The Response of Common Bean to Sulphur and Molybdenum Fertilization

Faculty of Agrobioengineering, University of Life Sciences in Lublin, 15 Akademicka Street, 20-950 Lublin, Poland Institute of Animal Breeding and Biodiversity Conservation, University of Life Sciences in Lublin, 13 Akademicka Street, 20-950 Lublin, Poland Institute of Animal Nutrition and Bromathology, University of Life Sciences in Lublin, 13 Akademicka Street, 20-950 Lublin, Poland Department of Plant Physiology, Faculty of Horticulture and Landscape Architecture, University of Life Sciences in Lublin, 15 Akademicka Street, 20-950 Lublin, Poland


Introduction
Leguminous crops are unique in the high protein content of their seeds and their ability to fix atmospheric nitrogen [1].Legume seeds are also very good sources of proteins and amino acids in human and animal nutrition [2,3].According to Sulieman et al. [4], plants that acquire N by biological fixation (BNF) have a higher sulphur (S) requirement than those which use only soil N. In the physiology of legumes, sulphur has a dual role: it is necessary, as a component of methionine, cystine, and cysteine, for the biosynthesis of proteins and for the biological reduction of molecular nitrogen [5,6].Sulphur can influence BNF by modulating nodule growth and function or by affecting the growth of the host plant [7].Deficiency of sulphur reduced BNF in pulse plants by decreasing ferredoxin and leghemoglobin concentrations and ATP supply [6].According to Tabatabai [8], many crops around the world now require fertilization with sulphur.e main factors responsible for the greater need for sulphur application are increased use of high-analysis fertilizers containing little or no sulphur, increased crop yields, decreased use of sulphur as a pesticide, and decreased acquisition of atmospheric sulphur by soils and plants.In various countries, including Poland, some symptoms of sulphur deficiency in plant production have been recently observed [9].e most sulphur-deficient soils occur in North America, Australia, New Zealand, Africa, and Canada, and also more and more soils in sulphur-poor soil meet in Europe [10].e deficiencies of this component in the arable soils may decrease the yield and quality of seeds of legumes, by increasing the content of protein, macroelements, and sulphur amino acids [3,5,11].
e nitrogen-fixing process depends on numerous factors, associated with the plant, the bacteria, their symbiosis, and the environment.
e limited availability of certain nutrients is the most important factor limiting the efficiency of BNF [12].Molybdenum (Mo) is an essential micronutrient for BNF, and its deficiency in soil can decrease this process.Molybdenum deficiency may result from continuous cropping, soil erosion, a reduction in soil organic matter, and acidification. is element stimulates BNF by nodules, living in symbiosis with them.e primary role of molybdenum is to enhance flavoprotein enzymes associated with nitrogen metabolism and to participate in the enzymatic activation of molecular hydrogen, resulting in a reduction in nitrogen.Molybdenum plays a role as a cofactor of the proteins responsible for electron transfer in the synthesis of the nitrogenase complex, which is responsible for the conversion of N 2 to ammonium [13].
Common bean (Phaseolus vulgaris L.) ranks third among all the major food legumes worldwide, after soybeans and peanut, and comprises 50% of the grain legumes consumed worldwide.It plays a significant role in human nutrition, as an economically viable source of protein, dietary fibre, and minerals [14,15].e aim of the study was to assess the impact of S and Mo fertilization on the yield and composition of common bean seeds.

