The significance of Plasmodiophora brassicae Woronin and clubroot disease which it incites in members of the family Brassicaceae is reviewed as the focus for this special edition of the Journal of Plant Growth Regulation. This is a monographic treatment of recent research into the pathogen and disease; previous similar treatments are now well over half a century old. Vernacular nomenclature of the disease indicates that it had a well-established importance in agriculture and horticulture from at least the Middle Ages onward in Europe and probably earlier. Subsequently, the pathogen probably spread worldwide as a result of transfer on and in fodder taken by colonists as livestock feed. It is a moot point, however, whether there was much earlier spread by P. brassicae into China and subsequently Japan as Brassica rapa (Chinese cabbage and many variants) colonized those lands in archaeological time. Symptoms, worldwide distribution, and economic impact are briefly described here to provide a basis for understanding subsequent papers. Clubroot disease devastates both infected field and protected vegetable and agricultural Brassica crops. Particular importance is placed on recent reports of crop losses in tropical countries, albeit where the crops are grown in cooler altitudes, and in the Canadian prairie land canola crops. The latter is of enormous importance because this crop is the single most important and essential source of vegetable oils used in human foodstuffs and in industrial lubricants where mineral oils are inappropriate.

The Occurrence and Economic Impact of Plasmodiophora brassicae and Clubroot Disease

Plasmodiphora brassicae is a soil-borne obligate parasite. The pathogen has three stages in its life cycle: survival in soil, root hair infection, and cortical infection. Resting spores of P. brassicae have a great ability to survive in soil. These resting spores release primary zoospores. When a zoospore reaches the surface of a root hair, it penetrates through the cell wall. This stage is termed the root hair infection stage. Inside root hairs the pathogen forms primary plasmodia. A number of nuclear divisions occur synchronously in the plasmodia, followed by cleavage into zoosporangia. Later, 4–16 secondary zoospores are formed in each zoosporangium and released into the soil. Secondary zoospores penetrate the cortical tissues of the main roots, a process called cortical infection. Inside invaded roots cells, the pathogen develops into secondary plasmodia which are associated with cellular hypertrophy, followed by gall formation in the tissues. The plasmodia finally develop into a new generation of resting spores, followed by their release back into soil as survival structures. In vitro dual cultures of P. brassicae with hairy root culture and suspension cultures have been developed to provide a way to nondestructively observe the growth of this pathogen within host cells. The development of P. brassicae in the hairy roots was similar to that found in intact plants. The observations of the cortical infection stage suggest that swelling of P. brassicae-infected cells and abnormal cell division of P. brassicae-infected and adjacent cells will induce hypertrophy and that movement of plasmodia by cytoplasmic streaming increases the number of P. brassicae-infected cells during cell division.

Life Cycle of Plasmodiophora brassicae

Clubroot disease is a major threat to crops belonging to the Brassicaceae. It is controlled most effectively by the use of resistant cultivars. Plasmodiophora brassicae, the causal agent, shows a wide variation for pathogenicity, which can be displayed by using differential host sets. Except for Brassica juncea and B. carinata, resistant accessions can be found in all major crops. Most resistance sources are race-specific, despite some race-independent resistant accessions which can be found in B. oleracea. European field isolates from P. brassicae display great variation and show a tendency to overcome different resistance sources from either B. rapa or B. oleracea. At present, resistance genes from stubble turnips (B. rapa) are most effective and most widely used in resistance breeding of different Brassica crops. Resistance to P. brassicae from turnips was introduced into Chinese cabbage, oilseed rape, and B. oleracea. Although most turnips carry more than one resistance gene, the resistant cultivars from other crops received primarily a single, dominant resistance gene having a race-specific effect. Populations of P. brassicae that are compatible against most of the used resistance sources have been present in certain European areas for many decades. Such pathogen populations appeared in Japanese Chinese cabbage crops only a few years after the introduction of resistant cultivars. As the spread of virulent P. brassicae pathotypes seems to be slow, resistant cultivars are still a very effective method of control in many cropping areas. Mapping studies have revealed the presence of several clubroot-resistance genes in the Brassica A and C genomes; most of these genes are showing race specificity. Only in B. oleracea was one broad-spectrum locus detected. Two loci from the A genome confer resistance to more than one pathotype, but not to all isolates. Progress made in the determination of resistance loci should be used to formulate and introduce an improved differential set. Future efforts for breeding P. brassicae resistance will focus on durability by broadening the genetic basis of clubroot resistance by using either natural variation or transgenic strategies.

