Soybean Nodulation and Plant Response to Nitrogen and Sulfur Fertilization in the Northern US

Soybean [Glycine max (L.) Merrill] seed yields in the northern United States may increase with the application of fertilizers; however Nitrogen (N) may decrease root nodulation. This study was conducted to understand the impact of N and sulfur (S) fertilization on soybean nodulation, plant, shoot and root biomass. Two cultivars were planted in experiments across ten site-years during 2015-2016. Plant observations took place at the V4 and R4 soybean growth stages. There were 41% more nodules per plant at R4 compared to V4 (38.3 vs 27.2 nodules, respectively). Cultivars responded differently to N and S fertilizer. The nodules per plant between the cultivars (30.3 vs 24.4) were different as well as the percent medium and large-sized nodules, which indicates the need to evaluate additional genotypes. Adding N decreased root nodulation (from 31.8 to 23.7 nodules per plant) and decreased nodule size but had no effect on plant, shoot or root mass. Averaged across N rates total plant mass was 2.26 and 11.36 g per plant at V4 and R4, respectively. Shoot mass, average across N rates was 1.77 and 9.65 g per plant at V4 and R4, respectively, and root mass, average across N rates was 0.49 and 1.71 g per plant at V4 and R4, respectively. Sulfur did not have an effect on nodules per plant but increased the percent medium size nodules at the R4 observation. There was no N by S interaction observed for nodule number, size of the nodules, and plant, root and shoot mass. As cultivars differed in their nodulation response to N and S, additional research would be helpful to screen other cultivars.


Introduction
Over the last two decades soybean [Glycine max (L.) Merrill] cultivars have been developed, which are adapted to be planted in the northern soybean-growing region of the United States. Soybean yields have increased due to new cultivars, application of fertilizers and pesticides, and more intensive crop management practices. For example, the average North Dakota and Minnesota soybean yields have increased by 23.5 and 28.1 kg•ha −1 •yr −1 , respectively, from 1970 through 2019 [1].
In Minnesota and the northern Great Plains (NGP), which includes eastern Montana, northeastern Wyoming, North Dakota and South Dakota, soybean is planted in mid-May and harvested in late September or early October. Improving fertilization practices could increase Minnesota and NGP average soybean yield. Presently, no N application is recommended for North Dakota and northern Minnesota, except as a rescue treatment [2].
As newly developed soybean cultivars with higher yield potentials are being grown, there are concerns to whether or not biological N 2 fixation is capable of meeting increased plant N demands [3] [4]. Current knowledge states that soybeans acquire about 44% -72% of their N from N 2 fixation and the remainder from residual soil fertilizer and N mineralization [5].
Based on a meta-analysis using peer-reviewed papers, N fertilized soybean field experiments had 44% lower biological N 2 fixation compared to the unfertilized control [6]. Fertilizer N decreased soybean nodulation and increased aboveground plant dry matter [7]. Additionally, Heatherly [8] concluded that the application of 35 kg•N•ha −1 in the form of granular ammonium nitrate applied before V2 [9] did not increase yield. However, low rates of N at seeding have increased soybean yield and nodulation in some regions [7] [10] [11], possibly due to limited N fixation at the beginning of the season. The main source of N during early vegetative development is the utilization of 3 NO -N − from the soil, which can be from soil mineralization or application of fertilizer [12]. However, N as incorporated ammonium sulfate (AMS) and broadcast incorporated urea at planting may not increase soybean yield in certain environments [13] [14]. Thus, identifying soil limiting environments and targeting those for N application may provide some increase in yield potential.
Sulfur is becoming deficient in soils due to the introduction of high yielding crop cultivars, the use of high-grade S free fertilizers, and the reduced emission of S from industrial processes [15]. Soil S levels have decreased as S removal and yields of all major agricultural crops have increased, and deposition of SO 4 -S via rainfall, fertilizer, and pesticides has decreased [16] [17]. Although dry and wet S deposition rates in the northern United States have remained fairly steady [18], above normal precipitation since 1993 has caused some S deficiencies in corn (Zea mays L.) in North Dakota [19]. Soils typically at risk for S deficiency include coarse-textured soils, soils low in organic matter, soils experiencing large amounts of rainfall in the fall or spring, and soils located on higher landscape positions [20]. Since many of the soils and parent materials in the NGP have gypsum (CaSO 4 •2H 2 O) within them, S deficiencies, if they occur, will not likely be widespread [21]. Between deposition, soil-derived S from secondary minerals, and mineralization of soil organic matter, there has not previously been a need to apply S to row crops within the NGP. However, not unlike N, if supply of soil S is not keeping up with plant demands there is a likelihood of deficiency and reduction in yield potential.
It  [24]. In Tennessee 11, 23, and 34 kg•S•ha −1 as AMS, were applied to soybean in an S deficient soil. Soybean seed S content was significantly increased, but not yield [25].
The application of gypsum in Ohio, (where S deposition has greatly declined since 1990) at rates of 16 kg S and 67 kg•S•ha −1 , increased soybean yield by 4.8% and 11.6%, respectively [20]. In India, application of gypsum alone, up to a rate of 40 kg•S•ha −1 30 d after planting, increased the yield of soybean nearly 40%, and yield continued to increase with the additional application of farm yard manure which, also contains N [26] In other legumes, additions of S have shown positive responses to nodulation.
Fertilizing with S increased average nodule number and average nodule mass of white clover (Trifolium repens L.) compared to white clover grown under S deficient conditions [27]. Application of S in the form of CaSO 4 significantly increased dry mass (10%) and nodule number (45%) of garden pea (Pisum sativum L.) [28]. Ammonium sulfate depressed nodulation but addition of S increased nodules plant −1 by 36% in soybean [29]. Although S fertilization may not be necessary for all soils, its need has been well documented and thus warrants more investigation for some regions.
Given the dominance of soybeans within farmers' rotations in Minnesota and the NGP and the genetic potential for higher yields, the objectives of this study were to determine the effect of N and S fertilization on soybean nodulation, nodule size, above ground plant mass, and root growth. No previous regional research has evaluated a potential interaction of fertilizer N and S on nodulation and plant mass. The null hypothesis for this study is that the application of N and S do not influence these metrics. Except the Fargo location, all experiments were conducted on farmers' fields.

