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Microbiological Research 166 (2011) 207—215

Available online at www.sciencedirect.com

www.elsevier.de/micres

Antifungal activity of zinc oxide nanoparticles

against Botrytis cinerea and Penicillium expansum

Lili He 1, Yang Liu 1, Azlin Mustapha, Mengshi Lin ∗

Food Science Program, Division of Food Systems & Bioengineering,

University of Missouri, Columbia, MO 65211-5160, United States

R e c e i v e d 2 8 O c t o b e r 2 0 0 9 ; r e c e i v e d i n r e v i s e d f o r m 1 0 M a r c h 2 0 1 0 ; a c c e p t e d 1 1 M a r c h 2 0 1 0

KEYWORDS

Z i n c o x i d enano par ticles;A n t i f un g a l a c t i vi t y ;Botrytis cinerea;Penicillium

expansum;Raman spectr o sco py

AbstractA n ti f un ga l a ct i vi ti e s o f z i nc o xi de n an op ar ti cl es ( Zn O N Ps ) a nd t he ir m od e o f a c ti o n a g ai n st t w o p o st h ar v es t p a th o ge n ic f u n gi (Botrytis cinerea and Penicilliumexpansum) w er e i n ve st ig at ed i n t hi s s tu dy. Z nO N Ps w it h s iz e s o f 7 0± 1 5 n m a ndc on ce nt ra ti on s o f 0 , 3 , 6 a nd 1 2 m m ol l−1 w e r e u s e d . Tr a d i ti o n a l m i c r ob i o l o gi c a lp l a t i n g , s c a n n i n g e l e c t r o n m i c r o s c o p y ( S E M ) , a n d R a m a n s p e c t r o s c o p y w e r e u s e d t os t ud y a n ti f u ng a l a c ti v i ti e s o f Z n O N P s a n d t o c h ar a ct e ri z e t h e c h an g e s i n m o rp h ol -o g y a n d c e ll u la r c o mp o si t i on s o f f u ng a l h y ph a e t r ea t ed w i th Z n O N P s. R es u lt s s h ow

t h at Z n O N P s a t c o nc e nt r at i on s g r ea t er t h an 3 m m ol l−1 c a n s i g n i fi c a n tl y i n h i b i t t h eg r o w t h o f B. cinerea and P. expansum. P. expansum w a s m o r e s e n s i t i v e t o t h e t r e a t -m en t w it h Z nO N Ps t ha n B. cinerea. S E M i m a g es a n d R a m a n s p e c tr a i n di c at e t w od i f f e re n t a n t i f u n g a l a c t i v i t i es o f Z n O N P s a g a i n s t B. cinerea and P. expansum. Z n ON P s i n h i b i t e d t h e g r o w t h o f B. cinerea b y a f f e c t i n g c e l l u l a r f u n c t i o n s , w h i c h c a u s e dd e f o rm a t i on i n f u n g a l h y p h a e . I n c o m pa r i s on , Z n O N P s p r e v en t e d t h e d e v e l op m e n to f c o ni d io p ho r es a n d c o ni d i a o f P. expansum, w h ic h e v en t ua l ly l e d t o t h e d e a t h o f f u ng al h yp ha e. T he se r es ul ts s ug ge st t ha t Z nO N Ps c ou ld b e u se d a s a n e ff ec ti vef u n g i c id e i n a g r i cu l t u r al a n d f o o d s a f e t y a p p l ic a t i on s .© 2 0 1 0 E l s e v i e r G m b H . A l l r i g h t s r e s e rv e d .

1. Introduction

G ro wt h o f f un ga l p at ho ge ns i s t he m ai n c au seo f c o ns i de r ab l e e c on o mi c l o ss d u ri n g p o st h ar v es t

∗C o r re s p on d i n g a u t h or. Te l . : + 1 5 7 3 8 8 4 6 7 1 8;f a x : + 1 5 7 3 8 8 4 7 9 6 4 .

E-mail address: [email protected] ( M . L i n ) .1 B o t h a u t h o r s c o n t r i b ut e d e q u a ll y t o t h i s a r t i c l e .

h an dl in g o f f ru it s (S pa da ro e t a l. 2 00 4). Botrytiscinerea and Penicillium expansum c a n c a u s e s e v e r ep o st h ar v es t f r ui t d i se as e s i n cl u di n g g r ey a n d b l uem o ld e v en w he n t h e m o st a d va n ce d p o st h ar v es tt e c hn o l o gi e s a r e a p p l i ed (S p ad a ro e t a l . 2 0 0 4). B.cinerea i s c on si de re d a s o ne o f t he m os t i mp or -t a nt d i se a se s o f t a bl e g r ap e s (L a t o r r e e t a l . 1 9 9 4 ),while P. expansum p ri ma ri ly c au se s t he r ot o f s t o r e d a p p l e s a n d p e a r s (C a b a n a s e t a l . 2 0 0 9). F u r -thermore, P. expansum i s r eg ar de d a s t he m aj or

0 9 4 4- 5 0 1 3/ $ – s e e f r o n t m a t te r © 2 0 1 0 E l s e v i er G m b H. A l l r i g h t s r e s e r v e d.doi:10.1016/j.micres.2010.03.003

http://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.micres.2010.03.003http://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.micres.2010.03.003http://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.micres.2010.03.003mailto:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.micres.2010.03.003http://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.micres.2010.03.003mailto:[email protected]://localhost/var/www/apps/conversion/tmp/scratch_2/dx.doi.org/10.1016/j.micres.2010.03.003

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208 L. He et al .

p ro du ce r o f a m yc ot ox in , p at ul in , w hi ch i s c om -m o nl y f o un d i n r o tt i ng a p pl e s. U . S. F o od a n d D r u gA dm in is tr at io n ( FD A) l im it s p at ul in t o 5 0 m g / l i na p p l e j u i c e s ( M o a k e e t a l . 2 0 0 6 ).

