Molybdenum supply and biological fixation of nitrogen by two Brazilian common bean cultivars

O feijao apresenta baixa capacidade de fixacao biologica de nitrogenio, o qual pode ser mais eficiente com o fornecimento de molibdenio (Mo). O objetivo deste estudo foi avaliar doses de Mo no crescimento e na fixacao biologica de nitrogenio em duas cultivares de feijoeiro usando a tecnica de diluicao isotopica. O experimento foi realizado em 2014 em delineamento inteiramente casualizado e em esquema fatorial 5 x 3, correspondendo a cinco doses de Mo (controle, 40, 80, 120 e 240 g ha-1), cultivares de feijoeiro Apore, Ouro Negro e NORH-54, cultivar de feijao nao nodulante e tres repeticoes. A aplicacao de Mo e a inoculacao das sementes com uma estirpe de rizobio contribuiram para melhorar a fixacao de nitrogenio e o peso de graos. A cultivar Ouro Negro apresentou maior numero e peso de nodulos do que a cultivar Apore e maiores valores de nitrogenio proveniente da atmosfera. A fixacao biologica do nitrogenio foi mais dependente da aplicacao de Mo na cultivar Apore. Esses resultados indicaram que a inoculacao com rizobio e o fornecimento de Mo contribuiram com a fixacao biologica de nitrogenio e producao de graos.


Introduction
The common bean (Phaseolus vulgaris L.) is a staple food in many Latin American and African countries, regarded as a primary source of daily proteins and minerals.The carioca and black common bean group are the two most consumed types in Brazil (Vogt et al., 2013), cultivated mainly by small holder farmers.
It is well established that leguminous plants in association with Rhizobium sp. can use atmospheric N 2 (Marschner, 2012;Boyd & Peters, 2013).However, the common bean has been considered to have low biological N 2 fixation (BNF) capacity compared to other legumes, such as soybean (Piha & Munns, 1987).
Although the common bean can form symbiosis with a wide range of indigenous rhizobia, these rhizobia have been considered low efficiency for BNF, in addition to having a short vegetative fixation period (Piha & Munns, 1987).However, inoculating the common bean with selected rhizobia strains could increase the N 2 fixation, as expressed by higher N content in plant tissues, demonstrated by Hungria et al. (2003) and Kimura et al. (2004).
In addition to inoculation with selected strains, the BNF in the common bean can be made more effective by molybdenum (Mo) supplementation.Mo plays an essential role in many biochemical processes in plants and is a constituent of nitrogenase (Fageria et al., 2011;Marschner, 2012), the enzyme that catalyzes the reduction of atmospheric nitrogen into ammonia, and a cofactor in nitrate reductase.
Due to the role of Mo in the mechanisms associated with the regulation of N 2 fixation in legumes, Mo supplementation has increased N accumulation and grain yield in common beans, as reported by several authors (Fageria et al., 2015;Rocha et al., 2011;Araújo et al., 2009).However, there are differences among the genotypes of common beans with regard to Mo responses, mainly due to seed size and Mo concentration (Vieira et al., 2014).
Considering that much more research is required to improve yield and N 2 fixation in legumes, mainly in the common bean (Bambara & Ndakidemi, 2010), the objective of this study was to evaluate the influence of Mo rates on the growth and amount of N fixed from the atmosphere in two Brazilian common bean cultivars, using the 15 N isotope dilution technique.

