Improved Maize Growth in Condition Controlled by PGPR Inoculation on Ferruginous Soil in Central Benin

The use of microbial technologies in agriculture is rapidly expanding with the discovery of new bacterial strains effective in improving plant growth. In this study, we tested and highlighted the efficacy of PGPR (Plant Growth Promoting Rhizobacteria) alone or in a consortium on maize growth. For this purpose, a greenhouse experiment was carried out in pots containing sterilized ferruginous soil for 30 days. The corn seeds of the EVDT 97 SRT C1 variety were inoculated with bacterial suspensions of concentration 10 UFC/ml. The experimental device was a random block of 16 three-repeat treatments. The incidence of PGPR inoculated strains is assessed on the biomass growth and yield parameters of maize. At the end of the trial, the results showed that inoculation stimulated plant growth and development and resulted in a significant increase in the height, diameter at the collar, leaf surface and dry weight of aerial biomass of 20.15%, 21%, 32.77% and 37.73% respectively compared to controls, especially in corn plants inoculated with B. thurengiensis + B. panthéthonicus + S. marcescens and Pseudomonas cichorii + Pseudomonas putida + Pseudomonas syringae. These results show the potential of using these rhizobacteria as biological inoculants to improve maize productivity in Benin. How to cite this paper: Amogou, O., Agbodjato, N.A., Dagbénonbakin, G., Noumavo, P.A., Sina, H., Sylvestre, A.A., Adoko, M.Y., Nounagnon, M., Kakaï, R.G., Adjanohoun, A. and Baba-Moussa, L. (2019) Improved Maize Growth in Condition Controlled by PGPR Inoculation on Ferruginous Soil in Central Benin. Food and Nutrition Sciences, 10, 1433-1451. https://doi.org/10.4236/fns.2019.1012102 Received: November 16, 2019 Accepted: December 28, 2019 Published: December 31, 2019 Copyright © 2019 by author(s) and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
Some bacteria that live in the rhizosphere define how the volume of soil influenced by the roots has the ability to promote plant growth [1] [2]. These microorganisms referred as PGPR drew attention to the need to reduce chemicals particularly in the context of sustainable agriculture and environmental protection [3]. PGPRs influence plant health and productivity through a variety of mechanisms that involve mineral solubilization, nitrogen fixation, phytohormone synthesis, hydrolytic enzyme synthesis and balance modulation plant hormone by deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) [4] [5]. These rhizobacteria can improve the elongation and branching of the root system which promotes the absorption of water and minerals from the soil to the host plant including wheat, barley, corn and rice that are necessary for plant survival [6]. The benefits of PGPR inoculation on plant growth and productivity are well documented and have been correlated with phytohormone production and higher nutrient intake [7] [8] [9]. Various species of PGPR have been considered PGPR to stimulate plant growth and some of them marketed in recent years belong to the genera Pseudomonas, Azospirillum, Azotobacter, Bacillus, Enterobacter, Rhizobium, Mycobacterium, Enterobacter, Caulobacter, Serratia, Flavobacterium, Actinobacteria sp. [10] [11].
Maize is a staple crop in Benin but soils are poor and inappropriate use of mineral fertilizers prevents producers from achieving the potential yield of this crop. Several research studies have reported that the excessive use of chemical fertilizers in agronomic systems aiming at improving yield, is less efficient in terms of stabilizing chemical elements that are quickly leached just after their application at ground level [12] [13]. This represents a deficit of absorption and nutritional assimilation in plants.
In Benin, the phenomenon of soil degradation is increasing especially in central regions dominated by ferruginous soils. This contributes to the depletion of organic matter in the soil as well as the nutrients essential for plant development [14]. The role of PGPRs in resolving several constraints such as soil degradation, water stress, and declining soil fertility that limit agricultural production has been widely proven [15]. The use of plant growth-promoting bacteria (PGPR) can be a promising alternative that can reduce the application of chemicals and improve crop yields and plant health [16] [17] [18]. For example, [19] has shown that PGPRs can increase nutrient bioavailability in the rhizosphere. They fix nutrients and prevent them from washing. In the work undertaken [20], the improvement in wheat yield was attributed to the co-inoculation of Bacillus thuringiensis and Serratia sp. Similarly, [21] [22] noted that inoculation of corn Food and Nutrition Sciences seeds with strains of Bacillus polymyxa, Pseudomonas alcaligenes and Actinomycetes sp. (O19-AHB12) significantly improved the dry weight of maize plants by 19% to 52%, total maize biomass by 38% and the weight of 1000 corn seeds by 74.72% compared to controls. To date, little work has been done in Benin on the use of PGPRs to improve maize growth and yield. The objective of this study is to investigate the effects of nine (09) isolated rhizobacteria (PGPR) identified in central and northern Benin on the growth of greenhouse maize on ferruginous soil in central Benin.

