Methods for Assessment of Viability and Germination of Plasmodiophora brassicae Resting Spores

Clubroot caused by the obligate biotrophic parasite Plasmodiophora brassicae is a destructive soil borne disease of cruciferous crops. Resting spores of P. brassicae can survive in the soil for a long period without hosts or external stimulants. The viability and germination rate of resting spores are crucial factors of the inoculum potential in the field. The accurate assessment of viability and germination rate is the foundation to evaluate the effect of control methods. In this study, we evaluated several methods for the assessment of viability and germination rate of P. brassicae resting spores. Dual staining with calcofluor white-propidium iodide (CFW-PI) or single stain with Evans blue showed reliable accuracy in estimating viability. CFW-PI was capable of reliably determining the viability within 10 min, while Evans blue required overnight incubation to obtain accurate results. Due to DNA degradation of heat treatments, acetone was selected to evaluate the efficiency of propidium monoazide (PMA)–quantitative PCR (qPCR) used for the quantification of DNA from viable cells. The staining with 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI) and the use of differential interference contrast microscopy were suitable for the determination of resting spore germination rates. The latter method also allowed recording individual germination states of spores. Alternatively, dual staining with CFW-Nile red was successfully used to assess the germination rate of resting spores with a lethal pre-treatment. This study evaluates and confirms the suitability of various microscopic and molecular genetic methods for the determination of viability and germination of P. brassicae resting spores. Such methods are required to study factors in the soil regulating survival, dormancy and germination of P. brassicae resting spores causing clubroot disease in Brassicaceae hosts and therefore are fundamental to develop novel strategies of control.

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An Improved Evans Blue Staining Method for Consistent, Accurate Assessment of Plasmodiophora brassicae Resting Spore Viability

Plant Disease ◽

10.1094/pdis-05-18-0855-re ◽

2019 ◽

Vol 103 (9) ◽

pp. 2330-2336 ◽

Cited By ~ 1

Author(s):

M. W. Harding ◽

T. B. Hill ◽

Y. Yang ◽

G. C. Daniels ◽

S. F. Hwang ◽

...

Keyword(s):

Evans Blue ◽

Staining Method ◽

Plasmodiophora Brassicae ◽

Blue Staining ◽

Inoculum Potential ◽

Spore Viability ◽

Modified Method ◽

Resting Spore ◽

Resting Spores ◽

Vital Stains

Clubroot caused by Plasmodiophora brassicae is an important disease of brassica crops. The use of vital stains to determine the viability of P. brassicae resting spores can provide useful information regarding spore longevity, inoculum potential, or the efficacy of antimicrobial treatments. Evans blue is one example of a vital stain that has been reported to differentially stain viable and nonviable resting spores. Some previously published protocols using Evans blue to stain P. brassicae resting spores have not provided accurate or consistent results. In this study, we modified the Evans blue method by increasing the staining time to 8 h or more and evaluated P. brassicae resting spores after heat treatment at various combinations of temperature and time. Extending staining times significantly increased the numbers of stained resting spores up to 7 h, after which the numbers of stained spores did not change significantly (R2 = 96.88; P ≤ 0.001). The accuracy of the modified method to discriminate viable and nonviable spores was evaluated in repeated experiments and by comparing the staining data with those derived from inoculation assays and propidium monoazide quantitative PCR (qPCR). The results demonstrated that the modified Evans blue staining method improved the accuracy and consistency of measurement of P. brassicae resting spore viability. Additionally, it was equivalent to the qPCR method for differentiating viable and nonviable spores (R2 = 99.84; P ≤ 0.001) and confirmed in canola infection bioassays.

