Biological Mode of Action of the Fungicide, Flusulfamide, Against Plasmodiophora brassicae (clubroot)

Flusulfamide (2, 4-dichloro-,,-trifluoro-4-nitro-m-toluenesulfonanilide) was investigated for its mode of action against Plasmodiophora brassicae Woronin. Seedlings of Chinese cabbage (Brassica rapa L. subsp. pekinensis) were grown for 14 and 21 days in soil infested with P. brassicae and then transplanted into soil containing flusulfamide (0.9µg a.i.g^–1 dry soil). Clubroot was not suppressed by this treatment, indicating that the fungicide is ineffective against P. brassicae established within cortical cells of the host root. Where seedlings were grown in soil infested with resting spores which had previously been treated with flusulfamide, root-hair infection and club formation were suppressed. This indicates that flusulfamide directly acts against resting spores. When placed in root exudates of Chinese cabbage, untreated resting spores germinated at a high frequency while flusulfamide-treated resting spores hardly germinated at all. Use of the Evan's blue staining assay indicated that flusulfamide-treated resting spores remained viable. Flusulfamide was detected by high performance liquid chromatography on resting spores treated with flusulfamide for 30min. This indicates that the chemical is adsorbed onto resting spores. These results suggest that flusulfamide suppresses clubroot disease by inhibiting germination of P. brassicae resting spores through adsorption onto their cell walls.     

Microscopical observations of the primary zoospores of Spongospora subterranea f.sp. subterranea

A solution culture test system with Spongospora subterranea f.sp. subterranea spore ball inoculum and tomato bait plants was used to create a pulse of primary zoospore production and subsequent host-root infection. Spore balls and zoospores were examined by light, fluorescence, and transmission and scanning electron microscopy. Most of the resting spores with a developing exit pore did not show any changes in cytoplasmic content typical of zoospore formation. A few empty resting spores and some with developing exit pores were also observed in the absence of host-root exudates. The average diameter of exit pores of empty resting spores was 1.5 μm and they were often encircled by a ring-like fusion of wall layers. Mature zoospores were never found inside resting spores. Primary and secondary zoospores are identical in morphology. The infection process is similar to that of other Plasmodiophoromycetes with internal 'Rohr'-like structures in encysted zoospores which were attached by an adhesorium to tomato root hairs. Post-infection papillae and uninucleate plasmodia were observed.     

Fluorescent staining of resting spores of Polymyxa betae as a fungal vector of rhizomania disease of sugar beet in soil

When the resting spores of Polymyxa betae were pretreated with 2% sodium dodecyl sulfate (SDS) and then stained with various fluorochromes including 3,3′-dihexyloxacarbocyanine iodide [DiOC6(3)], calcofluor, and a fluorescein isothiocyanate (FITC)-conjugated lectin, such as wheat germ lectin (WGA) or caster bean lectin, most spores fluoresced brightly. FITC-WGA mainly stained the cell surface, while DiOC6(3) stained the cytoplasm. After pretreatment with SDS and addition of FITC-WGA or DiOC6(3) to a soil suspension containing resting spores, the resting spores were distinguishable from the soil particles. This staining method is easy to use for the detection of resting spores in the soil.     

Effect of conditions during storage of infested soil on infection of bait plants by Polymyxa betae and beet necrotic yellow vein virus

Infectivity of resting spores ofPolymyxa betae in soil stored air-dry or moist was determined by assessing infection of bait plants that were exposed to the soil. Storage of soil under air-dry conditions at room temperature resulted in a delayed onset of germination of resting spores compared to germination in soil stored under moist and cool conditions, as inferred from the infection of the bait plants. Bait plants had to be exposed for more than 12 h to flooded infested soil before germination and infection had occurred. However, when soil was prewetted for 24 h before exposing bait plants, germination, infection and transmission of beet necrotic yellow vein virus (BNYVV) were accomplished within 12 h, but only with the moistly stored soil. When resting spores isolated from roots were stored for 4 and 8 weeks under dry conditions at 22°C, germination of viruliferous spores, as measured by detection of BNYVV in bait plants exposed for 48 h to the spores, was less than that of spores stored in moist soil at 22°C. Approximately 100% of bait plants were infected after exposure to resting spores that were frozen in demineralized water or stored cool (5°C) in water or moist soil for 42 weeks. Air-dry cool storage for 42 weeks resulted in a low percentage of infection. Storage conditions of soil influence the results of bioassays for detection of rhizomania when short baiting periods are applied, whereas differences in infectivity were not detected using a bioassay with long duration.     

