The role of bacteria in the root and crown rot complex of irrigated sainfoin (Onobrychis viciifolia
Scop.) in Montana
by Denis Andrew Gaudet
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in PLANT PATHOLOGY
Montana State University
© Copyright by Denis Andrew Gaudet (1978)
Abstract:
Root and crown rot is believed to limit stand longevity and forage yield in sainfoin grown in Montana.
Primary objectives of this study were to: a) describe the progression of root and crown deterioration in
irrigated sainfoin, b) survey and identify the organism(s) associated with the disease complex, and c)
further describe some of the complex host-parasite interactions which exist in the root and crown rot of
sainfoin. Four different bacteria were consistently isolated from two, three and five year old naturally
infected sainfoin roots exhibiting typical symptoms. Three of these bacteria caused typical symptoms
of root and/or crown rot when reinoculated into greenhouse grown sainfoin. The three bacteria,
Pseudomonas marginal is, Pseudomonas syringae and Erwinia amylovora, appeared equally capable of
pro-ducing symptoms in the inoculated plants. Fusarium solani could not be repeatedly isolated from
symptomatic tissue. Histological evidence indicated the presence of bacterial cells within discolored
vessels and decayed cavities in the root xylem. The bacteria were closely associated with tissue
degeneration in naturally infected three and four year old sainfoin. Fungal mycelium was observed in
the crown of two year old sainfoin, but the roots exhibited only limited tissue damage. Symptoms in
greenhouse grown sainfoin inoculated with F. solani were insufficiently severe to account for the
extensive vascular degeneration associated with this disease in the field. Consequently, the causal
organisms involved in the root and crown deterioration in irrigated sainfoin appear to be caused by one
or more bacteria, rather than a single fungal pathogen. STATEMENT OF PERMISSION TO COPY
In p r e s e n t i n g t h i s t h e s i s 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 i r e m e n t s f o r an advanced d e g r e e a t Montana S t a t e U n i v e r s i t y ,
I a g r e e t h a t t h e l i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r
inspection.
I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e copying
o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by my m a jo r
p r o f e s s o r , o r , in h i s a b s e n c e , by t h e D i r e c t o r o f L i b r a r i e s .
I t is
u n d e r s t o o d t h a t any copyi ng o r p u b l i c a t i o n o f t h i s t h e s i s f o r
f i n a n c i a l g a i n s h a l l n o t ba all ow ed w i t h o u t myi w r i t t e n p e r m i s s i o n .
Sig nature_
Date
THE ROLE OF BACTERIA IN THE ROOT.AND CROWN ROT COMPLEX OF
IRRIGATED SAINFOIN ( ONOBRYCHIS: VICIIFOLIA SCOP.) IN MONTANA
by
DENIS ANDREW GAUDET
A t h e s i s submitted in p a r t i a l f u l f i l l m e n t
of th e requirem ents f o r th e degree
of
MASTER OF SCIENCE
in
PLANT PATHOLOGY
Approved-:
C h a i r p e r s o n , G r ad u at e Committee
Head, Major Department
Graduate^Dean
MONTANA STATE UNIVERSITY
Bozeman, Montana
December, 1978
iii
ACKNOWLEDGMENTS
I wish t o e x p r e s s my s i n c e r e a p p r e c i a t i o n t o t h e f o l l o w i n g :
Dr. D . ' E . Mathre f o r h i s g u id a n c e and f r i e n d s h i p d u r i n g t h e c o u r s e
o f t h e s t u d y and p r e p a r a t i o n o f t h i s t h e s i s .
Dr. R. D i t t e r l i n e f o r h i s f r i e n d s h i p and a s s i s t a n c e in c o n d u c t i n g
t h i s study.
Dr. D. Sands f o r h i s a s s i s t a n c e in i s o l a t i n g and c h a r a c t e r i z i n g
the b a c te r ia .
Dr. I . W. C a r r o l l and Dr. E. L. Sharp f o r s e r v i n g on my g r a d u a t e
com mittee.
My w i f e , M i c h e l l e , f o r h e r p a t i e n c e , c o o p e r a t i o n and lo v e which
s u p p o r t e d me t h r o u g h o u t my g r a d u a t e w or k.
TABLE OF CONTENTS
Page
V I T A ............................................................................... .... .......................................................
ACKNOWLEDGMENTS...................................................................................
TABLE OF CONTENTS
ii
iii
.......................................................................... ...........................
iv
LIST OF TABLES .......................................................................... vi
LIST OF F I G U R E S ................................ ..............................................................................vi Tl
ABSTRACT.......................................................................... .............................................
INTRODUCTION .......................................... ....
. . ...................................................
x,
. .
I
.
3
D e s c r i p t i o n and Agronomic C h a r a c t e r i s t i c s o f S a i n f o i n
. . . .
.
D i s e a s e s o f O th er Forage Legumes, Fungal D i s e a s e s
. . . . . . . .
D i s e a s e s o f Oth er Forage Legumes, B a c t e r i a l D i s e a s e s . . . . . .
3
LITERATURE R E V I E W ....................................................................
EXPERIMENT ONE:
6
7
THE PATTERN OF ROOT AND CROWN ROT
DETERIORATION IN: IRRIGATED SAINFOIN
PLANTS AND ITS PREVALENCE IN FIRST AND
SECOND YEARSAINFOINSTANDS ...............................................
9
M a t e r i a l s and M e t h o d s ....................... .
.......................................................
R e s u l t s and D i s c u s s i o n ..............................................................................
C o n c l u s i o n s ....................................................................................................................
9
1.0
17
EXPERIMENT TWO:
ISOLATION AND IDENTIFICATION OF FUNGAL
AND BACTERIAL ORGANISMS ASSOCIATION WITH
ROOT AND CROWN ROT OFS A I N F O I N ....................................
Isolation:
M a t e r i a l s and Methods
; .............................................. . . . . . . . .
R e s u l t s and D i s c u s s i o n .........................................................................
20
20
22
Identification:
M a t e r i a l s and M e t h o d s .................. ........................... .........................................
29
R e s u l t s and D i s c u s s i o n ................... . . . . . . . . .
....................... 36
Concl u s i o n s . ............................................................................................
44
V
Page
I
EXPERIMENT THREE:
EVALUATION OF THE PATHOGENICITY OF
REPRESENTATIVE BIOTYPES OF THE DIFFERENT
BACTERIAL SPECIES ISOLATED FROM DISEASED
SAINFOIN ROOTS TO GREENHOUSE REARED SAIN­
FOIN AND THE COMPARISON OF THE RELATIVE
PATHOGENICITY OF FOUR ERWINIA AMYLOVORA
STRAINS WITH THAT OF F. SOLANI ........................47
M a t e r i a l s and Methods ............................................................................................
47
R e s u l t s and D i s c u s s i o n
........................................................................................ 51
C o n c l u s i o n s ....................... , .
.......................................................................... 55
EXPERIMENT FOUR:
HISTOLOGY OF SAINFOIN ROOTS DISPLAYING
TYPICAL ROOT AND CROWN ROT SYMPTOMS
. . . .
M a t e r i a l s and Methods . ' ......................
R e s u l t s and D i s c u s s i o n ..............................................
C o n s l u s i o n s .....................................
EXPERIMENT FIVE:
EVALUATION OF THE PATHOGENICITY OF
DIFFERENT F. SOLANI FORM SPECIES AND
ISOLATES ON PEAS, BEANS, SQUASH AND .
SAINFOIN...................................................
56
56
57
64
66
M a t e r i a l s and Methods ............................................................................................. 66
R e s u l t s and D i s c u s s i o n
........................................................................................ 69
C o n c l u s i o n s ......................................................................................................................... 74
EXPERIMENT SIX:
INVESTIGATION INTO THE ROLE OF F. SOLANI
IN THE ROOT AND CROWN COMPLEX IN SAIN­
FOIN .........................................................................
77
M a t e r i a l s and M e t h o d s ................................................................................... .... . 77
R e s u l t s and D i s c u s s i o n
............................................................................................. 78
C o nc lu si on s .......................................... ........................... . . . . ............................ 83
SUMMARY AND CONCLUSIONS......................................................................
LITERATURE CITED
APPENDIX
85
. ........................................................................................................... 89
. . ' . ..........................
97
vi
LIST OF TABLES
Tab! e
1-1.
2-1.
2-2.
2-3.
2-4.
2-5..
2-6.
2-7.
3- 1 .
Page
A s u r v e y o f common r o o t and crown d e g e n e r a t i o n symp­
tom t y p e s o c c u r r i n g in n a t u r a l l y i n f e c t e d 3 and 15
months s t a n d s o f 1E s k i 1 s a i n f o i n l o c a t e d a t t h e F i e l d
Resea rc h L a b o r a t o r y , Bozeman, Montana ..........................................
11
B a c t e r i a and f un gi i s o l a t e d from s p e c i f i c symptom ty p e s
found i n 2 y e a r o l d s a i n f o i n p l a n t s c o l l e c t e d a t t h e
F i e l d Research L a b o r a t o r y , Bozeman, Montana ............................
24
B a c t e r i a and f un gi i s o l a t e d from s p e c i f i c symptom t y p e s
found in 3 y e a r o l d s a i n f o i n p l a n t s c o l l e c t a t t h e
F i e l d Research L a b o r a t o r y , Bozeman, Montana ............................
25
B a c t e r i a and fun gi i s o l a t e d a t d i f f e r e n t i n t e r v a l s
al o ng t h e c e n t r a l decay r e g i o n in t h e t a p r o o t s o f
s e v e re ly d iseased 5 y e a r old s a in f o i n c o l l e c t e d a t
t h e F i e l d Research L a b o r a t o r y , Bozeman, Montana ...................
27
Comparison o f bio c he mi c al and p h y s i o l o g i c a l c h a r a c t . e r i s t i c s o f t h e o x i d a s e n e g a t i v e Pseudomonads
i s o l a t e d from d i s e a s e d s a i n f o i n p l a n t s t o t h o s e
- p u b l i s h e d f o r Pseudomonas s y r i n g a e ..................................... ....
37
.
Comparison o f b io c h em ic al and p h y s i o l o g i c a l t e s t s
between t h e o x i d a s e p o s i t i v e Pseudomonads i s o l ­
a t e d from d i s e a s e d s a i n f o i n w i t h t h o s e p u b l i s h e d
f o r _P. m a rg i n al i s v a r . a l f a l f a e and P_. m a rg in al i s . . . .
40
Comparison o f b io ch em ic al and p h y s i o l o g i c a l c h a r a c t ­
e r i s t i c s of th e w hite b a c t e r i a with 2 s e le c te d p la n t
p a t h o g e n i c Erw ini a amylovora s p ..........................................................
43
Comparison o f b io ch em ic al and p h y s i o l o g i c a l t e s t s
o f t h e mucoid b a c t e r i a i s o l a t e d from d i s e a s e d
s a i n f o i n r o o t s w i t h t h o s e p u b l i s h e d f o r Erwinia
h e r b i c o l a v a r . h e r b i c o l a .....................................................................
45
E i g h t d i f f e r e n t b a c t e r i a l i s o l a t e s and t h e i r o r i g i n
r e p r e s e n t i n g t h e 4 b a c t e r i a l s p e c i e s used i n t h e
g r e e n h o u s e i n o c u l a t i o n s t u d y ............................................................
50
vii
Table
3-2.
3- 3 .
5- 1 .
5- 2 .
5 - 3.
6 - 1.
Page
The p a t h o g e n i c i t y o f 8 b a c t e r i a l i s o l a t e s on s a i n f o i n
s e e d l i n g s as measured by t h e e x t e n t o f d i s c o l o r a t i o n ,
from t h e i n o c u l a t i o n p o i n t and by t h e mean d i s e a s e
seven" t y ................................................................................................. * .
52
The p a t h o g e n i c i t y o f 4 i s o l a t e s o f E. amylovora and
one i s o l a t e o f £ . s o l a n i in s a i n f o i n s e e d l i n g s
as measured by t h e e x t e n t o f d i s c o l o r a t i o n from t h e
i n o c u l a t i o n p o i n t and t h e mean d i s e a s e s e v e r i t y . . . . . .
54
The o r i g i n o f t h e d i f f e r e n t formae s p e c i e s and i s o l a t e s
o f £ . s o l a n i used in. t h e p a t h o g e n i c i t y t e s t s on p e a s , ■
b e a n s , squash and s a i n f o i n
.................................................................
67
The p a t h o g e n i c i t y o f v a r i o u s formae s p e c i e s and i s o l a t e s
o f £ . s o l a n i on p e a s , b e a n s , squash and s a i n f o i n as
measured by t h e mean d i s e a s e s e v e r i t y and % i n f e c t i o n . .
70
P a t h o g e n i c i t y on peas o f £ . s o l a n i ' s a i n f o i n ' i s o l a t e s
c o l l e c t e d from s a i n f o i n p l a n t s growing in d i f f e r e n t
a r e a s i n North A m e r i c a ............................................................ ....
The e f f e c t o f removal o f t h e bark on t h e i s o l a t i o n
frequency of £ . solani in n a t u r a l l y in f e c te d 4
y e a r o l d s a i n f o i n r o o t s e c t i o n s ........................................................82
75
viii
LIST OF FIGURES
'
'
:
.
;
F ig u r e
1-1.
1-2.
Page
L a t e r a l r o o t d i s c o l o r a t i o n which e x t e n d s i n t o t h e
t a p r o o t and tow ard s t h e crown in second y e a r
s a i n f o i n ...................................................................................................
12
Black crown d i s c o l o r a t i o n i n i t i a t e d a t t h e base
o f t h e o l d dead stem in second y e a r s a i n f o i n . . . . . . .
12
1-3.
P r o g r e s s i o n o f t h e red crown d e t e r i o r a t i o n down t h e
t a p r o o t in second y e a r s a i n f o i n . ..........................................................14
1-4.
P r o g r e s s i o n o f t h e red crown d e t e r i o r a t i o n down t h e
t a p r o o t in second y e a r s a i n f o i n . . . ..........................................
14
P r o g r e s s i o n o f t h e r e d crown d e t e r i o r a t i o n down t h e
t a p r o o t i n second y e a r s a i n f o i n .......................................................
14
Black v a s c u l a r d i s c o l o r a t i o n i n i t i a t e d a t t h e base
o f two o l d dead stems in second y e a r s a i n f o i n
. . . . . .
15
Black v a s c u l a r d i s c o l o r a t i o n e x t e n d i n g from two
o l d dead stems down t h e t a p r o o t i n t h i r d y e a r
s a i n f o i n ..................................................................... ' ......................... ....
15
1-5.
1-6.
1-7.
1-8.
1-9.
Removal o f t h e bark r e v e a l s crown d i s c o l o r a t i o n and
decay which i s a f f e c t i n g t h e newly i n i t i a t e d
xylem in t h i r d y e a r s a i n f o i n ; .....................................................................
16
E x t e n s i v e r o o t and crown d i s c o l o r a t i o n and decay in
f o u r t h y e a r s a i n f o i n ...........................................................................................
16
1-10. Larval f e e d i n g g a l l e r i e s on t h e s u r f a c e on s a i n f o i n
r o o t s caus ed by t h e i n s e c t S i t o n i a s i s s i f r o n s
" ( D a y ) ...........................................................................................................
18
1-11. Larval f e e d i n g g a l l e r i e s on t h e s u r f a c e on s a i n f o i n
r o o t s caus ed by t h e i n s e c t S i t o n i a s i s s i f r o n s
(Day)
18.
1-12. Larval f e e d i n g g a l l e r i e s on t h e s u r f a c e on s a i n f o i n
r o o t s ca u se d by t h e i n s e c t S i t o n i a s i s s i f r o n s
( D a y ) ..................................... .............................................................................
18
Ix
Figure
4- 1 :
4-2.
4-3.
4-4.
4-5.
4-6.
6-1.
6-2.
'
.
,
Page
Cross s e c t i o n s o f n a t u r a l l y i n f e c t e d f o u r y e a r o ld
s a i n f o i n r o o t s d i s p l a y i n g t y p i c a l r o o t and crown
r o t symptoms . . .......................................................
58
Cross s e c t i o n s o f n a t u r a l I y i n f e c t e d f o u r y e a r o l d
s a i n f o i n r o o t s d i s p l a y i n g t y p i c a l r o o t and crown
r o t s y m p t o m s ..............................................................................
53
Cross s e c t i o n s o f n a t u r a l l y i n f e c t e d f o u r y e a r o l d
s a i n f o i n r o o t s d i s p l a y i n g t y p i c a l r o o t and crown
r o t s y m p t o m s .............................................. .... . ..........................................
61
Cross s e c t i o n s o f n a t u r a l l y i n f e c t e d f o u r y e a r o l d
s a i n f o i n r o o t s d i s p l a y i n g t y p i c a l r o o t and crown
r o t s y m p t o m s ............................................................................................
61
Cross s e c t i o n o f a n a t u r a l I y i n f e c t e d f o u r y e a r
s a i n f o i n r o o t d i s p l a y i n g t y p i c a l r o o t and crown
r o t symptoms ............................................................................................
63
Cross s e c t i o n o f n a t u r a l Iy i n f e c t e d two y e a r o l d ■
s a i n f o i n r o o t e x h i b i t i n g b l a c k crown d i s c o l o r a t i o n . . . .
63
Cross s e c t i o n s o f s i x month o l d s a i n f o i n r o o t s
a r t i f i c i a l l y i n o c u l a t e d w it h £ . s o l a n i in t h e
g r ee n ho u s e . . . ........................................................................................ ....
80
Cross s e c t i o n s o f s i x month o l d s a i n f o i n r o o t s
a r t i f i c i a l l y i n o c u l a t e d w it h F. s o l a n i in t h e
g r e e n h o u s e ..........................................................................................................
80
ABSTRACT '
Root and crown r o t i s b e l i e v e d t o l i m i t s t a n d l o n g e v i t y and f o r a g e
y i e l d in s a i n f o i n grown in Montana. Prima ry o b j e c t i v e s o f t h i s s tu d y
were t o : a) d e s c r i b e t h e p r o g r e s s i o n o f r o o t and crown d e t e r i o r a t i o n
in i r r i g a t e d s a i n f o i n , b) s u r v e y and i d e n t i f y t h e o r g a n i s m ( s ) a s s o ­
c i a t e d w it h t h e d i s e a s e complex, and c) f u r t h e r d e s c r i b e some o f th e
complex h o s t - p a r a s i t e i n t e r a c t i o n s which e x i s t in t h e r o o t and crown
rot of sainfoin.
Four d i f f e r e n t b a c t e r i a were c o n s i s t e n t l y i s o l a t e d
from two, t h r e e and f i v e y e a r o l d n a t u r a l l y i n f e c t e d s a i n f o i n r o o t s
e x h i b i t i n g t y p i c a l symptoms. Three o f t h e s e b a c t e r i a ca us ed t y p i c a l
symptoms o f r o o t a n d / o r crown r o t when r e i n o c u l a t e d i n t o greenhouse
grown s a i n f o i n .
The t h r e e b a c t e r i a , Pseudomonas ma rg in al i s . Pseudo­
monas s y r i n g a e and Erw inia a m y l o v o r a , a p pe a r ed e q u a l l y c a p a b l e o f p r o du ci n g symptoms in t h e i n o c u l a t e d p l a n t s .
Fusarium s o l a n i coul d n o t be
r e p e a t e d l y i s o l a t e d from symptomatic t i s s u e .
H is to lo g ic a l evidence
i n d i c a t e d t h e p r e s e n c e o f b a c t e r i a l c e l l s w i t h i n d i s c o l o r e d v e s s e l s and
decayed c a v i t i e s i n t h e r o o t xylem. The b a c t e r i a were c l o s e l y a s s o ­
c i a t e d w i t h t i s s u e d e g e n e r a t i o n in n a t u r a l l y i n f e c t e d t h r e e and f o u r
y e a r old s a in f o i n .
Fungal mycelium was o b s e r v e d in t h e crown o f two
y e a r o l d s a i n f o i n , b u t t h e r o o t s e x h i b i t e d only l i m i t e d t i s s u e damage.
Symptoms in g r ee n ho u s e grown s a i n f o i n i n o c u l a t e d w ith F. s o l a n i were
i n s u f f i c i e n t l y severe to account fo r th e exten siv e v ascu la r degenera­
ti o n a s s o c ia t e d w ith t h i s d is e a s e in th e f i e l d .
Consequently, the
c a u s a l organ ism s i n v o l v e d in t h e r o o t and crown d e t e r i o r a t i o n in i r r i ­
g a t e d s a i n f o i n a p p e a r t o be caus ed by one o r more b a c t e r i a , r a t h e r th a n
a s i n g l e fu nga l p a t h og en .
INTRODUCTION
.
''
.
S a i n f o i n ( Onobrychis v i c i i f o l i a S c o p . ) , a deep r o o t e d p e r e n n i a l
legume, was r e i n t r o d u c e d i n t o Montana as a f o r a g e cr o p in t h e e a r l y
1960's.
D e s i r a b l e c h a r a c t e r i s t i c s such as n o n - b l o a t , d r o u g h t r e s i s ­
t a n c e , e x c e l l e n t f o r a g e q u a l i t y and r e s i s t a n c e t o t h e a l f a l f a weevil
( Hypera p o s t i c a Hyll e n h a l ) won s a i n f o i n w id e s p r e a d p o p u l a r i t y as an
a l t e r n a t i v e cr op t o a l f a l f a .
A f t e r s e v e r a l y e a r s in u s e , i t became e v i d e n t t h a t s a i n f o i n was
p a r t i c u l a r l y s u s c e p t i b l e t o r o o t and crown r o t which s e v e r e l y reduced
s t a n d p e r s i s t e n c e and f o r a g e y i e l d .
Initial
i n v e s t i g a t o r s were l e d t o
t h e c o n c l u s i o n t h a t r o o t and crown d e t e r i o r a t i o n was i n c i t e d by t h e
f u n g a l or g an is m Fusarium s o l a n i
( M a r t . ) Appel & Wr.
O th er fungal
or ga ni s m s o c c a s i o n a l l y i s o l a t e d from d i s e a s e d s a i n f o i n p l a n t s such as
Fusarium oxysporum S c h l e c t . and R h i z o c t o n i a s o l a n i Kuehn
were r u l e d
o u t as s u s p e c t p a th og en s in s u b s e q u e n t p a t h o g e n i c i t y t r i a l s .
Conse­
q u e n t l y , a s c r e e n i n g program t o s e l e c t f o r r e s i s t a n c e t o £ . s o l a n i .
was i n i t i a t e d .
C e r t a i n i n c o n s i s t e n c i e s in t h e p r e v i o u s i n v e s t i g a t i o n s , p a r t i c u ­
l a r l y t h e i n a b i l i t y t o c o n s i s t e n t l y i s o l a t e IF. s o l a n i from t i s s u e s
d i s p l a y i n g t y p i c a l symptoms, n e c e s s i t a t e d f u r t h e r i n v e s t i g a t i o n i n t o
t h e problem.
In o r d e r t o d e v el o p a more e f f e c t i v e s c r e e n i n g program
f o r r e s i s t a n c e t o r o o t and crown r o t in s a i n f o i n , a com prehensive s tu d y
o f t h e m o r p h o l o g ic a l and h i s t o l o g i c a l a s p e c t s o f t h e r o o t d e c l i n e and
t h e i d e n t i f i c a t i o n o f t h e d i s e a s e o rg an is m s r e s p o n s i b l e f o r t h e d e c l i n e
2
was r^ecess^ry. . Thi s i n v e s t i g a t i o n a t t e m p t s t o more t h o r o u g h l y d e s c r i b e
some o f t h e complex h o s t - p a r a s i t e i n t e r a c t i o n s which e x i s t in t h e r o o t
and crown r o t complex i n s a i n f o i n .
The o b j e c t i v e s o f t h i s s t u d y were t o :
I) d e s c rib e , f u l l y , the
p r o g r e s s i o n o f t h e r o o t and crown d e t e r i o r a t i o n in s a i n f o i n th r o u g h t h e
use o f ph o to g r a p h s and r e c o r d e d f i e l d o b s e r v a t i o n s ; 2 ) s u r v e y and
i d e n t i f y t h e fu nga l and b a c t e r i a l o rg an is m s c l o s e l y a s s o c i a t e d with
n a t u r a l l y i n f e c t e d s a i n f o i n r o o t s as t h e r o o t s p r o g r e s s e d th r o u g h t h e
d i f f e r e n t s t a g e s o f d e t e r i o r a t i o n ; 3) d e t e r m in e t h e p a t h o g e n i c i t y o f
t h e or ga n is m s i s o l a t e d from d i s e a s e d s a i n f o i n r o o t s i n greenh ous e
r e a r e d s a i n f o i n s e e d l i n g s ; 4) i n v e s t i g a t e r o o t and crown d e t e r i o r a t i o n
in s a i n f o i n , , , h i s t o l o g i c a l l y , u s i n g n a t u r a l l y i n f e c t e d f i e l d m a t e r i a l ;
5) d e t e r m i n e t h e p a t h o g e n i c i t y o f d i f f e r e n t f o r m - s p e c i e s and i s o l a t e s
o f £_. s o l a n i c o l l e c t e d from d i f f e r e n t r e g i o n s o f North America on s a i n ­
f o i n ; and 6 ) i n v e s t i g a t e t h e r o l e o f £_. s o l a n i in t h e r o o t and crown
r o t complex o f s a i n f o i n .
