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CEPHALOSPORIUM LEAF STRIPE OF WHEAT:
DECLINE AND MODE OF INFECTION

By
Jack Eugene Bailey

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology
l980

ABSTRACT
CEPHALOSPORIUM LEAF STRIPE OF WHEAT: DECLINE AND MODE OF INFECTION
By
Jack Eugene Bailey

PART I: Decline of Cephalosporium leaf stripe

A field previously reported to be exhibiting declining disease
incidence was selected for this work and was studied in its eighth and
ninth years of wheat monoculture. Three similar fields with O, 3 and 4
years of wheat monoculture were also studied. Soil was reciprocally

transferred between these fields, and CephalOSporium gramineum soil

 

populations as well as disease incidence were monitored. Both were low
in the newest and oldest fields, and high in the fields of intermediate
age. Addition of C, gramineum, as oat seed inoculum, increased disease
in the newest and oldest fields only and resulted in an inverse relation-
ship between the years of wheat monoculture and disease incidence.
Results in the second year of testing were identical except that addi-
tional inoculum produced uniformly high levels of disease in all four
fields. In the second year, population levels were too variable to
allow the substantially larger populations in the two fields of inter-
mediate age to be statistically significant. There was no reduction in
disease incidence from reciprocal transfers of soil. Fields supporting
4 or 5 years of wheat monoculture did not appear to show spontaneous

disappearance of disease. It was concluded that the field with 9 years

J. E. Bailey

of continuous wheat is maintaining its decline status, but that disease

decline may not result in other fields under continuous wheat monoculture.

PART II: Mode of Infection
Various methods of measuring the effect of root exudates on

Cephalosporium gramineum were developed. It was found that exudates

 

from frozen roots increased germination, growth, and conidiogenesis

above that of non-frozen root exudate. Conidia held in a fungistatic
condition, due to the influence of non-sterilized soil, were able to
germinate and penetrate root tissue when exudate from roots that had been
frozen was present. Hyphae were shown to grow deep into host root tissue.
Various methods of freezing roots allowed 9, gramineum to colonize plants
and cause symptom production. Evidence is presented that such roots

were unbroken, thereby implicating an active penetration process. Roots
held at low temperatures (2 C), but not frozen, did not become infected.
An inexpensive and easy-to-use apparatus was designed to simulate field
freezing. This freezer allowed non-vernalized plants to be frozen
without crown death because only root tissue came into contact with
frozen soil. Freezing and inoculating grain varieties with known resis-
tances resulted in disease incidence similar to those found in the field.
It is concluded that active penetration after freeze stress is an important
means of infecting plants. Field observations tended to support this

conclusion.

To my wife, son, parents, and brothers

ii

ACKNOWLEDGEMENTS

I would like to thank Dr. John Lockwood for his extremely valuable
comments and hours of dedication in guiding my research and in the
preparation of this manuscript.

I would also like to thank Dr. Maurice Niese for his help in
initiating this research on good, sound, scientific footing. Also,
thanks go to Al Ravenscroft who answered endless questions and helped
in this work in many ways.

I very much appreciate the work done by Drs. D. w. Fulbright,

G. R. Hooper, and J. M. Tiedje in serving on my committee and editing
this manuscript.

A very special thanks goes to my wife Becky. Her inspiration and
understanding was of immeasurable importance to the success and completion

of this dissertation.

TABLE OF CONTENTS

 

Page

List of Tables ......................... vi
List of Figures ........................ viii
Literature Review ....................... 1
PART I---Decline of Cephalosporium Leaf Stripe ......... ll
Introduction ......................... l2
Materials and Methods .................... l2
Field selection ...................... l2

Field maintenance ..................... l3
Planting ......................... l3
Inoculum preparation ................... 13
Monitoring Cephalosporium gramineum p0pulations ...... l3

Soil transfers and infestation . . . ........... 14
Disease rating ...................... 15
Results ........................... l5
Discussion .......................... 22
PART II-——Mode of Infection ................... 25
Introduction ......................... 26
Materials and Methods .................... 26
Fungus culture ...................... 26
Source of wheat seed ................... 27
Sterilization of seeds .................. 27

Media ........................... 27
Method for studying soil-imposed fungistasis ....... 28
Electron Microsc0py .................... 3l
Artificial wheat freezing methods ............. 3l
Cation exchange chromatography and bioassay procedures . . 32
Quantification of sugar in wheat root exudate ....... 38

iv

Results ...........................

The develOpment of a sterile agar disk method for studying
Cephalosporium gramineum conidia and soil fungistasis .

 

Nheat roots as a source of nutrition for Cephalosporium
gramineum . . . ....................

a) Effect of total wheat root contents on
conidiogenesis. . . ..............

b) Effect of exudates from non- injured wheat roots
on conidial germination .............

c) Effect of exudates from wheat seedling roots
subjected to freezing and mechanical injury on

 

conidial germination ..... . .........
d) Root exudate fractionation ............
Disease incidence in relation to mechanical and freeze-

induced injury. . . ............. . . . . .
a) Mechanical severing of roots ...........
) Artificial freeze-induced injury .........
c) Natural freeze-induced injury . . ........

) Cultivar screening procedures for resistance
determination ..................
Discussion ..........................
List of References .......................

Page
38

38
43
43

45

47
52

59
59
64
67
68

76

Table

10.

LIST OF TABLES

Effect of number of years of continuous wheat on
natural Cephalosporium gramineum p0pulations and
infection caused by natural and oat seed inoculum

of C, gramineum ......................

Effect of soil transfers between fields with different
periods of wheat monoculture on populations of

 

Cephalosporium gramineum .................

 

Effect of soil transfers between fields with different
periods of wheat monoculture on disease caused by

natural populations of Cephalosporium gramineum ......

 

Effect of soil transfers between fields with different
periods of wheat monoculture on disease caused by
natural populations of Cephalosporium gramineum

 

supplemented with oat seed inoculum. . ..........

Effect of period of wheat monoculture on natural
Cephalosporium gramineum populations and on disease

 

caused—by natural and supplemental inoculum of C,

gramineum. . . ..... . ................

Effect of incorporating wheat soil from a field
exhibiting Cephalosporium leaf stripe decline into
a field not in disease decline, as measured by disease

incidence and population levels ..............

Germination of Cephalosporium gramineum conidia
after 24 hr incubation on agar disks covering soil

 

Germination of Cephalosporium gramineum conidia on
agar disks covering soil after various incubation

 

periods ...... . . . . . . ..............

Numbers of Cephalosporium gramineum conidia produced
on wheat root agar and water agar after 9 days

 

incubation . . . . . . ..... . ............

Effect of exudate from wheat roots treated in various
ways on conidial germination and growth of Cephalosporium

 

gramineum on sterile depression slides ..........

Vi

Page

l6

T7

18

19

20

Zl

42

44

46

49

Table
ll.

12.

l3.

l4.

l5.

l6.

l7.

l8.

Effect of fractionated exudate from frozen or
non-frozen wheat roots on germination of

Cephalosporium gramineum conidia ............

Response of Cephalosporium gramineum to exudates
from frozen and non-frozen, axenically grown

 

wheat roots ......................

Leaves colonized with Cephalosporium gramineum after
cut wheat roots were either dipped in a conidial
suspension or placed in soil infested with C,

 

gramineum conidia ...................

Effect of exposure of wheat plants growing in
sand to freezing conditions on devel0pment of

CephalOSporium leaf stripe symptoms ..........

Effect of exposing wheat plants to various periods
of winter conditions in relation to development of

Cephalosporium leaf stripe symptoms ..........

Evaluation of a modified chest freezer as a method

to screen plants for Cephalosporium leaf stripe . . . .

Effect of various concentrations of Cephalosporium
gramineum conidia used as a root dip on wheat plants
of known resistances after mechanical severing of

 

their roots ......................

Comparison of active and passiVe infection

processes .......................

vii

Page

58

63

65

66

69

LIST OF FIGURES

Figure Page
l. Sterile chamber used to study soil-imposed
fungistasis ....................... 3O
2. Chest freezer method of simulating freeze stress ..... 34

3. Soil temperature dynamics 2.5 cm from bottom center
of cups containing a greenhouse mix undergoing
freezing and thawing in the chest freezer ........ 36

4. Standard Optical density curve for various
concentrations of glucose ................ 4O

5. Light and electron micrographs of Cephalosporium
gramineum germination on agar covering soil and
growth through wheat root tissue ............. Sl

 

6. Electron micrographs of Cephalosporium gramineum
penetration into wheat root epidermis following
freezing (-lO C) in syringes and l0 days' incubation
with C, gramineum conidia ................ 54

 

7. Electron micrograph of Cephalgsporium gramineum
growth over the surface of wheat root endodermal
tissue (476 X) following freezing (-l0 C) in
syringes and l0 days' incubation with C, gramineum
conidia ......................... 56

 

viii

LITERATURE REVIEW

General
In 1934 Nisikado gt_al,, described a new disease of wheat in Japan

(57), caused by an undescribed species of Cephalosporium. Nisikado and

 

Ikata named the fungus Cephalosporium gramineum N15. and Ika. and the

 

disease CephalOSporium leaf stripe. Cephalosporium leaf stripe (CLS)
has since been described in many grasses, including the genera Qagtylis,

Avena, Bromus, Elymus, Secale, Hordeum, Triticum, Agrgpyron, and

 

Arrhenatherum (8, 36). Cephalosporium leaf stripe is a serious problem

 

in Japan (57), Europe (29, 75), and the United States (52, 55, 69, 79,
81). In the United States, Cephalosporium leaf stripe is of particular
importance in Washington (7), Kansas (94), and Montana (53); in Montana
it is the most important soil-borne disease of wheat (52). In 1966,
CLS was observed in Michigan by N. A. Smith, R. P. Scheffer, and A. H.
Ellingboe (79).

In 1963, Bruehl showed that C, gramineum also formed a sporodochial
stage on dead, infested wheat stubble in the field (9). Colonies of
this fungus on agar were identical to those formed by C, gramineum and
inoculation of wheat produced the symptoms of Cephalosporium leaf stripe.

Bruehl identified the fungus as Hymenula cerealis Ellis and Everh. and

 

stated that this name should have precedence over 9, gramineum (9). He
did, however, recommend that Cephalosporium leaf stripe remain the name

of the disease. Therefore, the causal organism has two imperfect names:

Cephalosporium gramineum for its parasitic stage, and Hymenula cerealis

 

 

for its saprophytic stage. To date, no perfect stage has been described
for this fungus. For clarity, C, gramineum will be retained as the name

of the fungus in this paper.

