Short-term fertilizer application alters phenotypic traits of symbiotic nitrogen fixing bacteria

Natural history of study system

The field experiment was conducted in a recently disturbed old field habitat with dense populations of the legume Medicago lupulina growing in the Koffler Scientific Reserve (www.ksr.utoronto.ca, 44.0300°N, 79.5275°W) in Southern Ontario, Canada. M. lupulina is an annual exotic that forms facultative mutualistic interactions with symbiotic nitrogen fixing bacteria, Ensifer meliloti and Ensifer medicae in loamy soils stereotypical of Southern Ontario soil profiles (Prévost & Bromfield, 2003; Bromfield et al., 2010). Interactions between Medicago and Ensifer occur in the early spring during plant germination when symbiotic bacteria infect plant roots and induce nodule formation. During nodule formation between Medicago and Ensifer, a portion of rhizobia cells differentiate into specialized cells that fix atmospheric nitrogen (Oke & Long, 1999). When plant seed set occurs in August and September, nodule plant tissue begins to senesce, releasing rhizobia cells into the soil—the undifferentiated fraction of the cells survive in a free-living state until the following growing season (Hirsch, 1996).

Testing for field fertilizer treatment on host performance using whole-soil inoculations

We randomly positioned 16 plots (0.25 × 0.25 m) over two large M. lupulina population sites at the Koffler Scientific Reserve (44.0300°N, 79.5275°W). Within each population, half of the plots were randomly selected for fertilizer application. We applied 1 tbsp. (13 g) of Osmocote Miracle-Grow slow release fertilizer beads containing macro and micronutrients (in an N:P:K ratio of 19:6:12; Micromax^® by Scotts brand) over each plot in the early spring (May). When Medicago lupulina plants seeded and began to senesce by mid-August, soil was sampled at each plot and stored at 4 °C for further experimentation.

We initially tested for differential mutualistic effects of fertilized and unfertilized field soil on host plants by inoculating potted plants with whole soils in the glasshouse. We included three host genotypes (Cote-d’Or, France [FR]; Nebraska, USA [US] and Ontario, Canada [CA]; USDA germplasm repository: P1 234953-96i-SA19792, P1 215243-93i-53416, W6 4578-99i-2044) and a slow-release fertilizer application (same as field treatment) to determine whether the effects of field soil treatments were consistent across host genotype and fertilizer environment. In total, our design included field fertilizer treatment, glasshouse fertilizer treatment and host genotype in a full factorial design (i.e., 2 Field fertilizer treatments × 8 plots per field fertilizer treatment × 2 glasshouse fertilizer treatments per field plot × 3 plant genotypes per pot × 5 replicate pots = 480 total plants).

Each pot contained steam sanitized low nutrient soil (1:4; turface:sunshine mix #2) and a band of field soil applied (30 ml volume) at mid-depth and covered with a layer of autoclaved sand. To reduce effects on host performance as a result of chemical differences between fertilized and unfertilized field soil (as opposed to differences driven by microbial communities), we added autoclaved soil from the opposing field treatment to each pot in equal proportion. The opposing soil was prepared by autoclaving a soil mixture containing a subset of soil from all plots that received the same fertilizer treatment. For example, pots assigned with the unfertilized field soil treatment received 15 ml of unfertilized field soil from a given plot and 15 ml of autoclaved soil from all other fertilized field plots. We also added 10 control pots (which received 15 ml of autoclaved soil sample from each field treatment).

Prior to planting, seeds were scarified, sterilized in commercial bleach, stratified in the dark on 1.5% agar at 4 °C and pre-germinated at 22 °C for 12 h for radicle growth. Pre-germinated radicles from each host genotype were planted in each 6 inch pot (3 plant genotypes/pot). Plants were grown for 53 days and each pot was carefully top watered to minimize cross-contamination. At harvest, we recorded plant mortality, total dried plant biomass for each plant and haphazardly selected 15–20 nodules for dried preservation in tubes containing Drierite desiccant (Somasegaran & Hoben, 1994). Dried nodules were weighed to obtain a mean nodule mass measurement and stored at 4 °C in desiccant tubes for further experimentation. Plant performance was measured using plant biomass, which has been found to be strongly positively correlated with fruit and seed production in previous glasshouse experiments on Medicago lupulina in similar growing conditions (see Simonsen, Chow & Stinchcombe, 2014). Control plants showed significantly lower amounts of nodulation (11.2 nodules/plant) compared to experimental plants (127.2 nodules/plant; t = 8.6196, p < 0.0001), indicating that any low-level contamination that occurred is unlikely to explain experimental inoculation treatments.

