Europ. J.

Agronomy 30 (2009) 296303

Contents lists available at ScienceDirect

European Journal of Agronomy

journal homepage: www.elsevier.com/locate/eja

Effect of Gluconacetobacter diazotrophicus and Trichoderma viride on soil health,

yield and N-economy of sugarcane cultivation under subtropical climatic
conditions of India
R.L. Yadav a, , Archna Suman b , S.R. Prasad c , O. Prakash d
a

Indian Institute of Sugarcane Research, P.O. Dilkusha, Rae-barely Road, Lucknow, UP 226 002, India
Soil Microbiology Laboratory, IISR, Lucknow, India
c
Division of Crop Production, IISR, Lucknow, India
d
Krishi Vigyan Kendra, Lucknow, India
b

a r t i c l e

i n f o

Article history:
Received 12 June 2008
Received in revised form 22 January 2009
Accepted 22 January 2009
Keywords:
Bioagent
Sugarcane
Soil health
Organic carbon
Gluconacetobacter
Trichoderma
Soil microbial biomass
N-economy

a b s t r a c t
Intensive cropping and exhaustive nature of sugarcanewheatrice cropping system in the Indo-Gangetic
Plains of South Asia have led to the depletion of soil organic carbon content and inherent soil fertility
resulting in a serious threat to the sustainability of these production systems. Bioagents like Gluconacetobacter diazotrophicus and Trichoderma viride have great potential to restore soil fertility and promote
sugarcane growth. Field experiments, therefore, have been conducted to study the integrated effect of
bioagents (G. diazotrophicus and T. viride), Farm Yard Manure (FYM) and fertilizer N on sugarcane rhizosphere, crop yield and N economy for two crop cycles during 20042006 and 20052007 crop seasons at
Lucknow, in the middle Indo-Gangetic plain region. Both bioagents could survive and colonize sugarcane
rhizosphere and FYM improved their colonization. Enhanced soil microbial population and microbial
carbon (SMC) and nitrogen (SMN) with increasing N level were probably due to more available N in the
soil. FYM/bioagents amendment further enhanced the microbial carbon. The uniform increase in the
fraction of SMC and SMN of total organic carbon indicated that immobilization/mineralization was being
maintained in the soil where enhanced microbial biomass might act later as a source of nutrients.
Bioagents ammended FYM enhanced the uptake of N, P and K in sugarcane at all the levels of fertilizer
N. It was mainly due to the enhanced nutrient availability in the rhizospheric soil as the soil organic C and
available N, P and K content increased with the application of bioagents/FYM. A saving of 76.3 kg N ha1
was envisaged by the use of G. diazotrophicus inoculated FYM with marginal (2.4 t ha1 ) decline in the
cane yield. Application of T. viride enriched FYM, however, brought economy in the use of fertilizer N by
45.2 kg ha1 and also increased the yield by 6.1 t ha1 compared to the control treatment. Overall, strategic
planning in terms of an integrated application of these bioagents/manures with fertilizer N will not only
sustain soil fertility but will also benet farmers in terms of reducing their dependence and expenditure
on chemical fertilizers.
2009 Elsevier B.V. All rights reserved.

1. Introduction
Sugarcane is an important industrial crop of the Indo-Gangetic
Plain region of South Asia, with an approximate 4.2 million hectare
area in India where ricewheatsugarcane crop rotation is the
major production system. The extensive cereal-based cropping, lack
of legumes in the crop rotations and poor soil manuring have led
the soils of the Indo-Gangetic plains become poor in organic carbon
content. Sugarcane is a very demanding crop as for a cane yield of
100 t ha1 it removes about 205 kg N, 55 kg P2 O5 , 275 kg K2 O and

Corresponding author. Tel.: +91 522 2480726; fax: +91 522 2480738.
E-mail address: rattanlal.yadav@gmail.com (R.L. Yadav).
1161-0301/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.eja.2009.01.002

a large amount of micronutrients from the soil (Yaduvanshi and

Yadav, 1990). In order to sustain productivity, major nutrients are
provided each year at the recommended application rates, which
in the subtropical part of India are 150 kg N ha1 for the sugarcane
plant crop and 220 kg N ha1 for its ratoon crop and 60 kg each of
P2 O5 and K2 O ha1 for both the plant and ratoon crops. However,
the efciency of sugarcane to utilize N ranges between 16% and
45% as large quantities of applied N leach down through the soil
layers due to irrigation (Yadav and Prasad, 1992). Deterioration in
the physico-chemical and biological properties of the soil is considered to be the prime reason for the declining sugarcane yield and
productivity (Garside, 1997; Speir et al., 2004).
Integrated nutrient management (INM) through balanced use
of chemical fertilizers, manures and biofertilizers is considered a

