AECV93-R5

% t ,

Blackleg of Canola
in Alberta

Investigations on Biology,

Epidemiology and Management

Prem D. Kharbanda, Ph.D., P.Ag.

S3

ENVIRONMENTAL CENTRE

M National Library Bibliothgque nationate
of Canada du Canada

BLACKLEG OF CANOLA IN ALBERTA:

Investigations on Biology, Epidemiology
and Management

by

P. D. Kharbanda, Ph.D., P.Ag.
Alberta Environmental Centre
Bag 4000, Vegreville, Alberta
T9C 1T4

November 1993

DISCLAIMER

This report contains results of research and does not imply that the uses of products discussed
have been registered by federal and provincial authorities. Mention of a product does not
constitute or imply endorsement or guarantees by the Alberta Environmental Centre, nor does it
imply endorsement over similar products not mentioned.

Additional copies of this publication can be obtained by writing to:

Communications

Alberta Environmental Centre

Bag 4000

Vegreville, AB T9C 1T4

This publication may be cited as:

Kharbanda, P.D. 1992. Blackleg of Canola in Alberta: Investigations on Biology, Epidemiology
and Management. Alberta Environmental Centre, Vegreville, AB. AECV93-R5. 85 pp.

ISBN 0-7732-1253-1

u

ACKNOWLEDGEMENTS

This research on control of blackleg disease of canola was funded in part by the Canola
Council of Canada, the Alberta Canola Producers Commission, and the Alberta Agricultural
Research Institute. Technical assistance of Mrs. R.R. Stevens, Mr. G.D. Turnbull, Mr. S.P.
Werezuk, Mr. M.J. Ostashewski and Ms. S.L. Cox, and cooperation of farm owners Mr. and Mrs.
T. Zeschuk is gratefully acknowledged.

The province-wide survey of blackleg was coordinated by Dr. I.R. Evans, Alberta
Agriculture. Professional input into surveys was also provided by several colleagues and plant
pathologists: Ms. L. Harrison, Dr. R.J. Howard, Dr. H.C. Huang, Mr. D.A. Kaminski,

Mr. S.A. Slopek, Dr. J.P. Tewari.

I am grateful to Dr. A. Petrie, Agriculture Canada Research Station, Saskatoon, for help
in identifying virulent strains of Leptosphaeria maculans , providing a virulent strain prevalent
in Saskatchewan for comparisons, and for providing scientific information on the blackleg disease
on several occasions, to Dr. P.H. Williams and Dr. A. Mengistu, University of Wisconsin for
providing representative L. maculans isolates of different pathogenicity groups, and to Dr. G.
Stringam, University of Alberta, for seed of several Brassica napus lines for differentiating
pathogenicity reaction of various L. maculans isolates. The credit for characterizing citrinin
isolated from Penicillium verrucosum goes to Dr. J.S. Dahiya who helped me with the biocontrol
of blackleg project.

I am also thankful to Mr. D. Macyk, Director, Plant Industry Division, Mr. R. Berken,
Field Services Director, Region 4, Mr. A. Hall, Field Services Director, Region 3, and Mr. P.
Thomas, Oilseed Crops Specialist, all of Alberta Agriculture, Food and Rural Development, to
Mr. A. Muchka, and to Mr. D. Elliot, Alberta Canola Producers Commission for their overall
support of the blackleg research program at the Alberta Environmental Centre.

Grateful thanks are also due to Mr. M. Herbut who took most of the photographs for this
monograph.

I also express sincere thanks to Drs. F.A. Qureshi and M.P. Sharma and Mr. H.G. Philip
for their support and encouragement during the course of the research and writing of the
manuscript, and to Drs. L.M. Dosdall, I.R. Evans and R.J. Howard for reviewing the manuscript.

in

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TABLE OF CONTENTS

PAGE

ACKNOWLEDGEMENTS in

LIST OF TABLES vii

LIST OF FIGURES ix

LIST OF PLATES x

ABSTRACT xi

1 INTRODUCTION 1

2 SYMPTOMS OF BLACKLEG OF CANOLA 2

3 THE PATHOGEN 6

3.1 Cultural and Pathogenic Variability 7

3.1.1 Cultural Variability 8

3. 1.1.1 Determination of Cultural Characteristics 8

3.1.2 Pathogenic Variability . 9

3. 1.2.1 Susceptibility of Cruciferous Weeds and Cultivated Vegetable Crops 13

4 DISEASE EPIDEMIOLOGY 21

5 OCCURRENCE AND SPREAD OF BLACKLEG IN ALBERTA 22

5.1 Survey: Methodology 22

5.2 Survey: Results 23

5.3 Survey: Role of Ascospores 28

5.3.1 Field Surveys to Search for Pseudothecia 28

5.3.2 Laboratory Tests to Induce Mating of Leptosphaeria maculans Isolates 29

6 DISEASE MANAGEMENT 34

6.1 Cultural Control 34

6.2 Resistant Cultivars 34

6.2.1 Cultivar Tolerance: Field Evaluation 35

6.2.2 Cultivar Tolerance: Results and Discussion 36

6.2.2. 1 Cultivar Susceptibility to Blackleg 36

6.2.2.2 Cultivar Response in Yield 36

6.2.2.3 Effect of Blackleg on Oil and Protein Content 37

6.3 Chemical Control 44

6.3.1 Seed Treatments 45

6.3.1. 1 Laboratory Tests 46

6.3. 1.2 Growth Chamber Tests 46

6.3. 1.3 Field Tests 47

6.3. 1.4 Results: Seed Treatments 47

6.3.2 Foliar Sprays 49

v

6.3.2. 1 Growth Chamber Tests 49

6.3.2.2 Field Tests 51

6.3.2.3 Results: Foliar Sprays 51

6.3.2.4 Ascospore Population and Timing Fungicidal Foliar Spray 57

6.3.2.5 Integration of Fungicidal Control with Disease Resistance 58

6.3.3 Chemical Control: Discussion 59

6.4 Biological Control 71

6.4.1 Isolation and Characterization of Pencillium verrucosum Metabolite 72

6.4.2 Spectral Analysis 73

6.4.3 Biological Activity of the Metabolite 73

6.4.3. 1 Effect of Heat on Metabolite 74

6.4.3. 2 Inhibitory Effect on Blackleg of Canola 74

6.4.3. 3 Biological Control: Results and Discussion 74

7 REFERENCES 80

vi

LIST OF TABLES

PAGE

Table 1. Cultural characteristics of 13 Leptosphaeria maculans isolates recovered
in Alberta and an isolate from Saskatchewan on potato dextrose agar after

21 days incubation at 21°C 10

Table 2. Reaction of cruciferous weeds to infection by Leptosphaeria maculans

strains: Foliar Inoculation 18

Table 3. Reaction of cruciferous weeds to infection by Leptosphaeria maculans

strains: Soil Inoculation 19

Table 4. Reaction of vegetable crops to infection by three Leptosphaeria maculans

strains applied with two inoculation techniques 19

Table 5. Reaction of canola cultivars ( Brassica napus) to infection by
Leptosphaeria maculans isolates belonging to known and unknown

pathogenicity groups (PG) 20

Table 6. Growth of different strains of Leptosphaeria maculans on potato dextrose

agar plates amended with one of several fungicides tested 20

Table 7. Cropping history and other features of fields infested with Leptosphaeria

maculans in north-eastern Alberta, 1984 30

Table 8. Cropping history and other features of fields infested with Leptosphaeria

maculans in north-eastern Alberta, 1985 31

Table 9. Cropping history and other features of fields infested with Leptosphaeria

maculans in north-eastern Alberta, 1986 32

Table 10. Resistance to blackleg of canola cultivars recommended for Alberta -

1993 33

Table 11. Performance of Brassica napus and Brassica rapa cultivars in a blackleg-

infested field near Sedgewick, 1992 38

Table 12. Performance of Brassica napus and Brassica rapa cultivars in a blackleg-

infested field near Lloydminster, 1992 38

Table 13. Effect of blackleg disease severity on percent oil contents in canola seeds

of several Brassica napus cultivars grown at Sedgewick, 1992 42

Table 14. Effect of blackleg disease severity on percent oil contents in canola seeds

of several Brassica napus cultivars grown at Lloydminster, 1992 42

Table 15. Inhibition by fungicidal seed treatments of Leptosphaeria maculans
growth from artificially infected canola seed on potato dextrose agar +

streptomycin medium 21 days after incubation at 21°C 48

Table 16. Analysis of variance of percent Leptosphaeria maculans colonies data

presented in Table 15 48

Table 17. Mean percent healthy seedlings of canola cv. Westar from seed treated
with different fungicides and planted in a soil mix covered with perlite

infested with Leptosphaeria maculans in a growth chamber 49

Table 18. Blackleg severity on canola plants cv. Westar following artificial
inoculation of stem or leaves and application of fungicidal foliar sprays

in a growth chamber 55

Table 19. Analysis of variance of blackleg severity on stem (i) data presented in

Table 18 55

vii

Table 20. Analysis of variance of blackleg severity on leaves data in Table 18 56

Table 21. Analysis of variance of blackleg severity on stem (ii) data in Table 18 ... 56

Table 22. Mean number of healthy plants and mean yield per plot in canola cv.

Westar artificially inoculated with Leptosphaeria maculans at growth
stage 2.4 and sprayed with different fungicides at growth stage 3.1 in

field in 1988 56

Table 23. Mean number of healthy plants and mean yield per plot of canola cv.

Westar artificially inoculated with Leptosphaeria maculans at growth
stages 2.3 and 3.5, and sprayed with different fungicides at growth stage

2.5 and 4.1 in field in 1989 57

Table 24. Effect of prochloraz (Sportak) sprays applied once at weekly intervals
during the growing season on mean disease severity and mean yield per
plot of Brassica napus cv. Legend grown in a blackleg-infested field,

1990 65

Table 25. Effect of prochloraz (Sportak) sprays applied once on different dates, at
weekly intervals, during the growing season on mean disease severity and
mean yield per plot of Brassica napus cv. Legend grown in a blackleg-

infested field, 1991 65

Table 26. Mean disease severity and mean yield per plot of two cultivars of
Brassica napus , cvs. Legend and Westar, grown in a blackleg-infested
field and sprayed once or twice with iprodione (Rovral) or prochloraz

(Sportak), 1990 66

Table 27. Analysis of variance of blackleg severity data in Table 26 66

Table 28. Mean number of healthy plants resulting from prochloraz (Sportak) or

iprodione (Rovral) sprays on Legend and Westar plots in a blackleg-

infested field 67

Table 29. Analysis of variance of number of healthy plants data shown in Figure

12 67

Table 30. Influence of heat on the biological activity Penicillium verrucosum

metabolite against Rhizoctonia solani 76

Table 31. Severity of blackleg on canola leaves inoculated with Leptosphaeria
maculans conidia alone or in a mixture with conidia of Penicillium
verrucosum 76

vm

LIST OF FIGURES

PAGE

Figure 1. Growth rate of Leptosphaeria maculans isolates recovered in Alberta at

21°C on potato dextrose agar 12

Figure 2. Blackleg of canola distribution in Alberta, 1984-87 24

Figure 3. Blackleg of canola distribution in Alberta, 1990 25

Figure 4. Blackleg of canola distribution in Alberta, 1991 26

Figure 5. Blackleg of canola distribution in Alberta, 1992 27

Figure 6. Yield response of Brassica napus and Brassica rapa cultivars in a

blackleg-infested field near Sedgewick, 1992 40

Figure 7. Yield response of Brassica napus and Brassica rapa cultivars in a

blackleg-infested field near Lloydminster, 1992 41

Figure 8. Regression of percent oil deviation from reported values and mean disease

severity in five Brassica napus cultivars, grown at two locations, 1992. ... 43
Figure 9. Mean disease severity on leaves inoculated with Leptosphaeria maculans
conidia and sprayed with a fungicide once, 4 days before inoculation or

twice, 4 days before and 6 days after inoculation 53

Figure 10. Daily counts of Leptosphaeria maculans ascospores, temperature and
precipitation measured in a blackleg-infested canola field near

Lloydminster, Alberta, 1990 62

Figure 11. Daily counts of Leptosphaeria maculans ascospores, temperature and
precipitation measured in a blackleg-infested canola field near

Lloydminster, Alberta, 1991 63

Figure 12. Number of healthy plants resulting from one or two sprays of either
iprodione or prochloraz in two cultivars of canola in a blackleg-infested

field, 1990 64

Figure 13. Chemical structure of citrinin 72

Figure 14. HPLC trace of Penicillium verrucosum metabolite (B) and of citrinin

(A) 77

Figure 15. Mass spectrum of Penicillium verrucosum culture filtrate (M+ 250) 78

IX

LIST OF PLATES

PAGE

Plate 1. Blackleg of canola symptoms (A) 3

Plate 2. Blackleg of canola symptoms (B) 4

Plate 3. Blackleg of canola symptoms (C) 5

Plate 4. Colony characteristics of five virulent Leptosphaeria maculans isolates

and one weakly virulent isolate referred to in the text as: BL-S (1), BL-N

(2), BL-B (3), BL-F(4), BL-P (5), and weakly virulent (x) 11

Plate 5. Symptoms of blackleg on cruciferous weeds artificially inoculated with

Leptosphaeria maculans (BL-A) 16

Plate 6. Symptoms of blackleg on cruciferous weeds artificially inoculated

with Leptosphaeria maculans (BL-A) 17

Plate 7. Ten day old cotyledons of Brassica napus cultivars showing differential
susceptibility to different isolates of Leptosphaeria maculans : 0(R) =

resistant; L = low; M = moderate; S = severe 18

Plate 8. Performance of Brassica napus (A) and Brassica rapa (B) cultivars in a
blackleg-infested field, 1992. Note premature ripening and lodging in

Westar severely affected with blackleg 39

Plate 9. Effectiveness of iprodione (Rovral) (a), and prochloraz (Sportak) (b),
sprayed on 6-week old canola plants 4 days after stem inoculation with

Leptosphaeria maculans 54

Plate 10. Burkard 7-day volumetric spore trap (A) and a cage (B) housing a

thermograph installed in a blackleg-infested field, 1990 60

Plate 11. Portion of tape from spore trapping showing deposits of fungal spores

including an ascospore (X) of Leptosphaeria maculans 61

Plate 12. Blackleg lesions on canola leaves inoculated with Leptosphaeria maculans
(isolate BL-A) conidia alone or in a mixture with conidia of Penicillium
verrucosum 79

x

ABSTRACT

Blackleg, caused by the highly virulent strain of Leptosphaeria maculans (Desmaz.) Ces.
& de Not. [conidial state= Phoma lingam (Tode: Fr.) Desmaz.], is a destructive disease of canola
and has inflicted serious losses to Brassica crops around the world. In Canada, blackleg was first
recorded in Saskatchewan in 1975. In Alberta, we first discovered the disease in one field near
Vermilion in 1983. Our surveys conducted over the past several years have revealed that the
disease has gradually spread throughout Agricultural Regions 4 and 3 in north-central Alberta.

