Received: 21 September 2016

| Accepted: 1 March 2017

DOI: 10.1002/jobm.201600588

RESEARCH PAPER

Population densities of indigenous Acidobacteria change

in the presence of plant growth promoting rhizobacteria
(PGPR) in rhizosphere

Sadaf Kalam | Subha Narayan Das | Anirban Basu | Appa Rao Podile

Department of Plant Sciences, School of Life

Sciences, University of Hyderabad, Hyderabad, Rhizosphere microbial community has diverse metabolic capabilities and plays a
Telangana, India
crucial role in maintaining plant health. Oligotrophic plant growth promoting
Correspondence rhizobacteria (PGPR), along with difficult-to-culture microbial fractions, might be
Prof. Appa Rao Podile, Department of Plant
involved synergistically in microbe-microbe and plant-microbe interactions in the
Sciences, School of Life Sciences, University of
Hyderabad, P.O. Central University, Gachibowli, rhizosphere. Among the difficult-to-culture microbial fractions, Acidobacteria
Hyderabad, Telangana 500 046, India.
constitutes the most dominant phylum thriving in rhizospheric soils. We selected
Email: [email protected];
[email protected] effective PGPR for tomato and black gram and studied their effect on population
densities of acidobacterial members. Three facultatively oligotrophic PGPR were
Funding information
Department of Science and Technology, Ministry identified through 16S rRNA gene sequencing as Sphingobacterium sp. (P3),
of Science and Technology, Grant number: SR/
Variovorax sp. (P4), and Roseomonas sp. (A2); the latter being a new report of PGPR.
WOS-A/LS-294/2012(G)
In presence of selected PGPR strains, the changes in population densities of
Acidobacteria were monitored in metagenomic DNA extracted from bulk and
rhizospheric soils of tomato and black gram using real time qPCR. A gradual increase
in equivalent cell numbers of Acidobacteria members was observed over time along
with a simultaneous increase in plant growth promotion by test PGPR. We report
characterization of three effective PGPR strains and their effects on indigenous,
underexplored difficult-to-culture phylum-Acidobacteria. We suggest that putative
interactions between these two bacterial groups thriving in rhizospheric soils could be
beneficial for plant growth.

KEYWORDS
Acidobacteria, metagenomics, plant growth promoting rhizobacteria, real time qPCR

1 | INTRODUCTION Plant-associated bacteria play key roles in maintaining

ecological integrity and have subsequent agrobiotechnolog-
Rhizosphere supports complex plant-associated microbial ical applications in crop management. Plant growth promot-
communities that play a vital role in maintaining plant health. ing rhizobacteria (PGPR) are a heterogeneous group of
free-living soil bacteria that aggressively colonize plant roots
and benefit plants by growth promotion via production and
Abbreviations: ACC, 1 aminocyclopropane-1-carboxylic acid; CBli, secretion of an arsenal of regulatory chemicals in the
commercial Bacillus licheniformis; CMC, carboxymethyl cellulose; IAA,
rhizospheric environs [1,2]. PGPR stimulate plant growth
indoleacetic acid; MOLIM, modified oligotrophic isolation medium; PGP,
plant growth promotion; PGPR, plant growth promoting rhizobacteria;
by one or many direct or indirect ways [3]. Indirect
qPCR, quantitative polymerase chain reaction; TRE, tomato root exudates; stimulation of plant proliferation includes suppression of
TRhE, tomato rhizospheric extract. either deleterious microorganisms or root pathogens by
J Basic Microbiol. 2017;9999:1–10. www.jbm-journal.com © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim | 1
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| KALAM ET AL.

