Sugar Tech (Jan-Mar 2013) 15(1):36–43
DOI 10.1007/s12355-012-0191-8
RESEARCH ARTICLE
Effects of Diverse Habitat Biofertilizers on Yield and Nitrogen
Balance in Plant–Ratoon Crop Cycle of Sugarcane in Subtropics
Archna Suman • Pushpa Singh • Menhi Lal
Received: 13 June 2012 / Accepted: 1 November 2012 / Published online: 27 November 2012
Ó Society for Sugar Research & Promotion 2012
Abstract Three nitrogen fixing biofertilizers of diverse
habitat; endophytic Gluconacetobacter diazotrophicus,
free living Azotobacter chrococcum and associative Azo-
spirillum brasilense were tested in field condition for a
complete sugarcane crop cycle of one plant and a ratoon
crop at different levels of N input for enhancing crop
productivity and soil N balance. Different biofertilizers
increased the crop yield at all nitrogen levels but the
maximum increase was recorded at N75 as compared to
N150.G. diazotrophicus based biofertilizer was found to
be the best in terms of crop yield production followed by
its combination with A. chrococcum and A. brasilense.
PFP for nitrogen was more in plant crop (9–38%) than
the ratoon crop (4–24%) due to different biofertil-
izers. Apparent N balance indicated a contribution of
52.3–216.7 kg N ha-1 in plant crop and from 65 to
225.3 kg N ha-1 in ratoon crop due to biological nitrogen
fixation in different biofertilizer 9 N treatments. The
maximum N contribution was recorded with G. diazotro-
phicus and minimum with A. brasilense in a plant-ratoon
cycle of sugarcane crop. Overall field inoculations
of sugarcane by diazotrophic biofertilizers are highly
A. Suman
Division of Microbiology, Indian Agricultural Research
Institute, New Delhi, India
e-mail: archsuman@yahoo.com
P. Singh (&)
Organic Chemistry Laboratory, Division of Plant Physiology
and Biochemistry, Indian Institute of Sugarcane Research,
Lucknow, India
e-mail: parampushpa@yahoo.com
M. Lal
Division of Crop Production, Indian Institute of Sugarcane
Research, Lucknow, India
123
beneficial and of tested biofertilizers G. diazotrophicus
based biofertilizer is most efficient in terms of higher cane
yield, commercial cane sugar production, efficient N uti-
lization and maintaining positive N balance.
Keywords Biofertilizer 
Gluconacetobacter diazotrophicus  Azotobacter 
Azospirillum  Sugarcane  BNF  N use efficiency 
N-balance
Introduction
Integrated nutrient management (INM) through balanced
use of chemical fertilizers and biofertilizers is considered a
promising agro-technique to sustain crop yields, increase
fertilizer use efficiency and to restore the soil fertility.
Biofertilizers are the 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). Through plant growth promoting and nutrient
mobilization mechanism, these biofertilizers retain more
soil organic N and other nutrients in the plant–soil system,
thus reducing the need for chemical fertilizers (Vessey
2003). Non symbiotic nitrogen fixing diazotrophic bacteria
like Azotobacter, Azospirillium and Gluconacetobacter
(Acetobacter) having different habitat are being used as
biofertilizer for different crop plants of Poaceae family.
Azotobacter species are free living, aerobic, heterotrophic
diazotrophs. Inoculations with Azotobacter have shown to
increase yields of rice by 20 % (Yanni and El-Fattah 1999),
cotton by 15–25 % (Iruthayaraj 1981) and saving of 50 %
urea-N in wheat (Hegazi et al. 1998). Azospirillium species
are associative, aerobic heterotrophs that fix N2 under
Sugar Tech (Jan-Mar 2013) 15(1):36–43
micro-aerophilic condition (Kennedy and Tchan 1992). It
grows extensively in the rhizosphere of graminaceous
plants, constituting about 1 % of total aerobic hetero-
trophs in rice soils (Ladha et al. 1987). Inoculations with
Azospirillium isolate increased yields of rice by 22 %
(Balandreau 2002) and wheat by 30 % (Okon and Laban-
dera-Gonzalez 1994). It also significantly increased cane
yield in both plant and ratoon crops (Shankariah and
Hunsigi 2001). Gluconacetobacter diazotrophicus, an
endophytic diazotroph of sugarcane is mainly responsible
for high biological N fixation potential (30–80 %) of sug-
arcane (Boddey et al. 2001). The natural population of G.
diazotrophicus in subtropical Indian sugarcane varieties
was more at less fertilized conditions (Suman et al. 2008).
