Improving the short-term efficiency of rock phosphate-based fertilizers in pastures by using edaphic biostimulants

Abstract

Background

The use of reactive rock phosphate (RP) in acidic soils as a phosphate (P) source for pastures and crops presents attractive economic advantages with respect to soluble phosphate. However, some studies have demonstrated that the short-term (1-year) efficiency of RP, compared with that of water-soluble P, is relatively poor. This fact penalizes not only the yield and quality of the earlier harvests, but even the whole final yield when the crop is affected by some abiotic or biotic stress at the beginning of the cycle. In the present study, we investigated the ability of new edaphic biostimulants to increase the short-term efficiency of RP-based fertilizer as a P source for pastures cultivated in acid soils. To this end, we have granulated rock phosphate with two edaphic biostimulants: tryptophan and a heteromolecular organic complex formed by humic acid and tryptophan through iron bridges, and compared their short-term P fertilizer efficacy with that of single superphosphate and rock phosphate.

Results

Soil incubation studies showed that the heteromolecular complex humic acid-tryptophan and Tryptophan were able to increase soil CO2 production compared with native soil, rock phosphate, and superphosphate. Likewise, the presence of humic acid-tryptophan complex and Trp significantly increases plant-available phosphate compared with rock phosphate, up to levels similar to those of superphosphate. Plant (ray grass)-soil-pot studies showed that rock phosphate/(humic acid-tryptophan) formulation yielded values for both ray grass dry matter production and shoot P concentration, clearly higher than those of rock phosphate and rock phosphate/tryptophan. In addition, the results associated with rock phosphate/(humic acid-tryptophan) were similar to those for superphosphate, after 3 months of cultivation.

Conclusions

Taken together, these results showed the suitability of the use of specific humic acid-based edaphic biostimulants to improve the short-term effect of rock phosphate fertilizers as a phosphate source for pastures cultivated in acid soils.[Figure not available: see fulltext.]

Fernndez et al. Chem. Biol. Technol. Agric. (2016) 3:5 DOI 10.1186/s40538-016-0057-5

RESEARCH

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Web End = L. Fernndez1,2, R. Baigorri2*, O. Urrutia2, J. Erro2, P. M. AparicioTejo1, J. C. Yvin3 and J. M. GarcaMina2,4*

Abstract

Background: The use of reactive rock phosphate (RP) in acidic soils as a phosphate (P) source for pastures and crops presents attractive economic advantages with respect to soluble phosphate. However, some studies have demon strated that the shortterm (1year) efficiency of RP, compared with that of watersoluble P, is relatively poor. This fact penalizes not only the yield and quality of the earlier harvests, but even the whole nal yield when the crop is aected by some abiotic or biotic stress at the beginning of the cycle. In the present study, we investigated the ability of new edaphic biostimulants to increase the shortterm efficiency of RPbased fertilizer as a P source for pastures cultivated in acid soils. To this end, we have granulated rock phosphate with two edaphic biostimulants: tryptophan and a heteromolecular organic complex formed by humic acid and tryptophan through iron bridges, and compared their shortterm P fertilizer efficacy with that of single superphosphate and rock phosphate.

Results: Soil incubation studies showed that the heteromolecular complex humic acidtryptophan and Tryptophan were able to increase soil CO2 production compared with native soil, rock phosphate, and superphosphate. Likewise, the presence of humic acidtryptophan complex and Trp signicantly increases plantavailable phosphate compared with rock phosphate, up to levels similar to those of superphosphate. Plant (ray grass)soilpot studies showed that rock phosphate/(humic acidtryptophan) formulation yielded values for both ray grass dry matter production and shoot P concentration, clearly higher than those of rock phosphate and rock phosphate/tryptophan. In addition,the results associated with rock phosphate/(humic acidtryptophan) were similar to those for superphosphate, after 3 months of cultivation.

