Pathogenesis of Septoria nodorum (Berk) Berk. on wheat cultivars varying in resistance to glume
blotch
by Mary Louise Straley
A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF
PHILOSOPHY in Plant Pathology
Montana State University
© Copyright by Mary Louise Straley (1979)
Abstract:
Septoria nodorum, causal agent of glume blotch of wheat, was inoculated onto intact leaves of spring
and winter wheats that exhibited varying degrees of resistance to the pathogen. Inoculated leaves were
removed at 24 hour intervals up to 192 hours after inoculation for observations of nodorum
germination, penetration, and disease development. Germination and penetration by the spores on the
host tissue were the same regardless of the degree of host resistance with the exceptions of Fortuna and
Manitou. Spore germination on Fortuna, a susceptible cultivar, was significantly higher than on the
other cultivars. Spore germination on Manitou, a resistant cultivar, was significantly lower than on the
other cultivars. Contrary to previous reports stomatal penetration was observed. Sto-matal penetration
occurred either through open or closed stomata, but did not occur prior to 72 hours after inoculation.
Trichome penetration, also was observed although infrequently. Stained thin-sections, fluorescent
whole mounts, and scanning electron microscope examinations were used to monitor the development
of the pathogen within the leaf. Such development occurred both intra- and inter-cellularly with the
latter predominating. Hyphae could be distinguished within lesion areas in resistant cultivars and both
within and outside of lesion areas in susceptible cultivars. The degree of intercellular ramification and
cellular disintegration was correlated with host resistance. Wheat leaves were analyzed for the presence
of fungal growth stimulators and inhibitors. No active compounds were detected with the experimental
procedure used. The degree of water congestion in a cultivar was correlated with disease resistance and
the amount of free intercellular water was important in this disease. Several reasons for the importance
of this intercellular water were postulated. PATHOGENESIS OF SEPTORIA NODORUM (BERK) BERK. ON WHEAT
CULTIVARS VARYING IN RESISTANCE TO GLUME BLOTCH
by
■ MARY LOUISE STRALEY
A thesis submitted in p a rtia l fu lfillm e n t
of the requirements fo r the degree
of
DOCTOR OF PHILOSOPHY
in
Plant Pathology
Approved:
Chairperson, Graduate Committee
HeadyMajor Department
Graduate Dean
MONTANA STATE UNIVERSITY
Bozeman, Montana
September, 1979
'
ACKNOWLEDGMENTS
I wish to acknowledge Dr. A. L. Scharen, my major professor,
fo r his professional help, advice, and consideration during the prog­
ress of th is research p ro jec t.
I also wish to acknowledge Dr. R. L. D itte r lin e fo r his advice,
frie n d s h ip , and moral support during th is p ro je c t.
In addition I would lik e to thank Mr. Don F r it t s fo r helping me
with the photographic problems encountered during th is research
p ro je c t.
I would lik e to acknowledge Dr. D. C. Sands and Dr. G. A. Strobe!
fo r serving on my committee.
F in a lly , I would lik e to thank the Montana A gricu ltu re Experiment
S ta tio n , the United States Department o f A g ric u ltu re , and the Montana
Wheat Commission who supplied the funds th a t supported me during
th is p ro je c t.
TABLE OF CONTENTS
Page
V I T A ..............................................
ACKNOWLEDGMENTS
T1-
................
iii
TABLE OF CONTENTS.................................................................................................... iv
LIST OF TABLES............................................................................................................ vi
LIST OF FIGURES
. .
v ii
ABSTRACT ....................................................................................................................
x
INTRODUCTION
I
LITERATURE R E V IE W ...............................................................................■ . . . .
3
Disease Organism ...................................................................... . . . .
The D is e a s e ...............................................................................................
Disease Resistance .......................................................................... .
H is to lo g y .............................................................................................. .
MATERIALS AND METHODS..................................................................................
.
3
4
IO
14
21
General Procedures .................................................
21
S pecific Procedures . . . ..........................................................
23
Extended Dew P e r i o d s ............................................................................... 25
Spore G e rm in a tio n ...........................
26
H istolo gical E x a m in a tio n ..................... ....................• ......................26
Special S t u d ie s .....................' ................................................................ 29
V o la tile In h ib ito rs ......................................................
29
Whole Leaf M o u n ts.............................................
29
Secondary Spore P r o d u c t io n ............................. .................... .... . . 30
Stomata .............................................................................................................31
Water C o n g e s tio n .......................................................................... , . 32
Scanning Electron Microscope ................................. . . . . . .
33
RESULTS.........................................................................................................................34
Pre- and po st-inoculation dew p e r io d s .............................................. 34
Spore germination and p e n e tr a tio n .....................................
38
Whole le a f m oun ts....................................................................................... 44
V
Page
V o la tile In h i b i t o r s .....................................................
44
H is to lo g ic a l ........................................... . . . . ! ! ! ! . * ! . ' !
45
Secondary Spore P r o d u c t io n ................................................................... 61
Stomata ..................................................
. . . . . . .
65
Water C o n g e s tio n ........................
57
Scanning Electron M ic r o s c o p e ......................................! . " . * ! ! ! 71
DISCUSSION..................... . ' ........................ ........................ ....
81
SUMMARY.........................................................
90
REFERENCES.................................................................................
92
Vl
LIST OF TABLES
T ab le
I.
Page
C ultivars of wheat with habit and disease reaction to
Septoria nodorum ................. ...................................... . . . . . .
22
Wheat c u ltiv a rs used in pre- and post-inoculation dew
period studies ..........................................................
24
3.
Wheat c u ltiv a rs used fo r h isto lo g ic al studies .............................
28
4.
E ffe c t o f preinoculation dew periods on germination of
Septoria nodorum pycnidiospores on wheat c u ltiv a rs
varying I n r e s is t a n c e .................■.........................................................35
5.
E ffe c t o f po st-inoculation dew periods on germination of
Septoria nodorum pycnidiospores on wheat c u ltiv a rs varying
in r e s is ta n c e ................................................................................................ 36
6.
E ffe c t of extended dew periods on expression of symptoms in
three wheat c u ltiv a rs varying in Septoria nodorum
r e s i s t a n c e .................................................................................................... 37
7.
Germination of Septoria nodorum pycnidiospores on le a f
surfaces of 18 c u ltiv a rs of wheat varying in th e ir
disease resistance . . ..............................
2.
39
8.
Secondary spore production in three c u ltiv a rs of wheat
varying in disease r e s is ta n c e ..................................... ' .................64
9.
Numbers of stomata on adaxial le a f surfaces of three c u l t i ­
vars of ten day old wheat seedlings grown under id e n tic a l
e n v iro n m e n ts .............................................................
66
E ffe c t o f disruption of le a f surface structure on ex­
pression of disease resistance to Septoria nodorum . . . .
72
10.
vi i
LIST OF FIGURES
F ig u re
1.
Page
Septoria nodorum pycnidiospores e x h ib itin g e a rly
germination patterns on inoculated wheat leaves .................
40
2.
Septoria nodorum pycnidiospore e x h ib itin g germination
from one end and from the side o f the s p o r e .....................40
3.
Branching of germination hypha o f Septoria nodorum
on wheat l e a f .......................................................................................
42
Germinating Septdria nodorum pycnidiospore showing the
formation o f an appressorium in an epidermal crack
and hypha extending beyond the appressorium ...........................
42
Stomata! penetration by the hypha o f Septoria nodorum
without the formation o f an a p p re s s o riu m .....................
43
Photomicrograph o f the i n i t i a l stage o f disease develop­
ment in a susceptible wheat c u ltiv a r .....................................
47
Photomicrograph o f increasing disease development
in a susceptible wheatc u ltiv a r .....................................................
49
8 . . Photomicrograph o f disease development in a susceptible
wheat c u ltiv a r 72 hours a fte r inoculation .............................
49
4.
5.
6.
7.
9.
10.
11.
Photomicrograph showing in t e r - and in t r a - c e llu la r
hyphae of Septoria nodorum w ithin a wheat le a f .................50
Photomicrograph o f safranin-stained m aterial
accumulated on mesophyll c e ll w alls o f a wheat
le a f in response to invasion by Septoria nodorum, . . . .
52
Photomicrograph of safranin-stained m aterial accumulated
in the in t e r - c e llu la r spaces o f a wheat le a f in
response to invasion .by Septoria n o d o ru m .............................
52
v iii
F ig u re
12.
13.
14.
15.
16.
17.
18.
Page
Photomicrograph o f a lesion extending to .th e opposite
epidermal surface on a susceptible wheat c u ltiv a r .................
53
Photomicrograph o f stomata! penetration by a germin­
ating pycnidiospore o f Septoria nodorum ......................................
54
Photomicrograph o f triehome penetration by a germin­
ating pycnidiospore o f Septoria nodorum ......................................
55
Photomicrograph o f a ty p ic a l Septoria nodorum induced
lesion contained by a vascular bundle . ........................... ....
57
Photomicrograph o f a lesion caused by Septoria nodorum
th a t is beginning to surround a small vascular bundle . . .
57
Photomicrograph showing to ta l c e llu la r collapse in a
susceptible wheat c u ltiv a r . . . . .
..............................................
58
Photomicrograph showing to ta l c e llu la r collapse in a
re s is ta n t wheat c u ltiv a r
................................................................
58
19.
Photomicrographs showing the morphology and o rie n ta tio n
of Septoria nodorum pycnidia in a susceptible wheat
c u l t i v a r ........................................................................................................ 60
20.
Photomicrograph of an inoculated susceptible wheat c u l­
tiv a r showing no evidence of pathogen invasion .......................
62
21. ' Fluorescent photomicrograph o f the le a f tissue in Figure
20 showing evidence of pathogen invasion ................. . . . . .
62
22.
Fluorescent photomicrograph showing fluorescence be­
tween epidermal c e ll walls in d ic a tin g pathogen
p e n e tr a tio n .................................................................................................... 63
23.
Photomicrograph showing water congestion in a susceptible
c u ltiv a r o f wheat a fte r 48 hours in the mist chamber . . .
68
Photomicrograph showing water congestion in a re s is ta n t
c u ltiv a r o f wheat a fte r 48 hours in the mist chamber . . .
69
24.
ix
Figure
Page
25.
Photomicrograph showing water congestion in a very
re s is ta n t c u itiv a r o f wheat ( f . timopheevi) a fte r
48 hours in the mist c h a m b e r .......................................................... 70
26.
Scanning electron micrograph of a germinating pycnidiospore o f Septoria nodorum w ith a penetration peg
emerging d ire c tly from the spore, a podi cal appressorium, and another penetration peg .........................................
74
27.
Scanning electron micrograph o f a germinating pycnidiospore o f Septoria nodorum showing a ty p ic a l germination
hyp ha and a rounded d is tin c t appressorium ................................. 75
28.
Scanning electron micrograph o f a penetration peg of
Septoria nodorum emerging from the side of a germina­
tio n hypha ...................................................... ................................ . . .
76
Scanning electron micrograph o f an in fe c tio n hypha of
Septoria nodorum emerging d ire c tly from a pycnidiospore . .
77
29.
30.
Scanning electron micrograph o f a germination hypha
o f Septoria nodorum growing around a wheat le a f
t r i c h o m e ................................................................................................... 78
31.
Scanning electron micrograph of a d is in te g ra tin g
Septoria nodorum hypha in a re s is ta n t c u itiv a r of
wheat 96 hours a f t e r i n o c u l a t i o n ......................................... ...
32.
Scanning electron micrograph of an in ta c t Septoria
nodorum hypha in a susceptible c u itiv a r of wheat
96 hours a fte r i n o c u l a t i o n ..................................................'. . .
.
79
80
X
ABSTRACT.
Septoria nodorum, causal agent o f glume blotch o f wheat, was
inoculated onto in ta c t leaves o f spring and w inter wheats th a t ex­
h ib ite d varying degrees o f resistance to the pathogen. Inoculated
leaves were removed a t 24 hour in te rv a ls up to 192 hours a fte r in ­
oculation fo r observations o f Sv nodorum germination, penetration,
and disease development. Germination and penetration by the spores
on the host tissue were the same regardless o f the degree o f host
resistance with the exceptions o f Fortune and Manitou. Spore germ­
in atio n on Fortuna, a susceptible c u lt iv a r , was s ig n ific a n tly higher
than on the other c u ltiv a rs . Spore germination on Manitou, a re s is ­
ta n t c u ltiv a r , was s ig n ific a n tly lower than on the other c u ltiv a rs .
Contrary to previous reports stomata! penetration was observed. Sto­
mata! penetration occurred e ith e r through open or closed stomata,
but did not occur p rio r to 72 hours a f t e r in o cu latio n . Trichome
penetration also was observed although in fre q u e n tly . Stained th in sections, fluorescent whole mounts, and scanning electron micro­
scope examinations were used to monitor the development o f the path­
ogen w ithin the le a f. Such development occurred both in t r a - and
in t e r - c e llu la r ly with the la t t e r predominating. Hyphae could be d is ­
tinguished w ithin lesion areas in re s is ta n t c u ltiv a rs and both w ithin
and outside o f lesion areas in susceptible c u ltiv a rs . The degree of
in te r c e llu la r ra m ific a tio n and c e llu la r d is in te g ra tio n was correlated
with host resistance. Wheat leaves were analyzed fo r the presence
o f fungal growth stim ulators and in h ib ito rs . No ac tiv e compounds ■
were detected with the experimental procedure used. The degree of
water congestion in a c u ltiv a r was correlated with disease re s is ­
tance and the amount o f free in te r c e llu la r water was important in
th is disease. Several reasons fo r the importance of th is in t e r ­
c e llu la r water were postulated.
INTRODUCTION
Glume blotch of. wheat (Triticum aestivum) caused by the fungus
Leptosphaeria nodorum M u lle r, is an economically important disease
throughout the wheat growing areas of the world.
As more areas are
converted to wheat production th is disease could have an even greater
impact on p o te n tia l crops.
The correct name fo r the fungus is
Leptosphaeria nodorum M uller ( a f t e r it s perfect s ta g e ), although the
pathogen is commonly re fe rred to by it s imperfect name, S e p to ria
nodorum (Berk) Berk.
The disease is commonly called Septoria glume
b lo tc h .
In recent years th is disease has, become increasingly prevalent
due to the phenotypic, c u ltu r a l, and genotypic v u ln e ra b ility of the
cuI t i vars grown.
PhenotypicalIy shorter and le a f ie r cuItiv a r s are
more susceptible to the disease because of increased canopy moisture
(9 0 ).
Increased usage of f e r t i l i z e r s often combined with ir r ig a tio n
has also contributed to the increase in incidence of th is disease.
These same factors combine to increase the density of the canopy and
hence increase canopy m oisture.
U n til recently there has been a v a il­
able l i t t l e genetic resistance to S ep to ria glume b lo tch , and most
cuI t i vars grown have been susceptible under proper environmental con­
d itio n s .
G e n e tic a lly controlled disease resistance has been in v e s ti­
gated extensively in recent years and sources of resistance have
been found (7 5 ).
In the case of glume b lo tc h , disease resistance is
2
of a general types with no major dominant genes being found to d a te .
This study was undertaken to understand more f u l ly the glume
blotch resistance mechanisms functioning in wheat.
Comparisons of
the. disease progression in r e s is ta n t, in term ed iate, and susceptible
cuItiv a r s of wheat were made.
These comparisons involved monitoring
the a c tiv ity of the pathogen on the le a f surface as w ell as w ithin
the le a f tis s u e .
I t is hoped from th is study th a t the mechanism(s)
of resistance by wheat to
and defined.
nodorum may be more c le a rly understood
LITERATURE REVIEW
DISEASE ORGANISM
The i n i t i a l id e n tific a tio n of the pathogen causing glume blotch
of wheat was by Berkley in 1845 ( 86 ) .
Subsequently, the disease
and pathogen were reported from a ll major wheat growing areas of the
world.
I t was not u n til 1898, however, th a t economic losses a ttrib u ­
ta b le to the disease were studied and published (8 0 ).
