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This article was downloaded by: [University of Newcastle (Australia)]On: 30 September 2014, At: 17:45Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Archives of Agronomy and Soil SciencePublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/gags20
Impact of the use of biofertilizers oncotton ( Gossypium hirsutum ) cropunder irrigated agro-ecosystemNeeru Narula Prof. Dr , BS Saharan , Vivek Kumar , RanjanaBhatia , LK Bishnoi , BPS Lather & K Lakshminarayanaa Department of Microbiology, College of Basic Sciences &Humanities , Haryana Agricultural University , Hisar, IndiaPublished online: 06 Oct 2011.
To cite this article: Neeru Narula Prof. Dr , BS Saharan , Vivek Kumar , Ranjana Bhatia , LKBishnoi , BPS Lather & K Lakshminarayana (2005) Impact of the use of biofertilizers on cotton (Gossypium hirsutum ) crop under irrigated agro-ecosystem, Archives of Agronomy and Soil Science,51:1, 69-77, DOI: 10.1080/03650340400029275
To link to this article: http://dx.doi.org/10.1080/03650340400029275
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Impact of the use of biofertilizers on cotton (Gossypium
hirsutum) crop under irrigated agro-ecosystem
(Einfluss von Biodungemitteln auf Baumwolle (Gossypium
hirsutum) im bewasserten Agro-Okosystem)
NEERU NARULA, B. S. SAHARAN, VIVEK KUMAR, RANJANA BHATIA,
L. K. BISHNOI, B. P. S. LATHER & K. LAKSHMINARAYANA
Department of Microbiology, College of Basic Sciences & Humanities, Haryana Agricultural University,
Hisar, India
(Received 25 August 2004; accepted 4 December 2004)
AbstractHigh nitrogen fixing, phosphate solubilizing, phytohormones producing isolates of Azotobacter,Azospirillum, Acetobacter and Pseudomonas were used as inoculants for cotton. Important cultures wereselected on the basis of their effect on root/shoot length and chemotactic behaviour. Selectedbioinoculants were earlier tested for their beneficial properties like nitrogen fixation (ARA), ammoniaexcretion, IAA production etc. These bio-inoculants were further tested for phosphate solubilizationproperty. Various chosen strains were tested with Desi (HD 123) and American (H 1098) cotton underfield conditions (as for wheat). Plant height and boll weight were determined at the time of harvestingwhereas survival rate of inoculated bacteria was determined after 30, 80 and 130 days respectively. In theyear 2000 – 01, on the basis of boll number and boll weight plant7 1 AVK 51 (36; 76.2 g plant7 1), HT57 (27; 56.9 g plant7 1), AC18 (33; 61.5 g plant7 1), Ala 27 (36; 61.4 g plant7 1) and Pseudomonas (34;71.3 g plant7 1) were identified as significant both for American and desi cotton varieties. Highestsurvival rate was observed with Mac 68 (33.4 x 105) followed by HT54 (31.5 x 105) after 30 days ofsowing, which decreased after 80 days and remained constant up to 130 days. This trend was observedwith all the cultures. Similar results were observed in 2001 – 02. 25 kg ha7 1 N saving was observed withA. chroococcum (AVK51) bioinoculant for cotton crop.
Keywords: Bio-inoculants, azotobacter chroococcum, azospirillum, pseudomonas, gossypium hirsutum,antifungal activity, high temperature tolerance
Introduction
Cotton is one of the most important commercial crops in India. In the last five years,
however, production and yield in the country has declined as compared to the yield
Correspondence: Prof. Dr Neeru Narula, Department of Microbiology, CCS Haryana Agricultural University, Hisar-125 004, India.
