effect of temperature, biofungicides and fungicides …
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EFFECT OF TEMPERATURE, BIOFUNGICIDES AND FUNGICIDES ON
CLUBROOT OF SELECTED BRASSICA CROPS
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
KALPANA KC ADHIKARI
In partial fulfillment of requirements
for the degree of
Master of Science
August, 2010
© Kalpana KC Adhikari, 2010
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ABSTRACT
EFFECT OF TEMPERATURE, BIOFUNGICIDES AND FUNGICIDES ON CLUBROOT OF SELECTED BRASSICA CROPS
Kalpana KC Adhikari Advisors: University of Guelph, 2010 Dr. Mary Ruth McDonald
Dr. Bruce D. Gossen
Clubroot is an economically important disease of Brassica crops caused by the
soil-borne protist Plasmodiophora brassicae Woronin. Shanghai pak choy was identified
as a model crop for the study of clubroot on canola. The Rapid Cycling Brassica lines of
Brassica carinata and B. juncea, and two canola lines 46A65 and 46A76 were
susceptible to pathotype 6 in Ontario. Controlled environment and field trials
demonstrated that low temperature (< 17° C) reduced initial infection and clubroot
development. Severe clubroot was observed at temperatures 19.6°-25.5° C. Drench
application of the bio fungicides Mycostop and Actinovate® and the fungicides Allegro®
5 OOF and Ranman® 400 SC reduced clubroot severity in Shanghai pak choy under
controlled conditions. Ranman application was effective when disease pressure was
moderate to high. Fungicide application is not needed when there is a low risk of clubroot
as a result of cool soil temperatures early or late in the season.
ACKNOWLEDGEMENTS
I would like to give my sincere thanks to my advisor Dr. Mary Ruth McDonald
and co-advisor Dr. Bruce D. Gossen for their guidance, support and encouragement
during my master degree. I am greatly indebted to my advisors for offering this
wonderful opportunity and opening the door of success in my academic career. I could
not have accomplished this without your constructive suggestions and your inspiration.
I would also like to thank Dr. Laima Kott and Dr. Sean M. Westerveld for their
great input, inspiration and immense support as members of my advisory committee. I
want to acknowledge the funding agencies to make this project successful including
University of Guelph, Ontario Ministry of Agriculture and Rural Affairs and Pest
Management Centre of Agriculture and Agri-Food Canada.
I would like to thank all the staff of the Muck Crops Research Station for their
time, technical support and assistance. I am especially thankful to Kevin, Shawn, Laura,
Michael, Catarina and Derk for their great help and support in many ways. Thanks to
Vanessa and Kristen for taking me from Guelph to Muck Crops Research Station and
your kind cooperation. I am greatly thankful to my lab and office members for having
good friendship and your help.
I would like to extend my great appreciation to all the graduate students and staff
in the department of Plant Agriculture who helped me during my graduate studies.
Special thanks to Dr. Victor who helped me in sending clubroot infested soil from
Alberta for growth cabinet trials in canola. Thank you for your great contribution. I
would like to thank Dr. Gary Peng from Agriculture and Agri-Food Canada for your
guidance in conducting the biofungicide trials.
1
/ I would also like to express my deep and sincere thanks to my family and friends
for your support and encouragement. To my parents - thank you so much for your love,
care and support throughout my life. Your efforts and desire in making me a successful in
every steps of life are always invaluable. Finally, to my husband, I am so grateful for you
for your great support, understanding and help during my busy time.
11
TABLE OF CONTENTS
Acknowledgements i
Table of Contents iii
List of Tables v
List of Figures vii
General Introduction and Objectives 1
Chapter One : Literature Review 6
1.1 Brassica crops 6 1.1.1 Asian Brassica vegetables in Ontario 7 1.1.2 Canola 7 1.1.3 Diseases of Brassica crops 8
1.2 Clubroot of Brassica crops 9 1.2.1 History 10 1.2.2 Causal agent 11 1.2.3 Host range 11 1.2.4 Pathotypes 12 1.2.5 Symptoms 13 1.2.6 Disease dissemination 14 1.2.7 Disease cycle 15
1.3 Factors influencing infection and development of clubroot 21 1.3.1 Temperature 21 1.3.2 Soil moisture 22 1.3.3 SoilpH 23 1.3.4 Light 24 1.3.5 Spore load 24
1.4 Disease management ...24 1.4.1 Synthetic fungicides and surfactants 25 1.4.2 Cultural and biological control 29
Chapter Two : Screening host lines for reaction to Plasmodiophora brassicae 37 2.1 Introduction 37 2.2 Materials and methods 39
2.2.1 Data Analysis 43 2.3 Results 43
2.3.1 Weather 43 2.3.2 Incidence and severity assessment 44
2.4 Discussion 47 Chapter Three : Effect of temperature on infection and symptom development of clubroot 51
3.1 Introduction 51 3.2 Materials and methods 55
3.2.1 Plant materials 55 3.2.2 Controlled environment trials 55 3.2.3 Seeding date trials 59
iii
3.2.4 Zoosporangia development in root hairs 62 3.2.5 Data analysis 65
3.3 Results 66 3.3.1 Temperature differences in a controlled environment 66 3.3.2 Impact of seeding date on clubroot development 73 3.3.3 Zoosporangia development in root hairs 89
3.4 Discussion 93 Chapter Four : Evaluation of efficacy of fungicides and biofungicides for clubroot management on Asian vegetables and other brassica crops 104
4.1 Introduction 104 4.2 Materials and methods 105
4.2.1 Plant materials, fungicides and biofungicides 105 4.2.2 Growth cabinet studies 107 4.2.3 Field trial 109 4.2.4 Data analysis 110
4.3 Results 110 4.3.1 Growth cabinet trials... 110 4.3.2 Field trial 111
4.4 Discussion 112 Chapter Five : General discussion and conclusions 118
References 132
Appendix 1: ANOVA Tables for Chapter Two 143
Appendix 2: Supplementary Tables and Figures for Chapter Three 145
Appendix 3: ANOVA Tables for Chapter Three 154
Appendix 4: ANOVA Tables for Chapter Four 170
Appendix 5: Raw Data for Chapter Two 172
Appendix 6: Raw Data for Chapter Three 174
Appendix 7: Raw Data for Chapter Four 196
IV
LIST OF TABLES
Table 2.1 Lines of Brassica species assessed for susceptibility to clubroot in naturally infested soil in field trials at the Holland Marsh, ON, 2008 and 2009 42
Table 2.2 Mean air temperature and rainfall during the growing period of Brassica crops for clubroot screening at the Holland Marsh, ON, 2008 and 2009 44
Table 2.3 Clubroot incidence (CI %) and disease severity index (DSI) on Brassica crops or lines grown in organic soil naturally infested with clubroot at the Holland Marsh, ON, 2008 and 2009 46
Table 3.1 Target and actual temperatures achieved in Trials 1 and Trial 2 under controlled conditions 69
Table 3.2 The interaction of host (Shanghai pak choy vs. Chinese flowering cabbage) and fungicide application (Ranman vs. control) with seeding date on clubroot incidence and severity, summarized as area under the disease progress curve (AUDPC), in a field study at the Holland Marsh, ON (combined data from 2008 and 2009) 79
Table 3.3 Monthly mean air temperature and rainfall at the Muck Crops Research Station, Holland Marsh, ON, in 2008 and 2009 and long term means 85
Table 3.4 Linear correlation between clubroot incidence and severity and selected weather variables (mean air and soil temperatures and rainfall) during the interval before harvest for Shanghai pak choy and Chinese flowering cabbage grown at the Holland Marsh, ON, 2008 and 2009 88
Table 4.1 Bio fungicides and fungicides treatments for clubroot management applied to Shanghai pak choy in growth cabinet trials at the University of Guelph, Guelph, ON, 2008 and 2009 108
Table 4.2 Efficacy of fungicides and biofungicides for the management of clubroot on Shanghai pak choy grown under controlled conditions 111
Table 4.3 Evaluation of clubroot incidence, severity (Disease Severity Index) and percentage of marketable heads of cabbage treated with fungicides and biofungicides in a field trial at the Holland Marsh, ON, 2008 112
Table A 2.1 Shanghai pak choy 2008: Efficacy of Ranman® 400 SC application (Ran) on clubroot incidence and severity compared to a nontreated control (C) in Shanghai pak choy grown in organic soil naturally infested with the clubroot pathogen at the Holland Marsh, ON in 2008 145
Table A 2. 2 Chinese flowering cabbage 2008: Efficacy of Ranman® 400 SC application (Ran) on clubroot incidence and severity compared to a nontreated control (C) grown in organic soil naturally infested with the clubroot pathogen at the Holland Marsh, ON in 2008 146
Table A 2. 3 Shanghai pak choy 2009: Efficacy of Ranman® 400 SC application (Ran) on clubroot incidence and severity compared to a nontreated control (C) grown in organic soil naturally infested with the clubroot pathogen at the Holland Marsh, ON in 2009. ..147
v
Table A 2. 4 Chinese flowering cabbage 2009: Efficacy of Ranman application (Ran) on clubroot incidence and severity compared to a nontreated control (C) grown in organic soil naturally infested with the clubroot pathogen at the Holland Marsh, ON in 2009. ..148
Table A 2. 5 Correlation between clubroot levels (incidence/severity) and components of air and soil temperatures during the interval before harvest of Shanghai pak choy and Chinese flowering cabbage grown at the Holland Marsh, ON 2008 and 2009 152
Table A 2. 6 Effect of Ranman 400 SC application on top weight (g) of Shanghai pak choy and Chinese flowering cabbage at optimum harvest grown at the Holland Marsh, ON, 2008 and 2009 153
Table A 2. 7 Autocorrelation among mean air temperatures, mean soil temperatures at a depth of 5-cm and cumulative rainfall throughout the growing period of crops the Holland Marsh, ON, 2008 and 2009 153
VI
LIST OF FIGURES
Figure 1.1 Life cycle of Plasmodiophora brassicae 16
Figure 3.1 Planting Shanghai pak choy and Chinese flowering cabbage at the Muck Crops Research Station, Holland Marsh, ON, 2009 61
Figure 3.2 Effect of temperature on clubroot incidence and symptom development over time on Shanghai pak choy grown under controlled conditions 68
Figure 3.3 Effect of temperature on clubroot incidence and severity in Shanghai pak choy grown under controlled conditions 71
Figure 3.4 Effect of temperature on clubroot incidence and severity in canola grown under controlled conditions 72
Figure 3.5 Clubroot incidence (%) and severity (disease severity index) on Shanghai pak choy planted at monthly intervals in organic soil naturally infested with the clubroot pathogen at the Holland Marsh, ON, in 2008 and 2009 75
Figure 3.6 Clubroot incidence (%) and severity (disease severity index) on Chinese flowering cabbage planted at monthly intervals in organic soil naturally infested with the clubroot pathogen at the Holland Marsh, ON, in 2008 and 2009 76
Figure 3.7 The effect of seeding date on clubroot incidence and severity summarized as area under the disease progress curve (AUDPC) and combined across host, fungicide treatment and year, at the Holland Marsh, ON, 2008-2009 78
Figure 3.8 Clubroot incidence (%) on Shanghai pak choy seeded in May in soil naturally infested with clubroot at the Holland Marsh, ON, in 2008 and 2009.. 82
Figure 3.9 Clubroot incidence (%) and severity on Shanghai pak choy seeded in August in soil naturally infested with clubroot pathogen at the Holland Marsh, ON, in 2008 and 2009 84
Figure 3.10 Root hairs of Shanghai pak choy grown in sand-liquid culture medium under controlled conditions 90
Figure 3.11 Incidence (%) and intensity index of zoosporangia in root hairs on Shanghai pak choy 10 and 14 days after inoculation (DAI) 91
Figure 3.12 Root hairs with zoosporangia (%) based on counts of 100 root hairs at the mid section of each root on Shanghai pak choy at 10 and 14 days after inoculation (DAI).
92
Figure A 2.1 Clubroot incidence (%) on Shanghai pak choy seeded in June in soil naturally infested with clubroot at the Holland Marsh, ON, in 2008 and 2009 149
Figure A 2.2 Clubroot incidence (%) and severity on Shanghai pak choy seeded in July in soil naturally infested with clubroot at the Holland Marsh, ON, 2008 and 2009 150
Figure A 2.3 Clubroot incidence (%) on Shanghai pak choy seeded in September in soil naturally infested with clubroot pathogen at the Holland Marsh, ON, in 2008 and 2009
151
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GENERAL INTRODUCTION AND OBJECTIVES
Clubroot, caused by soil-borne protist Plasmodiophora brassicae Woronin, is an
important disease of Brassica crops. In Canada, this disease has long been reported as an
established disease of Brassica vegetables in Ontario, Quebec, British Columbia and
Atlantic Provinces (Howard et al. 2010; Tewari et al. 2005). Clubroot is a major threat in
canola {Brassica napus L. and B. rapa L.) production in western Canada. It was first
identified in commercial canola fields in central Alberta in 2003 (Tewari et al. 2005) and
has spread rapidly within the province (Cao et al. 2009). Clubroot is a limiting factor for
successful production of Brassica vegetables and oil crop species, causing 10-15% crop
loss worldwide (Dixon 2006) and 30-100% yield loss in canola in highly infested areas
in Alberta (Strelkov et al. 2007; Tewari et al. 2005).
Clubroot is a serious soil-borne disease, which is easily disseminated through
movement of contaminated soil (Karling 1968) including infected transplants and
seedling trays (Donald et al. 2006), farm machinery and equipments (Cao et al. 2009) and
any other sources that carry contaminated soil. The pathogen can persist in soil for many
years as resting spores (Karling 1968; Wallenhammar 1996). The disease is generally
more severe in wet and poorly drained soil with moisture content of 70-80% of its
maximal water holding capacity (Monteith 1924) and that are acidic with pH from 5.4-
7.1 (Myers and Campbell 1985).
Temperature is an important environmental factor that influences on clubroot
incidence and severity. Temperatures of 16°-21° C are required for germination of
resting spores of P. brassicae (Chupp 1917). Soil temperatures of 18°-25° C were
reported as optimal for clubroot incidence and severity (Colhoun 1952) and low levels of
1
clubroot were observed when temperatures were below 14° C (Thuma et al. 1983). It was
also demonstrated that mean air temperatures 10 days before harvest had a strong
correlation with clubroot incidence and severity on short-season vegetables grown in
Ontario (McDonald and Westerveld 2008). There is limited numbers of research on effect
of temperature on clubroot development, which mainly focused on the impact of
temperature on clubroot incidence and severity at harvest. The effect of temperature on
the specific stage(s) in the infection cycle of this pathogen is unknown. Thus a study to
identify the critical period of infection and symptom development of clubroot in relation
to temperature is important for a better understanding of host-pathogen interaction and
proper management of this disease.
Several strategies of clubroot management have been recommended and practiced
for many years. Clubroot incidence and severity can often be reduced by crop rotation
with non-host species at least for 5-7 years (OMAFRA 2008a), soil amendment by
liming (Dobson and Gabrielson 1983; Klasse 1996; Murakami et al. 2002) to increase
soil pH to 7.2 or over, avoidance of production of susceptible crops in high risk areas and
sanitation measures to limit the spread of P. brassicae from one place to other. But these
strategies of clubroot management have some limitations. Crop rotation that is long
enough to minimize inoculum level to reduce severity is not feasible for the growers who
grow Brassica crops in the rented land. An increase in soil pH can reduce clubroot, but
high soil pH is not suitable for some crops (Hildebrand and McRae 1998) and it can be
prohibitively costly to raise the desired level of soil pH, especially in high acreage for
relatively low value crops like canola (Howard et al. 2010). Clubroot can also be
2
managed by resistant cultivars but most of the sources of resistance are race specific and
have generally not been durable (Diederichsen et al. 2009).
In this context, finding a sustainable and effective method of clubroot
management is critical. This research will contribute to the management of clubroot by
determining the crucial period of infection and symptom development in relation to
temperature and evaluating potential biofungicides and fungicides to provide alternative
options to reduce clubroot severity when disease pressure is high. In addition, screening
of several Brassica species identified small, fast growing crops that can be used as model
crops for future research, and these crops have potential to develop the Canadian
differential sets for effective differentiation and classification of P. brassicae available in
Canada.
This research was initiated to test several hypotheses. The hypotheses were:
1. The reaction to P. brassicae pathotype 6 is different among various Brassica lines.
2. Low temperatures during plant growth delay infection, symptom initiation and
severity of the clubroot.
3. The fungicide Ranman is effective for the management of clubroot under a wide
range of temperature regimes and disease pressure in the field
4. Microbial biofungicides (Mycostop, Prestop, RootShield, Actinovate and
Serenade) and fungicides (Ranman 400SC and Allegro 500F) are effective for
reducing clubroot severity both under controlled conditions and in the field.
The overall objective of this research was to identify effective and sustainable
options to manage clubroot on Brassica crops. The hypotheses reflect the specific
3
objectives of this research. The objectives were; to assess the reaction of selected
Brassica crops to P. brassicae pathotype 6, to determine the effect of temperature on
infection and symptom development of clubroot on Shanghai pak choy (B. rapa L. subsp.
Chinensis (Rupr.) var. communis Tsen and Lee), Chinese flowering cabbage (B. rapa L.
subsp. Chinensis (Rupr.) var. utilis Tsen and Lee) and canola, to assess the efficacy of
Ranman fungicide across a range of temperatures and a range of disease pressure, and to
evaluate the efficacy of commercially available microbial bio fungicides and registered
and potential fungicides in reducing clubroot on cabbage (B. oleracea var. capitata cv.
Saratoga) and Shanghai pak choy.
The field trials were established in organic soil naturally infested with clubroot
pathogen pathotype 6 to assess the reaction of various Brassica hosts; Rapid Cycling
Brassica Collection lines, Asian vegetables and canola lines to P. brassicae. Shanghai
pak choy was used as a susceptible control to identify model crops for subsequent
clubroot studies. The effects of varying range of temperatures on symptom expression
were investigated in the field, where several planting dates were used to provide different
temperature regimes. Plants were harvested at weekly intervals to track symptom
development. The effects of a wide range of temperatures (10°-30° C) were studied in
controlled environment. Canola and Shanghai pak choy were used to identify the host
reaction to P. brassicae in relation to temperature. Plants were grown at one temperature
for three weeks then transferred to another temperature to represent wide range of
temperatures in the field throughout the growing season. Shanghai pak choy seedlings
were grown using sand-liquid culture to study the effect of temperature on zoosporangia
development in root hairs.
4
The fungicide Ranman was applied as drench on Shanghai pak choy and Chinese
flowering cabbage at various seeding dates to identify its efficacy at wide range of
temperatures and disease pressure. Efficacy of fungicides (Allegro and Ranman) and
commercially available biofungicides (Mycostop, Prestop, RootShield, Actinovate and
Serenade) were evaluated both under controlled conditions on Shanghai pak choy.
Inoculum concentrations of lO5 and 106 resting spores/mL were used to identify the
efficacy of these selected products under moderate to high disease pressure. Two
fungicides (Allegro and Ranman) and two biofungicides (RootShield and Serenade) were
evaluated on cabbage under field conditions.
5
CHAPTER ONE
LITERATURE REVIEW
1.1 Brassica crops
The genus Brassica includes 35 species of mostly annual and some perennial
herbs and small shrubs. Crops in this genus are grown throughout the world for their
edible and economically important roots, stems, leaves, buds, flowers and seeds. These
crops are used mainly as sources of oil, vegetables, condiments and fodder. Brassica
crops are becoming popular because of their anticarcinogenic properties, health
promoting compounds and other health benefits (Rimmer et al. 2007). The Brassica crops
include six economically important species: Brassica nigra (L.) W.D.J. Koch, B.
oleracea L., B. rapa L., B. napus L., B. carinata (L.) A. Braun and B. juncea (L.) Czern
(Rimmer et al. 2007).
Most of the Brassica species are cool-season crops and can be grown in a wide
range of soil types. These crops require relatively high moisture and grow well in sandy
loam soil (OMAFRA 2008a). The optimal pH for production of these crops generally
ranges from 6.0-6.5 (Rimmer et al. 2007). In Ontario, cabbage (B. oleracea var.
capitaia), broccoli (B. oleracea var. italica) and cauliflower (B. oleracea var. botrytis)
are the major Brassica vegetable crops grown, with an area of production of 1532, 1435
and 530 ha respectively in 2008 (OMAFRA 2008b). Seedlings are generally transplanted
in the field during May and June and the crop is harvested during August and September
for the main season crops. Direct seeding is also an option for production of these
vegetables, but transplants are widely used to ensure uniform size and maturity
(OMAFRA 2008a).
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1.1.1 Asian Brassica vegetables in Ontario
The majority of Asian vegetables grown in Ontario are Brassica crops. Recently,
the area of production of Asian vegetables has increased in organic soil (organic matter ~
69%) in Ontario (McDonald et al. 2004). The reason may be the immigration of people
from Asian countries and a general increase in consumption of these oriental vegetables.
The area of production of Asian vegetables was estimated at 900 ha in Ontario and 1300
ha in Canada in 2008 (J. Chaput, personal communication). In 2008, napa cabbage (B:
rapa subsp. pekinensis) was grown in 299 ha with a farm gate value of 2.5 million in
Ontario (OMAFRA 2008b). Among the Asian Brassica vegetables, Shanghai pak choy
(B. rapa subsp. Chinensis (Rupr.) var. communis Tsen and Lee) and Chinese flowering
cabbage (B. rapa subsp. Chinensis (Rupr.) var. utilis Tsen and Lee) are common. These
crops are short-season vegetables. In Ontario, they are mainly grown in organic soil and
are direct seeded in the field during the season. Seeding can start in late April to early
May and continued to September (McDonald and Westerveld 2008). The optimal
temperature for seed germination of Shanghai pak choy and Chinese flowering cabbage is
15-29° C and these crops grow well in the temperature range of 12-24° C (Martin 2008).
The crops become ready to harvest within 4-6 weeks, depending on the planting month
of growing season (McDonald and Westerveld 2008).
1.1.2 Canola
Canola is an oilseed crop that was developed by Canadian plant breeders R. K.
Downey and B. R. Stefansson in the early 1970's using traditional plant breeding
techniques (Canola Council of Canada 2009). The name 'canola' is a Canadian trademark
for rapeseed {B. napus and B. rapa) cultivars or lines in which the processed oil contains
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less than 2% erucic acid and the residual meal contains less than 3 mg/g glucosinolates
(Daun 1986a; Daun 1986b). Canola was registered in 1979 by the Canola Council of
Canada as a high quality vegetable oil with low levels of saturated fats, suitable for
human and animal consumption (Rimmer et al. 2007).
Canola is a cool season crop. The optimal temperature for seed germination is 10°
C and growth and development is 21° C. Canola seed starts to germinate at soil
temperatures of 5° C and grows well in the range of temperatures 12-30° C (Canola
Council of Canada 2003). Canola is widely grown mainly as spring canola in Canada in
which crop is generally seeded during the month of May (Kutcher et al. 2010) and is
harvested 80-120 days after seeding depending on the location and canola varieties
(Canola Council of Canada 2003). In western Canada, growing winter canola is not
possible because of extreme weather conditions, which are not suitable for this crop
(Canola Council of Canada 2003). Canola is grown on about 6.5 million ha in Canada,
each year, with about 48% of the acreage in Saskatchewan, 32% in Alberta, 19% in
Manitoba and 0.3% in Ontario (Statistics Canada 2008). The canola industry contributes
more than $13 billion annually to the Canadian economy (Canola Council of Canada
2009).
1.1.3 Diseases of Brassica crops
There are number of diseases of Brassica crops, including Alternaria diseases
{Alternaria brassicae (Berk.) Sacc, A. brassicicola (Schwein.) Wiltshire and A. japonica
Yoshii), black leg (Leptosphaeria maculans (Sowerby) P. Karst), clubroot
{Plasmodiophora brassicae Woronin), downy mildew (Peronospora parasitica (Pers.) de
Bary) and sclerotinia blight {Sclerotinia sclerotiorum (Lib.) de Bary). Clubroot is one of
8
the most economically important diseases of Brassica crops (Faggian and Strelkov 2009)
because yield loss can be very high when symptoms are severe. It was estimated that the
annual yield loss caused by this pathogen worldwide is 10-15% of total production
(Dixon 2006). In Alberta, yield losses of 30-100% have been reported in severely
infested canola fields (Strelkov et al. 2007; Tewari et al. 2005).
1.2 Clubroot of Brassica crops
Clubroot is caused by the soil-borne protist Plasmodiophora brassicae Woronin.
After infection, roots of susceptible Brassica crops swell and become club-like in
appearance and severely distorted. As a result of the clubbing, dislocation of vascular
bundles takes place, leading to a disruption in the uptake of nutrients and water from the
soil (Mithen and Magrath 1992). In severe cases, plants can become wilted and stunted.
This disease can cause severe economic losses in Chinese cabbage, broccoli and other
cruciferous crops (Mitani et al. 2003). A 91% reduction in yield of canola grown in a
field severely infested with P. brassicae was reported (Xue et al. 2008) and there was
100% yield loss in one infested field in Alberta when canola was grown year after year
(Strelkov et al. 2007).
Clubroot is an endemic disease of cole crops grown in organic (muck) soils in
Ontario. This disease has become an important limiting factor for successful production
of Brassica crops including Asian vegetables. Among the short-season Asian vegetables,
Shanghai pak choy was found to be even more susceptible than Chinese flowering
cabbage, which is highly susceptible to clubroot (McDonald et al. 2004).
9
1.2.1 History *
The origin of clubroot is unknown (Karling 1968), but P. brassicae was first
identified as the causal organism of clubroot by Woronin in 1878 (Cook and Schwartz
1930; Tommerup and Ingram 1971). Clubroot was first identified in Europe in the 13th
century and it had spread to most of the parts of that continent by the 18th century (Hirai
2006). Clubroot is known by different names in different countries. In Australia and New
Zealand, it is known as clubroot; in Belgium as kwab, knol, bosse, kanker; in France as
gros pied; in Germany, Switzerland and Austria as kohlhernie, kohlkropf; in Italy as
ernia; in South Africa as clubroot, finger and toe, club foot and in North America as
clubroot, finger-and-toe, clump foot and clubbing (Karling 1968).
It is unknown when clubroot was first introduced in Canada but this disease has
long been reported on Brassica vegetables production areas of Ontario, Quebec, British
Columbia and the Atlantic Provinces (Howard et al. 2010). Clubroot was first reported in
Ontario in 1923 and 15-20% disease incidence was occurred in cauliflower in 1930 in
Lincoln County (Dominion of Canada Department of Agriculture 1930). In 1953,
clubroot in Brassica crops grown in the Hamilton-Toronto area resulted in total loss in
some heavily infested sites. The clubroot pathogen was widely disseminated over the
Bradford Marsh from infested areas due to extensive flooding that took place in 1954 and
moderate to severe clubroot incidence was observed in cabbage and cauliflower grown at
the Muck Crops Research Station (Conners et al. 1956).
Clubroot has been reported occasionally in home gardens and commercial
vegetable fields in Alberta and Manitoba over the past 80 years (Tewari et al. 2005;
Howard et al. 2010). However, the disease was detected in canola for the first time in the
10
fields near St. Albert in Alberta in 2003, and extensive surveys of adjacent fields
confirmed 12 commercial canola fields infested with this disease (Strelkov et al. 2005;
Tewari et al. 2005). Additional surveys in 2005 and 2006 reported more than 40 fields
infested with clubroot (Strelkov et al. 2007). Annual surveys from 2005 to 2008
confirmed more than 400 clubroot infested commercial canola fields in central and
southern Alberta (Cao et al. 2009). This indicates that the disease is spreading quickly in
the province. The pathogen was recently found in one field in Saskatchewan (Dokken-
Bouchard et al. 2010).
1.2.2 Causal agent
Clubroot of Brassica crops is caused by the obligate biotroph Plasmodiophora
brassicae Woronin. Plasmodiophora brassicae is a protist under the phylum
Plasmodiophoromycota (Barr 1992). It is an intracellular pathogen that infects roots in
the Brassicaceae family and cannot be grown in a pure culture (Bulman et al. 2006). It is
a soil-borne pathogen and survives in the soil in the absence of hosts as resting spores
that are not easily degraded by other components of the soil micro-flora (Dixon 1996).
Resting spores remain viable in soil for at least 7 years (Karling 1968). The half-life of
the P. brassicae spore inoculum was determined to be 3.6 years and 17.3 years were
required to reduce the level of infestation in soil from 100% to levels below the disease
causing threshold (Wallenhammar 1996).
1.2.3 Host range
Plasmodiophora brassicae occurs worldwide mainly in the areas where Brassica
crops are grown extensively. In addition to infecting many economically important crops
in the Brassicaceae, it can also infect and produce symptoms in Arabidopsis thaliana, the
11
most extensively studied member of the Brassicaceae (Schuller and Ludwig-Miiller
2006). Plasmodiophora brassicae has a wide host range in the family Brassicaceae. It
can infect more than 300 species in 64 genera of crucifers and can be found on in both
cultivated and wild species of this family. Most economically important hosts include
cabbage, collards (B. oleracea L. var. viridis L.), mustard (B. nigra L.), Brussels sprouts
(B. oleracea var. gemmifera DC), radish (Raphanus sativus L.), turnip (B. rapa L. subsp.
rapa), rutabaga (B. napus L. subsp. napobrassica (L.) Jafri), cauliflower, broccoli (B.
oleracea var. italica Plenck), rape {B. napus subsp. napus (L.) Hanelt) and kohlrabi (B.
oleracea var. gongylodes L.). Plasmodiophora brassicae can also infect the root hairs of
a number of non-crucifers and form zoosporangia and zoospores, but no other stages of
this pathogen have been observed (Macfarlane 1952). All cultivars of cabbage,
cauliflower, broccoli and Brussels sprouts recommended for commercial production in
Ontario were susceptible to clubroot in 1974 (Reyes et al. 1974) but resistant cultivars are
now becoming available commercially. The clubroot resistant rutabaga varieties;
Kingston and York, and cabbage cultivars; Richelain and Acadie are available in Canada
(Howard etal. 2010).