Since then
minor to severe epidemics have been reported in a ll wheat growing
regions of the world with losses ranging from 5-50% (4 0 ).
The causal organism was described by Berkley and named Septoria
nodorum in 1945 (8 0 ).
O rg in ally Berkley suggested th a t the organism
attacked only the nodes and internodes of the host p la n t.
Later
P asserinii ( 66 ) described a s im ila r organism which appeared on the
glumes of wheat.
P asserin ii named th is organism Septoria glumarum.
I t was la t e r determined by Grove ( 86 ) th a t S_. nodorum and S^ glumarum
were the same organism and the e a r lie r name, SL nodorum, was retained
Although p e rith e c ia were found associated with the fungus as e a rly as
1904, i t was not u n til 1952 th a t M u lle r described Leptosphaeria
nodorum as the p erfect stage of SL nodorum (8 0 ).
The mycelium of SL nodorum is ty p ic a lly branched, s e p ta te, and
hyaline although la te r i t may turn a dark olivaceous color (1 9 ).
In
a r t i f i c a l culture there is considerable v a ria tio n in growth h a b it,
c o lo r, speculation, and pathogenicity (7 4 ).
Pycnidia and pycnidio-
spores form re a d ily e ith e r in cultu re or on host m a te ria l.
The
4
pycnidia are irre g u la r in shape, subepidermal, and dark in color
(7 4 ), although recently a e ria l pycnidia have been seen (7 3 ).
Pyc-
nidiospores, released through the o s tio le are b a c illa r y , 1-3 septate,
h yaline, and average 3 x 26 u in size (6 3 ).
spore contains one nucleus (7 9 ).
Each c e ll w ithin the
The spores exude from the pycnidia
in long pink serpentine strands (53, 86 ) .
P e rith e c ia, formed on host plant m a te ria l, contain numerous
club-shaped, eight-spored, b itunicate asci (5 3 ).
The ascospores are
s tra ig h t to s lig h tly curved, th re e -s e p ta te , fusoid, with the second
c e ll from the apex enlarged.
A ll o f the c e lls contain one or two
prominent g u ttu le s , are hyaline to pale-yellow brown, and average 5
x 27 u.
(5 3 ).
P erithecia have been formed in c u ltu re on oat e x tra c t agar
M icropycnidia, producing microspores, have also been observed
(7 7 ).
Septoria nodorum has been reported on plants in 17 genera (78,
82) including wheat, b a rle y , oats, and various w ild grasses.
is l i t t l e evidence of s t r i c t host s p e c ific ity (8 0 ).
There
However, i t has
been reported th a t is o la te s from one host are usually less v iru le n t
on other hosts (44, 8 0 ).
THE DISEASE
On wheat the organism is pathogenic on a ll above ground plant
parts (63) and can in fe c t anytime from seed germination to plant
)
5
m aturity (8 0 ).
Septoria ntidorum may be seed-borne and cause charac­
t e r i s t ic seedling in fe c tio n (4 , 14, 29, 8 7 ).
and deformed co le o p tile s (4 ) .
Symptoms include stunted
Also, there may be brown streaks
a ris in g from the base o f the c o le o p tile s .
When browning is severe
enough death o f the seedling may re s u lt (7 1 ).
vive although y ie ld is often decreased (8 0 ).
Plants generally sur­
Under high r e la tiv e
humidity, the in fe c tio n on the c o le o p tile s has been reported to pro­
duce pycnidia which can act as an inoculum source la t e r in the growing
season (3 1 ).
Other researchers could fin d no evidence o f pycnidia in
c o le o p tile lesions (14, 2 9 ).
In fec tio n re su lts when subperic a rp ic
mycelium emerges from under the seed-coat and penetrates the epidermal
c e lls o f the c o le o p tile (4 ).
The more ty p ic a l disease cycle begins with in fe c tio n o f growing
plants by pycnidiospores produced in plan t debris (2 8 , 33, 88 ).
The
c ir r h i are produced during wet periods and the pycnidiospores are
spread by splashing or wind-blown rain (71, 72).
Spore germination
and penetration are maximum between 15° and 25°C with a minimum of
six hours of wetness (98% RH) necessary fo r good in fe c tio n (9 0 ).
Secondary in fe c tio n takes place during periods of wet, windy weather
and prolonged favorable environmental conditions may cause epidemics.
The primary source o f inoculum is from pycnidiospores produced on
plant debris with secondary hosts and the perfect stage playing a
6
r e la tiv e ly minor ro le in th is disease.
Infected plant debris may
produce several crops o f pycnidiospores with a lte rn a te wet and dry
periods (7 2 ), thus providing fo r an e f f ic ie n t continual source of
primary inoculum.
Light green to yellow ish pathces between veins on the le a f or
le a f sheath are ty p ic a lly the f i r s t symptoms o f S_. nodorum in fec tio n
(7 8 ).
Later symptoms on infected leaves are yellow to brown elongate
lesions which tend to be s lig h tly darker in the center (5 , 8 0 ).
Lesions may appear 2-22 days a fte r inoculation depending on environ­
mental conditions (9 0 ).
There is evidence fo r toxin production by the
fungus as the necrosis extends well beyond the colonized c e lls (9 , 4 8 ).
The toxin produced by
nodorum is non-specific in nature a ffe c tin g
both susceptible and re s is ta n t c u ltiv a rs as well as non-host plants.
When a r t i f i c a l l y inoculated onto plants th is toxin produces charac­
t e r i s t ic yellow elongate elsions with dark brown centers.
Pycnidia
normally occur in the center of the lesio n s, although they may be
scattered throughout the le a f tissue (1 9 ).
On the culm, sheath, and
rachis the lesions are generally dark and coalesce re a d ily to form
elongate brown to dark brown colored areas.
Pycnidia may also form
on these areas in 10-21 days a fte r inoculation (8 0 ).
Y ield loss from Septoria in fec tio n s is due to improper f i l l i n g
o f the seed, with a decrease in seed numbers playing a less important
ro le (5 , 51, 7 9 ).
Very severe e a rly in fec tio n s may r e s u lt in decreased
7
t i l l e r numbers thus reducing y ie ld (4 ) .
Ecnonmic losses from
Septoria range from 1-2% fo r lig h t in fec tio n s and up to 50% fo r severe
in fec tio n s i f in fe c tio n occurs before heading (80, 90).
occuring a f t e r heading cause no decrease in y ie ld .
re s u lt in infected seed.
In fectio n
However, they may
Several methods have been developed fo r
assessing the degree o f in fe c tio n and y ie ld losses due to Sz nodorum
(51, 52, 55, 76).
The influence of age o f plant tis s u e , temperature, lig h t , r e la tiv e
humidity, and spore concentration on the incidence of Sz nodorum have
a ll been studied.
(7 0 ).
Typical in fectio n s may occur in tissue o f any age
However, more y ie ld loss is associated with in fectio n s in older
tissue (16, 17, 32, 46, 8 0 ).
In fectio ns in seedlings (re s u ltin g from
infected seed) can cause death, although plants freq uently outgrow the
disease (3 , 4 ).
Invasions by the pathogen in plants o f various growth
stages re s u lt in in fectio n s which may or may not be damaging to y ie ld
o f grain depending upon subsequent environmental conditions (16, 17,
22, 32, 6 1 ).
Light is said to be necessary fo r pycnidiospores to germ­
in ate (61) and Baker and Smith ( 6 ) report th a t lig h t is necessary fo r
f u l l development o f symptoms subsequent to successful invasion by the
pathogen.
L i t t l e work has been done concerning the e ffe c ts of d i f f e r ­
ent lig h t q u a n titie s or q u a litite s on Sz nodorum invasion and in fec tio n
processes, although Benedict (7) studied th is problem with the related
S. t r i t i c i , and Cooke (15) studied the e ffe c t of n e a r -u ltr a v io le t
8
lig h t on
nodorum sporulation in c u ltu re . •
Considerable more work has been done on the e ffe c t o f dew or post­
inoculation wetness periods oh the disease development ( 11 , 22 , 32,
61, 7 1 ).
I t has been found th a t th is is a c r it ic a l c o n tro llin g fa c ­
to r in the in it ia t io n o f the disease (71) as well as the continued de­
velopment o f the disease w ithin infected tissue (22, 32, 6 1 ).
Even
a fte r successful invasion and colonization of host tissue by
nodorum
fu rth e r progression o f the disease is lim ite d by the r e la tiv e humidity
surrounding the in fected plan t (32, 6 1 ).
D iffe rin g dew .period
lengths can g re a tly influence the reaction of a c u ltiv a r to the dis­
ease (22, 7 5 ).
Determining the proper length of dew period fo r suc­
cessful in fe c tio n is necessary when screening plants fo r disease re ­
sistance.
I t is necessary to increase the lengths o f post-inoculation
dew periods as resistance le vels are increased in order to detect
even higher levels o f resistance in plant populations (7 5 ).
In sev­
eral other le a f spotting fungal diseases, resistance to the pathogen
is d ir e c tly correlated with po st-inoculation dew periods (3 4 , 35, 36,
37, 3 8 ).
For some ho st-parasite in te ra c tio n s , the "expression of sus­
c e p t ib ilit y or resistance to le a f spotting was associated with the
duration of the wet period" (3 6 ).
Another important aspect of the disease etiology is the e ffe c t
o f spore concentration on disease development.
There is general
agreement th a t increasing spore concentrations increases disease
9
development (5 , 12).
Increasing the spore concentration w i l l , how­
ever, have an unequal e ffe c t on re s is ta n t and suceptible c u ltiv a rs
(5 , 12).
The q u a n tita tiv e differences between reactions on the sus­
c e p tib le and re s is ta n t c u ltiv a rs w ill become g reater.
With decreasing
spore loads, i t is possible to lower the concentration to a point
where no reaction is e lic it e d in re s is ta n t c u ltiv a rs (5 ) .
From a r t i ­
fic e ! inoculation studies the amount of surfactant or s tic k e r has been
shown to play an important ro le .
The addition of a surfactant may
allow a s im ila r degree o f symptom development to remain the same while
the to ta l spore concentration in the inoculum is lowered (4 5 ).
The
most freq uently used concentrations are between I x IO^ and I x IO^
spores/ml and plants are generally sprayed with th is suspension to
"ru n -o ff" (11, 22, 4 6 ).
Some degree of control fo r th is 'd is e a s e has. been achieved by
c u ltu ra l practices (stubble and volunteer c o n tro l), use of diseasefre e seed, seed treatm ent, and crop ro tatio n s (19, 9 0 ).
Disease re ­
s is ta n t c u ltiv a rs are also c u rre n tly being used, although none can be
c la s s ifie d as being highly re s is ta n t (49, 7 4 ).
Lines and c u ltiv a rs
having increased le v e ls of disease resistance are being developed fo r
future.use (5 , 7 5 ).
The type of resistance being incorporated
c u rre n tly in to wheat lin e s is described as general resistance rather
than s p e c ific resistance.
10
DISEASE RESISTANCE
The terms general and s p e c ific resistance can be correlated with
Van der Plank's (85) concepts of horizontal and v e rtic a l resistance
re s p e c tiv e ly .
As.o r ig in a lly defined by Van der Plank the term h o ri­
zontal is used when resistance is evenly spread against a l l races of
the pathogen while v e rtic a l is when a v a rie ty is more re s is ta n t to
some races of a pathogen than others and d iffe r e n tia l in teractio n s are
found (8 5 ).
These types of resistance are d iffe re n tia te d from t o l ­
erance in th a t tolerance implies the a b il it y of a plan t to y ie ld nor­
mally even while severly infected (6 9 ).
S pecific and general re s is ­
tance imply some type of r e s tric tio n of the pathogen's a b il it y to
e ith e r I ) successfully invade the host or 2 ) develop normal disease
expression in the p la n t .(5 7 ).
In most disease resistance breeding
e ith e r of the two "resistance" types are used in preference to t o l ­
erance as i t is more d i f f i c u l t to measure the tolerance levels while
re s is ta n t reactions are usually more dramatic (6 9 ).
Also, i t is the
fe e lin g of some researchers th a t to le ra n t v a rie tie s provide fo r a con­
tinuous source of inoculum which could then in fe c t other c u ltiv a rs
(10, 8 4 ).
S p ecific resistance has often been equated with the presence of
a single major gene which is e ffe c tiv e against a p a rtic u la r race of
pathogen.
Once a gene fo r s p e c ific resistance has been incorporated
11
in to the c u ltiv a r the p a rtic u la r race o f the pathogen is no longer
able to cause disease (1 0 ,.8 5 , 86 ) .
This type of resistance is easy
to fo llo w g e n e tic a lly since i t is simply in h e rited and is r e la tiv e ly
in s e n s itiv e to environmental changes (56, 8 5 ).
A disadvantage of
s p e c ific resistance is th a t genetic changes in the pathogen often
re s u lt in a "new" physiological race which is then able to in fe c t
previously re s is ta n t plants (10, 56, 86 ).
General resistance is more d i f f i c u l t to work with since it s
e ffe c ts do not f a l l in to e a s ily categorized classes, but rath er
show a lessening o f lesion development, a lessening o f spore pro­
duction, or some other re s tr ic tio n placed on pathogen growth and re ­
production (10, 86 ) .
Thus increases or tran sfers of th is resistance
often confer small changes which are d i f f i c u l t to measure and/or
q u an tify .
General resistance is usually more e ffe c tiv e against a ll
races (or pathogenic virulence types) o f the pathogen (8 5 ).
Also,
i t is considerably more stable in it s reaction to genetic changes in
the pathogen (10, 85, 86 ) .
The expression of general resistance,
however, shows a high degree of s e n s itiv ity to environmental changes.
Some researchers fe e l th a t general resistance is contro lled by poly­
genic action with many "minor genes" contributing to the overall re ­
s is ta n t reaction (10, 56).
Recently Nelson (56) has disagreed with the terms h o riz o n ta l,
.
12
v e r tic a l, major gene, and minor gene in re la tio n to disease re s is ­
tance.
He says th a t v e rtic a l (major gene, s p e c ific ) resistance may
confer elements of horizontal (minor gene, general) re s is ta n t re ac t­
ions to.some races of the pathogen and th a t horizontal resistance
factors are not necessarily spread evenly over a ll races of a patho­
gen.
Also, th a t a single major gene in a d iffe r e n t genetic back­
ground may act lik e a minor gene or the converse.
Because of these
factors Nelson (56) has proposed th a t the two types of resistance be
c a lle d by new terms.
He re fe rs to horizontal resistance as a ra te -
reducing resistance and v e rtic a l resistance as a resistance th a t re ­
duces the amount of e ffe c tiv e i n i t i a l inoculum.
He contends th a t
there are no major or minor genes fo r resistance but simply re s is ­
tance genes which may act in a "h o riz o n ta l" manner when combined
together and/or in a " v e rtic a l" manner when appearing singly in the
appropriate genetic background.
Resistance to
nodorum has been sought by researchers in a ll
wheat growing areas of the world (2 , 5 , 47, 49, 50, 59, 7 5 ).
Var­
ie t a l d iffe re n c e s , although they occur, are influenced by environ­
mental changes which makes u t iliz a b le h e rita b le reactions hard to
d e fin ite ly estab lish (5 9 ).
Among those te s te d , spring wheats are
more susceptible than w inter wheats and I . aestivum is more suscep­
t ib le than some of the other Triticum spp (4 9 ).
Various inoculation
13
and screening techniques have been tr ie d in order to standardize con­
d itio n s so th a t repeatable resu lts w ill allow fo r the selection of
plants which show true resistance ( 2 , 11 , 47, 49, 5 0 ).
Greenhouse
screening of seedlings a t the three le a f stage (Peekes' Scale I ) has
proven e ffe c tiv e and correlates w ell with f ie ld resu lts (49, 50, 75).
This type of screening procedure gives more repeatable results be­
cause i t is possible to duplicate the environmental conditions.
It
also reduces the number of plants th a t need to be screened in the
f i e l d (4 9 ).