I t i s d i ffi c ul t t o c o nt r ol f u ng a l g r ow t h b e ca u sef un gi h av e d ev el op ed r es is ta nc e t o m an y c on -v e nt i on a l f u ng i ci d es s u ch a s b e nz i mi d az o le s a n d

d i c a rb o x i mi d e s (El ad et a l. 1 99 2). To o v e rc o m et hi s r es is ta nc e, i t i s i mp or ta nt t o e xp l or e n ov ela n t if u n g al a g e nt s , w h ic h m a y r e p la c e c u rr e n t c o n -t r ol s t ra t eg i es . I n r ec e nt y ea r s, n a no p ar t ic l e ( N P)m a te r ia l s h a ve r e ce i ve d i nc r ea s in g a t te n ti o n d u et o t h ei r u ni q ue p h ys i ca l a n d c he m ic a l p r op e rt i esw h i ch d i f f er s i g n ifi c a nt l y f r o m t h e ir c o n v en t i on a lcounte rparts (S t o i m e n o v e t a l . 2 0 0 2). Re ce nt stud-i e s h a ve d e mo n st r at e d a n ti m ic r ob i al a c ti v it i es o f v a ri o us N P m a te r ia l s, i n cl u di n g s i lv e r (Ki m e t a l.2 0 08 a ,b ; K um a r e t a l . 2 0 08), c o pp e r (C io ffi e t a l.2005), t i t a n i u m d i o x i d e ( K w a k e t a l . 2 0 0 1), a n d z i n co x i d e (L i u e t a l . 2 0 0 9).

H i gh l y i o ni c n an o pa r ti c ul a te m e ta l o x id e s s uc ha s z i nc o x id e n an o pa r ti c le s ( Z nO N P s) a r e u ni q uei n t ha t t he y c an b e p ro du ce d w it h h ig h s ur fa cea r e a s a n d w i t h u n u s u a l c r y s t a l s t r u c t u r e s ( Klabundeet al. 1996). C om pa re d t o o rg an ic m at er ia ls ,i n or g an i c m a te ri a ls s uc h a s Z n O p o ss e ss s u pe r io rd u r ab i l i ty, g r e at e r s e l ec t i vi t y, a n d h e a t r e s is t a nc e(P ad m a v at h y a n d V i ja y a ra g h av a n 2 0 0 8). Moreover,z i n c i s a m i n e r a l e l e m e n t e s s e n t i a l t o h u m a n h e a l t ha nd Z nO is a f orm i n th e d ail y s up pl emen t f orz in c. Z nO N Ps a ls o h av e g oo d b io co mp at ib il it yt o hu ma n cel ls (P ad m a v at h y a n d V i ja y a ra g h av a n

2008). T h e a n t ib a c t er i a l a n d a n t i fu n g al a c t iv i t y o f b u lk Z n O p o wd e rs h a s b e en d e mo n st r at e d a l re a dy(Ya m am o to 2 0 01 ; S a wa i a n d Yo s hi k aw a 2 0 04). Ina gr ic ul tu re , z in c c om po un ds a re m ai nl y u se d a sf u ng i ci d es (Wa x m an 1 9 9 8). T he 5 0% l et ha l d os e(L D50 ) o f o r a l t o x i c i t y f o r Z n O i s 2 4 0 m g / k g f o r r a t s(S o u t h 2 0 0 2). R e c e n t i n t e r e s t l i e s i n t h e i r N P f o r m s .I t i s b e li ev e d t h a t t h e s m al l er t h e s i z e o f Z n O, t h es t ro n ge r a n ti m ic ro b ia l a c ti v it y i t h a s (Yamamoto2001). P r el i mi n ar y s t ud i es s h ow t h at t h e a n ti b ac -t er ia l a ct iv it y o f Z nO N Ps m ig ht b e r el at ed t o t hef o r m a t i o n o f f r e e r a d i c a l s o n t h e s u r f a c e o f t h e N P s ,

a n d t h e d a m a g e t o t h e l i p i d s i n b a c t e r i a l c e l l m e m -b r a ne b y f r e e r a d i c al s , w h i ch c o n se q u en t l y l e a d t ot h e l e ak a ge a n d b r ea k do w n o f b a ct e ri a l c el l m e m-b ra ne (B ra yn er e t a l. 2 00 6; Re dd y e t a l. 2 00 7).H o we v er, t o t h e b e st o f o u r k n ow l ed g e, t h e e f fe cta nd m od e o f a ct io n o f Z nO N Ps o n t he g ro wt h o f f u ng i s u ch a s B. cinerea and P. expansum h a v e n o tb e e n s t u d ie d .

I n t hi s s tu dy, w e i nv es ti ga te d t he a nt if un ga la c ti v it i es o f Z n O N P s a g ai ns t t w o i m po r ta n t p l an tpathoge nic fungi, B. cinerea and P. expansum. T h em od e o f a ct io n o f Z nO N Ps o n t he g ro wt h o f f un -

g a l h yp h ae w a s a l so s t ud i ed b y s c an ni n g e l ec t ro nm i c ro s c op y ( S E M) a n d R a m an s p e ct r o s co p y.