R.L. Yadav et al. / Europ. J. Agronomy 30 (2009) 296303

promising agro-technique to sustain crop yields, increase fertilizer use efciency and to restore soil fertility (Kennedy et al.,
2004). The application of organic matter from animal manures,
crop residues and green manuring has been shown to replenish
organic C and improve soil structure and fertility. Biofertilizers
are products containing living cell of microorganisms having the
ability to convert nutritionally important elements from unavailable to available form through biological processes (Vessey, 2003;
Kennedy et al., 2004). Trichoderma spp. is common inhabitants of
the rhizosphere and is well recognized as plant growth hormone
producing and biocontrol agents (Chet, 1987). Gluconacetobacter
diazotrophicus (earlier known as Acetobacter diazotrophicus) a nitrogen xing bacteria, associated with sugarcane as an endophyte
exists in high numbers (as high as 106 counts g1 plant tissue) in
root, shoot and leaves (Cavalante and Dobereiner, 1988). It is primarily responsible for biological N xation and seems to contribute
substantially to nitrogen nutrition of the plant (James et al., 1994;
Dbereiner et al., 1995). G. diazotrophicus inoculation experiments
involving micro-propagated plants suggest the positive colonization and its contribution to plant growth and development in
terms of improved plant height, nitrogenase activity, leaf nitrogen,
biomass and yield (James et al., 1994; Sevilla et al., 2001; Oliveira
et al., 2002; Muthukumarasamy et al., 2002). Suman et al. (2001)
reported that the native occurrence of G. diazotrophicus in sugarcane varieties of subtropical India is very low but through the
inoculation of efcient indigenous isolates, their number, plant N
uptake and nutrient use efciency could be increased (Suman et al.,
2005). However, high N fertilization causes a negative effect on the
population of such endophytic diazotrophic bacteria in sugarcane
(Suman et al., 2008). Apart from nitrogen xation, other properties associated with G. diazotrophicus are P-solubilization, plant
growth hormone indole acetic acid (IAA) production and suppression of red rot disease (Muthukumarasamy et al., 1999; Suman et al.,
2001).
Field inoculation of G. diazotrophicus and Trichoderma with different organic manures indicated a positive nutrient balance in
sugarcane soils (Singh et al., 2007; Shukla et al., 2008). However,
information regarding their integrated use with fertilizer N in terms
of sugarcane crop productivity and N economy is lacking. Therefore,
the present study was planned to evaluate the effect of inoculation
of G. diazotrophicus and Trichoderma viride with and without organic
manure: (i) on soil chemical and biological activity, (ii) the survival
and colonization of introduced bioagents and ultimately, (iii) on
crop productivity, N-economy and yield response.
The ultimate goal of the present study is to develop a strategy
wherein nutrient needs of sugarcane crop be met in a sustainable
way by utilizing suitable bioagents and/or organic manure with N
fertilizer. This shall not only help the farmer community economically by lowering down the production costs but also improve the
soil quality.

2. Materials and methods

2.1. The experimental site, climate and soil
A eld experiment was conducted during two crop seasons
(20042006 and 20052007) at the Research Farm of the Indian
Institute of Sugarcane Research, Lucknow. The climate of the experimental site is semi-arid subtropical with hot dry summers and
cold winters. The average monthly minimum and maximum temperatures during summer (AprilJune) range from 18.4 to 39.1 C
and in winter (NovemberFebruary) from 7.4 to 29 C. The average
annual rainfall is 1045.5 mm and cumulative open pan evaporation 1750 mm, nearly 72% of the total rainfall is received through
north-west monsoons during JulySeptember.

