E F F E C T O F C YC LO H EX IM ID E (A C T I-D IO N E ) ON TH E G E R M IN A T IO N O F S P O R E S O F S E V E R A L PH Y T O PA T H O G E N IC FU N G I By ANDREW THANOS A TH ESIS S u b m i t t e d to th e S c h o o l o f G r a d u a t e S t u d i e s o f M i c h i g a n S ta te C o l l e g e o f A g r i c u l t u r e and A p p l i e d S c i e n c e in p a r t i a l f u l f i l l m e n t o f th e r e q u i r e m e n t s f o r th e d e g r e e o f D O C T O R O F P H IL O S O P H Y D e p a r t m e n t o f B o t a n y an d P l a n t P a t h o l o g y 1952 A C K N O W LED G M EN TS T h e w r i t e r w i s h e s to e x p r e s s h i s s i n c e r e g r a t i t u d e and th a n k s to D r . J . R . V a u g h n , u n d e r w h o s e g u i d a n c e t h i s i n v e s ­ tig a tio n w a s co n d u cted . c o n str u ctiv e c r it ic is m He a l s o w i s h e s to th a n k h i m f o r th e o f f e r e d in th e p r e p a r a t i o n o f t h i s t h e s i s . M a n y th a n k s a r e a l s o due to D r . C. J . A l e x o p o u l o s f o r h i s a d ­ v i c e a n d i n s p i r a t i o n g i v e n s o w i l l i n g l y t h r o u g h o u t th e c o u r s e of t h is i n v e s t i g a t i o n a n d f o r h i s c a r e f u l c r i t i c i s m A l s o m a n y th a n k s a r e due D r . G. B . W i l s o n B. o f th is t h e s is . and D r. W illia m D r e w f o r t h e i r a d v i c e d u r in g th e w r i t i n g o f t h i s m a n u s c r i p t . In a d d it io n , th e w r i t e r w i s h e s to th a n k th e U p j o h n C o m p a n y o f K a l a m a z o o , M i c h ig a n , f o r s u p p ly in g th e A c t i - d i o n e w h i c h w a s u s e d a s a b a s is fo r th is e n tir e in v e s tig a tio n . M a n y th a n k s a r e a l s o due th e m a n y o t h e r m e m b e r s o f th e f a c u l t y and f e l l o w g r a d u a t e s t u d e n t s f o r h e l p a n d i n s p i r a t i o n g iv e n so w illin g ly w h en r e q u e ste d . F i n a l l y , th e w r i t e r w i s h e s to th a n k th e c i t i z e n s o f th e U n it e d S t a t e s and t h e V e t e r a n s A d m i n i s t r a t i o n f o r th e e x c e l l e n t f i n a n c i a l a s s i s t a n c e p r o v i d e d t h r o u g h th e P u b l i c L a w N o . 16, b y w h i c h th e w r i t e r w a s a s s i s t e d in c o m p l e t i n g h i s t r a in i n g . TABLE OF CONTENTS Page IN T R O D U C T IO N A ND R E V IE W O F L IT E R A T U R E . 1 M A T E R I A L S A N D M E T H O D S ......................................................... 8 E X P E R I M E N T A L R E S U L T S ................................................................ 22 ........................................... 23 C l a d o s p o r i u m c u c u m e r i n u m .................................................. 23 B o t r y t i s c i n e r e a .............................................................................. 38 M o n i l i n i a f r u c t i c o l a ................................................................ 59 R a m u l a r i a s p ........................................................................................ 79 D i r e c t Im m e r sio n E x p e r im e n ts H u m id ity E x p e r im e n ts ......................................................... 98 ...................................................................................... 1 14 C O N C L U S I O N S ............................................................................................. 122 S U M M A R Y ........................................................................................................... 125 L IT E R A T U R E 129 D IS C U S S IO N . . . ; . C I T E D ........................................................ B I B L I O G R A P H Y ............................................................................................. 133 IN T R O D U C T I O N A N D R E V I E W O F L I T E R A T U R E C y c l o h e x i m i d e , b e t t e r k n o w n b y th e b r a n d n a m e A c t i d io n e ,^ i s a b y - p r o d u c t r e s u l t i n g f r o m t h e p r o c e s s o f S t r e p t o ­ m y c in p ro d u ctio n . A c t i - d i o n e w a s f i r s t r e p o r t e d i n th e l i t e r a t u r e b y W h iff e n , B o h o n o s an d E m e r s o n in 19 47 (48) a s a s u b s t a n c e fo u n d to e x h i b i t a n t if u n g a l b u t n o t a n t i b a c t e r i a l a c t i v i t y . W h if f e n e t a l . c o n d u c t e d a n u m b e r o f t e s t s w it h it a n d f o u n d th a t it i n ­ h i b i t e d a n u m b e r of d e r m a t o p h y t e s a n d s e v e r a l y e a s t s , b u t h ad a b s o l u t e l y no a c t i o n a g a i n s t b a c t e r i a . A l s o in 1 9 4 7 , L e a c h , F o r d an d W h iffe n (21) r e p o r t e d on th e c h e m i c a l f o r m u l a C H L* I N _0 * C* Cd p o i n t o f 115 . T h e y fo u n d t h i s s u b s t a n c e to h a v e a m e l t i n g o ,o to 116 C ., a m o l e c u l a r w e i g h t o f 5 0 7 a n d i s | m o s t a c t i v e in th e a c i d r a n g e b e i n g r a p i d l y i n a c t i v a t e d b y d i ­ lu te a lk a lie s at r o o m te m p e r a tu r e s . A c t i - d i o n e i s a p a t e n t e d n a m e b y th e U p joh n C o m p a n y o f K a l a m a z o o , M i c h ig a n , a n d a l l o f th e m a t e r i a l u s e d in t h e s e s t u d i e s w a s s u p p l i e d b y th e U p jo h n C o m p a n y . 2 E a r l y in 1 9 4 8 F e l b e r a n d H a m n e r (8) o f th e D e p a r t m e n t of H o r t i c u l t u r e a t M i c h i g a n S t a t e C o l l e g e , s t a r t e d a s e r i e s o f e x p e r i m e n t a l s t u d i e s i n v o l v i n g th e u s e o f C y c l o h e x i m i d e ( A c t i d io n e) in o r d e r to d e t e r m i n e i t s g r o w t h e f f e c t s u p on h i g h e r p la n ts. D u r in g t h e i r i n v e s t i g a t i o n t h e y fo u n d A c t i - d i o n e to b e t o x i c , to y o u n g b e a n p l a n t s an d o a t s e e d l i n g s , in c o n c e n t r a t i o n s h i g h e r th a n 100 p . p . m . ( p a r t s p e r m i l l i o n ) . In th e p r o c e s s o f t h e s e i n v e s t i g a t i o n s t h e y o b s e r v e d a r a t h e r s e v e r e i n f e c t i o n of p o w d e r y m i l d e w E r y s i p h e p o l y g o n i D . C ., o n t h e i r o l d e r R e d K id n ey b e a n p la n t s . T h is w a s q u i c k l y e r a d i c a t e d , w it h in 4 8 h o u r s b y s p r a y i n g t h e i n f e c t e d p l a n t s w it h a 10 p . p . m . s o l u ­ t io n o f A c t i - d i o n e . T he r e s u lt s of th is in v e s t ig a t io n in d ic a te th a t th e s u b s t a n c e m a y b e e f f e c t i v e l y u s e d a s a f u n g i c i d e f o r p ow d ery m ild e w of b e a n s. T h e s e fin d in g s s e r v e d a s a s t im u ­ l u s to th e i n i t i a t i o n o f m a n y n e w s t u d i e s i n v o l v i n g th e a p p l i c a ­ t io n o f t h i s a n t i b i o t i c in c o n t r o l l i n g p l a n t d i s e a s e s . A d d i t io n a l i n v e s t i g a t i o n s w e r e s t a r t e d w it h th e o b j e c t o f s tu d y in g th e v a ­ rio u s p h y sio lo g ic a l e ffe c ts of th is su b sta n ce on a n u m ber of p a t h o g e n i c f u n g i an d a l s o th e c y t o l o g i c a l e f f e c t s o f t h i s s u b ­ s t a n c e o n th e c e l l s o f s e v e r a l p l a n t s (1 1 , 5 1 ). 3 L a t e in 1 9 4 8 , V a u g h n a n d h i s c o - w o r k e r s (45) c o n d u c t e d a s e r i e s o f e x p e r i m e n t s to d e t e r m i n e th e e f f e c t o f A c t i - d i o n e o n v a r i o u s p l a n t t i s s u e s a n d o n th e v e g e t a t i v e g r o w t h o f s e v ­ e r a l fu n g i. T h e ir c o n c lu s io n s w e r e th at A c t i- d io n e c o n tr o ls m ild e w on b e a n s and r o s e s , under g r e e n h o u s e co n d itio n s. M il­ d e w o n b e a n s w a s c o n t r o l l e d a t 10 p . p . m . w it h n o a p p a r e n t p h yto to x ic r e s u lt s . cen tra tio n s l e s s M ild e w on r o s e s w a s c o n tr o lle d w ith c o n ­ th a n 5 p . p . m . They a ls o s t a t e th a t i n j u r y v a r i e d b e t w e e n v a r i e t i e s , b u t th a t y o u n g e r f o l i a g e w a s m o r e s u s c e p t i b l e th a n o l d e r f o l i a g e . In a d d it io n to t h i s , t h e y a l s o fo u n d th a t " g r o w t h o f p a t h o g e n i c f u n g i o n a c u l t u r e m e d i u m c o n t a in in g a c t i d i o n e i n d i c a t e s s t r o n g in h i b i t i o n o f 10 d i f f e r e n t p a t h o g e n e s a t c o n c e n t r a t i o n s o f 100 p . p . m . o f a c t i d i o n e . " In 1 9 4 9 , V a u g h n a n d H a m n e r (46) r e p o r t e d o n th e e f f e c t o f A c t i ­ d io n e a s a f u n g i c i d e in c o n t r o l l i n g sev era l com m on d ise a se s, and c o n c l u d e d th a t th e A c t i - d i o n e i s w o r t h y o f f u r t h e r e x t e n ­ s iv e te s tin g on v a r io u s c r o p s a g a in s t fungus d i s e a s e s . A lso in 1 9 4 9 , de Z e e u w (6) an d V a u g h n r e p o r t e d th a t A c t i - d i o n e p r o ­ d u c e d th e b e s t c o n t r o l o f c u c u m b e r s c a b , C l a d o s p o r i u m c u c u m e r in u m . F r o m R ic h m o n d , In d ia n a , c a m e r e p o r t s , v i a c o r r e ­ s p o n d e n c e to V a u g h n ( 4 2 ), th a t g r e e n h o u s e t e s t s w it h t h is m a t e r i a l 4 have g iv e n s a t is f a c t o r y c o n tr o l of p o w d e r y m ild e w of r o s e s w h e n th e l a t t e r w e r e s p r a y e d w it h c o n c e n t r a t i o n s b e l o w 5 p . p . m . K l o m p a r e n s (19) c o n d u c t e d a n i n v e s t i g a t i o n to d e t e r m i n e th e e f ­ fic ie n c y of th is su b stan ce a s a w ood p r e s e r v a t iv e . H e fo u n d t h a t A c t i - d i o n e w a s v e r y t o x i c to a l l o f th e w o o d - r o t t i n g b a s i d i o m y c e t e s , w h i c h h e u s e d in h i s s t u d i e s , e v e n a t v e r y l o w c o n ­ cen tra tio n s. b y th e t i s s u e s H is c o n c l u s i o n w a s th a t A c t i - d i o n e w a s a d s o r b e d o f t h e w o o d e n b lo c k s a m p l e s a n d h i s a t t e m p t s to l e a c h o u t th e t o x i n , r e s u l t e d in a v e r y l o w r e c o v e r y . In 1 9 5 0 , W h if fe n (48) r e p o r t e d o n e x p e r i m e n t s in w h i c h s h e s t u d i e d th e a c t i v i t y o f A c t i - d i o n e i n v i t r o o n 33 s p e c i e s an d s t r a i n s o f p h y t o p a t h o g e n ic f u n g i. T h e s e w e r e in h ib ite d by A c t i- d io n e v a r y i n g in r a n g e o f c o n c e n t r a t i o n f r o m 0 . 1 2 5 to 1 0 0 .0 m i c r o ­ gram s per m illilite r . P e t e r s e n an d C a t io n (38) r e p o r t e d t h e i r e x p e r i m e n t a l r e s u l t s o n th e u s e o f A c t i - d i o n e f o r th e c o n t r o l o f P e a c h B r o w n R o t ( M o n il in ia f r u c t i c o l a ) , a n d C h e r r y L e a f S p o t (H i g g i n s i a h y e m a l i s ) . T h e i r c o n c l u s i o n w i t h r e s p e c t to th e b r o w n r o t i s th a t in v i e w o f th e f a c t t h a t s e v e r e in j u r y to n e a r - r i p e f r u i t w a s e v i d e n t , A c t i - d i o n e i s o f d o u b tfu l v a lu e a s a p ea ch brow n rot sp ray. O n th e o t h e r h a n d , in th e c a s e o f c h e r r y l e a f s p o t , e x c e p t i o n a l l y g o o d r e s u l t s w e r e o b t a in e d a s 5 an e r a d i c a n t w i t h no a p p a r e n t i n j u r y to th e f o l i a g e . In 1950 G o tt lie b e t a l . (10) r e p o r t e d on e x p e r i m e n t s w i t h A c t i - d i o n e a s a p la n t p r o t e c t a n t . T h e y t e s t e d i t at v a r i o u s c o n c e n t r a t i o n s a g a i n s t 6 o r g a n i s m s a n d fo u n d th a t a l l e x c e p t S c l e r o t i n i a f r u c tic o la w e r e c o n t r o l l e d a t 100 p . p . m . w e r e c o n t r o l l e d at 1 ,0 0 0 p .p . m . A ll 6 o r g a n is m s , h o w e v e r , In a d d it io n t h e y t e s t e d 5 e c o ­ n o m i c p l a n t s in o r d e r to d e t e r m i n e p o s s i b l e t o x i c i t y . r e su lts T h e ir s h o w i n j u r y to a l l of th e p l a n t s e x c e p t s t r a w b e r r i e s , a t 100 p . p . m . an d 1 ,0 0 0 p . p . m . In p r i v a t e c o n v e r s a t i o n , R. H. F u lt o n , D e p a r t m e n t of B o t a n y an d P l a n t P a t h o l o g y , M i c h ig a n S ta te C o l l e g e , h a s t o ld t h i s a u th o r t h a t h i s e x p e r i m e n t s w it h A c t i - d i o n e on s t r a w b e r r y p la n t s h a v e r e s u l t e d in in j u r y e v e n at c o n c e n tr a tio n s as lo w as 2 p .p .m . E a r l y in 1 9 5 1 , H e n r y e t a l . (12) r e p o r t e d t h a t " A c t i - d i o n e p r o v e d p r o m i s i n g , p a r ­ t i c u l a r l y f o r th e t r e a t m e n t of o a t s f o r th e c o n t r o l of s m u t c a u sed b y U stila g o k o lle r i W ille ." A t th e 4 2 n d a n n u a l m e e t ­ in g of th e A m e r i c a n P h y t o p a t h o l o g i c a l S o c i e t y , V a u g h n (4 7 ), r e p o r t i n g on h i s e x p e r i m e n t s w it h A c t i - d i o n e on t u r f d i s e a s e s , sta te d that " D o lla r S p o t" S c le r o t in ia h o m e o c a r p a w as c o n s i s ­ t e n t l y l e s s p r e v a l e n t in c y c l o h e x i m i d e - t r e a t e d p l o t s th a n in t h o s e t r e a t e d w it h c o m m o n t u r f f u n g i c i d e s ; an d t h a t " M e l t i n g - 6 o u t " (H e l m i n t h o s p o r i u m s p .) w a s c o m p l e t e l y c h e c k e d b y c y c l o ­ h e x im id e . A t th e s a m e m e e t i n g , K l o m p a r e n s (20) r e p o r t e d o n th e t o x i c i t y o f c y c l o h e x i m i d e to c e r t a i n w o o d - r o t t i n g f u n g i, an d on h is p r e lim in a r y s tu d ie s on the a b s o r p tio n o f th is a n tib io tic by tis s u e s. A t th e p r e s e n t t i m e a n u m b e r o f i n v e s t i g a t o r s a r e c o n ­ d u c tin g t e s t s on v a r i o u s c r o p s an d d i s e a s e s in a t t e m p t s to l e a r n m o r e a b o u t t h i s p r o d u c t w h i c h h a s u n t il n o w c r e a t e d m u ch w id e sp r e a d in t e r e s t am ong p a th o lo g ists and e v e n so m e g r o w e r s of fr u it and v e g e ta b le c r o p s . C u r ren tly , s t u d i e s a r e b e i n g m a d e a t M i c h ig a n S ta te C o l l e g e b y V a u g h n an d K l o m p a r e n s o n t u r f d i s e a s e s , d e Z