INTRODUCTION

Slow-release fertilizers(SRFs)are condensation products obtained by reacting urea,the most common mineral fertilizer characterized by a high nitrogen(N)content and relatively low cost,with several aldehydes.These SRFs release N at slower rates compared to conventional N-fertilizers,such as urea releasing N rapidly by hydrolysis;thus,in theory SRFs facilitate better N uptake and utilization by crop plants.Potential benefits from SRFs include improved N use effi-ciency(NUE),reduced volatilization loss and nitrate leaching,increased N availability during plant growing season,and reduced costs of application(i.e.,multiple applications of conventional N-fertilizers vs.a single application of SRFs)(Allen,1984).Among SRFs,urea-formaldehyde(UF),isobutylidene diurea(IBDU),and crotonylidene diurea(CDU)have gained popularity(Trenkel,1997).Urea-formaldehyde is obtained by reacting urea with formaldehyde and consists of a mixture of chain polymers with different lengths.The degradation and subsequent N release from UF is driven by the size and activity of soil micro flora(Alexander and Helm,1990)and factors that influence microbial activity,such as soil moisture and temperature.Crotonylidene diurea,a ring-structured compound,is produced by condensation of urea with acetic aldehyde.Both microbial activity and hydrolysis drive the degradation of CDU.Thus,soil moisture,temperature,and pH in fluence the CDU-N release(Trenkel,1997).Isobutylidene diurea is a single oligomer compound,formed by the reaction of urea and isobutyraldehyde.The IDU-N release occurs by chemical hydrolysis,and its decomposition in soil is affected by soil temperature,moisture,and pH(Guertal,2009).

Slow-release fertilizers have been largely studied under different temperature and soil moisture regimes(Engelsjord et al.,1997;Agehara and Warncke,2005;Fan and Li,2010;Fan et al.,2011),whereas the effect of size and activity of soil micro flora on SRF degradation has received less attention with contrasting results observed(Benedetti,1983;Koivunen and Horwath,2004).For example,in the case of UF,a significant positive relationship between microbial activity and N release was found under laboratory conditions(Trinchera et al.,2010).However,Koivunen and Horwath(2004)observed that both size and activity of soil micro flora have no significant effects on N release.In addition,due to the role played by the pH in regulating hydrolysis reactions,the evaluation of N release from SRFs under different soil pH has implications on their performance in different soil types.To date,the question of whether N release from SRFs proceeds faster in soils with higher microbial activity is still open.The aim of this study was therefore to better understand such relationships by determining N release from UF,IBDU,and CDU in soils with different microbial activities.Given that N release from UF should be mainly driven by microbial activity,we hypothesized a faster N release from UF in soils with higher microbial activity.

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MATERIALS AND METHODS

Soils and fertilizers

Four soils with different physicochemical and biological characteristics were selected for this research.Soils were all classified according to the World Reference Base for Soil Resources(FAO,2015),as reference European classification.The first soil was a Gleyc Cambisol with sandy loam topsoil(43°18′31.28′′N,11°49′15.02′′E),developed on recent alluvial deposits with seasonal herbaceous crop land use inside the Cesa Research Centre for Agricultural Technologies and Extension Services(CATES)in the Province of Arezzo,Italy.The second soil was a Calcaric Fluvisol with loam topsoil(43°40′59.05′′N,10°20′32.06′′E),developed on the reclaimed alluvial floodplain of the Arno River with seasonal herbaceous crop land use inside the Interdepartmental Centre for Agro-Ecological Research Enrico Avanzi(CIRAA),located near the city of Pisa,Italy.The third soil was a Cambic Umbrisol with clay loam topsoil(42°22′6.83′′N,12°19′44.03′′E),developed on a volcanic tuffplateau of the Vico complex with permanent crop(hazelnut orchards)land use,near Viterbo,Italy.Finally,the fourth soil was a Relictigleyc Calcaric Cambisol with a loamy sand topsoil(49°24′50.27′′N,8°23′56.76′′E),developed on recent sandy alluvial deposits of the Rhine River with seasonal herbaceous crop land use,near Limburgerhof,Germany.Topsoil samples were collected from the 0–15 cm depth(Ap horizon),dried at room temperature(approximately 25°C)for 2 weeks,and then passed through a 2-mm sieve.Main soil physicochemical parameters are presented in Table I.The N contents in the SRFs were 380,320,and 320 g kg−1for UF,IBDU,and CDU,respectively,and the urea with 460 g kg−1 N was used as a control.