1.2.4 Pathotypes
Different systems have been proposed for pathotype designation of P. brassicae
(Xue et al. 2008). Among them, the differential set of Williams and the European
Clubroot Differential (ECD) have been commonly used to characterize pathogen
populations from Canada (Reyes et al. 1974; Strelkov et al.,2006). The word pathotype
has replaced the earlier term race for the clubroot pathosystem as suggested by Voorrips
(1995) because of the lack of genetic uniformity and stability both in the populations of
12
pathogen and the differential host to fulfill the requirements to be a race (Parlevliet
1985). The European Clubroot Differential set is one of the most commonly used systems
for classification and characterization of pathotypes of P. brassicae. The differential set
comprises of five lines of each of B. rapa (syn. B. campestris L.), B. napus and B.
oleracea (Buczacki et al. 1975). The differential set of Williams is another system to
classify pathotypes of P. brassicae. This is based on the reaction to infection in two
cabbage varieties 'Jersey Queen' and 'Badger Shipper' and two rutabaga varieties
'Laurentian' and 'Wilhelmsburger' (Williams 1966).
Populations of P. brassicae often consist of a combination of pathotypes. In
Canada, a more diversified pathotype composition of this pathogen was reported when
single-spore isolates were examined (Xue et al. 2008). Pathotype 6 or ECD 16/0/14 as
classified on the differential set of Williams and the ECD sets respectively, was found to
be the predominant pathotype in Ontario (Reyes et al. 1974; Strelkov et al. 2006).
Similarly, pathotypes 2 and 6 were identified in populations of P. brassicae obtained
from Quebec and British Columbia respectively (Strelkov et al. 2006; Williams 1966).
Pathotypes 3 and 5, or ECD 16/15/12 and ECD 16/15/0, were initially identified in the
population from Alberta (Strelkov et al. 2006). In subsequent studies, pathotype 3 was
identified to be predominant in canola in the Alberta and other pathotypes 2, 5, 6 and 8
were also detected using single-spore isolates off. brassicae population in the province
(Xue et al. 2008; Cao et al. 2009).
1.2.5 Symptoms
The wilting of leaves of infested plants is common when temperature is high and
soil moisture level is low (Cheah et al. 2006; Karling 1968). The wilted plants initially
13
)
recover their turgidity overnight and appear normal and fresh in the morning, but when
disease is more severe the leaves turn yellow and the plants are stunted. The increase in
total leaf area and total dry weight is slower in affected plants which have fewer and
smaller leaves than healthy ones (Karling 1968). In severe cases, dislocation of vascular
bundles takes place, resulting in disruption of water and nutrient uptake from the soil
(Buhariwalla et al. 1995).
Swelling or enlargement of the infected roots leading to gall formation is the main
characteristic symptom of clubroot (Cheah et al. 2006). Galls can form on both tap and
lateral roots, and occasionally on the base of the stem in affected plants (Cao et al. 2007).
Morphological changes such as hyperplasia and hypertrophy occur in the early stages of
infection (Devos et al. 2005; Mithen and Magrath 1992). The increase in concentration of
the plant hormones auxins and cytokinin, which are involved in cell division and cell
elongation, were found to be associated with the development of root galls (Mithen and
Magrath 1992).
1.2.6 Disease dissemination
Dispersal of clubroot is primarily associated with movement of infested soil. It is
easily disseminated by numerous agents once it has become established in soil. Resting
spores of P. brassicae can be dispersed through the transport of infested soil on farm
tools, farm equipments, animals and humans (Karling 1968). Agricultural practices"such
as movement of shared machinery from farm to farm, use of infected transplants, and
irrigating fields with water contaminated with P. brassicae, can contribute to spreading
the clubroot pathogen from one place to another (Donald et al. 2006). Soil erosion by air
and water is another important method of pathogen dispersal. Resting spores remain
14
viable after passage through the digestive tract of animals, so fertilizing fields with
composted manure from livestock fed on diseased roots could be another means of
disease dissemination (Karling 1968). Surveys on clubroot in canola fields in Alberta
identified a high frequency of incidence at the field entrance, which decreased with
increase in distance from the entrance. This pattern indicates that the rapid spread of
clubroot likely occurred as the result of movement of infested soil with or on agricultural
machinery (Cao et al. 2009). Therefore, effective farm hygiene and nursery management
are necessary to limit the spread of clubroot and minimize its impact in regions where P.
brassicae is present (Donald et al. 2006).
1.2.7 Disease cycle
The lifecycle of P. brassicae consists of two phases, the primary phase that is
restricted to root hairs of a wide range of plant species, not all of which are susceptible
hosts, and the secondary phase that occurs in the root cortex leading to abnormal
development of roots and initiation of club formation (Ingram and Tommerup 1972).
The life cycle of P. brassicae is very complicated. There have been many studies on the
life cycle of this pathogen but certain parts are still not completely understood. It is not
known whether primary zoospores directly infect the cortical cells or secondary
zoospores reinfect the root hairs. It is also unknown if the secondary zoospores released
from non-host plants can infect Brassica crops leading to symptom development
(Kageyama and Asano 2009). There is also no information about the secondary
zoospores released from zoosporangia in root hairs and epidermal cells can directly infect
the root cortex without being released into the soil, to continue the secondary phases of
lifecycle.
15
Figure 1.1 Life cycle of Plasmodiophora brassicae. a Resting spore, b Primary zoospore, c Primary plasmodium in root hair, d Zoosporangial cluster in root hair, e Empty zoosporangium. f, g Secondary plasmodia in cortical cells, h, i Resting spores in cortical cells (Adapted from Kageyama and Asano, 2009).
16
The lifecycle of P. brassicae begins with the germination of resting spores
released from decayed galls, which are the primary source of inoculum. Resting spores of
P. brassicae are round, have a diameter of 3.0 to 5.0 jam and are haploid (Ingram and
Tommerup 1972; Tommerup and Ingram 1971). Based on observations made using
scanning electron microscopy, young spores are surrounded with fibrous materials and
mature spores have numerous small spines (Ingram and Tommerup 1972; Kageyama and
Asano 2009). The cell wall composition of resting spores are reported to have 25% chitin,
>2.5% other carbohydrates, 34% protein and >17.5% lipid (Moxham and Buczacki
1983).
The first sign of germination of the resting spore is the disappearance of refractile
globules, which are characteristic of dormant resting spores (Macfarlane 1970).
Absorption of calcium ions may be necessary for germination of resting spores
(Kageyama and Asano 2009). Resting spores extracted from old, decaying galls have a
higher germination potential than the spores extracted from freshly harvested young galls
(Macfarlane 1970). During the early stages of zoospore emergence, the central spore
nucleus enlarges slightly and becomes peripheral (Ingram and Tommerup 1972). During
periods of cool, wet weather when the soils become saturated with water, resting spores
germinate to produce primary zoospores, which have two flagella. The flagella are
unequal in length (Ingram and Tommerup 1972). The shorter fiagellum has a blunt end
and the longer fiagellum has a whiplash or tail piece (Kageyama and Asano 2009).
Sometimes germination of resting spores occurs in the absence of host plants, but usually
germination takes place in the presence of hosts because exudates from roots stimulate
germination (Friberg et al. 2005).
17
The primary phase of the life historyhfP. brassicae occurs in the root hairs and
epidermal cells of roots (Ingram and Tommerup 1972). Primary zoospores, released from
the resting spores, swim to the root by means of flagella and penetrate root hairs of young
roots. Once they reach the surface of the root hair, the zoospores encyst and then enter the
host cytoplasm by penetrating the cell wall and forming primary plasmodia. This stage is
called the primary infection stage or root hair infection stage (Kageyama and Asano
2009). The primary plasmodia undergo several mitotic nuclear divisions and then divide
to form multinucleate structures called zoosporangia (Ingram and Tommerup 1972).
Zoosporangia have been observed in root hairs at 4-6 days after inoculation (Matsumiya
et al. 1992). The zoosporangia undergo two or three synchronous mitotic divisions and
cytoplasm within the zoosporangia starts to divide and form 4-16 secondary zoospores
(Ingram and Tommerup 1972). The zoosporangia form clusters in root hairs and empty
structures are visible after they release the secondary zoospores (Kageyama and Asano
2009). When the secondary zoospores are released from zoosporangia, they are similar to
the primary zoospores and also have two flagella of unequal length (Matsumiya et al.
1992).
Root hair infection by primary zoospores results in deformation and curling of the
root hairs (Samuel and Garret 1945). There was a linear relationship between the
logarithm of root hair infection and spore concentration, but high disease incidence can
occur in seedlings with low numbers of root hair infections (Macfarlane 1952)
Root hair infection has been observed in non-host plants from different families,
but there was no evidence of continuation to cortical infection (Macfarlane 1952). It is
18
not known if secondary zoospores released from nonhost plants can infect Brassica crops
leading to symptom development (Kageyama and Asano 2009).
The secondary phase of the life history of P. brassicae begins with infection of
the root cortex by secondary zoospores. Infected cells undergo hyperplasia and
hypertrophy leading to characteristic gall formation. Binucleate secondary plasmodia,
which were believed to develop through fusion of secondary zoospores, are visible in the
cortical cells of the roots at the beginning of this stage (Ingram and Tommerup 1972).
The secondary plasmodia, which have five haploid chromosomes, enlarge and become
multinucleate through a number of mitotic divisions. At the end of the development of
multinucleate secondary plasmodia, the haploid nuclei come together in pairs and fuse to
form the diploid nuclei (10 chromosomes). The fusion is closely followed by meiosis of
the diploid nuclei and then cleavage of the plasmodium to yield numerous haploid resting
spores (Ingram and Tommerup 1972).
As the secondary plasmodia grow and develop, they take up proteins and sugars
from the plant cells. They also stimulate the plant cells to divide and enlarge (Ingram and
Tommerup 1972). Continuous division of these plant cells results in disorganization of
the structural arrangement in the cortex (Mithen and Magrath 1992). The cortical stages
of development can occur in resistant hosts, but resistant hosts prevent the degradation of
cell wall materials in the xylem that are required for symptom development (Donald et al.
2008). The abnormal growth and development of the cell results in gall formation.
Mature galls are attacked by soil micro-organisms, decay, and the resting spores are
released into the soil (Ingram and Tommerup 1972).
19
Stages of the life cycle of A. brassicae inside the host can be studied using a range
of stains and stained preparations. Viability of resting spores can be differentiated using
Evan's blue, which stains the cytoplasm of dead resting spores (Tanaka et al. 1999). The
stain propionic orcein can be used to observe the germination of resting spores (Friberg et
al. 2005; Ingram and Tommerup 1972). It is possible to visualize the zoosporangial
stages of P. brassicae inside the root hairs using aniline blue (Agrawal et al. 2009),
phloxine B (Donald and Porter 2004) and aceto-carmine (Samuel and Garret 1945).
Aceto-carmine stains nuclei and chromosomes (Dapson 2007). The secondary stages of
the life cycle of P. brassicae in the root cortex of susceptible host can be stained with
toluidine blue (Donald et al. 2008), methylene blue and fast green (Kobelt et al. 2000).
Toluidine blue stains the nucleus, nucleolus and chromosomes of P. brassicae and this
stain can be used to study various phases of mitosis of this pathogen inside the host
(Garber and Aist 1979). The secondary multinucleate plasmodia of P. brassicae inside
the cortex of the host are also clearly visible using toluidine blue stain (Grsic-Rausch et
al. 2000). Methylene blue in combination with Azur II and basic fuchsin stains the
cytoplasm, nuclear membrane, nucleoli, chromosomes and wall of resting spores
(Buczacki and Moxham 1979). Fast green stain can be used to study the hypersensitive
reaction of a host after secondary infection by P. brassicae using fluorescence
microscopy (Kobelt et al. 2000). This stain autoflurescences a yellowish colour for
necrotic tissues of the host and green for the pathogen inside the host roots.
20
1.3 Factors influencing infection and development of clubroot
1.3.1 Temperature
Air temperature and soil temperature are important environmental factors
influencing clubroot incidence and severity. Air temperature does not influence clubroot
but easily measured and highly correlated with soil temperature. Air temperatures are the
better indicator of the clubroot development which can be used when the soil temperature
is not available (McDonald and Westerveld 2008). Temperature has an effect on resting
spore germination and severity of clubroot on various Brassica crops. Some studies have
been done on clubroot incidence and severity in relation to soil temperature and air
temperature. The air temperatures required for germination of resting spores were
reported as 16°-219 C in the presence of a suitable host (Chupp 1917). Einhorn and
Bochow (1990) demonstrated that resting spores can germinate easily when soil
temperature is at or above 14° C. Monteith (1924) found that clubroot development took
place between temperatures of 9°-30° C but disease was severe at 25° C. He also
concluded that the temperature range over which the disease occurred was more or less
the same as that required for host growth. According to Wellman (1930), there was no
clubroot development below 12° C and above 25° C but the optimal temperature of
infection was at a range of18°-24° C. In alkaline soil, high disease severity was found
when there was a fluctuation of air temperature between points of much higher and lower
temperature than the mean temperature of 23° C (Colhoun 1953). Optimal soil
temperature for clubroot development was reported to be 18°-25° C (Colhoun 1952;
Colhoun 1953) and a positive correlation was found between soil temperature and
severity of the disease throughout the growth of the host crops. Mean air temperature of
21
19.5° C was required for^high percentage of disease to occur in a greenhouse study
(Buczacki et al. 1978). Clubroot severity on cabbage, Chinese cabbage, mustard and
radish was minimal below 14° C and maximum between 20° and 22° C (Thuma et al.
1983).
Mean air and soil temperatures during vegetative growth can have a great
influence on the development of clubroot symptoms on Asian Brassica crops, which are
short duration vegetables. Mean air temperature in the 10 days before harvest was highly
correlated with clubroot incidence and severity in Shanghai pak choy and Chinese
flowering cabbage grown in organic soil (McDonald and Westerveld 2008). A stronger
correlation was found between clubroot incidence and mean air temperature than with
soil temperature. However, soil temperature at 5 cm was more highly correlated with
symptom development than temperatures at 10 cm or deeper. Little or no clubroot
developed when mean air temperature over the last 10 days before harvest was less than
12° C. Clubroot incidence and severity were highest when the temperature was between
20°-22° C during the final 10 days before harvest (McDonald and Westerveld 2008).
1.3.2 Soil moisture
Soil moisture is another important factor influencing clubroot incidence and
severity. Soil moisture content of 70-80% of the maximum water holding capacity in
acid soil was reported to be very favourable for infection and development of clubroot
(Monteith 1924). However, continuous high moisture content is not necessary for
infection and development of clubroot. Plant roots that had been exposed for at least 18
hrs to infested soil with 80% of maximum water holding capacity can become highly
diseased (Wellman 1930). It was reported that the cumulative rainfall for the first 2-3
22
weeks after seeding and interaction of soil moisture and temperature were strongly
correlated with clubroot development (Thuma et al. 1983). It was suggested that a heavy
rain or a few moderate rains for short intervals raised the moisture content sufficient to
increase infection on cabbage plants (Wellman 1930).
1.3.3 Soil pH
Soil pH is also an important factor influencing clubroot development and severity.
There is a close relationship between clubroot incidence and soil pH (Tremblay et al. .
2005). Low soil pH favours the development of the disease. High clubroot incidence was
reported in the pH ranges 5.4-7.1 (Myers and Campbell 1985). Clubroot incidence can be
observed in alkaline soil at relatively high temperature and high spore loads (Colhoun
1953). Application of lime to increase soil pH for control of clubroot has been practiced
by farmers for many years (Dixon and Page 1998). However, application of lime to
manage clubroot is ineffective when the spore load in the soil is high (Colhoun 1953).
Soil pH may affect clubroot incidence through its influence on infection. For
example, maximal root hair infection in broccoli was observed at pH 5.5 (Donald and
Porter 2004). They also reported that the number of infected root hairs decreased with
increasing pH and there were only 5-25% infected root hairs 10 days after inoculation at
pH 8. In another study, reduction in primary infection and clubroot incidence was
observed at pH 7.2 or above, attributed to abortion of primary thalli prior to the release of
secondary zoospores (Myers and Campbell 1985). Finally, clubroot incidence was
suppressed by application of farm yard manure or compost. The authors concluded that
application of organic matter plays an important role in clubroot suppression by
increasing soil pH (Niwa et al. 2007).
23
1.3.4 Light
Light can influence clubroot levels in Brassica crops. In a glass house study, high
light intensity during the second and third week after seeding increased clubroot severity
in cabbage seedlings. The reason might be an increase in the concentration of
glucobrassicin, a precursor to clubroot development (Buczacki et al. 1978). In an earlier
study, clubroot incidence was influenced by light intensity only at low inoculum levels
(Colhoun 1961).
1.3.5 Spore load
There is a consistent relationship between high levels of inoculum and high
incidence and severity of clubroot. A spore load of at least 1000 spores/g of soil is
suggested as the minimum threshold level for symptom development of clubroot
(Faggian and Strelkov 2009). Disease severity of Chinese cabbage plants increased and
top weights decreased with increasing inoculum level from 0 to 10 spores/g of soil
(Hildebrand and McRae 1998).
1.4 Disease management
Several strategies have been developed to manage this disease. Traditionally, crop
rotation with non-Brassica crops and application of agricultural lime to raise soil pH has
been practiced (Donald et al. 2006). In Canada, the control measures recommended for
clubroot management in Brassica vegetables include crop rotation with non-Brassica
crops for 5 to 7 years, soil amendment to raise soil pH to 7.2 or higher and avoiding
growing susceptible crops in poorly drained soil (OMAFRA 2008a).
In Australia, an integrated approach to manage clubroot has been developed based
on detection and quantification of P. brassicae in the field, improvement of farm and
24
nursery hygiene and strategic application of lime (calcium oxide), calcium, boron and
fluazinam (Donald et al. 2006). Their experience indicates that this integrated control of
clubroot can be a cost effective and sustainable tool to manage clubroot in vegetable
crops. Farm and nursery hygiene, together with application of lime, calcium, boron and
the fungicide fluazinam have provided some success in clubroot management (Donald et
al. 2006). Other available strategies such as prediction of disease risk, crop rotation, use
of resistant cultivars and fungicide application can be used in integrated ways to reduce
incidence in the areas where disease pressure is moderate to high (Donald et al. 2006).
In Australia, the particular combination of treatments that is recommended as an
integrated approach to manage clubroot is based on the risk of clubroot development.
Lime application prior to transplanting and calcium nitrate and boron application at
transplanting and post transplanting is recommended at low risk sites. An application of
fluazinam is also recommended at high risk sites to manage clubroot on vegetable fields
(Donald et al. 2006).
1.4.1 Synthetic fungicides and surfactants
Fungicides with various modes of action have been evaluated against clubroot,
including fluazinam (Allegro® 5 OOF, ISK Biosciences Corporation) and cyazofamid
(Ranman® 400SC ISK Biosciences Corporation). The effective fungicide should be
active against at least one of the stages of disease development such as resting spore
germination, root hair infection, cortical infection or pathogen multiplication and
subsequent symptom expression to achieve a desirable level of control (Naiki and Dixon
1987). Knowledge of the mode of action and site of action of a fungicide is important to
control disease adequately (Tanaka et al. 1999) because it provides an indication of which
25
fungicide will be effective against which stage(s) of P. brassicae and will assist in
selecting the proper timing of fungicide application.
In the early 1970s, fungicides such as benomyl, mercurous chloride and
pentachloronitrobenzene were tested against clubroot in peat and sandy loam soil infested
with P. brassicae in greenhouse experiments. Mercurous chloride was effective both in
peat and sandy loam soils whereas benomyl and pentachloronitrobenzene were able to
reduce clubroot incidence only in sandy loam soil (Finlayson and Campbell 1971).
Mercurous chloride was widely used at the time but its use has been discontinued because
mercury was an undesirable environmental hazard (Naiki and Dixon 1987).
Buczacki (1973) examined several systemic fungicides and showed that benomyl
was effective against clubroot. Naiki and Dixon (1987) studied the impact of benomyl,
calcium cyanamide, pentachloronitrobenzene and trichlamide on root hair infection,
symptom development, host growth and clubroot severity. Benomyl and trichlamide were
effective against root hair infection, and benomyl also inhibited cortical stages of
infection by secondary zoospores. Germination of resting spores was prevented by
calcium cyanamide, which also reduced spore viability. All of these fungicides also
promoted the growth of the host (Naiki and Dixon 1987). Pentachlronitrobenzene
reduced clubbing by 90% and zoosporangial clusters by 95%. It had a limited effect on
resting spore germination, but was found to be effective against P. brassicae at the
cortical stages of infection (Naiki and Dixon 1987). In Canada, pentachloronitrobenzene
(Quintozene 75 WP) is registered to control clubroot and recommended for transplant
treatment for Brassica vegetables (OMAFRA 2008a).
26
Various surfactants were evaluated for clubroot control in the late 1980s and
1990s. Two formulations of the surfactants sodium dioctyl sulphosuccinate (Aerosol OT,
Monawet MO70) and alkyl phenyl ethylene oxide (Agral) reduced clubroot when applied
as a pre-planting compost soak. Monawet MO70 and Agral also increased the top weight
of treated plants (Humpherson-Jones 1993). Hildebrand and McRae (1998) tested the
nonionic surfactants Agral, Citowett Plus (50% octylphenoxypolyethoxy ethanol) and
AquaGro 2000-L for control of clubroot in both greenhouse and field trials. These liquid
formulations reduced clubroot severity and increased yield when applied to the transplant
hole or as a split application to the transplant hole followed by a surface drench 10 days
later (Hildebrand and McRae 1998). However, none of these surfactants were registered
in Canada to control clubroot because of the phytotoxicity to the crops (Howard et al.
2010).
Currently, the fungicide cyazofamid (Ranman® 400 SC) is registered for clubroot
control for Brassica vegetable crops in many countries including Japan (Ohshima et al.
2004) and fluazinam (Allegro® 5 OOF) is registered in Canada to control clubroot in
Brassica vegetables (PMRA 2008). However, there are no fungicides registered to control
clubroot in canola. Research is underway to identify cost-effective methods of clubroot
management for canola. A drench application of any fungicide is not practical in
commercial canola production because of the high cost of fungicide and application as
compared to returns per acre (S. Strelkov, personal communication). It would be best to
develop a method of clubroot control by seed treatment.
Fluazinam fungicide (3-chloro-N-(3-chloro-5-trifluoro-methyl-2 pyridyl)-a,a,a-
trifluoro-2,6-dinitro-p-tolidine) is currently recommended for clubroot control of
27
• vegetables in Canada. It is effective against plant diseases caused by Botrytis,
Colletotrichum, Phytophthora, Pseudoperonospora, Pyricularia and others (Komyoji et
al. 1995). It is a pyridinamine fungicide and is sold under various trade names such as
Allegro®500F in Canada, Omega in the USA and Shirlan, Shogun, and Altima in other
countries. Fluazinam has a multi-site mode of action and acts to interrupt the production
of energy in the fungal pathogen by an uncoupling effect on oxidative phosphorylation
(Guo et al. 1991). This fungicide has protective action, but little curative or systemic
activity. Fluazinam controls P. brassicae by inhibiting germination of resting spores, root
hair infection and cortical infection in Chinese cabbage. It has no effect after the cortex is
infected (Suzuki et al. 1995).
Fluazinam was introduced in the Japanese market in 1990 (Komyoji et al. 1995),
was registered in Australia in 1996 for use as a soil drench to control clubroot on Brassica
crops (Donald et al. 2001), and was registered by the Pest Management Regulatory
Agency (PMRA) in Canada in 2008 as Allegro®500F (40% fluazinam) for clubroot
control of Brassica vegetable crops either as a pre-transplant or transplant treatment. The
most effective method of application identified in Australia is incorporation of fluazinam
into the soil in bands of 23 cm wide along the transplant row to a depth of about 15 to 20
cm before transplanting. Banded soil incorporation increased the marketable yield of
broccoli and cauliflower and reduced the volume of water to apply fluazinam by 80%
compared to other commercial methods of application (Donald et al. 2001).
Cyazofamid (4-chloro-2-cyano-N, N-dimethyl-5-p-tolylimidazole-1 -sulfonamide)
is a fungicide in the phenylimidazole group that has activity against a broad spectrum of
Oomycetes and Plasmodiophoromycetes at very low use rates. This fungicide was
28
discovered and developed by Ishihara Sangyo Kaisha, Ltd. and is specifically active for
the control of late blight on potatoes and tomatoes, and downy mildew on grapevines,
cucumbers and melons (Ohshima et al. 2004). It is sold under various trade names such
as Ranman® 400 SC, Mildicut and Docious. It was registered in Japan for the control of
clubroot in Chinese cabbage in 2001 (Mitani et al. 2003). Ranman® 400 SC (34.5%
cyazofamid) is registered in Canada to control late blight of potatoes, downy mildew of
cucurbits and disease caused by Pythium spp. Its registration for clubroot control is
pending.
Cyazofamid inhibits all stages in the life cycle of Oomycetes. Cyazofamid has a
different site of action than other fungicides. Cyazofamid inhibits mitochondrial
respiration in Oomycetes and Plasmodiophora by blocking electron transfer to the bci
complex of mitochondrial cytochrome by binding to the Qj center of the mitochondrial
respiratory chain enzyme (Ohshima et al. 2004).
Cyazofamid inhibits resting spore germination of P. brassicae by 80% and when
applied to soil, it also inhibited root hair infection and club formation in Chinese cabbage
(Mitani et al. 2003).
1.4.2 Cultural and biological control
Cultural control. There are several recommended cultural practices for management of
clubroot such as crop rotation with non-Brassica crops, soil amendment to raise soil pH,
sanitation and improved drainage. These have long been recognized and are still widely
practiced today. Application of these cultural practices has had a great influence on the
severity of clubroot and the longevity of resting spores in the soil (Donald and Porter
2009).
29
' Crop rotation is one of the most recommended practices to manage clubroot.
Resting spores released from decayed galls are the source for inoculum build-up in the
soil in areas where Brassica crops are grown repeatedly (Cao et al. 2007). Resting spores
can persist and remain viable in soil up to 18 years (Wallenhammar 1996) and still cause
disease in susceptible hosts. Crop rotation has some limitations. Some growers do not
have enough land to rotate crops for extended periods of time and others grow Brassica
crops in rented land for one season only (Hildebrand and McRae 1998). Thus crop
rotation is not always feasible for all growers. However, a long crop rotation is
considered crucial to minimizing the level of inoculum in the soil (Donald and Porter
2009).
Liming the soil before planting is the most widely used control method in
clubroot infested areas (Colhoun 1953; Dobson and Gabrielson 1983; Murakami et al.
2002). Lime application does not eradicate the pathogen responsible of clubroot but it
creates unfavorable conditions for disease development (Belec et al. 2004).
Liming not only inhibits disease but also reduces spore density in the soil. Lime
application, which raises both calcium levels and pH, can reduce the number of root hair
infections and symptom development of clubroot (Webster and Dixon 1991b). The effect
of calcium on P. brassicae was found to be pH dependent (Myers and Campbell 1985).
The efficacy of lime can be increased by using finely ground lime which has a greater
surface area to volume ratio and can react more quickly in the soil up on contact with
moisture (Dobson and Gabrielson 1983).
Application of calcium cyanamide is one of the many forms of liming. It produces
hydrogen cyanamide and hydrated lime on decomposition, and hydrogen cyanamide has
30
fungitoxic properties (Klasse 1996). Calcium cyanamide has an effect on viability of
resting spores and initial infection by P. brassicae (Klasse 1996). It reduced spore density
of P. brassicae by 17-31% in comparison to nontreated control (Murakami et al. 2002).
Calcium cyanamide (Perlka, 50% calcium oxide, 19.8 % nitrogen, 1.5% magnesium
oxide) applied at three rates; 1000, 500kg/ha broadcast and 333 kg/ha in a 20 cm band 7
to 14 days before seeding, significantly reduced clubroot incidence and severity
compared to nontreated control on Shanghai pak choy grown in organic soil in Ontario
(McDonald et al. 2004).
Liming fields to raise soil pH to control clubroot has some limitations. Yearly
application of lime to maintain a high soil pH may not be suitable for cultivation of crops
other than Brassica crops (Hildebrand and McRae 1998). In Ontario, many Asian
Brassica vegetables are produced in organic soil that has relatively low pH (5.5 to 6.5)
and are highly buffered. It requires a large amount of lime to raise the soil pH and it is
costly for the growers who grow vegetables in rented land (McDonald et al. 2004). Lime
application is highly expensive for canola production to manage clubroot in terms of
economic return (Howard et al. 2010).
Farm and nursery hygiene is another important practice for the management of
clubroot and to prevent the spread of P. brassicae from infested areas to non-infested
areas. In production of transplanted vegetables, plastic trays returned to the nursery for
reuse from infested fields can also be the source of contamination (Donald et al. 2006). It
was also recommended not to use dam water contaminated with P. brassicae to irrigate
nursery stock, particularly if the nursery is located within Brassica growing regions
(Donald et al. 2006). It is important to avoid infested fields for production of susceptible
31
crops; and to use growing media that is free of pathogen inoculum (Faggian and Strelkov
2009).
Some success has also been achieved to manage clubroot disease by using
Brassica crops that have high levels of glucosinolates such as B. rapa and B. napus as
break crops (Cheah et al. 2006). Reduction in clubroot severity in Chinese cabbage was
observed when these break crops were incorporated and decomposed for three months
before planting the main crops (Cheah et al. 2006). Brassica crops having high level of
glucosinolates produce isothiocyanates up on decomposition, which has biocidal effect
against many organisms including fungi (Sawar et al. 1998). It is likely that
isothiocyanates has effect to control clubroot on Brassica crops.
Application of boron is another effective method of cultural control that reduces
the maturation rate of different stages of P. brassicae inside root hairs, and subsequent
gall formation. It inhibits sporangial maturation and suppresses the cortical colonization
by P. brassicae. Application of 30 ppm boron at pH 6.2 significantly reduced disease
development in Chinese cabbage (Webster and Dixon 1991a).
