Plant Soil (2012) 358:143–154

DOI 10.1007/s11104-012-1293-5

REGULAR ARTICLE

Responses of Cajanus cajan and rhizospheric N-cycling

communities to bioinoculants
Rashi Gupta & David Bru &
Virendra Swarup Bisaria & Laurent Philippot &
Shilpi Sharma

Received: 30 September 2011 / Accepted: 9 May 2012 / Published online: 29 June 2012
# Springer Science+Business Media B.V. 2012

Abstract Results A significant increase in growth of C. cajan

Background and aims Bioinoculants are commonly was observed when treated with mixture of three bio-
used for enhancing crop productivity but little informa- inoculants with dry biomass and grain yield increase
tion is available on their effect on key microbial commu- by 330 % and 238 %, respectively. The combination
nities such as those involved in the cycling of nitrogen, a of three bioinoculants also increased the abundance of
major plant nutrient. Here we developed a formulation nitrogen fixers and denitrifiers towards the flowering
combining different bioinoculants (Bacillus megaterium, and maturity stages.
Pseudomonas fluorescens and Trichoderma harzianum) Conclusions The consortium of three bioinoculants
and examined their effects on both Cajanus cajan growth increased plant growth and grain yield of C. cajan.
and N-cycling microorganisms. These bioinoculants also had a positive effect on the
Methods Seven bioinoculant combinations were eval- abundance of several N-cycling microbial communi-
uated in pots under field conditions, and their effects ties stressing the importance of understanding non-
on plant growth were measured using various biomet- target effects of bioinoculants together with their im-
ric parameters. The abundances of the total bacterial pact on plant growth.
and crenarchaeal communities along with those in-
volved in N-cycling were monitored by qPCR at veg- Keywords Nitrification . Denitrification . Nitrogen
etative, pre-flowering, flowering and maturity stages fixation . Microbial communities . Soil . Plant growth
of the crop. promotion . qPCR

Introduction
Responsible Editor: Hans Lambers.

R. Gupta (*) : V. S. Bisaria : S. Sharma The cultivation of legumes and their rotation with other
Department of Biochemical Engineering and crops is of great importance for soil nitrogen (N) enrich-
Biotechnology, Indian Institute of Technology Delhi,
ment, especially in sustainable agriculture. Cajanus ca-
Hauz Khas,
New Delhi 110016, India jan is an important legume crop with high N-fixation
e-mail: [email protected] ability (79 %N derived from the atmosphere) (Espana et
al. 2006). To enhance crop productivity, farmers in India
D. Bru : L. Philippot
treat C. cajan seeds with a local mixture of bioinoculants,
INRA, Université de Bourgogne, Soil and Environmental
Microbiology, Beejamrut, a plant based inoculant, containing bacteria,
BP 86510, 21065 Dijon Cedex, France fungi, actinomycetes, phosphate solubilizing organisms,
144 Plant Soil (2012) 358:143–154