E-mail: [email protected] and [email protected]
Archives of Agronomy and Soil Science
February 2005; 51(1): 69 – 77
ISSN 0365-0340 print/ISSN 1476-3567 online # 2005 Taylor & Francis Ltd
DOI: 10.1080/03650340400029275
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observed in leading cotton- growing countries. It is well recognized that microbial
inoculants constitute an important component of integrated nutrient management system
that would lead to sustainable agriculture. Biofertilization is known to help in the expansion
of root system and better seed germination. Plant associated rhizobacteria (PGPRs) such as
Azotobacter, Azospirillum, Acetobacter, Pseudomonas, etc. are able to colonize roots and can be
classified into beneficial, deleterious and neutral groups on the basis of their effects on plant
growth. PGPRs also include the diazotrophs which have the ability to convert atmospheric
nitrogen to ammonia which can be used by the plant as a source of nitrogen. Because of
their competitive advantages in a carbon rich, nitrogen poor environment, diazotrophs
become selectively rich in the rhizosphere putting them in a good position to promote plant
growth. These types of bacteria are able to stimulate growth and increase the yield of non-
legumes (Biswas et al., 2000; Yanni et al., 2001). Therefore, the present attempt was
undertaken to test selected Azotobacter strains which are high temperature tolerant (488C),
fix high amounts of nitrogen, P solubilizers and phytohormone producers and also possess
antifungal properties (Kumar & Narula, 1999; Verma et al., 2001). Selected (from pot
house studies) strains of Azotobacter, Acetobacter, Pseudomonas and Azospirillum were
evaluated on two varieties of cotton, Desi HD123 and American H1098 under field
conditions (irrigated agro ecosystem) for two years followed each year by wheat crop in the
same field.
Materials and methods
Bacterial strains
Soil isolates/mutants used during the studies were earlier isolated from rhizospheric soil of
wheat and cotton, characterized and identified according to Bergey’s Manual of Systematic
Bacteriology (Staley et al., 2001).
Bioinoculants’ effect on germination
Cotton seeds were sterilized by the conventional method using 0.1% acidified mercuric
chloride followed by 5 – 6 washings with sterilized distilled water. Sterilized seeds were treated
with freshly grown cultures for 30 minutes and kept on 0.8% water agar and incubated at
258C. Root and shoot length was measured after seven days.
Chemotactic studies
Chemotactic behaviour of some bioinoculants was studied using the exudates of five-day-old
seeds. The exudates were collected by placing 10 seeds in a test tube for five days. A sterilized
capillary tube was filled with the seeds exudates and placed for 30 minutes in the culture
broth. The contents of the capillary tube were then plated on Jensen media (JM) plates to
determine the number of bacteria attracted towards the seed exudates. For control, only water
or phosphate buffer was taken in place of exudates.
Phosphate solubilization
P solubilization property was studied in all the selected strains in Jensen’s broth containing
0.25% tricalcium phosphate (TCP). P solubilization was determined by colorimetric method
of John (1970) and colour development was measured at 600 nm.
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Field trials – for cotton
Field trials were laid out in the randomized block design (RBD) with three replications at the
university farms, CCS HAU, Hisar, India. The soil was sandy loam. The experiment was
carried out continuously for two years i.e., 2000 – 2001 and 2001 – 2002 in the same field.
During cotton season the temperature varies from 32.7 – 478C (maximum) and 23.5 – 30.28C(minimum). The mean rainfall during July – September was 53 and 58.1 mm. The
recommended dose of urea and 60 kg P2O5 ha7 1 (in the form of single super phosphate,
SSP) were added as mentioned in the package of practices, CCS HAU, Hisar.
During both years, nitrogen dose of 45 and 80 kg N ha7 1was used along with chosen
strains. In 2001 – 02, the experiment was done with six high temperature tolerant isolates/
mutants using both American (H 1098) and Desi (HD 123) varieties.
Treatment of seeds
Cotton seeds were first soaked overnight and then coated with molasses/sugar solution.
These seeds were treated with charcoal based cultures (109cfu g7 1 charcoal), mixed
properly and kept in the shade for 30 – 45 min. By the application of molasses, the charcoal
based cultures are able to stick well to the seeds. Treated seeds were then sown under field
conditions.
