Antonie van Leeuwenhoek (2011) 100:161–170

DOI 10.1007/s10482-011-9570-5

SHORT COMMUNICATION

Genetic and functional diversities of bacterial communities

in the rhizosphere of Arachis hypogaea
Shyamalina Haldar • Susanta Roy Choudhury •

Sanghamitra Sengupta

Received: 29 September 2010 / Accepted: 25 February 2011 / Published online: 5 March 2011
Ó Springer Science+Business Media B.V. 2011

Abstract Bioinoculants are environmentally friendly, their targeted application in the groundnut production
energy efficient and economically viable resources in system.
sustainable agriculture. Knowledge of the structure and
activities of microbial population in the rhizosphere of a Keywords Arachis hypogaea  Rhizosphere 
plant is essential to formulate an effective bioinoculant. Plant growth promoting rhizobacteria  Bioinoculant 
In this study, the bacterial community present in the Proteobacteria  Bacillus
rhizosphere of an important oilseed legume, Arachis
hypogaea (L.) was described with respect to adjoining
bulk soil as a baseline control using a 16S rDNA based
metagenomic approach. Significantly higher abundance Introduction
of Gamma-proteobacteria, a prevalence of Bacillus and
the Cytophaga-Flavobacteria group of Bacteroidetes Peanuts are rich sources of important nutrients and
and absence of the Rhizobiaceae family of Alpha- bioactive constituents such as arginine-rich proteins,
proteobacteria were the major features observed in the high levels of soluble and insoluble fiber, beneficial
matured Arachis-rhizosphere. The functional charac- fatty acids, vitamins including high folate and vitamin
terization of the rhizosphere-competent bacteria was E, phenolics and phytosterols, all of which provide a
performed using culture-dependent determination of wide range of health benefits (Kris-Etherton et al.
phenotypes. Most bacterial isolates from the groundnut- 2008). According to a report of the USDA-Foreign
rhizosphere exhibited multiple biochemical activities Agricultural Service (2008–2009), China leads in the
associated with plant growth and disease control. production of Runner, Virginia, Spanish and Valencia
Validation of the beneficial traits in candidate bioinoc- market-type peanuts (Arachis hypogaea L.) having a
ulants in pot-cultures and field trials is necessary before share of about 33% of overall world production,
followed by India (18%) and the United States of
America (7%). In rain-fed areas of Asia, soil fertility
S. Haldar  S. Sengupta (&)
is an important constraint for high pod yields. The
Department of Biochemistry, University of Calcutta,
35 Ballygunge Circular Road, Kolkata 700019, India majority of the small-scale farmers in these regions
e-mail: [email protected] are reluctant to invest in chemical fertilizers and other
agrochemicals because of the unassured crop returns
S. Roy Choudhury
owing to the high incidence of fungal diseases and
Molecular & Human Genetics Department, Indian
Institute of Chemical Biology, 4 Raja S. C. Mullick Road, unpredictable monsoon (Kishore et al. 2005). Besides,
Kolkata 700032, India application of inorganic fertilizer debilitates the soil’s

