Effect of Chitosan on Growth and Toxin Production by ...

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EVect of Chitosan on Growth and Toxin Production by Alternaria alternata f. sp. lycopersici M. V. BHASKARA REDDY , JOSEPH ARUL , ESSAID AIT-BARKA , PAUL ANGERS , CLAUDE RICHARD & FRANCOIS CASTAIGNE Published 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 Chitosan on Growth and Toxin Production by Alternaria alternata f. sp. lycopersici, Biocontrol Science and Technology, 8:1, 33-43, DOI: 10.1080/09583159830414 To link to this article: http://dx.doi.org/10.1080/09583159830414

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

E V ect of C hitosan on Growth and Tox in P roduction by A lternaria alternata f. sp. lycopersici M . V. BH A SK A R A R E D D Y, 1 JO SE P H A RU L , 1 E SSA ID A I T-BA R K A , 1 PAU L A N G E R S, 1 C L AU D E R I C H A R D 2 and F R A N C O IS C A STA I G N E 1


1 D epartm ent

of Food S cience and N ut rit ion and H orticultural R esearch C ent er, L aval U niversity, S ainte-Foy, Q uebec, C anada G1K 7P 4; 2 A griculture and A gri-Food C anada , S oils and C rops R esearch and D evelopm ent C enter, 2560 H ochelaga B oulevard, S ainte-Foy, Q uebec, C anada G1V 2J 3 (R eceived for publication 16 June 1997; revised m anuscript accepted 1 S eptem ber 1997)

T he antif unga l activity of chitosan , a biopoly m er of b -1-4 glucosa m ine, agains t A lt er n a ria a lt er n a ta f. sp. lyco p er sici, causa l agen t of black m old of tom at o, was investigated. C hitosan 21 was incorpor at ed into potat o±dex trose broth at concen trations of 100 ± 640 0 l g m l , and t he grow th and tox in produc tion by the fungus were assesse d af ter 15 day s of incuba tion. A t the higher concen trations, chitosan signi® cant ly a Vected both fungal grow th and tox in produc tion. H owever, at lower concen trations tox in produc t ion was a Vected m ore than growth. T he fungu s sporula ted ex cessivel y in the presenc e of chitosan , but the spore s were less viable. C hitosan also induce d aggrega tion, abnorm al shape, ex cessiv e branchin g and hy pha l contortion of funga l cells, and leak age of proteins. T he virulenc e of the tox in in culture ®lt rates of the fungu s grown on di Verent concen trations of chit osan was assesse d by adm inisterin g tox in on tom at o disk s. T he phospho lipid content, electrolyte leak age and activities of x y lanase and pectin m et hy lesterase were m easure d in the tom at o tissu e adm inistered wit h culture ® lt rat es containin g fungal t ox in. D ecrease d trends in the tendency t o caus e elec troly te leak age, phospho lipid degrada tion and activation of x y lanas e and pectin m et hy lesterase in the tom at o tissue were observe d wit h increasin g concen trations of chitosan . T he results showed that tox in produce d in the presenc e of chitosan was less e Vective in causin g degrada t ion of tom at o tissu e com pare d wit h the control. T hus, chitosan is a potential antif unga l agen t which can interfere with the pathogenic factors of the fungus. Keywords: chitosan, black m old, t om at o, A lt er na ria tox ins, elec troly te leak age, phospho lipids, x y lanase, pectin m et hy lesteras e IN T R O D U C T IO N A lt ernaria sp ecies in gen er al a re ®eld p ath ogen s wh ich a lso ca use st o rage lo sses in a va riety o f fr u it s. A . alt ernat a (F r.) K eissler f. sp. ly copersic i is th e ca u sa l a gen t of st em ca n ker C o r respo n dence to : J. A r u l. Tel: 418 656 2839; F a x: 418 656 3353. 0958 ±3157/98/010033 ±11 $9.00

� 1998 C a rfax P ub lishin g L td


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M . V. BH A SK A R A R E D D Y E T A L .

