Characterization of Potential Plant Growth Promoting Rhizobacteria

Research ArticleCharacterization of Potential Plant GrowthPromoting Rhizobacteria Isolated from Maize (Zea mays L) inCentral and Northern Benin (West Africa)

Nadegravege A Agbodjato1 Pacocircme A Noumavo1 Farid Baba-Moussa2

Hafiz A Salami1 Haziz Sina1 Alphonse Segravezan3 Honoreacute Bankoleacute4

Adolphe Adjanohoun5 and Lamine Baba-Moussa1

1Laboratoire de Biologie et de Typage Moleculaire en Microbiologie Departement de Biochimie et de Biologie CellulaireFaculte des Sciences et Techniques Universite drsquoAbomey-Calavi 05 BP 1604 Cotonou Benin2Laboratoire de Microbiologie et de Technologie Alimentaire Faculte des Sciences et Techniques Universite drsquoAbomey-Calavi04 BP 1107 Cotonou Benin3Laboratoire de Biomembrane et de Signalisation Cellulaire Faculte des Sciences et Techniques Universite drsquoAbomey-Calavi01 BP526 Abomey-Calavi Benin4Section Hygiene des Eaux et Aliments Laboratoire National de Sante Publique 01 BP 418 Cotonou Benin5Centre de Recherches Agricoles Sud Institut National des Recherches Agricoles du Benin BP 03 Attogon Benin

Correspondence should be addressed to Lamine Baba-Moussa laminesaidyahoofr

Received 5 April 2015 Accepted 21 July 2015

Academic Editor Marco Trevisan

Copyright copy 2015 Nadege A Agbodjato et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Our study aims to characterize Plant Growth Promoting Rhizobacteria (PGPR) isolated from maize roots in five agroecologicalzones of central and northern Benin Sixty samples were collected at the rate of four samples per village and three villagesper agroecological zone Rhizobacteria strains were isolated from these samples and biochemically characterized These strainswere analyzed for some of their PGPR traits like ammonia production and hydrogen cyanide following conventional methodsMicrobiological investigation of these samples has shown that maize rhizospheres in central and northern Benin contain a highdiversity of microorganisms A total of nine species of maize Plant Growth Promoting Rhizobacteria were identified Those PGPRinclude five Bacillus species (B polymyxa B pantothenticus B anthracis B thuringiensis and B circulans) three Pseudomonasspecies (P cichorii P putida and P syringae) and Serratia marcescens The microbial diversity does not depend on the soil typesThe 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 7777 of Pseudomonas produced this metabolite The hydrogen cyanidewas produced by all isolates (100) independent of their genus These results suggest the possibility to use these rhizobacteria asbiological fertilizers to increase maize production

1 Introduction

The first aim of agriculture was to ensure survival by produc-ing the necessary for feeding It was subsistence farming Butnowadays due to continued and worrying growth of worldpopulation this primary objective of agriculture changedcompletely Indeed the world population is estimated around7 billion people and may reach 8 billion by 2020 [1] So it is

urgent to considerably increase the agricultural productionto reply to the strong food demand to reduce the risk ofmalnutrition and the increasing of poverty

Therefore the new cereal varieties of high yield weredeveloped In addition agrochemical products such as chem-ical fertilizers herbicides fungicides and insecticides werecurrently improperly and excessively used in order to increasecrop yield The direct consequence of these agrochemical

Hindawi Publishing CorporationApplied and Environmental Soil ScienceVolume 2015 Article ID 901656 9 pageshttpdxdoiorg1011552015901656

2 Applied and Environmental Soil Science

products use is the environment pollution through groundwater and crop products contamination by heavy metals thatare contained in these agricultural inputsThese heavymetalsare known to be a public health problems because transferredto humans they are involved in the cancer occurrence[2] Apart from medical damages other consequences inagricultural area such as natural ecological nutrient cyclinginterruption and soil biological communities destruction arefrequently reported [3] Regarding the damages caused by theexcessive use of agrochemical products other research pathsare explored worldwide Among the explored paths the useof microorganism currently called Plant Growth PromotingRhizobacteria (PGPR) is in pole position

Indeed the PGPR is a group of bacteria capable ofcolonizing actively plant roots system and improving theirgrowth and yield [4] The expression ldquoPGPRrdquo was firstlyproposed by Kloepper et al [5] and was used especially forthe fluorescent Pseudomonas involved in biological controlof pathogens and the improvement of plant growth LaterKapulnik et al [6] extended this expression to rhizobacteriacapable of promoting directly the plant growth Nowadaysthis expression is used to refer to all bacteria living in therhizosphere (around roots) improving plant growth by oneor several mechanisms [7] A large range of species belongingto the genus Pseudomonas Azospirillum Azotobacter Kleb-siella Enterobacter Alcaligenes Arthrobacter BurkholderiaBacillus and Serratia were reported to be PGPR [8]

Yazdani et al [9] asserted that the PGPR use can reducethe application of phosphorus to 50 without affecting themaize (Zea mays L) seed yield Several authors reported theincrease 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 andidentify the potential PGPR frommaize (most cultivated andconsumed cereal in Benin) rhizosphere in the central andnorthern Benin The medium-dated objective of this studyis to propose for farmers the biological fertilizers based onnative PGPR for increasing maize production

2 Materials and Methods

21 Geographical Characterization of Study Area This studywas carried in five agroecological zones (I II III IV andV) located in the central and northern Benin West Africa(Figure 1) Indeed Benin is localized in West Africa (southof Sahara) in the tropical zone between Equator and Tropicof Cancer precisely between the parallel 6∘ 301015840 and 12∘ 301015840 ofnorth latitude and meridians 1∘ and 30∘ 401015840 of east longitude

