Effect of temperature on primary infection by Plasmodiophora brassicae and initiation of clubroot symptoms

A study was conducted to assess the effect of temperature on infection and development of Plasmodiophora brassicae in root hairs of Shanghai pak choi (Brassica rapa subsp. chinensis) and on initiation of clubroot symptoms. Ten‐day‐old seedlings were grown in liquid‐sand culture, inoculated with resting spores and maintained in growth cabinets at 10, 15, 20, 25 and 30°C. Seedlings were harvested at 2‐day intervals, starting 2 days after inoculation (DAI) and continuing until swelling of the tap root was observed (maximum 28 days). Roots were assessed for root hair infection (RHI), stage of development of infection (primary plasmodia, zoosporangia, release of zoospores, secondary plasmodia), symptom development, and for clubroot severity at 24 DAI. Temperature affected every stage of clubroot development; RHI was highest and visual symptoms initiated earliest at 25°C, intermediate at 20 and 30°C, and lowest and latest at 15 and 10°C. Root hair infection was observed at every temperature, but clubroot symptoms developed only above 15°C. A substantial delay in the development of the pathogen was observed at 10 and 15°C. No symptoms were observed at 28 DAI in plants grown at 10°C. Swelling of the tap root was visible at 28 DAI in plants at 15°C, 14 DAI at 20 and 30°C, and 10 DAI at 25°C. These results support and explain the observation in companion studies that cool temperatures result in slower development of clubroot symptoms in brassica crops.     

土壤pH对芸薹根肿菌侵染及病害发生的影响

通过设置不同pH梯度,研究土壤pH对根肿菌侵染及病害发生的影响。结果表明:土壤酸性时病菌侵染速度快,碱性时慢,而强酸性和碱性土壤条件则抑制孢子萌发;pH为6.0时最有利于根肿菌休眠孢子萌发,萌发率最高,为53.96%;碱性条件可使初级原生质团变形凝结成球状,不能正常分裂或延迟形成游动孢子囊,从而不利于根肿菌侵染。白菜发病率与病情指数随pH升高,呈先上升后下降趋势。其中,pH为5.0时,发病率和病情指数最高,pH 7.0~8.0时发病轻。因此,适宜的偏酸性环境条件下,通过作用于病菌休眠孢子萌发和侵染,提高病害危害程度,而中性或碱性条件干扰该过程并降低病害发生。     

Plasmodiophora brassicae resting spore dynamics in clubroot resistant canola (Brassica napus) cropping systems

The soilborne pathogen Plasmodiophora brassicae, causal agent of clubroot of canola (Brassica napus), is difficult to manage due to the longevity of its resting spores, ability to produce large amounts of inoculum, and the lack of effective fungicides. The cropping of clubroot resistant (CR) canola cultivars is one of the few effective strategies for clubroot management. This study evaluated the impact of the cultivation of CR canola on P. brassicae resting spore concentrations in commercial cropping systems in Alberta, Canada. Soil was sampled pre-seeding and post-harvest at multiple georeferenced locations within 17 P. brassicae-infested fields over periods of up to 4 years in length. Resting spore concentrations were measured by quantitative PCR analysis, with a subset of samples also evaluated in greenhouse bioassays with a susceptible host. The cultivation of CR canola in soil with quantifiable levels of P. brassicae DNA resulted in increased inoculum loads. There was a notable lag in the release of inoculum after harvest, and quantifiable P. brassicae inoculum peaked in the year following cultivation of CR canola. Rotations that included a ≥2-year break from P. brassicae hosts resulted in significant declines in soil resting spore concentrations. A strong positive relationship was found between the bioassays and qPCR-based estimates of soil infestation. Results suggest that CR canola should not be used to reduce soil inoculum loads, and crop rotations in P. brassicae infested fields should include breaks of at least 2 years away from B. napus, otherwise the risk of selecting for virulent pathotypes may increase.     

