Potential of Biostimulants Based on PGPR Rhizobacteria Native to Benin’s Soils on the Growth and Yield of Maize (<i>Zea mays </i> L.) under Greenhouse Conditions

The application of biostimulants in agriculture represents an environmentally friendly alternative while increasing agricultural production. The aims of the study were to develop solid biostimulants based on five rhizobacteria native to Benin’s soils and to evaluate their efficacy on the growth and biomass yield of maize under greenhouse conditions on ferrallitic and ferruginous soils. Clay and peat were used as a conservation binder for the preparation of the biostimulants. These binders were used alone or combined in the different formulations with maize flour and sucrose. 10 g of biostimulants were applied at sowing in pots containing five kilograms of sterilised soil. The experimental design was a completely randomised block of 24 treatments with three replicates. The results obtained showed significant improvements ( P < 0.001) in height (49.49%), stem diameter (32.7%), leaf area (66.10%), above-ground biomass (97.12%) and below-ground biomass (53.98%) on ferrallitic soil with the application of the clay + Pseudomonas putida biostimulant compared to the control. On the other hand, the use of the peat biostimulant + Pseudomonas syringae was more beneficial for plant growth on ferruginous soil. The height, stem diameter, leaf area, above-ground biomass and below-ground biomass of the plants under the influence of this biostimulant were improved by 83.06%, 44.57%, 102.94%, 86.84% and 42.68%, respectively, compared to the control. Therefore, these results confirm that Rhizobacteria express their potential through biostimulants formulated on maize. The formulated biostimulants can later be used by producers to improve crop productivity for sustainable agriculture.

• Biostimulants
• Plant Growth Promoting Rhizobacteria
• Soil Fertility
• Binder
• Corn
• Benin

1. Introduction

Maize is a staple food crop for many people in West African countries. In Benin, it is the leading cereal crop, accounting for 52.6% of total land area and 78.3% of all cereal production [1]. It is also used for animal feed and industrial uses in the form of starch, flour, ethanol and cooking syrup [2]. More than 90 maize-based foods and drinks have been identified and documented by Adjadi et al., 2015 [3]. Despite the importance of this speculation for food security, its productivity faces many constraints, including the constant decline in the fertility of cultivated land due to its degradation [4]. This situation forces producers to make heavy use of chemical inputs. The high use of these products causes them to accumulate in soils and food products, thus leading to an imbalance in the nutrient cycle [5]. This is an ecological concern whose consequences are felt by the population. It is imperative to find a realistic, cheap, better and safer way to maximise agricultural productivity. The application of biostimulants in crop production is an important ecological alternative to achieve all these criteria [6]. A plant biostimulant is a substance or micro-organism applied to plants to improve the nutritional efficiency, tolerance to abiotic stress and/or quality characteristics of crops, regardless of their nutrient content. The use of microorganisms as biostimulants is not a widespread practice. This is due to the particular conditions of use of each microbial group [7]. The application of biostimulants makes it possible to reduce the use of chemicals that have generated a serious environmental impact [8] [9].

In Benin, several studies have been carried out using inocula of PGPR rhizobacteria native to Benin soils to improve maize growth and yield [10] [11]. In the light of these studies, several strains have shown their effectiveness and in particular P. putida incontrolled environments [12] [13] [14] and in farming environments [10]. During this work, the inoculation of bacterial suspension was a real problem raised by these different authors. Moreover, the conservation of the liquid biostimulant in natural conditions was also a difficulty for the authors as well as the growers who hosted the trials. Further research is needed to formulate effective biostimulants that can help us overcome these constraints. The aims of the study were to develop solid biostimulants based on five rhizobacteria native to Benin’s soils and to evaluate their efficacy on the growth and biomass yield of maize under greenhouse conditions on ferrallitic and ferruginous soils.

