1. '
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Bio-Science Research Bulletin. Vot. l6 (No.2) 2000: p.5t_j6
EFFECT OF VITAMINS OF ENDOPOLYGALACTURONASE PRODUCTION IN
ALTERNARIA CEPULAE
B. Annadurai', l!I. Shanmuganr and D,B. Motlag
Department of Biochemistry and Molecular Biology. LJniversity of Madras, Ch-ennai - 600025. lndia
'Presently : Reader and Head, Department of Botany and Biochemistry,. C.A.ll. College, Melvisharant
,. (Vellore)-632-s09. India.
Departrnent ol Applied Zoology, Kuvenrpu University. Jnana Sahyadr.i. (Shimoga Dist.) - 57745 l. India.
ABSTRAC'[
When Pectin rncdiunr rvas supplcnrcntcd *ith'itatnin. the actiit of
cndopolygalacturonase lEndo I'c) production and growth of.4rternaria ,'elturue
was estimatDd, The effcct ol vitarnins on endopc production rvas remariable.
Except CalciLrm pantothenate. oihcr vitatnins increase the production of
cndoPG. When all the vitamins are adclctl togetlrer. maxintum production of
endoPc and grorvth of._l. ceplrlae is obscrved inrj prescnted.
Keywords : Endopolygalacruronase (Endo pG..4hernaria cepnlae. Mycelial dry weight
INTRODUCTION
Endopolygalacturonase (Poly o, I,4 garacturonide grycanohydrolase, EC 3.2.1.15) plays a
significant role in pathoge'esis of many plant diseases (cooper, I9g0, Boothby, 19g4, Mills.
1985)' Endopolvgalacturonase is one of-the prime nraceratingenzyme produced by Alternaria
cepulae during ieafbright disease of onion (Annadurai et ar.,ldg6. r"oet, ieesy.
Endopolygalacturonase is produced both constitutively and adaptivery by different
microorganisms (Anrradtrrai, 1987). In most instance, the enzymes are produced adaptively rather
than constitutively (Keen and Horton. 1966) Fusariunt nroniliforme (Biehn, l97l)and Sclerotium
rolfsii (Puwa.et al, 1985)-produce it adaptively in the presence olpectic substrates (Bilgrami,
1982)' A smaf l number of pathogens llke-Piricitoriafilanrctosa(Ay"o unJ fupuuizas, 1966) and
Colletotrichunrfalcatum (Singh and Hussain, 1964), Verticiiliuni aiboatrurr(Mussel and Strouse,
1972).and Aphanomyces cutcic'es (Ayers, I965) are known to produce po)ygaracturonase in a
constitutive rnanner.
Pectin induces greater e,zyme production in adaptive enzymes(crant, r9g5). The pathogen in
nature confronts witlr pectin substances not in isolation, but in
'combination
with other
carbohydrates (Annadurai et al, 1998. I999). These carbohydrates are reported to control the
g1o-!u1tr-o1t.of nectic enzyrnes (Keen and Horton. r966, patir and Dimond, r96g. Moran and Staff,1969' Maldonado et al .1986.). Catabolic repression of the synthesis of pectic enzyrnes by sugars
is a common phenornenon in many phyropathogenic organisnrs lBraihwaite & Dickey, r970,Biehn and Dimond, l 97 r . Sparding ; i,t.' tsil,CJoper and woou, i gzs. Alnadurai et ar, 1999).
Systernatic investigations were carriecl out on various
'rrri,ir*r'i".;;;, ;;ilrlture conditionsinfluencing the production of endoPc of A.rrpuiau. The effect of various vitamins on endopGproductiorr and growth of Alternariu cepulae rvas ascertained.

2. B. Annadurai, M. Shanmugam and D.B. Motlag
MATERIALS AND METHODS
MYCELIAL DRY WEIGHT DETERMINATION
Mycelial dryweight r'vas deternrined by follorving the rnethod of Annadurai et al,(1998).
After l6 days of inoculation of A.capulae in different physicochernical environment the contents
of the erlenrneyer flask rvas filtered through a glassfunrreifitted with a coarse grade sintered glass
filter and washed thoroughly with water. The mat was pressed in filter paper tJremove the excess
of moisture. This was transferred to a previously weighed filter paper. Iiwas dried in an oven at
70oc overn iglrt. It w,as cooled to room iemperature (:zt t "c) in a desicator and weighed.
ESTIMATION OF ENDOPOLYGALACTURONASE
EndoPG activity was estimated by reducing sugar nlethod according to Nelson (1944) and
Somogyi( 1952).
The incLrbation mixture containirrg.t.c rnl of sodiurn polypectate at pH 5.0.0.5 ml of enzyme
were incubated al (321luc) for I hour. Adding 2.0 nil oi'alkaline copper reagent stopped the
reaction. The tubes were kept in a boiling water bath for 30 rninutes. The control treatments were
carried out in the sarne manner except was added after adding the alkaline copper reagent. The
tubes were then cooled to room temperature (32+l"C) and l.-0 ml of Arsenomolybdati reagent
was added and then it was read at 530nrn in uv 260 shimadzu spectrophotorneter.
ESTIMATION OF PROTEIN
Protein in the culture filtrate was estimated by employing the nrethod of Lowry era(1951) using
crystalline bovine serum albumin as starrdard.
INFLUENCE OT THE VITAMINS ON ENDOPG ACTTVITY
20ml of Pectirr rnedium was taken in l00ml Erlenmeyer flasks. The medium was supplemented
with I rng of all the B-complex vitamins. To evaluateihe significarrce of the B-complex vitamins
Tlriamine hydrochloride, calciumpantotheriate, p-arninobenioicacid, Nicotinamide, ionositol and
Riboflavin, one of these vitamins was omitted at a time in the flask. pectin rnedium with the B-
complex vitamins served as control I and pectin medium without any vitamins served as control
II' The flasks were sterilised in an autoclive at l5 lb pressure for,io minut"s. They wercther
cooled to room temperature at 320C. Alternaria cepulai was then inoculated in the medium in a
sterile chamber. After 16 days of growth mycelial mats were removed and the contents were
filtered through ctreese cloth. The fiitrate was centrifuged at 20,000 rpm for i0 minutes at the
supernatant was used for the estimation ofendopG activity.
RI,SULTS
When the leafblight causing fungus.A.cepulae is grown in synthetic pectin medium the mycelial
growth as indicated by dryweight slowly increases and it is significant upto 20 oays 1p>o.ooty.
The difference is not significant after 20 days. The protein content also increases from 54gpg to
638 pg/ml. The differenceis significant on g,r,clay + l2,r,day. It is significantat 5% level from l6
days to 30 days. EndopG acrivity slowry decieases tiorn 80 to*564 units on*;;']il';;
lltele,after the activit,v- slorvly decrea."r. Th" <iifference of endopG activity is significant
(p<0.001).
Bio-Science Research Bulletin. Vot. l6 (No. 2) 2000
52

