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BIOJOURNAL, VOL. 11 NO. 1 & 2, T55-160 JUNE & DECEMBER, lggg

ESTIMATION OF PHYTOHORMONES IN NORMALAND INFECTED ONION LEAVES BY

ALTERNARIA CEPTILAE

B. Annadurai and D. B. Moilag1Department of Biochemistry and Molecular Biology, University of Madra, Madras- 600 025

ABSTRACTAuxin content, which has a maior role in disease resistance, was estimated in normalas wellas in infected onion leaves. Auxin content in normal leaves slowly decreases when

the groMh advances. ln diseased onion leaves, the auxin content in the lesion area increasestill the 16th day after inoculation and decreases thereafter.The effect of f ive phytohormoneson the mycelial growth of A. cepulae in the culture medium and on the activity of endo pGwas examined in 100-ppm concentration of Naphthalene acetic acid. lndole butyric Acidand lndole acetic acid showed inhibitory effect on pectic enzymes. Effect of phytohormonesin disease resistance and hyperauxiny due to pathogenesisis discussed.

INTRODUCTIONPhytohormones occur in traces and are constantly maintained at low level by the plants

(Albersheim et.al, 1959, Bateman, 1966 and Cooper !t.at, tszs;.This constani levet of aphytohormone is disturbed by different factors.The chief one is infection. During infectionphytohormones either directly or indirectly prevent the action of the parasites.The phytohormones may also counter the various physiological manifestations

triggered by parasites. Guiscafre-Arillago,1949a reported that2-4aicnloro phenoxy Aceticacid (2,4D) inhibited directly the growth ot Penicillium dicitatum and phomopsis citri.

It was reported that in several plant diseases. Phytohormones and phenols inhibit theactivity ol cellwall degrading enzymes (Dekkar, 1g63, Desai et.al., 1974, Dubey et.al. 1976and Fehrman et.al. 1967)- Bateman (1966) suggested that the phytohormones have a rote indisease resistance- sanderson (1965) reported that the o*ioition producls of phenoliccompounds also act as enzyme inhibitors. Plant pathologists have been trying to controlthe plant diseases with the help of various phytohormones (Guiscatre-Rrilllgo, tg49u,Hancock et.al, 1964 and Jacobs, 1979). Oku and Nakanishi (1962) reported that lnd-ole AceticAcid sprayed on rice plants stimulated phytoalexin synthesis. Fehrman and Dimond (1967)reported that lndole Acetic Acid trated Potato tubers and their phenol levels increased.Thehigher concentration of auxin influenced the phytoalexin production in the tissue culturesot Phaseolus vulgaris.

Department of Botany and Biochemistry, c. Abdul Hakeem colleg;, M;lv[haram_632509, Vellore District, Tamilnadu.

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ANNADIJRAI AND MOTLAG

Garg and Mehrotra ('lgr7)reported that gibberellin suppresses the activity of cellulases

and polygalacturonase ol Fusarium sotanif pisr. Mehta and Mehta (1979) observed that GA

inhibited polygalacturonases, pectin trans eliminase and pectin methylesterase' Dekker

(1963) oOservea that kinetin inhibited the development of powdery midew' ln this paper the

auxin levels in normal and diseased levels estimated'The effect of phytohormones on crude

enzyme preparation and on purified EPG was studied'

i nuxtN coNTENT lN NoR..,iAL otllottLEAVE6

?50

150

100

50

.04 Days I days 12 dalrs 16 daYs 20 daYs

AGE OF LEAVESI

The Fungus

lsolates ol Alternaria cepulaeobtained from the diseased onion leaves were used for

the interaction study.

Estimation of auxin

The auxin content was estimated by adopting the methods of Mahadevan and sridhar

(1e82).

Effect of PhYtohormones on endoPc

1ml of the purified enzyme (7-ml/ml w/v) was incubated with equal volume of 10 ppm,

and 100-ppm concentration of lndole Acetic Acid. !ndole Butyric Acid. Naphthalene Acetic

Acid, GA and Kinetin 0.5 ml of the incubated enzyme was tested for reducing sugar by the

methods of Nelson (1941) and Somogyi(1952)'

Estimation of endoPG activitY

EPG activity was estimated using the Nelson (1944) and Sonnogyi (1952) method'

RESULTS AND DTSCUSSION

Estimation of auxin content in normal onion Ieaves is shown inTable 1.The auxin

content was maximum on 4th day while it slowly decreases'from the 8th day onwards'

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BIOJOURNAL, JUNE & DECEMBEB,1999

Table - 1

Estimation of auxin content in normal onion leaves :

Auxin contents SignificanceSl.No. Age of Leaves

1.

