Soybean Root Exudates Research Articles

Metabolite profiling of soybean root exudates under phosphorus deficiency

Root exudates are derived from plant metabolites and their composition is affected by the plant nutritional status. The purpose of this study was to examine soybean root exudates under phosphorus (P) deficiency using a metabolite profiling technique. Glycine max L. (cv. Suzuyutaka) was grown in a culture solution at P concentrations of 0 (P0) and 8 (P8) mg P L−1 for 1, 5, 10 and 15 days after transplanting. Shoot extract, root extract and root exudates were collected and their metabolites were determined by capillary electrophoresis/time-of-flight mass spectrometry (CE-TOF MS). The shoot P concentration and dry weight of soybean plants grown at P0 were lower than those at P8. One hundred and eight, 116 and 79 metabolites were identified in the shoot extract, root extract and root exudates, respectively. The concentrations of several metabolites including amino acids and organic acids in root exudates were higher at P0 than at P8, irrespective of the P concentration in the shoot or root extract. These findings suggest that soybean roots actively release metabolites in response to P deficiency.

Effect of the presence of the plant growth promoting rhizobacterium (PGPR) Chryseobacterium balustinum Aur9 and salt stress in the pattern of flavonoids exuded by soybean roots

In this work we studied how biotic and abiotic stresses can alter the pattern of flavonoids exuded by Osumi soybean roots. A routine method was developed for the detection and characterization of the flavonoids present in soybean root exudates using HPLC-MS/MS. Then, a systematic screening of the flavonoids exuded under biotic stress, the presence of a plant growth promoting rhizobacterium, and salt stress was carried out. Results obtained indicate that the presence of Chryseobacterium balustinum Aur9 or 50 mM NaCl changes qualitatively the pattern of flavonoids exuded when compared to control conditions. Thus, in the presence of C. balustinum Aur9, soybean roots did not exude quercetin and naringenin and, under salt stress, flavonoids daidzein and naringenin could not be detected. Soybean root exudates obtained under saline conditions showed a diminished capacity to induce the expression of the nodA gene in comparison to the exudates obtained in the absence of salt. Moreover, lipochitooligosaccharides (LCOs) were not detected or weakly detected when Sinorhizobium fredii SMH12 was grown in the exudates obtained under salt stress conditions or under salt stress in the presence of C. balustinum Au9, respectively.

Signaling from soybean roots to Rhizobium

Legume plants have a unique ability to fix atmospheric nitrogen via symbiosis with rhizobia. For the establishment of symbiosis, legume plants secrete signaling molecules such as flavonoids from root tissues, leading to the attraction of rhizobia and the induction of rhizobial nod genes. Genistein and daidzein are found in soybean root exudates and function as signal molecules in soybean – Bradyrhizobium japonicum chemical communication. Although it is more than 20 years since these signal flavonoids were identified, almost nothing has been characterized concerning the membrane transport process of these molecules from soybean roots. To elucidate the transport mechanism we performed membrane transport assays with plasma membrane-enriched vesicles and various inhibitors. As a result, we concluded that an ATP-binding cassette-type transporter is involved in the secretion of genistein from soybean roots. The possible involvement of a pleiotropic drug resistance-type ABC transporter in this secretion is also discussed.

Isolation and identification of alectrol as (+)-orobanchyl acetate, a germination stimulant for root parasitic plants

Alectrol, a germination stimulant for root parasitic plants, was purified from root exudates of red clover ( Trifolium pratense L.) and identified as a strigolactone, (+)-orobanchyl acetate [(3a S,4 S,8b S, E)-8,8-dimethyl-3-((( R)-4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)methylene)-2-oxo-3,3a,4,5,6,7,8,8b-octahydro-2 H-indeno[1,2- b]furan-4-yl acetate], by 1D and 2D NMR spectroscopy and ESI- and EI-MS spectrometry. Orobanchyl acetate afforded an [M–42] + ion in EI-MS and thus had been recognized as an isomer of strigol. Orobanchyl acetate was detected in root exudates of soybean ( Glycine max L.) and cowpea ( Vigina unguiculata L.) along with orobanchol.

