Chemotaxis of Bradyrhizobium japonicum to soybean exudates.

The chemotactic response of Bradyrhizobium japonicum toward soybean seed and root exudates was examined. Assays using various isoflavones and fractionated exudate indicated that isoflavones are not the principal attractants in exudates. Likewise, induction of nod genes with isoflavones or seed exudate before assay did not enhance chemotaxis. Screening of numerous compounds revealed that only dicarboxylic acids and the amino acids glutamate and aspartate were strong attractants. The presence of glutamate, aspartate, and dicarboxylic acids in appreciable concentrations in soybean seed and root exudates indicates that these compounds likely represent natural chemoattractants for B. japonicum.     

Effect of lectin on nodulation by wild-type Bradyrhizobium japonicum and a nodulation-defective mutant.

The nodulation characteristics of wild-type Bradyrhizobium japonicum USDA 110 and mutant strain HS111 were examined. Mutant strain HS111 exhibits a delayed-nodulation phenotype, a result of its inability to initiate successful nodulation promptly following inoculation of the soybean root. Previously, we showed that the defect in initiation of infection leading to subsequent nodulation which is found in HS111 can be phenotypically reversed by pretreatment with soybean root exudate or soybean seed lectin. This effect is not seen after pretreatment with root exudates and lectins obtained from other plant species. Treatment of strain HS111 with as little as 10 soybean seed lectin molecules per bacterium (3.3 X 10 (-12) M) resulted in enhancement of nodule formation. Pretreatment of wild-type B. japonicum USDA 110 with soybean root exudate or seed lectin increased nodule numbers twofold on 6-week-old plants. Wild-type strain USDA 110 cells inoculated at 10(4) cells per seedling exhibited a delay in initiation of infection leading to subsequent nodulation. Wild-type cells pretreated in soybean root exudates or seed lectin did not exhibit a delay in nodulation at this cell concentration. Mutant strain HS111 pretreated in seed lectin for 0 or 1 h, followed by washing with the hapten D-galactose to remove the lectin, exhibited a delay in initiation of nodulation. Phenotypic reversal of the delayed-nodulation phenotype exhibited by strain HS111 was seen if incubation was continued for an additional 71 h in plant nutrient solution following 1 h of lectin pretreatment. Reversal of the delayed-nodulation phenotype of HS111 through lectin pretreatment was prevented by chloramphenicol or rifampin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lectin on nodulation by wild-type Bradyrhizobium japonicum and a nodulation-defective mutant

The nodulation characteristics of wild-type Bradyrhizobium japonicum USDA 110 and mutant strain HS111 were examined. Mutant strain HS111 exhibits a delayed-nodulation phenotype, a result of its inability to initiate successful nodulation promptly following inoculation of the soybean root. Previously, we showed that the defect in initiation of infection leading to subsequent nodulation which is found in HS111 can be phenotypically reversed by pretreatment with soybean root exudate or soybean seed lectin. This effect is not seen after pretreatment with root exudates and lectins obtained from other plant species. Treatment of strain HS111 with as little as 10 soybean seed lectin molecules per bacterium (3.3 X 10 (-12) M) resulted in enhancement of nodule formation. Pretreatment of wild-type B. japonicum USDA 110 with soybean root exudate or seed lectin increased nodule numbers twofold on 6-week-old plants. Wild-type strain USDA 110 cells inoculated at 10(4) cells per seedling exhibited a delay in initiation of infection leading to subsequent nodulation. Wild-type cells pretreated in soybean root exudates or seed lectin did not exhibit a delay in nodulation at this cell concentration. Mutant strain HS111 pretreated in seed lectin for 0 or 1 h, followed by washing with the hapten D-galactose to remove the lectin, exhibited a delay in initiation of nodulation. Phenotypic reversal of the delayed-nodulation phenotype exhibited by strain HS111 was seen if incubation was continued for an additional 71 h in plant nutrient solution following 1 h of lectin pretreatment. Reversal of the delayed-nodulation phenotype of HS111 through lectin pretreatment was prevented by chloramphenicol or rifampin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bradyrhizobium japonicum nodD1 can be specifically induced by soybean flavonoids that do not induce the nodYABCSUIJ operon.

