plate 1 - Pondicherry University DSpace Portal

ANTIFUNGAL EFFECTS OF AQUEOUS BULB EXTRACT 

OF AKiom sativum L. ON Colletotrichum capsici 

(Syd,) BUTLER & BlSBY INFECTING 

Capsicum annum 1. 

Thesis submitted to the Pondicherry University 

For the award of rhe degree of 

DOCTOR OF PHILOSOPHY 

IN 

Llrc SCIENCES (BOTANY) 

DEPARTMENT OF BIOLOGICAL SCIENCES, 

PONDICHERRY UNIVERSlTY, 

PONDICHERRY-605 014, 

INDIA. 

DECEMBER - 2002

Dr. U. KANNABIRAN, M.Sc. I'h.1). 

Reader in Plant Sciences 

Department of Biological Sciences 

Pondicherry University 

I'ondicherry-605 014. 

CERTIFICATE 

I liereby certif! that this thesis elititled '.ANTIIWNGAI, EI'I~I

DECLARATION 

I hereby declare that this thesis entitled '.AN'I'IPUN(;AI. EFFIX"1S 01; 

AQUEOUS BULB EXTRACT OF Allium sarivunl L. ON Collcrorriclturii cirl~sici 

(Syd.) BUTLER & BISBY INFECTING Capsicunr irrrnunt I.." sub~iiiued Tor ~hc 

award of degree of Doctor of Philosophy in Life Sciences (Botaii!) i\ in! original 

~ t r and ~ l has not previously formed the basis for the ,\\lard of ail! I>egtec. 1)iploina. 

Asrociateship. Fellowsh~p or other similar titles.

I wuli to express my profound sen ofgratttude to (Dl Q qantrablran, 

Qader, (Department of~~lzohgualSctences, Ponddeny 'Llntver ril\ 

Jorg~utr~ me an 

opportunity to do my Ph \.I) under film I also thank 61mfor liic 1 aha68 gurdance, 

patience, gcrrc~ostty, rasv approachabrhty andabofor helpcng nrc 111 i ome ~lircru~qh the 

fiuras ofCLngIuli 

I owe a hep srtisc ofgratttde to Or (E Vyayan, (l'rofissor and /Ted 

Departrnrnt o/ ~iholb~ual Sciences, Ponduheny Vnivers~t~ cuho war very 

unhrstandng andorsu~cdlhe use offacthtus tn the dipadment i ~lian( lirn~for tfiu 

from the depth of niy heafl 

1 am cxtrerncly thankjuful to the faculty memberr cfDepa~.trtrertt of3u1logtcal 

Scunces, ~?oonhcher.ry I/nrvcrsity, Dr ?( Snkumar, Prof ? I' Natfiu~ and 

or 3 8.janracliandra @.edy 

andfo~ the11 .culuaGlc he&? 

for [tndy allownu me to use thcrr jil~joralo~yjarrht~es 

I tliankprojl~sely a~ldstncerely Dr 5 ~ayacfiandran, 1'1oj1sso1 arrd /[cad; 

Dcpartn~cnL OJ diotcchnohgy, PonLcfiefiy Vntwrszty, far hls r ,ihil~6(c suqqcrlions, 

flenerosrt) constiuclt~~~ suilflesttons, t&alutlc metfiodobgial oI7ifl~ilt10n rn nri~kng 

tfiupiolect, 14 dream come true" andforprovdng me necessaiy Lihn~ntotyfarrhtles 

I ako (hilnijlilsfarnlh members for aterythln'q 

I L I gratefuf ~ to Dr ?( Ja(tfitvef Lecturer, rL)epaflnreril ill 

Pondtchrn~ L'r~:oers~ty,for hu fructfi(stqp~tions andtlmcly adr I' LA 

, 

/irotcchrroi(yy, 

I 11 ~sli LO express my specla[ wliobhartcdlhan~ to my soilo~ I)r V $omattit, 

for her frurlful suggesttons, cntual comments, ~tniely dvtce, c" orri~mual support,

ncedrdtnqirafrorr andforproundi:ng me the ncceoay fileratrrns dunrlg thr rorirsc 

thrs coot.(., 

I would h(e to place on records my 1ndc6tcdnes.r to Or, 7. qanc.ran, 

(bartment of Botany, Tanchi %tarnunwar Center for il'ost - (iraduale .~ludus 

(~yv)~.\), firthcheny, /or hls valua6h suggesltons, elaborate drscu.rsrorrs, 

ertcouragcments andfor prvudng me t h necessay Ltcratures reGvant to my ruo4 

7fu rrrolstbb Tmnd~~ thre in tlie scnpt andpresentatiotl I am much ~han/$d to hrm. 

1 evrtss nly humbh gratttude to fi$ A,$. qarnachandran Natr and 

Prof 'I{ Sunya O'rakah qao, Depaitmcnt of Chcnlu~ry, Porrdicheny Urr'nrventty, 

(01 Gunasegaran and Or. K, ,Subrananr, "n:$f,i16,\, o)ondtcheny, 

Mr .S Thtagaralan, MSc, Public Nealth Laboratoty, tPonduherry, %/r. Janlhana 

xnrhnan, M.Sc., qfficer, Pesttcde @stdue Jdyzmng Laboratory, Podulieny, for 

thr~ oaluablk sugdestions and cnttcaf comments rtl the acllvt pnnctph rsoLtlron 

.stltdici 

I qress my stncere thnk to Dr. 3: Par~hasaralht, qcadrt, .(ahrnJh .I'rhoof 

'Llnlverstty, Q40j 5, Wadana~unjzthanr. ~'ro/ x. Kadavul; 

o/,Ltclhyy, Po~rhchcny 

~L)I '7' $(anrassamy, Dr. (;, l(unlaravelu, mPG4'$, ~i)crndrr/i~ny, a~rd 

01 4 d'ragasam, Ifcad; Department oj$olany, ~~liaralhldasarr ~lr~r~crnn~eirt (,'olGagc 

~(JI 11 cllrian, Ponhchcny, for thetr valuabl;' suggesttons In the Idii~l+~atmoll o/phrrts 

arld'lrrscjy heks 

1 wrsh to record my thank to the members of the ,I)clrlor.al Commtttet, 

iDr. M.P. R,amanujam, qedr tn (Botany, mL?$j, (i)ondu/icny and 

Or. J. Prugasam, Department ofBotany, Bhrathdasan (jou~emment Cobage 

for rc oman, Porduhevy

&tng 

I wod&& LO thank Ofie staff~~epattrncnt ofBwlogtcal.~caencesfor 

me to use the dipartment computers andfor tLtr vdua6h Lijs 

I eqress my sincere thank to the Ltbraty Jutliontles and the StafjCojSuG - 

BIG, ~~onduheieny Unzverslty for thetr ttmeCy helps. 

wy warm apprecrat~ons to establihers of; New hnk, %&wd .Corn and 

NJXComputer centers for havlng brought out tau thesrr in thepresenljorm 

I am grateful to or, Bolan, Dr. Pavan andor. Clandrasekar, /JcmiCR 

Ponhrheny, who helped me the way I hob$ 

ahrudcs through thelrpatunt andconstant advlces. 

at life and strengthened my menlal 

I rail never thank my parenls and brother enough jor aB their sup pot^ and 

love ficy were always wtth mc dung the di$cuCt tlmes and many 1hzng.r wouu 

have 6cei1 kft lncomphte wlthout their heks 

.$a many others have contn6utedzn makng my dreams conie true 111 lhls u1or.k 

I tak this opportunity to express my sinceye thank to alofthenl 

~ G L Id O ~ I submlt myself to the almghty who had Gccn wrth me in lhrs 

cndeaooi oridstioweredme wlth has bounttfulbhsszngs 

Bate. 3 1 ~i)ecember 2002 

PIhcee %rrhifre~.

ABBREVIATIONS 

niiti 

% 

BOD 

PP"' 

"C 

I1 

rnl 

a.nl 

rpm 

Ctll 

11 I 

Ill g 

8 

lllll 

\ !\ 

\?I\ 

N aOl-I 

DNA 

RNA 

I\ 1 

110 

111111 

hl 

11s 

1 rib 

SIX 

- minute 

- percentage 

- biological oxygen dcniand 

-parts per millio~i 

- degree centigrade 

- hour 

- millilittre 

- anti meridieli 

- revolution per minute 

- centimeter 

- microlitrc 

- milligram 

- gram 

- nanometer 

- volume by volume 

- weight by volume 

- sodium hydroxide 

- deosy ribonucleic acid 

- ribonucleic acid 

- weight 

- numhcr 

- mtllitiieter 

- molar 

- microgram 

- tris (I~ydroxymcthyl) ami~ioctanc 

- sodiunl dodecyl sulplicrc olyacrilanl~dc 

gel electrophoresis 

- litter 

- pheny 1 methyl sulpho~~! 1 Iluoridc 

- hcta mcrcaptoethanol 

- ('uomasivc brilliant blur. 

- tetratncthyl ethylene diaminc 

- Volts 

- milli ampers 

- ctliylcne diamino trlra acytale 

- sodium chloride 

- optical density 

- Lris acetic acid ethylenc dianiinc tetra 

acetalc buffer 

- acceleration duc Lo gra\ il)

- polyvinyl pyrrolidone 

- micrograms 

- kilogl.am 

- ~lllr~l~~iolel 

- Souricr transform infi.alcd spcctroscop!, 

spcclroscopy 

- revcrsc phase high prcssurc liquid 

cht.omatograpIiy 

- alpha 

- potassi~irn dihydrogen orthopliospliatc 

- sodium nitrate 

- potassium chloride 

- ferrous sulphatc 

- seconds 

- standard errol. 

- sodium dodecyl sulphate 

- chlorophyll 

- milligra~i~igram fresh weight 

- milligram/ gram dry weight 

- 3- (3.4 Dihydroxyphenyl) L- Alaninc 

- proton iiuclear iilagnetic resonance 

- magnesium sulphate 

- carbon nuclear ~nagnetic resonance

CONTENTS 

1. INTRODUCTION 1 - 10 

2. MATERIALS AND METHODS 11 - 46 

3. OBSERVATIONS 47 - 56 

4. DISCUSSION 57 - 66 

5. SUMMARY 67 - 69 

6. BIBLIOGRAPHY 70 - 86 

7. APPENDIX

INTRODUCTION 

Plant pathogens adversely affect crop y~eld and crcdtc

and four are nematodes Among the ma101 fungal d~sedse\ anthtatnose aiie~ts the vleld 

directly by ~nfect~ng fni~ts Anthracnose (llu~t rot) IS d 5erlous dljedsc 111 L ~ I I I I dnd 111 

surtahle weather, ~t may cause 12-15 per cent of damdgc to the crop (Go~iidtli~ 2001) 

In ~h1l11 plants, the d~sease anthracnose (Ilterally means ~odl~kc ) 15 LCILI~C~ 

hy 

('ollcro~~r~hrrm cops~cr (syd ) Butler and 81shy It belong5 to the oldel Mcldnconlulcs 

contalnlng a s~ngle fam~ly Melanconlaceae 

The llpe frult rot of ch1l11 caused by thls fungus Ir a very serlous dlsedse I he 

rlpe fru~ts, turnlng red, show ell~ptlcal lesions whrch are green~sh bldch (or) d~lly glay 

rn color showing numerous dot-l~ke black stluctures, the aceivul~ of thc lungus rlic 

fungus spreads to the central cavlty of the fru~t and Infects the seeds ( ~ul)srcr produces 

dense ~hrt~sh to dark gray aer~al mycelium, reverse of the colony 15 ddrk brown 

Con~d~al mdsses are pale buff to salmon colored hut lnd~v~dual con~dla arc hyallne, 

falcatc lus~form, aplces dcute. 18-24 x 3 5 p, setae numerous trlch~forln brow11 to 

blachl5h blown, 2-4 septate, 50 - 100 x 2 6p Applesorla are abunddlltly produced by 

the gclmlnatlng conld~a 

D~schson (1 925) made a comprehens~ve review on the Colletotr~chum spp 

H~gp~ns (1926) descr~bed the morphology of C tup\to 1 Ing and 1111 (1944) 

desc~ bed con~d~a of d~fferent spp of C oile~oirrchurn Dura~ral (1956) ~eported thdt 

suclose and ammonium sulfate were found to be su~table for the g~owth dnd 8porulatlon 

of tlic fungus C tuprlcl 

C rr~psrc~ requlres a temperature a~ound 28"C, thc relatne huniid~ty ol 02% dnld 

opt1nl~1111 pH of 5 6 for ~ts best growth 

(Chowdhury 1957) h41~1dnd Mdhniood 

(1961) ~epo~ted the effect of v~tam~ns and Iio~mones on the g ~ o of ~ C ~ cupro h Ndla~n 

dnd [la, (1970) reported the colletotln tox111 ,produ~t~on by C CU/?~LI, wh1c.h kllled the 

host t~ssuc Rout and Rath (1972) established the seed borne natulc of C ir~po~r

Colle/urrichum spp. was reporled to inhib~t photosynthesis and physiolugq of ilic 

host plant (Grewal and Grower, 1974). 

An important feature in the pathogenesis ol' fruit rot IS the cl~e~nical ~aeclsanism 

of penetration of pathogens into the host and colonization of host tissue. It has been 

already reported that many Collerorichurn spp secrete pectinolyt~c and ccllulolytic 

enzymes. which play a significant role in pathogenesis (Cliacko PI ol.. 197X, hilanjee! 

Kaur and Deshpande, 1980; Cervone ct ul.. 198 1 ). 

The effect of different cult~tre niedia on thc growth and sposulatio~l of ('cor/)sici 

and ('cr~~.cumue was studied by Palarpawer (1987). Muruganandam el a1 (1987) 

reported the influence of some nitrogen sources and asparagine induced mauiniuni 

appressorium formation. Cellophane favors the formation of appressorium than agar 

and glycine. An intriguing finding was the replacement of contact st~n~ulus by glycinc 

in the formation of appressorium by augmenting DNA synthesis, which is a prerequisite 

for appressorium formation in an anthracnose fungus. 

Jindal el 01. (1994) reported seed borne nature of C capsici and 11s transnlission 

in bell pepper. Datar (1995) conducted a survey to evaluate the daniagc of ch~lli Srtr~ts in 

!lie markct. He reported that two species of Fu.sorium, 1)rcchslcrcc ccrir~rul~e~~~r~ and 

('LUIJ~IL~ are responsible for post harves! damage. Among them (' [(I/I\ICI 

serious damage. 

caused 

Manandhar ei a/. (1995) examined thc conidial germination and appressorial 

formation of Crapsici and Cgloeosporiordes on pepper fruits. 'The requircnient of 

different nitrogen sources for the growth and sporulation of Collcrorric~ll~~m spp was 

investigated by Palatpawar and Ghrude (1997). 

Conidia of Collerotrichum spp. as soon as dispersed rapidly adhere to aerial 

parts of the plants, to initiate disease. The'conidia are embedded in wcr-soluhle 

mucilage. composed of high molecular mass glycoproteins, which is rr.sin~nsihlc for 

initial attachment of conidia to hydrophobic substrate (Hughes ci oi.. 1999). Following 

3

germiruition. the spores produce short genntube, *ch 

required for initial penet~ation ofthe plant cuticle and cell wall. 

in turn develops apprcssonum. 

Pmveen and Purohit (2001) reported the ultrastructure of conidium ontogeny in 

CoNrro/rrchum cup~icl. 

Chelnical factors involved in the conidial germination of Ccup.\irl on the 

surface ofcliilli pods were reported by Rajapakse (2002). 

Fungicide development has been driven not by the occasional cr regional l'ungal 

problems of crops but by their global value to the manufacturing industry. 

discovery and development of effective chemical control emerged only in the mid-19~ 

century Currently. ~iiethods of agriculture and horticulture rely heavily upon the usc of 

fungicides to the extent that some crops cannot be grown in their absence. 

'The 

4 good number of reports are available on the chemical control of C cupsici 

Cho\vdhury (1957) conducted spraying trials to control fruit infection of chillies 

by Ccrlpslo and concluded that 3 to 4 sprajs of poerenox, bordeaux mixture. ditliatie 

2-78 and !ellow cuproxide at 15 to 21 days intervals controlled the disease to a great 

extent and helped production of a higher percentage of healthy fruit,. 

Sarain and Panigrahi (1971) evaluated the efficacy of alltifungal activity of 

eight s!steinic co~ilpounds against C.capsio They further reported that agritiiyci~l I00 

(terranl!c~n and streptomycin), aureofungin. hio one, actidiolie at 10 ppm were Ibund to 

havc I I ~tililbltor! effeci on collidial ger~iiination of C'capsicl. Lira111 was Ibu~id to 

inhibit cirti~dlal gerlnination of Ccap~ici and inhibit its infec11011 on thc fruits of 

C'upoc rr1,i ~IIII~IIIII. 

Rqi~ el (11. (1982) recommended capton as the effecthe fungicide for this 

disease due to its compatibility with pesticides like quinalphose, dilnathoate, phosolone 

and carbar!l. Spraying with paushamycin at 200 ppm along with 0.3% blitox, 0.25%

and dithane M-45 controlled the incidence of Cxapsici and Xanthomonas (Raju and 

Rao, 1984). 

At present chemical fungicides dithane M 45, copper oxychloride, benomyl, 

capton, bavistin, ziram etc., are used to control C,capsici infection on chilli fruits. 

(Chakravarty ct al., 1981; Raju et al., 1982; Hewitt, 1999). 

It (Anonymous, 2002 a) was reported that the control of C cup,sici infection of 

chilli was possible through the use of disease free seeds, hot water treatment at 5Z°C for 

30 millutes and by crop rotation. 

Sugunagar Reddy e/ 01. (1980) reported that C cups~cisolates tolerant to copper 

sulphate are 1-5 fold more resistant to zineb. They concluded that even alternative use 

of different fungicides would be of no effect in controlling fungicides tolerant pathogen. 

Sariah (1989) reported benornyl resistance in C cupsici in the chilli growing field sites 

in Ma1a)sia 

Lvaluation of plant products againat fungal pathogens in the reccnt past showed 

that the! could be successfully used as effective alternative to currently used synthelic 

Cunglcides. Some of the research works on antifungal activity of plant extracts against 

fungal pathogens in general are given below. 

The tirst authentic report on the antifungal activity of secondary nielfiholitcs was 

reported h! Walker in 1925 (Seliapandy, 2001) 

Diiit el ai. (1976) observed that flower extract of llosu indictr strongly inhibited 

spore gern~~nation as well as radial groMh of ('ur~luluriu pullescc.~r\. ('epholospurium 

.socc hu1.1 and Fusurium nrl'ule. 

Dhauale and Kolmelwar (1978) repolled that seed leachatrs of chillies wcre 

fbund to inhibit C:capsici.

Eflcct ol flower extracts of' five plants on con~dtal gclmltlatlon dttd tny~cllnl 

glow111 wd5 studied by Kapr el ul (1981) They observed Uiat Cvolvulu~ uhtnoidec 

and ('an~~olvulus plurtcaulo almost completely lnh~b~ted growth of 

hi tic \,toe I hr ~I.Y~ILO/~ dnd Fusar~um oxyrporum 

Alrctnurru 

Es$c~itldl otl fiom Hyplts suuveolens was found lo be fungttox~c w~th broad- 

speitlum d~tl\t~ty (Pandey el ul, 1982) Leaf extract of Dalrtrcr nlha and (hnnuhrc 

\~IIIIU ctieihvcly tcduced the seed mycoflora of Eleuslne corucunc~ and thcrr populatton 

a1 lo%, conlcnlratlon (Pandey, 1982) 

M,l~ootl el a1 (1984) reported that among the 43 plant$ cc~ceticd, wdiet extrall 

ol I~iitri~rnrI~ri\ iprnosuv was the only effectlve extract both unde~ laborato~y dnd field 

io~ltltttoni ~ l~e~lt~lg growth and Cercospora leaf spot development 111 mung bcdn 

F%cnt~dl o ~l extract from the roots of Curculrgo or~li~~rdes showed both 

'111itlungai diid aottbdcterlal actlvity (Ja~swal el a[, 1984) 

.Ailr~l~rngdl assaq of 10 medlclnal plants agamst Aspetgrllr~~ sp dnd I'cnr~illrunt 

511 ilio\~cd [Ilat only Argemone mexrcana reduced growill of dl1 tile reel fungr (Nan11 

'i~id h,~du 1'187) 

Sr'~~tl~~l~i~~tlid~l 

dlld Na~dstrnhan (1987) repo~tcd the dtIll~~ll~d~ cflcct ol glolcln 

p'111 ill 'lilu~o~li C'\traLt$ of Aegie murnle/or dnd Pio\ol?r\ /lr/r/ioi~r ~1g~nn5t t/lo~~tirrrr 

IClllil\\~lllll 

Bdtlritid llands when d~pped In 1% nee111 011 for tlirec 111111utri recorded only 12% 

III~LC~IOII h\ ( li1111ae even after I0 day5 whe~eas control I I L I I ~ ~ecorded ~ 924%1 

~nle~t~oll \\ttl~tli C days (leyarajdn, 1988) 

\ .~po~\ 01 t~oenrculum vulgure and P~nzl~tnellu uni\lrm l~u~tt \Iio\+'cd ~omplclc 

~t~li~l~~ttoti 011 lie spore germlnatlon of C tupsrcr (ShuLld el L// . 19801

'riwari er 01. (1990) observed that only vapors of ('rir.rr\ nlctiiccr atid ('1er111rc~ 

vitcnsu completely inhibited radial growth of two storage lung! \I/ ,I //ol.i,\ end 

P oxul~cum 

bsseat~al 011 from Ocrmum canum, l'rnu, rt~xb~rr$!hrr atiil It utt cplciirp (11 

('111 II\ 

mcdrcu reduced germination and viability of sclerol~a of :M~~I~~~/IIJIIIIIILI l~/~o\co/rttci 

(Dube, 1991). 

Ilhanapal tr ul. (1993) reported that aqueous extracts ol ticcln Ical. untlpcd 

fru~cs, seeds and other cornmercral neeni products effectivel! ~nlirht!cti iadlal toutll 01 

Ithi.-ooon~n sulunl and P/~!~/opthora mcudrl S~ngh c/ a1 (1993) \tudlcd tile cllicacy (11' 

aqueous eytracts of 1 I 

niedicinal plants to control banana frutt rot i.caf c\lracts of 

A:odir~~c~/iilr lndicu. Ociitium sancrum and R~cinus conzniunrc wcre lourid to hc most 

ef'li.ctl\e In controlling the fruit rot 

Kala~chelvan and Sumathi (1994) reported that Solo~rri~~i ~ir,ql.ri~i~ lias 

solaniargtne, a steroid, xhich is inhibitory ro the test fungi /Ircth\l~~~rr 01 ~:oe and 

( 'ti~.~~t/ur I ~ lrtnuru I 

Ganesan arid Kstshnaraju (1995) observed that D or:)cuc cotii~t~al gci.liirnatloli 

\\as tnluhrted by 23 out of 36 plants tested Banibwale e/ ol (1995) also reported that 

onl! one plaqt. Luuwunici ~nermis, out of 14 medicinal plants tested. ~nhihitcd conldral 

perriilnartoli and rnycelial growth of 2 imponant cotton pah)gcns 1 rircicroc/~orw and 

\h~rcii/iet ~I~III roridum 

('l~~omolaenu odorola leaf extract of 2% concentratron r~ili~b~ted gro~tli. 

sporul,iuotl. sporanglal gemmination, zoospore release and germlnatloli of fJh)'~o/~hrhor(t 

i(ip\ii i ( Inandaraj and Lrela, 1996). 

Srivastava and Lal (1997) reported that out of aten plants trsted, fresh leaf 

culracts of Aladrruhra indka, Calo/ropis procera, 0 ~UJIII~IIIII slopped mycclial 

grout11 01' 

(' lirhcrcula~a and A alternata. Gehlot arid Bohra (1997) ~eported antifungal 

7

activity of certain halophytes against A, solani. The bark and leal' extracts ol' 7Bnrur.i~ 

uphyllu, leal' and stel11 c;tracts of Sulsolu buryo.sma. stem and root extracts (11' A~rrlder 

len/i/orn~i\ and only stem extract of Huloxylon recurvum showed total inhibition 01' tlrc 

potato blight pathogen. 

'The antifungal effect of grape proteins against fungal pathogs~ls (i~ri~nicr,ditr 

hcdclli and Uotryris cincrcu was reported by Salzman el a/. (1 997). 

Amodiolia (1998) reported that leaf extracts of papaya I~il\e the potcnt~al 10 

control yo\jdcrq mildcw disease of ('. unnum in the field. 

Garg and Jail1 (1998) reported than an essential 011 Sru~ii the rhi/.omc of 

Curcunrii CLISS~~LI exhibited strong inhibitory effect towards all the bacteria and fungi 

used b! 

tl~eni. 

Fi\ s conlpounds isolated from Ecliplu ulba and Wedelia /rr/ohri/u wcre tested for 

their antifungal activity against nine fungal species. The mixture of coumcstans froin 

Ealbo shoi\ed maximum activity at 200, 100 and 50 ppm concentrations ('fliqagaraian 

and Krislina~iloo~~hq.. 1999). 

\'apo~~r toxicit! 01' 1 I plants against A.niger, Pes/ulo~iit poliiriii~tiir~ and 11 oi:l,ztrcJ 

~n),cclial gro\\~li was studied by Ganasan (2000). Praveen and Ki11113r (7000) evaluated 

aqueous exrracts of I5 plants, bulb extracts of two plants and r111i.onic cstract of onc 

plant for n~itifi~ngal actility against A.lrilicina. Among the 18 plants tesled I'o/j~uI/hio 

lottgifolici gue maxiniuni inhibition (70%) followed by ,4, indicic. 0' iirirt~rl \rrnc/ltni, ('. 

i.o.\cl~.\ and %in,yiher officinalis. Other plants were least effecti\e !I11 tlic sterili~cd 

!extracts lost act~vity 

.A11tlin!,cotic acti\ iry of D.innoxio extracts against ('cul~cii.~ \bas rcported by 

t'llitra and Lanabiran (2001). Kavitha e/ a/. (2001) eveluatcd Ical'r.\trac~s of 13 spccics' 

101' ('ii.c.\ict g gain st four fungal pathogens. All the plants produced more than 50%

~nll~h~t~on In mycelial growth of A colunr Fox,~\l~orurn I' nrro~rc~f~ and Sclerolirm 

rolf\~r 

IlakL~ c/ a1 (2002) reported the antlfungal effects ol' glucose dci~vak~vcs of 

Qucrc~ic rn/tc,orru against Allernur~u alterna~u, Cundidu ulhrcun\ etc 

I'hopp~l (2002) reported ant~ni~crob~al acl~vrty of esacnt~dl o ~l ol Ar/cmr\rtr 

nrlgriictr. a strongly scented weedy herb of Asteraceac The o ~l wa, toxic to all the slx 

bacter~a and seven fungi In vrlro ant~fungal actlvlty of T1norpor.a cord~folr~ was studled 

hy H11t1o el ul (2002) uslng chloroform and ethanol extract\ 

I hey oh\e~vcd that 

mycchal g~owth of both the test fung~ was affected and the ~nli~hltor? ralc ~ncrca+cd 

with Illcleaslng concentrauon Among the two solvents chlorofo~m was lound to be 

\upc~~o~ o\'el ethanol 

111 OUI labo~atnry. several stud~cs wele undcrldhen to b1111g out tllc ~I~III"UII~CII 

effe~t ot botan~cal pestlc~des and antagonlstlc m~crohes (.[asni~ne, 1997 Scnth~l 

kunidl~n 1998, go math^, 2001, Pugazhenthi, 200 1 )) 

t

4. To investigate host patliogcn interactions and efficacy of thc selcclcd 

antifungal .aqueous plant extract to control anthracnose in thc potted 

conditions. 

5. To find out the antifungal active principles present in the selected a~~tifungal 

aqueous plant exlract through pal.tial spectral, chromatographical and 

phytochen~ical tests.

MATERIALS AND METHODS 

In thc present study experirnenls were conducted to find ( I ~ I ~lic potential 

fungitoxic aqueous plant extract to control the anthracnosc fungus in chilli plants 

I. ISOLATION OF THE PATHOGEY 

The infected fruits of Cap.sirum onnum L. werc collected lionl chilli plants 

growing in the fields in and around Pondicherry. Fruits wcre surface - s!crilizcd u~th 

0.2% mercuric chloride solution for 1 niin, washed thoroughly in stcrilc distilled nater 

for four times and the infected portions of the fruits were cut into sn~cill hi~s and placed 

on C~apek's medium with 50 ppm of streptomycin and incubated in HOD incubator at 

28 i 2 OC. 

1.1. Identification 

I he pathogen was maintained in potato dextrosc agar (t'1)A) ~nied~unl at 

optiriiuni temperature in BOD incubator at 28 f 2°C. Cultu~al characters and 

morphology of reproductive structures (fruit bodies), conidia and setae were recorded. 

Identification of the fungus was done on the basis of reports of Dastur (1021 ). Stitton 

(1973). \on Arx (1975), Kulshrestha cr a/ (1976), Sutton (1980) and Mchrotra and 

4neja (1990). Identification and confirmation of C'capsiri was donc 11: 

Ad\vanced Studies in Botany, University of Madras. 

1.2. Pathogcnieity Study 

the Centcr Ibr 

The pathogenicity of the isolate was tested on l'alur 1 variety of cl~~lli. grown in pots. 

I'hc conidial suspension of the isolate was prepared in sterile W~IL'I li.0111 7 days old 

culrure grown on PDA slants. The conidial suspension was diluted \\irl~ stcrilc dis~illcd 

\rarer in such a way that it contains approximately 2000 to 3000 con~dla ml 

The chilli plants of 120 to 135 days after sowing (DASI werc used for 

inoculation. The plaits were kept in a humid atmosphere for 24 Ii h! covering thcui 

i 

wit11 lioled polythene bags. 'he ripened red chilli fruits werc injured \\ith the help of 

surgical needle. 100 ml of the conidial suspension was sprayed with ~hr. hand sprayer on 

the fruits a1 7.30 a.m. Control plants were sprayed with the same \olume of sterile 

distilled water. All .the phts were icdiptely cuvered with blcdplylhme bags 

4: 

sprinkled with .smile water on the inner side to rnaiha high hmiiidity a d kcp

PLATE 1 

Anthracnose disease of chilli fruits 

I. Fruits of Capsicum annum showing symptoms of 

anthracnose disease caused by C.capsici. 

