European Journal of Soil Biology 75 (2016) 15e23

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European Journal of Soil Biology

journal homepage: http://www.elsevier.com/locate/ejsobi

Original article

Cyanobacteria-based bioinoculants influence growth and yields by

modulating the microbial communities favourably in the rhizospheres
of maize hybrids
Radha Prasanna a, *, Amrita Kanchan a, Balasubramanian Ramakrishnan a, Kunal Ranjan a,
Siddarthan Venkatachalam a, Firoz Hossain b, Yashbir S. Shivay c, Prameela Krishnan d,
Lata Nain a
a
Division of Microbiology, ICAR- Indian Agricultural Research Institute, New Delhi, 110012, India
b
Division of Genetics, ICAR- Indian Agricultural Research Institute, New Delhi, India
c
Division of Agronomy, ICAR- Indian Agricultural Research Institute, New Delhi, 110012, India
d
Division of Agricultural Physics, ICAR- Indian Agricultural Research Institute, New Delhi, 110012, India

a r t i c l e i n f o a b s t r a c t

Article history: Interactions between ten maize hybrids (Prakash, HM4, HM8, HM9, HQPM1, HQPM5, PEHM7, PEHM3,
Received 26 February 2016 PEHM5 and VH27) and five cyanobacteria based inoculants, along with uninoculated control, were
Accepted 3 April 2016 evaluated for identifying the promising combinations for improved soil microbial activities and crop
production. The rhizosphere soil samples analyzed at mid-crop stage revealed significant interactive
Handling Editor: C.C. Tebbe effects of hybrids and inoculants on the concentrations of glomalin and polysaccharides and dehydro-
genase activity. Acetylene reduction (assayed as an estimate of nitrogenase activity) was three folds
Keywords: higher in the inoculated treatments. In the phospholipid fatty acid based analysis of selected rhizosphere
Community profiles soils, the concentrations of total PLFAs were found to range from 163 to 398 nmol g
1 soil with lowest at
Microbial consortia H1B1 (control) and highest at H5B2 (HQPM1 inoculated with Anabaena þ Nostoc consortium). The
Plant growth promotion principal component analysis suggested that H1B1 (control) and H4B2 (HM9 inoculated with Anabaena
Nutrient availability -Nostoc consortium) shared common characteristics of community profiles with higher abundance of AM
Phospholipid fatty acid profiles fungi and Eukaryotes. Among the inoculants, Anabaena-Azotobacter, Anabaena-Trichoderma biofilms, and
Yield
the Anabaena þ Providencia consortium were identified as the most promising for all the hybrids tested,
while the combination of hybrid Parkash and Anabaena-Trichoderma biofilm was superior to other
combinations, exhibiting highest values for both crop and microbiological parameters. Maize plant
height, was positively influenced by the cyanobacteria-based formulations, though the cob yields were
statistically at par with control (no inoculation), albeit with 20e30% increases in all inoculated treat-
ments. These analyses suggest the promise of these bioinoculants as plant growth promoting options in
the integrated nutrient management strategies for maize hybrids.
© 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction
Maize is an agronomically important crop that is cultivated for
the preparation of human food, animal feed and fodder, and raw
materials for industries. New food habits among the humans and
higher requirements for the production of oil and starch by the
industries have led to an increasing growth rate in the cultivation of
* Corresponding author.
E-mail address: radhapr@gmail.com (R. Prasanna).
maize in many countries, including India [1]. Both the conventional
and molecular approaches have been deployed for increasing pro-
duction and productivity. However, this crop has a heavy reliance
on the chemical fertilizers for increasing its productivity [1]. But,
chemical fertilizers have been found to be hazardous for soil health
in the long-term as well as for the well-being of human and animal
populations. The contemporary agriculture emphasizes on the
reduction of the use of pesticides and inorganic fertilizers; the
search for alternative ways to sustain the agricultural production is
very intensive [2]. The introduction of biological agents is now
preferred to the application of chemical fertilizers as they are not