producing antagonistic substances like cyanide, proteases, PGPR inoculation enhance the population densities of native
chitinases, antibiotics, competing for nutrients and space in difficult-to-culture bacteria thriving in rhizospheric soils with
the rhizospheric region, and/or inducing systemic resistance concomitant increase in plant growth.
in plants against a broad-spectrum of plant pathogens [1,4].
Direct stimulation involves providing plants with compounds
such as fixed nitrogen, phytohormones like indole-3-acetic 2 | MATERIALS AND METHODS
acid, indole butyric acid, cytokinins, gibberellins or solubi-
lized nutrients like iron, phosphorous, zinc from the soil [1,5]. 2.1 | Seed material
Soil harbors a plethora of microorganisms which include
oligotrophs inhabiting low nutrient environments. Oligo- Tomato (cv. Arka Vikas) seeds were obtained from Indian
trophs are microorganisms exhibiting sluggish growth, low Institute of Horticulture Research (IIHR), Bangalore, India.
metabolic rates, and maintaining low population densities [6]. Black gram (cv. LBG 623) seeds were procured from Central
A small minority (<1%) of the microorganisms in the Research Institute for Dryland Agriculture (CRIDA),
environment can be readily cultivated in vitro under standard Hyderabad, India.
conditions [7]. The terms “difficult-to-culture” and “as yet
uncultivated” are often used to describe the larger fraction of 2.2 | Isolation of rhizobacteria
the microbial community which might be oligotrophs and yet
2.2.1 | Sample collection
to be grown on artificial media [8]. Cultivation-independent
and cultivation-dependent methods have revealed the Tomato plants were collected from fields of Ranga Reddy
predominance of seven bacterial phyla, viz., Proteobacteria, District, Telangana, India where tomato was grown in crop
Actinobacteria, Acidobacteria, Verrucomicrobia, Plancto- rotation with legumes like beans and green gram. The fields
mycetes, Bacteroidetes, and Firmicutes in the rhizosphere of with red sandy loam soil have been treated with full to half
plants. Among these phyla, Acidobacteria members tend to doses of recommended NPK fertilizers viz., nitrogen (urea) @
be far and widely distributed possessing high abundance in 120 ha−1, phosphate (P2O5) @ 50 ha−1 and potash (K2O) @
the soil environment [9], but are considered to be “hard-to- 50 ha−1. No specific permission was required for sampling in
culture” or “difficult-to-culture” [8]. Several reports indicate these locations, and the field studies did not involve any
that Acidobacteria are often associated with plants [10–12] endangered or protected species. For collection of rhizospheric
and recently Kielak et al. [13] confirmed that certain members soil, plants were uprooted using sterile gloves and transferred
of this enigmatic phylum establish successful Acidobacteria- to sterile plastic cover. Collection of non-rhizospheric soil was
plant interactions resulting into efficient plant growth also done followed by proper storage in separate sterile sample
promotion in Arabidopsis thaliana. The concealed diversity containers. The samples were transported to the lab in ice
of difficult-to-culture rhizospheric microbial communities boxes and were stored at 4 °C until further use.
could be studied using gene-targeted metagenomics, coa-
lesced with real time qPCR. The combination offers a 2.2.2 | Preparation of tomato rhizospheric extract
valuable tool to determine target gene copy numbers leading (TRhE) and root exudates (TRE)
to the rapid determination of abundances of specific Preparation of tomato rhizospheric extract (TRhE) and root
microbial lineages in soil [14,15]. exudates (TRE) was done using protocols by da Rocha
Most PGPR exhibit multiple PGP activities [3,16]. The et al. [17] and Dutta et al. [18], respectively. TRhE was
focus so far has been on PGPR taxa that are easy to grow and prepared from tomato rhizosphere soil by incubating 40 g of
amenable to genetic and genomic analyses. Irrespective of tomato roots with tightly adhering soil in 2 L of sterilized
high abundance of Acidobacteria in soil environment, the distilled water for overnight at 4 °C. The extract was filtered
rhizospheres of crop plants have not yet been explored for through Whatman no.1 filter paper. To prepare TRE, 40 g of
such difficult-to-culture groups. The Acidobacteria members tomato roots were washed by rinsing with tap water. The roots
might possess PGP potential analogous to PGPR and could were completely immersed in 1 L of sterile distilled water for
influence each others’ population dynamics in the rhizo- 48 h at 30 ± 2 °C. The extract containing TRE was filtered
spheric niche and thereby contribute to the plant health. through Whatman no.1 filter paper, followed by centrifuga-
Although, reports indicate Acidobacteria members to be tion at 10,000 × g for 10 min, and was sterilized by filtration
associated with plants, the possible intriguing interactions through a 0.22 μm filter (Pall, India).
between Acidobacteria members and other PGPR that thrive
in rhizosphere are not yet known. In the present study, we 2.2.3 | Sample preparation
report isolation and characterization of three native rhizo- Approximately 1 g of tomato rhizospheric soil tightly adhering
bacterial strains from tomato rhizosphere, with potential for to the roots was shaken for 10 min and dispensed in 200 ml of
plant growth promotion, including a novel PGPR. Using 0.1% sodium pyrophosphate solution and 1 mM dithioerythri-
culture-independent approach, we also show that selected tol [17]. The tomato rhizosphere suspension was centrifuged
KALAM ET AL.
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twice for 1 min at 12,000 × g, serial dilutions (10−2, 10−4, and activity, chitinase activity, biofilm formation [16], zinc
10−6) were plated on three different media described below. solubilization [22], and phytase production [23].

