Inoculation of Rhizobiums sp Strains to Improve Soil Fertility: A Peanut Trial in Covè and Ouessè (Benin)

Thought the increasing demand Arachis hypogaea L. (groundnut), its yields remain low with increasingly using chemical fertilizers. To reduce the costs for chemical fertilizers inquisition and their long-term toxic effects on soils, microbial bio-fertilizers could be an accessible alternative to peanut farms. Thus, the aim of this study was to assess the performance of rhizobia strains on peanut varieties production. The experiments were conducted in two agro-ecological zones of Benin, in a peasant environment peasant-researcher control or under peasant and researcher control. The experimental device used was a complete random block with nine repetitions and two factors namely inoculation (with Rhizobium sp and without Rhizobium sp) and mineral fertilizer (with N15P15K15 and without N15P15K15). The effects of these factors divided into four treatments were evaluated on the plants vegetative, symbiotic and production parameters. In addition, an evaluation of each treatments’ comparative advantages was carried out. The results showed that the association Rhizobium sp and N15P15K15 induced groundnut plants best vegetative and productive parameters. The best comparative advantages in economic terms were also recorded with the same combination (Rhizobium sp + N15P15K15). Considering the technical performance, the recorded treatments effects can be classified as follows: Control < Rhizobium sp < N15P15K15 < Rhizobium sp + N15P15K15. Thus, the association Rhizobium sp + N15P15K15 induced both the best plants vegetative and productive parameters and the best comparative advantage from an economic point of view. The results also How to cite this paper: Adjile, A., Didagbe, O., Ayelo, C., Boko, F., Houngnandan, P. and Mongbo, R. (2020) Inoculation of Rhizobiums sp Strains to Improve Soil Fertility: A Peanut Trial in Covè and Ouessè (Benin). Agricultural Sciences, 11, 448-463. https://doi.org/10.4236/as.2020.114027 Received: February 17, 2020 Accepted: April 20, 2020 Published: April 23, 2020 Copyright © 2020 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/ Open Access


Abstract
Thought the increasing demand Arachis hypogaea L. (groundnut), its yields remain low with increasingly using chemical fertilizers. To reduce the costs for chemical fertilizers inquisition and their long-term toxic effects on soils, microbial bio-fertilizers could be an accessible alternative to peanut farms. Thus, the aim of this study was to assess the performance of rhizobia strains on peanut varieties production. The experiments were conducted in two agro-ecological zones of Benin, in a peasant environment peasant-researcher control or under peasant and researcher control. The experimental device used was a complete random block with nine repetitions and two factors namely inoculation (with Rhizobium sp and without Rhizobium sp) and mineral fertilizer (with N 15 P 15 K 15 and without N 15 P 15 K 15 ). The effects of these factors divided into four treatments were evaluated on the plants vegetative, symbiotic and production parameters. In addition, an evaluation of each treatments' comparative advantages was carried out. The results showed that the association Rhizobium sp and N 15 P 15 K 15 induced groundnut plants best vegetative and productive parameters. The best comparative advantages in economic terms were also recorded with the same combination (Rhizobium sp + N 15 P 15 K 15 ). Considering the technical performance, the recorded treatments effects can be classified as follows: Control < Rhizobium sp < N 15 P 15 K 15 < Rhizobium sp + N 15 P 15 K 15 . Thus, the association Rhizobium sp + N 15 P 15 K 15 induced both the best plants vegetative and productive parameters and the best comparative advantage from an economic point of view. The results also