Some other cultural methods of clubroot management can be soil amendment by
organic matter and use of trap crops. Incorporation of farmyard manure or food factory
sludge compost to conducive soil reduced clubroot infection (Niwa et al. 2007). They
concluded that soil amendment by these organic matter sources increases the soil pH
from 5.7 to 7.4, and that the change in pH was the primary cause of clubroot suppression.
The spore concentration off. brassicae in the soil can be reduced by using trap crops
that stimulate the germination of resting spores. In glass house experiments, the nonhost
plant species leek {Allium porrurri), winter rye (Secale cereale) and perennial ryegrass
32
(Lolium perenne) reduced clubroot incidence on Chinese cabbage (Friberg et al. 2006),
but did not provide effective clubroot reduction in the field.
Biological control. In recent years, attempts have been made to develop biological
controls of clubroot. Microorganisms, especially plant endophytes and rhizosphere
colonizers, are potential candidates for the control of P. brassicae (Narisawa et al. 2000).
Several biological control agents that are already registered in Canada may have potential
to suppress clubroot, and many more are being assessed (G. Peng, personal
communication)
Trichoderma species have a broad spectrum of activity as biocontrol agents.
There are strains of this genus that provide effective biocontrol of a wide range of plant
pathogens via mechanisms such as parasitism, antibiosis and competition for resources
and space (Harman 2006). These fungi colonize the epidermal and cortical layer of roots.
The mechanisms of activity of strains that utilize mycoparasitism include directed growth
toward target pathogens, attachment and coiling around hyphae of the target pathogen
and production of a range of antifungal extracellular enzymes (Harman 2006). As a
result, Trichoderma spp. may have potential to control P. brassicae. In one study, 25
isolates of Trichoderma spp. were screened for biocontrol activity against P. brassicae on
Chinese cabbage in a glasshouse experiment. Among them, 17 isolates reduced clubroot
severity compared to the nontreated control (Cheah and Page 1997). In a field
experiment, 2 isolates reduced clubroot incidence (Cheah and Page 1997). RootShield®
Drench ™ WP (1.15% Trichoderma harzianum Rifai strain KRL-AG2) is registered in
Canada to control root diseases caused by Pythium, Rhizoctonia and Fusarium in
greenhouse tomatoes, cucumbers and ornamental plants.
33
Another promising group of organisms for biological control are Streptomyces
spp. They are naturally occurring soil bacteria belonging to the family Stretomycetaceae
in the order Actinomycetales. These species are very effective in biocontrol of many
kinds of plant diseases including clubroot of Brassica crops (Cheah et al. 2001). The
mode of action of these species is based on a combination of mechanisms including root
colonization, hyperparasitism and production of antifungal metabolites (Lahdenpera
2000). Some strains of Streptomyces produce small quantities of an antifungal polyene
compound that is antagonistic against many fungal pathogens in the rhizosphere
(Tahvonen 1993). Streptomyces spp. isolated from Sphagnum peat was effective against
damping-off and foot rot of Brassica crops and root rot disease of cucumber (Tahvonen
1988). The biocontrol activity of Streptomyces spp. against clubroot in Brassica
vegetables has been tested and an effective isolate (S99) was identified (Cheah et al.
2000). However, the mechanism underlying the biocontrol efficacy of this agent is
unknown. There are two Streptomyces biofungicides registered in Canada. Mycostop
WP (30% S. griseoviridis Anderson et al. strain K61) is registered for the control of
damping-off, root and stem rot, and wilt caused by Fusarium on greenhouse cucumbers,
tomatoes, peppers and greenhouse ornamentals. Actinovate® SP (0.371% S. lydicus De
Boer et al. strain WYEC 108) is registered for suppression of fungal diseases in
strawberries and peppers (PMRA 2007b).
Another potential biofungicide, Bacillus subtilis (Ehrenberg) Cohn, is naturally
occurring and can be used to control a number of fungal and bacterial diseases. It
produces complex lipopeptides that are directly involved in destroying spores and
mycelium and can suppress disease through nutrient competition, site exclusion and
34
colonization. It also induces systemic acquired resistance and induced resistance in plants
(PMRA 2007a). Serenade® ASO™ (1.34% B. subtilis QST 713) is registered in Canada
to control fungal diseases in asparagus, cole crops, and various fruits and vegetables
(PMRA 2007a).
Gliocladium catenulatum Gilman and Abbott, another potential biofungicide, is a
fungus that can grow in organic matter and is commonly found in the soil worldwide. It
suppresses other diseases through hyperparasitism and enzymatic activity. Prestop® WP
(32% G. catenulatum strain J1446) is registered in Canada for suppression of soil-borne
fungal diseases in greenhouse grown vegetables, herbs, and ornamentals (PMRA 2009).
It is highly effective against many kinds of fungal diseases.
The final potential biocontrol in this list, Heterochonium chaetospira (Grove)
M.B. Ellis, is a dark, septate, endophytic fungus that has been isolated from deciduous
trees, millipede droppings, arable soils and alpine habitats, and recently it has been
isolated from roots of Chinese cabbage grown in wheat field soil (Ohki et al. 2002). It is
also present in the humus-rich woodland soil of western Canada (Narisawa et al. 2007).
This fungus was effective in suppressing clubroot and verticillium yellows on Chinese
cabbage in vitro and under field conditions (Narisawa et al. 2000). The initial process of
host infection by this endophyte involves the formation of appressoria in the root
epidermal cells and subsequent growth of hyphae within cortical cells of the host plant
(Narisawa et al. 2000). This fungus, when grown as a root endophyte, inhibited the
development of clubroot and sometimes induced systemic acquired resistance in Chinese
cabbage (Hashiba and Narisawa 2005). A 52-97% reduction in clubroot incidence was
reported in Chinese cabbage preinoculated with H. chaetospira (Narisawa et al. 2000)
35
and two isolates of H. chaetospira (M5018 and H4027) were identified as the most
effective (Hashiba and Narisawa 2005). However, this potential biocontrol agent is not
registered for use in Canada and so could not be included in field trials of registered
agents.
Plant Resistance. Use of resistant cultivars is one of the most effective ways of
managing a wide range of diseases. Unfortunately, Brassica cultivars resistant to all P.
brassicae pathotypes are not yet available because this pathogen shows a wide variation
in pathogenicity (Diederichsen et al. 2009). Currently, resistance genes from turnip (B.
rapa) are widely used in various Brassica crops. Resistant cultivars of three Brassica
species, B. napus, B. oleracea and B. rapa, have recently been released (Diederichsen et
al. 2009). The clubroot resistance from these sources is regarded as race specific
(Diederichsen et al. 2009) but has not been studied detail. Developing cultivars with
durable and non-specific resistance to the pathotypes of P. brassicae is a major challenge.
Recently, Western Canadian Canola/ Rapeseed Recommending Committee
recommended a canola variety (45H29, Pioneer Hi-Bred) with clubroot resistance for
registration in Canada (Cao et al. 2009).
36
CHAPTER TWO
SCREENING HOST LINES FOR REACTION TO PLASMODIOPHORA
BRASSICAE
2.1 Introduction
Clubroot caused by Plasmodiophora brassicae Woronin, is an economically
important disease of Brassica crops in Canada and worldwide. Most Brassica crops are
highly susceptible to this disease, which causes 10-15% crop loss throughout the world
(Dixon 2006). Severe clubroot infestation in canola {Brassica napus L.) production areas
in Alberta, Canada, resulted in 30-100% yield loss in affected fields (Strelkov et al.
2007; Tewari et al. 2005). Control of this pathogen is especially problematic because it
produces abundant and persistent resting spores, which are easily disseminated through
movement of contaminated soil (Karling 1968).
Clubroot severity can often be reduced using strategies that have been
implemented in Brassica crops production for many years. However, these methods are
not feasible or effective in every situation, and can be prohibitively expensive. In
addition, consistent and desirable levels of clubroot control are rarely achieved by relying
on a single approach to clubroot management. Recently in Australia, integrated control of
clubroot has been emphasized, resulting in reduction in clubroot incidence and severity in
the field (Donald et al. 2006; Donald and Porter 2009).
The development of cultivars with resistance to P. brassicae would provide a
simple and environmentally friendly option for clubroot management. Breeding a cultivar
with durable resistance to P. brassicae is a major challenge because there is a diverse
range of pathotypes and most sources of resistance are race specific (Diederichsen et al.
37
2009). However, some sources of resistance appear to be effective against a range of
pathotypes. For example, swede or rutabaga (B. napus subsp. napobrassica (L.) Reichb)
is reported to have resistance to P. brassicae (Karling 1968). Similarly, some sources of
resistance were identified in European turnips (B. rapa var. rapifera) and employed to
develop a clubroot-resistant cultivar of Chinese cabbage (B. rapa var. pekinensis) (Hirai
2006). Recently, Pioneer Hi-Bred released a hybrid canola cultivar '45H29' that appears
to have nonspecific resistance to the clubroot pathotypes present in western Canada (Cao
et al. 2009).
Identification of highly susceptible and resistant hosts is important to the study of
host-pathogen interaction. Host screening of lines of Brassica crop species against P.
brassicae can identify differences in susceptibility to this pathogen. Plants in the Rapid
Cycling Brassica Collection, also known as Wisconsin Fast Plants, were developed by
selecting and breeding early flowering lines of Brassica species that have a short
lifecycle and can produce up to 10 generations of seeds per year (Williams and Hill
1986). Screening of these Brassica Fast Plants against P. brassicae could identify lines
that would be used for research under controlled conditions where results could be
obtained more quickly than with conventional crop cultivars.
The overall objective of this study was to assess the disease reaction of selected
Brassica crops to P. brassicae pathotype 6, which is the predominant pathotype at the
testing site in Ontario (Cao et al. 2009; Reyes et al. 1974). However, single-spore
assessments have indicated that pathotypes 3 and 5 may also be present in clubroot
infested soil in this province (Xue et al. 2008). One objective of this research was to
identify Fast Plant lines that were as susceptible to P. brassicae as Shanghai pak choy,
38
which might be used in research conducted on a temperature gradient plate. The
temperature gradient plate is a precise and costly piece of equipment specifically
designed for research related to temperature (B. D. Gossen, personal communication).
Using a small short-cycle crop can expedite the research, minimize the space required for
assessments under controlled conditions, and reduce the time for each cycle, thereby
minimizing the cost of the research. Another objective of this research was to identify
Fast Plant lines that might be used as models for canola and other commercial crops in a
broad range of studies. A third objective was to identify species or lines that could be
used to develop a Canadian series of host differentials to identify pathotypes of P.
brassicae. Identification of a Canadian set of host genotypes is important because the
differential sets developed for effective differentiation and classification of P. brassicae
does not provide consistent results to classify the pathotypes predominant in Canada.
Also, it is getting difficult to obtain seeds of the one important species in Differential sets
of the Williams series, Granaat napa cabbage. The (S. E. Strelkov, personal
communication).
2.2 Materials and methods
Diverse Brassica crops were selected for clubroot screening to identify the
reaction to P. brassicae pathotype 6 in field soils in Ontario. These trials were conducted
at the University of Guelph, Muck Crops Research Station (MCRS), Holland Marsh, ON,
Canada (44° 15' N, 79° 35' W) in 2008 and 2009, where there is organic soil (pH 6.7,
69% organic matter) naturally infested with P. brassicae.
Nine Brassica Fast Plant lines obtained from the Rapid Cycling Brassica
Collection (RCBC), Wisconsin, U.S.A. were assessed in these trials. The Fast Plants were
39
Brassica carinata A. Braun, B.juncea L., B. napus L., B. nigra L., B. oleracea L., B.
rapa L. astroplant, B. rapa standard rapid cycling, B. rapa atrazine resistant and
Raphanus sativus L. Other crops tested were: four lines of canola: 46A76 IMI
(Imidazolinone tolerant), 46A65 (conventional), 45H21 RR (Canola Roundup Ready
hybrid) and Invigor 5020 LL (Liberty Link), and the Asian vegetables Shanghai pak choy
(two cultivars), two cultivars of Chinese flowering cabbage and three cultivars (Deneko,
Bilko and Yuki) of napa cabbage. Seed sources and cultivar names are listed in Table 2.1.
In these trials, Shanghai pak choy, which is highly susceptible to P. brassicae (McDonald
and Westerveld 2008), was used as a susceptible control. Some of the hosts tested in the
trials in 2008 were not included in 2009. The host reaction to P. brassicae was similar
within each species for two lines of Shanghai pak choy, the two lines of Chinese
flowering cabbage and the three cultivars of napa cabbage. Therefore, only one line from
each of these groups was assessed in the trials conducted in 2009.
All of the crops were direct seeded on 09 July in 2008 and 2009. The seeding date
was selected to ensure that crop growth occurred during that portion of the growing
season when there is a high risk of clubroot development (McDonald and Westerveld
2008). A randomized complete block design was used in all trials. In 2008, a limited
amount of seed of the Fast Plants was available, so they were seeded in two 1.5-m-long
rows per plot with three replications. The other crops were seeded in single 5.4-m long
rows per plot with four replications. In 2009, there were two 3-m-long rows per plot and
four replications for all crops. The trials in 2008 and 2009 were conducted within 100 m
of each other. The Fast Plants, canola and Asian vegetables were harvested 6 weeks after
seeding and the napa cabbages at 10 weeks after seeding to obtain their optimum harvest
40
maturity. All of the plants in each plot were assessed for clubroot incidence and severity.
Disease severity was rated using a 0-3 scale based on Kuginuki et al. (1999), where: 0 =
no galling, 1 = a few small galls (small galls on less than one-third of roots), 2 =
moderate galling (small to medium-sized galls on one-third to two-thirds of roots), and 3
= severe galling (medium to large-sized galls on more than two-thirds of roots (Fig.
3.IB). A disease severity index (DSI) was calculated using the following equation
(Kobriger and Hagedorn 1983), as:
X [(class no.)(no. of plants in each class)]
DSI= ______ ; xioo (total no. plants per sample)(no. classes -1)
Weather parameters were measured at a weather station located at the Muck
Crops Research Station within 100 m of the experimental plots. Daily air temperatures
were measured using a HMP35C probe and rainfall data using a tipping bucket rain
gauge (Campbell Scientific, Edmonton, AB, Canada). The data for temperatures and
rainfall were recorded every hour using a CR21X data logger (Campbell Scientific).
Daily maximum, minimum and mean temperatures and total rainfall were calculated for
the period between the day after seeding and the day before harvest for each trial.
41
Table 2.1 Lines of Brassica species assessed for susceptibility to clubroot in naturally infested soil in field trials at the Muck Crops Research Station, Holland Marsh, ON, 2008 and 2009.
Crop and line Scientific name Seed source
Shanghai pak choy
Generic
Mei Qing Choi
Chinese flowering cabbage
Generic
Tsoi-sim
Napa cabbage
Deneko
Bilko
Yuki
Canola
45H21
46A76
46A65
Invigor 5020 LL
B. rapa L. subsp. Chinensis (Rupr.) var. communis Tsen and Lee
B. rapa L. subsp. Chinensis (Rupr.) var. utilis Tsen and Lee
B. rapa L. subsp. pekinensis (Lour.) Hanelt
B. napus L.
Chan Man Hop Seeds Co., Hong Kong
Stokes Seeds Ltd., ON, Canada
Chan Man Hop Seeds Co., Hong Kong
Stokes Seeds Ltd., ON, Canada
Bejo Seeds Inc., New York, U.S.A.
Bejo Seeds Inc., New York, U.S.A.
Stokes Seeds Ltd., ON, Canada
Pioneer Hi-Bred, ON, Canada
Pioneer Hi-Bred, ON, Canada
Pioneer Hi-Bred, ON, Canada
Bayer Crop Science, ON, Canada
42
2.2.1 Data Analysis -
All of the statistical analyses were performed using SAS software (version 9.1
SAS Institute, Cary, NC). A mixed-model analysis of variance for the data in each trial
was conducted using PROC MIXED procedure. The fixed effect was species/lines and
the random effects were year and replication. The data set for each trial was tested for
normality using the Shapiro-Wilk test of residuals and outliers were identified using
Lund's test of standardized residuals (Lund 1975). Mean comparisons were completed
using Tukey's Multiple Mean Comparison Test. Differences were significant at P < 0.05
unless otherwise noted.
2.3 Results
2.3.1 Weather
Air temperature and rainfall during the growing period of the crops were recorded
for both years and compared with the long-term (10-year) average for the site (Table 2.2).
The long-term average was the mean of 10-years data including the year when the trial
was conducted. The mean air temperature during July was substantially lower and August
was higher in 2009 compared to July and August in 2008. Temperatures in September
were similar for both years. Air temperatures during August in 2008 and July in 2009
were below the long-term average. Rainfall during July was substantially higher for both
years compared to other months, and above the long-term average (Table 2.2).
43
Table 2.2 Mean air temperature and rainfall during the growing period of Brassica crops for clubroot screening at the Muck Crops Research Station, Holland Marsh, ON, 2008 and 2009.
iviontn
2008
July
August
September
2009
July
August
September
Temperature (°C)
LTA1
20.3
19.2
15.7
19.9
19.3
15.5
Actual
20.4
17.9
14.7
17.9
19.4
14.9
Rainfall (mm)
LTA
69
56
80
76
57
72
Actual
137
63
82
135
89
62
Long-term average (10-year mean) (Source: Muck Vegetable Cultivar and Research Report 2008 and 2009)
2.3.2 Incidence and severity assessment
There were differences in clubroot incidence and severity among the Brassica
crops in both years and the year by host interaction was significant. Clubroot incidence
and severity were higher in 2008 compared to 2009. However, the general pattern of the
host reaction to P. brassicae was similar in both years, although there were differences in
reaction of some of the Fast Plants in 2009 that did not show up in 2008.
In 2008, the Fast Plant lines of Raphanus sativus and B. napus had a relatively
high level of resistance to P. brassicae; incidence and severity for these crops were less
than 10%. Most of the other Fast Plant lines were highly susceptible to clubroot, with
levels similar to that of the susceptible control, Shanghai pak choy in 2008 (Table 2.3).
Clubroot incidence and severity were numerically higher in B. carinata and B. juncea
than in Shanghai pak choy even though they were not significantly different. The two
cultivars of Shanghai pak choy were equally susceptible to P. brassicae and there were
44
also no difference between the two cultivars of Chinese flowering cabbage in the 2008
trial (Table 2.3). Among the four canola lines, two (46A76 and 46A65) were relatively
susceptible to pathotype 6 with incidence of 52% and 33% respectively in 2008. The two
other lines (Invigor 5020LL and 45H21) were completely resistant at this site (Table 2.3).
Clubroot incidence and severity on the three cultivars of napa cabbage, which were sold
as resistant to clubroot, were very low and did not differ from the resistant lines of
canola.
The trial in 2009 was conducted within 100-m of the trial in 2008. Clubroot
incidence and severity in 2009 were relatively low in comparison to 2008, and the most
susceptible crop had roughly 50% of the incidence of 2008. Most of the lines had a
similar pattern of response in both years (Table 2.3). Among the Fast Plant lines, B.
carinata and B. juncea had the highest incidence and severity and there was low or no
clubroot on the B. napus and Raphanus sativus lines. The canola lines responded in the
same manner in 2009 as in 2008. Correlation analysis showed that there was positive
correlation between the two years of data for clubroot incidence (r = 0.67, p = 0.005) and
severity (r = 0.80, p = 0.0002).
45
Table 2.3 Clubroot incidence (CI %) and disease severity index (DSI) on Brassica crops or lines grown in organic soil naturally infested with clubroot at the Muck Crops Research Station, Holland Marsh, ON, 2008 and 2009.
Crops/lines
Rapid cycling Brassica crops
Brassica carinata
B. juncea
B. nigra
B. rapa; astroplant
B. rapa; standard rapid cycling
B. rapa; atrazine resistant
B. oleracea
Raphanus sativus
B. napus
Asian vegetables
Shanghai pak choy (generic)
Mei Qing Choi
Flowering cabbage (generic)
Tsoi-sim
Napa cabbage
Deneko
Bilko
Yuki
Canola
46A76
46A65
Invigor 5020LL
45H21
2008
CI (%)
97 f1
96 f
89 ef
84 ef
82 ef
75def
57c-f
10 ab
4ab
89 ef
80 ef
38 bed
37 bed
6ab
3ab
l ab
52 cde
33abc
0a
0a
DSI
97 f
95 f
66def
69 ef
74 ef
67 ef
37b-e
7ab
1 a
71 ef
56 cde
23 abc
23 abc
2 a
3a
l a
44b-e
26a-d
0a
0a
2009
CI (%)
45 c
30 be
3a
2a
19 abc
8ab
10 ab
0a
1 a
nd
9ab
nd
4ab
0a
nd
nd
l l a b
4a
0a
0a
DSI
19c
16 be
1 a
1 a
8 abc
7 abc
3ab
0a
0.4 a
nd
3ab
nd
2ab
0a
nd
nd
4ab
l a
0a
0a
Values within a column followed by the same letter do not differ at P = 0.05, Tukey's Multiple Mean Comparison Test, nd = not done
46
2.4 Discussion '
There were differences in clubroot reaction among the Brassica crops and lines
assessed in 2008 and 2009. Among the four canola lines, two (45H21 and Invigor
5020LL) were completely resistant at this site and two others (46A76 and 46A65) were
susceptible. The study is the first to evaluate the reaction of Brassica Fast Plants to P.
brassicae. Most of the Fast Plant lines were highly susceptible to P. brassicae pathotype
6. Two Fast Plant lines, B. napus and Raphanus sativus, and all the cultivars of napa
cabbage showed relatively high levels of resistance to pathotype 6, with clubroot
incidence and severity below 10% in both trials. The Fast Plant lines of B. carinata, B.
juncea and B. nigra had similar reaction to that of the susceptible control, Shanghai pak
choy. Thus these Fast Plant lines are potential candidates for host-pathogen interaction
studies.
Clubroot incidence and severity were lower in 2009 than in 2008. Most of the
highly susceptible and highly resistant Brassica lines showed a similar pattern in both
years. However, the B. nigra and B. rapa astroplant lines, which had high disease in
2008, had low disease in 2009 compared to the B. rapa standard rapid cycling, B. rapa
atrazine resistant and B. oleracea lines of Fast Plants. One possible explanation for the
differences in clubroot levels between the years is a difference in distribution of the
resting spore density in the soil, even though the trials in 2008 and 2009 were conducted
within 100 m of each other. A spore load of at least 1000 spores/g of soil has been
reported as a minimum threshold level for symptom development (Faggian and Strelkov
2009). Root hair infection (Naiki et al. 1978) and clubroot severity (Hildebrand and
McRae 1998) increase with increasing spore loads. It is possible that the spore
47
concentration was lower at the site of the 2009 trial than the one in 2008. The possible
reason of reduced level of spore load in 2009 plot may be due to the growing of non-host
crop (onion) during the year before the trial was conducted. Wallenhammar (1996) also
reported significant1 decrease in clubroot incidence (49% to 7%) with time (1986-87 to
1990-92) when Brassica crops were not grown in the infested field. An efficient method
to determine inoculum concentration would be useful to test the distribution and
concentration of inoculum before a trial was established and qPCR techniques are being
developed to address this need (S.E. Strelkov, personal communication).
Several Brassica Fast Plant lines were as susceptible to P. brassicae pathotype 6
as Shanghai pak choy. Crete and Chiang (1980) also reported high susceptibility (DSI
52-100) when they evaluated 109 Brassica genotypes to P. brassicae pathotype 6. The
Fast Plant lines B. carinata and B. juncea can be used as model crops for subsequent
studies on clubroot. The Fast Plant line B. rapa has also potential to be as model crop for
canola since some canola cultivars are B. rapa. This might be also useful to compare the
yield in relation to clubroot severity, and in research situations where time and space are
limited. The Fast Plants are small and have a short generation time, which can be grown
at high densities (2500 plants per square meter) (Williams and Hill 1986). These plants
facilitate completion of research within a short period relative to regular lines of the same
crop where space is limited, such as containment facilities or temperature gradient plate.
The resistant lines that were identified in these trials might be utilized as sources
of genetic resistance. Highly resistant lines can be further tested with different pathotypes
of clubroot to determine their reaction. The susceptible canola lines (46A76 and 46A65)
can be used to study the biology of host-pathogen interaction at the field site in Ontario
48
without having to introduce pathotype 3 from western Canada at this site. All of the
cultivars of napa cabbages (Deneko, Bilko and Yuki) that were marketed as being
clubroot resistant were found to be highly resistant, but were not immune to P. brassicae
pathotype 6. The result of cultivar Bilko in the trial in 2008 was similar with Hasan
(2010) who reported 89-100% of resistance reaction of this cultivar to all pathotypes (2,
3,5,6 and 8) of P. brassicae from Canada. He also evaluated seventy seven B. nigra
genotypes against these Canadian pathotypes, and reported sixty genotypes with high
level of resistance and two with susceptible reactions. In the current trial, the Fast Plant
line B. nigra was highly susceptible to P. brassicae pathotype 6 in the2008 trial. It is
possible that the genotype of this line may be similar with the susceptible genotypes of B.
nigra that reported by Hasan (2010).
In the current trials, the Fast Plant line Raphanus sativus showed a high level of
resistance to P. brassicae pathotype 6 in both years. This crop is in the Brassica family
and a close relative of the genus Brassica (Williams and Hill 1986) and the introduction
of clubroot resistance genes from this crop to Brassica crops were reported to be possible
through somatic hybridization (Hagimori et al. 1992). Thus the resistant Fast Plant line R.
sativus identified in these trials may have potential for breeding resistant Brassica
cultivars. However, evaluation of reaction of this host to wide range of pathotypes of P.
brassicae is crucial to develop non-specific and durable resistant cultivar.
Two systems have been widely used for pathotype classification of P. brassicae;
the differential set of Williams (1966) and the European Clubroot Differential (ECD) set
(Buczacki et al. 1975). These systems have been used to characterize the populations of
P. brassicae pathogen from Canada (Strelkov et al. 2006). However, the current cultivars
49
in the ECD differential set do not provide a consistent reaction that can be used for the
differentiation and classification of P. brassicae strains in Canada (Strelkov et al. 2006;
Howard et al. 2010). A previous study conducted in Japan to classify the populations of
P. brassicae also reported intermediate results using these differential hosts (Kuginugi et
al. 1999). It is also a problem to locate commercial sources of several of the lines in these
differential sets such as the seeds of Granaat napa cabbage (S. E. Strelkov, personal
communication). Thus, development of Canadian differential sets to classify P. brassicae
available in Canada is important for resistance breeding and clubroot management. The
differences in susceptibility that were identified in these current trials could be utilized in
developing a Canadian set of differential plants to determine pathotypes of P. brassicae.
In this study, canola lines Invigor 5020 LL and 45H21, which were known to be
susceptible to pathotype 3 (Strelkov et al. 2006), showed a resistant reaction to pathotype
6. Thus either of these lines can be used to differentiate pathotype 6 from pathotype 3 of
P. brassicae.
In summary, Rapid Cycling Brassica lines B. carinata and B. juncea can be used
as models for commercial cultivars in clubroot studies under controlled conditions.
Canola cultivars 46A76 and 46A65 susceptible to P. brassicae pathotype 6 can be used
for subsequent clubroot studies in Ontario without introduction of pathotype 3 from
western Canada. Canola cultivars Invigor 5020 LL and 45H21 susceptible to pathotype 3
and resistant to pathotype 6 can be used to separate pathotype 6 from 3. The Rapid
Cycling Brassica lines Raphanus sativus and B. napus which showed a high level of
resistance to pathotype 6 may be the potential source of resistance breeding but further
investigation with other pathotypes is required to confirm the results.
50
CHAPTER TtiREE
EFFECT OF TEMPERATURE ON INFECTION AND SYMPTOM
DEVELOPMENT OF CLUBROOT
3.1 Introduction
Temperature is an important environmental factor that influences clubroot
incidence and severity on Brassica crops. The optimal soil temperature for clubroot
development was reported to lie in the range of 18°-25° C (Colhoun 1953), and a mean
air temperature of 19.5° C was required for 100% infection of clubroot in a greenhouse
study (Buczacki et al. 1978). Thuma et al. (1983) observed very low levels of clubroot at
temperatures below 14° C and high levels at temperatures of 20°-22° C, but the specific
stage(s) in the complex infection cycle of this pathogen that is affected by temperature
was not identified. Identifying the time periods or host developmental stages where soil
and air temperature have the greatest impact on infection and symptom development of
clubroot will enhance our understanding of the biology of this host-pathogen interaction
and could be useful for management of this disease.
Mean air temperature in the 10 days before harvest was positively correlated with
clubroot incidence and severity on short-season Brassica vegetables grown in organic soil
(McDonald and Westerveld 2008). The highest clubroot incidence and severity was
observed for crops harvested during July and August, when temperatures ranged from
20°-22° C during the growing period, and the lowest incidence and severity was
observed for crops harvested in October when mean air temperatures during the final 10
days before harvest were below 12° C. This indicates that one potential approach for
utilizing information on temperature relationships in clubroot management would be for a
51
producer whose land base is heavily contaminated with clubroot resting spores to select
planting dates that avoid the warm conditions that this pathogen requires, and thereby
minimize clubroot incidence and severity. However, these observations have not been
substantiated in research trials designed specifically to study this response.
Much of the research on the impact of temperature has focused on clubroot
incidence and severity in relation to soil and air temperatures in the field and greenhouse,
and only a few studies on the impact of temperature have been conducted in controlled
environments. A report from 1917 first indicated that temperatures of 16°-21° C were
required for germination of resting spores of P. brassicae in the presence of the host
(Chupp 1917). Similarly, resting spores germinated readily when soil temperature was at
or above 14° C (Einhorn and Bochow 1990). However, there are no studies on the impact
of temperature on root hair infection, or to demonstrate a relationship between resting
spore germination and root hair infection. In contrast, the relationship between root hair
infection and subsequent clubroot incidence and severity has been examined several
times, but the results were not consistent. A linear relationship was reported between root
hair infection and spore concentration in the inoculum in one report (Macfarlane 1952)
but root hair infection was not related to clubroot incidence in a subsequent study (Naiki
et al. 1978). Macfarlane (1952) also observed that a high incidence of clubroot was
occasionally associated with lower numbers of root hair infections on cabbage seedlings.
