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EVect of Chitosan on Growthand Toxin Production byAlternaria alternata f. sp.lycopersiciM. V. BHASKARA REDDY , JOSEPH ARUL , ESSAIDAIT-BARKA , PAUL ANGERS , CLAUDE RICHARD &FRANCOIS CASTAIGNEPublished online: 28 Jun 2010.

To cite this article: M. V. BHASKARA REDDY , JOSEPH ARUL , ESSAID AIT-BARKA ,PAUL ANGERS , CLAUDE RICHARD & FRANCOIS CASTAIGNE (1998) EVect of Chitosanon Growth and Toxin Production by Alternaria alternata f. sp. lycopersici,Biocontrol Science and Technology, 8:1, 33-43, DOI: 10.1080/09583159830414

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B iocontrol S cience and Technology ( 1998) 8, 33±43

EVect of Chitosan on Growth and Toxin Production byAlternaria alternata f. sp. lycopersici

M . V. BH A SK AR A R E D DY,1 JOSEPH ARU L,1 ESSAID AIT-BA R K A,1

PAU L A N G ER S,1 CLAU D E R ICH AR D 2 and F R AN CO IS CA STAIG N E 1

1 Department of Food S cience and N ut rit ion and H orticultural R esearch Center,L aval University, S ainte-Foy, Quebec, Canada G1K 7P4; 2 A griculture and

A gri-Food Canada, S oils and Crops R esearch and D evelopment Center,2560 H ochelaga Boulevard, S ainte-Foy, Quebec, Canada G1V 2J3

(R eceived for publication 16 June 1997; revised m anuscript accepted 1 S eptember 1997)

T he antifunga l activity of chitosan , a biopolymer of b -1-4 glucosam ine, agains t A lterna ria

a lter na ta f . sp. lycopersici, causa l agen t of black m old of tom ato, was invest igated. C hitosanwas incorporated into potato±dex trose broth at concen trations of 100±6400 lg m l2 1, and thegrowth and tox in produc tion by the fungus were assessed af ter 15 days of incuba tion. A t thehigher concen trations, chitosan signi®cant ly aVected both fungal growth and tox in produc tion.H owever, at lower concen trations tox in product ion was aVected more than growth. T he fungussporula ted ex cessivel y in the presence of chitosan , but the spores were less viable. C hitosanalso induced aggrega tion, abnorm al shape, ex cessive branchin g and hy phal contort ion of funga lcells, and leak age of proteins. T he virulence of the tox in in culture ®ltrates of the fungus grownon diVerent concen trations of chitosan was assessed by adm inistering tox in on tom atodisk s. T he phospho lipid content , electrolyte leak age and activit ies of x y lanase and pectinm ethy lesterase were measured in the tom ato tissue adm inistered with culture ® ltrat es containingfungal tox in. Decreased trends in the tendency to cause elec troly te leak age, phospho lipiddegrada tion and activation of x y lanase and pectin m ethy lesterase in the tom ato tissue wereobserved wit h increasin g concen trations of chitosan . T he results showed that tox in produce d inthe presence of chitosan was less eVective in causing degrada t ion of tom ato tissue com paredwith the control. T hus, chitosan is a potential antifunga l agen t which can interfere with thepathogenic factors of the fungus.

Keywords: chitosan, black mold, t om ato, Alter na ria tox ins, elec troly te leak age, phospho lipids,x y lanase, pectin methy lesterase

INTRODU CTION

A lternaria sp ecies in gener al a re ®eld pathogens wh ich a lso ca use sto rage lo sses in a va riety

o f fr u its. A . alt ernat a (F r.) K eissler f. sp. ly copersic i is th e ca usa l agen t of st em canker

Co rrespondence to : J. Aru l. Tel: 418 656 2839; F ax: 418 656 3353.

