Can silicon applied to correct soil acidity in combination with Azospirillum brasilense inoculation improve nitrogen use efficiency in maize?

Alternative management practices are needed to minimize the need for chemical fertilizer use in non-leguminous cropping systems. The use of biological agents that can fix atmospheric N has shown potential to improve nutrient availability in grass crops. This research was developed to investigate if inoculation with Azospirillum brasilense in combination with silicon (Si) can enhance N use efficiency (NUE) in maize. The study was set up in a Rhodic Hapludox under a no-till system, in a completely randomized block design with four replicates. Treatments were tested in a full factorial design and included: i) five side dress N rates (0 to 200 kg ha-1); ii) two liming sources (Ca and Mg silicate and dolomitic limestone); and iii) with and without seed inoculation with A. brasilense. Inoculation with A. brasilense was found to increase grain yield by 15% when N was omitted and up to 10% when N was applied. Inoculation also increased N accumulation in plant tissue. Inoculation and limestone application were found to increase leaf chlorophyll index, number of grains per ear, harvest index, and NUE. Inoculation increased harvest index and NUE by 9.5 and 19.3%, respectively, compared with non-inoculated plots. Silicon application increased leaf chlorophyll index and N-leaf concentration. The combination of Si and inoculation provided greater Si-shoot accumulation. This study showed positive improvements in maize growth production parameters as a result of inoculation, but the potential benefits of Si use were less evident. Further research should be conducted under growing conditions that provide some level of biotic or abiotic stress to study the true potential of Si application.

Introduction

Maize (Zea mays L.) is among of the most relevant and cultivated grain crop used for human and animal consumption [1]. In Brazil, the average yield (e.g., 5,476 kg ha-1) is still low compared with high yielding areas of the USA (USDA average 10,840 kg ha-1) and Europe (Eurostat average 8,399 kg ha-1) [2-5], despite technological improvements in plant nutrition, soil management and field equipment. This low maize yield observed in Brazil is usually attributed to low-fertility soils that require high fertilizer inputs for optimum productivity [6]. Grass crops, such as maize, wheat (Triticum aestivum) and rice (Oryza sativa), consume approximately 50% of all N-based fertilizer produced [7]. It has been estimated that as much as 15% of the total operating profit of the maize produced in the Brazilian Savannah is due to side-dress N application [8]. It is well known that production and over application of N fertilizer contribute to greenhouse gas emission and water contamination [9-11]. Management practices that improve N use efficiency (NUE) are therefore needed to assure that maize productivity is maximized while reducing the environmental impacts resulting from excessive nutrient application in the Brazilian Savannah [8, 12, 13].

The use of biological agents such as plant growth-promoting bacteria (PGPB) can improve NUE, crop development, and grain yield in cereal crops growing in tropical regions [6, 8, 10]. Several PGPB genera associate with different species of agricultural importance, such as Azospirillum, Arthobacter, Azotobacter, Bacillus, Bradyrhizobium, Burkholderia, Clostridium, Gluconacetobacter, Herbaspirillum, Pseudomonas, Rhizobium and Streptomyces [14]. In Latin America, the use of PGPB in many different crops has increased significantly over the last decade [6, 15]. The Azospirillum spp. is one of the most studied plant growth promoting genus [16]. An analysis of field trials conducted worldwide for over 20 years, where various non-legume crops were inoculated with Azospirillum spp. under different weather and soil conditions, concluded that crop yield can increase up to 30% with inoculation [17]. Zeffa et al. [14] performed a meta-analysis on the influence of N side dress application associated with Azospirillum spp. inoculation in maize and reported an average yield increase of 652 kg ha-1.

These bacteria can stimulate plant growth by a series of mechanisms, including the production of phytohormones, such as indole-3-acetic acid (IAA), gibberellins, cytokinins and salicylic acid [18, 19], biological nitrogen fixation (BNF) [20], increase nutrient availability [13] and increase nitric oxide production [21]. In addition, Azospirillum spp. has been reported to reduce biotic and abiotic stresses, increase proline content in shoots and roots, improve water potential, increase apoplastic routes for water content, increase cell wall elasticity and chlorophyll content, increase photoprotection pigments and improve stomatal conductance [22]. Azospirillum spp. has been reported to increase plant resistance to pathogens, for example promoting disease resistance in rice crops [23] or inhibiting development of bacterial diseases on Prunus cerasifera [24]. The level of inoculation response has been described as wide ranging and is determined by plant-inoculant-environment interactions [25].

The use of Si-based fertilizers has been reported to improve N uptake and NUE in different crops [26, 27]. It has been reported that some crops (e.g., sugarcane—Saccharum officinarum, rice, wheat, and maize) can absorb Si quickly and in great quantity [28]. In recent years, the number of studies reporting the effects of Si application to crops has increased substantially, especially in grain crops. This increased interest in Si is likely due to the beneficial effects of Si application on plant resistance to abiotic and biotic stresses such as insects and pathogens [29], salt stress and drought [30], and heavy rain and winds [31]. Silicon has also been reported to improve crop yield [32], plant growth, plant architecture, erectness, and photosynthesis rate [33], to decrease transpiration rate [34, 35] and damage caused by pests and diseases [36], and to reduce water use [32]. Recently, Jang et al. [37] concluded that Si application could induce increased levels of bioactive hormones that participate in various physiological responses. For example, the authors reported an increase in the levels of hormones that are involved in the mitigation of abiotic stress (active endogenous gibberellin GA1, jasmonic acid JA, and salicylic acid SA). Calcium and magnesium silicate also can increase base saturation and extractable levels of P, Ca, Mg, and Si, as well as decrease the phytotoxic effect of Fe, Mn, Zn, Al, and Cd [38, 39] and correct soil acidity [40]. Galindo et al. [41] studying inoculation methods associated with Si application reported an average wheat grain yield increase of 6.7% when seed inoculation and Si application were performed.

The application of Ca and Mg silicate to supply Si in addition to seed inoculation with A. brasilense may increase maize grain yield; however, increases in yield are not always observed. Further research with inoculation associated with Si use is needed to determine how to maximize their benefits on NUE, plant development, and productivity. In addition, studies are needed to define how much N fertilizer needs to be applied when Si is applied in combination with A. brasilense for optimum grain yield. It would also be valuable to determine if Si utilization has any negative effect when maize is inoculated with A. brasilense. The hypothesis of this study was that the application of Si in combination with A. brasilense inoculation would improve NUE and reduce the amount of N required for maximum maize production in the Brazilian Savannah.

Materials and methods

Field site description

The study was conducted under field conditions in Selvíria (Brazilian Cerrado–Savannah region), state of Mato Grosso do Sul, Brazil (20°22′S and 51°22′W, 335 m above sea level (a.s.l.)) (S1 Fig), during the crop years of 2015/16 and 2016/17. The soil was classified as Clayey Oxisol (Rhodic Hapludox) according to the Soil Survey Staff [42]. Soil chemical and physical properties were determined from soil samples collected prior to lime application and analysed according to Raij et al. [43]. Total N was determined by the Kjeldahl method [44]. Si was determined after extraction in Ca chloride (0.01 mol L-1) according to the methodology of Korndörfer et al. [45] (Table 1). Particle size analysis was performed according to Embrapa, [46] and showed the soil at the site used had 471 g kg-1of sand, 90 g kg-1 of silt and 439 g kg-1 of clay at the 0–0.20 m depth; and 471 g kg-1of sand, 82 g kg-1 of silt and 447 g kg-1 of clay at the 0.20–0.40 m depth. The experimental area had been cultivated with annual cereal and legume crops for over 30 years. In addition, the area has been under no-tillage for the last 13 years. The crop sequence prior to maize in 2015/16 were maize (2014), soybean (2014/15) and maize (2015) and prior to maize in 2016/17 was wheat (2016). The 2015 maize crop received the application of 180 kg N ha-1. The maximum and minimum temperatures, rainfall, and air relative humidity observed during the study are presented in Fig 1.

Table 1

Soil chemical attributes in 0–0.20m and 0.20–0.40m layers before the application of liming sources.

Soil chemical attributes	0–0.20m layer	0.20m-0.40m layer
Total N	1.04 g kg-1	0.81 g kg-1
Si (CaCl2)	9.4 mg dm-3	10.2 mg dm-3
P (resin)	19 mg dm-3	17 mg dm-3
S (SO4)	10 mg dm-3	30 mg dm-3
Organic matter	21 g dm-3	16 g dm-3
pH (CaCl2)	5.0	4.8
K	2.1 mmolc dm-3	1.2 mmolc dm-3
Ca	19.0 mmolc dm-3	11.0 mmolc dm-3
Mg	13.0 mmolc dm-3	8.0 mmolc dm-3
H+Al	28.0 mmolc dm-3	28.0 mmolc dm-3
Al	1.0 mmolc dm-3	2.0 mmolc dm-3
B (hot water)	0.17 mg dm-3	0.11 mg dm-3
Cu (DTPA)	3.1 mg dm-3	2.1 mg dm-3
Fe (DTPA)	20.0 mg dm-3	10.0 mg dm-3
Mn (DTPA)	27.2 mg dm-3	10.7 mg dm-3
Zn (DTPA)	0.8 mg dm-3	0.2 mg dm-3
Cation exchange capacity (pH 7.0)	62.1 mmolc dm-3	48.2 mmolc dm-3
Base saturation (%)	55	42

Fig 1

Rainfall, air relative humidity, maximum and minimum temperatures obtained from the weather station located in the Education and Research Farm of FE / UNESP during the maize cultivation in the period November 2015 to April 2016 (A) November 2016 to April 2017 (B).