Involvement of a Soybean ATP-Binding Cassette-Type Transporter in the Secretion of Genistein, a Signal Flavonoid in Legume-Rhizobium Symbiosis

Legume plants have an ability to fix atmospheric nitrogen into nutrients via symbiosis with soil microbes. As the initial event of the symbiosis, legume plants secrete flavonoids into the rhizosphere to attract rhizobia. Secretion of flavonoids is indispensable for the establishment of symbiotic nitrogen fixation, but almost nothing is known about the membrane transport mechanism of flavonoid secretion from legume root cells. In this study, we performed biochemical analyses to characterize the transport mechanism of flavonoid secretion using soybean (Glycine max) in which genistein is a signal flavonoid. Plasma membrane vesicles prepared from soybean roots showed clear transport activity of genistein in an ATP-dependent manner. This transport activity was inhibited by sodium orthovanadate, a typical inhibitor of ATP-binding cassette (ABC) transporters, but was hardly affected by various ionophores, such as gramicidin D, nigericin, or valinomycin, suggesting involvement of an ABC transporter in the secretion of flavonoids from soybean roots. The K(m) and V(max) values of this transport were calculated to be 158 mum and 322 pmol mg protein(-1) min(-1), respectively. Competition experiments using various flavonoids of both aglycone and glucoside varieties suggested that this ABC-type transporter recognizes genistein and daidzein, another signaling compound in soybean root exudates, as well as other isoflavonoid aglycones as its substrates. Transport activity was constitutive regardless of the availability of nitrogen nutrition. This is, to our knowledge, the first biochemical characterization of the membrane transport of flavonoid secretion from roots.

Efeito de exsudatos radiculares em endósporos de Pasteuria penetrans e em juvenis do segundo estádio de Meloidogyne incognita

Juvenis do segundo estádio (J2) de Meloidogyne incognita foram incubados nos exsudatos radiculares de soja (Glycine max), tomateiro (Lycopersicon esculentum), cafeeiro (Coffea arabica), feijoeiro (Phaseolus vulgaris), mostarda (Brassica rapa), Crotalaria juncea e C. spectabilis e em água por 12 h. Em seguida, realizou-se o teste de adesão por centrifugação ou por borbulhamento. Em outro ensaio, endósporos de Pasteuria penetrans foram incubados por quatro dias a 26 ºC nos exsudatos e submetidos à adesão em J2 de M. incognita, sob borbulhamento constante por 24 h em tubos contendo água. Os J2 com endósporos aderidos pelo teste de borbulhamento foram inoculados em mudas de tomateiro. Verificou-se que a incubação dos J2 por 12 h nos exsudatos radiculares testados reduziu o número de endósporos de P. penetrans por J2 independentemente do método de adesão empregado. Os J2 incubados nos exsudatos radiculares testados proporcionaram menor número de fêmeas parasitadas em tomateiro em relação à testemunha (água), bem como menor número de galhas com exceção dos J2 incubados em exsudato do próprio tomateiro. A reprodução dos J2 incubados nos exsudatos radiculares não foi afetada quando comparada à testemunha. A incubação dos endósporos nos exsudatos das plantas testadas reduziu a adesão e a infetividade em J2, em relação à testemunha. Após 28 dias da inoculação, observou-se redução no número de fêmeas parasitadas resultantes da infecção desses J2 com endósporos incubados em exsudatos radiculares comparada com aqueles incubados em água. O parasitismo do J2 com endósporos tratados com exsudatos radiculares e a reprodutividade de fêmeas oriundas da infetividade desses J2 foram semelhantes aos incubados em água.

Adverse Effects of Soybeans on Subsequent Wheat and Triticale Crops

ABSTRACT Except where a good legume sod is plowed under or a heavy application of manure is used, N fertilizer applications are generally necessary for producing high yields from grain crops. A soybean (Glycine max (L.) Merr)-wheat (Triticum aestivum L.) rotation appears to be a logical opportunity to use legume N as a sustainable grain production resource. An experiment conducted to compare N from soybeans with fertilizer N resulted in a 46% reduction in wheat yield following soybeans where wheat was not fertilized with N. Subsequent research showed that when soybeans were allowed to grow to bloom stage or later preceding a wheat or triticale (× Triticosecale Wittmack) crop a significant reduction in grain yield resulted from the previous soybean crop. When the grain crop was fertilized with 80 lbs/acre of N this response did not occur. The experimental results suggest that soybean root exudates are responsible for the decrease in wheat and triticale growth and yield.