e e u w o n c u c u m b e r s c a b a n d C a t io n a n d F u l t o n o n f r u i t d i s e a s e s . In a d d it io n to t h e s e , o t h e r t u r f an d f r u i t i n v e s t i g a t i o n s , u s i n g A c t i ­ d io n e a r e b e i n g c o n d u c t e d in v a r i o u s e x p e r i m e n t s t a t i o n s t h r o u g h ­ o u t th e U n i t e d S t a t e s . S p o r e g e r m i n a t i o n t e s t s h a v e b e e n u s e d to a s s a y f u n g i ­ cid e s for m any y e a r s . A c c o r d i n g to H o r s f a l l (1 7 ), P r e v o s t w a s o n e o f th e e a r l i e s t r e s e a r c h e r s to u s e t h i s m e t h o d o f f u n g i c i d e assay. M any o th er in v e s tig a to r s have s in c e u s e d it and p u b ­ l i s h e d t h e i r r e s u l t s w it h m i x e d o p i n i o n s on t h e e f f i c a c y o f th e 7 m eth od . N e v e r t h e l e s s , th e m e t h o d i s w e l l e s t a b l i s h e d , w it h m any m o d ific a tio n s. M c C a l l a n an d W i l c o x o n o f th e B o y c e T h om p so n In stitu te h a v e u s e d it e x t e n s iv e ly a s e v id e n c e d by t h e i r m a n y p u b l i c a t i o n s on s p o r e g e r m i n a t i o n s t u d i e s . S i n c e n o p r e v i o u s i n v e s t i g a t i o n h a d b e e n m a d e o f th e e f fe c ts of A c t i- d io n e on p a th o g e n ic fu n g o u s sp o re g erm in a tio n , i t w a s c o n s i d e r e d t h a t s u c h a s t u d y w o u ld b e i n t e r e s t i n g an d o f p o s s i b l e v a l u e f r o m a p r a c t i c a l s t a n d p o in t . It i s th e h o p e o f the a u t h o r th a t a t s o m e f u t u r e t i m e , t h e s e r e s u l t s m a y p r o v e o f v a l u e in t h e e v e r - c o n s t a n t s t r u g g l e o f c o n t r o l l i n g p la n t d i s e a s e s . P e r h a p s s o m e i n v e s t i g a t o r w h o m a y w i s h to s tu d y th e e f f e c t s o f A c t i - d i o n e o n a n y o n e o r m o r e o f th e m a n y d ise a se s c a u s e d b y t h e o r g a n i s m s u s e d in t h i s s t u d y w i l l h a v e a c c e s s to s o m e i n f o r m a t i o n r e g a r d i n g th e a c t i o n of A c t i - d i o n e up on the s p o r e s o f t h e o r g a n i s m h e i s a b o u t to s tu d y . An investigation was started by the w riter in September, 1949, with the objective of studying the effects of A cti-dione on the germ ination of spores from sev era l pathogenic fungi, under the influence of several environmental fa cto rs. M A T E R IA L S A N D M ETH ODS P u r e c u l t u r e s o f e a c h o r g a n i s m u s e d in t h is i n v e s t i g a ­ tio n w e r e o b t a i n e d f r o m th e s t o c k c o l l e c t i o n o f f u n g i o f the D e p a r t m e n t o f B o t a n y and P l a n t P a t h o l o g y a t M i c h i g a n S t a t e C o l l e g e , e x c e p t f o r th e c u l t u r e o f B o t r y t i s c i n e r e a , w h i c h w a s iso la te d fr o m n a tu r a lly -in fe c te d P r e m ie r R o b e r t H. F u l t o n . w as r a th e r d iffic u lt, s t r a w b e r r ie s by M r. M on osp orou s is o la tio n of th e se o r g a n ism s so sin g le hyphal tip s w e r e t r a n s fe r r e d f r o m P e t r i d i s h c u l t u r e s to a g a r s l a n t s in t e s t t u b e s . The c o n id ia l s t a g e s of t h e s e o r g a n is m s w e r e n e e d e d and a l l e f f o r t s w e r e d i r e c t e d t o w a r d o b t a in in g th e m o s t d e s i r a b l e m e d ia fo r co n id ia l p ro d u ctio n . M o n ilin ia f r u c t ic o la w a s grow n on an a g a r m e d i u m o f th e f o l l o w i n g f o r m u l a : M a lt e x t r a c t 5 gram s Y e a s t e x t r a c t ................. 1 gram S o r b o s e ............................... 5 gram s G l y c i n e ............................... 1 gram B a c t o - A g a r ( D if c o ) D istille d w a t e r . .. 18 g r a m s 1000 m il lil it e r s T h is f o r m u l a w a s g i v e n to th e a u th o r b y D r . H. L . B a r n e t t , o f W est V ir g in ia U n iv e r sity . C la d o sp o r iu m c u c u m e r in u m , R a m u la r ia 9 sp. and B o t r y t is c i n e r e a w e r e c u ltu r e d on a g a r s la n ts of p otato d e x t r o s e a g a r p r e p a r e d in a c c o r d a n c e w i t h th e f o l l o w i n g f o r ­ m u la : L iq u id p otato e x t r a c t f r o m 200 g r a m s of p o ta to e s D e x t r o s e , C. P B a c t o - A g a r ( D if c o ) D istille d w a t e r 5 gram s . . . 17 g r a m s 100 0 m i l l i l i t e r s The pH of t h e s e m e d ia w a s d e t e r m in e d by a B e c k m a n pH m e te r (m od el H 2 ). M e d i u m X , w h i c h r e f e r s to th e m e d i u m u s e d f o r M o n il in ia f r u c t i c o l a , h a d a p H v a l u e of 5 . 6 . t a t o - d e x t r o s e a g a r h a d a p H o f 5 .9 a f t e r s t e r i l i z a t i o n . The p o ­ T hese m e d i a w e r e u s e d f o r p r o d u c in g th e c o n i d i a w h i c h w e r e l a t e r u s e d in th e e x p e r i m e n t s d e s c r i b e d in th e l a t t e r p o r t i o n o f t h is sectio n . In p r e l i m i n a r y t e s t s s te r ile d is tille d w ater w as u se d as th e s p o r e s u s p e n s i o n m e d i u m a n d a s th e m e d i u m i n w h i c h the s p o r e s w o u ld b e a l l o w e d to g e r m i n a t e . A fter s e v e r a l te sts w it h e a c h o r g a n i s m i t w a s f o u n d th a t th e s p o r e s , f r o m th e o r g a n i s m s u s e d in t h e s e t e s t s , d id n o t s h o w a h ig h p e r c e n t a g e of g erm in a tio n . S t i m u l a n t s w e r e in o r d e r , an d a s r e c o m m e n d e d b y M c C a l l a n (31) a n u m b e r o f t h e m w e r e t r i e d . D ifc o n u trien t b r o t h w a s d e c i d e d u p o n a s th e m o s t s u i t a b l e s i n c e i t g a v e g o o d 10 r e s u l t s , w a s r e a d i l y a v a i l a b l e , and w a s a l w a y s c o n s t a n t . liq u i d n u t r i e n t b r o t h w a s p r e p a r e d b y d i s s o l v i n g d e h y d r a t e d b r o t h in to in g at 121 o C. 8 g r a m s o f th e 1 ,0 0 0 m l . o f d i s t i l l e d w a t e r a n d a u t o c l a v - F o llo w in g s t e r i l i z a t i o n it w a s l a b o r a t o r y r e f r i g e r a t o r s u n t il u s e d . fe r e n t in te r v a ls, T h is ste r ile s t o r e d in the F o r c o m p a r i s o n , at d i f ­ d o u b l e - d i s t i l l e d w a t e r and s o m e t i m e s s t e r i l e tap w a t e r w a s u s e d . The w a te r , h o w e v e r , p ro v ed v e r y u n s a t i s f a c t o r y s i n c e l o w g e r m i n a t i o n w a s n o t e d e v e r y t i m e and its u s e w a s d isco n tin u ed . T h e s l i d e s u s e d in t h i s s t u d y w e r e p r e p a r e d a c c o r d i n g to th e m e t h o d d e s c r i b e d b y P . D. P e t e r s o n (3 7 ). C lea n g la s s s l i d e s w e r e l i g h t l y s m e a r e d w ith a v e r y t h i n l a y e r o f V a s e l i n e b y u s e o f th e i n d e x f i n g e r . Onto t h i s s u r f a c e w e r e a f f i x e d 1 2 -m m . c ir c u la r c o v e r g l a s s e s w h ich had p r e v io u s ly b e e n c l e a n e d in h o t w a t e r c o n t a i n i n g D r e f t , a g o o d d e t e r g e n t f o r t h i s pu rp ose. used. T h e g l a s s e s w e r e th e n s t o r e d in 70% a l c o h o l u n til E a c h c o v e r g l a s s , b e f o r e u s e , w a s q u i c k ly f l a m e d and p r e s s e d in to p l a c e , i . e . , The s lig h t p r e s s u r e on th e V a s e l i n e s u r f a c e of th e s l i d e . a p p l i e d to th e c o v e r g l a s s , r e s u l t e d in a r i d g e o f V a s e l i n e f o r m i n g a r o u n d th e e d g e of th e c o v e r g l a s s . T h is e d g e r i m s e r v e d to r e s t r i c t th e s p o r e s u s p e n s i o n w ith in 11 th e b o u n d a r i e s o f t h e c o v e r g l a s s a n d th u s p r e v e n t e d r u n n i n g o ff o r c o a l e s c i n g o f t h e s p o r e su sp e n sio n d rop s. T h is m e th o d w a s c h o s e n b e c a u s e , a s P e t e r s o n m e n tio n s , it n o t o n l y i s q u ic k a n d i n e x p e n s i v e in m a t e r i a l s , b u t a l s o p r o ­ v i d e s a d e f i n i t e s u r f a c e a r e a o v e r w h i c h th e s p o r e s u s p e n s i o n s w e r e p l a c e d a n d th u s a m o r e o r l e s s d e f i n i t e v o lu m e o f li q u i d . F u r t h e r m o r e e a c h a n d e v e r y r e p l i c a t e w a s d u p l i c a t e d in th a t e a c h c o v e r e d th e s a m e spore su rface area . T h e v o l u m e of li q u i d s u s p e n s i o n w a s q u ite c o n s t a n t in e a c h r e p l i c a t e a s an a t t e m p t w a s m a d e to p l a c e o n ly tw o e q u a l s i z e d r o p s o n e a c h cover g la ss. E a c h drop w a s a p p r o x im a te ly 1 /2 0 of a m l. as m e a s u r e d b y a f a ir l y a c c u r a te p ip e tte . B efo re th ese s l i d e s w e r e u s e d e a c h w a s p l a c e d in a c l e a n s t e r i l e P e t r i d i s h l i n e d w it h 2 f i l t e r p a p e r d i s k s an d th e n w e r e e x p o s e d to u l t r a v i o l e t i r r a d i a t i o n b y p l a c i n g t h e m u n d e r an u l t r a v i o l e t l a m p u n it c o n s i s t i n g o f 2 S l i m l i n e l a m p s , 6 i n c h e s f r o m th e l o w e r s u r f a c e o f t h e l a m p s . E a ch and e v e r y s l i d e w a s e x p o s e d f o r a p e r i o d o f 10 m i n u t e s . There w as a l ­ m o s t no co n ta m in a tio n u n d er th e s e c o n d itio n s . The spore s u s p e n s i o n s u s e d i n th e g e r m i n a t i o n s t u d i e s w e r e p r e p a r e d as fo llo w s: 7 m l. of e a c h s t e r il e n u trien t 12 b r o t h - A c t i - d i o n e c o n c e n t r a t i o n w e r e p o u r e d , a s e p t i c a l l y , in t o an a g a r s l a n t c u l t u r e , w h i c h w a s s p o r u l a t i n g a b u n d a t ly , a n d e a c h t e s t tu b e w a s r o l l e d v i g o r o u s l y b e t w e e n th e p a l m s o f th e h a n d s f o r a p p r o x i m a t e l y 100 t i m e s . sio n e v e r y t im e . T h is p r o d u c e d a g o o d s u s p e n ­ The s u s p e n s io n w a s then t r a n s f e r r e d i m m e ­ d i a t e l y to s t e r i l e c e n t r i f u g e t u b e s b y f i l t e r i n g t h r o u g h s e v e r a l la y e r s of s t e r i l e c h e e s e c lo t h w h ich had b e e n p r e v io u s ly p la c e d in th e c e n t r i f u g e t u b e s . A ll s u s p e n s io n s w e r e then c en trifu g e d f o r a p p r o x i m a t e l y 2 m i n u t e s f o l l o w e d b y d e c a n t i n g o f th e li q u i d w it h c a r e s o a s to k e e p t h e c l u m p e d s p o r e s at th e b o t t o m of th e c e n t r i f u g e t u b e s . T h e s p o r e s w e r e th e n r e s u s p e n d e d in f r e s h s t e r i l e n u t r i e n t b r o t h and c e n t r i f u g e d a g a in f o r a b o u t 2 m in u te s. T h e li q u i d m e d i u m w a s a g a in d e c a n t e d an d th e s p o r e s w e r e a g a in r e s u s p e n d e d in n u t r i e n t b r o t h . E a c h c e n t r i f u g e tube w a s th e n r o l l e d b e t w e e n th e p a l m s o f th e h a n d s to c a u s e an e v e n s u s p e n s i o n o f th e s p o r e s an d b r e a k a n y c l u m p s t h a t m a y h a v e f o r m e d d u r in g c e n t r i f u g a t i o n . To fu rth er in s u r e th is, a c l e a n s t e r i l e 2 - m l . p i p e t t e w a s p l a c e d in t o the l i q u i d s p o r e s u s p e n s i o n , a n d b y d r a w in g s o m e o f th e li q u i d in to th e p ip e t t e an d a l l o w i n g i t to f l o w b a c k in to th e t u b e , f u r t h e r d i s p e r s i o n o f th e s p o r e s w a s a c c o m p l i s h e d . U s i n g th e s a m e p i p e t t e a 13 d ro p o f th e s u s p e n s i o n w a s q u i c k l y p l a c e d up on a H o w a r d M o ld C o u n tin g C h a m b e r s l i d e an d th e s u s p e n s i o n w a s s t a n d a r d i z e d s o a s to c o n t a i n a p p r o x i m a t e l y 3 0 , 0 0 0 s p o r e s p e r m l . of l i q u i d , a s r e c o m m e n d e d b y th e A m e r i c a n P h y t o p a t h o l o g i c a l S o c i e t y C o m ­ m i t t e e o n S t a n d a r d i z a t i o n o f F u n g i c i d a l T e s t s (1 ). A ft e r e a c h s u s p e n s io n w a s sta n d a r d iz e d tw o d ro p s w e r e p l a c e d q u i c k l y on e a c h o f th e t w o c o v e r g l a s s su rfa ces borne o n th e s u r f a c e of th e s p e c i a l l y p r e p a r e d s l i d e s p r e v i o u s l y d e ­ s c r ib e d (P la te 1). E a c h s l i d e w a s th e n q u ic k l y c o v e r e d b y p l a c i n g th e l i d on e a c h P e t r i d i s h , th u s r e d u c i n g th e c h a n c e s f o r c o n t a m i n a n t s to g a in e n t r a n c e . To e a c h P e t r i d ish 2 .5 m l. o f s t e r i l e d i s t i l l e d w a t e r w a s a d d e d to m o i s t e n th e f i l t e r p a p e r d i s k l i n i n g it , s o a s to p r o d u c e an d m a i n t a i n a h u m i d i t y s u f ­ f i c i e n t l y h ig h s o th a t th e s p o r e s u s p e n s i o n s w o u ld n o t d r y o u t (P la te 1). E a c h e x p e r i m e n t c o n s i s t e d of 7 P e t r i d i s h e s c o n t a in in g o n e s l i d e e a c h and e a c h s l i d e c o n t a i n e d o n e c o n c e n t r a t i o n o f A c ti-d io n e . The c o n c e n tr a tio n s of A c t i- d io n e u s e d fo r th is i n v e s t i g a t i o n w e r e 0 .5 p a r t p e r m i l l i o n , 2 . 5 p . p . m . , 5.0 p . p . m . , 1 0 .0 p . p . m . , 2 5 .0 p . p . m . , 5 0 .0 p . p . m . , 7 5 . 0 p . p . m . and 100 p . p . m . 15 T h e l a t t e r tw o c o n c e n t r a t i o n s w e r e u s e d o n ly in th e e x p e r i ­ m e n ts w ith B o t r y t is c in e r e a and R a m u la r ia s p . The A c ti-d io n e co n cen tra tio n s w e r e p r e p a r e d b y c a r e ­ f u l l y d i s s o l v i n g 1 0 0 . 