For Review Only · Manuscript ID CJPS-2019-0126.R1 Manuscript Type: Article Date Submitted by the Author: 14-Aug-2019 Complete List of Authors: Cao, Tiesen Manolii, Victor; University

For Review OnlyEffect of canola (Brassica napus) cultivar rotation on

Plasmodiophora brassicae pathotype composition

Journal: Canadian Journal of Plant Science

Manuscript ID CJPS-2019-0126.R1

Manuscript Type: Article

Date Submitted by the Author: 14-Aug-2019

Complete List of Authors: Cao, TiesenManolii, Victor; University of Alberta, Agriculture, Food and Nutritional SciencesZhou, Qixing; Crop Diversification Centre North, Alberta Agriculture and Rural DevelopmentHwang, Sheau-Fang; University of Alberta, Dept. of Agricultural, Food and Nutritional Science; Strelkov, Stephen; University of Alberta, Agricultural, Food and Nutrional Science

Keywords: Canola, clubroot, oilseed rape, pathotypes, Plasmodiophora brassicae

Is the invited manuscript for consideration in a Special

Issue?:Not applicable (regular submission)

https://mc.manuscriptcentral.com/cjps-pubs

Canadian Journal of Plant Science

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Effect of canola (Brassica napus) cultivar rotation on Plasmodiophora

brassicae pathotype composition

Tiesen Cao, Victor P. Manolii, Qixing Zhou, Sheau-Fang Hwang and Stephen E. Strelkov

T. Cao, V.P. Manolii, S.F. Hwang, and S.E. Strelkov. Department of Agricultural, Food and

Nutritional Science, 410 Agriculture/Forestry Centre, University of Alberta, Edmonton AB T6G

2P5, Canada.

Q. Zhou. Crop Diversification Centre North, Alberta Agriculture and Forestry, Edmonton AB

T5Y 6H3, Canada.

Corresponding author: Stephen Strelkov (email: [email protected]).

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Abstract: In Canada, clubroot (Plasmodiophora brassicae) disease is managed mainly by

planting clubroot resistant (CR) canola (Brassica napus). New pathotypes of P. brassicae have

emerged recently, however, which are virulent on most CR canola cultivars. To understand the

impact of cultivar rotation on pathotype abundance, greenhouse experiments were conducted in

which different canola cultivar rotations were grown in a soil mix containing equal amounts of

pathotypes 5X and 3, which are virulent and avirulent, respectively, on CR canola. The rotation

treatments included: T1, the same susceptible cultivar planted over four cycles; T2, the same CR

cultivar planted over four cycles; and T3, different CR cultivars planted in each cycle. Clubroot

severity increased from cycles one to four in all treatments, with the exception of one CR

cultivar in T3 that may carry a different source of resistance. Pathogen populations were

recovered with a susceptible bait crop and pathotyped on the differentials of Williams plus a CR

host (B. napus ‘Mendel’). The percentage of galls classified as pathotype 5X in T1 declined from

50% to 6.7% over the course of the experiment, while galls classified as pathotype 5X increased

from 50% to 66.7% in both T2 and T3. Quantitative PCR analysis of the soil with pathotype 5X-

specific primers generally confirmed an increase in 5X DNA. The results suggest that continuous

planting of CR canola favours a rapid proliferation of virulent pathotypes of P. brassicae, as

indicated by the increases in pathotype 5X observed in this study.

Key words: canola, clubroot, oilseed rape, pathotypes, Plasmodiophora brassicae.

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Introduction

Plasmodiophora brassicae Woronin is a soil-borne pathogen that causes clubroot on

cruciferous oilseed and vegetable crops in Canada and other countries. Infection by P. brassicae

often results in the formation of clubs or galls on the roots of susceptible hosts, which interrupt

nutrient and water uptake by the plant and cause stunted growth and yellowing when the disease

is severe. Clubroot infection may significantly decrease yield, grain mass and oil content (Pageau

et al. 2006). On a global scale, yield losses associated with clubroot infestations are estimated to

be as high as 10-15% (Dixon 2006, 2009a).

Clubroot has long been recognized in Canada, where it has been a problem mainly on

cruciferous vegetables in Ontario, Quebec, British Columbia and the Atlantic Provinces (Howard

et al. 2010). In Alberta and Manitoba, clubroot has been reported sporadically in home and

market gardens since the 1920s (Howard et al. 2010). However, the disease was not described on

canola (oilseed rape; Brassica napus L.) in Alberta until 2003, when 12 clubroot infested fields

were detected near Edmonton (Tewari et al. 2005). Annual disease surveys since that time have

revealed > 3,000 clubroot infested fields by 2018 (Strelkov et al. 2019). Given its soil-borne

nature, the rapid spread of P. brassicae across canola fields has been attributed to the movement

of infested soil on farm machinery (Cao et al. 2009), some movement on wind-borne dust

(Rennie et al. 2015) and possibly dissemination on the surface of seeds or tubers (Rennie et al.

2011). The widespread occurrence of clubroot in Alberta, along with increasing reports of its

incidence on canola in Saskatchewan, Manitoba, Ontario and North Dakota (Cao et al. 2009;

Dokken-Bouchard et al. 2012; Chittem et al. 2014; Al-Daoud et al. 2017), suggest that it poses a

significant threat to the production of this crop in North America.

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Clubroot management is challenging given the fact that P. brassicae is able to survive as

resting spores in infested soil for extended periods of time (Karling 1968; Wallenhammar 1996).

