Powdery scab. Research in Switzerland

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1 Powdery scab. Research in Switzerland U. Merz, Federal Institute of Technology, Institute of Plant Sciences, Plant Pathology, 8091 Zurich, Switzerland There is a long tradition of powdery scab research in Switzerland. As long ago as the 1920 s Nora Wild conducted investigations into the morphology of tuber infection and the influence of different environmental factors on the occurrence of the disease (Wild, 1929). Many years later, Diriwächter began work to see if the causal organism, S. subterranea, which is an obligate parasite, could be cultured on artificial media. In addition, he tested the ability of a range of chemicals to control the pathogen (Diriwächter, 1981). Biology of the pathogen In 1984, I began my research with the hypothesis that the resting spores of S. subterranea survive in soil in a dormant state and release zoospores only in response to a host specific signal. Thus, induction of the zoospore stage in the absence of a host plant or in the presence of a control chemical could represent a valuable new control strategy. Different methods of quantifying the germination rate of zoospores where tested, but all failed due to the three dimensional spongy-like structure of the sporeballs (Fig 1). Consequently, a bioassay with tomato bait plants in nutrient solution was developed to quantify the infectivity of cystosorus inoculum or infested soil (Fig 2). 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 (Merz, 1989). Using this bioassay, a correlation was obtained between the intensity of root infection and the resting spore inoculum density. Varying the ph of the nutrient solution between 5.2 to 8.0 did not affect root infection. Infectivity of soil decreased with increasing time when untreated soil was stored. For improved identification, secondary zoospores of S. subterranea were observed during and after emergence from sporangia using a light microscope, video equipment and scanning electron microscopy. Freshly discharged zoospores showed a characteristic swimming pattern with rapid movements and sudden changes in direction. Electron micrographs showed a lateral insertion of two flagella at an angle of to each other (Fig 3). The long flagellum had a gradu- Fig 2 Bioassay with tomato bait plants in nutrient solution Fig 1 A sporeball of S. subterranea consisting of numerous single resting spores 67

2 Fig 3 A secondary zoospore of S. subterranea showing two, laterally inserted flagella 1993). The same solution culture test system was used to create a pulse of primary zoospore production and subsequent host root infection. Sporeballs, zoospores and host roots were examined by light, fluorescence, transmission and scanning electron microscopy (Merz, 1997). Many resting spores with developing exit pores were observed, but didn t show any of the changes in cytoplasmic content typical of zoospore formation. It was concluded that formation of zoospores may occur quickly just before release. Mature zoospores were never found inside resting spores. The average diameter of exit pores of empty ally tapering end, whilst the short flagellum showed an abrupt transition from wide to narrow (Merz, 1992). With a modified bioassay, the influence of root exudates on resting spore germination was investigated. A 5h-incubation of cystosori together with roots of tomato or wheat was sufficient to increase the intensity of root infection significantly. A high level of root infection was also found on tomato exudate plants but not on wheat (Merz, Fig 4 Fluorescence micrograph of encysted S. subterranea primary zoospores on tomato root surface with the bright Rohr -like structure inside. resting spores was 1.5µm. Primary and secondary zoospores are identical in morphology. Zoospores that were encysted and attached by an adhesorium to tomato root hairs, displayed the internal Rohr -like structure similar to that of other Plasmodiophoromycetes (Fig 4). Post-infection papillae and uninucleate plasmodia were also observed. In a study of the structure of S. subterranea cystosori, various methods of sporeball preparation for the TEM were tested. Rod-shaped particles with a mean diameter of 20,5 nm and a maximum length of 500nm, resembling rod-shaped virions (e.g. mop-top virus) were found inside freeze-fractured resting spores (Merz, 1995). Detection of the pathogen In 1991, a survey of soil samples from potato-producing areas was made using the bioassay. It was shown that the pathogen is more widespread than expected (Merz, 1993). The cultural practice of ploughing in spring, often following pasture as a pre-crop, was correlated with fields having powdery scab problems. In laboratory experiments it was confirmed that inoculum density in highly infested soils is higher than 500 cytosori/g soil. Control of powdery scab is difficult because of the persistence of the resting spores in soil and the difficulty in killing them. The only reliable form of control would be to plant clean seed into uncontaminated land. However, even though seed tubers with scabs may be identified by visual inspection, blemish-free tubers may be contaminated by contact with scabbed potatoes. Further, the determination of the contamination levels of field soils using a bioassay is very labour intensive and time consuming. Sensitive and rapid detection of resting spores both on tubers and in field soils would be a great aid to the development of disease management strategies and the determination of disease thresholds. Therefore, a polyclonal antiserum was developed against S. subterranea sporeballs (Walsh et al., 1996). This ELISA assay was able to detect 0.02 sporeballs. In spiked soil samples however, the antiserum discriminated the contamination 68

