1. ,.
BtoJouRNAL,VOL.l0 NO.1 & 2,"!73-178 JIJNE & DECEMBEB ',
1998
STUDIESoNTHERoLEoFGELLWALLDEGRADING
ENZYMES lN LEAF BLIGHT DISEASE OF ONION (ALLIUM
CEPA LINN) CAUSED BY ALTERNARIA CEPULAE
B. Annadurai, D' Gopinathl' and R' Palani2
Centre for Biotechnology' C' Abdul Hakeem College'
Melvisharam- 632 509, Vellore District
ABSTRACT
Afunguswhichcauseslealblightwasisolated.Thephysiologyofthediseased
state and the role of pectic enzymes in cell was studied' lt was found that poly-
galacturonase enzyme was responsible for this disease' similar activity was also seen at
pHT.0.Theoptimumtemperaturefortheactivitywas35C.MaximunnproductionofPG
was on the 16th day of the culture. Enzyme kinetics was also studied PG activity was
seen both at pH 5.0 and pH 7.0 in Napp substrate'The posibility that PG exists as isozyme
was discussed.
INTRODUCTION
Specimensofonionleaveswithleafblightdiseasewerecollectedfromthefieldsof
kaliyankadu Erode DistrictTamilnadu during November and December of the year' lt was
identified as Atternaria ceputae (ponnappa & Deshpande) by Dr. K. Ponnappa' similar
species was reported by Ayyangar (1928) and Pandotra (1964)'
The Biochemistry of the diseased state of the hosts when infected by fungi was
studiedbyWood,(1960),andBateman&Millar(1966).Pecticenzymeproducedby
phytopathogenswhichtakepartincellwalldegradationwasstudiedFahraeusand
I-jungern (1959 & 1969)'Wang & Pinekar (1971)'
cellwall degradation by proteolytic and cellulotytic enzyme were reported in other
species like a Porri (wasiy 1983) and olutiola 1982)' Sporulation was studied by Khare
(1981).tnthepresentstudiesanattemptismadetostudythepecticenzymesinAlternaria
cepulae in leaf blight disease of onion'
MATERIALS AND METHODS
1. FUNGUS:-
Thefunguswasisolatedfromthealfectedlealoftheonionplantandwasregularly
miahCollege,Vaniyambadi;VelloreDistrict'
(173)

2. -ANNADURAI et. al.
grown in PDA slant.The Erlen-meyerflask were sterlised at 15 1b pressure in an
autoclave containing the following growth media. The fungus was inoculated in a
dust free inoculation chamber for the production of pectic enzymes. The fotlowing
media were tried in allcases.
1. Potato deitrose Agar medium.
2- Czapeck's Dox medium.
3. NaturalOnion Medium.
4. NaturalOnion leaf Medium.
5. Pectin yeast extract medium.
6. GlycerolMedium.
7. Pectin Medium.
2. ASSAY OF PECTIC ENZYMES :-
2.1 ESTIMTION OF POLYGALACTURONASE :-
PG was assayed by the following three methods.
2.1.1 VISCOMETRIC METHoD :-
The eslimation of endo PG was done using the method of AtUersheim et. al.
(1969).Which has an absorption maximum at 500 nm.
2.1.3. REDUCING SUGAR METHOD:-
The increase in reducing group in reaction mixture was assayed by Nelson
and Somogyi method (1944).
2.2 ESTIMATION OFTRANSEI-IMINASE (OR LYASE) :-
The estimation was carried out with Napp & Pectin as substrate by viscometric
method (Bellet. al. 1955 and Bateman (1966).Thio barbutyric acid test with an absorption
maxium at 550 nm (Albersheim, 1969). Here the unsaturated products are released from
pectiic acid and pectin by layse absorbing ultraviolet light maximally at 230 nm respectively
(Ayers,1966).
2.g ESTIMATION OF PMG & PME :-
PMG was estimated by adopting the method suggested by Talboys & Bush
(1741