The N use efficiency (NUE) of sugarcane also improves
upon G diazotrophicus inoculation under pot-house con-
dition (Suman et al. 2005). It is highly desirable as NUE of
sugarcane is only 35–40 % of applied chemical N fertilizer
(150 kg ha-1 for sugarcane plant and 200 kg ha-1 for
ratoon crop) in subtropical India (Yadav 1981); the excess
N is contaminating the ground water and environment.
Under organic nutrient management system also, inocula-
tion by G. diazotrophicus maintains positive N balance in
the soil. Due to different habitat and metabolic sites, all
three Azotobacter, Azospirillum and Gluconacetobacter are
the potential bacterial agents which can be developed to
commercial biofertilizer for sugarcane but no where these
have been used simultaneously in a particular environment,
so as to conclude that which microbe–plant interaction is
most suited for sugarcane in terms of improving cane yield,
sugar productivity and apparent N-balance of sugarcane–
ratoon system. Therefore, the present study was planned to
(i) Study the effect of three diazotrophic biofertilizers
(alone or in combinations) on sugarcane yield and
sugar productivity at variable N input in a plant–
ratoon system and
(ii) Evaluate the NUE and apparent N-balance in differ-
ent treatment combinations.
Materials and Methods
Site, Soil and Climate
A field experiment was conducted on spring (February)
planted sugarcane (2006–2007) at the research farm of
Indian Institute of Sugarcane Research, Lucknow, India,
located at 26.5°N, 80.5°E and 120 m above mean sea level
in the state of Uttar Pradesh which contributes 51 % to
total cultivated area of sugarcane in the country. The cli-
mate of Lucknow is semi-arid sub-tropical with hot dry
summers and cold winters. The average monthly minimum
37
and maximum temperatures during summer (April to June)
ranges between 18.4 to 39.1 °C and in winter (November
to February) ranges between 7.4 to 29 °C. The average
annual rainfall is around 1,045.5 mm with cumulative open
pan evaporation of 1,750 mm. and nearly 72 % of the total
rainfall is received through north-west monsoons during
July to September. The soil of the experimental site
belongs to fine loamy non-calcareous mixed hyperthermic
Typic ustochrepts and are well drained, flat (about 1 %
slope). Before commencement of the experiment in both
the seasons, soil samples were collected from 0 to 15 cm
profile depth at 4 sites in the experimented field. The sub-
samples were mixed, bulked and representative sample
drawn, pulverized using wooden pestle and mortar and
sieved through 100-mesh sieve. The processed samples
were analyzed for available N (KMNO4-method), 0.5 M
NaHCO3 (pH 8.5) extractable P and 1 N NH4O Ac-
extractable K following Page et al. (1982). Available N, P
and K contents of the experimental field were 217.4, 18.4
and 214.2 kg ha-1, respectively and the soil pH was 8.2.
Treatment and Crop Culture
Three nitrogen fixing biofertilizers (G. diazotrophicus,
A. chrococcum and A. brasilense) alone and in different
combinations making a total of eight bacterial treatments
were evaluated at 3 N input levels (0, 75, 150 kg N ha-1).
