T Viridae
T Viridae
T Viridae
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
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.
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
298
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
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
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.
299
300
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
Table 2
Optimum nitrogen dose and yield response of sugarcane as inuenced by bioagents/FYM application.
Treatments
Yield curve
R2
Control
B1
B2
B3
B4
B5
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.
301
302
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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