High pH in the nutrient solution impairs water uptake in Lupinus angustifolius L.

Root growth in Lupinus angustifolius is greatly decreased when the nutrient solution has a pH above 6.0. This study examined the water relations in this species (cv. Yandee) in response to high pH in solution culture in a glasshouse.The dry weight of roots, the length of taproots and lateral roots and the number of lateral roots were significantly reduced at day 12 after transfer to solution with a pH of 7.5 compared to pH 5.2. This resulted in a marked reduction of total root surface area. However, shoot growth and total leaf area were not affected. In comparison with pH 5.2, plants grown at pH 7.5 in the nutrient solution had a 14–38% more-negative leaf water potential, and their stomatal resistance had increased by 67%.The observations indicate that the impairment of the water relations by high pH is mainly caused by decreased root growth.     

Development and implementation of a sequence-specific PCR marker linked to a gene conferring resistance to anthracnose disease in narrow-leafed lupin (Lupinus angustifolius L.)

Anthracnose caused by Colletotrichum gloeosporioides is the most serious disease of lupins (Lupinus spp). A cross was made between cultivars Tanjil (resistant) and Unicrop (susceptible) in narrow-leafed lupin (L. angustifolius). Analysis of disease reaction data on the F₂ population and on the resultant F₇ recombinant inbred lines suggested that Tanjil contained a single dominant gene (Lanr1) conferring resistance to anthracnose. The parents and the representative F₂ plants were used to generate molecular markers liked to the Lanr1 gene using the MFLP technique. A co-dominant MFLP polymorphism linked to the Lanr1 gene was identified as a candidate marker. The bands were isolated, re-amplified by PCR, cloned and sequenced. The MFLP polymorphism was converted into a co-dominant, sequence-specific, simple PCR-based marker. Linkage analysis by the computer program MAPMAKER indicated that the marker was 3.5 centiMorgans (cM) from the gene Lanr1. This marker is currently being implemented for marker assisted selection in the Australian National Lupin Breeding Program.     

The isolation and characterization of a cDNA clone encoding Lupinus angustifolius root nodule glutamine synthetase

Glutamine synthetase, purified from Lupinus angustifolius legume nodules, was carboxymethylated and succinylated prior to chemical or enzymatic cleavage. Peptides were purified and sequenced. An oligonucleotide probe was constructed for the sequence MPGQW. This probe was used to identify a glutamine synthetase cDNA clone, pGS5, from a lupin nodule cDNA library constructed in pBR322. pGS5 was sequenced (1043 bp) and computer-assisted homology searching revealed a high degree of conservation between this lupin partial cDNA clone and other plant glutamine synthetases at both the amino acid (>90%) and nucleotide (>80%) level. Northern and Southern analyses using pGS5 supported the conclusion that a multigene glutamine synthetase family exists in lupin which is differentially expressed in both an organ-specific and temporal manner. Western and Northern blot analyses indicated the accumulation of a glutamine synthetase specific mRNA species during nodule development corresponded to the appearance of a novel glutamine synthetase polypeptide between 8 and 10 days after rhizobial inoculation.     

Measurement of N2-fixation in field-grown pigeonpea [Cajanus cajan (L.) Millsp.] using15N-labelled fertilizer

Two experiments were carried out from 1981 to 1983 in Vertisol field at ICRISAT Center, Patancheru, India to measure N₂-fixation of pigeonpea [Cajanus cajan (L.) Millsp.] using the¹⁵N isotope dilution technique. One experiment examined the effect of control of a nodule-eating insect on fixation while another in vestigated the effect of intercroping with cereals on fixation and the residual effect of pigeonpea on a succeeding cereal crop. Although both experiments indicated that at least 88% of the N in pigeonpea was fixed from the atmosphere, one result is considered fortuitous in view of the differential rates of growth of the legume and the control, sorghum [Sorghum bicolor (L.) Moench]. The difference method of calculation in dieated negative fixation and the results emphasized the problem of finding a suitable nonfixing control. In a second experiment, when all plants were confined to a known volume of soil to which¹⁵N fertilizer was added in the field, these problems were overcome, and isotope dilution and difference methods gave similar results of N₂-fixation of about 90%. In intercropped pigeonpea 96% of the total N was derived from the atmosphere. This estimate might be an artifact. There was no evidence of benefit from N fixed by pigeonpea to intercropped sorghum plants. Plant tissue¹⁵N enrichments of cereal crops grown after pigeonpea indicated that the cereal derived some N fixed by the previous pigeonpea. Thus residual benefits to cereals are not only an effect of ‘sparing’ of soil N.     