Plant parameters
Plant height, dry matter (2001 – 02), boll number and boll weight plant7 1 were determined.
Survival rate of inoculants
Survival of the inoculated bacteria from rhizosphere soil was determined at three stages viz.,
30, 80 and 130 days after sowing of the crop by plate count method. Composite rhizospheric
soil samples were withdrawn from three plants per plot, suspended in sterile distilled water,
serially diluted and plated on respective agar medium plates. Plates were incubated at 308Cfor 48 – 96 hours and colony forming units (cfu) g7 1 soil was determined.
Results
Bioinoculants’ effect on root and shoot length
The effect of various representative cultures was tested on American cotton germination with
reference to root and shoot length of cotton seedlings. Azotobacter HT54 and Azospirillum FS
showed better shoot length as compared to control. Root length and the number of secondary
roots were best in HT54 followed by Azospirillum FS and Acetobacter 35 – 47 (see Table I).
Chemotactic response
Chemotactic behaviour of Azotobacter HT54, HT57, E12 and Azospirillum FS were studied
using the exudates of five-day-old cotton seedlings (see Table II). The cultures showed
attraction towards the exudates leading to movement of inoculants towards roots exudates.
This result shows better colonization of the bioinoculants in the cotton rhizosphere, leading to
greater plant growth.
Impact of Biofertilizers on cotton yield 71
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Phosphate solubilization
Phosphate solubilizing activity of the various bio-inoculants was studied on JM+ TCP plates.
P solubilization was highest in AVK42 (0.9555mg ml7 1) followed by Mac 68
(0.9220 mg ml7 1) and HT57 (0.9082 mg ml7 1). Maximum decrease in pH irrespective of
P solubilization (due to production of organic acids etc.) was observed in HT 57 (4.87). Most
of the cultures tested could solubilize P and decrease the pH which ranged from 4.87 to 5.43
from the original pH of 7.0 (see Table III).
Cotton experiment during 2000 – 01
Effect of selected isolates on one variety of cotton (HD 123) was investigated under field
conditions. The results are shown in Table IV. Plant height, dry matter and boll weight
plant7 1 were determined at the time of harvesting whereas survival rate was studied at
various intervals of time. Maximum boll weight was observed with AVK51 (76.2g plant7 1)
which is 48.16% more as compared to the control. It was followed by Pseudomonas (71.3 g
plant7 1), AC 18 (61.5g plant7 1) and Ala27 (61.4g plant7 1). Boll number per plant was
maximum with Ala27 (36 plant7 1) and AVK 51 (36 plant7 1) followed by Pseudomonas (34
plant7 1). Maximum plant height (149.7cm) as well as dry matter (592.7g per plant) was
obtained with Pseudomonas followed by AVK 51 (146.1 cm; 587.4 g plant7 1resp.)
In the same field, survival rate of inoculated bacteria was also determined after 30, 80 and
130 days of sowing of the cotton crop. After 30 days, highest survival was observed in Mac 68
(33.4 x 105) followed by HT 54 (31.5 x 105) which decreased after 80 and 130 days. After 80
days, the survival in case of Mac 68 was 33.8 x 104 while 29.7 x 104 with HT 54. The survival
rate further decreased after 130 days and was noted as 15.3 x 104 and 12.6 x 104 in cases of
Mac 68 and HT 54, respectively (see Table V).
Kharif (summer) 2001 – 02
Various chosen strains were tested with two varieties of cotton viz., Desi (HD123) and
American cotton (H1098) under field conditions. Plant height and yield were determined at
Table I: Effect of bioinoculants on root/shoot length of cotton seedlings.
Inoculant Shoot length Root length No. of secondary roots
Control 6.04+1.53 7.74+2.91 18.1+3.58
Azotobacter (HT54) 6.50+1.54 14.0+3.39 20.7+4.16
Azotobacter (HT57) 5.00+2.51 8.54+5.91 10.8+4.43
Acetobacter (35 – 47) 5.48+2.08 10.9+5.19 13.16+4.17
Azospirillum FS 7.44+2.59 12.34+1.82 14.75+4.65
Table II. Chemotaxis of bioinoculants towards cotton seed exudates.