Materials and Methods
Sites were selected to represent a normal farm condition and none of the experiments had known N or S limitations.
Each experiment at the Fargo site had four replications and each other site had two replications, each year. The site, soil and previous crop information are provided in Table 1. Soil samples were taken prior to the fertilizer application, and analyzed at the NDSU Soil Testing Laboratory (Table 2).
At each site, there were two cultivar by six fertilizer treatments observation   Each subplot received different rates of N and S (Table 3). Nitrogen was applied in the form of urea (46-0-0) and S was applied in the form of gypsum   [5].
Dates of field operation and measurement are provided in Table 4. Statistical analysis was conducted using standard procedures according to Carmer et al. [31] for a randomized complete block design with a two-factor factorial arrangement within a split plot, with cultivar being the main plot. All dependent variables were analyzed with a mixed model (PROC MIXED) on SAS 9.3 (SAS

Weather
In 2015, May precipitation at all locations was 2 to 2.8 times more compared with the long-term normal precipitation. However, precipitation in June through September tended to be below normal, which greatly reduced the cumulative seasonal precipitation and was below the long-term average in Lisbon, Mooreton, and Gwinner (

Nodulation
Levels of significance for cultivar, N, S and interactions for the first (V4) and second (R4) nodulation observations are presented in Table 6 and Table 7, respectively.    Hope, respectively ( Figure 1). Possible explanations for differential environmental response include factors such soil type, previous crop (Table 1), precipitation (Table 5), and level of soil N at the start of the experiment (Table 2).

Cultivar
Average nodule number per plant was significantly different at the V4 stage between cultivars. However, by the R4 stage cultivar had no significant impact on average nodule number per plant (Table 8). A significant difference in root mass between cultivars was also observed at the V4 growth stage, but evened out by the R4 growth stage (Table 8). Cultivars differed in average percent medium and large nodules at the R4 stage. PFS 15R07 had an average of 5.2% more large nodules per plant than PS 30 -80 at the R4 stage (Table 9).

Nitrogen
As the N rate increased at both observed growth stages, average nodule number per plant decreased and average nodule size decreased (Table 10)      this study confirm those reported by Hungria et al. [35] and Mendes et al. [36] who conclude that N fertilization at rates of 30 to 400 kg•N•ha −1 decreased nodulation and the contribution of biological N fixation. However, Mendes et al. [36] reported that average nodule number was 50% lower for plants treated with 40 kg•N•ha −1 compared to the control 15 d after emergence, but these significant differences in average nodule number per plant had disappeared by the R1 stage.
The significant difference between average nodule number at R4 with and without N fertilizer reported in this study could be attributed to the higher rates of N fertilizer used compared with Mendes et al. [36].
Our results also agree with those of Salvagiotti et al. [37], who showed a negative N fixation response as N fertilizer was added to the soil surface or incorporated in the topmost layers. Specifically, nitrate has been shown to decrease no-  [38] concluded that rapid inhibition of nodule activity was attributed to a decrease in transport of photosynthate to nodules from the shoot. Ohyama et al. [39] reported that soy-

Sulfur
At the V4 and R4 growth stage, S had no significant effect on nodules per plant, percent small or large nodules. However, at the R4 growth stage S significantly influenced medium nodule size with 57.2 and 60.5% of the total nodules being medium sized for the 0 and 112 kg•S•ha −1 , respectively. Results reported by Varin et al. [27] indicated that S significantly increased nodule size of white clover.
However, in our study the average percent of small and large sized nodules per plant did not change in response to S application.

Cultivar by Sulfur
The Anova showed a cultivar by S interaction at the V4 growth stage for the number of nodules per plant (

Nitrogen by Sulfur Interaction
The experiment was set up as a factorial to explore the interaction of N and S.  (Table 6 and Table 7). Overall, N fertilizer was a more dominant factor for influencing nodulation than S fertilizer.

Conclusions
Sulfur influenced nodule size at the R4 stage. However, the relationship remains unclear and more research is necessary to establish how S influences nodule size. Additionally, the interaction of cultivar and S influenced average nodule number at the V4 stage. The relationship indicates that the significance was due to a difference in magnitude for average nodules between cultivars and was not a true interaction. More research is needed to determine how S influences different soybean cultivars.
Nodulation and plant vegetation were more effected by fertilizer in early vegetative growth than reproductive growth, indicating that plant growth throughout the season made up for early differences between treatments and control.
Soil types varied by site-year and likely influenced the results of the study; however, in this study no interaction between N and S was observed. Environments played a key role in response to treatments; however, the experimental sites in this study were not specifically selected for limiting N and S conditions. Future research should focus on understanding genotypic variations in response to N and S fertilizer, soil-specific response to N and S application, and different forms of N and S fertilizer at various application timings to maximize the sustainability of soybean production practices in Minnesota and the NGP States.