Foliar Chlorosis in Symbiotic Host and Nonhost Plants Induced by Rhizobium tropici Type B Strains

Rhizobium tropici CIAT899 induced chlorosis in the leaves of its symbiotic hosts, common bean (Phaseolus vulgaris L.), siratro (Macroptilium atropurpureum Urb.), and Leucaena leucocephala (Lam.) de Wit. Chlorosis induction by strains CIAT899 and CT9005, an exopolysaccharide-deficient mutant of CIAT899, required carbon substrate. When the bacteria were added at planting in a solution of mannitol (50 g/liter), as few as 10³ cells of CIAT899 were sufficient to induce chlorosis in bean plants. All carbon sources tested, including organic acids and mono- and disaccharides, supported chlorosis induction. The addition of a carbon source did not affect the growth rate or the population density of CT9005 in the bean plant rhizosphere. Cell-free filtrates of cultures of CT9005 did not induce detectable chlorosis. All type B strains of R. tropici tested also induced chlorosis in common bean. Type A strains of R. tropici and all other species of bacteria tested did not induce chlorosis. Several lines of evidence indicated that nodulation was not required for chlorosis induction. Strain RSP900, a pSym-cured derivative of CIAT899, induced chlorosis in wild-type P. vulgaris. In addition, NOD125, a nodulation-defective line of common bean, developed chlorosis when inoculated with CIAT899, but did not develop nodules. CIAT899 consistently induced severe chlorosis in the leaves of the nonhost legumes alfalfa (Medicago sativa L.) and Berseem clover (Trifolium alexandrinum L.), and induced chlorosis in 29 to 58% of the plants tested of sunflower, cucumber, and tomato seedlings, but it did not induce chlorosis in the leaves of corn or wheat. Chlorosis induction in nonhost plants also required carbon substrate. The data are consistent with the hypothesis that R. tropici type B strains produce a chlorosis-inducing factor that affects a wide range of plant species.     

Response of common bean lines to inoculation: comparison between the <Emphasis Type="Italic">Rhizobium tropici</Emphasis> CIAT899 and the native <Emphasis Type="Italic">Rhizobium etli</Emphasis> 12a3 and their persistence in Tunisian soils

Since Phaseolus vulgaris (L) is poorly nodulated in all regions of Tunisia where this crop is grown, the response of common-bean lines CocoT and Flamingo to inoculation with reference Rhizobium tropici CIAT 899 or native rhizobia, namely Sinorhizobium fredii 1a6, Rhizobium etli 12a3, and Rhizobium gallicum 8a3, was studied in a field station. Since R. etli 12a3 was found to be the most effective native rhizobium, it was subsequently compared with R. tropici CIAT 899 in a broader study in two stations over 3 years. A significant interaction between bean and rhizobia was observed for nodule number, shoot dry weight, grain yield, and contents of nitrogen and chlorophyll. The native rhizobia was more efficient than CIAT899 for Flamingo, though not for CocoT. The Enzyme-linked immunosorbent assay technique was used with polyclonal antibody to assess the occupancy in nodule and persistence in soil of the inoculated rhizobia. For both stations the nodule occupancy was 100% during the first year for each rhizobium, but during the next 2 years, between 7 and 15% of nodules were formed by the rhizobia inoculated in the neighboring plot. It is concluded that the first-year inoculation is sufficient to maintain an adequate rate of nodulation during three growth cycles, and that the native R etli can be recommended for the common-bean inoculation in similar soils of Tunisia.     

Symbiotic performance of common bean and soybean co-inoculated with rhizobia and <Emphasis Type="Italic">Chryseobacterium balustinum</Emphasis> Aur9 under moderate saline conditions