Besides genistein and daidzein, which are active inducers of the nodYABCSUIJ operon in Bradyrhizobium japonicum, soybean seeds also excrete compounds that are not inducers of the nodYABCSUIJ genes but enhance induction of this operon in the presence of a suboptimal genistein concentration. This synergism was studied in detail, and specific compounds were identified in seed exudate which specifically induce the nodD1 gene but not the nodYABCSUIJ operon. Therefore, our current hypothesis is that the observed synergism is caused by a specific induction of nodD1. The specific nodD1 inducers from soybean seed extract have been purified and characterized chemically. They appear to be derivatives of genistein, glycitein, and daidzein with glucose, malonyl, and acetyl groups attached. Both root and seed exudate appear to contain these compounds, with the seed being the major source. No hydrolysis of these compounds to their aglycone forms was detected in the presence of B. japonicum. A model for nod gene induction in B. japonicum is discussed.     

Endogenous isoflavones are essential for the establishment of symbiosis between soybean and Bradyrhizobium japonicum

Legume iso/flavonoids have been implicated in the nodulation process, but questions remain as to their specific role(s), and no unequivocal evidence exists showing that these compounds are essential for nodulation. Two hypotheses suggest that the primary role of iso/flavonoids is their ability to induce rhizobial nod gene expression and/or their ability to modulate internal root auxin concentrations. The present work provides direct, genetic evidence that isoflavones are essential for nodulation of soybean roots because of their ability to induce the nodulation genes of Bradyrhizobium japonicum. Expression of isoflavone synthase (IFS), a key enzyme in the biosynthesis of isoflavones, is specifically induced by B. japonicum. When IFS was silenced using RNA interference in soybean hairy root composite plants, these plants had severely reduced nodulation. Surprisingly, pre-treatment of B. japonicum or exogenous application to the root system of either of the major soybean isoflavones, daidzein or genistein, failed to restore normal nodulation. Silencing of chalcone reductase led to very low levels of daidzein and increased levels of genistein, but did not affect nodulation, suggesting that the endogenous production of genistein was sufficient to support nodulation. Consistent with a role for isoflavones as endogenous regulators of auxin transport in soybean roots, silencing of IFS resulted in altered auxin-inducible gene expression and auxin transport. However, use of a genistein-hypersensitive B. japonicum strain or purified B. japonicum Nod signals rescued normal nodulation in IFS-silenced roots, indicating that the ability of isoflavones to modulate auxin transport is not essential to nodulation.     

Host recognition in the Rhizobium-soybean symbiosis : evidence for the involvement of lectin in nodulation

Rhizobium japonicum mutant strain HS111 was previously shown to be defective in the rate of initiation of infection leading to subsequent nodule formation (1984 Plant Physiol 74: 84-89). Mutant strain HS111's defect in nodulation can be phenotypically reversed to wild type levels by pretreatment with root exudates from all soybean varieties that have been tested. The data indicate that lectin-Rhizobium interaction is necessary for the phenotypic reversal of the nodulation characteristics of mutant strain HS111. Pretreatment of strain HS111 with soybean seed lectin mimics the effect of root exudate pretreatment. In addition, the presence of 30 millimolar d-galactose, a hapten of soybean seed lectin, in the root exudate or soybean seed lectin pretreatment solution prevents enhancement of nodulation of strain HS111. Pretreatment of mutant strain HS111 in soybean root exudate which has had galactose-specific lectin(s) removed by affinity chromatography (affinity eluate) results in no enhancement of nodulation by strain HS111. Lectin(s) subsequently removed from the affinity column possesses 100% of the stimulatory activity originally found in the root exudate. Pretreatment of strain HS111 in root exudate from a soybean seed line (T102) known to lack seed lectin due to an insertion in the structural gene results in the reversal of the defective nodulation phenotype. This latter result indicates that the lectin found in soybean root exudate is genetically distinct from the seed lectin. It is apparently this root lectin that is involved in nodulation.     