Rainfall, air relative humidity, maximum and minimum temperatures obtained from the weather station lon>an class="Chemical">cated in the Education and Research Farm of FE / UNESP during the maize cultivation in the period November 2015 to April 2016 (A) November 2016 to April 2017 (B).

Experimental design, treatment, and field management

The experimental design was a randomized complete block design with four replicates arranged in a 5 × 2 × 2 factorial scheme. There were five N rates applied as side dress (0, 50, 100, 150, and 200 kg ha-1); two liming materials Ca and Mg silicate, which was also the Si source (the composition was 10% of Si, 25% CaO, and 6% MgO) with effective neutralizing power (ENP) of 88%; and dolomitic limestone (the composition was 28% CaO, 20% MgO) with an ENP of 80%; and two inoculations: with or without seed inoculation with A. brasilense. The experimental plots were six 5-m maize rows spaced at a distance of 0.45 m, and the useful area of the plot considered was the central four rows, excluding 0.5 m from each end. Prior to the start of the study a blanket fertilizer application of 375 kg ha-1 of the granular fertilizer 08-28-16 (N-P2O5-K2O) had to be performed on the entire experimental site to supply phosphorus and potassium. During this blanket nutrient application 30 kg N ha-1 was applied to the entire experimental area. Therefore, the total amount of N applied in each treatment is the amount of N applied at the side dress (0 to 200 kg N ha-1 as indicated above) in addition to the blanket application of 30kg N ha-1. Split application of fertilizers is a general practice used by farmers growing maize in Brazil. Nitrogen treatments were applied manually to evenly distribute the fertilizer on the soil surface without incorporation. The amount of fertilizer needed per plot was applied between the maize rows on December 13, 2015, and December 10, 2016, when the plants were in the V6 stage (with six leaves completely unfolded). After side dress application the experimental area was irrigated with 14 mm of water to minimize ammonia volatilization. Harvest took place on March 15, 2016, and March 21, 2017.

Lime was broadcast applied in one single application at the rate of 1.76 t ha-1 for the silicate and 1.94 t ha-1 for the limestone 30 days before planting. No incorporation was done as the area was under no-tillage. This is also a common practice used by farmers growing maize in this region of Brazil. The amount of lime applied was based on the amount needed to increase the base saturation to 80% based on the initial soil analysis.

Azospirillum brasilense strains Ab-V5 Ab-V6 was inoculated at a rate of 300 mL of liquid inoculant per hectare (guarantee of 2×108 CFU [colony forming unity] mL-1). These are commercial strains used in Brazil with brand name AzoTotal®. These strains when used under similar conditions (Brazilian Savannah) have shown positive results in maize development [2, 8, 13, 16]. Seeds were inoculated one hour before planting and after seed treatment with fungicide and insecticide when the seeds were completely dry. The fungicides used were thiophanate-methyl + pyraclostrobin (56 g + 6 g of a.i. per 100 kg of seed) and the insecticide used was fipronil (62 g of a.i. per 100 kg of seed).

Maize DOW 2B710 PW simple hybrid was mechanically sown on November 13 and November 11 for the 15/16 and 16/17 crops, respectively, at the planting density of 3.3 seeds per metre (7.3 plants m-2). Seedling emergence occurred five days after sowing, on November 18, 2015, and November 16, 2016, respectively. Supplemental irrigation using a centre pivot sprinkling system was done when need at a water depth of 14 mm. The herbicides atrazine (1000 g ha-1 of a.i.) and tembotrione (84 g ha-1 of a.i.) in combination with vegetable oil adjuvant (720 g ha-1 of a.i.) were used for post-emergence weed control on December 4, 2015 and December 2, 2016, respectively. During the growing season, weeds were controlled by applying 2,4-D (670 g ha–1 of the active ingredient [a.i.]) and glyphosate (1800 g ha–1 of the a.i.). Insect control was made with triflumuron (24 g ha-1 a.i.) and methomyl (215 g ha-1 a.i.) on December 20, 2015, and December 17, 2016, respectively. The straw remaining from the previous crop was collected by removing the residue from 10 random points in the experimental area measuring 0.5 m2. The residue sampled was used for chemical tests to determine nutrient accumulation (Table 2).

Table 2

Nutrient accumulation in maize straw (2015/16 predecessor crop).

N	P	K	Ca	Mg	S	Si	B	Cu	Fe	Mn	Zn	C/N ratio
------------ (kg ha-1)------------							------------ (g ha-1) -----------
78.2	7.2	68.8	23.3	21.1	16.3	13.1	260.4	74.0	1018.1	709.6	185.1	38.3

Measurements collected

In season evaluations

During the growing season, leaf chlorophyll index (LCI) was indirectly measured in 10 plants using a portable non-destructive chlorophyll meter ClorofiLOG® model CFL-1030 [47]. Tissue N and Si concentration was determined by collecting 20 leaves from the main ear insertion during the female flowering stage from each plot. The leaves were then used to determine N and Si uptake by tissue. Nitrogen and Si concentration in tissue was determined according to Cantarella et al. [48]. In addition, N and Si concentration in shoot and roots were also quantified, and N analysis followed the methodology proposed by Malavolta et al. [49], while Si analysis followed the methodology proposed by Silva [50].

Productive components, NUE, and grain yield

Root and shoot dry matter were measured during the female flowering stage in each experimental plot by collecting 5 plants per plot. Plant height and diameter were measured at maturity in 10 plants per plot. Height was measured from the ground surface to the apex of the tassel, and stem diameter was measured in the second internode of the plant using a caliper. Ten ears were collected at harvest to determine ear diameter, ear length (determined from the base of the ear to the apex), number of rows per ear, number of grains per row, number of grains per ear, and mass of 100 grains adjusted to 13% moisture. Harvest index (HI) and nitrogen use efficiency (NUE) wer then calculated with the Eqs 1 and 2 (below). Grain yield was determined by harvesting the useful experimental area and adjusted to 13%.

Where, GYF = Grain Yield with pan class="Chemical">fertilizer and GYW = Grain Yield without n>an class="Chemical">fertilizer

Statistical analysis

All data were initially tested for normality using the Shapiro and Wilk [52] test which showed the data to be normally distributed (W≥0.90). The data was analysed by ANOVA in a 3-way factorial design with N application rates, liming material source and inoculation and their interactions considered fixed effects in the model using ExpDes package. Mean separation was done when significant factors or interactions were observed using the test Tukey. Regression analysis was used to discern whether there was a linear or non-linear response to N rates using R [53].

Results

Leaf chlorophyll index and plant nutrient uptake

Nitrogen leaf concentration responded linearly to N application rates in 2015/16 (Tables 3 and 4). In 2016/17, in the absence of inoculation, the application of limestone resulted in greater N-leaf concentration compared to silicate application; however, with inoculation, silicate provided greater concentration compared to limestone use (Tables 3 and 5). In 2016/17, the control treatment (0 N) showed greater N concentration when inoculated compared with non-inoculated treatments (Fig 2A). Furthermore, inoculation changed the behaviour of N concentration in tissue (Table 3). Nitrogen-leaf concentration in tissue responded linearly to N application rates in the presence of inoculation and non-linearly in the absence of inoculation (Fig 2A). Si-leaf concentration responded linearly to N application rates (Tables 3 and 4) and was greater in the plots with Ca and Mg silicate application in both years than in limestone amended plots (Tables 3 and 6).

Table 3

P-values for N and Si-leaf concentration, leaf chlorophyll index, N and Si-shoot and root accumulation, shoot and root dry matter, plant height, stem diameter, ear length and diameter, number of rows per ear, grains per row and grains per ear, mass of 100 grains, harvest index, nitrogen use efficiency, and corn grain yield affected by rates of nitrogen, liming sources, with or without inoculation with Azospirillum brasilense.

2015/16 and 2016/17.