N2 fixers (Sreenivasa et al. 2009) and Trichoderma vir- combinations, on C. cajan growth and grain yield,
ide. This is followed by application of Rhizobium and and the abundance of key functional microbial com-
phosphate solubilizing bacterial biofertilizer (www. munities in its rhizosphere. Since nitrogen is a crucial
moffindia.org/Downloadble/POP/RED%20GRAM. plant nutrient determining the fertility and productivity
doc). Co-inoculation of Pseudomonas fluorescens and of agricultural soils, we focused on microbial commu-
Rhizobium sp. has been reported to increase C. cajan nities involved in N-cycling, viz. nitrogen fixers, deni-
grain yield (40 %) and nodule occupancy (65–85 %) trifiers and ammonia-oxidizers. We hypothesized that
(Tilak et al. 2006). Other bioinoculants have been the dynamics of these microbial guilds in the rhizo-
reported to also enhance disease-free growth of C. cajan, sphere will be affected not only by the plant develop-
e.g. inoculation with Trichoderma harzianum against C. ment but also by the addition of bioinoculants. To test
cajan wilt (Prasad et al. 2002; Singh et al. 2002). Simi- this hypothesis, we conducted a pot experiment under
larly, Pseudomonas fluorescens, with roles in phosphate field condition, for monitoring by qPCR the changes
solubilization and production of indole-3-acetic acid, in abundances of N-cycling microbial communities in
siderophores and 2, 4-diacetyl phloroglucinol (Kumar et the rhizosphere of C. cajan inoculated with different
al. 2010), acts as a biocontrol agent protecting plant roots formulations of three bioinoculants. In addition, we
against parasitic fungi such as Fusarium and Pythium, as also monitored several parameters related to the
well as some phytophagous nematodes (Niranjana et al. growth of C. cajan such as grain yield, and shoot
2009; Siddiqui and Shakeel 2009). There are other and root biomass.
reports as well which prompted the use of Bacillus mega-
terium, Pseudomonas fluorescens and Trichoderma har-
zianum, along with Rhizobium in C. cajan (Rudresh et al. Materials and methods
2005; Niranjana et al. 2009; Anandaraj and Leema Rose
Delapierre 2010). Soil characteristics
Despite bioinoculants being used for enhancing
crop productivity over decades, little information is The soil used in this study was collected from an agri-
available on their non-target effects on microbial com- cultural field of Delhi, India with the following proper-
munities in the rhizosphere of crops. Some studies ties: clayey loam, organic matter content of 0.42 %, pH
investigated the effects of bioinoculants on the diver- of 7.2 (in water), electrical conductivity of 0.04 dS.m−1,
sity of microorganisms in plant rhizosphere using and water holding capacity of 14 %. The nitrogen,
cultivation-based approaches targeting either the total phosphorus, and potassium content of the soil were
microbial community or specific groups (Duponnois 242.4, 12.4 and 228.1 kg ha−1 respectively.
et al. 2005; Mishra et al. 2008; Bomberg and Timonen
2009). However, only a few reports have focused on Microbial strains, compatibility assay and preparation
the effect of bioinoculants on functional microbial of formulation
communities responsible for processes involved in soil
nutrient cycling. Zarea et al. (2009) found an increased The bioinoculants used in this study were Bacillus
yield of two clover plants (Trifolium alexandrinum megaterium MTCC 453, Pseudomonas fluorescens
and T. resupinatum) when inoculated with a combina- LPK2, and Trichoderma harzianum MTCC 801. Ba-
tion of Glomus mosseae and Pheretima sp., which cillus megaterium and T. harzianum cultures were
could be attributed to increased soil microbial bio- procured from the Institute of Microbial Technology,
mass, nitrogen uptake and nitrogenase activity of Chandigarh, India, while P. fluorescens was kindly
free-living N-fixing microbes. Thus, it is important provided by Prof. R. C. Dubey, Gurukul Kangri Uni-
to understand non-target effects of bioinoculants on versity, Haridwar, India.
key rhizospheric microbial communities as a part of Bacillus megaterium and P. fluorescens cultures
their development and release in agricultural soil, be- were maintained as 15 % glycerol stocks at −20°C in
sides their target effects on plants. Sperber medium (Molla and Chowdhury 1984) and
The present study assesses the effect of three bio- King’s B medium (Battu and Reddy 2009), respective-
inoculants (B. megaterium, P. fluorescens and T. har- ly. Trichoderma harzianum was maintained in Rose
zianum) individually and in their non-conventional Bengal Chloramphenicol medium (Gil et al. 2009).
Plant Soil (2012) 358:143–154 145

