Sinergii Din 1937

7/29/2019 Sinergii Din 1937

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O N T H E C U L T U R E B E H A V I O U R O F A S P EC I ES O FR O S E L L I N I A .

I I . F u r t h e r E x p e r i m e n t s o n t h e P r o d u c t i o n o s P e r i t h e c i a .BY ,~. N. DAS GUPTA.

(From the Departme~t of Bota~,y, University of Lucknow, Lucknow.)Received D ecember 8, 1937.

Introduction.IN an earlier paper (Das Gupta, 1937) an account has been given of theinhib itory effect of cert ain chemicals on the product ion of perithecia ina species of Rosellinia. It has been shown that the complete absence ofperithecia in a standard syntheti c medium which consisted of glucose,asparagin, magnesium sulphate, potassium phosphate (K~PO,), starch andagar was due to the combined effect of asparagin and potassium phosphate.although none of these t~r chemicals has any inhibitory influence individu-ally. It has been fur ther shown tha t glucose at any concentration beyond0.3 per cent. and potassium phosphate at about 0.6 per cent. completelyinhibit the formation of perithecia.

The present paper embodies the results of further nutritional work onthe problem which was carried out by growing Rosellinia in various concen-tra tions of acid and alkali and such chemicals as asparagiu, potassiumnitrate, etc.

Fungus.--The fungus Rosellinia as stated in the previous paper (DasGupta, 1937) was obtained by Horne and Ni timargi in the course of theirinvestigation into the fungal population in the air of the apple orchard ofthe Eas t Malling Exper imental Station (England) from whom it was obtainedby the present author. The fungus was cleaned and cultures derived froma single hyphal tip of uncontaminated mycelium were used throughoutthe experiment.

Method.~Usual culture method was employed in order to find out th2effect of various chemicals and the acidity of the medium on the productionof peritheeia. Plain agar which proved very suitable for perithecia produc-tion was taken as the basal medium, and the chemicals whose effects wereto be ascertained ~ere mixed to the agar medium in requi.~ite proportions.The media were autoclaved, poured in sterilized plates and inoculated withRosellinia. To ensure uniformity of growth, the mycelium used for inocu-lation was always taken from the advancing region of a culture three days22


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O n l he C u l l u r e g e h a v i o u r o ~ a S p e c ie s o ~ R o s e l l i n i a - - H 2 3o ld . T h e n u m b e r o f p e r i t h e c i a f o r m e d i n y o u n g c u l t u r e s w a s c o u n t e d b yp u t t i n g i n k d o t s o n t h e b a c k o r t h e t o p o f t h e p e t r i - d i s h a g a i n s t e a c h p e r i -t h e c i u m b u t f o r o l d e r c u l t u re s w h i c h h a d b e e n l e f t l o n g e n o u g h a n d h a db e e n a l l o w e d s u f fi c i e n t t i m e t o e x h a u s t a l l p o s s i b i l i ti e s o f f u r t h e r d e v e l o p -m e n t o f f r e sh p e r i th e e i a , t h e s e w e r e c o u n t e d b y p i c k i n g p e r i t h e c i a o u t o n ea f t e r a n o t h e r b y m e a n s o f a n e e d le . I t s h o u l d b e b o r n e i n m i n d t h a t t h ea c t u a l n u m b e r o f p e r i th e c i a f o r m e d v a r i e s f r o m e x p e r i m e n t t o e x p e r i m e n te v e n w h e n c ar ri e d o u t i n t h e s a m e m e d i u m a n d in i d e n t i c a l c o n d i t io n s .T h e p e r i t h e c i a n u m b e r o f t w o d i f f e re n t e x p e r i m e n t s i s n o t s t r i c t l y c o m p a r a b l e .A l l e x p e r i m e n t s w e r e c arrie d_ o u t a t 2 0 ~ C . . e x c e p t w h e r e s t a t e d o t h e r w i s e .

T h e c o m p o s i ti o n o f t h e s t a n d a r d m e d i u m w h i c h w a s a ls o u s e d i n t h ec o u r s e o f t h e e x p e r i m e n t , i s as f o l lo w s :

A s p a r a g i n 2 g m s . , g l u co s e 2 g i ns ., m a g n e s i u m s u l p h a t e 0 . 7 5 g m s . ,p o t a s s i u m p h o s p h a t e 1 . 2 5 g m s . , s t a r c h 1 0 g i ns ., a g a r 1 5 g m s . , i n a ! i t r e o fd i s t i l l e d w a t e r .

Effect of a Bacterium on the Formation of Peri thecia.W h e n Rose l l in ia w a s f i r st i s o l a t e d f r o m t h e a i r o f t h e a p p l e o r c h a r d ,

i t w a s f o u n d t o b e a s s o c i a te d w i t h a b a c t e r i u m . A f t e r t h e f u n g u s h a s b e e nf r e e d f r o m b a c t e r i u m i t w a s f o u n d t h a t t h e c l e a n e d Rose l l in ia d o e s n o tp r o d u c e p e r i th e c i a in t h e s t a n d a r d m e d i u m a l th o u g h a b u n d a n t p e r i th e c i af o r m e d i n p l a in a g ar . F o r m a t i o n o f p e r i th e c i a in t h e s t a n d a r d m e d i u mt h u s s e e m e d t o b e c o n d i t i o n a l t o t h e p r e s e n c e o f t h e b a c t e r i u m i n t h ec u l tu r e . E x p e r i m e n t s w e r e m a d e t o t e s t th i s p o i n t b y g r o w i n g Rosell iniaa n d t h e b a c t e r i u m i n d i ff e r en t d e g re e s o f p r o x i m i t y i n t h e s t a n d a r d m e d i u m .T w e l v e p l a t e s o f t h e s t a n d a r d m e d i u m w e r e p r e p a r e d . F o u r o f t h e s e w e r ei n o c u la t e d w i t h t h e b a c t e r i u m a n d t h e Rosell inia m y e e l i u m p l a c e d c o n t i g u -o u s l y a t t h e c e n t r e ( Se ri es A ). F o u r o t h e r p l a t e s w e r e i n o c u l a t e d i n t w op l a c e s ea c h , o n e o f t h e i n o e u l u m b e i n g o f t h e f u n g a l m y c e l iu r n , t h e o t h e r o ft i le b a c t e r i u m p l a c e d a b o u t 4 e ra . a p a r t ( S er ie s B ) . T h e r e m a i n i n g f o u rp ! a te s w e r e i n o c u l a t e d a t t h e c e n t r e w i t h Rosell inia o n l y ( S e r i e s C ) .