d isea se in to m ato (G ilch rist & G ro ga n , 1976). T h e fu ngu s a lso ca u ses b la ck m o ld o f rip e to m ato fr u it s, resu lt in g in su b st an t ia l lo sses. T h e d isea se is favo red by wa r m ra in y wea th er o r d ew fo r m at io n o n th e fr u it su rfa ce, a n d it s sever it y o f in fect io n is gr ea ter in th e rip e fr u it s th a n a t th e g reen st a ge (P ea rso n & H a ll, 1975) . A lt ernaria p ath ogen s a re k no wn to p ro d u ce h o st -sp eci®c to xin s (H STs) a s a gen ts o f vir u len ce o r p ath ogen icity fa ct or s; a t lea st n in e H ST -p ro d u cin g p at h o gen s h ave been rep or ted (O ta n i et al., 1995). A lt ernaria to xin s a lso h ave th e p o ten t ia l to b e h aza rd o u s to h um an h ea lt h (K in g & Sch ad e, 1984). T h ese to xin s a re lo w m o lecu la r weigh t secon d ar y m et ab o lites. T h ey a re ch em ica lly diverse, a n d m ay b e gr o up ed in to d ib en zo p yr en e d er ivat ives, su ch a s alt er n ar io l (A L ), a lt er n ario l m o n o m et h yl et h er (A M E ) a n d alt en u en e, an d tet ra m ic acid der ivat ives, su ch a s ten u a zo n ic a cid (TA ) a n d p er ylen e d er iva t ives (K o h m o to & O tan i, 1991; C h u lze et al., 1995) . T h e sp o ila ge o f to m at o b y A lt ernaria is o ft en acco m p an ied b y th e p ro du ct io n o f m yco toxins, a n d exten sive d et er io rat io n o f th e p ro d u ct resu lt s in eco n o m ic lo ss to co m m er cia l m a rk et er s (H a ssa n , 1995). It h a s b een r ep or ted th at th ese to xin s a ct o n th e p la sm a m em b ra ne of su scep t ib le t issu e a n d ca use elec tro lyte lo ss r esu lt in g in cell d ea th (O ta n i & K o hm o to, 1992) . F u r th er m o re, th ese to xin s a ct ivate p h o sp h o lip a se A 2 a nd in d u ce th e d egra d at io n of p h o sp h o lip id s in th e m icro so m e fr a ct io n s fro m su scep t ib le cells (L ee et al., 1992). Wea th er-b a sed fu n gicid e sp ray p ro gr am m es a re availab le to co n tro l A lt ernaria ®eld d isea ses o f to m a to (P it b la d o , 1989 ) a n d n on e o f th e fu ngicid es h ave b een a pp ro ved fo r po st h a rvest trea tm en t to in h ib it a lt er n a ria ro t. H o wever, gro wer s ar e co n fro n ted w it h th e p ro b lem s o f d er egistr at io n o f fu n gicid es d u e to h a r m fu l resid u es a n d in cr ea sed resist an ce of fu n ga l st ra in s to ch em icals ( Ja co b sen & Back m a n , 1993). Su ch envir o n m en ta l co n seq u en ces p ro m p ted wo r k er s to exp lo re a lt er n a t ive st rat egies fo r d isea se m an a gem en t (C het , 1987 ; K u c, 1987 ; D ea co n & B er r y, 1993). Bio co n tro l of p la n t p ath ogen s is o n e p o t en tia l o p t io n . C h it o sa n , a p o lycat io n ic p o lym er o f b -1,4-lin k ed d -glu co sa m in e, is on e b io a ct ive agen t k n o wn to b e a n t ifu n ga l (A lla n & H a d wiger, 1979 ; Sto ssel & L eu b a, 1984 ; H ir an o & N aga o, 1989 ; Ben h a m o u & T h er ia u lt , 1992 ; E l G h a o uth et al., 1992 a ). In a d d it io n , ch it o sa n ca n elici t p la n t d efen se m ech an ism s, su ch as the p ro d uct io n o f p h yt o a lexin s (H a d wiger & B eck m a n , 1980). C u ero et al. (1991 a) rep o rted th at ch it o sa n in h ib it ed a ¯atoxin p ro du ct io n b y A spergillu s ¯avus in p ea n u ts a n d m a ize. T h e o b ject ive o f th e p resen t st u dy was to in vest igate th e e Vect o f ch it o sa n o n th e grow th a n d p ro d u ct io n o f A L a nd A M E b y A . alt ernat a f. sp . ly copersic i, sin ce t hese to xin s a re co n sid er ed to b e viru len ce fa ct o rs in th e p at h o gen esis p ro cess a n d a re p ro d uced b y m o st st r ain s o f A . alt ernat a in rela t ively la rge q u a n t it ies. T h e p o ten cy o f cu lt u re ®lt rat es o f t he fu n gu s p ro d u ced in th e p resen ce of ch it o sa n wa s a lso st u d ied in ter m s o f th e ab ility to b rin g ab o u t d egra d at ive ch a nges in to m ato t issu e, su ch as electro lyte lea k a ge an d a ct ivat io n o f so m e o f th e cell wa ll-d egr ad in g en zym e system s. M AT ER IAL S A N D M E T H OD S Fungus C u lt u res o f A . alt ernat a f. sp. ly copersic i wer e o bta in ed fr om ca n ker s o f n atu ra lly in fected to m ato p la n ts wit h typ ica l st em -ca n k er sym p tom s. Sin g le sp o re iso la t io n s o f the cu lt u res u sed in th is st u d y wer e m a in tain ed o n p o ta to±d extro se aga r (P D A ) at 21± 23ëC . C hitosan Sh rim p -sh ell ch it o sa n was p u rch a sed fro m N ova ch em (H alifax, N ova Scot ia , C an a d a) a n d gro u n d to a ®n e p o wd er. P u ri®ed ch it osa n wa s p r ep ared b y d isso lvin g ch it o sa n in 0.25 n -H C l, a n d th e u n d isso lved p a r ticles wer e rem oved b y cen trifu ga t io n (15 m in , 10 000 3 g). T h e visco u s so lu t io n was th en b ro u gh t to p H 9.8 wit h 2.5 n -N a O H to p recip it ate th e ch it o sa n . T h e p recip it ated ch it o sa n wa s r ecovered b y ®lt r at io n , wa sh ed exten sively wit h d eio n ized water to rem o ve sa lt s a n d lyo ph ilized . C h it o sa n st o ck so lu t io n (10 m g m l- 1 ) was p rep a red b y d isso lvin g ch it o sa n in 0.05 n -H C l a n d the pH was ad ju st ed to 5.6. C h it o sa n

