Characterization of Potential Plant Growth Promoting Rhizobacteria Isolated from Maize ( Zea mays L . ) in Central and Northern Benin ( West Africa )

1Laboratoire de Biologie et de Typage Moléculaire en Microbiologie, Département de Biochimie et de Biologie Cellulaire, Faculté des Sciences et Techniques, Université d’Abomey-Calavi, 05 BP 1604 Cotonou, Benin 2Laboratoire de Microbiologie et de Technologie Alimentaire, Faculté des Sciences et Techniques, Université d’Abomey-Calavi, 04 BP 1107 Cotonou, Benin 3Laboratoire de Biomembrane et de Signalisation Cellulaire, Faculté des Sciences et Techniques, Université d’Abomey-Calavi, 01 BP526 Abomey-Calavi, Benin 4Section Hygiène des Eaux et Aliments, Laboratoire National de Santé Publique, 01 BP 418 Cotonou, Benin 5Centre de Recherches Agricoles Sud, Institut National des Recherches Agricoles du Bénin, BP 03 Attogon, Benin


Introduction
The first aim of agriculture was to ensure survival by producing the necessary for feeding.It was subsistence farming.But nowadays, due to continued and worrying growth of world population, this primary objective of agriculture changed completely.Indeed, the world population is estimated around 7 billion people and may reach 8 billion by 2020 [1].So, it is urgent to considerably increase the agricultural production to reply to the strong food demand to reduce the risk of malnutrition and the increasing of poverty.
Therefore, the new cereal varieties of high yield were developed.In addition, agrochemical products such as chemical fertilizers, herbicides, fungicides, and insecticides were currently improperly and excessively used in order to increase crop yield.The direct consequence of these agrochemical products use is the environment pollution through ground water and crop products contamination by heavy metals that are contained in these agricultural inputs.These heavy metals are known to be a public health problems because, transferred to humans, they are involved in the cancer occurrence [2].Apart from medical damages, other consequences in agricultural area such as natural ecological nutrient cycling interruption and soil biological communities destruction are frequently reported [3].Regarding the damages caused by the excessive use of agrochemical products, other research paths are explored worldwide.Among the explored paths, the use of microorganism currently called Plant Growth Promoting Rhizobacteria (PGPR) is in pole position.
Indeed, the PGPR is a group of bacteria capable of colonizing actively plant roots system and improving their growth and yield [4].The expression "PGPR" was firstly proposed by Kloepper et al. [5] and was used especially for the fluorescent Pseudomonas involved in biological control of pathogens and the improvement of plant growth.Later, Kapulnik et al. [6] extended this expression to rhizobacteria capable of promoting directly the plant growth.Nowadays, this expression is used to refer to all bacteria living in the rhizosphere (around roots), improving plant growth by one or several mechanisms [7].A large range of species belonging to the genus Pseudomonas, Azospirillum, Azotobacter, Klebsiella, Enterobacter, Alcaligenes, Arthrobacter, Burkholderia, Bacillus, and Serratia were reported to be PGPR [8].
In this context, the aim of our study was to isolate and identify the potential PGPR from maize (most cultivated and consumed cereal in Benin) rhizosphere in the central and northern Benin.The medium-dated objective of this study is to propose for farmers the biological fertilizers based on native PGPR for increasing maize production.

Geographical Characterization of Study
Area.This study was carried in five agroecological zones (I, II, III, IV, and V) located in the central and northern Benin, West Africa (Figure 1).Indeed, Benin is localized in West Africa (south of Sahara), in the tropical zone between Equator and Tropic of Cancer, precisely between the parallel 6 ∘ 30  and 12 ∘ 30  of north latitude and meridians 1 ∘ and 30 ∘ 40  of east longitude.