LITERATURE REVIEW
D e s c r i p t i o n and Agronomic C h a r a c t e r i s t i c s o f S a i n f o i n .
S a i n f o i n was r e i n t r o d u c e d i n t o Montana w it h t h e r e l e a s e o f t h e
. c u l t i v a r l E s k i l in 1964 ( 2 2 ) .
forage (28,39).
I t pr od uce s a high q u a l i t y , n o n - b l o a t i n g
When grown under op ti m al c o n d i t i o n s , which a r e in well
d r a i n e d , b a s i c s o i l s w it h good m o i s t u r e h o l d i n g c a p a c i t y , y i e l d s o f
s a i n f o i n a r e comp arab le t o t h o s e o f a l f a l f a (1 5, 28 , 3 9 ) .
Oth er a t t r i ­
b u t e s i n c l u d e w i n t e r h a r d i n e s s , d r o u g h t t o l e r a n c e and r e s i s t a n c e t o
both t h e a l f a l f a weevil
( Hyper p o s t i c a H y l l e n h a l ) and b a c t e r i a l w i l t
ca u se d by C o r y ne b ac te r iu m i n s i d i o s u m (McCull. ) ( 1 5 , 28, 39 ).
S a i n f o i n has a well d ev el op ed r o o t system which c o n s i s t s o f a
c e n t r a l t a p r o o t and p r o f u s e l y branched l a t e r a l
r o o t n o r m a l l y e x t e n d s t o a d e p t h o f 1-2 m e t e r s .
roots ( I ) .
The t a p
Despite the presence
o f n o d u le s on t h e small s ec o n d a r y r o o t s , e f f i c i e n t R h i z o b i urn s t r a i n s
have n o t been i s o l a t e d (9 , 65) .
S a i n f o i n pr od uc es t a l l , e r e c t stems which o r i g i n a t e from a
branc he d crown.
The stems a r e u s u a l l y hollow and b e a r 11-29 p i n n a t e l y
compound l e a v e s ( 5 3 ) .
The s ee ds a r e smooth, kidney shaped and a r e h e l d
w i t h i n a tough f i b r o u s pod (28, 7 4 ) .
Although s a i n f o i n i s n o r m a l ly c o n s i d e r e d long l i v e d ( 2 8 , 3 9 ) ,
i n v e s t i g a t o r s began w i t n e s s i n g s t a n d d e t e r i o r a t i o n a f t e r 3 o r 4 y e a r s ,
e s p e c i a l l y in i r r i g a t e d p a s t u r e s (10, 15, 3 9 ) .
In t h e i n i t i a l d e t a i l e d
i n v e s t i g a t i o n . S e a r s (61) found r o o t and. crown r o t in both d r y l a n d and
i r r i g a t e d s a i n f o i n f i e l d s t h r o u g h o u t Montana.
The problem has a l s o
4
been r e p o r t e d in New Mexico (50) and a p p e a r s p r e v a l e n t w he re v er s a i n ­
f o i n i s grown in t h e U n ite d S t a t e s .
Root and crown r o t r e l a t e d s t a n d
d e t e r i o r a t i o n has n o t been r e p o r t e d in Canada where s a i n f o i n i s s t i l l
e x t e n s i v e l y grown in some r e g i o n s ( 2 8 ) .
s e v e r e a t t h e crown (61, 6 2 ) .
Decay a p p e a r s t o be most
In i r r i g a t e d s a i n f o i n s t a n d s , v a s c u l a r
d i s c o l o r a t i o n and decay s p r e a d s from t h e crown down t h e t a p r o o t and
throughout the l a t e r a l
roots.
F o l i a r symptoms a r e n o t g e n e r a l l y
o b s e r v e d (61, 6 2 ) .
The or gan ism most f r e q u e n t l y i s o l a t e d from d i s e a s e d p l a n t s was
£_. s o l a n i
( M a r t . ) Appel & Wr.
This o rg ani sm was most r e a d i l y i s o l a t e d
from d i s e a s e d crowns b u t was r a r e l y i s o l a t e d from d i s c o l o r e d v a s c u l a r
r e g i o n s o f t h e t a p and l a t e r a l
roots.
S e a r s (61) p o s t u l a t e d t h a t a
m i c r o b i a l t o x i n may be r e s p o n s i b l e f o r t h e d i s c o l o r a t i o n .
S c h l e c h t . and R h i z o c t o n i a s o l a n i Kuehn
F_. oxysporum
were o n l y s p o r a d i c a l l y
r e c o v e r e d from a f f e c t e d r o o t s and a t t e m p t s t o i s o l a t e Cor yn eba ct eri um
i n s i d i o s u m and P h y t o p t h o r a megasperma ( D r e c h s . ) , common r o o t pathogens
o f a l f a l f a , were u n s u c c e s s f u l
(61).
I n f e c t i o n o f s a i n f o i n r o o t s by £ . s o l a n i and F_. oxysporum o cc u r s
s h o r t l y a f t e r s e e d i n g (6 1, 6 2 ) .
When l E s k i '1 and 'Remont' s a i n f o i n were
grown und er normal f i e l d c o n d i t i o n s , 60% and 40%, r e s p e c t i v e l y , were
i n f e c t e d w i t h £ . s o l a n i by t h e end o f t h e f i r s t growing s e a s o n .
t i o n l e v e l s f o r F. oxysporum were o n l y 15% f o r both c u l t i v a r s .
Infec­
Sears
(61, 62) p o s t u l a t e d t h a t t h e seed pod i n j u r y , which r e s u l t s when t h e
5
t o u g h , f i b r o u s seed pod c u t s i n t o t h e e n l a r g i n g r o o t , may a c c o u n t f o r
t h e h ig h i n c i d e n c e o f i n f e c t i o n by F. sol am’ in t h e f i r s t growing
season.
Symptoms a r e n o t a p p a r e n t in t h e f i r s t growing se as o n and begin t o
a p p e a r d u r i n g t h e second growing s e a s o n (61, 6 2 ) .
Symptoms begin as a
da r k en e d s t r e a k in t h e crown t i s s u e b e n e a th t h e o l d decayed stem from
t h e p r e v i o u s y e a r ' s growth.
Thi s c o i n c i d e s w it h t h e p h y s i o l o g i c
t e a r i n g o f t h e i n n e r crow n, a phenomenon which may r e s u l t from t h e
i n a b i l i t y o f t h e s a i n f o i n crown t o s u p p o r t t h e l a r g e number o f stems
produced ( 6 1 ) . . Thi s s i t e would be i d e a l f o r c o l o n i z a t i o n by. d i s e a s e
o r g a n is m s .
In a f o l l o w - u p s t u d y , Auld (3, 4) conf irm ed t h e p a t h o g e n i c i t y o f
F. sol a n i t o s a i n f o i n .
S a i n f o i n s e e d l i n g s were i n o c u l a t e d
by s o a k in g
t h e s e v e r e d r o o t s in a mi cro con i d i a l s u s p e n s i o n o f £ . sol a n i and
£_. oxysporum.
Plants inoculated
:
with £. solani disp la y ed exten siv e
v a s c u la r d i s c o l o r a t i o n of th e r o o t s , while those in o c u la te d
with
JF. oxysporum e x h i b i t e d o n l y s p o r a d i c v a s c u l a r d i s c o l o r a t i o n and deca y.
Few o t h e r r o o t d i s e a s e s o f s a i n f o i n have been r e p o r t e d .
V e r t i c i l l i u m a l b o - a t r u m Reinke and B er th . has been i s o l a t e d from p l a n t s
e x h i b i t i n g w i l t i n g symptoms in Germany and England ( 3 2 ) , b u t has no t
been i s o l a t e d from d i s e a s e d s a i n f o i n p l a n t s in Montana ( 6 1 ) .
S c l e r o t i n i a t r i f o l i o r u m E r i k s s , a fungus, which c a u s e s r o o t and stem, r o t
has been o b s e r v e d in Montana and Europe.
However, i t i s n o t c o n s i d e r e d
6
a major prpblem in t h i s s t a t e ( 4 8 ) .
Zhavoronkova (77) d e s c r i b e d a r o o t
r o t o f swept c l o v e r , a l f a l f a , l e n t i l s and s a i n f o i n in Ru ss ia caused by
t h e b a c t e r i u m , Pseudomonas r a d i c i p e r d a .
An a t t e m p t t o i s o l a t e b a c t e r i a ,
o t h e r t h a n C o ry n eb ac te riu m 'i n s i d i o s u m , from s a i n f o i n in Montana has n o t
been made p r e v i o u s t o t h i s i n v e s t i g a t i o n .
I n s e c t p e s t s can c a u s e s e v e r e damage t o f o r a g e p r o d u c t i o n in s a i n ­
fo in stan d s (73).
The r o o t f e e d i n g i n s e c t s , which a r e p o t e n t i a l l y t h e
most d e l e t e r i o u s t o s a i n f o i n , i n c l u d e common p e s t s such as t h e n a t i v e
S e t o n i a weevil
(Setonia s c i s s i f r o n s S ay)(73).
A d u l t s , which o v e r w i n t e r
i n f i e l d d e b r i s , become a c t i v e in l a t e s p r i n g and begin f e e d i n g on t h e
f o l i a g e and s te m s.
The eggs a r e d e p o s i t e d in t h e c r a c k s and c r e v i c e s
in t h e s o i l a t t h e b as e o f t h e s a i n f o i n r o o t s .
The l a r v a e , upon
h a t c h i n g , begin f e e d i n g on t h e n o d u le s and r o o t h a i r s and e v e n t u a l l y on
t h e ma tur e r o o t s ( 4 6 ) .
Th is form o f i n s e c t f e e d i n g has been demon­
s t r a t e d t o mark ed ly i n c r e a s e t h e o c c u r r e n c e and s e v e r i t y o f r o o t r o t in
a l f a l f a and red c l o v e r (25, 3 0 ) .
D is e a s e s o f O th e r Forage Legumes
Fungal D i s e a s e s .
Fusarium r o o t and crown r o t has been a s s o c i a t e d
w i t h e x t e n s i v e damage t o f o r a g e c r o p s such as r e d c l o v e r , a l f a l f a , t r e ­
f o i l and l e n t i l s
(11, 35, 36, 41, 4 5 , 60 , 68 ).
The p r i n c i p l e or ganisms
i m p l i c a t e d i n t h e s t a n d d e c l i n e o f t h e s e f o r a g e s a r e £ . oxyspo.rum,
£ . roseum and £ . sol a n i .
In an e x t e n s i v e s t u d y i n t o t h e r o l e o f t h e s e
or gan ism s in t h e r o o t and crown r o t o f f o r a g e legumes, Leath e t a l .
7
( 4 1 ) co n cl u d ed t h a t r o o t r o t s were a complex i n t e r a c t i o n o f t h e p h y s i ­
cal and b i o l o g i c a l e n v i ro n m e n ts which may i n v o l v e one o r more o f t h e s e
Fusarium s p e c i e s .
Fusarium sp.
a r e c a p a b l e o f i n v a d i n g n o n - i n j u r e d t i s s u e and can
p a r a s i t i z e both v a s c u l a r and c o r t i c a l t i s s u e (11, 6 3 ) .
The fun gi p r o ­
duce enzymes c a p a b l e o f d e g r a d i n g both p e c t i n and c e l l u l o s e (5 1 ) .
Although s e e d l i n g d a m p in g - o f f has been r e p o r t e d in some f o r a g e s ,
more commonly r o o t s become i n f e c t e d w it h Fusarium spp. a t an e a r l y
stag e but f a i l
t o e x p r e s s symptoms ( 4 1 ) .
Root and crown r o t symptoms
o f t e n o c c u r a t t h e end o f t h e f i r s t and t h r o u g h o u t t h e second growing
s e a s o n s (35, 36, 41, 68 ) .
N e c r o t i c a r e a s o f t e n a p p e a r on t h e s u r f a c e ,
o f t h e t a p and l a t e r a l r o o t s w i t h d i s c o l o r a t i o n and dec ay r e s t r i c t e d
t o t h e c o r t e x and o u t e r a r e a s o f t h e r o o t s o r t o t h e c e n t r a l r e g i o n s
n e a r t h e crown (41, 68 ) .
Root and crown r o t te n d s t o be more s e v e r e
in y e a r s o f high t e m p e r a t u r e s , low s o i l m o i s t u r e and when p l a n t s a r e
subjected to n u tr itio n a l s t r e s s
(1 2, 4 1 ) .
O th er f un gi have been i m p l i c a t e d i n t h e s t a n d d e c l i n e o f f o r a g e
crops.
P h y t o p t h o r a ( 6 0 ) , Aphanomyces (41, 68 ) , Pythium (2 7, 60) and
. R h i z o c t o n i a have been r e p o r t e d p a t h o g e n i c on a l f a l f a and r e d c l o v e r .
B acterial D iseases.
Cor y ne b ac te riu m in s i d i o s u m i s t h e most p r e ­
v a l e n t o f t h e b a c t e r i a l d i s e a s e s o f a l f a l f a and i s p e r h a p s t h e most
i m p o r t a n t r e c o g n i z e d d i s e a s e o f t h e c r o p in North America ( 5 5 ) .
b a c t e r i a p e n e t r a t e the. r o o t system and ca u s e e x t e n s i v e v a s c u l a r
The
8
d i s c o l o r a t i o n accompanied by a w i l t i n g o f t h e t o p growth (34, 4 0 ) .
A
p h y t o t o x i c gT y c o p e p ti d e i s produced by t h e b a c t e r i a and i s th o u g h t t o
c a u s e t h e w i l t i n g phenomenon ( 5 5 ) .
Pseudomonas m a rg i n al i s and Erwinia amylovora have r e c e n t l y , been
i s o l a t e d from d i s e a s e d a l f a l f a r o o t s in P e n n s y lv a n i a (57, 58, 59).
Pseudomonas r a d i c e r p e r d a , Erwinia c a r o t o v o r a and Pseudomonas
f l u o r e s c e n s (16) and Pseudomonas m e d ic ag in s (54) have been i s o l a t e d
■
■
from d i s e a s e d a l f a l f a r o o t s in R u s s ia .
■
■
.
.
Experiment One
THE PATT-ERN OF ROOT AND CROWN ROT DETERIORATION IN IRRIGATED
SAINFOIN PLANTS AND ITS PREVALENCE IN FIRST AND SECOND YEAR SAINFOIN
STANDS'.
MATERIALS AND METHODS
Al I p l a n t specimens were s e l e c t e d a t random from i r r i g a t e d s a i n ­
f o i n s t a n d s l o c a t e d a t t h e F i e l d Research L a b o r a t o r y , Bozeman, Montana.
S a i n f o i n r o o t s were e x t r a c t e d u s i n g a s h a r p s h o o t e r s h o v e l .
The r o o t
system u s u a l l y c o n s i s t e d o f t h e t a p r o o t and l a r g e r l a t e r a l
roots.
In
t h e l a b o r a t o r y , r o o t s were t h o r o u g h l y c l e a n e d and t h e v i s u a l symptoms
such a s i n s e c t wounds were n o t e d .
The b a r k , which c o n s i s t s o f phloem,
c o r t e x and p e r i d e r m , 'was removed t o uncov er t h e s u r f a c e xylem d i s c o l o r ­
ation.
The r o o t was th e n s p l i t l o n g i t u d i n a l l y t o r e v e a l i n t e r n a l v a s ­
c u l a r d i s c o l o r a t i o n and crown decay.
a)
S a i n f o i n p l a n t s were randomly sampled from f i r s t , seco nd ,
t h i r d , f o u r t h and f i f t h y e a r s a i n f o i n s t a n d s d u r i n g t h e summers o f 1977
and 1978.
Ph o to gr ap hs were t a k e n o f t h e d i f f e r e n t symptoms and n o t e s
were r e c o r d e d on t h e p r o g r e s s i o n o f t h e d i s e a s e symptoms.
b)
In t h e summer o f 1977, 50 t h r e e month and 50 f i f t e e n month o l d
s a i n f o i n r o o t s were s e l e c t e d a t random.
and d i s c o l o r a t i o n , t a p and l a t e r a l
The i n c i d e n c e o f crown decay
r o o t d i s c o l o r a t i o n and i n s e c t wound
r e l a t e d d i s c o l o r a t i o n were r e c o r d e d .
RESULTS AND DISCUSSION
In f i r s t s e a s o n s a i n f o i n s t a n d s , symptoms c o n s i s t e d p r e d o m i n a n tl y
o f v a s c u l a r d i s c o l o r a t i o n i n t h e l a t e r a l and t a p r o o t s (Tab le 1 - 1 ) .
The d i s c o l o r a t i o n a p p e a r s t o move from t h e l a t e r a l r o o t s i n t o t h e t a p
r o o t to w ar d s t h e crown ( F i g u r e 1-1) and rem ai ns r e s t r i c t e d t o a small
number o f v e s s e l s .
D iscoloration w ithin the tap ro o t consisted p r i ­
m a r i l y o f s p o r a d i c b l a c k s t r e a k i n g in t h e v a s c u l a r system though s e v e r e
d i s c o l o r a t i o n was sometimes o b s e r v e d .
The p e r c e n t a g e o f 3 month old
s a i n f o i n p l a n t s e x h i b i t i n g one o r more d i s c o l o r e d l a t e r a l and t a p r o o t s
was r e l a t i v e l y s m a l l , b u t n e a r l y 100% o f t h e 15 month p l a n t s d i s p l a y e d
t h e symptom (Tab le 1 - 1 ) .
t i s s u e in th e l a t e r a l
The r e s u l t i n g d e s t r u c t i o n o f t h e v a s c u l a r
and t a p r o o t s may be o f s e r i o u s con sequence t o
w a t e r t r a n s l o c a t i o n , p a r t i c u l a r l y in s e a s o n s o f m o i s t u r e s t r e s s , s i n c e
t h e d i s c o l o r a t i o n in a g iv e n p l a n t becomes more e x t e n s i v e as t h e
disease progresses.
These d a t a emphasize t h a t few p l a n t s d i s p l a y symp­
toms in t h e s e e d i n g y e a r , b u t a t t h e end o f t h e second growing s e a s o n ,
a l l p l a n t s e x h i b i t symptoms.
I n f e c t e d p l a n t s e x h i b i t crown d i s c o l o r a t i o n and decay a t t h e end
o f t h e f i r s t o r in t h e second growing se as o n d i r e c t l y below, t h e o ld
decayed stems ( F i g u r e 1 - 3 ) . . The a f f e c t e d t i s s u e i s r e d - o r a n g e in c o l o r
and i t s p r e s e n c e seems t o c o i n c i d e w it h t h e p h y s i o l o g i c crdwn s p l i t t i n g
d e s c r i b e d by S e a r s ( 6 0 ) .
E i g h ty p e r c e n t o f t h e 15 month s a i n f o i n . . 1-
p l a n t s p o s s e s s t h i s t y p e o f crown dec a y .
O ccasionally, black vascula r
d i s c o l o r a t i o n was o b s e r v e d a t t h e base o f t h e o l d dead stem
11
Ta b le 1-1,
A s u r v e y o f commbn, 'r o o t and crown d e g e n e r a t i o n symptom
t y p e s o c c u r r i n g . i n n a t u r a l l y i n f e c t e d 3 and 15 month s t a n d s
o f ' E s k i 1 s a i n f o i n l o c a t e d a t t h e F i e l d . Resea rc h L a b o r a t o r y
Bozeman, Montana.
% o f t h e P la n t s . E x h i b i t i n g ! S y m p t o m s I /
Symptom Type
3 month o ld
sainfoin
15 month o l d
sainfoin.
0
80
Vascular d i s c o l o r a t i o n
in t h e t a p r o o t
18
96
Vascular d i s c o l o r a t i o n
in t h e l a t e r a l r o o t s
24
.48
0
96
Crown d i s c o l o r a t i o n a t t h e
base o f t h e o l d dead stem
I n s e c t wound r e l a t e d
discoloration
^Based on o b s e r v a t i o n s o f 50 randomly s e l e c t e d p l a n t s from each
stand.
12
F ig u r e 1 - 1 : L a t e r a l r o o t d i s c o l o r ­
a t i o n which e x t e n d s i n t o t h e t a p r o o t
and towards t h e crown in second y e a r
sainfoin.
F ig u r e 1 - 2 : Black crown d i s c o l o r ­
a t i o n i n i t i a t e d a t t h e base o f th e
o ld dead stem in second y e a r s a i n ­
foin.
13
(Figure 1-2).
Seed pod wounding, which o c c u r s n e a r tfie crown, was n o t
a s s o c i a t e d w it h v a s c u l a r d i s c o l o r a t i o n o r d ec a y , s u g g e s t i n g t h a t i t i s
not a source o f vascu la r in f e c tio n .
1
•
The r e d crown dec ay a p p e a r s t o p r o g r e s s down t h e t a p r o o t .
( F i g u r e s 1-4 and 1 - 5 ) .
The c e n t r a l decay which i s p r e s e n t in most
s a i n f o i n r o o t s in advanced s t a g e s o f d e c l i n e , i s c l e a r l y i n i t i a t e d
t h r o u g h o ld dead s te m s.
tial
Moreover, each newly c u t stem becomes a p o t e n ­
source o f in f e c tio n .
F i g u r e s 1-6 and 1-7 i l l u s t r a t e t h e p r o g r e s ­
s i o n o f v a s c u l a r d i s c o l o r a t i o n o r i g i n a t i n g from two o l d c u t stems in
second y e a r s a i n f o i n r o o t s .
The v a s c u l a r d i s c o l o r a t i o n i s b la c k and
a p p e a r s in d e p e n d e n t o f t h e . r e d c e n t r a l dec a y .
Decay a s s o c i a t e d w ith
t h e s e dead stems co n v e r g e s in t h e crown t o c o l l e c t i v e l y produce an
e x t e n s i v e c e n t r a l r e g i o n o f t i s s u e d e g e n e r a t i o n . . , L a t e r a l movement o f
t h e decay i n t o newly i n i t i a t e d xylem t i s s u e o c c u r s in advanced s t a g e s
o f the d is e a s e (Figure 1-8 ).
In a s ur v ey o f 25 f o u r t h y e a r s a i n f o i n
p l a n t s , between 50% and 90% o f t h e v a s c u l a r t i s s u e in t h e crown was
decayed o r d i s c o l o r e d ( F i g u r e 1 - 9 ) .
I n s u f f i c i e n t v a s c u la r t i s s u e to
s u p p ly t h e w a t e r demands o f t h e p l a n t w i l l r e s u l t in t h e e v e n t u a l d e a t h
of the p lan t.
Decay o f t h e phloem and c o r t i c a l t i s s u e , a phenomenon
not o b s e r v e d i n f i r s t and second y e a r s a i n f o i n , may be e x t e n s i v e in
severely affected roots.
All s a i n f o i n r o o t s e x c e p t t h o s e i n t h e i r f i r s t growing se ason
showed s i g n s o f i n s e c t i n j u r y on t h e t a p and l a t e r a l r o o t s caus ed by
j
F i g u r e 1-3 t o 1 - 5 :
P r o g r e s s i o n o f t h e red crown d e t e r i o r a t i o n down t h e t a p r o o t in second
year sainfoin.
15
F ig u r e 1 - 6 : Black v a s c u l a r d i s ­
c o l o r a t i o n i n i t i a t e d a t t h e base
o f two o ld dead stems in second
year sainfoin.
F ig u r e 1 - 7 : Black v a s c u l a r d i s c o l ­
o r a t i o n e x t e n d i n g from two o l d dead
stems down t h e t a p r o o t in t h i r d
year sainfoin.
16
F i g u r e 1 - 8 : Removal o f t h e bark
r e v e a l s crown d i s c o l o r a t i o n and
decay which i s a f f e c t i n g t h e newly
i n i t i a t e d xylem in t h i r d y e a r s a i n ­
foin.
F ig u r e 1 - 9 : E x t e n s i v e r o o t and
crown d i s c o l o r a t i o n and decay in
fourth year sain fo in .
17
t h e S i t o n i a weevil
( S i t o n i a s i s s i f r o n s D a y ) ( F ig u r e 1 - 1 0 ) .
Xyletn d i s -
• 1V - '
c o l o r a t i o n r e s u l t e d when l a r v a l
f e e d i n g g a l l e r i e s e x t en d ed th r o u g h t h e
c o r t e x t o t h e o u t e r xylem ( F i g u r e 1 - 1 1 ) .
In t h e two y e a r o l d s a i n f o i n
p l a n t s , v a s c u l a r d i s c o l o r a t i o n g e n e r a l l y remained r e s t r i c t e d t o t h e
p o i n t o f i n s e c t f e e d i n g though d i s c o l o r a t i o n was sometimes o b s er v ed
s p r e a d i n g from t h e wound ( F i g u r e 1 - 1 2 ) .