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TABLE I Selected physicochemical properties of four soils used in this study,including a Gleyc Cambisol in the Province of Arezzo,Italy,a Calcaric Fluvisol near the city of Pisa,Italy,a Cambic Umbrisol near Viterbo,Italy,and a Relictigleyc Calcaric Cambisol near Limburgerhof,Germany

a)EC=electrical conductivity;OM=organic matter;CEC=cation exchange capacity. b)According to USDA Soil Taxonomy.

Propertya) Arezzo Pisa Viterbo Limburgerhof Sand(%) 66 40 39 84 Silt(%) 16 38 32 10 Clay(%) 18 22 29 6 Soil textureb) Sandy Loam Clay Loamy loam loam sand pH 7.8 7.8 5.2 7.4 EC(dS m−1) 0.19 0.24 0.10 0.22 OM(g kg−1) 10.30 20.20 34.40 15.10 CEC(cmol kg−1) 15.43 17.74 16.47 9.22 Organic C(µg g−1) 6.0 11.7 19.9 8.7 N(µg g−1) 0.6 1.1 1.1 0.9 C/N ratio 9.9 10.5 18.3 9.7

Microbial biomass carbon(MBC)and basal soil respiration

SoilMBC wasmeasuredbythechloroform fumigation-extraction method(Vance et al.,1987)using air-dried soil samples conditioned by a 21-d incubation in open glass jars at 33 kPa water tension and 30°C to restore soil microbial activity.Two portions of 20 g(oven-dry basis)moist soil sample were weighed∶the first one(non-fumigated)was extracted with 80 mL of 0.5 mol L−1K2SO4for 30 min by oscillating shaking at 200 r min−1and then filtered using Whatman No.42,and the second one was fumigated for 24 h at 25°C with ethanol-free CHCl3.Following fumigant removal,the soil was extracted similarly as the non-fumigated soil sample.Organic carbon(C)in the extracts was determined after oxidation with 0.4 mol L−1K2Cr2O7at 100 °C for 30 min,and MBC was calculated as follows∶

Modified leaching method of Stanford and Smith(1972)(Benedetti et al.,1994)was used to measure N release from N sources.Briefly,50-g air-dried soils were mixed with quartz sand at a 1∶1 ratio(weight∶weight),followed by the addition of N-fertilizer at the ratio of 250 mg N kg−1.The mixtures(soil,quartz sand,and N-fertilizer)were then transferred to the B¨uchner funnels and incubated at 60%water-holding capacity(WHC)(pF=2.5)at 30°C.After 7,14,28,45,60,75,and 90 d,released inorganic N(Ning)was leached as NO−3-N and NH+4-N with 500 mL of 0.01 mol L−1CaSO4solution.To avoid nutrient limiting effects,after each leaching,a nutrient solution without N containing 0.002 mol L−1CaSO4,0.002 mol L−1 MgSO4,0.005 mol L−1Ca(H2PO4)2,and 0.0025 mol L−1K2SO4was added to the mixtures.During the incubation,B¨uchner funnels were covered with punctuated aluminium foil and weighed every 3 d to monitor soil moisture.Distilled water was added to maintain soil at 60%WHC.Non-fertilized soil was considered as the control.Leachates were filtered by Whatman No.42 filter paper and analyzed for NH+4-N and NO−3-N with a Technicon II autoanalyser(Bran&Luebbe,Sydney,Australia).Net NH+4-N and NO−3-N on time from fertilizer release in each soil were calculated after the subtraction of those of the control soil.Nitrogen concentrations were reported on a dry matter basis.Each treatment was replicated three times.

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It should be noted that soil MBC and basal respiration were only measured once,just prior to the soil incubation.We recognized that this may represent a limit of our approach as both MBC and microbial activity can change during the incubation.This excludes the possibility to determine causal links between microbial activity and N release from SRFs.On the other hand,our approach allows for the determination of the relationships between pre-existing microbial activity and fertilizer degradation.Therefore,it provided valuable information on the use of soil microbial activity as a putative indicator of fertilizer degradation.

