Plasmodiophora Brassicae Woron Research Articles

Plasmodiophora brassicae root hair interaction and control by Bacillus subtilis XF-1 in Chinese cabbage

Clubroot caused by Plasmodiophora brassicae Woron is one of the most destructive diseases of Chinese cabbage (Brassica napus). A highly potent strain of Bacillus subtilis, XF-1, which was isolated from the rhizosphere of Chinese cabbages with severe clubroot in Guandu District of Kunming, Yunnan Province, China, had strong inhibitory effects on both the survival and germination of resting spores of P. brassicae. Several strategies were evaluated to efficiently manage clubroot in Chinese cabbage, and different mechanisms were investigated. The ability of Chinese cabbage root hairs to reduce infection by P. brassicae in the growth and efficiency of colonization of XF-1 on seedling roots was also studied. Responses to the suppression of clubroot increased with increased seedling age in pot and field conditions, and the rate of noninfectious root hair was enhanced by the inoculation of pathogens, suggesting that seedling age impacts the infection by P. brassicae. The application of B. subtilis XF-1 liquid culture for treatment during the developmental stages of infected Chinese cabbage seedlings gradually decreased the control effect. Moreover, soaking seeds with B. subtilis XF-1 culture reduced clubroot disease incidence rate and disease incidence by 40% and 69%, respectively. These results indicate that prevention and treatment by B. subtilis XF-1 at an early stage is the best way to reduce the damage caused by clubroot disease.

Deciphering the bacterial and fungal communities in clubroot-affected cabbage rhizosphere treated with Bacillus Subtilis XF-1

Clubroot is an infection of cruciferous crops which results in considerable yield losses, caused by Plasmodiophora brassicae Woron. Bacillus subtilis XF-1 isolated from the rhizosphere of Chinese cabbages with severe clubroot in Guandu District of Kunming, Yunnan Province, China, has strong inhibitory effects on the resting spores of P. brassicae. However, its potential effects on the soil bacterial and fungal communities are still unknown. In this study, B. subtilis XF-1 was inoculated into cabbage rhizosphere and incidence of clubroot disease was surveyed, furthermore, an effect of this strain on soil microbial community in cabbage rhizosphere was investigated using Biolog™ MicroPlates and 454 pyrosequencing. B. subtilis XF-1 reduced the disease index (DI) by 17.14% and the control efficiency was 76.92%. The results of Biolog analysis and high-throughput pyrosequencing demonstrated that Proteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria, Firmicutes, and Gemmatimonadetes were the dominant taxonomic phyla found among bacteria in ten samples and Ascomycota, Basidiomycota and early diverging fungal lineages were reported among fungi. The soil bacterial and fungal communities were reduced greatly at the beginning, but they were recovered gradually with the growth of plant. However, there was little difference between treatment and control at the mature stage. The present study demonstrated that the effect of B. subtilis XF-1 on soil fungal community in cabbages rhizosphere was just transient and that the high-throughput 454 pyrosequencing is a suitable method for the characterization of microbial communities of rhizosphere soil of cabbage.