	N-leaf concentration			Si-leaf concentration			Leaf chlorophyll index		N-shoot accumulation
	---------- (g kg-1 of D. M.) ---------								--------- (kg ha-1) ---------
P-values	2015/2016	2016/2017		2015/2016	2016/2017		2015/2016	2016/2017	2015/2016	2016/2017
Rates (R)	0.009**	0.001**		0.949ns	0.026*		0.643ns	0.001**	0.026*	0.009**
Sources (S)	0.596ns	0.594ns		0.001**	0.007**		0.002**	0.032*	0.300ns	0.209ns
Inoculation (I)	0.915ns	0.056ns		0.062ns	0.648ns		0.009**	0.041*	0.465ns	0.606ns
R × S	0.231ns	0.942ns		0.135ns	0.268ns		0.002**	0.001**	0.765ns	0.980ns
R × I	0.364ns	0.001**		0.942ns	0.946ns		0.955ns	0.631ns	0.043*	0.794ns
R × I	0.914ns	0.001**		0.172ns	0.197ns		0.008**	0.875ns	0.855ns	0.183ns
R × S × I	0.342ns	0.135ns		0.604ns	0.776ns		0.311ns	0.739ns	0.431ns	0.870ns
	Si-shoot accumulation			N-root accumulation			Si-root accumulation		Shoot dry matter
	---------------- (kg ha-1) ---------------
P-values	2015/2016	2016/2017		2015/2016	2016/2017		2015/2016	2016/2017	2015/2016	2016/2017
Rates (R)	0.10ns	0.008**		0.001**	0.001**		0.001**	0.015*	0.116ns	0.044*
Sources (S)	0.935ns	0.311ns		0.351ns	0.513ns		0.001**	0.001**	0.763ns	0.299ns
Inoculation (I)	0.577ns	0.553ns		0.356ns	0.575ns		0.949ns	0.538ns	0.506ns	0.801ns
R × S	0.997ns	0.749ns		0.012*	0.014*		0.139ns	0.242ns	0.986ns	0.868ns
R × I	0.559ns	0.378ns		0.015*	0.004**		0.707ns	0.622ns	0.666ns	0.535ns
R × I	0.384ns	0.004**		0.692ns	0.508ns		0.052ns	0.695ns	0.242ns	0.085ns
R × S × I	0.472ns	0.810ns		0.648ns	0.804ns		0.309ns	0.221ns	0.391ns	0.903ns
	Root dry matter			Plant height			Stem diameter		Ear lenght
	------ (kg ha-1) -----			----- (m) -----			------ (cm)) -----
P-values	2015/2016	2016/2017		2015/2016	2016/2017		2015/2016	2016/2017	2015/2016	2016/2017
Rates (R)	0.001**	0.044*		0.444ns	0.001**		0.006**	0.001**	0.001**	0.001**
Sources (S)	0.041*	0.773ns		0.240ns	0.523ns		0.016*	0.530ns	0.107ns	0.332ns
Inoculation (I)	0.299ns	0.592ns		0.084ns	0.711ns		0.267ns	0.737ns	0.142ns	0.234ns
R × S	0.002**	0.521ns		0.869ns	0.614ns		0.113ns	0.139ns	0.567ns	0.280ns
R × I	0.096ns	0.785ns		0.864ns	0.960ns		0.064ns	0.475ns	0.177ns	0.573ns
R × I	0.324ns	0.537ns		0.586ns	0.088ns		0.377ns	0.001**	0.385ns	0.270ns
R × S × I	0.358ns	0.598ns		0.886ns	0.573ns		0.236ns	0.267ns	0.228ns	0.678ns
	Ear diameter			Number of rows per ear			Number of grains per row		Number of grains per ear
	--------- (cm) ---------
P-values	2015/2016	2016/2017		2015/2016	2016/2017		2015/2016	2016/2017	2015/2016	2016/2017
Rates (R)	0.386ns	0.003**		0.733ns	0.919ns		0.225ns	0.001**	0.001**	0.005**
Sources (S)	0.640ns	0.013*		0.222ns	0.306ns		0.339ns	0.668ns	0.746ns	0.438ns
Inoculation (I)	0.932ns	0.233ns		0.812ns	0.011*		0.794ns	0.190ns	0.023*	0.014*
R × S	0.392ns	0.515ns		0.086ns	0.734ns		0.152ns	0.571ns	0.121ns	0.930ns
R × I	0.124ns	0.201ns		0.898ns	0.200ns		0.344ns	0.110ns	0.015*	0.116ns
R × I	0.545ns	0.894ns		0.504ns	0.309ns		0.606ns	0.054ns	0.299ns	0.340ns
R × S × I	0.632ns	0.572ns		0.901ns	0.329ns		0.171ns	0.441nsns	0.339ns	0.198ns
	Mass of 100 grains			Harvest index			Nitrogen use efficiency		Grain Yield
	---------- (g) ----------			------- (%) -------			-- (kg grains kg N applied-1) --		----- (kg ha-1) -----
P-values	2015/2016		2016/2017	2015/2016		2016/2017	2015/2016	2016/2017	2015/2016	2016/2017
Rates (R)	0.002**		0.001**	0.045*		0.009**	0.001**	0.001**	0.001**	0.001**
Sources (S)	0.010*		0.260ns	0.031*		0.258ns	0.918ns	0.467ns	0.840ns	0.995ns
Inoculation (I)	0.081ns		0.234ns	0.027*		0.004**	0.007**	0.193ns	0.006**	0.033*
R × S	0.941ns		0.211ns	0.920ns		0.667ns	0.249ns	0.001ns	0.008**	0.001**
R × I	0.608ns		0.509ns	0.708ns		0.562ns	0.422ns	0.489ns	0.003**	0.001**
R × I	0.445ns		0.220ns	0.748ns		0.482ns	0.380ns	0.752ns	0.118ns	0.324ns
R × S × I	0.588ns		0.528ns	0.616ns		0.589ns	0.063ns	0.122ns	0.115ns	0.781ns

**, * and ns: significant at p<0.01, p<0.05, and not significant, respectively.

Table 4

2015/2016 and 2016/2017 crop season significant regression equations as a function of N rates.

Independent variable	2015/16 crop season
N-leaf concentration	Ŷ = 28.1655 + 0.0077x (R2 = 0.84**)
Si-root accumulation	Ŷ = 5.1999 + 0.0683x - 0.0002x2 (R2 0.99**)
Stem diameter	Ŷ = 2.0797 + 0.0004x (R2 = 0.71**)
Ear length	Ŷ = 16.8721 + 0.0043x (R2 = 0.71**)
Mass of 100 grains	Ŷ = 30.3150 + 0.0079x (R2 = 0.98**)
Harvest index	Ŷ = 41.0715 + 0.0236x (R2 = 0.55*)
Nitrogen use efficiency	Ŷ = 20.7759–0.0592x (R2 = 0.87**)
Independent variable	2016/2017 crop season
Si-leaf concentration	Ŷ = 16.8082 + 0.0070x (R2 = 0.65*)
N-shoot accumulation	Ŷ = 152.5325 + 0.3496x (R2 = 0.99**)
Si-shoot accumulation	Ŷ = 24.5345 + 0.0707x (R2 = 0.96**)
Si-root accumulation	Ŷ = 6.9308 + 0.0146x (R2 = 0.54*)
Shoot dry matter	Ŷ = 10123.4143 + 15.0346x (R2 = 0.96*)
Root dry matter	Ŷ = 768.6313 + 1.2807x (R2 = 0.73*)
Plant height	Ŷ = 2.4589 + 0.0019x - 0.000005x2 (R2 = 0.95**)
Stem diameter	Ŷ = 2.0323 + 0.0007x (R2 = 0.90**)
Ear length	Ŷ = 15.3472 + 0.0126x (R2 = 0.92**)
Ear diameter	Ŷ = 5.4497 + 0.0014x (R2 = 0.78**)
Number of grains per row	Ŷ = 32.9052 + 0.0243x (R2 = 0.92**)
Number of grains per ear	Ŷ = 605.9887 + 0.3856x (R2 = 0.89**)
Mass of 100 grains	Ŷ = 30.0132 + 0.0141x (R2 = 0.97**)
Harvest index	Ŷ = 42.2123 + 0.0269x (R2 = 0.79**)
Nitrogen use efficiency	Ŷ = 26.2053–0.0787x (R2 = 0.94**)

** and *: significant at p<0.01 and p<0.05, respectively.

Table 5

Interaction between inoculation and liming sources in leaf chlorophyll index, N-leaf concentration, Si-shoot accumulation and stem diameter.

2015/16 and 2016/17.

Source	2015/16 crop season
	Limestone	Silicate
	Leaf chlorophyll index
With A. brasilense	69 aA ± 2.9†	71 aA ± 2.1
Without A. brasilense	66 bB ± 3.0	70 aA ± 3.6
	2016/17 crop season
	N-leaf concentration (g kg-1)
With A. brasilense	27 bB ± 2.9	29 aA ± 2.8
Without A. brasilense	28 aA ± 2.4	26 bB ± 3.1
	Si-shoot accumulation (kg ha-1)
With A. brasilense	23 bB ± 0.7	38 aA ± 1.1
Without A. brasilense	36 aA ± 1.0	28 aA ± 0.5
	Stem diameter (cm)
With A. brasilense	2.0 bB ± 0.07	2.1 aA ± 0.09
Without A. brasilense	2.1 aA ± 0.08	2.0 aB ± 0.09

†The letters correspond to a significant difference at 5% probability level (p ≤ 0.05). Uppercase letters indicate difference between inoculation or not with A. brasilense, and lowercase letters indicate differences between liming sources, respectively. Means are followed by the standard deviation (n = 4).