Chemotaxis to aromatic and hydroaromatic acids: comparison of Bradyrhizobium japonicum and Rhizobium trifolii

Rhizobia are bacteria well known for their ability to fix nitrogen in symbiosis with leguminous plants. Members of diverse rhizobial species grow at the expense of hydroaromatic and aromatic compounds commonly found in plant cells and plant litter. Using a quantitative capillary assay to measure chemotaxis, we tested the ability of hydroaromatic acids, selected aromatic acids, and their metabolites to serve as chemoattractants for two distantly related rhizobial species, Bradyrhizobium japonicum and Rhizobium trifolii. Slow-growing B. japonicum I-110 demonstrated positive chemotaxis to shikimate, quinate, protocatechuate, and vanillate; threshold concentrations for the compounds were as low as 10(-6) M. The dicarboxylic acids succinate and beta-ketoadipate, metabolites in the catabolism of many aromatic compounds, were positive chemoattractants with low threshold concentrations as well. Taxis to beta-ketoadipate occurred constitutively and, of the tested compounds, beta-ketoadipate gave the strongest peak response. Taxis to shikimate or quinate was induced by growth on either substrate but not by growth on protocatechuate or succinate. In contrast, fast-growing R. trifolii 2066 was only weakly attracted to quinate and other aromatic and dicarboxylic acids that were strong attractants for B. japonicum. The R. trifolii strain exhibited positive chemotaxis to shikimate, but the threshold concentration of shikimate required to elicit a response (10(-4) M) was 2 orders of magnitude higher than that for the B. japonicum strain.     

Relationships between cell membrane stability, exudate content and infectivity of Bradyrhizobium japonicum strain 639 to boron starved soybean plants

The influence of boron starvation on the root exudates content in soybean seedlings (Glycine max. L. Merr.) and the effect of exudates pretreatment on the pre-infection processes in symbiotic system Br. japonicum strain 636 and soybean were investigated. Root cell membrane stability of boron starved soybean plants (-B) decreased compared to the control. The concentrations of all analyzed metabolites (reducing sugars, free amino acids, organic acids, soluble phenols and total flavonoids) from root exudates of -B plants were lower than the control concentrations. Analysis of polyphenols after HPLC chromatography of root exudates showed significant difference of peak numbers between chromatograms of exudates obtained from boron starved and from control plants. Bacterial culture treatment with root exudates from -B plants showed decreased growth, chemotaxis and attachment ability toward the host root compared to the control exudate treatments. These changes were accompanied by decreased nodulation and acetylene reduction activity of boron starved soybean plants.     

Assessment of the Role of Chemotaxis and Biofilm Formation as Requirements for Colonization of Roots and Seeds of Soybean Plants by <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> BNM339

This article correlates colonization with parameters, such as chemotaxis, biofilm formation, and bacterial growth, that are believed to be connected. We show here, by using two varieties of soybean plants that seeds axenically produced exudates, induced a chemotactic response in Bacillus amyloliquefaciens, whereas root exudates did not, even when the exudates, also collected under axenic conditions, were concentrated up to 200-fold. Root exudates did not support bacterial cell division, whereas seed exudates contain compounds that support active cell division and high cell biomass at stationary phase. Seed exudates of the two soybean varieties also induced biofilm formation. B. amyloliquefaciens colonized both seeds and roots, and plant variety significantly affected bacterial root colonization, whereas it did not affect seed colonization. Colonization of roots in B. amyloliquefaciens occurred despite the lack of chemotaxis and growth stimulation by root exudates. The data presented in this article suggest that soybean seed colonization, but not root colonization, by B. amyloliquefaciens is influenced by chemotaxis, growth, and biofilm formation and that this may be caused by qualitative changes of the composition of root exudates.     