The disease spreads with infected seeds and by the wind-borne ascospores and
pycnidiospores of the fungus that develop on the overwintering infected stubble. Our survey
results also indicate that the disease was introduced into Alberta most likely with infected seed
and that ascospores did not play a major role in the disease spread in Alberta until 1989.

The fungus exhibits considerable variability in cultural characteristics; 350 isolates
collected in Alberta could be grouped into five categories on the basis of overall cultural
characteristics and rate of growth on potato dextrose agar. One representative isolate from each
of the five categories was further tested for pathogenic variability and determination of race
structure in L. maculans. None of the plant species belonging to the several genera of
Crucifereae tested were found suitable to differentiate races of L. maculans. All commonly
cultivated Brassica cultivars were found highly susceptible to blackleg, whereas most of the
common Cruciferous weeds tested developed resistance to the fungus soon after emergence.
Erucastrum gallicum (dog mustard) was found as a new host of L. maculans.

To provide information on blackleg tolerance of the recently licensed canola cultivars
under Alberta conditions, research demonstration plots were set up at two farms naturally infested
with blackleg in Alberta Agriculture Regions 3 and 4 in the summer of 1992. Under heavy
blackleg disease pressure, Brassica napus cultivars Cyclone and Legend provided good yield and
disease resistance, whereas Westar was totally destroyed by the disease. Compared with Westar,
Tobin (B. rapa) developed significantly less blackleg. There was a significant negative
correlation between blackleg disease severity and loss and in percent oil content of seeds; the loss
could be estimated with the equation Y = 0.1151 - 0.2511 X, where X represents the disease
severity on a scale of 0 to 5.

xi

To develop a fungicidal control program of the disease, several fungicides were evaluated
in the laboratory, growth chamber and field as seed treatments and foliar sprays for their
protective and curative action. Seed treatment with either of the fungicides, benomyl (Benlate®
50 WP), carbathiin + thiram (UBI-2390-2, 33.3 FL), iprodione (Rovral® ST 16.7 FL), prochloraz
(Sportak® 20 SN), thiabendazole (Mertect® 45 FL), or tolclofos-methyl (Rizolex® 50 WP),
significantly suppressed seed-borne L. maculans in agar plates. In the growth chamber, iprodione
and prochloraz seed treatments effectively protected seedlings from infection up to 21 days after
seeding. In field tests, however, none of the seed treatments prevented infection in seedlings
artificially inoculated with the pathogen 15 days after seeding. Iprodione or prochloraz, sprayed
once either four days before foliage inoculation or four days after stem inoculation with L.
maculans in a growth chamber test, significantly controlled the disease. In field experiments
conducted over two years (1988 and 1989), prochloraz (500 g a.i./ha) was the most effective
fungicide under conditions of artificial inoculation. However, with natural infection and heavy
disease pressure, two sprays of prochloraz at growth stages 2.5 and 4.1 failed to control the
disease.

Field experiments were also conducted in 1990 and 1991 to develop control procedures
for the disease by determining the timing of a fungicidal foliar spray using an estimate of the
ascospore population in the air, and by utilizing partial resistance to blackleg present in the
recently licensed canola cultivar Legend ( B . napus). Foliar sprays consisted of the fungicides
prochloraz and iprodione.

The population of ascospores in the air was determined by using a Burkard 7-day
volumetric spore trap. Results of spore trapping indicated that i) ascospores were captured
between May and September each year, ii) the population of ascospores did not increase as the
growing season progressed and did not seem to peak at any particular time; however, the number
of ascospores captured was considerably higher on the days immediately following precipitation
of about 5 mm or more, and iii) numbers of ascospores captured were higher in 1991 than in
1990.

The disease severity of blackleg was higher in 1991 than in 1990. A significant control
of the disease was obtained only in 1990 when prochloraz was sprayed before the crop was six
weeks old. In 1991, spraying any time during the crop season did not control the disease. In
the cultivar Legend, which developed significantly less disease than Westar, iprodione was

xii

ineffective and two sprays of prochloraz were required to significantly reduce the disease;
however, there was no significant increase in yield due to any fungicide treatment in either
cultivar.

Biological control of the disease was attempted with Penicillium verrucosum recovered
as an aerial contaminant. High performance liquid chromatographic (HPLC) analysis of the
culture filtrate (6 weeks old) of the fungus P. verrucosum yielded an antifungal compound with
a retention time of 10.6 minutes. Based on high-performance liquid chromatography, nuclear
magnetic resonance, ultra-violet and mass spectral analysis, this compound was identified as
citrinin. The metabolite was inhibitory to L. maculans, Sclerotinia sclerotiorum , and Rhizoctonia
solani but not to Alter naria brassicae and Fusarium equiseti. Canola leaves from six- week-old
plants, when inoculated with a mixture of conidia of P. verrucosum and L. maculans , developed
blackleg which was much less severe than the disease which developed when inoculated with
conidia of L. maculans alone. However, the reported nephrotoxic nature of citrinin makes it a
non-candidate for biocontrol of blackleg and other diseases of canola.

xm

■

'

■c I

1 INTRODUCTION

Blackleg caused by Leptosphaeria maculans (Desmaz.) Ces. & de Not. [conidial state
Phoma lingam (ToderFr.) Desmaz.] has caused significant damage to Brassica crops in several
countries within the last two decades. In 1966, severe blackleg on cabbage ( Brassica oleracea
L. var. capitata L.) in several eastern states in the U.S.A was traced to infected seed produced
in Australia. In southern Australia, rapeseed was introduced as an alternative crop to wheat in
1968. Initial yields were promising, and the crop increased to about 200,000 ha in 1971.
However, blackleg occurred widely in all the main growing areas in 1971, and caused a serious
epidemic in 1972 (Bokor et al. 1975). Since then, the disease has become the major limiting
factor in the establishment of the rapeseed industry in Victoria, New South Wales and Western
Australia (Barbetti 1975a, Mcgee and Emmett 1977). From 1966 to 1968, prior to the
development of resistant cultivars, blackleg was the major disease of rapeseed in parts of France,
and caused a severe epidemic in 1966 in central France. Blackleg is one of the most important
diseases of winter rapeseed in England and Germany. In 1976-77, stem canker was damaging
in eastern and southeastern England (Gladders and Musa 1980).

In Canada, blackleg was confirmed first on oil rape [ Brassica napus L., B. campestris
(i rapa ) L.] in 1961, but it was considered of minor importance as strains that occurred were of
relatively low virulence and only produced superficial stem lesions which did not cause any yield
loss. However, in 1975, a virulent strain was isolated from rape stubble from two fields in east-
central Saskatchewan. Its prevalence and incidence increased rapidly during the following years
and severe localized stem canker outbreaks occurred in parts of the province in 1982 and 1984,
causing yield loses from 10 to 25% (Petrie 1978, 1985). The disease was reported from 92% of
Saskatchewan fields surveyed in 1989 (Jesperson 1989, 1990). The disease is quite widespread
in Manitoba where about 61% of the fields surveyed were found infested in 1989.

In Alberta, the virulent strain of L. maculans was found for the first time in 1983 in a
field near Vermilion (Kharbanda and Stevens 1983). The disease has since spread throughout
the northeastern region of the province.

The canola cultivars presently grown are all susceptible to blackleg but sources of
resistance have been identified and are currently being used in canola breeding programs. A
considerable amount of work has been done on occurrence, epidemiology and control of this
disease at the Alberta Environmental Centre, Vegreville. The purpose of this monograph is to

2

compile this information with a view to developing an integrated disease control strategy to
prevent and minimize crop losses.

2 SYMPTOMS OF BLACKLEG OF CANOLA

Leptosphaeria maculans attacks the leaves, stems and seed pods of several Brassica spp.,
and causes a variety of symptoms. Seedlings attacked early in the season develop seedling blight
(Plate la). On turnip and Swede bulbs, and on cabbage stems, it produces black dry rot in field
or storage.

On canola, leaf spots appear as dirty white, round to irregular lesions speckled with
numerous black pycnidia which in the presence of abundant moisture, exude a pink-amethyst
ooze containing spore masses (Plate lb).

On the stem, grey to tan-coloured lesions, usually with a black margin, develop near the
base of the scar remaining from fallen infected leaves. These lesions develop into crown canker
causing extensive tissue damage, and may expand in size until the plant is severed at ground
level and lodges. Less severely affected plants may remain standing but ripen prematurely; pods
fail to fill and develop shrivelled seeds. Pycnidia are frequently produced on the cankered area
and can also ooze pink spore masses (Plate 2a). Occasionally, some infected stems may not
show any external symptoms but internally there is marked blackening of the tissue (Plate 2b).
Infection of pods (Plate 2c) in field is not very frequent, but when it happens, it causes premature
pod splitting, resulting from unequal drying out of infected and uninfected portions of the halves.
Greyish mycelium may be present inside the pod. Seeds beneath the lesions are shrunken,
unsound and pale grey. Pycnidia of the fungus are sometimes visible to the naked eye on the
seed surface (Plate 3a).

A weakly virulent strain of the fungus infects plants near maturity, causing lesions which
tend to be shallow, do not have a distinct black margin and contain relatively fewer, scattered
pycnidia (Plate 3b). The weakly virulent strain causes only limited tissue damage. On the other
hand, the more aggressive (or highly virulent) strain produces deep lesions resulting in extensive
tissue damage. The lesions have a distinct black margin, and contain numerous pycnidia which
may be grouped together.

3

a. Young plants.

Plate 1. Blackleg of canola symptoms (A).

4

a. Stem

b. Stem (internal symptoms).

c. Pod.

Plate 2. Blackleg of canola symptoms (B).

5

b. Stem infected with weakly virulent Leptosphaeria maculans.

Plate 3. Blackleg of canola symptoms (C)

6

3 THE PATHOGEN

The taxonomic classification of L . maculans could be summarized as follows (Ainsworth
et al 1973):

Division: Eumycota (Filamentous fungi)

Sub-Division: Ascomycotina (Sexual reproduction resulting in the formation of

ascospores borne in asci)

Class: Loculoascomycetes (Asci bitunicate; ascocarp an ascostroma developing into
pseudothecium)

Order: Pleosporales (Locules generally polyascal; pseudoparaphyses present)

Family: Pleosporaceae (Pseudothecia mid-sized to large containing cylindrical asci
among persistent pseudothecia, superficial erumpment, or, in or on stroma, ascospores
one to many celled)

Genus: Leptosphaeria (Pseudothecial wall composed of a scleroplectenchyma of

isodiametric, thick-walled cells, pseudothecial beak short, ascospores three to many
septate brownish yellow to hyaline; mostly parasitic on stems of herbaceous plants)
The sexual stage of P . lingam was first discovered by Smith and Sutton (1964) as
L. maculans. The fungus is heterothallic (Venn 1979) and forms on dead leaves, stems, and roots
of crucifers. In culture, production of pseudothecia requires growing of opposite mating types
under specialized conditions of temperature, near ultra violet light, and organic substrate
(Mengistu et al. 1990).

The ascocarps (pseudothecia) on stems and leaves are immersed, becoming erumpent,
globose, black, with protruding ostioles, 300-500p diameter. Asci are cylindrical to clavate,
sessile or short stipitate, eight spored, 80-125xl5-22p; ascus wall bitunicated. Ascospores are
5- septate, biseriate, cylindrical to ellipsoidal, ends mostly rounded, yellow brown, slightly or not
constricted at the central septum, guttulate, 33-70x5-8p. Pseudoparaphysis filiform, hyaline and
septate (Punithalingam and Holliday 1972).

Phoma lingam , the asexual state belongs to the class Deuteromycetes or Imperfect Fungi,
order Sphaeropsidales (conidia in pycnidia), and family Sphaeropsidaceae.

Pycnidia on stems and leaves are immersed, becoming erumpment, gregarious, variable
in shape, convex, soon becoming depressed and concave, sometimes without any definite shape,

7

with narrow ostioles, 200-500p across, wall composed of several layers of cells, thick walled on
the outermost layer. Conidia hyaline, shortly cylindrical, mostly straight, some curved,
guttulated, with one guttule at each end of the conidium, unicellular 3-5x1. 5-2p (Punithalingam
and Holliday 1972).

3.1 Cultural and Pathogenic Variability

Leptosphaeria maculans exhibits considerable variability in virulence and in cultural
characteristics. Henderson (1918) noted variability in cultural characteristics of three L. maculans
isolates from Ohio and Wisconsin, U.S.A, cultured on several media and concluded that the
strains belonged to the same species. Cunningham (1927) examined pathogenicity and cultural
characteristics of about 400 isolates of P. lingam and classified them into two strains: (i)

relatively slow growing and strongly pathogenic on Brassica, and (ii) faster growing and weakly
pathogenic on Brassica . Pound (1947) also described a strain of P. lingam from Puget Sound
area of western Washington (USA) that grew rapidly in culture, was a weak pathogen on cabbage
plants, and produced a water-soluble yellow to brown pigment in culture. A number of other
investigators since reported similar phenomena and identified two strains, weakly virulent (non-
aggressive) and highly virulent (aggressive) (Bonman et al. 1981; Humpherson-Jones 1983;
Koch et al. 1989; McGee and Petrie 1978).

Recognition of strains varying in virulence is the backbone in the development of crop
cultivars with lasting resistance to that fungus in a disease management program. Not much
information on existence of distinct races of L. maculans is available because differential hosts
are not known. Koch et al. (1989) found that three B. napus cultivars, Westar, Quinta and
Glacier, could be used as differential hosts. Using these differential hosts, Mengistu et al. (1991)
could group about 100 isolates into four pathogenicity groups (PG), PG1, PG2, PG3 and PG4;
however, specific races of L. maculans could not be defined.

To explore the compositions of L. maculans populations in Alberta, I evaluated cultural
and pathogenic variability among L. maculans isolates collected between 1983-1988.

8

3.1.1 Cultural Variability

Isolates of L. maculans were obtained from infected plant stubble collected during annual
surveys in north-eastern Alberta conducted since 1983. Infected plant tissue was surface
sterilized in 1% NaOCl for 1 minute, and plated on V8-juice agar containing rose bengal and
streptomycin sulfate (McGee and Petrie 1978). The resulting L. maculans colonies were sub-
cultured on potato dextrose agar (PDA), single spored and preserved in PDA culture tubes at 4°C
until further use.

Over 350 isolates collected between 1983-1986 were single spored. The isolates varied
considerably in their morphological characteristics, amount of aerial mycelium, production of
pycnidia, color of pigmentation in the medium, and overall colony appearance. The isolates were
classified into 15 groups based on overall colony appearance and diameter (4 weeks growth).
One representative isolate was chosen for further study.

