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The isolation and identification of soft rot Erwiniafrom Saguaro (Carnegiea gigantea) flowers
Item Type text; Thesis-Reproduction (electronic)
Authors Schuyler, Mary Esther, 1943-
Publisher The University of Arizona.
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Download date 10/05/2021 03:59:01
Link to Item http://hdl.handle.net/10150/551992
THE ISOLATION AND IDENTIFICATION OF SOFT ROT ERWINIA
FROM SAGUARO (CARNEGIEA GIGANTEA) FLOWERS
by
M ary E s th e r S c h u y le r
A T h e s is S u b m it te d to th e F a c u l ty o f th e
DEPARTMENT OF MICROBIOLOGY
In P a r t i a l F u l f i l l m e n t o f t h e R e q u ire m e n ts F o r th e D eg ree o f
MASTER OF SCIENCE
In th e G ra d u a te C o l le g e
THE UNIVERSITY OF ARIZONA
1 9 6 8
STATEMENT BY AUTHOR
T h is t h e s i s h a s b e e n s u b m i t t e d i n p a r t i a l f u l f i l l ment o f r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t The U n i v e r s i t y o f A r i z o n a and i s d e p o s i t e d i n t h e U n i v e r s i t y L i b r a r y to be made a v a i l a b l e t o b o r r o w e r s u n d e r r u l e s o f th e L i b r a r y .
B r i e f q u o t a t i o n s f rom t h i s t h e s i s a r e a l l o w a b l e w i t h o u t s p e c i a l p e r m i s s i o n , p r o v i d e d t h a t a c c u r a t e a c k n o w l edgment o f s o u r c e i s made. R e q u e s t s f o r p e r m i s s i o n f o r e x t e n d e d q u o t a t i o n f rom o r r e p r o d u c t i o n o f t h i s m a n u s c r i p t i n w hole o r i n p a r t may be g r a n t e d by t h e h e a d o f t h e m a j o r d e p a r t m e n t o r t h e Dean o f t h e G r a d u a t e C o l l e g e when i n h i s ju d g m e n t t h e p r o p o s e d u s e o f t h e m a t e r i a l i s i n t h e i n t e r e s t s o f s c h o l a r s h i p . I n a l l o t h e r i n s t a n c e s , h o w e v e r , p e r m i s s i o n m u s t be o b t a i n e d f rom t h e a u t h o r .
SIGNED:
APPROVAL BY THESIS DIRECTOR
T h is t h e s i s h a s b e e n a p p r o v e d on t h e d a t e shown b e lo w :
ALCORNS. M.P r o f e s s o r o f P l a n t P a t h o l o g y
ACKNOWLEDGMENTS
I w ish to th a n k D r. S t a n le y A lc o rn f o r h i s
g u id a n c e , u n d e r s t a n d in g an d c o n t i n u a l a s s i s t a n c e d u r in g
t h i s s t u d y , and th e o t h e r m em bers o f my c o m m itte e , D r.
W aybum S . J e t e r an d D r. I r v i n g Y a l l , f o r t h e i r h e l p f u l
comm ents when r e v ie w in g th e m a n u s c r ip t . I w ish to
ack n o w led g e my g r a t i t u d e to S u p e r in te n d e n t o f th e S a g u a ro
N a t io n a l M onum ent, R o b e r t L . G i l e s , f o r p e r m is s io n to w ork
in t h a t a r e a , and to th e A g r i c u l t u r a l R e s e a rc h S e r v ic e
o f th e U n ite d S t a t e s D e p a r tm e n t o f A g r i c u l t u r e f o r th e
f i n a n c i a l s u p p o r t t h a t h e lp e d make t h i s s tu d y p o s s i b l e .
i i i
TABLE OF CONTENTS
Page
LIST OF TA B LES.......................................................................................... v
ABSTRACT......................................................................................................... v i
INTRODUCTION ......................... 1
MATERIALS AND METHODS ...................................................................... 9
P l a n t S e l e c t i o n ........................................................................... 9C o l l e c t i o n T e c h n iq u e ................................................................. 10I s o l a t i o n T e c h n i q u e s ............................. 12C u l t u r e s ............................................................................................... 16M e d i a .................................................................................................... 18A n t i b i o t i c s ..................................................................................... 20P a t h o g e n i c i t y ................................................................................ 20
R E S U L T S ......................................................................................................... 22
I s o l a t i o n s fro m F lo w e rs ....................................................... 22I d e n t i f i c a t i o n o f O rg an ism s ............................................. 25
D ISC U SSIO N .................................................................................................... 30
LITERATURE C IT E D ........................ 36
i v
LIST OF TABLES
T a b le Page
1 . S o u rc e s o f i s o l a t e s o f E rw in ia s p e c i e st e s t e d ............................................................................................... 17
2 . F r a c t i o n o f s a g u a ro p o s tb lo o m s c o n ta in i n gs o f t r o t t i n g b a c t e r i a .............................................. 24
3 . B io c h e m ic a l r e a c t i o n s o f E rw in ia c a m e g ia n aand s o f t r o t E r w i n i a ........................................................... 26
4 . Some d i f f e r e n c e s b e tw e e n E rw in ia c a r n e g ia n a and th e i s o l a t e s o f th e t h r e e s p e c i e s o f s o f t r o t E r w i n i a ................................................................. 28
v
ABSTRACT
The o b j e c t i v e s o f t h i s s tu d y w ere to d e te rm in e i f
E rw in ia c a m e g ia n a i s p r e s e n t i n s a g u a ro f lo w e r s and to
c l a r i f y th e ta x o n o m ic p o s i t i o n o f th e o rg a n is m . A
s e l e c t i v e b r o t h c o n ta in i n g p e c t i n , s a l t s , g lu ta m ic a c i d ,
and 4 p g /m l e ry th r o m y c in , an d a c a lc iu m p o l y p e c t a t e d i f f e r
e n t i a l s o l i d medium w ere em ployed in i s o l a t i o n s fro m
s a g u a ro f l o w e r s . E le v e n p e r c e n t o f p o s tb lo o m s (p e rm a n e n t ly
c lo s e d f lo w e r s ) w ere fo u n d to c o n ta in p e c t o l y t i c , s o f t
r o t t i n g , Gram n e g a t iv e b a c t e r i a . O nly two o f a t o t a l o f
189 C a c to b r o s i s l a r v a e fo u n d f r e e on th e arm s ( b r a n c h e s )
o r i n th e f lo w e r s o f th e s a g u a ro w ere fo u n d to c o n ta in s o f t
r o t t i n g b a c t e r i a . E . c a m e g ia n a i s v e ry s i m i l a r b i o
c h e m ic a l ly to th e s p e c i e s E. c a r o t o v o r a , E . a r o i d e a e , and
E. a t r o s e p t i c a . S even o f 12 i s o l a t e s o f E. c a m e g ia n a
p ro d u c e d s o f t r o t o f c a r r o t s l i c e s ; a l l o f them s o f t r o t t e d
to m ato f r u i t s . The o rg a n is m s h o u ld be p la c e d i n th e g enus
P e c to b a c te r iu m W aldee w i th th e o t h e r s o f t r o t E r w in ia ,
p r o b a b ly in th e same s p e c i e s .
v i
INTRODUCTION
The s a g u a ro o r g i a n t c a c tu s ( C a m e g ie a g i g a n t e a
B r i t t an d R o se ) i s th e m o s t o b v io u s an d b e s t known o f th e
S o n o ran D e s e r t p l a n t s i n th e Low er S o n o ran L i f e Z one. I t
h a s come to b e th e sym bo l o f th e w e s t and d e s e r t l i f e in
g e n e r a l , and o f A r iz o n a i n p a r t i c u l a r . I t s f lo w e r i s th e
s t a t e f lo w e r o f A r iz o n a .
P o s s i b l y th e f i r s t w r i t t e n a c c o u n t o f a s o f t
r o t t i n g d i s e a s e o f th e s a g u a ro was t h a t o f H ubbard i n 1899
( 1 1 ) ; H ubbard was p r o b a b ly th e f i r s t to i n o c u l a t e an
a p p a r e n t l y h e a l t h y p l a n t w i th t i s s u e from a r o t t i n g p l a n t .
On r e v i s i t i n g th e s i t e l a t e r , h e r e p o r t e d , " I fo u n d th e
p l a n t a lm o s t e n t i r e l y consum ed by th e r o t w i th w h ich I h ad
i n f e c t e d i t . O th e r t r u n k s w h ich I h ad c u t down a b o u t th e
same tim e w i th o u t i n o c u l a t i o n h av e n o t r o t t e d an d a r e s t i l l
sound and g r e e n ."
L i g h t l e , S t a n d r in g , and Brown (1 6 ) w ere th e f i r s t
to i s o l a t e E rw in ia c a m e g ia n a S ta n d r in g fro m r o t t i n g
t i s s u e s and to v e r i f y i t a s th e c a u s a t iv e a g e n t o f th e
n e c r o t i c d i s e a s e o f th e s a g u a r o . They gave th e f o l lo w in g
d e s c r i p t i o n o f th e d i s e a s e a s c a u se d by E . c a m e g i a n a :
The f i r s t symptom o f th e b a c t e r i a l n e c r o s i s o f C a m e g ie a i s a s m a l l , c i r c u l a r , l i g h t - c o lo r e d s p o t u s u a l l y w i th w a te r so a k e d m a rg in , on th e s u r f a c e o f th e t r u n k and b r a n c h e s .
1
2
U n d e rn e a th th e s u r f a c e d i s c o l o r a t i o n th e s o f t p a re n c h y m a to u s t i s s u e s becom e w a te r - s o a k e d a f t e r b a c t e r i a l i n v a s io n an d s u b s e q u e n t ly brow n to a lm o s t b l a c k . As th e i n f e c t i o n p r o g r e s s e s th e s p o t e n l a r g e s and a ssu m es a p u r p l i s h h u e .In c a s e s i n w h ich th e i n f e c t i o n a d v a n c e s r a p i d l y th e c e n t r a l p a r t o f th e s p o t b r e a k s and a brow n l i q u i d e x u d e s . R a p id d e s t r u c t i o n o f th e t i s s u e s i s th e n a cc o m p a n ie d by " b le e d in g " b u t s lo w e r i n t e r n a l d e c a y may p re c e d e e x u d a t io n .The r o t t e d t i s s u e s d r y , b r e a k up i n t o g r a n u l a r to lumpy p i e c e s and f a l l to t h e g ro u n d , l e a v in g th e woody s t e l a r s t r a n d s b a r e .