0 0 m g . o f c r y s t a l l i n e A c t i - d i o n e in t o 1 ,0 0 0 m l . of n u t r i e n t b r o t h (D ifc o ) in a 1 , 0 0 0 - m l . v o l u m e t r i c f l a s k . T h i s s o l u t i o n c o n t a i n e d 100 p . p . m . o f A c t i - d i o n e . U s i n g th is a s a s t o c k s o l u t i o n , b y a s e r i e s of d i l u t i o n s , t h e o t h e r c o n c e n ­ tr a tio n s w e r e p r e p a r e d . A f t e r th e s t e r i l e d i s t i l l e d w a t e r h a d b e e n a d d e d to e a c h P e t r i d i s h , e a c h o f th e l a t t e r w e r e m a r k e d a n d q u i c k l y p l a c e d a lo n g w it h the c o n t r o l , e i t h e r i n on e o f th e s e v e r a l in c u b a t o r s u s e d in t h i s s t u d y , o r l e f t a t r o o m t e m p e r a t u r e . At room te m ­ p e r a t u r e , th e P e t r i d i s h e s c o n t a in in g th e s l i d e s w e r e in c u b a t e d i n l a r g e f i n g e r b o w l s to w h i c h e n o u g h w a t e r w a s a d d e d to m a i n ­ ta in a h ig h r e la t iv e h u m id ity (P la te 2 ). in th is stu d y w e r e : The t e m p e r a t u r e s u s e d R o o m t e m p e r a t u r e , 2 2 ° C ., 2 4 ° C ., 2 6 ° C . , 2 8 ° C. a n d 3 0 ° C . M e t h o d an d T i m e I n t e r v a l s U s e d in M a k in g C o u n ts In th e c a s e o f C l a d o s p o r i u m c u c u m e r i n u m th e s u s p e n ­ s io n s w e r e c h e c k e d for g e r m in a tio n a s fo llo w s: The f i r s t c o u n t P la te 2 w as m ade 12 h o u r s a f t e r th e s t a r t o f th e e x p e r i m e n t , th e s e c ­ o n d c o u n t w a s m a d e a f t e r 24 h o u r s , th e t h ir d a f t e r 48 h o u r s and th e f o u r t h a f t e r 72 h o u r s . F o r M o n i l i n i a f r u c t i c o l a th e f i r s t c o u n t w a s m a d e a f t e r 12 h o u r s , th e s e c o n d in 24 h o u r s , th e t h i r d in 4 8 h o u r s and t h e f o u r t h in 72 h o u r s . The c o u n t s f o r R a m u l a r i a s p . w e r e m a d e s o o n e r a n d at s h o r t e r i n t e r v a l s b e c a u s e th is o r g a n is m g e r m in a te d m o r e v io u s o n e s . q u ic k ly th a n th e p r e ­ T h e c o u n t s w e r e m a d e a t 3, 6, 9, 1 2 , and 4 8 h o u r s . T h e c o u n t s f o r B o t r y t i s c i n e r e a w e r e m a d e a f t e r 9, 18, 36 an d 72 h o u r s . T o f a c i l i t a t e c o u n t in g , a s p e c i a l g r i d e y e p i e c e w a s u s e d in th e m i c r o s c o p e . A t o t a l o f 25 h i g h - p o w e r f i e l d s w e r e c o u n t e d in e a c h o f th e tw o d r o p s p e r p ow er fie ld s p e r slid e . s l i d e g i v i n g a t o t a l o f 50 h i g h - E a c h e x p e r i m e n t w a s r e p l i c a t e d 10 tim e s. H u m id i t y T e s t s S p ore s u s p e n s io n s w e r e p r e p a r e d a s p r e v io u s ly m e n tio n e d b u t i n s t e a d o f b e i n g p l a c e d u p o n th e s p e c i a l s l i d e in l i q u i d f o r m a s in t h e p r e v i o u s e x p e r i m e n t s , e a c h s u s p e n s i o n w a s s p r a y e d o n to t h e c l e a n s t e r i l e slid e b y m ea n s of a s m a ll h a n d -o p e r a te d 18 De V ilb is N o. ( P l a t e 3). 15 a t o m i z e r , c o m m o n l y u s e d f o r n a s a l s p r a y s A n a t t e m p t w a s m a d e to s p r a y e a c h s l i d e u n i f o r m l y b y s p r a y i n g e a c h 5 t i m e s , h o ld in g t h e n o z z l e 6 i n c h e s f r o m th e s l i d e su rfa ce. T h is r e s u l t e d i n w h a t a p p e a r e d t o be a u n i f o r m s p r a y w i t h a g o o d d i s t r i b u t i o n of t h e cover g la ss. s p o r e s on e a c h T h e s l i d e s w e r e t h e n a l l o w e d to a i r d r y f o l l o w ­ in g w h i c h t h e y w e r e p l a c e d in an a i r - t i g h t g l a s s - c o v e r e d c h a m ­ b e r ( P l a t e 3), th e l i d o f w h i c h w a s s e a le d w ith m a sk in g ta p e . To m a i n t a i n e a c h o f th e v a r i o u s h u m i d i t i e s a c c u r a t e l y , a s p e ­ c i f i c c h e m i c a l w a s p l a c e d in e a c h c h a m b e r a n d th e c h a m b e r w a s p l a c e d in an in c u b a t o r o f th e r i g h t t e m p e r a t u r e . r e s u lt s of th e s e e x p e r im e n ts w e r e The c h e c k e d a s in t h e p r e v i o u s e x p e r i m e n t s , n a m e l y , c o u n t s w e r e m a d e o f th e t o t a l n u m b e r o f s p o r e s g e r m i n a t e d p e r h i g h - p o w e r f i e l d a s c o m p a r e d to th e to ta l u n g er m in a te d sp o r e s: The c o u n t s w e r e m a d e a t th e s a m e tim e in te r v a ls m e n tio n e d p r e v io u s ly . W a s h in g E x p e r i m e n t s R e c a l l i n g th e p r o c e d u r e o f p r e p a r i n g e a c h s l i d e w it h its spore s u s p e n s i o n s it w i l l be n o t e d th a t n o t a l l o f the l i q u i d s p o r e s u s p e n s i o n w a s u s e d ; m o s t o f it r e m a i n e d in th e c e n t r i f u g e P la te 3 20 tu b es. E a c h c e n t r i f u g e tu b e w a s c a r e f u l l y m a r k e d and p l a c e d in th e s a m e i n c u b a t o r w it h t h e s l i d e s . A f t e r th e f i n a l c o u n t s w e r e m a d e , th e l o w e s t c o n c e n t r a t i o n o f A c t i - d i o n e w h i c h c o m ­ p l e t e l y in h i b i t e d g e r m i n a t i o n , p lu s th e n e x t l o w e r c o n c e n t r a ­ tio n , w a s s e l e c t e d f o r the w a s h i n g e x p e r i m e n t s . The p r o p e r c e n t r i f u g e t u b e s c o n t a in in g t h e l i q u i d s p o r e s u s p e n s i o n s th a t w e r e in h i b i t e d b y th e A c t i - d i o n e , w e r e r e m o v e d f r o m th e i n ­ c u b a t o r an d e a c h s u s p e n s i o n w a s c h e c k e d m i c r o s c o p i c a l l y f o r e v i d e n c e o f g e r m i n a t i o n , th e n if t h e e v i d e n c e c o r r e l a t e d w it h th e r e s u l t s o b t a i n e d f r o m t h e s l i d e s th e s p o r e s u s p e n s i o n s w e r e c a r e fu lly d ecan ted . T o e a c h c e n t r i f u g e tu b e 5 m l . o f s t e r i l e d i s t i l l e d w a t e r w a s a d d e d arid th e t u b e s w e r e r o l l e d in th e p a l m s of th e h a n d s a s in th e p r e v i o u s e x p e r i m e n t s . T h is w a s d on e to d i s p e r s e th e s p o r e s t h r o u g h th e w a t e r s o th a t a s m u c h o f th e A c t i - d i o n e c o u l d be w a s h e d o f f a s p o s s i b l e . The tu b es w e r e a g a in c e n t r i f u g e d f o r 2 m i n u t e s , th e l i q u i d w a s d e c a n t e d an d th e p r o c e s s w a s r e p e a t e d 5 t i m e s ; e a c h t i m e u s i n g f r e s h w ater. T h e s p o r e s w e r e t h e n r e s u s p e n d e d in 5 m l . o f th e n u ­ t r i e n t b r o t h s t i m u l a n t and p l a c e d o n c l e a n s l i d e s , in s t e r i l e P e t r i d i s h c h a m b e r s and t h e n p l a c e d b a c k in to th e i n c u b a t o r f r o m w h ich th ey w e r e o r ig in a lly ta k en . T h e s e w e r e th e n c h e c k e d 21 f o r g e r m i n a t i o n a s in t h e p r e v i o u s e x p e r i m e n t s and a t th e s a m e in te r v a ls. pH E x p e r im e n t s A n u m b e r of e x p e r im e n t s w e r e co n d u c te d in v o lv in g a s t u d y to d e t e r m i n e th e e f f e c t t h a t A c t i - d i o n e w o u ld h a v e on th e g e r m i n a t i o n o f s p o r e s , i f th e l i q u i d m e d i u m in w h i c h th e spores w ere s u s p e n d e d v a r i e d in h y d r o g e n io n c o n c e n t r a t i o n . F iv e d iffe r e n t pH v a lu e s w e r e a r b itr a r ily ch o sen : 6 .0 , 5 .0 , 4 .0 , and 3 .0 . p H 6 .8 , E ach ex p erim en t w as p rep a red a s p r e ­ v i o u s l y d e s c r i b e d an d c o n d u c t e d a t e a c h o f th e t e m p e r a t u r e s as sta ted p r e v io u sly . P r i o r to c o u n t in g th e r e s u l t s o f g e r m ­ in a t io n o n th e s l i d e s , th e p H w a s c a r e f u l l y m e a s u r e d b y u s e o f a B e c k m a n p H m e t e r , a n d a n y c h a n g e in p H v a lu e o f th e m e ­ d iu m w a s r e c o r d e d . E X P E R IM E N T A L R E SU L T S In p r e s e n t i n g th e r e s u l t s o f t h i s i n v e s t i g a t i o n , b o t h t a b l e s and g r a p h s w e r e u s e d . The t a b l e s s h o w t h e e x a c t p e r c e n t a g e o f in h ib i t i o n o b t a in e d w h e n s p o r e s u s p e n s i o n s o f e a c h o r g a n i s m w e r e e x p o s e d to th e v a r i o u s c o n c e n t r a t i o n s o f A c t i - d i o n e w h i c h h a d b e e n a d j u s t e d to p H v a l u e s o f 6 .8 , 6 .0 , 5 .0 , 4 .0 a n d 3 .0 u n d e r th e f o l l o w i n g t e m p e r a t u r e s : C. 2 2 ° , 2 4 °, 2 6 °, 2 8 ° , and 30° E a c h t a b le w a s t h e n p r e s e n t e d in a g r a p h in o r d e r to d e m ­ o n s t r a t e m o r e c l e a r l y th e e n t i r e p i c t u r e f o r e a c h o r g a n i s m a s it w as a ffe c te d b y A c t i- d io n e u n der the f a c t o r s m e n tio n e d ab ove. In a d d it io n to t h i s , d a ta o b t a in e d f r o m th e h u m i d i t y e x p e r i m e n t s , a r e p r e s e n t e d in t a b u la r and g r a p h ic f o r m . It w i l l b e n o t e d th a t a l o g a r i t h m i c s c a l e g r a p h p a p e r w a s u s e d f o r the c o n c e n t r a t i o n s of t h e A c t i - d i o n e , th u s i t b e ­ c a m e n e c e s s a r y to i n d i c a t e th e r e s u l t s o f th e c o n t r o l s in w h a t m a y b e c o n s i d e r e d an u n o r t h o d o x m e t h o d . The c o n t r o l s h a v e b e e n p l a c e d on th e e x t r e m e l e f t s i d e o f e a c h c h a r t , n e x t to the o r d in a t e . T h is w a s d o n e so th a t a c o m p a r i s o n c o u ld b e e a s i l y m a d e b e t w e e n th e c o n t r o l s a n d e a c h c o n c e n t r a t i o n of A c t i - d i o n e 23 used. T h e c o n c e n t r a t i o n s o f A c t i - d i o n e a r e e x p r e s s e d in p a r t s p e r m i l l i o n and a r e h e r e a f t e r d e s i g n a t e d b y th e a b b r e v i a t i o n p .p .m . It w i l l b e n o t e d t h a t th e r e s u l t s o f t h r e e e n v i r o n m e n t a l f a c t o r s a r e p r e s e n t e d in t h i s t h e s i s . v ir o n m en ta l fa c to r s w e r e A n u m b e r of o t h e r e n ­ s t u d i e d and r e s u l t s w e r e o b t a i n e d a s s t a t e d u n d e r th e h e a d in g M a t e r i a l s an d M e t h o d s . not p r e s e n te d h e r e . T h ese are T h e o t h e r e n v i r o n m e n t a l f a c t o r s c o u ld n o t e a s i l y b e c o n t r o l l e d in th e f i e l d an d w o u l d th u s b e o f no d ir e c t p r a c tic a l v a lu e . In v i e w o f t h i s f a c t i t w a s d e c i d e d to m e n t i o n t h e s e r e s u l t s b r i e f l y h e r e an d p e r h a p s p u b l i s h t h e m as a s e p a r a te p a p e r at a la t e r d ate. D ir e c t Im m e r sio n E x p erim en ts C la d o sp o r iu m C u c u m e rin u m In T a b le I a r e s h o w n th e r e s u l t s o b t a in e d w it h s p o r e s o f C l a d o s p o r i u m c u c u m e r i n u m e x p o s e d to s o l u t i o n s o f A c t i - d i o n e p r e v i o u s l y a d j u s t e d to th e f o l l o w i n g p H c o n c e n t r a t i o n s : 5 . 0 , 4 .0 a n d 3 . 0 . 6 .8 , 6 .0 , T h e s e p H v a l u e s in e a c h c o n c e n t r a t i o n of A c t i - d i o n e w e r e e x p o s e d to e a c h o f th e f o l l o w i n g t e m p e r a t u r e s : 24 TABLE I IN H IB IT IO N O F S P O R E G E R M IN A T IO N O F C L A D O S P O R IU M C U C U M E R IN U M B Y 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 A C T I-D IO N E A T F IV E D IF F E R E N T T E M P E R A T U R E S A N D F I V E H Y D R O G E N ION C O N C E N T R A T I O N S T em ­ pera­ tu res C oncen­ tr a tio n of A c ti-d io n e in p . p . m . P e r c e n t a g e o f I n h ib itio n pH 3.0 pH 4 .0 PH 5 .0 PH 6 .0 PH 6.8 22 0 .0 0 .5 2 .5 5 .0 1 0 .0 2 5 .0 5 0 .0 No G er­ m in a ­ t io n 6 2 .5 5 7 4 .0 2 8 9 .4 1 9 5 .0 4 1 0 0 .0 0 1 0 0 .0 0 1 0 0 .0 0 3 3 .9 3 5 0 .0 0 6 0 .1 8 6 5 .1 8 8 2 .0 0 1 0 0 .0 0 1 0 0 .0 0 5 .2 2 2 3 .1 6 2 8 .0 1 36 .3 1 7 9 .9 8 1 0 0 .0 0 1 0 0 .0 0 4 .6 5 8 .0 0 1 6 .3 3 1 9 .6 4 7 5 .5 6 1 0 0 .0 0 1 0 0 .0 0 No G er­ m in a ­ t io n 7 0 .3 2 24 0 .0 0 .5 2 .5 5,0 1 0 .0 2 5 .0 5 0 .0 5 3 .1 0 4 4 .7 7 4 0 .3 5 4 8 .7 0 7 6 .5 8 9 8 .2 0 1 0 0 .0 0 1 8 .0 0 1 2 .0 3 2 7 .1 4 5 4 .0 1 7 2 .1 1 1 0 0 .0 0 1 0 0 .0 0 1 7 .8 3 1 0 .9 0 2 4 .9 6 61.21 8 1 .9 2 1 0 0 .0 0 1 0 0 .0 0 26 0 .0 0 .5 2 .5 5 .0 1 0 .0 2 5 .0 5 0 .0 No G erm in a tio n (° C.) 6 1 .0 9 7 7 .3 3 88 .8 1 1 0 0 .0 0 1 0 0 .0 0 1 0 0 .0 0 4 3 .1 0 5 5 .0 0 6 2 .1 3 7 5 .1 3 8 4 .1 0 9 6 .1 2 1 0 0 .0 0 2 8 .8 3 4 7 .3 2 6 6 .9 6 7 2 .0 7 8 7 .3 9 9 6 .4 6 1 0 0 .0 0 1 0 .1 0 1 6 .1 8 4 2 .0 0 6 1 ,2 2 8 9 .4 2 9 8 .1 2 1 0 0 .0 0 8 .5 9 1 2 .6 9 3 4 .3 8 5 8 .7 8 9 1 .7 2 1 0 0 .0 0 1 0 0 .0 0 25 T A B L E I (C o n tin u ed ) Tem peratu res C oncen­ tr a tio n of A c ti-d io n e in p . p . m . P e r c e n t a g e of I n h i b i t io n PH 3 .0 PH 4 .0 PH 5 .0 PH 6 .0 PH 6 .8 28 0 .0 0 .5 2 .5 5 .0 1 0 .0 2 5 .0 5 0 .0 No G er­ m in a ­ t io n 8 7 .0 0 8 9 .2 3 9 5 .1 6 9 8 .0 2 1 0 0 .0 0 1 0 0 .0 0 1 0 0 .0 0 5 6 .8 8 5 7 .8 0 7 9 .1 3 8 6 .3 6 1 0 0 .0 0 1 0 0 .0 0 1 0 0 .0 0 3 8 .0 0 4 7 .0 0 6 2 .6 5 73 .5 1 1 0 0 .0 0 1 0 0 .0 0 1 0 0 .0 0 4 5 .2 3 4 8 .0 1 5 7 .3 2 6 8 .7 6 1 0 0 .0 0 1 0 0 .0 0 1 0 0 .0 0 30 0 .0 0 .5 2 .5 5 .0 1 0 .0 2 5 .0 5 0 .0 No G er­ m in a ­ t io n 9 7 .1 3 9 1 .0 6 9 8 .7 1 1 0 0 .0 0 1 0 0 .0 0 1 0 0 .0 0 1 0 0 .0 0 8 6 .2 4 7 4 .5 5 7 8 .5 7 9 4 .5 5 1 0 0 .0 0 1 0 0 .0 0 1 0 0 .0 0 5 1 .2 1 6 2 .1 4 7 4 .3 1 8 8 .9 1 1 0 0 .0 0 1 0 0 .0 0 1 0 0 .0 0 6 0 .3 1 6 6 .5 6 7 3 .3 3 8 5 .1 5 1 0 0 .0 0 1 0 0 .0 0 1 0 0 .0 0 (° c.) 26 2 2 ° C ., 2 4 ° C ., 2 6 ° C ., an d 3 0 ° C. T h e r e s u l t s o f T a b le I h a v e b e e n p l o t t e d on s e m i - l o g a r i t h m i c g r a p h p a p e r ( F i g u r e s 1, 2, 3, 4 ). It m u s t b e p o i n t e d o u t th a t in a n u m b e r o f c a s e s th e c o n c e n tr a tio n of A c t i- d io n e w h ich p r e v e n te d g e r m in a tio n of s p o r e s a f t e r 12 h o u r s w a s i n s u f f i c i e n t to m a i n t a i n th a t i n h i b i ­ t io n a f t e r 24 h o u r s an d in s o m e c a s e s a fter 48 h o u r s . No c h a n g e w a s n o t e d , h o w e v e r , a f t e r 72 h o u r s in a n y c a s e . It a l s o m a y b e n o t e d t h a t c o m p l e t e in h i b i t i o n w a s o b t a in e d a t 5 0 .0 p . p . m . in a ll c a s e s , r e g a r d l e s s o f t e m p e r a t u r e o r p H c o n - .cen tra tio n . In c e r t a i n c a s e s it a p p e a r s th a t b o t h t e m p e r a t u r e and p H w e r e i n f l u e n t i a l in th e c o m p l e t e in h ib i t i o n o f s p o r e g e r m in a tio n at lo w e r c o n c e n tr a tio n s of A c t i- d io n e . (F ig u r e A t p H 6 .8 I) in h ib it i o n w a s g r e a t e s t at 3 0 ° C. a t a l l c o n c e n t r a t i o n s o f A c t i - d i o n e and p r o g r e s s i v e l y l e s s e n e d w it h t h e d rop in t e m ­ p eratu re. A n e x c e p t i o n to t h is i s o b s e r v e d a t o n l y two p o i n t s w it h t e m p e r a t u r e s o f 24 o p o i n t e d o u t th at a d e f i n i t e ,o C . an d 26 C. It s h o u l d a l s o be s t i m u l a t i o n w a s o b s e r v e d in 0 .5 p . p . m . a t 2 4 ° C. a s c o m p a r e d to th e c o n t r o l . A t p H 6 .0 ( F i g ­ u r e 2) th e s a m e p a t t e r n o f in h ib it i o n i s o b s e r v e d a s in th e p r e v i o u s c a s e e x c e p t f o r the f a c t th a t a h ig h e r p e r c e n t o f 27 100 30 90 28 80 P ercentage of In h ib ition 70 24 22 60 50 F ig u r e 1. C la d o sp o r iu m c u c u m e r in u m - pH 4 . 