Despite these different observations, study of root hair infection may be useful to identify
various factors that inhibit infection (Samuel and Garrett 1945) because subsequent
stages of cortical infection and symptom development rarely if ever occur without root
hair infection.
52
Fungicide application is commonly used for clubroot management in Brassica
vegetable crops. One effective new product is cyazofamid fungicide (Ranman® 400 SC),
which has activity against P. brassicae (Donald and Porter 2009). It inhibits resting spore
germination, root hair infection and clubroot symptom development (Mitani et al. 2003).
Ranman is not currently registered for this use in Canada, so evaluation leading to
registration of this fungicide against clubroot will provide a new alternative for managing
this disease.
A study was undertaken to learn more about the effects of temperature on various
stages of the life cycle of P. brassicae. The first phase of infection by P. brassicae is root
hair infection (Ingram and Tommerup 1972). Primary plasmodial and zoosporangial
stages of root hair infection can be observed under magnification by staining infected
root hairs using 1% aceto-carmine (Samuel and Garrett 1945), phloxine B (Donald and
Porter 2004) or aniline blue (Agrawal et al. 2009). The aceto-carmine stains nuclei and
chromosomes (Dapson 2007). When primary plasmodia differentiate to multinucleate
zoosporangia, they take up stain well, making it easy to observe root hair infection
(Samuel and Garrett 1945). Phloxine B stains callose, nuclei and cell wall, and aniline
blue stains fungus spores and mycelium (Schneider 1981). Both stains enable observation
of primary stages of P. brassicae infection inside root hairs (Agrawal et al. 2009; Donald
and Porter 2004). The secondary stages of the life cycle of P. brassicae, which develop in
the root cortex of susceptible hosts, can be stained using toluidine blue (Donald et al.
2008), methylene blue or fast green (Kobelt et al. 2000). Toluidine blue stains the
nucleus, nucleolus and chromosomes of P. brassicae and this stain can be used to study
various phases of mitosis of this pathogen inside the host (Garber and Aist 1979). The
53
secondary multinucleate plasmodia of P. brassicae inside the cortex of the host are also
clearly visible using toluidine blue stain (Grsic-Rausch et al. 2000). Methylene blue in
combination with Azur II and basic fuchsin stains the cytoplasm, nuclear membrane,
nucleoli, chromosomes and wall of resting spores (Buczacki and Moxham 1979). Fast
green stain can be used to study the hypersensitive reaction of a host after secondary
infection by P. brassicae using fluorescence microscopy (Kobelt et al. 2000). This stain
autoflurescences a yellowish colour for necrotic tissues of the host and green for the
pathogen inside the host roots.
The main objective of this study was to identify the effect of temperature on
symptom development of clubroot over the first 6 to 7 weeks of plant growth. Specific
trials were conducted to determine the effect of temperature on clubroot incidence and
severity on canola and Shanghai pak choy under controlled conditions and to identify the
effect of temperature on symptom development and severity in the field using selected
seeding dates to provide a range of temperature regimes. A secondary objective was to
determine if temperature and disease pressure affected the efficacy of Ranman 400® SC
(34.5% cyazofamid) fungicide against clubroot on Shanghai pak choy and Chinese
flowering cabbage. Evaluating Ranman on several seeding dates to assess its efficacy
across a range of temperatures and hence a range of disease pressure will provide an
indication of the conditions when fungicide application is necessary for clubroot
management of Brassica crops grown in infested organic soil. An additional objective
was to identify the effect of temperature on root hair infection on Shanghai pak choy
under controlled conditions. This information could be useful in identifying the critical
period(s) of infection and pathogen development in relation to temperature. It will also
54
provide an insight into this holt-pathogen interaction, which in turn could be useful in
identifying the appropriate time to apply fungicide and other management tools to control
clubroot effectively.
3.2 Materials and methods
3.2.1 Plant materials
Three Brassica crops susceptible to P. brassicae were used for the field and
growth cabinet studies. Two short-season Asian vegetables were chosen for these trials
because they were known to be susceptible to clubroot pathotype 6, which occurs in
Ontario. The crops were Shanghai pak choy {Brassica rapa subsp. Chinensis (Rupr.) var.
communis Tsen and Lee), and Chinese flowering cabbage (B. rapa subsp. Chinensis
(Rupr.) var. utilis Tsen and Lee). Canola 34-65 RR (B. napus L.), which is susceptible to
pathotype 3, was used in the trials with soil from Alberta infested with pathotype 3.
3.2.2 Controlled environment trials
Trials were conducted in growth cabinets at the University of Guelph in 2008 and
2009 to identify the effect of temperature on infection and symptom development of
clubroot under controlled conditions and to compare the effects of temperature on
clubroot incidence and severity between Shanghai pak choy and canola.
Resting spores of pathotype 6 (Reyes et al. 1974; Strelkov et al. 2006) were
extracted from clubbed roots of cabbage {B. oleracea L. var. capitata L. cv. Saratoga)
plants grown in soil naturally infested with clubroot at the Muck Crops Research Station,
Holland Marsh, ON, Canada in 2008. After collection, the roots were washed and stored
at -20° C until use. At the time of spore extraction, 3 g of frozen galls were soaked in
distilled water at room temperature for 2 hr to soften the tissue. The roots were macerated
55
with 100 mL distilled water in a commercial waring blender at a high speed for 2
minutes. The resulting suspension was filtered through eight layers of cheese cloth. The
concentrations of resting spores in the filtered solution were estimated using a
haemocytometer (Fisher Scientific, Markham, ON) and diluted with distilled water to the
desired concentration for inoculation.
In the first experiment (Trial 1), Shanghai pak choy seeds were planted in organic
soil obtained from the Muck Crops Research Station that was naturally infested with
clubroot pathogen. The soil was placed in rhizotron boxes (Root Vue Farm, 2000 HSP
Nature Toys, North Hills, CA) and each seed was planted adjacent to the transparent
acrylic viewing window to facilitate observation of the development of clubroot galls
over time. There were three replicate rhizotrons per temperature treatment. Each
rhizotron box was seeded with eight seeds of Shanghai pak choy and two seeds of
Chinese flowering cabbage. Plants of flowering cabbage were grown with the pak choy to
provide an indication of the relative stage of maturity because flowering cabbage
produces flowers several weeks earlier than Shanghai pak choy (M.R. McDonald,
personal communication). Under cool temperature conditions, one cannot differentiate
the growth stages of the pak choy other than through plant size (S. M. Westerveld,
personal communication). The size of the plant is much different in rhizotron boxes or
other containers than in the field in response to growing conditions. As a result, flowering
cabbage is a better indicator of the effects of temperature on plant physiological
development of pak choy, and was used to estimate the physiologic growth stage of the
pak choy in the temperature treatments.
56
The inoculurA potential of the infested soil used in Trial 1 was supplemented by
inoculation with resting spores of P. brassicae at a concentration of 1 x 10 spores/mL
suspension, applied at 5 mL /seeding hole, before seeding, because clubroot incidence
was highly variable in a preliminary experiment that utilized infested soil alone.
Subsequently, the spore load of P. brassicae in soil samples from the area where the soil
was collected for Trial 1 was estimated at 1 x 106 resting spores/g of soil (M. T.
Tesfaendrias, personal communication).
In Trial 2, Shanghai pak choy seeds were sown in soil-less mix (Sunshine mix #4,
Sun Gro Horticulture Canada Ltd, Spruce Grove, AB) in individual tall plastic pots called
conetainers (164 mL, Stuewe & Sons, Inc. Corvallis, OR) with 10 plants per replicate and
four replicates per treatment. Each conetainer was inoculated with 5 mL resting spore
suspension with a concentration of 1 x 106 resting spores/mL. The change to inoculated
soil-less mix was made to ensure that there was a known and repeatable concentration of
inoculum.
The canola plants in Trial 1 and Trial 2 were grown in conetainers filled with
mineral soil from Alberta that was naturally infested with clubroot. The exact spore load
of P. brassicae in the soil used for these trials is not known. However, it was estimated at
between 106 and 107 resting spores/g of soil based on clubroot development on bait plants
grown in soil collected from the same area (S.E. Strelkov, personal communication). The
soil was mixed thoroughly to ensure uniform distribution of P. brassicae prior to filling
the soil in conetainers. There were 10 plants per replicate, with three replicates per
treatment in Trial 1 and four replicates in Trial 2. Shanghai pak choy and canola in both
57
trials were grown for 6 weeks and watered daily with water adjusted to pH 6.3 to
maintain high moisture and a slightly acidic pH in the soil.
There were two parts to each trial. In the first part of Trial 1 (Trial 1 A), plants
were grown in growth cabinets set at 14°, 17°, 20°, 23° or 26° C for the first 3 weeks and
then moved to 20° C for the final 3 weeks. In the second part of the experiment (Trial
IB), plants were grown at 20° C for the first 3 weeks and then transferred to growth
cabinets set at 14°, 17°, 20°, 23° or 26° C for the final 3 weeks. In Trial 2, a wider range
of temperatures were examined. Plants were grown at 10°, 15°, 20°, 25° and 30° C for the
first 3 weeks (Trial 2A) or for the final 3 weeks (Trial 2B). Plants in each experiment
were maintained under a 14-hr photoperiod with 65% relative humidity. A combination
of fluorescent and incandescent lights with an intensity of 200-250 umolm" s' was
provided.
Temperatures inside the growth cabinets were monitored using HOBO
temperature sensors (Model HOBO ProSeries Temp RH (c) 1998 ONSET, Agviro Inc.
Guelph, ON). Temperatures were recorded at 4-hr intervals and the daily mean
temperature was calculated from the day after seeding to the day before harvest.
Temperature data recorded from observation of a thermometer placed inside of each
growth cabinet was used to estimate the temperature in those instances where the HOBOs
failed to record the data.
Symptom development of clubroot in Shanghai pak choy was observed during the
growing period through the viewing window of the rhizotron boxes in Trial 1, starting 3
weeks after seeding and continuing twice a week until 6 weeks after seeding. The growth
stages of Chinese flowering cabbage were recorded while assessing the clubroot infection
58
biweekly lising the growth stage key developed for B. campestris and B. napus (Harper
and Berkenkamp 1975) in which 0 = pre-emergence, 1 = seedling, 2 = rosette, 3 = bud
and 4 = flower stage. The Shanghai pak choy and canola plants were harvested at the end
of week 6. The roots were thoroughly washed and assessed for clubroot incidence and
severity. Clubroot severity was rated using a 0-3 scale (Kobriger and Hagedorn 1983)
(Fig 3.IB) to separate plants into four classes, where: 0 = no galling, 1 = a few small galls
(small galls on less than 1/3 of roots), 2 = moderate galling (small to medium sized galls
on 1/3 to 2/3 of roots), and 3 = severe galling (medium to large sized galls on more than
2/3 of roots (Kuginuki et al. 1999). A disease severity index (DSI) was calculated using
the following equation.
X [(class no.)(no. of plants in each class)] DSI= . xlOO
(total no. plants per sample)(no. classes -1)
3.2.3 Seeding date trials
Plots were seeded at monthly intervals during the growing season in 2008 and
2009 at the University of Guelph, Muck Crops Research Station, Holland Marsh, ON to
study the effect of temperature on clubroot development in the field. These seeding dates
provided a range of temperature regimes. Shanghai pak choy and Chinese flowering
cabbage were direct seeded using a Stan Hay precision seeder (Stan Hay Co., Ashford,
UK) at the rate of 34 seeds/m (Fig. 3.1A). The seeding dates were 13 May, 11 June, 09
July, 06 August and 03 September in 2008 and 13 May, 11 June, 08 July, 05 August and
02 September in 2009. There were two treatments for both crops at each seeding date; a
nontreated control and a drench application of Ranman® 400SC (34.5% cyazofamid, 46 g
59
a.i./ha, 300 mL/m) in a 15-cm wide band over the seed row within 3 days of seeding.
Ranman® 400 SC was applied 14 May, 12 June, 10 July, 08 August and 05 September in
2008 and the same day as seeding in 2009. Each plot consisted of two rows, 42 cm apart
and 6 m in length. There were four replications per treatment arranged in a split-split plot
design in which seeding date was the main plot, host was the subplot, and fungicide was
the sub-subplot. In 2009, approximately 7 mm of irrigation was applied to each plot using
a sprayer within 24 hr after the drench application of Ranman" 400 SC to determine if
irrigation increased the efficacy of the fungicide.
Assessments of clubroot incidence and severity were conducted at weekly
intervals for each seeding date. Plants in 1 m of row were uprooted starting from 2-3
weeks after seeding, whenever the plants had three to four true leaves, to 5-6 weeks after
seeding, when the plants were at marketable size or 1 week after they reached the
marketable size. The roots were thoroughly washed and assessed as described previously.
60
Cfesv--:--",
Figure 3.1 (A) Planting Shanghai pak choy and flowering cabbage at the Muck Crops Research Station, Holland Marsh, ON, 2009. (B) Clubroot severity rating scale where: 0 = no galling, 1 = a few small galls (small galls on less than 1/3 of roots), 2 = moderate galling (small to medium sized galls on 1/3 to 2/3 of roots), and 3 = severe galling (medium to large sized galls on more than 2/3 of roots).
61
Weather parameters were measured in a weather station (Campbell Scientific,
Edmonton, AB) located at the Muck Crops Research Station within 100 m of the
experimental plots. Daily air temperatures were measured using a HMP35C probe and
rainfall data using a tipping bucket rain gauge. The data for air temperature and rainfall
were recorded every hour using a CR21X data logger. Soil temperature at a depth of 5-
cm in the cropped field was obtained by a HOBO temperature sensor (Model HOBO
ProSeries Temp RH (c) 1998 ONSET, Agviro Inc. Guelph, ON) buried at 5 cm depth in
the plot from the day of seeding to final harvest. Soil temperature was recorded at 4 hr
intervals. Daily mean, minimum, and maximum temperatures and total rainfall were
calculated for the period between the day after seeding and the day before harvest for
each trial.
3.2.4 Zoosporangia development in root hairs
A trial to assess the impact of temperature on zoosporangia development inside
root hairs was conducted in growth cabinets in 2009. This trial was conducted in parallel
with Trial 2 of the temperature study, in which plants of both species were grown in
conetainers. This allowed comparison of the effect of temperature on zoosporangia
development with clubroot incidence and severity in the conetainer trial. Plants were
grown using a modification of the sand-liquid culture method developed by Donald and
Porter (2004). Shanghai pak choy seeds were sown into 5 mL pipette tips containing
autoclaved sand. One seed was sown in each pipette tip and then placed inside 50 mL
Falcon tubes (Fisher Scientific, Markham, ON) containing nutrient solution, with three
tips per tube. A stock nutrient solution was prepared by mixing 80 g of 15:15:18 NPK
fertilizer to 1 L of water. The nutrient solution was prepared by adding 5 mL of stock
62
solution to 1 L of water and the resulting solution was adjusted to a pH of approximately
6.3 using commercial vinegar (5% acetic acid; 0.8 mL/L of deionized water). Nutrient
solution was added to the Falcon tubes as required. The level of nutrient solution in the
Falcon tube was determined by visual observation of the movement of solution to the
pipette tips to ensure sufficient moisture in the growing media. The nutrient solution
moved in the sand to the germinating seed by capillary action.
Resting spores were extracted as described previously from galls of cabbage
grown at the Muck Crops Research Station that had been stored -20° C. The inoculum
was applied to the sand before seeding to provide a condition similar to the field.
o
Inoculation was done by pipetting 300 uL of spore suspension (1x10 resting spores
/mL) onto the surface of the sand in each pipette tip. A 300 uL quantity of water was
applied for the control. After seeding, Falcon tubes were placed in growth cabinets
maintained at 10°, 15°, 20°, 25° and 30° C. Plants were grown with a 14-hr photoperiod
at 65% relative humidity. A combination of fluorescent and incandescent lights was used
in the growth cabinets to provide light intensity of 200-250 umolm" s" .
Plants were harvested at 10 days and 14 days after inoculation to observe the
incidence of root hair infection. There were six replications for each harvest date, each
consisting of five plants per treatment. No samples were obtained from the growth
cabinet at 10° C because the seed did not germinate at this temperature. Five plants from
two Falcon tubes per replication were harvested 10 days after inoculation and the roots
were washed to remove adhering sand particles. The cleaned roots were placed in a
fixative (50 mL glacial acetic acid and 50 mL ethyl alcohol) for at least 24-hr. Roots were
then placed individually in 2 mL centrifuge tubes containing aceto-carmine staining
63
solution. The root samples could be kept in this solution for prolonged periods without
deterioration or over-staining. Counts of the infected root hairs were made after staining
for 24 hr or more.
The aceto-carmine staining solution was prepared as follows: 45 mL glacial acetic
acid was mixed with 55 mL distilled water and heated to boiling, then 0.5g carmine red
powder was added to the solution and heated for 15-20 minutes while stirring. The
solution was cooled and filtered through Whiteman filter paper. A ferric oxide solution
was prepared separately by mixing 45 mL glacial acetic acid, 55 mL distilled water and
5g ferric oxide. The ferric oxide solution was added drop-wise to 50 mL of the aceto-
carmine solution until precipitation occurred. The remaining 50 mL solution was added
to the mixture and the resulting solution was filtered through filter paper. The prepared
aceto-carmine solution was stored in a dark glass bottle until required.
Stained root samples were assessed for root hair infection using a compound
microscope at 125x (objective lOx and eye piece 12.5x) magnification. Assessment was
performed using two methods. In the first method, roots were assessed using a 0 to 4
scale (Merz 1989), where 0 = no sporangia, 1 = only a few sporangia, 2 = several roots
with sporangia, 3 = sporangia regularly present, moderate infection, and 4 = sporangia
regularly present, heavy infection. An intensity index for root hair infection was
calculated using the formula for disease severity index by Kobriger and Hagedorn (1983)
described previously. In the second method, only three roots from each replication were
assessed, employing the technique of Donald and Porter (2004). In this method, 100 root
hairs from the midsection (approximately 2-3 cm from hypocotyls) of each root were
counted and used to calculate the percentage of root hair infection.
64
3.2.5 Data analysis
All statistical analyses were performed using SAS software (version 9.1 SAS
Institute, Cary, NC). The entire data set for each trial was tested for normality using the
Shapiro-Wilk test of residuals and outliers were identified using Lund's test of
standardized residuals (Lund 1975). Analysis of variance for all data in each trial was
conducted using PROC MIXED or PROC GLM. The impact of temperature in the
growth cabinet trials was assessed using single degree freedom contrasts to detect linear
and quadratic relationships. Where these assessments identified a significant effect of
temperature, that effect was described using regression. Residuals from the regression
analysis were graphed and assessed visually to ensure that these regression lines
represented robust estimates of the relationship. Means comparisons of clubroot
incidence and severity were conducted using Tukey's Multiple Mean Comparison Test.
To summarize the overall effect of seeding date, host and fungicide application on
the incidence and severity of clubroot in the field trials, the area under the disease
progress curve (AUDPC) was calculated for incidence and severity up to optimal harvest
maturity of the crops for each seeding date treatment using day as the time unit. A mixed
model analysis of variance of the combined data across two years was conducted. The
fixed effects were seeding date (5 seeding dates), host (Shanghai pak choy and Chinese
flowering cabbage) and fungicide (Ranman application and nontreated control); and the
random effects were year, block and their interaction with the fixed variables. There was
no main effect for year or interaction of year with seeding date, host or fungicide
application.
65
Pearson correlation analysis (PROC CORR) was used to examine the strength of
the relationship between weather variables (soil and air temperatures and rainfall) and
clubroot level (incidence and severity). Step-wise multiple regression was also performed
to assess the relative impact of rainfall over the growing season, early (1-15 days after
seeding), late (1-10, 11-20 days before harvest) and season mean air and soil
temperatures on clubroot incidence and severity at optimum harvest in the field trials. In
all of the analyses, differences were significant at P < 0.05 unless otherwise stated.
3.3 Results
3.3.1 Temperature differences in a controlled environment
The development of clubroot symptoms on Shanghai pak choy grown in rhizotron
boxes was strongly influenced by temperature in both the first 3 weeks (Trial 1 A, Fig. 3.2
A) and for the final 3 weeks (Trial IB, Fig. 3.2 B) of growth. Clubroot symptoms
developed earlier in plants grown at high temperatures compared to lower temperatures.
Symptoms were first observed at 21, 25, 28, 32 and 39 days after seeding in plants kept at
26°, 23°, 20°, 17° and 14° C respectively for the first 3 weeks and moved to a cabinet
maintained at 20° C for the final 3 weeks (Trial 1 A, Fig. 3.2 A). Clubroot symptoms were
first observed 28 days after seeding in 4 of 5 temperature treatments when plants were
kept at 20° C for the first 3 weeks (Trial IB, Fig. 3.2 B), and were present in the
remaining temperature treatment (26° C) at 32 days after seeding (Fig. 3.2 B).
Temperature also had an impact on subsequent symptom development in this trial
(Fig. 3.2). Clubroot incidence was high and different from other temperature treatments
at 25, 28 and 32 days after seeding when plants were grown at 26° C during the early
stage of crop growth (Fig. 3.2 A). Clubroot incidence progressed rapidly from 32 to 39
66
days after seeding in all of the temperature treatments except 14° C, which resulted in
similar level of clubroot incidence among these treatments by 42 days after seeding.
Clubroot development was delayed at 14° C compared to the other temperature
treatments (Fig. 3.2 A).
There were no differences on clubroot incidence among temperature treatments
until 32 days after seeding when plants were grown at 20° C for the first 3 weeks and
moved to the growth cabinets set at wide range of temperatures for the final 3 weeks (Fig.
3.2 B). Clubroot incidence increased with increasing temperatures at 35, 39 and 42 days
after seeding, which resulted in high clubroot incidence for the crops grown at 23° and
26° C and low clubroot incidence for the crops grown at 14° C during late growth stages
of the crop (Fig. 3.2 B).
In the growth cabinet trials, actual air temperatures were recorded in each growth
cabinet using HOBO temperature sensors to determine the fluctuation from target
temperatures (Table 3.1) because large differences could lead to misinterpretation of the
data on clubroot development in relation to temperature. In most of the growth cabinets,
actual temperatures were close to the target temperatures with overall fluctuation of mean
temperature less than 1° C. The exceptions were the growth cabinets set at 14° and 23° C
for the first trial, where actual mean temperatures were 16.3° and 21.3° C, respectively
(Table 3.1).
67
Figure 3.2 Effect of temperature on clubroot incidence and symptom development over time on Shanghai pak choy grown under controlled conditions. In Trial A, plants were grown for the first 3 weeks at 14°, 17°, 20°, 23° and 26° C and at 20° C for the final 3 weeks (A). In Trial B, plants were grown at 20° C for the first 3 weeks and moved to 14°, 17°, 20°, 23° and 26° C for the final 3 weeks (B). Values within a same day of assessment followed by same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test.
68
Table 3.1 Target and actual temperatures achieved in Trials 1 and Trial 2 under controlled conditions
Target
First 3 weeks
14
17
20
23
26
20
20
20
20
20
temperature (°C)
Final 3 weeks
Actual temperature (°C)
First 3 weeks
Trial 1A
20
20
20
20
20
16.3
17.4
20.1
21.7
26.9
Trial IB
14
17
20
23
26
20.6
20.6
20.6
20.6
20.6
Final 3 weeks
19.8
19.8
19.8
19.8
19.8
16.3
17.4
20.1
21.7
26.9
Target
First 3 weeks
10
15
20
25
30
20
20
20
20
20
temperature (°C)
Final 3 weeks
Actual temperature (°C)
First 3 weeks
Trial 2A
20
20
20
20
20
10.0
14.6
20.5
25.0
30.0
Trial 2B
10
15
20
25
30
20.5
20.5
20.5
20.5
20.5
Final 3 weeks
19.0
19.0
19.0
19.0
19.0
10.0
14.6
19.0
24.0
30.0
69
The response of clubroot incidence and severity on Shanghai pak choy and canola
at final harvest in relation to temperature was mostly quadratic for both early and late
growth stages of the crop (Figs. 3.3 and 3.4). The one exception for Shanghai pak choy
occured in Trial IB, where the response was linear rather than quadratic during the late
stages of growth (Fig. 3.3). In canola, clubroot incidence was so high for all temperature
treatments that there was no relationship between incidence and temperature in Trial 1 or
Trial 2A (Fig. 3.4). Also, there was no significant trend for clubroot severity in Trial IB .
However, clubroot severity exhibited a consistent quadratic relationship with temperature
in early stages of growth in Trial 1 and both early and late stages of growth in Trial 2 for
both Shanghai pak choy and canola (Figs. 3.3 and 3.4) even though these crops were
grown in different growing media. Regression analysis indicated that the highest clubroot
incidence and severity in Shanghai pak choy occurred at 22.4° and 24.1° C (Trial 1A) and
at temperatures 21.8° and 19.6° C (Trial 2B) respectively. In canola, clubroot severity
was highest at 23.8° C in Trial 1A and in Trial IB clubroot incidence and severity were
highest at 25.5° and 21.7° C respectively. Overall, the optimum temperatures of clubroot
incidence and severity during early and late growth stages of Shanghai pak choy and
canola ranged from 19.6° to 25.5°C.
70
120 -i
100
m
$ 60 a
40
20
0 -I
120
100
bso in
S 60 -
a 40
20
o -
Incidence : R2 = 0.96, Y = -239.17 + 30.97x - 0.69x2
Severity: R2 = 0.96, Y = -230.46 + 23.46x - 0.48x2
1A
1 , O Incidence
, • Seventy
/ t
• Severity
i i i i i i
Incidence : R2 = 0.98, Y= -131.05 + 14.57x -0.22x2
Severity: R2 = 0.98, Y = -91.70 + 9.50x - 0.11 x2
2A ~s^<>
o S , / * ' - ' •
/ S
/ • ' ' / r
/ / / /
f t / /
/ / / *
%// ft T ' ~ i i i i ^
) 10 15 20 25 30 35 '
Temperature ( C) for early growth stage
Incidence : R2 = 0.48. Y = 7.33 + 3.72x Severity: R2 = 0.63, Y = -21.14 +3.98x
1B
o y •
Incidence : R2 = 0.52, Y = 7.78 + 8.29x - 0.19x2
Severity: R2 = 0.98, Y = -53.63 + 12.15x - 0.31 x2
2B
j . ^
\ 4
•
t 10 15 20 25 30 35
Temperature (°C) for late growth stage
Figure 3.3 Effect of temperature on clubroot incidence and severity in Shanghai pak choy grown under controlled conditions in Trials 1 and 2, where temperature treatments were applied in the first 3 weeks (A) or the subsequent 3 weeks (B).
71
Incidence : not significant Severity: R2 = 0.90, Y = -34.26 +• 11.43x - 0.24x2
120
100
« v> 60 -
5 40
20 -
1A
* •
O Incidence • Severity
Severity
Incidence : not significant Severity: R2 = 0.83, Y = -23.73 + 8.29x - 0.12x2
120 -,
100
3?
V)
at u> 60 Q
40
20
n -
-
2A O 0 O 1
0 0 • •
t
f
•
Incidence and severity : not significant
1B O O
0 0
0 • • •
Incidence : R2 =0.83, Y =-88.99 + 15.32x -0.30x2
Severity: R2 = 0.87, Y =-34.26+9.11 x-0.21x2
2B ^
/
•
U "I I I I I 1 1 -1 1 , 1 , 1 1
5 10 15 20 25 30 35 5 10 15 20 25 30 35
Temperature (°C) for early growth stage Temperature ( C) for late growth stage
Figure 3.4 Effect of temperature on clubroot incidence and severity in canola grown under controlled conditions in Trials 1 and 2, where temperature treatments were applied in the first 3 weeks (A) and the subsequent 3 weeks (B).
72
The growth stages of Chinese flowering cabbage and canola in all trials were
similar across the range of temperature treatments. The timing of seed germination and
development of true leaves were similar between Shanghai pak choy and Chinese
flowering cabbage prior to the bolting stage of flowering cabbage. Low temperature (10°
C) delayed germination of the crops. Germination was completed (95-100%
germination) at 7 days after seeding at 10° C, at 5 days after seeding at 15° and 20° C,
and 3 days after seeding at 25° and 30° C. The growth of plants grown at 25° and 30° C
differed by only one leaf compared to plants grown at 15° and 20° C. For example, plants
grown at 10° C had four true leaves when the plants grown at 20° C had five true leaves.
There were no differences in growth stage in plants grown at 20° C for the first 3 weeks
and moved to the range of temperature treatments for the final 3 weeks.
3.3.2 Impact of seeding date on clubroot development
The timing of symptom development and final clubroot incidence and severity in
the nontreated control differed among the seeding dates in both 2008 and 2009. In 2008,
symptoms were first observed at 3 weeks after seeding in the July planting for both
Shanghai pak choy and Chinese flowering cabbage (Figs. 3.5 and 3.6). The first
symptoms appeared 4 weeks after seeding in crops planted in June, followed by
September (5 weeks) and May (6 weeks) plantings for both crops. Clubroot did not
develop on Shanghai pak choy seeded in August, but trace levels were observed on
Chinese flowering cabbage at 5 weeks after seeding. In addition, the rate of clubroot
development differed among the seeding dates in both years. In 2008, clubroot incidence
and severity increased between 3 and 4 weeks after seeding for both crops seeded in July
(Figs. 3.5 and 3.6). Clubroot incidence and severity also increased between 4 and 6 weeks
73
after seeding in the June planting in both crops and increased between 5 and 7 weeks in
the May planting of Chinese flowering cabbage (Figs 3.5 and 3.6). In 2009, clubroot
symptoms were first observed in the June and July plantings at 3 weeks after seeding,
followed by August (4 weeks), and May (5 weeks). No clubroot developed in September
on either crop (Figs. 3.5 and 3.6). The pattern of symptom development in 2009 at the
various seeding dates was similar to that in 2008, with the exception of the May and
August plantings. There was an increase in incidence and severity on Shanghai pak choy
from 3 to 4 weeks after seeding in July, 4 to 5 weeks after seeding in June, and 5 to 6
weeks after seeding in May (Fig. 3.5). The highest incidence of clubroot on Shanghai pak
choy was 90% in week 6 of the July seeding in 2008 and 99-100% by week 5 of the June
and July seedings in 2009. On Chinese flowering cabbage, clubroot incidence and
severity increased between weeks 5 and 6 in the August seeding and between weeks 6
and 7 in the May seeding (Fig. 3.6).