The effect of co-inoculating beans and soybeans with rhizobia and Chryseobacterium, a plant growth promoting bacteria (PGPR), was studied under conditions of mild saline stress. Chryseobacterium balustinum Aur9 was used with Rhizobium tropici CIAT899 or R. etli ISP42 to inoculate common bean (Phaseolus vulgaris L.), or jointly with Ensifer (Sinorhizobium) fredii SMH12 and HH103 to inoculate soybean (Glycine max (L.) Merrill). The effect of co-inoculation was studied by following nodule primordia initiation, nodulation kinetics and symbiotic performance in plants grown under moderate saline conditions (25 mM NaCl). In common bean, co-inoculation improved nodule primordia formation when compared with single inoculation (R. tropici CIAT899). However, co-inoculation did not provide benefits in the development of nodule primordia in soybean with E. fredii SMH12. The kinetic of nodulation in bean was also favored by double inocula resulting in a higher number of nodules. Long-term effects of co-inoculation on beans and soybeans depended on the rhizobial species used. In both, control and saline conditions, co-inoculation of R. tropici CIAT899 and C. balustinum Aur9 improved bean growth when compared with the single inoculation (CIAT899). However, the positive effect of double inocula on plant growth did not occur when using R. etli ISP42. Soybean plants receiving double inoculation (E. fredii SMH12 and C. balustinum Aur9) showed better symbiotic performance, mostly under saline stress, than with a single inoculation. The results indicate that co-inoculation with C. balustinum and rhizobia under mild saline conditions partially relieves the salt-stress effects, although do not always result advantageous for symbiotic N₂ fixation in legume plants.     

Discrimination against 15N among recombinant inbred lines of Phaseolus vulgaris L. contrasting in phosphorus use efficiency for nitrogen fixation

Although isotopic discrimination processes during nitrogen (N) transformations influence the outcome of ¹⁵N based quantification of N₂ fixation in legumes, little attention has been given to the effects of genotypic variability and environmental constraints such as phosphorus (P) deficiency, on discrimination against ¹⁵N during N₂ fixation. In this study, six Phaseolus vulgaris recombinant inbred lines (RILs), i.e. RILs 115, 104, 34 (P deficiency tolerant) and 147, 83, 70 (P deficiency sensitive), were inoculated with Rhizobium tropici CIAT899, and hydroaeroponically grown with P-sufficient (250 μmol P plant⁻¹ week⁻¹) versus P-deficient (75 μmol P plant⁻¹ week⁻¹) supply. Two harvests were done at 15 (before nodule functioning) and 42 (flowering stage) days after transplanting. Nodulation, plant biomass, P and N contents, and the ratios of ¹⁵N over total N content (¹⁵N/Nt) for shoots, roots and nodules were determined. The results showed lower ¹⁵N/Nt in shoots than in roots, both being much lower than in nodules. P deficiency caused a larger decrease in ¹⁵N/Nt in shoots (−0.18%) than in nodules (−0.11%) for all of the genotypes, and the decrease in shoots was greatest for RILs 34 (−0.33%) and 104 (−0.25%). Nodule ¹⁵N/Nt was significantly related to both the quantity of N₂ fixed (R² = 0.96***) and the P content of nodules (R² = 0.66*). We conclude that the discrimination against ¹⁵N in the legume N₂-fixing symbiosis of common bean with R. tropici CIAT899 is affected by P nutrition and plant genotype, and that the ¹⁵N/Nt in nodules may be used to screen for genotypic variation in P use efficiency for N₂ fixation.     

Oxidative stress in the root nodules of Phaseolus vulgaris is induced under conditions of phosphorus deficiency

One of the most adverse effects of phosphorus (P) deficiency on N₂-fixing legumes is the generation of harmful active oxygen species which cause oxidative stress. And although oxidative stress has been widely studied in roots and shoots of various plant species, it has not yet sufficiently been documented in bean nodules so far. In this study, two recombinant inbred lines RIL115 (P-deficiency tolerant) and RIL147 (P-deficiency sensitive) of common bean and Concesa (local variety) were inoculated separately with the reference strain R. tropici CIAT899, RhM₁₁ (R. gallicum) or RhM₁₄ (R. tropici); two local strains of the Marrakesh region of Morocco. Nodulated plants were grown under semi-hydroponic conditions with sufficient or deficient P supply and analyzed for their oxidative responses at the flowering stage. The results indicated that P-deficiency decreased the growth of shoots (48 %) and nodules (32 %), particularly with RhM₁₄ exhibiting the highest decrease (52 %) of nodulation. This constraint increased electrolyte leakage of nodules (40 %) as compared to leaves (20 %), especially for plants inoculated with RhM₁₄ and CIAT899. Moreover, high H₂O₂ and malondialdehyde contents were noticed in P-deficient nodules of RhM₁₄ and RhM₁₁. These variations were associated with peroxidase activity stimulation in P-deficient nodules induced by CIAT899 and RhM₁₄. In symbiosis with RIL115, these last strains exhibited the highest nodule phenol content. Overall, phenol content was mainly enhanced in P-deficient nodules (35 %) as compared to the leaves (16 %). It was concluded that the genotypes inoculated with CIAT899 and RhM₁₁ are relatively P-deficiency tolerant combinations as compared to those inoculated with RhM₁₄. Increase of oxidative stress in nodules rather than in leaves points to the need for further investigations of mechanisms that improve the root-nodule efficiency under adverse conditions.     

