Microbiological Research 188-189 (2016) 97–105

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Enhancement of plant growth and yields in Chickpea (Cicer arietinum

L.) through novel cyanobacterial and biofilmed inoculants
Ngangom Bidyarani a , Radha Prasanna a,∗ , Santosh Babu a , Firoz Hossain b ,
Anil Kumar Saxena a
a
Division of Microbiology, ICAR, Indian Agricultural Research Institute (IARI), New Delhi 110012, India
b
Division of Genetics, ICAR, Indian Agricultural Research Institute (IARI), 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: The use of Rhizobium inoculants in chickpea is well established; however, meagre efforts have been
Received 3 February 2016 directed towards the use of other microbial supplements for improving nutrient uptake and yields. A set
Received in revised form 6 April 2016 of novel cyanobacterial and biofilmed inoculants were evaluated in chickpea under field conditions. A
Accepted 10 April 2016
significant two-fold enhancement in leghaemoglobin content of nodules and plant biomass was recorded
Available online 13 April 2016
with Anabaena laxa treatment. The inoculants – Anabaena laxa and Anabaena – Rhizobium biofilmed
formulation proved to be the top-ranking treatments. Soil chlorophyll, nitrogen-fixation and available
Keywords:
N possessed high positive direct effects on grain yield through positive – correlations and – high direct
Biofilm
Chickpea
effects and also had high positive indirect effects through other component traits. The cumulative effect
Cyanobacteria of improved plant growth and nutrient uptake exhibited a positive correlation with microbiological
Nodules activity, especially nitrogen fixation, soil chlorophyll and soil available nitrogen. This may account for
Rhizobium the significantly higher yield parameters in the A. laxa treatment, which recorded 50% higher grain yield
Yield (1724 kg ha−1 ) as compared to control (847 kg ha−1 ).
© 2016 Elsevier GmbH. All rights reserved.

1. Introduction nutrient uptake, leading to for sustainable crop production (Uddin

et al., 2014; Welley et al. 2005). These biofertilizers have attracted
Chickpea (Cicer arietinum L.), is the world’s third most impor- much attention for their eco-friendliness and low-cost, besides
tant food legume, cultivated on about 11.5 million ha worldwide. effective and reduced use of non-renewable resources from var-
It is a major winter crop in India among pulses. Chickpea is a good ious agricultural systems across the world (Caliskan et al., 2013;
source of protein, carbohydrates, minerals (phosphorus, calcium, Gopalakrishnan et al., 2015).
magnesium, iron and zinc) and ␤-carotene; its protein quality is The rhizosphere of leguminous crops present a unique eco-
better than that of most other legume crops (Jukanti et al., 2012; logical niche in which nodule formation is an important facet of
Siddique et al., 2012). Chickpea cultivation is a good alternative for the metabolic activities; however, other inoculated microorgan-
rice in areas with poor irrigation to improve soil fertility and weed isms/PGPR are known to improve plant vigour and yields, especially
management (Singh and Mukherjee, 2009). Leguminous crops can in chickpea (Valverde et al., 2006; Verma et al., 2012, 2013). How-
fix nitrogen through symbiotic bacteria in their root nodules. The ever, not much work has been done on other biofertilizers except
use of plant growth promoting (PGP) organisms-based biofertil- for symbiotic associations with Rhizobium.
izers (Rhizobium with Phosphobacteria) increases crop yield by Several reports on the use of cyanobacteria, especially their
fixing the atmospheric nitrogen and improving the availability of role in flooded paddies, have shown that these organisms not only
phosphorus in leguminous crops (Selvakumar et al., 2012). Further- contribute to biological nitrogen fixation, but also improve nutri-
more, the application of PGP organisms-based biofertilizers, along ent uptake and availability, soil structure and function (Mandal
with inorganic phosphorus fertilizer, in leguminous crops signif- et al., 1999). Although the potential of cyanobacteria as inocu-
icantly reduces the amount of phosphate fertilizer and improves lants for crops, other than rice, is a less investigated area, their
promise as biofertilizers and biocontrol agents has been well estab-
lished in wheat, tomato (Solanum lycopersicum), cotton (Gossypium
spp.) and other vegetable crops in the last two decades (Kloepper
∗ Corresponding author. et al., 1991; Zahir et al., 2004; Prasanna et al., 2008, 2012, 2013a).
E-mail address: adhapr@[Link] (R. Prasanna).