3 levels more accurately at concentrations above 2000 sporeballs/g soil, whereas the bioassay gave good discrimination of levels containing less than 1000 sporeballs/g. The antiserum could detect spores from different cultivars and geographical origins. To improve the sensitivity of the assay, a monoclonal antiserum against resting spores was produced which showed no cross-reaction with several strains of Streptomyces ssp. This assay is now commercially available and is particularly recommended for testing seed tubers. An simple method for routine testing was developed using a commercial kitchen peeling machine (Merz, 2000a). The bioassay discriminates not only better at lower levels of soil contamination, it also detects only living resting spores, which in terms of epidemiology are the important fraction. It was therefore concluded that a combination of the bioassay with immunological assessment of root infection may provide the best solution and the development of a monoclonal antiserum against zoosporangia of S. subterranea is in progress. Control of the pathogen A reduction in powdery scab disease incidence and severity can be achieved by the application of pesticides to seed tubers or soil (Falloon et al., 1996; see also Merz, 2000b) and by the use of disease resistant cultivars and appropriate cultural practices (Falloon et al., 1997). Soil micronutrients, including boron, have been shown to reduce infection of brassica roots by Plasmodiophora brassicae (Webster and Dixon, 1991), which is closely related to S. subterranea. An experiment was carried out to measure the effect of boron on infection of tomato roots by S. subterranea. This study aimed to determine whether modification of the soil chemical environment had potential as a further control method for the integrated management of powdery scab (Falloon et al., 2001) and was carried out in collaboration with Crop&Food, Christchurch, New Zealand. No clear relationship between seedling shoot or root fresh weight and boron concentration was obtained, although the 20 mg/l boron treatment reduced these parameters in two of the three experiments. Infection severity declined with increasing rates of boron, with a 50% reduction in severity resulting from the addition of 12 mg boron/l. At 20 mg/l, very few zoosporangia were observed in seedling roots (Fig 5). In a infection stage experiment, zoosporangium infection was severe (mean score 3.8) for seedlings without added boron, but was low from each of the treatments that applied 20 mg boron/l at different bioassay stages. Almost no zoosporangia (mean score <0.1) occurred from the incubation and cultivation period treatments, and very few (mean 0.9) occurred from the baiting period treatment Fig 5 Tomato infection Boron concentration (mg/l) S. subterranea infection severity scores of tomato seedling roots at different boron levels (LSD indicated, mean of 3 reps) Fig 6 Micro-bioassay with potato tissue- culture plantlets Resistance screening of potato cultivars is usually carried out in expensive field trials, and infection by S. subterranea is not guaranteed due to variable environmental factors. A lab- based screening system has the advantage of being rapid, cost-effective and reproducible. On the base of the bioassay described earlier, a 69

4 5 Infection severity score (0-5) Erntestolz Sirtema Urgenta Charlotte Desiree Agria Bintje Gladiator Granola Nicola Ditta Fig 7 Root infection with zoosporangia of S. subterranea of potato tissue-culture plantlets from 11 cultivars in a micro-bioassay micro-bioassay was developed using potato tissue-culture plantlets as bait plants (Fig 6). The solution culture test could significantly discriminate between the two cultivars that showed highest and lowest tuber susceptibility to powdery scab (cv Erntestolz, cv Ditta) in Swiss field trials. Furthermore, the three cultivars with the lowest root infection levels in the micro-assay also had the lowest scores for tuber infection in the field (Fig 7). It was concluded that the test is effective as a pre-screen for resistant breeding lines and cultivars. In addition, it is useful for cultivar x pathogen strain experiments. When the micro-assay results were compared with field data, it became obvious that the use of a common scoring scale for tuber infection is crucial for accurate comparison. Therefore, an illustrated scoring scale was suggested with 7 infection levels (Fig. 8). This scale was tested successfully in Switzerland in 2000 and will be recommended for use in other countries in Europe. References Diriwächter G., Spongospora subterranea, Erreger des Pulverschorfes der Kartoffel: Entwicklung in Kulturmedien und Versuche zur Bekaempfung. Diss ETH Fig 8 Scoring scale for potato tuber infection with S. subterranea 70

5 Falloon R.E., A.R. Wallace, M. Braithwaite, R.A. Genet, H.M. Nott, J.D. Fletcher and W.F. Braam, Assessment of seed tuber, in-furrow, and foliar chemical treatments for control of powdery scab (Spongospora subterranea f. sp. subterranea) of potato. New Zealand Journal of Crop and Horticultural Science 24, Falloon, R.E., U. Merz, R.A. Genet, J.W. Marshall and M. Braithwaite, Powdery scab of potato the pathogen and progress toward control. Crop & Food Research Broadsheet No. 40: 6 pp. Falloon R.E., U. Merz, D. Curtin and R.C. Butler, Boron affects Spongospora subterranea infection of host roots; laboratory and glasshouse results. Proceedings of the 2 nd Australasian Soilborne Diseases Symposium, Melbourne (in press). Merz U., Infectivity, inoculum density and germination of Spongospora subterranea resting spores: a solution-culture test system. Bulletin OEPP 19, Merz U., Observations on swimming pattern and morphology of secondary zoospores of Spongospora subterranea. Plant Pathology 41, Merz U., Epidemiological aspects of powdery scab of potatoes caused by Spongospora subterranea. Proc. of the 2nd Symp. of the International Working Group on Plant Viruses with Fungal Vectors. Montreal, Canada, July 25-27, Merz U., PMTV-like particles inside resting spores of Spongospora subterranea. Journal of Phytopathology 143 (11-12), Merz U., Microscopical observations of the primary zoospores of Spongospora subterranea f.sp. subterranea. Plant Pathology 46 (5), Merz U., 2000a. Powdery scab control in Switzerland. Proceedings of the first European Powdery Scab Workshop, Aberdeen, July 20-22, Merz U., 2000b. Experiments on direct control and yield loss made in New Zealand. Proceedings of the first European Powdery Scab Workshop, Aberdeen, July 20-22, Walsh J.A., U. Merz and J.G. Harrison, Serological detection of sporeballs of Spongospora subterranea and quantification in soil. Plant Pathology 45 (5), Webster, M.A. and G.R. Dixon, Boron, ph and inoculum concentration influencing colonization by Plasmodiophora brassicae. Mycologocal Research 95, Wild, N., 1929: Untersuchung über den Pulverschorf der Kartoffelknollen Spongospora subterranea (Wallr.) Johnson. Phytopath. Z. 1,