0958±3157/98/010033±11 $9.00 � 1998 Carfax Pub lishing L td

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34 M . V. BH A SK AR A R ED D Y ET A L .

d isea se in tom ato (G ilchrist & G roga n , 1976). T he fungu s a lso ca uses b la ck mo ld o f ripe

tom ato fru its, result ing in su bstan t ia l lo sses. T he d isea se is favo red by warm ra iny weather

o r dew fo rm at ion on the fru it su rfa ce, and its sever ity o f in fect io n is grea ter in the rip efr u its th an a t the green stage (Pea rson & H a ll, 1975) . A lternaria pathogens a re known to

p roduce host -speci®c toxin s (H STs) a s agen ts o f viru lence o r pathogen icity fact or s; a t lea st

n in e H ST-p roducing pathogens have been rep or ted (O tan i et al., 1995). A lternaria toxin sa lso have the po ten t ia l to be hazardous to hum an hea lth (K ing & Sch ade, 1984). T hese

toxin s a re low molecu la r weight secondary metabo lites. T hey a re ch em ica lly diverse, and

m ay be grouped in to dib en zo pyren e der ivat ives, su ch a s alter nar io l (A L), a lter nario lm onomethyl ether (A M E) and alten uene, and tet ram ic acid der ivat ives, su ch a s tenuazo nic

acid (TA ) and perylene der iva t ives (K ohmo to & O tan i, 1991; C hu lze et al., 1995) . The

spo ilage o f tomato by A lternaria is o ften accompan ied by the p roduct io n o f myco toxins,and exten sive deter io rat io n o f the p roduct resu lts in econom ic loss to com mer cia l ma rketers

(H assan, 1995). I t h a s been rep or ted that these toxin s act on the p la sm a mem brane of

suscep t ib le t issue and ca use elec tro lyte lo ss r esu lt in g in cell d ea th (O tan i & K ohm o to, 1992) .F ur thermore, th ese toxin s act ivate phospho lipa se A 2 and induce the degradat ion of

phospho lip ids in the m icro so me fract ion s from su scep t ib le cells (L ee et al., 1992).

Wea ther-ba sed fungicide sp ray p rogram mes a re availab le to co n tro l A lternaria ®eld

d isea ses o f toma to (P itbla do, 1989) and none o f the fungicides have been app roved fo r post -ha rvest trea tm en t to inh ib it a lterna ria ro t . H owever, grower s ar e co n fron ted with the

p rob lems of deregistr at ion o f fungicides due to ha rmful residues and increa sed resist ance of

funga l st ra in s to ch em icals ( Ja co bsen & Backm an, 1993). Such environmen ta l co nsequen cesp rom pted workers to exp lo re a lt erna t ive st rat egies fo r disea se m anagemen t (C het , 1987 ;

K uc, 1987; D eacon & Berry, 1993). Bio co ntro l of p la n t pathogens is one poten tia l opt io n .

C hito sa n, a po lycat ion ic po lym er o f b -1,4-linked d-gluco sam ine, is one b ioact ive agen tknown to be ant ifunga l (A llan & H adwiger, 1979 ; Sto ssel & Leu ba, 1984; H irano & N aga o,

1989; Benhamou & Ther ia ult , 1992 ; E l G haouth et al., 1992a ). In add it io n , ch ito sa n ca n

elici t p la n t defen se mechan isms, such as the p roduct io n o f phytoa lexin s (H adwiger &Beckman , 1980). Cuero et al. (1991a) rep o rted that ch ito san inh ib ited a¯atoxin p roduct io n

by A spergillu s ¯avus in pea nu ts and ma ize. The ob ject ive o f the p resen t study was to

invest igate the eVect o f ch ito sa n on the growth and p roduct io n o f AL and AM E byA . alt ernat a f. sp . ly copersic i, since these toxin s a re co nsidered to be viru lence fa cto rs in the

pathogenesis p rocess and a re p roduced by most st r ain s o f A . alt ernat a in rela t ively la rge

quant it ies. T he po ten cy o f cu ltu re ®lt rat es of the fungus p roduced in the p resence ofch ito sa n was a lso stud ied in term s o f the ab ility to b ring abou t degradat ive ch anges in

tom ato t issue, su ch as electrolyte leakage and act ivat io n o f so m e o f the cell wa ll-degrad ing

enzym e system s.

M ATER IALS AN D M ETH OD S

FungusC ultu res o f A . alt ernat a f. sp. ly copersic i were obta in ed from ca nkers o f natu ra lly in fected

tom ato pla nts wit h typ ica l stem -canker sym p tom s. Single sp o re iso la t io n s of the cu ltu resu sed in th is study wer e ma in tain ed on po ta to±dextro se aga r (P D A) at 21±23ëC .