Bacterial culture CFU6102 cfu 100 ml7 1 (seed exudates)
HT54 1.4
HT57 1.3
E12 1.3
FS 1.3
72 N. Narula et al.
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the time of harvesting whereas survival rate was studied at various intervals of time (30, 80 and
130 DAS).
In case of American cotton (see Table VI), maximum boll weight was with AVK42 (68.3 g
plant7 1) followed by AVK 51 (57.3 g plant7 1), Ala27 (48.9 g plant7 1) and AC 18 (48.3 g
plant7 1). Boll number was maximum with AC18 (41 plant7 1) followed by AVK42 (32
plant7 1) and AVK 51 (28 plant7 1). Maximum plant height was obtained with Ala27
(193.4 cm) followed by AC 18 (143.1cm) and AVK51 (118.3 cm). After 30 days of sowing,
survival rate was observed maximum in case of HT 57 (8.29 x 106) followed by Ala 27 (3.71 x
106). Microbial population decreased with increase in time (see Table VII).
In Desi cotton, maximum boll weight was observed with Pseudomonas (62.9 g plant7 1)
followed by AVK51 (61.4 g plant71) and AC18 (56.8 g plant7 1). Boll number was
Table III. Phosphate solubilization by isolate/mutants of A. chroococcum.
Strain P solubilization (mg ml7 1) pH
Control 0.0830 6.90
Mac68 0.9220 5.21
AVK42 0.9555 4.92
Ala27 0.8710 5.20
IS16 0.8321 5.21
103 0.7987 5.22
HT54i 0.8230 5.23
HT54ii 0.8175 5.43
HT57 0.9082 4.87
AC18 0.8521 5.19
BNR-4 0.5522 5.67
For further studies Pseudomonas and Acetobacter were included.
Table IV. Effect of seed treatment with various bioinoculants on cotton (HD123) under field conditions (2000 – 01).
Treatment* Plant height (cm) Boll No. Plant – 1 Boll wt. Plant – 1 (g) Dry matter Plant – 1
(g)
Control 102.1 15.0 36.7 508.2
103 99.5 18.0 37.7 485.1
AC18 80.4 33.0 61.5 445.2
Mudhol 1 114.2 27.0 49.7 535.3
HT57 138.6 27.0 56.9 560.6
HT54 110.3 21.0 50.8 510.7
Mac68 112.7 26.0 37.8 523.4
Ala27 141.7 36.0 61.4 563.5
E12 133.4 23.0 53.6 551.7
AVK42 139.8 26.0 49.0 568.1
AVK51 146.1 36.0 76.2 587.4
AVK1 139.6 28.0 51.0 564.3
Pseudomonas 149.7 34.0 71.3 592.7
Acetobacter 35 – 47 109.4 28.0 43.1 504.8
CD 3.457 6.240 3.291 7.105
SE(d) 1.672 3.019 1.592 3.438
SE(m) 1.183 2.135 1.126 2.431
CV 1.670 13.694 3.706 0.786
*Nitrogen levels: Control with 80 kg N ha71; Bioinoculants + 45 Kg N ha7 1.
Impact of Biofertilizers on cotton yield 73
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maximum with BNR4 (43 plant7 1) followed by Pseudomonas (39 plant7 1) and Ala27 (29
plant7 1). Maximum plant height was obtained with BNR4 (198.0 cm) followed by AVK51
(197.0 cm) and Pseudomonas (190.4 cm) as shown in Table V. Survival rate (Table VII) was
also found significant with Acetobacter (9.35 x 106), HT57 (9.34 x 106) and HT54 (8.29 x 106)
after 30 days of sowing. Survival decreased after 80 days and remained almost constant till
130 days of sowing.