T h is d i s e a s e c a u s e d a 29.3% m o r t a l i t y i n a s t a n d o f
1380 c a c t i i n th e S a g u a ro N a t io n a l Monument e a s t o f T u c s o n ,
A r iz o n a i n th e 21 y e a r s fro m 1942 to 1961 ( 3 ) . I f m or
t a l i t y due to b a c t e r i a l n e c r o s i s c o n t in u e s a t i t s p r e s e n t
r a t e an d th e p ro b le m s r e l a t i n g to s e e d l i n g e s t a b l i s h m e n t
p e r s i s t , i t was p r e d i c t e d b y A lc o rn an d May ( 3 ) t h a t th e
s t a n d w i l l b e w ip ed o u t b y 2000 A. D. S in c e i n th e U . S .
s a g u a ro s n a t u r a l l y e x i s t i n A r iz o n a a n d , o n ly m a r g i n a l l y ,
i n C a l i f o r n i a , t h e r e l a t i o n s h i p o f b a c t e r i a l n e c r o s i s to
p o p u l a t i o n d e c r e a s e o f t h i s c a c tu s i s o f c o n t in u in g
c o n c e rn .
T h e re i s c o n f u s io n in th e l i t e r a t u r e c o n c e rn in g th e
p r o p e r ta x o n o m ic p o s i t i o n o f some m em bers o f th e g en u s
E rw in ia , i n c l u d i n g E . c a m e g i a n a . P h y to p a th o g e n ic
b a c t e r i a may be d iv id e d i n t o g ro u p s b a s e d on th e c h a n g e s
th e y make in p l a n t t i s s u e . S o f t - r o t p r o d u c in g , c o l i f o r m
b a c t e r i a p o s s e s s enzym es t h a t b r e a k down th e p e c t i c s u b
s t a n c e s i n th e m id d le l a m e l l a o f th e c e l l w a l l s o f
p a re n c h y m a to u s t i s s u e s , th u s m ak in g p l a n t t i s s u e s s o f t and
3
s l im y ( 9 ) . I n a d d i t i o n to p o s s e s s i n g p e c t i c e n zy m es , t h e s e
b a c t e r i a a r e Gram n e g a t i v e , p e r t r i c h o u s l y f l a g e l l a t e d , an d
f a c u l t a t i v e l y a n a e r o b i c . They re d u c e n i t r a t e to n i t r i t e ,
and f e rm e n t many c a r b o h y d r a te s to a c i d ( 9 ) . S in c e th e y a r e
s i m i l a r to e n t e r i c b a c t e r i a , th e y h av e b e e n p la c e d i n th e
gen u s E rw in ia .
The g en u s E r w in ia , h o w e v e r , a l s o c o n ta in s o t h e r
b a c t e r i a w h ich do n o t c a u s e " s o f t r o t " d i s e a s e s . One o f
th e s e s p e c i e s , th e ty p e s p e c i e s o f th e g e n u s , E rw in ia
a m y lo v o ra ( B u r r i l l ) W inslow e £ a l . , c a u s e s f i r e b l i g h t o f
p e a r and a p p le , a d ry n e c r o s i s . The r e c o g n iz a b le and
im p o r ta n t d i f f e r e n c e s b e tw e e n th e n o n - s o f t r o t t i n g m em bers
o f th e E r w in ia , r e p r e s e n t e d b y E . a m y lo v o ra , and th e s o f t
r o t t i n g s p e c i e s , E . c a r o to v o r a ( J o n e s ) H o l la n d , E. a r o id e a e
(T ow nsend) H o l l a n d , E . a t r o s e p t i c a (v a n H a l l ) J e n n i s o n , and
E. c h ry s a n th e m i B u rk h o ld e r e t a l . , h av e b e e n p o i n te d o u t by
s e v e r a l d i f f e r e n t a u t h o r s , f i r s t b y W aIdee ( 5 ) , l a t e r by
S u t to n ( 2 8 ) , Graham ( 9 ) , and L o c k h a r t an d K o en ig ( 1 7 ) . The
l a c k o f s i m i l a r b io c h e m ic a l c h a r a c t e r i s t i c s i s so d e f i n i t e
t h a t t h e s e a u th o r s b e l i e v e i t i s n e c e s s a r y to r e c o g n iz e th e
two g ro u p s a s d i s t i n c t g e n e r a b y rem o v in g th e p e c t o l y t i c
s p e c i e s fro m E rw in ia to th e g en u s P e c to b a c te r iu m W ald ee .
S u t to n (2 8 ) h a s s u g g e s te d t h a t th e s e b e re d u c e d to two
s p e c i e s o f P e c to b a c t e r iu m , i . e . , P . c a r o to v o r a and P .
c h r y s a n th e m i . The b io c h e m ic a l ly d e f in e d P . c a r o to v o r a
w o u ld , t h e n , in c lu d e E. a r o i d e a e and E. a t r o s e p t i c a .
4
H ow ever, Graham ( 9 ) r e c o g n iz e s o n ly one s p e c i e s , P .
c a ro to v o ru m . T h is h e d i v id e s i n t o f o u r v a r i e t i e s , P .
c a ro to v o ru m , i t s e l f , P . c a ro to v o ru m v a r . a t r o s e p t i c a , P .
c a ro to v o ru m v a r . a r o i d e a e , and P . c a ro to v o ru m v a r .
c h ry s a n th e m i♦
The o rg a n is m r e s p o n s i b l e f o r th e n e c r o s i s o f th e
s a g u a ro c a c t u s , E . c a m e g i a n a , was o r i g i n a l l y d e s c r ib e d
(1 6 ) a s b e in g a Gram p o s i t i v e , p e r i t r i c h o u s , e n c a p s u l a t e d ,
n o n sp o ro g e n u s r o d . I t was n o te d a s b e in g s i m i l a r to E.
c a r o to v o r a i n th e ty p e o f d i s e a s e p ro d u c e d , an d in
r e a c t i o n s to m o s t m ed ia t e s t s . H ow ever, when E . c a r o to v o r a
was i n o c u l a t e d i n t o th e s a g u a ro no d i s e a s e r e s u l t e d , and
when E . c a m e g i a n a was i n o c u l a t e d i n t o c a r r o t s and o t h e r
h o s t s o f E . c a r o t o v o r a no s o f t r o t t i n g o c c u r r e d .
A p p a r e n t ly , b e c a u s e o f th e d i f f e r e n c e i n Gram s t a i n
r e a c t i o n and p a t h o g e n i c i t y , t h e o rg a n is m was c o n s id e r e d a
new s p e c i e s .
The Gram r e a c t i o n was c o n f irm e d b y B o y le ( 4 ) who
n o te d , h o w e v e r , t h a t o ld l a b o r a t o r y s t o c k c u l t u r e s becam e
Gram n e g a t i v e . On th e o t h e r h a n d , B u rk h o ld e r fo u n d t h i s
o rg a n is m to be Gram n e g a t i v e , w i th o ld c u l t u r e s show ing
Gram p o s i t i v e g r a n u le s ( 5 ) . B o y le fo u n d E. c a m e g ia n a to
b e lo n g to th e s o f t r o t g ro u p on th e b a s i s o f p h y s i o l o g i c a l
and c u l t u r a l s i m i l a r i t i e s to E . c a r o t o v o r a . H ow ever, i t
was a l s o fo u n d to h a v e a th e r m a l d e a th p o i n t o f 59 C, 7 C
above t h a t p u b l i s h e d f o r E . c a r o t o v o r a , and to b e s e n s i t i v e
5
to p e n i c i l l i n , w h i le E . c a r o t o v o r a was n o t . S u t to n (2 8 )
and Graham (9 ) i n t h e i r s t u d i e s o f th e gen u s E rw in ia
exam ined c u l t u r e s o f E. c a m e g ia n a and fo u n d them n o t to
c a u se p e c t o l y s i s an d s t a t e d t h e r e f o r e t h a t i t c o u ld n o t be
a s o f t r o t t e r . In d e e d th e l i t e r a t u r e i s c o n fu s e d r e g a r d in g
E . c a m e g i a n a .
S e v e r a l d i f f e r e n t p o s s i b l e m odes o f t r a n s m i s s io n o f
E. c a m e g ia n a h av e b e e n p ro p o s e d b y B o y le ( 4 ) : r o o t to
r o o t t r a n s m i s s i o n , i n f e c t i o n fro m c o n ta m in a te d s o i l ,
f a l l i n g o f an i n f e c t e d p l a n t o r b ra n c h a g a i n s t a h e a l t h y
p l a n t , an d b y i n s e c t s . A lc o rn and May ( 3 ) s u b s t a n t i a t e d
th e o b s e r v a t i o n o f B o y le ( 4 ) t h a t p r o x im i ty o f h e a l t h y to
i n f e c t e d p l a n t s c o u ld b e a f a c t o r i n t r a n s m i s s i o n . T ak acs
(2 9 ) fo u n d t h a t i n j u r i e s m u s t b e p r e s e n t i n o r d e r f o r i n f e c
t i o n to o c c u r when s a g u a ro s e e d l i n g r o o t s a r e e i t h e r d ip p e d
in b a c t e r i a l s u s p e n s io n s o r p l a n t e d on s o i l i n f e s t e d w i th
b a c t e r i a . I n th e f i e l d , r o o t s may be i n j u r e d b y th e a c t i o n
o f w e a th e r , r o d e n t s , o r i n s e c t s ( 4 ) .
B o y le c o n s id e r e d th e n i g h t f l y i n g m o th , C a c to b r o s i s
f e m a l d i a l i s H u l s t to be th e m o s t im p o r ta n t v e c t o r co n
c e rn e d w i th th e s p r e a d o f b a c t e r i a l n e c r o s i s . When
d i s e a s e d c a c t i w ere c u t down, and s l i c e d t h r u th e m id d le
l o n g i t u d i n a l l y , many t u n n e l s w i th o c c a s i o n a l r o t p o c k e ts
o c c u r r in g a lo n g them w ere r e v e a l e d . E . c a m e g i a n a was
i s o l a t e d from th e t i s s u e s s u r r o u n d in g th e tu n n e l s an d th e
s u r f a c e and i n t e s t i n e o f th e C a c to b r o s i s f e m a l d i a l i s
6
l a r v a e r e s p o n s i b l e f o r th e t u n n e l s . On r e a r i n g th e s e
l a r v a e i n c a g e s , th e b a c te r iu m was i s o l a t e d from th e a d u l t
and th e s u r f a c e o f th e e g g s . When l a r v a l d e s c e n d a n ts o f
th o s e c o l l e c t e d fro m th e t u n n e l s w ere i n o c u l a t e d i n t o
c a c t i , t y p i c a l s o f t r o t c h a r a c t e r i s t i c o f th e d i s e a s e
r e s u l t e d .