C hitosanShrim p-shell ch ito san was pu rch a sed from N ova ch em (H alifax, N ova Scot ia , C anada)

and ground to a ®ne powder. Pu ri®ed ch itosan was prep ared by d isso lving ch ito sa n in

0.25 n-H C l, and the und isso lved pa r t icles were rem oved by cen tr ifuga t io n (15 m in ,10 000 3 g). T he viscous so lu t io n was then b rough t to pH 9.8 wit h 2.5 n-N aOH to p recip ita te

the ch itosan . T he p recip ita ted ch ito sa n was recovered by ®lt r at io n , wash ed extensively with

deion ized water to rem ove sa lts and lyoph ilized . Ch ito sa n stock so lu t io n (10 m g m l-1 ) wasp repa red by d isso lving ch it osa n in 0.05 n-H C l and the pH was ad ju sted to 5.6. C hito sa n

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AN T IF U N G AL AC TIVIT Y O F C H ITOSA N 35

solu t io n was au toclaved and added to po tato±dext ro se b ro th (PD B) to ob tain co ncen trat io ns

o f 100, 200, 400, 800, 1600 , 3200 and 6400 lg m l-1 o f the med ium .

Inoculation and IncubationT he ster ile med ia were inocu la t ed with 1 m l o f spo re su sp en sion co n ta in ing 2.5 3 105 sp o res

in ster ile wa ter, p repa red from 10-day-o ld cu ltu res grown on PD A. The funga l sp o reconcentr at io n was determ in ed using a hem ocytom eter (H ausser Scien t i®c, H or sh am , PA ,

U SA). The ¯ask s were in cu bated as a standing cu ltu re at 22ëC under a r t i®cia l ligh t (12 h );

500-m l ¯asks co nta ined a to tal o f 100 m l o f PD B en riched with ch ito sa n at diVeren tconcentr at io n s. A fter 15 days o f in cu ba t io n , the mycelium was ha rvested by vacu um

®lt rat io n on W ha tman (M a idstone, K ent, U K ) no. 1 ®lter paper. Spo re co unts were m ade

from the wash ings o f the mycelium by hem ocytom eter. Spore viab ilit y was determ ined byspread ing the sp o re su sp en sion prepa red from the wash ings on aga r p la tes and incubat in g

a t 15ëC fo r 12 h in the dark. About 100 sp o res wer e observed for each su sp en sion and the

per cen tage spo re germ ina tio n was determ in ed . Spo res were co nsidered to be germ in atedwhen the germ tube was longer than the wid th o f the spo re. T he mycelium was exam ined

under ligh t m icro scopy (3 400) fo r m orphologica l changes. The mycelia wer e p la ced in an

oven at 60ëC overnight , and the dry weigh t was determ in ed . The cu ltu re ®lt rates were

pa ssed th rough a m illipo re ®lter (0.45 lm) and sto red at 4ëC befo re toxin extra ct ion . Cell-fr ee ®lt rate (5 m l) from each tr ea tm en t was cen tr ifuged a t 10 000 3 g fo r 5 m in and the

super natan t was a ssayed for pro tein using Brad ford method (Brad fo rd, 1976).

P hospholipid and Enzyme AssaysM ature green tom ato fru its were ha rvested from a lo ca l greenhouse, washed , su rfa ce

ster ilized with 1% so d ium hyp ochlo rite fo r 3 m in and rin sed with ster ile water. D isk s ofper ica rp t issue, 10 mm in diameter and 2 m m th ick , were p rep a red using a sh a rp st er ile

cork bo rer. T he d isk s were pla ced in an iso tonic so lu t ion o f mann ito l a s soon as they were

m ade. F ive d isk s were a r ranged on two layer s of m oistened ®lt er paper in 8.5-cm Pet rip la tes. The d isks were punctu red a t the ep id erma l su rfa ce u sing a ster ile need le, and 50 l l

o f th e cu ltu re ®lt r ate wer e deposited on ea ch d isk to determ in e the eVect o f toxin . Cont ro ls

were ma in ta in ed with medium and ch ito sa n wit hou t the fungu s at a ll co ncen trat io ns. Theen t ire process was ca r ried ou t under ster ile lam in ar a ir ¯ow to avo id exter nal co ntam in at io n

o f tomato t issue. The Pet r i p la tes were sea led with Para®lm and in cu ba ted at room

tem pera tur e (22ëC ) fo r 48 h . T he d isks were then frozen in liqu id nit ro gen and stor ed at

2 80ëC un t il enzym e assay. T he phospho lip id con ten t was det erm in ed using the m ethod of

M arin et t i (1962) . T he t issue was hom ogen ized with methano l and ch lorofo rm (1 :1, v : v) at