A N T IF U N G A L AC T IVI T Y O F C H IT O SA N

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so lu t io n was a u to clave d a n d a d d ed t o p o tato ±d ext ro se b ro th (P D B) to o b tain co ncen trat io n s o f 100 , 200, 400, 800, 1600 , 320 0 a n d 6400 l g m l- 1 o f th e m ed iu m . Inoculation and Incubation T h e st er ile m ed ia wer e in o cu la t ed w it h 1 m l o f sp o re su sp en sio n co n ta in in g 2.5 3 10 5 sp o res in st er ile wa ter, p rep a red fro m 10-d ay-o ld cu lt u res gro wn o n P D A . T h e fu n ga l sp o re co n cen tr at io n wa s d et er m in ed u sin g a h em ocyt o m et er (H a u sser Scien t i®c, H o r sh am , PA , U SA ). T h e ¯ask s wer e in cu b ated as a st a n din g cu lt u re at 22ëC u nd er a r t i®cia l ligh t (12 h ); 500- m l ¯ask s co nta in ed a to tal o f 100 m l o f P D B en rich ed wit h ch it o sa n at d i V er en t co n cen tr at io n s. A ft er 15 d ays o f in cu b a t io n , the m yceliu m wa s h a rvested by va cu u m ®lt rat io n o n W ha tm a n (M a id st o n e, K en t, U K ) n o. 1 ®lt er pa p er. Sp o re co u nts wer e m ad e fr o m th e wa sh in gs o f th e m yceliu m b y h em ocyto m et er. Sp or e viab ilit y wa s d et er m in ed b y sp rea d in g th e sp o re su sp en sio n p rep a red fro m th e wa sh in gs on aga r p la tes a nd in cu b at in g a t 15ëC fo r 12 h in th e d ar k . A b o u t 100 sp o res wer e o b served fo r ea ch su sp en sion a n d th e p er cen ta ge sp o re ger m in a tio n was d et er m in ed . Sp o res wer e co nsid er ed to b e ger m in ated wh en th e ger m tu b e was lo n ger th an th e w id th o f th e sp o re. T h e m yceliu m was exam in ed u n d er ligh t m icro sco py ( 3 400 ) fo r m o rp h olo gica l ch a n ges. T h e m ycelia wer e p la ced in a n oven at 60ëC o ver n igh t , a n d t h e dr y weigh t wa s d et er m in ed . T h e cu lt u re ®lt rates wer e p a ssed th ro u gh a m illip o re ®lt er (0.4 5 l m ) a nd st o red at 4ë C b efo re to xin extra ct io n . C ellfr ee ®lt rate (5 m l) fro m ea ch tr ea tm en t was cen trifu ged a t 10 000 3 g fo r 5 m in a n d th e su p er n ata n t wa s a ssayed fo r p ro tein usin g Bra d fo r d m et h o d (Bra d fo rd , 1976). P hospholipid and Enzyme Assay s M a tu r e gr een to m ato fr u it s wer e h a rvested fro m a lo ca l gr een ho u se, wash ed , su rfa ce st er ilized wit h 1% so d iu m h yp o ch lo rit e fo r 3 m in a nd rin sed w it h st er ile water. D isk s of p er ica r p t issu e, 10 m m in d ia m et er a nd 2 m m th ick , were p rep a red u sing a sh a rp st er ile co r k b o rer. T h e d isk s were p la ced in a n iso to nic so lu t io n o f m a n n it o l a s so o n a s th ey wer e m ad e. F ive d isk s wer e a r ra n ged o n two la yer s of m o isten ed ®lt er pa p er in 8.5-cm P et ri p la tes. T h e d isk s wer e p u n ct u red a t t he ep id er m a l su rfa ce u sing a st er ile n eed le, a nd 50 l l o f th e cu lt u re ®lt r ate wer e d ep o sited o n ea ch d isk to d et er m in e th e e V ect o f t oxin . C o nt ro ls wer e m a in ta in ed w it h m ed iu m a nd ch it o sa n wit ho u t th e fu ngu s at a ll co ncen trat io n s. T h e en t ir e p ro cess wa s ca r ried o u t u n d er st er ile la m in ar a ir ¯ow to avo id exter n al co nt am in at io n o f to m ato t issu e. T h e P et ri p la tes wer e sea led wit h P ara ®lm an d in cu ba ted at ro o m tem p er a tu r e (22ëC ) fo r 48 h . T h e d isk s wer e th en fro zen in liq u id nit ro gen an d st or ed at 2 80ëC u n t il en zym e a ssay. T h e p h osp h o lip id co n ten t wa s d et er m in ed u sin g th e m et h o d of M a rin et t i (1962) . T h e t issu e wa s h om ogen ized wit h m et h a no l a nd ch lo rofo r m (1 : 1, v : v) at 10 g m l - 1 . T h e extra ct s wer e th en dried wit h a nh yd ro u s N a 2 SO 4 a n d th e o rga n ic ph a ses wer e rem o ved o n a ro t ar y eva p o rato r. T h e resid u e wa s recovered wit h ch lo ro fo r m (2 ± 3 m l) a n d co n cen trated u n d er n it ro gen ga s. T h e o il resid ue co nst it u ted th e tot al lip id extra ct a n d wa s k ep t a t 4ëC u n t il u se. F ro m th is extra ct , p o la r lip id s (m ain ly p h o sp h o lip id s) a n d n eu tra l lip id s wer e sep a rated b y p recip it at io n in a ceto n e a s d escrib ed by K ates (1986) . P er ch lo ric a cid (0.4 m l) wa s a d d ed to t h e r esidu e. T h e so lu t io n wa s a llowed to st a n d fo r 4 m in u n t il it wa s clea r, an d th is wa s fo llo wed b y th e a d dit io n o f 4.2 m l o f d ist illed water an d 0.2 m l ea ch o f a m m o n iu m m o lyb d ate (5% w /v) an d am id o l (1% w /v). T h e m ixtu r e was vo r texed , h ea ted fo r 7 m in an d co o led fo r 15 m in , th en th e op t ica l d en sity (O D ) wa s m ea su red at 830 nm . F o r en zym e a ssays, th e cr u d e extra ct was p rep a red b y h om o gen izin g th e to m at o d isks in 1 m l o f 60% su cro se in p h o sp h at e b u V er ( p H 6.5) a nd 1 m l of st er ile wa ter /g o f t issu e, u sin g a m o rta r a n d p estle. T h e m ixt u r e wa s cen tr ifu ged at 13 000 3 g fo r 3 m in . T h e p ellet o b ta in ed was wa sh ed t wice in 2 m l o f su cro se b u Ver so lu t io n an d cen trifu ged b et ween wa sh es. T h e co m b in ed su p er n a ta n t co n st it u ted th e cr u d e extra ct . T h e en t ir e p ro ced u re wa s ca r ried o u t at 4ë C . P ect in m et h ylester ase a ct ivity was d et er m in ed b y in cu b at in g 0.5 m l o f rea ct io n m ixtu re co n ta in in g 0.25% p ect in A , 50 m l n m -P IP E S b u V er an d 0.1 m l o f cr u d e extra ct a t pH 7.5