Alkaloid level in narrow-leafed lupin, Lupinus angustifolius, influences green peach aphid reproductive performance

Two viviparous parthenogenetic clones of the green peach aphid, Myzus persicae (Sulzer), one collected from Rydalmere, New South Wales (NSW), and the other from South Perth (SP), Western Australia, were reared on radish, Raphanus sativus L. cv. Scarlet Globe, under controlled conditions. The NSW clone was fed on simple artificial diets containing alkaloid extracted from narrow-leafed lupin, Lupinus angustifolius L. cv. Fest., and its reproductive performance monitored over 112 h. Forty (40) h into the experiment and thereafter, aphids on the control diet (sucrose solution) produced significantly more offspring (P<0.05) than those on diets containing alkaloid. In a separate experiment, apterae of each clone were caged on three lines (cv. Yorrel, cv. Danja and 84L:441) of narrow-leafed lupin, and allowed to reproduce. The first three offspring were retained, and all developed to 3rd or 4th instar stage. Two nymphs were removed, and the remaining nymph reared through. All three lines produced adults. The number of young produced were counted over 11 days. Fecundity of the SP clone was lower on line 84L:441, but there was no difference in the fecundity of the NSW clone. Phloem exudate and green tissue was concomitantly collected from all lines, and analysed by GC-MS for the alkaloids lupanine and 13-hydroxylupanine. Line 84L:441 contained the highest level of total alkaloids in both phloem and tissue. All experiments indicate that alkaloid level may suppress fecundity of green peach aphids.     

A strategy to develop molecular markers applicable to a wide range of crosses for marker assisted selection in plant breeding: a case study on anthracnose disease resistance in lupin (Lupinus angustifolius L.)

A key challenge in marker-assisted selection (MAS) for molecular plant breeding is to develop markers linked to genes of interest which are applicable to multiple breeding populations. In this study representative F₂ plants from a cross Mandalup (resistant to anthracnose disease) × Quilinock (susceptible) of Lupinus angustifolius were used in DNA fingerprinting by Microsatellite-anchored Fragment Length Polymorphism (MFLP). Nine candidate MFLP markers linked to anthracnose resistance were identified, then ‘validated’ on 17 commercial cultivars. The number of “false positives” (showing resistant-allele band but lack of the R gene) for each of the nine candidate MFLP markers on the 17 cultivars ranged from 1 to 9. The candidate marker with least number of false positive was selected, sequenced, and was converted into a co-dominant, sequence-specific, simple PCR based marker suitable for routine implementation. Testing on 180 F₂ plants confirmed that the converted marker was linked to the R gene at 5.1 centiMorgan. The banding pattern of the converted marker was consistent with the disease phenotype on 23 out of the 24 cultivars. This marker, designated “AnManM1”, is now being used for MAS in the Australian lupin breeding program. We conclude that generation of multiple candidate markers, followed by a validation step to select the best marker before conversion to an implementable form is an efficient strategy to ensure wide applicability for MAS.     

Phosphorus requirements and nitrogen accumulation by three mungbean (Vigna radiata (L) Welzek) cultivars

A promising approach for overcoming poor crop yields in phosphorus (P)-deficient soils is to exploit the genetic variation among plants to grow under low P conditions. We examined the P requirements of three mungbean cultivars, T-77, MI-5 and E-72, using four P rates, 0, 30, 60 and 90 mg P kg^-1 soil (designated P₀, P₁, P₂ and P₃, respectively). Nodulation was highest in T-77, and unlike the other cultivars, nodule numbers were not increased by P application. Similarly, growth of T-77 was the highest, and was not influenced by P rates. In contrast shoot yields of MI-5 and E-72 at P₀ were only 76 and 65%, respectively, of the maximum obtained under P application. Nodule dry weight and the amount of N fixed (Ndfa) in each cultivar was enhanced by P application, with T-77 generally giving the lowest response, and accumulating the highest Ndfa. The data suggest a higher P requirement for N₂ fixation (especially for T-77) than for growth. All plants increased their P uptake as P rates increased, with T-77 accumulating the highest amount of P at each P level. Differences in the physiological P use efficiency, PPUE (g shoot mg^-1 P) among genotypes were generally not significant, neither were there any consistent trends as P rates changed. The ability to absorb P therefore appeared to be more important than PPUE in enhancing growth. We conclude from our data that it is possible by selection to obtain plants capable of good growth and high N₂ fixation in soils of low P; cultivar T-77 is a good example.     

Yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.) in Peru

Two field experiments were performed to evaluate the nitrogen fixation potential of twenty common bean cultivars and breeding lines during summer and winter seasons of 1986 and 1988, respectively. The ¹⁵N isotope dilution method was used to quantify N₂ fixation. The cultivars and breeding lines were variable in terms of their N₂ fixation. The cv. Caballero was very efficient, with more than 50% N derived from the atmosphere and 60–80 kg N ha^–1 fixed in both seasons. Other cultivars were less efficient, since the poorest ones derived less than 30% of their nitrogen from the atmosphere and fixed less than 20 kg N ha^–1. After additional testing the best cultivars may be used directly by the farmers for cultivation. The experiments have provided information about which genotypes may be used to breed for enhanced fixation in common bean.     

Variability in nitrogen fixation of common bean (Phaseolus vulgaris L.) intercropped with maize

Thirty one selected bean lines were evaluated in the field for ability to support N₂ fixation when intercropped with maize which received 0, 30 and 60 kg N ha^–1 as ammonium sulphate. The amount of fixed N₂ was estimated using the natural variation of ¹⁵N and wheat as the standard non-fixing crop. Nitrogen as low as 15 kg N ha^–1 at sowing suppressed nodule weight and activity (acetylene reduction activity) but not nodule number, suggesting that the main effect of mineral N was on nodule development and function. ¹⁵N data revealed a high potential of the bean genotypes to fix N₂, with the most promising ones averaging between 50–60% of seed N coming from fixation. Bean lines CNF-480, Puebla-152, Mexico-309, Negro Argel, CNF-178, Venezuela-350 and WBR22-3, WBR22-50 and WBR22-55 were ranked as good fixers.     

Nitrogen fixation by soybeans (Glycine max L.) in Zambia

Soybeans (Glycine max L.) are being introduced as a cash crop to small scale farmers in Zambia for rotation in their farming systems. The objectives of this study were to compare and select the most approriate non-fixing reference crop for estimating N₂ fixation by soybeans and assess yields and N₂ fixation of soybeans in Zambia. Nitrogen isotope dilution techniques using¹⁵N-labelled organic or inorganic materials were utilized. Two nonnodulating soybean cultivars, Clark RJ1 and N77 or in their absence Pearl millet (Panicum glaucum L.) were judged to be appropriate reference crops. A local soybean fixing cultivar (Glycine max L. cv. Magoye) rated highest among three cultivars tested for its ability to support symbiotic N₂ fixation byB. japonicum under the experimental conditions. Values of percent N derived from atomosphere for this cultivar were in the order of 65 to 70%.deceased.Contribution no R531 of the Saskatchewan Institute of Pedology. Present address (REK): Esso Chemical Canada, P.O. Box 3010, Lethbridge, Alberta Canada T1J 4A9.     

Interactive effect of cytokinin and potassium on sink-source relationships in Lupinus angustifolius

This study examined whether increased K supply in conjunction with BAPcould increase lupin seed yield and harvest index by enlarging sink volume (podnumber), increasing assimilate and improving assimilate partitioning to filltheadditional pods induced by BAP treatment. Narrow-leafed lupin(Lupinusangustifolius, cv. Danja abs^– mutant) was grown inaglasshouse, in pots containing sandy soil with four K treatments (0, 15, 60 and120 mg K/kg soil). BAP (2 mM) was applied daily toallmain stem flowers throughout the life of each flower from opening to senesced.BAP application did not affect assimilate production (as measured by totalabove-ground biomass), but changed assimilate partitioning. On BAP-treatedplants, there were greater proportions of seed to pod wall dry weight on themain stem but smaller proportions on the branches, and an increased weightratioof seed to pod wall overall which meant more assimilate was used for seedgrowthrather than pod wall growth. BAP increased the number of pods per plant by35% and this more than compensated for the decreases in seeds per podandseed weight. Therefore, there was an increased harvest index (+11%)and seed yield per plant (+13%) in BAP-treated plants. BAP alsoincreased the number of pods with filled seeds (146%) on the main stemand main stem seed K⁺ concentration (from 0.81% to0.87%). Added K increased biomass but only slightly affected assimilatepartitioning. As applied K increased, relatively more assimilate was used forpod wall growth rather than seed growth. Added K increased seed yield per plantby about 14% due to increases in seed weight and the number of pods onthe main stem. Moreover, K⁺ concentration in seeds and shootsincreased with increasing level of applied K. Seed yield was enhanced more byBAP when K was supplied at high levels. Increasing K supply interactedpositively with added BAP by increasing narrow-leaf lupin seed yield andharvestindex through increases in assimilate supply and its partitioning into seeds.     

