This document summarizes a study on the growth of the fungus Alternaria cepulae and its production of endopolygalacturonase (Endo PG) enzyme in onion leaves and pectin medium. The study found that Endo PG activity was highest on the 16th day after inoculation of A. cepulae in both onion leaves and pectin medium. Endo PG plays a role in the pathogenesis of leaf blight disease in onions by degrading the plant cell wall and allowing the fungal hyphae to enter the plant tissues. The optimum conditions for A. cepulae growth and Endo PG production were determined to be 16 days after inoculation.

1. BIOJOURNAL,VOL. ll NO.1 & 2,161.165 JUNE & DECEMBER, l999
GROWTH OF ALTERNARIA CEPULAE IN
LEAFBLIGHT DISEASE IN ONION
B. Annadurail and D. B. Motlag
Department of Biochemistry, University of Madras, Chennai- 600 025
ABSTRACT
The activity of endopolygalactuonase (Endo PG) in infected Onion leaves and Pectin
medium by Atternaria cepulae, a leafblight causing f ungus was estimated. lt was found that
in both the cases, the activity of endo PG was maximum on the 1 6th day after the inocu lation
of A. cepulae from Onion leaves.
INTRODUCTION
Endopolygalacturonase (Poly o, 1,4 galacturonide glycanohydrolase, EC 3.2.1.15) plays
a significant role in pathogenesis of many plant diseases (Cooper, 1980, Boothby, 1984,
Mills, 1985). Endopolygalacturonase is one of the prime macerating enzyme produced by
Alternaria ceputae during leafblight disease of Onion (Annadurai,1996, 1998,1999).
Endopolygalacturonase is produced both constitutively and adaptively by different
microorganisms (Annadurai, 1987).ln most instances, the enzymes are produced adaptively
rather than constitutively (Keen & Horton, 1966). Fusarium monilitorme (Biehn, 1971) and
Scterotium rotfsii(Punja et. al., 1985) produce it adaptively in the presence of pectic
substrates. A small number of pathogens like Prrcularia filamefosa (Ayers and Papavizas,
1966) and Cotletorichum falcatum (Singh and Hussain, 1964) Verticillium alboatrum (Mussel
and Strouse, 1972) and Aphanomyces cutices (Ayers, 1965) are known to produce
polygalacturonase in a constitutive manner.
pectin induces greater enzyme production in adaptive enzymes (Grant, 1985). The
pathogen in nature confronts with pectin substances not in isolation, but in combination
with other carbohydrates (Annadurai, et al" 1998, 1999).These carbohydrates are reported
to control the productlon of Pectic enzymes (Patil and Diamond, 1968, Moran and Starr,
1969, Maldonado et. al. 1986). Catabolic repression of the endo PG levels in infected Onion
leaves and in pectin medium were ascertained (Annadurai et. al., 1999). Systematic
investigations were carried out on various nuitritional factors and culture conditions
influencing the production of endo PG of A. cepulae.
MATERIALS AND METHODS
Mycelial Dry Weight Deterrnination
Mycelial dry weight was determined by following the method of Annaduraiet al (1998).
After 16 days of inoculation ol A, cepulae in different physicochemical environment the
Head of the Department of Botany and Biochemistry, CAH College, Melvisharam-
632509, Vellore District, Tamilnadu
1.
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2. ANNADUBAI AND MOTLAG .
contents of the erlenmeyer flask was filtered through a glassfunnel fitted with a coarse
grade sintered glass f ilter and washed thoroughly with water. The mat was pressed in f ilter
paper to remove the excess of moisture.This was transferred to a previously weighed f ilter
paper. lt was dried in an oven at 700C Overnight. lt was cooled to room temperature
lSZt t0C) in a desicator and weighed.
Estimation of Endopolygalacturonase
Endo PG activity was estimated by reducing sugar method according to Nelson (1944)
and Somogyi(1952).
The incubation mixture containing 1.0 ml of Sodium polypectate at pH 5.0 0.5 ml of
enzlrme were incubated at 3Z r 10C for t hour.The reaction was stopped by adding 2.0 mlof
alkaline copper reagent. The tubes were kept in a boiling water bath for 30 minutes. The
control treatments were carried out in the same manner except was added after adding the
alkaline copper reagent.
The tubes were the.n cooled to room temperature (32 t 10C; and 1.0 ml of
Arsenomolybdate reagent was added and tlren it was read at 530 mm in uv 260 Shimadzu
spectrophotometer.