2. Materials and methods

2.1. Nanoparticle materials

Z n O N P s u s p e n s i o n s w i t h N P s i z e o f 7 0 ±1 5 n m w e r ep u r c h a s e d f r o m A l f a A e s a r ( W a r d H i l l , M A , U S A ) . A na li qu ot ( 10 m l) o f Z nO N P s us pe ns io n w as v ac uu mfi l te re d t h ro ug h a n a l um i nu m o x id e m e mb r an e fi l -t e r w i t h a 2 0 n m p o r e s i z e a n d 2 5 m m O D ( A n o d i s c ;W ha tm an I nc ., C li ft on , N J, U SA ), r es ul ti ng i n aN P -f r ee s o lu t io n . T h e c om p os i ti o n o f t h e N P -f r ees o l u t i o n w a s a n a l y z e d t o b e w a t e r a n d a d i s p e r s a n t ,a n d i t s e f fe ct o n b a ct e ri a l g r ow t h w as e x am i ne d .T he o ri gi na l Z nO N P s us pe ns io n ( 12 m ol l−1 ) andN P - f r e e s o l u t i o n w e r e t h e n d i l u t e d w i t h p o t a t o d e x -

t r os e a g ar ( P DA , c o nt a in in g t h e e x tr a ct f r om 2 0 0 gb oi le d p ot at o, 2 0 g g lu co se a nd 2 0 g a ga r i n 1 l o f d i st i ll e d w a te r) t o m a ke a s e ri e s o f m ed i a c o nt a in -i ng Z nO N Ps w it h c on ce nt ra ti on s o f 0 , 3 , 6 a nd1 2 m m o l l−1 a n d N P - f re e s o l ut i o n.

2.2. Antifungal test

Two pathoge nic fungi, B. cinerea and P. expansum,w er e o b ta i ne d f r om t h e c u lt u re c ol l ec t io n o f t h eF o od M i cr o bi o lo g y l a bo r at o ry a t t h e U n iv e rs i ty o f Missouri, C olumbia, MO. B. cinerea and P. expansum

w er e c ul tu re d o n P DA a t 2 5◦

C i n t he d ar k. A nt i-f u ng a l t e st s w er e p e rf o rm e d b y t h e a g ar d i lu t io nm e th o d (F r at e rn a le e t a l . 2 0 03) w i th s o me m o di -fi c at i on . T h e a ut o cl a ve d P D A m e di a w it h Z nO N P sa t c on ce nt ra ti on s o f 0 , 3 , 6 a nd 1 2 m mo l l−1 a n d aN P - f re e s o l ut i o n w e r e p o u r ed i n t o t h e P et r i d i s he s( 9 c m d i am e te r ). T h e f u ng i w er e i n oc ul a te d a f te rt h e P D A m e d i a s o l i d i fi ed . A d i s c ( 1 . 4 c m ) o f m y c el i a lm a te ri a l t a ke n f r om t h e e d ge o f 7 - da y -o l d f u ng a lc u l t u r e s w a s p l a c e d i n t h e c e n t e r o f e a c h P e t r i d i s h .T he Pe tr i d is h w it h t he i no cu lu ms w as t he n i nc u-b at ed a t 2 5 ◦C . T he e ffi ca cy o f Z nO N P t re at me ntw as e va lu at ed a t t he t im e i nt er va ls o f 2 , 4 , 6 , 9 ,a nd 1 2 d ay s b y m ea su ri ng t he d ia me te r o f f un ga lc o l o n i e s . A l l t e s t s w e r e p e r f o r m e d i n t r i p l i c a t e a n dt h e v a l u e s w e r e e x p r e s s e d i n c e n t i m e t e r s .

2.3. Morphological test of fungal hyphae

S E M w as u s ed t o e x am i ne m o rp h ol o gi c al c ha n ge sof B. cinerea and P. expansum h yp h a e b e f o re a n da f te r t r ea t me nt w it h Z n O N P s. Pi e ce s o f m yc e li a lm a te ri a l c ut f r om 7 - da y- o ld c ul t ur es w er e i n oc u -l a te d o n t o t h e P D A c o n ta i ni ng 1 2 m m ol l−1 Z n O N P s

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A n t if u n ga l a c t iv i t y o f z i n c o x i d e n a n op a r ti c l e s a g a i n s t Botrytis cinerea and Penicillium expansum 209

a n d t h e c o nt r ol ( t he P D A c o nt a in in g n o Z n O N P s ) ,f o l l o w e d b y i n c u b a t i o n f o r 1 2 d a y s . T h e n , p i e c e s o f m y c e l i a l m a t e r i a l w a s c u t f r o m t h e e d g e o f t h e f u n -g a l c u l t u r e s , a n d d i r e c t l y s u b j e c t e d t o S E M a n a l y s i su nd e r t h e e nv i ro n me n ta l m o de . S E M i m ag e s w er et a ke n b y F E I Q u an t a 6 0 0F E nv i ro n me nt a l S E M ( F EIC om pa ny, H il ls bo ro , O R, U SA ) a t a v ol ta ge o f 7 o r

1 0 k V a n d a p re ss ur e f r om 5 25 t o 6 1 9 Pa .

2.4. Raman instrumentation and dataanalysis

A R en i sh a w R M 10 0 0 R a ma n s p ec t ro m et e r s y st e m( G l o uc e s te r s hi r e , U K ) e q u ip p e d w i t h a L e i ca D M L Bm i c ro s c op e ( We t z l ar, G e r ma n y ) a n d a 7 8 5 n m n e a r-i n fr ar e d d i od e l a se r s o ur c e ( m ax i mu m a t 3 0 0 m W )w as u se d i n t hi s s tu dy. R am an s ca tt er in g s ig -na ls w ere d et ec ted b y a 5 78×3 85 p ix el s C CDa r r ay d e t ec t o r. R a m a n s p e c t r a w e r e a c q u i r ed f r o m

m y c el i a l m a t er i a l s i s o l at e d f r o m B. cinerea and P.expansum c u l t u r e s w i t h o r w i t h o u t t h e e x p o s u r e t o1 2 m m o l l−1 Z n O N P s a f t e r 1 2 - d a y i n c u b a t i o n . A 5 0×o b je c ti v e w as u s ed w it h a R a ma n d e te c ti o n r a ng ef ro m 6 0 0 t o 1 8 00 c m−1 i n t h e e x t e n d e d m o d e . T h em e as u re m en t w a s c o nd u ct e d w i th a 1 0 s e x po s ur et i m e a n d ∼1 0 m W l a s er p o w er.

