itl

t

EFFECT OF PITYTOHORMONES ON PURIFIED ENDOPOLYGALACTURONASEENZYME PRODUCED BY ALTERNARIA CEP(ILAE

B. Annadurai', M. Shanmugam-'and D.B. MotlagDepartment 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 innormal as well as in int'ected onion leavcs. Auxin content in norrnal leavesslowly dccreases when the grorvth advanccs. rn diseased onion reaves. theauxin content in the lesion area increascs till thc Idl'day alier inoculation anddecreases thereafter. The elrbct ol'five phytohornrures on the mycerial growthof A. ceparae in the culture medium and on the activity ot'endopG- ce'ilulaseambinasc xylanasr and protcinase rvas examined l0o ppm concentration ofNaptharene aceric acid (NAA), Indole butyric Acid ii Indor" acetic acidshowed inhibitory eflbct on these extracellular enzymes. E{Ibct ofphyobormones in disease resistance and hyperauxiny due io pathogenesis isdiscussed.

' 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 (Albersheimet al.l959; Bateman, 1966; cooper et a1.,1975). This constant level of a phytohormone isdisturbed 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 ofcell^wall degrading enzymes (Dekkar.l963; Desai er al.l975; ouuey el al.l976; Fehrmarr eraLl967).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 enzynreinhibitors- Plant pathologists have been trying to conrtol the plant diseases with the heli ofvarious 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 andDimond (1967) reported that Indole acetic acid treated potato tubers induces phenol levels. Thehigher concentration of auxin influ.enced the phytoaleiin production in the tissue cultures ofPhoseolus vulgaris.

Garg and Mehrotra (1977) reported that gibberellin suppresses the activity of cellulases andpolygalacturonnses of Fusarium solani Z pi.si. Mehta-and Mehta (1979) observed that CAinhibited polygalacturonases. Pectin trans eliminase and pectin methyl esterase.

B. Annadurai, M, Shanmugam and D.B. Motlag

Dekker (1963) observed that kinetin inhibited the development of powdery milderv. ln this paperthe auxin levels in rrorrnal and diseased levels are estinrated. The effect of phytolrormones orrcrude enzyme preparatiorr and on purified EPC was studied.The effects of phytohor.nrones onother extracellular enzyrres like cellulase, alabinase, xylanase and proteinase was alsoinvestigared.

MATERIALS AND METHODS

THE FUNGUS

Isolates of Alternaria cepulae obtained fi'orn the cliseased onion leaves wele used for. theinteraction 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 100ppln concentration of Indole acetic acid, Indole butyric acid, Napthatene acetic acid, GA andkinetin 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 pprnconcentration GA. Naphthalene acetic acid, Indole butyric acid did not suppresses the encloPGactivity very efTectivcly at 100 pprn concentrations. All phytohonnones suppresses the encloPGactivity 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 ofGal. Acid

rel/nrl/30 min.)

Relativeactivity(%)

Significance

lontrol 9 4280.26tt2.25 100.00

Indole acetic acid IO

1005

5

800.12+8.1625.32x4.O8

I8.690.58

++++

2 lndole butyric acid t0100

5

5

2740.20+12.0832.1$:1.24

64.000.75

++++

J NepthaleneAcetic acid

t0t00

5

5

r 850. I 614.7018.24rO.94

43.200.42

++++

Cibberellic acid l0t00

5

5

3320.32r r .90

220.42!3.4577.57

5. t4I+++

5 Kinetin t0t00

5

5

660.20!5.45204.t4x3.24

15.424.ll)

+++l'

58

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 l0ppril 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 tlieDepaftrrerll of Biotechnology,ClRl" Chennai, for Laboratory tacilities and useful suggestiotrs

and UCC.Nerv Delhi, for reseat'ch graut.

RBFtrRENCT]S

l. Albershein. P. and Bonner, J.(1959). Metabolisrn and hormonal control of p-ctic sttbstatrces.

J.lliol.('hem,234: p. 3 105-l 108.

7. Batemal, D. F. (1966). Hyclrolytic ancl transelirtrinative degradation of Pectic substances by

extracellular euzynles of Fttstrritrm solttni f Phs.teoli. I'hytopothol.56; p.238-244.

3. Cooper, R. M. and Wood, R.K.S. (1975). Regulation of synthesis of cellwall degrading

enzyme by Verticilliunt ulbotttrum and Ful;orium <txysponm f sp Lycopersici. Pfusiol. Pl.

Puth. 5'. p. I35- I 56.

4. Dekker.J.(1963).EffectofKinetinonPowderynrildew. NLttttre. 197:p. lA27-1028.

5. Desai. B. G.; Geypens, M. and Van, Assche C.(1974). lnfluence of three fungicides on the

ploductiorr of Cellulolytic Pectinolytic enzymes in the culture filtrates of Pythittnt spp.

Meded.Fac.Lund boutvet Rijks Univgent.38; p. 1455-1466.

6. Dubey, L. and Joshi, R.D.(1976). Effect of Phytohormones Boltytis spp. lndian.lournal o/Microbiol.l6; p.34.

7 . Fe lrnnan, H. and Dianroud, A. E. 1 I 967a). Stud ies on auxins in tl"rc Phytopthora disease of the

potatotuber.l. Role of Indole acetic acid in pathogenesis. P1ry1op117.2.591'p- 83-100.

Ilio-Science Resetu"ch Bttlletin. Vol. l6 (No. 2) 2000

59

B. Annadurai, M. Shanmugam and D.B, Motlag

8. Carg. D. K. and Mehrotra., R..S.(1.977). Elfect of firngicides and growth regularors onproduction of pectolytic artd cellulolytic erzynles by Fisarium solaii.f.sppisi in culture.hdian. Phytopoth,30; p. 546-548.

9' Cuiscafre--Arillago. ( l-949a). lnhibition of pectolyric enzyme by growth horrnones. Journul oJPhytopathol., 9l ; p. 39.

l0' Hancock' J. C'l Miller' R. L. and Lorbeer, L. W.(1964). Pectolytic and Celluloytic enzymesp"^qtlt:! by Bortrytis alli. B-cinereo and B. squannsa in vitro and in vivo.phytopatfutl.,54.: !p. 928-93 l.

ll' Jacobs' w' P. (1979). Plant hormones and Plant developrnent, Carnbridge University press,Canrbridge.

I2' Mehta' P' (1979). Plant growth regulators:lnhibitory agents of polygalactu ronase. phillipittc.t,Iournal of Science.106: p. 71 .

f 3' Mehta, P. and Mehta, A.(1979b). studies of transeliminases in Alternqriu.ll lnhibitoryeffects of plant growth regurators, phenorics and fungicides . Ind. phytopathi. rz.: p. s3s.

l4' Nelson, N: (-1944). A Photometric adaption of the Somogyi rnethod for the cetermination ofGlucose. J. Biol Chem.l 53; p. 375-j80.

15' oku, H. and Nakariishi, T.(1962). Relation of Phyoalexin-like antit'ungal substarrces toresistance of rice plants aqainst Helminthosporiuni leaf spot rlisease. Ain.Rept.TokuntinaLub,l4:p.120-128.

l6' Plich. M.( 1976). Resistance to reafspot diseases. F-ruit science Report.3.; p. 33.

l7' Sanderson, c.w. (1965)' The action of polyphenolic conrpounds on enzyrnes. Biochcntistry,,5; p.24.

I 8. Sornogyi, M. ( 1952). Notes on Sugar determin atiort..l. Biol. Chem, 195; p. 19_23 .

Bio-Science Research Bullerin. Vol. t6 (No.2) 200A

60