Introduction

The oomycete genus Phytophthora includes many plant pathogens that cause destructive diseases and severe commercial losses, not only to agricultural crops, but also to forest trees and nursery plants. Many new species of Phytophthora have been identified from diseased nursery trees and plants in natural ecosystems, and new species continue to be found (Brasier 2009). With the expansion of global plant trade, Phytophthora species have also spread globally (Brasier 2009). These pathogens usually cause little damage to their host plants in their native habitats and have coevolved with their hosts, achieving a natural balance. However, when introduced by human activity into other regions with a favorable environment that lacks natural enemies, they can be highly virulent and cause serious damage on genetically susceptible host plants (Brasier 2008).

More precise phytosanitary measures are required to prevent economic loss of crops on a global scale and to conserve endemic biodiversity. In Japan, P. ramorum was isolated during quarantine inspection from discolored leaves of rhododendrons imported from the United Kingdom in 2015 (Sakoda et al. 2017). In Japan, P. ramorum and P. kernoviae are considered the most important quarantined species, and only P. nicotianae is exempt from quarantine, since it is already a major pathogen. Molecular identification techniques are required for simultaneous detection of Phytophthora species; therefore, we developed a simple identification technique using loop-mediated isothermal amplification (LAMP) with a quenching probe (QProbe) (Hieno et al. 2020) that was designed for a sequence specific to P. nicotianae in the amplified region of a LAMP primer specific for Phytophthora. The total time from sampling to detection is approximately 2 h; thus, it is a time-saving technique for import/export quarantine. Other advantages include high tolerance to inhibitors from biological sources, and thus, easy DNA extraction methods are applicable (Hieno et al. 2019). However, the multiplex reaction in the LAMP method presents difficulties in that the strong amplification of more abundant DNA may overwhelm the amplification of less abundant DNA, and the sensitivity of multiple targets may be reduced (Hieno et al. 2021). In contrast, PCR can achieve high sensitivity in a simultaneous detection method (Li et al. 2011). In this study, we aimed to develop a multiplex PCR method using primers specific for Phytophthora at the genus level and primers specific for P. nicotianae (Li et al. 2011) as an accurate and time-saving method to detect multiple targets. This multiplex PCR can be used to inspect samples for the broader Phytophthora genus and for P. nicotianae whether the pathogens are present in the sample or subject to quarantine or not. Furthermore, the genus-specific primers are expected to be used for sequencing to identify the Phytophthora species detected in the sample. In previous studies on PCR techniques using primers specific for Phytophthora species, most primers were designed for the ITS region, but identical ITS sequences have been identified for 16 pairings of species from molecular phylogenetic clades 1, 5, 6, 7 or 8, making identification of the species difficult (Yang and Hong 2018). The introns of the Ypt1 gene are sufficiently polymorphic to discriminate all Phytophthora species and are located near the conserved coding regions, which is suitable for designing primers specific to the genus Phytophthora (Schena and Cooke 2006). In the present study, we designed genus-specific primers for Ypt1 by modifying the primers reported by Schena et al. (2008). The modified forward and reverse primers have been used for multiplex PCR detection of the kiwifruit pathogens Phytophthora cactorum, P. cinnamomi and P. lateralis (Bi et al. 2019). The modified primers have the potential to be used for detecting additional species of Phytophthora but have not been tested with enough strains of Phytophthora and with other genera, including closely related Pythium and Phytopythium.

In this study, we reevaluated the applicability of our modified genus-specific primers by using a sufficient number of strains, then developed a duplex PCR using the Phytophthora genus-specific primers and plant primers (Martin et al. 2004) as an internal control. We also developed a triplex PCR using additional P. nicotianae-specific primers (Li et al. 2011) for effective quarantine control in Japan. By combining this multiplex PCR method with a simple method to extract DNA from diseased plants, we aimed to establish a more practicable and user-friendly protocol for quarantine inspections.

Materials and methods

Isolates and mycelial DNA extraction

Isolates of Phytophthora spp., Phytopythium spp., Pythium spp. and other pathogens used in this study are listed in Table 1. For the mycelial DNA extraction, isolates were grown on V8 juice agar plates [Miller (1955) with the following modifications: 1 l including 162 mL V8 juice (Campbell Japan), 20 g agar, pH modified with CaCO3] at 25 °C until the mycelium reached the edge of the plates. The mycelium was scraped from the plates with inoculation needles into 1.5-ml Eppendorf tubes containing 100 µl of 50% PrepMan Ultra Reagent (Thermo Fischer Scientific, Waltham, MA, USA) and incubated at 100 °C for 10 min. After 3 min at room temperature, the sample was centrifuged at 15,000 rpm for 3 min. The supernatant was transferred to a new 1.5-ml tube. The DNA concentration was measured using the QuantiFluor dsDNA System (Promega, Madison, WI, USA) and adjusted to 100 pg/µl with Tris–EDTA buffer (TE buffer; 10 mM Tris–HCl, 1 mM EDTA, pH 8.0). The samples were stored at 4 °C until further use.