36



fo r 6 h a t 25ëC . T h e rea ct io n was th en ter m in ated b y a d d in g 0.5 m l o f 1 m -H 2 SO 4 , a n d th e a m o u n t o f m et h a n ol lib er ated wa s d et er m in ed b y th e m et h o d o f Wo od a nd Sid d iq u i (1971) . X yla n a se act ivity wa s d et er m in ed u sin g X yla n -R BB (Sigm a C h em ica l C o., St L o u is, M O , U SA ) a s th e su b st ra te. T h e rea ct io n m ixtu re co n ta in ed 0.3 m l of su b st rat e so lu tio n (0.1% , w /v), 0.6 m l o f 0.1 m -so d iu m a cetate ( p H 4.8) an d 0.3 m l o f cr u d e extra ct . T h e rea ct io n was ter m in ated a ft er 4 h b y ad d in g 0.3 m l o f 0.5 m -H C l. A ft er co o ling in a n ice water b a th fo r 15 m in , th e m ixtu re was cen trifu ged at 13 000 3 g fo r 5 m in a nd th e abso rb a n ce o f the dye co va len tly lin k ed to th e d egra d ed p o lysa cch a rid es was m ea su red a t 600 n m . E lectro lyte lea k a ge wa s m ea su r ed u sin g a co n d u ct ivity m et er (m o d el E S-14, H o rib a L td , K yo to , Ja p a n ). To m ato d isk s weigh in g 4 g wer e p la ced in 25 m l o f 0.4 m -m a n n it o l so lu t io n a n d in cu b ated fo r 3 h at 28ëC . T h e in it ia l co n d u ct ivity (I C ) of th e aliqu o t was m ea su red at ro o m tem p er atu re (25ëC ). F in a l co nd u ct ivity (F C ) wa s d et er m in ed aft er a u to clavin g th e t issu e at 120ëC fo r 10 m in . T h e resu lt s wer e exp ressed as (I C /F C ) 3 100. E x tractio n and Assay of Tox ins M illip o re-®lt er ed cu lt u re ®lt rates st o red at 4ëC wer e ext ract ed twice w it h 50 m l o f ch lo rofo r m , an d th e a q u eo u s a n d o rga n ic p h a ses wer e sep ar ated . T h e o rga n ic p ha se was t hen co n cen tr ated t o 5 m l u sin g a ro t ar y eva p o ra to r, th en dried un d er n it ro gen . Toxin was d er iva t ized b y a d d in g 100 l l o f ch lo rofo r m , 50 l l o f d im et h yl fo r m a m id e a n d 50 l l o f th e r ea gen t m et h yl tert -b u tyl silyl t ri¯u ro a ceta m id e (M T BST F A ), a nd th e rea ct io n m ixtu re wa s kep t at 65ëC fo r 2 h . A L an d A M E wer e a ssayed b y ga s ch rom ato gr ap h y u sin g a H ewlet t P ack a rd (Avo n d a le, PA , U SA ) m o d el 589 0 eq u ip p ed wit h a ¯am e io n izat io n d et ecto r. T h e ch ro m a togr a p h ic sep ara t io n wa s ca rried o u t o n a ca p illa ry co lu m n (H ewlet t P a ck a rd , m o d el D B5-30M ) 0.25 l m w id e a n d 30 m lo n g. H yd ro gen was u sed a s th e ca r rier ga s at 55 kP. T h e d et ecto r tem p er a tu r e wa s 325 ëC , th e in let a ir tem p er at ure wa s 300 ëC , th e in it ia l tem p er a tu re wa s 200ëC a n d th e ®n a l tem p er atu re wa s 300ëC . T h e in ject io n s wer e ca r ried ou t wit h a n a uto sa m p ler (H ewlet t P ack a rd , m o d el 7673). T h e d ata wer e p rocessed u sing H P 3365 so ft wa re. U n d er t h ese co n d it io n s, A L h ad a r et en t io n t im e o f 14 m in a n d A M E ha d a ret en t io n t im e o f 8 m in . To xin st a n d a rd s (Sigm a ) wer e used fo r id en t i®ca t io n. S tatistic al Analysis F o u r r ep licates wer e u sed fo r ea ch trea tm en t an d th e exp er im en t wa s rep ea t ed twice. A s th e va r ia n ces wer e h o m o gen eo u s, th e d ata fro m t h e two exper im en ts wer e po o led . Stat ist ica l a n alysis was ca rried o u t o n th e p o o led d a ta u sin g the Stat ist ical A n a lysis System (SA S, 1982). T h e d ata wer e su b jected to a n a lysis o f va ria n ce. M ea n s wer e co m p a red by D u n ca n’s m u lt ip le-ra n ge test . R E SU LTS E Vect of C hitosan on M ycelia l D ry Weight and Tox in P roductio n C h it o sa n sh o wed sign i®ca n t m a in e V ects on th e m ycelia l gr owth a n d sp o re viab ility o f A lt ernaria an d p ro d u ct io n o f to xin s b y th e fu n gu s a s well a s o n th e e V ect iven ess o f th e to xin s in in d u cin g cellu la r d a m age o f to m a to t issu e (P < 0.0001) . T h e m ycelia l weigh t d ecrea sed fro m 0.67 to 0.0 g at ch it o sa n co n cen trat io n s ran ging fro m 0 to 6400 l g m l - 1 (F igu re 1). H o wever , th e d ecrea se in weight wa s n o t sign i®ca n t u p to 200 l g m l- 1 , a nd sh o wed sign i®ca n t red u ct io n wit h ea ch in cr ea se in ch it o sa n co ncen trat io n o f 400 l g m l - 1 a n d u p wa rd s. T h er e wa s a 78.0 % red u ct io n in m ycelia l weight at 320 0 l g m l- 1 co m p ared w it h th e co n tro l. T h e p ro d u ct io n o f A L va ried fro m 484 t o 0 l g m l - 1 at ch it o sa n co n cen tra t io n s of 6400 l g m l - 1 . H o wever , lo w co ncen trat io n s o f ch it o sa n (100 a nd 200 l g m l- 1 ) d id n ot r esu lt in a n y sign i®ca n t d iV er en ce in A L an d A M E p ro d u ct io n co m p a red w it h t he co n tro l. Sign i®ca n t red u ct io n s in to xin p ro du ct io n wer e o b ser ved wit h ch it o sa n co n cen tra t io n s of



