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.     

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.     

Effect of temperature and host genotype on the production of inoculum by Phytophthora fragariae var. fragariae from the roots of infected strawberry plants

A bioassay was used to monitor the release of inoculum in drainage water from strawberry plants inoculated with zoospores of Phytophthora fragariae var. fragariae. The fungus was detected in drainage water from plants that had been held at temperatures between 2 and 20 C. but not from plants held at 26°C. The lag phase before secondary inoculum was first released, the maximum and total amounts of inoculum released, and the length of time over which inoculum was released were all greater at the lower temperature regimes, especially those below 10 C. The results were consistent with observations on the effect of temperature on zoospore production from agar discs and on zoospore motility: more zoospores were produced at lower temperatures and they remained motile for longer. From this it is concluded that the inoculum detected consists mainly of motile zoospores. In most experiments with standardized suspensions c. 10-15 were sufficient to initiate infection of the plants in the bioassay. In general, more inoculum was produced by host genotype/fungal isolate combinations in which there were marked root rot symptoms than in combinations in which the host was resistant.     

Detection and Quantification of Spongospora subterranea in Soil, Water and Plant Tissue Samples Using Real-Time PCR

A sensitive real-time polymerase chain reaction (PCR) assay was developed for the quantification of Spongospora subterranea, the cause of powdery scab and root galling in potato, and the vector of Potato mop top virus. A specific primer pair and a fluorogenic TaqMan® probe were designed to perform a quantitative assay for the detection of S. subterranea in soil, water and plant tissue samples. The assay was tested using DNA from cystosori, zoospores, plasmodia and zoosporangia of the pathogen. DNA was extracted directly from cystosori suspended in water and from clay soil with varying levels of added cystosori. DNA obtained from zoospores released into nutrient solution by cystosori in the presence of tomato bait plants was also tested, as was DNA from plasmodia and zoosporangia in infected tomato roots. In many cases, detection was successful even at low inoculum levels. This specific quantitative assay could therefore be a useful tool for studying the biology of S. subterranea, and for the optimisation of disease avoidance and control measures.     

Microscopic Studies on Attachment and Differentiation of Zoospores of the Phytopathogenic Fungus <Emphasis Type="Italic">Aphanomyces cochlioides</Emphasis>

The mode of aggregation, attachment and differentiation of zoospores of the phytopathogenic fungus Aphanomyces cochlioides when interacting with the host and a host-specific attractant and a G-protein activator, mastoparan, was studied by light and scanning electron microscopy. When a zoospore approached very close to the host root, it seemed to halt, then coiled its anterior flagellum on its body. The halted zoospore appeared to contact the host surface with its posterior flagellum, which gradually drew the encysting zoospore onto the root surface. The spore then docked precisely on the root surface at its ventral face with the help of the posterior flagellum and anchored itself by releasing some adhesive materials. The adherent spore became a spherical after shedding its flagella and rapidly turned into an expanded cyst forming a smooth cyst coat around it, and finally changed into a smaller cystospore covered with a wrinkled surface. In contrast, the mastoparan- or cochliophilin A-stimulated zoospores on artificial membranes aggregated by using their posterior flagella before encystment. These contrasting phenomena suggest that A. cochlioides zoospores may use their posterior flagella for successful docking on the host surface or for aggregation of encysting spores in the absence of the host. Received 30 August 2001/ Accepted in revised form 8 November 2001     

Infectivity,inoculum density and germination of Spongospora subterranea resting spores: a solution-culture test system1

Spongospora subterranea, causal agent of powdery scab of potatoes and vector of potato mop-top furovirus, survives in the soil as balls of resting spores (cystosori). So far, the factors affecting longevity, germination and infectivity of cystosori have not been investigated. A rapid and versatile bioassay with tomatoes as bait plants has been developed to quantify the infectivity of cystosorus inoculum or infested soil. The intensity of root infection, as a measure of infectivity, was determined by evaluating the quantity of zoosporangia present in epidermal cells and root hairs of the whole, stained root system. A correlation was obtained between the intensity of root infection and the cystosorus inoculum density in nutrient solution. Sterile soil suppressed the inoculum potential of pure cystosori. Infectivity of untreated soil decreased with increasing time of storage. Root infection was not influenced by the pH level of the nutrient solution.     