Conclusions: Taken together, these results showed the suitability of the use of specic humic acidbased edaphic biostimulants to improve the shortterm eect of rock phosphate fertilizers as a phosphate source for pastures culti vated in acid soils.

Keywords: Edaphic biostimulants, Rock phosphate, Phosphate fertilizer, Pastures, Plantavailable phosphate soil microbial activity, Humic acid, Humic acidbased heteromolecular complexes

Background

Water-soluble phosphate (P)-based fertilizers, mainly single superphosphate (SSP) and triple superphosphate

(TSP), are the main sources of P used for cultivated pastures, mainly in alkaline soils, but also in acidic soils [1, 2]. However, in pastures (and also other crops) cultivated in acidic soils, the direct application of rock phosphate (RP) (granule or powder)without previous reaction with sulfuric and/or phosphoric acidsmay be a suitable, less-expensive, alternative to water-soluble P fertilizers [1]. In addition, the slow solubilization of RP in acidic soils may also contribute to decrease environmental risks, such as the eutrophication of surface waters [3].

*Correspondence: [email protected]; [email protected]

2 Department of Environmental Biology (Agricultural Chemistry and Biology Group), Faculty of Sciences, University of Navarra, 31080 Pamplona, Spain

4 Department of Environmental Biology, Agricultural Chemistry and Biology Group, Faculties of Sciences and Pharmacy, University of Navarra, C/Irunlarrea 1, 31080 Pamplona, Navarra, SpainFull list of author information is available at the end of the article

2016 Fernndez et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/

Web End =http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/

Web End =http://creativecommons.org/ http://creativecommons.org/publicdomain/zero/1.0/

Web End =publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated.

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However, although some authors have reported that the efficiency of RP as a P source for pastures cultivated in acid soils is as eective as that of SSP or TSP in long-term studies (34years) [1, 4, 5], most studies have shown that RP efficiency is normally lower than that of water-soluble P fertilizers, with this fact being likely related to the lower short-term (1-year) efficiency of RP as a source of plant-available P with respect to that of water-soluble P (SSP or TSP) [1].

Historically, researchers have tried to improve plant-available P release rates from RP using several strategies. Some authors have prepared partially acidulated RP, or RP mixed with water-soluble P (TSP, SSP, or ammonium phosphates) [611]. Recently, other strategies included the use of stabilized phosphorus-solubilizing microorganisms (PSMs) [1216], or plant growth-promoting rhizobacteria (PGPR) [1719] along with or without RP.

Very recently, several studies have shown that specic enzyme-based hydrolyzed compost and protein residues, named edaphic biostimulants, are able to signicantly increase the activity of most soil enzymes [20, 21]. This action was accompanied by increases in the biodegradation of many organic contaminants and xenobiotics [21]. In this line, Renella et al. [22] showed that an edaphic biostimulant based on the heteromolecular complex between a sedimentary humic acid (HA) and tryptophan (Trp) (HATrp) was able to signicantly increase the activity of several enzyme families (phosphatases, organic hydrolases, and proteinases) in dierent soil types. In fact, previous studies had shown the stimulant action of Trp on the growth of soil cultivated plants, likely through the promotion of the biosynthesis of auxin in both the rhizosphere and plants [23, 24]. However, the presence of Trp complexed by the HA supra-structure assures and enlarges the invitro biostimulant action of Trp by delaying fast Trp degradation, thus promoting a more sustained eect [25].

In this framework, the aim of our study is to investigate whether the granulation of micronized RP along with (HATrp) complex is able to increase the short-term fertilizer action of RP by improving P solubilization rates in the rhizosphere. Our working hypothesis was that the potential increase in soil microbial activity, related enzyme activities, and local pH acidication in the physical rhizospheric environment surrounding RP granule triggered by HATrp might increase the rate of P mobilization from water-insoluble P in RP to the soil solution, thus enhancing short-term RP fertilizer efficiency. With this aim, we have investigated the eect of a granulated fertilizer made from RP and coated with (HATrp) (RP/ (HATrp)) and corresponding control treatments including RP coated with Trp alone (RP/Trp), RP, and SSP, on the early yield and P leaf concentration (13months) of

ray grass (Lolium perenne) cultivated in pots containing an acidic soil. These studies were complemented by soil incubation experiments carried out in order to evaluate the dierential action of all treatments on soil microbial activity and potentially plant-available P.

