Plant Soil DOI 10.1007/s11104-017-3405-8

REGULAR ARTICLE

Effects of microbial bioeffectors and P amendements on P forms in a maize cropped soil as evaluated by 31P–NMR spectroscopy Meng Li & Vincenza Cozzolino & Pierluigi Mazzei & Marios Drosos & Hiarhi Monda & Zhengyi Hu & Alessandro Piccolo Received: 8 February 2017 / Accepted: 28 August 2017 # Springer International Publishing AG 2017

Abstract Background and aims Identification of organic P species is important to understand their origin, turnover in soils and their effects on soil fertility. Attention has been recently devoted to microbial inocula, referred to as Bioeffectors, that are capable to increase P bioavailability and plant uptake. Nevertheless, little is known on the effect of Bioeffectors on soil P forms and their dynamics in agricultural soils upon different P fertilization. Methods We investigated the effects of the application of different commercial inocula strains (Trichoderma harzianum T 22, Pseudomonas sp., and Bacillus amyloliquefaciens) alone or in combination with different P fertilizers (triple superphosphate, rock phosphate, and both composted cow- and horse-manure) on soil organic P forms. P forms were characterized by liquidstate 31P–NMR spectroscopy, while plant P uptake from

P-treated soil was followed in a greenhouse pot experiment under maize cultivation. Results NMR spectra showed that the type of P fertilizer and bioeffectors inoculation, affected the abundance and the composition of organic P forms. The specific capacity of all bioeffectors, and especially Pseudomonas, was related to an increased content of diesters P forms. Pseudomonas, and, to a lesser extent, B. amyloliquefaciens showed the largest increase in combination with organic P amendments, which also provided the largest plant P uptake. This suggests a key role of Diester-P forms in determining P availability in agroecosystems. Conclusions Microbial inoculation plays an important role in the dynamics of soil P, inducing a rapid P cycling that prevents P fixation and losses from soils, thus enhancing the P fertilizer use efficiency in agricultural soils.

Responsible Editor: Daniel Menezes-Blackburn.

Keywords Solution-state 31P–NMR . Soil fertility . Phosphorus fertilizer . Plant growth promoter . Plant P . Diester phosphates

M. Li : Z. Hu College of Resources and Environment, Sino-Danish College, University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China V. Cozzolino (*) Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy e-mail: [email protected] P. Mazzei : M. Drosos : H. Monda : A. Piccolo (*) Interdepartmental Research Centre CERMANU, University of Naples Federico II, Portici, Italy e-mail: [email protected]

Introduction Phosphorus (P) is a critical resource for agricultural productivity and plays a vital role in the soil-plant system (Condron and Newman 2011). A sustainable global food production requires to improve the efficacy of the plant P uptake by advancing knowledge on the nature of P species in soils, and their turnover rates and bioavailability. The large variety of inorganic and organic P

Plant Soil

forms in soil differ in terms of behavior and bioavailability (Vincent et al. 2010). Orthophosphate (Ortho-P) represents the main inorganic P form and the most important P source for plant and microbes uptake, whereas pyrophosphate (Pyro-P) and inorganic polyphosphates (Poly-P) content in soil is usually small, but they can be rapidly hydrolyzed to Ortho-P, thus becoming a potentially available P source (TorresDorante et al. 2005; Cheesman et al. 2010). Soil organic P (OP) forms represents a large share of total P, although their content may vary considerably (Turner and Newman 2005). In most mineral soils, OP occurs as a mixture of monoesters (Mono-P) and diesters (Di-P) phosphates, with smaller amounts of phosphonates (Phos-P) and organic polyphosphates (Turner and Engelbrecht 2011; Cade-Menun and Liu 2013). While Mono-P include a number of organic phosphates and inositol hexakisphosphates, diesters comprise phospholipids (PL) and DNA-P (Paraskova et al. 2014). Although most input of OP in soil originates from Di-P (Cosgrove 1967), DNA-P is easily hydrolyzed in soil and leaves PL and Mono-P to represent the prevailing form of soil organic P. OP compounds are continuously degraded into plant accessible phosphate by enzymes and acids produced by microbes and roots, thereby cycling between microbial fixation and release (Rodríguez and Fraga 1999; Richardon 2010; Richardson et al. 2011). Due to precipitation and immobilization processes, P availability is commonly suboptimal for vegetative growth for approximately 70% of arable land (HerreraEstrella and López-Arredondo 2016). In most soils, crops assimilate only 20–30% of applied P, since it is rapidly fixed in the soil due to its high reactivity with cations such as calcium and magnesium in calcareous soils or aluminum and iron in acidic soils (Brady and Weil 2008). Also microbial P immobilization and remineralization contribute to reduce P availability to plants (Frossard et al. 2000). Consequently, farmers in developed countries applied two to four times as much P as was removed in the crop harvest (Brady and Weil 2008). However, this overfertilization entails growing disadvantages in terms of dwindling P resources and enhance environmental pollution. (Withers and Haygarth 2007). The progressive decrease of rock phosphate as P source (Cordell et al. 2009) has called to integrate P additions with manure or manure compost, where OP forms account for a large part of their P composition. Recycling manure is of huge importance for optimizing P-resource use in the future (Shen et al. 2011), since,

unlike other non-renewable resources such as fossil fuels, for which alternative products are or will soon be available, inorganic P fertilizer has no alternative or replacement (Herrera-Estrella and López-Arredondo 2016). Several studies had been conducted to increase understanding of how the molecular quality of OP fertilizers affects composition of P in soil. For example, a crop field experiment that applied several types of manure amendment into a silt loam soil, reported that manure applications increased Mono-P content in soil (Shafqat et al. 2009). On the other hand, a recent fertility experiment monitored the effect of P fertilizers on the organic P composition and found that Mono-P was the prevailing P form (60–70%) in soil prior to any P addition, while the application of either mineral P alone or in combination with manure enhanced significantly the Ortho-P contribution to soil P forms (Ahlgren et al. 2013). However, an enhanced knowledge of the effect of P amendments on soil P composition is yet to be gained before fully understanding how P bioavailability to plants can be controlled and improved. Attention has been recently devoted to microbial inocula, referred to as bioeffectors, that are capable not only of root systems promotion and protection against pathogens, but also to P bioavailability and plant uptake enhancement (Richardson et al. 2011). Trichoderma harzianum Rifai 1295–22 (T-22) is known to protect from the attack of pathogens, such as Pythium and Fusarium (Harman and Björkman 1998). Furthermore, Trichoderma strains increase plant growth directly by promoting root development or indirectly by solubilizing P minerals (via acidification, redox, chelation, and hydrolysis reactions) (Li et al. 2015b). Similar results have been reported for bacteria Pseudomonas sp. DSMZ 13134 (Rodriguez et al. 2006; Fröhlich et al. 2012) and Bacillus amyloliquefacients FZB42 (Qiao et al. 2014). They populate the roots and protect them from root diseases caused by fungi, such as Fusarium oxysporum Schlecht f. sp. (Fröhlich et al. 2012). Pseudomonas sp. and Bacillus were shown to be among the most efficient phosphate-solubilizing bacteria in soil (Rodríguez and Fraga 1999; Vazquez et al. 2000; Fröhlich et al. 2012; Qiao et al. 2014), thereby enhancing nutrient plant uptake. Such microorganisms are commonly isolated from the rhizosphere and their capacity to solubilize P is generally reported to be associated with ability in culture to acidify growth media (particularly when evaluated on Ca phosphates) and release a number of organic anions (Fröhlich et al.