22 Collection of Rhizospheric Samples Three (3) villageswere selected by agroecological zone and four fields werechosen in each village Three maize plants distanced at least10 meters were dug up in each fieldTheir roots were cut withsoil adheres and mixed in a bucket Three hundred gramsof this mixture was packed in a sterile stomacher bag andlabeled correctly to form the sample of the field A total of 60samples were collected and immediately transported at 4∘Cto the laboratory for further analysis Once they are at the

laboratory the microbiological screening was immediatelyrealized or samples were kept at 4∘C until screening

Several other parameters (climate soil type annual plu-viometry and other crops grown except for maize) of thesampling sites were collected during the sampling

23 Isolation of Rhizobacteria According to Speck [17]method 10 g of each sample was mixed into Erlenmeyerflask containing 90mL of tryptone salt The mixture wasvigorously shaken for about 30 s to obtain 10minus1 dilutionThe previous dilution (1mL) was transferred into 9mL oftryptone salt to obtain 10minus2 dilution This operation wasrepeated until obtaining 10minus8 dilution Each dilution (01mL)was streaked on different specific isolationmediaThe aerobicmesophilic flora was enumerated on Plate Count Agar asrecommended by the French standard V08-051 Bacillussp and Serratia sp were isolated on nutrient agar afterincubation at 37∘C for 24 h and 30∘C for 48 h respectively[18 19] Pseudomonas sp was isolated on King A and KingB agar after incubation at 30∘C for 72 h [20]

24 Identification of Rhizobacteria The identification of iso-lated rhizobacteria consisted firstly in macroscopic (colonymorphology pigmentation etc) and microscopic (gramreaction mobility cell shape spores position etc) obser-vations This first identification was followed by severalbiochemical and enzymatic tests The performed tests areproduction of oxidase catalase indole urease and hydrogensulfide respiratory type acid and gas production on glucoseagar citrate and nitrate utilization hydrolysis of starchcaseinmannitol gelatin and lecithin fermentation of glucoseand lactose growth on MacConkey and Cetrimide agarVoges-Proskaur test growth at 42 45 55 and 65∘C andfluorescence at 360∘C [18 21ndash23]

25 Plant Growth Promoting Properties

251 Hydrogen Cyanide Production All isolated rhizobacte-ria were screened for hydrogen cyanide production followingthe method described by Lorck [24] Each rhizobacteriumwas streaked on nutrient agar medium added with glycine(44 gL) The agar was covered with a Whatman number1 filter paper previously soaked in a specific solution (05picric acid and 2 sodium carbonate wv) Plates were sealedwith parafilm paper and incubated at 36plusmn2∘C for 4 daysTheappearance of orange or red color indicates the production ofhydrogen cyanide

252 Ammonia Production To research the production ofammonia each identified rhizobacteria strain was grown inpeptone broth (10mL) and incubated at 36 plusmn 2∘C for 48 to72 h After incubation 05mL of Nesslerrsquos reagent was addedto bacterial suspension The development of brown to yellowcolor indicated ammonia production [25]

26 Statistical Analysis Microsoft Office Excel 2007 hadbeen used to create data base The different parametersevaluated were submitted to Analysis of Variance (ANOVA)

Applied and Environmental Soil Science 3

Kandi

Nrsquodali

Djougou

Bassila

Karimama

Banikoara

Malanville

Natitingou

Ina

Koua

Bagri

Monsey

NrsquodaliSakarou

Arbonga

Achakpa

Molla centre

Manigri Ikanni

Sonsoro

TomboutouBurkina-Faso

N

W

S

E

Togo

Niger

Atlantic Ocean

Nigeria

District capitalStudy villagesAEZ borderAEZ I

AEZ IIAEZ IIIAEZ IVAEZ V

Manigri Okeacute

Barieacutenou

0 25 50 100(km)

7∘30

9984000998400998400N

9∘09984000998400998400N

10∘30

9984000998400998400N

12∘09984000998400998400N

7∘30

9984000998400998400N

9∘09984000998400998400N

10∘30

9984000998400998400N

12∘09984000998400998400N

0∘36

9984000998400998400E 1

∘39

9984000998400998400E 2

∘42

9984000998400998400E 3

∘45

9984000998400998400E

0∘36

9984000998400998400E 1

∘39

9984000998400998400E 2

∘42

9984000998400998400E 3

∘45

9984000998400998400E

Source fond topographique IGN 1992

Realization LaCartoDGATFLASHUAC 2015

Benseacutekou

Sav e

Bembereke

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

at probability level of 5 following a mean separation(Student-Newman-Keuls test) by Statistical Analysis System(SAS) software Version 81 In this model soil types andagroecological zones were considered as a fixed factor whilereplicates were considered as a random factor

3 Results

31 Agroecological Characteristics of the Villages SurveyedTable 1 shows agroecological characteristics of villages sur-veyed by agroecological zone The Sudanese climate with


Table 1 Agroecological characteristics of villages surveyed

Agroecologicalzone Climate Annual

pluviometry (mm) Village Type of soil Other crops grown

I Far North Benin

Sudano-Sahelianwith one rainyseason

700 to 900years

Tomboutou Washing and No ConcretionTropical Ferruginous Soil

Rice sorghum small milletMonsey

Molla centre Washing and HydromorphicTropical Ferruginous Soil

II Cotton zone ofNorth Benin

Sudanese with onerainy season 800 to 900years

Bensekou Washing and Idurate TropicalFerruginous Soil

MilletSonsoro

Arbonga Washing and HydromorphicTropical Ferruginous Soil

IIIfood-producingzone ofSouth Benin


InaNrsquodaliSakarou

Washing and ConcretionTropical Ferruginous Soil

Sorghum cotton beancassava

IVWestAtacora zone


Barienou FewWashing TropicalFerruginous Soil

Sorghum bean bambaragroundnut cassavagroundnut yam

KouaBagri Few Developed Soil

VCotton Zone ofCentral Benin

Sudano-Guineanwith two rainyseasons

1100 to 1400yearsAchakpa Impoverished Tropical

Ferruginous SoilYam cassava pimentoManigri Oke Washing and No Concretion

Tropical Ferruginous SoilManigri Ikanni

A

A

A

A

A

AEZ I AEZ II AEZ III AEZ IV AEZ VAgroecological zones (AEZ)