[Link]
0944-5013/© 2016 Elsevier GmbH. All rights reserved.
98 N. Bidyarani et al. / Microbiological Research 188-189 (2016) 97–105

Recent reports on the benefits of cyanobacteria, e.g., improved bacterial partners) by using MPN (most probable number), involv-
yields, nutrient uptake, biofortification and soil fertility, in crops ing dilution and plating techniques (Triveni et al., 2013a,b). The
such as wheat (Triticum aestivum), cotton (Gossypium spp.), maize Anabaena-based biofilm was added to the carrier to prepare the
(Zea mays), legumes, and vegetables illustrate the resilience of formulation, maintaining the chlorophyll content as 100 ␮g g−1
cyanobacteria, under not only flooded conditions, but also under carrier (Prasanna et al., 2013a). The bacterial partner in cyanobac-
different terrestrial habitats/crop ecologies with various moisture terial biofilm was maintained as 107 –108 cfu ml−1 (Prasanna et al.,
levels (Karthikeyan et al., 2007; Prasanna et al., 2013a, 2014, 2015; 2011). Preliminary pot experiment was set up with a set of eight
Manjunath et al., 2011; Rana et al., 2012). treatments (Fig. 1a), including novel biofilmed and cyanobacte-
The newly developed concept of biofilmed formulations is based rial inoculants: T1:Control (No inoculation); T2: T. viride–M. ciceri
on the presence of extracellular mucilage in diazotrophic filamen- biofilm-based formulation; T3: T. viride–B. subtilis biofilm-based
tous cyanobacteria, which offers a nutrient—rich niche for bacteria. formulation; T4: T. viride–B. subtilis biofilm formulation; T5: A.
This has been used as a strategy by our group for generating useful torulosa–Pseudomonas sp. biofilm; T6: A. torulosa–Azotobacter sp.
combinations of cyanobacteria with agriculturally beneficial bac- biofilm formulation; T7: A. torulosa–[Link] sp. biofilm-based for-
teria under optimized conditions of growth (Prasanna et al., 2011, mulation and T8: A. laxa formulation. Application of fertilizer in all
2013a,b). Such biofilms showed plant growth-promoting activity pots using 100 kg diammonium phosphate (18% N and 46% P2 O5 )
and enhanced nutrient sequestration in various crops—rice (Oryza was done. This led to the identification of four promising inoc-
sativa), wheat, cotton, mungbean (Vigna radiata), soybean (Glycine ulants, which were further evaluated in a field experiment. The
max), tomato (Solanum lycopersicum), leguminous crops and spices pot experiment was conducted at the net-house of the Division
(Prasanna et al., 2009, 2013a,b, 2014, 2015; Kumar et al., 2013). of Microbiology, ICAR-Indian Agricultural Research Institute, New
Trichoderma is a well investigated biocontrol agent, which also Delhi. The variety of chickpea Pusa 1103 (recommended for the
possesses extensive mucilage and was also explored for biofilm northern region of India), was obtained from the Division of Genet-
preparation with agriculturally important bacteria (Prasanna et al., ics, IARI, New Delhi. Three replicates were used for each treatment,
2013b; Triveni et al., 2013a,b). The major aim of this investigation with 10 kg of soil taken per pot with six seeds/pot. The soil from
was to identify promising cyanobacteria or biofilmed biofertilizers the experimental field, whose details are given below were used
that could augment nutrient availability, crop growth and yields of for filling the pots. Twenty g formulation/pot was added after sow-
chickpea and promising microbiological indices which can relate ing of seeds. Nodule samples were taken at mid-crop stage (after
positively to enhanced yields in biofertilizer-based cropping sys- 30 days of sowing) and the number of nodules and leghaemoglobin
tem. content used as indices to select the promising treatments for field
experiment.

