Comparative salinity tolerance of symbiotically dependent and nitrogen-fed pigeonpea (Cajanus cajan) and its wild relative Atylosia platycarpa

Summary Once symbiosis between the pigeonpea cultivar ICPL 227 and the Rhizobium sp. strain IC 3024 is established, it is efficient in fixing N₂ under saline conditions and can support growth comparable to N-fed plants in growth media with up to 6 dS m^-1 salinity. However, the early stages of establishment of the pigeonpea-IC 3024 symbiotic system have proved sensitive to salinity. The present study showed that the number of nodules was markedly reduced at 8 dS m^-1 salinity; however, nodule development and functioning were not affected by salinity in the pigeonpea-IC 3024 symbiosis. The symbiotic system of Atylosia platycarpa and Rhizobium sp. strain IC 3087 was established successfully even at 12 dS m^-1 and supported growth comparable to that of N-fed plants. P levels in leaves were increased under saline conditions in N-fed and N₂-fixing pigeonpea and A. platycarpa. There were no consistent differences in the leaf Na and chloride levels between N-fed and N₂-fixing plants of pigeonpea and A. platycarpa. The present study suggests that the rhizobial symbiosis may not be a necessary factor for initial screening of pigeonpea and related wild species for salinity tolerance.Submitted as JA No. 964 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

Nodule-specific expression of Rhizobium meliloti symbiotic promoters P1 and P2 in chickpea-Rhizobium sp. symbiosis

Developmentally specific expression of Rhizobium spp. genes involved in symbiotic N₂ fixation is known to operate through cascade regulation of various nif and fix operons. Fusion constructs of lacZ under symbiotic promoters P1 (for nifHDK operon) and P2 (for fixABCX operon) of Rhizobium meliloti were mobilized into Rhizobium spp. (Cicer) strains Rcd301 and RCR13. The assays for -galactosidase activity to monitor the expression of lacZ under these promoters was performed in host backgrounds of Escherichia coli, R. meliloti, and Rhizobium spp. (Cicer). The enzyme assays indicated significant levels of expression from P1 and P2 promoters in chickpea rhizobia, specifically in symbiotic cells from nodules. However, as in R. meliloti, these promoters did not induce strong expression in free-living cells of Rhizobium spp. (Cicer). This indicates functional homology of R. meliloti promoters in rhizobium spp. (Cicer). Functional cross-reactivity of trans regulatory factors like NtrA, NtrC, and NifA between these rhizobia seems evident from the nodule-specific expression of P1 and P2 cis elements.     

Effect of high temperature on plasmid curing of Rhizobium spp. in relation to nodulation of pigeonpea [Cajanus cajan (L.) Millsp.]

Summary Fifty-six isolates of Rhizobium and Bradyrhizobium spp. (Cajanus) were studied for their plasmid profile and N₂-fixation efficacy. One to three plasmids were reproducibly detected in all the Rhizobium spp. strains but no plasmid was detected in the Bradyrhizobium spp. strains. Rhizobium sp. strain P-1 was mutagenized by Tn5 and three nod^– and six nod⁺fix^– were screened for symbiotic parameters. Neomycin-sensitive mutants were isolated by elevated temperatrue (40°C) from tranconjugants carrying Tn5 insertions. The high temperature cured these mutants from the single large plasmid present in the parent strain P-1. All these cured mutants were nod^–, indicating that the genes for nodulation were present on this plasmid, which is readily cured at a high temperature (40°C). The high temperature in the semi-arid zones of Haryana could be responsible for the low nodulation of pigeonpea because the plasmid carrying the nodulation genes is cured at 40°–45°C giving rise to non-nodulating mutants.     

Rhizobium sp. exopolysaccharide production,nodulation, and acetylene reduction activity (N2-fixation) in pigeon pea (Cajanus cajan L.)

Summary There was no correlation between the quantity of exopolysaccharide produced and acetylene reduction activity by Rhizobium spp. or by Bradyrhizobium spp. (Cajanus). The exopolysaccharide-defective mutants of Rhizobium sp. strain P 116 either failed to nodulate or showed a decrease in effectiveness. The deficiency in exopolysaccharide production was corrected by the addition of purified exopolysaccharide from the parent strain, or from Bradyrhizobium sp. strain P 149 or S24 isolated from pigeonpea (Cajanus cajan) and mungbean (Vigna radiata), respectively. However, the nodules so formed were not fully effective compared to those formed by the parent strain.     