0123456789

(108

CFU

g o

f soi

l)

(a)

BC

A

ABAB

BC

C C

CWTFS WNCTFS WHTFS WITFS FWTFS ITFS FDSTypes of soil

012345678

(108

CFU

g o

f soi

l)

(b)

Figure 2 Distribution of aerobic mesophilic flora according to (a) AEZ and (b) type of soil WCTFS Washing and Concretion TropicalFerruginous Soil WNCTFS Washing and No Concretion Tropical Ferruginous Soil WHTFS Washing and Hydromorphic TropicalFerruginous Soil WITFS Washing and Idurate Tropical Ferruginous Soil FWTFS Few Washing Tropical Ferruginous Soil ITFSImpoverished Tropical Ferruginous Soil FDS FewDeveloped SoilThemeans with different letters are significantly different with probabilitylevel of 5 according to Student-Newman-Keuls test

one rainy season predominates in the five agroecologicalzones Annual pluviometry varies from 700mmyear (zoneI) to 1400mmyear (zone V) Pluviometry increases whenwe come from northern to southern Benin The study areais characterized by 7 different types of soil These soiltypes are the Washing and Concretion Tropical FerruginousSoil (WCTFS) the Washing and No Concretion TropicalFerruginous Soil (WNCTFS) the Washing and Hydromor-phic Tropical Ferruginous Soil (WHTFS) the Washing andIdurate Tropical Ferruginous Soil (WITFS) the Few Wash-ing Tropical Ferruginous Soil (FWTFS) the ImpoverishedTropical Ferruginous Soil (ITFS) and the FewDeveloped Soil(FDS) Except for zone III all the other agroecological zones

contain at least two different types of soil Several other cropswere grown by farmers apart from the maize

32 Density of Mesophilic Microflora The agroecologicalzones and soil types investigated in this study present a variedmicrobial density The rhizosphere of agroecological zone Icontains mesophilic microflora (625 times 108 CFUg of soil)clearly abundant compared to the other agroecological zones(Figure 2(a)) The zone IV is the least loaded in mesophilicmicroflora (140 times 108 CFUg of soil) Our data displaythat the Washing and No Concretion Tropical FerruginousSoil (WNCTFS) contains the highest mesophilic microbial


ABA

BB

BA

A AA

A

B BC

A

BC


01234567

(106

CFU

g o

f soi

l)

Bacillus spPseudomonas sp

Serratia sp

(a)

BC B BCA

BCBC

CA A A A

B

AA

B BB B

A

B

A



Serratia sp

01234567

(106

CFU

g o

f soi

l)

(b)

Figure 3 Distribution of rhizobacteria density according to (a) AEZ and (b) type of soil WCTFS Washing and Concretion TropicalFerruginous Soil WNCTFS Washing and No Concretion Tropical Ferruginous Soil WHTFS Washing and Hydromorphic TropicalFerruginous Soil WITFS Washing and Idurate Tropical Ferruginous Soil FWTFS Few Washing Tropical Ferruginous Soil ITFSImpoverished Tropical Ferruginous Soil FDS FewDeveloped SoilThemeans with different letters are significantly different with probabilitylevel of 5 according to Student-Newman-Keuls test

population (554 times 108 CFUg of soil) The lowest mesophilicmicroflora charge was recorded with the Impoverished Trop-ical Ferruginous Soil (ITFS 099times 108 CFUg of soil) and FewDeveloped Soil (FDS 095 times 108 CFUg of soil) (Figure 2(b))

33 Density of Rhizobacteria Isolated The density of isolatedrhizobacteria according to agroecological zone and soil typeis shown in Figure 3The rhizospheres of agroecological zoneII contain the highest density of Bacillus sp (570times 106 CFUgof soil) andPseudomonas sp (313times 106 CFUg of soil) On thecontrary samples of the agroecological zone IV contain thelowest population ofBacillus sp (347times 106 CFUg of soil) andPseudomonas sp (140 times 106 CFUg of soil) Serratia sp is notfound in rhizosphere of agroecological zone I (Figure 3(a))but it is abundant in rhizosphere of agroecological zone IV(312 times 106 CFUg of soil)

The density of rhizobacteria strains varies also from asoil type to another (Figure 3(b)) The Washing and IdurateTropical Ferruginous Soil (WITFS) contains the largest pop-ulation of Bacillus sp (603 times 106 CFUg of soil) whereasthe Few Developed Soil (FDS) contains the least populationof Bacillus spp (32 times 106 CFUg of soil) ImpoverishedTropical Ferruginous Soil (ITFS) and Few Washing TropicalFerruginous Soil (FWTFS) contain respectively the largepopulations of Pseudomonas sp and Serratia sp In generalthe density of Bacillus sp is higher than Pseudomonas sp andSerratia sp Serratia sp is the least abundant in the majorityof soils

34 Rhizobacteria Species Identified Microbial investigationof samples collected from the 5 agroecological zones showedthe presence of several rhizobacterial species Five Bacillusspecies (B polymyxa B pantothenticus B anthracis Bthuringiensis and B circulans) 3 Pseudomonas species (Pcichorii P putida and P syringae) and Serratia marcescenswere identified The morphological and biochemical charac-teristics of these rhizobacteria are shown in Table 2

35 Ammonia andHydrogenCyanide Production byRhizobac-teria The production of ammonia (NH

3) and hydrogen

cyanide (HCN) by rhizobacteria isolated from soil samplescollected in the northern and central Benin is shown inTable 3 Our data suggested that all the rhizobacteria strainsproduce hydrogen cyanide Concerning the production ofammonia it was observed that all Serratia strains produceit against 80 of Bacillus sp and 7777 of Pseudomonas sp(Table 3)

