Enhancement of Biocontrol Activities and Cyclic Lipopeptides Production by Chemical Mutagenesis of Bacillus subtilis XF-1, a Biocontrol Agent of Plasmodiophora brassicae and Fusarium solani

Bacillus subtilis XF-1 has been used as a biocontrol agent of clubroot disease of crucifers infected by Plasmodiophora brassicae, an obligate pathogen. In order to maximize the growth inhibition of the pathogen, random mutagenesis using N-methyl-N′-nitro-N-nitrosoguanidine was applied to strain XF-1. The efficacy of 226 selected mutants was assessed against the growth of an indicator fungal pathogen: Fusarium solani using agar plate assay and the disruptive effects on the resting spores of P. brassicae. Four mutants exhibited inhibition activity significantly higher than the wild type. The cell extracts of these mutants and the XF-1 were subjected to matrix-assisted laser desorption ionization-time of flight mass spectra analysis, and three families of cyclic lipopeptides (CLPs) fengycin, surfactin and iturin were identified from the parental strain and the screened mutants. However, the relative contents and compound diversity changed after mutagenesis, and there was slight variation in the surfactin and fengycin. Notably, only 5 iturin components were discovered from the wild strain XF-1, but 13 were obtained from the mutant strains, and the relative CLPs contents of all mutant strains increased substantially. The results suggested that CLPs might be one of main biocontrol mechanisms of the clubroot disease by XF-1. The 4 mutants are far more effective than the parental strain, and they would be promising biocontrol candidates not only against P. brassicae but probably other plant diseases caused by fungi.     

Detection and Measurement of <Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis>

Clubroot, caused by Plasmodiophora brassicae, is one of the most important diseases of brassicas. Management of clubroot is difficult, and the best means of avoiding the disease include planting in areas where P. brassicae is not present and using plants and growing media free from pathogen inoculum. As P. brassicae is not culturable, its detection has traditionally relied on plant bioassays, which are time-consuming and require large amounts of glasshouse space. More recently, fluorescence microscopy, serology, and DNA-based methods have all been used to test soil, water, or plant samples for clubroot. The use of fluorescence microscopy to detect and count pathogen spores in the soil requires significant operator skill and is unlikely to serve as the basis for a routine diagnostic test. By contrast, serologic assays are inexpensive and amenable to high-throughput screening but need to be based on monoclonal antibodies because polyclonal antisera cannot be reproduced and are therefore of limited quantity. Several polymerase chain reaction (PCR)-based assays have also been developed; these are highly specific for P. brassicae and have been well-correlated with disease severity. As such, PCR-based diagnostic tests have been adopted to varying extents in Canada and Australia, but wide implementation has been restricted by sample processing costs. Efforts are underway to develop inexpensive serologic on-farm diagnostic kits and to improve quantification of pathogen inoculum levels through real-time PCR. Proper detection and quantification of P. brassicae will likely play an increasingly important role in the development of effective clubroot management strategies.     

Mechanisms of the biofungicide Serenade (Bacillus subtilis QST713) in suppressing clubroot

Clubroot is a serious threat to canola production in western Canada. The biofungicide Serenade® (Bacillus subtilis QST713) reduced the disease substantially in controlled environment, but showed variable efficacy in field trials. To better understand how this biofungicide works, two of the product components, i.e., B. subtilis and its metabolites (product filtrate), were assessed under controlled conditions for their relative contribution to clubroot control. The information may be used to optimize the product formulation. The bacterium or product filtrate alone was only partially effective against clubroot, reducing disease severity by about 60% relative to untreated controls. In contrast, Serenade controlled the disease by over 90%. This pattern of response was mirrored in quantitative PCR assessment on P. brassicae DNA within canola roots; the lowest and highest amounts of pathogen DNA were found in roots of Serenade treatment (0.02 and 0.01 ng/g) and controls (0.52 and 13.35 ng/g), respectively, at 2 and 3 weeks after treatment. During this period, the amount of DNA changed little in Serenade-treated roots but increased by almost 30-fold in the control. The product filtrate or B. subtilis also reduced the pathogen DNA substantially (0.03–1.16 ng/g). Serenade decreased the germination and viability of P. brassicae resting spores only marginally. It is suggested that biofungicide Serenade controls clubroot largely via suppressing root-hair and cortical infection by P. brassicae zoospores. The bacterial metabolites in the product formulation possibly assist B. subtilis in rhizosphere colonization and clubroot control by minimizing the competition from other soil microbes.     