123
162 Antonie van Leeuwenhoek (2011) 100:161–170

physical and chemical status and drastically alters plants. To manage the soil fertility, the planters grow
microbial diversity, which is vital to sustain the groundnut and other leguminous plants on a rotational
fertility of soil (Adesemoye and Kloepper 2009; Yang basis. However, there was no report of cultivation of
et al. 2000). any leguminous plants prior to collection of the soil for
As a step towards sustainable agriculture, there is a plant growth in pots (personal communication with
greater scope for development and popularization of local farmers). Immediately after collection of soil,
bioinoculants in the groundnut production system. physicochemical characterization of the soil was
The two most common groundnut plant growth performed and organic carbon, available nitrogen,
promoting rhizobacteria (PGPR) that enhance bio- phosphorous and potassium contents were determined
mass, nitrogen and phosphorous uptake and crop- to be 0.176%, 121.7, 29.2 and 38.99 kg/h, respectively.
yield are Pseudomonas and Bacillus sp. (Dey et al. Before sowing peanut seeds, the soil was sieved,
2004; Saravanakumar and Samiyappan 2007; Kishore homogenized and transferred into plastic pots
et al. 2005). Until recently, most studies aiming for (12 9 12 9 6 cm; 400 g soil/pot). The seeds were
the isolation and identification of PGPR were based sown in February, 2008 (average maximum and min-
on cultivation and phenotypic characterization of imum temperatures were 28 and 18°C) and harvested
bacterial isolates (Prosser et al. 2006). There is in May, 2008 (average maximum and minimum
growing body of evidence that diverse trophic and temperatures were 35 and 25°C). No plant protection
functional groups of microorganisms contribute to the methods were used. Initially five surface-sterilized
establishment and functioning of the microbial com- seeds were sown in each pot and twelve such pots were
munity in the rhizosphere (Kent and Triplett 2002). used. After 90 days of sowing, the time required for
Thus, a polyphasic approach which will provide a physiological maturation of A. hypogaea, a total of 50
reliable and wider identification, as well as a better plants were harvested by uprooting them from a depth
elucidation of functional attributes of members of the of 10 cm. The shoot system was cut out using sterile
bacterial community, is preferred. Given the eco- scissors and nodules were separated. The bulk soil was
nomic and nutritional importance of A. hypogaea in collected by shaking the roots to dislodge the loosely
the Indian sub-continent, the present report utilizes a attached soil in 50-ml centrifuge tubes whereas
combination of methods including 16S rDNA based rhizosphere sample was collected by rinsing the roots
molecular typing, microbial culture techniques, bio- in phosphate-buffered saline (PBS; 7 g/l NaCl, 0.7 g/l
chemical assays and statistical analyses to character- Na2HPO4, 0.2 g/l KH2PO4; pH 7.2) for 5 min, fol-
ize the bacterial community in the rhizosphere with lowed by centrifugation at 7,000 rpm for 5 min at 4°C.
respect to that in bulk soil and to decipher the Soil representing rhizosphere and bulk samples were
functional attributes related to plant growth promo- analyzed by mixing the soil samples from all the plants
tion and disease control towards systematic recon- and stored at 4 and -20°C for microbiological and
struction of a rhizosphere-competent inoculant for the molecular analyses, respectively.
groundnut plant. Soil DNA was extracted from 100 mg of rhizo-
sphere and bulk soil using a Soil Master DNA
Extraction Kit (Epicenter). The bacterial 16S rRNA
Materials and methods gene (partial) from the soil DNA was amplified using
degenerate primers 27F (50 -AGAGTTTGATCMTGG
Fresh, healthy groundnut seeds (AK-1224), obtained CTCAG-30 ) and 1492R (50 -ACGGYTACCTTGTTA
from West Bengal State Seed Corporation (Midnapur), CGACTT-30 ), following the PCR protocol described
were disinfected with 70% ethanol, washed in sterile by Zhang et al. (2006). PCR products were purified
water and were directly planted in pots containing loam using a QIAquick Gel Extraction Kit (Qiagen),
soil of composition 40% sand, 40% silt, 20% clay and ligated to TA-vector pTZ57R/T (InsTAclone PCR
of pH 8.17. The soil used was collected from an Cloning Kit, Fermentas) and transformed into Esch-
agricultural field located on the southern fringes of the erichia coli DH5-alpha cells. Plasmid DNA was
city of Kolkata, lying in the Gangetic belt of West isolated from each clone using the alkaline-lysis
Bengal, India (22°340 N and 88°240 E). The field was method (Birnboim and Doly 1979) followed by
primarily used for commercial production of flowering amplification of the insert with the 27F and 1492R

