High-Throughput Chemical Screening Identifies Compounds that Inhibit Different Stages of the Phytophthora agathidicida and Phytophthora cinnamomi Life Cycles

Abstract

Oomycetes in the genus Phytophthora are among the most damaging plant pathogens worldwide. Two important species are Phytophthora cinnamomi, which causes root rot in thousands of native and agricultural plants, and Phytophthora agathidicida, which causes kauri dieback disease in New Zealand. As is the case for other Phytophthora species, management options for these two pathogens are limited. Here, we have screened over 100 compounds for their anti-oomycete activity, as a potential first step toward identifying new control strategies. Our screening identified eight compounds that showed activity against both Phytophthora species. These included five antibiotics, two copper compounds and a quaternary ammonium cation. These compounds were tested for their inhibitory action against three stages of the Phytophthora life cycle: mycelial growth, zoospore germination, and zoospore motility. The inhibitory effects of the compounds were broadly similar between the two Phytophthora species, but their effectiveness varied widely among life cycle stages. Mycelial growth was most successfully inhibited by the antibiotics chlortetracycline and paromomycin, and the quaternary ammonium salt benzethonium chloride. Copper chloride and copper sulfate were most effective at inhibiting zoospore germination and motility, whereas the five antibiotics showed relatively poor zoospore inhibition. Benzethonium chloride was identified as a promising antimicrobial, as it is effective across all three life cycle stages. While further testing is required to determine their efficacy and potential phytotoxicity in planta, we have provided new data on those agents that are, and those that are not, effective against P. agathidicida and P. cinnamomi. Additionally, we present here the first published protocol for producing zoospores from P. agathidicida, which will aid in the further study of this emerging pathogen.

Keywords: Agathis australis; avocado root rot; high-throughput screening; kauri dieback; oomycetes; phenotype microarray; zoospore.

FIGURE 1

High-throughput screening for anti-oomycete compounds. (A) Diagram showing the standard test arrangement. An agar plug of mycelium is used to inoculate an agar plate with disks saturated either with water (control; marked 0) or increasing concentrations of a test compound (in a clockwise direction, marked 1–4). (B) Representative results from a typical experiment. Here, P. agathidicida mycelial growth is inhibited in the presence of increasing concentrations of paromomycin. (C) Control plate showing normal growth of P. agathidicida in the absence of test compounds.

FIGURE 2

Mycelial growth inhibition curves for P. agathidicida (green) and P. cinnamomi (blue). Each panel (A–H) shows the inhibition curves for a single compound, as identified in the label on the x-axis. Data points show the mean of triplicate inhibition assays.

FIGURE 3

Inhibition of zoospore germination in P. agathidicida (green) and P. cinnamomi (blue). Each panel (A–H) shows the inhibition curves for a single compound, as identified in the label on the x-axis. Dashed lines represent the germination rates on unamended agar. Data points are mean values from triplicate germination assays. Error bars are ± standard error of the mean. Where error bars are not visible, they are smaller than the symbol.