Resting-spore germination inSynchytrium endobioticum

The resting spores ofS. endobioticum function as a prosorus in germination. The contents of the resting spore are extruded as a vesicle. Within this vesicle one single sporangium develops. Germinating resting spores resemble resting spores which have been parasitized by certain fungi, but it is obvious from several facts presented that no parasites were present. On the basis of the mode of germination described,S. endobioticum should be transferred from the subgenusMesochytrium to the subgenusMicrosynchytrium.     

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.     

Germinate to exterminate: chemical stimulation of Spongospora subterranea resting spore germination and its potential to diminish soil inoculum

Hoagland's solution (HS), a defined nutrient supplement for plants, has been previously reported to stimulate zoospore release from resting spores of the potato pathogen Spongospora subterranea f. sp. subterranea. This study obtained direct empirical evidence for an increase in zoospore release with HS treatment, and identified Fe‐EDTA as the stimulant component of HS. Stimulation of resting spores by HS and Fe‐EDTA resulted in greater and earlier zoospore release compared to a distilled water control, and in the presence of a susceptible tomato host plant resulted in enhanced root infection. Given the labile nature of S. subterranea zoospores, it was postulated that stimulation of premature release of zoospores from the dormant resting spores in absence of susceptible hosts could reduce soil inoculum levels. In two glasshouse trials in the absence of host plants, both Fe‐EDTA and HS soil treatments reduced S. subterranea soil inoculum levels, providing proof of concept for the ‘germinate to exterminate’ approach to inoculum management.     

Methoxyfenozide and pyriproxifen alter the cellular immune reactions of Eurygaster integriceps Puton (Hemiptera: Scutelleridae) against Beauveria bassiana

Effects of the two insect growth regulators (IGRs) methoxyfenozide, 20-hydroxyecdysone (20E) agonist, and pyriproxifen, Juvenile hormone (JH) agonist, were examined on the cellular immune responses of the Sunn pest, Eurygaster integriceps versus the entomopathogenic fungus Beauveria bassiana. The simultaneous treatment with the IGRs and the fungal spores altered haemocyte count (total and differentiate), nodulation response and phenoloxidase (PO) activity in a dose- and time-dependent manner. It was observed that different concentrations of methoxyfenozide increased total and differentiate haemocyte numbers as well as B. bassiana-induced nodulation response. In contrast with the JH agonist, pyriproxifen significantly decreased total and differentiate haemocyte numbers and inhibited nodule formation in E. integriceps adults. The 20E agonist displayed major effects when injected at the doses 2.79 and 5.59 μg/mg adult. In contrast, injecting adults by pyriproxifen significantly impaired their ability to raise an efficacious response against the fungal spores. The ability of the two IGR analogues to interfere with activity of the PO system in haemolymph of E. integriceps adults was also investigated 6 h after injection by fungal spores. Methoxyfenozide had an excitatory effect on PO activity when the 5.59 μg/mg concentration was used against adults. Conversely, pyriproxifen had an inhibitory effect on PO activity when used at 1.49 μg/mg adult concentration. These findings demonstrate that pyriproxifen may interfere with cell-mediated immunity of E. integriceps. So, pyriproxifen could be a good candidate for the integrated control of the Sunn pest.     

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.     

Surface Disinfestation of Resting Spores of Plasmodiophora brassicae Used to Infect Hairy Roots of Brassica spp

ABSTRACT Resting spores of Plasmodiophora brassicae were surface-disinfested by treatment with 2% chloramine-T for 20 min and then with an antibiotic solution (1,000 ppm of colistin sulfate, 1,000 ppm of vancomycin hydrochloride, and 6,000 ppm of cefotaxime sodium) for 1 day. The disinfested resting spores were used to inoculate hairy roots of cabbage (Brassica oleracea var. capitata cv. Fuji Wase), Chinese cabbage (B. pekinensis cv. Musou Hakusai), turnip (B. rapa var. rapifera cv. Wase Okabu), and rape (B. napus line Dc 119). Differences among hosts in susceptibility to clubroot in hairy roots were evident. Chinese cabbage and turnip hairy roots supported the highest percentages of root hair infection (53.3 to 80%) and the greatest production of zoosporangial groups (8.5 to 32.5 per root). Moreover, gall formation was observed only on Chinese cabbage and turnip hairy roots. The morphology of zoo-sporangia, plasmodia, and resting spores in diseased hairy roots was found to be identical to that in infected intact plants by both light and scanning electron microscopy. Pathogenicity tests confirmed the infectivity of resting spores produced in hairy roots. Thus, the hairy root culture technique should prove useful as a dual culture system for P. brassicae.     