123
Antonie van Leeuwenhoek (2011) 100:161–170 163

primers to confirm integrity of the insert. Four accession numbers GU217722–GU217788; GU24
microliter of each PCR product was restriction 5895–GU245926; GU269387–GU269410, GQ410
digested separately with 10 U of HhaI and RsaI 441–GQ410442, GQ410548–GQ410549, HQ2043
(Fermentas) following manufacturer’s protocol. The 21–HQ204332, HQ222600–HQ222601, HQ670722,
digests were electrophoresed in 2% agarose gels and GQ375136, GQ375143, GQ410444, GQ410448, GQ
DNA fragment sizes were determined with respect to 410453, GQ410478, GQ410492, GQ410501, GQ410
external DNA-ladder using a TANON gel analysis 504, GQ410516, GQ410533, GQ410553, GQ410555
system (4100). Clones displaying indistinguishable and HQ326749.
pattern were grouped under a phylotype. DNA To isolate individual bacterial species, 1 g each of
sequencing were performed using a BigDye sequence rhizosphere and bulk soil was suspended in 9 ml
terminator kit v1.1 (Applied Biosystems) with the sterile PBS; centrifuged at 180 rpm for 10 min at
27F and a nested 515F (50 -GTGCCAGCMGCCG 30°C and serially diluted with PBS. One hundred
CGGTAA-30 ) primer (Papineau et al. 2005) and an microlitre aliquots from different dilutions were
ABI Prism 3100 genetic analyzer. The termini of the spread onto Luria-agar (HiMedia Laboratories) and
27F and 515F generated sequences of a clone were Pseudomonas Isolation Agar (Difco Laboratories)
trimmed by inspecting individual electropherograms and the plates were incubated for 48 h at 30°C
by two independent researchers (SH and SSG). (Jensen et al. 2001). Based on the colony character-
Sequence reads generated using primers 27F and istics, single colonies were selected and streaked at
515F for each clone were stitched after identification least three times to allow the isolation of pure
of overlapping region (at least 50 basepairs overlap). colonies. Stock cultures were prepared in Luria-broth
Approximately, 1 kb sequence data was available for containing 25% (v/v) glycerol and stored at -80°C.
each bacterial clone. Routine microbial tests, e.g. Gram-staining, catalase
Phylotype data was used to compare bacterial activity and morphology examination were per-
abundance and diversity in rhizosphere and bulk soils formed. Genomic DNA was extracted from the
in terms of phylotype richness (Margalef index and bacteria following the SDS-CTAB lysis method
Chao1 estimate), diversity (Shannon–Weiner and (Sambrook and Russel 2001). The 16S rRNA gene
Simpson’s indices), similarity (Sorensen’s index) was amplified, sequenced and assembled using
and evenness (Pielou index) using rarefaction anal- methods described by Han et al. Indole acetic acid
ysis (www.biome.sdsu.edu/fastgroup/cal_tools.htm). (IAA) production by the bacterial isolates in the
Binomial proportion testing and independent t-tests absence of amended L-tryptophan was assayed col-
were performed to calculate the significance of dif- orimetrically (JASCO V-630 spectrophotometer)
ference of the results between two samples using using the Salkowski reagent as described previously
SPSS for windows version 10.0 (SPSS Inc). Identity (Ali et al. 2009). The quantity of produced IAA was
of bacterial species in each sequenced clone was measured at 530 nm against IAA standards (HiMedia
deciphered by using the Basic Local Alignment Laboratories). Phosphate-solubilization and sidero-
Search Tool (www.ncbi.nlm.nih.gov/BLAST/) and phore production were qualitatively determined by
Classifier Tool of Ribosomal Database Project (RDP- inoculating the bacteria on to Pikovskaya (PVK) agar
II) Release 10 (www.rdp.cme.msu.edu/) after exam- medium containing precipitated tri-calcium phos-
ining for chimeras by the CHIMERA-CHECK online phate (Chaiharn and Lumyong 2010) and chrome
analysis program of RDP-II (http://rdp8.cme.msu. azurol S (CAS)-blue agar medium (Schwyn and
edu/cgis/chimera.cgi?su=SSU) (Han et al. 2009). The Neilands 1987), respectively. The presence of a
phylogenetic affiliation of soil bacteria was done clearing zone around bacterial colonies on PVK
following the classification system proposed by Tin- medium and a color change (orange/purple) on CAS
dall et al. (2006). Bacterial lineages were defined at medium after 3 days of incubation at 28°C were used
the genus level since 16S rDNA sequence analysis as the indicator for phosphate-solubilization and
may lack the power to uncover species diversity at a siderophore production, respectively.
finer scale. A total of 155 sequences (140 from To assay antifungal activity, 72 h old Macropho-
metagenomic and 15 from culture-based soil clone mina phaseolina fungal spores were seeded at a
libraries) were deposited in GenBank with the distance of 4 cm from 24 h grown bacterial colonies

123
164 Antonie van Leeuwenhoek (2011) 100:161–170

on Potato-dextrose agar medium. Bacterial strain and respectively. The numbers of different RsaI phylotypes
M. phaseolina were grown individually as controls. were 53 and 46 in rhizosphere and bulk soil libraries,
After 3 days of incubation at 28°C, radial growth of respectively. Rarefaction curves using phylotype fre-
M. phaseolina co-inoculated with the bacterium was quency data using HhaI on rhizosphere and bulk soil
examined for inhibition of growth. Each experiment samples plateaued at their right hand side, suggesting
for ascertainment of a plant growth promoting trait that the number and distribution of HhaI phylotypes
(PGPT) of an individual bacterial isolate was con- captured the diversity of the two specimens nearly
ducted in triplicate. completely (Fig. 1a). Twelve HhaI and ten RsaI
phylotypes were common between the libraries.
Therefore, more than 80% of phylotypes were exclu-
Results and discussion sive to each library, as elucidated from low Sorensen’s
similarity indices between the samples for both
Two libraries, one with 180 clones from rhizosphere enzymes (Table 1). A significantly higher proportion
(AFR) and the other with 190 clones from bulk soil of phylotypes (63%) obtained by HhaI restriction
(AFB), collected from matured A. hypogaea plants, digestion from rhizosphere-derived library were rep-
were constructed by integrating a 1.4 kb fragment of resented by single clones compared to those derived
the bacterial 16S rRNA gene amplified from soil DNA from bulk soil (44%); (v2 = 4.38, P = 0.036). The
into the TA vector. Phylotyping was performed by frequency of the remaining phylotypes varied between
digesting the cloned insert with HhaI and RsaI. 2 and 19 for HhaI and 2 and 31 for RsaI with a
Seventy-nine and 71 distinct HhaI phylotypes were preponderance of low-frequency phylotypes. The
detected from rhizosphere and bulk soil libraries, phylotype richness of the rhizosphere bacterial