2. Materials and Methods

2.1. Biological Material

The rhizobacteria that were used are Pseudomonas putida (South), isolated and characterized from the rhizosphere of maize from the different development poles of Southern Benin by [15]; Pseudomonas putida (North), Pseudomonas syringae, Bacillus thuringiensis and Serratiamarcescens isolated and characterized from the corn rhizosphere of different development poles of Central and Northern Benin by [16]. These rhizobacteria were kept at the Laboratory of Biology and Molecular Typing in Microbiology in Muller Hinton broth with added glycerol (10%) at −85˚C. The 2000 SYNEE maize variety supplied by National Agricultural Research Institute of Benin (INRAB) was used. It is an extra-early variety of 75 days, with a potential grain yield varying between 2 and 2.5 t/ha in rural areas [17]. Substrates such as peat and clay were used as binders in the preparation of the formulation.

2.2. Validation of the Best Binder

Starch, peat and clay were used to formulate the various biostimulants. These binders were sterilised using an autoclave at a temperature of 121˚C for 30 minutes. After the formulation, the evolution of the microorganisms was followed in boxes incubated at 25˚C for two months in an aseptic medium after which the strains were observed. The best binder was the one that maintained the bacteria during this time.

2.3. Development of Biostimulants

The adapted method of Connick et al., 1991 [18] has been used for the preparation of microbial biostimulants. For this purpose, 32 g maize flour, 30 ml bacterial suspension (10⁸ CFU/ml), 6 g binder and 2 g sucrose were filled into plastic boxes and mixed well with gloved hands under aseptic conditions until a soft dough was obtained. The control formulation was prepared with 32 g maize flour, 30 ml sterile distilled water, 6 g binder and 2 g sucrose. After mixing, the different formulations were spread on aluminium foil for two days at a temperature of 25˚C in an aseptic environment. After three days of drying in ambient air in an aseptic environment, the paste was crushed in a mortar and sieved.

2.4. Chemical Analysis of Soils

The soils used were sampled at 0 - 20 cm horizon using a marked shovel spade at the experimental sites. The ferrallitic soil was collected at the Agricultural Research Centre-South Niaouli and the ferruginous soil was collected in central Benin at the Reasearch & Development site of Miniffi in Dassa-Zoumè district. The chemical analyses were carried out at the Laboratory of Soil, Water and Environmental Sciences of INRAB in order to determine their characteristics. The chemical analyses consisted of the determination of carbon and organic matter by the [19]; water pH and KCl pH using a pH meter with (1/2.5) as soil-water ratio; assimilable phosphorus by the [20]; exchangeable cations (Ca, Mg, K and Na) by the ammonium acetate method using atomic absorption spectrophotometry [21].

2.5. Experimental Device

The experimental device was a randomized block of 24 treatments with three replicates per soil type. After sieving these soils, they were sterilized in an oven at a temperature of 120˚C for 30 minutes and then introduced into plastic jars of 5 dm³ volume. The different treatments were defined as follows: T₀: control (without PGPR), T₁: P. putida S, T₂: P. putida N, T₃: P. syringae, T₄: B. thuringiensis, T₅: S. marcescens, T₆: clay-peat (without PGPR), T₇: clay-peat + P. putida S, T₈: clay-peat + P. putida N, T₉: clay-peat + P. syringae, T₁₀: clay-peat + B. thuringiensis, T₁₁: clay-peat + S. marcescens, T₁₂: peat (without PGPR), T₁₃: peat + P. putida S, T₁₄: peat + P. putida N, T₁₅: peat + P. syringae, T₁₆: peat + B. thuringiensis, T₁₇: peat + S. marcescens, T₁₈: clay (without PGPR), T₁₉: clay + P. putida S, T₂₀: clay + P. putida N, T₂₁: clay + P. syringae, T₂₂: clay + B. thuringiensis, T₂₃: clay + S. marcescens.