10 g m l-1 . The extra cts were then dried wit h anhyd rous N a 2 SO 4 and the organ ic phaseswere rem oved on a rot ary evapo rato r. The residue was recovered with ch lo ro fo rm (2±3 m l)

and co ncen trated under n it rogen ga s. The o il residue co nst itu ted the tot al lip id extra ct and

was kep t a t 4ëC un t il use. F rom th is extra ct , po la r lip ids (m ain ly phosp ho lip id s) and neu tra llipid s were sepa rated by p recip ita t ion in acetone a s describ ed by K ates (1986) . Perch loric

acid (0.4 m l) was added to the residue. The so lu t io n was a llowed to stand for 4 m in un t il it

was clea r, and th is was fo llowed by the addit io n o f 4.2 m l o f dist illed water and 0.2 m l eacho f amm on ium molybdate (5% w /v) and am ido l (1% w /v). T he m ixture was vo r texed , hea ted

fo r 7 m in and co o led for 15 m in , th en the op t ica l den sity (O D ) was measured at 830 nm .

For en zyme assays, th e cr ude extra ct was p rep a red by hom ogen izing the tomato d isks in1 m l o f 60% sucro se in phosphat e buVer (pH 6.5) and 1 m l of ster ile wa ter /g o f t issue, using

a m orta r and pest le. The m ixture was centr ifuged at 13 000 3 g fo r 3 m in . T he pellet

ob ta in ed was wash ed twice in 2 m l o f su cro se buVer so lu t ion and cen tr ifuged betweenwash es. The co mb in ed su per na tan t co nst itu ted the crude extra ct . T he en t ire p ro cedu re was

car ried out at 4ëC.

Pect in methylester ase act ivity was determ ined by in cu bat ing 0.5 m l o f rea ct io n m ixtu recon ta in ing 0.25% pect in A , 50 m l n m-P IPES buVer and 0.1 m l of crude extra ct a t pH 7.5

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36 M . V. BH A SK AR A R ED D Y ET A L .

fo r 6 h a t 25ëC. The react io n was then term in ated by adding 0.5 m l o f 1 m-H 2 SO 4 , a nd the

amoun t o f methanol lib erated was determ ined by the m ethod o f Wood and Sidd iqu i (1971) .

Xylana se act ivity was determ in ed using Xylan -R BB (Sigma Chem ica l C o., St Lou is, M O,U SA) as the subst ra te. T he react io n m ixtu re con ta ined 0.3 m l of su bst rat e so lu tio n (0.1%,

w /v), 0.6 m l o f 0.1 m-sodium acetate (pH 4.8) and 0.3 m l o f cr ude extra ct . T he react io n was

term in ated a fter 4 h by add ing 0.3 m l of 0.5 m-H C l. A ft er co o ling in an ice water ba th for15 m in , th e m ixtu re was cen tr ifuged at 13 000 3 g fo r 5 m in and the absorbance o f the dye

cova lently linked to the degraded polysaccha rid es was mea su red a t 600 nm .

E lectrolyte leakage was m easured using a co nduct ivity m eter (m odel ES-14, H o riba L td ,K yoto , Ja pan). Tom ato d isks weigh ing 4 g wer e p la ced in 25 m l o f 0.4 m-m ann ito l so lu t io n

and in cu bated fo r 3 h at 28ëC. T he in it ia l co nduct ivity (IC ) of the aliquo t was mea su red at

room tem peratu re (25ëC ). F in a l co nduct ivity (F C ) was determ ined after au toclaving thet issue at 120ëC fo r 10 m in . T he resu lts wer e exp ressed as (IC /F C) 3 100.

Extractio n and Assay of Tox insM illipo re-®ltered cu ltu re ®lt rates sto red at 4ëC were ext racted twice with 50 m l o f ch loro-

fo rm , and the aqueo us and o rgan ic phases were separated . The o rgan ic phase was then

concentr ated to 5 m l using a rot ary evapora to r, th en dried under n it rogen . Toxin was

der iva t ized by add ing 100 l l of ch lo rofo rm , 50 l l o f d im ethyl fo rmamide and 50 l l o f th er eagen t methyl tert -b u tyl silyl t r i¯u ro acetam id e (M TBSTFA ), and the rea ct io n m ixtu re was

kept at 65ëC fo r 2 h .

