Twelve advanced lines of mustard (Brassica juncea L.) were evaluated for response to treatment with Azospirillum brasilense in yield and a few components. Two lines gave yield increases with concomitant increases in nitrogenase activity and Azospirillum population. In general, there were desirable and significant correlations between plot yield, nitrogenase activity and log, (Azospirillum population). The results point to the possibility of utilizing genotype-dependent response to Azospirillum in breeding programmes and also for exploring new avenues of Azospirillum-based cultural practices in sustaining improved productivity

Arunachalam, V.

Kesava Rao, P. S.

Tilak, K. V. B. R.

English

Publications of the IAS Fellows

-----,-- S TER GA TIVE 
Arunachalam, V., Kesava Rao, P. S. an 
Tilak, K.V.B.R. 
Brassica Juncea L. 
Current Science, 59 1990: 607-609. 
Record no. D-77 
• In 
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Genotype-dependent response to 
Azospirillum treatment in yield and 
nitrogenase activity in Brassica juncea 
L. 
P. s. Kesava Rao, V. Arunachalam and 
K. V. B. R. Tilak* 
Di\ isions of Genetics. *Microbiology. Indian Agricultural Research 
"Institute. New Delhi 110 012. India 
Twelve advanced lines of mustard (Brassica juncea L.) 
were evaluated for response to treatment with 
Azospirillum brasilense in yield and a few components. 
Two lines gave yield increases with concomitant increases 
in nitrogenase activity and Azospirillum population. In 
general, there were desirable and significant correlations 
between plot yield, nitrogenase activity and log, 
(Azospirillum population). The results point to the 
possibility of utilizing genotype-dependent response to 
Azospirillum in breeding programmes and also for 
exploring new avenues of Azospirillum-based cultural 
practices in sustaining improved productivity. 
IN the context or imperative need for increasing and 
, 
sustaining prodUCtivity in oleiferous Brassicae, the utility 
of bio-fertilizing agents assumes crucical importance. 
Az()spiri/lum has been reported to be a good microaerophilic 
nitrogen fixer in several plants I. 2. Its potential to increase 
grain yields has been recorded in crops like sorghum, 
pearl millet. ragi and barley, though the magnitude of 
improvement varied with the crop and genotypes. The . 
utility of such biofertilization in Brassica did not appear 
CURRENT SCIENCE VOL. 59, NO. 12, 25 JUNE 1990 
R.ESE,~RCH COMMUNICATIONS 
-_.. _ .. _- . ...... .. -... _.- ---
L) have been exammcd or reported so far. This paper 
sJrnmarizes the results of a pilot study on the response of 
Brassico juncea to seed treatment with A:ospirillum 
hrasiJense. 
Ten_ advanced lines of B. juncea developed in the Oilseeds 
Unit of the Indian Agricultural Research Institute, and the 
two checks~ Pusa Bold and Pusa Barani fonned the material. 
, 
~eeds of the lines were coated with A. brosilense following 
the procedure outlined by Subba 'Raoet 01.3 Thus in each 
line there were two treatments . control, and treated with 
Azospirillum. The material was grown in a split-plot design 
with three replications, with the lines allotted to main 
plots and the two treatments to sub-plots. The plot size 
was five rows of 5 m length, with inter-row space of 60 
cm and inter-plant distance of 10 to 15 cm. The crop was 
raised under normal agronomic and cultural practices. The 
crop was free of diseases; but aphids were controlled by 
timely application of the insectiside, Rogor. 
Observations were recorded on plot yield (g) and on the 
following characters on samples of five plants, with mean 
values being used for statistical analysis: plant height (cm), 