Discussion
Response of various crops to inorganic fertilizers is well understood. However a combination
of microbial inoculants and chemical fertilizers has shown variable results. Our studies were
aimed at testing the selected strains of Azotobacter, Acetobacter, Azospirillum and Pseudomonas
on two varieties of cotton (American H1098 and Desi HD123) continuously for two years
(2000 – 01 and 2001 – 02) under field conditions. These two varieties of cotton are genetically
different. HD123 is a Desi cotton variety which is diploid, with less nutrient uptake and has
less susceptibility to pests. H1098 is a tetraploid American cotton variety which has high
nutrient uptake ability and is highly susceptible to pests.
As cotton is a summer crop and temperature in summer rises up to 488C, selected cultures
were mostly high temperature tolerant. Azotobacter has the property of forming cysts. This
enables it to survive at high temperatures. Several reports have suggested that PGPRs (Plant
Growth Promoting Rhizobacteria) also stimulate plant growth by facilitating the uptake of
minerals such as N, P, K and other important micronutrients (Barea et al., 1976; Dobbelaere
et al., 2003). This uptake is suggested to be due to a general increase in the volume of the root
system (Gunarto et al., 1999; Biswas et al., 2000). Kumar and Narula (1999) have reported
that higher amount of IAA effects the seed emergence of wheat primarily because of the
production of growth regulators by bacteria. Better performance is attributed to capability of
high temperature tolerance of some cultures during the cotton crop season. It is also due to
the better proliferation, survival, ability to fix more N, antifungal properties of the inoculant
strains (Verma et al., 2001) and growth promoting substances which are also likely to
contribute to the beneficial effects on crops. The Azotobacter strains used in the present
investigation have also been tested for the above mentioned properties and it has been
Table V. Survival of bio-inoculants in cotton (HD 123) under field conditions (2000 – 01).
Strains *30 days *80 days *130 days
103 18.4 x 105 15.3 x 104 5.1 x 104
AC18 23.1 x 105 12.5 x 104 11.2 x 104
Mudhol – 1 23.3 x 105 28.9 x 104 18.0 x 104
HT57 19.0 x 105 18.3 x 104 5.8 x 104
HT54 31.5 x 105 29.7 x 104 12.6 x 104
Mac68 33.4 x 105 33.8 x 104 15.3 x 104
Ala27 19.4 x 105 21.8 x 104 14.1 x 104
E12 29.7 x 105 23.8 x 104 16.3 x 104
AVK42 25.7 x 105 27.1 x 104 21.4 x 104
AVK51 24.9 x 105 31.4 x 104 20.3 x 104
AVK 1 25.3 x 105 24.3 x 104 5.7 x 104
Pseudomonas 17.6 x 105 18.2 x 104 9.2 x 104
Acetobacter 35 – 47 12.4 x 105 12.9 x 104 2.3 x 104
*cfu g7 1 soil. Control = 103 to 104 cfu g7 1 soil.
74 N. Narula et al.
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observed that they have the ability to excrete ammonia, produce IAA, siderophores, have
antifungal property and are capable of fixing nitrogen (Narula et al., 2005).
Higher seed yield, plant growth and survival of the bio-inoculants may be attributed to
many factors most important being the favourable influence exerted by root exudates
(Vancura & Harizlikova, 1972; Leinhos & Vacek, 1994) that contain acids, organic acids,
carbohydrates and growth hormones like indole acetic acid. IAA synthesized by bacteria is
taken up by the plants and can stimulate cell proliferation. Nitrogen fixation and
solubilization of insoluble phosphate also contributes significantly to plant growth. Phosphate
solubilizers can exert a considerable influence on nutrient uptake (Barea et al., 1976).
Therefore, use of P solubilizing, IAA producing Azotobacter chroococcum may augment the
efficiency of applied and native P2O5 by reducing fixation by soil fraction.