Phenotypic variation of Pseudomonas putida and P. tolaasii affects the chemotactic response to Agaricus bisporus mycelial exudate.

The chemotactic response of wild-type Pseudomonas putida and P. tolaasii, and a phenotypic variant of each species, to Agaricus bisporus mycelial exudate was examined. Both P. putida, the bacterium responsible for initiating basidiome development of A. bisporus, and P. tolaasii, the causal organism of bacterial blotch disease of the mushroom, displayed a positive chemotactic response to Casamino acids and to A. bisporus mycelial exudate. The response was both dose- and time-dependent and marked differences were observed between the response time of the wild-type strains and their phenotypic variants. Phenotypic variants responded rapidly to both attractants and reached a maximum response after 10-20 min, whereas the wild-types took 45-60 min. The differences are partly explained by the more rapid swimming speed of the phenotypic variants. Both variants responded maximally to similar concentrations of Casamino acids and mycelial exudates. Investigations into the nature of the attractants contained in the mycelial exudate indicated that they are predominantly small (Mr less than 2000) thermostable compounds. Sugars present in the exudate did not elicit a chemotactic response in any isolate, but a mixture of 14 amino acids detected in the exudate accounted for between 50 and 75% of the chemotactic response of the fungal exudate.     

Chemotaxis of deleterious rhizobacteria to velvetleaf (Abutilon theophrasti Medik.) seeds and seedlings

Abstract Intact seeds and seed and seedling root exudates of velvetleaf ( Abutilon theophrasti Medik.) were used as chemoattractants in experiments to determine the relative importance of chemotaxis in spermosphere and rhizosphere colonization by selected rhizobacteria. Results for soft-agar, capillary tube and soil chemotaxis assays indicated that selected deleterious rhizobacteria were specifically attracted to seed and seedling root exudates. Several amino acids and sugars detected in exudates were chemoattractants for these rhizobacteria. Using soil-chemotaxis assemblies, migration of rhizobacterial isolates through 2-cm distances of soil toward velvetleaf seeds or exudates was detected within 24 h. Isolates were not detected at the same site in soils without seeds or exudates until 72 h after inoculation. These results suggest that attraction of delecterious rhizobacteria toward seeds and seedling roots mediated by exudates (chemotaxis) might be the first step in establishment and subsequent colonization of biological control bacteria on weed seeds and seedling roots in soil.     

Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates

The distribution of flavonoids, isoflavonoids, and their conjugates in developing soybean (Glycine max L.) seedling organs and in root and seed exudates has been examined. Conjugates of the isoflavones daidzein and genistein are major metabolites in all embryonic organs within the dry seed and in seedling roots, hypocotyl, and cotyledon tissues at all times after germination. Primary leaf tissues undergo a programmed shift from isoflavonoid to flavonoid metabolism 3 days after germination and become largely predominated by glycosides of the flavonols kampferol, quercetin, and isorhamnetin by 5 days. Cotyledons contain relatively constant and very high levels of conjugates of both daidzein and genistein. Hypocotyl tissues contain a third unidentified compound, P19.3, also present in multiple conjugated forms. Conjugates of daidzein, genistein, and P19.3 are at their highest levels in the hypocotyl hook and fall off progressively down the hypocotyl. These isoflavones also undergo a programmed and dramatic decrease between 2 and 4 days in the hypocotyl hook. All root sections are predominated by daidzein and its conjugates, particularly in the root tip, where they reach the highest levels in the seedling. Light has a pronounced effect on the distribution of the isoflavones; in the dark, isoflavone levels in the root tips are greatly reduced, while those in the cotyledons are higher. Finally, the conjugates of daidzein and genistein and several unidentified aromatic metabolites are selectively excreted into root and seed exudates. Analysis of seed exudates suggests that this is a continuous, but saturable event.     