T h i r d and f o u r t h y e a r s a i n f o i n
r o o t s e x h i b i t e d a c t i v e decay in t h e v i c i n i t y o f i n s e c t wounds.
These
wounds presu mab ly s e r v e as an i n f e c t i o n s i t e f o r d i s e a s e o r g an is m s.
CONCLUSION
Root and crown r o t i n s a i n f o i n a p p e a r s to be i n i t i a t e d below t h e
o l d c u t stem.
is created.
Each ti m e t h e f o r a g e i s h a r v e s t e d , a new i n f e c t i o n s i t e
The d i s e a s e o r g a n i s m ( s ) a p p e a r s t o move down t h e hollow
stems and co nv erg e in t h e crowns o f t h e 3 and 4 y e a r o l d s a i n f o i n
p l a n t s r e s u l t i n g in a s e v e r e decay o f t h e v a s c u l a r t i s s u e in t h i s
region.
D i s c o l o r a t i o n and decay a l s o s p r e a d down t h e c e n t e r o f t h e t a p
r o o ts , thereby f u r t h e r d is r u p ti n g water t r a n s p o r t .
Decay o f t h e v a s c u ­
l a r t i s s u e may d i r e c t l y k i l l t h e s a i n f o i n p l a n t o r p r e d i s p o s e i t t o
e n v i ro n m e n ta l s t r e s s e s such as d r o u g h t o r w i n t e r k i l l .
Cumulative l a t e r a l
r o o t d i s c o l o r a t i o n and i n s e c t w o u n d - r e l a t e d
decay and d i s c o l o r a t i o n a p p e a r s t o be p o s i t i v e l y c o r r e l a t e d w ith t h e
age o f t h e p l a n t and i s d i s t i n c t from t h e crown decay p r e v i o u s l y
described.
The r o l e o f t h e s e phenomena i n t h e a c t u a l d e c l i n e o f
F i g u r e s 1-10 t o 1 - 1 2 :
Larval f e e d i n g g a l l e r i e s on t h e s u r f a c e o f s a i n f o i n r o o t s caused by
t h e i n s e c t S i t o n i a s i s s i f r o n s ( D ay ) ( F ig ur e 1-10) and t h e c o r r e s p o n d ­
ing d i s c o l o r a t i o n and decay on t h e xylem s u r f a c e a f t e r removing the
bark ( F i g u r e 1 - 1 1 ) .
F ig u r e 1-12 d e p i c t s xylem d i s c o l o r a t i o n moving
both up and down from t h e i n s e c t wound.
19
•
. ■,
•'.
.
. ' ■
s a i n f o i n s t a n d s i s n o t known.though t h e y p r o b a b l y s e r v e as an i n f e c t i o n
s i t e f o r d is e a s e organisms.
The d e s t r u c t i o n . o f w a t e r c o n d u c t i n g t i s s u e
a d v e r s e l y a f f e c t s s a i n f o i n p l a n t s , e s p e c i a l l y in t i m e s o f m o i s t u r e
stress.
E x p erim en t 2
ISOLATION AND IDENTIFICATION OF FUNGAL AND BACTERIAL ORGANISMS
ASSOCIATED WITH ROOT AND CROWN ROT OF SAINFOIN.
I.
ISOLATION
. MATERIALS AND METHODS
During t h e month o f J u l y , 1978, 50 second y e a r , 50 t h i r d y e a r and
25 f i f t h y e a r s a i n f o i n p l a n t s were randomly s e l e c t e d from i r r i g a t e d
s a i n f o i n f i e l d s l o c a t e d a t t h e F i e l d Research L a b o r a t o r y , Bozeman,
Montana.
A s h a r p - s h o o t e r shovel was used t o d i g up t h e p l a n t s .
t h e r o o t s were t h o r o u g h l y washed t o remove a d h e r i n g s o i l
A fter
p a r t i c l e s , the
bark was removed, t h e r e b y ex po sin g t h e c e n t r a l v a s c u l a r c o r e .
The
r o o t s were th e n s p l i t l o n g i t u d i n a l l y t o expose t h e crown decay and
vascular d isco lo ratio n .
I s o l a t i o n s from t h e symptom t y p e s d e s c r i b e d in Experiment One
were c a r r i e d o u t as f o l l o w s :
a) p o r t i o n s o f t h e d i s c o l o r e d xylem a s s o ­
c i a t e d w it h i n s e c t a c t i v i t y , and t h u s d i r e c t l y exposed t o t h e s o i l
e n v i r o n m e n t , were removed w i t h a s t e r i l e s c a l p e l .
e x p os ur e o f t h e s u b s u r f a c e d i s c o l o r a t i o n .
Th is r e s u l t e d in
A small p i e c e o f a f f e c t e d
t i s s u e was a s e p t i c a l l y removed f o r i s o l a t i o n ,
b) D i s c o l o r e d l a t e r a l
r o o t s were sampled by e x c i s i n g a small p i e c e o f t i s s u e a t t h e p o i n t
where t h e l a t e r a l
root jo in s the tap ro o t,
c) I s o l a t i o n s from d i s e a s e d
crown t i s s u e were u s u a l l y made a t t h e l e a d i n g edge o f decay and d i s ­
coloration.
d) Dead stem a s s o c i a t e d decay and d i s c o l o r a t i o n were
21
sampled in t h e crown t i s s u e a t t h e bas e o f t h e dead stem.
In 5 y e a r
o l d p l a n t s , crown decay was u s u a l l y e x t e n s i v e and t a p r o o t decay n o r ­
m a ll y e x t e n d e d 10-30 cm below t h e crown.
I s o l a t i o n s were t a k e n a t t h e
l e a d i n g edge o f t h e decay 2, 5, 10 and 20 cm from t h e crown.
The p r o c e d u r e f o r i s o l a t i n g b a c t e r i a c o n s i s t e d o f p l a c i n g a small
p i e c e o f a f f e c t e d t i s s u e i n t o a t e s t t u b e c o n t a i n i n g I ml o f s t e r i l e
d i s t i l l e d water.
A f t e r i n c u b a t i n g t h e p r e p a r a t i o n a t 21 C f o r 15-17
h o u r s , a small d ro p o f w a t e r was removed from t h e t u b e u s i n g a flamed
p l a t i n u m loop and s t r e a k e d on p l a t e s c o n t a i n i n g K in g ' s B a g a r (Appen­
d ix I ) , a g e n e r a l medium f o r i s o l a t i o n o f b a c t e r i a .
The p l a t e s were
i n c u b a t e d a t 21 C f o r 72 hours and su rv ey ed f o r b a c t e r i a l c o l o n i e s .
Because Fusarium sp. had been p r e v i o u s l y i m p l i c a t e d in t h e r o o t
and crown r o t o f s a i n f o i n and o t h e r f o r a g e legumes, Komada1s medium
( 3 8 ) , a s p e c i f i c medium f o r t h e i s o l a t i o n o f Fusarium s p . , was used.
A
small p i e c e o f d i s c o l o r e d t i s s u e was p l a c e d d i r e c t l y on t h e media w i t h ­
out p rio r surface s t e r i l i z a t i o n .
S u r f a c e s t e r i l i z a t i o n was no t deemed
n e c e s s a r y bec a us e o f t h e h i g h l y s p e c i f i c n a t u r e o f t h i s medium.
Plates
were i n c u b a t e d a t 21 C f o r 10 d a y s , th e n su rv ey ed f o r t h e p r e s e n c e o f
fu nga l c o l o n i e s .
Random i s o l a t i o n s from d i s e a s e d t i s s u e were a l s o made
on A c i d i f i e d P o t a t o D ex tr o s e Agar (HPDA)^ a g e n e r a l p u r p o s e medium f o r
t h e i s o l a t i o n o f fun gi
(Appendix I ) .
RESULTS AND DISCUSSION
Fusarlum i s o l a t e s were i d e n t i f i e d t o s p e c i e s u s in g t h e p r o c e d u r e
o u t l i n e d by Toussoun and Nelson ( 7 1 ) .
t h e common b a c t e r i a l typps
o b t a i n e d from d i s e a s e d 2, 3, and 5 y e a r o l d s a i n f o i n p l a n t s were c l a s ­
s i f i e d i n t o t h r e e groups based on c o l o n y morphology, growth and pigmen­
t a t i o n produced by t h e b a c t e r i a on K i n g ' s B a g a r .
B a c t e r i a l c o l o n i e s , which r e s u l t e d when t h e t i s s u e p r e p a r a t i o n s
were s t r e a k e d on BC a g a r , were numerous.
a p p ea r ed on a p l a t e .
O f t e n , 200-400 c o l o n i e s
The col on y t y p e s were u n if o r m , in many i n s t a n c e s ,
in growth and morphology, i n d i c a t i n g t h a t t h e b a c t e r i a in many d i s e a s e d
t i s s u e s belong t o one b a c t e r i a l s p e c i e s .
As t h e decay p r o g r e s s e d ,
e s p e c i a l l y in t h e crown o f 5 y e a r o l d s a i n f o i n p l a n t s , t h e b a c t e r i a l
f l o r a was found t o be h i g h l y h e t e r o g e n e o u s .
One group o f b a c t e r i a (Group I ) c o n s i s t e d o f Pseudomonas ty p e s
which formed ro u n d , y e l l o w c o l o n i e s and produced a pigment which
f l u o r e s c e d und er u l t r a v i o l e t l i g h t .
Group 2 produced round w h ite
c o l o n i e s and a brown d i f f u s a b l e pigment a f t e r 8-10 d a y s .
They a l s o
s o l u b i l i z e d t h e MgSO4 p r e c i p i t a t e which n o r m a l ly c l o u d s K in g ' s B a g a r ,
t h e r e b y p r o d uc in g a c l e a r zone around c o l o n i e s a f t e r 4 - 6 d ay s .
Group 3
formed i r r e g u l a r mucoid c o l o n i e s and produced a y e l l o w n o n - d i f f u s a b l e
pigment.
23
Second Year S a i n f o i n
Very few Fusarium sp. were i s o l a t e d from symptomatic t i s s u e .
There w e r e , however, a p re p o n d e r a n c e o f b a c t e r i a , p a r t i c u l a r l y Pseudomonas s p p . , in t h e s a i n f o i n p l a n t s e x h i b i t i n g symptoms (Ta b le 2 - 1 ) .
N i n e t y -o n e p e r c e n t o f t h e p l a n t s d i s p l a y i n g t h e red crown d e t e r i o r a t i o n
c o n t a i n e d t h e Pseudomonas t y p e s .
These b a c t e r i a were a l s o abun dan t in
t h e t a p r o o t d i s c o l o r a t i o n , in t h e decay below t h e o l d dead stems and
in i n s e c t wounds.
The mucoid b a c t e r i a and t h e w h i t e b a c t e r i a were
i s o l a t e d i n f r e q u e n t l y , a l t h o u g h 25% o f t h e b la c k d i s c o l o r a t i o n in t h e
t a p r o o t s c o n t a i n e d t h e w h i t e b a c t e r i a (Table 2 - 1 ) .
T h i r d Year S a i n f o i n
Fusarium s p e c i e s , p a r t i c u l a r l y JF. s o l a n i , were most f r e q u e n t l y
i s o l a t e d from i n s e c t wounds, b u t r a r e l y from o t h e r a f f e c t e d t i s s u e s
(Table 2 - 2 ) .
Pseudomonas spp. a g a i n p r ed om in ate d in t h e m a j o r i t y o f
the diseased ti s s u e types.
The w h i t e b a c t e r i a were a l s o f r e q u e n t l y
i s o l a t e d i n t h e r e d c e n t r a l decay r e g i o n , though t h e y a p p e a r t o be more
c l o s e l y a s s o c i a t e d w it h v a s c u l a r d i s c o l o r a t i o n o f t h e t a p and l a t e r a l
r o o t s and w i t h i n s e c t i n j u r y .
The mucoid b a c t e r i a were i s o l a t e d from
a l l symptom t y p e s , b u t a p p e a r most f r e q u e n t l y in t h e crown decay and in
i n s e c t wounds (Ta b le 2 - 2 ) .
.
T able 2-1.
B acteria
sainfoin
and f u n g i i s o l a t e d f r o m s p e c i f i c symptom t y p e s f o u n d i n 2 y e a r o l d
p l a n t s c o l l e c t e d a t ,the F i e l d R e s e a r c h L a b o r a t o r y , Bozeman, M ontana.
O
O
O
91
2
11
Black d i s c o l o r a t i o n
b e n e a th t h e s e c o n d a r y
o l d dead stem
23
2
2
2
70
4
15
Vascular d i s c o l o r a ­
t i o n in t h e t a p r o o t
11
O
O
O
50
25
0
I n s e c t wound r e l a t e d
discoloration
14
O
O
O
70
7
0
Mucoid
Bacteria
(Group I I I )
F. roseum
35
White
Bacteria
(Group I I )
F. oxysporum
Red crown decay
Symptom Type
Number
o f symptom
t y p e s sampled
for isolation
Pseudomonas
spp.
(Group I)
F. s o l a n i
% o f th e i s o l a t i o n s with I /
■'"Based on i s o l a t i o n s made on Komada' s medium f o r Fusarium spp. and on K in g 's B a g a r f o r
the b a cteria.
T able 2-2.
B a c t e r i a a n d f u n g i i s o l a t e d fr o m s p e c i f i c symptom t y p e s f o u n d i n 3 y e a r o l d
s a i n f o i n p l a n t s c o l l e c t g d a t t h e F i e l d R e s e a r c h L a b o r a t o r y , Bozeman, Montana
% o f t h e i s o l a t i o n s p r o d u c in g I /
E
3
S-
Symptom Type
Number
o f symptom
t y p e s sampled
for isolation
Red crown decay
35
Black d i s c o l o r a t i o n
b e n e a th t h e o l d
s e c o n d a r y stem
23
Vascular d is c o !o ra t i o n in th e ta p ro o t
.
10
.
.
«
o
u_|
O
CL
E
3
V)
QJ
CO
O
O
u l \
S-
Lu|
CO
fd
C
O
E
Q.
O
3
TJ
3
• O
QJ C l S CO C LC D
"
CL CO
QJ
-W
•1—
- C
S
(O I—I
e.rS - CL
QJ 3
-W O
O Sr e o
CQ —
-O
CL
• r - QJ 3
O - W O
O O S 3 fO CU
s : cq
O
O
O
60
60
24
2
.2
O
66
73
45
O
O
O
20
60
20
Vascular d i s c o l o r a ­
tio n in the l a t e r a l
roots
12
O
O
O
58
66
25
I n s e c t wound r e l a t e d
discoloration
14
23
o.
O
66
73
45
^Based on i s o l a t i o n s made on Komada1s medium f o r Fusarium spp. and on K i n g ' s B a g a r f o r
the b a c te ria .
'
26
F i f t h Year S a i n f o i n
£_. s o l a n i was i s o l a t e d in a p p r e c i a b l e l e v e l s o n l y from t h e
d iseased t i s s u e s of 5 y e a r old sa in fo in p l a n t s .
Th is o rg ani sm was most
f r e q u e n t l y found in t h e decayed crown r e g i o n s ; 32% o f p l a n t s examined
had t h i s f u n g u s .
The f r e q u e n c y d e c r e a s e d a s t h e decay and d i s c o l o r a ­
t i o n p r o g r e s s e d down t h e t a p r o o t .
readily isolated at all
C o n v e r s e l y , Pseudomonas spp. were
lev els of the tap ro o ts.
In many i n s t a n c e s ,
t h e s e b a c t e r i a were i s o l a t e d in a h i g h l y p u r i f i e d s t a t e a t t h e l e a d i n g
edge o f t h e d e ca y , which i n d i c a t e d t h a t t h i s o r g a n i s m ( s ) may be r e s p o n ­
s i b l e f o r t h e t i s s u e d e s t r u c t i o n o c c u r r i n g in t h i s r e g i o n .
Both w h it e
and mucoid b a c t e r i a were i s o l a t e d most f r e q u e n t l y i n t h e t a p r o o t n e a r
t h e crown (Table 2 - 3 ) .
B a c t e r i a a p p e a r t o be c l o s e l y a s s o c i a t e d w it h r o o t and crown r o t
symptoms i n s a i n f o i n grown under i r r i g a t i o n .
They a r e c o n s i s t e n t l y
i s o l a t e d from a l l o f t h e symptom t y p e s d e s c r i b e d in t h e p r e v i o u s
experiment.
The Pseudomonas spp. a r e t h e pr edom inant b a c t e r i a l forms.
I n i t i a l l y , w h i t e b a c t e r i a seem t o be-more c l o s e l y a s s o c i a t e d w ith t h e
b l a c k v a s c u l a r d i s c o l o r a t i o n in t h e t a p and l a t e r a l r o o t s , b u t th e y
a p p e a r e q u a l l y d i s t r i b u t e d in a l l t h e symptom t y p e s in t h e more ma ture
sainfoin ro o ts.
all
Although t h e mucoid b a c t e r i a coul d be i s o l a t e d from
symptom t y p e s , t h e y a p p e a r most o f t e n in t h e i n s e c t wounds and in
t h e d i s c o l o r e d and decayed crown t i s s u e s .
Table 2-3.
B a c t e r i a and fu n g i i s o l a t e d a t d i f f e r e n t i n t e r v a l s a lo n g th e c e n t r a l decay
re g io n in ta p r o o ts o f s e v e r e ly d is e a s e d 5 y e a r old s a i n f o i n c o l l e c t e d a t th e
F i e l d R e s e a rc h L a b o r a t o r y , Bozeman, M ontana.
% of the is o la tio n s y ie ld in g I /
C
fO
E
=3
SO
CL
V)
> 5
Number sampled
for isolation
O
V)
Li-
Xj
O
U-
E
3
0)
I/)
O
S•
U-
.
(Z)
(0
C
O
-E
CL
O
3
-o
3
• O
CL
S_
CU
V) CLCD
O - CO ""—'
I-H
(d H-H
<D
4->
•i-c
3
SCD
-H
U
re
CO
Q3
O
So
'
(T3
HH
-Q S -C •1— 0) 3
O H O
O U S 3 (13 CD
s : c o ■—
C e n t r a l decay
region a t d i f f e r e n t
d i s t a n c e s from t h e
crown
2 cm
25
33
O
O
80
36
44
5 cm
24
20
O
O
83
40
33
10 cm
23
13
O
O
82
13 •
13
20 cm
15
13
O
O
66
6
O
^Based on i s o l a t i o n s made on Komada1s s e l e c t i v e medium f o r Fusarium spp . and on K ing 's B
agar fo r the b a c te r ia ."
28
” ■I,- sol an i and o t h e r Fusarium spp. do n o t a p p e a r t o be d i r e c t l y
a s s o c i a t e d w i t h symptomatic t i s s u e s . . Although t h e o rg an is m wa,s i s o ­
l a t e d from t h e crowns o f a p p r o x i m a t e l y o n e - t h i r d o f t h e decayed 5th
y e a r s a i n f o i n crowns, i t i s n o t a s s o c i a t e d w ith t h e l e a d i n g edge o f t h e
decay which e v e n t u a l l y e x t e n d s deep i n t o t h e r o o t and c o n t r i b u t e s t o '
the eventual death o f the p la n t.
C o n s e q u e n t ly , t h e a b s e n c e o f Fusarium
cou pl ed w i t h t h e overwhelming p r e s e n c e o f b a c t e r i a , p a r t i c u l a r l y t h e
Pseudomonas s p p . , s u g g e s t s t h a t r o o t and crown d e t e r i o r a t i o n in i r r i ­
g a t e d s a i n f o i n s t a n d s in Montana i s ca us ed by a b a c t e r i a l p a t h o g e n ( s )
r a t h e r t h a n a fungus pat h og en .
II.
IDENTIFICATION
MATERIALS AND METHODS
The b a c t e r i a were i d e n t i f i e d on t h e b a s i s o f t h e i r b e h a v i o r ip a
wide r an g e o f bio c he mi c al and p h y s i o l o g i c a l t e s t s as o u t l i n e d in
B e r g e y ' s Manual o f D e t e r m i n a t i v e B a c t e r i o l o g y
(17, 4 3 ) .
Initial tests
s u g g e s t e d t h a t t h e t h r e e p r e v a l e n t b a c t e r i a l t y p e s i s o l a t e d from
d i s e a s e d s a i n f o i n r o o t s bel onged t o t h e b a c t e r i a l g e n e r a . Pseudomonas
and E r w in ia .
C o n s e q u e n t l y , t h e t e s t s were d e s i g n e d t o d i f f e r e n t i a t e
between t h e major taxonomic groups b e l o n g i n g t o t h e s e g e n e r a .
The f o l l o w i n g i s o l a t e s were used in t h e bioc hem ic al and p h y s i o - .
logical ch ara cterizatio n t e s t s :
6 t y p i c a l and 2 a t y p i c a l w h i t e b ac­
t e r i a , 4 t y p i c a l mucoid b a c t e r i a and 22 t y p i c a l and a t y p i c a l Pseudo­
monads.
A ty p i c a l w h i t e b a c t e r i a f a i l e d t o c a u s e a c l e a r i n g o f t h e
c l o u d y p r e c i p i t a t e in BC a g a r and d id n o t produce t h e brown pigment on
t h e same a g a r .
A ty p i c a l Pseudomonads produced c o l o n i e s which d i f f e r e d
in growth and morphology from t h e m a j o r i t y o f i s o l a t e s .
were randomly s e l e c t e d from t h e b a c t e r i a l
All b a c t e r i a
i s o l a t i o n s made from t h e 2,
3 and 5 y e a r o l d s a i n f o i n p l a n t s .
All b a c t e r i a l
i s o l a t e s used in t h e c h a r a c t e r i z a t i o n t e s t s were
s i n g l e - c e l l e d a minimum o f two t i m e s p r i o r t o s t o r a g e i n s t e r i l e d i s ­
t i l l e d w a t e r a t 4 C.
Unless o t h e r w i s e i n d i c a t e d , t h e w a t e r c u l t u r e s
were s t r e a k e d on p l a t e s c o n t a i n i n g BC a g a r and i n c u b a t e d f o r 48-72
30
hours.
The c o l o n i e s growing on, t h e s e p l a t e s were s u b s e q u e n t l y used t o
i n o c u l a t e t h e t e s t media.
■'
Moti I i t y
M o t i l i t y was d e t e r m in e d by p l a c i n g t h e b a c t e r i a in a drop o f d i s ­
t i l l e d w a t e r c o n t a i n e d on a g l a s s s l i d e .
A f t e r a f i v e m in ute i n t e r v a l ,
a c o v e r - s l i p was p l a c e d on t h e drop and t h e b a c t e r i a were viewed a t
400X m a g n i f i c a t i o n ' un d er phase o p t i c s f o r m o t i l i t y .
Biochemical and P h y s i o l o g i c a l T e s t s
(
Al I med ia, u n l e s s o t h e r w i s e i n d i c a t e d , were s t e r i l i z e d f o r 15
mi nu te s a t 121 C.
The carb on and n i t r o g e n s o u r c e s u t i l i z e d in t h e
n u t r i t i o n a l t e s t s were s t e r i l i z e d by M i l l i pore f i l t r a t i o n .
Oxidase P r o d u c t i o n
The t e s t f o r t h e p r e s e n c e o f cytochrome o x i d a s e was
a c c o r d i n g t o t h e method d e s c r i b e d by S t a n i e r e t a l . ( 6 7 ) .
performed
A lo o p f u l
o f b a c t e r i a t a k e n from 24 hour c u l t u r e s was smeared on a f i l t e r p ap er
p r e v i o u s l y soaked in an aqueous s o l u t i o n o f N, N1- d i m e t h y l - p - p h e n y l en e d i amine.
A p o s i t i v e r e a c t i o n , which c o n s i s t e d o f a d a r k p u r p l e c o l o r
produced a f t e r 15 s e c o n d s , i n d i c a t e d t h e p r e s e n c e o f cytochrome o x i d a s e .
H y p e r s e n s i t i v e R ea c ti o n on Tobacco
A s u s p e n s i o n c o n t a i n i n g a p p r o x i m a t e l y 8 X 10
8
b a c t e r i a l c e l l s/ml
was i n j e c t e d i n t o t h e i n t e r v e i n a l r e g i o n s o f l e a v e s o f N i c o t i n l a .
31
tobaccum L. ■A p o s i t i v e h y p e r s e n s i t i v e r e a c t i o n c o n s i s t e d o f a t o t a l
c o l l a p s e and n e c r o s i s o f t h e i n j e c t e d a r e a a f t e r 2-4 days ( 3 7 ) .
G elatin H ydrolysis
G e l a t i n h y d r o l y s i s was d e t e r m in e d u s i n g t h e t e c h n i q u e d e s c r i b e d by
S ta n i e r e t . a l . (67).
B a c t e r i a were s t r e a k e d on p l a t e s c o n t a i n i n g t h e
g e l a t i n medium (Appendix I ) and were s u b s e q u e n t l y i n c u b a t e d a t 27 C.
P l a t e s were su rv ey ed f o r c l e a r i n g o f t h e n o r m a l ly c l o u d y a g a r around
b a c t e r i a l c o l o n i e s a f t e r 2 and 5 d ay s .