2. Materials and methods

2.2. Field experiment details
2.1. Development and maintenance of biofilms and inoculants
The experiment was carried out in the field at the Division of
The individual strains/organisms used in this study, viz., Agronomy, ICAR-IARI, New Delhi, with the variety Pusa 1103, which
Anabaena torulosa (BF1), Bacillus subtilis (RP24), Mesorhizobium is recommended for the northern region of India, obtained from the
ciceri (thereafter referred to as M. ciceri) and Anabaena laxa (RPAN8), Division of Genetics, ICAR-IARI, New Delhi was used. Delhi is sit-
were obtained from the germplasm of the Division of Micro- uated at a latitude of 28◦ 40 N and longitude of 77◦ 12 E, having an
biology, ICAR-Indian Agricultural Research Institute, New Delhi. altitude of 228.6 m above the mean sea level (Arabian Sea); with
Trichoderma viride (ITCC 2211) was obtained from the Indian Type a mean annual rainfall of 650 mm, and more than 80% generally
Culture Collection (ITCC), Division of Pathology, ICAR-IARI, New occurs during the south-west monsoon season (July–September).
Delhi, India. The strains/organisms were grown in their respective The temperature ranges from 16 to 25 ◦ C, with relative humidity
media—potato dextrose broth for T. viride, nutrient broth for B. sub- ranging from 85 to 90% during the growth period from November
tilis, yeast extract mannitol broth (Fred et al., 1932) for M. ciceri and to March. The fertilizer dose used was 100 kg diammonium phos-
nitrogen-free BG11 medium (Stanier et al., 1971) for A. torulosa phate added per hectare (18% N and 46% P2 O5 ). The soils of the
and A. laxa. Cyanobacterial strains were axenized by standard pro- experimental field had 220 kg ha−1 alkaline permanganate oxidiz-
cedure, employing a set of antibiotics (Kaushik, 1987), grown and able N applied, 14.0 kg ha−1 available P, 260 kg 1 N ammonium
maintained in nitrogen free BG11 medium (Stanier et al., 1971), at acetate exchangeable K, and 0.50% organic carbon, as measured by
a temperature of 28 ± 2 ◦ C. following standard protocols, as given in Kalra and Maynard (1991)
For the development of biofilms, the bacterial cultures of M. and earlier investigations (Karthikeyan et al., 2007; Prasanna et al.,
ciceri and B. subtilis were cultured separately at 30 ± 2 ◦ C in a shak- 2012, 2013a). The pH of the soil was 7.5 (1:2.5 soil and water ratio).
ing incubator, whereas T. viride was maintained as static culture The treatments were arranged as a randomized block design (RBD).
at 30 ◦ C for 1 week. Trichoderma-based biofilms with M. ciceri and The experiment was performed in triplicate plots of 10 m2 each,
B. subtilis were grown in yeast extract mannitol broth and nutri- with 10 rows of 2 m length each and 30 cm spacing between rows.
ent broth, respectively (Triveni et al., 2013a). The biofilms, after The details of treatments used for further field experiment are
2 weeks of growth, were extracted from flasks and thoroughly T1: T. viride–M. ciceri biofilm-based formulation; T2: T. viride–B.
washed with distilled water, centrifuged and vortexed to gener- subtilis biofilm-based formulation; T3: A. torulosa–M. ciceri biofilm-
ate a uniform suspension for analyses (Triveni et al., 2013a,b). based formulation; T4: A. laxa-based formulation and T5: Control,
For the development of Anabaena-based biofilms, 48 h-old bac- i.e., carrier-based, without additional microbial inoculants. The
terial culture of M. ciceri was co-inoculated into 7 d-old culture seeds were coated with 5 g formulation/plot using 1% carboxy
of A. tolorusa in nitrogen free BG11 medium (Prasanna et al., methyl cellulose (CMC) as the sticking agent. Paddy straw compost
2010, 2011) and grown under L:D cycle (16:8 h) with white light was mixed with vermiculite (1:1) and used as carrier (Chaudhary
(50–55 ␮mol photons m−2 s−1 ) at 28 ± 2 ◦ C for a period of 3 weeks et al., 2012; Prasanna et al., 2013a). Sowing was done on 13th
under stationary conditions, as was optimized earlier (Prasanna November 2013 and a mid-term sampling was done after 75 d
et al., 2011, 2013a). The Trichoderma-based biofilms were ana- (7th February 2014) during flowering stage (Fig. 1b). The crop was
lyzed for their colony forming unit (cfu) ml−1 (107 –1010 of the harvested after 150 d of sowing (12th April 2014). A pre-sowing
 N. Bidyarani et al. / Microbiological Research 188-189 (2016) 97–105 99

Fig. 1. (a, b) Layout of pot and field experiment. (c) Nodules on the control and A. laxa treated plants from pot experiment. (d) Representative plants from control and
Anabaena torulosa–Mesorhizobium ciceri biofilm treatments from field experiment.

irrigation followed by two pre-flowering irrigations was given, fol- 539 nm was subtracted from 559 nm and the values of the reduced
lowed by one at pod development stage; all were light irrigations. hemochrome were expressed as ␮M (equivalent to nmoles) g−1
fresh nodules, using the formula given by Appleby and Bergersen
2.3. Biometrical and yield observations (1980).

