Stimulation by caffeic acid,coumalic acid,and corilagin of the germination of resting spores of the clubroot pathogen Plasmodiophora brassicae

Some chemicals were examined for their effects on the germination of resting spores of the clubroot pathogen Plasmodiophora brassicae, and on the control of clubroots in Chinese cabbage. Caffeic acid, coumalic acid, and corilagin stimulated the germination of Plasmodiophora spores and prevented the formation of clubroots in Chinese cabbage. Clubroot might be controlled by agents with germination-stimulating effects.     

The host range of Plasmodiophora brassicae and its relationship to endogenous glucosinolate content

The host range of the soilborne obligate biotroph, Plasmodiophora brassicae was investigated. Evidence is presented that infection by P. brassicae might occur in non- Brassica species, leading to the potential formation of resting spores. Structures resembling P. brassicae were found in the root cortex of Tropaeolum majus , Carica papaya , Reseda alba and Beta vulgaris as demonstrated by scanning electron microscopy. Inoculation of Brassica rapa roots with spores extracted from either T. majus or B. vulgaris roots which had been previously inoculated with P. brassicae led to development of clubroot in the roots of B. rapa . It was also shown that the development of the symptom might be correlated with glucosinolate content, although other host factors are implicated in the B. vulgaris interaction with P. brassicae . In the glucosinolate-containing non-Brassicas, T. majus and C. papaya , the concentrations of benzylglucosinolate increased markedly in roots inoculated with P. brassicae , compared with the controls. There were also increases in concentrations of benzylglucosinolate in leaves of T. majus after P. brassicae infection. However, in R. alba roots, the total glucosinolate content decreased after inoculation with P. brassicae compared with the controls. High root concentrations of 2-OH-2-phenylethylglucosinolate (glucobarbarin) compared with low root indole glucosinolates in this species might limit P. brassicae infection and development. The importance of our investigations in relation to cultivation of non- Brassica species on fields infested with P. brassicae is discussed.     

甘蓝根肿病菌的生物学特性研究*

对甘蓝根肿病菌生物学特性研究表明,该菌休眠孢子萌发的最适温度24℃,最适pH值6.0～6.7,致死温度45℃,肿根腐烂处理可以显著提高萌发率,光对休眠孢子萌发有明显抑制作用。该菌休眠孢子在感病寄主的根分泌物溶液中萌发率最高,达75%。耐病甘蓝品种及番茄的根分泌物均能刺激休眠孢子萌发。通过电镜观察,根肿病菌休眠孢子为近球形,孢壁有乳状突起,直径2.1～3.1mm(平均直径2.5mm)。游动孢子为近球形或椭圆形,大小为1.6～3.6mm,同侧着生不等长尾鞭式双鞭毛。     

Plasmodiophora brassicae-induced Cell Death and Medium Alkalization in Clubroot-resistant Cultured Roots of Brassica rapa

Plasmodiophora brassicae causes clubroot in the turnip, Brassica rapa L. We used organ cultures of adventitious roots from B. rapa seedlings to investigate the initial response of resistant and susceptible cultivars to P. brassicae infection. Primary plasmodia of P. brassicae were observed in root hairs of both susceptible and resistant cultured roots. On the other hand, secondary plasmodia were able to proliferate only in the susceptible root culture but not in the resistant one. Root cultures from the susceptible cultivar all developed clubroot 4 weeks after treatment with 10⁴, 10⁵ or 10⁶ spores/ml, but roots from the resistant cultivar did not develop clubroot under the same conditions. Cell death, as measured by Evans blue and TTC dye methods, was observed in cultured roots from the resistant cultivar but did not occur in roots from the susceptible cultivar after exposure to P. brassicae spores. Cell death was inhibited almost completely by EGTA and verapamil but not by the calmodulin antagonist W7. These results suggest the involvement of Ca²⁺ in P. brassicae‐induced cell death. Alkalization of the root culture medium of the resistant cultivar was observed 2 days after treatment with P. brassicae spores but was not observed in root culture medium from the susceptible strain. We conclude that our root culture system must be a useful tool for further studies of the molecular mechanism of clubroot resistance.     

Germination of surface-disinfected resting spores ofPlasmodiophora brassicae and their root hair infection in turnip hairy roots

Germination of surface-disinfected resting spores ofPlasmodiophora brassicae and its infection of turnip hairy root hairs were studied. Surface-disinfected resting spores showed higher germination than non-disinfected resting spores. Root hair infection was most frequent in the section of root formed 1 d before inoculation. Root hair infection began 4 d after inoculation, increased up to 6 d, and continued to increase more slowly until 10 to 12 d after inoculation. Growth ofP. brassicae in the root hair of hairy roots was observed serially. Most primary plasmodia differentiated to mature zoosporangia 8–10 d after inoculation. The secondary zoospores were initially released 6 d after inoculation.     

