Plant Soil (2012) 356:291–293 DOI 10.1007/s11104-012-1267-7

LETTER

John Featherstone Witty 1944 to 2009 Frank Minchin & Murray Unkovich

Received: 7 March 2012 / Accepted: 20 April 2012 / Published online: 12 May 2012 # Springer Science+Business Media B.V. 2012

John Witty died suddenly on 24th February, 2009. Those who knew John will have memories of a generous, sociable man who was gifted with an excellent, if somewhat irreverant, sense of humour. Beyond these attributes John was also an extremely able scientist who combined an incisive analytical mind with an extraordinary innovative talent, backed up by a detailed understanding of biology, chemistry, physics, F. Minchin Rivermead, Penrhyncoch, Aberystwyth, Ceredigion, SY23 3EQ, UK M. Unkovich (*) School Agriculture, Food and Wine, The University of Adelaide, Glen Osmond, SA 5074, Australia e-mail: [email protected]

mathematics and electronics. His papers always had a high novelty value and his insight often allowed him to identify areas where research was leading up a blind alley. Thus, his work on the measurement of N2 fixation and the physiology of legume nodules is still very relevant and heavily cited today. The 1970’s–1990’s provided significant advances in our knowledge of N2 fixation measurement, at field, glasshouse and organ scales, primarily due to technological advances with stable isotope applications and microelectrodes. The key UK work in measurement techniques was done at Rothamstead Experimental Station, the Institute for Grassland Research (Hurley) and the Institute for Grassland and Environmental Research in Wales (Aberystwyth and North Wyke). John Witty worked across all of these institutions and left a valuable legacy to those continuing in this field of research. John’s varied academic background started with a B. Sc. in zoology and an M.Sc. in water management, followed by a Ph.D. in nitrogen fixation by cyanobacteria, based at Rothamsted Experimental Station (RES). This lead to a Postdoctoral Fellowship at the International Rice Research Institute (IRRI), from which he returned to Rothamsted in 1974 to work on nitrogen fixation with Peter Dart, John Day and Ken Giller. He soon turned his analytical skills to the problems of nitrogen fixation measurement using the 15N dilution technique, where he demonstrated significant errors due to differences in N uptake patterns by legumes and non-fixing control plants, coupled with decreasing 15N enrichment of the available soil N pool (Witty 1983).

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This demonstrated that the choice of reference plant could make large differences in the resulting estimates of N2 fixation. . Further work showed that reference plant-dependent errors could be reduced by the application of slow release 15N fertilizer, but the efficacy of this approach varied with the type of fertilizer used (Witty and Ritz 1984). The answer was to immobilize the slow release 15N fertilizer by applying it to soil, along with a sucrose solution, several months prior to the start of fixation measurements (Giller and Witty 1987). John was also interested in the measurement of nitrogen fixation using the acetylene reduction assay. He initially showed measurement errors for soil-bourne free-living microbes due to acetylene inhibition of ethylene oxidation by soil microbes, resulting in enhanced ethylene production during the assay (Witty 1979). Then, in collaboration with Frank Minchin and John Sheehy, he discovered major errors in the measurement of N2 fixation by legume nodules due to substantial decreases in nitrogenase activity following exposure to either acetylene or argon/oxygen (Minchin et al. 1983) or disturbance of nodulated roots (Minchin et al. 1986). This discovery relied upon the development and construction of an open, flow-through gas system, for which John had the lead role. Those who knew John will not be surprised that he also proposed using this system to expose nodulated roots to an explosive gas stream of 10 % acetylene, with an oxygen concentration that was gradually increased to 80 %. These studies on nitrogen fixation measurement continue to inform the use of appropriate techniques for both asymbiotic (Unkovich and Baldock 2008) and symbiotic (Unkovich et al. 2008) N2 fixation. Indeed, by establishing rigorous protocols for measuring legume BNF he paved the way for similar studies in non-legumes, such as sugarcane N2 fixation in Brazil (Urquiaga et al. 1992). The spin-offs from the acetylene reduction work also had impacts on the understanding of legume nodule physiology. This began with the realisation that the strong correlation between acetylene-induced declines in both nitrogenase activity and nodule/nodulated root respiration could provide an accurate measurement of the respiratory costs of fixation (Witty et al. 1983); a goal of legume physiologists for several years (Minchin and Witty 2004). However, the major spin-off was the demonstration of a variable oxygen diffusion barrier (ODB) in legume nodules (Sheehy et al. 1983; Witty et al. 1984).

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The ODB was to form the major part of John’s work for the next 15 years, although he still found time to act as a consultant for the International Atomic Energy Agency (IAEA) to advise on setting up 15Nbased fixation measurement systems in developing countries. In addition, his detailed knowledge of legume and nodule physiology led to numerous invitations to speak at international meetings, including the International N2 Fixation Congress at Knoxville in 1990. John followed up the initial discovery of the ODB with the development of an oxygen microelectrode which was more sensitive and far less intrusive than previous electrodes (Witty et al. 1987). This was later modified to measure internal hydrogen concentrations in nodules (Witty 1991), which, when coupled to measurements of hydrogen production, provided proof of a variable ODB within the cortex of legume nodules (Witty and Minchin 1998a). These studies were followed by the development of novel systems for oxygen and hydrogen detection, which allowed for continuous measurements over a period of days, or even weeks (Witty and Minchin 1998b). But perhaps John’s most “left of field” idea was to use the different diffusion rates of oxygen through helium and air to demonstrate the closure of a continous network of gasfilled pores in stressed nodules (Witty and Minchin 1994). This body of work continues to be an important theme in discussions of the role of oxygen in legume nodules (eg. Minchin et al. 2008) and the microelectrodes he developed have also found significant application in studies of gut function in invertebrates (Ebert and Brune 1997). However, not all of John’s innovative ideas were so successful, and attempts to dry magnesium perchlorate in a microwave oven, or cook a meat pie in the oven of a gas chromatogram, must be counted as failures. Although John’s studies were able to survive the disruption of a transfer, in 1986, from Rothamsted to the Welsh Plant Breeding Station (later to morph into the Institute for Grassland and Environmental Research, IGER) they were not able to withstand the financial pressures which continued to affect UK science during the 1990s. Funding for nitrogen fixation research at IGER was curtailed during 1997/1998. While this greatly saddened John it did not dampen his enthusiasm for innovative research, and he soon obtained funding to look at the effects of white clover on soil structure and to develop apparatus for