Flo oding o f t h e p l a t e s with
m e r c u r i c c h l o r i d e t o p r e c i p i t a t e t h e un hy dr oly ze d g e l a t i n as p r e s c r i b e d
by Skerman (66) was n o t deemed n e c e s s a r y .
Zones o f c l e a r i n g confirm ed
t h e b a c t e r i u m ' s c a p a c i t y t o produce e x t r a c e l l u l a r p r o t e a s e s ( S t a n i e r
e t a l . (6 7 ) .
N i t r a t e Re duction
The a b i l i t y o f t h e b a c t e r i a t o grow a n a e r o b i c a l l y by u s in g n i t r a t e
i n s t e a d o f oxygen as t h e t e r m i n a l e l e c t r o n a c c e p t o r , was t e s t e d u s in g
t h e method o u t l i n e d by S t a n i e r e t a l . ( 6 7 ) .
Tubes c o n t a i n i n g 5 mis o f
n i t r a t e medium (Appendix I ) were i n o c u l a t e d w ith t h e b a c t e r i a and s u b ­
s e q u e n t l y o v e r l a y e r e d w it h m el ted 2% Difco a g a r .
i n c u b a t e d a t 27 C f o r 5 d a y s .
The t u b e s were the n
The p r e s e n c e o f a dens e t u r b i d i t y in t h e
medium i n d i c a t e d a p o s i t i v e r e a c t i o n (5 2 , 67 ).
32
Levan ' P r o d u c t! o n
'
P l a t e s o f n u t r i e n t a g a r c o n t a i n i n g 5% s u c r o s e were s t r e a k e d w ith
b a c t e r i a and i n c u b a t e d a t 27 C f o r 5 d a y s . .
Levan p r o d u c e r s formed
l a r g e w h i t e , mucoid c o l o n i e s (44, 52 ).
Li pas e
P l a t e s c o n t a i n i n g S i e r r a ' s l i p a s e medium (64) (Appendix I ) were
s t r e a k e d w it h t h e b a c t e r i a and i n c u b a t e d a t 27 C f o r 5 d a y s .
The p r e ­
s en ce o f an opaque h al o i n t h e medium s u r r o u n d i n g t h e b a c t e r i a l
colonies in d ic a te d a p o s iti v e t e s t f o r the production o f e x t r a c e l l u l a r
l i p a s e s by t h e b a c t e r i a ( 5 2 ) .
Pectin L iq u ific a tio n
A medium c o n t a i n i n g 2.2% sodium p o l y p e c t a t e was a d j u s t e d t o p H '7 . 0
a c c o r d i n g t o t h e method d e s c r i b e d by H il d e b ra n d (29) (Appendix I ) .
p l a t e s were a l lo w e d t o a i r d r y f o r 3 d a y s .
The
B a c t e r i a were s p o t t e d oh
t h e medium and i n c u b a t e d a t 27 C f o r 3 d ay s . . The o c c u r r e n c e o f d e p r e s ­
s i o n s in t h e medium around t h e b a c t e r i a l c o l o n i e s was i n d i c a t i v e o f
p e c t i c enzyme a c t i v i t y .
P o t a t o S o f t Rot
Washed and p e e l e d p o t a t o t u b e r s w ere s l i c e d i n t o s e c t i o n s I cm
thick.
P o t a t o d i s c s were made u s i n g a I cm cork b o r e r and p l a c e d in a.
p e t r i d i s h , f o u r d i s c s p e r p l a t e , h a l f immersed in s t e r i l e d i s t i l l e d
water.
A l o o p f u l o f b a c t e r i a was s p r e a d o v e r t h e c e n t e r o f t h e d i s c s ;
33
3 d i s c s in each p l a t e were i n o c u l a t e d w h i l e t h e 4 t h s e r v e d as a con­
tro l.
The p l a t e s were i n c u b a t e d f o r 3 d a y s . a t 21 C.
The t e s t was co n­
s i d e r e d p o s i t i v e i f t h e i n o c u l a t e d d i s c s were c o m p l e t e l y s o f t as com­
par ed t o t h e c o n t r o l s .
Catalase
A drop o f 10% hydrogen p e r o x i d e was p l a c e d on a 4 8 - hour o ld b a c ­
t e r i a l co l o n y .
The p r o d u c t i o n o f bu b b le s was i n d i c a t i v e o f t h e
presence o f c a t a l a s e (66).
T em per at ure R e l a t i o n s h i p s
The a b i l i t y o f t h e b a c t e r i a t o grow a t 4 , 37 and 41 C was d e t e r ­
mined in t e s t t u b e s c o n t a i n i n g 5 mis o f Yeast E x t r a c t Medium ( D i f c o ) .
Tubes were i n o c u l a t e d w i t h a l o o p f u l o f b a c t e r i a and p l a c e d in i n c u b a ­
to r s s e t a t the d i f f e r e n t tem peratures.
Growth, as measured by th e
t u r b i d i t y o f t h e medium, was r e c o r d e d a f t e r 5 days f o r t h e t u b e s i n c u ­
b a t e d a t 27 and 37 C and a f t e r 10 days f o r t h o s e i n c u b a t e d a t 4 and
41 C.
Erwinia S p e c i f i c T e s t s
Anaer obic F e r m e n t a t i o n o f Glucose *I)
Tubes c o n t a i n i n g 5 mis o f Hugh and L e i f s o n ' s medium (31) (Appendix
I ) were s t a b i n o c u l a t e d w it h a l o o p f u l o f b a c t e r i a and o v e r l a y e r e d w i t h
m e lt ed 2% a g a r ( D i f c o ) .
The t u b e s were th e n i n c u b a t e d a t 27 C f o r 5
34
day s.
The c o n v e r s i o n o f t h e i n d i c a t o r dy e, c o n t a i n e d in tfie medium,
from gr een t o y e l l o w i n d i c a t e d a p o s i t i v e t e s t f o r t h e a n a e r o b i c f e r ­
mentation o f glucose.
N u tritio n al Tests
To d e t e r m i n e t h e n u t r i t i o n a l v e r s a t i l i t y o f t h e o r g a n i s m s , t h e
p r o d u c t i o n o f a c i d on c a r b o h y d r a t e and r e l a t e d carb on s o u r c e s was
t e s t e d by t h e t e c h n i q u e o u t l i n e d by Dye ( 1 8 ) .
Tubes c o n t a i n i n g \%
( v / v ) s o l u t i o n o f n-methyl g l u c o s i d e , g l y c e r o l , c e l l o b i o s e , m a l t o s e ,
x y l o s e , s a l i c i n , l a c t o s e and rhamnose in Dye's medium (18) (Appendix I)
were i n o c u l a t e d w i t h b a c t e r i a and i n c u b a t e d , unshaken, a t 27 C f o r 6
da ys .
A change in t h e c o l o r o f t h e a c i d i n d i c a t o r c o n t a i n e d in t h e
n u t r i e n t s o l u t i o n , from g ree n t o y e l l o w , was s c o r e d as a p o s i t i v e t e s t
f o r a n a e r o b i c growth on t h e v a r i o u s s u b s t r a t e s .
Pseudomonas S p e c i f i c T e s t s
Arginine Dihydrolase
A r g i n i n e d i h y d r o l a s e p r o d u c t i o n was t e s t e d u s in g T h o r n l e y 1s method
(66).
Tubes c o n t a i n i n g T h o r n l e y ' s medium (Appendix I ) were s t a b i n o c u ­
l a t e d w it h t h e b a c t e r i a and s u b s e q u e n t l y o v e r l a y e r e d w i t h mel ted 2%
Difco a g a r .
The t u b e s were th e n i n c u b a t e d a t 27 C f o r 5 d ay s .
The
t e s t was c o n s i d e r e d p o s i t i v e i f t h e bas e i n d i c a t o r c o n t a i n e d w i t h i n t h e
medium changed in c o l o r from l i g h t p in k t o deep r e d .
35
N u tritio n al Tests
The f o l l o w i n g ca rb on and c a r b o n - n i t r o g e n compounds were used as
t h e s o l e o r g a n i c s u b s t r a t e s f o r growth o f t h e b a c t e r i a :
alpha-L-
a l a n i n e , b e t a - a l a n i n e , s o r b i t o l , s u c r o s e and t r e h a l o s e .
The c a r b o n -
n i t r o g e n s o u r c e s and s u g a r s were added 0.1% ( v / v ) and, 0.2% ( v / v ) ,
r e s p e c t i v e l y , to Dye's M i n e r a l Medium (18) (Appendix I ) .
were s o l i d i f i e d w it h 1.2% Nobel a g a r .
4 bacterial
The p l a t e s were s p o t i n o c u l a t e d ,
i s o l a t e s p e r p l a t e , and i n c u b a t e d a t 27 C.
scored f o r b a c t e r ia l
Both media
growth a f t e r 5 and 10 days.
The p l a t e s were
36
RESULTS AND DISCUSSION
The f l u o r e s c e n t Pseudomonads were c h a r a c t e r i z e d u s i n g t h e d e t e r ­
m i n a t i v e scheme p r o pos ed by L e l l i o t e t a l . ( 4 4 ) .
Of p r im a r y d e t e r m i n a ­
t i v e v a l u e a r e t h e l e v a n , o x i d a s e , p o t a t o s o f t r o t , a r g i n i n e d i hy dr o­
l a s e and to b a c c o h y p e r s e n s i t i v i t y t e s t s
(LOPAT).
Subsidiary te s t s
i n c l u d e t h e l i p a s e and n i t r a t e r e d u c t i o n t e s t s .
The Pseudomonads i s o l a t e d from d i s e a s e d s a i n f o i n r o o t s f e l l
into
two d i s c r e t e g r o u p s , t h e o x id a s e p o s i t i v e and o x i d a s e n e g a t i v e Pseudo­
monads.
Oxidase N eg at i v e Pseudomads
R e a c t i o n s t o t h e b io c h em ic al and p h y s i o l o g i c a l t e s t s were uniform
among t h e t e n i s o l a t e s t e s t e d (Tab le 2 - 4 ) .
S l i g h t d i f f e r e n c e s wer'e
e n c o u n t e r e d i n t h e r e a c t i o n o f t h e i s o l a t e s on b e t a - a l a n i n e .
Although
t h e m a j o r i t y o f t h e i s o l a t e s d i s p l a y e d no growth on t h e s u b s t r a t e , a
few i s o l a t e s e x h i b i t e d s l i g h t growth a f t e r 10 d ay s .
Re sidual o r g a n i c
i m p u r i t i e s in t h e p e t r i d i s h e s o r in t h e s u b s t r a t e may have acc ou n te d
f o r t h e ma rg in al growth o f t h e s e i s o l a t e s .
The o x i d a s e n e g a t i v e Pseudomonads were s i m i l a r , b i o c h e m i c a l l y and
p h y s i o l o g i c a l l y , t o Pseudomonas s y r i n g a e in 93% o f t h e c h a r a c t e r i z a t i o n
t e s t s p u b l i s h e d by Misaghi and Grogan (52) f o r t h i s b a c t e r i u m (Table
2-4).
The o n l y d i s c r e p a n c y between t h e s a i n f o i n i s o l a t e s and t h e t y p e
s p e c i e s o f P_. s y r i n g a e was in t h e p o s i t i v e l i p a s e t e s t f o r t h e s a i n f o i n
37
Tabl e 2- 4 .
Comparison o f b io ch em ic al and p h y s i o l o g i c a l c h a r a c t e r i s t i c s
. o f t h e o x i d a s e n e g a t i v e Pseudomonad^'i s o l a t e d , from d i s e a s e d
s a i n f o i n p l a n t s t o t h o s e p u b l i s h e d f o r Pseudomonas
• syringae.
Oxidase N eg at i ve Pseudomonads
Test
Oxidase
H ypersensitivity
Arginine d i hydrolase
M otility
Gelatin hydrolysis
N itra te reduction
Catalase
Potato s o f t ro t
Sodium p o l y p e c t a t e (pH .7)
Li pa s e
Levan
Carbon s u b s t r a t e :
Sorbitol
Suc ro se
Trehalose
Al p h a - a l a n i n e
B eta-alanine
T em per at ure
Relationships:
4 C
27 C
37 C
41 C
sainfoin iso la te s I/
+
-
+
■+
P. s y r i n g a e 2 /
+
-
+
+
-
-
+
+
-
-
-
-
+
+
+
+
+
-
+
+
+
+
-
-
+
+
+
-
d 4/
+
-
1Based on 10 i s o l a t e s c o l l e c t e d from d i s e a s e d s a i n f o i n r o o t s and crowns.
P
^ R e s u lt s .taken from Misaghi and Grogan ( 5 2 ) .
O
- = n e g a t i v e , + = p o s i t i v e and + = weak growth.
^d = 21-79% o f t h e s t r a i n s t e s t e d were p o s i t i v e .
38
isolates.
Because t h e p r o p e r t i e s o f t h e s a i n f o i n i s o l a t e s conform
a l m o s t i d e n t i c a l l y t o t h o s e p u b l i s h e d f o r P_. s y r i n g a e , i t i s proposed
t h a t t h e o x i d a s e n e g a t i v e Pseudomonads i s o l a t e d from d i s e a s e d s a i n f o i n
<■
r o o t s and crowns be. r e f e r r e d t o as Pseudomonas s y r i n g a e .
Whether o r
not s u f f i c i e n t d i f f e r e n c e s e x i s t between t h e s a i n f o i n i s o l a t e s and t h e
d i f f e r e n t p a t h o v a r s o f F\ s y r i n g a e w i t h r e s p e c t t o h o s t r a n g e , remains
t o be d e t e r m i n e d .
The e x t e n t t o which P_. s y r i n g a e o c c u r s in t h e r o o t s and crowns o f
d i s e a s e d s a i n f o i n grown un der i r r i g a t i o n i s unknown b ec a u se t h e o x i d a s e
t e s t was no t r o u t i n e l y performed on a l l t h e Pseudomonas i s o l a t e s
e n c o u n t e r e d in t h e i s o l a t i o n s .
However, in a random s u r v e y o f 33 BC
a g a r p l a t e s c o n t a i n i n g Pseudomonas spp. o b t a i n e d from t h e 2, 3 and 5
y e a r o l d s a i n f o i n p l a n t s , 1/3 o f t h e i s o l a t e s were o x i d a s e n e g a t i v e .
Because t h e r e i s a high c o r r e l a t i o n between t h e i d e n t i t y o f a Pseudo­
monas i s o l a t e as b ei ng _P. s y r i n g a e and a n e g a t i v e o x i d a s e t e s t (43, 44 ,
5 2 ) , t h i s i n d i r e c t e v i d e n c e s u g g e s t s t h a t t h i s o rg ani sm o c c u r s f r e ­
q u e n t l y in t h e symptomatic t i s s u e in d i s e a s e d s a i n f o i n r o o t s .
P_. s y r i n g a e i s g e n e r a l l y c o n s i d e r e d a l e a f s p o t t i n g pathogen (56,
52) and i s n o t n o r m a l l y a s s o c i a t e d w i t h r o o t t i s s u e .
There a r e no
r e p o r t s i n t h e l i t e r a t u r e o f £_. s y r i n g a e being i s o l a t e d from d i s e a s e d
r o o t s o f f o r a g e legumes and c o n s e q u e n t l y , t h e a s s o c i a t i o n between t h i s
or gan ism and s a i n f o i n r o o t s i s a uniq ue f i n d i n g .
P_. s y r i n g a e has been
39
i m p l i c a t e d in a v a s c u l a r b l a c k e n i n g d i s e a s e o f s u g a r b e e t t a p r o o t s in
Germany ( 4 9 ) .
O x i d a t i v e P o s i t i v e Pseudomonads
C o n s i d e r a b l e h e t e r o g e n e i t y e x i s t e d i n t h e r e a c t i o n s among t h e 11
o x i d a s e p o s i t i v e Pseudomonads in t h e c h a r a c t e r i z a t i o n t e s t s
(Table 2 - 5 ) .
Between 10% and 20% o f t h e i s o l a t e s behaved ab n o r m a ll y in t h e g e l a t i n ,
n i t r a t e r e d u c t i o n and le v a n t e s t s .
Sev ent y-t wo o f t h e i s o l a t e s p r o ­
duced a s o f t r o t in p o t a t o e s .
Seven o f t h e e l e v e n i s o l a t e s were in ac c o r d a n c e w it h LOPAT r e q u i r e ­
ments o u t l i n e d by L e l l i o t e t a l .
( 4 4 ) , f o r t h e b a c t e r i u m Pseudomonas
margi nal i s , a group o f p l a n t p a t h o g e n i c s o f t r o t t i n g o r g a n i s m s .
Nega­
t i v e r e a c t i o n s e n c o u n t e r e d in t h e s u b s i d i a r y c h a r a c t e r i s t i c s , l i p a s e and
n i t r a t e r e d u c t i o n , f o r t h e s a i n f o i n i s o l a t e s as compared t o t h e t y p e
s t r a i n o f P_. ma rg in al i s may r e p r e s e n t a f u r t h e r d e t e r i o r a t i o n in t h e
n u tr i tio n a l v e r s a t i l i t y of the s a in fo in i s o l a t e s .
This o bservation i s
common among p l a n t p a t h o g e n i c Pseudomonads (52, 56, 6 7 ) .
The o x i d a s e
p o s i t i v e s a i n f o i n i s o l a t e s were 88% s i m i l a r t o Pseudomonas marginal i s
w ith r e s p e c t t o t h e i r b e h a v i o r in t h e c h a r a c t e r i z a t i o n t e s t s .
I t is
t h e r e f o r e prop os ed t h a t t h e o x i d a s e p o s i t i v e , s o f t r o t t i n g Pseudomonads
i s o l a t e d from d i s e a s e d s a i n f o i n p l a n t s be i n c l u d e d in t h e Pseudomonas
margi nal i s gro up.
The s a i n f o i n i s o l a t e s a p p e a r d i s t i n c t , b i o c h e m i c a l l y
and p h y s i o l o g i c a l l y , from F\ margi nal i s v a r . a I f a l f a e which were o n ly
T ab l e 2 - 5 .
Comparison o f b io ch em ic al and p h y s i o l o g i c a l t e s t s between t h e o x i d a s e p o s i ­
t i v e Pseudomonads i s o l a t e d from d i s e a s e d s a i n f o i n r o o t s w it h P_. margi nal i s
v a r . a l f a l f a e and t h e c h a r a c t e r i s t i c s p u b l i s h e d f o r P_. marginal i s .
Isolates
Test
Oxidase
H ypersensitivity
Arginine d i hydrolase
M otility
Gelatin hydrolysis
N itrate reduction
Catalase
Potato s o f t r o t
Sodium p o l y p e c t a t e (pH 7)
Li pa s e
Levan
+
+
+
+
+
+
+
+
Carbon s u b s t r a t e :
Sorbitol
S uc r o s e
Trehalose
A lpha-alanine
Beta-alanine
+
+
+
+
+
P. ma rg in al i s
v a r . a l f a l f a e 2/
K L
M X 8
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
-
+
-
+
-
-
-
+
-
+
+
-
+
+
+
+
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Group I I / I
•—. + +
+
+
+
+
+
+
+
+
P. marginal i s 3/
Table 2-5
(C en t.).
,
Comparison o f b io c h e m ic a l and p h y s i o l o g i c a l t e s t s between t h e o x id a s e
p o s i t i v e P s e u d o m o n a d s i s o l a t e d f r o m d i s e a s e d s a i n f o i n r o o t s w i t h P_.
m a rg in a l i s v a r . a l f a l f a e and t h e c h a r a c t e r i s t i c s p u b l i s h e d f o r
P_. m a r g i n a l i s .
'
Test
Te m per at ure
Relationships
i
p
Group I I / I
4
27
37
41
C
C
C
C
+
+
K L
Isolates
P_. ma rg in al i s
M X 8 . var. a l f a l f a e 2/
+ + + + + +
+ + + + ■ + +
- - - - - - - - - - -
P_. marginal i s 3/
+
+
-
04/
0
0
0
Group c o n s i s t s o f 4 i s o l a t e s .
I s o l a t e s o b t a i n e d from Dr. F. L. Lukezic a t P e n n s y l v a n i a S t a t e U n i v e r s i t y .
^Data from L e l l i o t e t a l . ( 4 4
) , and D oud ero ff and P a l l e r o n i
( 17
).
^Data on t h e t e m p e r a t u r e r e l a t i o n s h i p s o f P_. ma rg in al i s was n o t found i n l i t e r a t u r e .
42
56% s i m i l a r t o t h e s a i n f o i n i s o l a t e s in t h e c h a r a c t e r i z a t i o n t e s t s .
Whether o r n o t t h e s e i s o l a t e s a r e s u f f i c i e n t l y d i f f e r e n t t o w a r r a n t
s p e c i a l p a t h o v a r d e s i g n a t i o n must be d e t e r m in e d i n f u t u r e h o s t r an ge .
tests.
The n o n - s o f t - r o t t i n g Pseudomonads i s o l a t e d from t h e d i s e a s e d s a i n ­
f o i n p l a n t s p r o b a b l y belo ng t o t h e s a p r o p h y t i c Pseudomonad groups which
c o n s t i t u t e a la rg e proportion o f th e so il m icro flo ra (67).
I t is
l i k e l y t h a t t h e s e o r gan ism s a r e s e c o n d a r y i n v a d e r s in t h e de ca y in g
tissu e.,
White B a c t e r i a The w h i t e b a c t e r i a were i n i t i a l l y d i v i d e d i n t o 2 groups based on
t h e i r a b i l i t y t o produce a brown d i f f u s a b l e pigment and c a u s e a s o l u ­
b i l i z a t i o n o f t h e c l o u d y p r e c i p i t a t e in K in g ' s B a g a r .
Group A, which
were p o s i t i v e in both t h e s e t e s t s and were t h e p re d o m in an t w h i t e b a c ­
t e r i a l forms i s o l a t e d , behaved u n i f o r m l y in t h e m a j o r i t y o f t h e c h a r a c ­
terization te sts
(Tab le 2 - 6 ) .
Some v a r i a b i l i t y e x i s t e d in t h e t e s t s
i n v o l v i n g a c i d p r o d u c t i o n on t h e d i f f e r e n t o r g a n i c s u b s t r a t e s .
Group A i s o l a t e s were i d e n t i c a l
in 78% o f t h e c h a r a c t e r i z a t i o n
t e s t s t o t h o s e p u b l i s h e d f o r Erwinia a m y lo v o r a , t h e c a u s a l a g e n t o f
f i r e b l i g h t i n many f r u i t and or namental t r e e s ( 1 9 ) .
The ma jor d i f f e r ­
ence between t h e b a c t e r i a l t y p e s r e s i d e d in t h e a b i l i t y o f t h e s a i n f o i n
i s o l a t e s t o a n a e r o b i c a l l y r e d u c e n i t r a t e and t h e i r i n a b i l i t y t o induc e
th e h y p e r s e n s iti v e r e a c t i o n in tobacco.
However, t h e w h i t e b a c t e r i a
43
Tabl e 2^6.
Comparison o f bio ch em ic al and p h y s i o l o g i c a l c h a r a c t e r i s t i c s
o f t h e w h i t e b a c t e r i a w it h 2 s e l e c t e d p l a n t p a t h o g e n i c .
Erwinia amylovora spp.
Sainfoin I s o la te s
Erwinia amylovora Erwinia
Group A V Group B 2 / v a r . a l f a l f a e 3 / amyIovora 4 /
Test
Oxidase
H ypersensitivity
Glucose f e r m e n t a t i o n
M otility
G elatin hydrolysis
N itra te reduction
Catalase
Potato s o f t r o t
Sodium p o l y p e c t a t e
(pH 7)
Li pase
Levan
Acid P r o d u c t i o n on:
n-methyl-glucoside
Gly cer ol
Cellobiose
Malt o s e
Xylose
Salicin
La c to s e
Rhamnose
Tem perature
Relationships
4
27
37
41
C
C
C
C
- 5/
-
+
+
+
+
+
+
-
+
+
+
+
+
+
-
-
-
+
-
d
o I/
O
O
O
+
O
-
+
+
+
+.
d
-
-
-
+
+
+
+
d
+
-
-
+
-
—
+
+
+
-
+
+
-
+
. -
1
+
+
+'
+
O
+
O
O
^Group A c o n s i s t e d o f 6 t y p i c a l i s o l a t e s o f t h e w h i t e b a c t e r i u m .
oGroup B c o n s i s t e d o f 2 a t y p i c a l i s o l a t e s o f a w h i t e b a c t e r i u m ,
a Data from Shinde and Lukezic ( 58 ).
nData from Dye ( 18 ).
z.- = n e g a t i v e , + = p o s i t i v e , and + = weak growth.
° d = between 21-79% o f t h e s t r a i n s t e s t e d were p o s i t i v e .
O = d a t a c o n c e r n i n g t h i s t e s t was n o t found in t h e l i t e r a t u r e .
-
.