Parasitic stage

 

It is generally agreed that C, gramineum invades its hosts through
the root system, (4, 8, 53, 56, 57, 62, 76). Once in the vascular tissue,
systemic colonization of all vegetative portions of the plant occurs;
however, the fungus is restricted to the xylem as long as the plant is
alive (86). When introduced directly into root xylem, at room tempera-
ture, the fungus moves through the crown into the lower leaf sheaths
within 4-6 days (86). Stripe development begins approximately one week
after (86), when long chlorotic bands extend the length of the leaves
(7, 8, 57) encompassing darkened vascular bundles. Acropetal symptom
develOpment follows an early senescence of lower leaves resulting in
stunting, poorly filled or unfilled seed heads, and decreased flour
quality (53, 55). Cephalosporium leaf stripe can result in a 70-80%
yield reduction in severely infected plants (40, 67, 76).

In 1938, Ikata and Kawai (38) showed that certain culture filtrates
of C, gramineum stunted wheat seedlings, while others produced stripe
symptoms which later became necrotic. Bruehl (8) suggested that symptom
development was most likely a result of xylary plugging by mycelium
and physiological disfunction due to toxic metabolites produced by the
fungus. Spaulding et_al, (80) found that C, gramineum produces pectinases
and cellulases, and were able to demonstrate pectinase activity in
diseased but not healthy plants. They also found that diseased tissues

had a lessened water content and attributed this to increased viscosity

of the water and xylary plugging by a polysaccharide produced by the fungus.
They felt that hyphae and pectin plugs in the xylem elements further
inhibited water movement, since acropetal and lateral dye movement could
not occur in stripe tissue. They concluded that such "vascular distress”
may contribute to cellular dysfunction and death. Work by Pool and
Sharp (61) supported this conclusion by showing that a polysaccharide
produced by C, gramineum cultures restricted dye movement in the xylem.

Wiese (86) suggested that a diffusable substance(s) and not pectin
or hyphal plugging of the xylem was responsible for interveinal stripe
development. He found that such occlusions followed rather than preceded
lateral extension of leaf striping. Electron micrographs of infected
vascular bundles showed an accumulation of an electron dense material
surrounding conidia, which, after liberation, was found to line the walls
of infected vessels. Wiese felt this may be visual evidence of the dif-
fusable product responsible for the restriction of lateral water and/or
nutrient movement.

Kobayashi and Vi (41) identified a toxin from culture filtrates of
Q, gramineum which caused symptoms much like those of naturally infected
plants. This compound, Graminin A, was also found to have antibiotic
pr0perties effective against some bacteria and fungi. Creatura (21)
found that Graminin A caused stomates to open wider and respond more
slowly to water potential changes than stomates not treated with this
compound. This reSponse preceded stripe development and was more pro-
nounced under conditions of water stress. She showeithat differences
in stomatal activity were not a function of differences in the leaf
water status. Her work indicates that a toxin, Graminin A, and not

diffusable metabolites are responsible for the early pathogenesis.

Saprophytic stage

 

After plant scenescence, C, gramineum begins its Hymenula cerealis

 

stage characterized by random saprophytic growth from the xylem and the
colonization of cortical tissues. The hyphae emerge through openings
(i.e. stomates) in the plant residues, and phialides form at hyphal tips
(92). Rapid conidiogenesis along with mucopolysaccharide production
results in a mass of tightly adhering phialospores (13, 15, 61, 92).
Wiese and Ravenscroft (89) found these conidia to have a half-life of
0.5 to 2.5 weeks in moist field soil held at 25 C, or 26 weeks when held
at 7 C. When colonized plant material was present the p0pu1ation was
renewed as long as the integrity of the host material was maintained.

For example, infested wheat straw on the soil surface was able to renew
the colony forming units (probably conidia) in soil each fall and winter,
for approximately three years. Such straw was less efficient with each
succeeding year. Buried straw, however, was a source for population
renewal for only one year. The population density of C, gramineum
buried in infested straw was approximately 11-32% of the original p0pu1a-
tion after 18 months (42). Pool and Sharp (62) observed survival for as

long as 40 months in infested residue. Trichoderma spp. were predominant

 

colonizers of degenerating C, gramineum-infested straw; however, C,

gramineum can exclude Trichoderma and other fungi for at least 13 weeks

 

(12). Thus, 9, gramineum is an effective substrate possessor (11, 12,
43). There is some evidence that antibiotic production may play a role
in excluding other fungi from previously infested wheat straw (14).
Effective substrate possession is not unusual among fungi and may be of

particular importance to pathogens of weak saprophytic ability (12).

Survival during the fall and winter until host infection occurs is,
therefore, a function of Spore production and longevity as well as
effective substrate possession.

The develOpment of a selective medium (green wheat agar) in 1973
(88) allowed for the first time quantitative detection of C, gramineum
prepagules in the soil. Wiese and Ravenscroft (89) showed that colony
forming units in the field rose from near zero in July to a peak of
approximately 100,000 colony forming units/g soil by mid-winter
(November-January). This was followed by a decrease to near zero by
May. They found that removal of plant residues or treatments which
hastened residue decay greatly reduced detectable Q, gramineum popula-
tions. Population levels were progressively lower when wheat residue

was disked, plowed, or removed, respectively, from the field.

Disease decline

 

Soils are known to vary in their 'hOSpitality' to microorganisms.
Chemical (l7, 18, 48), physical (19, 30, 65), and microbial (3, 18, 20,
47, 48, 49) factors all influence the success of a fungal species to
establish and maintain its presence in the soil ecosystem. In the case
of soil-borne pathogens, these factors can affect disease incidence and
severity. It is well known that sterilized soil stimulates growth of
introduced, weakly-pathogenic fungi. This usually results in an increase
in disease severity, as compared with non-sterilized soils which are
suppressive to such unchecked growth. Consequently, all soils exhibit
biological control of disease, to a greater or lesser extent, when com-
pared to sterilized soil. 0f more practical significance are those
naturally occurring soil systems which are indistinguishable from fields

with high disease incidence, yet suppress soil-borne diseases to

economically acceptable levels. There are several good examples of
soils which have evolved to a suppressive state (1, 5, 28, 31, 33, 34,
71, 72, 82, 84, 93).

Once a field becomes infested with C, gramineum, continued wheat
monoculture normally results in a build-up of Q, gramineum populations.
Recently, Wiese and Ravenscroft (91) showed a long-term decrease in
pathogen p0pulation and disease incidence with continued wheat mono-
culture. This phenomenon was observed over an eight-year period and
resulted in disease levels too low to be used for disease studies.
Addition of infested straw, the naturally occurring source of inoculum,
was ineffective in significantly changing the disease status of this
field (Personal communication, A. V. Ravenscroft). Soils exhibiting a
Spontaneous decrease in disease incidence with continued monoculture are
sometimes referred to as 'decline' soils, and the disappearance of the
disease may be called 'disease decline.‘ The most studied example of
this phenomenon is take-all decline (TAD) of wheat.

Gaeumannomyces graminis is the causal organism of take-all disease

 

of wheat, a disease of great significance in temperate climates where
wheat is intensively cultured (83, 87) particularly in the Pacific
Northwest (18). As early as 1898, Roediger noted a decrease in infection
in previously infested land (34). Glynne et al. in 1935 (25) observed
that soil continually planted to wheat was not conducive to take-all.

It wasn't until 1964 that Slope and Cox (34) obtained experimental
evidence that there was a decline of take-all over time. In 1972, Pope
showed a decrease in infectivity in soils which 12.5% 'decline' soil

had been added (63). Pearson et a1. (60) showed that as little as 0.0001%

(by wt.) of decline soil could decrease disease in non-decline soils.

In the field, fumigation eliminated the decline factors, but it was
reinstated by adding 1% (by wt.) suppressive soil (73).

Attempts to link the suppressiveness to specific factors has so
far met with only mixed success. Early work attributed TAD to the micro-
biological status of the soil (34). Antagonistic microorganisms were
thought to be responsible for decline since exposure to 60 C for 30 min.,
or chemical sterilants removed the decline factor (73). Vojinovic (34)
found that populations of bacterial colonies and actinomycetes antagonistic
to G, graminis increased after the decay of host tissue. This was
particularly true for diseased host tissue.

9, graminis is known to respond tropically to wheat root exudates
(69), increasing the effectiveness of a given inoculum concentration.
Pope and Jackson showed that the efficiency of this response decreased
in decline soils, as compared with non-decline soils (64). They attri-
buted this to either a depletion of exudates due to increased microbial
competition, or to 'signal jamming' by metabolites produced by a new
rhizosphere population. 2099 and Jaggi (96) demonstrated an increase
in numbers of bacteria and actinomycetes in soil supplemented with G,
graminis hyphae. Sixty percent of the microorganisms isolated were

antagonistic to g, graminis. Fluorescent Pseudomonas were suspected of

 

being responsible for TAD since they : 1) are common in wheat rhizospheres
(particularly in TAD soil) (51) and on the rhizoplane of g, graminis-
1esioned roots (20), 2) have the same cardinal temperature of inactivation
as decline soil (20), 3) are as effective at reinstating decline conditions
in bioassays (73) as known decline soil (20), and 4) are efficient

antagonists of G, graminis on agar (68).

Reduced virulence and viral infection of G, graminis were hypo-
thesized as factors in decline. Cunningham suggested that less virulent
strains of the pathogen were important in TAD (34). Lapierra et a1. (44)
and Lemaire et a1. (45) found that g, graminis isolates from decline
fields were infected with a virus causing reduced virulence and poor
culturability. However, Rawlinson et a1. (66) in a follow-up study
could not demonstrate causality between viral infection of g, graminis
and virulence, sectoring, growth, or perithecia formation. Virus infec-
tion is not now thought to be important in the establishment of
hypovirulence (3, 66).

Other explanations for TAD such as volatile, plaque-forming
substances (74), saprobic/pathogenic population shifts (34), changes in
the NH+4 -N:N0-3-N ratios (6), and cross protection by weakly- or
non-pathogenic fungi (22, 95) have not received general acceptance.

Cephalosporium leaf stripe decline has not been reported in any of
the major wheat growing areas outside of Michigan, where wheat mono-
culture is commonly practiced. Its only documented occurrence is at the

Michigan State University farms in East Lansing, Michigan.