Introduction
Peanuts are annual oilseed legume often produced in rotation or in association with cereal crops in Benin. However, in last years, the yield of peanut has decreased drastically. According to UDA [1], Benin recorded a peanut yield of 0.93 t/ha against 1.2 t/ha in Nigeria, 1.10 t/ha in Ghana and 1.13 t/ha in Côte d'Ivoire.
The world average is 1.5 t/ha and more than 3 t in the United States and China [2]. Although this peanuts production yield decrease is imputed to several factors, the lowering soil fertility appears to be non-negligible. According to Igué et al. [3], 68% of the soils in Benin have suffered a severe decrease in its agricultural potential specially the southern and central regions. This is, we according to these authors, to the soil's low nitrogen, phosphorus and potassium content and its cation exchange capacity.
To improve their peanuts production yields then, producers often make use of chemical fertilizers. However, due to their high cost, the use of chemical fertilizers is restricted to a small number of peanuts producers. Although chemical fertilizer seems to improve their production yield, their impact on the environmental is disastrous at long term.
Actually, the success of groundnut cultivation is mainly due to its ability to fix atmospheric nitrogen thanks to the presence of root nodules hosting colonies of Rhyzobium sp strains, which allow a slight improvement on the production yield on poor soils, and this, with minimal intervention [4]. The success of this crop therefore requires the establishment of an efficient association between the host plant and the bacteria of the genus Rhizobium. Inoculating the peanut with effective rhizobia has been proven a beneficial practice for improving the peanuts productivity [5] [6] in Benin. This practice would also constitute a prospect for promoting a sustainable and environment-friendly farming system.
The present study was initiated, in the same framework, and, conducted in a peasant environment to assess the response of peanut seeds (Arachis hypogaea) grown in Ouèsse-and on the Agonli uplands in the south and center of Benin to inoculation with a rhizobium and to compare its advantage to other fertilization options in these environments.

Effects of the Factors Studied and Their Interactions on the Different Parameters
The study was carried out from August 2015 to January 2016 in peasant farms in the sub-districts of Houéhounta (district of Covè) and Gbanlin (district of Ouèssè) (Figure 1).

Physical Characteristics of the Experiments Sites (Ouèssè and Covè)
Ouèssè is located in the fifth agro-ecological zone of Benin in the heart of the humid tropical zone. It is beneficiary of a tropical climate intermediate between the Guinean climate and the Sudanese climate [7]. The annual rainfall varies between 1100 mm and 1200 mm. It has lands and lowlands appropriate to the good development of agricultural crops and an agricultural diversity estimated at around 1500 km 2 of cultivable land. On the soil level, almost all of Ouèssè is covered with ferruginous soils [8].
Located in the sixth Agro-Ecological Zone of Benin, Covè covers an area of 525 km 2 in the Southern-East of the Department of Zou. It has a Sudan-Guinean or subequatorial type climate with two rainy seasons alternated by two dry seasons. The average rainfall recorded in the area varies between 900 mm to 1100 mm. More than 60% of the district is covered with ferralitic soils characterized by a dominance of iron oxides due to a still incomplete alteration of the primary minerals [9].
The first rainy season which extends from March to July, experienced the rainiest month in March in the two municipalities with a height of rain of 116mm in Covè and 162.3 mm in Ouèssè. A drop in precipitation, more pronounced in Ouèssè than in Covè, immediately followed this moderately wet month. A gradual recovery was observed in May, June and July.
As for the second rainy season, mid-August to November, the period during which the tests were conducted, we note that the month of August was very wet due to the height of the rains recorded in Ouèssè unlike Covè. However, there has been a gradual fall in precipitation during the months of September, October and November in Ouèssè. In Covè, October was an extremely humid month with precipitation ranging to 174.5 mm before declining in November with a transition to the dry season. In sum, for this growing season, a total of 162.6 mm and 218.3 mm of rain was obtained in 3 months (September to November) of rain respectively in Ouessè and Covè.

Biological and Chemical Materials
The main materials used are: 1) farmer seeds obtained from producers' harvests.

Isolation of Bacteria Nodulating Peanut Roots
The collected nodules from the underground part of the peanut plants were ste-rilized with 8% bleach for 10 min. These nodules were crushed using the sterilized glass rod to obtain a milky suspension of bacteroids. This was streaked on the Yeast Extract Mannitol Agar (YEMA) medium + 0.0025% (w/v) of Red Congo and incubated at 30˚C ± 1˚C for 24 -48 h [10]. The strains obtained after spreading on culture medium were purified after successive subculturing on YEMA medium [11].