Several practices, including liming the soil to increase pH (Murakami et al. 2002), soil fungicide

applications (Donald et al. 2001, 2004; Hwang et al. 2014), growing bait crops to reduce soil

spore load (Murakami et al. 2000, 2001), the manipulation of seeding date to minimize infection

(Gossen et al. 2009, 2012; Hwang et al. 2012), boron application (Deora et al. 2011), crop

rotation (Peng et al. 2015), and the sanitization of field equipment/machinery to limit field-to-

field disease spread (Howard et al. 2010) have been suggested to minimize the adverse impact of

clubroot. Since P. brassicae inoculum can build up quickly in the soil, the deployment of

clubroot resistant (CR) cultivars has been the most efficient and widely accepted measure for

canola disease management in western Canada (Gossen et al. 2014; Peng et al. 2014; Rahman et

al. 2014). The first CR canola cultivar was released in 2009, and as of fall 2018 there were 28

varieties with clubroot resistance available on the Canadian market. Continuous cropping of

resistant cultivars can, however, exert selection pressure on P. brassicae populations, resulting in

shifts in pathogen virulence (Seaman et al. 1963; LeBoldus et al. 2012). In 2013, only four years

after the introduction of the clubroot resistance trait, a new pathotype of P. brassicae capable of

overcoming resistance in most canola cultivars was detected in central Alberta (Strelkov et al.

2016). This pathotype, classified as pathotype 5 on the differentials of Williams (1966) or as

pathotype X on the Canadian Clubroot Differential Set (Strelkov et al. 2016; Strelkov et al.

2018), is known widely as pathotype ‘5X’. It differs from older pathotypes, including pathotype

3, which was the most prevalent before the introduction of CR canola, by its ability to cause

severe clubroot on most ‘resistant’ canola (Strelkov et al. 2016). Since 2013, multiple other new

pathotypes capable of overcoming resistance have been identified (Strelkov et al. 2018).

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In the current study, the effects of three different cultivars on the abundance of two P.

brassicae pathotypes (5X and 3) were examined over multiple cycles of rotation. Upon the

completion of a fourth cycle of rotation, P. brassicae resting spores in the soil were recovered by

growing a bait crop and the resulting galls were pathotyped on the differentials of Williams

(1966) plus the CR oilseed rape cultivar ‘Mendel’. Soil samples also were collected at the end of

each rotation cycle and the amount of pathotype 5X DNA was quantified with a 5X-specific

primer set and probe. The major objective of this study was to understand how canola cultivar

rotation treatments affect P. brassicae pathotype composition.

Materials and Methods

Inoculation methods and treatments

A black chernozemic soil, collected from a field at the Edmonton Research Station,

University of Alberta, with no history of clubroot (i.e., no history of cultivation of canola or

cruciferous vegetables), was used in this study. Testing with the P. brassicae-specific primers

TC1F/TC1R (Cao et al. 2007) confirmed the absence of pathogen DNA. The soil was passed

through a 10-mm metal sieve and air-dried in the laboratory at room temperature for about 1

month. The dried soil then was passed through a 2-mm sieve, and 6 kg of the sieved soil was

placed in each of nine polyethylene tubs (52 cm × 37 cm × 21 cm).

Two P. brassicae field isolates representing pathotypes 3 and 5X (Strelkov et al. 2016, 2018)

served as inoculum. The field isolates were maintained as resting spores in frozen (-20°C) root

galls on the universally susceptible Chinese cabbage (Brassica rapa var. pekinensis (L.) Lour.)

‘Granaat’. A total of 350 g of fresh galls of each pathotype were homogenized in 2 L sterile

distilled water (sdH2O) in a large blender for 3 min at high speed. The resulting spore suspension

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was adjusted to 2.5 L with sdH2O, mixed evenly, quantified with a haemocytometer and poured

into each of the nine polyethylene tubs. A total of 5 L of spore suspension was added per tub,

representing 2.5 L of each of pathotypes 3 and 5X. The soil in each tub was mixed manually with

the spore suspension for 1 min with a small shovel to generate an even, muddy mixture. The tubs

containing the homogenized soil inoculated with the spore suspension were placed on a bench in

the greenhouse and allowed to air dry for 2 weeks. The average infestation level after drying was

equivalent to 2 × 107 resting spores g-1 soil of each pathotype. After the soil had dried, any

clumps were homogenized in a mortar with a ceramic pestle and again passed through a 2-mm

sieve. The sieved soil in each tub was mixed evenly with two volumes of Sungro Professional

Growing Mix (Sun Gro Horticulture Canada Ltd., Seba Beach, Canada). The soil mixes were

transferred to nine polyethylene tubs with holes on the bottom, which were ready for subsequent

seeding.

Plant materials included the CR canola cultivars ‘45H29’, ‘6056CR’, ‘1960’, ‘9558C’, the

clubroot susceptible cultivar ‘‘45H26’, and the aforementioned Chinese cabbage ‘Granaat’.

About 160 seeds of a specific canola cultivar were planted in four rows in each of the nine

polyethylene tubs, which were placed in large polyethylene trays (115 cm × 198 cm × 9.5 cm).

The seeded tubs were watered carefully until full saturation of the soil was achieved. Three

weeks after seeding, the seedlings in each tub were thinned to 60 - 70 plants. The cultivar

rotation treatments included: T1, continuous cropping of the same susceptible canola cultivar

(‘45H26’); T2, continuous cropping of the same resistant canola cultivar (‘45H29’); and T3,

alternating resistant canola cultivars in the rotation. Each of the three rotation treatments was

replicated three times and one polyethylene tub was regarded as one experimental unit. All the

canola cultivars included in each rotation are listed in Table 1.