26 40 30 20 10 J 0 .5 2 .5 I L 5 10 25 C o n c e n tr a tio n o f A c t i- d io n e in p .p .m . 50 28 100 90 30 80 In h ib itio n 50 P ercen tage 60 of 70 28 24 40 22 30 F i g u r e 2. 26 C la d o sp o r iu m c u c u m e r in u m - pH 5. 20 10 0 .5 2 .5 J L _L 5 10 25 C o n c e n tr a tio n o f A c t i- d io n e in p .p .m . 50 100 80 G O 30 *4-1 o 40 28 30 20 C la d o sp o r iu m c u c u m e r in u m F ig u r e 24 pH 6. ------ 26 22 0 .5 2 .5 5 10 25 C o n c e n tr a tio n o f A c t i- d io n e in p .p .m . 50 30 100 80 P ercen tage of In h ib ition 70 50 28 40 30 20 24 F ig u r e 4. C la d o sp o r iu m c u c u m e r in u m --------26 22 0 .5 2 .5 5 10 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 75 31 in h ib it io n w a s o b t a i n e d at 0 . 5 p . p . m . at 2 2 ° C . th an a t 2 4 ° C. and 2 6 ° C. a s w a s the c a s e at pH 6 . 8 . A lso an a n o m a l y to the g e n e r a l p a t t e r n of p r o g r e s s i v e l y l o w e r e d i n h ib it i o n a s th e te m p e r a tu r e is l o w e r e d m a y be o b s e r v e d b e tw e e n t e m p e r a t u r e s 2 2 ° C ., 2 4 ° C. a n d 2 6 ° C., i n s t e a d o f 24 ° C . and 2 6 ° C. a s in th e p r e v i o u s c a s e - - p H 6 .8 . A s in t h e p r e v i o u s c a s e h e r e a g a in s t i m u l a t i o n o c c u r r e d in 0 .5 p . p . m . a t 2 5 to th e c o n t r o l . f e c t i v e a t 22 o A t pH 5.0 ( F i g u r e C. th a n at 2 4 o C. o C. a s c o m p a r e d 3) A c t i - d i o n e w a s m o r e e f A l s o , a s lig h t stim u la tio n w a s o b s e r v e d in 0 .5 p . p . m . an d 2 .5 p . p . m . at 2 5 ° C ., a n d in 0 .5 p . p . m . a t 3 0 ° C. a s c o m p a r e d to t h e c o n t r o l . u r e 4) i t w i l l b e n o t e d t h a t in h ib i t i o n at 2 2 ° th a n a t 24 o p . p . m . a t 30 th a n a t 26 o .o C. o r 2 6 C. ° C. C. a n d 24 ° A t p H 4 .0 ( F i g ­ C. w a s g r e a t e r A s t i m u l a t i o n w a s o b s e r v e d in 0 .5 C . and a t t h e l a t t e r it w a s g r e a t e r In s u m m a t i o n it m a y be s a i d that a s th e pH c o n c e n t r a t i o n w a s l o w e r e d , 22 o C. a n d 24 o C. b ecam e m ore e f f e c t i v e t e m p e r a t u r e s a s a f a c t o r i n i n h ib it i o n . No g erm in a ­ t io n o c c u r r e d at p H 3.0 a t a n y t r e a t m e n t i n c l u d i n g th e c o n t r o l , th u s no r e s u l t s c o u l d be p r e s e n t e d . i 32 In F i g u r e s 5, 6 , 7, 8, a n d 9 d a t a a r e p r e s e n t e d to s h o w th e r e l a t i o n s h i p b e t w e e n the p H c o n c e n t r a t i o n s in r e l a t i o n to con stan t te m p e r a tu r e s. A t 2 2 ° C. th e g e n e r a l p a t t e r n a p p e a r s to b e t h a t i n h i b i ­ t i o n p r o g r e s s i v e l y i n c r e a s e s w i t h th e l o w e r i n g o f p H c o n c e n t r a ­ tio n . A t 2 4 ° C. the s a m e g e n e r a l p a t t e r n p r e v a i l s e x c e p t th a t a s l i g h t s t i m u l a t i o n i n g e r m i n a t i o n i s n o t e d in 0 .5 p . p . m . a t a ll pH c o n c e n tr a tio n s. A f u r t h e r s t i m u l a t i o n of g e r m i n a t i o n i s n o t e d in 2 . 5 p . p . m . a t pH 5 . 0 . A ls o at h ig h e r c o n c e n t r a ­ t i o n s th a n 2 . 5 p . p . m . m o r e i n h i b i t i o n i s n o t e d a t pH 6 .8 th a n 6 .0 o r 5 .0 . A s w e p r o c e e d to 2 6 ° C . i t i s n o t e d th a t p H 6 .8 n o t o n ly s u p e r c e d e s p H 6.0 a n d 5.0 a s in th e p r e v i o u s c a s e , b u t pH 4 .0 a s w e l l . p H 4 .0 a n d 5 .0 e x c h a n g e p o s i t i o n s in d e ­ g r e e s o f i n h ib it i o n s e v e r a l t i m e s b e t w e e n 0 .5 p . p . m . an d 5 0 .0 p .p .m . O t h e r w i s e , h e r e a l s o , w e n o t e th e g e n e r a l p a t t e r n d e ­ sc r ib e d p re v io u sly . A t 28° C. th e r e a p p e a r s no s ig n ific a n t d if ­ f e r e n c e f r o m th e p r e v i o u s f i g u r e s e x c e p t f o r th e a b s e n c e o f s t i m u l a t i o n in g e r m i n a t i o n a n d a d e f i n i t e t e n d e n c y t o w a r d a h i g h e r d e g r e e of i n h ib it i o n in a l l c o n c e n t r a t i o n s o f A c t i - d i o n e and at a l l p H l e v e l s . A l s o to b e n o t i c e d i s th e l a c k o f p o s i t i o n e x c h a n g e in d e g r e e o f in h ib it i o n b y t h e v a r i o u s g r a p h l i n e s of 100 80 P e r c e n ta g e of I n h ib itio n 70 4 .0 50 40 5 .0 30 20 6.0 F ig u r e C la d o sp o r iu m c u c u m e r in u m 6. 8 22 0 .5 2 .5 5 10 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 34 100 80 In h ib itio n 4 .0 P e r c e n ta g e of 5.0 40 6.0 20 6.8 F i g u r e 6. 0 .5 2 .5 5 10 C la d o sp o r iu m c u c u m e r in u m 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 100 /f- 80 70 fi o xa VH O c u V P* 50 40 30 20 6.8 4 .0 F ig u re 25. 3.0 0 .5 M o n il in ia F r u c t i c o l a 22 2 .5 5 10 25 C. 50 C o n c e n tr a tio n of A c t i - d i o n e in p .p .m . 71 100 90 60 of 60 50 P e r c e n ta g e I n h ib itio n 70 40 30 F ig u r e 26. 20 M o n il in ia F r u c tic o la 10 4 .0 0 .5 2 .5 5 10 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 72 100 90 80 P e r c e n ta g e of I n h ib itio n 70 60 50 40 F ig u r e 27. 30 20 M o n il in ia F r u c tic o la 3 .0 4 .0 10 5 .0 0 .5 2 .5 5 10 25 C o n c e n tr a tio n o f A c t i- d io n e in p .p .m . 50 73 F igu re y i-dione of A c ti C oncen tr a tio n 28 M o r u la n ia i*1 P - P ' 74 100 80 a o +> ‘H V| O v bo 4n ->i C v u u v & 50 40 F ig u r e 29. M o n il in ia F r u c t i c o l a 20 3 .0 5 .0 4 .0 6.8 0 .5 2 .5 5 10 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 75 th a t a t t h i s t e m p e r a t u r e , a l l p H v a l u e s r e a c h e d t h e i r m a x i m u m e f f e c t a t 2 5 .0 p . p . m . A l s o to b e n o t e d i s th e a l l i g n m e n t o f the c o n t r o l r e s u l t s a s c o m p a r e d to th e t r e a t m e n t s . T em p era tu re 24° C . at p H 6 .8 ( F i g u r e 2 6 ) . A g a in , A c t i- d io n e i s m o s t e f f e c tiv e E x c e p t f o r th e v e r y m a r k e d s t i m u l a t i o n o f g e r m i n a t i o n at 2 .5 p . p . m . a t p H 6 .8 a n d th e r e a r r a n g e m e n t o f th e c o n t r o l r e s u l t s th e p a t t e r n o f b e h a v i o r w a s s i m i l a r to th e p r e v i o u s (F ig u r e 25). A ll pH v a lu e s c o n v e r g e at 2 5 .0 p .p .m . in d ica tin g m a x im u m in h ib itio n . A l s o th e l i n e r e p r e s e n t i n g pH 6 .8 c r o s s e s p H v a l u e s 6 .0 an d 5 .0 at tw o d i f f e r e n t p o i n t s o f co n cen tra tio n . T h e l i n e r e p r e s e n t i n g p H 4 .0 c r o s s e s p H 3.0 and i n d i c a t e s l e s s e f f e c t o r s i m i l a r e f f e c t a s p H 3 .0 b u t at h ig h er c o n c e n tr a tio n s of A c t i- d io n e . T e m p e r a t u r e 2 6 ° C. T h e p H v a l u e o f 6 .8 i n d i c a t e s m a x ­ i m u m e f f e c t f o l l o w e d in s e q u e n c e b y p H 6 . 0 , 5 . 0 , 4 . 0 , a n d 3.0 ( F i g u r e 2 7 ). A s in th e p r e v i o u s t e s t s , th e c o n t r o l r e s u l t s do n o t f o l l o w t h e p a t t e r n o f th e t r e a t m e n t s . T h ere w as an a b sen ce of s t i m u l a t i o n of g e r m i n a t i o n a t p H 6 .8 a n d t h e p r e s e n c e at p H 4 .0 a n d p H 3 .0 a t 10.0 p , p . m . slig h t stim u la tio n n oted at 0 .5 of it T h ere w as a lso a v e r y p .p .m . a t pH 3 .0 ., a p p ro x im a tely 76 2.00% . A ll lin e s r e p re se n tin g pH v a lu e s converge a t 2 5 .0 p .p .m . T em p eratu re 28° C . The m o s t s t r i k i n g d i f f e r e n c e n o t e d h e r e i s t h a t the g r a p h l i n e s do n o t a l l m e e t a t 2 5 .0 p . p . m . a s i n the t h r e e p r e v i o u s e x p e r i m e n t s . F u r th e r m o r e it a p p ea rs t h a t a t t h i s t e m p e r a t u r e p H 5.0 r e a c h e d i t s m a x i m u m at a l o w e r c o n c e n t r a t i o n o f A c t i - d i o n e th an p H 6 .0 a s w a s the c a s e p r e v i ­ o u sly . A t lo w e r c o n c e n tr a tio n s ( 2 .5 p . p . m . ) , h o w e v e r , pH 4 .0 w a s s u p e r i o r in in h i b i t i o n to b o th p H 6 .8 an d 5 .0 . p eratu re, A t t h is t e m ­ s o m e r e d u c t i o n o f i n h ib it i o n o c c u r r e d at a l l pH v a l u e s e x c e p t p H 6 .0 . A l s o n o t e th a t th e s t i m u l a t i o n o c c u r r e d a t a l o w e r c o n c e n t r a t i o n o f A c t i - d i o n e th a n a t 26 T em p eratu re 30 o t h i s t e m p e r a t u r e a n d 28 C. o C. The p r i n c i p l e d i f f e r e n c e b e t w e e n C. w a s that a ll pH v a lu e s r e a c h e d t h e i r m a x i m u m e f f e c t a t 1 0 .0 p . p . m . ( F i g u r e 2 9 ) . A noth er d if­ f e r e n c e o f i n t e r e s t w a s t h e f a c t th a t p H 3 .0 w a s s u p e r i o r in in h ib i t i o n to p H 5 .0 and 4 . 0 . B o t h o f th e l a t t e r , h o w e v e r , w e r e s u p e r i o r to pH 3 .0 at 0 .5 p . p . m . A ls o of c o n s id e r a b le in t e r e s t w a s th e c o m p l e t e e x c h a n g e o f p o s i t i o n s b y p H 6 .8 an d 3.0 in th e c o n t r o l r e s u l t s . 77 T A B L E III I N H IB IT IO N O F S P O R E G E R M I N A T I O N O F M O N IL IN IA F R U C T IC O L A BY D IF F E R E N T C O N C EN TR A TIO N S OF A C T I-D IO N E AT F IV E D IF F E R E N T T E M P E R A T U R E S A N D F I V E H Y D R O G E N ION C O N C E N T R A T I O N S T em ­ pera­ tu res (° C.) 9C 9t C oncen­ t r a t i o n of A c ti-d io n e in p . p . m . PH 3.0 pH 4 .0 pH 5.0 pH 6.0 pH 6 .8 C ontrol 0 .5 2 .5 5 .0 1 0 .0 2 5 .0 5 .2 6 4 .6 3 7 .8 4 1 7 .5 9 3 0 .6 3 1 0 0 .0 0 6 .1 2 2 3 .0 1 1 9 .8 9 3 9 .7 2 5 4 .4 9 1 0 0 .0 0 7 .8 5 6 0 .4 8 5 8 .2 3 7 1 .3 2 8 0 .0 0 1 0 0 .0 0 8 .3 3 8 1 .2 2 7 7 .0 0 8 4 .4 9 8 9 .1 2 1 0 0 .0 0 1 0 .2 8 8 9 .7 2 7 8 .5 7 9 5 .5 0 99 .1 1 1 0 0 .0 0 C ontrol 0 .5 5.0 1 0 .0 2 5 .0 1 0 .1 0 1 .8 9 7.21 1 4 .8 5 7 6 .9 9 1 0 0 .0 0 7.51 2 6 .0 0 3 1 .4 0 4 5 .8 8 5 6 .4 2 1 0 0 .0 0 1 5 .1 5 6 9 .8 3 6 2 .0 0 6 5 .5 2 7 8 .2 2 1 0 0 .0 0 1 1 .0 5 7 3 .1 0 6 8 .1 4 7 5 .0 2 8 0 .1 2 1 0 0 .0 0 1 4 .2 9 8 4 .4 0 3 7 .2 7 8 1 .0 8 8 7 .3 9 1 0 0 .0 0 C ontrol 0 .5 2 .5 5.0 1 0 .0 2 5 .0 2 0 .1 9 1 7 .7 6 4 6 .3 0 3 0 .1 0 9 .4 8 1 0 0 .0 0 1 8 .0 0 2 0 .0 1 6 .5 0 2 8 .2 5 5 6 .9 8 6 0 .8 8 7 0 .0 0 1 0 0 .0 0 4 .5 1 1 7 .6 9 5 8 .6 2 69.31 8 8 .1 2 1 0 0 .0 0 8 .7 7 4 3 .7 5 6 4 .5 5 8 3 .3 3 9 0 .0 0 1 0 0 .0 0 P e r c e n t a g e of I n h ib itio n 4 8 .1 9 3 5 .0 0 2 6 .3 3 1 0 0 .0 0 78 T A B L E III (Continued) Tem ­ pera­ tu res (° C.) 9L a ft O 30 C oncen­ t r a t i o n of A c ti-d io n e in p . p . m . PH 3.0 pH 4 .0 pH 5 .0 pH 6 .0 pH 6 .8 C ontrol 0 .5 2 .5 5 .0 1 0 .0 2 5 .0 3 .7 7 5.41 8 .4 9 5 .9 4 5 .6 6 1 0 0 .0 0 2 .0 3 1 9 .5 0 2 2 .1 4 2 1 .0 0 3 0 .7 1 1 0 0 .0 0 10 .9 1 3 7 .1 1 2 7 .1 0 7 6 .2 1 1 0 0 .0 0 1 0 0 .0 0 4 .5 1 3 3 .4 0 4 1 .9 9 6 5 .0 0 8 5 .0 0 1 0 0 .0 0 12.61 4 2 .2 0 3 3 .0 3 8 7 .9 6 1 0 0 .0 0 1 0 0 .0 0 C o n tr o l 0 .5 14.81 1 7 .9 2 4 0 .5 7 8 4 .4 0 1 0 0 .0 0 1 0 0 .0 0 1 1 .5 6 2 3 .2 6 2 8 .0 0 6 0 .1 0 1 0 0 .0 0 1 0 0 .0 0 1 2 .1 0 26 .00 3 6 .5 0 4 5 .2 3 1 0 0 .0 0 1 0 0 .0 0 6 .0 0 6 2 .1 8 8 3 .1 1 9 1 .0 0 1 0 0 .0 0 1 0 0 .0 0 6.9 6 6 5 .4 9 8 6 .7 3 9 8 .1 8 1 0 0 .0 0 1 0 0 .0 0 li 5 .0 1 0 .0 2 5 .0 P e r c e n t a g e of I n h ib it io n 79 R a m u la r ia sp. T h e s p o r e s o f t h is o r g a n i s m w e r e p r e p a r e d f o r e a c h e x p e r i m e n t , a s p r e v i o u s l y d e s c r i b e d in the s e c t i o n e n t i t l e d M a ­ t e r i a ls and M eth o d s. T h e c o u n t in g , h o w e v e r , w a s m a d e a t s h o r t e r i n t e r v a l s t h a n t h o s e u s e d f o r th e o t h e r o r g a n i s m s due to th e f a c t t h a t th e s p o r e s g e r m i n a t e d a f t e r 6 h o u r s . w e r e m a d e a f t e r 6, 12, 2 4 an d 36 h o u r s . C o n seq u en tly cou n ts A t the e n d o f t h is p e r i o d c o u n t in g w a s d i s c o n t i n u e d . It s h o u l d b e p o i n t e d ou t th a t n o d i s t o r t i o n o r d e s t r u c t i o n of g e r m t u b e s o r t h e i r c y t o p l a s m i c c o n t e n t o c c u r r e d in the e x ­ p e r i m e n t s i n v o l v i n g t h is o r g a n i s m . In c o n c e n t r a t i o n s of 5.0 p . p . m . a n d h i g h e r , i t w a s c o n s i s t e n t l y o b s e r v e d th a t th e g e r m t u b e s of t h e g e r m i n a t e d s p o r e s w e r e c o n s i d e r a b l y s h o r t e r th a n th o se of th e c o n t r o ls . F u r t h e r m o r e , t h e y a p p e a r e d s h o r t e r in e a c h su c c e e d in g h ig h e r co n c e n tr a tio n . pH 3 .0 . T h r e e p e a k s of m a x i m u m i n h ib it i o n a r e to b e n o t e d a t t h i s h y d r o g e n io n c o n c e n t r a t i o n , t h e f i r s t b e i n g a t 5 .0 p . p . m . a t a t e m p e r a t u r e o f 22 a t a t e m p e r a t u r e o f 26 C ., th e s e c o n d at 1 0 .0 p „p .m . C ., and t h e t h ir d a t 5 0 .0 p . p . m . b y a ll o f th e t e m p e r a t u r e s u s e d i n t h i s i n v e s t i g a t i o n ( F i g u r e 30). A 80 100 of 50 P ercen tage In h ib ition 80 40 VI I 30 22 26 20 F i g u r e 30. 24 R a m u la r ia s p . 28 pH 3. 