Mitigation of salt stress in white clover (Trifolium repens) by Azospirillum brasilense and its inoculation effect

Salt stress negatively influences the morphological, ionic, and physiological as well as biochemical characteristics in plants (Chen et al. 2013; Abbasi et al. 2015). It has been reported that increasing the salt level, reduces the osmotic potential, which results in cell dehydration, due to increased water efflux from cell (Amjad et al. 2014). Beneficial soil bacterial mediated growth enhancing attributes have been documented in enormous plant species (Gray and Smith 2005; Xie et al. 2009; Paré et al. 2011). The growth amplification of several plants, such as wheat (Triticum aestivum), maize (Zea mays), tomato (Lycopersicon esculentum), dwarf salt wort (Salicornia bigelovii), chickpea (Cicer arietinum), and alfalfa (Medicago sativa) by beneficial soil bacteria have also been observed at varying salt exposures (Bashan et al. 2000; Mayak et al. 2004; Ibragimova et al. 2006; Bano and Fatima 2009; Tiwari et al. 2011). A significant growth improvement of white clover by A. brasilense inoculated soil recorded in the present study was comparable with previous reports in Arabidopsis (Zhang et al. 2007, 2008, Zhang et al. 2010a, b; Xie et al. 2009; Paré et al. 2011). Noticeably, our findings revealed that A. brasilense in white clover has more active shoot growth promoting role compared to its root growth, particularly under salt stress conditions (40, 80, and 120 mM NaCl concentration). Similar results for salt preventing potentiality of Azospirillum strains have been documented previously by several researchers evaluated the bio-control and salt stress alleviating perspective of A. lipoferum in wheat crop and found that A. lipoferum displayed the good potential to promote the growth of wheat under saline conditions (up to 150 mM of NaCl) (Bashan and Levanony 1990). Synthesis of growth promoting substances (GPS) and adaptation to current environmental conditions may confer increased germination percentage and alleviation under prevailing salt conditions (Rueda-Puente et al. 2007; Nadeem et al. 2013). Leaf development and/or enlargement play a significant role in plant production since it is strongly associated with plant growth and biomass accumulation (Gutiérrez-Boem and Thomas 1998; Bacilio et al. 2004; Battie-Laclau et al. 2013).

A significant augmentation in leaf area and leaf number per plant was also noted in A. brasilence inoculated white clover plants. In another study, Han et al. (2014) reported that GB03 inoculation significantly enlarged leaf area and number per plant, and thus average leaf area in white clover. Likewise, photosynthetic pigment, leaf chlorophyll content is also an important physiological/biological attribute directly related to photosynthesis rate in plants (Ma et al. 2012). Inoculation of A. brasilence considerably improved the leaf chlorophyll content in white clover plant, cultivated under both non-saline as well as variable salt concentrations of 40, 80, and 120 mM NaCl. Similar to our study, Zhang et al. (2008) described that B. subtilis GB03 enhances photosynthetic efficiency by increasing chlorophyll content in Arabidopsis. In contrast, some reports highlighted that plants grown under salinity environments produced a smaller amount of chlorophyll and dry matter than those without salt exposure presumably due to chlorophyll per-oxidation (Lunde et al. 2007; Tuna et al. 2008; Barry 2009).

Soils with salinity contain an array of several cation–anion pairs (i.e., CaSO₄, MgSO₄, MgCl₂, Na₂CO₃, and Na₂SO₄), with Na⁺ ions being the predominant species (Zhang et al. 2010a). Growth is impeding, a common plant response to salt stress is often related to elevated intracellular Na⁺ concentration and low K⁺/Na⁺ ratio in the plant (Zhang et al. 2010a). Researchers have reported that plants grown under saline conditions can curtail sodium toxicity by limiting Na⁺ uptake, re-directing Na⁺ from shoots to roots, and also extruding Na⁺ loadings from root cells (Tester and Davenport 2003; Munns and Tester 2008; Zhang et al. 2010a, 2011; Kronzucker and Britto 2011). In a previous study, (Zhang et al. 2010a, b) reported that B. subtilis significantly reduced (54%) the plant Na⁺ content in Arabidopsis compared with control plants by down-regulating and up-regulating HKT1 expression in roots and shoots, respectively. In this study, soil inoculated with A. brassilence considerably reduced the Na⁺ level and increased K⁺ to Na⁺ ratio in both roots and shoots of the experimental plant at all levels of tested salt stresses.