response of solar radiation bioconversion on medicago sativa l. silage

RESPONSE OF SOLAR RADIATION BIOCONVERSION ON MEDICAGO SATIVA L. SILAGE POTENTIAL D. DUNEA1, N. DINCA2*, C. RADULESCU3,4*, C. MIHAESCU5, I.D. DULAMA4, S. TEODORESCU4 1

Valahia University of Targoviste, Faculty of Environmental Engineering and Food Sciences, 13 Sinaia Alley, 130004 Targoviste, Romania, E-mail: [email protected]; 2 University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59 Marasti Blvd, District 1, Bucharest, Romania, E-mail: [email protected]; 3 Valahia University of Targoviste, Faculty of Sciences and Arts, 13 Sinaia Alley, 130004 Targoviste, Romania, E-mail: [email protected]; 4 Valahia University of Targoviste, Institute of Multidisciplinary Research for Science and Technology, 13 Sinaia Alley, 130004 Targoviste, Romania, E-mail: [email protected], [email protected]; 5 *

University of Pitesti, 1 Targu din Vale Street, Pitesti, Romania.

Corresponding authors: [email protected]; [email protected]. Received October 31, 2017

Abstract. Medicago sativa L is an important perennial plant species, especially in temperate regions, having large requirements for light, heat and water. Dry matter accumulation (DM) and forage qualitative parameters are directly correlated to the amount of photosynthetically active radiation (PAR) intercepted by the canopy. The objective of the study was to assess the solar radiation bioconversion to DM and silage quality in Medicago sativa L during three years of cropping in Targoviste Piedmont Plain, Romania. The experiments were carried out on pseudogleic brown alluvial soil using two Romanian synthetic cultivars (i.e., traditional species Roxana and Mihaela). The cultivars were sown in a Latin rectangle design with four replicates. For ensiling, wilted Medicago sativa and ¼ green maize leaves were chopped at 2-3 cm, mixed together and compacted in 2-L containers with gas release valve for 35 days. Forage chemical composition of silage was determined using Attenuated Total Reflection - Fourier Transform Infrared Spectrometry (ATRFTIR). Organic acids were determined by gas chromatography (GC). Relative feed value (RFV) was computed. The multiannual average of Radiation Use Efficiency (RUE) ranged between 1.3 and 1.4 g MJ-1 m-2 in tested cultivars. It was found that increasing of RUE determines the decreasing of CP content (% DM) of the silage (Pearson r = -0.985; p < 0.001). More efficient bioconversion processes may occur with advancing in maturity and crop aging that increase the DM content, but the quality of DM may decrease pointing out that stand management is a key factor to insure optimal nutritional value of the resulted fodder. The RFV average of Medicago sativa silages mixed with ¼ maize leaves ranged between 137.3% (year 2014) and 141.9% (year 2012) confirming the relative reduction of forage quality with crop aging. Based on RFVs, both cultivars showed good quality of obtained silages that were superior to the value reported for corn silage-well eared (133%). This type of experiments may provide key milestones for Medicago sativa stand management for maintaining the nutritional quality in the context of climate change.

Romanian Journal of Physics XX, XYZ (2017)

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Key words: biometeorology, radiation use efficiency, Medicago sativa, crude protein, relative feed value, alfalfa.

