The clubroot pathogen Plasmodiophora brassicae: A profile update

Background

Plasmodiophora brassicae is the causal agent of clubroot disease of cruciferous plants and one of the biggest threats to the rapeseed (Brassica napus) and brassica vegetable industry worldwide.

Disease symptoms

In the advanced stages of clubroot disease wilting, stunting, yellowing, and redness are visible in the shoots. However, the typical symptoms of the disease are the presence of club-shaped galls in the roots of susceptible hosts that block the absorption of water and nutrients.

Host range

Members of the family Brassicaceae are the primary host of the pathogen, although some members of the family, such as Bunias orientalis, Coronopus squamatus, and Raphanus sativus, have been identified as being consistently resistant to P. brassicae isolates with variable virulence profile.

Taxonomy

Class: Phytomyxea; Order: Plasmodiophorales; Family: Plasmodiophoraceae; Genus: Plasmodiophora; Species: Plasmodiophora brassicae (Woronin, 1877).

Distribution

Clubroot disease is spread worldwide, with reports from all continents except Antarctica. To date, clubroot disease has been reported in more than 80 countries.

Pathotyping

Based on its virulence on different hosts, P. brassicae is classified into pathotypes or races. Five main pathotyping systems have been developed to understand the relationship between P. brassicae and its hosts. Nowadays, the Canadian clubroot differential is extensively used in Canada and has so far identified 36 different pathotypes based on the response of a set of 13 hosts.

Effectors and resistance

After the identification and characterization of the clubroot pathogen SABATH-type methyltransferase PbBSMT, several other effectors have been characterized. However, no avirulence gene is known, hindering the functional characterization of the five intercellular nucleotide-binding (NB) site leucine-rich-repeat (LRR) receptors (NLRs) clubroot resistance genes validated to date.

Important Link

Canola Council of Canada is constantly updating information about clubroot and P. brassicae as part of their Canola Encyclopedia: https://www.canolacouncil.org/canola-encyclopedia/diseases/clubroot/ .

Phytosanitary categorization

PLADBR: EPPO A2 list; Annex designation 9E.     

Evaluation of Brassica germplasm for field resistance against clubroot (Plasmodiophora brassicae Woron)

Of the 124 germplasm accession of oil seed Brassicas screened under field condition against clubroot disease (Plasmodiophora brassicae), 80% were susceptible and 17, 3, 1 and 1 of Brassica juncea, Brassica rapa var. toria, B.rapa var. yellow sarson and B. rapa, respectively, were resistant.     

Molecular Cloning of PbSTKL1 Gene from Plasmodiophora brassicae Expressed during Clubroot Development

Infection of crucifers by the obligate plant pathogen Plasmodiophora brassicae Woron. results in the formation of clubroot disease in these plants. Plasmodiophora brassicae gene expression during disease development was studied by differential display analysis of total RNA extracted from the roots of Chinese cabbage inoculated with the pathogen. In a series of experiments, 30 differentially expressed bands of cDNA were detected, and the expression of clone no. 17 was confirmed in clubbed roots. Southern blot analysis showed that this clone was a single‐copy gene in the P. brassicae genome. Putative amino acid sequence analysis of the full‐length cDNA of clone no. 17 (4.6 kb, designated PbSTKL1) revealed a serine/threonine kinase‐like domain at the C‐terminal region and a coiled‐coil structure in the middle region of the putative protein. PbSTKL1 expression increased strongly beginning 30 days after inoculation and was coincident with resting spore formation.     

Selection for resistance to clubroot (Plasmodiophora brassicae) in marrowstem kale (Brassica oleracea var. acephala L.)

Ninety-six cultivars of Brassica oleracea were screened for clubroot resistance in a seedling test using two populations of Plasmodiophora brassicae. The most resistant cultivars were kales. Sixteen resistant marrowstem kale cultivars of diverse geographical origin were used to start a selection programme for clubroot resistance. Four generations of selection, involving single plants, half-sib and full-sib families, reduced a disease index averaged over six clubroot populations from 41.2 to 12.5. This was lower than the most resistant cultivar in the original population, cv. Mixti 28.8, and as good as a German landrace of cabbage noted for its resistance, Bohmerwaldkohl 10.5. In comparison, the mean of five kale controls, cvs Bittern, Canson, Condor, Kestrel and Merlin, was 61.1 and the value for the most susceptible control, cabbage cv. Septa, was 89.3. In the final assessment, there were no clubroot population x B. oleracea genotype interactions and in the initial assessment of cultivars there were only small interactions which could be removed by an angular transformation of the data. It was concluded that a high level of non-differential resistance had been achieved and that it may prove durable. It was also concluded from a small field trial that this level of resistance would prevent serious yield losses in practice.     

