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.     

Visual and PCR assessment of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) cultivars

Methods to assess light leaf spot ( Pyrenopeziza brassicae ) on winter oilseed rape cultivars were compared in laboratory, controlled-environment and field experiments. In controlled-environment experiments with seedling leaves inoculated at GS 1,4, the greatest differences in percentage area affected by P. brassicae sporulation were observed with inoculum concentrations of 4 × 10³ or 4 × 10⁴ spores mL⁻¹, rather than 4 × 10² or 4 × 10⁵ spores mL⁻¹, but older leaves had begun to senesce before assessment, particularly where they were severely affected by P. brassicae . In winter oilseed rape field experiments, a severe light leaf spot epidemic developed in 2002/03 (inoculated, September/October rainfall 127·2 mm) but not in 2003/04 (uninoculated, September/October rainfall 40·7 mm). In-plot assessments discriminated between cultivars best in February/March in 2003 and June in 2004, but sometimes failed to detect plots with many infected plants (e.g. March/April 2004). Ranking of cultivar resistance differed between seedling experiments done under controlled-environment conditions and field experiments. The sensitivity of detection of P. brassicae DNA extracted from culture was greater using the PCR primer pair PbITSF/PbITSR than using primers Pb1/Pb2. P. brassicae was detected by PCR (PbITS primers) in leaves from controlled-environment experiments immediately and up to 14 days after inoculation, and in leaves sampled from field experiments 2 months before detection by visual assessment.     

Antifungal activity of bergenin, a constituent of Flueggea microcarpa

The antifungal activity of bergenin against some plant pathogenic fungi, namely, Alternaria alternata , A. brassicae , A. carthami , Fusarium udum , F. oxysporum f.sp. ciceri , Curvularia lunata and Erysiphe pisi , was studied. Bergenin as its monosodium salt was effective against all the fungi and the effective dose for complete inhibition of spore germination varied from 15 μg mL⁻¹ for F. udum to 125 μg mL⁻¹ for E. pisi . Experiments on the effect of bergenin on powdery mildew development under glasshouse conditions revealed that it can control powdery mildew of pea at 2000 μg mL⁻¹ by postinoculation treatment, the results being comparable with those of carbendazim (1000 μg mL⁻¹) and wettable sulfur (2000 μg mL⁻¹). It affected hyphal elongation and the number of primary and secondary branches.     

The incorporation of pathogen spores into rain-splash droplets: a modelling approach

Simple, theoretical, physical principles and existing experimental data were used to derive an analytical model to describe the incorporation of plant pathogen spores into splash droplets. Data were obtained from experiments on splash dispersal of spores of Pseudocercosporella herpotrichoides (cereal eyespot), Pyrenopeziza brassicae (oilseed rape light leaf spot) and Septoria nodorum (wheat glume blotch). In these experiments, incident drops of diameter 4–5 mm were allowed to fall onto spore suspensions 0.5 mm deep with 1.2 × 10⁵ to 6.5 × 10⁵ spores/mL. The analytical model was constructed as the product of three functions of droplet diameter which described, respectively, the frequency distribution of droplet diameters, the proportion of droplets carrying spores and the mean number of spores in spore-carrying droplets in each diameter category. The frequency distribution of droplet sizes was described by a log-normal distribution, the proportion of droplets carrying spores was described by an exponential function and the adimensional spore concentration in spore-carrying droplets was described by a power law. The cumulative proportions of spores in droplets in diameter categories of increasing diameter were calculated to compare observed and fitted data.     

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.     

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。     

Berry infection and the development of bunch rot in grapes caused by Aspergillus carbonarius

The effects of different levels of inoculum of Aspergillus carbonarius and time of inoculation on berry infection and the development of aspergillus bunch rot on grapevines (cv. Sultana) were studied under field conditions. Inflorescences at full bloom were inoculated with aqueous spore suspensions of A. carbonarius containing 0 or 1 × 10⁶ spores mL⁻¹ in 2004/05 and 0, 1 × 10² or 1 × 10⁵ spores mL⁻¹ in 2005/06. In both years, the incidence of infection in inoculated berries was significantly higher than in uninoculated berries. Incidence of infection in berries from veraison until harvest was higher than at earlier stages of bunch development (berry set to berries that were still hard and green). Inoculation of bunches at veraison did not significantly increase A. carbonarius infection prior to harvest, at harvest, 6 days after harvest or when berries were over-ripe. Bunches inoculated at harvest did not significantly increase infection 6 days after harvest or when berries were over-ripe. Aspergillus carbonarius was isolated more frequently from the pedicel end (53·1%) than from the middle section (37·5%) and distal end (35·0%) of berries that were inoculated with 10⁵ spores mL⁻¹.     