297

The soil of the experimental site belongs to ne loamy noncalcareous mixed hyperthermic Udic ustochrepts and is well
drained, at (about 1% slope). Before commencement of the experiment in both the seasons, soil samples were collected from 0 to
15 cm prole depth at 4 sites in the experimental eld. The subsamples were mixed was bulked and representative sample drawn,
pulverized using wooden pestle and mortar and sieved through
100-mesh sieve. The processed samples were analyzed for organic
carbon (Walkley and Black method), available N (KMNO4 -method),
0.5 M NaHCO3 (pH 8.5) extractable P and 1N NH4 OAC-extractable
K following Page et al. (1982). Organic carbon, available N, P
and K contents of the experimental eld were 0.44%, 258.0,
18.4 and 220.0 kg ha1 in 20042006, and 0.42%, 260.0, 22.5 and
218.0 kg ha1 in 20052007, respectively. The pH of the soil was 8.2.
The moisture content of the soil was 21.5% at 0.3 bar atmospheric
pressure and 6.3% at 15 bar.
2.2. Treatment and crop cultivation
There were 24 treatment combinations having 4 levels of N and
6 modes of bioagents/FYM application. Four levels of N were 0, 100,
200 and 300 kg of N ha1 supplied through urea and were termed as
N0 , N100 , N200 and N300 , respectively. Six modes of bioagents/FYM
application included: (i) B1 : Farm Yard Manure (FYM) 10 t ha1 , (ii)
B2 : G. diazotrophicus alone (Gd), (iii) B3 : T. viride (Tv) alone, (iv) B4 :
FYM + Gd, (v) B5 : FYM + Tv and (vi) C: control where neither bioagents nor FYM was applied. The treatments were replicated thrice
in a split plot design having mode of bioagents/FYM application
as a main plot treatment and levels of N application as sub-plot
treatments. The size of sub-plots was 10 m 10 m. Sugarcane variety CoSe 92423 was used for planting using ring-pit technique as
described by Singh et al. (1984) and Yadav and Kumar (2005). FYM
used was containing 0.5% N, 0.27% P and 0.25% K.
The crop was planted using 100,000 2-bud cane setts ha1 on 24
November 2004 and again on 22 October 2005. The respective crops
were harvested on 22 February 2006 and 28 January 2007. The crop
under all the treatments received a uniform dose of 60 kg P2 O5 and
80 kg K2 O ha1 . Urea (46.4% N), Single super phosphate (16% P2 O5 )
and muriate of potash (60% K2 O) were used to supply nitrogen,
phosphorus and potassium, respectively. Entire dose of P and K,
FYM, bioagents and one-third dose of nitrogen as per treatment
were applied at the time of planting beneath cane setts. Remainder
nitrogen was top dressed in June. The crop received 4 pre-monsoon
and 2 post-monsoon irrigations.
2.3. Preparation and application of G. diazotrophicus and T. viride
based culture
Isolate IS100 of G. diazotrophicus (Suman et al., 2005) was used
for mass inoculation production. Starter culture was prepared from
single colony of G. diazotrophicus in 100 ml of sterilized LGI broth
(Cavalante and Dobereiner, 1988) and after incubating at 30 C for
3 days, the starter inoculum was used to inoculate 5 l of LGI broth.
After 5 days of incubation at 30 C, the culture broth having approximately 9.2 108 cells ml1 was mixed with 5 kg of carrier (Press
mud:FYM: 1:1). The carrier was prepared by mixing equal quantities of press mud, a sugar factory byproduct and FYM, ground, sieved
and sterilized at 121 C (5 lb pressure) for 1 h for 3 consecutive days.
Nutrient composition of FYM (0.5% N, 0.3% P and 0.5% K) and press
mud (1.5% N, 1.0% P and 1.2% K) was also determined. The prepared carrier based inoculum contained approximately 4.0 108
Gd counts g1 and 15 kg ha1 of it was used by sprinking over the
sugarcane setts in furrows at plant crop initiation.
Mass inoculum of T. viride was prepared by preparing starter culture on Potato-dextrose agar medium plates and incubated at 30 C
for 10 days. Well sporulated culture was scrapped from the media

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R.L. Yadav et al. / Europ. J. Agronomy 30 (2009) 296303

plates and mixed with sterilized corn grain powder and incubated
for 15 days at 30 C, after which it was mixed with sterilized FYM
carrier and further incubated for 15 days at 30 C. The prepared FYM
carrier based inoculum containing approximately 5 107 Tv counts
g1 and 20 kg ha1 was used as mentioned above.
2.4. Enumeration of total bacteria and fungi population
For enumerating microbial population in the rhizosphere of sugarcane, soil samples were drawn from 0 to 15 cm depth near root
zone along rows of standing crop by a core sampler of 8 cm diameter
during August which coincides with the maximum (called grand)
growth stage of the crop. Serial dilutions of soil samples of different treatments using saline buffer blanks were prepared and plated
on Tryptic Soy agar and Tryptone Yeast-extract Mannitol (TYM)
agar for aerobic heterotrophic bacteria and added to semisolid LGI
medium vials with 0.3% agaragar for putative diazotrophic population (Cavalante and Dobereiner, 1988). Similarly dilutions were
plated on Rose-Bengal Agar for total fungi counts and Trichoderma
spp. among fungal colonies were identied by characterizing them
microscopically for suitable characteristics (Pitt and Hocking, 1985).
2.5. Soil and plant analysis
After harvest of the crop, soil samples were collected again from
0 to 15 cm prole in each plot by a core sampler of 8 cm diameter. Soil samples were analyzed for organic C, available N, P and
K as described above. Soil microbial C and N were determined
using the chloroform fumigationincubation method (Jenkinson
and Powlson, 1976).
For dry matter and nutrient uptake studies, plants from 1 m row
length at harvest were uprooted from each plot with intact root,
shoot, dry and green leaves. Fresh weight was recorded after separating different plant components and a representative sample was
drawn for drying in hot air oven at 70 C. The dried samples were
ground in stainless steel Wiley mill and wet digested in concentrated H2 SO4 for determination of total N and in di-acid mixture
(HNO3 and HClO4 : 4:1) for determination of total P and K. The N
content was determined by Kjeldahl method using Kjeltec autoanalyser, P by Vanado molybadate Yellow colour method (Piper,
1966) using an UVvis spectrophotometer and K by ame photometer.
2.6. Statistical analysis of data
The data of each crop season were statistically analyzed separately. Then the homogeneity of error variance was tested using
Bartletts X2 test. As the error variance was homogenous, pooled
analysis was done. Since the variation between the two crop seasons was not signicant, the mean data of two crop seasons are
discussed here.
To compare the treatments, two-way analysis of variance for a
split plot factorial design was used. For analysis of variance (ANOVA)
the degrees of freedom (d.f.) were partitioned as: replications2, bioinoculants/manure-5, error (a) 10, N levels-3, interaction
of bioinoculants/manure X N-level-15, error (b) 36. Means were
compared using Duncans multiple-range test as a post hoc analysis
(Snedecor and Cochran, 1967).
The economic optimum doses of N for sugarcane under different
modes of N application were worked out by formula.
X=