2.

3.

4.

5.

4 days 5

8 daYs 5

12 days 5

16 days 5

200.25 x,12-24

80.50 t 4.50

56.75 t 4.11

28.25 t3.434.50 t 1.10

++

++

++

++

++

Values expressed are the mean values 6 SD

d.f. = Degrees of freedom = n' 1 observation

Auxin content is expressed as pgof IAA present in one mlof ethanolextract as assessed

Table - 2from standard IAA graPh.

AUXIN CONTENT tN LEAFBLIGHT AREA300250200150100

500

I

I

Il=su.lTE

zoaulJz

FzUJFzoo2

=f

E:l-l

"r.""o$t*".""*.,S"*"oSu*CDAYS AFTER TNOCULATICN

Estimation of auxin content in leafblight area of onion leaves affected by A' cepulae

Sl.No. Age of Leavesinoculation

Auxin content in lesionarea Per ml 6 SD

Significance

1.

2.

3.

4.

5.

6.

4 days

8 daYs

12 days

16 daYs

20 daYs

24 daYs

5

5

5

5

5

5

50.2566.53

120.5068.18

170.7566.33

240.25612.25

180.75610.27

6.0061.25

++

++

++

++

++

++

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ANNADURAI AND MOTLAG

Values expressed are the mean value 6 SD

f. = degrees of freedom = n-1 observations.

Auxin content is expressed as pg of tAA present in one ml of ethanot extract.The results reveal that there is a positive correlation between resistance and

concenlration of phytohormones by the host plant against the leaf blight causing fungus A.cepulae.

The estimation of auxin in normal leaves (Table - 1) indicates that the auxin leveldeceases as growth advances. Leopol et.al. (1983) stated that this is due to the transport olauxin to the actively growing regions.

Studies on the estimation of auxin content in the blight area affected by A. cepulaeshow that from the 4th day after inoculation, the auxin content increases upto 50 pg. On16th day the auxin content is found to be 240-pg ml. Pilet (1960) suggested that this increasebe due to the translocation of auxin from active site to the site of infection. The resultsobtained are similar to the results of Page and Selman (1959). Pilet (1960) and Sepueira andKelman (1962).

The effect of phytohormones on mycelial growth ol A. cepulae suggests that almostall hormones inhibited the growth at 100-ppm concentration. Out of two differentconcentrations, 100-ppm concentration of Naphthalene Acetic Acid, Kinetin, and lndoleButyric Acid and lndole Acetic Acid inhibited the growth very well. The results agree withthe f indings of Guiscaf re Arillago (1949) and Davis and Dimond (1953). Results of the effectof phytohormones on endo PG activity indicaie that Naphthalene Acetic Acid, lndote ButyricAcid and lndole Acetic Acid inhibited the endo PG activity at 100-ppm concentration.Theinhibitory effect of Kinetin.and Gibberellic acid is upto 40% the results are in agreementwith the findings of Sinha and Wood (1967, 1968). Thakur and Chenula (1974), Plich (1926)and Mehta (1979).

When the effect of phytohormones on the EPG activity was tested invitro was foundthat at 10-ppm concentration GA and Indole Butyric Acid are less effective. At 100-pmconcentration almost all phytohormones have inhibitory effect on EPG upto 95%.The slightdifference in inhibition may be due to the intermediate compounds formed in the culturemedium.

ACKNOWLEDGEMENT

The author B.A. is gratefulto Dr. S. C. Dhar and Dr. R. Puvana Krishnan, Scientists atthe Department of Biotechnology, CLRI, Chennai for laboratory facilities and usefulsuggestion and UGC, New Delhifor research grant.

REFERENCES

Albersheim P. & Bonner J. 1959. Metabolism and hormonal control of pectic substances. J.Biol. chem, 234 : 3105-3108.

Bateman D. F., 1966 Hydrolytic and transeliminative degradation of Pectic substances byextracellular enzymes ot Fusarium solanif Phaseoli. Phytopathol,56:238-244.

