Nodulation,nitrogen fixation and nitrogen uptake in pigeonpea (Cajanus cajan (L.) Millsp) of different maturity groups

Summary The seasonal patterns of nodulation, acetylene reduction, nitrogen uptake and nitrogen fixation were studies for 11 pigeonpea cultivars belonging to different maturity groups grown on an Alfisol at ICRISAT Center, Patancheru, India. In all cultivars the nodule number and mass increased to a maximum around 60–80 days after sowing and then declined. The nodule number and mass of medium- and late-maturing cultivars was greater than that of early-maturing cultivars. The nitrogenase activity per plant increased to 60 days after sowing and declined thereafter, with little activity at 100 days when the crop was flowering. At later stages of plant growth nodules formed down to 90 cm below the soil surface but those at greater depth appeared less active than those near the surface. All the 11 cultivars continued to accumulate dry matter until 140 days, with most biomass production by the late-maturing cultivars (up to 11 t ha⁻¹) and least by the early-maturing determinate cultivars (4 t ha⁻¹). Total nitrogen uptake ranged from 69 to 134 kg ha⁻¹. Nitrogen fixation by pigeonpea was estimated as the difference in total nitrogen uptake between pigeonpea and sorghum and could amount to 69 kg N ha⁻¹ per season, or half the total nitrogen uptake. Fixation by pigeonpea increased with crop duration, but there were differences within each maturity group. The limitations of the methods used for estimating N₂ fixation by pigeonpea are discussed. Submitted as J.A. No. 552 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).     

The effect of aluminium on growth of and nitrogen fixation in vegetable soybean germplasm

Vegetable soybeam germplasm was screened for its tolerance to 0, 50 and 100 μM Al in solution culture. Plants were inoculated with prescreened acid-Al tolerantBradyrhizobium japonicum strain USDA 110 and a localRhizobium isolate SM867. Aluminum concentrations of 0, 50, and 100 μM affected the root lengths of all germplasm lines in the first few weeks of their growth. At 100 μM, the plants had severely stunted roots throughout the growing period of 35 days, but at 50 μM the initial stunting of the roots was overcome after the third week of growth, and there were no significant differences between the root lengths of these plants and of the controls. The appearance of the first nodule was delayed for 2–3 and 4–5 days at 50 μM and 100 μM Al, respectively. There was a significant reduction in nodule numbers and acetylene reduction activity (ARA) at 100 μM Al. At 50 μM Al, even though the number of nodules was decreased significantly, nodules were larger in size, so there was no significant reduction in nodule fresh weight and ARA. No significant differences in nitrogen fixing abilities of the soybean lines were observed between the twoRhizobium strains. Germplasm line Kahala showed the greatest tolerance to 50 μM Al, and Kahala, Kim and Wolverine tolerated 100 μM Al better than other germplasm lines.     

Alleviation of Salt Stress in Common Bean (Phaseolus vulgaris) by Exogenous Abscisic Acid Supply

In this work the effect of abscisic acid (ABA) and 100 mM NaCl on common bean (Phaseolus vulgaris var. Coco) growth, nitrogenase activity, and nodule metabolism was studied. Experiments were carried out in a controlled environmental chamber and plants, at the vegetative growth stage (16 days old), were treated with ABA (1 μM and 10 μM) and 48 h later were exposed to saline treatment. Results revealed that plant dry weight, nodule dry weight, nitrogen fixation (acetylene reduction activity and ureides content), and most enzymes of ammonium and ureides metabolism were affected by both ABA and NaCl. The addition of 1 μM ABA to the nutrient solution before the exposure to salt stress reduced the negative effect of NaCl. Based on our results, we suggest that ABA application improves the response of Phaseolus vulgaris symbiosis under saline stress conditions, including the nitrogen fixation process and enzymes of ammonium assimilation and purine catabolism.     

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.     

Effect of fungicides on survival of Rhizobium on seeds and the nodulation of bean (Phaseolus vulgaris L.)