1
2c

P

PS

where X is the economic optimum dose (N kg ha1 ), b and c are

the constants of quadratic curve, PN and Ps represent the price of

Table 1
Microbial population in the sugarcane rhizosphere soil as inuenced by bioagents/FYM and N levels.
Treatments

Control
B1
B2
B3
B4
B5
N0
N100
N200
N300

Microbial population (counts g1 soil)

Total bacteria

Gd

Total fungi

Tv

2.2 106a
3.6 106cd
3.3 106c
2.6 106b
2.6 106b
3.1 106c
1.3 106a
2.9 106b
4.6 106d
2.8 106b

50.0b
78.8c
124.5d
26.3a
247.5f
29.0a
87.0c
196.0e
76.5c
10.8a

3.2 103b
2.7 103a
2.7 103a
5.7 103c
2.7 103a
2.7 103a
1.5 103a
3.8 103b
5.7 103c
2.2 103a

5.3a
6.5a
5.3a
248.8b
8.3a
950.0c
166.0b
204.7b
254.5b
190.8b

Control: urea alone; B1 : Farm Yard Manure (FYM); B2 : Gluconacetobacter diazotrophicus (Gd); B3 : Trichoderma viride (Tv); B4 : FYM + Gd; B5 : FYM + Tv; N0 : no nitrogen;
N100 : 100 kg nitrogen ha1 ; N200 : 200 kg nitrogen ha1 ; N300 : 300 kg nitrogen ha1 .
Means with different letters are signicantly different at P 0.05 by the Duncans
multiple range test.

nitrogen (Indian rupees 10.5 kg1 ) and sugarcane (Rs. 12000 t1 )

respectively. Yield response curves were calculated as
Y = bN + cN 2
where Y is expected response, N is the dose of nitrogen (kg ha1 )
b and c are the constants of the tted response curve. All data are
expressed on an oven-dry soil weight basis.
3. Result
3.1. Colonization of inoculated bioagents in the sugarcane
rhizosphere
Population of culturalable bacterial and fungal isolates and colonization of G. diazotrophicus and T. viride in sugarcane rhizosphere
was studied using selective growth medium. Population of bacteria and fungi increased with increasing level of N up to N200 , after
which the microbial population declined (Table 1). Colonization of
Gd was affected by N fertilizer as Gd counts increased only up to
N100 and thereafter, decreased with increase in N fertilizer. Population of Gd in sugarcane was affected by the addition of FYM and
bioagents. It increased signicantly with the addition of FYM and
Gd inoculum but decreased with the inoculation of Tv alone or with
FYM compared to control treatment. The native occurrence of Tv in
sugarcane ranged from 5 to 8 g1 soil in control, FYM and Gd treated
plots. Inoculation with bioagent Tv indicated its colonization in
the rhizospheric soil as the Tv population increased to approximately 249 counts g1 in treatment B3 . Inclusion of Tv enriched FYM
enhanced its colonization as the Tv population increased approximately 4 times to 950 counts g1 soil in B5 treatment. Addition
of fertilizer N affected the population of Tv and it increased from
166 to 254 counts g1 when N was increased from N0 to N200 but
at higher dose of fertilizer N (N300 ) decline in Tv population was
recorded.
3.2. Soil chemical and microbial properties as inuenced by
bioagents/FYM
Soil organic carbon (SOC) content increased only where T.
viride was used and was signicantly highest in plots treated with
FYM + Tv compared to control plot (Fig. 1). SOC content increased
with increasing levels of fertilizer N and was statistically at par up to
200 kg N ha1 . The enhanced availability of N and P with increasing
levels of fertilizer was statistically at par. Availability of K was significantly higher at higher levels of fertilizer N (200 and 300 kg ha1 ).
Among bioagents and FYM, the availability of N was enhanced only