2.6. Sowing and Application of Biostimulant

The different strains were applied individually to each type of soil in the form of a bacterial suspension at a rate of 10 ml (10⁸ CFU/ml). In the control pots, 10 ml of sterile distilled water was used instead of the bacterial suspension. Following the opening of the approximately 5 cm deep holes, two maize seeds were inserted and the holes were immediately closed again; then 10 g of formulated microbial biostimulant was applied according to the treatments and mixed with the upper part of the soil in the pots. The control pots received 10 g of control formulations.

2.7. Data Collection

The height and stem diameter of the plants were measured every 72 hours, on the 10th, 13th, 16th, 19th, 22nd, 25th, 28th and 31st days after sowing (DAS). The height (distance between the crown and the last ligule) of the maize plant was measured with a tape measure; the stem diameter was measured with a sliding foot. The leaf area was measured only on day 31 after sowing. It was estimated by the product of leaf length and width affected by the 0.75 coefficient [22]. The aerial and underground biomass were collected by treatment and by repetition and stored in an envelope designed for this purpose. The envelopes were placed in a 65˚C oven for 72 hours until constant dry weight was obtained [23]. The weights were taken using a precision balance.

2.8. Statistical Analysis

The analyses were performed with R 3.6.0 software [24] using nlme packages [25], lsmeans, car, lattice, ggplot2, FctoMine R and factoextra [26]. Normality and homoscedasticity of the data were verified using Ryan-Joiner and Levene tests respectively [27] prior to performing ANOVA. Post-hoc or multiple comparison (SNK) tests were performed to assess statistical differences. The hierarchical classification of main components (HCPC) [28] was performed in order to identify the model of discrimination of treatments taking into account their performance. A Major Component Analysis (PCA) [29] [30] and a hierarchical classification were performed on these variables to classify treatments into homogeneous groups. The combination of these two analyses is useful to show the pattern across the data [31].

3. Results

3.1. Validation of the Best Binder

Two months after formulation, the biostimulants formulated with Serratiamarcescens were presented in the different aspects of Figure 1. These strains had grown well in clay formulations, moderately with peat and very weakly with cooked starch. This growth was observed by the red coloration on these biostimulants. Clay and peat were the best binders.

3.2. Chemical Characteristics of Soils

The chemical properties of the two soil types before the tests were installed (Table 1) generally showed that the soil in Niaouli (ferrallitic) was slightly acidic and that in Miniffi (ferruginous) was alkaline. These soils showed a low level of fertility characterised by high C/N ratios. The Miniffi soil was richer in exchangeable K+ than the Niaouli soil. However, this soil had a low level of exchangeable organic carbon, assimilable phosphorus, Ca²⁺ and Mg²⁺.

3.3. Evolution of the Height of Maize Plants on Ferrallitic Soil

The evolution over time of the height of maize plants on ferrallitic soil is illustrated by the curves in Figure 2. The best height was recorded with the solid 31 DAS.

Table 1

Chemical properties of soils prior to test installation

Sites	Villages	Depths (cm)	pH (eau)	C-org (g/kg)	N-total (g/kg)	C/N	P-Bray1 (mg/kg)	B.E (cmol/kg)
								Ca2+	Mg2+	K+
Dassa	Miniffi	0 - 20	7.8	8.0	0.6	13.3	47.5	33.3	2.3	2.2
Allada	Niaouli	0 - 20	5.9	10.6	0.7	14.5	35.5	6.1	4.2	0.7

C-org: Organic Carbon; N-total: total nitrogen; P-Bray1: Assimilable phosphorus; B.E: Exchangeable Bases.

biostimulant T₁₅: peat + P. syringae. It induced an increase of 83.06% compared to the control formulation (T₁₂: peat without PGPR). A highly significant difference (p < 0.001) existed between treatments at 31 DAS.

