1. r_
BIOTOURNAL, VOL. B, No I & 2, IOS.IO, TUNE & DECEMBER, 1996
EXTRACELLUI-AR ENZYMES OF ALTERNARIA CEPULAE
IN LEAF BLIGHT DISEASE OF ONION
B. Annadurai and D.B. Motlag
Department of Biochemistry University of Madras
ABSTRACT
A purc strain of A.lternaria cepulac was isolated from leatblight area of onion. The
orgatiisnr was lirunil to grrtw rvell in the natural onion medium. czapeck and linciberg rne{ium,
Bacons mcdium, Ray's medium and Pectin medium as indicated by the growth rate. Thc
acti'.'ity ril' cell wall degrailing enrrymcs iikc cellulase. arabinase, xylanase and prgteinasc is
cst i ntatccl.
INTRODUCTION
Extcnsivc studics on varicus diseases in onion (Allium cepa Linn) have been rcportcel
(Nlichail cl al 198i: Maltitz ct al 1984: Tanaka ct al 1985; Ramsc'y et al 1986). Lcaf blighr
discase clus to Alieruaria spp was reportcd as early as 1!]23 by Ajrckar. It was also rcprlrterl in
coinrbalttne (Ayyangar. t92li) ancl in Poona (Uppal et al. 19.1j). A purple hlofch disease in
Onicin duc i() Alternaria porri (Ellis) was reported in Bihar (Thirur*alaehar and Mi:sra. l9-i3l
and rn Puniab tPandotra. 1U641.
The lealblight diseasc iu cnion occurred in and around kaliyangadu ol'pcriyar districr of
TLmil hiadu since t981.The cpidemologv o[ the diseasc was studieil by khare ancl Ncma. ( lgli I ].
Thc l'ungus Alternaria cegr:"llae rvas isolated fiom this diseas$ and used in r-his inves[igation.
lt is prtipi;scd [o isolate the lcaf blight causirg lung:s A. ccpulac sLucly its c:ulture
eharactcrist.ics and to csl-inralc the activity olthe cxtracellular Gnzyn-res.
MATERIALS AND METHONS
ESTIM.4TION ()F CELLULASE {EC 3.2.1.4'l
ccilulase activity was. determined by the methotl cl' Hane ock et al ( I964)
Estimatiorr of Arabinase (EC 3.2.1.55) and rylanase (EC 5.3.i.5).
Arahinase and rylanase activities wcre determined according to the procedure gt C6op,ir
ancl Wrrocl (1975). Estimation of proteinase :
Proteinase acrivity was estir.natecl according to tht: method of Kunitz (194?).
Statistical analy".ris :
The rnean (X) and standar<l deviatirrns were calculated using thc procedurc of Bailey
984). The difference in results obtained was examined with "t"tests for small samples.
t05

2. AN NADII RAI and M OTL.tlG
RESULTS AND DISCUSSION "
The Growth of Alternaria cepulae expressed as dry weight in grams in different culture
media is shown in fig. l It was observed that czapeck and lindeberg mcdium was most
favt-rurable tbr the growth. Becons medium, Natural onion medium also support good growth.
The activity of celluiase (C1) during different periods of incubation and at different pH is
shown in Fig 1. From the results it is seen that cellulase (Ct) activity was maximum at pH 5.()
and the enzyme production was maximum on the 1-6th day of culture. The activity of cellulase
(Cx) during 16th the rlay of culture at different pH is shown in frg Z.It is observed that the
cellulase (Cx) activity was optimum at pH 5.5'
55.56
fi93 fig3
CL,LTURE t4EDtun fr,g s
o''d
g'6
ll
23 L 1,5
i-ao iI?
i:oo'yln
f rmg
l!d
frTo t
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u
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let i.
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to!
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).
F
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(
>.3
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'(
1fi
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F
o
fl-
t".5 5556 I
Plt Fig I
CUI.TURE MEDIUI,I
t06
CULTUFE M[OIUI'4
0.?.