3. 1.1.1 Determination of Cultural Characteristics

Colony characteristics and rate of growth were observed on Difco PDA. A mycelial agar
disc (4 mm) from the margin of a 10-day-old colony was placed in the centre of a 90 mm plate
containing 20 mL of medium and incubated at 21° C. There were four replications for each
isolate arranged in a completely randomized design. Two diameters at right angles were
measured for each colony at 3-day intervals. The amount of aerial mycelium, color,
concentration, and size of pycnidia, and pigmentation in the medium were noted after 21 days.
The experiment was repeated once. For sporuiation assessment, 90 mm plates containing 20 mL
PDA were flooded with a 3 mL suspension containing lxlO7 conidia/mL and incubated for 10
days at 21° C. There were four replications for each isolate. Concentrations of harvested spores
(final volume 100 mL) were determined by counting four samples from each replication with a
hemacytometer. The experiment was repeated once.

Cultural characteristics of the 15 single- spored isolates tested are presented in Table 1.
Rate of growth (over 21 days) was categorized from very slow to fast using the following scale:
Very slow: colony diameter less than 40 mm
Slow: colony diameter between 41 mm and 60 mm
Moderate: colony diameter between 61mm and 80 mm
Fast: colony diameter over 81 mm

9

All weakly virulent (WV) isolates were fast growing. Several virulent isolates varied
considerably in terms of pycnidial production, aerial mycelium, colony morphology, rate of
growth and production of pigment in the culture medium. Considering all these characteristics,
the virulent isolates could be classified into five categories (Plate 4, Figure 1):

1. Very slow growing; fluffy, white mycelium; pycnidial production little (Isolate S)

2. Moderately fast growing; compact, white mycelium; very few pycnidia (Isolate N)

3. Slow growing; flat, grey mycelium; pycnidial production abundant (Isolates A,B,C,M)

4. Moderately fast growing; flat, white or grey mycelium; pycnidial production mostly
abundant (Isolates F,G,HJ,K,L)

5. Moderately fast growing; compact, white mycelium; pycnidial production abundant
(Isolates P,D)

Attempts were also made to study cultural characteristics and to group the isolates
collected in 1987 and later years. All isolates exhibited enormous cultural variability; however,
these could be placed in one of the five categories stated above. Due to the similarities of these
isolates with those found in the previous years described above, their data are not presented in
this report.

3.1.2 Pathogenic Variability

One representative isolate from each of the five categories (based upon morphological
characteristics) was tested for virulence on B. napus cv. Westar and B. rapa cv. Tobin. A
modified method of Helms and Cruickshank (1979) was followed:

Seeds of the cultivar to be tested were placed on the moistened surface of the soil mix
in a 15 cm pot and covered with 150 mL perlite mixed with 50 mL conidial suspension (5 x 106
conidia/mL). The seeded pots were covered individually with plastic bags to maintain high
humidity for 4 days. All treatments were replicated four times and arranged in a completely
randomized design in a growth chamber. The number of infected seedlings (with typical
pycnidia) in each pot were recorded at various time intervals until 28 days after seeding.

In both cultivars, the percentage of plants infected averaged between 60-80% for each
isolate indicating that both of these cultivars are equally susceptible to the L. maculans isolates
used under growth chamber conditions, and that these cultivars were not of much use as
differential hosts to identify different races of L. maculans . Therefore, attempts were made to

Table 1. Cultural characteristics of 13 Leptosphaeria maculans isolates recovered in Alberta and an isolate from Saskatchewan
on potato dextrose agar after 21 days incubation at 21°C.

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Plate 4.

Colony characteristics of five virulent Leptosphaeria maculans isolates and one
weakly virulent isolate referred to in the text as: BL-S (1), BL-N (2), BL-B (3),
BL-F(4), BL-P (5), and weakly virulent (x).

12

Figure 1. Growth rate of Leptosphaeria maculans isolates recovered in Alberta at 21°C on potato dextrose agar.

13

test virulence of the five morphologically distinct L. maculans isolates on different genera of
Cruciferae including cruciferous weeds commonly prevalent in Alberta as well as several
cruciferous vegetable crops; these plant species were chosen to obtain information on their
susceptibility to blackleg and their role in the disease epidemiology.

3. 1.2.1 Susceptibility of Cruciferous Weeds and Cultivated Vegetable Crops

The weeds and vegetables tested were: Neslia paniculata (ball mustard), Lepidium
densiflorum (common peppergrass), Erucastrum gallicum (dog mustard), Descurainia sophia
(flixweed), Capsella bursa-pastoris ( shepherd ’s-purse), Thlaspi arvense (stinkweed), Brassica
kaber var. pinnatifida (wild mustard), Raphanus raphanistrum (wild radish), Erysimum
cheiranthoides (wormseed mustard), Brassica oleracea var. botrytis (broccoli), Brassica oleracea
var. capitata (cabbage), Brassica oleracea var. botrytis (cauliflower), Brassica napus var.
napobrassica (rutabaga).

To manage the growth chamber space and time used for inoculations, the experiment was
conducted in several batches each including only two of the weeds and all the L. maculans
isolates. Each batch also included B. napus cv. Westar as a susceptible check. To avoid
problems associated with poor germination of weed seeds, the seeds were germinated in petri-
plates and only viable seedlings were placed on the soil-mix at 10 seedlings per pot. Inoculations
were done using two different techniques: (i) the germination inoculation technique described
earlier (modified from Helms and Cruickshank 1979), and (ii) a foliar inoculation technique of
Wood and Barbetti (1977) in which germinated seeds of cruciferous weeds, or seeds of other
cruciferous species, were planted at 10 per pot in 15-cm-diameter fibre pots. At the two to three
leaf growth stage (GS 2.3) (Harper and Berkenkamp 1975), seedlings were sprayed with a
pycnidial suspension of L. maculans in 0.5% gelatin containing 5 x 106 pycnidia/mL. The pots
were covered individually with plastic bags for 48 hours. The seedlings were examined for
blackleg infection 7 days after inoculation; the final data were taken 21 days after inoculation.

Results are summarized in Tables 2-5. In the foliar inoculation tests, all the weeds
tested showed resistance to most of the L. maculans isolates, except wild mustard which showed
low susceptibility to isolates BL-N and BL-P, and stinkweed which showed low susceptibility

14

to BL-A, BL-N and the Saskatchewan isolate BL-S (Table 2). Wild mustard was previously
reported to be a host of L. maculans (Petrie and Vanterpool 1968).

In the soil inoculation tests which provided heavy disease pressure on the seedlings,
cotyledons and root of several weeds developed infection (Table 3; Plate 5, 6). Dog mustard
was the most susceptible of all the weeds tested; the cotyledons on some plants were severely
infected by all the isolates except BL-S (Plate 5a). This is the first report on susceptibility of
the genus Erucastrum to L. maculans. Cotyledons of wild radish were also susceptible to several
isolates (Plate 5b). The significance of these weeds in the epidemiology of blackleg is uncertain,
however, because the weeds seem to have developed resistance to blackleg soon after emergence
as evident from the foliar inoculation test in which leaves did not develop any infection.

Variable expression of disease symptoms by the weeds to the single-spored isolates tested
shows genetic variability among different L. maculans isolates as well as variability in the genetic
make-up of the individual weed populations. Genetically homogenous populations of some of
the weeds such as dog mustard, flixweed, stinkweed, and wild radish may be useful to distinguish
L. maculans races.

All the cultivated cultivars (vegetables) belonging to several Brassica spp., except
rutabaga, were found to be extremely susceptible to the three L. maculans isolates that were
tested by either soil or foliar inoculation techniques (Table 4); rutabaga developed infection only
when planted in inoculated soil. These vegetable crops could play an important role in blackleg
epidemiology because blackleg-infected seed could spread the disease to new uninfested locations
hundreds of miles away. In a region intensively cultivated with canola, such as the Peace Region
of Alberta, this source of infection could be extremely important in introducing the disease.

Due to the extreme susceptibility of the cultivated genera of the Brassica spp., their use
as differential hosts to determine races of L. maculans is only limited. To further investigate if
more than one race of virulent strain of L. maculans exists in Alberta, the pathogenicity of the
six selected isolates was tested on genetically homogenous populations of B. napus cultivars
Westar, Quinta, and Glacier. These cultivars were recently used by Mengistu et al. (1991) to
differentiate pathogenicity groups (PG), PG1, PG2, PG3, PG4. Reaction of these pathogenistic
groups as described by Mengistu et al. (1991) is summarized below:

15

PG1

We star

Ouinta

Glacier

PG2

+

--

--

PG3

+

+

-

PG4

+

+

+

In our tests, a representative isolate of each group, obtained from the University of
Wisconsin, was included to serve as a check. A cotyledon inoculation technique described by
Mengistu et al. (1991) was followed. The disease severity was rated from low to severe based
on the lesion size on cotyledons (Plate 7). The results presented in Table 5 show that the
representative isolates belonging to different PGs did not give the reaction as reported (Mengistu
et al. 1991). Therefore, these cultivars used in our studies did not prove to be truly differential
hosts. The results indicate that further work is required to locate differential hosts to identify L.
maculans races.

In further attempts to identify L. maculans races, their differential susceptibility to
various fungicidal groups was explored. Fungicides tested were: Benlate (benomyl), HWG 1608
(ethyltrianol), Rovral (iprodione), San 619 (cyproconazole), Sportak (prochloraz), and Tilt
(propiconazole). The requisite amount of a fungicide was dissolved in 1 mL of acetone and
incorporated into 19 mL of PDA (50° C) in an 90 mm petri-plates so as to obtain a final
concentration of 50 mg/mL. On the solidified agar, a 4 mm mycelial plug of actively growing
L. maculans culture was placed in the centre of each plate. Colony diameters were measured 21
days after incubation at 21° C in the dark.

Results are presented in Table 6. On the basis of growth attained on Sportak and
Rovral, the L. maculans isolates used could be classified into four genetically diverse groups: (i)
WY, (ii) BL-A, (iii) BL-N, and (iv) BL-B, BL-F, and BL-S. Further work is required on using
such fungicidal markers to identify genetically diverse races of L. maculans.

16

a. Dog mustard ( Erucastrum gallicum).

b. Wild radish ( Raphanus raphanistrum).

Plate 5,

Symptoms of blackleg on cruciferous weeds artificially inoculated with
Leptosphaeria maculans (BL-A).

17

a. Wild mustard root ( Brassica kaber) in infested soil.

b. Stinkweed ( Thlaspi arvense) reaction to foliar inoculation.

Plate 6.

Symptoms of blackleg on cruciferous weeds artificially inoculated with
Leptosphaeria maculans (BL-A).

18

Plate 7. Ten day old cotyledons of Brassica napus cultivars showing differential
susceptibility to different isolates of Leptosphaeria maculans : 0(R) = resistant;
L = low; M = moderate; S = severe.

Table 2. Reaction of cruciferous weeds to infection by Leptosphaeria maculans strains:
Foliar Inoculation *

SPECIES

BL-A

BL-B

BL-F

BL-N

BL-P

BL-S

Wild mustard

R

R

R

L

L

R

Shepherd ’s-purse

R

R

R

Flixweed

R

R

R

R

R

Common peppergrass

R

R

R

Dog mustard

R

R

R

R

R

R

Ball mustard

R

R

R

R

R

R

Wormseed mustard

R

R

Stinkweed

L

R

R

L

R

L

Wild radish

R

R

R

R

R

R

Disease rating: R = 0% plants infected (PI); L = 1-30%.

19

Table 3. Reaction of cruciferous weeds to infection by Leptosphaeria maculans strains: Soil
Inoculation*

SPECIES

BL-A

BL-B

BL-F

BL-N

BL-P

BL-S

Wild mustard

L

L

L

L

R

L

Shepherd’ s-purse

R

R

R

Flixweed

L

R

L

R

L

R

Common peppergrass

R

R

R

Dog mustard

S

S

S

M

S

L

Ball mustard

R

R

R

R

R

R

Wormseed mustard

R

R

Stinkweed

L

L

R

R

R

R

Wild radish

M

M

R

S

L

* Disease rating:
100% PI.

R = 0% plants infected (PI); L = 1-30% PI; M

= 31-70% PI; S = 71-

Table 4. Reaction of vegetable crops to infection by three Leptosphaeria maculans strains

applied with two inoculation techniques *

Soil Inoculation

SPECIES

BL-A

BL-F

BL-P

Broccoli

S

i

S

<

j

Cabbage

s

»

S

<

j

Cauliflower

s

j

S

<

5

Rutabaga

s

i

S

<

j

Foliar Inoculation

SPECIES

BL-A

BL-F

BL-P

Broccoli

S

i

S

<

j

Cabbage

s

i

S

<

5

Cauliflower

s

i

M

<

j

Rutabaga

F

R

R

* Disease rating: R = 0% plants infected (PI); L = 1-30% PI; M = 31-70% PI; S = 71-100% PI.

20

Table 5. Reaction of canola cultivars ( Brassica napus ) to infection by Leptosphaeria
maculans isolates belonging to known and unknown pathogenicity groups (PG) *

ISOLATE

We star

Glacier

Ouinta

PG-4

S - M

S

S

PG-3

S - M

S - M

M

PG-2

S

S

S

BL-A

S

M

S

BL-B

L

L

L

BL-F

S

S

L

BL-N

L

L

M

BL-P

L

M

S

BL-S

S

L - M

L

* Disease rating: L =

low; M = medium; S

= severe (See Plate 7).

Table 6.

Growth of different strains of Leptosphaeria maculans on
plates amended with one of several fungicides tested.

potato dextrose agar

Fungicides Tested’

ISOLATE

I

II

IS

iv

V

VI

VII

Colony

mean diameter (mm)

WV

13.5

8.75

5.5

4.25

11.75

26

67.5

BL-A

4

4

4

4

4

9

44.5

BL-B

4

5.5

4

5

5

19.75

40.5

BL-F

4

5

5.5

4.5

5.25

14.75

45.25

BL-N

0

0

4.75

0

0

0

30.75

BL-P

5

7

6

6.5

7.75

24.25

45.75

BL-S

4.5

6.75

5.25

4.25

5.5

29.75

40

I = cyproconazole; II = propiconazole; HI = prochloraz; IV = benomyl; V = ethyltrianol;
VI = iprodione; VTI = Check (no fungicide).

21

4 DISEASE EPIDEMIOLOGY

The fungus overwinters on crop stubble and within and on infected seed. All infected
seeds do not give rise to infected seedlings and usually the infection is very low, about 0.6%.
However, depending upon host susceptibility and suitable environmental conditions, even 0.6%
seedling infections could result in serious disease levels. In areas where the virulent strain is
established, seed infection is of little importance to disease development but it could be the
means of introducing the pathogen in infested regions.