Methods

Physicochemical features ofRP, SSP, HA, andTrp

The RP and SSP granulated samples (average size distribution 90 % between 2 and 4 mm of granule diameter) were obtained from Timac Agro Fertilizer plant in Lodosa (Spain). Samples of granulated RP/Trp and RP/ (HATrp) were obtained by coating RP granules with a solution of vegetal oil containing talc and the edaphic biostimulant, while RP and SSP were coated with vegetal oil and talc. The concentration of P expressed as P2O5 of

the dierent fertilizers were RP (29 %), RP/Trp (29 %), RP/(HATrp) (29%), and SSP (17%). In the case of RP-based fertilizers, P is not soluble in water (it is mainly apatite), while SSP contains water-soluble P (mainly monocalcium phosphate).

HATrp heteromolecular complex was obtained by reaction of potassium-iron humate and Trp at pH 6 and room temperature as described in [25].

The leonardite HA employed for the preparation of HATrp complexes was extracted, puried, and characterized as described in [26, 27]. Elemental analysis revealed that the average chemical composition of HA was 51% C, 1.2% N, 2.6% H, and 45.2% O. Regarding the distribution of the dierent functional C-types, 13C

NMR studies indicated that HA contained 32% alkyl C, 9 % O-alkyl C, 43 % aromatic C, 13 % phenolic C, and 16 % carbonyl C. Concerning the size distributions of the dierent humic samples, the HPSEC study showed a main peak with a maximum corresponding to an apparent MW of 2.3 104 Da, a shoulder corresponding to an apparent MW of 5.8103Da, and a third minor peak related to a fraction with average apparent MW of 1.1103Da.

Samples of Trp (99.9 %) were obtained from Timac

Agro Spain.

Physicochemical features ofthe acid soil employed insoil incubation andsoilplant studies

An acidic soil from Egozkue (Navarra, Spain), with low potential plant-available P concentration, was used in the experiments. Egozkue is a small village placed in the north of Navarra. Soils in Egozkue are mainly acidic and poor in organic matter; pluviometry is around 1500 l per year and day/night winter temperature is 10 C on average. The soil was air-dried and sieved at 2mm. The nal sample was analyzed using Spanish-official analytical methods [1, and references therein] (Table1). Briey,

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Table 1 Physico-chemical features ofEgozkue soil

Conductivity (S cm1) 24.0

pH 5.60 Extractable P (mg kg1)a 4.02

K (mmol kg1)a 1.20

Mg (mmol kg1)a 3.50

Ca (mmol kg1)a 17.4

Na (mmol kg1)a 2.50

Fe (mmol kg1)b 0.49

Mn (mmol kg1)b 0.35

Cu (mmol kg1)b 0.003

Zn (mmol kg1)b 0.003

Mo (mmol kg1)b Under detection limits Organic Matter (g kg1) 0.10

Total CaCO3 (g kg1) 0.000 Sand (%) 15.0

Silt (%) 44.0 Clay (%) 41.0

a Mehlich-1

b DTPA

soil particle size composition was determined by densitometry (Bouyoucos method); total N was determined by LECO CHN elemental analyzer; K, Mg, Ca, and Na were extracted with a 40 mM HCl and 10 mM H2SO4 solution (Mehlich I extractant) and further analyzed using ICP-OES; micronutrients (Fe, Cu, Zn, and Mn) were analyzed following extraction with a solution of DTPA 5mM [28] using ICP-OES; organic matter was determined by dichromate oxidation method [29]; total carbonates were measured with a Bernard calcimeter method. The pH and electrolytic conductivity were measured using specic electrodes in water (1:2.5 soil/water ratio). Molibdate was analyzed in the DTPA-water extract using ICP-OES.