2. C.capsici - Pure culture

PLATE 1

' 

u~id~st~~rhcil as such for 24 11. I'cr~odical obscrv;~~icins o~i dl>casc dc\~loprncnt \\crc 

lll~~ll~, 

Scvci~ days after inoculation. 

the fruit\ were crhscrvcd Sor the disease 

dc\clopnicnt (Plate I). 'l'he pathogen was reisolatcd ii.onl rl~c inkc~cd arca of' the 

inoculated liuits and comparcd with the original isolalc. 

1.3. Maintenance of pathogen 

I llc ~)"thogc~i (,'olle~o~ric.hutn cul)sici (Syd.) Butlc~ & Dishy (I'latc I) was 

p~tr~licd b! single conidium isolation method. 'l'lic purified culture was stored in the 

sl.1111~ oi' I'IIA. 

2. SCREENING OF AQUEOUS PLANT EXTRACTS 

2.1. I'reparation of aqueous plant extracts 

Thc plant parts were freshly collected and washed in running tap water and 

\+i.rc iurtacc - sterilised w~th 0.2% mercuric chloridc solution for I min and then rinsed 

\+ill1 slerilc distilled water for 4 - 5 tinics to cnsurc that all thc cliloride ions ucre 

\razhc;i a\+ay. The aqueous plant extracts were preparcd accordung to the ~nic~liod 

descr~hcd hy Saspal Singh and Tripathi (1993). Plant extracts were prepared under 

azcplli' co~ldition. I'he surface sterilized plant parts \\ere ground 111 a stcrilc blender \vitli 

etlu.il \oIi~me of sterile distilled water b) weigiit. Illis slurr! Mas iiltcrcd th~.oupli a 

doi~blc la!crrd 

sterile cheese cloth and \\as cen1ri1'uyc.d at 50(l0 rpm lilr 10 Inln and the 

clear supernatant was saved. This serled as concentrated crude extract and required 

ct)~lccntralions were obtained. The extracts wcre freslil) preparcd as and wlicii nceded. 

2.2. Screening of plant extracts 

2.2.1. Conidial germination studics 

Conidial ger~nination studies ~ crcarried out In cavil! slidcs by ~ncubatin; 111 a 

n>iob.;r cllambrr at room temperature. Conidial suspension of 8000 - 12000 conidi;~ ' 1111 

\\ .iz ~vel~arcd in sterile dis~illed water \I ill1 the help of Ilaemoc! tonic.tc.r. 1 ml of conldial 

suspi;l~sion was mixed well with I n~l (i1'2OY0 plant extract, so that tlic rcsulti~ig solution 

\\ 111 1.1: of 10% concentration of plant extract \vith 4000 - 6000 conidla 1 1111. i:or control. 

stcriic distilled water was added with the conidial suspenslun instead ol' the plant 

nlracl. Triplicates were maintained [or each exgerimcnt. 

Slides were observed for conidial germination after 24:$ 

and 18 h. Percentage of 

inlrihition over control was calculated b! usi11g the fornlula of L'incent (1927).

I=- 

C-T 

* 100 

C 

whele. I = Inh~b~tlon over control 

C = %of gelm~natton ~n control 

1 = ?4 of gerliilndtlon in treated 

The aqueous plant extracts those produced above 50% ~nliibi~loii on ~ hconld~al 

germlnatlon at 48 h were further selected to find out their cllc~r on thc indldi ~iiycel~al 

plo\\tll of ( LOlItiLi 

2.2.2. Kad~rl m)celial growth stud~e\ 

Ihc lddldl g~owth of tlie liiy~el~uni or dnth~drnort I~l~igu\ (( icil)tici) ud4 

mcdiuled by po15oned food techiilque wltli PDA (Nene el ril 

1971) After the 

~~c~ilizat~o~i of pct~iplates (9 cm), PDA niedlum. cork bore~s and othci glas wales In an 

autocla\e at 121 j°C fo~ 15 mln wlth 15 Ib I sq ~nch pressure tlic plant extlacts were 

ddilcd to thc \\alni PDA to obtain 10% concentration F~nally tlic pldtei \rcii ~no~uldted 

b\ placing 9 ruin d~scut from the growing tlp of 7 day5 old cultule ol C cuptro All 

theye \\ele done undei tlie larnlnar flow cliamber PDA plates \\~lhouthe pldnt extlact 

sci\cd ns ~ontiol 'I he contlol and treated pldtes were niain~~iilied In illpli~dte lhc 

iiio~l~ldted plates Mete sealed w~th palalilm dnd ~n~ubated 111 1301) iii~~lb~~ro~ rl~ 78 i 

2°C 011 thc 8 "'dd\ ot ~n~ubdtlon. the mi.dsu~cmelits ln ill1 NLIL Idh~11 dlolig llic iddid1 

liiii. ot the ~liy~eliai giowtli In the pcti~pldtes l'hc pcrccnt+i 

111liih111on \&ds cdl~~lldled by ~lbiiig tlic li~liiiuld ~i'Vlil~ellt (19371 

(11 1iij~~l1'11 g1ov.111 

v.heie I = Inhtb~tton ol mycel~al g~owh 

C = d~ameter gro\\tli In control 

'I = d~alneter gro\\th In treattilent 

Thc plant extract (Allrum satlvurr~ dqueous bulb extra~t) \sh~ih sho~vcd the bcst 

ie.ultb both on the lnhlblt~an of conldial gernilnatlon and rdulnl mycel~al giowth was 

selected for tirrther In xtro and In vrvo studies to confirm its an~lCurrgal effect5 

13

2.3. I)etcrminstion of minimum inhibitory conccntrstiun (MIC) 

To find'out MIC. required for the inhibition ol'the pathogcn both on ihc conidial 

gcrmi~lation and rad~al tiiycelial growth, the test plant extract was tcstcd at 10. 5. 2.5. 1. 

0.5.0. I. 0.05 and 0.02% concentrations. 

2.4. I)ctcrmination of LI) 5,) 

l,I)c,l value (lethal dose at which 50%) reduction 111 gl.owtli occurs) was 

deteriilined on dry weight basis. Graded series ol'the test plant extract at tlie rate 01' 100. 

;00. 500. 700 and 900 pl were added to 250 mi Erlenmeyer conical flasks conpaining 

('/,apch's-Dos broth to get 0.1. 0.3. 0.5. 0.7 and 0.9% concentrations respcctivcly. 

Med~uni u~tliout extract served as control. Eacli llask was inoculated with two 0 mm 

diamctcr niycelial discs cut from the growing tip of the scvcn days old culture (I!' 

('coj~~ic,i Triplicates were maintained lor control and treatment 

I'hc llasks wcre 

tnci~bated at static condition for seven days at 28 k 2°C under laboratory condition. 

Ilo\$c\r'i. the flasks \\ere agitated at 24 I1 interlais. On the 8 'I' &a), 

each llnsh was filtered separately through a pic-\rciglied Whatman KO. l 

dfx dnii dr~ed in a hot air oicn at 60°C for 24 li 

mycelial mat in 

lilter paper 

The drj weight ol'tlle nijccl~unl 1Y 

;axis) \\CIS plotted agaltist concentration ofthe aqilcolis plant extract (X - as~s) l,ro~ii tllc 

rrsulti~lg growth curve. I.Dla value (0.7%) was determined. 

2.5. 1lc;it tolerance ot'the pant extract 

I lie rest plant

mantile Culttvdt~on practices dnd application of fc~Ltl~/e~$ wcle donc dj I L L ~ I I ~ I I ~ I L I ~ ~ L ~ 

by I'dlu~ Vegetable Resear~h Statlon Conhol pld111\ wcle 5pld!~d ~1111 L I ~ L \dine 

volu~nc oC stcr~le d~st~lled water On the 8 'I' d+ of thc \ ~ I G I \ L ~ L ~ L L ~ ~ I IWCIC 

I ~ L , 

5clc~tcd lot pllytotox~ctty stud~es 

2.7.1. Morpholog~cal parameters 

2.7.1 1 Length nt the rhoot and root 

I he length of the root and shoot were nlcdcu~ed w~th d Ihill~ ol '1 \L~IIL 

2.7.1.2 Fresh and Dry we~ght of the +hoot and root 

l lle pidnls wele sepaidted tluo shoot dnd loot 101 dl). \\'etglil L ~ pld11l L 1)dtls 

\WIL L L ~ 111 I the electr~ oven at 80°C for 24 h file lleah dtld dl! we~glil of thi: plant 

\\'I, found oul by usrng electrl~ slngle pan halan~c 

2.7.2. Blochernlcal parameters 

2 7 2 1 Photosynthet~c p~gments (H~scox and Isrdelstam. 1979) 

4n amount of 100 mg leaf tlssue was kept In a tcst t~~be Lontdlnlng 7 mi of 

dl~nctll\ I wlphoxtde (DMSO) and kept In an ~ncubator 111 tile dat h dt 65°C until the leaf 

rnate~~,ll\ became colorless The extract i~qutd was transferred to d ;!ddu'it~d l~ihc and 

[IIL 

\(llk111ie\\as made up to 10 ml wtth DMSO and qto~ed ai 4°C uni~l ILL~LIIIL~ fb~ 

~ L I I ~ I ~ L ';I~~~\SIF 

I lhe absorbance wd\ medsuted at 480 04~ dr~d 00; 11111 

111 a 

~p~~t~~pl~otonieter dgatllst d i(dgent blanh 1 he IIISIIILIII cot~ttn~\ UCIL L \ ~ I I ~ I ~ I L L'15 I PCI 

,llc tiu~~lul,~ StLen by Harborne (1984) 

I he plgnient content5 were expressed as 11i:'g itch \~rtplil 01 IIIL l~dl 

2 7 2 2 C~rhohydrnte\ 

I hc ~drbohyd~ate frd~t~ons vt/ totdl \ L I ~ ~ I r~duc~nf 

\ 

\LI~JI\ '111d ion-~cducing 

\up,i~*,ind \td~~h hele cxtld~ted 1ro111 o~en - d~~ed powdctcd nldtclrdl+ (11 llic iont~ol 

dnd t1c.11ed ieedltngs, followrng the stditdard methods

Alcoholic extraction 

350 11ig of po\vdered \dnlple uJr ~xll'lited in I0 ~nl ol XO'% ctll,~nol dnd ho~lcd 

I hc honiogenate wds centriruged at 6000 1p11l 101 15 111111 l llc \ L I ~ L I I ~ L I wa5 ~ ~ I ~ rdvcd I 

dnd made up to 20 ml wlth XOK ethyl dlcohol 1 hls ~ ~ L O ~ O ~\lld~t 

~ I C wd\ LIU~ 101 tllc 

qudntltattve esl~mdl~on of reducing sugd~\, told1 su&rls, non-~educlng augdls, phenol\ 

dnd total free anilno ac~ds The res~due i\da sd\ed for \lnrch estlmatlon 

2.7.2.2.1. lotal sugars (Dublo\ el ui lc)51 

Reagent 

Method 

I. Anthrone reagent: 2 y of cinthlone wds dl\\oI\cd 111 1 I 01 ~i)ncellttatcd 

sulphuric ac~d (cone H~SCIII lhl\ reage111 wdj plcp'tred (~csli lust hclo~c 

use 

'lo 0 5 in1 of dlcohol~~ ~\tlait 0 5 1111 01 d~rl~llid wdlcr way added I o 

the dbove solut~on, 4 ml of old dntluoile lcdgent wnc ddded Ihls has hedted fo~ 10 

111111 111 a bolllng water bath To preirlif loss due to evapolatlon, the te\t tubes weic 

cloqed with glass marbles The test tuhcs wels cooled and the abso~hance was ~e.~d at 

620 nni In a spectrophotometer agdlnsr a ledgcnt blank The standard graph wds d~d\rn 

v, ltli Lnown Loncentratlons of glucose 

2.7.2.2.2. Reduclng Sugars (Wang i.1 iii 1997) 

lleagcn t 

Mctliotl 

I. l)~n~trosallcylate reagent I g of i 5 d~~utro\nl~i\ldlr (DN\) 30: 

sod~um potasslum tallaldte 111d 1 6 g of sod~um h\dio\ldc ncle rll\.;oI\~d In 

80 ml of dlst~lled wdter and \nude up to 100 1111 

10 ml of alcohol~c extract was ~ddcd lo 2 0 nil 01 DNS liagellt 1 IIC tube\ \\clv 

~ove1L.d wlth glass marbles and kept In .i boiling watel bath fo~ 10 lnln Then the) nelc 

coolcil and d~luted \\lth 10 rnl ol walela I hc olange led ~ olo~ lormed was lueds~llcd a( 

575 Illn in n spectrophotometcr dgalnbl GI redgent blank I he sldiiddrd gldph was d~d\\ll 

1t11 Luown concentrations of glu~ose 

2.7.2.2.3. Non-reduc~ng Sugars (Looiii~s and Sliull, 19i7) 

The amount of non-reduc~ng sugars was deterouneQ.'h! the tollowuig lormuln 

ol 

Non-reduc~ng sugar = TomI sdgnn - Free rtducrng sugars o 95 

16

2.7.2.2.4. Estimation of Stnrch (ML Creddy el (11 1950) 

flie rcs~duc left hehrnd afier alcoholrc extractloll w,t\ u\cd lor the c\t~~ndt~on 

~td~cli 

Reagent% 

I. Anthronc Itengent: ./\nthrone 20 mg was dts\ol\cti 111 100 1111 01 old 05% 

Mctllod 

H2S04 

11. I'erchloric acid (PCA). I8 nil of d~itrlled water 52 1111 ol LOIIIIIILILI~~ I'CA 

(70%)) wele nilxed io gct 52?4 PCA 

Distilled wdtei and PCA (52%) 5 nil of each wcrc added io thc ~c~duc lelt dltc~ 

dlcoholii extlactlon Ihe mlxture wds incubated for 30 mln 'ind liltered I lie lilt~atc was 

ii?ade up to 300 nil with d~strlled water in a volulnelrlc fla\k 

lo 0 5 1111 01 the above extract 2 5 nil of d~st~lled \\CILCI 

'lnd 5 1111 of cold 

dnth~one iedgent were added dnd heated loi 7 5 mln In '4 boil~iig \\~[LI hdili I Iic lube5 

ucle ~nimedra~el) cooled lo rooni ternps~dtt~~c and nilxcd ~Iioicruglil) 111 ,I c\clom~\iurc 

Ihe bluish yeen colnr was read at 630 nnl dgrlili\t 

'I IL~I~LI~~ hldnk 111 

ipcci~oplioto~iiete~ A stdndard culve uac d1,1u11 w~th ILno\l~i L~LI (11 91dlill WdS cdlculated b\ lll~lIl}l~)'lll~ t h flll~iir~ ~ ~ijill\~llclll ~lli\illt 111 

tlli ~,~inpli h) 0 U 

2.7.1.3. Arn~no ac~d, (Moore and Stem 1948) 

Ite,~gents 

rlic nlnhydrln reagent was p~epa~ed b!. lresh ml\iti; ol L~IU.II il~~rl~il~trc\ 01 

~oi~~ltoil A dnd iolullon B 

Rlerliod 

Strlutlon A: 800 mg of stannous chlot~de wdi d~s\ol\id 111 500 rlil ol 0 2 M 

cltlalc buffci (pH 5) 

Solutron U: 20 gof nlnhydrin \43\ dlscolved In 500 1n1 01 

~lli'tli\ I CCII(I\OIVL 

1)tlut;lnt solution: I'h~s was piepnred h) lnlxlng equ.11 L~LI~III~I~IC~ 01 \\iltcr ,111d 11 

}"op"lol (\!v) 

I o I ml of the dl~oholic extract 1 ml of freshly pieprl~cd ~unllydr 111 redgent via\ 

ddded It was healed in a water bath at 60°C tor 20 mln 5 mi 01 d~lutant solut~on was 

ddded to il wlien 11 was st111 In the waier bath It was coo~cu'dnd the color developed 

\\a> lead at 570 nm in a spectrophotometer agalnst a reagent hlaiih The amount of total

lrec amino aclds prcsenl 111 Ihe sa~nple was calculated by u\ing tllc s~andard grdph 

prepared by usi~ig !lie amlno acld, leuc~ne 

2.7.2.4. I'henols 

2.7.2.4.1. Est~mation of topal phenol (Bray and I'horp. 1954) 

Itengents 

Mcthod 

ii. 

I. Sodium carbonate 20%: 20 g of sod~um carbo~i~itc was dlssol\cd rn 100 

ml of dist~lled water 

Folin-ciocnlteu reagent: Fohn-c~ocalteu wd\ d~lutcd i411Ii d~\t~lled wa\cl 

at l 

I ~allcl (viv) 

1 o I 1111 01 alcoliol~~ extract I 1111 of fohn-c~oialteu Ic,ipcnt dnd ?. 1111 of 70% 

iod~~~iii carbonale were ddded and m~xed well In a cycloni~\ti~rc I lic nil\ture was 

hedted In d hoillng water bath for I niln and cooled Ilic sol~~t~o~i ud\ d~lutcd to 2.5 mi 

uith d~~t~lied ikaier The Inlenslty oi'result~iig blulsh wlicle colot i\d\ lead dl 650 nm 111 

,I \pi'c~~opliotoniete~ agalnst a reagent blank. A standard LLIIIL iini p~c.lu~ed u~tl~ 

Ikno~~i qudntlties of catechol as phenol equi;alent 

2.7.2.4.2. Estimation ol ortho-dihydror! phenol (Mdliade\,il? i ~ 511dlid1 ~ ~ 1006) ~ i 

Reagent+ 

Metliod 

I. Arnow's reagent I0 g ol rod~u~n llltrltc d~id l0; 111 \O~ILIIII ~~lr~ljhildl~ 

\\el? d~qsolved 111 100 ml of distilled watcl 

b~oi\ 11 hottlc It I\ \table IOI oil? yea1 

11. i ICI. O 5 N 

111. %OH, 1 h 

I 

Ihc t~,l~pit wd\ \~oltd 111 ,I 

1111 of thc alcohol extlact was p~pstted Into a tcst tube r ~~i~i i 1111 01 

0 5 3 1 l('1. I 

1111 01 311io\\'\ leagelit 10 ml of d~st~lled ivater and 2 till 01 I \ \ctO1l \\cii. 'idded 

Soon atte~ tlic add~t~on of thc alkal~, the p~nh color developed :\ 1c.dyc111 hlCilih w~thout 

tlic ~ \ I I ~ Iisas L ~ ~nauitalncd I lien, 11 wds illluted to 25 ml dnil ilic r~b\~irhr~~i~~ or \lit 

solution \ids lead at 515 nni In a spectrophotonieter 0 I) 

sdlnp1r.r \\ere calculdted from the standa~d curve p~epared \c.~tIi c,~ttcIic~l 

plicnols prcjclit In tlic

2.7.2.5. Nucleic acids and proteins (Schneider, 1945) 

Extraction of nucleic acids and protein 

Nucleic acids and protein were extracted from the frcsh leaf tissucs of control 

and treated seedlings. 500 mg of fresh leaf tissue was weighed; to this 5 ml of IO'% cold 

trichloro acetic acid (TCA) was added in ice. This was homogcnizcd with a 

homogenizer and then allowed to stand for 30 min. The supernatant was discarded. To 

the pellet, 3 ml of 10% cold TCA was added and mixed thoroughly in a cyclomixer. It 

\vas centrifuged at 2500 rpm for 10 min. The supernatant was discarded. 1'0 the pellet 3 

1111 of iso-propanol was added and mlxed tl~oroughly in a cyclonlixcr. 'l'his was 

centr~fuged at 2500 rpm fbr 10 olin. 'The supernatant was discarded. Iso-propanol 

washing was repeated thrice. To the precipitate, 5 1111 of 5% PCA was added. mixed and 

licatcd in a boiling water bath for 1.5 niin. The tubes were centrifuged at 3000 rpm for 

15 1i11n. The supei.,iitanl was saved and used for the estiluatioli of DNA and RNA. 

The residual pellet was d~ssol\,c.d in 5 ml of 0.1 N NaOll and centrifuged at 

3000 rpln for 10 min. ?'he supernatant uaa used for protein csumatlon 

1.7.2.5.1. DNA (Bul-ton. 1956) 

Rcagents 

Mctliod 

i. I)iphen!.lamine Reagent: l .j g diplienylaminc \\its dissolved in 100 1111 of 

redlstillrd acetic acid. To 1111s. I .5 1111 of COIIC. I12SOi \\ah addcd and stored 

at 4'C or ill dark colo~.ed hottlc l'o every 20 ml of thc reagent. 0.1 ml of' 1.6 

D/o aqueous acetaldehyde \\as added just before 115c to potentiate the color 

de\,elopnient. 

ii. Acetaldehyde solution: Itcd~stilled acetaldeli!dc nt a conccntratioli of 1 .O 

'Yo was prepared as an aqucous solution and stored at 4°C' 

iii. I'crchloric acid (PCA): 3% PCA was prcparcd b! dissolving 5 ml of 73 %I 

'1'0 

l'C.4 11168 n1l ofd~stillcd \\atcr. 

1 5 ml of nucleic acids extract, 3 ml of diphen! lamine reagent was added. 

I he ~bcs bere Lrpt in a water bath maintained at 70°C for 10 mln and then cooled. 'Thc 

color development was read at 640 nni in a spectrophoton~etcr against a reagent blank. 

.4 standard curve was prepared b! ust~ig knoyi conc;cnirwons of calf thymus DNA. 

The DNA content was expressed in III~ of DNAIg fresh WI. 

of~he leaf tissue.

Ilcngcnt* 

Uetliod 

In 

I. Reagent A: I g of orclnol wd5 d~ssolved in 100 ml ol d~qt~lled wutc~ and 

staled In a refrlge~dto~ 

11. Reagent U: Co~l~entrated hydrochlor~c JCI~ (ronc Ii

III 

Ileagent C: Terrl~ chlor~dc (I cC'I3 GH-0) 10% solution 

\I Orclnol Reagent lo 10 nil of' reagent A and 40 nil of ledgent 13. 1 1n1l (11 

leagent C %'a< ddded Orc~liol leafcnt %as frcshl) plcpd~cd dl th ~IIIIL 01' 

LISL 

I o 0 5 nil of nuclelc actds extract, 3 rnl of orcinol reagent wds added dnd liedted 

\+'itel bath I ~ 20 I lnln at 90°C and then cooled 1 he color development wds read al 

665 ~uii 111 GI spcct~ophotomete~ agalnst a reagent hlank A standdrd curve was prepa~cd 

b\ using Lnown concentrdtlonc ot purltied RK4 The RNA content N ~ expressed S In 

Ing of Ill\! 41g fiesh wt of the leaf ttsaue 

2 7 2 5.7 I'roteln ( Loury 195 1 ) 

Ilc,~gcntq 

I 

II 

III 

Metl~od 

Ilc.lgent A 0 5 g ioppc~ slllphdte (CLI~OI 5H20) dnd 1 y ol scidl~lrn ilttclt~ 

d~\$ol\cd tn 100 nil of d~~tillcd wdtei (I urlong el ul Ic)7;1 

Itcrgcnt B: 70 g ol sod~unl calhotiate (Na2C01) dnci 4 p \od~itlil li>d~o\~dc 

~cre dlssol~ed In I I of \later 

lledgent C: To 50 1111 of reagent B I nil of reagent A uas ddded 

I\ Keagent D: Folln-c~ocaltedu reagent \sds prepaled by add~ng equdl voiume 

of dtstllled \\ater to the conirnerc~al reagent 

Reagents C and L) werc prepdred fresh at the tlme ol use 

lii 0 5 ml of the protclli extrau 2 5 nil of lcayent C vd5 dddcci '111d th~ IIII\IUIC 

t~ic~lh:+ted lo1 5-10 nilti dliii tlicn 015 ti11 [it ledgelit I) wd\ r~dd~ii I l i ~ 1111\tu1c iws 

I I ~ L L I ~ ~ lor ~ I Ldnothcl L ~ 20-30 mlti 1 Iic ~olor deleloped way ledd dr .I \\~~\c.lingtli ol 660 

nm 111 n ~pectrophotonieler against a redgent blank Stdndard cclrte ol prolecn wds 

prepdred b\ usllig known concentrations ol bovlne seru~album~n (H{A) I'roteln 

content \\as expressed ln mg of proteinlg offrrsh wt of leaf tlssue

2.7.2.6. O\idativc en/! inch 

Prep~ration of the salnplc extract 

I 

g of fresh leaf sdniplc rids waslicd w~th lunillng tap \\alci and 1111scd wttll 

dislllled water 'Thc \\aslied t~csll leaves werc cut 111 to small p~cccs slid 5 nil ol 

phosphate buffer p1.i 7 was added and gronnd to a fine paste uslng a homogcnizel. I'he 

extract bas centrifuged a1 4000 ~pni tor 10 IIIIII dl 4'(' 

and the cleai wpclnatanl was 

collected 'I'he collccted extrtct w,\r kepi under tol~iene at O°C lo1 one wceL 

2.7.2.6.4. Laccase (Mali'idevan and Sridha~. 1996) 

Itcagent 

Method 

i. Phosphbte hufii (0 I bl) p116 ~ontaln~ng I 0 11iM g~i~rl~ol. 

5 ml of sodu~m pl~ospli~ilc burfei conta~~ung guiacol \\.I\ p~lxttccl Into a (c'il 

tiihc 0 1 nil of thc enzynlr rource was added to it 

After 5 niin tlie ah5oihancc was 

dctcim~ncd at 470 n~n m a \pect~opliotomctci agdinst n leagciii hlaiih Suitrihlc con~~ol 

w~th bolled enzyme rids ~na~iitaiiied The enLylne actlvlty was e\pressed 111 un~ts One 

unlt = changes In the absorbance of the leacrion nnxtu~e ! mln 

2.7.2.6.2. Polyphenol oxidase (Mahade\an and S~idha~. 19961 

Itcagenth 

Rlctl~od 

i. Phosphate huffei (0 I M) pi-I 6 

ii. I. - Dopa (0 01 7 h4) 

3 5 in1 of phosphate hulfei was p~prtted inlo a tcsr tube .111d 0 5 1111 01 1.- 1)opa 

\\'I\ .iddcii ro 1t Tiit\ mixttilc war cal~hei,~tcd 111 'I \&dtcr hatii CII YO'( I (I tlli\ 0 5 111101 

tlie enc!lnr source was added and the Isactlor1 ciiixturc was I ~ L L I 111 ~ ,I ~ \\atel C ~ bath 

lo1 5 lnim at 30°C 

Absorbance of the solut~on was deternulitd at 175 nm in a 

~pectiopliotometer Su~rable control was mamta~ned w~tli boiled cnr! nic 

I lic enzyme 

dcti\it! \\as expresaed'in urits One unit = changes In the absoth~ilcr. ol rhc Icaciion 

ii11\1~1e Inin 

2.7.2.6.3. I'eroxidase (Hampton, 1962) 

Iteagen ts 

i. Bho&iite buffer (0.05 M) pH 6. j

Metllod 

ii. l'liyrogallol (0 00 1 M) 

iii. I-lydrogen pcroxidc (1120?)(0.588 M) 

-1.0 1 ml of0.001 M pyrogallol in 0.05 M phosl7liatc buil;.~ 01 pi l 0 5. 1 X till 01' 

distilled water was addcd In a cuvette and the ahsorb;illcc was idlitstcd to /cro at 470 

ntn tn a spectropliotoii~ctcr Inimcdiatelq 0 I ml 01' ?'I,;) (0.5KR \ I) tl.0. and O.inil o(' 

enqme was added. 'flit. contents wcre mi\cd wcll anci placed ill ~ I I C spcctstijil1tito1liclcr 

'flic changes in the ahsorhance a1 e\:ery -30 second\ tntcrvals llri ; mlti \\,I\ mcasurcd 

Sultable control with boiled enzyme was maintained 

The ctllyrnc acttvlty was 

csp~csscd in units. Ons unit == changes in thc absorhancc of the ~c.lct~oti Iilluturc i min. 

2.7.2.6.4. Catalase (Chancc and Malchlq. 1955) 

Reapents 

Metl~od 

i. Phosphate huCli.r (0 1 M) 100 ml contaming 0 16 1111 01 li,O: 

? till of II20? - phiisphatc buffer (pH 7) was pipetted into a ri\t t~llic I (1 tli~s. 20 pI 

of cti/!nic source \ I ~ S addcd and thc breakdown ol'H!O- was ~ll;,~r~~rcd ;it 240 nil1 111 ii 

s ~ I ~ L I ~ ~ :III ~ eqi~ivalctit ~ ~ ~ o amount ~ o ~ or ~ buffet ~ c ~ cc)nt.l~niny c ~ 11.0, \\.I\ 11>cd as blank 

I'lit. c~i/!'tnc activit! \\as expressed in enzyme units i nig ot I~I(IICIII 011~ L I I I ~ ~ 

c11'iti~i.s In the ahsorb~~ice of'the reaction mixture! nit11 

2.8. Sced gcrrnination studies 

\'lahle chilll weds of Palur variety I wcre surface - .tc~tli/c~i O 1% 

Inclclnlc chloride solut~on fbt one min and washed repeatedl! 

i\~tli \tcr~I~/cd dtsttlled 

\4a1c1 I he seeds. ar rile rate of 100 1 plate, were transl'crred IOLII I i el11 dtanietcr 

pct~~lll.ites lined with I\\O laqcrs of Whatman No I iil~c~ papcl LIIL~C\ I lie iiltc~ ~papcr\ 

uc~c ~iio~slened \*,it11 15 ml ofA ,$utiijun~ aqueous bulh cxtracr ('I,IICI 

~CCCIVC~ 

d~st~llcli watt1 I'he pldtes werc kept in dark at 78 F- 2 "(' Em~.~g;ticc 01 thc ~adicei wub 

tahi.11 .IS tllc criteria To1 gernilnation. Thc nu~:lbcl. of'gcrniinatcd \:LYIS \\as countcd aficr 

90 Ii .~tiii tlic per cci~t ol'gcrln~nation was calc~tlatrd hy us~ng the lk>~~ii~~lrl 

(IS 1 A. 1070). 