②《医疗事故处理条例》第四条 根据对患者人身造成的损害程度，医疗事故分为四级：一级医疗事故：造成患者死亡、重度残疾的；二级医疗事故：造成患者中度残疾、器官组织损伤导致严重功能障碍的；三级医疗事故：造成患者轻度残疾、器官组织损伤导致一般功能障碍的；四级医疗事故：造成患者明显人身损害的其他后果的。 具体分级标准由国务院卫生行政部门制定。

Isobutylidene diurea released N at very low rate during the first week of incubation,with net Ningconcentration consistently less than 17 mg N kg−1soil,with non-significant(P ＞ 0.05)differences among soils(Fig.2).Conversely,approximately 68%,76%,and 79%of the applied N was recovered in Arezzo,Pisa,and Limburgerhof soils,respectively,during the next two incubation periods,i.e.,days 7–14 and 15–28.Distinct peaks in net Ningconcentration were also found for the Viterbo soil during the same incubation periods,but the amount of N released was significantly lower(P＜0.05)compared to the other three soils.During days 8–14,net Ningconcentration increased 10-fold to 83 mg N kg−1soil in Arezzo soil and 64 mg N kg−1 soil in Limburgerhof soil,7-fold to 16 mg N kg−1soil in Pisa soil,and approximately 4-fold to 32 mg kg−1 soil in Viterbo soil(P＜0.001)compared to those during days 0–7.From days 15 to 28,net Ningconcentration increased 2-fold in Limburgerhof and Viterbo soils(P＜0.001),but it remained almost constant in Arezzo soil(P=1.000),and significantly decreased in Pisa soil(P＜0.001)compared to the former duration.After 28 d,net Ningconcentration significantly decreased in all soils and became negligible for the remaining period.Nitrogen was nitrified faster in Arezzo,Pisa,and Limburgerhof soils,but persisted in NH+4-N in Viterbo soil.

Soil incubation procedure

where EC is the difference between organic C extrac-ted from fumigated and non-fumigated soils and kEC,the conversion factor,is 0.38(or 1/2.64)(Vance et al.,1987).

Statistical analysis

使用外量子效率和暗场J-V曲线对太阳能电池性能变化作进一步分析.如图2(c)和2(d)所示，对波长小于800 nm的光子，所有电池的外量子效率基本相同；但是，当引入MgO介质层后，器件在大于800 nm波段的外量子效率得到明显提高，并随MgO厚度有先增加后减少的趋势，这与JSC以及光电转换效率的变化吻合.Gr/Si太阳能电池的暗场正向电流J可表示为[18]

where Nrel(mg N kg−1soil)is the cumulative N released from each fertilizer at time t(d),N0(mg N kg−1soil)is the potentially mineralizable N in the fertilizer,and K0(d−1)is the mineralization rate constant.All data points were used in the regression analysis,although data were reported as mean values.The N0and K0were considered statistically different at P ＜ 0.05,if the 95%con fidence intervals did not overlap.Data were analyzed using SPSS version 22.0 software.

RESULTS

Soil MBC and basal respiration

Among the four studied soils,MBC was significantly the lowest(P ＜0.05)in Arezzo soil(52.04µg C g−1soil)and significantly higher in Pisa(115.57 µg C g−1soil)and Viterbo(144.31 µg C g−1soil)soils(Fig.1).All soils showed significant differences in basal respiration in the range from 1.78 µg CO2-C g−1soil in Limburgerhof soil to 12.39 µg CO2-C g−1soil in Viterbo soil.

Soil effects

At the end of the incubation period,CDU released approximately 51%of the applied N with Ningconcentrations being 110.3 and 138.9 mg N kg−1soil for Arezzo and Viterbo soils,respectively(Table II).However,the difference was not statistically significant(P ＞0.05).Isobutylidene diurea released more than 80%of the applied N with a significantly lower amount of N detected from the Viterbo soil(147.9 mg N kg−1soil;P＜0.05).The recovered urea-N ranged from 89%to 100%of the applied N,with non-significant differences among soils.Nitrogen released from UF was signi ficantly lower in Viterbo soil(116.4 mg N kg−1soil;P＜0.05)than the other soils.

