The effect of Fe-EDDHA on shoot multiplication and in vitro rooting of Carlina onopordifolia Besser

The effect of two different iron chelates and iron concentration on multiplication, shoot growth, chlorophyll content and rooting of Carlina onopordifolia were studied in in vitro culture. FeEDTA presented in MS basal medium was replaced by FeEDDHA, which was applied in three concentrations: 93.5, 187.0 and 280.5?mg?dm^?3 (5.6?mg?dm^?3, 11.2 and 16.8?mg?dm^?3 Fe ions, respectively). Changing chelate or iron concentration in the medium had no effect on axillary shoot number proliferation, but growth of shoots was significantly inhibited by a two- and three-fold increase in concentration of FeEDDHA in the medium. Supplementation of the medium with FeEDDHA as Fe source significantly increased the level of chlorophyll in the leaves. After treatment of shoots with IBA for 5?s and growing them on the MS medium supplemented with FeEDTA, the number of roots per shoot was significantly higher than on medium containing FeEDDHA. Increasing the concentration of Fe ions in the medium after a short pulse (5?s) of IBA had no effect on shoot rooting. After 30?s of 1-g?dm^?3 IBA treatment, growth of roots on medium with FeEDDHA was stimulated. The survival rate was relatively low and did not depend on the type and concentration of iron chelate in the rooting medium.     

Improved shoot multiplication of mature hazelnut (Corylus avellana L.) in vitro using glucose as a carbon source

Shoot cultures established from mature trees of hazelnut (Corylus avellana L.) cvs. Nonpareil and Tonda Gentile Romana were used to determine the effects of basal media, carbon sources and concentrations, pH and cytokinins on shoot multiplication. All factors except pH affected the multiplication rate. Shoot multiplication was the best on a modified Driver and Kuniyuki medium for Paradox walnut (DKW) supplemented with 6-benzylaminopurine (BA) (1.5–3 mg/l). Plants grown on 3% glucose or fructose medium produced more and longer shoots than those on sucrose. The general appearance and growth habit of shoots were better on medium with glucose than fructose. Nonpareil shoots elongated better than those of Tonda Gentile Romana. Changes in medium pH from 4.7 to 5.7 did not significantly affect the multiplication rate. More than 10 genotypes propagated well on modified DKW medium with glucose. This is the first report of the effect of carbon sources on shoot multiplication of hazelnut and provides a basis for further research in the improvement of hazelnut micropropagation.Abbreviations MWPMC modified woody plant medium for chestnut (Yang et al. 1986) - DKW Driver and Kuniyuki (1984) medium for Paradox walnut - BA 6-benzylaminopurine - Z zeatin - 2iP N6-(2-isopentenyl) adenine - K kinetin - IBA indole-3-butyric acid     

Impact of nitrogen form on iron uptake and distribution in maize seedlings in solution culture

Comparative studies on the effect of nitrogen (N) form on iron (Fe) uptake and distribution in maize (Zea mays L. cv Yellow 417) were carried out through three related experiments with different pretreatments. Experiment 1: plants were precultured in nutrient solution with 1.0×10^–4 M FeEDTA for 6 d and then exposed to NO₃–N or NH₄–N solution with 1.0×10^–4 M FeEDTA or without for 7 d. Experiment 2: plants were precultured with ⁵⁹FeEDTA for 6 d and were then transferred to the solution with different N forms, and 0 and 1.0×10^–4 M FeEDTA for 8 d. Experiment 3: half of roots were supplied with ⁵⁹FeEDTA for 5 d and then cut off, with further culturing in treatment concentrations for 7 d. In comparison to the NH₄-fed plants, young leaves of the NO₃-fed plants showed severe chlorosis under Fe deficiency. Nitrate supply caused Fe accumulation in roots, while NH₄–N supply resulted in a higher Fe concentration in young leaves and a lower Fe concentration in roots. HCl-extractable (active) Fe was a good indicator reflecting Fe nutrition status in maize plants. Compared with NO₃-fed plants, a higher proportion of ⁵⁹Fe was observed in young leaves of the Fe-deficient plants fed with NH₄–N. Ammonium supply greatly improved ⁵⁹Fe retranslocation from primary leaves and stem to young leaves. Under Fe deficiency, about 25% of Fe in primary leaves of the NH₄-fed plants was mobilized and retranslocated to young leaves. Exogenous Fe supply decreased the efficiency of such ⁵⁹Fe retranslocation. The results suggest that Fe can be remobilized from old to young tissues in maize plants but the remobilization depends on the form of N supply as well as supply of exogenous Fe.     