The three microbial strains were assessed for compat- seeds exhibited a reduction of cfu count by a factor of
ibility prior to their use as bioinoculants using Cross 10 as compared to the inorganic carrier-based formu-
Streak Assay method (Anandaraj and Leema Rose lations used for seed bacterization. The seeds for con-
Delapierre 2010). All strains were compatible with trol (C) were treated with formulation without any
each other and able to grow together without any bioinoculant.
inhibition in growth. For seeding, 144 cylindrical pots (9 treatments × 4
The broths containing 1×108 cfu ml−1 of each mi- sampling points × 4 replicates) of approximate height
crobial strain was used for the preparation of formula- and diameter of 40 cm were filled with soil collected
tions. Five hundred grams of autoclaved-sterilized in the field and mixed well with host specific Rhizo-
talcum powder was used as inorganic carrier. bium strain as recommended by seed producer (pro-
The formulations were prepared as described pre- cured from IARI, New Delhi). The control and the
viously (Sarma et al. 2009). The product was dried in seeds treated with the formulations, individually as
shade to reduce the moisture content. For the present well as in combinations, were sown in each pot at a
study, the combination of two or three bioinoculants depth of about 4–5 cm. Four replicates were set up for
was made by mixing equal quantity of individual each treatment and for each sampling point. The de-
bioinoculant’s formulation. The formulation contained sign used for the experiment was completely random-
2×107 cfu g−1 of each bioinoculant. ized design. The pots were kept under sunlight
(approximate 16/8 photoperiod during the experiment)
Seed sterilization, bacterization and sowing in a field, which experienced a maximum temperature
ranging from 34°C (minimum 26°C) at the start of the
Seeds of Cajanus cajan variety UPAS 120 (an early experiment to 23°C (minimum 8°C) at the end. Pots
maturing variety) were procured from National Seed were irrigated with uniform amount of water at regular
Corporation (NSC), Indian Agricultural Research In- intervals to maintain constant moisture level (approx-
stitute (IARI), New Delhi. Seeds were surface- imately 14 %) in the soil.
sterilized with 70 % ethanol for 30 s followed by
sterilization with NaClO (0.01 %) for 2 min. The seeds Sampling
were then washed with 0.01 N HCl to remove sodium
hypochlorite (Abdul-Baki 1974), and then rinsed Four samplings were performed at different develop-
8 times with sterile water. Seeds of approximately mental stages of crop namely, vegetative (1 month
similar size and shape were selected for seed bacteri- after sowing), pre-flowering (2 months after sowing),
zation (by visual observation and by passing through a flowering (3 months after sowing), and maturity stage
coarse sieve of approximately 0.8 cm mesh size). Seed (4 months after sowing). Sample of roots and above
bacterization was done by mixing a fixed number of ground parts of four randomly selected C. cajan plants
seeds with the formulation prepared for eight treat- were taken for each treatment, and control. The roots
ments including control. The nomenclature used for were shaken vigorously to separate soil not tightly
the treatments were: B. megaterium (B), P. fluores- adhering to the roots. The soil, tightly adhered to the
cens (P), and T. harzianum (T) individually; B. meg- roots, was collected using a soft brush without dam-
aterium + P. fluorescens (BP), B. megaterium + T. aging the root and root nodules and this was termed as
harzianum (BT), and P. fluorescens + T. harzianum “rhizosphere soil”. These rhizosphere samples were
(PT) in combination, and BPT as mixed consortium stored at −20°C (after shock freezing in liquid nitrogen)
(B. megaterium + P. fluorescens + T. harzianum); for DNA extraction.
control i.e. seeds without any inoculation (C), un-
planted (and no inoculation) soil (US). Biometric observations
Before seed sowing, the colony forming units (cfu)
were counted in the formulations using standard dilu- The plants harvested at each time point were assessed
tion and plate count technique and it was found to be for their growth parameters. The root and shoot
~1×106 seed−1 for each of the three bioinoculants. lengths were measured. Root length was measured
Constant cfu of each bioinoculant was maintained in from the point of attachment of the stem base to the
individual as well as combined treatments. Bacterized tip of the tap root. Shoot length was measured from the
146 Plant Soil (2012) 358:143–154

stem base to the tip. Nodulation was recorded by used as molecular markers to assess the abundances of
carefully uprooting the plants at harvest and counting nitrogen fixers, bacterial (AOB) and crenarchaeal
the number of nodules per root system. The composite (AOA) ammonia oxidizers, and denitrifiers, respective-
dry weights of stems and roots were estimated after ly (Table 1). Amplification was carried out in an ABI
drying in oven at 70°C for 24 h (Ruiz et al. 2000). Prism 7900 HT sequence detection system (Applied
Grains were collected from the pods and the grain Biosystems, USA) in a reaction mixture of 15 μl con-
yield per plant was recorded. taining 10 ng of total DNA, SYBR green as dye (Abso-
lute QPCR SYBR Green Rox, Thermo, France), 1 μM
DNA extraction for each primer, and 250 ng of T4gp32 (MP Biomed-
icals, France). At least two independent qPCR assays
DNA was extracted from rhizosphere samples using were performed on each of the four biological replicate
the FastDNA® SPIN Kit for Soil and FastPrep® In- samples and an average technical variability of 16 %
strument (MP Biomedicals, Santa Ana, CA, USA), was observed. Serial dilutions of a known amount of the
according to manufacturer’s instructions and eluted plasmid DNA containing the targeted genes were used
in 40 μl of elution buffer. The concentration of the to generate the standard curves (r2 >0.984 for all assays;
extracted DNA was estimated at 260 nm using a Bio- intercepts ranged between 37.2 and 46.7 and thresholds
Photometer (Eppendorf, Germany) and ranged 180– between 0.05 and 0.55 depending on the targeted
301 μg ml−1. The DNA was then stored at −20°C until genes). PCR efficiency for the different assays ranged
further use. between 86 and 100 %.
Possible inhibitory effect of the co-extracted sub-
Real time PCR assay stances in qPCR amplifications was tested by mixing a
known amount of pGEM-T plasmid with the soil
The total bacterial and crenarcheal communities were DNA extracts or water with the plasmid-specific T7
quantified using 16S rRNA primer-based qPCR assays and SP6 primers, before running qPCR assays. In all
employing primers as described by Muyzer et al. (1993) cases, no inhibition was detected as the measured
and Ochsenreiter et al. (2003), respectively. Additional- cycle threshold (Ct) values were not significantly dif-
ly, genes encoding the catalytic enzymes responsible for ferent between the DNA extracts and the controls
nitrogen-fixation (nifH), ammonia oxidation (amoA), with water. No Template Controls (NTC) gave null
and denitrification (narG, napA, nirK, nirS, nosZ) were or neglectable values.