Plant Soil

2012; Khan et al. 2009). Gluconate is a major component of Pseudomonas organic anion production and may therefore play an important role in the solubilization of inorganic P as rock phosphate (Rodriguez et al. 2006) as well as organic P forms such as calcium-phytate (Giles et al. 2014). Various Pseudomonas strains can also degrade organic P compounds, such as phytate, phosphonates and phosphites through the excretion of different extracellular phosphatase enzymes, thereby improving soil P fertility by facilitating the remineralisation of organic P into inorganic P (Lidbury et al. 2017; Richardson et al. 2001; Rodríguez and Fraga 1999; Shen et al. 2011; Tarafdar and Jungk 1987; White and Metcalf 2004). Managing soil P in either deficient or excess conditions requires detailed information about both P concentrations and chemical forms which will determine P bioavailability and environmental reactivity (Condron et al. 2005; Pierzynski et al. 2005). Nevertheless, little is known so far on the effect of bioeffectors on soil P forms and their dynamics in agricultural soils upon different P fertilization. The aim of this work was to study the combined effect of either inorganic or organic P fertilizers and bioeffectors on soil P forms composition in a greenhouse experiment under maize. 31P–NMR spectroscopy was employed on alkaline extracts from soil samples to speciate and quantify P forms in alkaline extracts of treated soil samples.

Materials and methods

weighed into a porcelain crucible and ignited for 1 h at 550 °C. The soil residue, and an Bunignited^’ soil sample (2.0 g), were extracted with 50 mL of 0.5 M H2SO4 solution and shaken for 16 h The extracts were then centrifuged at 1610×g for 20 min and the supernatant collected after filtration through Whatman no. 42 filter paper. The supernatant was subsequently analyzed for inorganic P using the molybdenum blue colorimetric method of Murphy and Riley (1962). The inorganic P concentrations of the ignited and unignited extracts are referred to as total and inorganic P, respectively. The difference between total and inorganic P determined by the ignition–H2SO4 extraction technique represents the organic P. Fertilizers and microbial inoculants Triplesuperphosphate (TSP) and rock phosphate (RP) were provided by Landor AG S.r.l (Auhafen, Muttenz, Switzerland). Two composts were employed as organic P fertilizers, from cow- and horse-manure, both produced at the BonFarm^ composting plant at the experimental station of Castel Volturno (CE). Compost properties are given in Table 2. As for Bioeffectors, B1 Trichoderma harzianum, strain T-22, was used as Trianum®, by Koppert B.V. (The Netherlands), B2 Pseudomonas sp. DSMZ 13134 was that of Proradix®, by Sourcon-Padena GmbH & Co. KG (Germany), and, B3 Bacillus amyloliquefaciens was obtained as Rhizovital FZB42®, by ABiTEP GmbH (Germany).

Soil collection and characterization

Greenhouse experiment with maize growth

The surface layer (0–20 cm) of a farmland Vertic Xerofluvent clay-loam soil was collected, at Castel Volturno (CE) Experimental Station of the University of Naples Federico II. Soils were air dried, sieved at 5 mm and mixed before experiment. Soil chemical and physical properties were determined on soil after collection and at the harvest. The main physical and chemical characteristics of soils are given in Table 1. Soil pH was measured by a pH meter (HANNA, Italy) in a 1:2.5 soil/ water ratio. Total C and N were determined by an elemental analyzer (Eager 200, Fisons). Total soil P was measured by the ignition–H2SO4 extraction technique of Saunders and Williams (1955), as modified by Walker and Adams (1958). In brief, 2.0 g of soil was

For the greenhouse pot experiment under maize growth, a soil substrate was prepared by mixing the clay-loam farmland soil with a quartz sand at a 2:1 soil/sand (w/w) ratio and thoroughly homogenized. This soil substrate was incubated in covered plastic boxes at 20 ± 2 °C during 30 days prior to planting. Maize (Zea mays, cv Colisee, KWS) plants were grown in pots (3 L) filled with 2.5 kg of soil substrate. The experimental trial consisted of a factorial combination of five soil P treatments: 1) P0, no P addition; 2) P1, addition of TSP; 3) P2, addition of RP; 4) P3, addition of composted buffalo manure; 5) P4, addition of composted horse manure, with four soil inoculations with microbial Bioeffectors: 1) B0, no inoculation; 2) B1, Trichoderma harzianum; 3) B2, Pseudomonas sp.; 4)

Plant Soil Table 1 Soil properties from greenhouse pot experiment. TN, total nitrogen; TC, total carbon; TP, total phosphorus; TPi, total inorganic P; TPo total organic P; CT-FA, untreated farmland soil; B0, no inoculation; B1, Trichoderma harzianum; B2,

Pseudomonas sp.; B3, Bacillus amyloliquefaciens; P0, no P fertilization; P1, triple superphosphate; P2, Rock phosphate; P3, Buffalo-manure compost; P4, horse-manure compost