Thus there were 24 treatments (8 biofertilizers 9 3 N
levels) which were tested in a randomized block design
with three replications. Moist hot air treated 8–10 month
old canes of sugarcane variety CoSe 92423 were used as
seed cane. The plot size was 3.6 9 5 m with 4 rows at
90 cm row-spacing. The fertilizers used were urea (46 % N),
super-phosphate (6.8 % P) and murate of potash (46.2 %
K) and the recommended application of fertilizer for sug-
arcane (kg ha-1) in subtropical belt is 150:60:60, respec-
tively. As per the N treatment, one third of N dose and full
P and K dose were applied as basal dose in the furrow
beneath cane-setts and the remainder N was top-dressed in
two equal splits at 45 and 90 days after planning. The crop
was grown under irrigated conditions with 6 pre-monsoon
and 3 post monsoon irrigations. Twelve months old plant
canes were harvested close to ground level manually by
garasa in the month of February 2006 and the entire above-
ground biomass was removed from the field. For ratoon
initiation, the field was cleared and inter-row spacing was
deep cultivated and irrigated. Soon after fertilizer for P and
K (60 kg ha-1) and half of treatment N were placed near
the cane stubble and earthed up. At 30 days after root
initiation biofertilizer as per the treatment plot were placed
10–15 cm deep near the stubble and one hoeing was done
to cover up the soil. Remaining N was used at 90 days of
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38
Table 1 Bacterial isolates, their source and characteristics
S no. Bacterial isolate Source
1 Gluconacetobacter diazotrophicus IS100 Suman et al. (2005)
2 Azotobacter chrococcum Ac3 Suman et al. (2002)
3 Azospirillum brasilense CdzA Division of Microbiology
IARI, New Delhi
ratoon initiation. Ratoon cane was harvested at 11 months
old stage in the month of January 2007.
Biofertilizer Preparation and Its Application
Three bacterial isolates belonging one each to G. diazo-
trophicus, A. chrococcum and A. brasilense were used for
the preparation of biofertilizers. Source, growth conditions
and general characteristics of these organisms have been
given in Table 1. The starter culture of each bacterium was
prepared by inoculating single colony of bacterium in
100 ml of sterilized respective broth medium (Table 1) and
incubating on shaker (120 rpm) at 30 °C for 3 days. Starter
culture having approximately 108 cells ml-1 was used to
inoculate 5 liters of same broth and after 5 days of incu-
bation at 30 °C the cells from culture broth were harvested
by centrifugation at 5,000 rpm at 4 °C. Harvested cells
were suspended in saline phosphate buffer to get a cell
suspension containing approximately 9.2 9 108 cells ml-1
which was mixed with 5 kg of carrier (press mud ? farm
yard manure 1: 1) to a final approximate count of
3.0–4.6 9 108 cells g-1 of carrier. The rate of biofertilizer
application was 15 kg ha-1 and for uniform spreading 30 g
of biofertilizer for each plot was mixed with 70 g of farm
yard manure (20 % water holding capacity) and the pre-
pared 100 g biofertilizer mixture was spread over cane-setts
in the rows (25 g 9 4 rows) at planting and 10–15 cm deep
near stubble in ratoon crop and was covered with the soil.
Post Harvest Soil and Plant Analysis
After harvest of the crop, soil samples were collected again
from 0 to 15 cm profile in each plot by a core sampler of
8 cm diameter. Post harvest sample after processing ana-
lyzed for available N content. For dry matter, five plants
having intact dry and green leaves were collected randomly
from whole plot harvest of each plot and were chopped into
1.0 mm pieces. Fresh weight of chopped pieces was
recorded and representative samples were drawn for drying
in a hot air oven at 70 °C. The dried samples were ground
in a stainless steel Wiley mill, and wet digested in con-
centrated H2SO4 for determination of N. The N content
was determined by the Kjeldahl method using a Kjeltec-
auto-analyser.
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Sugar Tech (Jan-Mar 2013) 15(1):36–43
Habitat Growth medium
Endophytic LGI (Cavalcante and Döbereiner 1988)
Free living Ashby’s broth
Associative JNFb (Baldani et al. 1992)
Quality Analysis of Cane Juice
For juice analysis, 10 sugarcanes from whole plot harvested
lot were selected randomly. These were crushed in hori-
zontal steel crusher to extract juice. Percentages of purity,
brix and sucrose in juice were determined by using Autopol
880 sucrose analyzer (Rudolph research analytical system).
Commercial cane sugar recovery (t ha-1) was calculated as:
CCS % ¼ ½S  0:4ðB  SÞ  0:73
 
CCSðt ha1 Þ ¼ CCS % in juice  cane yield (t ha1 Þ =100
where S is sucrose % and B is brix (%) in juice.
Statistical Analysis and Calculations
To determine the effects of different treatments and their
interactions, analysis of variance was performed using
GenStat statistical package and means were compared at
5 % level on LSD basis. To assess the inter-relationships
between different parameters correlation coefficients and
regression equations were calculated using MS-Excel Data
Analysis statistical tool. For measuring NUE partial factor
productivity (PFPN) was computed which is an integrated
index that quantifies total economic output relative to the
utilization of nutrient resource applied in the system.