Screening and isolation of mutants of Bacillus megateriumB153-2-2 for motility, chemotaxis, antagonism, and sporulation

Cells of Bacillus megateriumstrain B153-2-2 from the exponential growth stage were treated with 0.01 methylmethane sulphonate for 1h at 30°C. Mutagenized cells were screened for motility and/or chemotaxis mutation based on swarm morphology. Sixty-eight putative mutants for chemotaxis and/or motility were isolated. The chemotactic phenotype of all mutants was confirmed by capillary assays for their chemotactic response to soybean ( Glycine max) root exudates. These mutants were tested further for growth rate, sporulation, and antagonism against Rhizoctonia solani. Eight mutants and a previous nitrosoguinadine-induced mutant, M2144, had the same growth rate as original B153-2-2 strain but differed in motility (Mot), chemotaxis (Che), sporulation (Spo), and/or antagonism to R. solani(Ant). By comparison with B153-2-2 (Mot +Che +Ant ++Spo +), nine mutants were grouped as follows: SD12 and SD20 (Mot −Che −Ant ++Spo ++), SF8 and SF21 (Mot −/+Che −Ant ++Spo ++), M2144 (Mot +Che +Ant +Spo +), R1013 (Mot ++Che ++Ant +Spo +), R518 (Mot ++Che ++Ant +Spo −), and LR1013 and LR518 (Mot +++Che +++Ant −Spo −). Mutant cells with an increase in motility and chemotaxis generally had decreased sporulation and/or in antagonism to R. solani.

Reduction of structural iron in selected iron‐bearing minerals by soybean root exudates grown in anin vitrogeoponic system

Abstract Research on the reduction of iron (Fe) by plant‐root exudates has been conducted using hydroponic solutions containing Fe salts or chelates. These solutions, however, fail to reflect the true soil environment because plants derive their majority requirement from the solid Fe(III) sources. An in vitro Geoponic system (IVGS) is developed to study the reduction of Fe‐bearing clay minerals, i.e., Upton and SWa‐1 (smectite), and Si‐containing amorphous Fe oxide by soybean‐root exudates. Surface sterilized soybean seeds, [Glycine max (L.) Men.] cv. Williams (marginally susceptible to Fe chlorosis), were germinated in presterilized glass culture tubes containing semi‐solid agar media (sucrose free) and Fe minerals. These tubes were placed in an incubator programmed for a white‐fluorescent light cycle for 16 h and temperature setting of 25±2°C. After 15 d of plant growth, the system was analyzed for Fe2+ and total Fe. The amount of structural Fe reduction was 0.012, 0.095 and 0.182 mmol/g for Upton, SWa‐...

Isoliquiritigenin, a strong nod gene- and glyceollin resistance-inducing flavonoid from soybean root exudate.

Isoflavonoid signal molecules from soybean (Glycine max (L.) Merr.) seed and root exudate induce the transcription of nodulation (nod) genes in Bradyrhizobium japonicum. In this study, a new compound with symbiotic activity was isolated from soybean root exudate. The isolated 2',4',4-trihydroxychalcone (isoliquiritigenin) is characterized by its strong inducing activity for the nod genes of B. japonicum. These genes are already induced at concentrations 1 order of magnitude below those required of the previously described isoflavonoid inducers genistein and daidzein. Isoliquiritigenin is also a potent inducer of glyceollin resistance in B. japonicum, which renders this bacterium insensitive to potentially bactericidal concentrations of glyceollin, the phytoalexin of G. max. No chemotactic effect of isoliquiritigenin was observed. The highly efficient induction of nod genes and glyceollin resistance by isoliquiritigenin suggests the ecological significance of this compound, although it is not a major flavonoid constituent of the soybean root exudate in quantitative terms.

Identification and cloning of Bradyrhizobium japonicum genes expressed strain selectively in soil and rhizosphere.

The growth of Bradyrhizobium japonicum USDA 110 and USDA 438 in soil extract-supplemented medium led to transcription of a large amount of DNA not expressed in basal medium. Strain USDA 438 was more competitive for the nodulation of soybean than strain USDA 110. To identify and isolate DNA regions which were expressed specifically in strain USDA 438 but not in strain USDA 110 in response to soil extract or soybean root exudate, we developed a subtractive RNA hybridization procedure. Several cosmid clones which showed strain-specific gene expression were isolated from a USDA 438 gene library. Two clones enhanced competitive nodulation when mobilized to USDA 110. The method described may be useful for identifying genes expressed in response to environmental stimuli or genes expressed differently in related microbial strains.