http://dx.doi.org/10.1016/j.ejsobi.2016.04.001
1164-5563/© 2016 Elsevier Masson SAS. All rights reserved.
16 R. Prasanna et al. / European Journal of Soil Biology 75 (2016) 15e23
only eco-friendly and economical but are also involved in
improving and maintenance of soil biodiversity and health [3].
Grain yield in maize is a complex trait yet there are breeding
programmes to develop hybrids with desired maturity and grain
yield. Since these plants depend on the rhizosphere microbiome for
making the nutrients accessible to them or for phytohormones,
which improve growth [4], different maize genotypes may influ-
ence the composition and diversity of microbial communities in
their rhizospheres differently. Peiffer et al. [5] observed the
enrichment of the rhizosphere microbial members belonging to the
orders Burkholderiales, Oceanospirillales and Sphingobacteriales of
the Proteobacteria and also a heritable variation in the bacterial
diversity in 27 modern maize inbreds, planted across fields. The
bacterial enrichments in the maize rhizospheres can change
dynamically from early to late growth stages [6] and can also serve
as protectants against pathogens. Gaining a better understanding of
these ecological interactions is vital to the breeding and manage-
ment for the sustainable maize production [7].
The use of plant-growth-promoting rhizobacteria (PGPR) to
improve crop production has become an integral part of sustainable
agriculture. Plant growth is directly influenced by PGPR producing
and releasing phytohormones or indirectly influenced by PGPR
limiting the detrimental effects of phytopathogenic organisms in
the rhizosphere or facilitating the availability and uptake of certain
nutrients from the root environment [8,9]. The selected strains of
PGPR are used as seed inoculants as there are increases in seed
emergence, plant weight, crop yields and disease control [10]. The
beneficial effects of cyanobacterial inoculation on crops including
rice, wheat and maize have been recently observed [11e13].
Nevertheless, the alterations in the ecological interactions due to
the application of bioinoculants are not clearly understood. Though,
the colonization of plants, functional activities and their effect on
plant growth are evaluated, it is not known whether there are
changes in the composition of native microbial communities or
enrichments due to the application of bioinoculants in the rhizo-
spheres of maize hybrids. This information is essential to identify
the maize hybrids that can favour better enrichments of beneficial
microorganisms or colonization by the introduced bioinoculants. In
the present study, we examined the changes in the rhizosphere
microbial communities as influenced by the microbial formulations
that consist of plant growth promoting cyanobacteria and their
combinations with rhizobacteria or fungi. The evaluation of
selected plant parameters, soil microbial activities and the
compositional changes in the microbial communities aided in the
selection of promising inoculants and specific inoculants-hybrid
combinations.
2. Materials and methods
2.1. Selection and maintenance of microorganisms
The cyanobacterial strains-BF1 Anabaena torulosa; BF2 Nostoc
carneum; BF3 Nostoc piscinale; BF4 Anabaena doliolum, Anabaena sp.
(CW1), Anabaena sp. (CR1) and bacterial strains, viz. Providencia sp.
(PW5), Azotobacter chroococcum (W5), and Providencia sp. (PR3)
were collected from the germplasm of the Division of Microbiology,
ICAR-Indian Agricultural Research Institute (IARI), New Delhi. The
salient characteristics of these strains include higher nitrogen
fixing ability and IAA production, their ability to promote plant
growth and enhance nutrient mobilisation in soil as well as uptake
by crop plants including maize. The details of their potential for
plant growth promotion in rice and maize are provided in
Refs. [12,13]. Trichoderma viride (ITCC 2211) used for preparing
biofilm was supplied by the Indian Type Culture Collection (ITCC),
Division of Pathology, IARI, New Delhi, India. Cyanobacterial
biofilms- Anabaena -T. viride and AnabaenaeAzotobacter sp. were
developed and characterized, as given in Prasanna et al. [14,15]. The
selected organisms used for this study have been evaluated in
riceewheat cropping system, legumes and in maize hybrids
[12,13,16]. The growth media used were Jensen's medium for
Azotobacter sp., nutrient broth for Providencia sp., and potato
dextrose broth for T. viride (HiMedia Laboratories Pvt. Ltd., Mumbai,
India). The above mentioned organisms were incubated at 30 ± 2  C
in a shaking incubator, except for T. viride, which was processed as
the static culture at 30  C. The standard procedures were used for
axenizing the selected cyanobacterial cultures [17] and grown in
Haffkine flasks using nitrogen-free BG11 medium [18], under a
temperature of 27 ± 1  C (light:dark cycles; 16: 8) and white light
(50e55 mmol photons m
2 s
1).
2.2. Preparation and incorporation of formulations
Paddy straw compost after amendment with vermiculite (1:1)
was used as a carrier, by amendment with the respective cultures
[12,13]. For the cyanobacterial cultures and their respective bio-
films, the chlorophyll content was measured according to the
method of MacKinney [19] and the concentration of chlorophyll
pigments in all the bioinoculants was normalized at 100 mg g
1
carrier. Likewise, the colony forming units were normalized as
107e108 CFU for each bacterial or fungal partner.
2.3. Experiment with the maize crop
The field experiment was conducted at the research farm of
Indian Agricultural Research Institute, New Delhi (28 400 N, 77120
E and 228.6 m above the mean sea level). The mean annual evap-
oration in Delhi is about 850 mm while the mean annual rainfall is
650 mm. More than 80% of rainfall generally occurs during the
southwest monsoon season (JulyeSeptember). The soil is a well-
drained old alluvium soil, classified as a coarse sandy loam mem-
ber, non-acidic, a mixed hypothermic family of Typic Haplustept.
The experimental field soils contained 225 kg ha
1 alkaline per-
manganate oxidizable N, 16 kg ha
1 available P as estimated by
standard protocols [20e22] and a pH of 7.5 (1: 2.5 soil-and-water
ratio). The chemical sources of N, P, and K fertilizers were prilled
urea, single super phosphate, and muriate of potash. The applica-
tion of recommended dose of fertilizers (120:80:60 NPK kg ha
1)
was based on the soil test values, measured and considered as
control; all the microbial inoculant treatments received 50%
N þ full dose of P and K fertilizers. The treatments included: B1-
Control (no inoculation); B2- Cyanobacterial consortium (BF1- 4 of
BF1 Anabaena torulosa; BF2 Nostoc carneum; BF3 Nostoc piscinale;
BF4 Anabaena doliolum); B3- Anabaena sp.eTrichoderma sp. biofilm;
B4- Anabaena sp.e Azotobacter sp. biofilm,; B5- Anabaena
sp. þ Providencia sp. (CW1þPW5) and B6- Anabaena
sp. þ Providencia sp. (CR1þPR3); and ten different maize hybrids
(H1-Parkash, H2-HM-4, H3-HM-8, H4-HM-9, H5-HQPM-1, H6-
HQPM-5, H7-HQPM-7, H8-PEHM-3, H9-PEHM-5 and H10-VH27).
The experimental design was split plot design with three replica-
tions. In the main plots, different bioinoculants were applied, while
in the subplots, half of the recommended dose of fertilizers was
applied to different maize hybrids tested. Each maize hybrid was
grown in 3 m row with 20 cm plant-to-plant and 75 cm row-to-row
spacing. The soil samples were collected at the tasseling and silking
stages.
