Soil-free bioassays for testing novel control agents against Phytophthora cinnamomi root rot

Phytophthora cinnamomi is considered as one of the world's worst plant pathogens, infecting about 5,000 plant species including those of agricultural and environmental significance. Disease management is largely dependent on chemical control, particularly synthetic fungicides such as phosphonic acid-based fungicides, e.g., phosphite/potassium phosphonate. While phosphonic-acid-based fungicides have been highly effective for more than 40 years, their prolonged use has led to the development of tolerance and decreased sensitivity in P. cinnamomi. Novel control agents that are effective but environmentally sustainable are therefore urgently needed. RNA-based biopesticides, which use exogenously applied double-stranded RNA (dsRNA) specific to the target pest or pathogen to avoid off-target effects on other organisms in the environment including beneficials, have emerged as a potential novel disease management strategy against P. cinnamomi. Due to the limited availability of bioassays to study the efficacy of this novel control agent against P. cinnamomi, we developed water-based lupin and pineapple bioassays using readily available plastic cups and glassware with mycelial plugs as inoculum. Infection rate was assessed 3 to 7 days post-inoculation (dpi) for lupin and 7 to 14 dpi for pineapple by measuring root lesion length and rating root rot. Potassium phosphonate (Agri Fos 600) and dsRNA were tested as example control agents, with dsRNA uptake tested via northern blotting. The bioassays were found suitable for P. cinnamomi pathogenicity assays, with one mycelial plug an effective inoculum; fungicide sensitivity testing, with doses as low as 0.45 g L^-1 Agri Fos® 600 providing protection; and exogenous dsRNA studies targeting root pathogens, with dsRNA able to be taken up by germinating lupin seeds. Overall, the assays are soil-free and thus overcome dsRNA stability issues in the soil and enable the collection of intact clean roots for molecular analyses. Furthermore, the bioassays are non-destructive, allowing root lesion symptoms to be visually monitored and repeatedly measured across different timepoints.