Soil incubation studies forthe evaluation ofplantavailable P insoilTreatments

Five repetitions for each treatment and a control without P treatment were used. The test consisted of SSP, RP, and RP complemented with two dierent edaphic biostimulants incorporated during the granule coating process. The two edaphic biostimulants considered in the study were as follows:

Trp, a precursor of auxin biosynthesis in soils and plants (1% in the formulation with RP);

(HATrp), a heteromolecular complex of HA and Trp through electropositive bridges [25] (2:1 of HA:Trp ratio). The nal dose of (HATrp) in the formulation was 3%.

Control treatments included native soil without any treatment (blank, B), soil plus RP, and soil plus SSP. Preliminary studies conducted in our laboratory showed that control treatments including the soil plus Trp or (HATrp) without RP at concentrations equivalent to those involved in RP/Trp and RP/(HATrp) treatments did not present results dierent from those of the control without any treatment concerning soil-related parameters and plant growth rates. For this reason, we have not included these controls (soil plus Trp or (HATrp), without RP) in data presentation and further discussion. The concentration of P applied to the soil in the experiments is described below.

Soil incubation experiments forthe evaluation ofplantavailable P insoil

A mixture of 100g of soil and 7g of perlite was placed in 150-mL plastic pots. The dierent treatments were added to the pot and the content was intensively mixed. A fertilizer rate of 150 mg P kg1 soil was used for all treatments and positive and negative controls. A control without added P was also used (B). Treated soil samples were homogenized and supplied with type I de-ionized water to reach soil eld capacity, which was previously determined by moistening a soil column and allowing it to drain freely. Pots were closed and allowed to stand at ambient temperature in the dark. Samples corresponding to ve replications were taken after 10, 20, and 30days from the onset of the treatments and air-dried for analysis. Pots were opened every day to avoid microbial life inhibition and anaerobic processes.

Analysis ofplantavailable P fractions incontrol andtreatments

The total potentially plant-available P in samples of incubated soil was evaluated using the anion-exchange resin-extractable P [30, 31]. Soil samples were taken for analysis after 10, 20, and 30days from the onset of treatments. The amount of P desorbed by an anion-exchange resin was determined using the method of Sibbesen [30] with slight modications. The resin used was 2050 mesh Dowex 14 anion-exchange material in chloride form. An amount of 0.6g of air-dried soil was placed in a 50-mL plastic tube. A volume of 30mL of de-ionized water and 2.2g of resin, held in a nylon bag, were added to the soil ample. Following shaking at the maximum possible speed for 2.5h, the resin bag was withdrawn, the soil suspension centrifuged, and the solution discarded. The nylon bag was then rinsed with water and P eluted with 30mL of 0.5M HCl with shaking at the maximum speed for 30min. Another 30mL of 0.5M HCl was then added and shaking applied to complete P elution. The

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two HCl solutions were ltered and analyzed by ICPOES. This P fraction was designated plant-available P.

Soil incubation studies forthe evaluation ofsoil microbial activity

Both the fertilizer treatments and the concentration of fertilizer used were similar to those employed for the evaluation of plant-available P, and described above.

Two dierent assays were carried out in order to measure microbial activity: the FDA method and CO2 soil production [32, 33].

Soil incubations were carried out as described below. Closed 0.5-L pots with a mixture of 100g of soil7g of perlite and the dierent controls and treatments were irrigated with 43g of water (at eld capacity) and maintained at 25C. Samples for analysis were taken after 7, 14, 21, and 28 days. Two replicates for each treatment were used.

