Bacillus subtilis QST713 and cellulose amendment enhance phosphorus uptake while improving zinc biofortification in wheat

Applied Soil Ecology 142 (2019) 81–89

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Bacillus subtilis QST713 and cellulose amendment enhance phosphorus uptake while improving zinc biofortification in wheat

T

Aurora Moreno-Lora, Ramiro Recena, Antonio Delgado

⁎

Dpto. Ciencias Agroforestales, ETSIA, Universidad de Sevilla, Ctra. Utrera km 1, 41013 Sevilla, Spain

ARTICLE INFO

ABSTRACT

Keywords: Phosphorus Zinc nutrition Biofortification Grain quality Soil enzymatic activities Wheat

Phosphorus (P) in an essential non-renewable resource in agriculture and zinc (Zn) deficiency is an important micronutrient imbalance in calcareous soils. Inoculation with Bacillus subtilis QST713 and amendments with readily available C for soil microorganisms is known to enhance the uptake of some nutrients by plants. We hypothesized that these practices may also improve P and Zn uptake. However, since Zn and P are antagonistic nutrients, it is also relevant to assess if a potential enhancement of P nutrition worsens Zn uptake by plants. An experiment with wheat (Triticum durum L) was performed involving three factors: (i) treatments affecting rhizospheric microorganisms: control without treatment, cellulose as readily C source for microorganisms, and inoculation with B. subtilis QST713, (ii) soil type: Vertisol and Alfisol, and (iii) P source: unfertilized control, and rock phosphate. The supply of cellulose improved total P uptake by plants in both soils independently of the P supply. Overall, inoculation with B. subtilis also provided benefits on P uptake, despite this effect varied depending on soils and P supply. B. subtilis improved Zn concentration in grains by 24% relative to untreated control, while the effect on total Zn uptake by plants was non-significant. This explained the increased Zn harvest index with B. subtilis. The improvement of P uptake by B. subtilis and cellulose did not lead to an antagonistic effect on Zn, whose uptake by plants increased linearly with increased P uptake (R2 = 0.68; P < .001). These results provide evidence of the benefits of agricultural practices affecting microbial properties of the rhizosphere in improving P supply to plants. B. subtilis increased Zn accumulation in edible parts, thus promoting a biofortification effect.

1. Introduction Phosphorus is an essential but inefficiently used resource in agriculture (Schröder et al., 2016; Recena et al., 2017) whose future scarcity may constraint global food security (Cordell et al., 2009; Cordell and Neset, 2014). Its inefficient use involving excessive application rates not only provokes relevant environmental problems due to eutrophication of waterbodies (Ryan et al., 2012; Recena et al., 2017), but also relevant agronomic problems through the promotion of nutritional antagonisms such as that with Zn; i.e., an increased P availability decreases Zn uptake by plants (Verma and Minhas, 1987; Zhang et al., 2012a; Zhang et al., 2017). In particular, these risks of nutritional imbalances due to excessive P supply may be more relevant under soil conditions prone to the deficiency of metallic micronutrients, i.e. calcareous soils with basic pH (Ryan et al., 2013; Sánchez-Rodríguez et al., 2013). Thus, it is crucial to consider these interactions between P and metallic micronutrients for sustainable fertilization management in soils with basic pH.

⁎

Zn deficiency causes substantial, but often unrecognized, losses in crop yield, and it is assumed to be the most important micronutrient imbalance in alkaline-calcareous soils (Rashid and Ryan, 2004; Ryan et al., 2013). This deficiency is a well-documented problem in staple food crops with possible health hazards in humans by the decreased supply of Zn in cereal-based diets (Cakmak, 2008; Borrill et al., 2014; Wang et al., 2014; Zhao et al., 2014). Based on several studies, the average concentration of Zn in whole grain of wheat in many countries ranged from 20 to 35 mg kg−1, and it should be increased approximately 10 mg kg−1 to achieve a minimum nutritional quality for humans (Cakmak, 2008; Pfeiffer and McClafferty, 2007). Mobility and availability of Zn to plants in soil depends on many factors such as soil pH, total Zn content, quantity and quality of organic matter, and soil mineralogy (Roholla Mousavi, 2011). P fertilization and availability in soil also affect Zn uptake as a result of a combination of several processes, including decreasing mycorrhizal colonization (Ova et al., 2015), reduced plant-availability of Zn in the rhizosphere, reduction in Zn uptake per unit of root weight, diminished root-to-shoot

Corresponding author. E-mail address: [email protected] (A. Delgado).

https://doi.org/10.1016/j.apsoil.2019.04.013 Received 8 October 2018; Received in revised form 21 March 2019; Accepted 14 April 2019 Available online 27 April 2019 0929-1393/ © 2019 Elsevier B.V. All rights reserved.

Applied Soil Ecology 142 (2019) 81–89

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translocation of Zn, and yield-induced dilution effect (Chen et al., 2017). Soil organic matter is crucial contributing to the solubility and transport of Zn to plant roots (Catlett et al., 2002; Obrador et al., 2003). Organic amendments may increase the content of soluble organic matter in soil that is able to bind to metals, thus increasing the solubility and transport of Zn to roots (Herencia et al., 2008; Maqueda et al., 2015). On the other hand, these amendments may contribute to metal immobilization because increased organic matter provides new exchange sites for metal adsorption (Díaz-Barrientos et al., 2003; Maqueda et al., 2015). Inoculation with rhizospheric microorganisms is a relevant strategy for improving plant nutrition, and evidences are clear for other micronutrients such as Fe (Marschner et al., 2011). In the case of Zn, microorganisms may increase Zn uptake by plants in contaminated soils (Marques et al., 2013), but evidences in non-polluted soils are scarce (e.g. Ramesh et al., 2014; Ghavami et al., 2016). Organic amendments may also change microbial activity, which in turns may influence Zn uptake by plants. Overcoming Zn deficiencies involves the application of Zn fertilizers (Cakmak, 2008; Zhang et al., 2012a, 2012b; McBeath and McLaughlin, 2014). In addition, Zn fertilizers may be the mean for increasing Zn concentration in grains for enhanced nutritional quality, the so called biofortification (Alloway, 2009; White and Broadley, 2009; Zhang et al., 2012b). To these ends, Zn fertilizers are however expensive and not always efficient, and other more sustainable agricultural practices are required, such as the use of microbial inoculants able to improve Zn uptake by plants and biofortification (Wang et al., 2014; Khande et al., 2017). The use of some microbial inoculants proved effective for increasing P and Fe uptake by plants (Viruel et al., 2014; García-López and Delgado, 2016); however, the effect on Zn uptake by plants of these microorganisms was never proven. In addition, to assess their potential benefits, the antagonism between nutrients such as P and Zn should be taken into account, since the improvement in one of the nutrient may negatively affect the other. The use of microbial inoculants able to provide several benefits for crops (e.g. disease control, plant growth promotion, or nutrient mobilization) may contribute to the sustainability and cost-effectiveness of agricultural practices (García-López and Delgado, 2016). In this sense, Bacillus subtilis is a plant growth promoting bacteria with disease biocontrol capabilities able to increase the uptake of P and Fe by plants in calcareous growing media (García-López and Delgado, 2016, who used B. subtilis QST713). Organic amendments supplying readily available Csources affect microbial activity and community structure, which may affect nutrient uptake by plants. Positive evidences of these strategies on Zn nutrition would provide additional arguments for their involvement in sustainable integrated practices for biofortification. Furthermore, potential benefits will provide additional tools for biological agriculture production where the use of synthetic fertilizers is restricted. We hypothesized that nutrient mobilizing mechanisms by B. subtilis QST713 and other microorganisms may increase P and Zn mobilization and availability to plants. Thus, this work aimed at studying the effects of the inoculation with B. subtilis QST713 and the supply of a readily available C source for soil microorganisms on the uptake of P and Zn by wheat plants. However, since Zn and P are antagonistic nutrients, it is also relevant to assess if the potential enhancement of P nutrition worsen Zn uptake by plants. Particular focus should be put on the accumulation of Zn in grains to assess biofortification effects. The study was performed in two different soils to assess how the potential benefits of inoculation and organic amendment are affected by soil properties. These soils had different Zn availability index values, enzyme activity, and texture. In the study, rock phosphate (mainly composed of hydroxyapatite) was used as P source to assess the effect of inoculation or organic amendment in the use by plant of major inorganic P fraction in soils with basic pH (Ca phosphates) and if these treatments can increase the efficiency in the use of non-soluble P fertilizers.