Treatments

pH

TN g Kg −1DW

TC

TP

TPi

TPo

CT-FA

7.94 ± 0.02abc

0.56 ± 0.06a

14.71 ± 0.84a

0.620 ± 0.020a

0.580(93.6)§bcd

0.040(6.4)§h

B0P0

7.99 ± 0.01a

0.56 ± 0.08a

14.87 ± 1.12a

0.640 ± 0.013a

0.600(93.8)ab

0.040(6.3)h

B0P1

7.95 ± 0.01ab

0.66 ± 0.04a

16.63 ± 1.09a

0.661 ± 0.012a

0.617(93.4)a

0.044(6.6)h

B0P2

7.98 ± 0.01a

0.61 ± 0.06a

16.65 ± 2.39a

0.619 ± 0.012a

0.574(92.3)bcd

0.045(7.3)h

B0P3

7.93 ± 0.03abc

0.68 ± 0.05a

15.40 ± 0.99a

0.648 ± 0.007a

0.595(92)ab

0.053(7.9)h

B0P4

7.94 ± 0.04abc

0.65 ± 0.06a

16.95 ± 1.34a

0.639 ± 0.016a

0.593(93.0)ab

0.045(7.0)h

B1P0

7.95 ± 0.04ab

0.54 ± 0.09a

14.50 ± 2.97a

0.622 ± 0.012a

0.549(88.3)def

0.073(11.7)ef

B1P1

7.98 ± 0.02a

0.54 ± 0.07a

14.90 ± 0.56a

0.612 ± 0.039a

0.539(88.1)defg

0.083(13.5)cde

B1P2

7.96 ± 0.01ab

0.63 ± 0.07a

13.65 ± 0.78a

0.589 ± 0.014a

0.523(88.9)efg

0.066(11.2)fg

B1P3

8.00 ± 0.03aa

0.65 ± 0.04a

14.93 ± 0.42a

0.613 ± 0.018a

0.510(83.2)fg

0.103(16.8)ab

B1P4

7.97 ± 0.02a

0.63 ± 0.04a

15.21 ± 0.42a

0.588 ± 0.017a

0.505(85.9)g

0.083(14.1)cde

B2P0

7.86 ± 0.01 cd

0.61 ± 0.05a

15.32 ± 1.27a

0.623 ± 0.011a

0.546(87.6)defg

0.077(12.4)def

B2P1

7.88 ± 0.01bcd

0.62 ± 0.02a

15.44 ± 0.85a

0.632 ± 0.008a

0.556(88.0)cde

0.077(12.0)def

B2P2

7.82 ± 0.03d

0.63 ± 0.12a

15.35 ± 3.04a

0.616 ± 0.019a

0.543(88.1)def

0.073(11.8)ef

B2P3

7.84 ± 0.04d

0.67 ± 0.09a

16.52 ± 4.67a

0.612 ± 0.007a

0.506(82.7)g

0.106(17.3)a

B2P4

7.87 ± 0.02bcd

0.65 ± 0.06a

17.43 ± 4.23a

0.607 ± 0.021a

0.512(84.3)g

0.095(15.7)abc

B3P0

7.85 ± 0.00 cd

0.56 ± 0.06a

13.33 ± 2.83a

0.588 ± 0.016a

0.511(87.0)fg

0.077(13.0)def

B3P1

7.85 ± 0.03 cd

0.59 ± 0.08a

14.55 ± 0.07a

0.585 ± 0.015a

0.517(88.5)efg

0.078(11.5)def

B3P2

7.85 ± 0.02 cd

0.54 ± 0.07a

13.29 ± 2.07a

0.591 ± 0.018a

0.513(86.8)fg

0.078(13.2)def

B3P3

7.87 ± 0.04 cd

0.64 ± 0.04a

13.95 ± 1.34a

0.618 ± 0.014a

0.514(86.4)fg

0.104(13.6)ab

B3P4

7.86 ± 0.03 cd

0.68 ± 0.05a

16.42 ± 3.25a

0.601 ± 0.013a

0.510(85.0)fg

0.091(15.0)bcd

§

In parentheses: Percent of inorganic and organic P to total P. Data are presented as mean values ± SD (n = 5) and have been analyzed by one-way analysis of variance. Means followed by the same letter within columns are not significantly different by Tukey’s test at the 5% level

B3, Bacillus amyloliquefaciens. All treatments were replicated five times, for a total of 100 pots. The P-containing materials were all applied to the substrate soil of each pot at the rate of 50 mg P kg−1 dry soil substrate and mixed thoroughly. However, P3 and P4 composts were applied 15 days before sowing. A basal nutrients addition provided nitrogen (N) as Ca(NO3)2 at the rate of 100 mg N kg−1 dry substrate, Table 2 Properties of the organic fertilizers applied in the pot experiment Compost

C %

N

P g kg−1

C/N

Composted cow manure (P3)

30

2.3

7.75

15.24

Composted horse manure (P4)

31

1.4

10.1

25.80

and potassium (K) as Kalimagnesia (30% K2O + 10% MgO) at the rate of 166 mg K kg−1 dry substrate. Three maize seeds per pot were sown prior to microbial inoculation. The microbial Bioeffectors were inoculated on soil surface at sowing by spraying their suspension in a 2.5 mM CaSO4 solution of demineralized water at the rate of 2.5 × 104 spores, 2 × 106 cfu and 2 × 106 cfu per g of substrate for B1, B2 and B3 respectively. Daily demineralized water was added manually to maintain the soil between 40 and 70% of water holding capacity throughout the experiment. Maize plants were harvested 8 weeks after sowing. The shoots were cut, oven-dried at 65 °C for 48 h, weighed, and milled. The soil adhering to the root segments, having a 1–2 mm diameter, after a gentle shake was considered to be rhizosphere soil and was removed by brushing and stored at −20 °C. The soil falling from roots and the

Plant Soil

remainder of the collected soil, were regarded as bulk soil and two subsamples were stored at 4 and −20 °C respectively. Soil samples were freeze-dried, ground to pass through 1-mm sieve, and stored at −20 °C prior to P extraction and analysis. To determine plant P concentration and total P uptake, shoots were digested in a mixture of 7 ml of HNO3 (65%) and 2 ml H 2O 2 (30%) in a microwave (Milestone, Digestor/Dring Ethos 900). Digests were analyzed by the molybdenum blue assay method (Murphy and Riley 1962). Soil phosphorus speciation by solution-state 31P–NMR spectroscopy Species of P in soils and compost were identified by 31P −NMR spectroscopy as by a method optimized earlier (Xu et al. 2012; Li et al. 2015a). In brief, about 4 g of sample was mixed with 32 mL of a 0.25 M NaOH and 0.05 M EDTA solution and shaken at 20 °C for 16 h. The mixture was then centrifuged at 12,000 g (20 °C) for 30 min. The clear supernatant solution was collected into a 50-mL centrifuge tube, to which 3.55 mL of a 3% 8-hydroxyquinoline solution were added to remove Fe and Mn metals (Ding et al. 2010). The solution pH was adjusted to 9.0 ± 0.1, kept steady for at least 30 min, and again centrifuged at 12,000 g (20 °C) for 30 min. The supernatant was separated, frozen at −20 °C, and freezedried. This extract was then re-dissolved in 2 mL of a 1 M NaOH solution for at least 2 h by Vortex shaking, and the suspension centrifuged at 12,000 g (20 °C) for 30 min. An aliquot (940 μL) of the supernatant was transferred into 5-mm NMR tube, and added with a deuterated aqueous solution of methylenebisphosphonic acid-P, P′-disodium salt (MDP, Epsilon Chimie, Brest) as internal standard (δ = 16.62 ppm), to reach a final 2.65 mM concentration. Three replicate pots for each treatment were prepared for NMR analyses. The NMR tube was placed on a 400 MHz Bruker Avance spectrometer, equipped with a 5 mm Bruker BBI (Broadband Inverse) probe, operating at 31P resonating frequency of 161.81 MHz. 31P spectra were obtained by using 6 s initial delay, a 45° pulse length ranging between 8.5 and 9.5 μs (−2 dB power attenuation), and an acquisition time of 15 h. The spectra comprised 9000 transients, 16,384 time domain points and a spectral width of 250 ppm (40,650 Hz). An inverse gated pulse sequence, with 80 μs length Waltz16 decoupling scheme and around 15.6 dB as power level, has been

employed to decouple phosphorous from proton nuclei (Mazzei and Piccolo 2012). All sixty 31P spectra were baseline corrected and processed by MestReC software (v. 4.9.9.9). The free induction decays (FID) for solution-state 31P–NMR spectra were transformed by applying a 4 folds zero filling and a line broadening of 6 Hz. Signal areas were calculated by integrating the individual peaks resulting from a deconvolution process. The MDP internal standard was contained in the solution used to dissolve samples for NMR analyses and served also to calibrate the frequency axis, standardize data and perform a quantitative assessment of P forms. Signals were assigned according to related references (Cade-Menun 2005; Turner 2008; Condron and Newman 2011; CadeMenun and Liu 2013; Li et al. 2015a; McLaren et al. 2015). No detailed assignment of singular peaks ranging within the monoester region was possible because of uncertainty deriving from peaks overlapping. Statistical analysis Analysis of variance (ANOVA) was performed to evaluate differences among P fertilizers and Bioeffectors treatments using Statgraphics Centurion (Version XV) and treatment means compared by Tukey’s test (P < 0.05). All data were tested for normality before analysis and, where required, skewed data were transformed before analysis. Principal component analysis and linear regression were conducted with XLStat 2014 (Addin software).