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.     

The potential for the Rapid Screening of Potato Cultivars (Solanum tuberosum) for Resistance to Powdery Scab (Spongospora subterranea) using a Laboratory Bioassay

Powdery scab of potato, once established in a field, is difficult to control because of the longevity of the resting spores (cystosori) of the causal organism, Spongospora subterranea f.sp. subterranea. Host resistance is likely to be the most efficient in a long-term control strategy for preventing build-up of field inoculum and spread of the disease. Resistance screening of potato cultivars is mostly done in laborious field trials where disease development is likely to be unpredictable. A bioassay with potato tissue cultured plantlets and cystosori as inoculum is described and was tested for its potential to screen potato cultivars at an early stage for their relative susceptibility to powdery scab by comparing the lab results with field data. With cystosori inoculum of Swiss origin, the laboratory test showed clear differences between the potato cultivars in the severity of zoosporangial root infection which correlated better with ranked tuber infection data, compared to root galling. There are apparent differences in the relative trends in susceptibility between roots and tubers of five selected cultivars when using naturally infested soil instead of prepared cystosori as inoculum in the lab bioassay. Furthermore, differences in the severity of zoosporangial root infection of two selected cultivars were found when cystosori from different countries where used as inoculum. A possible host genotype × pathogen interaction is discussed. The bioassay has the potential to screen and select for resistant material at an early breeding stage thus making field trials not unnecessary but more economical. It will allow the use of a standard set of pathogen collections and facilitate testing for inoculum virulence in infested soils.     

Polymyxa betae zoospores as vectors of beet necrotic yellow vein furovirus1

When beet seedlings exposed as bait plants in infested soil were placed in a nutrient solution, they released Polymyxa betae zoospores, infected with beet necrotic yellow vein furovirus. The roots produced the first zoospores 5 days after the start of soil baiting. When seedlings were inoculated with zoospore suspensions, infection occurred within 5 min and reached a maximum in 30 min. The suspensions remained infectious for at least 2 h after removal of the bait plants from which the zoospores were released. So many spores were released into the suspension that disease transmission could be obtained within half an hour from an infected plant to a healthy plant, placed together into fresh medium. Suspensions could be diluted 1/16 with nutrient solution without any loss of infectivity, whereas 1/4 dilution with tap water resulted in a complete loss of infectivity.     

Use of zoospores of Polymyxa betae in screening beet seedlings for resistance to beet necrotic yellow vein virus

A system to culture viruliferousPolymyxa betae and to produce zoospores is described. The zoo spores were used for inoculation of beet seedlings, grown in nutrient solution, in tests for resistance to beet necrotic yellow vein virus (BNYVV). On most occasions in a time course experiment, and with various zoospore cultures, the partially resistant cultivar Rima and the accession Holly-1–4 had virus concentrations similar to the susceptible cultivar Regina, but the virus concentration inBeta vulgaris ssp.maritima accession WB42 was significantly lower (P<0.05). ‘Regina’ could be distinguished from various resistant accessions by a significantly higher virus concentration (P<0.05) shortly after inoculation, or after transplanting the seedlings from the nutrient solution into sand. Results of screening for resistance to BNYVV, using zoospores for inoculation, did not correspond with results of a test in which infested soil was used.Tests in which seedlings are grown in nutrient solution and inoculated with zoospores are suitable for the detection of accessions with a high level of resistance to BNYVV. To obtain virus infection in all plants, the optimal density of the zoospore suspension should first be determined and plants should be assayed shortly after inoculation.     

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.     