3.4. Evolution of the Height of Maize Plants on Ferruginous Soil

Figure 3 illustrates the evolution over time of the height of maize plants on ferruginous soil. The advantage of applying biofertilizer on this soil was most noticeable with T₁₁: clay-mud + S. marcescens. This treatment showed an improvement of 89.72% compared to the control (T₆: clay-mud without PGPR). There was a highly significant difference between the treatments (p < 0.001) at

3.5. Evolution of the Stem Diameter of Maize Plants on Ferrallitic Soil

The curves in Figure 4 illustrated the evolution over time of stem diameter of maize plants on ferrallitic soil. The best diameter was recorded with the biostimulant T₁₅: peat + P. syringae. This biostimulant had an increase of 44.57% compared to the control (T₁₂: peat without PGPR). A highly significant difference (p < 0.001) existed between treatments at 31 DAS.

3.6. Evolution of the Stem Diameter of Maize Plants on Ferruginous Soil

The curves in Figure 5 illustrate the evolution over time of stem diameter of maize plants on ferruginous soil. The best stem diameter was recorded with the solid biostimulant T₁₀: peat-clay + B. thuringiensis with an overrun of 66.27% of the control. There was a highly significant difference (p < 0.001) between the different treatments at 31 DAS.

3.7. Leaf Area, Aerial Biomass and Underground Biomass on Ferrallitic Soil

Table 2 provides data on leaf area, aerial biomass and underground biomass on ferrallitic soils. The best leaf area (154.68 cm²) was recorded with the application of the biostimulant T₁₉: clay + P. putida S which had an increase of 66.10% compared to the control formulation (T₁₈: clay without PGPR). The highest aerial biomass was recorded with the application of biostimulant T₁₉: clay + P. putida S which exceeded the control formulation (T₁₈: clay without PGPR) by 63.49%. The highest underground biomass was obtained with the biostimulant T₁₉: clay + P. putida S with an increase of 53.98% compared to the control (T₁₈: clay without PGPR). A highly significant difference (p < 0.001) existed between the different treatments at 31 DAS for all parameters.

3.8. Leaf Area, Aerial Biomass and Underground Biomass on Ferruginous Soil

Table 3 provides data on leaf area, aerial biomass and underground biomass on ferruginous soils. The biostimulant formulated with the combination of peat-clay + S. marcescens (T₁₁) gave the best leaf area (188.28 cm²) with an excess of 112.36% compared to the control formulation (T₆: peat-clay without PGPR). The highest value of aerial dry biomass was recorded with the application of the biostimulant T₁₉: clay + P. putida S which exceeded the control formulation (T₁₈: clay without PGPR) by 97.12%. On ferruginous soil, it is with the biostimulant T₁₅: peat + P. syringae that we have a better underground biomass of maize plants. This formulation exceeded the control (T₁₂: peat without PGPR) by 42.68%. However, a highly significant difference existed between the different treatments at 31 DAS (p < 0.001) for all parameters.

3.9. Correlation between Growth and Yield of Maize Plants on Ferrallitic Soils

Principal Component Analysis (PCA) on the different maize plant growth and yield parameters showed that the first two axes retain 85.99% of the total variance. The projection of individuals indicated three groups of treatments (Figure 6). The first group (G₁) consisted of the four controls T₀, T₆, T₁₂ and T₁₈. The plants maintained under these treatments were characterised by an average height of 15.3 cm ± 1.06; an average stem diameter of 0.83 cm ± 0.02; an average leaf area of 86.25 cm² ± 8.32; an average aerial biomass of 27.07 g ± 1.35 and an average underground biomass of 16.45 g ± 0.4. The second group (G₂) consisted of 10 treatments T₂, T₃, T₄, T₅, T₁₀, T₁₄, T₁₆, T₁₇, T₂₁ and T₂₂. The plants that benefited from the treatments of this group G2, had an average height of 19.58 cm ± 1.3; an average stem diameter of 0.98 cm ± 0.05; an average leaf area of 106.64 cm² ± 11.99; an average aerial biomass of 29.05 g ± 2.75 and an average underground biomass of 19.86 g ± 2.36. The third group (G₃) consisted of 10 treatments T₁, T₇, T₈, T₉, T₁₁, T₁₃, T₁₅, T₁₉, T₂₀ and T₂₃. The plants subjected to these