The survival of Rhizobium leguminosarum biovar phaseoli on seeds of bean was tested, using the cultivar Carioca. The seeds were treated seven days before inoculation with Benlate, Vitavax, Banrot, Difolatan or Ridomil fungicides. The rhizobial strains used were: CIAT 899, CPAC 1135 and CIAT 652. Strain CIAT 899 showed greater survival on the seed with fungicide than the other strains. Two hours after the contact with fungicides strains CIAT 652 and CPAC 1135 had significantly lower numbers of rhizobia than the treatment without fungicide. The Benlate and Banrot fungicides had the greatest effect on survival of rhizobial strains. There was a drastic mortality of the two strains, CIAT 652 and CPAC 1135, on seeds treated with Benlate and Ridomil. Under field conditions, granular inoculation produced fewer nodules, but a similar total nodule weight as seed inoculation. Serological tests (ELISA) showed that seed treatment with Benlate in connection with seed inoculation reduced drastically the occurrence of inoculated strains in nodules, while the same fungicide treatment and inoculation applied in the seed furrow did not affect the survival of the inoculated strain.     

Quantitative trait locus analysis of symbiotic nitrogen fixation activity in the model legume <Emphasis Type="Italic">Lotus japonicus</Emphasis>

Many legumes form nitrogen-fixing root nodules. An elevation of nitrogen fixation in such legumes would have significant implications for plant growth and biomass production in agriculture. To identify the genetic basis for the regulation of nitrogen fixation, quantitative trait locus (QTL) analysis was conducted with recombinant inbred lines derived from the cross Miyakojima MG-20 × Gifu B-129 in the model legume Lotus japonicus. This population was inoculated with Mesorhizobium loti MAFF303099 and grown for 14 days in pods containing vermiculite. Phenotypic data were collected for acetylene reduction activity (ARA) per plant (ARA/P), ARA per nodule weight (ARA/NW), ARA per nodule number (ARA/NN), NN per plant, NW per plant, stem length (SL), SL without inoculation (SLbac−), shoot dry weight without inoculation (SWbac−), root length without inoculation (RLbac−), and root dry weight (RWbac−), and finally 34 QTLs were identified. ARA/P, ARA/NN, NW, and SL showed strong correlations and QTL co-localization, suggesting that several plant characteristics important for symbiotic nitrogen fixation are controlled by the same locus. QTLs for ARA/P, ARA/NN, NW, and SL, co-localized around marker TM0832 on chromosome 4, were also co-localized with previously reported QTLs for seed mass. This is the first report of QTL analysis for symbiotic nitrogen fixation activity traits.     

Symbiotic nitrogen fixation and vegetative growth of cowpea (Vigna unguiculata (L.) walp.) in waterlogged conditions

Summary Various periods of waterlogging (up to 32 days duration) were imposed upon cowpea plants grown in pots under controlled glasshouse conditions. Particular attention was paid to treatment effects on nodule cortication, nitrogenase activity and fixation efficiency, and the consequent differences in plant dry weight and nitrogen content.All waterlogging treatments increased nodule cortication as compared with the unstressed controls; a 16-day stress period being of critical duration with respect to the bi-phasic nature of this anatomical response. Conspicuous lenticel-type protuberances were present on nodules formed under waterlogged conditions but were markedly reduced, or indeed absent, in the controls. Total dry weight of nodules per plant was reduced by 60 per cent after only 8 days waterlogging, but nitrogen fixation efficiency of nodules which persisted was only 18 per cent less than those on control plants; mean nodule cortex having increased from 39.8 (control) to 51.5 per cent. After 16 days waterlogging, total plant dry weight was decreased by ca 60 per cent as compared with control plants; reflecting similar adverse changes in leaf, stem and root dry weight. The most severe treatment (32 days waterlogging) did not further reduce plant dry weight but mean nodule cortex area increased from 55.9 (16 days) to a maximum of 59.3 per cent. With the exception of nodules, percentage nitrogen content of various plant components was unaffected by the treatments imposed.Both the formation of enlarged lenticles and increased nodule cortication are regarded as adaptive anatomical responses which facilitate continued symbiotic nitrogen fixation and vegetative growth of this legume under waterlogged conditions.One of a series of papers describing work undertaken in a collaborative project with the International Institute of Tropical Agriculture, Nigeria, sponsored by the U.K. Ministry for Overseas Development.     