B o y le b e l i e v e d t h a t C a c to b r o s i s l a r v a e h a d one l i f e
c y c le a y e a r w i th th e l a r g e s t p a r t s p e n t t u n n e l i n g i n th e
c a c t u s . She o b s e rv e d l a r v a e l e a v in g p l a n t s to p u p a te u n d e r
d e b r i s on th e g ro u n d from May th ro u g h A u g u s t. I n
l a b o r a t o r y s t u d i e s , p u p a t io n r e q u i r e d 28 -33 d a y s ; th e a d u l t
n o c t u r n a l m oth l a i d i t s eg g s an d d ie d w i t h in a t h r e e day
p e r i o d . The e g g s r e q u i r e d 30 d a y s to h a t c h .
B u t l e r an d W ern er ( 6 ) c o l l e c t e d C a c to b r o s i s a d u l t s
in a b l a c k l i g h t t r a p i n th e S a g u a ro N a t io n a l Monument
d u r in g 1960 an d 1 9 6 1 . A d u l ts f i r s t a p p e a re d i n M arch i n
s m a ll n u m b e rs . They w ere num erous i n A p r i l and May, b u t
w ere m o s t num erous i n J u n e . S m a ll num bers w ere fo u n d in
J u l y , A u g u s t, and S e p te m b e r .
From o u r own o b s e r v a t i o n s l a r v a e ch an g e c o l o r a s
th e y d e v e lo p : a t 2 mm th e y a r e p in k , from 3 to 15 mm
bro w n , an d from 18 to 24 mm b l u e . S m a ll p in k l a r v a e can
e n t e r s m a l l g re e n h o u s e r e a r e d p l a n t s a n y w h e re , h o w ev er th e y
can o n ly e n t e r l a r g e c a c t i n e a r th e a p e x ( t h e g ro w in g
p o i n t ) due t o h e a v y c u t i n i z a t i o n e ls e w h e re ( 4 ) . L a rv a e o f
a l l s i z e s , h o w e v e r , h a v e b e e n fo u n d i n s i d e th e f lo w e r s t h a t
7
fo rm a crow n a ro u n d th e a p e x i n May an d J u n e (A lc o rn and
B u t l e r , u n p u b l is h e d 1 9 6 7 ) . O f te n l a r v a e a r e s e e n e a t i n g
t h e i r way o u t o f f lo w e r s ; th e y c o u ld e n t e r th e m ain body o f
th e c a c tu s a t t h i s t im e .
B o y le ( 4 ) r e c o v e r e d E . c a m e g ia n a fro m p a r t i a l l y
r o t t e d f lo w e r s and f r u i t s fo u n d on th e g ro u n d a ro u n d c a c t i .
No l a r v a e w ere fo u n d i n th em , b u t h o le s th o u g h t to h a v e
b e e n made b y th e l a r v a e w ere fo u n d . B o y le (4 ) and T ak acs
(2 9 ) c a u s e d f lo w e r s to r o t b y s p r a y in g them w i th s u s p e n
s io n s o f E . c a m e g i a n a . T hese f i n d in g s r a i s e d th e q u e s t io n
o f w h e th e r th e f lo w e r s c o n t a in i n g E. c a m e g ia n a fo u n d on
th e g ro u n d b y B o y le w ere i n f e c t e d on th e p l a n t o r a f t e r
f a l l i n g , s i n c e th e b a c te r iu m c an s u r v iv e i n s o i l ( 4 , 2 9 ) .
The q u e s t io n a r o s e , a l s o , w h e th e r th e C a c to b r o s i s l a r v a e
w ere r e s p o n s i b l e f o r th e i n f e c t i o n o f f lo w e r s o r i f
p o s s i b l y th e r e v e r s e m ig h t be th e c a s e .
The p u rp o s e o f t h i s i n v e s t i g a t i o n was f o u r f o l d :
1 . To d e te rm in e i f E . c a m e g ia n a i s p r e s e n t i n an y o f
th e f lo w e r s t a g e s — p re b lo o m ( b u d ) , b loom (o p en
f l o w e r ) , o r p o s tb lo o m (p e rm a n e n t ly c lo s e d f lo w e r )
on th e p l a n t .
2 . I f th e f lo w e r s a r e i n f e c t e d on th e p l a n t , th e n to
d e te rm in e w h a t r e l a t i o n s h i p e x i s t s b e tw e e n th e
p r e s e n c e o f C a c to b r o s i s l a r v a e i n th e f lo w e r s an d
th e p r e s e n c e o f E . c a m e g i a n a ; t h a t i s , to d e te rm in e
i f l a r v a e c a r r y th e b a c t e r i a to th e f l o w e r s .
3 . I f t h e C a c to b r o s i s l a r v a e a r e n o t c a r r y i n g th e
b a c te r iu m to th e f l o w e r s , th e n to d e te rm in e i f
f lo w e r s a r e b eco m in g c o n ta m in a te d b y f l y i n g i n s e c t s
o r d u s t - b o r n e b a c t e r i a .
4 . To m ore f u l l y b io c h e m ic a l ly c h a r a c t e r i z e E.
c a m e g ia n a an d to com pare i t w i th some o f th e
o t h e r s p e c i e s o f E rw in ia i n h o p e s o f c l e a r i n g up
some o f th e c o n f u s io n i n th e l i t e r a t u r e a b o u t E.
c a m e g i a n a .
8
MATERIALS AND METHODS
P l a n t S e l e c t i o n
S a g u a ro c a c t i i n th e E a s t D iv i s io n o f th e S a g u a ro
N a t io n a l Monument an d i n th e W est D iv i s io n (3 0 m i le s away
on th e w e s t s lo p e s o f th e T ucson M o u n ta in s ) w ere s e l e c t e d
f o r r e c o v e r y e x p e r im e n ts i n 1 9 6 6 . In a l l , 110 p l a n t s w ere
c h o se n t h a t h a d f lo w e r s on th e a p ic e s o f t h e i r m ain s tem s
o r b r a n c h e s (a rm s ) t h a t w ere a c c e s s i b l e e i t h e r fro m th e
g ro u n d o r fro m a d e c k on th e r o o f o f a p a n e l t r u c k . T h ree
o f th e 110 w ere o b v io u s ly d i s e a s e d . Of th e p l a n t s i n th e
E a s t D iv i s io n t e n w ere t r e a t e d a s c o n t r o l s ; i n o r d e r to
k e ep o u t i n s e c t s , one f lo w e r in g arm was c o v e re d w i th t h r e e
to f o u r l a y e r s o f c h e e s e c l o th and a n o n - s y s te m ic i n s e c t i
c i d e , ORTHO-KLOR-44 (O rth o D i v i s i o n , C a l i f o r n i a C h em ica l
Company, San F r a n c i s c o , C a l i f . ) was p a i n t e d a ro u n d th e arm
b e lo w th e c h e e s e c l o th to fo rm a band a p p ro x im a te ly an in c h
w id e . A lth o u g h th e i n s e c t i c i d e was a p p l i e d to p r e v e n t
c r a w l in g i n s e c t s fro m v i s i t i n g th e f lo w e r s o f th e s a g u a r o ,
a n t s and C a c to b r o s i s l a r v a e w ere s u b s e q u e n t ly s e e n on m o s t
a rm s .
T w e n ty - f iv e s a g u a ro c a c t i i n th e n o r t h c a c tu s
f o r e s t o f t h e E a s t D iv i s io n o f th e S a g u a ro N a t io n a l Monu
m ent w ere s e l e c t e d f o r th e r e c o v e r y e x p e r im e n ts i n 1967 .
The p l a n t s w ere a l l 20 to 25 f e e t t a l l w i th two to f i v e
9
10
arm s e x c e p t f o r one a p p ro x im a te ly t e n f e e t t a l l w i th o n ly
one a rm . P l a n t s o f t h i s d e s c r i p t i o n w ere c h o se n a t
random ; t h a t i s , p l a n t s w ere c h o se n t h a t c o u ld b e re a c h e d
from th e ro a d b y t r u c k and h a d two f lo w e r in g t r u n k a n d /o r
b r a n c h t i p s t h a t c o u ld b e r e a c h e d when th e c o l l e c t o r s to o d
on th e to p o f th e t r u c k . None o f th e p l a n t s was o b v io u s ly
d i s e a s e d ; d i s e a s e d p l a n t s w ere i n th e a r e a b u t n o n e was
c l o s e r th a n 70 f e e t . On e a c h p l a n t one f lo w e r in g a p e x was
t r e a t e d w i th c h e e s e c l o th and i n s e c t i c i d e a s d e s c r ib e d f o r
1966 .
C o l l e c t i o n T e c h n iq u e
C o l l e c t i o n s w ere made fro m a p p ro x im a te ly 7 AM to
10 AM. The c o l l e c t o r ' s h a n d s w ere w ashed w i th 2% AMPHYL
(L ehn an d F in k P ro d u c ts C o r p . , B lo o m f ie ld , N. J . ) b e f o r e
m oving from one p l a n t to a n o th e r , and f r e q u e n t l y m o is te n e d
w i th AMPHYL b e tw e e n f lo w e r c o l l e c t i o n s fro m a g iv e n p l a n t .
Each f lo w e r s t a g e rem oved was p la c e d i n a new , s m a l l , brow n
p a p e r b a g on w h ich was r e c o r d e d th e p l a n t n u m b er, w h e th e r
o r n o t t h e a p e x was c o v e re d w i th c h e e s e c l o t h , th e s t a g e o f
th e f lo w e r , and i f t h e r e w ere s ig n s o f l a r v a e b e in g
p r e s e n t . I f a C a c to b r o s i s l a r v a was v i s i b l e , i t was p la c e d
in a s t e r i l e , s c re w c a p p e d v i a l w i th th e a i d o f AMPHYL
t r e a t e d f o r c e p s . Any l a r v a e fo u n d c ra w l in g f r e e on th e
b ra n c h w ere a l s o p la c e d i n a s e p a r a t e , s t e r i l e v i a l .
11
I n 1966 c o l l e c t i o n s w ere made fro m one u n c o v e re d
f lo w e r in g a p e x on e a c h p l a n t e x c e p t f o r th e t e n p l a n t s t h a t
h a d c o v e re d a p i c e s ; from t h e s e , f lo w e r s w ere c o l l e c t e d from
two a p i c e s , one c o v e re d an d one u n c o v e re d . From e a c h o f
th e p l a n t s up to t h r e e b loom s an d t h r e e p o s tb lo o m s w ere
rem oved from e a c h a rm . C o l l e c t i o n s w ere m ade on s e v e n
d i f f e r e n t d ay s i n th e p e r i o d o f May 6 to Ju n e 1 4 . E ach
p l a n t was c o l l e c t e d fro m o n ly o n c e , e x c e p t f o r th e c o v e re d
and n o n c o v e re d arm s on th e same p l a n t s , two c o l l e c t i o n s
w ere made fro m t h e s e . A t o t a l o f 323 p o s tb lo o m s an d 250
b loom s w ere c o l l e c t e d from u n c o v e re d a p i c e s ; 32 p o s tb lo o m s
and 20 b loom s fro m c o v e re d a p i c e s .