1. INTRODUCTION

Medicago sativa L. is widely considered “the queen of forage crops” because of its ecological plasticity, fodder quality, resistance to drought, important break crop in the rotation, and symbiotic fixation of nitrogen in soil. Medicago sativa is estimated to fix 83–594 kg N ha-1yr-1 [1]. Crop yield varies with climate and length of total growing period. Good yields after the first year are in the range of 2 to 2.5 tons/ha per cut (hay with 10-15% moisture) of about 25 to 30 day cutting interval [2]. It has valuable fodder properties due to its high content of proteins, vitamins and minerals. Traditionally, Medicago sativa is preserved as hay, but the process of obtaining the hay is influenced by the weather conditions and also delays the regrowth period if the harvested material is not removed from the field. Wilting is usually performed after harvesting to increase DM content and decrease the growth of clostridial bacteria [3]. The weather pattern for a growing season, especially temperature-induced lignification of the neutral detergent fiber (NDF) and leaf loss from damaging rains can dramatically change the NDF level, crude protein content or digestibility of the forage even though the fiber content can remain relatively stable [4]. Furthermore, the hay production is characterized by significant loses e.g., 21% from dry matter (DM), 28% from crude protein (CP) and up to 40% from the weight of leaves [5]. Such loses also affects significantly the nutritional quality. Silage is an alternative method to retain the forage quality. The assessment of silage quality is typically based on determining the fermentation qualities and changes in microbial compositions. The most common indicators include the silage DM weight and content, water-soluble carbohydrate concentration, and target bacterial counts [6]. In the mass of ensiled fodder, there is mainly lactic fermentation, but also secondary fermentations: acetic, butyric and alcoholic may occur. The predominance of one or the other depends on several factors, of which a decisive role is the presence or absence of oxygen, the reaction of the environment and the content of the silage plants in soluble carbohydrates. Lactic fermentation has the main importance because it leads to the accumulation of lactic acid, a preservative, on which depends the quality of the silage. Lactic fermentation is produced by lactic bacteria (e.g., Streptococcus, Leuconostoc, Lactobacillus, Pediococcus), which convert carbohydrates to lactic acid and small amounts of acetic, succinic, formic and propionic acid. Lactic bacteria support a stronger acidic environment than butyric bacteria, i.e., pH less than 4.5. At these pH values, butyric fermentation bacteria as well as

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other bacteria leading to unwanted secondary fermentations cease their activity. The mold develops to the reaction of the medium corresponding to a pH between 1.2 and 1.8, but does not support anaerobiosis [7]. An important role has the content of soluble carbohydrates. Thus, perennial grasses, harvested in the optimum stage for silage, have soluble carbohydrate content up to 20%, depending on the species and cropping technology, while in legumes, the proportion of soluble carbohydrates is only 9-10% of the DM [8], which is insufficient to achieve the required amount of lactic acid. Medicago sativa can be preserved in silage with some difficulties due to its low content of fermentable sugars requiring extra carbohydrates and lactic acid bacteria (LAB)-containing additives for proper fermentation [9]. The fermentation of Medicago sativa silage with LAB additives results in a decrease in pH due to the production of organic acids during the process [6]. Growth of Clostridia increases proteolysis and butyric acid in legume silages [10, 11]. Butyric acid is considered to be responsible for reducing silage palatability. In Romania, the traditional method uses the direct harvest and silage of harvested material after a preliminary chopping and mixing with green maize [12], Sudan grass, straws and preservatives [7]. In these conditions, the fermentation process runs slow because of the low content of soluble sugars (7-8%). To support the fermentation process, the lowering of the humidity is required up to 55-65% for the harvested material, followed by its chopping and depositing in silages [5]. Later on, the stored material must be compacted to obtain the anaerobic conditions that favor the multiplication of lactic ferments. Hence, the fodder conservation is realized in short time and loses (especially the percentage of leaves) are significantly diminished. The butyric fermentation may occur at > 65% humidity, while a humidity < 55% favors aerobic fermentation because the compaction is hindered. DM accumulation and allocation in morphological organs as well as the qualitative parameters of forage are directly correlated to the amount of intercepted photosynthetically active radiation (PARi) by the canopy of species [13]. Radiation use efficiency (RUE) is a key indicator of biological efficiency of a species regarding the conversion of light in DM (εb) [14]. In Medicago sativa, Justes et al. [15] found a RUE of 1.72 g DM MJ−1 irrespective of sowing date (spring or summer), and Allirand [16] 1.76 g DM MJ−1, respectively. Brown et al. [17] showed that estimated radiation use efficiency (RUE) in Medicago sativa had a distinct seasonal pattern, increasing from 0.80 g DM MJ-1 in early spring to 1.60 g DM MJ-1 in late summer before decreasing to 0.80 g DM MJ-1 in late autumn. In dry and full sunlight conditions, Varella [18] observed mean RUEs over the experimental period of 1.06 g MJ-1. Stanciu et al. [19] found RUEs of 1.82 g DM MJ-1 m-2 for orchard grass in mixture with Medicago sativa. Generally, mixtures of perennial grasses with Medicago sativa provided increased RUEs during growth