Effect of pH on competition for nodule occupancy by type I and type II strains ofRhizobium leguminosarum bv.phaseoli

The effect of soil pH on the competitive abilities of twoRhizobium leuminosarum bv.phaseoli type I and one type II strains was examined in a nonsterile soil system.Phaseolus vulgaris seedlings, grown in unlimed (pH 5.2) or limed (pH 7.6) soil, were inoculated with a single-strain inoculum containing 1 × 10⁶ cells mL^–1 of one of the three test strains or with a mixed inoculum (1:1, type I vs. type II) containing the type II strain CIAT 899 plus one type I strain (TAL 182 or CIAT 895). At harvest, nodule occupants were determined. In a separate experiment, a mixed suspension (1:1, type I vs. type II) of CIAT 899 paired with either TAL 182 or CIAT 895 was used to inoculateP. vulgaris seedlings grown in sterile, limed or unlimed soil. The numbers of each strain in the rhizosphere were monitored for 10 days following inoculation. The majority of nodules (> 60%) formed on plants grown in acidic soil were occupied by CIAT 899, the type II strain. This pattern of nodule occupancy changed in limed soil. When CIAT 899 was paired with TAL 182, the type I strain formed 78% of the nodules. The number of nodules formed by CIAT 899 and CIAT 895 (56% and 44%, respectively) were not significantly different. The observed patterns of nodule occupancy were not related to the relative numbers or specific growth rates of competing strains in the host rhizosphere prior to nodulation. The results indicate that soil pH can influence which symbiotype ofR. leguminosarum bv.phaseoli will competitively nodulateP. vulgaris.     

A Rhizobium tropici DNA region carrying the amino-terminal half of a nodD gene and a nod-box-like sequence confers host-range extension

Rhizobium tropici CIAT899 is a broad-host-range strain that, in addition to Phaseolus, nodulates other plant legumes such as Leucaena and Macroptilium. The narrow-host-range of Rhizobium leguminosarum biovars phaseoli (strain CE3) and trifolii (strain RS1051) can be extended to Leucaena esculents and Phaseolus vulgaris plants, respectively, by the introduction of a DNA fragment 521 bp long, which carries 128 amino acids of the amino-terminal region of a nodD gene from R. tropici, as well as a putative nod-box-like sequence, divergently oriented. The 521 bp fragment, in the presence of L. esculenta or P. vulgaris root exudates, induced a R. leguminosarum bv. viciae nodA-lacZ fusion in either a CE3 or RS1051 background, respectively.     

Burkholderia phymatum Strains Capable of Nodulating Phaseolus vulgaris Are Present in Moroccan Soils