Table 2

Leaf area, aerial biomass and underground biomasson ferrallitic soils

Treatment	Leaf surface (cm2)	Treatment	Aerial Biomass (g)	Treatment	Underground biomass (g)
	Mean ± sd		Mean ± sd		Mean ± sd
T19	154.68 ± 3.97a	T19	45.32 ± 0.76a	T19	25.50 ± 1.99a
T8	152.47 ± 6.23a	T7	36.40 ± 0.52b	T17	23.65 ± 1.05b
T7	147.73 ± 4.46ab	T13	35.32 ± 0.33bc	T16	16.64 ± 0.70gh
T15	137.56 ± 1.15bc	T11	34.72 ± 0.35bcd	T20	21.37 ± 0.96c
T20	137.17 ± 23.50bc	T8	34.17 ± 0.45bcd	T7	21.28 ± 0.43c
T13	128.25 ± 4.34cd	T14	33.35 ± 0.85cde	T3	21.08 ± 0.04c
T1	127.17 ± 5.88cd	T17	32.80 ± 0.33def	T13	20.87 ± 2.52c
T23	125.05 ± 6.08cd	T16	32.34 ± 0.66def	T22	20.63 ± 0.43cd
T11	124.94 ± 5.58cd	T9	32.34 ± 0.66def	T23	19.81 ± 0.64cde
T3	124.43 ± 2.03cd	T23	31.57 ± 1.81ef	T21	19.80 ± 1.32cde
T9	121.33 ± 0.39cd	T20	31.04 ± 0.56efg	T1	19.58 ± 1.01cdef
T5	118.10 ± 10.00de	T1	30.50 ± 1.12fgh	T8	19.23 ± 0.53cdefg
T21	115.90 ± 0.13de	T21	29.04 ± 1.32ghi	T11	18.94 ± 0.28cdefg
T22	113.98 ± 1.80de	T6	28.60 ± 2.01hi	T2	18.68 ± 0.39cdefgh
T2	112.37 ± 7.74def	T22	28.38 ± 1.74hi	T5	18.62 ± 0.10cdefgh
T17	104.92 ± 0.28efg	T2	27.94 ± 1.01ij	T14	18.04 ± 2.01defgh
T14	99.40 ± 0.88fgh	T18	27.72 ± 1.32ijk	T4	17.94 ± 1.29defgh
T18	95.53 ± 3.22gh	T5	27.06 ± 0.66ijk	T15	17.64 ± 0.72efgh
T10	94.98 ± 0.51gh	T10	26.66 ± 0.27ijk	T12	16.95 ± 0.17fgh
T16	92.50 ± 5.07gh	T4	26.52 ± 1.33ijk	T10	16.64 ± 0.70gh
T4	89.81 ± 7.09gh	T12	26.40 ± 1.32ijk	T9	16.60 ± 0.68gh
T12	87.45 ± 0.60hi	T3	26.39 ± 0.17ijk	T18	16.56 ± 0.53gh
T6	86.72 ± 5.04hi	T0	25.57 ± 1.57jk	T6	16.16 ± 0.32h
T0	75.29 ± 5.64i	T15	25.03 ± 0.05k	T0	16.10 ± 0.35h
DF	23	DF	23	DF	23
F value	32.78	F value	58.28	F value	18.71
Pr(>F)	<0.001***	Pr(>F)	<0.001***	Pr(>F)	<0.001***

T₀: control, T₁: P. putida S, T₂: P. putida N, T₃: P. syringae, T₄: B. thuringiensis, T₅: S. marcescens, T₆: clay-peat (without PGPR), T₇: clay-peat + P. putida S, T₈: clay-peat + P. putida N, T₉: clay-peat + P. syringae, T₁₀: clay-peat + B. thuringiensis, T₁₁: peat-clay + S. marcescens, T₁₂: peat (without PGPR), T₁₃: peat + P. putida S, T₁₄: peat + P. putida N, T₁₅: peat + P. syringae, T₁₆: peat + B. thuringiensis, T₁₇: peat + S. marcescens, T₁₈: clay (without PGPR), T₁₉: clay + P. putida S, T₂₀: clay + P. putida N, T₂₁: clay + P. syringae, T₂₂: clay + B. thuringiensis, T₂₃: clay + S. marcescens.