D a ta a n al y si s w as p e rf o rm e d u s in g D e li g ht v e r-sion 3.2.1 (D-Square d De ve lopme nt Inc., L aGrande ,O R, U SA ) s of tw ar e. Pr e- pr oc es si ng a lg or it hm ss u ch a s s m oo t hi n g a n d p o ly no m ia l s u bt r ac t w er ee mp lo ye d t o e li mi na te i ns tr um en ta l n oi se s a nd

r e m o v e b a s e l i n e o f f s e t s . P r i n c i p a l c o m p o n e n t a n a l -y si s ( PC A) w as u se d t o a na ly ze t he s pe ct ra l d at a.P CA i s a m ul ti va ri at e a na ly si s m et ho d u se d t oe x t r a c t p a t t e r n s o r t o e s t a b l i s h r e l a t i o n s h i p s i n t h ed a t a s e t s (G o o d a c r e e t a l . 1 9 9 8).

3. Results

3.1. Antifungal effect of ZnO NPs

Z n O N P s us p en s io n s w er e e x am i ne d b y t r an sm i s-

s i o n e l e c t r o n m i c r o s c o p y ( T E M ) ( F i g . 1). T h e s h a p e so f Z nO N Ps w er e m os tl y r od -l ik e s tr uc tu re . T hes i ze i s i n a g re em e nt w i th t h e p r od u ct d e sc ri p ti o n,t h a t i s 7 0±1 5 n m i n l e ng t h. T h e Z n O N P -f r ee s o lu -t i on w as o b ta i ne d b y fi l tr a ti o n a n d i t s c o mp o si t io ni n cl u de d w a te r a n d a d i sp e rs a nt (L iu e t a l. 2 00 9).T h e Z n O N P -f r ee s o lu t io n h a d n o e f fe ct o n f u ng a lg r o wt h a n d n o r ma l c o l on y f o r m at i o n w a s o b s er v e d( d at a n o t s h ow n) . Fig s. 2 a nd 3 s ho w t h e e f fe c tof ZnO NP s o n t he g row th of B. cinerea and P.expansum t ha t w er e c ul t iv at ed o n P DA c on ta in -i ng d if fe re nt c on ce nt ra ti on s o f Z nO N Ps ( 0, 3 , 6 ,

Fig. 1. T E M i m ag e o f z i nc o x id e n a n o pa r ti c le ( Z n O N P )s u s p en s i o n (L i u e t a l . 2 0 0 9).

Fig. 2. A n t i f u ng a l a c t iv i t i e s o f Zn O N P s a g ai n s t Botrytiscinerea o n P D A i n t h e p r es e nc e o f d i ff e re n t c o nc e nt r a-t i on s o f Z n O N P s. D a ta a r e s h o wn w i th m e an v a lu e s a n ds t an d ar d e r ro r s o f b a ct e ri a l c o un t s. E a ch p o in t r e pr e se n tsa n a v e r ag e o f t r i p l ic a t e m e a s ur e m e nt s .

Fig. 3. A n ti f u ng a l a c t i vi t ie s o f Z n O N P s a g ai n st Peni-cillium expansum o n P DA i n t he p re se nc e o f d if fe re ntc o nc e nt r at i on s o f Z n O N P s. D a ta a r e s h o w n w i th m e anv a l u e s a n d s t a n d a r d e r r o r s o f b a c t e r i a l c o u n t s . E a c h p o i n tr e p re s e n t s a n a v e r ag e o f t r i p li c a t e m e a s u r e m en t s .

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210 L. He et al .

Fig. 4. S E M i m a g e s o f Botrytis cinerea w i t h o u t ( A a n d B ) o r w i t h ( C a n d D ) t h e t r e a t m e n t o f Z n O N P s u s p e n s i o n .

1 2 m m o l l−1 ) a nd i nc ub at ed a t 2 5◦C f or 1 2 d ay s.G e n e r a l l y , t h e u s e o f Z n O N P s u s p e n s i o n w a s e f f e c -t i ve i n i n hi b it i ng f u ng a l g r ow t h f o r b o th B. cinereaand P. expansum. T h e a v e r a g e g r o w t h o f B. cinereaw a s i n h i b i t e d b y f r o m 6 3 % t o 8 0 % i n t e r m s o f c o l o n y

g ro wt h d ia me te rs a ft er 1 2 d ay s o f i nc ub at io n a st h e c o n ce n tr a ti o n o f Z n O N P s i n cr ea s ed f r om 3 t o1 2 m m o l l−1 (F ig . 2). Fo r P. expansum, t he r ed uc -t io n r at e o f f un ga l g ro wt h v ar ie d f ro m 6 1% t o 9 1%a s t he c on ce nt ra ti on o f Z nO N Ps i nc re as ed f ro m3 to 12 mmol l−1 w it h a n e ar c o mp l et e i n hi b it i onat 6 mmol l−1 (F ig . 3). S i gn ifi c an t d i ff e re nc e w asf o u n d f o r d i f f e r e n t c o n c e n t r a t i o n s o f Z n O N P t r e a t -m en t (P < 0 .0 5) . T he se r es ul ts i nd ic at e t ha t Z nON Ps a t c on ce nt ra ti on g re at er t ha n 3 m mo l l−1 cans i gn i fic a nt l y i n hi b it t h e g r ow t h o f B. cinerea andP. expansum; a nd Z nO N Ps w er e m or e e ff ec ti veagainst P. expansum than B. cinerea.