4 Discussion

Several studies have reported the benefit of seeds inoculationby Plant Growth Promoting Rhizobacteria This growthpromoting effect is influenced by biotic and abiotic factorsincluding bacterial species and the soil types It is in thiscontext that this prospective study was realized in prelude ofthe promotion of microbial biofertilizers based on native rhi-zobacteriaThe agroecological characteristics of sites samplescollected were presented in Table 1 Each agroecological zonecontains at least two soil typesThe soil types encountered areWashing and Concretion Tropical Ferruginous Soil Washingand No Concretion Tropical Ferruginous Soil Washingand Hydromorphic Tropical Ferruginous Soil Washing andIdurate Tropical Ferruginous Soil Few Washing TropicalFerruginous Soil Impoverished Tropical Ferruginous Soiland Few Developed Soil This result is different to thoseobtained in the southern Benin by Adjanohoun et al [26]Indeed our result reflects the large soils diversity in Benin asAdjanohoun et al [26] reported other types of soil such asVertisols Degraded Bar Land and No Degraded Bar Land

Apart from maize many other crops are growing indifferent villages surveyed in this study These crops are ricesorghum small millet millet cotton bean cowpea cassavabambara groundnut yam and pimento In southern Beninexcept maize Adjanohoun et al [26] had identified cottongroundnut sweet potato cowpea and cassavaThese cultureswere mostly found in northern and central Benin Firstly


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

Test Bacillus Pseudomonas Serratiapolymyxa pantothenticus anthracis thuringiensis circulans cichorii putida syringae marcescens

Bacteria shape Rod Rod Rod Rod Rod Rod Rod Rod RodGram reaction + + + + + minus minus minus minus

Catalaseproduction + + + + + + + + +

Spore position Central Terminal Central Central Central nd nd nd ndGrowth onanaerobiccondition

+ + + + + nd nd nd nd

Acid fromglucose + + + + + nd nd nd nd

Gas fromglucose + minus minus minus minus nd nd nd nd

Mobility + + minus + minus + + + +Delay onglucose minus minus minus minus minus nd nd nd nd

Voges-Proskauer + minus + + minus nd nd nd nd

Indoleproduction minus minus minus minus minus nd nd nd minus

Citrateutilization minus minus minus minus minus nd nd nd +

Mannitolutilization nd nd nd nd nd + + + +

Starchhydrolysis + + + + + nd nd nd nd

Caseinhydrolysis + + + + minus nd nd nd +

Gelatinliquefaction + + + + + nd nd nd +

Lecithinhydrolysis + minus + + minus + minus minus nd

Ureasehydrolysis minus minus minus minus minus nd nd nd minus

DNAse activity nd nd nd nd nd nd nd nd +Nitratereduction + + + + minus nd nd nd nd

Growth at 45∘C + + minus + + nd nd nd ndGrowth at 55∘C minus minus minus minus minus nd nd nd ndGrowth at 65∘C minus minus minus minus minus nd nd nd ndFluorescence a360 nm nd nd nd nd nd + + + nd

Colony onnutrient agar nd nd nd nd nd Whitish-Shiny Whit-Shiny Whitish-Shiny nd

Oxidaseproduction nd nd nd nd nd + + minus nd

Glucosefermentation nd nd nd nd nd minus minus minus +

Lactosefermentation nd nd nd nd nd minus minus minus minus

Gas production nd nd nd nd nd minus minus minus minus

H2

S production nd nd nd nd nd minus minus minus minus


Table 2 Continued


Growth onCetrimide(37∘C)

nd nd nd nd nd minus + + nd


nd nd nd nd nd minus + minus nd

Growth onMacConkey nd nd nd nd nd nd nd nd +

Pigmentproduction nd nd nd nd nd nd nd nd Red

+ = positive minus = negative nd = no determined

Table 3 Microbial production of NH3

and HCN

RhizobacteriaProduction of

NH3

()

Production ofHCN()

Bacillus sp 80 100Pseudomonas sp 77 77 100Serratia sp 100 100

we can think there are more crops associated with maizein northern and central Benin than in southern Benin Butit is important to indicate that Adjanohoun et al [26] hadnoted the crops sown in fields beforemaize sowing while thisstudy registered the crops sown in the study zone during thesampling

The soil aerobic mesophilic microflora has greatly variedfrom an agroecological zone to another (Figure 2(a)) but thisdifference is not significant (119901 gt 005) at probability level5 This result can be explained by the large variability ofmicrobial density in a same agroecological zone due to thesoil heterogeneity existing in each zone On the contrarythe density of aerobic mesophilic microflora has also greatlyvaried from a soil type to another but the difference ishighly significant (119901 lt 0001) at probability level 5(Figure 2(b)) The variability of microbial density is probablydue to physicochemical properties of the different soil typeswhich certainly impact the microbial activity in rhizosphereThese results are similar to those obtained by Adjanohounet al [26] in southern Benin when they observed a largedifference of microbial density between Vertisols DegradedBar Land and Not Degraded Bar Land Indeed Schoenbornet al [27] reported that rhizosphere contains a greatmicrobialpopulation between 108 and 109 CFUg of soilThis microbialabundance is explained by the richness of rhizosphere innutrients such as sugars amino acids organic acids hor-mones and other smallmolecules derived from root exudates[28] The microorganisms find in rhizosphere the energysubstrates required for their metabolism [29] Converselyin stressed ecosystem the microorganism population can beless than 104 CFUg of soil [30] So in spite of the differentenvironmental stress (climate change) themaize rhizosphere

in central and northern Benin still contains an abundantmicrobial population

The density of isolated rhizobacteria has varied accordingto the agroecological zone and the soil types (Figure 3) Thedensity difference between the soil types is significant forBacillus sp (119901 gt 005) and highly significant (119901 lt 0001)for Pseudomonas sp and Serratia sp The microbial densityvaried from 32 to 603 times 106 CFUg of soil (Pseudomonassp) 105 to 333 times 106 CFUg of soil (Bacillus sp) and 067to 313 times 106 CFUg of soil (Serratia spp) These microbialdensities are inferior to those obtained by Joseph et al [31]on chickpea (Cicer arietinum L) in India During their workthese authors counted about 05 to 21 times 109 CFUg and 11to 21 times 109 CFUg of soil for Bacillus sp and Pseudomonassp respectively In our study Bacillus sp population is mostabundant than Pseudomonas sp and Serratia sp This remarkwas earlier done by Saharan andNehra [8]when they assertedthat Bacillus sp is the most abundant genus in their studiedrhizosphere In addition Garbeva et al [32] had concludedthat a majority of soil gram positive bacteria (95) aremember of the genus Bacillus (B mycoides B pumilus Bmegaterium B thuringiensis and B firmus etc) similar toPaenibacillus

