In March of 2004, stratified ginseng seeds from commercial Wisconsin gardens were planted in sterilized silica sand in a research greenhouse at Michigan State University. Following emergence, seedlings exhibiting wilting, damping off, and black stem lesions were observed. In the laboratory, symptomatic seedlings were rinsed with distilled water. Tissue samples were excised and embedded in water agar amended with ampicillin (100 mg/liter) and incubated at 25°C. In addition to the isolation of Phytophthora cactorum, a known pathogen of ginseng, P. citricola, (five isolates) also was identified from single-zoospore cultures based on morphology (2). One-week-old, dilute V8 agar cultures were used to obtain single zoospores. Cultures were flooded with 20 ml of sterilized distilled water chilled to 10°C and incubated at 25°C for 25 min to induce zoospore release. Zoospore suspensions were spread onto water agar plates, and after 24 h at 25°C, single germinating zoospores were selected at random and transferred to benomyl, ampicillin, rifampicin, and pentachloronitrobenzene (BARP)-amended V8 agar plates. Sequence analysis of the internal transcribed spacer (ITS) region 1 and 2 of the rDNA was also used to distinguish P. citricola from P. cactorum. A representative sequence for the isolates of P. citricola (NCBI Accession No. FJ217388) matched (100% similarity) a P. citricola isolate deposited in GenBank (Accession No. DQ486661). To screen P. citricola for in vitro response to mefenoxam, agar plugs (7-mm diameter) from 1-week-old V8 agar cultures incubated at 25°C under fluorescent lighting were placed in the center of each of two V8 agar plates amended with 0 and 100 ppm of mefenoxam (Ridomil Gold EC, 48% a.i., suspended in sterile distilled water and added to V8 agar cooled to 49°C). The plates were incubated at 25°C for 3 days under fluorescent lighting. Isolates were assigned a mefenoxam sensitivity rating based on the percentage of radial mycelial growth on the amended V8 agar when compared with the unamended control. Each of the five isolates was scored as mefenoxam resistant with growth on 100-ppm plates >30% of the controls. Koch's postulates were conducted for the isolates of P. citricola recovered from ginseng seedlings to confirm pathogenicity. Previously, P. citricola was reported as nonpathogenic to ginseng (1). Three-week-old, healthy ginseng seedlings were planted into 89- × 64-mm pots filled with autoclaved medium-particle vermiculite and maintained in the greenhouse under 63% shade cloth with temperatures between 18 and 26°C. Pots were arranged in a completely randomized block design with eight seedlings per isolate as replicates and watered as needed. A 2-ml inoculum suspension (approximately 104 zoospores) was injected into the potting medium at the stem base of each seedling. All of the isolates were pathogenic to ginseng seedlings with 60% of inoculated seedlings per isolate exhibiting wilting, damping off, and blackened stems within 3 weeks after inoculation. P. citricola was reisolated from all inoculated plants. To our knowledge, this is the first report of P. citricola pathogenic on ginseng.
References: (1) T. W. Darmono et al. Plant Dis. 75:610, 1991. (2) D. C. Erwin and O. K. Ribeiro. Page 96 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN. 1996.