UCLA UCLA Previously Published Works Title Exopolysaccharide production in Ensifer meliloti laboratory and native strains and their effects on alfalfa inoculation. Permalink https://escholarship.org/uc/item/1803018m Journal Archives of microbiology, 202(2) ISSN 0302-8933 Authors Primo, Emiliano D Cossovich, Sacha Nievas, Fiorela et al. Publication Date 2020-03-01 DOI 10.1007/s00203-019-01756-3 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Archives of Microbiology https://doi.org/10.1007/s00203-019-01756-3 ORIGINAL PAPER Exopolysaccharide production in Ensifer meliloti laboratory and native strains and their efects on alfalfa inoculation Emiliano D. Primo1 · Sacha Cossovich1 · Fiorela Nievas1 · Pablo Bogino1 · Ethan A. Humm2 · Ann M. Hirsch2,3 · Walter Giordano1 Received: 20 August 2019 / Revised: 7 October 2019 / Accepted: 22 October 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Bacterial surface molecules have an important role in the rhizobia-legume symbiosis. Ensifer meliloti (previously, Sinorhizo- bium meliloti), a symbiotic Gram-negative rhizobacterium, produces two diferent exopolysaccharides (EPSs), termed EPS I (succinoglycan) and EPS II (galactoglucan), with diferent functions in the symbiotic process. Accordingly, we undertook a study comparing the potential diferences in alfalfa nodulation by E. meliloti strains with diferences in their EPSs produc- tion. Strains recommended for inoculation as well as laboratory strains and native strains isolated from alfalfa felds were investigated. This study concentrated on EPS-II production, which results in mucoid colonies that are dependent on the presence of an intact expR gene. The results revealed that although the studied strains exhibited diferent phenotypes, the diferences did not afect alfalfa nodulation itself. However, subtle changes in timing and efcacy to the efects of inocula- tion with the diferent strains may result because of other as-yet unknown factors. Thus, additional research is needed to determine the most efective inoculant strains and the best conditions for improving alfalfa production under agricultural conditions. Keywords Alfalfa · Sinorhizobium meliloti · Ensifer meliloti · Exopolysaccharides · Symbiosis · Biofertilization Introduction Horwath 2016). Although legume agriculture has expanded in Argentina, it has been recently infuenced by a dramatic Alfalfa (Medicago sativa L.) is one of the most important increase in soybean cropping (Caviglia and Andrade 2010), forage legumes for agriculture because it is well adapted to leading to a decrease in the land area allocated to forage diverse environmental conditions (Avci et al. 2013). Conse- crops such as alfalfa. As a result, forage crop systems based quently, it has become an important crop in many parts of on the use of double crops where summer crops are used for the world including the Pampean region of central Argen- silage and winter crops for pasture have become increasingly tina (Garcia et al. 2014) and also California (Geisseler and common (Abdelhadi et al. 2004; Arelovich et al. 2011). Nev- ertheless, the increasing demand for livestock feed in Argen- tina is negatively afected by suboptimal forage production Communicated by Erko Stackebrandt. levels, which is attributed to low nitrogen (N) fertilization Electronic supplementary material The online version of this rates (Agnusdei et al. 2010). Therefore, an increase in the article (https ://doi.org/10.1007/s0020 3-019-01756 -3) contains efciency of animal food production is needed to meet the supplementary material, which is available to authorized users. growing demand within the constraints of the land area cur- * Walter Giordano rently cultivated (Evans 1993). [email protected] Biofertilization of legume seeds with nitrogen-fxing bacteria collectively known as rhizobia started many years 1 Departamento de Biología Molecular, Universidad Nacional ago. Collectively, the legume-rhizobia symbiosis contributes de Río Cuarto, Río Cuarto, Córdoba, Argentina over 40 million tons of N per year to agricultural systems 2 Department of Molecular, Cell and Developmental Biology, (Herridge et al. 2008). Thus, the contribution of N by sym- University of California-Los Angeles, Los Angeles, USA biotic fxation via the legume-rhizobia symbiosis is the most 3 Molecular Biology Institute, University of California-Los important source of N in agroecosystems (Ohyama 2017). Angeles, Los Angeles, USA Vol.:(0123456789)1 3 Archives of Microbiology Enhancing the efectiveness of rhizobial inocula is desirable for the Mesorhizobium loti and Lotus japonicus N-fxing because bacteria can alleviate the requirement for synthetic associations (Kawaharada et al. 2015). Some conditions, for N fertilizers