3.2. Morphological analysis of fungal growth

To i nv es ti ga te t he m ec ha ni sm b y w hi ch Z nO N Psa ff ec t t he g ro wt h o f B. cinerea and P. expan-sum, S EM a na ly si s w as e mp l oy ed t o e xa mi ne t hes t ru ct u ra l c ha n ge s o f f u ng a l s a mp l es a f te r Z n O N Pt r ea t me nt . P D A m e di a c o nt a in i ng 1 2 m m ol l−1 ZnON P s w er e p r ep a re d , a n d f u ng a l s a mp l es w er e t h eni no cu la te d o nt o t he P DA p la te a nd i nc ub at ed a t25 ◦C f or 1 2 d ay s. F ig . 4A a nd B s ho ws t he i ma ge s

o f m yc el ia o bt ai ne d f ro m t he e dg e o f B. cinereac u l t u r e i n t h e c o n t r o l ( u n t r e a t e d s a m p l e s ) , d e m o n -s t ra t in g h yp h ae w i th t y pi c al n et s t ru ct u re a n ds m oo t h s u rf a ce . A f te r t r ea t me nt w i th 1 2 m m ol l−1

Z nO N Ps , t he h yp ha e l os t t he ir s mo ot hn es s a nd

f or me d u nu su al b ul g es o n t he s ur fa ce o f f un ga lh yp h ae (F ig . 4C a nd D ), i nd ic at in g t ha t Z nO N Psi n hi b it e d t h e g r ow t h o f B. cinerea b y d e fo rm i ngt h e s t r u c t u r e o f f u n g a l h y p h a e . S E M o b s e r v a t i o n s o f e xte rnal my ce lia from P. expansum colonie s de mon-s t r at e b r a nc h e d c o n i d i a w i t h r o u n d - a n d c h a in - l ik es t r uc t u re s (F ig . 5A a n d B ). A ft er t re at me nt w it h1 2 m m o l l−1 Z nO N Ps f or 1 2 d ay s, g er mi na ti on o f P. expansum c o ni d i a w a s c o m p le t e l y i n h i b i te d a n dc o ni d ia l d e ve l op m en t o f P. expansum w as s up -p r e s s e d (F i g . 5C a n d D ) . T h es e r es u lt s s u gg e st t h atZ nO N Ps d is to rt ed a nd d am ag ed t he c on id ia o f P.expansum. C o ns e qu en t ly, t h e f u ng a l g r ow t h w a sgre atly inhibite d.

3.3. Study of mode of action of ZnO NPs byRaman spectroscopy

R a ma n s p ec t ro s co p y w a s u s ed t o f u rt h er i n ve s ti g at et he m od e o f a ct io n o f Z nO N Ps a nd t he ir a nt if un -gal me chanisms. B. cinerea and P. expansum werei n c u b a t e d o n P D A w i t h o r w i t h o u t Z n O N P s a t 2 5 ◦Cf o r 1 2 d a ys . T h en f u ng a l c o lo n ie s w e re e xa m in e db y R a m a n s p e c t r o sc o p y. T h e s p e ct r a o f B. cinerea

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Fig. 5. S E M i m a g e s o f Penicillium expansum w i t h o u t ( A a n d B ) o r w i t h ( C a n d D ) t h e t r e a t m e n t o f Z n O N P s u s p e n s i o n .

and P. expansum e x h i b it e d d i s t in c t iv e a b s o rp t i o nb an ds i n t he r an ge b et we en 6 00 a nd 1 80 0 c m−1 ,r e v ea l i n g v i b r at i o n al i n f o rm a t i on o n c a r bo h y dr a t es ,p ro te in s, l ip id s a nd n uc le ic a ci ds . M aj or R am anp e ak s a c qu i re d f r om t h e f u ng a l h y ph a e a n d t h ei r

b a n d a s s i g n m e n t s a r e s h o w n i n Table 1 (Schuste r e ta l. 2 00 0; M aq ue li n e t a l. 2 00 2; C ha n e t a l. 2 00 7).Fi g. 6 s ho ws t he a ve ra ge s pe ct ra o f B. cinereac ul t iv at ed o n P DA w it h o r w it ho ut 1 2 m ol l−1 ZnON P s . F o r e x a m p l e , b a n d s a r o u n d 7 4 3 , 7 7 2 , 9 3 8 a n d1 0 82 c m−1 w er e a s si g ne d t o n uc l ei c a c id s ; b a nd sa ro un d 8 48 , 8 68 , 8 95 , 9 65 a nd 1 04 8 c m−1 werea ss ig ne d t o c ar bo hy dr at es ; b an ds a ro un d 1 00 5,1 2 54 a n d 1 6 6 8 c m−1 w e r e a s s i g n e d t o p r o t e i n s ; a n db a nd s a r ou nd 1 4 16 a n d 1 4 6 0 c m−1 w e r e a s s i g n e d t o

Fig. 6. R a ma n s p e ct r a o f Botrytis cinerea w it h ( A) o rw i th o ut ( B ) t h e t r ea t me n t o f Z n O N P s u sp e ns i on . M e a-s u re m en t s w e r e t a ke n f r om 6 0 0 t o 1 8 00 c m−1 u n d e r 1 0 sand ∼1 0 m W l a s e r p o w e r.

l i pi d s. I t i s o b se rv e d t h at t h e i nt e ns i ty o f n u cl e icacid and carbohy drate bands incre ase d significantlyi n f un ga l h yp ha e t re at ed w it h Z nO N Ps ; w hi le n oo bv io us c ha ng e o f R am an s ig na ls o f p ro te in s a nd

Table 1. B a nd a s si g n me n t o f R a m a n p e ak s i n t h e r a ng eo f 6 0 0— 1 80 0 c m−1 (S c hu s te r e t a l . 2 0 0 0 ; M a q u el i n e t a l .2 0 0 2 ; C h a n e t a l . 2 0 0 7 ).

