North Dakota dry bean (Phaseolus vulgaris L.) classes black, navy, and pinto averaged 33,600, 40,400, and 142,100 planted ha, respectively, from 2011 through 2015 which represents approximately 14%, 18%, and 62% of total North Dakota planted hectares, respectively [1] . With 33% of the national planted hectares in 2017, North Dakota is the largest dry bean producing state followed by Michigan, Minnesota, Nebraska, and Idaho [1] .

In North Dakota, the use of N fertilizer is recommended when the dry bean yield goal is greater than 2200 kg ha⁻¹, or the residual soil-N level is less than 56 kg ha⁻¹. Contrastingly, production recommendations are to inoculate the seeds with Rhizobium phaseoli bacteria if the yield goal is lower than 2200 kg ha⁻¹ and the residual N in the soil is less than 56 kg ha⁻¹ [2] [3] . On average, 95% of North Dakota and Minnesota dry bean farmers [4] apply 56 kg N ha⁻¹. Studies show that N increases the yield components number of pods per plant, number of seeds per pod, seed weight, and final grain yield expression [5] . Excessive N can increase lodging, inhibit nodule formation, delay maturity, and promote excessive leaf canopy growth [6] . Nitrogen is an essential nutrient for plant growth [7] , and dry bean plants have been shown to fix between 22 and 90 kg N ha⁻¹, with an average of 44 kg ha⁻¹ [8] . Dry bean N₂ fixation is generally considered lower than for faba bean (Vicia faba L.), field pea (Pisum sativum L.), and lentil (Lens culinaris Medik.) [9] .

Nitrogen fixation alone may not provide enough N for a legume’s needs throughout a growing season. However, N fertilizer application may reduce N₂ fixation in root nodules [10] . A balance between soil available N, application of N fertilizer and biological N₂ fixation, may be required for optimal yields [11] . Assuming the right strain of Rhizobium is available in the soil, legume plants only make the energy investment required for symbiotic N₂ fixation when there are short supplies of mineral N [12] . Since inoculation is inexpensive, environmentally friendly, and sustainable compared to supplemental N fertilizer applications, finding a way to consistently increase N₂ fixation may benefit farmers. Graham [13] found that applications as low as 15 kg N ha⁻¹ may reduce rates of N₂ fixation of dry bean by as much as 40%. Compared with other legumes dry bean is not an efficient N₂ fixing species and there appears to be a cultivar specificity in the strain of Rhizobia for nodulation [14] .

Low and variable dry bean nodulation and N₂ fixation have been attributed to many factors including, high temperature, drought, soil acidity [15] , high soil residual N, a short growing season, seasonal weather variation [16] [17] , and differences in growth habit between cultivars [10] . Edje et al. [10] researched the effect of six fertilizer levels (0, 40, 80, 120, 160, and 200 kg N) on dry bean and reported that seed yield increased with addition of 40 kg N ha⁻¹ (2704) compared to the non-fertilized control (2150 kg ha⁻¹). However, N rates from 40 to 160 kg N ha⁻¹ produced similar seed yield and seed yield was only higher at the 200 kg N ha⁻¹ (3779 kg ha⁻¹) rate compared to the 40 kg N ha⁻¹ rate. Leaf area index, total dry matter, and general plant vigor increased as N rate increased. Eckert et al. [18] conducted research on pinto bean cultivars and N fertilization. They reported that increasing N level did not influence seed yield or seed weight.

According to Kellman et al. [19] , most soils sown to dry bean contain indigenous rhizobia. However, their results found that it should be possible to increase nodulation and seed yield of dry beans by combining suitable cultivars with an appropriate strain of rhizobia. Fageria et al. [20] reported that inoculation with a mixture of three Rhizobium tropici strains improved dry bean grain yield. Work done by Weiser et al. [21] observed dry bean nodulation in Fargo, ND on soil that had not previously been grown to dry bean or been inoculated. The study found that nodulation can be enhanced by inoculation of commercial strains of Rhizobium phaseoli in soils containing indigenous strains of rhizobia and relatively high levels of N. Inoculation resulted in significantly greater nodule numbers relative to indigenous soil strains, however significant seed yield increases were not found from the increase in inoculation.