To measure soil basal respiration,25 g(oven-dry basis)moist soil sample was weighed in a vessel and then placed inside a 1-L stoppered glass jar.The CO2 evolved was trapped,and 2 mL of 1 mol L−1NaOH was placed in another vessel inside the jar.After 1,2,4,7,10,14,17,and 21 d,CO2was determined by titration of the excess NaOH with 0.1 mol L−1HCl(Alef,1995).The average of the 21-d measurements was used as the basal respiration value for each soil.

Fertilizer effects

In all soils,fertilizers released a decreasing amount of N in the order∶urea＞ IBDU＞UF＞ CDU(Table II).Net cumulative concentration of Ningwas signi ficantly different(P ＜ 0.05)among fertilizer treatments for the Arezzo soil.In Limburgerhof and Pisa soils,net cumulative Ningconcentration was significantly higher(P＜0.05)for urea and IBDU treatments than UF and CDU treatments,with no differences between the former two fertilizer treatments.In Viterbo soil,the net cumulative Ningreleased from CDU and UF was not statistically different(P ＞ 0.05).

贮藏初期因薯块刚入窖，窖温和湿度可能高一些，这是正常现象，但一般不会超过20℃，20天后窖温下降。长期贮藏的块茎，温度在2～4℃之间最合适，这样块茎不会发芽。湿度维持在85%～90%为宜，可使块茎不致抽缩，保持新鲜状态。

Dynamics of N concentration as affected by soil type,fertilizer,and incubation time

A two-way ANOVA was performed to evaluate the effects of soil and fertilizer on net Ningat the end of incubation(90 d)(Table III).However,the main effects of soil(F=29.40,P ＜ 0.001)and fertilizer(F=177.05,P＜0.001)were both significant and superseded by significant soil and fertilizer interactions(F=6.54,P＜0.001).The three-way mixed betweenwithin subject ANOVA showed that incubation time was the within-subject factor of Ningconcentration,whereas fertilizer and soil type,factorially combined with incubation time,were the between-subject factors.For NH+4-N and NO−3-N,as well as for Ning,there were significant main effects for both within-and bet-ween-subject factors.However,these effects were obtained in the context of significant three-way interactions∶F=197.47,P ＜ 0.001 for NH+4-N,F=76.67,P＜0.001 for NO3N,and F=16.27,P＜0.001 for Ning.

Fig.1 Microbial biomass C and basal respiration in four soils prior to the incubation.Values are means with standard deviations shown by vertical bars(n=3).Different letters indicate statistically different means between soils at P ＜ 0.05.

TABLE II Net cumulative concentrations of inorganic N(NH+4-N+NO−3-N)in four soils amended with slow-releas fertilizers(crotonylidene diurea(CDU),isobutylidene diurea(IBDU),urea,and urea-formaldehyde(UF))at the end of the incubation period(90 d)

a)Means±standard errors(n=3). b)Means followed by different lowercase letters within a column and uppercase letters within a row are statistically different for a given soil and fertilizer,respectively(P＜0.05).

Soil Net cumulative concentration of inorganic N CDU IBDU Urea UF mg N kg−1soil Arezzo 127.8±3.4a)aAb) 217.7±5.8aB 249.8±17.7aC 168.1±5.8aD Limburgerhof 132.6±10.4aA 220.8±8.9aB 222.6±8.6aB 181.8±8.7aD Pisa 138.9±5.5aA 235.8±3.2aB 249.5±5.6aB 175.8±4.8aD Viterbo 110.3±3.8aA 147.9±3.2bB 241.2±2.8aC 116.4±6.3bA

TABLE III Three-way mixed analysis of variance results for the effects of slow-release fertilizers(crotonylidene diurea,isobutylidene diurea,urea,and urea-formaldehyde),soil type(Arezzo,Limburgerhof,Pisa,and Viterbo),and incubation time(7,14,28,45,60,and 90 d)on soil net N concentrations

a)Degree of freedom.