(i~.rnmtnation percentage = 

Nuniber of seeds gsrmina~tii 

f 

,!- * 100 

Total number of seeds so\\ n

3. IN VITRO STUDlES 

The Czapek's liquid media (broth) was used for in virro studres. '1.0 50 ml of 

sterilized media in a 250 ml Erlenmeyer conical flask the test plant ex(ract was added at 

LD 50 concentration (treatment). Medium devoid of plant extract served as control. Two 

discs of 9 mm each in size were cut with the help of a cork borer froill the growing tip 

of the 7 days old culture of c'caps~ci. They were inoculated in the flask and incubated 

in a BOD incubator at 28 t 2°C. The flasks were incubated Tor 8 days. The ct~trol and 

trcated flasks were all maintamed in triplicate. After incubation. on 4 'I' and X 'I' day the 

fungal mycelial mat and the liquid media were separated by double layered Whatman 

No 1 filter paper placed in Buckner funnel under suction by vacuum pump. On 8 "day 

of liltration, the filtrates were further used for the analysis of ex~racellular protein 

profiles of the pathogen and also as enzyme source after centrifugation at 5000 rpm for 

T!ic mycelial mat of the pathogen grown in Czapek's broth with sucrose as 

carbon sdul.ce was separated and utilized for the estimation of nucleic acids, protein and 

total lipid contents. Cell permeability and analysis of genomic DNA profiles based on 

ditt'eient ~.estrlction enzymes were carr~ed out only on mycel~al mat 01' [he pathogen 

collected on 8 'I' da!,. 

For pectinolytic en/!,mes production the pathogen \\as grown in Czapck's 

~lii'diii~ii supplemented with pectin as carbon source replacing sucrosc I:ol oxidative 

civ! tnes. amylase and invertase sucrose was used. Sinillarl! for celluloytrc enzymes 

c21 ho\!ruethyl cellulose (CMC) was used 

3.1. Cell permeability 

I 

g of fresh mycelial mat was d~pped in 25 ml or distilled water Alicr 24 h and 

-18 11. the conductivity of the bathing solution was recorded on Control Dynamics 

conductivity Bridge. Results are expressed in milli Seimcni (mS) 1 cm2 of bathing 

s011111o11 

3.1. Estractiun of nucleic acids and protein (Schneider. 1945) 

Nucleic acids and protein were estracted from the fresh m!.celial mat. 1:xtraction 

\\as carried out with similar procedure as mentioned in *tion 2.7.2.5. 

3.2.1. DNA 

Estiiiiated with sirniiar &ure as mntioned in section 2.7.2.5 

23

3.2.2. RNA 

Estimated w~th s~mllar procedure as ment~oned In sectlon 2 7 2 5 

3.2.3. Proteln 

Est~iiiated w~th si~li~ldr procedure as meiit~oned 111 sectlon 2 7 2 5 

3.3. I'otal l~prds (Tsuda er a1 , 1972) 

I 

g of fresh mycelium was extracted after suspending overn~ght In 10 1111 of 

chlo~oform methanol (2 1 viv) at room teniperature 

Then the myceltum was 

hoillogenized in a morter with pestle 111 5 ml of cllloroforn~ methanol (2 1 vlv) Aftei 

filt~at~on the ~esidue was resuspended overn~glit 111 10 ml ~hlorotorm methaiiol (2 1 

vlv) and then extracted 

The chloroform 

methanol extracts were pooled and the crude lip~d thus 

obta~iied was pur~fied by the method of Bllgh and Dyer (1959) To tli~s crude lip~d 

soli~t~on, 15 ml of chloroform water (1 1 vI\) wa, added in a separallng lunnel After 

thoroiigh mlxlng, the lower chloroform phase wa\ withdrdwn dlluted w~th ben7ene lo 

ienlovc traLes of water and brought to near dnness Thc ~esidue was immedi,ttely 

diiscilved 111 ~hloroform methanol (I 1 vlv) solut~on and made to a known volume with 

chloiofoim I lie total l~pid content wds obtained by determin~ng the d~y weight in 

al~quots of this solut~on The total l~pid content uas exprefsed 111 nig I g fiesh wt of 

ni\cc11~1111 

3 1 En~y me Assaj s 

3.4 1 Est~matron of Pectrnolytrc enzymes 

1. Pol\ nlethyl esterase (PMt) 

r Elid product esllriiatioll 

2 I1oI\ ~iietliyl galacturonase (PMG) 

d VISLOSI~~ dssay 

b. 1 11d pioduct estlmdtion 

3.1\55d\ of Pectin tiails el~minase (I' 1 i-.) 

a.Viscosity assay 

b.End product estlrndlioil 

3.4.1.1. I'oly methyl esterase 

methanol 

Poly methyl esterase (PME) cleaves pectin result~nq in pep ti^ dud and 

lnclease in free carboxyl groups was n~od~tored In a Control - Dynamu pH 

meter (Mahadevan and Sndhar, 1996) 

24

Reagents 

Method 

I. 1 5 g of pectin In 100 ml of 0 2 M NaCl 

ii. 0 02 N NaoH 

20 ml of pectin solut~on was p~petted out In a 50 ml beahc~ I01111 of (he cnlynie 

sourLe was added to th~s pectln solution and the ptl was ~m~ned~dtely adltc~tcd lo 7 by 

ddding 1 N NaOH Thls IS the zero tlnie Contro! was malntauned with h11i.d en~yiiie 

The cnzyme substrate 1nlxtu.e was Incubated for 24 I1 and the pl-l was readlusted nlth 

O 02 N NdOH to pH 7 The enzyme aLtivlty was exp~esscd as jpcc~fic dir~~~ty 

LIIII~F 

(SAIJ) One unlt = ml of 0 02 N NaOH requlred to malntaln pl l 7 111 

3.4.1.2. Polymethyl galacturonase (Mahadevan and Sr~dhdr 1096) 

Endo PMG actlvlty was determ~ned by measurlng the loss 111 vlscoslty and exo 

PML actlvlty was determ~ned by measurlng the reduc~ng wgars p~oduced dr the end ol 

leactlon (Wang el a/, 1997) 

3.4.1.2.a. Viscosity assay 

V~scos~ty loss was determ~ned w~th the Ostwald vlscometrl 150 at intervals ol 

30 111111 staltlng fiom 0 to I80 mln after preparing the reactloll mi\rurc 

Reagents 

I 

Method 

pipet1:d 

II 

Sodlum acetate - acetlc ac~d buffer, pH 5 2 

Substrate preparation: 1 g of pectln wa5 dissolved In 100 mi ol acctale 

buffer, pH 5 2, heated to 50-60°C In a wdtci bath d~id 11il\ed w~rll rlic hclp of 

a homogenizer and then passed through two layered cheese cloth The pll 

wds adjusted by using 1 N HCI or I 

toluene was added to the substrate and stored at 4°C 

h' NaOH to pH 5 2 l i\\ d~op? ol 

4 nil of the substrate, I ml of the buffer and 2 1111 of tlic c'lizynic SOLIIC~ *ere 

into the Ostwald vlscometer 150 The eftlux tame oS th? ied~tlon ml\ture was 

mea\u~ed at every 30 mln interval for 3 h and the loss In v~scosrt \\A\ ~dlc~ldled by Ilie 

torinul~

Wl1cl.i.. . 

V - percent loss in viscosity 

Ti, = flow time of reaction mixture at 0 min 

1'1 - flow time ofreaction misturc at a particular time interval 

T,,= flow time of distilled water 

3.4.1.2.b. Assay of exo I'M(; 

Ileagents 

Method 

i. Dinitrosalicylatc Reagent: I g of 1.5 DNS, 30g of sodium potassium 

tartarale and I .6 g of sodium hydroxide were dissolied in 80 nil of distilled 

water and made up to I00 1111 

ii. Sodium acetalc acetic acid buffer, pH 5.2 

iii. Standard glucose. Img 1 ml solution 

From the 3 h incubated reaction mixture of the previous reaclion 2 ml oi'aliquot 

was pipetted in to a test tube. To this 2 mi of DNS reagent was added and heated in a 

boil~ng water bath for 10 min, then cooled and then diluted with 10 ml of distilled 

&atrl 

I'hc orange red color was read at 575 nm in a spectrophotometer, with 

appropriate blank Control was maintained with boiled enzyme reactlon mixlure. The 

cnr!mi. activity was expressed as spec~fic activity units. Onc ulnl .- mg of glucose 

reIt;~\ed i hr, 

3.4.1.2. Pectin trans eliminnse 

1;iido Pl't activity was determined by measuring the loss in \.iscosll> of rcaction 

I~~I\~LIIS and eso PTE activ~ty by determining the production of tliiobarhituric acid 

( I U \ i ~ractiiig substances (Mahadevan and Sridhar, 1996). 

3.1.1.3.a. Viscosity assay 

\!iscosity loss was determined with the Ostwald viscometrr 150 at intervals of30 

inin s~nrting from 0 to 180 min after preparing tlie reaction niiilurc 

Ileagents 

i. Boric acid - borax buffer, pH 8.7 

ii. Substrate preparation: I 

g of pectin was Aissol\ed in 100 1111 of borax 

buffcr pH 8.7, heated to 50-60°C in a watcr bath and mifed with the help of a

homclgenii.cr atid then passed through two lahcred chccsc clot11 and t!ic 1111 was adius~cd 

to 8.7. Few drops of toluene was added to thc \ilhs~ratc and SILII.CL~ ;it J"(' 

Mcthod 

4 ml of the substrate. I 1111 of the hull'er and 1 1111 ol' tlic CIII~IIIC source \vcrc 

prpctted in to thc Ostwald viscoliieter IS0 '1 Iic eflu\ 111i1c trl'tlic rcacuoll mixturc was 

mei~sured at every 30 min interval tijr i Ii and llic rcducuoll 111 tl~c \~';cosit! \+:is 

calculated by the for~iiula of thc \.iscosity as\q ol'clido I'M(; 

3.1.1.3.h. Assay of eso PTE 

Itcagents 

Mctl~od 

i. 0.01 M TBA 

ii. 0,j N I-ICI 

3 rnl of the reaction mixture incubated for 3 h. was plpctted into a 25 nil tesl 

tuhC lo this. I0 ml of 0.01 M TBA and 5 nil oi'O.5 Ti tICI \rclc ~iddcd and placed In a 

ho~lilig water bath for 60 min. This was coolrd uilder tunning tnjl \\atel and thc voiunic 

(:!' rlic solution was adjusted to 18 ml w~tll distilled water llis nhsorhance ol the 

>upclllatant was measured between 480 to 580 nm in a ~~~~.~tr~:~>Iiu~o~iicter. 

I'lic 

11121\111111111 abs~rbancc of the solutioti was obscl.vcd at 547 11111 I 11/!1iic substrate 

~ilr\t~~rc' drawn at Lelo ti~iie incubation and ho~lcd en/ymc \\.it 

u\cd as blank. I'lic 

ell/>llir activlty was expressed as spccitic acti\.ity units. 0111. ittilt - changes ill thc 

ahcotharice of0 001 hr. 

3.4.2. Estimation of cellulolytic enzymes 

1. I .4 H-Exo-giilcanase (cl) 

a. End product estimation. 

2. I .J [i-Fndo-glucanase (cx) 

a. viscosity assay 

2.1. I-'so f:ndo P 1.4 - gulcai-,case (y) 

3. Cellobiasc 

a. End product esttmatioii 

a. End product estimation

Z 4 2 1 Measuretncnt of 1,4 11- Exo-gluernn\c (CI) (Mrl~~dcva~l and Sndhrr, 

I W6) 

TIIL ~LIIVI~Y 01 C 

produced h\ CLLI~\ILI wds d\\d\ed by nica\urlng the 

l~duclllg \LlgdlS rclcdbed by the blcdi\do\~~~ of d\ Ice1 with dllllllOl1~ lCdgelll 

Itedgcnl\ 

Mctliod 

I 5od1um acetate - dcetlc dctd hufter, pl l 5 0 

11 1% avlcel (M~c~ocryctall~~ie crllulose) \~~\pended 111 hulle~ 

111 Orc~nol reagent. I g ol oli~nol wa\ d~b\olved 111 .O 

1111 01 d1\11IIcd WdlCl 

(~~ddually 20 ml ol 67%) H7{O, wds added w~tli cool~ng I lie volumc wa\ 

~dlaed to 100 ml \\'1tI1 dlstlllid \\ater 

I\ Anthrone leaKent 200 tiif of anthronc wdi d15soI\~d In 100 ml 01 

ionc HlbO, 

1 1111 of encytlic coulce I ml of buffet and 0 5 mi 01 \ubstrale uele added rn d 

te\i tuht atid lnc~~bdted at room tempelarure fol 3 11 ]lie IC~LIIOII I~IIX~LIIC \vd? lulxed 

wcll \\lth iyclomixture dt ~.rgular ~nter\dly of 30 Inln 

At the end nl the tedilton the volume ol tht Icactlon nu\tule nil\ ddju5tcd lo 5 

lnl \\1tI1 dlitllled ~.dte~ I he tubes werc iintr~fi~pid 101 I5 nl,li ,it 2000 g to d~pos~l the 

IL\I~U~II II\ILLI CLIILII~\L

3.4.2.2.a. V~rco\ity Measurcrnent 

O\twdld vl\colneter 150 wds uscd to ~CILIIIIII~L I ~ L \ I\L~I\II! 01 LLIIUIO~L 

I

4. Cataldsc 

I strrllLited wrth srriiridr pro~edurc\ mentroned In wctron 2 7 ? 0 

3.4. Amylase (Mahadevan and Srrdliar. 1996) 

Reagcnts 

I. Sodlunl dcetdte - dcetlL acrd buffer ( I M) pl l 6 

Metl~od 

11. 1% soluble starch solution: I g of starch WAS d~ssol\ cd 111 100 1111 01 

d~jt~lled hatel 

III. Urn~trosal~cylatc reagent: I g of 3.5 1)NS. 3) g ol \oiI~tl~n ~01~1\\111111 

tarialdtc dlid I h g of s~druli~ hydroxide N8elc dr~sol\cii 11180 1111 01 d1\111lcd 

water and made up to 100 ml 

5 1111 01 the enzyme source was plpetted Into a 100 1111 flil\l\ I ( I [Ill\ I0 1111 ol I 

M dcclale butter pll 6 and 2 ml of 1 percent soluble stdr~li \~ILIIIOI~ \rcIL 'lddcd I lie 

redctlon riil\ture W ~ S Incubated at room temperature for 21 li lllc 1cdit1011 W ~ F 

term~nd~ed b! the add~tron ol 1 ml of DNS reagent to I nil of tli~ Isdctlon rnr\~u~c I hr 

dliiount ol reduclng sugars liberated was estlniated ds In the L~\L 01 ?\(I I'M(I 1 lie 

cn/\riic actnlt) was expreswd as specrfic actlvltg uli~t\ Oric LIIIII lily 0 1 ~c~l~~irrig 

5ug.11\ I~heidtcd I g 01 I~eili wt of the tissue 

3.1. In\ ertrsc (h'tahddcvdn dnd Srldhdr, 1996) 

Ilcdgent6 

Mctliod 

1. Aqueous sucrose solut~on 2.5%: 2 5 g of suLrose \ha, d~\\olvcd 111 100 nil 

ol dlst~lled uater 

11. Sodturn dcrtate - dcetlc acrd buffer (I M) pH 5 

111. Urnitrosalrcylaie reagent: 1 g of ?,5 DNS 30 g ot ~odii~rii potd$\~u~ii 

tdrtdldte and 1 6 g of sodrum h!drox~de Rere dissoccd 111 110 rnl ol d~\tllled 

bdter arid made up to 100 nil 

i 1111 01 thc enzyme extract was pipctted Into a 100 n11 t1d4 10 1841 ol dLetatc 

bull~i ( I \I) pli 5 and 5 ml of 2 5% sucrose solution were addcd I lic IC~L~IOI~ 

1111\llrre 

wd\ u~ctlbated dl loom temperature tor 24 1 Thr rcdctroli \\&I\ Icrn~rririlcd h! the 

addruon ot I ml ol DNS reagent to lnll of the reactron mr\ture 

I hc mount of 

reducrng sugars Itberated was eshmated as In the eve of exo PMG The enzynle actlvlty

wa\ cxp~cssed as spcc~ii~ actrv~ty units One unrl = Ing ol'reduc~ny sitgals II~K.THIL~ / g 

of Iircsh'wt ol'thc tlssuc 

3.6. S1)S - I'A(;E nnrlysis of crtraecllulnr proteins (I..tcni~nl~. 1970. M;~lradcv;l~l and 

Sr~dhar. 1996) 

I'rupwntion of samplc 

C~~lture filtrates of cont~ol and treatment were liltcred tlirougli What~iiatn No. I 

liltc~ papel using Buchner funnel, w~th suctlon. Two volumes ol chilled acctonc was 

added to thc culture filtrate kept 111 a bcake~ placed on ~cc hatli Incuhalcd lor 1 11 and 

ccnt~~filgcdl 10.000 g fo~ 30 mln at 4°C The supernatalit Mas dlscaldcd and the 

Icllldlnlng dcctone was evaporated I he pellet was d~\solved 111 mlnlmunl qilantlty 01 

III\ 

HC'I butte1 (pll 7) l'hc proteln content was estlnrated \\~th s~~ii~la~ p~occdurc 

nicnr~oncd 111 2 7 2 .i and adjusted ro around 180 pg In cach samplc 

Ilcrgcnts 

I. .4crylamidc stock solution (30%): 30 g 01 acrylam~de and 0 8 g ol' b ~r 

ac~ylam~de were dissolved 111 100 nil of d~srllled nater and filtered through 

V.'hatmati No I 

filter paper liir filtrate was \torcd 111 '3 h1ow11 hor{lc a1 4" (' 

2. Itcsolvin:: gel buffer: 18 17 f of Tr~s (1 5 M) was d~ssolied 111 100 nil of 

warel and the pH was adlusred to 8 8 w~th con^ llCl 

lilte~ed and stored at 4 ' ~ 

I'hc soli~tlon \+as 

3. Stacking gel huffer: 12 11 g of I'rts (I Mi waq d~\\ol\cd 111 10(1 1111 ol' 

d~st~llsd \vatrr and tlic pll \\,I\ ddji~stcd to 0 X U I ~ I I L~~IIL 1 [('I I lic ~OIUIIOII 

\rdi liltcied and srolcd at 411( 

4. SI)S Solution (10'Y~): 10 g 01 SLIS \\as dr\\olvcd 111 100 ml ol d~s~~lltd 

\\ale! filtered and storcd at ~oolu temperatulc 

5. APS (10%) - Initiator: 10 f of al~imon~urn persulphate way d~ssolved 111 10 

1111 ofdlsulled water I he solut~on was p~epa~cd lic\li 

6. I

8. Coumassie staining solution: 0.25 g ofCH13 11-250 was Jissol\ed in 9Oml of 

methanol : water (1 : 1) and 10 ml of glacial acetic acid and then tiliered 

through Whatmanil No. l filter paper. 

9. 1)estaining solution: YO rnl of methanol : water ( I : I ) and 10 1111 of glncidl 

3.6.1. (icl casting 

acetic acid were mixed. 

'I'he electrophoresis glass plates. spacers and comb were cleaned with acetone lo 

remove an) grease. The spacers werc placed between the two pla~es. 'l'he plates wcrc 

fixed perfectly on to the gel casting apparatus. The resolving gel (running gel) was 

pourcd using a Pasteur pipette between the glass plates ensuring no leakage 

acrylamidc solution \\.as overlaid with ethanol. This overlaying prevents oxygcn Crom 

difi'using into the gel and inhibits polymerization. After the polymerization was 

completed, tlic overla!, uas poured olT and tlie Lop was washed >cvcral times with 

deionized water to reniove acy unpolymerized acrylamide. Care was takcn to drain as 

niuch fluid as possible from the top of tlic gel and then. tlie remaining water was 

~relilo\ cd \\it11 the edge of paper towel. Over tlic polymerized resol! Ing gel. the slilcklng 

gcl \\CIS 

applied, lhc ell leniplatc (coluh) was imrnediatel! piaccd on tiir gel to form 

\\ells, (cure has ~aheli to avoid an! trapping of air bubbles). Alicr ~lic luil!~llcri;ljttion 

was complete. tlie Teflon Comb %as removed carefully and the wells were illllnediately 

\\ashcd \\ itli dcioiiired waler to relno\ c the unpolymerized acl.ylarnidi.. 

'l'he 

I 

lie composit~oll ol'thc resol! in: gcI and tlie stacking gcl arc .I\ gi~c11 below: 

. ..~ -- -- 

~ Solutions - 1 I

1.6.2. 'I he Electrophoretic Run 

I'he prolein sample wds [hen ddded on to [lie wells 

I lie resctvolt bullet wd\ 

filled In both the electrode chambers and the electrodes were ~onnectcd to the powel 

pa~k Ihe condlttons were set lnttidlly dt 90 V and 25 mA unt~l the dye Iiont red~hed 

the resolving gel and later at 100 V tlll the dye front reached the end After runntng, the 

gel wa5 removed, waqhed In dtsttlled water and was stalned w~di thc Coomdssie blue 

iolution 

3.6.3. Stalnlng with coomastve blue 

fhe gel was tmtnersed In at least 100 nil of statnlng solutton dnd kept ovetn~ghl 

dt roo111 temperature The gel was then destained by soaklng ~t In the dc\tdinln$ rolu[ion 

3 4 tlmes 

3 7 DNA gel electrophores~s 

I'reparat~on of sample 

25 mg of lyophlltzed ~ iiy~~i~al po\~dcr of ( CCI~FICI 

\vd\ ~u~p~tidcd 111 500 pI of 

the extraction buffer, (I00 mM Trts - HCI pll 8 , I00 mM ED lA , 250 mM NaC12) In a 

I 

3 ml mtcrocentr~fuge tube To thts, 50 PI 

oi 105/0 SDS was added and the nilxture wds 

iniuhdted tot 30 niln at 65°C Then 100 111 of 10 % potasslum acetdte solutlon (3 M 

potdss~uni and 5 M acetate , pH 4 8) was added dtld the tube \vat kept on tct tor 15 tntn 

I lic ni!\lut~ nds ientrituged at 13 000 ~pni lot I? mtn and llie '~upi~ndlrl~~t ~otitdi~iiiig 

gillomlc DN.4 wds transferled In lo 

tilne w~th clilo~ofoini iso-dln!l 

fterll tube Th~supetndtant udb c\LtCtcted onc 

al~ohol (24 li mt\ture Alk~ ceiilttl~tgdltot~ 01 tlii 

~mul\toii to1 7 mtn the uppet aqueouv pli'i,~ ~ ~tli the DNA \\d\ t~dn\lotrncd lo dnothcr 

tuhc containlug 50 PI ot [so-propanol I hc iontcni\ &ere ~ I I \ L g~ntl) ~ dtld th~ lube Wdb 

plt~icd on ILL lo1 DNA plecipttdtton IINA \\.I\ p~ll~ied at 13 000 lpni lot 111111 dnd tlic 

pellet was alr dried and dissolved In 50 111 of TE (Irts - EDTA) buffet Purity and the 

concentlation ul DNA Mas estitnated by using llic ~pectrophotomc~ct 

37.1. Quantlficatlon of DNA and estlmatlon ot purity 

'5 111 of the Dh4 sdniple wds dllured to i ml and the oplical dcliiit! wd\ 

llleds~lled ai 760 11111 and at 280 nni The residue ~ L I I I ~ of \ the sdmple nd5 cdli~i~ated by 

tllc for~nula

I he amount of DNA was measured by ustng a standard valuc 

I (4 D ?,,I, corresponds to 50 pg 1 ml of DNA 

3.7.2. Restriction digestion of geni~rnic DNA (Sambrc~)k ci trl . 19x0) 

1. rhe (ienoniic DNA ~solated con^ control dnd treated niyccl~al nisi wele 

digested us~iig EcoR1, Hlnd Ill. Sal 1. Pst I. and Not I 111 d~fTc~cnt comh~natlon 

1 lie rwcrlon mixture for the digestion ofgeno~n~c DNA I20 PI] contained 

2. Genorn~c DNA 5 pl 

3. I0 X l3uKer 2 111 

1. kcoR1 I 

5. Iilnd 111 1 PI 

6. D~st~lled water 

Il ul 

7. I he reactlon mlxture was gently nilxed and incubated at 37'C lor 90 mln 

I lien slniilar reactions were done w~th different comb~nat~on\ or iestrlcuon 

digcst~oii such as EcoRl and Not 1. Hind Ill and Not I Not 1 dnc! Sat 1. EcoIZl 

'tnd Sal 1 respect~vely w~th the concentrations as glven by the guppller Illen the 

d~gestcd DNA was run on 0 7% agalose gel to check the d~ge

15 mln I o th~tloltc~~ 

'I~~IO\L SOX I Al W ~ d(1ilcd F IO dtt'l~li d lin,tl ~o~i~c~i~~dt~on 

of 

1X and nyxed well I he dgdrOSe solution was po~t~cd on to 4 t~dy li\cd wlth a colnb 

(clearance bet\+een ~onib and tcmplatc ud\ dboul 1 m~n) dnd \\,I\ dllo\rcd to \el lo1 30 

Inin I Iic comb WCI~ 

then removed and the gel wa\ pl't~ed on ,I ho~i/onl,tl gcl tdiih lillcd 

wlth suflic~enl IX I Al- bufler to subme~gc the agaro\c gel DNA .;ample\ ronrdtnlng 

IX loading dye Mcre loaded Into the slot\ of the gel I-lectrophores~c \\d\ cdltled out at 

50V lo^ lcqu~ted tinlc 

LI~II~ILIII~ 

IINA hmd\ ~esolbrd on the dgd~(~w gel\ Melt \,~\ualt/cd by ~lta1111tlg tllc ~ cI 111 

htonltde I he stdllllng solutton \\as ptepa~ed by addlnp d 10 n~yl~iil sto~h ol 

~~li~d~u~ri h1on11d~ to d~\l~llcd wale! to d l111al LonLcllltdtlon ol 0 7 ug ml IIlc gel uas 

\ubnielged In thc 5tdln lor 30 - 45 min and then dcsralned ~n d~sttlled ualer lor 15 Inln 

I hc sta~ncd gel ad\ observed under short \\dvelength (300 nni) LV t~d~l\tllun~tnato~ 

4. IN VIVO STUDIES (POTTED EXPERIMENTS) 

I'ottcd cipcrllnents \rere conduct2d In the I'ond~cherr) I'mverstt~ c'inipuc I',llur 

I \d~let$11 C~II[I seeds wrle obtalned ltotn Palul \'egetable lleaed~cli Stdtlon I',ilur 

\'ldble 'IL'C~S 01 uli~lorni uzc color and \\sight were ~electcd lo1 rn rrio studlc\ Sccds 

\\ctc rCll\ed In dpproplldte field cond~tlo~l 111 earthenuarc pots I Ilr pot5 \$ere filled 141th 

7 1 1 t,itlo 01 cdnd led \otl and farni!arti manure On 4.5 "' da\ 2 to 4 \ecdl~ng\ of 

LIIIIIOII~I \i/c wet' ltdllrpldlllcd 111 ea~ll pc11 ol 30 c1i1 ' stzc ( LII~I\~I~IOII PIC+L~ILC\ rind 

,ippl~cn~~t~ll 111 Ictttll/e~s \\rlc done d\ ~~~on~n,ctldcd h! f',lltii \'cg~tahl~ II~C~I~~IJ~IOI~ 011011~ 

\LI 01 1111ccled pldnt+ (IIC~II~~ pldnt\) OIC \ct 

of 1ntectc.d plants welt. left '1, 

\u~h I or ~ot~ttol, t~cdted and tntecrcd pldntc pot\ uele

4.2.4. Pknolr 

Eslimated with iimilar procedure iu: mentioned In sectlor1 2 7.2 

4.2.5. Amino acids 

lis~imaled with similar procedure as nie~~tioned 111 scctlon 2 7 ? 