Nitrogen nutrition influences some biochemical responses to iron deficiency in tolerant and sensitive genotypes of Vitis

The effects of nitrogen source on iron deficiency responses were investigated in two Vitis genotypes, one tolerant to limestone chlorosis Cabernet Sauvignon (Vitis vinifera cv.) and the other susceptible Gloire de Montpellier (Vitis riparia cv.). Plants were grown with or without Fe(III)-EDTA, and with NO₃⁻ alone or a mixture of NO₃⁻ and NH₄⁺. Changes in pH of the nutrient solution and root ferric chelate reductase (FC-R) activity were monitored over one week. We carried out quantitative metabolic profiling (¹H-NMR) and determined the activity of enzymes involved in organic acid metabolism in root tips. In iron free-solutions, with NO₃⁻ as the sole nitrogen source, the typical Fe-deficiency response reactions as acidification of the growth medium and enhanced FC-R activity in the roots were observed only in the tolerant genotype. Under the same nutritional conditions, organic acid accumulation (mainly citrate and malate) was found for both genotypes. In the presence of NH4⁺, the sensitive genotype displayed some decrease in pH of the growth medium and an increase in FC-R activity. For both genotypes, the presence of NH₄⁺ ions decreased significantly the organic acid content of roots. Both Vitis genotypes were able to take up NH₄⁺ from the nutrient solution, regardless of their sensitivity to iron deficiency. The presence of N-NH₄⁺ modified typical Fe stress responses in tolerant and sensitive Vitis genotypes.     

Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes

Neutral carrier-based liquid membrane ion-selective microelectrodes for NH₄⁺ and NO₃⁻ were developed and used to investigate inorganic nitrogen acquisition in two varieties of barley, Hordeum vulgare L. cv Olli and H. vulgare L. cv Prato, originating in cold and warm climates, respectively. In the present paper, the methods used in the fabrication of ammonium- and nitrate-selective microelectrodes are described, and their application in the study of inorganic nitrogen uptake is demonstrated. Net ionic fluxes of NH₄⁺ and NO₃⁻ were measured in the unstirred layer of solution immediately external to the root surface. The preference for the uptake of a particular ionic form was examined by measuring the net flux of the predominant form of inorganic nitrogen, with and without the alternative ion in solution. Net flux of NH₄⁺ into the cold-adapted variety remained unchanged when equimolar concentrations (200 micromolar) of NH₄⁺ and NO₃⁻ were present. Similarly, net flux of NO₃⁻ into the warm-adapted variety was not affected when NH₄⁺ was also present in solution. The high temporal and spatial resolution afforded by ammonium- and nitrate-selective microelectrodes permits a detailed examination of inorganic nitrogen acquisition and its component ionic interactions.     

Ammonium under solution culture alleviates aluminum toxicity in rice and reduces aluminum accumulation in roots compared with nitrate

Al stress and ammonium–nitrogen nutrition often coexist in acidic soils due to their low pH and weak nitrification ability. Rice is the most Al-resistant species among small grain cereal crops and prefers NH₄ ⁺ as its major inorganic nitrogen source. This study investigates the effects of NH₄ ⁺ and NO₃ ^? on Al toxicity and Al accumulation in rice, and thereby associates rice Al resistance with its NH₄ ⁺ preference. Two rice subspecies, indica cv. Yangdao6 and japonica cv. Wuyunjing7, were used in this study. After treatment with or without Al under conditions of varying NH₄ ⁺ and NO₃ ^? supply, rice seedlings were harvested for the determination of root elongation, callose content, biomass, Al concentration and medium pH. The results indicated that Wuyunjing7 was more Al-resistant and NH₄ ⁺-preferring than Yangdao6. NH₄ ⁺ alleviated Al toxicity in two cultivars compared with NO₃ ^?. Both NH₄ ⁺-Al supply and pretreatment with NH₄ ⁺ reduced Al accumulation in roots and root tips compared with NO₃ ^?. NH₄ ⁺ decreased but NO₃ ^? increased the medium pH, and root tips accumulated more Al with a pH increase from 3.5 to 5.5. Increasing the NO₃ ^? concentration enhanced Al accumulation in root tips but increasing the NH₄ ⁺ concentration had the opposite effect. These results show NH₄ ⁺ alleviates Al toxicity for rice and reduces Al accumulation in roots compared with NO₃ ^?, possibly through medium pH changes and ionic competitive effects. Making use of the protective effect of NH₄ ⁺, in which the Al resistance increases, is advised for acidic soils, and the hypothesis that rice Al resistance is associated with the preferred utilization of NH₄ ⁺ is suggested.     