I n 1967 c o l l e c t i o n s w ere made fro m one c o v e re d and
one u n c o v e re d a p e x on e a c h p l a n t . On May 11 , 46 c o v e re d
and 45 u n c o v e re d p re b lo o m s , 8 c o v e re d an d 15 u n c o v e re d
b lo o m s , and 9 c o v e re d an d 15 u n c o v e re d p o s tb lo o m s w ere
c o l l e c t e d from a t o t a l o f 16 p l a n t s . N o t m ore th a n t h r e e
o f e a c h s t a g e w ere c o l l e c t e d from e a c h a p e x . On th e
s u c c e e d in g c o l l e c t i o n d a te s May 16 , 2 0 , 2 3 , an d 2 6 , o n ly
c o v e re d an d u n c o v e re d p o s tb lo o m s w ere c o l l e c t e d . No m ore
th a n s i x o f e a c h came fro m e a c h a p e x . L a b o r a to r y
p r o c e s s in g p r o c e d u r e s n e c e s s i t a t e d r e s t r i c t i n g th e c o l l e c
t i o n s to 250 p o s tb lo o m s o r l e s s on a g iv e n d a t e . B e c a u se
o f t h i s , and th e p l a n t to p l a n t v a r i a t i o n o f s u i t a b l e
f lo w e r s t a g e s , a t t im e s m a t e r i a l was c o l l e c t e d fro m a s few
a s 18 p l a n t s . I n a l l , 401 c o v e re d p o s tb lo o m s an d 405
12
u n c o v e re d p o s tb lo o m s w ere c o l l e c t e d . I n a d d i t i o n to t h e
l a r v a e c o l l e c t e d on th e c o v e re d an d u n c o v e re d a rm s , 146
m ore l a r v a e w ere c o l l e c t e d fro m o t h e r p l a n t s b e tw e e n May
20 an d Ju n e 9 .
I s o l a t i o n T e c h n iq u e s
In 1966 , i n th e l a b o r a t o r y , e a c h f lo w e r was p la c e d
i n a s t e r i l e , g l a s s , p e t r i d i s h , and s l i c e d l o n g i t u d i n a l l y
w i th a f la m e d s c a l p e l . One s e c t i o n was p la c e d i n a t e s t
tu b e c o n ta in i n g 10 m l o f s t e r i l e d i s t i l l e d w a te r . I f a
l a r v a was fo u n d i n a f l o w e r , i t was p la c e d i n a s e p a r a t e
t u b e . A f t e r n o t m ore th a n o n e - h a l f h o u r , th e tu b e c o n te n t s
w ere m ixed on a V o r te x s t i r r e r and th e n one l o o p f u l was
s t r e a k e d f o r i s o l a t i o n on a p o t a t o d e x t r o s e p e p to n e (PDF)
a g a r p l a t e . T h is m edium c o n ta in s 39 g B a c to - p o ta to
d e x t r o s e a g a r , 10 g B a c to - p e p to n e , 5 g B a c to - a g a r p e r
l i t e r o f d i s t i l l e d w a te r ; t h i s was a d j u s t e d to pH 7 .5
b e f o r e a u to c l a v i n g a t 15 p s i f o r 15 m in u te s . A f t e r 24 h r
i n c u b a t i o n a t 30 C an y c o lo n i e s r e s e m b l in g E . c a m e g ia n a
w ere p ic k e d o f f an d s t r e a k e d on a PDF s l a n t . A f t e r 24 h r
in c u b a t io n th e b a c t e r i a on th e s e s l a n t s w ere su s p e n d e d i n
3 m l o f s t e r i l e d i s t i l l e d w a te r an d up to 1 /4 m l o f t h i s
s u s p e n s io n was i n o c u l a t e d w i th a s t e r i l e , d i s p o s a b l e
s y r in g e i n t o a two y e a r o ld s a g u a ro s e e d l i n g . A f t e r 48
h r , b a c t e r i a w ere r e i s o l a t e d fro m d i s e a s e d s e e d l i n g s b y
s t r e a k i n g a s m a l l am ount o f a s o p t i c a l l y i s o l a t e d , s o f t
13
r o t t e d t i s s u e on PDF p l a t e s . S u b s e q u e n t ly , b a c t e r i a l
g ro w th was c h e c k e d f o r c o lo n y m o rp h o lo g y an d Gram r e a c t i o n .
To f a c i l i t a t e th e i s o l a t i o n o f E. c a m e g i a n a from
s a g u a ro f lo w e r s a new i s o l a t i o n te c h n iq u e was d e v e lo p e d in
1967 . The a b i l i t y to p ro d u c e p e c t i c enzym es d o e s n o t
n e c e s s a r i l y mean an o rg a n is m i s a p l a n t p a th o g e n ( 1 9 ) ;
h o w e v e r , s o f t r o t t i n g E rw in ia p ro d u c e p e c t i c enzym es m ore
r a p i d l y an d i n g r e a t e r v a r i e t y th a n n o n -p a th o g e n s (1 4 , 1 9 ) .
F o r t h i s r e a s o n a s e l e c t i v e b r o t h was d e v e lo p e d b y
m o d ify in g N asuno and S t a r r ' s medium f o r p e c t i c enzyme
p r o d u c t io n b y E. c a r o to v o r a (2 2 ) by th e a d d i t i o n o f
e ry th r o m y c in , a m a c r o l id e a n t i b i o t i c a c t i v e a g a i n s t Gram
p o s i t i v e b a c t e r i a i n v e ry low c o n c e n t r a t i o n s ( 1 2 ) . S in c e
e ry th ro m y c in i s a b a s i c m o le c u le and i t s a c t i v i t y i n c r e a s e s
f i v e f o l d f o r an i n c r e a s e o f one u n i t o f pH i n th e ra n g e
from 6 .5 to 7 .5 ( 1 2 ) , t h e medium was a d j u s t e d to pH 8 .0 .
The s e l e c t i v e m edium c o n ta in e d 2 s e p a r a t e l y a u to c la v e d
p a r t s . P a r t A was a 1% s o l u t i o n o f S u n k i s t P e c t i n N. F .
# 3 4 6 6 . P a r t B was 0.24% KHPO^, 0.08% N a2HP04 , 0.05%
MgS04 *7 HgO, 0.3% (NH4 ) 2S04 , 0.3% g lu ta m ic a c i d . To p a r t B
was a d d ed enough 10 N NaOH to make th e pH o f th e co m b in a
t i o n o f A an d B 8 .0 . One m l o f a s to c k 4 m g/m l e ry th ro m y c in
s o l u t i o n was made up to a volum e o f 25 m l w i th s t e r i l e pH 8
p h o s p h a te b u f f e r an d th e n a d d ed to th e pH 8 .0 c o m b in a t io n
o f p a r t s A and B . The s t o c k e ry th ro m y c in s o l u t i o n was p r e
p a re d by d i s s o l v i n g 4 m g/m l e ry th ro m y c in b a s e i n a s m a l l
14
q u a n t i t y o f r e a g e n t g ra d e m e th a n o l and th e n m ak ing up to
volum e w i th s t e r i l e pH 8 p h o s p h a te b u f f e r . The s o l u t i o n
can b e k e p t f o r no m ore th a n one week a t 4 C.
In o r d e r to d e te rm in e th e maximum c o n c e n t r a t i o n o f
e ry th ro m y c in t h a t c o u ld b e u s e d w i th o u t i n h i b i t i n g th e
g ro w th o f E . c a m e g i a n a , tu b e s w i th t e n m l a l i q u o t s o f th e
s e l e c t i v e m edium c o n ta in i n g r e s p e c t i v e l y 0 , 2 , 4 , an d 8
p g /m l e ry th ro m y c in w ere i n o c u l a t e d w i th 0 .1 m l p h y s i
o l o g i c a l s a l i n e c o n ta in i n g a p p ro x im a te ly 2 x 10^ c e l l s o f
E. c a r n e g i a n a . A d u p l i c a t e s e t o f tu b e s was i n o c u l a t e d
w i th 0 .1 m l p h y s i o l o g i c a l s a l i n e c o n ta in i n g a p p ro x im a te ly
2 x 10^ c e l l s o f S ta p h y lo c o c c u s a u r e u s . A f t e r i n c u b a t io n
f o r 24 h r a t 30 C, t e n f o l d s e r i a l d i l u t i o n s i n p h y s i
o l o g i c a l s a l i n e w ere made o f th e b r o t h i n e a c h tu b e . T e n th
m l p o r t i o n s o f e a c h d i l u t i o n w ere p l a t e d t h r e e t im e s and
th e num ber o f c o lo n i e s d e v e lo p in g a f t e r 24 h r a t 30 C w ere
c o u n te d . From th e i n o c u l a t e d tu b e s c o n ta in i n g 0 , 2 , 4 , an d
8 p g /m l o f e r y th r o m y c in , t h e r e w ere r e s p e c t i v e l y i s o l a t e d
an a v e ra g e o f 38 + 5 x 1 0 ^ , 38 + 6 x 1 0 ^ , 210 + 34 x 1 0 ^ ,
and 204 + 24 x 10^ c e l l s / m l o f E . c a r n e g i a n a . T h e re w ere
no S . a u re u s c e l l s i n an y o f t h e tu b e s c o n ta in i n g e r y t h r o
m y c in ; i n t h e tu b e c o n ta in i n g no e ry th ro m y c in t h e r e w ere
202 + 21 x 10^ c e l l s / m l . F o u r p g /m l th u s a p p e a re d to b e
th e h i g h e s t c o n c e n t r a t i o n o f e ry th ro m y c in a t t h i s pH t h a t
can b e u s e d to i n h i b i t Gram p o s i t i v e o rg a n is m s and s t i l l
e f f e c t i v e l y i n h i b i t E . c a r n e g i a n a .
15
A m o d i f i c a t i o n o f th e s o l i d c a lc iu m p o l y p e c t a t e g e l
d e v e lo p e d by S t a r r (2 6 ) was u s e d a s a s o l i d , d i f f e r e n t i a l
medium f o r s o f t r o t E r w in ia . I t c o n ta in e d 15 g sod ium
p o l y p e c t a t e ( S u n k i s t ) , 2 .2 5 m l 1 N NaOH, 3 m l f r e s h 10%
C a C l2 '2 H2 O, 0 .5 m l 1.6% brom th y m o l b lu e ( i n 95% e th a n o l )
and 500 m l d i s t i l l e d w a t e r . The i n g r e d i e n t s w ere m ixed in
a W aring B le n d o r f o r one m in u te and th e n a u to c la v e d i n an
u n p lu g g e d f l a s k f o r 15 m in u te s a t 15 p s i . To th e 500 m l o f
medium 50 m l o f a m e l te d 4% a g a r s o l u t i o n was a d d e d , an d
th e m ix tu r e p o u re d im m e d ia te ly . The g e l c a n n o t be
r e m e l te d ; th e f i n a l c o l o r i s g r a s s g re e n w i th a pH o f 6 . 5 .