Cinnamate toxicity expression on phenylalanine ammonia-lyase activity,germination and growth of cucumber (Cucumis sativis) seedlings

Low pH (5.2) decreased nodule number and acetylene reduction. Aluminium further depressed those parameters in theRhizobium leguminosarum-Pisum sativum associations examined. In the Al-treated plants nodule formation by strains 128C53 and 128C30 was not affected by 3 or 15 and 30 or 60 μM Al, respectively, as compared with the number of nodules on plants grown at pH 5.2 in the absence of Al. However, improved nodulation rates by those strains did not enhance plant dry weight or reduced nitrogen content. No differences in nitrogenase activity were found among strains of nodulating plants grown at the same aluminium level. These results suggest that Al-ions affected specifically nitrogenase activity and that this effect was primarily responsible for the reduction in plant growth.     

The effects of salinity on growth,nodulation and nitrogen fixation ofCasuarina equisetifolia

The growth, nodulation and nitrogen fixation ofCasuarina equisetifolia were compared at six levels (0–500mM NaCl) of salinity in sand culture. Dry weight of nodules, shoots and roots and N content of shoots increased at intermediate levels of salinity (50–100 mM) but decreased at 500 mM NaCl. Nodulation occurred at all NaCl levels, but at 500mM NaCl level, the nodule dry weight declined by 50% from the control. Increasing NaCl concentration of up to 200mM had little effect on the N₂-fixation rate, but at 500mM NaCl level the rate decreased to 40% of the control value.     

Nodulation and growth response ofAllocasuarina andCasuarina species to phosphorus fertilization

To examine how soil phosphorus status affects nitrogen fixation by the Casuarinaceae —Frankia symbiosis,Casuarina equisetifolia and two species ofAllocasuarina (A. torulosa andA. littoralis) inoculated or fertilized with KNO₃ were grown in pots in an acid soil at 4 soil phosphate levels. InoculatedC. equisetifolia nodulated well by 12 weeks after planting and the numbers and weight of nodules increased markedly with phosphorus addition. Growth ofC. equisetifolia dependent on symbiotically fixed nitrogen was more sensitive to low levels of phosphorus (30 mg kg^–1 soil) than was growth of seedings supplied with combined nitrogen; at higher levels of phosphorus, the growth response curves were similar for both nitrogen fertilized and inoculated plants. The interaction between phosphorus and nitrogen treatments (inoculated and nitrogen fertilized) demonstrated that there was a greater requirement of phosphorus for symbiotic nitrogen fixation than for plant growth when soil phosphorus was low.WithAllocasuarina species, large plant to plant variation in nodulation occurred both within pots and between replicates. This result suggests genetic variation in nodulation withinAllocasuarina species. Nodulation ofAllocasuarina species did not start until 16 weeks after planting and no growth response due toFrankia inoculation was obtained at the time of harvest. Addition of nitrogen starter is suggested to boost plant growth before the establishment of the symbiosis. Growth ofAllocasuarina species fertilized with nitrogen responded to increasing levels of phosphorus up to 90 mg P/kg soil after which it declined by 69% forA. littoralis. The decrease in shoot weight ofA. littoralis, A. torulosa, C. equisetifolia andC. cunninghamiana at high phosphorus was confirmed in a sand culture experiment, and may be atributable to phosphorus toxicity.     

Effect of salinity on nodulation,nitrogen fixation and growth of common bean (<Emphasis Type="Italic">Phaseolus vulgaris)</Emphasis> inoculated with rhizobial strains isolated from the Haouz region of Morocco