青花菜根肿病田间防治技术研究

2018年开展青花菜根肿病田间防治试验, 移栽后用5种药剂灌根处理?分别进行了5种药剂灌根次数试验?3种药剂灌根剂量试验和3种药剂灌根时期试验?试验结果表明, 5种药剂中500 g/L氟啶胺SC是防治青花菜根肿病的最佳药剂, 但使用不当会有药害风险, 移栽当天用2 250 g/hm2?移栽后3 d用3 375 g/hm2和4 500 g/hm2灌根, 花球成熟期推迟, 株高?最大叶面积均不同程度受抑制, 尤其在苗期使用会明显抑制生长, 表现为植株矮小?叶片黄化?无法正常盘根?建议用2 250 g/hm2分别在移栽后3~7 d和移栽后30 d各灌根1次?其次采用100 g/L氰霜唑SC, 338 g/hm2在移栽后0~3 d和30 d灌根也可取得较好防效?75%百菌清WP 8 440 g/hm2与100 g/L氰霜唑SC 338 g/hm2防效相当?20%噻唑锌SC防效较低, 62.5 g/L精甲·咯菌腈FSC防效最差, 不适宜用在根肿病防治上?     

Effects of cyazofamid against Plasmodiophora brassicae Woronin on Chinese cabbage

Cyazofamid (4-chloro-2-cyano-N,N-dimethyl-5-p-tolylimidazole-1-sulfonamide) is a novel fungicide with high levels of activity against Oomycetes fungi and Plasmodiophora brassicae Woronin. The effects of cyazofamid were investigated against P. brassicae, the causal agent of clubroot disease in Chinese cabbage. Cyazofamid at 0.3 mg litre(-1) inhibited resting spore germination of this pathogen by about 80%. Cyazofamid at 3-10 mg litre(-1) exhibited fungicidal activity to resting spores of P. brassicae 1-10 days after treatment. When cyazofamid was applied to infested soil, both root-hair infections and club formation caused by P. brassicae were strongly inhibited at 1-3 mg kg(-1) dry soil. These results suggest that cyazofamid directly inhibits resting spore germination, thereby leading to the inhibition of root-hair infection and club formation. Cyazofamid at 3 mg kg(-1) dry soil also exhibited complete control of clubroot disease. The effect of broadcast soil application using a dust formulation at 2 kg AI ha(-1) (equivalent to 1.3 mg AI kg(-1) dry soil), and plug seedling tray application by a suspension concentrate formulation at 200 and 400 mg AI tray(-1) (30 x 60 x 4 cm3) against P. brassicae was also evaluated. Cyazofamid exhibited good efficacy against the pathogen. The sequential treatment including plug seedling tray application with cyazofamid and pre-plant broadcast soil application with the fungicide fluazinam also exhibited excellent levels of control. These results indicate that cyazofamid has a high potential to be an effective fungicide for the control of clubroot disease.     

Reduction of Spore Density of Plasmodiophora brassicae in Soil by Decoy Plants

The severity of clubroot (Plasmodiophora brassicae) on Chinese cabbage was reduced by growing plants such as oats, spinach and leafy daikon prior to Chinese cabbage in pot experiments. Resting spore densities of P. brassicae in the soil were 29–62%, depending on the pervious crop, as compared to unplanted control plot after ploughing under the previously cultivated plants. Root hairs of the preceding plants were infected with P. brassicae, but clubbed roots were not formed on these plants. The results indicate that these plants functioned as decoy plants reducing the resting-spore density in soil and thereby suppressing disease severity. Received 21 February 2000/ Accepted in revised form 5 September 2000     

Effect of host and non‐host crops on Plasmodiophora brassicae resting spore concentrations and clubroot of canola