Results Soil properties and major P fractions Soil pH varied relatively little among the different treatments ranging from 7.82 to 8.0, although were slightly lowered after B2 and B3 application (Table 1). Total phosphorus (TP) in soils ranged from 0.59 to 0.65 g kg−1, and showed no significant difference after addition of triple superphosphate and both compost materials, with and without treatments with Bioeffectors (Table 1). Compared with P0, P1 and P2, organic P amendments addition increased slightly the total organic P, in particular in B2 inoculated soils (B2P3, B2P4). The soils that were inoculated by bioeffectors showed a significant general decrease in total inorganic

Plant Soil

(TPi), while concomitantly increasing total organic phosphorus (TPo) (Table 1). There was no significant effects on the total N (TN) and total C (TC), although TC slightly increased in the P4 treatment (Table 1). The NaOH-EDTA extraction recovered 32–42% of the total P from these soils (Table 3). Out of the P extracted with NaOH-EDTA, 8–12% was organic and 88–92% was inorganic (Table 3). Effect of different P treatments on soil P composition Solution 31P NMR spectra of NaOH-EDTA extracts are shown in Fig. 1. The dominant inorganic compound in all spectra was inorganic orthophosphate which ranged from 169 to 261.4 mg P kg−1 and accounted for between 87.5 and 91.2% of the NaOH–EDTA extracted P (Table 3). Inorganic pyrophosphate (Pyro-P) followed with a much smaller amount (0.31 mg P kg−1) and only 0.2% of the extracted soil P. Most of the organic P was present as orthophosphate monoesters (82–90% of the extracted soil organic phosphorus). Phosphomonoesters occurred at concentrations between 16.2 and 23.3 mg P kg−1 and accounted for between 7 and 11% of the NaOH–EDTA extracted P, followed in the order by orthophosphate diesters (Di-P) (0.9–1.8% of extracted P), and phosphonates (Phos-P) (Table 3). The organic P amendments decreased significantly the ratios of monoesters to diesters (Table 3). Different trends were observed with specific P materials and microbial Bioeffectors (Table 3 and Fig. 1). The Ortho-P content increased slightly with P fertilizers, in particular with TSP addition (P1), while Pyro-P and Poly-P responded less to P and Bioeffectors additions. The variation of organic P forms in the treated soils were of a smaller extent, except for Di-P content that was generally larger in all inoculated soils, especially with B2 and B3 addition. The P3 and P4 compost additions emphasized even more this trend. The effects of soil treatments on soil P composition were evaluated by elaborating NMR results with the Principal Component Analysis (PCA), that is an unsupervised multivariate statistical method. PCA datasets were prepared by varying the bioeffectors treatments while keeping P-amendment constant (Score-plots in Fig. 2) or viceversa (Score-plots in Fig. 3). Each scoreplot reports only PCA loading vectors, which, based on the ANOVA test, significantly differentiate soil P composition according to treatment (Figs. 2b and 3b). The PC1 and PC2 axes in the PCA score-plots reported in Fig. 2a explained 60.2, 45.9, 44.6, 46.2, 40.8%

and 20.0, 31.7, 24.3, 24.8, 25.8% of the overall variance for the P0, P1, P2, P3, and P4 treatments, respectively. For the unamended cultivated soil (P0), the scores for the untreated original soil (CT-FA) and treated soils were well separated, thus suggesting that bioeffectors changed the soil P composition even in soil without P addition. Amendments with either inorganic or organic P fertilizers altered significantly the distribution of P forms, depending on the employed bioeffector. The scores for the triple superphosphate fertilizer (P1) were well separated among bioeffectors, whereas P forms produced a poor separation along the PC1 of the rock phosphate plot (P2). A neat separation of P forms in both PC1 and PC2, was observed in both compost score plots (P3 and P4), as a function of bioeffector type. The PCA score-plot permits to explore dense data matrixes by easily undescoring similarities and dissimilarities among studied samples, whereas the related PCA loading plot enables identification of the variables associated to the PCA axes. These variables determine the score placement in the PCA plane, and reveal their involvement in samples differentiation. On this basis, Fig. 2b reports the loading vectors which passed the ANOVA test and are significantly responsible for groups differentiation, and, thus, reflect the response of P forms to soil treatments. With the exception of the P2 treatment, phospholipids was the main responsible variable for the differentiation along the PC1 of the response to bioeffectors, though to a different extent, under different P amendments. In particular, while the addition of triple superphosphate (P1) to soil promoted a PC1 differentiation due only to PL, the addition of rock phosphate (P2) differentiated a positive bioeffectors response in the same PC1 only for Ortho-P and Pyro-P forms. For both the P3 and P4 compost treatments the PC1 showed a net positive differentiation mainly due to the Di-P forms (Fig. 2b). The differences were statistically significant as indicated by Tukey’s test in Table 3. As for the PC2 loading vectors (Fig. 2b), the variables that mostly contributed to differentiate soil P composition as a function of P amendments, were Phos-P, DNA-P and Poly-P for the P1 and P2 treatments, OrthoP for the P3 compost, and both Pyro-P and Phos-P for soils treated with P4 compost. Effects of bioeffectors on soil P composition The PCA score-plots reported in Fig. 3a describe the effect of P amendments when varying the Bioeffectors

207.7(90.4)bcd 227.1(90.7)cd

218.6(90.4)bcd 230.6(89.6)cd

261.4(40)

223.4(36)

233.4(36)

220.8(35)

229.7(33)

250.3(37)

238.3(32)

221.5(32)

227.8(34)

241.8(39)

257.3(41)

223.6(36)

234.7(35)

237.7(36)

222(39)

243.6(42)

227.1(38)

234.3(38)

217.1(36)

B0P1

B0P2

B0P3

B0P4

B1P0

B1P1

B1P2

B1P3

B1P4

B2P0

B2P1

B2P2

B2P3

B2P4

B3P0

B3P1

B3P2

B3P3

B3P4

0.3(0.2)

0.9(0.4)e

0.6(0.3)cd

0.6(0.2)abcd

0.6(0.3)cd

0.5(0.2)abcd

0.5(0.2)abc

0.7(0.3)d

0.5(0.2)abc

0.5(0.2)abc

0.6(0.2) bcd

0.5(0.2)abc

0.6(0.2)abcd

0.6(0.3)bcd

0.5(0.2)abc

0.4(0.2)ab

0.5(0.2)abc

0.5(0.2)abc

0.6(0.3)abcd

0.5(0.2)abc

0.4(0.2) ba

0.2(0.1)