3. BIOTOURNAL, JUNE & DECEMBER, 1996
UC
200
r60
2Jqto.
CULruRE MEUUM
30
)
Aao
;::_
E10
6
(}
(L
0.14
!0.12
E0'1
Pb{B
z.
co,oq
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i o.s
:
! o.a
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u
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loo;
iro!
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c
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ieo
Fig l.lQFig 1.9
. 70oYs
'al/Jals
a 6Days
Fig 1.12
r a D^lJ
irecrYc
a 3 g^Ja
a&gNa
Fig l'11
o Pectin.H9'CF
FH figt.t4
Figr.13
J 'l J' I
'
cuL?urEMEDrutr Flgl.16
P{' Fig 1.15
i IILTURE MEDIUM
107

4. ANNADURAI and MOTI- O
I ig. I Growth of Alternaria cepulae atler 16 days ol' incubation in dill'crent eulture media
l. Potato dcxtrose agar 2. Czapeckdox Agar Mediun:
3. Natural Onion Leavcs 4. Natural C)nion Bulbs
-5. Pcctin Yeast Extract 6. Glycerolmedium
7. Pectin metlium 8. Rey's medium
t). Czapeck's antt Linrlberg 10. Bacons rnedium
$'ig. 2 Estimatign of Cellulase (C1) in different days of culture at dill'erent pfl.
Ccllulase (Cl) activ"ityis expressed in relative viscosity units RVU:10{l/T-50 is the timc
takcn lirr .5()'Z losr of viscosiLy.
Valur:s givcn arc the mean + SD
Procluction of cellulasc at pH 5.0 is significzrnt at 5'/c lov-el(P < 0.05)
l'ig. 3 Estinration of crlluiase tCX) aftrr t6 days of growth at ili{fererit pH by rtducing -sugar
mrthrxl.
L.ellulase activitl, {Cx) is expressed as mg ol glucose relesecl/rnilhr',:alues uxprcssed arc
thc rni:an + SD
f ig. 4. !-stimation of Arabinase activity of A. crpular in differeni €ulture media after 16 days
of growth
l. Czapcck rnedium 2. Natural onion lcaves medium
3. Natural onion trulbs medium 4. Pectin ycast extract
.5. t-'lyc:erol mediurn 6. Pectin meiiium .
7. Rav's medium 8. Czapeck and lindherg medium
9. Bacuns medium
Bars arc thc mean value of Arubinase ac.tivity in diffcrent culture filtrate + SD
i-ig. 5. Estimation of Xylanase activity of A. cepulae iri differenlculture unedia after 16 ilays of
growth
l. Llzapcck medium 2, Natural onion leaves medium
3. Natural onion bulbs me dium 4. Pectin ycast cxlract
-5.
(ilyccrol medium 6. Pectin medium
7. Ray's N{cdium 8. Czapcck and lindtrerg medium
(). Bacons medium
' Burs arc lhe mcan value of rylanase activity in diffe rcnt culture filtrate + SD
108

5. BIOJO|iRNAL, lllNE & DECEMB{R, 19eo
!-ig. 6 Estimation of proteinase activity of A. ctpulase in different eulture media alter 16 days
ol grrlw'th
!. Clzapcck rne dium 2. Natural onion le aves metlium
3. Natural onion bulbs mcclium 4' Pect'in yeast exlract
-5. Gl-v-ccrol metlium 6' Pcctin meelium
7. Ray's rretlium 8. Czapeck ancl linclberg mediu
9. Bacttns rneclittnr
Bar-s arc thc rrre an valuc of prol.einase activitV in dilTercnt culture filtrate SD'
Thc activity ot arabinase ii mrurimum in natural onion medium (Fig. 3). Fig' 4 inrlicatcs
that the ry-lanase acriviti; is maximum in natural onion meclium and pectin medium'
Proteinase activity {Fig -5) was maxinlum in Ray's medium' Activity is also observed in
natural onitrn rncdium and Bactlns mcdium'
Ccllulase (C1 aniJ Cx). Arahinasc. Xvlanase and Preiteinase are cell wall and membranc
cLgrading enzylrcs li.runtl to hc r.vell markctl in ihese studics. There results are in support ol'
thc rrbscrvation ill'sanclu, (191i2) and Mc' Carrul (19ti5)"
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