A 2004 study by Henson and Halvorson at the North Dakota Agricultural Expt. Station Carrington Research Extension Center (Carrington, ND) and North Central Research Extension Center (Minot, ND) evaluated dry bean with seven levels of N without Rhizobium inoculation [22] . Additional treatment comparisons with and without inoculation at lower N levels and evaluation of the response to split N applications were studied. Soil test data (0 - 61 cm depth) indicated 17 kg NO₃-N ha⁻¹ at Carrington and 57 kg NO₃-N ha⁻¹ at Minot. An increase in seed yield was observed at Carrington as total N increased from 17 to 56 to 84 kg N ha⁻¹. Contrastingly, no significant (p < 0.05) differences in seed yield were observed at Minot.

Nitrogen-limited conditions without extreme pH (not acidic or alkaline), no salinity and appropriate plant available water are considered necessary for symbiotic relationships between legumes and rhizobia, but the effects of N rich conditions on this symbiotic status remains poorly understood. Research conducted by Nanjareddy et al. [23] examined rhizobial symbiosis with dry bean under different N conditions. They found that high levels of N impaired nodule maturation and nodule numbers. Low N conditions increased nodule number, biomass, and extended the duration of N₂-fixing activity, consistent with observations made by Edje et al. [10] .

Scientists are exploring tools such as optical sensors to determine the need for in-season N application to increase N use efficiency (NUE). The overall NUE of a cropping system can be increased by achieving greater uptake efficiency from applied N inputs and reducing the amount of N lost from soil organic and inorganic N pools [24] . In order to maximize seed yield it is important to find an efficient N management system [25] .

The chlorophyll carotenoid pigments in plant leaves absorb visible light from 400 to 700 nm for photosynthesis and reflect green light. Bullock and Anderson [26] found the Minolta SPAD 502 (SPAD) chlorophyll N meter reading to be useful as a diagnostic aid rather than a tool for N management in corn (Zea mays L.). The SPAD reading could provide a measure of the relative greenness of living leaves at a specific point in time and could possibly be useful in detecting N deficiencies in growing dry bean when compared to a non N limiting standard in the field.

Fritschi and Ray [27] assessed genotypic variation in soybean [Glycine max (L.) Merrill] chlorophyll content with the use of SPAD meter readings to see if these data could be used as a rapid screening method to predict genotypic variation in leaf tissue N content. They found that chlorophyll content was related to SPAD readings and leaf N content; however, the relationships found were not consistent for chlorophyll to be useful as a predictive tool for leaf N content in soybean. If SPAD readings were correlated to yield in dry bean, it could be a useful management tool.

The relationship between dry bean and strains of Rhizobium phaseoli bacteria associated with N fixation is highly variable and specific to environmental conditions. Dry hot weather conditions, periods of soil water saturation, and cold weather, will result in an inconsistency of the symbiotic relationship in supplying N nutrition during the growth cycle. No recent research in North Dakota has evaluated the effect of inoculum on the yield of the main dry bean classes grown in the state and the effect of fertilizer N on nodulation. Only about 19% of the surveyed growers in North Dakota used inoculum in 2010 [4] .

The objectives of this dry bean research were to: 1) compare multiple types of inoculant and fertilizer management in relation to seed yield; 2) compare nodule formation among treatments and its association with seed yield; and 3) record observations on plant growth, utilizing visual scores, plant measurements, and optical sensors in order to better understand dry bean growth in relation to seed yield.