T h e r e s u l t s h o w e d t h a t : - -1. Rosell inia f r e e d "fro m t h e b a c t e r i u m c a n n o t p r o d u c e p e r i t h e c i a i n t h e

s t a n d a r d m e d i u m .2 . T h e p e r i t h e c i a a r e r e l a t i v e l y m o r e n u m e r o u s i n c u l t u r e s w h e r e t h e

b a c t e r i u m a n d t h e f u n g u s a r e g ro w n t o g e t h e r t h a n i n cu l tu r e sw h e r e t h e g r o w t h s f r o m b o t h a r e a l lo w e d t o i n t er m i n g l e . I n t h el a t t e r c a s e o n l y a f e w p e r i t h e c i a a r e f o r m e d a l o n g th e l in e o f c o n t a c to f t h e b a c t e r i u m a n d t h e f u n g u s .


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24 S .N . D a s G uptaT h e r a t e o f a d v a n c e o f Rosel l inia i n S e r i e s A i s r e l a t i v e l y s l o w e r t h a n

t h a t i n e i t h e r S e ri e s B o r C . A b r o w n s u b s t r a t a l c o l o u r is f o r m e d i n c u l t n r e so f s e ri es A e x t e n d i n g b e y o n d t h e a d v a n c i n g m a r g i n o f t h e f u n g u s i n f o r mo f a z o n e w h i c h a t t a i n e d a w i d t h o f 2 - 2 . 5 c m . i n c u l t u r e s o f 2 w e e k s g r o w t h .T t ' e e o l o u r i s n o t p r o d u c e d i n t h e S e r i e s C c u l t u r e s . I n B i t i s o n l y f a i n t l ye v i d e n t i n t h a t r eg i on w h e r e t h e f u n g al g r o w t h m i n g l e d w i th t h e b a c t e r iu m .

A s t h e c h a n g e d a c i d i t y o f t h e m e d i u m d u e t o b a c t e ri a l a c t i v i t y m a y b er e s p o n s ib l e f o r t h i s p r o d u c t i o n o f p e r i t h e c i a t h e p i t v a l u e o f t i le m e d i a i nw h i c h Rosel l inia x ~ as g r o w i n g w a s t e s t e d u s i n g B . D . t I . C a p i l l at o r .

O n e p l a t e e a c h f r o m S e r ie s A a n d C w a s e m p l o y e d a n d d i f fe r e n t r e g io n so f c u l t u r e s o f t w o w e e k s g r o w t h w e r e t e s t e d t h e r e s u lt s a r e a s f o ll ow s : - -l . T h e s t a n d a rd m e d i u m w i t h p u re g r o w t h o f Rosel l inia ( n o p e r i -

t h e c i u m ) . . . . . . . . . . . . p t I 7 .42 . S u p e r f ic i a l l a y e r o f t h e m e d i u m ( 5 r a m . d e e p ) c o n t a i n i n g t h e

b a c t e r i u m Rosel l inia m y c e l i u m a n d p e r i t h e c i a . . . . . . 7 .73. A l a ye r o f t h e m e d i m n (5 r a m . d e e p ) b el o w 2 . . . . ,, 8 .24 . S u p e r fi c ia l l a y e r o f t h e m e d i u m f r o m t h e b r o w n z o n e b e y o n d

t h e a d v a n c i n g m a r g i n o f t h e f u n g u s (5 m m . d e e p ) . . ,, 7 . 75. A l a y e r o f t h e m e d iu m (5 ra m . d e e p ) b e l o w 4 . . . . . . 7 ,9T h e f ol lo w i n g t w o p o i n t s a r e a t o n c e e v i d e n t : - -1. R e l a t i v e l y g r e a t e r a lk a l i n i ty o f t h e l o w e r s t r a t u m o f t h e m e d i u m

t h a n t h e u p p e r .2 . R e l a t i v e l y g r e a t e r a l k a l i n i t y o f t h e l a y e r o f t h e m e d i u m p r o d u c i n g

p e r i t h e c i a .A s p e r i t h e c ia w e r e f o u n d i n t h a t l a y e r o f t h e m e d i t m l w h e r e a l k a l i n i t y

w a s r e l a t i v e l y h ig h e r , i t s e e m e d n o t u n l i k e l y t h a t f o r m a t i o n o f p e r i t h e c i ai n t h e s t a n d a r d m e d i u m c a n b e in d u c e d b y a l t e ri n g it s a l k a li n i ty b u t a l la t t e m p t s i n t h i s d i r e c t i o n p ro v e d u n s u c c e s sf u l .

Effect of Certain Chemicals on the Formation of Perithecia.E ffe ct o f A g a r . ~ I t h a d b e e n o b s e rv e d t h a t a b u n d a n t p e r i t h e c i a d e v e l o p e d

w h e n Rosel l inia w as gr ow n in 1 .5 OZ;op l a in a g a r . S o m e e x p e r i m e n t s w e r et h e r e f o r e m a d e i n o r d e r t o a s c e r t a i n t h e m o s t f a v o u r a b l e a g a r c o ~ . c e n t ra t io nf o r p r o d u c t i o n o f p e r i t h e c i a . T h e f o l lo w i n g c o n c e n t r a t i o n s w e r e e m p l o y e d : - -

0 . 5 , 1 . 0 , 2 .0 , 4 . 0 , 8 - 0 a n d 1 2 . 0 p e r c e n t .T h e r e s u lt s a r e s h o w n i n T a b l e I .


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O n t he Cu l t u re Behav i our o f a Spec i e s o / Rose l l i n i a - -H 25TABLE I .Showing the Effect of Agar Concentrations on

the Production of Perithecia.D i a m e t e r o f t h e P e r i t h e c i aC u l t u r e s i n m m .A g a r i n g i n s.

p e r c e n t . T i m e r e q u i r e d f o r C h a r a c t e rd e v e l o p m e n t i n d a y s

0.5

1.0

2.0

4.0

8-0

12.0

4 8 h r s . 7 2 hrs.