Preparation of the Bacterial Suspension
Bacterial culture was carried out according to the modified method described by Montage and Beunard [12]. Each 250 ml Erlenmeyer flask containing approximately 100 ml of sterile YEM is inoculated using a loop under a host, a bacterial strain stored on YEMA medium contained in a sealed petri dish by Para film.
The obtained culture was stirred (200 rpm) at 28˚C for 7 days. The control of the bacterial growth was ensured by samples of an aliquot in the liquid inoculum in preparation according to the dilution method and counting [13], making the gram staining and pH control (the pH is adjusted by aseptically adding 5N KOH).

Inoculation Support and Seed Sterilization
The peat used as a carrier for the inoculant underwent several stages (dried, crushed, sieve and neutralized) of preparation. This support was packaged (30 g per bag) in heat-resistant polypropylene bags (10 cm × 15 cm). The latter underwent three successive sterilizations each 24 hours [14]. The methodology adopted for seed sterilization is an adaptation to that of Ngo Nkot et al. [15]. Thus, the peanut seeds have been surface sterilized in 3.3% calcium hypochlorite solution for 3 minutes and washed with sterile distilled water. After sterilization, some seeds were incubated to germinate in petri dishes containing 0.9% (w/v) TYA for 2 to 3 days at 28˚C for viability tests.
The appearance of the radicle shows that the seeds selected are of good quality.

Experimental Design
The system set up in each farmer field is a two factor Complete Random Block with nine (09) repetitions of four (04) treatments on experimental units of 20 m 2 each. These treatments are distributed randomly as follows: Seeds not inoculated and without fertilizer (T1), seeds with only N 15 P 15 K 15 (T2), seeds only inoculated by Rhizobium sp (T3) and seeds inoculated Rhizobium sp + N 15 P 15 K 15 (T4).

Symbiotic Parameters: Number and Dry Weight of Nodules
The nodules are removed from the peanut roots at 42 days after sowing. After counting, the dry weight of the nodules was measured after drying in an oven at 65˚C for 72 hours.

Production Parameters: Yield in Full Pods
The yield was evaluated by treatment for each trial on a 9 m 2 unit of production area (kg/ha). The peanut plants were harvested at 90 days after sowing.

Economic Performance Evaluation
The analysis of the economic performance of the treatments was made from the yield of pods and expenses of production.

Statistical Analysis
The data were processed with the Excel spreadsheet 2010 version. Statistical Analysis System software version 9.1 was then used for the statistical analysis.
One-way analysis of variance has been used to assess the effect of the treatments on the various parameters.

Results
In general, the highest values for all the parameters observed are recorded on the plants having received the combined contribution of Rhizobium sp and N 15 P 15 K 15 . A higher mean value significantly different to plants having benefited from all the other types of treatment regardless of the experimental site has been recorded. Table 1  rather significant difference at the 0.05 threshold. The interaction experiment site and treatments reveals non-significant differences effects on the dry weight of root biomass but high different was recorded on the total biomass per plant and very high on the other measured parameters.  all other types of treatments. The same trends were observed in the commune of Ouèssè. However, there was no significant difference between the average dry weight values in root biomass of the four treatments including the control treatment. The average values obtained in Covè are globally higher and significantly different from those obtained in Ouèssè for the dry weights (DW) of the aboveground (AB), root (RB) and total (TB) biomass per peanut plant.