Influence of Protein Synthesis on NO(3) Reduction, NH(4) Accumulation, and Amide Synthesis in Suspension Cultures of Paul's Scarlet Rose

Changes in the concentrations of NH₄⁺ and amides during the growth of suspension cultures of rose (Rosa cv. Paul's Scarlet) cells were examined. When cells were grown in medium possessing only NO₃⁻ as a nitrogen source, the concentrations of NH₄⁺ and amides increased to 4.0 × 10⁻¹ and 5.9 micromoles per gram fresh weight, respectively. The amounts of both constituents declined during the later stages of growth. When a trace amount of NH₄⁺ was added to the NO₃⁻ base starting medium, the concentration of NH₄⁺ in the cells was increased to 7.0 × 10⁻¹ micromoles per gram fresh weight.     

Short-term studies of 15NO3 - and 15NH4 + uptake by micropropagated strawberry shoots cultured with or without CO2 enrichment

The uptake of ¹⁵NO₃ ^- and ¹⁵NH₄ ⁺ has been examined in 5-,10- and 28-day-old micropropagated strawberry (Fragaria x ananassa Duch. cv. Kent) shoots rooted in one-half strength Murashige and Skoog (MS) liquid medium on cellulose plugs (Sorbarods). The results indicated that the plantlets absorbed both NO₃ ^- and NH₄ ⁺ during the culture with a greater uptake of NH₄ ⁺ at 5 days of culture. Furthermore, a pronounced reduction in NO₃ ^- and NH₄ ⁺ uptake at 10 and 28 days of culture was observed within 6 h of the short-term uptake study. This reduction could be explained by the low CO₂ concentration in test tubes during the photoperiod, since no reduction in nitrogen uptake occurred in the CO₂ enriched condition. The results are interpreted as an indication of the important role for photosynthetic CO₂ fixation in the process of nitrogen uptake by the plantlets during the rooting stage.Contribution No. CRH 82, Centre de Recherche en Horticulture, F.S.A.A., Université Laval, Québec.     

Interactive effect of salt (NaCl) and nitrogen form on growth, water relations and photosynthetic capacity of sunflower (Helianthus annum L.)

The effects of the ammonium (NH₄⁺) and nitrate (NO₃^-) forms of nitrogen and NaCl on the growth, water relations and photosynthesis performance of sunflower (Helianthus annuus L.) were examined under glasshouse conditions. Eight-day-old plants of cv. Hisun 33 were subjected for 21 days to Hoagland's nutrient solution containing 8 mol m^-3N as NH₄⁺or NO₃^-, and salinised with 0, 60, or 120 mol m^-3NaCl. Fresh weights of shoots and roots, and leaf area of NO₃^-supplied non-salinised plants were significantly greater than those of NH₄⁺-supplied non-salinised plants. But addition of NaCl to the rooting medium of these plants had more inhibitory effect on the growth of NO₃^--supplied plants than on NH₄⁺-supplied plants. Both leaf water and osmotic potentials of plants grown with NH₄⁺were lower than those of plants given NO₃^-under both non-saline and saline conditions. Chlorophylls a and b concentrations were higher in plants grown with NH₄⁺than N0₃^--supplied plants at the lower two levels of salinisation. The rate of photosynthesis in plants was considerably higher in non-salinised plants grown with NO₃^-than with NH₄⁺, but with increase in salinisation the photosynthesis rate decreased in NO₃^--supplied plants, but not in those given NH₄⁺. The rate of transpiration was increased significantly by salinisation in NO₃^--supplied plants, but not consistently so in NH₄⁺-supplied plants. The stomatal conductances were much higher in plants given NO₃^-than with NH₄⁺when grown under non-saline conditions, but not when salinised. As a consequence, water-use efficiency in NO₃^--supplied control plants was better than in NH₄⁺-supplied under non-saline conditions, but worse under saline conditions. The different forms of nitrogen and the addition of NaCl to the growing medium did not affect the relative intercellular concentrations of CO₂ (C_i/C_a). Overall, the NH₄⁺form of nitrogen inhibited the growth of sunflowers under non-saline conditions, but NO₃^-and NaCl interacted to inhibit growth more than did NH₄⁺under saline conditions.     