Root exudates of plants

Summary Comparisons have been made of the amount and composition of seed and seedling exudates in barley, wheat, cucumber, and bean. Except in the case of wheat a greater proportion of the total nitrogen content in the seed exudates was formed by protein and peptide nitrogen than by nitrogen of free amino acids. In contrast, the greater part of the total nitrogen in the exudates of seedlings was formed by free amino acid nitrogen, except in the case of barley. Peptides represented 8 to 26 per cent of the protein and peptide fraction in seed exudates, the highest amount being found in bean. On an equal weight basis, the spectrum of amino acids released from seeds and seedlings differed little between barley, wheat and cucumber; greater differences were observed in bean exudates. Seedlings exuded reducing substances to a considerably greater extent than seeds. The spectrum of reducing sugars in seed and root exudates differed greatly, especially in the case of keto sugars. Differences in the organic acid spectra were small, except for bean plants, the seedling exudate of which contained more organic acids with a richer spectrum than seed exudate. Both seeds and seedlings of cucumber exuded a small quantity and a poor spectrum of organic acids.     

Studies on the rhizosphere mycoflora of broad bean and cotton

Summary Seed and root exudates from three varieties of both broad bean and cotton grown aseptically were analyzed for amino acids and sugars. Broad bean varieties were found to excrete considerable amounts of amino compounds with 15 different amino acids, while cotton varieties excreted less consisting of 12 amino acids. Five sugars were identified in the seed and root exudates from broad bean varieties, while three were present in the seed and root exudates from cotton varieties. Seed and root exudates stimulated spore germination and growth of fungi isolated from the rhizosphere.     

Recognition of dominant attractants by key chemoreceptors mediates recruitment of plant growth-promoting rhizobacteria

Chemotaxis to plant root exudates is supposed to be a prerequisite for efficient root colonization by rhizobacteria. This is a highly multifactorial process since root exudates are complex compound mixtures of which components are recognized by different chemoreceptors. Little information is available as to the key components in root exudates and their receptors that drive colonization related chemotaxis. We present here the first global assessment of this issue using the plant growth-promoting rhizobacterium (PGPR) Bacillus velezensis SQR9 (formerly B. amyloliquefaciens). This strain efficiently colonizes cucumber roots, and here, we show that chemotaxis to cucumber root exudates was essential in this process. We conducted chemotaxis assays using cucumber root exudates at different concentrations, individual exudate components as well as recomposed exudates, taking into account their concentrations detected in root exudates. Results indicated that two key chemoreceptors, McpA and McpC, were essential for root exudate chemotaxis and root colonization. Both receptors possess a broad ligand range and recognize most of the exudate key components identified (malic, fumaric, gluconic and glyceric acids, Lys, Ser, Ala and mannose). The remaining six chemoreceptors did not contribute to exudate chemotaxis. This study provides novel insight into the evolution of the chemotaxis system in rhizobacteria.     

Isoflavonoid-inducible resistance to the phytoalexin glyceollin in soybean rhizobia.

The antibacterial effect of the soybean phytoalexin glyceollin was assayed using a liquid microculture technique. Log-phase cells of Bradyrhizobium japonicum and Sinorhizobium fredii were sensitive to glyceollin. As revealed by growth rates and survival tests, these species were able to tolerate glyceollin after adaptation. Incubation in low concentrations of the isoflavones genistein and daidzein induced resistance to potentially bactericidal concentrations of glyceollin. This inducible resistance is not due to degradation or detoxification of the phytoalexin. The inducible resistance could be detected in B. japonicum 110spc4 and 61A101, representing the two taxonomically divergent groups of this species, as well as in S. fredii HH103, suggesting that this trait is a feature of all soybean-nodulating rhizobia. Glyceollin resistance was also inducible in a nodD1D2YABC deletion mutant of B. japonicum 110spc4, suggesting that there exists another recognition site for flavonoids besides the nodD genes identified so far. Exudate preparations from roots infected with Phytophthora megasperma f. sp. glycinea exhibited a strong bactericidal effect toward glyceollin-sensitive cells of B. japonicum. This killing effect was not solely due to glyceollin since purified glyceollin at concentrations similar to those present in exudate preparations had a much lower toxicity. However, glyceollin-resistant cells were also more resistant to exudate preparations than glyceollin-sensitive cells. Isoflavonoid-inducible resistance must therefore be ascribed an important role for survival of rhizobia in the rhizosphere of soybean roots.     