6.4.2.6. Estimation of nucleic acids and protein 

listimated with similar procedure as niclitioned In xcllon 2 7 2 5 

4.2.7. Aminonitrogen (Moore and Stem. 1948) 

I\ .1~ld1112 0 1 2 1 

111 I I tr I 1111 tlic ahovc extract I ~ nl of nlnhydr~n rcdgeni \\.I\ aci(1c.d I 111'11. 11 \\,I\ 

lic.r~~~I lo1 3 1 111111111 a bo~l~ng \\atel hath and coolcd 5 ~nl ol Ll~,:~llcd \\,i[cr \\A .~ddc~l 

and [lit abw~hdnce was measured at 475 nm In a spectropho1t111li.1t.1 .Igalnbt .i iii1s1'111 

blank St.iii~l.~~~I curve \\as prepared \\ II~ 

I\no\rn quantltles (11 :I~I~.II~IIL.ILICI 

4.2.8. Ascorbic acid (Sadas~~ani and \lan~cla~n. 1091 ) 

Itcagent$ 

Mcfltod 

i. O\nlir arid -ioh: 4 g ol o\al~c ac~d wa.\ d~haol\cd 111 lot1 1111 01 dlsl~llcd 

\\ JlCl 

ii. l)!c solution: 42 1i1g ol'snd~u~i~ b~carho~ia[c \4,,\ ~I\~oI\L~LI 

111 \11iaIl\~I~IIIIL, 

ol d1stil1t.d \\ater lo th~s 52 mg of 2.0 ci~cllloro ~II~cI~oI ~~i~iophcnol 

~Jdcd and thc. volunlc ua\ ~nadc up to 200 1111 \\lth J~,i~llcii \\die1 

0 5 g ol' the wmple was extracted ~ lth 4% oxal~c ac~d .~nd 11li1d' upto IOU 1111 111 

a volunietr~c Ilash. From this. 5 ml of the solutiorl Has pipetted 1nO ;I 100 1111 COIIICJI 

3 7

flash TO this 10 ml 4% oxal~c dc~d wds ddded dnd (Itrated agalnc,t the dyc \c14(111cln I Ire 

end pornt 1s tht dppearance of blue color Amount of ascnrh~~ dcld plcwnc 111 llic g1tc.11 

xcm~ple traa ~dl~uldted by the lollow~ng formuld 

0 5 my v> I00 nil 

* - * * 100 

Vl ml 5 ml wt of (he sample 

VI nil = \,olu~iic of the dye consumed by standard ascorb~c ac~d ,elution 

V, 1111 Volunie of thc dle consumed hy the ascorb~c ac~d present In the sdnipl~ 

1.2.9. Capsalc~n l?adasl\dm and Mdnlckdnr 1991 ) 

1teu:cntr 

\ltthod 

\\,I\ 

I 0.4% rodlum hbdroxlde: 0 4 g of sodium hydroxide uas dissol\cd 111 100 

nil 01 dlst~llcd \rater 

11. 3% phosphomolyhd~c ac~d: 7 g of phosphomolbd~c d ~ HA\ ~ d d~\\(d\cd 111 

100 1111 ord~stllled water 

111. Dn acetone: 25 g of anhydrous sodlum sulphate was added to 500 rill of 

dcetolr~ one da\ bclore use 

0 5 g 01 dl\ ch~ll~ powder \rd\ tdlrn In d screw cap tube '0 nil ol dl\ '~ccroli~ 

.~ddcd to 11 .lnd pldicd m d niechdli~~dl shdh lor 3 h and centlilr~ged dl 10 000 1pnl 

101 10 umn I lnl of clsd~ \upern ltant wac p~petcd In to a test tuhc and c\ ~ptn~lc~l to 

I I I I r e 

h i Ihe rcs~duc \\'I\ d~isol\ed In i 1111 ol 04",~ \od~~inl 

Ii\d~o\~,i~ so1~1101r 1 ci tht\ > 1111 or j0/~, plit~~pho~ii~~lyhd~c acld \\'I\ .idtled ,111d\I~.I~LII 

,~nd,~IIin\cd 

10 \~dlid lor I 11 

1p1~i IOI 

rhc resull~ng rolu11011 \\as lillercd dud cc11trilugcd ,I! '000 

10-15 111111 TIic CIC~I hlue colored \olullon \rd\ dl~ectl, t~d~i\fe~~td I!) 'I L~I\LIIL 

.rnd lhc .rhso~hatr~c \4d\ read at 650 nm 111 a \peclrophotonieter ngdln\t J ledgenl hl'lnh 

\t,~nd.~~,i grdpli \\A\ plotlcd \\~tIi hnown LoliccnllLillons of cap\dILIIi 

4.2 10. ( cllulosr (S.~d,i\t\~~~ii and Man~cl~rln 1001 ) 

Ilragrnt\ 

I Acet~clN~tr~c reagent: I50 nil ol 8Pi0 acetlc dc~d uas m ~wd \\~th I? lnl 01 

coli~~tll~dled IL 

dud (conL I-INO:)

Method 

ii. Anthronc reagent: '00 111g ofd~itt~r~ne wd\ d~ss~lvcd 111 100 1111 01 COI~C 

tizS04. Freshly prep4 andch~lled for 2 h before u\e 

3 ml of awttcinllr~c ledgent was addcd lo I g ol the sd~nplc 111 '4 IC\I tuhc dnd 

n~lxd ucll In a vOnek mlxture and placed In a wale1 hdth dt 10OU( lu~ 30 mi11 dnd tk11 

cctrtr~lugcd at 3000 rpm for 15-20 mln I he supernatant Has d~\~d~dcd I he rc\iduc wd\ 

washcd w~th d~st~lled water 

lo th~s 10 rill ol 67% \ulphu~lc d ~ wd, ~ d dddcd and 

.~IIoned 10 stand for I h I ml ofthc above solut~on wd\ d~luted to lo() n ~l w1tl1 d~\t~llcd 

watel 1 o ow ml of the d~luted solut~on 10 ml oianthronc reapeni \\d\ added and 1111\cd 

well 1111s was heated In a bo~l~ng water bath lbr 10 min and cooled under running tap 

wdter and the absorbance was read at 630 nnl In d \pectrophotonlctc~ 4nth1onc rcdgent 

along \\IIII dist~lled water was used as blank Standdrd graph \rd\ plotted s~tli Lnciwn 

Loncenlrdtlons of cellulobe 

1.3. En11 mc Assays 

I'rcparation of acetone powder 

Ilragcnts 

Mrtl~otl 

I. Acetone 

11. I)I~II~>I cthri 

111. I'hosphdtr bullcr. pli 7 

1 he t~ssues sere weighed and cut ~nto p~eccs of 1-2 LII~ cdcll dnd then 

~I,III\~~II~~ to a hlendor Ch~llsd acetone (-10 "\\L~CI \\'I\ \p~cdd 011 

dr~cd tor r~h~~ul 1 11 l lhi. lpt~\\il~i \\a\ \IIOIC~ 111

Enzymc prcprralion 

(I I 2 or Inc powder was wclghed and ground in 5 1111 01 ~>ho\pI~a~c hullel (0 I 

M) pl l 7 ;11 -1°C' lib1 10 lo 15 nltn rn a homogenizer l'hc extract was cc~~tr~lugcd a1 I(KW) 

g C~I 20 111111 at 4°C' and the supernalan1 was used as tlic cn/ynic wulcc 

4.3.1. b:nr! mc asrays 

4.3.1.1. I

I Jcca\c. polyplicnol oxidasc. pcroxldasc and catalasc act11 ~tlcs wclc c\t~m,~lc

'lkc~n~posilion OF drc resolving gel and the slacking gel arc as glven klow. 

)-.-Solutionl-I- 

- -- -- 

Resalvmg Gel (I2 "/a)--]- 

Actylatnide Stock 6.0 m l - 7 

- 

1.5 M Iris. pll 8.8 -1----.------3:8 lnl -- -.I 

-- -. - - -. . - --. - - . - ---A. 

10% SDS 7.- 

o.ir,l 

..-. -- .. Deioni/cd itater 

--I-- 

-- - 

I 

491111 1 

(;el cltstlng. electrophoretic run and stalnic~g 01' [lie gel \\elr. done as ~nsnliond in 

scc~l~lll : 6 

5. ACTIVE: PKlNClPLES ISOLATION 

5.1. F.\-traction of the active principles 01' A.snlivutr~ aqueou\ hulh cxtract 

(I Iarhorlic. 1984) 

('rude .A crrtn~rim extract Has prepared h! 

solvent c\ti~ct~o~i procedure I'lle 

P~IIICII~IC 111\oI\cd I\ that the sol\cnr takes thC soluhle comp~~u~ld., ~prcst~it 111 the plant 

dclltl?.iillg ~1x111 rlis~r solubility facto~ I'hc sol\snt used lo1 111s c\uactmn was 05"" 

rcct~li~il \iv~t ;111il dcli!dratcd alcohol 1111\1urc (I I ) 

ICII 101 

\\,I 

I is ol'l1gli1l! cn~shcd fresh garlic hulhs \&as soakc~i 111 2 I 01 tile aolvcn[ a~ici 

t\\o 111o11111\ nith agitat~on at ~xirliculai tlitcr\al.r. Alt?~ i\ro ~n(~litli$. thc chtract 

~il~srcJ tlirough double la!crcd cl~cc.;~, clo~li slid CL\II~L,IIII,II~L~ 10 IOU 1111 b! 

3.2. \ntil'ungaI nsss! or the crudr pli~nt cxtract 

111 i~tro tcbtc were carried out to conti1.111 tlw ant~I'l~~ig,il actl\~t! ol'tl~c cruds 

5.2.1. ('anitlid grrmination studies 

('ollldial gcrniination st~~dics \\us car1.1cd out III ca\ 11) 41ric.\ h! ~llcuhallng 111 .I 

Iiiolet cli;~mhcr at room teli~peratllre 100 p1 of thc crude plnlil c\ll.acl was ddded to tI?c 

c;~\ IIICS 

ultl illlol\ied to cvaporatc I n rll~s 100 511 oi of cci~i~di,ii \LIS~)~I~~ICIII conii111111lg 

400(~-600() con~d~a I ml was added. Lor control cltraction ~~I!$III \\:I\ addsd ~nslsitd oi 

12

thc crude plant extract dnd allowed to c\dp~~dtc 1 (I thi~ I00 111 01 

LIIL CI>I~I~I,,~ 

suspension was added Contdtal gernundtloti \tudtc\ a~id per cent inh~htllon ovc~ control 

were carr~ed out as descr~bed in sectton 2 2 1 

5.2.2. Radial mycelial growth studies (Goh~l dnd Vdl,~ 1996) 

Agar plates of PDA wcre uniformly \prwd pl'itcd w~tli ( ~ ( I ~ ~ con~d~rl 

I L I Well, 

wcre made In the plate\ by u.;ing 9 mm hnier 100 111 of the c~ude pl,~ri~ c\tr,~ct ~.i\ 

added to the wells The plate5 were ~ncuhatcd at 1ooii1 tcnlperatuic 1 or ~o~itrol I00 111 

of the solvent was added The plate5 were sc~eened for ant~funfal actlblty dftet 

6 I:! 18.24 and 48 h of tncubation 

5.2. I'urification of the bioactive compounds of A.trrlrvum crude plant eltract I]\ 

sil~ra gel column chromatography (brthlhdi alu 2002) 

5.3.1. Packing of the column 

'The column was packed by slurrv method In 11i1\ 

method ~IIIL~ gel .\lur~) ~ d \ 

prepared 111 chloroform and poured ~ns~dc the ~olumn and allo\\ed to XI wit11 

chlorolorm on the top of the mluntn 

5.2.2. I.uading and elutlnn of tt~r ant~fungal compounds It.on~ thc crude plant 

erlnlcf 

I hs rude plant extract to be stparaled \\a\ lo'ided on IIIC top ot [lie ~olumn 

dic11 Ihrec ml of thc sample contatntns round 200 ni: 

ol the co~npound\ v,a\ 

lo,idcJ on the column and allowed to settle oil rlie ~ut~ning \ol\cti~ ~hlotolortii Al~ct 

the e\tmct entered the column gradlent eluticln ot the cornp~inti~ \\rrri petlormcd I hi. 

\ol\cnt\ (25 ml) used for elut~on weir 100% ~hlorolaiii~ 9 I clii~~tolorrn nisthdnol 8 7 

chlorotorni 

methanol 7 1 chlorolo~m nltthdnol h 4 ~hl~~~ilo~ili ~~ieth.~ii~l 5 1 

clllotolorni methanol. 4 6 clilorolo~nl nlttlianol ; 7 chlori~torill ~ilcthatiol 2 8 

chlorolorm methanol. 1 9 chlorolo~ri~ rncrll,~nol and 100% u~li~ill~~iiol I I~ILIIOI~\ 

W~IC 

cc~llc~tcd and dssa!ed lor antilungdl dcli\it\ hot11 011 ~onid13l ,'_LII~III~~~IOI~ dnd iddi~l 

n~\ccli.~l growth as descrlhed in Yccrion 5 l I,.~nd 5 2 : 

01 the dhsdted lrd~t1011\ onI\ tll~rd (\.~rilpl~ I \ %111d lo~~~tli 

6 

(\,II~I~~L 

eluted u ~th 20% methanol w~th chlo~olorn~ \Ilo\~ed ttcihle an~~tungdl .ILII\ 

7) Ird~Iton\ 

it\ al 200 ~il 

conccntratlon on the conid~al germinalton ol the ( cup\rcr hut not o11 tlic IJJI~II 111\cdl1dl

5.3.3. Solub~l~ty tot& for active I'rncticmh (bnmplc 1 and 2) 

llle 20% methdnol exlrdci of the sample I and ? dftc~ C\~IP(II.IIIOII (11 lIIC 

uilvcnt\ ucre tested to lind out tlie~r wluh~l~ty 111 d~llc~c~it 'IOIVC~I\ I Iic VII\LI~I\ tl\rit 

were d~\t~llcd wdter 0 1 N \od~uni hydrox~de dehydrdted dlcoliol 1iictl1d1101 .ILLIIL 

ilcld. dlmethyl sulphox~de. d~chloroniethane, ethyl acetate !so-hutyl ,IILoI~oI 11 

propdnol. 150-propdnol. carbon tctrachlor~de, acetone dlnmonld solu~~oti d~clli!l LII~LI 

bcn/cnc Iicxane and xylcne Among the solvents tested saniple 1 dnd !. \rclL lotlnd I~I 

hc wluhle ~onipletely only In d~sr~lled water and O IN sodium Iivdrox~dc 

5.1 Spcctrul, chromntugruph~cal and phytochem~cal nnalj\l\ of \aniplr I ~ ntl ? 

\d~iiplc I dnd 2 w~th promising dnt~fungdl ~LIIVI~) 

were dgdln \uhlect~d 10 (I\ 

I. I IIt 'H-NMR "c-NMI< spectral and RP-HI'L< analysls I he te\t\ 101 \ugdl\ 

p10tc111\ dnd dmlno ac~d\ were alao carr~cd out 

5.4.1. Mol~sh a - naphthol test for sugars (Wlnton and Wlnton 1999) 

5letl1ud 

I. 10% a - naphthol solut~on: 10 g ol a - naphthol \+as d~\sol\ed 111 100 ml 01 

dehydrdted alcohol 

11. ( ollc l I7504 

I Ii~ic n~l ot the \,i~iiplc I and 2 were nu\cd \cpdra~cl\\\it11 1 (0 1 ' d~op\ 01 

5.4.2 1 c\t fur protcln\ 

I\ ~ies~r~hcd 111 \cction 2 7 2 5 3 O ; 1111 of saliiplc 1 and I \\elc tr\cd lo1 th~, 

5.4.3. 'Test for umlno acldv 

L'\11Cl 

4\ descr~hed 111 section 2 7 2 ? 100 p1 of ~aniplc 1 and 2 \\elc uwd lo^ th~\ 

1111c11t 

6. STATISTICAL ANALYSIS 

I hc ddta oh~alncd wele buhlectcd to palred student t tc\t ,111,11\\1\

ANNEXURE 

1. Preparation of Growth Medium 

1. I. I'olalo-l)cxtrosc-agar medium (l'l)A) 

'1.0 

prepare I 1. ol'the I'DA medium. 250 g of peclcd potalo was made into 

thin chips and boiled in 500 nil of distilled water. 'l'o the extracl. 20 g ol'glucosc was 

added. 15-18 g of agar (solidifying agent) wa~ 

melted in 500 ml of distilled watcr and 

then mixed with potato-glucose solution and the iolume was ~iiadc up to I I. and thc pl l 

was adjustcd lo 6.8 to 7.0 with a pli nlcler using IN IiCI and IN NaOli. 

1.2. Czaprk's medium 

I:ol. I I. of the medium the Ibllowing chemicals were d~ssol~ed in 1000 ml of 

distillcd uater and [he pH was adjusted to 6.8 to 7.0. 

2. Stains 

2.1. ('otron hlur lactophenol 

('atton blue (1OU/u aqueous soluriol~) : 5 ~nl 

I'licnol c~ystals 

I.;~ct~c ncid 

70 g 

10 g 

(il! ccrinc 40 $ 

3. 1)isinfectants 

3.1. hlcrcuric chloride solution 

Slocli solulitrn 

hlcrcur~c chloride 

: 20 g 

('\)nc. HCI : I(K) 1111 

i ml oftlie stock solution wasdiluted to I I. with distillcd \\atel

4. Cleaning solution for glassware 

Dilute roln. Conc. Soln. 

Potassium chrolnate : 60 g 60 g 

Water : II 300 rnl 

Conc. H2S04 : 60ml 460 nil 

I)icliromate was dissolved in warm water. cooled and acid was added slowl! 

All the above mentioned reagents werc preparcd by [lie nicthods descrihcd h) 

h4ali;ldcvan and Sridhar ( 1996).

OBSERVATIONS 

The rcsults ofthe present work arc prchcnted uudct thc li)llr~wt~~p Iicadlng.\ 

I. GENERAL STUDIES 

I 

Screening of' aqueous plant extracts on thc conld~al ~C'I.~~III~C~IIOII 

01 ( it~j~\ii I 

2 Screening of aqueous plant extracts those produced dbovc 50 %I ~nhtir~[tot~ 

on the conidial germinalion of ('cu/j.trc~ at 4X h 

growth of C capsici 

(111 thc ~'~dlel ln!ccll,tl 

7 Selection of potential aqueous plant extract (thc tesl plan1 c\tract) aylr\l 

C' t.Upslt.1 

4 I)elcrrn~nal~on of MIC ol'the lest plan1 extracl 

5 Ilctcrm~nauon of Ll>~o 

6 L)erenn~nat~on of heat tolerance 

7 I)c~rrrn~nduon of' shelf l~le 

11. PHYTOTOXICITY STUDIES 

X 

Morphological stud~es 

A 

I1 

Seed germination 

Shoo~ length and root Icngth 

C 1 resh weight of the shoot and loot 

1) [)I? weight of thc shoot and loor 

0 I11~l1cn11i'al studies 

I 

I 

I'll~~tosynthctic pign~ents 

thohyd hydrates 

(i I'llcnols 

t4 

I 

huslc~c acids and prntcln 

Ammo acids 

J. O\~dat~ve enzymes 

111.1.Y L'ITRO STUDlES 

10 H~ochem~cal changes in the pathogen 

A Cell permeability 

17

t3. Nuclcic acids. protein and total lipid synthesis 

C. Enzymatic studies 

IV. 1N VIVO STUDIES 

1 I. 1'111 

a. I'cctinolytic 

b. ('ellulolytic 

c. Amylase 

d. Invcrtase 

e. Osidative enzymes 

I), lixtracellular protein profiling studies 

E. Restriction enzymes based genomic DNA profiling studies 

I ?. ('ell pcrmeabilit) 

13. H~ochen~ical changes 

A 

Photosynthetic plgments 

13 ('arhohydratcs 

C 

Phenols 

I) Kucle~c acids. proteln and amino ac~ds 

1. ('apsalcin. cc.llulosc. ascorh~c acid and ami~lo nit1.o~r11 

I 

I'roluic 

(i. I.n/y~natic >ttidics 

a 

I'ectinolytic 

b. ('cllulolytic 

c. ,An~ylasc 

d. In\.ertase 

c 

Oxtdative en/! Inch 

I{ I .saf protein profiling stud is^ 

1'. AC'.I.I\'E PHINCII'LE ISOLATION STC'III ES

I. Gcneri~l studies 

I. Serrming of aquaour planl catract$ on cenidial germination. 

Aqueous extracts ol 275 plant 9pp were tcstcd for thc~r cflect\ 011 the. L~UII~I~~ 

germmation of ('LUPAILI 

(Table 1) 

A. Plants with fungicidal ctrect 

Based on the~r cflccts the) were class~fied Into Iou~ group, 

These planl extracts were found to produce cent pcr cent ~nli~b~tion 

thc ionld~dl 

germlnatlon both at 24 h and 48 I1 (I-~g 1 B) 

U. I'lsnts with fungistatic effect 

Ihc per cent ~nh~bit~on produced by thn group of plants \\crc found to hc 

rcdu~cd at 48 h compared w~th 24 h 

C. Germtube abnormal germ~nation Inducing plants 

Thts group of plants induced stout branched and beaded dhllOr111d~ gslnundtlon 

ol thc con~dra (Fig 1 C) 

L). Plants with no inhibitoiy effect 

Ill15 group of plants were not round to produce ~nh~b~t~on 

the conldlal 

2. Screcnlng of aqueous plant extracts on rad~al mycelial growth 

I how aqueous extracts of 27 plants p~oduced dhove 5O0o ~nh~h~t~on 

thc 

~onldldl gernlrndtlon of ( LU~J~ILI here fi~rrhei screened to find out tlle~r eflc~l on the 

rddldl I~~\L~II.~I glowth ol C ~UIJ\ILI 

(Idblc I) Of thc 27 tested dquc.ou\ plat11 cxtrdct\ 

111111 ~111~1/~1 / I/( ll/~~/ll~ ~l!lt~/llOl Ill\ /ill1\lllllll IIlL'l /?I/\ tll?l/ /ll~~lh~'l fl//ll lllll/l\ 11 L'l L' 

/~JIII~~/ ro /)I otlrrtr 100. 26. 26. 26. 26. 100 dnd 26% ol lnh~blt~on respect~vcl! on ~dd~dl 

I~I\LC'II,II g~o\\tIi Ihe remalnlng 20 pldnt\ \\c~e not lou~ld to produ~c An\ IIII~I~I~IOII 

A ctvril~~n hulh and L rnermlr leal aqueous extracts were found to produce maxlmum 

~nh~hit~un (1004.0) both on the ~onldldl gc.llnrndtlon and rddldl 111\~c'lldl glowtll 01 

( ~~I/I+ICI I Ilc cent per cent ~nh~h~roc ctfc~t of I. 111crn11\ on the ~dd~al ~nyccl~.~l g~outll 

ol ( ~(I/HI~I \\as Sound ro bc ~nconslstent Hencc 1 ctr/~i,r~m dqueou, hulh citlnct (thc 

tc\t pl.1111 C\II;I~~) was selec[cd lor furthe1 111 IJIIO 

'111d 111 i 110 qtuii~c\

Fig. 1. Effect of aqueous plant extracts on the conidial germination of 

C. capsici 

A. Normnly germinated conidia ofC.capsici 

B. Inhibition of germination of conidia of C.capsici by aqucous extracts of 

Alliitm sativum and Lawsonia inermis 

C. Conidia treated with Mangifera indica and Anacardiunr occidentnle 

shoa4ng abnormal germination

Table 1. Effect of aqueous plant extracts on the conidial germination 

and radial mycelial growth of C.capsici. 

/ 

S.No. 

Plant name 

Parts 

used inhibition on 

the conidial 

m 

Group 1 Plants with fungici 

germination 

al effects 

%or 

inhibition on 

the radial 

growth on 8" 

day 

18. 

Helio~ropicum indicum L. 

Leaves 

100 

100 

0 

19. 

LowSonia imrmis L. 

Leaves 

100 

I00 

100 

20. 

Par~hsnium hysrerophorus L. 

Leaves 

100 

100 

0 

' 2 1. 

Psdalium murex L. 

Leaves 

100 

100 

0 

22. 

Plumeria alba L. 

Lerva 

100 

100 

0

Willd. Ex Klotzsch

orcuuro (Wight 8: Am.)

I 81 

. 182 

hplodsnra rqcuiala (ReU) 

Wlghr k Am 

Leprsan~hes retraphylla 

Leava 

Leaves 

0 

0 

0 

0 

0 

-: 

. 

. , , "fl 

I

Table 2. Determination of MIC of A.sativum aqueous bulb extract 

Control 

F 

Table 3. Determination of shelf life of A.sativum aqueous bulb extract 

Mean + S.D

w. Metmination of LDa vallre of A.salivum aqueous bulb 

extract against C.epsici 

0 0.1 0.3 0.5 0.7 0.9 

CONCENTRATION OF THE PLANT EXTRACT IN %

3. Determination of MlC 

The test plant extract diluted to 10, 5, 2 5, 1, 0 5, 0 1, 0 05 and 0 02% of 

concentmttons were fourid to produce 100, 100, 100, 100, 100, 100. 18 66 and 0% of 

lnh~bltlon respectlvely on conldlal germlnat~on at 24 h and 100. 100. 100. 100. 100. 

100, 13 66 and 0% of lnhlbltlon respect~vely on con~dial germlnatlon dt 48 h and 100 

81. 69. 58, 43 66, 32 33, 0 and 0% of ~nhlbltlon respectlvely on radldl mycellal growth 

of C' cupsrcr (Table 2) 

4. Determination of LDso 

The test plant extract diluted to 0 1, 0 3, 0 5, 0 7 and 0 9% of concentrations 

were used to find out the LDso of the test plant extract agalnst C'capsrcr 

percentage of lnhlblt~on produced were 25 21, 36 72, 42 12, 50 and 61 48 respectlvely 

The LD5" concentration of the test plant extract was found to be 0 7% (Fig 2) 

5. Determination of heat tolerance 

The test plant extract heated at 100uC for 10 mln was not found to lose 11s cent 

per cent lnhlb~tory effect both on conldlal germlnatlon and rad~al mycellal growth. 

The 

6. 1)eterminntron of shelf life 

The test plant extract tested at 7, 14, 21, 28. 35, 42 and 49 days intervals were 

found to produce 100, 100, 100, 100, 84, 32 and 0% of lnhlb~t~on respectlvely on 

conldlal gennlnatlon at 24 h and 100, 100, 100, 100, 61, 19 and 0% of lnhlbitlon 

respectlvely on con~dlal genlnation at 48 h and 100, 100, 100, 43. 15. 0 and 0% 

~nhlbltlon respectively on radial mycellal growth (Table 3) 

11. Phytotoxicity Studies 

The effect of the test plant extract on the seed germ~nat~on and 25 days old chllll 

seedlings arc glven below 

7. Morphological studies 

A. Seed gemins tion 

The test plant extract was found to lnhlblt seed gerrnlnatlon (91 30%) compared 

with that of control seeds (98 33%) (Table 4)

Table 4. Effect of A.sativum aqueous bulb extract on the seed 

germination, morphologicrl and biochemical changes in the chilli 

seedlings 

Parameters 

Seed gemiination 

Shoot len* f;- --I 

_Root len! th (in cm) 

Fresh we1 

(in g) 

Fresh weight of the Root 

(in g) 

Dry weight of the Shoot 

Contd 

SEED GERMINATION 

98.33 i 1.24 

MORPH0UX;ICAL CHANGES 

B..' 1". 

1 

".I, I 

?nlQ L I QC 

L".IO Z c.0, 

12.01 tlL7 a.ur 

0.10 

ght of the Shoot 1 1.17 + 

0.74 i0.10 

0.25 * 0.02 

A. sarivwn-- 

91.30 t 1.24.' 

24.77 i 1.44* 

15.60 t 0.61' 

2.12 t 0.04. 

1.12 *0.03** 

0.36 i 0.02**

It. Sh1n11 and root length 

I hc nrilxlmum shoot and root length wa\ lound In the test pla~lt extr'ict t~c,~t~d 

\c.cdl~ngs(24 77 d~id I5 GO cni I~L~CLII\CIY) co~iil~~i~cd w~th thdt 01' c01it101 \CC~IIII~\ 

(20 18 and 12 01 cm respct~vely) (l'ahlt. 4) 

( . Fresh wcight of the shoot and root 

I he maxlmum shoo1 dnd root \\SI;II~ 

wd\ found ~n 1r~~ilt.d \eedl~~lg\ (7 I :! and 

1 12 g respect~vely) colnpdred \81th tli~~t of cont~ol seedl1ng5 (1 17 and O 74 g 

I cspect~\ cly) ( l able 4) 

I). I)ry wc~ght of thc shoot and root 

I hc Ilir~XlIll~Ill \hoot dnd 1001 \\LI~~II 

Iound In trcdtcd jccdl~ng\ I0 

000 f ~cspecl~\cl!) colllpdrcd u~tii llldl ol ~o1111oI seedll~igs (025 ,~nd 00; 2 

~c\pcct~\c.l! ) ( I dble 4) 

X. Biochemical changes 

I-. I'hotosj nthetr pigments 

'111d 

1 hc mahlmum Chi 'a'. ('111 h lotdl Chi dnd cdrotena~d contents wcrc lound in 

11cdtcd secdllng\ (I 38. 0 48. I 91 dnd 0 78 nig ~syxct~vely) compared w~tll thow 01 

control secdllngs (1 18.0 78. 1 62 and 0 57 ntg respcct~uely) (Table 4) 

Is. Carboh\dratcs 

1 he mahrmum rcducmg sugal\ 1oi.11 \Li:al> lion-~uduc~~ig iugd15 dnd \t'i~ch 

Lolltents \\z~c found In tredtcd \ccd1111;\ (2; 15 40 75 1 h 62 dnd 56 4 l 111; 

IC\I~CCII\CI\) ~~lnpdrcd \\~tIi ~Iio\c ot ~o111101 beedlulg\ (1941 32 84. 12 76 d11d 44 I(() 

111~ rc\pcct~\cl!) ( I ahle 4) 

(8. I1henol\ 

I hc nld\lnluni 0 D phs~iols dlid io1.11 plic~iols \\ere found 111 trea~ed \ct.dh~lg\ 

(; 64 nnd 9 05 mg respectlvcl!) compa~cd iiltli those of control seedl~ngs (1 75 'ind 

5 10 nip ~c~puct~vel!) (7able 4) 

II. Nuclcic acids and protcln 

nit. motmum DNA. RN.4 dnd ~IOLCIII colilents ive~c found 111 trcd~cd \ecdl~~ig\ 

(0 17, 0 80 ,411d 0 51 nig respect~\cl)) ~o~iqi.i~td \\1t11 II~O\C 01 COIIIIOI \ccdl~~if\ (024 

0 58 and 0 !.: mg rcspect~iely) ( I dhlc 4)

Table 5. Effect of A.sativum aqueous plant extract on the cell 

permeability (rn~lcrn~) of ccapsici - in vitro 

Treatment 

Cell permeability 

Control 

A. sativum 

Mean iS.D 

* fl.05; **p

I. Amino acids 

'['he maximum annino acid5 contents ~4ct.e li)~i~id 111 treatcd seedlilnyh (0.36 my) 

compared with those of control seedlings (0.26 ~ng) ('I ahlc 4 J. 

.I. Oxidative enzymrs 

'l'hc maximum actlvitlss ol laccase. p)lyphcnol oxidase. pcro\idac illit1 cat~tlilsc 

were found in treated seedlings (0.04. O.lj. 0.003 and 11.01 ul~i~\ ~c\pccti\cl~j 

conipared with those ot'conirol seedlings (0.02. 0.10. 0 001 and X 70 u~iirs ~.cspecrivcl~) 

(I ahlc 4). 

