Associated callus culture technique for in vitro growth of rust fungi

Abstract

Rust fungi are obligate parasites which require a living host for growth, reproduction and completion of their life cycle (Eckardt 2006). There have been a few reports of in vitro culturing of rust fungi but these studies could not be repeated by other workers (Scott & Maclean 1969; Bose & Shaw 1974; Wiethölter et al. 2003). The inability to culture rust fungi in defined conditions has been a major impediment to studying host-pathogen interactions and performing detailed biochemical and molecular analysis. The present study was undertaken to investigate the competence of rust fungus Uromyces hobsoni Vize to grow with host tissues in artificial culture media. Material and methods Collection of plant material and tissue explant preparation: The leaves of Jasminum officinale var. grandiflorum (L.) Bailey, both healthy and heavily infected by rust fungus Uromyces hobsoni Vize. (Image 1 ad) were chosen for culture experiments. The leaves were collected from the same host plant in the field and preserved in brown paper bags (storage in polythene bags leads to development of moisture and hyperparasites on the tissues). Leaves were deemed infected if they contained telial pustules. Explants were washed thoroughly under running tap water to remove dust and other debris, further washed with an aqueous suspension of 0.01% (v/v) Tween 20 (a liquid detergent) for 10 minutes followed by aqueous suspension in Dettol disinfectant for 10 minutes. Further processing of explants was carried out under aseptic conditions in a horizontal laminar air flow cabinet. The explants were washed with sterile distilled water, treated with 70% alcohol for 30 seconds, surface sterilized with an aqueous solution of 0.1% mercuric chloride for two minutes and inoculated with fungus in test tubes containing sterile Murashige and Skoog's growth medium (Murashige & Skoog 1962) supplemented with 2 mg/L 2, 4-D with 3% sucrose (w/v) and 0.8% agar (w/v) at pH 5.5-5.7. Incubation and Measurement: Cultures were incubated at 27±2 0 C at 1200 lux fluorescent white light at 16 hours photoperiod. Cultured explants were observed regularly for contamination and callus development. Growth of callus tissue was analyzed at an interval of four days after the initiation of culture. Ten cultures from each experiment were used to determine the fresh weight of callus, which was then dried in a hot air oven at 40 0 C until a constant dry weight was obtained. Microscopic observation of calli involved looking for fungal structures including mycelia and haustoria via cotton blue mounting. Final development was assessed on the 32 nd day post inoculation for 10 callus cultures each from healthy and infected tissues. Each experiment was done in triplicate and the number of cultured explants per replicate was 25.