Dry plant weight (g) was measured at the mid-crop stage sam-
pling and expressed on a per-plot basis. Plant dry biomass (in kg
plot−1 ), number of pods per plant, total yield (kg ha−1 ) and the 2.6. Analysis of soil biochemical and microbiological parameters
number of nodules per plant were measured at the harvest stage.
Dehydrogenase activity was assayed using the method of Casida
2.4. Acetylene reduction activity (ARA) et al. (1964). The values were expressed as ␮g triphenyl formazan
(TPF) g−1 soil d−1 . Microbial biomass carbon (MBC) was estimated
Acetylene reduction activity, as an index of nitrogenase activity using the method of Nunan et al. (1998), after dichromate diges-
of the nodules of chickpea crop, was determined by using gas chro- tion of K2 SO4 extracts. The MBC was measured after back titration
matograph (GC), Model Bruker 450 with Flame Ionisation Detector. with ferrous ammonium sulfate and calculated using the following
Nodules from roots of each treatment were placed in glass test equation:
tubes and the tubes were sealed with stoppers. Ten percent air
space (v/v) of the tube was replaced with acetylene and the tubes
were incubated for 1 h using the methodology, as optimized by BiomassC = 2.64 × CE
Prasanna et al. (2014). The nodules of control treatment (T5) was
included. The activity was performed in triplicate for each treat-
ment. After incubation, 0.1 ml of gas sample from the tubes was where CE = (organic C from fumigated soil) − (organic C from non-
injected into GC for analysis. The ethylene produced was calculated fumigated soil). Microbial biomass C was expressed as ␮g C g−1
and expressed as nmoles of ethylene g−1 fresh nodules h−1 . soil.
Soil chlorophyll was assayed after adding dimethyl sulfoxide
2.5. Leghaemoglobin content of nodules (DMSO) and acetone in 1:1 ratio, at the rate of 4 ml g−1 soil. The
contents were thoroughly shaken and incubated for 48–96 h in
Leghaemoglobin was estimated from fresh nodules (500 mg) by dark at room temperature. Regular shaking was done every 24
macerating with 5 ml 0.1 M of sodium phosphate buffer (of pH 7.4), to extract chlorophyll completely. Data were recorded at 663,
following the methodology of Appleby and Bergersen (1980). The 645, 630, 775 nm, and chlorophyll a concentration was deter-
extract of fresh nodules was filtered and centrifuged at 10000g for mined using a spectrophotometer according to Nayak et al. (2004).
15 min. 5 ml of alkaline pyridine reagent was added and mixed Total polysaccharides were estimated using the method of Lin
with 5 ml of supernatant collected after centrifugation. The solu- (2005) and expressed as mg g−1 soil. Available nitrogen and phos-
tion turned into greenish-yellow color. To the 2.5 ml of solution, a phorus in soil were estimated by following the protocols given
few crystals of sodium dithionate were added to reduce the forma- in Kalra and Maynard (1991). Data on the above-mentioned soil
tion of ferric hemochrome and absorbance was observed at 559 nm parameters were collected at the mid-stage crop sampling, i.e.,
after 2–5 min. A few crystals of potassium hexacyanoferrate were 75 d after sowing. Plant N% was estimated using Kjeldahl method
added to oxidize the hemochrome to the other 2.5 ml of solution and plant P% was estimated via vanadomolybdophosphoric acid
and absorbance was read at 539 nm. The obtained absorbance at method (Jackson, 1967).
100 N. Bidyarani et al. / Microbiological Research 188-189 (2016) 97–105

a 100 3.1. Mid crop stage

90
Leghaemoglobin μM/g nodule fresh wt.

80 Dry weight was significantly higher in T. viride–M. ciceri biofilm

70
(40.53 g) based formulation at mid crop stage (Table 1), while A.
laxa based formulation recorded almost five folds higher ARA, as
60
compared to control. In the present study, cyanobacteria based
50
treatments, both as individual strain or biofilm exhibited better
40
performance for most of the parameters evaluated.
30
Anabaena–M. ciceri biofilm based formulation showed the high-
20 est activity in terms of soil dehydrogenase activity, polysaccharide
10 content and healthy growth of plants vis a vis control (Fig. 1d).
0 Dehydrogenase activity ranging from 4.46 to 3.79 ␮g TPF g−1 soil
T1 T2 T3 T4 T5 T6 T7 T8 d−1 was observed in all the treatments (Table 1). In terms of
Treatments polysaccharide content of soil, Anabaena–M. ciceri biofilm based
b formulation showed the highest value with 71.80 mg g−1 soil,
35.00

30.00 which represented a tremendous increase of 7 folds increase

over the control, followed closely by A. laxa based formulation
25.00 (63.13 mg g−1 soil). Polysaccharide content, which can be a direct
Number of nodules