Effects of Rhizobium and vesicular-arbuscular mycorrhiza inoculations on nodulation,symbiotic nitrogen fixation and soybean yield in subtropical-tropical fields

Summary Field experiments were carried out to determine the effects of single and mixed inoculations with Rhizobium and vesicular-arbuscular mycorrhiza (VAM) on nodulation, symbiotic N₂ fixation and yield of soybeans in six Taiwan subtropical-tropical sites. Inoculation with Rhizobium alone significantly increased nodulation, nodule weight and nitrogenase activity of nodules in three out of six experimental fields, and affected soybean yields in the range –13% to + 134%. Inoculation with VAM fungi alone did not have a significant effect on nodulation and nitrogenase activity. Mycorrhiza inoculation affected soybean yields in the range –13% to + 65%, but only the yield increases at one out of six sites with N application were statistically significant. Mixed inoculation with Rhizobium and mycorrhiza affected yields in the range –8% to + 145% A synergistic effect from mixed inoculation of Rhizobium-mycorrhiza on soybean yields was found in one out of six experimental fields. The yield response to N application (40 kg N ha^–1) in these six paddy-field trials was not significant. These results suggest that single or mixed inoculation of rhizobia can greatly assist soybean grain yields and can replace N fertilizers.     

Effect of nitrogen levels and Rhizobium strains on symbiotic N2 fixation and grain yield of Phaseolus vulgaris L. genotypes in normal and saline-sodic soils

Summary Following screening, selection, characterization, and symbiotic N₂ fixation with 12,5, 25.0, and 40.0 mg N kg^–1 in normal and saline-sodic soils, only two Phaseolus vulgaris genotypes (HUR 137 and VL 63) and two Rhizobium spp. strains (ND 1 and ND 2) produced maximum nodulation, nitrogenase activity, plant N contents, and grain yields in saline-sodic soil, with 12.5 mg N kg^–1, compared with the other strains. However, interactions between strains (USDA 2689, USDA 2674, and ND 5) and genotypes (PDR 14, HUR 15, and HUR 138) were significant and resulted in more nodulation, and greater plant N contents, nitrogenase activity, and grain yields in normal soils with 12.5 mg N kg^–1 compared with salt-tolerant strains. Higher levels of N inhibited nodulation and nitrogenase activity without affecting grain yields. To achieve high crop yields from saline-sodic and normal soils in the plains area, simultaneous selection of favourably interacting symbionts is necessary for N economy, so that bean yields can be increased by the application of an active symbiotic system.     

AFLP Fingerprinting in Pigeonpea (Cajanus cajan (L.) Millsp.) and its Wild Relatives

Detection of DNA polymorphism in cultivated pigeonpea (Cajanus cajan) and two of its wild relatives Cajanus volubilis and Rhynchosia bracteata is reported here for the first time using amplified fragment length polymorphism (AFLP) fingerprinting. For this purpose, two EcoRI (three selective nucleotides) and 14 MseI (three selective nucleotides) primers were used. The two wild species shared only 7.15% bands with the pigeonpea cultivars, whereas 86.71% common bands were seen among cultivars. Similarly, 62.08% bands were polymorphic between C. volubilis and pigeonpea cultivars in comparison to 63.33% polymorphic bands between R. bracteata and pigeonpea cultivars, and 13.28% polymorphic bands among pigeonpea cultivars. The cluster analysis revealed low polymorphism among pigeonpea cultivars and very high polymorphism between cultivated pigeonpea and its wild relatives. The AFLP analysis also indicated that only one primer combination (EcoRI + ACT and MseI + CTG), at the most any four primer pair combinations, are sufficient for obtaining reliable estimation of genetic diversity in closely related cultivars like pigeonpea material analyzed herein. AFLP analysis may prove to be a useful tool for molecular characterization of pigeonpea cultivars and its wild relatives and for possible use in genome mapping.     

Impact of nodule damage by Rivellia angulata on N2-fixation,growth, and yield of pigeonpea (Cajanus cajan L. Millsp.) grown in a Vertisol