We tested for field fertilizer treatments using a generalized linear mixed model on plant biomass, nodule size (log-transformed, PROC MIXED dist = Gaussian; SAS institute v9.3) and nodule number (PROC GLIMMIX, dist = Poisson). Our model included field fertilizer treatment, greenhouse fertilizer treatment, host genotype, block, final plant density in each pot (since some mortality occurred during the experiment), field site and harvest date as fixed effects, and pot and plot as random effects.

Testing for field fertilizer treatment on host performance using single-isolate inoculations

We obtained individual rhizobia isolates using preserved nodules from the whole-soil inoculation and used them in a single-isolate inoculation experiment to provide a measure of each isolate’s mutualistic benefit towards its host and thus disentangle whole-soil effects from the rhizobia community on host traits. A preserved nodule was randomly selected from each unfertilized plant in the whole-soil inoculation experiment (described above), rehydrated in sterile water, and subcultured on Tryptone-Yeast (TY) agar media (Somasegaran & Hoben, 1994) until clean isolates were obtained. Field soil treatments that received the additional glasshouse fertilizer application were excluded from the rhizobia isolation procedure. A total of 191 isolates were successfully cultured (101 from unfertilized and 90 from fertilized field plots).

The US genotype was selected for use in this second experiment based on overall vigour, reduced mortality and since analysis showed no indication of host genotype*field fertilizer interaction in the initial experiment (see “Results”). Seeds were germinated as described above and planted in autoclaved turface:sunshine #2 mix (4:1) in 4 inch pots (1 plant/pot) in a randomized blocked glasshouse design (191 isolates × 5 replicate pots per isolate distributed over 5 blocks). Wild rhizobia isolates were grown in TY media for roughly 36–48 h and diluted to equalize cell inoculation densities (OD600 = 0.1; ∼10⁶ cells/ml); 5 ml of inoculant was applied to each pot. Preliminary culturing indicated that most isolates neared stationary phase of growth after 36 h. Plants were given nitrogen-free Fahraeus nutrient solution (Somasegaran & Hoben, 1994) once weekly until harvest at 80 days. We measured host performance as the sum of total fruit and flower production per individual plant at harvest. None of the un-inoculated control plants (n = 36) flowered at harvest and were also significantly smaller prior to harvest (5.7 leaves/plant compared to 26.8 leaves/plant on inoculated plants; t = 20.39, p < 0.0001).

We determined the field fertilizer effect on all measures of host performance using a generalized linear mixed model (PROC GLIMMIX, dist = over-dispersed Poisson for fruit and flower production; SAS institute v9.3). Field fertilizer treatment, genotype of origin host (where preserved nodule was obtained), origin field site, greenhouse block and harvest date were included as fixed effects, while field sample plot (of original field soil sample) was included as a random factor. We tested for isolate effects using a log-likelihood ratio test between the full mixed model containing isolate and reduced model excluding the isolate term.

Testing for field fertilizer treatment on rhizobia isolate growth

We selected a random sub-set of isolates from each field fertilizer treatment for in vitro growth assays—31 from fertilized field soil (n_[CA] = 8, n_[FR] = 7, n_[US] = 16 from each host genotype) and 27 isolates from unfertilized field soil (n_[CA] = 8, n_[FR] = 11, n_[US] = 8. Isolates were grown in TY media containing three different plant fertilizer concentrations: no fertilizer (control), 0.25 tbsp. of fertilizer/500 ml media (low) and 0.5 tbsp. of fertilizer/500 ml media (high). All media was prepared using sterile filtered stock fertilizer solutions containing the same slow release fertilizer brand that was applied in the field plots (5 tbsp. of dissolved fertilizer per liter of distilled water). In total, our in vitro assay evaluated 58 isolates over 3 nutrient conditions with 8 replicates for each [isolate]*[fertilizer media] treatment combination.