Effects of macro elements and nitrogen source on adventitious root growth and ginsenoside production in ginseng (Panax ginseng C. A. Meyer)

We investigated the effects on ginseng adventitious root growth and ginsenoside production when macro-element concentrations and nitrogen source were manipulated in the culture media. Biomass growth was greatest in the medium supplemented with 0.5-strength NH₄PO₃, whereas ginsenoside accumulation was highest (9.90 mg g^-1 DW) in the absence of NH₄PO₃. At levels of 1.0-strength KNO₃, root growth was maximum, but a 2.0 strength of KNO₃ led to the greatest ginsenoside content (9.85 mg g^-l). High concentrations of MgSO₄ were most favorable for both root growth and ginsenoside accumulation (up to 8.89 mg g^-1 DW). Root growth and ginsenoside content also increased in proportion to the concentration of CaCI₂ in the medium, with the greatest accumulation of ginsenoside (8.91 mg g^-1 DW) occurring at a 2.0 strength. The NH₄/NO₃ ^-- ratio also influenced adventitious root growth and ginsenoside production; both parameters were greater when the NO₃ ^- concentration was higher than that of NH₄ ⁺. Maximum root growth was achieved at an NH₄ ⁺/NO₃ ^- ratio of 7.19/18.50, while ginsenoside production was greatest (83.37 mg L^-1) when NO₃ ^- was used as the sole N source.     

Influence of trace elements and nitrogen sources on versicolorin production by a mutant strain of Aspergillus parasiticus

A mutant strain of Aspergillus parasiticus blocked in aflatoxin biosynthesis accumulates versicolorin A and versicolorin C. The effect of trace elements on the growth and versicolorin production by this strain was studied in a defined medium. The omission of manganese was slightly stimulatory to versicolorin production; when zinc was omitted from the medium, no detectable versicolorins were produced. Experiments on nitrogen sources in a highsucrose medium indicated that fourfold to fivefold increases in versicolorin yields could be obtained by substituting 3 ml/l corn steep liquor or 0.1 M NH₄NO₃ for the 0.023 M (NH₄)₂SO₃ used previously as the nitrogen source in studies on versicolorin production by this strain. These improved yields will facilitate attempts to accumulate enough versicolorin A and versicolorin C for toxicity and carcinogenicity testing. Chromatographic profiles of mycelial extracts of cultures grown in a defined medium with 0.1 M NH₄NO₃ as the nitrogen source revealed 2 previously unrecognized compounds. The accumulation of these new metabolites in a mutant blocked in aflatoxin production may indicate that they are biosynthetically related to aflatoxin.     

Nitrate and ammonium as nitrogen sources for lupins prior to nodulation

Two cultivars of Lupinus angustifolius L. were grown in a glasshouse in solutions containing NO₃ ^-, NH₄ ⁺ or NH₄NO₃ with a total nitrogen concentration of 2.8 M m^-3 in each treatment. One cultivar chosen (75A-258) was relatively tolerant to alkaline soils whereas the other (Yandee) was intolerant to alkalinity. Controlled experiments were used to assess the impact of cationic vs. anionic forms of nitrogen on the relative performance of these cultivars. Relative growth rates (dry weight basis) were not significantly different between the two cultivars when grown in the presence of NO₃ ^-, NH₄ ⁺ or NH₄NO₃. However, when NO₃ ^- was supplied, there was a modest decline in relative growth rates in both cultivars over time. When plants grown on the three sources of nitrogen for 9 days were subsequently supplied with ¹⁵NH₄NO₃ or NH₄ ¹⁵NO₃ for 30 h, NH₄ ⁺ uptake was generally twice as fast as NO₃ ^- uptake, even for plants grown in the presence of NO₃ ^-. Low rates of NO₃ ^- uptake accounted for the decrease in growth rates over time when plants were grown in the presence of NO₃ ^-. It is concluded that the more rapid growth of 75A-258 than Yandee in alkaline conditions was not due to preferential uptake of NH₄ ⁺ and acidification of the external medium. In support of this view, acidification of the root medium was not significantly different between cultivars when NH₄ ⁺ was the sole nitrogen source.     

Influence of nitrogen source and concentration on nitrogen isotopic discrimination in two barley genotypes (Hordeum vulgare L.)