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Disease evaluation

The plants were uprooted gently with a spatula 6-7 weeks after seeding, and the roots were

washed in water. Clubroot severity on the roots of each plant was rated on a 0 to 3 scale

(Kuginuki et al. 1999), where: 0 = healthy (no galling), 1 = small galls on less than one-third of

the roots, 2 = small to medium galls on one-third to two-thirds of the roots, and 3 = medium to

large galls on more than two-thirds of the roots. An index of disease (ID) was calculated based

on the disease severity rating data using the formula of Horiuchi and Hori (1980) as modified by

Strelkov et al. (2006):

%1003

)3210((%)

Nnnnn

ID

where n is the number of plants in a class; N is the total number of plants; and 0, 1, 2, and 3 are

the symptom severity classes.

Quantification of P. brassicae pathotype 5X

About 120 g of soil was collected prior to the start of the first rotation (Dec. 19 2016) and at

the end of each rotation cycle (Feb. 21 2017, Apr. 13 2017, Jun. 09 2017, and Aug. 02 2017).

The soil samples were air-dried at room temperature in brown paper bags for 7 - 10 days,

homogenized in a mortar with a pestle, and passed through a 2-mm sieve to remove perlite

granules from the potting mix. Genomic DNA was extracted from 250 mg of the sieved soil

using a PowerSoil® DNA Isolation Kit (Qiagen Inc., Toronto) as per the manufacturer’s

instructions.

The quantitative polymerase chain reaction (qPCR) primers P5XF3, P5XR3 and minor groove

binding (MGB) probe P5XP3 (Zhou et al. 2018) were used to measure the amount of pathotype

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5X DNA in each soil sample. The qPCR assay was conducted in a Step One Plus Real-Time

PCR System (AB Applied Biosystems) in a 15-µL total reaction volume consisting of 0.4 µM

each forward or reverse primer, 0.2 µM TaqMan MGB probe, 1× TaqMan Environmental Master

Mix 2.0 (Thermo Fisher Scientific, Waltham, MA), and 5 µL sample DNA. The amplification

cycle was modified slightly and consisted of an initial step at 95oC for 10 min, followed by 40

cycles of 95°C for 15 s, and 60°C for 1 min. A total of 45 independent DNA samples were

included in the qPCR assay. For each qPCR run, a set of known quantities of P. brassicae

pathotype 5X DNA (1 × 108 to 1 × 101 fg) was included in triplicate on the 96-well plate to

generate a standard curve for each plate. The correlation equation generated from the known

amount of DNA (i.e., log10(DNA)) and threshold values (Ct) were used to calculate the amount

of pathotype 5X DNA present in each unknown sample on the same 96-well plate. The DNA

calculated according to each standard curve was normalized on a per gram soil basis, which was

subsequently used for mean comparisons of the treatments.

Pathotyping of galls recovered from the soil mixture

Resting spores of P. brassicae were recovered from the soil mix following the final cycle of

canola by growing the universally susceptible Chinese cabbage ‘Granaat’ as a bait crop. The

roots of the bait plants were collected after 6-weeks as described above, and single galls were

homogenized in ca. 5 ml sdH2O with a pestle in a mortar and filtered through 4 layers of

cheesecloth. The spore concentration was measured with a haemocytometer and adjusted to ca.

1 × 107 resting spores mL-1 with sdH2O. The recovered isolates were pathotyped on the

differentials of Williams (1966), which include the cabbages (B. oleracea var. capitata L.)

‘Badger Shipper’ and ‘Jersey Queen’ and the rutabagas (B. napus var. napobrassica (L.) Rchb.)

‘Laurentian’ and ‘Wilhemsburger’. The CR oilseed rape ‘Mendel’ also was included in the

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differentials, to identify isolates that could overcome resistance. Twelve seedlings of each host

were inoculated with inoculum from each gall following Strelkov et al. (2006), and inoculations

were replicated four times. The inoculated seedlings were grown in 6 cm × 6 cm × 6 cm plastic

pots (Kord Products Inc., Brampton, ON) filled with Sungro Professional Growing Mix at a rate

of one seedling per pot, and were maintained in a greenhouse at 20oC ± 2oC with a 16 h

photoperiod at a light intensity of 300 µmol m-2s-1. The growing mix was kept saturated with

water for the first week after inoculation, and then was watered and fertilized as required.

Clubroot development was evaluated as described earlier. Differential hosts were considered

resistant if the mean ID of the four replicates was < 50% and the 95% confidence interval did not

overlap 50% (LeBoldus et al. 2012). A total of 90 single root galls were evaluated for pathotype

classification, representing 10 single root galls from each of the nine combinations of rotation

treatment and replication.

Data analysis

Data were analyzed for statistical significance using the Analysis of Variance Procedure of

SAS (Statistical Analysis System; SAS Institute, Cary, NC), and Tukey’s honest significance test

was used for mean comparisons. The SAS LOGISTIC Procedure was used for analyzing

categorical data such as disease ratings. When comparing IDs, mean IDs (± 1 SE) are presented

for simplicity with statistical significance levels generated from the contrast of the SAS

LOGISTIC Procedure using the ordinal rating data.

Results

Effect of canola cultivar rotation on clubroot severity

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At the end of the first cycle of each rotation, the highest ID (91.5%) was observed in T1 on

the canola ‘45H26’, followed by IDs of 78.0% and 69.1% on ‘45H29’ in T2 and T3, respectively

(Table 2). All three IDs were significantly different from each other. At the end of cycle 2, the

IDs were 93.4% on ‘45H26’ in T1, 92.2% on ‘45H29’ in T2, and 91.3% on ‘6056CR’ in T3. At

the end of cycle 3, the IDs were 97.6% on ‘45H26’ (T1), 97.9% on ‘45H29’ (T2) and 98.2% on

‘1960’ (T3). The IDs at the end of cycles 2 and 3 showed no significant differences between

treatments (Table 2). At the end of cycle 4, IDs of 99.7% and 98.4% were observed on ‘45H26’

and ‘45H29’ in T1 and T2, respectively, values that were significantly greater than the ID of

14.1% observed on the cultivar ‘9558C’ in T3 (Table 2). Following each of the canola rotations,

the Chinese cabbage ‘Granaat’ was grown as a bait crop in all of the treatments. The highest ID

developed on the ‘Granaat’ plants in T1 (99.8%), while a significantly lower ID (98.7%)

developed on ‘Granaat’ in T3; in T2, the bait plants developed an ID of 99.1%, which was not

significantly different from the other two treatments (Table 2).