Plasmodiophora brassicae, causal agent of clubroot of crucifers, poses a serious threat to Canadian canola production. The effects of fallow (F) periods and bait crops (clubroot‐susceptible canola (B) and perennial ryegrass (R)) on clubroot severity and P. brassicae resting spore populations were evaluated in five sequences: R–B, B–R, R–F, B–F and F–F. Both host and non‐host bait crops reduced clubroot severity in a subsequent crop of a susceptible canola cultivar compared with fallow. Resting spore and P. brassicae DNA concentrations decreased in all treatments, but were lowest for the R–B and B–R bait crop sequences. In addition, two studies were conducted in mini‐plots under field conditions to assess the effect of rotation of susceptible or resistant canola cultivars on clubroot severity and P. brassicae resting spore populations. One study included three crops of susceptible canola compared with a 2‐year break of oat–pea, barley–pea, wheat–wheat or fallow–fallow. The other study assessed three crops of resistant canola, two crops of resistant canola with a 1‐year break, one crop of resistant canola and a 2‐year break, and a 3‐year break with barley followed by a susceptible canola. The rotations that included non‐host crops of barley, pea or oat reduced clubroot severity and resting spore concentrations, and increased yield, compared with continuous cropping of either resistant or susceptible canola. Growing of a susceptible canola cultivar contributed 23–250‐fold greater gall mass compared with resistant cultivars.     

Influence of cultivar resistance and inoculum density on root hair infection of canola (Brassica napus) by Plasmodiophora brassicae

The impact of cultivar resistance and inoculum density on the incidence of primary infection of canola root hairs by Plasmodiophora brassicae, the causal agent of clubroot, was assessed by microscopy. The incidence of root hair infection in both a resistant and a susceptible cultivar increased with increasing inoculum density, but was two‐ to threefold higher in the susceptible cultivar; the relationship between root hair infection and inoculum density was also substantially stronger and more consistent in the susceptible cultivar. In the susceptible cultivar, the root hair infection rate peaked between 6 and 8 days after sowing and then declined. In the resistant cultivar, it increased over the 14‐day duration of each study. It appears that examination of root hair infection by microscopy in a bait crop of susceptible canola could serve as a useful tool for estimating P. brassicae inoculum levels in soil. In a separate trial, the relationship between inoculum density and clubroot severity, plant growth parameters, and seed yield was assessed under greenhouse conditions. Inoculum density in the susceptible genotype was strongly and positively correlated with clubroot severity and negatively correlated with plant height and seed yield. In addition, a single cropping cycle of the susceptible cultivar contributed significantly higher levels of resting spores to the soil in a greenhouse test than did a cycle of the resistant cultivar, as assessed by quantitative PCR and microscope analysis.     

百菌清防治大白菜根肿病应用技术研究

直播大白菜于播种后即用百菌清1000倍液每穴300mL药液淋施．移栽大白菜于移栽时即用百菌清1000倍液每株300mL药液灌根1次．2种防治技术均对根肿病有显著的控病保产效果，对大白菜根肿病根瘤的抑制率分别达86．2％和71．7％。     

不同杀菌剂对油菜根肿病的防效及对油菜产量损失的影响

本研究使用5种杀菌剂和1种微肥于油菜苗期2~3片真叶期进行灌根处理,以传统的石灰处理方法比较,评价不同处理对油菜根肿病的防治效果和对油菜产量损失的影响。5种杀菌剂处理、微肥以及石灰处理均对油菜根肿病有较好的防效,其中50%多菌灵可湿性粉剂防效达90.82%;从产量损失方面比较,10%氰霜唑悬浮剂、50%多菌灵可湿性粉剂,75%百菌清可湿性粉剂和60%硫磺·敌磺钠可湿性粉剂处理可显著减少根肿病引起的产量损失;50%氟啶胺悬浮剂处理较对照增产不显著,而且对油菜生长有抑制作用;油菜根肿病的病情指数与油菜产量呈显著的负相关性,相关系数r=-0.796 2,油菜产量(y)与病情指数(x)的回归方程为y=-32.65x+2 372。     

芸薹根肿菌（Plasmodiophora brassicae）单孢分离接种及生理小种的鉴定

芸薹根肿菌(Plasmodiophora brassicae)单孢分离接种方法曾有报道,本试验改进了国外单孢分离技术.将休眠孢子悬浮液稀释到1个/0.5 μL,滴在一块消过毒的载玻片上,放置在显微镜下反复检查,以确保它精确只含有一个孢子.用微管将孢子吸入接种在2日龄白菜幼苗根毛上,营养液培养3周.为使其更接近田间发病环境条件,后期将疑似发病根在无菌土中继续进行病菌增繁.该方法能够有效地加快病菌繁殖感染,提高单孢分离接种成功率.对不同地区病菌进行大量单孢分离接种.本试验对3个地区的病菌单孢接种共950株,接种成功27株,使用Williams根肿病菌鉴别寄主鉴定,共9个生理小种.     