Effects of Treatments on Production Parameters
The results of the production parameters from the different treatments are recorded in Table 4. The application of the different treatments generally led to an improvement in the yield of aerial and ground peanuts compared to the controls.  In terms of yields of solid peanut pods for this experiment site, there is a positive increase in yields between the control plots and all the treatments with mean values statistically different from each other. The optimal yield is obtained with the combination Rhizobium sp + N 15 P 15 K 15 (1388.5 ± 108.13 Kg/ha). Furthermore, the average yield of full pods from inoculated plots, although higher than that of control plots, remains lower than that of plots amended only with N 15 P 15 K 15 fertilizer (Figure 2).    However, by integrating the costs of fertilization and the manpower or labour cost for spreading the fertilizer, the use of mineral fertilizer is more costly and therefore presents an economic gain lower than the balance of the application of the inoculum. Therefore, treatment with Rhizobium sp would be more cost-effective than the single supply of mineral fertilizer.

Economic Evaluation of Treatments
In the experiment site of Ouèssè, we observe a trend opposite to that of Covè. The capital gain in FCA and the capital gain balance of the plots amended with mineral fertilizer are higher than those inoculated are. However, the combined contribution of the two treatments (Rhizobium sp + N 15 P 15 K 15 ) would be better regardless of the experiment site.

Discussion
The results showed that the nutrients provided by the various treatments have a variable and significant influence on the different parameters evaluated.
From the results of our investigation on the growth parameters (dry weights of aerial biomass, root biomass and total biomass), a homogeneity of effects was observed overall on all the plots except for the plots sowed with seeds inoculated and then amended with NPK fertilizer. This would be due to the growing conditions (rainfall) which, overall, were not favorable to a good plant productivity. The non-significant responses thus obtained especially between the three treatments (T1, T2, T3) are similar to the observations made by Ndiaye [16] on the growth of the plant on which the biomass yield also depends. Thus, "the drought occurred in full flowering period probably caused a slowing down of the vegetative development and mostly on the inoculated plots; which must have reduced the differences in growth between treatments (particularly at Ouèssè)". Indeed, for the flowering-formation of pods phase observed between 30 to 70 days after sowing, Covè seems to have been favored compared to Ouèssè by the drop in rainfall.
The highest values observed for the treatment combining Rhizobium sp + N 15 P 15 K 15 are in accordance with the observations made by Didagbé et al. [17].
According to their investigation carried in the Center and North Benin, overall, the treatments by Rhizobium strains supplemented with phosphorus produced the highest aerial biomass. This would imply the positive response (p < 0.05) to the addition of NPK at inoculation observed on the peanut plants growth parameters. These same trends were observed for the symbiotic parameters and the production parameters. In reality, in addition to the phosphorus demand from the host plant, nodules require greater amounts of P and energy than other plant tissues [18]. Phosphorus contributes to root growth [19]; nitrogen being a quality of and growth factor [20] [21] and potassium not only improves the plant's water regime but also increases its tolerance to drought, frost and salinity [20].
Furthermore, the number of nodules observed on controls revealed the presence of Rhizobium specific for peanuts in the soil at each site. Indeed this parcel has been used for peanuts crop cultivation. However, the small quantities of spontaneous nodules obtained could be due to the low strains population density of these soils [22]. On the inoculated plots, the formation of nodules and the symbiotic activities in the different peanut populations were therefore carried out with the native strains of Rhizobium present in the soil as well as the Rhizobium sp strains. The number of nodules formed (transformed value) differ significant to (p < 0.05) from the inoculated plants to the control plants. However, the confident interval of these differences is narrower compared to the number of nodules of the plants fertilized with N 15 P 15 K 15 only. This could be due to the competitiveness capacity of the strains. The native competitive but efficient less strain [23] can affect the inoculation of a given legume by a selected rhizobium with high efficiency. Nevertheless, the inoculation were important because the native Rhizobium strains would have been infectious but not efficient for the most part. The difference on the number of nodules counted in the control plots and in the inoculated plots is small was statistically different on the two experimental sites and no depressive effect of the inoculation on the nodulation was observed. This would indicate that inoculation has a favorable effect on the nodulation of legumes [16] [24].