2. Material and methods 2.1. Experimental design An experiment involving the cultivation of wheat plants (Triticum durum cv Amilcar) was performed under controlled environmental conditions following a completely randomized design with five replications. The experiment involved three factors: (i) treatments affecting rhizospheric microorganisms, with three treatment levels, control without treatment, cellulose as readily C source for microorganisms, and inoculation with B. subtilis QST713, (ii) soil type, with two different soils, an Alfisol (Typic Haploxeralf) and a Vertisol (Chromic Haploxerert) according to the Soil Taxonomy (Soil Survey Staff, 2014); and (iii) P source, with an unfertilized control, and applying rock phosphate as P source for crop at a rate of 0.2 g P kg−1. The two treatment levels in the latter factor was intended to assess the potential benefits of treatments in the use by plants of P from soil or from a non-soluble fertilizer which is an inefficient P source since rock phosphate was mainly constituted by non-soluble Ca phosphate (hydroxyapatite). The P rate applied with rock phosphate, which was equivalent to 200 kg P ha−1 under no tillage, is a high rate according to usual P rates (30–80 kg P ha−1 depending on the crop when applied as soluble P fertilizer), but in the same order of magnitude. The high P rate used was intended to study the potential as P fertilizers of low efficient non-soluble sources that require higher rates than soluble fertilizers, and to check the mobilization and use by plants of the major inorganic P fraction in soils with basic pH. In this regard, 0.2 g kg−1 of P precipitated as non-soluble Ca phosphate is usual in soils under Mediterranean climate (Recena et al., 2017). 2.2. Soils Both soils were selected in south Spain (Alfisol, 37°43′34.00″ N, 5° 4′43.08″ W; Vertisol, 37°24′07″N, 5°35′10″W), as representative soils for usual irrigated and rainfed field crop production under Mediterranean climate. Sampling was done in the first week of December 2013 by taking surface soil samples (till 20 cm depth; 30 subsamples per soil of approximately 0.5 kg, which were mixed for its characterization and use in the experiment). After that, soil were airdried and sieved to < 4 mm. A portion of them was sieved to < 2 mm and used for soil characterization. Soils were selected to encompass representative soils from the Mediterranean region where the problem of Zn deficiency is frequent, with low available Zn (below 0.5 mg kg−1 of DTPA extractable Zn according to Lindsay and Norvell, 1978), and with basic pH. In addition, soils showed similar levels of P availability as assessed by the Olsen P (Olsen et al., 1954); these levels being below the threshold level for fertilizer response (Recena et al., 2016). Main physic-chemical soil properties are shown in Table 1. 2.3. Plant growing conditions Wheat seeds were pre-germinated on a petri dishes and grown on a seedbed to the stage Z1.3 of Zadoks scale (three true leaves) in a growing chamber (Zadoks et al., 1974). Then, plants were transplanted into 350 mL (15 cm high × 5.5 cm–diameter polystyrene cylinders) pots containing 0.3 kg of air dry soil. After transplanting, pots were placed in a growing chamber with a photoperiod of 16 h, with 22 W m−1 of light intensity, a temperature of 25/20 °C and 45/60% of relative humidity, day/night. Plants were irrigated with a Hoagland type solution without P and micronutrients (total volume in the cycle of 1.25 L), containing (mmol L−1): Ca(NO3)2 (5), KNO3 (5), MgSO4 (2), KCl (0.05). The experiments concluded after 11 weeks when plants were at stage Z9.2 of the Zadoks scale. 82

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Table 1 Properties of studied soils. Soil

Sand

Silt

Clay

SOC

Total N

ACCE

pH

EC

CEC

Exchangeable cations Ca

g kg−1 Vertisol Alfisol

50 421

250 241

700 338

6.2 5

0.96 0.42

11 21

7.7 7.9

dS m−1

cmoclc kg−1

0.17 0.18

36.4 12.4

29.1 10.5

Mg

K

DTPA extractable Na

Fe

Olsen P

Cu

Mn

Zn

1.3 0.8

34.7 27.9

0.3 0.5

mg kg−1 5.2 1.6

0.8 0.3

0.2 0.1

34.0 21.3

6.6 7.1

SOC, soil organic carbón; ACCE, active calcium carbonate equivalent; EC, electrical conductivity; CEC, cation exchange capacity; DTPA, diethylenetriaminepentaacetic acid.

2.4. Soil treatment

recovery of nutrients. P concentration in the digest was determined colorimetrically (Murphy and Riley, 1962) and Zn concentration by atomic absorption spectrophotometry (TJA Solutions, 1999). Total uptake of P and Zn by plants was calculated as the sum of the product of DM by its concentration in each organ minus the amount of nutrient present in the seed. The Zn harvest index (ZnHI) was calculated as the ratio of total Zn accumulated in grains to that accumulated in the whole aerial parts of plants. P in grains is assumed to be mainly as phytate, to which metals such as Zn are bound reducing its absorption during digestion in humans and animals. The P to Zn molar ratio was calculated as an index of potential availability to humans and animals of Zn as affected by phytate content of grains (Miller et al., 2007).