37

F IG U R E 1.

E V ect o f ch itosa n concen tr atio ns on m ycelia l gr owth ( m ) an d sp o r ulat ion ( d ) of A . alternata f. sp . ly copersici a fter 15 days of growt h in P D B; m ea ns 6 st a n da r d erro r m ea n (SE M ) fo r eigh t r ep licates.

F IG U R E 2.

E V ect of chito sa n concent rat ion s o n a lter na rio l (A L ) ( m ) a nd a lter nar io l m o n o m ethyl ether (A M E ) ( d ) p r odu ct ion b y A . alternata f. sp. ly copersici after 15 days o f growt h in P D B; m ea ns 6 SE M fo r eigh t r ep licates.

400 l g m l - 1 an d u p wa rd s; t h e d i V er en ces wer e sign i®ca n t w it h in cr ea ses in ch it o sa n co n cen tr at io n (F igu re 2). A ch it o sa n co n cent rat io n o f 640 0 l g m l- 1 p ro ved to b e leth a l t o th e fu n gu s, an d n eith er gr o wth n o r to xin p ro d u ct io n wer e ob ser ved. T her e wa s a 86.73 % r ed u ct io n in A L p ro d u ct io n at 320 0 l g m l- 1 o f ch it o sa n co m p a red w it h th e co n tro l. Sim ila rly, th e p ro d u ct io n o f A M E va ried fro m 45.36 to 12.4 4 l g m l - 1 at ch it o sa n co n centr a-