Through various screening procedures disease to le ra n t
or re s is ta n t cuItiv a r s have been detected and studies are presently
being done to determine the type of gene ac tio n (s ) and mode of inher­
itance they e x h ib it (5 , 7 5 ).
with extensively ( 5 ) .
Tolerance to _S. nodorum has been worked
Other workers have reported resistance th a t is
due to dominant, p a r t ia lly dominant, or recessive gene action (5 , 49,
80). ' Progeny of d ia lle l crosses of re s is ta n t and susceptible lines
have been studied (50, 7 3 ).
Laubscher e t aj_, (50) found there was a
large number of re s is ta n t types in the progenies of the most re s is ­
ta n t parent except when crossed with the most suscep tible.
Suscep­
t ib le in d ivid u als comprised the m a jo rity of the progenies from the
susceptible parent except when i t was crossed to the most re s is ta n t.
They concluded from t h e ir study there was workable h e rita b le re s is ­
tance to Sl. nodorum in wheat.
Scharen (75) and Krupinsky (49) have
14
both shown th at through a s e le c tio n , crossing, and reselectio n pro­
gram th a t levels of resistance may be increased in succeeding genera­
tions of wheat lin e s .
None of these studies have shown evidence fo r
major dominant gene a c tio n , but instead show a q u a n titia tiv e type of
inheritance fo r resistance to S. nodorum.
Because of the resistance
levels found in Triticum spp other than I .
aestiyum, e ffo rts are
being made to tra n s fe r resistance genes in to T. aestivum lin e s .
This
is being done by the s e le c tio n , crossing, s e lfin g , reselectio n
scheme mentioned above (7 5 ).
uated under f ie ld cond itio ns.
Agronomic t r a i t s also are. being e v a l­
Evidence fo r transgressive segregation
in c u ltiv a rs being studied (49, 73) suggests th a t th is phenomenon
may be of major importance in the development of re s is ta n t lines fo r
the fu tu re .
HISTOLOGY
Several h is to lo g ic a l studies have been made on non-haustoria
forming, non-obligate le a f spotting fungi ( I , 8 , 13, 30, 39, 58, 81,
8 3 ).
Penetration was d ire c t (through the c u t ic le ) , between adjacent
epidermal c e ll w a lls , or through the stoma, and accompanied or not by
the formation of appressoria depending upon the pathogen.
The a c tiv ­
it ie s of pathogens on le a f surfaces have been monitored ( I , 58, 83)
and a v a rie ty of techniques have been developed to study in t r a - and
in te r c e llu la r fungal growth (18, 24, 60, 64, 65, 68 ) .
Also,
15
researchers have looked at the d if fe r e n t ia l responses of susceptible
and re s is ta n t c u ltiv a rs to invasion and colonization by pathogens (23,
24, 26, 58, 81).
These papers have described re s is ta n t reactions in
main categories.
The f i r s t type of resistance mechanism is the hyper­
sense reaction in which the host c e lls die rap id ly with successful
fungal invasion lim itin g fu rth e r spread (1 0 ).
The second type of
mechanism re su lts in the slowing down of the pathogen's invasion,
c o lo n iza tio n , and speculation processes in the re s is ta n t c u ltiv a r .
(1 0 ).
There are instances where both types of resistance mechanisms
are manifested in the same host-pathogen complex (8 9 ).
With S ep to ria, less h is to lo g ic a l work has been done.
However,
in 1957 Green and Dickson (23) published a thorough cyt o Io g ical
study of j). p a s s e r in ii, causal agent of Septoria le a f blotch of bar­
le y .
They found, in contrast to previous reports ( 86 ) , th a t Sv
p a s s erin ii ra re ly exhibited d ire c t c u tic le penetration and the p r i­
mary mode of penetration was through stomata.
They reported th at
the disease seemed to progress along a "stomata! g radient".
Reac­
tions to th is organism by susceptible and re s is ta n t v a rie tie s were
studied and i t was found th a t the susceptible plants allowed the
pathogen to grow fr e e ly through the le a f tis s u e .
Host c e ll death
did not a ffe c t the pathogen's a b ilit y to spread.
In co n tra s t, the
ho st-p arasite in te ra c tio n in re s is ta n t plants was deleterious to both
e
16
the host and the pathogen.
The Interm ediate reaction showed aspects
of both susceptible and re s is ta n t reactions.
Some of the hyphae were
able to spread, beyond the areas of c e llu la r d is ru p tio n , although not
as freq u en tly as in the susceptible re ac tio n .
Benedict (7) reported
th a t Sy t r t t i c i p rim a rily penetrated through the stomata of wheat
leaves, but did not study fu rth e r development of the pathogen w ithin
host tissu e.
The i n i t i a l cytolog ical study of S. podorum in fe c tio n of wheat
was done by Weber. ( 86 ).
He found th a t Septoria entered the host by
d ire c t penetration of the c u tic le in the depressions d ir e c tly above
adjacent epidermal c e lls .
Shipton (80) and Brown (12) la t e r in dica­
ted th a t stomata! entry had never been observed.
A fte r penetration
the hyphae grew between the epidermal c e lls and did not branch u n til
they had come in contact with the "parenchymatous c e lls " below the
epiderm is, Weber reported ( 86 ) .
The hyphae then grew p a r a lle l to the
le a f surface and often occurred in the crevices formed by the e p i­
dermal c e lls running p a ra lle l with the vascular bundles.
Host c e lls
in the v ic in it y of the invading hyphae appeared normal u n til a ll the
in te r c e llu la r spaces had been f i l l e d with hyphae.
He also found
aggregations of hyphae in the substomatal c a vitie s which in it ia t e d the
formation of pycnidia.
The pycnidia were formed so th a t t h e ir o s ti-
oles were d ire c tly beneath the opening of the stoma.
17
Baker and Smith ( 6 ) recen tly have completed a study on the devlopment of re s is ta n t and susceptible reactions in wheat when inocu­
lated with
nodorum.
They used lig h t and scanning electron micros­
copy in in te rp re tin g the reactions e lic it e d by the pathogen in the
two wheat c u ltiv a r s , Maris Ranger (Susceptible) and Engelen (re s is ­
t a n t).
Engelen, however, has re ce n tly been found to be susceptible
when screened against is o la te s used by Shcaren (7 3 ).
Detached leaves
were observed, by Baker and Smith, a t s p e c ific in te rv a ls of time
a fte r inoculation fo r pathogen development and host c e ll response.
They evaluated the e ffe c ts of various spore concentrations, lig h t
regimes, and temperature regimes on disease progression.
They found .
th a t lesion development was in d ire c t c o rre la tio n to the spore con­
centration in both v a r ie tie s .
A lower lim it of spore concentration
was eventually reached in the re s is ta n t c u ltiv a r .
At a ll three temp­
eratures studied (1 8 , 20, 25°C) lesions developed, but 20°C was o p ti­
mum fo r to ta l numbers and speed of development.
Incubation in dark­
ness gave more re s tric te d lesion development and was necessary fo r
development of the disco lo ratio n re ac tio n .
The disco lo ratio n reaction
occurred in both wheat lin es although i t occurred f i r s t and most in ­
tensely in the re s is ta n t c u lt iv a r .
The major differences between the
re s is ta n t and susceptible reactions noted are summerized in the
follow ing chart:
18
Resistant
Spore germination
Same
Spore penetration
Lesser
Necrosis
Noted f i r s t , but
lesser in f in a l
degree
Same
Greater
Eventually greater in
degree and spread
Lesser
Hyphal development
Sporulati on
Susceptible
Greater
Negative (fo r
time period
studied)
,'
P ositive
No differences in spore germination on the le a f surfaces were
observed between the two c u ltiv a r s , although penetration occurred
more freq uently on the susceptible le a f pieces.
This was demonstrated
by a d iffe r e n tia l degree of epidermal c e ll s ta in in g .
P enetration,
rep o rte d ly , took place d ir e c tly beneath appressoria which formed at
the junctions of c e ll w a lls .
Necrotic browning was noticed in the re s is ta n t v a rie ty as e arly
as 72 hours a fte r in o c u la tio n .
The necrosis which did not a ffe c t
epidermal c e lls was observed in the in t e r c e llu la r spaces of the subepidermal c e lls .
This necrosis appeared to be in conjunction with
the veins, although the vascular bundles themselves seemed to be nor­
mal.
The necrosis in the susceptible le a f d iffe re d by involving whole
c e lls , not ju s t in t e r c e llu la r spaces, and w ithin 96 hours a fte r inoc­
u latio n extended from the inoculated le a f surface to the opposite
side.
In te r c e llu la r hyphal development appeared less extensive in
19
the re s is ta n t re ac tio n , although th is was not q u a n tifie d .
Hyphae in
the re s is ta n t reaction tended to ramify in te r c e llu la r ly along the
length o f the le a f a x is .
A fte r 144 hours o f incubation no hyphae
could be detected w ithin or surrounding the lesio n .
In contrast,
hyphal development in the susceptible le a f occurred both in t e r - and
in tr a c e llu la r ly , was heavily branched, and could be detected in ad­
vance of susceptible lesions a fte r 240 hours.
Recent h isto lo g ic al work with four c u ltiv a rs o f wheat has shown
no observable reactions to in fe c tio n in the leaves o f the susceptible
c u ltiv a rs (2 6 ).
The researcher postulated th a t the fungus may rap­
id ly disrupt the normal physiological/biochem ical processes and thus
stop the formation of a normal h isto lo g ic al defense mechanism.
Following penetration, colonization o f the host was in te r c e llu la r
and ty p ic a lly was characterized by chlorosis and necrosis of colonized
tis s u e .
Tissue in advance o f the zone o f colonization also exhibited
c h lo ro tic and necrotic reactions.
In contrast to previous reports.
Narrower (26) did observe penetration through open or closed stomata.
Although resistance to S^. nodorum has been found and u tiliz e d i t
was not known what exact mechanism o f resistance was functioning.
progression of the disease in wheat c u ltiv a rs of varying resistance
was therefore studied to determine what these mechanisms were.
The
pathogen-host in te ra c tio n was studied a t both the le a f surface as
The
20
well as the in tern a l level to determine exactly when and where the
re s is ta n t reaction occurred.
Often when s p e c ific defense mechanisms
are defined and understood i t is possible to make more e f f ic ie n t use
o f these mechanisms in developing disease re s is ta n t c u ltiv a rs .
Also
knowledge gained about defense mechanisms operating in one host-para­
s ite in te ra c tio n may have im plications in understanding other host
parasite in te ra c tio n s .
p ro ject was undertaken.
I t was fo r these reasons th a t th is research
MATERIALS AND METHODS
GENERAL PROCEDURES
Eighteen wheat c u ltiv a rs (Table I ) were grown in 7.5mm p la s tic
pots f i l l e d with washed sand and watered with non-nutrient water u n til
ten days a f t e r seeding ( Feekes Scale I ) .
Plants were maintained in
a growth chamber w ith a 12 hour day temperature of IS0C and night
temperature of 23°C.
Plants were watered from the bottom o f the pot
in order to minimize in terferen ce from other f o l ia r pathogens.
lo ts were the same throughout the study.
Seed
Eighteen c u ltiv a rs were
used as the base population and selected c u ltiv a rs were used in in d i­
vidual experiments.
The SL nodorum c u ltu re used in th is study was obtained from a
single-spore is o la te o f a known v iru le n t c u ltu re .
The is o la te was
chosen fo r it s a b il it y to maintain s u ffic ie n t, uniform spore produc­
tio n through repeated tra n s fe rs .
Reculturing from infected Fortuna
(Cl 13596) leaves was done occasionally to make sure th a t the contin o u sly tran sferred cultures remained the same as the o rig in a l iso ­
la te .
Yeast-m alt agar
plates were inoculated with S. nodorum by
flooding with a suspension of pycnidiospores obtained from one week
old c u ltu res.
These were maintained under 20 hours o f lig h t a t 15°C
fo r seven days p rio r to u t iliz a t io n .
Inoculum was prepared by gently
scraping the mycelia and pycnidia from the surface o f seven-day old
p la te s , mixing with 60ml d is t ille d water fo r one minute in a Waring
22
Table I .
C u ltivars of wheat with habit and disease reaction to
S. nodorum
C u ltiv a r
Triticum
Species
C .I.
Number
Disease I /
Reaction
Fortuna
Newana
Manitou
Redhart
Red Chief
Cl 12373
Cl 14032
Oasis
Arthus 71
Centurk
Michigan Amber
Hadden
Blueboy
Mt 7406
Mt 74394
CVLO 2204
Yamhill
Z e n a ti-B o u tie lIe
aestivum
aestivum
aestivum
aestivum
aestivum
aestivum
aestivum
aestivum
a e s tivurn
aestivum
aestivum
aestivum
aestivum
aestivum
aestivum
aestivum
aestivum
durum
13596
17430
13775
8898
12109
S
I
■
R
• R
R
R
R
R
R
S
S
I
I
I
I
R
R
VR
15929
15282
15075
11379
13488
14031
13554
Habit - /
Spr
Spr
Spr
W
W
W
W
W
W
W
W
W
W
W
W
W
W
Spr
—/ S = susceptible, I = in term ediate, R = re s is ta n t, VR = very
re s is ta n t
2/
- Spr = spring, W = w inter
23
Blender, and f il t e r in g through doubled cheesecloth.
Spore counts
were made with the aid o f a haemocytometer and the fin a l concentra­
tio n was adjusted to 20 x IO^ spores/ml (allow ing ten mis per pot o f
wheat).
One drop of surfactant (Iv o ry soap) was added to the spore
suspension which was atomized into the plan ts.
Unless otherwise stated a ll plants were allowed a pre-inoculation
dew period of four hours and a po st-inoculation dew period of 48
hours.
When possible plants were inoculated in place in the dew'or
mist chamber i t s e l f .
I t was necessary during the germination studies
to remove the plants from the dew chamber before inoculating them.
In th is case a ll plants were moved as a u n it with special a tten tio n
being paid to cause as l i t t l e of the dew to run o ff as possible. : Dis­
ease ratings were made seven days a fte r in oculatio n.
In addition
some m aterial was kept fo r 25 days to study the production o f pycnidia.
In a ll cases appropriate control plants were used and assessed fo r
disease reactions a t the same time as inoculated plants.
SPECIFIC PROCEDURES .
The optimum length o f time fo r pre- and post-inoculation dew
periods was determined fo r four c u ltiv a rs (Table 2 ).
Pre-inoculation
dew periods were assessed by placing ten day old seedlings in a Percival Dew Chamber with a water bath temperature of 35°C and a wall
temperature o f 7°C ( a ir temperature was 20°C ).
Plants were given
24
Table 2.
Wheat c u ltiv a rs used in pre- and po st-inoculation dew
period studies.
C u ltiv a r
Triticum .
Species
C .I.
Number
Fortuna
Manitou
Cl 14032
Blueboy
aestivum
aestivum
aestivum
aestivum
13596
13775
14031
D isease
Reaction
Habit
S
R
R
I
Spr
Spr
W
W
25
preinoculation dew periods o f 0 , 2 , 4 hours, inoculated, and then sam­
ples were removed a t 4 , 12, and 24 hours a f t e r in ocu latio n .
Germina­
tio n counts were made on the oldest le a f from each of three seedlings
in each pot and re p lic a te d twice in a completely random design.
Leaves were cut into IOmm pieces ( t ip and base ends were discarded),
placed in a lactophenol-trypan blue solution (43) fo r four hours, and
mounted in glycerine on microscope s lid e s .
A ll germinated and un­
germinated spores were counted on fiv e seperate areas on each le a f.
Each area consisted of a section 0.516 mm across the width o f the le a f
blade.
Dew length periods a f t e r inoculation were studied using the same
four c u ltiv a rs and techniques as above.
A four hour pre-ino cu lation
dew period was used and samples were removed a t 24, 48, and 72 hours
of post-inoculation dew periods.
EXTENDED DEW PERIODS
Sixteen pots each of ten day old Fortuna, Newana, and Manitou
seedlings (15 p la n ts /p o t) were subjected to extended pre- and p o s t-in ­
oculation dew periods in each of two re p lic a te d experiments in a com­
p le te ly random design.