R a m an s h i f t(cm−1 )

Assignment

∼743 Nuclei c acids (A)∼772 Nuclei c acids (C, T)∼848 Car bohydr ate (C—C stretching,

C — O — C 1 , 4 g l y c os i d i c l i n k )∼868 Car bohydr ate∼895 Car bohydr ate (C—O—C str etch ing)∼938 DNA backbone

∼965 Car bohydr ate (C—C)∼1005 Phenylalanine∼1048 Car bohydr ates (C—C defor matio n)∼1082 DNA (O—P—O− str etching)∼1198 Car bohydr ate (—C—C—, C—O, C—O—H)∼1254 Ami de III∼1368 Li pid (C—H2 defo r matio n)∼1416 Li pid (C—H2 , C — H 3 defo r matio n)∼1454 Li pid (C—H2 , C — H 3 defo r matio n)∼1603 Ami de II∼1668 Ami de I, u nsaturated lipi d (C C

str etching)

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212 L. He et al .

Fig. 7. R a ma n s p e ct r a o f Penicillium expansum with( A ) o r w i th o ut ( B ) t h e t r ea t me n t o f Z n O N P s u sp e ns i on .M e as u re m en t s w e re t a ke n f r om 6 0 0 t o 1 8 00 c m−1 under1 0 s a n d ∼1 0 m W l a s e r p o w e r.

Fig. 8. P CA p lo t b as ed o n R am an s pe ct ra o f f un ga lsamples: Botrytis cinerea w it h t re at me nt o f Z nO N Ps us pe ns io n ( B Z ) a nd t he c on tr ol ( B C ); Penicillium expan-sum w i th t h e t r e a tm e nt o f Z n O N P s u s p en s io n ( P Z ) a n dt h e c o n t ro l ( P C ) .

l i p i d s w a s o b s e r v e d . F i g . 7 sh o w s t h e a v e r a g e s p e c -t r a o f P. expansum g r o w i n g o n P D A w i t h o r w i t h o u t1 2 m m o l l−1 Z n O N P s . F o r i n s t an ce , a b a nd a r ou n d1 1 98 c m−1 w a s a s si g ne d t o c a rb o hy d ra t es ; b a nd sa r ou nd 1 3 68 a n d 1 4 5 4 c m−1 w e r e a s s i g n e d t o l i p i d s ;a n d a b a nd a r ou nd 1 6 03 w as a s si g ne d t o p r ot e in s .N o o b v i o u s R a m a n p e a k s w e r e o b s e r v e d f r o m f u n g a lh yp h ae o f P. expansum a f t er Z n O t r ea t me nt . P C Aw a s p e r f o r m e d o n t h e R a m a n s p e c t r a o f B. cinereaand P. expansum i nc ub at ed o n P DA w it h o r w it h-o ut 1 2 m m ol l−1 Z n O N P s. A t w o- d im e ns i on a l ( 2 D)P CA p lo t w it h t he fi rs t t wo P C s co re s i s s ho wn i nF i g . 8. C l e a r s e g r e g a t i o n s w e r e o b s e r v e d a m o n g t h ed a t a c l u s t e r o f B. cinerea c o nt r ol ( B C ) , B. cinereat re at ed w it h Z nO N Ps ( B Z ) , P. expansum control( P C ) , a n d P. expansum t r ea t ed w i th Z nO N P s ( P Z ) .

4. Discussion

Z n O N P s a r e u s u a l l y p r e s e n t i n a f o r m o f a g g l o m e r -a t e s d u r i n g i t s m a n u f a c t u r i n g p r o c e s s (Z h a n g e t a l .2007). Tw o m e t ho d s , u l t r as o n ic a t io n a n d a d d i ti o no f d is pe rs an ts , a re o ft en u se d t o b re ak d ow n N P

a g g l om e r at e s . C o m m on l y u s e d d i s p er s a nt s i n c lu d epoly viny lpy rolidone , poly e thy le ne gly col, and othe rc h em i c a ls (B r ay n er e t a l . 2 0 06). F ig . 1 s h o ws t h a tZnO NPs used in this study (Liu et al. 2009).I n fo r ma t io n r e ga r di ng t h e d i sp e rs a nt u se d i n t h isp r op r ie t ar y c o mm e rc i al N P p r ep a ra t io n w as n o ta v ai l ab l e f r om t h e m a nu f ac t ur er. Z nO N P s w er e

u ni f or m ly d i sp e rs ed i n P D A, w h ic h w a s p r ov e n b yc o n si s t e nt i n h ib i t i on r e s ul t s o b t a in e d i n t h i s s t u d y.

T h e s i g ni fi c an t l y a n t if u n g al a c t iv i t y a g a in s t B.cinerea and P. expansum w as f o un d u s in g t h e Z n ON P s a s l o w a s 3 m m o l l−1 . A s t h e c o n c en t ra t io n o f Z n O N P s i n c r e a s e d f r o m 3 t o 1 2 m m o l l−1 , t h e e f fi -c a c y o f Z nO N P t r e at m e nt w a s e n ha n c ed . C o m p ar e dto B. cinerea, P. expansum i s m o r e s e n s i t i v e t o Z n ON P t r ea t me nt . H o we v er, t h e d i ff e re nt a n ti f un g ale f f e c t s m a y r e s u l t f r o m d i f f e r e n t g r o w t h m o r p h o l o -g i e s o f t h e s e t w o f u n g i . P. expansum t e n d s t o g r o wm o re d e ns e ly o n t h e s u r fa c e o f a g ar p l at e t h an B.cinerea, s o t ha t i t h as m or e e xp os ur e t o Z nO N Psc o m pa r e d w i t h B. cinerea. A n ot h er p o ss i bl e r ea -s o n f o r t h e d i ff e re nc e c o ul d b e i nn a te t o le ra n ceo f e ac h f un gu s t o Z nO N Ps . S a w ai a n d Yo s h ik a w a(2004) r e p or t ed t h e m i ni m um i n hi b it o ry c o nc e n-t r at i on o f b u lk Z n O p o wd e r a g ai n st Saccharomycescerevisiae, Candida albicans, Aspergillus niger, andRhizopus stolonifer w as o ve r 1 0 0 m g m l−1 (∼1 . 2 m o ll−1 ) b y a n i n d i r e c t c o n d u c t i m e t r i c a s s a y (S a w a i a n dYoshikawa 2004). Z n O N P s i n o u r s t u d y s h o w g r e a te nh a nc e me n t i n t h e a nt i mi cr o bi a l a c ti v it y d u e t ot h e ir u n i qu e p r o p er t i es s u c h a s l a r g e s u r f a ce a r e a.However, K as e me t s e t a l . ( 2 00 9 ) f o un d n an o a nd

b u lk Z n O w er e o f c o mp a ra b le t o xi c it y a g ai ns t S.cerevisiae.