This study compared growth, nodulation, nitrogen fixation, and nodular enzyme activities in response to salinity in some common bean-rhizobia symbiotic combinations. Seeds of Paulista and Efequince, two varieties of the common bean (Phaseolus vulgaris) were germinated and seedlings were transferred to pots containing vermiculite inoculated with the reference Rhizobium strain CIAT899 or with RhM11 or RhM14, two local strains. Plants were grown in a temperature-controlled glasshouse at 28°C and irrigated with a nutrient solution without NaCl (control) or supplemented with 25 mM NaCl (stressed). Plants were harvested at the flowering stage. The results showed that in controls, inoculation with RhM11 improved plant and nodule growth compared with those inoculated with RhM14 and CIAT 899. NaCl treatment generally had a negative affect on plant and nodule growth. Under the saline treatment, symbiotic nitrogen fixation was not significantly affected in the CIAT899-Paulista, CIAT899-Efequince and RhM11-Paulista combinations. Plant mineral nutrition was negatively affected under salt treatment for all of the tested symbiotic combinations. Inoculation with CIAT899 and RhM11 conferred more plant tolerance to salinity than inoculation with RhM14. The nodular phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) exhibited higher activities and were less affected by salinity in plants inoculated with the reference strain CIAT899 than those inoculated with local strains. We conclude that plants inoculated with CIAT899 and RhM11 showed more salinity stress tolerance than those inoculated with RhM14.     

Changes in ascorbate peroxidase, catalase, guaiacol peroxidase and superoxide dismutase activities in common bean (Phaseolus vulgaris) nodules under salt stress

To analyse nodular antioxidant enzyme expression in response to salt stress, Phaseolus vulgaris genotype BAT477 was inoculated with reference strain CIAT899, and treated with 50 mM NaCl. Plant growth, nodulation and nitrogen fixing activity were analysed. Results showed that: (1) all parameters, particularly in nodules, were affected by salt treatments, and (2) confirmed preferential growth allocation to roots. The ARA was significantly decreased by salt treatments. Protein dosage confirmed that nodules were more affected by salt treatment than were roots. We analysed superoxide dismutase, catalase, ascorbate peroxidase and peroxidase in nodules, roots and a free rhizobial strain. Our results indicated that SOD and CAT nodular isozymes had bacterial and root origins. The SOD expressed the same CuZn, Fe and Mn SOD isoforms in nodules and roots, whereas in free rhizobia we found only one Fe and Mn SOD. APX and POX nodule and root profiles had only root origins, as no rhizobial band was detected. Under salt stress, plant growth, nitrogen fixation and activities of antioxidant defense enzymes in nodules were affected. Thus, these enzymes appear to preserve symbiosis from stress turned out that NaCl salinity lead to a differential regulation of distinct SOD and POX isoenzyme. So their levels in nodules appeared to be consistent with a symbiotic nitrogen fixing efficiency hypothesis, and they seem to function as the molecular mechanisms underlying the nodule response to salinity.     

Genetic variability in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> strains nodulating soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill]

Brazil has succeeded in sustaining production of soybean [Glycine max (L.) Merrill] by relying mainly on symbiotic N₂ fixation, thanks to the selection and use in inoculants of very effective strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. It is desirable that rhizobial strains used in inoculants have stable genetic and physiological traits, but experience confirms that rhizobial strains nodulating soybean often lose competitiveness in the field. In this study, soybean cultivar BR 16 was single-inoculated with four B. japonicum strains (CIAT 88, CIAT 89, CIAT 104 and CIAT 105) under aseptic conditions. Forty colonies were isolated from nodules produced by each strain. The progenitor strains, the isolates and four other commercially recommended strains were applied separately to the same cultivar under controlled greenhouse conditions. We observed significant variability in nodulation, shoot dry weight, shoot total N, nodule efficiency (total N mass over nodule mass) and BOX-PCR fingerprinting profiles between variant and progenitor strains. Some variant strains resulted in significantly larger responses in terms of shoot total N, dry weight and nodule efficiency, when compared to their progenitor strain. These results highlight the need for intermittent evaluation of stock bacterial cultures to guarantee effective symbiosis after inoculation. Most importantly, it indicates that it is possible to improve symbiotic effectiveness by screening rhizobial strains for higher N₂ fixation capacity within the natural variability that can be found within each progenitor strain.     