+
+
+
+
O
+
-
44
i s o l a t e d from d i s e a s e d s a i n f o i n res po nde d i d e n t i c a l l y t o t h e c h a r a c :
t e r i z a t i o n t e s t s p u b l i s h e d f o r Erwinia amyldvora v a r . a l f a l f a e ( 5 8 ) . .
■
'
'
'
' .
;
.
•
C o n s e q u e n t ly , i t i s proposed t h a t t h e w h i t e b a c t e r i a which comprised
Group A be i n c l u d e d in t h e Erwinia amylovora s p e c i e s .
In l i g h t o f t h e
e x i s t i n g d i f f e r e n c e s between t h e s a i n f o i n i s o l a t e s and t h e ty p e s t r a i n s
o f t h e F i r e b l i g h t o r g a n is m , s a i n f o i n i s o l a t e s s ho u ld p r o b a b l y be given
a separate pathovar designation.
F u r t h e r c o m p a r a ti v e c h a r a c t e r i z a t i o n
and p a t h o g e n i c i t y t e s t s would be r e q u i r e d t o d e t e r m i n e i f t h e s e i s o ­
l a t e s s h o ul d be i n c l u d e d in w it h Erw ini a amylovora v a r . a l f a l f a e .
Mucoid B a c t e r i a
The 4 i s o l a t e s o f t h e mucoid b a c t e r i a behaved i d e n t i c a l l y in t h e
characterization te s ts
(Ta b le 2 - 7 ) .
These b a c t e r i a a p p e a r t o be
Erw inia h e r b i c o l a v a r . h e r b i c o l a , a y e l l o w pigmented b a c t e r i u m commonly
found on t h e s u r f a c e o f most p l a n t s ( 1 8 ) .
Most r e s e a r c h e r s b e l i e v e
t h i s b a c t e r i u m t o be a s ec o n d a r y i n v a d e r o f decayed stems and r o o t s and
is of l i t t l e
im p o r ta n c e as a p r im ar y pat ho gen .
CONCLUSIONS
B a c t e r i a a p p e a r t o be t h e p r e d om in an t organ ism s i n t h e decayed and
d i s c o l o r e d r o o t s o f s a i n f o i n grown un der i r r i g a t i o n , whereas £ . s o l a n i
a p p e a r s l e s s i n t i m a t e l y a s s o c i a t e d w i t h d i s e a s e d t i s s u e th a n was p r e ­
v io u sly thought.
Because t h i s fungus i s p r i m a r i l y a s s o c i a t e d w ith
d e c a y i n g crowns o f t h e o l d e r d i s e a s e d p l a n t s and i s n o t commonly found
45
Ta b le 2-7
Comparison o f bioc hem ic al and p h y s i o l o g i c a l t e s t s o f t h e
mucoid b a c t e r i a i s o l a t e d from d i s e a s e d s a i n f o i n r q p t s w it h
t h o s e p u b l i s h e d f o r Erwlnia h e r b i c o l a . v a r . h e r b i c o l a .I
. Mucoid I /
Bacteria
Tests
Oxidase
H ypersensitivity
Glucose f e r m e n t a t i o n
M otility
G elatin hydrolysis
N itrate reduction
C a ta lase
Potato s o f t r o t
Li pas e
Levan
Acid P r o d u c t i o n on:
n-m ethyl-glucoside
Gly cer ol
Cellobiose
Maltose
Xylose
Salicin
La c to s e
Rhamnose
Tem perature
Relationships
I
4
27
37
41
C
C
C
C
'
- j '+
.+
.+
+
-
■
+
+
+
+
+
+
+
+
Erw ini a h e r b i c o l a 2 /
. var. herbicola
+
+
+
+
+
d 4/
T
+
+
' +
d
+
O 5/
+
+
Data based on 4 i s o l a t e s o b t a i n e d from d i s e a s e d s a i n f o i n r o o t s and
^crowns.
= n e g a t i v e , + = p o s i t i v e , + = weak growth.
^Data o b t a i n e d from Dye ( 19 J .
(id = between 21-79% o f t h e i s o l a t e s t e s t e d were p o s i t i v e .
bO = d a t a c o n c e r n i n g t h i s t e s t was n o t found in l i t e r a t u r e .
46
in t h e l e a d i n g edge o f t h e c e n t r a l dec ay r e g i o n , i t a p p e a r s t h a t n e i ­
t h e r £ . soTani nor o t h e r Fiisarium spp. can be s o l e l y r e s p o n s i b l e f o r
t h e r a p i d s t a n d d e c l i n e in i r r i g a t e d s a i n f o i n .
B a c t e r i a a r e c l o s e l y and c o n s i s t e n t l y a s s o c i a t e d w i t h t h e sympto­
m a ti c t i s s u e s a t a l l s t a g e s o f t h e d e c l i n e .
P_. s y r i n g a e , commonly a
l e a f s p o t t i n g o r g a n is m , has n o t been a s s o c i a t e d w i t h t h e r o o t s o f
f o r a g e legumes and, c o n s e q u e n t l y , i t s a s s o c i a t i o n w it h s a i n f o i n r o o t s
is of considerable in t e r e s t .
E., amylovora and P. m arg in al i s have been
i m p l i c a t e d as t h e c a u s a l a g e n t s in a r o o t r o t o f a l f a l f a in P e n n s y l­
vania.
The r e s u l t s o b t a i n e d from t h e s e e x p e r i m e n t s s t r o n g l y s u g g e s t t h a t
t h e r o o t arid crown r o t complex in i r r i g a t e d s a i n f o i n s t a n d s in Montana
i s a b a c t e r i a l , r a t h e r th a n a f un gal problem.
An e x t e n s i v e s ur v ey o f
t h e i r r i g a t e d s a i n f o i n in Montana and North America would be r e q u i r e d
t o d e t e r m i n e t h e e x t e n t t o which t h e s e org ani sm s o c c u r in d i s e a s e d
sainfoin roots.
The. high m o i s t u r e c o n d i t i o n s in i r r i g a t e d f i e l d s
cou pl ed w it h t h e heavy Montana s o i l s p r o v i d e s an i d e a l envi ron m ent f o r
t h e growth and p r o l i f e r a t i o n o f b a c t e r i a i n t h e s e r o o t s .
Whether o r
n o t t h e s e or ga n is m s a r e r e s p o n s i b l e f o r t h e d r y l a n d crown r o t in s a i n ­
foin s t i l l
rem a in s t o be d e t e r m i n e d .
Experim ent 3
•
•
. ' ■EVALUATION,OF THE PATHOGENICITY OF REPRESENTATIVE BIOTYPES OF THE
DIFFERENT BACTERIAL SPECIES ISOLATED FROM DISEASED SAINFOIN ROOTS TO
GREENHOUSE REARED SAINFOIN AND THE COMPARISON OF THE RELATIVE PATHOGENI
CITY OF FOUR ERWINIA AMYLOVORA STRAINS WITH THAT OF F. SOLANI.
MATERIALS AND METHODS
The e v a l u a t i o n o f t h e p a t h o g e n i c i t y o f t y p i c a l b i o t y p e s o f t h e d i f
f e r e n t b a c t e r i a l s p e c i e s and t h e comparison o f t h e r e l a t i v e p a t h o g e n i ­
c i t y o f f o u r i s o l a t e s o f E. amylovora and £ . s o l a n i were c a r r i e d o u t in
two s e p a r a t e g r ee n h o u s e t r i a l s .
The e f f e c t o f combined i n o c u l a t i o n
w i t h E. amylovora and £ . s o l a n i in s a i n f o i n was a l s o examined i n t h e
second e x p e r i m e n t .
Remont s a i n f o i n s ee d s were p l a n t e d in 15 cm p l a s t i c p o t s con­
t a i n i n g unused v e r m i c u l i t e .
r e d uc e d t o s i x p e r p o t .
A f t e r g e r m i n a t i o n , s e e d l i n g numbers were
The p l a n t s were grown in t h e g ree n h o u se u s in g
a 16 hour p h o t o p e r i o d p r o v id e d by s up p lem en tal l i g h t i n g from f l u o r e s ­
cent lights.
(Appendix I ) .
P l a n t s were w a t e r e d w i t h a d i l u t e n u t r i e n t s o l u t i o n
Both t r i a l s were c o n d u ct ed as a random b lo c k d e s ig n
using th r e e r e p l i c a t i o n s .
b ot h t r i a l s .
S i m i l a r e x p e r i m e n t a l c o n d i t i o n s p r e v a i l e d in
Data were a n a l y z e d by a n a l y s i s o f v a r i a n c e and t h e means
were s e p a r a t e d u s in g Duncan's M u l t i p l e Range T e s t ( 6 9 ) .
48
B a c t e r i a l i s o l a t e s were s t o r e d in s t e r i l e d i s t i l l e d w a t e r a t 4 C.
Four days p r i o r t o i n o c u l a t i o n , t h e b a c t e r i a were s t r e a k e d on K in g 's B
a g a r and i n c u b a t e d a t 21 C.
The £ . s o l a n i s a i n f o i n i s o l a t e was s t o r e d
in s o i l a c c o r d i n g t o t h e p r o c e d u r e o u t l i n e d by Toussoun and Nelson
(71).
A small amount o f s o i l was s p r e a d on p e t r i d i s h e s c o n t a i n i n g
HPDA and i n c u b a t e d a t 21 C f o r f o u r d a y s .
A s t e r i l e loop was dipped
i n t o t h e c u l t u r e and s t r e a k e d on a c i d i f i e d w a t e r a g a r p l a t e s (Appendix
I ) and i n c u b a t e d 48 hours a t 21 C.
Using a d i s s e c t i n g m i c r o s c o p e , a
s i n g l e g e r m i n a t i n g s p o r e was t r a n s f e r r e d t o PDA and i n c u b a t e d f o r 10
d a y s , u s i n g f l u o r e s c e n t lamps t o produce a 12 hour p h o t o p e r i o d .
The same i n o c u l a t i o n t e c h n i q u e was used f o r both t h e b a c t e r i a and
£. solani.
A 26 gauge n e e d l e was dippe d i n t o t h e b a c t e r i a l o r fungal
c o lo ny and pushed cros swa ys th r o u g h t h e crowns o f t h e s a i n f o i n s e e d ­
lings.
Ap pr o xi ma te ly 8 X IO^ b a c t e r i a and IO^ m ac ro con idi a were i n o ­
c u l a t e d i n t o t h e crowns in t h i s f a s h i o n .
A f t e r t h e i n c u b a t i o n p e r i o d , t h e p l a n t s were h a r v e s t e d and th e
r o o t s s p l i t l o n g i t u d i n a l l y t h r o u g h t h e crowns.
The s c o r i n g p r oc ed u r e
c o n s i s t e d o f m e a su r i n g t h e d i s t a n c e (mm) which t h e d i s c o l o r a t i o n had
s p r e a d from t h e p o i n t o f i n o c u l a t i o n .
d i s c o l o r a t i o n was r a t e d as f o l l o w s :
In a d d i t i o n , t h e s e v e r i t y o f t h e
I = l i g h t , 2 = m o d e ra te and 3 =
s e v e r e , depend ing on t h e i n t e n s i t y and t h e l a t e r a l
t i o n d i r e c t l y below t h e i n o c u l a t i o n p o i n t .
spread o f d is c o lo r a ­
49
Symptomatic r o o t s e c t i o n s from each t r e a t m e n t were 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 i n u te s in 0.5% NaOCl and p l a c e d on PDA and HPDA.
I s o l a t i o n f o r b a c t e r i a on K in g 's B a g a r was c a r r i e d o u t u s i n g t h e
method d e s c r i b e d in t h e p r e v i o u s e x p e r i m e n t .
inoculated
Fungi r e i s o l a t e d from t h e
s e c t i o n s were i d e n t i f i e d m i c r o s c o p i c a l l y .
B a c t e r i a were
i d e n t i f i e d by comparing t h e r e i s o l a t e d c o l o n y t y p e s w i t h t h o s e o f t h e
original is o la te s .
In a d d i t i o n , t h e o x i d a s e t e s t was performed on t h e
Pseudomonas i s o l a t e s .
T e s t I _______
Eight b a c t e r ia l
i s o l a t e s a s s a y e d i n t h e p a t h o g e n i c i t y t e s t s and
t h e i r o r i g i n a r e l i s t e d i n Tabl e 3 - 1 .
i s o l a t e o f P_. margi nal i s v a r . al f a l f a e .
I n c lu d e d in t h e t e s t s was an
The same b a c t e r i a were b i o ­
c h e m i c a l l y and p h y s i o l o g i c a l l y c h a r a c t e r i z e d in t h e p r e v i o u s ex p e r i m e n t
The b a c t e r i a were i n o c u l a t e d
i n t o t h e crowns o f two month o l d s a i n f o i n
s e e d l i n g s and i n c u b a t e d in t h e g ree n h o u se f o r a p e r i o d o f f o u r weeks.
S t e r i l e d i s t i l l e d w a t e r was i n o c u l a t e d
in the control s e r i e s .
T e s t 2_______
The f o u r i s o l a t e s o f E_. amylovora and t h e F. s o l a n i i s o l a t e used
in t h e i n o c u l a t i o n s
were o r i g i n a l l y i s o l a t e d from d i s e a s e d f o u r y e a r
o ld s a i n f o i n p l a n t s growing a t t h e F i e l d Research L a b o r a t o r y , Bozeman,
Montana.
%
Fusarium s o l a n i s t r a i n FS-9 was s e l e c t e d b e c a u s e i t was
50
judge d t h e most v i r u l e n t s a i n f o i n i s o l a t e e n c o u n t e r e d on s a i n f o i n and
peas in p r e v i o u s p a t h o g e n i c i t y t e s t s .
Table 3 - 1 .
Bacterial
E i g h t d i f f e r e n t b a c t e r i a l i s o l a t e s and t h e i r o r i g i n r e p r e ­
s e n t i n g t h e 4 b a c t e r i a l s p e c i e s used in t h e greenho us e
in o c u la tio n study.
Isolate
Origin
#1.
#2.
#3.
P. s v r i n q a e
P. s v r i n q a e
-Er. h e r b i c o l a
#4.
#5.
E. amylovora
E. amylovora
#6.
#7.
#8.
E. amylovora
P. m a r q i n a l i s
P. m a rq in al i s v a r . a l f a l f a e
2 y e a r o l d s a i n f o i n , Bozeman, MT
5 y e a r o l d s a i n f o i n , Bozeman, MT
4 y e a r o ld s a i n f o i n , . S a s k a to o n ,
Sa sk atc he wa n, Canada
3 y e a r o ld s a i n f o i n , Bozeman, MT .
7 y e a r o ld s a i n f o i n . S a s k a to o n ,
Sa sk atchewan, Canada
3 y e a r o l d s a i n f o i n , Bozeman, MT
2 y e a r o l d s a i n f o i n , Bozeman, MT
Dr. F. L. Lu kez ic , P e n n s y lv a n i a
S tate U niversity
The f o u r i s o l a t e s o f £ . amylovora and t h e £_. s o l a n i i s o l a t e were
each a s s a y e d i n d i v i d u a l l y .
In a d d i t i o n , t h e f o u r b a c t e r i a l
strains
were combined in equal volumes and t h e combined c e l l s i n o c u l a t e d w ith .
t h e £ . s o l a n i i s o l a t e i n t o t h e s a i n f o i n crowns.
f u l l o f each b a c t e r i a l
mixed.
To i n o c u l a t e :
a.loop-
i s o l a t e was p l a c e d in a s t e r i l e b e a k e r and
The 26 gauge n e e d l e was dip p ed i n t o t h e s u s p e n s i o n and pas sed
th r o u g h t h e s a i n f o i n crowns.
The n e e d l e was s u b s e q u e n t l y dippe d i n t o
t h e F. s o l a n i c u l t u r e and pa s s ed t h r o u g h t h e same i n o c u l a t i o n h o le .
The i n o c u l a t e d p l a n t s were i n c u b a t e d f o r a p e r i o d o f s i x weeks and
then harvested.
RESULTS AND DISCUSSION
The symptoms e n c o u n t e r e d in t h e p l a n t s i n o c u l a t e d w i t h 1b a c t e r i a ..
were s i m i l a r t o t h o s e e n c o u n t e r e d in n a t u r a l l y i n f e c t e d s a i n f o i n grown
under i r r i g a t i o n .
In most i n s t a n c e s , t h e i s o l a t e s p r o d u c e d . a b l a c k ,
v a s c u l a r d i s c o l o r a t i o n moving away from t h e p o i n t o f i n o c u l a t i o n .
bacterial
One
i s o l a t e , _P. s y r i n g a e #1, produced a red d i s c o l o r a t i o n and ,
decay in t h e i n o c u l a t e d s a i n f o i n p l a n t s .
Symptoms i n n a t u r a l l y
i n f e c t e d s a i n f o i n p l a n t s a r e p r e d o m i n a n t l y red crown d eca y and b la c k
vascular d isco lo ratio n .
F u r t h e r i n v e s t i g a t i o n s t o d e t e r m i n e which
or ga ni s m s a r e r e s p o n s i b l e f o r t h e d i f f e r e n t symptoms a r e n e c e s s a r y in
o r d e r t o more f u l l y u n d e r s t a n d t h e complex n a t u r e o f t h e d i s e a s e .
B a c t e r i a r e i s o l a t e d from t h e i n o c u l a t e d crowns were s i m i l a r in
c o l o n y morphology and t y p e t o t h o s e o r i g i n a l Iy i n o c u l a t e d .
In t h e
t r e a t m e n t i n v o l v i n g JE. amylovora and IF. s o l a n i s i m u l t a n e o u s l y i n o c u ­
l a t e d i n t o i n d i v i d u a l p l a n t s , both o r g a n is m s were r e i s o l a t e d .
F. oxy-
s porum was f r e q u e n t l y i s o l a t e d from i n o c u l a t e d t r e a t m e n t s i n c l u d i n g
the controls.
Of t h e f o u r b a c t e r i a l
s p e c i e s a s s a y e d , P. sj/ringae^ a p pe a r ed t o be
t h e most v i r u l e n t , f o ll o w e d by E. amylovo r a and P. marg i n a l i s (Table
3-2).
D i f f e r e n c e s in t h e mean s e v e r i t y s c o r e s and t h e e x t e n t o f t h e
d i s c o l o r a t i o n were i n s u f f i c i e n t t o s t a t i s t i c a l l y d i f f e r e n t i a t e between
t h e v i r u l e n c e l e v e l s o f E. amylovo r a and F\ s y r i n g a e .
However, P_.
s y r i n g a e was s i g n i f i c a n t l y more e f f e c t i v e in pr o du ci n g d i s e a s e symptoms
52
Tabl e 3 - 2 ,
The p a t h o g e n i c i t y o f 8 b a c t e r i a l i s o l a t e s on s a i n f o i n s e e d ­
l i n g s as measured by t h e e x t e n t o f t h e d i s c o l o r a t i o n from
t h e i n o c u l a t i o n p o i n t and by t h e mean d i s e a s e s e v e r i t y .
Isolate
Extent o f D iscolo ratio n
Mean Distance(mm) _!/
Mean d i s e a s e
Severity s c o r e V
P. s y r i n g a e (#1)
P. s y r i n g a e (#2)
18.5 A 3/
1 8 .8 A
P. ma rg in al i s (#7)
P. m a rg in al i s v a r . a l f a l f a e
15.3 AB,
11 .9 BC
1.58 B
1 . 50 B
E. amylovora (#4)
E. amylovora (#5)
E. amylovora (#6)
17,2 AB
15.2 AB
14 .8 AB
2.08 A
1.58 B
1.67 B
E. h e r b i c o l a (#3)
8 . 5 CD
1.0 0 C
Control - HgO
6.2 D
1 .0 0 C
2 .1 7 A
■ 2.33 A
■
—/ The e x t e n t o f movement o f b la c k d i s c o l o r a t i o n from t h e i n o c u l a t i o n
r,, p o i n t .
— S e v e r i t y measured as f o l l o w s :
I = l i g h t , 2 = mo d e ra te and 3 = s e v e r e
—
Column means f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y
d i f f e r e n t (P = 0 . 0 5 ) .
53
in i n o c u l a t e d r o o t s th a n was _P. mar g i n a l i s .
The symptoms produced by
t h e t h r e e b a c t e r i a were s i g n i f i c a n t l y more e x t e n s i v e (by both p a r a ­
m e t e r s ) t h a n t h o s e in t h e c o n t r o l s .
The symptoms produced by t h e E.
h e r b i c o l a i s o l a t e were s i m i l a r t o t h o s e o f t h e c o n t r o l s .
Thi s f i n d i n g
h e l p s c o n f ir m t h e s a p r o p h y t i c r o l e o f t h i s or g an is m in t h e r o o t and
crown r o t complex in s a i n f o i n and i s c o n s i s t e n t w it h t h e l i t e r a t u r e
p u b l i s h e d on t h i s or ga n is m ( 1 9 ) .
Because t h e o n l y i s o l a t e o f t h i s ,
o r g a n is m a s s a y e d d i d n o t o r i g i n a t e l o c a l l y , f u r t h e r s t u d y i n t o t h e
v i r u l e n c e o f Montana i s o l a t e s o f EL h e r b i c o l a on s a i n f o i n needs t o be
u n d e r t a k e n in o r d e r t o c o m p l e t e l y e l i m i n a t e t h i s o rg an is m as a p o t e n ­
tial
p a t ho ge n .
The symptoms caus ed by P_. ma rginal i s v a r . a l f a l f a e were
s t a t i s t i c a l l y more s e v e r e th a n t h o s e in t h e c o n t r o l s i n both d i s e a s e
p a r a m e t e r s , though v i r u l e n c e l e v e l s were low (Tab le 3 - 2 ) .
The f o u r JE. amylovora i s o l a t e s were s i g n i f i c a n t l y more e f f e c t i v e
in r e p r o d u c i n g d i s e a s e symptoms as measured by t h e e x t e n t o f d i s c o l o r a ­
t i o n t h a n t h e IF. s o l a n i
(Tab le 3 - 3 ) .
Combining both t h e b a c t e r i a and
fungus in t h e crowns had no e f f e c t on e i t h e r d i s e a s e p a r a m e t e r .
Conse­
q u e n t l y , a s y n e r g i s t i c r e a c t i o n i n v o l v i n g a complex o f t h e s e two
or ga ni s m s i s u n l i k e l y .
Such a r e a c t i o n may e x i s t between JF. s o l a n i and
t h e two r e m a i n i n g b a c t e r i a o r among t h e t h r e e b a c t e r i a l s p e c i e s and
s ho u ld be examined in a g r ee n ho u s e s t u d y .
P l a n t s i n o c u l a t e d w i t h £ . s o l a n i a l o n e d id n o t a p p e a r s i g n i f i ­
c a n t l y d i f f e r e n t t h a n t h e c o n t r o l s i n term s o f s e v e r i t y o f symptoms
54
i
,
.
T ab l e 3- 3 .
The p a t h o g e n i c i t y o f 4 i s o l a t e s o f £ . amylovora and one
i s o l a t e o f £_. s o l a n i in s a i n f o i n s e e d l i n g s as measured by
t h e e x t e n t o f t h e d i s c o l o r a t i o n measured from t h e i n o c u l a ­
t i o n p o i n t and t h e mean d i s e a s e s e v e r i t y s c o r e .
Organism
E. amylovora
Extent o f D isc o lo ra tio n I /
Mean D i s t a n c e (mm)
Isolate
15-5
15-9
19-1
20-1
F. s o l a n i +
E. amylovora
F. s o l a n i
Control - HgO
23.8 A
2 2 . 5 AB
18 .6 AB
18. OAB
16 .9 B
3/
Mean d i s e a s e
s e v e r i t y score 2/
2 .6 7
2 .7 5
2.08
1.9 2
A
A
BC
BC
2 . 4 0 AB .
ES-9
8.4 C
1.58 C
>
7 . SC
I . OOC
— The e x t e n t o f t h e b l a c k d i s c o l o r a t i o n from t h e i n o c u l a t i o n p o i n t .
~ S e v e r i t y measured as f o l l o w s :
I = l i g h t , 2 = m od era te and 3 = s e v e r e
— Column means f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y
d i f f e r e n t (P = 0 . 0 5 ) k
55
pr oduced.
The r e l a t i v e l y s h o r t i n c u b a t i o n p e r i o d o f s i x weeks may have
n o t been s u f f i c i e n t l y lo ng t o a l l o w f o r a d e q u a t e growth and development
o f t h e fu n g u s .
CONCLUSION
B a c t e r i a a r e c a p a b l e o f p r o d u c in g t h e symptoms n o r m a l l y a s s o c i a t e d
w i t h r o o t and crown d e t e r i o r a t i o n i n i r r i g a t e d s a i n f o i n i n Montana.
Moreover, t h e b a c t e r i a a p p e a r more a g g r e s s i v e i n p r o d u c i n g symptoms
th a n £_. s o l a n i .
Symptoms produced by t h e b a c t e r i a c o n s i s t e d p r i m a r i l y
o f b l a c k v a s c u l a r d i s c o l o r a t i o n , a l t h o u g h a r e d crown decay was p r o ­
duced by one P_. s y r i n g a e i s o l a t e .