74
Figure 3.5 Clubroot incidence (%) and severity (disease severity index) on Shanghai pak choy planted at monthly intervals in organic soil naturally infested with the clubroot pathogen at the Holland Marsh, ON, in 2008 and 2009. A solid line denotes development to optimum harvest maturity and a dotted line indicates crop beyond optimum harvest maturity. Values for a single seeding date followed by the same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test.
75
1
100 i
50
80 -
- 7 0 -
| 50 -
1 40 < - 30 -
20 •
10 -
0 H
too -
90 •
I 70 H
£ 50 . lb
1 50-
« g 30 • 4ft
Q 20 -
10 -
ft -
2008
. c
«««•-Angus! h / j;
-•—Sspl. / J*
2008
c c , , - *
2 3 4 5 6
Weeks after seeding
b
b
7
2009
a
2089
a
2
c
•
/ /
j j ab'
be , ' ^ 4 /
J'
3 4 5 6 7
Weeks after seeding
Figure 3.6 Clubroot incidence (%) and severity (disease severity index) on Chinese flowering cabbage planted at monthly intervals in organic soil naturally infested with the clubroot pathogen at the Holland Marsh, ON, in 2008 and 2009. A solid line denotes development to optimum harvest maturity and a dotted line indicates crop beyond optimum harvest maturity. Values for a single seeding date followed by the same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test.
76
Analysis of AUDPC combined over two years showed that seeding date had an
important impact (P < 0.0001) on clubroot incidence and severity, but there were
interactions with both host and fungicide application (see ANOVA tables, Appendix 3).
Clubroot incidence and severity were most severe in the July planting followed by the June
planting. Clubroot levels were minimal in the May, August and September plantings and
there were no differences among these months (Fig. 3.7).
Seeding date also had an effect on the expression of host reaction to P. brassicae.
Clubroot incidence and severity on Shanghai pak choy were higher than on Chinese
flowering cabbage (Table 3.2), especially in the June and July plantings where clubroot
levels were highest. In the August planting, clubroot incidence was higher in Shanghai pak
choy than Chinese flowering cabbage, but there was no difference in severity. Shanghai
pak choy and Chinese flowering cabbage had similar levels of clubroot in the May and
September plantings, when clubroot levels were very low (Table 3.2).
A drench application of Ranman reduced clubroot incidence in all plantings
compared to the nontreated control, except in the September seeding when only a trace
amount of clubroot developed. Similarly, Ranman reduced clubroot severity compared to
the nontreated control in the May, June and July plantings, but had no measurable impact
in August or September, when clubroot severity was very low (Table 3.2). The efficacy of
Ranman application on crop yield at optimum harvest was also evaluated on both Shanghai
pak choy and Chinese flowering cabbage. There was no difference in yield of both crops
between nontreated control and treated with Ranman for any seeding date either for either
year (Table A 2.6).
77
600 -
500
400 A
UD
PC
3 i
100
n -
600 -
500 -
400
g 300 Q => < 200 -
100 -
a
c
Incidence (%)
a
b
Disease Severity Index
b
V
a
c
May June July
Seeding month
a
a
August
a
a
Sept.
Figure 3.7 The effect of seeding date on clubroot incidence and severity summarized as area under the disease progress curve (AUDPC) and combined across host, fungicide treatment and year, at the Holland Marsh, ON, 2008-2009. Bars with the same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test.
78
Table 3.2 The interaction of host (Shanghai pak choy vs. Chinese flowering cabbage) and fungicide application (Ranman vs. control) with seeding date on clubroot incidence and severity, summarized as area under the disease progress curve (AUDPC), in a field study at the Holland Marsh, ON (combined data from 2008 and 2009).
Host Seeding month shanghai pak Flowering
choy cabbage
Fungicide
Control Ranman
AUDPC (Incidence %)
May 126
June 314*
July 739*
Aug. 126*
Sept. 24
AUDPC (Disease severity index)
May 57
June 160*
July 399*
Aug. 50
Sept. 13
45
170
393
2
1
22
70
194
1
1
119*
296*
725*
103*
22
67*
156*
407*
41
12
26
189
407
24
3
11
74
186
9
2
*Indicates that pairs of means (Shanghai pak choy vs. flowering cabbage, Ranman vs. control) for a specific seeding date differed at P = 0.05 based on Tukey's Multiple Mean Comparison Test.
79
The relationship between daily mean air and soil temperatures and infection and
symptom development of clubroot was investigated for Shanghai pak choy grown in
2008 and 2009. Daily mean air temperature was more variable than soil temperature, but
showed a similar pattern to soil temperature. A benchmark of 17° C was chosen for the
analysis of the effect of temperature on clubroot development in the field trials because
results from earlier controlled environment trials demonstrated that clubroot development
was slow or non-existent on Shanghai pak choy at temperatures below 17° C. This
temperature is also close to but below the temperature range of 18°-25° C that Colhoun
(1953) reported to be optimal for clubroot development.
In the May plantings in 2008 and 2009, the mean air and soil temperatures were
below 17° C for the first 3 weeks in 2008 and for the first 4 weeks in 2009, although air
temperatures went above 17° C for a short period in the first 3 weeks for both years (Fig.
3.8). In 2008, soil and air temperatures started to increase at 3 weeks, and were above
17° C between 3 and 5 weeks after seeding. The air and soil temperatures during the final
10 days before harvest were above 17° C, and similar for both years (Fig. 3.8). In 2008,
clubroot symptoms were first observed at 6 weeks after seeding and reached only 9%
incidence at final harvest despite warm temperatures (>17° C) between 3 and 5 weeks
after seeding and during the final 10 days before harvest. In contrast, clubroot symptoms
were first observed at 5 weeks after seeding in 2009 and increased to 95% incidence at
final harvest, even though temperatures were cooler (<17° C) up to 4 weeks. However,
mean air (20° C) and soil (21° C) temperatures were warm during the final 10 days
before harvest (Fig. 3.8). It is likely that the temperatures did not explain all of the
80
variation in clubroot incidence on Shanghai pak choy in the May seeding between the
two years.
Daily mean air and soil temperatures were above 17° C in the June seeding for
both years except the second week of the month in 2008. Clubroot symptoms in the June
planting in 2008 developed at 4 weeks after seeding and incidence at final harvest was
64% (Fig. A 2.1). In contrast, soil and air temperatures from 2 to 3 weeks in the June
seeding in 2009 ranged from 20° to 24° C. Clubroot symptoms developed at 3 weeks
after seeding and incidence reached 99% at final harvest. There was a sharp increase in
clubroot incidence from 3 to 4 weeks after seeding in June 2009 (Fig. A 2.1).
In the July seeding in 2008 and 2009, daily mean air and soil temperatures were
above 17° C most of the times throughout the growing period and clubroot symptoms
were first observed at 3 weeks after seeding in both years. The clubroot incidence at final
harvest was 87% in 2008 and 100% in 2009 (Fig A 2.3).
81
tyy It
24 -
_ 21 • U *~ 18 V w 3 15 , m £ 12
1-6
3 -
0
-•*- Soil temperature
May seeding 2008
• \ * A. * ^ I 1
1 : 1
—*— Air temperature
1 *"•!
JL if 5
a a — • • -
J* ~
—•— lncidence(%)
•s»
ML * * * » V * - -
W^*~——¥:-
-_ a a
-*•*?— 1 -L
3 (B I) Q. E «
100
90
7 0 ' - -
60 ~ w
50 § 40 |
c 30 -
20
10
0
3 4
Week after seeding
Figure 3.8 Clubroot incidence (%) on Shanghai pak choy seeded in May in soil naturally infested with clubroot at the Holland Marsh, ON, in 2008 and 2009. A solid line denotes clubroot development to optimum harvest maturity and a dotted line indicates development beyond optimum harvest maturity. Values followed by the same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test.
82
In the August plantings in 2008 and 2009, mean air and soil temperatures
throughout the growing season were above 17° C, and were similar in 2008 and 2009. In
2008, there was more fluctuation in air temperatures compared to soil temperatures (Fig.
3.9). The air and soil temperatures were above 17° C from the end of the first week to 4
weeks after seeding, except that the air temperature went below 17° C for a short period
during this time. In 2009, air and soil temperatures were above 17° C for the first 3
weeks, then went below for week 4, above 17° C for week 5, and then dropped again.
Mean air and soil temperatures during the final 10 days before harvest were below 17° C
for both years. In 2008, no clubroot symptoms developed on Shanghai pak choy. In 2009,
clubroot symptoms were first observed at 3 weeks after seeding and incidence increased
to 87% at final harvest (Fig. 3.9).
In the September seeding in 2008 and 2009, daily mean air and soil temperatures
were below 17° C for most of the growing period after seeding and were well below than
this temperature range for the 2 weeks before harvest in both years. Clubroot incidence
was low: 8% in 2008 and 0% in 2009 (Fig. A 2.4).
83
Soil temperature •Air temperature •Incidence (%)
3
|
I
27
24 +
21
18 t-15 -
12 -
9 -
6 -
3 -
0 -
August seeding 2008
100
w o c
2 '5 c
2 3 4
Week after seeding
Figure 3.9 Clubroot incidence (%) and severity on Shanghai pak choy seeded in August in soil naturally infested with clubroot pathogen at the Holland Marsh, ON, in 2008 and 2009. A solid line denotes clubroot development to optimum harvest maturity and a dotted line indicates development beyond optimum harvest maturity. Values followed by the same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test.
84
Weather
The 2008 field season was hot and dry while the 2009 field season was
comparatively cool and wet. In 2008, the mean air temperatures were below the long-
term (10-year) averages for August and September, near normal for July and above
average for June. Total rainfall values were below the long-term averages for May and
June, above average for July and August and near normal for September. In 2009, the air
temperatures were below the long-term average values for June and July, and near normal
for May, August and September. Total rainfall values were above the long-term averages
for May, July and August and below average for June and September (Table 3.3).
Table 3.3 Monthly mean air temperature and rainfall at the Muck Crops Research Station, Holland Marsh, ON, in 2008 and 2009 and long term means.
Month
2008
May
June
July
August
September
2009
May
June
July
August
September
Temperature (°C)
LTA1
12.6
18.4
20.3
19.2
15.7
12.1
18.2
19.9
19.3
15.5
Actual
10.7
19.2
20.4
17.9
14.7
12.6
16.5
17.9
19.4
14.9
Rainfall
LTA
80
76
69
56
80
86
74
76
57
72
(mm)
Actual
48
68
137
63
82
117
49
135
89
62
Long-term average (10-year mean) (Source : Muck Vegetable Cultivar and Research Report 2008 and 2009)
85
Correlation analysis was used to identify the association between clubroot
incidence and severity on Shanghai pak choy and Chinese flowering cabbage and
selected weather parameters. There was a positive correlation between clubroot incidence
on Shanghai pak choy and mean air and soil temperature at 5-cm depth calculated across
the growing period (Table 3.4). However, clubroot severity on Shanghai pak choy and
incidence and severity on Chinese flowering cabbage were not correlated with seasonal
means of either air or soil temperature. Instead, cumulative rainfall during the growing
period of the crop was highly correlated with clubroot severity in Shanghai pak choy and
Chinese flowering cabbage. Rainfall was also correlated with clubroot incidence on
Chinese flowering cabbage and weakly correlated with incidence on Shanghai pak choy
(Table 3.4). Total rainfall during the first 2 weeks after seeding was not correlated with
clubroot incidence and severity in Shanghai pak choy and Chinese flowering cabbage.
Total rainfall during the first 3 weeks was only correlated with clubroot severity on
Chinese flowering cabbage (Table 3.4). Similarly, stepwise multiple regressions also
indicated that the total rainfall during the growing period of the crop was the most
important weather variable affecting clubroot incidence and severity for both Shanghai
pak choy and Chinese flowering cabbage.
A more detailed analysis was performed by dividing the growing period into pre
selected time intervals. The time intervals were 1 to 5, 6 to 10, 1 to 10, 11 to 15, and 11 to
20 days before optimum harvest and 1 to 15 days after seeding. There was no correlation
between temperature and clubroot incidence or severity for any of the selected time
intervals (Table 3.4). The relationships among weather variables were also tested to
86
determine the degree of autocorrelation. The strongest correlation (r = 0.96, p = <.0001)
occurred between season mean air temperatures and soil temperatures at a depth of 5 cm.
The correlation analysis was also performed to identify the relationship between
clubroot severity and crop yield at harvest for the data from field trials. There was no
correlation between clubroot severity and crop yield on both Shanghai pak choy and
Chinese flowering cabbage (Table 3.4).
87
Table 3.4 Linear correlation between clubroot incidence and severity and selected variables (mean air and soil temperatures, rainfall and top weight) during the various time intervals for Shanghai pak choy and Chinese flowering cabbage grown at the Holland Marsh, ON, 2008 and 2009.
Time interval and variables
Season mean Air
Soil, 5-cm
Rainfall Season total
First 2 weeks
First 3 weeks
Top weight Root weight
Correlation with clubroot
Pak
r
0.64
0.65
0.63
0.21
0.54
0.19
0.91
incidence
choy
P
0.04
0.04
0.05
NS
NS
NS
0.0002
1 to 5 days before harvest Air
Soil, 5-cm
0.47
0.55
NS
NS
6 to 10 days before harvest Air
Soil, 5-cm
0.47
0.52
NS
NS
1 to 10 days before harvest Air
Soil, 5-cm
11 to 20 days Air
Soil, 5-cm
11 to 15 days Air
Soil, 5-cm
0.50
0.55
NS
NS
before harvest 0.45
0.38
NS
NS
before harvest 0.55
0.60
NS
NS
1 to 15 days after seeding Air
Soil, 5-cm
0.46
0.38
NS
NS
Flowering cabbage
r
0.57
0.54
0.75
0.19
0.56
0.19
0.32
0.37
0.43
0.49
0.53
0.46
0.49
0.45
0.34
0.55
0.58
0.36
0.27
P
NS
NS
0.013
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
Correlation with clubroot severity
Pak choy
r
0.60
0.61
0.74
0.24
0.61
0.13
0.91
0.40
0.47
0.47
0.51
0.46
0.51
0.43
0.33
0.54
0.58
0.45
0.36
P
NS
0.05
0.013
NS
NS
NS
0.0002
NS
NS
NS
NS
NS
NS
NS
NS'
NS
NS
NS
NS
Flowering cabbage
r
0.58
0.56
0.83
0.32
0.68
0.30
0.48
0.30
0.36
0.49
0.52
0.42
0.46
0.44
0.34
0.54
0.58
0.44
0.33
P
NS
NS
0.003
NS
0.03
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
88
3.3.3 Zoosporangia development in root hairs
The impact of temperature on zoosporangia development in root hairs of
Shanghai pak choy was similar for both techniques of root hair assessment. No
zoosporangia were found in the noninoculated controls (Fig. 3.10 A). The zoosporangial
stage of the pathogen was observed in root hairs (Fig. 3.10 C) and in the root epidermis
(Figure 3.10 D).
The seedlings grown in sand-liquid culture medium were assessed at 10 and 14
days after inoculation on a 0-4 scale. No zoosporangia were observed at either
assessment date in plants grown at 15° and 20° C. Zoosporangia were observed in root
hairs at 25° and 30° C as early as 10 days after inoculation (Fig. 3.11). The intensity of
root hair infection at 25° C was higher (64 %) than at 30° C (45%) at 14 days after
inoculation (Fig. 3.11).
Zoosporangia in root hairs at 10 and 14 days after inoculation were also assessed
by counting 100 root hairs at the midsection (2-3 cm from hypocotyl) of the root. The
percentage of infected root hairs at 25° C was higher than at 30° C at both 10 and 14 days
after inoculation (Fig. 3.12).
89
*
Figure 3.10 Root hairs of Shanghai pak choy grown in sand-liquid culture medium under controlled conditions. A) Healthy root hairs on a noninoculated plant, B) Root hairs infected with Plasmodiophora brassicae, C) Close-up of zoosporangia in root hairs, and D) Zoosporangia in root epidermal cells.
90
100 -i
and
in
ten
sity
<J
> 00
o
o
1 1
§S 40 -In
cide
nce
D
O
100 -
* w 80 -01
TJ 60 -c re
£. 40 -
Inci
denc
e
3 O
u
10 DAI
a Incidence {%)
• Intensity index
a x a x
b
y a
X i
i • " " i i i
b b
14 DAI
a x a x
• - z
y
i i i i
15 20 25 30 Temperature (°C)
Figure 3.11 Incidence (%) and intensity index of zoosporangia in root hairs of Shanghai pak choy 10 and 14 days after inoculation (DAI). Bars followed by the same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test.
91
""TP*
£ g £3 » ( .£ . i
!
o
*
50 -
45 -
40 -
35 -
30 -
25 -
20 -15 -
10 -
5 -n u
ia10 DAI
014DAI
X a i
15
c
y
x a l l l l 20 25
Temperature (°C)
b
X i i
30
Figure 3.12 Root hairs with zoosporangia (%) based on counts of 100 root hairs at the mid section of each root on Shanghai pak choy at 10 and 14 days after inoculation (DAI). Bars capped with the same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test.
92
3.4 Discussion
Across all of the studies conducted under controlled conditions and in the field,
temperature had a substantial influence on root hair infection, initiation of symptom
development, and the subsequent severity of clubroot. Clubroot symptoms developed
earlier and were most severe at temperatures above 17° C.
In the growth cabinet studies, clubroot severity in both Shanghai pak choy and
canola generally exhibited a quadratic response to temperature even though the two crops
were grown in different growth media and the source of inoculum differed. In Trial 1,
canola was grown in mineral soil heavily infested with P. brassicae and Shanghai pak
choy was grown in naturally infested soil amended with spores (1 x 106 spores/mL, 5
mL/seedling). In Trial 2, the study was standardized by growing the plants in soil-less
mix and applying a known number of spores at planting. The incidence of clubroot on
canola was higher than in Shanghai pak choy and approached 100% in most of the
temperature treatments. It was reported that clubroot severity increased with increasing
inoculum level on Chinese cabbage (Hildebrand and McRae 1998). It is possible that the
high spore load in the mineral soil may have overwhelmed any response to temperature.
However, clubroot severity on canola exhibited the same strong response to temperature
to that of Shanghai pak choy in most assessments.
In Trial 1, clubroot incidence and severity in the 14° C treatment would
presumably have been even lower if the target temperature had been achieved, but there
were fluctuations in the temperature in the growth cabinet and the mean value was
substantially higher at 16.3° C. However, the broader temperature regime assessed in
Trial 2 provided the same pattern of response; clubroot incidence and severity were very
93
low when plants were kept at 10° and 15° C for the first 3 weeks and moved to 20° C for
the final 3 weeks. This indicates that temperatures below 17° C restrict development of
clubroot on seedlings.
Shanghai pak choy seedlings were grown in rhizotron boxes under controlled
conditions in Trial 1. We found that plants could be grown for at least 6 weeks in these
boxes. It was possible to observe clubroot development through the transparent acrylic
viewing window of the rhizotrons without disturbing the plants. However, it was not
possible to visualize all of the roots from outside, and incidence was often higher when
the harvested roots were assessed that based on visual assessment of plants in-situ. The
clubroot development in these boxes was less consistent among replications in our
preliminary trial and in Trial 1. To reduce this variability, tall plastic pots (conetainers)
were used in Trial 2 instead of the rhizotron boxes. These conetainers could not be used
to observe symptom development without destructive sampling, but plants could be
maintained for 7 weeks or more to reach optimum maturity of the crops and to provide
more time for disease development. There is also an advantage in that their long (21cm),
narrow shape allows for the development of more normal root architecture as compared
to shallower standard-shaped pots. It also makes it possible to grow plants very close
together, and hence to conduct large trials using limited space under controlled
conditions. In the current trials, conetainers were found to be effective compared to
rhizotrons for conducting trials on clubroot.
Based on the response of Shanghai pak choy and canola in these growth cabinet
trials, we conclude that Shanghai pak choy can be used as model crop for canola in
subsequent studies of temperature, but further work is required to determine if the
94
response of inoculum concentration is the same. Shanghai pak choy may be good model
for other kinds of studies as well. Shanghai pak choy is a small plant with a short
generation cycle (McDonald and Westerveld 2008). The use of pak choy, which is
susceptible to pathotypes of P. brassicae that do not attack canola, as a model would
make it feasible to conduct controlled environment research on clubroot outside of
containment facilities. This would be a substantial benefit to the growing number of
researchers located in areas of western Canada where the pathotypes that attack canola
are not yet established, who are constrained from working with the pathogen by concerns
about the possibility of inadvertent release of the pathogen. However, Shanghai pak choy
as a model crop for canola may not be suitable to compare yield loss due to clubroot
severity because this is a leafy vegetable and optimum maturity is based on the maturity
of edible leaves. The yield portion of canola is the mature seed. To reach this stage,
plants must grow for a much longer period of time, and the stress as a result of clubroot
infection could have a much greater effect on seed yield.
In the field trials, temperature differences as a result of different seeding dates had
a substantial influence on clubroot incidence and severity on Shanghai pak choy and
Chinese flowering cabbage. This supports the findings in a previous study (McDonald
and Westerveld, 2008), in which clubroot incidence and severity were higher in these
crops planted in June and July than either earlier or later in the growing season. Another
study conducted on canola demonstrated that early May seeding reduced clubroot
severity by 10-15% and increased yield by 30-58% compared to seeding in the late May
(Gossen et al. 2009). The results from these current trials confirmed those of the
McDonald and Westerveld (2008) that clubroot on short-season Brassica vegetables can
95
* be managed in part by only growing susceptible cultivars on infested soil early or late in
the season to avoid warm conditions and minimize disease risk.
The difference in clubroot incidence and severity between the crops seeded in
May in 2008 and 2009 offers a useful insight into the interaction of temperature and
rainfall on clubroot development. Clubroot in Shanghai pak choy was relatively severe
(56% DSI) in the May planting in 2009 compared to the same month (6% DSI) in 2008.
The results from these trials were similar to the results obtained from previous trials
conducted in 2001 and 2002 on Shanghai pak choy (McDonald and Westerveld 2008).
They also observed high clubroot severity (99.2%) in May seeding in 2002 compared to
the clubroot severity (38.9%) in 2001 in the same seeding month. In these current trials,
mean air and soil temperatures during the growth period for the May planting were
similar for both years, but total rainfall in May 2009 (117 mm) was substantially higher
than in 2008 (48 mm). The higher clubroot incidence and severity in 2009 appears to be
associated with higher soil moisture, which may have been more conducive for infection
or symptom development. Unfortunately, soil moisture data for the research site was not
available for comparison.
In the field trials, mean air and soil temperatures in the August planting were
above 17° C for both years. No clubroot developed on Shanghai pak choy and very
minimal symptoms developed on Chinese flowering cabbage in the August seeding in
2008, but substantially more clubroot developed in 2009. Clubroot development in 2008
may have been inhibited by drier than normal conditions during the growth period. It has
been reported that soil moisture content of 60%) or above is required for successful
clubroot formation and that no symptoms developed when soil moisture was below 45%)
96
even when plants were grown at optimum range of temperatures (Monteith, 1924).
Thuma et al. (1983) demonstrated that the interaction of soil temperature and soil
moisture was strongly correlated with clubroot development on radish. The results from
the field trials in the current study also indicate that moisture level may influence
infection and symptom development of clubroot.
In a previous study, mean air and soil temperatures 6 to 10 days before harvest
exhibited a strong positive correlation with clubroot incidence and severity on short-
season Brassica vegetables (McDonald and Westerveld 2008). In contrast, clubroot
incidence and severity were not correlated with air and soil temperatures at these time
intervals in 2008 and 2009, but there were positive correlations with clubroot incidence
and season-long temperatures on Shanghai pak choy. Also, all of the correlation
coefficients between clubroot levels and temperature were positive, which may indicate
that a relationship between temperature and clubroot levels was present in this study as
well but that the number of years assessed was not sufficient to provide a consistent
picture of the results. The correlation coefficients were consistently high (0.54 or higher)
for both clubroot incidence and severity for air and soil temperatures in the time period
between 11 to 15 days before harvest for both crops. In the current study, the crop was
not always harvested in its optimum harvest maturity because sampling was based on
weekly assessments. This difference in sampling may have also contributed to differences
from the previous study. However, it is more likely that rainfall had an impact in these
trials, which confounded the effect of temperature on clubroot development.
In the field trials, soil temperatures throughout the growing season were recorded
by burying temperature sensor at a depth of 5-cm in the experimental plot because
97
' strongest correlation was obtained between soil temperatures at this depth and clubroot
development in a previous trial. The correlation coefficient for these variables decreased
with increasing depth (McDonald and Westerveld 2008). Thuma et al. (1983) also
observed strong correlation between clubroot development and soil temperatures at a
depth of 10-cm compared to temperatures recorded at 15-cm depth. They did not have the
soil temperatures data at a depth Of 5-cm. In the current trial, daily mean air temperature
was highly correlated with soil temperatures of 5-cm depth. This indicates that air
temperatures can be used to predict clubroot development when soil temperatures are not
available.
Ranman 400 SC (a.i. cyazofamid) was evaluated at each seeding date in 2008 and
2009 to assess its impact across a wide range of temperature regimes and disease
pressure. Mitani et al. (2003) demonstrated that cyazofamid has activity against resting
spore germination, root hair infection, and symptom development of clubroot. However,
it is not known how long this fungicide remains effective once it has been applied in the
field. A drench application within 3 days of seeding reduced clubroot incidence and
severity on Shanghai pak choy and Chinese flowering cabbage in all of the seeding dates
except September, when there was only a trace amount of clubroot development. This
indicates that Ranman was effective against clubroot even when disease pressure was
high. Therefore, a drench application of Ranman fungicide should be considered when
the growing conditions are expected to favour severe clubroot development, e.g. in fields
where inoculum levels are high and plantings are planned in June and July. Although
Ranman was demonstrated to effectively reduce clubroot in Brassica vegetables, it is not
feasible to use this product as a drench in commercial canola production because the cost
98
of both the fungicide and the application method are prohibitively high relative to
economic return for canola. However, if this fungicide could be made available in a
granular formulation at a lower cost, it might represent an option for commercial canola
production.
The sand-liquid culture method developed by Donald and Porter (2004) was
found to be the most useful method for observation of root hair infection. In this method,
seedlings can be maintained in very small containers (5 mL pipette tips) until galls are
formed (Donald and Porter 2004). Seed of Shanghai pak choy sown directly into the sand
failed to germinate at 10° C in these small containers, but there were no problems in
germination of seeds at 15° C or higher. This problem could be overcome by allowing
seeds to germinate at optimum temperatures before the Falcon tubes with seedlings are
moved to the desired temperature treatment.
This study in sand-liquid culture was undertaken to investigate the effect of
temperature on the initial stages of the life cycle of P. brassicae (Ingram and Tommerup
1972). Previous studies on clubroot in relation to temperature were focussed on the effect
of temperature on resting spore germination (Chupp 1917; Einhorn and Bochow 1990) or
the effect of temperature on final clubroot incidence and severity (Colhoun 1953;
Buczacki 1978; Thuma et al. 1983). To my knowledge, this is the first study on the
impact of temperature on zoosporangia development in root hairs. Root hair infection as
described as zoosporangia development in root hairs were observed in Shanghai pak choy
as early as 10 days after inoculation at 25° and 30° C. Zoosporangia in the epidermis and
empty zoosporangia in the root hairs were observed at these same temperatures at 14 days
after inoculation. No zoosporangia had developed at 15° or 20° C at 14 days after
99
inoculation. As there are no previous studies on effect of various temperatures on root
hair infection by P. brassicae to compare the present findings directly with others.
However, the results from 20° C are consistent with the results of Agrawal et al. (2009),
who observed zoosporangia development inside root hairs of Arabidopsis only 17 days
after inoculation in which plants were grown at 20° C under controlled conditions.
Donald and Porter (2004) observed primary plasmodia to fully differentiated
zoosporangia at 10 days after inoculation when plants were grown at temperatures of day
25° C and night 20° C under controlled conditions. This result was similar to the result
from this current trial in which zoosporangia were observed in Shanghai pak choy
seedlings were grown at 25° C and 30° C 10 days after inoculation. Other researchers
observed fully differentiated zoosporangia in root hairs at 7 days after inoculation when
plants were grown at 25° C under controlled conditions (Samuel and Garrett 1945; Naiki
et al. 1978). In this current trial, it might have been possible to observe zoosporangia in
root hairs in the plants grown at 25° C at 7 days after inoculation but the assessments
were started 10 days after inoculation.
Across all of these trials, clubroot developed more slowly and was less severe
when temperatures were lower than 17° C. The results from growth cabinet trials
identified 19.6°-25.5° C as optimum temperatures for symptom development and
clubroot severity, and 25° C for zoosporangia development in root hairs. The results from
this study support the findings of Colhoun (1953) who reported temperatures 18-25° C as
optimum for clubroot development and Monteith (1924) who observed high clubroot
severity on cabbage at temperatures 20° and 25° C. Buczacki et al. (1978) also
demonstrated that the minimum temperature of 19.5° C was required to get close to
100
100% clubroot infection. The temperature reported here is similar to the lower limit of
optimum temperature that was identified in the current trials under the controlled
conditions. Thuma et al. (1983) demonstrated that the optimum temperatures for clubroot
in radish were 20°-22° C. This result was somewhat different from the results from these
current trials. However, closer examination of the data points in the graph presented in
the paper showed that the clubroot levels were high in the temperature range from 18.9°-
26.7° C. This indicates that these data are consistent to these current results on clubroot
development in relation to temperature.