Collection of Rhizospheric
Samples.Three (3) villages were selected by agroecological zone and four fields were chosen in each village.Three maize plants distanced at least 10 meters were dug up in each field.Their roots were cut with soil adheres and mixed in a bucket.Three hundred grams of this mixture was packed in a sterile stomacher bag and labeled correctly to form the sample of the field.A total of 60 samples were collected and immediately transported at 4 ∘ C to the laboratory for further analysis.Once they are at the laboratory, the microbiological screening was immediately realized or samples were kept at 4 ∘ C until screening.
Several other parameters (climate, soil type, annual pluviometry, and other crops grown except for maize) of the sampling sites were collected during the sampling.

Isolation of Rhizobacteria.
According to Speck [17] method, 10 g of each sample was mixed into Erlenmeyer flask containing 90 mL of tryptone salt.The mixture was vigorously shaken for about 30 s to obtain 10 −1 dilution.The previous dilution (1 mL) was transferred into 9 mL of tryptone salt to obtain 10 −2 dilution.This operation was repeated until obtaining 10 −8 dilution.Each dilution (0.1 mL) was streaked on different specific isolation media.The aerobic mesophilic flora was enumerated on Plate Count Agar as recommended by the French standard V08-051.Bacillus sp. and Serratia sp. were isolated on nutrient agar after incubation at 37 ∘ C for 24 h and 30 ∘ C for 48 h, respectively [18,19].Pseudomonas sp. was isolated on King A and King B agar after incubation at 30 ∘ C for 72 h [20].

Identification of Rhizobacteria.
The identification of isolated rhizobacteria consisted firstly in macroscopic (colony morphology, pigmentation, etc.) and microscopic (gram reaction, mobility, cell shape, spores position, etc.) observations.This first identification was followed by several biochemical and enzymatic tests.The performed tests are production of oxidase, catalase, indole, urease and hydrogen sulfide, respiratory type, acid and gas production on glucose agar, citrate and nitrate utilization, hydrolysis of starch, casein, mannitol, gelatin and lecithin, fermentation of glucose and lactose, growth on MacConkey and Cetrimide agar; Voges-Proskaur test, growth at 42, 45, 55, and 65 ∘ C, and fluorescence at 360 ∘ C [18,[21][22][23].

Plant Growth Promoting Properties
2.5.1.Hydrogen Cyanide Production.All isolated rhizobacteria were screened for hydrogen cyanide production following the method described by Lorck [24].Each rhizobacterium was streaked on nutrient agar medium added with glycine (4.4 g/L).The agar was covered with a Whatman number 1 filter paper previously soaked in a specific solution (0.5% picric acid and 2% sodium carbonate w/v).Plates were sealed with parafilm paper and incubated at 36 ± 2 ∘ C for 4 days.The appearance of orange or red color indicates the production of hydrogen cyanide.

Ammonia Production.
To research the production of ammonia, each identified rhizobacteria strain was grown in peptone broth (10 mL) and incubated at 36 ± 2 ∘ C for 48 to 72 h.After incubation, 0.5 mL of Nessler's reagent was added to bacterial suspension.The development of brown to yellow color indicated ammonia production [25].at probability level of 5%, following a mean separation (Student-Newman-Keuls test), by Statistical Analysis System (SAS) software Version 8.1.In this model, soil types and agroecological zones were considered as a fixed factor while replicates were considered as a random factor.

Agroecological Characteristics of the Villages Surveyed.
Table 1 shows agroecological characteristics of villages surveyed by agroecological zone.The Sudanese climate with     .Serratia sp. is not found in rhizosphere of agroecological zone I (Figure 3(a)), but it is abundant in rhizosphere of agroecological zone IV (3.12 × 10 6 CFU/g of soil).The density of rhizobacteria strains varies also from a soil type to another (Figure 3(b)).The Washing and Idurate Tropical Ferruginous Soil (WITFS) contains the largest population of Bacillus sp.(6.03 × 10 6 CFU/g of soil) whereas the Few Developed Soil (FDS) contains the least population of Bacillus spp.(3.2 × 10 6 CFU/g of soil).Impoverished Tropical Ferruginous Soil (ITFS) and Few Washing Tropical Ferruginous Soil (FWTFS) contain, respectively, the large populations of Pseudomonas sp. and Serratia sp.In general, the density of Bacillus sp. is higher than Pseudomonas sp. and Serratia sp.Serratia sp. is the least abundant in the majority of soils.