Fig 2

Interaction between inoculation and nitrogen rates in N-leaf concentration in 2016/17 (a), interaction between liming sources and nitrogen rates in LCI in 2015/16 (b) and 2016/17 (c), interaction between inoculation and nitrogen rates in N-shoot accumulation in 2015/16 (d), interaction between liming sources and nitrogen rates in N-root accumulation in 2015/16 (e) and 2016/17 (f). The letters correspond to a significant difference at 5% probability level (p ≤ 0.05). ** and *: significant at p<0.01 and p<0.05, respectively. Error bars indicate the standard deviation of the mean (n = 4). P.M. = point of maximum response to N rates.

Table 6

Si-leaf concentration, Si-root accumulation, stem diameter, mass of 100 grains, ear diameter and harvest index as a function of liming sources.

2015/16 and 2016/17.

Source	2015/16 crop season	2016/17 crop season
	Si-leaf concentration (g kg-1)
Limestone	11.1 b ± 1.4†	16.9 b ± 3.4
Silicate	12.7 a ± 1.5	18.1 a ± 4.2
	Si-root accumulation (kg ha-1)
Limestone	7.6 b ± 2.2	6.6 b ± 2.5
Silicate	9.7 a ± 2.9	10.1 a ± 2.4
	Stem diameter (cm)
Limestone	2.1 b ± 0.09	-
Silicate	2.2 a ± 0.10	-
	Mass of 100 grains (g)
Limestone	31 b ± 1.3	-
Silicate	32 a ± 1.1	-
	Ear diameter (cm)
Limestone	-	5.7 a ± 0.2
Silicate	-	5.5 b ± 0.2
	Harvest index (%)
Limestone	45 a ± 4.6	-
Silicate	41 b ± 5.1	-

†The letters correspond to a significant difference at 5% probability level (p ≤ 0.05). Error bars indicate the standard deviation of the mean (n = 4).

**, * and ns: signifin>an class="Chemical">cant at p<0.01, p<0.05, and not significant, respectively.

** and *: pan class="Chemical">signifin>an class="Chemical">cant at p<0.01 and p<0.05, respectively.

†The letters correspn>ond to a n>an class="Chemical">significant difference at 5% probability level (p ≤ 0.05). Uppercase letters indicate difference between inoculation or not with A. brasilense, and lowercase letters indicate differences between liming sources, respectively. Means are followed by the standard deviation (n = 4).

†The letters pan class="Chemical">correspn>ond to a n>an class="Chemical">significant difference at 5% probability level (p ≤ 0.05). Error bars indicate the standard deviation of the mean (n = 4).

LCI response to N application changed based on the liming materials used and also in the cropping season (Table 3, Fig 2B and 2C). In 2015/16, LCI did not respond to N application when limestone was used, while it showed a non-linear response to increasing N rates when Ca Mg silicate was used. The highest LCI measured was observed when 93 kg N ha-1 was applied (Fig 2B). In 2016/17, LCI response to N application was found to be non-linear for both liming sources with Ca and Mg silicate tending to provide higher LCI at lower N application rates (Fig 2C). In addition, in 2015/16, in the absence of inoculation, the use of silicate led to higher LCI levels compared to the use of limestone (Tables 3 and 5). Also, in 2016/17 inoculation resulted in greater LCI compared to non-inoculated plots (Tables 3 and 7).

Table 7

Leaf chlorophyll index, number of rows per ear, grains per ear, harvest index, and N use efficiency as a function of inoculation.

2015/16 and 2016/17.

Source	2015/16 crop season	2016/17 crop season
	Leaf chlorophyll index
With A. brasilense	-	70 a ±2.9†
Without A. brasilense	-	67 b ± 2.7
	Number of rows per ear
With A. brasilense	-	18.5 a ± 0.7
Without A. brasilense	-	17.9 b ± 0.6
	Number of grains per ear
With A. brasilense	-	662 a ± 52
Without A. brasilense	-	627 b ± 70
	Harvest index (%)
With A. brasilense	45 a ± 5.3	47 a ± 3.8
Without A. brasilense	42 b ± 4.1	43 b ± 4.7
	N use efficiency (kg grains kg N applied-1)
With A. brasilense	14.8 a ± 3.6	-
Without A. brasilense	11.9 b ± 3.1	-

†The letters correspond to a significant difference at 5% probability level (p ≤ 0.05). Error bars indicate the standard deviation of the mean (n = 4).

†The letters pan class="Chemical">correspn>ond to a n>an class="Chemical">significant difference at 5% probability level (p ≤ 0.05). Error bars indicate the standard deviation of the mean (n = 4).

In 2015/16, N shoot acn>an class="Chemical">cumulation tended to respond linearly to N application rates when plots were inoculated with no trends for non-inoculated plots (Table 3, Fig 2D). In 2016/17, N-shoot accumulation respond linearly to N application regardless of inoculation (Tables 3 and 4). It was observed that in 2016/17 the application of limestone increased Si-shoot accumulation in non-inoculated plots (Tables 3 and 5). Silicon accumulation in tissue was found to respond linearly to N application rates in 2016/17 (Tables 3 and 4).

Nutrient accumulation in the root varied based on N application rates, liming materials, inoculation, and cropping seasons (Table 3). In 2015/16, N-root accumulation was found to respond linearly to N application rates when limestone was used and non-linearly with the application of Ca and Mg silicate (Table 3). It was observed that the highest N accumulation was achieved when 143 kg N ha-1 was applied in combination with Ca and Mg silicate (Fig 2E). Contrasting results were observed for the 2016/17 season; the N accumulation in root response to N application rates was linear for the Ca and Mg silicate and non-linear for the limestone (Fig 2F). Although, the highest accumulation was observed when Ca and Mg silicate was used, root N accumulation tended to be greater when plots were treated with limestone (Fig 2E and 2F). Similarly, N accumulation in root response to N rates showed contrasting results under the different inoculation treatments (Fig 3A and 3B). Root N accumulation as a function of N application rates in plots receiving inoculation was non-linear in the 2015/16 season and linear in the 2016/17 season (Fig 3A and 3B). In contrast, root N accumulation as a function of N application rates in non-inoculated plots was linear in the 2015/16 season and non-linear in the 2016/17 season (Fig 3A and 3B). Although there were significant differences, the results of the study do not allow for a clear understanding of the effects of liming materials and inoculation on nutrient accumulation in maize roots. Si-root accumulation showed different responses to N application rates and was found to respond non-linearly in 2015/16 and linearly in 2016/17 (Tables 3 and 4). In general, Si-root accumulation was greater in the plots with Ca and Mg silicate application in both years (Tables 3 and 6).

Fig 3

Interaction inoculation and nitrogen rates in N-root accumulation in 2015/16 (a) and 2016/17 (b), interaction between liming sources and nitrogen rates in root dry matter in 2015/16 (c) and in number of grains per ear in 2015/16 (d). The letters correspond to a significant difference at 5% probability level (p ≤ 0.05). ** and *: significant at p<0.01 and p<0.05, respectively. Error bars indicate the standard deviation of the mean (n = 4). P.M. = point of maximum response to N rates.

Productive components and NUE

In 2015/16, root biomass dry matter showed a non-linear respn>onse to N applin>an class="Chemical">cation rates and the application of limestone tended to result in greater root dry matter accumulation than Ca and Mg silicate, but only at low N application rates (Table 3, Fig 3C). In the second season, root biomass dry matter was found to respond linearly to N rates regardless of inoculation of silicate sources (Tables 3 and 4).

In 2016/17, inoculation had opposite effects on stem diameter based on liming materials (Tables 3 and 5). Stem diameter increased under inoculation when Ca and Mg silicate was used compared with non-inoculated; in contrast, stem diameter was greater in non-inoculated plots when limestone was used (Tables 3 and 5). In both years, stem diameter showed a linear response to N rates (Tables 3 and 4).

The number of grains per ear response to N application rates showed different behaviour based on inoculation in the 2015/16 season (Table 3). In 2015/16, the number of grains per ear in plots receiving inoculation showed a non-linear response to N application rates while non-inoculated plots showed a linear response to N rates (Fig 3D). Although there were different responses based on inoculation, there was no clear evidence of the effect of inoculation on the number of grains per ears in the first season. In 2016/17, the number of grains per ear was found to respond linearly to N application rates and also was greater when plots were inoculated compared with non-inoculated plots (Tables 3, 4 and 7).

Ear length, mass of 100 grains and harvest index were found to respond linearly to increasing N rates in both cropping years (Tables 3 and 4). Shoot dry matter, ear diameter and number of grains per row were found to respond linearly to increasing N rates in 2016/17 but showed no response in 2015/16 (Tables 3 and 4). Also, in 2016/17, plant height showed a non-linear response to increasing N application rates (Tables 3 and 4). In contrast, NUE was found to decrease linearly to increasing N application rates in both years (Tables 3 and 4). The use of Ca and Mg silicate lead to greater 100 grain mass in 2015/16; however, this liming source decreased ear diameter in 2016/17 and harvest index in 2015/16 (Tables 3 and 6). Inoculation with A. brasilense lead to greater harvest index in both years, NUE in 2015/16 (2.84 kg grains kg N applied-1, an increase that is equivalent to 23.7%), and number of rows per ear in 2016/17 compared with non-inoculated plots (Tables 3 and 7).

