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

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1 Fernández et l. Chem. Biol. Technol. Agric. (2016) 3:5 DOI /s RESEARCH Open Access Improving the short term efficiency of rock phosphte sed fertilizers in pstures y using edphic iostimulnts L. Fernández 1,2, R. Bigorri 2*, O. Urruti 2, J. Erro 2, P. M. Apricio Tejo 1, J. C. Yvin 3 nd J. M. Grcí Min 2,4* Astrct Bckground: The use of rective rock phosphte (RP) in cidic soils s phosphte (P) source for pstures nd crops presents ttrctive economic dvntges with respect to solule phosphte. However, some studies hve demonstrted tht the short-term (1-yer) efficiency of RP, compred with tht of wter-solule P, is reltively poor. This fct penlizes not only the yield nd qulity of the erlier hrvests, ut even the whole finl yield when the crop is ffected y some iotic or iotic stress t the eginning of the cycle. In the present study, we investigted the ility of new edphic iostimulnts to increse the short-term efficiency of RP-sed fertilizer s P source for pstures cultivted in cid soils. To this end, we hve grnulted rock phosphte with two edphic iostimulnts: tryptophn nd heteromoleculr orgnic complex formed y humic cid nd tryptophn through iron ridges, nd compred their short-term P fertilizer efficcy with tht of single superphosphte nd rock phosphte. Results: Soil incution studies showed tht the heteromoleculr complex humic cid tryptophn nd Tryptophn were le to increse soil CO 2 production compred with ntive soil, rock phosphte, nd superphosphte. Likewise, the presence of humic cid tryptophn complex nd Trp significntly increses plnt-ville phosphte compred with rock phosphte, up to levels similr to those of superphosphte. Plnt (ry grss) soil pot studies showed tht rock phosphte/(humic cid tryptophn) formultion yielded vlues for oth ry grss dry mtter production nd shoot P concentrtion, clerly higher thn those of rock phosphte nd rock phosphte/tryptophn. In ddition, the results ssocited with rock phosphte/(humic cid tryptophn) were similr to those for superphosphte, fter 3 months of cultivtion. Conclusions: Tken together, these results showed the suitility of the use of specific humic cid-sed edphic iostimulnts to improve the short-term effect of rock phosphte fertilizers s phosphte source for pstures cultivted in cid soils. Keywords: Edphic iostimulnts, Rock phosphte, Phosphte fertilizer, Pstures, Plnt-ville phosphte soil microil ctivity, Humic cid, Humic cid-sed heteromoleculr complexes Bckground Wter-solule phosphte (P)-sed fertilizers, minly single superphosphte (SSP) nd triple superphosphte *Correspondence: rigorri@timcgro.es; jgmin@timcgro.es 2 Deprtment of Environmentl Biology (Agriculturl Chemistry nd Biology Group), Fculty of Sciences, University of Nvrr, Pmplon, Spin 4 Deprtment of Environmentl Biology, Agriculturl Chemistry nd Biology Group, Fculties of Sciences nd Phrmcy, University of Nvrr, C/Irunlrre 1, Pmplon, Nvrr, Spin Full list of uthor informtion is ville t the end of the rticle (TSP), re the min sources of P used for cultivted pstures, minly in lkline soils, ut lso in cidic soils [1, 2]. However, in pstures (nd lso other crops) cultivted in cidic soils, the direct ppliction of rock phosphte (RP) (grnule or powder) without previous rection with sulfuric nd/or phosphoric cids my e suitle, less-expensive, lterntive to wter-solule P fertilizers [1]. In ddition, the slow soluiliztion of RP in cidic soils my lso contriute to decrese environmentl risks, such s the eutrophiction of surfce wters [3] Fernández et l. This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pulicdomin/zero/1.0/) pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Fernández et l. Chem. Biol. Technol. Agric. (2016) 3:5 