Rhizobacteria Species
Identified.Microbial investigation of samples collected from the 5 agroecological zones showed the presence of several rhizobacterial species.Five Bacillus species (B.polymyxa, B. pantothenticus, B. anthracis, B. thuringiensis, and B. circulans), 3 Pseudomonas species (P.cichorii, P putida, and P. syringae), and Serratia marcescens were identified.The morphological and biochemical characteristics of these rhizobacteria are shown in Table 2.

Ammonia and Hydrogen Cyanide Production by Rhizobacteria.
The production of ammonia (NH 3 ) and hydrogen cyanide (HCN) by rhizobacteria isolated from soil samples collected in the northern and central Benin is shown in Table 3.Our data suggested that all the rhizobacteria strains produce hydrogen cyanide.Concerning the production of ammonia, it was observed that all Serratia strains produce it against 80% of Bacillus sp. and 77.77% of Pseudomonas sp.(Table 3).

Discussion
Several studies have reported the benefit of seeds inoculation by Plant Growth Promoting Rhizobacteria.This growth promoting effect is influenced by biotic and abiotic factors including bacterial species and the soil types.It is in this context that this prospective study was realized in prelude of the promotion of microbial biofertilizers based on native rhizobacteria.The agroecological characteristics of sites samples collected were presented in Table 1 [26].Indeed, our result reflects the large soils diversity in Benin as Adjanohoun et al. [26] reported other types of soil such as Vertisols, Degraded Bar Land, and No Degraded Bar Land.
Apart from maize, many other crops are growing in different villages surveyed in this study.These crops are rice, sorghum, small millet, millet, cotton, bean, cowpea, cassava, bambara groundnut, yam, and pimento.In southern Benin, except maize, Adjanohoun et al. [26] had identified cotton, groundnut, sweet potato, cowpea, and cassava.These cultures were mostly found in northern and central Benin.Firstly,   [26] had noted the crops sown in fields before maize sowing, while this study registered the crops sown in the study zone during the sampling.
The soil aerobic mesophilic microflora has greatly varied from an agroecological zone to another (Figure 2(a)), but this difference is not significant ( > 0.05) at probability level 5%.This result can be explained by the large variability of microbial density in a same agroecological zone due to the soil heterogeneity existing in each zone.On the contrary, the density of aerobic mesophilic microflora has also greatly varied from a soil type to another, but the difference is highly significant ( < 0.001) at probability level 5% (Figure 2(b)).The variability of microbial density is probably due to physicochemical properties of the different soil types, which certainly impact the microbial activity in rhizosphere.These results are similar to those obtained by Adjanohoun et al. [26] in southern Benin when they observed a large difference of microbial density between Vertisols, Degraded Bar Land, and Not Degraded Bar Land.Indeed, Schoenborn et al. [27] reported that rhizosphere contains a great microbial population between 10 8 and 10 9 CFU/g of soil.This microbial abundance is explained by the richness of rhizosphere in nutrients such as sugars, amino acids, organic acids, hormones, and other small molecules derived from root exudates [28].The microorganisms find in rhizosphere the energy substrates required for their metabolism [29].Conversely, in stressed ecosystem the microorganism population can be less than 10 4 CFU/g of soil [30].So in spite of the different environmental stress (climate change), the maize rhizosphere in central and northern Benin still contains an abundant microbial population.
The density of isolated rhizobacteria has varied according to the agroecological zone and the soil types (Figure 3).The density difference between the soil types is significant for Bacillus sp.( > 0.05) and highly significant ( < 0.001) for Pseudomonas sp. and Serratia sp.The microbial density varied from 3.2 to 6.03 × 10 6 CFU/g of soil (Pseudomonas sp.), 1.05 to 3.33 × 10 6 CFU/g of soil (Bacillus sp.), and 0.67 to 3.13 × 10 6 CFU/g of soil (Serratia spp.).These microbial densities are inferior to those obtained by Joseph et al. [31] on chickpea (Cicer arietinum L.) in India.During their work, these authors counted about 0.5 to 2.1 × 10 9 CFU/g and 1.1 to 2.1 × 10 9 CFU/g of soil for Bacillus sp. and Pseudomonas sp., respectively.In our study, Bacillus sp.population is most abundant than Pseudomonas sp. and Serratia sp.This remark was earlier done by Saharan and Nehra [8] when they asserted that Bacillus sp. is the most abundant genus in their studied rhizosphere.In addition, Garbeva et al. [32]  In order to identify Plant Growth Promoting Rhizobacteria among isolated rhizobacteria in the central and northern Benin, we have screened all the strains for ammonia and hydrogen cyanide production.Thus, all the Serratia strains followed by 80% of Bacillus and 77.77% of Pseudomonas produced ammonia.These rates of ammonia production are lower than the 95% and 94% obtained, respectively, for Bacillus sp. and Pseudomonas sp. by Joseph et al. [31].Likewise, all Bacillus and Pseudomonas isolated by Yadav et al. [33] from chickpea rhizosphere in India have also produced ammonia.Ammonia production is an important characteristic of PGPR, which indirectly influences plants growth [33].
All strains produced hydrogen cyanide (100%).Our results seem higher than the 75% of hydrogen cyanide production by Bacillus sp.strains isolated from rice rhizosphere [34] and 40% of bacteria (Bacillus sp., Pseudomonas sp., Enterobacter sp., Acinetobacter sp., and Micrococcus sp.) isolated from the beans rhizosphere [35] in India.Indeed, the hydrogen cyanide is part of powerful antifungal compounds produced by PGPR and involved in pathogens biological control [36].