number of primary branches, number of secondary branches 
and biomass (g). The presence of Azospirillum in the 
rhizosphere soil was detected by the ~ost probable number 
(MPN) method4 • The bacterial population in the 
endorhizosphere was estimated by macerating one gram 
of fresh roots after surface-sterilizing the roots with 0.1 % 
chloramine-T for 30 min and making to 10-fold serial 
dilutions in sterile distilled water. The MPN counts were 
taken according to AlexanderS and the.population expressed 
as per g dry soil or per g fresh weight of roots. The cell 
count values were transformed to log' scale to remove 
non-nonnality for further analysis. The acetylene reduction 
assay suggested by Hardy et 01.6 was used to assess the 
nitrogenase activity in excised roots. 
The variation all!ong the lines was significant for plot 
yield and the three characters logl' (Azospirillum population 
in rhizosphere, RHS), logt (Azospirillum population in 
endorhizosphere, ERS) and nitrogenase activity (NGA). 
Treatment variation and the line x treatment interaction 
were significant for biomass, ERS and NGA; they were 
not significant for plot yield. 
The range of variation among the lines was high in the 
control !ines for all characters except primary and seconeary 
branches (fable 1). Azospirillum treatment enhanced the 
mean ~nd the range of variation still further. Overall, plot 
yield showed a little but non-significant depression under 
treatment. In general, the coefficient of variation (CV) for 
the treated lines was much higher than the corresponding 
values for control, though for Azospirillum cell count and 
nitrogenase activity there were either no differences or a 
nominal decrease in the CV of treated lines. 
However, a few cultures responded to the Azospirillum 
treatment and could show significant improvement over 
control for primary branches, ,biomass and plot yield (Table 
2). The improvement was relatively higher for other 
607 
• 
) 
RESEARCH COMMUNICATIONS 
------>_._--_._-.:.....-_-
Table 1. Mean <:HlU 1<1l1t:C 01 ;,'anatlOfl for vanoo~ traits 10 B.)UflC·ea 
ConLroi Treated 
Genetic 
vananCt' 
----------- among 
Character Range Mean CV Range Mean CV lines 
...... _>-- '" ""'-'--' --,._. --- ... __ ._----.,. ---- ... --.-.. --
Plant height (em) 
Primary branches 
Secondary branches. 
Biomass (g) 
Plot yield (g) 
log, (A::ospirillum 
population) 
Rhizosphere (per g 
soil) 
Endorhizosphere (per 
g fresh rOOI weight) 
Nitrogenase activilY 
(umol C;!HJhlg fresh 
root weight) 
148-204 
5-7 
10-17 
68-127 
1220-2987 
9-14 
1-10 
4-13 
173 9 
6 8 
13 15 
106 18 
2391 22 
12 15 
8 I I 
6 
151-208 174 10 228.27 
6-7 6 7 0.0012 
(}9-18 13 20 0.55 
78-176 110 27 ~.96 
1267-3060 2291 25 0.27 
15-23 18 12 2.95 
11-16 12 12 0.43 
6-22 I I 12.10 
Table 2. Signifi<:ani improvement ('*) in some characters with applic;ation of 
A. brasilenle in B. junceo. 
Treated> Control Treated < Conlrol 
------ ----
Character Cultivar Improvement Cultivar Improvement 
Primal)" brdJlche~ DIRA 326 30 
DIRA 329 20 
DIRA 333 22 
Secondarv brdn(~-; 
Biomass 
Plot vidd 
DIRA 326 
DIRA 329 
DIRA 333 
DIRA 337 
DIRA 326 
DIRA 313 
DIRA 335 
characters than for plot yield .. The yieJd increase was nol 
accompanied by simultaneous impro\'ement in other 
characters. Some lines showed significant decrease in the 
values of secondary branches. biortlb'S and plot yield under 
Azospirillum treatment. The results are thus suggestive of 
a genotype-d~pendent response to Azospirillum treatment. 
The observed improvements could be associated with 
significant and desirable correlation between yield (YLD) 
and some of the component characters defining increased 
nitrogenase activity. In particular. RHS. NGA and. ERS 