Phylogenetic analysis of 16S rRNA, nodC, and nifH genes of four bacterial strains isolated from root nodules of Phaseolus vulgaris grown in Morocco soils were identified as Burkholderia phymatum. All four strains formed N₂-fixing nodules on P. vulgaris and Mimosa, Acacia, and Prosopis species and reduced acetylene to ethylene when cultured ex planta.Until 2001 all known bacteria involved in root nodule symbioses with leguminous plants were classified as members of the order Rhizobiales of the Alphaproteobacteria, including Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (28, 36, 38). Moulin et al. (21), however, described two Burkholderia nodule-forming strains isolated from Machaerium lunatum in French Guiana and Aspalathus carnosa in South Africa, respectively, this being the first report on the presence of a betaproteobacterium within root nodules of legumes. Later, the strains were formally classified as Burkholderia phymatum STM815^T and Burkholderia tuberum STM678^T, respectively (33). Burkholderia species are the predominant isolates from nodules of mimosoid legumes from Panama (2), Costa Rica (3), Taiwan (4, 6), Brazil (5, 7), Venezuela (5), and Madagascar (24), which indicates a high affinity of Burkholderia for forming effective symbioses with Mimosa. Diazotrophy is well represented in Burkholderia; among the more than 55 species presently classified as Burkholderia, 9 have been shown to fix N₂ ex planta by using either the acetylene reduction activity (ARA) assay or the presence of nifH genes encoding nitrogenase reductase (3, 5, 11, 24) and more recently by ¹⁵N₂ isotopic dilution experiments (17).Common bean (Phaseolus vulgaris) is an herbaceous leguminous plant which establishes N₂-fixing symbiosis with at least 5 species of the genus Rhizobium. Rhizobium etli is the predominant species in America (29) and is also detected in Europe and Africa (13, 20). Rhizobium leguminosarum bv. phaseoli is commonly found in Europe (13) and has also been reported to be present in Tunisia (20) and Colombia (10). Rhizobium tropici is found in acid soils of South America and is also present in Europe and several African countries (18). Rhizobium giardinii has been detected only in European and Tunisian soils (1, 20), and Rhizobium gallicum has been found nodulating beans in Europe, North Africa, and Mexico (1, 13). In this study we report on the isolation and characterization of B. phymatum from root nodules of P. vulgaris grown in alkaline soils from Morocco. Our results show that strains formed effective nodules on species of Mimosa, Acacia, and Prosopis and fixed atmospheric N₂ under free-living conditions.Soil was taken from a field near Oulade Mansour (34°47′N, 2°15′W, Oujda province, Morocco) where maize and common bean have traditionally been grown as rotational crops without N fertilization. Soil had a sandy-clay texture and the following characteristics: pH (in water), 8.1; 55.18% sand; 17.17% silt; 27.65% clay; 6.1% carbonates; 7.69% organic carbon; 0.069 total nitrogen. Seeds of P. vulgaris cv. Flamingo were surface sterilized (96% ethanol for 30 s followed by immersion in 15% [vol/vol] H₂O₂ for 8 min), washed several times with sterile water, germinated in the dark, and planted in 1-kg pots containing soil and sterile sand (1:1, vol/vol). Plants were grown for 30 days in controlled environmental chambers under conditions previously described (9). Nodules were collected, pooled together, surface sterilized with 2.5% HgCl₂ for 5 min, and rinsed thoroughly with sterile distilled water. Then, 12 nodules were placed independently on petri dishes and crushed in a drop of sterile water with a sterile glass rod. The resulting suspension was streaked onto petri dishes containing either yeast extract-mannitol (YEM) medium (35) or peptone-mineral salts-yeast extract (PSY) medium (25). After incubation of the plates at 30°C for 7 days, CFU which represented all of the colony types that could be distinguished by microscopic observation of living cells were chosen. After identification, Burkholderia strains were routinely grown in BAc medium (12).For DNA extraction and PCR amplifications, genomic DNA was isolated from bacterial cells using the RealPure genomic DNA extraction kit (Durviz, Spain) according to the manufacturer''s instructions. Repetitive extragenic palindromic (REP) fingerprinting was performed using primers REPIR-I and REP2-I according to the method of de Bruijn (8). PCR amplifications of the 16S rRNA gene fragments were done with the