Nitrogen fixation in breeding lines of Phaseolus vulgaris L.

Nitrogen fixation in nine common bean (Phaseolus vulgaris L.) lines was estimated using the ¹⁵N isotope dilution method at two locations in two seasons. In the first season at one location no N₂ fixation was detected while in the second season up to 51 kg N ha^–1 were estimated. There were significant differences between lines and correlations between trials were significant for the amounts of N₂ fixed, but not for total shoot nitrogen. The plants that fixed the most nitrogen nodulated rapidly after germination. Differences in maximum nodule mass, but not specific nodule activity, were detected also.     

Potassium nutrition effects on seed alkaloid concentrations,yield and mineral content of lupins (Lupinus angustifolius)

To ensure that narrow-leafed lupin (Lupinus angustifolius L.) meets feed quality standards, the concentration of alkaloids must be kept under the maximum acceptable limit of 200 mg kg^–1 DM. One of the factors that may affect seed alkaloid concentration is soil nutrient deficiency. In this paper, we report the results of glasshouse and field experiments that tested the effect of potassium (K) deficiency on seed alkaloid concentrations. In the glasshouse, seed alkaloid concentrations increased by 385, 400 and 205% under severe K deficiency in sweet varieties (Danja, Gungurru and Yorrel, respectively) of L. angustifolius. The concentration of alkaloids in Fest, the bitter variety, was always high regardless of soil K status. At all levels of applied K (0–240 mg kg^–1 soil), lupanine was the predominant alkaloid in sweet varieties, whereas 13-hydroxylupanine prevailed in the bitter variety. Seed yield of all varieties increased exponentially with increasing amounts of applied K, reaching a maximum at 60 mg K kg^–1 soil. In the field, application of K to deficient soils decreased seed alkaloid concentration at Badgingarra, Western Australia (WA) but not at Nyabing, WA, in 1996. In both field trials, seed yield and mineral content were not affected by the amounts of K fertiliser applied. These findings highlighted the need for adequate K fertilisation of deficient soils in WA to avoid the risk of producing low quality lupin seed with high alkaloid concentrations. K deficiency is involved in stimulating alkaloid production in sweet varieties of L. angustifolius.     

15N estimates of nitrogen fixation by white clover (Trifolium repens L.) growing in a mixture with ryegrass (Lolium perenne L.)

The ¹⁵N isotope dilution technique and the N difference method were used to estimate N₂ fixation by clover growing in a mixture with ryegrass, in a field experiment and a controlled environment experiment. Values obtained using N difference were approximately 25% lower than those estimated using ¹⁵N isotope dilution. In the field experiment there was a measured N benefit to grass growing with clover, equivalent to 42.7 kgN ha^-1. The grass in the mixture had a lower atom %¹⁵N content and a higher N content than grass in a monoculture; therefore values for N₂ fixation were different depending on choice of control plant i.e. monoculture or mixture grass. In the controlled environment experiment there were no significant differences between either the atom %¹⁵N contents or the N contents of monoculture grass and grass growing in a mixture with clover. It is concluded that there is a long term indirect transfer of N from clover to associated grass which can lead to errors in estimates of N₂ fixation.     