AL and AM E were a ssayed by ga s chrom atography using a H ewlet t Pack a rd (Avonda le,PA, U SA) model 5890 equ ipped with a ¯am e ion izat ion det ecto r. The ch roma tographic

separa t io n was ca rried ou t on a ca pilla ry co lumn (H ewlet t Pack a rd , m odel D B5-30M )

0.25 lm wide and 30 m long. H ydrogen was used a s the car rier ga s at 55 kP. T he detecto rtem pera tur e was 325ëC, the in let a ir tem perature was 300ëC , the in it ia l tem pera tu re was

200ëC and the ®na l tem peratu re was 300ëC. The in ject io n s were car r ied ou t wit h an auto

sam pler (H ewlet t Packa rd , model 7673). The data were p rocessed using H P3365 so ftwa re.U nder these co ndit io n s, A L had a reten t io n t im e of 14 m in and AM E had a reten t io n t im e

o f 8 m in . Toxin standa rd s (Sigma) were used fo r iden t i®ca t io n.

Statistical AnalysisFou r r ep licates were u sed fo r ea ch trea tmen t and the exper im en t was rep ea t ed twice. A s the

var ia nces wer e hom ogeneo us, th e data from the two exper im en ts were poo led . Stat ist ica lanalysis was ca rried ou t on the poo led da ta using the Stat ist ical Ana lysis System (SA S,

1982). T he data were sub jected to ana lysis o f va riance. M eans wer e co mpa red by D unca n’s

mu lt ip le-range test .

R ESULTS

EVect of C hitosan on M ycelia l D ry Weight and Tox in P roductionC hito sa n sh owed sign i®can t ma in eVects on the mycelia l growth and sp o re viab ility o f

A lternaria and p roduct io n of toxin s by the fungu s a s well a s on the eVect iveness o f thetoxin s in inducing cellu la r dam age of toma to t issue (P < 0.0001) . T he mycelia l weigh t

decrea sed from 0.67 to 0.0 g at ch itosan co ncen trat io n s ranging from 0 to 6400 lg ml-1

(F igu re 1). H owever , th e decrea se in weight was no t sign i®can t up to 200 lg m l-1 , a ndshowed sign i®ca n t red uct ion with each in crea se in ch ito sa n co ncen trat ion o f 400 lg ml-1

and upward s. Ther e was a 78.0% red uct io n in mycelia l weight at 3200 lg m l-1 com pared

with the co ntrol.The p roduct io n o f AL va ried from 484 to 0 lg m l-1 at ch ito sa n co ncen tra t io n s of

6400 lg ml-1 . H owever , low co ncen trat io ns o f ch it osa n (100 and 200 lg m l-1 ) d id not

r esu lt in any sign i®can t d iVerence in AL and A M E product io n co mpa red with the co n tro l.Sign i®ca nt red uct ion s in toxin p roduct io n were observed wit h ch ito sa n concen tra t io n s of

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AN T IF U N G AL AC TIVIT Y O F C H ITOSA N 37

F IG U R E 1. EVect o f ch itosan concen trations on mycelia l growth (m ) and sporulat ion (d ) of A . alternataf. sp . lycopersici a fter 15 days of growth in PD B; means6 standa rd error m ean (SEM ) fo reigh t rep licates.

F IG U R E 2. E Vect of chitosan concent rat ion s on a lterna rio l (AL) (m ) and a lternar io l monomethyl ether(AM E) (d ) product ion by A . alternata f. sp. lycopersici after 15 days o f growth in PD B;m eans6 SE M for eigh t rep licates.

400 lg ml-1 and upwards; t he diVeren ces were sign i®can t with in crea ses in ch ito sa nconcentr at io n (F igu re 2). A ch ito sa n concent rat ion o f 6400 lg m l-1 p roved to be letha l t o

the fungus, and neith er growth no r toxin product ion were observed. T here was a 86.73%

red uct io n in AL product io n at 3200 lg m l-1 o f ch ito sa n co mpa red with the co n tro l.Sim ila rly, th e p roduct io n o f AM E va ried from 45.36 to 12.44 lg m l-1 at ch ito sa n concentr a-

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38 M . V. BH A SK AR A R ED D Y ET A L .

