Economic trade-offs with canola-based crop rotations in the presence of clubroot (Plasmodiophora brassicae)

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.

Rhizosphere microbiomes are constantly mobilized during plant–pathogen interactions, and this, in turn, affects their interactions. However, few studies have examined the activities of rhizosphere microbiomes in plants with different susceptibilities to soil-borne pathogens, especially those that cause clubroot disease. In this study, we compared the rhizosphere bacterial community in response to infection of Plasmodiophora brassicae among the four different clubroot susceptibility cultivars of oilseed rape (Brassica napus). Our results revealed obvious differences in the responses of rhizosphere bacterial community to the P. brassicae infection between the four cultivars of oilseed rape. Several bacterial genera that are associated with the nitrogen cycle, including Limnobacter, Thiobacillus, Anaeromyxobacter, Nitrosomonas, Tumebacillus, and Halomonas, showed significantly different changes between susceptible and resistant cultivars in the presence of P. brassicae infection. Moreover, increased connectedness and robustness were exhibited in the rhizosphere bacterial community co-occurrence network in clubroot-susceptible cultivars that were infected with P. brassicae, while only slight changes were observed in clubroot-resistant cultivars. Metagenomic analysis of microbial metabolism also indicated differences in the rhizosphere bacterial community between susceptible and resistant cultivars that were infected with P. brassicae. Functional analysis of the nitrogen cycle showed that genes related to nitrification (nxrB) were upregulated in susceptible cultivars, while genes related to assimilatory nitrate reduction (nasA, narB, and nirA) were upregulated in resistant cultivars that were infected with P. brassicae. These findings indicate that the synthesis and assimilation process of NO3- content were promoted in susceptible and resistant cultivars, respectively. Our study revealed differences in the characteristics of rhizosphere bacterial communities in response to P. brassicae infection between clubroot-susceptible and clubroot-resistant cultivars as well as the potential impact of these differences on the plant–P. brassicae interaction.

Clubroot disease caused by Plasmodiophora brassicae is one of the most serious diseases in Brassica crops worldwide. In this study, the pathotypes of 12 Korean P. brassicae field isolates were determined using various Chinese cabbage including 22 commercial cultivars from Korea, China, and Japan, and 15 inbred lines. All P. brassicae isolates exhibited the typical clubroot disease on non-clubroot resistant cultivar, indicating that the isolates were highly pathogenic. According to the reactions on the Williams’ hosts, the 12 field isolates were initially classified into five races. However, when these isolates were inoculated onto clubroot-resistant (CR) cultivars of Chinese cabbage, several isolates led to different disease responses even though the isolates have been assigned to the same race by the Williams’ host responses. Based on the pathogenicity results, the 12 field isolates were reclassified into four different groups: pathotype 1 (GN1, GN2, GS, JS, and HS), 2 (DJ and KS), 3 (HN1, PC, and YC), and 4 (HN2 and SS). In addition, the CR cultivars from Korea, China, and Japan exhibited distinguishable disease responses to the P. brassicae isolates, suggesting that the 22 cultivars used in this study, including the non-CR cultivars, are classified into four different host groups based on their disease resistance. Combining these findings, the four differential hosts of Chinese cabbage and four pathotype groups of P. brassicae might provide an efficient screening system for resistant cultivars and a new foundation of breeding strategies for CR Chinese cabbage.

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 clubroot severity, seedling emergence and yield of canola. Can. J. Plant Sci. 92: 1175–1186. The infestation of seeds by Plasmodiophora brassicae can result in the transmission of clubroot disease in canola. Five fungicides, including Dynasty 100 FS (azoxystrobin), Helix Xtra (thiamethoxam+difenoconazole+metataxyl+fludioxonil), NebijinTM5SC (flusulfamide), Prosper FX (clothianidin+carbathiin+trifloxystrobin+metalaxyl), and Vitavax RS (carbathiin+thiram), were evaluated under greenhouse conditions using artificially infested canola seeds for their efficacy in eliminating seed-borne inoculum. All of the fungicides significantly reduced clubroot relative to the non-treated control, but NebijinTM5SC and Dynasty 100 FS were the most effective. However, in field trials with Cruiser 5 FS (thiamethoxam), Helix Xtra, Dynasty, Prosper and Sedaxane (pyrazole anilide) applied alone or as a mixture, none of the treatments reduced clubroot severity or improved seedling emergence or yield compared with the insecticidal control (Cruiser 5 FS) in the susceptible cultivar. Clubroot severity was lower in early-seeded canola compared with the late-seeded crops in 2 site-years. The younger seedlings had greater disease severity and reduced plant height and yield than did older seedlings in both resistant and susceptible canola cultivars. We conclude that a combination of approaches including seed treatments and manipulation of seeding dates in conjunction with deployment of resistant cultivars is necessary for the sustainable management of clubroot in canola.