Materials and Methods
Field experiments were conducted in years 2012, 2013, and 2014 in private farm located in powiat Zamość (50 °73′N, 23 °65′E), in southeastern Poland.In each year of research, the field experiment was assumed as one-factor in the arrangement of completely randomized blocks, in 4 replications.e experiment was located on brown soil of the group Cambisols [16] with neutral pH (7.0 in KCl), average organic matter content (20 g•kg −1 according to Tiurin), high available of P and K content, average Mg content, and low S content.e subject of the study was the cultivar Aura of common bean (Phaseolus vulgaris L.).
e scheme of the study included the following treatments: O-control (without sulphur and molybdenum), Momolybdenum (100 g•ha Before common bean sowing, N at 30 kg•ha −1 (ammonium nitrate), P at 31 kg•P•ha −1 (as granular triple superphosphate), and K at 82 kg•ha −1 (as potassium nitrate) were applied.e size of the plots was 15 m 2 , spacing between rows was 45 cm, and density was 220.000 plants per hectare.
e occurring weeds were controlled by chemical methods compliant with the recommendations of the Institute of Plant Protection in Poznań [17].Herbicide Afalon Dyspersyjny 450 SC 1.5 dm 3 •ha −1 (linuron 675 g•ha −1 ) was applied after sowing, and Basagran 480 SL 1.25 dm 3 •ha −1 (bentazon 600 g•ha −1 ) and Targa Super 05 EC 1 dm 3 •ha −1 (chizalofop-P-etylu 50 g•ha −1 ) were applied after emergence, when the first pair of trifoliate leaves had unfolded in beans plants ( .Beans were grown on-site, where the previous crop was spring barley.In the subsequent years of the study, beans were sown on 29th and 30th April and on 2nd May.Beans were grown for dry seeds and were harvested in the first week of September.After harvesting, seed yields were determined. In dry mass (d.m.) of seeds, the following macronutrients were determined: nitrogen by Kjeldahl's methods, total sulphur by the Bradley-Lancaster nephelometric method (after wet mineralization with concentrated sulfuric acid with 30% perhydrol), phosphorus by spectrophotometry, potassium and calcium by flame photometry, and magnesium by flame atomic absorption spectrometry (FAAS).e analyses of macronutrients were performed at the Regional Chemical and Agricultural Station in Lublin.Content of S-containing protein amino acids (cysteine and methionine) was determined by the HPLC method in an INGOS AAA 400 amino acid analyser.e analyses of amino acids were performed at the Central Laboratory of Agroecology, University of Life Sciences in Lublin.Total protein content in the seeds was calculated as 6.26 × N tot content.e results were analyzed statistically by analysis of variance using Statistica 13 PL (StatSoft Poland).Analysis of variance (one-way) was carried out for each year of experiment separately.Twofactor analysis of variance (treatment and years) was also performed.
ere was no significant interaction between years of research and treatments.Differences between the means were assessed by protected Fischer's test LSD values calculated for P < 0.05.