Pre-inoculation dew periods of 4 , 12, 24, and
48 hours were used with pots removed 24, 48, 72, and 96 hours a fte r
in ocu latio n .
The plants were a ll inoculated a t the same time with the
standard inoculum previously described.
Ten days a fte r inoculation
26
a ll pots were scored fo r s everity o f disease symptoms on the primary
le a f.
Disease s e ve rity was recorded as average percent necrosis on
ten primary leaves o f each c u ltiv a r .
Percent necrosis was recorded
as the length o f the necrotic region o f each le a f /t o t a l length of the
le a f x 100.
SPORE GERMINATION
Al I 18 c u ltiv a rs o f wheat were used in spore germination studies.
Pre- and post-inoculation dew periods were achieved in a Percival Dew
Chamber set as described above.
Germination counts were made a t 2 , 4,
8 , 12, 24, and 48 hours a fte r inoculation and were re p lic a te d three
times in a completely random design.
During germination observations
irre g u la r germination, degree o f germ tube elongation, branching, and
formation o f appressorial structures were a ll noted.
HISTOLOGICAL EXAMINATIONS
For the h is to lo g ic a l experiments, which were re p lic a te d tw ice,
a greenhouse bench m ist chamber provded the pre- and post-inoculation
dew periods.
The mist chamber was constructed of a wooden frame
surrounded by a covering of c le a r vinyl p la s tic .
An a ir. seal was
achieved on three sides of the plastic-greenhouse bench junction by
burying the end of the p la s tic in the sand.
The remaining side of the
p la s tic was loosely fastened.
Mist was provided by three "Walton-
type" cold water hu m idifiers.
Leaf samples were removed from c u ltiv a rs
27
lis te d in Table 3 a t 24 hour in te rv a ls from 24 and 196 h o u rs.after
inoculation these were.cut in to 5mm sections and immediately immersed
in FAA fix in g solution (4 1 ).
Other samples were removed 25 days
a fte r inoculation to study formation o f the pycnidia.
Leaf pieces in
FAA were v a cu u m -in filtra te d to assure complete fix a tio n .
A fte r f i x ­
ing overnight, samples were taken through a t-b u ty l alcohol graded de­
hydration series (4 1 ), equlibrated overnight in a t-b u ty l alcoholp a ra ffin o il solution (50-50 v /v ) , in f ilt r a t e d with Paraplast (four
changes), and subsequently Paraplast embedded in Tissue-Tek p la s tic
mounts.
A fte r i n i t i a l trimming of the blocks, samples were allowed to
soften fo r two weeks in a solution o f g lycerin-w ater (50-50 v /v ) be­
fore sectioning on an A-O Model 820 ro ta ry microtome.
The ten u s e ria l
sections were a ffix e d to slides with Haupt's adhesive (4 1 ), dried
overnight on a warming tra y (43°C) and stained with one o f the fo llo w ­
ing I ) a Safranin-Fast Green combination; 2) Johansen's Quadruple
S tain ; or 3) Conants1 Quadruple Stain (4 1 ).
A fte r stain in g cover
s lip s were a ffix e d with Permount and slid es were examined with a Zeiss
Binocular Research Microscope Model Standard 14 equipped with a 35mm ■
camera and o p tiv a r m agnification changer.
Extachrome Tungsten 160 and
Panatomic X film were used to record development o f the pathogen w ith­
in le a f tis s u e .
28
Table 3.
Wheat cult.ivars used fo r h isto lo g ic al studies.
C u ltiv a f
Fortune
Newana
Manitou
Centurk
Michigan Amber
Mt 7406
Mt 74394
Arthur 71
Triticum
r Species
aestivum
aestivum
aestivum
aestivum
aestivum
aestivum
aestivum
aestivum
C ,I.
Number
13596
17430
13775
15075
11379
Disease
Reaction
Habit
S
I
R
S
S
I
I
R
Spr
Spr
Spr
W
W
W
W
W
29
SPECIAL STUDIES
VOLATILE INHIBITORS
Inoculated and uninoculated le a f pieces were placed in close
proxim ity to the Sz nodorum seeded agar surface o f a 55mm p e tri dish
in order to te s t fo r production o f v o la t ile in h ib ito rs .
Leaf sections
(35mm) were removed from leaves o f the d iffe r e n t wheat c u ltiv a rs and
were taped with double stic k y tape to the li d o f the p e tri dish in
the form o f an X.
The p e tri dishes were sealed with P arafilm and
allowed to incubate upside down a t 15°C with a 12 hour photoperiod.
At 96 and 196 hours a f t e r sealing , the plates were examined fo r any
differences in Sz nodorum growth patterns although the P arafilm wrap­
ping was not removed u n til a fte r 196 hours.
WHOLE LEAF MOUNTS
Various whole le a f mount analyses were done to demonstrate the
processes occurring during in fe c tio n o f wheat by Sz nodorum.
Leaf
pieces (5mm) from Fortune (S ), Newana ( I ) , and Manitou (R) spring
wheats were used in these studies.
Plants were grown and inoculated
under standard conditions and samples were removed a t 24 hour in te r ­
vals fo r T20 hours.
Leaf pieces were fix e d in GAA-ETOH solution overnight, cleared
overnight in lactophenol, and stained fo r one to 24 hours in 0.1%
acid fuchsin in lactophenol (w/v) (5 4 ).
Excess stain was removed by
careful b lo ttin g and pieces were counter stained in fa s t green fo r
30
fo r fiv e minutes.
This procedure allowed fungal structures on the
le a f surface to stain green while those in the in te r io r o f the le a f
stained red (5 4 ).
In a seperate set of studies le a f pieces were fix e d in e ith e r
GAA-ETOH, FAA3 or lactophenol-ETOH and then stained with the Periodic
S c h iff Acid stainin g procedure (6 0 ), lactophenol cotton blue (1 2 ),
or a c etic a n ilin e blue ( 6 ) .
A technique using C alcofluor was also attempted (6 5 ).
An e p i-
fluorescent attachment fo r the Zeiss microscope was u t iliz e d .
The
f i l t e r set in the attachment consisted o f E xciter f i l t e r BP 450490, Beam s p lit t e r FT 510, and B a rrie r F i lt e r LP 520.
This f i l t e r
set did. not permit the fluorescence o f Calcofluor to be expressed and
th erefo re i t was modified by replacement o f the E xciter f i l t e r (BP
450-490) by a BG-3 f i l t e r mounted in an accessory f i l t e r holder.
The
B a rrie r f i l t e r and the Beam s p lit t e r were not removed.
SECONDARY SPORE PRODUCTION
Fortuna,.Newana, and Manitou were inoculated w ith a standard
spore suspension in the usual manner and were then allowed to grow
under greenhouse conditions fo r 25 days.
were removed from inoculated leaves.
Four 25mm le a f segments
One end of the leaves was
placed in a small metal c lip which was suspended in a t ig h t ly stop­
pered j a r .
One inch o f deionized d is t ille d water was placed in the
31
j a r and a f i l t e r paper c ylin d er was inserted so i t rested on the bot­
tom and around the inside perimeter o f the j a r to act as a wick.
P rio r to placement in the ja r the le a f pieces were dipped in deionized
d is t ille d water to wet the pycnidia.
A ll three o f the wheat c u ltiv a rs
could be placed in the same j a r insuring uniform conditions.
The ja r
O
was placed in an incubator a t 15 C with a 12 hour photoperiod.
Spores
were harvested a t 48 hours in a manner s im ila r to th a t described by
Gough (2 4 ).
A fte r removal o f the spores the number of pycnidia in
the le a f segments were determined by observation o f the le a f pieces
under a dissescting microscope.
The number of pycnidiospores per
pycnidium was determined fo r the three d iffe r e n t c u ltiv a rs ;
STOMATA
2
The number o f stomata per cm was determined in ten day old Fortuna, Newana, and Manitou seedlings.
Stomata! condition (open or
closed) was determined during d iffe r e n t dew periods.
Epidermal le a f
peelings were made of the upper surfaces o f the three wheat c u ltiv a rs
a fte r exposure to 24 and 48 hours of m ist and were mounted in la c to phenol cotton blue.
These epidermal le a f peels were removed from
plants o f s im ila r age and s im ila r le a f expansion.
In order to deter­
mine the exact stomata! condition, the peelings were immediately fixed
in 100% ETOH th a t contained 0.1% cotton blue (6 2 ).
Peelings were
examined m icroscopically and stomata! numbers and condition were
32
recorded.
Two re p lic a tio n s o f th is completely random designed ex­
periment were made.
WATER CONGESTION
Six pots each o f Fortune, Newana9 Manitou 9 Frondoso9 Klein Toledo,
and T. timopheevi were grown to ten day old seedling stage (15 p lan ts/
p o t).
Fronodoso9 Klein Toledo, and T. timopheevi a ll show an extremely
high degree o f resistance to in fe c tio n by jS. nodorum.
T. timopheevi
is considered to be the most re s is ta n t of a ll Triticum lin e s (4 9 ).
One-half the pots were sprayed with a standard inoculum dose and oneh a lf were l e f t as healthy controls.
One pot of each c u ltiv a r was re ­
moved from the m ist chamber a t 48, 72, and 96 hours a f t e r in oculation.
Leaf samples were removed from both the inoculated and uninoculated
pots and were assayed fo r degree o f water congestion (4 2 ).
were read fo r disease symptoms seven days a f t e r in o cu latio n .
Plants
In add­
itio n four plants in each pot were tagged and the le a f blades were
pulled through a thumb and fo re fin g e r which had been dipped in water
or chloroform.
The water and chloroform treatments were used with the
in ten tio n o f disrupting the wheat le a f triehomes and/or the waxy c u t i­
cle o f the le a f .
This two structures are possible b a rrie rs to in fe c t­
ion by fungi so the e ffe c t o f th e ir disruption was studied.
These
four leaves were then assayed fo r any differences th a t th is treatment
made in degree o f in fe c tio n or degree o f water congestion.
SCANNING ELECTRON MICROSCOPE
Two pots each of Fortunag Frondosog Klein Toledo, and T.
timOpheevi were grown and inoculated under standard conditions.
Pots
contained 15 plants and a completely random experimental design was
used.
Leaf samples (7mm) were removed a t 24, 48, and 96 hours a fte r
in oculatio n; fix e d in 2.5% glutaraldehyde in 0.1 M phosphate buffer
pH 7 .0 ; and dehydrated in a graded ethanol s e ries .
Dehydrated le a f
pieces were gradually in f ilt r a t e d with liq u id Freon, c r it ic a l point
dired in a Bomar Model SPC-900 c r it ic a l point drying apparatus, and
coated with fold-pallad ium in a SPI S putter-Triode Coater.
An ETEC
Autoscan Scanning Electron Microscope was used fo r examining the
coated le a f pieces; p o sitiv e and negative images were recorded
simultaneously by means o f an attached Polaroid camera.
RESULTS
PRE- AND POST-INOCULATION DEW PERIODS
Results from the pre- and po st-inoculation dew.period experiments
(Tables 4 and 5) indicated th at a four hour p re-ino cu lation and a 48
hour po st-inoculation dew period allowed the expression o f a suitable
range of symptoms on c u ltiv a rs studied.
In th is study, and in work by
other researchers with d iffe r e n t organisms, both p re -inoculation and
po st-inoculation dew periods have an e ffe c t on disease expression
(22, 3 7 ).
The longer the dew period , p rim a rily p o st-in o cu latio n , the
greater the disease reaction on c u ltiv a rs o f a ll reaction types.
Results from extended dew periods are given in Table 6 .
Percent
necrosis increases in Fortuna and Newana during the f i r s t 24 hours of
the pre-in o cu latio n dew period.
In co n tra s t, the percent necrosis in
Manitou remains f a i r l y constant u n til a f t e r 48 hours o f the pre-inoc­
u latio n dew period is reached.
The po st-inoculation dew period is o f major importance in develop­
ment o f the disease.
iods there is l i t t l e
With short (24 hours) post-inoculation dew per­
disease development on re s is ta n t c u ltiv a r s , in
contrast to the disease development on susceptible lin e s .
When post­
inoculation dew periods were extended beyond 48 hours the re sista n t
c u ltiv a rs appeared more susceptible (necrosis greater than 50%).
Other researchers (22) have found th a t the most re s is ta n t c u ltiv a rs
continue to show resistance u n til a f t e r 96 hours o f dew chamber
35
Table 4.
C u ltiv a r
E ffe c t o f preinoculation dew periods on germination of
Septoria nodorum pycnidiospores on wheat c u ltiy a rs varying
in resistance.
Samples ] _ /
(hours a fte r
in oculatio n)
Germination percentages 2/
Hours o f preinoculation dew period
O
2
4
Fortune
4
12
24
8
14
20
7
22
45
20
47
57
Manitou
4
12
24
5
9
15
10
20
31
17
38
41
Cl 14032 .
4
12
24
8
10
23
10
17
25
21
40
39
9
27
33
20
31
43
BI ueboy
4
12
24
.
11
15
19
'
V
Based on two re p lic a tio n s of three leaves per re p lic a tio n .
2/
No s t a t is t ic a lly s ig n ific a n t differences between treatments
were found.
36
Table 5.
E ffe c t o f post-inoculation dew periods on germination of
Septoria nodorum pycnidiospores on wheat c u ltiv a rs varying
in resistance.
C iiltiv a r
Germination percentages I /
Hours o f post-inoculation dew period
24
48
72
Fortuna
BIueboy
Cl 14032
Manitou
51a
41b
35b
30c
60a
50ab
39bc
35c
. 64a
60a
48b
43b
I / Based on two re p lic a tio n s o f three leaves per re p lic a tio n
2 / Column numbers followed by d iffe r e n t le tte r s are s ig n ific a n tly
d iffe r e n t (Duncan's M u ltip le Range Test; P = 0.05)
37
Table 6 .
E ffe c t o f extended dew periods on expression of symptoms in
three wheat c u ltiv a rs varying in Septoria nodorum resistance.
Disease se ve rity (percent necrosis) ] / 2 /
C u ltiv a r
Hours o f p o st-inoculation dew period
24
48
72
96
Fortune
Newana
Manitou
55a
30b
IOc
75a
45b •
IOc
' 70a
60ab
50b
90a
90b
70 b
Disease s e ve rity (oercent necrosis)
(twelve hour p re -in o cu latio n dew period)
Hours of po st-inoculation dew period
24
48
72
96
Fortuna
Newana
Manitou
70a
50b
IOc
65a
70a
15b
90a
85a
55b
90a
90a
75 b
Disease s e v e rity (percent necrosis)
(tw enty-four hour pre-•inoculation dew period)
Hours of p o st-inoculation dew period
48
96
24
72
Fortune
Newana
Manitou
75a
55b
20c
90a
80a
80a
IOb
90a
.
55b
90a
90a
90a
Disease s e ve rity (percent necrosis)
(fo r ty -e ig h t hour pre-■inoculation dew period)
Hours of po st-inoculation dew period
96
48
72
24
Fortune
Newana
Manitou
75a
65a
45b
90a
80a
40b
90a
90a
75a
90a
90a
90a
I/
Based on percent necrosis 10 days a fte r in oculatio n.
Inoculum con­
centration o f 2 x 10? spores/ml with 10 mis of inoculum per pot of
10 plan ts.
2/
Based on two re p lic a tio n s of three leaves per re p lic a tio n .
3/
Column numbers followed by d iffe r e n t le tte r s are s ig n ific a n tly
d iffe r e n t.
(Duncan's M u ltip le Range Test; P = 0.05)
38
conditions a t which tim e.resistan ce s ta rts to break down (2 2 ).
The
f
e ffe c t o f longer dew periods may influence disease development by
providing I ) a more favorable environmental condition fo r maximum
germination and penetration by S. nodorum and 2) longer periods of
fre e moisture on leaves which places the plan t under a stress condi­
tio n .
This condition may lead to water-congestion and increased d is ­
ease development.
Resistance to _S. nodorum thus depends on environ­
mental conditions and the physiological condition o f the host during
inoculation and incubation.