On the first year crop stubble, L. maculans overwinters as mycelium and pycnidiospores
and forms perfect stage pseudothecia some time before the following spring. It is not known
what conditions trigger the formation of pseudothecia. The fungus is heterothallic and, therefore,
(+) and (-) strains of the fungus need to cross for successful formation of pseudothecia (Venn
1979). Ascospores are ejected and are wind-borne over long distances. These wind-borne
ascospores are the major source of infection and their release occur at intervals following rainy
periods (Barbetti 1975b, Gladders and Musa 1980).

Ascospores and pycnidiospores primarily infect young rapeseed plants, resulting in
cotyledon, leaf and stem infection. The earlier the infection takes place, the more severe the
disease in adult plants; canola plants are most susceptible to infection from seedlings to the six-
leaf stage of growth (Barbetti 1975b, McGee and Petrie 1979).

In crops sown on or adjacent to fields in which rapeseed was grown in the previous year,
the disease sometimes becomes severe enough to cause crop failure (Gladders and Musa 1980,
McGee and Emmett 1977). Even a very small amount of infected stubble may be sufficient to
initiate an epidemic by ascospores. Pycnidiospores produced subsequently in these primary
lesions can be carried in a moisture film or by rain splash from leaf spots to the leaf axil or by
spread of mycelium down the petiole to the stem (Hammond et al. 1985). Although the
pycnidiospores do not become airborne, they can be responsible for leaf, stem and crown
infections, where they can then play a major role in the spread and establishment of the disease
within a rape crop (Barbetti 1976, McGee 1977). High rainfall in conjunction with long periods
of continuously wet days favour the spread of disease. The lack of sporulation at low
temperatures may reduce the rate of spread of the pathogen in the field, and so slow down
secondary disease spread within a crop (Barbetti 1976).

22

The pathogen can survive on crop residue for about five years and it has been shown that
ascospores can be discharged for at least three years after the original crop was grown. Under
normal field conditions, however, only a small proportion of crop residue survives more than one
year after harvest (Barbetti 1975b, McGee 1977).

5 OCCURRENCE AND SPREAD OF BLACKLEG IN ALBERTA

Field surveys to monitor blackleg of canola were initiated by the Alberta Agriculture
after virulent strains of L. maculans was found in Saskatchewan in 1976. Alberta Environmental
Centre assumed the responsibility for the disease surveys in the north eastern region (Alberta
Agriculture, Region 4) in 1981. Since the disease had spread in Saskatchewan up to about 20
km east of Lloydminster in 1981, probability of it continuing to spread westward was certain.
As well, large quantities of seed were being imported into Alberta by farmers in the Lloydminster
area. The purpose of the surveys was to determine the presence of the virulent strain of L.
maculans. (Evans et al. 1990, 1991, 1992, 1993; Kharbanda and Stevens, 1983 and Kharbanda
et al. 1988 and 1989.)

5.1 Survey: Methodology

Unless mentioned otherwise, canola fields were surveyed soon after swathing during the
first two weeks in August of each year. One field was chosen at random about every 15 km
along the secondary highways in north-central Alberta. Each field was sampled by traversing
along the path of an inverted V and examining canola plants at five spots about 30 m apart. At
each spot, 10 plants were examined visually, and the plants suspected to be infected with
blackleg were collected for laboratory testing. The virulent nature of blackleg was confirmed by
cultural methods (McGee and Petri 1978). Blackleg severity was assessed from low to severe
based upon the depth and size of stem lesions: healthy = no lesions; low = small basal lesions;
moderate = lesion up to several cm long; severe = stem girdled but not severed at base; very
severe = stem severed, plant lodged. From 1989 onward, most of the surveys were carried out
by Agricultural Fieldmen who forwarded all samples suspected of blackleg infection to the
nearest Alberta Agriculture Regional Crop Laboratories in Brooks, Fairview or Olds, or at
Vegreville (Alberta Environmental Centre); however, final diagnosis was made at Vegreville.

23

5.2 Survey: Results

The spread of blackleg of canola in Alberta is depicted in Figures 2-5. In 1983, blackleg
was found for the first time near Vermilion, about 60 km west of the Saskatchewan border at
Lloydminster, Alberta (Kharbanda and Stevens 1983). The disease was found in six fields (11%
of fields surveyed) in 1984, and in twelve fields (33%) in 1985, around Paradise Valley about
60 km south-west of Lloydminster. In 1986, the disease was found in 50% of the fields surveyed
in the same general area as in the previous years. In 1987, environmental conditions were quite
dry and the disease was found in 30% of the fields surveyed; however, it had dispersed to a
wider area in the Region (Figure 2).

In 1988, blackleg was discovered in 90% of the fields surveyed and it covered the area
from Bonnyville and Provost in the east and to Smoky Lake and Camrose in the west. The
disease was particularly severe around Paradise Valley, Viking and Sedgewick. One of the farms
lost over 60% yield. This was the first year the significant economic losses due to blackleg was
observed on a farm scale in Alberta. In 1989, in contrast to previous years results, the disease
was found only in 54% of the fields, even though the environmental conditions for blackleg
development were very conducive due to frequent rains. The most probable reason for this
reduction in number of infested fields appears to be that in response to the intensive blackleg
awareness program launched by Alberta Agriculture a majority of the farmers in this region did
not grow canola on fields where canola was cropped in the past 2-3 years and had adopted other
cultural control measures for the disease.

Our 1990 and 1992 survey results show that the disease has gradually spread in most of
Region 4 and Region 3; however, the zone of greatest disease incidence has shifted from Paradise
Valley to Sedgewick where the disease has been located mostly on farms where canola was
planted more frequently than once in three years (I.R. Evans, personal communication).

To determine the primary source of blackleg infection in Alberta, pertinent information
was collected on infected fields in 1984, 1985, and 1986. Results are presented in Tables 7, 8,
9. Since the seed planted was treated, and infested fields were very close to the Saskatchewan
border, it was presumed that the primary source of infection was ascospores that had blown
westward. However, because wind directions mostly prevail from the north-west in this region,
it was suspected that perhaps infected seed had also played some role in the disease spread. In
1985, therefore, several seed samples from co-operating farmers were tested at the Environmental

1985 BLACKLEG OF CANOLA SURVEY
INCIDENCES OF VIRULENT STRAIN

24

Figure 2. Blackleg of canola distribution in Alberta, 1984-87.

25

VIRULENT BLACKLEG
OF CANOLA
IN ALBERTA, 1991

ENVIRONMENTAL CENTRE

Computer Graphics by M. Herbut

Figure 4. Blackleg of canola distribution in Alberta, 1991.

27

28

Centre. The results were quite revealing as percent infected seeds in the infested samples varied
between 9 - 23%. This is considerably higher than that found in Saskatchewan. Petrie and
Vanterpool (1974) found a maximum seed infection of 6% in 1426 samples tested between 1969
and 1973 and concluded that diseased seed might have contributed significantly to the spread of
blackleg into new areas of production. Although the most prevalent strain in Saskatchewan in
those years was the weakly virulent strain, the numbers do indicate the explosive nature of the
disease. It is conjectured, therefore, that the virulent strain was brought into Alberta with infested
seed 2 or 3 years before it was discovered near Vermilion in 1983. To determine the role of
ascospores in the disease spread, surveys were started in 1985.

5.3 Survey: Role of Ascospores

To determine the role of ascospores in development of blackleg in Alberta, two studies
were carried out: Field surveys to search for pseudothecia and laboratory tests to induce mating
of L. maculans isolates.

5.3.1 Field Surveys to Search for Pseudothecia

Field surveys of known infected fields in south-central part of the Agriculture Region
4 were started in 1985. Each year, until 1990, stubble samples from crops found infected in the
past four years were collected. A minimum of five fields were surveyed for each infection year
and a minimum of five basal stem/root pieces per field were examined under the microscope for
the presence of pseudothecia and ascospores. No pseudothecia and ascospores were discovered
until 1988. In 1989, pseudothecia were suspected on one stubble sample from a crop found
infected in 1988 but no ascospores were observed. In 1990, pseudothecia containing immature
asci were observed on 1989 crop stubble. In 1991, however, abundant pseudothecia and asci
were observed on 1989 crop stubble collected from two separate fields.

The survey results gave clear indication that ascospores were not the source of primary
infection in the area surveyed within Agriculture Region 4 until the 1990 crop year.

In a separate field study (details under Chemical control, Section 6.3.2.4), I have been
able to trap ascospores using a Burkard spore trap in 1990 and 1991. However, very few spores
were trapped between August and October 1989; it is uncertain whether, affected by the
environmental conditions, ascospores were not released from pseudothecia or were not present.

29

Environmental conditions responsible for the development of pseudothecia in the field
are not known. The fungus is reported to be heterothallic (Venn 1979), and pseudothecia are
formed only when opposite strains of L. maculans cross. Laboratory tests were conducted to
determine if opposite strains were prevalent in the region heavily infested with blackleg in
Alberta.

5.3.2 Laboratory Tests to Induce Mating of Leptosphaeria maculans Isolates

Laboratory Tests: Ten highly virulent and five weakly virulent strains of L. maculans
collected between 1984 and 1989 from various fields distantly located in the infested region
(southern half of the Alberta Agriculture Region 4) were crossed. The isolates were crossed with
themselves, and with every other isolate using Sach’s Agar and sterilized canola stem pieces
(Petrie and Lewis 1985). The plates were incubated at 18°C under continuous near- ultraviolet
light for 70 days. No ascospores developed in any plate indicating that all isolates used in our
studies were most likely of the same strain.

In conclusion, our field surveys and laboratory studies indicate that:

(i) Blackleg was introduced into Alberta most likely with infected canola seed.

(ii) Until 1989, ascospores of L. maculans did not play a major role in the disease
spread.

(iii) With the discovery of ascospores in Alberta in the past two years, the disease
could spread at a faster rate, and the disease control might become more difficult.

30

Table 7. Cropping history and other features of fields infested with Leptosphaeria maculans
in north-eastern Alberta, 1984.

Virulent Blackleg Isolated

Field No.

Cultivar

Seed Source

Seed

Treatment

Cropping History (4 vr)
1983 Previous Canola*

OS84-35

Westar

Manitoba (Foundation)

yes

Fallow

1980

OS84-39

Westar

Sask. Wheat Pool

yes

Fallow

1980

OS 84-40

Tobin

United Oil Company

yes

Fallow

n/a

OS 84-63

Westar

United Grain Growers

yes

Fallow

n/a

Weakly Virulent Blackleg Isolated

OS 84-30

Tobin

Cargill

no

Wheat

1982

OS84-33

Candle

Seed Grower

yes

Wheat

n/a

OS 84-41

Tobin

Sask. Wheat Pool

yes

Fallow

1981

OS 84-42

Tobin

Lloyd. Wheat Pool

yes

Oats

1981

OS 84-49

Westar

United Oilseed

yes

Fallow

n/a

* na: information not available

31

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Table 9. Cropping history and other features of fields infested with Leptosphaeria maculans in north-eastern Alberta, 1986.

Virulent Blackleg Isolated

Seed treatment Cropping history (4 yr)

Field no. Severity* Cultivar Seed source 1985 Previous canola

32

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33

Table 10. Resistance to blackleg of canola cultivars recommended for Alberta - 1993.

Brassica navus

Blackleg resistance*

Brassica camvestris Blackleg resistance*

Westar

5

Tobin 4

Alto

5

AC Parkland 4

Bounty

4

Colt 4

Celebra

3

Eldorado 4

Crusher

3

Goldrush 4

Cyclone

2

Horizon 4

Delta

3

Reward 4

AC Elect

3

AC Excel

3

Garrison

2

Global

2

HC 120°

4

Hyola 401

4

Legend

3

Profit

3

Seville

3

Vanguard

3

Stallion

3

AC Tristar

5

* Disease resistance: 5-highly susceptible;
2-moderately tolerant; 1 -tolerant.

4- susceptible; 3 -moderately susceptible;

34

6 DISEASE MANAGEMENT

Since the blackleg-causing fungus is seed-bome and also overwinters in infected stubble,
most attempts to control the disease have been targeted at these two sources of primary inoculum.

6.1 Cultural Control

Cultural practices may be the most economical and practical means of controlling
blackleg. The isolation of newly sown crops from the previous season’s stubble can reduce
disease incidence, but this may be of limited practical value because few farmers have their crops
sufficiently isolated from neighbours’ infested fields to escape severe canker infection initiated
with ascospores.

Ploughing infected canola stubble is effective for reducing the amount of debris on the
soil surface, and this can apparently reduce the spread of infection to newly sown crops (Gladders
and Musa 1980, McGee and Petrie 1979).

A three to four year crop rotation is one of the best methods for controlling the disease
because the natural microflora of the soil will destroy the infested stubble and the pathogen. The
time required for stubble decomposition, however, depends upon the availability of moisture;
micro-organisms are less active under drier conditions. For the crop rotation to be effective,
fields should be kept free of volunteer canola and weeds that are susceptible to this disease.

6.2 Resistant Cultivars

All presently grown cultivars of canola in Canada are susceptible to blackleg. Although
during the past three years several moderately susceptible cultivars of B . napus have been
licensed (Table 10), there is an urgent need for developing early-maturing, blackleg tolerant
cultivars of B. napus and B. campestris .

To provide information on blackleg tolerance of the recently licensed canola cultivars
under Alberta conditions, research demonstration plots were set up at two farms naturally infested
with blackleg, one each in Alberta Agriculture Regions 3 and 4, in the summer of 1992. An
interim progress report on this project was submitted to the agencies that funded the research
(Kharbanda 1993).

35

6.2.1 Cultivar Tolerance: Field Evaluation

Two identical experiments were set up at the following two sites found infested with
blackleg in 1991:

Sedgewick: NE 16 - 45 - 11 - W4
Lloydminster: NW12 - 48 - 1 - W4
Canola cultivars used were:

B. napus - Celebra, Cyclone, Delta, Legend, Westar.

B. rapa - Horizon, Tobin.

All seed was treated with Vitavax RS® at the normal recommended rate (30mL/kg seed)
to control seed-borne blackleg, seed decay, seedling blight and flea beetles. Soil was also treated
with Furadan (1 g/6 m row) to control flea beetles and aphids.

Canola seeds were planted 2 cm deep in 1.6 m X 6 m plots containing 6 rows, 20 cm
apart, at a rate of 200 seeds per row. The experimental design was a split plot with 4
replications. The two main plots consisted of B. napus and B. rapa. Adequate fertilizer (17-20-0
15% S) was applied with seed as determined by the soil test at each location.

Seeding Dates: Lloydminster- May 26, 1992. Sedgewick- May 19, 1992.

Harvest Dates: Lloydminster- B. napus- September 1, 1992; B. rapa - August 18, 1992.