Effect of canola cultivar rotation on amount of pathotype 5X DNA

The amount of pathotype 5X DNA quantified by qPCR analysis was regarded as a proxy for

the size of the 5X population in the soil mix. In T1, this amount (on a per gram air-dried soil

basis, mean ± 1 SE) fluctuated from 7.4 × 102 ng ± 1.8 × 102 ng at the beginning of cycle 1 to 6.5

× 102 ng ± 1.4 × 102 ng by the end of cycle 4 (Fig. 1), with no statitistically significant

differences at any time. In T2, the amount of pathotype 5X DNA also was fairly constant from

the beginning of cycle 1 (5.9 × 102 ng ± 9.7 × 101 ng) to the end of cycle 3 (6.1 × 102 ng ± 8.9 ×

101 ng), but increased significantly to 2.0 × 103 ± 3.1 × 102 ng by the end of cycle 4 (Fig. 1). In

the case of T3, the amount of pathotype 5X DNA ranged from 5.8 × 102 ng ± 1.4 × 102 ng to 8.4

× 102 ng ± 2.8 × 102 ng over most of the cycles, but peaked at 1.1 × 103 ng ± 1.1 × 102 ng at the

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end of cycle 3 (Fig. 1). Across treatments, 5X DNA levels were highest for T3 at the end of cycle

3 and for T2 at the end of cycle 4 (i.e., at the end of the canola rotational sequence).

Effect of canola cultivar rotation on pathotypes recovered

Pathotype 5X was recovered at a high frequency (66.7%) from the root galls of bait plants

grown at the end of the rotations in T2 and T3 (Table 3). In contrast, the percentage of galls

classified as 5X was significantly lower (6.7%) in T1. Conversely, while pathotype 3 was most

commonly recovered (63.3%) in T1, it was rare in galls recovered from T2 and not found at all in

galls from T3. In addition to the two pathotypes originally (5X and 3) used to inoculate the soil

mix, several other pathotypes were recovered at lower frequencies in the three treatments. These

included pathotypes 5 (6.7%) and 8 (23.3%) in T1, pathotypes 5 (6.7%), 6 (10%) and 8 (10%) in

T2, and pathotypes 5 (16.7%), 6 (6.7%) and 8 (10%) in T3 (Table 3).

Discussion

Given the widespread and intensive cultivation of CR canola to manage clubroot (Peng et al.

2014), there has been significant selection pressure on P. brassicae populations to overcome host

resistance (Strelkov et al. 2016, 2018). Only four years passed between the release of the first CR

canola cultivar in Alberta and the detection of a new pathotype able to overcome this resistance

(Strelkov et al. 2016). This pathotype, 5X, is now just one of many novel pathotypes reported in

the province that can attack CR canola (Strelkov et al. 2018). The emergence of new pathotypes

represents one of the biggest challenges to clubroot management, since the planting of CR canola

is the most effective and widely used tool to control this disease (Peng et al. 2014; Strelkov and

Hwang 2014). Uncertainties with respect to pathotype shifts or pathogen population dynamics

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can complicate the sustainable production of canola in P. brassicae-infested fields. The current

report provides some insights into the nature of pathotype shifts.

An earlier study under greenhouse conditions found that field and single-spore isolates of P.

brassicae can adapt quickly to Brassica host genotypes, resulting in significant increases in

clubroot severity after only a few cycles of repeated exposure to the same hosts (LeBoldus et al.

2012). That study, however, did not compare the abundance of specific pathotypes in mixtures

exposed to different host rotations. Indeed, while it has been postulated that virulent components

of a P. brassicae population are selected preferentially in response to a resistance source

(Howard et al. 2010; Tanaka and Ito 2013), the present study shows how rapidly, after just a few

cycles, one pathotype can become predominant over another. In the absence of selection

pressure (i.e., T1, continuous susceptible canola), pathotype 3 was recovered most commonly

from the ‘Granaat’ bait plants at the end of the rotation. In contrast, when one (T2) or more (T3)

CR canola cultivars were rotated, pathotype 5X became predominant, while pathotype 3 was rare

(T2) or absent (T3).

In addition to the two ‘initial’ pathotypes 3 and 5X inoculated at the start of each rotation,

various other pathotypes (5, 6 and 8) were recovered at low frequencies at the end of the

experiment. Pathotype designations were based on the system of Williams (1966), with the

addition of the CR oilseed rape ‘Mendel’ to detect isolates able to overcome resistance. As such,

the full spectrum of pathotypes that can be distinguished with the recently developed CCD Set

(Strelkov et al. 2018) could not be identified. Nevertheless, aside from those classified as

pathotype 5X, none of the field isolates recovered at the end of the experiment were virulent on

‘Mendel’; this suggests that most if not all could not break clubroot resistance, which was

expected given that pathotypes other than 5X were recovered mainly from T1 (continuous

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susceptible canola). The identification of additional pathotypes is interesting and may reflect the

fact that field (vs. single-spore) isolates were used as the starting inoculum. Field isolates often

consist of pathotype mixtures, as has been shown in a number of earlier studies (see for example

Somé et al. 1996; Xue et al. 2008), and it is likely that some of the other pathotypes identified in

addition to 3 and 5X were present as minor components in the original inoculum. Moreover, the

possibility of additional diversity resulting from reproduction by the pathogen over the course of

the experiment cannot be ruled out, since karyogamy and meiosis occur in the P. brassicae life-

cycle (Tommerup and Ingram 1971; Buczacki 1983).