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BIOJOURNAL, JUNE & DECEMBER, 1999

Cooper R. M. & Wood R.K.S. 1975 Regulation of synthesis of Cellwall degrading enzyme byVerticillium alboatrum and Fusarium oxysporum f sp Lycopersicr. Physiol [P] Path,5 :

1 35-1 56.

Davies D. & Dimond A.E., 1953 lnducing disease resistance with plant growth regutators.Phytopathol 43, 137-1 40.

Dekker J. 1963, Effect of Kinetin on Powdery mildew. Nature, 1g7,1027-1028.

Desai B. G-, Geypens M. &Van Assche C,1974,lnfluence of three fungicides on the productionof Cellulolytic Pectinolytic enzymes in the culture tiltrates ol Pythium spp. Meded.Fac. Land bouw wet Rilks Univgent,38, 1455-1466.

Dubey L. & Joshi R. D,1976, Effect of phytohormones Botrytis spp,lnd. J. Microbiol,16,34.

Fehrman, H. & Dimond A.E. 1967a. Studies on auxins in the Phytopthora disease of thePotatotuber !. Role of lndole acetic acid in pathogenesis, Phytopath 2,59 : 83-100.

Garg D. K. & Mehrotra B. S. 1977,Ettect of fungicides and growth regulators on productionof Pectolytic and Cellulolytic enzymes by Fusarium solani. f . sp. pisiin culture. lndianPhytopath, 30, 546-548.

Guiscafre-Arillago, (1949a),lnhibition of pectolytic enzymes by growth hormones. Journalof Phytopalhol,9l,39,

Hancock J. G. Miller R. L. & Lorbeer J.W. 1954 Pectolytic and Cellulolytic enzymes producedby Botrytis alli, B. cinerea and B. squamosa invitro and invivo. Phytopathol,54 : 928-93'1.

JacobsW P.,1979, Plant hormones and Plant development, Cambridge University of press,Cambridge.

Leopold A. C. & Friedmann P. E. 1983 Plnat growth and development Tata Mc Graw Hillpublishing company, New Delhi.

Mahadeven A. & Sridhar R., 1982, Methods of Physiological PIant Pathology, SivakamiPublication, Madras.

Mehta P, 1979 Plant growth regulators : lnhibitory agents of polygalacturonase. PhilippinesJournal of science 106,77.

Mehta P. & Mehta A.,1979b. Studies on transeliminases in Alternaria.l! lnhibitory effects ofplant growth regulators, phenolics and fungicides. lnd. Phytopath,32, 538.

Nelson N. 1944. A Photometric adaptation of the Somogyi method for the determination ofGlucose. J. Biol Chem, 153 : 375-380.

Oku H. & Nakanish T. 1962. Relation of Phytoalexin-like antif ungal substances to resistanceof rice plants against Helminthosporium spot disease. Ann. Hept.Tokamine Lab. 14,120-128.

Pegg G. F. & Selman 1.W.,1959. An analysis of the growth response of young tomato plantsto infection by Verticillium albo-atrum. IlThe production of growth substances. Ann.Appl. Biol, 47, 222-231.

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ANNADURAI AND MOTLAG

Pilet P' E' 1950' Auxin content and auxin catabolism of the stems ot Euphorbia cyparissiasL. infected by Uromyces pisi (pers.) phytopathoir. +OlzS-so.Plich M, (1976). Resistance to reafspot diseases. Fruit science Report. 3, 33.sanderson G' w' 1965'The action of polyphenolic componds on enzymes. Biochemistry, 5,24.

sequeira L' & Kelman A, 1962.The accurnulation of growth substances in plants infected byp s e u d o m o n a s s o I a n a c e a r u m. pirytopairro t;d ;;; ;;;:;48.tt"^rrfuI;t.wood R.K-S. 1968. Resistance to vascurar wirt parasites, Neth_ J. pr. path,74 :

Sinha A' K' & wood R'K's', 1967. The effect of growth substances on verticilliumwitt oftomato plants. Ann. Appl. Biol, 59, 112-12g.somogyi M. 1952, Notes on sugar determination. J. Bior chem. r95 : 1g-23.Thakur D. P. & Chenutu V. V., tnd phytopath, 2Z (1914) 325.

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