0 .5 2 .5 5 10 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 75 81 g l a n c e a t F i g u r e 30 w i l l q u i c k l y r e v e a l th e a n o m a l o u s b e h a v i o r o f t h i s o r g a n i s m o r th e e r r a t i c e f f e c t o f th e A c t i - d i o n e upon th e s p o r e s o f t h i s o r g a n i s m . In 0 .5 p . p . m . th e s p o r e s w e r e m o s t s u s c e p t i b l e t o the t o x i n a t 3 0 ° C. b u t q u i c k ly th e p i c t u r e c h a n g e d an d 2 8 ° C . b e c a m e th e t e m p e r a t u r e w h i c h w a s m o s t effectiv e. T h is c h a n g e , h o w e v e r , d id n o t p e r s i s t s i n c e a t a p - p r o x i m a t e l y 3.0 p . p . m . , 2 2 m a x i m u m in 5 .0 p . p . m . o C. t o o k th e l e a d and r e a c h e d i t s T h is m a r k e d r e v e r s a l of in h ib it i o n m a y be in te r p r e te d as stim u la tio n . On th e o t h e r h a n d i t m a y b e a m a t t e r of t h e t o x in h a v in g l e s s e f f e c t at t h a t p a r t i c u l a r c o n c e n t r a t i o n u n d e r th e g i v e n c o n d i t i o n s . A s o n e p r o c e e d s up th e s c a l e o f A c t i - d i o n e c o n c e n t r a t i o n , it b e c o m e s e v i d e n t th a t th e t o x i c e f f e c t i s g r e a t e s t at 30 o C. H e r e a l s o a r e tw o i n s t a n c e s o f d e f in it e s t i m u l a t i o n w h i c h o c c u r r e d in 0 .5 p . p . m . a n d 1 0 .0 p . p . m . a t 2 4 ° C. o n l y c a s e o f s t i m u l a t i o n i n th is e x p e r i m e n t . T h is w a s th e W hat h a s b e e n d e s c r i b e d a b o v e r e g a r d i n g th e v a r i o u s t e m p e r a t u r e s w h i c h w e r e m o s t effe ctiv e at v a rio u s co n cen tra tio n s of A c ti-d io n e is a lso t r u e in t h e c a s e o f o t h e r t e m p e r a t u r e s ; t h e y c r o s s e a c h o t h e r at s e v e r a l p la c e s . the r e s u l t s at 2 4 o It i s i n t e r e s t i n g to n o te th a t in th e c o n t r o l s , C. a n d 28 o C. a r e i d e n t i c a l . 82 pH 4 . 0 . A s in th e p r e c e d i n g e x p e r i m e n t , t h e r e a r e h e r e t h r e e p e a k s o f m a x i m u m i n h ib it i o n o b s e r v e d . 5 .0 p . p . m . a t 24 o T h e f i r s t w a s in C ., th e s e c o n d in 5 0 .0 p . p . m . at 2 4 o C. a n d 2 8 ° C.; f o r t h e l a t t e r t e m p e r a t u r e t h i s r e p r e s e n t s t o t a l g e r m ­ in a tio n . T h e t h ir d p e a k r e p r e s e n t s t o t a l i n h ib it i o n b y a l l t e m ­ p e r a t u r e s , e x c e p t 2 8 ° C ., in 75 p . p . m . T h e e r r a t i c b e h a v i o r of th e o r g a n i s m o r a s p r e v i o u s l y s t a t e d th e a c t i o n of th e to x in u p o n th e s p o r e s , i s pH 5 . 0 . show n by F ig u r e 31. The p r in c ip a l d iffe r e n c e h e r e a s c o m p a r e d to th e p r e v i o u s e x p e r i m e n t s i s th e f a c t th a t t h e m a x i m u m a m o u n t o f i n h ib it i o n w a s in 7 5 .0 p . p . m . a t a l l t e m p e r a t u r e s { F ig u r e 32). T h e o n ly p e a k of i n h i b i t i o n o t h e r th a n th e a b o v e i s in 5.0 p . p . m . a t 2 2 ° C . w h i c h i s f o l l o w e d b y a s h a r p r e d u c t i o n o f in h i b i t o r y a c t i o n at 1 0 .0 p . p . m . a t 2 4 ° C. a n d 3 0 ° C . A t th is pH, A c t i- d io n e w as m o s t e ffe c tiv e The f o r m e r t e m p e r a t u r e w a s m o s t e f ­ f e c t i v e in 0 . 5 p . p . m . and 2 .5 p . p . m . w h i l e th e l a t t e r w a s m o s t e ffe c tiv e at a ll h ig h er c o n c e n tr a tio n s. N o t e w o r t h y i s th e s t i m ­ u l a t o r y e f f e c t o f th e A c t i - d i o n e in 0 .5 p . p . m . at 26 and 3 0 ° C. C ., 28 C. N o o t h e r s t i m u l a t i o n i s n o t e d a t a n y o f th e h i g h e r c o n c e n tr a tio n s of A c t i- d io n e . C o^c 84 100 90 80 of 60 50 P ercen tage In h ib itio n 70 40 30 26 F ig u r e 32. R a m u la ria 20 sp. 10 0 .5 2 .5 5 10 25 C o n c e n tr a tio n o f A c t i- d io n e in p .p .m . 50 75 85 pH 6 .0 . T h e m o s t s t r i k i n g d i f f e r e n c e n o t e d h e r e i s the v e r y c o n sp ic u o u s s o l i t a r y p e a k o f i n h ib it i o n a t 2 2 ° C. in 2 .5 p . p . m . an d th e f a c t t h a t a t 30 fectiv e (F ig u r e 33). o C . A c t i - d i o n e i s th e l e a s t e f ­ In c o n n e c t i o n w ith t h i s o b s e r v a t i o n i t is i n t e r e s t i n g t h a t in th e c o n t r o l r e s u l t s th e l e a s t a m o u n t o f g e r m i n a t io n o c c u r r e d at 30 i s th e a b s e n c e o C . an d t h e m o s t a t 22 o C. N otew orthy of th e v e r y e r r a t i c p a t t e r n o b s e r v e d in t h e p r e ­ v i o u s e x p e r i m e n t s w it h t h is o r g a n i s m . T he o n ly s tim u la tio n of s p o r e g e r m i n a t i o n o b s e r v e d h e r e w a s in 0 . 5 p . p . m . at 2 6 ° C. and 3 0 ° C. p H 6 .8 . T he m o s t n o t e w o r t h y o b s e r v a t i o n in t h i s se r ie s o f e x p e r i m e n t s w a s th e v e r y h i g h i n h i b i t o r y a c t i o n o f A c t i d io n e a t th e l o w c o n c e n t r a t i o n o f 2.5 p . p . m . and th e i m m e d i a t e r e d u c t i o n of i n h i b i t i o n a t the n e x t h i g h e r c o n c e n t r a t i o n u s e d - 2 .5 p . p . m . A s in a l l p r e v i o u s e x p e r i m e n t s , th e o n ly s t i m u l a ­ t o r y a c t i o n o c c u r r e d a t 2 6 ° C . a n d 30 C . in 0 . 5 p . p . m . To b e n o t e d i s th e e x t r e m e i n f l u e n c e of t e m p e r a t u r e u pon t h e e f ­ f e c t o f A c t i - d i o n e in i t s i n h i b i t o r y a c t i o n a s d e m o n s t r a t e d b y th e g r a p h l i n e r e p r e s e n t i n g 2 2 ° C. and a n y o f th e o t h e r t e m ­ p e r a t u r e s at any g iv e n c o n c e n tr a tio n of A c t i- d io n e . 86 100 - F ig u r e 33. R a m u la r ia sp. 80 of 50 P ercentage I n h ib it io n 70 40 30 28 20 .24 22 10 0 .5 2 .5 5 10 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 75 87 F ig u r es 3 5, 3 6 , 3 7, 38 a n d 39 p r e s e n t th e s a m e d a ta d i s c u s s e d a b o v e but in a d i f f e r e n t m a n n e r . The attem p t is m ade to s h o w the r e l a t i o n s h i p s an d d i f f e r e n c e s b e t w e e n e a c h pH v a l u e a t e a c h of th e t e m p e r a t u r e s u s e d in t h i s s t u d y . T e m p e r a tu r e 22 of A c ti-d io n e e x c e p t 6 .0 . o C. H ere th ere are stim u la to ry e ffe c ts in 0 .5 p . p . m . a t e v e r y h y d r o g e n io n c o n c e n t r a t i o n T h e l a r g e s t a m o u n t o f s t i m u l a t i o n i s in 0 .5 p . p . m . at p H 5 f o l l o w e d b y p H 4 . 0 . A l s o to b e n o t e d i s t h e s t i m u l a ­ t io n o f g e r m i n a t i o n i n 2 5 .0 p . p . m . at e a c h an d e v e r y h y d r o g e n io n c o n c e n t r a t i o n . From 5.0 p . p . m . a n d h i g h e r th e a m o u n t o f i n h i b i t i o n a t h y d r o g e n io n c o n c e n t r a t i o n s o f 6 . 8 , 5 .0 an d 5.0 a l m o s t p a r a l l e l e a c h o t h e r u n t i l a t 1 0 0 .0 p . p . m . t o t a l i n h ib it i o n a re the r e s u lt s fo r a ll th r e e . T o t a l in h ib i t i o n o f g e r m i n a t i o n w a s a t 7 5 .0 p . p . m . a t p H 4 .0 a n d 3 .0 . T e m p e r a t u r e 2 4 ° C. T h e b e s t r e s u l t s in in h ib it in g th e g e r m i n a t i o n o f s p o r e s w e r e a t t a in e d a t p H 6 .8 a l m o s t at e v e r y c o n c e n t r a t i o n o f A c t i - d i o n e up to 5 0 .0 p . p . m . ( F i g u r e 3 6 ). The n e x t b e s t p H v a l u e , a c c o r d i n g to th e r e s u l t s , w a s p H 5 .0 . T hree o f t h e l i n e s r e p r e s e n t i n g h y d r o g e n io n c o n c e n t r a t i o n s m e e t a t 7 5 .0 p . p . m . to i n d i c a t e th e m a x i m u m a m o u n t o f in h ib it i o n a n d th e o t h e r tw o m e e t a t 1 0 0 .0 p . p . m . 88 100 90 80 of 60 50 P e r c e n ta g e I n h ib itio n 70 40 30 , 26 R a m u l a r i a sp. 20 22 pH 6 . 8 . 10 0 .5 2 .5 5 10 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 75 89 100 80 of 50 P e r c e n ta g e I n h ib itio n 70 40 30 4 .0 F ig u r e 35. 20 R a m u la r ia sp. 22 10 0 .5 2 .5 5 10 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 75 C. 90 « // 91 100 F ig u r e 37. R a m u la r ia sp. 90 80 of 60 50 P e r c e n ta g e I n h ib itio n 70 40 30 20 10 -4 .0 -3 .0 "5.0 0 .5 2 .5 5 10 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 75 92 100 F ig u r e 3a. R a m u la r ia sp. 80 of 50 P ercentage In h ib itio n 70 40 30 20 10 3 .0 4 .0 6.0 5 .0 6.8 0 .5 2 .5 5 10 25 C o n c e n tr a tio n o f A c t i- d io n e in p .p .m . 50 75 \ -J O' 94 D e f i n i t e s t i m u l a t i o n o c c u r r e d in 0 .5 p . p . m . a t e v e r y pH e x c e p t 3.O.. A t p H 6 .8 t h e r e w a s e v i d e n t a v e r y m a r k e d s t i m u ­ l a t i o n in 0 . 5 p . p . m . o f A c t i - d i o n e . A d d itio n a l stim u la tio n s o c ­ c u r r e d a t 2 .5 p . p . m . , 5.0 p . p . m . a n d 2 5 .0 p . p . m . H e r e th ere i s the c o m p l e t e a b s e n c e o f s t i m u l a t i o n at p H 6 .0 . T e m p e r a t u r e 26 o C. T he m o s t n o t e w o r t h y d i f f e r e n c e a t t h i s t e m p e r a t u r e w a s th e w id e d i f f e r e n c e i n c o n c e n t r a t i o n of A c t i - d i o n e r e q u i r e d in e f f e c t i n g 30% o r m o r e in h ib it io n , e . g . , a t p H 6 .0 o n l y 0 .5 p . p . m . o f A c t i - d i o n e w a s r e q u i r e d at pH 3 .0 . A v e r y s l i g h t s t i m u l a t i o n o c c u r r e d a t pH 6 .8 in 0 .5 p . p . m . a n d a v e r y m a r k e d s t i m u l a t i o n at p H 5.0 in th e s a m e c o n c e n t r a t i o n of A c t i - d i o n e . 5 .0 ( F i g u r e In f a c t the l i n e r e p r e s e n t i n g p H 37) i n d i c a t e d 0% in h i b i t i o n o r t o t a l g e r m i n a t i o n . A l s o a r a t h e r s i g n i f i c a n t a m o u n t o f s t i m u l a t i o n in 2 5 .0 p .p . m . o f A c t i - d i o n e at t h e pH c o n c e n t r a t i o n s of 4 . 0 and 3 . 0 . To b e n o t e d i s th e f a c t t h a t a l l b u t on e o f th e p H g r a p h l i n e s t e r m ­ in a t e a t 1 0 0 .0 p . p . m . ; p H 6 .0 i s t h e o n ly l i n e t e r m i n a t i n g at 7 5 .0 p . p . m . o f A c t i - d i o n e c o n c e n t r a t i o n s . . 95 T em p eratu re 28° C. The p r in c ip a l d iffe r e n c e b e tw e e n t h is a n d th e p r e v i o u s t e m p e r a t u r e i s th a t a ll p H g r a p h l i n e s t e r m i n a t e a t 7 5 .0 p . p . m . ( F i g u r e 3 8 ). In a d d it io n a h i g h e r p e r ­ c e n t a g e o f i n h i b i t i o n i s a t t a i n e d a t p H 6 .8 th a n p r e v i o u s l y in 0 .5 p . p . m . And th e r e is p .p .m . of A c t i- d io n e and 6 . 0 . s t i m u l a t i o n of g e r m i n a t i o n in 0 . 5 a t th e p H c o n c e n t r a t i o n s of 3 . 0 , 4 . 0 , 5.0 A l s o t h e r e w e r e m a r k e d s t i m u l a t i o n s o c c u r r i n g at 5 .0 p . p . m . a t e v e r y h y d r o g e n io n c o n c e n t r a t i o n b u t o n e a n d at 2 5 .0 p . p . m . a t p H 4 . 0 and p H 3 .0 . A c ti-d io n e w a s m o st e f ­ f e c t i v e h e r e a t p H 6 . 8 a s in th e p r e v i o u s e x p e r i m e n t s . T em p eratu re 3 0 ° C. A s n o t e d in th e p r e v i o u s t h r e e t e s t s th e r e s u l t s o f th e c o n t r o l s p a r a l l e l e a c h o t h e r in a r r a n g e ­ m e n t o n th e g r a p h s , i . e . , th e l a r g e s t a m o u n t o f g e r m i n a t i o n w a s o b s e r v e d a t pH 3 .0 an d th e l e a s t a t p H 6 .8 . T h e r e s u l t s of a l l o t h e r p H v a l u e s f o l l o w e d in s e q u e n c e . O t h e r th a n t h e s i m i l a r i t y in th e c o n t r o l r e s u l t s , th e g e n e r a l p a ttern of b e h a v io r is s i m i l a r a t t h is h y d r o g e n io n c o n c e n t r a t i o n a s in th e p r e v i o u s t e s t . A n o t h e r e x c e p t i o n to be n o t e d i s th e a b s e n c e o f th e m a r k e d s t i m u l a t i o n s o c c u r r i n g in 2 5 .0 p . p . m . o f A c t i - d i o n e . 96 T A B L E IV IN H IB IT IO N O F S P O R E G E R M IN A T IO N O F R A M U L A R IA S P . B Y D IF F E R E N T C O N C E N T R A T IO N S O F A C T I-D IO N E A T F I V E D I F F E R E N T T E M P E R A T U R E S A N D F IV E H Y D R O G E N ION C O N C E N T R A T I O N S T em ­ pera­ tr\u r e s ( C oncen­ tr a tio n of A c ti-d io n e in p . p . m . PH 3 .0 pH 4 .0 pH 5.0 22 C ontrol 0 .5 2 .5 5.0 1 0 .0 2 5 .0 5 0 .0 7 5 .0 11.0 1 1 3 .1 6 2 7 .8 3 9 4 .4 4 11.7 1 5 6 .0 0 1 0 0 .0 0 1 0 0 .0 0 6 .0 6 8 .9 5 1 9 .0 0 3 5 .4 8 2 .0 5 3 2 .4 0 5 0 .2 5 1 0 0 .0 0 3 .2 5 10.50 2 0 .0 0 4 0 .9 5 5 .2 6 4 5 .0 0 59.51 1 0 0 .0 0 24 C ontrol 0 .5 . 2 .5 5 .0 1 0 .0 2 5 .0 5 0 .0 7 5 .0 1 0 .0 0 3 .9 5 3 0 .6 3 8 2 .3 0 5 .3 6 4 2 .0 0 1 0 0 .0 0 1 0 0 .0 0 8 .9 2 6 .4 8 4 2 .5 5 6 7 .0 0 1 6 .3 7 5 5 .5 4 8 6 .1 2 1 0 0 .0 0 7 .0 5 1 4 .8 6 49.21 4 5 .3 0 5 4 .1 0 69 .0 0 C on trol 0 .5 2 .5 • 5.0 1 0 .0 2 5 .0 5 0 .0 10.71 1 5 .6 0 1 2 .5 5 1 3 .5 2 2 1 .1 0 C.) 26 P e r c e n t a g e of I n h ib it io n 2 6 .0 9 4 5 .8 7 8 0 .9 1 3 8 .1 0 1 0 0 .0 0 3 3 .0 9 57.81 6 2 .1 5 7 7 .0 0 PH 6.0 PH 6 .8 2.5 0 1.82 2.00 2 .7 0 6 9 .4 6 9 8 .0 0 13.71 ■ 1 5 .9 3 26.21 3 6 .9 4 7 0 .2 7 35 .0 9 70 .2 5 9 7 .2 5 1 0 0 .0 0 1 0 0 .0 0 8 3 .9 9 1 0 0 .0 0 3.51 4 .0 5 8 .1 2 6 .5 3 11.5 6 2 1 .1 5 6 7 .2 8 1 0 0 .0 0 0 .9 2 2 .7 0 5.61 5.61 10.71 2 6 .1 3 7 2 .0 7 1 0 0 .0 0 9 .12 3.00 16.41 14.20 3 7 .2 8 57 .8 0 61 .0 0 4 .6 8 2 .7 5 8.5 6 9 .1 3 1 5 .1 7 3 4 .0 6 6 0 .2 4 5.50 1.77 6.96 5 .4 5 1 2 .7 3 2 4 .3 2 70.91 97 T A B L E IV (Continued) T em ­ pera­ tu res n ( C.) C oncen­ t r a t i o n of A c ti-d io n e in p . p . m . 28 C on trol 0 .5 2 .5 5 .0 1 0 .0 2 5 .0 5 0 .0 7 5 .0 4 5 .1 9 7 1 .1 7 1 0 .1 9 3 5 .2 5 1 0 0 .0 0 1 0 0 .0 0 C on trol 0 .5 2 .5 11.1 1 1 8 .5 2 2 8 .1 8 S '° 1 0 .0 2 5 .0 5 0 .0 7 5 .0 2 9 .9 1 1 2 .8 4 6 3 .0 0 1 0 0 .0 0 1 0 0 .0 0 30 P e r c e n t a g e o f I n h i b i t io n PH 3.0 1 0 .0 0 1 5 .0 5 PH 4 .0 PH 5.0 PH 6 .0 PH 6 .8 9 .3 0 4..18 2 4 .5 0 3 9 .2 8 2 6 .1 4 4 7 .8 8 1 0 0 .0 0 1 0 0 .0 0 4 .1 6 2 .5 0 1 4 .6 7 2 5 .0 0 3 7 .2 8 6 0 .5 2 7 0 .3 4 1 0 0 .0 0 5.51 9.0 1 2 2 .5 1 3 4 .1 3 4 2 .2 7 7 8 .2 0 1 0 0 .0 0 1 0 0 .0 0 2 .0 5 3 .1 5 3 .5 0 8 .5 7 1 7 .1 3 2 2 .2 8 6 0 .1 0 1 0 0 .0 0 7 .1 2 1 9 .0 0 3 1 .0 0 4 9 .8 2 4 6 .2 2 65.11 1 0 0 .0 0 1 0 0 .0 0 - 5.41 4 .0 2 4 0 .2 8 7 0 .5 0 7 5 .0 3 8 1 .9 9 95 .2 1 1 0 0 .0 0 7 .2 5 2 .0 2 7 .1 3 2 .7 0 1 .7 9 2 .7 0 7 .3 4 4 .4 2 9 .7 3 6 8 .4 2 1 0 0 .0 0 4 .5 9 1 0 .3 2 1 8 .0 0 5 7 .4 5 1 0 0 .0 0 98 H u m id i t y E x p e r i m e n t s E a c h o r g a n i s m w a s t e s t e d in t h r e e d i f f e r e n t r e l a t i v e h u m i d i t i e s (98%, 95%, 93%) a t t h e h y d r o g e n i o n c o n c e n t r a t i o n s u s e d in the p r e v i o u s e x p e r i m e n t s . in a t e in 93% r e l a t i v e h u m i d i t y , The s p o r e s fa ile d to g e r m ­ s o r e s u l t s w e r e o b t a in e d in th e 98% a n d 95% r e l a t i v e h u m i d i t y t e s t s . In 98% h u m id it y the g e r m t u b e s o f a l l o r g a n i s m s w e r e c o n s i d e r a b l y s h o r t e r , c o n s i d e r a b l y t h in n e r a n d m o r e i r r e g u l a r th a n t h o s e g e r m i n a t e d d i r e c t l y in liquid.