Pge 2 of 9 However, lthough some uthors hve reported tht the efficiency of RP s P source for pstures cultivted in cid soils is s effective s tht of SSP or TSP in long-term studies (3 4 yers) [1, 4, 5], most studies hve shown tht RP efficiency is normlly lower thn tht of wter-solule P fertilizers, with this fct eing likely relted to the lower short-term (1-yer) efficiency of RP s source of plntville P with respect to tht of wter-solule P (SSP or TSP) [1]. Historiclly, reserchers hve tried to improve plntville P relese rtes from RP using severl strtegies. Some uthors hve prepred prtilly cidulted RP, or RP mixed with wter-solule P (TSP, SSP, or mmonium phosphtes) [6 11]. Recently, other strtegies included the use of stilized phosphorus-soluilizing microorgnisms (PSMs) [12 16], or plnt growth-promoting rhizocteri (PGPR) [17 19] long with or without RP. Very recently, severl studies hve shown tht specific enzyme-sed hydrolyzed compost nd protein residues, nmed edphic iostimulnts, re le to significntly increse the ctivity of most soil enzymes [20, 21]. This ction ws ccompnied y increses in the iodegrdtion of mny orgnic contminnts nd xenoiotics [21]. In this line, Renell et l. [22] showed tht n edphic iostimulnt sed on the heteromoleculr complex etween sedimentry humic cid (HA) nd tryptophn (Trp) (HA Trp) ws le to significntly increse the ctivity of severl enzyme fmilies (phosphtses, orgnic hydrolses, nd proteinses) in different soil types. In fct, previous studies hd shown the stimulnt ction of Trp on the growth of soil cultivted plnts, likely through the promotion of the iosynthesis of uxin in oth the rhizosphere nd plnts [23, 24]. However, the presence of Trp complexed y the HA supr-structure ssures nd enlrges the in vitro iostimulnt ction of Trp y delying fst Trp degrdtion, thus promoting more sustined effect [25]. In this frmework, the im of our study is to investigte whether the grnultion of micronized RP long with (HA Trp) complex is le to increse the short-term fertilizer ction of RP y improving P soluiliztion rtes in the rhizosphere. Our working hypothesis ws tht the potentil increse in soil microil ctivity, relted enzyme ctivities, nd locl ph cidifiction in the physicl rhizospheric environment surrounding RP grnule triggered y HA Trp might increse the rte of P moiliztion from wter-insolule P in RP to the soil solution, thus enhncing short-term RP fertilizer efficiency. With this im, we hve investigted the effect of grnulted fertilizer mde from RP nd coted with (HA Trp) (RP/ (HA Trp)) nd corresponding control tretments including RP coted with Trp lone (RP/Trp), RP, nd SSP, on the erly yield nd P lef concentrtion (1 3 months) of ry grss (Lolium perenne) cultivted in pots contining n cidic soil. These studies were complemented y soil incution experiments crried out in order to evlute the differentil ction of ll tretments on soil microil ctivity nd potentilly plnt-ville P. Methods Physico chemicl fetures of RP, SSP, HA, nd Trp The RP nd SSP grnulted smples (verge size distriution 90 % etween 2 nd 4 mm of grnule dimeter) were otined from Timc Agro Fertilizer plnt in Lodos (Spin). Smples of grnulted RP/Trp nd RP/ (HA Trp) were otined y coting RP grnules with solution of vegetl oil contining tlc nd the edphic iostimulnt, while RP nd SSP were coted with vegetl oil nd tlc. The concentrtion of P expressed s P 2 O 5 of the different fertilizers were RP (29 %), RP/Trp (29 %), RP/(HA Trp) (29 %), nd SSP (17 %). In the cse of RPsed fertilizers, P is not solule in wter (it is minly ptite), while SSP contins wter-solule P (minly monoclcium phosphte). HA Trp heteromoleculr complex ws otined y rection of potssium-iron humte nd Trp t ph 6 nd room temperture s descried in [25]. The leonrdite HA employed for the