In general, the amount of pathotype 5X DNA in the treatments, as determined by qPCR

analysis, was consistent with the pathotyping results. The level of 5X DNA in T1 (continuous

susceptible canola) was similar throughout the trial, presumably because proliferation of

pathotype 5X was not favored in this treatment and no pathotype shift was observed. In contrast,

the amount of quantifiable 5X DNA increased significantly in T2 (repeated cultivation of

‘45H29’), which was consistent with the predominance of this pathotype among the field isolates

recovered at the end of the experiment. The only apparent discrepancy between the pathotyping

and qPCR results was observed in T3 at the end of the fourth cycle of the rotation, when the

amount of 5X DNA measured by qPCR was lower than expected. The CR canola ‘9558’, which

appeared to be fairly resistant to pathotype 5X, was grown in the fourth cycle of T3. It is

possible, therefore, that the inclusion of a resistant host resulted in a reduction in the quantity of

5X DNA in the soil mix when it was sampled immediately afterwards. This hypothesis is

supported by the observation that the amount of 5X DNA in T3 was highest (relative to T1 and

T2) at the end of the third cycle, when the canola ‘1960’ had been grown and developed severe

clubroot. Since the primers and probe used in the qPCR analysis were specific for pathotype 5X

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(‘pathotype 5-like strains’; Zhou et al. 2018), this assay could not be used to monitor the amount

of P. brassicae DNA corresponding to other pathotypes, including pathotype 3. Nonetheless, a

recent study indicated fairly rapid changes in soil inoculum loads under field conditions after the

cropping of a CR canola variety (Ernst et al. 2019).

Clubroot severity increased across all treatments over the course of the rotations; these

increases were most pronounced in T2 and T3, where the resistance-breaking pathotype 5X

initially represented half of the pathogen population but became more common after repeated

exposure to one (T2) or multiple CR canola cultivars (T3). Although the basis for resistance in

most cultivars is not in the public domain, a recent study suggested that the majority of CR

canola cultivars in Canada derived their resistance from the European Clubroot Differential

(ECD) 04 (Brassica rapa subsp. rapifera (L.) Metzg.) or the winter oilseed rape ‘Mendel’

(Fredua-Agyeman et al. 2018). It appears that resistance in ‘9558’, however, may be distinct,

given the low ID that developed on that genotype relative to all others. A generally lower

clubroot severity on ‘9558’ has been reported previously (Strelkov et al. 2018). Without definite

knowledge of the genetics of resistance in specific cultivars, however, it is difficult to draw

conclusions regarding the potential for rotation of resistance sources as a clubroot management

strategy.

It is worth noting that the ID which developed on ‘45H29’ in cycle 1 of T2 (78%), while in

the same general range as the ID on ‘45H29’ in cycle 1 of T3 (69%), was nonetheless

significantly higher at P < 0.05. In theory at least, the two values should have been nearly

identical, since it was the same host in the initial cycle for both of these treatments. It is

important to recall, however, that this experiment was conducted using field isolates of P.

brassicae. As discussed earlier, it is well established that field isolates may consist of pathotype

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mixtures (Somé et al. 1996; Xue et al. 2008), and indeed several additional pathotypes were

recovered in this experiment beyond the two (3 and 5X) that were inoculated originally.

Therefore, some limited fluctuation in ID would be expected and likely reflected the presence of

these minor components of the pathogen population.

Canola cultivar rotation had a significant impact on P. brassicae pathotype composition in the

current study. While farming practices and environmental conditions may mitigate this impact

under field conditions, it is clear that continuous cropping of CR canola can result in a rapid shift

towards a more virulent pathotype. This may help to explain the rapid emergence of new

pathotypes of P. brassicae that has been observed in Canada, and underscores the need for an

integrated approach to the management of clubroot of canola.

Acknowledgements

The authors acknowledge financial support from Agriculture and Agri-Food Canada and the

Canola Council of Canada via the Canadian Agricultural Partnership (CAP), as well as from

Alberta Canola, SaskCanola and the Manitoba Canola Growers Association. In-kind

contributions from Alberta Agriculture and Forestry and the University of Alberta are also

acknowledged. The authors also wish to thank H. Askarian, A. Botero, N. Fox, R. Fredua-

Agyeman, L. Galindo-González, C.P.A. Jayasinghege, E. Perez-Lara, X. Ma, M.M. Rahaman, R.

I. Strelkov, B. Wei, and Q. Zhou for technical assistance. Any mention of a commercial brand or

name does not constitute an endorsement by any of the funders.

References

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Al-Daoud, F., Moran, M., Gossen, B.D., and McDonald, M.R. 2017. First report of clubroot

(Plasmodiophora brassicae) on canola in Ontario. Can. J. Plant Pathol. 40: 96-99.

Buczacki, S.T. 1983. Plasmodiophora: an inter-relationship between biological and practical

problems. Pages 161-191 in S.T. Buczacki, ed. Zoosporic plant pathogens. Academic

Press, London, UK.

Cao, T., Manolii, V.P., Hwang, S.F., Howard, R.J., and Strelkov, S.E. 2009. Virulence and

spread of Plasmodiophora brassicae [clubroot] in Alberta, Canada. Can. J. Plant Pathol.