PFP ¼ Y=Fn
N contribution from biological nitrogen fixation (BNF)
or other soil sources was measured by following equation
Nbnf=others ¼ ½Pn þ FSN  ½ISN þ Fn
where Y is cane yield in fertilizer N treated plot (Fn), Fn is
amount of applied fertilizer N, Pn is N uptake by the plant
and ratoon crop, ISN is the initial status of soil available N
at the start of the experiment, FSN is final status of soil N
after sugarcane crop cycle of plant and ratoon crop. All are
expressed in kg ha-1.
Results and Discussion
Cane Yield and Commercial Cane Sugar
Sugarcane yield measured as t ha-1 was significantly
affected by bacterial inoculation (P \ 0.05), nitrogen dose
Sugar Tech (Jan-Mar 2013) 15(1):36–43 39

Table 2 Effect of
Treatment Plant crop Ratoon crop
biofertilizer 9 N interactions
on cane yield (t ha-1) of N0 N75 N150 N0 N75 N150
sugarcane plant–ratoon system
T1 76.6 90.5 97.6 75.3 81.7 90.0
T2 71.0 74.6 82.6 72.3 75.3 82.0
T3 72.9 81.5 82.4 68.3 71.7 73.0
T4 76.3 83.3 90.3 71.7 78.6 84.0
T5 74.6 84.8 88.3 69.3 75.3 76.0
N0: No nitrogen input; N75: T6 72.7 78.1 82.2 66.9 69.0 75.0
75 kg N ha-1; N150: T7 69.8 73.2 74.8 73.7 75.3 88.3
150 kg N ha-1; T1:
Gluconacetobacter T8 60.1 65.5 71.2 57.0 65.7 72.3
diazotrophicus; T2: Azotobacter F value LSD (P \ 0.05) F value LSD (P \ 0.05)
chrococcum; T3: Azospirillum
brasilense; T4: T1 ? T2; T5: Bacterial treatment (T) 45.05 2.96 32.01 2.88
T1 ? T3; T6: T2 ? T3; T7: Nitrogen (N) 98.24 1.81 76.12 1.76
T1 ? T2 ? T3; T8:
uninoculated control T9N 1.84 5.13 2.17 4.99

(P \ 0.05) and their interaction (P \ 0.05) both in plant
(cv = 4 %) and ratoon (4.1 %) crop. The minimum and
maximum yield recorded was 55 and 102 t ha-1 in plant
crop, respectively whereas it was 56 and 93 t ha-1in ratoon
crop (Table 2). Different biofertilizer treatments signifi-
cantly increased the yield for both plant and ratoon crop at
3 N levels; however, the increase was more in plant crop.
Increase in mean yield of plant crop due to biofertilizers was
found to be 16–27, 12–35 and 5–37 % as compared to control
at N0, N75 and N150 respectively. The highest yield was
recorded in T1 (G. diazotrophicus treatment) while the
lowest yield was in T7 (combination of three biofertilizers).
Application of biofertilizers also improved ratoon cane
yield and it was highest in G. diazotrophicus treatment (T1)
and lowest in T6 (combination of Azotobacter and Azospirillum).
Commercial cane sugar (CCS) was significantly influ-
enced by bacterial treatment (P [ 0.05), N application
(P [ 0.05) and their interaction (P [ 0.05) and ranged
from 6.7 to 11.3 t ha-1 in plant crop (cv = 2.3 %) and 6.3
to 10.3 t ha-1 in ratoon crop (cv = 2.1 %) (Table 3). CCS
of plant crop was 9.32 t ha-1 (N150), 8.69 t ha-1 (N75) and
7.95 t ha-1 N0) with N application while with biofertilizer
treatments, CCS was grouped as T1 [ T2, T4, T5 [ T7, T3,
T6 [ control. Increase in CCS of plant crop due to bio-
fertilizers over control was 20–36 % at N0, 11–33 % at N75
and 6–23 % at N150 with highest due to G. diazotrophicus
treatment (T1). CCS of ratoon crop ranged from 9.5 t ha-1
(N150) [ 8.7 t ha-1 (N75) [ 8.2 t ha-1 (N0) while CCS
with biofertilizer treatments were grouped as T2, T1,
T4 [ T7, T5 [ T6 [ T3, control.