were worth mentioning {r: YLD-RHS = 0.546*; YLD-
NGA ~ 0.457*; RHS~ = 0.896*). Fmther. the.legJession 
. / I 
of plot y'ield on ~itrogenase activity "'as significant (at 
P = 0.05) under Azospirillum treatment (b = 1.638*) and 
not significant under control (b = 2.027. not signifICant). 
In tum, the regression of the increase in nilrOgenase activity 
in lines on the i~ in RHS was signiflCallt (b = 5.428*). 
Thus the results would suggest that A:ospirilfum count in 
rhizosphere could have accentuated nitrogenase activity 
resulting in better nitrogen remobilization and thereby in 
increased yields. Such a view is also ,upported by the 
608 
55 
", 
-. 
24 
160 
I I 
R 
DIRA 335 
Pusa Bold 
Pusa Barani 
DJRA 335 
Pusa Bold 
DIRA 18 
DIRA 344 
24 
19 
29 
31 
30 
26 
12 
desirable .and positive increases in (he values of RHS. 
ERS and NGA in cultures that recorded increases in plol 
yield (Table 3). But such increases were not observed in 
the relatively high-yielding checks~ Pusa Bold and Pusa 
Barani, which recorded high .increases in nilrogemtSe activity. 
It is possible that the Azospirillum population had already 
reached the required threshold in the check varieties: as a 
consequence, further increases in cell count and henCe 
nitrogenase activity could not result in proportionate 
increases in yield. By the same logic, lines like DIRA 
3) 3, DIRA 335 which did not possibly reach such thresholds 
in A:ospirillum population and nitrogenase activity could 
record appreciable > yield increases (Table 3). Thus the 
observation that there is genotype-dependent response for 
A:ospiri/lum treatment in B. juncea gains further ground. 
I.t Earlier work has also shown differential response to 
A:ospirillum in varying growth environments and various 
crops 7.". In particular, Azospirillum treatment combined 
with low doses of nitrogenous fenilizer gav~ responses 
equivalent to those obtainable under high doses of N 
application. entailing considerab1e saving in fossil fuel 
CU RRENT SCIENCE VOl. 59, NO 12. 25 JUNE 1990 
Table 3. Increase in mean \'alue~ of some chardnt'f~ In A~'(lSfJtriliLlm tn:at-
menl over control in ~ific lines of 11 Junau 
Log 1.4 :o5piri/lun' 
r 
population) Endorhl-
zos~re 
Ploc Rhizosphere (per g fJnh Nitrogenase 
Line Biomass yield (pcr g soil) roo( weight) activity 
DIRA 313 21.8 0.21 5.92 4.94 4.75 
DIRA 335 - 34.7 0.23 6.36 ,3.30 7.50 
Pusa Bold - 32.9 - 0.21 6.61 6.32 6.59 
Pusa Barani - 11.0 - 0.14 9.05 8.60 9.17 
Isd 43.78 0.38 2.44 2.05 2.87 
depleting chemical fertilizers. With such possibilities, 
breeding for higher productivity in B. juncea begets an 
expanded horizon in which (i) increases due to combined 
application (of low doses) of N with Azospirillum~ and 
(ii) exploitation of genotype environment-dependent 
A:ospirillum response becomes feasible propositions to 
reckon with . 
. __ ._---- .. _------_._- ---------- -.. - . 
1. Lc1shmi-Kumari. M .. Kavimandan. S. K. and Subba Rao. r". S .. lndian 
I Exp. BioI., 1976. 14.638. 
2. Boddey. R. M. and Dobereiner. L in Nom .... mbuHic. ""·lIfo.~t·n Fualion 
and Organic Maller in the Tr<lpics. XII International (ongres~ Soil 
SCI.. New Delhi. India. 19M2. p. ~X. 
3. Subba Rao. N. S .• Tilak. K. \'. B. R.. Sin~h. C S. and Lakshmi-
Kumari. M .. Curro Sci .. 1979.48. 133. 
4. Cochran. W. G .. Biomt'1riCJ. 19:'0.6. 105. 
S Alexander. M .. in Methods oj Soil Allaly.Sl5. Pan II. I~(h A. Black 
el al.), American Society Soil &l~nces. 1467. 
6. Hardy. R. W. F .. Holsten. R-D .. KackMln. E. K. and Bums. R. C._ 
PlanT PhYJiol.. 43. 1185. 
7 Tila~. K. V. B. R .. S( I RCI'. 19,"7. 24. 16~. 
X Tilak. K V. B R .. anJ Subba RJn. ~. S .. RUII I-t" .. 1 \,.//\ 19~7. 4 
~7 
• 
-.. 