SPORE GERMINATION AND PENETRATION
Spore germination re su lts are given in Table 7.
There are no
d ifferences 48 hours a f t e r inoculation in germination o f Sv nodorum
pycnidiospofes on le a f surfaces among the 18 c u ltiv a rs with the ex­
ceptions o f Fortuna and Manitou.
Germination of spores on Fortuna
was s ig n ific a n tly higher 48 hours a fte r inoculation than on other
c u ltiv a rs while germination on Manitou was s ig n ific a n tly lower.
Other researchers working with _S. nodorum ( 6 ) and other le a f spotting
fungi (23, 58) have not found differences in germination percentages
on le a f surfaces.
There were no differences among the 18 c u ltiv a rs in development
and morphology o f germination hyphae.
T y p ic a lly , a t 48 hours, the
spores would be germinated out of one or both ends (Fig I . ) although.
39
Table '7.
Germination o f Septoria nodorum pycnidiospores on le a f sur­
faces of 18 c u ltiv a rs of wheat varying in th e ir disease
resistance.
Germination percentages (hours a fte r in o cu latio n ) ] _ /
4
8
12
24
48
C u ltiv a r
2
Fortune
Manitou
Newana
Redhart
Red Chief
Cl 12373
Cl 14032
Oasis
Arthur 71
Centurk
Michigan Amber
Z en atiB o u tie lle
Hadden
Blueboy
Mt 7406
Mt 74394
CVLO 2204
Yamhill
5
2
4
5'
3
2
8
5
4
6
5
28
18
20
23
21
27
19
21
25
21
18
4
6
8
6
4
8
3
19
24
22
41
28
36
39
39
38
26
25
22
43
34
36
36
38
40
36
35
37
39
.
37
38
40
61
68a - /
38b
51c
44c
39b
40b
40 b
50
43
48c
49c
44c
77a
37c
54b
48b
48b
43b
43b
51b
47 b
53b
51b
41
44
48
49
42
39
48
40b
50c
50c
59c
54c
47c
51c
46b
57b
61b
58b
51b
50b.
59b
35
40
36
39
43
40
38
36
43c
I/
Based on three re p lic a tio n s o f three leaves per r e p lic a tio n .
2/
Column numbers followed by d iffe r e n t le tte r s are s ig n ific a n tly
d iffe r e n t (Duncan's M u ltip le Range Test; P = 0 .0 5 ).
\
40
Figure I .
Septoria nodorum pycnidiospores e xh ib itin g e arly germin­
ation patterns on inoculated wheat leaves. Spores are
ty p ic a lly germinating from one end. Bar represents 15
microns.
Figure 2.
Septoria nodorum pycnidiospore e x h ib itin g germination from
one end and from the side of the spore (arrow ). Bar rep­
resents nine microns.
41
germ tubes would occasionally a ris e from the side of the spore (Fig.
2 ).
The m ajo rity of spores lodged in depressions made by the jo in in g
of two epidermal c e lls and germ tubes extended along these depressions,
the germ tubes from spores located outside these depressions grew at
random over the le a f surface with occasional branching (F ig . 3 ).
Sm all, dark s ta in in g , enlarged areas o f the germination hyphae
were formed freq uently in the junctions between two epidermal c e lls .
These structures have been re fe rred to by other researchers as appressoria ( 6 , 86 ) .
Appressorial structures are formed in freq u en tly dur­
ing the f i r s t 24 hours, but were much in evidence by 48 hours a fte r
in o cu latio n .
There were no differences in appressorium formation
e ith e r in time o f in it ia t io n or to ta l numbers produced among the 18
c u ltiv a rs studied.
S im ilar to re su lts of Green and Dickson (23) there was frequent
evidence of germination hyphae extending beyond appressoria (F ig . 4 ).
Attempts were made to show th a t penetration took place via a penetra­
tion peg beneath these appressoria, but even in vaccum stained whole
mounts there was a lack of epidermal c e lls staining as described by
Baker and Smith ( 6 ) .
Since p o sitiv e hyphal penetration could not be
demonstrated i t was sought in the th in section staining experiments.
Penetration of stomata! openings occurred 72 hours a fte r inoculation
(F ig . 5 ).
This was s im ila r to work done by Green and Dickson with
S. p a s s erin ii (2 3 ).
42
Figure 3.
Branching of germination hypha of Septoria nodorum on
wheat le a f surface. Bar represents 50 microns.
Figure 4.
Germinating Septoria nodorum pycnidiospore showing the
formation of an appressorium in an epidermal crack (arrow)
and hypha extending beyond the appressorium. Bar
represents 25 microns.
43
Figure 5.
Stomata! penetration (arrow) by the hypha of Septoria
nodorum without the formation of an appressoriurn. Bar
represents ten microns.
44
WHOLE LEAF MOUNTS
Whole le a f samples o f Fortuna 9 Newana9 and Manitou were examined.using a v a rie ty o f staining procedures ( 6 , 12, 54, 6 0 ).
Samples were
removed a t 24 hour in te rv a ls u n til 144 hours a fte r inoculation to
study fungal development w ithin le a f tis s u e .
were modified by u t iliz in g
The stain in g procedures
vacuum i n f i l t r a t i o n , longer s tain in g times,
or stronger solutions in order to obtain stained fungal tissue w ithin
cleared le a f segments.
A ll such procedures and th e ir m odifications
gave negative re su lts and the only fungal structures th a t stained were
the spores and germination hyphae on the le a f surface.
A technique described using C a lc o fluor, an o p tic al brightner
(6 5 ), was also attempted.
The spores on the le a f surface fluoresced
weakly, but were often obscured by the fluorescence o f the le a f mat­
e r ia l i t s e l f .
Attempts made to combine the C alcofluor with standard
staining procedures in order to quench the fluorescence o f the le a f
m aterial allowed fo r a greater d iffe r e n tia tio n of fungal hyphae, but
only surface fungal structures were v is ib le .
Development o f disease
progression therefo re had to be monitored by th in -s e c tio n h isto lo g ic al
examination ra th e r than on cleared whole le a f mounts.
VOLATILE INHIBITORS
There were no apparent v o la t ile pre-formed or inducible in h ib ito rs
present in the wheat tissu es.
The m ycelial growth of
nodorum and
subsequent pycnidia formation were not v isab ly a ffected in the area
45
immediately opposite the leaves.
There was no change in growth
patterns or to ta l amount o f growth o f
nodorum on the sealed
plates th a t contained leaves as opposed to those plates th a t d id n 't
contain leaves.
Other workers have reported antifungal compounds
in wheat to be a c tiv e against S>. nodorum,
but these compounds were
I ) in minute q u a n titie s , 2 ) not correlated with re s is ta n t and suscep­
t ib le v a rie tie s and/or 3) not present in mature or f ie ld grown plants .
(.5, 6 ).
HISTOLOGICAL
Disease development was s im ila r among the eight c u ltiv a rs studied,
although there were occasional h isto lo g ic al d ifferen ces.
For ease of
description the h is to lo g ic a l reactions w ill be grouped in to suscep­
t i b l e , in term ediate, and re s is ta n t classes.
The only reaction d i f f ­
erence between w inter and spring wheat types was a m atter o f degree
o f in fe c tio n rath er than a d ifferen ce in s p e c ific defense reactions.
R esistant, interm ediate and susceptible w inter wheat c u ltiv a rs typ­
ic a lly had less in fe c tio n than the respective spring wheat lin e s under
the same environmental conditions and inoculum concentrations.
12 HOURS
Twelve hours a fte r inoculation none of the c u ltiv a rs showed e v i­
dence o f reaction to the presence o f the pathogen oh the surface.
Occasional spores were noticed on the surface, but no penetration
46
structures were seen.
There were no differences between the healthy
checks and inoculated controls in c e ll staining reactions and c e llu la r
in te g r ity .
In the s a fra n in -fa s t green stainin g method the c e ll w a lls ,
chlo ro plasts, and cytoplasm stained with fa s t green, w hile the fungal
spores and the vessel element walls stained with safran in .
No c e ll­
u la r changes were noted in tissue stained with Johnsen1s Quadruple
and Conant's Quadruple staining procedures.
24 HOURS
Twenty-four hours a f t e r inoculation there was l i t t l e
c e llu la r morphology among the wheat lin e s .
change in
No apparent penetration
structures were noted on any of the slid es examined, athough there
was an occasional c e llu la r reaction in the susceptible lin e s .
The
c e ll w alls of the mesophyll c e lls lin in g some of the substomatal
c a v itie s stained, a dark blue (F ig . 6 ) .
u la r appearance in .th e s e c e lls .
This was accompanied by a gran­
This reaction occurred only in
Michigan Amber, Centurk, and Fortuna.
Uninoculated controls and inoc­
ulated plants showed th is reaction in d ic a tin g th a t i t was due to an
environm entally induced physiological response, rath er than a disease
reactio n .
In the other wheat c u ltiv a rs there was no evidence of any
type of c e llu la r reaction to e ith e r the pathogen or the environment.
- 48 HOURS
In susceptible c u ltiv a rs there was an increase in the disruption
47
Figure 6.
Photomicrographs of the i n i t i a l stage of disease
development in a susceptible wheat c u ltiv a r . Mesophyll c e lls lin in g the substomatal cavity show
typ ical accumulation of blue granulation. Reaction
occurred 24 hours a fte r in oculatio n. Bar represents
30 microns.
48
of c e lls lin in g substomataI c a v itie s .
There was more granulation and
increasing evidence o f c e llu la r collapse (F ig . 7 ).
MesophylI c e lls
were affected and epidermal c e lls did not appear to be affected a t th is
stage.
( 6 , 8 5 ).
This is consistent with re su lts reported by other researchers
There was no evidence o f c e llu la r disruption in areas away
from the substomata l c a v itie s and no hyphae could be discerned w ithin
the a ffected tis s u e .
the sections examined.
No penetration structures were evident in any of
The healthy controls of the susceptible class,
although s t i l l showing s lig h t c e llu la r disruption did not have the
same degree o f reaction as did the inoculated plants.
There was a
granulation o f the mesophyll c e lls surrounding the substomatal c a v ity ;
to a lesser extent in the healthy controls and to a greater extent in
the inoculated plants.
There was no v is ib le reaction to the disease
or environment in the re s is ta n t class o f c u ltiv a rs .
Although spores
and germination tubes could be seen on the surface there was no e v i­
dence o f penetration in the interm ediate and re s is ta n t classes.
72 HOURS
By 72 hours a fte r inoculation there was considerable damage in
the susceptible class o f wheat c u ltiv a rs ;
In addition to the granu­
la tio n o f the mesophyll c e lls there was also c e llu la r collapse (F ig . 8 )
the presence of in t e r - and in t r a - c e llu la r hyphae (F ig . 9 ) , and a large
amount o f safranin staining m aterial deposited on the c e ll walls (F ig .
49
Figure 7.
Figure 8.
Photomicrograph of dis­
ease development in a
susceptible c u ltiv a r 72
hours a fte r inoculation.
In ternal c e ll destruc­
tio n has occurred. Bar
represents 30 microns.
8
Photomicrograph of in ­
creasing disease develop­
ment in a susceptible c u lt iv a r . Cells beneath
granulated c e lls (arrow)
are beginning to show d is ­
ease e ffe c ts . Bar repre­
sents 15 microns.
50
Figure 9.
Photomicrograph of in te r - and in t r a - c e llu la r hyphae
(arrows) of Septoria nodorum w ithin a wheat le a f. Sus­
c ep tib le c u ltiv a r of wheat with hyphae both w ithin and
outside of lesion area. Bar represents 30 microns.
'51
10) and in the in te r c e llu la r spaces (F ig . 11).
The hyphae could only
be seen c le a rly in tissues where c e llu la r disruption was minimal.
Hyphae o f :S. nodorum stained p rim a rily with safranin and the safranin
staining m aterial in conjunction with the c e ll walls and in the in te r ­
c e llu la r spaces often masked the presence o f the hyphae.
This masking
occurred wherever there was moderate c e llu la r d is in te g ra tio n .
In
addition to the involvement of the mesophyll c e lls , the epidermal c e lls
were affected and occasionally lesions extended through the e n tire le a f
to the opposite side (F ig . 12).
Since s e ria l sections were used
throughout th is study, i t was possible to follow the lesions in the
long axis of the le a f .
ded from stoma to stoma.
The lesions
p a ra lle l to the le a f v e in , exten­
The lesions began to extend la t e r a lly at
th is stage, but not to the degree of lo ngitu din al spread.
No penetra­
tio n stuctures in d icatin g d ire c t penetration of the c u tic le were
seen.
However, stomata! penetration was observed (F ig . 13) as well as
ocassional penetration o f le a f trichomes (F ig . 14).
The in term ed iate,
reactions in te n s ifie d and a t 48 and 72 hours a fte r inoculation resem­
bled the susceptible reaction a t 24 and 48 hours re sp ec tiv e ly .
The
re s is ta n t class started to show a reaction to the pathogen a t th is
tim e.
The reaction was confined to the c e lls lin in g the substomataI .
c a v ity , but in the re s is ta n t reaction there was no granulation o f the
c e lls and the reaction was ty p ifie d by an accumulation of a safranin
52
Figure 10.
Photomicrograph of safranin-stained m aterial accumulated
on mesophyll c e ll walls (arrow) of a wheat le a f in res­
ponse to invasion by Septoria nodorum. Bar represents
25 microns.
Figure 11.
Photomicrograph of safranin-stained m aterial accumulated
in the in te r c e llu la r spaces of a wheat le a f in response
to invasion by Septoria nodorum. Bar represents 20
microns.
53
Figure 12.
Photomicrographs o f le s io n s on a s u s c e p tib le wheat c u l t i v a r extending to the opposite epidermal s u r fa c e .
Large
amounts o f the blue g r a n u la t io n r e a c tio n and s a f r a n i n s ta in e d m a t e r ia l are p r e s e n t.
Bar re p res e n ts 30 microns.
54
Figure 13.
Photomicrograph of stomataI penetration by a germinating
pycnidiospore of Septoria nodorum. Bar represents
6 microns.
55
Figure 14.
Photomicrograph of trichome penetration (arrow) by a
germinating pycnidiospore of Septoria nodorum. Bar
represents 10 microns.
56
stainin g substance deposited on the c e ll w a lls .
No hyphae could be
seen in the re s is ta n t class of wheat c u ltiv a rs e ith e r in the affected
area or in adjacent c e lls .
There was a lack of the blue staining
m aterial in the re s is ta n t reaction a t th is stage. .
96 HOURS
N in e ty -s ix hours a f t e r inoculation the reaction in a ll three
classes o f wheat in te n s ifie d .
In the susceptible class lesions exten­
ded through the width of the le a f and had moved la t e r a lly to involve
a ll the tissue between vascular bundles.
The lesions were normally
confined to the areas bwtween the vascular bundles (F ig . 1 5 ), although
th is phenomenon depended on the size of the bundle.
I f bundles were
small and sclerenchyma tissue was minimal the pathogen seemed able to
spread around the vascular bundle and in to the next in te rv e in a l area
(F ig . 16).
However, i f the sclerenchyma tissue was well defined there
was no movement by the pathogen around the bundle.
This is in agree­
ment with Green and Dickson's findings in th e ir work w ith
(2 3 ).
passerinii
C e llu la r collapse a t th is ,s ta g e included the epidermal c e lls
(F ig . 17) and there was a greater deposition of the safranin stainedm aterial in and around the c e lls .
The interm ediate reaction also in ­
creased in in te n s ity and a fte r 96 hours no discernable differences
in the appearance of in divid ual lesions between the interm ediate class
and the susceptible class could be determined.
There was a visual
57
Figure 15.
Figure 16.
Photomicrograph of
lesion caused by
Septoria nodorum
th a t is beginning
to surround a
small vascular
bundle. Bar rep­
resents 25 microns.
Photomicrograph of
a ty p ic a l Septoria
nodorum lesion con­
tained by a vascu­
la r bundle. Bar
represents 35
microns.
58
Figure 17.
Figure 18.