Suppression of clubroot (Plasmodiophora brassicae) development in Arabidopsis thaliana by the endophytic fungus Acremonium alternatum

This study investigated the ability of an endophytic fungus Acremonium alternatum to reduce clubroot formation in the model plant Arabidopsis thaliana, which is highly susceptible to Plasmodiophora brassicae . Quantitative PCR demonstrated that A. alternatum colonized the P. brassicae -infected roots and shoots of the host plant. When Arabidopsis plants were co-inoculated with P. brassicae and A. alternatum , gall formation was reduced as shown by the reduction of the disease index (DI) by up to 50% compared to plants only infected with P. brassicae, whereas the infection rate was lowered by about 20% only in several, but not all, experiments. Clubroot was similarly suppressed when plants were inoculated with autoclaved A. alternatum spores or spore extracts, showing that viable spores were not needed. However, A. alternatum spores did not inhibit P. brassicae resting spore germination. Compared to the normal root galls, the smaller root galls on A. alternatum -inoculated plants contained fewer resting spores of the clubroot pathogen. It was thus hypothesized that inoculation with A. alternatum delayed the development of P. brassicae . Using quantitative RT-PCR to monitor the expression of P. brassicae genes differentially expressed during the development of the disease, a delayed pathogen development was corroborated. Furthermore, greenhouse experiments identified a time window in which the endophyte had to be administered, where the latest effective time point was 5 days before inoculation with P. brassicae and the optimum treatment was to administer A. alternatum and P. brassicae at the same time. These results indicate that A. alternatum and perhaps similar endophytes could be useful for the management of clubroot disease.     

Control of soilborne clubroot disease of cruciferous plants by epoxydon from Phoma glomerata

Culture broth from an isolate of Phoma glomerata (no. 324, = JCM 9972) from the leaves of Viola sp., controlled the soilborne pathogen Plasmodiophora brassicae which causes clubroot disease of cruciferous plants. This effect was caused by epoxydon (5-hydroxy-3-(hydroxymethyl)-7-oxabicyclo[4.1.0]hept-3-en-2-one). Although this substance was known to have antitumour activity, phytotoxicity and antiauxin activity, no plant disease reduction had been reported previously. Epoxydon possessed neither strong antimicrobial activity nor did it induce acquired resistance. It protected crucifers from clubroot disease at 250  μ g mL⁻¹ following addition to the soil. Several antiauxins were tested for similar properties resulting in the suppression of clubroot disease and one, 2,3,5-triiodobenzoic acid, was effective at 10  μ g mL⁻¹. Clubroot reduction by epoxydon may result from antiauxin activity. This opens opportunities for a new group of agrochemicals.     

A qualitative quick-test for detection of herbicide resistance to tribenuron-methyl in Papaver rhoeas

A qualitative seed-based method useful for the detection of resistance to the herbicide tribenuron-methyl in Papaver rhoeas L. is described. Seeds were germinated on 35 mL of a 1.3% agar medium containing 2 g KNO₃ L^–1 in 8.5 cm Petri dishes in a growth chamber under 20 μmol s^–1 m^–2 of fluorescent light. When 0.24 μM tribenuron-methyl or more was added, growth in susceptible plants stopped after the cotyledon stage and they turned chlorotic. The resistant plants continued developing new leaves. The same effect was achieved when 0.2 g gibberellin (GA₃) L^–1 and 7.68 μM tribenuron-methyl or 0.5 g GA₃ L^–1 and 61.44 μM tribenuron-methyl were added. Germination percentage rose with gibberellin in the presence or absence of the herbicide. Plants developed rapidly, with only about 14 d needed to finish the test but sometimes root growth was reduced because of the addition of gibberellin. In the absence of gibberellin but in the presence of the herbicide, plants grew more slowly and developed smaller leaves with a 17-d evaluation period requirement. The test was validated with pot experiments in a greenhouse and also with field trials. The best combination was found to be 0.2 g GA₃ L^–1 and 7.68 μM tribenuron-methyl, assuring homogenous germination and testing of dormant seeds but avoiding root inhibition associated with too much gibberellin.     