2.4. Evaluation of soil microbial parameters
The concentrations of Glomalin Related Soil Proteins (GRSPs)
were assayed by adding 20 mM citrate buffer (pH 7.0) at 121  C for
 R. Prasanna et al. / European Journal of Soil Biology 75 (2016) 15e23
90 min [23]. The supernatant was removed by centrifugation at
3000 rpm for 15 min. The extracted GRSPs were diluted again and
mixed with the Bradford dye; the absorbance was recorded at
590 nm and the concentration of GRSPs was expressed in
mg g
1 soil. The total polysaccharides in soils were quantified by the
method of Lin [24], and the concentrations of total polysaccharides
were expressed in mg g
1 soil. The soil dehydrogenase activity was
assayed using 3% triphenyl tetrazolium chloride (3%) and, after
extraction with methanol, the absorbance was measured at 485 nm
[25]. The available N in the soil was quantified using the alkaline
permanganate method as described by Subbiah and Asija [22]. The
acetylene reducing assay was done using the soil samples collected
at the flowering stage according to Prasanna et al. [26].
2.5. Activity of defense enzymes in maize leaves at the flowering
stage
The leaf samples were homogenized with 50 mM Tris HCl buffer
and the homogenates were centrifuged at 12,000 rpm for 20 min at
4  C. Then, the supernatants were stored at
20  C until further
analysis. The activity of polyphenol oxidase (PPO) was assayed
using catechol as the substrate with the modification of the method
of Jennings et al. [27]. The enzyme extract (100 ml each) were
collected in the cuvette (2 ml) containing 0.02 M citrate phosphate
buffer (pH 6.0), proline (5 mg ml
1), and catechol (2 mg ml
1).
Before the addition of catechol, the mixture was aerated, and the
enzyme activity was determined spectrophotometrically at 546 nm
by recording the difference in absorbance at 30 s intervals for 2 min.
One unit of enzyme was defined as the change in absorbance of
0.01 IU min
1.
For the phenylalanine ammonia-lyase activity, the L-phenylala-
nine (2%) in 50 mM TriseHCl (pH 5e6) buffer solution was used.
The reaction mixture was incubated at 40  C for 60 min as
described by Beaudoin-Eagan and Thorpe [28]. The amount of
trans-cinnamic acid formed from L-phenylalanine was measured
spectrophotometrically at 290 nm against the blank of distilled
water. One unit of enzyme (IU) was defined as the change in
absorbance min
1 g
1 fresh weight.
2.6. Plant biometric analyses
At the flowering stage, canopy reflectance of the maize crop was
observed to find the Normalised Difference Vegetation Index
(NDVI) to characterise the extent of green vegetation in an area. The
measurements were taken with the Green Seeker Handheld Optical
Sensor unit (NTech Industries Inc, USA) in all the plots. The bio-
metric measurements and the number of days to 50% male and
female flowering time were recorded for each hybrid. The ear
height and cob yield were recorded at the time of harvest.
2.7. Community profiling by phospholipid fatty acids (PLFAs) in the
rhizosphere soils
The standard protocol of Buyer et al. [29] was employed to
prepare the PLFAs from the soil samples collected at the flowering
stage of the selected treatments (H1B1, H1B2, H1B3, H1B4, H1B5,
H1B6, H2B2, H2B3, H2B6, H4B2, H4B3, H4B6, H5B2, H5B3, H5B6,
H9B2, H9B3, and H9B6). The selection of treatments was based on
their superior performance in terms of soil microbial parameters
and enzyme activities of leaves. The lipid fractions in the fresh soil
samples (5 g each) were extracted using a modified Blight-Dyer
extraction. The stream of nitrogen was used for evaporation; the
lipid fractions were then separated on a solid-phase extraction
column. The methanol was used to elute these fractions, which
were then evaporated under nitrogen. Later, the phospholipids
17
were trans-esterified to fatty acid methyl esters, extracted with
hexane, evaporated and analyzed by gas chromatography. Fatty
acid methyl esters (FAMEs) were separated on a 25 m
long  0.2 mm internal diameter  0.33 mm film thickness column.
The analytical conditions were as follows: temperature of the oven
was 190  C initially, with ramping to 285  C at the rate of
10  C min
1, and then to 310  C at 60  C min
1, followed by a hold at
310  C for 2 min; temperature of the injector was 250  C while that
of the flame ionization detector was 300  C. The system was
controlled using the MIS Sherlock (MIDI, Inc, Newark, DE, USA). The
identification of FAMEs was performed using the MIDI PLFAD1
calibration mix and naming table. The concentrations of FAMEs
were calculated using an internal standard of methyl
nonadecanoate.
Different fatty acid types such as straight, branched, hydroxyl,
monounsaturated fatty acids (MUFA), polyunsaturated fatty acids
(PUFA), dimethyl acetal and other mixed functional groups were
expressed as nmol g
1 soil. These fatty acids were also summed
notionally into different ‘microbial groups' as suggested by Fros-
tegard and Baath [30], Zelles [31] and Ringelberg et al. [32]. The
concentrations of PLFAs representing different microbial groups
were ordinated using principal component analysis based on the
correlation to explore the variability among the treatments using
the statistical analysis package of XL STAT (version 2014.5.3).
2.8. Statistical analyses
The data for the various parameters were analyzed by ANOVA
using WINDOWSTAT 8.0 statistical package. The mean performance
of inoculants (B2eB6 treatments) over uninoculated treatment
(control B1) was calculated individually and denoted as microbial
inoculation efficacy for each hybrid. Pearson's correlation analyses
were performed using the Microsoft Excel.
3. Results
3.1. Changes in the soil functions
The concentrations of GRSPs in the rhizospheres of maize hy-
brids tested with different bioinoculants ranged from 8.7 to
10.8 mg g
1 soil (Table 1). The highest concentration was recorded
in the B2 treatment (Anabaena eNostoc consortium, BF1þ2þ3þ4),
followed by B5 (Anabaena sp. þ Providencia sp., CW1þPW5). The
bioinoculant treatments led to significant increases in the con-
centrations of GRSPs, as compared to control. Total polysaccharides
in the rhizosphere soils were between 4.4 and 5.0 mg g
1. The
highest concentration was recorded in the B5 (Anabaena
sp. þ Providencia sp., CW1þPW5) treatment, while the lowest was
in the B6 treatment (Anabaena sp. þ Providencia sp., CR1 þ PR3).
The concentration of polysaccharides in the control B1 (no inocu-
lation) treatment was at par with that of the B3 treatment.
The soil dehydrogenase activity was significantly higher due to
the application of cyanobacteria-based formulations than the
control B1. While the dehydrogenase activity ranged from 15.2 to
18.2 mg TPF g
1d
1 in these soils, the highest activity was recorded
in the B6 Anabaena sp. þ Providencia sp. (CR1þPR3) treatment. The
inoculated treatments B2eB5 also recorded significantly higher
values as compared to control B1. The acetylene reduction assay
(ARA as an index of nitrogen fixation) showed a three fold higher in
the activity due to the application of cyanobacteria-based formu-
lations, about that of the control. The B4 treatment of Anabaena sp.-
Azotobacter sp. biofilm showed the highest activity, followed by the
B5 Anabaena sp. þ Providencia sp.(CW1þPW5). The rhizosphere
soils had the concentrations of available phosphorus ranging from
34.7 to 46.8 mg kg
1. All the cyanobacteria-based inoculants led
18 R. Prasanna et al. / European Journal of Soil Biology 75 (2016) 15e23