0.2(0.1)cde

0.17(0.1)c

0.3(0.1)fg

0.31(0.1)fg

0.29(0.1)fg

0.19(0.1)cd

0.18(0.1)c

0.57(0.3)l

0.65(0.3)l

0.41(0.2)hi

0.17(0.1)c

0.44(0.2)i

0.26(0.1)def

0.32(0.1)fg

0.15(0.1)ab

0.27(0.1)efg

0.17(0.1)c

0.08(0.0)ab

0.35(0.1)gh

0.06(0.0) ba

20.2(9.3)efgh

19.6(8.4)defg

17.6(7.7)bc

21.8(9.3)hil

16.2(7.0)b

20.3(8.6)fgh

23.3(9.9)l

18.1(8.1)cd

22.0(8.6)hil

18.5(7.6)cde

20.0(10.1)efgh

20.4(9.2)fgh

19.7(7.9)defg

19.5(7.8)defg

18.9(8.2)cde

20.7(9.4)ghi

17.2(7.4)bc

17.9(8.0)bcd

22.3(8.5)il

13.0(8.4) ba

18(10.9)

a

2.0(0.9)gh

2.1(0.5)gh

1.1(0.5)bc

1.2(0.5)bcd

1.1(0.5)bc

1.9(0.8)fgh

2.2(1.0)h

1.2(0.5)bcd

1.6(0.6)def

1.6(0.6)def

1.4(0.6)cde

1.9(0.8)fgh

1.4(0.6)cde

0.9(0.4)b

1.1(0.5)bc

1.4(0.6)cde

1.2(0.5)bcd

1.1(0.5)bc

1.2(0.5)bcd

0.3(0.2) ba

0.7(0.4)

PL

Di-P

1.7(0.8)cde

1.6(0.7)bcde

1.6(0.7)bcde

1.3(0.6)abc

1.3(0.6)abc

2.1(0.9)g

2.1(0.9)g

1.2(0.5)ab

1.4(0.5)abcd

1.3(0.5)abc

1.0(0.4)a

1.4(0.6)abcd

1.4(0.6)abcd

1.8(0.7)efg

1.1(0.5)ab

1.4(0.6)abcd

1.3(0.5)abc

1.0(0.4)a

1.2(0.5)ab

1.0(0.7) ba

1.0(0.8)

DNA

3.8(1.7)ef

3.7(1.2)ef

2.7(1.2)bcd

2.5(1.1)bc

2.4(1.1)bc

4.1(1.7)f

4.3(1.8)f

2.4(1.1)bc

3.1(1.2)cd

2.9(1.2)cd

2.4(1.0)bc

3.3(1.5)de

2.7(1.1)bcd

2.8(1.1)bcd

2.2(0.9)b

2.8(1.1)bcd

2.5(1.1)bc

2.1(1.0)b

2.4(0.9)bc

1.3(0.9) ba

1.7(1.1)

Total Di-P

0.5(0.2)bcd

0.5(0.2)abcd

0.4(0.2)abcd

0.5(0.2)bcd

0.8(0.4)e

0.8(0.3)e

0.6(0.3)de

0.5(0.2)bcd

0.5(0.2)bcd

0.8(0.3)e

0.2(0.1)a

0.5(0.2)bcd

0.5(0.2)cde

0.2(0.1)abc

0.4(0.2)abcd

0.6(0.3)de

0.3(0.1)abc

0.2(0.1)ab

0.5(0.2)abcd

b

0.4(0.3) babcd

0.5 (0.2)

Phos-P

5.45a

5.29a

6.51b

8.72e

6.75b

5.08a

5.41a

7.54 cd

7.33bc

6.73b

8.33e

6.18ab

7.03bc

7.22bc

8.5e

7.39bc

6.88ab

8.52e

9.29f

10.0f

10.54f

M:Di

Values in columns represent mean concentration (n = 3 replicate pots) of P extracted in NaOH-EDTA. In parentheses: percent of P recovered by NaOH-EDTA to total soil P; percent of inorganic and organic P compounds to total soil P extracted in NaOH–EDTA. Different letters in each column indicate significantly different means by Tukey’s test at the 5% level

191.5(88.2)b

209.7(89.9)bcd

205.9(90.5)bc

217.9(89.0)bcd

209.0(91.2)bc

211.9(89.1)bcd

205.6(87.6)bc

201.5(90.1)bc

198.5(88.5)bc

196.3(88.6)b

214.8(90.4)bcd

195.9(88.8)b

212.7(91.2)bcd

202.5(90.6)bc

235.3(90.0)d

168.5(87.5) 169.7(90.2)ba

189.2(34)

192.7(32)a

CT-FA

Mono-P

Poly-P

Ortho-P

Pyro-P

Organic-P

untreated farmland soil; M:Di, the ratio of orthophosphate monoesters to orthophosphate diesters. B0, no inoculation; B1, T.harzianum; B2, Pseudomonas sp.; B3, B. amyloliquefaciens; P0, no P fertilization; P1, TSP; P2, RP; P3, Buffalo-manure compost; P4, horse-manure compost

Inorganic-P

B0P0

NaOH-EDTA Total P

Treatments

Table 3 Content (mg kg−1 DW) of P forms, as measured by solution-state 31P–NMR, in NaOH-EDTA extracts of soils from greenhouse pot experiment. Ortho-P: orthophosphate; Pyro-P: pyrophosphate; Poly-P: polyphosphates; Mono-P: orthophosphate monoesters; Di-P: orthophosphate diesters; PL: phospholipids; Phos-P: phosphonates; CT-FA,

Plant Soil

Plant Soil

Fig. 1 31P–NMR spectra of soils amended with different P fertilizers and microbial inocula under greenhouse pot cultivation. B0, no inoculation; B1, Trichodema harzianum; B2, Pseudomonas sp.; B3, Bacillus amyloliquefaciens. P0, no fertilization; P1, Triple superphosphate; P2, Rock phosphate; P3, Buffalo manure

compost; P4, Horse manure compost. a, phosphonates; b, MDP, Methylenebisphosphonic acid-P, P′-disodium salt, as internal standard; c, orthophospate; d, orthophosphate monoesters; e, phospholipids; f, DNA; g, pyrophosphate; h, polyphosphates

addition. The PC1 and PC2 explained 49.8, 35.1, 41.2, 38.0% and 30.0, 23.0, 28.7, 26.2%, of the overall variance for the B0, B1, B2, and B3 treatments, respectively. In the case of the untreated soils (B0), a distinct separation among samples classes was detected and ascribed to a different soil P composition, that reflected the P distribution in the different amendments. With inoculation of Trichoderma harzianum (B1), the all P-based treatments showed a smaller but still detectable differentiation, except for amendments with triplesuperphosphate (P1) and rock-phosphate (P2). In particular, P4 showed a larger amount of Ortho-P accompanied by a relatively smaller content of DNA-P, whereas no differences were observed between P1 and P2 treatments. This suggests that B1 was similarly affecting both P1 and P2 inorganic fertilizer, thus leading to the same soil P composition (Fig. 3b). Pseudomonas sp.(B2) induced a neat differentiation on soil P-composition of the organic P3 and P4 treatments in comparison to P1 and P2. Moreover, these organic P treatments provided the largest content of Phos-P, DNA-P and Pyro-P, as indicated by the association of P3 and P4 samples to the positive PC1values (Fig. 3b). Interestingly, the largely positive placement of