that are economically and environmentally example, limitations of N and sulfur, high phosphate con- costly. In Argentina, a high percentage of legumes including centrations, and hyperosmotic stress stimulate EPS-I synthe- soybeans, peanuts and alfalfa are inoculated (Vicario et al. sis, whereas others such as phosphate starvation induce EPS- 2015; López et al. 2018). However, very little information on II production (Janczarek et al. 2014). EPSs are produced the biological signifcance of biofertilization for Medicago in two diferent forms, of high and low molecular weights, sativa (alfalfa) is available. and are termed HMW and LMW, respectively. The LMW E. meliloti is a soil-inhabiting N-fxing Alphaproteobac- fraction is an active biological form of EPS that is essential terium, which under conditions of N limitation, establishes for successful infection of leguminous plants that establish a root nodule symbiosis with legume plants, especially indeterminate-type nodules (Skorupska et al. 2006). alfalfa, but also with other E. meliloti host legumes such Under controlled laboratory conditions, Rm1021, a wild- as Melilotus alba (Giordano and Hirsch 2004). As a result type strain of E. meliloti, synthesizes detectable quantities of this interaction, the bacteria provide ammonia to their of EPS-I but does not synthesize EPS-II. Synthesis of EPS- hosts and receive nutrients, mainly carbohydrates, from them II in Rm1021 can be induced by environmental conditions (Jones et al. 2007). The process of symbiotic N fxation is and mutations, for example under low-phosphate conditions carried out in specialized root structures, i.e. nodules. The (Zhan et al. 1991) and in a mucR mutant (Keller et al. 1995). interaction of the rhizobia and legume plants is highly host- The presence of a functional expR gene is necessary to pro- specifc (Denarié et al. 1996), which is one of the many mote the synthesis of symbiotically active EPS-II in strain reasons agricultural legume seeds are coated with specifc Rm8530, which has an intact expR and is termed expR+ rhizobial inoculants. While rhizobia were once thought to (Glazebrook and Walker 1989). However, strain Rm1021 be the only N-fxing inhabitants of legume nodules, other carries an insertion sequence (IS) element within the expR bacteria, which are distinct from rhizobia, are often iso- gene, which prevents EPS-II synthesis (Pellock et al. 2002). lated from surface-sterilized nodules obtained from soil, EPS-II production in E. meliloti facilitates autoaggrega- thus revealing the existence of a phytomicrobiome where tion, a mucoid phenotype, and bioflm formation (Rinaudi the interaction among the individuals is not only complex and González 2009; Sorroche et al. 2010). A positive cor- but also likely to afect the behavior and ftness of the host relation between bioflm formation and autoaggregation for plant (Martíınez-Hidalgo and Hirsch 2017). Previously, we E. meliloti strains isolated from alfalfa root nodules in the reported on the complexity of the bacterial communities feld led us to conclude that a functional EPS-II is needed isolated from alfalfa rhizospheric soils in the agricultural for both bacterial-bacterial and bacterial-surface interactions region of the Argentinean Pampas (Bogino et al. 2013), but (Sorroche et al. 2012). In addition, a mutation in LPS in the whether these or nodule microbiome bacteria infuence the presence or absence of EPS-II leads to important changes symbiosis is as yet unknown. in the adhesive properties of E. meliloti as well as in the Rhizobial surface components, especially exopolysaccha- symbiont’s interaction with alfalfa (Sorroche et al. 2018). rides (EPSs), fagella, and lipopolysaccharides (LPSs), as well We previously demonstrated by PCR analysis that the as bacterial and environmental signal molecules play impor- expR gene does not exhibit an obvious insertion in either tant roles in symbiosis, particularly in the formation of meta- indigenous E. meliloti strains isolated from Argentinean bolically active root nodules (Hirsch 1999). These molecules soils or the reference strain Rm8530 (Sorroche et al. 2012). are also crucial for rhizobial bioflm formation (Fujishige et al. However, relatively little is known about the efcacy of the 2006; Rinaudi et al. 2006; Rinaudi and Giordano 2010) and inoculation response of alfalfa to many of the rhizobia used for attachment to legume roots (Hirsch et al. 2009). Strains of as inoculants. The objectives of this study were (1) to com- E. meliloti produce two diferent EPS molecules: succinogly- pare the genetic (expR gene structure) and biochemical (EPS can (EPS-I) and galactoglucan (EPS-II) (Fraysse et al. 2003). production) features of two inoculant strains (E. meliloti