The detection of Spongospora subterranea by bioassays, molecular and serological methods

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1 The detection of Spongospora subterranea by bioassays, molecular and serological methods K. Bouchek-Mechiche, D. Ruer, D. Andrivon and B. Jouan, INRA, UMR BIO3P, Domaine de la Motte, Bp35F Le Rheu Cédex, France Introduction Spongospora subterranea (Wallroth) Lagerheim f. sp. subterranea Tomlinson (subsequently referred to as S. subterranea) is the causative agent of powdery scab, which constitutes a major disease problem for potato growers in many areas of the world. The disease is still uncommon in France, even though, during cold and humid years, some occasional attacks have been observed. Nevertheless, increasing importation of contaminated seed from northern Europe constitutes a major risk of introduction and spread of the disease in our country. As this pathogen has never been studied in France, the main aim of our research has been to develop sensitive methods of detection and quantification of the fungus, in order to evaluate the inoculum possibly to be present on the seed and in the soil. Assessment of methods used for the detection of S. subterranea I. Detection of fungus using bioassays We compared 2 methods described in the literature (Fig 1): Nutrient solution method The protocols of Merz (1989) and Fornier (1997) which involve the use soil of nutrient solution were performed. Firstly, soil or cystosori were suspended in a nutrient solution in containers or flasks and then incubated in a growth chamber (15 C night / 18 C day) in the dark (incubation period). Secondly, tomato bait plants were added to the suspension and grown for 3 to 6 days (baiting period). Thirdly the roots were washed and the tomato bait plants were cultivated in fresh nutrient solution for 7 days (cultivation period). Finally the roots were stained and observed through a microscope or stereo microscope and then rated according to the modified scale of Kole (1954). The differences between Merz s (1989) and Fornier s (1997) protocols lie in the duration of incubation and baiting periods (Fig 1). Fig 1 Comparative schematic representation of the various bioassays for detection of S. subterranea (IP: incubation period; BP: baiting period; CP: cultivation period; NS: nutrient solution; MO: microscope observation; D: day; DD: direct detection) 61

2 Direct detection of the fungus in the soil for 3 days (Fornier, 1997). The tomato seeds were sown in the soil and grown To compare the sensitivity of the three protocols, three soils from production tunnels contaminated with imported peat substrates were used. In addition, in this experiment we used both non-inoculated (sterilised soil Tu8,1 h at 12 C)and inoculated controls (sterilised Tu8 soil inoculated with 1 sporeballs/g soil), referred to as UC and IC respectively. Table 1 Incidence and severity of root infection by S. subterranea using the various bioassays Protocols Fornier, 1997, NS c Merz, 1989 Fornier, 1997, DD d Samples N a ID b N ID N ID UC IC Tu6 Tu9 Tu8 /8 1/1 /8 1/8 / /8 8/8 5/8 3/8 / /6 1/ a N: number of plants infected b ID: Degree of infection of bait plants (average of scores of all plants on a -4 scale- Kole 1954) c NS: nutrient solution d DD: direct detection The results obtained in this experiment (Table 1) showed that Fornier s (1997) protocol (NS) allowed the detection of the fungus in only one (Tu9) of the 3 test soils, and produced a low degree of infection compared with that in the inoculated control. Merz s protocol allowed the detection of the fungus in 2 (Tu9 and Tu6) of the 3 soils tested and the number of infected plants was higher than with Fornier s (1997) protocol. These differences in sensitivity can be explained by the duration of the tests, and the presence or absence of the incubation period. Furthermore, in the case of Fornier s protocol (1997), the soils were not dried before the experiment. Direct detection of the pathogen in the soil with Fornier s protocol (1997) is most sensitive, since the fungus was detected in all soils tested, and produced high level of root infections in Tu9 and Tu6 (Table 1). The increased sensitivity of direct detection compared to the other protocols can lead to be explained by the longer of the baiting period. The disease observed on the roots is certainly overestimated compared to the initial sporeballs present in the soil because of several secondary infections. For this reason, Merz s protocol seems most appropriate to evaluate the infectivity of the soils. 62

