Dual inoculation withRhizobium sp. andGlomus fasciculatum enhances nodulation,yield and nitrogen fixation in chickpea (Cicer arietinum Linn.)

Summary Seed inoculation with Rhizobium and soil inoculation withGlomus fasciculatum increased nodulation, nitrogen and phosphorus concentration in plants and yield of chickpea (Cicer arietinum) var. BG 212 in pots containing unsterilized soil especially with 50kgP₂O₅ ha⁻¹ in the form of superphosphate. Inoculation with Rhizobium orG. fasciculatum separately or in combination significantly increased the N₂ fixed in straw and grain than uninoculated controls as determined by¹⁵N atom percent excess of plants grown in soil amended with labelled ammonium sulphate (¹⁵NH₄)₂SO₄) at the rate of 20kg N ha⁻¹. These increases were most pronounced when P was applied at 50kgP₂O₅ ha⁻¹.     

Fast chlorophyll fluorescence transient and nitrogen fixing ability of chickpea nodulation variants

High nodulating (HN) selections of the cultivars ICC 4948 and ICC 5003 had the highest nodule number and nodule dry mass followed by low nodulating (LN) selections of the same cultivar. Both non-nodulating (NN) selections of cv. ICC 4993 and ICC 4918 did not show any nodule. Using N-difference method the HN selection of cv. 1CC 4948 was able to meet 73 % of its demand of N through biological fixation of N₂ [P(fix)], while 27 % of N demand was met by uptake from the soil, whereas its LN selection was able to meet only 54 % of its demand of N through biological fixation of N₂. Similarly in cv. ICC 5003 HN and LN selections the P(fix) was 76 and 64 %, respectively. Fast chlorophyll (Chl) fluorescence transient data analysis showed that performance index PI(abs) was 62.0 in cv. ICC 4948 HN selection and 44.5 in its respective LN selections. Corresponding values for cv. ICC 5003 were 32.4 and 28.4. In NN selections of ICC 4993 and ICC 4918 it was 12.6 and 30.7, respectively. Structure function index of the plants SFI(abs) and driving force for photosynthesis (DF) were highest in the HN selections followed by LN selections and lowest in the NN selections. The total uptake of N by chickpea plants was significantly and positively correlated with the density of reaction centres ABS/CS₀, TR₀/CS₀, and DI₀/CS_M, whereas total N uptake by chickpea seeds was significantly positively correlated with N and TR₀/CS₀. The percentage of P(fix) was highly significantly positively correlated with N, the so-called turnover number which indicates how many times Q_A has been reduced in the time span from 0 to t_Fmax and TR₀/CS₀. Fast Chl a fluorescence measurement can be used as a model system to assess the N fixation ability in chickpea.     

Improving chickpea yield by incorporating resistance to ascochyta blight

Ascochyta blight [Ascochyta rabiei (Pass.) Lab.] is the most destructive disease of chickpea (Cicer arietinum L.), but it can be managed effectively by the use of resistant cultivars. Therefore, a breeding programme was initiated during 1977–78 at ICARDA, Syria, to breed blight-resistant, high-yielding chickpeas with other desirable agronomic traits. Crosses were made in main season at Tel Hadya, Syria, and the F₁s were grown in the off season at Terbol, Lebanon. The F₂, F₄ and F₅ generations were grown in a blight nursery in the main season where blight epidemic was artificially created. The plants and progenies were scored for blight resistance and other traits. The F₃ and F₆ generations were grown in the off season under normal day length to eliminate late-maturing plants. The pedigree method of breeding was followed initially, but was later replaced by the F₄-derived family method. The yield assessment began with F₇ lines, first at ICARDA sites and later internationally. A total of 1584 ascochyta blight-resistant chickpea lines were developed with a range of maturity, plant height, and seed size not previously available to growers in the blight-endemic areas in the Mediterranean region. These included 92 lines resistant to six races of the ascochyta pathogen, and 15 large-seeded and 28 early maturity lines. New cultivars produced 33% more seed yield than the original resistant sources. The yield of chickpea declined by 340 kg ha^-1, with an increase in blight severity by one class on a 1–9 scale, reaching zero yield with the 8 and 9 classes. Development of blight-resistant lines made the introduction of winter sowing possible in the Mediterranean region with the prospect of doubling chickpea production. Twenty three cultivars have been released so far in 11 countries.Joint contribution from ICARDA and ICRISAT. ICRISAT Journal Article no. JA 1886.     

