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

Authors: Nadège A. Agbodjato; Pacôme A. Noumavo; Farid Baba-Moussa; Hafiz A. Salami; Haziz Sina; Alphonse Sèzan; Honoré Bankolé; Adolphe Adjanohoun; Lamine Baba-Moussa
Journal: Applied and Environmental Soil Science
 (2015)

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## Abstract

Our study aims to characterize Plant Growth Promoting Rhizobacteria (PGPR) isolated from maize roots in five agroecological zones of central and northern Benin. Sixty samples were collected at the rate of four samples per village and three villages per agroecological zone. Rhizobacteria strains were isolated from these samples and biochemically characterized. These strains were analyzed for some of their PGPR traits like ammonia production and hydrogen cyanide following conventional methods. Microbiological investigation of these samples has shown that maize rhizospheres in central and northern Benin contain a high diversity of microorganisms. A total of nine species of maize Plant Growth Promoting Rhizobacteria were identified. Those PGPR include fiveBacillus species (B. polymyxa, B. pantothenticus, B. anthracis, B. thuringiensis, and B. circulans), three Pseudomonas species (P. cichorii, P. putida, and P. syringae), and Serratia marcescens. The microbial diversity does not depend on the soil types. The microbial density, generally high, varies according to both soil types and agroecological zones. All Serratia strains (100%) have produced ammonia, whereas 80% of Bacillus and 77.77% of Pseudomonas produced this metabolite. The hydrogen cyanide was produced by all isolates (100%) independent of their genus. These results suggest the possibility to use these rhizobacteria as biological fertilizers to increase maize production.


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## Body

## 1. 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 fluorescentPseudomonas 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 genusPseudomonas,Azospirillum,Azotobacter,Klebsiella,Enterobacter,Alcaligenes,Arthrobacter,Burkholderia,Bacillus, andSerratia were reported to be PGPR [8].Yazdani et al. [9] asserted that the PGPR use can reduce the application of phosphorus to 50% without affecting the maize (Zea mays L.) seed yield. Several authors reported the increase of maize yield [10, 11], Tea [12], soybeans [13], alfalfa [14], wheat [15], and onion [16] simply by PGPR inoculation.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.


## 2. Materials and Methods

### 2.1. 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 (Figure1). 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.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.


### 2.2. 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.


### 2.3. 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. andSerratia 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].


### 2.4. 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, 21–23].


### 2.5. 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.


#### 2.5.2. Ammonia Production

To research the production of ammonia, each identified rhizobacteria strain was grown in peptone broth (10 mL) and incubated at36±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].


### 2.6. Statistical Analysis

Microsoft Office Excel 2007 had been used to create data base. The different parameters evaluated were submitted to Analysis of Variance (ANOVA) 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.


## 2.1. 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 (Figure1). 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.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.


## 2.2. 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.


## 2.3. 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. andSerratia 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].


## 2.4. 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, 21–23].


## 2.5. 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.


### 2.5.2. Ammonia Production

To research the production of ammonia, each identified rhizobacteria strain was grown in peptone broth (10 mL) and incubated at36±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].


## 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.


## 2.5.2. Ammonia Production

To research the production of ammonia, each identified rhizobacteria strain was grown in peptone broth (10 mL) and incubated at36±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].


## 2.6. Statistical Analysis

Microsoft Office Excel 2007 had been used to create data base. The different parameters evaluated were submitted to Analysis of Variance (ANOVA) 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.