P_. s y r i n g a e , P_. m a rg in al i s and IE.
amylovora a p p e a r e q u a l l y v i r u l e n t in i n o c u l a t e d s a i n f o i n , whereas E_.
h e r b i c o l a a p p e a r s t o be n o n - p a t h o g e n i c .
A s y n e r g i s t i c r e a c t i o n was n o t
o b s e r v e d between £_. s o l a n i and E. amylovora.
Experim ent 4
HISTOLOGY OF SAINFOIN ROOTS DISPLAYING TYPICAL ROOT AND CROWN ROT
SYMPTOMS..
MATERIALS AND METHODS
All p l a n t s sampled were from i r r i g a t e d s a i n f o i n s t a n d s s i t u a t e d a t
t h e F i e l d Res earch L a b o r a t o r y , Bozeman, Montana.
a) During t h e summer o f 1977, f i v e f o u r y e a r o l d s a i n f o i n p l a n t s
exhibiting typical
r o o t and crown r o t symptoms were sampled.
Tissue
s e c t i o n s were ta k e n from 3 d i f f e r e n t p l a n e s in t h e t a p r o o t in t h e
r e g i o n between 6 cm and 15 cm below t h e crown.
Three l a t e r a l
root sec­
t i o n s were sampled from each p l a n t a p p r o x i m a t e l y I cm from t h e t a p r o o t
b) During t h e summers o f 1977 and 1978 t e n p l a n t s , each w ith t y p i ­
cal r o o t and crown r o t symptoms, were sampled from ea ch o f a second and
a th ir d year sain fo in stand.
Tap r o o t s e c t i o n s were removed, one p e r
p l a n t , from t h e a f f e c t e d t i s s u e s .
S a i n f o i n r o o t s were removed from t h e ground u s i n g a s h a r p s h o o t e r
shovel and p l a c e d im m e d i a te l y i n t o a p l a s t i c bag t o i n s u r e specimen
integrity.
The r o o t s were th e n washed t h o r o u g h l y t o remove t h e s o i l
particles.
Root s e c t i o n s 0 . 5 cm t h i c k were c u t and p l a c e d in a forma­
l i n a c e t i c - a c i d a l c o h o l f i x a t i v e (33) (FAA) (Appendix I ) f o r a minimum
o f 48 h o u r s .
fixative.
L a r g e r r o o t s e c t i o n s were vacuum i n f i l t r a t e d w ith t h e .
The t i s s u e s e c t i o n s were t h e n d e h y d r a t e d u s i n g J o h a n s e n ' s
t - b u t y l a l c o h o l s e r i e s and embedded in p a r a p l a s t ( 3 3 ) .
Specimen b lo c k s
57
were trimmed and p l a c e d in a s o l u t i o n c o n t a i n i n g 320 mis o f 60% e t h a n o l ,
20 mis o f s a t u r a t e d g l y c e r o l and 80 mis o f g l a c i a l a c e t i c - a c i d f o r
t h r e e weeks a t 30 C t o s o f t e n t h e t i s s u e s .
Specimen b l o c k s were s t o r e d a t - 9 . 0 C f o r a t l e a s t 4 hours p r i o r
t o s e c t i o n i n g and t h e t i s s u e was s e c t i o n e d a t 8 mic ron s in t h i c k n e s s .
S e c t i o n s were a f f i x e d t o g l a s s s l i d e s u s i n g H a u p t1s a d h e s i v e (33) and.
were d r i e d on a s l i d e warming t r a y f o r 48 hours a t 42 C.
Johansen's
q u a d r u p l e s t a i n i n g p r o c e d u r e was employed on t h e s e c t i o n s . ( 3 3 ) .
S t a i n e d s e c t i o n s were mounted in H i s t o c l a d and oven d r i e d f o r 48 hours
a t 42 C.
S l i d e s were viewed w i t h a Z e i s s m i cr o s co p e and p h o to m ic r o ­
gr ap hs were ta k e n u s i n g Kodak Panatomic f i l m .
RESULTS AND DISCUSSION
B a c t e r i a were o b s e r v e d in t h e t a p r o o t s in a l l o f t h e f o u r y e a r
o l d s a i n f o i n p l a n t s sampled.
The b a c t e r i a were r e s t r i c t e d p r i m a r i l y t o
t h e v e s s e l s and open c a v i t i e s in t h e xylem in a l l p l a n e s o f s e c t i o n
between 6 cm and 15 cm below t h e crown ( F i g u r e s 4 - 1 and 4 - 2 ) .
Lateral
r o o t s in f o u r o f t h e f i v e p l a n t s sampled a l s o c o n t a i n e d t h e b a c t e r i a .
No fu nga l mycelium was o b s er ve d in any o f t h e d i s e a s e d r o o t s e c t i o n s .
S u r r o u n d in g t h e v e s s e l s c o n t a i n i n g b a c t e r i a were chromophylic
parenchyma c e l l s , v e s s e l s and f i b e r s .
These chromophyli c a r e a s in t h e
v a s c u l a r system c o r r e s p o n d t o t h e d a r k v a s c u l a r d i s c o l o r a t i o n o b s er v ed
in d i s e a s e d s a i n f o i n r o o t s grown un der i r r i g a t i o n .
In a s u r v e y o f t h e
58
F i g u r e s 4-1 t o 4 - 2 : Cross s e c t i o n s o f n a t u r a l Iy i n f e c t e d f o u r y e a r
o ld s a i n f o i n r o o t s d i s p l a y i n g t y p i c a l r o o t and crown r o t symptoms.
4- 1) P r e s e n c e o f b a c t e r i a (B) in v e s s e l el em en t s (x 5 7 5) . 4-2)
B a c t e r i a o c c l u d i n g i n f e c t e d v e s s e l s and a p p e a r t o be a s s o c i a t e d
w i t h t h e d e g r a d a t i o n o f a d j a c e n t parenchyma c e l l s (x 55 0) .
Bars
r e p r e s e n t 20 m i c r o n s .
59
f o u r y e a r o ld r o o t s s e c t i o n e d , between 50% and 90% o f t h e v a s c u l a r
r e g i o n s e x h i b i t i n g t h e chrom op hy li c r e g i o n s were a s s o c i a t e d w ith t h e
presence of b a c te r ia .
Those a f f e c t e d r e g i o n s which were n o t a s s o c i a t e d
w i t h b a c t e r i a o f t e n c o n t a i n e d g e l s , gums and c e l l wall f r a g m e n ts w i t h i n
the v e s s e ls (Figure 4 - 3 ) .
D i f f u s a b l e enzymes and t o x i n s produced by
p a th og en s such as £_. s o l a n i f . sp . phas eol i , Pseudomonas t a b a c i , Pseudo­
monas s y r i n g a e and Erw ini a amylovora have been shown t o c a u s e a s i m i l a r
d i s r u p t i o n i n c e l l s l o c a t e d a t a d i s t a n c e from t h e c e l l s o f t h e p a t h o ­
gen ( 8 ) .
Py s y r i n g a e i s known t o produce t h e t o x i n , s y r i n g o m y c i n , a com­
pound which p o s s e s s e s both p h y t o t o x i c and a n t i b i o t i c a c t i v i t y (14, 76 ).
_P. s y r i n g a e i s o l a t e d from d i s e a s e d s a i n f o i n p l a n t s may a l s o produce a
sim ila r substance.
In t h e l a b o r a t o r y , PDA p l a t e s were s p o t t e d w ith P_.
s y r i n g a e and s u b s e q u e n t l y i n c u b a t e d f o r 72 h o u r s .
The p l a t e s were th e n
s p r a y e d w i t h a c e l l s u s p e n s i o n o f R h o d o to r u la , a s o i l - b o r n e y e a s t , o r
Fy s o l a n i and were i n c u b a t e d f o r f i v e d a y s .
I n h i b i t i o n zones r a n g i n g
from 0 . 2 - 0 . 8 cm f o r £ . s o l a n i and 0 . 8 - 2 . 0 Cm f o r R ho d o to ru la were o b ­
s e r v e d around t h e _P. s y r i n g a e c o l o n i e s .
Thi s s u g g e s t s t h a t fungi t o x i c
s u b s t a n c e s such a s s y r ing om yc in a r e produced by t h e s a i n f o i n i s o l a t e s
of
syringae.
Amylovorin, a high m o l e c u l a r w e i g h t t o x i n produced by E_. a m y lo v o r a ,
has been a s s o c i a t e d w i t h t h e p r o d u c t i o n o f f i r e b l i g h t symptoms in f r u i t
t r e e s (24, 7 0 ) .
There i s i n d i r e c t e v i d e n c e t h a t a t r a n s l o c a t e d compound
60
such a s amylo vo rin i s r e s p o n s i b l e f o r symptom p r o d u c t i o n i n greenhouse
r e a r e d s a i n f o i n p l a n t s i n o c u l a t e d w i t h E_. am y lo v o r a .
In a h i s t o l o g i c a l
s t u d y i n v o l v i n g t h e s a i n f o i n p l a n t s i n o c u l a t e d w it h £ . amyl o v o r a , t h e
b a c t e r i a a p p e a r e d t o remain r e s t r i c t e d t o a r e g i o n w i t h i n 0 . 5 cm o f t h e
p o i n t o f i n o c u l a t i o n , whereas t h e d i s c o l o r a t i o n had s p r e a d as f a r as
25 cm from t h i s p o i n t .
H i s t o l o g i c a l l y , t h e symptoms o f d i s c o l o r a t i o n
c o n s is te d o f chromophyllic regions surrounding d is c o lo re d v e s s e ls .
Although t h e r e i s no h ar d e v i d e n c e t h a t e i t h e r . s y r i n g a e o r E. amylovora prod uce p h y t o t o x i c s u b s t a n c e s , i t i s p o s s i b l e t h a t s u b s t a n c e s such
as syr in g om y ci n and am ylo vor in a r e a s s o c i a t e d w i t h t h e v a s c u l a r d i s ­
c o l o r a t i o n in d i s e a s e d t h r e e and f o u r y e a r o l d s a i n f o i n .
F r e q u e n t l y o b s e r v e d in t h e r a y parenchyma c e l l s a d j a c e n t t o v e s ­
s e l s c o n t a i n i n g b a c t e r i a was a zone o f c e l l p r o l i f e r a t i o n ( F i g u r e 4 - 4 ) .
This phenomenon may r e p r e s e n t an i n t e r n a l form o f wound r e s p o n s e w h er e­
by t h e h o s t c e l l s a t t e m p t t o wall o f f t h e i n v a d i n g o r g a n is m s .
The decayed c a v i t y which e x t e n d s from t h e crown r e g i o n down t h e
c e n t e r o f t h e t a p r o o t i n p l a n t s showing advanced s t a g e s o f d e c l i n e
a l s o c o n t a i n e d a bu n da n t b a c t e r i a .
B a c t e r i a were a l s o o b s e r v e d in newly
c r e a t e d b r ea k s in t h e v a s c u l a r system ( F i g u r e 4 - 2 ) .
Though b a c t e r i a
were o b s e r v e d w i t h i n h e a l t h y a p p e a r i n g parenchyma c e l l s ( F i g u r e 4 - 5 ) ,
more commonly, t h e membranes a d j a c e n t t o a b a c t e r i a l c o n c e n t r a t i o n
a p p e a r t o c o l l a p s e and become c h r o m o p h y li c.
The b a c t e r i a th e n ap p e a r
t o in va d e t h e s e a f f e c t e d a r e a s and d eg r ad e t h e c e l l components.
61
F i g u r e s 4-3 and 4 - 4 : Cross s e c t i o n s o f n a t u r a l Iy i n f e c t e d f o u r y e a r
o ld s a i n f o i n r o o t s d i s p l a y i n g t y p i c a l r o o t and crown r o t symptoms.
4 - 3 ) The a c c u m u la ti o n o f g e l s (G) and c e l l f r ag m e n ts (CF) in v e s s e l s
in d i s c o l o r e d r o o t r e g i o n s n o t a s s o c i a t e d w ith t h e immediate p r e s ­
ence o f b a c t e r i a (x 3 10 ) . 4 -4 ) Su rr ou nd ing v e s s e l s c o n t a i n i n g b a c t ­
e r i a (B) a r e chrom op hyl ic parenchyma, v e s s e l and f i b e r c e l l s .
A
zone o f c e l l p r o l i f e r a t i o n (ZP) f r e q u e n t l y oc cu rs in t h e r a y p a r ­
enchyma c e l l s a d j a c e n t t o v e s s e l s c o n t a i n i n g b a c t e r i a (x 2 8 0 ) . Bars
r e p r e s e n t 50 m i c r o n s .
62
In many t a p r o o t s e c t i o n s , b a c t e r i a were o b s e r v e d in c r a c k s and
b r e a k s in t h e phellum and c o r t e x .
Because t h i s phenomenon was only
observed in th e 4 y e a r old s a in f o in r o o t s e c t i o n s , th e occurrence of
b a c t e r i a in t h e s e r e g i o n s i s p r o b a b l y i n d i c a t i v e o f advanced s t a g e s o f
root decline.
B a c t e r i a were a l s o numerous in t h e d i s c o l o r e d v a s c u l a r r e g i o n s
in t h e t h r e e y e a r o l d s a i n f o i n p l a n t s .
In n i n e o f t h e t e n r o o t s s e c ­
t i o n e d , b a c t e r i a were a s s o c i a t e d w i t h t h e decay and c e l l u l a r , d i s r u p t i o n
o f xylem e l e m e n t s .
C o n v e r s e l y , o n l y two o f t h e t e n r o o t s e c t i o n s in
t h e second y e a r s a i n f o i n were o b s er v ed t o c o n t a i n b a c t e r i a .
More f r e ­
q u e n t l y o b s e r v e d in t h e s e r o o t s e c t i o n s was fungal mycelium which
a p p e a r e d t o be a s s o c i a t e d w it h t h e b l a c k v a s c u l a r d i s c o l o r a t i o n in t h e
crown r e g i o n s .
The mycelium seemed r e s t r i c t e d t o t h e v e s s e l e l e m e n t s ,
and was g e n e r a l l y a s s o c i a t e d w i t h ch ro mo ph yl ic t i s s u e s s u r r o u n d i n g t h e
v e sse ls (Figure 4 -6 ).
S e c t i o n s o f s a i n f o i n r o o t s e x h i b i t i n g t h e r e d crown decay con­
t a i n e d n e i t h e r fu nga l n or b a c t e r i a l c e l l s , a l t h o u g h b a c t e r i a , p a r t i c u ­
l a r l y t h e Pseudomonas s p p . , a r e r e a d i l y i s o l a t e d from t h i s r e g i o n in
two y e a r o l d s a i n f o i n crowns.
I t i s p o s s i b l e t h a t t h e b a c t e r i a in
t h e s e open c a v i t i e s were washed f r e e d u r i n g t h e d e h y d r a t i o n s e r i e s in
the h is to lo g ic a l preparation o f the root ti s s u e .
An oth er e x p l a n a t i o n
i s t h a t p e r h a p s t h e r e d crown .decay o b s e r v e d in t h e second y e a r s a i n ­
f o i n p l a n t s i s a p h y s i o l o g i c a l phenomenon due t o t h e a c c u m u l a t i o n and
63
F i g u r e 4 - 5 : Cross s e c t i o n o f a
n a t u r a l l y i n f e c t e d f o u r y e a r old
sainfoin root displaying typical
r o o t and crown r o t symptoms. B a c t ­
e r i a (B) p a r a s i t i z i n g r a y parenchyma
c e l l s (RP)(x 55 0) .
F ig u r e 4 - 6 : Cross s e c t i o n o f n a t u r ­
al Iy i n f e c t e d two y e a r o l d s a i n f o i n
r o o t e x h i b i t i n g b l a c k crown d i s c o l ­
oration.
Fungal mycelium (M) in i n ­
f e c t e d v e s s e l s a s s o c i a t e d w ith chromop h y l i c v e s s e l s and f i b e r s (x 200 ).
Bars r e p r e s e n t 50 m i c r o n s .
64
o x i d a t i o n o f p h e n o l i c compounds d u r i n g t h e crown s p l i t t i n g p r o c e s s .
Decay c a u s i n g o rg an is m s may th e n c o l o n i z e t h i s r e g i o n and b e g i n ' a c t i v e
d e s tru c tio n of the root ti s s u e .
B a c t e r i a a s s o c i a t e d in r o o t and crown r o t in s a i n f o i n may p o s s i b l y
form a t r u e complex whereby d i f f e r e n t b a c t e r i a a r e r e s p o n s i b l e f o r t h e
d i f f e r e n t degradation processes.
E. amylovora and P. s y r i n g a e a r e
c a p a b l e o f p r o d u c i n g e x t r a c e l l u l a r p r o t e a s e s and l i p a s e s in v i t r o as
r e v e a l e d in t h e p h y s i o l o g i c a l and bioc hem ic al c h a r a c t e r i z a t i o n t e s t s .
They may a l s o pr od uc e p h y t o t o x i n s w hi ch , t o g e t h e r w i t h t h e enzymes,
a f f e c t t h e r e g i o n o f c e l l s s u r r o u n d i n g t h e v e s s e l s r e s u l t i n g in t h e
d i s r u p t i o n o f t h e membranes in t h e s e c e l l s .
P_. ma rg in al i s , a known
p r o d u c e r o f p e c t i n a s e , may f o l l o w t h e s e or ganisms c a u s i n g t h e d i s s o l u ­
t i o n o f t h e r e m a in in g t i s s u e .
The n o t i o n o f a t r u e complex between
t h e s e or gan ism s rem ai ns t o be t e s t e d i n gre en h o u se e x p e r i m e n t s .
CONCLUSIONS
The h i s t o l o g i c a l
i n v e s t i g a t i o n r e v e a l e d t h a t b a c t e r i a were numer­
ous in t h e v e s s e l s and decayed c a v i t i e s in t h e t h i r d and f i f t h y e a r
sainfoin roots.
No f ung al mycelium was o b s er v ed in any o f t h e r o o t
s e c t i o n s o f t h e more ma ture s a i n f o i n .
Because s t a n d d e c l i n e and y i e l d
l o s s e s become e v i d e n t in t h e t h i r d and f o u r t h y e a r s o f p r o d u c t i o n in
s t a n d s s i t u a t e d in Montana, b a c t e r i a become t h e p r im ar y s u s p e c t p a t h o ­
gens in t h e r o o t and crown r o t complex o f i r r i g a t e d s a i n f o i n .
Although
65
f ung al mycelium was o b s e r v e d in t h e d i s c o l o r e d crown t i s s u e s in second
y e a r s a i n f o i n r o o t s , t h e e x t e n t o f t h e d e t e r i o r a t i o n o f t i s s u e s was
lim ited;
I
E xperim ent 5
EVALUATION.OF THE PATHOGENICITY OF DIFFERENT F. SOLANI FORM SPE• CIES AND ISOLATES OM PEAS, BEANS, SQUASH AND SAINFOIN.
.
;
•
.
'
•
>
The p r up o s e o f t h i s e x p e r i m e n t was t o d e t e r m i n e t h e h o s t r a n g e s o f
14 i s o l a t e s and formae s p e c i e s o f JF . s o l a n i on p e a s , b e a n s , squash and
sainfoin.
MATERIALS AND METHODS
Isolates
( T ab l e 5) were s t o r e d in s o i l a c c o r d i n g t o t h e t e c h n i q u e
d e s c r i b e d by Toussoun and Nelson (71) and s i n g l e s p o r e d . i s o l a t e s were
o b t a i n e d u s in g t h e method d e s c r i b e d i n Experiment T h r e e .
An a g a r plug
from a s i n g l e s p or ed c u l t u r e was p l a c e d i n a t e s t t u b e c o n t a i n i n g s t e r ­
i l e d i s t i l l e d w a t e r , and t h e s p o r e s were shaken i n t o s u s p e n s i o n .
Fif­
t e e n cm p e t r i d i s h e s c o n t a i n i n g N a tu r a l P o t a t o D ex tr o s e Agar were
f l o o d e d w i t h t h e s p o r e s u s p e n s i o n and i n c u b a t e d a t 21 C u s in g a 12 hour
p h o t o p e r i o d p r o v i d e d by cool w h i t e f l u o r e s c e n t l i g h t s .
After three
weeks, t h e p l a t e s were f l o o d e d w it h s t e r i l e d i s t i l l e d w a t e r , and t h e
s p o r e s were s c r a p e d i n t o s u s p e n s i o n u s in g a g l a s s r o d .
The s u s p e n s i o n
was c o l l e c t e d and d i l u t e d t o a f i n a l c o n c e n t r a t i o n o f 2 . 5 x 10® macro­
c o n i d i a/ml .
One hundred mis o f t h e s p o r e s u s p e n s i o n were t h o r o u g h l y
mixed i n t o t h e t o p 10 cm o f a sandy loam s o i l m i x t u r e c o n t a i n e d in
s t e r i l e 25 cm p o t s .
The s o i l had been p r e v i o u s l y a u t o c l a v e d u s i n g
2
steam s t e r i l i z a t i o n f o r 72 hours a t .42 kg/cm .
The p o t s were soaked
67
Ta b le 5 - 1 .
The o r i g i n o f t h e d i f f e r e n t formae s p e c i e s and i s o l a t e s o f
F\ s o l a n i used in t h e p a t h o g e n i c i t y t e s t s on p e a s , b e a n s ,
squa sh and s a i n f o i n .
Isolate
Host
Investigator
Origin
F-47
F-77
#246
W-8
VI-10
74-4
Cn-56
L-8
P l-8 10
#280
FS-9
K-I
#296
C-559
Pea
Pea
Pea
Bean
Bean
Bean
Squash
Al fa ! fa
Al f a ! fa
Sainfoin
Sainfoin
Sainfoin
Cotton
Cotton
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
Dr.
P r o s s e r , Washington
P r o s s e r , Washington
Pu llman, Washington
P r o s s e r , Washington
P r o s s e r , Washington
Berkeley, C alifo rn ia
Berkeley, C alifo rn ia
P e n n s y lv a n i a
Pennsylvania
Bozeman, Montana
Bozeman, Montana
Kansas
A th e n s , Georgia
A th e n s , Georgia
I.
J.
L.
J.
J.
J.
J.
F.
F.
M.
M.
A.
M.
M.
G.
G.
L.
L.
Kraft
Kraft
Hadwiger
Kraft
Kraft
Hancock
Hancock
Lukezic
Lukezic
Dr. D. R. Surmer
Dr. D. R. Surmer
68
in 1 . 0 % ' NaOCl f o r two m i n u te s f o r s t e r i l i z a t i o n . •
Seeds o f 1A la s k a ' p e a s . Red Kidney Beans, 1Golden Summer' Crookneck squash and Remont s a i n f o i n , which had been 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 i n u t e s in 0.5% MaOCl, were p l a n t e d i n t h e i n o c u l a t e d s o i l , 12
seeds/pot/species.
The p o t s were a r r a n g e d in a s p l i t p l o t randomized
c o m pl et e b lo c k d e s i g n w i t h t h r e e r e p l i c a t i o n s .
The f o u r h o s t p l a n t s
were a s s i g n e d t o main p l o t s , and t h e 14 £ . s o l a n i i s o l a t e s were
assigned to s u b p lo ts .
The p e a s , beans and squash were grown in t h e
gr een ho u se f o r 22 days d u r i n g t h e month o f J u l y u s in g t h e ambient tem­
p e r a t u r e and p h o t o p e r i o d and were w a te r e d u s i n g a d i l u t e n u t r i e n t s o l u ­
t i o n (Appendix I ) .
The s a i n f o i n p l a n t s were i n c u b a t e d a t o t a l o f 8 . 5
months in t h e g r ee n h o u s e u s in g a 16 hour p h o t o p e r i o d p r o v id e d by f l u o r ­
escent lig h ts .
The p l a n t s were s c o r e d f o r d i s e a s e s e v e r i t y im me di ate ly
a f te r harvest.
Data were a n a l y z e d u s in g a n a l y s i s o f v a r i a n c e and t h e
means were s e p a r a t e d u s i n g L e a s t S i g n i f i c a n t D i f f e r e n c e s (LSD) and
Duncan's M u l t i p l e Range T e s t (6 9 ) .
S e c t i o n s o f s q u a s h , bean and pea hypocotyl t i s s u e and s a i n f o i n t a p
r o o t t i s s u e were p l a c e d on. Komada' s medium and HPDA t o d e t e r m i n e p e r c e n t
infection.
Based on t h e r e s u l t s o b t a i n e d in t h i s e x p e r i m e n t , a s u b s e q u e n t
e x p e r i m e n t was d e s i g n e d t o d e t e r m i n e t h e p a t h o g e n i c i t y and v i r u l e n c e o f
F. s o l a n i
' s a i n f o i n ' on p e a s .