Chemoattraction of zoospores of the soybean pathogen, Phytophthora sojae, by isoflavones

The main infective agents in plant diseases caused by species of the fungal genera Phytophthora and Pythium are zoospores that are attracted chemotactically to plant surfaces. Here we demonstrate that the simple isoflavones daidzein and genistein, which occur in soybean root exudates, are highly effective chemoattractants for zoospores of Phytophthora sojae, an economically important pathogen of soybeans. When added to suspensions of actively swimming zoospores, daidzein and genistein also cause rapid encystment and germination. The isoflavones are active at concentrations down to 10 nM but are inactive with zoospores of several other species of Phytophthora and Pythium, non-pathogenic on soybeans. Daidzein and genistein are also inducers of nodulation genes in Bradyrhizobium japonicum, the nitrogen-fixing bacterial symbiont of soybeans. Thus, both the pathogen and the symbiont identify their host by recognizing the same chemical signals.

Competitiveness of indigenous populations of Bradyrhizobium japonicum serocluster 123 as determined using a root-tip marking procedure in growth pouches

Soil Bradyrhizobium populations limit nodule occupancy of soybean by symbiotically-superior inoculant strains throughout much of the American midwest. In this study, the competitiveness of indigenous populations of B. japonicum serocluster 123 from Waukegan and Webster soils was evaluated in growth pouches using a root-tip marking procedure. The native rhizobia were from soils incubated 0–8 h in soybean root exudate (SRE) or plant nutrient solution (PNS) prior to inoculation. Populations of serocluster 123 strains in soil and nodule occupancy by these strains were assessed using fluorescent antibodies prepared against B. japonicum USDA 123. There were no significant differences in populations that came from SRE or PNS incubated soils: both populations increased in number over the incubation period. Nodule occupancy by both populations in growth pouches was similar to that previously encountered in field studies with these two soils. With the Waukegan soil, the serocluster 123 population dominated nodulation forming 69 and 62% of taproot nodules above and below the root tip mark, respectively. However, for the more alkaline Webster soil, serocluster 123 strains were much less competitive, producing only 9 and 13%, respectively, of the nodules formed above and below the root tip mark. In growth pouches, soil populations of bradyrhizobia from the Webster soil produced significantly more nodules than those from the Waukegan soil, but both strains and a pure culture of USDA 110 had a similar distribution of nodules.

Nodulation of soybean roots by an isolate of Bradyrhizobium japonicum with reduced firm attachment capability

A field isolate of Bradyrhizobium japonicum which failed to attach polarly or firmly to soybean roots was compared with the laboratory isolate I-110 for its relative rate of growth, piliation, attachment and nodulation. Both isolates grew at a comparable rate in yeast extract-gluconate medium as well as in soybean root exudate, produced comparable amounts of soybean lectin binding polysaccharide, infected through curled root hairs and developed effective nodules. Approximately 5% of cells in cultures of 110 possessed pili but none were detected in cultures of 1007 by electron microscopy. Light microscopic observations of root hairs from roots exposed to 1007 and 110 inoculum showed no polarly attached cells of 1007 and approximately 100 cells of 110 polarly attached per mm root hair length. Plate counting of firmly bound cells released by sonication indicated that the number of 1007 cells firmly adhering was at least 1000-fold lower than the number of 110 cells attached. The significance of polar, firm and weak attachment in the initiation of symbiotic interactions is discussed.

Study of soybean and lentil root exudates

The effect of two isoflavonoids, coumestrol and daidzein which are present in aseptically grown roots and root exudates of soybean, was tested on some rhizospheric microorganisms. It was found that coumestrol promotes the growth ofR. japonicum USDA 138 (about 30%) andR. leguminosarum (about 15%) whereas it inhibits the growth ofAgrobacterium tumefaciens (about 50%) andPseudomonas sp. (about 20%). The following microorganisms were unaffected by this molecule:R. japonicum W505,Agrobacterium radiobacter, Micrococcus luteus andCryptococcus laurentii. It was found that daidzein promotesR. japonicum USDA 138 growth (about 20%) and inhibitsPseudomonas sp. growth (about 20%); other microorganisms were unaffected. In addition, coumestrol favoured the formation of ‘coccoids’ cells byRhizobium japonicum USDA 138 which could be the infective state of this strain. It seems that this compound is able to help nodulation of soybean by aRhizobium strain. This result supports the work of Peterset al. (1986) and Redmondet al. (1986) who show that flavones present in plant exudates induces expression of nodulation genes in Rhizobium.