Sedgewick- B. napus- August 26, 1992; B. rapa - August 14, 1992.

To determine yield, middle 4 rows of each plot were trimmed to 5 m, harvested, dried
in bags and threshed with a small-plot thresher.

The data on blackleg severity was recorded after harvesting for each plot; the middle four
rows were used for disease evaluation. To estimate disease severity, 10 stubbles were pulled
from each of the four middle rows, and rated on a scale of 0 - 5. The disease severity scale
was: 0 = Healthy, 1 = Trace of blackleg, 2 = up to Vz stem girdled, 3 = Vz to Vz stem girdled, 4
= stem mostly girdled, plant not lodged, 5 = stem girdled, plant lodged.

To determine the effect of blackleg on oil and protein contents of the individual cultivars,
the harvested seed from each plot was analyzed for these components.

36

6.2.2 Cultivar Tolerance: Results and Discussion

6.2.2. 1 Cultivar Susceptibility to Blackleg

The blackleg severity was greater, and cultivar reaction to blackleg more clear, at the
Sedgewick than at the Lloydminster site. Cyclone was the most tolerant among all the cultivars
and developed significantly less disease than in any other cultivar tested at both the locations
(Tables 11 and 12). Cultivars Celebra, Delta and Legend were moderately susceptible and did
not differ significantly in their reaction to blackleg. On the other hand, Westar was the most
susceptible and developed sever stem cankers; at Sedgewick, it developed mean disease severity
(MDS) of 4.3, which was significantly higher than in any other B. napus or B. rapa cultivars
(Table 11), resulting in lodging and premature ripening of the plants (Plate 8).

There was no significant difference in disease susceptibility of the two B. rapa cultivars,
Tobin and Horizon; these cultivars were as tolerant as Legend (B. napus ) to blackleg (Tables 11
and 12). This is the first report demonstrating that between the two most widely grown cultivars
in Alberta, the cultivar Tobin is not as susceptible as Westar to blackleg under field conditions.

6. 2.2. 2 Cultivar Response in Yield

At Sedgewick, where the disease severity was high, Westar (4 bushels/acre) yielded
significantly less than any other cultivar (Table 11, Figure 6). Under practical farming
conditions, it would not have been possible to swath such severely affected lodged crop and
would have been a total loss. It is noteworthy that Tobin yielded better than Westar under
conditions of high disease pressure. There was no significant difference in yields of the rest of
the cultivars, however, cultivars Cyclone (30 bushels/acre) and Legend (28.8 bushels/acre)
seemed to have performed better than rest of the cultivars.

At Lloydminster, where the disease pressure was low, Westar was one of the highest
yielders confirming the high yield potential of this cultivar (Table 12, Figure 7). Celebra, on the
other hand, was the poorest yielder. At this site, yield might have been affected by snow that
fell in August 1992.

37

6. 2.2.3 Effect of Blackleg on Oil and Protein Content

Results on effect of blackleg on oil and protein contents in seed of the cultivars tested
are presented in Tables 13 and 14. There was no significant difference between cultivars in these
two characters. In the absence of completely disease-free comparative plots for each cultivar.
It is not certain how much oil or protein loss occurred due to blackleg. However, using the long-
term average percent oil content of Westar (44.3), and the average units by which each other
cultivar differs from Westar, as reported in Western Co-op Tests for canola candidate cultivars
conducted by the Western Canada Canola/Rapeseed Recommending Committee, deviation
between the expected values for each cultivar and the percent oil obtained in our tests at both the
locations was determined. The data was also analyzed using a simple linear regression procedure
(Figure 8). The results show that deviation from expected percent oil contents had significant
negative linear relationship with the disease severity and could be expressed by the equation
Y = 0.1150 - 0.251 1 X, when X represent disease severity on a scale of 0-5. The results confirm
earlier suspicions that premature ripening in blackleg affected plants might have negative impact
on oil contents of seed. The results reveal that the loss in percent oil increases with the increase
in blackleg disease severity.

In conclusion, Cyclone was the most tolerant to blackleg among the canola cultivars
tested. Under heavy disease pressure, Cyclone and Legend provided good yield and disease
resistance, whereas, Westar was extremely susceptible. However, when blackleg pressure was
low, Westar yielded as good as Cyclone and Legend. Compared with Westar, Tobin developed
significantly less blackleg. There was a significant negative correlation between blackleg disease
severity and loss in percent oil content of seeds. These conclusions are, however, based on one
year’s research and need to be confirmed for at least one more year.

38

Table 11. Performance of Brassica napus and Brassica rapa cultivars in a blackleg-infested
field near Sedgewick, 1992.

Cultivar

Mean

disease

severity*

Mean yield
(grams/plot)*

Bushels/

acre

Oil %*

Protein %*

Brassica napus

Celebra

2.612 B

451.1 A

20.1

43.44 A

26.71 A

Cyclone

1.175 C

646.6 A

28.8

43.16 A

26.16 A

Delta

2.325 B

481.0 A

21.5

43.01 A

26.12 A

Legend

2.025 B

663.5 A

30.0

43.31 A

26.34 A

Westar

4.325 A

89.2 B

4.0

43.01 A

25.34 A

Brassica rapa

Horizon

2.300 B

206.9 B

9.2

42.47 A

24.21 A

Tobin

2.350 B

232.6 B

10.4

42.13 A

24.08 A

* Mean of four replications; mean in columns followed by the same letter do not differ
significantly according to Duncan’s Multiple Range Test, (P=0.05).

Table 12. Performance of Brassica napus and Brassica rapa cultivars in a blackleg-infested
field near Lloydminster, 1992.

Cultivar

Mean

disease

severity*

Mean yield
(grams/plot)*

Bushels/

acre

Oil %*

Protein %*

Brassica napus

Celebra

1.350 B

385.2 C

17.2

43.55 A

27.48 A

Cyclone

0.700 C

517.0 ABC

23.1

43.23 A

26.77 AB

Delta

1.362 B

541.4 AB

24.2

43.16 A

26.24 B

Legend

1.237 B

540.6 AB

24.1

43.53 A

27.70 A

Westar

2.900 A

627.2 A

28.0

43.59 A

27.29 A

Brassica rapa

Horizon

1.200 B

399.4 BC

17.8

42.44 A

25.86 A

Tobin

1.237 B

367.7 C

16.4

42.47 A

25.86 A

* Mean of four replications; mean in columns followed by the same letter do not differ
significandy according to Duncan’s Multiple Range Test, (P=0.05).

39

Plate 8. Performance of Brassica napus (A) and Brassica rapa (B) cultivars in a blackleg
infested field, 1992. Note premature ripening and lodging in Westar severely
affected with blackleg.

40

41

ajoy/jaqsng

42

Table 13. Effect of blackleg disease severity on percent oil contents in canola seeds of
several Brassica napus cultivars grown at Sedgewick, 1992.

Cultivar

Mean

disease

severity*

Reported
% oil

Observed
% oil*

Deviation from
reported % oil

Brassica napus

Celebra

2.612

43.6

43.44

- 0.16

Cyclone

1.175

43.7

43.16

- 0.54

Delta

2.325

43.5

43.01

- 0.49

Legend

2.025

43.3

43.31

+ 0.01

We star

4.325

44.3

43.01

- 1.29

Brassica rapa

Horizon

2.300

43.2

42.47

- 1.74

Tobin

2.350

41.9

42.13

+ 0.23

* Mean of four replications.

Table 14. Effect of blackleg disease severity on percent oil contents in canola seeds of
several Brassica napus cultivars grown at Lloydminster, 1992.

Cultivar

Mean

disease

severity*

Reported
% oil

Observed
% oil*

Deviation from
reported % oil

Brassica napus

Celebra

1.350

43.6

43.55

- 0.05

Cyclone

0.700

43.7

43.23

- 0.47

Delta

1.362

43.5

43.16

- 0.34

Legend

1.237

43.3

43.53

+ 0.23

Westar

2.900

44.3

43.59

- 0.71

Brassica rapa

Horizon

1.200

43.2

42.44

- 0.56

Tobin

1.237

41.9

42.47

+ 0.57

* Mean of four replications.

43

Figure 8.

Regression of percent oil deviation from reported values and mean disease severity
in five Brassica napus cultivars, grown at two locations, 1992.

44

6.3 Chemical Control

Since the incidence of serious crop damage due to blackleg has increased steadily in
recent years (Hopkin et al. 1989, Jesperson 1989, Kharbanda et al. 1989, Petrie 1985, Platford
& van den Berg 1989), it has generated demand to control the disease with fungicides. Though
some fungicidal seed-slurry and seed-soak treatments have been employed successfully to control
seed-bome L. maculans (Gabrielson et al. 1977, Jacobsen & Williams 1971, Maude et al. 1984),
seed treatments have failed to control the disease under field conditions (Barbetti 1975; Brown
et al. 1976, McKenzie & Verma 1988, Rawlinson & Muthyalu 1979). In Canada, there are no
foliar fungicides registered to control blackleg. Since canola cultivars resistant to the disease are
not available for commercial use, research was initiated to develop fungicidal control of the
disease by combining seed treatments with promising foliar fungicides and partial crop resistance
to blackleg available in some recendy licensed cultivars. Also, the role of ascospores as primary
inocula to initiate development of blackleg of canola in spring has not been previously
investigated in Alberta. In this province, ascospore-producing fruiting bodies of the fungus,
pseudothecia, were not found until 1989. It is commonly believed that early spring infections
are initiated by ascospores and pycnidiospores. Recent studies in Saskatchewan show that the
fungus produces ascospores on canola stubble as early as June, about 10 months after crop
harvest. However, it is not known when, under natural conditions, the ascospore population in
the environment is at its peak during the season. This information would assist in establishing
proper timing for the fungicidal spray which should coincide with the earliest heavy build-up of
ascospores when the environmental conditions for the disease development are also favourable.

The initial objectives of this study were: (i) to determine the length of time seed

treatment fungicides provide protection against blackleg to the emerging seedlings, and (ii) to
determine protective and curative effects of fungicidal sprays against blackleg. Experiments were
conducted in controlled environments and in the field between 1986 and 1989. From all
fungicides evaluated, two of the most promising ones were selected for further field-testing in
1990 and 1991, and the study was expanded to include the following objectives: (iii) to

determine the most suitable timing of fungicidal foliar sprays to maximize control, and (iv) to
integrate fungicidal control of blackleg with resistance to the disease in a moderately susceptible
canola cultivar. Since the timing of a fungicidal spray could be affected by the number of
ascospores of L. maculans present in the environment, their concentration was monitored with

45

a spore trap in 1990 and 1991. A report was submitted to the agencies that funded the research
(Kharbanda 1992b). A part of the research results have been published (Kharbanda 1992a).

To determine the protective and curative action of fungicides, several fungicides were
screened in preliminary petri-plate tests for toxicity towards L. maculans conidia and only those
found to be toxic were evaluated further as a seed treatment, foliar spray or both. All laboratory
and growth chamber tests were repeated at least once.

A single-spore isolate of a highly virulent strain of L. maculans , BL-A, recovered from
naturally infested plants in Alberta, was used. Plants were inoculated with a conidial suspension
prepared in 0.5% gelatin solution.

The planting medium used in growth chamber tests was a pasteurized soil mix containing
peat: sand: soil (1:1:1, by volume) in 15-cm-diameter fibre pots. To ensure high humidity for
seedling infection, the pots were watered before inoculation and covered individually with plastic
bags for 48 hours after inoculation. The light and temperature regime in the growth chamber was
16 hours light/2 1°C and 8 hours dark/15°C. Light intensity was 300 mEm'V1 provided by cool
white fluorescent tubes and incandescent lamps.

Unless specified otherwise, B. napus cv. Westar was used. This and B. rapa cv. Tobin
have been widely grown in western Canada and are highly susceptible to blackleg. Westar
generally develops a heavier canopy than does Tobin, and therefore is probably more susceptible
to infection under field conditions.

6.3.1 Seed Treatments

The fungicides used for seed treatment studies were: benomyl (Benlate® 50 WP,

DuPont); carbathiin + thiram (UBI-2390-2, 33.3 FL, Uniroyal); iprodione (Rovral® ST 16.7 FL,
Rhone-Poulenc); prochloraz (Sportak® 20 SN, Elanco); propiconazole (Tilt® 25 EC,
Ciba-Geigy), thiabendazole (Mertect® 45 FL, Chipman); and tolclofos-methyl (Rizolex® 50 WP,
Sandoz).

Seed of cvs. Tobin and Westar was inoculated with L. maculans following a modified
method of Maude et al. (1984). The seed was surface-sterilized by soaking in a 1.0% solution
of NaOCl for 5 minutes, rinsed once with sterile distilled water, and then soaked for 18 hours
in a conidial suspension (107 conidia/mL) three times its volume. The soaked seeds were spread

46

on a sterile cheesecloth to dry under a fumehood for 24 hours, and stored in paper bags at 4°C
until used. The seed infestation was nearly 100%.

The inoculated or healthy seed was treated with individual fungicides at the
manufacturers’ recommended rates in batches of 100 gm in 500-mL erlenmeyer flasks. The
flasks were agitated briskly to obtain uniform coverage of the seed. The efficacy of seed
treatments was evaluated in laboratory, growth chamber and field tests.

6.3. 1.1 Laboratory Tests

Agar plate tests were conducted to determine the direct effect of seed treatments on the
seed-bome fungus and to select the more effective ones for further work. Fifty artificially
infested seeds of the two cultivars, untreated or treated with one of the fungicides, were plated
on potato dextrose agar (PDA) amended with streptomycin sulfate (150 mg/L) in 90-mm-diameter
petri-plates at 10 seeds per plate. The plates, arranged in a completely randomized design with
five replications, were incubated at 21°C. After 7 days incubation in the dark, the plates were
irradiated with near-UV lights for 16 hours per day to promote pycnidial production. The
number of L. maculans colonies on each plate was recorded after 21 days. The experiment was
repeated once, and the data were pooled. Since the percentage data varies between 0-100, arcsin
transformation was done prior to analysis of variance. The experiment was analyzed as 7
(treatments) x 2 (cultivars) factorial.

6.3. 1.2 Growth Chamber Tests

These were conducted to study protection against L. maculans conferred to young
emerging seedlings by fungicidal seed treatments. A modified method of Helms & Cruickshank
(1979) was followed: 25 healthy seeds of cv. Westar, untreated or treated with one of the
fungicides, were planted in the pasteurized soil mix. The seeds were placed on the moistened
surface of the soil mix and covered with 150 mL perlite mixed with 50 mL conidial suspension
(5xl06 conidia/mL). The seeded pots were covered individually with plastic bags to maintain
high humidity for four days. All treatments were replicated four times and arranged in a
completely randomized design in a growth chamber. The number of infected seedlings (with
typical pycnidia) in each pot were recorded at various time intervals until 28 days after seeding.