Bphym-F and Bphym-R species-specific primer pair (37).After isolation from root nodules, 52 strains forming morphologically different colonies were obtained and grouped in two main clusters after REP-PCR fingerprinting (data not shown), a technique that is extensively used to group bacteria at subspecies or strain level (8, 34) and has proven to be a powerful tool for studies of microbial ecology and evolution (14). The nearly complete sequence of the 16S rRNA gene from a representative strain of each REP-PCR group was obtained and compared with those held in GenBank. Forty-six strains in cluster I were members of the family Rhizobiaceae from the Alphaproteobacteria. Another 4 strains, here referred to as GR strains (GR01 to GR04), grouped in cluster II and were classified into the family Burkholderiaceae within the Betaproteobacteria. The remaining two strains have not been clearly classified as yet. The four GR strains have almost identical 16 rRNA gene sequences, and BLAST searches showed that they were phylogenetically close (99% identity) to B. phymatum STM815^T, a strain originally isolated from the papilionoid legume Machaerium lunatum (21, 33). A phylogenetic analysis including 30 Burkholderia reference strains showed that strains from root nodules of P. vulgaris form a tight cluster with B. phymatum STM815^T (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Phylogenetic tree showing the positions of four P. vulgaris-isolated strains, GR01, GR03, GR05, and GR06, within the genus Burkholderia based on 16S rRNA gene sequence comparisons. One thousand bootstrap samplings were performed. The NCBI GenBank accession number for each strain is shown in parentheses. The bar represents one nucleotide substitution per 1,000 nucleotides. The multiple alignments of the sequences were performed with CLUSTAL W software (30). The tree topology was inferred by the neighbor-joining method (27), based on 1,310 DNA sites, and the distance matrix method was performed according to the method of Jukes and Cantor (15) using the program MEGA version 2.1 (16).The nodC gene was amplified with the primer pairs and conditions previously described (11). Amplification of the nodC gene from each GR strain yielded a DNA fragment of about 0.4 kb (data not shown) whose nucleotide sequences were identical for all four strains and showed 99% identity to those of B. phymatum strains STM815^T and NGR195A (11). A phylogenetic tree inferred from NodC sequences from B. phymatum strains and members of the order Rhizobiales is shown in Fig. ​Fig.2.2. Primers IGK (23) and NDR-1 (31) were used for amplification of the nifH genes as indicated earlier (22). PCR amplifications of the nifH gene and further sequencing from each GR strain revealed that they all had almost identical DNA sequences, which were 99% identical to those of B. phymatum STM815^T and NGR195A (11). A phylogenetic tree based on NifH sequences showing the relationships between B. phymatum and other Burkholderia and rhizobial species is shown in Fig. ​Fig.33.Open in a separate windowFIG. 2.Phylogenetic tree inferred from NodC sequences shows the positions of four P. vulgaris-isolated strains, GR01, GR03, GR05, and GR06, within the genus Burkholderia. The tree topology was inferred by the neighbor-joining method (27) based on 195 sites. The bar represents the number of amino acid substitutions per site. One thousand bootstrap samplings were performed. The NCBI GenBank accession number for each strain is shown in parentheses.Open in a separate windowFIG. 3.Phylogenetic tree inferred from NifH sequences shows the positions of four P. vulgaris-isolated strains, GR01, GR03, GR05, and GR06, within the genus Burkholderia. The tree topology was inferred by the neighbor-joining method (27) based on 195 sites. The bar represents the number of amino acid substitutions per site. One thousand bootstrap samplings were performed. The NCBI GenBank accession number for each strain is shown in parentheses.For nodulation tests, seeds of Glycine max, Cicer arietinum, Pisum sativum, Lens culinaris, Lotus corniculatus, and Medicago sativa were surface sterilized as described above for common beans. Seeds of Mimosa, Leucaena, Prosopis, and Acacia were surface sterilized with concentrated sulfuric acid for 10 min followed by 3% sodium hypochlorite for 10 min and then washed thoroughly with sterile water. Plant cultivation was carried out as indicated above. Acetylene reduction activity (ARA) by nodulated plants was assayed on detached root