determinant of biofilms in soil showed a significant positive

20.00
correlation (r = 0.74) with dehydrogenase activity. Interestingly,
15.00 dehydrogenase activity recorded a significant positive correlation
of (r = 0.95) with microbial biomass carbon, available N (r = 0.92),
10.00 available P (r = 0.82), number of pods (r = 0.56), grain yield and
dry biomass (r = 0.65). This illustrates the significant role of the
5.00
biofilmed inoculants in mobilizing nutrients in soil, improving
0.00 plant growth and thereby yields. Microbial biomass carbon (MBC)
T1 T2 T3 T4 T5 T6 T7 T8 was significantly highest in Anabaena–M. ciceri biofilm formula-
Treatments
tion (485.49 ␮g C g−1 soil), followed by A. laxa based formulation
(373.05 ␮g C g−1 soil) and Trichoderma–Bacillus biofilm based for-
Fig. 2. Influence of microbial inoculation on (a) Leghaemoglobin content of
nodules; (b) number of nodules per plant in the various treatments (TI
mulation (325.48 ␮g C g−1 soil). A significant increase of 38–66%
Mesorhizobium ciceri); T2, Anabaena torulosa–Mesorhizobium ciceri biofilm; T3, were recorded in inoculants formulated as compared to that in the
T. viride–Mesorhizobium ciceri biofilm; T4, T. viride–Azotobacter sp. biofilm; T5 control.
Anabaena torulosa–Pseudomonas sp. biofilm;T6 Anabaena torulosa–Azotobacter sp. Nitrogenase activity is an important factor for a leguminous
biofilm; T7, T. viride–B. subtilis biofilm; T8 A. laxa).
crop through which the activity of nodules can be determined,
which ranged from 977.3 to 5736.4 n moles of ethylene g−1 fresh
2.7. Statistical methods nodules h−1 (Fig. 3a), with highest activity in A. laxa treated formu-
lation followed by Anabaena–M. ciceri based biofilm formulation
The experiment was undertaken using a randomized block (3405.1 nmol of ethylene g−1 fresh nodules h−1 ). This illustrated
design (RBD), which contained five treatments, including the con- an increase of 38–80% over control treatment. Leghaemoglobin is
trol. The data obtained in triplicate on the measured parameters an oxygen carrying protein in nodules, which is an important indi-
were analyzed via ANOVA (analysis of variance) using MSTAT-C cator of the nitrogen fixing ability. An increase from 2 to 50% in
statistical package and the critical difference (CD) was calculated at leghaemoglobin content over the control was recorded (Fig. 3b).
5% probability for treatments (Ai-Aj) or T1–T5. Standard deviations In treatments involving the use of A. laxa based formulation
(SD) are denoted by error bars in graphs. Coefficient of variation, (126.35 ␮M g−1 fresh nodules) a two folds increase was recorded,
which represents the ratio of the standard deviation () to the while the treatments—Trichoderma–Bacillus biofilm (65.81 ␮M g−1
mean ␮, was calculated, as an index of the reproducibility and pre- fresh nodules), Trichoderma–M. ciceri biofilm (64.10 ␮M g−1 fresh
cision of the measurements. Pearson’s correlation analyses were nodules) also showed much higher values as compared to control
computed using Microsoft Excel package. Path coefficient analyses treatment (62.54 ␮M g−1 fresh nodules). Soil chlorophyll content
(Dewey and Lu, 1959; Kang, 2015) were carried out as per the stan- was highest in treatment receiving A. laxa based formulation
dard formula (r1k = P1k + r12 P2k + . . .. . .. . .. . .r1n Pnk , where 1 to n are (3.97 ␮g g−1 soil), which was 6–7 folds higher, as compared to
independent variables, k is dependent variable, P and r represent the control treatment (0.60 ␮g g−1 soil). Anabaena–M. ciceri biofilm
direct effects and correlations between traits, respectively). They based formulation showed the next higher values of 2.98 ␮g g−1
were computed using WINDOWSTAT 8.1. soil chlorophyll content (Fig. 4a). Soil chlorophyll is an index
of photosynthetic biomass. A very significant positive correla-
3. Results tion was observed among the following parameters—ARA and
leghaemoglobin (0.97); ARA and soil chlorophyll (r = 0.97); soil
Preliminary pot experiment with eight treatments was under- chlorophyll and leghaemoglobin (0.96). Also, a significant positive
taken (Fig. 1a). This helped in identifying the statistically superior correlation was recorded between soil chlorophyll and dehydro-
treatments in terms of number of nodules and leghaemoglobin con- genase activity (r = 0.82), illustrating that cyanobacteria, being
tent of nodules (Figs. 1c; 2 ). A. laxa based formulation recorded the photosynthetic organisms are contributing to the positive effects
highest values for both these parameters. Dehydrogenase activity on microbial activity and nodule activity.
ranged from 2.6 to 3.5 ␮g TPF/g soil/d, while polysaccharide con- Available N in soil showed values ranging from 203.63 to
tent of 225–430 mg/kg soil were recorded (data not shown). The 87.17 kg ha−1 in which Anabaena–M. ciceri biofilm based formula-
top four three treatments (T. viride–M. ciceri biofilm, T. viride–B. tion showed 30–57% increased over the control (Fig. 4b). Available
subtilis biofilm, A. torulosa–M. ciceri biofilm and A. laxa-based for- N in soil was positively correlated with dehydrogenase activity
mulations), along with control were taken up for field evaluation. (0.91) soil organic carbon (0.70), MBC (0.99) and dry biomass of
 N. Bidyarani et al. / Microbiological Research 188-189 (2016) 97–105 101

Table 1
Influence of microbial inoculants on soil and plant parameters at mid-crop stage of chickpea.

Treatments Dry Polysaccharide Dehydrogenase MBC Available P Plant N% Plant P%

weight/plot (g) (mg/g soil) (␮g TPF/g (␮g C/g (mg/kg soil)
soil/d) soil)

T1 Trichoderma–[Link] biofilm 40.53 59.80 3.92 264.98 9.32 3.75 0.30

T2 Trichoderma–Bacillus biofilm 26.87 46.47 3.79 325.48 10.33 3.29 0.27
T3 Anabaena–[Link] biofilm 33.34 71.80 4.46 485.49 10.70 3.15 0.30
T4 Anabaena laxa (RPAN8) 29.12 63.13 4.17 373.05 10.59 3.63 0.34
T5Control 19.77 57.80 3.50 163.34 8.40 2.41 0.33
a
CD 5% (Ai − Aj) 2.72 3.80 0.11 6.71 0.54 0.37 –
b
% SS Factor A (Treatments) 96.81 94.98 98.15 99.90 91.39 88.91 29.93
CV% 4.83 3.37 1.42 1.11 2.93 6.12 13.31
a
Critical difference at 5% within the treatments (Ai − Aj) or T1–T5.
b
Fraction of Sum of square of treatments (Ai − Aj)/Total Sum of Squares × 100 reflects proportion of total variation caused by treatments; CV, coefficient of variation
represents the ratio of the standard deviation to the mean ␮ and is an index of the reproducibility and precision of the measurements.