Development of a PCR-based Assay for the Detection of Plasmodiophora brassicae in Soil

A single-tube nested polymerase chain reaction (STN PCR) method was developed for detecting the causal agent of clubroot disease, Plasmodiophora brassicae. Outer primer PBTZS-2 (5′-CCGAATTCGCGTCAGCGTGA-3′) to amplify a 1457 bp-fragment from P. brassicae DNA and nested primers, PBTZS-3 (5′-CCACGTCGATCACGTTGCAAT-3′) and PBTZS-4 (5′-GCTGGCGTTGATGTACTGGAA-TT-3′), to amplify a 398 bp-fragment internal of the 1457 bp-fragment were used for the STN PCR. The 398 bp-fragment was amplified from as little as 1 fg of P. brassicae DNA with the STN PCR. A protocol for extracting P. brassicae DNA directly from soil was developed. By using the protocol, DNA was extracted from artificially infested soil containing various numbers of P. brassicae resting spores and the resulting DNA was used as template for the STN PCR. As little as one resting spore of P. brassicae per g of soil was detectable with the STN PCR. The STN PCR was applied to naturally infested soil from 3 fields and one canal bed. The 398 bp-fragment was amplified from soil of 2 fields and the canal bed. To improve the detection of P. brassicae, the STN PCR products were subjected to second PCR amplification (double PCR) using the nested primers PBTZS-3 and PBTZS-4. The double PCR amplification generated a single 398 bp-DNA band which was visualized clearly on the agarose gel for all the 4 soil samples tested. A combination of the STN PCR and the double PCR appears a useful assay method for detecting P. brassicae resting spores in field soil.     

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.     

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.     

Glasshouse evaluation of some systemic fungicides for control of clubroot of brassicae

Of nine systemic fungicides screened as soil mixes against clubroot, only the precursors of methyl benzimidazol-2-ylcarbamate (MBC) or ethyl benzimidazol-2-ylcarbamate (EBC) gave promising results. Benomyl was the most effective, usually giving control at 250 mg/kg dry soil. Most fungicides were less effective against an isolate of Plasmodiophora brassicae from Brussels sprouts than against one from rape. Disease control was slightly better on cabbage than on a highly susceptible rape variety.     

A novel methyltransferase from the intracellular pathogen Plasmodiophora brassicae methylates salicylic acid

The obligate biotrophic pathogen Plasmodiophora brassicae causes clubroot disease in Arabidopsis thaliana, which is characterized by large root galls. Salicylic acid (SA) production is a defence response in plants, and its methyl ester is involved in systemic signalling. Plasmodiophora brassicae seems to suppress plant defence reactions, but information on how this is achieved is scarce. Here, we profile the changes in SA metabolism during Arabidopsis clubroot disease. The accumulation of SA and the emission of methylated SA (methyl salicylate, MeSA) were observed in P. brassicae‐infected Arabidopsis 28 days after inoculation. There is evidence that MeSA is transported from infected roots to the upper plant. Analysis of the mutant Atbsmt1, deficient in the methylation of SA, indicated that the Arabidopsis SA methyltransferase was not responsible for alterations in clubroot symptoms. We found that P. brassicae possesses a methyltransferase (PbBSMT) with homology to plant methyltransferases. The PbBSMT gene is maximally transcribed when SA production is highest. By heterologous expression and enzymatic analyses, we showed that PbBSMT can methylate SA, benzoic and anthranilic acids.     

Integrated Control of Clubroot

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.     

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.     

根际微生态调控白菜根肿病发生的机制研究进展

白菜根肿病是由芸薹根肿菌(Plasmodiophora brassicae Woron)引起的一种常见土传病害,主要危害白菜的根部。根际是土壤-植物-微生物相互作用最活跃的关键微域,根际微生态系统中的微生物失衡是导致土传病害的重要因素,深入探究根际微生态与土传病害互作机制,有利于从根际微生物、抑病物质和功能代谢等方面挖掘防控土传病害安全高效的方法。本文综述了根际微生态与白菜根肿病的发生机制关系,从该病害的危害、发生的根际微生态机制及生防菌防治研究等方面综合分析了根际微生物调控白菜根肿病发生的机制,以期为白菜根肿病防控、促进土壤健康和维持根际微生态系统稳定提供理论依据。     

Identification of two loci for resistance to clubroot (<Emphasis Type="Italic">Plasmodiophora brassicae</Emphasis> Woronin) in <Emphasis Type="Italic">Brassica rapa</Emphasis> L.

In an analysis of 114 F₂ individuals from a cross between clubroot-resistant and susceptible lines of Brassica rapa L., 'G004' and 'Hakusai Chukanbohon Nou 7' (A9709), respectively, we identified two loci, Crr1 and Crr2, for clubroot (caused by Plasmodiophora brassicae Woronin) resistance. Each locus segregated independently among the F₂ population, indicating that the loci reside on a different region of chromosomes or on different chromosomes. Genetic analysis showed that each locus had little effect on clubroot resistance by itself, indicating that these two loci are complementary for clubroot resistance. The resistance to clubroot was much stronger when both loci were homozygous for resistant alleles than when they were heterozygous. These results indicate that clubroot resistance in B. rapa is under oligogenic control and at least two loci are necessary for resistance.Communicated by H.C. Becker     

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 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.     

Revision of the species of the Tychus rufus group (Coleoptera: Staphylinidae: Pselaphinae)

The species belonging to Tychus rufus group are revised. Eleven species are recognized, described and illustrated and a key to their identification is provided. Nine taxa are new to science: Tychus carpathius n. sp. from Karpathos island (Greece); T. torticornis n. sp. from Lesbos Island (Greece); T. pisidicus n. sp. and T. inermis n. sp. from southwestern Turkey; T. antiocheus n. sp. and T. effeminatus n. sp. from southeastern Turkey; T. artvinensis n. sp. from northeastern Turkey, and T. sidonicus n. sp. and T. libanus n. sp. from Lebanon.