Estimation of Protein
Protein in the culture filtrate was estimated by employing the method of Lowry et. al.
(1951) using crystalline Bovine serum albumin as standard,
RESULTS AND DISCUSSION
Estimation of endopolygalacturonase activity in blight affected Onion leaves is shown
inTable 1.lt indicates that the lesion area and area around lesion in the inoculated leaves
shows optimum endo PG activity. ln both the areas an endo PG activity starts from the 8th
day of inoculation. The optimum endo PG activity was observed on the 16th day after
inoculation of A. cepulae.The difference in endo PG acitivty on 12th and 16th day is signif icant
p < 0.01 in the lesion area and in the area around lesion.The endo PG activity is significant
al0.1'/.level on 8th day and 2o/" level on 20th day.
When the leaf blight causing fungus A. cepulae is grown in synthetic pectin medium
(Table 2), the mycelial growth as indicated by dry weight slowly increases and it is signif icant
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 12th day. lt is
significant at 5% Ievelfrom 16th days to 30th day. Endo PG activity slowly decreases to B0
to 564 units on the 16th day.Thereafter the activity slowly decreases.The difference of endo
PG acitivity is significant (p < 0.001).
The results presented inTable 1, indicates that the EPG activity in lesion area and
around lesion area began on the 16th day in synthetic pectin medium also (Table 2).The
EPG activity is more in the area around the lesion than in the lesion itself.This suggests that
endo PG enzymes initiate the pathogenesis by the cell wall and pave way for the entry of the
hyphae (Bateman, DF and Basham, H.G.1976). After the 16th day of growth of the fungus,
the EPG activity slowly decreases.
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3. BIOJOURNAL, JIJNE & DECEMBER, lggg
Table - 1
Estimation of endo PG of in blight affected onion leaves caused by A. cepulae
sl.
No.
Days after d.f.
inoculation
Control treatments lnoculated leaves
Wounded Healthy
area area
Lesion area Around lesion Away
from
Lesion
area
Units
RVU/ml
Unit
RVU/ml
1. 4 days
2. I days
3. 12 days
4. 16 days
5. 20
5
5
5
5
5
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL
NIL NIL
17
=
3.27 25 + 1-7
29
=
2.45 34 t 2.1
45 t 1.8 ++ 40 x. 1.4
23 t 1.41 NS 46 x.2.62
++
+
+
NIL
NIL
NIL
NIL
NIL
Values given are the mean value of EpG activity in RVU unit * SD.
EPG activity is expressed in Belative viscosity units (RVU - 100/Ts') whereTuo is the time
taken in seconds tor 5oo/o loss of viscosity. The procedure is explai-nld in the iJxt.
d.f. - Degrees of freedom =.number of observation - 1.
Significance = ++ P < 0.001, + = P < 0.001 (N = 1)
Table - 2
Estimation of endo PG of A. cepulae in synthetic pectin medium
Sl. Culture d.f.
No. Age
Mycelialdry wt. Protein Endo PG activity
ln gms/ Signi-
flask t SD ficance
Sd Signi
ficance
Units t SD!g,I Signi-
ficance
1. 4 days 5 0.35 t 0.01 549.00 * 9.70 80.00 t 12.00
2- 8 days 5 0.58 t 0.02 ++ s64.00 + 4.10 ++ 144.00
=
1.76 ++
3. 12 days 5 0.85 r 0.03 ++ 604.00 * 5.73 ++ 2}4.oo r 10.6 ++
4. 16 days 5 1.40 t0.03 ++ 612.00 t 4.52 + 364.00 t 1.85 ++
5. 20 days 5 1.90 t 0.07 ++ 620.00 x. 4.s2 + 300.00 r 1.85 ++
6. 25 days 5 1.92 r 0.07 NS 624.00 t 1.85 + 284.00 t 8.98 ++
7. 30 days 5 1.94 t 0.08 NS 628.00 t 1.54 + 188.00 t 8.58 ++
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4. ANNADUNAI AND MOTLAG
Protein content is expressed in pg/ml by adopting the method of Lowry et aL, with
BSA as standard.
EFG activity is expressed in units as pg of galacturonic acid released from one ml in
30 minutes from galacturonic acid standard.
Mycelial dry weight is in grams t Sd
Allvalues expressed are the mean value (X) of e individualexperiments * SD
Significance : ++ = p < 0.001, * = p < 0.05, NS = Not significant
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