Characterisation and pathogenicity of scab isolates
3.1 Introduction
3.1.4 Pathogenicity Tests
One of the major obstacles to defining the taxonomic relationships between scab-causing microorganisms has been the lack of reliable and rapid methods for testing pathogenicity. Traditionally, seed potatoes were planted in scab infested soil either in the field or glasshouse and scored for scab symptoms once the plants have died down and the crop lifted. The problems with this type of test include, difficulties associated with maintaining a constant environment, in the field this is virtually impossible with workers reporting huge variations due to differences in annual precipitation. Attempts to overcome these problems have included covering the newly emerged plants for the first 9 weeks and while susceptible to infection with polythene tunnels (Jellis, 1975).
Many workers prefer the more controlled environment of the glasshouse (Booth, 1970; Gunn et al., 1983). However even within the glasshouse it is difficult to eradicate the often large gradients in temperature, light and humidity that occur, even within the pot. The inclusion of sufficient replication is used to compensate for a certain proportion of this variation. It is generally possible to have more replication in the field than in the confines of a glasshouse. For these reasons the use of sterile mini tuber assays are attractive, since they eliminate a number of the problems associated with environmental variables. Lawrence & Barker (1963) reported the development
of such a system of pathogenicity screening, where in vitro potato plantlets were induced to produce mini tubers on elevated levels of sucrose (8%). They were unable to reproducibly induce scab symptoms on the tubers in tissue culture conditions, but found that by transferring plants bearing mini tubers to a moist vermiculite environment, reproducible induction of scab symptoms upon inoculation with a spore suspension of a plant pathogenic streptomycete was achieved. Lawrence et al. (1990) have subsequently applied this system to their studies of the vivotoxin, thaxtomin.
A number of methods have been devised for scoring virulence of isolates on susceptible tubers, though unfortunately a universal standard has not been adopted and different workers have used different criteria in their assessments. The problem is exaggerated by the number of different types of scab symptoms and confusion generated by the different names assigned to them (McKee, 1958). For instance superficial scab is used to include a number of minor types of infection including those described elsewhere as russet (Harrison, 1962) and netted scab (Scholte & Labruyere, 1985). Generally, the two indices of pathogenicity used are the percentage coverage of the tuber by scab lesions and the severity of the lesions. Many workers have indicated a good correlation between these two criteria, such that badly scabbed tubers are covered extensively with severe lesions (Leach et al., 1938; Stevenson et al.,
1942; Jellis, 1977) although a few exceptions were noted. Percentage coverage has been frequently scored using keys (Large & Honey, 1955; McKee, 1963; Lapwood & Dyson, 1966; Dowley, 1972).
An alternative to the keys is the spot sampling method developed from the point quadrat method for determining botanical species composition in grasslands and introduced by Lowlings & Ridgeman (1959). The method
involved placing a marked strip longitudinally around the potato and noting the number of points on the strip that were covered by scab lesions. This estimate expressed as a percentage cover of tubers was found to be quite accurate when compared to the percentage scabbed surface area calculated by measuring the area of scabbed lesions using dividers and expressing this as a percentage of the whole surface area established by measuring the area of the potato peelings. However, the accuracy of this method was found to be off set by its laborious time consuming nature (Langton, 1972). Finally Leach et al.
(1938) and Lauer & Eide (1963) have considered assessing common scab infection using the 'highest scab' method. This method involved scoring the severity of infection according to the severest lesions on the tubers. Although the method was liable to greater test error (Leach et al., 1938) it was found to be a rapid and effective method for assessing clonal resistance (Lauer & Eide, 1963). Many workers have combined both criteria of percentage coverage and lesion type to produce a scab index (Marais & Vorster, 1988). Bjor & Roer (1980) obtain a pathogenicity score by multiplying lesion severity (identified from a key) by the percentage cover times 100/ 27. The figure 27 relates to the product of 9 categories of percentage cover and 3 categories of lesion severity.
3.2 Alms
3J.1 To investigate the taxonomic position of a group of putative pathogenic streptomycetes with respect to the numerical phenetic classification studies of the Streptom yces genus (Williams e t aln 1983a) using a probabilistic identification matrix (Williams et al-, 1983b). To establish whether these strains have a common identity which is phenotypically definable as proposed by Lambert & Loria (1989).
3.2.2 To assess the value of 16S rRNA targeted oligonucleotide probes described for S.scabies ISP5078 (W itt et al., 1989) in the characterization of plan t pathogenic streptomycetes. Furtherm ore to assess w hether the p atterns of hybridization from the 16S rRNA targeted probes correlate with the phenotypic identifications.
3.2.3 To establish the pathogenicity of isolates under glasshouse conditions. In order to check pathogenicity of strains received as putative pathogens and assess the degree of virulence of isolates. To relate this d ata to the identification data, to gain fu rth er understanding of the identity of the causal agent of common scab of potatoes.
3.2.4 Preliminary studies to investigate the application of an axenic mini tu b e r assay for testing the pathogenicity of scab-causing streptomycetes. Problem s surround the traditional methods for assessing pathogenicity u n der field or glasshouse conditions. These include variable environm ental factors and the difficulties of working in a non-sterile environm ent, in term s of establishing whether disease symptoms are a pro du ct of the inoculant o r other members of the soil microflora. Sterile plan t tissue culture systems might offer a definitive and rapid solution.