The experimental design was a RCBD 3 × 2 × 3 factorial arrangement of cultivar, N fertility level, and inoculant treatment with four replicates. Three cultivars each from a different dry bean market class (black, navy, and pinto) were used: black, ‘Eclipse’ [28] ; navy, ‘Vista’ (Gen-Tec Seeds Ltd., South Woodslee, ON, released 1989); and pinto, ‘Lariat’ [29] . The three market classes are the dominant classes in North Dakota and the cultivars the most commonly grown for a market class during the study period. There were two inoculant treatments alongwith a control (non-inoculated), Rhizobium leguminsarum bv. phaseoli 2 × 10⁹ viable cells g⁻¹ combined with Bacillus subtilis 2 × 10⁸ viable spores g⁻¹ (Hi-Stick, Becker Underwood Inc., Ames, Iowa), and Rhizobium legumnosarum bv. phaseoli 2 × 10⁸ viable cells g⁻¹ (RhizoStick, Becker Underwood Inc., Ames, Iowa). These peat-based inoculants were applied to seed prior to planting. In order to avoid inoculant treatment contamination, the non-inoculant treatments were planted first, then all plots containing RhizoStick were planted. After the planting cones were sanitized, the cones were coated with HiStick and the remaining plots which contained HiStick were planted. There were two fertilizer treatments of no fertilizer (control) and 56 kg N ha⁻¹ in the form of urea at emergence. Pure live seeds were packaged to obtain the regionally recommended population of 222,400 plants ha⁻¹ for Eclipse and Vista and 173,000 plants ha⁻¹ for Lariat [30] .

The cultivars were planted in four-row plots, 7.6 m long with a 45.7 cm row spacing. Plots were planted with a John Deere 71 flex four-row planter with a cone seed distribution system (John Deere, Moline, IL). Seeds were planted at a depth of 2.5 to 3.8 cm. At all locations and years, the previous crop was wheat (Triticum aestivum L. emed. Thell.) and fields with a history of no recent dry bean production were chosen. The research was done at Prosper, ND, in 2010 and 2012. In 2013 and 2014, experiments were planted at Prosper and near Park River, ND, However, only the pinto cultivar was planted in 2014 and not the black or navy dry bean cultivars.

Soil tests were collected in the spring of the growing seasons (Table 1). Soil tests were conducted for nitrate-N (NO₃-N) at the NDSU Soil Testing Lab in Fargo, ND.

Dry nitrogen urea fertilizer (46-0-0) treatments were applied at emergence. Furrows were hoed about 5 cm deep and 5 cm to the side along each row, with the fertilizer hand spread into the furrows and immediately covered with soil.

Dimethenamid-P: (S)-2chloro-N-[(1-methyl-2-methoxy) ethyl]-N (2, 4-dimethyl- thien-3-yl)-acetamide (Outlook, BASF, Research Triangle Park, NC) was applied for weed control immediately after planting in all years and locations using a backpack sprayer with 8001 VS nozzles at 275 KPa at a rate of 941 g ha⁻¹. Hand weeding was conducted as needed during the season for additional weed control. On July 2^nd of 2014, Prosper and Park River were also sprayed with bentazon: (3-(1-methylethyl)-1H-2, 1, 3-benzothiadiazin-4 (3H)-one 2, 2-dioxide)) (Basagran, BASF, Research Triangle Park, NC) at 0.3 L ha⁻¹ of a.i. and imazamox: 2-(4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1HOimidazol-2-yl)5-(methoxymethyl)-3-pyridinexarboxylic acid) (Raptor, BASF, Research Triangle Park,

^†USDA soil survey data [31] . ^‡No experiment in 2010 and 2012.

NC) at 8.4 g ha⁻¹ of a.i. In 2013 and 2014, preventative fungicide was applied using boscalid: (3-pyridinecarboxamide, 2-chloro-N-(4-chloro (1,1-biphenyl)-2-yl)) (Endura, BASF, Research Triangle Park, NC) at a rate of 539 g ha⁻¹ of a.i. during early reproductive growth stages.

A vigor score, scale of 1 - 9 with 9 being the most vigorous, was used during the growing season along with a visual greenness score, with a scale of 1 - 5 with 5 being greener (Table 2). Chlorophyll readings on a scale from 0 to 99.9 with higher readings being greener, were taken with a handheld SPAD-502 meter (Spectrum Technologies, Aurora, IL). The SPAD readings were taken from the upper-most fully developed leaf and readings were averaged over 6 plants within the two center plot rows.