31: 321–329.

Cao, T., Tewari, J., and Strelkov, S.E. 2007. Molecular detection of Plasmodiophora brassicae

Woronin, causal agent of clubroot of crucifers, in plant and soil. Plant Dis. 91: 80–87.

Chittem, K., Mansouripour, S.M., and del Río Mendoza, L.E. 2014. First report of clubroot on

canola caused by Plasmodiophora brassicae in North Dakota. Plant Dis. 98: 1438.

Deora, A., Gossen, B.D., Walley, F., McDonald, M.R. 2011. Boron reduces development of

clubroot in canola. Can. J. Plant Pathol. 33: 475-484.

Dixon, G.R. 2006. The biology of Plasmodiophora brassicae Wor. – A review of recent

advances. Acta Hortic. 706: 271–282.

Dixon, G.R. 2009a. The occurrence and economic impact of Plasmodiophora brassicae and

clubroot disease. J. Plant Growth Regul. 28: 194–202.

Dixon, G.R. 2009b. Plasmodiophora brassicae in its environment. J. Plant Growth Regul. 28:

212-228.

Donald, E.C., Porter, I.J., and Lancaster, R.A. 2001. Band incorporation of fluazinam (shirlan)

into soil to control clubroot of vegetable Brassica crops. Aust. J. Exp. Agric. 41: 1223-

1226.

Page 16 of 26



For Review Only

17

Donald, E.C., Lawrence, J.M., and Porter, I.J. 2004. Influence of particle size and application

method on the efficacy of calcium cyanamide for control of clubroot of vegetable

brassicas. Crop Prot. 23: 297-303.

Dokken-Bouchard, F.L., Anderson, K., Bassendowski, K.A., Bouchard, A., Brown, B., Cranston,

R., Cowell, L.E., Cruise, D., Gugel, R.K., Hicks, L., Ippolito, J., Jurke, C., Kirkham, C.L.,

Kruger, G., Miller, S.G., Moats, E., Morrall, R.A.A., Peng, G., Phelps, S.M., Platford,

R.G., Schemenauer, I., Senko, S., Stonehouse, K., Strelkov, S., Urbaniak, S., and

Vakulabharanam, V. 2012. Survey of canola diseases in Saskatchewan, 2011. Can Plant

Dis. Surv. 92: 125–129.

Ernst, T.W., Kher, S., Stanton, D., Rennie, D.C., Hwang, S.F., and Strelkov, S.E. 2019.

Plasmodiophora brassicae resting spore dynamics in clubroot resistant canola (Brassica

napus) cropping systems. Plant Pathol. 68: 299-408.

Fredua-Agyeman, R., Hwang., S.F., Strelkov, S.E., Zhou, Q., and Feindel, D. 2018. Potential

loss of clubroot resistance genes from donor parent Brassica rapa subsp. rapifera (ECD

04) during doubled haploid production. Plant Pathol. 67: 892-901.

Gossen, B.D., Adhikari, K.K.C., and McDonald, M.R. 2012. Effect of seeding date on

development of clubroot in vegetable Brassica crops. Can. J. Plant Pathol. 34: 516–523.

Gossen, B.D., Deora, A., Peng, G., Hwang, S.F., and McDonald, M.R. 2014. Effect of

environmental parameters on clubroot development and the risk of pathogen spread. Can.

J. Plant Pathol. 36 (Suppl. 1): 37-48.

Gossen, B.D., McDonald, M.R., Hwang, S.F., and Kalpana, K.C. 2009. Manipulating seeding

date to minimize clubroot (Plasmodiophora brassicae) damage in canola and Brassicas.

Phytopathology. 99: S45 [Abstr.].

Page 17 of 26



For Review Only

18

Howard, R.J., Strelkov, S.E., and Harding, M.W. 2010. Clubroot of cruciferous crops –

perspectives on an old disease. Can. J. Plant Pathol. 32: 43–57.

Hwang, S.F., Ahmed, H.U., Zhou, Q., Strelkov, S.E., Gossen, B.D., Peng, G., and Turnbull, G.D.

2014. Efficacy of Vapam fumigant against clubroot (Plasmodiophora brassicae) of

canola. Plant Pathol. 63: 1374-1383.

Hwang, S.F., Cao, T., Xiao, Q., Ahmed, H.U., Manolii, V.P., Turnbull, G.D., Gossen, B.D., Peng,

G., and Strelkov, S.E. 2012. Effects of fungicide, seeding date and seedling age on

severity, seedling emergence and yield of canola. Can. J. Plant Sci. 92: 1175-1186.

Jones, D.R., Ingram, D.S., and Dixon, G.R. 1982a. Factors affecting tests for differential

pathogenicity in populations of Plasmodiophora brassicae. Plant Pathol. 31: 229–238.

Jones, D.R., Ingram, D.S., and Dixon, G.R. 1982b. Characterization of isolates derived from

single resting spores of Plasmodiophora brassicae and studies of their interaction. Plant

Pathol. 31: 239–246.

Karling, J.S., 1968. The Plasmodiophorales. Hafner Publishing Company, Inc., New York.

Kuginuki, Y., Hiroaki, Y., and Hirai, M. 1999. Variation in virulence of Plasmodiophora

brassicae in Japan tested with clubroot-resistant cultivars of Chinese cabbage (Brassica

rapa L. spp. pekinensis). Eur. J. Plant Pathol. 105: 327–332.

LeBoldus, J.M., Manolii, V.P., Turkington, T.K., and Strelkov, S.E. 2012. Adaptation to

Brassica host genotypes by a single-spore isolate and population of Plasmodiophora

brassicae (clubroot). Plant Dis. 96: 833-838.