Grain yield

Maize grain yield showed difn>an class="Chemical">ferent behaviours in the two years of the study based on liming source (Table 3). In the 2015/16 season, application of Ca and Mg silicate caused a non-linear response to N application rates, while the application of limestone led to a linear response to N application rate (Fig 4A). In 2016/17, maize grain yield responded linearly to N rates, with plots treated with limestone showing a faster rate of increase in grain yield per kg of N applied than plots treated with Ca and Mg silicate (Fig 4B).

Fig 4

Interaction between liming sources and nitrogen rates in grain yield in 2015/16 (a) and 2016/17 (b). Interaction between inoculation and nitrogen rates in grain yield in 2015/16 (c) and 2016/17 (d). The letters correspond to a significant difference at 5% probability level (p ≤ 0.05). ** and *: significant at p<0.01 and p<0.05, respectively. Error bars indicate the standard error of the mean (n = 4). P.M. = point of maximum response to N rates.

The effect of inoculation and N application rates on grain yield was similar to what was observed for liming source and N application rates (Table 3). In the first season, plants receiving inoculation showed a linear response to increasing N rate, while non-inoculated plants showed a non-linear response to N rates (Fig 4C). Although different responses to N rates were observed for the two inoculation treatments, the most significant difference was observed at the highest N rate (Fig 4C). In 2016/17, plots that did not receive N yielded 7,955 kg ha-1 when inoculated and 6,905 kg ha-1 when not inoculated (Fig 4D). In addition, in 2016/17 grain yield was found to respond linearly to N application rates with non-inoculated plots having a greater rate of increase per kg of N applied than inoculated plots (Fig 4D). The results of the study showed that in 2016/17 each kg of N added resulted in an increase of 7.6 kg of grain for inoculated plots and 12.9 kg of grain for non-inoculated plots (Fig 4D). The lower rate of response observed for inoculated plots likely reflects the fact that inoculation provided some N to the maize during the growing season.

Discussion

N is the nutrient that is most demanded by maize plants and directly affects crop development and yield. The higher N availability as a function of the N application likely favoured the development of a well established root system leading to improved shoot development and maize grain yield. Increased root dry matter can positively influence root scavenging, that is important for the interception of N and Si in crop systems. Although the exact mechanisms underlying the PGPB effect on N acquisition by maize was not evaluated in the present study, it is possible that the greater NUE observed in inoculated plants was due to its ability to promote plant growth [6, 16, 19, 25]. Specifically, it has been demonstrated in draft genome sequences that the strains Ab-V5 and Ab-V6 of A. brasilense, carry similar nif and fix genes that confer their ability to fix atmospheric N [54]. Although the strains differ in their capacity to synthesize phytohormones [19, 55], both share the same genes related to the synthesis of auxins. This growth promotion mechanism might have improved the ability of plants to explore the soil more efficiently, as indicated in previous studies using A. brasilense [6, 55–59]. Also, according to Cormier et al. [60], two strategies may be devised for N use efficiency improvement: increasing the yield at a constant N supply and/or maintaining high yield when reducing N supply. In the first season, our data showed that grain yield in inoculated plots at the highest N rate tested yielded 10% more than non-inoculated plots. In the second season, a different behaviour was observed and a residual effect or NBF was likely observed (Fig 4D). Plots that were inoculated showed a slower rate of response to applied N compared with plots that were not inoculated. This was very evident in the control treatment where no N was added and yields were 15% greater in the inoculated plots. The lower rate of response for grain yield to N applied in inoculated plots suggests that N was being supplied from sources other than the fertilizer added. Similar results were reported elsewhere when side dress N application was found to improve maize yield between 3.8 to 27% in inoculated plants compared with non-inoculated plants [16, 61–63]. The results of our research show that inoculation with A. brasilense has potential as a strategy to improve NUE.

In this study, most of the Si taken up by the plant accumulated in the leaf tissue because Si deposits are known to occur more frequently in tissues where water is lost in large amounts due to plant transpiration [64, 65]. Silicon translocation rate varies among plant species, but once deposited in the cell wall it becomes immobile [66]. Some grass species, such as maize, can take up and redistribute large amounts of Si in the aboveground tissue due to specific Si transporters [67]. The amount of Si accumulated in the roots was greater than the amount of N accumulated in the roots. The N/Si ratio in the leaf tissue and shoot accumulation averaged 1.9 and 9.6, respectively, in both seasons, while the N/Si ratio in the root accumulation was 0.64. In the second season, Si appeared to be more soluble, or a residual effect was observed, and greater uptake was detected, which affected the N/Si ratio in leaves, shoots, and roots. The N/Si ratio decreased from 2.4 (2015/16) to 1.6 (2016/17) in leaves and 17.0 (2015/16) to 5.9 (2016/17) in shoot, but consequently, the N/Si ratio in roots increased from 0.53 (2015/16) to 0.75 (2016/17). The Si concentration in tissue (11.95 and 17.51 g Si kg-1) was near the suitable concentration range for K (17–35 g kg-1 D.M.) and above the suitable concentration for Ca (2.5–8.0 g kg-1 of D.M.), which are the second and third nutrients most absorbed by maize according to Cantarella et al. [48]. The fact that a significant amount of Si is absorbed by maize suggests that this nutrient could have a more significant role in crop production than has been realized by researchers and deserves further investigation.

In this study, the use of Si had little to no quantifiable effect on maize development and grain yield. The Si benefits are more frequent in hyperaccumulator crops [34, 35], which contain SiO2 concentrations above 5% of shoot dry matter [68, 69]. In addition, increased grain yield is unlikely when available Si in soil is above 10.0 mg kg-1 [40, 70]. In this study, the available soil Si contents was close to this range (9.4 mg kg-1 at 0–0.20 m and 10.2 at 0.20–0.40 m). In addition, more Si became available as straw decomposed during the growing season (13.1 kg ha-1 Si, and 38.3 of C/N ratio, Table 2). Some studies have reported Si can be beneficial under biotic and abiotic conditions [71, 72]. For example, Galindo et al. [41] studying inoculation methods associated with Si application reported an average wheat grain yield increase of 6.7% when seed inoculation and Si application were performed. Guével et al. [71] observed that foliar applications of three different Si products (Kasil 26.5% SiO2; Silamol 2% SiO2 and 46% K2O; and MRD-250 43.2% of SiO2) did not promote wheat plant growth compared to the control treatment under greenhouse conditions, except when the plants were infected by powdery mildew. The fact that adequate amounts of Si were present in the soil used for this study and the lack of a positive response to added Si suggest that little to no biotic or abiotic stress was present during the growing seasons studied.

Conclusions

Inoculation with A. brasilense was found to increase N shoot and root accumulation, number of grains per ear, harvest index, NUE, and grain yield and also LCI when limestone was used. Yield increased by as much as 15% when plants were inoculated and fertilizer application was omitted and 10% when N was applied, showing the potential for nitrogen fixation by the PGPB used in this study. The utilization of Ca and Mg silicate as a Si source had small effects on plant development and grain yield and mostly increased Si uptake by the plant. The amount of Si removed was comparable to the amount of K removed and greater than the amount of Ca removed by maize grain. This study used an irrigated field which could have created conditions that hindered our ability to fully investigate the potential benefits of Si application. Therefore, studies conducted under conditions that challenge the crop in terms of stress, biotic or/and abiotic, are necessary to better understand the role of Si, applied alone or in combination with growth-promoting bacteria.

Study area at the Selvíria, state of Mato Grosso do Sul, Brazil (20o22′S, 51o22′W, the altitude of 335 m above sea level).

Image obtained in Google Earth program. Google pan class="Chemical">company (2019).

(TIF)

Clin>an class="Chemical">ck here for additional data file.

(XLSX)

Clin>an class="Chemical">ck here for additional data file.

14 Opan class="Chemical">ct 2019

PONE-D-19-24172

Can n>an class="Chemical">silicon applied to correct soil acidity in combination with Azospirillum brasilense inoculation improve nitrogen use in maize?

PLOS ONE

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The manuscripn>t "n>an class="Chemical">Can silicon applied to correct soil acidity in combination with Azospirillum brasilense inoculation improve nitrogen use in maize? is interesting but need revision as suggested by the reviewers.

i

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Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: The manuscript entitled “Can silicon applied to correct soil acidity in combination with Azospirillum brasilense inoculation improve nitrogen use in maize” reported the interactive influence of silicon, Azospirillum brasilense inoculation and nitrogen application on maize growth, yield and NUE. Although, the theme of ms is interesting and falls in the scope of journal, yet I have serious concerns in various section of the draft.

The title of the artipan class="Chemical">cle needs to be revised. It may pan class="Chemical">cover your key findings.

Abstract is poorly strun>an class="Chemical">ctured.. Results are poorly drawn, conclude the section with your key findings.. I personally could not understand the treatment’s effects after reading the abstract section.

L36: optimized the N pan class="Chemical">fertilization, inn>an class="Chemical">creasing harvest index (HI), NUE and grain yield in 9.5, 19.3 and 5.5%, respectively????

Language needs substantial improvement. There are numerous grammatin>an class="Chemical">cal and typo mistakes throughout the manuscript.

Introduction: Should be fon>an class="Chemical">cused on the topic. Highlight research gap, and add clear cut objectives. I suggest adding recent studies on the interaction of Si and seed inoculation, Si and N, and N and seed inoculation.