We conducted the growth assays in 96 well plates, each containing 150 uL of liquid media and initially inoculated with 10 uL of diluted cell culture, grown initially in standard TY media for 36–48 h (OD₆₀₀ = 0.1, roughly 10⁶ cells/ml). Initial cell density measurements were taken immediately following inoculation and at 36 h. Each plate assayed 4 isolates in all 3 media treatments (n = 8 wells/isolate in each media treatment), and 8 additional un-inoculated wells containing blank TY media. Isolates were randomly assigned over 6 trials and cell density was estimated by measuring optical density (OD₆₀₀). We found no indication of contamination in un-inoculated controls (as indicated by unchanging optical density measurements during growth assays and a lack of cell growth when subsequently cultured on TY agar plates).

We evaluated the effect of field fertilization on optical density after 36 h using a generalized linear model (PROC MIXED, dist = Gaussian) containing field fertilizer, media fertilizer, origin host genotype and origin site as fixed effects. Optical density at initial inoculation was included as a covariate to account for any absorbance differences caused by fertilizer media treatment and initial inoculation. Isolate, trial, plate and field sample plot were included as random effects. We tested for isolate effects as above.

Testing for associations between rhizobia growth and mutualism benefit traits

We calculated mean growth and host performance traits for each isolate using fixed- effect lsmeans from a mixed model output. We obtained means for cell density counts (at 36 h) across each fertilizer media treatment from a mixed model that included fertilizer media treatment and isolate as fixed effects, and trial and plate as random effects; lsmeans were obtained from [fertilizer media]*[isolate] term (PROC MIXED, dist = Gaussian). Additionally, we calculated a growth plasticity index as the ratio of growth response in fertilizer and no fertilizer after 36 h for each isolate (see ‘tolerance index’ in Thrall et al., 2009): [OD_{high,36 h} − OD_high,initial]/(OD_{control,36 h} − OD_{control,initial}] and [OD_{low,36 h} − OD_{intermedite,initial}]/(OD_{control,36 h} − OD_{control,initial}], where ‘control’, ‘low’ and ‘high’ refer to fertilizer concentration. For isolate means in host performance, total fruit and flower production was modelled by block, harvest date and isolate as fixed effects and lsmeans were obtained from the isolate term (PROC GLIMMIX, dist = over-dispersed Poisson). We tested for associations between in vitro growth assays and mutualistic benefit using a general linear model, with host performance as the response and cell density, field fertilizer treatment and host genotype as predictors. We repeated the model for each type of growth assay (cell density count in control, low and high nutrient and the growth plasticity index).

Hosts performance in field soil inoculations

Plant biomass was larger when hosts were grown in unfertilized field soil compared to fertilized field soil (Fig. 1; F_1,418 = 471, p = 0.0306; Table S1). Expectedly plants were larger when fertilizer was applied to pots in the greenhouse (F_1,418 = 121.36, p < 0.0001; Table S1). Host genotype also explained variation in plant size (Fig. 1; F_2,418 = 42.47, p < 0.0001). However, host genotype and greenhouse fertilizer application did not alter rank effects of field fertilizer treatment, as indicated by a lack of interactive effects between the field and greenhouse fertilizer treatment (F_1,418 = 0.7595, p = 0.7595; Table S1) and between field fertilizer and host genotype (F_2,418 = 0.56, p = 0.5714). Nodule number and mean nodule size was 3.49% and 15.28% larger in fertilized field soil, but not significantly so (F_1,272 = 2.73, p = 0.0997; F_1,13.11 = 2.71, p = 0.1232 resp.). Generally, these results indicate that the higher host performance in unfertilized field soil was consistent across host genotypes and greenhouse fertilizer treatments.

Hosts performance in single isolate inoculations

Full model results are presented in Table S2. In contrast to whole-soil inoculation effects, plants had higher performance (measured by fruit and flower production) when inoculated with isolates originating from fertilized field soil (Fig. 2; F_1,152 = 4.35, p = 0.0386). Biomass was non-significant, but trended in the same direction as fruit and flower production, being higher when inoculated with isolates from fertilized field soil (not shown). Host performance differed significantly among isolates (χ² = 20.9, p < 0.0001), indicating genetic variation in rhizobia partner quality among our isolates. Rhizobia partner quality, or partner quality response to field fertilization, were not affected by the host genotype from which the isolate was originally collected (in the whole soil inoculum experiment), as indicated by non-significant Host genotype and Field Fertilizer*Host genotype effects (F_1,148.7 = 1.01, p = 0.37 and F_1,148.7 = 0.64, p = 0.5266; Table S2). These results indicate that isolates isolated from unfertilized field soil provided lower mutualism benefits to their host compared to isolates from fertilized field soil.