t io ns o f 0±3200 lg m l-1 (F igure 2). There was no signi®ca nt d iVeren ce between the co n tro l

and 100 lg ml-1 t rea tmen t, but a 72.55% red uct io n in the p roduct io n of A M E was observed

a t a ch it osa n co ncen tra t io n o f 3200 lg ml-1 .The ana lysis o f the resu lts sh owed that ch ito sa n aVected toxin p roduct io n a t rela t ively

lower co ncen trat ion s than tho se a t which it aVected funga l growth . T he m in im um inhib ito ry

concentr at io n (M IC) o f ch ito sa n fo r toxin p roduct io n and mycelia l growth was determ inedby p lo t t ing V 0 /V i aga inst I , where V 0 is th e toxin co ncen tra t io n o r mycelia l growth at zero

concentr at io n of ch ito san , V i is the toxin product ion o r mycelia l growth at va rio us

concentr at io n s o f ch ito sa n and I is th e inh ib ito r co ncen trat io n . T his p lo t was describ ed bythe eq uat io n V 0 /V i 5 I /K i, where I /K i represen ts the slope o f the lin ea r plo t and K i is the

M IC. T he der ived M IC of ch itosan was 493.34 lg m l-1 for AL , 675.43 lg m l-1 fo r AM E

and 937.65 lg ml-1 for mycelia l growth, em phasizing that toxin p roduct io n was aVect edm ore than mycelia l growth at lower concen tra t io n s o f ch ito san .

M icro scop ic observa t io n of the mycelium exposed to va rio us ch itosan co ncen trat ion s

r evea led aggregat io n o f funga l cells. H yp hae appea red to be abno rm al in sh ape and growth ,showing swelling and excessiv e branch ing of con to r ted hyp hae. T he hyphae also appea red

to be more hya lin e, and lysis of the cell wa ll was no t iced . Such ch anges were most p rom inent

a t 3200 lg m l-1 o f ch ito sa n (F igure 3). A t su b -letha l concen tra t io n s o f ch ito sa n , excessiv e

spo rula t io n o f the fungu s was observed , su ggest ing that fu nga l cells wer e st ressed (F igu re1). H owever , th e sp ore viab ility decreased with in crea ses in ch ito sa n co ncen tra t io n . I t

d ecrea sed from 92.66% in the co n tro l to 0% at 3200 lg ml-1 of ch ito sa n (F igure 4). The

decrea se in sp o re viab ility was no t sign i®ca n t up to a co ncen trat io n of 200 lg m l-1 .

Induction of Fungal Cellular Leakage by ChitosanC hito sa n induced cellula r leakage o f p ro tein s in to the medium . T he amount of pro teinr ecovered from the m ed ium va ried from 3.32 to 13.48 lg m l-1 at ch itosan co ncent rat ion s

o f 0±3200 lg ml-1 . T here was a sign i®ca n t (P < 0.05) increa se in leakage o f p ro tein s with

an increa se in ch itosan co ncen tra t io n (F igure 4).

P hospholipid Content and Electrolyt e Leakage from Tomato TissueT he p lan t t issue dam age in response to trea tm en t with cu ltu re ®lt rate co nta in ing funga ltoxin s was a ssessed by extra ctab le phospho lip id co n ten t and electrolyte leakage o f the t issue.

T he phosp ho lipid co nten t of toma to t issue trea ted with cu ltu re ®lt rates when the fungu s

was grown with ch ito san was sign i®can tly h igher than that from ®lt rat es withou t ch ito sa n(P < 0.05) (F igu re 5). T he d iVeren ces wer e more eviden t from ch ito sa n concentr at ion s of

400 lg ml-1 and h igher. The electr ica l co nduct ivit y of tomato d isk s trea ted with cu ltu re

®lt rates of the fungu s decreased sign i®ca n tly (P < 0.05) with in crea sing co ncen tra t io n s ofch ito sa n (F igu re 5). T he d iVeren ces were m ore sign i®ca n t from the ch ito san concentr at ion

o f 200 lg m l-1 and h igher co m pa red with the co n tro l. The eVect of ch ito sa n per se on bo th

phospho lip id co nten t and elec tro lyte leakage o f toma to t issue was not sign i®can t (F igu re5). T hese resu lts su gges t th at th e toxin s p roduced in ch ito sa n -free med ium were more po ten t

a t a ct ivat in g phosp ho lipa se in the mem brane and ca using m em brane damage than toxin

p roduced in the p resence o f ch itosan .