2.2.4 | Media 2.4 | Glasshouse evaluation of selected PGPR

Three different low nutrient media were used, viz., VL55 (pH for plant growth promotion
5.5) and VL70 (pH 7.0) [19], and MOLIM (Modified
2.4.1 | Seed treatments
Oligotrophic Isolation Medium; MgSO4–5, KCl–5,
K2HPO4–10.8, NH4Cl–0.4, sodium pyruvate–0.4 mg L−1, Seeds of tomato (Arka Vikas) and black gram (LBG 623)
pH 6.8). The media were amended with penicillin were surface sterilized with 2% sodium hypochlorite (NaOCl)
(100 µg ml−1) and nystatin (100 µg ml−1), and 3% phytagel solution and were dried under laminar airflow in a sterile
was used as the solidifying agent. To mimic rhizospheric atmosphere. Bacterization was done using 1 × 108 CFU ml−1
conditions, the media were also supplemented with TRhE and culture suspensions prepared in 1% sterile carboxymethyl
TRE. All the plates inoculated with tomato rhizosphere cellulose (CMC). Commercially available strain of Bacillus
suspension were incubated at 30 °C (optimum temperature) licheniformis, designated as CBli, was used as a positive
for 48–96 h. Based on colony morphology and character- control along with other test bacterial suspensions for seed
istics, visibly distinct isolates were transferred to fresh bacterization. CMC was employed as a negative control.
medium and maintained as pure cultures. Bacterized seeds of tomato (ten) and black gram (five) were
shaken on a vortex mixer for 3 min with autoclaved distilled
water, and serial dilutions (10−2, 10−4, and 10−6) were plated
2.3 | Characterization of the isolates
onto LB agar plates to determine CFU/seed treated.
2.3.1 | Molecular characterization
2.4.2 | Plant growth promoting activity
Bacteria were grown in LB broth at 30 °C and 160 × g, for 12 h.
Pot trials were conducted in glasshouse. Soil physical and
Genomic DNA was isolated using a standard protocol by Sharma
chemical properties were determined using Soil Testing Kit®
and Singh [20]. One hundred nanogram of genomic DNA was used
(HIMEDIA, India) for the upper soil layer (0–20 cm) at the
as the template for amplification of 16S rRNA gene in a
start of the glasshouse experiment. Plant growth promoting
thermocycler (Eppendorf Mastercycler Gradient, Germany) using
activity of the bacterial isolates was tested using plastic pots
the universal primers (SIGMA, USA): 27F (5′-
(15 cm diameter) filled with soil in the glasshouse. The plants
GTTTGATCCTGGCTCAG) and 1494R (5′-ACGGC-
were maintained in a controlled growth room (16/8 h
TACCTTGTTACGACTT). The PCR mixture contained 2 μl of
photoperiod, 30 ± 2 °C, and 70% relative humidity). The
each primer (10 mM), 15 μl of Emerald Mastermix (TAKARA
pots were watered daily with the same volume of tap water to
Bio, Inc., Japan), 2 μl of DNA template, and 9 μl of autoclaved
keep the soil moist at the same time when needed, and no
MilliQ water in a final volume of 30 μl. DNA amplification was
other nutrients or PGPR inocula were supplied. Initially, PGP
performed with the following thermocycler regime: 4 min at
activity of the test isolates were confirmed in sterilzed soil
95 °C (initial denaturation), followed by 32 cycles for 1 min at
(data not shown) and further experiments were conducted
94 °C (denaturation), 50 s at 50 °C (annealing), 2 min at 72 °-
three times with triplicates in non-sterilized soil.
C (extension), and a single step at 72 °C for 10 min (final
extension). A total of 5 μl of each PCR product was loaded and
visualized after electrophoresis in 1.5% agarose gel. The PCR 2.4.3 | Plant growth measurements
products were purified using Nucleospin® Extract II spin columns After 15, 30, 45, and 60 days, three plants from each treatment
from Macherey Nagel (Germany) following the instructions were randomly selected, uprooted and were then compared
provided by the manufacturer. The eluted PCR products were with the negative control (CMC) in terms of total length (root
sequenced using ABI PRISM 3730XI Genetic Analyzer (Applied length + shoot length) and dry weight of the plants.
Biosystems, USA) at First Base, Malaysia. The isolates were
identified using the EzTaxon server (http://www.ezbiocloud.net/ 2.5 | Soil metagenomic studies
eztaxon; [21]) on the basis of 16S rRNA sequences. The 16S rDNA
sequences of the bacterial isolates were deposited in GenBank and 2.5.1 | Metagenomic DNA extraction
accession numbers were also obtained from NCBI. Metagenomic DNA extraction from bulk soil, rhizosphere
soil (of tomato and black gram grown in non-sterilized soils)
2.3.2 | Plant growth promotion traits at different time points, viz., 15, 30, 45, and 60 days, was done
Plant growth promotion (PGP) activities (in vitro) of the using the PowerSoil® DNA Isolation Kit (MO BIO
bacterial isolates were determined employing standard Laboratories, Inc., CA, USA) following manufacturer’s
protocols for phosphate solubilization, siderophore produc- instructions. Concentration and purity of DNA were checked
tion, HCN production, IAA production, ACC deaminase in Nanodrop spectrophotometer (Thermo Scientific, UK).
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| KALAM ET AL.