Effect of soil organic matter on chickpea inoculated withAzospirillum brasilense andRhizobium leguminosarum bv.ciceri

Azosprillum inoculated withRhizobium improved the nodulation of chickpea-Cicer arietinum. This interaction was further enhanced by organic matter present in the growth medium.     

Glucose uptake by free living and bacteroid forms of Rhizobium leguminosarum

Free living cells of Rhizobium leguminosarum contain a constitutive glucose uptake system, except when they are grown on succinate, which appears to prevent its formation. Bacteroids isolated from Pisum sativum L fail to accumulate glucose although they actively take up ¹⁴C-succinate. Glucose uptake in free living cells is an active process since uptake was inhibited by azide, cyanide, dinitrophenol and carbonyl-m-chlorophenyl hydrazone but not by fluoride or arsenate. The non-metabolizable analogue -methyl glucose was extracted unchanged from cells, showing that it was not phosphorylated during its transport. Galactose also appears to the transported via the glucose uptake system. Organic acids, amino acids and polyols had no effect on the actual uptake of glucose. The K _m for -methyl glucose uptake was 2.9×10^-4 M.     

The effect of soil pH on chickpea (Cicer arietinum) genotype sensitivity to isoxaflutole

A glasshouse experiment was conducted to investigate the effect of soil pH on chickpea (Cicer arietinum) tolerance to isoxaflutole applied pre-emergence at 0, 75 (recommended rate) and 300 g a.i. ha⁻¹. For this study, the variables examined were two desi chickpea genotypes (97039-1275 as a tolerant line and 91025-3021 as a sensitive line) and four pH levels (5.1, 6.9, 8.1, and 8.9). The results demonstrated differential tolerances among chickpea genotypes to isoxaflutole at different rates and soil pH levels. Isoxaflutole applied pre-emergence resulted in increased phytotoxicity with increases in soil pH and herbicide rate. Even the most tolerant chickpea genotype was damaged when exposed to higher pH and herbicide rates, as indicated by increased leaf chlorosis and significant reductions in plant height, and shoot and root dry weight. The effects were more severe with the sensitive genotype. The susceptibility of chickpea to this herbicide depends on genotype and soil pH which should be taken into account in breeding new lines, and in the agronomy of chickpea production.     

Effect of Rhizobium andAzospirillum lipoferum inoculation on the nodulation,yield and nitrogen uptake of peanut cultivars

Peanut (Arachis hypogaea Linn.) Cvs. Robut 33-1 and JL 24 were inoculated with Rhizobium strain NC 92 and a strain ofAzospirillum lipoferum singly and as mixed inoculum. Seed inoculation with these bacteria enhanced nodulation, N content and yield of these cultivars under field conditions. While a mix inoculation of these two diazotrophic cultures had an adverse effect on these parameters as compare to single inoculation.     

Effects of supplemental nitrate and thermal regime on the nitrogen nutrition of chickpea (Cicer arietinum L.)