3. t;
1.
2.
BIOJOUBNAL, JUNE & DECEMBER,1998
(1970). PME was estimated by the method of Kertez (1951).
3. ENZYME KINETICS :-
3.1 EFFECT OF PH :-
The effect of pH was studied by the viscosity experiments on the 16th day of
culture filtrate with different hydrogen concentration ranging pH 3,4,5,6,7,8
&9.
3.2. EFFECT OFTEMPERATURE :-
The 16th day culture filtrates were taken for viscosity experiments at
temperatures ranging lrom 20, 25,30,35, 40, 45 and 50 C were carried out at
pH 5.0.
RESUTTS AND DISCUSSIONS
FUNGUS:.
The production of pectic enzyme was high in natural onion medium and in Pectin
medium.The assay ol Pectic enzymes were carried out both in Natura! as well as in
pectin medium.The growth was good in still culture when compared to shake culture
filtrateshows maximum activity of PG (Fig.1).
ASSAY OF PECTIC ENZYMES:-
2.1. ESTIMATION OF PG :-
ln all the three methods activity of PG was found to be maximum in Napp
substrate at pH 5.0 and at pH 7.0 (Fig. 2, 3 & 4).This may be due to the existence
of two isoenzyme (isoenzymes) in the culture filtrate. Enzyme activitity was
expressed in RelativeViscosity Units RVU) (i.e., 100 to 50 time in min. for 50%
loss of Viscosity of reaction mixture)
2.2. ESTIMATION OFTRANSELIMINASE :-
No activity was seen in all the methods at diflerent pH. This confirms the
absence of lyase or Transeliminase.
2,3 &2.4 PMG & PME :.
No activity was observed.
ENZYME KINETICS:-
3.1 EFFECT OF pH :-
ln all assays there was a rise of PG activity from pH 3 to pH 5.0 After that the
activity declines and again there was an activity at pH 7.0. Then the activity
declines in pH 8.0 and pH 9.0 in the same substrate Napp (fig.2)
3.
(17s)

4. OF TIIE CULTI,RE FILTRATE
&
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210
ts
lm
ANNADURAIet. al.
trlCIrBE I : ltlE ACflVITy OF pc AT DIFFERET{I AGE
l23t . rceoannrntlnevssl 6 7
EGURE2: vtlloounrucelSryotr*irmrFEcroFm
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E
E
a
t
=N
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ffiilEr: tmTis
7t l,
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FIG.
$He. .{FifEsrED LE{F CAITi ERA LUCBA DI.{GR.I}I
of,'coNlDtArfi(}u.tfe$T
(176)

5. BIOJOURNIIL, JUNE & DECEMBER, 1gg8
s.2 EFFECT OFTEMPERATURE :- '
PG activity was gradually increased from 25 C and maximunl activity was
seen al 35 C. At higher temperature like 40 C and 45 C activity rernained
constant (Fig.5).
3.3. ENZYME AND SUBSTRATE cONCENTRATTON :-
3"/" of substrate Napp (Sigma Chemical U.S.A.) was found to be suitable for
easy flow through the capillary of the Viscometer. Aclivity was seen in S"/o
concentration of 4 ml of the subtrate and with 1 ml of enzyme concentration.
This substrate was taken for all types of assays pectin was not as active at
Napp.
The results in the present study indicates that pectic enzymes play a
key role in macerating the barriers of cellwall and membrane for the microbial
Invasion.
ln some 'cases like Rhizocotonia so!ani (Bateman, 1g66) both
polygalcturonase and Transeliminase were responsible for the cellwall
degradation.
ln our present study it was found that only endo pG at pH 5.0 and
presumably an isozyme at pH 7.0 were responsible for the cellwall degradation
of leaf blight desease in onion.The enzyme activity was rnaximum in the 16th
day of the culture.
T[re result of the present work agrees with the early reports in Rhizoctonia
fragariae (Cervone, 1978), Botrydiplodia theobramae (Arinze, 1979), Botrytis
cinerea, B. squamosa (Hancock et. a!., 1962), Pyrenochaeta terrestris (Keen &
Florton, 1966), Alternaria porii (Wasf y, 1977) that PG activity was responsible
for the cellwall degradaton.
Alternaria cepulae produces at least two hydrolytic enzymes which
degrade a "l-4 glyeosidic linkags in pectic acids. Both of them are endoenzyme
attack on the same substrate Napp at p!-l 5.0 and pH 2.0. Therefore !t is
presunred to be an isozyme. This enzyme did not prefer pectin as substrate
when compared to sodium polypectate.These enzyme r,.rere also inhibitecl by
ca(*iu'r-r chroride (Ayers et. al" 1g66, Bateman & Millar, 1966).
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