1. J.Ecotoxicol. Environ.Monit.l0(1) 37-41 (2000)
OPalani Paramount Publications, Printed in India ,-'$=
ISSN :0971 -0965-0 I 0-37
EFFECT OF VARIOUS CARBON SOURCES ON
PRODUCTION OF ENDOPOI YGALACTURONASE OF
ALTERNARIA CEPULAE
B ANNADURAI* AND D B MOTLAG
DEPARTMENT OF BIOCHEMISTRY, UMVERSITY OF MADRAS
CHENNAI600 O25INDIA
*DEPARTMENT OF BOTA].IY AND BIOCHEMISTRY, C A H COLLEGE'
MELVISHARAM 635 509 TAMIL NADU, INDIA
ABSTRACT
The activity ofendopolygalactuonase (Endo PG) in infected onion leaves and pectin medium
by Alternaria cipulae was estimated. The effect of various carbon sources like starch, iellulose,
gum arabic and glutamic acid are favourable for the growth of A. cepulae and endo PG production.
INTRODUCTION
Endopolygalacturonase @oly a 1,4 galcturonide glycanohydrolase, EC 3.2.1.15)
plays a significant role in pathogenesis of many plant diseases (Cooper 1980; Boothby
igti+; tvtiits tlas;. Endopolygalacturonase is one of the prime macerating enzymes
produced by Alternaria cepulae during leaf-blight disease of onion (Annadurai et al
1996;1998;1999).
Endopolygalcturonase is produced both constitutively and adaptively by different
miero organisms (Annadurai 1987). In most instances, the enzymes are produced
adaptively rather than constitutively (Keen & Horton 1966). Fusarium moniliforme
(Bi;hn 1p7l) and Sclerotium rolfsii (Punja et al 1985) produce it adaptively in the
presence of pectic substrates (Bilgrami 1982). A small number of pathogens like
biriculariay'iamentosa(Ayers &Papavizas 1966) andColletotrichumfalcatum(Singh
& Hussain 1964), Verticillium alboartrum (Mussel & Strouse 1972) and Aphanomyces
cutices (Ayers 1965) known to produce polygalcturonase in a constitutive manner.
pectin induces greater enzyme production in adaptive enzymes (Grant 1985).
The pathogen in nature confronts with pectic substances not in isolation, but in
combination with other carbohydrates. These carbohydrates are reported to control the
production of pectic enzymes (Keen & Horton 1966; Patil & Dimond 1968; Moran &

2. 38 B ANNADURAI AND D B MOTLAG
Starr 1969; Maldonado et al1986) carried out on various nutritional factors and culture
conditions influencing the production of endo PG of A.cepulae.
MATERIALS AND METHODS
Mycelial dry weight determination : Mycelial dry weight was determined by adopting the method of Annadurai
et al (1998). After 16 days of inocul ation of A.cepulae in different physico-chemical environments the contents
of the erlenmeyer flask was filtered through a glass funnel fitted with a coarse grade sintered glass filter and
washed thoroughly with water. The mat was pressed in a filter paper to remove the excess of moisture. Thrs was
transferred to a previously weighed filter paper. It was dried in an oven at 70 'C overnight. It was cooled to room
temperature 32+ l" C in a desicator and weighed.
Estimation of endopolygalacturonase : Endo PG activity was estimated by reducing sugar method according to
Nelson(1944)andSomogyi(1952). Theincubationmixturecontainingl.0mlofSodiumpolypectateatpH5.0;
0.5 ml of enzyme was incubate d at 32! I
o
C and I .0 ml of Arsenomolybdate reagent was added and then it was
read at 530 nm in uv 260 Shimadazu spectrophotometer.
Estimation of protein : Protein in the culture filtrate was estimated by employing the mthod of Lowry et al
(1951) using crystalline bovine serum albumin as standard.
Effect of addition of different sources of carbon on the activity of endo PG in culture {iltrate: The effect of
addition of different sources of carbon on endo PG activity was studied according to the method of Singh and
Tandon ( I 966). The synthetic pectin 20 ml was taken in I 00 ml Erlenmeyer flask. The medium was supplemented
with 1 gm of different carbon sources (5% w/v) like Glyceraldehyde, L-Arabinose, D-Arabiose, D-Ribose, D-
Xylulose, Rhamnose, D-Glucose, D-Fructose, D-Mannose, Sucrose, Lactose, Maltose Starch, Cellulose, Carboxy
methyl cellulose, Pectin, Gum acacia, Gum tracacanth, Agar, D-Calacturonic acid, Lactic acid, Oxalic acid. Maleic
acid, Ciric acid, Ascorbic acid, Sorbose, Ethylene Glycol, Ethanol, Ethylene acetate and Glutamic acid. The
flasksweresterilisedinanautoclaveandcooled. Thefungusl.cepulaewastheninoculatedandincubatedforl6
daysat32+l"C.Afterl6days,themycelialmatwasremoved. Thecontentoftheflaskwasfiltered. Thefiltrate
was contrifuged at 25,000 rpm for 15 minutes. The endo PG activity and the protein content were estimated and
recorded.
RESULTS DISCUSSION
When the leaf blight causing fungus Alternaria cepulae is grown in synthetic
pectin medium (Table Z),the mycelial growth as indicated by dry weight 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 pg/ml. The difference is significant
on 8th day and l2thday. It is significantat5o/o level from 16 days to 30 days. Endo PG
activity slowly decreases to 80 to 564 units on 16th day. Thereafter, the activity slowly
decreases. The difference of endo PG activity is significant (P<0.001).
Among different carbons tested for the effect on endo PG activity (Table 1), the
mycelial gowth significantly increases by glutamic acid, DL Galacturonic acid (P<
0.02), Carboxy methylcellulose (P<0.05) and gum tracacanth (P<0.02). Other carbon
sonrces do not influence the growth of A.cepulae.

3. 39EFFECT OF CARBON SOURCES ON ALTERNARIA
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4. B ANNADURAI AND D B MOTLAG
Endo PG activity is increased by starch, cellulose, gum tracacanth, DL
galacturonic acid, ascorbic acid, ethanolamine and glutamic acid (P<0.001). Other
carbon sources are ineffective.
Protein content is increased by D-Arabinose, D-Fructose, Lactose, Maltose,
starch, Pectin, Gum tacacanth, DL Galacturonic acid, ascorbic acid, Sorbose and
Ethanolamine (P<0.001). Other carbon sources are ineffective.
The media, which the pathogen grows detennine the tlpes quan'tities of cell
wall degrading enyzmes @ateman 1966;Bateman & Basham 1976). Living organisms
are known to utilise 40 elements among which carbon plays a significant role. As a
component ofboth structural and functional cell constituents, carbon accounts for fifty
percent of the total mycelial dry weight (Bilgrami & Verma 1982). Al1 important
components of the cell wall like cellulose, chitin and pectic substances contain carbon
in varying forms of ooncenhations. The results of various carbon soruces are shown in
Table I and suggest that starch, cellulose, gum fracacanth, DL galacturonic acid, ascorbic
acid and glutamic acid are the sources of carbon which influence the increase of growth
and endo PG.
ACIC{OWLEIIGEMENT
The author (B.A) is a grateful to Dr.S.C.Dhar and Dr.R.Puvanakrishnan, Scientists at the Department of
Biotechnology, Central Leather Research Institute, Chennai for laboratory facilities, useful suggestions and UGC,
New Delhi for research grant.
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