Enzymatic Activity in Tomato TissueT he act ivit ies o f xylanase and pect in m ethyles tera se were a ssessed in response to trea tmen twith cu ltu re ®lt rate co nta in ing funga l toxin s. The enzym at ic act ivity in cr ude extra cts o f

tom ato d isk s d iVered when the d isk s had been t rea ted with funga l cu ltu re ®lt ra tes of

d iVeren t ch itosan co n ten t (F igure 6). F iltra t es o f the fungu s grown withou t ch ito san sh owedthe h ighest detect ab le act ivit ies of xylana se and pect in methylester ase. T here was a 50%

red uct io n in bo th pect in m ethylestera se and xylana se act ivit ies at a ch it osa n co ncen trat io n

o f 3200 lg ml-1 co mpared with the co n tro l. Th is observa t io n revea led that t hough thefungus ha s the ab ility to p roduce toxin at su b -letha l levels o f ch ito sa n, th e po ten cy of

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AN T IF U N G AL AC TIVIT Y O F C H ITOSA N 39

F IG U R E 3. L ight micrograph s o f mycelia of A . alternata f. sp. lycopersici grown in (a ) PD B and (b) brothamended with chitosan at 3200 lg ml-1 .

th e toxin in act ivat ing cell-wall degrading enzym es decreased with increa ses in ch ito san

concentr at io n . T he levels o f en zymat ic act ivit ies were low in tom ato t issue trea ted withch ito sa n per se (without fungu s), in d icat in g tha t th e st im u la t ion of cell-wall degrading

enzym es in the p lan t t issue is at tr ib utab le to the funga l toxin s (F igu re 6).

D ISCU SSION

T he resu lt s o f th is study dem onst rate that ch ito sa n inh ib ited bo th funga l growth and toxinp roduced by A . alt ernat a. T he an t ifunga l act ivity of ch ito san is well docum en ted . C hito sa n

in ter feres with funga l m em brane funct io n , cau sing a lterat io ns in the permeab ility o f the

m em brane and p romo t ing in terna l o sm ot ic im ba lances (L eu ba & Sto ssel, 1986). Theobserved ch anges in protein leakage from mycelia in the presen t study also su ggest

in ter feren ce o f ch ito sa n with the funga l mem brane. Th is co u ld a lso be due to a lterat io n s o f

the funga l cell wa ll in the presence o f ch ito san . C hito sa n is known to in crease the act ivit yo f ch it in dea cetylase, wh ich co nver ts fu nga l cell wa ll ch it in in to ch ito sa n . Th is ha s been

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40 M . V. BH A SK AR A R ED D Y ET A L .

F IG U R E 4. Spo re germina tion (m ) and leakage o f p roteins (d ) from A . alternata f. sp. lycopersici grownin PD B amended with diVerent concentrat ions o f chito san; means6 SEM for eight replicates.

F IG U R E 5. Phospho lip id con ten t (m ) and electrolyte leakage (d ) in toma to tissue treated with cu lture®ltra te of A . alternata f. sp. lycopersici grown with diVerent concen tra tion s of chitosan .Phospho lip id content (n ) and electro lyte leakage (s) in tomato tissue treated with diVerentconcen trations of chitosan from the uninoculated medium . M eans6 SEM fo r eight replicates.

d em onst rated in R hizopus stolonifer (E l G haouth et al., 1992b ). I t h a s been sh own previously(Bar tn ick i-G arcia , 1973) that ch it osa n co uld in tera ct with the freshly form ed p la sm a lemm a

a t the hyp hal apex, ca u sing form at ion of po res in the mem brane which lead s to mem brane

leakage. A cco rd ing to H adwiger and Loschke (1981) , ch itosan may enter the funga l cellth rough loosened walls and in ter act with funga l D N A and m R N A to ca use an t ifunga l

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AN T IF U N G AL AC TIVIT Y O F C H ITOSA N 41

F IG U R E 6. Xylanase (m ) and pect in m ethylestera se (PM E ) (d ) activity in tomato t issu e treated withcu lture ®ltrate of A . alternata f. sp . lycopersici from diVerent concentrat ions of chitosan.Xylanase (n) and PM E (s) act ivity in tom ato t issue treated with diVerent concentrat ions ofch ito san from the un inoculated m edium. M ean s6 SEM fo r eight replicates.

eVects. T he posit ively ch a rged glu co sam in e un its of the po lym er co u ld in tera ct with

negat ively cha rged macromo lecu les of the exposed funga l cell su rfa ces. Ch itosan in ter fereswith ch it in bio synthesis (Bar tn icki-G a rcia & L ippman, 1972) or cr ystalliza t io n o f ch it in

(Vannin i et al., 1986) in the cell wa ll.

