeast extract had been supplied to the agar, a clear zone of bacterial growth was observed surrounding the roots of all the legumes tested (fig. 4. 10), except with peas where no zone of stimulated growth occurred.In chapter 5 the composition of a synthetic medium, suitable for the growth of Rhizobium leguminosarum, strain PRE, is given. This strain, frequently used in the present investigations, did not grow on regular Rhizobium media. It was found that sulfhydryl compounds together with uracil or cytosine had to be added to the basal medium to achieve optimal yields (table 5.1). A large number of Rhizobium strains tested in this medium showed excellent growth. The nutrient solution, by omitting certain constituents, was used to investigate the action of certain specific root exudates on the growth of Rhizobium spp. in vitro.In chapter 6 the exudation of nucleic-acid derivatives by roots of pea seedlings, grown in water, is described. Apparently two U-V-absorbing compounds were exuded by the growing roots (fig. 6.3), UV 1 and UV 2. The former consists of uracil and a derivative of uracil which after hydrolysis gave uracil (figures 6.4 and 6.5). UV 2, in subsequent experiments, appeared to be also ninhydrinpositive (see chapter 7).The amounts of UV 1, exuded by roots without lateral roots, were found to be proportional to time of exudation while a considerable increase of the exudation of this compound was noticed after the formation of lateral roots (table 6.1). This compound is presumably synthesized in the root-tip region (of the main root as well as of the lateral roots), possibly as an intermediate in nucleicacid metabolism: exudation being a result of overproduction. This agrees with the fact that UV 1 was found in relatively small amounts in pressure juices of whole seedling roots, but in clearly higher concentrations in pressure juices of root tips.The amount of UV 2 exuded depends on the length of roots being dipped in water at the start of an experiment. Roots of which the tips only were dipped exuded much less compound UV 2 than those which were immersed more deeply (table 6.2). This shows that UV 2 is released by the entire root surface, rather than by the growing root tips. Relatively high concentrations of UV 2 were found in pressure juices of whole roots. Furthermore, root scrapings (including root hairs, epidermal cells and outer cortex cells) contained large amounts of UV 2. When the roots of pea seedlings (no lateral roots present), grown in humid air, were transferred to water, large amounts of UV 2 were released within a few hours, indicating the presence of UV 2 on the root surface and in epidermal cells, becoming damaged upon transfer to water (table 6.3).Chapter 7 deals with the free ninhydrin-positive compounds (n.p.c.) of pea seedlings and the exudation of these compounds by the roots. Extracts of roots of 5 or 6-days-old pea seedlings were found to contain 24 n.p.c., two of which were unknown: 'unknown X' and 'unknown Y' (figures 7.3 and 7.4). As the peaks of these unknown compounds on the chromatograms corresponded with those of glutathione and aspartic acid as obtained after separation of mixtures of known n.p.c., it was initially believed that these compounds represented glutathione and aspartic acid (cf. the literature survey in 7.3). However, a more detailed investigation showed that this hypothesis was not correct (fig. 7.5 A-F). 'Unknown X' was found to be a dipeptide, most likely consisting of glutamine and alanine and 'unknown Y' was identical with the U-V-absorbing compound UV 2, which most likely is a pyrimidine-amino acid.Homoserine was quantitatively the most important free n.p.c. in the root extracts. As this amino acid is specific to the genus Pisum , other legumes were tested for the presence of such specific compounds. In some of the legumes studied, this was indeed the case. In all of the legume seedlings tested, except peas and Lathyrus, the amides glutamine and asparagine were the predominant amino compounds (fig. 7.6 A-I).Since it might be expected that the exudation of certain compounds by roots of pea seedlings is closely connected with the occurrence of these compounds in the roots, a number of experiments were carried out in which the free amino compounds in different parts (cotyledons, roots, tops) of the seedlings were estimated during a period of approximately 1 month after the start of germination. The pea plants were grown under different environmental conditions, including nutrition.Homoserine, hardly present in dry pea seeds, increased rapidly in the germinating seed. In the cotyledons, its maximum value was reached 6 or 7 days after wetting the seed (table 7.2 and fig. 7.7). From the cotyledons the amino acid was transported mainly to the developing root system.Approximately 70 % of the total amount of the free n.p.c. of the seedling root consisted of homoserine at 7 days after the start of germination when the concentration of free n.p.c. had reached its maximum. During the subsequent 2-3 weeks the concentration of homoserine in the roots dropped, owing to increased root weight, but the total amount of this amino acid in the roots decreased only slightly. On the 24th day after the start of germination still 50 %of the free n.p.c. of pea roots was present as homoserine. Hereafter a pronounced decrease of the amount of homoserine (as well as of other n.p.c.) of the roots occurred in those plants which had not been supplied with nitrogen (table 7.3 and fig. 7.8). The soluble amino compounds of the roots of these plants, including homoserine, were needed to supply the growing top with nitrogen.Plants supplied with nitrate did not utilize the soluble nitrogen compounds of the roots for the growth of the tops. As a result of this, the amount of homoserine remained more or less constant from the 7th day after the start of germination until the experiments