1. i
tl
t
EFFECT OF PITYTOHORMONES ON PURIFIED ENDOPOLYGALACTURONASE
ENZYME PRODUCED BY ALTERNARIA CEP(ILAE
B. Annadurai', M. Shanmugam-'and D.B. Motlag
Department of Biochemistry and Molecular Biology, University of Madras, Ciennai - 600025, lndia
Prcrcltly : Reader and Head, Department of Botany and Biochemistry, c.A.H. college, Melvisharam
(Vellore!.632509, lndia.
Department of Applied Zoology, Kuvempu university Jnana Sahyadri, (Shimoga. DisrF57745 l,
Karnarak4 lndia.
ABSTRACT
Auxin content rvhich hzs a ma.ior role in disease resistance was estimated in
normal as well as in int'ected onion leavcs. Auxin content in norrnal leaves
slowly dccreases when the grorvth advanccs. rn diseased onion reaves. the
auxin content in the lesion area increascs till thc Idl'day alier inoculation and
decreases thereafter. The elrbct ol'five phytohornrures on the mycerial growth
of A. ceparae in the culture medium and on the activity ot'endopG- ce'ilulase
ambinasc xylanasr and protcinase rvas examined l0o ppm concentration of
Naptharene aceric acid (NAA), Indole butyric Acid ii Indor" acetic acid
showed inhibitory eflbct on these extracellular enzymes. E{Ibct of
phyobormones in disease resistance and hyperauxiny due io pathogenesis is
discussed.
' Keywords : Phyrohormones, IAA, lBA. NAA. GA, Kinetin. ;l/t ernaria cepulae. EpG.
INTRODUCTION
Phytohormones occur in traces and are constantly maintained at low level by plants (Albersheim
et al.l959; Bateman, 1966; cooper et a1.,1975). This constant level of a phytohormone is
disturbed by different factors, the chief one being infection. During infrction phytoholnon",
either directly or indirectly prevent the action of the parasites. The- phytohgrmones may also
!9I1!". the various physiological manifestations triggered by parasiLs.
-Cuiscafre-Aiillago
(1949a) reported that 24 dichloro phenoxy acetic acid (2.4D) inhibited directly the growth
-of
Penicillium dictatum and Phonopsis citri.
It was reported that in several plant diseases, Phytohormones and phenols inhibit the activity of
cell^wall degrading enzymes (Dekkar.l963; Desai er al.l975; ouuey el al.l976; Fehrmarr er
aLl967).Bateman (1966) suggested that the phytohonnones have a role in disease resistance.
F3:o (1965) reported that the oxidation products of phenolic compounds also act as enzynre
inhibitors- Plant pathologists have been trying to conrtol the plant diseases with the heli of
various phytohormones (Hancock et al.l964; Jacobs, 1979). ok; and Nakanishi (1962) reported
1!1 in{ole acetic acid sprayed on rice plants stimulated phytoalexin synthesis. Fehnnan and
Dimond (1967) reported that Indole acetic acid treated potato tubers induces phenol levels. The
higher concentration of auxin influ.enced the phytoaleiin production in the tissue cultures of
Phoseolus vulgaris.
Garg and Mehrotra (1977) reported that gibberellin suppresses the activity of cellulases and
polygalacturonnses of Fusarium solani Z pi.si. Mehta-and Mehta (1979) observed that CA
inhibited polygalacturonases. Pectin trans eliminase and pectin methyl esterase.

2. B. Annadurai, M, Shanmugam and D.B. Motlag
Dekker (1963) observed that kinetin inhibited the development of powdery milderv. ln this paper
the auxin levels in rrorrnal and diseased levels are estinrated. The effect of phytolrormones orr
crude enzyme preparatiorr and on purified EPC was studied.The effects of phytohor.nrones on
other extracellular enzyrres like cellulase, alabinase, xylanase and proteinase was also
investigared.
MATERIALS AND METHODS
THE FUNGUS
Isolates of Alternaria cepulae obtained fi'orn the cliseased onion leaves wele used for. the
interaction study.
EFFECT OF PIIYTOHORMONES ON ENDO PG
I ml of the purified enzyme(1 ml/ nrl w/v) was incubated with equal volurne of l0 ppm. ancl 100
ppln concentration of Indole acetic acid, Indole butyric acid, Napthatene acetic acid, GA and
kinetin 0.5 rnl of the incubated enzyme was tested for redLrcing sugar by the nrethods of Nelson
( I 94 I ) and Sornogyi( I 952).
ESTIMATION OF ENDO PG ACTIVITY
EPG activity was estirnated using the Nelson ( I94l) and Sornogyi( 1952) rnetlrod.
RESULTS
The ef'fect of phytohornrones on purified EPG is shown in table l. It reveals that 100 pprn
concentration GA. Naphthalene acetic acid, Indole butyric acid did not suppresses the encloPG
activity very efTectivcly at 100 pprn concentrations. All phytohonnones suppresses the encloPG
activity very effectively. It is significant(p<0.001).'
TABLf, - I : Effect ofphytohornrones on EPG (purified) activity of A. ceptilae.
EPG used was EPG which was obtained after purifying the activity EpG I fractions.
Bio-Science Research Bullerin. Vot. l6 (No. 2) 2000
st.
No
Phytohormones Concentration
(ppm)
d.ll EPC units (pg of
Gal. Acid
rel/nrl/30 min.)
Relative
activity(%)
Significance
lontrol 9 4280.26tt2.25 100.00
Indole acetic acid IO
100
5
5
800.12+8.16
25.32x4.O8
I8.69
0.58
++
++
2 lndole butyric acid t0
100
5
5
2740.20+12.08
32.1$:1.24
64.00
0.75
++
++
J Nepthalene
Acetic acid
t0
t00
5
5
r 850. I 614.70
18.24rO.94
43.20
0.42
++
++
Cibberellic acid l0
t00
5
5
3320.32r r .90
220.42!3.45
77.57
5. t4
I+
++
5 Kinetin t0
t00
5
5
660.20!5.45
204.t4x3.24
15.42
4.ll)
++
+l'
58

3. Effect of phytohormones on purilied entlopolygalzrcturonase enzyme """
EPC activity is expressecl in units as pg ofgalacturonic acid released for one nrl in 30 tlinutes.
Values given zrie tlre niean (X) t SD.
d.f.:ciegrees of freedonr = n-l obsel'vatiolts.
Relative activity is expr.essed in percentage by taking cotrtrol as cerlt precent.
Significance and confidence iirtrits are fi'onr 0. i% to l0%
r+ .p<0.001 * - p< 0.01 .
DISCUSSION
When the etlect of phytohornrones on the EPG activity u,as tested in vitro was fotrnd that at l0
ppril concentration CA and lndole trutyric acid are less effective. At 100 ppm corlcentration
inlribition nray be clue to the interrnediate conrpouuds tblnted in the culture mediutr.
CONCLUSION
T[e resLrlts presenled in this paper srrggest that these phytohormones are effective as inhibitory
effect is nruch less for x1'lanase, arabinasc and protcinases.
ACKNOWLEDGEMENT
T'he author B.A is gratetul to Dr. S.C.Dhar and Dr. R.Puvanakrishan. Scientists, at tlie
Depaftrrerll of Biotechnology,ClRl" Chennai, for Laboratory tacilities and useful suggestiotrs
and UCC.Nerv Delhi, for reseat'ch graut.
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