Peroxidase and chitinase isoenzyme activities during root infection of Chinese cabbage with Plasmodiophora brassicae

Roots of two Chinese cabbage (Brassica campestris L. ssp. pekinensis) varieties, one tolerant and one susceptible, were inoculated with Plasmodiophora brassicae in liquid medium and in soil. Chitinase and peroxidase activities were determined in roots and shoots 1–21 days after inoculation with resting spores of Plasmodiophora and the enzyme activities compared with healthy tissue of the same age. In infected roots of the susceptible variety ‘Granat’ chitinase activity was higher than in the control 10 days after inoculation with spores. In the tolerant variety ‘Parkin’ we detected an increase in chitinase activity at the same time, which was about twice that of ‘Granat’. Chitinase activity in ‘Granat’ was also enhanced on day 13, 14 and 17 after inoculation, whereas chitinase activity in ‘Parkin’ was lower in the infected roots than in the controls during that period. In the shoots no correlation between chitinase activity and infection in the two varieties was observed. Chitinase from Chinese cabbage was further characterized and showed a pH optimum at pH 4.5–5.5 and a temperature optimum at 35–45°C. After isoelectric focusing 7 isoenzymes were discovered, but there were almost no differences between infected and healthy root extracts. Two isoenzymes with pI 8.7 and 8.8 showed cross-reactivity with an antiserum against bean chitinases. The molecular mass of these isoenzymes was determined as 33 kDa. Total peroxidase activity was generally higher in root tissue of both varieties than in the shoots. Peroxidase activity was increased most prominently in infected ‘Granat’ roots on day 13 after inoculation and of both varieties on day 17 compared to the controls. In clubbed tissue of ‘Granat’ a specific peroxidase isoenzyme appeared the first time 21 days after inoculation and was most prominent 28–30 days after inoculation. This isoenzyme had a molecular mass of ca 24 kDa and a pI of ca 8.8. With respect to our results the strategy of the Plasmodiophorales for plant attack is discussed.     

Cytology of infection,development and expression of resistance to Plasmodiophora brassicae in canola

The timing and expression of resistance to four isolates of Plasmodiophora brassicae, collected from research sites where pathotypes 2, 3, 5 and 6 (Williams' system) had been dominant when characterised in 2006, were assessed in four new commercial cultivars of canola (Brassica napus) with resistance to clubroot. Each of the resistant cultivars was highly resistant to all four of the isolates, and there was no difference in their response to infection. Root hair infection occurred at high levels, but pathogen development occurred more slowly than in a susceptible cultivar (control). Secondary infection and development in cortical cells was severely inhibited in each of the resistant cultivars; only a few bi‐nucleated plasmodia were observed at 12 days after inoculation (DAI), and plasmodia were rarely observed at 18 and 24 DAI. In contrast, development in the susceptible cultivar had progressed to resting spores by 24 DAI. A dense ring of accumulated reactive oxygen species (ROS) was observed in the endodermis, pericycle and vascular cambium of non‐inoculated controls and inoculated plants of the resistant cultivars. However, the ROS ring disappeared rapidly in infected plants of the susceptible cultivar. Plasmodia invaded the stele of susceptible roots by preferentially colonising the xylem parenchyma cells. Expansion and enlargement of lignified xylem cells was observed by 35 DAI. The absence of any specific points of ROS accumulation or lignification of epidermal or cortical cells in the resistant cultivars indicates that a hypersensitive response is not the main mechanism of resistance in these lines. The uniform response of these resistant cultivars to the four isolates of P. brassicae indicates that the resistance in each cultivar may be conditioned by a gene(s) from a single source that confers broad resistance, because most sources of resistance to P. brassicae are pathotype specific.     