Nitrogen Fixation, Assimilation and Transport During the Initial Growth Stage of Phaseolus vulgaris L

In nodulated common bean (Phaseolus vulgaris L.), there is typicallya period of N stress between 15 and 20 d after emergence (DAE),due to a lack of synchronization between the depletion of Nin the cotyledons and the beginning of N² fixation and transport.Screening trials identified some Rhizobium leguminosarum bv.phaseoli strains with which symptoms of N deficiency were notvisible (‘precocious’ strains). Cultivar Negro Argelwas then inoculated with two ‘traditional’ strains(C-05 and CIAT 727) and two ‘precocious’ strains(CNPAF 146 and CNPAF 512), and plants were harvested from 8to 30 DAE. There were no differences between the two groupsof strains in nodule dry weight or in the acetylene reductionrates between 8 and 16 DAE. However, nodules induced by the‘precocious’ strains showed earlier onset of glutaminesynthetase (GS) (EC 6.3.1.2 [EC] ) and glutamate synthase (GOGAT)(EC 1.4.1.14 [EC] ) activities, and ureide synthesis. The N concentrationin the nodules formed by ‘precocious’ strains variedfrom 4.2 to 4.5%, whereas with the ‘traditional’strains, it increased from 3.2% at 8 DAE to 65% at 18 DAE, atwhich time plants exhibited N-deficiency symptoms. By 21 DAE,GS and GOGAT activities in ‘traditional’ noduleswere increased, as well as the ureide-N-concentration in thexylem sap, nodule N content declined to 4.5% and the leavesbecame green. These results suggest that the N stress with ‘traditional’strains is not a limitation in early N² fixation activity butrather in the rates of expression of the processes of N assimilationand transport. Key words: Glutamate synthase, glutamine synthetase, nitrogen fixation, Phaseolus vulgaris, Rhizobium     

Physiological and biochemical studies on senescing tap root nodules of soybeans.

Senescence of soybean (Glycine max L. Merr.) tap root nodules was investigated by comparing changes in various physiological and biochemical activities with changes in capacity to fix nitrogen. Field-grown Beeson and Calland varieties of soybeans of various ages were sources of tap root nodules. With both varieties, the number of tap root nodules per plant remained constant between 56 and 86 days after planting but fresh weight, dry weight, and mass of tap root nodules increased duing this period. Nitrogen (C2H2)fixation by attached tap root nodules was maximum on a fresh weight, dry weight, or nitrogen basis about 56 days after planting for either variety. Metabolic activities of bacteroids as measured by carbon dioxide evolution from glucose and succinate did not appear to vary among nodules of different ages. There was also no indication of mobilization or deposition or deposition of iron, molybdenum, calcium, zinc, and nitrate in aging tap root nodules. Nitrate levels in the aerial portion of the plants decreased significantly after the initial decline in acetylene reduction. Nicotinamide deamidase activity in the cytosol and in extracts of bacteroids did not change significantly as tap root nodules aged. However, significant and consistent changes were observed in initial pH values of nodule breis and the initial decline occurred before (Calland) or concurrently (Beeson) with the initial decline of nitrogen fixation.     

Inoculation and production of container-grown red alder seedlings

Summary The inoculation ofAlnus rubra (red alder) withFrankia sp. can lead to a highly efficient symbiosis. Several factors contribute to the successful establishment of nitrogenfixing nodules: (1) quantity and quality ofFrankia inoculant; (2) time and method of inoculation; (3) nutritional status of the host plant.Frankia isolates were screened for their ability to nodulate and promote plant growth of container-grown red alder. Inoculations were performed on seedlings and seeds. Apparent differences in symbiotic performance could be seen when seeds or seedlings were inoculated. Plants inoculated at planting performed significantly better than those inoculated four weeks later in terms of shoot height, nodule number and shoot dry weight. If inoculation was delayed further, reduction in shoot height, nodule number and shoot dry weight resulted. The effect of fertilizer was also investigated with regard to providing optimal plant growth after inoculation. Plants receiving 1/5 Hoagland's solution minus nitrogen showed maximal plant growth with abundant nodulation. Plants receiving 1/5 Hoagland's solution with nitrogen showed excellent plant growth with significantly reduced nodulation.     

