KL BAYLISS^1,5, J KUO², K SIVASITHAMPARAM^1,5, M BARBETTI^3,5 and ES LAGUDAH⁴

¹Department of Soil Science and Plant Nutrition, Faculty of Agriculture, University of Western Australia, Nedlands, WA 6907, Australia; ²Centre for Microscopy and Microanalysis, University of Western Australia, Nedlands, WA 6907, Australia; ³Agriculture Western Australia, South Perth, WA 6151, Australia; ⁴CSIRO Plant Industry, Canberra, ACT 2601, Australia; ⁵Centre for Legumes in Mediterranean Agriculture, University of Western Australia, Nedlands, WA 6907, Australia

Background and objectives
Kabatiella caulivora, the causal organism of clover scorch disease, is a serious pathogen of clover species (Trifolium spp.) throughout the world. The fungus causes lesions on the leaves, petioles and petiolules, causing plants to wilt as the moisture supply becomes restricted. During favourable weather conditions, entire swards of clover may collapse from infection.

Until recently, the disease was controlled with resistant varieties. However, in 1990, a 'new', resistance-breaking race emerged and caused severe disease on some previously highly resistant varieties of subterranean clover in Western Australia [1]. We examined the infection process of the 'new' race in comparison to that of the 'old' race on two varieties of subterranean clover (T. subterraneum).

Materials and methods
Infected leaves, petioles and petiolules of cv. Woogenellup, susceptible to both races of K. caulivora, and cv. Daliak, resistant to the 'old' race but susceptible to the 'new', were fixed and embedded in glycol methacrylate. Sections (2 µm) were stained for structural components, lignin, proteins, lipids and carbohydrates. Spore attachment and germination percentage were determined on inoculated tissue by fluorescence microscopy after staining with 0.05% aniline blue.

Results and conclusions
Inoculated spores attached to the plant surface near trichomes, often germinating and growing along them. Significantly greater numbers of germinating and non-germinating spores were counted on susceptible than on resistant plants. A larger number of spores of the 'new' race were found on Daliak compared to the 'old' race, with no differences on Woogenellup.

Plants susceptible to K. caulivora developed large, water-soaked lesions. In contrast, on resistant plants the lesions were restricted to minute black specks. The 'new' race also caused large lesions to develop on Daliak (previously resistant). Loss of chlorophyll and starch grains in cells ahead of invading hyphae was the first evidence of infection. A hyphal network then developed inter- and intracellularly, before cell lysis occurred. Cells in uninfected plants stained positively for hemicellulose and pectin. Negative staining occurred in cells infected with the pathogen, indicating the degradation and loss of structural components of the cell wall. Both races infected all tissues in leaves, petioles and petiolules in susceptible plants. Only the 'new' race could extensively invade the variety previously resistant to K. caulivora (Daliak), in which infection by the old race was limited to the epidermal cells and two to three layers of mesophyll cells. Histochemical staining did not reveal any distinct plant resistance responses in leaves, petioles or petiolules.

Apart from the resistance of Daliak to the old race, there was no difference in the infection process of K. caulivora on subterranean clover for either race. It is possible that infection of Daliak by the new race is a consequence of the plant failing to recognise this new race, thus preventing or delaying the resistance response.

References
1. Barbetti MJ, 1995. Australian Journal of Agricultural Research 43, 645-653.