Control

No chemical means of controlling this disease are currently available;
nor does chemical control appear likely in the near future, due to the
low cash value of the cr0p and the intravascular nature of the fungus.
Proper residue management (e.g., deep plowing) and crop rotation have
proven to be quite effective in reducing C, gramineum populations. How-
ever, in some states crop rotation is not economically feasible due to

the limited number of cr0ps which can be grown. In addition to saving

fuel and unnecessary machinery wear, wheat residues must be left on the
surface of some soils to prevent excessive water loss and to prevent
erosion.

In lieu of residue management, resistance is the best potential
method of controlling the disease. In Montana, where CLS is the most
important soil-borne disease of wheat, over 1,000 lines have been
screened for resistance, using artificially infested field plots (52).
No immune varieties have been found, but various levels of resistance

have been identified.

Mode of infection and disease resistance

 

Several papers have focused on the nature of resistance to CLS.
Inoculation for these studies usually involves root severing followed
by root dip, or direct injection of conidia via a hypodermic needle into
the stem (8, 39, 40, 54, 56). This circumvention of extra-xylary tissues
is thought to mimic the natural infection process since it is generally
agreed that C, gramineum is incapable of direct penetration of roots
and that wounding is first necessary. Although root feeding wireworms
have been shown to effect such injury, Spring heaving due to diurnal
freezing and thawing is regarded as the single most important source
of wounding. Spring wheat, although susceptible, is virtually disease
free, even in infested fields. Likewise, susceptible wheat grown under
greenhouse conditions in infested soil rarely becomes infected unless
roots are mechanically severed and drenched with a conidial suspension.
That the predisposition factor occurs during winter field conditions was
shown by Bruehl in 1957 (8). After germinating wheat seeds on cultures

of g, gramineum and planting them outdoors in the fall, 100% infection

10

resulted by the following spring. Only 6% of the plants became infected
when grown in the greenhouse.

Early fall planting and fertilization have been shown to increase
the size of the root systems and, consequently, the number of roots
broken per plant due to heaving (10, 62). Such treatment increased the
level of Cephalosporium leaf stripe when rated the following summer (62).
More recently, it was observed that the earlier wheat was sown, the more
disease occurred up to a point, after which disease incidence began to
decrease (90). Mathre and Johnson (54) were unable to demonstrate active
growth by C, gramineum from soil to newly cut or crushed roots which
presumably simulated naturally occurring heaving damage. To explain
the mode of fungal entry from the soil into the xylem, they proposed
that conidia were 'vacuumed up' by mass flow with water as it entered
the broken xylem elements. Resistance, therefore, would be an intra-
xylary phenomenon.

Morton and Mathre (56) identified three types of resistance to CLS:
1) a reduction in the number of diseased plants in a ponulation, 2) a
reduction in the number of diseased tillers within a plant, 3) a reduction
in the rate and severity of disease development within a plant. The

latter two responses have been observed in only one wheat cultivar.

PART I: DECLINE OF
CEPHALOSPORIUM LEAF STRIPE

11

12

INTRODUCTION

Cephalosporium leaf stripe, caused by the soil-borne fungus

Cephalosporium gramineum, affects winter wheat in many of the wheat

 

growing regions of the world including the United States (52, 55, 69,

79, 81). Among the commercial wheat cultivars, levels of resistance are
insufficient to combat the disease and avoid yield losses. Cultural
practices, however, can play a major role in the survival of C, gramineum
in the soil. Recently, Wiese and Ravenscroft (91) reported that extended
wheat monoculture may lead to a Cephalosporium leaf stripe 'decline'.
During the latter part of their eight year study, C, gramineum popula-
tions and leaf stripe symptoms decreased dramatically. Similar phenomena
have been observed for take-all of wheat (25, 34, 35, 71, 93) and various
other soil-borne fungal diseases (28, 31, 33, 72, 82, 84). The purpose
of this study was to describe the relationship between leaf stripe
incidence,_§. gramineum p0pulations, and years of continuous wheat
production and to determine if disease suppression may be a transferable

trait.

MATERIALS AND METHODS

Field selection

 

Fields at Michigan State University, East Lansing, as previously
described (91) with 0, 3, 4 and 8 years of continuous wheat production
were used. Soybeans were previously grown in the 0 and 3-year-old wheat
fields, oats in the 4-year-old field and corn in the 8-year-old field. I

The wheat cultivars (Triticum aestivum L.) grown in each of the fields

 

were Ionia, Genesee, or Yorkstar, all equally susceptible to Cephalosporium

leaf stripe (91).

13

Field maintenance

 

The pH of all soils was maintained between 6.0 and 6.8 as recommended
by the Michigan State University Soil Testing Laboratory. No irrigation
or pesticides were applied except for 2,4-dichloro—phenoxyacetic acid
(2,4,-D) in the Spring to reduce broad—leaf weeds. Each autumn, preplant
tillage operations included plowing the previous wheat stubble 20-25 cm

deep with a fixed moldboard plow followed by a spring-tooth harrow.

Planting

Planting was done in October. The cultivar 'Genesee' was used

exclusively. Seeds were planted 3.6 cm deep in 15.5 cm rows.

Inoculum preparation

 

Isolates of Cephalosporium gramineum Nis. & Ika. (=Hymenula cerealis

 

 

Ellis & Everh.) from Montana (supplied by D. Mathre) and Michigan were
used in the 1977-78 and 1978-79 field experiments, respectively.

One hundred and fifty g of oat seeds were autoclaved in 100 ml
water for 30 min. Ten m1 of a conidial suspension grown in liquid
culture for 7 days were added to each jar. After shaking and incubation
at room temperature for 1-2 months, the inoculum was allowed to dry at

room temperature (53).

Monitoring Cephalosporium gramineum_populations

 

Soil samples were taken in all uninoculated plots during the winters
of 1977-78 and 1978-79. Three samples, taken from the t0p 7.5 cm (3 in.)
of soil from each plot, were mixed and assayed in the laboratory using
wheat leaf agar (88). C, gramineum prOpagules/g soil were determined by
adding approximately one to two 9 soil from the subsample mixture to one

liter of water and shaking vigorously. One ml aliquots of this suspension

14

were pipetted onto each of two assay plates and the number of C,
gramineum colonies determined after 4-7 days incubation at 22 C. These
two values were averaged and adjusted to represent the number of

prOpagules per dry g of soil.

Soil transfers and infestation

 

1977-78 season

A completely randomized, split block design was used in these
experiments. Fields with 0, 3, 4 and 8 years of wheat were divided into
12, 1.2 x 6.1 m (4 x 20 ft) plots. Nine of these were amended with soil
from the other fields and three plots remained unaltered. For example,
field 0 had three plots amended with soil from field 3, three plots
amended with soil from field 4, and three plots amended with field 8
soil. Fields 3, 4, and 8 were treated likewise. Soil was transferred
at a rate of 1.3 x 102 kl/ha (95 liters/plot). If soil amendments did
not change disease incidence or population levels from that in unamended
plots, these data were combined to increase the number of replications
for further analysis. One half of each plot was infested with C,
gramineum grown on oat seed. Oat inoculum (4.7 g m row) was placed

3.6 cm deep between the rows using a Planet Junior hand planter 2 weeks

after planting.

1978-79 season

The same fields, now with l, 4, 5, and 9 years of continuous wheat,
were used. All fields had 9, 1.2 x 6.1 m plots which were monitored for
C, gramineum populations and rated for disease incidence. Half of 3
plots in each field had added oat seed inoculum. Soil transfers were

made from field 9 to field 4 only at two rates; 1.3 x 102 and 3.9 x 102

15

kl/ha. Three plots of each transfer rate was rototilled about 20 cm
deep, and half of each plot was infested with oat inoculum one week
after planting (4.7 g/m row). This inoculum was placed directly over

the furrow, on the soil surface, then covered with a thin layer of soil.

Disease rating

 

Disease incidence was assessed at anthesis and reported as percen-
tage of plants with leaf stripes. One hundred plants were rated for

each treatment.

RESULTS

In 1977-78, the natural C, gramineum population was significantly
lower in field 8 than in fields 3 and 4, whereas field 0 was intermediate
(Table 1). Leaf stripe caused by natural C, gramineum inoculum was
correlated with the inoculum density (Table 1). When additional ino-
culum was added (in the form of colonized oat seed), there was no increase
in disease in fields 3 and 4; however, fields 8 and 0 showed marked
disease increases. This apparently reflects a difference in the
conduciveness to disease of these soils, since field 0, a field with
a very low population of C, grgmjneum, exhibited the greatest amount of
disease (Table 1).

Natural C, gramineum populations were not affected by the introduced
soils (Table 2), nor was the resultant disease incidence, either with or
without supplemental oat seed inoculum (Tables 3 and 4).

In the 1978-79 season population change in relation to years of
wheat monoculture had too much variability to be significantly different
though the same tendency to higher populations in fields 4 and 5 was

seen (Table 5). However, in fields with only natural inoculum, disease

16

TABLE 1. Effect of number of years of continuous wheat on natural
Cephalosporium gramineum populations and infection caused by
natural and oat seed inoculum of C, gramineum.

 

 

Plants showing stripe symptoms, %a

 

 

. b Natural populationC . Natural plus oat
Field (X 103/gdry 5011) Natural inoculum seed 1noculum

0 2.7 ab 9 a 33 d

3 10.2 b 15 bC 21 C

4 7.5 b 17 bC 18 be

8 1.0 a 6 a 13 b

 

aMean of 12 replicates with 100 plants per replicate. Values followed
by the same letter do not differ significantly (E_= 0.05).

bThe field is represented by the number of years of wheat monoculture
in that field.

cMean of 6 soil bioassays made from each treatment between November

1977, and June 1978. Samples were taken from plots without supplemental
inoculum.

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20

TABLE 5. Effect of period of wheat monoculture on natural Cephalosporium
gramineum populations and on disease caused by natural and
supplemental inoculum of C, gramineum.a

 

 

 

Plant showing stripe symptoms, %

 

 

Natural populationb Natural Natural plus oat
Field (X 103/g dry soil) inoculation seed inoculumd
l 3.2 a 8.3 a 92.3 c
4 60.0 a 59.6 b 92.0 c
5 17.4 a 58.0 b 96.3 c
9 1.9 a 8.3 a 97.0 c

 

aValues followed by the same letter do not differ significantly (E_= 0.05).

bMean of three soil bioassays from November 1978 to March 1979, from plots

without supplemental inoculum.

cMean of nine replicates with 100 plants per replicate.

dMean of three replicates with 100 plants per replicate.