Hydrolysis of uorescein diacetate [3,6-diacetyluorescein (FDA)] [32]: Two grams of air-dried soil was placed in a 125-ml Erlenmeyer ask. 50 ml of 60mM sodium phosphate buer, pH 7.6, and 0.50ml of 4.9mM FDA lipase substrate solution (20mg FDA lipase substrate in 10 ml acetone) were added. After mixing, samples were incubated for 1h at 25C. Then 10ml of acetone was added to the suspension to stop FDA hydrolysis. Samples were ltered through What-man No. 2 lter paper. The absorbance was measured on a spectrophotometer (HP-8453) at a wavelength of 490nm.

Measurement of CO2 release from soil by titration method according to Anderson [33]: A vial of 5 ml of 1M KOH was placed in each closed 0.5-L pots. The alkali traps were changed and titrated at 7, 14, 21, and 28 days. Unreacted alkali in the KOH traps was back-titrated with 0.4 M HCl to determine the CO2 release by microbial respiration.

Soilplant experiments

Experiments were carried out in a greenhouse under controlled temperature and lighting conditions. A 24/18C day/night temperature regime and a relative humidity of 4060% were used.

Five replicates of 500 g of soil were blended with the dierent treatments: control without added P, SSP, RP, RP/Trp (Trp), and RP/(HATrp). In order to prepare the soilfertilizer mixture, a Thermomix at maximum power for 5 s was used prior to the placement of the soil in plastic pots. Then, each soilfertilizer mixture was carefully blended with 50 g of perlite and supplied with 10 seeds of ray grass (Lolium sp.) on pot surface (113cm2). The P addition rates used were 10, 30, and 50mg P kg1

soil. As for the other nutrients, 200mgkg1 soil N and

200 mg kg1 K soil were added as urea and potassium chloride in order to complete macronutrient fertilization. Finally, 0.4mgkg1 soil of Mo as sodium molibdate was also added to prevent a potential deciency of Mo. The shoots corresponding to each pot were consecutively harvested at the end of the month for 3months after seed germination. Once analyzed for fresh matter, shoots were dried in an oven at 40C for 3days to determine dry matter. Next, the dry shoots were homogenized in a mill and sub-samples attacked with HNO3 and H2O2, and digested in a microwave oven, to determine P by ICP-OES as described in [27].

Statistical analyses

All experimental results were subjected to multiple pairwise comparisons between treatments, using Fishers least signicant dierence (LSD) post hoc test in a oneway ANOVA method with the overall -level set at 0.05.

Results anddiscussion

Association ofRP withHATrp or Trp increased bothsoilavailable P andsoil microbial activity, compared withRP

Our results clearly show that SSP, RP/Trp, and RP/ (HATrp) caused a prompt (after 10days) and signi-cant increase in the concentration of plant-available P in the soil with respect to RP and native soil (Table2). This increase, however, was transient and decreased after 20 and 30days to RP levels (Table2). In principle, this decrease in plant-available P was expected as the absence of a sink for available P in the soil system (such as plant roots) leads to an increase in plant-available P in soil solution that, in turn, may cause a feedback inhibition of those processes and enzyme activities involved in the P mobilization mechanisms activated by these formulations [22]. In fact, some authors have reported that an increase in the concentration of P in water-soluble P was associated with a decrease in CO2 soil production by an inhibition of soil microbial activity [3436]. This fact was also linked to signicant decreases in soil enzymatic activities, including those related to dierent types of soil phosphatases [37]. However, other mechanisms, such as phosphate precipitation or phosphate absorption, can also contribute to the plant-available P decline with time.