Status and Perspectives of Clubroot Resistance Breeding in Crucifer Crops

Clubroot, caused by Plasmodiophora brassicae, was identified in a number of canola (Brassica napus) fields in central Alberta in 2003. To characterize the virulence of the pathogen in the province, field populations from a number of locations in the Edmonton region were tested on the two most widely used sets of differential hosts, those of P.H. Williams and the European Clubroot Differential (ECD) series. Populations from British Columbia and Ontario were included for comparison. While the reaction of some hosts could be clearly defined as either resistant or susceptible, others showed intermediate disease index scores. If disease indices of 0%–49% and 50%–100% were regarded as resistant and susceptible, respectively, then populations from Alberta were classified as ECD 16/15/12 and 16/15/0 on the ECD set, or pathotypes 3 and 5, respectively, on the hosts of Williams. The population from British Columbia was classified as ECD 16/2/12 or pathotype 6, and the Ontario population was classified as ECD 16/0/1...

Characterization of Plasmodiophora brassicae populations from Alberta, Canada

Clubroot caused by Plasmodiophora brassicae affects the Brassicaceae family of plants, including many important vegetable and broadacre crops. In the last 20 years increasing intensity of vegetable production and the rapid growth in popularity of oilseed rape as a broadacre or arable break crop have increased the severity of clubroot and the area of land affected in both the vegetable and broadacre industries. Resting spores of P. brassicae are long-lived in soil, but the number of spores can be reduced through crop rotation, fallowing, chemical application, and management of brassica weeds. The host-pathogen system is responsive to a range of control measures, including calcium and boron amendments, manipulation of soil pH, and fungicide application. Molecular tests have been developed to predict disease and resistant cultivars are available for some crops. Increasingly, a multifaceted or integrated approach is being used to manage clubroot. This approach has been particularly successful in vegetable production systems.

Integrated Control of Clubroot

During 1986–87 the presence of clubroot in soils sampled from 190 fields was assessed using a bioassay method, based on baiting the soils with Brassica campestris spp. pekinesis cv. Granaat. Clubroot was detected in 148 (72%) of the fields investigated and, on average, 49.2% of the plants were infected according to the bioassay. Subsequent testing of fields in 1990 and 1992 (54 and 81 fields, respectively) where no further Brassica crops had been grown indicated a significant decrease in the degree of infestation to 7.1% in 1992. Clay soils showed, on average, the highest degree of infestation, and high infestation was recorded for a wide range of pH values (5.2–6.6). The highest degree of infestation was recorded on fields where oilseeds were grown five times during the period 1965–85. The results presented show that, in a field with 100% infestation, the level of infestation declined to below the detection level after a period of 17.3 years. The half-life of the spore inoculum was determined to be 3.6 years. During the sensitivity testing of different Brassica species and cultivars, it was found that Chinese cabbage showed a low percentage of infestation in two soils, whilst some oilseed rape and spring oilseed turnip rape cultivars showed high degrees of infestation in these soils.