2.5.2 | PCR amplification of metagenomic DNA 2.6.3 | Absolute quantification of gene copy
Metagenomic DNA was used as a template for amplification of numbers
partial gene sequences of 16S rRNA gene in a thermocycler The Ct values obtained from the qPCR analyses were used to
(Eppendorf Mastercycler Gradient) using Acidobacteria quantify the corresponding log equivalent cell numbers of the
phylum-specific primers viz., Acd31F (5′ GATCCTGGCT- Acidobacteria members in different rhizosphere samples,
CAGAATC 3′) [24] and 338R (5′ GCTGCCTCCCGTAG- from the standard curve.
GAGT 3′) [25]. The PCR mixture (15 μl) contained 1 μl of
each primer (Acd31F/338R; 10 mM), 7.5 μl of Emerald 2.7 | Statistical analyses
Mastermix (TAKARA Bio, Inc.), 2 μl of metagenomic DNA
Data were analyzed for significant mean differences via two-
template and 3.5 μl of autoclaved MilliQ water. The reaction
way Analysis of Variance (ANOVA) using GraphPad Prism
was set to an initial denaturation step for 3 min at 95 °C,
statistical software (Version 6.0). Whenever required,
followed by 32 cycles of denaturation for 30 s at 94 °C, primer
multiple mean comparisons were performed using Dunnett’s
annealing for 30 s at 55 °C, and elongation for 45 s at 72 °C.
multiple comparisons test. Statistical significance was
The reaction was completed with a final elongation step for
determined at the critical α-level of 0.05.
5 min at 72 °C followed by a hold at 4 °C. The PCR products
were analyzed on 2% agarose gel to detect the presence of
Acidobacteria members in the metagenomic DNA of the 3 | RESULTS
rhizosphere samples.
3.1 | Isolation of rhizobacteria
2.6 | Monitoring changes in population Bacteria were isolated from tomato rhizospheric soil using
densities using real time qPCR culture dependent standard plating technique employing
three different oligotrophic isolation media, viz., VL55 (8
2.6.1 | Preparation of standard curve for
isolates), VL70 (15 isolates), and MOLIM (11 isolates),
calibration of real time qPCR systems
amended with antibiotics, tomato rhizospheric extracts, and
Cell numbers were estimated using DNA extracts made from root exudates. Out of these 34 isolates, three isolates
10-fold serially diluted suspensions of Acidobacterium exhibited distinct and varied biochemical characteristics.
capsulatum (DSMZ 11244) starting at the highest density These isolates were designated as A2 (MOLIM), P3 (VL70),
of 109 cells ml−1. Real time qPCR employing Acd31F/338R and P4 (VL70) and selected for further studies.
primer pair was run in triplicate, and the measured threshold
cycle (Ct) values were plotted against the log of cell numbers 3.2 | Molecular characterization
for each reaction.
Genomic DNA was used as a template for PCR amplification
2.6.2 | Real time quantitative PCR of the 16S rRNA gene using the 27F and 1494R universal
Real time qPCR was performed using metagenomic DNA primer pair resulting in amplicons of approximately 1500 bp.
from tomato and black gram rhizospheres and bulk soil for The similarity of the 16S rRNA gene sequences was obtained
absolute quantification of Acidobacteria members. The by using NCBI and EzTaxon databases. Isolate A2 was
qPCR analysis was carried out in triplicates and repeated identified as Roseomonas sp. with 98.97% 16S rRNA gene
twice. One negative control (containing DNA from a Bacillus sequence similarity with Roseomonas ludipueritiae 170/96
strain as a template to check primer pair specificity) and (T). Isolate P3 was closely related to the members of the
another negative control, with sterile nuclease-free water genus Sphingobacterium and had 97.77% 16S rRNA gene
(Sigma–Aldrich) only, were used in each run. sequence similarity to Sphingobacterium mucilaginosum
Each 15 µl reaction mixture contained the following THG-SQA8(T). Isolate P4 was identified as Variovorax sp.,
ingredients: 7.5 µl of SYBR Premix Ex Taq 2X (TAKARA having 16S rRNA gene sequence similarity of 99.50% with
Bio, Inc.) containing ROX Reference Dye II 50X (TAKARA Variovorax guangxiensis GXGD002(T). The GenBank
Bio, Inc.), 0.5 µl of each primer (10 mM; Sigma–Aldrich), Accession numbers for the 16S rRNA gene sequences of
3.5 μl of autoclaved MilliQ water, and 3.0 µl of template the isolates A2, P3, and P4 are KP789481, KM077439, and
metagenomic DNA (10 ng μl−1). All reactions were run in a KM077440, respectively.
thermocycler (Eppendorf) using Eprealplex software for one
cycle at 50 °C for 2 min; one cycle at 95 °C for 1 min; 40
cycles at 95 °C for 15 s, 55 °C for 20 s, and 72 °C for 30 s.
3.3 | Characterization for plant growth
Furthermore, melting curves were scrutinized to rule out the
promoting traits
possibility of primer dimer formation or any other non- The plant growth promoting traits of the bacterial isolates
specific amplification. were determined in vitro, as shown in Table 1. All the three
KALAM ET AL.
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TABLE 1 Plant growth promoting traits of bacterial isolates