Factor(s) Inorganic N NH+4-N NO−3-N dfa) F value P value df F value P value df F value P value Between subjects Soil type(ST) 3 29.40 ＜0.0001 3 592.22 ＜0.0001 3 488.74 ＜0.0001 Fertilizer 3 177.05 ＜0.0001 3 43.51 ＜0.0001 3 174.45 ＜0.0001 ST×fertilizer 9 6.54 ＜0.0001 9 134.76 ＜0.0001 9 39.33 ＜0.0001 Error 32 32 32 Within subjects Incubation time(IT) 3.21 1172.3 ＜0.0001 3.85 651.22 ＜0.0001 2.78 633.12 ＜0.0001 IT×ST 9.64 22.7 ＜0.0001 11.55 203.19 ＜0.0001 8.35 100.08 ＜0.0001 IT×fertilizer 9.64 951.5 ＜0.0001 11.55 369.75 ＜0.0001 8.35 618.48 ＜0.0001 IT×ST×fertilizer 28.90 16.3 ＜0.0001 34.65 197.47 ＜0.0001 25.04 77.67 ＜0.0001 Error 103 32 32

Crotonylidene diurea-amended soils

Net Ningconcentrations were quite low during thefirst 14 d in all soils amended with CDU with the exception of Pisa soil,and then increased significantly from days 15 to 28 in all the soils,but generally decreased afterwards(Fig.2).In particular,on day 7,net Ningconcentration was in the range of 14%,6%,5%,and 3%of the applied N in Pisa,Arezzo,Limburgerhof,and Viterbo soils,respectively,with significant differences between Pisa and Limburgerhof soils(P＜0.05)and between Pisa and Viterbo soils(P＜0.01).The net Ningconcentration significantly decreased(P＜0.01)in Pisa soil by day 14.From days 15 to 28,net Ningconcentrations increased 3.5-fold to 27.5 mg N kg−1soil,11-fold to 43.3 mg N kg−1soil,15-fold to 53 mg kg−1soil than the former period(from days 8 to 14)in Pisa,Limburgerhof,and Viterbo soils,respectively(P＜0.001).On day 28,Arezzo soil exhibited the significantly lowest net Ningconcentration(P＜0.001)compared to the other three soils.From days 29 to 45,net Ningconcentration significantly decreased in Viterbo soil(P＜0.05)and remained quite constant in Limburgerhof and Pisa soils.Nitrogen dynamics differed among soils.Pisa soil,with the exception of the first week,was clearly dominated by the prevalence of NO−3-N during the incubation period.Yet,NO−3-N was prevalent in Arezzo soil for at least the first three-incubation periods and in Limburgerhof soil from days 29 to 45 and 46 to 60.In Viterbo soil,NO−3-N was almost absent during the first two weeks,but increased afterwards.

Isobutylidene diurea-amended soils

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Urea-amended soils

As expected,urea released N very fast,and almost all N added was recovered during the first 2 weeks of incubation(Fig.2).The remaining N was recovered on day 14,with no difference(P ＞ 0.05)among soils.

Soil MBC and basal respiration were subjected to one-way analysis of variance(ANOVA).A two-way ANOVA was performed to test differences in net cumulative Ning(NH+4-N+NO−3-N)at the end of incubation(90 d).We performed a 7×4×4 threeway mixed between-within subject ANOVA to test the effect of soils from Arezzo,Limburgerhof,Pisa,and Viterbo,fertilizers(CDU,IBDU,Urea,and UF),and incubation time on soil Ningconcentrations during 90-d incubation.In the mixed ANOVA model,time was the within-subject factor,whereas soil and fertilizers were considered as the between-subject factors.The Mauchly’s test of sphericity indicated a violation of the sphericity assumption for N concentrations;therefore,the Greenhouse-Geiser adjusted degrees of freedom of F-distribution was used(von Ende,1993).In the twoway ANOVA,as well as in the three-way ANOVA,main effects were superseded by significant interactions.To break down these interactions,we performed a simple effect analysis with Bonferroni-adjusted simple effect tests,holding the alpha level at 0.05.We used non-linear regression to fit N leaching rate to a first-order model(Agehara and Warncke,2005)as follows∶

Urea-formaldehyde-amended soils

In soils amended with UF,net Ningconcentration decreased with incubation time in Pisa,Arezzo,and Limburgerhof soils(Fig.2).The UF-N recovered on day 7 was 26%,24%,and 19%of the applied N in Pisa,Arezzo,and Limburgerhof soils,respectively,with no significant difference.During the same incubation period,approximately 10%of the applied N was recovered in Viterbo soil with significant differences(P ＜ 0.01)compared to Pisa and Arezzo soils.During days 15–28,a significantly higher net Ningconcentration(52.5 mg kg−1soil)was released in Viterbo soil than Arezzo and Limburgerhof(P＜0.001)and Pisa(P＜0.05)soils.The released N was nitrified very fast in Pisa soil,as NO−3-N was the prevalent N form during the incubation period.Arezzo soil followed similar dynamics,with NO−3-N concentration always higher than that of NH+4-N.The balance between the two N forms was different in Limburgerhof soil because of the prevalence of NH+4-N during days 0–7 and the presence of both NH+4-N and NO−3-N during days 8–14,whereas NO−3-N dominated the remaining incubation period.As observed for the other treatments,Viterbo soil showed different N dynamics,with NH+4-N as the dominant form during the first 28 d of incubation but higher NO−3-N concentration during days 29–45 and 46–60.

