The strategic role of 15N in quantifying the contribution of endophytic N2 fixation to the N nutrition of non-legumes

Methods based on the use of the stable isotope N-15 have been used to confirm active endophytic biological N-2 fixation (BNF) in non-legumes, to obtain quantitative estimates of the proportion of plant N obtained from the atmosphere (P-atm), and to assess the response of plants to inoculation with diazotrophic bacteria. The purpose of this review is to describe these methods, the assumptions on which they are based, the information that they provide and the situations where they are most appropriately used. The review will concentrate mainly on the literature published during the past 25 years, which follows on from an earlier review by the author. The host range of endophytic BNF is much wider than previously known and now includes cereals, forages, industrial crops, fruits, vegetables and woody forest species. Many new species of diazotrophs that inhabit non-legumes have been identified. The experimental scales range from in vitro studies to glasshouse and field investigations, and include the direct N-15(2) technique and indirect N-15 enrichment or N-15 natural abundance methods. All N-15 methods are yield-independent and the indirect methods can provide an assessment of P-atm over a plant's life cycle. There is continuing interest in quantifying endophytic BNF as evidenced by documented studies in 22 countries worldwide since 1991. Estimates of P-atm varied across species, cultivars, inoculants, experimental scale and environmental-edaphic conditions, from low (< 15 %), to moderate (15-30 %) to high (> 30 %) values. High estimates were most often obtained under optimal ambient conditions. Relatively few experiments have been conducted in field settings, but there is convincing evidence based on N-15 and N balance studies that some Brazilian sugar cane cultivars can contribute in excess of 40 kg N ha(-1) yr.(-1) via endophytic BNF. N-15-based estimates of P-atm are essential in order to identify if a positive yield response to inoculation is due to enhanced BNF or to additional uptake of soil N.