Table 1 Primers and thermal profiles used for real-time PCR quantification of different functional genes

Target gene Primers Thermal profile No. of cycles Reference

nifH PolF 95°C-15 s/60°C-30 s/72°C-30 s/80°C-30 s 35 Poly et al. 2001

PolR
B-amoA amoA1F 95°C-60 s/55°C-60 s/72°C-30 s 45 Leininger et al. 2006
amoA2R
A-amoA crenamoA23f 94°C-30 s/55°C-30 s/72°C-60 s 10 Tourna et al. 2008
crenamoA616R 92°C-30 s/55°C-30 s/72°C-60 s 25
napA napA3F 95°C-15 s/61°C-1°C/cy-30 s/72°C-30 s/80°C-30 s 6 Bru et al. 2007
narA4R 95°C-15 s/56°C-30 s/72°C-30 s/80°C-30 s 35
nirK nirK 876F 95°C-15 s/63°C-1°C/cy-30 s/72°C-30 s/80°C-30 s 6 Henry et al. 2004
nirK 1040R 95°C-15 s/58°C-30 s/72°C-30 s/80°C-30 s 35
nirS nirS4QF 95°C-15 s/63°C-1°C/cy-30 s/72°C-30 s/80°C-30 s 6 Kandeler et al. 2006
nirS6QR 95°C-15 s/58°C-30 s/72°C-30 s/80°C-30 s 35
narG narGF 95°C-15 s/63°C-1°C/cy-30 s/72°C-30 s/80°C-30 s 6 Bru et al. 2007
narGR 95°C-15 s/58°C-30 s/72°C-30 s/80°C-30 s 35
nosZ nosZ1840F 95°C-15 s/65°C-1°C/cy-30 s/72°C-30 s/80°C-30 s 6 Henry et al. 2004
nosZ2090R 95°C-15 s/60°C-30 s/72°C-30 s/80°C-30 s 35
Plant Soil (2012) 358:143–154 147

Statistical analysis Even individual applications of bioinoculants stimu-

lated plant growth, though the increase was not as high
The experiments were carried out in a completely ran- as mixed consortium (BPT) treatment. While Bacillus
domized design. Standard deviations for each treatment had a maximum effect on root length of C. cajan (21 %
were calculated. The data were subjected to analysis of increase) when applied individually, Trichoderma sp.
variance (ANOVA) using SPSS Statistical System (133 %) had the strongest positive effect on nodulation
(SPSS 16.0 for Windows) with dependent variables as compared to other individual treatments. Amongst the
the respective ‘plant growth parameter’ or ‘gene copy individual treatments, maximum increase in dry mass
number’, and independent variable as either ‘treatment’ (235 %) and grain yield (108 %) was obtained with
or ‘time point’. Comparison between means was made Pseudomonas. Also, both Bacillus and Pseudomonas
using Duncan’s multiple range test (DMRT) at P<0.05 in individual treatments led to an increase in shoot
(Little and Hills 1978). length by approximately 37 % over control.