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Effect of pathogen virulence on pathogenicity, host range and reproduction of Plasmodiophora brassicae, the causal agent of clubroot disease

Plant Disease ◽

10.1094/pdis-02-21-0410-re ◽

2021 ◽

Author(s):

Nazanin Zamani-Noor ◽

Sinja Brand ◽

Hans-Peter Soechting

Keyword(s):

Plasmodiophora Brassicae ◽

Brassica Species ◽

Post Inoculation ◽

Clubroot Disease ◽

Alopecurus Myosuroides ◽

Pathogen Virulence ◽

Iberis Amara ◽

Thlaspi Arvense ◽

Resting Spores ◽

Pathogen Dna

A series of greenhouse experiments was conducted to evaluate the effect of Plasmodiophora brassicae virulence on clubroot development and propagation of resting spores in 86 plant species from 19 botanical families. Plants were artificially inoculated with two isolates of P. brassicae, which were either virulent on clubroot-resistant oilseed rape cv. Mendel (P1 (+)) or avirulent on this cultivar (P1). Clubroot severity and the number of resting spores inside the roots were assessed 35 days post inoculation. Typical clubroot symptoms were observed only in the Brassicaceae family. P1 (+)-inoculated species exhibited more severe symptoms (2 to 10–fold more severe), bigger galls (1.1 to 5.8 fold heavier) and higher number of resting spores than the P1-inoculated plants. Among all Brassica species, Bunias orientalis, Coronopus squamatus and Raphanus sativus were fully resistant against both isolates, while Camelina sativa, Capsella bursa-pastoris, Coincya momensis, Descurainia sophia, Diplotaxis muralis, Erucastrum gallicum, Neslia paniculata, Sinapis alba, S. arvensis, Sisymbrium altissimum, S. loeselii and Thlaspi arvense were highly susceptible. Conringia orientalis, Diplotaxis tenuifolia, Hirschfeldia incana, Iberis amara, Lepidium campestre and Neslia paniculata were completely or partially resistant to P1-isolate but highly susceptible to P1 (+). These results propose that the basis for resistance in these species may be similar to that found in some commercial cultivars, and that these species could contribute to the build-up of inoculum of virulent pathotypes. Furthermore, the pathogen DNA was detected in Alopecurus myosuroides, Phacelia tanacatifolia, Papaver rhoeas and Pisum sativum. It can concluded that the number and diversity of hosts for P. brassicae are greater than previously reported.

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First Report of Clubroot on Capsella bursa-pastoris Caused by Plasmodiophora brassicae in Sichuan Province of China

Plant Disease ◽

10.1094/pdis-04-13-0395-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 687-687 ◽

Cited By ~ 2

Author(s):

L. Ren ◽

X. P. Fang ◽

C. C. Sun ◽

K. R. Chen ◽

F. Liu ◽

...

Keyword(s):

Plasmodiophora Brassicae ◽

Tap Water ◽

Disease Incidence ◽

Morphological Characteristics ◽

Pcr Amplification ◽

Sichuan Province ◽

Clubroot Disease ◽

Cruciferous Vegetable ◽

First Report ◽

Resting Spores

Shepherd's purse (Capsella bursa-pastoris (L.) Medicus) is an edible and wild medicinal plant widely distributed in China. This plant has been cultivated in Shanghai, China, since the end of the 19th century. Infection of C. bursa-pastoris by Plasmodiophora brassicae, the causal agent of clubroot disease on Brassica spp. has been reported in Korea (2), but is not known to occur in China. In February of 2011, stunted and wilted shepherd's purse (SP) plants were observed in a field planted to oilseed rapes (B. napus) in Sichuan Province of China. Symptomatic SP plants also exhibited root galls. Disease incidence was 6.2% and 100% for SP and B. napus, respectively. Root galls on diseased SP plants were collected for pathogen identification. Many resting spores were observed when the root galls were examined under a light microscope. The resting spores were circular in shape, measuring 2.0 to 3.1 μm in diameter (average 2.6 μm). PCR amplification was conducted to confirm the pathogen. DNA was extracted from root galls and healthy roots (control) of SP. Two primers, TC2F (5′-AAACAACGAGTCAGCTTGAATGCTAGTGTG-3′) and TC2R (5′-CTTTAGTTGTGTTTCGGCTAGGATGGTTCG-3′) were used to detect P. brassicae (1). No PCR amplifications were observed with the control DNA as template. A fragment of the expected size (approximately 520 bp) was obtained when DNA was amplified from diseased roots of SP. These results suggest that the pathogen in the galled roots of SP is P. brassicae. Pathogenicity of P. brassicae in SP was tested on plants of both SP and Chinese cabbage (CC) (B. campestris ssp. pekinensis). A resting spore suspension prepared from naturally infected SP roots was mixed with a sterilized soil in two plastic pots, resulting in a final concentration of 5 × 106 spores/g soil. Soil treated with the same volume of sterile water was used as a control. Seeds of SP and CC were pre-germinated on moist filter paper for 2 days (20°C) and seeded into the infested and control pots, one seed per pot for planted for CC and four seeds per pot for SP. The pots were placed in a chamber at 15 to 25°C under 12 h light and 12 h dark. Plants in each pot were uprooted after 4 weeks and the roots of each plant were washed under tap water and rated for clubroot disease. No disease symptoms were observed in the control treatments of SP or CC. Plants of both species showed symptoms of clubroot, with the disease incidence of 62.5% and 100% on SP and CC, respectively. The pathogen was isolated from diseased roots of each plant and confirmed as P. brassicae based on morphological characteristics and PCR detection. To our knowledge, this is the first report of clubroot disease on C. bursa-pastoris in Sichuan Province of China. This finding suggests that it may be necessary to manage C. bursa-pastoris in cruciferous vegetable (cabbage, turnip) and oilseed rape production fields. References: (1) T. Cao et al. Plant Dis. 91:80, 2007. (2) W. G. Kim et al. Microbiology 39:233, 2011.