Synchytrium endobioticum – risk from biogas plants?

Synchytrium endobioticum is one of the most important pathogens of potato and is known for its persistent propagation structures. Under favourable conditions, infection of highly susceptible potato cultivars leads to clearly visible cauliflower‐like tissue warts, the typical symptom of potato wart disease. However, unfavourable infection conditions or low infection pressure may result in symptoms being overlooked. Thus, the introduction of pathogen structures into stages of the processing industry cannot be ruled out. As the amounts of processed potato products continue to rise, phytosanitary risks from processing discarded potatoes and potato waste in biogas plants have to be considered. Hence, the resilience of resting spores against mesophilic anaerobic digestion was analysed in stirred tank reactors. Laboratory‐scale results show that S. endobioticum not only withstands mesophilic anaerobic digestion but also subsequent storage of the digestate for at least 4 weeks. Large numbers of viable resting spores were detectable by microscopic assessment in all samples. Viability was proved and verified additionally by bioassay. Consequently, potatoes, potato waste and processing water from potato processing industries used in biogas plants pose a phytosanitary risk if the accruing digestates are returned as fertilizer to arable land.     

Effects of growing leafy daikon (Raphanus sativus) on populations of Plasmodiophora brassicae (clubroot)

Control of some soilborne pathogens may be achieved by use of decoy or catch crops. These stimulate the germination of resting spores, resulting in limited expression of disease symptoms. Results achieved using this approach are reported here using leafy daikon (radish, Raphanus sativus var. longipinnatus ) for control of Plasmodiophora brassicae , the cause of clubroot disease of Brassicaceae. Disease indices of Chinese cabbage plants grown in pots that had previously contained leafy daikon were lower compared with pots where no plants had been grown before (control pots). Numbers of resting spores of P. brassicae in soil in pots after cultivation with leafy daikon were reduced by 71% compared with control pots when resting spores were recovered and counted directly. In a field experiment, numbers of resting spores were reduced by 94% compared with the start of the experiment when leafy daikon was grown in advance of Chinese cabbage, but there was no reduction in disease severity in the Chinese cabbage. Plasmodiophora brassicae infected the root hairs of leafy daikon and those of Chinese cabbage, but no clubs were found on leafy daikon roots. The results from pot trials indicate that leafy daikon may be useful as a decoy crop for the control of clubroot disease in field crops.     

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.     

Effect of sugar beet cultivars with different levels of resistance to beet necrotic yellow vein virus on transmission of virus byPolymyxa betae

The effect of resistance of sugar beet cultivars to beet necrotic yellow vein virus (BNYVV) on virus content of resting spore clusters of the vectorPolymyxa betae was studied in controlled environments and in naturally infested fields. The total number of resting spore clusters formed in roots of a partially resistant and a susceptible cultivar did not differ when assessed 6 and 12 weeks after inoculation with viruliferous resting spores. Transmission experiments showed that in partially resistant plants, having a low virus content in the roots, the population of resting spores formed was less viruliferous than that in susceptible plants with a high virus content. Consequently, growing a resistant cultivar can be expected to delay the build-up of virus inoculum in soil.In a trial field sampled in 1991, the inoculum potential of BNYVV (most probable number of viruliferousP. betae propagules) in soil was lower after growing a partially resistant cultivar than after growing a susceptible one. On the other hand, in four sites sampled in 1990, inoculum potential in soil was hardly increased by growing sugar beet and was not significantly affected by the cultivar grown.     

Susceptibility of hairy root lines of Brassica species to Plasmodiophora brassicae and in an in vitro subculture system

To investigate the susceptibility of hairy root lines of Brassica species to Plasmodiophora brassicae, hairy roots were induced in a number of Brassica species with Agrobacterium rhizogenes. Turnip hairy root was highly susceptible to P. brassicae; infection rates were high and large galls formed. In contrast, the rates of root hair infection and gall formation on intact Brassica plants did not differ significantly from the control. To induce resting spore formation, turnip hairy roots were incubated at 15°, 20°, or 25°C after 3 weeks of incubation at 25°C. The number and fresh mass of the galls per hairy root were higher and formation of resting spores was greatest after a 7-week incubation at 20°C. To subculture P. brassicae using turnip hairy root, turnip hairy roots were reinoculated with resting spores and gall with resting spores then formed on the hairy roots. In this way, P. brassicae using hairy roots could be subcultured in vitro two or three times on three single-spore isolates of P. brassicae. This is the first report of in vitro subculture of P. brassicae using hairy root.     