Isolate PS PP ZnS SP HCN ChA IAA ACCd BF
Roseomonas sp. (A2) − − − + − − +++ + +
Sphingobacterium sp. (P3) − − + − − − + + +
Variovorax sp. (P4) − − + + − − + − +

Plant growth promoting (PGP) traits of the rhizobacterial isolates viz., A2, P3, and P4 were determined in vitro using standard protocols.
PS, phosphate solubilization; PP, phytase production; ZnS, zinc solubilization; SP, siderophore production; HCN, hydrogen cyanide production; ChA, chitinase activity;
IAA, indole acetic acid production; ACCd, ACC deaminase activity; BF, biofilm formation; +, positive; −, negative result for the test. For IAA production: +, absorbance
<0.1; ++, absorbance between 0.1 and 0.3; +++, absorbance >0.3.

isolates were negative for phosphate solubilization, phytase seed bacterization with P3, P4, and A2, along with a well-
activity, HCN production and chitinase activity. Zinc characterized PGPR, CBli on the growth of tomato and black
solubilizing capacity was exhibited by P3 and P4 while the gram was assessed in terms of total length (Fig. 1) and dry
isolates A2 and P4 had the ability to produce siderophores. A2 weight (Fig. 2) of seedlings. Tomato and black gram
and P3 were potent ACC deaminase producers while all the responded differently to seed bacterization with respect to
three isolates were positive for IAA production and biofilm total length and dry weight over 60 days at 15 days interval
formation. Out of the three isolates, A2 produced consider- (15, 30, 45, and 60 days).
ably higher amounts of IAA. In tomato plants, the variations in total length with respect
to CMC were non-significant at the initial time points but
were significant at 60 days. CBli exhibited a highly
3.4 | Growth responses of tomato and black significant increase in total length of tomato seedlings.
gram to the selected bacterial isolates in Significant changes (with respect to CMC) were observed in
glasshouse the dry weight of tomato seedlings treated with CBli and P3
The red sandy soil used for glasshouse studies had the over the four time points. The total length and dry weight of
following properties: pH = 7.5, oxidizable organic-C = 1– tomato seedlings was CBli>P3>CMC>P4>A2 at 60 days. In
1.5%, ammoniacal-N (NH4+-N) = 10–15 kg ha−1, nitrate-N response to seed bacterization, the length of seedlings was not
(NO3−-N) = 10–20 kg ha−1, available p = 56–73 kg ha−1, significantly different from each other over the four time
and available potassium (K) = 112–280 kg ha−1. Effect of points in black gram. Isolate A2 was able to significantly

F I G UR E 1 Plant growth responses in glasshouse with respect to total length. Effect of the three rhizobacterial test isolates (A2, P3, and P4) along with
a positive control (CBli) and a negative control (CMC) on total length (cm) of tomato and black gram seedlings were assessed in glasshouse (A) 15 days,
(B) 30 days, (C) 45 days, and (D) 60 days. Values are the means of three replicates and the vertical lines represent ± standard error of the mean. Statistical
analysis was performed using two-way ANOVA for growth followed by Dunnett’s multiple mean comparison test (p < 0.05). a: highly significant
(p < 0.0001), b: moderately significant (p < 0.001), c: relatively less significant (p < 0.05) compared to CMC control
6
| KALAM ET AL.

F I G UR E 2 Plant growth responses in glasshouse with respect to dry weight. Effect of the three rhizobacterial test isolates (A2, P3, and P4) along with a
positive control (CBli) and a negative control (CMC) on dry weight (mg) of tomato and black gram seedlings were assessed in glasshouse (A) 15 days,
(B) 30 days, (C) 45 days, and (D) 60 days. Values are the means of three replicates and the vertical lines represent ± standard error of the mean. Statistical
analysis was performed using two-way ANOVA for growth followed by Dunnett’s multiple mean comparison test (p < 0.05). a: highly significant
(p < 0.0001), b: moderately significant (p < 0.001), c: relatively less significant (p < 0.05) compared to CMC control