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seasons due to a better interception of solar radiation in the heterogeneous canopy [20]. Consequently, a higher quantity of light and a better cropping management including proper hay and silage production are expected to increase RUEs and improve forage quality. Solar cycle, nebulosity and climate change are independent factors that are determining PAR availability and thus, Medicago sativa growth. In this context, the objective of the study was to assess the solar radiation bioconversion to DM accumulation and silage quality in Medicago sativa during three years of cropping in Targoviste Piedmont Plain, Romania. Farmers may use this type of information to maximize the maintenance of the nutritional quality for obtained fodders in the context of climate change.

2. MATERIALS AND METHODS

The experiments were carried out in Targoviste Piedmont Plain, Romania (N44°46¹.905, E25°43¹.045, 179-m altitude) between 2012 and 2014 on pseudogleic brown alluvial soil using two Romanian synthetic cultivars of Medicago sativa (i.e., Roxana and Mihaela species) developed at NARDI Fundulea [21]. The cultivars were sown in a Latin rectangle design with four replicates. The plants were given nitrogen fertilizer in all experimental variants at one rate (25 kg N ha-1) to support crop establishment and avoid nutrient limiting growth. Irrigation was not applied to comply with the common cropping practices used by farmers in the region. Three cutting cycles were performed each year according to the recommended phenophases for Medicago sativa harvesting [5] i.e., first cutting cycle: at the beginning of flowering stage; second: +7 weeks from the first cut; third cutting: +6 weeks after second cutting. Samples were collected before each cutting cycle using a quadrate of 50×50 cm in two points of each variant and each repetition to determine DM accumulation (g·m-2). The continuous energetic fluxes of solar radiation at the location were determined using a PAR Quantum Sensor (range = 0 2000 µmol m2 -1 s ) connected to a data logger. Meteorological parameters at the experimental field were acquired using a Delta-T Devices automatic weather station (Table 1). For ensiling, wilted Medicago sativa from both cultivars (50-60% moisture) and ¼ green maize leaves were chopped at 2-3 cm, mixed together and compacted in 2-L containers with gas release valve for 35 days. The silage samples (2 for each replicate; n = 16) were made only in the first cropping cycle of each year. The containers were opened on 36 day for determination of pH, DM, CP, and total acids content. The silage pH was determined using a WTW pH-meter. Molecular identification of chemical functional groups of organic/inorganic compounds (i.e. forage chemical composition CP, Acid Detergent Fiber - ADF,

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Neutral Detergent Fiber - NDF, and mineral content) was performed by Attenuated Total Reflection - Fourier Transform Infrared Spectrometry (ATR-FTIR) using Vertex 80v spectrometer (Bruker), which absorbs infrared radiation in 350-8000 cm-1 range, equipped with diamond attenuated total reflection accessory, as well as with Hyperion IR microscope [22-26]. Table 1. Meteorological parameters recorded during the experiment in Targoviste Piedmont Plain between 2012 and 2014 (annual averages)

Meteorological parameter

2012

2013

2014

Average

Temperature (°C) Relative humidity (mm) Sum of precipitations (mm) Days without precipitations Global radiation (MJ/m2/day) Total global radiation (MJ/m2/yr)