양배추 및 브로콜리 뿌리혹병에 대한 효율적인 저항성 검정 방법 확립

Plasmodiophora brassicae Woron.에 의한 뿌리혹병은 배추과 작물에 발생하는 전 세계적으로 가장 중요한 식물병 중 하나이다. 본 연구는 Brassica oleracea에 속하는 양배추와 브로콜리의 뿌리혹병에 대한 효율적인 저항성 검정 방법을 확립하기 위하여, 레이스 9인 GN1 균주를 이용하여 접종원의 농도와 P. brassicae 접종 후 배양 기간 등의 발병 조건에 따른 양배추와 브로콜리 뿌리혹병 발생을 조사하였다. 접종원의 농도는 14일 재배한 유묘에 포트 당 <TEX>$2.5{\times}10^9$</TEX>개의 포자를 접종하는 것이 적당하였으며, 안정적인 발병을 위하여 접종한 유묘는 3일 동안 <TEX>$20^{\circ}C$</TEX> 생육상에서 하루에 12시간 광을 처리하며 배양하고, 그 후 온실(<TEX>$20{\pm}5^{\circ}C$</TEX>)에서 5주 동안 재배한 후에 발병 정도를 조사하는 것이 요구되었다. 위의 검정법을 이용하여 레이스가 다른 P. brassicae 4개 포장 균주에 대한, 시판 중인 양배추 16종과 브로콜리 17종 품종의 저항성 정도를 실험한 결과, 레이스 9인 GN1, GN2 그리고 GS 균주에 대해서는 중도저항성을 보이는 몇 가지 품종이 있었다. 그러나 레이스 2인 YC 균주에 대해서는 실험한 모든 품종이 고도의 감수성 반응을 나타내었다. 따라서 이 방법은 양배추와 브로콜리 뿌리혹병 저항성을 검정하기 위한 효율적인 검정법이라는 것을 알 수 있었다. Clubroot caused by Plasmodiophora brassicae Woron. is one of the most important diseases in Brassica crops worldwide. To establish more simple and reliable screening method for resistant cabbage and broccoli to P. brassicae, the development of clubroot on the plants according to inoculum concentration and incubation period after inoculating with the pathogen was investigated using P. brassicae GN1 isolate (race 9). To facilitate and acquire precise result of resistance screening of cabbage and broccoli to clubroot, 14-day-old seedlings were inoculated by drenching roots with the spore suspension of P. brassicae to give inoculum density of <TEX>$2.5{\times}10^9$</TEX> spores/pot. To develop the disease, the inoculated seedlings were incubated in a growth chamber at <TEX>$20^{\circ}C$</TEX> for 3 days, and then cultivated in a greenhouse (<TEX>$20{\pm}5^{\circ}C$</TEX>) for five weeks. Under the optimum conditions, 16 cabbage and 17 broccoli cultivars were tested for resistance to four field isolates (GN1, GN2, GS and YC) of P. brassicae collected from four regions in Korea. Among them, some cabbage and broccoli cultivars showed different resistance response to three isolates (GN1, GN2 and GS) determined as race 9 by using the differential varieties of Williams. On the other hand, all the tested cultivars were highly susceptible to YC isolate (race 2). The results suggest that this method is efficient screening method of cabbage and broccoli for resistance to P. brassicae.

효율적인 무 뿌리혹병 저항성 검정법 확립

To establish simple and reliable screening method for resistant radish to Plasmodiophora brassicae Woron. using soil-drenching inoculation, the development of clubroot on radish seedlings inoculated with P. brassicae GN-1 isolate according to several conditions such as inoculum concentration, plant growth stage and incubation period after inoculation was studied. To select resistant radish against clubroot, 10-day-old seedlings were inoculated with P. brassicae by drenching the roots with the spore suspension of the pathogen to give spores/pot. The inoculated seedlings were incubated in a growth chamber at for 3 days then cultivated in a greenhouse () for 6 weeks. Under the optimum conditions, 46 commercial cultivars of radish were tested for resistance to YC-1 (infecting 15 clubroot-resistant cultivars of Chinese cabbage) and GN-1 (wild type) isolates of P. brassicae. Among them, thirty-five cultivars showed resistance to both isolates and one cultivar represented susceptible response to the pathogens. On the other hand, the other cultivars showed different responses against the tested P. brassicae pathogens. The results suggest that this method is an efficient system for screening radish with resistance to clubroot.

Factors affecting the pattern of the dose response curve of clubroot disease caused by Plasmodiophora brassicae

The severity of clubroot disease caused by Plasmodiophora brassicae Woron. depended on the density of resting spores in soil, soil type, soil pH, and host susceptibility. The dose response curve (DRC) was determined to indicate the relationship between the disease index and these parameters for predicting the disease severity for each field. DRC patterns varied with the soil types and even among soils of the same type depending on the sampling areas. Disease incidence was lower in the soils adjusted to a higher pH than in those at the initial pH. DRC patterns were also influenced by plant species or cultivars. The DRC is useful for estimating the potential disease severity in agricultural fields with different soils and different plants and essential for the development of an integrated pest management (IPM) strategy for the control of clubroot disease.