- T h e g e r m t u b e s in 95% w e r e e v e n s h o r t e r and q u ite i r r e g u l a r . A c o n sid e r a b le a m o u n t o f p l a s m o l y s i s w a s e v i d e n t in b o th g e r m t u b e s and u n ­ g e r m i n a t e d s p o r e s a f t e r 24 h o u r s . N o d i s o r g a n i z a t i o n of c y t o ­ p l a s m w a s n o t e d at t h e l o w e r c o n c e n t r a t i o n s o f A c t i - d i o n e in any o r g a n is m . S o m e b r e a k d o w n c o u ld b e o b s e r v e d a t th e h i g h e r c o n cen tra tio n s. C la d o s p o r iu m C u c u m e rin u m O n ly th e r e s u l t s f r o m th e 98% r e l a t i v e h u m id it y t e s t w i l l b e p r e s e n t e d s i n c e no g e r m i n a t i o n w a s o b s e r v e d a t 95%. The b e s t i n h ib it i o n w a s fo u n d in 1 0 .0 p . p . m . o f A c t i - d i o n e at pH 4 , 0 . N o a n o m a l i e s w e r e o b s e r v e d a s w it h s o m e o f the o t h e r 99 s p o r e s w h e n i m m e r s e d in s o l u t i o n ( F i g u r e 4 0 ). The p o o r e s t r e s u l t s w e r e o b t a i n e d a t p H 6 .8 a n d s t i m u l a t i o n o f g e r m i n a t i o n o c c u r r e d o n l y at p H 4 .0 in 0 .5 p . p . m . B o tr y tis C in e re a 98% r e l a t i v e h u m i d i t y . T h e b e s t in h ib it i o n w a s o b s e r v e d in 5 0 .0 p . p . m . of A c t i - d i o n e an d th e n e x t b e s t i n 1 0 ,0 p . p . m . at p H 6 .8 ( F i g u r e 4 1 ) . 5 .0 T h e p o o r e s t r e s u l t s w e r e o b t a in e d a t p H in 0 . 5 p . p . m . a n d a t p H 3 ,0 a t the h i g h e r c o n c e n t r a t i o n s of A c ti-d io n e . T h is p a t t e r n o f b e h a v i o r a s c o m p a r e d to th e C la d o - sp o r iu m , w a s so m e w h a t a n o m a lo u s. In a d d it io n t h e r e w a s som e s t i m u l a t i o n o f g e r m i n a t i o n in 0 .5 p . p . m . a t pH c o n c e n t r a t i o n s of 4 .0 , 5 .0 and 6 .8 . A b n o r m a l i t i e s o b s e r v e d , c o m p a r e d w ith th e c o n t r o l s , w e r e c o n f i n e d to th e g e r m t u b e s and u n g e r m i n a t e d s p o r e s .- The a b n o r m a l i t i e s w e r e th e s a m e a s l i s t e d p r e v i o u s l y f o r t h i s o r ­ g a n ism . In a d d it io n to t h i s , th e g e r m t u b e s w e r e v e r y th in and q u ite s h o r t . 95% r e l a t i v e h u m i d i t y . The p r in c ip a l d iffe r e n c e b e tw e e n t h e s e r e s u l t s and a t 98% r e l a t i v e h u m id it y i s th a t a m o r e o r 100 100 4 .0 90 80 5 .0 of 60 50 P ercentage I n h ib itio n 70 40 30 F ig u r e 40. C la d o sp o r iu m c u c u m e r in u m 98% h u m id it y . 20 10 0 .5 2 .5 5 10 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 101 100 80 70 - • I / 40 — 3 .0 5 .0 20 F ig u r e 41. B o tr y tis c in e r e a 98% h u m id it y . 0 .5 2 .5 5 10 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 102 l e s s b e t t e r i n h i b i t i o n r e s u l t e d at a g i v e n c o n c e n t r a t i o n o f A c t i d io n e a t a g i v e n p H c o n c e n t r a t i o n ( F i g u r e 4 2 ). M o n ilin ia F r u c t i c o la 98% r e l a t i v e h u m i d i t y . m id ity a r e T h e r e s u l t s at t h is r e l a t i v e h u ­ s t r i k i n g l y s i m i l a r to t h o s e o b t a in e d w h e r e the s p o r e s w e r e i m m e r s e d d i r e c t l y i n th e l i q u i d A c t i - d i o n e . The p r in c ip a l d i f f e r e n c e i s th a t a h i g h e r p e r c e n t of in h ib it i o n w a s fo u n d in a g i v e n A c t i - d i o n e c o n c e n t r a t i o n and pH th a n in the d i r e c t i m m e r ­ sio n e x p e r im e n t s . T h e d i s t o r t i o n an d o t h e r m o r p h o l o g i c a l a b ­ n o r m a l i t i e s th a t w e r e p r e s e n t in th e d i r e c t i m m e r s i o n e x p e r i ­ m e n t s w e r e a l s o p r e s e n t h e r e , p l u s the v e r y th in g e r m t u b e s t h a t w e r e o b s e r v e d in th e c a s e of th e B o t r y t i s o r g a n i s m . T h e m o s t f a v o r a b l e p H w a s 6 .8 in 0 .5 p . p . m . and 1 0 .0 p .p .m . T h e p o o r e s t o r m o s t u n f a v o r a b le p H w a s 3 .0 ( F i g u r e 43). 95% r e l a t i v e h u m i d i t y . The r e s u lts at t h is p H a r e v e r y s i m i l a r to the a b o v e , e x c e p t f o r a s l i g h t d i f f e r e n c e in a m o u n t o f i n h ib it i o n e x h i b i t e d at 2 .5 p . p . m . In th e p r e v i o u s t e s t t h e l o w e s t in h i b i t i o n w a s a t t a i n e d at p H 3.0 w h e r e a s h e r e th e l o w e s t 103 100 I— 90 80 70 4 .0 6.8 60 3.0 50 40 6.0 / 30 F ig u r e 42. 20 B o tr y tis c in e r e a 95% h u m id it y . 10 0 .5 2 .5 J 5 L 10 _L 25 < C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 104 100 90 60 5 .0 4 .0 of 60 50 P ercen tage I n h ib itio n 70 40 3 .0 30 F ig u r e 43. M o n il in ia F r u c t i c o l a 98% h u m id it y . 20 10 0 .5 2 .5 5 10 25 C o n c e n tr a tio n o f A c t i- d io n e in p .p .m . 50 i s a t p H 4 .0 ( F i g u r e 4 4 ) . A l s o to b e n o t e d h e r e i s t h e a b s e n c e of g e r m in a tio n at pH 6 .8 . R a m u l a r i a Sp. 98% r e l a t i v e h u m i d i t y . The h i g h e s t p e r c e n t o f in h ib it io n a t t h is r e l a t i v e h u m i d i t y w a s o b s e r v e d in 2 .5 p . p . m . a t pH 6 .8 and a g a i n at a l l h y d r o g e n io n c o n c e n t r a t i o n s in 50 p . p . m . ( F i g ­ u re 45). N o s t i m u l a t i o n s w e r e o b s e r v e d i n 0 .5 p . p . m . a s in p rev io u s e x p e r i m e n t s , b u t a c o n s i d e r a b l e r e d u c t i o n o f in h ib it io n w a s o b s e r v e d in 5 .0 and 1 0 .0 p . p . m . of A c t i - d i o n e . A c tu a lly , t h e r e w a s a s l i g h t s t i m u l a t i o n in 1 0 .0 p . p . m . at pH 3 .0 and 4 . 0 . 93% r e l a t i v e h u m i d i t y . The h y d r o g e n - i o n c o n c e n t r a t i o n m o s t f a v o r a b l e in t h i s r e l a t i v e h u m id it y w a s 6.8 in th e A c t i d io n e c o n c e n t r a t i o n o f 2 .5 p . p . m . ( F i g u r e 4 6 ) . T o t a l in h ib it i o n o f g e r m i n a t i o n o c c u r r e d a t th e s a m e pH b u t at 50 p . p . m . n e x t m o s t f a v o r a b l e h y d r o g e n io n c o n c e n t r a t i o n w a s A c t i - d i o n e c o n c e n t r a t i o n o f 5 .0 p . p . m . The 3 .0 in th e The c o m p le x b eh a v io r p a t t e r n o f t h is o r g a n i s m u n d e r th e v a r i o u s t r e a t m e n t s at t h is r e l a t i v e h u m id it y w a s n o t i c e a b l e . T here a r e a nu m ber of r e ­ d u c t i o n s o f in h i b i t i o n in c o n c e n t r a t i o n s w h e r e n o r m a l l y an i n ­ crease o f in h ib i t i o n of s p o r e g e r m i n a t i o n w a s e x p e c t e d . T hese 106 100 6.0 90 5 .0 4 .0 80 3 .0 of 60 50 P ercentage I n h ib itio n 70 40 F ig u r e 44. M o n il in ia F r u c t i c o l a 30 95% h u m id it y . 20 10 0 .5 2 .5 5 10 25 C o n c e n tr a tio n of A c t i- d io n e in p .p .m . 50 107 80 P ercen tage of I n h ib itio n 70 50 3 .0 40 4 .0 5 .0 30 20 F ig u r e 45. 10 R a m u l a r i a sp. 98% h u m id it y . 0 .5 2 .5 5 10 25 C o n c e n t r a t i o n of A c t i - d i o n e in p . p . m . 50 108 100 |- 90 80 70 In h ib itio n 3.0 4 .0 60 of 5 .0 50 P ercentage 3 6.0 6.5 40 30 F ig u r e 46. 20 R a m u la r ia sp. - 95% h u m id it y . 10 0.5 J 2 .5 L 5 10 I 25 C o n c e n t r a t i o n of A c t i - d i o n e in p . p . m . I 50 109 a n o m a l i e s w e r e o b s e r v e d a t 5 .0 and 1 0 .0 p . p . m . c o n c e n t r a t i o n s . S t i m u l a t i o n s o f s p o r e g e r m i n a t i o n a p p e a r in 0-. 5 p . p . m . a t pH 3 .0 , 4 . 0 and- 5 . 0 . F u r t h e r s t i m u l a t i o n s w e r e o b s e r v e d in 10.0 p . p . m . a t p H 4 . 0 and 5 . 0 . 110 TABLE V IN H IB IT IO N O F S P O R E G E R M IN A T IO N O F C L A D O S P O R IU M C U C U M E R IN U M B Y D I F F E R E N T C O N C E N T R A T IO N S O F A C T I - D I O N E A T F I V E D I F F E R E N T H Y D R O G E N ION C O N C E N T R A T I O N S A N D TWO R E L A T I V E H U M ID IT IE S A T 2 0 ° C. R e la tiv e H u m id ­ it y (%) 98 C oncen­ tr a tio n of A c ti-d io n e in p . p . m . 0 .0 0 .5 2 .5 5.0 1 0 .0 2 5 .0 5 0 .0 P e r c e n t a g e o f In h ib itio n pH 3 .0 pH 4 .0 PH 5 .0 pH 6.0 PH 6 .8 No G er­ m in a ­ t io n 94 .5 1 86 .6 1 9 0 .8 4 9 7 .3 8 1 0 0 .0 0 1 0 0 .0 0 1 0 0 .0 0 6 3 .4 5 7 6 .4 2 8 4 .4 9 8 8 .9 5 9 4 .2 8 9 8 .7 8 1 0 0 .0 0 3 5 .8 4 5 4 .6 5 6 1 .3 0 7 1 .4 5 9 3 .2 8 9 6 .1 0 1 0 0 .0 0 3 3 .0 0 4 0 .0 9 4 9 .3 1 5 2 .7 5 8 8 ,3 2 9 2 .2 0 1 0 0 .0 0 Ill T A B L E VI IN H IB IT I O N O F S P O R E G E R M IN A T IO N O F B O T R Y T IS C I N E R E A BY D I F F E R E N T C O N C E N T R A T IO N S O F A C T I-D IO N E A T F IV E D IF F E R E N T HYDROGEN ION C O N C E N T R A T I O N S A N D TWO R E L A T I V E H U M ID IT IE S A T 2 0 ° C. R e la tiv e H u m id ­ it y (%) C oncen­ t r a t i o n of A c ti-d io n e in p . p . m . PH 3.0 . PH 4 .0 PH 5.0 PH 6.0 PH 6 .8 98 0 .0 0 .5 2 .5 5 .0 1 0 .0 2 5 .0 5 0 .0 3 2 .0 0 3 3 .2 6 4 1 .0 0 5 3 .8 5 65 .1 0 6 0 .1 2 1 0 0 .0 0 4 4 .1 8 4 2 .3 5 4 4 .9 2 6 1 .6 5 6 6 .5 0 6 6 .0 0 1 0 0 .0 0 2 4 .0 0 1 1 .0 0 3 2 .5 0 4 4 .3 5 7 5 .1 6 6 9 .1 2 1 0 0 .0 0 2 7 .5 0 4 5 .3 2 55.21 7 0 .3 0 8 4 .7 6 8 4 .0 0 1 0 0 .0 0 4 3 .0 0 3 0 .5 2 3 9 .2 1 7 9 .9 9 9 4 .6 5 8 3 .6 0 1 0 0 .0 0 95 0 .0 0 .5 2 .5 1 0 .0 2 5 .0 5 0 .0 5 0 .4 9 57 .0 0 6 1 .0 0 7 6 .7 8 6 6 .0 0 1 0 0 .0 0 7 0 .6 2 5 8 .1 3 6 3 .5 0 78 .9 0 7 3 .1 2 1 0 0 .0 0 4 8 .5 1 3 6 .2 3 2 4 .2 5 7 8 .8 5 7 6 .2 2 1 0 0 .0 0 4 1 .0 0 6 4 .3 9 6 9 .8 0 8 7 .7 8 8 6 .0 0 1 0 0 .0 0 6 6 .2 2 3 8 .6 0 4 5 .2 7 9 2 .0 0 8 8 .3 4 1 0 0 .0 0 P e r c e n t a g e o f I n h ib itio n I 112 T A B L E VII IN H IB IT IO N O F S P O R E G E R M IN A T IO N O F M O N IL IN IA F R U C T I C O L A B Y D I F F E R E N T C O N C E N T R A T IO N S O F A C T I - D I O N E A T F I V E D I F F E R E N T H Y D R O G E N IO N C O N C E N T R A T IO N S A N D TWO R E L A T I V E H U M ID IT IE S A T 2 0 ° C. R e la tiv e H u m id ­ i t y (%) 98 C oncen­ t r a t i o n of A c ti-d io n e in p . p . m . 0 .0 0 .5 2 .5 5 .0 1 0 .0 0 .0 0 .5 2 ,5 5 .0 P e r c e n t a g e of I n h ib itio n PH 3.0 4 2 .2 5 4 7 .8 7 6 2 .5 0 7 7 .2 0 1 0 0 .0 0 . 7 4 .3 5 7 5 .9 8 8 3 .2 5 9 2 .3 0 PH 4 .0 PH 5.0 PH 6.0 PH 6 .8 6 3 .3 5 6 5 .1 8 6 3 .7 5 8 9 .8 2 1 0 0 .0 0 8 0 .8 5 8 3 .4 6 8 0 .0 4 8 4 .9 0 1 0 0 .0 0 9 0 .1 3 9 1 .0 0 8 7 .5 0 9 7 .0 0 1 0 0 .0 0 94.00 9 7 .3 5 8 9 .2 3 98.19 1 0 0 .0 0 8 1 .8 2 8 3 .2 8 8 0 .7 6 9 3 .6 0 88 .0 9 9 0 .8 5 8 9 .9 0 9 7 .4 5 98 .0 0 9 9 .4 8 9 7 .3 5 99 .5 0 113 T A B L E VIII IN H IB IT IO N O F S P O R E G E R M IN A T IO N O F R A M U L A R IA S P B Y D I F F E R E N T C O N C E N T R A T IO N S O F A C T I - D I O N E A T F I V E D I F F E R E N T H Y D R O G E N ION C O N C E N T R A T IO N S A N D TWO R E L A T I V E H U M ID IT IE S A T 2 0 ° C. R e la tiv e H u m id ­ C oncen­ t r a t i o n of A c ti-d io n e in p . p . m . P e r c e n t a g e of I n h ib itio n PH 3 .0 pH 4 .0 pH 5 .0 PH 6 .0 PH 6 .8 98 0 .0 0 .5 . 2 .5 5.0 10 .0 2 5 .0 5 0 .0 4 6 .0 0 4 6 .5 5 5 4 .7 2 8 9 .0 0 5 8 .5 0 8 2 .2 5 1 0 0 .0 0 3 9 .8 8 4 3 .1 0 4 5 .1 2 5 5 .4 9 3 4 .1 5 4 8 .1 1 1 0 0 .0 0 35 .3 1 3 7 .2 2 4 1 .0 0 59.7 1 35.01 5 0 .1 8 1 0 0 .0 0 3 0 .5 2 3 8 .2 0 7 0 .0 2 5 0 .1 5 6 1 .4 6 6 5 .1 2 1 0 0 .0 0 2 8 .0 0 3 3 .2 5 9 9 .5 0 5 0 .0 1 7 1 .4 5 8 4 .5 0 1 0 0 .0 0 95 0 .0 0 .5 2.5 5 .0 0 10.0 2 5 .0 5 0 .0 6 7 .8 2 58.01 6 3 .7 6 9 1 .5 1 6 3 .4 2 8 4 .1 5 1 0 0 .0 0 6 4 .3 5 5 6 .1 5 5 8 .1 4 6 2 .1 8 4 8 .0 0 5 2 .0 0 1 0 0 .0 0 5 2 .6 5 5 1 .0 0 5 8 .1 4 6 4 .9 3 4 8 .8 9 5 3 .5 0 1 0 0 .0 0 4 5 .9 6 5 2 .1 8 7 1 .1 6 5 6 .4 9 6 5..90 7 4 .0 0 1 0 0 .0 0 4 2 .0 0 4 9 .3 3 9 7 .0 0 57 .2 1 6 9 .5 0 8 5 .0 0 1 0 0 .0 0 i t y (%) M ■ 'I D IS C U S S IO N T h e o b s e r v a t i o n s m a d e d u r in g t h is i n v e s t i g a t i o n in d i c a t e s e v e r a l p a t t e r n s o f b e h a v i o r b y th e o r g a n i s m s in r e s p o n s e to a n u m b e r o f t r e a t m e n t s b y A c t i - d i o n e u n d e r th e i n f lu e n c e of t e m ­ p e r a t u r e , p H and m o i s t u r e . A c t i - d i o n e w a s u s e d in c o n c e n t r a t i o n s f r o m 0 .5 p . p . m . to 1 0 0 .0 0 p ..p .m . A t the l o w e s t c o n c e n t r a t i o n , it w a s c o n s i s t e n t l y o b s e r v e d t h a t t h e r e w a s f r e q u e n t l y a s t i m u l a t o r y r a t h e r th a n an in h i b i t o r y e f f e c t u p o n s p o r e g e r m i n a t i o n . T h is i s q u ite a n o m a ­ l o u s to th e g e n e r a l p a t t e r n o f b e h a v i o r , o f th e f o u r o r g a n i s m s stu d ied , w h ich in d ic a te d th e te n d e n c y to w a rd a h ig h er p e r c e n ta g e o f in h ib i t i o n o f g e r m i n a t i o n a s th e c o n c e n t r a t i o n of th e A c t i d io n e w a s i n c r e a s e d . T h e s t i m u l a t i o n o c c u r r i n g in 0 .5 p . p . m . of A c ti-d io n e ranged fr o m 1.09% a t pH 4 .0 a t 24 o C . w ith B o t r y t i s to 14.41% a t p H 5 .0 at 2 6 ° C . w i t h th e s a m e o r g a n ­ ism . In th e e x p e r i m e n t s w it h C l a d o s p o r i u m , i t i s i n t e r e s t i n g to n o t e , th a t s t i m u l a t i o n o f g e r m i n a t i o n o c c u r r e d s i x t i m e s in 0 . 