Therefore, selection of isolates with high temperature tolerance, P solubilization,
phytohormone production and high N fixation has expanded the possibilities of using free
living nitrogen fixers to cereals and other non-legume crops. Our studies suggest that
microbial inoculants can be used as an economic input to increase crop productivity and
Table VI. Effect of seed treatment with various bio-inoculants on cotton under field conditions (2001 – 02).
Bio-inoculant Plant height (cm) Boll no. Plant 7 1 Boll wt. Plant 7 1 (g)
American cotton (H 1098)
N0 89.4 15.0 38.9
N1 95.6 24.0 43.7
N2 123.9 37.0 66.2
AC18 143.1 41.0 48.3
HT57 117.0 26.0 39.5
Ala27 193.4 23.0 48.9
AVK42 118.3 32.0 68.3
AVK51 118.3 28.0 57.3
BNR 4 101.3 19.0 47.3
CD 4.493 5.819 3.258
SE(d) 2.101 2.722 1.524
SE(m) 1.486 1.925 1.078
CV 2.105 12.245 3.664
Desi cotton (HD 123)
N0 113.4 15.0 34.9
N1 137.2 32.0 38.4
N2 188.3 39.0 55.9
103 184.0 26.0 43.9
AC18 139.0 23.0 56.8
HT57 130.8 27.0 35.2
Ala27 179.4 29.0 37.4
AVK51 197.0 24.0 61.4
BNR4 198.0 43.0 45.3
HT54 145.9 23.0 49.7
Pseudomonas 190.4 39.0 62.9
Acetobacter 140.3 28.0 52.3
CD 5.495 3.944 1.473
SE(d) 2.633 1.890 0.706
SE(m) 1.861 1.336 0.499
CV 1.991 7.980 1.807
N0=No nitrogen; N1=45 kg N ha7 1; N2=80 kg Nha71. Bio-inoculants + 45 kg N ha7 1.
Impact of Biofertilizers on cotton yield 75
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lowering the fertilizer level along with harvesting more nutrients from the soil. But a lot of
research work is still left to be done on aspects of phytohormone production and increased
nutrient uptake which is an important parameter in plant- microbe interaction.
Acknowledgement
We thank National Agricultural Technology Project (NATP) for the financial assistance
provided to carry out the research work.
References
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Table VII. Survival of bioinoculants on cotton under field conditions (2001 – 02).
Bioinoculants *30 days *80 days *130 days
American cotton (H 1098)
N0 4.89 x 103 5.92 x 104 2.89 x 103
N1 3.90 x 103 3.01 x 104 2.16 x 103
N2 3.25 x 103 4.29 x 104 7.89 x 103
AC18 1.58 x 106 2.97 x 105 2.92 x 105
HT57 8.29 x 106 7.92 x 105 9.34 x 105
Ala27 3.71 x 106 7.82 x 105 4.38 x 105
AVK42 1.93 x 106 3.57 x 105 3.56 x 105
AVK51 1.03 x 106 3.78 x 105 1.45 x 105
BNR4 1.46 x 106 1.78 x 105 5.89 x 105
Desi cotton (HD 123)
N0 4.92 x 103 2.51 x 104 7.90 x 103
N1 7.09 x 103 9.54 x 104 5.78 x 103
N2 5.20 x 103 5.67 x 104 4.35 x 103
103 2.57 x 106 2.58 x 105 2.45 x 105
AC18 1.01 x 106 4.67 x 105 4.67 x 105
HT57 9.34 x 106 3.67 x 105 7.90 x 105
Ala27 2.89 x 106 7.89 x 105 3.21 x 105
AVK51 7.34 x 106 2.45 x 105 6.48 x 105
BNR4 7.90 x 106 2.67 x 105 8.69 x 105
HT54 8.29 x 106 2.90 x 105 5.43 x 105
Pseudomonas 3.67 x 106 3.89 x 105 2.90 x 105
Acetobacter 9.35 x 106 8.59 x 105 8.89 x 105
*cfu g7 1 soil.
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