preprtion of HA Trp complexes ws extrcted, purified, nd chrcterized s descried in [26, 27]. Elementl nlysis reveled tht the verge chemicl composition of HA ws 51 % C, 1.2 % N, 2.6 % H, nd 45.2 % O. Regrding the distriution of the different functionl C-types, 13 C NMR studies indicted tht HA contined 32 % lkyl C, 9 % O-lkyl C, 43 % romtic C, 13 % phenolic C, nd 16 % cronyl C. Concerning the size distriutions of the different humic smples, the HPSEC study showed min pek with mximum corresponding to n pprent MW of D, shoulder corresponding to n pprent MW of D, nd third minor pek relted to frction with verge pprent MW of D. Smples of Trp (99.9 %) were otined from Timc Agro Spin. Physico chemicl fetures of the cid soil employed in soil incution nd soil plnt studies An cidic soil from Egozkue (Nvrr, Spin), with low potentil plnt-ville P concentrtion, ws used in the experiments. Egozkue is smll villge plced in the north of Nvrr. Soils in Egozkue re minly cidic nd poor in orgnic mtter; pluviometry is round 1500 l per yer nd dy/night winter temperture is 10 C on verge. The soil ws ir-dried nd sieved t 2 mm. The finl smple ws nlyzed using Spnish-officil nlyticl methods [1, nd references therein] (Tle 1). Briefly,

3 Fernández et l. Chem. Biol. Technol. Agric. (2016) 3:5 Pge 3 of 9 Tle 1 Physico-chemicl fetures of Egozkue soil Conductivity (µs cm 1 ) 24.0 ph 5.60 Extrctle P (mg kg 1 ) 4.02 K (mmol kg 1 ) 1.20 Mg (mmol kg 1 ) 3.50 C (mmol kg 1 ) 17.4 N (mmol kg 1 ) 2.50 Fe (mmol kg 1 ) 0.49 Mn (mmol kg 1 ) 0.35 Cu (mmol kg 1 ) Zn (mmol kg 1 ) Mo (mmol kg 1 ) Under detection limits Orgnic Mtter (g kg 1 ) 0.10 Totl CCO 3 (g kg 1 ) Snd (%) 15.0 Silt (%) 44.0 Cly (%) 41.0 Mehlich-1 DTPA soil prticle size composition ws determined y densitometry (Bouyoucos method); totl N ws determined y LECO CHN elementl nlyzer; K, Mg, C, nd N were extrcted with 40 mm HCl nd 10 mm H 2 SO 4 solution (Mehlich I extrctnt) nd further nlyzed using ICP-OES; micronutrients (Fe, Cu, Zn, nd Mn) were nlyzed following extrction with solution of DTPA 5 mm [28] using ICP-OES; orgnic mtter ws determined y dichromte oxidtion method [29]; totl crontes were mesured with Bernrd clcimeter method. The ph nd electrolytic conductivity were mesured using specific electrodes in wter (1:2.5 soil/wter rtio). Molidte ws nlyzed in the DTPA-wter extrct using ICP-OES. Soil incution studies for the evlution of plnt ville P in soil Tretments Five repetitions for ech tretment nd control without P tretment were used. The test consisted of SSP, RP, nd RP complemented with two different edphic iostimulnts incorported during the grnule coting process. The two edphic iostimulnts considered in the study were s follows: Trp, precursor of uxin iosynthesis in soils nd plnts (1 % in the formultion with RP); (HA Trp), heteromoleculr complex of HA nd Trp through electropositive ridges [25] (2:1 of HA:Trp rtio). The finl dose of (HA Trp) in the formultion ws 3 %. Control tretments included ntive soil without ny tretment (lnk, B), soil plus RP, nd soil plus SSP. Preliminry studies conducted in our lortory showed tht control tretments including the soil plus Trp or (HA Trp) without RP t concentrtions equivlent to those involved in RP/Trp nd RP/(HA Trp) tretments did not present results different from those of the control without ny tretment concerning soil-relted prmeters nd plnt growth rtes. For this reson, we hve not included these controls (soil plus Trp or (HA Trp), without RP) in dt presenttion nd further discussion. The concentrtion of P pplied to the soil in the experiments is descried elow. Soil incution experiments for the evlution of plnt ville P in soil A mixture of 100 g of soil nd 7 g of perlite ws plced in 150-mL plstic pots. The different tretments were dded to