1. {t.
ErrnCr OF DIFFERENT NITROGEN SOURCES ON PRODUCTION OF
INDOLE ACETIC ACID O)ilDASE OF ATTENNINU CEP(il.118.
B. Annadunai, *R. Jayaprakash and ** R. Puvanakrishnan
*Departnent of Plant Biolory and Biotechnolory, C. Abdul Hakeem College, Melvishararr 632 50g.
**Departrnent of Biotechnology, Cental Leather Research Institute, Adyar, Chennai- 600 200.
Accepted for publication - l4th September, 2(N4-
Abstract
Indole Acetic Acid Oxidase IAAO, an auxin degrading en4rne fuom Alternaria cepulae was
estimated in infected leaves of onion as well as in culture filtrate of the pathogen. The
activity of lndole Acetic Acid Oxidase (IAAO) when supplemented with various nitrogen
sources like, Ammonium Phosphate, Arginine are favourable for the growth of Alternaria
cepulae and Ammonium nitrate, Ammonium Phosphate, Magnesium Nitrate, Aspargine,
Gelatin, Yeast extract, Groundnut flour and soybean meal favours the IAAO production
whereas Arginine, Aspargine, Gelatin, Groqndnut flour, Peptone, Soyabean meal and Sodium
Nitate favours protein production..
Key words z Alternaria cepulae,Indole Acetic Acid Oxidase (IAAO), Nitogen sources.
lntroduction
During Leaf blight disease of Onion,
IAA concentration was increased in
the blight areas of Onion leaves
(Annadurai,l 989, 1996, 1998, 1999, 2000).
It occurred during the .first i 6 days of
infection. Shaw and Hawkins ( 1958 )
found 214 fold increase of auxin in wheat
infected by Puccinia graminis and 5 fold
increase of IAA in barley infected by
Erisiphe graminis (Fric 1975). Pilet
(1953) reported that the ratio of IAA
equivalents of diseased and healthy
tissues per kg fresh weight was 39.7;
7.6. This excess production of auxin is
known pathogenesis (Pegg and Selman,
1959, Sequeira and Kelman,1962, Matta
and Gentile, 1968). Hyperauxiny is not
conductive to the growth of pathogen and
hence the pathogen Produces auxin
degrading erWme and hence the pathogen
produces auxin degrading enzyme for its
survival (Annadurai, 1989). Indole acetic
acid oxidase is one of the auxin
de grading eflzy me (Krupasag ar,I9 69 ;
Annadurai, 1989).
The mould Omphalia flavida was
reported to produce IAAO while infecting
on Coffea arabica and Coleus blumei
(Sequeira and Steeves 1954). Verticillium
*presently as Reader and llead Department of Botany & Biochemistry, CAH College, Melvisharam -632 509.

2. Effect of various N, sources on production of IAAO of Alternaria epulae
dahliae on Capsicum annum (Srivastava
et al., 1962), Fusarium oxYsPorum ol
Mangifera indica (Kumar et al., 1960),
Protontyces macrosporus on Coriandrum
saliwrn (Tayal et al., l98l). Plasmodiophora
brassicae on Lycopersicum esculentum
(Reuveni et a.,1985) and Plasmodio-
phora brassicae on Grapes (Boerner
et at.,l97 4). Since auxin 'content
decreases in the infected host tissue due
to the action of IAA oxidase IAA
oxidase level in culture filtrate of
Alternaria cepulae and in the.blight
, areas of Onion leaves (Annadurai &
.Motlag, 1999).The cultire-filtrate obtained
from the Ray's medium was purified and
analyzed is presented in this paper.
1AAO plays.a significant ro.le in
pathogenesis of many p)ant d'rseases.
Endopolygalacturonase is one of the
prime macerating enzyme produced by
Alternaria cepulae (Annadurai et al.,
1998-99) duriqg leafblight disease of '
Onion.
Endopolygala'cturonase is produted
both constitutively and adaptively by
different microorSanisms. In most
instances, tlle- gn-zyme.s are produced
adaptiv-ely raiher than con.stitutively.
Fuiarium mon ilydr me and Sclerafiu'm
zolfiil (Bilgrdmi,1992) proddce it
adaptively in the presence of pectic
substrates. A small number of pathogens
like Piricularia ftlamenlosa (Ayers and
Papavizas, 1966) and Colletotrichum
faleatum (Singh,1964), Verticillium
alboatrum and Aphanomyces cwtices
(Ayers, 1965) are known to Produce
polygalacturonase in a constitutive
manner. These carbohYdrates ate
reported to control the production of IAA
oxidase enzymes carried out on various
nuitritional factors and culture conditions
influencing the production of IAA oxidase
of Alternaria cePulae.
Materials and Methods
Crude enzyme PreParation
Crude enzyme was obtained from the
medium suggested by RaY (1956)'
Mycelial dry weight determination
It is donb following the method of
Annadurai et at., (1999, 2000).
Estimation of IAA oxidase activity
IAA oxidase activity was estimated
according to the rffethod of Sequeria and
Mtrro (966). t usi t[ A'A' sxi(ass
enzyme was defined as mg of IAA
destroyed per ml of enzyme in one hour
(Imbert and Wilson, 1972).
Estimation of Protein
The iroteih-content was estimated
according to the niethod of Lowry et al
(1951) using crystallilne Bovine Serum
Albumin as the reference Protein.
Effect of addition of different sources
.of'Nitrogen on the activity EndoPG
in culture filtrate : The effeet of
addition of differerfi sources of Nitrogen
on IAA oxidase activity was studied
according to the rnethod of Singh and
Tandon (i966).
The synthetio pectin 20 ml was taken
in 100 ml Erlenrneyer flask. The 20 rnl
of the synthetic R.ays medium was taken
in 100 rnl Erlenmeyer flask. The rnedium
was supplemented with lgn of different