Dissemination of Plasmodiophora brassicae in livestock manure detected by qPCR

The present study was performed to investigate whether Plasmodiophora brassicae can be disseminated by livestock manure. A quantitative PCR (qPCR) assay was developed and used to detect and quantify P. brassicae in manure samples from naturally and artificially infested chickens and pigs. In naturally infested manure, quantifiable levels of infestation were observed in 7 out of the 28 samples, ranging from 10³ to 10⁷ resting spores per g of manure. The vast majority of the resting spores (76–91%) were viable, as determined by a dual fluorescence viability assay. Clubroot symptoms developed on plants inoculated with P. brassicae resting spores isolated from all seven qPCR‐positive samples. Artificially infested manure samples were produced by feeding chickens and pigs on P. brassicae‐contaminated feed. The levels of infestation were 10³ resting spores per g of manure for both chicken and pig manure sampled 24 and 48 h after feeding, respectively. Spore viability was >80% for both samples, and the disease severity indices were both >40 as indicated by bioassay. This showed that resting spores can survive the digestive tracts of chickens and pigs, and retain strong pathogenicity. The findings indicate that manure dissemination is possible for P. brassicae. Farmers should avoid feeding livestock on P. brassicae‐contaminated feed or applying infested manure as fertilizer on land intended for crucifer crops.     

江西油菜根肿菌生理小种鉴定及品种抗病性鉴定

油菜根肿病在江西省多市均有分布, 尤以上饶婺源、吉安永新、九江瑞昌等地发生严重。为明确江西省油菜主产区根肿菌的生理小种, 采用SCD鉴定系统对江西油菜主产区根肿菌进行了生理小种鉴定, 结果共鉴定出3个生理小种类型:Pb1、Pb5和Pb12。为筛选出适于江西根肿病发病地区种植的油菜材料, 开展了油菜材料对根肿菌的抗性测定。室内盆栽抗性测定表明, 育种中间材料‘P360429001’‘2018362481’‘P360924009’‘P360281040’‘中油杂12’‘华油杂62R’和‘华双5R’的病情指数均低于或等于30.00, 其中‘华油杂62R’和‘华双5R’表现为中抗水平。田间抗性测定结果表明, ‘华油杂62R’‘秦优DK4’‘阳光50’和育种中间材料‘P360429001’‘ZS115R’的根肿病病情指数均低于30.00, 但只有‘华油杂62R’对Pb1生理小种表现出中抗水平。本研究针对性地筛选出适于江西油菜根肿病发病地区种植的油菜材料, 对江西油菜根肿病的防控和创制抗病性油菜资源提供了科学依据。     

Flooding affects the development of Plasmodiophora brassicae in Arabidopsis roots during the secondary phase of infection

Clubroot, a disease of Brassicaceae species, is caused by the soilborne pathogen Plasmodiophora brassicae. High soil water content was previously described to favour the motility of zoospores and their penetration into root cells. In this study, the effect of irrigation regimes on clubroot development during the post‐invasive secondary phase of infection was investigated. Three irrigation regimes (low, standard, high) were tested on two Arabidopsis accessions, Col‐0 (susceptible) and Bur‐0, a partially resistant line. In Col‐0, clubroot symptoms and resting spore content were higher under the ‘low irrigation’ regime than the other two regimes, thus enhancing the phenotypic contrast between the two Arabidopsis accessions. Clubroot severity under high and low irrigation regimes was evaluated in near‐isogenic lines derived from a Col‐0 ×  Bur‐0 cross, to assess the effect of soil moisture on the expression of each of four quantitative trait loci (QTL) controlling partial resistance. The presence of the Bur‐0 allele at the QTL PbAt5.2 resulted in reduced severity only under low irrigation, whereas the Bur‐0 allele at QTL PbAt5.1 was associated with partial resistance only under high irrigation. QTL PbAt4 reduced the number of resting spores in infected roots, but was not associated with reduced clubroot symptoms. The results indicated that soil moisture could have consequences for the secondary phase of clubroot development, depending on plant genotype. Future genetic studies may benefit from using combinations of watering conditions during the secondary stage of infection, thus opening up the possibility of identifying genetic factors expressed under specific environmental conditions.     