Inoculation with B. subtilis strain QST 713 (Serenade Max, Bayer Cropscience) was carried out as described by (García-López and Delgado, 2016), by adding 104 colony forming units (CFU) per gram of soil. To this end, a volume of 20 mL of aqueous suspension containing 1.5 × 105 CFU per mL was applied to the soil surface in each pot at five points (4 mL of inoculant suspension per point) around the plants after transplanting; each point was at 1.5 cm from the plant shoot. Application of a labile C source was performed using β-cellulose. Soils were mixed with β-cellulose (CF11, Whatman) at a rate of 200 mg kg−1 before transplanting by irrigating with a suspension containing 2.72 g cellulose per L of water (the content of C was 0.44 g per g of β-cellulose; 20 mL of suspension applied per pot).

2.7. Statistical analysis

2.5. Soil analysis after harvest

A three-way analysis of variance was performed to assess the effect of soil and treatments affecting microbial activity in rhizosphere. Previously, normality according to the Kolmogorov-Smirnov test, and homocedasticity according to Levene test, were checked. Potential transformations were performed if required to fully meet these both criteria. Analysis of variance was performed with the General Linear Model procedure in Statgraphics Centurion XVI (StatPoint Technologies, 2013). When the effect of a factor was significant, means for each factor level were compared via Tukey's test (P < .05), except when the interaction between factors was significant. In the case of significant interactions, the effect of main factors cannot be assessed and only the interaction can be discussed since the effect of one factor depends on the level of the other (Acutis et al., 2012). In these cases, the combined effect of both factors was assessed with interaction figures. Linear regressions and Pearson correlation coefficients were calculated, and comparison of the slopes of regressions performed, using the same software mentioned above.

Population of B. subtilis in the rizhospheric soil [sampled as described Wang et al., 2009] was measured through dilution plating after Na-pyrophosphate extraction using a nutrient–agar medium after heating the suspension at 80 °C for 10 min according to Tuitert et al. (1998). The count of CFU was carried out at 48 h after plating. Characterization of the CFU counted and confirmation of B. subtilis presence was performed a week after plating according to the method of Gospodarek et al. (2009). Enzymatic activities were measured at 35 (Z4.0-Z5.0 stage) and 65 (Z9.0 stage) days after transplanting. To this end, soil from pots was sampled to obtain a portion of about 4 g of soil from the surface layer (0–5 cm) of the pot using 5 mm-diameter cylinders at 1.5 cm from the main plant tiller. Dehydrogenase activity was measured using a modification of the method of Casida Jr et al. (1964): a mixture of 2 g of soil and 0.02 g of CaCO3 was placed in a 50 mL polyethylenecentrifuge tube, adding 0.35 mL of 3% 2,3,5-triphenyl-tetrazolium chloride (TTC) and 0.85 mL of water (a control without TTC was included). The samples were mixed and incubated at 37 °C during 24 h. Then, triphenylformazan produced was extracted three times with 10 mL of ethanol each one, the total amount of extract was mixed, and the concentration was determined colorimetrically. β-Glucosidase activity was determinated by measuring the amount of p-nitrophenol produced after the addition of 0.05 M p-nitrophenyl-β-D-glucopyranoside as substrate according to Eivazi and Tabatabai (1988). Enzyme activity was expressed as the amount of compound (in mg or g depending on the enzyme) produced in the corresponding incubation per unit of mass of dry soil (kg) and time (h).

3. Results The shoot and total dry matter yield was higher in the Alfisol than in the Vertisol (Table 2). Higher P concentration in all the organs, Zn concentration in shoots, and higher total P and Zn uptake were observed in plants grown in the Alfisol when compared with those in the Vertisol (Table 2). This latter soil showed the highest enzymatic activities, except dehydrogenase at 65 days when differences were not significant between both soils (Table 2). Inoculation with B. subtilis QST713 significantly increased the concentration of P and Zn in grains and Zn harvest index (Fig. 1) when compared with control and cellulose, meanwhile P concentration in grains with cellulose was higher than in control (Table 2). P supply as rock phosphate increased P concentration in roots (Table 2). This fertilization increased β-glucosidase activity 35 after transplanting and the Zn harvest index, while it decreased the molar P to Zn ratio relative to the unfertilized control (Table 2). Significant interactions between treatments affecting microorganisms in the rhizosphere and P source were observed for root and total DM (Table 2). B. subtilis significantly increased these both variables

2.6. Plant harvest and analysis After 11 weeks, plants were harvested, and grains, shoots and roots were separated and dried at 65 °C during 48 h in a forced-air oven to determinate dry matter (DM). For plant analysis, 0.25 g of dried and milled (< 1 mm) plant material were mineralized at 550 °C during 8 h in a muffle furnace. After calcination, ashes were dissolved in 10 mL 1 N HCl; this solution was heated at 100 °C for 15 min for ensuring the full 83