Prevalence of Plasmodiophora brassicae in a spring oilseed rape growing area in central Sweden and factors influencing soil infestation levels

A protocol using real-time polymerase chain reaction (PCR) for the direct detection and quantification of Plasmodiophora brassicae in soil samples was developed and used on naturally and artificially infested soil samples containing different concentrations of P. brassicae. Species-specific primers and a TaqMan fluorogenic probe were designed to amplify a small region of P. brassicae ribosomal DNA. Total genomic DNA was extracted and purified from soil samples using commercial kits. The amount of pathogen DNA was quantified using a standard curve generated by including reactions containing different amounts of a plasmid carrying the P. brassicae target sequence. The PCR assay was optimized to give high amplification efficiency and three to four copies of the target DNA sequence were detected. Regression analysis showed that the standard curve was linear over at least six orders of magnitude (R 2 >0 AE99) and that the amplification efficiency was >92%. The detection limit in soil samples corresponded to 500 resting spores g )1 soil. The intersample reproducibility was similar to, or higher than, that of assays for other pathogens quantified in soil samples. Bait plants were used to validate the real-time PCR assay. The protocol developed was used to investigate the spatial distribution of P. brassicae DNA in different fields and a significant difference was found between in-field sampling points. The reproducibility of soil sampling was evaluated and showed no significant differences for samples with low levels of inoculum, whereas at higher levels differences occurred. Indicator kriging was used for mapping the probability of detecting P. brassicae within a2 -ha area of afi eld. At hreshold level of 5 fg plasmid DNA g )1 soil, corresponding to approximately 3 · 10 3 P. brassicae resting spores g )1 soil, is suggested for growing resistant cultivars. The results provide a robust and reliable technique for predicting the risk of disease development and for assessing the distribution of disease within fields.

https://bsppjournals.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1365-3059.2011.02477.x

In‐field distribution of Plasmodiophora brassicae measured using quantitative real‐time PCR

Sclerotinia stem rot of spring oilseed rape (Brassica napus) is caused by Sclerotinia sclerotiorum. In Sweden, the disease leads to severe crop damage that varies from year to year. A real-time PCR assay was developed and used to determine the incidence of S. sclerotiorum DNA on petals and leaves of spring oilseed rape as well as in air samples, with the aim of finding tools to improve precision in disease risk assessment. Five field experiments were conducted from 2008 to 2010 to detect and study pathogen development. Assessments of stem rot showed significant differences between experimental sites. The real-time PCR assay proved fast and sensitive and the relationship between percentage of infected petals determined using a conventional agar test and the PCR assay was linear (R2 > 0·76). There were significant differences in S. sclerotiorum incidence at different stages of flowering. The incidence of S. sclerotiorum DNA on the leaves varied (0–100%), with significantly higher incidence on leaves at lower levels. In one field experiment, S. sclerotiorum DNA was not detected on petals during flowering, whereas the pathogen was detected on leaves, with a corresponding stem rot incidence of 7%. The amount of S. sclerotiorum DNA in sampled air revealed that spore release did not coincide with flowering on that experimental site. Thus, using a real-time PCR assay to determine the incidence of S. sclerotiorum on oilseed rape leaves, rather than on petals, could potentially improve disease risk assessment.

https://bsppjournals.onlinelibrary.wiley.com/doi/pdf/10.1111/ppa.12230

Monitoring of plant and airborne inoculum of Sclerotinia sclerotiorum in spring oilseed rape using real-time PCR

Clubroot, caused by Plasmodiophora brassicae, is a crucial oilseed rape disease worldwide. Information on the virulence of P. brassicae populations is essential to apply disease control with proper clubroot-resistant cultivars. In 2016–2020, 84 isolates of P. brassicae were collected in the Czech Republic (CZ), Germany (DE), Poland (PL), and Sweden (SW). Pathotypes were designated using 17 Brassica hosts, including the European Clubroot Differentials (ECD), Somé set, and clubroot-resistant oilseed rape cv. Mendel. According to the ECD set, virulence analyses differentiated the isolates into 42 pathotypes. The most common pathotypes were 16/31/31 (in DE, PL, and SW) and 16/06/12 (in CZ, DE, and PL). Six pathotypes were found according to the Somé set, including 1–4 pathotypes per country. P1 was most prevalent in DE, PL, and SW, while P3 was abundant in CZ, DE, and PL. The current study provides clear evidence for a shift towards increased virulence in P. brassicae populations compared to previous studies. Several isolates overcame the resistance of cv. Mendel and of Brassica rapa genotypes ECD 01 to ECD 04. Considering all investigated samples, significant negative correlations were found between clubroot incidence and the frequency of oilseed rape in crop rotation, as for clubroot incidence and soil pH.