Results and Discussion
e weather in different growing seasons and the sulphur and molybdenum application had a significant effect on the seed yield of common bean and on its protein content (Tables 1 and 2).
e highest yield, but with the lowest content of protein, was achieved in 2013, which was characterized by high and uniformly distributed precipitation (Figure 1).
In this growing period, the seeds had the highest content of methionine and cysteine (Table 3).e lowest yield of common bean seeds was recorded in the 2014 growing 2 International Journal of Agronomy    season, which had excessive rainfall in May and August. is may have adversely affected initial growth and impeded the maturation of common bean [18].e highest content of protein, sulphur, phosphorus, potassium, magnesium, and calcium was observed in the year with the lowest sum of rainfall in the growing season.is result is supported by other authors, who reported that water stress increased content of prolamin in faba bean seeds [19] or content of protein in red been seeds [20].e protein content in legumes depends not only on the agronomic practices and genetic properties of the variety but also on the climatic conditions prevailing during the vegetation period.e accumulation of protein in legumes is favoured by a higher average daily temperature and a light rainfall shortage [21].
rough its role in fixation of N by legumes, molybdenum not only has a significant impact on the yield of the crop but can also increase the content of protein, chlorophyll, and vitamins.Togay et al. [22] reported that molybdenum application at a rate of 6 g•kg −1 seeds significantly increased the yield of lentil seed, by 31%, and protein content, by 8.4%.In our study, application of molybdenum increased seed yield by 8.0%, nitrogen and protein content by 5.2%, and protein yield by 13.8%, on average for the experiment (Tables 1, 2, and 4).
According to Campo et al. [12], foliar application of molybdenum increased soybean seed yield by 7-16% and protein content by 5-6%.In addition, in the cited work, grain yield gains obtained with molybdenum complementation depended on the molybdenum content in the seeds used for sowing and were more pronounced for Mo-poor seeds than for Mo-rich seeds.
Sulphur fertilization, regardless of the method of application, was more effective than fertilization of molybdenum.Application of 50 kg•ha −1 of sulphur had a positive effect on seed yield (14.5% increase), nitrogen, and protein content.Differences between the method of sulphur application were small and statistically insignificant.e highest yield of bean seeds, protein, and nitrogen content in seeds was found after the use of molybdenum and sulphur, and the way sulphur was brought had no significant effect.e greatest increases in sulphur content, as compared to the control treatment, were identified as a result of the application of molybdenum and sulphur used in the foliar applications (Tables 1, 2, and 4).Numerous studies confirm the beneficial effect of sulphur on the yield of leguminous plants, i.e., for narrow-leaf lupin [5], broad bean, [7] common bean [3].Similarly, the positive effect of sulphur fertilization of legumes on nitrogen content and uptake is also confirmed by other authors [23,24].However, this is not the rule because according to Barłóg et al. [25], fertilization with sulphur did not have a significant impact on the horse bean yield and did not increase the sulphur content in the seeds.According to various authors, high yield effectiveness of sulphur fertilization can be achieved on soils characterized by a deficit of this element [5,26,27].Sulphur fertilization has a positive effect on the binding of atmospheric nitrogen by root nodules of plants from the Fabaceae family and better utilization of mineral nitrogen, and thus higher production of protein and plant biomass.Shock et al. [28] observed that following correction of soil S-deficiency, the yield of subclover increased threefold and the amount of fixed N increased more than 12-fold.us, dry matter and BNF can be adversely affected by severe nutrient deficiencies [29].An analysis conducted by Divito and Sadras [30] revealed that S starvation may directly impair nodule productivity.However, positive or neutral effects are also possible, depending on the combination of plant species, the severity of the nutrient stress, and the ratio between N demand and nodule mass.Barczak et al. [23] reported that the uptake of nitrogen in narrow-leaf lupin was significantly higher as a result of foliar application of sulphur, as compared with the application into soil.In our study, the method of sulphur application did not affect nitrogen uptake by the bean.But the largest nitrogen uptake was found in combinations where molybdenum and sulphur were used (Figure 2).
Plants also require sulphur for structurally and physiologically important disulphide linkages and sulfhydryl groups, as well as for activation of certain enzymes.Beans require one part of sulphur for every 12-17 parts of nitrogen to ensure maximum production [8].In our study, the N : S ratio was decreased by molybdenum or sulphur application.
e narrowest proportion of these nutrients was found in the conditions of the use of sulphur foliar application (SFA) or molybdenum and sulphur foliar application (Mo + SFA).Been seeds as the most other plant foodstaff are characterized by a S : N ratio situated between 1 : 20 and 1 : 35, meaning well below human tissue requirements (1 : 14.5) [31].
Grain legumes are considered a major source of dietary proteins, but the amino acid profiles of proteins in leguminous seeds are unbalanced.Essential sulphur-containing amino acids, i.e., methionine and cysteine, are considered the most critical limiting components of legume proteins [2,11].e most plants used for human consumption reveal limiting sulphur amino acids content in relation to human requirements.e notable exception is soy products because of its unusual N and sulphur amino acids content.Soy adequately fulfills human tissue requirements and does not necessitate supplemental methionine to reach the biological value of animal foodstuffs [31].Sulphur plays an important role in the synthesis of amino acids, proteins, vitamins, and coenzymes [6].Recent studies indicate that lack of sulphur to meet plant requirements may reduce not only yields but also the quality of grain legumes (reduction of cysteine and methionine) by changing the gene expression of storage proteins in developing seeds [3,8].Molybdenum is also important for sulphur metabolism, so it can have a positive impact on the biological value of protein [32].In our study, the effect of molybdenum application on the content of sulphur-containing amino acids was inconclusive.Content of methionine was increased significantly, while cysteine content was decreased in relation to the control.Application of sulphur (before sowing or as foliar application) significantly increased the methionine and cysteine content in comparison with the control and with molybdenum application.Most preferably, the content of the analyzed sulphur amino acids was influenced by the fertilization with both components (Table 3).
is result is supported by International Journal of Agronomy Klikocka et al. [26], who reported that sulphur significantly increased the content of cysteine (by 6.0%) and methionine (by 16.5%) in spring wheat seeds.In the study conducted by Pandurangan et al. [3], the changes in the concentration of the sulphur amino acids after sulphur application were different between genotypes of common bean.e cited authors stated that adequate sulphur nutrition is required to maximize the concentration of sulphur amino acids and therefore protein quality in genotypes lacks phaseolin and major lectins.erefore, as deposition of sulphur due to atmospheric pollution decreases, sulfate fertilization might become necessary for common bean production [3], as well as for other legumes [33].
Common bean is an important source of minerals for humans.It provides 25% of the daily requirement for magnesium and 15% of potassium [34].Analysis of variance confirmed a significant effect of the fertilization tested on nutrient accumulation in bean seeds.Molybdenum application significantly increased P, Mg, and Ca content and uptake in the seeds of common bean (Tables 5 and 6; Figures 3 and 4).Togay et al. [22] also reported that application of Mo increased the content of K and P in lentil seeds.ese results are in agreement with glasshouse and field studies conducted by Makoi et al. [15], where it was shown that there were significant differences in the uptake of P, K, Ca, and Mg by common bean after molybdenum application.According to the cited authors, greater plant nutrient requirement during the N 2 fixation by legumes such as P. vulgaris has similarly necessitated greater uptake of such macroelements from the rhizosphere to the plant.Application of sulphur (SBS or SFA) significantly increased Ca content as compared to the control (O) and to molybdenum (Mo) fertilization.Barczak et al. [23] reported that foliar sulphur application, as compared with its application into soil, showed a significantly better effect on the potassium content in lupin seeds.In our study, sulphur fertilization did not affect the potassium content, but the foliar sulphur application significantly increased the phosphorus content in common bean seeds in comparison with control.But effectiveness of sulphur was smaller than molybdenum.Application of molybdenum and sulphur (Mo + SBS or Mo + SFA) significantly increased P and Mg content and uptake in common bean seeds.Klikocka and Głowacka [35] reported that sulphur fertilization increased calcium and magnesium content in potato tubers.Similarly, Brodowska and Kaczor [36] demonstrated that fertilizing with sulphur, irrespective of its form, clearly increased the uptake of magnesium and calcium by the plants.But Barczak et al. [23] observed that the narrow-leafed lupin fertilised with sulphur showed a significantly lower content of calcium and magnesium than the control.
According to Baudoin and Maquet [11], the correlation between seed yield of beans and protein content is small but positive, and the correlation between seed yield and specific amino acid contents is significant.Moreover, the negative correlation between total protein concentration and sulphur-containing amino acid content is largely due to the differential accumulation of storage proteins, with different proportions of amino acids.Our experiment also found significant positive correlations between seed yield and the   content of methionine and cysteine.No correlation was found between total protein content and yield as well as between protein content and cysteine and methionine content in bean seeds (Table 7).