1 8 4 0 . 02 5 . 0 5 1 . 03 1 . 0 6 5 . 03 3 . 0 7 2 . 04 0 . 0 8 2 . 04 0 . 0 0 8 0 . 0

101 0765

5

A f e w sca t t e r edm o s t l y i m m a t u r e .A l a r g e n u m b e rm a t u r e .A b u n d a n t , b i g , m a -t u r e .A b u n d a n t , s m a 1 1m a t u r e .A b u n d a n t , v e r ym i n u t e , m a t u r e .A b u n d a n t , v e r y m i -n u t e , m a t u r e .

T a b l e I s h o w s t h a t t h e r a t e o f m y c e l i a l g r o w t h i n c r e a s e s w i t h t h e i n c r e as ei n t h e c o n c e n t r a t i o n o f a g a r. A s r e g a r d s p e r i t h e c i a f o r n l a t io n , a t th e l o w e s tc o n c e n t ra t io n e m p l o y e d (0 . 5 % ) p e r i t h e c i a a r e f ew a n d m o s t o f t h e mi m m a t u r e . W h e n t h e c o n c e n t r a t io n i s i n c re a s e d t o 1 . 0 % a l ar g e n u m b e r o fp e r i t h e c ia a r e f o r m e d w h i c h ar e m o s t l y m a t u r e . W i t h t h e f u r t h e r i n c re a s eo f t h e c o n c e n t r a t i o n to 2 . 0 % a b u n d a n t , b i g, m a t u r e p e r i t h e c ia a r e o b ta i n e d .I n 4 . 0 a n d 8 . 0 % t h e p e r i t h e c i a a re s t il l m o r e n u m e r o u s b u t sm a l l e r i n s iz et h a n t h o s e f o u n d in 2 % a g a r. A t 1 2 % t h e p e r i t h e c i a a r e i n n u m e r a b l e a n dm i n u t e . A s w i ll b e s e e n f r o m t h e ta b l e t h e c o n c e n t r a t i o n a l s o a f fe c t s t h et i m e r e q u i r e d f o r t h e d e v e l o p m e n t o f p e r i t h e c i a . A t l o w e r c o n c en t ~r a ti o n s( 0 . 5 a n d 1 . 0 % ) l o n g e r t i m e is r e q u i r e d t h a n a t h ig h e r c o n c e n t r a t i o n s .

T h e e f f e ct o f a g a r c o n c e n t r a t io n m u s t b e d u e t o t h e i n c r e a s e o f t h e n u t r i -t i v e s al ts p r e s e n t in t h e a g a r a s i m p u r i ti e s . T h e d e c r e a s e i n t h e w a t e r - c o n t e n tand. t h e r e l a t iv e h a r d n e s s o f t h e s u b s t r a t u m m a y a ls o b e t h e c o n t r i b u t o r yc a u s e s .Effect o] Nitrogen.---Two s o u r c e s o f n i t ro g e n , a s p a r a g i n a n d p o t a s s i u mn i t r a t e , w e r e i n v e s t i g a t e d f o r t h e i r p o s s i b l e ef fe c t.

Asparagin . - -Rosel l in ia w a s g r o w n i n a g a r m e d i u m h a v i n g d i f f e r e n tc o n c e n t r a t io n s o f a s p a r a g i n . T h e p l a te s w e r e e x a m i n e d p e r i o d i c a ll y , b u t t h en u m b e r o f p e r i t h e c i a f o rm e d w a s n o t c o u n t e d e x c e p t i n g f o r p la t e s h a v i n ga v e r y s m a l l n u m b e r . T h e r e s u l ts o b t a i n e d w i t h c u l t u r e s fi ve w e e k s o ld ar eg i v e n i n T a b l e n .


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26 S. N. Das GuptaTABLE I I .

Showing the Ef fec ts o f Asparagin on the Forma t ion o f Perithecia.A s p a r a g i n i n g m s , N u m b e r o f N a t u r e o fp e r 1 0 0 c .c . p e r i t h e c i a p e r i t h e c i a R e m a r k s

0 . 00 .10 . 20 . 20 . 51 . 02 . 05 . 0

1 0 . 0

M o d e r a t eA b u n d a n t

77

S ix9 9

E i g h t

M a t u r e

M a t u r eM e d i u m f a i le d t o s e t . V e r ys l i g h t g r o w t h .N [ e d iu m f a i le d t o s e t p r o p e r l y .T h e a s p a r a g i n c r y s t a l l i s e d o u t .

V e r y s l o w g r o w t h o f t h ef u n g u s .

I t w i l l b e s e e n f r o m T a b l e I I t h a t w i t h t h e s l ig h t a d d i t i o n o f a s p a r a g i n( 0 . 1 % ) t h e r e i s a n in c r e a s e i n t h e n u m b e r o f p e r i t h e c i a f o r m e d w h i c h c o n t i n u e sf o r a ll c o n c e n t r a t io n s u p t o 1 . 0 % . P e r i t h e c i a w h i c h d e v e l o p i n a ll t h e s e m e d i aa r e m a t u r e a n d p r o d u c e n o r m a l a s c o s p o r es . A t th e h i g h e r c o n c e n t r a t i o n s ,h o w e v e r , t h e re s u l ts ar e d i f fe r e n t. O n l y si x p e r i t h e c i a a p p e a r e d a t 2 - 0 %a s p a r a g i n , a l l o f t h e m m a t u r e . . at 5 - 0 % t h e m e d i u m f a i le d t o s e t a n d th eg r o w t h o f t h e fu n g u s w a s g r e a t l y r e t a rd e d a n d n o p e r i t h e c i a d e v e lo p e d . A tt h e s t il l h i g h e r c o n c e n t r a t i o n o f ! 0 % a s p a r a g i n - - t h e h i g h e s t c o n c e n t r a t i o ne m p l o y e d - - t h e r e s u lt i s a n o m a l o u s . T h e m e d i u m d o e s n o t s e t, t h e fu n g u sg r o w s a t a s lo w e r r a t e a n d a fe w n o r m a l p e r i th e c i a a r e f o r m e d - - t h e n u m b e rb e i n g m o r e t h a n t h a t o b t a i n e d w i t h 2 % a s I )a r ag i n. T h i s a n o m a l o u s re s u l tis p r o b a b l y t o b e e x p l ai n e d b y t h e f ac t t h a t d u e t o o v e r - s a t u r a ti o n a s p a r ag i ni n t h i s m e d i u m e r y s t a l l i s e d o u t .