P4 samples along the PC1 axis in the B3 score-plot, indicates that the Phos-P, DNA-P and Pyro-P forms were also relatively more with Bacillus amyloliquefaciens (B3) inoculation, when the horsemanure compost (P4) was supplied to soil. A positive correlation was found in the linear regression (R = 0.76898; P < 0.0001) between TPo calculated by the ignition-H2SO4 method and the Di-P forms extracted by NaOH-EDTA and determined by NMR (Fig. 4), whereas no correlation was found for the Mono-P form (R = 0.42733; P = 0.060). A moderate positive correlation was also found between TPo extracted in NaOH-EDTA and TPo by the ignition-H2SO4 method (R = 0.5755; P < 0.01). P uptake in shoots Shoot growth was significantly (P < 0.001) affected by different treatments, whereby plants grown in both P-fertilized and inoculated soils were larger than for the P-unfertilized and not-inoculated treatments (data not shown). In particular, the application of bioeffectors increased plant growth in P1 and compost (P3 and P4) fertilization.

Plant Soil Fig. 2 Greenhouse pot experiment. PCA score-plots (a) for P forms in soils treated with different P amendments (P0, no fertilization; P1, Triple superphosphate; P2, Rock phosphate; P3, Buffalo manure compost; P4, Horse manure compost) and varying Bioeffectors (CT-FA, untreated soil, ◇; B0, no inoculation, □; B1, Trichoderma harzianum, ×; B2, Pseudomonas sp., ○; B3, Bacillus amyloliquefaciens, +). The corresponding PCA loading vectors are shown in (b): a, phosphonates (Phos-P); c, orthophospate (Ortho-P); d, orthophosphate monoesters (Mono-P); e, phospholipids (PL); f, DNA; g, pyrophosphate (PyroP); h, polyphosphates (Poly-P)

Plant Soil Fig. 3 Greenhouse pot experiment. PCA score-plots (a) for P forms in soils amended with different Bioeffectors (B0, no inoculation; B1, Trichodema harzianum; B2, Pseudomonas sp.; B3, Bacillus amyloliquefaciens) and varying P amendments (CT-FA, untreated soil, ◇; P0, no fertilization,□; P1, Triple superphosphate, △; P2, Rock phosphate, ○; P3, Buffalomanure compost, ×; P4, Horsemanure compost,+). The corresponding PCA loading vectors are shown in (b): a, phosphonates (Phos-P); c, orthophospate (Ortho-P); d, orthophosphate monoesters (Mono-P); e, phospholipids (PL); f, DNA; g, pyrophosphate (PyroP); h, polyphosphates (Poly-P)

Concentration and uptake of P in shoots were strongly affected by P amendments and microbial strains (Fig. 5, a,b). An increased P concentration and uptake

were observed after B2 and B3 applications, especially under compost fertilization, representing the largest values of all treatments (Fig. 5). In the case of P3

Plant Soil 140

Ignition -e xtrac table total organic P mg kg-1

Fig. 4 Linear regression between TPo (ignition method) and Di-P forms (NaOH-EDTA extraction) amounts. The solid, the dotted and thin lines represent the linear regression, the 95% confidence interval and 95% prediction interval, respectively

R2 =0.5913 y=28,033x+0,747

120

100

80

60

40

20

0 1

1,5

2

2,5

3

NaOH-extractable Di-P

fertilization, B2 and B3 inoculation increased P concentration and uptake by about 68 and 42%, respectively, as compared to B0. Although to a lesser extent, a similar trend was observed for P4 fertilization. However, there was no significant response in P uptake to the application of B1, B2 and B3 strains for either P1 or P2 amendments, whereas there was a significant increase in P concentration for B2P0 treatments (Fig. 5). The values for plant P uptake were reported in Thonar et al. (2017), provided by SEM (standard error of the means), while in the present paper by +/−SD (standard deviation).

Discussion Soil properties and total P Changes in soil pH observed especially with B2 inoculation (Table 1) were in agreement with previous studies showing acidification of the soil or media due to production of organic anions by Pseudomonas spp. (Antoun 2012; Giles et al. 2014; Oteino et al. 2015). In the rhizosphere, bacteria may secrete organic acids (e.g., gluconate, citrate, oxalate, acetate) which results in phosphate solubilization from insoluble complexes, making it available for plant uptake (Richardson et al. 2011). These organic acids can chelate the cation bound

3,5

4

4,5

mg kg -1

to phosphate with their hydroxyl and carboxyl groups (Kpomblekou-a and Tabatabai 1994). A large share (88.5% on average) of total P detected by the ignition–H2SO4 extraction technique was identified as inorganic P (Table 1), being the majority of P (60%) bound to calcium compounds, as determined in this farmland soil by the Hedley P fractionation (Cozzolino et al. 2013). This is consistent with the presence of Ca-phosphates as the main form of soil P in different alkaline soils (Piccolo and Huluka 1985; Cross and Schlesinger 1995; Hinsinger 2001). This suggests that much of the inorganic P (TPi) in the topsoil layer derives from apatites and fertilizer degradation products (Hedley et al. 1982; McLaren et al. 2014). Although only 32–45% of total soil P was extracted by NaOH-EDTA (Table 3), our results are in line with previous findings that reported similar extraction efficiencies for semiarid and alkaline soils (Turner et al. 2003a; McLaren et al. 2014). On average, inorganic P extracted by NaOH-EDTA accounted for 40% of that determined by the ignition–H2SO4 extraction method, while organic P represented only 27% (Tables 1 and 3). It is reported that the ignition extraction method may overestimate organic P content, thus contributing to the low efficiency of organic P extraction by NaOH-EDTA (Cade-Menun and Lavkulich 1997). However, this discrepancy may be explained with a limited solubility of soil organic P in NaOH–EDTA (Bünemann et al. 2008).

Plant Soil

A 1.8

B0

B1

B2

B3 m

lm

1.6 1.4

ghi

m lm

ikl

1

defg

klm

hik efgh

fghi

1.2

gP kg -1DW

Fig. 5 Concentration (a) and total P uptake (b of maize plants at harvest, in all treatments. Data are expressed as mean values ± SD (n = 5). Different letters above the bars indicate significantly different means by Tukey’s test at the 5% level. B0, no inoculation; B1, Trichoderma harzianum; B2, Pseudomonas sp.; B3, Bacillus amyloliquefaciens; P0, no P fertilization; P1, triple superphosphate; P2, Rock phosphate; P3, Buffalo-manure compost; P4, horse-manure compost. P uptake data were reported in Thonar et al. 2017 in slightly different way since, in the present manuscript, data are provided by+/−SD whereas in the cited paper SEM had been given

efgh

defg bcde defg

cdef

abcd a ab abc

0.8 0.6 0.4 0.2 0

P0

B

P1

P2

P3

40

35

i

30

mg P plant-1

P4

h

gh fg ef

25

def def 20

def cd

gh fg

f cde

cd bc

b

a

ab ab

15

a 10

5

0

P0

In fact, it has been already noted that the tight P binding to Ca in Vertisols reduces the extraction efficiency in the NaOH-EDTA solution (Cross and Schlesinger 1995; McLaren et al. 2014). Effects of P fertilizers and Bioeffecttors on soil P forms and plant P uptake Solution 31P NMR analysis of the NaOH–EDTA extracts of these soils showed the predominance of orthophosphate, that represented by far the most intense peak in NMR spectra, while Mono-P was the most abundant form of organic P (81–90% of organic P extracted by NaOH-EDTA (Fig. 1; Table 3). These results are