Conclusion
The maize rhizospheres in central and northern Benin contain high diversity of microorganisms.The bacterial density is generally high and varies according to both the agroecological zones and the type of soils.Nine species of potentially maize plants growth promoting rhizobacteria (B.polymyxa, B. pantothenticus, B. anthracis, B. thuringiensis, B. circulans, P. cichorii, P putida, P. syringae, and Serratia marcescens) were identified during this study.All isolates have produced hydrogen cyanide, while 86.66% of them produced ammonia.In perspective, these rhizobacteria will be assessed to promote maize seeds germination and plant growth.

Figure 1 :
Figure 1: Agroecological zones surveyed.AEZ: agroecological zone; AEZ I = Far North Benin; AEZ II = Cotton zone of North Benin; AEZ III = food-producing zone of South Benin; AEZ IV = West Atacora zone; AEZ V = Cotton zone of Central Benin; AEZ IV = Bar Land zone.

Figure 2 :
Figure 2: Distribution of aerobic mesophilic flora according to (a) AEZ and (b) type of soil.WCTFS: Washing and Concretion Tropical Ferruginous Soil; WNCTFS: Washing and No Concretion Tropical Ferruginous Soil; WHTFS: Washing and Hydromorphic Tropical Ferruginous Soil; WITFS: Washing and Idurate Tropical Ferruginous Soil; FWTFS: Few Washing Tropical Ferruginous Soil; ITFS: Impoverished Tropical Ferruginous Soil; FDS: Few Developed Soil.The means with different letters are significantly different with probability level of 5% according to Student-Newman-Keuls test.