While the increase in plant-available P is easily explained for SSP since it has water-soluble phosphate, in the case of RP/Trp and RP/(HATrp) this fact has to be related to some kind of process leading to P solubilization from RP. These processes are normally related to changes in the activity of P-solubilizing microbiota [18]. Our studies showed that both Trp and HATrp associated with RP caused a signicant increase in soil

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Table 2 Time-course variation of soil plant-available P (mgkg1) measured bythe resin method

Time/days Treatments

Days B RP SSP RP/Trp RP/(HATrp)

10 3.7aA 19.7bA 33.6cB 46.9cB 33.2cB

20 11.5aB 24.2cA 21.7cB 16.0bcA 13.5bA

30 5.8aA 17.3bA 14.6bA 12.3bA 17.0bA

Data followed by the same lowercase letter in each row or by the same uppercase letter in each column are not statistically dierent from each other (Fishers test, P<0.05). Capital letters are used for dierences with time within each treatment. Lowercase letters are used for dierences among treatments within each time

microbial activity compared with the native soil, SSP, and RP (Table 3). This fact was very clear when CO2

soil production was evaluated, while the results for FDA hydrolysis were less conclusive (Table 3). These dierences between methods have also been described by other authors, and seem to be related to the fact that FDA hydrolysis method only includes specic families of soil hydrolases [3840] and, therefore, it can give an incomplete evaluation of soil microbial activity in some soil types. It is for this reason that traditional methods such as the analysis of soil CO2 production might be more sensitive for the evaluation of whole soil microbial activity changes. In any case, these results suggest that the increase in soil microbial activity plays an important role in the P-solubilizing action of Trp and HATrp from RP.

Association ofRP withHATrp, butnot withTrp alone, produced a sustained increase inshoot dry matter production upto levels similar tothose ofSSP

The results clearly showed that the short-term action of RP on both shoot dry matter and P fertilizer efficiency is signicantly lower than that of SSP for all harvests and higher P doses (30 and 50mgkg1) (Fig.1). In line with these results, shoot dry matter values for RP only presented a doseresponse pattern with added P rates at the rst harvest, while SSP maintained a doseresponse pattern for all harvests (Fig. 1). These results conrm that our experimental model is adequate to investigate the main issues posed by the study.

As for the ability of the dierent fertilizers to enhance shoot dry matter production, although both RP/Trp and RP/(HATrp) presented very similar results to each other in soil incubation studies, HATrpbut not Trp alonepresented a benecial action of the efficiency of RP to enhance shoot dry matter production when all harvests are considered (Fig.1). In fact, even though RP/Trp caused a prompt and signicant increase in shoot dry matter production at the rst harvest and for the higher doses of P (30 and 50mgkg1), this eect disappeared at the following harvests. Conversely, both SSP and RP/ (HATrp) presented higher increases in shoot dry matter production than the control and RP for the second and third harvests (Fig.1). Thus, the association of RP with (HATrp) caused a signicant and sustained increase in shoot dry matter production compared with RP for all harvest times, with this increase being similar to that

Table 3 Time-course variation ofFDA hydrolysis andCO2 soil production

g FL g1h1

7days 14days 21days 28days

FDA hydrolysis

B 1.88 2.76 3.71 4.08a RP 1.80 2.73 3.59 4.02a SSP 1.58 2.76 3.55 3.98a RP/Trp 2.04 3.03 4.00 4.41c RP/(HATrp) 1.71 2.74 3.70 4.15b mg CO2 kg1 soil

7days 14days 21days 28days

CO2 soil production

B 83.6 123 147 165a RP 117 154 170 203a SSP 81.4 128 152 178a RP/Trp 96.8 154 187 229b RP/(HATrp) 123 167 202 240b

Data followed by the same lowercase letter in each column are not statistically dierent from each other (Fishers test, P<0.05)