P1

P2

P3

P4

consistent with NMR studies of P forms from cropped soils in semiarid environments (Bünemann et al. 2008; McLaren et al. 2014; Turner et al. 2003a). Contrary to earlier indications (Ahlgren et al. 2013), P fertilization did not enhance the Ortho-P contribution to soil P forms in this study. In fact, only a general slightly increase of Ortho-P content was observed with triple superphosphate (P1) addition, thus suggesting that differences in total P are due to the variability of inorganic P extracted from the diverse P materials amended to soil (Table 3). The low amount of Di-P in all samples, represented by less than 2% of total P and 9.7–16% of organic P extracted in NaOH-EDTA, is also consistent with other

Plant Soil

findings for alkaline soils (Turner et al. 2003a; McLaren et al. 2014) (Table 3). The Di-P forms include nucleic acids, phospholipids and other compounds, that characterize a labile and readily mineralized soil organic fraction. In fact, the low charge densities and the shielded phosphate group of Di-P forms enhance their availability to microbial or enzymatic attack, thus accounting for their low content in soil (Tate 1984). Furthermore, most Mono-P in NaOH-EDTA extracts are overestimated and those of Di-P underestimated due to the complete hydrolysis of RNA and partial hydrolysis of some phospholipids to monoesters during alkaline extraction (Makarov et al. 2002; Turner et al. 2003a; Murphy et al. 2009; Vincent et al. 2013). Nevertheless, our results show that despite the small amount of these organic P forms in soil, they changed significantly according the different treatments, as it is shown by the values of total Di-P and Mono-P/Di-P ratios (Table 3), and by multivariate analyses (Fig. 2 a,b; Fig. 3 a,b). Although NMR spectra indicate a general increase in the content of all P forms for both P fertilizers and Bioeffectors treatments, the Di-P form was particularly enhanced by inoculation with Bioeffectors in combination with inorganic P1 and both P3 and P4 composts. Conversely, rock phosphate (P2) amendment generally showed the smallest content of Di-P, Ortho-P, and Mono-P forms (Table 3). This response may be due to the poor water solubility of the rock phosphate, resulting in a consequent low P availability. On the contrary, the 90% water-soluble triple superphosphate treatment (P1) provided relative larger inorganic P forms, that are rapidly uptaken by plants or, when immobilized in microbes, transformed into organic P forms in soil (Fig. 2b). However, the largest values of Di-P forms were found in soils treated with organic P amendments (Table 3). It is noteworthy that the amount of Di-P forms appeared strongly correlated to the TPo determined by the ignition-H2SO4 method (R = 0,7690; P < 0.0001) (Table 1; Fig. 4), thereby suggesting that Di-P extracted by NaOH-EDTA was a consistent and significant fraction in all treatments. These findings are quite similar to those reported by Guggenberger et al. (1996), who found a close functional relation between the labile Po determined by a resin extraction and the Di-P form measured by NMR, thus suggesting that Di-P may contribute significantly to the labile Po pool and represent a readily available form for microbial mineralization. Moreover, while total soil P did not vary among

treatments, its organic and inorganic P components varied complementary showing a significant and concomitant increase and decrease, respectively, in all inoculated soils, and especially upon compost addition (Table 1). This could suggest bioeffectors not only promoted organic P mineralization, but they also must have uptaken inorganic P during their growth. It was reported that biological activity changes in a grass-legume rotation in comparison to a grass system, showed a greater turnover of organic matter, and larger P cycling and availability (Oberson et al. 1999). Our results could indicate that microbial communities in inoculated soils enhanced the interconversion rate between labile or non-labile inorganic Pi, mobilized from soil or added with compost, into labile or nonlabile organic Po. This suggests a microbial synthesis of Po, of which a portion may be partially retained in microbial cells and in associated pool of microbial metabolites (Ayaga et al. 2006; Gichangi et al. 2009; Malik et al. 2012). Thus, immobilization of P by microorganisms and its gradual release through microbial turnover protects P from sorption onto the soil components and favors a slow release of orthophosphate for useful plants and microbes uptake (Oberson et al. 2001). This behavior was more pronounced for B2, and, to a lesser extent, B3 inoculation (Table 3; Fig. 2 a, b; Fig. 3 a, b). In fact, soils inoculated with Pseudomonas spp. (B2) and Bacillus a. (B3) revealed the largest Di-P content and the smallest Mono-P/Di-P ratios, especially in combination with compost treatments (P3 and P4) (Table 3). Although no microbial biomass indexes nor mineralization rates were provided here to support the suggested P cycling, our results are consistent with those reported elsewhere (Condron et al. 1990; Frossard et al. 2000; Turner et al. 2003b), which inferred that Di-P forms are indicators of microbial P-cycling. Di-P were shown to be more readily mineralized in soil than Mono-P (Condron et al. 1990), and strongly correlated with microbial biomass (Turner et al. 2003b; Vincent et al. 2013). Greater concentrations of Di-P forms in grassland and agricultural soils are indicative of greater biological P turnover (Frossard et al. 2000; Turner et al. 2003b), and an active population efficient in turnover of soil Po in conditions of limited biovailability of inorganic P (Bünemann et al. 2012; Cross and Schlesinger 1995; Makarov et al. 2002). In contrast, Di-P was reported to accumulate when conditions for microbial decomposition were limited by climate (Makarov et al. 2002). Mono-P seem to represent only a small fraction

Plant Soil

of soil Po available to plants and microbes, since their charge density favours reactivity in soil with clays, metals and organic matter (Celi et al. 1999; Turner et al. 2002). While this behavior protects Mono-P from biological degradation, it may concomitantly restrict their contribution to the short-term organic matter turnover (Turner et al. 2003b). In fact, large amounts of water-extractable organic P in some Australian pasture soils occurred as phosphate diesters, with labile monoesters being only negligible (Turner et al. 2002). Based on differences of the processes controlling concentrations of Mono-P and DNA-P, it was suggested that only a small fraction of the soil organic P is actively involved in the short-term soil P cycle (Stewart and Tiessen 1987). Previous reports on 31P NMR measurements of P forms in soil failed to univocally assess whether an increase in the amount of soil Di-P had to account to high turnover rates coupled to an enhanced microbial activity or an accumulation of labile organic P due to limited microbial activity (Guggenberger et al. 1996; Makarov et al. 2005). Here, we found that both P concentration and content in plant were largest for the B2 and B3 treatments under compost addition (Fig. 5 a, b). This may suggest that B. amyloliquefaciens (B3), and even more Pseudomonas (B2), were capable to exploit organic P amendments more than the indigenous microbial communities alone (B0) or Trichoderma (B1), by promoting an effective mineralization of the organic P forms, particularly Di-P, and making P quickly available to plants (McDowell et al. 2007; Murphy et al. 2009). The B2 and B3 treatments may have thus favored a fast P cycling between inorganic and organic P forms, thereby ensuring more bioavailable soil P to plants and microbes. In fact, significant amounts of P may be released by soil microorganisms without a net change in microbial biomass P content due to recycling and turnover. Recently, an accelerated turnover of microbial P without any change in microbial P content has been reported in calcareous soils when available P was low (Schneider et al. 2017). These authors stated that rapid microbial P turnover may help to provide P to plants under low Pi conditions, through a rapid uptake and release of P from microbial P pools. It is known that soil microorganisms produce a range of phosphatases which foster the enzymatic transformation of organic P forms (Tarafdar and Jungk 1987; Tate 1984). The inoculation with Pseudomonas spp. with high phytase activity was already shown to increase the use of phytate P by grass species (Richardson et al. 2001). The