Evaluation of French Brassica oleracea landraces for resistance to Plasmodiophora brassicae

A total of 240 kale, 38 cabbage and 126 winter cauliflower French landraces from the B. oleracea genepool of INRA were assessed for resistance to clubroot caused by Plasmodiophora brassicaeWoron. Two French isolates of the pathogen (K and SJ) were used in the experiments under controlled conditions. The reaction of the 126 cauliflower accessions to naturally occurring clubroot was also evaluated in field trials. Kales exhibited considerable variation for expression of disease resistance and high levels of resistance were found in several accessions. In this group, single resistant plants were observed in most of the morphological types and from quite different geographical origins. Cabbage accessions were moderately to highly susceptible to both isolates. All cauliflower populations proved to be highly susceptible to K isolate and moderately susceptible to SJ isolate. In field trials, cauliflowers were also severely infected. Two lines selected from a resistant kale population were highly resistant against a large range of pathotypes of the pathogen. These lines presented a sufficient level of resistance to be directly useful in the breeding program in order to develop cauliflower and broccoli hybrids resistant to clubroot.

Perspective on ecological studies of Plasmodiophora brassicae, an unculturable microorganism pathogenic on brassica crops.

Clubroot incited by Plasmodiophora brassicae Woron. causes serious yield losses in a wide range of brassica crops such as Chinese cabbage (Brassica rapa var. pekinensis). This pathogen is a root-invading organism belonging to the Plasmodiophorales, whose members are mostly obligate parasites. This pathogen does not grow in axenic culture, and can survives in soil as resting spores for a long period of time. These facts imply that the assessment of pathogenic activity of resting spores in soil is essential to study the ecology and control of the disease. The detection method using calcofluor white M2R and ethidium bromide has recently been developed. Other recent advances include in vitro growth of the pathogen and the inclusion of host-DNA in resting spores etc. A review of in vitro growth studies on Plasmodiophora brassicae was discussed here.

Genetic analysis of resistance to clubroot (Plasmodiophora brassicae Woron) in two Brassica oleracea groups (ssp. acephala and ssp. botrytis) through diallel analysis

AbstractClubroot, caused by Plasmodiophora brassicae Woron, is a major disease of cruciferous crops and few sources of resistance have been detected and genetically studied in the Brassica oleracea species. In this study, two diallel mating systems using resistant kale lines and susceptible cauliflower lines were performed. Resistance was assessed from a disease index evaluated on young plants artificially inoculated with local isolates of the parasite.Both general and specific combining ability effects (GCA and SCA) and partly reciprocal effects were demonstrated. Resistance inheritance was, however, characterized by a predominance of additive genetic effects (GCA). Three groups of parent lines could be separated; kale lines with very high resistance, kale lines with intermediate resistance and susceptible cauliflower lines. In the two kale groups, two genetically different resistance types were suggested and various recurrent selection procedures are proposed following the diallel results.

Detection and analysis of QTLs based on RAPD markers for polygenic resistance to Plasmodiophora brassicae Woron in Brassica oleracea L.

Resistance to Plasmodiophora brassicae Woron, the causal fungus of clubroot, was examined in an F2 population of a cross between a clubroot-resistant kale (Brassica oleracea L. var. acephala) and a susceptible cauliflower (Brassica oleracea L. var. botrytis). QTL detection was performed with RAPD markers. Two resistance notations, carried out at different times after inoculation, were used. Three markers were associated with these two notations and three were specifically linked to only one notation. QTL analysis suggests the existence of at least two genetic mechanisms implicated in the resistance phenomenon.

Detection and analysis of QTLs based on RAPD markers for polygenic resistance to Plasmodiophora brassicae Woron in Brassica oleracea L.

Detection and analysis of QTLs based on RAPD markers for polygenic resistance to Plasmodiophora brassicae Woron in Brassica oleracea L.

Screening of Portuguese cole landraces (Brassica oleracea L.) with Peronospora parasitica and Plasmodiophora brassicae

Forty-four landraces of Portuguese coles (Brassica oleracea L.), representing different morphotypes, were screened at the cotyledon stage for downy mildew resistance, caused by Peronospora parasitica. Two isolates of the pathogen, PHW 630 and PHW 828, were used in the experiment. The mean disease interaction phenotype varied among the landraces. Overall, the Portuguese coles have shown resistance to both isolates, particularly to isolate PHW 828. Some landraces were identified with differential reaction to the two P. parasitica isolates. Some of the Portuguese coles are, therefore, good sources of resistance genes to P. parasitica, and could be used in breeding programs or in the development of a downy mildew host differential set. Among the morphotypes tested, Couve Algarvia (ISA 207), Couve Galega (ISA 243 and 187) and Couve Gloria de Portugal (ISA 84) have presented the highest resistance interaction. For the development of host differential sets, the landraces ISA 2 (Penca de Mirandela), ISA 35 (Couve de Valhascos) and ISA 600 (Penca Espanhola) could be further selected for the differentiation of P. parasitica isolates. They are resistant to PHW 828 and susceptible to PHW 643. The same morphotypes and landraces were also screened at the seedling stage for resistance to clubroot, caused by Plasmodiophora brassicae Woron.. Although some variation for resistance could be noticed in some the landraces tested, mainly Galega kale types, none of them presented a sufficient level of resistance as to be directly be useful in breeding for resistance to race 6 of P. brassicae.