Conclusion
e results of the study indicated that molybdenum and sulphur application increased the yield of common bean    6 International Journal of Agronomy seeds and the content and yield of total protein, as well as nitrogen and sulphur.Moreover, sulphur improved the biological value of protein by increasing the content of methionine, cysteine, and some macronutrients.e most beneficial effects were obtained when both molybdenum and sulphur were used in fertilization.In the case of nitrogen, sulphur, methionine, cysteine, and some macroelements, the foliar application of sulphur was more favourable than its soil application before common bean sowing.Considering the yield-producing effect and the impact on the biological quality of the protein, sulphur fertilization should be included in the crop management practices for common bean.

Figure 1 :
Figure 1: Rainfall and air temperature in months 4-9 as compared to the long-term means (1971-2010), according to the Meteorological Station in Zamość.

Table 2 :
e influence of Mo and S application on the yield of total protein and N : S ratio.
Values with different letters differ significantly (P � 0.05); small letters in the column: treatments; capital letters in line: years.

Table 3 :
e influence of Mo and S application on the methionine and cysteine content.
Values with different letters differ significantly (P � 0.05); small letters in the column: treatments; capital letters in line: years.

Table 1 :
e influence of Mo and S application on the seeds yield and total protein content.

Table 4 :
e influence of Mo and S application on the nitrogen and sulphur content.

Table 5 :
e influence of Mo and S application on the phosphorus and potassium content.

Table 6 :
e influence of Mo and S application on the calcium and magnesium content.
Values with different letters differ significantly (P � 0.05); small letters in the column: treatments; capital letters in line: years; d.m., dry matter.