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determining soil quality in terms of gaseous emissions. Lesser people may have been content with a device which measured 2 or 3 gases; John’s “sniffer” simultaneously detected 11 gases, in most cases with a sensitivity of less than 1 ppm. During this period he also developed a technique for determining soil permeability, for which a patent was obtained in 2002. Throughout John’s long career he provided help, advice and encouragement to a large number of fellow scientists. Indeed, such was his generousity in terms of time and help that John’s colleagues soon came to regard him as a true friend. John retired in October 2004 to concentrate on his love of boat building and sailing. It is a great sadness that this retirement lasted for a little over four short years, but John’s legacy of scientific work will continue to impact on nitrogen fixation research for many years to come.

References Ebert A, Brune A (1997) Hydrogen concentration profies at the oxic-anoxic interface: a microsensor study of the hindgut of the wood-feeding lower termite Reticulitermes flavipes (Kollar). Appl Environ Microbiol 63:4039–4046 Giller KE, Witty JF (1987) Immobilized 15N-fertilizer sources improve the accuracy of field estimates of N2-fixation by isotope dilution. Soil Biol Biochem 19:459–463 Minchin FR, Witty JF (2004) Carbon costs of nitrogen fixation. Chapter 11. In: Lambers H, Ribas-Carbo M (eds) Plant respiration: from cell to ecosystem. Springer, Dordrecht, pp 192–205 Minchin FR, Witty JF, Sheehy JE, Muller M (1983) A major error in the acetylene reduction assay: decreases in nodular nitrogenase activity under assay conditions. J Exp Bot 34:641–649 Minchin FR, Sheehy JE, Witty JF (1986) Further errors in the acetylene reduction assay: effects of plant disturbance. J Exp Bot 37:1581–1591 Minchin FR, James EK, Becana M (2008) Oxygen diffusion, production of reactive oxygen and nitrogen species, and antioxidants in legume nodules. Chapter 11. In: Dilworth

293 MJ, James EK, Sprent JI, Newton WE (eds) Nitrogenfixing leguminous symbioses. Springer, Dordrecht, pp 321–362 Sheehy JE, Minchin FR, Witty JF (1983) Biological control of the resistance to oxygen flux in nodules. Ann Bot 52:565–571 Unkovich M, Baldock J (2008) Measurement of asymbiotic N2 fixation in Australian agriculture. Soil Biol Biochem 40:2915–2921 Unkovich M, Herridge D, Peoples M, Cadish G, Boddey B, Giller K, Alves B, Chalk P (2008) Measuring plantassociated nitrogen fixation in agricultural systems. ACIAR, Canberra, p 258 Urquiaga S, Cruz KHS, Boddey RM (1992) Contribution of nitrogen fixation to sugar cane: nitrogen-15 and nitrogen balance estimates. Soil Sci Soc Am J 56:105–114 Witty JF (1979) Acetylene reduction assay can overestimate nitrogen fixation in soil. Soil Biol Biochem 11:209– 210 Witty JF (1983) Estimating N2-fixation in the field using 15Nlabelled fertilizer: some problems and solutions. Soil Biol Biochem 15:631–639 Witty JF (1991) Microelectrode measurements of hydrogen concentration and gradients in legume nodules. J Exp Bot 42:765–771 Witty JF, Minchin FR (1994) A new method to detect the presence of continuous gas-filled pathways for oxygen diffusion in legume nodules. J Exp Bot 45:967–978 Witty JF, Minchin FR (1998a) Hydrogen measurements provide direct evidence for a variable physical barrier to gas diffusion in legume nodules. J Exp Bot 49:1015–1020 Witty JF, Minchin FR (1998b) Methods for the continuous measurement of O2 consumption and H2 production by nodulated legume root systems. J Exp Bot 49:1041– 1048 Witty JF, Ritz K (1984) Slow release 15N fertilizer formulations to measure N2-fixation by isotope dilution. Soil Biol Biochem 16:657–661 Witty JF, Minchin FR, Sheehy JE (1983) Carbon csosts of nitrogenase activity in legume root nodules determined using acetylene and oxygen. J Exp Bot 34:951–963 Witty JF, Minchin FR, Sheehy JE, Minguez MI (1984) Acetylene-induced changes in the oxygen diffusion resistance and nitrogenase activity of legume root nodules. Ann Bot 53:12–20 Witty JF, Skot L, Revsbech NP (1987) Direct evidence for changes in the resistance of legume root nodules to O2 diffusion. J Exp Bot 38:1129–1140