Murakami, H., Tsushima, S., Akimoto, T., Murakami, K., Goto, I., and Shishido, Y. 2000.

Effects of growing leafy daikon (Raphanus sativus) on populations of Plasmodiophora

brassicae (clubroot). Plant Pathol. 49: 584-589.

Page 18 of 26



For Review Only

19

Murakami, H., Tsushima, S., Akimoto, T., and Shishido, Y. 2001. Reduction of spore density of

Plasmodiophora brassicae in soil by decoy plants. J. Gen. Plant Pathol. 67: 85–88.

Murakami, H., Tsushima, S., Kuroyanagi, Y., and Shishido, Y. 2002. Reduction of resting spore

density of Plasmodiophora brassicae and clubroot disease severity by liming. Soil Sci.

Plant Nutr. 48: 685-691.

Pageau, D., Lajeunesse, J., and Lafond, J. 2006. Impact de l’hernie des crucifères

[Plasmodiophora brassicae] sur la productivité et la qualité du canola. Can. J. Plant

Pathol. 28: 137-143.

Peng, G., Lahlali, R., Hwang, S.F., Pageau, D., Hynes, R.K., McDonald, M.R., Gossen, B.D.,

and Strelkov, S.E. 2014. Crop rotation, cultivar resistance, and fungicides/biofungicides

managing clubroot (Plasmodiophora brassicae) on canola. Can. J. Plant Pathol. 36

(Suppl. 1): 99–112.

Peng, G., Pageau, D., Strelkov, S.E., Gossen, B.D., Hwang, S.F., and Lahlali, R. 2015. A > 2-

year crop rotation reduces resting spores of Plasmodiophora brassicae in soil and the

impact of clubroot on canola. Eur. J. Agron. 70: 78-84.

Rahman, H., Peng, G., Yu, F., Falk, K.C., Kulkarni, M., and Selvaraj, G. 2014. Genetics and

breeding for clubroot resistance in Canadian spring canola (Brassica napus L.). Can. J.

Plant Pathol. 36 (Suppl. 1): 122–134.

Rennie, D.C., Holtz, M.D., Turkington, T.K., LeBoldus, J.M., Hwang, S.F., Howard, R.J., and

Strelkov, S.E. 2015. Movement of Plasmodiophora brassicae resting spores in

windblown dust. Can. J. Plant Pathol. 37: 188-196.

Page 19 of 26



For Review Only

20

Rennie, D.C., Manolii, V.P., Cao, T., Hwang, S.F., Howard, R.J., and Strelkov, S.E. 2011. Direct

evidence of surface infestation of seeds and tubers by Plasmodiophora brassicae and

quantification of spore loads. Plant Pathol. 60: 811-819.

Seaman, W.L., Walker, J.C., and Larson, R.H. 1963. A new race of Plasmodiophora brassicae

affecting Badger Shipper cabbage. Phytopathol. 53: 1426–1429.

Somé, A., Manzanares, M., Laurens, F., Baron, F., Thomas, G., and Rouxel, F. 1996. Variation

for virulence on Brassica napus L. amongst Plasmodiophora brassicae collections from

France and derived single-spore isolates. Plant Pathol. 45: 432-439.

Strelkov, S.E., Hwang, S.F., Manolii, V.P., Cao, T., and Feindel, D. 2016. Emergence of new

virulence phenotypes of Plasmodiophora brassicae on canola (Brassica napus) in

Alberta, Canada. Eur. J. Plant Pathol. 145: 517–529.

Strelkov, S.E., Hwang, S.F., Manolii, V.P., Cao, T., Fredua-Agyeman, R., Harding, M.W., Peng,

G., Gossen, G.D., Mcdonald, M.R., and Feindel, D. 2018. Virulence and pathotype

classification of Plasmodiophora brassicae populations collected from clubroot resistant

canola (Brassica napus) in Canada. Can. J. Plant Pathol. 40: 284-298.

Strelkov, S.E., Manolii, V.P., Lageyre, J., Hwang, S.F., Harding, M.W., and Daniels, G.C. 2019.

The occurrence and spread of clubroot on canola in Alberta in 2018. Can. Plant Dis. Surv.

99: 160-163.

Strelkov, S.E., Tewari, J.P., and Smith-Degenhardt, E. 2006. Characterization of

Plasmodiophora brassicae populations from Alberta, Canada. Can. J. Plant Pathol. 28:

467–474.

Tanaka, S., and Ito, S. 2013. Pathogenic and genetic diversity in Plasmodiophora brassicae

(clubroot) from Japan. J. Gen. Plant Pathol. 79: 297-306.

Page 20 of 26



For Review Only

21

Tewari, J.P., Strelkov, S.E., Orchard, D., Hartman, M., Lange, R.M., and Turkington, T.K. 2005.

Identification of clubroot disease of crucifers on canola (Brassica napus) in Alberta. Can.

J. Plant Pathol. 27: 143–144.

Tommerup, I.C., and Ingram, D.S. 1971. The life-cycle of Plasmodiophora brassicae Woron. in

Brassica tissue cultures and in intact roots. New Phytol. 70: 327-332.

Wallenhammar, A.C. 1996. Prevalence of Plasmodiophora brassicae in a spring oilseed rape

growing area in central Sweden and factors influencing soil infestation levels. Plant

Pathol. 45: 710–719.

Williams, P.H. 1966. A system for the determination of races of Plasmodiophora brassicae that

infect cabbage and rutabaga. Phytopathol. 56: 624–626.

Xue, S., Cao, T., Howard, R.J., Hwang, S.F., and Strelkov, S.E. 2008. Isolation and varation in

virulence of single-spore isolates of Plasmodiophora brassicae from Canada. Plant Dis.