氟唑活化酯诱导大白菜抗根肿病的研究

本文研究了我国具有自主知识产权的新型化合物氟唑活化酯诱导大白菜对根肿病的抗病效果及诱导技术。荧光染色观察发现该化合物对根肿菌休眠孢子没有明显的杀死作用,但具有明显的诱导抗病效果。不同的诱导浓度、诱导间隔期和诱导次数对根肿病的诱抗效果均不同。温室盆栽试验结果显示,25 mg/L的氟唑活化酯间隔7 d诱导大白菜3次,对根肿病的防病效果最好,达到71.33%,与诱抗剂苯并噻二唑效果相当,显著高于杀菌药剂50%氟啶胺悬浮剂拌土的防效(63.32%)。田间从苗期开始用氟唑活化酯25 mg/L诱导大白菜3次,诱导间隔期为7 d,对根肿病的诱抗效果最好达69.99%±2.01%,略低于对照杀菌剂氟啶胺拌土施药。本文明确了新型诱抗剂氟唑活化酯诱导大白菜对根肿病的抗病效果,并系统研究了施药技术,为白菜根肿病的防治提供了新手段。     

Variation in pathotypes and virulence of Plasmodiophora brassicae populations in Germany

Between 2012 and 2015, 49 new clubroot‐infested fields were identified in 12 German federal states. Clubroot disease incidence varied within these fields from 22% to 92%. Field information revealed that in 85% of fields, oilseed rape was grown in rotation once every 2 or 3 years. Frequency of OSR in the rotation was significantly correlated with the incidence and prevalence of clubroot disease. The disease was detected in fields with soil pH ranging from 5.1 to 8.3, and a significant negative correlation was found between soil pH and the disease incidence of infested fields. Furthermore, more cases of disease and severe incidences were observed in sandy loam and loamy sand as compared with other soil types. Pathotype classification of the 49 Plasmodiophora brassicae populations was conducted on two differential sets, the European Clubroot Differential set and the set of Somé (1996). Additionally, the degree of virulence of the collected isolates was analysed on the clubroot‐resistant oilseed rape cv. Mendel. The results showed variation in pathotype distribution in different regions in Germany. The majority of isolates according to Somé were pathotypes 1 and 3, respectively, with pathotypes 2 and 5 in the minority. Detailed classification according to Buczacki showed the dominance of 16/31/31, 16/14/30 and 16/14/31 populations among 20 distinct virulence patterns of collected isolates. From all populations tested for virulence on cv. Mendel, 15 isolates were found to be moderately or highly virulent. These virulent populations were not restricted to a small geographical area in the country.     

Studies into primary and secondary infection processes by Plasmodiophora brassicae on canola

The early stages of infection of canola roots by the clubroot pathogen Plasmodiophora brassicae were investigated. Inoculation with 1 × 10⁵ resting spores mL^?1 resulted in primary (root hair) infection at 12 h after inoculation (hai). Secondary (cortical) infection began to be observed at 72 hai. When inoculated onto plants at a concentration of 1 × 10⁴ mL^?1, secondary zoospores produced primary infections similar to those obtained with resting spores at a concentration of 1 × 10⁵ mL^?1. Secondary zoospores caused secondary infections earlier than resting spores. When the plants were inoculated with 1 × 10⁷ resting spores mL^?1, 2 days after being challenged with 1 × 10⁴ or 1 × 10⁵ resting spores mL^?1, secondary infections were observed on the very next day, which was earlier than the secondary infections resulting from inoculation with 1 × 10⁷ resting spores mL^?1 alone and more severe than those produced by inoculation with 1 × 10⁴ or 1 × 10⁵ resting spores mL^?1 alone. Compared with the single inoculations, secondary infections on plants that had received both inoculations remained at higher levels throughout a 7‐day time course. These data indicate that primary zoospores can directly cause secondary infection when the host is under primary infection, helping to understand the relationship and relative importance of the two infection stages of P. brassicae.     