Table 1
Soil functional activities in the rhizosphere of maize hybrids as influenced by the application of cyanobacteria-based inoculants.

Treatment GRSPs Polysaccharide Dehydrogenase ARA nmol ethylene Available phosphorus Available nitrogen
mg g
1 soil mg g
1 soil mg g
1 d
1 g
1 soil h
1 kg ha
1 soil kg ha
1 soil

B1 Control-No inoculation 8.66 4.45 15.20 12.14 34.65 112.67

B2 Anabaena-Nostoc consortium 10.81 4.71 16.99 38.31 43.47 130.03
(BF1þ2þ3þ4)
B3 Anabaena sp. eTrichoderma sp. 9.67 4.42 16.85 37.48 46.78 136.35
biofilm
B4 Anabaena sp. e Azotobacter sp. 9.54 4.78 16.59 39.24 38.16 140.90
biofilm
B5 Anabaena sp. þ Providencia 9.73 5.07 17.82 39.05 40.03 136.00
sp.(CW1þPW5)
B6 Anabaena sp. þ Providencia sp. 9.28 4.13 18.21 35.41 41.98 142.17
(CR1þPR3)
CD (5%) 0.25 0.12 0.51 1.90 2.43 10.54
CV % 5.07 5.01 5.88 11.03 11.58 15.46

significant increases, compared to the control; highest was in the
B3 Anabaena sp.-Trichoderma sp. biofilm, followed by B2 Anabaena
eNostoc consortium (BF1þ2þ3þ4). Likewise, the concentrations of
available nitrogen were also positively influenced by the bio-
inoculants. The B6 Anabaena sp. þ Providencia sp.(CR1þPR3)
showed a significant increase, relative to the control.
3.2. Microbial community changes based on the PLFA profiles
and H4B2 (HM9 inoculated with Anabaena sp þ Nostoc consortium
B2) shared common characteristics of community profiles with
higher abundance of AM fungi and eukaryotes. The community
profiles of H1B5, H2B2, H2B3, H5B2, H9B3, and H9B6 were similar
while those of H1B4, H2B6, H4B3, H5B5 and H9B2 shared similar
characteristics. Likewise, the PLFA community profiles of H1B2,
H1B3, H1B6, H4B6 and H5B6 had comparable characteristics sug-
gesting their similarities.

The concentrations of total PLFAs ranged from 163 to
398 nmol g
1 soil (Table 2) with lowest at H1B1 (control) and
highest at H5B2 (HQPM1 inoculated with Anabaena þ Nostoc con-
sortium, B2). The fatty acid types differed characteristically in these
soils (Table 3). Straight chain fatty acids were negatively correlated
with branched chain fatty acids (
0.57) and poly unsaturated fatty
acids (PUFA) (
0.74 at a ¼ 0.05). The concentrations of 18:1 w9c
and 18:2 w6,9c were negatively correlated (0.9514). When these
fatty acid types were analyzed as biomarkers for the notional
groups of microorganisms, the abundance of Gram-negative bac-
teria was found to be positively correlated with those of actino-
bacteria and fungi but negatively correlated with eukaryotes. The
principal component analysis showed that both the principal
component 1 (PC1), and component 2 (PC2) represented 68.03% of
the variability (Fig. 1). The biplot chart showed that H1B1 (control)
3.3. Enzyme activities in maize leaves
Both the polyphenol oxidase and phenylalanine ammo-
niaelyase enzyme activities were analyzed using the leaf samples.
The phenylalanine ammoniaelyase (PAL) activity was higher than
the polyphenol oxidase activity (Table 4). The activity of PAL in the
leaf tissues was between 1.87 and 8.62 IU g
1 fresh tissue. The
values in the inoculated treatments were statistically at par,
although significantly higher than control B1. The B5 Anabaena sp.þ
Providencia sp.(CW1þPW5) recorded the highest activity, followed
by that of B4 Anabaena sp.-Azotobacter sp. biofilm. The polyphenol
oxidase activity ranged from 1.30 to 1.92 IU g
1 fresh tissue and
showed a positive and significant correlation with the number of
days to male and female flowering (r ¼ 0.36, 0.38, respectively). The
B2 Anabaena eNostoc consortium (BF1þ2þ3þ4) application

Table 2
Microbial groups notionally identified using PLFA biomarkers (nmol PLFAs g soil
1) in the rhizosphere of maize hybrids as influenced by the application of cyanobacteria-based
bioinoculants.