The occurrence of nitrogen isotope discrimination with absorption and assimilation of nitrate (NO₃^–) and ammonium (NH₄⁺) was investigated using two genotypes of barley, Hordeum vulgare L. cv. Steptoe and Az12 : Az70, the latter of which lacks the characterized nitrate reductase isozymes. Plants were grown under two situations: a closed system with limited nitrogen or an open system with unlimited nitrogen, to elucidate the conditions and processes that influence discrimination. There was no discrimination observed for Az12 : Az70 when supplied with limited nitrogen. Discrimination was observed for Steptoe seedlings at high external NO₃^– concentrations, but not with low NO₃^– when assimilation is probably rapid and complete. The same pattern was observed for Steptoe when NH₄⁺ was supplied; indicating that for both nitrogen forms discrimination is dependent upon the presence of the assimilatory enzyme and the external concentration. The implications of this study are that both internal (assimilatory enzyme distribution) and external (source concentration) factors may have a larger impact on tissue δ ¹⁵N than the form of nitrogen utilized. This suggests that tissue δ ¹⁵N may not always be a reliable indicator of a plant's integrated nitrogen nutrition.     

Physiological studies on Phymatotrichum omnivorum X. Influence of nitrogen sources on free and bound amino acid pools

The effect of nitrogen source on the free and bound amino acids of mycelium of Phymatotrichum omnivorum (Shear) Dugg was investigated. The largest free amino acid pool was present in the natural medium and the smallest in the synthetic medium. Phymatotrichum omnivorum was able to utilize different nitrogen sources with the best growth occurring with NH₄NO₃. The ratio of glycine to alanine and aspartic to glutamic was around 0.25 in the free amino acid pool and around 1 in the bound amino acid pool. The free pool of glutamic acid ranged from 5.6 % to 27.2 % depending upon the nitrogen source in the media. The free pool of alanine ranged from 35.7 % to 17.2 % in relation to the nitrogen source. Most other amino acid ratios did not vary significantly between the free amino acids and the bound amino acids.     

Effect of nutritional factors on lipase biosynthesis by Aspergillus niger

Summary Lipase biosynthesis occured in medium without lipids, but for improved production an inducer was needed. The source and concentration of an inducer had no signifficant effect. Starch as an additional carbon source stimulated lipase biosynthesis when used in small amounts. Addition of NH₄NO₃ as a nitrogen source, KH₂PO₄ as a phosphate source as well as Mg ions to the medium with inital pH 5.0 gave the best yield.     

The Influence of Ammonium Nitrate,pH and Indole Butyric Acid on Root Induction and Survival in Soil of Micropropagated <Emphasis Type="Italic">Eucalyptus globulus</Emphasis>

Rooting of Eucalyptus globulus shoots was influenced by the concentration of the indole butyric acid (IBA) and NH₄ ⁺ in the root-induction medium. Optimum plantlet vigor and survival were achieved using low concentrations (1 – 2.5 μM) of IBA and when NH₄NO₃ was removed. Removal of NH₄ ⁺ also had a significant effect on medium pH, its presence caused a decrease in pH as the culture period proceeded. When different nitrate compounds (excluding NH₄NO₃) were used as the nitrogen source, the medium pH was more stable and this was associated with higher root production. The higher root production, in association with appropriate IBA concentrations, produced plantlets with higher survival and better growth on transfer to soil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nitrogen metabolism of Spirodela oligorrhiza

Summary Spirodela oligorrhiza grown in axenic culture was able to use either ammonium, nitrate, or nitrite as sole source of nitrogen, although the morphology of the plants was affected. Plants utilizing ammonium contained higher levels of NH₄, arginine, asparagine and glutamine than did those utilizing NO₃, whereas concentrations of other amino acids were similar.The utilization of NH₄ inhibited that of NO₃ by inhibiting, at least partially, NO₃ uptake, and by inhibiting almost completely the reduction of NO₃ to NO₂. NO₂ also inhibited the utilization of NO₃. NH₄ and NO₂ were taken up and assimilated simultaneously when they were supplied together in the medium.     

RETRACTED ARTICLE: Effect of root-zone salinity and form of N on photosynthate partitioning in wheat (Triticum aestivum L.)