P o t a ss iu m N i t r a t e . - - T h e e f f e c t o f 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 K N O 3 w e r ea s c e r ta i n e d in t h e u s u a l m a n n e r . T h e e x p e r i m e n t w a s r e p e a te d , t h e n u m b e ro f p e r i t h e c i a v a r i e d e a c h t i m e , b u t t h e t r e n d o f t h e re s u l ts w a s t h e s a m e .T h e r e s u l ts o f o n e o f t h e e x p e r i m e n t s w i t h t !m c o n c e n t r a t i o n s e m p l o y e d


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O n t h e C u l / u r e B e h a v i o u r o f ct S p e c ie s o /" Rosellinia--D" 27are given in Table II! . The dimensions given are the average of 20 peri-thecia taken out at random.

TABL~ III.Showing the Ef fect of K N 0 3 on the Product ion of Peri thecia.

K N O ~ i ng ins . pe r100 e.r

0 . 0 00 . 0 30 . 0 60 . 11 .01 . 52 . 57 . 5

1 5 . 0

D i a m e t e ri n m m .4 d a y sg r o w t h8O7 88O7 88 89O8 442

, .

N a t u r eof pe r i -t h e e i at D i m e n s i o n o fI A v e r a g e A v e r a g el e n g t h b r e a d t h

p e r i t h e e i a i n y-N o . o fp e r i t h e -c i a

M a t u r e 4 5 0 2 9 0,, 4 1 8 2 7 5, , 418 275, , 410 290, , 420 280, , 400 291, , i 455 280

A v e r a g el e n g t h o fn e c kR a n g e o f v a r i a t i o nL e n g t h ] B r e a d t h

1 0 5 3 5 0 - 5 0 01 0 9 2 9 1 - 4 3 69 0 3 6 t - 4 7 31 2 5 3 6 4 - 4 5 5I! 125 346-4 731 2 5 3 0 4 - 4 5 5180 383-510

2 4 0 - 3 5 02 0 0 - 3 1 02 0 0 - 3 2 82 3 6 - 3 1 02 1 8 - 3 2 82 3 6 - 3 4 62 5 4 - 3 4 6

It will be seen from Table II I tha t the number of perithecia formedshows a gradual decrease with the increasing concentration of KNOa. Anappreciable drop in the number of perithecia occurs at a concentration of0.1%, another drop at 2.5% KNO3. At a still higher concentra tion of7.5% while the growth rate of the fungus is halved, the formation of peri-thecia stops altogether. The concentration of KNO3 seems to have littleeffect on the size of the perithecia formed. In all those cases where peritheciadevelop, they are mostly fertile, the smaller perithecia sometimes failingto produce the asci or ascospores. The laerithecia become visible on the7th day usually, but at higher concentrations these take longer to mature.

Ef f ec t o f C /N Ra t io . - -T he effect of the C /N ratio on the developmentof perithecia was determined by growing Rosellinia in all possible combina-tions of KNO3 (0.00%, 0.01%, 0.03%, 0.09%,0.27% , 0.8]% a nd 2. 45 %)and glucose (0.00%, 0.018%, 0.054%, 0..16%, 0.48%, 1.46% amt 4-37%).The combinations employed can be seen from Table IV.

The plates were examined in great detail and the rate of advance of thefungus, the formation of perithecia, their number, dimensions, the colour ofthe substratum and all such points were carefully noted.


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28 S. N. Das GuptaT h e n u m b e r o f p e r i t h e c i a f o r m e d a t d i f f e re n t c o n c e n tr a t io n s a n d t h e

a v e r a g e d i m e n si o n s f o r t w e n t y p e r it h e c ia t a k e n a t r a n d o m a re s h o w n inT a b l e I V . T h e i ta l i c i se d f ig u r es i n d i c a t e t h e p e r i t h e e i a n u m b e r .

TABI,F. IV .Showing the Re la tive Nu mbe r o f Per ithec ia formed in

D(l~erent Combinations of K N O ~ and G lucoseand their Average Dimenisons in ~.

G l u c o s e i n g m s . p e r l O 0 e . c .K N O ~ i ng m s . p e r I I0 0 e . c . 0 . 0 0 0 . 0 1 8 0 . 0 5 4 0 . 1 6 2 0 . 4 8 6 1 . 4 5 8 4 . 3 7 4a b c d e f 9

3 6 0 2 4 8 . . . . . ..00 A

0-01 B

0.03 C

0 . 0 9 D

0 . 2 7 E

0 . 8 2 F

2 4 5 G

7 . 3 5 H

521 1876

5 5 0 x2911220

3 8 8 X 2 5 71160

3 9 3 980

4 4 X 2 7 31060

466 280656

5 3 5 X 3 1 3542

4 8 2 1775

4 5 8 2 8 61880

4 3 3 1580

4 4 8 1610

4 5 5 1880

4 6 7 X 2 7 0 "1808

4 9 1 1220

4 1 2 1938

571 2080

4 5 5 X 2 6 52300

4 8 4 1980

4 9 5 X 2 81584

4 9 1 X 2 6 1 "1550

561 1558

331 210

1 9 8 I n n u m e r a b l em i n u t e2 2 2 1 8 0I n n u m e r a b l e

m i n u t e1 9 2 X 1 6 8I n n u m e r a b l em i n u t e157 140 "I n n u m e r a b l em i n u t e *1 5 0 1 0 9 "I n n u m e r a b l em i n u t e

* I n d i c a t e p e r i t h e c ia w i t h o u t a s c i .I t w i ll b e s e e n f r o m T a b l e I V t h a t C / N r a t i o h a s p r a c t i c a ll y n o e f fe c t o n

t h e p r o d u c t i o n o f p e r i th e c i a . T h e n u m b e r o f p e r it h i c i a fo r m e d s h o w s as l i g h t in c r e a se w i t h t h e i n c r e a s e i n g lu c o se c o n c e n t r a t i o n . T h e g r e a t e s t in -c r e a s e is , h o w e v e r , o b t a i n e d i n m e d i a c o m b i n i n g 0 . 1 6 2 % g l u co s e c o n t e n tw i t h r e l a ti v e l y h i g h K N O 3 c o n t e n t ( 0 . 0 3 % - 7 . 3 5 % ) w h e r e i n n u m e r a b l e m i n u t ep e r i t h i c i a d e v e lo p . I t i s t h u s a p p a r e n t t h a t t h e e f fe c t i s n o t so m u c h d u e t ot h e C J N r a ti o a s t o t h e c o m b i n e d e f f ec t o f t h e h i g h c o n c e n t r a t io n s o f t h e t w oc h e m i c a l s .