Ill. In ~itro studies 

A. ('ell permeability 

'l'hc maximum cell pcrlncability \a> loul\d 111 t l \cs\ ~ plan1 c\tract trcaled 

myccl~al (treatment) ma1 (11' ('co,,~ici hotli a! 21 h (0.37 rn~~cm') and 48 1 (0.4; 

mS ~111') compared w~rh ~l~ar of control (0.30 id 0 36 ~ii~lcni' ~.cspt.crl\i.l! at 14 h and 

48 I1 I I I ahlc 5 ). 

H. Nucleic acids, prutein and totrl lipid 

flic rfl'ecl 

of the lest plant extract on tlit nucle~c acids. protcili and toral lipid 

contents of treated ('c,u/,sir~ on fourth and r~glith da! are given helo\\. 

Fourtb da! 

I'hc masin~u~n I?NA. KM. prorein and [oral lipid collrclnc.; \ii.lc lburid in 

control ~n!cc.lial mar (0.23. 0.86. 0 65 and 0.05 mg rtspectivel! ) col?lpari.ii \~itll ~liat 01' 

trcatcii ~nl!ct.llal mat (0.10. 0 XO. O.sX and 0.02 mg rt.spcctively) ('I,lbli. 01 

Eigi~tll dr! 

I'Iic ~niasimu~n I)N:\. 

IINA. protciii .11i~1 total lipid contcni\ 

~irund in 

coritrirl ~n!cchal mat (0.26, 091. 0.72 and 0 Oh mg rcspectivcl!) ct~rnpal.c~i \\ith \ha\ 111' 

II.~;II~~ ~in!ccllaI ma1 (0.27. O 71. 0.53 and 0.01 111; 

('. k:ntymatic studies 

;im!lasc. 

rc.spccr~\lely) (labit. 01 

Et'i~ct of the test plant extract on rlic acti~ities ol' pecri~it~l!ti~~. cclluloly~ic. 

~li\cllasc and "sidaiive cnlynlcs oi ('c.oln~c.~ ikas sliid~cd. I IIC rc

Fig 3 Actlv~ty of PME of C,'.cap.s~c~ 

- in vitro 

Control

F1g.4. Activity of endo-PMG of ( ' L,ap.s,a 

- ~n vrlro 

0 >O 60 90 I20 150 180 

Time interval (in mill) 

Fig.5 Activity of exo-PMG of C.capsici 

- In villa

0 3 0 60 90 120 150 180 

Time interval ( ~n min) 

Fig.7. Activity of eso-PTE of C.cap.vici 

- n7 lv/lro 

: Control 

1 W A.sa/r~*t~n~

0 30 60 00 170 1.50 1 SO 

Time inte~val (in min)

Flg 10 Actrvlt~es of Exo 1,4 B-glocal~ase dnd celloblase 

of (' ~ap~lct - m vilro 

Exo- 1,4 B glucanase 

Cellob~ase 

Fig 1 1 Act~v~tles of amylase and lnvertase of 

C capsla - m vrlro 

0181 

016- 

5 014- 

012- 

F Ol- 

C 

oos- 

006- 

1 OM- 

002- 

0 1 

1 

Amylase 

r 

lnvertase 

t

Fig. 12. Activities of laccase and perox~dase of 

(:.cap.srci- m vllro 

Laccase 

Peroxidase 

Fig 13 Activities of polyphenol oxidase and 

catalase of C'. capsic1 - 111 vltro 

Polyphenol oKidase 

Catalase

Fig. 14. Effect of A..valrvun~ aqueous bulb 

extract on tile PDI 

of C:.cap.s~cr 

Control 

F3 Infected

a. Peetfndyticcnzymer 

i Peetin methyl estcraae 

The h u m PME activity 

of treatment (5.13~~) (Fig. 3). 

ii. Polymethyl galrcturonare 

found In ~~~~trol(l 1.68%~) compared with that 

The maximum endo-PME and exo-PME activities wen found in control 

(56.84% and 0 .23~~ respectively) compared with that of treatment (8.87% and 0.02uu 

respectively) (Figs. 4 and 5). 

iii. Pectin trans eliminrse 

The maximum endo-PTE and exo-PTE activities were found in control (59.65% 

and 38.27~~~ respectively) compared with that of treatment (15.27% and 0.0SsAu 

respectively) (Figs. 6 and 7). 

b. Cellulolytic enzymes 

i. 1,4 p - endo-glucanase and exo endo P 1,4 - glucanase 

The maximum 1, 4 0-endo-glucanase and exo endo P 1,Cglucanase activities 

were found in control (57.43% and 0.27~~~ respectively) compared with that of 

treatment (13.39% and 0.02s~u respectively) (Figs. 8 and 9). 

ii. 1,4, p-exo glucannse and cellulobinse 

The maximum 1,4, P-exo-gluwulse and cellulobiase activities were found in 

control (0.38%" and 0.19 sAu respectively) compared yith that of treatment (0.09s~~ 

and 0.02sAu resespcctively) (Fig. 10). 

c. Amylase and invertase 

The maximum amylase and invertase activities were found in control ( 0.15~~~ 

and O.l&,,u respectively) compared with that of treatment (0.07~~~ and 0.08~~~ 

respcctively) (Fig. 1 1). 

d. Oxidative enzymes 

activitja 

The maximum activities of laccase, polyphenol oxidasc, pcroxidase and catalase 

found in treatment (0.005, 0.03, 0.004 and 0.14 units mpe~tivel~) 

corn@ with that of control (0.002,0.01,0.002 and 0.12 units respectively) (Figs. 12 

and 13).

Tabk 8. Effect of A.s&vum aqueous bulb extract on the biochemical 

changei of chilli fruits infected with C.capsici - in vivo

D. Edracelluar protein profiling studies 

Studies on the effects of the test plant cxtract on the extraccllulal. protein 

profiling suggested that there was marked difference in the extracellular protein proliles 

between control and treatment. 

E. Restriction enzymes based genomic DNA profiling studics 

Studies on the effect of the test plant extract on the gcnomic ONA proiilrng 

suggested that there was no marked difference in the restriction cclzyrnr: bascd geiloii~ic 

DNA profiles between control and treatment. 

IV. In vivo studies 

11. PDI 

The minimum PDI was found in treated fruits (2.5%) cornpal-td with tllose of 

infected fruits ( I 7 %) (Fig. 14). 

12.Cell permeability 

The maximum cell permeability was found in infected frsits horh at 24 11 arid 48 

h (0.47 and 0.54 ms/cm2 respect~vely) followed by treated (0.39 and 0.43 ~IS'C~I' 

respectively) and control fruits (0.36 and 0.40 m~/cm' respect~vely) (Tablc 7). 

13. Biochemical changes 

A. Photosynthetic pigments 

The ~naximum Chl 'a' Chl 'b', total Chl and calaterioid conlenls trcrc lburid in 

control fnrits (3.50. 2.22. 5.46, and 2.20 mg respectively) comparcd \\it11 thosc of 

treated (3.02. 2.03. 5.08 and 1.97 mg respectively) and infected li.uits ( 1.71. 0.70. 2.17 

and (1.84 mg respectively) (l'ahle 8). 

B. Carbohydrates 

The maximum reducing sugars. total sugars. non-reducing sugars and aarcli 

contents were found in control fruits (39.76. 53.78, 13.51 and 94.06 Ing respect~\el!.) 

cornpitred with those of treated (37.76. 51.17. 12.73 and 91.65 rug rr.specti\el!.) and 

infected fruits (27.81. 30.88, 2.9 1 and 5 I .3J nig respectively) (.['able 8) 

C. Phenols 

l'he maximum 0.D. Phenols and total phenols werc found in infected ti.uits 

(12.89 e& 31.56 mg respectively) followed by treated (7.78 and 18.49 mg ~espccti\el!') 

and control fruits (5.77 and 14.71 mg rcsptctively) ('l'ablc. 8). 

54

Fig. 15. Activity of PME of C.cap.vrcl 

- m vivo

Fig. 16. Act~vity of endo-PMG of (',capsrcr - 117 vrvo 

(4- Infected 

I 

0 30 60 90 120 150 180 

Time interval (in min) 

Fig.17. Activity of exo-PMG of C.capsici - /I? vivo

F;~. 1 8. Actlvlty of endo-PTE of C. capslci - in vivo 

0 30 60 90 120 150 180 


Fig. 19. Activity of exo-PTE of C. capsici - in vivo 

. A.sativum 

El Infected

Fig.20. Activity of endo-Cx of (.'.capsici - in vivo 

30 60 90 120 150 IRO 


Fig.21. Activity of exo-Cx of C.capsici - in vivo 

O Control 

A.satiwrm 

Infected

I-$22. 

Activities of exo 1,4 B-glucanase and cellobiase 

of (.'.cap.~ici - 111 vlvo 

/ El Infected 

Eso- 1.4 B glucanase 

Cellobiase 

Fig.23. Activities of arnylase and invertase of 

C.capsici - in Irvo 

I A. sativt~~?i 

0.35 1 I Infected 

Amylase 

lnvertase

i;'ig 24. Activities of laccase and peroxidase of 

t,'. cap.ricr - in vivo 

I- 

D Control 

m A. sa/r\~~mi 

H lnfected 

Peroxidase 

Laccase 

Fig.25. Activities of polyphenol oxidase, 

catalase and ascorbic acid oxidase of 

C.capsici - in vivo 

B Infected 

Polyphenol Catalase Ascorbic acid 

oxidase 

oxidase

D. Nucleic acids, protein and amino acids 

I'he maximum DNA, RNA, protein and amlno ac~ds were found In control fruit5 

(0 79, 1 56. 1 16 and 0 85 mg respectively) compared with those of treated (0 77, 1 Si, 

1 12 and 0 80 mg respect~vely) dnd infected fru~ts (0 39, 0 87, 0 69 and 066 mg 

respect~vely) (Table 8) 

E. Capsaicin, cellulose, amino nitrogen and ascorbic acid 

The maxrmum capsalcrn, cellulose, ammo nttrogen and ascorb~c ac~d contents 

were found In control fruits (0 07, 0 22. 33 88 and 21 97 mg respect~vely) compared 

with thox of treated (0 06, 0 20, 32 03 and 19 74 mg respectively) and infected fru~ts 

(0 03.0 l I. 20 49 and 8 08 mg respect~vely) (Table 8) 

F. Proline 

The niaximum prol~ne content was found in infected plants (0 26 mg) followed 

by treated (0 I3 mg) and control plants (0 10 mp) (1 able 8) 

G. Enzymatic stud!es 

a. Pectinolytic enzymes 

I. Prct~n methyl esterase 

The maximum PME actlvlty was found In ~nfected fru~ts (25 525~~) 

followed by 

tredted (I692\A,,) and control fru~ts (I404\41 ) ( FI~ 15) 

ii. Pol! methyl galacturonase 

1 he nlaxlmum endo-PMG and exo-PMG actnltles were found In ~nfected fru~ts 

(54% and 0 19SAl respect~vely) followed by treated (13 16% and 0 035~~1 

~espcct~vely) 

and control fruits (I0 54% and 0 02jAk! respect~vely) (Figs 16 and 17) 

ii~. Pect~n trans eliminasc 

I he maxlnlum cndo-I'I'C and exo-PI t d ~t~\ !ties were found in ~nfec~cd frwts 

(5 I 67% ~ n d 35 6IsA1 reqpectlvely) followed b! treated (23 37% dnd 0 12%~ 

respectnel! ) and control fru~ts (20 79% and 0 0 9 respect~vely) ~ (F~gs 18 and 19) 

b. Cellulolytic enzymes 

i. 1,4, p - endo glucanase and exo endo P 1,4-glucanase 

I IIC. mailmum I I f3-endo-glucanase and eio endo P I,4-glucandsc dct~vttles 

were lound In rnfected fru~ts (63 96% and 0 27~~~1 

respect~vely) followed by treated

Fig. 26.UV - SPECTRUM OF SAMPLE 1 

Nanometer = 223 Absorbance = 1 A 18 

Fig. 27. UV- SPECTRUM OF SAMPLE 2 

Nanometer = 2 19 AMwbance = 1 A 12

N 

r;; 

cl 

r 

2 6E,& E 

2 L60G E 

I8955 E 

91895 E 

0 E6L5 E 

5 :; : 

g 9E959 E 

+ 61099 E 

15519 E- 

y 66E89 E 

b 6%2L E 

REEL I 

I L5629 V 

g 69969 V 

E z 

I 

E 

i

I801 POI 1 

U 

3

Fig. 34 RP - HPLC - SI'I

Fig. 35. RP - HPLC SPECTIWM OF SAMPLE 2 

SatnpIcNanLl: 

VlVl 

l",CCllO" 1 

I",CCl,O" VPIIICIII. 

Channel 

RU" nnic 

,..>#11f,lc4 

I 

lo 00 ill 

>nBiCl~ann~I I 

70 o M ~~I~TCS 

Sampla Type 

Dalc Acquired 

Unknoun 

1012812002 3 35 H PM 

AcQ Method Scl WA-MEW 

PIOCLSSIIIQ Melhod ENW 

Dale Plaeerred 

1112812002 4 03 46 PM 

-- 

Peak Rcwlt. 

7 .. 

, . .l.n 1 KT 1 Arc0 / M.'

(22.13% and 0.06~~~ respmlivel~) and control liu~ts (10,4i1X, 2nd 0.05, ,: rcsl,cctivcly, 

(Figs. 20 ~d 2 1 ). 

ii. 1,4 p - Exo-glucana~e and ~ellulobias~ 

The maximum 1, 4 P ex0 - giucanase and celluli,hiasc ,lctivilics 

f;ound in 

infected fruits (0.62 and 0.43\~1: respectively) lbllowed h) rl.cstcd (0 50 and 0.i j,,l, 

respectively) and control fruits 10.44 and 0. I&,, 

c. Amylase and invertase 

respecr~vcl) I 

Maximum amylase and invertase activities wcrc hunt1 ill infccled ii.tllls (0.25 

and 0.?0$4\1 respectively) followed by treated (0.17 and 0 

control fruits (0.18 and 0.17~A1 respectively) (FI~. 23). 

22) 

rsspecti\cly) and 

d. Oxidative enzymes 

. . 

Ihe niaximum activities of laccase, polyphencil oxida,c. perci\~dasc. catalase 

and ascorbic acid oxidase activities were found in infected fruits (0.09.0.7-0, 0,008.0.1 7 

and 0.17 units respectively) followed by treated (0.05. 0 16. 0.005. 0.15 and 0 I3 units 

respectively) and control fruits (0.04. 0.15. 0.004. 0 14 and 0.1 l uu~th respectively) 

(Figs. 24 and 25). 

c. Leaf protein profiling studies 

[.car protein 

studies suggested that thcrc was difference. In the pl.otcin 

pl.i)lilcs hetv,\.rell [he leaves of control. treated arid in1'c.cti.d plallrs 

V. Active principle isolation studies 

UV (Figs. 26 and 27). FTIR (Figs. 28 and 29). H I 

NMR (Figs. 30 and 31) and 

C" NMR (Figs, 32 and 33) spectral, RP-HPLC' (Figs. 3 and 35) anaI!>is and 

i~~l\~oc~lr.nllcal tests sowed the presence of mixture of sugars. proteins and anlilio ~tcids 

In rhc acti\e fractions (sample 1 and 2).

DISCUSSION 

The present study brings out the effect oiaqueous extracts of 275 plant spp, on 

the conidial germination of Cq.cupsici and those of 27 aqueous plant extracts which 

produced above 50% inhibition on the conidial germillation both at 24 h and 48 h, on 

the radial mycelial growth of C.capsici and that of A.sa/ivum aqueous bulb extract 

which produced cent per cent inhibition both on the conidial germination and radial 

mycelial growth. on the in vitro and in viva physiological and biochemical activities of 

('. capsici. 

Aqueous extracts of 275 plant spp. based on their effects on the conidial 

germination of ('capsici were divided into 4 groups. 

Group 1: Aqueous extracts of plants completely inhibited the conidial 

germination of ('cupsici both at 24 h and 48 h. which showed their fungicidal effect on 

the conidial germination of C cupsici. 

Chitra and Ka~abiran (2002) reported the fungicidal effect of aquzous plant 

extracts on the conidial germination of C'cupsrci. 

Group 2: Per cent of ~nhibition produced by the aqueous plant cstracts on the 

conidial ger~iiinaiion of c cup sic,^ was found to be reduced at 48 11 compared w~th 24 h. 

which slio\\s their fungistatic effect on the conidial germination of(' ctrp.~icr. 

Gaticsali and Naidu (1999) reported the fungisraric effect of petal e\tracts on the 

conidial germination of D ory:ucJ. 

Group 3. Aqueous extracts of plants induced stout. kaded atid branched 

abnornial getmination of I' capsici conidia. 

;\h~iomial gc~mination inducing effect of aqueous leaf estracts on the cotiidial 

germination of ('.capsici has reported by Gomathi and Kannabirdri (1000).

Group 4 Aqueous extracts of plants were not found to have lnh~b~tory effect on 

the con~d~al germlnatlon of C capstcr 

Ch~tra and Kannablran (2001) reported aqueous plant extracts w~th out any 

lnhtb~tory effect on the con~d~al germlnatlon of C capsrcr 

Of the aqueous extracts of 275 plant spp tested on the con~d~al germlnatlon of 

C tapstcr, only those of 27 plant spp were found to produce above 50% lnh~blt~on 

the con~d~al germlnatlon of C capsrcr both at 24 h and 48 h They were further screened 

for thew effects on the rad~al mycellal growth of C capsrcr 

Eventhough aqueous extracts of Adharoda vasrca, Asparagus racemosus, 

Bauhrnra purpurea, Carrca papaya, Cassra fistula, Casuarrna eqursetrfolra. Cesrrum 

drurnum. Coleus barbatus, Cordra sebesrena, Cor~andrum satrvum, Courouptra 

gurunemts, Dol~chos rrrlobus, Helrorroptum rndrcum, Parrhenrum hysrerophorur 

Pedalrum murex, Plumerta alba and Solanum melongena produced cent per cent 

lnh~b~t~on 

the con~d~al germlnatlon of Ccapslcr they faded to produce ~nh~b~tory 

effect on the radlal rnycel~al growth of C capsrct 

In the present study the rate of lnhlb~t~on of con~d~al germlnatlon could not be 

correlated wlth radlal mycellal growth Because of the avrulabillty of more tlme and 

nutrients the plant extract could not act upon the growth of the fungus depending upon 

thew chemlcal const~tuents (SenUul Kumaran, 1998) Of the aqueous extracts of 27 

plant spp screened for thelr effects on the rad~al mycelral growth of Ccapsrcr 

maxlmum lnhrb~t~on (100%) both on the con~d~al germlnatlon and rad~al mycel~al 

growth were produced by L tnermts leaf and A sanvum bulb aqueous extracts (Plate 2) 

The ant~fungal actlvlty of L tnermrs was found to be ~ncons~stent agrunst Ccapsrcr 

However the antlfungal actlvlty of A satrvum was found to be consistent Hence ~t was 

selected for further m vrtro and m vrvo studes Numerous prel~m~nary reports are 

avlulable on ;he antlfungal actlvlty of A sot~vum (Mlsra and D~xlt, 1976, Lakshmanan, 

1990, Gohll and Val& 1996, Nandan and Anup, 2002)

PLATE 2 

Effect of plant extracts on C.capsici 

1. Control -Culture of C.capsici 

2. Effect of A.salivum aqueous bulb extract on the radial 

mycelial growth of C cupsici 

3 Effect of L.inermis aqueous leaf extract on the radial 

mycelial growth of C.cup.~ici 

I 

Effect of three weeks stored A.sarivum aqueous bulb 

extract on the radial mycelial growth of C.capsici 

5. Effect of hear treated A surivum aqueous bulb extract 

on the radial mycelial growth of Ccapsici

PLATE 2

. . 

Ihe active principles of A.suril:um were found to be thennostable against 

C'.cup.~tci (piate 2). 

Gomathi e/ ul. (2001) reported the thermostable nature of the aqueous leaf 

extracts of Soianum rorvum, Daturu melul and Prosupis julifloru against Ccupsici and 

C:giorosporioides. 

Chitra er a/. (1999) reported the thermostable nature of D.innoxla flower extract 

against Ccapsici. 

The shelf life of A.sa/ivum aqueous bulb extract was found to persist upto 6 

weeks on the conidial germination and 5 weeks on the radial mycelial growth (Plate 2) 

of (' ctr/~sici. 

Active principles present in Foeniculum vulgarue and Pimpinella unisum were 

found to be stable upto 240 days in stored materials (Shukla er ul., 1989). 

'The antifungal effect of Mangifera irtdica against Bi~alaris sorokiniunu wa 

found to persist upto 50 days in stored materials (Muhesh Kumar e/ al., 2001). 

.4 srr/i~~lmr aqueous bulb extract was found to significantly inhibit the seed 

germination of chilli compared with control. 

It \\as reported earlier that ..lrrcrho/rp hexuperulo~cs leal' and Slc~iercniu 

muhctgonr bark extracts besides being strongly fungitoxic. severel! inhibited seed 

germination of' brassica (Grainge and Alvarez, 1987) and paddy seeds (Sellapandi, 

2001) respectively. 

011 the contrary. when sprayedon 25 days old chilli seedlings (Plate 3) the same 

extract \\.as found to stimulate the growth. synthesis of photosynthetic pigments. 

carbohydrates. nucleic acids, protein, amino acids, phenols and the activities of 

oxidatitc enzymes similar to that of groNh hornione compared with co~ltrol.

PLATE 3 

Effect ofA.sativum aqueous bulb extract on chilli 

seedlings - in vivo 

1. Control seedlings 

2. Seedlings sprayed with A.salivurn aqueous bulb extract

PLATE 3

The psltrve effect of Lcuca~ o5prru root extract on the growth d11d 

photosynthetic plgments contents of paddy and green gram seedlings was repned by 

Sangodldmmalle (2000) 

Rajeswarr dnd Marlappan (1991) reported the Increased d~trvrt~es 01 ox~ddt~v~ 

enLyme.i In I'yrrculuria oryzuc - inoculated paddy plants sprayed with A ~~u\rtu dnd 

Prosopr~ /ullfloru extracts 

Antifungal compounds In general Interfere wlth any one 01 the several 

h~osynthet~c pathways (or) energy production pdthway5 Otren antrtungal agent5 

Interfere strongly at a spec~fic stage In a metabolrc pathway, whlclr soan spreads to 

other pathways, as the metabol~c pathways are all In one way (or) other ~nterconnected 

to each other leadrng to total arrest of the cellular activities and even klll~ng of the 

organism Slna the queous bulb extract of A sativum exlub~ted strong dntlfungal 

act11 I[\ the b~ochemlcal Impact of the lnh~b~t~on 

the pathogen was invest~gated 

I he In vrrro blochemrcal analvsis of' treated mycelldl mat of C cupsro on fourth 

dnd c~phth dav was found to show lns~gnlficant rrductlon in the DNA and s~gn~firdnt 

reductron In the RNA pmteln and lipid synthes~s compared w~th control benom~c 

DNA prolillng studres based on different restrlctlon enzymes suggest that there wds no 

s~gr~ificant drllerence In the restriction enzimes based genornic DNA profile., between 

colurol ,~nd treatcd m\cel~al mat of ( cup3icr (Plate 4) 

A feu workers have reported the alterat~ons In the nucle~c acrds protelns dnd 

lrprd a\ nthes~s of fungal mycellurn 

\ataralan and : allthakumarl (1987) repnrred the rnh~b~tor\ effect of I ~nernilc 

lcal e\lr,ict on the nuclelc aclds dnd proteln ,)nthes~a of D oryIup 

I'atcha1vd71arnman (2002) reported the 111111b1tory etTect of bdih extract of Aglulcc 

eIueogritirde\ on the nuclelc actds, protern and total lrptd synthesrs of R solanr

PLATE 4 

Effect of A.sativum aqueous bulb extract on the restriction enzymes based 

genomic DNA profiles of C. capsici 

Lane 1: 1 Kb Ladder 

Lane 2: Control: Hind IIIMot I digest 

Lane 3: Treatment: H i IIUNot 1 digest 

Lane 4: Control: Not YSal I digest 

Lute S: Tnatment: Not YSal I digest 

Lane 1: 1 Kb Laddm 

Lane 2: Conml: EcoRlMind 111 digest 

Lane 3: Treatment: EcoRlMInd 111 digest 

Lane 4: Control: EcoRlMotl digest 

Lane 5: Treatment: EcoRlMotl digest 

Luc 6: Control: EcoRllSal I digest 

k c 7: Tnatment: EcoRllSal I digest

llew~tt (1999) rcported the ~nliihitory effe~t of the fuiig~~~de Iiymcx'w~i on 111' 

nucleic acids and protein synthesrs of plant pathogens Fungic~de melalaxyl ~nhibited 

the plant pathogens by specrfically ~nh~biting the synthes~s of nbosomdl RNA via the 

KNA plymer~~e I - template complex result~ng in the disrupt~on of protein \ynrhesis 

(Dav~dse and van der Beiy - Valthius, 1989) 

In the present study the processes of transcription and tranaldtion ueic h~glily 

affected compdred with DNA synthes~s as a result of which there wds \~giiificant 

reducr~on in the synthes~s ofI1NA and proteln 

I)i~function (or) mallunction of the memhrdne wds evrdeut from th~ lii~redsed 

ele~trolyt~c leakage of treated C capsrc~ mycelium compared with control 

Portelvy (2000) reported the increased electrolyt~c leakage of R solu~~r mycel~al 

mat tr~ared with bark extract of Anacardrum occ~dentale 

'I he Increased eltctrolyt~c leakage from the treated mycelrum may be attributed 

ro thc arrong ~nhib~tion of protein and hpid synthe~is This observation derites support 

from rhe observations of Rao (1984), who reported that all the funglc~des thar ~edu~ed 

l~p~d content In D oryzae also induced electrolyt~c leakage 

I n7tnies produced by plant pathogens play a very unportant role in thc 'ntr! 

rlie pdtliogcnc Into the host tissue by degrading the cell wall Any inh~b~t~on 

tli~ aboie 

rnenr~c~ned process would cause a deletenous effect on the pathogen 

of 

In the present stud) the activities of pect~nolyt~cellulolyt~c am\lase dild 

Intertdse enzymes of C'cupcprro were srgnificantly (P

PLATE 5 

Effect of A.sativum aqueous bulb extract on the extracellular protein profile 

of C.capsici - in viiro. 

Lrnc 1: Protein Marka (BSA, 66 kDa) 

hat 2: Control 

Lrpc 3: Treetmk~lt

Inhibitory effect of Daturcr innoxia fruit and flower extracts on tllc cnzymallc 

activities of Ccapsici was reported by Chitra el ul. (2000). 

Kiraley el al. (1985) reported the inhibitory effect of the fungicide fenitropan on 

the protein synthesis and enzymatic activities of plant pathogens. 

The fungicide tilt was found to inhibit the enzymatic activities of C.capsici in in 

vitro condition (Gopinath et a/., 1999). 

On the contrary, in the present study the activities of oxidative enzymes were 

found to increase in in vifro condition. 

Decreased activities of call wall degrading enzymes and increased activities of 

ox~dative enzymes of Cgloeosporioides treated with Ainnoxia aqueous extract was 

already reprted by Pugazhenthi (2001). 

This shows that the activities of oxidative enzymes of plant pathogens were 

found to increase under stressed condition (Rajaswari, 2002). 

SDS-PAGE analysis of extracellular protein profiling studies showed significant 

difference between control and treated I'cupsici extracellular protein profiles. In the 

case of treatment the intensity and width of the protein bands were reduced while some 

of' the bands were missing compared with control (Plate 5). This shows the toxic effect 

of tllr .4 .\[r~ir,um aqueous bulb extract on the extracellular protein profile of (:cu/~\ici 

(Kuruchc\ e and Padmavathi, 1997). 

h4illl) lungicides were reported 2 inhibit the protein synthesis of fungal plant 

pathogens (Buchenaur, 1978; Mahrotra. 1996). 

In in viw condition Ccapsici - inoculated chilli fruits treated with A.sa/ivum 

aqueous hulh extract showed significant reduction in the PDI (P

PLATE 6 

Effect of A.sntivum aqueous bulb extract on 

C.cnpsici - in vivo 

1. Control - Healthy chilli plants 

2, infected chilli plants sprayed with A sa/l~.um aqueous bulb extract 

3, Infected plants

PLATE 6

Kaoialakannan (2001) reporled he antifungal erfect of Prosopi.c ,jul~/lorcr, 

Ziziphus tujuhu and Aitrdiruchla indtcu in reducing the PDI of t'yrrcuiuriu grisco - 

~noculeied paddy plants. 

S~ngli el ul (2001) reported the effect of' the fungicide ridomil in reducing the 

PDI of'I'l~y~o/~hrhoru cinnamoni on pointed guard. 

I lierc was significant increase in the electrolytic leakage of infcctcd fruits 

coniparcd with control fruits due to the disruption of the cell plasma membrane and cell 

wall b! 1111. penetration process of C cupslri (Mahadevan and Sridhar, 1996). 

Singh L'I ul (2001) reported the increased electrolytic leakage ofthc paddy seeds 

infected \\~tll different fungal spp. 

I Ile reduction in the electrolytic leakage of the treated fruits could be attributed 

to the I'unglc~dal compounds present in the test plant extract. which nii~ht have ~nhibited 

the groutll and penetration process of C cnpsici (Singh er ul.. 1993). 

In rlie infected fruits compared with control fruits there was significant reduction 

In tlie pl~otos~nthcti pigments. carbohydrates. nucleic acids. protein. amino acids. 

capsaic111. ccllulosr. amlno nitrogen and ascorbic acid contents. 

'1 hi. decreased level of biochemical constituents in the infected fruits lilight be 

du' to IIIC tos~c metabolites produced b) the C.cupsrci. which affect tlie host 

1net3holl.1n (V~rpn~a el ~rl.. 1997). 

ltl rrtated liuits insignificant decrease uas noticed in all the aho\ementioned 

hiocIirn~i~~,il cc~nstituenrs compared with control fruits. As the PLII \\as found to hc 

reduced 111 treated fiuits. there was insignificant reduction In the hlocliemical 

cons~ituclits compared \\ ith conlrol I'ruits (V~dhyesekaran el crl.. 10971

I'arirnalan (1999) reported the decreased PDI and increased synthesis of 

biochemical constituents in Ccapsici - inoculated chilli seedlings treated with aqueous 

plant extracts compared with infected seedlings. 