21

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22

incidence showed a pattern identical to that of the 1977-78 season, i.e.,
increasing then decreasing disease levels with years of monoculture
(Table 5). Without oat inoculum, fields 4 and 5 had significantly greater
disease than fields 1 and 9. The oat-inoculated plots were so heavily
diseased that no differences could be distinguished between fields
(Table 5).

In the 1978-79 season, soil transfers were made only from field 9
to field 4 to further test if the apparent suppressiveness in field 9 was
a transferable trait. Soil transfers did not change the population
level or disease incidence except for an enhancement of disease when soil
at the rate of 3.9 x 102 kl/ha was transferred to plots in field 4 (Table
6). The oat seed inoculated plots gave uniformly high levels of disease.
The natural pathogen populations were unaffected by these soil transfers

(Table 6).

DISCUSSION

Fields that had been planted to wheat for 3 or 4 years had higher

Cephalosporium gramineum populations and disease incidence than fields

 

sown to wheat for 0 or 8 years. The low pathogen density in field 8
resulted from a long-term population decline (91). Field 0, not
previously planted to wheat, supported a low p0pulation of C, gramineum
due to the lack of a susceptible host. This trend appeared to be
repeated in the following season, in the same fields, since disease
incidence was again higher in the two fields of intermediate age.
However, the population differences were not statistically significant
due to great variability in the data.

Cephalosporium gramineum populations have two characteristics which

 

make their quantification difficult; l) extreme fluctuations in numbers

23

over relatively short periods of time, and 2) localization of spore

production. Cephalosporium gramineum propagules increase from near

 

zero propagules/g soil in mid-summer, to peak populations by mid-winter
(89). These progagules are produced in foci (sporodochia) which, until
Spores become disseminated by rain, are highly localized. This makes
soil population determinations highly variable for sites even inches
apart. For example, in one plot sampled on January 9, 1979, and again
on March 1, 1979, the population changed from zero to 1.0 x 106 propagules/
9 soil, respectively. Apparently no infested straw was in the first
sample and straw with sporodochia was in the second. Since these samples
were collected from frozen ground with the aid of an axe to loosen the
soil, it was difficult to collect a more homogenous sample.

It may be assumed that in 1978 the high level of disease developing
in field 0, when oat seed inoculum was used (Table 1), was caused by a
population density equal to, or less than, that established in fields 3,
4 and 8. This assumption can be made since the native pathogen p0pu1ation
was low in field 0 and the amount of artificial inoculum was uniform
between all fields. Therefore, in the summer of 1978 the field with the
fewest years of continuous wheat gave the greatest disease reSponse to
a given amount of added inoculum. This implies that disease incidence
is inversely related to years of wheat monoculture, a correlation
supportive of the idea of disease decline. This trend was not observed
the following year due to uniformly high levels of disease in all oat
inoculum-supplemented plots (Table 5). Interestingly, this indicates
that a high incidence of disease can be established, even in fields

exhibiting a 'decline'.

24

The transferability of a 'decline factor' was not demonstrated.
Moreover, the increase in disease (but not pathogen population) when
'decline' soil was transferred to a 'conducive soil' is not easily
explained since the 'decline' soil had a low p0pulation of the pathogen.

There appear to be several factors involved in determining
Cephalosporium leaf stripe incidence in any given year, viz. i) the
previous winter's population density, ii) environmental conditions and,
iii) the suppressiveness of the soil. The field which had nine years of
continuous wheat maintained the characteristics of field exhibiting a
disease decline. However, there are not enough data to understand the
long term disease pattern of Cephalosporium leaf stripe, or indeed
if there is a generalized pattern. It is possible that pathogen p0pu-
1ations and disease fluctuations over time may be a characteristic of

individual fields regardless of seemingly identical cultural practices.

PART II: MODE OF INFECTION

25

26

INTRODUCTION

Cephalosporium leaf stripe is a disease of winter wheat caused by

the soil-borne fungus Cephalosporium gramineum. This disease is present

 

in many of the major wheat growing regions of the world (7, 8, 16, 29,
57, 69, 75, 79, 94) including the United States. Cephalosporium leaf
stripe is particularly destructive in Washington (7, 8) and Kansas (99),
and is the most serious of all soil-borne wheat diseases in Montana (52).
Over 1,000 wheat lines have been screened for disease resistance to
Cephalosporium leaf stripe in Montana alone (52). Most of the basic
studies on resistance assume that C, gramineum enters passively through
severed roots, as conidia, drawn into the plant by transpiration water
(54). That winter stresses predispose wheat to infection is now well
established (8, 58, 59, 62, 90); however, other forms of winter injury
have not been studied in regard to Cephalosporium leaf stripe. Previous
work at Michigan State University has uncovered evidence that other forms
of winter stress may play a major role in the predisposition of plants

to infection (2). This paper presents evidence that increased root
exudation resulting from freeze stress caUses C, gramineum to germinate

and actively penetrate wheat roots, resulting in disease.

MATERIALS AND METHODS

Fungus culture

 

Cephalosporium gramineum Nis. and Ika., (=Hymenula cerealis Ellis

 

 

& Everh.) was used throughout this work. The isolate came from a
naturally infested wheat field in East Lansing, Michigan, unless other-
wise stated. Stock cultures were maintained on potato-dextrose agar
(PDA) at 4 C. The fungus was routinely passed through wheat plants to

insure against loss of pathogenicity.

27

Source of wheat seed

 

Winter wheat seed (Triticum aestivum (L.) cv. 'Genesee') was obtained

 

from the Michigan Cr0p Improvement Association.

Sterilization of seeds

 

Wheat seeds were sterilized by treating for 5 min. in 7 % ethanol
followed by 5 min. in 5.25% sodium hypochlorite amended with 0.5 m1 of
Tween 20 per 100 ml solution. After four washes in sterile distilled
water, seeds were germinated in approximately 5 ml of sterile 0.1%

nutrient broth in petri dishes.

The pathogen was cultured on the following media: 1) potato-dextrose
agar, ii) water agar prepared with 2% agar, and iii) peptone/yeast
extract medium (PYM) (18.0 9 glucose, 3.0 g yeast extract, 5.0 g
peptone, 1.4 g KZHP04, 1.7 g KH2P04, 0.5 g M9304, and one liter of
water). This medium was used for liquid culture. Solid PYM was prepared
with 2% agar.

Green leaf agar (GLA), as developed by Wiese and Ravenscroft (88),
was used throughout this work for the selective isolation of C,
gramineum from soil and plant material. Leaves from 30-day-old wheat
plants grown in the greenhouse or growth chamber were autoclaved for
5 min at 130 C and 20 lb pressure (100 9 leaves: 1 liter water). The
liquid was poured through two layers of cheesecloth and the volume was
adjusted to one liter. This extract was stored at -10C until needed
or was used immediately. Two percent agar was added to solidify the medium.

Prior to solidification, 0.9 or 1.1 g CuSO4 - 5H20 was added per liter.

28

Method for studying soil-imposed fungistasis

 

A method was devised utilizing agar on soil to test the effect of
different soils (or other planting media) and root treatments on conidial
fungistasis. Soil was passed through a 1 mm sieve, and ca. 10 g was
spread on the bottom of a petri dish. A filter paper (Whatman #1) was
placed over the soil and enough distilled water was pipetted on the
filter paper to wet the soil thoroughly and to assure good contact
between the soil and filter paper. A 6 cm x 6 cm x 1 mm square of 2%
water agar was placed on t0p of the filter paper. Conidia applied to
the surface of the agar were observed by taking sample 8 mm diam. disks
from the agar with a cork borer and examining them under the microscope.
Wheat seedling roots were placed on the agar to assess the effect of
root exudates on conidial germination.

In some studies, it was desirable to have a system in which non-
sterile field soil could be used while maintaining sterility of the
conidia and wheat roots (Figure 1). The center of a 'ring‘ out from
10 m1 of solidified 2% water agar, with a central Opening 2.5 cm diam.,
was filled with saturated (31% moisture), non-sterile field soil. A
sterile Millipore filter (0.22 um, 4.7 cm diam.) was placed over the
soil with the edge extending beyond the outer portion of the agar ring.
Disks of agar (8 x 1 mm) bearing conidia of C, gramineum on the upper
surface were placed on the center portion of the filter over the soil.
Conidia treated in this way did not germinate as long as the disks
remained in contact with the filter-covered soil (Table 1). The agar
disks did not become contaminated with other microorganisms. Conidia
placed on water agar alone without soil readily germinated, producing

copious numbers of conidia.

29

FIGURE 1. Sterile chamber used to study soil-imposed fungistasis.
All components within the petri dish are sterile except for
the soil. The bacterial-proof membrane filter maintained
the sterility of the agar disks.

 

 

 

conidia

 

 

\& (dmrdisc

 

 

fillers;

l I

1 1

 

 

 

 

FIGURE 1.

31

Electron microscopy

 

Root segments were cut into short (1-2 cm) pieces and placed in a
5% gluteraldehyde solution at pH 7.2 overnight. After two washes in
0.1 M phosphate buffer (pH 7.2) the root tissue was soaked in 1. %
osmium tetroxide overnight. Two washes in 0.1 M phosphate buffer
(pH 7.2) preceded an ethanol dehydration series. The tissue was
freeze-fractured by placing in liquid nitrogen and striking repeatedly
with a razor blade until all pieces were approximately 2-3 mm in length.
The tissue was critical-point dried using a Sorval critical-point dryer
(Ivan Sorvall, Inc., Norwalk, Conn.), and was fixed to a stub using
double stick tape. Specimens were gold coated to a thickness of
approximately 120 nm using a sputter coater (Mini-coater, Film-Vac,
Inc., Englewood, NJ). A Super Mini II scanning electron microscope
(International Scientific Instruments, Japan) was used to visualize

the samples.

Artificial wheat freezing methods

 

The freeze chamber used was supplied through the courtesy of
C. R. Olien, Department of Crop and Soil Sciences, Michigan State
University. It consisted of an insulated cabinet coupled with a large
refrigeration unit. Temperatures were controlled by setting a time
clock to trigger a motor driven valve which allowed increased amounts
of coolant to be released. Fans were used to transfer cold air from
the refrigeration unit to the freeze chamber cabinet. The minimum
_temperature desired was then held for the appropriate amount of time
before the timer turned off the cooling unit, allowing the material to

thaw.