Flavonoids in seed and root exudates of Lotus pedunculatus and their biotransformation by Mesorhizobium loti

Analysis of the flavonoid content of seed and root exudates of Lotus pedunculatus was undertaken using multiple coupled analytical techniques: capillary zone electrophoresis coupled to a UV spectral array detector (CZE-UV), high performance thin-layer chromatography with densitometry (HPTLC-UV) and gas chromatography with mass spectrometry (GC-MS). These procedures provided separation, identification and structural confirmation of the previously unidentified flavonoids in this plant's seed and root exudates and were particularly applicable to samples from a small seeded legume. Catechin, naringenin, kaempferol, quercetin aglycone and 3 different glycosides of quercetin were detected in seed exudate. Sterile root exudates contained catechin, naringenin and quercetin in addition to apigenin, kaempferol and other flavones and flavanones for which partial identifications were obtained. When sterile root exudate was incubated with Mesorhizobium loti , changes in its flavonoid content were detected. Analysis of bacterial cells after incubation revealed the presence of quercetin, kaempferol and one other flavone. The monocyclic aromatics protocatechuic acid and phloroglucinol were detected in both the incubated root exudate and its bacterial cells.     

Levels of ACC and related compounds in exudate and extracts of canola seeds treated with ACC deaminase-containing plant growth-promoting bacteria

It was previously proposed that plant growth-promoting bacteria that possess 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase could utilize ACC that is present in the exudate of germinating canola seeds. The uptake and cleavage of ACC by these bacteria would lower the level of ACC, and thus ethylene within the plant, and reduce the extent of its inhibition on root elongation. To test part of the above mentioned model, ACC levels were monitored in canola seed tissues and exudate during germination. Lower amounts of ACC were present in the exudate and tissues of seeds treated with the plant growth-promoting bacterium Enterobacter cloacae CAL3, than in control seeds treated with MgSO4. The ACC-related compounds, alpha- and gamma-aminobutyric acids, both known to stimulate ethylene production, were also measured in the canola seed exudate and tissues. Approximately the same levels of alpha-aminobutyric acid were present in the exudates of the bacterium-treated seeds and the control seeds, but the amount of alpha-aminobutyric acid was lower in the tissues of the bacterium-treated seeds than in the control seeds. Smaller quantities of gamma-aminobutyric acid were seen in both the exudate and tissues of the E. cloacae CAL3-treated seeds than in the control seeds.     

Comprehensive chemical profiling of gramineous plant root exudates using high-resolution NMR and MS

Root exudates released into soil have important functions in mobilizing metal micronutrients and for causing selective enrichment of plant beneficial soil micro-organisms that colonize the rhizosphere. Analysis of plant root exudates typically has involved chromatographic methods that rely on a priori knowledge of which compounds might be present. In the research reported here, the combination of multinuclear and 2-D NMR with GC-MS and high-resolution MS provided de novo identification of a number of components directly in crude root exudates of different plant types. This approach was applied to examine the role of exudate metal ion ligands (MIL) in the acquisition of Cd and transition metals by barley and wheat. The exudation of mugineic acids and malate was enhanced by Fe deficiency. which in turn led to an increase in the tissue content of Cu, Mn, and Zn. The presence of elevated Cd maintained at a free activity pCd of 8.8 (10(-8.8) M), resulted in reduced phytosiderophore production by Fe deficient plants. The buffer morpholinoethane sulfonate (MES), which is commonly used in chelator-buffering nutrient solutions, was detected in the root exudate mixture, suggesting uptake and re-secretion of this compound by the roots. The ability to detect this compound in complex mixtures containing organic acids, amino acids, and other substances suggests that the analytical methods used here provide an unbiased method for simultaneous detection of all major components contained in root exudates.