R.L. Yadav et al. / Europ. J. Agronomy 30 (2009) 296303

299

3.3. Sugarcane yield and nutrient uptake as inuenced by

bioagents/FYM
Fertilizer-N application to the sugarcane crop increased the cane
yield signicantly as the N level increased from N0 to N300 but
the differences in yield between N200 and N300 were not signicant (Fig. 3). Application of FYM and bioagents (Gd and Tv) alone or
in combination enhanced the cane yield signicantly (716 t ha1 )
compared to control where only fertilizer N was used (Fig. 3). Treatments, where bioagents alone were used (B2 and B3 ) increased the
cane yield by 6.49.4% compared to control (C). These treatments
were statistically at par with FYM alone (B1 ) in improving the cane
yield (8.6%). On the other hand the application of FYM enriched with
bioagents (treatments B4 and B5 ) improved the cane yield maximum (1013.5%) compared to control and were signicantly better
than the FYM alone; with maximum increase in FYM + Tv treatment.
On average, the crop removed 176 kg N, 29 kg P and 158 kg K per
hectare. In general, N, P and K uptake was least in N-unfertilized
plots (Fig. 3). However, when N was applied at 100 kg ha1 , N uptake
increased by 24.2 kg ha1 , P uptake by 2 kg ha1 and K uptake by
2.8 kg ha1 . When level of N raised from N100 to N200 N uptake was
increased by 30.7 kg ha1 , P uptake by 3.4 kg ha1 and K uptake by
4.9 kg ha1 . Raising the level of N from 200 to 300 kg ha1 enhanced
N uptake by 31.3 kg ha1 , P uptake by 2.7 kg ha1 and K uptake
by 3.3 kg ha1 . Application of FYM and bioagents improved the
N-uptake at all levels of N, the greatest uptake was found in the
plots treated with FYM + Tv. Application of FYM alone increased the
uptake of P and K but was statistically at par with the control treatment, however, the application of bioagents alone or enriched FYM
signicantly improved the uptake of P and K.
3.4. N optimum and crop yield response
From the quadratic curve and equation between cane yield
and fertilizer N level (Fig. 4) the N optimum was calculated for
each bioagent/FYM treatment as the interaction of the bioagent

Fig. 1. Soil organic C and available NPK content as inuenced by bioagents/manure

treatment and levels of N application. C: urea alone; B1 : Farm Yard Manure (FYM);
B2 : Gluconacetobacter diazotrophicus (Gd); B3 : Trichoderma viride (Tv); B4 : FYM + Gd;
B5 : FYM + Tv. N0 : no nitrogen; N100 : 100 kg nitrogen ha1 ; N200 : 200 kg nitrogen ha1 ;
N300 : 300 kg nitrogen ha1 . Treatment bars of bioinoculant/manures and N application sections labeled with different letters are signicantly different at P 0.05 by
the Duncans multiple range test.

when FYM was used. The bioagents alone or in combination with

FYM did not improve the availability of N in soil. However, the
availability of P and K was signicantly enhanced using FYM and
bioagents.
Soil microbial activity measured as soil microbial carbon (SMC)
and nitrogen (SMN) increased with increasing levels of fertilizer N
(Fig. 2). SMC increased signicantly with inclusion of bioagents and
FYM with maximum being in plots treated with FYM and FYM + Tv
compared to the control plot. SMN did not differ signicantly due
to different treatments of FYM and bioagents and was less as compared to the control treatment.
Increasing levels of fertilizer N increased SMC and SMN as a fraction of OC (SMC or SMN/OC). The SMC fraction ranged from 4.89%
at N0 to 9.09% at N300 and SMN fraction ranged from 0.04% at N0 to
0.08% at N300 . Inclusion of bioagents and FYM invariably increased
the fraction of SMC and SMN of OC compared to control but did not
differ signicantly among different treatments.

Fig. 2. Soil microbial C and N (SMC and SMN) as inuenced by bioagents/manure

and different levels of N application. C: urea alone; B1 : Farm Yard Manure (FYM); B2 :
G. diazotrophicus (Gd); B3 : T. viride (Tv); B4 : FYM + Gd; B5 : FYM + Tv. N0 : no nitrogen;
N100 : 100 kg nitrogen ha1 ; N200 : 200 kg nitrogen ha1 ; N300 : 300 kg nitrogen ha1 .
Treatment bars of bioinoculant/manures and N application sections labeled with
different letters are signicantly different at P 0.05 by the Duncans multiple range
test.

300

R.L. Yadav et al. / Europ. J. Agronomy 30 (2009) 296303

Fig. 4. Yield response curves for N application rates as inuenced by bioagents/FYM

application C: urea alone; B1 : Farm Yard Manure (FYM); B2 : G. diazotrophicus (Gd);
B3 : T. viride (Tv); B4 : FYM + Gd; B5 : FYM + Tv.