32

The equipment used in the previous method is expensive in cost,
time, maintenance, and Space. Consequently, an inexpensive and easy-
to-use method was developed (Figure 2). A lid was constructed for a
527.5 liter (18.61 cu. ft.) chest style freezer (Frigidaire) from 3/4“
plywood in which 33, 10 cm (4 in.)-diameter holes were drilled. Plants
grown in one liter plastic cups (32T Sweethart Plastics, Inc., Wilmington,
MA 01889) containing greenhouse mix as a growth medium, were inserted in
these holes so that approximately two inches of the cup remained above
the plywood. All cups were saturated with water before the freezer was
turned on. Decreasing pot temperatures were monitored using a thermister
with a strip-chart recorder (Esterline Minigraph temperature recorder,
George R. Peters Assoc., Troy, MI 48084).

When the freezer was set at -18 C, soil temperatures fell at well
within the rates established for simulating field conditions (Figure 3).
Soil temperature within the freeze chamber was equilibrated at -5 C :_
1.5 approximately 2.5 cm above the bottom center of the cups. The soil
was frozen to within 7.5 cm of the soil surface, but not above, thus
allowing the crown tissues to remain unfrozen. Hardened and unhardened
plants were tested in this chamber without plant death occurring, which
allowed continued incubation and disease rating after exposing plants

to freeze stress.

Cation exchange chromatography and bioassay procedures

 

Procedures followed were similar to those of McKeen (37). Pasteur
pipettes (0.5 cm diam.) were packed with 1 cm of glass wool followed
by 1 m1 of wet Dowex 50-8 cation exchange resin. Ten ml of l N
hydrochloric acid was used to saturate the column followed by a water

wash. When the pH of the water remained unchanged after passage through

33

FIGURE 2. Chest freezer method of simulating freeze stress.

34

 

9.5.03 39.3 IV

 

.N mmawmm

3.210

.5489? 9:32.

 

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Goa «08

3397820

35

FIGURE 3. Soil temperature dynamics 2.5 cm from bottom center of cups
containing a greenhouse mix undergoing freezing and thawing
in the chest freezer. The data are from a typical freeze
experiment.

36

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37

the column, the root exudate sample was applied followed by five bed
volumes of water. The material which passed through constituted the
neutral and anionic fraction. The column was subsequently eluted with
five bed volumes of 2M ammonium hydroxide. This eluate comprised the
cationic fraction. The different fractions were vacuum evaporated to
dryness at 40 C before resuspension.

Root exudates were aseptically collected from wheat seedlings grown
axenically in syringes for 11 days before freezing to -1.0 C. Roots
remained alive as determined by vital staining with (3-(4,5-dimethylthiazol-
Z-Yl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT). Before staining,the
frozen roots were white and succulent, similar to unfrozen roots.

Freezing was effected in the chest freezer. The plywood lid was modified
to accept 2.5 cm diam. syringes rather than pots. Exudates collected 24 h
before and 24 h after freezing constituted the non-frozen and frozen root
exudates, respectively. The exudates were incubated overnight in methanol
(2 x the volume of exudate) and vacuum evaporated to dryness at 40 C in

a water bath. The dried material was resuspended in 1 ml water and passed
through the cation exchange column then redried under vacuum at 40 C.

The exudates were resuspended in 0.1 M phosphate buffer per ml exudate

to yield a solution of pH 6.5 containing exudate equivalent to that from
0.027 g dry root/m1. Five pl of exudate were placed with 5 pl of a

washed conidial suspension (ca. 106 conidia/ml) on an agar disk, incubated
1 hr on a sterile membrane filter covering soil, then incubated for 12 hr
with no soil contact. Results were reported as percent germination based
on the number which germinated on PDA without the influence of soil

(Table 5).

38

Quantification of sugar in wheat root exudate

 

Thirty m1 plastic disposable syringes containing 3 m1 sterile
Hoagland's solution were used as containers for the seedlings. Sterile
serum bottle caps covered the small orifice and sterile cotton plugged
the large orifice. Seedlings were grown until the first true leaf touched
the cotton plug. The cotton was removed, and the seedlings were elevated
so that the seed and root remained within and the shoot was outside the
syringe. The cotton was then aseptically replaced and syringes were
placed in conetainer trays (Ray Leach's Conetainer Nursery, Canby, OR
97013). Twenty-five m1 of sterile Hoagland's solution was injected into
each syringe, through the serum bottle cap. Fresh sterile Hoagland's
solution was periodically added to maintain a constant level.

The concentration of sugars in root exudate was determined by the
procedure of Dubois et a1. (23). Two ml of root exudate was combined
with 1 ml of 5% phenol in water in 16 mm diameter test tubes. Five m1
of concentrated (95%) sulfuric acid was quickly added the hot liquid
was left to stand for 10 min. before shaking and incubating in a 25 C
water bath for 25 min. Optical density was read at 480 mu and compared
to a standard curve made from known concentrations of glucose (Figure 4).

Results are reported in glucose equivalents (pg/ml).

RESULTS

The development of a sterile agar disk method for studying Cephaldsporium

 

gramineum conidia and soil fungistasis

 

In order to study the effect of Nutrients (i.e., root exudates) on
conidial germination, a system which allowed indefinite sterility and

intimate contact of conidia with non-sterilized soil was developed (Figure 1).

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41

The conidia were placed on water agar disks (8 x 1 mm) which had previously
been placed on a membrane filter (0.22 pm) covering non-sterilized soil.
Conidia were placed on agar disks immediately or five days after the

disks were placed on the filter. In half of the five-day treatments,

disks were removed prior to addition of conidia to determine if there was

a residual effect from the soil contained in the agar. Conidia in sterile
distilled water and on water agar disks alone (without soil) served as
controls. The conidia were incubated for 24 hr on all disks before
germination was counted. Because there was poor germination in this
experiment, germination was determined by counting the number of germinated
conidia per microscope field (Table 7). This method allowed for rapid
counting of large numbers of conidia since each field contained approxi-
mately 200 conidia.

Non-sterilized soil imposed fungistasis upon the test conidia whether
or not there was a prior incubation of the disks on the soil. Disks
incubated for five days on non-sterile soil, then removed, retained the
capacity to limit germination even though they were not in contact with
the soil during the germination period. Incubation on sterilized soil
or on water agar which had not been in contact with soil allowed signi-
ficantly more germination than non-sterilized soil. There was no
marked effect of varying the length of contact with sterile soil. Sterile
distilled water did not support germination.

Another experiment was designed to more precisely define the
relationship between the time of incubation of agar disks on soil and
the expression of fungistasis. Disks were incubated on soil for 0, 2,

6, 12 or 24 hr, after which they were aseptically placed in sterile

plastic petri plates without soil. One-half of the disks carried conidia

42

TABLE 7. Germination of Cephalosporium gramineum conidia after 24 hr
incubation on agar disks covering soil.

 

 

 

Treatment Soil Germinationa

 

l. Agar in contact with soil 5 days Natural 0 a
before addition of conidia; agar
remained on soil for conidial

incubation.
2. As 1, except agar removed after Natural 6 a
5 days Sterile 88 b
3. Conidia applied to agar at same Natural 0 a
time agar was applied to soil Sterile 68 b
4. Water agar contol -- 211 c
5. Sterile distilled water -- 0 a

 

aThe numbers represent the number of conidia germinated per microsc0pic
field (approximately 1 cm2). Values followed by the same letter do
not differ significantly (E_= 0.05).

43

before incubation on soil, the remainder had conidia placed on them at
the conclusion of these incubation periods and were allowed to germinate
for 24 hr (Table 8). The longer disks were incubated on soil, the greater
was the inhibition. Except for the longest incubation period (24 hr),
there were no differences in germination between conidia which were on
the agar disks while in contact with soil and those which were not.
When the conidia, which had been on the disks during the soil incubation,
were allowed to germinate an additional 24 hr after removal from the soil,
significantly more germination occurred. This shows the conidia were
still viable.

In another experiment the effect of a richer nutritive substrate
on germination of conidia in contact with agar disks over soil was investi-
gated. A conidial suspension (0.5 ml, 106 conidia/ml) was added to the
surface of agar disks prepared from 2% PDA, water agar, and washed
water agar. These were immediately placed on a membrane filter which
covered field soil as above. Disks prepared as above and incubated on a
membrane filter without soil served as controls. After 24 hr incubation
on soil, the percentage germination and germ tube lengths were determined.

Germ tube length, as well as percentage germination, were greater
on PDA disks than on water agar or washed water agar disks. When non-
autoclaved field soil was present, 0, 2, and 13% of the conidia germinated
on the washed water agar, water agar, and PDA, respectively. Control

disks gave 43, 53, and 56% germination, respectively.

Wheat roots as a source of nutrition for Cephalosporium gramineum

 

a. Effect of total wheat root contents on conidiogenesis
To determine the effect of wheat root contents on C, gramineum

conidiogenesis, 10 g of roots from 67-day-old wheat plants were blended

44

.psoosoo cospossesom mssssEsmpou sos sp3osm sous oos n 02s

 

o
.Amo.o ".mv asusoossssmsm Losssu pos ou sopuos mEom osp xo uozossos msoo532o
s mm W mu mu sospooooss s; um
sopso mos sass uousoeq
us sq om sospooooss ss 0
sooso mas susz uousoE<
ou ms 0o
on s we um um
sou 0s us
on 0 o o «N ms
as om we
uo ms o m um 0
so GM as
sou ms so 0N om N
nozs mu
ou us so um «N o

 

usussoo us“ so
sosasuuo op Losso ssom sosuoozoss ssom
so uoponuoss mxmso msssou mxmwu so usussou
us cospossesoa

ss .xmsu so sospooooss
sosussoo so spasms

ss .Psom so sosuooooss
xmsu so spasm;

 

 

.muossoo sospooooss moosso>
souso ssom mssso>oo mxmsu sumo so osussoo Eoosssosm sossoomosommou so sosuossssow .0 mom<s

 

45

in a Serval Omnii mixer in 50 ml distilled water at high speed for 5
minutes. The slurry was filtered through two layers of cheesecloth and
the final volume was adjusted to 100 ml with distilled water. Two 9 of
agar were added before autoclaving, then this wheat root extract agar was
poured into petri plates (10 ml per plate) and allowed to solidify.
Conidia were spread evenly over the surface of the agar. After nine
days incubation (ca. 22 C) conidial numbers were determined. Five ran-
domly selected disks of agar (8 x 1 mm) were placed in 0.25 ml of 5 N HCl
in test tubes and heated in a water bath at 80 C for l min. After
liquification of the agar, conidia were counted using a haemocytometer.
TWO percent water agar treated as above but without wheat root extract
served as a control.

