Rhizobium leguminosarum strain combination effects on nodulation and biological nitrogen fixation with Vicia villosa
Introduction
Cover crops are often used to conserve valuable soil resources. They benefit agroecosystems by stabilizing soil to reduce erosion (Clark et al., 1995), providing weed suppressing ground cover (Blackshaw et al., 2001), limiting nutrient leaching (Wyland et al., 1996), and adding plant residues that mineralize into potential nutrients for successive crops (Ranells and Wagger, 1997). Leguminous cover crops can additionally improve soil fertility through biological nitrogen fixation (BNF), which is the conversion of atmospheric dinitrogen gas (N2) into plant available ammonium (NH4+) inside legume root nodules occupied by symbiotic rhizobia N-fixing bacteria (Prell and Poole, 2006). Because of BNF, legumes have a consistent source of N, and legume plant residue is a valued N source in agriculture. Legume cover crops have been especially beneficial to organic farmers, who are prohibited by the National Organic Program certification law from using synthetic N fertilizers (SARE, 2007). Although N fixation in legume cover crops provides a substantial benefit to soil fertility, a farmer survey conducted by the United States Department of Agriculture in 2012 reports that only 3% of U.S. farmland was cover cropped (USDA, 2014). Also, several agencies advocate for increased legume cover crop adoption because of the soil and environmental conservation benefits they provide (SARE, 2007).
Vicia villosa (hairy vetch) is a broadly adapted legume cover crop with the potential to improve N fertility in agroecosystems (Bamford and Entz, 2016; Ebelhar et al., 1984; Hanson et al., 1993; Kuo et al., 1997). Vetch is winter-hardy in U.S. plant hardiness zones 4 to 6. It is typically planted in late summer or early fall, and harvested in early spring, which is similar to other winter cover crops. Studies have shown vetch to provide greater total N (100 to 135 kg N ha−1) than Austrian winter pea (Pisum sativum subsp. arvense) and white clover (Trifolium repens), two other common winter-hardy legume cover crops (Lichtenberg et al., 1994; Parr et al., 2011). Additional benefits of vetch include fast mineralization of residues (Azam et al., 1993; Brandsæter et al., 2008) and substantial groundcover formation (Campiglia et al., 2010; Teasdale et al., 2004).
For vetch, BNF is initiated when bacteria of the species Rhizobium leguminosarum biovar viciae (Rlv) form N-fixing root nodules after penetrating root tissue of the host plant (Wielbo et al., 2010). Vetch and Rlv co-evolved to form this symbiosis, and specific nodulation is observed throughout the Leguminosae family (Andrews and Andrews, 2017). A newly established vetch cover crop will have more difficulty forming nodules with a sparse Rlv soil population (Mothapo et al., 2013b), so farmers may inoculate legume seeds or soil (via liquid or peat) with compatible rhizobia to enhance nodulation and BNF. Legume yields may be improved after inoculation (Buttery et al., 1992; Carter et al., 1994; Denton et al., 2013; Kurchak and Provorov, 1995), and even after a large population of compatible rhizobia is established, inoculation can be used to maintain a diverse rhizobia community in soil (Trabelsi et al., 2011).
Previous research has attempted to establish a connection between rhizobia applied as inoculant and competition for nodule occupancy (Amarger, 1981). Inoculant rhizobia sometimes fail to occupy a substantial number of nodules when competing with resident rhizobia (Denton et al., 2002; Spriggs and Dakora, 2007), and this changes expectations of N supplied by BNF. Despite known capacities of individual rhizobia strains, the efficacy of inoculation varies because inoculant rhizobia must first compete with resident rhizobia for resources and nodule occupancy, and edaphic factors may also limit competitiveness in different environments (de Castro Pires et al., 2018; Rathi et al., 2018). Field studies have shown that rhizobia strains from inoculants can be predominant in nodules (Beyhaut et al., 2006; Denton et al., 2003) and also fail to occupy more nodules than resident rhizobia (Denton et al., 2002; Grossman et al., 2011; Malek et al., 1998). Although a specific strain can have measurable effects on BNF in legumes under controlled conditions (Ballard et al., 2004), when several strains are simultaneously present it is difficult to attribute changes in nodulation or BNF to the presence of a specific strain without knowing which strains are occupying root nodules. This limitation exists in part because tracking individual rhizobia strains has historically been difficult (Hirsch, 2005); however, the application of molecular biology techniques (e.g., DNA fingerprinting) to rhizobia studies facilitates tracking and identification of strains within nodules (Sarita et al., 2005; Thies et al., 2001; Wongphatcharachai et al., 2015). To better understand factors affecting rhizobia-plant dynamics in agricultural fields, exploration of rhizobia competition for nodulation, as well as the effect of rhizobia strain combinations on growth and BNF in legumes is critical.
Inoculants may be beneficial for legume growth, but relative nodule occupancy and BNF by individual strains, particularly when competing with other rhizobia, is not well understood. The goal of this study is to improve our understanding of Rlv strain competition effects on hairy vetch growth, nodulation, and BNF. The specific objectives were to (1) determine the additive effects of Rlv strain combinations on vetch BNF and nodulation based on individual strain characteristics, and (2) assess if characteristics of strain BNF can be explained by nodule occupancy.
Section snippets
Rhizobia culture selection
For several years, a library of rhizobia strains collected from agricultural fields throughout North Carolina was maintained at North Carolina State University. Three soil isolates collected during previous experiments were selected from this library for this research (Mothapo et al., 2013a, Mothapo et al., 2013b; Parr et al., 2011). Strains NCSU332 and NCSU478 were isolated from hairy vetch nodules collected from the Piedmont Research Station in Salisbury, North Carolina, USA (35°41′49.9″N
Plant growth and nodulation
Cell counts of cultures from the selected rhizobia strains measured at OD600 = 0.5 were 6.5 × 107, 6.8 × 107, 7.9 × 107, and 8.6 × 107 colony forming units per mL for strains C10, NCSU332, NCSU435, and NCSU478, respectively. Cell counts were comparable to those observed in other rhizobia studies with similar incubation protocols (Malek et al., 1998; Yahalom et al., 1987). Each cell culture was diluted to 1 × 107 CFU mL−1 and mixed separately for each treatment to facilitate inoculation.
Masses
Conclusion
This study found that inoculating hairy vetch with single-strain and multi-strain combinations of Rlv did not result in significantly different BNF. DNA fingerprinting indicated that the strains were not competitive for nodule occupancy. We also observed a wide range of BNF between vetch and Rlv used as single-strain and multi-strain inoculants, but BNF was rarely less than what was predicted based on results from single-strain trials. The rhizobia did not display any consistent synergistic or
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was performed in part at the Environmental and Agricultural Testing Service laboratory (EATS) in the Department of Crop and Soil Sciences at North Carolina State University. We thank the NC State phytotron for assistance with plant growth facilities. Any mentions of product names are to provide detail on materials used and does not imply specific endorsements by the researchers.
Funding
Funding was provided by the USDA NIFA Organic Transitions Program (2010-51106-21872) and the Dean's Graduate Research Assistantship from North Carolina State University. This work was supported by the USDA National Institute of Food and Agriculture, Hatch projects NC02440 and NC02713.
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