Several rhizobacteria species were isolated namely Bpolymyxa B pantothenticus B anthracis B thuringiensisB circulans P cichorii P putida P syringae and Serratiamarcescens (Table 2) In southern Benin Adjanohoun etal [26] isolated from maize rhizosphere B coagulans Bthuringiensis B pumilus B polymyxa B licheniformis Blentus B circulans B firmus P fluorescens P aeruginosaP putida S hygroscopicus S rimosus S fasciculatus andA lipoferum These results still indicate the large microbialdiversity of maize rhizosphere in Benin

In order to identify Plant Growth Promoting Rhizobacte-ria among isolated rhizobacteria in the central and northernBenin we have screened all the strains for ammonia andhydrogen cyanide production Thus all the Serratia strainsfollowed by 80 of Bacillus and 7777 of Pseudomonasproduced ammonia These rates of ammonia productionare lower than the 95 and 94 obtained respectivelyfor 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 alsoproduced ammonia Ammonia production is an importantcharacteristic of PGPR which indirectly influences plantsgrowth [33]

All strains produced hydrogen cyanide (100) Ourresults seem higher than the 75 of hydrogen cyanideproduction by Bacillus sp strains isolated from rice rhizo-sphere [34] and 40 of bacteria (Bacillus sp Pseudomonassp Enterobacter sp Acinetobacter sp and Micrococcus sp)isolated from the beans rhizosphere [35] in India Indeed thehydrogen cyanide is part of powerful antifungal compoundsproduced by PGPR and involved in pathogens biologicalcontrol [36]

5 Conclusion

The maize rhizospheres in central and northern Benincontain high diversity of microorganisms The bacterialdensity is generally high and varies according to both theagroecological zones and the type of soils Nine species ofpotentially maize plants growth promoting rhizobacteria (Bpolymyxa B pantothenticus B anthracis B thuringiensisB circulans P cichorii P putida P syringae and Serratiamarcescens) were identified during this study All isolates haveproduced hydrogen cyanide while 8666 of them producedammonia In perspective these rhizobacteria will be assessedto promote maize seeds germination and plant growth

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors acknowledge the International Foundation ofSciences (IFS research Grant no C5252-1) and West AfricaAgricultural Productivity Programme (WAAPPPPAAO) forfunding this work

References

[1] B RGlick ldquoPlant growth-promoting bacteriamechanisms andapplicationsrdquo Scientifica vol 2012 Article ID 963401 15 pages2012

[2] S-Y Koo and K-S Cho ldquoIsolation and characterization ofa plant growth-promoting rhizobacterium Serratia sp SY5rdquoJournal of Microbiology and Biotechnology vol 19 no 11 pp1431ndash1438 2009

[3] P Karuppiah and S Rajaram ldquoExploring the potential ofchromium reducingBacillus sp and there plant growth promot-ing activitiesrdquo Journal of Microbiology Research vol 1 no 1 pp17ndash23 2011

[4] S C Wu Z H Cao Z G Li K C Cheung and M H WongldquoEffects of biofertilizer containing N-fixer P and K solubilizersand AM fungi on maize growth a greenhouse trialrdquoGeodermavol 125 no 1-2 pp 155ndash166 2005

[5] J W Kloepper M N Schroth and T D Miller ldquoEffects ofrhizosphere colonization by plant growth promoting rhizobac-teria on potato plant development and yieldrdquo Ecology andEpidemiology vol 70 pp 1078ndash1082 1980

[6] Y Kapulnik Y Okon J Kigel I Nur and Y Henis ldquoEffectof temperature nitrogen fertilization and plant age on nitro-gen fixation by Setaria italica inoculated with Azospirillumbrasilense (strain cd)rdquo Plant Physiology vol 68 no 2 pp 340ndash343 1981

[7] B J Haghighi O Alizadeh and A H Firoozabadi ldquoThe roleof plant growth promoting rhizobacteria (PGPR) in sustainableagriculturerdquo Advances in Environmental Biology vol 5 no 10pp 3079ndash3083 2011

[8] B S Saharan and V Nehra ldquoPlant growth promoting rhizobac-teria a critical reviewrdquo Life Sciences and Medicine Research vol21 pp 1ndash30 2011

[9] M Yazdani M A Bahmanyar H Pirdashti and M A EsmailildquoEffect of phosphate solubilization microorganisms (PSM) andplant growth promoting rhizobacteria (PGPR) on yield andyield components of corn (Zea mays L)rdquo World Academy ofScience Engineering and Technology vol 49 pp 90ndash92 2009

[10] P A Noumavo E Kochoni Y O Didagbe et al ldquoEffect ofdifferent plant growth promoting rhizobacteria on maize seedgermination and seedling developmentrdquo American Journal ofPlant Sciences vol 4 no 5 pp 1013ndash1021 2013

[11] A Adjanohoun P Noumavo R Sikirou et al ldquoEffets desrhizobacteries PGPR sur le rendement et les teneurs enmacroelements du maıs sur sol ferralitique non degrade auSud-Beninrdquo International Journal of Biological and ChemicalSciences vol 6 pp 279ndash288 2012