Two g r ee nh o u s e t r i a l s were d e v i s e d f i r s t
t o d e t e r m i n e t h e p a t h o g e n i c i t y o f F\ s o l a n i i s o l a t e d from s a i n f o i n
\
69
growing a t t h e F i e l d Research L a b o r a t o r y , Bozeman, Montana, an a r e a
known t o be p r e v i o u s l y a s s o c i a t e d w i t h pea p r o d u c t i o n , and s e c o n d l y , t o
d e t e r m i n e t h e p a t h o g e n i c i t y o f £ . s o l a n i i s o l a t e d from s a i n f o i n growing
in A l b e r t a , N ebr as ka, and Kansas, a r e a s n o t known t o be p r e v i o u s l y
a s s o c i a t e d w i t h pea p r o d u c t i o n .
The e x p e r i m e n t a l c o n d i t i o n s and p r o c e ­
d u r e s f o r a s s a y and r e i s o l a t i o n o f b o th s e t s o f £ . s o l a n i
1s a i n f o i n 1
i s o l a t e s were t h e same a s t h o s e used in Experiment I i n v o l v i n g t h e £ ,
s o l a n i i s o l a t e s on p e a s , b e a n s , squash and s a i n f o i n .
The pea se eds
were p l a n t e d f i v e p e r p o t and t h e p o t s were a r r a n g e d in a c o m p l e t e l y
randomized b lo c k d e s i g n .
RESULTS AND DISCUSSION
£. s o l a n i f .
sp. p i s i , £ . s o l a n i f . s p . p h a s e o l i and £ . s o l a n i f .
sp. c u c u r b i t a e e x h i b i t e d a high d e g r e e o f v i r u l e n c e a s measured by t h e
d i s e a s e s e v e r i t y s c o r e s on t h e h o s t s p e c i e s from which t h e y were i s o ­
l a t e d and a low d e g r e e o f v i r u l e n c e on a l l o t h e r h o s t s p e c i e s (Table 2)
These d a t a d i r e c t l y s u p p o r t t h e formae s p e c i a l i s d e s i g n a t i o n in £ .
s o l a n i , a c o n c e p t which i s e s t a b l i s h e d on t h e b a s i s o f a l i m i t e d o r
h i g h l y s e l e c t i v e p a t h o g e n i c i t y in c e r t a i n b i o t y p e s o f t h e genus
Fusarium ( 2 ) .
These c o n c l u s i o n s a r e d i r e c t l y c o n t r a d i c t o r y t o t h o s e
proposed by Auld e t a l .
( 3, 4 ) , who s u g g e s t e d t h a t l i t t l e s p e c i f i c i t y
i n p a t h o g e n i c i t y e x i s t e d in t h e p i s i and p h a s e o l i form s p e c i e s o f £ .
solani.
I t i s l i k e l y t h a t t h e e r r o n e o u s c o n c l u s i o n s a r o s e as a r e s u l t
Ta b le 5- 2.
The p a t h o g e n i c i t y o f v a r i o u s forma e s p e c i e s and i s o l a t e s o f £_. s o l a n i on
p e a s , b e a n s , squa sh and s a i n f o i n as measured by t h e mean d i s e a s e s e v e r i t y
and- % in fec tio n .
Host
Peas
Beans
2/ S ev erity % Inf.
% Inf.
Severity
100
100
100
0 .7 9
0.8 6
0 .7 9
100
100
100
1.00
1.30
1.10
12
0
55
3 .7 0
3.66
3 .3 0
100
100
100
0.64
0 .1 8
0.8 8
0
0
0
0.66
1:00
0.5 4
12
0
0
12
0.1 7
75
0.4 3
0
4.53
100
100
100
100
1.65
1.33
0 .0 0
100
100
100
0 .8 4
0.81
0 .6 4
100
100
100
0.8 0
0.6 0
1.08
12
50
37
100
100
0 .5 4
1.1 6
100
100
0.17
100
0.43
ioo
1.08
. 1.30
.44
12
Severity I/
% Inf.
Pea
F-47
F-77
#246
3.79
2.29
2 .4 1
100
100
100
1.4 9
0 .9 9
0 .7 9
Bean
W-8
W-IO
74-4
0 .6 6
0.40
0.6 1
0
0
0
Squash
Cn-56
0.79
Sainfoin
#280
FS-.9 .
K-I
2 .6 2
2 .9 1
0.71
Cotton •
#296
C-559
0 .0 8
0 .2 1
\
Squash 4 /
S e v e r i t y 3/
Isolate
'
Sainfoin
% Inf
Tabl e 5 - 2 .
(C o n 't.)
Host
Beans
Peas
Sainfoin
Squash
Isolate
Severity
% Inf.
Severity % Inf.
Severity
% Inf.
Severity
% Inf.
Al f a ! f a
P l-8 10
L-8
1.2 4
1.0 0
100
100
0.9 5
1.21
100
100
0.5 7
0 .4 5
100
100
0.93
1.00
25
25
Control
H2O
0 .2 1
12
0.00
12
0 .1 4
0
0.7 3
0
LSD
0 .1 8
0. 33
0 .3 3
0.4 5
— The d i s e a s e s e v e r i t y f o r p e a s , beans and squash was d e t e r m in e d as f o l l o w s :
0 = no
symptoms, I = f l e c k i n g on t h e h y p o c o t y l , 2 = small n e c r o t i c l e s i o n s on t h e h y p o c o t y l ,
3 = l a r g e n e c r o t i c l e s i o n s on t h e h y p o c o t y l , 4 = n e c r o s i s c o m p l e t e l y c o v e r i n g t h e
2 /hypo co tyl and 5 = dead p l a n t .
— The % i n f e c t i o n was based on i s o l a t i o n s from symptomatic t i s s u e s e c t i o n s p l a c e d on
q , Komada' s medium and HPDA.
— The d i s e a s e s e v e r i t y f o r t h e s a i n f o i n was based on t h e e x t e n t o f v a s c u l a r d i s c o l o r a t i o n
as follow s:
I = l i g h t s t r e a k i n g in t h e t a p a n d / o r l a t e r a l r o o t s , 2 = Between 5 and 15%
„ , o f t h e t a p r o o t d i s c o l o r e d and 3 = between 15 and 25% o f t h e t a p r o o t d i s c o l o r e d .
— The p o r t i o n o f t h e e x p e r i m e n t i n v o l v i n g squa sh was r e p e a t e d a t a l a t e r d a t e .
72
o f wounding t h e h o s t p l a n t a t t h e ti m e o f i n o c u l a t i o n .
Because t h e
d i s e a s e p r o d u c in g c a p a b i l i t y o f Fusafium s p e c i e s i s b as ed on i t s a b i ­
l i t y to p e n e t r a t e th e t i s s u e o f the host (7 ), i t is im portant t h a t
natural
inoculation conditions prevail
in t h e s t u d y o f t h e h o s t ran ge
o f FY s o l a n i . b i o t y p e s .
A g r e a t deal o f v a r i a b i l i t y was e n c o u n t e r e d in t h e d i s e a s e
s e v e r i t y s c o r e s w i t h i n and between b l o c k s in t h e p o r t i o n o f t h e e x p e r i ­
ment i n v o l v i n g t h e 14 FY s o l a n i i s o l a t e s on s qu as h .
C o n s e q u e n t ly , t h i s
p o r t i o n o f t h e e x p e r i m e n t was r e p e a t e d a t a l a t e r d a t e .
Although t h e
r e s u l t s o f 'the r e p e a t e d e x p e r i m e n t a r e l i s t e d in Ta bl e 5 - 2 , t h e y may
n o t be d i r e c t l y compared t o t h e p er fo rm a nc e o f t h e 14 i s o l a t e s on p e a s ,
b e a n s , and s a i n f o i n .
The same e x p e r i m e n t a l p r o c e d u r e s used in t h e main
e x p e r i m e n t p r e v a i l e d e x c e p t t h a t 15 cm p o t s were u t i l i z e d and t h e
s e e d i n g r a t e was red uc e d t o f i v e p e r p o t .
F_. s o l a n i f . sp . p i s i
i s o l a t e s and t h e c o t t o n , a l f a l f a and s a i n ­
f o i n i s o l a t e s p o s s e s s a wide h o s t r a n g e as r e f l e c t e d i n t h e p e r c e n t
i n f e c t i o n o f t h e s e i s o l a t e s on o t h e r h o s t s p e c i e s ( T ab l e 5 - 2 ) .
v e rse ly , £. solani f.
Con­
s p . p h a s e o l i and £ . s o l a n i f . s p . c u c u r b i t a e
d e m o n s t r a t e a narrow h o s t r a n g e .
Because n e a r l y a l l
is o la te s exhibit a
lowered v i r u l e n c e on n o n - h o s t s p e c i e s , t h e high p e r c e n t i n f e c t i o n
e n c o u n t e r e d w i t h £ . s o l a n i f . sp . p i s i and t h e c o t t o n , a l f a l f a and
s a i n f o i n i s o l a t e s on o t h e r h o s t s p e c i e s may r e f l e c t t h e a b i l i t y o f
t h e s e i s o l a t e s t o s u r v i v e on h o s t e x u d a t e s and in t h e o u t e r c e l l l a y e r s
73
of the ro o t without a c t u a l l y d i r e c t l y p e n e tra tin g in to the root.
Thi s
i n c r e a s e d s a p r o p h y t i c c a p a c i t y on n o n - h o s t t i s s u e s may, c o n s e q u e n t l y ,
be o f s u r v i v a l v a l u e in a b s en ce o f t h e h o s t cr o p .
Although most o f t h e i s o l a t e s were o n l y a b l e t o i n c i t e mild symp­
toms in s a i n f o i n a f t e r an i n c u b a t i o n p e r i o d o f 8 . 5 months, t h e pea ,
beans and s a i n f o i n i s o l a t e s were most s u c c e s s f u l in p r o d u c in g t h e b l a c k
v a s c u l a r d i s c o l o r a t i o n in s a i n f o i n t a p r o o t s f o l l o w e d by t h e s q u a s h ,
a l f a l f a and c o t t o n i s o l a t e s .
The a b i l i t y o f t h e m a j o r i t y o f F_. s o l a n i
i s o l a t e s t o i n c i t e some symptoms i n s a i n f o i n r o o t s r a i s e s some i n t e r ­
e s t i n g q u e s t i o n s r e g a r d i n g t h e r e l a t i o n s h i p between £ . s o l a n i and th e
s a i n f o i n r o o t , p a r t i c u l a r l y when some i s o l a t e s such a s t h e bean and
squa sh i s o l a t e s were n o t r e c o v e r e d from t h e r o o t s e c t i o n s d i s p l a y i n g
typical vascular d isco lo ratio n .
I t a p p e a r s . t h a t £. so lan i i s able to
d i r e c t l y p e n e t r a t e t h e s a i n f o i n r o o t s s i n c e u n n a t u r a l wounding p r e ­
sumedly d i d n o t o c c u r .
Another e x p l a n a t i o n f o r t h e development o f
symptoms i s t h a t t h e fungus may grow s a p r o p h y t i c a l l y on t h e r o o t s u r ­
f a c e and r e l e a s e t o x i c s u b s t a n c e s which a r e ab so rb ed by t h e r o o t and
t r a n s l o c a t e d through th e v a s c u la r system, thereby causing th e r e s t r i c t e d
black s tre a k in g .
When t h e r o o t s o f s a i n f o i n p l a n t s a r e soaked in c u l ­
t u r e f i l t r a t e s o f £ . s o l a n i f . sp . pi s i
( I s o l a t e # 2 4 6 ) , t y p i c a l dark
d i s c o l o r a t i o n accompanied by gel f o r m a t i o n i s o b s er v ed a f t e r 24 hours
(G aud et, u n p u b l i s h e d d a t a ) .
Thi s s u g g e s t s t h a t £ . s o l a n i may be c a p a ­
b l e o f c a u s i n g symptoms w i t h o u t d i r e c t l y e n t e r i n g t h e s a i n f o i n p l a n t .
74
The o b s e r v a t i o n t h a t n e i t h e r t h e squa sh n o r t h e bean i s o l a t e co u ld be
r e i s o l a t e d from symptomatic t i s s u e s would te n d t o s u p p o r t t h i s c o n t e n ­
tion.
I f t h e fungus does a c t u a l l y p e n e t r a t e t h e s a i n f o i n r o o t , i t may
be r e s t r i c t e d t o t h e e n t r y p o i n t and s u b s e q u e n t l y r e l e a s e t o x i c su b­
s t a n c e s i n t o t h e v a s c u l a r system.
been i s o l a t e d and i d e n t i f i e d
Toxins produced by £ . s o l a n i have
( 7 6 ) , a l t h o u g h t h e i r mode o f a c t i o n
rem ai ns t o be e l u c i d a t e d .
IF. s o l a n i
1s a i n f o i n 1 i s o l a t e s a p p e a r s i m i l a r t o t h o s e o f £_. s o l a n i
f . sp . pi s i in t h e i r h o s t ran ge and v i r u l e n c e on peas (Tab le 5 - 2 ) .
In
the subsequent experiment, the m a jo rity o f the £. so lani
iso­
'sainfoin'
l a t e s e x h i b i t e d a mo de rat e t o high v i r u l e n c e on peas ( T ab l e 5 - 3 ) .
o b s e r v a t i o n t h a t F. s o l a n i
' sainfoin'
The
is o la te s , i n i t i a l l y collected
from s a i n f o i n growing i n a r e a s n o t p r e v i o u s l y a s s o c i a t e d w i t h pea p r o ­
d u c t i o n , a r e m o d e r a t e l y p a t h o g e n i c on peas s u g g e s t s t h a t genes f o r
p a t h o g e n i c i t y f o r both £ . s o l a n i
' s a i n f o i n ' and £ . s o l a n i f . sp. pi si
may be s i m i l a r .
CONCLUSIONS
The d i f f e r e n t form s p e c i e s o f £ . s o l a n i e x h i b i t e d a high d e g r e e o f
v i r u l e n c e on t h e h o s t s p e c i e s from which t h e y were o r i g i n a l l y i s o l a t e d
and a low d e g r e e o f v i r u l e n c e on a l l o t h e r s p e c i e s .
i s o l a t e s produced o n l y mild symptoms on s a i n f o i n .
pi s i and £ . s o l a n i
The m a j o r i t y o f
£ . s o l a n i f . sp.
' s a i n f o i n ' were s i m i l a r in t h e i r h o s t range and
75
t a b le 5-3.
P a t h o g e n i c i t y t o peas o f F_. s o i a n j 1s a i n f o i n 1 i s o l a t e s c o l ­
l e c t e d from s a i n f o i n p l a n t s growing in d i f f e r e n t a r e a s in
North America.
Experiment I
Source o f I s o l a t e
Isolate
Severity I /
Bozeman, MT
ES-9
FS-22
ES-17
FS-19
FS-16
FS-15
F S -14
3.60
3 .4 7
3.13
2.86
2 .7 5
2.60
1.47
a 3/
ab
abc
bed
bcde
cde
f
0.06 g
Controls-HgO
% In f e c tio n 2/
.
100
100
100
100
100
100
100
a
a
a
a
a
a
a
12 b
Experiment 2
L ethbridge, A lta.
LE-83
L E -I l
LE-22
3 .5 3 ab
3 . 3 0 abc
2.70 b
100 a
100 a
100 a
O d e l l , Nebraska
OD-2
00-9
00-6
00-8
3.30
2.30
1.60
1.0 0
100
100
100
100
Mound V a l l e y , Kansas
K-I
K-2
1.33 f
0.88 f
100 a
100 a
Check
FS-9
4.06 a
100 a
0.00 g
0 b
Control HgO
— Dis e a se s e v e r i t y was measured as f o l l o w s :
abed
de
ef
f
a
a
a
a
0 = no symptoms, I =
f l e c k i n g on t h e h y p o c o t y l , 2 = n e c r o t i c l e s i o n s on t h e h y p o c o t y l , 3 =
l a r g e n e c r o t i c l e s i o n s on t h e h y p o c o t y l , 4 = n e c r o s i s c o m p l e t e l y
, , , c o v e r i n g t h e hypocotyl and 5 = dead p l a n t .
— The % i n f e c t i o n based on i s o l a t i o n s from symptomatic t i s s u e s e c t i o n s
g . p l a c e d on Komada1s medium and H+PDA.
- C o l u m n means f o l l o w e d by t h e same l e t t e r a r e n o t s i g n i f i c a n t l y
d i f f e r e n t (P = 0 . 0 5 ) .
76
v i r u l e n c e on both s a i n f o i n and p e a s , i n d i c a t i n g t h a t t h e two b i o t y p e s
are c lo s e ly r e l a t e d , i f not i d e n t i c a l .
Experim ent 6
INVESTIGATION INTO THE ROLE OF £ . SOLAN! IN THE ROOT AND CROWN
COMPLEX IN SAINFOIN.
The o b j e c t o f t h e s e s t u d i e s was t o d e f i n e , more c l e a r l y , t h e r o l e
o f £_. s o l a n i i n t h e r o o t and crown d e t e r i o r a t i o n in i r r i g a t e d s a i n f o i n
P r e v io u s i n v e s t i g a t o r s were l e d t o t h e c o n c l u s i o n t h a t £ . s o l a n i was
t h e c a u s a l o rg an is m in t h e d i s e a s e bec a u se I ) .£. s o l a n i was c o n s i s ­
t e n t l y i s o l a t e d from r o o t and crown s e c t i o n s d i s p l a y i n g t y p i c a l symp­
toms and b) symptoms such as t h e b l a c k v a s c u l a r d i s c o l o r a t i o n were
rep ro du ce d when t h i s o rg ani sm was i n o c u l a t e d i n t o t h e wounded r o o t s o f
g r e e n h o u s e grown s a i n f o i n ( 3 , 4 , 61, 6 2 ) .
The f o l l o w i n g s t u d i e s were
d e s i g n e d t o compliment t h e r e s e a r c h c a r r i e d o u t in t h e p r e v i o u s e x p e r i ­
ments and, h o p e f u l l y , would a l l o w a more d e f i n i t i v e d e s c r i p t i o n o f t h e
symptoms produced by t h i s org ani sm .
MATERIALS AND METHODS
A h i s t o l o g i c a l s t u d y was co n du ct ed on gre en h o u se r e a r e d s a i n f o i n
in o c u la te d with £. so lan i using the ro o t- c u t - s o a k technique (3).
The
t a p r o o t s o f f o u r month o l d s a i n f o i n p l a n t s grown in v e r m i c u l i t e were
c u t a p p r o x i m a t e l y 5 cm.below t h e crown and soaked in a m i c r o c o n i d i a l
6
s u s p e n s i o n o f £ . s o l a n i c o n t a i n i n g 10 s p o r es /m l f o r 15 m i n u te s and
78
t r a n s p l a n t e d i n t o p a s t e u r i z e d s o i l f i l l e d bench es.
The p l a n t s were
i n c u b a t e d f o r 2 months, th e n sampled f o r h i s t o l o g i c a l e x a m i n a t i o n s .
The t a p r o o t s o f f i v e i n o c u l a t e d s a i n f o i n p l a n t s were washed and
p r e p a r e d f o r h i s t o l o g i c a l s t u d y u s in g t h e methods d e s c r i b e d in E x p e r i ­
ment Four.
Root s e c t i o n s were t a k e n a t I cm i n t e r v a l s t o e n a b l e d e t e r ­
m i n a t i o n o f t h e d i s t a n c e t r a v e l l e d by t h e fu ngal c e l l s from t h e i n o c u ­
la tio n point.
b) F o ur th y e a r s a i n f o i n p l a n t s d i s p l a y i n g t y p i c a l r o o t and crown
r o t symptoms, were c o l l e c t e d from t h e F i e l d Research L a b o r a t o r y ,
Bozeman, Montana.
P l a n t s were washed and 4-6 cm s e c t i o n s o f t a p and
l a t e r a l r o o t s were s u r f a c e s t e r i l i z e d i n 1.0% NaOCl f o r 5 m i n u te s .
r o o t s e c t i o n s were t h e n s e r i a l l y c u t in 0 . 5 - 1 . 0 cm s e c t i o n s .
The
The bark
was a s e p t i c a l l y removed from e v e r y s ec on d s e c t i o n and a l l s e c t i o n s were
p l a c e d s e r i a l l y on p l a t e s c o n t a i n i n g Komada1s . medium (Appendix I ) .
P l a t e s were i n c u b a t e d a t 21 C f o r 8 days and su rv eye d f o r t h e p r e s e n c e
o f Fusarium spp.
I s o l a t e s were i d e n t i f i e d t o s p e c i e s a c c o r d i n g t o t h e
t e c h n i q u e d e s c r i b e d by Toussoun and Nelson (7 1) .
RESULTS AND DISCUSSION
In t h e h i s t o l o g i c a l s t u d y , t h e fu ng al hyphae e x t e n d e d up 1-2 cm
from t h e i n o c u l a t i o n p o i n t whereas t h e v a s c u l a r d i s c o l o r a t i o n e x t en d ed
3-4 cm i n advance o f t h e fu ngal c e l l s .
t o t h e v e s s e l s in a l l
The mycelium remained r e s t r i c t e d
i n s t a n c e s and a p p e a r e d t o move v e r t i c a l l y tow ards
79
t h e crown in a r e s t r i c t e d manner w i t h i n a few v e s s e l s o n l y .
The triyce-
.
Iium n e a r t h e i n o c u l a t i o n p o i n t o c c a s i o n a l l y o c c l u d e d t h e v e s s e l s ,
whereas t h e mycelium in t h e v e s s e l s 1-2 cm from t h e i n o c u l a t i o n p o i n t
c o n s i s t e d o f o n l y one o r two branched hyphal s t r a n d s .
In most i n s t a n c e s , t h e d i s c o l o r e d r e g i o n s were a s s o c i a t e d w ith
e x t e n s i v e a c c u m u l a t i o n o f g e l s , gums and w i t h chromophyll i e v e s s e l s and
fibers.
Although a g e n e r a l c e l l u l a r d e g r a d a t i o n and decay was ob se rv ed
d i r e c t l y above t h e i n o c u l a t i o n p o i n t , t h e r e was no e v i d e n c e o f c e l l
damage a d j a e d n t t o v e s s e l s c o n t a i n i n g t h e fu ngal c e l l s
(Figure 6-1).
Gels i n i t i a l l y a r i s e as small p r o t u b e r a n c e s in t h e v e s s e l w a l l s
and a p p e a r t o expand u n t i l t h e y o c c l u d e t h e e n t i r e v e s s e l
(Figure 6 -2 ).
B ec au se .m os t g e l s and d i s c o l o r a t i o n a p p e a r in advance o f t h e fungal
c e l l s , i t i s p o s s i b l e t h a t fungal m e t a b o l i t e s , such a s enzymes and
t o x i n s , a r e produced by t h e or gan ism and t r a n s l o c a t e d up t h e r o o t s .
These s u b s t a n c e s c o u l d i n t e r a c t w i t h t h e h o s t c e l l s t o r e s u l t i n g e l a ­
t i n and d i s c o l o r a t i o n o f t h e t i s s u e .
Q u e s ti o n s c o n c e r n i n g t h e o r i g i n and n a t u r e o f t h e v a s c u l a r d i s ­
c o l o r a t i o n and g e l s i n d is e a s e d t i s s u e have n o t . b e e n c o m p l e t e l y . r e ­
solved..
Some i n v e s t i g a t o r s (5, 6, 47) b e l i e v e t h a t h y d r o l y t i c enzymes
produced by t h e i n v a d i n g pathogen ca u s e d i s f u n c t i o n and s u b se q u e n t r e ­
l e a s e o f p h e n o l i c compounds from s p e c i a l i z e d parenchyma c e l l s s c a t t e r e d
t h r o u g h o u t t h e xylem.
These ph eno ls a r e s u b s e q u e n t l y o x i d i z e d by enzymes
80
F i g u r e s 6-1 and 6 - 2 : Cross s e c t i o n s o f s i x month o l d s a i n f o i n r o o t s
a r t i f i c i a l l y i n o c u l a t e d w ith £ . s o l a n i in t h e g r e e n h o u s e . 6 -1 ) Fungal
mycelium (M) in t h e v e s s e l does no t a p p e a r t o cau se c e l l u l a r damage
in t i s s u e s a d j a c e n t t o t h e v e s s e l (x 5 5 0) . 6 -2 ) D i s c o l o r a t i o n in
i n o c u l a t e d s a i n f o i n r o o t s c o n s i s t s p r i m a r i l y o f g e l s (G) which o r i g ­
i n a t e as p r o t u b e r a n c e s (P) in t h e v e s s e l w a l l s and d i s c o l o r e d f i b e r
e l em en t s (DF)(x 160). Bars r e p r e s e n t 20 microns in F i g . 6-1 and
50 micr on s in F i g . 6 - 2 .
81
released
both t h e o rg an is m and t h e h o s t c e l l s t o c a u s e t h e v a s c u l a r
browning ( 5, 6, 4 7 ) .
Wong and P r e e c e (75) emphasized t h e complex n a t u r e o f t h e p lu g g i n g
s u b s t a n c e s in wood i n f e c t e d w it h Erw inia s a l i c i s which t h e y found t o be
composed o f p o l y p h e n o l s and s u b s t a n c e s r e l a t e d t o p e c t i n , l i g n i n and
cellulose.