Strain growth assays on differing media fertilizer concentrations

We found no main effect of field fertilization on strain growth in culture (F_1,1118 = 1.14, p = 0.2862; Table S3). Media fertilizer had a consistent and positive effect on rhizobia growth, causing intermediate cell density at low fertilizer concentrations, and high cell density counts at high media fertilizer concentrations (Fig. 3A, 3B and Table S3). We also found significant differences in growth among isolates (χ² = 408.2, p < 0.0001; Table S3). Isolates originating from fertilized field soil exhibited the highest increase in growth as fertilizer media content increased, as indicated by a significant overall effect of [field fertilizer]^*[media fertilizer] (Fig. 3A; F_2,1114 = 4.08; p = 0.0172; Table S3). Host genotype origin also affected growth reaction norms, with isolates from FR hosts exhibiting the highest increase in growth across media fertilizer concentrations (Fig. 3B; F_4,1114 = 9.40; p < 0.0001). These results indicate that field fertilizer treatment, rhizobia nodule isolates, and the genotype of the origin host all affect the degree of rhizobia plasticity in response to plant fertilizer in the liquid growth media.

Correlations between free-living growth and mutualistic benefit

We did not detect any broad association patterns between mutualistic benefit (measured by host performance) and cell growth measures using cell density estimates in any media fertilizer treatment (indicated by no significant main effect of cell density; see Table S4), nor did we detect any field fertilizer treatment specific associations between host performance and cell density estimated in any fertilizer culture media (indicated by no significant cell density*field fertilizer interaction; see Table S4). However, the low fertilizer growth plasticity index (the ratio of growth responses in low fertilizer vs. no fertilizer after 36 h) did show field treatment specific reaction norms, exhibiting a positive correlation between traits in the presence of field fertilizer and a negative correlation in unfertilized treatments (Fig. 4A; field fertilizer*growth plasticity index; F_1,43 = 6.25, p = 0.0163 in Table S4). The high fertilizer growth plasticity index (the ratio of growth responses in high fertilizer vs. no fertilizer after 36 h) trended in similar but non-significant reaction norm patterns (Fig. 4B and Table S4).

Growth responses of rhizobia to plant fertilizer

Our data show that, even after a single season of fertilizer application, isolates from fertilized field soil grew faster than isolates from unfertilized field soil when the growing media contained the original plant fertilizer used on field soil. These results suggests that field fertilizer application caused a community shift or within-species evolutionary change, favouring lineages or genotypes (or even alleles at specific genes) that are more capable of utilizing higher dosages of plant fertilizer for growth in the free-living state (in the soil or in culture) in more nutrient-rich conditions. Given that rhizobia isolates from both field soil treatments responded positively to fertilizer in agar media, indicating that plant fertilizer provide nutrients that ordinarily limit free-living growth, our data support the hypothesis that fertilizer affected rhizobia fitness components related to free-living persistence in the soil. However, we cannot exclude the possibility that fertilizer application affected symbiosis fitness components, favouring rhizobia isolates that are competitive for Medicago nodulation, thus gaining higher fitness through host feedbacks.

We found no preliminary evidence of a fitness trade-off for growth in higher fertilizer—rhizobia from fertilized field soil did not have lower growth rates than rhizobia from unfertilized field on control media containing no plant nutrients supplement (Fig. 3A). However, previous empirical studies have found fitness costs (affecting free-living persistence) to adaptation to salt (Thrall et al., 2009) and metal (Porter & Rice, 2012) in soil. It is possible that the fitness costs observed for higher metal and salt concentrations occurs because these factors are generally detrimental to rhizobia growth and may require physiological trade-offs for survival (i.e., minimizing uptake of harmful elements while maintaining acquisition of beneficial elements). A lack of a fitness cost observed in our experiment may be because plant fertilizer stimulates growth and thus does not require physiological trade-offs to survive in low or high nutrient conditions.

To our knowledge, this is the first study to provide evidence that plant fertilizer (containing all macro and micro nutrients) is directly beneficial for rhizobia growth and favours isolates that have higher growth response to fertilizer.