84

0.10 0.10

P source (P) 0 RP

0.4317 0.0009 0.4583 0.3934 0.1318 0.5082 0.6739

0.2494 0.1297 0.2888 0.2814 0.1002 0.3647 0.4305

0.56 0.58

0.56 0.59

0.56 0.59 0.56

0.4411 0.0051 0.9725 0.9939 0.0418 0.9506 0.6048

1.53 1.54

1.48b 1.59a

1.50 1.56 1.55

0.0005 0.0031 0.0066 0.0019 0.2388 0.0867 0.1589

482b 580a

470 595

656 482 454

mg kg−1

0.3666 0.0136 0.1256 0.3637 0.1472 0.4100 0.0159

244 223

221 246

242 235 222

Shoot

0.0000 0.0000 0.5550 0.4664 0.0638 0.0028 0.1559

2740 2694

2535 2903

2999a 2676b 2461c

Grain

0.0000 0.0000 0.7982 0.8181 0.0392 0.1004 0.0120

1.82 1.82

1.67 1.96

1.92 1.83 1.67

mg plant−1

P

Total

0.1323 0.4661 0.8735 0.6403 0.5452 0.3451 0.0989

25 23

23 24

24 24 23

mg kg−1

Root

0.1417 0.0000 0.9305 0.2393 0.0873 0.0457 0.0810

14 14

13 16

13 15 14

Shoot

Zn concentration

0.0000 0.058 0.0842 0.1626 0.4410 0.0129 0.5628

36 38

35 38

41a 36b 33b

Grain

0.1421 0.0000 0.2262 0.4092 0.2391 0.0654 0.0993

35 36

32b 39a

37 36 34

μg plant−1

Zn

Total DHA2

0.9334 0.0001 0.1992 0.2600 0.5056 0.4666 0.3311

3.4 3.8

4.4a 2.8b

3.5 3.6 3.7

0.4448 0.5357 0.0395 0.2727 0.2871 0.0259 0.7231

5 4

4.3 4.6

4.5 4.9 4.5

mg kg−1 h−1

DHA1

Enzyme activity βgluc2

0.1717 0.0000 0.0380 0.8250 0.2455 0.1504 0.4114

108b 117a

131a 93b

110 118 109

0.2258 0.0000 0.1713 0.8350 0.7204 0.0528 0.0962

118 126

145a 98b

127 114 125

mg kg−1 h−1

βgluc1

0.0000 0.0216 0.0457 0.2114 0.8257 0.4627 0.7545

0.58b 0.60a

0.61a 0.58b

0.64a 0.58b 0.55b

ZnHI

0.4121 0.1127 0.0118 0.3657 0.5961 0.6906 0.4581

161a 151b

153 159

153 159 156

P/Zn grain

Molar ratio

DHA1, and DHA2, Dehydrogenase activity at 35 and 65 days after transplanting; βgluc1, and βgluc2, β-glucosidase activity at the same times; ZnHI, Zn harvest index; PHI, P harvest index. Total P and total Zn, total uptake of P and Zn by plants, respectively; calculated as the product of DM in each organ and its concentration of the nutrient, minus the amount of nutrient present in the seed. Potential transformations for P and Zn concentration in shoot and root. Mean followed by different letters in the same column for each treatment within a factor are significantly different according to the test of Tukey at P < .05. Significant effects are marked in italics. a If interactions are significant the effect of main factors cannot be discussed and only the interaction between both factors should be discussed; effects in bold and italics are the only ones that can be discussed.

ANOVA (P value) Treatment 0.7066 Soil 0.7202 P source 0.4035 T ∗ Sa 0.6672 T∗P 0.0130 S∗P 0.2565 T∗S∗P 0.3936

0.82b 0.90a

0.10 0.10

Soil (S) Vertisol Alfisol

0.87 0.85

0.84 0.86 0.87

g plant−1

Total

Root

Grain

Root

Shoot

P concentration

Dry matter

Treatment (T) B. subtilis 0.10 Cellulose 0.10 Control 0.11

Factor

Table 2 Effect of treatments, soil, and P source on wheat and soil enzyme activities.a

A. Moreno-Lora, et al.

Applied Soil Ecology 142 (2019) 81–89

Applied Soil Ecology 142 (2019) 81–89

A. Moreno-Lora, et al.

-1

P concentration in shoot (mg kg )

0,7 0,6

Zn HI

0,5 0,4 0,3 0,2 0,1

350 Control B. subtilis Cellulose

300 250 200 150 100 50 0

P0

0,0 Control

B. Subtilis

Cellulose

Treatment Total P uptake (mg plant )

-1

RP Alfisol

Control B. subtilis Cellulose

-1

0,14

Root DM (g plant )

P0

2,5

Fig. 1. Effect of treatments affecting soil microorganism in the rhizosphere on the Zn harvest index (Zn HI), estimated as the ratio between Zn content in grains and Zn content in the aerial parts of wheat plants. Mean (n = 20) for Bacillus subtilis was significantly higher than that of untreated control and that with cellulose application according to the Tukey test (P < .05).

Control B. subtilis Cellulose

0,12

RP Vertisol

2,0

1,5

1,0

0,5

0,10

0,0

0,08

P0

Vertisol

0,06

RP

P0

RP

Alfisol

Fig. 3. Interaction between treatments affecting soil microorganism in the rhizosphere, P P source (P0, unfertilized control, PR, rock phosphate supply), and soil type on P concentration in shoots (a) and total P uptake by plants (b). Error bars indicate standard error (n = 5).

0,04 0,02 0,00

P0

RP

The supply of P as rock phosphate increased P concentration in grains relative to unfertilized control, but only in the Vertisol (2632 vs 2440 mg kg−1). In the Alfisol, this supply did not promoted positive effects on this concentration (not shown). Zn concentration in shoots increased with rock phosphate in the Vertisol (38 vs 33 mg kg−1 without fertilizer), while it was unaffected in the Alfisol (not shown). Zn concentration in grains improved in the Vertisol with rock phosphate (from 28 to 32 mg kg−1), meanwhile it was unaffected by this fertilizer in the Alfisol. All this explains the significant interaction observed between soil and P source for these variables (Table 2). Interactions between the three factors were observed for P concentration in shoots and total P uptake by plants (Table 2). This reveals that the effect of inoculation and organic amendment varied depending on the soil and on the P source. In the Vertisol without P supply, B. subtilis and cellulose increased P concentration in shoots when compared with control. In this soil, rock phosphate supply lead to higher P concentration in shoots and total P uptake by plants than unfertilized control without inoculation or cellulose (Fig. 3). In the Alfisol, B. subtilis, cellulose, and rock phosphate supply did not lead to significant effects on P concentration in shoots of wheat plants (Fig. 3). However, in this soil, B. subtilis and cellulose increased total P uptake by plants relative to control (Fig. 3). Positive correlations were found between Zn concentration in shoots and grain, and total Zn uptake with P concentration in shoots and grains, and with total P uptake (Table 3). In shoots, Zn concentration increased with increased P concentration; similarly occurred for the uptake of Zn and P by plants (Fig. 4). On the contrary, the Zn harvest index was negatively correlated with P concentration in shoots. Overall, P and Zn concentration in the different organs and their total uptake were not correlated with dehydrogenase activity (DHA), by

P source 1,8 Control B. subtilis Cellulose

-1

Total DM (g plant )

1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 P0

RP

P source Fig. 2. Interaction between treatments affecting soil microorganism in the rhizosphere and P source (P0, unfertilized control, PR, rock phosphate supply) on root dry matter (a), and total dry matter (b) in wheat plants; DM, dry matter. Error bars indicate standard error (n = 10).

when rock phosphate was supplied, meanwhile both were increased by cellulose without P supply (Table 2; Fig. 2). The effect of the treatments on P concentration in roots varied depending on the soil, as revealed by the significant interaction between both factors (Table 2). B. subtilis was the only treatment increasing this concentration, but only in the Alfisol, where it near doubled the P concentration in roots when compared with untreated control (819 vs 595 mg kg−1). 85

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Table 3 Correlation coefficients for nutritional variables and enzymatic activity in the rhizosphere for both sources of P.