## 3. Results

### 3.1. Agroecological Characteristics of the Villages Surveyed

Table1 shows agroecological characteristics of villages surveyed by agroecological zone. The Sudanese climate with one rainy season predominates in the five agroecological zones. Annual pluviometry varies from 700 mm/year (zone I) to 1400 mm/year (zone V). Pluviometry increases when we come from northern to southern Benin. The study area is characterized by 7 different types of soil. These soil types are the Washing and Concretion Tropical Ferruginous Soil (WCTFS), the Washing and No Concretion Tropical 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. Several other crops were grown by farmers apart from the maize.Table 1
Agroecological characteristics of villages surveyed.
Agroecological zone
Climate
Annual pluviometry (mm)
Village
Type of soil
Other crops grown
I :Far North Benin
Sudano-Sahelian with one rainy season
700 to 900/years
Tomboutou
Washing and No Concretion Tropical Ferruginous Soil
Rice,   sorghum,   small millet
Monsey
Molla centre
Washing and Hydromorphic Tropical Ferruginous Soil
II :Cotton zone of North Benin
Sudanese with one rainy season
800 to 900/years
Bensékou
Washing and Idurate Tropical Ferruginous Soil
Millet
Sonsoro
Arbonga
Washing and Hydromorphic Tropical Ferruginous Soil
III:food-producing zone of South Benin
Sudanese with one rainy season
900 to 1300/years
InaN’dali Sakarou
Washing and Concretion Tropical Ferruginous Soil
Sorghum,  cotton,   bean,   cassava
IV:West Atacora zone
Sudanese with one rainy season
800 to 1300/years
Bariénou
Few Washing Tropical Ferruginous Soil
Sorghum,   bean,  bambara groundnut,  cassava,  groundnut,  yam
Koua
Bagri
Few Developed Soil
V:Cotton Zone of Central Benin
Sudano-Guinean with two rainy seasons
1100 to 1400/years
Achakpa
Impoverished Tropical Ferruginous Soil
Yam,   cassava,  pimento
Manigri Oké
Washing and No Concretion Tropical Ferruginous Soil
Manigri Ikanni


### 3.2. Density of Mesophilic Microflora

The agroecological zones and soil types investigated in this study present a varied microbial density. The rhizosphere of agroecological zone I contains mesophilic microflora (6.25 × 108 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 × 108 CFU/g of soil). Our data display that the Washing and No Concretion Tropical Ferruginous Soil (WNCTFS) contains the highest mesophilic microbial population (5.54 × 108 CFU/g of soil). The lowest mesophilic microflora charge was recorded with the Impoverished Tropical Ferruginous Soil (ITFS, 0.99 × 108 CFU/g of soil) and Few Developed Soil (FDS, 0.95 × 108 CFU/g of soil) (Figure 2(b)).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.
(a)
(b)


### 3.3. Density of Rhizobacteria Isolated

The density of isolated rhizobacteria according to agroecological zone and soil type is shown in Figure3. The rhizospheres of agroecological zone II contain the highest density ofBacillus sp. (5.70 × 106 CFU/g of soil) andPseudomonas sp. (3.13 × 106 CFU/g of soil). On the contrary, samples of the agroecological zone IV contain the lowest population ofBacillus sp. (3.47 × 106 CFU/g of soil) andPseudomonas sp. (1.40 × 106 CFU/g of soil).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 × 106 CFU/g of soil).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.
(a)
(b)The density of rhizobacteria strains varies also from a soil type to another (Figure3(b)). The Washing and Idurate Tropical Ferruginous Soil (WITFS) contains the largest population ofBacillus sp. (6.03 × 106 CFU/g of soil) whereas the Few Developed Soil (FDS) contains the least population ofBacillus spp. (3.2 × 106 CFU/g of soil). Impoverished Tropical Ferruginous Soil (ITFS) and Few Washing Tropical Ferruginous Soil (FWTFS) contain, respectively, the large populations ofPseudomonas sp. andSerratia sp. In general, the density ofBacillus sp. is higher thanPseudomonas sp. andSerratia sp.Serratia sp. is the least abundant in the majority of soils.