Summary Damage caused by Rivellia angulata larvae to pigeonpea root nodules at the ICRISAT center in India was greater in the crop grown on Vertisols (up to 86%) compared to that on Alfisols (20%). Attempts to quantify the field effects of nodule damage on growth and yield of pigeonpea in a Vertisol, involving many heavy applications of soil insecticides (aldrin and hexachlorocyclohexane) failed because the insecticides did not control the pest and adversely affected the growth of the pigeonpea and the subsequent crop of sorghum (Sorgorum bicolor L. Moench). The impact of nodule damage on pigeonpea growth, yield and nutrient uptake was successfully studied in greenhouse-grown plants at three N levels. In this pot study, artificial inoculation with Rivellia sp. led to substantial nodule damage (70%). The results of this damage were a significant overall reduction in nodule dry weight (46%), acetylene reduction activity (31%), total leaf area (36%), chlorophyll content of leaves (39%) and shoot dry weight (23%) 68 days after sowing. At maturity, Rivellia sp. infestation caused significant reductions in top dry weight (22%), root and nodule dry weight (27%), seed dry weight (14%), and total N (29%) and P uptake (19%). The problems and prospects of manipulating nodule damage so as to reduce N losses in pigeonpea are discussed.Submitted as JA No. 756 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

Variation in nodulation and N2 fixation by the Gliricidia sepium/Rhizobium spp. symbiosis in a calcareous soil

Summary Variation in nodulation and N₂ fixation by the Gliricidia sepium/Rhizobium spp. symbiosis was studied in two greenhouse experiments. The first included 25 provenances of G. sepium inoculated with a mixture of three strains of Rhizobium spp. N₂ fixation was measured using the ¹⁵N isotope dilution method 12 weeks after planting. On average, G. sepium derived 45% of its total N from atmospheric N₂. Significant differences in fixation were observed between provenances. The percentage of N derived from atmospheric N₂ ranged from 26 to 68% (equivalent to 18–62 mg N plant^-1) and was correlated with total N in the plant (r=0.70; P=0.05). The second experiment included six strains of Rhizobium spp. and two methods of inoculation and the plants were harvested 14,35 and 53 weeks after planting. In the first harvest significant differences were found between the number of nodules and the percentage and amount of N₂ fixed. There was also a significant correlation between the number of nodules and the amount of N₂ fixed (r=0.92; P=0.05). In the final harvest no correlation was observed, although there were significant differences between the number of nodules and the percentage of N derived from the atmosphere. The amount of N₂ fixed increased with time (from an average of 27% at the first harvest to 58% at the final harvest) and was influenced by the Rhizobium spp. strain and the method of inoculation. It ranged from 36% for Rhizobium sp. strain SP 14 to 71% for Rhizobium SP 44 at the last harvest. Values for the percentage of atmosphere derived N₂ obtained by soil inoculation were slightly higher than those obtained by seed inoculation.     

Survival and competitiveness of Bradyrhizobium sp. in the rhizosphere of pigeonpea (Cajanus cajan)

Summary The survival of Bradyrhizobium Cajanus strain CC1021 in the soil and rhizosphere of pigeonpea [Cajanus cajan (L.) Millsp] was determined by most probable number and fluorescent antibody techniques. The survival of strain CC1021 was poor under field compared with pot-culture conditions. Although the rhizosphere of pigeonpea promoted the growth of native pigeonpea rhizobia, although no increase in the number of rhizobia was observed with the inoculant strain. Under similar conditions of rhizosphere colonization the competitiveness of strain CC1021 with cv UPAS 120 and ICPL 312 was 10% and 66016 during the first year and 8.4% and 33.3%, respectively, during the second year.     

Effect of inoculum rate on the performance of chickpea (Cicer arietinum L.) Rhizobium strains in the field

Summary The influence of three inoculum rates on the performance of three chickpea (Cicer arietinum L.) Rhizobium strains was examined in the field on a Mollisol soil. Increasing amounts of inoculum improved the performance of the strains. A normal dose (10⁴ cells per seed) applied at different intervals gave non-significant increases in nodulation, nitrogenase activity (acetylene reduction assay), nitrogen uptake and grain yield. A ten-fold increase in inoculum increased nodule number, shoot dry weight, nitrogenase activity (ARA) and grain yield, but increases over the control were significant only for nodule dry weight and nitrogen uptake by shoot and grain. The highest level of inoculum (100 × normal) significantly increased nodule dry weight, grain yield, total nitrogenase activity (ARA) and nitrogen uptake by shoot and grain. Strain TAL 620 was more effective than the other two. Combined nitrogen (60 kg N ha^–1) suppressed nodulation and nitrogenase activity (ARA).Research paper No. 4345 from the Experiment Station, G. B. P. U. A. & T., Pantnagar, Nainital, U. P.     