Inoculation of pearl millet with the downy mildew pathogen, Sclerospora graminicola: chilling inoculum to delay zoospore release and avoid spray damage to zoospores

Inoculation of plants by spraying with a suspension of zoospores (released from sporangia of Sclerospora graminicola ) induced immediate encystment of the zoospores and drastically reduced their ability to cause disease. The incidence of infection in spray-inoculated pearl millet seedlings was reduced from 90% when sporangia were sprayed, to less than 30% when zoospores were sprayed. Rapid encystment was observed when zoospores were sprayed from both a hand-pumped sprayer and a compressed-gas sprayer and was probably caused by shearing forces. Chilling suspensions of sporangia prior to spraying delayed zoospore release and was an effective method for maintaining infection potential. Disease incidence was higher when sporangia were chilled to 0·2°C rather than to 4°C. Chilling resulted in some abnormal zoospore structures being released from sporangia when suspensions were returned to 20°C. The frequency of these structures increased with increasing chilling time. For seedlings sprayed with sporangia before zoospore release, a small reduction in disease incidence was observed when chilled inoculum was used, probably due to cold disruption of zoosporogenesis. For large-scale disease resistance screening, this reduction is outweighed by the benefit of a uniform and adequately high disease pressure that can be obtained over many hours using chilled spore suspensions.     

Effects of hydrostatic pressure,agitation and CO2 stress on Phytophthora nicotianae zoospore survival

BACKGROUND: Phytophthora nicotianae Breda de Haan is a common pathogen of ornamental plants in recycled irrigation systems. In a previous study, annual vinca (Catharanthus roseus Don) inoculated with zoospore suspensions using a CO₂‐pressurized sprayer had less foliage blight than plants inoculated using a hand sprayer. Here, the impact of hydrostatic pressure, agitation and aeration with CO₂ on the survival of P. nicotianae zoospores was examined. RESULTS: Exposure of zoospores to 840 kPa hydrostatic pressure for 8 min or agitation at a mixing intensity (G) of 6483 s^?1 for 4 min at 22–23 °C did not kill zoospores, but resulted in viable cysts. Motile and forcefully encysted zoospores of P. nicotianae were equally infectious on vinca or lupine (Lupinus polyphylus Lindl.). Bubbling CO₂ into zoospore‐infested water at 110.4 mL (0.2 g) min^?1 for 5 min caused 81% reduction in the number of germinated zoospores. Pressure at 630 kPa (16.3 g CO₂) or 70 kPa (3.85 g CO₂) facilitated CO₂ injection and shortened the zoospore inactivation time to 30 s. When air was bubbled through the suspension, germination was similar to the control. CONCLUSIONS: Exposure to CO₂ killed P. nicotianae zoospores in water. Neither pressure nor agitation had an effect on zoospore viability or infectivity. Based on results of this study, the authors designed a recycling CO₂ water treatment system that is currently under evaluation. Copyright © 2010 Society of Chemical Industry     

Effect of susceptible and resistant canola plants on Plasmodiophora brassicae resting spore populations in the soil

Clubroot, caused by Plasmodiophora brassicae, has become a serious threat to canola (Brassica napus) production in western Canada. Experiments were conducted to assess the effect of growing resistant and susceptible canola genotypes on P. brassicae soil resting spore populations under greenhouse, mini‐plot and field conditions. One crop of susceptible canola contributed 1·4 × 10⁸ spores mL^?1 soil in mini‐plot experiments, and 1 × 10¹⁰ spores g^?1 gall under field conditions. Repeated cropping of susceptible canola resulted in greater gall mass compared to resistant canola lines. It also resulted in reduced plant height, increased clubroot severity in susceptible canola, and increased numbers of resting spores in the soil mix.     

Effects of inoculum density,plant age and temperature on disease severity caused by pythiaceous fungi on several plants

Alfalfa, maize, sorghum and sugarbeet plants were inoculated with zoospores ofPhytophthora andPythium species in order to assess the effects of inoculum density, plant age and temperature on disease severity. Seedlings were grown axenically in test tubes and inoculated with zoospore suspensions. Disease severity was assessed by measuring the root growth and discoloration of treated and control seedlings. The incremental root length of all plants decreased and root discoloration increased as inoculum concentration of the pathogen increased. Changes were more intensive among low levels of zoospore concentrations and no significant differences in disease severity were found for inoculum densities higher than 10⁴ zoospores ml^-1. Disease severity was negatively related to plant age. Disease development on sugarbeet seedlings infected withPythium andPhytophthora species was affected by temperature, but the pattern of response was determined by the pathogen’s temperature preferences. The incremental root length decreased as temperature increased up to 25°C. The effect ofPythium dissimile andPhytophthora cactorum on root length was significantly lower at 35°C than at 25°C, whereasPythium aphanidermatum andPhytophthora nicotianae caused significant damage to roots even at 35°C. http://www.phytoparasitica.org posting Dec. 3, 2001.     