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O n t he C u l t u r e B e hav i our o f a Spec ie s o f Rosel l inia--M 29At a concentration of 7.5% KNO3 no perithecia develop irrespective of

the glucose content and that glucose inhibits the perithecia production ata very. moderate concentration of 0.486% irrespective of the concentrationsof KNO~. It should be noted in this connection that in all those combinationswhere perithecia are not produced, the fungus grows quite actively. In fact,there is hardly any difference in the growth rate of the fungus at any combina-tion of glucose and KNOs employed.

As regards the naturg of perithecia produced C/N ratio has hardly anyeffect. Normal and large peritheeia were produced in all the following media :Aa, Ba, Ca, Da, Ea, Fa and Ga ; Ab, Bb, Cb and Db ; Ac, Bc, Ce, De and Ec ;Ad, Bd, Cd, Dd and Ed. In all other combinations Eb, Fb, Gb ; Fc, Gd theperithecia proved to be mostly immature or half mature. A scru tiny of theseresults shows that in the absence of glucose the increase in KNO3 content doesnot have any effect on the maturi ty of the perithecia, that is, asci andascospores found in the perithecia are the same as in normal agar culture.The immaturity and the semi-maturity of the perithecia are seen in media withhigh content of KNO3 (0.27%, 0.82% and 2.4.~%) to which glucose (0. 018~0.054% and 0-162%) is added. At 0.162% of glucose, in the absence of KNO3or when KNO3 is added in the proportion of 0.01%, the perithecia producedare normal. When KNO3 concentration is increased (0-03%, 0.09% and0.27%), the perithecia produced are distinctly smaller and the number iscorrespondingly large and almost all of these are mature . With still higherconcentration (0.82% and 2.45%) innumerable small immature peritheciawithout asci and ascospores are formed.

Rosdl inia does not produce any colour in ordinary culture medium. Whileworkingwiththe C/N ratio series, it was observed that in all the media contain-ing 0.486, 1.458 an~t 4-374 per cent. of glucose in combination with variousconcentrations of KNO3 showed brown and yellow colour in the substratum,the colour being more intense in the marginal regions of the cultures.

Effect of Ac idi ty and A lkal in i tv . - -Th e effect of the acidity and alkalinityof the medium on the prgduction of perithecia was found out by alteringthe acid and alkali content of agar by the addition of malic acid and ammo-nium carbonate respectively.

Acid Series.---Rosellinia was grown in agar with differen~ concentra-tions of malic acid, and the plates were examined for perithec{a periodically.Wherever per itheeia developed tl~.e first visible sign appeared usually aftersix days growth. Most of the peri thecia were superficial, a few being com-pletely immersed in the medium. Cultures of 44 days growth were usedfor final counting. The various concentrations employed and the resultobtained are shown in Table V along with the diameter of the cultures of


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3 0 S . N . D a s G u ptat h r e e d a y s g r o w t h . T h e n u m b e r o f p e r i t h e c i a i s g i v e n i n r o u n dT h e p t t w a s t a k e n b y t h e B . D . t t . c a p i ll a to r m e t h o d .

TABLe. V .Showing the Ef fect of Acid on the Product ion of Peri thecia.

f i gure .

Acid Cone . i ng ins . C .C .

0 . 0 0 20 . 0 0 40 . 0 0 80 . 0160 . 0 3 20 . 0 6 40 . 1 2 80 . 2 5 6

I n i t i a l p H o f t h em e d i u m

6 . 15 . 95 . 35 . 04 . 74 . 13 . 93 . 1

D i a m e t e r o f c u l t u r e s i n m m .( 3 d a y s g r o w t h )

5 65 85 85026"2 4

4

N u m b e r o f p e r i t h e c i a i ncu l tu re s o f 44 days g rowth

5 4 05 6 05 0 06 4 085060 03 00

T h e a c i d i t y o f t h e m e d i u m h a s v e r y l i tt l e e f f e c t o n th e n u m b e r o f p e r i-t h e c i a f o r m e d b y t h e f u n g u s . U n t i l a v e r y h i g h d o s e of m a l i c a c id i s a d m i n i -s t e r e d t h e p e r i t h e c i a a r e q u i t e a b u n d a n t .

T h e t y p e o f p e r i t h e c i a f o r m e d , h o w e v e r , s h o w e d a g o o d d e a l of d i f-f e r e n c e . I n g e n er a l, t h e p e r i t h e c i a a re m a t u r e b u t a t h i g h c o n c e u t r a t i o n so f a c i d t h e s e t e n d t o b e c o m e v e r y s m a ll , in f e r ti l e, a n d i m m e r s e d .

A c o m p a r i s o n o f g r o w t h r a t e o f t h e f u n g u s a t d i f f e r en t c o n c e n t r a t i o n ss h o w s t h a t u p t o t h e c o n c e n t r a t i o n o f 0 . 0 1 6 % ( p H 5 . 0 ) t h e e ff e ct o f a c ido n t h e r a t e o f t h e g r o w t h o f Rosel l inia i s v e r y l i t tl e . A d i s t i n c t r e t a r d a t i o ni s, h o w e v e r , s e e n a t 0 . 0 3 2 % (p I-I 4 . 7 ) a n d 0 . 0 6 1 % ( p H 4 - 1 ) . A t a s t i ll h i g h e rc o n c e n t r a t i o n of 0 . 1 2 8 % ( p H 3 . 9) t h e g r o w t h is e x t r e m e l y f ee b l e a n dr e a c h es o n l y 4 m m . o n t h e t h i r d d a y a s o p p o s e d to t h e 2 4 m m . a t 0 - 0 6 4 % .N o g r o w t h w a s o b s e rv e d a t 0 . 2 5 6 % c o n c e n t r a t i o n ( p H 3 -1 ).