### 3.4. Rhizobacteria Species Identified

Microbial investigation of samples collected from the 5 agroecological zones showed the presence of several rhizobacterial species. FiveBacillus species (B. polymyxa,B. pantothenticus,B. anthracis,B. thuringiensis, andB. circulans), 3Pseudomonas species (P. cichorii,P putida, andP. syringae), andSerratia marcescens were identified. The morphological and biochemical characteristics of these rhizobacteria are shown in Table 2.Table 2
Morphological and biochemical characteristics of rhizobacteria isolated from samples collected in the central and northern Benin.
Test
Bacillus
Pseudomonas
Serratia
polymyxa
pantothenticus
anthracis
thuringiensis
circulans
cichorii
putida
syringae
marcescens
Bacteria shape
Rod
Rod
Rod
Rod
Rod
Rod
Rod
Rod
Rod
Gram reaction
+
+
+
+
+
−
−
−
−
Catalase production
+
+
+
+
+
+
+
+
+
Spore position
Central
Terminal
Central
Central
Central
nd
nd
nd
nd
Growth on anaerobic condition
+
+
+
+
+
nd
nd
nd
nd
Acid from glucose
+
+
+
+
+
nd
nd
nd
nd
Gas from glucose
+
−
−
−
−
nd
nd
nd
nd
Mobility
+
+
−
+
−
+
+
+
+
Delay on glucose
−
−
−
−
−
nd
nd
nd
nd
Voges-Proskauer
+
−
+
+
−
nd
nd
nd
nd
Indole production
−
−
−
−
−
nd
nd
nd
−
Citrate utilization
−
−
−
−
−
nd
nd
nd
+
Mannitol utilization
nd
nd
nd
nd
nd
+
+
+
+
Starch hydrolysis
+
+
+
+
+
nd
nd
nd
nd
Casein hydrolysis
+
+
+
+
−
nd
nd
nd
+
Gelatin liquefaction
+
+
+
+
+
nd
nd
nd
+
Lecithin hydrolysis
+
−
+
+
−
+
−
−
nd
Urease hydrolysis
−
−
−
−
−
nd
nd
nd
−
DNAse activity
nd
nd
nd
nd
nd
nd
nd
nd
+
Nitrate reduction
+
+
+
+
−
nd
nd
nd
nd
Growth at 45°C
+
+
−
+
+
nd
nd
nd
nd
Growth at 55°C
−
−
−
−
−
nd
nd
nd
nd
Growth at 65°C
−
−
−
−
−
nd
nd
nd
nd
Fluorescence à 360 nm
nd
nd
nd
nd
nd
+
+
+
nd
Colony on nutrient agar
nd
nd
nd
nd
nd
Whitish-Shiny
Whit-Shiny
Whitish-Shiny
nd
Oxidase production
nd
nd
nd
nd
nd
+
+
−
nd
Glucose fermentation
nd
nd
nd
nd
nd
−
−
−
+
Lactose fermentation
nd
nd
nd
nd
nd
−
−
−
−
Gas production
nd
nd
nd
nd
nd
−
−
−
−
H2S production
nd
nd
nd
nd
nd
−
−
−
−
Growth on Cétrimide (37°C)
nd
nd
nd
nd
nd
−
+
+
nd
Growth on Cétrimide (42°C)
nd
nd
nd
nd
nd
−
+
−
nd
Growth on MacConkey
nd
nd
nd
nd
nd
nd
nd
nd
+
Pigment production
nd
nd
nd
nd
nd
nd
nd
nd
Red
+ = positive; − = negative; nd = no determined.


### 3.5. Ammonia and Hydrogen Cyanide Production by Rhizobacteria

The production of ammonia (NH3) 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 allSerratia strains produce it against 80% ofBacillus sp. and 77.77% ofPseudomonas sp. (Table 3).Table 3
Microbial production of NH3 and HCN.
Rhizobacteria
Production of NH3 (%)
Production of HCN(%)
Bacillus sp.
80
100
Pseudomonas sp.
77, 77
100
Serratia sp.
100
100


## 3.1. Agroecological Characteristics of the Villages Surveyed

Table1 shows agroecological characteristics of villages surveyed by agroecological zone. The Sudanese climate with one rainy season predominates in the five agroecological zones. Annual pluviometry varies from 700 mm/year (zone I) to 1400 mm/year (zone V). Pluviometry increases when we come from northern to southern Benin. The study area is characterized by 7 different types of soil. These soil types are the Washing and Concretion Tropical Ferruginous Soil (WCTFS), the Washing and No Concretion Tropical 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. Several other crops were grown by farmers apart from the maize.Table 1
Agroecological characteristics of villages surveyed.
Agroecological zone
Climate
Annual pluviometry (mm)
Village
Type of soil
Other crops grown
I :Far North Benin
Sudano-Sahelian with one rainy season
700 to 900/years
Tomboutou
Washing and No Concretion Tropical Ferruginous Soil
Rice,   sorghum,   small millet
Monsey
Molla centre
Washing and Hydromorphic Tropical Ferruginous Soil
II :Cotton zone of North Benin
Sudanese with one rainy season
800 to 900/years
Bensékou
Washing and Idurate Tropical Ferruginous Soil
Millet
Sonsoro
Arbonga
Washing and Hydromorphic Tropical Ferruginous Soil
III:food-producing zone of South Benin
Sudanese with one rainy season
900 to 1300/years
InaN’dali Sakarou
Washing and Concretion Tropical Ferruginous Soil
Sorghum,  cotton,   bean,   cassava
IV:West Atacora zone
Sudanese with one rainy season
800 to 1300/years
Bariénou
Few Washing Tropical Ferruginous Soil
Sorghum,   bean,  bambara groundnut,  cassava,  groundnut,  yam
Koua
Bagri
Few Developed Soil
V:Cotton Zone of Central Benin
Sudano-Guinean with two rainy seasons
1100 to 1400/years
Achakpa
Impoverished Tropical Ferruginous Soil
Yam,   cassava,  pimento
Manigri Oké
Washing and No Concretion Tropical Ferruginous Soil
Manigri Ikanni