Control of Penicillium expansum by plant volatile compounds

Nine plant volatiles were tested for their activity in vitro and in vivo against Penicillium expansum , the cause of blue mould of pear. In vitro spore germination and mycelial growth assay showed a consistent fungicidal activity by trans -2-hexenal, carvacrol, trans -cinnamaldehyde and citral, while hexanal (-)- carvone, p -anisaldehyde, eugenol and 2-nonanone exhibited a progressively lower inhibition. trans -2-Hexenal was the best inhibitor of conidial germination [MIC (minimum inhibitory concentration) = 24·6  µ L L⁻¹; ED₅₀ = 10·2  µ L L⁻¹], while carvacrol was the best inhibitor of mycelial growth (MIC = 24·6  µ L L⁻¹; ED₅₀ = 9  µ L L⁻¹). The four most active compounds in in vitro studies were tested in vivo as fumigants against blue mould on pear cv. Conference. Best control was achieved by trans -2-hexenal vapour treatments (12·5  µ L L⁻¹) when applied over a 24-h period, beginning 24 h after inoculation. In contrast, carvacrol (12·5–200  µ L L⁻¹), and trans -cinnamaldehyde (50–400  µ L L⁻¹) were ineffective and citral (200  µ L L⁻¹) showed only slight effect.     

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.     

Real-time PCR assay for quantification of Tilletia caries contamination of UK wheat seed

A quantitative real-time PCR assay using TaqMan chemistry has been developed to quantify the level of Tilletia spp. contamination in wheat-seed lots. In the UK wheat seed is predominantly contaminated with Tilletia caries (syn. Tilletia tritici ), and the probability of detecting other Tilletia spp. is negligible. DNA standards, prepared from T. caries spores, were calibrated using a set of 26 seed samples, with T. caries contamination levels ranging from 0 to 1000 spores per seed. The linear calibration model obtained by the regression of log₁₀ (number of spores per seed + 1) on mean log₁₀ DNA ( µ g) produced a coefficient of determination ( R ²) of 0·904. The calibration model was tested using 226 seed samples; of these, 91% fell within the 95% confidence intervals. Of the 21 samples that were outside the limits, 16 were overpredictions and five underpredictions. The five underpredictions were all from seed samples where contamination was less than one spore per seed. The model predicts that samples with 44 pg of DNA will be below one spore per seed with 95% probability. Of the 226 test samples compared with this threshold, 99 contained less than 44 pg DNA, and these were found to have less than one spore per seed by microscopic assay. This real-time assay allows an increase in test throughput and provides the sensitivity required for an advisory threshold of one spore per seed.     

Prestorage heat treatment reduces pathogenicity of Penicillium expansum in apple fruit

Penicillium expansum is one of the main postharvest pathogens of apples in Israel. Heating apple fruit inoculated with P. expansum for 96 h at 38°C completely inhibited decay development. Fruit held for 24 h at 42°C or 12 h at 46°C had significantly reduced decay after an additional 14 days incubation at 20°C, compared with unheated inoculated control fruit. Mycelial growth and percentage spore germination in vitro were inversely proportional to length of time of exposure to various temperatures. The ET₅₀ for spore germination was 42, 34 and 20 h at 38, 42 and 46°C, respectively, while the ET₅₀ for mycelial growth was 48, 44 and 36 h at those temperatures. When Penicillium spores were incubated on crude extract prepared from the peel of apple fruits held 4 days at 38°C, germ tube elongation was significantly reduced, while the walls of the tubes were thicker, compared with germ tubes from spores incubated on crude extract prepared from peel of non-heated fruit. The evidence presented here supports the hypothesis that the effect of heating on the decay of apples caused by P. expansum is not only the result of direct inhibition of fungal germination and growth by high temperature, but is also partly due to the formation of an inhibitory substance in the heated peel.     

Germination, emergence, and dormancy of Mimosa pudica

Mimosa pudica (common sensitive plant) is a problematic weed in many crops in tropical countries. Eight experiments were conducted to determine the effects of light, seed scarification, temperature, salt and osmotic stress, pH, burial depth, and rice residue on the germination, seedling emergence, and dormancy of M. pudica seeds. Scarification released the seeds from dormancy and stimulated germination, though the germination of the scarified seeds was not influenced by light. The scarification results indicate that a hard seed coat is the primary mechanism that restricts germination. The germination increased markedly with the exposure to high temperature "pretreatment" (e.g. 150°C), which was achieved by placing non-scarified seeds in an oven for 5 min followed by incubation at 35/25°C day/night temperatures for 14 days. The germination of the scarified seeds was tolerant of salt and osmotic stress, as some seeds germinated even at 250 mmol L⁻¹ NaCl (23%) and at an osmotic potential of −0.8 MPa (5%). The germination of the scarified seeds was >74% over a pH range of 5–10. The seedling emergence of the scarified seeds was 73–88% at depths of 0–2 cm and it gradually decreased with an increasing depth, with no seedling emergence at the 8 cm depth. The rice residue applied to the soil surface at rates of ≤6 t ha⁻¹ did not influence the seedling emergence and dry weight. The information gained from this study identifies some of the factors that facilitate M. pudica becoming a widespread weed in the humid tropics and might help in developing components of integrated weed management practises to control this weed.     