Quantification of total and functional microbial

Results communities in the rhizosphere

The sampling of rhizosphere and plants were per- Total bacterial and crenarcheal communities
formed at four different growth stages of the plant.
There was an increase in soil pH at the first sampling The qPCR data reveals that the abundances of the
point varying from 7.2 for un-planted soil to 8.3 for bacterial and crenarcheal communities were stimulat-
soils with B and BT treatments. The soil pH then ed in the rhizosphere of C. cajan in all treatments at
remained stable (approximately 8.3–8.7) in all treat- 3rd and 4th time points, respectively (Fig. 2). Com-
ments till the plants attained maturity. parison of the treatment with mixed consortium (BPT)
to the control soil (C) at plant maturity did not show
Effect of bioinoculants on plant growth any significant difference for the total bacterial com-
munity while the abundance of the crenarchaeal com-
Monitoring various plant properties revealed that the munity decreased to almost half.
treatments (mixed consortium, dual, and individual
inoculations) showed stimulation of plant growth as Nitrogen fixers
compared to the control plants at the maturity stage
(4th time point). The mixed consortium of Bacillus sp., The rhizosphere samples collected at different time
Pseudomonas sp. and Trichoderma sp. (BPT) was the points contained nifH gene copies ranging from 4.6×
most effective in enhancing the shoot length (44 %), root 106 to 1×108 copies per gram dry soil (Fig. 3). The
length (80 %), nodulation (100 %), dry mass (330 %) and application of bioinoculants and the successional age
grain yield (238 %) of C. cajan plants (Fig. 1a–e). of the plants were found to have a strong positive
Nodulation was the highest at pre-flowering stage (2nd impact on the abundance of the nitrogen fixers as
time point) followed by significant decrease at later time evidenced by the highest nifH gene copy numbers in
points in all the treatments. BPT treatment at the flowering and maturity stages of
Dual inoculations with different combinations of the the plants with an increase of approximately, 1091 %
three bioinoculants (BP, BT and PT) also led to an en- and 386 % over US and C soils at maturity, respec-
hancement in all the plant parameters over control plants, tively. In general, the highest abundance of nitrogen
though it was up to a lesser extent than the mixed con- fixers was observed towards the end of the plant
sortium. Application of BP, BT and PT led to an increase development for most treatments.
in shoot and root length (approximately 21–25 %). Nod-
ulation was enhanced by all three combinations (BP, BT Nitrifiers
and PT) with maximum increase in case of BT treatment
(100 %). However, amongst the three dual inoculations, The bacterial amoA gene copy number per gram dry soil
dry mass (260 %) and grain yield (182 %) were enhanced ranged from 2.4×106 to 1.5×107. As shown in Fig. 4,
to their maximum by PT and BP treatments, respectively. the number of AOB did not significantly vary with time
148 Plant Soil (2012) 358:143–154

Fig. 1 Growth parameters of Cajanus cajan var. UPAS 120 (a) columns, and among treatments for the same time point are
shoot length (b) root length (c) number of nodules (d) dry mass marked by lower case letters (a to d). C 0 Control, B 0 B.
(e) grain yield, at four time points, viz. vegetative growth stage megaterium, P 0 P. fluorescens, T 0 T. harzianum, BP 0 B.
(1 month after sowing), pre-flowering stage (2 months after megaterium + P. fluorescens, BT 0 B. megaterium + T. harzia-
sowing), flowering stage (3 months after sowing), maturity num, PT 0 P. fluorescens + T. harzianum, BPT 0 B. megaterium
stage (4 months after sowing). Significantly different values + P. fluorescens + T. harzianum, and US 0 un-planted soil. Error
(P<0.05) between different sampling time points in the same bars represent standard deviations
treatment are marked by uppercase letters (A to C) under the

in most treatments. In contrast, in the control soil the to their bacterial counterpart, with crenarchaeal amoA
number of AOB increased after 2 and 3 months before gene copy numbers g−1 dry soil ranging from 9.4×107
returning to the initial abundance at month 4. No signif- to 2.87×108 (Fig. 4). Unlike the AOB, the plant devel-
icant effect of the bioinoculant treatments was observed opment stage had significant effect on the abundance of
at the 4th time point. the AOA with strongest increases of 158–205 % in the
Irrespective of sampling time point and treatment, the treatments with Bacillus (B), Pseudomonas (P) and
number of ammonia-oxidizing archaea was higher (al- Trichoderma (T) individually at 3rd and 4th time points
most two orders of magnitude) in all samples compared compared to the earlier stage of the plant growth.
Plant Soil (2012) 358:143–154 149

Fig. 2 Gene copy numbers

of 16S rRNA g−1 dry soil
under different treatments of
bioinoculants for: (a) total
bacterial community (b) total
crenarcheal community.
Details of sampling points are
same as in legend to Fig. 1.
Significantly different values
(P<0.05) between different
sampling time points in the
same treatment are marked
by capital letters (A to C)
under the columns, and be-
tween different treatments for
the same time point are
marked by lower case letters
(a to d). Error bars represent
standard deviations. For
abbreviations please see leg-
end to Fig. 1