Alka l in i t y . - -Rose l l i n ia w a s g r o w n in d i f f e re n t c o n c e n t r a t i o n s o f a m m o -n i u m c a r b o n a t e a n d t h e e f fe c t o f t h e c o n c e n t r a t io n w a s f o u n d o u t b y c o u n t i n gt h e n u m b e r o f p e r i t h e c ia i n c u l t u re s o f 4~ d a y s g r o w t h i n th e s a m e w a y a si n t h e a c i d s e ri es . T h e r e s u l ts o f t h e c o u n t w h i c h i s a v e r a g e f o r t w o p l a t e sa r e g iv e n in T a b l e V I , M o n g w i t h t h e d i a m e t e r o f t h e f u n g u s g r o w t h . T h ep K v a l u e o f t h e a l k a l i n e s e r ie s is n o t g i v e n s in c e a t h i g h e r c o n c e n t r a t i o n s o f


10/14

0~ t/z e Cu l ture Behav iour o f a Specie s o f R os e l l i n ia - - I [ 31a l k a l i t h e m e d i a d e v e l o p e d a c o l o u r w h i c h i n t e r f e r e d w i t h t h e f i n d i n g o f p Hb y c o l o r im e t r i c m e t h o d .

T A B L E V I .Sh ow ing the Effect o f A lk al i on tl'be Production of Perithecia.A m m o n . c a r b . i n g i n s .p.c.

0 . 0O. 001 60 . 0 0 3 5

D i a m e t e r o f c o lo n y i nm m . ( 3 d a y s g r o w t h )

565 25 6

N u m b e r o f p e r i t h e c i a i ncu l tu re s o f 44 days g rowth

80 078064 00 . 0 0 7

O. 0150 . 0 30 . 0 60 . 1 20 . 2 50 . 51 . 02 . 0

"5 456525 6565 44416

96072 05 2 03 6 04 0 02 4 040 0480

T h e r e s u l t i n d i c a t e d t h a t i n s l i g h t l y a l k a l i n e m e d i u m t h e p e r i t h e c i a ld e v e l o p m e n t i s f a v o u r a b l e , b u t a t h i g h e r c o n c e n t r a t io n s t h e n u m b e r f a ll so ff . T h e s iz e of t h e p e r i t h e c i a t e n d t o b e c o m e s m a l l er a s t h e c o n c e n t r a t i o ni n c re a s e s a n d m a n y o f t h e m a p p e a r i n f e r ti l e a n d i m m a t u r e .I t i s r e a li s e d t h a t b o t h i n a c i d a n d a l k a li s e r ie s th e n u m b e r o f p e r i t h e c i ais b o u n d t o v a r y f r o m p l at e t o p la t e a n d w i t h e a c h e x p e r i m e n t . N e v e r t h e -l es s t h e r e s u l t s p r o b a b l y c o r r e c t l y i n d i c a t e t h e r e l a t iv e a b u n d a n c e o f p e r i-t h e c i a a t 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 i d a n d a l k a li .

Regional Distribution of Perithecia in Culture.I n t h e c o u r s e o f t h e e x p e r i m e n t s d e s c r i b e d in t h e f o re g o i n g p a g e s , i t w a s

o b s e r v e d t h a t i n c u l t u r e s w h e r e p e r i t h e c i a w e r e p r o d u c e d t h e f e r ti le a n di n fe r ti le r e g io n s v a r i e d b o t h i n e x t e n t a n d i n t h e i r r e l a t i v e p o s i ti o n s , w i t ht F . e t y p e o f t h e m e d i u m e m p l o y e d .


11/14

3 2 S.N. Das GuptaI n p l a in a g a r m e d i u m ( 1 . 5 % ) t h e p e r i t h e c i a w e re i n v a r i a b l y fm t n d t o

g r o w a t t h e c e n t r a l r eg i o n o f t h e c u l t u r e w h i l e t h e p e r i p h e r a l r e g i o n re m a i n e dc o m p l e t e l y i n fe r ti le . S i m i l a r w a s t h e c a se w h e n t h e c o n c e n t r a t i o n s e m p l o y e dw e r e 0 ...~~ a n d 1 . 0 % a g a r . B u t a s t h e c o n c e n t r a t i o n i n c r e a s e d f r o m 1 . 5 %t o 1 2 . 0 % , t h e f e rt il e z o n e g ra d u a l l y r e c ed e d t o w a r d s t h e p e r i p h e r y l e a v i n ga n i n f e rt i le re g i o n a t t h e c e n t r e . A c lo s e r i n s p e c t i o n s h o w e d t h e p r e s e n c eo f a v e r y n a r r o w i n f e rt i le z o n e a t t h e p e r i p h e r y a s w e ll.

A d d i t i o n o f g lu c os e to t h e p l a i n a g a r m e d i u m b r o u g h t a b o u t t h e s a m er e s u l t . A t 0 - 0 1 8 % g l u c o s e a d i s t i n c t i n [ e r t i! e r e g i o n a p p e a r e d i n t h e c e a t r ew h i c h i n cr e 0 s ed i n si z e a s m o r e a n d m o r e g l u c o se w a s a d d e d ( 0 . 0 5 L 0 . 1 62 ) ;t h e f e r t il e re g i o n b e c o m i n g c o r re s p o n d i n g l y n a r r o w . A l m o s t s im i l a r r e su l t sw e r e o b t a i n e d w i t h t h e l o w e r c o n c e n t r a t i o n s o{ a s p a r a g i n ( 0 . 1 % - - I - 0 % ) .A t h i g h e r c o n c e n t r a t i o n s ( e x c e p t i n g a t 5 . 0 % w h e r e n o p e r i t h e c i a a p p e a r e d )o n l y a v e r y f ew p e r i t h e c i a d e v e l o p e d a n d a l l t h o s e w e r e r e s t r i c t e d a t t h ec e n t r a l r e g io n o f t h e c u l t u r e .