These compounds a r e t h o u g h t t o o r i g i n a t e from t h e a c t i v i t y
o f p e c t o l y t i c and c e l l u l o l y t i c enzymes, produced by t h e p a t h o g e n , on
t h e c e l l wall components ( 5 , 6 ) .
Gels r e p r e s e n t a p h y s i c a l b a r r i e r and
r e s t r i c t t h e s p r e a d o f c e r t a i n v a s c u l a r pat h o ge n s w h i l e phenol r e l a t e d
compounds a r e g e n e r a l l y t o x i c t o most m i c r o - o r g a n is m s ( 5 , 4 7 ) .
Conse­
q u e n t l y , t h e p r e s e n c e o f g e l s and v a s c u l a r d i s c o l o r a t i o n a r e i n h i b i t o r y
t o t h e s p r e a d o f p a t h o g e n i c o r gan ism s and r e p r e s e n t a d e f e n s e mechanism
in t h e h o s t p l a n t a g a i n s t v a s c u l a r s p r e a d o f pat h o ge n s ( 5 ) .
When t h e bark i s removed from s a i n f o i n r o o t s e x h i b i t i n g t y p i c a l
v a s c u l a r symptoms, t h e i n c i d e n c e o f F\ sol.ani in t h e r o o t s e c t i o n s i s
d r a s t i c a l l y red uc e d (Ta b le 6 - 1 ) .
Th is e v i d e n c e s u g g e s t s t h a t , in most
in s ta n c e s , £ . so lani is a s s o c ia te d with the o u te r regions of the ro o t
t i s s u e s and n o t w i t h t h e v a s c u l a r system e x h i b i t i n g t h e symptoms.
When
fre sh free-hand s e c tio n s of tap ro o ts o f four y ear old sa in fo in p la n ts
a r e viewed under t h e m i c r o s c o p e , m a c r o c o n id i a s i m i l a r in morphology and
shape t o t h o s e o f F. s o l a n i a r e seen in abundance on t h e r o o t s u r f a c e .
Because mycelium was n o t o b s e r v e d on t h e r o o t s u r f a c e o f n a t u r a l l y
i n f e c t e d f i e l d m a t e r i a l p r o c e s s e d f o r h i s t o l o g i c a l e x a m in a t io n
82
. Tabl e 6 - 1 .
The e f f e c t o f t h e removal o f t h e bark on t h e i s o l a t i o n
f r e q u e n c y o f F\ sol am' in n a t u r a l l y i n f e c t e d 4 y e a r old
sainfoin root sections.
No. sampled
for iso la tio n
Tap r o o t s e c t i o n s
Bark i n t a c t
Bark removed
19
17
Lateral root sections
Bark i n t a c t
Bark removed
43
36 .
% of the sectio n s
w i t h F. sol a n i I /
100
35
.
91
25
I Based on i s o l a t i o n s on Komada' s medium, a h i g h l y s e l e c t i v e medium f o r
t h e i s o l a t i o n o f Fusarium sp.
83
(Ex periment 4 ) , i t i s p r o b a b l e t h a t t h e fungus grows s u p e r f i c i a l I y on
t h e o u t e r l a y e r s o f t h e phellum which a r e c o n t i n u a l l y b e i n g s lo u g h ed .
T h i s p r o c e s s i s a r e o c c u r r i n g one in t h e r o o t s o f most f l e s h y d i c o t y l e ­
dons as s o i l c o n d i t i o n s promote t h e r a p i d decay o f . t h e o u t e r m o s t l a y e r s
o f the ro o t (21).
Lack o f a c l o s e a s s o c i a t i o n between IF. s o l a n i and t h e d i s c o l o r a ­
t i o n in t h e d i s e a s e d s a i n f o i n r o o t s was i n i t i a l l y n o te d by S ear s (61,
62 ) .
Because t h e fungus i s o c c a s i o n a l l y i s o l a t e d from t h e v a s c u l a r
system o f t h e more m at ur e s a i n f o i n r o o t s , i t may be r e s p o n s i b l e f o r
v a s c u l a r d i s c o l o r a t i o n and g e l a t i o n which i s no t a s s o c i a t e d w i t h t h e
presence o f b a c t e r ia .
B a c t e r i a have a l s o been a s s o c i a t e d w i t h t h e
p r o d u c t i o n o f v a s c u l a r g e l s (75) an d , c o n s e q u e n t l y , may a l s o be
r e s p o n s i b l e f o r t h e v a s c u l a r p lu g g i n g and d i s c o l o r a t i o n seen in
n atu ra lly infected sainfoin root sections.
CONCLUSIONS
The symptom complex which r e s u l t e d when g r ee nh o u s e grown s a i n f o i n
p l a n t s were a r t i f i c i a l l y i n o c u l a t e d w i t h £ . s o l a n i c o n s i s t e d p r i m a r i l y
o f g e l a t i o n in t h e v e s s e l s and d i s c o l o r a t i o n o f t h e v e s s e l s , f i b e r s and
parenchyma c e l l s .
Very l i t t l e c e l l d i s r u p t i o n and decay was ob se rv ed
e x c e p t in t h e r e g i o n d i r e c t l y above t h e i n o c u l a t i o n p o i n t .
The p r e s e n c e o f £ . s o l a n i was n o t h i g h l y c o r r e l a t e d w i t h t y p i c a l
symptoms i n f o u r y e a r o l d s a i n f o i n .
R a t h e r , t h e fungus seems t o grow
84
s a p r o p h y t i c a H y on t h e o u t e r c e l l l a y e r s o f t h e bark on t h e r o o t s u r ­
face.
These o b s e r v a t i o n s . s u g g e s t t h a t F\ sol a n i i s n e i t h e r p r e s e n t in
t h e symptomatic t i s s u e s o f p l a n t s e x h i b i t i n g t y p i c a l r o o t and crown
r o t symptoms n or i s i t c a p a b l e o f p r o d u c in g t h e e x t e n s i v e r o o t and
crown decay in i r r i g a t e d s a i n f o i n grown in Montana.
■
85
SUMMARY AND CONCLUSIONS,
The r o o t and crow n r o t complex in s a i n f o i n i s l i m i t i n g t o s t a n d
l o n g e v i t y and f o r a g e y i e l d o f
s a i n f o i n grown in Montana.
decay o f t h e v a s c u l a r t i s s u e in t h e crown reg io n ,
E x t e n s iv e
which consumes
between 50% and 90% o f t h e crown a r e a i n f o u r y e a r o l d s a i n f o i n p l a n t s ,
i s t h e ma jor c o n t r i b u t o r y f a c t o r in t h e e v e n t u a l d e a t h o f t h e p l a n t .
Damage
a s s o c i a t e d w i t h l a t e r a l r o o t s and i n s e c t wounds i s
p r o b a b ly
o f r e l a t i v e minor im p o r t a n c e , a l t h o u g h t h e s e r e g i o n s p r o b a b l y s e r v e as
e n t r y p o i n t s f o r d i s e a s e o r g a n is m s .
Decay c a u s i n g o r gan ism s a p p e a r t o e n t e r t h e s a i n f o i n crown th r o u g h
hollow stems which remain a f t e r h a r v e s t o f t h e f o r a g e .
stem i s c u t , a new i n o c u l a t i o n p o i n t i s c r e a t e d .
Each time a new
These hollow stems
a r e c o n t i n u o u s w i t h v a s c u l a r t i s s u e in t h e crown an d , c o n s e q u e n t l y ,
t h e y p r o v i d e an i d e a l s i t e f o r e n t r y o f d i s e a s e o r g a n i s m s .
As t h e s e
organ ism s c on v er g e in t h e crown from t h e d i f f e r e n t i n o c u l a t i o n s i t e s ,
s e v e r e decay o f t h e crown and t a p r o o t r e s u l t s .
The o r gan ism s most f r e q u e n t l y a s s o c i a t e d w it h a l l s t a g e s of. r o o t
and crown r o t in i r r i g a t e d s a i n f o i n were t h e b a c t e r i a Pseudomonas
s y r i n g a e , Pseudomonas marginal i s , Erw inia amylovora and Erwinia h e r b i cola.
In g r ee n ho u s e p a t h o g e n i c i t y t r i a l s , _P. s y r i n g a e , P_. marginal i s .
and JE. am y lo v o r a. a p p e a r e q u a l l y c a p a b l e o f p r o d u ci n g symptoms which
c o n s is ts p rim a rily o f black v a sc u la r d i s c o l o r a t i o n .
I
IE. h e r b i col a was
86
I
>
1
’
found t o bp n o n - p a t h o g e n i c .
Al I t h r e e b a c t e r i a were s i g n i f i c a n t l y more
a g g r e s s i v e th a n £_. sol an i in p r o d u c in g symptoms in s a i f i f p i n crowns.
The h i s t o l o g i c a l
i n v e s t i g a t i o n in to th e pathology o f n a t u r a l l y i n ­
f e c t e d t h r e e and f o u r y e a r o ld s a i n f o i n r e v e a l e d t h a t b a c t e r i a were i n ­
t i m a t e l y a s s o c i a t e d w i t h r e g i o n s o f b l a c k v a s c u l a r d i s c o l o r a t i o n and in
t h e c e n t r a l decay r e g i o n s in t h e t a p and l a t e r a l r o o t s .
B acteria,
however, were no t o b s e r v e d in t h e r o o t s o f second y e a r s a i n f o i n p l a n t s .
£_. sol an i was r a r e l y a s s o c i a t e d w i t h decayed r o o t t i s s u e e x c e p t in
t h e crowns o f t h e f i v e y e a r o l d s a i n f o i n p l a n t s .
Because t h i s o rg ani sm
i s n o t a s s o c i a t e d w i t h t h e l e a d i n g edge o f decay which e x t e n d s deep in
t h e t a p r o o t , i t i s d o u b t f u l w he th er e x t e n s i v e decay and d i s c o l o r a t i o n
i n t h e crown and t a p r o o t s o f t h e s e p l a n t s can be ca us ed by t h i s f u n ­
gus.
In a d d i t i o n , fungal mycelium was n o t o b s er v ed in any o f t h e r o o t
s e c t i o n s in t h e t h r e e and f o u r y e a r o l d s a i n f o i n r o o t s .
In second y e a r
s a i n f o i n crow ns , fungal c e l l s were a s s o c i a t e d w ith b l a c k v a s c u l a r d i s ­
c o l o r a t i o n , a l t h o u g h t h e c e l l u l a r damage, which c o n s i s t e d o f chromop h y l l i c t i s s u e s s u r r o u n d i n g v e s s e l s c o n t a i n i n g fu ngal mycelium,
a p p e a r e d t o be l i m i t e d .
In g r ee n h o us e grown s a i n f o i n i n o c u l a t e d w ith
F. s o l a n i , t h e b l a c k v a s c u l a r symptoms produced were p r e d o m i n a n tl y
a s s o c i a t e d w it h t h e f o r m a t i o n o f g e l s , gums and d i s c o l o r a t i o n o f t h e
v e s s e l s and f i b e r s .
L i t t l e c e l l d e t e r i o r a t i o n and decay was o bs erv ed
in a s s o c i a t i o n w i t h t h e p r e s e n c e o f t h e fungal c e l l s .
,
87
A s y n e r g i s t i c r e a c t i o n r e s u l t i n g i n enhanced s e v e r i t y o f symptoms
was n o t o b s e r v e d when E_. amylovora and £_. sol an i were combined in t h e
s a i n f o i n crowns.
I t i s a l s o d o u b t f u l w h e th e r s u c h . a r e l a t i o n s h i p be­
tween F_. s o l a n i and P.. s y r i n g a e e x i s t s bec a u se F\ s y r i n g a e was shown
t o produce s u b s t a n c e s i n h i b i t o r y t o t h e growth o f JF. s o l a n i on PDA.'
The p r o d u c t i o n o f t h e s e i n h i b i t o r y s u b s t a n c e s may e x p l a i n t h e s c a r c i t y
o f fu nga l o rg an is m s such as F. s o l a n i in t h e decayed r o o t s and crowns
in s a i n f o i n .
Because F_. s o l a n i was n o t o b s e r v e d in symptomatic t i s s u e s in d i s ­
e a s e d s a i n f o i n nor was i t i s o l a t e d e x t e n s i v e l y from r o o t s e x h i b i t i n g
t y p i c a l r o o t and crown r o t symptoms in d i f f e r e n t s t a g e s o f d e t e r i o r a ­
t i o n , t h e a s s o c i a t i o n o f t h i s or gan ism w it h s a i n f o i n r o o t s may be t h a t
o f a weak p a r a s i t e w it h a l i m i t e d c a p a c i t y f o r p r o d u c in g symptoms in
irrig a te d sainfoin roots.
Whether o r n o t e i t h e r t h e b a c t e r i a , o r £_.
s o l a n i a r e a s s o c i a t e d w i t h t h e more g r ad u al r o o t d e c l i n e i n d r y la n d
s a i n f o i n i s n o t known.
c o n d i t i o n s in
irrigated
B a c t e r i a a r e more s u i t e d t o t h e high m o i s t u r e .
s a i n f o i n and t h e r e f o r e , t h e i r r o l e in t h e
d r y l a n d crown r o t i s q u e s t i o n a b l e .
F u r t h e r s t u d i e s on t h e i d e n t i f i c a ­
t i o n o f t h e m i c r o b i a l f l o r a a s s o c i a t e d w i t h t h e s e r o t t e d crowns s hou ld
be u n d e r t a k e n u s i n g t h e i s o l a t i o n and c h a r a c t e r i z a t i o n t e s t s o u t l i n e d ,
in t h i s t h e s i s .
The devel opment o f s a i n f o i n c u l t i v a r s r e s i s t a n t t o t h e organisms
i n v o l v e d in t h e r o o t and crown r o t complex in s a i n f o i n i s r e q u i r e d
88
b e f o r e stptnd l o n g e v i t y and s u s t a i n e d y i e l d s can be a t t a i n e d .
A green­
house s c r e e n i n g t e c h n i q u e would be o f q u e s t i o n a b l e u s e f u l n e s s
bec a us e a) r e s i s t a n c e t o a l l t h r e e b a c t e r i a l o r gan ism s may be s c a r c e
and c o n s e q u e n t l y l a r g e p o p u l a t i o n s would have t o be s c r e e n e d ; b) th e
c o n d i t i o n s f o r maximum devel opment o f t h e d i s e a s e complex i n v o l v i n g
t h e b a c t e r i a have n o t been d e t e r m i n e d ; and c) i t i s p o s s i b l e t h a t
o t h e r o r g a n i s m s , i n c l u d i n g F. s o l a n i , may be a s s o c i a t e d w i t h r o o t and
crown r o t o f s a i n f o i n i n o t h e r r e g i o n s in North America.
F u t u r e s t u d i e s s h o u ld c o n c e n t r a t e on a) e l u c i d a t i n g t h e complex
n a t u r e o f t h e i n t e r a c t i o n s between t h e s e b a c t e r i a in g ree n h o u se i n o c u ­
latio n studies.
H i s t o l o g i c a l and h i s t o c h e m i c a l i n v e s t i g a t i o n s s hou ld
accompany t h e g r ee n h o u s e s t u d y t o d e t e r m i n e t h e n a t u r e o f t h e c e l l u l a r
p a t h o l o g y ca u s ed by each o f t h e s e o r g a n i s m s ; b) s u r v e y s o f both i r r i ­
g at ed and d r y l a n d s a i n f o i n s t a n d s i n Montana and North America sh oul d
be made t o d e t e r m i n e t h e e x t e n t t o which t h e s e b a c t e r i a and o t h e r
or ga ni s m s a r e i n v o l v e d i n t h e r o o t and crown r o t o f s a i n f o i n ,
c) F i n a l l y , a long term s c r e e n i n g program in t h e f i e l d where high i n o c u ­
lum c o n d i t i o n s a p p e a r t o e x i s t s hou ld be u n d e r t a k e n .
Wounding th e
s a i n f o i n r o o t s , p o s s i b l y th r o u g h t h e use o f a r o o t p r u n e r , may speed up
t h e p r o g r e s s i o n symptom development i n s a i n f o i n s t a n d s ; and d) s e l e c ­
t i o n f o r c e r t a i n a n a t o m ic a l f e a t u r e s such as s o l i d stems and crowns
which may c o n f e r some d i s e a s e r e s i s t a n c e i n a g r ee nh o u s e o r f i e l d
s c r e e n i n g scheme may be u s e f u l .
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53.
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1971. S p e c i f i c c o m p o s i ti o n o f t h e pat ho gen s o f
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Is o la tio n , pathogenicity
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S c h in d e , P . A . , and F.L. L u k e z i c . 1974. C h a r a c t e r i z a t i o n and
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S e a r s , R. G. , R.L. D i t t e r l i n e , and D.E. Mathre.
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r o o t r o t t i n g o rg an is m s a f f e c t i n g s a i n f o i n (Onobrychis v i c i a e f o l i a ) in Montana.
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S id di u i , W.M., and P.M. H a l i s k y .
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d i e s o f r e d c l o v e r i n f e c t e d by Fusarium roseum.
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S i e r r a , G. 1957. A s im p l e method f o r t h e d e t e c t i o n o f l i p o l y t i c
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i n e f f e c t i v e r h i z o b i a and i t s r e l a t i o n t o t h e n i t r o g e n n u t r i t i o n
in Montana, p. 8 - 1 2 .
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P h y t o t o x i n s and
APPENDIX
98
Appendix Ta b l e I .
I.
The c o m p o s i ti o n o f media and s o l u t i o n s used in t h i s
thesis.
Fungal Media:
I.
Fre sh Potato. D ex tro se Agar (FPDA)( 7 1 ) :
Fresh u n pe el ed p o t a t o e s
25 0 .0 g
D ex tro se
20.0 g
Agar ( D if c o )
2 0 .0 g
D i s t i l l e d water
Preparation:
1000.0 ml
P o t a t o e s were t h i n l y s l i c e d and steamed f o r
one hour i n an a u t o c l a v e w i t h t h e e x h a u s t
open and f i l t e r e d t h r o u g h two l a y e r s of
cheesecloth.
Agar i s added t o t h e f i l t r a t e .
The s o l u t i o n i s a u t o c l a v e d and poured i n t o
s t e r i l e p e tri dishes.
2.
A c i d i f i e d P o t a t o D extrose Agar (HPDA):
P o t a t o d e x t r o s e a g a r ( D if c o )
D i s t i l l e d water
1000.0 ml
L a c t i c a c i d (25%) .
Preparation:
3.
39.0 g
2 . 0 ml
L a c t i c a c i d added a f t e r a u t o c l a v i n g .
Komada1s Mediurn (3 8 ) :
a) Basel medium
K2HPO4
LO g
99
3.
Komada1s Medium:
(continued)
KCl
0.5 g
MgSO4 r 7H20
0.5 g
Fe-Na-EDTA
0 .0 1 g
L-asparagine
2.0 g
D-galactose
/
20.0 g
Agar ( D if co )
.15.0 g
D i s t i l l e d water
1OO0.O ml
b) A n t i m i c r o b i a l supplement
P e n t a c h l o r o n i t r o b e n z e n e (PCMB)1 75% WP
1.0 g
Oxgall
( D if co )
0.5 g
Na2B4 O7 -IOH2O
LO g
S t r e p t o m y c i n S u l p h a t e (SigmA).
0.3 g
Preparation:
A u to cl av e t h e basal medium f o r 15 m i n u te s a t
121 C and a l l o w t o cool t o 5 0 , C.
Then add
t h e a n t i m i c r o b i a l supp lem ent and a d j u s t pH
t o 3 . 8 - 4 . O u s i n g 10% p h o s p h o r i c a c i d .
B a c t e r i a l Media:
I.
K in g 's B Agaf + Cyclohexamine
P r o t e o s e p e p t on e ( D if c o )
20.0 g
K2 HOP4 -BH2O
. 2.5 g
MgSO4 1TH2O
6 .0 .g
Cyclohexamine (Sigma)
0.1 g
100
1.
K in g ' s B Agar + Cyclohexamine:
(continued)
Nobel a g a r ( D if co )
G lyc ero l
D i s t i l l e d water
2.
12 .0 g
. 1 5 . 0 ml
.
1000.0 ml
G e l a t i n H y d r o l y s i s Medium ( 6 7 ) :
G e l a t i n (Sigma)
4.0 g
N u t r i e n t b r o t h ( D if co )
8.0 g
Nobel a g a r ( D if co )
D i s t i l l e d water
Preparation:
1 2 .0 g
1000.0 ml
A u to c l a v e t h e g e l a t i n s e p a r a t e l y in 100 ml
o f d i s t i l l e d w a t e r and mix w i t h medium
before pouring.
3.
Dye's Mineral Medium ( 1 9 ) :
Stock S o l u t i o n s :
(NH4 ) 2PO4 (10% aqueous s o l u t i o n )
1 3 .0 ml
KCl (2% aqueous s o l u t i o n )
1 3 .0 ml
MgSO4 *7H20 (2% aqueous s o l u t i o n )
13 .0 ml
D i s t i l l e d water
4.
96 1 .0 ml
N i t r a t e Reduction Medium ( 6 7 ) :
D ye's m i n er al medium (13 ml o f each s t o c k s o l u t i o n )
Y east E x t r a c t ( D ifc o )
KNOg '
Nobel a g a r ( D if co )
3 9 . 0 ml
5.0 g
1 0 .0 g
1.0 g
N i t r a t e Reduction Medium ( 6 7 ) :
(continued)
G ly ce ro l
1 0 .0 ml
D i s t i l l e d water
1000.0 ml
S i e r r a ' s L i p a s e Medium ( 6 4 ) :
B a c t o - P e p to n e ( D ifc o)
10 .0 g
NaCl
5.0 g
CaCl2 -ZH2O
0.1 g
Tween 20 ( p o l y o x y e t h y l e n e s o r b i t a n m o n o o l ea te )
10 .0 ml
Tween 80 ( p o l y o x y e t h y l e n e s o r b i t a n m o no o le at e )
10.0 ml
D i s til le d water
Preparation:
1000.0 ml
A u to cl av e Tweens s e p a r a t e l y , th e n add t o
medium.
A d ju s t medium t o pH 7 . 4 w ith 10%
HCl b e f o r e a u t o c l a v i n g .
H i l d e b r a n d s ' s P e c t i n Medium ( 2 9 ) :
Sodium p o l y p e c t a t e (NBC)
22.0 g
CaCl2 1ZH2O
0.1 g
Bromothymol b l u e (1.5% a l c o h o l i c s o l u t i o n )
1 . 0 ml
Agar ( D if co )
4.0 g
D i s t i l l e d water
P reparation:
1000.0 ml
Bring d i s t i l l e d w a t e r t o b o i l i n g , the n add
sodium p o l y p e c t a t e , c a l c i u m c a r b o n a t e and
bromothymol b l u e and a u t o c l a v e .
Aut ocl ave
a g a r s e p a r a t e l y in 100 ml d i s t i l l e d w a t e r ,
102
th e n add t o medium.
7.
Hugh and L i e f s o n 1s F e r m e n t a t i o n Medium ( 3 1 ) :
B ac to Pe pto ne ( D ifc o )
2.0 g
NaCl
5.0 g
KgHOP,
0.3 g
Bromothymol b l u e (1.5% a l c o h o l i c s o l u t i o n )
0 . 3 ml
Glucose
10 .0 g
Nobel a g a r ( D if co )
3.0 g
D i s t i l l e d water
Preparation:
1000.0 ml
A d j u s t pH t o 7 . 1 u s in g 10% NaOH.
S t e r i l ize
g l u c o s e s e p a r a t e l y in 100 ml d i s t i l l e d w a t e r
th e n add t o medium.
P i p e t t e medium i n t o
s t e r i l e t e s t tubes.
8.
T h o r n l e y 1s A r g i n i n e D ih y d r o l a s e Medium ( 6 6 ) :
Peptone ( D if co )
,0.25 g
NaCl
1.25 g
K2 HPO4
0.075 g
Phenol red
'
0.0025 g
Arginine
2.5 g
Nobel a g a r ( D ifc o )
0.7 5 g
D i s t i l l e d water
250 .0 ml
103
Preparation:
A d j u s t pH t o 7 . 2 u s in g 10% NaOH, p i p e t t e
i n t o t e s t t u b e s and a u t o c l a v e .
III.
N utrient Solution:
Normal N u t r i e n t S o l u t i o n :
N-
2.3 g
P2°5
2.3 g
K2O
2.3 g
Tap w a t e r
IV.
18 .9 I
H isto lo g ical Solution:
I.
Formalin Alcohol A c e t i c Acid (50% a l c o h o l ) ( 3 3 ) :
Solution
A: Formal in
95% Ethanol
Solution
B: G l a c i a l a c e t i c a c i d
. D is til le d water
P reparation:
12 .0 ml
8 8 . 0 ml
10 .0 ml
9 0 . 0 ml
The two s o l u t i o n s a r e mixed i n equal p a r t s
j u s t p r i o r to use.
MONTAW STATE
USRAHES
1762 10013801
G au d et, D e n is A
The r o l e o f "b acteria
in t h e r o o t and crown
r o t com p lex o f i r r i g a t e d
s a in fo in
N 378
G 233
cop. 2
IS SU E D TO
DATE
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