Agar made from wheat root extract greatly stimulated conidial pro-
duction. There was a 665-fold increase in conidial numbers in the wheat
root extract agar compared to a 160-fold increase in water agar alone
(Table 9).

b. Effect of exudates from non-injured wheat roots on conidial
germination

Roots are known to exude nutrients which can stimulate the germina-
tion of Spores under the influence of soil imposed fungistasis (49, 70).
The following experiments were designed to measure the effect of wheat
seedling root exudation on C, gramineum conidial germination under such
conditions. Four-day-old wheat seedlings, grown aseptically, were
placed on the agar surface at the same time conidia were added, or in
another experiment, the roots were allowed to grow under the agar for
five days before addition of the conidia to the surface of the agar.

Both systems were designed to allow exudates from the roots to diffuse

46

TABLE 9. Numbers of Cephalosporium gramineum conidia produced on wheat
root agar and water agar after 9 days incubation.

 

 

 

Conidia per petriplatea

 

 

Incubation time (days) Wheat root agar Water agar
o 2.1 x 103 2.1 x103
9 1.4 x 106 3.4 x 105

 

aConidial numbers were determined using a haemocytometer after
liquification of agar disks.

47

into the agar and be available to C, gramineum conidia. Non-sterilized
soil or sand was used to induce fungistasis in these experiments. The
conidia were examined daily for four days by removing agar disks (8 x 1
mm) and observing under the microscope. The results Of both experiments
gave identical results.

Conidial germination over the soil or sand was limited to short germ
tubes (<30 um), regardless of the proximity to roots. Germination
occurred within 12 hr on water agar alone, producing COpious growth. By
24 hr new conidia were being produced.

c. Effect of exudates from wheat seedling roots subjected to
freezing and mechanical injury on conidial germination.

Roots placed under stress such as freezing soil temperatures, are
known to exude more than non-stressed roots (50). The effect Of nutrients
from frozen roots on conidial germination was examined by removing 14-day-
Old wheat plants from the sand potting medium, severing the roots from
the shoots, and freezing the roots (-11 C) for 15 hr in distilled water.
After a 9 hr thaw at 22 C, the roots were removed and the concentration
of the resulting exudate solution was adjusted to be equivalent to that

from 3.3 x 10'3

g dry root per ml water. Roots obtained as above from
,identical plants were either left intact or were cut into 1 cm lengths
after severing from the shoot. These roots were soaked for 24 hr in
distilled water at 22 C without freezing and, as before, adjusted to
3.3 x 10-3 g dry root equivalents per ml water. Equal volumes (ca.

1.0 m1) of each Of these three exudates were added to 1 m1 of a conidial
suspension (106/ml) and placed in individual wells of acid-washed,

sterilized depression slides. After incubating,overnight germination

was determined. Sterile distilled water served as a control.

48

A marked stimulation of germination occurred with frozen exudate as
compared with exudate prepared from cut or uncut roots or sterile distilled
water (Table 10).

The effects of exudate from frozen wheat roots on the germination
of spores in soil-imposed fungistasis was also investigated. Sterile,
4-day-old wheat seedlings were placed on water agar disks so that roots
came into contact with C, gramineum conidia previously placed on the
upper surface. The agar disks rested on a membrane filter which covered
non-sterile field soil (Figure l). Seedling roots were frozen immediately
before placing them on the agar disks. After various incubation times,
the conidia were observed under the microscope for germination.

Freezing was effected by adding enough sterile distilled water to
cover the roots of the seedlings which had previously been placed in
sterile 50 ml beakers. The beakers were covered with Parafilm and
placed into an ethanol bath. The temperature of the ethanol was lowered
by the addition of liquid nitrogen. A stirring bar kept the temperature
changes uniform throughout the ethanol. The water temperature dropped
from 20 C to -5 C in 12 min. After an additional 3 min at -5 C, all
beakers were removed and allowed to thaw at room temperature. Due to
contact of the crown with the frozen water, the frozen plants were killed.
Non-frozen roots were treated as above, except that they were incubated
at 20 C for 15 min. Conidia placed on disks without frozen roots served
as controls.

Frozen wheat seedling roots placed on agar disks over soil stimu-
lated abundant germination of C, gramineum conidia and vigorous growth
of mycelium (Figure 5). Non-frozen roots stimulated Sparse germination,

whereas, there was no germination in the absence Of roots, even after a

49

TABLE 10. Effect of exudate from wheat roots treated in various ways on
conidial germination and growth of Cephalosporium gramineum

on sterile depression slides.

 

 

 

 

Exudate sourcea Germination, %b Germ tube length, mb
Frozen intact roots 23 b 27.0 d
Non-frozen cut rootsC 5a 2.5 c
Non-frozen intact roots 3a 0.5 b

Water 0a 0.0a

 

aExudate was collected from l4-day-old wheat plants grown in sand.
Roots were frozen for 15 hr at -11 C; all other roots were held at
22 C. All roots were in water for a total of 24 hr.

bValues in each column followed by the same letter do not differ

significantly (P = .05)

CRoots were cut with a razor blade into 1 cm lengths before placing in

water for 24 hr.

50

FIGURE 5 (A to G). Light and electron micrographs of Cephalosporium

gramineum germination on agar covering soil ana'growth through
wheat root tissue.

 

(A to D). Nomarski interference contrast micrographs of C.
gramineum conidia on agar disks which were placed on a membrane
filter covering unsterilized soil and incubated for 24 hr.

A) No wheat roots present; conidia remain ungerminated (145 X).

B) As A, except that no soil was present below the filter (145 X).
C) As A, but with a sterile, non-frozen wheat root stimulating
some conidial germination (145 X). D) As C, except that the

wheat root had been frozen (145 X). Note the vigorous hyphal
growth.

(E to G). Scanning electron micrographs of C. ramineum penetra-
tion and growth within a frozen wheat root after 3 Hays' incubation.
E) Cross section of a frozen wheat root. Hyphae have penetrated
the epidermis and cortex, but the stele has not been colonized
(117 X). F) Hyphae penetrating the cell walls of the cortex
(118 X). G) Hyphal tips displaced from their original point

of contact (arrows) with a cortical cell wall during sample
preparation, showing apparent sites of penetration (4880 X).

51

 

FIGURE 5.

52

six-day incubation. Electron micrographs of freeze-fractured, lO-day-

old frozen wheat roots showed hyphal penetration and growth deep into

the root tissues. Therefore, not only was there stimulation Of germina-
tion and growth, overcoming soil-imposed fungistasis, there was active
penetration and colonization of roots. Non-frozen roots were not examined
with the electron microscope.

Further electron micrography studies were conducted on roots frozen
to -10 C in syringes (Figures 6 and 7) into which conidia were introduced.
After germination, hyphae grew randomly along the surface of the root
before penetrating the epidermis, either intra- or intercellularly.
Penetration of the epidermis was usually preceded by a swelling of the
hypha, probably serving as an appressorium at the point of entry. In
some cases a somewhat pointed projection from the hyphae, apparently
serving as a penetration peg, was observed to enter the epidermal cell
first. Entry made by hyphae without such a structure was also seen.

Once in the epidermis, growth was more directed through the cortex

toward the axis. Upon endodermal contact the hyphae Spread over the
surface of this tissue layer in a manner similar to that before epidermal
penetration. Growth through the endodermis was not Observed at the time
intervals sampled. Either few entry points into the stele were made,

and were therefore missed, or they occurred after the tenth day of
incubation. Non-frozen roots were also examined, but hyphal penetration
was not found to extend beyond the epidermis.

d. Root exudate fractionation.

In order to quantitate the effect Of freezing on exudation from
wheat roots a chemical index of exudation was sought. Common and

abundant components of root exudate are amino acids and sugars (70).

,5
IV

netrat1on
in
conidia.

F pie
C;

 

like

oriu*-

S
no a
rbn-

a
-r
hr

8
‘13:?

.- D
”an

v. .3

e a;;res

of ertry (arrows).

tion

ra
ry po1n

O
a'
v

P
H

:CTGIS

Ora

. In

.2

I.
O

a:

t

a»
I
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en

.
.3
E
Z

ane other

.Ql.

Hi

.3
u‘r .

.rarr
-yi

h
u '

fl
. .

54

 

FIGURE 6.

'11. I'll

I)

q.

S

‘
m 111 (11 111

‘

1
11'

I 3

l 111

In N {n ‘.‘
‘

111

u-‘a k ‘

11

U1

- a-

” 0";- (nn ‘PF .

i. | vvrvirzy O O-
a

‘a:e 0* n'EEt r
3 '-13 C, in s;
.* S'ldla. Tn
.al1zaticn of t

()1

(Jl

 

raminesm growth over
sue (475 X) following

3
's incubation with C.

stripped away to allow

Ce:talcs:crium

:t ercszerial

inges and 13 d

cortex has be

is pnase of co nization.

 

FIGURE 7 .

57

Measuring the changes in these compounds should reflect changes of mem-
brane permeability. Therefore, exudates were separated into two fractions,
i) the cationic and ii) the anionic plus neutral fractions, and these were
tested for ability to stimulate conidial germination. The procedure
used Should separate the amino acids into the cationic fraction and the
sugars into the neutral-anionic fraction.

Results indicated that the anionic-neutral fraction contained the
stimulatory component in both frozen and non-frozen root exudate. The
cationic fraction stimulated no more germination than the water control
(Table 11).

Root exudation after artificially imposed freeze stress should be
similar to that occurring under natural conditions if the freeze kinetics
closely approximate those found in the field. Wheat seedlings were
aseptically grown in syringes containing sterile Hoagland's solution for
20 days at 18 C and hardened for 20 days at 2 C. The seedlings were
then frozen follOwing established freeze-stress procedures (59) in the
chest freezer at a rate of -0.5 C/hr. A minimum temperature of -10 C
was followed by a slow thaw (0.5 C/hr). Root exudates were collected
three days before and three days after freezing. These solutions were
adjusted to contain the equivalent of exudate from 0.002 g dry root per
ml water. Two percent agar (w/v) was then added before autoclaving
and pouring into petri plates (10 ml/plate). One ml of conidia (approxi-
mately 105 conidia/m1) was added to disks (8 x 1 mm) of exudate agar and
incubated at 22 C for 1.5 days. Germination, length Of germlings, hyphal
branching, and conidial production were determined at the end of the

incubation period. Glucose equivalents were determined in the exudate.