Infection of Arabidopsis thaliana by Plasmodiophora brassicae

AbstractThree populations of Arabidopsis thaliana (L.) Heynh. were inoculated with three isolates of Plasmodiophora brassicae Woron. Inoculation of young Arabidopsis plants caused clubbing of roots and, in the late flowering population CrGC 9–4, infection of shoots. In this population, the number of inoculated plants reaching the flowering stage was reduced, and the majority of plants died prematurely. Symptoms of shoot infections were compressed rosettes with thickened and stunted leaves containing resting spores of P. brassicae. The results showed clearly that A. thaliana is susceptible to P. brassicae.

Soil solarisation combined with low rates of soil fumigants controls clubroot of cauliflowers, caused by Plasmodiophora brassicae Woron

The effect of solarisation combined with low rates of soil fumigants on the severity of clubroot and yield of cauliflowers was determined at 2 locations in southern Victoria. The effectiveness of treatments was shown to be dependent on location; on the type, water content and temperature of soil; and on the population density of Plasmodiophora brassicae. Yields were reduced depending upon the disease severity, usually within 60 days after transplanting. Propagules of P. brassicae could survive for more than 28 days in ovens at 45�C when in dry soil but died within 14 days at 40�C in moist soil. At Werribee in 1985 on a red brown earth, solarisation combined with dazomet (100 kg dazomet/ha) gave significantly better control than either treatment alone. This treatment reduced P. brassicae in the 0-10 cm layer, reduced the disease rating from 2.7 to 0.9 (0-3), and increased yield from 2.4 to 47 t/ha compared with controls. In 1986, solarisation combined with 98% methyl bromide-2% chloropicrin (100 and 250 kg/ha) reduced the population density of P. brassicae in the 0-10 and 10-20 cm layers of soil, reduced the disease rating from 3 to 1.8, and increased yield from 0 to 22 t/ha. These treatments were more effective than solarisation and dazomet used alone or in combination. At Keysborough in 1985 on a grey sand, separate treatments of solarisation or dazomet (100 and 250 kg dazometha) were as effective as combined treatments and significantly reduced disease and increased yields compared to controls. Solarisation combined with either fumigant significantly reduced the distribution and total number of weeds at all sites and was generally more effective than separate treatments.

アブラナ科野菜根こぶ病菌（Ｐｌａｓｍｏｄｉｏｐｈｏｒａ ｂｒａｓｓｉｃａｅ Ｗｏｒｏｎ．）休眠胞子の蛍光抗体法による土壌および植物体からの検出について

Indirect fluorescent-antibody technique was applied for diagnosis of the clubroot of Brassicaceae caused by Plasmodiophora brassicae Woron. IgG (γ-globulin; 0.91 mg/ml) was purified from antiserum against resting spores of P. brassicae prepared from a rabbit. Infested soil and root were stained by fluorescent-antibody technique with the IgG and FITC conjugated antirabbit IgG-sheep IgG. (Cappel Lab.) Resting spores were effectively detected, and also clearly differentiated from small particles of soil and tissues of plant in the reflected light fluorescence microscope (Olympus BHS-RF-A, B-excitation). This method is considered to be suitable for the detection of P. brassicae.

Karyotypic analysis of Polymyxa betae (Plasmodiophoromycetes) based on serial thin sections of pachytene nuclei

Three pachytene nuclei of Polymyxa betae Keskin were reconstructed from serial thin sections. Thirty synaptonemal complexes (SCs) were counted, indicating a haploid chromosome number of 30. SCs of Polymyxa were similar to those of Sorosphaera veronicae Schroeter and Membranosorus heterantherae Ostenfeld and Peterson but differed from SCs of Plasmodiophora brassicae Woron. and Woronina pythii Goldie-Smith.