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Plasmodiophora brassicae (club root).

10.1079/cpc.41865.20210102194 ◽

2021 ◽

Author(s):

Graham McGrann

Keyword(s):

Crop Rotation ◽

Plasmodiophora Brassicae ◽

Brassica Species ◽

Infested Soil ◽

Yield Losses ◽

Varietal Resistance ◽

Weed Species ◽

Clubroot Disease ◽

Local Spread ◽

Resting Spores

Abstract Plasmodiophora brassicae is a root-infecting protist pathogen that causes clubroot disease in brassica species. The organism is soil-borne and has long-lived resting spores that can survive in soil for more than 15 years. Local spread of motile zoospores can be facilitated by wet conditions but most dispersal of the pathogen is through the movement of infested soil. P. brassicae has a wide host range in the brassica family including numerous weed species. Control of the disease is difficult but clubroot can be managed by a combination of crop rotation, varietal resistance, improved agronomic practice such as improved drainage and the application of lime of related products to raise pH which can limit the effects of the disease. There are currently no effective fungicides for the widespread control of clubroot. Yield losses range from 10 to 15% but can exceed 50% under disease conducive environmental conditions.

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MAPKK Inhibitor U0126 Inhibits Plasmodiophora brassicae Development

Phytopathology ◽

10.1094/phyto-07-17-0240-r ◽

2018 ◽

Vol 108 (6) ◽

pp. 711-720 ◽

Cited By ~ 2

Author(s):

Tao Chen ◽

Kai Bi ◽

Yanli Zhao ◽

Xueliang Lyu ◽

Zhixiao Gao ◽

...

Keyword(s):

Plasmodiophora Brassicae ◽

Transcriptional Profiling ◽

Specific Inhibitor ◽

Mapk Signaling ◽

Cellular Growth ◽

Mitogen Activated Protein Kinase ◽

Clubroot Disease ◽

Mapk Kinase ◽

Mapk Cascades ◽

Resting Spores

Mitogen-activated protein kinase (MAPK) cascades play a central role in cellular growth, proliferation, and survival. MAPK cascade genes have been extensively investigated in model plants, mammals, yeast, and fungi but are not characterized in Plasmodiophora brassicae, which causes clubroot disease in cruciferous plants. Here, we identified 7 PbMAPK, 3 PbMAPKK, and 9 PbMAPKKK genes in the P. brassicae genome. Transcriptional profiling analysis demonstrated that several MAPK, MAPK kinase (MAPKK), and MAPK kinase kinase (MAPKKK) genes were preferentially expressed in three different zoosporic stages. Based on yeast two-hybrid assays, PbMAKKK7 interacted with PbMAKK3 and PbMAKK3 interacted with PbMAK1/PbMAK3. The PbMAKKK7-PbMAKK3-PbMAK1/PbMAK3 cascade may be present in P. brassicae. U0126, a potent and specific inhibitor of MAPKK, could inhibit the germination of P. brassicae resting spores. U0126 was used to treat the resting spores of P. brassicae and coinoculate rapeseed, and was proven to significantly relieve the severity of clubroot symptoms in the host plant and delay the life cycle of P. brassicae. These results suggest that MAPK signaling pathways may play important roles in P. brassicae growth, development, and pathogenicity.