The results of field trials were similar to those of McDonald and Westerveld
(2008) although the correlations were not as strong, probably as a result of the very
different levels of rainfall in the two years. These data indicate that low temperatures
early or late in the growth of the crop delay the development of P. brassicae, reducing
clubroot incidence and severity. This is the first study to identify the relationship of
temperature on infection and clubroot development during the first three weeks of growth
(0-3 weeks) and second three weeks (4-6 weeks) of crop growth. It also demonstrated
the efficacy of Ranman to manage clubroot when disease pressure was high, and
provided data on clubroot control with Ranman that has been submitted as part of the
minor use registration application for this fungicide in Canada.
In the field trials, cumulative rainfall throughout the growing season of the crop
was positively correlated with clubroot severity on Shanghai pak choy and Chinese
flowering cabbage. In a previous study on Shanghai pak choy and Chinese flowering
cabbage, McDonald and Westerveld (2008) did not find any correlation between season
long rainfall to clubroot incidence or severity. In another study on radish, Thuma et al.
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(1983) demonstrated that the total rainfall during the first 2-3 weeks after seeding was
highly correlated with clubroot development. This result was consistent with the results
from the current study only on Chinese flowering cabbage in which clubroot severity was
correlated with total rainfall for the first 3 weeks after seeding. However, there was no
correlation between clubroot incidence and severity to total rainfall for the first 2 or 3
weeks after seeding on Shanghai pak choy.
Under controlled conditions, aceto-carmine stain was used to study the effect of
temperature on zoosporangia development in root hairs on Shanghai pak choy. Aceto-
carmine was chosen for this study because several previous researchers; Samuel and
Garrett (1945), Macfarlane (1952) and Tanaka et al. (1999) successfully observed the
developmental stages of P. brassicae in root hairs using this stain. In addition, this stain
was also suggested by Hildebrand (P. D. Hildebrand, personal communication). In some
cases, it was very difficult to distinguish plasmodial stages of root hair infection in
inoculated root hairs from that cytoplasm in noninoculated root hairs using this stain.
Naiki et al. (1978) also experienced the same problem when using cotton blue for study
on root hair infection on Chinese cabbage by P. brassicae. Thus in this study, root hair
infection mainly focussed on developmental clusters of zoosporangia because this stage
of the pathogen was stained well and clearly visible under the microscope.
In growth cabinet trials, plants were grown at one temperature for the first 3
weeks (0-3) weeks and moved to the growth cabinets maintained at another temperature
for the final 3 weeks (4-6) to identify the effect of temperature on root hair infection and
possible symptom initiation during the early growth stage of the crop and subsequent
severity during late growth stage of the crop. These sets of temperatures were chosen to
102
represent the temperature variations in the field at various seeding dates. Previous study
identified that temperatures were highly correlated with clubroot incidence or severity 6-
10 days before harvest (McDonald and Westerveld). This current study was the first
study to demonstrate the effect of temperature during early growth stage of the crop on
symptom initiation. Both growth cabinet and field trials confirmed that low temperatures
(<17° C) during first 3 weeks of crop growth delayed root hair infection and symptom
initiation and low temperatures during 4-6 weeks of growth reduced the rate of clubroot
development, resulting lower incidence and severity at harvest.
Future research should focus on the effect of temperature at various stages of the
life cycle of P. brassicae to identify the effect of temperature on growth and
development, which lead clubroot incidence and severity. The impact of inoculum
concentration and moisture at various temperatures should be evaluated to identify the
interaction among these variables and their contribution to clubroot incidence and
severity.
103
CHAPTER FOUR
EVALUATION OF EFFICACY OF FUNGICIDES AND BIOFUNGICIDES FOR
CLUBROOT MANAGEMENT ON ASIAN VEGETABLES AND OTHER
BRASSICA CROPS
4.1 Introduction
Strategies of clubroot management have been recommended and applied for many
years, but are not always effective. Biological controls may be a useful management tool.
In recent years, research in several laboratories has focused on identifying
microorganisms that have potential to reduce clubroot. Soil microorganisms from a wide
range of sources have been tested against plant pathogens to identify alternative options
of disease management in many host-pathogen systems (Vannacci and Gullino 2000).
Microorganisms that colonize roots and are highly rhizosphere competent may be
potential candidates to manage clubroot (Narisawa et al. 2000).
Isolates of Trichoderma and Streptomyces (Cheah et al. 2000) were identified that
reduce clubroot incidence on Chinese cabbage and cauliflower, respectively, in
greenhouse and field conditions. Other soil microorganisms, including Phoma glomerata
(Corda) Wollenw. and Hochafel (Arie et al. 1998) and Heteroconium chaetospira
(Grove) M.B. Ellis (Narisawa et al. 2000), have been reported to reduce clubroot in
Brassica vegetables. Other biocontrol agents registered in Canada (PMRA 2007a; PMRA
2009) are Bacillus subtilis (Ehrenberg) Cohn and Gliocladium catenulatum Gilman &
Abbott, which have activity against wide range of fungal pathogens. Recent studies
conducted on canola in western Canada have evaluated several commercially formulated
biocotnrol agents against P. brassicae and identified Serenade® (B. subtilis QRD137),
104
Prestop (G. catenulatum J1446) and Mycostop (S. griseoviridis strain K61) to be
effective in reducing clubroot severity under controlled conditions (Agriculture and Agri-
Food Canada 2009).
Application of synthetic fungicides to control clubroot has been practiced for
many years and recently new materials have become available. The fungicides fluazinam
(Allegro® 500 F) and cyazofamid (Ranman® 400 SC) have activity against P. brassicae.
Fluazinam (Suzuki et al. 1995) and cyazofamid (Mitani et al. 2003) inhibited germination
of resting spores and the root hair and cortical stages of infection by P. brassicae,
resulting in reduced gall formation in Chinese cabbage. Both fungicides reduced clubroot
severity on broccoli (Everest and Green Magic) grown in the field compared to
nontreated control (Miller et al. 2007). Another fungicide, flusulphamide, is registered in
New Zealand, where it is widely used to manage clubroot in Brassica vegetable crops
(Donald and Porter 2009). In Canada, pentachloronitrobenzene (Quintozene 75 WP) and
fluazinam (Allegro® 500 F) are registered to manage clubroot on Brassica vegetable
crops (OMAFRA 2008a; Howard et al. 2010).
The objective of this study was to evaluate the efficacy of registered and potential
biofungicides and fungicides in reducing clubroot incidence and severity under controlled
conditions and in field studies on Shanghai pak choy and cabbage. The impact of
inoculum concentration on the efficacy of these agents was also assessed.
4.2 Materials and methods
4.2.1 Plant materials, fungicides and biofungicides
Two Brassica crops susceptible to P. brassicae pathotype 6 were selected for
these studies. Shanghai pak choy (B. rapa L. subsp. Chinensis (Rupr.) var. communis
105
Tsen and Lee) was used for all experiments in the growth cabinet studies conducted at the
University of Guelph in 2008 and 2009. Cabbage (B. oleracea var. capitata cv. Saratoga)
was used for the field trial at the Muck Crops Research Station in 2008. These crops
species were selected because Shanghai pak choy is a small, fast growing and short-
season crop. It is suited for studies under controlled conditions and it is a potential model
crop for canola. Cabbage is a long-season crop under field conditions, with growing
period similar to canola.
Biofungicides and synthetic fungicides were evaluated in both controlled
environment and field trials. The commercially formulated biofungicides evaluated in
these trials were: Mycostop® WP (30% S. griseoviridis, strain K61; Verdera Oy, Espoo,
Finland), Prestop® WP (32% G. catenulatum strain J1446; Verdera Oy, Espoo, Finland),
RootShield® Drench™ WP (1.15% T. harzianum Rifai strain KRL-AG2; Bioworks, Inc.
Geneva NY), Serenade® ASO™ (1.34% B. subtilis QST 713; AgraQuest, Inc. Davis,
CA) or Serenade® MAX™ (14.6% B. subtilis QST 713 strain; AgraQuest, Inc. Davis,
CA) and Actinovate® SP (0.371% S. lydicus De Boer et al. 1956 strain WYEC 108;
Natural Industries, Inc. Houston, TX). Each of these products is registered in Canada but
is not currently registered for clubroot control. These biofungicides were selected for this
initial evaluation to identify microbial agents that have potential to reduce clubroot on
Brassica vegetables. The synthetic fungicides tested were AllegnT500F (40% fluazinam;
ISK Biosciences Corp. Concord, OH), which is registered in Canada to control clubroot
on Brassica vegetables, and Ranman 400 SC (34.5% cyazofamid; ISK Biosciences
Corp. Concord, OH), which has activity against clubroot and is expected to be registered
for this use in the near future. The biofungicides Mycostop, Prestop and Actinovate were
106
evaluated only under controlled conditions, but the other biofungicides and fungicides
were evaluated both in controlled environments and in the field.
4.2.2 Growth cabinet studies
Experimental design and growth condition
Shanghai pak choy was grown in soil-less mix (Sunshine Mix #4, Sun Gro
Horticulture Canada Ltd, North Hills, AB) using individual tall plastic (conetainers, 164
mL, Stuewe & Sons, Inc. Corvallis, OR) with one seedling in each conetainer. Trial 1
was seeded on 15 December, 2008, Trial 2 on 03 March, 2009 and Trial 3 on 20 July,
2009. There were nine treatments in each trial: five biofungicides, two fungicides (Table
4.1), one noninoculated control treatment (negative control) and an inoculated but
nontreated control treatment (inoculated control). Each study was arranged in a complete
randomized design with 10 plants per replicate and four replicates per treatment. Plants
were grown for 7 weeks in growth cabinets maintained at temperatures of 23°/18° C
(day/night), withl4-hr photoperiod, and 65% RH. A combination of fluorescent and
incandescent lights was used in the growth cabinets with an intensity of 200-250 umolm"
V1.
Product rates and application timing
The biofungicides were applied at five times the label rate for these initial
evaluations because there was no prior data for these microbial fungicides against P.
brassicae. The biofungicides were applied 5 days after seeding and 3 days before
inoculation to allow sufficient time for the biocontrol agents to colonize the roots and
increase in numbers in the rhizosphere. The fungicides were applied at the label rate 8
days after seeding and one hour after inoculation. Each treatment was applied as a soil
107
drench at the rate of 50 mL application volume per plant (conetainer) to saturate the
growth medium, using a pipette. The nontreated control and inoculated controls were
treated with 50 mL of water.
Table 4.1 Biofungicides and fungicides treatments for clubroot management applied to Shanghai pak choy in growth cabinet trials at the University of Guelph, Guelph, ON, 2008 and 2009.
Treatment
Biofungicides
Mycostop®
Prestop®
Root Shield®
Serenade® ASO
Actinovate®
Fungicides
Allegro® 500F
Ranman® 400SC
Company
Verdera OY
Verdera OY
Bioworks Inc.
Agraquest Inc.
Natural Industries Inc.
ISK Bioscience Corp
ISK Bioscience Corp
Label rate (g/L water)
0.5
1.5
2.4
l%v/v
0.4
0.5
0.54
Application rate (g/L water)
2.5
7.5
12
5% v/v
2
0.5
0.54
Plant inoculation and disease assessment
Resting spores were extracted from clubs of cabbage plants as described
previously and diluted with distilled water to prepare a solution of desired concentration
for inoculation. Plants were inoculated with 5 mL/plant of spore solution at lx 105 or 1 x
106 resting spores/mL. A concentration of lx 10 resting spores/ mL suspension was used
in Trial 1, lx 105 resting spores/ mL suspension in Trial 2 and both concentrations were
evaluated in the Trial 3 to determine if inoculum concentration had an impact on the
efficacy of fungicides and biofungicides. The inoculated plants were watered with
acidified water (pH 6.3) for 2 weeks after inoculation to ensure a high percentage of
108
infection. Soil moisture was maintained at a high level in each conetainer for 1 week after
inoculation by setting each tray of conetainers in a plastic tray of water deep enough to
cover the bottom of the conetainers. At 2 weeks after inoculation, plants were watered
with tap water (pH 7. 3). The plants were grown for a total of 7 weeks and then
destructively harvested for disease assessment. Roots were thoroughly washed and
assessed for clubroot incidence and severity. Disease severity was rated (0 to 3 scale) and
a disease severity index (DSI) was calculated as described previously.
4.2.3 Field trial
The trial was conducted in 2008 at the University of Guelph, Muck Crops
Research Station, Holland Marsh, Ontario, in organic soil (pH ~ 6.7, organic matter
-69%) naturally infested with P. brassicae. The spore load of P. brassicae in the
experimental plot was estimated at 1.3 x 106 spores/g of soil in 2009 (M.T. Tesfaendrias,
personal communication). Cabbage seeds were planted on 25 May in 128-cell plastomer
plug trays and hand transplanted on 25 June. Each replicate plot consisted of two rows,
86 cm apart and 6.2 m in length, with an in-row spacing of 45 cm. A randomized
complete block design with four replicates per treatment was used. The treatments were:
drench application of Ranman® 400 SC (134 mL/lOOL), RootShield® (354 gm/lOOL),
Serenade® MAX ™ (475 g/lOOL), Allegro® 500F (50 mL/lOOL), and a nontreated
control. The treatment solutions, at a rate of 100 mL per plant, were applied as a drench
to the base of each plant on the day of transplanting. A seed treatment of Serenade (2.5
g/kg of seeds) was also evaluated to identify if there was a difference between seed
treatment and drench application in reducing clubroot. On 24 September, the heads of 12
cabbage plants per plot from the middle segment of both rows were harvested, weighed
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and assessed for marketable and unmarketable yield. The heads that were firm and at
least 15 cm in diameter were considered marketable. The roots of all of the plants in each
plot were destructively harvested, washed and assessed for clubroot incidence and
severity.
4.2.4 Data analysis
Data were analyzed using SAS software (version 9.1; SAS Institute Inc., Cary,
NC, USA). Analysis of variance (ANOVA) was conducted using a general linear model
procedure Proc GLM for incidence, severity and percentage of marketable head. All data
were tested for normality using the Shapiro-Wilk test and outliers were identified using
Lund's test of standardized residuals (Lund 1975). Means comparison was done using
Tukey's multiple mean comparison test. A type I error rate of P = 0.05 was used for all
statistical analyses.
4.3 Results
4.3.1 Growth cabinet trials
The fungicides were more effective than the biological controls tested in growth
cabinet trials conducted at different times and inoculum concentrations. The synthetic
fungicides Allegro 500F and Ranman 400 SC were highly effective and reduced clubroot
severity by 100% in all these trials (Table 4.2). Among the bio fungicides, Mycostop
consistently reduced clubroot severity under moderate (lxlO3) and high (lxlO6 resting
spores/mL) inoculum concentrations. This biofungicide reduced clubroot severity by 40 -
63% in Trials 2 and 3 compared to the inoculated control. Differences in the disease
severity index between the positive control and plants treated with RootShield were
found only in Trial 2 which was inoculated at lxlO5 resting spores/mL. Actinovate was
110
also effective in Trials 2 and 3 but not in Trial 1. Serenade and Prestop did not suppress
clubroot development in any trial (Table 4.2). A phytotoxic effect (stunted seedlings) was
observed on Shanghai pak choy seedlings treated with Serenade ASO, but the plants
recovered 1 week after application. There were no phytotoxic effects to the crop treated
with the other fungicides and biofungicides.
Table 4.2 Efficacy of fungicides and biofungicides for management of clubroot on Shanghai pak choy grown under controlled conditions.
Disease severity index
Trial 1 Trial 2 Trial 3 Fungicide,treatment [Q5 j^3 r^s y j^
spores/mL spores/mL spores/mL spores/mL Fungicides Ranman 400 SC
Allegro 500F
Biofungicides
Mycostop
Actinovate
RootShield
Serenade ASO
Prestop
Controls Negative control
Inoculated control
0 a1
0a
88 b
92 be
95 be
92 be
98 be
Nd
100 c
0 a
0a
38 be
24 b
43 c
69 d
68 d
0a
66 d
0 a
0a
36 b
38 b
48 be
48 be
51 be
0a
60 c
0 a
0a
16b
19b
21 be
30 be
25 be
0a
35 c
[Means in a column followed by the same letter do not differ at P = 0.05 based on Tukey's Multiple Means Comparison Test, nd = not done.
4.3.2 Field trial
Clubroot incidence and severity were high in all of the plants treated with
biofungicides and fungicides and the nontreated control. There were no differences
among the treatments for clubroot incidence, DSI or percentage of marketable heads
111
(Table 4.3). The high incidence and severity observed in this trial probably occurred as a
result of the high inoculum density at this site, which may have reduced the efficacy of
both the fungicides and biofungicides.
Table 4.3 Evaluation of clubroot incidence, severity (Disease Severity Index) and percentage of marketable heads of cabbage treated with fungicides and biofungicides in a field trial at the Holland Marsh, ON, 2008.
Fungicide treatment
Fungicides
Allegro® 500F
Ranman® 400SC
Biofungicides
RootShield® Serenade® Max soil drench Serenade® Max seed treatment Control
Nontreated control
Clubroot incidence (%)
77 ns1
89
-
75
89
99
96
Disease severity index (%)
42 ns
38
48
53
59
47
Marketable heads (%)
50 ns
77
60
48
56
81 Not significant. There were no difference among treatments at P = 0.05 based on
Tukey's Multiple Mean Comparison Test.
4.4 Discussion
Evaluation of selected synthetic fungicides and biofungicides both under
controlled conditions identified several potential options for clubroot management on
Brassica crops. The fungicides Allegro and Ranman, and the biofungicides, Mycostop
and Actinovate, effectively reduced clubroot severity on Shanghai pak choy. However,
none of the fungicides and biofungicides reduced clubroot severity in the cabbage trial
conducted in the field. The fungicides and biofungicides that were effective in reducing
clubroot in a controlled environment have high potential to manage this disease in
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Brassica crops. However, further research on range of hosts is required to validate the
results and maximize the efficacy of these products in the field.
Under controlled conditions, the fungicides cyazofamid and fluazinam completely
inhibited clubroot development on Shanghai pak choy at both concentrations (lO3 or 106
resting spores/mL) of inoculum. This result supports the findings from previous research,
in which clubroot development was inhibited as a result of cyazofamid application
(Mitani et al. 2003) and fluazinam had activity against P. brassicae in Chinese cabbage
(Suzuki et al. 1995). Recent studies on canola conducted in a controlled environment also
reported that these fungicides were highly effective to reduce clubroot even under high
disease pressure (Agriculture and Agri-Food Canada 2009). Results showed that these
fungicides have high potential to manage clubroot, and indicate that these treatments may
be useful when applied at seeding or transplanting to protect seedlings from the early
stages of infection by P. brassicae.
The efficacy of the biofungicides was lower when disease pressure was high in
the trials conducted under controlled conditions. In a previous report, Streptomyces spp.
and Trichoderma spp. exhibited the efficacy against clubroot on Chinese cabbage (Cheah
and Page 1997). In another trial, Cheah et al. (2000) also identified an isolate of a
Streptomyces spp. (S99) and three isolates of Trichoderma (TC32, TC45 and TC63) that
reduced clubroot severity on Chinese cabbage grown in infested soil (inoculum
concentration unknown) in glasshouse and field trials. The results from the current
growth cabinet trials were similar to these previous findings. In the recent trials, S.
griseoviridis strain K61 (Mycostop® WP) consistently reduced clubroot severity in all of
the trials. Trichoderma harzianum Rifai strain KRL-AG2 (RootShield®) also effectively
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reduced clubroot only in one trial, in which Shanghai pak choy was inoculated with spore
suspension of lxl05 resting spores/mL. The biofungicide Actinovate could not reduce
clubroot effectively in Trial 1 when disease pressure was high (100%) in nontreated
control plants. It was also reported that root endophytic fungus Heteroconium
chaetospira reduced clubroot severity only at inoculum concentration of lxl05 resting
spores/mL or lower and was ineffective at high inoculum level/disease pressure
(Narisawa et al. 2005). It was also reported that biofungicides Mycostop, RootShield and
Prestop were effective to control Fusarium and Pythium root rot on cucumbers under low
disease pressure in the greenhouse trials (Rose et al. 2004). From these results we can
conclude that the biocontrol agents are more effective in reducing clubroot when disease
pressure is low.
Data on evaluation of B. subtilis QST 713 (Serenade®) and G. catenulatum strain
J1446 (Prestop®) against clubroot of Brassica vegetable crops are limited. However, the
study conducted in canola reported that Serenade and Prestop reduced clubroot severity
by 91% and 81% respectively in controlled environment studies (Agriculture and Agri-
Food Canada 2009). Another study conducted in organic soil showed that these
biofungicides were ineffective to control clubroot on Shanghai pak choy (McDonald and
Vander Kooi 2009). In the current study, these biofungicides did not reduce clubroot
severity on Shanghai pak choy compared to the nontreated control in growth cabinet
trials. The efficacy of these biocontrol agents were consistent with the trials conducted on
Shanghai pak choy but different from that on canola. It is possible that the root
colonization ability of these products may differ in various hosts resulting difference in
efficacy to control disease. It was reported that the root colonization potential of S.
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griseoviridis was high (72%) in turnip rape roots and low (1%) on* carrot roots (Kortemaa
et al. 1994).These results indicate that these biocontrol agents may not be effective to
control clubroot on Shanghai pak choy inoculated with P. brassicae pathotype 6.
However, further research is required to identify the interactions between host and
biocontrol agents and its impact on clubroot control.
In the field trial, the biofungicides and fungicides did not reduce clubroot
incidence or severity on cabbage compared to the nontreated control. These products
were chosen for this study because they were most effective in efficacy trials conducted
in canola under controlled conditions (G. Peng, personal communication). The results
from the field study were very different from those of the growth cabinet trials, especially
for the fungicides. However, microbial biofungicides that were not effective on Shanghai
pak choy under controlled conditions were also not effective on cabbage in the field. But
the reason for the poor performance of the fungicides in the field trial may be that the
application volume might not have been sufficient to cover the rhizosphere and protect
the seedling roots against clubroot infection. In contrast, a fungicide application volume
of about 50 mL per conetainer was sufficient to saturate the growth medium in the
growth cabinet studies, which resulted effective reductions in clubroot. Previous research
on broccoli (B. oleracea L.) cv. Everest Green Magic in organic soil showed that
application of Allegro and Ranman followed by irrigation immediately after transplanting
reduced clubroot (McDonald and Vander Kooi 2006). In another study, Ranman and
Allegro (Omega) exhibited significant clubroot reduction (40-63%) in the field on
broccoli when transplants were irrigated within 1-3 days after fungicide application
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(Miller et al. 2007). Thus, it is likely that the efficacy of these fungicides can be increased
in the field by increasing drench volumes or irrigation after fungicide treatment.
In Australia, fluazinam reduced clubroot severity on broccoli when applied as a
soil drench in a band of 23-cm band over the transplant rows to a depth of 15 to 20 cm
after transplanting (Donald et al. 2001). This method of fungicide application was more
effective in reducing clubroot in comparison to individual transplant treatment at the rate
of 100 mL/plant (Donald et al. 2001). In the current field trial, cabbage transplants were
treated individually with a soil drench with 100 mL of solution at the day of
transplanting. Thus it is expected that the efficacy of the fungicides in reducing clubroot
can be improved by employing a method of application which uniformly distributes the
product around the root zone to a desired depth to protect roots from infection.
In these studies, a single application of the fungicide was made at the time of
transplanting in the field trials and 1-hr after inoculation in growth cabinet studies. It is
not known how long the fungicides remain effective once they have been applied. Long
season Brassica crops like cabbage stay in the field for a longer period of time than
Shanghai pak choy in conetainers, so it is likely that cabbage is exposed to infection and
clubroot symptom development for a much longer period than Shanghai pak choy. Thus
additional application might be required to ensure adequate protection over a longer
duration for long-season Brassica crops.
In the growth cabinet trials, seedlings of Shanghai pak choy were stunted after
application of Serenade ASO but recovered 1 week after application. It is possible to
minimize the phytotoxic effect on Shanghai pak choy by lowering the application rate.
Similarly, phytotoxicity was reported when Serenade ASO was applied at a higher rate
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(7.56 L/378 L of water) on delphinium to control leaf spot caused by Pseudomonas
delphini, but there was no phytotoxicity at a lower rate (PMRA 2007a). In a previous
study, Cheah et al. (2000) observed stunted seedlings after application of Streptomyces
spp. and Trichoderma spp. on Chinese cabbage. They also reported that seedlings had
recovered by 3 weeks after the treatment. In the current trials, no phytotoxic effects were
observed from the application of fungicides and biofungicides except Serenade.
In the controlled environment trials, the system of growing plants using tall
plastic conetainers was effective for efficacy trials of fungicides and biofungicides
because plants can be maintained up to 7 weeks, which allow a time for the plants
achieve optimum maturity to develop clubroot symptoms. These narrow individual
conetainers prevent cross contamination of applied products and provide an opportunity
to conduct a large trial using limited space in growth cabinets. There is also an advantage
in that the shape allows for the development of a more normal root architecture that
standard shape pots.
In summary, the fungicides Allegro® 500F and Ranman® 400 SC were highly
effective and reduced clubroot severity by 100% on Shanghai pak choy for all trials under
controlled conditions. However, they need to be investigated further to increase their
efficacy under field conditions. The potential biocontrol agents Mycostop® and
Actinovate® should be evaluated further to validate these results. Research on the mode
of action of these potential biocontrol agents may provide an insight into the interaction
between host and antagonist, and so provide a better understanding that could lead to
improved clubroot control.
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CHAPTER FIVE
GENERAL DISCUSSION AND CONCLUSIONS
Clubroot caused by Plasmodiophora brassicae is an economically important
disease of Brassica crops throughout the world. In Canada, it is an established and
devastating disease of Brassica vegetables in Ontario, Quebec, and British Columbia and
has become an economic threat to the canola industry in central Alberta (Tewari et al.
2005). The long-term persistence of the resting spores and their easy dissemination
through movement of infested soil (Karling 1968) has resulted in a rapid increase in
numbers of clubroot infested fields in Alberta since its introduction in 2003, even in 2009
when severe drought limited the initial growth of canola crops (Gossen et al. 2010). Also,
a single club can release millions of viable spores to the soil, which contribute to
inoculum build up and result in severe economic loss.
Effective and sustainable tools to manage clubroot are required to minimize yield
loss in Brassica crops. Recommendations for clubroot management on vegetable crops
include long rotations out of susceptible crops, soil amendment to increase soil pH to 7.2
or over, and avoiding production of susceptible crops in high risk areas (OMAFRA
2008). These approaches are widely accepted and practiced to minimize disease pressure
in vegetable production, but these methods have limitations. Crop rotations that are long
enough to reduce the inoculum level in soil below the minimum threshold required to
cause disease under conducive weather conditions are generally not feasible for vegetable
growers, and are not economical for canola producers in the Canadian prairies. An
increase in soil pH can reduce clubroot, but high pH is not suitable for some crops and it
can be prohibitively costly to raise the desired level of pH in the soil (Hildebrand and
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McRae 1998), especially in high acreage, relatively low value, crops like canola.
Breeding durable and non-specific cultivar resistant to P. brassicae is also a challenge
because the resistance sources that are available generally race specific (Diederichsen et
al. 2009). Researchers are seeking effective management options to minimize crop loss
from clubroot over the long-term, which will also be economical for growers and fit into
existing cropping systems. In this context, this study focused on assessing the clubroot
reaction of various host species and identifying resistant lines, determining the critical
period of infection and symptom development in relation to temperature, and identifying
fungicides and biofungicides that provide effective management of clubroot.
The differential systems (Williams 1966; Buczacki et al. 1975) that are commonly
used to characterize the P. brassicae populations provide intermediate results from
several of the differential lines in Canada (Howard et al. 2010), which indicates that the
entire range of pathotype diversity in the Canadian population of this pathogen is not
being accounted for by these differentials. Therefore, development of a new system of
differential hosts to cover the range of diversity that occurs in Canada is needed to
identify novel sources of resistance, and to develop lines with durable resistance. Various
host genotypes evaluated in this study can be used to develop such a system. For
example, B. carinata or B. juncea lines from the Fast Plants collection can be used to
identify pathotype 6. In addition, evaluation of clubroot resistance in lines of various
Brassica spp. can be a useful tool to identify potential sources of resistance for breeding
resistant cultivars. However, further trials are needed to identify the reaction of the
cultivars and lines assessed in this trial to the range of P. brassicae pathotypes available
in Canada.
119
This was the first study to evaluate lines of Rapid Cycling Brassica Collection
(RCBC, also known as Wisconsin Fast Plants) for reaction to P. brassicae pathotype 6
under field condition. Use of these short-generation lines as model crops for the study of
many aspects of this host-pathogen interaction could reduce the cost and duration of
many types of experiments. Their small stature would also facilitate studies in situations
where space is restricted, such as containment facilities. The RCBC lines B. carinata and
B. juncea were highly susceptible to P. brassicae pathotype 6 and had a similar reaction
to the susceptible control Shanghai pak choy. The RCBC line of B. napus was resistant to
pathotype 6.
Screening trials in the field identified canola lines that were susceptible and
resistant to pathotype 6. Among the canola lines from commercial companies, two 46A76
and 46A65 were moderately susceptible and two Invigor 5020 LL and 45H21 were
completely resistant. Identification of commercial cultivars that are susceptible to
pathotype 6 is important because they can be used in studies of host-pathogen interaction
on canola at the field site in Ontario without having to introduce pathotype 3 from
western Canada.
Clubroot incidence and severity in Shanghai pak choy and canola in relation to
temperature were assessed under controlled conditions. Clubroot severity on both crops
showed a similar pattern of response in relation to temperature during the early stages of
growth in Trial 1 and both early and late stages of growth in Trial 2. In both crops,
clubroot levels were lowest at temperatures at or below 17° C, increased to a maximum at
20°-26° C, and declined slightly when the temperature was increased to 30° C. These
results indicate that Shanghai pak choy can be used as a model crop for canola in
120
subsequent studies of temperature. Shanghai pak choy will be useful for studies outside
of containment facilities in western Canada because it is possible to utilize pathotype 6
which does not attack most commercial canola cultivars. However, further research is
required to confirm the results from these trials using the same growing media and same
inoculation technique with a known concentration of pathogen inoculum. Quantification
of viable resting spores of the inoculum would be useful to get consistent results across
the trials.