On t h i s m edium , i s o l a t e d c o lo n i e s o f E . c a m e g ia n a and
o t h e r s o f t r o t t i n g E rw in ia h y d r o ly z e th e p e c t a t e so t h a t
c y l i n d r i c a l h o l e s a r e fo rm ed w h ich e x te n d to th e b o tto m o f
th e p e t r i d i s h i n 48 to 72 h r a t 30 C; o t h e r b a c t e r i a grow
on th e s u r f a c e o n ly , o r fo rm m ore s h a l lo w d e p r e s s io n s i n
th e g e l i n th e sam e p e r i o d o f t im e .
In 1967 , th e b loom and p o s tb lo o m s w ere s l i c e d
l o n g i t u d i n a l l y an d p la c e d i n 50 m l o f s e l e c t i v e m edium .
A f t e r 24 h r a t 30 C 0 .1 m l o f th e b r o t h was rem oved w i th a
s t e r i l e d i s p o s a b l e 1 m l p i p e t t e , and s p r e a d ( w i th a f la m e d
b e n t g l a s s r o d ) on th e s o l i d d i f f e r e n t i a l m edium . E ach
l a r v a fo u n d i n a f lo w e r was rem oved an d p la c e d i n a s t e r i l e
tu b e c o n ta in i n g s e l e c t i v e b r o t h and s u b s e q u e n t ly t r e a t e d in
th e same m anner a s th e f l o w e r s . I f th e l a r v a was i n th e
b a s e o f th e f lo w e r , th e b a s e was c u t o f f and p la c e d i n a
s e p a r a t e tu b e o f s e l e c t i v e b r o t h .
B a c t e r i a fro m th e h o l e s o r l i q u i f i e d a r e a s o f th e
d i f f e r e n t i a l p l a t e s w ere s t r e a k e d w i th a s t e r i l e lo o p f o r
i s o l a t i o n on a c o m p a ra b le p l a t e . B a c t e r i a fro m i s o l a t e d
h o le s on th e l a t t e r p l a t e s w e re s t r e a k e d on PDF p l a t e s ,
i n c u b a te d , and o b s e rv e d f o r c o lo n y a p p e a ra n c e . C o lo n ie s
w i th m o rp h o lo g y i n th e ra n g e o f v a r i a t i o n o f th o s e o f th e
s o f t r o t t i n g E rw in ia w ere Gram s t a i n e d (1 5 ) an d s t r e a k e d
on PDF s l a n t s . T hese s l a n t s w ere u s e d f o r d e te r m in in g
p a t h o g e n i c i t y i n s a g u a ro s e e d l i n g s a s was done i n 1 9 66 .
C u l tu r e s
Tw elve i s o l a t e s o f E . c a m e g ia n a w ere e x am in e d :
t h r e e from r o t t i n g s a g u a r o s , 1 -1 2 , 106 , an d 6 4 -2 8 , i s o l a t e d
i n 1957 , 1958 , an d 1964 r e s p e c t i v e l y ( s t o c k s from D e p a r t
m en t o f P l a n t P a t h o l o g y ) , and n in e from s a g u a ro f l o w e r s ,
6 5 -1 6 4 , 6 5 -1 4 5 , an d 6 5 -2 1 4 i s o l a t e d i n 1965 , and 66 -185
th ro u g h 6 6 -190 i s o l a t e d in 1966 . In a d d i t i o n , t h r e e
i s o l a t e s e a c h o f E. a r o i d e a e , E . c a r o t o v o r a , an d two
i s o l a t e s o f E . a t r o s e p t i c a w ere t e s t e d (T a b le 1 ) . O rg an ism s
u se d a s c o n t r o l s on th e p h y s i o l o g i c a l t e s t s w ere o b ta in e d
from th e D e p a r tm e n t o f M ic ro b io lo g y a t th e U n i v e r s i t y o f
A r iz o n a ; th e y in c lu d e d E s c h e r i c h i a c o l i (M ig u la ) C a s t e l l o n i
and C h a lm e rs , S e r r a t i a m a rc e s e e n s B i z i o , P r o te u s v u l g a r i s
16
H a u s e r , Pseudom onas a e r u o g in o s a ( S c h r o e t e r ) M ig u la ,
T a b le 1 . S o u rc e s o f i s o l a t e s o f E rw in ia s p e c i e s t e s t e d
S p e c ie s Code No. S o u rc e D ate
E. a r o id e a e Ea 14 D ic k e y , C o r n e l l 1964
E. a r o id e a e Ea 6 D ic k e y , C o r n e l l 1964
E. a r o i d e a e Ea 139 S t a r r , U .C . a t D a v is 1965
E. c a r o to v o r a Ec 6 D ic k e y , C o r n e l l 1964
E. c a r o to v o r a Ec 14 D ic k e y , C o r n e l l 1964
E . c a ro to v o r a E c^ lS F r ie d m a n , USDAa 1964
E. a t r o s e p t i c a E 28 D ic k e y , C o r n e l l 1965
E. a t r o s e p t i c a E 70 D ic k e y , C o r n e l l 1964
a The l a t e D r. F rie d m a n was a t t h e USDA M a rk e tP a th o lo g y L a b . , N .Y .C . H is code n o . f o r th e i s o l a t e E c^ lS was E S 4 -7 . The o t h e r code num bers a r e th e num bers t h a t th e s e n d e r s u s e d .
18
S ta p h y lo c o c c u s a u re u s R o se n b a c h , and A e r o b a c te r a e ro g e n e s
(K ru s e ) B e i j e r i n c k .
M edia
S tu d ie s w ere done to d e te rm in e w h e th e r rh a m n o se ,
d u l c i t o l , s a l i c i n , s o r b i t o l , s u c r o s e , d e x t r o s e , l a c t o s e ,
and m a n n i to l w ere f e r m e n te d . The m ed ia c o n ta in e d 10 g
B a c to - p e p to n e , 10 g c a r b o h y d r a t e , and 1 m l o f a 1.6% s o l u
t i o n o f B a c to -b ro m th y m o l b lu e ( i n 95% e th a n o l ) p e r l i t e r
o f d i s t i l l e d w a te r an d w ere s t e r i l i z e d b y f i l t r a t i o n
th ro u g h EKS2 f i l t e r s h a v in g 0 .1 m ic ro n p o re s i z e ( M ic r o c e l l
F i l t e r C o ., C ro s s e P o in te F a rm s, M ic h . ) . They w ere
in c u b a te d one w eek a t 30 C to c h e c k f o r s t e r i l i t y b e f o r e
i n o c u l a t i o n . The r e s u l t s w ere r e a d e v e ry o t h e r d ay f o r th e
f i r s t w eek and tw ic e a w eek a f t e r t h a t f o r a t o t a l o f 3
w e e k s .
O th e r b io c h e m ic a l s t u d i e s i n c lu d e d : t e s t s f o r
h y d r o l y s i s o f g e l a t i n ( u s in g n u t r i e n t a g a r p l a t e s co n
t a i n i n g 0.4% g e l a t i n ) , th e u s e o f c i t r a t e a s a s o l e s o u rc e
o f c a r b o n , th e p r o d u c t io n o f ( u s in g 1% t r y p to n e b r o t h
and le a d a c e t a t e p a p e r s ) , and th e r e d u c t io n o f n i t r a t e to
n i t r i t e . T hese w ere done a s s p e c i f i e d i n th e M anual o f
M ic r o b io l o g i c a l M ethods ( 2 5 ) . P o s i t i v e c o n t r o l s w e re :
S . m a rc e s c e n s on th e n u t r i e n t g e l a t i n , A. a e ro g e n e s f o r
c i t r a t e u t i l i z a t i o n on B a c to -K o s e r c i t r a t e m edium , an d E.
19
c o l i f o r th e r e d u c t i o n o f n i t r a t e to n i t r i t e on B a c to -
N i t r a t e a g a r .
I n d o le p r o d u c t io n an d m o l i t i t y w ere c h e c k e d w i th
B ac to -S IM m edium w i th P . v u l g a r i s a s th e p o s i t i v e c h e c k .
U re a se p r o d u c t io n was d e t e c t e d b y th e u s e o f C h r i s t e n s e n 's
u r e a a g a r m edium and p h e n y la la n in e d e am in a se b y B a c to -
p h e n y la la n in e d e a m in a se b y B a c to - p h e n y la la n in e a g a r ; i n
b o th c a s e s P . v u l g a r i s s e r v e d a s th e p o s i t i v e c h e c k . The
p r e s e n c e o f o x id a s e was d e te rm in e d b y S t e e l e ' s (2 7 )
m o d i f i c a t i o n o f K o v a k 's (1 3 ) te c h n iq u e u s in g P . a e r u g in o s a
a s th e p o s i t i v e c o n t r o l . P ro c e d u re s u s e d f o r d e te r m in in g
th e o x i d a t i o n o f g lu c o n a te to 2 k e to - g l u c o n a t e w ere th o s e
o f Graham and Dowson ( 1 0 ) , w i th A. a e ro g e n e s a s th e p o s i
t i v e c o n t r o l . The p r e s e n c e o f p e c t i c enzym es was d e t e c t e d
w ith S t a r r ' s c a lc iu m p o l y p e c t a t e g e l ( 2 6 ) . The p r o d u c t io n
o f a r g i n i n e , l y s i n e , o r n i t h i n e and g lu ta m ic a c id
d e c a rb o x y la s e s was c h e c k e d by i n c o r p o r a t i n g th e s e am ino
a c id s i n t o s e p a r a t e l o t s o f B a c to - d e c a r b o x y la s e b a s e
m edium . P . a e r u o g in o s a was th e p o s i t i v e c h e c k f o r
a r g i n i n e d e c a r b o x y la s e , and £ . m a rc e s c e n s f o r ly x in e and
o r n i t h i n e . P . v u l g a r i s was th e p o s i t i v e c h e c k f o r g lu ta m ic
a c i d d e c a r b o x y l a s e , a c c o r d in g to th e m ethod o f M o e l le r
( 20) .
F o r th e m e th y l r e d an d V o g e s -P ro s k a u e r r e a c t i o n s ,
o rg a n ism s w ere grow n two d a y s a t 30 C i n Bacto-M R-VP b r o t h .