S EM h as b ee n s uc ce ss fu ll y u se d t o a ss es s m or -p h ol o gi c al c h an g es o f m i cr o bi a l c e ll s i n du ce d b yZnO NP s (B ra yn er e t a l. 2 00 6; Z ha ng e t a l. 2 00 7)a n d f u ng a l h yp h ae t r ea t ed w i th o t he r c h em i ca l s(S h ar m a a n d S ha r ma 2 0 08 ; Ye n e t a l . 2 0 08). S o m es tu di es p ro po se d t ha t Z nO N Ps m ay c au se s tr uc -t u r al c h a ng e s o f m i c ro b i al c e l l m e m b r a ne , c a u si n gc y t o p l a s m l e a k a g e a n d e v e n t u a l l y t h e d e a t h o f b a c -t er ia l c el l s (S a wa i a n d Yo s hi k aw a 2 0 04 ; B r ay ne re t a l. 2 00 6). I n t h is s t ud y, P. expansum produce d

c o n id i a w h i le B. cinerea b i o ma s s w a s m a in l y c o m-p o se d o f h yp h ae . Z nO N P t r ea t me nt i n hi b it e d t h ec o ni d ia l d e ve l op m en t a nd d i st o rt e d t h e c o ni d io -p h o re s o f P. expansum. C o mp a re d t o P. expansum,B. cinerea a p pe ar ed t o b e m or e r es is ta nt t o Z nON P s. T h e fi n e s t ru c tu re o f B. cinerea m y c el i a w a sp r es e rv e d, a l th o ug h t h e s u rf a ce o f f u ng a l h y ph a ew a s d e f o r m e d . T h e r e f o r e , Z n O N P s m a y e x h i b i t d i f -fe re nt antifungal activitie s against P. expansum andB. cinerea.

V ib r at i on a l s p ec t ro s co p ic t e ch ni q ue s , s uc h a sR a m a n s p e c t r o s c o p y , a r e u s e f u l f o r m o n i t o r i n g s u b -

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t l e c h a n g e s i n m i c r o b i a l c e l l s a s t h e t e c h n i q u e s a r er a pi d , n o n- d es t ru c ti v e a n d r eq u ir e m i ni m al s a m-p l e p r ep a ra t io n (D e G us se m e t a l. 2 00 6). Ramans p e c t r o s c o p y h a s b e e n a p p l i e d e x t e n s i v e l y f o r m o n -i t or i ng a n d c h ar a ct e ri z in g b a ct e ri a a n d f u ng i (DeG us se m e t a l. 2 00 5; S en gu pt a e t a l. 2 00 6; C ha net al. 2007; Szeghalmi et al. 2007), and for

i n ve st i ga t in g t h e m o de o f a c ti o n o f a nt i mi cr o bi a la ge nt s (L op ez -D ie z e t a l. 2 00 5; N eu ge ba ue r e ta l. 2 00 6; S id er ou di e t a l. 2 00 6). R a ma n s c at t er -i n g s h if t s i n a m a nn er o f c h ar a ct e ri s ti c s m o le cu l arv i br a ti o ns (Kn ei pp e t a l. 1 99 9). T h er e f or e , t h e fi n ge r pr i nt - li k e s p ec t ra c a rr y t h e o v er a ll a n ds p ec ifi c i nf o rm a ti o n o n v a ri o us c h em i ca l a n d b i o-c h e m i c a l c o m p o u n d s i n c o m p l e x s y s t e m s ( Naumann2000). R a ma n s p ec t ro s co p y h a s b e en s u cc e ss f ul l yu s ed i n s t ud y in g t h e m e ch a ni s m o f a nt i mi cr o bi a le f fe ct o f a n ti b io t ic s . N eu ge ba ue r e t a l. ( 20 06 )r e p or t e d t h a t c i p ro fl o xa c i n, a fl u o ro q u in o l o ne d r u g,i n fl u e n c e d t h e g r o w t h o f Bacillus pumilus b y i n t e r -a c ti ng w it h t h e g y ra s e— DN A c o mp l ex . T h is r e su l tw as i nf er re d f ro m t he c ha ng es o f R am an b an dsof t he nu cl ei c a cid s an d th e p rot ein b ui ld ingb lo ck s. S im il ar m ec ha ni sm o f a mi ka ci n a ga in stPseudomonas aeruginosa w as a ls o r ep or ted b yL o p e z - D i e z e t a l . ( 2 0 0 5 ) using Raman spe ctroscopy .