## 3.2. Density of Mesophilic Microflora

The agroecological zones and soil types investigated in this study present a varied microbial density. The rhizosphere of agroecological zone I contains mesophilic microflora (6.25 × 108 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 × 108 CFU/g of soil). Our data display that the Washing and No Concretion Tropical Ferruginous Soil (WNCTFS) contains the highest mesophilic microbial population (5.54 × 108 CFU/g of soil). The lowest mesophilic microflora charge was recorded with the Impoverished Tropical Ferruginous Soil (ITFS, 0.99 × 108 CFU/g of soil) and Few Developed Soil (FDS, 0.95 × 108 CFU/g of soil) (Figure 2(b)).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.
(a)
(b)


## 3.3. Density of Rhizobacteria Isolated

The density of isolated rhizobacteria according to agroecological zone and soil type is shown in Figure3. The rhizospheres of agroecological zone II contain the highest density ofBacillus sp. (5.70 × 106 CFU/g of soil) andPseudomonas sp. (3.13 × 106 CFU/g of soil). On the contrary, samples of the agroecological zone IV contain the lowest population ofBacillus sp. (3.47 × 106 CFU/g of soil) andPseudomonas sp. (1.40 × 106 CFU/g of soil).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 × 106 CFU/g of soil).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.
(a)
(b)The density of rhizobacteria strains varies also from a soil type to another (Figure3(b)). The Washing and Idurate Tropical Ferruginous Soil (WITFS) contains the largest population ofBacillus sp. (6.03 × 106 CFU/g of soil) whereas the Few Developed Soil (FDS) contains the least population ofBacillus spp. (3.2 × 106 CFU/g of soil). Impoverished Tropical Ferruginous Soil (ITFS) and Few Washing Tropical Ferruginous Soil (FWTFS) contain, respectively, the large populations ofPseudomonas sp. andSerratia sp. In general, the density ofBacillus sp. is higher thanPseudomonas sp. andSerratia sp.Serratia sp. is the least abundant in the majority of soils.


## 3.4. Rhizobacteria Species Identified

Microbial investigation of samples collected from the 5 agroecological zones showed the presence of several rhizobacterial species. FiveBacillus species (B. polymyxa,B. pantothenticus,B. anthracis,B. thuringiensis, andB. circulans), 3Pseudomonas species (P. cichorii,P putida, andP. syringae), andSerratia marcescens were identified. The morphological and biochemical characteristics of these rhizobacteria are shown in Table 2.Table 2
Morphological and biochemical characteristics of rhizobacteria isolated from samples collected in the central and northern Benin.
Test
Bacillus
Pseudomonas
Serratia
polymyxa
pantothenticus
anthracis
thuringiensis
circulans
cichorii
putida
syringae
marcescens
Bacteria shape
Rod
Rod
Rod
Rod
Rod
Rod
Rod
Rod
Rod
Gram reaction
+
+
+
+
+
−
−
−
−
Catalase production
+
+
+
+
+
+
+
+
+
Spore position
Central
Terminal
Central
Central
Central
nd
nd
nd
nd
Growth on anaerobic condition
+
+
+
+
+
nd
nd
nd
nd
Acid from glucose
+
+
+
+
+
nd
nd
nd
nd
Gas from glucose
+
−
−
−
−
nd
nd
nd
nd
Mobility
+
+
−
+
−
+
+
+
+
Delay on glucose
−
−
−
−
−
nd
nd
nd
nd
Voges-Proskauer
+
−
+
+
−
nd
nd
nd
nd
Indole production
−
−
−
−
−
nd
nd
nd
−
Citrate utilization
−
−
−
−
−
nd
nd
nd
+
Mannitol utilization
nd
nd
nd
nd
nd
+
+
+
+
Starch hydrolysis
+
+
+
+
+
nd
nd
nd
nd
Casein hydrolysis
+
+
+
+
−
nd
nd
nd
+
Gelatin liquefaction
+
+
+
+
+
nd
nd
nd
+
Lecithin hydrolysis
+
−
+
+
−
+
−
−
nd
Urease hydrolysis
−
−
−
−
−
nd
nd
nd
−
DNAse activity
nd
nd
nd
nd
nd
nd
nd
nd
+
Nitrate reduction
+
+
+
+
−
nd
nd
nd
nd
Growth at 45°C
+
+
−
+
+
nd
nd
nd
nd
Growth at 55°C
−
−
−
−
−
nd
nd
nd
nd
Growth at 65°C
−
−
−
−
−
nd
nd
nd
nd
Fluorescence à 360 nm
nd
nd
nd
nd
nd
+
+
+
nd
Colony on nutrient agar
nd
nd
nd
nd
nd
Whitish-Shiny
Whit-Shiny
Whitish-Shiny
nd
Oxidase production
nd
nd
nd
nd
nd
+
+
−
nd
Glucose fermentation
nd
nd
nd
nd
nd
−
−
−
+
Lactose fermentation
nd
nd
nd
nd
nd
−
−
−
−
Gas production
nd
nd
nd
nd
nd
−
−
−
−
H2S production
nd
nd
nd
nd
nd
−
−
−
−
Growth on Cétrimide (37°C)
nd
nd
nd
nd
nd
−
+
+
nd
Growth on Cétrimide (42°C)
nd
nd
nd
nd
nd
−
+
−
nd
Growth on MacConkey
nd
nd
nd
nd
nd
nd
nd
nd
+
Pigment production
nd
nd
nd
nd
nd
nd
nd
nd
Red
+ = positive; − = negative; nd = no determined.