Treatment Gram-positive bacteria Gram-negative bacteria Anaerobe Actinobacteria AM fungi Fungi Methanotrophs Eukaryote

H1B1 18.76cde 46.38c 3.15c 1.55d 0a 5.72d 0a 24.44h

H1B2 19.57def 49.83cd 5.38fg 0.71b 0a 5.72d 0a 18.78g
H1B3 20.66efg 36.33b 3.09b 0a 0a 4.94c 0a 34.99j
H1B4 22.55gh 49.31cd 5.69g 1.00c 0a 7.41ef 0a 14.04f
H1B5 18.10cd 58.86e 4.85de 3.10i 0a 9.55i 1.46c 4.09a
H1B6 20.80fg 51.01cd 10.48j 0a 0a 2.90a 0a 14.80f
H2B2 17.10c 53.44d 5.8g 2.28g 0a 9.40i 0.080b 11.19de
H2B3 13.93b 61.92e 3.64c 2.47h 0a 8.60h 0a 9.43cde
H2B6 21.42fg 52.17d 4.80de 1.76e 0a 8.41h 0a 11.45e
H4B2 11.84a 27.34a 2.30a 0.81b 1.46d 3.31ab 0a 52.94l
H4B3 23.96h 51.12cd 6.84hi 3.78j 0.35c 7.45ef 0a 6.50b
H4B6 20.16ef 31.29a 4.57d 1.04c 0a 4.95c 0a 37.99k
H5B2 18.83cde 63.17e 5.10ef 1.95f 0.29b 7.72fg 0a 2.14a
H5B3 24.22h 51.24cd 7.10i 3.13i 0a 7.02e 0a 7.30bc
H5B6 22.12g 36.16b 6.61h 0a 0a 3.67b 0a 31.43i
H9B2 19.59def 58.64e 5.11ef 4.27k 0a 3.41ab 0a 8.98cd
H9B3 17.15c 53.46d 5.82g 2.19g 0a 9.48i 0.83b 11.07de
H9B6 12.93ab 62.96e 3.61c 2.65h 0a 8.21gh 0a 9.03cd
CD (5%) 8.60 10.17 4.24 1.08 e 1.09 e 7.84

n. d. (not detected-Below the detection limit of the instrument) was considered as 0 for statistical analysis. Means followed by the same letter(s) are not significant by Duncan's
Multiple Range Test.
 R. Prasanna et al. / European Journal of Soil Biology 75 (2016) 15e23 19

Table 3
Concentrations of different fatty acid types (nmol g soil
1) in maize rhizospheres as influenced by the cyanobacteria-based inoculants.

Treatment Total Straight Branched Hydroxy Cyclo MUFA PUFA DMA 18:w9c 18:2w6, 9c 10-methyl

H1B1 163.41c 36.38a 11.94j 0a 0a 12.06j 15.55g 2.00def 17.44j 3.64j 0.98f
H1B2 325.69g 50.16c 10.12i 0.22b 0.65b 10.77i 9.28e 2.66g 12.98fg 2.83fg 0.35b
H1B3 207.32d 35.13a 12.81k 0a 0a 9.16gh 21.09i 1.86de 12.75e 2.98g 0a
H1B4 105.16a 53.45cde 10.50i 0a 0a 8.93fgh 6.53d 2.65g 14.03gh 3.45ij 0.47c
H1B5 131.95b 62.81h 6.73de 0a 0a 7.86cde 1.52ab 1.8d 14.57hi 3.55j 1.15h
H1B6 292.47f 57.42efg 8.86gh 0a 0a 16.72l 6.30d 4.46i 5.01a 1.24ab 0a
H2B2 200.05d 35.23a 11.56j 0a 0a 7.01bc 20.01i 1.54c 10.34d 1.45bc 0a
H2B3 385.91h 61.79gh 5.56c 0a 0a 9.52h 3.58c 1.38bc 13.97gh 3.26hi 0.94f
H2B6 202.14d 52.00cd 10.28i 0.22b 0a 6.78b 5.50d 2.95h 17.39j 4.04k 0.84e
H4B2 295.15f 44.55b 6.79de 0a 0a 8.68efg 29.22j 1.27ab 7.21b 1.83d 0.45c
H4B3 375.37h 71.54i 7.02de 0a 0a 8.13def 1.83ab 1.93def 6.39b 2.10e 1.07g
H4B6 174.65c 54.26cde 9.22h 0a 0a 7.86cde 17.38h 2.09f 6.46b 2.26e 0.47c
H5B2 398.64h 59.97fgh 7.59ef 0a 0a 10.12hi 1.18a 2.04ef 15.24i 3.09gh 0.78de
H5B3 260.90e 63.04h 8.95gh 0a 0a 10.51i 2.70bc 2.62g 8.42c 2.59f 1.16h
H5B6 224.20d 55.86def 1.08a 0a 0a 10.50i 13.74f 2.89h 5.31a 1.61cd 0a
H9B2 382.01h 68.34i cd 0a 0a 13.74k 2.83bc 1.16a 4.75a 1.07a 1.50i
H9B3 199.63d 51.97cd 8.24fg 0a 0a 5.52s 5.52d 2.80gh 20.56k 4.55l 1.05g
H9B6 380.05h 60.21fgh 4.23b 0a 0a 7.34bcd 2.47abc 1.12a 12.45e 3.01gh 0.72d
CD (5%) 9.44 5.74 4.51 e e 2.28 5.14 0.98 2.36 1.10 0.54

n. d. (not detected-Below the detection limit of the instrument) was considered as 0 for statistical analysis. Means followed by the same letter(s) are not significant by Duncan's
Multiple Range Test.