the pot nd the content ws intensively mixed. A fertilizer rte of 150 mg P kg 1 soil ws used for ll tretments nd positive nd negtive controls. A control without dded P ws lso used (B). Treted soil smples were homogenized nd supplied with type I de-ionized wter to rech soil field cpcity, which ws previously determined y moistening soil column nd llowing it to drin freely. Pots were closed nd llowed to stnd t mient temperture in the drk. Smples corresponding to five replictions were tken fter 10, 20, nd 30 dys from the onset of the tretments nd ir-dried for nlysis. Pots were opened every dy to void microil life inhiition nd neroic processes. Anlysis of plnt ville P frctions in control nd tretments The totl potentilly plnt-ville P in smples of incuted soil ws evluted using the nion-exchnge resin-extrctle P [30, 31]. Soil smples were tken for nlysis fter 10, 20, nd 30 dys from the onset of tretments. The mount of P desored y n nion-exchnge resin ws determined using the method of Siesen [30] with slight modifictions. The resin used ws mesh Dowex 1 4 nion-exchnge mteril in chloride form. An mount of 0.6 g of ir-dried soil ws plced in 50-mL plstic tue. A volume of 30 ml of de-ionized wter nd 2.2 g of resin, held in nylon g, were dded to the soil mple. Following shking t the mximum possile speed for 2.5 h, the resin g ws withdrwn, the soil suspension centrifuged, nd the solution discrded. The nylon g ws then rinsed with wter nd P eluted with 30 ml of 0.5 M HCl with shking t the mximum speed for 30 min. Another 30 ml of 0.5 M HCl ws then dded nd shking pplied to complete P elution. The

4 Fernández et l. Chem. Biol. Technol. Agric. (2016) 3:5 Pge 4 of 9 two HCl solutions were filtered nd nlyzed y ICP- OES. This P frction ws designted plnt-ville P. Soil incution studies for the evlution of soil microil ctivity Both the fertilizer tretments nd the concentrtion of fertilizer used were similr to those employed for the evlution of plnt-ville P, nd descried ove. Two different ssys were crried out in order to mesure microil ctivity: the FDA method nd CO 2 soil production [32, 33]. Soil incutions were crried out s descried elow. Closed 0.5-L pots with mixture of 100 g of soil 7 g of perlite nd the different controls nd tretments were irrigted with 43 g of wter (t field cpcity) nd mintined t 25 C. Smples for nlysis were tken fter 7, 14, 21, nd 28 dys. Two replictes for ech tretment were used. Hydrolysis of fluorescein dicette [3,6 -dicetylfluorescein (FDA)] [32]: Two grms of ir-dried soil ws plced in 125-ml Erlenmeyer flsk. 50 ml of 60 mm sodium phosphte uffer, ph 7.6, nd 0.50 ml of 4.9 mm FDA lipse sustrte solution (20 mg FDA lipse sustrte in 10 ml cetone) were dded. After mixing, smples were incuted for 1 h t 25 C. Then 10 ml of cetone ws dded to the suspension to stop FDA hydrolysis. Smples were filtered through Whtmn No. 2 filter pper. The sornce ws mesured on spectrophotometer (HP-8453) t wvelength of 490 nm. Mesurement of CO 2 relese from soil y titrtion method ccording to Anderson [33]: A vil of 5 ml of 1 M KOH ws plced in ech closed 0.5-L pots. The lkli trps were chnged nd titrted t 7, 14, 21, nd 28 dys. Unrected lkli in the KOH trps ws ck-titrted with 0.4 M HCl to determine the CO 2 relese y microil respirtion. Soil plnt experiments Experiments were crried out in greenhouse under controlled temperture nd lighting conditions. A 24/18 C dy/night temperture regime nd reltive humidity of % were used. Five replictes of 500 g of soil were lended with the different tretments: control without dded P, SSP, RP, RP/Trp (Trp), nd RP/(HA Trp). In order to prepre the soil fertilizer mixture, Thermomix t mximum power for 5 s ws used prior to the plcement of the soil in plstic pots. Then, ech soil fertilizer mixture ws