Didagbé et al. [17] have shown that Rhizobium strain is one of the best performing exogenous strains of Bradyrhiobium used in their investigation. However, our results on plant nodulation are relatively lower than those previously obtained [17]. The competition between native and introduced strains could be low. As Ndiaye [16] observed on the CB 756 strain, the increases in yield following inoculation seem to indicate that the strain Rhizobium sp has a fixing potential greater than the native soil strains, but the competitiveness of the native strains and the poor survival conditions make it (very) difficult to increase the yields.
According to Tombozara [25], the possibility that the introduced rhizobia have not competed with the microorganisms present in the tested parcels must be considered. Other factors such as the quality of the plant material could also have had an impact on the low performance observed. Notwithstanding the probable competitiveness effect between the strains, the analysis of the dry weights of the nodules indicates that the difference between the average values are insignificant between the controls, the N 15 P 15 K 15 and Rhizobium sp + N 15 P 15 K 15 at both Covè and Ouèssè. It is therefore possible that the rainfall regime mentioned above contributed to this reduction in the differences of the effects between the treatments. A water deficiency would induce a significant decrease in the number and yield of nodules [26]. This water deficit is the consequence of pockets of drought occurring at the beginning of the plant development cycle and in full bloom. This could partly explain the fact that the results obtained at Covè are better than at Ouessè.
In addition, the physicochemical properties including among others the phosphorus deficiency affect the multiplication of rhizobia in the soil. It has been established that the effectiveness of endomychorizial symbiosis depends on the level of assimilable phosphorus in the soil [19]. For Mandimba and Djondo [27] phosphorus could increase nodulation in peanuts from 57% to 176% and, by extension, the yield. Phosphorus deficit would result in a reduction in the growth of nodules. Thus, the addition of N-P-K fertilizer has considerably improved the efficiency of the Peanut/Rhizobium symbiosis. N-P-K fertilization alone had more influence on yields than inoculation alone despite the non-significant difference observed. In accordance with our results and those obtained by other researchers [28] [29] [30], like all crops, legumes have nutrient requirements that, depending on their availability, influence their yields. According to these same authors, even nitrogen fertilizer is necessary to improve the yields of legumes. Therefore, the increases in the yield of full pods obtained indicate that the low dose of nitrogen provided by the mineral fertilizer made it possible to increase the groundnut yields from 26% to 36% [30].
The pod yield is one of the first parameters on which all the changes affecting the plant are reflected [31]. Analysis of the average yields in full pods revealed that the local variety "Monto" responded favorably to inoculation Rhizobium sp. This positive effect of inoculation with exogenous Rhizobium on the yields of legumes has been demonstrated by several studies on peanuts, soybeans, cowpeas and other legumes [6] [18] and [26].
However, by including production costs, the use of mineral manure costs more than the inoculum. Therefore, the treatment with Rhizobium sp would be more profitable than the single contribution of the mineral fertilizer especially at Covè. In addition, the use of this type of fertilization also helps to protect the environment by limiting the pollution by nitrites.

Conclusion
The experiment conducted in a peasant environment made it possible to evaluate and compare the response of peasant seed to inoculation with the Rhizobium strain. Compared to Ouessè, the results obtained at Covè were significantly better. It was observed that the nitrogen nutrition of the peanut could be ensured by the inoculation of the seeds and mineral fertilizers could provide the nutritive elements in particular urea, triple superphosphate (TSP) and potassium chloride. However, these two simple factors separately induced a significantly slight improvement in the vegetative parameters (growth, nodulation) compared to the control. The effect of the fertilizer factor was more favorable on the symbiotic activity of the peanut than the growth of the plant thanks to the moderate supply of both N, P and K. As for inoculation alone, despite the increase in nodulation, it had beneficial but not very remarkable effects on the production parameters, certainly due to the poor survival conditions of the bacteria introduced. Regarding the performance of the treatments, the vegetative and productive behaviors of the plants were more influenced by the combined effects of inoculum and mineral fertilizer. The best results in terms of comparative advantages and from the economic point of view were obtained by combining the