Differential responses of chickpea (Cicer arietinum L.) — Rhizobium combinations to saline soil conditions

Summary Chickpea cultivars (Cicer arietinum L.) and their symbiosis with specific strains of Rhizobium spp. were examined under salt stress. The growth of rhizobia declined with NaCl concentrations increasing from 0.01 to 2% (w : v). Two Rhizobium spp. strains (F-75 and KG 31) tolerated 1.5% NaCl. Of the 10 chickpea cultivars examined, only three (Pusa 312, Pusa 212, and Pusa 240) germinated at 1.5% NaCl. The chickpea — Rhizobium spp. symbiosis was examined in the field, with soil varying in salinity from electrical conductivity (EC) 4.5 to EC 5.2 dSm^-1, to identify combinations giving satisfactory yields. Significant interactions between strains and cultivars caused differential yields of nodules, dry matter, and grain. Four chickpea — Rhizobium spp. combinations, Pusa 240 and F-75 (660 kg ha^-1), Pusa 240 and IC 76 (440 kg ha^-1), Pusa 240 and KG 31 (390 kg ha^-1), and Pusa 312 and KG 31 (380 kg ha^-1), produced significantly higher grain yields in saline soil.     

Patterns of diversity in pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> (L.) Millsp.) germplasm collected from different elevations in Kenya

Pigeonpea germplasm accessions collected from low (<500 m), medium (501–1000 m), high (1001–1500 m) and very high elevation zones (>1500 m) of Kenya were evaluated for 15 agronomic traits and seed protein content at ICRISAT, Patancheru, India. There were significant differences (P < 0.001) among elevation zones for the number of primary and secondary branches, days to 75% maturity, pod length, seeds per pod, 100-seed weight and seed yield. Mean values indicated that the accessions from low elevation zone were significantly different from those collected in higher elevation zones for early flowering and maturity, number of primary branches, pod length, number of pods per plant, seeds per pod, 100-seed weight, seed yield and harvest index. None of the accessions collected in Kenya belonged to extra early (<80 days to 50% flowering) and early (80–100 days to 50% flowering) maturity groups, as defined by time to flowering at Patancheru, India. Mean diversity index based on all characters indicated that accessions from the low elevation zone are more diverse than those from the higher elevation zones. Frequency distribution for trait extremes indicated that the accessions from the low elevation zone were early to flower and mature, short statured, produced more primary and secondary branches with high pod bearing length, long pods, more pods per plant, more seeds per pod, a high seed yield and harvest index. Accessions from the very high elevation zone were late flowering, with a large number of tertiary branches, large seeds and a high shelling percentage and could be a source for cold tolerance and the breeding of vegetable types. Results suggest that the elevation of collection sites is therefore a very important determinant of variation patterns of pigeonpea in Kenya.     

Symbiotic effectiveness of streptomycin-resistant Rhizobium spp. mutants of chickpea (Cicer arietinum L.)

Summary Antibiotic-resistant Rhizobium spp. strains have been used in ecological studies of legumerhizobia symbiosis. It has been suggested that in the course of acquiring resistance against high doses of antibiotics, rhizobia might lose their symbiotic effectiveness. Evidence both for and against this argument has been presented (Kremer and Peterson 1982; Materon and Hagedron 1983). This communication reports our experience with streptomycin-resistant (Str⁺) mutants of chickpea Rhizobium spp. strains. Parent strains were used as controls.Research paper No. 5233 from the Experiment Station, G.B.P.U.A.&T. Pantnagar, Nainital     

Establishment and effectiveness of added pigeonpea (Cajanus cajan) rhizobia in different soils of narrow abiotic variability

Summary Pot and laboratory experiments were conducted to study the establishment and effectiveness of a streptomycin-sulphate-resistant (1 mg/ml of medium) pigeonpea rhizobia strain (RM₇) in sterile sand and non-sterile soils. Strain RM₇ increased the drymatter yield of pigeonpea plants (Cajanus cajan) by 106% over control plants under sterile conditions. However, when the rhizobia strain was introduced into 14 different non-sterile soils with a narrow abiotic variability, the comparable beneficial effect was observed only in one soil inoculated with log 6.70 cells/pot. At this inoculum rate, the percentage increase in yield over control plants varied from –1 to 140 in different soils. Rhizobium (RM₇), applied at log 3.70 cells/pot (3 kg soil), showed less than 5% establishment in four soils. However, establishment varied from 8% to 72% at a higher level of inoculation (log 6.70 cells/pot). Displacement of native rhizobia and creation of new sites for nodulation by the introduced rhizobia were also affected by soil properties. The increase in shoot dry-matter yield compared with control plants was positively correlated with the percent establishment of RM₇ (r = 0.60^*) in these soils. Experiments showed that some biotic stresses led to poor survival, proliferation and establishment of the added alien in the soil. Therefore, any culture that is efficient in one soil may not produce similar results under all situations.     