Fig. 1. Principal component analysis of abundances of biomarker PLFAs representing different microbial groups in the rhizosphere soils of maize hybrids, as influenced by the
inoculation of cyanobacterial bioinoculants. The details of selected treatments are provided in Table 1.

resulted in higher activity than the control. Both the B3 Anabaena
sp.-Trichoderma sp. biofilm and the control had the polyphenol
oxidase activities that were statistically at par.
3.4. Plant biometric and yield parameters
The number of days to male flowering was about 50e51; the B6
Anabaena sp.þ Providencia sp.(CR1þPR3) showed late flowering by
one day. But the inoculants such as B2 (Anabaena þ Nostoc con-
sortium, BF1þ2þ3þ4), B3 (Anabaena sp.-Trichoderma sp. biofilm)
and B3 (Anabaena sp.-Trichoderma sp. biofilm) showed similar
values as B1 (control), in terms of days to male flowering. The
number of days to female flowering was more than that of the male
flowering. It took about 53.4e54.5 days. The B6 Anabaena sp.þ
20 R. Prasanna et al. / European Journal of Soil Biology 75 (2016) 15e23

Table 4
Phenylalanine ammonia lyase and polyphenol oxidase activities in leaves of maize hybrids as influenced by the application of cyanobacteria-based inoculants.

Treatment Phenylalanine ammonia lyase (IU g
1leaves f.w.) Polyphenol oxidase (IU g
1leaves f.w.)

B1 Control-No inoculation 1.87 1.30

B2 Anabaena-Nostoc consortium (BF1þ2þ3þ4) 4.85 1.92
B3 Anabaena sp. eTrichoderma sp. biofilm 5.79 1.39
B4 Anabaena sp. e Azotobacter sp. biofilm 7.29 1.59
B5 Anabaena sp. þ Providencia sp. (CW1þPW5) 8.62 1.47
B6 Anabaena sp. þ Providencia sp. (CR1þPR3) 6.86 1.26
CD (5%) 2.03 0.17
CV% 67.42 22.16

Providencia sp.(CR1þPR3) and the control treatments took a day
more than all other inoculants. A significant and positive correla-
tion was recorded between days to male/female flowering with
yield (r ¼ 0.44 & 0.48, respectively). The Normalised Difference
Vegetation Index (NDVI) values ranged from 0.614 to 0.646 due to
different cyanobacterial inoculants, and these indices were statis-
tically at par with B1 control (Table 5). Among the bioinoculant
treatments, the B5 Anabaena sp. þ Providencia sp.(CW1þPW5) had
the highest index while the B2 Anabaena þ Nostoc consortium
(BF1þ2þ3þ4) had the lowest. NDVI showed a significant correla-
tion with number of days to male and female flowering and yield
(r ¼ 0.54, 0.54, & 0.46, respectively).
Highest plant height (168.5 cm) was recorded in the B6 Ana-
baena sp. þ Providencia sp.(CR1þPR3), followed by the B3 Anabaena
sp.-Trichoderma sp. biofilm and the B5 Anabaena sp. þ Providencia
sp. (CW1þPW5) treatments. The plant heights in the inoculated
treatments were significantly higher than that of the control (B1).
The values of ear height, another index of plant growth, ranged
between 81.8 and 87.8 cm in the maize hybrids tested. Plant height
and ear height showed a strong positive correlation (r ¼ 0.82).
There were no significant differences among the cyanobacteria-
based inoculants; only B6 Anabaena sp. þ Providencia
sp.(CR1þPR3) recorded significantly higher values as compared to
control B1. The yield was between 6.45 and 6.88 g plant
1; the
different cyanobacterial inoculants showed statistically non-
significant increase compared to the B1 control treatment. The
yield also positively correlated with plant height and ear height
(r ¼ 0.34 & 0.35, respectively).
4. Discussion
Maize is cultivated year after year in many cropping systems and
the interactions between the plants and soil microorganisms are
critical to plant health and productivity. Dohrmann et al. [33]
retrieved more than 500, 000 bacterial 16S rRNA genes from the
rhizosphere of the field grown maize. They also found that maize
plants selected a core bacterial community, despite the differences
in varietal characters, age or soil. But, the application of chemical
fertilizers in the monoculture of maize to enhance the yield pro-
ductivity, can affect the core microbial community that is essential
for the plant health and productivity.
The rhizosphere niches have greater functional diversity than
the bulk soil [34]. In the present study, we estimated the GRSPs that
are known to contribute towards the aggregation and sequestration
of organic matter. These proteins are of microbial origin, and their
chemical compositions depend largely on the diversity of micro-
organisms including those of arbuscular mycorrhizal fungi [35]. The
recent proteomic studies suggest that this fraction may also contain
significant amounts of other soil-related heat-stable proteins,
mainly of non-mycorrhizal origin [36]. These fractions are closely
related to net primary productivity and constitute a substantial
portion of the terrestrial carbon pool [23]. An enhancement of
7e10% over control was recorded in terms of GRSPs. Analyses of
GRSPs in cyanobacterial cultures used in this study, including the
biofilms, revealed values of 200e300 mg g
1 wet weight or
2400e3900 mg GRSPs g
1 dry weight (pers. commun.), which
provide support to the observed enhancement. The results of the
present study were in agreement with our earlier reports with
maize as well as other crops [13,37] in which more than 4 folds
enhancement in GRSPs relative to the control was recorded. Both
the B2 (Anabaena eNostoc consortium, BF1þ2þ3þ4) and B5 (Ana-
baena sp. þ Providencia sp., CW1þPW5) led to higher concentra-
tions of GRSPs in the present study. These results suggest that the
combination of bioinoculants is an important determinant for the
production of GRSPs, which can subsequently influence the soil
aggregation. Another factor involved in soil aggregation is the
polysaccharide content of soil. This was significantly enhanced only
in B5 (Anabaena sp. þ Providencia sp., CW1þPW5), B2 (Anabaena
eNostoc consortium, BF1þ2þ3þ4) and B4 (Anabaena-Azotobacter
biofilm) over the control B1, illustrating the significance of speci-
ficity and selectivity in microbe-genotype interactions.
The soil enzymes mediate many transformation processes of
elements and are an important determinant of soil fertility [38].
When compared to the enumeration of microbial members from
the soil using the culture medium or microscopical observations,
the activity measurements of soil enzymes are rapid and sensitive
[39]. Since the microbial activity and soil fertility are closely related,
they are often considered to be an early and sensitive indicator of
soil fertility status. Soil dehydrogenase activity was significantly
higher due to the application of cyanobacteria-based formulations,