Plasmodiophora brassicae causes clubroot disease in cruciferous plants, and is an emerging threat to Canadian canola (Brassica napus) production. This review focuses on recent studies into the pathogenic diversity of P. brassicae populations, mechanisms of pathogenesis and resistance, and the development of diagnostic tests for pathogen detection and quantification. Plasmodiophora brassicae is a soil-borne, obligate parasite within the class Phytomyxea (plasmodiophorids) of the protist supergroup Rhizaria. Clubroot development is characterized by the formation of club-shaped galls on the roots of affected plants. Above-ground symptoms include wilting, stunting, yellowing and premature senescence. DISEASE CYCLE: Plasmodiophora brassicae first infects the root hairs, producing motile zoospores that invade the cortical tissue. Secondary plasmodia form within the root cortex and, by triggering the expression of genes involved in the production of auxins, cytokinins and other plant growth regulators, divert a substantial proportion of plant resources into hypertrophic growth of the root tissues, resulting in the formation of galls. The secondary plasmodia are cleaved into millions of resting spores and the root galls quickly disintegrate, releasing long-lived resting spores into the soil. A serine protease, PRO1, has been shown to trigger resting spore germination. PHYSIOLOGICAL SPECIALIZATION: Physiological specialization occurs in populations of P. brassicae, and various host differential sets, consisting of different collections of Brassica genotypes, are used to distinguish among pathotypes of the parasite. DETECTION AND QUANTIFICATION: As P. brassicae cannot be cultured, bioassays with bait plants were traditionally used to detect the pathogen in the soil. More recent innovations for the detection and quantification of P. brassicae include the use of antibodies, quantitative polymerase chain reaction (qPCR) and qPCR in conjunction with signature fatty acid analysis, all of which are more sensitive than bioassays. RESISTANCE IN CANOLA: Clubroot-resistant canola hybrids, recently introduced into the Canadian market, represent an important new tool for clubroot management in this crop. Genetic resistance must be carefully managed, however, as it has been quickly overcome in other regions. At least three resistance genes and one or two quantitative trait loci are involved in conferring resistance to P. brassicae. Root hair infection still occurs in resistant cultivars, but secondary plasmodia often remain immature and unable to produce resting spores. Fewer cell wall breakages occur in resistant hosts, and spread of the plasmodium through cortical tissue is restricted. More information on the genetics of clubroot resistance in canola is needed to ensure more effective resistance stewardship. http://www.canolacouncil.org/clubroot/resources.aspx, http://tu-dresden.de/die_tu_dresden/fakultaeten/fakultaet_mathematik_und_naturwissenschaften/fachrichtung_biologie/botanik/pflanzenphysiologie/clubroot, http://www.ohio.edu/people/braselto/plasmos/

Select biofungicides and fungicides, used alone or with cultivar resistance or crop rotation, were assessed for their potential in integrated management of clubroot disease. The synthetic fungicides pentachloronitrobenzene, fluazinam and cyazofamid showed activities against Plasmodiophora brassicae. The biofungicides Serenade® and Prestop® also suppressed the disease on canola via antibiosis and induced host resistance under controlled-environment conditions. Granular and seed-treatment formulations were developed to facilitate the delivery of biofungicide in field trials. Where P. brassicae resting spore populations were large in the soil, neither biofungicides nor synthetic fungicides were sufficiently effective when applied in the seed furrow. They occasionally reduced clubroot severity on Chinese cabbage. More than 5000 soil microbial isolates indigenous to the Canadian prairies were screened for potential clubroot control, but none showed consistent efficacy. Resistant cultivars reduced clubroot severity and canola yield losses significantly. A 2-year break from canola reduced P. brassicae resting spore concentrations by 90% relative to growing continuous canola or only a 1-year break in heavily infested field plots. This 2-year break alleviated disease impact on plant growth and development in a susceptible canola cultivar. Despite the substantial inoculum reduction after 2 years, the levels were still too high to obtain commercially acceptable yields in a susceptible cultivar. In a resistant cultivar, >2-year breaks increased yields by up to 25% relative to continuous growing of canola. A 2-year interval with non-hosts between canola crops, together with use of resistant cultivars, is recommended to reduce the inoculum load of P. brassicae in soil and achieve maximum yields of canola.