Table 3

Leaf area, aerial biomass and underground biomasson ferruginous soils

Treatment	Leaf surface (cm2)	Treatment	Aerial Biomass (g)	Treatment	Underground biomass (g)
	Mean ± sd		Mean ± sd		Mean ± sd
T11	188.28 ± 3.97a	T19	52.79 ± 2.05a	T15	21.26 ± 0.47a
T10	181.28 ± 1.91ab	T13	50.28 ± 6.27ab	T11	19.48 ± 0.81b
T15	173.25 ± 2.78bc	T15	49.01 ± 0.87ab	T20	19.16 ± 1.14bc
T19	170.35 ± 5.36bcd	T11	46.50 ± 0.66b	T10	19.00 ± 0.91bc
T7	165.22 ± 5.11cd	T20	39.23 ± 1.14c	T19	18.84 ± 1.37bcd
T23	163.31 ± 10.07cd	T14	39.18 ± 3.01c	T4	18.51 ± 0.99bcd
T8	162.55 ± 9.47cd	T10	39.01 ± 2.63c	T13	18.39 ± 0.77bcd
T13	155.82 ± 3.11de	T4	38.94 ± 1.72c	T8	17.88 ± 1.02bcde
T20	154.84 ± 7.57de	T17	37.21 ± 1.05cd	T7	17.80 ± 0.91bcdef
T4	154.28 ± 1.88de	T23	36.68 ± 3.45cd	T1	17.42 ± 0.57bcdefg
T16	153.60 ± 5.53de	T7	35.17 ± 2.84cd	T14	17.40 ± 0.60bcdefg
T1	145.64 ± 10.52ef	T8	32.48 ± 1.32de	T17	16.80 ± 1.20bcdefgh
T2	136.50 ± 9.87fg	T1	30.57 ± 0.63ef	T23	16.80 ± 1.20bcdefgh
T14	130.30 ± 8.47g	T22	29.80 ± 0.91ef	T22	16.44 ± 1.76bcdefghi
T17	124.48 ± 11.20gh	T16	27.99 ± 2.84efg	T2	16.37 ± 1.26cdefghi
T5	123.22 ± 1.24gh	T5	27.28 ± 2.77efg	T3	15.89 ± 2.61defghi
T9	114.01 ± 0.11h	T3	27.06 ± 2.89efg	T16	15.00 ± 0.60efghij
T21	100.96 ± 6.73i	T18	26.78 ± 1.41efg	T12	14.90 ± 0.14fghij
T18	96.04 ± 0.21ij	T2	26.60 ± 0.91efg	T5	14.78 ± 0.70ghij
T22	95.63 ± 2.07ij	T12	26.23 ± 1.47fg	T9	14.60 ± 1.24ghij
T3	92.59 ± 5.43ij	T21	26.00 ± 1.83fg	T0	14.00 ± 0.69hij
T6	88.66 ± 6.79ij	T6	25.20 ± 1.58fg	T21	13.97 ± 0.37hij
T12	85.37 ± 10.41ij	T9	25.20 ± 1.20fg	T6	13.60 ± 0.69ij
T0	82.55 ± 1.06j	T0	22.60 ± 0.92g	T18	13.36 ± 0.96j
DF	23	DF	23	DF	23
F value	79.88	F value	44.5	F value	11.72
Pr(>F)	<0.001***	Pr(>F)	<0.001***	Pr(>F)	<0.001***