enhance total length followed by other isolates (except P4) in value. Thus, the corresponding cell numbers of Acidobacteria
comparison to CMC. At 15 and 30 days, there was moderate members was quantified using the following equation: log
to highly significant increase in dry weight in comparison to cell number = (37.253−Ct)/3.2255.
CMC, but the isolate A2 was able to enhance significantly dry Optimization of real time qPCR lead to determination of
weight of black gram seedlings up to 60 days. Bacterial the equivalent number of Acidobacteria cells in tomato and
treatments affected total length and dry weight of black gram black gram rhizosphere samples and bulk soil. After real time
seedlings in the following order — A2>CBli>CMC>P3> P4 qPCR analysis, the melting curves were scrutinized for
at 60 days. confirming the specificity of amplification. For controls, PCR
was also performed in triplicates, and only late Ct values
(>30) were observed. Single melting peaks at the same
3.5 | Soil metagenomic studies
melting temperature indicated no amplification of non-
Partial 16S rDNA sequences of Acidobacteria members were specific products with the primer sets.
amplified using Acd31F/338R primer pair from metagenomic
DNA of tomato and black gram rhizosphere soils and bulk soil. 3.7 | Effect of bacterial isolates on indigenous
Successful amplification through conventional PCR yielded Acidobacteria population densities
approximately 300 bp amplicons in all the test samples,
An overall increase in equivalent cell numbers of Acidobacteria
confirming the presence of Acidobacteria members in the test
members was observed (Fig. 3) in tomato rhizospheric soil up to
crop rhizospheres and bulk soil. PCR reaction did not yield any
60 days. The number of Acidobacteria members significantly
product with the same primer sets from DNA of non-
increased in the rhizosphere of tomato seedlings treated with
corresponding strain (Bacillus sp.) used as a negative control.
CBli at 60 days, in comparison to other isolates and CMC
(Fig. 3A). Similarly, in black gram rhizosphere, out of all the
3.6 | Real time qPCR enumeration of
isolates, the isolate A2 resulted in a significant increase in
Acidobacteria members numbers of Acidobacteria members at the 60 days time point
The efficiency, slope and correlation coefficient of the qPCR (Fig. 3B). There was a gradual increase of the Acidobacteria
assay using phylum-specific primers were determined based population in the crop rhizospheres over time when compared to
on a standard curve obtained (data not shown). The standard the bulk soil. The pattern of accumulation of Acidobacteria
curve presented a suitable linear correlation as calculated by members was as follows: tomato—CBli>P3>CMC>P4>A2;
regression analysis. The regression equation obtained was: black gram—A2>CBli>CMC>P3>P4, which might be corre-
y = −3.2255x + 37.253, where x is log cell number and y is Ct lated to the plant growth promotion by the isolates.
KALAM ET AL.
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4 | DISCUSSION growth enhancing trait [22,26]. Two isolates A2 and P4

produced siderophores, while A2 and P3 produced ACC
Rhizosphere microbiome plays a cardinal role in community deaminase. Bacteria possessing ACC deaminase break the
dynamics and soil health. Diverse groups of bacteria ethylene precursor ACC into ammonia and α-ketobutyrate,
including PGPR inhabit the soil. Effects of PGPR inoculation thus leading to plant growth [27]. Production of the
on population densities of abundant but difficult-to-culture phytohormone IAA was also implicated in growth promotion
rhizospheric Acidobacteria members is little known. Here, by PGPR. All the three isolates A2, P3, and P4 produced IAA,
we isolated and characterized three facultatively oligotrophic although A2 produced more effectively. Growth regulators