Genotype dependent response to Azospirillum treatment in yield and nitrogenase activity in Brassica juncea L.

Indian Academy of Sciences

-----,-- S TER GA TIVE 
Arunachalam, V., Kesava Rao, P. S. an 
Tilak, K.V.B.R. 
Brassica Juncea L. 
Current Science, 59 1990: 607-609. 
Record no. D-77 
• 
In 
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---
---
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------
---
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---
---
~ --
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---
---
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-----W -------
~ ---------UI:: 
----, --0\ --------
~ --------00 ------- --<.0= ------.... --= ------.... --.... ------.... --
N ------... --
W ----- --... --
~ ---------
Genotype-dependent response to 
Azospirillum treatment in yield and 
nitrogenase activity in Brassica juncea 
L. 
P. s. Kesava Rao, V. Arunachalam and 
K. V. B. R. Tilak* 
Di\ isions of Genetics. *Microbiology. Indian Agricultural Research 
"Institute. New Delhi 110 012. India 
Twelve advanced lines of mustard (Brassica juncea L.) 
were evaluated for response to treatment with 
Azospirillum brasilense in yield and a few components. 
Two lines gave yield increases with concomitant increases 
in nitrogenase activity and Azospirillum population. In 
general, there were desirable and significant correlations 
between plot yield, nitrogenase activity and log, 
(Azospirillum population). The results point to the 
possibility of utilizing genotype-dependent response to 
Azospirillum in breeding programmes and also for 
exploring new avenues of Azospirillum-based cultural 
practices in sustaining improved productivity. 
IN the context or imperative need for increasing and 
, 
sustaining prodUCtivity in oleiferous Brassicae, the utility 
of bio-fertilizing agents assumes crucical importance. 
Az()spiri/lum has been reported to be a good microaerophilic 
nitrogen fixer in several plants I. 2. Its potential to increase 
grain yields has been recorded in crops like sorghum, 
pearl millet. ragi and barley, though the magnitude of 
improvement varied with the crop and genotypes. The . 
utility of such biofertilization in Brassica did not appear 
CURRENT SCIENCE VOL. 59, NO. 12, 25 JUNE 1990 
R.ESE,~RCH COMMUNICATIONS 
-_.. _ .. _- . ...... .. -... _.- ---
L) have been exammcd or reported so far. This paper 
sJrnmarizes the results of a pilot study on the response of 
Brassico juncea to seed treatment with A:ospirillum 
hrasiJense. 
Ten_ advanced lines of B. juncea developed in the Oilseeds 
Unit of the Indian Agricultural Research Institute, and the 
two checks~ Pusa Bold and Pusa Barani fonned the material. 
, 
~eeds of the lines were coated with A. brosilense following 
the procedure outlined by Subba 'Raoet 01.3 Thus in each 
line there were two treatments . control, and treated with 
Azospirillum. The material was grown in a split-plot design 
with three replications, with the lines allotted to main 
plots and the two treatments to sub-plots. The plot size 
was five rows of 5 m length, with inter-row space of 60 
cm and inter-plant distance of 10 to 15 cm. The crop was 
raised under normal agronomic and cultural practices. The 
crop was free of diseases; but aphids were controlled by 
timely application of the insectiside, Rogor. 
Observations were recorded on plot yield (g) and on the 
following characters on samples of five plants, with mean 
values being used for statistical analysis: plant height (cm), 
number of primary branches, number of secondary branches 
and biomass (g). The presence of Azospirillum in the 
rhizosphere soil was detected by the ~ost probable number 
(MPN) method4 • The bacterial population in the 