Phenols, proline contents and activities of oxidative enzymes (p

PLATE 7 

Effect of A.sativum aqueous extract on the total soluble leaf protein profile 

of C.annum plants infected with C.capsici - in vivo. 

Lane 1: Protein Marker (BSA, 66 kDa) 

Lsac 2: Control 

Lane 3: TRatmmt 

h 4: Infected

iosynthesis of lytic fungal enzymes i~~volved in cell lysis (or) their secretion at rl~c 

point of fungal penetration (Daniel and I,ucas, lYY5; Milling and Richardson, 1995). 

SDS-PAGE analysis of total soluble leaf protein profiling studies did 11ot shou 

any significant difference between leaf protein profiles of control and treated plants 

(Plate 7). This shows the alleviating effect of the test plant extract on the harmful eRi.ct 

of C'cupsici on the protein synthesis of treated plants (Mutliulakshmi and Sectharaniaci. 

1987). However, in the infected plants the protein synthesis was found to be inhibited. 

which is etident from the lesser intensity and width of the Icaf protein bands compared 

with control and treated plants (Plate 7). 

'Thc inhibition of foliar prote~n synthesis during pathogenesis has already 

reported (Jabakuniar rt ul., 2002: Chakrabony et 01.. 2002). 

'I'lle antifungal compounds present in the A saiivum aqueous bulb extract were 

Sound lo he tnixture of sugars. proteins and amino acids. 

In tlic present study both the crude .l .vurivum solvent extract and the purilied 

active fractions (sample 1 and 2), which produced cent per cent inhibition on the 

conid~al germination of ('cupsici failed to. produce an!, 

inhibition on the radial 

~nycelial pro~h This could be attributed to the sufficient concentrations oi' the active 

princip1e.i presenr in the crude extract and the activc fractions. which could bring out the 

inhihit~on on conidial germination but not 011 rhe radial mycelial grou~li (Chitra. 1998). 

l'lic heat stablc nature of Mirtrhrl~\ julcipu antiviral protein \(as reported b! 

IiabuLi~ i3/ 111. (I%9). 

I'lic ant~li~tigal activity of peptides from Heliunrhus co1nu.s flower against 

Sclerorotlrtr sclcroirr~m was reported by Mariana ei a/. (1997). 

It (Anonymous. 2002 b) was reported that sugars and proteins of A saiilstrm 

showed antifungal and antibacterial activit! against yeast and Pediococcus.

Antifungal amino acids was reported earlier from whey protein of milk 

(Anonynlous. 2002 c). 

The results or the present study suggest that the A.sulivun~ aqueous bulb extract 

because of'its strong antifungal activity against C'.cqsici both in in vicro and in vi,ro 

conditions with non phytotoxic effect ma! prove to be an effectiic eco-friendly phyto 

fungicide for controlling the fruit rot of chilli.

SUMMARY 

The present study brings out the cfleci ol'aquuous extracis 01'275 plan1 spp otl 

the conidial germination of chilli Fruit rot Fungus ('cupsic; and thc cl'kcts of aqucous 

extracis of 27 plant spp. which produced above 50% inhibition on the conidial 

gernlination both at 24 h and 48 h, on the radial mycelial growl11 01' C'.cul~sici, 01' the 

aqueous extracts of 27 plant spp, screened only thosc of L.incrmi.~ leaf and .I su/ii~um 

bulb aqueous extracts were found to produce nlaxlnium inhibition ( IOOU/o) ho~h 011 the 

conidial gerniination and radial mycelial grohth ol' ('.iul7src1. I'hc rcs~~lt ol' I. iner-1111s 

has found to be inconsistent. Hence. A.suli!~uni aqueous bulh extraci (the icst plant 

extraci) was selected for further in viiro and in i'irbo studies against ( ' [.U/,SIL,I 

Thc shelf life of the test plant exiracr was Sound to persist upio 6 ~ccks agai~ist 

('cupalcr and the antirungal compounds were Uound to be stahlc on heat trcatmcnt. 

Regarding phyiotoxicity studies. the test plant extract was lound to inhibit the 

seed germ~nation of chilli. On the contrary. when sprayed on 25 days old chilli 

seedlings it was found to stimulate the gro~.th and the s!.nthesis ol' photosynthetic 

pigments. carhnhydrates. nucleic ac~ds. protein. amino acids. phellols and the acti\,ities 

r)So\~daii\cn/!nlcs. 

studics. 

At I.l)i,, (0.7%) concentration the rest plant extract \+as uscil lilr ti~rrhcr in vi~ln 

'I'licrc \+as signilicant increaw in the cell pcrmeahilii! 01' 

rrsarcd ('cccl)r~r; 

compared \\~rh control. The DNA s!~nthesis was I'ound to he insig~nlica~ltl! afictcd in 

trcaied ('~~,psrcr. Similarly there werc insignificant changes in iOc d~l'l'crcnt rcsiliction 

enzynles based gcnoniic DNA profiles ol' treated i'.capsrcr compared with control. 

However the synthesis of RNA. protein, I'ipid cc3ntents and the activities of pectinolytic. 

cellulol!\ic. 

alnylase and invertase enzymes of treated ('r(ri).\ic.r \+ere lbutld to be 

significa,jtl! inhihilcd colnpared with control. On the contrar! thc acti\ iticb of'oxidative

enzymes of treated C.capsici were found to be significantly increased compared with 

control. 

Extracellular protein profiling studies of treated C capsici showed significant 

difference in the protein profile in terms of width and intensity ofthe protein bands and 

even some of the bands were fbund to he missing compared with the protein prolile of 

control. 

10% concentration of the test plant extract was used for in vita studies 

There was significant reduction in the PDI of treated chilli fruits compared with 

infected chilli fruits. There was significant and insignificant increase in the cell 

permeability of infected and treated fruits respectively compared with control fruits. 

l'he synthesis of photosynthetic pigments, carbohydrates. nucleic acids, protein, 

amino acids, capsaicin. cellulose, amino nitrogen and ascorbic acid were found to be 

significantly inhibited in infected fruits compared with control fruits. In lreatd fruits 

there was insignifiiant decrease in the above mentioned biochemical constituents 

conlpared with control fruits. 

Phenols. prol~ne contents and the activities of hydrolytic and 0xidatij.e enzymes 

werc found to he iiioderatelg'. highly and slightly significantly increased respec~ively in 

infected fruits compared with control fruits. where as in the treated fruits only in the 

case of h!drolytic enzymes a slight significant increase was observed. Phenols. prol~nc 

contents and thc activities of oxidative enzymes were found to be insignificantly 

increased co~nparcd with control fruits. 

'I'otal soluble leaf protein profiling studies showed signtlicant differencc in thc 

protcln hands of inkcled plants leaves in terms of lesser width and intensit! of protein 

bands compared with control plants leaf protein profile. 'l'herc was ~nsignificanl 

dilkrcilct. In thc protein profiles betwocn treated and control plank.

, . 

I hc active conlpsunds present in thc A sulivuni aqueous bulb extract were found 

10 be the mixture of sugars, protein and amino acids. 

The results of the present work establishes the antifungal effect or A swrvum 

aqueoui bulb extract against C'cupsici both in in vilro and in 1i18o condition with non 

phytotoxic effects. Hence A solivum aqueous bulb extract could be used as an 

alternative. hiodegradable and environmnentally salcr bioh~lgicide against the chilli 

anthracnose fungus C cupsici.

REFERENCES 

Anbazhagan.I!. 

(1985). Alrernuriu leaf'sp~t of brin,ial. Al I'hil, l)i,\ter/~trron, Annamalai 

University. Annamalai Nagar. India 

Anonymous (2002 c).http:/www.custornprobiotics.com/whey.htm. 

Agarual.M.1 Satish Kumar. Goe1.A.K. and Taya1.M.S. (1982). Biochemical analysis 

of turmeric: some hydrolysing and oxidative enzymes and related chemical 

metabolites. Indian Phyiopath.. 35: 438-441 

Amodioha.A.C. (1998). Control of powder! mildeu in pepper (('al~srruril annurri L.) h! 

leuf cxtrdcls of papaya (Curicu /)u/?tr~.rr L.). J Herbs. Spree.\ h !tdrcinul PIonr\. 

6: 4 1-44. 

Anandarq.hl, and Lee1a.N.K. (1996), losic effec~ of somc plant extracts oil 

l'l!~ ioll~hihoru cupsicr, the foot rot pathogen of black pepper. lntlran Phi,ro~~ctrh.. 

19. 181-184. 

Hanibaualc.0.M. M0han.P. and Charahart!.M. (1995). Ellicac! ol' some medicinal 

plants against cotton pathogens. Ad Pluni Sci., 8: 224-229. 

'Bligh.1. C; and I1ycr.W.J. (1959). A rapid rnethod ol'toial extl.ac~io~i an~i pur~lica~ion 

('err .I Biochem Ph~:vrol.. 37. 01 1-01 7. 

*Bora.R.S.. Murthy.M.Ci., Shenbagarithai.~~. and Sekar.V. (1994). Introduction to ;I 

l.cpidop1cra11 specific iasecticidal cr! stal protcill gcnc of Il~rc~illtr~ ihuri11girnrir 

sub sp. ktrrsraki by conjugal transfer in to a Bacillus ntc~lctcrirtni strain that 

persists in the cotton phyllosphere. .4ppl. Environ Microhiol.. 60: 114-222. 

70

*Bray,G.H. and Thrope, W.V. (1954). Analysis of phenolic components of interest in 

metabolism. Method in Bio. Chem. Anal., 1: 27-52. 

Britto,S.J., Leo,M.B.N., NatarajqE. and Arochiasamy,D.I. (2002). In vi~o antifuagal 

properties of Tinospora cordifolia (Willd) Hookf and Thomm l Swalny Bot. 

CI., 19: 35-36. 

Buchenaur,H. (1 978). Anology in the m& of action of fluotrimaarle and chlotrimamle 

in Ustilago avenae. Pest.Biochem. Physiol., 8: 15 

'Burt0nA.J. (1956). The study of the condition and mechanism of the diphenylamine 

reaction for the calorimetric estimation of DNA. Biochem. J., 62: 315-323. 

Ce~one,F., Andebrhan,T., Coutts,R.H. and Wood,R.K.S. (1981). Effect of french bean 

tissue and leaf protoplast on CoNetotrichum Iindemuthiunum polygalacturonase. 

Phyroparh. Z., 102: 238-246. 

Chacko,S., Khare,M.N. and Aganval,S.C. (1978). Cellulolytic and pectinolytic enzymes 

production by Collerorrichum demarium f. sp. fruncatum causing anthracnose of 

soybean. Indian Phyropath., 31: 69-72 

Chakraborty,B.N., Dutta,S. and Chakraborty,U. (2002). Biochemical responses of tea 

plants induced by foliar infection with Exobaridium vexans. Indian Phylopath., 

55: 8-13. 

Chakravarty,D.K., Khanna,D.C. and Giri,D.N. (1981). Evaluation of some chemical 

dips for the post harvest anthnicnose of chilli fruits. Indian l Mycol . PI. Prrthol., 

10: 243-245. 

*Chance,B. and Malchly,A:C. (1955). Assay of cataleses and pxidases. Method! 

Enrymologv, 2: 764-775. 

ChitqH. (1998). In viko studies on the effect of e mts of the flower, fruit and root 

parts of the Dafura innoxia M. on the anthracnosc fungas, Co~leto~ichrun

cap.sici (Syd.) Butler and Bisby infecting Capsicum annum I*. 

Dissrrrulion, Pondicherry University, India. 

Ml'hil 

(:hitra.ll. and ~alinabiran,~. (2001). Antifungal effect of L)rrturcr innoriu M. on the 

anthracnose fungas Colle!orrichum cupsici, in 17irro. Ad. Piunt Scr., 14: 3 17-320 

Chi1ra.H. and Kannabiran-B. (2001). Effects of aqueous leaf extracts on the spore 

germination of Colle~otrichum cupsici (Syd.) Butler and Bisby. fiol En),. & 

('on.\.. 7: 53-56. 

Ch11ra.l-1, and Kannabiran,B. (2002). Screening of aqueous extracIs of some plants on 

the conidial germination and mycelial growth of Colletotrichum capsici (Syd.) 

Butler and Bisby. Gcohios, 29: 185-186. 

('liitra.1-I.. Golnall1l.V. and Kannabiran,D. (1909). Effccts of extracts of Duturo innoxirr 

Miller on thc spore germination and mycelial growth of C'olletotricltum ccrpsici 

(S!d.) Hurler and Bisby. Geohros. 26: 177-178. 

Chitra.H.. Gon1athi.V. atid Kamabiran.B. (2000). Inhibitory effect of Da~uru innoxio 

h4iller on the enzymatic activities of the anthracnose fungus ('oilr/r~/ric.hrr~,i 

c'ri/l.%ii'l iii ~.//ro. lndiun Ph,vtoputh.. 54: 353-255 

('llo\\dIiur! .S 1 1957). Studies on the dcvelop~nrnt and control of fruit rol of chillics 

1ir~i1~111 l'li~~opa/h.. 10: 55-61 

lo lo^. 1 ..1. Lorh. \.A, and Chandler.l'.M. (1989). A c-DNA based conlparison 01' 

deli!dr,~tiol\ induced proteins (dehydran) in barley and corn. Plfrn~ A401 

13 10-108 

Danirls.:\, alld I.ucas.J.A 

Biol.. 

(1995). Mode of action studies of the anilinopyrimidint. 

f~~~igicide p! rimethanil on Borrytis s/vlq,. 1. br virro activily against B./irhoi, 011 

hroad k an (I 'icia fuhu) leaves. Pesricide Scieni.r. 45: 33-4 l

Uastur.J.l:.M.S. (1921). Die hack ofchilllcs (Cu/>sicrrm spp.) in Bihar. Men? /)el)/ Ayri 

lndiu Uor Soc., 11: 129-144. 

l)atar.V.V.(1995). Pathogenicity and effect of temperature on six fungi causing fruit rot 

ol'chilli. 1ndron.J Mycol. 1'1. I'ulhoi.. 25: 195-197. 

I)avidsc.l..G. 

and van der Berg-Valthius.(j.C.M. (1989). Biochemical and molecular 

aspects of the phenylamide l'ungicide receptor interaction in plant pathogenic 

l'h~~/ophlhoru spp. In: Lugtenberg.0.J.J. (ed.) Signal Molecules in I'lon~s ond 

I'lim/-Microhe ln/eru~/ion.r. Springer-Verlag. I3erlin. 

1)hanapal.K.. Joseph,'l', and Naidu,k. (1993). Use of neem in integrated management of 

rot diseases of small cardarnon) (Ele/iuria cardornomum). World Neem 

('onfcrcnce. India. kern and En~r/.unmen/. 2: 793-798. 

1)hawale.S.D. and Kodmelvan.R.V. ( 19781 Studies on mycoflora of seed. Seer/ Rea.. 6: 

23-30. 

1)lsli.S U . 1'npathi.S.C. and Ilpadh!.ay.ll K.. (1076). The antifungal substance of rosc 

tlo\\lers (Hosu rndrcrr). Don Bortrrii . 30: 37 I -3 74. 

I)uhc K C ( 1991). I ungicldal effect of es\i.ntial oils of three higher plants (111 sclen~ia 

of .\t(rc~.up/to~nr~tir phlrceolinu. 1n(lr(111 I'h~~folxtrh.. 44: 741 -714. 

I)uhios.ll . Gi1lies.K \I.. 1lamilton.l.K.. 1l;ihers. P.K. and Smith.1:. (1951). Calorimeiric 

nlethod for the deternrination of'suyrs and related substances .-lnrrl Chcm.. 28: 

310-356. 

I)urairql.\ 

(1950). Growl1 of ('oll(%~it~ic.hurr, ctrpsici in ptcrc culrurc. Inilior~ 

I'ln./r~l)i~/h.. 10: 109-4 13.

*I urlong,C I . C~rakaglu.(' Willrs II C and Sanly. I' A (1973) A jnnplc ptcpalatlvc 

polyacrylarn~dc gel clectrophoresl~ apparatus pur~ficatlon 01 

three branched 

chtl~n ?mlnoacld b~ndlng protelns lion Etcherrthru colr A1iu1 Urotiioil. 51 

297-3 I1 

Ganesan.1 (2000) M!cotouc volatlle omp pounds ol plant orlgln (~cohro\. 27 134- 

135 

(ianesru1.7 

and Krlshnara~u,J (1995) Antlrungal properties of w~ld piant5 -11 Ad11 

PIUI~ISLI.~ 194-196 

(ianesan.1 

and Lal~th S Naldu (1999) I flcct o! petal extracts all iot~~dtal gcmlnatlon 

of'Drcthsleru oQzue Geobiot, 18 71 -73 

(ido.S . and Shaln,l 

( 19%) Actlv~ty of pol!falacturonaw produced by Crvllhonccirru 

piirrr\~/r~u In chestnut bark and I(\ ~nh~bltlon h) extracts llorn amerlcan and 

chlncse chehtnut Ph,z.\ro/ Ado/ PI I'ci/hol .46 199-21 3 

(;arg.S C and .lain, R K (1998) Ant~m~~lob~al efficacq ol essc.ilt~aI o~l from ('ut~urnu 

ccrccrcr Indrun .I Idrcrobrol . 38 169-1 70 

(iehlot.1) and Hohra.4 (1997) Eflect 01 r.\tlacts of \omc haloph!tei on the growth ol 

Ilrr~nurra co1[111r 1ndron.l ih

G0mathi.V. (2001). Studies on the antifungal effects ol'plant extracts r~n (i~lle/o/richtrrn 

spp. infecting Cbpsicum annum L. Oh 11. Thesis. I'ondicherr! I~I~Iv~~sIL!. India 

G0mathi.V. and Kannabiran.R. (1999). Effects of plant extracts on the cull wall 

degrading enzymes of Coilerofrichum cupsici (Syd.) Butler and Iiish! 

111 the 

host plant Capsicum onnum 1.. Proc. In11 ('on/ I;i.oct/iers 111 /.'11nyr11 Ilirj/c,cl~ K. 

I'bnr Parhogen lnlerucrion. Osmania linivcrsity. I ljderahad. India. 

Ci0mathi.V.. Chitra,H. and Kannabiran,U. (2001). Effect of licat and organic soi\cnts on 

the antifungal activity of leaf extracts against ('ollr~orrichuii~ spp. (irohioc. 28. 

12.5-128. 

Ciopinath,K.. Jayaraj,J., Senthil,N. and Kadhakrishnan. (1999). It? l,irr-o evaluation of the 

fungicide 'tilt' against the growth and enrynie produciorl of C'ollcro/ric~ht~~rr 

ccrpsici, incitant of anthracnose of chillies. Proc. lt~rl. C'ot7f F1.ontiei.r III l.'tr~?,oal 

lliorech & I'lonl Pathogen In/eruci~on, Os~nan~a l Jni\ersit!. H!derahad. India. 

(;raingr.bl.I), 

and A1varze.A.M. (1987). Antibacterial and antifungal acri\i~! of 

.41~1trhorr)~s hexopeiolous leaf extracts. Inr .I 

179. 

7i.n/~ical I'1oi11 Discrnc,~. 5: 17i- 

(ircual.:\.S and (.rover.K.II (1974). Changes in amino acids. carhoh!dr,ttc.s and 

capsaicin contents of red fruits of chillir> infrcted \\lrli 

pcpei.u/~tn~. Indian Phyroparh., 27: 003-604. 

('olle~(~i~.~chtrn~ 

Iiabul\a..l.l:.. Murakarni.Y.. Noma.M.. Kudo.'l'. and Horiloshi.K, I loS9) ..\tiuno acid 

sequence of Mirabili, ,jalapu antiv~ral protrln. total s).nthi.sil;. its sene and 

rsprcssion in Eschrrichlu coli. J. Biol. ('hr~ti.. 264. 1629-163-. 

Hakki.A.h,l.. 1lcim.A. and Sm.S. (2002). Anlihal.lerial ,and antifungal elf'ects ol' various 

plant extracts. http:llwww.gec~itcs,co1111n1dig~ah.gc~~~yayi1 Ilr~nl.

*Hampton.l

infecting Cap.~iici~rit annum L. M.lJhil. Dissertalion., I'ondicherry linivenity. 

India 

Jaspal singh and 'fripatl1i.N.N. (1993). Eficacy 01' plant extracts agalnsl I~ir.\uriun~ 

axysporum f. sp. I~ntis on kn.\ esculeniu. .I Indian hoi Soc.. 72. 5 1-53, 

Jeyarajan.8. (1988). Use of botanicals for [he control of fungal and viral discases of 

crops in rice-based cropping systems. Finn1 WorkFhop qf IRRI-ADB-EWC 

Projeci on Bottrnicul Pe~r l~anirol in Ilice-hosed ('rorpprng Sysrcms. 

International Rice Research Institute. Man~la. Phillippines. 

Jindal,K.K., Gupta,S.K. and Shyam,K.R. (1994). Studies on gernilnation. vigour and 

microflora on bell pepper seeds from healthy and diseased fruits, lndiun J 

Mycol PI Pathul.. 24: 227-228. 

Johal.G.S., Gray,l.. Gruis.D and Rriggs.S f'. (1995). Convcr~cnt insights into 

mechanism deetermining disease and resistance response in plant fungal 

interactions. Chn .I Boi., 73: 461-474. 

Kalaichelvan.P.'i'. and Sumathi.L. (1994) A fungitoxic subslancc from Solununi 

rtrgrctr". Indiun Phr.ri~path.. 15: 112-25.; 

Kama1akannan.A.. Shann~ugam,V.. Surendran.M. and Srin1vasan.R. (1001 ). Antifungal 

propenlrs of planr extracts against I?rrccrlirrro grrsco. thc I I ~ C blast pathogen. 

lnditrn I'h,v~opuih.. 54: 490-491. 

Kapoor.A.. Kekha Mahor. Vaishampaya11.N. and Gaut11am.N. \ 198 I). 

spcctruni or some plant extracls. (;erlhro.\. 8: 66-67. 

Andfungal 

Karthikayalu.S. (2002). S4icrobiological and b~ochemical isolat~o~l. charactcri~iicion 

and purification of antibacterial. ani~fungal and anticancer compounds from 

~uedical plants. M.Tc Thesi.~. Pondiche17-y [iniversi~!. I~dia.

Klrdl! I 

1akucs.E , Race,[, Idmes.1. and Las7tlty.D (1985) Effect of the fung~~~dc 

lcnrtropan on the metabol~sm of some hrgher plants ~ nd I;usurrum oxj~,por rrin 

I'etrrc,dc ,Science. 16 1-9 

Kul\hre?tha,[) D , Mathur,S B and Neergdard,P (1976) Identification 01 seed bornc 

'Kuruc1ieve.V 

spe~~cs of Col/e~o/rrchum / rre\ru. 11 1 16-125 

and Padmavath1.1l (1997) l'ung~tox~c~ty of wlected plant products 

agdlnst Pyrhrum aphanldermu~um Indian Phyroputh .SO 529-535 

'lde~ii~nl~ li K (1970) Clevage of structu~al protellis dur~ng the assembly of the head 

of hdctenophage 14 Nulure. 222 680-685 

1 nlshnianan P (1990) 1 ffect of certaln plant extracts against (orynrsporu tutrrcolo 

I 

III~ I 

Intirun J A4vcol PI Purhol 20 267-669 

dnd I in I< R (1944) 011 the occuielice of Co//erorrrth~rrn cupsrcr In Chlnd 

I~~ttirrn ~RIU Stre~ice\ 11 102 167 

l ooliii\ I\ L dild SIiuII C A ( I 937) In \krhollc rn Plan/ Phj \rolog~(Mc Cirdu 11111 

t3oiA ( o) Neu ! ork 

'1 oi\~\ O H Rosenbergh N 1 l arl A L and Randall K I (1951) Protc~n 

drtern~lnatron using 1 ol~t~-c~ocal~sau reagent J B~ol C hen1 . 193 265-27.5 

Mc h~~int-\ Ii I4 (1923) A ncu $>\tern of grad~~ig of plant diseases J 4grrc Kc\ .26 

105-218 

Mahadtian./\ and Slid11dr.R (1096) hk~hotl\ In /'h)~&ro/ogrc~r/ plunl Pu~holog~j (4 "' 

l di ) SnaLam~ Publ~cauon Chennd~

Mahrotm,R.S. (1996). Plan1 Physiology. Tats Mc Graw llill Pub. Com. Lim~td. New 

Deihi. 

Munandhar.J.B:, 

Hartmsn,G.L, and Wang,T.C (1995). Conidial germination and 

appresorial formation of (.'olletorrichum cul~.~ici and (:doeosl~oriordc.s isolates 

from pepper. Phnt Diseu.rc.s. 79: 361-366. 

Manjeet Kaur and Deshpande.K.U. (1980). Studies on pectinolytic enzymes of certain 

fungi causing leaf spot diseases. lnditrn Phyropurh., 33: 333-334. 

Mariana.R.(:., Bruno,C.P.A., Wil1iam.F.B. and Laura.D.L.C. ( 1997). 'l'hr anii(irnga1 

activity of peptides from Heliunrhus unnus flower against Sclerotiniu 

scleroliorum. http:/labstract.aspb.org/aspb1997/j4-20k. 

Mar0on.S.. C1arissa.J.. 0pina.O.S. and Mo1ina.Jr.A.B. (1984). Evaluation of crude plant 

cxtracts as fungicides for the control of C'crco.~poru leaf spot of Munghean 

(I'i~niradiufu (L,.) welzeck.). Phil Phyopurh.. 20: 27-38. 

Mc Creadg.K.M., Guggolz,J., Si1viera.V. and 0wens.H.S. (1950). Determination of 

starch and amylases in vegetables. Applicatio~~ to peas. Ind DI~. ('hen1 ilncrl 

1:'iI.. 22: 1 156. 

Mc1irc1~ra.R S and Ane1a.K.R. (1990). An I~rr~~o~lut~~ror~ to Mycoloa,. Wile! 1:astern I.id. 

*Millinp.K.J. and Kichardson.C.J. (1995). Mode of action studies of the 

anilinopyrimidine fungicide pyrimethanil on Borrytis spp. 2. Effects on enzyme 

secretion. Pesticide Science. 45: 43-48, 

Misra.A.P, and Mahm0od.M. (1961). Effect or vita~nins and horrllones on growtlr and 

sporulation of C'ollerofrich~rm caps/t,r. Iitdiun ph!,topath., 14: 20-22. 

Mism.S.B. and Dixi1.S.N. (1976). Fungicidal spectrum of the leaf extract of Allium 

~u~rrum. Inrlicln Phylopurh., 29: 448-1.19.

*Moore,S. and Stein.W.tl (1948). I'hotometric ilinhydrin method Ibi II\C ill tllc 

chromatography ofamino acids ,I Biol. C'hmi.. 176 367-388 

Mukesll Kumar pandey. Singll.A K and Singh.1t.R. (2001 ) Mycoto\ic stc~dic, (11 \olnc 

lugher plants. lndrun IJ/i,yiolxr~h.. 54; SOY 

Murugaaandan~,V., Suryanarayana.R S and Venkatacl1alam.S. ( 1987) I:orili,~tion of 

infection structure ~n Colle/o/rrr.hum cu/nrci as influenced b! nirogen sources 

and cellophane ('rrrr Br . 13 671-675 

MuthulaLslimi.l' and Seuthaian1an.K (1087) \ sr of plaiit extract.; 111 the nlanagcmcnl 

of fruit rot disease of chilli caused b! Alrerrictr~rtr len~ric hces I'ro~ Brol 

('ortlrol of Plunr I>r\c~cicor. Natio~lal WorLshop. Coln1batol.e India 

Nanddn.K.J and Anup.K.J (2002) Allrrrrn ,~u/~~~irm Garllc I.ashun IJliv/op/ior.r~i. 3 h- 

I h 

Nan1r.S I' and Kadu.H I3 (1987) H'l'ci.[ ol' somc med~cinal plant< cutracl on ,oilre 

tungi Arra Uorunrcu Ilidru. 15 170-175 

Narai~l.\ and I)as.I)S (1970) luxiii ~voduction during ~~~I~O~CI\C? 

01 

('oll~~orrrcbirrn CCI/~\~CI causlnf ,inthracno\t. nt' chillies Irltli~oi Ph)'r~l~~r~h 23 

484-390 

Narain.A and I'anlgrah1.C ( 197 1 ) hfficac! of' wnr. fuiigtc~dal cc~nilioundi to coiitrol 

('olle~orrrchum cup~crrn ~r\c,crses. \ar~trnal \\orh\lrop. 

Cormbatore. India 

Natarqlail.M.K. and La1ithaLumari.I) (1987) L.mf extracts of Lcn~ \orsru riii,r.nii\ as 

a~~~iiungal agent. ('un. Scr.. 56 1021- 1022

I'alqmwer.M.Y. (19117). Growth and sporulation of C.capsici and C'.curcuniue on 

different culture media. Indian J. Mycol PI Pathi., 17: 208. 

Pa1arpawer.M.Y: and Ghrude,V.K. (1997). Influence of different nitrogen sources on 

growth and sporulation of C.capsici and C.curcumae. lndiun J. Mycd. 1'1. 

P~/hol., 27: 227-228. 

l'andey.l).K.. l'ripathi,N.N., T'tripathi.R.D. and Dixit,S.N. (1982). t:ungitoxic and 

Pande!,K.K. 

phytotoxic properties of the essential oil of Hptis suuveolens. Phytoplh.Z.. 

344-$49. 

(1983). Antifungal activity of some medicinal plants on stored seeds of 

Eleusine corucana. Indian Phylopath., 35: 499-501. 

I'arr~nala~l:I. (1999) Studies on the effects of culture filtrate of C'olle~orrrchum cccp,\icr 

(S!d ) Butler and Bisby on seedlings of Cupsicunl rtnnoni L. A4.Phil. 

I)r\\c~r~u/roti. Pondicherry University. India. 