systems excised at the cotyledonary node as previously described (19). The GR strains are true symbionts of P. vulgaris as, after nodule isolation, they were able to establish new effective symbiosis with common beans, with values of ARA ranging from 492 to 525 μmol ethylene/plant/h. B. phymatum STM815^T also infected P. vulgaris, but the efficiency of the symbiosis, determined as plant dry weight (1.06 ± 0.18 g/plant/h), was half of that found in plants nodulated by the GR strains. These strains also nodulated Mimosa pigra, Acacia cochliacantha, Acacia bilimeki, Leucaena glauca, and Prosopis laevigata but were unable to form nodules on P. sativum, L. culinaris, L. corniculatus, M. sativa, G. max, and C. arietinum.Diazotrophy is common among Burkholderia species, as shown recently by N₂ isotopic dilution studies (17). Under free-living conditions, ARA by the GR strains was tested in semisolid JMV medium as indicated earlier (26). At the end of the experiments, the culture purity was routinely checked by plating to verify uniform colony morphology. Like strain STM815^T, strains isolated from P. vulgaris also had nitrogenase activity when grown ex planta. Values of activity, however, were about half of that detected in strain STM815^T (83 ± 15 nmol C₂H₄/h).Preparation of whole-cell proteins and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) assays were performed as described previously (12). Protein profiles were compared with type and reference strains of legume-nodulating Burkholderia species. P. vulgaris-isolated strains showed SDS-PAGE protein profiles (evaluated by visual comparison) almost identical to those from the STM815^T type strain of B. phymatum but clearly different from those of other legume-nodulating Burkholderia species (Fig. ​(Fig.4).4). No differences were found when API 20 NE and API 50 CH strips were used to check for differences in nitrogen and carbon sources between B. phymatum STM815^T and the GR strains (data not shown).Open in a separate windowFIG. 4.Protein electrophoregrams (SDS-PAGE) of P. vulgaris-isolated strains and type strains of known legume-nodulating Burkholderia species. Lane 1, B. tuberum STM678^T; lane 2, B. phymatum STM815^T; lanes 3 to 6, B. phymatum GR01, GR03, GR04, and GR06, respectively; lane 7, B. mimosarum PAS44^T; lane 8, B. nodosa Br3437^T.Based on 16S rRNA gene sequences and protein profiles, which provide strong evidence for the delineation of bacterial species (32), the Phaseolus-isolated strains could be assigned to the species B. phymatum. Moreover, analysis of the phylogenetic relationships of such sequences and other Burkholderia species showed that they formed a robust clade with B. phymatum STM815^T. In addition, sequencing of the nodC and nifH genes revealed that the phylogenetically closest bacterial species was B. phymatum STM815^T. All these results support the affiliation of Phaseolus-isolated strains as B. phymatum. Since genomic DNAs from the GR strains had the same DNA band pattern after REP-PCR fingerprinting and extremely similar 16S rRNA and nifH gene sequences, as well as identical nodC sequences, the four strains could be derived from a single clone. Our results also suggest that strains of B. phymatum isolated from Mimosa and Phaseolus have acquired their symbiosis genes either from a common ancestor or by lateral transfer between them, the direction of transfer being unknown. Although limited to three isolates, strains NGR114 and NGR195A from Mimosa invisa and Mimosa pudica in Papua New Guinea, respectively (11), and STM815^T from M. lunatum in French Guiana (21, 33), and four strains from P. vulgaris in Morocco, our results raise questions concerning the biogeographical, environmental, and host taxon distribution of B. phymatum nodule symbionts. B. phymatum was originally discovered in nodules from M. lunatum in French Guiana, and most other strains in this lineage have been found associated with host legumes in the genus Mimosa, primarily in the Neotropics. Thus, the current results extend both the known host distribution and geographic range of this group of nodule symbionts. Whether B. phymatum is prevalent on rhizobial species within nodules of P. vulgaris in the geographic site where soil samples were taken cannot be elicited from the present results. Accordingly, it will be important in future work to survey additional sites both within the native geographic range and elsewhere to understand the consistency of the association between Phaseolus and Burkholderia.     