Bacillus megaterium and P. fluorescens bioinoculants genes, except nosZ was highest at the flowering stage
also stimulated the abundance of the AOA but when (Figs. 5 and 6). This trend was particularly clear for the
applied as a consortium (BPT), a reduction of the AOA nirS-denitrifiers for which a 3 to 10 fold increase was
(by 38–46 %) was observed compared to individual observed in all the treatments after 3 months compared to
applications of the bioinoculants after 4 months. the initial growth stage. At the maturity stage, the abun-
dance of the nirS-denitrifiers decreased to that observed at
Denitrifiers the first sampling date. In contrast, a negative effect of
time was observed for the nosZ denitrifiers (Fig. 6c) in
Gene copy numbers of narG ranged from 1.6×108 to majority of treatments with a decrease of 66 % in un-
7.05×108 copies per gram dry soil, napA from 3.9×107 planted soil at maturity stage compared to the initial stage
to 5.6×108, nirK from 4.15×107 to 1.84×108, nirS from of the plants. Only inoculation with BPT had a positive
2.5×107 to 7.3×108, and nosZ from 1.7×107 to 1.77× effect on copies of all denitrification genes compared to
108 per gram dry soil. The abundance of all denitrification the control treatment after 4 months.

Fig. 3 Gene copy numbers g−1 dry

soil of nitrogenase (nifH) in the rhizo-
sphere of Cajanus cajan. Significantly
different values (P<0.05) between
different sampling time points in the
same treatment are marked by capital
letters (A to C) under the columns, and
between different treatments for the
same time point are marked by lower
case letters (a to d). Error bars repre-
sent standard deviations. For abbrevi-
ations please see legend to Fig. 1
150 Plant Soil (2012) 358:143–154

Fig. 4 Gene copy numbers

g−1 dry soil of (a) bacterial
ammonia-monooxygenase
(amoA-B), and (b) archaeal
ammonia-monooxygenase
(amoA-A) in the rhizosphere
of Cajanus cajan. Signifi-
cantly different values (P<
0.05) between different
sampling time points in the
same treatment are marked
by capital letters (A to B)
under the columns, and be-
tween different treatments
for the same time point are
marked by lower case letters
(a to c). Error bars represent
standard deviations. For
abbreviations please see
legend to Fig. 1

Discussion microbial guilds. The three multi-trait bioinoculants se-

lected for the present study were Bacillus megaterium
Cajanus cajan being an important legume crop at global (B), Pseudomonas fluorescens (P), and Trichoderma har-
level, the present study developed an efficient consortium zianum (T). The inoculants employed in the present study
to improve growth of C. cajan, and assessed the non- have also been reported to enhance plant growth in
target effects of this novel association on indigenous different combinations in other plants. The mixed
Fig. 5 Gene copy numbers
g−1 dry soil of (a) periplas-
mic nitrate reductase
(napA), and (b) membrane-
bound nitrate reductase
(narG) in the rhizosphere of
Cajanus cajan. Significantly
different values (P<0.05)
between different sampling
time points in the same
treatment are marked by
capital letters (A to C) under
the columns, and between
different treatments for the
same time point are marked
by lower case letters (a to e).
Error bars represent standard
deviations. For abbrevia-
tions please see legend to
Fig. 1
Plant Soil (2012) 358:143–154 151

Fig. 6 Gene copy numbers

g−1 dry soil of (a) Cu-
containing nitrite reductase
(nirK), (b) cytochrome cd1-
containing nitrite reductase
(nirS), and (c) nitrous oxide
reductase (nosZ) in the rhi-
zosphere of Cajanus cajan.
Significantly different val-
ues (P<0.05) between dif-
ferent sampling time points
in the same treatment are
marked by capital letters (A
to D) under the columns,
and between different treat-
ments for the same time
point are marked by lower
case letters (a to c). Error
bars represent standard
deviations. For abbrevia-
tions please see legend to
Fig. 1

inoculation of Rhizobium sp., P. fluorescens and B. mega- wherein a decrease in number of nodules with soybean
terium enhanced shoot (30 %) and root (85 %) growth of plant age was reported. Amongst the three individual
Vigna radiate (Anandaraj and Leema Rose Delapierre treatments, Trichoderma sp. (T) yielded the highest
2010). Similar increase in plant height, plant biomass nodulation in C. cajan. Similar enhancement in number
and grain yield per plant have been observed in Cicer of nodules by T. harzianum was reported in Rhizobium
aritenium (Rudresh et al. 2005). Dual inoculation of T. treated soil (Shaban and El-Bramawy 2011). These
harzianum and P. fluorescens increased seedling emer- authors suggested that the increase in nodulation in
gence and reduced fusarium wilt disease in Cajanus legumes was due to antagonistic effect between biocon-
cajan (Niranjana et al. 2009). trol agents and plant pathogenic fungi. Using the same
All the plant growth parameters were enhanced by strain, P. fluorescens LPK2, Kumar et al. (2010) ob-
the treatments at maturity, except nodulation, which served an increase in shoot length (23 %), dry mass
reached its maximum at pre-flowering stage. The de- (60 %) and 26 % increase in number of nodules in C.
crease in nodulation at later time points is consistent cajan. The higher nodulation (66 %) in this study is
with an earlier study by Larson and Siemann (1998), likely to be due to difference in crop varieties, soil and
152 Plant Soil (2012) 358:143–154