3. Effect of vitamins of endopotygaracturonase production in Arternuia cepurae
The influence of vitarnins on
-endoPc
activity is shown in Table I . All vitanrins of the B conrplex
group increase the growth ofmycelium lpiO.OOt), except calcium pantothenate. other vitanrins
1i:rp.the activity of endopc *hen aii the viianrins are added iog.th"r, maxinrum endopG
aclrvtty ls observed'.
Table I: Influence of vitamirrs on endo pc activitl,of the reafurigrrt causing fungus r. cepukte
Values displayed are the mean vatue (x. ) of 6 individual experiments.
d.f. = degree of fieedom = n - I observations
Significance ++ = p < 0.001;
NS = Not significant
Mycelial dry weight is expressed in grams.
EPG
.activity
is expressed in. units as pg of galacturonic acid released per ml in 30 minutesRelative activity is expressed in percentafe by iaking contror as cent percent.
DISCUSSION
The medium, in which the pathogen grows deterrnine the types arrd quantities of cell walldegrad.ing enzymes (Bateman, t966, BaLrnan and Basham, rqibl-r-i"i,rg
"lsuni'n,
ur" kno*]to utilise 40 eiements among which carbon prays a significant rore. as- u iornpor"n, of both
Bio-Science Research Bulletin. vol. t6 (No. 2) 2000
st.
No.
Vitamins omittetl d.f. Mvcelial dn,wt. EndoPG activitv Relative
Activity
("/")
Gms/flask+ Sf Signili-
cance
Units/ml + SD signifi
cance
I Ihiamine hydrochloride 5 0.65 + 0.02 ++ 308.00 t 2.56 ++ t93
2 Pyridoxine hydro chloride 5 0.62r0.02 ++ 284.00*2.95 ++ 178
3 Calcium Pantothenate 5 0.61 r 0.02 -t+ r32.00 i 2.50 NS 83
4 p-arnino benzoic acid 5 0.68 r 0.07 l+ 191.00 + I .95 ++ 183
5 Nicotinamide 5 ).72!0.02 ++ 272.00x2.2s ++ l'70
6 lonositol 5 0.71 r 0.02 ++ 392.00!2.82 ++ 245
7 Riboflavin 5 0.691 0.02 ++ 248.00 !2.35 ++ 155
Control I (All vitamins) 5 0.75 !0.02 ++ 404.0012.6s ++ 253
Control II (No vitamins) 5 0.27 t0.02 160.00 r 2.15 r00
53

4. B. Annadurai, M. Shanmugam and D.B. Motlag
structural and functional cell constituents, carbon accoullts for tifty percent ofthe total rnycelial
dry weight (Bilgrarni and Verma, 1982). All important components of the cell wall like cellulose,
chitin and pectic substances contain carbon in varying forms and concentrations. The results slrow
that fungi requires vitamins for the production of Enzymes which are involved in various
nretabolic activites. Some fungi secrete vitamirrs by themselves while others require it from
external sources (Schopfer,
,l
943): The results of the effect of various vitar.nins are supplemented
together there is luxuriant growth of the fungus and rnaximurn EPG production. When one
vitamin is ornitted frorn tl-re mediurr, it is observed that except calciurn pantothenate all other
vitamins have no effect on the rnycelial growth and EPC activity when compared to the control.
CONCLUSION
The precise role of vitarr-rins itr etrzytnes production is still a matter of controversy.
ACKNOWLEDGMENTS
The author B.A is gratefirl to Dr'. S.C.Dhar and Dr. R.Puvana Krishnan, Scientists at the
Department of Biotechnology, CLRI, Chennai, for laboratory facilities and useful suggestions and
UGC, New Delhi for research grant.
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