[12] U Chakraborty B N Chakraborty P R Chowdhury C Tong-den and M Basnet ldquoInvestigation on plant growth promotingrhizobacteria of tea rhizosphererdquo in Proceedings of the 6th Inter-national Workshop on Plant Growth Promoting Rhizobacteria(PGPR rsquo06) pp 78ndash82 IISR Calicut India 2006

[13] MHAbd-Alla ldquoUse of organic phosphorus byRhizobium legu-minosarum biovarviceae phosphatasesrdquo Biology and Fertility ofSoils vol 18 no 3 pp 216ndash218 1994

[14] H Rodrıguez and R Fraga ldquoPhosphate solubilizing bacte-ria and their role in plant growth promotionrdquo BiotechnologyAdvances vol 17 no 4-5 pp 319ndash339 1999

[15] M A Whitelaw T J Harden and G L Bender ldquoPlant growthpromotion of wheat inoculated with Penicillium radicum spnovrdquo Australian Journal of Soil Research vol 35 no 2 pp 291ndash300 1997

[16] N Vassilev M Toro M Vassileva R Azcon and J M BarealdquoRock phosphate solubilization by immobilized cells of Enter-obacter sp in fermentation and soil conditionsrdquo BioresourceTechnology vol 61 no 1 pp 29ndash32 1997

[17] M L Speck Compedium of Methods for Examination of FoodMicrobiological American PublicHealthAssociationWashing-ton DC USA 1976

[18] Y Aparna and J Sarada ldquoMolecular characterization and Phy-logenetic analysis of Serratia sp-YAJSmdashan extracellular Dnaseproducer isolated from rhizosphere soilrdquo Science ResearchReporter vol 2 no 1 pp 104ndash109 2012

[19] A T Wahyudi R P Astuti A Widyawati A Meryandiniand A A Nawangsih ldquoCharacterization of Bacillus sp strainsisolated from rhizosphere of soybean plants for their use aspotential plant growth for promoting rhizobacteriardquo Journal ofMicrobiology and Antimicrobials vol 3 pp 34ndash40 2011


[20] E O KingM KWard andD E Raney ldquoTwo simplemedia forthe demonstration of pyocyanin and fluoresceinrdquoThe Journal ofLaboratory and Clinical Medicine vol 44 pp 301ndash307 1954

[21] J Guiraud and P Galzy Lrsquoanalyse microbiologique dans lesindustries alimentaires LrsquoUsine Nouvelle Paris France 1980

[22] D Clauss and R C W Berkeley ldquoGenus Bacillusrdquo in BergeyrsquosManual of Determinative Bacteriology Williams amp WilkinsBaltimore Md USA 1986

[23] F M Olajuyigbe and J O Ajele ldquoProduction dynamics ofextracellular protease from Bacillus speciesrdquo African Journal ofBiotechnology vol 4 no 8 pp 776ndash779 2005

[24] H Lorck ldquoProduction of hydrocyanic acid by bacteriardquo Physi-ologia Plantarum vol 1 no 2 pp 142ndash146 1948

[25] J C Cappuccino and N ShermanMicrobiology ldquoA LaboratoryManualldquo BenjaminCummings New York NY USA 1992

[26] A Adjanohoun L Baba-MouSSA R Kakai et al ldquoCar-acterisation des rhizobacteries potentiellement promotrices dela croissance vegetative du maıs dans differents agrosystemesdu Sud-Beninrdquo International Journal of Biological and ChemicalSciences vol 5 no 2 pp 433ndash444 2011

[27] L Schoenborn P S Yates B E Grinton P Hugenholtz and PH Janssen ldquoLiquid serial dilution is inferior to solid media forisolation of cultures representative of the phylum-level diversityof soil bacteriardquo Applied and Environmental Microbiology vol70 no 7 pp 4363ndash4366 2004

[28] D V Badri T L Weir D van der Lelie and J M VivancoldquoRhizosphere chemical dialogues plant-microbe interactionsrdquoCurrent Opinion in Biotechnology vol 20 no 6 pp 642ndash6502009

[29] A Adam Elicitation de la resistance systemique induite chez latomate et le concombre et activation de la voie de la lipoxygenasepar des rhizobacteries non-pathogenes [These de Doctorat]Universite de Liege Liege Belgium 2008

[30] S Timmusk V Paalme T Pavlicek et al ldquoBacterial distributionin the rhizosphere of wild barley under contrasting microcli-matesrdquo PLoS ONE vol 6 no 3 Article ID e17968 2011

[31] B Joseph R Ranjan Patra and R Lawrence ldquoCharacteriza-tion of plant growth promoting rhizobacteria associated withchickpea (Cicer arietinum L)rdquo International Journal of PlantProduction vol 2 pp 141ndash152 2007

[32] P Garbeva J A Van Veen and J D Van Elsas ldquoPredominantBacillus spp in agricultural soil under different managementregimes detected via PCR-DGGErdquo Microbial Ecology vol 45no 3 pp 302ndash316 2003

[33] J Yadav J P Verma and K N Tiwari ldquoEffect of plant growthpromoting Rhizobacteria on seed germination and plantgrowthChickpea (Cicer arietinumL) under in vitro conditionsrdquoBiological Forum vol 2 no 2 pp 15ndash18 2010


[35] A Kumar A Kumar S Devi S Patil C Payal and S NegildquoIsolation screening and characterization of bacteria fromRhizospheric soils for different plant growth promotion (PGP)activities an in vitro studyrdquo Recent Research in Science andTechnology vol 4 no 1 pp 1ndash5 2012

[36] D Haas and G Defago ldquoBiological control of soil-bornepathogens by fluorescent pseudomonadsrdquo Nature ReviewsMicrobiology vol 3 no 4 pp 307ndash319 2005

Submit your manuscripts athttpwwwhindawicom

Forestry ResearchInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Environmental and Public Health