Table 1 PCR with Phytophthra genus-specific primers
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DNA preparations from inoculated and from naturally infected plants

The host–pathogen combinations used in the inoculation tests are shown in supplementary Table S1. P. nicotianae (GK10Eg1 and 13ASP1-1), P. capsici (CH01CUCU10 and CH02UE0202), P. hedraiandra (TGTA1-1) and P. melonis (CH00ME21-21) were grown on V8 juice agar plates at 25 °C until mycelial growth reached the edge of the plate, and 6- or 10-mm-diameter mycelial disks were taken from actively growing colonies. For tomato, eggplant, pumpkin and cucumber, the smaller mycelial discs were placed on the fruit. Inoculated materials were placed on wet paper towels in plastic trays, which were covered with polyethylene bags to maintain high humidity and incubated in a growth chamber (12 h light/12 h dark) at 25 °C for 5–7 days until symptoms were obvious. For hydrangea in pots, the larger mycelial disks were placed on a needle wound on the basal stem, then wrapped in parafilm. The whole plant was covered with a polyethylene bag to maintain humidity and kept at room temperature (approximately 25 °C) in the laboratory for 10 days until symptoms were seen. For ivy, P. citrophthora (CH94HE11) was cultured in V8 juice broth in a 6 cm Petri dish until the entire surface was covered with hyphae (~ 4 days). The broth was then removed with a filter paper, and the mycelia were homogenized with 100 mL of sterilized distilled water at 3,000 rpm for 5 min. Young ivy leaves were then detached and placed in the mycelial suspension for 7–10 days at 20 °C until symptoms were obvious.

Detached leaves of rhododendron, Japanese andromeda and camellia were inoculated with one or two mycelial disks (7 mm) P. ramorum (CBS 101553), P. kernoviae (P19875) and P. lateralis (P3361) in a plastic box as described by Hieno et al. (2021) with 12 h light at 20 °C/12 h dark at 15 °C for 3–5 days until symptoms were seen.

Symptomatic stems of periwinkle from Gifu, Japan and tobacco plants from Java island, Indonesia were used as naturally infected samples.

For DNA extraction from symptomatic leaves or stems, a 5 × 5 mm leaf piece or 0.2 g of epidermis shaved from the stem was shredded with a blade, then all material for the test plant was placed in a 1.5-ml Eppendorf tube. DNA was extracted using the Kaneka Easy DNA Extraction Kit version 2 (Kaneka, Tokyo, Japan) and the manufacturer’s protocol. All extracts were diluted 20 times with TE buffer and stored at 4 °C until further use.

Primers specific for Phytophthora

In our previous study (Bi et al. 2019), we designed a primer pair specific for Phytophthora (Yph1F_mod2: CGACCATKGTGGACTTTG, Yph2R_mod2: ACGTTCTCRCAGGCGTATCTG) based on the Phytophthora-specific primers (Yph1F and Yph2R) of Schena et al. (2008). In the present study, we further tested the specificity and applicability of our genus-specific primer pair (Bi et al. 2019) using 222 Phytophthora isolates that represented 155 taxa and 104 isolates that belonged to other genera (Table 1). We then used this primer pair to develop the multiplex PCR assay (described later) to detect Phytophthora species from infected plants.

Simplex PCR using genus-specific primers for Phytophthora

The reaction mixture contained 0.5 µM of each primer (Yph1F_mod2, Yph2R_mod2), 0.625 U Taq HS DNA polymerase (Takara Bio, Kusatsu, Shiga, Japan), 0.2 mM dNTP mixture, 1 × PCR buffer (10 mM Tris–HCl pH 8.9, 50 mM KCl and 1.5 mM MgCl2), 10 ng of bovine serum albumin (Merck KGaA, Darmstadt, Germany), and 0.1 ng of DNA template, in a total volume of 25 µl. The PCR was run in a BioRad T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) at 95 °C for 2 min; 40 cycles of denaturation at 94 °C for 30 s, annealing at 62 °C for 45 s, an extension at 72 °C for 30 s; and a final extension at 72 °C for 10 min.

