Phytophthora erythroseptica was isolated from infected potato tubers in Maine in 2013. The isolates were confirmed by sequence analysis using internal transcribed spacer 1 region of rDNA. The culture was maintained on V8 agar Erwin and Ribeiro, 1996) throughout the study. Preparation of zoospore suspension was conducted based on the methods described by others (Fitzpatrick-Peabody, 2011) with slight modification. Briefly, mycelial plugs on 2% V8 agar medium were placed in 3% lima bean broth and incubated 3 days at 22°C in the dark for vegetative growth. For sporangial production, the mycelial mats were transferred to 10% soil extract solution after rinsing with sterile distilled water. To maintain a low nutrient condition, the seedling plugs were removed before being incubated at 18°C for 4 days under fluorescent light. Zoospores could be harvested after the mycelial mats were flooded with chilled sterile distilled water at 4°C for 1 h. Zoospore suspension was achieved after filtration through one layer of Miracloth to remove mycelium, sporangia, and other structures. The concentration was determined using a hemocytometer.

Zoospore exudate (ZE) was defined as the exudate from zoospore suspension at a concentration of >5 × 10⁵ zoospores/ml. To test the effects of ZE on zoosporic behaviors, ZE must be separated from zoospores. The zoospore suspension obtained as described above was filtered through a 0.2-μm filter membrane to remove zoospores and cysts. The filtrate was used to investigate its effects on zoosporic germination and infection. To confirm that the exudates come from zoospores instead of other P. erythroseptica structures, liquid culture exudates at different stages, including exudates before chilling and zoospore release, were collected for use in the experiments described below.

The method of Kong and Hong (2010) was used for the zoosporic germination assay with slight modification. Zoospore suspension of P. erythroseptica at a concentration of 5 × 10⁵ zoospores/ml was diluted to 5 × 10² spores/ml using either tested solution or sterile distilled water and incubated at 22°C on depression well slides. The tested solutions include ZE from 5 × 10⁵ spores/ml suspension and its dilution series, liquid exudates of P. erythroseptica at different stages: 0 and 1 h after chilling, before and after zoospore release; dilution series of V8 broth, lima bean broth, and CaCl₂ solution (concentration at 0.01, 0.1, 1, 10 mM). Sterile distilled water was used as control. The original zoospore suspension without dilution was used as positive control. The zoospore suspension on depression wells were incubated at 22°C in 10 cm Petri plates over a water-soaked filter paper to maintain the moisture. After 4 h of incubation, zoospore germination was examined for 50 spores from each well. Zoosporic aggregation was recorded not only at different spore concentration but also at multiple temperatures, including 4, 15, 18, and 22°C. The experiment was conducted twice.

To determine the correlation between zoospore density and host infection, two trials were conducted. In the first trial, healthy “Russet Norkotah” (pink rot susceptible variety) tubers were surface disinfested with 0.6% sodium hypochlorite. Three eyes on each tuber were inoculated with 10 μl of zoospore suspension at different densities: 0, 1, 5, 10, 20, 50, 100, or 600 zoospores per site. Three tubers were tested for each treatment. The tubers treated with sterile distilled water were used as control. The treated tubers were incubated at 22°C on top of a plastic grid positioned over a water-soaked paper towel to maintain the moisture. After 8 days of incubation, the treated tubers were cut and exposed to the air for 15–20 min, resulting in the infected tuber tissues turn into pink color, which is a typical symptom caused by P. erythroseptica. In the second trial, surface disinfested tubers were vertically cut into 5 mm thick slices. Zoospore suspension was diluted with either ZE or sterile distilled water to different concentrations. Ten microliters of diluted zoospore suspension were inoculated onto the potato tuber slice at various densities as above. Five tuber slices were tested for each treatment. The tubers treated with sterile distilled water were used as control. The treated tubers were incubated at 22°C on top of a plastic grid positioned over a water-soaked paper towel to maintain the moisture. After 6 days of incubation, the treated tuber slices were examined for necrotic lesions. The experiments were conducted twice.

To confirm whether P. erythroseptica zoosporic germination was induced by Ca²⁺ in ZE, CaCl₂ solution at different concentrations was used to treat zoospore suspension at 1 × 10³ spores/ml. After 4-h incubation, the zoosporic germination ratio was evaluated. Sterile water was used as negative control, and ZE from zoospore suspension at 1 × 10⁵ spores/ml was used as positive control. To confirm whether the nutrient residue in ZE may contribute to zoosporic germination, diluted V8 and lima bean broth were also used to treat zoospores. The germination results were compared with that from ZE treatment.