Table 5
Yield and yield related parameters of maize hybrids as influenced by the application of cyanobacteria-based inoculants.

Treatment Plant height (cm) NDVI Days to male flowering Days to female flowering Ear height (cm) Yield g
1 plant

B1 Control-No inoculation 160.2 0.622 50.7 54.1 81.8 6.45

B2 Anabaena-Nostoc consortium (BF1þ2þ3þ4) 166.7 0.614 50.2 53.6 83.2 6.79
B3 Anabaena sp. eTrichoderma sp. biofilm 166.3 0.629 50.1 53.4 85.0 6.71
B4 Anabaena sp. e Azotobacter sp. biofilm 168.5 0.634 50.6 54.1 85.0 6.77
B5 Anabaena sp. þ Providencia sp. (CW1 þ PW5) 167.7 0.646 50.8 54.1 84.5 6.66
B6 Anabaena sp. þ Providencia sp. (CR1þPR3) 169.7 0.631 51.1 54.5 87.8 6.89
CD (5%) 4.0 0.019 0.7 0.8 3.7 0.52
CV% 4.65 5.823 2.88 2.90 8.6 15.16
 R. Prasanna et al. / European Journal of Soil Biology 75 (2016) 15e23
than that of the control (without any bioinoculant application). The
soil dehydrogenase activity is a good indicator of microbial activity
as these enzymes are of intracellular nature [25] and earlier studies
have shown an increase of 10e25% over the control [11e13,15,16].
In the present study, the use of Anabaena sp. þ Providencia sp.
formulation (B6) showed the best performance, with an enhance-
ment of 20% in the dehydrogenase activity compared to that of
control. Besides these biochemical changes, the cyanobacteria-
based formulations brought about 20e22% enhancement in the
concentrations of available nitrogen and available phosphorus in
the rhizospheres of the maize hybrids tested. In an earlier report,
Shaharoona et al. [40] showed that the combination of rhizo-
bacterium and nitrogen improved the N availability by 60%
compared to that of rhizobacterium alone, and our results of the
present study are in agreement. This is very significant as the bio-
inoculants tested improved the available forms of N and P, more so
in the case of N, which was only applied at the half dose of the
recommended levels.
The maize rhizosphere may support numerous microorgan-
isms for sustaining the functional activities. In a recent study,
Peiffer and Ley [7] reported that there was heritable variation in
the microbial communities of the rhizosphere of 27 field grown
modern maize hybrids, and concluded that maize genotype
significantly influences a and b diversity across fields. In the
present study, the microbial community changes were investi-
gated by profiling the PLFAs of the selected maize genotypes and
bioinoculants. These analyses of biomarker PLFAs for the notional
groups of microorganisms suggested that there were differences
in the abundance of these microbial groups due to the interactions
of maize genotypes and bioinoculants. Some of the maize geno-
types (H4 when applied with the B2 and B3 and H5 with B2
bioinoculant) supported the arbuscular mycorrhizal fungi in their
rhizospheres, evidently from the PLFAs in detectable amounts
while others were not. The actinobacteria were not detected in the
rhizospheres of H1B3, H1B6 and H5B6. The principal component
analysis showed that both the principal component 1 (PC1), and
component 2 (PC2) represented 68.03% of the variability. The
community profiles of these maize hybrids treated with bio-
inoculants formed three different groups: Group I of H1B5, H2B2,
H2B3, H5B2, H9B3, and H9B6; Group II of H1B4, H2B6, H4B3, H5B5
and H9B2; and Group III of H1B2, H1B3, H1B6, H4B6 and H5B6.
The present analysis using the selected rhizosphere samples and
the following groupings indicate the stronger influences of both
the maize genotypes and bioinoculants. More importantly, the
modulation of the effect of genotype by the inoculant was clearly
perceived, which was also reported earlier by Aira et al. [4]. The
causal relationship between the changes in communities and
maize yield is hard to demonstrate with the relatively low reso-
lution method of PLFA profiling used in the present study and also
due to the dynamic changes in the microbial communities during
the plant growth period [34,41].
The identification, screening and characterisation of rhizo-
bacterial isolates have shown their abilities to promote plant
growth and development, and subsequently yield in many crops
including maize [13,42,43]. The presence of a high diversity of
microorganisms capable of plant growth promotion makes the
search for unique species difficult. Besides, the breeding of maize
hybrids had often aimed at enhancing the yields. Hence, the utili-
zation of proven microbial bioinoculants can provide the oppor-
tunities for identifying those interactions that are useful.
Cyanobacterial inoculation, alone or as in consortia is known to
bring out significant enhancement in soil fertility and yields of
several crops [37,44,45]. Our earlier study showed that cyano-
bacterial formulations trigger high levels of plant defense enzyme
activity, especially in tomato plants; this is one of the bioprotection
21
mechanisms against Fusarium wilt [15]. The polyphenol oxidase has
broad substrate specificities, with the capability of oxidizing ortho-
diphenolics, such as caffeic acid and its conjugates, catechol de-
rivatives, and dihydroxyphenylalanine (DOPA). In the present
experiment, the phenylalanine ammoniaelyase (PAL) activity was