38


t io n s o f 0 ± 320 0 l g m l - 1 (F igu re 2). T h er e wa s n o signi®ca n t d i Ver en ce b et ween th e co n tro l a n d 100 l g m l - 1 trea tm en t, b u t a 72.55 % red u ct io n in th e p ro d u ct io n of A M E wa s o b served a t a ch it o sa n co n cen tra t io n o f 320 0 l g m l - 1 . T h e an a lysis o f th e resu lt s sh owed th at ch it o sa n a Vected to xin p ro d u ct io n a t rela t ively lo wer co n cen trat io n s th a n th o se a t wh ich it a V ected fu n ga l gro wth . T h e m in im u m in hib it o r y co n cen tr at io n (M IC ) o f ch it o sa n fo r to xin p ro d u ct io n a n d m ycelia l gr owth was d et er m in ed b y p lo t t in g V 0 /V i a ga in st I, wh er e V 0 is th e to xin co n cen tra t io n o r m ycelia l gro wth at zero co n cen tr at io n o f ch it o sa n , V i is th e t o xin p rod u ct io n o r m ycelia l gr o wth at va rio u s co n cen tr at io n s o f ch it o sa n a n d I is th e in h ib it o r co n cen trat io n . T h is p lo t wa s describ ed b y th e eq u at io n V 0 /V i 5 I /K i , wh er e I /K i rep r esen ts th e slo pe o f th e lin ea r plo t a n d K i is th e M I C . T h e d er ived M IC o f ch it o sa n wa s 493.3 4 l g m l- 1 fo r A L , 675.4 3 l g m l- 1 fo r A M E a n d 937.65 l g m l - 1 fo r m ycelia l g ro wt h, em p h a sizin g th at to xin p ro du ct io n wa s a V ect ed m o re th a n m ycelia l gro wth at lo wer co n cen tra t io n s o f ch it o sa n . M icro sco p ic o b ser va t io n o f th e m yceliu m exp o sed to va rio u s ch it osa n co ncen trat io n s r evea led a ggregat io n o f fu n ga l cells. H yp h a e a p p ea red to b e ab no r m al in sh ap e an d grow th , sh o win g swellin g a n d excessiv e b ra n ch in g of co n to r ted hyp ha e. T h e hyp h a e also ap p ea red to b e m o re h ya lin e, an d lysis o f th e cell wa ll wa s n o t iced . Such ch a n ges wer e m o st p ro m in en t a t 3200 l g m l- 1 o f ch it o sa n (F igur e 3). A t su b -leth a l co n cen tra t io n s o f ch it o sa n , excessiv e sp o r u la t io n o f th e fu n gu s wa s o b ser ved , su ggest in g th at fu n ga l cells wer e st resse d (F igu re 1). H o wever , th e sp o re viab ility d ecrea sed wit h in cr ea ses in ch it o sa n co n cen tra t io n . It d ecrea sed fro m 92.66% in th e co n tro l to 0% at 320 0 l g m l - 1 of ch it o sa n (F igure 4). T h e d ecrea se in sp o re viab ility was n o t sign i®ca n t u p to a co n cen trat io n of 200 l g m l- 1 . Induction of Fungal C ellular Leakage by C hitosa n C h it o sa n in d u ced cellu la r lea k a ge o f p ro tein s in to th e m ed iu m . T h e a m o u n t of pro tein r eco vered fr o m t h e m ed iu m va ried fr om 3.32 to 13.4 8 l g m l- 1 at ch it o sa n co ncen t rat io n s o f 0 ± 3200 l g m l - 1 . T h er e wa s a sign i®ca n t (P < 0.05 ) in cr ea se in lea k a ge o f p ro tein s wit h a n in cr ea se in ch it o sa n co n cen tra t io n (F igu r e 4). P hospholipid C ontent and Electrolyt e Leakage from Tomato Tissue T h e p la n t t issu e d a m age in resp o nse to trea tm en t wit h cu lt u re ®lt rate co n ta in in g fu n ga l to xin s was a ssesse d b y extra ct ab le p h o sp h o lip id co n ten t an d electro lyte leak a ge o f th e t issu e. T h e p h o sp h o lip id co n ten t o f to m a to t issu e trea ted wit h cu lt u re ®lt rates when th e fu n gu s wa s gro w n wit h ch it o sa n was sign i®ca n tly h igh er th a n th at fro m ®lt rat es w it h o u t ch it o sa n (P < 0.05 ) (F igu re 5). T h e d i V er en ces wer e m o re evid en t fro m ch it o sa n co n cen tr at io n s of 400 l g m l - 1 a n d h igh er. T h e electr ica l co n d u ct ivit y o f to m ato d isk s trea ted w it h cu lt u re ®lt rates o f th e fu n gu s d ecrea sed sign i®ca n tly (P < 0.05 ) wit h in cr ea sin g co n cen tra t io n s of ch it o sa n (F igu re 5). T h e d i V er en ces wer e m o re sign i®ca n t fro m th e ch it o sa n co n cen tr at io n o f 200 l g m l- 1 a n d h igh er co m p a red w it h the co n tro l. T h e e V ect o f ch it o sa n per se o n b o th p h o sp h o lip id co n ten t an d elec tro lyte leak a ge o f to m a to t issu e was n ot sign i®ca n t (F igu re 5). T h ese resu lt s su gges t th at th e t o xin s p ro d u ced in ch it o sa n -free m ed iu m wer e m o re po ten t a t a ct ivat in g p h o sp h o lip a se in th e m em b r an e an d ca u sin g m em b ra n e d a m a ge th a n toxin p ro d u ced in th e p resen ce o f ch it osa n . E nzymatic Activity in Tomato Tissue T h e a ct ivit ies o f xyla n ase a n d p ect in m et hyles ter a se wer e a ssesse d in resp o n se to trea tm en t w it h cu lt u re ®lt rate co n ta in in g fu n ga l to xin s. T h e en zym at ic a ct ivity in cr u de extra ct s o f to m ato d isk s d i Ver ed wh en t h e d isk s h a d b een t rea ted wit h fu n ga l cu lt u re ®lt ra tes of d i V er en t ch it o sa n co n ten t (F igu r e 6). F iltrat es o f th e fu n gu s gr own wit h ou t ch it o sa n sh o wed th e h igh est d et ect ab le a ct ivit ies of xylan a se a n d p ect in m et hylester ase. T h er e wa s a 50% r ed u ct io n in bo th p ect in m et h ylester a se a n d xyla n a se a ct ivit ies at a ch it osa n co ncen trat io n o f 3200 l g m l - 1 co m p ar ed wit h th e co n tro l. T h is o b ser va t io n revea led th at t h o u gh t h e fu n gu s h a s t h e ab ility to p ro d u ce to xin at su b -letha l levels o f ch it o sa n, th e p o ten cy of



F IG U R E 3.