Fig.2 Net concentrations of inorganic N(Ning)containing NH+4-N and NO−3-N in four soils(Arezzo,Limburgerhof,Pisa,and Viterbo)amended with slow-release fertilizers(crotonylidene diurea(CDU),isobutylidene diurea(IBDU),urea,and urea-formaldehyde(UF))during the 90-d incubation.

Nonlinear regression

The first-order model generally fit the obtained data(Fig.3,Table IV),with the only exception of Arezzo soil fertilized with CDU,where the model predicted an unrealistic N0.The analysis of residuals(data not shown)revealed systematic patterns in their distribution;thus indicating that the observed data for CDU-amended Arezzo soil were not well described by the model.Therefore,both N0and K0predicted for Arezzo soil amended with CDU were excluded and not compared with the other regression coefficients.Nonsignificant differences(P ＞ 0.05)in both N0and K0 were observed in the other soils treated with CDU(Table IV).For IBDU-amended soils,N0was significantly lower(P＜0.05)in Viterbo soil than the other three soils,whereas no statistical differences were detected for K0.In soils amended with urea,Limburgerhof soil showed the lowest N0,and K0values were not statistically different among the four soils.Interestingly,for UF-amended soils,both N0and K0were signi ficantly lower in Viterbo soil(P＜0.05)than the other soils,and the highest K0was observed in Arezzo soil,followed by Pisa and Limburgerhof soils,with statistical differences between Arezzo and Limburgerhof soils(Table IV).

DISCUSSION

Fig.3 Nonlinear regressions of net cumulative inorganic N(Ning)in four soils(Arezzo,Limburgerhof,Pisa,and Viterbo)amended with slow-release fertilizers(crotonylidene diurea(CDU),isobutylidene diurea(IBDU),and urea-formal dehyde(UF))against incubation time according to the first-order model.

It should be noted that in this study,we were primarily interested in the elucidation of the relationships that exist between soil microbial activity and N release from slow-release fertilizers.Regarding the effects of the size and activity of soil micro flora on UF degradation,the following can be discussed.Soil microbial activity was in the following order∶Viterbo＞ Pisa＞Arezzo＞Limburgerhof.If microbial activity was the main driving factor of UF decomposition,N release would be expected to decrease from Viterbo to Limburgerhof soils,according to the scheme above.Interestingly,net cumulative Ningwas the highest in Limburgerhof soil,followed by Pisa and Arezzo soils.Most importantly,it was significantly lower in Viterbo soil,which was characterized by the largest and more active soil micro flora.These findings were additionally supported by K0predicted for Arezzo(K0=0.062)and Viterbo(K0=0.031)soils.Although the in fluence of microbial activity on UF degradation has been well-documented(Benedetti,1983;Nicolardot et al.,1994),our results generally agree to those of Koivunen and Horwath(2004),who did not observe a direct link between N release from methylene urea,a slowrelease fertilizer,and soil microbial activity.This lack of consistency could be explained as follows.A mixture of methylene urea with different long-chain polymers forms UF,which could be broken-down by both soil bacteria and fungi(Alexander and Helm,1990).To date,different bacterial methylene urea-degrading enzymes have been purified(Jahns et al.,1997;Jahns and Kaltwasser,2000;Koivunen et al.,2004),and these were structurally different,depending on the specific microorganisms and geographic locations.A limit of our approach was that we neither performed enzymatic assays,nor identified UF-degrading microorganisms.However,it is reasonable to conclude that the degradation of this fertilizer in soils was driven by the presence of specific UF-degrading microorganisms providing methylenediurea deaminase activity,and thus be higher in soils harboring such microorganisms.Therefore,specific enzymatic activities rather than total soil microbial activity may better represent a predictor of UF degradation in soils.Another important aspect characterizing UF was that a relatively fast N release occurred during the first 2 weeks of incubation,during which the applied N was recovered from 30%to 40%.Although this could be explained by the presence of unreacted urea in the UF,it is noteworthy that in Pisa soil,and to some extent also in Arezzo soil,the N released was rapidly nitrified,creating the potential for N losses through NO−3leaching or denitrification.Therefore,the use of this fertilizer does not necessarily avoid environmental problems.