bacteria, including a novel Roseomonas sp. PGPR strain. like ethylene and IAA are known to regulate various stages of
These rhizobacteria were isolated on oligotrophic isolation plant growth commencing at seed germination, aiding
media, and subsequently purified and maintained on nutrient processes of tissue differentiation finally culminating into
rich LB medium, and hence considered as facultative leaf senescence [28]. Thus, the production of ACC deaminase
oligotrophs. and IAA by root colonizers impart better growth of
Potential PGPR exhibit multiple PGP traits. Our results plants [29,30].
on in vitro PGP traits indicated that the rhizobacterial isolates Biofilms enhance rhizobacterial root colonization by
P3 and P4 were positive for zinc solubilization. Zinc retaining moisture and protecting plant roots from phyto-
solubilizing ability by PGPR was regarded as an auxiliary pathogens [31]. All the three isolates exhibited biofilm
forming capacity. We, thus, identified A2 (Roseomonas sp.),
P3 (Sphingobacterium sp.), and P4 (Variovorax sp.) which
exhibited typical PGP traits. The results were in accordance
with those reported by Vaikuntapu et al. [16]. Rhizobacterial
isolates exhibiting multiple PGP activities have been reported
to considerably enhance the growth of different crop
plants [32].
Isolation and exploitation of novel PGPR which are
metabolically and functionally more competent than the
reported and commercial PGPR is essential [33]. Use of
oligotrophic isolation media resulted in the isolation of three
facultatively oligotrophic rhizobacterial isolates (Roseomo-
nas sp., Sphingobacterium sp., and Variovorax sp.) showing
pronounced PGP effect on tomato and black gram. Rose-
omonas spp. are known to exist widely in nature and have
frequently been detected during molecular surveys from
various environments including soil [34,35]. Roseomonas
soli sp. nov. has been isolated from soil in which Chinese
cabbage has been cultivated [35] but was not reported as
PGPR. Whereas, Variovorax sp. stimulated root elongation in
vitro in Brassica plants [36]. Chen et al. [37] examined the
positive effects of Variovorax paradoxus with the ability to
produce ACC deaminase on the growth and development of
A. thaliana. Another ACC deaminase producing rhizobacte-
rium, V. guangxiensis sp. nov. was isolated from banana
rhizosphere [38]. Two species of the genus Sphingobacterium
(S. pakistanensis and S. canadense) isolated from crop
rhizospheres were reported as potent PGPR solely based on
F I G UR E 3 Changes in population densities of Acidobacteria members
over time. Changes in population densities of Acidobacteria members in
one or two in vitro PGP attributes like IAA production and
bulk soil and tomato (A) and black gram (B) rhizospheres in the presence mineral phosphate solubilization [39,40]. We report for the
of test bacteria and CMC was monitored employing real time qPCR over first time detailed in vitro and in planta PGP activities with
the 15 days, 30 days, 45 days, and 60 days time points. The values are Sphingobacterium and Variovorax isolates and the identifi-
means of six replicates (three from each set), and the vertical lines cation of a novel PGPR (at 16S rRNA gene level),
represent ± standard error of the mean. Statistical analysis was performed
Roseomonas sp., exhibiting PGP activities in black gram.
using two-way ANOVA for log cell numbers followed by Dunnett’s
multiple mean comparison test (p < 0.05). a: highly significant
Glasshouse experiments were designed to evaluate plant
(p < 0.0001), b: moderately significant (p < 0.001), c: relatively less growth responses to the three PGPR isolates viz., P3, P4, and
significant (p < 0.05) compared to bulk soil control A2 along with a commercially available strain CBli and a
8
| KALAM ET AL.

