2. Materials and Methods

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 2nd 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,

Table 1. Soil test results for NO3-N at Prosper, ND, 2010, 2012, 2013 and Park River, ND, in 2013 and 2014.

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

Table 2. Dates of application and observations for the cultivar, N-level, and rhizobacteria inoculant study conducted at two North Dakota locations from 2010 to 2014.

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.

3. Results and Discussion

3.1. Weather for Nitrogen Fertilization and Inoculation Effects on Dry Bean

Air temperatures were close to historical 30-yr averages each year of this research; however, there was noticeable variation in rainfall (Table 3 and Table 4). There was above average rainfall during several month periods at Prosper recorded in July of 2010 (103.4 mm), and May (105.2 mm) and June (192.5 mm) of 2013. Park River had above average rainfall in August of 2014 (88.4 mm). These differences in rainfall may have impacted how the dry bean plants responded to each treatment and with heavy rainfall, root nodule activity may have been reduced.

3.2. 2010, 2012, and 2013 Factorial Observing Effects on Cultivar, Nitrogen Fertilizer, and Inoculant


The probability levels for significant factors for individual environments and

Table 3. Monthly mean temperature and rainfall for Prosper, ND, in 2010, 2012, 2013 and 2014.

†Historical data represents a 30-yr average from 1981-2010 (NDAWN, 2017).

Table 4. Monthly mean temperature and mean rainfall for Park River, ND, in 2013 and 2014.

†Historical data represents a 30-yr average from 1981-2010 (NDAWN, 2017). NDAWN station, about 17.5 km from Park River, ND, experiment site. (NDAWN, 2017).

all probabilities for the combined analysis are provided in Table 5.

For each environment and when the data from Prosper 2010, 2012, and 2013 along with Park River 2013 were combined, differences were found among the cultivars of dry bean for number of nodules plant−1, plant height, plant vigor, and 1000 seed weight (Table 6). On average the cultivar Lariat had more nodules plant−1 (19) than both Eclipse (13) and Vista (9). At the various environments nodule numbers varied from 6 to 33 for Lariat, 5 to 28 for Eclipse, and 3 to 17 for Vista (Table 6).

Lariat was 3.6 cm taller than Vista; however, Eclipse did not differ in height from either cultivar. Lariat had a higher 1000 seed weight (300.6 g), typical for pinto bean, than the Eclipse (180.5 g) and Vista (169.3 g). Eclipse had a higher 1000 seed weight and number of nodules plant−1 than Vista when locations and years were combined (Table 6). At Prosper in 2010 Vista was yielding more than Eclipse and Lariat. At Prosper in 2012 Lariat yielded the highest (Table 6). Across all environments the seed yields were not significantly different at the 0.05 level, however the probability for differences in seed yield was 0.09 (Table 5).

Table 5. Probability level for significant factors for nodules per plant−1, plant vigor, plant height, 1000 seed weight, and yield for 2010, 2012 and 2013 in Prosper, ND and 2013 at Park River, ND, and complete probabilities for combined analysis.

In this research, only one cultivar per class was evaluated. Future research should look into the nodulation ability of more cultivars per class.


Across cultivars and inoculants there were no seed yield differences with and without application of N. Addition of 56 kg ha−1 N reduced the number of nodules per plant at Prosper 2012 and Park River 2013 (Table 7). Addition of N increased the height of the plants and decreased the 1000 seed weight at Park River in 2013 (Table 7). When data was combined across locations, application of 56 kg N ha−1 across all cultivars of dry bean had an increase in vigor score and decrease in 1000 seed weight (Table 7). The N applied may have put more of the

Table 6. Mean trait values for nodules plant−1, plant vigor, plant height, 1000 seed weight, and yield averages of cultivars across inoculants and N treatments averaged over four environments: Prosper, ND, in 2010, 2012, 2013, and Park River, ND, in 2013.

Visual score (1 - 9) with 1 being poor plant vigor and 9 indicating best plant vigor. Within columns, means followed by the same letter are not significantly different at (p ≤ 0.05), ns = not significant.

Table 7. Nodules per plant, height, 1000 seed weight and plant vigor for N treatments for across cultivars and inoculants for Prosper 2012, Park River 2013 and four environments; Prosper, ND, in 2010, 2012, 2013, and Park River, ND, in 2013.

†Visual score (1 - 9) with 1 being poor plant vigor and 9 indicating best plant vigor. ‡Within columns, means followed by the same letter are not significantly different at (p ≤ 0.05).

Table 8. Seed yield and nodule count for inoculants across N levels and cultivars of dry bean at Park River, ND, in 2013.

†Within columns, means followed by the same letter are not significantly different at (p ≤ 0.05).

plants’ energy toward vegetative growth or possibly more pods, causing a lower 1000 seed weight as less energy would be going towards pod fill.