I'atclia~\;c/~nmman.S. (2002). In 11i1ro studies on antifungal acti\it! of Aglriu 

~,I~r~~~r,ytior~Ic~' \ ar, coro.1~1lensr.s Gamble, A! Phil ~~.\.\c~f'/i~l~0li. Pondichcrry 

\ ln\crsit!. India. 

I'at~l.l

I'ug;~/.licntlii.S. (2001 ). IYfecl of aqueous leaf extract of I).innoxiu. culture til~ra~es of 

lirchodermu spp. and bavistin on Cb~leiolrichum gloco.sl~urioides (I'enz.) l'enz. 

And Sacc. Mphrl. Disserto/ion, Pondicherry Ul~iversity. India. 

RadI1a.M.. Mala.S.K., Revathy,G. and Solayappan,S.R. (1998). In ~fitro antifungal 

properties of garlic (Allium .sa!ivum L.) extracts against Colle~o~richumjhlcarum 

kcnt. .I. Hiol C'onlrol. 12. XI -83. 

Ralapaise. R.G.A.S. (2002). http:llw.gov.lk/AgricultutelAgridep~1~ordi/Pub-Absl 

f

*Kawal.V.M.. 1'atel.U.S.. Ra0.G.N. and Dcsai.K.It. (1977). Chemical and biochemical 

siudics oe cataracts md human lenses 111. Quantitative study ol' proteins, I

Senthil Kurnaran,R. (1998). In ilr1r.o phys~olog~cal studlea on the rllecls ol w~ld pldnl 

extracts on Cbllerorrrchum cap~~cr (Syd 1 IJutler and Utsb! causing antli~aenose 

111 Ctrpsrcunr unrtum L (cli~ll~) M lJlrrl I)r~.serro~ron. I'ond~clicr~) (Jn~tcrs~t! 

India. 

Sentht1nathan.V and Naras1rnhan.V (1987) Effect of plant exiract3 on mycelr~l you~l~ 

and spore germtnatlon of Allcrnurru ~enutc\rmu lnc~tlng the bl~ght ol' onion and 

the nature of the ant~fungal compounds I'roc Niriror~ul Mi11 k\hop on Ij~olo~~r~ul 

('onrrol of I'lunl drreme 1'NAU Colmbatorc, lnd~a 

Shuhla.11 S . Chaturved~.R V and I ripath1.S (' ( 1989) h4ycoto\1c ploperrlea oi Iru~ts 

of I.oenrculum vulgure and IJrmj~rnel!tr itnrrrrm J Ir~dran hot ';oc . 68 113-144 

S1ngh.l l N P . Prasad.M M and S1nha.K K ( 1993) E\ aluat~cin ol mcdlc~nal plant 

eltracts agalnst banana rot .I Indrr~ri hol Soc . 72 163-164 

Slt~gh.M I . S1ngh.M S and Pandc).R R (1001) Lffrct ot storage t'ilng~ on elcct~ol~tic 

lenbayc from I Ice seeds Indron I'ht II~INIII ,51 12;- 125 

S1ngh.V . 1'athak.S P . Chakrabo~-t~.D K and S1ngh.S 13 (7001 I tfl'ect of some 

lut~g~c~des. orgnlc ammdnients and h~oagent on rnallagclilrnt of vine rot ol 

po1111r.d gourd lndrun Philoporb. 54 508 

Srl\asia\a.4 K and 1.al.R (1997) Srud~cs on blotuny~c~dai propi.lrlc\ 01 lcal c\tr,cct ol 

sonic plants Indrttn Phi~/o/xr/h . 50 408-4 I 1 

Sugundlal Keddg.M . Karnapandu.S . and Appa Rao.A (1980) Cross reslstalice ol 

ILng~c~dc resistant slralns G'ioeo\lx~r irtm ornprloplrirgrrm ( o!!c~~o/rrchwir c[rp\rc i 

nnd I.rr.\crrrtm~ ox,vsporu~i~ I'rp 

I'lr~/o~/,t~r,/h . 33 450-455 

I~ropcr.\rr~ to olhc~ lu~~g~c~dc\ Irrd/in~ 

Sutto11.B (' ( 1973) Coelo~nycctes spp 111 th~, hingr Vol IVA I ds C C A~n\\ci~?Ii. 

Sparrow.F.K and Suss~uan.A.S., Acadern~c Press New YorA

Sutton.B.C. (1980). The Coelomycetes. Commenfiealth Mycological Ins~~rutc Keu. 

England. 

Thoppil,l.E., DeenqM.J., Tajo,A., Sreeranjini.K., Kochuthressia.M.V and Leeja.1. 

(2002). Antimicrobial potential of the essential oil of Arlin~i.,irr n11:;1.1c.i1 (('larkc) 

Pamp. Geohios, 29: 181-182. 

. . 

1hyagarajan.R. and Krishnamoorthy,N.R. (1999). Antifungal actit ~ty ol' sonic 

compounds isolated from Eclipta alha Hask. and Wedeliu /riloho/tr (I..) 

Geohios. 26: 91 -94. 

Hitche. 

I iuar~.R.. IJpadhyaya,P.S. and Mishra, D.N. (1990)..l.oxicity of vapours of some green 

p~ants against Aspergilusjlavus and Penici//iccm oxulicum. (;eoh~)s: 1.7: 63-66. 

'l'r1patl1i.M.N.. Shuk1a.D.N and Dwivedi.B.K. (1999). EfTect of some aqueous exlracts 

of leaves of sonic medicinal plants on spore germination of k'~rs~o.ium spp. 

8to1,rd.. 10: 43-44. 

* I suda.M.. Ileyama,A.. Nakana.M. and Fujino.Y (1972). Chemical components of the 

l~pid ol'fielmin~hosporium oy:uc 

.inn l'ln,~o/>urh. Soc.. .lapan. 38 60-07 

*l ,ma.S.. Ravisl1ankar.K.V.. Prasad,P.G.. Reid..l.l>, and Uda! akumal..M. (1993). 

Abscisic acid responsive proteins induce salinity stress tole~.a~ice in lingermillet 

( E~~,u.vIII~, coructrrm Gaerth.) ('urr Sc.i.. 65 514-554. 

I lpadli!aya.M.L.. and Gupta.R.C. (1990). tltects of txtracts oi'snmc mcdlclnal plants 

(111 thc groulh of ( 'ur~~rrlurio 11mu1cr Ir~dirtrl .I \!11col 1'1 I'uilrol.. 20 111- 145. 

v ii~~hnc! .. 

.\'.(2001). f'llect of plant extracts on I)~.c~~~hs/rrrr ~runlirwtr. the caubal agent 

of stripc disease of barley. lndiun 1'/1~,1(1~xrrh.. 51: 88-90, 

Vcc.ra~nolian.li.. (iovindarajulu,'f. atid Kanms\\anl!.V. 

(1994) Uiochemical and 

ph!.siological changes in chilli leaves inoculated with Alternuria solani. 

.kt P~JIII Scr.. 8: 414-416.

Vidhyisekara~i,l'., Rabindran,R., Muthamilan,M., Nayar,K.. Io~h . 54: 393-298.

LIST OF PUBLICATIONS 

C11itrt1.l I.. (ic~mathi.\', and Kan11abiran.B. (1 999). Effects of extracts of I)tr/trrtr rn~~o.~irr 

\llllCr on the spore gemmination and mycelial growth of' ('ol/c~ro~r.~i~/~rr,rr i r~/)\rc.t 

(S!d. I Uutlrr and f3isb). C;eobio.c. 26: 177-1 78. 

(.'hitra.li . (jomathi,V, and Kannabiran.B. (2000). Inhibitory rt'frci of l)rr/uro rnr~orro 

\lillcl on the en~ymatic activities of the anrhracnosc Sunyus ('o//~~o~rrr III~I~I 

~'1/1\11 1 111 1 ,1/~11. 111diun f'l~,v~~~p~r/l~.. 54: 353-255 

( i(l~ima[l~~.\ . ('1litri1.l I . itnd Kanr~ahiran.B. (2001) Efkct oChcat and organic \~rl\cnt> oil 

~lic ;r~~iiti~ngal act~vity of' leaf extracts against C'o/l~iorricAirr~t spp. L",ohior. 28. 

l25-l28. 

l11tra.tl 31111 lii~nni~hiran,B. (1001). ESSects of' aqueous leal' e\lraci.: on [hc aporr: 

;;:~il~~l.~liti~i (11' ( ~~l/~~~orr.r~~/i~rm 

CCIIJ~~L~~ (Syci.~ B~~tler a11d BI+! 

( ,!,I! , - j?-5(1, 

1;iol 1.111 d 

( Il~ir,~.l l .~iid ha111iah1r31m.B. (2002). Screening of aqueous extract2 of' sow plalit3 on 

I!); con~d~al germination and rnycclial growth of ('ollcrorr r'.lrrrr~r trr/J\rr r (S!d. 1 

RittlL.1 .!!id Rish\ G'eohio\. 29, 185-186.

Vd. 26{4), 1998 177 

GEO610S26~l77-178,1999 

EFFECTS OF EXTRACTS OF DATURQ INNOXIA MILLER ON THE SPORE GERMINATION AND 

MYQLIAL GROWTH OF COlLElULRICHUM CAPSICI ISYD.) BUTLER & BlSBY 

H CHITRA, V GOMATHI and 8 KANNABIRAN 

School 01 1/19 Suences, Pondidreny UniyBrs~ly, PondrchenydO5 014, India 

(Rwived Febmary 27, Rewsed July 26, 1999) 

Key wds Extra& of Datum tmwa, spore gemmallon, rad~al growth of ColIelomd,um apsm 

ABSTRACT 

While all the extracts of Datum ~nnar~a lnhmbfted spore pernunatton ot Cdleotndrum 

CBP~CI, only flower hol waler exEnct (BOOC), fmtt ethanol extnrct and Rower methanol 

extracl mnhtblted the radlal growth of gemmnated spores 

INTRODUCTION Spore suspension of about 8000-12000 

Fruit rot of chilly caused by Cd/etotnchum sporeslml of sterile distilled Water was 

capsln is one of the important and destructive prepared by using the haemocytometer. The 

diseases which causes substantial loss CaVlty slrdes Were maintained Ill tnpllcates 

In recent years, several phytoextracts are lnSSlde a moist chamber with 20 per cent 

being used as funglc~des for the cdntrol of extract and equal Of Wore 

various plant pawens (Gohi1& Vah, 1996). SUSPenslOn. The conlrol was made up of 

There are a number of reports on the ~ ~ \ ~ ~ $ 

antifungal nature of different species of (Nene 1971, ;vas followed to study the 

Dau@ ('J~dhyaYa 4 Gu~ta, 1990; Gomathi 

of all the exbacts on the 

Kannabiran! lgQ9). There Mtems to be no grM of C, 

For this purpose PDA 

lnfcnation warding the '%Iicida1 

Of (Potato dextrose agar) plates containing 10% 

9~tura innoxla so far in this connection. em were used, The percentage of 

the present investi@tion was taken spore gemination inhibition and mycelial 

up lo W out the effect of different exbacts gm was by using the 

of D. On ti^ and fm& of (1927). 

RdialgrowlhdC.~. 

Only fruit, !lower and root of D, imoxia I 1 ~ x 9 

were studied for their antifungal awb and I = InhWition of spore gerrninationlmycel~al 

their aqueous extracts were prepared by g m ~ c , = gemination I growth In control, 

using the method of Senthihathan & and T = gemnation I growth in treatment. 

Nuasimhan (1993) and for hot water extrads 

ol huit and flower, thek aqueous extracts were RESULTS AND DISCUSSION 

heated to different temperatures (40°C ,a the tested extracts of D, bmoxia were 

12%) (Narayana Bhat 8 Sivaprakasam, fwnd to produoe varying degrims of inhibition 

1803). Ethanol, methanol and acetone in spore gemhation of C. cap& 

UWb Offdl and fbW ~rsDared by (We 1). Among them, flower hot water 

ruhq the mothod of Natarajan il Lalitha- &~CI (4@i2i°C) produced cent per cent 

kumui (1987b). inhibition d spore gmination both after 24

GEOBlOS 

178 

~~w 1. -01 differant exirec$ of 0. won REFERENCES 

the spore gadnatbn ndidgmh ~ohil, V.P. and Vala, 0.G. Effect of axlnrcts of 

~tornvm capaidpaid 

somemedldrulphntsonthegrowthof 

Nam of e m Pereentape inm 

F ,,,,, Fwrkun m.wMm6. Indian J. myod. PI. 

gemstion wh Palhd., 1996, 26, 110.111. 

24h 48h 

Gomathi, V, and Kan~biran, 8. Effects of 

Flower aqueous exiract 45.73 36.47 31.13 aqueous leaf extracts on spore 

FWR aqueous extract 38.61 33.77 22.78 germination and mycelial growth of 

w t aqueous extract 37.56 36.66 - 

FWr ethanol extract 46.64 44.78 - 

anthracnose fungi infecting Capsicum 

~de(heno)em 52.74 51.58 51.22 annum L. Proc. Indian Sci. Cong, 86th 

Flower methanol exhad 41.65 41.14 51.22 Session, Chennai, 1999, pp. 43-44. 

FN& methanol exiract 29.82 29.22 17.07 

vhro on the 

Flower &&one emcf 36.61 33.69 38.08 JabminB' R' In ' 

FwH acetone extract 28.08 26,82 11.90 efficacy of aqueous plant extracts against 

Fruit aqueous emct heated to 4: 

the anthracnose fungi, Colletotrichum 

40' 27.08 26.52 2.46 

60' 30.24 29.64 3.70 

capsici (Syd.) Butler 8 Bisby infecting 

80' 32.38 30.54 1.23 Capsicum annum L. M. Phil. Thesis, 

1W 29.12 28.47 4.93 Pondiiny University, 1997. 

121° 31.1627.54 7'40 Narayana Bhat, M. and Sivaprakasam, K. 

Flower aqueous extrecf heated to C 

40' loo loo 34.37 Antifungal activity of some plant extracts. 

60' 100 100 35.93 Proc. Biological Pest Control Conf., 

80' 

1W 

loo 100 loo 100 51'56 46.87 Madurai, 1993, pp. 335-339. 

121" 100 100 23.43 Natarajan, M.R. and Lalithakumari, D. Leaf 

extracts of Lawsonia inemis as antifungal 

h and 48 h as compared to other extracts. 

agent. Curr. Sci., 1987a, 56, 1021-1022. 

Thisindicates thefungitoxicandthermwtable Nalarajan, M.R, and Lalithakumari, D. 

nature of the active compounds present in it. 

The fact that the inhibition rate was reduced Antifungal activity of the leaf extract of 

after 24 h in all other extracts indicates their 

inennis on Drechslem Owe. 

fungistatic nature (Jasmine, 1997). Indian Phytopelh.. 1087b, 40, 390.395. 

Higher rate of inhibition of radial mycelial 

Nene, Y. L. Fungicrdes in Plant Disease 

growlh of C, capsici was brougM about by . Contd. IBH PUN. Co., New Delhi, 1971. 

flower hot water extract (w), fndt ethanol Sen'ilnathan~ V, and Narasimhan+ V. Effect 

extract and flower methanol extract as 

Of plan' extracts I products On 

compared to all the other em& (Table 1). growth and spore germination of 

This might be due to the e m potentiality Ahemaria tenuiSSima inciting the blight of 

of the solvent used and 480 on the m n . onion and the nature of the antifungal 

tration of the active principles present in them wmponent8. Pw~ BMogicalPest Cpntrol 

(Nataralan 8 Lalithakumari, 1987a). In the mf., Madural, 1993, pp. 307-313. 

prosent study, the rate of inhibition of spore Upadhyaya, M.L and Gupta, R.C. Effect of 

germination could not be correlated with the OXtracts of some medidnal planta on the 

radial growth, which might be due to the ability growth of Curvuleda lunsta. Indian J. 

of wmPouM5 of the concerned extracts to mpd, Pl, POW,, lm, m, 144.145. 

inhihi spore gemination but not radial growlh Vincent, J.M. DisImon of f ~wl h m e In 

and availability of time and nutrient for the lhe prim of own imm, 

mymri groM of pathogen (Jasmine, 1997). igg, 159, 850, 

&urn

Inhibitory effect of Datum innoxia on the enzymatic activities of 

the anthracnose fungus Colktotrichum capsici in vitro 

hymes produced by tix pathogens play a pmnuncnt 

role In Lsease development (3, 11) Therefore 

hbluon of these enzymes IS rmportaat in dlsease 

mtml lnhlblt~on of Ihs prcduchon of enzymes by 

pathogens, by plant pmducts has alnady k n reported 

by many workers (6 9) Earher stwies m our labora 

tory (unpbllshed repon) on the effect of extracts of 

fru~c, flower and rmt of D mnmo thowed that fruit 

(dhanol) and flower (hot w*) e x showed ~ s~gdcant 

mhbmon of s p gmrunahon and mycel~al 

gmvlh of C capstch Funher stuhes have been con 

dud now on the effect of h ~ (ethanol) t and flower 

OMt water ) extracts of D IIUIOXU~ on the pecmlyuc 

and ccllulolyuc enzyme producnon of C capslc~ under 

m vltm condItlon 

Flve g mature fresh h t (calyx and h~t) of D 

IM~M was macerated wlth 20 ml of ethanol In a ster- 

11c blender after surface stenllzatlon In 0 2% mercunc 

chlonde solutron Ihe ground nuxtun was squeezed 

through double layer chtese cloth and the res~due was 

&d The solvent fracuon was cenmfuged at 3000 

rpm for 10 nun at mm tempvnture The clear supernatant 

was dned m a desiccator m remove the solvent 

The dned extract was dssolved agun in 5 ml of stenle 

Lstllled water (4) 

Rcpvltko of Lwer bot water cdnd 

P~ve p fresh flower (calyx and corolla) of D 

IIWUIU wu\ mraccJ wlth Sml cilcnk d~st~llal wukr 

m a rtenk blendcr nRu surfre slenluatloo m 02% 

mCtnrric chlonde solution 7he $round mture was 

q d through dwble hyu dvcrc cloth md the 

nridue wu dkdd Tbe wlwt fncam WIS mPI- 

~I(MOOrpaforlOmtnule,ItroomtPmpnaae 

Tbcpcll#wdiarrdcduidtbdur~twr 

hwvd#WCfnamarbuh(8) 

Reparah of peetiadytic enzgws of C C~SICI 

For the purpose of m vltro tests, the pathogen 

C cwrn was grown m Czapek's mtdtum contatrung 

2% pechn Two Lscs of 9 nun from the growlng t!~ of 

seven days old culture of C C~SICI wm ~noculated In 

250 ml Erlenmeyer flasks wntauung 45 ml of Czapek's 

medlum All these flasks contatrung Czapek's med~a 

were autoclaved at 15 p s I pressure for 20 nunutes 

before moculahng wrth dws of C capsrcl In order to 

detect the effect of hit (ethanol) and flower (hot water) 

extracts of D mnnoxu on ttLe enzyme pmductron by the 

pathogen these extracts were added (treated) to the 

flasks separately at 10% concentmuon Med~um devo~d 

of exaacts served as control Ihe flasks were 

incubated at 24-26" for seven days On the eighth 

day, the culture filtrates were o b ~ by d papsrng the 

l~qu~d cultures through Whatman No-1 filter paper 

placed In Buchner ftnnel The culture filtrates were 

stored at 2-4" under a layer of toluene In order to 

avo~d contanunatlon and used wrth m a wak These 

culture filtrates were used for enzymauc stud~es 

Reparatian d duldytie mzp~s of C C~JSICI 

The enzyme scum was prepared In the same way 

iw m the case of pcct~nolyuc enzymes But ~nsteed of 

addmg 2% pecun to the CEspeL's medium, I% car. 

boxymethyl cellulose (CMC) was added as carbon 

SOUlCe 

ihihuh d enzymatic Ktlvllle cf C, capin' 

Tbe ncunt1et of paturolytlc cnzylm polymrhyl 

gal- (PMG) snd pea pans d~rmnrse (PTE) 

Wac daanuKd by cahmamg &e I p of ~lscoslty m 

thcnrtioPmmrebj.~mcuhcdrt~~tervrls 

of Mmta&utmefmmOto 180 muw. pfta 

~thc~onmoxbnePaPMOmenMoa 

mix~tecoMuvd4mlofl%pechnd~,Imld

acetate buffer of pH 5 2 and 2 ml of culture fdm In 

the ease of PIZ m a bon to vlscomemc method, rhe 

enzyme actlvlty was detected by calculatmg the amount 

of tluobarbitunc acid (TEA) m n g substnnces pro 

ducod by the culture filtrate The W o n mmtun for 

PIZ conwed 4 ml of I% pectin soluuon, I mi of 

bonc acid-borax buffer of pH 8 7 and 2 ml of culture 

filtrate (7) 

The cellulolybc enzyme cellulese mnry was also 

detmrumd by the same wscomehlc method ar In the 

case of pcctlnolpc enzymes 'he m o n mixture here 

eontuned 4 ml of 0 5% CMC soluaon. I ml of acetate 

buffer of pH 52 and 2 ml of culture filtrate (7) In 

akl~bon to the vscometnc method the amount of re 

ducmg sugars liberated by the culw filtrate dunng 

Ihr~ hours of reaCUon Ume (I e 0 lo 180 mnutes) was 

also calculated to dekmme the acuvtty of cellulase 

(5) In all the caus the loss In vlscos~ty 1s expressed as 

percentage loss In v~scos~ry and calculated by the formula 

What, V =Per cent loss m v~scos~ty. To = Flow 

tlme of w o n INXNR at zao ume. T = Flow bme 

of muon matun at a patucular mtcrvd, and T. = 

Flow ume of dlstllled water 

Eauocu of fm~t and flower of D mnnoxma were 

fcund to uhblt bolh endo @MG. PTE and cellulese) 

and exo (PR and cellulase) act~vlues of pecunolpc 

and cellulolybc enzymes of C cqs~cm U&I ln vlfro 

condibon (Tables 1, 2 and 3) Umalkar and ius assoc~am 

(10) rcporlcd that the ~ntub~twy effect of exuocts 

of Acaclcl nmlor~co on the enzyme producuon of some 

Tabk I Effeet of cxmts of D nnoxu on the acbvlry of 

pechn uan~lim~nase (PTE) of Ccops~cl 

Tlmc ~nlclval 

Lar In v~scos~ly In per LO[ 

Control FNI~ Flower 

ethanol hot wsar 

extan exm~ 

0 0 0 0 

30 5 2 05 18 

M) 8 6 I8 18 

90 12 7 3 8 34 

120 16 1 5 9 5 2 

I50 21 2 7 6 69 

180 269 119 8 9 

TBA rtacung 3 7 2 7 13 

substances pmdund 

(In un~l) 

Mean of 3 rcpl~cates 

UNI = Changes m absorbandm1 of emymelh x 101 

Table 3. WKI of exuacu of D imxu on the actlvlty of 

cellulase of C copsrc~ 

T I interval ~ 

-- 

Loss in v~scos~ty m pn cent 

Conml Fmlt Flown 

ethanol hot watcr 

exmt CXWC( 

Rcducmg sugars 73 3 53 3 23 3 

lrtuated @gM 

of cmYldl) 

Mean of 3 rcphw 

Tlmc fntcrvll 

Imc 10 vlsamily ~n went 

Conmrl FNII Rower 

&nol hotwucr 

exwt c x t ~ 

0 0 0 0 

30 7 9 2 3 16 

60 16 8 5 3 1 

#) 24 7 86 45 

120 308 102 49 

Is0 354 13 

6 1 

180 413 158 9 

Mcnd3nplii 

pPthogeluc fun@ mght be due to the phenolic compounds 

pnsent m them Byrde (1) has reponed that the 

vanetal res~slaace of cum vanehes of cldar apples to 

brown rot was due to their hgh content of polyphenols 

Chona and h~s assoctaW (2) repoRed the tlbb 

tory effect of magncslum Ions and polassturn phmphate 

la potsto tuber e m and m&c acld m ~ l e 

jua on a number of plmt *I Th hblto~y 

dfectoffrutandQowaextm3dD urnmybe 

ductothcpresvlccof~MmpouDds~.ek~~ 

& ~ompouads, which naods fur the^ mvcsnga- 

Uon

Of the extracts of D. ~nnoxio teatcd, flower (hot) 

water exmct is more efficient in inhibiting fhe exo 

activities of enzymes of C. cap~ici compand with fruit 

(uhmol) extract. This could be attributd to the variation 

among the compounds present with in them and 

also to Lhe extracting ability of the solvents used. Since 

~nhibition of exo activities of enzymes of pathogens 

play a vital role in plant pathogenesis, funher in vivo 

lcsting of flower (hot) water extract of D.innoxio against 

anthracnow fungus will lead to the development d 

ecolricndly fung~cidc. 

REFERENCES 

I. B& R.J.W. (1957) J Hon. Sci. 32: 227-238. 

2. Cbora, BL 11932). AnnBor 46 1033-1050 

3. Dl Pletro, A. and Roncero, M.I.C. (1996). 

Phylopyholo~y 66: 1324-1330. 

4 Cawan, T. (1993). Gtobios M: 261-266. 

5. Jsyararnan, 1. (1985) Lnbororory Manual In 

Bioekmistry 2"d Ed. Wilcy blem Ltm~ud, Chemai. 

6. Mshsdcvm, A. Kuc, J, and WUUIm, E.B. (1965). 

Phytopothology 55. IWO-1003. 

7. Mahdev~ A, and Sr&Jhar, R (1986) Mcfiudi in 

Phys~ological Plan1 Parhofogy. 3' Ed S~vakami 

Publ~cations, chcnnu. 

8 Narayans Bhat, M. and Sivapraham, K. (1987). 

Pmc, of Nano~l Workshop on Biol. Conrrol of pht 

Diseases. Coirnbatore, 322-326. 

9. ~RK"PateI,ICD,ShumqA.andPlthrlqV.N. 

(1992). Indian I Mycol PI. Porhol. W2): 199-200. 

10. UmslLsr, G.V, Sdyl Begun and Arua Nebrmhh, 

K.M. (1976). Indian Phyopdh 29: 469-470. 

II. Ylkoby, N, Fmmm, S, Diwor, k, l(ren, N.T. and 

Pnaky, D. (2WO). Mol. Plnnr Microk lnrtmcr. 1H81: 

887-891. 

Rece~ved for pubhcatlon March 12, 1999

EFFECT OF HEAT AND ORWlC SOUEUTS ON THE ANTIFUNGAL ACTIVITY OF LEAF UTRAm 

MST COUElPTRlCHUY SPR 

P-W 

July 11,2044: Rmbd JMwy 90,2W1) 

Kly : h WW baf exWb, wlwnts, heat treatments, Cdletotridrum ~ppid, 

c. funoiloxic adivlty 

ABSTRACT 

COW OemJruh of C. capsidand C. gkmwiidues was totally Mlblled by bat 

.treal6d aqueoa exttWa of dwMlm D. m#e/ and F! jdhkm. Hhbr percent inhlbitkn of 

redid gmwlh of C. cawid and &fmpWm was btwght about by ethanol extnrct of 0. 

W, ((84.52%& 875%) lolkwedby heat treated aqueous e W of S. lorn (82% & 79.43%). 

INTRODUCTION 

Anthracnose d chilli is one of the important 

and destructive diseases caused by 

CdletotfMum cepsici (Syd.) Butler and Bisby 

growing fields in and around Pondicherry. 

Cultures were identified as Colleiotrichum 

gloeosporioidesand C. capsiciand tested for 

their pathogenicily. 

and Cdle@M~um *ma Penzig. In Aqueous leaf extracts of S, torvum, D. 

India Mder'rngenial weather ~~ the mete1 and R juII~lil were prepared by using 

disease MY cause even 12.25% loas in the the method of Singh & Tripathi (199.3). Hot 

crop (Kannan et al.8 1998)- In recent Years, mter extma were prepared by heating the 

phytoextracts were proved be aqueous extracts to desired temperatures 

funOiddaltomanyplantpathoge&fyngi(Mista (40-121%) for 10 minutes (Singh & Varma, 

& Ml 1g76; et *.I 1g91). Asthana et 1981). Ethanol, methanol, acetone and 

(1984) reported that C, capsidi and C, benzene extracts were prepared by the 

-ddes were tdally by method of Nataajan & Laliiakumari (1887). 

e-1 eugend (200 ppm) and citral Conidial suspnsicn about 400MWM0 

(300&400ppm).Itwasalreadyprovedthat 

the 

Sdanwn lowm Sw,, ~~,,,d conidiialml in 5% conc, leaf extracts was 

mefelLandmjm(Sw.)Dc.~ prepared by using the haemocytometer. 

C.+ Conidial germination studies were carried out 

dronO 

C, mddeS (oomathi & in cavity slldes inside moist cham'bers, 

lw). me study was to 

gemination was obsetved after 24 and 48 h. 

detmine the of w e leaf extmcts, To evaluate the effect8 of leaf extracts on the 

obtained in different solvents and heat dial m~celial gmwth* Potato Dbar 

treatments on conidial germination and (PDA) m n g 5% 

rnyodirl the ~thogens. leaf extmds were used. PDA PW without 

leaf extracts served as control. Triplicates 

MATERIALS AND METHODS were maintained for each treatment. The 

~a caw d~lll~~ ahowing typical percent inhlbltion of conidlal genlnation 

rot w, wm from M I (PICG) and percent lnNbition of radial m~ceu

126 

GEOBlOS 

gmwth (PIRMG) were calculated by the (a) wCvmk#tbn:wmd 

fotmula of Vincent (1927). 

allthe~planbeatracM~dihn! 

phyoice-chemlorl OOMMOM wen found to 

RESULTS AND DlSCUSSlON 

wyinO drgne, d khbtktl on 

Effects of dmerent leaf e~tri?CtB of S. conldlal gertnlnatlon of C. ap8IcI and C. 

torvum, D, metel and P. juliflora'on the gbwspom Ceini percent Wbitjon on 

conidial gemination and Inycelial growth of conidial pflthdh of both the pathogem 

C. capsici and C, gloeosporlodes are was broughl about by aqwu and heatpresented 

in Table 1. treated lurbacls (BOPC, 100°C & 121%) of S. 