Photomicrograph show­
ing to ta l c e llu la r co­
llapse in a susceptible
wheat c u ltiv a r . Bar
represents 12 microns.
Photomicrograph showing
to ta l c e llu la r collapse
in a re s is ta n t c u ltiv a r .
Bar represents 50
mi crons.
1 8
•w
59
d iffe re n c e in the to ta l amount of le a f tissue affected between the
two classes although th is was not q u a n tifie d .
The re s is ta n t class
also reacted more in ten sely to the pathogen although c e llu la r granula­
tio n o f the mesophyll c e lls was not apparent.
S a fran in -p o sitiv e
m aterials around c e ll w alls accumulated to s ig n ific a n t le v e ls .
The
percentage of tissue involved in the re s is ta n t class was le s s , a l ­
though in divid ual lesions could progress to to ta l c e ll collapse as in
the susceptible reaction (F ig . 1 8 ). .
120 HOURS
By 120 hours a f t e r inoculation there was T i t t l e i f any difference
in appearance of in divid ual lesions in a ll three classes of c u ltiv a rs .
A ll showed densely stained areas of c e llu la r d is in te g ra tio n and d is ­
ruption.
In divid ual hyphae could not be distinguished w ith in the
areas o f c e ll collapse.
The differences between the three classes of
wheat c u ltiv a rs were in the percentage of le a f tissue involved.
Formation o f pycnidia was the same in the various c u ltiv a rs .
Hyphae accumulated in the area below the substomatal c a v ity and even­
tu a lly gave ris e to pycnidia (F ig . 1 9 ).
The formation and enlargement
o f the pycnidia! mass caused fu rth e r c e ll disruption and d is in te g ra ­
tio n from pressure applied to adjacent c e lls .
Tissue stained with the s a fra n in -fa s t green stainin g method
fluoresced with the e p i-flu o re s c e n t equipment previously mentioned
60
Figure 19.
Photomicrograph showing the morphology and o rie n ta tio n
of Septoria nodorum pycnidia (arrows) in a suscep­
t ib le wheat c u ltiv a r . Bar represents 150 microns
in Figure 19a and 50 microns in 19b.
61
(standard f i l t e r s e t).
The epidermal c e ll walls fluoresced a dark
purple c o lo r, chloroplasts a dull red, vessel element walls a bright
ye llo w -red , fungal structures brig h t red, and c e lls in the immediate
proxim ity of fungal tissue were w hite.
C ells ringed with a w hitish
fluorescence could be observed before any evidence o f plan t response
with a normal lig h t source (F ig . 20 and 2 1 ).
White fluorescence
often appeared between two epidermal c e lls (F ig . 22) in d ir e c tly
cating pathogen penetration a t th is p o in t.
in d i­
Examination of these areas
under o il immersion and phase contrast did not in dicate definable
fungal s tru ctu res.
As lesions enlarged, surrounding adjacent c e lls
were ringed with a white fluorescence although they appeared healthy
under normal lig h tin g .
The f i r s t c e lls to show evidence of a reaction
were the mesophyll c e lls lin in g the substomata l c a v ity .
Uninoculated
susceptible controls also evidenced a fluorescence in the substomatal
c a v itie s , but not to the extent of the inoculated plan ts.
This reac­
tio n did not occur in the re s is ta n t plants.
SECONDARY SPORE PRODUCTION
The re su lts of the secondary spore production experiments are
given in Table 8 .
There was a s ig n ific a n t reduction in the number
and size o f pycnidia formed in the re s is ta n t le a f m a te ria l.
As a con­
sequence of the size reduction there was also a concurrent reduction in
the number of spores/pycnidium.
In divid ual pycnidiospores sizes
)
62
Figure 20.
Photomicrograph of an inoculated susceptible wheat cult iv a r showing no evidence of pathogen invasion (arrow)
Bar represents 25 microns.
Figure 21.
Fluorescent photomicrograph of the le a f tissue in
Figure 20 showing evidence of pathogen invasion (arrow ).
Bar represents 25 microns.
63
Figure 22.
Fluorescent photomicrograph showing fluorescence between
epidermal c e lls walls (arrow) in dicating pathogen pene­
tr a tio n . Bar represents 25 microns.
64
Table O'.
C u ltiv a r
Fortuna
Newana
Manitou
Secondary spore production in three c u ltiy a rs . of wheat
varying in disease resistance.
Average number
o f pycnidia
produced/cnr I /
41a 3 /
30ab
24b
Average size 2 /
of pycnidia
204 x 163 u
179 x 140 u
171 x 134 u
Average number of
pycnidiospores/
pycnidium x IO3 I /
6 . 1a
4.2ab
2 . 2b
]_/
Based on three subsamples with two re p lic a tio n s .
2/
Based on 50 dry pycnidia chosen.at random from three subsamples
with two re p lic a tio n s .
3/
Column numbers followed by d iffe r e n t le t t e r are s ig n ific a n tly
d iffe r e n t (Duncan's M u ltip le Range Test; P = 0 . 0 5 ) .
65
remained the same, regardless o f the disease.reaction o f the c u ltiv a r . .
Although, not q u a n tifie d , there was a trend fo r pycnidia to in it ia t e
formation sooner (1-2 days) in the re s is ta n t p lan t.
These results
agree with work by Gough (24) and Eyal (21) in studying the e ffec ts of
host resistance on pycnidia and pycnidiospore formation by S. t r i t i c i .
Gough (24) found th a t between 60-80% o f to ta l pycnidiospores w ithin a
pycnidium are released w ithin the f i r s t wetness period and a lte rn a te
drying and rehydration periods w ill cause the release o f more spores.
Some o f the spores are never discharged from the pycnidium.
pycnidia of
T e rtia ry
nodorum are formed with a lte rn a tin g periods o f wetness
and dryness.
I t has been estimated th a t a minimum of 25,000 spores/cm^
o f le a f tissue may be released (7 2 ).
STOMATA
Number o f stomata varied extensively w ithin and between c u lt i vars and th erefo re could not be correlated with c u ltiv a r disease
reactio n .
The numbers o f stomata ranged from 3000/cm
to 5400/cm .
The susceptible c u ltiv a r appeared to have a greater percentage of
open stomata a t the end of 48 hours in the mist chamber, but the num­
ber was not s ig n ific a n tly d iffe r e n t from re s is ta n t c u ltiv a rs (Table
9 ).
N in e ty -s ix percent o f the stomata were open in the susceptible
c u ltiv a r as opposed to 92% and 93% in the interm ediate and re s is ta n t
c u ltiv a rs re sp ec tiv e ly .
66
Table 9.
C u ltiv a r
Numbers o f stomata on adaxial le a f surfaces o f three
c u ltiv a rs o f ten day old wheat seedlings grown under
id e n tic a l environments.
Stomata/cm^ I /
Fortuna
Newana
Manitou
4800 2 /
3000
5400
Percent stomata open
24 hours
48 hours
95
93
93
I / Based on two re p lic a tio n s .
2 / No s ig n ific a n t differences were found among c u ltiv a rs .
96
92
93
. WATER CONGESTION
Water congestion is defined by Johnson (.42) as "the accumulation
o f excessive water in the in te r c e llu la r spaces as the r e s u lt of in ­
tern al water pressures."
This water congestion may be induced by
c e rta in mist chamber conditions or may occur n a tu ra lly under f ie ld
conditions (excessive periods of high r e la tiv e humidity accompanied
by co o l, cloudy days).
with wind-blown ra in .
Water congestion may also occur in conjunction
This type o f water congestion may be accompanied
by wounding o f the epidermal layer of c e lls which f a c ilit a t e s invasion
by pathogens.
Differences in water congestion among the c u ltiv a rs
a fte r 48 hours in the m ist chamber were apparent (F ig . 23 and 24).
The degree o f water congestion was greater in the susceptible c u ltiv a rs
than in the re s is ta n t c u ltiv a r s .
%. timopheevi appeared e s s e n tia lly
immune to water congestion under the same conditions (F ig . 25).
Water
congestion i n i t i a l l y occured in the in te r c e llu la r spaces o f the c e lls
surrounding the substomatal c a v ity , but ra p id ly spread along the le a f
axis in the areas between the vascular bundles.
When plants were re ­
moved from the mist chamber most o f the water congestion disappeared
w ithin six hours.
In uninoculated Fortune, however, there was some
c e llu la r damage in the substomatal c a vity area.
This was evidenced by
a browning reaction in these c e lls a f t e r all I water congestion had d is ­
appeared.
Inoculated plants showed more c e llu la r disruption and small
68
Figure 23.
Photomicrograph showing water congestion in a susceptible
c u ltiv a r of wheat a fte r 48 hours in the mist chamber. 231 a non-stained le a f photographed with transm itted lig h t
(c le a r areas are water congested). 23-2 a le a f treated
with rose bengal dye (dark areas are water congested).
69
Figure 24.
Photomicrograph showing water congestion in a re sista n t
c u ltiv a r a fte r 48 hours in the mist chamber. 24-1 a nonstained le a f photographed with transm itted lig h t (clear
areas are water congested). 24-2 a le a f treated with
rose bengal (dark areas are water congested).
70
Figure 25.
Photomicrograph showing water congestion in a very re sis ­
tan t c u ltiv a r of wheat ( I . timopheevi) a fte r 48 hours in
the mist chamber. The no n -stained le a f was photographed
with transm itted lig h t (c le a r areas are water congested).
71
necrotic areas a t th a t tim e.
The re s is ta n t plants and T. tiriiopheevi
evidenced no reaction to water congestion on a c e llu la r . le v e l.
Leaves
th a t were pulled through the thumb and fo re fin g e r dipped in water or
chloroform had increased incidence o f disease when compared to controls
(measured as.percent necrosis) (Table 10).
The chloroform treated
leaves were more severely affected than water treated ones.
A p o r -.*.•
tio n o f the increase in incidence o f disease may be a ttrib u te d to
mechanical wounding o f the epidermal la y e r allowing fo r a greater in ­
vasion by Sy nodorum.
The treated plants also had a greater degree of
water congestion than controls e s p e cia lly in the case o f T. timopheevi.
This increase in water congestion may also be due to the wounding or
removal of epidermal h a ir s .. T. timopheevi has a large number of long
trichomes which e f f ic ie n t ly keeps drop o f dew (or spore suspensions)
o ff the surface of the le a f .
When the surface hairs o f T. timopheevi
are removed mechanically an increased number of.w ater drops or spore
suspension drops re s t on the surface.
This increases the incidence
o f both water congestion and pathogen invasion.
The chloroform
treatm ent may increase both water congestion and in fe c tio n in the
c u ltiv a rs studied by dissolving portions o f the c u tic le and removing
some the plants natural defense against water absorbtion.
SCANNING ELECTRON MICROSCOPE
Scanning electron micrographs of Sy nodorum on the le a f surface
.
72
Table 10.
E ffe c t of disruption of le a f surface stru ctu re on ex­
pression o f disease resistance to Septoria nodorum.
C u ltiv a r
Fortuna
Newana
Manitou
Frondoso
Klein Toledo
T. timopheevi
y
Disease s e ve rity (as percent necrosis) I / 2/
Water
Chloroform .
Treatment
Control
Treatment
70
40
10
5
O
O
90
70
60
60
70
40
90
90
70
. 70
80
50
Based on percent necrosis 10 days a fte r in ocu latio n .
2 / Based on three re p lic a tio n of two leaves per re p lic a tio n .
73
showed the presence o f various penetration structures:
penetration pegs, and in fe c tio n hyphae.
ia b le in morphology (F ig . 26 and 2 7 ).
appressoria,
Appressoria were highly var­
The morphology seemed to be
correlated with the position o f the appressorium.
I f the appressorium
was in the center o f an epidermal c e ll i t tended to be rounded and dis
t in c t while i f i t occurred in the epidermal cracks or stomata! open­
ings i t was more irre g u la r and had many "pods" or projections assoc­
iated with i t .
Penetration pegs arose from appressoria, d ir e c tly
from a spore, or from the germination hyphae (F ig . 26 and 2 8 ).
Pene­
tra tio n pegs were approximately 1/10 the size o f appressoria (3-5 u)
and 1/5 the size of a germination hypha (2 u ) .
In fe c tio n hyphae
were observed emerging d ir e c tly from the spore (F ig . 29) or from the
base o f an appressorium (F ig . 30).
These in fe c tio n hyphae were approx
im ately 1/5 o f the size o f the normal germination hyphae (2 u ) .
Within the le a f tissue hyphae could be distinguished.
N inety-six
hours a fte r in oculation the hyphae w ith in lesions in re s is ta n t c u ltiv a rs started to d is in te g ra te (F ig . 31) while the hyphae w ithin a
susceptible c u ltiv a r remained in ta c t (F ig . 3 2 ).
d iffe re n c e noted between
This was the only
nodorum in fe c tio n on re s is ta n t and suscep­
t ib le c u ltiv a rs by scanning electron microscope examination.
74
Figure 26.
Scanning electron micrograph of a germinating pycnidiospore of Septoria nodorum with a penetration peg emerging
d ire c tly from the spore (arrow I ) , a podical appressorium
(arrow 2 ) , and another penetration peg (arrow 3 ). Bar
represents 1.0 micron.
75
Figure 27.
Scanning e le c t r o n micrograph o f a germ inating p y c n id io spore o f S e p to ria nodorum showing a t y p i c a l germ ination
hypha (emerging from the s id e ) and a rounded d i s t i n c t
appressorium ( a r r o w ).
Bar re p re s e n ts 1 .0 m icron.
76
Figure 28.
Scanning e le c t r o n micrograph o f a p e n e tr a tio n peg (arrow )
o f S e p t o r i a nodorum emerging from the side o f a germina­
t i o n hypha.
Bar re p re s e n ts 1 .0 micron.
77
Figure 29.
Scanning e le c t r o n micrograph o f an i n f e c t i o n hypha o f
S e p to ria nodorum emerging d i r e c t l y from a pycnidiospore
( a r r o w ) . Bar re p res e n ts 1 .0 micron.
78
Figure 30.
Scanning electron micrograph of a germination hypha
(arrow I ) of Septoria nodorum growing around a wheat
le a f trichome. An appressorium has formed and an in ­
fectio n hypha has emerged from it s base (arrow 2 ).
Bar represents 10.0 microns.
79
Figure 31.
Scanning electron micrograph of a d isin te g ra tin g Septoria
nodorum hypha (arrow) in a re s is ta n t c u ltiv a r of wheat
96 hours a fte r in oculation. Hyphae are located w ithin
a lesion area. Bar represents 10 microns.
80
Figure 32.
Scanning e le c t r o n micrograph o f an i n t a c t S e p to ria
nodorum hypha (a rro w ) in a s u s c e p tib le c u l t i v a r o f wheat
96 hours a f t e r i n o c u l a t i o n .
Hyphae are lo c a te d w i t h i n
a le s io n a r e a . Bar re p re s e n ts 1 .0 micron.
DISCUSSION
S ig n ific a n t differences in germination o f pycnidiospores
occurred on leaves o f Fortuna and Manitou.
No explanation fo r th is
reaction was observed, but the possible presence o f v o la t ile fungal
spore germination in h ib ito rs was studied.
No v o la tile in h ib ito rs or
stim ulators were detected w ith the procedure used as spore germination
and mycelial growth were not affected by close proxim ity to le a f
m a te ria l.
Germination hyphae development and formation o f appressoria
were not affected by disease reaction o f the c u ltiv a r .
Spores f r e -
quently lodged in cracks between adjacent epidermal c e ll w alls with
th e ir hyphae extending lo n g itu d in a lly along these cracks.
Hyphae
branched on le a f surfaces in contrast to Weber's findings ( 86 ).
This
branching occurred most freq uently when the germination hyphae did not
l i e in epidermal cracks.
Twenty-four hours a fte r in o c u la tio n , appressoria! structures
were seen in a ll c u ltiv a rs .
Formation o f appressoria! structures
could not be correlated with any s p e c ific anatomical features of the
le a f surface, although they occurred most frequently in the epidermal
cracks.