The m e th y l r e d t e s t was p e r fo rm e d a s s p e c i f i e d i n th e
20
M anual o f M i c r o b io l o g i c a l M e th o d s; th e V o g e s -P ro s k a u e r t e s t
was done a c c o r d in g to th e " S ta n d a rd M eth o d s" o f th e APHA
(1 9 4 6 ) a s g iv e n i n th e same m an u a l ( 2 5 ) . E . c o l i and A.
a e ro g e n e s w ere u s e d i n b o th t e s t s a s c o n t r o l s .
A n t i b i o t i c s
S e n s i t i v i t y to p e n i c i l l i n G was a s s a y e d u s in g th e
p e t r i p l a t e m ethod (1 2 ) e m p lo y in g 1 /2 in c h p a p e r d i s k s (N o.
7 4 0 -E , C a r l S c h l e i c h e r an d S c h u e l l C o ., K eene , N . Y . ) on
w h ich was p i p e t t e d .0 9 m l o f th e p e n i c i l l i n s o l u t i o n . The
" b a s e " l a y e r was 20 m l o f G and R a g a r m edium #1 and th e
" s e e d " l a y e r was 4 m l o f G and R a g a r m edium #2 ( 1 2 ) .
F iv e r e p l i c a t e d i s k s w ere t e s t e d on e a c h o rg a n is m a t e a c h
c o n c e n t r a t i o n o f p e n i c i l l i n .
P a t h o g e n i c i t y
P a t h o g e n i c i t y to c a r r o t s , to m a to f r u i t s , and
s a g u a ro s e e d l i n g s was c h e c k e d . C a r r o t s w ere s u r f a c e
s t e r i l i z e d f o r f i v e m in u te s w i th 1 :1 0 0 0 H g C ^ and th e n
r i n s e d t h r e e t im e s w i th s t e r i l e d i s t i l l e d w a t e r . F o u r
1 cm t h i c k s l i c e s w ere p la c e d i n s t e r i l e , g l a s s p e t r i
d i s h e s on p i e c e s o f s t e r i l e m o is te n e d f i l t e r p a p e r . T h ree
o f th e s l i c e s w ere i n o c u l a t e d w i th 0 .0 5 ml o f a s u s p e n s io nq
c o n ta in i n g 5 -1 0 x 10 c e l l s p e r m l; th e c o n c e n t r a t i o n o f
th e s u s p e n s io n was d e te rm in e d s p e c t r o p h o t o m e t r i c a l l y ( 2 9 ) .
On th e f o u r t h s l i c e 0 .0 5 m l o f s t e r i l e d i s t i l l e d w a te r was
p i p e t t e d . I f t h e c a r r o t s w ere s o f t r o t t e d a g l a s s ro d
c o u ld be e a s i l y p u sh e d th ro u g h th e s l i c e a f t e r 24 h r a t
30 C.
Tomato f r u i t s , two y e a r o ld s a g u a ro s e e d l i n g s (1 to
1 -1 /2 in c h ) and f o u r y e a r o ld s a g u a ro s e e d l i n g s (3 to 4
in c h e s ) w ere s u r f a c e s t e r i l i z e d w i th 2% AMPHYL 15 m in u te s
p r i o r to i n o c u l a t i o n w i th b e tw e e n 1 /4 and 1 /2 m l o f th e
b a c t e r i a l s u s p e n s io n by m eans o f a s t e r i l e d i s p o s a b l e
s y r i n g e . T h ree o f e a c h w ere i n o c u l a t e d ; one was i n j e c t e d
w i th 1 /4 to 1 /2 m l s t e r i l e d i s t i l l e d w a te r .
21
RESULTS
I s o l a t i o n s from F lo w e rs
In May an d Ju n e o f 1966 , 4 o f 323 u n c o v e re d p o s t
b loom s c o n ta in e d E . c a m e g i a n a . Of 32 c o v e re d p o s tb lo o m s
2 c o n ta in e d th e b a c t e r iu m . The p o s tb lo o m s from w h ich
th e s e i s o l a t i o n s w ere made d id n o t c o n ta in C a c to b r o s i s
l a r v a e . E. c a m e g ia n a c o u ld n o t be i s o l a t e d fro m 250
u n c o v e re d and 20 c o v e re d b lo o m s .
The s t r i k i n g l y low in c id e n c e o f E. c a m e g ia n a
r e c o v e r e d fro m u n c o v e re d p o s tb lo o m s c a s t some d o u b t on th e
e f f i c i e n c y o f th e i s o l a t i o n te c h n iq u e u s e d . U s in g th e new
te c h n iq u e an a t t e m p t was made i n 1967 to d e te rm in e how few
c e l l s o f E . c a m e g ia n a i n f lo w e r s c o u ld b e d e t e c t e d . S in c e
num erous a t t e m p t s i n 1966 h a d f a i l e d to a l lo w i s o l a t i o n o f
E. c a m e g ia n a fro m b loom s c o l l e c t e d a t 7 AM a f t e r h a v in g
b e e n open s i n c e a b o u t 10 PM th e p r e c e d in g e v e n in g , b loom s
w ere s e e d e d and th e n r e c o v e r y by th e new te c h n iq u e
a t t e m p te d . Ten b loom s e a c h w ere i n o c u l a t e d w i th 560 + 90
c e l l s , 56 + 9 c e l l s , an d 4 -6 c e l l s o f E. c a m e g ia n a
su sp e n d e d i n 0 .1 m l o f p h y s i o l o g i c a l s a l i n e . Ten b loom s
w ere l e f t a s u n i n o c u l a t e d c o n t r o l s . A f t e r 4 h r th e b loom s
w ere s l i c e d l o n g i t u d i n a l l y , and p u t i n 125 m l f l a s k s
c o n ta in i n g th e s e l e c t i v e b r o t h . A f t e r 24 h r a t 30 C,
0 .1 m l o f th e b r o t h was rem oved from e a c h f l a s k w i th a
22
s e p a r a t e , s t e r i l e d i s p o s a b l e 1 m l p i p e t t e and s p r e a d ( w i th
a f la m e d b e n t g l a s s ro d ) on th e s o l i d d i f f e r e n t i a l m edium .
T y p ic a l su n k en c o lo n ie s w ere o b t a in e d a f t e r 48 h r fro m a l l
i n o c u l a t e d b lo o m s . W hile i t was n o t c e r t a i n t h a t th e
i n o c u l a t e d f lo w e r s d id n o t a l r e a d y c o n ta in E. c a m e g i a n a ,
no t y p i c a l su n k e n c o lo n ie s w ere fo u n d on th e p l a t e s s p r e a d
w i th th e b r o t h c o n ta in i n g th e t e n c o n t r o l b lo o m s .
U sin g th e new i s o l a t i o n te c h n iq u e t h r e e o f th e 15
u n c o v e re d p o s tb lo o m s c o l l e c t e d May 1 1 , 1967 w ere fo u n d to
c o n ta in E. c a m e g i a n a - l i k e b a c t e r i a ( i . e . , r a p i d p e c t a t e
h y d r o ly z e r s p a th o g e n ic to s a g u a ro s e e d l i n g s ) . H ow ever,
n o n e o f th e 45 u n c o v e re d p re b lo o m s o r 15 u n c o v e re d b lo o m s ,
46 c o v e re d p r e b lo o m s , 8 c o v e re d b lo o m s , o r 9 c o v e re d
p o s tb lo o m s c o n ta in e d th e b a c t e r iu m . S in c e th e l o n g e r th e
tim e th e b loom s a r e o p e n , th e g r e a t e r th e c h an c e th e
b a c t e r i a m ig h t m u l t i p l y and make i s o l a t i o n e a s i e r , p o s t
b loom s o n ly w ere s u b s e q u e n t ly c o l l e c t e d . Of a t o t a l o f
401 p o s tb lo o m s fro m c o v e re d a rm s , o n ly two w ere fo u n d to
h av e E. c a m e g i a n a - l i k e b a c t e r i a ( .5 2 % ); o f a t o t a l o f 405
p o s tb lo o m s from u n c o v e re d arm s 45 w ere fo u n d to h a r b o r
s i m i l a r b a c t e r i a (1 1 % ). The p e r c e n t o f p o s tb lo o m s t h a t
c o n ta in e d th e s e b a c t e r i a v a r i e d w i th c o l l e c t i o n d a te
(T a b le 2 ) .
Seven p e r c e n t (2 9 /4 0 1 ) o f th e c o v e re d p o s tb lo o m s
and 4.4% (1 8 /4 0 5 ) o f th e u n c o v e re d p o s tb lo o m s e i t h e r
c o n ta in e d C a c to b r o s i s l a r v a e o r h a d t u n n e l s r e v e a l i n g t h a t
23
24
T ab le 2 . F r a c t i o n o f s a g u a ro p o s tb lo o m s c o n ta in i n g s o f t r o t t i n g b a c t e r i a .
D ate
C o v e re d a U n c o v e re d 13
PBC PBLd PB PBL
May 11 0 /6 0 /3 3 /1 1 0 /7
May 16 1 /5 3 0 /1 9 /5 0 0 /2
May 20 0 /1 0 4 1 /1 3 1 4 /1 0 5 1 /4
May 23 0 /9 0 0 / 4 7 /9 8 0 /5
May 26 1 /1 1 9 0 /8 1 2 /123 0 /0
^ F lo w e r in g t i p c o v e re d w i th c h e e s e c l o th an d arm s b a n d ed w i th 44% ORTHO-KLOR-4 4 .
-^No c h e e s e c lo th c o v e r in g ; no i n s e c t i c i d e a p p l i e d .
^ F r a c t io n o f p o s tb lo o m s t h a t h a d no s ig n s o f l a r v a e t h a t c o n ta in e d s o f t r o t t i n g b a c t e r i a .
^ F r a c t io n o f p o s tb lo o m s t h a t h a d l a r v a e i n them o r s ig n s o f l a r v a e t h a t c o n ta in e d s o f t r o t t i n g b a c t e r i a .
25
l a r v a e h a d e n t e r e d th e b a s e o f th e f l o w e r . Of t h e s e , o n ly
th e l a r v a e i n th e s e two c a s e s w ere l a r g e an d b l u e ; s i n c e
th e y w ere i n a d v e r t e n t l y s l i c e d when th e p o s tb lo o m s w ere
c u t , th e w ho le f lo w e r and th e s l i c e d l a r v a w ere c u l t u r e d
t o g e t h e r . F o r t h i s r e a s o n , i t c o u ld n o t b e d e te rm in e d
w h e th e r th e l a r v a e a lo n e h a d th e b a c t e r i a o r i f t h e
b a c t e r i a w ere i n th e u p p e r p o r t i o n o f th e p o s tb lo o m o n ly .