Fig. 1 Diversity and abundance of bacterial population in abundance of phylotypes obtained upon restriction digestion
rhizosphere and bulk soil of Arachis hypogaea based on of 16S rDNA clones with HhaI (1 and 2) and RsaI (3 and 4)
phylotype and sequence data. a Rarefaction curves based on from rhizosphere (1 and 3) and bulk soil (2 and 4) libraries of
phylotype-frequency obtained upon restriction digestion of Arachis hypogaea, c rarefaction curves based on frequency of
16S rDNA clones with HhaI (1 and 2) and RsaI (3 and 4) the genus detected by 16S rDNA sequencing of clones from
from rhizosphere (1 and 3) and bulk soil (2 and 4) libraries of rhizosphere (5) and bulk soil (6) libraries of Arachis
Arachis hypogaea, b rank-abundance curves based on the hypogaea

123
Antonie van Leeuwenhoek (2011) 100:161–170 165

Table 1 Bacterial distribution, abundance and diversity based on HhaI and RsaI restriction analyses of rhizosphere (AFR) and bulk
soil (AFB) libraries
Restriction enzyme No. of phylotypes Phylotype richness Diversity indices
AFR/AFB AFR/AFB AFR/AFB
Total Singleton Margalef index Chao1 Simpson’s Shannon’s Sorensen’s Pielou
estimate similarity

HhaI 79/71 42/21 14.83/13.35 128/86* 0.974/0.972 4.03/3.95 0.22 0.927/0.924

RsaI 53/46 21/18 10.01/8.58 75/66* 0.950/0.943 3.47/3.30 0.16 0.875/0.866
* indicates difference between the estimates is statistically significant

population was remarkably higher as per Margalef colonies) with colony-forming-unit (CFU) in the
index and Chao1 estimates; (P = 0.004). The shallow range of 106/g soil. The two major bacterial phyla
slopes of the rank-abundance curves (Fig. 1b) and high identified from the rhizosphere by the culture-depen-
Pielou indices revealed an even distribution pattern of dent method were Proteobacteria and Firmicutes.
most bacterial species in rhizosphere and bulk soils. The species under Proteobacteria belonged to the
Shannon–Weiner, Simpson’s and Pielou diversities genera Acinetobacter, Aeromonas, Enterobacter,
were also comparable between the samples (Table 1). Klebsiella, Pseudomonas, Xanthomonas (Gamma-
For identification of individual bacterial species proteobacteria) and Achromobacter (Beta-proteobac-
present in the rhizosphere and bulk soil of A. hypo- terium), while those under Firmicutes belonged to
gaea, all RsaI phylotypes and multiple clones repre- Bacillus and Staphylococcus (Bacilli) (Table 2).
senting different HhaI sub-classifications from a Agrobacterium and Bacillus were the two genera
given RsaI phylotype were selected for sequencing. detected in bulk soil. Thus, Pseudomonas, Bacillus,
Altogether 140 clones, 74 clones from rhizosphere Xanthomonas and Agrobacterium were detected
and 66 clones from the bulk soil library were using culture-dependent and independent methods
sequenced. Based on 16S rRNA gene analysis, the from the same soil source. The very small overlap of
two major bacterial phyla detected in both soils were bacterial taxa observed between culture-based and
Proteobacteria (43% in rhizosphere and 41% in bulk culture-independent methods can be attributed to the
soil) and Bacteroidetes (34% in rhizosphere and 24% fact that only 1% of the soil microbes are estimated to
in bulk soil). The comparative profile of bacterial be cultivable and only 38% (140 out of 370) of the
species detected in the rhizosphere and bulk soil total clones were sequenced in this study. Rarefaction
associated with A. hypogaea is presented in Table 2. analysis (Fig. 1c) implicated that 16S rDNA based
Few notable observations were: (i) a significant sequencing revealed the diversity of bacterial popu-
difference in proportion of Gamma-proteobacteria lation present in Arachis-rhizosphere only partially
between rhizosphere and bulk soil samples (24% in (Zhang et al. 2007).
rhizosphere and 6% in bulk soil; P \ 0.0001), (ii) a The majority of the bacterial genera isolated from
complete absence of Alpha-proteobacteria belonging rhizosphere were found to produce IAA (Table 3).
to Rhizobiaceae family and (iii) a remarkably high Strains belonging to the genera Achromobacter, Aci-
abundance of Cytophaga-Flavobacteria (CF-bacte- netobacter, Pseudomonas and Staphylococcus were
ria) particularly Flavobacterium sp. (15%), belonging shown to produce siderophores. Achromobacter sp., in
to Bacteroidetes in the rhizosphere. addition, could solubilize inorganic phosphates while
To utilize the information on bacterial composition isolates from the genera Bacillus, Pseudomonas and
of groundnut-rhizosphere towards development of Xanthomonas demonstrated strong antifungal activity
bioinoculants, four PGPTs such as ability to produce against the plant pathogen M. phaseolina which causes
IAA and siderophores, to solubilize inorganic phos- crown-rot disease in groundnut plants. Our results
phates and to exert anti-fungal activity were assayed implied that Achromobacter sp., which showed
in individual bacterial isolate. A total of 60 colonies positive results for three out of four PGPTs examined,
were obtained from rhizosphere soil on Luria (20 is a strong candidate to be used as rhizoinoculant in
colonies) and Pseudomonas-isolation agar (40 addition to Pseudomonas and Bacillus species. The