## 3.5. Ammonia and Hydrogen Cyanide Production by Rhizobacteria

The production of ammonia (NH3) 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 allSerratia strains produce it against 80% ofBacillus sp. and 77.77% ofPseudomonas sp. (Table 3).Table 3
Microbial production of NH3 and HCN.
Rhizobacteria
Production of NH3 (%)
Production of HCN(%)
Bacillus sp.
80
100
Pseudomonas sp.
77, 77
100
Serratia sp.
100
100


## 4. 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 Table1. 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. [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, we can think there are more crops associated with maize in northern and central Benin than in southern Benin. But it is important to indicate that Adjanohoun et al. [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 (Figure2(a)), but this difference is not significant (p>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 (p<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 108 and 109 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 104 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 (Figure3). The density difference between the soil types is significant forBacillus sp. (p>0.05) and highly significant (p<0.001) forPseudomonas sp. andSerratia sp. The microbial density varied from 3.2 to 6.03 × 106 CFU/g of soil (Pseudomonas sp.), 1.05 to 3.33 × 106 CFU/g of soil (Bacillus sp.), and 0.67 to 3.13 × 106 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 × 109 CFU/g and 1.1 to 2.1 × 109 CFU/g of soil forBacillus sp. andPseudomonas sp., respectively. In our study,Bacillus sp. population is most abundant thanPseudomonas sp. andSerratia sp. This remark was earlier done by Saharan and Nehra [8] when they asserted thatBacillus sp. is the most abundant genus in their studied rhizosphere. In addition, Garbeva et al. [32] had concluded that a majority of soil gram positive bacteria (95%) are member of the genusBacillus (B. mycoides,B.pumilus,B.megaterium,B.thuringiensis, andB.firmus, etc.) similar toPaenibacillus.Several rhizobacteria species were isolated, namely,B.polymyxa,B.pantothenticus,B.anthracis,B.thuringiensis,B.circulans,P.cichorii,P.putida,P.syringae, andSerratia marcescens (Table 2). In southern Benin, Adjanohoun et al. [26] isolated from maize rhizosphereB.coagulans,B.thuringiensis,B.pumilus,B.polymyxa,B.licheniformis,B.lentus,B.circulans,B.firmus,P.fluorescens,P.aeruginosa,P.putida,S.hygroscopicus,S. rimosus,S. fasciculatus, andA.lipoferum. These results still indicate the large microbial diversity of maize rhizosphere in Benin.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 theSerratia strains followed by 80% ofBacillus and 77.77% ofPseudomonas produced ammonia. These rates of ammonia production are lower than the 95% and 94% obtained, respectively, forBacillus sp. andPseudomonas sp. by Joseph et al. [31]. Likewise, allBacillus andPseudomonas 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 byBacillus sp. strains isolated from rice rhizosphere [34] and 40% of bacteria (Bacillus sp.,Pseudomonas sp.,Enterobacter sp.,Acinetobacter sp., andMicrococcus 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].


## 5. 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, andSerratia 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.



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*Source: 901656-2015-09-06.xml*