A resistant type of small, spheroid clubroot galls (SSGs) containing resting spores formed on the root surface of clubroot-resistant (CR) cultivars of Chinese cabbage (Brassicarapa L. subsp. pekinensis) inoculated with an avirulent population of Plasmodiophora brassicae. Populations isolated from the SSGs severely affected a common (susceptible) cultivar but did not have the typical pathogenicity on CR cultivars, indicating similarity in pathogenicity between SSG and original spore populaions. Populations pathogenic on CR cultivars were not detected from SSGs. Therefore, the ability of the avirulent population among the SSGs to form resting spores may need to be considered to achieve clubroot control in common cultivars of crucifers.

In order to breed broccoli and other Brassica materials to be highly resistant to clubroot disease, 41 Brassicaceae varieties were developed and identified between 2020 and 2021. Seven known clubroot genes were used for screening these materials. In addition, the resistant and susceptible broccoli cultivars were designed for observing their differences in the infection process with Plasmodiophora brassicae. The results showed that 90% of total materials had carried more than two clubroot resistance genes: one material carried two disease resistance genes, four materials carried seven genes for clubroot resistance, two materials carried six genes for clubroot resistance, and in total 32% of these materials carried five genes for clubroot resistance. As a result, several new genotypes of Brassicaceae germplasm were firstly created and obtained based on distant hybridization and identification of loci conferring resistance against Plasmodiophora brassicae in this study. We found and revealed that similar infection models of Plasmodiophora brassicae occurred in susceptible and resistant cultivars of broccoli, but differences in infection efficiency of Plasmodiophora brassicae also existed in both materials. For resistant broccoli plants, a small number of conidia formed in the root hair, and only a few spores could enter the cortex without forming sporangia while sporangia could form in susceptible plants. Our study could provide critical Brassica materials for breeding resistant varieties and new insight into understanding the mechanism of plant resistance.

Clubroot disease, caused by Plasmodiophora brassicae, poses a significant global threat to cruciferous crops. The epidemic area of clubroot disease is expanding rapidly. In response to this pressing issue, there is a compelling need for the development of clubroot disease-resistant radish cultivars. China boasts an extensive array of radish varieties and germplasm resources. However, a comprehensive assessment of their resistance to clubroot has not yet been carried out, thereby impeding the effective utilization of germplasm and clubroot-resistant breeding. Therefore, it is urgent to systematically evaluate the clubroot resistance of the radish germplasm and identify resistant resources. In this study, clubroot resistance evaluations were conducted on 268 excellent radish varieties derived from 30 provinces in China, as well as seven accessions from Russia, North Korea, France, South Korea, and Germany. The resistance evaluation revealed a diverse range of resistance indices, with a mean disease index (DI) ranging from 0.6 to 58.5, showing significant disparities in clubroot resistance among these radish resources. A total of six accessions were characterized as highly resistant to clubroot, and a further 50 accessions were characterized as resistant. The disease-resistant radishes showed diversity in horticultural traits. Provinces in South China contributed significantly more resistance germplasm than those of North China. These materials are of great value for both genetic investigation and the crop breeding of clubroot resistance. Furthermore, we employed a previously established clubroot-resistance-linked SSR marker to analyze the clubroot-resistant resources. The accessions exhibited dissimilar genetic profiles from known clubroot-resistant germplasm, suggesting their potential status as novel sources of clubroot resistance. Conclusively, these newly identified accessions enriched the genetic diversity within the clubroot-resistant gene pool and may contribute to the future cloning of previously undiscovered clubroot-resistant genes.