Materipan class="Chemical">als and Methods:

Experimental den>an class="Chemical">sign: The experimental design was a completely randomized block design? To my knowledge, CRD is recommend only for controlled conditions.

L141: When N was applied?

Results: Too lengthy, can easily be brief. Parameters (e.g. yield and yield components) with similar trend can be combined. Be consistent regarding treatment description and the use of abbreviations. Better to add the numeric description of results (% variations) instead of just adding the data values for easy understanding of the readers. Various statements are confusing and unclear. It not enough to simply state the significant and non-significant effect of treatments..

Discussion should be merely based on the observed findings. Discussion on the interactive influence of silicon, Azospirillum brasilense inoculation and nitrogen application is poorly drawn. Conclusion: Report the key findings, consistent with the objectives.. It should not be the general summary. You may report some key genes.

Chen>an class="Chemical">ck whether the format of all references is according to the journal format.

Qupan class="Chemical">ality of Figures 2-6 needs to be impn>roved.

Reviewer #2: The experiment has a certain novelty and workload. The manusn>an class="Chemical">cript is also well written and presented well. The data are presented clearly for the most part. Keeping in view of these study the follow justification needed:

1. More information about the cultivars should be provided in the Materin>an class="Chemical">als and Methods part including the breeding organization and why exactly these two cultivars had been chosen.

2. Acn>an class="Chemical">curate soil data in experiment site such as organic matter content, total nitrogen content, total potassium content and total phosphorus content should be provided.

3. The manuscripn>t has a n>an class="Chemical">few grammatical errors which should be attended. A proofreading is suggested.

Reviewer #3: Please refer to the attached file for detailed point-wise comments and address/answer properly. Overall the manuscript is technically sound and conducted with viable research objectives and hypothesis. However, authors should need to consider related factors (i.e. N application in rotational crop) in expressing the study results.

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Reviewer #1: No

Reviewer #2: Yes: Xiangru Tang

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Submitted filename: pan class="Chemical">comments_24172 - n>an class="Chemical">Copy.docx

Clin>an class="Chemical">ck here for additional data file.

30 Nov 2019

Dear Apan class="Chemical">cademin>an class="Chemical">c Editor,

We would like to express our gratitude for the reviewers who took the time to provide such a thorough review of our manuscript. We believe that the changes suggested have made our manuscript much more direct and much easier to follow. We have addressed all of the concerns raised and provide a point by point answer on how we handled each comment provided. Our answers will be in italic and underlined right after each comment.

Because too many authors have revised this vern>an class="Chemical">sion it is possible that some language is still conflicting with some of our previous work. However, should you find this to be the case in any sentence of the revised document we will make every effort possible to fix it again.

Again, our most sincere gratitude to you and the reviewers who took time from their busy schedule to help us making this manuscript a better paper. We hope that we have answered every inquiry to your satisfaction and also hope that you will find this version of publishable quality. Should you find that further work is needed we will also gladly do it in a timely manner.

Very best,

Authors

PONE-D-19-24172

Can n>an class="Chemical">silicon applied to correct soil acidity in combination with Azospirillum brasilense inoculation improve nitrogen use in maize?

PLOS ONE

Dear Author,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Thank you for submitting your manuspan class="Chemical">cripn>t to n>an class="Chemical">PLOS ONE. After careful consideration, we feel that it has merit, but is not suitable for publication as it currently stands. Therefore, my decision is "Major Revision."

We invite you to submit a revised verpan class="Chemical">sion of the manusn>an class="Chemical">cript that addresses all the comments raised by the reviewers.

==============================

We would apprepan class="Chemical">ciate ren>an class="Chemical">ceiving your revised manuscript by Nov 28 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your finann>an class="Chemical">cial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

pan class="Chemical">Please inn>an class="Chemical">clude the following items when submitting your revised manuscript:

• A rebuttal letter that respn>onds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tran>an class="Chemical">cked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your respn>onse, if your artin>an class="Chemical">cle is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to repan class="Chemical">ceiving your revised manusn>an class="Chemical">cript.

Kind regards,

Dr. pan class="Chemical">Umair Ashraf

Apan class="Chemical">cademin>an class="Chemical">c Editor

PLOS ONE

Journpan class="Chemical">al Requirements:

1. When submitting your revipan class="Chemical">sion, we need you to address these additionn>an class="Chemical">al requirements.

pan class="Chemical">Please ensure that your manusn>an class="Chemical">cript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/n>an class="Chemical">PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

R: The authors apprepan class="Chemical">ciate the information. Thank you!

2. We noticed you have some minor on>an class="Chemical">ccurrence of overlapping text with the following previous publication(s), which needs to be addressed:

http://www.scielo.br/sn>an class="Chemical">cielo.php?lng=en&pid=S0006-87052018000300493&script=sci_arttext&tlng=en

https://mra.asm.org/pan class="Chemical">content/6/20/e00393-18.long

https://www.mdpi.pan class="Chemical">com/2223-7747/6/3/35/htm

In your revipan class="Chemical">sion ensure you n>an class="Chemical">cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed

R: We have made our best attempts to address those issues. However, since most of this is our previous published work, we might have rephrased things back to similar language due to several authors revising the manuscript. Should you find any further occurrence of overlapping text we will gladly fix those right away.

We would pan class="Chemical">also like to point out that after the revin>an class="Chemical">sion, the turnitin program was used to verify similarity and only two articles showed similarity above 1%:

1. Galindo FS, Rodrigues WL, Biagini n>an class="Chemical">ALC, Fernandes GC, Baratella EB, da Silva Junior CA, Buzetti S, Teixeira Filho MCM. Assessing forms of application of Azospirillum brasilense associated with silicon use on wheat. Agronomy. 2019;9:678.

2. Galindo FS, Teixeira Filho Mn>an class="Chemical">CM, Buzetti S, Pagliari PH, Santini JMK, Alves CJ, et al. Maize yield response to nitrogen rates and sources associated with Azospirillum brasilense. Agronomy Journal. 2019;111:1985-1997.

Both studies were developed and published by our team and were properly pan class="Chemical">cited and no n>an class="Chemical">data was reused, as can be verified by the lack of similarity mainly in the results and conclusions.

3. Our editorial staff has assessed your submission, and we have concerns about the grammar, usage, and overall readability of the manuscript. We therefore request that you revise the text to fix the grammatical errors and improve the overall readability of the text before we send it for review. We suggest you have a fluent, preferably native, English-language speaker thoroughly copyedit your manuscript for language usage, spelling, and grammar.

If you do not know anyone who can do this, you may wish to consider employing a professional scientific editing service.

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) and enter referral code PLOS15 for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmispan class="Chemical">sion, please provide the following:

• The name of the colleague or the details of the professional service that edited your manuscript

R: Professor Dr. Paulo Pagliari from the University of Minnesota Soil, Water, and Climate department has provided an in-depth review of this version of the manuscript. In addition, a communication specialist (Mrs Emily Evans (eneperma@umn.edu)) who works closely with Dr. Pagliari has also provided a thorough revision of this version of the manuscript focusing on language.

• A pan class="Chemical">copy of your manusn>an class="Chemical">cript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

• A pan class="Chemical">clean n>an class="Chemical">copy of the edited manuscript (uploaded as the new *manuscript* file)

Please note that PLOS ONE does not copyedit accepted manuscripts and that one of our criteria for publication is that articles must be presented in an intelligible fashion and written in clear, correct, and unambiguous English (http://www.plosone.org/static/publication#language). If the language is not sufficiently improved, we may have no choice but to reject the manuscript without review.

R: The authors apprepan class="Chemical">ciate the information. Thank you!

Additionpan class="Chemical">al Editor n>an class="Chemical">Comments (if provided):

The manuscripn>t "n>an class="Chemical">Can silicon applied to correct soil acidity in combination with Azospirillum brasilense inoculation improve nitrogen use in maize? is interesting but need revision as suggested by the reviewers.

R: The authors appreciate the editor n>an class="Chemical">comments and restructured the entire paper as suggested by the reviewers. Thank you!

pan class="Chemical">Comments to the Author

1. Is the manuscripn>t technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

________________________________________

2. Has the statistipan class="Chemical">cn>an class="Chemical">al analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

3. Have the authors made pan class="Chemical">all n>an class="Chemical">data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

4. Is the manuspan class="Chemical">cripn>t presented in an intelligible fashion and written in stann>an class="Chemical">dard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

5. Review pan class="Chemical">Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

REVIEWER 1

Reviewer #1: The manuscript entitled “Can silicon applied to correct soil acidity in combination with Azospirillum brasilense inoculation improve nitrogen use in maize” reported the interactive influence of silicon, Azospirillum brasilense inoculation and nitrogen application on maize growth, yield and NUE. Although, the theme of ms is interesting and falls in the scope of journal, yet I have serious concerns in various section of the draft.

The title of the artipan class="Chemical">cle needs to be revised. It may pan class="Chemical">cover your key findings.

R: The authors thank pan class="Chemical">all the great n>an class="Chemical">considerations of the reviewer. The title has been restructured and we followed your suggestion.