Frequently hyphae extended beyond these structures and there
was occasional branching a t the appressoria! structure i t s e l f .
Evi­
dence fo r epidermal c e ll penetration under these appressoria! struc­
tures although lacking with conventional lig h t microscope examinations,
82
was confirmed with scanning electron microscope examinations.
Forty-
eigh t hours a f t e r inoculation there was no fu rth e r increase in the
formation of appressoria beyond what had been present a t 24 hours.
Stomata! penetration was re a d ily observed 72 hours a f t e r in oculation.
Stomata were penetrated e ith e r in the open or closed s ta te with or
without the formation of appressoria, as also seen by Narrower (2 6 ).
One of the resistance mechanisms possessed by the c u ltiv a r
Manitou is a s ig n ific a n t reduction in spore germination and penetra­
tio n of leaves.
A ll other c u ltiv a r s , whether re s is ta n t or susceptible,
did not e x h ib it reduction in spore germination on the le a f surface.
Scanning electron micrographs demonstrated the presence of var­
ious penetration and in fe c tio n stru ctu res. ■ Penetration pegs arose
from germination hyphae, appressoria, and d ire c tly from the spore i t ­
s e lf.
In addition to the penetration pegs, the presence o f in fec tio n
hyphae was observed.
These hyphae, which had been assumed to occur,
had not previously been photographed due to th e ir small s iz e .
These
hyphae, approximately 1/5 the size of normal germination hyphae and
1/10 the size of an appressorium, are reported to enter between two
epidermal c e lls .
A fte r p e n e tra tio n 'o f the host tissue they enlarge .
to approximately 2 u in width ( 86 ) .
In fe c tio n hyphae were observed
o rig in a tin g from appressoria or d ire c tly from the spore i t s e l f .
The
in fe c tio n hyphae could not be seen with conventional lig h t microscope
83
techniques due
to the small size and "masking" by adjacent c e llu la r
stainin g reactions.
In fec tio n hyphae were seen by Nomarsky in t e r f e r -
ence-constrast op tic examinations although they were not s u ffic ie n tly
c le a r to permit photographs.
Wherever S .. nodorura..penetrated the le a f
tissue there was a fluorescent reaction in adjacent c e lls .
Scanning
electron microscope observations of fungal penetration structures and
epidermal holes showed th a t d ire c t c u tic le penetration occurred most ■
frequently 24 to 48 hours a fte r in o c u la tio n .
D ire ct c u tic le penetra­
tio n was fu rth e r substantiated by the observation of in fe c tio n hyphae
with Nomarsky in te rfe re n c e -c o n tra s t o p tic
examination and typ ical
c e llu la r staining and fluroescent reactions in c e lls th a t were in d ir ­
ect contact with Sv nodorum hyphae.
Hyphae were also seen w ithin the
le a f tissue p rio r to observation o f stomata! penetration.
Once the pathogen was inside the le a f tis s u e , the progression of
events in formation of in d ivid u al lesions was s im ila r in a ll c u ltiv a rs
The major d iffe re n c e between a susceptible and a re s is ta n t.re a c tio n ,
as noted in the h is to lo g ic a l s ta in in g , was a blue granulation in c e lls
of the susceptible host in addition to the safranin stainin g reaction
of the c e lls walls th a t occurred in both reaction types,
the suscep­
t ib le plan t often apeared to have less safranin stained m aterial and
more o f the blue granulation.
The safranin stained m aterial was ass­
ociated w ith lig n if ie d and suberized c e ll walls and was assumed to be
phenolic in character.
Certain phenols function in the formation of
84
o f c e ll p ro te c tiv e tissue ( lig n in , suberin, e tc ) (6 7 ).
tissues are a b a rrie r to the spread of most fu n g i.
Suberized
The blue granu­
la tio n appeared to be a clumping of the c e llu la r contents and an
accumulation of c e llu lo s e ra th e r than an elaboration of chemical com­
pounds.
Hyphae could be distinguished in a ll c u ltiv a r classes, a l­
though in the re s is ta n t host no hyphae could be found outside the le s ­
ion area i t s e l f .
Hyphae could be found freq uently in advance of the
lesion area in susceptible c u ltiv a rs and to a moderate extent in the
interm ediate classes.
Under scanning electron microscope examination,
hyphae in the re s is ta n t c u ltiv a r began to show d is in te g ra tio n w ithin
the lesion area 96 hours a fte r in o cu latio n .
c u ltiv a r did not d is in te g ra te .
Hyphae in the susceptible
Th is, evidence,
plus the staining
reactio ns, indicates th a t there may be a chemical defense mechanism
in the re s is ta n t c u ltiv a rs studied which is detrim ental to S_. nodorum
and r e s tr ic ts the fungus to more defined lesion areas.
The individual
lesions a ll begin the the c e lls lin in g the substomatal c a v itie s and
progress inward through the le a f to the other epidermal surface.
tu a lly the e n tire area between vascular bundles collapses.
Even
C hlorotic
areas, perhaps due to toxin a c t iv it y , did extend from one in terv e in a l
area to the next.
Previous researchers have reported the presence of
non-specific toxin production by S. nodorum (9 , 4 8 ).
The toxin may .
function in disease development or in a ffe c tin g host c e llu la r defense,
85
but the actual ro le of th is toxin has not been determined.
Under nor­
mal conditions lesions progressed lo n g itu d in a lly along the in te rv e in a l
area.
Several adjacent lesion areas often combined to resemble one
le s io n , but most freq uently there was no evidence of hyphae surround­
ing the vascular bundle.
Formation of pycnidia was in it ia t e d w ith in 25 days o f inoculation
under the experimental conditions.
There were no morphological d i f f ­
erences between c u ltiv a rs in pycnidia development.
Hyphae accumulated
in the substomatal area, followed by development of a pycnidium.
Pyc­
n id ia were oriented with th e ir o stio les pointing toward the stomata!
opening.
Secondary spore production from these pycnidia was shown to
be highly correlated with disease resistance.
The re s is ta n t c u ltiv a rs
had s ig n ific a n tly fewer pycnidia and pycnidiospores which would g re a tly
influence the secondary spread of the pathogen under usual f ie ld con­
d itio n s .
The re s is ta n t c u ltiv a rs are able not only to r e s t r ic t the
extent of lesion development, but also are able to r e s t r ic t the quan­
t i t y o f secondary inoculum.
Resistance to water congestion of leaves was correlated with dis­
ease s u s c e p tib ility or resistance.
C ultivars that water congested
e a s ily also were susceptible to in fe c tio n by Sl. nodorum w hile c u lt i­
vars re s is ta n t to water congestion also were disease re s is ta n t.
the surface c u tic le la y e r o f re s is ta n t c u ltiv a rs was. mechanically
When
86
disrupted in order to increase th e ir s u s c e p tib ility to water conges­
tio n , th e ir s u s c e p tib ility to the disease also was increased.
When
non-treated re s is ta n t plants were l e f t in the mist chamber fo r ex­
tended periods o f time both the degree o f water congestion and disease
s e v e rity increased.
Waiter congestion has been im plicated as a pre­
disposing fa c to r in other diseases (27, 4 2 ).
A number o f le a f-s p o ttin g diseases o f wheat depend on the length
o f post-inoculation dew periods fo r in fe c tio n (35, 36, 37, 38).
Heggestad (27) has shown th a t there is a v a rie ta l response to degree
o f water congestion arid th a t breeding fo r increased resistance to wa­
te r congestion is fe a s ib le .
A portion o f the resistance o f wheat c u l-
tiv a rs to in fe c tio n by Sv nodorum may be due to th e ir inherent a b ilit y
to withstand water congestion.
The presence of free in te r c e llu la r wa­
te r w ithin the host tissue may a lt e r the metabolism o f the host suf­
f ic ie n t ly to allow successful colonization by the pathogen.
Further­
more, when an infected susceptible plant is placed in an atmosphere not
conducive to the maintenance of fre e in te r c e llu la r water the progress­
ion of the disease stops (2 2 ).
When the same plant is placed under
conditions which allow fo r some free in te r c e llu la r water (high r e la ­
tiv e humidity) the pathogen w ill s ta r t to spread again.
The presence,
th e re fo re , o f fre e in te r c e llu la r water plays.an important ro le in the
host's response to invasion and colo nizatio n by Sv nodorum.
Since
87
v a rie ta l differences in s u s c e p tib lity to water congestion are known
i t seems lik e ly th a t a t le a s t a portion o f resistance to
is due to resistance to water congestion.
nodorum
Free in te r c e llu la r water
may influence disease development in a number o f d iffe r e n t ways.
In the re s is ta n t c u ltiv a rs d is in te g ra tio n o f hyphae occurs s t a r t ­
ing approximately 96 hours a fte r in o c u la tio n .
the susceptible c u ltiv a r s .
This does not occur in
This may be due to the accumulation of
plan t produced substances such as phenols or other antifungal com­
pounds.
I t is impossible to sta te what these might be, i f present,
since they were not s p e c ific a lly analyzed.
Water congestion may play
a ro le here as any antifungal substance produced by stressed c e lls
may be d ilu te d away in the susceptible c u ltiv a r so th a t they are not
as e ffe c tiv e as in the re s is ta n t c u ltiv a r where they remain concen­
trate d in a lo c a lize d area and are able to in h ib it fungal growth.
Water congestion may also p lay 'a d ire c t
ro le in stressing host
plan t c e lls thus weakening them which allows fo r increased pathogen
c o lo n iza tio n .
Septoria nodorum is a very e f f ic ie n t colonizer of weak,
dying, or dead tis s u e , but does not appear to be able to invade heal­
th y , a c tiv e ly m etabolizing
tissue to any s ig n ific a n t level ( 86 ).
Water congestion would change osmotic conditions w ithin the plant
allowing fo r the leaching of m etabolites and ions out of the affected
c e lls and in h ib itin g those reactions which require aerobic conditions.
88
Water congestion e s s e n tia lly places the environment w ith in the le a f
tissue in an anaerobic s ta te which would g re a tly a ffe c t the metabolic
a c t iv it ie s o f associated c e lls .
These stressed c e lls would thus be
more e f f ic ie n t ly colonized by the pathogen.
In addition to the stress fa c to r, the leakage of host c e ll
m etabolites and ions would have to be considered as these would be
b e n e fic ial fo r increased growth of SL nodorum.
Septoria nodorum is
able to germinate on water agar, but i t is not able to sustain normal
growth without n u trien ts from it s surrounding environment.
The leak­
age o f m aterials from host c e lls in to the in te r c e llu la r water is the
main source o f.n u trie n ts fo r SL nodorum since th is fungus does not pro
duce a haustorum fo r n u trie n t e x tra c tio n .
Septoria nodorum produces a non-specific toxin and i t may be the
ro le of th is toxin to k i l l or severely stress the plant c e lls so th at
they are more re a d ily colonized.
This toxin is non-specific with re ­
gard to re s is ta n t and susceptible c u ltiv a rs (48) and w ill cause ty p i­
cal chlorosis when concentrated frac tio n s are a r t if ic ia lly applied.
In
the highly water-congested susceptible c u ltiv a r s , the in te r c e llu la r
water may act as a transporting substance fo r the to x in , allowing i t
to spread through the tis s u e .
The increased number o f toxin affected
c e lls would allow fo r increased tissue colonization by JL nodorum.
the re s is ta n t c u ltiv a rs (non-water congested), or in susceptible
In
89
c u ltiv a rs under non-water congesting conditions, there is l i t t l e
in ­
t e r c e llu la r water and thus the e ffe c ts o f the toxin are lim ite d to
small areas surrounding fungal hyphae.
I f the fungus is unable to
colonize healthy, unstressed host tis s u e , then lesions must be s e lf lim itin g .
With a subsequent period o f high r e la tiv e humidity
causing an increase in in te r c e llu la r water in the susceptible c u ltiv a r ,
there would again be a spread of the toxin and an increase in the ex­
te n t o f the tissue colonized.
Those c u ltiv a rs which take prolonged
periods o f time under extremely high le v e ls o f r e la tiv e humidity to
show water congestion would thus show l i t t l e or no e ffe c ts with the
same conditions th a t allow disease progression in susceptible c u l t i ­
vars.
SUMMARY
The main differences found in th is study with regard to the de­
fense mechanisms in wheat to invasion and colonization by SL hodorum
were:
I ) a d iffe re n c e in germination o f spores on the le a f surfaces
o f Fortuna and Manitou and the re s t o f the c u ltiv a rs , and 2) d i f f ­
erences among the three d ise a s e-resistan t classes o f c u ltiv a rs as to
the in tern a l progression o f the disease.
The germination d iffe re n c e is not explained by v o la t ile com­
pounds emitted from the leaves since germination was n e ith er in h ib i­
ted or stim ulated under in v itr o conditions.
germinate well on water agar or on membranes.
Septoria nodorum spores
Therefore, the d i f f ­
erence does not appear to be n u tritio n a l unless m etabolites were pre­
sent on the leaves o f Fortuna and Manitou which stim ulated or in h ib ite d
metabolic processes necessary fo r germination ( ie . feed-back in h ib itio n
type mechanisms).
I t is probable th a t in h ib ito ry or stim ulatory com­
pounds were present in the two c u ltiv a rs th a t exhibited s ig n ific a n t
d iffe re n c e s , but they were not concentrated enough to be evident in
th is experiment or else they were not v o la t ile in nature.
The m ajority
o f c u ltiv a rs did not influence spore germination percentages.
Spore
germination on Manitou never reaches the level found on Fortune, but
disease se ve rity may be as great when plants are held in the mist
chamber fo r extended periods of time.
The major defense mechanism in the wheat c u ltiv a rs studied was
t
91
re la te d to the c o m p a tib ility o f the pathogen with the in tern a l envi­
ronment o f the host.
Here, the degree of water congestion played a
s ig n ific a n t ro le in disease progression.
Water congestion may func­
tio n in I ) the spreading o f a to x ic substance produced by the fungus;
2 ) placing plan t c e lls in an unfavorable metabolic environment fo r
adequate disease resistance; 3) the leaching of n u tritio n a l sub­
stances from the host tis s u e ; or 4) combining one or more o f the above
fa c to rs .
The primary defense mechanism observed in th is study was the
e ffe c t o f water congestion on the progression of th is disease.
Three
possible e ffe c ts of th is water congestion on disease progression have
been postulated although i t is lik e ly th a t they act in conjunction
with one another and not separately.
V a rie ta l differences in water
congestion have been noted by others (27) and incorporation of water
congestion resistance in to a
may be of value.
nodorum resistance breeding program
REFERENCES
1.
A yesu-O ffei, E. N ., and B. G. C lare. 1969. Processes in the
in fe c tio n o f barley leaves by Rhynchosporium s e c a lis . Aust.
J. b io l. Set. 23:299-307.
2.
Baker 9 C. J. 1970. V a rie ta l reaction of wheat t o .le a f in fe c ­
tio n by Leptosphaeria nodorum and Septoria t r i t i c i . Trans.
Br. mycol. Soc. 54:500-504.,
3.
Baker 9 C. J. 1970. Influence of environmental factors on devel^
opment o f symptoms on wheat seedlings grown from seed, infected
with Leptosphaeria nodorum. Trans. Br. mycol. Soc. .55:443^447.
4.
Baker 9 C. J. 1971. Morphology o f seedling in fe c tio n by
Leptosphaeria nodorum. Trans. Br. mycol. Soc. 56:306-309.
5.
Baker 9 E. A. 1978. Septoria - the lurking th re a t to wheat
y ie ld s . EPPO B u ll. 8:9 -2 0 .
6.
Baker 9 E. A,., and I . M. Smith. 1978. Development o f re s is ta n t
and susceptible reactions in wheat on inoculation with Septoria
nodorum. Trans. Br. mycol. Sqc. 71:475-482.
7.
Benedict, W. G. 1971. D iffe re n tia l e ffe c t o f lig h t in te n s ity on
the in fe c tio n o f wheat by Septoria t r i t i c i Desm. under con­
tr o lle d environmental conditions. P hysiol. Plant Pathol. I :
55-66.
' .
■
•
8.
