We examined 43 isolates of P. infestans randomly collected from different geographic areas in China, 29 isolates from 15 different Phytophthora spp., along with 15 isolates from other oomycetes and fungi. The origin, host, and number of isolates examined in current study are listed in Table 1.

The isolates and fungi were carefully amended on rye agar (Erwin and Ribeiro, 1996) or potato dextrose agar. Pathogen mycelia were grown in potato dextrose broth at 18–20°C (temperature varied depending on the pathogen) for 4–5 days, and mycelia were harvested and freeze dried. Total DNA extraction was done via DNA extraction kit (BioTeke Corp., Beijing, China). Extracted DNA concentration was checked using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, United States) and aliquots were diluted to 100 ng/μL using RNA–free, sterilized with double-distilled water, followed by storing at -20°C.

Total DNA was extracted from P. infestans-infected potato tissues (leaf and stem) with the Cetyl-Trimethylammonium Bromide (CTAB) procedure, as described earlier (Zhang et al., 2006). DNA samples from infected potato tissues were extracted rapidly, as described previously (Tooley et al., 1997) but with minor modifications. A 10-mg sample from a diseased potato plant (stem or leaf) and healthy tissues were excised and added to freshly prepared 0.5 M NaOH with a volume of 10 μl in each 1.5 mL EP (Eppendorf tube). The samples were crushed with a glass pestle and then subjected to centrifugation for approximately 5 min at 12,000 × g. After centrifugation, 5 μL of the liquid phase was diluted directly with 195 μL of 100-mM Tris (pH 8.0 adjusted). The extracted DNA samples were used directly for the LAMP reaction, or were frozen at -20°C for future usage. The products were tested further by electrophoresis in 2% agarose gel stained with ethidium bromide.

In this study, Ypt1 (Ras-related protein) was used as a target gene to detect P. infestans isolates collected from various regions of China. We collected sequence information for P. infestans in the Ypt1 gene, which had been analyzed in a previous study and documented in GenBank (accession number JN678988); we then performed a BLAST search of the National Center for Biotechnology Information (NCBI) database using sibling species to identify the Ypt1 conserved region. PCR universal primers, comprising forward and backward primers, were selected from a previous study of Phytophthora spp. based on sibling target genes (Chen et al., 2013). The P. infestans fragments that were isolated from pure cultures of the same species were cloned and sequenced. Based on the P. infestans Ypt1 target sequence, four LAMP primers were designed, consisting of two outer primers (F3 and B3) and two inner primers (FIP and BIP) using PrimerExplorer V4 software¹ (Eiken Chemical Co., Tokyo, Japan) as shown in Figure 1. Primers for nested PCR were constructed via Primer Premier 5 software, and real-time PCR primers were constructed using Beacon Designer software with the Ypt1-based sequence. Information on the primers used in the current study is presented in Table 2 and Figure 1.

The reaction mixtures (final volume: 25 μL) consisted of 12.5 μL of 2× TaqPCR MasterMix (TIANGEN, Beijing, China), 10 pmol of each primer, 1 μl of 100-ng template DNA, and sterilized double distilled water to create a final volume of 25 μl. All reactions were completed in a PTC200 Thermo Cycler (MJ Research, Watertown, MA, United States) and preset for an initial denaturation step at 95°C for 3 min, followed by 30 cycles of denaturation with a temperature of 95°C for 1 min, annealing for 1 min at 54°C, extension for 1 min at 72°C, with a final extension temperature of 72°C for 5 min to evaluate the universal primers (Yph1F/Yph2R). Nested PCR consisted of two amplification rounds, thereby using Phytophthora spp. universal primers (Yph1F/Yph2R) as outer primers in the first round and P. infestans-specific primers nPCRF/nPCRR were used in the second amplification round (Table 2); by using 1 μL of amplified product from the first round as a template DNA by amplifying a product of 203-bp. The second round PCR protocol started with a preliminary denaturation step at 95°C for 3 min, followed by 32 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 66°C, extension for 1 min at 72°C, and a final extension temperature of 72°C for 5 min. The sensitivity of primer pairs from different PCRs was tested using 10-fold serial dilution of the template DNA, ranging from 100 ng to 10 fg of isolated, purified DNA as a template. Negative controls devoid of template DNA were used in each experiment to check for contaminated reagents. All reagents used in this experiment for PCR amplification were purchased from TaKaRa Bio Inc. (Dalian, China). All products were then subjected to electrophoresis on 2% gel stained with ethidium bromide. All experiments were repeated at least three times.