growth conditions. Enhancement in biometric parame- gene copy numbers observed in the BTP treatment
ters, due to combined applications of PGPR, has been after 2 and 3 months may be attributed to cumulative
observed for other legumes including Vigna mungo and effects of introduced bioinoculants. These effects may
Cicer arietinum (Sarma et al. 2009; Verma et al. 2009). be enhanced supply of P and other nutrients, increased
The increase in growth and yield parameters in C. cajan efficiency of N fixation by Rhizobium, and also better
through the inoculation of Bacillus sp., Pseudomonas plant health due to the antagonism between biocontrol
sp. and Trichoderma sp. may be due to increased P agent (T. harzianum) and the plant pathogenic fungi
solubilization, production of growth promoting substan- (Vinale et al. 2008).
ces, and the biocontrol of soil borne plant pathogens Ammonia oxidation is a key step in nitrification, an
(Alagawadi and Gaur 1988; Rudresh et al. 2005). important process for plant growth as it increases the
Cultivation-independent molecular surveys showed nitrate availability in soil. In the present study, archae-
that the Crenarchaeota within the domain Archaea al ammonia oxidizers have been found to be more
represent an important component of microbial com- abundant in soils than their bacterial counterparts as
munities in aquatic and terrestrial environments (Nicol observed by Leininger et al. (2006). Inoculation with
and Schleper 2006). Their significance in various bio- P. fluorescens and B. megaterium individually resulted
geochemical processes makes it necessary to observe in higher AOA abundance after 4 months while mixed
the impact of bioinoculants on the indigenous cren- inoculation of the bacterial and fungal strains resulted
archeal community along with the bacterial communi- in a significant decrease of the AOA compared to
ty. In this study, we found that the number of individual bioinoculants. Since none of the bioinocu-
crenarchaea increased at month 4 in the control treat- lants contain AOA, this result demonstrates that bio-
ment compared to the unplanted soil (US), suggesting inoculants can indirectly affect functional guilds in the
that similar to bacteria, crenarchaea are also stimulated rhizosphere through competitive interactions or changes
in the plant rhizosphere. In contrast, a study performed in the plant physiology.
in rhizospheres of endemic plants Leucospermum Denitrification is the process that causes loss of
truncatulum and Leucadendron xanthoconus reported nitrogen from agricultural soils. Moreover, it is the
that Crenarcheaota were excluded from plant rhizo- main process responsible for the emission of N2O, an
spheres while being present in non-rhizospheric soil important greenhouse gas contributing to global
(Stafford et al. 2005). As no increase in amplification change (Conrad 1996). The ranges of denitrification
were observed even with increasing template target gene copies g−1 dry soil were in accordance with those
(of Archaea), and the possibility of PCR inhibition previously reported in arable soil systems (Enwall et
by inhibitory root exudates had been ruled out by al. 2010; Bru et al. 2011; Dandie et al. 2011). The
employing bacterial primers, it was suggested that higher abundance of most denitrification genes at the
crenarchaea were out-competed by the more diverse flowering stage could be due to stimulation of bacte-
bacteria in the rhizospheric soil. This discrepancy rial growth in the rhizosphere through increased car-
with our results, indicating that crenarchaea were bon availability by root exudation (Bürgmann et al.
not only present in the rhizosphere but also stimulated 2005). Similar to the nitrogen fixers, addition of the
by C. cajan roots, underlines the need for a better three bioinoculants had the strongest effect on the
understanding of the ecology of crenarchaea in the plant abundance of the denitrifiers. However, since some
rhizosphere. of the inoculated bacterial strains may carry one or
To determine the dynamics of nitrogen cycling more of the targeted denitrifiers genes, it is not possible
microbial communities in soil in response to inocula- to rule out the contribution of the introduced denitrifying
tion with different formulations, we used a cultivation- inoculants, together with an indirect effect due to bene-
independent approach based on qPCR analysis. Our ficial interactions between bioinoculants and the indig-
results clearly show that both plant growth stage, as enous denitrifying community.
well as introduction of non-conventional combinations This study emphasizes the non-target effects of
of bioinoculants significantly affected the targeted mi- selected bioinoculants. This has hitherto been a largely
crobial guilds. N-fixers in rhizosphere samples were ignored aspect in microbial ecology. Significant mod-
more abundant in the treated soils and at later stage of ifications in the microbial communities involved in N-
plant development. The strongest increase in nifH cycling, apart from their direct effects on plant growth,
Plant Soil (2012) 358:143–154 153