(0.86 t kg1 N) inoculation alone was also high as compared to

control and FYM treatment. In the control plot the optimum dose
of N was 182.5 kg N ha1 and the yield response was 0.72 t kg1 ,
whereas in FYM treatment the yield response was the lowest
(0.64 t kg1 N).
4. Discussion

Fig. 3. Cane yield and uptake of NPK by sugarcane as inuenced by bioagents/manure treatment and levels of N application. C: urea alone; B1 : Farm Yard
Manure (FYM); B2 : G. diazotrophicus (Gd); B3 : T. viride (Tv); B4 : FYM + Gd; B5 :
FYM + Tv; N0 : no nitrogen; N100 : 100 kg nitrogen ha1 ; N200 : 200 kg nitrogen ha1 ;
N300 : 300 kg nitrogen ha1 . Treatment bars of bioinoculant/manures and N application sections labeled with different letters are signicantly different at P 0.05 by
the Duncans multiple range test.

X N interaction was signicant for yield (R2 > 0.96 for different
bioagents/FYM treatments). The optimum economic dose of N
for sugarcane resulted to be the lowest (106.25 kg ha1 ) when
Gd supplemented FYM was applied to the crop and the highest
(221 kg ha1 ) when FYM alone was applied (Table 2). At these optimum N doses, the cane yield response per kg of N used was highest
(1.21 t kg1 N) where FYM + Gd was used, followed by FYM + Tv
(1.00 t kg1 N). The yield response to Gd (0.83 t kg1 N) and Tv

Indiscriminate high use of fertilizer N by the farmers in the

Indo-Gangetic Plains, to tackle declining crop yields and factor productivity, is the major concern for sustaining agriculture, soil health
and environment pollution. In recent years, microbial bioagents
have emerged as an important component of integrated nutrient
management of a crop due to their multiple benets in terms of
N-xation, P-solubilization, plant growth hormone production and
controlling pest and disease infestations etc. The use of bioagents
has been identied as an alternative to chemical fertilization to
increase soil fertility and crop production in sustainable farming
(Vessey, 2003; Kennedy et al., 2004). In this perspective the present
study was planned to examine whether soil health of sugarcane
elds could be improved by using bioagents like G. diazotrophicus
(Gd) and T. viride (Tv) along with FYM and chemical nitrogen which
may in turn improve N use efciency of the crop and result into
improved crop yields and N saving.
4.1. Soil properties
Our experiments showed that FYM along with bioagents
improved the soil organic carbon and available nutrients in sugarcane rhizosphere. The soluble and intermediate C pool is easily

Table 2
Optimum nitrogen dose and yield response of sugarcane as inuenced by bioagents/FYM application.
Treatments

Yield curve

R2

N optimum (kg ha1 )

Cane yield at N optimum (t ha1 )

Yield response (t kg1 N)

Control
B1
B2
B3
B4
B5

104.67 + 0.1976N 0.0003N2

111.54 + 0.1759N 0.0002N2
109.5 + 0.1492N 0.0002N2
113.257 + 0.1483N 0.0002N2
116.84 + 0.13N 0.0002N2
119.35 + 0.1699N 0.0002N2

0.946
0.981
0.980
0.960
0.980
0.980

182.5
221.0
153.7
152.0
106.2
137.3

130.7
140.6
127.7
131.1
128.3
137.0

0.72
0.64
0.83
0.86
1.21
1.00

Control: urea alone; B1 : Farm Yard Manure (FYM); B2 : G. diazotrophicus (Gd); B3 : T. viride (Tv); B4 : FYM + Gd; B5 : FYM + Tv.