Carbon-14 pulse labeling technique was used to study the effect of rooting medium salinity and form and availability of N on growth and rhizodeposition of wheat (Triticum aestivum L.). Thirty days old plants grown in continuously aerated Arnon and Hoagland nutrient solution were subjected to ¹⁴C pulse labeling for 24 h and transferred to aqueous rooting medium containing 0, 150, and 300 mM NaCl in all combinations with different forms (calcium nitrate, ammonium sulphate, and ammonium nitrate) and amounts (0.5, 1.0, 1.5, and 2.0 times the standard N concentration (150 ppm) of Arnon and Hoagland plant growth medium). Plant samples immediately after pulse labeling, following 7 days of growth under different rooting medium conditions, and the freeze-dried rooting medium were analyzed for total C and ¹⁴C. Length and fresh/dry weight of root and shoot portions and calculated values of unaccounted ¹⁴C were determined. Presence of NaCl in the rooting medium led to a decrease in root and shoot portions. However, NO₃ ⁻-fed plants showed better growth than NH₄ ⁺-fed plants at all the three salinity levels. Salinity in rooting medium led to higher rhizodeposition and lower loss of ¹⁴C. Relatively higher proportion of ¹⁴C was released as rhizodeposits and retained in root/shoot portions of plants fed with NH₄ ⁺ or NH₄ ⁺+NO₃ ⁻, than those with NO₃ ⁻, while less was respired. The specific activity of the rhizodeposits (kBq ¹⁴C g⁻¹ C) was also higher under saline conditions. The rhizodeposits in NH₄ ⁺-fed plants were more highly labeled as compared to NO₃ ⁻-plants.     

The role of nitrate and ammonium ions and light on the induction of nitrate reductase in maize leaves

Corn seedlings (Zea mays cv W64A × W182E) were grown hydroponically, in the presence or absence of NO₃⁻, with or without light and with NH₄Cl as the only N source. In agreement with earlier results nitrate reductase (NR) activity was found only in plants treated with both light and NO₃⁻. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer of the proteins to nitrocellulose paper and reaction with antibodies prepared against a pure NR showed that crude extracts prepared from light-grown plants had a polypeptide of approximately 116 kilodaltons (the subunit size for NR) when NO₃⁻ was present in the growth medium. Crude extracts from plants grown in the dark did not have the 116 kilodalton polypeptide, although smaller polypeptides, which reacted with NR-immunoglobulin G, were sometimes found at the gel front. When seedlings were grown on Kimpack paper or well washed sand, NR activity was again found only when the seedlings were exposed to light and NO₃⁻. Under these conditions, however, a protein of about 116 kilodaltons, which reacted with the NR antibody was present in light-grown plants whether NO₃⁻ was added to the system or not. The NR antibody cross-reacting protein was also seen in hydroponically grown plants when NH₄Cl⁻ was the only added form of nitrogen. These results indicate that the induction of an inactive NR-protein precursor in corn is mediated either by extremely low levels of NO₃⁻ or by some other unidentified factor, and that higher levels of NO₃⁻ are necessary for converting the inactive NR cross-reacting protein to a form of the enzyme capable of reducing NO₃⁻ to NO₂⁻.     

The effect of source,concentration and time of application of nitrogen on the growth,nodulation and nitrogen fixation ofLotus pedunculatus andTrifolium repens

Summary Studies under growth cabinet conditions investigated the effect of source and concentration of nitrogen and timing of nitrogen application on the growth and nitrogen fixation byLotus pedunculatus cv. Maku andTrifolium repens cv. S184. KNO₃, NaNO₃ and NH₄NO₃ were added at transplanting at the following rates: 3.33, 7.78 and 13.33 mg N/plant. KNO₃ was added at 3.33 and 7.78 mg N/plant at 0, 6, 12, 18, 24 or 30 days after transplanting.Lotus shoot weight increased with all increasing nitrogen sources but clover only responded to KNO₃ and NaNO₃. The root weight of both species increased with increasing KNO₃ and NH₄NO₃. The percentage increase in lotus and clover shoot growth was greater than that of root growth when KNO₃ was added within a week of transplanting. Increases in growth by both species resulted from added nitrogen except with lotus when NaNO₃ was applied where increased nitrogen fixation also contributed to increased growth.Weight and number of effective nodules on both species were increased with 3.33 mg N per plant as KNO₃ but nitrogen fixation was not affected. Addition of 13.33 mg N as NaNO₃ reduced weight and number of effective nodules in both species and also nitrogen fixation by lotus.KNO₃ increased growth and nodulation of both species when applied within one week after transplanting. Nodulated lotus plants responded to KNO₃ by increasing growth but not nodulation.KNO₃ appeared to affect infection and development of nodules on lotus and may affect the growth of existing nodules on clover.