Clubroot disease caused by Plasmodiophora brassicae is one of the serious threats to canola (Brassica napus L. subsp. napus) production. The evolution of new pathotypes rendering available resistances ineffective compel the introgression of new resistance into canola and extend our understanding of the genetic and molecular basis of the resistance. In this paper, we report the genetic and molecular basis of clubroot resistance in canola, introgressed from a rutabaga (B. napus L. subsp. rapifera Metzg. 'Polycross'), by using a doubled-haploid (DH) mapping population. Whole-genome resequencing (WGRS)-based bulked segregant analysis followed by genetic mapping and expression analysis of the genes in resistant and susceptible DH lines at 7 and 14 d after inoculation were carried out. Following this approach, two major quantitative trait loci (QTL) located at 14.41-15.44 Mb of A03 and at 9.96-11.09 Mb of A08 chromosomes and their interaction was observed to confer resistance to pathotypes 3H, 3A, and 3D. Analysis of the genes from the two QTL regions suggested that decreased expression of sugar transporter genes (BnaA03g29290D and BnaA03g29310D) may play an important role in resistance conferred by the A03 QTL, while increased expression of the toll/interleukin-1 receptor (TIR)-nucleotide binding (NB)-leucine rich repeat (LRR) (TNL) genes (BnaA08g10100D, BnaA08g09220D, and BnaA08g10540D) could be the major determinant of the resistance conferred by the A08 QTL. Single-nucleotide polymorphism (SNP) allele-specific polymerase chain reaction (PCR)-based markers, which could be detected by agarose gel electrophoresis, were also developed from the two QTL regions for use in breeding including pyramiding of multiple clubroot resistance genes.

Plasmodiophora brassicae causes a serious threat to cruciferous plants including radish (Raphanus sativus L.). Knowledge on the pathogenic regularity and molecular mechanism of P. brassicae and radish is limited, especially on the metabolism level. In the present study, clubroot-susceptible and clubroot-resistant cultivars were inoculated with P. brassicae Race 4, root hairs initial infection of resting spores (107 CFU/mL) at 24 h post-inoculation and root galls symptom arising at cortex splitting stage were identified on both cultivars. Root samples of cortex splitting stage of two cultivars were collected and used for untargeted metabonomic analysis. We demonstrated changes in metabolite regulation and pathways during the cortex splitting stage of diseased roots between clubroot-susceptible and clubroot-resistant cultivars using untargeted metabonomic analysis. We identified a larger number of differentially regulated metabolites and heavier metabolite profile changes in the susceptible cultivar than in the resistant counterpart. The metabolites that were differentially regulated in both cultivars were mostly lipids and lipid-like molecules. Significantly regulated metabolites and pathways according to the P value and variable important in projection score were identified. Moreover, four compounds, including ethyl α-D-thioglucopyranoside, imipenem, ginsenoside Rg1, and 6-gingerol, were selected, and their anti-P. brassicae ability and effects on seedling growth were verified on the susceptible cultivar. Except for ethyl α-D-thioglucopyranoside, the remaining could inhibit clubroot development of varing degree. The use of 5 mg/L ginsenoside Rg1 + 5 mg/L 6-gingerol resulted in the lowest disease incidence and disease index among all treatments and enhanced seedling growth. The regulation of pathways or metabolites of carbapenem and ginsenoside was further explored. The results provide a preliminary understanding of the interaction between radish and P. brassicae at the metabolism level, as well as the development of measures for preventing clubroot.

Clubroot disease, caused by Plasmodiophora brassicae Woronin, affects various cruciferous crops. Variations in pathogenicity and virulence are present among field populations of P. brassicae. Many races (pathotypes) have been reported in Japan as well as in other countries using various differential systems. Populations can be classified into four pathotypes using two clubroot-resistant (CR) cultivars of Chinese cabbage as differential hosts in Japan. However, it was recently indicated that each population is often heterogenic and composed of multiple genotypes (races or pathotypes). Breakdown in CR cultivars of Chinese cabbage is a problem in some areas of Japan and may contribute to the selective propagation of minor pathogenic genotypes on the CR cultivars. Clubroot has also been recorded on five species of cruciferous weeds in Japan. In particular, clubroot of Cardamine flexuosa is widely distributed in Japan. Some populations of C. flexuosa are often moderately pathogenic on Chinese cabbage and turnip. Therefore, the epidemiological relationship between clubroot of cruciferous crops and that of the weed has been noted but not thoroughly clarified. The relationship between pathogenic and genetic variations has also been examined among populations from cruciferous crops and weeds in Japan. The result implies an interesting genetic relationship among Williams’ races, among pathotypes determined using CR cultivars of Chinese cabbage and among populations from crops and C. flexuosa. This review includes an introduction of the status of studies on pathogenic and genetic diversity in P. brassicae from Japan.

