The effect of acidity on the production of signal molecules by Medicago roots and their recognition by Sinorhizobium E.L.J. Watkin a, b, * , L.A. Mutch a, b , S. Rome a , W.G. Reeve a , J.M. Castelli a , Y. Gruchlik c , W.M. Best c , G.W. O’Hara a , J.G. Howieson a, d a Centre for Rhizobium Studies, Division of Science and Engineering, Murdoch University, South Street, WA 6150, Australia b School of Biomedical Sciences, Curtin University of Technology, GPO Box U1987, Perth, WA 6845, Australia c Medicinal and Biological Chemistry Section, Chemistry Centre WA, East Perth, WA 6004, Australia d Agriculture Western Australia, Baron-Hay Court, South Perth, WA 6151, Australia article info Article history: Received 17 June 2008 Received in revised form 3 October 2008 Accepted 7 October 2008 Available online 5 November 2008 Keywords: Acidity Medicago nod gene inducing compounds Root exudates Signal induction Sinorhizobium medicae abstract The Medicago sativa–Sinorhizobium symbiosis is challenged by acidity, resulting in generally poor nodulation and production. Medicago murex, however, can nodulate and grow at low pH. The effect of low pH on signal exchange in the Sinorhizobium–Medicago symbiosis was studied to gain a greater understanding of the basis for poor nodulation of M. sativa compared to M. murex. Root exudates from M. sativa and M. murex grown in buffered nutrient solution at pH 4.5, 5.8 and 7.0, were collected to measure the expression of nodB induction in Sinorhizobium.A nodB–gusA fusion was constructed and inserted into Sinorhizobium medicae strains WSM419 (acid tolerant) and CC169 (acid sensitive). We identified greater induction by root exudates from both Medicago spp. collected at pH 4.5 than at pH 5.8 and 7.0, less induction by M. murex than M. sativa and less induction of WSM419 than CC169. The same major inducing compounds, 4 0 ,7-dihydroxyflavanone (liquiritigenin), 4 0 ,7-dihydroxyflavone, and 2 0 ,4 0 ,4-trihy- droxychalcone (isoliquiritigenin), were identified in exudates of M. murex and M. sativa at all pH values, although in increasing amounts at lower pH. Poor nodulation of M. sativa relative to M. murex under acid conditions is not the consequence of decreased induction of Sinorhizobium nodB by chemical inducers present in the root exudates of both species at low pH. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Acid soils constrain legume production (Aarons and Graham, 1991; Graham and Vance, 2000) and the symbiosis between Med- icago sativa and Sinorhizobium spp. is considered to be the most acid sensitive in the Leguminosae so far studied (Coventry and Evans, 1989). Soil acidity can limit growth and persistence of Sinorhizobium spp. (Robson and Loneragan, 1970; Brockwell et al., 1991), the nodulation process of Medicago spp. per se (Munns, 1968) as well as host plant growth (Coventry and Evans, 1989). By selection of acid-soil tolerance in both symbiotic partners (Howieson and Ewing, 1986, 1989), annual medics such as Medicago murex can be grown symbiotically on soils as acidic as pH 4.3 (Cheng et al., 2002). Whilst the genetic control of acid tolerance in Sinorhizobium is becoming increasingly understood (Dilworth et al., 2001), there is little information on the mechanisms contributing to enhanced nodulation at low pH in host species such as M. murex in comparison to M. sativa (D’Haeze and Holsters, 2002). Cheng et al. (2002) reported that the acid-sensitive species M. sativa exhibited delayed nodulation under acid stress relative to the acid- tolerant species M. murex, but that nodules eventually formed on both species in the same section of the root. The establishment of legume symbioses requires the interaction of specific recognition signal molecules produced by both bacterial and plant partners (Denarie et al., 1996). It has been shown that pH affects the exchange or recognition of these signal molecules by both plant and bacterial partners in both the medic symbiosis (Howieson et al., 1992) and the clover symbiosis (Richardson et al., 1988a,b; Howieson et al., 1992). Howieson et al. (1992) noted that root exudates collected at decreasing pH from acid-tolerant species of Medicago resulted in increased nod gene induction up to a critical pH when nod gene expression was then drastically reduced. The root exudates of acid-sensitive species of Medicago demonstrated a general reduction in nod gene inducing capacity with reducing pH. The more acid-tolerant clover symbiosis showed increasing nod gene inducing capability of root exudates with reducing pH to a lower critical pH level than seen in the Medicago symbiosis, but also showed species specific differences in the critical pH (Richardson et al., 1988a). * Correspondence address: E.L.J. Watkin, School of Biomedical Sciences, Curtin University of Technology, GPO Box U1987, Perth, WA 6845, Australia. Tel.: þ61 8 9266 2955; fax: þ61 8 9266 2342. E-mail address: e.watkin@curtin.edu.au (E.L.J. Watkin). Contents lists available at ScienceDirect Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio 0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2008.10.007 Soil Biology & Biochemistry 41 (2009) 163–169