endorhizosphere was estimated by macerating one gram 
of fresh roots after surface-sterilizing the roots with 0.1 % 
chloramine-T for 30 min and making to 10-fold serial 
dilutions in sterile distilled water. The MPN counts were 
taken according to AlexanderS and the.population expressed 
as per g dry soil or per g fresh weight of roots. The cell 
count values were transformed to log' scale to remove 
non-nonnality for further analysis. The acetylene reduction 
assay suggested by Hardy et 01.6 was used to assess the 
nitrogenase activity in excised roots. 
The variation all!ong the lines was significant for plot 
yield and the three characters logl' (Azospirillum population 
in rhizosphere, RHS), logt (Azospirillum population in 
endorhizosphere, ERS) and nitrogenase activity (NGA). 
Treatment variation and the line x treatment interaction 
were significant for biomass, ERS and NGA; they were 
not significant for plot yield. 
The range of variation among the lines was high in the 
control !ines for all characters except primary and seconeary 
branches (fable 1). Azospirillum treatment enhanced the 
mean ~nd the range of variation still further. Overall, plot 
yield showed a little but non-significant depression under 
treatment. In general, the coefficient of variation (CV) for 
the treated lines was much higher than the corresponding 
values for control, though for Azospirillum cell count and 
nitrogenase activity there were either no differences or a 
nominal decrease in the CV of treated lines. 
However, a few cultures responded to the Azospirillum 
treatment and could show significant improvement over 
control for primary branches, ,biomass and plot yield (Table 
2). The improvement was relatively higher for other 
607 
• 
) 
RESEARCH COMMUNICATIONS 
------>_._--_._-.:.....-_-
Table 1. Mean <:HlU 1<1l1t:C 01 ;,'anatlOfl for vanoo~ traits 10 B.)UflC·ea 
ConLroi Treated 
Genetic 
vananCt' 
----------- among 
Character Range Mean CV Range Mean CV lines 
...... _>-- '" ""'-'--' --,._. --- ... __ ._----.,. ---- ... --.-.. --
Plant height (em) 
Primary branches 
Secondary branches. 
Biomass (g) 
Plot yield (g) 
log, (A::ospirillum 
population) 
Rhizosphere (per g 
soil) 
Endorhizosphere (per 
g fresh rOOI weight) 
Nitrogenase activilY 
(umol C;!HJhlg fresh 
root weight) 
148-204 
5-7 
10-17 
68-127 
1220-2987 
9-14 
1-10 
4-13 
173 9 
6 8 
13 15 
106 18 
2391 22 
12 15 
8 I I 
6 
151-208 174 10 228.27 
6-7 6 7 0.0012 
(}9-18 13 20 0.55 
78-176 110 27 ~.96 
1267-3060 2291 25 0.27 
15-23 18 12 2.95 
11-16 12 12 0.43 
6-22 I I 12.10 
Table 2. Signifi<:ani improvement ('*) in some characters with applic;ation of 
A. brasilenle in B. junceo. 
Treated> Control Treated < Conlrol 
------ ----
Character Cultivar Improvement Cultivar Improvement 
Primal)" brdJlche~ DIRA 326 30 
DIRA 329 20 
DIRA 333 22 
Secondarv brdn(~-; 
Biomass 
Plot vidd 
DIRA 326 
DIRA 329 
DIRA 333 
DIRA 337 
DIRA 326 
DIRA 313 
DIRA 335 
characters than for plot yield .. The yieJd increase was nol 
accompanied by simultaneous impro\'ement in other 
characters. Some lines showed significant decrease in the 
values of secondary branches. biortlb'S and plot yield under 
Azospirillum treatment. The results are thus suggestive of 
a genotype-d~pendent response to Azospirillum treatment. 
The observed improvements could be associated with 
significant and desirable correlation between yield (YLD) 
and some of the component characters defining increased 
nitrogenase activity. In particular. RHS. NGA and. ERS 
were worth mentioning {r: YLD-RHS = 0.546*; YLD-
NGA ~ 0.457*; RHS~ = 0.896*). Fmther. the.legJession 
. / I 
of plot y'ield on ~itrogenase activity "'as significant (at 
