Effects of Multiple Reference Plants,Season, and Irrigation on Biological Nitrogen Fixation by Pasture Legumes using the Isotope Dilution Method

Abstract

The popular and widely used ¹⁵nitrogen (N)–isotope dilution method for estimating biological N fixation (BNF) of pasture and tree legumes relies largely on the ability to overcome the principal source of error due to the problem of selecting appropriate reference plants. A field experiment was conducted to evaluate the suitability of 12 non‐N₂‐fixing plants (i.e., nonlegumes) as reference plants for estimating the BNF of three pasture legumes (white clover, Trifolium repens L.; lucerne, Medicago sativa; and red clover, Trifolium pratense L.) in standard ryegrass–white clover (RWC) and multispecies pastures (MSP) under dry‐land and irrigation systems, over four seasons in Canterbury, New Zealand. The ¹⁵N‐isotope dilution method involving field ¹⁵N‐microplots was used to estimate BNF. Non‐N₂‐fixing plants were used either singly or in combination as reference plants to estimate the BNF of the three legumes. Results obtained showed that, on the whole, ¹⁵N‐enrichment values of legumes and nonlegumes varied significantly according to plant species, season, and irrigation. Grasses and herb species showed higher ¹⁵N‐enrichment than those of legumes. Highest ¹⁵N‐enrichment values of all plants occurred during late summer under dry‐land and irrigation conditions. Based on single or combined non‐N₂‐fixing plants as reference plants, the proportion of N derived from the atmosphere (% Ndfa) values were high (50 to 90%) and differed between most reference plants in the MSP pastures, especially chicory (Cichorium intybus), probably because it is different in phenology, rooting depth, and N‐uptake patterns compared to those of legumes. The percent Ndfa values of all plants studied also varied according to plant species, season, and irrigation in the MSP pastures. Estimated daily amounts of BNF varied according to pasture type, time of plant harvest, and irrigation, similar to those shown by percent Ndfa results as expected. Irrigation increased daily BNF more than 10‐fold, probably due to increased dry‐matter yield of pasture under irrigation compared to dry‐land conditions. Seasonal and irrigation effects were more important in affecting estimates of legume BNF than those due to the appropriate matching of N₂‐fixing and non‐N₂‐fixing reference plants.