crefully lended with 50 g of perlite nd supplied with 10 seeds of ry grss (Lolium sp.) on pot surfce (113 cm 2 ). The P ddition rtes used were 10, 30, nd 50 mg P kg 1 soil. As for the other nutrients, 200 mg kg 1 soil N nd 200 mg kg 1 K soil were dded s ure nd potssium chloride in order to complete mcronutrient fertiliztion. Finlly, 0.4 mg kg 1 soil of Mo s sodium molidte ws lso dded to prevent potentil deficiency of Mo. The shoots corresponding to ech pot were consecutively hrvested t the end of the month for 3 months fter seed germintion. Once nlyzed for fresh mtter, shoots were dried in n oven t 40 C for 3 dys to determine dry mtter. Next, the dry shoots were homogenized in mill nd su-smples ttcked with HNO 3 nd H 2 O 2, nd digested in microwve oven, to determine P y ICP-OES s descried in [27]. Sttisticl nlyses All experimentl results were sujected to multiple pirwise comprisons etween tretments, using Fisher s lest significnt difference (LSD) post hoc test in onewy ANOVA method with the overll α-level set t Results nd discussion Assocition of RP with HA Trp or Trp incresed oth soil ville P nd soil microil ctivity, compred with RP Our results clerly show tht SSP, RP/Trp, nd RP/ (HA Trp) cused prompt (fter 10 dys) nd significnt increse in the concentrtion of plnt-ville P in the soil with respect to RP nd ntive soil (Tle 2). This increse, however, ws trnsient nd decresed fter 20 nd 30 dys to RP levels (Tle 2). In principle, this decrese in plnt-ville P ws expected s the sence of sink for ville P in the soil system (such s plnt roots) leds to n increse in plnt-ville P in soil solution tht, in turn, my cuse feedck inhiition of those processes nd enzyme ctivities involved in the P moiliztion mechnisms ctivted y these formultions [22]. In fct, some uthors hve reported tht n increse in the concentrtion of P in wter-solule P ws ssocited with decrese in CO 2 soil production y n inhiition of soil microil ctivity [34 36]. This fct ws lso linked to significnt decreses in soil enzymtic ctivities, including those relted to different types of soil phosphtses [37]. However, other mechnisms, such s phosphte precipittion or phosphte sorption, cn lso contriute to the plnt-ville P decline with time. While the increse in plnt-ville P is esily explined for SSP since it hs wter-solule phosphte, in the cse of RP/Trp nd RP/(HA Trp) this fct hs to e relted to some kind of process leding to P soluiliztion from RP. These processes re normlly relted to chnges in the ctivity of P-soluilizing microiot [18]. Our studies showed tht oth Trp nd HA Trp ssocited with RP cused significnt increse in soil

5 Fernández et l. Chem. Biol. Technol. Agric. (2016) 3:5 Pge 5 of 9 Tle 2 Time-course vrition of soil plnt-ville P (mg kg 1 ) mesured y the resin method Time/dys Tretments Dys B RP SSP RP/Trp RP/(HA Trp) A 19.7A 33.6cB 46.9cB 33.2cB B 24.2cA 21.7cB 16.0cA 13.5A A 17.3A 14.6A 12.3A 17.0A Dt followed y the sme lowercse letter in ech row or y the sme uppercse letter in ech column re not sttisticlly different from ech other (Fisher s test, P < 0.05). Cpitl letters re used for differences with time within ech tretment. Lowercse letters re used for differences mong tretments within ech time microil ctivity compred with the ntive soil, SSP, nd RP (Tle 3). This fct ws very cler when CO 2 soil production ws evluted, while the results for FDA hydrolysis were less conclusive (Tle 3). These differences etween methods hve lso een descried y other uthors, nd seem to e relted to the fct tht FDA hydrolysis method only includes specific fmilies of soil hydrolses [38 40] nd, therefore, it cn give n incomplete evlution of soil microil ctivity in some soil types. It is for this reson tht trditionl methods such s the nlysis of soil CO 2 production might e