58

.zmo. u

Mg Asusoossssmsm Losssu nos ou smppos oEom ms» xo umzoP—os msooe:z

.&mm u <00

so sosuossssom soopo< .<0s so msspossEsom omosp so omopsoosoo o op uopmouuo ms sosuossEsmwo

uopoossoo oFQEom

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soNoss msoNoss use; muoos ososoo ss um umpoo—soo osoEum u

so~ossusoz

a

.0 0._- ou soNoss
uso sous: ss mzou ss szosm mmsssuoom poms: Eoss umpoossoo Assoosuoomo osoz mowouoxm poomo

 

 

on mm uOm: sassoo oz mpoos soosusz som< souo:
no sm uom: sassoo oz mpoos poospsz sosgsoo soss:m
o _m uOm: sassoo oz mpoos poospsz sospsou Lopez
no sm sospzoz + ossossq mpoos pzozpsz sopoz
e um assosoeo modes usages: Lopez

o ms suspooz + ossossq soNoss opousxm
no mu ossospou soNoss muouzxm
o _s suspooz + ossoss< soNossusoz mpouoxm
no _u ossospou smwosslsoz mpousxm
us sospossEsoo sosuooss opsmEpoosu osossouoe
poem “was

 

 

sospossEsom so mpoos poms: soNossusos so

 

.osussoo soossEosm Eossommososmou so
sm~oss Eoss opouoxo uoposospooss so poossm

.FP mzm<s

59

The percentage germination was unusually low for all samples, and
no differences were detected (Table 12). The lengths of germlings were
not statistically different due to excessive variability. Vigor of
growth, as measured by hyphal branching and conidial production were
significantly greater in the exudate from frozen roots than in that
collected before freezing. There was approximately a 77-fold increase
in glucose equivalents in exudate from frozen roots as compared with

before freeze exudate.

Disease incidence in relation to mechanical and freeze-induced injury

 

Root severing due to winter heaving has been cited as the major
predisposing factor for infection of wheat by C, gramineum (54). Other
forms of freeze-induced root injury which cause an increase in root
exudation, but no root breakage, have not been examined in regard to
disease incidence.

The following experiments were designed to partition several
components of winter injury and evaluate them as predisposition factors
for disease.

a. Mechanical severing of roots (heaving simulation).

Wheat seedlings grown for 10 days in sand were carefully removed
and washed in tap water. One-half Of the root system was removed by
cutting with a razor blade perpendicular to the axis of the plant. The
plants were transplanted into sieved (5 mm) field soil which had 1-6 x
103 colony forming units (cfu) of C, gramineum per 9 soil. The popula-
tion consisted of the naturally occurring C, gramineum propagules with
additional conidia added from liquid cultures grown in the laboratory.

After eight weeks incubation in the greenhouse, no symptoms were observed.

6O

.muee use: mpsooo ososoa
mzeu m._ epeoooss op psos use seme opeuoxm poos so mxmsu opso uoppooso use; esussoo uesmezo

.z.pz an emv same cps; easssespOm use ._s\pooe see a Noo.o

eoss opeuoxo so psope>sooo esp ssepsoo op uopmouue use; sass .0ssNooss sepse mxeu mossp
use ososoo meeu mossp uopoospoo osoz mopeuoxo pooz .0 0s- so esopesooeop e 00 uooouss
we: mmospm oNomss .mpseso pews: uosouses .szosm xpseospoome eoss uopoo—soo osoz mopeuoxuo

.Amo. ".mv xspseossssmsm sesssu pos ou soppop maem esp so uozo—pos ssosoo soeo ss moose>e

 

 

Aseme
. Lopezv
-1 o 00.0 e 0.0 e 0.00_ e 0._ -1 pospsou
mpoos
uommospm
e 0.0 e 00.0 e 0.0 e _._ms e s.m 0e -oNoosslsoz
oNomss
0 0.00s o 00.0 o 0.N e 0.000 e 0.0 0e Lops<
eNooss
e 0.0 0e sm.0 e 0.p e s._ms e 0._ sm ososom
mpoos
uommospm
-oNomss
mpe\0 xouss .oz E; o& sospessEsoo mxeu noosoom
.mpso_e>s:oe sospwouoso .mososeso .mmsssesom .ome opeuoxm
emoospe peseseoo pegs»: so zpmcaA peeps

 

 

e,mpoos pews; szosm Aspeussmxe
.soNoss1sos use soNoss Eoss mopeuoxo op EoossEesm Sassoomosessou so omsosmoz .N_ 000<s

 

61

sesp osoa

.pmop osspmeososoo usoe ossossom-_ososo esp 00 uossEsopou m<m

.0ssssso0 smo esussoo 3o: 0—

m use .osum u m .010 u _ .esussoo 3m: 0 u 0 ”use; moosuss sospoouoss pesussouu

 

zeezsspeoov .N_ memes

62

In another experiment, lO-day-Old wheat seedlings were grown and
root-injured as above and the seedlings were i) planted with no additional
conidia in unsterilized field soil; ii) root-dipped in a conidial sus-
pension (2.4 x 106 conidia per m1) and planted in field soil; iii) planted
directly in an area (ca. 2.5 cm diam. x 1.0 cm deep) of field soil to
which five ml of the conidial suspension had been mixed; and iv) planted
in autoclaved soil treated as in (iii). The naturally occurring
populations of C, gramineum were approximately 8.3 x 102 CFU per 9 soil.
Plants were grown in a greenhouse and Observed for symptom development.

Root severing was most effective as a predisposing factor when roots
were subsequently dipped in a conidial su5pension. Placing cut roots
into naturally or artificially infested soil, either autoclaved or non-
autoclaved, gave similar low rates of infection (Table 13).

6. Artificial freeze-induced injury

Six-week-old wheat plants grown in one liter pots Of sand at 20 C,
were hardened for three weeks at 2.5 l C in a lighted growth chamber.

Cephalosporium gramineum conidia were added as a drench to each pot

 

(67 x 103 CFU per g sand). Pots were then maintained either in the
growth chamber at 2.5 1 C, or were subjected to decreasing temperatures
for 24 hr until the air temperature reached -15.0 C. The soil temperature
reached was not measured. The refrigeration was then turned off and
the pots allowed to thaw in the closed freeze chamber. All plants were
moved to a greenhouse and observed for symptom development. This
procedure has been established as an accurate simulation of winter stress
encountered in Michigan (C. R. Olein, personal communication).

Plants with cut roots had the highest percentage infection, with

those held at 2.0 C having more infection (77%) than those at -15.0 C

63

TABLE 13. Leaves colonized with Cephalosporium gramineum after cut
wheat roots were either dipped in a conidial suspension
or placed in soil infested with C, gramineum conidia.

 

 

 

Inoculation
procedurea

Soil
treatment

Leaves
colonized, %

 

Natural infestation

Natural infestation and
plant roots dipped in
conidial suspension

Additional inoculum

Additional inoculum

Non-sterile

Non-sterile

Sterilized

Non-sterile

2 a

 

aRoots of 10-day-old wheat seedlings were cut in half (perpendicular
to the axis) and plants were transplanted in soil which was either

sterilized or non-sterilized.
soil. Plant roots dépped
SUSpenSion (2.4 x 10 CFU/ml) for 3 min before pla
inoculum = 5 ml of a conidial su5pension (2.4 x 10

Natural infestation
cut roots were immersed in a conidial

8 x 102 CFU/g

ing. Additional

conidia/m1) was

mixed into a small area of soil (ca. 2.5 cm diam. x 1.0 cm deep).

bPercent colonization was determined by leaf isolations on green leaf
agar. Values followed by the same letter do not differ significantly,

(3_= .05).

64

(39%) (Table 14). In addition, symptoms developed more rapidly on plants
with cut roots. Plants exposed to 2 C showed symptoms after 21 days and
those exposed to -15.0 C after 32 days. Of plants with non—cut roots,
only those subjected to -15.0 C gave symptoms (14%) which appeared after
46 days. Such cold temperature treatments had no significant effect on
numbers of C, gramineum colony forming units. Populations were assayed
using GLA 24 hrs after 18 C or -15 C. The means of three experiments
were 13 x 104 and 8 x 104 CFU/g soil for the 18 C and -15 C treatments,
respectively.

c. Natural freeze-induced injury

Seeds were sown October 6, 1978, 3.7 cm deep in a field which had
had three consecutive wheat crops. When the plants were 2.5 to 5.0 cm
tall they were removed from the soil and placed in 10 cm diameter plastic
pots. Five plants were placed in each pot using the field soil as a
planting medium. The pots were then buried so that the soil in the pots
was level with the surrounding field soil. After various incubation
periods, pots were removed from the field, placed in a growth chamber
and incubated for 50 days. Symptom develOpment was recorded as the
percentage of plants which yielded symptoms (Table 15). The first
disease appeared in the January 13 sample after which infection increased
with each successive sampling date. The soil temperature was monitored
using a thermister at a depth of 5.0 to 7.5 cm and a strip chart
recorder (Model M00206A4-5-002, Esterline Angus, Box 24000, Indianapolis,
IN 46224). In addition, soil temperature data collected by Dr. D. E.
Linvill, Michigan State University from an area approximately 0.8 km
from the experimental field were also consulted. The elevation and soil

type were similar to those found in the experimental plots.

65

.30. n .00 328E205 Less; pa: 8 .632 2:3
esp >0 uezopsos sEosoo soeo ss moose> .mpsoessmoxo opeseoom mossp so mseoe esp use msmaeszn

.uosoeos we; AesopesoQEop ssev 0 0.m_- ssps: ss\0 0.0 apepeesxossse pe

esopesoQEop psosaee esp 0ssoouos >0 smNoss use; mpseps .sonEeso 0ssNooss esp opss mpseso esp

0ssoep0 ososoo pmou esussoo spsz uosososu use; mpoom .esussoo spsz 0sssososu ososon poa esp ss
ssom esp ossoesses sosp .oue_0 soNes e spsz ssea poos\usem esp so w 0ssso>om >0 poo use: mpooze

 

 

0 00 o 00 0.0_. p00
o 00 0 es 0.m_- pooisoz
e _0 u ms 0.0+ p30
1... e 0 . 0.0+ psoisoz
sosmmosoxo Eopsezm pmsss op 0 .mo>em_ uopoossp 0o .osopeseQEos epsoEpeosp poom

.0

esmssoo so sospsuue sopse mze0

 

.msopoezm ossspm seep Eossoomosessou so psoEoosm>ou
so msospsusoo ossNeoss op usem ss 0sszos0 mpseso pews: so osomosxo so poossm .ep 000<s

66

TABLE 15. Effect of exposing wheat plants to various periods of winter
conditions in relation to development of Cephalosporium leaf
stripe symptoms.a

 

 

Date pots were transferred from

 

field to growth chamber Plants with symptoms, %
11/20 0
l/13 2
3/7 11
5/3 28
Controlb 34

 

aSeeds were sown on October 6, 1978

bPlants were left in the field and rated for disease at anthesis.