Summary Nodulated chickpea plants were grown in pots in a glasshouse programmed to simulate either hot (32.5°C day/18°C night) or warm (25°/18°C) thermal regimes characteristic of those experienced by crops grown in different seasons or locations in the semi-arid tropics. The plants were irrigated with nutrient solution either devoid of inorganic nitrogen or containing 0.71, 1.43 or 2.86 mM nitrate. Increasing concentrations of supplemental nitrate stimulated the rate of dry matter production by vegetative plants in both thermal regimes. Differences between vegetative dry weight of plants given nitrate and those relying exclusively on symbiotic dinitrogen fixation were greatest in the hot regime where the durations of vegetative growth were shorter. However, symbiotically-dependent plants and those given 0.71 mM nitrate continued to produce branches throughout the reproductive period, particularly in the warm regime. As they matured, these plants became more comparable in vegetative stature to those which had received greater concentrations of nitrate and had established final branch numbers earlier (i.e prior to main pod-fill). Potential seed yields were determined primarily by the number of potential reproductive sites (nodes) available (i.e. by the extent of branching) which largely determined the number of seeds harvested. Since final branch numbers in all nitrate treatments were greatest in the warm regime, yields were also larger than those at 32.5°C. The implications of these data for the nitrogen economy of chickpea crops is discussed.One of a series of papers resulting from a collaborative project with the International Crops Research Institute for the Semi-Arid Tropics, India; sponsored by the UK Overseas Development Administration.     

Field response of chickpea to inoculation withGlomus versiforme

The response ofCicer arietinum to inoculation withGlomus versiforme under field conditions was investigated in a phosphorus deficient sandy loam soil. Inoculation with the mycorrhizal fungusGlomus versiforme increased the rate of VAM development in chickpea. The weight of nodules and the number of nodules per plant were higher in inoculated than in uninoculated plants. The phosphorus content of the shoots and its total uptake, were increased by either the application of single super-phosphate, or by inoculation withG. versiforme. Inoculation increased shoot dry weights and grain yields by 12% and 25% respectively, as compared with the 33% and 60% increases respectively produced by P-treated plants.     

Ultrastructure of chickpea nodules

Summary Developing and senescing chickpea (Cicer arietinum L.) nodules formed byRhizobium sp. (Cicer) CC 1192 have been shown by light and electron microscopy to have general morphological and ultrastructural features that are characteristic of indeterminate nodules. These features included the presence of persistent meristematic tissue at the distal ends of the multi-lobed nodules, and a gradient of cells at different stages of development towards the proximal point of attachment of the nodules to the parent root. The cytoplasm of infected cells in the nitrogen-fixing region of the nodules was densely packed with symbiosomes, most of which contained a single bacteroid. Infection threads containing bacteria were noted in invaded cells from the nitrogen-fixing region of the nodules. Other features that were observed in chickpea nodules included the presence of electron-dense occlusions in intercellular spaces in the nitrogen-fixing region, and plasmodesmata that connected infected cells with other infected cells and with uninfected cells. No poly--hydroxybutyrate granules were noted in the bacteroids. In later stages of development, infected cells became enlarged and highly vacuolated, and eventually lost their contents. Uninfected cells in the central region were smaller than infected cells and were also highly vacuolated. Some of the degenerative processes that take place in senescing bacteroids were noted.     

Multiple shoot induction by benzyladenine and complete plant regeneration from seed explants of chickpea (Cicer arietinum L.)

The efficacy of benzyladenine (BA) to induce multiple shoots from seed explants of chickpea (Cicer arietinum L.) was assessed. Shoot differentiation was influenced by the type of seed explant, genotype and concentration of BA. Orientation of the explant also strongly influenced the shoot regeneration response. The optimum BA concentration for shoot/shoot bud regeneration was genotype dependent. Two types of BA-induced response were observed: (1) at less than 7.5 gm BA, direct shoot differentiation (2 to 4-cm-long shoots) was observed within 30 days; (2) at higher BA concentrations (75–100 m), shoot/shoot bud differentiation was achieved in 45–90 days. A high BA concentration inhibited subsequent rooting of shoots. Roots, however, could be easily induced on shoots derived from <12.5 m BA. Following transfer to soil, 80% of the regenerants developed into morphologically normal and fertile plants.Abbreviations BA Benzyladenine     