3 To improve the sensitivity of Merz s protocol, we tested the following modifications (Table 2): T: Merz (1989) bioassay; T1: increased incubation period (from 7 days to 15 days); T2: addition of 3 ml of tomato root exudate solution at the beginning of the baiting period; T3: T1 +T2; T4 : 1 humid period and 1 drying period before incubation; T5: 2 humid periods and 2 drying periods before incubation; T6: T4 + T2; T7 : T5+ T2. Table 2 Incidence and severity of root infection by S. subterranea obtained with different modifications of Merz s bioassay. Plants Treatments Infection degree % plants infected T T T T T T T T The results of this experiment showed that lengthening the incubation period (comparison between T/T1 and T4/T5) enhances root infection. Conversely, the addition of 3 ml of exudate solution at the beginning of the baiting period (T/T2; T4/T6; T5/T7) has little effect on the infection of the roots. Comparing T with T4 and T1 with T5, which have the same incubation period, reveals that constant humidity is more favourable for the root infections than the cyclical humid/dry period. II. Molecular detection Specificity of the primers Two specific PCR primers pairs are described in literature: SPO8/SPO9 (Bullman and Marshal, 1998) and SP1/SP2 (Bell et al., 2). To validate the use of these primers in our laboratory, we tested their specificity and sensitivity. The specificity of primers was tested against genomic DNA from 2 samples. The specific primers for S. subterranea amplified a product of the correct size (391 bp for SP1/SP2- fig 2; 372 bp for SPO8 / SPO9- data not shown) only for the samples contaminated 63

4 with S. subterranea. The primers allowed the detection of all the forms of the pathogen (zoosporangia, plasmodia, sporeballs). The specificity of the primers was confirmed by the absence of PCR amplification products with DNA from non-infected tubers or roots (Fig 2). Fig 2 Assessment of S. subterranea primers, SP1 and SP2 specificity on DNA extracted from: 1 = non-infected tomato roots; 2 = infected tomato roots; 3 and 6 = non-infected potato periderm; 4 and 7 = infected potato periderm; 5 = purified sporeballs. Sensitivity of the primers Serial dilutions ranging from 4 ng µl -1 to 4x1-9 ng µl -1 of known quantities of genomic DNA from sporeball inoculum were used as a template to assess the sensitivity of PCR detection with the 2 primers. The sensitivity of these primers proved similar, as both primers pairs allowed the detection of 4 pg of DNA per reaction. However, DNA amplification with SP1/SP2 was more homogeneous than with SPO8/SPO9. Sensitivity of the primers was also tested against DNA from roots infected to different degrees (rated microscopically to 4). The primers were able to detect a low level of root infection (rate 1) corresponding to only a few sporangia observed microscopically. III. Serological detection The DAS - Elisa kit for the detection of S. subterranea cystosori (BIOREBA-MERZ) was tested against powdery scab inoculum prepared from different cultivars, scabbed tubers, healthy tubers, pathogenic Streptomyces strains (S. acidiscabies T, S. europaeiscabies T, S. stelliscabiei T, S. reticuliscabiei T, S. turgidiscabiei T, S. scabies T ), potato with symptoms of common and netted scab, infected and healthy tomato roots, contaminated soil and non-contaminated soil. Positive reactions were observed for all samples contaminated with powdery scab. Negative reactions were obtained with all Streptomyces species tested and with samples showing common or netted scab symptoms. Negative reactions were also obtained with infected tomato roots or contaminated soils. This was as expected, as the monoclonal antiserum is specific only to sporeballs and does not recognise the other forms of the pathogen such as zoosporangia or zoospores. This serum allowed the detection of.2 cystosori per well. Conclusions Molecular tests allowed the detection of all forms of the pathogen (zoosporangia, zoospores, plasmodia, sporeballs) in various substrates (tubers, soils, roots). Conversely serological detection with the monoclonal antiserum recognised only one life-cycle stage of the pathogen (sporeballs), but is more appropriate and efficient for the routine detection of powdery scab on tubers since some symptoms are easily confused with those of common scab. Coupling PCR with suitable baiting tests can result in an easier, faster and more accurate method of assessing plasmodial infection of root hairs. This is because counting plasmodia under the microscope is time consuming and laborious and recognition of different stages of 64

5 development of plasmodia can be difficult. However it is important to develop an automated and quantitative PCR system to quantify the fungus in the roots and to relate this to the cystosori concentration in the soil sample to give an accurate quantitative risk assessment for a particular field. References Fornier N, Epidemiology of Spongospora subterranea, the cause of powdery scab of potatoes. PHD Thesis of Aberdeen University. Kole A.P., A contribution to the knowledge of Spongospora subterranea (Wallr.) Lagerh., the cause of Powdery scab of potatoes. Tijdschrift over plantenziekten 6, Merz U., Infectivity, inoculum density and germination of Spongospora subterranea resting spores: a solution-culture test system. Bulletin OEPP 19,