Fig. 4. Effect of selected inoculants on (a) Soil chlorophyll; (b) available N in

Fig. 3. Effect of selected inoculants on (a) Nitrogen-fixing potential, measured
soil. Treatments include: T1: T. viride–Mesorhizobium ciceri biofilm; T2: T. viride–B.
as ARA of nodules; (b) Leghaemoglobin content of nodules. Treatments include:
subtilis biofilm; T3: A. torulosa–Mesorhizobium ciceri biofilm; T4: A. laxa and T5:
T1: T. viride–Mesorhizobium ciceri biofilm; T2: T. viride–B. subtilis biofilm; T3: A.
control—only carrier.
torulosa–Mesorhizobium ciceri biofilm; T4: A. laxa and T5: Control—only carrier.

plant (0.76). Available P showed no significant differences among

the treatments with values ranging from 10.71 to 8.40 mg kg−1 soil.
and 4.43 kg plant dry biomass followed by Anabaena–M. ciceri
Available P showed a positive correlation with available N (0.91)
biofilm (63.33) and Trichoderma–Bacillus biofilm (53.00) in num-
and microbial biomass carbon (0.88).
ber of pods per plant and also in plant dry biomass with values
Total plant N and P% showed no significant differences among
4.27 kg and 3.73 kg respectively (Table 2). Grain yield increased
the treated formulations, with values ranging from 3.76 to 2.41%
by 24–50% over control treatment (T5), in all treatments receiving
for plant N% and 0.34 to 0.28% in plant P%. Although inoculated
microbial inoculation. Trichoderma–M. ciceri biofilm based biofer-
treatments showed significantly higher values as compared to con-
tilizer showed the least (36.00) number of pods, even lower than
trol, statistical analyses of plant P revealed that the values of all
control treatment (46.35).
treatments were non-significant. Plant N% recorded a significant
Analysis of variance for plant and soil parameters (Supple-
positive correlation with grain yield (r = 0.62).
mentary Tables 1–3) illustrated the significant effect of microbial
inoculants on all parameters, except% Plant P. Harvest stage anal-
3.2. Harvest stage yses further strengthened the basis for illustrating their positive
influence on dry weight, number of pods and yields. Path analy-
At the harvest stage, the number of pods showed a significant ses illustrated that dry biomass of plants at harvest stage showed
positive correlation with plant dry biomass (r = 0.89) and grain yield a strong correlation with availability of nutrients in soil, including
(r = 0.82), which illustrates that better growth in the presence of carbon and chlorophyll content, clearly establishing the significant
microbial inoculants led to improved yields. A. laxa showed maxi- role of cyanobacteria (Fig. 5). The correlation between the parame-
mum number of pods per plant (87.33), grain yield (1724 kg ha−1 ) ters of ARA, Soil chlorophyll, dehydrogenase, leghaemoglobin, MBC,
102 N. Bidyarani et al. / Microbiological Research 188-189 (2016) 97–105

Table 2
Yield related parameters of chickpea at harvest stage of chickpea.

Treatments Dry weight (g) No. of pods/plant Yield (kg ha−1 )

T1 Trichoderma–[Link] biofilm 2.23 36.0 1110.00

T2 Trichoderma–Bacillus biofilm 3.73 53.0 1469.66
T3 Anabaena–[Link] biofilm 4.26 63.3 1393.66
T4 Anabaena laxa (RPAN8) 4.43 87.3 1724.00
T5 Control 2.83 46.4 847.00
a
CD 5% (Ai − Aj) 0.39 5.77 57.35
b
% SS Factor A (Treatment) 95.69 98.33 99.38
CV% 5.89 5.36 2.33
a
Critical difference at 5% within the treatments (Ai − Aj) or T1–T5.
b
Fraction of sum of square of treatments (Ai − Aj)/total sum of squares × 100 reflects proportion of total variation caused by treatments; CV, coefficient of variation
represents the ratio of the standard deviation to the mean ␮ and is an index of the reproducibility and precision of the measurements.

AA
1.20
0.97
0.52

SC

0.65
1.26
0.72

0.82
0.54

PO

0.51
0.28

-0.43
0.97

0.23
DE
0.59

0.96
-0.45

0.70
0.65

FW

0.69
0.54

-0.14
0.24

0.42
DW

0.26
0.63

-0.06

0.74
0.87

LH
0.67

0.88
-1.60
YLD

0.77
SN

0.91
2.19

0.42
SP

-0.15
-0.10

0.99
PN

0.56
0.05

0.76
PP
-0.11

MB
-1.42

NP
0.44

DB
-0.23

Residual effect
U

Fig. 5. Path diagram representing the relationships among the most significant parameters (AA, SC and SN) evaluated with yield of chickpea. Correlation values between
various traits with AA (ARA; nitrogen-fixing potential), SC (soil chlorophyll) and SN (Available nitrogen in soil), having positive effects are only illustrated. Abbreviations
include: AA, ARA (nitrogen-fixing potential); SC denotes soil chlorophyll; PO, denotes soil polysaccharides; DE, Dehydrogenase activity; FW, fresh weight; DW, dry weight,
LH, leghaemoglobin; SN, available nitrogen in soil; SP, available phosphorus in soil; PN, total N in plants; PP, total phosphorus in plants; MB, microbial biomass carbon; NP,
number of pods; DB, dry weight at harvest stage. Values in bold indicate direct effects, and the other values indicate the correlation between the traits.