Preparing the PGPR Inoculum
The method described by Guiraud and Galzy, 1994 was used to rejuvenate by transplanting the three (3) strains of Pseudomonas on King B medium. The Bacillus and Serratia strains were revived on Nutritive Agar [26] [27]. The inoculum of each PGPR was obtained by culture in a nutrient medium (liquid HD) for 24 hours at 30˚C. The method described by [28] allowed us to adjust bacterial cultures to a concentration of approximately 1 × 10 8 UFC/ml (OD 0.45 to 610 nm) with a spectrophotometer 24 hours after incubation.

Experimental Device
The device adopted was a complete random block comprising sixteen (16)

Processing the Substrate and Filling the Jars
The soil used consisted of depleted tropical ferruginous composite soil from the experimental site of the Agricultural Research Centre (CRA-Centre) in Gobé. This substrate is removed at a depth of 0 to 20 cm using a graduated shovel spade and is dried in the sun. It is then sifted to 2 mm and then doubly sterilized at 120˚C for 20 minutes at a 24-hour interval [29]. Then, 3 kg of the sterilized soil was weighed and distributed in each pot.

Description of the Chemical Characteristics of the Soil Used
The soil was analyzed in a previous study published in 2017 [30]. The data is in Table 1. This soil was moderately acidic, with a pH (water) of 6.5. The analysis also showed that the nitrogen (0.076%), phosphorus (5 ppm), potassium (0.16 meq)/100g) levels are very low. The sum of the exchangeable bases (3.66 meq/100g) is very small. On the other hand, the cationic exchange capacity (8 meq/100g) was average. These chemical properties reflect limited fertility of the study soil due to its low reserves of major nutrients (N, P and K) recorded.

Semi, Inoculation and Pot Maintenance
The pots were arranged inside the greenhouse and then watered at 2/9th of their maximum water retention capacity (CMR) 24 hours before sowing [31]. After opening the hole, two (02) seeds were deposited per pot and then inoculated with 10 ml of suspension of each bacterial strain of concentration 10 8 UFC/ml. The hole was immediately closed. A quantity of 500 ml water, or 1/9th of the soil's maximum water retention capacity (CRM), was brought to the plants every 48 hours after seed germination. Thinning to one plant per pot was performed one week after germination. The experiment was conducted in May 2019 under a greenhouse located within the grounds of the University of Abomeycalavi (West Africa, Republic of Benin) at an altitude of 22 m between latitude 6-258'N and longitude 2-20'E. The average temperature inside the greenhouse is 25.1˚C and 27.23˚C during the trial period.

Data Collection
Morphometric measurements were made 7 days after sowing. These morphometric  [32] using the measurement of the length and width of the last two leaves ligulate of the plants. In addition, the young corn plants were gently dug up and the roots were immersed in a large bowl of tap water to rid them of soil particles. The freshly weighed biomass was sun-dried at 100˚C for 72 hours to determine the dry weight of aerial and underground biomass.