more sensitive for the evlution of whole soil microil ctivity chnges. In ny cse, these results suggest tht the increse in soil microil ctivity plys n importnt role in the P-soluilizing ction of Trp nd HA Trp from RP. Assocition of RP with HA Trp, ut not with Trp lone, produced sustined increse in shoot dry mtter production up to levels similr to those of SSP The results clerly showed tht the short-term ction of RP on oth shoot dry mtter nd P fertilizer efficiency is significntly lower thn tht of SSP for ll hrvests nd higher P doses (30 nd 50 mg kg 1 ) (Fig. 1). In line with these results, shoot dry mtter vlues for RP only presented dose response pttern with dded P rtes t the first hrvest, while SSP mintined dose response pttern for ll hrvests (Fig. 1). These results confirm tht our experimentl model is dequte to investigte the min issues posed y the study. As for the ility of the different fertilizers to enhnce shoot dry mtter production, lthough oth RP/Trp nd RP/(HA Trp) presented very similr results to ech other in soil incution studies, HA Trp ut not Trp lone presented eneficil ction of the efficiency of RP to enhnce shoot dry mtter production when ll hrvests re considered (Fig. 1). In fct, even though RP/Trp cused prompt nd significnt increse in shoot dry mtter production t the first hrvest nd for the higher doses of P (30 nd 50 mg kg 1 ), this effect disppered t the following hrvests. Conversely, oth SSP nd RP/ (HA Trp) presented higher increses in shoot dry mtter production thn the control nd RP for the second nd third hrvests (Fig. 1). Thus, the ssocition of RP with (HA Trp) cused significnt nd sustined increse in shoot dry mtter production compred with RP for ll hrvest times, with this increse eing similr to tht Tle 3 Time-course vrition of FDA hydrolysis nd CO 2 soil production μg FL g 1 h 1 7 dys 14 dys 21 dys 28 dys FDA hydrolysis B RP SSP RP/Trp c RP/(HA Trp) mg CO 2 kg 1 soil 7 dys 14 dys 21 dys 28 dys CO 2 soil production B RP SSP RP/Trp RP/(HA Trp) Dt followed y the sme lowercse letter in ech column re not sttisticlly different from ech other (Fisher s test, P < 0.05)

6 Fernández et l. Chem. Biol. Technol. Agric. (2016) 3:5 Pge 6 of 9 First hrvest Third Hrvest 0,180 0,400 g 0,160 0,140 0,120 0,100 0,080 0,060 c g 0,350 0,300 0,250 0,200 0,150 0,040 0,100 0,020 0,050 0, mgkg-1 30 mgkg-1 50 mgkg-1 0, mgkg-1 30 mgkg-1 50 mgkg-1 dded P dded P g 0,400 0,350 0,300 0,250 0,200 Second hrvest g 1,000 0,800 0,600 Cumulted Dry Weight 0,150 0,400 0,100 0,200 0,050 0, mgkg-1 30 mgkg-1 50 mgkg-1 dded P 0, mgkg-1 30 mgkg-1 50 mgkg-1 Fig. 1 First, second, nd third hrvests s well s ccumulted shoot dry mtter production (g per pot; verge of five replictions) for RP dotted r, SSP gry shded r, RP/Trp cross-lined r, nd RP/(HA Trp) lck shded r. Columns followed y the sme lowercse letter re not sttisticlly different from ech other (Fisher s test. P < 0.05) dded P cused y SSP (Fig. 1). These results were clerer for the highest dose of P dded to the soil (50 mg kg 1 ) (Fig. 1). Differences etween RP/Trp nd RP/(HA Trp) might e consequence of n effect of HA on RP soluiliztion either directly or through n increse in soil microil ctivity. However, preliminry studies using P fertilizers contining 1 % HA did not show ny effect on oth P iovilility nd shoot growth in severl soil types [41]. In some wy, these results were expected since the concentrtion of P pplied to soil (50 mg Kg 1 ) involved the soil ppliction of very low concentrtions of HA (lower thn 40 mg kg 1 ). However, these studies showed tht when HA ws pplied forming stle nd solule complexes with Zn or Cu, cler increse in plnt growth ws oserved resulting from n improvement in the plnt uptke of oth micronutrients [42]. Although in our study we do not use