Journal of



EcosystemsJournal of


MeteorologyAdvances in

EcologyInternational Journal of


Marine BiologyJournal of


Hindawi Publishing Corporationhttpwwwhindawicom

Applied ampEnvironmentalSoil Science

Volume 2014

Advances in


Environmental Chemistry

Atmospheric SciencesInternational Journal of



Waste ManagementJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal of

Geophysics


Geological ResearchJournal of

EarthquakesJournal of


BiodiversityInternational Journal of


ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

OceanographyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of Computational Environmental SciencesHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


ClimatologyJournal of


products use is the environment pollution through groundwater and crop products contamination by heavy metals thatare contained in these agricultural inputsThese heavymetalsare known to be a public health problems because transferredto humans they are involved in the cancer occurrence[2] Apart from medical damages other consequences inagricultural area such as natural ecological nutrient cyclinginterruption and soil biological communities destruction arefrequently reported [3] Regarding the damages caused by theexcessive use of agrochemical products other research pathsare explored worldwide Among the explored paths the useof microorganism currently called Plant Growth PromotingRhizobacteria (PGPR) is in pole position

Indeed the PGPR is a group of bacteria capable ofcolonizing actively plant roots system and improving theirgrowth and yield [4] The expression ldquoPGPRrdquo was firstlyproposed by Kloepper et al [5] and was used especially forthe fluorescent Pseudomonas involved in biological controlof pathogens and the improvement of plant growth LaterKapulnik et al [6] extended this expression to rhizobacteriacapable of promoting directly the plant growth Nowadaysthis expression is used to refer to all bacteria living in therhizosphere (around roots) improving plant growth by oneor several mechanisms [7] A large range of species belongingto the genus Pseudomonas Azospirillum Azotobacter Kleb-siella Enterobacter Alcaligenes Arthrobacter BurkholderiaBacillus and Serratia were reported to be PGPR [8]

Yazdani et al [9] asserted that the PGPR use can reducethe application of phosphorus to 50 without affecting themaize (Zea mays L) seed yield Several authors reported theincrease 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 andidentify the potential PGPR frommaize (most cultivated andconsumed cereal in Benin) rhizosphere in the central andnorthern Benin The medium-dated objective of this studyis to propose for farmers the biological fertilizers based onnative PGPR for increasing maize production

2 Materials and Methods

21 Geographical Characterization of Study Area This studywas carried in five agroecological zones (I II III IV andV) located in the central and northern Benin West Africa(Figure 1) Indeed Benin is localized in West Africa (southof Sahara) in the tropical zone between Equator and Tropicof Cancer precisely between the parallel 6∘ 301015840 and 12∘ 301015840 ofnorth latitude and meridians 1∘ and 30∘ 401015840 of east longitude

22 Collection of Rhizospheric Samples Three (3) villageswere selected by agroecological zone and four fields werechosen in each village Three maize plants distanced at least10 meters were dug up in each fieldTheir roots were cut withsoil adheres and mixed in a bucket Three hundred gramsof this mixture was packed in a sterile stomacher bag andlabeled correctly to form the sample of the field A total of 60samples were collected and immediately transported at 4∘Cto the laboratory for further analysis Once they are at the

laboratory the microbiological screening was immediatelyrealized or samples were kept at 4∘C until screening

Several other parameters (climate soil type annual plu-viometry and other crops grown except for maize) of thesampling sites were collected during the sampling

23 Isolation of Rhizobacteria According to Speck [17]method 10 g of each sample was mixed into Erlenmeyerflask containing 90mL of tryptone salt The mixture wasvigorously shaken for about 30 s to obtain 10minus1 dilutionThe previous dilution (1mL) was transferred into 9mL oftryptone salt to obtain 10minus2 dilution This operation wasrepeated until obtaining 10minus8 dilution Each dilution (01mL)was streaked on different specific isolationmediaThe aerobicmesophilic flora was enumerated on Plate Count Agar asrecommended by the French standard V08-051 Bacillussp and Serratia sp were isolated on nutrient agar afterincubation at 37∘C for 24 h and 30∘C for 48 h respectively[18 19] Pseudomonas sp was isolated on King A and KingB agar after incubation at 30∘C for 72 h [20]

24 Identification of Rhizobacteria The identification of iso-lated rhizobacteria consisted firstly in macroscopic (colonymorphology pigmentation etc) and microscopic (gramreaction mobility cell shape spores position etc) obser-vations This first identification was followed by severalbiochemical and enzymatic tests The performed tests areproduction of oxidase catalase indole urease and hydrogensulfide respiratory type acid and gas production on glucoseagar citrate and nitrate utilization hydrolysis of starchcaseinmannitol gelatin and lecithin fermentation of glucoseand lactose growth on MacConkey and Cetrimide agarVoges-Proskaur test growth at 42 45 55 and 65∘C andfluorescence at 360∘C [18 21ndash23]

25 Plant Growth Promoting Properties

251 Hydrogen Cyanide Production All isolated rhizobacte-ria were screened for hydrogen cyanide production followingthe method described by Lorck [24] Each rhizobacteriumwas streaked on nutrient agar medium added with glycine(44 gL) The agar was covered with a Whatman number1 filter paper previously soaked in a specific solution (05picric acid and 2 sodium carbonate wv) Plates were sealedwith parafilm paper and incubated at 36plusmn2∘C for 4 daysTheappearance of orange or red color indicates the production ofhydrogen cyanide

252 Ammonia Production To research the production ofammonia each identified rhizobacteria strain was grown inpeptone broth (10mL) and incubated at 36 plusmn 2∘C for 48 to72 h After incubation 05mL of Nesslerrsquos reagent was addedto bacterial suspension The development of brown to yellowcolor indicated ammonia production [25]

26 Statistical Analysis Microsoft Office Excel 2007 hadbeen used to create data base The different parametersevaluated were submitted to Analysis of Variance (ANOVA)