Clubroot disease in Latin America: distribution and management strategies

Clubroot caused by Plasmodiophora brassicae is an important disease of cruciferous crops worldwide. In Latin America (from Mexico to Chile, including the Caribbean), most of the area in cruciferous crops is devoted to oilseed rape (Brassica napus; c. 230 600 ha) in Brazil, Chile, Paraguay, Uruguay and Argentina, while cruciferous vegetables such as cabbage, cauliflower, broccoli and Brussels sprouts (40 900 ha) are cropped intensively on small acreages across the region. Although clubroot is present in most Latin American countries, there have been very few studies of P. brassicae. Clubroot research in Latin America has focused mainly on adapting disease management strategies developed in temperate climates to tropical climates, including liming, biological control and genetic resistance. This review summarizes the management strategies used in Latin America to reduce the impact of clubroot, including novel strategies when compared with temperate regions, such as a crop rotation with aromatic plant species and the use of biological control with Trichoderma spp. Latin America has unique characteristics relative to temperate countries such as high humidity, warm temperatures and acidic soil that impact the interaction between P. brassicae and its plant hosts, so more research is required.     

Comparison of techniques for estimation of resting spores of Plasmodiophora brassicae in soil

Clubroot (Plasmodiophora brassicae) is an important disease of canola (Brassica napus) and other brassica crops. Accurate estimation of inoculum load in soil is important for evaluating producer risk in planting a susceptible crop, but also for evaluation of management practices such as crop rotation. This study compared five molecular techniques for estimating P. brassicae resting spores in soil: quantitative polymerase chain reaction (qPCR), competitive positive internal control PCR (CPIC-PCR), propidium monoazide PCR (PMA-PCR), droplet digital PCR (ddPCR) and loop-mediated isothermal DNA amplification (LAMP). For ddPCR and LAMP, calibrations were developed using spiked soil samples. The comparison was carried out using soil samples collected from a long-term rotation study at Normandin, Québec, with replicated plots representing 0-, 1-, 2-, 3-, 5- and 6-year breaks following susceptible canola infested with clubroot. CPIC-PCR and ddPCR provided repeatable estimates of resting spore numbers in soil compared with estimates from qPCR or LAMP alone. CPIC-PCR provided the most robust measurement of spore concentration, especially in the 2 years following a crop of susceptible canola, because it corrected for effects of PCR inhibitors. PMA-PCR demonstrated that a large proportion of the DNA of P. brassicae detected in soil after the susceptible canola crop was derived from spores that were immature or otherwise not viable. Each assay provided a similar pattern of spore concentration in soil, which supported the conclusion of a previous study at this site that resting spore numbers declined rapidly in the first 2 years after a susceptible crop, but much more slowly subsequently.     

Usefulness of nonhost plants in managing Plasmodiophora brassicae

Germination of resting spores of Plasmodiophora brassicae , causal agent of clubroot in crucifers, may be stimulated by certain nonhost plants. Without a host plant to infect, such germination would lead to a reduced persistence of resting spores in the soil. The effect of four nonhost plants on P. brassicae was investigated in a 3-year field experiment and a 14-month glasshouse experiment. Three of the plant species used, leek ( Allium porrum ), winter rye ( Secale cereale ) and perennial ryegrass ( Lolium perenne ), have been reported to stimulate resting spore germination, while the fourth, red clover ( Trifolium pratense ), does not. In the field experiment, none of the plant species reduced the concentration of P. brassicae in soils when tested with bioassay plants (Chinese cabbage, Brassica rapa var. pekinensis ). In the glasshouse experiment, there was a lower disease level in all plant treatments compared with the plant-free control following incorporation and decomposition of plant roots. At this time, pH in the soils with plant treatments was higher than that in the control soil. There were no indications of a species-specific interaction between any of the nonhost plants investigated and P. brassicae , and it cannot be concluded that any of them would be useful in the sanitation of P. brassicae -infested soils within short time periods.