Zoospore exudate was examined for its activity to various factors, including multiple temperature and enzymes. To test the effect of temperature, 10 ml ZE was incubated in a 15-ml tube, which was placed in water bath (VWR Scientific Inc., Radnor, PA, United States) at temperatures of 40, 60, 80, and 100°C for 20 min, or in an autoclave (Amsco Lab 250 sterilizer, Steris Inc., Mentor, OH, United States) at 121°C for 15 min. Proteinase K (Sigma-Aldrich, Inc., St. Louis, MO, United States, 37°C, pH 7.5), and catalase (MP Biomedicals, 25°C, pH 7.0) were tested for their effect on activity by incubating ZE with 10 mg/ml of each enzyme for 2 h at the optimal temperature according to the manufacturer’s instruction. Zoospore germination and tuber infection were tested using treated ZE as described above. Enzymes and solvents only were tested at the same time as negative controls. Four replicates were set for each treatment, and the experiments were conducted twice.

To prepare the samples for high performance liquid chromatography (HPLC) analysis, 200 ml ZE harvested from a high-density zoospore suspension was concentrated using a lyophilizer (Labconco, Kansas City, MO, United States). The residual powder was dissolved in 500 μl HPLC grade water. Prior to HPLC analysis, the samples were centrifuged at 12,000 g for 10 min and the supernatant was filtered through a 0.45 μm syringe filter (PTFE; 13 mm; 0.45 μm pore size; Fisher Scientific, Pittsburgh, PA, United States). Samples were analyzed using an Agilent 1100 series HPLC (Agilent Technologies, Santa Clara, CA, United States) coupled with an auto-sampler, a column thermostat, and a diode-array detector (DAD). Chromatographic separations were performed on a NH₂ column with a particle size of 5 μm Hypersil (4.6 mm × 250 mm, Phenomenex, Torrance, CA, United States). For gradient elution, mobile phase A (water) and B (acetonitrile) were employed. The following gradient was used: 0–5 min, 90% A, 10% B; 5–15 min, from 90% A, 10% B to 50% A, 50% B, linear gradient; 15–25 min, 50% A, 50% B; 25–30 min, from 50% A, 50% B to 90% A, 10% B, linear gradient; 30–40 min, 90% A, 10% B. The sample injection volume was 40 μl. The flow rate was 1 ml/min, and the column temperature was 25°C. Compounds were separated and detected by retention times and UV spectra (230 nm wave). The components of ZE were collected separately at different retention times from the HPLC. Water only was injected as control. The eluate fractions were collected at the same retention time point. The collected liquid was concentrated and dissolved in HPLC grade water and their effect on zoosporic germination was tested as described above. The HPLC fractions that had signal activity were further identified using liquid chromatography-mass spectrometry (LC-MS). Chromatographic separations were performed on a Kintex C18 column (4.6 mm × 150 mm) with a particle size 5 μm, equipped with a 4.6 mm × 12 mm guard column (Phenomenex, Torrance, CA, United States). The column compartment was maintained at 25°C. The flow rate was 0.4 mL/min, and the injection volume was 40 μL. For gradient elution, mobile phase A (0.1% formic acid) and B (acetonitrile with 0.1% formic acid) were employed. The following gradient was used: 0–5 min, 90% A, 10% B; 5–30 min, from 90% A, 10% B to 50% A, 50% B, linear gradient; 30–35 min, from 50% A, 50% B to 90% A, 10% B, linear gradient. Instrument operation and data analysis were performed using the Agilent LC-MSD Chemstation software.

Further identification was conducted by LC-MSD-ES ion trap mass spectrometry. The ion trap was operated using the ESI source in positive mode to focus on compounds producing a pseudo-molecular ion at m/z 50–500. The ion charge control target was set to 10,000 with a maximum accumulation time of 200 min. The MS conditions had been optimized with a capillary temperature of 350°C, a dry gas (nitrogen) flow rate of 9 L/min, and a nebulizer pressure with helium gas of 50 psi. The capillary voltage and trap driver voltage were maintained at 84 and 36.5 V, respectively.