I n t h e c a s e of t h e p o t a s s i u m p h o s p h a t e (K aP O4 } s e r ie s ~ h i c h h a s b e e nd e a l t ~ i t h i n a n e a r l i e r p a p e r ( D a s G u p t a , 1 93 7), t h e p o t a s s i u m n i t r a t es e r ie s , t h e a c i d s e ri e s a n d t h e a l k a l i se r ie s , t h e r e s u l t s w e r e d i f f e r e n t . I tw a s f o u n d t h a t t h e s e d i d n o t h a v e a n y a p p r e c i a b l e e f fe c t o n t h e r e g i o n a ld i s t r i b u t i o n o f p e r i t h e c ia . I n t h e e a s e o f K a P O 4, f o r e x a m p l e , t h e p e r i t h e c i ad e v e l o p e d m o s t l y in t h e c e n t r a l r e g io n a t t h e l o w e s t c o n c e n t r a t i o n e m p l o y e d( 0 . 1 0 % ) , o n l y a f e w b e i n g f o u n d a t t h e p e r i p h e r a l r eg i o n . A t h i g h e r c o n c e n -t r a t io n s a m u c h l a rg e r n u m b e r o f p e ri t h e c ia w a s f o u n d a t t h e p e r ip h e r a lr e g io n a n d a c o m p a r a t i v d y f ew a t t h e c en t re . O n l y a t t h e c o n c e n t r a t i o no f 0 . 3 % o f K a P O 4 d i d t h e c e n t r a l r e g io n p r o v e c o m p l e t e l y i n f e rt i le . N e i t h e rt h e a d d i t i o n o f a c i d h a d a n y a p p r e c ia b l e e f fe c t o n t h e d i s t r i b u t i o n o f p e r i-t h e c i a , t h a t i s t o s a y , t h e p e r i t h e c i a r e m a i n e d r e s t ri c t e d t o t h e c e n t r e a s inp l a in a g a r m e d i u m . W i t h t h e a d d i t io n o f a l ka l i t o t h e a g a r m e d i u m t w of e r t il e re g io n s a p p e a r e d i n t h e c u l t u r e o n e c e n t r a l , t h e o t h e r p e r i p h e r a l,s e p a r a t e d b y a n a r r o w , i rr e g u l a r , i n f e r ti l e z o n e .

S o m e e x p e r i m e n t s w e r e m a d e t o a s c e r t a i n t b e b e h a v i m l r o f t h e f u n g u sw h e n la r g e r a n d s m a l l e r a r e a a r e g i v e n f o r g r o w t h . P e t r i - d is h e s o f t h r e es i z e s w e r e e m p l o y e d , n a m e l y , l a era. , 1 0 e r a. ( n o r m a l ) a n d 7 e ra . d i a m e t e r ,a n d m e d i a u s e d w e r e t h o s e i n w h i c h i n f e r t i l e z o n e w a s k n o w n t o a p p e a r .I t w a s f o u n d t h a t i n l a r g e r d i s h e s c o r r e s p o n d i n g l y la r g e r i n f e r ti l e c e n t r a lz o n e a p p e a r e d i n d i c a t i n g t h a t t h e r e g io n a l d i s t r i b u t i o n o f p e r i t h e c i a i s p r o b -a b l y r e l a t e d to t h e a c c u m u l a t i o n o f s t a li n g p r o d u c t s .

Discussion.T h e r e s u lt s o b t a i n e d f r o m th i s i n v e s t i g a t i o n s u p p l e m e n t t h o s e a l r e a d y

p u b l i s h e d i n a p r e v i o u s p a p e r ( D a s G u p t a , 1 93 7). I t h a s b e e n d e f i n i t e l y s h o w n


12/14

On the Cul ture Behaviour of a Species of R o s e l l i n i a - - I I 3 3t h a t i n t h e s t a n d a r d s v n t he t ic m e d i u m t h e Rosel!inia s p. e m p l o y e d fo ri n v e s t i g a t i o n c a n n o t p r o d u c e p e r i t h e c i a u n l e ss a s s o c i a te d w i t h a s p e c ie s o fb a c t er i u m . T h e p e ri t h e ci a a r e m o s t a b u n d a n t w h e n t h e fu n g u s a n d t h eb a c t e r i u m a r e g r o w n t o g e t h e r b u t w h e n ti le t w o g r o w i n g si d e b y s id e co m ei n c o n t a c t w i t h e a c h o t h e r o n l y a fe w p e r i t h e c i a a r e i rr e g u l a r l y [ o r m e d a tt h e l in e o f j u n c t i o n . T h i s fa c t o f t h e s t i m u l a t i n g e f f e c t o f b a c t e r i a o n t h ep r o d u c t i o n o f f r u i t- b o d i e s is w e ll k n o w n a n d h a s b e e n n o t e d b y n l a n y a u t h o r s .A t t e m p t s t o a s c e r t a i n t h e c a u s e o f t h e s t i n m l a t i n g e f f e ct , h o w e v e r , p r o v e du n su c c es sf u l. I t is s h o w n t h a t t h e c o n t a m i n a t e d m y e e l i u m f o r m i n g t h ep e r it h e c ia g i v e h i g h e r a lk a li n e r e a ct i n n t h a n u n c o n t a m i n a t e d m y c e l i u m n o tp r o d u c i n g p e r i t h e c i a. T h a t a lk a l i n i t y is n o t t h e f a c t o r c o n t r o l li n g t h ep e r it h e c ia f o r m a t i o n h a s b e e n s h o w n e x p e r i m e n t a l l y b y g r o w i n g t h e u n c o n -t a m i n a t e d f u n g u s i n a l k a li n e c o n d i t io n . T h e c o n t r o l l in g f a c t o r is p r o b a b l ya n u t r i t i o n a l o n e .

T h e i n c r e a se in t h e n u m b e r o f p e r i t h e c i a w i t h t h e i n c r e a s e in a g a r c o n c e n -t r a t i o n m i g h t b e d u e t o t h e r e l a t iv e i n c r ea s e of n u t r i t i v e s a l ts p r e s e n t i na g a r . T h e d e c r e a s e i n t h e s iz e o f t h e p e r i t h e c i a s e em s , o n t h e o t h e r h a n d ,t o b e d u e t o t h e r e d u c e d w a t e r c o n t e n t a n d t h e f i r m n e s s o f t h e s u b s t r a t u m .

N i t r o g e n c o n t e n t o f t h e m e d i u m d o e s n o t p l a y a p r o m i n e n t p a r t i n t h ep r o d u c t i o n o f p e r i t h e c ia . B o t h a s p a r a g i n a n d p o t a s s i u m n i t r a t e a s s o u r c eso f n i t r o g e n b e h a v e s i m i l a r ly i n t h i s r e s p e c t. T h e p e r i t h e e i a n u m b e r d e c r e a se sa s t h e c o n c e n t r a t i o n is in c r e a s e d e x c e p t a t a l o w c o n c e n t r a t i o n o f 1 % a s p a r a g i nw h e r e a s l i g h t i n c r e a s e i n t h e p e r i t h e c i a n u m b e r i s o b s e r v e d . T h i s i s i n s p i t e o ft h e f a c t t h a t t h e v e g e t a t i v e g r o w t h o f t h e f u n g u s as m e a s u r e d b y i ts r a t e o fa d v a n c e r e m a i n s p r a c t i c a l l y t h e s a m e in al l c o n c e u t r a t i o n s e x c e p t i n v e r yh i g h o n e s .