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Plasmodiophora brassicae infection threshold – How many resting spores are required for infection of canola (Brassica napus)

10.1101/2021.05.21.445124 ◽

2021 ◽

Author(s):

Kher Zahr ◽

Yalong Yang ◽

Snezana Dijanovic ◽

Heting Fu ◽

Michael Harding ◽

...

Keyword(s):

Plasmodiophora Brassicae ◽

Management Strategies ◽

Detection Methods ◽

Single Spore ◽

Clubroot Disease ◽

Highly Sensitive ◽

Resting Spores ◽

Low Probability ◽

Highly Sensitive Detection ◽

Important Disease

Clubroot, caused by Plasmodiophora brassicae, is an important disease of canola and other brassica crops. Improved understanding of host and pathogen biology is frequently useful in guiding management strategies. In order to better understand infection thresholds, seven-day old seedlings of canola cultivar Westar were inoculated with serially diluted concentrations of P. brassicae resting spores. Controlled soil and plant inoculation assays were performed and the plants maintained in a greenhouse for 42 days and clubroot disease severity evaluated visually. Clubroot symptoms were observed in soils containing as low as one spore/mL soil and on plants inoculated with as few as ≤ 100 resting spores. These thresholds were lower than any previously reported. The results indicated the importance of highly sensitive detection methods for P. brassicae diagnosis and quantification methods for clubroot risk prediction in soils. Furthermore, these results highlighted the low probability of obtaining P. brassicae single spore isolates.

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ClubrootTracker: A Resource to Plan a Clubroot-Free Farm

Plant Health Progress ◽

10.1094/php-01-20-0003-rs ◽

2020 ◽

Vol 21 (3) ◽

pp. 185-187 ◽

Cited By ~ 1

Author(s):

Kevin Muirhead ◽

Christopher D. Todd ◽

Yangdou Wei ◽

Peta Bonham-Smith ◽

Edel Pérez-López

Keyword(s):

Plasmodiophora Brassicae ◽

Intracellular Parasite ◽

13Th Century ◽

Clubroot Disease ◽

Online Tool ◽

Obligate Intracellular ◽

Obligate Intracellular Parasite ◽

The World ◽

Resting Spores ◽

Ready Access

Clubroot is a devastating disease affecting cruciferous crops worldwide. Clubroot was first described in the 13th century in Russia and from that moment has been affecting European, Asian, and American brassica production. Plasmodiophora brassicae is the clubroot causal agent, and it is an obligate intracellular parasite that, as soil-borne resting spores, can remain viable in soil for many years. This persistence in the soil is a major negative contributing factor to the management of clubroot disease and highlights the importance for brassica growers to have ready access to current information on the distribution of the pathogen. The interactive online tool ClubrootTracker ( http://clubroottracker.ca ) has been developed to enable users to view pathogen and disease presence in geographic locations across the world. ClubrootTracker, as described in this manuscript, has been developed to provide brassica farmers a tool that will contribute to clubroot management and aid in planning a clubroot-free farm. This tool is an open resource that has the main goal of acquisition of GPS information in reporting the pathogen or the disease by the researchers working with it around the world.

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Germination of Plasmodiophora brassicae resting spores stimulated by a non-host plant

European Journal of Plant Pathology ◽

10.1007/s10658-005-2797-0 ◽

2005 ◽

Vol 113 (3) ◽

pp. 275-281 ◽

Cited By ~ 25

Author(s):

Hanna Friberg ◽

Jan Lagerlöf ◽

Birgitta Rämert

Keyword(s):

Host Plant ◽

Plasmodiophora Brassicae ◽

Resting Spores

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Propagation of Pistacia lentiscus var. Chia Genotypes and Determination of Their Ornamental Traits Combined with a Genetic Analysis Using ISSR Markers

Agronomy ◽

10.3390/agronomy11020205 ◽

2021 ◽

Vol 11 (2) ◽

pp. 205

Author(s):

Stefanos Kostas ◽

Stefanos Hatzilazarou ◽

Elias Pipinis ◽

Anastasios Vasileiadis ◽

Panagiotis Magklaras ◽

...