3. S/
B. Annadurail ef. a/., 339
Tabte 1 : Influence of Nitrogen source on IAAO production by leafblight causing fungus
A. cepulae.
S.
N(
Nitrogen source r Mycelial dry weight IAAO
Activity
RA
Vo
Protein
gms+SD SEM s Units
pgms/
ml.
s pgms/ml s
Control 5 0.66*4.072 0.0038 32+1.14 2.01 10c 184+9.44 1.15
l Ammonium Nitrate 5 0.65*0.08i 0.054 + 96+4.49 1.25 NS 30( l8GI8.2 1.05 NS
2. Ammonium phosphatt 5 0.9010.092 ).00089 NS 39+1.04 0.97 NS tzt 160*6.45 1.26 +
3. Ammonium oxalate 5 1.0r0.24 s.8t ++ 8+0.64 t.75 NS 25 156+5.76 1.56 +
4. Arginine 5 0.92+0.09: 0.0053 +++ 28+1.24 1.45 NS 8i z9al2.2: 1.26 NS
5. Magnesium nitrate' 5 1.07+0.02 5.8r + 80*2.25 8. l3 + 254 120+7.24 0.94 +
6. Urea 5 0.040+0.04 0.01 NS 8*,2.42 t.l5 NS 25 106*6.14 t.27 +
1. Aspargine 5 1.0+0.0{ 4.47 NS 80+3.34 1.45 N$ 25( 244+.t2;M 1.59 NS
8. Gelatin 5 0.41*0.05 6.26 NS 40+2.2 1.09 NS 12: 26(}!13.8( r.2l +
9. Groundnut flour 5 t.4910.421 9.39 + 52+5.7 1.85 + 475 214*ll.9t t.43 +
10 Peptone 5 1.3Ga0.5' 4.91 + 8+0.75 1.46 + 2: t96d9.45 0.93 +
1l Soybean meal 5 r.03+0.24 8.94 ++r 40*1.72 1.83 NS 125 314+15.6t 0.83 +
Values expressed are the mean value (X ) of 6 individual experiments + SD
d.f. : degrees of freedpm : n-l
SEM : Standard elror mean 1 SD/n
Sigrificance ++ = p < 0.001
+ = p <o.05
+++=p<0.002
NS : Not significant
Mycelial <iry weight in grarns.
IAA oxidase aativity is expressed as pgms of IAA destroyed/rnl&r'l
Relative activity is expressed in percentage by taking control as cent percent.
Protein content is expressed in rnicrograms per ml by the method of Lowry e, aL, with BSA standard.

4. Effect of various N, sources on production of IAAO ol Altemaia cepulae
Nitrogen sources (solowA/) like Ammonium
chloride, Ammonium nitrate, Ammonium
phosphate, Ammonium oxalate, Ammonium
sulphate, Magnesium nitrate, urea,
Aspargine, Gelatin, Groundnut flour,
peptone, soyabean meal. The flasks were
sterilised in an autoclave and cooled. The
fungus A. cepulae was theninoculated
and incubated for 16 days at 32*1"C.
After 16 days, the mycelial mat was
removed. The content of the flask was
filtered. The filtrate was centrifuged at
20,000 rpm for 15 minutes. The IAA
oxidase activity and the protein content
were estimated and recorded.
Results and Discussion
When the leaf blight causing fungus
Alternaria cepulae is grown in synthetic
pectin medium (Table l), the mycelial
growth as indicated by dryweight slowly
increases and it is significant upto 20 days
(p<0.001). The difference is not
significant after 20 days. The protein
content also increases from 548 mg to
638 mg/ml. The difference is significant
on 8th day and 12th dafl.It is significant
at 5Yo level from 16 days to 30 days.
IAAO activity slowly decreases to 80 to
564 units on the 16th day. Thereafter the
activity slowly decreases. The difference
of IAAO actrvity is significant (P<0.001).
Among different nitrogen tested for
the effect on IAAO activity the mycelial
growth significantly increased by
Ammonium phosphate, Arginine,
(P<0.001). Other Nitrogen sources do not
influence the growth of A. cepulae.
IAAO activity is increased by
Ammonium nitrate, Ammonium phosphate,
Magnesium nitrate, Aspargine, Gelatin,
Groundnut flour, Soyabean meal, yeast
extract (P<0.001). Other nitrogen
sources are ineffective- Protein content
is increased by Arginine, Aspargine,
Gelatin, Groundnut flour, Peptone,
Soyabean meal and Sodium nitrate.
(P<0.00i). Other nitrogen sources are
inaffective.
The medium, in which the pathogen
grows determine the types and quantities
of auxin degrading enzymes. Living
organisrns are known to utilize 40
elements among which nitrogen are the
component of both structural and
functional cell constituents, Nitrogen
accounts for fifty percent of the total
mycelial dry weight. AII important
oomponents of the cellwall contain
nitrogen in varying forms and
concentrations. The results of various
nitrogen sources shown in Tablel
suggest that Ammonium PhosPhate,
Arginine favours the growth and
Ammonium nitrate, Ammonium phosphate,
Magnesium nitrate, Aspargine, Gelatin,
Groundnut flour, Soyabean meal, yeast
extract favours IAA oxidase activity.
Hence these nitrogen sources are
suggested for industrial production of IAd
oxidase enzJme.
Acknowledgements
The author B.A is thankful to
Dr.S.C.Dhar, Dr.R.Puvanakrishnan and
Dr.S.Chandrasekar of CLRI for
laboratory facilities and critical discussion.

5. B. Annadurail et al., u1
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