47

6.3. 1.3 Field Tests

The experiments were carried out during the summers of 1988 and 1989. Healthy seeds
of cv. Westar, untreated or treated with one of the fungicides, were planted in 6 m x 0.9 m plots,
replicated four times in a randomized complete block design. Each plot was divided into four
quadrants and seedlings in one of the quadrants in each plot were inoculated by spraying with
a conidial suspension of L. maculans (5xl06 conidia/mL). Inoculation was done 21 days after
seeding in 1988, or 15 days after seeding in 1989. Immediately after inoculation, the entire
quadrant was covered with a plastic sheet which was pegged to the ground to maintain high
humidity and removed early next morning. A second quadrant in each plot was inoculated
28 days after seeding in 1988, and 21 days after seeding in 1989. Infected seedlings were
detected in the first quadrants when observed 12 days after inoculations each year. Since the
fungicides did not appear to protect the seedlings from infection, inoculation of the remaining
two quadrants was considered unwarranted and the quadrants were abandoned.

6.3. 1.4 Results: Seed Treatments

In agar plates, all the fungicides effectively inhibited seed-bome L. maculans in both
Westar and Tobin. Prochloraz was the most effective fungicide and completely prevented fungus
recovery from infected seeds of both cultivars (Table 15). The ANOVA of this factorial
experiment (Table 16) revealed that fungicide x cultivar interaction was highly significant. The
interaction obviously was due to the decrease in effectiveness of thiabendazole and
tolclofos- methyl on Tobin relative to Westar; the interaction were not significant when the data
were analyzed without these two fungicidal treatments.

In the growth chamber test, iprodione and prochloraz effectively protected the seedlings
from blackleg infection up to 15 days after seeding in infested perlite (Table 17); benomyl and
carbathiin + thiram were the only other treatments that protected more than 50% of seedlings
from infection. Twenty-one days after seeding, iprodione was the only treatment that prevented
infection in more than 90% of the seedlings; prochloraz was the next most effective with 45%
healthy seedlings. On the 28th day, almost all seedlings in the check were infected and the
percentage of healthy seedlings for iprodione and prochloraz declined to 38 and 22, respectively,
indicating that the effectiveness of these fungicides was also reduced considerably.

48

In the field test, none of the seed treatments prevented emerged seedlings from infection
when inoculated with L. maculans conidia 21 days after seeding in 1988, or 15 days after seeding
in 1989. All inoculated seedlings in each fungicide-treated and check plot were infected.

Table 15. Inhibition by fungicidal seed treatments of Leptosphaeria maculans growth from
artificially infected canola seed on potato dextrose agar + streptomycin medium
21 days after incubation at 21°C.

Rate

% L. maculans colonies

Fungicide

(g, a.i./kg seed)

cv. Westar

cv. Tobin

Benomyl

2.0

0

6

Carbathiin + thiram

1.0 + 2.0

2

4

Iprodione

5.0

6

4

Prochloraz

2.5

0

0

Thiabendazole

2.0

0

26

Tolclofos-methyl

3.0

16

52

Check

100

100

Table 16. Analysis of variance of percent Leptosphaeria maculans colonies data presented
in Table 15.

Source

Degree of
Freedom

Sum of
Squares

F-ratio

Cultivars

1

647.81

35.02**

Fungicides

6

28938.97

260.73**

Fungicides x Cultivars

6

1014.52

9.14**

Error

14

258.99

Total

27

30860.29

** Significant at P=0.01; analysis was performed on arcsin-transformed data.

49

Table 17. Mean percent healthy seedlings of canola cv. Westar from seed treated with

different fungicides and planted in a soil mix covered with perlite infested with
Leptosphaeria maculans in a growth chamber.

Fungicide

Rate

(g, a.i./kg seed)

Percent Healthy Seedlings
Days after seedings

*

15

21

28

Benomyl

2.5

52 be

22 c

11 c

Carbathiin + thiram

1.0 + 2.0

64 b

19 c

10 c

Iprodione

5.0

100 a

91 a

38 a

Prochloraz

2.5

99 a

45 b

22 b

Propiconazole

0.025

8 d

3 d

3 c

Tolclofos-methyl

3.0

46 c

7 d

2 c

Check

10 d

5 d

1 c

* Means within a column followed by the same letter do not differ significantly (P = 0.05)
according to Duncan’s Multiple Range Test. Analyses performed on arc sin- transformed data;
values in the columns were back-transformed after analyses.

6.3.2 Foliar Sprays

Some of the fungicidal formulations evaluated were the same as for the seed treatments.
Others were: cyproconazole (SAN-619F 100 SL, Sandoz); ethyltrianol (HWG-1608 3.6 FL,
Chemagro); iprodione (Rovral® 25 FL, Rhone-Poulenc); mancozeb (Manzate-200® WP, DuPont);
prochloraz (Sportak® 40 EC, Elanco). An adjuvant was added to each fungicide except Rovral
25 FL, which contains a surfactant in its formulation. The adjuvant used with prochloraz was
Enhance® (Fatty amine ethoxylate 78%, Elanco) 0.1% (v:v), and with all other formulations it
was Agral 90® (nonylphenoxy polyethoxyethanol 90%, Chipman) 0.1% (v:v) in 1987, 1988, and
Assist® (an oil concentrate, BASF) 1.0% (v:v) in 1989. Experiments were conducted in the
growth chamber and in the field.

6.3.2. 1 Growth Chamber Tests

Untreated, healthy seed of cv. Westar was planted in pasteurized soil mix in fibre pots.
Seedlings were thinned to five per pot after emergence.

50

To determine protective action of the test fungicides, 6- week-old seedlings were sprayed
with a conidial suspension to the point of run-off four days prior to foliage inoculation. Each
fungicide was also evaluated as two sprays; the second spray was applied six days after
inoculation. The fungicide treatments were replicated four times and the pots arranged in a
completely randomized design in the growth chamber. The disease severity (DS) was evaluated
for leaves and stems four weeks after the fungicides were sprayed.

To evaluate DS on leaves, a scale was developed based on extent of leaf and petiole
infection and presence of pycnidia: 0 = no lesions; 1 = no leaf wilted, one or more

leaves/petioles with small lesions up to 1 mm in diameter, pycnidia absent; 2 = one leaf wilted
or severed from stem, other leaves with small lesions without pycnidia; 3 = up to three leaves
wilted, other leaves with small lesions without pycnidia; 4 = up to three leaves wilted, several
other leaves with lesions and pycnidia; 5 = extensive defoliation; lesions with pycnidia present
at leaf scars.

The DS on stems was evaluated using the scale: 0 = no infection, 1 = small infected
lesion, no pycnidia; 2 = lesion less than 1/3 of the stem diameter, pycnidia present; 3 = lesion
up to 2/3 of the stem diameter, plant not wilted; 4 = stem mostly girdled, plant not wilted; 5
= stem mostly girdled, tissue damage extensive, plant wilted.

All five plants in a pot (replication) were rated individually and mean disease severity
(MDS) was calculated using the formula:

wn. y (no. of plants in category x DS numerical value)

MDS = -= —

Total number of plants rated

To determine curative actions of fungicides, seedlings were sprayed either once, four
days after stem inoculation, or twice, four and 10 days after inoculation. To inoculate, a one-mm
mycelial plug from an actively growing L. maculans culture on PDA was inserted in a slit made
with a sterile scalpel at the base of a 6-week-old seedling. The wound was then covered with
a wet cotton plug and wrapped with parafilm to provide high humidity. The parafilm was
removed after 72 hours. The check plants were subjected to the same procedure without insertion
of the fungus.

51

The DS was evaluated for stems and MDS was calculated as described for foliage
inoculation experiments.

The experiments were analyzed as 6 (main treatments) x 2 (# of sprays) factorials. The
disease severity data on stem and leaf were analyzed separately by analysis of variance. When
fungicide x spray interaction was not significant, Duncan’s multiple range test was used to
separate means of different treatments.

6. 3. 2. 2 Field Tests

These were conducted with either artificial inoculation or natural infestation. All field
plots were 6 m x 0.9 m consisting of four rows 15 cm apart; the treatments were replicated four
times in a randomized complete block design. Plots were separated from each other with a
0.9 m-wide strip of barley to reduce interplot interference.

In 1988, the plots were inoculated once at the four-leaf stage, growth stage (G.S.) 2.4
of Harper and Berkenkamp (1975). In 1989, two experiments were conducted: plots in one
experiment were inoculated twice, once at G.S. 2.3 and again seven days after the fungicides
were sprayed (G.S. 2.5). The other experiment was conducted in a naturally infested field and
was not artificially inoculated.

Fungicides were sprayed at the manufacturers’ recommended rates with a knapsack
sprayer. In the experiment in 1988, the fungicides were sprayed once only at the bud stage
(G.S. 3.1). In 1989, in both experiments fungicides were sprayed either early (G.S. 2.5), or early
and late (G.S. 4.1). Check plots were sprayed with water.

6.3.2.3 Results: Foliar Sprays

In the growth chamber test, fungicide x spray interaction was significant only for the data
from inoculated leaves (Tables 18, 19, 20, 21). A graph between MDS on leaves due to various
fungicides vs. numbers of sprays revealed that the interaction was probably due to the check
treatment (water sprays) (Figure 9). This interaction disappeared when the data were analyzed
excluding the check indicating that fungicidal effects were independent of the number of sprays:
the overall trend was a decrease in mean disease severity with a second spray application.

Significant disease reduction was achieved in stem-inoculated plants by a single spray
for all treatments except benomyl which was effective only when sprayed twice (Table 18).

52

Ethyltrianol and propiconazole were, however, phytotoxic at the rates evaluated, and caused
stunting, leaf scorching and puckering. Plants sprayed even once with either iprodione or
prochloraz looked vigorous and healthy (Plate 9). Generally, the reduction in DS with two sprays
was not significantly improved relative to that achieved with one spray. Similar results were
obtained in foliage-inoculated plants. The untreated, inoculated check plants were severely
defoliated and eventually developed basal stem cankers, whereas those treated with iprodione or
prochloraz were healthy and had minimal disease on either leaves or stems (Table 18).

In the 1988 field test, under artificial inoculation, ethyltrianol and prochloraz used with
an adjuvant were the most effective treatments (Table 22). These treatments resulted in
significantly more healthy plants and better grain yield than in the check. Considerable variation
occurred among replications of the iprodione treatment and some plots looked as healthy as those
treated with ethyltrianol + adjuvant or prochloraz + adjuvant. Propiconazole + adjuvant did not
control the disease but increased yield significantly. Use of an adjuvant improved the
performance of prochloraz and propiconazole, the only two fungicides that were also tested
without an adjuvant. Benomyl was the least effective fungicide and did not significantly increase
the number of healthy plants nor increase yield. Ethyltrianol and propiconazole which were
phytotoxic in growth chamber test were less phytotoxic in the field. Some leaf scorching and
puckering was evident at an early stage of crop development but the injury declined as the crop
aged and was not noticeable in adult plants.

In 1989, when the plots were inoculated twice, prior to and seven days after the first
fungicide application, two sprays of prochloraz + adjuvant significantly increased the number of
healthy plants over the untreated check (Table 23). Among other treatments, however, no
statistically significant differences were observed.

Under conditions of natural infection in 1989, neither iprodione nor prochloraz +
adjuvant were effective, and no significant disease reduction or yield increase over the check was
obtained; since there were no significant differences, data are not presented.

53

C/D

CD

>

03

<d

c

o

>s

(D

>

<D

CO

cd

C/5

03

CD

C/5

Q

c

03

CD

2

Number of Sprays

Figure 9.

Mean disease severity on leaves inoculated with Leptosphaeria maculans conidia
and sprayed with a fungicide once, 4 days before inoculation or twice, 4 days
before and 6 days after inoculation.

54

Plate 9.

Effectiveness of iprodione (Rovral) (a), and prochloraz (Sportak) (b), sprayed on
6-week old canola plants 4 days after stem inoculation with Leptosphaeria
maculans.

55

Table 18. Blackleg severity on canola plants cv. Westar following artificial inoculation of
stem or leaves and application of fungicidal foliar sprays in a growth chamber.

Rate

Number

Mean Disease Severitv (Max. 5)t
Stem inoculation Foliage inoculation

Fungicide

(g a.i./L)

of sprays

Sterr^j)

Leaves

Stem^ii)

Benomyl

0.6

1

3.9 ab

4.8 a

2.5 b

2

3.6 b

4.1 b

2.4 b

Ethyltrianol

0.3

1*

2.1 de

1.5 c

0.5 cd

2*

1.9 de

1.0 de

0.9 c

Iprodione

0.6

1

2.0 de

0.4 fg

0.1 d

2

1.6 e

0.1 g

0.1 d

Prochloraz

0.6

1

2.3 cd

0.6 ef

0.5 cd

2

2.1 de

0.5 fg

0.3 d

Propiconazole

0.15

1*

2.7 c

1.3 cd

0.4 d

2*

2.0 de

1.0 de

0.3 d

Check

1

4.2 a

4.8 a

3.0 a

2

4.4 a

5.0 a

3.0 a

t Two 6x2 factorial experiments (stem or foliage inoculation) with 6 levels of fungicides and 2
levels of sprays. Data analyzed separately for Stem (i), (ii) and leaves. Means within a column
followed by the same letter do not differ significantly (P = 0.05) according to Duncan’s
Multiple Range Test.

* Phytotoxic.

Table 19. Analysis of variance of blackleg severity on stem (i) data presented in Table 18.

Source

Degrees of Freedom

Sum of Squares

F-ratio

Sprays

1

0.9633

9.17**

Fungicides

5

43.3367

82.55*

Sprays x Fungicides

5

0.7167

1.33NS

Error

36

3.7800

Total

47

48.7967

** Significant at P=0.05; NS, not significant.

56

Table 20. Analysis of variance of blackleg severity on leaves data in Table 18.

Source

Degrees of Freedom

Sum of Squares

F-ratio

Sprays

1

0.9918

13.54**

Fungicides

5

165.6544

452.21**

Sprays x Fungicides

5

1.1393

3.11*

Error

36

2.6375

Total

47

140.4230

*, ** Significant at P=0.05 and P=0.01, respectively.

Table 21. Analysis of variance of blackleg severity on stem (ii) data in Table 18.

Source

Degrees of Freedom Sum of Squares F-ratio

Sprays

1

0.0300

0.34NS

Fungicides

5

68.8467

156.87**

Sprays x Fungicides

5

0.5200

1.18NS

Error

36

3.1600

Total

47

72.5567

** Significant at P=0.01; NS, not significant.

Table 22. Mean number of healthy plants and

mean yield per plot in

canola cv. Westar

artificially inoculated with Leptosphaeria maculans at growth stage 2.4 and

sprayed with different fungicides at growth stage 3.1 in field

in 1988.