negative control CMC on tomato and black gram. Tomato and Thus, the three facultatively oligotrophic rhizobacterial
black gram showed significant variations in their responses to isolates viz., Sphingobacterium sp., Variovorax sp., and
different treatments in the glasshouse. In tomato, CBli Roseomonas sp. exhibited multiple PGP traits and proved to
enhanced plant growth remarkably higher than the other be effective PGPR for tomato and black gram. Change in
isolates and control over the four time points. While in the population densities of Acidobacteria representatives, in the
presence of isolate A2, black gram showed a significant presence of selected PGPR in plant-soil ecosystems, is
increase in plant growth in comparison with other treatments reported the first time. Further investigations are required to
over the four time points. Similar in planta studies using unveil the core interactions of rhizospheric Acidobacteria
PGPR with different crops were reported by Pedraza members which might be crucial for plant growth promotion.
et al. [41] and Vaikuntapu et al. [16].
Recently, researchers evaluated Acidobacteria-plant ACKNOWLED GMENTS
interactions and fruitfully reported for the first time the in SK acknowledges Department of Science and Technology (DST), Govern-
vitro plant growth promoting potential of few Acidobacteria ment of India (GoI) for Women Scientist fellowship and funding [Grant no.
members [13]. But, population densities of the phylum SR/WOS-A/LS-294/2012(G)]. AB acknowledges University Grants Com-
Acidobacteria in the presence of PGPR have not been studied mission (UGC) for fellowship. We extend our sincere thanks to Prof. Ch.
yet. In presence and absence of selected PGPR, we studied the Venkataramana for his critical inputs in the study. We thank DST, GoI, Funds
changes in population density of rhizospheric Acidobacteria for Infrastructure in Science and Technology (FIST), Level II and University
members over time. Acidobacteria constitutes the most Grants Commission Special Assistance Programme (UGC-SAP) support to the
abundant “difficult-to-culture” phylum and comprises a Department of Plant Sciences at University of Hyderabad. The funders had no
major fraction of native soil microbiota as observed from role in study design, collection/ analysis/ interpretation of data, writing of the
RDP data [8,42]. Earlier reports also document the distribu- report, and in the decision to submit the article for publication.
tion of Holophagae (Acidobacteria) and Verrucomicrobia
(Sub-division 1) in bulk and rhizospheric soils [10,11], thus
CONFLICTS OF I NTEREST
strengthening the existence and some possible ecological
The authors declare that they have no conflict of interest.
roles of difficult-to-culture phyla in crop rhizospheres.
Whether or not any changes occur in the population of
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