T h e m e ch a ni s m o f t h e i n hi b it o ry e f fe c t o f Z n ON Ps o n m ic ro or ga ni sm s i s n ot f ul l y u nd er st oo d.S ev e ra l s t ud i es r ep o rt e d t h at i n te g ra t io n o f Z n ON Ps i nt o b ac te ri al c el ls m ay i nd uc e c on ti nu ou sr el ea se o f m em br an e l ip id s a nd p ro te in s, w hi chc ha n ge s t h e m e mb r an e p e rm e ab i li t y o f b a ct e ri a l

c el l s (A mr o e t a l. 2 00 0; B ra yn er e t a l. 2 00 6). Ass ho wn i n t he R am an s pe ct ra o f B. cinerea, thei n te n si t y o f n uc l ei c a c id a n d c a rb o hy d ra t e b a nd si nc re as ed s ig ni fic an tl y d ue t o t he Z nO N P t re at -m en t w hi le n o o bv io us c ha ng es i n R am an s ig na lso f p r ot e in s a n d l i pi d s w er e o b se rv e d. T h e d i ff e r-e n c es b e t we e n B. cinerea s i gn a ls w i th o r w i th o utZ n O N P t r e a t m e n t w e r e f u r t h e r p r o v e n b y P C A t o b es t a t is t i c al l y s i g ni fi c an t . T h e se r e s ul t s s u g g es t t h a tZ n O N P s m a y a f f e c t c e l l f u n c t i o n s a n d fi n a l l y c a u s et h e i n c r e a s e o f n u c l e i c a c i d c o n t e n t s . T h e i n c r e a s eo f n uc l ei c a c id m a y d u e t o s t re s s r e s po n se o f f u n-

g a l h y ph ae . T h e i nc r ea s e o f c a rb o hy d ra t es m a y b ed u e t o t h e s e lf - pr o te ct i ng m e ch a ni s m a g ai n st t h eZ n O N P s (A l v a r e z - P e r a l e t a l . 2 0 0 2). O b s e r v a t i o n o f i n cr ea s ed c a rb o hy d ra t es i n f u ng i t r ea t ed w it h N Pw as a ls o r ep or te d b y o th er s (K im e t a l . 2 0 08 a ,b).T he d ef or me d s tr uc tu re s o f t he h yp ha l c el ls i nF i g . 4C a n d D m a y b e d u e t o e x c e s s i v e a c c u m u l a t i o no f n u c l e i c a c i d a n d c a r b o h y d r a t e s . T h e s e d a t a s u g -g e s t a d i f f e r e n t m e c h a n i s m f o r t h e i n h i b i t o r y e f f e c to f Z nO N Ps o n f un gi c om pa re d t o t ho se r ep or te dp r ev i ou s ly f o r b a ct e ri a (L iu e t a l. 2 00 9). O n t heo t h e r h a n d , n o o b v i o u s R a m a n p e a k s w e r e o b s e r v e d

f r o m f u n g a l h y p h a e o f P. expansum a f t e r Z n O t r e a t -m e n t. T h e b a n d s a s s i g n ed t o c a r b oh y d ra t e s, l i p i dsa n d p r o t ei n s f r o m P. expansum de crease d dramat-i ca l l y d u e t o t h e t r e a tm e nt o f Z n O N P s, i nd i ca t in gt ha t t he g ro wt h o f P. expansum w a s c o m p le t e lyi nh i bi t ed . T h e i nh i bi t ed f u ng i d i d n o t h a ve s u ffi c ie n ts ub st an ce t o g en er at e R am an s ig na ls s o t ha t n o

o b vi o us R a ma n s i gn a ls w er e d e te c te d . T h es e r e su l tsa g r ee w i t h m i c r o b i ol o g i ca l p l a t in g a n d S E M r e s u l t s(F i g . 5C a n d D ) t h a t t h e g r o w t h o f P. expansum weregre atly suppre sse d.

5. Conclusion

I n c o n c l u s i o n , Z n O N P s w i t h s i z e s o f ∼7 0 n m p o ss es ss i g ni fi c an t a n t if u n ga l p r o p er t i es a g a in s t B. cinereaand P. expansum, a n d t h e i n hi b it o ry e f fe c ts i nc r ea s ea s t h e c o n c e n t r a t i o n s o f Z n O N P s i n c r e a s e . Z n O N P s

a t c o nc en t ra t io n g r ea t er t h an 3 m m ol l−

1 c a n s i g-n ifi c an t ly i n hi b it t h e g r ow t h o f B. cinerea and P.expansum. Z nO N P s a r e m o re e f fe ct i ve a g ai n st P.expansum than B. cinerea. T h e d a t a a c q u i r e d f r o mS EM a n d R a ma n s p ec t ro s co p y i nd i ca t e t h at t h er em a y e x i s t d i f f e r e n t m e c h a n i s m s o f Z n O N P s a g a i n s tt w o d i ff e re nt f u ng a l s p ec i es . Z nO N P s i n hi b it t h eg r ow t h o f B. cinerea b y i n te rf e ri n g c el l f u nc t io na n d c a u s i n g d e f o r ma t i on i n f u n ga l h y p ha e . I n c o m -p a ri s on , Z n O N P s w i th c o nc e nt r at i on s h i gh er t h an6 m m o l l−1 l ea d t o a c om pl et e i nh ib it io n o f t heg r o wt h o f P. expansum b y p r e ve n t in g t h e d e v el o p -m en t o f c on i di o ph o re s a n d c on i di a . T h es e r e su l tss u g g e s t t h a t Z n O N P s c o u l d b e u s e d a s a n e f f e c t i v ef u ng i ci d e i n a g ri c ul t ur a l a n d f o od s a fe t y a p pl i ca -t i o n s . F u r t h e r s t u d i e s a r e n e e d e d t o i n v e s t i g a t e t h ef e as i bi l it y o f i n co r po r at i ng Z n O N P s i n to fi l ms a ndo t h e r p a c k a g i n g m a t e r i a l s , a n d t h e u s e o f Z n O N P sf o r s o l v in g f o od s a fe t y i s s ue s i n a s a fe a n d r e s p on -sible fashion.

Acknowledgements

T h is r es e ar c h w a s s up p or t ed b y t h e U n iv e rs i ty o f M i ss o ur i R es e ar ch B o ar d p r oj e ct # R B 0 8 -0 3 6. S p e-c ia l t ha nk s a re g iv en t o t he E le ct ro n M ic ro sc op yC o r e F a c i l i t y a t t h e U n i v e r s i t y o f M i s s o u r i f o r a s s i s -t a n c e i n t h i s s t u d y .

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