67

Temperatures never dropped below freezing at the depths monitored.
The first freezing temperature occurred on December 31, and the soil
remained frozen for the next 31 days. The soil froze periodically until
March 17 after which no further freezing occurred. It is important to
note that the first freezing event occurred before the first symptom
development (Table 15). In addition, snow covered the ground during the
period preceding the first disease occurrence. These conditions are not
conducive to heaving Since the buffering capacity of the snow prevents
diurnal freeze/thaw cycles. In addition, disease incidence increased
steadily with time and did not appear to be related to conditions
favorable to heaving. These results agree with those of Wiese and
Ravenscroft (unpublished) who found in a four year study that disease
incidence appeared to progress linearly with time and was not necessarily
related to Spring heaving.

d. Cultivar screening procedures for resistance determination

One of the most important potential applications of freeze tests
is to allow screening of cultivars for resistance. An inexpensive
apparatus was designed for this purpose which effects rapid and repro-
ducible soil freeze profiles and, at the same time, Operates within the
established soil temperature rate changes. This 'chest freezer method:
termed as such since the freeze apparatus is constructed from a chest
freezer with a plywood pot rack, was evaluated using three cultivars Of

wheat and one Of Agrotriticum. Seeds were obtained from D. E. Mathre

 

from the University of Montana and were planted in 10 cm plastic cups.
A greenhouse soil-oat seed inoculum mixture (20:1, v/v) was used as the

planting medium and the plants were grown for one month in the greenhouse

68

before freezing (-5.0 C) the cups. Plants were then incubated in a
growth chamber at 18 C for 50 days and Observed for symptoms.

There was a good correlation between infection percentages in this
test and those found in the field (personal communication, 0. E. Mathre)

(Table 16). The most resistant plant, Agrotriticum, had significantly

 

less disease than did the most susceptible cultivar (WKP3496). A cultivar
with an intermediate level Of resistance (WKP3526) fell between the least
and most susceptible cultivars, as did WKP3512. The latter was reported
to be as susceptible as WKP3496, but low germination produced poor stands
which may have affected the results. Genesee, previously untested for

its resistance level in comparison to the cultivars in this test,

appeared to be quite resistant.

In another experiment, roots of 24-day-old seedlings, previously
grown in sand, were severed and dipped in one of various concentrations
of conidia. Concentrations ranged from 4.2 x 106 to 4.2 x 102 conidia
per ml. Subsequently, plants were placed in greenhouse soil and rated
for symptom development after 25 days (Table 17).

Disease incidence differed among the varieties. As in the freeze

test, Agrotriticum and Genesee Showed the fewest symptoms. However, the

 

remaining cultivars did not seem to correspond to their respective, field

determined, resistance levels.

DISCUSSION
Mathre and Johnston (54) concluded that hyphae from infested wheat
straw was unable to grow and infect wheat roots due to insufficient
response to root exudate. They hypothesized that passive entry of conidia
into the root system took place in the spring, after heaving had severed

these roots. Once root xylem was exposed, conidia, presumed to be drawn

69

TABLE 16. Evaluation of a modified chest freezer as a method to
screen plants for CephalOSporium leaf stripe.

 

 

 

Known relative Plants with
Cultivar resistancea symptoms, %
Agrotriticum High resistance 14 a
WKP3526 Intermediate 38 ab
resistance
WKP3512 Intermediate 34 ab
resistance
WKP3496 Low resistance 54 b
Genesee Unknown 20 a

 

aResistance levels were determined by D. E. Mathre, University
of Montana, in fields supplemented with oat inoculum of
Cephalgsporium gramineum.

 

70

TABLE 17. Effect of various concentrations of Cephalosporium gramineum

 

conidia used as a root dip on wheat plants of known
resistances after mechanical severing of their roots

 

 

Number Of plants showinggsymptomsa

 

 

 

 

° b . Unknown
Conidial conc. Most res1stant Least res1stant resistance
per ml x 4.1 Agrotriticum WKP3526 WKP3512 WKP3496 Genesee
106 0 5 5 3 0
105 0 4 2 0 1
1o4 0 1 o 0 o
103 1 1 0 0 o
102 o 0 o 0 o

 

aNumbers represent the number of plants showing symptoms per pot with
five plants per pot and one pot per treatment.

bResistance levels were determined by D. E. Mathre, University Of

Montana in fields supplemented with oat seed inoculum of C, gramineum.

71

in with transpiration water, would enter the vascular tissue, reproduce,
and colonize the plant. Such a mechanism would, in a fundamental way,

separate Cephalosporium gramineum from other vascular fungal pathogens

 

such as Fusarium Sp. and Verticillium sp. which gain entry by active

 

penetration and growth into root tissue.

The present work has shown that active growth and penetration of
wheat roots can occur in natural, unsterilized soil without root breakage.
Table 18 compares various aspects of active and passive infection.

Fungistasis was Shown to be overcome by the increase in root
exudation following freeze stress. Root breakage was not the cause of
exudation enhancement since microscopic examination did not reveal such
injury; in addition, cut roots did not release sufficient exudates to
account for this effect.

Changes in the nutrient content Of root exudate as a result Of
freezing resulted not only in increased germination but, perhaps more
importantly, increased vigor of growth and conidium production. The
end result was an increase in inoculum potential. Garrett defines inocu-
lum potential as "the energy of growth Of a parasite available for
infection of a host, at the surface Of the host organ to be infected"
(24). In this case the energy of growth goes from near zero, in the
completely fungistatic condition, to an energy level sufficient to
effect infection.

After conidial germination, runner hyphae grew over the root surface
which initiated penetration points on the epidermis. Once penetration
occurred, the epidermis and root cortex did not appear to serve as
barriers to colonization. Details of endodermal and xylary penetration

are not known since the process was not microscopically visualized.

72

 

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sopse ppm: .mssssm ss zpseom:
.0ss>eos sopse Asso szooo se0
Asospesssmsesp

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moess oxep p.seo apnenoss

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op uopesossoo Ms aspsoseoo<

uossoo

sospesooos xeoo 0ssmmeoeooso
xspeELos .oNomss sespsss
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Asospessomsesp

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3osm sous: moeps oxep se0

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usesaoNsss osspsm

mosepmsmom

sees soppe 00pm
EzessEes .0
6p asemeespepes

 

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zosm op assmsospesoz

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op sospsmoomsuoso Eoss
oespv uossoo psmpe0
sopoes sospsmosmsuoss

psooo sospoossp

 

m>Hmm<0

m>ps0<

spams

 

 

.mommoooss sospoosss o>smmeo use o>spoe so somsseseo0

.0— m0m<b

73

Therefore, the potential infection court in active penetration is the
entire rhizoplane, and the volume of soil which can contribute propagules
is that distance from the root surface in which the root exudates can
effectively support hyphal growth to the root. This volume would be
much larger than that sampled by passive infection, in which each root
can be broken in only one location to form an infection court. The
volume of soil sampled in passive infection would be that distance from
the xylem elements that transpiration water can draw in conidia. Wheat
crowns have been shown to screen out colloidal particles of gold (personal
communication, C. R. Olien) moving through xylem from the roots to the
shoots. If broken roots are unselectively sampling soil particles at
least the size of C. gramineum conidia, it would be of interest to
know how long a newly broken root would have adequate negative pressure
before becoming plugged with debris.

Smith and Olien (77) found that recovery in winter barley from

winter stress was reduced by disease incited by Fusarium roseum f. sp.

 

cerealis. They showed that freezing prediSposed the crown tissue to
disease resulting in less adventitious root development. Crown lesions,
caused by ice crystal damage, were believed to be the site of penetration.
In the present work it is not known if increased root exudation alone
is re5ponsible for disease occurrence, or if structural or physiological
changes due to freeze injury were also involved.

C, gramineum populations in soil change radically with time, reaching
a peak between December and January (95). Therefore, the likelihood of
a given volume of soil yielding infective propagules also changes with
time. Snow cover, which is common in Michigan wheat fields during

periods of peak C, gramineum p0pulations, greatly reduces an already

74

slow transpiration rate. Consequently, this would reduce the efficiency
of an infection mechanism depending on conidial uptake via the trans-
piration stream. Conversely, snow cover allows the soil to warm,
increasing the proportion of the root which is at temperatures compatible
for growth of C, gramineum. Although C, gramineum is described as a

slow grower even at its optimal temperature (20 C), its ability to grow
at low temperatures (5-7 C (9)) may afford it a competitive advantage
during the periods in which the soil is beginning to thaw. Therefore,

in Michigan, environmental and population parameters are more favorable
for active penetration than for passive entry.

In my work, wheat seedlings with severed roots, planted in an
infested greenhouse mix, yielded very low infection. However, plants
with severed roots were readily infected by applying a conidial suspen-
sion to saturated sand. Apparently, the soil type as well as the
moisture content of the soil is important to the success of passive
infection.

The freeze screening technique used in this study separated one

variety of Agrotritichum Sp. and three wheat cultivars according to

 

their field determined resistances. A test utilizing root severing as
the predisposition factor also resulted in differences in resistance;
however, the latter results were not as well correlated with field
observations.

Germination and growth in response to an external source Of nutrients
is common among soil fungi whether acting as saprophytes or pathogens.
However, successful saprophytism for C, gramineum is dependent upon
prior parasitic possession of substrate, not infestation by conidial

germination from soil. Germination would be counter-productive for

75

passive infection since this would increase the size of propagules and
make them less easily taken-up in the transpiration stream. Consequently,
germination and growth in soil in response to exudates is useful only as

a mechanism for active infection of plants. Therefore, the development
and retention of characteristics which allow germination, growth, and
infection in natural soils argues that there is a selective advantage

to these traits. Hence, active penetration is an important component

in the life cycle of C, gramineum.

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