Effect of soybean root exudates on Bradyrhizobium japonicum

Abstract Effects of soybean root exudates on the growth and survival of Bradyrhizobium japonicum (USDA 110) in artificial medium as well as in a Chester silt loam soil were studied. Exudates were added to a yeast extract‐mannitol medium at concentrations of 0.00, 0.01, 0.02, and 0.04% (w/v). Results showed that the lowest exudate concentration did not affect the growth of B. japonicum, while the highest level (0.04%) reduced the population in the medium. Exudates at a concentration of 0.02% enhanced the. population of B. japonicum in the medium. A study of the survival of B. japonicum in soil amended with exudates at 0.0, 0.1, 0.2, and 0.4% (w/w) was also conducted. Results indicated that exudates at 0.1 and 0.2% did not affect the population of rhizobia during the first week of incubation. The 0.4% concentration exhibited a rapid reduction in the population of B. japonicum during this time. After the first week, the population of B. japonicum declined for all treatments. By the end of the study, the soil...

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)

Host Recognition in the Rhizobium-Soybean Symbiosis

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.

Host Recognition in the Rhizobium-Soybean Symbiosis

The mechanism of host-symbiont recognition in the soybean-Rhizobium symbiosis was investigated utilizing mutants of R. japonicum defective in nodulation. Soybeans were grown in clear plastic growth pouches allowing the identification of the area on the root most susceptible to Rhizobium nodulation; the area between the root tip (RT) and smallest emergent root hair (SERH). The location of nodules in relation to this developing zone is an indication of the rate of nodule initiation. Nodules were scored as to the distance from the RT mark made at the time of inoculation. Seventy-eight per cent of the plants nodulate above the RT mark when inoculated with the wild type R. japonicum strain 3I1b110 with the average distance of the uppermost nodule being approximately 2 millimeters above the RT mark. These data indicate that the wild type strain initiates nodulation rapidly within the RT-SERH zone following inoculation. However, inoculation with the slow-to-nodulate mutant strain HS111 resulted in 100% of the plants nodulating only below the RT mark with the average distance of the uppermost nodule being approximately 56 millimeters below the RT mark. Thus, mutant strain HS111 is defective in the ability to rapidly initiate infection leading to nodulation within the RT-SERH zone. The location of the nodules suggest that stain HS111 must ;adapt' to the root environment before nodulation can occur. To test this, strain HS111 was incubated in soybean root exudate prior to inoculation. In this case, 68% of the plants nodulated above the RT mark with the average distance of the uppermost nodule being approximately 1 millimeter below the RT mark. Experiments indicated that the change in nodule initiation by strain HS111 brought about by incubation in soybean root exudate was due to a phenotypic, rather than a genotypic change. The half-time of root exudate incubation for strain HS111 necessary for optimal nodulation enhancement was less than 6 hours. Heat sensitivity and trypsin sensitivity of the nodulation enhancement factor(s) in soybean root exudate indicate a protein was involved in the reversal of the delay in nodulation by mutant strain HS111.

Root exudates in relation to growth and nitrogenase activity of Rhizobium japonicum.

Root exudates of cowpea and soybean were collected in aerated water cultures. The ability of these exudates to support nitrogenase activity of R. japonicum was studied on defined media. When a complete nitrogenase-inducing medium was supplemented with concentrated root exudate, there was an increased nitrogenase activity. When the cowpea root exudate was substituted for glutamine or sodium succinate in the medium, nitrogenase activity was not detected. Growth was good when the root exudate was substituted for sodium succinate but was poor when the exudate replaced glutamine. There was nitrogenase activity in R. japonicum when the cowpea root exudate was substituted for arabinose in the 20E medium. Gas chromatographic analyses of cowpea root exudates revealed that ribose, arabinose, glucose, and sucrose were the main constituents. The significance of this finding in the asymbiotic nitrogen fixation by Rhizobium spp. is discussed.