Biosynthesis of compatible solutes in rhizobial strains isolated from <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> nodules in Tunisian fields

Background  

Associated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. Rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or NaCl, two abiotic constraints frequently found in semi-arid lands. Osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. Whereas most studies on rhizobial osmoadaptation have focussed on the model species Sinorhizobium meliloti, little is known on the osmoadaptive mechanisms used by native rhizobia, which are good sources of inoculants. In this work, we investigated the synthesis and accumulations of compatible solutes by four rhizobial strains isolated from root nodules of Phaseolus vulgaris in Tunisia, as well as by the reference strain Rhizobium tropici CIAT 899^T.     

Localization of the <Emphasis Type="Italic">Bacillus subtilis</Emphasis> beta-propeller phytase transcripts in nodulated roots of <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> supplied with phytate

Soil organic phosphorus (Po) such as phytate, which comprises up to 80 % of total Po, must be hydrolyzed by specific enzymes called phytases to be used by plants. In contrast to plants, bacteria, such as Bacillus subtilis, have the ability to use phytate as the sole source of P due to the excretion of a beta-propeller phytase (BPP). In order to assess whether the B. subtilis BPP could make P available from phytate for the benefit of a nodulated legume, the P-sensitive recombinant inbred line RIL147 of Phaseolus vulgaris was grown under hydroaeroponic conditions with either 12.5 μM phytate (C₆H₁₈O₂₄P₆) or 75 μmol Pi (K₂HPO₄), and inoculated with Rhizobium tropici CIAT899 alone, or co-inoculated with both B. subtilis DSM 10 and CIAT899. The in situ RT-PCR of BPP genes displayed the most intense fluorescent BPP signal on root tips. Some BPP signal was found inside the root cortex and the endorhizosphere of the root tip, suggesting endophytic bacteria expressing BPP. However, the co-inoculation with B. subtilis was associated with a decrease in plant P content, nodulation and the subsequent plant growth. Such a competitive effect of B. subtilis on P acquisition from phytate in symbiotic nitrogen fixation might be circumvented if the rate of inoculation were reasoned in order to avoid the inhibition of nodulation by excess B. subtilis proliferation. It is concluded that B. subtilis BPP gene is expressed in P. vulgaris rhizosphere.     

Use of DNA marker to select well-adapted <Emphasis Type="Italic">Phaseolus</Emphasis>-symbionts strains under acid conditions and high temperature

Soil acidity and high temperature contribute to the failure of nodulation in the common bean. It is therefore urgent to select strains with a high competitive ability under these stress conditions. Two Egyptian Rhizobium etli strains, EBRI 2 and EBRI 26, were examined against Rhizobium tropici CIAT 899G labeled with the gus (β-glucuronidase) reporter gene. EBRI 2 and EBRI 26 were less competitive than CIAT 899G under acid conditions with both the Egyptian cultivar Giza 3 and the Colombian cultivar Rab 39. However, EBRI 2 and EBRI 26 gave higher nodule occupancy (78% and 62.5, respectively) than the nodule occupancy (18.5% and 35%) obtained by CIAT 899G at 35°C with cultivar Giza 3. Soil acidity (pH 5.8) was less detrimental to the nodule occupancy of EBRI 2 than EBRI 26 when they tested in competition with CIAT 899G.     

Combined inoculation with Glomus intraradices and Rhizobium tropici CIAT899 increases phosphorus use efficiency for symbiotic nitrogen fixation in common bean (Phaseolus vulgaris L.)

This study compared the response of common bean (Phaseolus vulgaris L.) to arbuscular mycorrhizal fungi (AMF) and rhizobia strain inoculation. Two common bean genotypes i.e. CocoT and Flamingo varying in their effectiveness for nitrogen fixation were inoculated with Glomus intraradices and Rhizobium tropici CIAT899, and grown for 50 days in soil–sand substrate in glasshouse conditions. Inoculation of common bean plants with the AM fungi resulted in a significant increase in nodulation compared to plants without inoculation. The combined inoculation of AM fungi and rhizobia significantly increased various plant growth parameters compared to simple inoculated plants. In addition, the combined inoculation of AM fungi and rhizobia resulted in significantly higher nitrogen and phosphorus accumulation in the shoots of common bean plants and improved phosphorus use efficiency compared with their controls, which were not dually inoculated. It is concluded that inoculation with rhizobia and arbuscular mycorrhizal fungi could improve the efficiency in phosphorus use for symbiotic nitrogen fixation especially under phosphorus deficiency.     