Rate

Mean number of

Fungicide

(g, a.i./ha)

healthy plants*

Mean yield (g)

Benomyl + adjuvant

500

22.0 b

102 cd

Ethyltrianol + adjuvant

250

39.0 a

146 c

Iprodione

500

32.0 ab

157 be

Prochloraz

500

27.0 ab

159 be

Prochloraz + adjuvant

500

40.0 a

204 ab

Propiconazole

125

20.0 b

158 be

Propiconazole + adjuvant

125

33.0 ab

248 a

Check

Nil

21.0 b

78 d

* Mean of 4 replications; means within a column followed by the same letter do not differ

significantly (P = 0.05) according to Duncan’s Multiple Range Test.

57

Table 23. Mean number of healthy plants and mean yield per plot of canola cv. Westar

artificially inoculated with Leptosphaeria maculans at growth stages 2.3 and 3.5,
and sprayed with different fungicides at growth stage 2.5 and 4.1 in field in 1989.

Fungicide

Rate

(g, a.i./ha)

Number of sprays

Mean number of
healthy plants*

Mean yield
i (g)*

Benomyl +
mancozeb

500 +
1800

1

24.0 b

365.2 ab

2

16.0 b

344.5 ab

Cyproconazole

150

1

23.0 b

392.7 ab

Cyproconazole**

150

1

14.0 b

183.7 b

2

20.0 b

398.8 ab

Ethyltrianol

125

1

22.0 b

354.0 ab

2

21.0 b

377.4 ab

Iprodione

500

1

23.0 b

288.0 ab

2

26.0 ab

461.0 ab

Prochloraz

500

1

25.0 b

363.2 ab

2

46.0 a

434.3 ab

Propiconzaole

125

1

20.0 b

357.1 ab

2

34.0 ab

489.6 a

Check

Nil

2

23.0 b

431.5 ab

* Mean of four replications; means within a column followed by the same letter do not differ
significantly (P = 0.05) according to Duncan’s Multiple Range Test.

** No adjuvant was added.

6.3.2.4 Ascospore Population and Timing Fungicidal Foliar Spray

The fungicide prochloraz, which was found most promising in the laboratory and growth
chamber experiments, was further evaluated to determine the most suitable time of spraying for
maximum control of blackleg. Experimental procedures and design were the same as described
under foliar sprays above. The cultivar used was Legend which is moderately susceptible to
blackleg. Each week, from June 13 to July 27, 1990 a set of four plots, one in each block
(replication) was sprayed once with prochloraz. A plot received only one spray during the course

58

of the experiment; unsprayed plots served as checks. The experiment was repeated in 1991; the
sprays were applied between June 19 and July 31.

To monitor the L. maculans ascospore population in air, a Burkard 7-day volumetric
spore trap (Plate 10) was set up on May 1, 1990 and 1991, and operated until October 25 in 1990
and October 7 in 1991. This machine utilizes adhesive-coated plastic tape to draw, under
vacuum, deposits from the air on a continuous basis, seven days a week. A new tape was
installed once a week, and the one removed cut up and mounted on slides for viewing under
microscope to determine the population of ascospores in the field (Plate 11). A seven-day
thermograph to check temperature variations and a rain gauge were also set up close to the spore
trap to collect supplementary data on environmental conditions that influence ascospore release
and blackleg development.

In 1990, significant control of blackleg was achieved with prochloraz fungicide only
when canola was sprayed before it was six weeks old (July 6, Table 24). None of the sprays,
however, improved yield. The daily spore count data show (Figure 10) that ascospores were
caught between May 15 and September 15 but relatively larger numbers of spores were caught
within a day or so after there was a precipitation of over 5 mm; the daily maximum temperature
on these days was below 15°C.

In 1991, the disease severity was generally higher than in 1990, and spraying of
prochloraz any week during the growing season did not cause a significant reduction in mean
disease severity or increase yield (Table 25). As well, the spore trap data show that frequency
of occurrences of large numbers of ascospores was greater in 1991 than in 1990
(Figures 10 & 11).

6.3.2.5 Integration of Fungicidal Control with Disease Resistance

In 1990, seed of a susceptible (Westar) and a moderately susceptible (Legend) cultivar
were planted in field plots keeping the procedure and experimental design the same as specified
above under foliar sprays. Fungicide, iprodione or prochloraz, was sprayed either once (early,
mid- June) or twice (early and late, mid-June and early July) on the two cultivars.

DS and seed yield were determined using all plants in 5-m-sections of the middle two
rows of each plot. The DS was evaluated on the scale described for stem infection in growth
chamber experiments. To determine disease control achieved due to fungicides, the numbers of

59

healthy plants for each treatment were compared. Plants with severities of 0, 1 or 2, which have
a minimal effect on canola yield (unpublished data), were pooled as healthy plants. To determine
yield, plants were hand-pulled and threshed.

The data were analyzed as 2 (cultivars) x 3 (fungicides) x 2 (sprays) factorial. Two
separate analyses were done using MDS or number of healthy plants resulting from various
treatments (Tables 26, 27, 28, 29; Figure 12); however, the conclusions drawn from the two
analyses were essentially the same. ANOVA of blackleg severity data show that none of the
various interactions were significant, but the differences within varieties and fungicides were
highly significant (Table 27). Further data analysis with Duncan’s multiple range test showed
that the mean disease severity in unsprayed Legend was significantly less than that in unsprayed
Westar confirming that Legend has superior blackleg tolerance. A significant reduction in disease
severity occurred in Westar with a single spray of either of the two fungicides. In Legend, which
developed considerably less disease than Westar to begin with, iprodione was ineffective and it
required two sprays of prochloraz to significantly reduce the disease severity compared with the
untreated check (Table 26).

In Westar, two sprays of prochloraz caused a significantly greater reduction in mean
disease severity of blackleg than two sprays of iprodione (Table 26). When data for Legend and
Westar were pooled, the disease reduction achieved with prochloraz was significantly greater than
with iprodione (Table 28; Figure 12).

6.3.3 Chemical Control: Discussion

Studies reported here demonstrate good protective and curative properties of iprodione
and prochloraz against L. maculans on canola under controlled environmental conditions. When
used as seed treatments these fungicides effectively suppressed seed-borne inoculum and also
protected seedlings from infection from external sources for about three weeks after seeding in
the growth chamber. Benzimidazole fungicides, e.g. benomyl and thiabendazole, were also quite
effective for controlling the seed-borne fungus (Table 15), which confirms earlier reports of
Brown etal. (1976) and Maude et al. (1984) on effectiveness of benomyl seed treatments against
L. maculans. It is noteworthy that benomyl, which showed acute fungitoxicity to seed-bome L.
maculans (Table 15), was not very effective for protecting seedlings (Table 17).

60

Plate 10. Burkard 7-day volumetric spore trap (A) and a cage (B) housing a thermograph installed in a blackleg-infested field,
1990.

61

Plate 11. Portion of tape from spore trapping showing deposits of fungal spores including an ascospore (X) of Leptosphaeria
maculans.

62

(lulu) uopjidpejd

63

(ujlu) uojjBjidpaid

(CL) ajrnBJadiuei

lunoo aiodsoosv

00

£

64

Figure 12. Number of healthy plants resulting from one or two sprays of either iprodione or prochloraz in two cultivars of canola
in a blackleg-infested field, 1990.

65

Table 24. Effect of prochloraz (Sportak) sprays applied once at weekly intervals during the

growing season on mean disease severity and mean yield per plot of Brassica
napus cv. Legend grown in a blackleg-infested field, 1990.

Timing of spray*

Mean disease severity**

Mean yield (grams/plot)**

June 13

0.7 BC

208 A

June 20

0.8 BC

217 A

June 27

0.7 BC

223 A

July 6

0.6 C

199 A

July 13

0.9 ABC

195 A

July 20

0.8 ABC

193 A

July 27

1.1 AB

211 A

No spray

1.2 A

220 A

* Fungicide prochloraz was

sprayed at 500 g ai/ha. The first date represents canola growth stage

2.4.

** Mean of four replications; means in columns followed by the same letter do not differ
significantly according to Duncan’s Multiple Range Test (P=0.05).

Table 25. Effect of prochloraz (Sportak) sprays applied once on different dates, at weekly

intervals, during the growing season on mean disease severity and mean yield per
plot of Brassica napus cv. Legend grown in a blackleg-infested field, 1991.

Timing of spray*

Mean disease severity**

Mean yield (grams/plot)**

June 19

2.1 A

334 A

June 26

2.3 A

314 A

July 3

2.2 A

345 A

July 10

2.2 A

286 A

July 17

2.0 A

288 A

July 24

2.3 A

305 A

July 31

2.4 A

295 A

No spray

2.4 A

324 A

* The fungicide prochloraz was sprayed at 500 g ai/ha. The first date represents canola growth
stage 2.4.

** Mean of four replications; means in columns followed by the same letter do not differ
significantly according to Duncan’s Multiple Range Test (P=0.05).

66

Table 26.

Mean disease severity and mean yield per plot of two cultivars of Brassica napus ,
cvs. Legend and Westar, grown in a blackleg-infested field and sprayed once or
twice with iprodione (Rovral) or prochloraz (Sportak), 1990.

Cultivar

Fungicide*

# of
sprays**

Mean disease
severity#

Mean yield
(grams/plot)#

Legend

Check

0

1.7 DE

220 AB

Legend

Iprodione

1

1.8 DE

315 A

Legend

Iprodione

2

1.7 DE

262 AB

Legend

Prochloraz

1

1.4 EF

302 A

Legend

Prochloraz

2

0.9 F

271 AB

We star

Check

0

3.9 A

159 B

We star

Iprodione

1

2.8 BC

192 AB

We star

Iprodione

2

3.3 AB

189 AB

We star

Prochloraz

1

2.6 C

214 AB

Westar

Prochloraz

2

2.2 CD

264 AB

* Each fungicide was sprayed @ 500 g ai/ha.

** First spray was applied at the growth stage 2.4; the second two weeks later.

# Mean of four replications; mean in columns followed by the same letter do not differ
significantly according to Duncan’s Multiple Range Test (P=0.05).

Table 27. Analysis of variance of blackleg severity data in Table 26.

Source

Degree of Freedom

Sum of Squares

F-Ratio

VAR

1

21.2576

108.44**

FUN

2

6.5034

16.59**

SPRAY

1

0.1405

0.72 NS

VAR*FUN

2

1.4928

3.81 NS

VAR*SPRAY

1

0.2178

1.11 NS

FUN* SPRAY

1

0.7021

3.58 NS

VAR*FUN* SPRAY

1

0.1653

0.84 NS

Error

30

5.8807

Total

39

36.3602

** Significant at P = 0.01; NS = not significant.

67

Table 28. Mean number of healthy plants resulting from prochloraz (Sportak) or iprodione

(Rovral) sprays on Legend and Westar plots in a blackleg-infested field.

Fungicide

Mean # of healthy plants*

Prochloraz

37 A

Iprodione

24 B

Check

21 B

* Means of four replications; means in columns followed by the same
significantly according to Duncan’s Multiple Range Test (P=0.01).

letter do not differ

Table 29. Analysis of variance of number of healthy plants data shown in Figure 12.

Source

Degree of Freedom

Sum of Squares

F-ratio

VAR

1

5428.90

32.70**

FUN

2

1881.54

5.67**

SPRAY

1

124.03

0.75 NS

VAR*SPRAY

2

117.54

0.35 NS

VAR*FUN

1

344.53

2.08 NS

FUN* SPRAY

1

34.03

0.21 NS

VAR*FUN* SPRAY

1

52.53

0.32 NS

Error

30

4980.00

Total

39

12963.10

** Significant at P = 0.01; NS = not significant.

Several workers reported failure of fungicidal seed treatments to control blackleg of
canola (McKenzie & Verma 1988, Verma & McKenzie 1982, Brown et al. 1976). Although
Brown et al. (1976) observed that benomyl seed treatments (1.1% w/w) completely protected
rapeseed cotyledons against L. maculans for 10 days after germination under greenhouse
conditions, they did not investigate the protection afforded by benomyl beyond 10 days. One
objective of the present study was to determine the longevity of several seed treatment fungicides
in seedlings to determine if their effectiveness lasted until the crop reached the six-leaf stage and

68

acquired some natural resistance to blackleg (McGee & Petrie 1979). Subsequently, a foliar
spray with benomyl or iprodione, registered for use against sclerotinia stem rot, might give
additional control of blackleg and so integrate control of the two important diseases. Although
iprodione and prochloraz were reasonably effective for reducing blackleg incidence in canola
until 21 days after seeding in the growth chamber tests, in field trials all fungicides when tested
15 days after seeding were found ineffective. No information on the breakdown of these
fungicides in canola is available in the scientific literature. It is likely that the concentration of
these fungicides in the expanded cotyledons is too low to protect the seedling from infection by
L. maculans. Evidently, benomyl, carbathiin or iprodione, presently registered for seed treatment
on canola in Canada, do not protect seedlings from blackleg infection for a desirable period of
5-6 weeks if canola is planted on blackleg-infected stubble.

Thiabendazole and tolclofos-methyl used as seed treatments to control seed-borne L.
maculans performed better on cv. Westar than on cv. Tobin in laboratory tests (Table 15).
Significant differences between the responses of the cultivars to some of the fungicidal seed
treatments is also evident from the results of ANOVA (Table 16). Perhaps the larger seeds of
cv. Westar facilitate uptake of more fungicide than do the smaller seeds of cv. Tobin.

Conflicting results have been reported by several workers on control of blackleg with
fungicidal foliar sprays (Barbetti 1975; Brown et al. 1976; Rawlinson et al. 1984). My results
with artificially inoculated plants in field and growth chamber tests indicate that when inoculum
pressure is not continuous over the growing season one spray of prochloraz + adjuvant
significantly reduced the DS resulting in an increased number of healthy plants and increased
yield. This fungicidal spray could be very useful to control blackleg in uninfested regions where
the disease is introduced with infected seed. However, in fields where the source of primary
inoculum is infested stubble, providing conidia and ascospores throughout the growing season
(as confirmed by my spore-trap studies), foliar-applied fungicides may not give a satisfactory
control of the disease. This is also supported by recent results of Morrall et al. (1988) and
Rawlinson et al. (1984) showing poor control of blackleg with multiple applications of prochloraz
in the field.

In the ANOVA for the disease severities on parts of the plant developing infection due
to direct inoculation (Tables 19, 20), the highly significant F-value for the sprays revealed that
over all the fungicides tested, two sprays caused a consistently greater reduction of the disease

69

severity relative to one spray; individually, however, each fungicide when sprayed twice did not
significantly reduce the disease severity compared with that due to one spray (Table 18). The
results suggest that two sprays were more effective than one spray in restricting the lesion size,
but had little effect on previously established lesions. As well, the non-significant F-value for
the sprays in the ANOVA results of the stem lesions, developing as a result of foliar inoculation
(Table 21), indicate that the second spray had minimal effect on the fungus deep seated in the
leaf tissue and systemically progressing towards the stem base (Hammond et al. 1985).