5 p ,.p .m ., f o u r o f t h e s e w e r e o b s e r v e d a t e v e r y h y d r o g e n io n c o n c e n t r a t i o n at 2 4 ° C. w h i l e th e o t h e r tw o o c c u r r e d o n ly a t p H 4 .0 a n d 5 .0 a t 3 0 ° C. S t i m u l a t i o n in 2 .5 p . p . m . w a s 115 c o n s i d e r a b l e l e s s p r e v a l e n t ; o n l y tw o o c c u r r e n c e s w e r e o b s e r v e d , o n e a t p H 5 .0 a t 2 4 ° C . an d th e o t h e r a t p H 5 .0 at 3 0 ° C . ( T a ­ b le I a n d F i g u r e 1); F r o m t h e s e r e s u l t s it i s o b v io u s th a t th e m a j o r i t y of s t i m u l a t i o n s o c c u r r e d at p H 5 .0 . T h ese r e su lts m a y t h e r e f o r e i n d i c a t e th a t th e c o n i d i a o f C l a d o s p o r i u m c u c u m e r i n u m a r e p r o n e to s t i m u l a t i o n at t h i s p H a t two d i f f e r e n t t e m p e r a t u r e s , 2 4 ° C. a n d 3 0 ° C. In the B o t r y t i s e x p e r i m e n t s s t i m u l a t i o n a t 0 .5 p . p . m . w a s c o n s i d e r a b l y m o r e p r o n o u n c e d th a n in t e s t s w ith C l a d o ­ sp o r iu m . A t o t a l o f 38 s t i m u l a t i o n s w e r e o b s e r v e d . S tim u ­ la t i o n w a s o b s e r v e d a t e v e r y t e m p e r a t u r e an d a t a l m o s t a ll h y d r o g e n io n c o n c e n t r a t i o n s ( T a b le II). A t o t a l o f 17 s t i m u l a ­ t io n s o c c u r r e d in 0 .5 p . p . m . a s c o m p a r e d to 6 w ith C_. c u c u m erin u m . Of f u r t h e r i n t e r e s t w a s t h e o b s e r v a t i o n th a t s o m e s t i m u l a t o r y e f f e c t s o c c u r r e d in A c t i - d i o n e c o n c e n t r a t i o n s a s h ig h a s 2 5 .0 p . p . m . ; t h e s e , o f c o u r s e , w e r e l e s s p r e v a l e n t . In c o n t r a s t to C . c u c u m e r i n u m , th e h y d r o g e n io n c o n c e n t r a t i o n s fa v o r in g stim u la tio n w e r e 3 . 0 , 4 .0 an d 6 .8 i n s t e a d o f p H 5.0 ( T a b le II). A d d itio n a l s tim u la to r y e f f e c t s w e r e m en ts o b s e r v e d in e x p e r i ­ w ith M o n ilin ia f r u c t i c o l a and R a m u la r ia sp . In t h e f o r m e r 116 o n l y 5 s t i m u l a t i o n s w e r e o b s e r v e d w h i l e in t h e l a t t e r t h e r e w e r e 15. A l l of th e 5 o c c u r r i n g in M. f r u c t i c o l a w e r e a t p H 3.0 a t o o ,o 2 2 , 24 a n d 26 C. ism s W h en c o m p a r e d w ith t h e o t h e r 3 o r g a n ­ M. f r u c t i c o l a w a s t h e l e a s t s u s c e p t i b l e to s t i m u l a t i o n b y e x p o s u r e to A c t i - d i o n e . In th e R a m u l a r i a s p . a s in t h e B o t r y t i s e x p e r i m e n t s , s t i m u l a t i o n o f g e r m i n a t i o n o c c u r r e d in e v e r y p H an d a t e v e r y t e m p e r a t u r e . The p r in c ip a l d iffe r e n c e , h o w ev er, i s th a t w i t h R a m u l a r i a th e g r e a t e s t s t i m u l a t o r y t e n d e n c y w a s a t p H 3 .0 , 4 .0 and 6 .8 w h e r e a s w it h B o t r y t i s it w a s a t pH 4 . 0 , 5 .0 and 6 . 0 . Of c o n s i d e r a b l e i n t e r e s t , w a s th e s i m i l a r i t y to the r e ­ s u l t s a b o v e in th e r e l a t i v e h u m id it y t e s t s ( T a b l e s V , VI, a n d V II). S t i m u l a t i o n w a s o b s e r v e d 27 t i m e s w i t h a ll f o u r o r g a n ­ i s m s in a l l h u m i d i t i e s an d h y d r o g e n io n c o n c e n t r a t i o n s . Of t h i s t o t a l, h o w e v e r , o n ly 10 o c c u r r e d at 0 . 5 p . p . m . an d 10 a t 2 .5 p . p . m . o f A c t i - d i o n e e x p e r i m e n t s , th e o t h e r s w e r e fou n d in A c t i - d i o n e c o n c e n t r a t i o n s up to 2 5 .0 0 p ^ p .m . Of a d d it io n a l i n t e r e s t is th e f a c t t h a t in th e h u m id it y t e s t s , th e C l a d o s p o r i u m s p o r e s w e r e to s t i m u l a t i o n th a n in th e p r e v i o u s sig n ific a n tly l e s s p ron e ex p erim en ts. A ls o a slig h t s t i m u l a t i o n w a s n o t e d h e r e a t p H 4 . 0 in 2 .5 p .p . m . th at w a s 117 a b s e n t in t h e p r e v i o u s t e s t s . B o tr y tis sp o r e s a lso resp ond ed to s t i m u l a t i o n in a s i m i l a r m a n n e r a s in th e p r e v i o u s t e s t s b u t in a l e s s e r 0 .5 p . p . m . am o u n t. M o s t of th e s t i m u l a t i o n o c c u r r e d at In the h u m i d i t y t e s t s w i t h t h is o r g a n i s m , t h e r e w a s a n a b s e n c e o f a n y s t i m u l a t i o n a t p H 3 .0 w h i c h w a s f a v o r a b l e to t h i s a c t i o n in th e d i r e c t i m m e r s i o n e x p e r i m e n t s . In the h u m i d i t y t e s t s th e M . f r u c t i c o l a s p o r e s w e r e no m o r e t i b l e to s t i m u l a t o r y a c t i o n th a n p r e v i o u s l y . su scep ­ It w a s i n t e r e s t i n g to o b s e r v e t h a t the s t i m u l a t i o n w h i c h d id o c c u r w a s n o t in 0 .5 p . p . m . o r a t p H 3 .0 , b u t a t p H 4 . 0 , 6 .0 an d 6 .8 in 2 .5 p .p . m . ( T a b le VII). M o s t o f th e s t i m u l a t i o n , in th e d i r e c t i m m e r s i o n t e s t s w it h R a m u l a r i a o c c u r r e d at p H 6 .0 ( T a b le VIII), th e c o m ­ p l e t e a b s e n c e o f s t i m u l a t i o n a t t h is p H in th e h u m id it y t e s t s i s n otew orth y. The s t i m u l a t i o n s w h i c h d id o c c u r w e r e fo u n d m o s t l y a t p H 4 .0 in 95% r e l a t i v e h u m id it y . In a d d it io n to th e s t i m u l a t o r y e f f e c t s o b s e r v e d , o th er o b serv a tio n s w e r e of in te r e s t. several A ll 4 o r g a n is m s w ere e x ­ p o s e d to A c t i - d i o n e a d j u s t e d to a l l o f th e h y d r o g e n i o n c o n c e n ­ t r a t i o n s a s d e s c r i b e d in th e M a t e r i a l s and M e th o d s s e c t i o n . S o m e g e r m i n a t i o n o c c u r r e d in e v e r y e x p e r i m e n t e x c e p t a t p H 3 .0 w it h t h e s p o r e s o f C. c u c u m e r i n u m . 118 T h e m i n i m u m l e t h a l d o s e a t w h i c h c o m p l e t e in h ib it i o n o c c u r r e d v a r i e d b e t w e e n o r g a n i s m s an d w a s d e f i n i t e l y i n f l u e n c e d to s o m e e x t e n t b y th e v a r i o u s t e m p e r a t u r e s a n d H io n c o n c e n ­ tra tio n s. In C. c u c u m e r i n u m th e l e t h a l d o s e v a r i e d w ith b o t h the p H a n d th e t e m p e r a t u r e , b u t in n o c a s e w a s i t m o r e t h a n 5 0 .0 0 p . p . m . ( T a b le I). A g la n c e at t h e s e r e s u lt s in d ic a te th at t e m ­ p e r a t u r e a n d p H h a d w h a t c o u ld b e t e r m e d a s y n e r g i s t i c e f f e c t , n e i t h e r o n e b e i n g a b le to w o r k i n d e p e n d e n t ly i n a c c o m p l i s h i n g th e s a m e e f f e c t s . G e n e r a l l y th e t e n d e n c y i n d i c a t e d w a s th a t a s th e p H w a s l o w e r e d an d th e t e m p e r a t u r e w a s r a i s e d l e s s A c t i - d i o n e w a s n e c e s s a r y to e f f e c t c o m p l e t e i n h ib it i o n . le th a l d o se v a r ie d fr o m 5 0 .0 p . p . m . to 5 .0 p . p . m . The There w ere d e f i n i t e e x c e p t i o n s to t h i s a s m a y be o b s e r v e d in T a b le I. T h e f a c t t h a t p l a s m o l y t i c d i s t o r t i o n of t h e s p o r e s and g e r m t u b e s w a s a b s e n t in t h is o r g a n i s m m a y b e due to th e sev era l fa ctors. It i s p o s s i b l e th a t th e c y t o p l a s m i c c o n t e n t of th e s p o r e s an d t h e i r g e r m t u b e s w a s in o r n e a r e q u i l i b r i u m w it h th e A c t i - d i o n e s o l u t i o n s in w h i c h t h e y w e r e i m m e r s e d . T r u e , in th e h u m i d i t y t e s t s t h e r e w a s n o i m m e r s i o n , b u t a f i l m o f h y g r o s c o p i c l i q u i d w h i c h c o n t a i n e d A c t i - d i o n e p l u s th e 119 n u t r i e n t s t i m u l a n t w a s p r e s e n t at a l l t i m e s , c o n s e q u e n t l y th e s a m e co n d itio n m a y have p r e v a ile d . In a d d itio n , i t i s p o s s i b l e t h a t th e s p o r e w a l l o f t h i s o r g a n i s m w a s o f s u c h a s t r u c t u r e a s to n o t a l l o w t h e f r e e p a s s a g e o u t w a r d o f th e c y t o p l a s m i c s a l t s , th u s p r e v e n t i n g p l a s m o l y s i s . A th ir d fa c to r w h ich m a y b e c o n s i d e r e d i s th e p o s s i b i l i t y th a t the c y t o p l a s m i c c o n c e n t r a ­ t i o n , in b o t h th e s p o r e s a n d the g e r m t u b e s , w a s s l i g h t l y l e s s t h a n th e A c t i - d i o n e s o lu t io n , so i n s t e a d o f p l a s m o l y s i s th e r e ­ v e r s e w a s th e r e s u l t , but n o t e n o u g h to c a u s e p l a s m o p t y s i s . T h e r e a s o n f o r m e n t i o n i n g t h is f a c t o r i s t h a t the o n l y a b n o r ­ m a l i t y o b s e r v e d i n th e r e s u l t s of t h i s o r g a n i s m w a s a t e n d e n c y for sh orter, s t o u t e r g e r m t u b e s w h i c h i n c r e a s e d w it h th e i n ­ c r e a s e in th e c o n c e n t r a t i o n o f A c t i - d i o n e . T h e g e n e r a l p a t t e r n o b s e r v e d in th e B o t r y t i s t e s t s w a s a t e n d e n c y to i n c r e a s e t o x i c i t y w it h the i n c r e a s e in A c t i - d i o n e c o n c e n t r a t i o n an d th e l o w e r i n g o f th e pH o f th e s o l u t i o n ( T a b le II). A n e x c e p t i o n to t h is w a s o b s e r v e d at p H 6 .8 a t 30 w h e r e t o t a l i n h i b i t i o n r e s u l t e d in 5 0 .0 p . p . m . C. A l s o h e r e , i t is o b v i o u s th a t t e m p e r a t u r e an d pH w o r k e d t o g e t h e r to b r in g about the o b s e r v e d r e s u l t s . In th e h u m id it y t e s t s w ith t h i s o r g a n i s m i t w a s o b s e r v e d th a t no s t i m u l a t i o n o c c u r r e d at p H 3.0 w h e r e a s 120 in t h e i m m e r s i o n t e s t s t h is p H d e f i n i t e l y f a v o r e d s t i m u l a t i o n . T h e r e a s o n f o r t h is i s u n k n o w n . B o th d i s t o r t i o n , a n d w h a t a p p e a r e d to b e c y t o p l a s m i c d i s o r g a n i z a t i o n , w a s o b s e r v e d i n t h is an d in th e s p o r e s o f M. fr u c tic o la . th ose The r e a s o n s fo r th is a re p o s s i b l y the r e v e r s e of s t a t e d f o r Cb c u e u m e r i n u m w h i c h e x h ib it e d n o p l a s m o l y t i c d isto rtio n . T h e w a l l s t r u c t u r e o f th e B o t r y t i s a n d th e M o n il in ia s p o r e s m a y b e c o n s t r u c t e d to a l l o w f r e e p a s s a g e o f w a t e r f r o m th e c y t o p l a s m ou t to a l e s s d e n s e l i q u i d w it h w h i c h th e s p o r e w a ll m a y b e su rro u n d ed . T h i s , o f c o u r s e , p r e s u p p o s e s the c o n c e n t r a t i o n o f th e w a t e r in th e c y t o p l a s m to b e h i g h e r th an th a t o f th e A c t i - d i o n e , o t h e r w i s e p l a s m o l y s i s w o u ld n o t tak e p la c e . ism s The m o s t s t r i k i n g d i f f e r e n c e b e t w e e n t h e s e tw o o r g a n ­ i s the a m o u n t o f s t i m u l a t i o n ( T a b l e s I and II). In B o t r y t i s n o t o n l y d id s t i m u l a t i o n o c c u r m o r e f r e q u e n t l y th a n it d id in M o n i l i n i a b u t a n u m b e r of s h a r p r e d u c t i o n s in a m o u n t of g e r m ­ i n a t i o n w e r e n o t e d w h e r e n o r m a l l y n o n e w o u ld b e e x p e c t e d . The r e s u l t s o f R a m u l a r i a s p . r e s e m b l e t h o s e o f Ck c u c u m e r i n u m i n th at th e u n g e r m i n a t e d s p o r e s in h i g h e r c o n c e n ­ t r a t i o n s of A c t i - d i o n e and th e g e r m i n a t e d in th e l o w e r c o n c e n ­ t r a t i o n s w e r e f r e e f r o m d i s t o r t i o n s an d c y t o p l a s m i c d i s o r g a n i z a t i o n . 121 O n th e o t h e r han d, A c t i - d i o n e w a s m o r e e f f e c t i v e a t a h ig h e r p H a g a i n s t CL c u c u m e r i n u m th a n R a m u l a r i a w h o s e m o s t e f f e c t i v e r e s p o n s e to th e A c t i - d i o n e w a s in pH 3 .0 ( T a b le IV). A noth er d i f f e r e n c e b e t w e e n th e tw o o r g a n i s m s i s th e a m o u n t o f s t i m u ­ l a t i o n o c c u r r i n g in 0 . 5 p . p . m . o f A c t i - d i o n e , i t w a s c o n s i d e r a b l y h i g h e r w i t h th e s p o r e s o f R a m u l a r i a . T h e p H w h ic h f a v o r e d t h e m o s t s t i m u l a t i o n s w a s pH 6 .0 , e v e n th o u g h s o m e s t i m u l a t i o n , a s w ith B o t r y t i s , o c c u r r e d w ith e v e r y pH a n d a t e v e r y t e m p e r a t u r e . T h e m o s t n o t e w o r t h y o b s e r v a t i o n in th e h u m i d i t y t e s t s i s th a t a lt h o u g h in m a n y i n s t a n c e s no c h a n g e w a s n o t e d in i n ­ h i b i t i o n a t a g i v e n A c t i - d i o n e c o n c e n t r a t i o n a n d a t a g i v e n pH , th e t e n d e n c y w a s t o w a r d h i g h e r i n h ib it i o n at 95% th a n a t 98% r e la t iv e h u m id ity . T h i s i n d i c a t e s the l i m i t i n g i n f l u e n c e o f th e h u m id it y u p o n g e r m i n a t i o n in g e n e r a l . The c o n t r o l r e s u l t s at 95% h u m i d i t y a l s o w e r e g e n e r a l l y h i g h e r th an at 98%. C O N C L U S IO N S T h e r e s u l t s o f t h i s i n v e s t i g a t i o n h a v e s h o w n th at A c t i d io n e i s d e f i n i t e l y t o x i c to th e s p o r e s o f t h e p h y t o p a t h o g e n ic f u n g i u s e d in t h i s stud y. The t o x i c i t y v a r i e d w it h th e p r e s e n c e o f t h r e e f a c t o r s w h i c h h a d an i n f l u e n c e u p o n th e r e s p o n s e o f th e o r g a n i s m s to th e A c t i - d i o n e . The fo u r fa c to r s w ere: con­ c e n t r a t i o n o f A c t i - d i o n e , t e m p e r a t u r e , p H o f the s p o r e s u s p e n ­ s i o n m e d i u m an d h u m i d i t y . T h e s e f a c t o r s p r o b a b l y e x e r t e d an i n f l u e n c e in d e p e n d e n t l y o f e a c h o t h e r b u t a l s o in c o m b i n a t i o n w it h o n e a n o t h e r . A s a r e s u l t of t h i s i n f l u e n c e , c e i 't a in t r e n d s w ere observed. T h e g e n e r a l t e n d e n c y w a s a g r e a t e r i n h ib it i o n a s th e c o n c e n t r a t i o n o f A c t i - d i o n e w a s i n c r e a s e d u n t il a l e t h a l c o n c e n ­ tr a tio n w a s r e a c h e d . The c o n c e n tr a tio n at w h ich th is w a s o b ­ s e r v e d v a r i e d w i t h th e o r g a n i s m an d th e c o m b i n a t i o n o f th e t h r e e f a c t o r s p r e s e n t , b u t th e r a n g e of t h i s e f f e c t v a r i e d f r o m 5 .0 p . p . m . to 1 0 0 .0 p . p . m . ( s e e t a b l e s a n d g r a p h s ) . A n o th e r c h a r a c t e r is t ic of A c ti-d io n e w a s it s stim u la to r y e f f e c t u p o n th e g e r m i n a t i o n o f th e s p o r e s , b o th in the i m m e r ­ s i o n and t h e h u m i d i t y e x p e r i m e n t s . In g e n e r a l , th e g r e a t e s t 123 a m o u n t o f s t i m u l a t i o n o c c u r r e d at 0 . 