Nitrogen Uptake and Soil Available Nitrogen (SAN)
Nitrogen uptake by the crop, measured, as kg ha-1 was
more in the plant crop compared to its ratoon crop. It
ranged from 100.5 to 310.5 kg ha-1 in plant crop
(cv = 4.3 %) and 86 to 296 kg ha-1 in ratoon crop
(cv = 6 %) (Table 4). N-uptake due to N application in
plant crop was grouped as N150 (244.17 kg ha-1) [ N75
(219.7 kg ha-1) [ N0 (165.5 kg ha-1) and different bio-
fertilizer treatments were grouped as T1 [ T6, [ T7 [ T4,
T5 [ T2 [ control. On overall mean basis, increased
N uptake due to N application ranged as N150 (238
kg ha-1) [ N75 (201 kg ha-1) [ N0 (147 kg ha-1) and
due to biofertilizers, it ranged as T7, T1 [ T4, T5 [ T2, T3,
T6 [ control.
Extractable soil nitrogen measured as kg ha-1 ranged
from 125 to 202 kg ha-1 in plant crop (cv = 4.2 %) and
170 to 230 kg ha-1 in ratoon crop (cv = 1.7 %) (Table 5).
At the start of the experiment, the soil available N was
186 kg ha-1. On mean basis, there was decline in SAN
after harvesting plant crop except in treatments T1 and T7
where it was at par with the initial level for both biofer-
tilizer and N treatments. During ratooning at N75 and N150,
SAN increased by 7–12 % compared to its initial level at
the start of the experiment. Among different biofertilizers
the increase in SAN ranged from 2 to 16 % compared to
initial status with maximum in treatment T1 (215 kg ha-1)
and minimum in T7 (190 kg ha-1) whereas treatments T2
and control were at par with the initial level. The data on
available nitrogen (N) content of soil (Table 4) indicated
that its values were higher in soils under organic farming
than conventional farming, irrespective of cropping system
followed. The increase in SAN can be explained to be due
to production of appreciable quantities of carbonic acids by
the treatments during decomposition of underground bio-
mass, which mineralize the complex organic substances
and in turn contribute to N pool. The increase in soil
available nitrogen is also attributable to the greater multi-
plication of soil microbes caused by the addition of root

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Table 3 Effect of
Treatment Plant Crop Ratoon Crop
biofertilizer 9 N interactions
on commercial cane sugar N0 N75 N150 N0 N75 N150
production (t ha-1) of
sugarcane plant–ratoon system T1 8.77 9.98 10.23 8.66 9.20 10.13
T2 8.50 8.93 9.67 9.02 9.43 10.10
T3 8.04 8.33 8.80 7.83 7.76 8.60
T4 8.17 9.20 9.67 8.50 9.46 10.00
T5 8.07 8.87 9.80 8.30 8.83 9.73
N0: No nitrogen input; N75: T6 7.80 8.27 9.00 8.03 8.46 9.46
75 kg N ha-1; N150: T7 7.87 8.45 9.03 8.60 9.06 9.76
150 kg N ha-1; T1:
Gluconacetobacter T8 6.47 7.50 8.33 6.70 7.76 8.66
diazotrophicus; T2: Azotobacter F value LSD (P \ 0.05) F value LSD (P \ 0.05)
chrococcum; T3: Azospirillum
brasilense; T4: T1 ? T2; T5: Bacterial treatment (T) 151.48 0.19 108.85 0.18
T1 ? T3; T6: T2 ? T3; T7: Nitrogen (N) 261.71 0.12 318.17 0.11
T1 ? T2 ? T3; T8:
uninoculated control T9N 4.97 0.328 3.93 0.31

Table 4 Effect of
Treatment Plant crop Ratoon crop
biofertilizer 9 N interactions
on N uptake (kg ha-1) by N0 N75 N150 N0 N75 N150
sugarcane plant–ratoon system
T1 211.7 290.3 305.7 184.6 256.3 271.3
T2 145.0 155.0 233.3 174.6 187.0 194.3
T3 136.3 192.7 244.0 124.3 182.3 229.7
T4 174.3 205.6 280.3 143.3 205.3 280.7
T5 193.7 202.6 235.0 143.3 221.00 242.6
N0: No nitrogen input; N75: T6 177.3 263.7 287.0 108.0 187.6 218.7
75 kg N ha-1; N150: T7 171.3 242.7 281.7 200.3 222.0 293.0
150 kg N ha-1; T1:
T8 114.3 126.6 164.7 96.3 143.6 178.3
Gluconacetobacter
diazotrophicus; T2: Azotobacter F value LSD (P \ 0.05) F value LSD (P \ 0.05)
chrococcum; T3: Azospirillum
brasilense; T4: T1 ? T2; T5: Bacterial treatment (T) 191.36 8.55 75.02 11.07
T1 ? T3; T6: T2 ? T3; T7: Nitrogen (N) 561.82 5.24 373.69 6.78
T1 ? T2 ? T3; T8:
T9N 15.02 14.81 8.00 19.17
uninoculated control

biomass, which mineralize organically bound N to inor-
ganic form.