P = 0.05) under Azospirillum treatment (b = 1.638*) and 
not significant under control (b = 2.027. not signifICant). 
In tum, the regression of the increase in nilrOgenase activity 
in lines on the i~ in RHS was signiflCallt (b = 5.428*). 
Thus the results would suggest that A:ospirilfum count in 
rhizosphere could have accentuated nitrogenase activity 
resulting in better nitrogen remobilization and thereby in 
increased yields. Such a view is also ,upported by the 
608 
55 
", -. 
24 
160 
I I 
R 
DIRA 335 
Pusa Bold 
Pusa Barani 
DJRA 335 
Pusa Bold 
DIRA 18 
DIRA 344 
24 
19 
29 
31 
30 
26 
12 
desirable .and positive increases in (he values of RHS. 
ERS and NGA in cultures that recorded increases in plol 
yield (Table 3). But such increases were not observed in 
the relatively high-yielding checks~ Pusa Bold and Pusa 
Barani, which recorded high .increases in nilrogemtSe activity. 
It is possible that the Azospirillum population had already 
reached the required threshold in the check varieties: as a 
consequence, further increases in cell count and henCe 
nitrogenase activity could not result in proportionate 
increases in yield. By the same logic, lines like DIRA 
3) 3, DIRA 335 which did not possibly reach such thresholds 
in A:ospirillum population and nitrogenase activity could 
record appreciable > yield increases (Table 3). Thus the 
observation that there is genotype-dependent response for 
A:ospiri/lum treatment in B. juncea gains further ground. 
I.t Earlier work has also shown differential response to 
A:ospirillum in varying growth environments and various 
crops 7.". In particular, Azospirillum treatment combined 
with low doses of nitrogenous fenilizer gav~ responses 
equivalent to those obtainable under high doses of N 
application. entailing considerab1e saving in fossil fuel 
CU RRENT SCIENCE VOl. 59, NO 12. 25 JUNE 1990 
Table 3. Increase in mean \'alue~ of some chardnt'f~ In A~'(lSfJtriliLlm tn:at-
menl over control in ~ific lines of 11 Junau 
Log 1.4 :o5piri/lun' 
r 
population) Endorhl-
zos~re 
Ploc Rhizosphere (per g fJnh Nitrogenase 
Line Biomass yield (pcr g soil) roo( weight) activity 
DIRA 313 21.8 0.21 5.92 4.94 4.75 
DIRA 335 - 34.7 0.23 6.36 ,3.30 7.50 
Pusa Bold - 32.9 - 0.21 6.61 6.32 6.59 
Pusa Barani - 11.0 - 0.14 9.05 8.60 9.17 
Isd 43.78 0.38 2.44 2.05 2.87 
depleting chemical fertilizers. With such possibilities, 
breeding for higher productivity in B. juncea begets an 
expanded horizon in which (i) increases due to combined 
application (of low doses) of N with Azospirillum~ and 
(ii) exploitation of genotype environment-dependent 
A:ospirillum response becomes feasible propositions to 
reckon with . 
. __ ._---- .. _------_._- ---------- -.. - . 
1. Lc1shmi-Kumari. M .. Kavimandan. S. K. and Subba Rao. r". S .. lndian 
I Exp. BioI., 1976. 14.638. 
2. Boddey. R. M. and Dobereiner. L in Nom .... mbuHic. ""·lIfo.~t·n Fualion 
and Organic Maller in the Tr<lpics. XII International (ongres~ Soil 
SCI.. New Delhi. India. 19M2. p. ~X. 
3. Subba Rao. N. S .• Tilak. K. \'. B. R.. Sin~h. C S. and Lakshmi-
Kumari. M .. Curro Sci .. 1979.48. 133. 
4. Cochran. W. G .. Biomt'1riCJ. 19:'0.6. 105. 
S Alexander. M .. in Methods oj Soil Allaly.Sl5. Pan II. I~(h A. Black 
el al.), American Society Soil &l~nces. 1467. 
6. Hardy. R. W. F .. Holsten. R-D .. KackMln. E. K. and Bums. R. C._ 
PlanT PhYJiol.. 43. 1185. 
7 Tila~. K. V. B. R .. S( I RCI'. 19,"7. 24. 16~. 
X Tilak. K V. B R .. anJ Subba RJn. ~. S .. RUII I-t" .. 1 \,.//\ 19~7. 4 
~7 
• 
-.. 


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Genotype dependent response to Azospirillum treatment in yield and nitrogenase activity in Brassica juncea L.

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