Mutualism benefit responses of rhizobia to plant fertilizer

Initial whole field soil inoculations showed that Medicago had lower biomass in fertilized field soil. Since we expected higher biomass in fertilized field soil due simply to the presence of additional nutrients, these results suggest that plant fertilizer addition altered the microbial community composition in ways that are relevant to the aboveground productivity of Medicago. However, legume hosts were larger when inoculated directly with the rhizobia isolates cultured from fertilized soil, which suggests that the rhizobia community is unlikely to be responsible for the differences in plant performance observed in the initial whole-soil inoculations. In contrast, Weese et al. (2015) found the host partner quality decreased as a result of long term addition of nitrogen. We explored the possibility that the increase in host partner quality observed in our study may be due to a positive genetic correlation with free-living tolerance to fertilizer (i.e., isolates with higher mutualism benefit were observed due to a positive trait correlation with fertilizer tolerance). While we did detect a positive genetic correlation between host partner quality and plasticity in growth response to fertilizer, the direction of the correlation only occurred in fertilized field soil, being more strongly positive in the presence of fertilizer. The application of field fertilizer may have induced a positive correlation due to environment specific expression of genes that had pleiotropic effects on free-living growth and mutualism benefit traits, and may explain why both traits changed in our study. Generally, our data show that growth responses to fertilizer and mutualism benefit are not independent, and that the presence of fertilizer alters the degree of trait dependency for traits relevant to rhizobia fitness.

Another possible mechanism for increased host partner quality is that different host feedback dynamics occurred in our experiment compared to Weese et al. (2015). The addition of fertilizer containing excessive nitrogen (as the case with Weese et al., 2015) typically supresses associations with rhizobia, which is expected to alter host feedback dynamics by reducing fitness benefits towards beneficial symbiotic rhizobia. However, different host feedback dynamics may be induced if fertilizer contains increasing concentrations of all other macro macronutrients (i.e., N, P and K). For example, an increased nitrogen and phosphorus supply can increase symbiotic associations with beneficial rhizobia partners, which would then actually increase fitness benefits towards beneficial rhizobia partners.

Alternatively, fertilizer input in our experiment could have reduced selective pressure from plant feedbacks, which would cause host partner quality traits to increase by drift. However, replicated randomized plots allowed us to ascribe any differences among fertilizer types to the treatments we applied, making drift a less likely explanation. Furthermore, drift is expected to randomly exacerbate plot level differences between rhizobia populations, but plot had no explanatory effect in either host partner quality or in vitro growth traits in our study.

Further experimentation will be required to delineate the suite of causes that produced lower Medicago performance on the whole-soil inoculations and identify other microbial taxa relevant for plant productivity. It is possible that our experiment did not adequately capture the diversity of rhizobia present in the field soil. It is also possible that that strains occurring in mixtures have different effects on host fitness then single strain inoculations alone, which has been found in Medicago (Simonsen, Chow & Stinchcombe, 2014) and Acacia (Barrett et al., 2015). Decreased host productivity in fertilized whole soil inoculations could also result from changes in the abundance or diversity of beneficial symbiotic mycorrhizal fungi as a result of altered host feedbacks from excessive soil phosphorus addition (Broghammer et al., 2012). Previous experiments by Weese et al. (2015) and Thrall et al. (2007) also observed differences in whole-soil versus individual rhizobia isolate results on host condition and fitness. These studies, along with ours, highlight the challenge of focusing on individual taxa for inferring processes on complex non-additive effects of microbial communities on plant-microbe interactions, plant productivity and fitness.

Host origin affects rhizobia traits

Legume hosts have an important influence on rhizobia fitness in soils. The particular host genotype and species also has an influence on rhizobia abundance and composition (Coelho et al., 2009). Our experiment further shows that the origin of the host genotype is also associated with colony growth phenotypes of individual rhizobia, regardless of the fertilizer treatment applied on the field soil (Fig. 3B). Specifically, we found that isolates from hosts originating from France (i.e., FR) had a larger response to growth on fertilized agar media compared to isolates obtained from hosts from Canada and United States. Interestingly, the French host performed the poorest in initial whole soil inoculation (Fig. 1). Isolates from the French host genotype also trended towards providing the highest mean mutualism benefit (Fig. 2). Our results suggest that the host genotype or the host genotype reaction to the soil affected the assemblage of symbiotic interacting rhizobia during the whole soil inoculation. Generally, these results are consistent with previous studies (Coelho et al., 2009; Heath & Tiffin, 2009; Lafay & Burdon, 2001; Hoque, Broadhurst & Thrall, 2011), which have found that host genotypes have strong effects on the assemblage of rhizobia isolates inhabiting the nodules and, therefore, subsequent effects on the genetic composition of the soil communities after plant senescence.