39

L igh t m icrogr aph s o f m ycelia of A . alternata f. sp. ly copersici gro wn in (a ) P D B an d (b ) br o th a m ended with chito sa n at 3200 l g m l- 1 .

th e to xin in a ct ivat in g cell-wall d egra din g en zym es d ecr ea sed w it h in cr ea ses in ch it o sa n co n cen tr at io n . T h e levels o f en zym at ic a ct ivit ies wer e lo w in to m ato t issu e trea ted wit h ch it o sa n per se (w it h o u t fu n gu s), in d icat in g th a t th e st im u la t io n of cell-wall d egra din g en zym es in th e p la n t t issu e is at trib u tab le to th e fu n ga l t oxin s (F igu re 6).

D ISC U SSION T h e r esu lt s o f th is st u d y d em o n st rate th at ch it o sa n in h ib it ed b o th fu n ga l gr owth a n d toxin p ro d u ced by A . alt ernat a. T h e an t ifu n ga l a ct ivity of ch it o sa n is well d ocu m en ted . C h it o sa n in ter feres w it h fu n ga l m em b ra n e fu n ct io n , ca u sin g a lt er at io ns in th e p er m eab ility o f th e m em b ra n e a n d p ro m o t in g in ter n a l o sm ot ic im b a la n ces (L eu b a & Sto ssel, 1986). T h e o b ser ved ch a nges in p ro tein lea k a ge fro m m ycelia in the presen t st u d y also su ggest in ter feren ce o f ch it o sa n w it h th e fu n ga l m em b ra n e. T h is co u ld a lso b e d u e to a lt er at io n s o f th e fu n ga l cell wa ll in th e p r esen ce o f ch it o sa n . C h it o sa n is k n own to in cr ea se th e act ivit y o f ch it in d ea cetylase, wh ich co n ver ts fu n ga l cell wa ll ch it in in t o ch it o sa n . T h is h a s b een


40


F IG U R E 4.

Sp o r e ger m ina tio n ( m ) a n d lea k a ge o f p ro teins ( d ) fro m A . alternata f. sp. lycopersici gro wn in P D B a m ended with diV erent con centr at ion s o f chito sa n; m ean s 6 SE M fo r eight r eplicates.

F IG U R E 5.

P h o sp ho lip id con ten t ( m ) an d electro lyte leak age ( d ) in tom a to tissue tr eated with cu ltur e ®ltra te of A . alternata f. sp. ly copersici grown with d iV eren t concen tra tion s o f chito sa n . P h o sp ho lip id content ( n ) an d electro lyte lea k a ge ( s) in to m ato tissu e tr eated with diV erent concen tr atio ns of chito sa n fro m the u nin oculated m edium . M ea ns 6 SE M fo r eight r eplicates.

d em o n st rated in R hizopus st olonifer (E l G ha o ut h et al., 1992b ). It h a s b een sh own p revio u sly (B ar tn ick i-G ar cia , 1973) th at ch it o sa n co uld in ter a ct wit h th e fr eshly fo r m ed p la sm a lem m a a t th e h yp h al ap ex, ca u sin g fo r m at io n of p o res in th e m em br an e which lea d s to m em br an e lea k age. A cco rd in g to H ad wiger a nd L o sch ke (1981) , ch it osa n m ay en ter t he fu n ga l cell th ro u gh lo o sen ed walls a n d in ter act wit h fu n ga l D N A an d m R N A to ca u se an t ifun ga l



F IG U R E 6.

41

X ylan ase ( m ) an d p ect in m ethylestera se (P M E ) ( d ) activity in to m ato t issu e treated with cu ltur e ®ltr ate of A . alternata f. sp . lycopersici fro m diV erent co n centr at ion s o f chit o sa n . X ylan ase ( n ) a nd P M E ( s) act ivity in tom ato t issu e tr eated with d iV eren t con centr at io ns of ch ito sa n from the u n inoculated m ediu m . M ean s6 SE M fo r eigh t r eplicates.