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First harvest

Third Harvest

0,180

0,400

c

0,160

0,350

b

0,140

ab

a a

b

0,300

0,120

b b

a a a a

b

0,250

b

0,100

a ab

g

g

0,200

0,080

a a a

a

0,150

0,060

0,100

0,040

0,020

0,050

0,000

0,000

10 mgKg-1 30 mgKg-1 50 mgKg-1

10 mgKg-1 30 mgKg-1 50 mgKg-1

added P

added P

Second harvest

Cumulated Dry Weight

0,400

1,000

b

0,350

b

b

a

b

0,800

0,300

a

b b b

a

a

a

a

0,250

a a a

a

0,600

b

a ab ab

g

0,200

g

0,400

0,150

0,100

0,200

0,050

0,000

0,000

10 mgKg-1 30 mgKg-1 50 mgKg-1

10 mgKg-1 30 mgKg-1 50 mgKg-1

added P

added P

Fig. 1 First, second, and third harvests as well as accumulated shoot dry matter production (g per pot; average of ve replications) for RP dotted bar, SSP gray shaded bar, RP/Trp cross-lined bar, and RP/(HATrp) black shaded bar. Columns followed by the same lowercase letter are not statistically dierent from each other (Fishers test. P < 0.05)

caused by SSP (Fig.1). These results were clearer for the highest dose of P added to the soil (50mgkg1) (Fig.1). Dierences between RP/Trp and RP/(HATrp) might be a consequence of an eect of HA on RP solubilization either directly or through an increase in soil microbial activity. However, preliminary studies using P fertilizers containing 1% HA did not show any eect on both P bio-availability and shoot growth in several soil types [41]. In some way, these results were expected since the concentration of P applied to soil (50mgKg1) involved the soil application of very low concentrations of HA (lower than 40 mg kg1). However, these studies showed that when HA was applied forming stable and soluble complexes with Zn or Cu, a clear increase in plant growth was observed resulting from an improvement in the plant uptake of both micronutrients [42]. Although in our study we do not use humic metal complexes, some action of HA in RP fertilizers improving micronutrient plant nutrition cannot be ruled out. Another factor inuencing the dierential eect of HATrp compared

with Trp alone might result from the presence of Fe in HATrp complexes. However, taking into account that the amount of plant-available Fe in Egozcue soil is quite high (Table1), it is rather improbable that the Fe added with HATrp in RP/(HATrp) treatment can cause signicant changes in plant growth. Finally, another possible explanation for these results may relate to some type of synergic action of HA and Trp when applied together. In fact, Trp complexation in HA supramolecule might delay its conversion in indolacetic acid (IAA) (and, therefore, IAA degradation) by favoring a slow release of Trp to the rhizospheric environment [2325]. This hypothesis might also contribute to explain the results obtained, but further experiments using this experimental model are needed in order to establish its validity.

Taking into account that the eect of RP/(HATrp) on shoot dry matter production was consistent with the increase in soil microbial activity caused for this fertilizer in soil incubation studies (Table3), this eect might be linked to some kind of secondary action of (HATrp) on

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local soil features in granule environment resulting from the enhancement in soil microbiota activity, which in turn favor P solubilization from RP.

Regarding the relationships between shoot dry weight production and the concentration of P in the shoot, the values of shoot P concentration showed that RP/ (HATrp) increased this parameter with respect to RP. In addition, these eects tended to be lower than those associated with SSP only at the rst harvest (Fig.2). The fact that the dierences of shoot dry matter production between RP/(HATrp) and SSP were not dierent from each other led to the fact that the fertilizer efficiency of RP/(HATrp) and SSP was quite similar to each other and signicantly higher than that of RP principally for the highest P dose (50mgkg1) and third harvest (Table4).

The fact that plants treated with RP/Trp have the highest concentration of P in the shoot for the second and third harvests probably derives from a concentration eect associated with the low production of shoot dry

matter produced by this fertilizer (Figs. 1, 2). This fact was also reected in the low values of RP/Trp fertilizer efficiency parameters (Table4).

Finally, we did not observe clear dierences between the efficiency of plant P utilization in the shoot for SSP and RP/(HATrp), although in both cases this parameter was higher than those for RP and RPTrp, principally for the highest P dose (50mgkg1) (Table5). This fact indicated that plant metabolism was not dierently aected by SSP and RP/(HATrp), thus suggesting that HATrp complex had not direct eect on plant metabolism at the doses associated with RP/(HATrp) fertilizer rates.