Root excavation was performed to count nodules per root two weeks after the R2 to R3 growth stage [32] (Table 2). Five plants per plot were extracted with shovels; the roots were cleaned in a bucket of water to remove soil before nodules were counted [33] . Plants for nodule counts were taken from the end of the two center plot rows before alleys were cut. Alleys were cut at the R5 growth stage in between the replicates, perpendicular to the planted plots, resulting in a harvested area of 6 m × 1.8 m. Plant height was measured before harvest from the soil surface to the upper main stem node of three random standing plants within the two center rows of every plot (Table 2). In 2010 and 2012, two rows per plot were harvested with a Hege 125B combine (Hege Company, Waldenberg, Germany). In 2013 and 2014 one row per plot was harvested by hand and threshed with a Hege 125B combine. The harvested samples were dried to about 14% moisture content and cleaned (Allan Machine Company, Nevada, IA) to remove foreign material. All samples were weighed and analyzed for seed yield, moisture, and 1000 seed weight. Moisture and test weight were measured using a GAC 2100 moisture tester (DICKEY-John Corp., Minneapolis, MN) and

observations were adjusted to 13.5% moisture content. For the Lariat data an economic analysis was performed using a farm gate price for pinto beans in ND of 0.61 ct kg⁻¹, which was multiplied by the yield to obtain the value in US $ for each research unit. The inoculant treatment was valued at $24.70 ha⁻¹. The 50 kg N ha⁻¹ was valued at $44.90 and application cost of $11.12 ha⁻¹ (spreading of the fertilizer) was used. Cost of inoculant and or fertilizer + application cost, based on the treatment, was deducted from the price x yield number to obtain a dollar value for each research unit.

Weather data was obtained from the North Dakota Agricultural Weather Network [34] .

Statistical analyses were conducted using standard procedures for a randomized complete block design. Data was analyzed as a factorial using analysis of variance with SAS 9.3 (SAS Institute Inc., Cary, NC). PROC MIXED procedure and Type 3 ANOVA tests were used to analyze treatment data. Each environment was analyzed separately. After testing for homogeneity of variance, research data with the complete set of treatments with all three cultivars from 2010 and 2012 at Prosper and Prosper and Park River 2013 were combined for analysis. In the combined analyses, the unique location and year combined into the term environment, and was considered a random effect in the sources of variation. Fixed effects in the analysis of this experiment were cultivar, inoculant, and application of N with all other factors considered random effects. All means were separated using a paired t-test at the 5% level of significance.

Additionally, due to Lariat only being planted in 2014 without the other two cultivars, the Lariat cultivar data from 2013 and 2014 at both Prosper and Park River was analyzed for each environment separately and combined across environments. All means were separated using a paired t-test at the 5% level of significance.

Across locations there was no N management practice that increased seed yield consistently among the different locations and under varying weather conditions. Application of N did not result in differences in yield, which conflicted with the results of Edje et al. [10] , who documented a yield increase due to N application but are consistent with the observations by Eckert et al. [18] conducting N research in North Dakota. Addition of fertilizer reduced nodule count by 40% and 25% at Prosper in 2012 and Park River in 2013, respectively. Although adding N did not increase seed yield, soil available N and rhizobacteria already in the soil may have been confounding factors. Cultivars differed in number of nodules, yield, height, and 1000 seed weight when averaged over four environments but the combined data did not show that interactions occurred among cultivar and inoculant and fertilizer factors.

The results of this study indicate that best management practices for dry bean N management will depend on location and environmental conditions. In farmer’s fields it may be beneficial to observe test strips of dry bean with and without inoculant and depending on soil N levels looking at varying levels of fertilizer applications, and adjust future management of fields on observed yield differences.

Buetow, R., Mehring, G.H., Kandel, H., Johnson, B. and Osorno, J.M. (2017) Nitrogen Fertilization and Inoculation Effects on Dry Bean. Agricultural Sciences, 8, 1065-1081. https://doi.org/10.4236/as.2017.810077