Nitrogen release from IBDU occurred mostly during the first 28 d of incubation with a lag phase in the first week,followed by a quick release during days 14–28.Among parameters in fluencing IBDU-N release,granule particle size,temperature,soil water content,and pH are considered the most important.In addition,as hydrolysis is faster in acidic condition,N release should increase with lowering pH(Trenkel,1997).Our results showed that N release did differ among Arezzo,Limburgerhof,and Pisa soils,which were characterized by similar pH.However,the expected higher N release in Viterbo soil due to its lower pH was not observed.Conversely,IBDU degradation was found to be higher in soils with the higher pH,where hydrolysis was presumed to be slower.However,the discovery of the soil bacteria Rhodococcus erythropolis,a strain able to degrade IBDU through the activity of a specific enzyme designated as an IBDU-amidinohydrolase,challenged the view that IBDU degradation occurs only abiotically(Jahns and Scheep,2001).In addition,the optimum pH for the IBDU-amidinohydrolase activity has been shown to be in the range of 8.0 to 8.5,which is closer to those of Arezzo,Pisa,and Limburgerhof soils.Therefore,the biotic pattern of IBDU degradation and,most importantly,the optimum pH for enzymes could explain our finding of higher IBDU degradation in the soils with a higher pH.Finally,regarding CDU degradation,it involves chemical hydrolysis and microbial attack.However,throughout the incubation period,neither the amount of the released N nor thepredicted regression coefficients differed significantly among the studied soils.Yet,our findings stand in contrast to previous studies reporting a faster N release in acidic than alkaline soils(Varadachari and Goertz,2010).Moreover,as for UF and IBDU,CDU-degrading microorganisms have been isolated from soils(Jahns et al.,2003).Our results indicate that both total microbial activity and pH are weak indicators of CDU behavior in soil,which led us to speculate that the presence and activity of specific microorganisms capable of degrading CDU may represent the key to predicting the N release rate.

TABLE IV Potentially mineralizable N in the fertilizer(N0)and mineralization rate constant(K0)in four soils amended with slow-release fertilizers(crotonylidene diurea(CDU),isobutylidene diurea(IBDU),urea,and urea-formaldehyde(UF))estimated by nonlinear regressions,assuming first-order kinetics

a)Statistical significance was not reported,because data did not fit the model used.b)Means followed by the same letter(s)within a column are not significantly different.

Soil CDU IBDU Urea UF N0 K0 N0 K0 N0 K0 N0 K0 mg N kg−1soil d−1 mg N kg−1soil d−1 mg N kg−1soil d−1 mg N kg−1soil d−1 Arezzo 1025.7a) 0.002a) 235.7a 0.038a 250.4a 0.482a 158.4a 0.062a Limburgerhof 192.6ab) 0.014a 242.0a 0.038a 222.8c 0.368a 179.0a 0.037bc Pisa 155.4a 0.024a 246.2a 0.049a 249.6ab 0.432a 163.5a 0.053ab Viterbo 147.3a 0.018a 170.3b 0.030a 241.3b 0.407a 131.1b 0.031c

CONCLUSIONS

We did not observe significant relationships between soil microbial activity and N release from UF-amended soils.It is possible that the latter occurs mainly due to the activity of microorganisms able to produce UF-degrading enzymes.Therefore,soil microbial activity appears to be a poor indicator of the UF behavior in soils.The effect of pH on IBDU degradation was of secondary importance because higher N release in acidic conditions was not observed.Yet,N release from CDU-amended soils was neither directly affected by microbial activity nor by soil pH.Therefore,although we recognize the importance of microbial activity for N release from SRFs,the results of the present study suggest that the size and the total activity of soil micro flora had marginal effects on fertilizer degradation.

ACKNOWLEDGEMENT

We thank the reviewers for the insightful comments and suggestions.

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