Data were analyzed using JMP statistical software (version 9.0, SAS Institute Inc., Cary, NC, United States). Analysis of variance (ANOVA) was conducted to analyze treatment effects. Mean separation was performed using Fisher’s protected least significant difference. An alpha level of 0.05 was used for all analysis. In analyzing the relationship between zoosporic germination and the concentration of ZE dilution factors, regression was performed by combining the data of two independent trials, with each having three replicates. All the experiments were conducted twice. If there was no interaction between repeated trials (P > 0.05), data were combined from all trials.

Zoospores did not germinate at concentrations of ≤1 × 10³ zoospores/ml, but germination was observed when the concentration was equal to or higher than 2 × 10³ spores/ml (Figure 1, 2A,B), where the frequency of germination increased as the concentration of zoospores increased (Figure 1). No specific aggregation was observed at any concentrations or temperatures. For the tuber infection assay, necrotic lesions were obtained only on the tuber slices treated with 100 or more zoospores (Figure 3C1). Tuber slices treated with 100 zoospores had disease incidence as high as 100% (P < 0.05). The same results were observed in the whole tuber infection test (Figure 3C2). Zoosporic infection results were correlated with germination, which illustrated that germination is more critical during zoosporic host infection compared with auto-aggregation for P. erythroseptica.

Non-treated zoospores of P. erythroseptica germinated at 2 × 10³ spores/ml or higher densities (Figure 1). However, when treated with ZE, zoospores at lower concentrations (<5 × 10² spores/ml) germinated (Figure 2C). The germination rate was as high as that of zoospores at 2 × 10³ spores/ml or higher concentration (Figure 4). When ZE was serially diluted, the induced effects declined as the dilution factor increased (Figure 4). ZE did not induce zoosporic germination at 1/50 dilution, which was at the same concentration as ZE from 1 × 10³ spores/ml zoospore suspension. The liquid exudates before chilling and zoospore release did not have the same effects on zoospores of P. erythroseptica, which indicated that the signaling molecules were secreted, by the zoospores instead of by other structures of P. erythroseptica (Table 1). All those results illustrated that P. erythroseptica zoosporic germination was triggered by extracellular signal molecules released by zoospores themselves. CaCl₂ started to induce zoosporic germination of P. erythroseptica at a concentration of 10 mM (Table 1). This concentration was higher than the 10⁵ fold of Ca²⁺ in ZE tested by Warburton and Deacon (1998). Neither V8 nor lemma bean broth induced zoosporic germination at the test concentration (Table 1).

Low-density zoospore suspensions (5 zoospores/inoculation per site) did not cause symptom on tuber slice or whole tuber (Figure 3A1,A2). Zoospores at higher density (100 zoospores/inoculation per site) caused symptom on both tuber slice, as brown necrosis lesions, and on whole tuber as pink color on the cut surface (Figure 3C1,C2). However, when zoospore suspension was treated with ZE, even 5 zoospores could cause brown necrosis lesions on tuber slice and whole tuber (Figure 3B1,B2). The disease incidences had no difference between zoospores treated with ZE and non-treated zoospores at high density (P > 0.1) (data were not show).

Zoospore exudate was stable when treated with high temperature. Autoclaved ZE still induced zoosporic germination (Table 2). The germination ratio of zoospores in treated ZEs had no significant difference compared with untreated ZE, except ZE treated with 40°C, which had even higher induced activity (Table 2). Similar results were achieved in the tuber infection assay. The ZE activity was significantly reduced after treatment with proteinase K. However, catalase did not have any effect on ZE (Table 2).

The chromatogram of ZE analyzed by HPLC-DAD illustrated peaks at 3, 5, 10, 14, 15, and 16 min (Supplementary Figure S1A). Eluate fractions were collected separately into five groups at the following retention times: 2–5 min, 10–12 min, 12–14 min, 14–16 min, and 16–20 min. Compared with the control, fractions from time ranges of 12–14 min and 14–16 min induced zoosporic germination, with the 12–14 min fraction having the highest rate of germination (Supplementary Figure S1B). The eluate from 12 to 16 min was collected separately and further analyzed using LC-MS. Two amino acids were detected and identified as phenylalanine (Supplementary Figure S2A), leucine (Supplementary Figure S2B). Standard samples of phenylalanine and leucine were purchased and tested in the germination assay. The result showed that leucine induced zoosporic germination compared with control (Table 1). The length of zoospore germ tubes induced by leucine was not significantly different compared with control, which indicated that leucine induced zoosporic germination as a signaling molecule instead of as nutrient. However, phenylalanine did not have any effect on zoosporic germination.