The pea ( Pisum sativum L.) genes sym33 and sym40 control infection thread formation and root nodule function

Two novel non-allelic mutants that were unable to fix nitrogen (Fix⁻) were obtained after EMS (ethyl methyl sulfonate) mutagenesis of pea (Pisum sativum L.). Both mutants, SGEFix⁻–1 and SGEFix⁻–2, form two types of nodules: SGEFix⁻–1 forms numerous white and some pink nodules, while mutant SGEFix⁻–2 forms white nodules with a dark pit at the distal end and also some pinkish nodules. Both mutations are monogenic and recessive. In both lines the manifestation of the mutant phenotype is associated with the root genotype. White nodules of SGEFix⁻–1 are characterised by hypertrophied infection threads and infection droplets, mass endocytosis of bacteria, abnormal morphological differentiation of bacteroids, and premature degradation of nodule symbiotic structures. The structure of the pink nodules of SGEFix⁻–1 does not differ from that of the parental line, SGE. White nodules of SGEFix⁻–2 are characterised by “locked” infection threads surrounded with abnormally thick plant cell walls. In these nodules there is no endocytosis of bacteria into host-cell cytoplasm. The pinkish nodules of SGEFix⁻–2 are characterised by virtually undifferentiated bacteroids and premature degradation of nodule tissues. Thus, the novel pea symbiotic genes, sym40 and sym33, identified after complementation analysis in SGEFix⁻–1 and SGEFix⁻–2 lines, respectively, control early nodule developmental stages connected with infection thread formation and function. Received: 12 June 1998 / Accepted: 25 June 1998     

Selection of Competitive Strains of Rhizobium Nodulating Phaseolus Vulgaris and Adapted to Environmental Conditions in Egypt,Using the Gus-Reporter Gene Technique

Two Rhizobium etli strains, EBRI 2 and EBRI 26, isolated from Egypt were tested for nodulation competitiveness on beans using Rhizobium tropici CIAT 899G as the competing strain. The insertion of the gus-reporter transposon mTn5ssgusA30 did not alter the nodulation or nitrogen fixation capacity of mutant strain CIAT 899G compared to the wild type. At neutral pH, R. etli strains EBRI 2 and EBRI 26 were more competitive than CIAT 899G with the bean cultivar Saxa. These two strains gave nodule occupancies of 52.1 and 61.1% competing with equal cell numbers of CIAT 899G. Nodule occupancies from these two native strains increased with the bean cultivar Giza 6 from Egypt to 66 and 67.5%. Based on these results, cultivar Giza 6 was used to select the most competitive strains under stress of salinity or alkalinity as a major problem for a large part of Egyptian soils. Under stress of salinity (0.2% NaCl or 34.2 mM NaCl), the salt-sensitive strain EBRI 2 was more competitive than the salt-resistant strain EBRI 26. Strain EBRI 2 gave 87.4% but strain EBRI 26 gave 63.7% nodule occupancy against CIAT 899G. The same trend of results was observed under stress of alkalinity (pH 8). Strain EBRI 2 occupied 83% while Strain EBRI 26 occupied 53.2%.     

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.     

Effect of seed size and plant growth on nodulation and nodule development in lima bean (Phaseolus lunatus L.)

Lima bean (Phaseolus lunatus L.) cultivars vary widely in their growth habit and seed size. Preliminary experiments indicated that a large-seeded pole cultivar (King of the Garden) formed many more nodules than a small-seeded bush cultivar (Henderson). The relative importance of seed size and shoot mass in determining nodule number and mass was assessed in five lima bean cultivars differing in seed size and growth habit. Between cultivars, significant positive correlations between initial seed mass, plant weight and nodule number and mass were observed during the first four weeks after planting. Comparisons within cultivars indicated a strong correlation between nodule mass and shoot dry weight. The influence of plant morphology on nodule formation and mass was secondary to the effects of seed and shoot mass. As plants matured, the increase in nodule mass paralleled the increase in plant mass, while nodule number was relatively stable after day 18. These results suggest that the highly regulated process of nodule formation was under the influence of seed derived factors, while the continued accumulation of nodule tissue was related to shoot growth.