Of 20 l a r v a e fo u n d f r e e on th e o u t s i d e o f th e
c o v e re d a rm s , 23 fo u n d s i m i l a r l y on u n c o v e re d a rm s , an d th e
146 l a r v a e fo u n d on p l a n t s ( o t h e r th a n th e 25 fro m w h ich
f lo w e r s w ere c o l l e c t e d ) , no s o f t r o t t i n g b a c t e r i a w ere
r e c o v e r e d .
I d e n t i f i c a t i o n o f O rg an ism s
A l l th e i s o l a t e s fro m f lo w e r s an d c a c t i and th e
i s o l a t e s o f E. c a r o t o v o r a , E . a r o i d e a e , an d E. a t r o s e p t i c a
w ere fo u n d to b e m o t i l e , Gram n e g a t iv e ro d s t h a t f e rm e n te d
d e x t r o s e , rh am n o se , s a l i c i n , s u c r o s e , m a n n i to l , and
s o r b i t o l w i th o u t g a s . D u l c i t o l was f e rm e n te d by n o n e o f
them . They a l l g av e th e same r e a c t i o n s on 14 a d d i t i o n a l
p h y s i o l o g i c a l t e s t s (T a b le 3 ) . E s p e c i a l l y n o te w o r th y i s
th e p e c t a t e l i q u e f a c t i o n s i n c e t h i s i s th e " h a l lm a rk " o f
th e " s o f t r o t t e r s . " The c o n t r o l s on e a c h Gram s t a i n e d (1 5 )
s l i d e , Sy a u re u s an d E. c o l i , w ere Gram p o s i t i v e and Gram
n e g a t iv e r e s p e c t i v e l y . A l l o rg a n is m s i n o c u l a t e d i n t o t e s t
m ed ia a s p o s i t i v e c o n t r o l s p ro d u c e d p o s i t i v e r e a c t i o n s .
26
T ab le 3 . B io c h e m ic a l r e a c t i o n s o f E rw in ia c a m e g ia n a and s o f t r o t E r w in ia .
B io c h e m ic a l T e s t E. c a m e g i a n a aS o f t R o t E rw in ia "
I n d o le p r o d u c t io n - -
H2 S p r o d u c t io n 0 + +
G e l a t in h y d r o l y s i s + +
P e c ta t e l i q u e f a c t i o n + +
U re a se - -
N i t r a t e r e d u c t io n + +
G lu c o n a te t e s t - -
O x id a se t e s t - -
G row th in c i t r a t e + +
L y s in e d e c a rb o x y la s e - -
O r n i th in e d e c a rb o x y la s e - -
G lu ta m ic a c i d d e c a rb o x y la s e - -
A rg in in e d e c a rb o x y la s e - -
P h e n y la la n in e d e a m in a se - -
^ R e s u l t s o f one r e p l i c a t i o n o f t e s t s on 12 i s o l a t e s o f E. c a m e g i a n a .
^ R e s u l t s o f one r e p l i c a t i o n o f t e s t s on t h r e e i s o l a t e s o f E. a r o i d e a e an d E . c a r o t o v o r a , and two o f E. a t r o s e p t i c a T T a b le 1 ) .
CH2 S p r o d u c t io n was d e t e c t e d on l e a d a c e t a t e p a p e r s o n ly ; n o n e c o u ld b e d e t e c t e d w i th SIM m edium .
27
A ll o f th e o rg a n is m s p ro d u c e d s o f t r o t o f two and
f o u r y e a r o ld s a g u a ro s e e d l i n g s and o f to m a to f r u i t s . How
e v e r o n ly s e v e n o f th e tw e lv e c a c tu s i s o l a t e s t e s t e d w ere
a b le to p ro d u c e c o n s i s t e n t , t y p i c a l s o f t r o t o f c a r r o t s
(T a b le 4 ) . Ten o f th e tw e lv e c a c tu s i s o l a t e s p ro d u c e d a c id
d u r in g th e f e r m e n ta t i o n o f l a c t o s e o n ly a f t e r two to t h r e e
w eeks i n c o n t r a s t to a c i d p r o d u c t io n w i t h in two d a y s b y th e
t h r e e s p e c i e s o f s o f t r o t t i n g E rw in ia (T a b le 4 ) . T h e re was
g r e a t v a r i a t i o n in t h e m e th y l r e d and V o g e s -P ro s k a u e r
r e a c t i o n s g iv e n b y th e c a c tu s i s o l a t e s (T a b le 4 ) .
The c o l o n i a l m o rp h o lo g y o f E. c a m e g i a n a was
com pared w ith t h a t o f E . c a r o t o v o r a , E . a r o i d e a e , and E.
a t r o s e p t i c a . A f t e r 24 h r i n c u b a t i o n a t 30 C on PDF a g a r
p l a t e s th e c o lo n ie s o f m o s t i s o l a t e s o f E. c a r n e g ia n a w ere
s m a l l , c o n v e x , e n t i r e , sm o o th , g l i s t e n i n g , g r a y i s h o r
som ew hat o p a l e s c e n t , d e p e n d in g on th e a n g le o f th e m ic r o
sc o p e l i g h t , an d t r a n s l u c e n t b eco m in g w h i te an d m ore op aq u e
w i th a g e . L in e s o r " b a r s " c o u ld b e s e e n i n th e c o l o n i e s .
The c o lo n ie s o f th e o t h e r t h r e e s p e c i e s w ere l e s s c o n v e x ,
w i th i r r e g u l a r e d g e s an d m ore p ro n o u n c e d " b a r s . " The
c o lo n ie s o f E. c a r n e g ia n a i s o l a t e s , h o w e v e r , v a r i e d som e
w hat in th e r e g u l a r i t y o f edge an d d e g re e o f c o n v e x i ty , and
some o f them a p p ro a c h e d th e c o l o n i a l m o rp h o lo g y o f th e
t h r e e o t h e r s p e c i e s .
The minimum i n h i b i t o r y c o n c e n t r a t i o n o f p e n i c i l l i n
was fo u n d to be 0 .5 p g /m l f o r S . a u r e u s , 100 p g /m l f o r
28
T ab le 4 . Some d i f f e r e n c e s b e tw e e n E rw in ia c a m e g ia n a and th e i s o l a t e s o f t h r e e s p e c i e s o f s o f t r o t E r w in ia .
I s o l a t e No. VP MR C a rro t a L a c to s e 13
66-185 - + — — — (+ )
66-186 - + +++ (+ )
66-187 - - — — — (+ )
66-188 + - "H— (+ )
66-189 + + ++- (+ )
66 -190 + - +++ (+ )
106 ; + + +++ (+ )
1 -12 + + +++ (+ )
64-28 + - +++ (+ )
6 5 -2 1 4 - - — — — (+ )
6 5 -1 6 4 + - +++ +
65-145 + + +++ +
Ec 6 , Ec 14 - + +++ +
Ea 139 , E c118 + + +++ +
Ea 14 , Ea 6 - + +++ +
E 2 8 , E 70 - + +++ +
a C a r r o t p a t h o g e n i c i t y t e s t ; + m eans s o f t r o t o f one o f t h r e e s l i c e s i n o c u l a t e d .
k L a c to s e f e r m e n ta t i o n ; + m eans a c i d p r o d u c t io n in two d a y s ; (+ ) m eans a c i d p r o d u c t i o n d e la y e d .
i s o l a t e 6 6 -188 o f E . c a m e g i a n a , 100 y.g/ml f o r Ec 1 4 , an
i s o l a t e o f E. c a r o t o v o r a , and 10 n g /m l f o r Ea 6 , a n i s o l a t e
29
o f E. a r o i d e a e .
DISCUSSION
S a g u a ro f lo w e r s d e f i n i t e l y can b e i n f e c t e d w h ile on
th e p l a n t ; s o f t r o t t i n g b a c t e r i a can b e i s o l a t e d from p o s t
b loom s , b u t n o t fro m b loom s o r p re b lo o m s . I t seem s
im p ro b a b le t h a t th e C a c to b r o s i s f e m a l d i a l i s l a r v a i s
r e s p o n s i b l e f o r th e p r e s e n c e o f th e s e b a c t e r i a i n f lo w e r s
s in c e th e b a c te r iu m c o u ld n o t be i s o l a t e d from th e 189
l a r v a e fo u n d on th e s u r f a c e o f th e a rm s , i r r e s p e c t i v e o f
th e t r e a tm e n t o f th e a rm . F u r t h e r , o n ly 2 o f th e 47 p o s t
b loom s w i th l a r v a e w ere a l s o fo u n d to h a r b o r su c h b a c t e r i a .
S in c e 11% o f t h e u n c o v e re d p o s tb lo o m s w ere fo u n d to c o n ta in
s o f t r o t t i n g b a c t e r i a , i t i s p o s s i b l e t h a t i n s t e a d o f th e
C a c to b r o s i s l a r v a e b r i n g in g th e s e to th e f l o w e r s , th e
l a r v a e m ig h t t h e r e becom e c o n ta m in a te d . S in c e B o y le (4 )
o b s e rv e d t h a t l a r v a l t u n n e l s a p p e a re d to g e n e r a l l y
o r i g i n a t e a t o r n e a r t h e b r a n c h a p i c e s , th e l a r v a e m ig h t
c a r r y b a c t e r i a i n t o th e body o f a s a g u a ro im m e d ia te ly a f t e r
l e a v in g th e f l o w e r s , th u s c a u s in g th e d i s t r i b u t i o n o f r o t
p o c k e ts a lo n g t u n n e l s , a s d e s c r ib e d by B o y le .
E rw in ia c a m e g i a n a h a s b e e n r e c o v e r e d fro m s o i l
( 4 , 2 9 ) , t h e r e f o r e th e p o s s i b i l i t y e x i s t s t h a t f lo w e r s
c o u ld becom e c o n ta m in a te d b y w in d -h o m e d u s t . The p r e s e n t
t e s t s d id n o t s p e c i f i c a l l y i n c lu d e e x p e r im e n ts to d e te rm in e
w h e th e r o r n o t d u s t c o u ld s i f t th ro u g h th e t h r e e to f o u r
3 0
3 1
l a y e r s o f c h e e s e c l o t h . P re su m in g , h o w e v e r , t h a t d u s t
c o u ld p e n e t r a t e to th e f l o w e r s , th e f a c t t h a t o n ly o n e -
h a l f p e r c e n t o f th e p o s tb lo o m s on th e c h e e s e c l o th c o v e re d
arm s c o n ta in e d th e b a c te r iu m com pared w i th e le v e n p e r c e n t
o f th e p o s tb lo o m s from u n c o v e re d a rm s , s u g g e s t s t h a t
c o n ta m in a t io n o f f lo w e r s w i th th e b a c te r iu m i s c a u s e d b y
o t h e r m e a n s .