Co-inoculation with Glomus intraradices and Rhizobium tropici CIAT899 increases P use efficiency for N2 fixation in the common bean (Phaseolus vulgaris L.) under P deficiency in hydroaeroponic culture

Common bean (Phaseolus vulgaris L.) genotypes CocoT and Flamingo were inoculated with Rhizobium tropici CIAT899 and Glomus intraradices (Schenck & Smith) and grown under sufficient versus deficient phosphorus supply for comparing the effects of double inoculation on growth, nodulation, mycorrhization of the roots, phosphorus use efficiency and total nitrogen. Although the double inoculation induced a significant increase in all parameters whatever the phosphorus supply in comparison to control, significant differences were found among genotypes and treatments. Nevertheless, the highest phosphorus use efficiency and plant total nitrogen were found under P deficiency in combination with arbuscular mycorrhizal fungi. It is concluded that inoculation with rhizobia and arbuscular mycorrhizal fungi could improve symbiotic nitrogen fixation even under phosphorus deficiency.     

Root interactions between intercropped legumes and non-legumes—a competition study of red clover and red beet at different nitrogen levels

Aims

To investigate root competition in a legume/non-legume mixture, and how root growth of the legume is affected by the competition at increasing nitrogen (N) supply.

Methods

Red beet (Beta vulgaris L.) and red clover (Trifolium pratense L.) were grown in transparent rhizotron tubes either in mixture or as sole crop at N supplies of 0, 75 or 150 kg ha^-1. The root growth was evaluated by the root intensity on the rhizotron surface, root depth and plant uptake of ¹⁵N injected into the soil at the deeper part of the red clover root system.

Results

Competition with red beet decreased clover root intensity in deeper soil layers compared to clover grown as sole crop. The difference between clover in sole crop and in mixture was not evident at the highest N supply because the root growth of clover in sole crop appeared to be lowered at high N level. Increased N supply increased the dominance of red beet, but generally did not alter the root growth and distribution of the two species grown in mixture.

Conclusions

Clover root growth and rooting depth were inhibited by competition with red beet but the effect was not enhanced by increased N supply; hence the increased dominance of red beet at higher N level was likely due to its increased growth and competitiveness for other soil resources.     

<Emphasis Type="Italic">Rhizobium etli</Emphasis> maize populations and their competitiveness for root colonization

Rhizobium etli, which normally forms nitrogen-fixing nodules on Phaseolus vulgaris (common bean), is a natural maize endophyte. The genetic diversity of R. etli strains from bulk soil, bean nodules, the maize rhizosphere, the maize root, and inside stem tissue in traditional fields where maize is intercropped with P. vulgaris-beans was analyzed. Based on plasmid profiles and alloenzymes, it was determined that several R. etli types were preferentially encountered as putative maize endophytes. Some of these strains from maize were more competitive maize-root colonizers than other R. etli strains from the rhizosphere or from bean nodules. The dominant and highly competitive strain Ch24-10 was the most tolerant to 6-methoxy-2-benzoxazolinone (MBOA), a maize antimicrobial compound that is inhibitory to some bacteria and fungi. The R. tropici strain CIAT899, successfully used as inoculant of P. vulgaris, was also found to be a competitive maize endophyte in inoculation experiments.     

Modulation of symbiotic efficiency and nodular antioxidant enzyme activities in two <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> genotypes under salinity

To analyse nodular expression of antioxidant enzymes depending on plant genotype and salinity, two Phaseolus vulgaris genotypes, tolerant BAT477 and sensitive COCOT, were inoculated with the reference strain Rhizobium tropici CIAT899 and grown under 25 and 50 mM NaCl. Plant growth, nodulation and nitrogen fixing activity measured by the acetylene reducing activity (ARA) as an indicator of nitrogenase (E.C. 1.7.9.92) activity were more affected by salt concentrations in COCOT than in BAT477, particularly with 50 mM NaCl. Electrophoresis analysis of antioxidant enzymes in nodules, roots and free-living rhizobia showed that only catalase (CAT E.C. 1.11.1.6) isoenzymes varied with genotype. The sensitive genotype showed lower antioxidant enzyme activities than tolerant genotype and it was more affected by salinity. In the tolerant genotype catalase and ascorbate peroxidase (APX, E.C. 1.11.1.11) were inhibited by salt stress, whereas superoxide dismutase (SOD, E.C. 1.15.1.1) and peroxidase (POX, E.C. 1.11.1.7) were activated by salinity. Statistical analysis allowed suggesting that tolerance to salinity is associated with a differential regulation of distinct superoxide dismutase and peroxidase activities.