This was the first study to identify the effect of temperature on symptom initiation
under controlled conditions and in field trials. Identification of the relationship between
clubroot infection and temperature can be used to improve disease forecasting, and to
plan measures ahead of time to minimize economic loss of crops at harvest. This study
also evaluated the impact of temperature on development of clubroot symptoms during
the early (0-3 weeks) and later (4-6 weeks) crop growth stages. Results from both
growth cabinet and field trials revealed that low temperatures (<17° C) during the first 3
weeks of crop growth delayed root hair infection and symptom initiation. Low
temperatures during weeks 4-6 of growth reduced the rate of clubroot symptom
development, resulting in lower incidence and severity at harvest. Field studies confirmed
the results from controlled environment studies. Therefore, it is possible to manage
clubroot by planting crops early or late in the season to avoid warm conditions, as
previously suggested by McDonald and Westerveld (2008).
In the field studies, seeding dates provided a wide range of temperature regimes,
which had a substantial impact on clubroot incidence and severity on Shanghai pak choy
and Chinese flowering cabbage. A previous study conducted at the same site
121
demonstrated that mean air and soil temperatures 6-10 days before harvest were strongly
correlated with clubroot incidence and severity on short-season Asian vegetables
(McDonald and Westerveld 2008). The current study showed a positive correlation with
clubroot incidence and season-long temperature on Shanghai pak choy. Total rainfall
during the growing season was highly correlated with clubroot incidence and severity.
However, there was no correlation between air or soil temperatures and clubroot
development at various time intervals before optimum harvest or after seeding. The
correlation coefficient was always positive and usually around 0.50, which may indicate
that a relationship exists between these variables but two years of field data may not have
been sufficient to demonstrate this relationship.
Previous studies on the effect of temperature on clubroot have mainly focused on
symptom incidence and severity at harvest. In these studies, the optimal soil temperature
for clubroot development was about 21° C (18°-25° C, Colhoun 1953; 20°-22° C,
Thuma et al. 1983), and the mean air temperature required for severe clubroot
development was reported to be 19.5° C (Buczacki et al. 1978). Clubroot severity below
14° C was minimal on radishes grown in muck soil (Thuma et al. 1983). The results from
the current study support these findings. Clubroot infection, incidence and severity under
both controlled environment and field conditions were higher when temperatures were
above 17° C and minimal below this temperature. In addition, this study demonstrated
that low temperatures during early growth (0-3 weeks) delayed symptom initiation, and
that low temperatures during later growth (4-6 weeks) delayed clubroot development,
resulting in minimal clubroot severity at harvest. This finding is important because it
facilitates forecasting clubroot development based on temperature. This may be useful to
122
manipulate seeding date! to avoid warm conditions to minimize disease pressure, and to
plan on fungicide application or other management options when there is a high risk of
clubroot development. The results from controlled environments were somewhat
different from conclusions of Thuma et al. (1983) who reported an optimum of 20-22° C,
and closer to those of Colhoun (1953) in that the optimum temperature for clubroot
development was in the range of 19.6°-25.5° C, and 25° C was the optimum for root hair
infection in the one trial with Shanghai pak choy. However, close inspection of the
graphs in the Thuma et al. (1983) paper shows that data points at 25° C are as high
clubroot severity as those at 18.9° C and 22.8° C and that disease severity does not
decrease until the 26.7° C treatment. Thus, their data are consistent with these current
results. The results from the McDonald and Westerveld (2008) also indicate that 25° C
may be closer to the optimum temperature that 22° C. That study showed a positive
correlation between clubroot severity and air temperatures in the 10 days before harvest
up to 24° C, the highest temperatures recorded, but soil temperatures in the same period
did not exceed 22° C.
Future research should continue to examine the interaction between temperature
and spore load on infection and symptom development. Quantification of viable resting
spores in the inoculum would be beneficial to get consistent results. Trials should be
conducted to identify the effect of constant and fluctuating temperatures using precision
equipment, such as temperature gradient plates, to determine the effect of fluctuating
temperatures on clubroot development. The fluctuating temperatures under controlled
conditions should correspond to the range of soil and air temperatures that can occur at
various seeding dates in the field. Also, weather variables such as rainfall and soil
123
moisture level should be assessed to determine the interaction between temperatures and
rainfall. Rainfall was measured in our trials and was the most important factor related to
clubroot incidence and severity in the field trials. Thuma et al. (1983) found that rainfall
was always a factor in clubroot severity in the field, although in one year it was total
rainfall in the first 2 weeks after seeding and in the second year the significant factor was
total rainfall in the first 3 weeks after seeding. They found that soil temperature
throughout the growing season (calculated as day degrees) and soil moisture in the
seedling stage were the most important factors affecting clubroot on radish in muck soils.
They did not provide the raw data on rainfall, so it is not possible to compare levels of
rainfall in these trials to theirs.
Quantification of spore load present in the naturally infested soil is another
important aspect that should be done for each research trial because it can be difficult to
interpret the results when research is conducted in soil where the concentration of resting
spores is not known. Finally, research is also needed to compare the effect of temperature
on clubroot incidence and severity between naturally infested soil and artificially
inoculated soil under controlled conditions to identify if the impact of temperature on
clubroot development differs with different growing media.
Seedlings of Shanghai pak choy were grown in sand-liquid culture, modified from
Donald and Porter (2004), to identify the effect of temperature on zoosporangia
development in root hairs. This method was used to prepare clean root samples for
microscopic observation. Root samples were assessed using two techniques; root scoring
using a 0-4 scale (Merz 1989), and counts of 100 root hairs at the midsection of
inoculated roots to calculate the incidence of zoosporangia in root hairs (Donald and
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Porter 2004). Results from both assessments showed the similar pattern of zoosporangia
development at the range of temperatures. However, counting 100 root hairs in the
midsection of roots was time consuming because Shanghai pak choy roots have many
branches with numerous root hairs. The pattern of infection was not always uniform
throughout the roots. Sometimes infection was highly concentrated on some roots, with
little or no infection on other roots of the same plant. The scoring technique was a more
useful method of assessment because it was quicker and could summarize the intensity of
root hair infection across the entire root system. To our knowledge, this was also the first
study to examine the effect of temperature on root hair infection under controlled
conditions.
Previous studies have assessed the effect of pH (Donald and Porter 2004), boron
(Weber and Dixon 1991a) and calcium (Weber and Dixon 1991b) on infection and
symptom development by P. brassicae. Studies on germination of resting spores of P.
brassicae reported that temperatures of 16°-21° C (Chupp 1917) and soil temperatures >
14° C (Einhorn and Bochow 1990) are required. However, there was no prior data on the
effect of temperature on root hair infection. In the current study, production of
zoosporangia after initial root hair infection was observed on Shanghai pak choy as early
as 10 days after inoculation at 25° and 30° C, and was not observed at 15° C or 20° C
when the trial was terminated at 14 days. The percentage of root hair infection as
described zoosporangia development in root hairs was higher at 25° C than 30° C at both
10 and 14 days after inoculation. In an earlier study, primary staged of infection from
primary Plasmodia to fully differentiated zoosporangia were observed on broccoli and
Chinese cabbage at 10 days after inoculation when plants were grown at temperatures
125
day 25° C and night 20° C under controlled conditions (Donald and Porter 2004). This
result was similar to the results obtained from current trial on Shanghai pak choy grown
at 25° C. In another study on Arabidopsis, zoosporangia were observed in roots at 17
days after inoculation where plants were grown using sand-liquid culture at 20° C under
controlled conditions (Agrawal et al. 2009). In the current trial, 14 days after inoculation
may be early to observe fully differentiated zoosporangia inside root hairs at 20° C.
Repetition of this trial over longer time up to swelling of roots is required to confirm the
results. It is possible that earlier root hair infection at optimal temperature resulted in
earlier symptom development and high clubroot severity at harvest. The results from this
study provide an insight on influence of temperature from early stages of P. brassicae
leading to clubroot development.
Research is underway to identify the effect of temperature on the development of
the various stages of pathogen inside the host, such as root hair infection and cortical
stages of infection, and on the interaction of pH and temperature (K. Sharma, personal
communication).
Short-season Asian vegetables can be grown repeatedly in the same site during
the growing season in Ontario (McDonald and Westerveld 2008). Growers need to have
options for clubroot management and information to assist them in identifying when
fungicide application is necessary. This study evaluated the efficacy of Ranman® 400 SC
fungicide (cyazofamid) against clubroot on Shanghai pak choy and Chinese flowering
cabbage at various seeding dates to provide a wide range of temperature regimes and
disease pressure during the growing season over two years. Seeding date had a substantial
impact on clubroot incidence and severity, fungicide efficacy, and need for fungicide
126
application. A drench application of Ranman (46 g a.i./ha) within 3 days of seeding
reduced clubroot incidence and severity even when disease pressure was high, but had
little or no impact when temperatures were below 17° C and clubroot severity was low.
Therefore, it was concluded that fungicide application is necessary only when growing
conditions are expected to favour severe clubroot development on short-season Brassica
vegetables. There was no correlation between crop yield and clubroot severity on either
Shanghai pak choy and Chinese flowering cabbage, and no yield increase with Ranman
treatment, even though there were differences in clubroot severity. These trials
demonstrated the efficacy of Ranman but indicate that there may be little or no need to
applying fungicide to short-season crops grown on muck soils.
There are a limited number of synthetic fungicides that are effective for the
control of clubroot on Brassica crops. Among them, only a few are available to growers
because of registration issues (Donald and Porter 2009) and cost compared to the
economic return of Brassica crops. Two fungicides (Allegro 500F, which is registered in
Canada to control clubroot on Brassica vegetables, and Ranman 400 SC, which is
expected to be registered for this purpose in the near future) were evaluated for clubroot
control in the field and under controlled conditions. A drench application of either
fungicide reduced clubroot severity by 100% on Shanghai pak choy in all growth cabinet
trials, but did not reduce clubroot on cabbage in a field trial on organic soil. However, as
mentioned above, Ranman reduced clubroot incidence and severity on Shanghai pak choy
and Chinese flowering cabbage in a study over two years and multiple seeding dates at
the same field site. It is possible that the volume of drench application (100 mL/seedling).
was not sufficient to completely cover and protect the rhizosphere of the cabbage
127
transplants. Also, cabbage plants take longer to mature in the field (4 months), and so are
exposed to the pathogen for a much longer time period than short-season crops like
Shanghai pak choy. More research is required to determine if fungicides efficacy can be
further improved by modification of drench volume or application method.
Previous studies of the impact of fungicides for management of clubroot have
shown that cyazofamid and fluazinam have activity against P. brassicae (Mitani et al.
2003; Suzuki et al. 1995). Similarly, studies on canola in western Canada have
demonstrated that these fungicides reduce clubroot severity under controlled conditions
(Agriculture and Agri-Food Canada 2009). The results from the current trials conducted
in growth cabinets and at various seeding dates in the field on Shanghai pak choy support
these results. These results indicate that the fungicide cyazofamid is often effective and
support registration in Canada to control clubroot, once registered will provide growers
with an additional management option. However, drench application of these fungicides
at the day of transplanting did not reduce clubroot severity on cabbage grown in organic
soil. So either application methods need to be changed or long-season crops left off the
label.
Further research is required to identify the application method or carrier volume
of drench application to maximize the efficacy of these products in the field for various
Brassica crops. Seed treatment of the fungicides should be evaluated to manage clubroot
because seed treatment would be a more feasible approach to applying these products in
commercial canola production than drench application, which is costly and impractical.
Biological control of clubroot is another potential option for management of
clubroot in the field. Intensive studies on potential microorganisms to control clubroot are
128
t in progress (G. Peng, personal communication). Studies on the impact of microbial
biofungicides for management of clubroot on Chinese cabbage have demonstrated that
isolates of Streptomyces spp. (Cheah et al. 2000) and Trichoderma spp. (Cheah and Page
1997) effectively reduced clubroot severity under greenhouse and field conditions. Some
success has been achieved to manage clubroot using the endophytic fungus
Heteroconium chaetospira at low spore inoculum and high soil moisture conditions
(Narisawa et al. 2005). The results from the current study showed that the biofungicide
Mycostop (S. griseoviridis strain K61) reduced clubroot severity on Shanghai pak choy
by 46-60% under controlled conditions. However, Rootshield (Trichoderma harzianum
Rifai strain KRL-AG2 and Actinovate (S. lydicus strain WYEC 108) were less effective.
Identification of these potential biofungicides may offer an alternative clubroot control
strategy on Brassica crops. A study conducted on canola to control clubroot reported that
two other biofungicides, Serenade (Bacillus subtilis QST 713 strain) and Prestop
(Gliocladium catenulatum strain J1446), reduced clubroot severity by 91% and 81%
respectively (Agriculture and Agri-Food Canada 2009). However, in the current studies
neither of these products was effective on Shanghai pak choy. It is possible that different
host and pathotype combinations have a different impact on the efficacy of these
microbial biofungicides.
No previous research is available on the mode of action of biocontrol agents to P.
brassicae. The mechanism by which Mycostop and Actinovate suppressed P. brassicae
in this study is not known and investigation in this area is required. However, there have
been studies on the bioactivity of these biofungicides on other crops. Their mode of
action is based on a combination of mechanisms including root colonization for
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rhizosphere competence, hyperparasitism and production of antifungal metabolites
(Lahdenpera 2000). After introduction of biocontrol agents into the soil, their survival,
reproduction and colonization of the rhizosphere and host roots should be monitored to
determine the conditions that are required to produce populations of microbial antagonist
that are adequate to suppress the clubroot. In the current trials, a single application of
biocontrol agents was made at the time of transplanting or prior to seedling inoculation.
This was to allow the biocontrol to colonize the roots prior to contact with the pathogen.
It is possible that a second application of biofungicides is required to provide protection
against P. brassicae. Application of microbial fungicides to transplants may be another
potential option in which they can colonize roots first and may be able to protect
seedlings from infection by P. brassicae.
Finally, an integrated approach to clubroot management should be considered to
identify the best combinations of management practices to control clubroot because none
of the available methods of clubroot management can reduce disease severity effectively
by itself in the field conditions. Manipulation of seeding date such as planting crops early
or late in the season can be done to avoid the warm conditions and minimize disease
pressure. A drench application of fungicides Allegro or Ranman should be incorporated
when there is a risk of high clubroot development. Fungicide application can be
integrated with other cultural practices like crop rotation and soil amendment, to
minimize the inoculum level in the soil and maximize the efficacy of these fungicides in
the field. It is also possible to integrate the fungicide application with biocontrol agents to
achieve synergistic effect but these microbial agents must be resistant to the applied
130
' fungicides. The management of clubroot in an integrated and sustainable way is very
important to minimize the economic loss from this disease.
131
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APPENDIX 1: ANOVA TABLES FOR CHAPTER TWO
Clubfoot incidence (data combined over years)
Random Effects
Year Block Year x Host Residual Fixed Effects Host
Estimate
814 10
420 156
Numerator 19
df
Standard Error
1193 12
170 23
Denominator 15
df
Z Value
0.68 0.83 2.47 6.85
F Value 2.51
Pr>Z
0.25 0.20 0.007
<.0001 P r>F 0.04
Clubroot severity index (data combined over years)
Random Effects
Year Block Year x Host Residual Fixed Effects Host
Estimate
670 4
420 108
Numerator df 19
Standard Error
987 6
165 16
Denominator df 15
Z Value
0.68 0.70 2.55 6.85
F Value 1.80
Pr>Z
0.25 0.24
0.005 <.0001 P r>F 0.13
Clubroot incidence (Year 2008)
Random Effects Estimate Standard Error Z Value Pr>Z
Block Block x Host
Residual Fixed Effects
26 196
i i
Numerator df
31 .41 0
Denominator df
0.83 4.78
F Value
0.20 <.0001
P r > F Host 19 46 23.65 <.0001
143
Clubfoot severity index (Year 2008) 1
Random Effects
Block Block x Host Residual Fixed Effects Host
Estimate
13 181
1 Numerator df
19
Standard Error
20 38 0
Denominator df 46
Z Value
0.66 4.78
F Value 20.54
Pr>Z
0.25 <.0001
Pr>F <.0001
clubroot incidence (Year 2009)
Random Effects
Block Block x Host Residual Fixed Effects Host
Estimate
0 108
1 Numerator df
15
Standard Error
22 0
Denominator df 45
Z Value
4.86
F Value 5.86
Pr>Z
<.0001
Pr>F <.0001
Clubroot severity index (Year 2009)
Random Effects
Block Block x Host Residual Fixed Effects Host
Estimate
0 27 1
Numerator df 1$
Standard Error
6 0
Denominator df 45
Z Value
4.73
F Value 4.93
Pr>Z
<.0001
P r > F <.0001
144
APPENDIX 2: SUPPLEMENTARY TABLES AND FIGURES FOR CHAPTER
THREE
Table A 2.1 Shanghai pak choy 2008: Efficacy of Ranman® 400 SC application (Ran) on clubroot incidence and severity compared to a nontreated control (C) in Shanghai pak choy grown in organic soil naturally infested with the clubroot pathogen at the Holland Marsh, ON in 2008.
Weeks after seeding
13 May
C Ran
Clubroot incidence (%)
2
3
4
5
6
7
AUDPC
—
0 ns
0
0
8
9
91
—
0
5
0
2
0
44
Disease severity index
2
3
4
5
6
7
AUDPC
—
0 ns
0
0
3
6
39
—
0
2
0
0.5
0
15
11 June
C
—
0 ns
10
36
64
549
—
0 ns
4
21
42
320
Ran
—
0
9
13
59
486
—
0
3
14
34
237
Seedinj gDate
08 July
C
0 ns
14
58
81
87
1376
0 a1
5a
39 a
69 b
81 a
1072
Ran
0
3
18
49
62
705*
0a
0.9 a
6a
29 a
42 a
402*
05 August
C
0 ns
0
0
0
0
0
0 ns
0
0
0
0
0
Ran
0
0
0
0
0
0
0
0
0
0
0
0
02 September
C
—
0 ns
2
8
4
84
—
0 ns
0.7
4
2
42
Ran
—
2
0
1
2
13
—
0
0
0.3
20
9
Means of the control and Ranman within the same seeding date and day of assessment followed by same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test, ns = not significant. indicates that pair of means (control vs. Ranman) for each seeding date differed at P = 0.05 based on Tukey's Multiple Mean Comparison Test. AUDPC = Area under the disease progress curve final harvest.
145
Table A 2. 2 Chinese flowering cabbage 2008: Efficacy of Ranman® 400 SC 'application (Ran) on clubroot incidence and severity compared to a nontreated control (C) grown in organic soil naturally infested with the clubroot pathogen at the Holland Marsh, ON in 2008.
Weeks after seeding
13 May
C Ran
11 June
C Ran
Seeding Date
08 July
C Ran
05 August
C Ran
02 September
C Ran
Clubroot incidence (%)
2
3
4
5
6
7
AUDPC
—
0 a1
0a
0a
7a
12b
91
. . .
0a
0a
0.7 a
3a
2a
33
Disease severity index
2
3
4
5
6
7
AUDPC
—
0a
0a
0 a
3a
9b
48
—
0a
0a
0.2 a
l a
0.7 a
11
—
0 ns
4
18
36
—
282
—
0 ns
1
10
24
—
158
—
0
6
11
29
—
215
—
0
2
5
14
—
98
0a
7a
39 a
61b
64 a
—
970
0a
2a
25 a
42 b
48 a
—
650
0a
2a
25 a
36 a
48 a
—
611*
0a
0.7 a
14 a
19a
34 a
—
355*
0 ns
0
0
0.9
0.8
—
10
0ns
0
0
0.3
0.4
—
4
0
0
0
0
0
—
0
0
0
0
0
0
—
0
—
0 ns
0
0.5
0.2
—
5
—
0 ns
0
0.2
0.8
—
4
—
0
0
0
0
—
0
—
0
0
0
0
—
0
Means of the control and Ranman within the same seeding date and day of assessment followed by same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test, ns = not significant. indicates that pair of means (control vs. Ranman) for each seeding date differed at P = 0.05 based on Tukey's Multiple Mean Comparison Test. AUDPC = Area under the disease progress curve final harvest.
146
Table A 2. 3 Shanghai pak choy 2009: Efficacy of Ranman" 400 SC application (Ran) on clubroot incidence and severity compared to a nontreated control (C) grown in organic soil naturally infested with the clubroot pathogen at the Holland Marsh, ON in 2009.
Weeks after seeding
13 May
C Ran
Clubroot incidence (%)
2
3
4
5
6
7
AUDPC
—
0 a1
0a
22 a
36 b
95 b
740
—
0a
0a
4a
6a
69 a
310*
Disease severity index
2
3
4
5
6
7
AUDPC
—
0a
0a
13 a
24 b
53 b
449
—
0a
0a
2 a
4 a
26 a
133*
11 June
C
—
8 ns
20
99
99
—
1288
—
3a
10a
70 b
81b
—
879
Ran
—
0
0
84
99
—
937*
—
0a
0a
34 a
62 a
—
450*
Seeding Date
08 July
C
0a
2 a
96 b
100 a
100 a
—
1732
0a
l a
40 b
74 b
88 b
—
1109
Ran
0a
0a
70 a
94 a
94 a
—
1476*
0a
0a
24 a
53 a
69 a
—
783*
05 August
C
0a
16 a
19 a
46 b
87 b
—
872
0a
5a
8a
18a
38 b
—
357
Ran
0a
5a
3a
12 a
34 a
—
258*
0a
2a
l a
5a
13 a
—
101*
02 September
C Ran
—
0 ns 0
0 0
0 0
0 0
—
0 0
—
0 ns 0
0 0
0 0
0 0
0 0
Means of the control and Ranman within the same seeding date and day of assessment followed by same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test, ns = not significant. * Indicates that pair of means (control vs. Ranman) for each seeding date differed at P = 0.05 based on Tukey's Multiple Mean Comparison Test. AUDPC = Area under the disease progress curve final harvest.
147
Table A 2. 4 Chinese flowering cabbage 2009: Efficacy of Ranmarl® application (Ran) on clubroot incidence and severity compared to a nontreated control (C) grown in organic soil naturally infested with the clubroot pathogen at the Holland Marsh, ON in 2009.
Weeks after
seeding
13
C
May
Ran
Clubroot incidence (%)
2
3
4
5
6
7
AUDPC
—
0 a1
0a
11a
18a
76 b
473
—
0 a
0a
0 a
0a
44 a
154*
Disease severity index
2 '
3
4
5
6
7
AUDPC
—
0a
0a
5a
10 a
45 b
265
—
0a
0a
0a
0a
17 a
59*
i
11 June
C
—
2 ns
8
60
68
—
744
—
1 a
3a
29 b
43 b
—
384
Ran
—
4
0
46
48
—
546*
—
l a
0a
17 a
27 a
—
228*
Seeding ] Date
08 July
C
0a
l a
48 b
58 a
60 a
—
957
0a
0.4 a
16b
26 a
36 a
—
423
Ran
0a
0a
6a
39 a
44 a
—
467*
0a
0a
2 a
15a
20 a
—
191*
05 Av
C
0a
0a
0a
l a
17b
—
68
0a
0a
Oa
0.3 a
6b
—
23
igust
Ran
Oa
Oa
Oa
Oa
7a
-—
23
Oa
Oa
Oa
Oa
2a
—
8
m VZ
September C
—
Ons
0
0
0
—
0
—
Ons
0
0
0
—
0
Ran
—
0
0
0
0
—
0
—
0
0
0
0
—
0
Means of the control and Ranman within the same seeding date and day of assessment followed by same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test, ns = not significant. * Indicates that pair of means (control vs. Ranman) for each seeding date differed at P = 0.05 based on Tukey's Multiple Mean Comparison Test. AUDPC = Area under the disease progress curve final harvest.
148
- * - Soil temperature —•—Air temperature •Incidence (%)
3
a E
o 3
E
27
24
21
18
15
12
3
6
5
<^\s / t
Y
June seeding 2009
¥
4-
100
§0
80
70 * 3"-
SO
50
40
SO
20
10
c
'u c
2 3
Week after seeding
Figure A 2.1 Clubroot incidence (%) on Shanghai pak choy seeded in June in soil naturally infested with clubroot at the Holland Marsh, ON, in 2008 and 2009. A solid line denotes clubroot development to optimum harvest maturity and as dotted line indicates beyond optimum harvest maturity of the crop. Values followed by the same letter do not differ at P - 0.05 based on Tukey's Multiple Mean Comparison Test.
149
Figure A 2. 2 Clubroot incidence (%) and severity on Shanghai pak choy seeded in July in soil naturally infested with clubroot at the Holland Marsh, ON, 2008 and 2009. A solid line denotes clubroot development to optimum harvest maturity and a dotted line indicates beyond optimum harvest maturity of the crop. Values followed by the same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test.
150
Figure A 2. 3 Clubroot incidence (%) on Shanghai pak choy seeded in September in soil naturally infested with clubroot pathogen at the Holland Marsh, ON, in 2008 and 2009. A solid line denotes development to optimum harvest maturity and a dotted line indicates beyond optimum harvest maturity of the crop. Values followed by the same letter do not differ at P = 0.05 based on Tukey's Multiple Mean Comparison Test.
151
Table A 2. 5 Correlation between clubroot levels (incidence/severity) and components of air and soil temperatures during the interval before harvest of Shanghai pak choy and Chinese flowering cabbage grown at the Holland Marsh, ON 2008 and 2009.
Time interval and weather variable
Correlation with clubroot incidence
Correlation with clubroot severity
Pak choy Flowering cabbage
Pak choy Flowering cabbage
1 to 7 days before harvest
Air 0.50 NS
Soil, 5-cm 0.55 NS
I to 10 days before harvest
Air 0.50 NS
Soil, 5-cm 0.55 NS
II to 15 days before harvest
Air 0.55 NS
Soil, 5-cm 0.60 NS
I to 15 days before harvest
Air 0.53 NS
Soil, 5-cm 0.58 NS
II to 20 days before harvest
Air 0.45 NS
Soil, 5-cm 0.38 NS
16 to 20 days before harvest
Air 0.29 NS
Soil, 5-cm 0.48 NS
1 to 20 days before harvest
Air 0.50 NS
Soil, 5-cm 0.59 NS
1 to 15 days after seeding
Air 0.46 NS
Soil,5-cm 0.38 NS
0.42
0.46
0.46
0.49
0.55
0.58
0.50
0.53
0.45
0.34
0.28
0.40
0.48
0.54
0.36
0.27
NS NS
NS
NS
NS
NS
N S •
NS
NS
NS
NS
NS
NS
NS
NS
NS
0.43
0.49
0.46
0.51
0.54
0.58
0.50
0.54
0.43
0.33
0.24
0.42
0.46
0.55
0.45
0.36
NS NS '
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
0.37
0.41
0.42
0.46
0.54
0.58
0.47
0.51
0.44
0.34
0.25
0.39
0.45
0.51
0.44
0.33
NS NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
152
Table A 2. 6 Effect of Ranman 400 SC application on top weight (g) of Shanghai pak choy and Chinese flowering cabbage at optimum harvest grown at the Holland Marsh, ON, 2008 and 2009.
Treatment
Ranman
Nontreated control
Shanghai
2008
951ns
933
pak choy
2009
1853 ns
1809
Chinese flowering
2008
1128 ns
955
cabbage
2009
670 ns
604
ns = not significant. No seeding date by treatment interaction. There were no difference among treatments at P = 0.05 based on Tukey's Multiple Mean Comparison Test.Data were pooled over all five seeding dates for each year.
Table A 2. 7 Autocorrelation among mean air temperatures, mean soil temperatures at a depth of 5-cm and cumulative rainfall throughout the growing period of crops the Holland Marsh, ON, 2008 and 2009.