Nitrogen fixation,transfer and turnover in upland and lowland grass-clover swards,using15N isotope dilution

The stable isotope¹⁵N is particularly valuable in the field for measuring N fixation by isotope dilution. At the same time other soil-plant processes can be studied, including¹⁵N recovery, and nitrogen transfer between clover and grass. Three contrasting sites and soils were used in the present work: a lowland soil, an upland soil, and an upland peat. Nitrogen fixation varied from 12 gm^–2 on lowland soil to 2.7 gm^–2 on upland peat. Most N transfer occurred on upland soil (4.2 gm^–2) which, added to nitrogen fixed, made a total of 8.7 gm² input during summer 1985.¹⁵N recovery for the whole experiment was small, around 25%.Measurement of dead and dying leaves, stubble and roots, suggests that plant organ death is the first stage in N transfer from white clover to ryegrass, through the decomposer cycle. Decomposition was fastest on lowland soils, slowest on peat. On lowland soil this decomposer nitrogen is apparently subverted before transfer, probably by soil microbes.Variations in natural abundance of¹⁵N in plants were found in the two species on the different soils. These might be used to measure nitrogen fixation without adding isotope, but the need for many replicates and repeat samples would limit throughput.     

Quantitative effects of soil nitrate,growth potential and phenology on symbiotic nitrogen fixation of pea (Pisum sativum L.)

The influence of soil nitrate availability, crop growth rate and phenology on the activity of symbiotic nitrogen fixation (SNF) during the growth cycle of pea (Pisum sativum cv. Baccara) was investigated in the field under adequate water availability, applying various levels of fertiliser N at the time of sowing. Nitrate availability in the ploughed layer of the soil was shown to inhibit both SNF initiation and activity. Contribution of SNF to total nitrogen uptake (%Ndfa) over the growth cycle could be predicted as a linear function of mineral N content of the ploughed layer at sowing. Nitrate inhibition of SNF was absolute when mineral N at sowing was over 380 kg N ha^–1. Symbiotic nitrogen fixation was not initiated unless nitrate availability in the soil dropped below 56 kg N ha^–1. However, SNF could no longer be initiated after the beginning of seed filling (BSF). Other linear relationships were established between instantaneous %Ndfa and instantaneous nitrate availability in the ploughed layer of the soil until BSF. Instantaneous %Ndfa decreased linearly with soil nitrate availability and was nil above 48 and 34 kg N ha^–1 for the vegetative and reproductive stages, respectively, levels after which no SNF occurred. Moreover, SNF rate was shown to be closely related to the crop growth rate until BSF. The ratio of SNF rate over crop growth rate decreased linearly with thermal time. Maximum SNF rate was about 40 mg N m^–2 degree-day^–1, equivalent to 7 kg N ha^–1, regardless of the N treatment. From BSF to the end of the growth cycle, the high N requirements of the crop were supported by both SNF and nitrate root absorption but, of the two sources, nitrate root absorption seemed to be less affected by the presence of reproductive organs. However, since soil nitrate availability was low at the end of the growth cycle, SNF was the main source of nitrogen acquisition. The onset of SNF decrease at the end of the growth cycle seemed to be first due to nodule age and then associated to the slowing of the crop growth rate.     

Nitrogen retranslocation in plants of maize,lupin and cocklebur

Summary In a split root experiment translocation of N from shoot to root was studied using¹⁵NO ₃ ^– . The three plant species selected for this experiment differed significantly with respect to root NRA. For lupin, maize and cocklebur about 80, 50 and 6% of all absorbed NO ₃ ^– was assmilated in the roots, respectively.Although NO ₃ ^– was reduced in the roots of lupin and maize plants to a greater extent than required for the roots' demand for organic N, a significant phloem flow of N from shoot to roots was found in these plants. Unexpectedly, for cocklebur, the plant with the very low root NRA, the fraction of total N present in the root that has been imported from the shoot was only half that as found for lupin and maize.     

Optimising biological N2 fixation by legumes in farming systems

Whether grown as pulses for grain, as green manure, as pastures or as the tree components of agro-forestry systems, the value of leguminous crops lies in their ability to fix atmospheric N₂, so reducing the use of expensive fertiliser-N and enhancing soil fertility. N₂ fixing legumes provide the basis for developing sustainable farming systems that incorporate integrated nutrient management. By exploiting the stable nitrogen isotope ¹⁵N, it has been possible to reliably measure rates of N₂ fixation in a wide range of agro-ecological field situations involving many leguminous species. The accumulated data demonstrate that there is a wealth of genetic diversity among legumes and their Rhizobium symbionts which can be used to enhance N₂ fixation. Practical agronomic and microbiological means to maximise N inputs by legumes have also been identified.