humic metl complexes, some ction of HA in RP fertilizers improving micronutrient plnt nutrition cnnot e ruled out. Another fctor influencing the differentil effect of HA Trp compred with Trp lone might result from the presence of Fe in HA Trp complexes. However, tking into ccount tht the mount of plnt-ville Fe in Egozcue soil is quite high (Tle 1), it is rther improle tht the Fe dded with HA Trp in RP/(HA Trp) tretment cn cuse significnt chnges in plnt growth. Finlly, nother possile explntion for these results my relte to some type of synergic ction of HA nd Trp when pplied together. In fct, Trp complextion in HA suprmolecule might dely its conversion in indolcetic cid (IAA) (nd, therefore, IAA degrdtion) y fvoring slow relese of Trp to the rhizospheric environment [23 25]. This hypothesis might lso contriute to explin the results otined, ut further experiments using this experimentl model re needed in order to estlish its vlidity. Tking into ccount tht the effect of RP/(HA Trp) on shoot dry mtter production ws consistent with the increse in soil microil ctivity cused for this fertilizer in soil incution studies (Tle 3), this effect might e linked to some kind of secondry ction of (HA Trp) on

7 Fernández et l. Chem. Biol. Technol. Agric. (2016) 3:5 locl soil fetures in grnule environment resulting from the enhncement in soil microiot ctivity, which in turn fvor P soluiliztion from RP. Regrding the reltionships etween shoot dry weight production nd the concentrtion of P in the shoot, the vlues of shoot P concentrtion showed tht RP/ (HA Trp) incresed this prmeter with respect to RP. In ddition, these effects tended to e lower thn those ssocited with SSP only t the first hrvest (Fig. 2). The fct tht the differences of shoot dry mtter production etween RP/(HA Trp) nd SSP were not different from ech other led to the fct tht the fertilizer efficiency of RP/(HA Trp) nd SSP ws quite similr to ech other nd significntly higher thn tht of RP principlly for the highest P dose (50 mg kg 1) nd third hrvest (Tle 4). The fct tht plnts treted with RP/Trp hve the highest concentrtion of P in the shoot for the second nd third hrvests proly derives from concentrtion effect ssocited with the low production of shoot dry Pge 7 of 9 mtter produced y this fertilizer (Figs. 1, 2). This fct ws lso reflected in the low vlues of RP/Trp fertilizer efficiency prmeters (Tle 4). Finlly, we did not oserve cler differences etween the efficiency of plnt P utiliztion in the shoot for SSP nd RP/(HA Trp), lthough in oth cses this prmeter ws higher thn those for RP nd RP Trp, principlly for the highest P dose (50 mg kg 1) (Tle 5). This fct indicted tht plnt metolism ws not differently ffected y SSP nd RP/(HA Trp), thus suggesting tht HA Trp complex hd not direct effect on plnt metolism t the doses ssocited with RP/(HA Trp) fertilizer rtes. Conclusion Summrizing, the results here descried show tht the ssocition of RP with n orgnic complex formed y HA nd Trp linked to ech other through electropositive ridges (minly, proton or metl, in this cse Fe) voided the short-term differences in gronomicl efficcy Fig. 2 First, second, nd third hrvests shoot P concentrtion (mg kg 1 dry weight) for RP dotted r, P gry shded r, RP/Trp cross-lined r nd RP/(HA Trp) lck shded r. Columns followed y the sme lowercse letter re not sttisticlly different from ech other (Fisher s test. P < 0.05)