Statistical Analysis
The Hierarchic Classification on Main Components (HCPC) preceded by a Main Component Analysis (ACP) was conducted to identify the pattern of discrimination caused by the effect of the PGPR and their combination on the growth and productivity parameters. The combination of these two consecutive multivariate analyzes is useful for showing the pattern across the data set [33]. The different classes obtained were described using descriptive statistics (mean, standard deviation). These multivariate analyzes were performed in the R 3.6.0 software [34] and required the use of the FctoMine R and factoextra packages. Within each group, the effect of PGPR and their combination on neck height and diameter was assessed for each parameter by fitting a linear mixed effects model to longitudinal data. In each model, the groups obtained were considered as fixed factors and time as a random factor. Adjusted averages were also calculated to represent the evolutionary trends of each growth parameter in each group. These analyzes were done with the R software using the packages nlme (for the fit of the model), lsmeans (for the computation of the adjusted averages), and ggplot2 (for the representation of the curves). The impact of PGPR and their combination on leaf area and plant yield parameters were assessed using an analysis of variance after verification of normality and homoscedasticity of the data respectively by Ryan-Joiner and Levene tests [35]. Post hoc or multiple comparison tests (SNK test) were performed to assess the statistical differences in averages when anova results are significant. Adjusted averages were also calculated to represent the averages of each parameter for each group. The car and lsmeans packages were respectively used to perform the anova, and the calculation of the adjusted averages.

Identifying Homogeneous Groups of Treatments
The ACP carried out on the different parameters of plant growth and yield shows that the first two axes retain 78.67% of the total variance. All growth parameters and yield parameters are strongly correlated with axis 1 and only the BSS variable (dry underground biomass) which is strongly correlated with axis 2 (Figure 1(a)).

Effect of PGPRs and Their Combination on the Height of Corn Plants
Based on the results of the mixed-effect linear model (Table 2), it appears that time has a significant effect on plant height growth (p-value < 0.0001). However, the treatment (groups) (p-value = 0.62) and the interaction between time and treatment (p-value = 0.45) are not significant, indicating that the variations ob-

Effect of PGPR and Their Combination on the Diameter at the Collar of Corn Plants
Analysis of mixed-effect variance ( Table 3) applied to plant collar diameter averages shows that time has a significant effect on the growth of plant collar diameter (p-value < 0.0001). The treatment (groups) (p-value = 0.02) and the interaction between time and treatment (p-value < 0.01) are also significant, indicating that the variations observed over time depend on treatment (group). The evolution curves of the diameter at the plant collar vary significantly from one collection period to another than from one treatment to another (Figure 3). In     to the control and group 3 (G3) plants.

Effect of PMPRs and Their Combination on the Biomass Yield Parameters of Corn Plants
In Figure 1, group 4 (G4) rhizobacteria inoculated plants produced the largest dry aerial biomass, an increase of 30.73% over the control. Based on the results of the variance analysis (Table 5), there was a significant difference (p < 0.001) between the different treatment groups. In addition, a highly significant improvement (p < 0.001) was observed between the treatment groups on dry underground biomass of the plants ( Table 6). The best productions (3.42 ± 0.42 g) were recorded at the level of the plants of pin 3 (G3) followed by those of group 4 (G4) (3.06 ± 0.45 g). Non-inoculated plants of group 1 (G1) were the least productive.

Discussion
Various research has focused on the assessment of rhizobacteria for their ability to induce improvements in growth and yield parameters on controlled cereals    [45] justify the broadest leaves carried by corn plants under the influence of rhizobacteria combinations by better development of the root system of the plants; this is conducive to better absorption not only of nutrients but also of water and therefore of better productivity.
In terms of biomass yield, the dry weight of the aerial and underground biomass of the plants measured during our harvest study was significantly improved with inoculation compared to control plants ( Figure 5). The results of the variance analysis applied to the average weights at the level of each group indicated a significant effect (Table 5) and a highly significant effect (Table 6) [45] reported that the combination of 4 rhizobacteria (Bacillus megaterium, Pseudomonas aeruginosa, Serratia spp., and Food and Nutrition Sciences   [47] reported that dry matter is used for the production of new roots, their proliferation (root volume), their lengthening (length growth) and their maintenance. The classification obtained on the basis of the dendrogram (Figure 1(b)) shows that most individually tested strains, for example P. putida, P. cichorii, P.  [25] has demonstrated the ability of the majority of strains tested in our study to solubilize inorganic phosphate and produce metabolites of agricultural interest including acetic indole acid (AIA). The best results recorded on the parameters measured during our work would be related to the ability of PGPR strains to produce AIA, the siderophore, to solubilize phosphorus as it has been the case in many

Conclusion
PGPRs are used in agriculture as soil biofertilizers. These microorganisms have