e Vects. T h e p o sit ively ch a rged g lu co sa m in e u n it s o f th e p o lym er co u ld in ter a ct wit h n egat ively ch a rged m a cro m o lecu les o f th e exp o sed fu n ga l cell su rfa ces. C h it osa n in ter feres w it h ch it in b io syn t h esis (B ar tn ick i-G a rcia & L ip p m a n, 1972 ) or cr ystalliza t io n o f ch it in (Va n n in i et al., 1986 ) in th e cell wa ll. C h it o sa n wa s n o t o n ly e V ect ive in in h ib it in g fu n ga l gr o wth b u t a lso a Vect ed th e p ro d u ct io n o f A L an d A M E b y the fu n gu s. A sim ila r in hib it o r y e Vect o f ch it o sa n o n A . ¯ avus a n d a¯at o xin p ro d u ct io n wa s o b ser ved in gro u nd n u t b y C u ero et al. (1991b ). F u r th er m o re, t h e e V ect o f ch it o sa n wa s sim ilar to p est icid es, as O m a r a n d M o h a m o u d (1995) sh o wed th a t a d d it io n o f th e fu ngicid e C u p ro sa n a n d su m i o il, a n in sect icid e, in h ib it ed gr o wt h a n d to xin pro d u ct io n b y A . alt ernat a in cu lt u re m ed iu m . T h e resu lt s o f th e p resent st u d y sh o w th a t ch it o sa n a t su b -leth a l co n cen tr at io n s a llow s the m yceliu m to gro w bu t to xin p ro d u ct io n is a Vected ; t h e M IC va lu es fo r to xin p ro d u ct io n wer e less th a n th o se fo r m ycelia l gr o wth . T h is co u ld b e d u e to m et ab o lic regula t or y a ct io n o f ch it o sa n o n th e syn th esis o f to xin . T h is wa s a lso o b ser ved b y C u ero et al. (1991 b) in th e p ro d u ct io n o f a ¯ato xin s b y A . ¯ avus in ch it o sa n -t rea ted gro u n d nu t seeds. T h e d ecrea se in t oxin pro du ct io n w it h in cr ea sin g ch it o sa n co n cen trat io n co u ld be d u e t o ch elat io n of C u , Z n a n d M n b y ch it o sa n (M u zza relli et al., 1980), b eca u se th ese io n s a re essen tia l fo r to xigen ic fu nga l gr o wt h an d toxin p ro d u ct io n (C u ero et al., 1987; C uero, 1989). C h it o sa n a t su b -leth al co n cen trat io n s a lso in d u ced in cr ea sed sp o r u la t io n o f the fu n gu s (F igu re 1). Su ch an e V ect co u ld b e d u e to st ress ca u sed b y ch it osa n . W h en an t ifu nga l co m p o u n d s ar e in co r p o ra ted in to th e m ed iu m o r fu n gi a re gr o wn o n n ut rien t-d e®cien t m ed iu m , in cr ea sed sp o r u la t io n is seen (Wo t to n & Stra nge, 1987). H o wever, th e sp o re viab ility d ecrea sed wit h in cr ea se in ch it o sa n co n cen trat io n, which is fu r th er evid en ce th at ch it o sa n ca n in h ib it m ycelia l gr o wth a n d sp o re ger m in a t io n . It h a s been sh o w n th at ch it o sa n ca n in h ib it sp o re ger m in at io n a n d ger m tu b e len gt h in fu n gi (E l G h a ou th et al., 1992a ). C u lt u re ®lt rates co n t ain in g toxin s o f A lt ernaria gro w n on d i V er en t co n centr at io n s o f ch it o sa n va r y in th eir ab ilit ies to ca u se d egra d at ive ch an ges in to m a to t issu e (e.g . extract ab le


42


m em b ra n e p h o sp h o lip id co n t en t, elec tro lyte lea k a ge a n d a ct ivat io n o f cell-wa ll degra d in g en zym es, su ch a s xylan a se an d p ect in m et h ylester a se). T h e e V ect o f th e to xin fr o m cu lt u re ®lt rates wh en ch it o sa n wa s p resen t o n th e t issu e decrea sed w it h in cr ea ses in ch it o sa n co n cen tr at io n . I t h as b een d em o n st ra ted th a t A lt ernaria to xin s d istu rb m em b ra ne in tegrit y in co m p a t ib le h o st s (H a & T h o m p so n, 1991). W h en su sp en sio n -cu lt u red cells o f st rawb er r y wer e trea ted w it h a lfa to xin 1 fro m A . alt ernat a, a n en h a n ced leve l o f ph o sp h olip a se (P L A -2) act ivity wa s o b served in th e m icro so m es at rela t ively lo w co n cen tra tio n s of th e to xin th at a re k n o w n to r ed u ce cell viability (L ee et al., 1991). It h as b een hyp o thesize d th a t to xin a V ect s cyto so lic C a 2 + co n cen tra t io n wh ich , in tu r n , en ha n ces P L A -2 (L ee et al., 1992). H o wever , th e d ecrea sed ability o f th e cu lt u re ®lt ra te co n ta in in g fu n ga l to xin s t o ca u se t issu e d a m age wit h in cr ea sed ch it o sa n co n cen trat io n s co u ld b e d u e to t he in ter feren ce o f ch it o sa n p rim a rily w it h fu n ga l gro wth a n d p ossib ly to xin b io syn th esis. In co n clu sio n , th e resu lt s o f th is st u d y d em o n st ra te t h a t ch it o sa n act s aga in st th e p ro d u ct io n of vir u len t fa ct o r s su ch a s to xin s b y p a th o gen s; th u s it m ay h ave in ter est in g ap p lica tio n s in t h e b io lo gica l co n t ro l o f p la n t d iseases.

AC K N OW L E D G E M E N TS T h e a u th o rs a ck n o wled ge ®n a n cial su p p o rt fr om th e N atu ra l Scien ces a n d E n gineerin g R esear ch C o u n cil (N SE R C ) o f C an a da Stra tegic G ra n t s P ro gr am .

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