Available N, Available P and number of pods with grain yield was nitrogen fixation (1.20), illustrating that cyanobacterial inoculation
very high (0.57–0.87) (Table 3). improved the availability of this element in soil and build up of
The influence of component traits on grain yield was analyzed photosynthetic biomass, thereby leading to improved yields. Fur-
through path coefficient analysis. Path diagram depicting the rela- ther, nitrogen fixation also contributed to grain yield by indirect
tionship between grain yield and selected component traits is effects through all the component traits, of which soil chlorophyll,
presented as Fig. 5, and the detailed diagram is given as Supple- leghaemoglobin, and numbers of pods are important. Soil chloro-
mentary Fig. 1. Available nitrogen in soil exhibited highest direct phyll also possessed high positive indirect effects on grain yield
effect (2.19) on grain yield, followed by soil chlorophyll (1.26) and through nitrogen fixation, leghaemoglobin and number of pods.
 N. Bidyarani et al. / Microbiological Research 188-189 (2016) 97–105 103

Table 3
Comparative performance of treatments, represented in terms of top three ranking treatments for all the parameters evaluated in chickpea.

Parameters Top ranking treatments

Trichoderma–[Link] biofilm Trichoderma–Bacillus biofilm Anabaena–[Link] biofilm Anabaena laxa Control

√ √ √
Fresh weight (g) – –
√ √ √
Dry weight (g) – –
√ √ √
ARA (n moles of ethylene/g fresh nodules/h) – –
√ √ √
Leghaemoglobin (␮M/g fresh nodules) – –
√ √ √
Soil chlorophyll (␮g/g soil) – –
√ √ √
Polysaccharides (mg/g soil) – –
√ √ √
Dehydrogenase (␮g TPF/g soil/d) –
√ √ √
MBC (␮g C/g soil) – –
√ √ √
Available N (kg ha−1 ) – –
√ √ √
Available P (mg/kg soil) – –
√ √ √
Plant N% – –
√ √ √
Plant P% – –
√ √ √
Dry weight (g) – –
√ √ √
No. of pods – –
√ √ √
Yield (kg ha−1 ) – –
7 9 13 15 1