Ch ito sa n was no t on ly eVect ive in inh ib it ing funga l growth bu t a lso aVect ed thep roduct ion o f AL and AM E by the fungu s. A sim ila r in hib ito ry eVect o f ch ito sa n on

A . ¯avus and a¯atoxin p roduct io n was observed in groundnu t by C uero et al. (1991b ).

F ur thermore, t he eVect o f ch ito sa n was sim ilar to pest icides, as O mar and M ohamoud(1995) sh owed tha t addit ion of the fungicide Cup ro san and su m i o il, an in sect icide, in h ib it ed

growth and toxin product io n by A . alt ernat a in cu ltu re medium . The resu lts o f the p resent

study sh ow tha t ch ito sa n a t sub -letha l concentr at io n s a llows the mycelium to grow bu ttoxin p roduct ion is aVected ; the M IC va lues fo r toxin p roduct io n wer e less than tho se for

mycelia l growth . T h is cou ld be due to metabo lic regula tory act ion o f ch ito sa n on the

syn thesis o f toxin . Th is was a lso observed by C uero et al. (1991b) in the product io n o fa¯atoxin s by A . ¯avus in ch ito san -t rea ted groundnu t seeds. The decrease in toxin product io n

with in creasing ch ito san co ncen trat io n co u ld be due to ch elat ion of Cu , Zn and M n by

chito sa n (M uzza relli et al., 1980), because these ions a re essen tia l fo r toxigen ic funga lgrowth and toxin product io n (C uero et al., 1987; C uero, 1989).

Ch ito sa n a t su b -lethal co ncen trat ion s a lso induced in crea sed sp oru la t io n o f the fungu s

(F igu re 1). Such an eVect co uld be due to st ress ca used by ch itosan . W hen an t ifunga lcom pounds are in co rpo ra ted in to the m ed ium or fungi a re grown on nut rient-de®cien t

m ed ium , in crea sed spo rula t io n is seen (Wot ton & Strange, 1987). H owever, th e sp o re

viability decrea sed with increa se in ch itosan co ncen trat io n, which is fu r ther eviden ce thatch ito sa n ca n inh ib it mycelia l growth and sp o re germ ina t io n . I t ha s been sh own that ch ito sa n

can inhib it sp ore germ in at ion and germ tube length in fungi (E l G haou th et al., 1992a ).

Cu lt ure ®lt rates co ntain ing toxin s o f A lternaria grown on d iVeren t concentr at ion s o fch ito sa n vary in their ab ilit ies to cause degradat ive ch anges in toma to t issue (e.g . extractable

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42 M . V. BH A SK AR A R ED D Y ET A L .

m em brane phosp ho lipid co nten t , elec tro lyte leakage and act ivat io n o f cell-wa ll degrad ing

enzym es, su ch a s xylana se and pect in methylestera se). T he eVect o f the toxin from cu ltu re

®lt rates when chito sa n was p resent on the t issue decrea sed with increa ses in ch ito sa nconcentr at io n . I t h as been dem onst ra ted tha t A lternaria toxin s distu rb mem brane in tegrity

in co m pa t ib le hosts (H a & Thompso n, 1991). W hen su sp en sion -cu ltu red cells o f st rawber ry

were trea ted with a lfa toxin 1 from A . alt ernat a, a n enhanced level o f phosp holipa se(P L A-2) act ivity was observed in the m icrosom es at rela t ively low co ncen tra t io n s of the

toxin that a re known to reduce cell viability (L ee et al., 1991). I t h as been hypo thesized

tha t toxin aVect s cyto so lic Ca 2 + co ncen tra t io n wh ich , in tu rn, en hances PLA-2 (L ee et al.,1992). H owever , th e decrea sed ability o f the cu ltu re ®lt ra te co nta in ing funga l toxin s to

cause t issue dam age with in crea sed ch itosan co ncen trat ion s co u ld be due to the in ter feren ce

o f ch ito sa n p rim a rily with funga l growth and possib ly toxin b iosyn thesis. In co nclu sion ,th e results of th is study dem onst ra te tha t ch ito san acts aga in st th e p roduct ion of viru len t

fa ct or s such a s toxin s by pa thogens; thu s it may have in ter est ing app lica t io n s in the

b io logica l co nt ro l of pla nt d iseases.

ACK NOWLEDG EM EN TS

T he autho rs ack nowledge ®nancial su ppo rt from the N atu ra l Sciences and EngineeringR esearch Council (N SER C) o f C anada Stra tegic G rant s P rogr am .

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