Kandi

Nrsquodali

Djougou

Bassila

Karimama

Banikoara

Malanville

Natitingou

Ina

Koua

Bagri

Monsey

NrsquodaliSakarou

Arbonga

Achakpa

Molla centre

Manigri Ikanni

Sonsoro


N

W

S

E

Togo

Niger

Atlantic Ocean

Nigeria



Manigri Okeacute

Barieacutenou

0 25 50 100(km)

7∘30

9984000998400998400N

9∘09984000998400998400N

10∘30

9984000998400998400N

12∘09984000998400998400N

7∘30

9984000998400998400N

9∘09984000998400998400N

10∘30

9984000998400998400N

12∘09984000998400998400N

0∘36

9984000998400998400E 1

∘39

9984000998400998400E 2

∘42

9984000998400998400E 3

∘45

9984000998400998400E

0∘36

9984000998400998400E 1

∘39

9984000998400998400E 2

∘42

9984000998400998400E 3

∘45

9984000998400998400E



Benseacutekou

Sav e

Bembereke



3 Results






I Far North Benin


700 to 900years







MilletSonsoro







IVWestAtacora zone










A

A

A

A

A


0123456789

(108

CFU

g o

f soi

l)

(a)

BC

A

ABAB

BC

C C


012345678

(108

CFU

g o

f soi

l)

(b)






ABA

BB

BA

A AA

A

B BC

A

BC


01234567

(106

CFU

g o

f soi

l)


Serratia sp

(a)

BC B BCA

BCBC

CA A A A

B

AA

B BB B

A

B

A



Serratia sp

01234567

(106

CFU

g o

f soi

l)

(b)







3) and hydrogen


4 Discussion









+ + + + + nd nd nd nd




















H2



Table 2 Continued










and HCN


NH3

()

Production ofHCN()











5 Conclusion




Acknowledgments


References










































Journal of












Volume 2014

Advances in









Geophysics















Kandi

Nrsquodali

Djougou

Bassila

Karimama

Banikoara

Malanville

Natitingou

Ina

Koua

Bagri

Monsey

NrsquodaliSakarou

Arbonga

Achakpa

Molla centre

Manigri Ikanni

Sonsoro


N

W

S

E

Togo

Niger

Atlantic Ocean

Nigeria



Manigri Okeacute

Barieacutenou

0 25 50 100(km)

7∘30

9984000998400998400N

9∘09984000998400998400N

10∘30

9984000998400998400N

12∘09984000998400998400N

7∘30

9984000998400998400N

9∘09984000998400998400N

10∘30

9984000998400998400N

12∘09984000998400998400N

0∘36

9984000998400998400E 1

∘39

9984000998400998400E 2

∘42

9984000998400998400E 3

∘45

9984000998400998400E

0∘36

9984000998400998400E 1

∘39

9984000998400998400E 2

∘42

9984000998400998400E 3

∘45

9984000998400998400E



Benseacutekou

Sav e

Bembereke



3 Results






I Far North Benin


700 to 900years







MilletSonsoro







IVWestAtacora zone










A

A

A

A

A


0123456789

(108

CFU

g o

f soi

l)

(a)

BC

A

ABAB

BC

C C


012345678

(108

CFU

g o

f soi

l)

(b)






ABA

BB

BA

A AA

A

B BC

A

BC


01234567

(106

CFU

g o

f soi

l)


Serratia sp

(a)

BC B BCA

BCBC

CA A A A

B

AA

B BB B

A

B

A



Serratia sp

01234567

(106

CFU

g o

f soi

l)

(b)







3) and hydrogen


4 Discussion









+ + + + + nd nd nd nd




















H2



Table 2 Continued










and HCN


NH3

()

Production ofHCN()











5 Conclusion




Acknowledgments


References










































Journal of












Volume 2014

Advances in









Geophysics


















I Far North Benin


700 to 900years







MilletSonsoro







IVWestAtacora zone










A

A

A

A

A


0123456789

(108

CFU

g o

f soi

l)

(a)

BC

A

ABAB

BC

C C


012345678

(108

CFU

g o

f soi

l)

(b)






ABA

BB

BA

A AA

A

B BC

A

BC


01234567

(106

CFU

g o

f soi

l)


Serratia sp

(a)

BC B BCA

BCBC

CA A A A

B

AA

B BB B

A

B

A



Serratia sp

01234567

(106

CFU

g o

f soi

l)

(b)







3) and hydrogen


4 Discussion









+ + + + + nd nd nd nd




















H2



Table 2 Continued










and HCN


NH3

()

Production ofHCN()











5 Conclusion




Acknowledgments


References










































Journal of












Volume 2014

Advances in









Geophysics















ABA

BB

BA

A AA

A

B BC

A

BC


01234567

(106

CFU

g o

f soi

l)


Serratia sp

(a)

BC B BCA

BCBC

CA A A A

B

AA

B BB B

A

B

A



Serratia sp

01234567

(106

CFU

g o

f soi

l)

(b)







3) and hydrogen


4 Discussion









+ + + + + nd nd nd nd




















H2



Table 2 Continued










and HCN


NH3

()

Production ofHCN()











5 Conclusion




Acknowledgments


References










































Journal of












Volume 2014

Advances in









Geophysics




















+ + + + + nd nd nd nd




















H2



Table 2 Continued










and HCN


NH3

()

Production ofHCN()











5 Conclusion




Acknowledgments


References










































Journal of












Volume 2014

Advances in









Geophysics















Table 2 Continued










and HCN


NH3

()

Production ofHCN()











5 Conclusion




Acknowledgments


References










































Journal of












Volume 2014

Advances in









Geophysics

















5 Conclusion




Acknowledgments


References










































Journal of












Volume 2014

Advances in









Geophysics




































Journal of












Volume 2014

Advances in









Geophysics


















Journal of












Volume 2014

Advances in









Geophysics














Documents

Characterization of Potential Plant Growth Promoting Rhizobacteria