我国禾谷多粘菌地理分布和生理分化研究

 调查我国26个省、市、自治区,93个县市,183个取样点,1 373个大、小麦根样本。根据休眠孢子出现频率(有休眠孢子样本数/总样本数)和每克麦根休眠孢子数,将我国禾谷多粘菌的分布区分为3个,多菌区包括江、浙、沪、皖、赣、湘、鄂和鲁的大部分和川、陕小部分,冀个别地区;少菌区包括豫大部和晋、甘小部或个别地区;其余地区为无菌区。分布区主要在长江、黄河和淮河中下游流域。禾谷多粘菌的分布范围比大麦黄花叶病等3种病毒病的分布更广泛。本研究还探讨了我国各地禾谷多粘菌的分离物存在生理分化现象     

Direct evidence of surface infestation of seeds and tubers by Plasmodiophora brassicae and quantification of spore loads

Using quantitative PCR, DNA of Plasmodiophora brassicae, the causal agent of clubroot, was detected and quantified on canola, pea and wheat seeds, as well as on potato tubers, all harvested from clubroot‐infested fields in Alberta, Canada. Quantifiable levels of infestation were found on seven of the 46 samples analysed, and ranged from <1·0 × 10³ to 3·4 × 10⁴ resting spores per 10 g seeds; the vast majority (80–100%) of resting spores on these samples were viable, as determined by Evan’s blue vital staining. However, the levels of infestation found were generally lower than that required to cause consistent clubroot symptoms in greenhouse plant bioassays. While the occurrence of P. brassicae resting spores on seeds and tubers harvested from clubroot‐infested fields suggests that seedborne dissemination of this pathogen is possible, practices such as commercial seed cleaning may be sufficient to effectively mitigate this risk.     

枯草芽孢杆菌BS80-6对苹果采后炭疽病的控病效果及作用机制

为提高枯草芽孢杆菌的生防效果,通过离子束诱变筛选出BS80-6菌株.利用生物测定方法研究BS80-6菌株及不同处理方法在不同贮藏温度下对采后苹果炭疽病的控制效果和作用机制.结果表明,BS80-6能显著提高对苹果炭疽菌的抑制作用.枯草芽孢杆菌BS80-6对苹果果实炭疽病的防治效果以活菌液效果最好,菌丝生长抑制率为80.14%,孢子萌发抑制率为98.65%,控病效果为60.34%;灭菌液和过滤液对苹果炭疽菌也有一定的抑制效果.贮藏温度明显影响BS80-6对苹果炭疽病的防治效果;接种方式对控病效果影响显著,先接拮抗菌再接病菌的防效好于先接病菌再接拮抗菌.     

Effect of temperature on cortical infection by Plasmodiophora brassicae and clubroot severity

A study was conducted to assess the effect of temperature on infection and development of Plasmodiophora brassicae in the root cortex of Shanghai pak choy (Brassica rapa subsp. chinensis) and on subsequent clubroot severity. Ten-day-old seedlings were grown individually, 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 8 days after inoculation (DAI) and continuing until 42 DAI. Roots were assessed at 4-day intervals for the incidence of cortical infection and stage of infection (young plasmodia, mature plasmodia, and resting spores), at 2-day intervals for symptom development and clubroot severity, and at 8-day intervals for the number of spores per gram of gall. Temperature affected every stage of clubroot development. Cortical infection was highest and symptoms were observed earliest at 25?C, intermediate at 20 and 30?C, and lowest and latest at 15?C. No cortical infection or symptoms were observed at 42 DAI in plants grown at 10?C. A substantial delay in the development of the pathogen was observed at 15?C. Resting spores were first observed at 38 DAI in plants at 15?C, 26 DAI at 20 and 30?C, and 22 DAI at 25?C. The yield of resting spores from galls was higher in galls that developed at 20 to 30?C than those that developed at 15?C over 42 days of assessment. These results support the observation in companion studies that cool temperatures result in slower development of clubroot symptoms in brassica crops, and demonstrate that the temperature has a consistent pattern of effect throughout the life cycle of the pathogen.     

Production and characterization of single-spore isolates of Plasmodiophora brassicae

An improved procedure for inoculating Brassica seedlings with single resting spores of Plasmo-diophora brassicae is described. Using this procedure, eight out of a total of 600 seedlings inoculated with single spores developed severe symptoms of clubroot disease. Seven isolates of P. brassicae derived from single spores were characterized using the European Clubroot Differential (ECD) series. Four distinct pathotypes were obtained. The original populations of P. brassicae were shown t o be heterogeneous; this supports the findings of other workers. The isolates have been used to establish a reference collection of single-spore isolates of P. brassicae of known pathotype for use in Brassica breeding.