5 p . p . m . i r r e s p e c t i v e o f th e p H o r t e m p e r a t u r e . The o r g a n is m s m o st prone to s tim u ­ l a t i o n a t t h i s c o n c e n t r a t i o n w e r e B o t r y t i s c i n e r e a f o l l o w e d in o r d e r b y R a m u la r ia s p . T h e l e a s t a m o u n t of s t i m u l a t i o n a t t h is c o n c e n t r a t i o n o f A c t i - d i o n e o c c u r r e d w it h M o n il in ia f r u c t i ­ c o la and C la d o sp o r iu m c u c u m e r in u m . A d d i t io n a l s t i m u l a t i o n s w e r e o b s e r v e d at 2 . 5 , 5 .0 , 1 0 .0 and 2 5 .0 p . p . m . p r i m a r i l y w it h th e s p o r e s o f B_. c i n e r e a an d to a l e s s e r e x t e n t w it h R a m u l a r i a sp. A n i n v e s t i g a t o r d e s i r i n g to s t u d y the e f f e c t s o f A c t i d io n e u n d e r f i e l d c o n d i t i o n s w o u ld h a v e s o m e p r e v i o u s k n o w l ­ e d g e o f i t s b e h a v i o r u n d e r th e in f l u e n c e of th e fo u r f a c t o r s u s e d in t h i s s t u d y . He c o u l d th u s a c c o r d i n g l y p la n h i s e x p e r i ­ m e n t s m o r e e f f i c i e n t l y an d p o s s i b l y a v o id s o m e e r r o r s w h ic h m i g h t o t h e r w i s e p r e s e n t t h e m s e l v e s in the a b s e n c e o f t h is i n ­ fo r m a tio n . If t h is i n v e s t i g a t i o n w a s to b e c o n t in u e d it w o u ld b e o f r e a l i n t e r e s t and o f p o s s i b l e p r a c t i c a l i m p o r t a n c e to c o n d u c t a d d it io n a l e x p e r i m e n t s of th e s a m e t y p e on m o r e s p o r e s of p h y t o p a t h o g e n ic fu n g i.. The i n f o r m a t i o n o b t a in e d m i g h t p r o v e o f g r e a t v a l u e in c o m b a t i n g a n y n u m b e r of d i s e a s e s f o r w h ic h at 124 p r e s e n t w e la c k su ita b le f u n g ic id e s . A l s o , it w o u ld b e n o t o n ly of i n t e r e s t but o f p o s s i b l e p r a c t i c a l v a l u e to d e t e r m i n e e x a c t l y h o w th e A c t i - d i o n e in h i b i t s th e g e r m i n a t i o n o f s u s c e p t i b l e s p o r e s . If th is i n f o r m a t i o n w a s k n o w n , s o m e a d j u s t m e n t o f the i n f l u e n c ­ in g f a c t o r s c o u ld b e m a d e s o t h a t it w o u l d b e c o m e m o r e e f ­ f e c t i v e o v e r a w i d e r r a n g e of o r g a n i s m s . T h is m ig h t m e a n a d d it io n o f o t h e r c h e m i c a l s to c o n t r o l e f f e c t o f p H and th e a p ­ p lic a tio n of s p r a y u nder su ita b le t e m p e r a t u r e s . SUM MARY 1. The s p o r e s of C la d o sp o r iu m c u c u m e r in u m , B o tr y tis c i n e r e a , M o n i l i n i a f r u c t i c o l a and R a m u l a r i a s p . w e r e s u b j e c t e d to a s e r i e s of t e s t s to d e t e r m i n e t h e i r r e s p o n s e to A c t i - d i o n e in v a r i o u s c o n c e n t r a t i o n s a s i n f l u e n c e d b y s e v e r a l e n v i r o n ­ m e n t a l c o n d i t i o n s ; t e m p e r a t u r e s , p H and h u m i d i t y . 2. 30 o C. T h e t e m p e r a t u r e s u s e d w e r e 2 2 ° , 2 4 ° , 2 6 ° , 2 8 ° and The h y d ro g en io n c o n c e n tr a tio n s u s e d w e r e 5 . 0 , 6 .0 an d 6 . 8 . 95% an d 93%. 3. 3 .0 , 4 . 0 , T h e r e l a t i v e h u m i d i t i e s t e s t e d w e r e 98%, The la tte r p ro d u ced no r e s u lt s . In g e n e r a l th e l e t h a l a c t i o n i n c r e a s e d a lo n g w it h the in c r e a s e of A c ti-d io n e c o n c e n tr a tio n . S tim u la tio n w a s p r e v a le n t in a l l t e m p e r a t u r e s and H - i o n c o n c e n t r a t i o n s but v a r i e d w it h the o r g a n ism s. M o s t s t i m u l a t o r y e f f e c t s w e r e o b s e r v e d at 0 .5 p . p . m . and w e r e e s p e c i a l l y p r e v a l e n t w it h th e s p o r e s o f B o t r y t i s a n d R a m u l a r i a s p . , C. c u c u m e r i n u m and M . f r u c t i c o l a w e r e s t i m u l a t e d b u t n o t to th e e x t e n t o f th e o t h e r two o r g a n i s m s . 126 4. C l a d o s p o r i u m c u c u m e r i n u m w a s s u s c e p t i b l e to A c t i - d io n e in a l l o f th e c o n c e n t r a t i o n s ; l e t h a l r e s u l t s w e r e o b t a in e d in c o n c e n t r a t i o n s f r o m 5 .0 p . p . m . to 5 0 .0 p . p . m . d e p e n d in g u pon th e t e m p e r a t u r e s an d p H . A t 2 2 ° C. A c t i - d i o n e w a s m o r e e f ­ f e c t i v e a t p H 4 .0 w h e r e a s at 3 0 ° C. it w a s m o r e e f f e c t i v e a t pH 6 .8 . No d is to r tio n or c y to p la s m ic p la s m o ly s is w a s n oted e v e n a f t e r 72 h o u r s e x p o s u r e . 5. T h e s p o r e s o f B . c i n e r e a w e r e s u s c e p t i b l e to th e le th a l a c tio n o f A c t i- d io n e but g e n e r a lly at a h ig h er c o n c e n t r a ­ t i o n th a n t h o s e o f th e o t h e r t h r e e o r g a n i s m s . The m in im u m l e t h a l d o s e f o r t o t a l k i l l v a r i e d f r o m 5 0 .0 to 1 0 0 .0 p . p . m . o f A c ti-d io n e . T h e l e t h a l d o s e o f 5 0 ,0 p . p . m . w a s a t p H 6 .8 at 3 0 ° C . w h e r e a s th e 1 0 0 .0 p . p . m . w a s o b s e r v e d th r o u g h o u t a l l o f th e o t h e r t e m p e r a t u r e s and b y h y d r o g e n io n c o n c e n t r a t i o n s . S t i m u l a t i o n s o c c u r r e d f r e q u e n t l y in s e v e r a l c o n c e n t r a ­ t i o n s o f A c t i - d i o n e a t a l l h y d r o g e n io n c o n c e n t r a t i o n s and t e m ­ p eratu res. T h e c o n c e n t r a t i o n o f A c t i - d i o n e at w h i c h m o s t o f th e s t i m u l a t i o n s o c c u r r e d w a s a t 0 .5 p . p . m . The m o s t f a v o r ­ a b le h y d r o g e n io n c o n c e n t r a t i o n s f o r th e o c c u r r e n c e o f s t i m ­ u la t io n w e r e 4 . 0 , 5 .0 a n d 6 .8 . C o n s i d e r a b l e d i s t o r t i o n o f th e 127 u n g e r m i n a t e d and g e r m i n a t e d s p o r e s an d t h e i r g e r m t u b e s w a s ev id en t. 6. M o n i l i n i a f r u c t i c o l a w a s s u s c e p t i b l e to l e t h a l d o s e s of A c ti-d io n e v a ry in g fr o m 1 0 .0 to 2 5 .0 p . p . m . T otal k ill at 1 0 .0 p . p . m . w a s o b s e r v e d at 3 0 ° C. a t a ll h y d r o g e n io n c o n ­ c e n tr a tio n s , w h e r e a s at a ll o th e r te m p e r a tu r e s and h yd rogen io n c o n c e n t r a t i o n s t o t a l k i l l w a s at 2 5 .0 p . p . m . o f A c t i - d i o n e . B o t h d i s t o r t i o n and p l a s m o l y s i s w e r e o b s e r v e d , in th e h i g h e r c o n c e n t r a t i o n s , in the u n g e r r n in a t e d and g e r m i n a t e d s p o r e s and th e ir g e r m tu b es. S t im u la t io n d id o c c u r but o n ly a t p H 3 .0 a n d a t 2 2 ° , 2 4 ° and 2 6 ° C. 7. The s p o r e s o f R a m u l a r i a s p . w e r e s u b j e c t to i n h i ­ b i t i o n an d l e t h a l a c t i o n a s t h o s e of th e p r e v i o u s t h r e e o r g a n ­ i s m s , b u t w e r e f r e e f r o m d i s t o r t i o n in th e h ig h e r c o n c e n t r a t i o n s of A c ti-d io n e . A c ti-d io n e . T otal k ill v a r ie d fr o m 5 0 .0 to 7 5 .0 p . p . m . o f S t i m u l a t i o n s o c c u r r e d f r e q u e n t l y b u t n o t to th e e x t e n t t h e y o c c u r r e d w it h B o t r y t i s . M o st stim u la tio n s o c c u r r e d a t 0 .5 p . p . m . b u t w e r e a l s o o b s e r v e d at o t h e r c o n c e n t r a t i o n s of A c t i - d i o n e . N o o n e p H w a s m o r e f a v o r a b l e th a n a n o t h e r in in f l u e n c i n g s t i m u l a t i o n s . 128 8. In g e n e r a l th e r e s u l t s o f th e h u m id it y t e s t s w e r e c o r r e l a t e d w it h th e i m m e r s i o n t e s t s . A s o m e w h a t h ig h e r p e r ­ c e n t a g e o f i n h ib it i o n w a s n o t e d h o w e v e r , a n d it i s b e l i e v e d th a t t h i s w a s due to th e l a c k o f a d e q u a t e m o i s t u r e r a t h e r th a n th e e f f e c t o f th e h u m i d i t y u p o n th e A c t i - d i o n e . A n u m b e r of s t i m ­ u l a t i o n s d id o c c u r w it h e v e r y o r g a n i s m u s u a l l y at the s a m e c o n c e n t r a t i o n o f A c t i - d i o n e , p H an d t e m p e r a t u r e a s o b s e r v e d in th e i m m e r s i o n t e s t s . S o m e d i s t o r t i o n d id o c c u r but w a s p r i m a r i l y a s tu n t in g of th e g e r m t u b e s w h i c h a p p e a r e d v e r y t h in a n d s h o r t . 9. The w a s h i n g e x p e r i m e n t s in w h i c h t r e a t e d s p o r e s o f th e o r g a n i s m s w e r e w a s h e d a f t e r e x p o s u r e to A c t i - d i o n e p r o ­ duced v e r y poor r e s u lt s . O n ly a t r a c t o f th e A c t i - d i o n e c o u ld b e r e c o v e r e d f r o m th e t r e a t e d s p o r e s . So s m a l l w a s th e q u a n ­ t i t y o f r e c o v e r e d A c t i - d i o n e th a t i t w a s n e g l i g i b l e . L IT E R A T U R E C IT E D A m e r i c a n P h y t o p a t h . S o c . , C o m m i t t e e on S t a n d a r d i z a t io n of F u n g i c i d a l T e s t s . The s l i d e g e r m i n a t i o n m e t h o d of e v a lu a tin g p r o te c ta n t fu n g ic id e s . P h y t o p a t h . 33: 6 2 7 - 6 3 2 .1943. _________ . T e s t tube d ilu t i o n t e c h n iq u e f o r u s e w ith th e s lid e g e r m in a tio n m eth o d o f ev a lu a tin g p ro tecta n t fu n g ic id e s. P h ytop ath . 3 7 :3 5 4 -3 5 6 . 1947 . C la y t o n , C. N . T h e g e r m i n a t i o n o f f u n g o u s s p o r e s in r e ­ la t i o n to c o n t r o l l e d h u m i d i t y . P h y to p a t h . 3 2 : 9 2 1 943. 1942. d e Z e e u w , D o n a ld , a n d J . R . V a u g h n . C ucum ber scab co n ­ t r o l in M i c h ig a n in 1 9 4 9 . P la n t D is e a s e R ep orter. F e l b e r , I r m a M ., a n d C. L . H a m n e r . C o n t r o l of m i l d e w on b e a n p l a n t s b y m e a n s o f a n a n t i b i o t i c . B o t . G a z . 110 ( 2 ) : 3 2 4 - 3 2 5 . 1948. 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A p a p e r - d i s k p l a t e m e t h o d f o r th e q u a n t i­ t a t i v e e v a l u a t i o n o f f u n g i c i d e s and b a c t e r i c i d e s . P h ytopath. 4 0 :4 1 9 -4 2 9 . 19 5 0 . T o g a sh i, T ogo. B io lo g ic a l C h a ra cters of P la n t P ath ogen s T e m ­ p era tu re R e la tio n s. 478 p p . T okyo, Japan. 1949. E F F E C T O F C Y C L O H E X IM ID E ( A C T I -D IO N E ) ON T H E G E R M IN A T IO N O F SPO R E S O F SE V E R A L PH Y T O PA T H O G E N IC FU N G I By A n d rew Thanos A N ABSTRACT S u b m i t t e d to th e S c h o o l o f G r a d u a te S t u d i e s o f M i c h ig a n S t a t e C o l l e g e o f A g r i c u l t u r e and A p p l ie d S c i e n c e in p a r t i a l f u l f i l l m e n t o f th e r e q u i r e m e n t s f o r th e d e g r e e of D O C T O R O F P H IL O S O P H Y D e p a r tm e n t of B otan y and P la n t P a th o lo g y Y ea r 1952 A pproved 1 ANDREW THANOS A B ST R A C T A n u m b e r of in v itr o t e s t s w e re c o n d u c te d in o r d e r to d e te r m in e th e e f f e c t of A c ti-d io n e u n d e r v a r io u s m ic r o - e n v ir o n ­ m e n ta l c o n d itio n s . S p o re s u s p e n s io n s , in c o r p o ra tin g v a r io u s c o n c e n tr a tio n s of A c ti-d io n e , w e re p r e p a r e d and s tu d ie d u n d e r a s e r i e s o f t e m p e r a t u r e s ra n g in g f r o m 22° C. to 30° C. A d d i­ tio n a l t e s t s w e re m a d e to d e te r m in e th e e ffe c t of pH in c o n ­ n e c tio n w ith e a c h of th e ab o v e te m p e r a tu r e s . A s e r i e s of w a sh in g e x p e r im e n ts w e re m a d e in an a tte m p t to d e te r m in e how m u c h of th e A c ti-d io n e c o u ld be r e c o v e r e d f r o m th e t r e a t e d sp o re s. In a d d itio n to th e s e , a s e r i e s of h u m id ity t e s t s w e re c o n d u c te d to d e te r m in e th e e f f e c t of A c ti-d io n e upon th e s p o r e s of th e s a m e o r g a n is m s u n d e r v a ry in g h u m id itie s and w ith v a r i ­ o u s h y d r o g e n -io n c o n c e n tr a tio n s . T he r e s u l t s in d ic a te th a t in th e c a s e of C la d o s p o riu m c u c u m e rin u m , A c ti- d io n e w a s m o s t e ffe c tiv e in in h ib itin g g e r m ­ in a tio n a t 2 2 ° C. a t pH 4.0 a n d a t 30° C. a t pH 6.8 . in a tio n o c c u r r e d a t pH 3.0. No g e r m ­ S om e s tim u la tio n of g e r m in a tio n o c c u r r e d a t 0 .5 , 2.5 an d 5.0 p .p .m . of A c ti-d io n e a t 2 4 ° C. and 30° C. No s tim u la tio n w as o b s e r v e d a t a ll of th e o th e r te m p e r ­ a tu re s . N o d is to r tio n w as o b s e r v e d a t a n y tr e a tm e n t. 2 ANDREW THANOS A B ST R A C T T he s p o r e s o f .B. c i n e r e a w e re k ille d a t 50.0 and 100.0 p .p .m . d ep en d in g u p o n th e t e m p e r a t u r e and pH . S tim u la tio n of g e r m in a tio n o c c u r r e d f re q u e n tly a t a l l t e m p e r a t u r e s a n d h y d r o ­ g e n io n c o n c e n tr a tio n s b u t p r in c ip a lly a t 0 .5 p .p .m . A c o n s id ­ e r a b le a m o u n t o f d is t o r t i o n an d c y to p la s m ic d is o r g a n iz a tio n w as n o te d in b o th g e r m in a te d a n d u n g e r m in a te d s p o r e s . M . f r u c t i c o l a s p o r e s w e r e c o m p le te ly in h ib ite d a t c o n ­ c e n tr a tio n s of A c ti-d io n e v a r y in g f r o m 10.0 to 25.0 p .p .m . The f o r m e r c o n c e n tr a tio n w as e ffe c tiv e a t 28 ° a n d 30° C. w h e re a s th e l a t t e r w a s m o s t e ffe c tiv e a t 2 2 °, 2 4 ° a n d 26° C . S om e d is to r tio n a n d d is o r g a n iz a tio n w a s o b s e r v e d in th e h ig h e r c o n ­ c e n tr a tio n s of th e t r e a t m e n ts u s e d . The s p o r e s o f R a m u la r ia sp . w e r e f r e e f r o m an y of th e d is to r tio n s o b s e r v e d w ith th e o th e r o r g a n is m s . Som e s tim - a u la tio n s w e re o b s e r v e d a t e v e r y te m p e r a tu r e and h y d ro g e n io n c o n c e n tr a tio n . T o ta l k ill o c c u r r e d a t 50.0 a n d 100.0 p .p .m . d e p en d in g u p o n th e t e m p e r a tu r e an d th e pH o f th e s o lu tio n s in w h ich th e s p o r e s w e r e im m e r s e d .