were observed. However, the present study is restricted Anandaraj B, Leema Rose Delapierre A (2010) Studies on
to the genetic potential of the system and to one season influence of bioinoculants (Pseudomonas fluorescens, Rhi-
zobium sp., Bacillus megaterium) in green gram. J Biosci
of the crop. As there are various factors affecting the Tech 1:95–99
expression of genes, it is often not sufficient to deter- Battu PR, Reddy MS (2009) Siderophore-mediated antibiosis of
mine the abundance of microorganisms genetically ca- rhizobacterial fluorescent pseudomonads against rice fun-
pable to perform a biogeochemical process to estimate gal pathogens. Int J Pharm Tech Res 1:227–229
Bomberg M, Timonen S (2009) Effect of tree species and
their actual activity. Therefore, though the abundance of mycorrhizal colonization on the archaeal population
N-cycling communities was affected by the bioinocu- of boreal forest rhizospheres. Appl Environ Microbiol
lants, it still remains to be assessed whether the activity 75:308–315
of these microbial guilds is affected as well. Bru D, Sarr A, Philippot L (2007) Relative abundances of
proteobacterial membrane-bound and periplasmic nitrate
reductases in selected environments. Appl Environ Micro-
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Conclusions Bru D, Ramette A, Saby NPA, Dequiedt S, Ranjard L, Jolivet C,
Arrouays D, Philippot L (2011) Determinants of the distri-
bution of nitrogen-cycling microbial communities at the
A non-conventional consortium consisting of B. meg- landscape scale. ISME J 5:532–542
aterium, P. fluorescens and T. harzianum was found to Bürgmann H, Meier S, Bunge M, Widmer F, Zeyer J (2005)
be the most efficient in terms of growth and grain yield Effects of model root exudates on structure and activity of
of C. cajan. This mixed consortium also stimulated the a soil diazotroph community. Environ Microbiol 7:1711–
1724
abundance of the nitrogen-fixers and denitrifiers to- Conrad R (1996) Soil microorganisms as controllers of atmo-
wards the later plant developmental stages, which can spheric trace gases (H2, CO, CH4, OCS, N2O, and NO).
be due either to direct or indirect effect. However, Microbiol Rev 60:609–640
changes in the AOA abundance in response to individ- Dandie CE, Wertz S, Leclair CL, Goyer C, Burton DL, Patten
CL, Zebarth BJ, Trevors JT (2011) Abundance, diversity
ual or combined inoculations clearly demonstrate that and functional gene expression of denitrifier communities
bioinoculants can have significant non-target effects on in adjacent riparian and agricultural zones. FEMS Micro-
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mycorrhizal fungus Glomus intraradices and rock phos-
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indigenous soil microbial community besides their tar- activity in the rhizosphere of Acacia holosericea. Soil Biol
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Acknowledgments RG wishes to acknowledge the French ment. Appl Environ Microbiol 76:2243–2250
Government for supporting her stay in INRA, France, for two Espana M, Cabrera-Bisbal E, Lopez M (2006) Study of nitrogen
months. The authors would like to express their gratitude to fixation by tropical legumes in acid soil from Venezuelan
Prof. R. C. Dubey, Gurukul Kangri University, Haridwar, India Savannas using 15 N. Interciencia marzo ano 31:197–201
for kindly providing the isolate Pseudomonas fluorescens LPK2 Gil SV, Pastor S, March GJ (2009) Quantitative isolation of
and to Dr. Suseelendra Desai, Central Research Institute for biocontrol agents Trichoderma spp., Gliocladium spp.
Dryland Agriculture (CRIDA), Hyderabad, India for measuring and actinomycetes from soil with culture media. Microbiol
the soil properties. Dr M. V. R. K. Sarma’s help with statistical Res 164:196–205
analysis is acknowledged. The comments and suggestions of Henry S, Baudouin E, Lopez-Gutierrez JC, Martin-Laurent F,
two anonymous reviewers helped enhance the manuscript Brauman A, Philippot A (2004) Quantification of denitri-
considerably. fying bacteria in soils by nirK gene targeted real-time PCR.
J Microbiol Methods 59:327–335 (Erratum 61:289–290,
2005)
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