Discrimination of Rhizobium japonicum,Rhizobium lupini,Rhizobium trifolii,Rhizobium leguminosarum and of bacteriods by uptake of 2-ketoglutaric acid,glutamic acid and phosphate

Rhizobium strains (one each of Rh.japonicum, Rh. lupini, Rh. leguminosarum) take up 2-ketoglutaric acid in general much faster and from lower concentrations in the medium than strains of Escherichia coli, Bacillus subtilis and Chromobacterium violaceum. A strain of Enterobacter aerogenes, however, is more similar to some Rhizobium strains. The same strains of Rhizobium take up also phosphate much faster and from lower concentrations than the other bacteria tested. 4 strains of Rh. lupini proved to be significantly different from 4 strains of Rh. trifolii in taking up l-glutamic acid from three to ten times lower concentration within 5 h. A similar difference was noticed between 5 strains of Rh. leguminosarum and 2 strains of Rh. japonicum for the uptake of 2-ketoglutaric acid and of l-glutamic acid. Isolated bacteriods from nodules of Glycine max var. Chippeway have a reduced uptake capacity for glutamic acid and for 2-ketoglutaric acid during the first 10–12 h, but reach the same value after 24 h as free living Rh. japonicum cells. The differences in the uptake kinetics are independent of cell concentration. The group II Rhizobium strains (Rh. japonicum and Rh. lupini, slow growing Rhizobium) are characterized by a rapid uptake of glutamic acid to a lowremaining concentration of 1–3×10^-7 M and an uptake of 2-ketoglutaric acid to a remaining concentration of 2–5×10^-7 M. The group I Rhizobium strains (Rh. trifolii and Rh. leguminosarum, fast growing Rhizobium), can be characterized by a much slower uptake of both substances with a more than ten times higher concentration of both metabolites remaining in the medium after the same time.     

Associative effect ofAzospirilium brasilense withRhizobium japonicum on nodulation and yield of soybean (Glycine max)

Summary Azospirillum was associated with nodules of soybean. In general, seed inoculation with a broth culture ofAzospirillum brasilense alone significantly increased nodulation and grain yield of soybean grown in pots in unsterilized soil with different levels of urea ranging from 0 to 80 kg N/ha. This trend was significantly reproducible in a second experiment when a carrier based inoculant of the bacterium was used for seed inoculation.Inoculation withRhizobium japonicum andA. brasilense in combination generally increased grain yield in both the experiments, although the data were not significant.     

Effect of salinity on antioxidant responses of chickpea seedlings

The changes in the activity of antioxidant enzymes, like superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase, and growth parameters such as length, fresh and dry weight, proline and H₂O₂ contents, chlorophyll fluorescence (Fv/Fm), quantum yield of PSII and the rate of lipid peroxidation in terms of malondialdehyde in leaf and root tissues of a chickpea cultivar (Cicer arietinum L. cv. Gökçe) under salt treatment were investigated. Plants were subjected to 0.1, 0.2 and 0.5 M NaCl treatments for 2 and 4 days. Compared to controls, salinity resulted in the reduction of length and of the fresh and dry weights of shoot and root tissues. Salinity caused significant (P < 0.05) changes in proline and MDA levels in leaf tissue. In general, a dose-dependent decrease was observed in H₂O₂ content, Fv/Fm and quantum yield of photosynthesis under salt stress. Leaf tissue extracts exhibited three activity bands, of which the higher band was identified as MnSOD and the others as FeSOD and Cu/ZnSOD. A significant enhancement was detected in the activities of Cu/ZnSOD and MnSOD isozymes in both tissues. APX and GR activities exhibited significant increases (P < 0.05) in leaf tissue under all stress treatments, whereas no significant change was observed in root tissue. The activity of CAT was significantly increased under 0.5 M NaCl stress in root tissue, while its activity was decreased in leaf tissue under 0.5 M NaCl stress for 4 days. These results suggest that CAT and SOD activities play an essential protective role against salt stress in chickpea seedlings.     