Figure 3 :
Figure 3: Distribution of rhizobacteria density according to (a) AEZ and (b) type of soil.WCTFS: Washing and Concretion Tropical Ferruginous Soil; WNCTFS: Washing and No Concretion Tropical Ferruginous Soil; WHTFS: Washing and Hydromorphic Tropical Ferruginous Soil; WITFS: Washing and Idurate Tropical Ferruginous Soil; FWTFS: Few Washing Tropical Ferruginous Soil; ITFS: Impoverished Tropical Ferruginous Soil; FDS: Few Developed Soil.The means with different letters are significantly different with probability level of 5% according to Student-Newman-Keuls test.
Impoverished Tropical Ferruginous Soil; FDS: Few Developed Soil.The means with different letters are significantly different with probability level of 5% according to Student-Newman-Keuls test.Several other crops were grown by farmers apart from the maize.
soil.WCTFS: Washing and Concretion Tropical Ferruginous Soil; WNCTFS: Washing and No Concretion Tropical Ferruginous Soil; WHTFS: Washing and Hydromorphic Tropical Ferruginous Soil; WITFS: Washing and Idurate Tropical Ferruginous Soil; FWTFS: Few Washing Tropical Ferruginous Soil; ITFS: Ferruginous Soil (WNCTFS), the Washing and Hydromorphic Tropical Ferruginous Soil (WHTFS), the Washing and Idurate Tropical Ferruginous Soil (WITFS), the Few Washing Tropical Ferruginous Soil (FWTFS), the Impoverished Tropical Ferruginous Soil (ITFS), and the Few Developed Soil (FDS).Except for zone III, all the other agroecological zones contain at least two different types of soil.contains mesophilic microflora (6.25 × 10 8 CFU/g of soil) clearly abundant compared to the other agroecological zones (Figure 2(a)).The zone IV is the least loaded in mesophilic microflora (1.40 × 10 8 CFU/g of soil).Our data display that the Washing and No Concretion Tropical Ferruginous Soil (WNCTFS) contains the highest mesophilic microbial soil.WCTFS: Washing and Concretion Tropical Ferruginous Soil; WNCTFS: Washing and No Concretion Tropical Ferruginous Soil; WHTFS: Washing and Hydromorphic Tropical Ferruginous Soil; WITFS: Washing and Idurate Tropical Ferruginous Soil; FWTFS: Few Washing Tropical Ferruginous Soil; ITFS: Impoverished Tropical Ferruginous Soil; FDS: Few Developed Soil.The means with different letters are significantly different with probability level of 5% according to Student-Newman-Keuls test.
population (5.54 × 10 8 CFU/g of soil).The lowest mesophilic microflora charge was recorded with the Impoverished Tropical Ferruginous Soil (ITFS, 0.99 × 10 8 CFU/g of soil) and Few Developed Soil (FDS, 0.95 × 10 8 CFU/g of soil) (Figure 2(b)).3.3.Density of Rhizobacteria Isolated.The density of isolated rhizobacteria according to agroecological zone and soil type is shown in Figure 3.The rhizospheres of agroecological zone II contain the highest density of Bacillus sp.(5.70 × 10 6 CFU/g of soil) and Pseudomonas sp.(3.13 × 10 6 CFU/g of soil).On the contrary, samples of the agroecological zone IV contain the lowest population of Bacillus sp.(3.47 × 10 6 CFU/g of soil) and Pseudomonas sp.(1.40 × 10 6 CFU/g of soil) . Each agroecological zone contains at least two soil types.The soil types encountered are Washing and Concretion Tropical Ferruginous Soil, Washing and No Concretion Tropical Ferruginous Soil, Washing and Hydromorphic Tropical Ferruginous Soil, Washing and Idurate Tropical Ferruginous Soil, Few Washing Tropical Ferruginous Soil, Impoverished Tropical Ferruginous Soil, and Few Developed Soil.This result is different to those obtained in the southern Benin by Adjanohoun et al.

Table 2 :
Morphological and biochemical characteristics of rhizobacteria isolated from samples collected in the central and northern Benin.

Table 3 :
Microbial production of NH 3 and HCN.