10.9 71.4 612 308 13.9

8.1 78.3 553 235 14.2

10.8 79.8 1039 300 12.9

9.9 76.5 734.7 281 13.7

Coeff. of var.(%) 16 5.9 36.1 14.2 5

5110

5161

4892

5054.3

2.8

ADF is the fibrous component representing the least digestible fiber portion of forage, while NDF is an estimate of the total fiber components (lignin, cellulose, hemicellulose, tannins, cutins and silica). The organic acids (i.e. lactic, acetic and butyric) were determined by gas chromatography (GC) method [27]. Analyses were performed with an Agilent 6890N GC Chromatograph equipped with Flame Ionization Detector (FID) and Autosampler. A J&W DB-624UI GC column, 30mx0.32 mm, 1.8 µm was held at the temperature of 260 ºC. Hydrogen (38 cm/s) was used as carrier gas. Injection volume was 1.0 mL/min, constant flow mode; n-propanol was used as internal standard. Nutritive value of forages depends on their DM digestibility and voluntary DM intake. Relative feed value (RFV), which is a widely accepted forage quality index that combines the estimates for forage digestibility and intake into a single number, was computed. RFV for legumes and legumes mixtures is calculated from estimation of ADF and NDF [28], as follows: RFV = (65.5+0.975 ADF–0.0277 ADF2) ⋅ (39.0+2.68 NDF–0.041 NDF2) ⋅ 0.025 The indicators of fodder quality were correlated with the amount of available PAR during various crop cycles using Pearson correlation. Statistical analysis was carried out using Statgraphics (StatPoint Inc., 2005) [29]. Significance between

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individual means was identified using Least Significant Difference (LSD) multiple comparison test. Mean differences were considered significant at p < 0.05. Regarding the climatic variability during experiment, the year 2013 recorded the lowest annual average temperature (8.1 °C) with more than 2.5 °C lower compared to 2012 and 2014. The year 2012 showed the highest amplitude of temperature regime, while year 2014 was characterized by the significant amount of annual precipitations and increased relative humidity. The highest amount of solar radiation was recorded in 2013, the second year of Medicago sativa cropping and the lowest in 2014, which was a year with increased nebulosity and rainfalls exceeding the multiannual average. Global radiation showed the lowest variance, while the sum of precipitations reached the highest coefficient of variation (36.1%).

3. RESULTS AND DISCUSSION 3.1. SOLAR RADIATION BIOCONVERSION TO DRY MATTER IN MEDICAGO SATIVA

Medicago sativa L. is a perennial crop that produces its highest yields during the second year of growth [2]. However, in this experiment the highest yields were obtained in the third year of cropping mainly due to the higher amounts of precipitations that were optimally distributed during the cropping cycles of year 2014. Although it is resistant to drought, Medicago sativa provides profitable yields only in regions where the annual precipitations sum exceeds 500 mm, which are well distributed during the growth season [9]. Medicago sativa required 750-800 °C for each cutting cycle. Temperatures > 35 °C occurred in the first cycle of year 2013 reducing the yield and corresponding RUE. Table 2 shows the RUEs recorded during the field experiment. Mihaela cultivar showed higher RUEs than Roxana excepting the 3rd cutting in 2012. No statistical difference (p > 0.05) was observed between cultivars. Mihaela cultivar showed an improved bioconversion of solar radiation to DM throughout growth seasons recording RUEs of 1.6-1.77 g MJ-1 m-2 in the 1st cycle, 1.4-1.66 g MJ-1 m-2 in the second cycle, and 0.78-1.02 g MJ-1 m-2 in the third cycle. Roxana cultivar provided similar RUEs i.e., 1.47-1.74 g MJ-1 m-2 in the 1st cycle, 1.27-1.61g MJ-1 m-2 in the second cycle, and 0.79-0.98 g MJ-1 m-2 in the third cycle. The similar responses occurred because the cultivars were obtained from the recombination of foreign and Romanian germplasm [21]. Both cultivars presented closed canopies, rapid spring growth, faster regrowth after cutting, good resistance to common diseases occurring in Romania, and improved winter hardiness. RUE

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had a reduced variance between years in the same cropping cycle, the highest being observed for the 3rd cutting cycle (Coeff. of var. = 13.8%).

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Table 2. -1

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Radiation use efficiency (g MJ m ) of Medicago sativa cultivars grown in Targoviste Piedmont Plain, Romania between 2012 and 2014 (three cutting cycles/year)

Cultivar Roxana 1st cutting 2nd cutting 3rd cutting Annual average Mihaela 1st cutting 2nd cutting 3rd cutting Annual average LSD 95% diff.