T h e r e s u l t s o b t a i n e d w i t h a c i d a n d a l k a l i s e r i e s a r e v e r y s i m i l a r e x c e p tt h a t i n b o t h t h e c a se s p e r it h e c i a a r e p r o d u c e d e v e n a t t h o s e c o n c e n t r a t i o n sw h e r e m y c e l i a l g r o w t h s a r e g r e a t l y r e d u c e d . H e r e t o o , a s i n o t h e r s a t h i g hc o n c e n t r a t i o n s , t h e p e r i t h e c i a t e n d t o b e c o m e s m a l l a n d i n f e r t i l e a n d i n a c i ds e ri es p a r t i c u l a r l y t o g r o w i m m e r s e d in t h e m e d i u m .

W h e n c a r b o n n i t r o g e n r a t i o i s a l t e r e d t h e e f f ec t o n t h e p e r i t h e c i a d e v e lo p -m e n t d o e s n o t s e e m t o b e v e r y g r e a t . W h a t e v e r e f f ec t s a r e o b s e r v a b let h e y a r e n o t s o m u c h d u e t o t h e C/N r a t i o , a s t o t h e t h e c o m b i n e d e f f e c t o f t h eh i g h c o n c e n t r a t i o n s of t h e tw o c h e m i c a l s . A n a p p r o p r i a t e e x a m p l e is t h ep e r i t h e c i a f o r m e d a t 0 . 1 6 2 % g ! u co s e i n c o m b i n a t i o n w i t h 0 . 0 3 , 0 . 0 9 , 0 - 2 7 ,0 . 8 2 a n d 2 .4 5~ K N O 3 . I n t h e f ir s t t h r e e c o m b i n a t i o n s t h e p e r i t h e c i a f o r m e da r e m i n u t e a n d i n n u m e r a b l e b u t n e v e r t h e l e s s m a t u r e . I n f a c t , i t is a r e d u c e df o r m o f n o r m a l p e r i t h e c i a w h e r e t h e f e r t i l i t y o f t h e f r u i t - b o d i e s , t h a t i s t h ec a p a c i t y o f p r o d u c i n g a s ci a n d a s c os p o r es , h a s n o t b e e n a f f e ct e d . B u t

B3


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3 4 S .N . D a s G u p tain thelast twocombina tions {0-162% glucose with 0-82 and. 2.~5% KNO~)the reduct ion proceeds still fu rther and the l~erithecia produced are both smalland infertile.

The infertile and semi-infertile condition of the perithecia are also foundin media having similar concentrations of KNOa (0.82% and 2.45%) but lessof glucose (0.018% and 0.05'~%). The size of the perithecia, however, is inthese cases comparatively bigger. It should be further noted tha t in thecomplete absence of glucose, KNO 3 does no t ~ffect the production of asci andascospores. Glucose therefore seems to exert a deleterious effect not onlyon the production of perithecia (Das Gupta, 1937) but also on the fer tili ty ofthose which are produced when combined with certain concentrations ofKNOa. The immature and iufertile condition of the fruit-bodies have beencorrelated to the increase in the glucose content together with the high KNO:,content in the medium. It is to be noted, however, t hat both in the acid andin the alkali series at high concentrations employed the perithecia appear assmall infertile bodies usually immersed in the medium ; but even in these smallperithecia the asci and ascospores do not show any deviation from the normalone, excepting that the y take longer to mature. Similarly in high agarconcentrations; however small the perthecia, lhey are usually mature.

A consideration of facts regarding the regional distribution of peritheciasuggests that the phenomenon is in some way connected with the alteredcondition of the media due to the accumulation of staling products in theadvancing region of culture. Fur the r work wiil be required before it is possibleto arrive at any definite conclusion.

S u m m a r y .The present 1Japer deals mainly with the effect, of various chemicals

(asparagin, KNOa, acid, alkali and C/N ratio) on the production of peritheciain a species of Rosellinia and supplements the results given in a previouspaper (Das Gupta, 1937). The stinmlating effect of a bacterium on the forma-tion of perithecia is briefly deo]t with.

The following are the main points : ~The number o fper i th ic ia formed in a medium decreases with the increase

in the concentra tion of asparagin, potassium nitrate, acid and alkali. Insimple agar medium the higher agar concentration the greater the number ofperithecia formed. INnumerable peritheeia appear in media combining rela-tively h~gh content of potassium nitrate with moderate amount of glucose.

The size of the perithecia is only slightly reduced in media combiningre!atively high concentration of asparagin and potassium nitrate with low


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O n t h e C u l l u r e t ? e h a v i o u r o / a S p e c i es o / R o s e l l i n i a - - / / 3 5c o n c e n t r a t i o n s of g l u c o se . T h e s iz e i s g r e a t l y r e d u c e d i n t h e m e d i a w h e r eK N O 3 c o n t e n t a n d g l u c o s e c o n t e n t , a r e b o t h r e l a t i v e l y h i g h .

A d d i t i o n o f g l uc o s e i n a m e d i u m h a v i n g r e l a t iv e l y h ig h K N O 3 c o n t e n tha s a deleterious effect on the production o[ asci an d a scospores.

D e v e l o p m e n t o f p e r i th e c i a is i n h i b i t e d m u c h b e f o re t h e v e g e t a t i v e g r o w t ho f t h e f u n g u s i s r e t a r t e d d u e t o h i g h c o n c e n t r a t i o n s o f c h e m i c a l s e x c e p t i n t h ec a se s o f t h e a c i d a n d a l k a li s e ri es w h e r e t h e p e r i t h c c i a d e v e l o p a s l o n g a sv e g e t a t i v e g r o w t h i s n o t t o t a l ly c h e c k e d .

R e g i o n a l d i s t r i b u t i o n o f p e r i t h e c i a i n d i f f e re n t m o d i f i e d m e d i a is b r ie f lyd e a l t w i t h .

LI TE RA TUR E CITED.Das Gupta, S. N., "On the culture behaviour of a species of RoseIlinia--I. Inhibitory effectof certain chemicals on the production of perithecia, " .Tour. Ind. Bot . Soc., 1937, 16.

Sinergii Din 1937

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