Material and Methods
The experiment was conducted in the year 2014, in a greenhouse at the Center of Nuclear Energy in Agriculture (CENA), in Piracicaba (Sao Paulo state, Brazil), using a completely randomized design in a factorial scheme (5 x 3) with three replications.
The treatments comprised four Mo doses (equivalent to 40, 80, 120 and 240 g ha -1 ), an untreated control (no Mo addition) and three cultivars of common bean: Aporé, which belongs to the carioca group; Ouro Negro, which belongs to the black group; and one non-nodulating common bean cultivar, NORH-54.This cultivar was used as non-fixing standard because it has the same root pattern, N absorption of sources available in the soil and similar maturity cycle.
The non-nodulating cultivar was used as a non-fixing reference plant to calculate the percentage of N derived from the atmosphere (%Ndfa) in the nodulated cultivars.Both cultivars Aporé (carioca group) and Ouro Negro (black group) have an indeterminate growth habit, and the total crop cycle from sowing to maturity is 80 -100 days.
The lime was thoroughly mixed with the soil (calcium carbonate equivalent = 100%) to raise the base saturation to 60%, according to the official recommendation of São Paulo State (Raij et al., 1997).The soil was watered to 70% of the maximum water holding capacity with distilled water and incubated for 30 days.
Phosphorus and potassium fertilization was accomplished by applying 300 mg kg -1 of superphosphate triple and potassium chloride.Fertilization with micronutrients was performed by applying a water solution containing 0.5 mg kg -1 of boron (B) obtained from Na 2 B 4 O 7 .10H 2 O, 1.5 mg kg -1 of Cu from CuSO 4 .5H 2 O, and 3.0 mg kg -1 of Zn as ZnSO 4 .7H 2 O.
The seeds received a liquid inoculant containing the strain SEMIA 4080 of Rhizobium on the day of sowing.The seeds were not treated with insecticide or fungicide.Three seeds were sown in each pot and thinned to one plant five days after emergence.The 15 N was applied as ammonium sulfate with 2.46 atm % 15 N in excess to each pot, corresponding to 125 mg of N per pot.
The dose of Mo used in the experiment was calculated according to the common bean plant density.For the cultivars Aporé and Ouro Negro, the recommendation is 240,000 plants per hectare.The rates evaluated were equivalent to 0, 40, 80, 120 and 240 g Mo for 240,000 plants per hectare, corresponding to the following rates: 0, 0.17, 0.33, 0.67 and 1.33 mg of Mo per pot (one plant pot -1 ).
The Mo was applied 25 days after plant emergence, at the stage of the fourth fully developed trifoliate, in the form of sodium molybdate (Na 2 MoO 4 .2H 2 O) at the soil surface.The irrigation was performed daily, manually, using distilled water, in the amount required to maintain substrate moisture near 70% of field capacity.
The plants were harvested at the stage of seed physiological maturity.The grains were separated from the shoot, and both parts were oven dried, weighed and finely ground using a roll-mill.The root systems were recovered by soil washing through a 2 mm aperture sieve, and the nodules were counted and weighted.
The 15 N isotope composition was measured in the shoots and grains by coupling gas chromatography-mass spectrometry (GC-MS).The percentage of N derived from the atmosphere (%Ndfa) was estimated by Eq. 1: These findings suggest that inoculation with the selected Rhizobium strain is an effective way to increase nodules on these cultivars of common bean and improve the N supply from biological fixation, which could be an alternative for smallholders farmers who cannot afford to purchase N fertilizers.However, it is important to emphasize that these results were obtained for only two Brazilian cultivars, Aporé and Ouro Negro, with all biotic and abiotic factors controlled.Therefore, it is necessary to validate these results with experiments in field conditions and in other cultivars.
These results corroborate with other reports.Pacheco et al. (2012) observed that seed inoculation with rhizobia markedly increased the nodulation in common beans.Hungria et al. (2003), in six field experiments performed in Oxisols in Southern Brazil, observed that a selection program of rhizobia strains was able to increase nodulation, BNF and the yield of common bean.Melo & Zilli (2009) evaluated the BNF in five cowpea cultivars, observed increase in grain yield as a consequence of the inoculation.
The Ndfa in the shoots increased progressively with the applied Mo doses (Figure 1C, 1D).The Ndfa in the Aporé shoots ranged from 4.04 mg plant -1 (control) to 58.34 mg plant -1 (240 g ha -1 of Mo), corresponding to 5.6 and 52.83% of the total N content in the plant, respectively.For the cultivar Ouro Negro, the Ndfa ranged from 37.53 to 103.32 mg plant -1 , corresponding to 35.34 and 66.2% of the total N.
The Ndfa values were greater in the grains than in the shoots.For Aporé, the values of Ndfa in the grains ranged from 11.06 mg plant -1 (control) to 336.57mg plant -1 (240 g ha -1 of Mo), corresponding to 13.48 and 78.27% of the total N, while for Ouro Negro, the values ranged from 119.3 to 389.36 mg plant -1 , corresponding to 62.16 to 83.15% of the total N (Figure 2C, 2D).The non-nodulating cultivar NdfA values were 1.23 and 1.21% in the shoot and in the grain, respectively.
These BNF results for the shoots and grains of the inoculated cultivars demonstrated the importance of Mo application to increasing and maintaining nitrogenase and nitrate reductase activities throughout the plant growth cycle (Vieira, 1998b).Mo is a constituent of nitrogenase, a key enzyme in all N 2 fixing microorganisms; consequently, nitrogen-fixing plants have a high Mo requirement, particularly in the root nodules (Hille, 1999;Marschner, 2012).Araújo et al. (2009) observed an increase in grain yield with Mo supplementation up to a rate of 80 g ha -1 of Mo.Fageria et al. (2015) also found that Mo application significantly increased the yield and yield components of common bean grown on a Brazilian Oxisol.Rocha et al. (2011) observed that Mo fertilization increased the total N in the leaves and the grain yield for common beans cultivated under both conventional and no-tillage systems.
Analysis of variance was performed.When the significance was verified by the F test, the Dunnet test (p < 0.05) was used to compare the means of all doses of Mo (0, 40, 80, 120, 240 g ha -1 ) of inoculated cultivars with the non-nodulating cultivar.Linear and exponential regressions were used to evaluate the effect of the Mo doses.The values of R 2 followed by the symbol (*) were significant at the 0.05 probability level.