2012

2013

2014

Average

Coeff. of var.(%)

1.51 1.33 0.84 1.23

1.47 1.27 0.79 1.18

1.74 1.61 0.98 1.45

1.6 1.4 0.9 1.3

9.3 12.9 11.3 11.2

1.61 1.47 0.78 1.29 ±0.90

1.60 1.40 0.86 1.29 ±0.83

1.77 1.66 1.02 1.48 ±0.91

1.7 1.5 0.9 1.4 -

5.7 8.9 13.8 8.1 -

The main factor determining the Medicago sativa growth, including the rate of biomass accumulation, is the amount of carbon assimilated through photosynthesis, which in turn is dependent on the amount of light intercepted by the canopy. Once carbon is assimilated though photosynthesis, biomass can be partitioned to aboveground morphological components (leaves and stems) or perennial belowground organs (crowns and roots) [30]. The canopy architecture of Medicago sativa provides efficient light capture because of the leaf area distribution of flat leaves in the lower layers of the canopy and vertical leaves in the top [31]. Medicago sativa detains an optimal light extinction coefficient per unit of leaf area between 0.8 and 0.9. The RUE results of Romanian cultivars were in agreement with data reported for Medicago sativa in [15, 16]. Moot [31] pointed out that in Medicago sativa, RUE for total biomass, as a proxy for net canopy photosynthesis, is approximately 1.8 g MJ-1 (total solar radiation). Under water stress, the RUE of a Medicago sativa-grass mixture was 1.4 g MJ-1 [32]. An increased RUE value does not necessarily imply an improved quality of the forage because by advancing towards maturity the proportion of lower quality stem material (lignification) increases and the overall leaf to stem ratio declines [33]. Leaf/stem ratio is a reliable indicator regarding the fodder quality in Medicago sativa, but was not determined in this study.

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3.2. SILAGE COMPOSITION AND RELATIONSHIP WITH BIOCONVERSION EFFICIENCY

The quality of herbage in Medicago sativa is directly related to the fraction of leaf and palatable stem compared with lower quality lignified stem [31]. Traditional hay production determines high loses of leaves due to shaking, which drops significantly the fodder quality. Furthermore, unfavorable weather conditions during field drying in rows may compromise the forage yield. Proper ensiling may maintain the nutritional quality preserving crude protein, minerals and vitamins. Table 3 shows the silage composition determined for the harvested and ensiled Medicago sativa material mixed with ¼ maize leaves at the first cycle of each year of cropping. The DM content was almost constant between years recording a multiannual average of 30.4%. CP showed a diminishing towards the third year of cropping with 5% DM. A fodder rich in protein can be obtained from an early harvesting but this could reduce the longevity of Medicago sativa [9]. In this experiment, crop aging and later cutting applied in the third year were responsible for the diminishing of CP and increasing of cellulose content in Romanian cultivars. After 35 days of ensiling, total acids ranged between 4.9 and 6.8% DM from 2012 to 2014 first crop cycles showing the highest variance of the tested variables (Coeff. of var. = 17.3%). This increment suggests that the quality of fodder has diminished with the crop aging, increasing the concentration of butyric acid from 0.4 (2012) to 1.5% DM (2014) and acetic acid from 1.3 to 2.6% DM. Acetic acid is associated with undesirable fermentations, while butyric acid favors protein degradation, toxin formation, and significant losses of DM and net energy. Lactic acid bacteria (LAB) utilize water-soluble carbohydrates to produce lactic acid, which is the primary acid responsible for decreasing the pH in silage [34]. The concentration of lactic acid remained almost constant (2.7-3.2% DM). The lowest pH (5.2) was determined in the silage obtained in the third cropping year, while the maximum was in the first year (5.8). The drop in pH was mainly determined by the lactic acid formation during fermentation process. Low pH values are favorable for better preserved and more stable Medicago sativa silages. However, Medicago sativa has a high buffering capacity compared to maize requiring increased acid production to lower the pH in Medicago sativa, which is difficult to obtain. The DM content of the forage can also have major effects on the ensiling process due to a number of various mechanisms [35]. First, drier silages do not pack well making difficult to extract all the air from the forage mass. Second, as DM content increases, growth of LAB is reduced followed by the reduction of the rate and extent of fermentation (slower acidification process and less total acids). In this experiment, addition of green maize leaves has increased the fodder quality, ensiling potential and silage stability. A very significant inverse correlation was observed between RUE and silage CP content of each replicate (Pearson r=-0.985; p