Canola is reported to be most susceptible to blackleg before it reaches the six-leaf stage
(Gladders and Musa 1980, McGee and Petrie 1979). My results show that significant control of
blackleg may not be obtained if application of an effective fungicide is delayed beyond the early
stages of plant growth. Perhaps the blackleg fungus, which is present in the leaves during the
early stages of infection, advances deeply enough into stem where it is not affected by the
delayed application of a fungicide. As well, the known systemic fungicides do not move
downwards within the plant after entering plant tissue. Therefore, there is a greater possibility
that the blackleg fungus which moves down within the infected stem (Hammond et al. 1985)
could escape action of a fungicide that is sprayed after the fungus has penetrated the stem.

Ascospores were observed as early as May 15 and as late as September 15. There was
some indication in 1991 that their population increased as the growing season progressed. Canola
is reported to develop some natural tolerance to the disease as the season progresses. Therefore,
the influence of a larger population of ascospores in the latter half of the growing season might
have been offset by the tolerance developed by the maturing crop.

The spore trapping results also suggest that the ascospore release from pseudothecia is
influenced by precipitation. These results support earlier reports from Australia (McGee 1977)
that rainfall of more than 1.0 mm was required for large ascospore discharges. Since canola is
more susceptible to blackleg during the early stages of plant growth, precipitation during that
period could appreciably increase the disease severity. Therefore, to achieve acceptable blackleg
control, farmers in an infested region may have to consider spraying a fungicide when practicable
within a day or so after rain showers during the first six weeks of plant growth.

Ascospore discharges have been reported to be greater in 2-year-old stubble than in
1 -year-old stubble (McGee and Petrie 1979). This increased ascospore activity during the early
part of growing season most likely was responsible for increased blackleg severity and poor

70

disease control in 1991. The findings reaffirm that crop rotation of at least 3 years, preferably
4, is required to minimize the chances of severe disease occurrence (Petrie 1986).

Blackleg of canola progresses at a faster rate at 18°C than at 12°C (Barbetti 1975). It
is not known what temperature is most conducive to the ascospore discharge from pseudothecia.
Most large ascospore discharges were recorded when the air temperature was about 15°C. These
discharges also coincided well with rainfalls of over 5 mm. More work is required to determine
the effect of temperature on survival and functioning of pseudothecia in aging blackleg-infected
canola stubble. Spore trap data show that temperature more than 20°C was not very conducive
to ascospore build-up.

The extent of disease reduction with a single spray of prochloraz was considerably more
in Westar (from disease severity of 3.9 in check to 2.6) than in Legend (from 1.7 to 1.4)
(Table 26). The results suggest that the systemic fungicides used may have limitations in fully
arresting the fungal activity once the disease has been reduced to certain low levels. Also, no
significant difference in disease control was found between the number of sprays; the results
agree with those of growth chamber studies mentioned earlier and suggest that the second spray
had little effect on the previously established lesions.

Results of two years of field studies confirm that Legend, although not totally resistant,
has superior tolerance to blackleg compared to Westar which is highly susceptible. Until a
suitable fungicidal control program is developed, canola growers in infested regions may have
to depend upon the use of cultivars such as Legend and crop rotation of four years or more to
reduce yield losses due to blackleg.

More work is needed to determine if the high fungitoxicity of some fungicides such as
ethyltrianol, prochloraz or propiconazole could be used against L. maculans to reduce primary
inoculum in an infested field by spraying the stubble.

Complete protection of young seedlings for 21 days with iprodione seed treatment
(Table 17) revealed its systemicity in canola, although it is not claimed to be so by the
manufacturer. It is not certain why iprodione gave good control of blackleg under growth
chamber conditions but not in the field. Inactivation due to weather under field conditions, which
has been reported for other fungicides (Woodcock 1977), should not be the case since iprodione
is used as a foliar spray to control sclerotinia stem rot on canola.

71

Improved disease control was achieved when fungicides were used with an adjuvant.
Adjuvants with properties of wetting agents possibly assist in the coverage of waxy plant surfaces
of canola leaves resulting in increased fungicidal protection against extraneous fungi. Surfactants
have also been reported to enhance penetration of stomata by pesticides (Prasad et al. 1967). The
phytotoxic effect of ethyltrianol and propiconazole in these studies may have been due to then-
increased uptake by leaves with the use of adjuvants.

6.4 Biological Control

Biological control is environmentally the most prudent way of managing plant diseases.
Development of new, fungicide-resistant strains of pathogens is a constant threat to any disease
control program. Therefore, investigations were conducted to identify organisms that inhibit
development of L. maculans . Of the various fungi and bacteria tested, the fungus Penicillium
verrucosum and the bacterium Bacillus macerans looked very promising. Research on B.
macerans is still in the preliminary stages, whereas we have completed the intended research on
isolation, identification and biological activity of the P. verrucosum metabolite, citrinin, which
was found to be inhibitory to L. maculans. The work on characterization of the metabolite was
done by J.S.Dahiya, Plant Science Department, University of Alberta, Edmonton.

Citrinin is known to be a secondary metabolite of several penicillia and aspergilli. This
mycotoxin, first isolated from Penicillium citrinum in 1931 (Hetherington and Raistrick 1931),
has strong antifungal but weak antibacterial activity. It was found associated with heated grains
in Saskatchewan (Scott et al., 1970; Scott 1978). Its biological activity against Sclerotinia
sclerotiorum and Rhizoctonia solani has been reported by Melouk and Akem (1987). In the
present study we report isolation of this mycotoxin from the fungus, P. verrucosum ; its biological
activity was studied against several fungi pathogenic on canola, including Alter naria brassicae,
Fusarium acuminatum, L. maculans , R. solani (AG2-1), and S. sclerotiorum with a view to use
this Penicillium sp. to develop an integrated biocontrol program for canola diseases. A report
on a part of this study has been published (Kharbanda and Dahiya 1990).

72

6.4.1 Isolation and Characterization of Pencillium verrucosum Metabolite

The fungus P. verrucosum used in the present study was recovered as an aerial
contaminant inhibiting L. maculans (Kharbanda and Dahiya 1990). A single-conidial culture of
the Penicillium isolate was identified as P. verrucosum by M.A. Klich, USD A, New Orleans,
Louisiana, USA and by the International Mycological Institute, Bakeham Lane, Egham, Surrey,
England.

The fungus was gTown at 23 ± 2°C in potato dextrose broth on an orbital shaker set at
150 rpm for six weeks. At the end of the growth period, the fermented growth was filtered
through cheesecloth, centrifuged @ 4000 rpm for 15 minutes and finally filtered through a 0.2
pm Millipore bacterial filter. The filtrate (500 mL) was dried in vacuo, at 30°C. The residue
was redissolved in distilled water (50 mL) and partitioned against ethyl acetate (100 mL, 3x).
Ethyl acetate fractions were pooled and dried in vacuo. The residue was re-dissolved in
methanol.

The methanolic residue was purified by HPLC analysis using RP-18 (NOVA PAK
column. Waters Associate) column (25.0 cm x 1.00 cm id) using a solvent CH3CN:H20, 65:35,
v/v. The LC-UV detector was set at X 254 nm and a solvent flow rate of 2.5 mL/min. Samples
corresponding to peaks on the chart recorder were collected, dried, and subjected to spectral
analysis.

Figure 13,

Chemical structure of citrinin.

73

6.4.2 Spectral Analysis

UV spectrum in absolute methanol was recorded on Varian XL-200 spectrophotometer.
1HNMR (CDC13400 MHz) was recorded on Brukar Model WH-90 using TMS as an internal
standard. The mass spectrum (70 eV) was recorded on VG-analytical 11-250J by direct probe
insertion. IR spectrum was recorded on Beckman Spectrometer using nujol.

6.4.3 Biological Activity of the Metabolite

The inhibitory effect P. verrucosum was tested against A. brassicae, F. acuminatum, S.
sclerotiorum, L. maculans (virulent strain), L. maculans (weakly-virulent strain), and R. solani
(AG2-1). These fungi were originally isolated from infected canola plants. Two experiments
were conducted: In the first, 100 mL petri plates containing 20 mL of potato dextrose agar (PDA)
were flooded with about 3 mL of a macerated culture suspension containing conidia and/or
mycelial fragments of the test fungus. The plates were gently swirled to cover the agar surface
evenly, and the excess suspension was decanted; two hours later, a 4-mm-diameter mycelial,
PDA-plug from an actively growing culture of P. verrucosum was placed at the centre of each
plate. Four replications for each fungus tested were arranged in randomized complete block
design and incubated at 23 °C in the dark for seven days. The fungi tested were then observed
for zone of inhibition around the P. verrucosum colony in each plate.

In the second experiment, the P. verrucosum metabolite purified and concentrated in
methanol was dried under vacuum and resuspended in sterile distilled water resulting in a
concentration of 2mg/mL. One mL aliquots of this concentrated suspension were dispensed into
20 mL petri plates and mixed thoroughly with 19 mL of molten PDA at about 45 °C. Sterile
distilled water without chemical or a suspension containing pure citrinin (Aldrich Chemical Co.,
Milwaukee, Wisconsin, USA) in sterile distilled water were used as control. On the solidified
agar, a 4 mm mycelial PDA-plug from an actively growing culture of a test fungus was placed
at the centre of each plate. Colony diameters were measured 21 days after incubation at 21°C
in the dark.

74

6.4.3. 1 Effect of Heat on Metabolite

Five mL aliquots of the purified metabolite (cone. 1 mg/mL) were autoclaved at 121°C
for 20 minutes. The biological activity of the autoclaved metabolite was tested against R. solani
and L. macularts following the procedure described for the experiment above.

6.4.3. 2 Inhibitory Effect on Blackleg of Canola

A conidial suspension of P. verrucosum (2 x 106"7 conidial /mL) was mixed with an
equal amount of a suspension of pycnidiospores of L. maculans (2 x 106 pycnidiospores/mL) so
as to obtain a final concentration of 50 mg/mL of the metabolite in sterile distilled water. The
second fully expanded leaves of canola plants cv. Westar (. B . napus) were inoculated at four
isolated spots by puncturing the leaf with a hypodermic needle and depositing either a mixture
of conidiospores + pycnidiospores, pycnidiospores alone, conidiospores alone, or sterile distilled
water; four leaves on separate plants were inoculated for each treatment. The inoculated leaves
were individually covered with plastic bags to maintain high humidity, and incubated at 21°C for
48 hours in a growth chamber. Final data on disease development were recorded 21 days after
incubation.

6.4.3. 3 Biological Control: Results and Discussion

The antifungal substance was obtained as yellow crystals from aqueous methanol fraction
of crude extract. On mass spectral analysis the purified compound gave a molecular ion M+ 250
(89.0) C13H1405, 250.0342) with other fragments (M+ -18,H20) 217(65) (M+ -CH), 206(100),
191(22.8), 175(19), 161(9), 146(7), 133(5.8), 103(3.9), 91(32), 77(28), 65(18), and 51(22.5)
(Figure 14,15). UV spectral analysis in absolute methanol gave (nm), 251, and 331.

LH NMR spectral analysis in CDC13 gave a doublet at q 1.26 and q 2.08. A quintet at q 4.10 and
triplet at 4.82 were due to methyl (-CH3) protons. A singlet at q 15.89 confirms the -COOH
proton. Presence of >*C=0 was further confirmed by IR spectral analysis data of 1610 cm'1.
Presence of aromatic protons was given by singlets at q 7.26 and q 7.42. Based on UV, IR
NMR, and mass spectral data the compound was identified as citrinin (Figure 13) already
described from Aspergillus terreus and P. citrinum (Hetherington and Raistrick 1931).

75

This is the first report on citrinin production by P. verrucosum in North America.
Citrinin has been associated with mycotoxicoses in farm animals (Scott et al. , 1970; Scott 1978).
Under natural field conditions, as happens with Fusarium spp., there may be certain factors that
trigger production of mycotoxin by P. verrucosum. Therefore, the animal feed contaminated with
this fungus could be potentially hazardous to livestock and unfit for consumption.

The metabolite was heat stable and autoclaving at 121°C for 15 minutes did not destroy
its inhibitory effect on growth of Rhizoctonia solani (Table 30). It exhibited strong antifungal
activity against L. maculans in petti plate tests. The mean colony diameters of virulent and
weakly virulent isolates of L. maculans on PDA mixed with metabolite were 35 mm and 45 mm,
respectively, compared with 42 mm and 52 mm on the unamended substrate. As well, the
severity of blackleg lesions on leaves was significantly less when canola leaves were inoculated
with a mixture of conidia of both L. maculans and P. verrucosum compared with the disease
severity caused by L. maculans alone (Table 31, Plate 12). Evidently, P. verrucosum effectively
controlled blackleg. Citrinin has also been reported to inhibit R. solani , and certain strains of
Sclerotinia (Melouk and Akem 1987, Kharbanda and Dahiya 1990). However, the reported
nephrotoxic nature of citrinin (Scott et al ., 1970) makes it a non-candidate for biocontrol of
blackleg and other diseases of canola.

76

Table 30. Influence of heat on the biological activity Penicillium verrucosum metabolite
against Rhizoctonia solani.

Treatment

Mean colony diameter (mm)*

Autoclaved (121°C, 15 mins.)

61 b

Non-autoclaved

62 b

No metabolite

76 a

* Column mean followed by the same letter are statistically not different according to Duncan’s
Multiple Range Test (P=0.05).

Table 31. Severity of blackleg on canola leaves inoculated with Leptosphaeria maculans

conidia alone or in a mixture with conidia of Penicillium verrucosum.

Treatment

Mean blackleg severity*

Leptosphaeria maculans alone

2.7 a

Leptosphaeria maculans + Pencillium
verrucosum

2.1 b

Pencillium verrucosum alone

0 c

check (water only)

0 c

* Blackleg severity scale: 0 = pycnidia absent; 1 = pycnidia present, lesion < 5 mm; 2 =
pycnidia present, lesion > 5 mm <10 mm; 3 = pycnidia present, lesion > 10 mm. Column
means followed by the same letter are not significantly different according to Duncan’s Multiple
Range Test (P=0.05).

77

Figure 14. HPLC trace of Penicillium verrucosum metabolite (B) and of citrinin (A).

78

Figure 15. Mass spectrum of Penicillium verrucosum culture filtrate (M+ 250).

Plate 12.

Blackleg lesions on canola leaves inoculated with Leptosphaeria maculans (isolate
BL-A) conidia alone or in a mixture with conidia of Penici Ilium verrucosum.

80

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