R.L. Yadav et al. / Europ. J. Agronomy 30 (2009) 296303

affected by cultivation, amendments, and weather conditions

(Staben et al., 1997; Salinas-Garcia et al., 1997; Zaman and Chang,
2004). Higher soil organic matter following the application of
manure like sulphitation press mud has been reported by Dee et al.
(2003) under sugarcane growing conditions which often witness a
loss of soil organic matter under conventional agriculture (Haynes
and Hamilton, 1999). Slow release of nutrients in the presence
of FYM improves the soil fertility compared to chemical fertilizer
applications (Chowdhary et al., 2004) and the use of bioagents by
immobilization retain soil nutrients in the plantsoil system and
reduces leaching losses (Kennedy et al., 2004). Increased soil carbon due to application of bioagents and FYM sustains soil health
for a longer period than the chemical fertilization (Jeyabal and
Kuppuswamy, 2003; Amlinger et al., 2003). Wu et al. (2005) have
shown that the dual inoculation of rhizobacteria and mycorrhizae
with manure resulted in a signicant increase of soil organic matter content in the maize rhizosphere. The availability of P was also
higher in the treatments where bioagents amended FYM was used.
The improved availability of P due to manuring could be due to the
release of P from the decomposition of amended residue, reduced P
sorption due to blocking of P-sorption sites by organic compounds,
complexing of Al by organic compounds leading to P release from
AlP complexes, or increased soil pH leading to an increased P concentration in the soil solution (Cong and Mercky, 2005; Gelsomino
et al., 2006). Enhanced availability of P in sugarcane rhizosphere
by recycling residues of intercrops (pulses/cereals) and press mud
incorporation could improve the uptake of P and other nutrients
(Suman et al., 2006; Singh et al., 2007). Wani (1990) reported that
the use of suitable farmyard manures, green manures and other
organic manures and fertilizers may enhance the benets of bioagents inoculation as the energy required for various metabolically
benecial activities is met through C content of the soil.
Soil microbial biomass (SMC and SMN) is a sensitive indicator
of soil health and is only 14% of soil organic C (Jenkinson and
Ladd, 1981; Anderson and Domsch, 1989). Due to its fast turnover
rate, it plays a key role in controlling nutrient cycles (immobilization/mineralization) and thus the availability of nutrients for
plant uptake (Li and Chen, 2004). Increase in both SMC and SMN
with increasing N level was mainly due to easily available N for
microbial activity. Graham and Haynes (2005) have also indicated
the role of readily available nutrients through NPK application
for enhanced microbial population and biomass. FYM/bioagents
amendment further improved the microbial biomass and the nutrients availability for the plant uptake. SMN was at par among
different bioagents and FYM treatment. Soil microorganisms consume a considerable amount of organic matter to generate energy
for maintenance and growth; hence some organic carbon is lost
with the production of carbon dioxide. Here, the uniform increase
in the fraction of SMC and SMN of total SOC indicated that immobilization/mineralization equilibrium is maintained in the soil and
enhanced microbial biomass shall act later as a source of nutrients. Singh et al. (2007) also found higher microbial biomass in
sugarcane rhizosphere in the plots receiving bio-manures than
the control plots. Long-term studies on sugarcanewheatrice
cropping system in subtropical Indian climate also revealed superiority of integrated nutrient supply (chemical fertilizer + FYM) over
chemical fertilizer alone for sustaining crops yield and improving soil health in terms of SMC and SMN and activities of soil
enzymes like dehydrogenase, urease and alkaline phosphatase
(Yadav et al., under publication). The application of FYM along
with recommended rates of chemical fertilizers have been advocated for accumulation and sequestration of C, improved biological
activity and soil fertility (Zaller and Kopke, 2004; Dwivedi and
Dwivedi, 2007) and enrichment with bioagents increased the efciency of different organic amendments (Jeyabal and Kuppuswamy,
2003).

301

4.2. Microbial activity and inoculum establishment in the

sugarcane rhizosphere
Soil harbors high population density and enormous natural microbial diversity under a tremendous range of physical
and chemical conditions, of which only <10% are culturable
(Handelsman, 2004; Leininger et al., 2006). Temporary variations
in the microbial communities in terms of population and species
occur due to variations in the cover plant, soil type, cultivation
practices, residue/fertilizer and bioagents incorporations (Mittal
and Johri, 2007), but the overall microbial activity in the plant rhizosphere determines the nutrient cycling and their availability to
the plant system. Changes in bacterial and fungal populations in
the rhizosphere indicate the variable nutrient ratios, their immobilization (sink) and mineralization (source) potential, which in
turn affects the availability of nutrients for the plant uptake. In
this study, microbial population was inuenced signicantly by
the addition of FYM, bioagents and fertilizer N in the soil and it
increased up to N200 (Fig. 1), which was mainly due to the readily
available nutrients for microbial population (Graham and Haynes,
2005). Decline in the culturable bacterial and fungal population
at N300 possibly refers to either high ammonium toxicity to the
population or shift in microbial communities better suited for denitrication etc., which could not be cultured on growth media used
here. This is in agreement with Wani (1990) and Wu et al. (2005)
who noted that the population size of N-xing bacteria in soil
deceased signicantly after N fertilizer was used and the propagation of A. chroococcum was seriously inhibited when the ammonium
N concentration exceeded 200 mg kg1 . There might be several
archeal/bacterial associations operating in the sugarcane rhizosphere which should be analyzed using metagenome approach as
is being done in several other crop systems (Handelsman, 2004;
Leininger et al., 2006).
For a bioagent to benet the plant, it should establish, colonize
and compete with the native heterotrophic bacterial population
and should acclimatize the local climatic conditions. Although the
population of total bacteria increased with increasing N, population of G. diazotrophicus (Gd) increased only up to 100 kg ha1
(Fig. 1) and further addition of N could not increase the number of Gd in the sugarcane rhizosphere. This is in conrmation
with earlier reports that Gd could be isolated easily where no
or low N input conditions are prevailing (Fuentes-Ramirez et al.,
1999; Suman et al., 2008). At high fertilizer N input, proliferation of non-nitrogen xing bacteria due to rich nutrient availability
may reect on the poor multiplication of Gd where as at low N
the growth of Gd remains unaffected due to its ability to sustain its N requirement through N xation (Muthukumarasamy et
al., 2002; Suman et al., 2005). Tv colonization in Tv treated plots
was signicant and probably was inuenced by the poor competition from native Tv population, which was quite low in our
soils. Improved colonization of both bioagents in the presence
of FYM was mainly due to availability of organic C which acts
as C and nutrient source for the multiplying microbial population.
4.3. Nutrient uptake and yield response
Sugarcane nutrient uptake was enhanced following inclusion
of FYM and bioagents at all levels of N. It was mainly due to the
enhanced nutrient availability in the rhizospheric soil as in the
present study, soil organic C and available N, P and K content
also increased when the soil was treated with bioinoculants/FYM
(Fig. 3). Singh et al. (2007) also indicated a positive balance of soil
N, P and K in the plots treated with bio-manures in multi-ratooning
sugarcane system. The conspicuous improvement in the soil health
including soil microbial activity upon treatment with bioinocu-