Infection of Chinese cabbage by Plasmodiophora brassicae leads to a stimulation of plant growth: impacts on cell wall metabolism and hormone balance

The importance of plant hormones in clubroot infection has long been recognized. The morphological changes, such as cell division and cell elongation leading to gall formation are triggered in the early stages of infection. We analysed cell expansion by localizing Xyloglucan endoTransglucosylase/Hydrolase (XTH)-action and screened the endogenous concentrations of several classes of phytohormones by mass spectrometry in the early stages of Plasmodiophora brassicae infection in Chinese cabbage (Brassica rapa spp. pekinensis). Infected plants showed a general transient growth promotion early in infection. Furthermore a clear XTH action was visible in the epidermal layer of infected roots. Complex changes in the endogenous phytohormone profile were observed. Initially infection resulted in an increased total auxin pool. The auxin increase, together with an increased XTH action, results in wall loosening and consequently cell expansion. When the first secondary plasmodia are formed, thirteen days after infection (DAI), can be considered a switch point in phytohormone metabolism. Twenty-one DAI the plasmodia might act as a plant hormone sink resulting in a reduction in the active cytokinin pool and a lower indole-3-acetic acid content in the infected plants.     

Transfer of resistance to clubroot (Plasmodiophora brassicae) to swedes {Brassica napus L. var. napobrassica Peterm) from B. rapa

In 1975, tests with UK populations of Plasmodiophora brassicae not only revealed a lack of effective clubroot resistance in swedes (Brassica napus), but also the outstanding resistance of the European Clubroot Differential (ECD)04 (B. rapa). It was, therefore, decided to transfer the resistance genes from ECD04 to swedes, using the most pathogenic UK population of clubroot (C56) available for screening purposes. An autotetraploid form of ECD04 was crossed with tetraploid kale (B. oleracea) using the latter as female parent. One of the euploid, 2n = 38, hybrids secured by embryo rescue in 1976 was crossed to the swede cultivars Marian and Ruta Øtofte. Three further backcrosses of clubroot resistant plants to lines derived from modern swede cultivars were made over the period 1980 to 1982. Selfing commenced in 1983 to produce F₂ populations. From F₃ to F₅ there was family selection for yield and agronomic characters, as well as single plant selection for clubroot resistance. In 1991, the six most promising F₅ families were multiplied for subsequent evaluation in replicated yield trials in Dundee. The most promising family entered official trials at the beginning of 1993 and, 2 years later, was added to the National List as cv. Invitation and granted Plant Breeders' Rights. The first certified seed was sold in 1996, 20 years after the original synthetic B. napus was produced. The breeding programme provided evidence for only one of the three postulated dominant genes in ECD04 being required for resistance to C56 and also good evidence of differential resistance from tests with other clubroot populations. Hence, whilst the differential resistance in cv. Invitation should prove useful in the UK in the immediate future, it may not be durable in the longer term. It is, therefore, argued that the next and more difficult goal to achieve should be to introduce high levels of non-differential resistance from B. oleracea.     

Proteogenomic Analysis to Identify Missing Proteins from Haploid Cell Lines

Chromosome‐centric Human Proteome Project aims at identifying and characterizing protein products encoded from all human protein‐coding genes. As of early 2017, 19 837 protein‐coding genes have been annotated in the neXtProt database including 2691 missing proteins that have never been identified by mass spectrometry. Missing proteins may be low abundant in many cell types or expressed only in a few cell types in human body such as sperms in testis. In this study, we performed expression proteomics of two near‐haploid cell types such as HAP1 and KBM‐7 to hunt for missing proteins. Proteomes from the two haploid cell lines were analyzed on an LTQ Orbitrap Velos, producing a total of 200 raw mass spectrometry files. After applying 1% false discovery rates at both levels of peptide‐spectrum matches and proteins, more than 10 000 proteins were identified from HAP1 and KBM‐7, resulting in the identification of nine missing proteins. Next, unmatched spectra were searched against protein databases translated in three frames from noncoding RNAs derived from RNA‐Seq data, resulting in six novel protein‐coding regions after careful manual inspection. This study demonstrates that expression proteomics coupled to proteogenomic analysis can be employed to identify many annotated and unannotated missing proteins.