T₀: control, T₁: P. putida S, T₂: P. putida N, T₃: P. syringae, T₄: B. thuringiensis, T₅: S. marcescens, T₆: clay-peat (without PGPR), T₇: clay-peat + P. putida S, T₈: clay-peat + P. putida N, T₉: clay-peat + P. syringae, T₁₀: clay-peat + B. thuringiensis, T₁₁: peat-clay + S. marcescens, T₁₂: peat (without PGPR), T₁₃: peat + P. putida S, T₁₄: peat + P. putida N, T₁₅: peat + P. syringae, T₁₆: peat + B. thuringiensis, T₁₇: peat + S. marcescens, T₁₈: clay (without PGPR), T₁₉: clay + P. putida S, T₂₀: clay + P. putida N, T₂₁: clay + P. syringae, T₂₂: clay + B. thuringiensis, T₂₃: clay + S. marcescens.

treatments had an average height of 22.82 cm ± 1.9; an average stem diameter of 1.05 cm ± 0.08; an average leaf area of 135.64 cm² ± 12.28; an average aerial biomass of 33.64 g ± 5.22; and an average underground biomass of 20.09 g ± 2.44. The G₃ group gave the best performance in both growth and yield parameters while the G₁ group gave the lowest performance. On ferrallitic soil the biostimulant T₁₉: clay + P. putida S is the best of all the others in group G₃.

3.10. Correlation between Growth and Yield of Maize Plants on Ferruginous Soils

Principal Component Analysis (PCA) on the different maize plant growth and yield parameters showed that the first two axes retained 89.86% of the total variance. The projection of individuals indicated three groups of treatments (Figure 7). The first group (G₁) consists of 11 treatments T₀, T₂, T₃, T₅, T₆, T₉,

T₁₂, T₁₆, T₁₈, T₂₁ and T₂₂. The plants maintained under these treatments are characterised by an average height of 22.69 cm ± 1.63; an average stem diameter of 1.05 cm ± 0.16; an average leaf area of 106.29 cm² ± 22.90; an average aerial biomass of 26.43 g ± 1.82 and an average underground biomass of 14.81 g ± 1.06. The second group (G₂) consists of nine treatments T₁, T₄, T₇, T₈, T₁₀, T₁₄, T₁₇, T₂₀ and T₂₃. The plants that have benefited from the treatments in this group G₂ have an average height of 27.38 cm ± 1.27; an average stem diameter of 1.23 cm ± 0.09; an average leaf area of 153.58 cm² ± 17.86; an average aerial biomass of 36.50 g ± 3.17 and an average underground biomass of 17.86 g ± 0.87. The third group (G₃) consists of four treatments T₁₁, T₁₃, T₁₅ and T₁₉. The plants subjected to these treatments have an average height of 32.67 cm± 1.74; an average stem diameter of 1.34 cm ± 0.12; an average leaf area of 171.93 cm² ± 13.31; an average aerial biomass of 49.64 g ± 2.62 and an average underground biomass of 19.50 g ± 1.26. Group G₃ gives the best performance in both growth and yield parameters while group G₁ gives the lowest performance. On ferruginous soil, the biostimulant T₁₅: peat + P. syringaeis the best.

4. Discussion

Chemical fertilizers are generally used to provide essential nutrients to plants. However, the high cost, availability and environmental concerns related to chemical fertilizers are real problems for agriculture [6]. The use of microbial biostimulants, including PGPR, to improve sustainable agricultural production is a practice that is becoming more widely accepted in intensive agriculture. PGPR are free-living soil bacteria that colonise plant roots and improve growth and yield when applied to seeds or crops [32]. The aims of the study were to develop solid biostimulants based on five rhizobacteria native to Benin’s soils and to evaluate their efficacy on the growth and biomass yield of maize under greenhouse conditions on ferrallitic and ferruginous soils.

The first activity was the validation of the best binders in order to use them for the formulation. The bacterial load was constant in the clay and peat biostimulants after two months of incubation. Similar results were reported by [33] in Tunisia with biostimulants composed of Kaolin or talc-kaolin binder and PGPR P. trivialis X33d at room temperature. Clay and peat biostimulants maintained a high bacterial population for five to eight months at 25˚C [34] [35] [36], explains the survival of the bacteria by the availability of labile carbon and nitrogen and also the pH of the binders (clay, peat).