Results and Discussion
The Rhizobium inoculation had significant effects on the grain dry weight (GDW) and all the other measured parameters, which were significantly higher than in the nonnodulating cultivar, considering the means of all doses of Mo (Table 1).
The cultivars Aporé and Ouro Negro presented hundreds of nodules, while the non-nodulating cultivar formed an insignificant number (the number and size of nodules were very low, assumed to be zero) (Table 1), indicating that inoculation with the selected Rhizobium strain was effective for producing nodules in the common bean, contributing to N supply and helpful in improving grain production.
The common bean is considered a promiscuous host because there are many rhizobia species capable of producing nodules in the common bean, with a variety of bean-rhizobia interactions (Moreira & Siqueira, 2006).
Nevertheless, some studies indicate that indigenous rhizobia strains are not efficient in common bean BNF, and these strains could hinder the introduction of more efficient strains (Vieira, 1998a), which is usually attributed to the lack of competitiveness against indigenous rhizobia, environmental conditions and plant genetic factors (Piha & Munns, 1987;Hardarson et al., 1993).
Means of all doses equivalent to 0, 40, 80, 120, 240 g ha -1 of Mo shoot dry weight (SDW); total nitrogen in the shoot or grain (N total); N derived from atmosphere (Ndfa) and grain dry weight (GDW) measured in non-nodulating and nodulated cultivars of common beans, Aporé and Ouro Negro maintain the reduction of atmospheric N 2 by the nitrogenase system (Hille, 1999;Marschner, 2012).Vieira et al. (2010), studying the toxicity of high Mo rates sprayed on foliage, verified that doses as high as 4,000 g ha -1 were not toxic to the common bean.Leite et al. (2007) evaluated foliar Mo application and verified that doses up to 2,560 g ha -1 did not decrease the grain yield.Johansen et al. (2007) showed that 500 g ha -1 Mo improved plant growth and resulted in chickpea seed yield responses ranging from 58 to 173%.

Conclusions
1.The inoculation of the common bean with a recommended rhizobium strain in combination with the application of Mo contributed to improving biological nitrogen fixation and grain dry weight in two common bean cultivars.
2. The Ouro Negro cultivar obtained a higher amount of N derived from the atmosphere than Aporé, even with no Mo supply.
excess (shoot or grain) of non-fixing

Table 1 .
. Means followed by * are significantly different by the Dunnet test (p < 0.05) Nodule number (n° nod) and nodule weight (NW);