Efficient and rapid in vitro plant regeneration system for Indian cultivars of chickpea (Cicer arietinum L.)

Lacking of an efficient regeneration protocol for the recalcitrant crop chickpea is a limiting factor for adapting genetic engineering approaches for its improvement. The present study describes a rapid and efficient method for multiple shoot regeneration for three Indian cultivars, B115, C235, ICCV89314, using single cotyledons with half embryos as explant. Modified MS medium with 1.5 mg l⁻¹ 6-benzyladenine (BA) and 0.04 mg l⁻¹ α-naphthaleneacetic acid (NAA) induced a maximum of 26 shoots from a single explant after 20 days of culture. When cultured in modified MS medium containing 0.2 mg l⁻¹ indole-3-acetic acid (IAA), 80% of the shoots from each regenerating explant elongated in another 20–25 days. Following a root-grafting protocol, 90–95% of the elongated shoots survived in soil which subsequently produced seeds. The regeneration process from explant preparation to complete plants took 55–60 days. The presently optimized rapid regeneration method holds promise for facilitating the deployment of agronomically important components through genetic transformation for betterment of this important food crop.     

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N⁶-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 M to 100 M), although, maximum morphogenic response was observed at 10 M concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 M NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog [14] basal medium - NAA naphthaleneacetic acid - TDZ thidiazuron [N-phenyl-N(-1,2,3,-thidiazol-5-yl)-urea]     

Direct somatic embryogenesis and plantlet regeneration from immature leaflets in chickpea

Summary Direct somatic embryo formation and plantlet regeneration was achieved from immature leaflets of chickpea (Cicer arietinum L.). Optimal somatic embryogenesis was obtained when immature leaflets were exposed to media supplemented with 15 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for 7 d, to 2000 μM 2,4-D for 3 d, and to 50 μM 2,4-D for 10 d, followed by transfer onto Murashige and Skoog (MS) basal medium. Exposure of explants to high 2,4-D levels (200–2000 μM) for 3 d produced bottle-shaped embryos, while exposure to low 2,4-D levels (<50 μM) and 50–2000 μM for 10 d produced spherical-shaped embryos. Two percent of embryos converted into plants upon culture on MS medium containing 15 μM gibberellic acid and 1 μM 3-indolebutyric acid. All regenerated plants were phenotypically normal.     

Evidence for two uptake systems in Rhizobium leguminosarum for hydroxy-aromatic compounds metabolized by the 3-oxoadipate pathway

Rhizobium leguminosarum biovar viciae and Rhizobium leguminosarum biovar trifolii have separate uptake systems for 4-hydroxybenzoate and protocatechuate. The 4-hydroxybenzoate uptake system (pobP) is inhibited by a range of compounds with substitution at the 4-position on the aromatic ring whereas the uptake system for protocatechuate (pcaP) is markedly inhibited only by other dihydroxybenzoic acids. The rate of 4-hydroxybenzoate uptake is very low in Rhizobium leguminosarum and Rhizobium trifolii grown on protocatechuate but mutants defective in 4-hydroxybenzoate uptake transport protocatechuate at rates similar to the wild-type grown under similar conditions.     

Carbendazim alleviates effects of water stress on chickpea seedlings

Carbendazim (methyl-2-benzimidazole carbamate) promoted root growth of chickpea (Cicer arietinum L.) seedlings subjected to polyethylene glycol (PEG, osmotic potential −0.5 MPa) induced water stress. The relative water content, membrane stability index, 2,3,5-triphenyltetrazolium chloride reduction and contents of some osmolytes (proline, sucrose, glucose and fructose) enhanced significantly while the contents of lipid peroxides and hydrogen peroxide diminished effectively by addition of 0.05 % carbendazim into PEG solution. This revised version was published online in September 2005 with the corrected author information.