8 Fernández et l. Chem. Biol. Technol. Agric. (2016) 3:5 Pge 8 of 9 Tle 4 Fertilizer efficiency index defined y [(dry mtter production) 100]/(dded P to the soil) Tretments RP SSP RP/Trp RP/(HA Trp) Added P (mg kg 1 soil) Hrvest First Second c d d d 0.65 Third c 0.46d d 0.69e c 0.46d c 0.60e Dt followed y the sme lowercse letter in ech row re not sttisticlly different from ech other (Fisher s test, P < 0.05) Tle 5 Plnt P utiliztion efficiency index defined y [(dry mtter production) 10,000]/(shoot P concentrtion) Tretments RP SSP RP/Trp RP/(HA Trp) Added P (mg kg 1 soil) Hrvest First Second c c Third Dt followed y the sme lowercse letter in ech row re not sttisticlly different from ech other (Fisher s test, P < 0.05) existing etween RP nd SSP, thus improving the strter ction of RP-sed fertilizers. This issue is very relevnt ecuse the min negtive feture of RP s P source for pstures cultivted in cidic soils is its poor short-term (1-yer) effect in comprison with tht of wter-solule P fertilizers, thus negtively ffecting yield nd qulity [1]. However, the long-term ction (3 4 yers) of RP-sed fertilizers s P source for pstures cultivted in cidic soils my e even more efficient thn tht of wter-solule P fertilizers [1, 4, 5]. This fct indictes tht oth fertilizers (HA Trp ctivted RP nd SSP) my e rther complementry in whole fertiliztion pln for pstures in cidic soils. Authors contriutions LF principlly ut lso RB, OU, nd JE hve mde the experimentl work nd contriuted to MS preprtion; RB, OU, JE, nd PMA contriuted to the experimentl design; JCY contriuted to the discussion of results; nd JMG contriuted to the design of the experiments, discussion of results, MS preprtion, nd finl writing. All uthors red nd pproved the finl mnuscript. Author detils 1 Institute of Agro Biotechnology, Pulic University of Nvrr-CSIC-Government of Nvrr, Cmpus de Arrosdí, Pmplon, Nvrr, Spin. 2 Deprtment of Environmentl Biology (Agriculturl Chemistry nd Biology Group), Fculty of Sciences, University of Nvrr, Pmplon, Spin. 3 Centre Mondil d Innovtion (CMI), Roullier Group, Sint Mlo, Frnce. 4 Deprtment of Environmentl Biology, Agriculturl Chemistry nd Biology Group, Fculties of Sciences nd Phrmcy, University of Nvrr, C/Irunlrre 1, Pmplon, Nvrr, Spin. Acknowledgements This Reserch Project hs een supported y Grnt from CDTI nd Government of Nvrr, s well the Roullier Group. We would like to thnk Pul W. Miller for kindly improving the English of the mnuscript. Competing interests The uthors declre tht they hve no competing interests. Received: 7 Septemer 2015 Accepted: 1 Mrch 2016 References 1. Domínguez Vivncos A. Trtdo de fertilizción. Mdrid: Mundi-Prens; Vn Strten P. Rocks for crops, Agrominerls of su-shrn Afric. Guelph: University of Guelph; He ZL, Bligr V, Mrtens DC, Ritchey KD, Elrshidi M. Effect of yproduct, nitrogen fertilizer, nd zeolite on phosphte rock dissolution nd extrctle phosphorus in cid soil. Plnt Soil. 1999;208: Chien SH, Hmmond LL. Agronomic effectiveness of prtilly cidulted phosphte rock s influenced y soil phosphorus-fixing cpcity. Plnt Soil. 1989;120: Zoys AKN, Lognthn P, Hedley MJ. Comprison of the gronomic effectiveness of phosphte rock nd triple superphosphte s phosphte fertilisers for te (Cmelli sinensis L.) on strongly cidic Ultisol. Nutr Cyc Agroecosys. 2001;59: Hmmond LL, Chien SH, Roy AH, Mokwunye AU. Soluility nd gronomic effectiveness of prtilly cidulted phosphte rocks s influenced y their iron nd luminium oxide content. Fert Res. 1989;19: Ghni A, Rjn SSS. Differentil vilility of prtilly sulphuric nd phosphoric cidulted phosphte rocks I plnt response. Nutri Cyc Agroecosys. 1997;47: McSweeney G, Chrleston AG. Prtilly cidulted phosphte rocksrections with wter. Fert Res. 1985;8: Menon RG, Chien SH, Hmmond LL. Comprison of Bry I nd Pi tests for evluting plnt-ville phosphorus from soils treted with different prtilly cidulted phosphte rocks. Plnt Soil. 1989;114: Begum M, Nrynsmy G, Bisws DR. Phosphorus supplying cpcity of phosphte rocks s influenced y compction with wter-solule P fertilizers. Nutr Cyc Agroecosys. 2004;68:73 84.

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