123
166 Antonie van Leeuwenhoek (2011) 100:161–170

Table 2 Phylogenetic affiliation of bacterial clones and isolates based on 16S rRNA gene sequencing from the rhizosphere and bulk
soil of Arachis hypogaea
Representative clones Closest bacterial affiliation in GenBank Phylogenetic % of
group (class) identity

Bacterial species from rhizosphere identified using 16S rRNA gene sequencing (AFR)
AFR-34 Uncultured Methylocella sp. Alpha-proteobacteria 92
AFR-2 Uncultured Hyphomonas sp. 90
AFR-105 Uncultured Ralstonia sp. Beta-proteobacteria 91
AFR-67 Uncultured Methylibium sp. 93
AFR-31 Uncultured Variovorax sp. 96
AFR-5,66 Uncultured Janthinobacterium sp. 93,96
AFR-22,26,27,32,69,102 Uncultured Pseudomonas sp. Gamma-proteobacteria 90–99
AFR-9 Uncultured Cellvibrio sp. 95
AFR-113 Uncultured Nitrosococcus sp. 92
AFR-165 Uncultured Methylococcus sp. 93
AFR-6,18,83,84,178 Uncultured Xanthomonas sp. 91–96
AFR-154 Uncultured Gamma proteobacterium 90
AFR-172 Uncultured Thioalkalivibrio sp. 89
AFR-33,108 Uncultured Legionella sp. 98
AFR-91,19 Uncultured Sorangium sp. Delta-proteobacteria 91
AFR-112,177 Uncultured Geobacter sp. 90
AFR-29,68 Uncultured Pelobacter sp. 90
AFR-23 Uncultured Helicobacter sp. Epsilon-proteobacteria 78
AFR-3,14,43,44,47,62,103,109,124,156,176 Uncultured Flavobacterium sp. Bacteroidetes (CFB) 93–96
AFR-74,96,111,137,166 Uncultured Cytophaga sp. 88–90
AFR-30,41,54 Uncultured Chitinophaga sp. 83
AFR-45,60 Uncultured Candidatus Amoebophilus sp. 94
AFR-35 Uncultured Candidatus Desulforudis sp. 86
AFR-70 Uncultured Croceibacter sp. 86
AFR-10 Uncultured Pedobacter sp. 82
AFR-75 Uncultured Bacillus sp. Firmicutes 92
AFR-11,21,93 Uncultured Clostridium sp. 81–82
AFR-174 Uncultured Pelotomaculum sp. 77
AFR-17 Uncultured Solibacter sp. Acidobacteria 87
AFR-163 Uncultured Mycobacterium sp. Actinobacteria 97
AFR-99 Uncultured Nocardioides sp. 92
AFR-81 Uncultured Tsukamurella sp. 98
AFR-155 Uncultured candidate division TM7 bacterium Candidate Division 91
AFR-90 Uncultured Prochlorococcus sp. Cyanobacteria 75
AFR-28,48,159 Uncultured Gemmatimonas sp. Gemmatimonadetes 88–90
AFR-36,63 Uncultured Planctomyces sp. Planctomycetes 83,89
AFR-130,151 Uncultured bacterium Ellin514 Verrucomicrobia 91,92
Bacterial species from bulk soil identified using 16S rRNA gene sequencing (AFB)
AFB-96,150 Uncultured Agrobacterium sp. Alpha-proteobacteria 95–98
AFB-44,81 Uncultured Rhizobium sp. 97,98
AFB-160 Uncultured Azorhizobium sp. 89
AFB-23,91,184 Uncultured Mesorhizobium sp. 93–98