Growth and movement of secondary plasmodia of Plasmodiophora brassicae in turnip suspension-culture cells

Growth of secondary plasmodia of the clubroot pathogen Plasmodiophora brassicae was studied in dual culture of P. brassicae and turnip suspension cells. Suspension culture of P. brassicae -infected turnip cells was achieved by using P. brassicae -infected callus in Murashige and Skoog medium supplemented with 0·1 mg 2,4-D L⁻¹ and 0·02 mg kinetin L⁻¹. The shape of secondary plasmodia in suspension cells was spherical-to-subspherical. A few young plasmodia divided and became numerous spherical, small plasmodia which eventually formed a plasmodial cluster. The plasmodia fused and became vegetative plasmodia. Infected cells were significantly larger than noninfected cells. Secondary plasmodia moved within transformed turnip suspension host cells by cytoplasmic streaming of the host cells. Secondary plasmodia divided in synchrony with the transformed turnip cells.     

Effects of calcium on biocontrol activity of yeast antagonists against the postharvest fungal pathogen Rhizopus stolonifer

Calcium chloride (2% w/v) significantly inhibited the growth of the pathogen Rhizopus stolonifer , but did not affect the colony-forming units (CFU) of yeasts Candida guilliermondii and Pichia membranefaciens in potato dextrose broth. The concentration of yeast suspension influenced spore germination and germ tube growth of R. stolonifer in vitro, as well as disease incidence and lesion development in fruits. There were significant negative relationships between the suspension concentrations of the yeasts and the growth as well as infectivity of the pathogen. The addition of calcium resulted in lower spore germination rates and slower growth of germ tubes in vitro , as well as in lower disease incidences and smaller lesion diameters compared with treatments with yeast antagonists alone. When yeast cell suspensions reached a concentration of 5 × 10⁸ CFU mL⁻¹, growth of the pathogen was completely limited in vitro , and no infection was found in peach and nectarine fruits treated with or without calcium.     

Effect of inoculum type and timing of application of Coniothyrium minitans on Sclerotinia sclerotiorum: influence on apothecial production

The effects of different inocula of the mycoparasite Coniothyrium minitans on carpogenic germination of sclerotia of Sclerotinia sclerotiorum at different times of year were assessed. A series of three glasshouse box bioassays was used to compare the effect of five spore-suspension inocula of C. minitans , including three different isolates (Conio, IVT1 and Contans), with a standard maizemeal–perlite inoculum. Apothecial production, as well as viability and C. minitans infection of S. sclerotiorum sclerotia buried in treated soil, were assessed. Maizemeal–perlite inoculum at 10⁷ CFU per cm³ soil reduced sclerotial germination and apothecial production in all three box bioassays, decreasing sclerotial recovery and viability in the second bioassay and increasing C. minitans infection of sclerotia in the first bioassay. Spore-suspension inocula applied at a lower concentration (10⁴ CFU per cm³ soil) were inconsistent in their effects on sclerotial germination in the three box bioassays. Temperature was an important factor influencing apothecial production. Sclerotial germination was delayed or inhibited when bioassays were made in the summer. High temperatures also inhibited infection of sclerotia by C. minitans . Coniothyrium minitans survived these high temperatures, however, and infected the sclerotia once the temperature decreased to a lower level. Inoculum level of C. minitans was an important factor in reducing apothecial production by sclerotia. The effects of temperature on both carpogenic germination of sclerotia and parasitism of sclerotia by C. minitans are discussed.     

Identification of genes expressed during spore germination of Mycosphaerella pinodes

Mycosphaerella blight, caused by Mycosphaerella pinodes, is one of the major diseases of cultivated pea (Pisum sativum L.). To isolate the genes that are up- and down-regulated during spore germination, suppression subtraction hybridization (SSH) was performed between ungerminated and germinated spores. The 232 and 128 clones from forward and reverse libraries, respectively, were collected, sequenced, and analyzed with a BLASTX homology search. About 95% of the 32 selected clones were expressed during spore germination on a paper sheet and during infection of pea leaves. We discuss the applicability of the SSH libraries for analyzing M. pinodes genes involved in the early stage of infection.     

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.