role of P solubilizing microbes is particularly relevant for maize because this plant does not release phytase enzymes capable to mineralize soil phytates (Richardson et al. 2000). Since most organic P inputs to soil are represented by Di-P (Cosgrove 1967), more attention has been devoted to phosphodiesterase activity (Turner and Haygarth 2005). It has been hypothesized that phosphodiesterase and phosphomonoesterase activities act sequentially in soil to release orthophosphate (Nannipieri et al. 2011). There are evidence which indicate that nucleic acid degradation in soil is primarily mediated by Gram negative bacteria as Pseudomonas (Greaves and Wilson 1970). Interestingly, two Pseudomonas strains have been identified as capable to secrete two extracellular glycerolphosphodiesterases, which are involved in the cleavage of glycerolphosphorylcholine, the head group of phospholipids as in phosphatidylcholine (Lidbury et al. 2017). Based on our findings, we may suggest that the bioeffectors employed here enabled the hydrolysis of phosphodiesters, thus increasing the soil P forms available to plant growth. It is noteworthy that soil pH was slightly lowered in soils treated with B2 and B3 (Table 1). This could indicate a larger production of organic anions, such as gluconic acid, by these bioeffectors, and a consequent greater capability to solubilize organic and inorganic P forms (Giles et al. 2014; Richardson et al. 2001). However, B2 and B3 inoculation did not enhance plant P uptake in soils treated with P0, P1 and P2, whereas compost additions (P3 and P4) determined the greatest P uptake by maize plants (Fig. 5b). This may be explained considering that composts consist of highly humified and slowly decomposable organic matter, thus representing a constant source of C and nutrients which may favor microbial growth, activity and a related formation of organic P forms (Malik et al. 2012). In fact, the soil concentration of Di-P was previously shown by 31 P NMR spectroscopy to increase when soil conditions were more favorable to microbial activity, such as amendments with C-rich materials (Sumann et al. 1998). Other studies reported faster rates of turnover time of soil microbial biomass in C-amended soils (Oehl et al. 2004; Bünemann et al. 2007; Achat et al. 2010). Furthermore, the application of manure in field experiments was found to favour large alkaline phosphatase and phosphodiesterase activity, possibly because of stabilization of extracellular enzymes by soil humus (Colvan et al. 2001). It was also suggested that the availability of P in manure may be enhanced by

Plant Soil

inoculation with phytase-producing bacteria (MenezesBlackburn et al. 2016). It has been shown that soil microorganisms mobilize P at higher rates when receiving C-amendments, without changing the rate of plant P uptake (Spohn and Kuzyakov 2013). This results in a net P mineralization, since microbial P turn-over is driven by the microbial requirement for carbon rather than for P. In fact, microbial key players in organic P mineralization are known to be affiliated to phyla specialized in organic matter decomposition (Ragot et al. 2016). Other than enhanced microbial mineralization, P desorption from soils treated with organic matter may be further increased due to competition for P adsorption sites on soil particles exerted by the acidic functional groups of the humic components present in organic amendments (Magid et al. 1996; Shen et al. 2011). Moreover, the quality of soil organic matter appears to affect the microbial activity and the Mono-P/Di-P ratio in soil (Turrión et al. 2001). In this study, a larger plant P uptake was found for B2P3 and B3P3 than for B2P4 and B3P4 (Fig. 5), thereby confirming that not only the soil microbial activity but also the response of maize plants to organic P-fertilization depended on the specific molecular composition of added compost, as previously reported (Cozzolino et al. 2016).

Conclusions Here we applied 31P–NMR spectroscopy to follow the changes of P forms in maize-cropped soils undergone combined treatments of both inorganic and organic P fertilizers and P-solubilizing microbial bioeffectors. 31 P–NMR spectra allowed to concomitantly speciate and quantify organic and inorganic P forms in alkaline extracts from soils. While total P was not affected by P fertilizer and bioeffectors additions, microbial inoculation complementary decreased and increased total inorganic and organic P, respectively, particularly in soil treated with compost, and also enhanced plant P uptake. This indicates the importance of a combined amendment of organic matter and P solubilizing bacteria to improve soil P bioavailability to plants and microbes. Our findings revealed that the microbial treatments generally enhanced the capacity of P fertilizes to provide both inorganic and organic P forms. In particular, the content of diesters P forms was found significantly

larger than control for soils treated with compost. This indicates that, compared to inorganic P addition, the Pcontaining organic compounds are less prone to sorption and precipitation, and, by addition of C source, stimulate a fast microbial growth and cycling between the mineralized inorganic forms and the organic P forms immobilized in the living or dead microbial biomass. The statistical analysis based on 31P–NMR spectra of soil extracts indicated that bioeffectors differed in their capacity of exploiting various P materials added to soils. The specific effect of all bioeffectors was generally related to an increased content of diesters P forms. This was particularly evident with Pseudomonas sp. and Bacillus amyloliquefaciens which exerted a greater effect with organic P amendments (B2P3 and B3P3) than with inorganic fertilizers, whereas Trichodema harzianum was less active. It is noteworthy that the largest Di-P contents found in B2P3 and B3P3 was also related to the largest plant P uptake in these treatments. This finding may suggest that larger P content became available for cycling between labile organic P fractions and microbial biomass. Hence, it may be suggested that the bioeffectors employed here succeeded in hydrolyzing phosphodiesters and increasing P fractions available to plant growth. Thus, the turnover of microbial P pools may slowly release P that becomes more efficiently uptaken by plants than a single large pulse of mineral P fertilizer, since this is subjected to fixation and removal before the plants can use it adequately. These results underline the important role of Di-P as an indicator of P transformations in agroecosystem soils. Our study indicates that bioeffectors addition to soils enable the efficient exploitation of soil P amendments, promote a fast cycling between inorganic and organic P forms in soil, and increasing P availability to plants and soil microbes. Our results have important implications for the development of sustainable agronomic practices with the objective to improve use efficiency of P fertilizer, especially those in recycled biomasses. Acknowledgements This work was supported by the European’s Seventh Framework Programme (FP/2007-2013) under Grant Agreement no. 312117. The first author was sponsored by the National Natural Science Fund Projects of China (No. U1133604) and China Scholarship Council (CSC). We thank three anonymous reviewers for their constructive suggestions in helping to improve the manuscript.

Plant Soil

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