All quantitative real-time PCR assay reactions were performed using a CFX96 real-time detection system (Bio-Rad, Hercules, CA, United States) by adding Maxima^TM SYBR Green I qPCR Master Mix (Purchased from TaKaRa, Dalian, China) in 0.5-ml thin-walled, optical grade PCR tubes for a final reaction volume of 25 μl. The reaction mixture consisted of 12.5 μl of Maxima^TM SYBR Green I qPCR Master Mix, 1 μl (10 μM) of each primer (qPCR F and qPCR R), along with 1 μl of DNA template, with sterilized double distilled water added to create the final volume. The amplification conditions for the reaction were 95°C for 30 s, with 40 cycles of 95°C for 5 s, followed by 60°C for 30 s, 72°C for 20 s, and a fluorescence read at 72°C at the end of each cycle, with a final melting curve at 65–95°C with an addition of 0.1°C s^-1. DNA was serially diluted using Easy Dilution (TaKaRa) to calculate the amplification efficiency of sensitivity with an estimated initial concentration of 1.28 × 10² ng μL^-1, diluted in a 1:10 series from 1.28 × 10² ng μL^-1 to 1.28 × 10^-4 ng μL^-1. The results of the assay were analyzed by plotting the log of the template DNA concentration against cycle threshold (C_t) values, using the formula E = [10 (-1/slope) - 1] × 100 to calculate the PCR efficiency, where E is the amplification efficiency and the slope is derived from the plot of log of the template DNA concentration against C_t. Values of Ct, standard curves, and corresponding correlation coefficients (R²) were generated automatically by Sequence Detection System v.1.2 software, by interpolating C_t values against the logarithm of the initial DNA concentrations.

To identify the optimal reaction for visual LAMP amplification, a range of concentrations of FIP/BIP (0.8–2.4 mM), F3/B3 (0.1–0.5 mM), MgSO₄ (2–10 mM), dNTPs (0.2–2 mM), and Betaine (0.8–1.6 M) (Sigma–Aldrich, St. Louis, MO, United States), and different incubation times (30–70 min) and temperatures (57–67°C) were used. The LAMP reaction system used in this study consisted of 12.5 μl 2 × LAMP Buffer [10xThermopol Buffer, dNTPs (25 mM), Betaine (5 M), MgSO₄ (50 mM), and Tween20 (2.5%)], 4 μl of FIP/BIP and F3/B3 primers, 1 μl of 50 μM Calcein-500 μM MnCl₂, 1 μL of 8000 U/ml Bst DNA Polymerase (Purchased from BioLab^® Inc., New England), 1 μL of template DNA (100 ng/μL) and double distilled water to make the final volume of 25 μL. The LAMP products were visually inspected by naked-eye or by running gel electrophoresis to conclude the optimal reaction conditions. All reactions were performed in 0.2-mL sterilized microtubes in a temperature-controlled water bath. A positive reaction yielded a green color, and a negative reaction yielded a brown color, through the quenching effect of 50 μM Calcein-500 μM MnCl₂. Nuclease-free water was used in negative reaction tubes instead of DNA.

To determine the specificity of LAMP for the target Ypt1 gene, 43 isolates of P. infestans from different geographic areas, 29 isolates from 15 different Phytophthora spp., and 15 other oomycetes and fungi (Table 1) were subjected to LAMP assays. The amplified LAMP products were detected as described above. All the experiments in the present study were repeated at least three times.