Applicability of the primers was evaluated in simplex PCR for a wide range of strains with different molecular phylogenetic groups (André Lévesque and De Cock 2004; Abad et al. 2022) as shown in Table 1. The sensitivity of the simplex PCR was tested with serial dilutions (10 pg–1 fg per reaction) of mycelial DNA of P. ramorum (Pr-1), P. kernoviae (P19875) and P. nicotianae (CBS 305.29). The simplex PCR was also tested for detectability of DNA from inoculated and naturally infested plants (Supplementary Table S1).

Duplex and triplex PCR

Plant primer pair (FMPl-2b and FMPl-3b) reported by Martin et al. (2004), which amplifies cox1, was used as an internal control to determine the success or failure of DNA extraction and PCR of the test samples. The plant species used to test primer detectability are shown in Table 2. We extracted DNA from plant samples and used it in PCR tests as described for the simplex PCR. The same DNA thermal cycler was used, but thermocyling conditions were 95 °C for 8.5 min; 40 cycles at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min; and 72 °C for 10 min.

Table 2 List of various plants used for the versatility test of the plant PCR primers and amplification results
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Duplex PCR for plant–Phytophthora spp. was also done as described for the simplex PCR above except for the concentrations of MgCl2 and plant primers. MgCl2 and the plant primers were tested in combinations of 1.5, 2.0, 2.5 mM and 0.05, 0.125, 0.25, 0.5 µM, respectively, to determine the optimal concentration of each.

Triplex PCR for plant–Phytophthora spp.–P. nicotianae was performed as described above for the duplex PCR, but using the primer pair specific for P. nicotianae, Nic-F1/Nic-R1 reported by Li et al. (2011). The primers were tested at 0.05, 0.1, 0.25, 0.5, 1.0 μM to determine the optimal concentration.

To evaluate the sensitivity of the duplex and triplex PCRs, we used mixtures of plant DNA extracted from aseptically cultivated plants (tomato and cucumber) and mycelial DNA of P. nicotianae (CBS 305.29), P. capsici (P1319) or P. melonis (P6870), which was serially diluted (10 pg–1 fg per reaction). Tomato and cucumber seeds were soaked in 10% v/v H2O2 solution for 20 min and then washed three times with sterile distilled water. The sterilized seeds were sown in 75 × 75 × 10 mm plant boxes on Murashige-Skoog agar (Fujifilm Wako, Osaka, Japan; 0.8% agar). The plants were grown at 25 °C for 2 weeks in a growth chamber. For aseptically grown plants, DNA was extracted from 5-mm lengths of the hypocotyl for tomato and 5 × 5 mm piece of the main leaf for cucumber as described above.

The DNA detectability of the duplex or triplex PCR was tested using DNA extracted as described above from inoculated and naturally infected plants (Supplementary Table S1).

PCR products were separated by electrophoresis in 2.5% Agarose S (Fujifilm Wako, Osaka, Japan). Gels were stained with Gel Red (10,000 × , Biotium, Fremont, CA, USA) and photographed under ultraviolet light. All experiments were done at least twice.

Results

Specificity and sensitivity of Phytophthora genus-specific primers

The modified primers were tested with more genera, species, and strains to examine the reliability of the detection of the genus Phytophthora. The simplex PCR assay enabled the amplification of mycelial DNA of all 222 Phytophthora isolates among 155 taxa (Table 1). The amplification efficiency varied among species, and P. aquimorbida was the most difficult to detect (detectable in three of five replicate reactions using at least two DNA extractions from the same isolate).

None of the DNA was amplified from the 97 non-target isolates, representing 55 species of Phytopythium, Pythium, Globisporangium, Elongisporangium, and one isolate each of seven species of soil-borne pathogens (Table 1). However, DNA from some closely related species was nonspecifically amplified including Pythium adhaerens and Py. plurisporium, one of three isolates of Py. periilum, and six of seven isolates of Globisporangium heterothallicum, Phytopythium cucurbitacearum, two of nine isolates of Pp. vexans and one of three isolates of Pp. mercuriale (Table 1).

The detection limit of the modified primer pair using mycelial DNA of P. ramorum, P. kernoviae, and P. nicotianae was determined to be 100 fg (Supplementary Fig. S1).

Application of simplex PCR using diseased plants

The genus-specific simplex PCR assay using the modified primer pair was tested in symptomatic plants (ivy, tomato, hydrangea, rhododendron, Japanese andromeda, and camellia) that had been inoculated with or naturally infected (periwinkle and tobacco) with various Phytophthora species. Phytophthora was detected in all inoculated and naturally infected plants (Supplementary Table S1), and no amplicons were obtained using healthy plant tissues.