found to be higher than the polyphenol oxidase (PPO) activity and
both the activities increased due to the bioinoculants tested. Earlier
studies in maize and rice with these inoculants had illustrated the
positive correlation of micronutrient mobilisation to flag leaf/grains
and PAL/PPO activity [13,45]. However, in these hybrids, PPO ac-
tivity of leaves showed a positive correlation only with days to
male/female flowering, and a positive correlation of PAL/PPO ac-
tivity of roots with Zn concentration in flag leaf, which was
observed an earlier study [13]. The complex interactions between
the maize hybrids and bioinoculants involving the plant defense
enzymes warrant future studies on the changes in the cellular
mechanisms at the level of transcription and proteomics as re-
ported by Faleiro et al. [46] and Planchamp et al. [47] using single
species in maize.
Another important plant attribute which is used as a predictor
of yield is Normalised Difference Vegetation Index (NDVI) or the
greenness index, whose values ranged from 0.614 to 0.646. This is
an important parameter which is known to be a reliable predictor
of yield in maize [48] and in our study, indices were found to
correlate significantly with yield (r ¼ 0.46) and available P in soil
(r ¼ 0.275). Grain yield was observed to show a strong relation-
ship with a number of plant attributes, including ear height and
plant height. Such a positive interaction has been observed
earlier in studies undertaken with a large set of maize varieties,
in different agro-ecological zones in Kenya and Nigeria, which
revealed that grain yield was positively and strongly correlated
with ear height, plant height and days to male/female flowering
[49,50].
Intensive cultivation of crops using fertilizers and pesticides
often leads to the loss of biodiversity and disturbances in the
biologically mediated processes [51]. Kamaa et al. [52] observed
that the application of inorganic fertilizers (120 kg N and
52.8 kg P) resulted in the bacterial diversity loss in a thirty-two-
year-old long-term trial in Kenya. Lazcano et al. [53] reported
that the application of inorganic fertilizer decreased the abun-
dance of PLFA biomarkers for Gram-negative bacteria compared to
the manure application. In a recent study in the soil and rhizo-
sphere of field-grown sugarcane, Paungfoo-Lonhienne et al. [54]
found that both the composition and taxon richness of fungal
communities were modified by the nitrogen fertilizer application,
even promoting the fungal genera with known pathogenic traits.
The use of bioinoculants along with fertilizers at half of the rec-
ommended dose of N led to the marginal increases in the yield
over the control (B1) with the full recommended dose of N along
with P and K fertilizers in our study. This kind of integrated
strategy with appropriate selection of plant genotype, microbial
inoculants, and lower fertilizer quantities is vital to the long-term
sustainability of maize cultivation.
The application of bioinoculants to attract and support complex
microbial communities that contribute to plant health and yield is a
simpler approach than the breeding of several new maize geno-
types. Also, such a strategy comprises dual objectives of obtaining
high-yield and responsiveness to favouring only the beneficial
microbial interactions in the rhizosphere soils [3,4]. The cyano-
bacterial formulations tested are quite promising and the
complexity of these formulations can provide resilience and pro-
mote the functional activities in the maize rhizosphere, even under
the stress conditions. These combinations of bioinoculants need to
be tested further for their synergy in different agro-climatic con-
ditions where maize is intensively cultivated.
22 R. Prasanna et al. / European Journal of Soil Biology 75 (2016) 15e23
5. Conclusions
The cyanobacteria-based inoculants resulted in higher levels of
soil functional activities while fertilizers alone showed no similar
influences. The bioinoculation strategy led to the favourable
changes not only in the composition of microbial communities in
the rhizosphere but also enhanced plant growth and development.
The integrated application of bioinoculants along with a half dose
of N gave marginal increases in maize yields; the benefits accrued
from the bioinoculant application included a major savings on the
chemical fertilizers. The genotypic influences on the rhizosphere
microbial communities were characteristically altered by the
combinations of bioinoculants tested, emphasizing the importance
of selecting the right combinations of bioinoculants for a particular
genotype depending on the soil conditions.
Acknowledgements
This investigation was partially supported by the funds from the
Network Project on Microorganisms “Application of Microorgan-
isms in Agricultural and Allied Sectors” (AMAAS/2014-17/PF/40)
granted by Indian Council of Agricultural Research (ICAR), New
Delhi to RP and SERB project (SR/S0/PS/164/2010), DST, Govern-
ment of India to BR. We gratefully acknowledge the Division of
Microbiology, ICAR-IARI, New Delhi for providing the necessary
facilities to undertake this study. The authors state that they have
no conflicts of interest.
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