Abstract is poorly strun>an class="Chemical">ctured. Results are poorly drawn, conclude the section with your key findings. I personally could not understand the treatment’s effects after reading the abstract section.

R: The abstrapan class="Chemical">ct has been restrupan class="Chemical">ctured and we followed your suggestion. Thank you.

L36: optimized the N pan class="Chemical">fertilization, inn>an class="Chemical">creasing harvest index (HI), NUE and grain yield in 9.5, 19.3 and 5.5%, respectively????

R: This sentenpan class="Chemical">ce has been restrun>an class="Chemical">ctured and we followed your suggestion.

Language needs substantial improvement. There are numerous grammatin>an class="Chemical">cal and typo mistakes throughout the manuscript.

R: Two persons who are fluent in English (a US professor and a native speaker – communication specialist) have revised this version of the manuscript focusing on this aspect. We hope that this version is acceptable.

Introduction: Should be fon>an class="Chemical">cused on the topic. Highlight research gap, and add clear cut objectives. I suggest adding recent studies on the interaction of Si and seed inoculation, Si and N, and N and seed inoculation.

R: We have done our best to address this pan class="Chemical">comment. However, we n>an class="Chemical">feel that the introduction is already addressing what the reviewer would like to see in the introduction. Perhaps with the improved language our message will be easier to follow now.

Materipan class="Chemical">als and Methods:

Experimental den>an class="Chemical">sign: The experimental design was a completely randomized block design? To my knowledge, CRD is recommend only for controlled conditions.

R: This was a mistake by the student, the pan class="Chemical">corren>an class="Chemical">ct design was a RCBD, this has been better explained now. Thank you.

L141: When N was applied?

R: We have added the following information in the text: “The amount of fertilizer needed per plot was applied between the maize rows, on December 13, 2015, and December 10, 2016, when the plants were in the vegetative stage equivalent to V6 stage (with six leaves completely unfolded). After side dress application the experimental area was irrigated with 14 mm of water to minimize ammonia volatilization.”

Results: Too lengthy, can easily be brief. Parameters (e.g. yield and yield components) with similar trend can be combined. Be consistent regarding treatment description and the use of abbreviations. Better to add the numeric description of results (% variations) instead of just adding the data values for easy understanding of the readers. Various statements are confusing and unclear. It not enough to simply state the significant and non-significant effect of treatments.

Discussion should be merely based on the observed findings. Discussion on the interactive influence of silicon, Azospirillum brasilense inoculation and nitrogen application is poorly drawn. Conclusion: Report the key findings, consistent with the objectives. It should not be the general summary. You may report some key genes.

R: This was the section where most of the work was done to try and make the manusn>an class="Chemical">cript shorter and more direct to address all of the reviewers. We hope that this version has met the expectations of the reviewer.

Chen>an class="Chemical">ck whether the format of all references is according to the journal format.

R: Request made. Thank you.

Qupan class="Chemical">ality of Figures 2-6 needs to be impn>roved.

R: To attend the reviewer and editorial team request, we have uploaded the figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool to ensure that figures meet PLOS One standard and requirements. Also, some figures have been replaced by tables to improve readability. Thank you.

REVIEWER 2

Reviewer #2: The experiment has a certain novelty and workload. The manusn>an class="Chemical">cript is also well written and presented well. The data are presented clearly for the most part. Keeping in view of these study the follow justification needed:

1. More information about the cultivars should be provided in the Materin>an class="Chemical">als and Methods part including the breeding organization and why exactly these two cultivars had been chosen.

R: The authors thank pan class="Chemical">all the great n>an class="Chemical">considerations of the reviewer. We are a bit confused, but we think the reviewer refers to the bacteria strains used in the research. We have added more information regarding the strains used to answer this question.

2. Acn>an class="Chemical">curate soil data in experiment site such as organic matter content, total nitrogen content, total potassium content and total phosphorus content should be provided.

R: This information is presented in Table 1. Thank you.

3. The manuscripn>t has a n>an class="Chemical">few grammatical errors which should be attended. A proofreading is suggested.

R: Two persons who are fluent in English (a US professor and a native speaker – communication specialist) have revised this version of the manuscript focusing on this aspect. We hope that this version is acceptable.

REVIEWER 3

Reviewer #3: Please refer to the attached file for detailed point-wise comments and address/answer properly. Overall the manuscript is technically sound and conducted with viable research objectives and hypothesis. However, authors should need to consider related factors (i.e. N application in rotational crop) in expressing the study results.

PONE-D-19-24172

Can n>an class="Chemical">silicon applied to correct soil acidity in combination with Azospirillum brasilense inoculation improve nitrogen use in maize?

The titled manuscript was conducted to evaluate the interactive effects of Si and inoculation on maize yield and N uptake, the study provides good insights and the results will be useful in future on similar topic. However, there are few concerns which should be addressed (especially for data analysis) before the final consideration of manuscript, and the below comments should be responded properly and addressed carefully in revised draft.

The title can be modified by repn>lan>an class="Chemical">cing the term ‘nitrogen use’ with ‘nitrogen use efficiency’

R: The authors thank pan class="Chemical">all the great n>an class="Chemical">considerations of the reviewer. The title has been restructured and we followed your suggestion.

In abstract sen>an class="Chemical">ction, mostly the authors discussed about methods, and the results/conclusion part is too brief. Please describe the results more.

R: The abstrapan class="Chemical">ct has been restrupan class="Chemical">ctured and we followed your suggestion. Thank you!

If the authors have information for lime use practin>an class="Chemical">ce by farmers in maize crop in Brazil’s maize region, please quote

R: The best management prapan class="Chemical">ctin>an class="Chemical">ce for lime application in these regions of Brazil is now cited in the revised version. Thank you for the recommendation.

L150 – given the ratio of compound n>an class="Chemical">fertilizer (08-28-16, N-P2O5-K2O), the blanket dose of N must be 30 kg ha–1, could you please justify?

R: The reviewer is right. We have corren>an class="Chemical">cted the blanket dose of N to 30 kg ha-1 and a justification is now provided in the text.

In methods, please provide the general practice of fertilizer input rate in maize crop. And with reference to L126-128, authors are requested to add the details of fertilizer N input to the study site for at least previous crop before the maize plantation in 2015/16.

R: Information added. Thank you!

L170 – please mentioned the plant denpan class="Chemical">sity in ‘m–2’ ban>an class="Chemical">sis. In given dimensions, it should be 6.6 plants m–2.

R: Information added. However, the plant denpan class="Chemical">sity is 7.3 plants m–2 (spn>an>an class="Chemical">ce between rows is 0.45 m).

L188 – please provide the pan class="Chemical">sampling frequenpan class="Chemical">cy and time

R: The pan class="Chemical">sampling took plan>an class="Chemical">ce only at once. Date of sampling is now reported. Thank you for the recommendation.

L214 – please mention the statistical package name, which was employed to analyze the data? Also mention if the year effect was considered as random or fixed? Why the data was not analyzed across the years?

R: We have added the name of the statistical package used. The main reason we analyzed the data by year was because the crops grown before each of the maize crop studied differed in each season. In the first season maize was cropped in the winter of 2015 (June to September 2015) and in the second season wheat was grown in the winter of 2016. Therefore, we felt it would be more appropriate to analyze the data by year as opposed to combining both years.

L227 – please use proper pan class="Chemical">sign for interan>an class="Chemical">ction (×) using ‘insert > symbol’ option in word program. Same is suggested for rest of the draft, and in Table 3.

Please ren>an class="Chemical">ctify the errors on page 15 (L315) and page 19 (L432).

R: pan class="Chemical">Change made. Thank you.

Discussion: please discuss the possibilities of factors affecting the study results due to other rotational wheat crop in the year 2016 (L127-128), what was the N rate in wheat crop? Why not the fallow period was observed between two maize crops?? Please consider this factor in discussing overall results.

R: Wheat was fertilized an>an class="Chemical">ccording to recommended rates throughout the entire area, so that is would not affect our results. In Brazil it is a common practice to grow a crop in the winter, short season crops usually, between the main summer crops. Therefore, to keep the results applicable to real farming conditions we opted for following practices that the farmers in the region adopt.

How the y-axis scale could be same for the temperature and precipitation in Fig. 1, please recheck by adding separate scale for precipitation.

R: The figure has been restrupan class="Chemical">ctured and we followed your suggestion. Thank you!

From the figure data presented, it n>an class="Chemical">can be assessed that most of the times the differences between the two levels of a factor (N dose, lime sources, inoculation) are negligible or null. Please discuss this aspect in the results section or in the discussion. Secondly, mention the main effects of N dose affecting the grain yield or other salient parameters. The variance between each level shows no/slight differences. Please explain!!

R: We have tried to address this point throughout the text as we revised it. pan class="Chemical">Please advise if more pan class="Chemical">changes are needed

6. pan class="Chemical">PLOS authors have the option to publish the peer review history of their artin>an class="Chemical">cle (what does this mean?). If published, this will include your full peer review and any attached files.

If you pan class="Chemical">choose “no”, your identity will remain anonymous but your review may still be made publin>an class="Chemical">c.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Xiangru Tang

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

R: The authors apprepan class="Chemical">ciate the information and attend this request. Thank you!