A num ber o f n i g h t a n d /o r day f l y i n g w in g ed a n im a ls
v i s i t th e s a g u a ro b loom s f o r p o l l e n a n d /o r n e c t a r ( 2 1 ) .
Some o f t h e s e , a s th e bum ble b e e ( Bombus L a t r e i l l e ) and
C a rp o p h ilu s l o n g i v e n t r i s S h a r p , may n e s t o r p u p a te i n th e
s o i l ( 3 , 2 2 ) . S h o u ld s u c h s i t e s be i n a r e a s t h a t c o n ta in e d
a h ig h c o n c e n t r a t i o n o f b a c t e r i a due to th e r o t t i n g o f a
c a c tu s n e a r b y , t r a n s f e r o f b a c t e r i a to f lo w e r s c o u ld be
e f f e c t e d .
Many i n s e c t s o f th e D ip te r o u s g ro u p a r e known to b e
a s s o c i a t e d w i th r o t t i n g s a g u a r o s ; o f t h e s e , S a n a ta n a (2 4 )
fo u n d V o lu c e l l a i s a b e l l i n a W i l l s to n l a r v a e to b e th e m o s t
common i n r a p i d l y r o t t i n g s a g u a r o s . W h ile o n e o r two
a d u l t s w ere o b s e rv e d b y h im a t th e o p e n in g o f r o t p o c k e t s ,
m ore w ere commonly fo u n d on f lo w e r s (p re s u m a b ly s a g u a ro
f l o w e r s , a l th o u g h th e y w ere n o t s p e c i f i c a l l y i d e n t i f i e d ) .
B o y le (4 ) c o l l e c t e d V o l u c e l l a l a r v a e fro m r o t p o c k e ts and
was a b le to i s o l a t e E rw in ia c a m e g ia n a fro m t h e i r s u r f a c e s ,
h o w e v e r, sh e c o u ld n o t i s o l a t e th e b a c te r iu m from l a b o r a
t o r y r e a r e d a d u l t s . I t i s s t i l l p o s s i b l e h o w e v e r , t h a t
3 2
a d u l t s m ig h t becom e c o n ta m in a te d w i th th e b a c te r iu m w h ile
l a y in g eg g s i n r o t p o c k e ts an d th e n l a t e r t r a n s m i t th e
b a c t e r i a to f l o w e r s . A p a r t fro m th e i m p l i c a t i o n b y S a n ta n a
( 2 3 ) , V o lu c e l l a i s a b e l l i n a h a s n o t y e t b e e n r e p o r t e d
v i s i t i n g s a g u a ro f l o w e r s . D r o s o p h i la l a r v a e a l s o i n h a b i t
r o t p o c k e ts an d h a v e b e e n shown b y G ra f ( 7 ) to b e p o s s i b l e
v e c t o r s o f E. c a m e g i a n a . A lth o u g h s m a l l D ip te r o u s i n s e c t s
w ere n o te d i n th e p r o x im i ty o f f l o w e r s , th e s e w ere n o t
i d e n t i f i e d . R e p o r ts a r e a l s o n o t known o f t h e i r v i s i t s to
s a g u a ro f l o w e r s . T h u s , p r o o f o f th e r e l a t i o n s h i p o f r o t
v i s i t i n g i n s e c t s to s a g u a ro f l o w e r c o n ta m in a t io n y e t
r e m a in s .
S a g u a ro b loom s p ro d u c e a s much a s 4 to 5 m l o f
n e c t a r ; i t i s n o t known w h e th e r E . c a m e g i a n a i s a b le to
m u l t i p ly i n th e n e c t a r . Thomas and A rk (3 0 ) fo u n d t h a t
E . a m y lo v o ra c o u ld n o t m u l t i p l y i n th e f lo w e r s o f a p p le s
and p e a r s u n l e s s r a i n d i l u t e d th e n e c t a r b e c a u s e o f th e
h ig h o s m o tic p r e s s u r e . W h e th e r o r n o t E . c a m e g i a n a c an
m u l t i p l y i n th e n e c t a r , i t i s l o g i c a l t h a t i t w ould b e a b l e
to m u l t i p l y i n th e p a re n c h y m a to u s c e l l s o f th e f lo w e r a s i t
d o es i n th e p l a n t . A f t e r th e f lo w e r c lo s e s t h e r e i s a
g re e n h o u se e f f e c t , t h a t i s , m o is tu r e i s t r a p p e d i n s i d e an d
th e h e a t from th e su n m akes a f a v o r a b le s i t u a t i o n f o r th e
g ro w th o f E . c a m e g i a n a .
E rw in ia c a m e g i a n a i s a s o f t r o t c o l i f o r m . The 12
i s o l a t e s from th e s a g u a ro c a c tu s an d s a g u a ro f lo w e r s w ere
33
fo u n d to p o s s e s s p e c t i c e n z y m e s , th e m o s t d e f i n i t i v e
c h a r a c t e r i s t i c o f t h e " s o f t r o t t e r s " ; i n 1967 t h i s f a c t was
u se d to f a c i l i t a t e i n th e i s o l a t i o n o f E . c a m e g ia n a from
s a g u a ro f l o w e r s . T h ese 12 i s o l a t e s a l s o s t a i n e d Gram
n e g a t i v e , p o s s e s s e d m o t i l i t y , re d u c e d n i t r a t e s to n i t r i t e s ,
and fe rm e n te d many s u g a r s to a c i d , a s d id th e s o f t r o t t i n g
E rw in ia t e s t e d . The c u l t u r e t h a t S u t to n (2 8 ) ex am in ed was
o b ta in e d fro m D r. Brown o f th e U n i v e r s i t y o f A r iz o n a i n
1944; i t i s p o s s i b l e t h a t i n t h e i n t e r v e n i n g y e a r s i t
becam e c o n ta m in a te d w i th a n o n - p e c t o l y t i c o rg a n is m .
Graham (9 ) d id n o t s t a t e th e s o u r c e o r age o f h i s
s u p p o s e d ly a u t h e n t i c E . c a m e g i a n a c u l t u r e s .
E. c a m e g ia n a i s v e ry s i m i l a r b io c h e m ic a l ly to th e
t h r e e s p e c i e s o f s o f t r o t t i n g E rw in ia t e s t e d ; a l l t e s t
r e a c t i o n s w ere i d e n t i c a l (T a b le 3 ) e x c e p t f o r f o u r (T a b le
4 ) . A l l o f th e r e s u l t s o b t a in e d f o r E . c a r o t o v o r a , E .
a r o i d e a e , and E . a t r o s e p t i c a a g r e e w i th th o s e o b t a in e d b y
S u t to n (2 8 ) and Graham ( 9 ) e x c e p t f o r th e a c i d p r o d u c t io n
i n th e f e r m e n ta t i o n o f s o r b i t o l . Two d i f f e r e n t l o t s o f
s o r b i t o l from D ifc o and one fro m E astm an O rg a n ic Co. w ere
t r i e d w i th a c i d p r o d u c t io n r e s u l t i n g i n a l l c a s e s . S u t to n
m e n tio n s t h a t o t h e r s h a d fo u n d th e s e o rg a n is m s to p ro d u c e
a c id from th e f e r m e n ta t i o n o f s o r b i t o l and assum ed t h a t th e
s o r b i t o l was c o n ta m in a te d w i th g l u c o s e , s i n c e i t i s u s e d to
p ro d u c e s o r b i t o l c o m m e rc ia l ly . The t h r e e l o t s o f s o r b i t o l
on w h ich a c i d was p ro d u c e d w ere t e s t e d w i th B e n e d i c t 's
3 4
q u a l i t a t i v e r e a g e n t f o r r e d u c in g s u g a r s ( 7 ) ; a p o s i t i v e
r e a c t i o n was o b ta in e d when a s a t u r a t e d s o l u t i o n was t e s t e d ,
i n d i c a t i n g th e p r e s e n c e o f r e d u c in g s u g a r s , p r o b a b ly
g l u c o s e .
The i s o l a t e o f E. c a m e g i a n a t e s t e d was n o t fo u n d
to b e m ore s e n s i t i v e to p e n i c i l l i n th a n th e i s o l a t e s o f E .
c a r o to v o r a and E . a r o i d e a e t e s t e d ; t h i s i s i n d is a g r e e m e n t
w i th th e f i n d i n g o f B o y le ( 4 ) . She d id n o t s t a t e th e pH o r
c o n c e n t r a t i o n o f p e n i c i l l i n t h a t s h e u s e d o r th e num ber o f
i s o l a t e s c h e c k e d . M ore i s o l a t e s o f E . c a r n e g ia n a and th e
o t h e r s p e c i e s s h o u ld be t e s t e d b e f o r e an y c o n c lu s io n s can
be draw n a b o u t th e r e l a t i v e s e n s i t i v i t y o f E. c a r n e g ia n a
an d th e o t h e r t h r e e s p e c i e s to p e n i c i l l i n .
I n c o n t r a s t to th e f i n d i n g o f L i g h t l e , S ta n d r in g
and Brown (1 6 ) t h a t E . c a r o t o v o r a d id n o t p ro d u c e d i s e a s e
in th e s a g u a ro and t h a t E . c a r n e g ia n a d id n o t p ro d u c e
d i s e a s e in any o f t h e h o s t s o f E. c a r o t o v o r a , E . c a r o to v o r a
h a s b e e n fo u n d to s o f t r o t s a g u a ro s e e d l i n g s , and E.
c a r n e g ia n a h a s b e e n fo u n d to c a u s e d i s e a s e i n some h o s t s
o f E. c a r o t o v o r a . A lc o rn ( 1 ) fo u n d E . c a r n e g ia n a to s o f t
r o t tom ato and s q u a s h f r u i t s an d h o n e y dew m e lo n s . A l l o f
th e i s o l a t e s i n t h i s s tu d y s o f t r o t t e d to m a to f r u i t s ; s e v e n
o f them p ro d u c e d c o n s i s t e n t , t y p i c a l s o f t r o t o f c a r r o t s .
S in c e E rw in ia c a r n e g ia n a i s a s o f t r o t t e r i t s h o u ld
be p la c e d i n th e gen u s P e c to b a c te r iu m a lo n g w i th th e o t h e r
s o f t r o t t i n g s p e c i e s o f E r w in ia . B e c a u se o f th e few
d i f f e r e n c e s t h a t e x i s t b e tw e e n i t and th e t h r e e o t h e r
s p e c i e s , th e y p ro b a b ly s h o u ld be c o n s id e r e d a s th e same
s p e c i e s .
LITERATURE CITED
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