At Park River in 2013, the addition of inoculant had an increase in yield for the HiStick inoculant compared to the control but it was not different in yield with the RhizoStick inoculant (Table 8). The slightly higher yield from HiStick may be related to the Bacillus subtilis in the product, which has natural anti- fungal properties, or could be related to the slightly higher cell count g−1 of Rhizobium leguminsarum bv. phaseoli compared to RhizoStick. There were nodules on the control (no inoculant) that indicates natural rhizobacteria in the soil (Table 8). Combined across four environments (2010, 2012, and 2013) the main effect “inoculants” did not show significant differences for the traits measured (Table 5).

3.3. Cultivar Lariat 2013 and 2014 Agronomic and Economic Observations

The following section is describing the pinto cultivar Lariat with inoculant and N application at Prosper and Park River in 2013 and 2014, Eclipse and Vista were not tested in these experiments.

SPAD Readings

Application of N resulted in differences in the green score and SPAD readings at Park River in 2013 and 2014, Prosper 2014 and across the environments (Table 9).

The SPAD readings were objective measurements, while visual green score was subjective. Observations at Park River indicated an increase in green score in both 2013 and 2014 with addition of N fertilizer; however, the SPAD readings showed no difference between N treatments in 2014 (Table 10).

The lack of consistency between green score and SPAD readings may be caused by the method used for SPAD readings, as readings were taken from the uppermost developed leaf and may not have been representative of the whole plant. Park River in 2013 showed a decrease in green score and SPAD reading with application of N. The combined results indicate that the plants were greener

Table 9. Probability level for significant factors for visual green score and SPAD observations for Lariat 2013 and 2014 in Prosper and Park River, ND, and combined across environments.

Table 10. Visual green score and SPAD readings of Lariat pinto bean across inoculants at Prosper and Park River, ND, in 2013 and 2014 and combined.

†Visual score (1 - 5) with 1 as lighter green and 5 as darker green. ‡SPAD readings (0 - 99.9) with higher readings representing darker green. §Within columns, means followed by the same letter are not significantly different at (p ≤ 0.05), ns = not significant.

with the application of N but the SPAD score was lower. Differences in green score and SPAD between inoculants were recorded only in 2014 at Park River (Table 11). The seed with HiStick had the lowest SPAD score and significantly lower than no inoculant, but seed with RhizoStick was not different from no inoculant or the HiStick application. In contrast, in 2013 the HiStick inoculant without N had a higher SPAD score than the no inoculant without N, however the RhizoStick with 56 kg N ha−1 had a higher SPAD reading compared with no inoculant with the application of N (Table 11).

SPAD reading indicated differences in greenness of dry bean plant leaves, but the method used in this study was not consistently representative of the color of the whole plant as the SPAD reading did not always match the visual observation. The method involving observation of the uppermost fully developed leaf may need to be improved upon, and more research using a SPAD meter as a management tool in dry bean could be useful. Utilizing other imagery technologies such as NDVI may be more successful for in-season fertilizer recommendations.

Although no significant difference in yield or nodule count was found for the main effects N, inoculation, or the interaction, the trends were higher yield with N application, more nodules with RhizoStick and lower nodule numbers with addition of N (Table 12). Eckert et al. [18] also noted a non-significant trend for higher dry bean yield with the application of 56 kg N ha−1 in North Dakota. The RhizoStick tended to have higher nodule numbers and higher yield compared to the no inoculant control. This would warrant to further investigate the effect of both N rate, source and application timing in combination with a Rhizobia inoculant.


When observing each individual year and location, a clear best management practice for N fertilizer does not emerge. Economic factors tend to drive production practices. Table 12 includes data combined across 2013 and 2014 from Park River and Prosper for the pinto variety Lariat and includes information on net

Table 11. SPAD readings of Lariat pinto bean for two N rates and across N rates at Park River, ND, in 2013 and 2014.

SPAD readings (0 - 99.9) with higher readings representing darker green. Comparing any means in the two fertilizer rate columns followed by the same letter are not significantly different at (p ≤ 0.05). §Within the column, means followed by the same letter are not significantly different at (p ≤ 0.05).

Table 12. Lariat dry bean seed yield and nodules per plant for applied N and inoculant for 2013 and 2014 at Park River and Prosper, ND, combined across environments.

†ns, within columns is not significantly different at (p ≤ 0.05). ‡Comparing any means in the three inoculant columns followed by the same. §Comparing means within the row. Pinto bean 0.61 ct kg−1, inoculant $24.70 ha−1, 50 kg N ha−1 was valued at $44.90, and fertilizer application cost of $11.12 ha−1.

return. Net return was calculated by using current prices for inoculant and fertilizer. The return is only comparing the inoculant and N inputs and does not consider unrelated costs such as seed, fuel, or chemical costs.

In this analysis, it shows that with the input costs of fertilizer compared to inoculant there are no significant differences and no significant differences among inoculant treatments. One major factor to consider is the initial N content of the soil (Table 1). It might be possible that under N deficient conditions there might be yield differences among the treatments. In production practices, before making a fertilizer management strategy, all conditions should be considered including existing N, prior rhizobacteria inoculation within field, and economic variables. Future research observing N deficient conditions would be insightful.

4. Conclusions

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.

Cite this paper

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


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