123
Antonie van Leeuwenhoek (2011) 100:161–170 167

Table 2 continued
Representative clones Closest bacterial affiliation in GenBank Phylogenetic % of
group (class) identity

AFB-46 Uncultured Novosphingobium sp. 96

AFB-20 Uncultured Sphingomonas sp. 93
AFB-75 Uncultured Comamonas sp. Beta-proteobacteria 96
AFB-83 Uncultured Acidovorax sp. 94
AFB-39 Uncultured Variovorax sp. 97
AFB-41 Uncultured Methylibium sp. 95
AFB-170 Uncultured Lutiella sp. 96
AFB-28 Uncultured Cupriavidus sp. 93
AFB-63,100 Uncultured Aromatoleum sp. 91,92
AFB-32,98 Uncultured Pseudomonas sp. Gamma-proteobacteria 97
AFB-24 Uncultured Stenotrophomonas sp. 97
AFB-178 Uncultured Alkalilimnicola sp. 88
AFB-128 Uncultured Syntrophobacter sp. Delta-proteobacteria 83
AFB-168 Uncultured Desulfovibrio sp. 81
AFB-84,122 Uncultured Geobacter sp. 86,92
AFB-87 Uncultured Desulfonatronospira sp. 81
AFB-16,56,59,69,77,112,187 Uncultured Chitinophaga sp. Bacteroidetes (CFB) 86–90
AFB-8,25,167,180 Uncultured Cytophaga sp. 86–87
AFB-186 Uncultured Sphingobacterium sp. 93
AFB-15,31 Uncultured Dyadobacter sp. 92
AFB-54 Uncultured Pedobacter sp. 93
AFB-38 Uncultured Candidatus Amoebophilus sp. 84
AFB-19,30,58,60,90 Uncultured Bacillus sp. Firmicutes 90–95
AFB-175 Uncultured Thermoanaerobacter sp. 81
AFB-124 Uncultured Carboxydibrachium sp. 81
AFB-127 Uncultured Moorella sp. 84
AFB-51,172 Uncultured Candidatus Koribacter sp. Acidobacteria 87,90
AFB-115 Uncultured Rubrobacter sp. Actinobacteria 85
AFB-111 Uncultured Janibacter sp. 97
AFB-22 Uncultured Salinispora sp. 97
AFB-40 Uncultured Nakamurella sp. 94
AFB-36 Uncultured Bifidobacterium sp. 82
AFB-34 Uncultured candidate division TM7 bacterium Candidate division 85
AFB-37 Uncultured Elusimicrobium sp. Elusimicrobia 84
AFB-119 Uncultured Gemmatimonas sp. Gemmatimonadetes 86
AFB-3,78 Uncultured Rhodopirellula sp. Planctomycetes 86
AFB-82 Uncultured Planctomyces sp. 87
AFB-21 Uncultured bacterium Ellin514 Verrucomicrobia 91
AFB-121 Uncultured Thermodesulfovibrio sp. Thermodesulfovibrio 83
Cultured bacterial species from rhizosphere in Luria-agar media (RZL)
RZL-4 Acinetobacter sp. Gamma-proteobacteria 98
RZL-17 Aeromonas sp. 98
RZL-10 Enterobacter sp. 99

123
168 Antonie van Leeuwenhoek (2011) 100:161–170

Table 2 continued
Representative clones Closest bacterial affiliation in GenBank Phylogenetic group % of
(class) identity