[P] root [P] shoot [P] grain Total P [Zn] root [Zn] shoot [Zn] grain Total Zn Zn HI

[P] shoot

[P] grain

Total P

[Zn] root

[Zn] shoot

[Zn] grain

Total Zn

Zn HI

DHA1

DHA2

βgluc1

βgluc2

0.10 ns

0.39** 0.58***

0.92*** 0.42*** 0.69***

0.1 ns 0.13 ns 0.25 ns 0.07 ns

0.1 ns 0.55*** 0.47*** 0.44*** 0.29*

0.37** 0.33* 0.80*** 0.64*** 0.16 ns 0.18 ns

0.15 ns 0.35** 0.53*** 0.86*** 0.24 ns 0.62*** 0.61***

0.12 ns −0.34** 0.00 ns 0.11 ns −0.29* −0.71*** 0.35** −0.15 ns

−0.25 ns −0.27* −0.17 ns −0.24 ns 0.22 ns −0.25 ns −0.06 ns −0.22 ns 0.23 ns

0.07 ns −0.05 ns −0.00 ns −0.11 ns 0.10 ns 0.05 ns 0.00 ns −0.14 ns −0.09 ns

−0.25 ns −0.38** −0.45*** −0.45*** −0.13 ns −0.63*** −0.22 ns −0.52*** 0.46***

−0.22 ns −0.15 ns −0.29* −0.34** −0.09 ns −0.43*** −0.14 ns −0.35** 0.22 ns

[P] and [Zn], concentration of P and Zn, respectively (mg kg−1 of nutrient in each organ); Total P and Zn, total element in plants. Total P and total Zn, total uptake of P and Zn by plants, respectively; calculated as the product of DM in each organ and its concentration of the nutrient, minus the amount of nutrient present in the seed; DHA1, and DHA2, Dehydrogenase activity at 35 and 65 days after transplanting; βgluc1, and βgluc2, β-glucosidase activity at the same times; ZnHI, Zn harvest index; PHI, P harvest index; n = 60. *,**,***, significant at P < .05, 0.01, and 0.001, respectively.

decreasing trend of total P and Zn uptake with increased β-glucosidase activity was however different depending on P supply (Figs. 5 and 6). The regressions with negative slope was more significant without rock phosphate, and in the case of total Zn uptake, the slope was significantly different between no P supply and phosphate rock supply. Without P supply, β-glucosidase activity explained near 50% of the variation in total Zn uptake by plants.

Total P uptake (mg plant-1)

2,6 2,4 2,2 2,0 1,8 1,6

4. Discussion

1,4

B. sutbilis QST713 increased total P uptake by wheat plants except in the Vertisol with rock phosphate supply. Thus, environmental and management (fertilization) factors should be considered in the assessment of the effect of microbial inoculation on P nutrition. On the other hand, cellulose was effective increasing P uptake in both soils with and without P supply. This is an interesting novel result which corroborates our hypothesis; it is the first time that an organic amendment with compounds not interacting with P geochemistry in soil (e.g. through inhibition of adsorption or precipitation) is demonstrated to increase P uptake by plants. Total Zn uptake by plants was not affected by B. sutbilis QST713 or cellulose. Thus, it can be supposed that availability to plants is not increased by these treatments. This contradicts our hypothesis of potential benefits ascribed to an increased Zn mobilization by microorganisms. However, Zn concentration in grains and Zn harvest index were increased by B. subtilis when compared with control (by 24 and 18%, respectively). This means that this microorganism may boost a preferential accumulation of Zn in edible parts, thus promoting a biofortification effect.

1,2 1,0 20

30

40

50

Total Zn uptake (µg plant-1) Fig. 4. Relationship between total P uptake and total Zn uptake by wheat plants: Y = 0.04 + 0.05 X; R2 = 0.68; P < .001; n = 60.

except P concentration in shoots with DHA at 35 days after planting (negatively, Table 3). On the contrary, concentration of P in shoots and grains, and total P uptake correlated negatively with both measures of β-glucosidase activity (at 35 and 65 days after planting). Also, Zn concentration in shoots and total Zn uptake correlated negatively with this enzymatic activity. However, Zn concentration in grains were not correlated with any enzymatic activity, and Zn harvest index was positively correlated with β-glucosidase activity at both times, the correlation being more significant for this activity at 35 days (Table 3). The

Fig. 5. Relationship between total P uptake by plants and β-glucosidase activity at 35 days after transplanting in the soil; a) for unfertilized plants (n = 30), Y = 2.6–0.008 X; R2 = 0.38; P < .001; b) for plants fertilized with rock phosphate (n = 30); regression not significant. 86

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Fig. 6. Relationship between total Zn uptake by plants and β-glucosidase activity at 35 days after transplanting in the soil; a) for unfertilized plants (n = 30), Y = 53–0.18 X; R2 = 0.47; P < .001; b) for plants fertilized with rock phosphate (n = 30); Y = 44–0.07 X; R2 = 0.14; P < .05.

(Alfisol, higher DTPA extractable value) showed the best DM yield and nutritional parameters of plants. Biological properties of soils seemed relevant explaining differences between both soils. Dehydrogenase activity (DHA) has been usually ascribed as an intracellular enzymatic activity, meanwhile β-glucosidase has been deemed an indicator of general microbial activity in soils related to the C cycle which can remain adsorbed in the soil after cellular lysis (Moreno et al., 2015). Both activities were the highest in the soil where the lowest P and Zn uptake by plants were observed (Vertisol, Table 2). The concentration in aerial organs and total P uptake decreased with increased β-glucosidase activity in soil along the experiment, and with increased DHA at the middle of the crop cycle (35 days after planting). Similar correlations with β-glucosidase were observed for Zn, by except its concentration in grain. This explains that Zn harvest index correlated positively with βglucosidase at the middle of the crop cycle (Table 3). Thus, it seems that the general microbial activity negatively contributes to total P and Zn uptake, but it does not worsen Zn concentration in grains, thus increasing Zn harvest index. This likely reveals an altered pattern of Zn accumulation in plant organs related to microbial activity in the growing media. The observed correlations between nutritional traits of plants and biochemical properties of soils do not explain the effects of B. subtilis and cellulose since these treatments did not affect enzyme activities. Overall, both enzymatic activities were more significantly affected by soil type than by treatments which are supposed to affect microorganisms (Table 2). Consequently, it can be assumed that enzymatic activities were more affected by native microbial population than by the effects of both, organic C supply and B. subtilis inoculation. Thus, it cannot be discarded the potential effects of different microbial activity and communities on P and Zn uptake depending on the soil type. The large difference in the C to N soil ratio between both soils may be reflected in different microbial community structure (Thébault et al., 2014). Soil microorganisms may compete with plants for nutrients, and this may be more evident under conditions of limited nutrient availability (Marschner et al., 2011). Thus, a greater microbial activity which is reflected in higher enzyme activities in the Vertisol may contribute to explain a decreased Zn uptake by plants in this soil relative to the Alfisol. In addition, recent evidences reveal that worsened micronutrient uptake by plants may be ascribed to shifts in microbial community structure (de Santiago et al., 2019), perhaps related to changes in the C to N ratio in soil (Thébault et al., 2014). All this may contribute to explain the differences in P and Zn uptake by plants between both soils, but not the effect of B. subtilis and cellulose on plant nutrition. Potential differences in the soil microbial activity or community structure between fertilized and unfertilized soils, and its subsequent effect on nutrient supply, is supported by the fact that the slope of the regression between total P and Zn in plants and β-glucosidase activity