To mitigate the impact and dissemination of clubroot in western Canada, canola ( Brassica napus ) producers have relied on clubroot resistance traits. However, in 2013 and 2014, new strains of the clubroot pathogen, Plasmodiophora brassicae , emerged that are virulent on most clubroot‐resistant ( CR ) canola genotypes. Novel strains of the pathogen were inoculated onto two susceptible canola cultivars, one resistant line and six CR cultivars. Although all cultivars/lines showed a susceptible response to inoculation with the new strains of P. brassicae , the severity of disease reaction, root hair infection rates and the amount of P. brassicae DNA present in each canola genotype varied depending on the strain. In addition, the effect of inoculum density on disease severity and gall formation was recorded for one of these new strains on a universally susceptible Chinese cabbage cultivar and one susceptible and 10 resistant canola genotypes. Although root galls were observed at an inoculum density of 10 3 spores per mL of soil, clear differentiation of susceptible and resistant reactions among canola cultivars/lines was not observed until the inoculum density reached 10 5 spores mL −1 . At a spore density of 10 6 spores mL −1 and above, all cultivars/lines developed susceptible reactions, although there was some differentiation in the degree of reaction. This study shows the potential to develop a unique disease profile for emergent clubroot pathotypes and shows a useful range of spore densities at which to study new P. brassicae strains.

Clubroot disease, caused by Plasmodiophora brassicae, is a threat to Brassica crops; therefore, understanding of host-resistance is important for developing clubroot-resistant cultivars. Using multi-omics analysis of clubroot-resistant (CR) and -susceptible (CS) near-isogenic lines (NILs) of B. napus, carrying the resistance of turnip (B. rapa var. rapifera), we characterized the host resistance mechanisms. Through proteome analysis, we identified 6626 differentially abundant proteins (DAPs) (2353 in CR-NILs, 4273 in CS-NILs) (q < 0.05), of which 50 in CR- and 62 in CS-NILs were detected across the disease developmental stages. Notable proteins included those involved in reactive oxygen species scavenging (BnaA09T0647200WE)], cell-wall modifications (BnaA04T0244300WE) and glucosinolate biosynthesis (BnaA01T0266700WE) in the CR-NILs. Additionally, disease-resistance proteins like ENHANCED DISEASE RESISTANCE 2-like (BnaA03T0055600WE) and hairpin-induced family protein YLS9 (BnaA08T0237900WE) showed increased abundance in CR-NILs. In contrast, CS-NILs exhibited decreased abundance of defense-related proteins, including proteins containing CUPIN domain (BnaA09T0578800WE) and LACCASE (BnaA02T0019200WE). Integration of proteome data with transcriptome data revealed 33 genes in CR- and 32 in CS-NILs showing a consistent pattern, including the genes related to PLANT INVERTASE/PECTIN METHYLESTERASE INHIBITOR (BnaC04T0003100WE), KELCH MOTIF (BnaC02T0374800WE), LACCASE (BnaA02T0019200WE), and antioxidant-related transcripts [GLUTATHIONE S-TRANSFERASES (BnaA03T0280900WE) and 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE (BnaA09T0641500WE)]. Our findings offer valuable new targets for breeding clubroot-resistant B. napus.

Plasmodiophora brassicae , causal agent of clubroot of crucifers, poses a serious threat to Canadian canola production. The effects of fallow (F) periods and bait crops (clubroot‐susceptible canola (B) and perennial ryegrass (R)) on clubroot severity and P. brassicae resting spore populations were evaluated in five sequences: R–B, B–R, R–F, B–F and F–F. Both host and non‐host bait crops reduced clubroot severity in a subsequent crop of a susceptible canola cultivar compared with fallow. Resting spore and P. brassicae DNA concentrations decreased in all treatments, but were lowest for the R–B and B–R bait crop sequences. In addition, two studies were conducted in mini‐plots under field conditions to assess the effect of rotation of susceptible or resistant canola cultivars on clubroot severity and P. brassicae resting spore populations. One study included three crops of susceptible canola compared with a 2‐year break of oat–pea, barley–pea, wheat–wheat or fallow–fallow. The other study assessed three crops of resistant canola, two crops of resistant canola with a 1‐year break, one crop of resistant canola and a 2‐year break, and a 3‐year break with barley followed by a susceptible canola. The rotations that included non‐host crops of barley, pea or oat reduced clubroot severity and resting spore concentrations, and increased yield, compared with continuous cropping of either resistant or susceptible canola. Growing of a susceptible canola cultivar contributed 23–250‐fold greater gall mass compared with resistant cultivars.