/pdf/pathotype-characterization-of-plasmodiophora-brassicae-the-2xbaqlb9.pdf

Pathotype Characterization of Plasmodiophora brassicae, the Cause of Clubroot in Central Europe and Sweden (2016–2020)

Abstract Background Oilseed rape (OSR; Brassica napus L.) is a highly valued crop for food, feed and industrial use. It is primarily grown in temperate climates, and over recent decades, its area of production and profitability have increased. Concurrently, several diseases negatively impact OSR production. Diseases caused by soil-borne pathogens, pose a risk of substantial yield loss since crop rotation schemes have become narrow as the time lapse between OSR crops in a field has been shortened. The aims of this paper were to provide an overview of plant protection measures available for OSR production and to identify knowledge gaps and areas where more research is needed. Methods This systematic map builds on a previously published protocol and follows the ROSES reporting standard. The search strategy was developed in collaboration with stakeholders and designed to cover available scientific evidence for OSR disease management in climate zones relevant for Scandinavian crop production (Dfc, Dfb, Cfb and Cfa in the Köppen-Geiger climate classification). Five scientific databases were used to identify peer-reviewed literature, complemented by additional searches performed in grey literature. Articles were screened at three stages: the title, abstract and full text. The eligible publications included studies of OSR crops, and all measures to control crop disease in agricultural fields were considered eligible interventions. The comparator was intervention and no intervention, and the yield per unit area, disease suppression or an increase in crop quality were determined to be outcomes of interventions. A basic assessment of the experimental design of each study was performed to assess its eligibility. All articles were coded based on the following categories: the location and climate zone, disease, pathogen, intervention and management method, outcome and study design. Articles not reporting original data but judged to be relevant (i.e., review papers, books and notes of registration of cultivars) were saved in a separate category called “books, reviews and reports”. Review findings A total of 4633 articles were collected through systematic searches. After duplicates were removed, 3513 articles were included in the screening process. After screening at the title and abstract levels, 897 articles were evaluated at the full text level, and 118 articles comprised the studies that met the eligibility criteria of the systematic map. The country (Canada) and region (Europe) with the largest OSR crop production areas also contributed the highest number of articles. In total, 17 different diseases were reported, with black leg (syn. Phoma stem canker) being the most studied disease. Nineteen different intervention methods or management types were examined. Cultivar resistance and pesticide application were the most studied control measures. Conclusion We report scientific studies on plant disease protection measures for OSR based on field trials where the results are intended to be directly implemented in crop production management. The map clearly provides an overview of research progress throughout the time period chosen, and it identifies knowledge gaps regarding important diseases where only a few studies have been published, for example, diseases caused by viruses. 

/pdf/scientific-evidence-of-sustainable-plant-disease-protection-jcebhmtp.pdf

A.-C. Wallenhammar

Papers

Prevalence of Plasmodiophora brassicae in a spring oilseed rape growing area in central Sweden and factors influencing soil infestation levels

In‐field distribution of Plasmodiophora brassicae measured using quantitative real‐time PCR

Monitoring of plant and airborne inoculum of Sclerotinia sclerotiorum in spring oilseed rape using real-time PCR

Pathotype Characterization of Plasmodiophora brassicae, the Cause of Clubroot in Central Europe and Sweden (2016–2020)

Scientific evidence of sustainable plant disease protection strategies for oilseed rape (Brassica napus) in Sweden: a systematic map