Borges9 0. L ., E. H. Stanford 9 and R. K. Webster. 1976.
host-pathogen in te ra c tio n of a lf a lf a and Stemphyli urn
botryosum. Phytopathology 66:749-753.
9.
Bousquet9 J. F ., and M. Skajennik o ff. 1974. Isolement e t mode
d 'a c tio n d'une phytotoxine produite en culture, par Septoria
nodorum Berk. Phytopathol. Z. 80:355-360.
The
10.
Breeding Plants For. Disease Resistance. 1973.
. Pennsylvania State U niversity Press. 401pp.
Ed. R. R. Nelson.
11.
Bronnimann9 A ., B. E. S a lly , and E. L. Sharp. 1972. In vestig a­
tions on Septoria nodorum in spring wheat in Montana. Plant
D is . Rep. 56:188-191.
93
12.
Brown, J . F. Mechanisms o f resistance in wheat to Septoria
nodorum and Septoria t r i t i c i . Unpublished.
13.
C lark, C. A ., and J. W. Lorbeer. 1976. Comparative histopathology of B otrytjs squamosa and B. cinerea on onion leaves.
Phytopathology 66:1279-1289.
14.
Cooke, B. M ., and J. I . F. Fozzard. 1973. Development, assess­
ment and seed transmission of Septoria nodorum. Trans. Br.
myc o I. Soc. 60:211-222.
15.
Cooke, B. M ., and D. G. Jones. 1970. A f ie ld inoculation method
fo r Septoria t r i t i c i and Septoria nodorum. P I. Path. 19:7274.
16.
Cooke, B. M ., and D. G. Jones. 1970. The epidemiology of
Septoria t r i t i c i and S^ nodorum I I . Comparative studies of
head in fe c tio n by Septoria t r i t i c i and S_. nodorum on spring
wheat. Trans. Br. mycol. Soc. '54:395-404.
17.
Cooke, B. M ., and D. G. Jones. 1971. The epidemiology of ,
Septoria t r i t i c i and S^ nodorum I I I . the reaction of spring
. and w inter wheat v a rie tie s to in fe c tio n by Septoria t r i t i c i
and S. nodorum. Trans. Br. m yco l.S o c. 56:121-135.
18.
DeWit, P. J. G. M. 1977. A lig h t and scanning-electron micro­
scopic study of in fe c tio n of tomato plants by v iru le n t and
a v iru le n t races of Cladosporium fulvum. Neth. J . P I. Path.
,83:109-122.
19..
Dickson, J . G.
517pp.
20.
Esau, K.
735pp.
21.
E yal, Z. 1971. The kinetics of pycnosopre lib e ra tio n in Septoria
t r i t i c i . Can. J . Bot. 49:1095-1099.
22.
E y a l, Z ., J. F. Brown, J . M. Krupinsky, and A. L. Scharen. 1977.
The e ffe c t of postinoculation periods of le a f wetness on the
response of wheat c u ltiv a rs to in fe c tio n by Septoria nodorum.
Phytopathology 67:874-878.
1953.
1956.
Diseases of F ield Crops.
Plant Anatomy.
McGraw-Hill.
John Wiley & Sons, Inc.
94
23.
Green, G. O ., and 0. G. Dickson. 1957. Pathological his­
tology and v a rie ta l reactions in Septoria le a f blotch of bar­
le y . Phytopathology 47:73-79.
24.
Gough, F. J. 1978. E ffe c t of wheat host c u ltiy a rs on pycnidiospore production by Septoria t r i t i c i .
Phytopathology 68 :
1343-1345.
25.
Narrower, K. M. 1977. Estimation of resistance of three wheat
c u ltiv a rs to Septoria t r i t i c i using a chemical method fo r
determination of fungal mycelium. Trans. Br. mycol. Soc.
69:15-19.
26.
Narrower, K. M. 1978. Some aspects of the in fe c tio n process and
sporogenesis of Septoria nodorum and Sv t r i t i c i . Proc. Aust.
Septoria Workshop: 20.
27.
Heggestad, H. 1945. V a rie ta l v a ria tio n and inheritance studies
on natural water-soaking in tobacco. Phytopathology 35:754770.
28.
Hewett, P. D. 1969.
ears I I . on nodes.
29.
Hewett, P. D .■ 1975. Septoria nodorum on seedlings and stubble
of w inter wheat. Trans. Br. mycol. S o c ..65:7-18.
30.
Higgins, V. J . , and G. L. Lazarovits . 1978. Histopathological
and u ltra s tru c tu ra l comparison of Stemphylium sarcinaeforme
and S. botryosum on red clover fo lia g e . Can. J. Bot. 56:20972108.
31.
Holmes, S. J . I . , and J. Colhbun. 1971. In fec tio n of wheat
seedlings by Septoria nodorum in re la tio n to environmental
fa c to rs . Trans. Br. mycol. Soc. 57:493-500..
32.
Holmes, S. J. I . , and J. Colhoun. 1974. In fec tio n of wheat by
•. Septoria nodorum and Sv t r i t i c i . in re la tio n to plan t age, ■ .
a ir temperature and r e la tiv e humidity. Trans. Br. mycol. Soc.
62:329-338.
33.
Holmes, S. J . L ., and J . Colhoun. 1975. Straw-borne inoculum ■
of Septoria nodorum and !5. t r i t i c i in re la tio n to incidence of
disease on wheat plan ts. .Pl. Path. 24:63-66.
Septoria nodorum Berk, on wheat I . on the
J . natn; In s t, a g ric . Bot. 11:547-558.
95
34.
Hosford, R. M ., J r. 1971. A form o f Pyrenophora trichostoma
pathogenic to wheat and other grasses. Phytopathology 61:
2 8 -3 2 .
35.
Hosford, R. M ., J r. 1975. Platyspora pehtamera, a pathogen of
wheat. Phytopathology 65:499-500.
36.
Hosford1 R. M ., J r. 1975. Phoma glomerata, a new pathogen of
wheat and t i t i c a l e s , c u ltiv a r resistance re la ted to wet per­
iod. Phytopathology 65:1236-1239.
37.
Hosford 1 R. M ., J r. 197,6. The e ffe c t o f post inoculation wet
periods on resistance of wheat to Leptosphaeria ,
herpotrichoides le a f spotting. Proc. Am Phytopathol. Soc.
3:205 (A b s tr.)
38.
Hosford 1 R. M ., J r. 1978. Effects o f wetting period on r e s i ­
stance to le a f spotting o f wheat by Leptosphaeria microscopica
with conidial stage Phaeoseptopia u rv ille a n a . Phytopathology
68:908-912.
39.
Hughes, J . , and M. E. McCully. 1975. The use of an optical
brightner in the study of plan t s tru c tu re . Stain Tech. 50:
319-329.
40.
Jenkins, J. E. E ., and W. Morgan. 1969. The e ffe c t o f Septoria
diseases on y ie ld in w inter wheat. P I. Path. 18:152-156.
41.
Johansen, D. A.
523pp.
42.
Johnson, J. 1947. Water-congestion in plants in re la tio n to
disease. Univ. of Wisconsin Res. B u ll. 160:34pp. .
43.
Johnson, L. F ., and E. A. C url. 1972. Methods fo r Research on
the Ecology of Soil-borne Plant Pathogens. Burgess Publishing
Co. 247pp.
44.
Jones, D. G. 1975. Comparative e ffe c ts of Septoria nodorum
on spring wheat.and barley. P I. Path. 24:19.9-201.
1940.
Plant Microtechnique.
McGraw H i l l .
96
45.
Jones, D. G., and K. Odebunmi. 1971. The epidemiology of
Septoria t r i t i c i and Sl. nodorum IV . the e ffe c t o f inoculation
a t d iffe r e n t growth stages and on d iffe r e n t plant parts. Trans
Br. mycpl. Soc. 56:281-288.
46.
Jones, D. G., and K. Odebunmi. 1971. The epidemiology of
Septoria t r i t i c i and S. nodorum F. e ffe c t of mixed inocula on
disease symptoms and y ie ld in two spring wheat v a rie tie s .
Trans. Br. mycol. Soc. 57:153^159.
47.
Jones, D. G ., and R. -D. W. Rowling. 1976. The reaction of two
spring wheat v a rie tie s exposed to epidemics o f Septdria
nodorum and Sv t r i t i c i of varying in te n s ity and duration.
J. a g ric . S c i., Camb. 87:401-406.
48.
Kent, S. S. , and G.'A-. S tro b e l, 1976. Phytotoxin from Septoria
nodorum. Trans. Br. mycol. Soc. 67:354-358.
49.
Krupinsky, J. M ., J. C. Craddock, and A. L. Scharen. 1977.
Septoria resistance in wheat. Plant Dis. Rep. 61:632-636.
50.
Laubscher, F. X ., F. vonWechman, and van Schalkwyk. 1966.
H eritab le resistance o f wheat v a rie tie s to glume blotch
( Septoria nodorum B e rk .). Phytopathol. Z. 56:260-264.
51.
. Leijerstam , B. 1972. A method fo r assessing the degree o f in . fe e tio n caused by Septoria nodorum on wheat. Meddelanden
from Statens Vaxtskyddsanstatt 15:281-285.
52.
L i t t l e , R ., and J. D. Doddson. 1974. A technique fo r assessing
the reaction o f wheat v a rie tie s to Septoria nodorum (Berk)
in fe c tio n and the preparation o f recommended l i s t fig u re s .
J. natn. In s t, a g ric . Bot. 13:152-159.
53.
Lucas, M. T ., and J. Webster. 1967. Conidial states o f B ritis h
species of Leptosphaeria. . Trans. Br. mycol. Soc. 59:85-121.
54.
Myers, D. F ., and W. E. Fry. 1978. The development o f
Gloeocercospora sorqhi in sorghum. Phytopathology 68:11471155.
97
55.
Nelson, L. R ., M. R. Holmes, and B. M. Confer. 1976. M u ltip le
regression accounting fo r wheat y ie ld reduction by Septoria
nodorum and other pathogens. Phytopathology 66:1375-^1379.
56.
Nelson, R. R. 1978. Genetics o f horizontal resistance to plant
disease. Ann. Rev. Phytopathol. 16:359-378.
57.
Plant Pathology - An advanced T re a tis e . 1959.
and A. E. Dimond. Academic Press. 674pp.
58.
Pierson, C. F ., and J. C. Walker. 1954. Relation of
Cladosporium cucumerinum to susceptible and re s is ta n t cucumber
tis s u e . Phytopathology 44:459-465.
59.
Pirson, H. 1960. Prufung verschiedener winterweizensorten auf
a n fa llig k e it gegen Septoria nodorum Berk, m it h iIf e von
kunstlichen in fektio n en . Phytopathol. Z. 37:330-342.
60.
Preece, I . F. 1959. A staining method fo r the study of apple
scab in fe c tio n s . P I. Path. 8:127-129.
61.
R a p illy , F. 1975. Etudes sur 1 1inoculum de Septoria nodorum
Berk. Agent de la Septoriose du Ble. I I . Les pycmospores.
Ann. Phytopathol. 6:71-82.
62.
Rathajah, Y. 1976. In fec tio n o f sugarbeet by Cercospora
b etico la in re la tio n to stomata! condition. Phytopathology
Ed. J. G. H orsfall
6 6 :7 3 7 ^ 7 4 0 .
63.
Richardson, M. J . , and M. Noble. 1970. S eptoria species on
cereals - a note to aid th e ir id e n tific a tio n . P I. Path. 19:
159-163.
64.
Ride, J. P ., and R. B. Drysdale. 1972. A rapid method fo r the
chemical estim ation of filamentous fungi in plan t tis s u e .
Physiol. Plant Pathol. 2:7 -1 5 .
65.
Rohringer, R ., W. K. Kim, D. J, Samborski, and N. K. Howes..
1977. C a lc o fluor: an op tical brightener fo r fluorescence
microscopy o f fungal plant parasites in leaves. Phytopathology
67:808-810.
98
.
66 .
Rosen, H. R. 1 9 2 1 .
Septoria glume blotch o f wheat.
Arkansas B u ll. 175:17pp.
67.
Rubin, B. A ., and Ye. V. A r ts ikhoyskaya. 1963. Biochemistry
and physiology o f plant immunity. Pergamon Press. 358pp.
68 .
Ryan, C. C ., and B. G. C lare. 1974. Coating o f le a f surfaces
with agarose to re ta in fungal inoculum in s itu fo r stain in g .
Stain Tech... 49:15-18.
69.
Schafer, J. F. 1971. Tolerance to plant disease.
Phytopath. 9:235-252.
70.
Scharen, A. L. 1963. E ffe c t of age of wheat tissues on suscep­
t i b i l i t y to Septoria nodorum. Plant D is. Rep. 47:952-954.
71.
Scharen, A. L. 1964. Environmental influences on development
o f glume blotch in wheat. Phytopathology 54:300-303.
72.
Scharen, A. L. 1966. Cyclic production of pycnidia and spores
in dead wheat tissue by Septoria nodorum. Phytopathology 56:
580-581.
73.
Scharen, A. L.
74.
Scharen,.A. L ., and J. M. Krupinsky. 1970. C ultural and inocu­
la tio n studies o f Septoria nodorum, cause of glume blotch of
wheat. Phytopathology 60:1480-1485.
75.
Scharen, A. L ., and J. M. Krupinsky. 1978. Detection and manip
U lation of resistance to Septoria nodorum in wheat.
Phytopathology 68:245-248.
76.
S co tt, R. P ., and P. W. Benedikz. 1977. Field techniques fo r
assessing the reaction o f w inter wheat c u ltiv a rs to Septoria
nodorum. Ann. appl. B io l. 85:345-358.
77.
Shaw, D. E. 1953. Cytology o f Septoria and Selenophoma spores.
Proc. Linnean Soc. (New South Wales) I x x y i i i : 122-131.
78.
Shipton, W. A. 1966. Septofia le a f spot and glume blotch of
wheat. Plant Diseases:160-163.
Univ. of
Ann. Rev.
Personal Communication.
■
99
79.
Shipton, W. A. 1968. The e ffe c t o f Septoria diseases on wheat.
Aust. J. Exp. a g ric . An. Hush. 8:89-93.
80.
Shipton, W. A ., W. R. J. Boyd, A. A. Rosie l l e , and B. I . Shearer.
1971. The common Septoria diseases o f wheat. Bot. Rev. 37:
231-262.
81.
Sloropad, W. R ., and. D. C. Arny. 1956. H istologic expression of
s u s c e p tib ility and resistance in barley to s tra in s .o f
Helminthosporium gramineum. Phytopathology 46:289-292.
82.
Sprague, R. 1950. Diseases of Cereals and Grasses in North
America., Ronald Press. 538pp.
83.
TeBeest, D. O ., G. E. Templeton, and R. J. Smith, J r. 1978.
Histopathology o f Colletotrichum gloesoporioides f . sp.
aeschynomene on Northern Jointvetchl Phytopathology 68:12711275.
84.
Tomiyama, K. 1963. Physiology and biochemistry of disease re ­
sistance of plan ts. Ann. Rev. Phytopath. I :295-324.
85.
Van der Plank, J. E. 1968.
Academic Press. 206pp.
86 .
Weber, G. F. 1922.
12:449-589.
87.
vonWechman, M. B. 1965. Seed transmission o f Septoria nodorum
Berk, in the western cape province. S. A fr . J. a g ric . S c i.
8:737-744.
88 .
vonWechman, M. B. 1966. In ve s tig atio n on the survival of
Septoria nodorum on crop residues. S. A fr . J. a g ric . S c i.
9:93-100.
89.
Wheat and Wheat Improvement.
Soc. Agr. 560pp.
90.
Weise, M. V. 1977.
Soc. 106pp.
Disease Resistance in P lants.
Septoria diseases o f cereals.
1967.
Phytopathology
Ed. K. S. Quisenberry.
Compendium o f Wheat Diseases.
Am.
Am. Phytopath.
MONTANA STATE UNIVERSITY LIBRARIES
100
CM
762
CD
CO
CO
111Illl111I111III