Analysis of the results of the initial chemical properties of the test soils shows that ferrallitic soil is slightly acidic while ferruginous soil is alkaline. The ferrallitic soil also showed a high level of assimilable phosphorus compared to the ferruginous soil. In general, both types of soil presented a high C/N ratio; the sum of exchangeable bases and the cation exchange capacity are low, which reflects their low fertility, as reported by [15]. Moreover, potassium is globally deficient in relation to calcium and magnesium in both soils. Imbalances existed between calcium, magnesium and phosphorus. These results are in line with those reported by [37].

Many researchers have indicated that the use of biostimulants plays a key role in improving soil fertility, crop growth and final yield. Their application improves soil biological activity and reduces the use of chemical fertilizers [38]. Recently, [39] reported that the use of microbial biostimulants can provide an alternative for improved nutrient bioavailability in the soil and therefore good crop productivity. Statistical analysis was highly significant (p < 0.001) for growth parameters (height, stem diameter and leaf area). Solid biostimulants (peat + P. syringae (T₁₅) and clay + P. putida S (T₁₉)) proved to be more effective than simple inoculation. Similar performance was reported in the work of [40] in Parkistan. These authors found that the application of peat + B. Thuringiensis had significantly improved the growth and yield of beans. [41] Observed a highly significant increase in growth parameters and overproduction of chlorophyll a and b by maize plants treated with the biostimulant. The larger leaf area, more the plant achieves good photosynthesis, which is favourable to better growth [42] [43]. Our results on growth parameters can be explained by the stimulation of good photosynthesis by the formulated solid biostimulants. In general, formulated solid biostimulants improved maize plant growth in height, stem diameter and leaf area more than bacterial suspensions. This difference is due to the effects of binders and other biostimulant additives [39]. According to different authors [44] [45] [46] the increase in plant growth is due to the production capacity of auxin, gibberellic acid, biological nitrogen fixation and improvement of the root system by rhizobacteria. The rhizobacteria P. putida S and P. syringaecontained in these biostimulants are able to solubilize phosphate, produce indole acetic acid [47] [48] and promote better nutrient uptake by plants from the soil [11]; which justifies the results obtained with inoculated plants.

Several authors have demonstrated the efficacy of microbial biostimulants on plant yield parameters [49] [50] [51]. It was recently found by [41] that A. brasilense biostimulant HM053 significantly increased maize yield by 53% compared to the control. In our study, biostimulants improved yield parameters in a highly significant way (p < 0.001). The best biomasses were obtained with the application of biostimulants T₁₅: peat + P. syringae, T₁₉: clay + P. putida S on both soil types. These results are in line with those of [52] and [53], who had increases of 33.85% and 80% in the biomass of Poaceae by applying biostimulants. Thanks to a better implantation, good water infiltration, a well-fed plant ensures a good yield. The application of biostimulants is one of the appropriate methods to improve phosphorus availability [39] [54] [55]. On these soils, application of the formulated solid biostimulants T₁₅: peat + P. syringae and T₁₉: clay + P. putida S improves maize growth and yield. These increases can be due to better root growth, better nutrient absorption, high capacity of the clay and peat to retain and provide a large surface area for the PGPRs to survive and function [40] [56].

5. Conclusion

Microbial biostimulants are very important for crop improvement. They improve plant growth and yield. The parameters of vegetative growth and biomass yield of maize have been greatly improved under greenhouse conditions on ferrallitic and ferruginous soils in Benin by the biostimulants clay + P. putida and peat + P. syringae. These results show that these biostimulants can be used as a biological stimulant for environmentally friendly sustainable agriculture, promoting the use of organic methods to increase the productivity of the various speculations in Benin.

Acknowledgements

The authors would like to thank the national agricultural research institute of Benin (INRAB) for agreeing to finance this project. Special thanks go to the Director General and his deputy.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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