92: 456-462.

Zhou, Q., Hwang, S.F., Strelkov, S.E., Fredua-Agyeman, R., and Manolii, V.P. 2018. A

molecular marker for the specific detection of new pathotype 5-like strains of

Plasmodiophora brassicae in canola. Plant Pathol. 67: 1582-1588.

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Table 1. Cultivar rotation sequences grown in a soil mix inoculated with Plasmodiophora brassicae pathotypes 3 and 5X (1:1 ratio).Treatmenta Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5Treatment 1 45H26 45H26 45H26 45H26 GranaatTreatment 2 45H29 45H29 45H29 45H29 GranaatTreatment 3 45H29 6056CR 1960 9558C Granaat

a Treatment 1, continuous cropping of Brassica napus ‘45H26’ (clubroot susceptible); Treatment 2, continuous cropping of B. napus ‘45H29’ (clubroot resistant); Treatment 3, alternating different clubroot resistant B. napus cultivars (‘45H29’ – ‘6056CR’ – ‘1960’ – ‘9558C’). The universally susceptible Chinese cabbage (B. rapa var. pekinensis) ‘Granaat’ was grown in cycle 5 of all treatments to recover P. brassicae from the soil mix.

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Table 2. Clubroot index of disease (%, mean ± 1 standard error) at the end of each rotation cycle.Treatmenta Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5Treatment 1 91.5 ± 1.2 a 93.4 ± 2.5 a 97.6 ± 1.3 a 99.7 ± 0.3 a 99.8 ± 0.2 aTreatment 2 78.0 ± 3.3 b 92.2 ± 0.7 a 97.9 ± 0.8 a 98.4 ± 0.9 a 99.1 ± 0.9 abTreatment 3 69.1 ± 1.5 c 91.3 ± 2.3 a 98.2 ± 0.8 a 14.1 ± 1.2 b 98.7 ± 0.6 b

Note: Means followed by the same letter within each column are not significantly different at P < 0.05 according to the contrast with the SAS LOGISTIC Procedure.

a Treatment 1, continuous cropping of Brassica napus ‘45H26’ (clubroot susceptible); Treatment 2, continuous cropping of B. napus ‘45H29’ (clubroot resistant); Treatment 3, alternating different clubroot resistant B. napus cultivars (‘45H29’ – ‘6056CR’ – ‘1960’ – ‘9558C’). The universally susceptible Chinese cabbage (B. rapa var. pekinensis) ‘Granaat’ was grown cycle 5 of all treatments to recover P. brassicae from the soil mix.

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Table 3. Plasmodiophora brassicae pathotypes recovered from a soil mix after various crop rotations.

Pathotypes Recoveredc

TreatmentaNumber of single galls testedb 3 5 5X 6 8

Treatment 1 30 19 (63.3%) a 2 (6.7%) b 2 (6.7%) b 0 (0%) b 7 (23.3%) a

Treatment 2 30 2 (6.7%) b 2 (6.7%) b 20 (66.7%) a 3 (10%) a 3 (10%) a

Treatment 3 30 0 (0%) c 5 (16.7%) a 20 (66.7%) a 2 (6.7%) a 3 (10%) a

Note: Percentages followed by the same letter within a column are not significantly different at 5% significance level according to a t-test.

a Treatment 1, continuous cropping of Brassica napus ‘45H26’ (clubroot susceptible); Treatment 2, continuous cropping of B. napus ‘45H29’ (clubroot resistant); Treatment 3, alternating different clubroot resistant B. napus cultivars (‘45H29’ – ‘6056CR’ – ‘1960’ – ‘9558C’). The universally susceptible Chinese cabbage (B. rapa var. pekinensis) ‘Granaat’ was grown in cycle 5 of all treatments to recover P. brassicae from the soil mix.

b Resting spores extracted from a single gall were regarded as one field isolate. Thirty galls (field isolates) were tested from each treatment.

c Numbers indicate the total field isolates classified into each pathotype, with the percentages in parentheses indicating the percentage out of the total of 30; pathotype classifications were based on the reaction of the differentials of Williams (1966) plus the clubroot resistant B. napus ‘Mendel’; only those field isolates classified as pathotype 5 on Williams system (1966) and able to overcome resistance in ‘Mendel’ were denoted as pathotype 5X. None of the other pathotypes recovered were virulent on ‘Mendel’.

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Figure Legend

Fig. 1. Plasmodiophora brassicae DNA concentration in a soil mix as measured by quantitative PCR

analysis with a pathotype 5X-specific primer and probe set. Means ± 1 standard error labelled with the

same letters are not significantly different at P < 0.05 according to Tukey’s test. Means of DNA

concentrations extracted from the soil samples collected on Dec 19 2016, Feb 21 2017, and Apr 13 2017

are not significantly different based on Tukey’s test, and thus are not labelled. Treatment 1, continuous

cropping of Brassica napus ‘45H26’ (clubroot susceptible); Treatment 2, continuous cropping of B. napus

‘45H29’ (clubroot resistant); Treatment 3, alternating different clubroot resistant B. napus cultivars

(‘45H29’ – ‘6056CR’ – ‘1960’ – ‘9558C’). The start of the rotations was on Dec 19, 2016 (first sampling

date), with additional sampling on Feb. 21 2017, Apr. 13 2017, Jun 09, 2017 and Aug. 02 2017,

corresponding to the end of cycles 1, 2, 3 and 4 of the rotations, respectively.

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For Review Only · Manuscript ID CJPS-2019-0126.R1 Manuscript Type: Article Date Submitted by the Author: 14-Aug-2019 Complete List of Authors: Cao, Tiesen Manolii, Victor; University