Extracellular invertase is involved in the regulation of clubroot disease in Arabidopsis thaliana

Clubroot disease of Brassicaceae is caused by an obligate biotrophic protist, Plasmodiophora brassicae. During root gall development, a strong sink for assimilates is developed. Among other genes involved in sucrose and starch synthesis and degradation, the increased expression of invertases has been observed in a microarray experiment, and invertase and invertase inhibitor expression was confirmed using promoter::GUS lines of Arabidopsis thaliana. A functional approach demonstrates that invertases are important for gall development. Different transgenic lines expressing an invertase inhibitor under the control of two root-specific promoters, Pyk10 and CrypticT80, which results in the reduction of invertase activity, showed clearly reduced clubroot symptoms in root tissue with highest promoter expression, whereas hypocotyl galls developed normally. These results present the first evidence that invertases are important factors during gall development, most probably in supplying sugars to the pathogen. In addition, root-specific repression of invertase activity could be used as a tool to reduce clubroot symptoms.     

Expression and localization of nitrilase during symptom development of the clubroot disease in Arabidopsis

The expression of nitrilase in Arabidopsis during the development of the clubroot disease caused by the obligate biotroph Plasmodiophora brassicae was investigated. A time course study showed that only during the exponential growth phase of the clubs was nitrilase prominently enhanced in infected roots compared with controls. NIT1 and NIT2 are the nitrilase isoforms predominantly expressed in clubroot tissue, as shown by investigating promoter-beta-glucuronidase fusions of each. Two peaks of beta-glucuronidase activity were visible: an earlier peak (21 d post inoculation) consisting only of the expression of NIT1, and a second peak at about 32 d post inoculation, which predominantly consisted of NIT2 expression. Using a polyclonal antibody against nitrilase, it was shown that the protein was mainly found in infected cells containing sporulating plasmodia, whereas in cells of healthy roots and in uninfected cells of inoculated roots only a few immunosignals were detected. To determine which effect a missing nitrilase isoform might have on symptom development, the P. brassicae infection in a nitrilase mutant (nit1-3) of Arabidopsis was investigated. As a comparison, transgenic plants overexpressing NIT2 under the control of the cauliflower mosaic virus 35S promoter were studied. Root galls were smaller in nit1-3 plants compared with the wild type. The phenotype of smaller clubs in the mutant was correlated with a lower free indole-3-acetic acid content in the clubs compared with the wild type. Overexpression of nitrilase did not result in larger clubs compared with the wild type. The putative role of nitrilase and auxins during symptom development 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.     

Isomers of zeatin and zeatin riboside in clubroot tissue: evidence for trans-zeatin biosynthesis by Plasmodiophora brassicae

Cytokinin-like substances in both healthy and infected ( Plasmodiophora brassicae ) Wor. strain S) roots of Brassica campestris L. ssp. pekinensis cv. Granat have been tentatively identified and quantified by HPLC. The isomers of the cytokinins could be seperated on a reversed phase column using a gradient elution with increasing amounts of methanol. Secondary plasmodia were isolated mechanically from Plasmodiophora brassicae infected roots. The time course of adenine uptake and its conversion to cytokinins were investigated. Evidence is presented for the incorporation of [U-¹⁴C]-adenine into trans -zeatin by secondary plasmodia.     

Molecular Detection and Pathogenicity Assay of Plasmodiophora brassicae in Chicken Manure

To determine the relationship between animal excreta and the occurrence of clubroot disease of cruciferous crops caused by Plasmodiophora brassicae, chickens were fed with resting spores of the pathogen. Their faeces were collected and used to inoculate crucifers. This study proved that both fresh and composted manures could induce clubroot and the presence of the pathogen in the manure was confirmed by PCR amplification. However, composting had detrimental effects on the virulence of the resting spores in the manure. When the temperature was over 32°C, the incidence and severity of clubroot declined with the increase in the exposure time of resting spores to high temperature and the pathogenicity was completely lost when the spores were kept at 48°C for 6 h. The control measures for the clubroot disease were discussed.     

The development and endophytic nature of the fungus Heteroconium chaetospira

The root endophytic fungus Heteroconium chaetospira was isolated from roots of Chinese cabbage grown in field soil in Japan. This fungus penetrates through the outer epidermal cells of its host, passes into the inner cortex, and grows throughout the cortical cells, including those of the root tip region, without causing apparent pathogenic symptoms. There are no ultrastructural signs of host resistance responses. H. chaetospira has been recovered from 19 plant species in which there was no disruption of host growth. H. chaetospira has a symbiotic association with Chinese cabbage. The fungus provides nitrogen in exchange for carbon. These associations are beneficial for the inoculated plants, as demonstrated by increased growth rate. When used as a preinoculum, H. chaetospira suppresses the incidence of clubroot and Verticillium yellows when the test plant is post-inoculated with the causal agents of these diseases. H. chaetospira is an effective biocontrol agent against clubroot in Chinese cabbage at a low to moderate soil moisture range and a pathogen resting spore density of 10(5) resting spores per gram of soil in situ. Disease caused by Pseudomonas syringae pv. macricola and Alternaria brassicae on leaves can be suppressed by treatment with H. chaetospira. The fungus persists in the roots and induces systemic resistance to the foliar disease.     