Phenotypic diversity in the pigeonpea (<Emphasis Type="Italic">Cajanus cajan</Emphasis>) core collection

The pigeonpea core collection developed at ICRISAT genebank consists of 1,290 accessions from 53 countries. The core collection, which includes selected lines in extra early, early, medium and late maturity groups was evaluated for 18 qualitative and 16 quantitative characters during the 2004 rainy season, to assess the phenotypic diversity and determine the relative importance of different characters in evaluating pigeonpea germplasm accessions. The four maturity groups differed significantly for all characters under study. The medium maturity group showed significantly higher mean number of primary, secondary and tertiary branches, number of racemes, pod bearing length, pods per plant, shelling percent and plot seed yield. Late maturity group showed significantly higher mean for leaf size, plant height, pod length, seeds per pod and 100-seed weight, indicating this group as a good source of vegetable pigeonpea. Significant positive correlations were found between number of secondary branches and pods per plant in extra early group (r = 0.756), late maturity group (r = 0.776) and entire core (r = 0.728) and between number of racemes and pods per plant in all maturity groups and entire core. Principal coordinate and principal component analysis showed that seven qualitative and nine quantitative traits were important in explaining multivariate polymorphism. The Shannon–Weaver diversity index (H′) varied for different maturity groups and traits. Phenotypic diversity, averaged over all the 16 characters, increases from extra early group (0.36 ± 0.04) to late maturity group (0.42 ± 0.04) suggesting that medium and late maturity groups have greater diversity compared to extra early and early maturity groups.     

Competitiveness of Rhizobium leguminosarum bv. phaseoli strains in relation to environmental stress and plant defense mechanisms

Summary Six Rhizobium leguminosarum bv. phaseoli strains (Ciat 151, Ciat 895, Ciat 899, CE3, H2C, Kim5s) were tested for nodule occupancy in different bean cultivars at two field sites (one fertile, one acid tropical soil) and in the greenhouse. The effects of several environmental factors such as low pH, high temperature, Al and Mn toxicity, iron deficiency, bean tannins, and bean phytoalexins were tested in vitro. Strain Kim5s was competitive under all tested conditions while strains CE3 and H2C had consistently low nodule occupancy levels. Strain Ciat 151 was superior to the other inoculant strains in the acid soil but competed poorly in the fertile soil. Strain Ciat 895 was more competitive in the fertile soil. There was a decline in nodule occupancy for all strains tested from the first trifoliate leaf stage to the pod-filling stage. No plant genotype effect on nodule occupancy was observed. There were significant (P<0.05) plant genotype and location effects, but no significant strain effect on acetylene reduction activity, plant dry weight, and nodule number. The greenhouse experiments confirmed, at least partially, the results from the field trials. In Leonard jars with an acid soil, strains Ciat 151 and Kim5s were amongst the most competitive strains. In coinoculation experiments, Kim5s was the most competitive strain, followed by Ciat 899 and Ciat 895. The competitiveness of a given strain was affected by the coinoculant strain. Tolerance in vitro to low pH, high growth temperature, Al or Mn toxicity, or Fe limitation was not related to competitiveness of the inoculum strains. The sensitivity of the strains towards bean tannins or bean phytoalexins also was not correlated with their competitiveness.     

Genetic diversity and symbiotic efficiency of population of rhizobia of <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L. in Brazil

The genetic diversity and symbiotic efficiency among indigenous rhizobia isolates obtained from native field with or without organic fertilization and superficial mineral fertilization were investigated. Eighty-six indigenous rhizobia were isolated from these fields using four common bean varieties as trap-host. The common bean varieties Mexico 309 and Rio Tibagi selected the most efficient rhizobia strains because they showed the best yields and N contents results. The genetic characterization of 36 rhizobia isolates was evaluated by using electrophoretic profiles of amplification products using primers ERIC1-R and ERIC-2. Our results demonstrated that besides the large diversity in the indigenous rhizobial community, the genotype of the trap-host probably influences the selection of the most efficient strains.     

共生固氮在农牧业上的作用及影响因素研究进展

共生固N是生物固N的主体部分,具有固N效率高、应用范围广等特点。叙述了主要豆科作物年固N量及固N量占豆科作物本身所吸收N的比例,阐述了豆科作物在与非豆科作物间套轮作中固定N素的转移及对非豆科作物的影响,并介绍了影响豆科作物-根瘤菌共生体共生固N效率的主要因素。开展豆科作物-根瘤菌共生体系方面的研究对农业可持续发展具有重要意义。