Relationship between Spongospora subterranea f. sp. subterranea soil inoculum level,host resistance and powdery scab on potato tubers in the field

The relationship between initial soil inoculum level of Spongospora subterranea f. sp. subterranea (Sss) and the incidence and severity of powdery scab on potato tubers at harvest was investigated. In all experiments soil inoculum level of Sss (sporeballs/g soil) was measured using a quantitative real‐time PCR assay. Of 113 commercial potato fields across the UK, soil inoculum was detected in 75%, ranging from 0 to 148 Sss sporeballs/g soil. When arbitrary soil inoculum threshold values of 0, <10 and >10 sporeballs/g soil were set, it was observed that the number of progeny crops developing powdery scab increased with the level of inoculum quantified in the field soil preplanting. In four field trials carried out to investigate the link between the amount of inoculum added to the soil and disease development, disease incidence and severity on progeny tubers was found to be significantly (P < 0·01) greater in plots with increasing levels of inoculum incorporated. There was a cultivar effect in all years, with disease incidence and severity scores being significantly greater in cvs Agria and Estima than in Nicola (P < 0·01).     

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.     

Resistance to Polymyxa betae in wild Beta species

Resistance to Polymyxa betae , the fungal vector of beet necrotic yellow vein virus, was studied in two wild beet species. Beta patellaris and B. procumbens. Plants grown in naturally infested soil or exposed to zoospore suspensions were examined in order to determine the stage in the life cycle of the fungus at which resistance was operating. Resting spores were never observed in the resistant species. Microscopic examination of stained transverse sections of fibrous roots taken at intervals after inoculation showed no evidence of even the earliest infection structures, the plasmodia; these were detected frequently in the epidermal cells of the susceptible Beta vulgaris. Use of the fluorescent stain DiOC₆(3) to label zoospores showed that these were attracted to and attached themselves to the roots and root hairs of resistant species in the same way as to susceptible species. Maximum zoospore attachment was observed 1–6h after roots were exposed to zoospore suspensions. There was no obvious difference in the numbers attracted to resistant and susceptible hosts. Apparent infection of root hairs by encysted zoospores was observed in all three species. The resistance mechanism in the wild species must operate soon after this initial infection, possibly involving a hypersensitive response that prevents the subsequent development of plasmodia in epidermal cells.     

Spongospora subterranea root infection assessed in two potato cultivars differing in susceptibility to tuber powdery scab

Infection by Spongospora subterranea of roots of two potato (Solanum tuberosum) cultivars, either very resistant or very susceptible to powdery scab on their tubers, was studied in a glasshouse experiment. Plants grown in sand/nutrient solution culture were inoculated with S. subterranea sporosori 2 weeks after planting. Plant parameters, the intensity of zoosporangium infection in roots, numbers of Spongospora root galls and amounts of Spongospora DNA in roots, measured using quantitative PCR (qPCR), were assessed at sequential harvests. Inoculation with S. subterranea reduced water use (56 days after planting) by 26% in the tuber resistant cultivar compared with uninoculated plants, and by 60% in the susceptible cultivar. Inoculation did not affect growth of the resistant cultivar, nor shoot mass of the susceptible cultivar, but caused a 38% reduction in root mass of the susceptible cultivar. The intensities of zoosporangium development in both cultivars were similar. The susceptible cultivar had approximately four times more Spongospora root galls g^?1 root mass than the resistant cultivar. Quantitative PCR detected S. subterranea DNA in roots 1 week after inoculation, and indicated a twofold greater amount of pathogen DNA in roots of the susceptible than the resistant cultivar. This study suggests that the S. subterranea zoosporangium stage in host roots is affected differently by host resistance factors than the sporosorus (root gall and tuber scab) stages. The study has also demonstrated the usefulness of qPCR for sensitive and consistent detection of S. subterranea across the duration of potato root infection.     

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.