Available nitrogen, among other traits had high desirable indirect improvement in plant and soil microbiological parameters (Gupta
effects on grain yield through majority of component traits viz., et al. 2013; Prasanna et al., 2012, 2013a,b, 2015; Swarnalakshmi
nitrogen fixation, soil chlorophyll, dehydrogenase, fresh weight, et al., 2013; Triveni et al., 2015). Improving bioavailability of nutri-
leghaemoglobin, available phosphorus, microbial biomass, num- ents is an important trait of microbial inoculants, and in this
ber of pods and dry weight. Leghaemoglobin and microbial biomass context N is among the most critical, as it governs soil fertility.
possessed strong undesirable direct effects on grain yield, although Fixation of nitrogen and its mobilisation by different soil micro-
they showed a positive correlation (0.78. and 0.74 respectively) bial and plant enzymes regulate crucial metabolic processes in
with grain yield. However, available nitrogen, soil chlorophyll and plants (Uddin et al., 2014). Roger and Watanabe (1986) observed
available nitrogen had desirable indirect effects. a correlation between pH, available P content of the soil and
the relative abundance of cyanobacteria in rice-field soils. In
the present investigation, we observed a significant correlation
4. Discussion between soil chlorophyll (an index of photosynthetic biomass)
and leghaemoglobin content in nodules, which represents a novel
The rhizosphere of plants resembles a play ground-like report. Also, soil chlorophyll showed a positive correlation with
environment, in which there is competition among beneficial the available nitrogen, phosphorus levels, dehydrogenase activity,
and pathogenic microorganisms leading to mutualistic, para- plant N and plant yields and dry biomass. This emphasizes the
sitic or neutralistic associations. The inoculation of microbes is significant role played by the cyanobacterial inoculants in improv-
known to enhance plant growth and sequestration of micro ing growth and yields of chickpea. The increased uptake of N by
and/macronutrients and indirectly control plant pathogens, plants in plots receiving microbial inoculation and their positive
through selective enrichment and indirect mechanisms (Kloepper correlation with yields reflects the critical role of N fixation by
et al., 1991; Zahir et al., 2004; Prasanna et al., 2008, 2012, 2013a). plants, increased availability in soil and stimulation of uptake by the
The root nodules of chickpea represent a unique niche for inter- plant growth promoting microbes used in the present study. Simi-
actions among the various macro and micro flora and fauna in the lar responses have been recorded by Verma et al. (2013) in chickpea
rhizosphere of legumes. through the use of PGPR-Mesorhizobium combinations. Applica-
Chickpea is a potential rotational crop, which fits well in sev- tions of biofertilizers are known to increase seed yields of late
eral cropping sequences, including those with maize, wheat and sown chickpea (Singh and Mukherjee, 2009). Aparna et al. (2014)
barley cropping systems, especially in India (Verma et al., 2012, observed that the co-inoculation with PGPR in chickpea enhances
2013). It is generally grown in semiarid rainfed conditions, being the number of associative nitrogen-fixers, leading to better growth.
prone to water stress and exposed to increasingly variable and In our study, it was interesting to note that the available N and P
extreme weather conditions. Several plant growth promoting bac- in soil showed a significant correlation with not only soil microbial
teria are known to enhance tolerance to biotic and abiotic stress. activity related parameters, but also with plant dry biomass at both
The adaptability of chickpea crop can be enhanced by inoculation vegetative and harvest stages.
of effective N-fixing microbes in conjunction with improved cul- Several cyanobacteria function as nutrient supplements (biofer-
tural cropping practices or through the use of better cultivars (Gan tilizers), soil conditioners, carbon sequestering and bio remediating
et al., 2009, 2010; Wani et al., 2007). Despite the availability of agents. Research efforts earlier undertaken at IARI, have revealed
more than seventy seven cultivars based on ICRISAT (International the tremendous potential of cyanobacterial inoculants and their
Crops Research Institute for Semi-Arid Tropics)—bred germplasm synergistic interactions with other rhizobacteria for improving soil
in different parts globally, not many concerted efforts on the use fertility and crop yields in the rice-wheat cropping sequence, and
of other microbial inoculants, besides Rhizobium have been under- as biocontrol agents in various crops, including cotton and vegeta-
taken, and cyanobacteria can be promising options, in view of their bles (Venkataraman, 1972; Kaushik, 1987; Prasanna et al., 2012,
independence for carbon and nitrogen. 2013a,b; Babu et al., 2015b; Bidyarani et al., 2015).
The present investigation explores the multifaceted role of The promise of nitrogen-fixing blue green algae in non-flooded
cyanobacterial inoculants (more commonly employed in rice) temperate agricultural soils (Roger and Watanabe, 1986; Reynaud
and newly developed biofilmed biofertilizers, under field condi- and Metting, 1988) has often been a matter of debate. However,
tions. Our earlier work with these inoculants in various crops, recent studies undertaken by Prasanna et al. (2013a, 2014, 2015)
both in pot and field experiments, have shown significant
104 N. Bidyarani et al. / Microbiological Research 188-189 (2016) 97–105

found an increase in soil nutrient status along with increase in crop 5. Conclusions
growth and yields even under non-flooded conditions/intermittent
irrigation of rice crop or in crops receive moderate to low levels In the present investigation, the inter-relationships among var-
of intermittent irrigation, including cotton, wheat and vegetables. ious traits and grain yield revealed the importance of nitrogen
Soil algae are known to play a major role in nutrient cycling in fixation, soil chlorophyll and available nitrogen in soil, as the key
desert and semi desert ecosystem, especially in the cycling of nitro- determining factors towards enhancement of grain yield in chick-
gen, because of the presence of extensive mucilage (Jeffries et al., pea. These traits, through direct as well as indirect effects, can serve
1992). In this context, the cyanobacterial mucilage was harnessed as important component attributes for selection of lines with high
to develop a novel concept of cyanobacteria based biofilms as grain yield potential under biofertilizer-based cropping system.
biofertilizers. In such biofilms, agriculturally useful bacteria were
co-inoculated with cyanobacteria and the resultant biofilms are
Competing interests
used as inoculants in rice, wheat, maize, cotton, vegetables and
legumes, (Prasanna et al., 2011, 2014, 2015). In a similar man-
The authors declare that they have no competing interests.
ner, using Trichoderma as a matrix, inoculants were developed,
which improved plant growth and N savings, besides proving to be
effective biocontrol agents in cotton (Triveni et al., 2013a, 2015). Acknowledgements
Therefore, the growth promoting potential of the biofilms and the
cyanobacterial strains led to enhanced nitrogen fixation; activities This study was supported partly by funds from the ICAR AMAAS
of plant-related enzyme activities, increment in root length, shoot Network Project on Microorganisms granted by Indian Council of
length, soil chlorophyll and other microbiological activities (micro- Agricultural Research (ICAR), New Delhi. The authors also thank the
bial biomass carbon, available N, available P etc.) and uptake by Division of Microbiology, IARI, New Delhi for the facilities provided.
plants.
In the present investigation, a significant increase in both
Appendix A. Supplementary data
microbial biomass carbon and leghaemoglobin in the inoculated
treatments was recorded, compared to control. Although positive
Supplementary data associated with this article can be found, in
correlation of several yield traits with microbiological parame-
the online version, at [Link]
ters was recorded, path analyses, which considers the effect of
005.
all parameters on a single trait revealed interesting results. Both
microbial biomass carbon and leghaemoglobin did not influence
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