Thermal inactivation of Plasmodiophora brassicae Woron. and its attempted control by solarization in the Salinas Valley of California

Thermal inactivation of resting spores of P. brassicae Woron. in glasshouse soil depended on temperature, duration of treatment, inoculum concentration, and soil moisture. At 42, 44 and 50°C, the relationship between temperature and the time for thermal inactivation plotted on a semi-log scale was linear. Treatment times up to 45 days at 30°C and 37°C did not reduce infectivity. The detection threshold in the system was 10 0·5 spores/g of soil. At all temperatures tested, inactivation was achieved more rapidly in soil infested with 10 2 spores/g than with 10 6 spores/g. Heat treatment was more effective in saturated soil than in half-saturated soil. Soil temperatures in the field in the northern Salinas Valley were increased 11–14°C by tarping with clear, polyethylene plastic. The average weekly maximum temperature and minimum temperature at a 10 cm depth under tarps were 38°C and 29°C respectively. Solarization reduced disease development after a 10-week treatment but not after a 5-week treatment.

The use of an evolutionary scheme for cauliflowers in the screening of genetic resources

A hypothetical scheme is given for the evolution of the different types of cauliflower (Brassica oleracea var. botrytis L.). This has been useful in identifying sources of reduced susceptibility to cabbage root fly (Delia radicum (L.)), and may also be useful in the search for reduced susceptibility to clubroot (Plasmodiophora brassicae Woron.). It is argued that knowledge of the phylogeny of types within each crop species is of great importance in the exploitation of genetic resources.

Karyotypic analysis of Plasmodiophora brassicae based on serial thin sections of pachytene nuclei

Reconstructions based on electron microscopy of serial thin sections of three pachytene nuclei of Plasmodiophora brassicae Woron. showed 20 synaptonemal complexes (SCs) and thus indicated a haploid chromosomal number of 20. No recombination nodules were observed, but modified regions with lateral elements more electron opaque than the rest of the SCs were reported. Ends of SCs attached to the nuclear envelope and clustered near the two centriole pairs.

The Conversion of 3-Indolylmethylglucosinolate to 3-Indolylacetonitrile by Myrosinase, and its Relevance to the Clubroot Disease of the Cruciferae

[Methylene-14C]-3-indolylmethylglucosinolate (I4CTMG) was converted in vitro to [methylene-14C]-3 indolylacetonitrile (14C-IAN) by myrosinase over a pH range of 4-0-6-0 and this conversion was enhanced by ferrous ions. Other products of the reaction included 3-indolylmethanol, 3,3 '-diindolyl methane and ascorbigen A. Trace amounts of 14C-IAN were produced non-enzymically from 14C-IMG in the presence of ferrous ion over a similar pH range. Furthermore, swede tissues (Brassica napus cv. Danestone) infected with Plasmodiophora brassicae Woron. could convert l4C-IMG to 14C-IAN. These results were consistent with the hypothesis that the overgrowth symptoms of the clubroot disease are caused by the conversion of IMG to the auxin precursor IAN.

The occurrence of free and bound cytokinins in clubroots and Plasmodiophora brassicae infected turnip tissue cultures

AbstractThis paper deals with the quantitative determination of free and bound cytokinins in clubroot tissue and in Plasmodiophora brassicae Woron, infected Brassica campestris L. callus tissue. The fractions were separated in a butanol soluble fraction containing the free cytokinins such as zeatin and zeatin riboside and a water soluble fraction containing the bound cytokinins.The butanol fraction was extensively purified and analysed by high pressure liquid chromatography (HPLC). The butanol fraction contained cytokinins which cochromatographed with zeatin and zeatin riboside and not with dihydrozeatin. Zeatin and zeatin riboside were quantitatively determined by HPLC. Recovery of the cytokinins varied between 30–50%. Clubs contained 50–160 ng zeatin and 210–300 ng zeatin riboside per g dry weight. Callus tissue contained 133 ng zeatin and 169 ng zeatin riboside per g dry weight.Clubs, callus as well as healthy tissue contain large amounts of bound cytokinins. Upon treatment of the water soluble fraction first with alkaline phosphatase and then with β‐glucosidase biologically active fractions were found which coeluted with zeatin and zeatin riboside on Sephadex LH20 in 20% ethanol. Evidence is presented for a novel cytokinin in the water soluble fraction which yields free zeatin and glucose‐6‐phosphate after treatment with β‐glucosidase.