RZL-1,3,5 Pseudomonas sp. 93

RZL-2,20 Achromobacter sp. Beta-proteobacteria 98
RZL-6,7,8,9,11,12,13,15,16,18,19 Bacillus sp. Firmicutes 90–99
RZL-14 Staphylococcus sp. 98
Cultured bacterial species from rhizosphere in pseudomonas isolation agar media (RZPIA)
RZPIA-3,31 Aeromonas sp. Gamma-proteobacteria 98
RZPIA-23 Klebsiella sp. 96
RZPIA-4 Pseudomonas sp. 93
RZPIA-5,8,63,79 Xanthomonas sp. 96–98
RZPIA-6,7,13,14,15,19,25,28,29,37,38,44,59, Achromobacter sp. Beta-proteobacteria 98–99
60,62,67
RZPIA-9,11,12,16,17,34,35,39,40 Bacillus sp. Firmicutes 91–99
RZPIA-55,56,58 Staphylococcus sp. 98
Cultured bacterial species from bulk soil in Luria-agar media (BL)
BL-16 Agrobacterium sp. Alpha-proteobacteria 98
BL-10,33,36,59 Bacillus sp. Firmicutes 95–99

Table 3 Plant-growth-promoting attributes of the bacterial isolates from rhizosphere of Arachis hypogaea
Bacterial species IAA production Phosphate Siderophore Antifungal
(isolate ID/GenBank accession number) (lg/ml) solubilization production activity

Achromobacter sp. (RZPIA-62/HQ204322) 3.3 ± 0.17 ? ? –

Acinetobacter sp. (RZL-04/HQ204331) 4.2 ± 0.20 - ? -
Aeromonas sp. (RZPIA-31/HQ204324) 3.2 ± 0.25 - - -
Bacillus sp.
(RZL-09/HQ222601) 26.0 ± 1.8 - ? -
(RZPIA-11/HQ204328) 2.08 ± 0.66 ? ? -
(RZL-12/HQ222600) 1.5 ± 0.4 - - -
(RZPIA-34/HQ204325) 1.24 ± 0.58 - - ?
Enterobacter sp. (RZL-10/HQ204332) 25.6 ± 0.66 - - -
Staphylococcus sp. (RZPIA-56/HQ204326) 0.77 ± 0.05 - ? -
Klebsiella sp. (RZPIA-23/HQ204321) 10.6 ± 1.5 - - -
Pseudomonas sp. (RZPIA-04/HQ204329) 2.4 ± 0.40 - ? ?
Xanthomonas sp. (RZPIA-63/HQ204323) 0.56 ± 0.67 - - ?
?
indicates the presence of the phenotype and - indicates the absence of the phenotype in a bacterial isolate. Data represent the
means ± standard error of three independent observations

detection of Xanthomonas sp., one of the most Enterobacter strengthen the idea that association of
ubiquitous groups of plant-associated bacterial patho- these species in the groundnut-rhizosphere is benefi-
gens in the rhizosphere of A. hypogaea is not cial for plant growth (Ibáñez et al. 2009). The very
surprising (Kremer et al. 1990). The very high high abundance of CF-bacteria in the Arachis-rhizo-
abundance of Gamma-proteobacteria in the rhizo- sphere was presumably due to their ability to degrade
sphere and the presence of different PGPT in bacterial complex organic exudates thus contributing to the
isolates belonging to Pseudomonas, Klebsiella and turnover of carbon, nitrogen and phosphorous