Benefits observed on P nutrition did not promote an antagonistic effect with Zn. The concentration of Zn in shoots and grains and total Zn in plants increased with increased concentration of P in shoots and grains and total P uptake (Table 3; Fig. 4). Thus, although antagonistic effect of P on Zn has been widely described, in our case, improved P nutrition did not lead to a decreased Zn concentration in shoots or grains. This can be ascribed to a situation of P and Zn deficiency. When the availability of both nutrients is reduced, the antagonistic effect between them should be less evident. Consequently, improved P nutrition with organic C supply or B. subtilis inoculation did not lead to a decreased Zn concentration in grains. Even more, in the Vertisol which is the soil with lower P availability according to P concentration and uptake by plants, rock phosphate increased P uptake and concentration in shoots, and Zn concentration in shoots. Thus, overcoming P deficiency may have positive effects on plant Zn nutrition. The increased Zn concentration in grains and harvest index without increased Zn uptake by B. subtilis likely reveals an altered transport or mobilization of this nutrient in plant. This change in nutrient accumulation within plants is in agreement with the negative correlation between Zn harvest index and Zn concentration in shoots (Table 3). This may be ascribed to auxins production by this microorganism (García-López and Delgado, 2016). Auxins may regulate transporters involved in metal homeostasis in plant tissues (David-Assael et al., 2006). The production of auxins by this microorganism is supported by the increased root development induced by B. subtilis when rock phosphate was supplied. The highest increase in P uptake relative to untreated control was promoted by the microorganism in the Vertisol without P supply (c.a. 30%). The positive effect of this microorganism should be related to its P-mobilizing capacity, as observed by García-López and Delgado (2016). According to these authors, the mobilizing capacity is different depending on P availability and soil properties, which may explain differences between both soils. The lack of positive effect of B. subtilis in the Vertisol with rock phosphate may be explained by the improved behavior of untreated control and also by a diminished P uptake with the microorganism when P is supplied. This last may occur because P supply may affect microbial activity or community structure, particularly when soil P availability is not high such as in our soils (Koyama et al., 2014), making B. subtilis less competitive. In fact, rock phosphate increased β-glucosidase activity at 35 days after transplanting, as an evidence of the effect of P fertilization on soil microorganisms. Both, B. subtilis and cellulose amendment improved P nutrition in the soil with the lowest enzymatic activities and consequently with the lowest microbial activity (Alfisol) independently of the P supply. With lower microbial activity, the effects of cellulose affecting soil microorganisms, or the development of inoculated B. subtilis may be more marked. Overall, the soil with the less restrictive Zn availability level 87

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was significantly more negative without P supply than with P fertilization (Figs. 5 and 6). This may reveal a different effect of increased microbial activities in the soil depending on P fertilization. Neither soils nor treatments affected the molar P to Zn ratio in grains, which means that digestibility of Zn in grains was unaffected since there is not an increased ratio of phytate to Zn concentration in grains. Surprisingly phosphate rock supply decreased this ratio, which may be ascribed to its positive effect on Zn harvest index. Thus, P fertilization improved grain quality, a result that may be likely expected under conditions of low P availability in soil. Overall, the inoculation with B. subtilis was positive improving P nutrition of crops, while it increased Zn concentration in grains and consequently Zn harvest index. Cellulose, as a labile C source, was effective improving P uptake, without negative effects on Zn concentration in grains. These evidences reveal that the use of microbial inoculants or organic amendments with readily available C sources for microorganisms may be strategies enabling better crop nutrition an improved nutritional quality. Results provide evidence of the benefit of agricultural practices affecting microbial properties of rizhosphere on the acquisition of P by plants and on the concentration of Zn in edible parts. These effects are relevant for overcoming the consequences of P and Zn deficiencies, which should however be tested in a wider set of environments under field conditions.