To determine the sensitivity of LAMP, 10-fold dilutions ranging from 1.28 × 10² to 1.28 × 10^-4 ng μL^-1 (1.28 × 10² ng μL^-1; 1.28 × 10¹ ng μL^-1; 1.28 ng μL^-1; 1.28 × 10^-1 ng μL^-1; 1.28 × 10^-2 ng μL^-1; 1.28 × 10^-3 ng μL^-1; 1.28 × 10^-4 ng μL^-1) of purified P. infestans DNA were prepared. Based on the Ypt1 gene sequence, we designed a specific nested PCR primer pair, nPCR-F/nPCR-R, and the sensitivity of the primers was evaluated to compare the LAMP assays. Nested PCR was performed as described above using a PTC-200 PCR apparatus (MJ Research). The LAMP amplification products were detected as described above. Nested PCR amplification products were separated by electrophoresis on 2.0% agarose gels stained with ethidium bromide, and visualized under ultraviolet (UV) light. All experiments carried out were repeated at least three times.

An infection time course experiment was performed to evaluate the earliest time point when P. infestans was detected by LAMP. Potato plants were grown in a greenhouse in plastic pots filled with a mixture of equal proportions of loamy soil, sand and composted farmyard manure. The plants were maintained at a mean daily temperature of 22°C under a 12-h day photoperiod. A suspension of P. infestans sporangia were made consisting of 10 sporangia/μL (Hussain et al., 2005). The leaves of the 6-week-old each potato plant was spray inoculated with 50 μL of the suspension (10 sporangia/μL) allowing them to stay at room temperature for different time span (1, 2, 4, 8, 16, and 32 h) along with negative control using double distilled water inoculation. After the given time span, the leaves was detached and washed thoroughly with tap water and rinsed three times with sterile distilled water. Total DNA was extracted rapidly from plant tissues as described above (Tooley et al., 1997). The DNA extracted was then subjected to LAMP assays. The amplified LAMP products were detected as described above. All the experiments were repeated at least three times.

To evaluate the application of LAMP for P. infestans detection in field sample, healthy-looking (symptomless) but pathogen-infected potato were collected from three different locations for LAMP detection. Total DNA was extracted from diseased plant tissues (potato leaf and stem) following the CTAB procedure, with fast DNA extraction via the NaOH procedure described above. Healthy plant tissues were used as controls, and LAMP reactions were performed with a constant temperature of 65°C for 60 min in water bath. Amplified products were observed using 2% agarose gel electrophoresis. P. infestans infection was confirmed using the conventional culture method for the identification of plant pathogens based on morphological characteristics (Fry, 2008). All the experiments conducted were repeated at least three times.

We performed the LAMP assay using P. infestans DNA as a template to determine the optimum primer concentrations, time, temperature, and amount of dNTPs, betaine, and MgSO₄. The results revealed an optimal temperature of 65°C, and an optimal time of 60 min. In our experiment, 1.5 μM FIP/BIP primers, 0.7 mM dNTPs, 0.8M betaine, and 4 mM MgSO₄ represented the best conditions. As expected, a ladder-like pattern on 2% agarose gel was observed for all isolates showing a positive reaction, but no such pattern was seen for negative reactions (Supplementary Figure S1). The characteristics of the products amplified were confirmed by sequencing, as the sequences obtained perfectly matched the expected DNA sequences of P. infestans (data not shown). Visual inspection of LAMP products revealed a green color for all positive reactions and a brown color for negative reactions (Supplementary Figure S1). Green color appeared through the addition of Calcein-MnCl₂ in the LAMP assay due to its quenching effect. All reactions in this assay were performed at least three times.