Nitrogen Use Efficiency and BNF Contribution
Partial factor productivity (PFP) was more in plant crop as
compared to ratoon crop under different biofertilizer
treatments. At N75 mean PFP was approximately 7 % more
in plant crop as compared to ratoon crop, whereas it was at
par at N150 in both the crops (Table 6). On mean basis,
different biofertilizers increased PFP by 9–38 % in the
plant crop and by 4–24 % in the ratoon crop over control.
Based on the data of available N in soil after harvest of
plant or ratoon crop, total uptake of N by the crop and the
amount of fertilizer N added to the soil, an apparent N
balance was calculated which clearly indicated that the
amount of N added to the system by BNF or from other
sources like soil organic matter/total nitrogen was very
high and it varied from 52.3 to 216.7 kg N ha-1 in plant
crop and from 65 to 225.3 kg N ha-1 in ratoon crop under
different treatments (Fig. 1). Although soil organic matter
and total N during crop cycle has not been much affected
(data computed but not shown), we presume that this N
addition is mainly through BNF but still in subsequent
discussion we shall represent it by Nbnf/othersources. Under
absolute control conditions (T8 N0) where no biofertilizer
or nitrogen was added, an estimated quantity of 58 and
102 kg N ha-1 was added to the system by BNF/other
sources for plant and ratoon crop, respectively. The max-
imum N contribution was recorded with G. diazotrophicus
and minimum with A. brasilense in a plant–ratoon cycle of
sugarcane crop.
Since the zone of inhabitation and metabolism are dif-
ferent for 3 classes of N fixers under study, these alone and

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Table 5 Effect of
Treatment Plant crop Ratoon crop
biofertilizer 9 N interactions
on soil available N (kg ha-1) N0 N75 N150 N0 N75 N150
under sugarcane plant–ratoon
system T1 186.0 187.7 202.0 195.0 209.0 218.6
T2 156.3 175.0 175.0 179.0 188.0 196.7
T3 141.3 164.6 167.0 193.6 203.3 215.0
T4 175.0 176.7 180.3 194.6 206.0 212.0
T5 153.0 159.6 172.3 194.7 205.6 211.0
N0: No nitrogen input; N75: T6 161.3 175.3 185.0 190.3 194.3 209.0
75 kg N ha-1; N150: T7 165.7 190.7 194.3 190.7 188.3 192.0
150 kg N ha-1; T1:
T8 130.3 155.0 161.0 173.0 190.0 197.7
Gluconacetobacter
diazotrophicus; T2: Azotobacter F value LSD (P \ 0.05) F value LSD (P \ 0.05)
chrococcum; T3: Azospirillum
brasilense; T4: T1 ? T2; T5: Bacterial treatment (T) 28.15 6.90 73.74 3.26
T1 ? T3; T6: T2 ? T3; T7: Nitrogen (N) 52.48 4.23 163.97 1.99
T1 ? T2 ? T3; T8:
T9N 2.11 11.95 4.62 5.64
uninoculated control

Table 6 Effect of biofertilizer 9 N interactions on PFP of sugarcane plant–ratoon system

Treatment Plant crop Ratoon crop
N75 N150 Mean N75 N150 Mean

T1 1,206.3 650.5 928.4 1,088.8 600.0 844.4

T2 994.7 551.1 772.9 1,004.4 546.7 775.5
T3 1,087.1 549.5 818.3 955.5 482.2 718.8
T4 1,110.7 602.2 856.4 1,048.0 560.0 804.0
T5 1,130.3 588.5 859.4 1,004.0 506.7 755.3
T6 1,040.9 547.8 794.4 920.0 500.0 710.0
T7 976.4 498.7 737.5 1,004.4 588.9 796.6
T8 873.7 474.8 674.3 875.5 482.2 678.8
Mean 1,052.5 557.8 805.2 985.5 533.6 759.5
LSD 23.6 18.9 35.6 25.6
-1 -1
N0: No nitrogen input; N75: 75 kg N ha ; N150: 150 kg N ha ; T1: Gluconacetobacter diazotrophicus; T2: Azotobacter chrococcum; T3:
Azospirillum brasilense; T4: T1 ? T2; T5: T1 ? T3; T6: T2 ? T3; T7: T1 ? T2 ? T3; T8: uninoculated control

in combinations were assessed at different levels of N input
for enhancing sugarcane crop productivity and soil N bal-
ance, as benefits of biofertilizers can be achieved best when
used in INM mode with chemical fertilizer (Kennedy et al.