L.L., Ma, W.Q., He, M.R., Zhang, X.Y., Kou, C.L., Li, Y.T., Tan, D.S., Cakmak, I., Zhang, F.S., Zou, C.Q., 2017. Harvesting more grain zinc of wheat for human health. Sci. Rep. 7, 1–8. https://doi.org/10.1038/s41598-017-07484-2. Cordell, D., Neset, T.-S.S., 2014. Phosphorus vulnerability: a qualitative framework for assessing the vulnerability of national and regional food systems to the multi-dimensional stressors of phosphorus scarcity. Glob. Environ. Chang. 24, 108–122. https://doi.org/10.1016/J.GLOENVCHA.2013.11.005. Cordell, D., Drangert, J.O., White, S., 2009. The story of phosphorus: global food security and food for thought. Glob. Environ. Chang. 19, 292–305. https://doi.org/10.1016/ J.GLOENVCHA.2008.10.009. David-Assael, O., Berezin, I., Shoshani-Knaani, N., Saul, H., Mizrachy-Dagri, T., Chen, J., Brook, E., Shaul, O., 2006. AtMHX is an auxin and ABA-regulated transporter whose expression pattern suggests a role in metal homeostasis in tissues with photosynthetic potential. Funct. Plant Biol. 33, 661–672. de Santiago, A., Perea-Torres, F., Avilés, M., Moreno, M.T., Carmona, E., Delgado, A., 2019. Shift in microbia community structure influence the availability of Fe and other micronutrients to lupin (Lupinus albus L.). Appl. Soil Ecol (under review). Díaz-Barrientos, E., Madrid, L., Maqueda, C., Morillo, E., Ruiz-Cortés, E., Basallote, E., Carrillo, M., 2003. Copper and zinc retention by an organically amended soil. Chemosphere 50, 911–917. https://doi.org/10.1016/S0045-6535(02)00695-1. Eivazi, F., Tabatabai, M.A., 1988. Glucosidases and galactosidases in soils. Soil Biol. Biochem. 20, 601–606. https://doi.org/10.1016/0038-0717(88)90141-1. García-López, A.M., Delgado, A., 2016. Effect of Bacillus subtilis on phosphorus uptake by cucumber as affected by iron oxides and the solubility of the phosphorus source. Agric. Food Sci. 25, 216–224. https://doi.org/10.23986/afsci.56862. Ghavami, N., Alikhani, H.A., Pourbabaee, A.A., Besharati, H., 2016. Study the effects of siderophore-producing bacteria on zinc and phosphorous nutrition of canola and maize plants. Commun. Soil Sci. Plant Anal. 47, 1517–1527. https://doi.org/10. 1080/00103624.2016.1194991. Gospodarek, E., Bogiel, T., Zalas-Wiȩcek, P., 2009. Communication between microorganisms as a basis for production of virulence factors. Polish J. Microbiol. 58, 191–198. Herencia, J., Carlos Ruiz, J., Morillo, E., Melero, S., Villaverde, J., Maqueda, C., 2008. The effect of organic and mineral fertilization on micronutrient availability in soil. Soil Sci. 173, 69–80. https://doi.org/10.1097/ss.0b013e31815a6676. Khande, R., Sharma, S.K., Ramesh, A., Sharma, M.P., 2017. Zinc solubilizing Bacillus strains that modulate growth, yield and zinc biofortification of soybean and wheat. Rhizosphere 4, 126–138. https://doi.org/10.1016/j.rhisph.2017.09.002. Koyama, A., Wallenstein, M.D., Simpson, R.T., Moore, J.C., 2014. Soil bacterial community composition altered by increased nutrient availability in Arctic tundra soils. Front. Microbiol. 5, 516. https://doi.org/10.3389/fmicb.2014.00516. Lindsay, W.L., Norvell, W.A., 1978. Development of a DTPA soil test for zinc, iron, manganese, and copper. Soil Sci. Soc. Am. J. 42, 421–428. https://doi.org/10.2136/ sssaj1978.03615995004200030009x. Maqueda, C., Morillo, E., Lopez, R., Undabeytia, T., Cabrera, F., 2015. Influence of organic amendments on Fe, Cu, Mn, and Zn availability and clay minerals of different soils. Arch. Agron. Soil Sci. 61, 599–613. https://doi.org/10.1080/03650340.2014. 946409. Marques, A.P.G.C., Moreira, H., Franco, A.R., Rangel, A.O.S.S., Castro, P.M.L., 2013. Inoculating Helianthus annuus (sunflower) grown in zinc and cadmium contaminated soils with plant growth promoting bacteria – effects on phytoremediation strategies. Chemosphere 92, 74–83. https://doi.org/10.1016/j.chemosphere.2013.02.055. Marschner, P., Crowley, D., Rengel, Z., 2011. Rhizosphere interactions between microorganisms and plants govern iron and phosphorus acquisition along the root axis model and research methods. Soil Biol. Biochem. 43, 883–894. https://doi.org/10. 1016/j.soilbio.2011.01.005. McBeath, T.M., McLaughlin, M.J., 2014. Efficacy of zinc oxides as fertilisers. Plant Soil 374, 843–855. https://doi.org/10.1007/s11104-013-1919-2. Miller, L.V., Krebs, N.F., Hambidge, K.M., 2007. A mathematical model of zinc absorption in humans as a function of dietary zinc and phytate. J. Nutr. 137, 135–141. Moreno, M.T., Carmona, E., Santiago, A., Ordovás, J., Delgado, A., 2015. Olive husk compost improves the quality of intensively cultivated agricultural soils. L. Degrad. Dev. 27, 449–459. https://doi.org/10.1002/ldr.2410. Murphy, J., Riley, J.P., 1962. A modified single solution method for the determination of phosphate in natural waters. Anal. Chem. ACTA 27, 31–36. https://doi.org/10.1016/ S0003-2670(00)88444-5. Obrador, A., Novillo, J., Alvarez, J.M., 2003. Mobility and availability to plants of two zinc sources applied to a calcareous soil. Soil Sci. Soc. Am. J. 67, 564–572. https:// doi.org/10.2136/sssaj2003.0564. Olsen, S.R., Cole, C.V., Watanabe, F.S., Dean, L.A., 1954. Estimation of Available Phosphorus in Soils by Extraction With Sodium Bicarbonate, USDA Circular 939. US Government Printing Office, Washington DC. Ova, E.A., Kutman, U.B., Ozturk, L., Cakmak, I., 2015. High phosphorus supply reduced zinc concentration of wheat in native soil but not in autoclaved soil or nutrient solution. Plant Soil 393, 147–162. https://doi.org/10.1007/s11104-015-2483-8. Pfeiffer, W.H., McClafferty, B., 2007. HarvestPlus: breeding crops for better nutrition. Crop Sci. 47, 88–105. https://doi.org/10.2135/cropsci2007.09.0020IPBS. Ramesh, A., Sharma, S.K., Sharma, M.P., Yadav, N., Joshi, O.P., 2014. Inoculation of zinc solubilizing Bacillus aryabhattai strains for improved growth, mobilization and biofortification of zinc in soybean and wheat cultivated in Vertisols of central India. Appl. Soil Ecol. 73, 87–96. https://doi.org/10.1016/j.apsoil.2013.08.009. Rashid, A., Ryan, J., 2004. Micronutrient constraints to crop production in soils with mediterranean-type characteristics: a review. J. Plant Nutr. 27, 959–975. https://doi. org/10.1081/PLN-120037530. Recena, R., Díaz, I., del Campillo, M.C., Torrent, J., Delgado, A., 2016. Calculation of threshold Olsen P values for fertilizer response from soil properties. Agron. Sustain.

5. Conclusions B. sutbilis QST713 increased total P uptake by wheat plants except in the Vertisol with rock phosphate supply. Cellulose was effective increasing P uptake in both soils with and without P supply. B. subtilis was effective improving Zn concentration in grains while the effect on total Zn in plants was non-significant. Improvement of P uptake by B. subtilis and cellulose did not lead to an antagonistic effect on Zn, whose harvest index was improved by B. subtilis. These results provide evidence of the benefits of agricultural practices affecting microbial properties of the rhizosphere in overcoming P deficiency while improving Zn accumulation in grains. Acknowledgements This work was funded by the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund of the European Union through the National Research, Development and Innovation Programme (Plan Estatal I + d + i, Project AGL201457835-C2-1-R). Bacillus subtilis strain QST713 was kindly supplied by Bayer CrospScience. The authors thank the Institute for Agricultural and Fisheries Research and Training of Andalusia (IFAPA) for its cooperation and the Agricultural Research Service of the University of Seville (SIA) for technical assistance and access to their facilities. References Acutis, M., Scaglia, B., Confalonieri, R., 2012. Perfunctory analysis of variance in agronomy, and its consquences in experimental results interpretation. Eur. J. Agron. 43, 129–135. https://doi.org/10.1016/j.eja.2012.06.006. Alloway, B.J., 2009. Soil factors associated with zinc deficiency in crops and humans. Environ. Geochem. Health 31, 537–548. https://doi.org/10.1007/s10653-0099255-4. Borrill, P., Connorton, J.M., Balk, J., Miller, A.J., Sanders, D., Uauy, C., 2014. Biofortification of wheat grain with iron and zinc: integrating novel genomic resources and knowledge from model crops. Front. Plant Sci. 5, 53. https://doi.org/10. 3389/fpls.2014.00053. Cakmak, I., 2008. Enrichment of cereal grains with zinc: agronomic or genetic biofortification? Plant Soil 302, 1–17. https://doi.org/10.1007/s11104-007-9466-3. Casida Jr., L.E., Klein, D.A., Santoro, T., 1964. Soil dehydrogenase activity. Soil Sci. 98, 371–376. https://doi.org/10.1097/00010694-196412000-00004. Catlett, K.M., Heil, D.M., Lindsay, W.L., Ebinger, M.H., 2002. Soil chemical properties controlling zinc activity in 18 Colorado soils. Soil Sci. Soc. Am. J. 66, 1182–1189. https://doi.org/10.2136/sssaj2002.1182. Chen, X.P., Zhang, Y.Q., Tong, Y.P., Xue, Y.F., Liu, D.Y., Zhang, W., Deng, Y., Meng, Q.F., Yue, S.C., Yan, P., Cui, Z.L., Shi, X.J., Guo, S.W., Sun, Y.X., Ye, Y.L., Wang, Z.H., Jia,