123
Antonie van Leeuwenhoek (2011) 100:161–170 169

(Forsberg et al. 1981). Our inability to detect any Chaiharn M, Lumyong S (2010) Screening and optimization of
Bradyrhizobia from the rhizosphere of groundnut indole-3-acetic acid production and phosphate solubili-
zation from rhizobacteria aimed at improving plant
plants was unexpected and may be attributed to growth. Curr Microbiol. doi:10.1007/s00284-010-9674-6
following factors. First, the incubation period of 48 h Dey R, Pal KK, Bhatt DM, Chauhan SM (2004) Growth pro-
used to isolate the soil bacteria might be inadequate for motion and yield enhancement of peanut (Arachis hypo-
growth of rhizobial strains. Moreover, the agricultural gaea L.) by application of plant growth-promoting
rhizobacteria. Microbiol Res 159:371–394
field from which the soil was collected lacked Forsberg CW, Beveridge TJ, Hellstrom A (1981) Cellulase and
immediate groundnut cultivation record. Thus, it xylanase release from Bacteroides succinogenes and its
remains possible that different results would be importance in the rumen environment. Appl Environ
obtained if soil and plants were analyzed from a field Microbiol 42:886–896
Han J, Xia D, Li L, Sun L, Yang K, Zhang L (2009) Diversity
with groundnut growing history. of culturable bacteria isolated from root domains of Moso
To conclude our results show, that although soil bamboo (Phyllostachys edulis). Microb Ecol 58:363–373
harbours a diverse bacterial population, a character- Ibáñez F, Angelini J, Taurian T, Tonelli ML, Fabra A (2009)
istic pattern of microbiota, many of which display Endophytic occupation of peanut root nodules by oppor-
tunistic Gammaproteobacteria. Syst Appl Microbiol
plant growth promoting activities, is associated with 32:49–55
the rhizosphere of A. hypogaea. Validation of ben- Jensen LB, Baloda S, Boye M, Aarestrup FM (2001) Antimi-
eficial traits in the candidate microbial inoculants in crobial resistance among Pseudomonas spp. and the
pot-cultures and field trials is necessary before their Bacillus cereus group isolated from Danish agricultural
soil. Environ Int 26:581–587
application in the groundnut production system. Kent AD, Triplett EW (2002) Microbial communities and their
Finally, we acknowledge that though 16S rDNA interactions in soil and rhizosphere ecosystems. Annu Rev
sequence analysis is a useful tool to classify bacterial Microbiol 56:211–236
lineages, additional data using other fingerprinting Kishore GK, Pande S, Podile AR (2005) Phylloplane bacteria
increase seedling emergence growth and yield of field-
techniques, that show a high degree of intraspecies grown groundnut (Arachis hypogaea L.). Lett Appl
discrimination power, such as multilocus sequencing Microbiol 40:260–268
typing (MLST), RNA-based fluorescence in situ Kremer RJ, Begonia MF, Stanley L, Lanham ET (1990)
hybridization, or cultivation targeting and nutrient Characterization of rhizobacteria associated with weed
seedlings. Appl Environ Microbiol 56:1649–1655
utilization profiling are necessary for unequivocal Kris-Etherton PM, Hu FB, Ros E, Sabatè J (2008) The role of
assessment of bacterial phylogenies in these environ- tree nuts and peanuts in the prevention of coronary heart
mental samples. disease: multiple potential mechanisms. J Nutr 138:
1746S–1751S
Acknowledgments This research was supported by University Papineau D, Walker JJ, Mojzsis SJ, Pace NR (2005) Compo-
Potential for Excellence-Modern Biology Research Scheme sition and structure of microbial communities from stro-
(UGC/191/UPE/07), University of Calcutta. SH is supported by matolites of Hamelin Pool in Shark Bay, Western
research fellowship from Council of Scientific and Industrial Australia. Appl Environ Microbiol 71:4822–4832
Research, Govt. of India. Prosser JI, Rangel-Castro JI, Killham K (2006) Studying plant–
microbe interactions using stable isotope technologies.
Conflict of interest The authors declare that they have no Curr Opin Biotechnol 17:98–102
conflict of interest. Sambrook J, Russel DW (2001) Molecular cloning: a labora-
tory manual, 3rd edn. CSH Laboratory Press, Cold Spring
Harbor
Saravanakumar D, Samiyappan R (2007) ACC deaminase from
Pseudomonas fluorescens mediated saline resistance in
References groundnut (Arachis hypogea L.) plants. J Appl Microbiol
102:1283–1292
Adesemoye AO, Kloepper JW (2009) Plant–microbes inter- Schwyn B, Neilands JB (1987) Universal chemical assay for
actions in enhanced fertilizer-use efficiency. Appl the detection and determination of siderophores. Anal
Microbiol Biotechnol 85:1–12 Biochem 160:47–56
Ali B, Sabri AN, Ljung K, Hasnain S (2009) Auxin production Tindall BJ, Kämpfer P, Euzéby JP, Oren A (2006) Valid
by plant associated bacteria: impact on endogenous IAA publication of names of prokaryotes according to the rules
content and growth of Triticum aestivum L. Lett Appl of nomenclature: past history and current practice. Int J
Microbiol 48:542–547 Syst Evol Microbiol 56:2715–2720
Birnboim HC, Doly J (1979) A rapid alkaline extraction pro- Yang Y, Yao J, Hu S, Qi Y (2000) Effects of agricultural chem-
cedure for screening recombinant plasmid DNA. Nucleic icals on DNA sequence diversity of soil microbial commu-
Acids Res 7:1513–1523 nity: a study with RAPD marker. Microb Ecol 39:72–79

123
170 Antonie van Leeuwenhoek (2011) 100:161–170

Zhang R, Jiang J, Gu JD, Li S (2006) Long term effect of Zhang HB, Yang MX, Shi W, Zheng Y, Sha T, Zhao ZW
methylparathion contamination on soil microbial com- (2007) Bacterial diversity in mine tailings compared by
munity diversity estimated by 16S rRNA gene cloning. cultivation and cultivation-independent methods and their
Ecotoxicology 15:523–530 resistance to lead and cadmium. Microb Ecol 54:705–712

123