The specificity of the reaction was confirmed by direct visual observation through the addition of Calcein-MnCl₂ during the reaction, and the same products were confirmed for LAMP in electrophoresis in 2% agarose gel stained with ethidium bromide. The results showed that LAMP Ypt1 primer amplification yielded positive reactions when tested with 43 different isolates of P. infestans collected from different geographic areas in China; the same LAMP Ypt1 primers could not yield positive reactions when tested with 29 isolates from different Phytophthora spp., including two other closely related Clade 1 species of P. infestans, and 15 other oomycetes and fungi; thus, the high specificity of the primers was confirmed (Table 1 and Figure 2). Tubes showing a positive reaction exhibited a ladder-like structure, unlike tubes showing a negative reaction when subjected to gel electrophoresis (Figure 2B). All reactions were performed at least three times. Nested PCR yielded the same specificity as the LAMP assay. As expected, a positive reaction was observed for all P. infestans isolates; however, no positive reaction was observed for other species, oomycetes, or fungi (Supplementary Figure S2). The specificity of the LAMP reaction was in line with that of PCR, which was further confirmed by 2% agarose electrophoresis on gel stained with ethidium bromide. The results revealed that LAMP and PCR assays were highly specific for P. infestans, based on the Ypt1 gene.

Sensitivity was assessed using 10-fold serial dilution of genomic DNA by LAMP, nested PCR, and conventional PCR. The sensitivity of the LAMP assay was 1.28 × 10^-4 ng μL^-1 of genomic DNA per reaction. The resulting products were further analyzed through 2% agarose gel electrophoresis and visual inspection by the naked eye (Figure 3). Sensitivity was further assessed by nested PCR, and the primer pairs detected 1.28 × 10^-3 ng μL^-1 of purified DNA per 25 μL of reaction volume (Figure 3). Comparatively, the least detection limit for the PCR assay was 1.28 × 10^-1 ng of genomic DNA, revealing that LAMP and the nested PCR assay were far more sensitive than the conventional PCR assay. All assays in this experiment were repeated at least three times (Figure 3 and Table 3).

Real-time PCR reactions were performed using 10-fold dilution of target genomic DNA based on the Ypt1 target gene, yielding to P. infestans C_t values (22.51 ± 0.08–35.75 ± 0.39) and negative control de-voiding of template DNA, thereby quantifying the specificity of the reaction. The 10-fold dilution sensitivity of template DNA determined by quantitative real-time PCR ranged from 1.28 × 10² to 1.28 × 10^-2 ng μL^-1, amplifying the 89-bp product (Figure 4 and Supplementary Figure S3); the C_t value and target DNA were linearly correlated with an extremely high coefficient of determination (y = –3.281x + 38.877, R² = 0.9992).

To evaluate the earliest time point of detection of P. infestans, an time course experiment was performed after artificial infection of potato leaves with low quantities of inoculum. LAMP assays after 1, 2, 4, 8, 16, and 32 h showed that infected leaves at all different time points gave positive results, while the uninfected control showed no amplification (Supplementary Figure S4A). The amplified products were subjected to gel electrophoresis to further confirm the reaction shown in Supplementary Figure S4B. All reactions were repeated at least three times with consistent results.

To confirm the application of the LAMP assay in the field, pathogen-infected (but healthy-looking and symptomless) tissues collected from infected fields and healthy plants control were subjected to LAMP assays using two different DNA extraction methods. Both extraction methods showed positive reaction results, indicating pathogen-infected tissues and positive controls, but no reaction for healthy plants or negative controls. The positive reaction exhibited a ladder-like pattern when subjected to 2% electrophoresis on gel stained with ethidium bromide, whereas healthy plants and negative controls showed no bands, thereby confirming the specificity of the Ypt1 primer pairs. The results of the LAMP assay and gel electrophoresis are shown in Figure 5. The isolation of P. infestans from these samples was performed using conventional isolation methods. The positive-samples (healthy-looking but pathogen-infected) were confirmed by the traditional isolation method. This was not the case for the samples in which P. infestans was not detected by the conventional culture method. Samples obtained from the healthy control were negative for P. infestans. These findings indicate the high detection competency of the LAMP assay.