Weather Mean temperature (°C) Rainfall
variables An: Soil
r p r p r p
Air 0.96 <.0001 0.41 NS
Soil 0.96 <.0001 0.96 <.0001 0.45 NS
Rainfall 0.41 NS 0.45 NS
153
APPENDIX 3: ANOVA TABLES FOR CHAPTER THREE
ANOVA tables for growth cabinet trials
Clubroot incidence (Trial 1)
Source Replication Growth stage (G) Temperature (T) Host (H) G x T G x H T x H G x T x H Error
Df 2 1 4 1 4 1 4 4 38
Mean Square 128 1475 1493 636 124 183 639 201 174
F Value 0.73 8.45 8.56 3.64 0.71 1.05 3.66 1.15
P r>F 0.49 0.006
<0001 0.06 0.59 0.31 0.01 0.35
Clubroot severity index (Trial 1)
Source Replication Growth stage (G) Temperature (T) Host (H) G x T G x H T x H G x T x H Error
Df 2 1 4 1 4 1 4 4 38
Mean Square 470 3457 3484 3579 207 2371 952 250 207
F Value 2.27 16.70 16.83 17.29 1.00 11.46 4.60 1.21
P r>F 0.12
0.0002 <.0001 0.0002 0.42 0.002 0.004 0.32
Clubroot incidence (Pak choy: Trial 1 A)
Source Replication Temperature
Linear Quadratic Residual
Error
df 2 4
(1) (1) (2). 8
Mean Square 0
1214 2755 1641 229 26
F Value 0.0
46.6 105.8 63.0 8.8
P r>F 1
<.0001 <.0001 <.0001
0.01
154
Clubfoot Severity Index (Pak choy: Trial 1 A)
Source Replication Temperature
Linear Quadratic Residual
Error
df 2 4
(1) (1) (2) 8
Clubroot incidence (Pak choy: Trial
Source Replication Temperature
Linear Quadratic Residual
Error
df 2 4
(1) (1) (2) 8
Mean Square 73
3239 11929 601 213 61
IB)
Mean Square 31
942 3443 256 34 512
F Value 1.21
53.51 197.06 9.94 3.52
F Value 0.06 1.84 6.72 0.50 0.07
P r>F 0.35
<.0001 <.0001
0.01 0.08
P r>F 0.94 0.21 0.03 0.5
0.94
Clubroot Severity Index (Pak choy: Trial IB)
Source Replication Temperature
Linear Quadratic Residual
Error
Clubroot Severity Index
Source Replication Temperature
Linear Quadratic Residual
Error
df 2 4
(1) (1) (2) 8
Mean Square 12
1074 3610
12 337 320
(Canola: Trial 1A)
df 2 4
(1) (1) (2) 8
Mean Square 99
218 652 207
7 15
F Value 0.04 3.35 11.27 0.04 1.05
F Value 6.51 14.41 43.07 13.68 0.45
P r>F 0.96 0.07 0.01 0.85 0.39
P r>F 0.02
0.001 0.0002 0.006 0.65
155
Clubfoot incidence (CanolA Trial IB)
Source Replication Temperature
Linear Quadratic Residual
Error
df 2 4
(1) (1) (2) 8
Mean Square 505 302 319 790 49 189
F Value 2.68 1.60 1.69 4.19 0.26
P r > F 0.13 0.26 0.23 0.07 0.78
Clubroot Severity Index (Canola: Trial IB)
Source Replication Temperature
Linear Quadratic Residual
Error
df 2 4
0) (1) (2) 8
Mean Square 1741 362 725 462 130 223
F Value 7.80 1.62 3.25 2.07 0.58
P r > F 0.01 0.26 0.11 0.19 0.58
Clubroot incidence (Trial 2)
Source Replication Growth stage (G) Temperature (T) Host (H) G x T G x H T x H G x T x H Error
Df 3 1 4 1 4 1 4 4 57
Mean Square 65
2761 5114 9461 1864 3511 1964 2389 115
F Value 0.56
23.92 44.30 81.94 16.15 30.41 17.01 20.69
P r>F 0.64
<.0001 <.0001 <.0001 <.0001 <.0001 <.0001 <.0001
156
Clubroot severity index (Trial 2)
Source Replication Growth stage (G) Temperature (T) Host (H) G x T G x H T x H G x T x H Error
Df 3 1 4 1 4 1 4 4 57
Mean Square 29 781
5327 3968 3000 1311 419
2436 110
F Value 0.26 7.10
48.40 36.05 27.26
.11.91 3.80
22.14
P r > F 0.85 0.01
<.0001 <.0001 <.0001 0.0011 0.008
<.0001
Clubroot incidence (Pak choy: Trial 2A)
Source Replication Temperature
Linear Quadratic Residual
Error
df 3 4
(1) 0) (2) 12
Mean Square 45
9930 31360 1607 3376
53
F Value 0.84
186.19 588.00 30.13 63.31
Pr>F 0.50
<.0001 <.0001 0.0001 <.0001
Clubroot Severity Index (Pak choy: Trial 2A)
Source Replication Temperature
Linear Quadratic Residual
Error
df 3 4
(1) (1) (2) 12
Clubroot incidence (Pak choy: Trial
Source Replication Temperature
Linear Quadratic Residual
Error
df 3 4
(1) (1) (2) 12
Mean Square 24
7154 24681 419 1757 48
2B)
Mean Square 218 418 203 1302 83 181
F Value 0.51
148.43 512.11
8.70 36.46
F Value 1.21 2.31 1.12 7.20 0.46
P r > F 0.69
<.0001 <.0001
0.01 <.0001
Pr > F 0.35 0.12 0.31 0.02 0.64
157
Clubroot Severity Index (Pak choy: Trial 2B)
Source Replication Temperature
Linear Quadratic Residual
Error
Clubroot incidence (Canola:
Source Replication Temperature
Linear Quadratic Residual
Error
df 3 4
(1) (1) (2) 12
Trial 2A)
df 3 4 1 1
(2) 12
Mean Square 171 945 100
3405 137 101
Mean Square 258 168 490 29 76 121
F Value 1.70 9.40 1.00
33.86 1.37
F Value 2.14 1.39 4.06 0.24 0.63
P r>F 0.22 0.001 0.34
<.0001 0.29
P r>F 0.15 0.30 0.07 0.64 0.55
Clubroot Severity Index (Canola: Trial 2A)
Source Replication Temperature
Linear Quadratic Residual
Error
Clubroot incidence (Canola:
Source Replication Temperature
Linear Quadratic Residual
Error
df 3 . 4 1 1
(2) 12
Trial 2B)
df 3 4
(1) (1) (2) 12
Mean Square 401 1719 3738 761 1188 142
Mean Square 73 818 423 402 1223 61
F Value 2.83 12.14 26.39 5.38 8.39
F Value 1.21 13.44 6.95 6.60
20.10
P r>F 0.08
0.0004 0.0002
0.04 0.005
P r>F 0.35
0.0002 0.02 0.02
0.0001
158
Clubroot Severity Index (Canola: Trial 2B)
Source Replication Temperature
Linear Quadratic Lack of fit
Error
df 3 4
(1) (1) (2) 12
Mean Square 70
1365 668 1505 1642 73
F Value 0.96 18.71 9.17
20.64 22.53
P r>F 0.44
<.0001 0.0105 0.0007 <.0001
ANOVA tables for seeding date field trials
AUDPC incidence (Combined data, 2008 and 2009)
Random Effects
Year (Y)
Block within year
Y x Seeding date (
Y x Host (H)
Y x Fungicide (F)
Residual
Fixed Effects
SD
H
SDxH
F
F x S D
F x H
F x SD x H
B(Y)
[SD)
Estimate 1152
2566
2968
6123
1309
16803
Numerator df 4
1
4
1
4
1
4
Standard Error 10158
1969
2844
9253
2446
2109
Denominator df 4
1
127
1
127
127
127
Z Value 0.11
1.30
1.04
0.66
0.54
7.97
F Value
25.58
2.92
7.58
8.75
6.17
3.59
0.34
P r > Z 0.45
0.10
0.15
0.25
0.30
<.0001
P r>F
0.004
0.34
<0001
0.21
0.0001
0.06
0.85
159
AUDPt severity (Combined data, 2008 and 2009)
Random Effects
Year (Y)
Block within year B(Y)
Y x Seeding date (SD)
Y x Host (H)
Y x Fungicide (F)
Residual
Fixed Effects
SD
H
SDxH
F
FxSD
F x H
F x SD x H
Estimate 0
1012
886
1365
99
6164
Numerator df 4
1
4
1
4
1
4
Standard Error
750
883
1722
345
774
Denominator df 4
•1
127
1
127
127
127
Z Value
1.35
1.00
0.79
0.29
7.96
F Value
22.32
4.00
7.53
25.33
9.06
7.88
0.80
P r>Z
0.09
0.16
0.21
0.39
<.0001
Pr>F
0.005
0.30
<.0001
0.12
<.0001
0.006
0.53
Repeated measure analysis of variance by year, seeding date and host
Clubroot incidence (Pak choy: May seeding 2008)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 103 50 106 46 63 33
F Value 1.97 0.48
1.39 1.89
P r>F 0.51 0.54
0.30 0.18
160
Clubfoot severity index (Pak choy: May seeding 2008)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 25 16 25 12. 16 10
F Value 0.99 0.64
1.18 1.59
P r>F 0.50 0.48
0.37 0.24
Clubroot incidence (Chinese flowering cabbage: May seeding 2008)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 31 72 16 91 42 7
Clubroot severity index (Chinese flowering cabbage:
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Clubroot incidence
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
(Pak choy:
Df 3 1 3 3 3 9
Mean Square 11 34 7
33 24 3
June seeding 2008)
Mean Square 2704 66 46
6067 14 63
F Value 2.01 4.59
13.22 6.07
P r>F 0.29 0.12
0.0002 0.007
: May seeding 2008)
F Value 1.48 4.64
10.59 7.77
F Value 59.13 1.43
95.85 0.22
P r>F 0.38 0.12
0.0007 0.003
P r>F 0.004 0.32
<.0001 0.88
161
t Clubroot severity index (Pak choy: June seeding 2008)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 3 3 9
Mean Square 1138 133 41
2383 36 28
F Value 27.52 3.21
84.54 1.28
P r>F 0.01 0.17
<.0001 0.34
Clubroot incidence (Chinese flowering cabbage: June seeding 2008)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 3 3 9
Mean Square 1344 87 40
1641 42 30
Clubroot severity index (Chinese flowering
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 3 3 9
cabbage:
Mean Square 455 94 27 575 50 18
Clubroot incidence (Pak choy: July seeding
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
2008)
Mean Square 3011 4662 315
8731 515 196
F Value 33.68 2.19
55.07 1.41
: June seeding
F Value 1.6.73 3.46
31.14 2.69
F Value 9.57 14.81
44.51 2.63
P r>F 0.008 0.24
<.0001 0.30
2008)
P r>F 0.02 0.16
<.0001 0.11
P r>F 0.05 0.03
<.0001 0.09
162
Clubfoot severity index (Pak choy: July seeding 2008)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Clubroot incidence (Chinese
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 2380 5291 185
6025 759 180
F Value 12.84 28.55
33.55 4.23
flowering cabbage: July seeding 2008)
Mean Square 637 1396 124
5125 192 44
F Value 5.13 11.25
117.13 4.38
P r>F 0.03 0.01
<.0001 0.02
Pr>F 0.11 0.04
<.0001 0.02
Clubroot severity index (Chinese flowering cabbage: July seeding 2008)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 444 978 64
2581 177 16
F Value 6.91 15.21
157.22 10.75
Pr>F 0.07 0.03
<.0001 0.0006
Clubroot incidence (Chinese flowering cabbage: August seeding 2008)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 1 1 1 0 0 0
F Value 1.00 2.06
1.36 1.36
P r>F 0.50 0,25
0.31 0.31
163
• Clubroot severity index (Chinese flowering cabbage: August seeding 2008)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 0.1 0.2 0.1 0.1 0.1 0.1
Clubroot incidence (Pak choy: September seeding
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 3 3 9
Mean Square 25 65 19 32 17 10
F Value 1.00 1.79
1.10 1.10
2008)
F Value 1.34 3.42
3.02 1.59
P r > F 0.50 0.27
0.40 0.40
Pr>F 0.41 0.16
0.09 0.26
Clubroot severity index (Pak choy: September seeding 2008)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 3 3 9
Mean Square 9 11 6 10 5 5
F Value 1.44 1.74
2.07 0.97
P r>F 0.39 0.28
0.17 0.45
Clubroot incidence (Chinese flowering cabbage: September seeding 2008)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 3 3 9
Mean Square 0.1 0.3 0.1 0.1 0.1 0.2
F Value 1.00 2.51
0.67 0.67
P r > F 0.50 0.21
0.59 0.59
164
Clubroot severity index (Chinese flowering cabbage: September seeding 2008)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 3 3 9
Mean Square 0.3 0.5 0.3 0.3 0.3 0.4
F Value 1.00 1.62
0.83 0.83
Pr>F 0.50 0.29
0.51 0.51
Clubroot incidence (Pak choy: May seeding 2009)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Clubroot severity
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 112
2233 17
9231 417 64
index (Pak choy: May seeding
Df 3 1 3 4 4 12
Clubroot incidence (Chinese
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 50
1369 26
2149 285 30
flowering cabbage:
Mean Square 108
1526 84
5229 372 101
F Value 6.50
129.12
144.30 6.52
2009)
F Value 1.94
52.64
71.09 9.43
May seeding 2009)
F Value 1.29
18.23
51.94 3.70
P r>F 0.08
0.002
<.0001 0.005
P r>F 0.30
0.005
<.0001 0.0011
Pr>F 0.42 0.02
<.0001 0.03
165
Clubroot severity index (Chinese flowering cabbage: May seeding 2009)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 136 762 77
1378 274 49
F Value 1.76 9.85
28.27 5.62
P r>F 0.33 0.05
<.0001 0.009
Clubroot incidence (Pak choy: June seeding 2009)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 3 3 9
Mean Square 69 901 144
20978 147 39
Clubroot severity index (Pak choy: June seeding
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 3 3 9
Mean Square 128
2342 61
9607 417 29
F Value 0.48 6.25
536.93 3.75
2009)
F Value 2.11 38.57
334.23 14.52
Pr>F 0.72 0.09
<.0001 0.05
P r>F 0.28 0.008
<.0001 0.0009
Clubroot incidence (Chinese flowering cabbage: June seeding 2009)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 3 3 9
Mean Square 1065 772 109
7287 158 63
F Value 9.76 7.07
116.58 2.54
P r>F 0.05 0.08
<.0001 0.12
166
Clubfoot severity index (Chinese flowering cabbage: June seeding 2009)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Clubroot incidence
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 3 3 9
Mean Square 323 472 21
2226 124 15
(Pak choy: July seeding 2009)
Df 3 1 3 4 4 12
Mean Square 68
625 38
20477 204 52
F Value 15.16 22.19
144.14 8.05
F Value 1.79 16.36
393.50 3.92
P r>F 0.03 0.02
<.0001 0.006
P r > F 0.32 0.03
<.0001 0.03
Clubroot severity index (Pak choy: July seeding 2009)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4, 12
Mean Square 138
1263 26
10309 207 30
F Value 5.40
49.23
339.13 6.79
Pr>F 0.10
0.006
<.0001 0.004
Clubroot incidence (Chinese flowering cabbage: July seeding 2009)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 104
2432 43
4967 569 186
F Value 2.41 56.58
26.77 3.06
Pr>F 0.24
0.005
<.0001 0.05
167
Clubroot severity index (Chinese flowering cabbage: July seeding 2009)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 38
666 0.4
1240 111 24
F Value 100.72 1745.90
51.58 4.61
P r>F 0.002
<.0001
<.0001 0.02
Clubroot incidence (Pak choy: August seeding 2009)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 1249 5193 374
4544 847 116
F Value 3.34 13.90
39.06 7.28
P r>F 0.17 0.03
<.0001 0.003
Clubroot severity index (Pak choy: August seeding 2009)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 286 963 138 799 190 18
F Value 2.06 6.97
44.55 10.60
P r > F 0.28 0.08
<.0001 0.0007
Clubroot incidence (Chinese flowering cabbage: August seeding 2009)
Source Block Fungicide (F) B x F (error a) Harvest (H) F x H Residual
Df 3 1 3 4 4 12
Mean Square 11 56 10
224 45 13
F Value 1.11 5.63
16.57 3.33
P r>F 0.47 0.10
<.0001 0.05
168
Clubroot severity index (Chinese flowering cabbage: August seeding 2009)
Source Df Mean Square F Value P r>F Block 3 1 1.11 0.47 Fungicide (F) 1 6 5.63 0.10 B x F (error a) 3 1 Harvest (H) 4 25 16.57 <0001 F x H 4 5 3.33 0.05 Residual 12 1
ANOVA tables for root hair infection study
Incidence (Scoring: 10 days after inoculation) Source Replication Temperature Error
Df 5 3 15
Intensity of infection (Scoring: Source Replication Temperature Error
Df 5 3 15
Intensity of infection (Scoring:
Source Replication Temperature Error
Df 5 3 15
Root hair infection % (Countin
Source Replication Temperature Error
Df 2 3 6
Root hair infection % (Countin Source Replication Temperature Error
Df 5 3 15
Mean Square 296.7 8683.3 136.7
F Value 2.2 63.5
10 days after inoculation) Mean Square
38.5 981.6 27.4
F Value 1.4
35.8
14 days after inoculation)
Mean Square 65.0
6386.1 61.1
F Value 1.1
104.5
ig: 10 days after inoculation)
Mean Square 3.9
29.8 3.1
F Value 1.3 9.7
g: 14 days after inoculation) Mean Square
56.2 1338.5 47.4
F Value 1.2
28.2
P r>F 0.1124 <.0001
P r > F 0.2782 <0001
P r>F 0.4184 <.0001
P r>F 0.3452 0.0102
P r>F 0.3621 <.0001
169
APPENDIX 4: ANOVA TABLES FOR CHAPTER FOUR
Clubroot severity index (all growth cabinet trials)
Source df Mean Square F Value P r > F Replication Trial (T) Biofungicides (B) Concentration (C) T x B B x C Error
3 2 8 1
14 7
33.3 11376.2 10348.4 4556.6 809.1 241.2
0.7 250.4 227.8 100.3 17.8 5.3
0.5349 <.0001 <.0001 <.0001 <.0001 <0001
Clubroot severity index (growth cabinet trial 1)
Source Replication Biofungicides Error
Clubroot severity index (growth
Source Replication Biofungicides Error
df 3 7
21
Mean Square 20.6
7630.5 15.5
cabinet trial 2)
df 3 8
24
Mean Square 10.7
3510.1 47.0
F Value 1.3
493.4
F Value 0.2
74.7
P r > F 0.2906 <.0001
P r > F 0.8769 <.0001
Clubroot severity index (growth cabinet trial 3: both concentrations)
Source DF Mean Square F Value P r>F Replication Biofungicides (B) Concentration (C)
B x C
Error
3 7 1
7
52
83.6 2510.3 4556.6
241.2
53.8
1.6 46.6 84.6
4.5
0.2122 <.0001 <.0001
0.0006
Clubroot severity index (g
Source Replication Biofungicides Error
;rowth cabinet trial 3: 105 spores/mL)
df 3 8
24
Mean Square F Value 137.0 719.5 36.7
3.73 19.6
P r>F 0.0247 <.0001
170
Clubroot severity index (growth cabinet trial 3: 106 spores/mL)
Source Replication Biofungicides Error
df 3 8
24
Mean Square 62.87
2383.46 65.41
F Value 0.96
36.44
P r > F 0.4271 <.0001
Clubroot incidence (field trial: 2008)
Source Block Biofungicides Error
df 3 5 15
Mean Square 667.9 377.4 588.1
F Value 1.14 0.64
P r > F 0.3664 0.6717
Clubroot severity index (field trial: 2008)
Source Block Biofungicides Error
Dependent variable:
Source Block Biofungicides Error
df 3 5 15
Mean Square 1507.3 225.3 481.7
F Value 3.13 0.47
Marketable cabbage head percentage (field trial: 2008)
df 3 5 15
Mean Square 2000.5 779.2 719.1
F Value 2.78 1.08
P r > F 0.0571 0.7944
P r > F 0.0771 0.4087
171
APPENDIX 5: RAW DATA FOR CHAPTER TWO
Host
Brassica rapa
B. nigra
B. oleracea
B. juncea
B. napus
B. carinata
Raphanus sativus
B. rapa RCI
B. rapa atrazine resistant
Pak choy
Block
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
1
2 3 4 1 2
Year 2008 CI (%) l
92.3 82.4 78.6 nd3
77.8 100.0
nd 46.2 75.0 50.0 nd
100.0 86.7 100.0
nd 0.0 4.2 7.0 nd
91.7 100.0 100.0
nd 23.1 0.0 6.3 nd
57.1 94.1 95.0 nd
77.8
46.2 100.0
nd 81.3 97.8
DSI2
66.7 72.5 66.7 nd
70.4 61.9
nd 25.6 56.3 27.8 nd
97.9 86.7 100.0
nd 0.0 1.4 2.3 nd
91.7 100.0 100.0
nd 17.9 0.0 2.1 nd
44.0 86.3 91.7 nd
74.1
38.5 88.9 nd
65.6 87.4
Year 2009 CI (%)
4.3 7.1 0.0 0.0 11.8 2.4 18.8 6.7 19.3 53.8 23.3 25.0 1.8 2.6 0.0 0.0
47.6 72.2 22.7 38.5 0.0 0.0 0.0 0.0 8.9 12.1 3.6 50.0 0.0 0.0 8.3 0.0
0.0
0.0 33.3 0.0 nd
DSI 1.4 2.4 0.0 0.0 3.9 0.8 6.3 2.2 10.8 27.8 12.2 13.9 0.6 0.9 0.0 0.0
23.0 34.3 7.6 12.8 0.0 0.0 0.0 0.0 5.4 5.1 3.6 16.7 0.0 0.0 2.8 0.0
0.0
0.0 25.9 0.0 nd
172
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2A
2 3 1 2 3 1 2
Canola 3
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Shanghai 1
pak choy 2
3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 10 10 10 10 15 15 15 15 20 20 20 20 25 25 25 25 30 30 30 30
20 20 23 23 23 26 26 26 14 14 14 17 17 17 20 20 20 23 23 23 26 26 26 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
100.0
100.0
87.5
100.0
100.0
100.0
100.0
100.0
70.0
90.0
60.0
50.0
100.0
100.0
90.0
100.0
100.0
100.0
100.0
90.0
70.0
90.0
88.9
0.0 0.0 10.0
10.0
10.0
10.0
0.0 0.0 100.0
80.0
100.0
90.0
100.0
100.0
100.0
80.0
100.0
100.0
100.0
100.0
50.0
66.7
62.5
87.5
90.5
72.2
81.0
83.3
40.0
83.3
26.7
23.3
73.3
55.6
50.0
83.3
86.7
70.0
86.7
66.7
40.0
83.3
70.4
0.0 0.0 3.3 3.3 3.3 3.3 0.0 0.0 50.0
60.0
73.3
63.3
93.3
86.7
90.0
70.0
86.7
86.7
80.0
83.3
93.7
124.4
104.3
103.4
98.0
91.6
85.4
78.9
43.0
38.6
51.2
38.4
39.8
49.1
45.4
38.0
33.6
36.7
27.4
35.0
28.9
20.6
33.5
56.8
52.6
. 55.5
76.6
64.3
71.9
79.0
87.1
73.5
73.4
88.2
53.3
42.9
46.2
42.3
45.9
52.2
44.5
45.5
39.8
12.9
15.1
8.1 17.5
12.2
6.1 6.6 9.3 10.8
17.4
10.3
11.0 15.5
13.8
16.8
18.5
15.3
19.1
15.8
14.2
5.4 8.3 10.2
15.0
16.0
17.8
17.7
18.3
19.7
22.2
19.8
41.6
40.4
46.6
51.3
39.3
37.0
42.6
38.0
37.5
29.3
32.2
32.1
175
2B
Canola 1
2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Shanghai 1
pak choy 2
3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Canola 1
2 3
10 10 10 10 15 15 15 15 20 20 20 20 25 25 25 25 30 30 30 30 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 10 10 10 10 15 15 15 15 20 20 20 20 25 25 25 25 30 30 30 30 10 10 10
90.0
80.0
100.0
80.0
100.0
100.0
100.0
50.0
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100.0
100.0
100.0
100.0
100.0
100.0
90.0
100.0
100.0
100.0
100.0
70.0
70.0
80.0
70.0
90.0
90.0
50.0
100.0
80.0
100.0
100.0
100.0
100.0
100.0
90.0
90.0
60.0
100.0
70.0
80.0
100.0
100.0
100.0
50.0
36.7
43.3
26.7
80.0
80.0
73.3
26.7
86.7
80.0
96.7
93.3
70.0
53.3
66.7
63.3
96.7
83.3
96.7
76.7
33.3
36.7
43.3
26.7
60.0
70.0
43.3
76.7
50.0
56.7
60.0
66.7
60.0
76.7
43.3
53.3
20.0
40.0
26.7
30.0
53.3
43.3
43.3
64.5
59.8
59.1
42.9
37.7
25.3
39.9
35.9
20.9
16.5
16.4
18.1
48.0
55.9
43.7
55.0
21.9
24.8
14.9
20.8
51.3
46.5
53.0
50.9
39.8
41.6
45.0
44.4
55.8
48.4
60.0
56.0
77.3
67.9
76.1
75.7
78.2
80.6
70.0
76.2
24.7
23.3
27.3
14.4
9.4 9.7 11.0
23.2
28.9
22.1
15.7
19.8
18.0
18.5
23.0
29.1
33.9
19.5
29.6
27.6
21.2
15.5
18.5
14.1
16.0
18.6
13.8
24.9
25.3
18.4
21.9
34.0
24.7
36.7
34.9
11.7
19.1
7.3 9.5 6.6 7.9 5.3 6.7 15.2
11.5
13.5
176
4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
ling
20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20
2
10 15 15 15 15 20 20 20 20 25 25 25 25 30 30 30 30
CI (%) =
100.0 90.0 60.0 50.0 70.0 100.0 90.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
43.3 30.0 30.0 20.0 56.7 76.7 70.0 70.0 70.0 73.3 70.0 83.3 80.0 36.7 43.3 50.0 50.0
28.7 46.6 62.3 55.2 46.0 23.1 14.1 23.1 21.9 30.4 25.7 26.6 25.3 39.3 34.3 39.8 47.2
Clubroot incidence (%)
13.1 11.8 17.2 14.3 14.9 23.7 18.1 16.6 20.5 21.8 18.9 23.3 19.9 11.9 9.7 12.4 13.0
DAS= Days after seeding 3DSI = Disease severity index
Raw data for seeding date field trials 2008 and 2009
Sdate = Seeding month, Host= Pack choy (PC) and Flowering cabbage (FC), Trt = Treatment (R= Ranman fungicide and C— Control), CI (%) = Clubroot incidence
(%) and DSI= Disease Severity Index
Year Sdate Host Block Trt H a r v f CI (%) DSI Tf^ R ° ^ week (g) (g)
2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008 2008
May May May May May May May May May May May May May May May May
PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC
1 1 2 2 3 3 4 4 1 1 2 2 3 3 4 4
C R C R C R C R C R C R C R C R
3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 18.8
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 6.3
3.5 2.5 3.3 0.9 2.5 1.8 1.1 3.5 2.9 13.3 11.5 2.2 8.4 2.1 5.8 5.0
0.6 0.5 0.8 0.3 0.7 0.4 0.5 0.8 0.3 0.9 0.6 0.1 0.4 0.1 0.2 0.3
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July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July July
PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC PC FC FC FC FC
1 2 2 3 3 4 4 1 1 2 2 3 3 4 4 1 1 2 2 3 3 4 4 1 1 2 2 3 3 4 4 1 1 2 2 3 3 4 4 1 1 2 2
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13
e i I t ! 3
APPENDIX 7: RAW DATA FOR CHAPTER FOUR
Raw data for growth cabinet trials
Treatment
Mycostop
Prestop
Serenade
RootShield
Actinovate
Ranman
Allegro
Pathogen
Control
Rep.
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
Trial 1
106 spores/mL
DSI1
90.0 86.7 93.3 83.3 100.0 90.0 100.0 100.0 93.3 93.3 90.0 90.0 93.3 96.7
9.6.7 93.3 100.0
80.0 90.0 96.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
100.0 100.0 100.0 100.0
nd
Top wt (g) 89.4 99.7 102.1 78.1 113.7 127.5 125.4 96.0 106.8 84.2 93.2 115.4 97.9 107.8 64.3 62.2 75.9 79.5 79.9 58.9 121.2 117.6 122.0 146.2 140.3 133.0 112.9
112.6 116.2 108.5
1.13.6 122.8
nd
Trial 2
105 Spores/mL
DSI
53.3 46.7 23.3 26.7 70.0 63.3 66.7 70.0 66.7 66.7 70.0 73.3 40.0 46.7 36.7 46.7 23.3 16.7 26.7 30.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 53.3 63.3 73.3 73.3 0.0 0.0 0.0 0.0
Top wt (g)
78.5 83.6 89.3 83.0 89.9 109.5 108.6 107.0 71.6 108.7 74.1 101.8 75.7 83.5 85.5 65.5 55.1 84.1 86.4 75.3 79.7 83.7 76.7 81.0 66.9 48.6 79.0 85.6 96.1 83.9 69.5 78.8 143.1 105.4
115.8 100.0
Trial 3
105spores/mL
DSI
33.3 36.7 40.0 33.3 56.7 50.0 46.7 50.0 46.7 40.0 60.0 43.3 46.7 66.7 50.0 30.0 20.0 53.3 33.3 46.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 63.3 66.7 53.3 56.7 0.0 0.0 0.0 0.0
Top wt (g)
100.1 79.2 84.2 95.7 99.7 77.2 118.1 106.5 87.6 70.5 71.0 65.8 78.3 70.7 83.6 82.8 95.1 77.5 87.2 85.6 77.7 75.0 81.7 86.5 55.1 60.6 48.4 58.4
101.1 122.5 117.8 110.4
117.3 118.9 118.5 122.4
105 Spores/mL
DSI
10.0 10.0 6.7 36.7 13.3 33.3 16.7 36.7 23.3 33.3 26.7 36.7 23.3 . 20.0 23.3 16.7 13.3 20.0 20.0 23.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 30.0 33.3 33.3 43.3 0.0 0.0 0.0 0.0
Top wt (g)
97.5 83.3 97.4 76.9 116.1 102.1 113.9 117.2 118.7 92.1 100.8 105.6 92.7 90.7 92.4
90.6 93.8 97.2 82.7 87.7 78.8 63.0 97.5 83.7 47.4 49.2 57.7 58.6 105.2 128.6 110.4 107.1 120.1 115.6 123.4
117.3
196
f Raw data for field trial 2009
Treatment
Nontreated Control
Serenade drench
Serenade seed treatment
RootShield
Allegro
Ranman
Block
1
2 3 4
1
2 3 4
1
2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
CI
(%)
100
100 100 82.8
100
57 100 100
100
100 97 100 100 97 89 14 100 89 30 90 100 96 100 60
DSI
51.1
67.8 37.0 31.0
61.7
21.1 60.7 67.8
96.4
50.0 38.9 50.6 98.8 55.2 32.1 4.6 52.9 73.8 10.0 29.9 51.7 37.0 44.0 20.0
Root wt
(kg)
7.2
10.8 6.9 6.2
10.0
4.5 10.0 9.0
9.8
7.1 7.1 7.0 8.4 9.4 6.5 5.9 8.3 10.5 5.3 6.6 8.7 7.1 7.8 6.3
Marketable
No. Of heads
7
10 10 12
8
2 5 8
3
4 10 10 0 8 9 12 8 2 4 10 9 12 4 12
Weight (kg)
18.2
20.4 30.6 29.4
19.7
2.7 12.0 17.2
5.8
7.4 27.6 22.2 0.0 18.5 21.2 28.8 14.1 4.6 8.8
28.6 13.4 34.0 7.9
28.4
Unmarketable
No. Of heads
5
2 2 0
4
10 7 4
9
8 2 2 12 4 3 0 4 10 8 2 3 0 8 0
Weight (kg)
7
2 4 0
5
8 11 5
9
10 3 2 8 5 5 0 5 12 13 2 5 0 12 0
'Clubroot incidence (%) 2Disease severity index
197