~ a I. ~ EM e of some plant exbcb on tha pew WllMlion of C d k m WP. 

I 

c. cdasid C.gksosporddbg . 

Trea$nent C. gm. - R.QL C.gem. - R. 0. 

24h '4th Bthday 24h 48h Bthday 

S. m: 

100 7839 100 100 78.65 

41PC """2 992 79.84 100 100 77.38 

604: 8622 100 78.07 100 100 79.43 

804; 100 100 82 100 100 78.5 

100 100 80.57 100 100 n.52 

121% lorPC 100 100 79.94 100 100 74.16 

M8thad 100 100 76.58 88.45 885 67.45 

w!ald 94 88.m 69.09 96.77 88.N 678 

Acetone 77-79 EZ.7 74.34 827 89.m 0 

Benzene 88.96 90.8 46.13 100 83 0 

D. metel : 

100 80.09 100 100 

%? im lW im m 

g 

100 100 

804: 98.59 100 74.17 100 100 71.60 

BOC 100 99.25 72.64 100 100 66.65 

100 107 70.86 100 100 50.81 

121% lmc 100 100 47.12 100 100 58.56 

fWd a84 94.74 75.4 100 100 

Ethand WP 89.88 8452 100 100 073 

A&m 88.88 95.44 71.w 8s 100 0 

6 8 ~ 86.31 ~ 81.m 0 100 100 0 

Fjumkn: 

97.7 100 41.38 958 100 38.32 

2? 

- 

100 QE.57 45.81 100 

38.88 

B(K: 100 87.87 42.44 100 

m 

loo 4232 

100 100 42.97 100 

.lacK: 98.1 100 

100 47.39 

107 4628 100 

D.5 

121% 90.02 88.47 42.91 100 100 11.7 

wmnol 95.78 95.05 04.94 100 100 3.3 

Emci 90.52 %27 4688 100 

97.7 

48.7 

872 58.18 8962 100 loo 

B8lZme 91.7B 77& 0 100 HX) 0 

C . m g m h a t i m a n d ~ . ~ & ~

tolwm, aqueous and heat-treated extracts 

(40% & 121%) of 0, meteland heat-treated 

exbad (BOOC) d t? iulMbre both after 24h and 

48h. this obcervation indicates the water 

soluble, themroaable and fungbxic natwe 

of adive principles of the concerned extracts 

(Singh & Tripathi, 1993). Certain extracts d 

thew planb produced cent percent inhibition 

on the conMial germination of C. capsid only 

but not in C. gloeosporioides and vbversa. 

interestingly the percent inhibition of 

conidial gelinhalion of C. Capsici was found 

to hcrease at 48h than at 24h when treated 

with ethanol, acatone wl bemne extfacts 

of S. lwwm and e W extract of t? julhm. 

Ma~beduetothefungrddalnahrreof 

the concerned extracts. Disintegration of 

genniMted conidia (Fig. 1A) iqd production 

of highly vacudated (Fig. 10) and abnormally 

stunted gemrtubes (Fig. 1C) were observed 

in the treated conidial sospenslon of both the 

test fungi. Even ungerminated conidia 

showed vacuolation and shrinkage. The 

vacuolar appearance of the conidia and 

permtube was due to the toxic substances 

present in the plant extracts. Similar 

obwvatfon was made by Sariah k m (1994) 

in the aame CoUetobidwm spp. when treated 

with aquws cukum RLbate of WIk subtiis. 

Difference in the inhibitory activity of the 

same plant extract obtained under different 

physico-chemical conditiona was already 

reported (Agacwal et el., 1878). The akohok 

fndionofdldAsavmdFiwcrrkwwashiOhly 

active qaht Aqw$Uu8 IwnClurf but its 

wfrectiongdnothaveany~tyonthis 

fungus. Natarajan & Uithakumari (lQ87) 

hm already reported the difference In the 

probnt inhibition of spore gemination of 

~rn~inethanol,mUlandPrrd 

wa&uctnact,dbrninemris.lMsmlgM 

kdwtohsd3lferencehIhe~bation 

ofadiwprkdpktpmmfhthe W&and 

thexhdhgabil#ydtheedvents~. 

(b) Radial groowth : The results on the 

antifungal nature of different extracts of S. 

torvum, D. meteland I? iuliflora on the radial 

gmwth of C, cepsici and C. gloeospon'oides 

~.l,AC.~ofplsntexbPcbon~~ 

garminailon of Coll~to~chum rpp. C. 

&wpdddm corkla in mcthnndthrd~ 

of f! IuMoa (A), In aaunw# heat-hated

areprewm(ildIn~abkl.~ronll~kc~ REFERENCES 

~,~sxtndofD.Mmfound Arthana, A., b y , N.K, Md Dhdi, &,N. 

topmducemm-of~ 

Fufglbxmd WllOlhd 

8onledon(lrhngl. 

g l d l o f b o t h p a ~ . ~ ~ ~ - 

MJ. iubtdol., 

med (mi000C) exmb of S. 

1084,24,1Wl. 

Agaiwal, I., Mathelo, C.S. and Slnha, S. 

produced higher PlRMQ of both the 

Studiw on the antlfungal acttvity of 

pathogens as compared with it8 otganic terpenoldeo againrt kpergIUl. lndlan 

solvent extracts. Hence, water can be w., lU9, 32,104-105. 

considered as the best sdvent for emng 

the inhibitory compounds of S.t q v Tewari ~ ~ 

(1986) reported lhat the inhi#krry sdMty 'Jf, 

aqueous extract of A# mnndos was 

betterm the other treamwmts ltke ethanol, 

benzene, hexane, chlorofo?l, and acetom 

extracts. In the case of D. matel, organic 

solvent e x w wch as lhoae d ethenoi and 

methenol were most effedlve In M n g the 

radial growth of both the pethogens than the 

aqueoucl and heat-mted aqueous extracts 

(40%-121°C). Among those of P. julifbm, 

methanol extract was found to produce 

highest PIRMG of both the pathogens. Slight 

incmaseinthePlRMGofboththepathogens 

was observed in the heat-treeted aqueous 

extracts of S. tmm and F! juHtlonn This 

mightbeduetotheevepwEdkneffect,which 

concentrated the inhibitory compounds. 

Owic wtvm like ethanol and methanol 

were found to be suitable for extracling the 

inhibitory compounds from 0, metel and P. 

iulinon. 

Thermostable nature of the aqueous 

emL of 0. mefel, S. tom and P. juhm 

upto 80C, 1000C and 121"C, mopwllwb and 

ava#abillty of the leaves th- the year 

indicate W these plant extracb can be wed 

as fungicides by the farmen. Further 

rwaroh is in progress in our hbocatory to 

tdlh@cimcecyofthwpknt~hh 

fietd anduons to cmd the fnrit mt of chilli. 

ACKNOWLEDGEMENT 

Dube, S., UpeBrW, 0.P. and Trlpslthi, S.C. 

Fung#o#icwdinredrepllent~cyof 

some aptme. In& Phyhpalhd., 1991, 

44, 101-105. 

Qomathi, V, and Kannrblran, 8. Effects of 

equeouc leaf extract8 on conidtai 

gemination and myceilal ~rowth of 

anthraonone fungi infecting Capsicum 

mnum L Pm lndkn Sci. Cmg. 86th 

!3eadan! aennal, 1989, pp. 43-44, 

Kannan, , R., Ananthan, M. and 

Baiasubramani, P. 

Anthracnose-A 

menace in chlili cuttivation. Spice I&, 

1998, 11, 2-3. 

Misra, S.B. and Dixit, S.N. Fungicidal 

spectrum of the leaf extract of Allium 

sattvum. lndlan Phytopefhd., 1976, 29, 

448.449. 

Natarajan, M.R. and Lallthakumari, D. Leaf 

extra& of lawsonfa bwmls as antUungal 

agent Gun. Sd., 1987,513, 1021-1022 

Sariah, Meon. Pbtentlal of Wus qp. as a 

biocontrolagentforanth~fnrltrot 

of chilli. MhjS. Appl. Bid., 1994,23,53- 

80. 

Shrgh, Inder)it and Vm J.P. Vim inhl#tor 

from &tun motd, lndiw Phyiqwlhol., 

1981, 34, 452.457. 

Singh Jacpal and Triprthl, N.N. ElRclcy of 

plant extract8 againrt Fusarlum 

oxyrrpoMn f.~. knlEs of Lm 8$a&ta. 

I J, lndbn W Soc., 1993,12,51~63~ 

Tewari, S.N. A m Gdnklu, for bkarcly of 

notudDlpRprodudCvn.Sd.,1988,56, 

1187-1139. 

Vimnt, J.M. Okaoltkn of fungal hyphan In 

Authon thank UGC, New blhl for the thr d m m. Mm, 

financial arreistance. 1927, IN, 850.

Many specles of Detwe are Mely used for medicinal purposes as well as for the 

Malogical control of diseases (Bambawaie et el., l(is5; Ganesan, 1993). But there aman 

to be-no infonnetlon regarding the antifungal property of Datum innoxia so far. en& the 

wesent w~er deals with the elfed of fruil ethanol extract and flower hot water extract of D. 

imoxle in ihe physical and some metabolic adivities of the ~dlel~hm capici, 

cawing anthracnose dlsease of chllli. A pure culture of C, capsiciisolated from anthracnose 

infected chilli frutt was used thmughout thls study. 

Prepamtion of fruit ethanol extract : 

Five g metum fresh fruit of 0, hmda was well ground for about 15 minutes with 20 mL 

of ethanol in a sterile Mender afler surface sterilization in 0.2 per cent mercuric chloride 

solution. The ground mixlure wassqueezed through double layer cheese cloth and the resC 

duswasdiscarded. The solvent ftadionwas centrifuged at 3000 rpm for 10 minutes at mom 

temperature (28C). The dear supernatant was drled In a desiccator to remove the solvent. 

The dried adred thus obtained wi$s fully dissolved In 5 mL of sterile distilled water (Ganesan, 

lm), 

Pnpurtlon of ftowr hot wabr extract 

Five gram ltesh flower of D, /moxhwas well gmund for about 15 minutes with 5 mL of 

sterile distilled water in a sterile Mender afler surface sterilization in 0.2 per cent mercuric 

chloride solution. The gmund mMure was squeezed through double layer cheese cloth and 

Hle residue was discarded. The solvent fraction was centrffuged at 3000 rpm for 10 minutes 

amom temperstun (28%). The pellet wasdiscarded and the dear supematant was heated 

to 80% in a water bath (Nemyana Bhat and Slvaprakesam, 1987). 

Antirungal propr~ly of ha axtract8 : 

Forthe purpose of our experiment, the pathogen C. cdpsIclwas grown in Czepek's

FQ 1. ERECT OF FRUIT EMANOLMTRACT AND FLMR HOT WATER DmuCT OF 

0. modr ONTHE FRESH AND DRY WOWf OF C. cgll(hgn) 

iL-1 

n#m hot mler Mnol 

DNA RNA mYm4

chh 6 )(lmlMnn 

msdlum. Two discs of9 mm wen, cuf fmm ttm orowlng tlp of the seven days old wnure of C, 

cldosjcland they me 

inoculated in 250 mL Erlsnmeyer flasks wntalning 45 mL of Czepak's 

media. All those fWs amlalnlng CzapeKs msdhwere autodaved at 15 p.s.1. pressure for 

20 minutes Wm InOCUkrting with dkcs of C.mpsIcI. In orderto find out the effed of fruit 

ethanol sldrad end flower hot watucndtact of 0. hxls on the gmwth and metabolic actlvitles 

of C.cspsMthese wars m M ('treated) to the flasks separately at 10 per cent concen 

tretlon. Medium dsvokl ofexlracts sewed as wntml. Two sets oft~Iplicateswere maintained 

for each habnent as well as for contrd. These triplicates were incubated for seven days at 

mom tempemlure (28%). The mycellel matsfomred were collected separately by passing 

the llguld wlkwes along with mycellal mats thmugh whatman No 1 finer papen placed In 

Buchner funnels. 

Mycellal mats collected from one set of triplicates were used to determine the fresh 

and dry weight ofthe C. capslcl(l.e., growth of the pathogen) by using dried and pncwelghed 

Whatman No 1 fller papen. Mycelial mats colleded from another set of triplicates were 

wed for the estimation of DNA (Burton, iW), RNA (Mahadevan and Sridhar, 1986) and 

protein (LGWIY et al., 1951) cantentsof C. capsld. 

The redudlon In the fresh wdgM ( FW ) and dry weight ( DW ) of C, capsici was 

noticed when It was inhibited bv hit ethanol extract FW = 0.46~ DW = 0.210) end flower 

hot water extract (NVI0.38g; QW=O.I~~) of D, in&xia compagd to contmiiFVV=0.63g; 

DW80.27g) (Flg.1). This might be due to reduction in the rate of cell division, membrane 

eneqy metabolism and Mosynthesls of essential enzymes of C, capsici, as reporled earlier 

by Kalaichelvan and Sumathi (1904). 

The redudlon in the DNA content of the fungus (Fig.2) when it was inhibited by the 

extracts of D. hoxle QNII ethanol extrad=0.27 mg; Flower hot water exlrad=0.24 mg) 

cMnpared to control (0.34mg) could be attributed to the redudon in the rate of cell division 

and resplratlon (Hammerschlag and Sislar, 1973). Butthe RNA (Fruit ethanol mdrl.23 

mg; Flower hot w a t o r ~ .I3 l ma) and protein (FNI ethanol extracI=l.O2mg; Flower 

hot water sxtmct=lmg) contents of C. cepsldwere found to lnaease compared to contml 

(RNA.0.72mg; p(oleinn0.41mg) (Fig2). The Imm in pmteln synthesis can be atlributed 

to the tmlfng of lress Induced pmteln synthesis Mlch was also supported by the in 

crerss In RNA symh6sk. Similar resub have bean obtained In the CBSO of USMN mafls, 

when lws Inhibited by BCM (a derivative of bnomyl) (Clemons and Sislar, 1971). 

FurVlsr inveatbation of these exhds botq In the h v/fm and b, vhro wndttion WM lead 

to th9 developmsnt of mvlmnmentelly safer Mofungldde from Dstcrie lmxle a~alnsl 

combwtlnnCdps#ps#

~rtfdololbmhnodr 

REFERENCES 

flrmklc, ON. Punit Mohrn and Mukta CtYEurbyr l995.Effhqof @me medidnrl phna 

winrtcottonptho~M.PtD~SddO~M-ZZ). 

~~tton, K 1968 Detuminrllonof DNA-mtion dth dipb- tu Gmsmur,L ad 

hio~ddvt,K (Ed) Mclhobinq-b~NudaicdputB,bL,rS*pyNn 

York, P: 163-166. 

Ckmonr,C.F! mdSi~H9.1~Loalhrdmofth si~of~ofahngitmicbcao~duintirc.Pa~lochcm.phlri1:32 

Qneun,T. 1993.hryitprict~of*@nthf~~.~ML 

~cNn~RSdSi~Iar,HD.~~llod nwd+2-baalmldrcdaarbo~ (MBC): 

Biorhcmicrl,ryopbdold~rgcrrdtPrid~roUbflpagdbd~ 

dPbtHodum~3r(ll 

Kahicbch.n,ET.dStrmnM,Ll9H.A~adc~lmoc~~aQllllrMhn~th 

471424423. 

Lowq ( XH,Rprc~ N.J,Pln, HLand~~RJ. 

l?5l.RotdadetarniDltionurtylblia. 

aocalmu reagent J. BioLCbem. W3SSn. 

Mnhdmn, h and Slidha; R 19% bhhod~ ln pbpbb&al plot prtbololp PEd Shrkr mj 

Publhtiom,Cbad,P:l67 

N~BbrbM.1dSinpnhEL981~APtiTuoglwdri?.d~pb~P1#,Biob~ml 

Olntrol of Plant D i i Naliou~l ~dahop, mud^ ICU, Coimherr,R 335-339.

Ecd. Env. 6. Crms. 7 (1) : 2001; pp. (53-56) 

Effects of Aqueous Leaf Extracts on the Spore 

Germination of Colletotrichum capsic1 (S yd.) 

Butler and Bisby 

H. Chlbr ad 0. Kannabhrn 

School of.Lfi SEimm, Pond* 

Unmity, Pondlckeny-LO5 014. lndm. 

Introduction 

Recurrent and a n d t e use of mudes that 

hawpoaeda~dveattohumanhealthaswme 

obth haw already proved to beeither mutageruc, 

carcinogenic or teratogenic (Imes ct al., 1960; 

Ccorgopouh and Zaracovitis, 1%7; Ramel, 1972; 

Ligtor and Zimmering, 1975). Apart from this, 

~psltedandhdMminatewof~esfOrthe 

management of plant diseases led to the 

development of resistance in pathogens to 

fungicides, residual toxicity in produce and 

atmo~pheric pollution (Muthulakshmi and 

seahMvrL 1989). 

?hepnanaofm~mpoundsinmme 

higher planb has long been recognized as an 

Lnpatant hdor d k resistance (Mahadevah 

1982). Such compd~ being biodegradrble and 

&dive h W krxWy, ue addered valuibk for 

controlling come pht diseases (Fawcett and 

Spnar, lPM; Tmul d al., 1988). &eed on these 

bthcplantdhuh*to- 

57kdym1LMc~~Rkmretotheefkds 

of thtlr rqwour leaf extracts on the spore 

gcrmlnatIon of Collrtotrickum capsici causing 

rlnkumdd$il(~amurn). 

m o h W k P l ~ 

hlaterinh and methods 

The test fungus C. capski was obtained from pure 

culture stod in hboratow of School of Life %ences, 

Pondicheny University. 

Fresh leave, of diffemt plants were COW locally 

andsurIaceskrilizedwith~chlorideforlt0 

2 minutes. Again C leaves were washd repeatedly 

with distilled water for 4 to 5 times, to remove the 

traces of mercuric chloride. The leaves were 

pulverized well h a mixu with stedized dietilled 

water in the ratio of 1 ml/g of tissue and filtered 

through double layer sterilized cheese doth and 

finally the filtrate was cmbhged at 5000 rpm for 5 

minutes. This formed the standard plant extract 

(100%). The exhacl wo diluted by adding &rile 

distilled water to make 20% concentration 

(Mu* and Whmman, 1987; Jayti Singh 

and Triphi, 1993). 

~~tianstudieswencariJedaaincBvity 

slides incubated in moist chiunber at PC. Spore 

suspension of 4000-6000 sporer/ml in 10%

concentration of leaf extract was prepared. For 

mtml rpme swperrPioa was prepared in sterile 

distilled water. Slid- wen observed for spore 

prththafter24hand48h 

PaantrBpd~d~~tim~~ 

control we+# calculated by using the formula of 

V i (1927). 

by many worh (Jmmine, 1997; Gomathi and 

KaFIMbiran, 1999; Chiin d d., 1999). 

~oftheaqwousleafexhcbartheapon 

~timofColpskibothafterUhand43han 

prrasntedintheTabkl.AUthctestedpLnt~ 

were not found to equally inhibit aport 

~tloh~~plantscanbedgnedto4 

groups (Table 1) (Ganew, 1993; Gancsan and 

Krishnuaju, 1995). Group 1 plants inhibited the 

spore$mi~tbndCoPprkicompleklybo(hlfta 

24th and 48h, indicating their fung~ddrl mhur cn 

TABU 1 

EffcctofRmtBximc4mlhcSpatGsminrtbnnfCqsid 

S. pturt~mp punily ~ ~ f ~ n h i i 

No. Z(h

(XRA AND KANNABIRAN 55 

TABLE L Contd . 

S. PLntNme Pamily YO af Inhibition 

No. 24h 48h 

16 ClnodrndnrminmnLj- Vedxnsceae 0 0 

27 DatuminnoxLMiUn Solanaceae 0 0 

Ze Dalmuun-Lf. Sapiiaceae 

29 DdklolrlabL hbawe 0 0 

30 EnfoPlobhmrampn(IpalJRdn hkmaceae 0 0 

31 Em!& diwkau (L) Buddl Apqnaceae 0 0 

32 Erythrina indim h. Fabaceae 0 0 

33 FinrsglaMlfpRmb. Moraeeae 0 0 

34 Ficus nligiosa L Moraceae 0 0 

35 Hcmdmw indinu (L) RBr. Asclepiadaceae 0 0 

36 Hip@ k n w a) Kun Malphighiaceae 0 0 

37 lm CDECinm L. Rubiaceae 0 0 

38 jminum~k Vahl Oleaceae 0 0 

39 Mirnbilb jalap? L Nyctaginaceae 0 0 

40 Mill~ngfonio hortmpis Lf, Bignoniaceae 0 0 

41 Nycfmths arbkktia L Oleanae 0 0 

42 Pdilnnfhus tithy&& (L) Pd. Euphorbiaceae 0 0 

43 Phylhfhus ncidus (Lh5hl.s Euphorbiaceae 

44 Pagmnia flak Vent Fabaceae 0 0 

45 PsidiumpjumL. Myrtaceae 0 0 

46 Pnguhia damnin (FW) ChiDIl, Asclepiadaceae 0 0 

47 Quisqualia indim L. Combretaceae 0 0 

48 SNtiP mydim ~BunnJ) Kun Rhamnaceae 0 0 

49 ~plthodarampsnu~ P. h u . Bignoniaceae 0 0 

50 Thbuia msar (&rtol.)DC. Bignoniaceae 0 0 

51 Tawm jamirum Sost Asteraceae 0 0 

52 T'mn sfnna (LJ Kunth Bignoniaceae 0 0 

53 Tchcma mina lAndmI Cr& Asdepiadaceae 0 0 

!4 'IhcspaiP populnm (Lj Sd. a Cmr, Sm. Malvaceae 0 0 

55 ?7mtio prmui,u~~ (Pns.j Mm. Apocyanaceae 0 0 

56 Tridarpmcunbms L Asteaceae 0 0 

9 WalthniPindiceL Sterculiaceae 0 0 

58 Ziziphus jujuh (L.) Cwtnn, Mn Milk Rhamnaceae 0 0 

the spore germination of C.fapsin' (Jasmine, 1997). 

Group 2 plant9 inhibited the germ tube fannation of 

tested spores and they produced only small 

protrusions even after 48h. 7% a)so indicates the 

fungicidal nature of concerned plant extracts. 

Croup 3 planb ehongly induce the mahmation of 

gamtubesdgcrmlnatadaponsandtheywerenot 

found to produce any appresoria as in the case of 

nome systemic fungicides. Koch (1971) has 

suggested thitt distorted germ tubes may indicate 

systemic activity, because somesystemic fungicides 

have little effect on spore germination but are lughly 

active in preventing hyphal growth and cell 

division. Group 4 plants were not found to have any 

inhibitory effect and germinated as those of control 

(CaneMn 1993). 

Further studies on the biodremical activities of C. 

cnpsici in the presence of leaf extracts of plants 

belonging to first 3 groups will confirm antifungal 

effects of the concerned plants.

One of us (H.C.) thanks Dr. Ganesan, Lecturer, 

Department of Botany, K.M.P.G. Centre. 

Pondicherry, for his fruitful suggestiont. 

References 

Chm- R 1989. Pobon In your fwd. In : lndh Todry, 

Junr IS. 

Chh,H.CwnM,V.indKuvubinn.a 1599.Hlcmdamccldl)caumhmdoMiReronthr~pmhtdm 

Mdmy 

kr&8lrb/,C&a16(4): 177-178. 

FmcgCHmdSpnar,DH.19M.Rmdnmobvqwlh 

r p p v ~ p r o d u c a ~ ~ o : ~ : : ~ ~ ~ a 

bmmT. I993,Fw@tdd7ectdwildp!~cIdapu~. 

W 20 : 261.266. 

GmmT.MdKTldnUlpllJ. lWS.Anrdunplpmprc*rd 

w#dpbndlAd#mSd 8 (I): 194.196. 

~ 5 . G d l%7.T~&m~ed~ 

~ C 

m ~ ~ k u w . R n ! 109-130. ~ S : 

Gmm$~I,V.ndKum&m,B. 1999.~drqugu,Y 

avuDcnspwegennvutbnmdmlcrYJjr~d 

nhMMs~hlac*~~~vnLfmc(Mm 

Inh Sd Chanu, pp 43.44. 

kmayirn. ~ hd aM. v m M.G. ~emvdl L ~ lkh 

LHurERRUom.Al,t)lm.RRWH.Ltur

SCREENIN6 Of AWEOUS EX- OF SOME PUNTS ON COlSlOlAL GERMINATION AND 

MVCEUAL BROrm OF COmNCHUM CAPJTM (SYO.1 BUTLER & BlSBY 

Key w& : Aqu@ou planl e&imct8 wnidial germination, mpelial gmwth. CoWelolridrum capsro 

The use of synthetic pesticides has 

become indispensable for modem strategy 

of crop pmduclion in India, partiilarly under 

afl intensive agricultural program (Radder & 

Sarawad, 1996). The veritable explosion in 

the wide use of these chemicals has led to 

many problems in the environment. 

Biodegradable pesticides are gaming 

increased attentb currentlv because of their 

environmental friendliness: A large number 

ol plants have been screened and reported 

to be effective against several plant 

palhogens (Bhowmick & Choudary, 1982; 

Chary et al., 1984). Out of 57 plant species 

screened in our laboratory, 10 were found to 

have the potential to inhibit the conidial 

germinatio? of C. capsici (Chitra & 

Kannabiran, 2001). The pfesent work was 

undertaken to evaluate the effects of aqueous 

extracts of 60 specles on the conidial 

germhation and that of 22 species on mycelial 

growth of f%tcfm capski. 

The test tungua C, caplid was obtained 

from pure culture atored in laboratory of 

Department ol Biological Sciences, 

Pondicherry Unker8Ity. Aqueous plant 

extracts wen proparad by udng the mahods 

of MuthuMshmi 6 SeeWmn (1987) and 

Singh & Tripathi (1883). Conidial gemination 

and myeelial growlh cludirc wm, carried out 

by using msthod8 of None (18711 and 

Chitra et al. (1999). The perceit inhibitioq of 

midial germination and mycelial growth was 

calculated by using the method ol Vincent 

(1927). 

Effects of aqueous plant extracts on the 

conidial germination both after 24 h and 48 h 

and percenlage of inhibition on mycelial 

growlh are presented in Table 1. Based on 

the antifungal effects of the aqueous extracts 

the tested plants were assigned to four 

groups (Ganesan, 1993; Ganesan & 

Kr~shnaralu, 1995). Group I plants Inhibited 

conidial gemination totaly both alter 24 hand 

48 h, indicating their fungicidal nature 

(Jasmine. 1997). Group 2 plants inhibited the 

germtube fornation of tested conidia even 

after 48h. This also indicates the fungicidal 

nature of mcerned plant extracts. On the 

contrary, the antifungal effect of group 3 plants 

was found to be reduced afler 24 h which 

showed their fungistatic nature. Group 4 

plants were not found to have any inhibitory 

effect on conidial gemination. 

Based on the results obained from the 

present and previous studies (Chitra & 

Kannabiran, 2001) on conidial germination 22 

plants'belonging to groups 1 and 2 were 

further screened to find out Wir effect on 

radial (mpliat) grn* of C. caphi. hng 

them, bulb extract of A, sativum and leaf 

extract of lawsonia hmds poduced 100% 

inhibition. on the myceljal growth. Those of

Cm(rm 0 0 

Group 1 Germ~nat~w lnlwbnws 

UYnYdrwnL a& IW 1W 

lqvngvr R C ~ WllW ~ Laaver I 1W 1W 

&&9mmphwmL Leaves 1W 1W 

L~IVOS 1W tW 

-mpL C~WML Leaves IW IW 

-rrh'wL 

Leaves 

pnnnarAuMcl Leaves 

Eucr)plUr mblMxr Smtlh Loaves 

HMltlaun-L 

Leavas 

Grwp 2 Genntube IOmnbon WOn 

- 

MM&& ~ees Leaves 97 91 

* :::::: ;: : 

nurpym Rcxb Leaves % % 

CWkqbllbtUIBmml Laaws w w 

W m b s k ~ L Leaver % % 

&L Leaves 98 88 

m u r L Lelws 98 9-9 

-&L Loaves 94 95 

Grw 3 PI.nts vim 1unpraI.tr sllsns 

Mob. imsd hrm I Lasrer 41 35 

aJc 

EUophyllum inophyllum, 

M i a crenuMa,*~/yptus terethmis and 

Cassia marginata produced 26 and 16 

percent of inhibition, respectively (Table 1). 

The remaining 15 plants were not found to 

produce any inhibitory effect. 

Further studies on the biochemical 

activities of C. capsici in response to the 

applimlion of aqueous extracts of A. sativum 

and L. inermls are in progress in our 

laboratory to conf~n their antifungal effects 

against C capstcl. 

REFERENCES 

Bhowmick. B.N. and Choudary. B.K. lndian 

601. Reptr., 1982, 1, 164-165. 

Chary, M.?. Reddy, E.J.S. and Reddy. S.M. 

Pest~cides, 1984. 18,17- 18. 

Chitra. H.. Gomathi, V. and Kannabiran. B. 

Geob~os, 1999. 26. 177-178. 

Ch~lra, H and Kannabiran, B. Ecol.Env. & 

Cons., 2001. 7, 5556. 

Ganesan, T. Gaolnos. 1993. 20. 264-266. 

Ganesan, T, and Krishnaraju. J. Ad. Plant Scr.. 

1995.8. 194.196. 

Jasmine, R. M.Phll. Daseflation, Pondlcherry 

Unlverslty, 1997. 

Muthulakshmi, P. and Seetharaman. K. Prcc. 

Nahonal Workshop on 6101. Control of 

Plant D~seases. Co~rnbatore. 1987, 295- 

302. 

Nene, Y.L. Fung~c~des m Plant D~sease 

Control. IBH Publ. Col.. New Delhl. 1971. 

Radder. B.M. and Sarawad. I.M. Kian World, 

1996. 23. 54. 

Singh. J. and Tripathi, N.N. J. Indian bot. Scc.. 

1993. 72. 51-53. 

V~ncent. J.M. Nature, 1927, 159. 850.

plate 1 - Pondicherry University DSpace Portal

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?