302

R.L. Yadav et al. / Europ. J. Agronomy 30 (2009) 296303

lants/FYM resulted into enhanced uptake of nutrients. Suman et al.

(2005) and Srivastava et al. (2006) attributed enhanced uptake of
plant nutrients by inoculation with Gd and Tv to signicant changes
in various plant growth parameters such as plant height, tiller number and dry matter yield. Enhanced N uptake has also been shown
by the inoculation of various nitrogen xing and plant growth promoting bacteria (Kennedy et al., 2004). In addition to N xation,
the benecial effect of Gd and Tv inoculation may also be attributed
to the production of plant growth hormones (Sevilla and Kennedy,
2000).
Signicant increase in cane yield from N0 to N200 compared to
non-signicant increase from N200 to N300 indicated that the application of higher dose of chemical fertilizer N could not increase
the cane yield. It could be due to either enhanced N losses or poor
uptake because of un-proportionate ratios of N, P and K as only N
input was increased. Improvement in the cane yield at all nitrogen
levels due to the application of FYM and bioagents clearly indicated
the role of improved availability of P and K in these treatments. Statistically par yield levels in the treatments where bioagents alone or
FYM were used indicated that the positive role of enhanced microbial activity in the rhizosphere. Manures alone can also mimic the
effect of inoculated bioagents and vice versa. Therefore, addition of
organic C for spurting native microora and/or benecial microora
shall sustain the soil health and crop productivity. Shukla et al.
(2008) also indicated the role of Trichoderma and Gluconacetobacter
in improving sugarcane ratoon yields due to improved soil carbon
and nitrogen status.
The ultimate aim of the present study was to investigate whether
economy in the use of fertilizer N is possible by the application of
bioagents. In this respect, it may be stated that in control plots the
economic optimum N dose resulted to be 182.5 kg ha1 and compared to it the least economic optimum N dose was 106.2 kg N ha1
in plots treated with Gd inoculated FYM. Thus a saving of about
76.3 kg N ha1 may be envisaged with 2.4 t ha1 yield decline in this
treatment. On the other hand, application of Tv inoculated FYM not
only brought economy in the use of fertilizer N by 45.2 kg ha1 , it
also increased the cane yield by 6.1 t ha1 over control plots. Overall
inclusion of manures and bioagents for nutrient mobilization has
proved be to benecial for sugarcane productivity and saving N fertilizer; consequently the amount of N-fertilizer could be reduced
and these benecial bioagents should be recommended for their
use in integrated nutrient management.

5. Conclusion
In Middle Gangetic Plain importance of organic amendments
in improving soil quality, ecological conditions, sugarcane growth
and yield has been recognized. Application of Trichoderma and Gluconacetobacter inoculated farm yard manure improved soil organic
carbon which in turn helped in sustaining soil health for longer
period. FYM provided organic carbon for enhanced multiplication
of inoculated microbial agents and provided a suitable niche for
plantmicrobe interactions. G. diazotrophicus and T. viride due to
their plant growth promotion ability produced synergistic effect
with FYM as compared to control. Enhanced organic carbon not
only sustained crop health but also retained more N in plant rhizosphere due to immobilization by microbial population. This also has
relevance for minimizing N leaching losses and making nutrients
available for the crop growth. Improved microbial population in
FYM inoculated treatments clearly established their role in improving biological activity. T. viride and G. diazotrophicus enriched FYM
resulted in higher cane yields and economy in the use of fertilizer N.
Thus, application of bioagents and manures can help in sustaining
soil health and increasing sugarcane production in Indo-Gangetic
Plain region.

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