2004). Different biofertilizers increased the crop yield at all
nitrogen levels but the maximum increase was recorded at
N75 as compared to N150. It is in agreement with the reports
that N is probably the key factor for the association of
diazotrophic bacteria with the sugarcane plant system
(Baldani et al. 2002). The 15N natural abundance technique
established that the BNF contributes up to 60 % the total
assimilated N by sugarcane varieties not receiving fertilizer
N (Boddey et al. 2001) and the associated diazotrophs like
A. diazotrophicus, A. brasilense, Herbaspirillum spp etc.
along with other obligate and facultative diazotrophs can
fix up to 150 kg N ha-1 of atmospheric N (Dobereine
1997; Reis et al. 2000). Evidences from Brazil indicates
that fertilizer N can be reduced to half by exploiting BNF
system, which is mainly due to diazotrophic Glu-
conacetobacter and Herbaspirillum spp. (Boddey et al.
1995; Dobereiner 1997). The beneficial effect of the isolate
of G. diazotrophicus being used in the present study has
been demonstrated and compared with other isolates of
G. diazotrophicus in a pot house study (Suman et al. 2005).
In the present study, G. diazotrophicus based biofertilizer
was found to be the best in terms of crop yield production
followed by its combination with A. chrococcum and
A. brasilense but A. brasilense alone and in combination
with A. chrococcum did not do very well. For a bioinoc-
ulant to benefit the plant, it should establish and compete
with the native heterotrophic bacterial population as well as
other co-inoculated microflora for active sites on plant
roots. Possibly, in combined inoculation G. diazotrophicus
had to compete with other two isolates, which was not
there in the case of single inoculation. Best performance by
G. diazotrophicus must be due to its endophytic association

123
42
Fig. 1 The contribution of N through BNF and other sources as
affected by biofertilizer treatments T1: Gluconacetobacter diazotro-
phicus; T2: Azotobacter chrococcum; T3: Azospirillum brasilense;
T4: T1?T2; T5: T1?T3; T6: T2?T3; T7: T1?T2?T3; T8:
uninoculated control. dots: Plant crop; horizontal lines: Ratoon crop
with sugarcane where fixation of N2 and utilization of fixed
N operates efficiently. Oliveira et al. (2002) have indicated
that high BNF contribution observed for Saccharum spp.
could not be attributed only to one or few species. It might
be due to different endophytic species acting during dif-
ferent growth stages or an interaction between the host
plant and a complex endophytic community to promote
high transference of fixed nitrogen as well as the PGPB
effects. Soil application of Azospirillum has also been
reported to increase cane yield by 9 and 5 t ha-1 in plant
and ratoon crops, respectively (Shankariah and Hunsigi
2001). Plant crop responded better to biofertilizer appli-
cation than the ratoon crop as yield levels at all N levels
was lower in ratoon crop than that of in plant crop.
Although there was no significant change in pol % of
cane juice under different treatments but the total com-
mercial cane sugar (CCS) production which basically
depends on crop yield, was highly influenced by N and
biofertilizers (r2 = 0.847, P [ 0.05). The CCS production
and N uptake by the crop was found to be the maximum in
treatments where G. diazotrophicus, A. chrococcum and
their combination were used. Patil and Patil (1984)
observed that seed inoculation with A. chrococcum along
with 50–100 kg urea N ha-1 gave higher dry matter yield,
N uptake and soil N content than those obtained with N
alone in greenhouse condition using non-sterile soils.