Conclusion

Summarizing, the results here described show that the association of RP with an organic complex formed by HA and Trp linked to each other through electropositive bridges (mainly, proton or metal, in this case Fe) avoided the short-term dierences in agronomical efficacy

Fig. 2 First, second, and third harvestsshoot P concentration (mg kg1 dry weight) for RP dotted bar, P gray shaded bar, RP/Trp cross-lined bar and RP/(HATrp) black shaded bar. Columns followed by the same lowercase letter are not statistically dierent from each other (Fishers test. P < 0.05)

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Table 4 Fertilizer efficiency index dened by[(dry matter production)100]/(added P tothe soil)

Treatments RP SSP RP/Trp RP/(HATrp)

Added P (mgkg1 soil) 10 30 50 10 30 50 10 30 50 10 30 50

Harvest

First 5.83 0.36 0.25 5.49 0.27 0.24 5.95 0.52 0.29 6.37 0.39 0.27 Second 18.37a 0.80c 0.48 21.56b 1.02d 0.71 19.58b 0.92d 0.51 22.53b 0.95d 0.65 Third 18.70a 0.73c 0.46d 19.73b 0.90d 0.69e 21.04b 0.77c 0.46d 23.29b 0.79c 0.60e

Data followed by the same lowercase letter in each row are not statistically dierent from each other (Fishers test, P<0.05)

Table 5 Plant P utilization efficiency index dened by[(dry matter production)10,000]/(shoot P concentration)

Treatments RP SSP RP/Trp RP/(HATrp)

Added P (mgkg1 soil) 10 30 50 10 30 50 10 30 50 10 30 50

Harvest

First 0.35 0.51 0.45 0.29 0.28 0.37 0.39 0.68 0.56 0.44 0.54 0.47 Second 2.60 2.50 1.80b 2.65 3.06 2.37c 2.29 2.29 1.26a 2.35 2.53 2.04c Third 3.21 2.56 1.85a 3.14 2.86 2.76b 3.21 2.60 1.56a 3.55 2.89 2.39b

Data followed by the same lowercase letter in each row are not statistically dierent from each other (Fishers test, P<0.05)

existing between RP and SSP, thus improving the starter action of RP-based fertilizers. This issue is very relevant because the main negative feature of RP as a P source for pastures cultivated in acidic soils is its poor short-term (1-year) eect in comparison with that of water-soluble P fertilizers, thus negatively aecting yield and quality [1]. However, the long-term action (34 years) of RP-based fertilizers as a P source for pastures cultivated in acidic soils may be even more efficient than that of water-soluble P fertilizers [1, 4, 5]. This fact indicates that both fertilizers (HATrp activated RP and SSP) may be rather complementary in a whole fertilization plan for pastures in acidic soils.

Authors contributions

LF principally but also RB, OU, and JE have made the experimental work and contributed to MS preparation; RB, OU, JE, and PMA contributed to the experi mental design; JCY contributed to the discussion of results; and JMG contrib uted to the design of the experiments, discussion of results, MS preparation, and nal writing. All authors read and approved the nal manuscript.

Author details

1 Institute of AgroBiotechnology, Public University of NavarraCSICGov ernment of Navarra, Campus de Arrosada, 31006 Pamplona, Navarra, Spain.

2 Department of Environmental Biology (Agricultural Chemistry and Biology Group), Faculty of Sciences, University of Navarra, 31080 Pamplona, Spain.

3 Centre Mondial dInnovation (CMI), Roullier Group, Saint Malo, France.

4 Department of Environmental Biology, Agricultural Chemistry and Biology Group, Faculties of Sciences and Pharmacy, University of Navarra, C/Irunlarrea 1, 31080 Pamplona, Navarra, Spain.

Acknowledgements

This Research Project has been supported by a Grant from CDTI and Govern ment of Navarra, as well the Roullier Group. We would like to thank Paul W. Miller for kindly improving the English of the manuscript.

Competing interests

The authors declare that they have no competing interests.

Received: 7 September 2015 Accepted: 1 March 2016

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