were finished (31 days after the start of germination; fig. 7.11).In the tops of 1-week-old pea plants, homoserine was also quantitatively the most important free amino compound. Without added nitrogen, it decreased rapidly from the 7th day after the start of germination owing to its utilization in the synthesis of insoluble plant material (table 7.4 and fig. 7.9). With added nitrogen, synthesis of homoserine took place in the tops from about the 24th day after the start of germination (fig. 7.12).The amides glutamine and asparagine were quantitatively the second most important amino compounds in the cotyledons of pea seedlings during the first two weeks after the start of germination. Hereafter a sharp increase of the amides took place so that these compounds became predominant and remained so until the cotyledons were exhausted (fig. 7.7). Almost all of the amides were transported to the tops, where they were used for the synthesis of insoluble cell compounds, mainly proteins. In the tops of etiolated plants this synthesis probably hardly occurred so that large amounts of the amides accumulated (fig. 7.14).Pea plants, inoculated with Rhizobium leguminosarum on the 12th day after the start of germination, and with nodules starting nitrogen fixation on approximately the 24th day after germination, rapidly increased in free amino compounds of both nodulated roots and tops. More than 90% of these N-compounds in the roots and nearly 60% in the tops consisted of glutamine and asparagine.Of the free amino compounds exuded by the root tip, 'unknown Y' was by far the most important compound while 'unknown X' was quantitatively the second most important (fig. 7.15). Homoserine was only present in minor quantities. When extracts were made of 2-mm slices of tips of young pea-seedling roots, 'unknown Y' was predominant in extracts of the first 2-mm slices, whereas homoserine was quantitatively most important in extracts of the second 2-mm slices (2 to 4 mm from the root tip; fig. 7.16 A and B). These results confirm the generally accepted view that the root tips are important sites of exudation.When roots, without laterals, grown in humid air, were transferred to water, large amounts of 'unknown Y' were released within a short time. (fig. 7.19). This was found to be mainly due to the presence on the root surface of 'unknown Y. It is assumed that under the conditions of this experiment, 'unknown Y', excreted by the root tip, is adsorbed by the root surface. After placing the root in water, the compound is released.Pea seedlings cultivated in nutrient solution during a period in which lateral roots had developed, released predominantly homoserine while 'unknown Y' came second (fig. 7.17). Lateral-root formation obviously cuased release of homoserine which was most likely liberated from the main root by the wounds originated by the formation of lateral roots. Further evidence concerning the importance of wounds for the release of homoserine was obtained by deliberately wounding seedling roots (grown in nutrient solution) shortly before the formation of lateral roots. After placing these roots in water, homoserine was by far the most important n.p.c. in the 'exudates'.Under the conditions of the present investigations, the first lateral roots emerged from the main root 6 or 7 days after the start of germination (fig. 7.18). In this period, the concentration of the free n.p.c., approximately 70 % of which consisted of homoserine, was maximal. Therefore, it is clear that during the formation of the first lateral roots, considerable amounts of homoserine were released.In chapter 8, experiments on the influence of homoserine on the growth of Rhizobium are summarized. The data recorded in chapter 7 suggested that homoserine would play an important role in the establishment of the rhizosphere microflora of pea plants.Compounds exuded by the tips of young roots in general will contribute to the development of a rhizosphere microflora, but the conditions on and close to the surface of intact seedling roots of pea plants are unfavourable for the multiplication of rhizobia owing to the release of inhibiting compounds (chapter 4).The experiments described in chapter 8 showed that R. leguminosarum grew equally well with homoserine as with glutamic acid as the nitrogen source or as the sole source of nitrogen, carbon and energy. Strains of R. trifolii and R. phaseo Ii behaved entirely differently. With glutamic acid as the only C and N-source, growth was similar to that of R. leguminosarum, but with homoserine growth was practically absent. Using this amino acid as the nitrogen source only, slight growth was observed. In the presence of homoserine and glutamic acid, the growth was strongly reduced as compared with glutamic acid as the sole N-source. For these strains homoserine was apparently toxic.The response of R. meliloti to homoserine was different from that of the other types of Rhizobium. When serving as both the nitrogen and carbon source, this amino acid gave considerably lower yields than glutamic acid, but when used as the nitrogen source only, it gave almost equally good growth as glutamic acid.These experiments show that homoserine, functioning as N, C and energy source, enables the growth of those Rhizobium strains which are capable of producing nodules with pea plants, i.e. strains belonging to the cross-inoculation group R. leguminosarum. This means that homoserine, released from the main root during the formation of the first lateral roots, selectively stimulates the growth of Rhizobium leguminosarum when a mixture of Rhizobium strains, belonging to different cross-inoculation groups, is present in the surroundings of the young pea root. However, definite proof of this assumption is awaiting inoculation experiments with pea seedlings using mixtures of Rhizobium strains belonging to different cross-inoculation groups.