Keyword(s):

Genetic Analysis ◽

Germination Rate ◽

Leaf Traits ◽

Issr Markers ◽

Pistacia Lentiscus ◽

Natural Habitats ◽

Indolebutyric Acid ◽

Single Male ◽

Asexual Propagation

Pistacia lentiscus L. is a dioecious shrub or small tree with high drought resistance, native in the entire Mediterranean basin. The variety ‘Chia’ (mastic tree) is cultivated for the production of mastic gum (a resin produced after wounding of the trunks and thick branches) in the Island of Chios (Greece), but it also has a significant ornamental value. In the present study, ten male and ten female genotypes of P. lentiscus var. Chia from the natural habitats of Chios were selected and examined with respect to the rooting efficiency of their shoot cuttings. The germination ability of the seeds was also tested. The 20 plants were morphologically described, mainly with respect to traits related to their ornamental value. Furthermore, leaves were collected from the 20 genotypes, from which genomic DNA (gDNA) was isolated, followed by amplification of gDNA fragments using the polymerase chain reaction (PCR) and inter simple sequence repeat (ISSR) primers. This was done aiming both at the determination of their genetic distance and the establishment of possible correlations between the amplified bands and certain morphological traits. The results of the study showed that there were differences among the genotypes for both methods of propagation. Regarding the rooting of the shoot cuttings, the best genotype was 8M (cv. ‘Psilophyllos’), achieving a rooting rate of 62.5% in winter with the application of indolebutyric acid (potassium salt of IBA), while regarding the seed germination capacity the genotype 2F exhibited the highest germination rate (57%). Genetic analysis using ISSRs separated the plants into four groups, one group consisting of male genotypes, one of the female genotypes, one consisting of members from both genders and a fourth containing a single male genotype. The genetic analysis of the male genotypes only produced a dendrogram showing the cultivars clustering in three different groups. Regardless of the genetic analysis, it seems that there were correlations between the ISSR markers and the leaf traits and also the gender and the asexual propagation. These correlations can assist future breeding programs of P. lentiscus var. Chia.

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Using seed respiration as a tool for calculating optimal soaking times for ‘on-farm’ seed priming of barley (Hordeum vulgare)

Seed Science Research ◽

10.1017/s0960258521000039 ◽

2021 ◽

pp. 1-9

Author(s):

Javier Carrillo-Reche ◽

Adrian C. Newton ◽

Richard S. Quilliam

Keyword(s):

Morphological Changes ◽

Low Cost ◽

Germination Rate ◽

Seed Priming ◽

Embryonic Axes ◽

Seedling Vigour ◽

Soaking Time ◽

On Farm ◽

Seed Respiration

Abstract A low-cost technique named ‘on-farm’ seed priming is increasingly being recognized as an effective approach to maximize crop establishment. It consists of anaerobically soaking seeds in water before sowing resulting in rapid and uniform germination, and enhanced seedling vigour. The extent of these benefits depends on the soaking time. The current determination of optimal soaking time by germination assays and mini-plot trials is resource-intensive, as it is species/genotype-specific. This study aimed to determine the potential of the seed respiration rate (an indicator of metabolic activity) and seed morphological changes during barley priming as predictors of the priming benefits and, thus, facilitate the determination of optimal soaking times. A series of germination tests revealed that the germination rate is mostly attributable to the rapid hydration of embryo tissues, as the highest gains in the germination rate occurred before the resumption of respiration. Germination uniformity, however, was not significantly improved until seeds were primed for at least 8 h, that is, after a first respiration burst was initiated. The maximum seedling vigour was attained when the priming was stopped just before the beginning of the differentiation of embryonic axes (20 h) after which vigour began to decrease (‘over-priming’). The onset of embryonic axis elongation was preceded by a second respiration burst, which can be used as a marker for priming optimization. Thus, monitoring of seed respiration provides a rapid and inexpensive alternative to the current practice. The method could be carried out by agricultural institutions to provide recommended optimal soaking times for the common barley varieties within a specific region.

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