Inoculation with Azospirillum significantly increased the N
content of sugarcane leaves in greenhouse conditions. The
Gluconacetobacter–sugarcane system has now become an
effective experimental model as the inoculation of seed-
lings with wild type G. diazotrophicus leads to greater
growth rates while nif- mutants were significantly less
effective proving that diazotrophic character (nif?) is
important for this system (Lee et al. 2002). Kennedy et al.
(2004) while reviewing the potential of non–symbiotic
bacterial diazotrophs as PGPR have stated that although the
magnitude of BNF from biofertilizers may account only for
123
Sugar Tech (Jan-Mar 2013) 15(1):36–43
a fraction of total crop N requirements but the effect of
reducing losses from an ecosystem may be equivalent to a
much more significant contribution to the N-economy of
crop production. Wu et al. 2005 demonstrated the positive
effect of biofertilizers containing N fixers, P and K sol-
ublizers and AM fungi on maize growth and have inferred
that microbial inoculum not only increased the nutritional
assimilation of plant (N, P and K) but also improved the
soil organic matter and total N in soil. Biofertilizer inoc-
ulations improved sugarcane N utilization efficiency as
indicated by increased PFP, which was higher at N75
compared to N150 both in plant and ratoon crops. Higher
PFP at N75 compared to N150 indicate that the plant system
efficiently utilized whatever soil nitrogen was present and/
or the inoculated biofertilizer established an efficient
microbe-plant association at low N input state leading
towards improved economic productivity. Proliferating
root biomass due to low N input might be providing more
sites for diazotrophic bacterial colonization and their entry
through cracks developed on at root initiation sites. At high
fertilizer N proliferation of non-nitrogen fixing bacteria due
to rich nutrient availability may inhibit the multiplication
of G diazotrophicus, whereas at low N the growth of
G diazotrophicus remains unaffected due to its ability to
sustain its N-requirement through nitrogen fixation
(Muthukumarasamy et al. 2002).
Apparent N balance under different biofertilizer treat-
ments for one crop cycle of sugarcane (plant ? ratoon)
clearly indicated that the crops accumulated more N than
the applied one. The probable sources of additional N are
(i) biologically fixed N by the biofertilizer or inherent
micro-flora associated with it and/or (ii) soil reserve N in
the form of organic matter. Since the use of 15N at field
level was not possible, indirectly to confirm the contribu-
tion of inoculated biofertilizers towards N economy
through BNF rather than mining of soil reserves, the soil
organic C was monitored throughout and it ranged from
0.47 to 0.5 %. This indicates that the added biofertilizers
established effective associations with sugarcane and
helped in N economy but due to no clear cut demarcation
available we used the additional N as N bnf/other sources. The
highest Nbnf/other sources contribution was achieved through
G. diazotrophicus based biofertilizer clearly indicating
high specify between G. diazotrophicus and sugarcane.
Muthukumarasamy et al. (1999) have indicated that com-
bination of G. diazotrophicus with vascular arbuscular
mycorrhiza can completely substitute the recommended
dose of 275 kg urea N ha-1 in tropical India.
Overall, it is concluded that the field inoculations of
sugarcane by diazotrophic biofertilizers are highly benefi-
cial and of tested biofertilizers, G diazotrophicus based
biofertilizer proved most efficient in terms of higher cane
yield, commercial cane sugar production and efficient N
Sugar Tech (Jan-Mar 2013) 15(1):36–43
utilization and at the same time maintaining positive N
balance; consequently chemical N fertilizers could be
saved by including G diazotrophicus based biofertilizers in
integrated management of sugarcane nutrition. However, it
seemed unlikely that a single strain of bacterium would be
capable of optimal activity and thus the diversity of habitat
and effectiveness might logically require more than one
bacterial strain to obtain the maximum biological effects on
plant growth.
Acknowledgments The authors thank Mr. Ashok and Mrs. Asha
Gaur for help in the experimentation and analysis. Financial assis-
tance by Department of Biotechnology, New Delhi, through INM
project is gratefully acknowledged.
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