88

Applied Soil Ecology 142 (2019) 81–89

A. Moreno-Lora, et al. Dev. 36, 54. https://doi.org/10.1007/s13593-016-0387-5. Recena, R., Díaz, I., Delgado, A., 2017. Estimation of total plant available phosphorus in representative soils from Mediterranean areas. Geoderma 297, 10–18. https://doi. org/10.1016/J.GEODERMA.2017.02.016. Roholla Mousavi, S., 2011. Zinc in crop production and interaction with phosphorus. Aust. J. Basic Appl. Sci. 5, 1503–1509. Ryan, J., Ibrikci, H., Delgado, A., Torrent, J., Sommer, R., Rashid, A., 2012. Significance of phosphorus for agriculture and the environment in the West Asia and North Africa region. Adv. Agron. 114, 91–153. https://doi.org/10.1016/B978-0-12-394275-3. 00004-3. Ryan, J., Ibrikci, H., Rashid, A., Sommer, R., 2013. Phosphorus in low-input dryland agriculture: the perspective from Syria. Commun. Soil Sci. Plant Anal. 44, 2378–2392. https://doi.org/10.1080/00103624.2013.794825. Sánchez-Rodríguez, A.R., Cañasveras, J.C., del Campillo, M.C., Barrón, V., Torrent, J., 2013. Iron chlorosis in field grown olive as affected by phosphorus fertilization. Eur. J. Agron. 51, 101–107. https://doi.org/10.1016/J.EJA.2013.07.004. Schröder, J.J., Schulte, R.P.O., Creamer, R.E., Delgado, A., van Leeuwen, J., Lehtinen, T., Rutgers, M., Spiegel, H., Staes, J., Tóth, G., Wall, D.P., 2016. The elusive role of soil quality in nutrient cycling: a review. Soil Use Manag. 32, 476–486. https://doi.org/ 10.1111/sum.12288. Soil Survey Staff, 2014. Keys to Soil Taxonomy, 12th ed. USDA Natural Resources Conservation Service, Washington, DC. StatPoint Technologies, 2013. Statgraphics Centurion XVI. Warrenton. Thébault, A., Clément, J-C., Ibanez, S., Roy, J., Geremia, R.A., Pérez, C.A., Butler, A., Estiene, Y., Lavorel, S., 2014 Nitrogen limmitation and microbial diversity in the treeline. Oikos 123, 729–740. https://doi.org: https://doi.org/10.1111/j.1600-0706. 2013.00860.x. TJA Solutions, 1999. SOLAAR M Series Operators Manual. Unicam Ltd, Cambridge, United Kingdom. Tuitert, G., Szczech, M., Bollen, G.J., 1998. Suppression of rhizoctonia solani in potting mixtures amended with compost made from organic household waste. Phytopathology 88, 764–773. https://doi.org/10.1094/PHYTO.1998.88.8.764. Verma, T.S., Minhas, R.S., 1987. Zinc and phosphorus interaction in a wheat-maize

cropping system. Fertil. Res. 13, 77–86. https://doi.org/10.1007/BF01049804. Viruel, E., Erazzú, L.E., Calsina, L.M., Ferrero, M.A., Lucca, M.E., Siñeriz, F., 2014. Inoculation of maize with phosphate solubilizing bacteria: effect on plant growth and yield. J. Soil Sci. Plant Nutr. 14, 819–831. https://doi.org/10.4067/S071895162014005000065. Wang, G., Xu, Y., Jin, J., Liu, J., Zhang, Q., Liu, X., 2009. Effect of soil type and soybean genotype on fungal community in soybean rhizosphere during reproductive growth stages. Plant Soil 317, 135–144. https://doi.org/10.1007/s11104-008-9794-y. Wang, Y., Li, J., Gao, X., Li, X., Ren, T., Cong, R., Lu, J., 2014. Winter oilseed rape productivity and nutritional quality responses to zinc fertilization. Agron. J. 106, 1349–1357. https://doi.org/10.2134/agronj14.0029. White, P.J., Broadley, M.R., 2009. Biofortification of crops with seven mineral elements often lacking in human diets – iron, zinc, copper, calcium, magnesium, selenium and iodine. New Phytol. 182, 49–84. https://doi.org/10.1111/j.1469-8137.2008. 02738.x. Zadoks, J.C., Chang, T.T., Konzak, C.F., 1974. A decimal code for the growth stages of cereals. Weed Res. 14, 415–421. https://doi.org/10.1111/j.1365-3180.1974. tb01084.x. Zhang, Y.Q., Deng, Y., Chen, R.Y., Cui, Z.L., Chen, X.P., Yost, R., Zhang, F.S., Zou, C.Q., 2012a. The reduction in zinc concentration of wheat grain upon increased phosphorus-fertilization and its mitigation by foliar zinc application. Plant Soil 361, 143–152. https://doi.org/10.1007/s11104-012-1238-z. Zhang, Y.Q., Sun, Y.X., Ye, Y.L., Karim, M.R., Xue, Y.F., Yan, P., Meng, Q.F., Cui, Z.L., Cakmak, I., Zhang, F.S., Zou, C.Q., 2012b. Zinc biofortification of wheat through fertilizer applications in different locations of China. Field Crop Res. 125, 1–7. https://doi.org/10.1016/j.fcr.2011.08.003. Zhang, W., Liu, D.Y., Li, C., Chen, X.P., Zou, C.Q., 2017. Accumulation, partitioning, and bioavailability of micronutrients in summer maize as affected by phosphorus supply. Eur. J. Agron. 86, 48–59. https://doi.org/10.1016/j.eja.2017.03.005. Zhao, A. qing, Tian, X. hong, Cao, Y. xian, Lu, X. chun, Liu, T., 2014. Comparison of soil and foliar zinc application for enhancing grain zinc content of wheat when grown on potentially zinc-deficient calcareous soils. J. Sci. Food Agric. 94, 2016–2022. https:// doi.org/10.1002/jsfa.6518.

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