Arabidopsis thaliana, a new tool to investigate Polymyxa betae-host interactions

Little is known about the genome of Polymyxa betae and its interactions with sugar beet, due partly to the obligate nature of the protist and the patents on Beta vulgaris sequences. The identification of an ecotype of Arabidopsis thaliana compatible with the protist would help to improve this knowledge. The infection and development of P. betae in 14 worldwide ecotypes of A. thaliana were studied. The detection of plasmodia and resting spores and the production of zoospores in the roots of A. thaliana were obtained in three bioassays, using automatic immersion systems and individual glass tubes. Detection was done using molecular detection and microscopy. Compatible interactions were established between 13 A. thaliana ecotypes of the 14 that were tested and the monosporosoric Belgian strain of P. betae, A26-41. The ecotype Cvi-0 (N1096), from the Cape Verde Islands, was the most compatible with the protist. This ecotype is also susceptible to Plasmodiophora brassicae, another plasmodiophorid. Polymyxa betae infection in A. thaliana was relatively very low compared with B. vulgaris, but every stage of the life cycle of the protist was present. The spore-forming phase was promoted at the expense of the sporangial phase, probably caused by the stress of this new environment. In addition, the protist revealed a new phenotype. This new model study will allow molecular tools available for A. thaliana to be used in order to gain a better understanding of the P. betae-plant interaction during the spore-forming phase.     

Randomly amplified polymorphic DNA (RAPD) profiling of Plasmodiophora brassicae

M. MÖLLER AND R. HARLING. 1996. A technique is described for the preparation of DNA suitable for use in RAPD analysis from pure, sterile resting spores of Plasmodiophora brassicae . Using this technique, random 10-base pair primers were applied to P. brassicae DNA from three single spore isolates. The resulting profiles were compared with the race classification based on inoculation of the European Clubroot Differential (ECD) series of Brassica hosts. Out of 40 primers tested, 23 gave amplification products, three gave isolate-specific profiles and one a profile which corresponded with the ECD race classification of the isolates. RAPD profiling can provide a faster means of race classification in P. brassicae .     

Life Cycle of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

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.     

甘蓝种质资源根肿病抗性鉴定及CRa、Crr1a同源基因分析

利用来源于湖北长阳、陕西太白、河南新野3个地方的根肿病菌对22份不同甘蓝材料进行抗病性鉴定。采用同源比对的方法,对甘蓝基因组中的大白菜抗根肿病同源基因CRa和Crr1a进行分析;同时对不同抗、感根肿病甘蓝材料中的CRa和Crr1a同源基因序列进行了扩增、测序和比对分析。结果表明:供试22份甘蓝材料对3份根肿病菌存在较大的抗感差异,推测来源于3个地区的菌种可能不是同一个生理小种;筛选出的抗性品种BDH3、Chou hybride Tekila、SW-110、CGL-8、先正达品种、SW-109将来可用作根肿病抗源和抗性基因挖掘;在甘蓝7号染色体上存在3个预测基因为CRa的同源基因,分别是Bo7g107710、Bo7g107730和Bo7g107740,其中,Bo7g107730基因在抗病材料SW-110存在较大的序列变异,推测可能与根肿病抗性相关;在甘蓝3号染色体上存在1个预测基因Bo3g164040为Crr1a的同源基因,所分析的抗、感病材料中Bo3g164040基因序列一致性极高,没有发现与抗根肿病有关的位点,说明甘蓝中Bo3g164040基因可能没有根肿病抗性功能。     

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.     

SnRK1.1-mediated resistance of Arabidopsis thaliana to clubroot disease is inhibited by the novel Plasmodiophora brassicae effector PBZF1

Plants have evolved a series of strategies to combat pathogen infection. Plant SnRK1 is probably involved in shifting carbon and energy use from growth-associated processes to survival and defence upon pathogen attack, enhancing the resistance to many plant pathogens. The present study demonstrated that SnRK1.1 enhanced the resistance of Arabidopsis thaliana to clubroot disease caused by the plant-pathogenic protozoan Plasmodiophora brassicae. Through a yeast two-hybrid assay, glutathione S-transferase pull-down assay, and bimolecular fluorescence complementation assay, a P. brassicae RxLR effector, PBZF1, was shown to interact with SnRK1.1. Further expression level analysis of SnRK1.1-regulated genes showed that PBZF1 inhibited the biological function of SnRK1.1 as indicated by the disequilibration of the expression level of SnRK1.1-regulated genes in heterogeneous PBZF1-expressing A. thaliana. Moreover, heterogeneous expression of PBZF1 in A. thaliana promoted plant susceptibility to clubroot disease. In addition, PBZF1 was found to be P. brassicae-specific and conserved. This gene was significantly highly expressed in resting spores. Taken together, our results provide new insights into how the plant-pathogenic protist P. brassicae employs an effector to overcome plant resistance, and they offer new insights into the genetic improvement of plant resistance against clubroot disease.