Submitted filename: Gpan class="Chemical">alindo et n>an class="Chemical">al_Authors response to reviewers_pp_11_07_19.docx

Clin>an class="Chemical">ck here for additional data file.

16 Jan 2020

PONE-D-19-24172R1

Can n>an class="Chemical">silicon applied to correct soil acidity in combination with Azospirillum brasilense inoculation improve nitrogen use efficiency in maize?

PLOS ONE

Dear Dr. Filho,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Apan class="Chemical">CADEMIn>an class="Chemical">C EDITOR: Reviewers raised concerns about the quality of the figures. Authors should improve the quality of the figures and upload again.

==============================

We would apprepan class="Chemical">ciate ren>an class="Chemical">ceiving your revised manuscript by Mar 01 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your finann>an class="Chemical">cial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

pan class="Chemical">Please inn>an class="Chemical">clude the following items when submitting your revised manuscript:

A rebuttal letter that respn>onds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tran>an class="Chemical">cked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your respn>onse, if your artin>an class="Chemical">cle is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to repan class="Chemical">ceiving your revised manusn>an class="Chemical">cript.

Kind regards,

pan class="Chemical">Umair Ashraf

Apan class="Chemical">cademin>an class="Chemical">c Editor

PLOS ONE

Additionpan class="Chemical">al Editor n>an class="Chemical">Comments (if provided):

Authors have done with the comments/questions asked by the reviewers., however, reviewers and myself too n>an class="Chemical">also have concerns about the quality of figures. Authors should improve the resolution of the figures to make it clear.

[Note: HTML markup is below. pan class="Chemical">Please do not edit.]

Reviewers' pan class="Chemical">comments:

Reviewer's Responses to Questions

pan class="Chemical">Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you n>an class="Chemical">feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: pan class="Chemical">All pan class="Chemical">comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: pan class="Chemical">All pan class="Chemical">comments have been addressed

**********

2. Is the manuscripn>t technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistipan class="Chemical">cn>an class="Chemical">al analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made pan class="Chemical">all n>an class="Chemical">data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuspan class="Chemical">cripn>t presented in an intelligible fashion and written in stann>an class="Chemical">dard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review pan class="Chemical">Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Although, the authors have respn>onded well to n>an class="Chemical">all the comments raised by me, the quality of figures is too poor. Authors should check the Journal's guidelines regarding the minimum quality of figures

Reviewer #2: The expression of the manusn>an class="Chemical">cript has been substantially improved after modification.The author also supplemented relevant information in the paper for my questions.

Reviewer #3: Authors have addressed all the comments in revised version, and now the revised version looks good. However, authors didn't address the comment No. 9 during 1st review round. Please use proper symbol for interaction. Also the figures quality is even poor than the 1st submission, the minimum resolution of all figures should be 300dpi, please consider revising.

Thanks

**********

7. pan class="Chemical">PLOS authors have the option to publish the peer review history of their artin>an class="Chemical">cle (what does this mean?). If published, this will include your full peer review and any attached files.

If you pan class="Chemical">choose “no”, your identity will remain anonymous but your review may still be made publin>an class="Chemical">c.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: pan class="Chemical">Saddam Hussain

Reviewer #2: No

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

29 pan class="Chemical">Feb 2020

Dear Apan class="Chemical">cademin>an class="Chemical">c Editor,

We would like to express our gratitude for the reviewers who took the time to provide such a thorough review of our manuscript. We believe that the changes suggested have made our manuscript much more direct and much easier to follow. We have addressed all of the concerns raised and provide a point by point answer on how we handled each comment provided. Our answers will be in italic and underlined right after each comment.

Again, our most sincere gratitude to you and the reviewers who took time from their busy schedule to help us making this manuscript a better paper. We hope that we have answered every inquiry to your satisfaction and also hope that you will find this version of publishable quality. Should you find that further work is needed we will also gladly do it in a timely manner.

Very best,

Authors

PONE-D-19-24172R1

Can n>an class="Chemical">silicon applied to correct soil acidity in combination with Azospirillum brasilense inoculation improve nitrogen use efficiency in maize?

PLOS ONE

Dear Dr. Filho,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Apan class="Chemical">CADEMIn>an class="Chemical">C EDITOR: Reviewers raised concerns about the quality of the figures. Authors should improve the quality of the figures and upload again.

R: The authors apprepan class="Chemical">ciate the editor n>an class="Chemical">comments and restructured all the figures. Thank you!

==============================

We would apprepan class="Chemical">ciate ren>an class="Chemical">ceiving your revised manuscript by Mar 01 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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pan class="Chemical">Please inn>an class="Chemical">clude the following items when submitting your revised manuscript:

• A rebuttal letter that respn>onds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tran>an class="Chemical">cked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your respn>onse, if your artin>an class="Chemical">cle is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to repan class="Chemical">ceiving your revised manusn>an class="Chemical">cript.

Kind regards,

pan class="Chemical">Umair Ashraf

Apan class="Chemical">cademin>an class="Chemical">c Editor

PLOS ONE

R: The authors apprepan class="Chemical">ciate the information. Thank you!

Additionpan class="Chemical">al Editor n>an class="Chemical">Comments (if provided):

Authors have done with the comments/questions asked by the reviewers., however, reviewers and myself too n>an class="Chemical">also have concerns about the quality of figures. Authors should improve the resolution of the figures to make it clear.

[Note: HTML markup is below. pan class="Chemical">Please do not edit.]

R: The authors apprepan class="Chemical">ciate the editor and reviewers n>an class="Chemical">comments and restructured all the figures. Thank you!

Reviewers' pan class="Chemical">comments:

Reviewer's Responses to Questions

pan class="Chemical">Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you n>an class="Chemical">feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: pan class="Chemical">All pan class="Chemical">comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: pan class="Chemical">All pan class="Chemical">comments have been addressed

2. Is the manuscripn>t technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

3. Has the statistipan class="Chemical">cn>an class="Chemical">al analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

4. Have the authors made pan class="Chemical">all n>an class="Chemical">data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

5. Is the manuspan class="Chemical">cripn>t presented in an intelligible fashion and written in stann>an class="Chemical">dard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

6. Review pan class="Chemical">Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Although, the authors have respn>onded well to n>an class="Chemical">all the comments raised by me, the quality of figures is too poor. Authors should check the Journal's guidelines regarding the minimum quality of figures

R: We have made our best attempts to address those issues. We replot pan class="Chemical">all the figures as suggested by the reviewer. We hope that we have answered every inquiry to your n>an class="Chemical">satisfaction and also hope that you will find this version of publishable quality. Should you find that further work is needed we will also gladly do it in a timely manner.

Thank you!

Reviewer #2: The expression of the manusn>an class="Chemical">cript has been substantially improved after modification. The author also supplemented relevant information in the paper for my questions.

R: The authors apprepan class="Chemical">ciate the reviewer n>an class="Chemical">comments. Thank you!

Reviewer #3: Authors have addressed all the comments in revised version, and now the revised version looks good. However, authors didn't address the comment No. 9 during 1st review round. Please use proper symbol for interaction. Also the figures quality is even poor than the 1st submission, the minimum resolution of all figures should be 300dpi, please consider revising.

Thanks

R: We have made our best attempts to address those issues. We replot pan class="Chemical">all the figures as suggested by the reviewer. n>an class="Chemical">Also, we have changed all “x” by “×” in the interactions as suggested by the reviewer. We hope that we have answered every inquiry to your satisfaction and also hope that you will find this version of publishable quality. Should you find that further work is needed we will also gladly do it in a timely manner.

Thank you!

7. pan class="Chemical">PLOS authors have the option to publish the peer review history of their artin>an class="Chemical">cle (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: pan class="Chemical">Saddam Hussain

Reviewer #2: No

Reviewer #3: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

R: The authors apprepan class="Chemical">ciate the information and attend this request. Thank you!

Submitted filename: Gpan class="Chemical">alindo et n>an class="Chemical">al_Authors response to reviewers_january.docx

Clin>an class="Chemical">ck here for additional data file.

13 Mar 2020

Can n>an class="Chemical">silicon applied to correct soil acidity in combination with Azospirillum brasilense inoculation improve nitrogen use efficiency in maize?

PONE-D-19-24172R2

Dear Dr. Filho,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Dr pan class="Chemical">Umair Ashraf

Apan class="Chemical">cademin>an class="Chemical">c Editor

PLOS ONE

Additional Editor n>an class="Chemical">Comments (optional):

Reviewers' pan class="Chemical">comments:

23 Mar 2020

PONE-D-19-24172R2

Can n>an class="Chemical">silicon applied to correct soil acidity in combination with Azospirillum brasilense inoculation improve nitrogen use efficiency in maize?

Dear Dr. Filho:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or pan class="Chemical">conn>an class="Chemical">cerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

pan class="Chemical">PLOS ONE Editorin>an class="Chemical">al Office Staff

on behpan class="Chemical">alf of

Dr. pan class="Chemical">Umair Ashraf

Apan class="Chemical">cademin>an class="Chemical">c Editor

PLOS ONE