Development of duplex PCR assay

As an internal control to determine the success or failure of DNA extraction and PCR detection, we developed a duplex PCR to combine our genus-specific primers with the plant primers reported by Martin et al. (2004). The plant primers amplified DNA from all 41 tested plant species of 33 families belonging to 21 orders (Table 2). In the tests for optimal concentrations of magnesium and each primer, in the assay, the higher the magnesium concentration, the higher the activity of the polymerase and the higher the amplification efficiency, but excessive polymerase activity resulted in a loss of specificity and non-target amplification. When we tested several combinations of magnesium concentrations (1.5, 2.0, 2.5 mM) and plant primer concentrations (0.05, 0.0625, 0.125, 0.025, 0.5 µM) using the same concentrations of genus-specific primers as in the simplex PCR reaction solution, DNA extracted from several diseased plant species were amplified stably using 2.5 mM MgCl2 and 0.05 µM of each plant primer (1/10 the concentration of the genus-specific primers) (data not shown). The detection limit of the duplex PCR with the mixture of DNA from aseptically grown plants and from mycelia was 10 to 100 fg for plants and 1 pg for Phytophthora spp. (Supplementary Fig. S2).

Development of the triplex PCR assay

Because P. nicotianae is already present in Japan and thus exempt from quarantine inspections, we needed a triplex PCR assay to simultaneously detect Phytophthora species and P. nicotianae in plant samples. In the tests of concentrations for the specific primers for P. nicotianae in the triplex PCR, the DNA for the three targets was amplified almost equally by using P. nicotianae specific primers at 1/10 the concentration of the genus-specific primers, as found for the plant primers. The detection limit of this triplex PCR using the mixture of DNA from aseptically grown plants and mycelia, was 1 pg for Phytophthora spp. and 100 fg for plants and P. nicotianae (Supplementary Fig. S3).

Detection of Phytophthora species in diseased plants using duplex and triplex PCR

The results of the PCRs using plants that were inoculated (eggplant, tomato, and pumpkin) or naturally infected (periwinkle and tobacco) are shown in Supplementary Table S1, with representative agarose gels of the amplicons in Figs. 1 and 2. The Phytophthora species were detected in all the diseased plant samples, and amplification of the plant DNA (cox1) was confirmed in all samples. These results indicate that DNA was extracted using the Kaneka Easy DNA Extraction Kit version 2 from diseased samples, and the presence or absence of Phytophthora was accurately determined. Triplex PCR results showed that the P. nicotianae-specific primers detected P. nicotianae from eggplant and tomato plants that were inoculated with the pathogen and from naturally infected periwinkle and tobacco; no amplicons were obtained from pumpkins inoculated with P. capsici. Thus, the duplex and triplex PCR tests confirmed the success of DNA extraction and PCR detection and determined whether the cause was, in fact, Phytophthora, and if the pathogen was determined to be in the triplex PCR, whether or not it was P. nicotianae, using only one tube.

Fig. 1
figure 1

Duplex PCR detection of Phytophthora spp. from various plant species inoculated with different pathogens. Plant–pathogen combinations: Eggplant 1 and tomato, P. nicotianae GK10Eg1; Eggplant 2, P. nicotianae 13Asp1-1; Pumpkin 1, cucumber 1 and 2, P. capsici CH01CUCU10; Pumpkin 2, P. capsici CH02UE0202; ivy, P. citrophthora CH94HE11; Hydrangea 1 and 2, P. hedraiandra TGTA1-1; Cucumber 3 and 4, P. melonis CH00ME21-21. PC: positive control, mycelial DNA of P. nicotianae CBS 305.29, P. capsici P0253. P. citrophthora P3693, P. hedraiandra P11725 and P. melonis P6870. NC: negative control, sterile distilled water. Black arrowhead: amplicons using Phytophthora genus-specific primers (around 470 bp). White arrowhead: amplicons using plant primers (approximately 140 bp)

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Fig. 2
figure 2

Triplex PCR detection of Phytophthora spp. and P. nicotianae using DNA from symptomatic plants that were inoculated or naturally infected. a, Inoculated plants. Eggplant 1 and tomato were inoculatsed with P. nicotianae GK10Eg1. Eggplant 2 was inoculated with 13Asp1-1. Pumpkin 1 and 2 were inoculated with P. capsici CH01CUCU10 and CH02UE0202, respectively. b, Naturally infected plants. PC, positive controls using mycelial DNA of P. nicotianae CBS 305.29 (PC 1) or P. capsici P0253 (PC 2). NC, negative control using sterile distilled water. Black arrowhead: amplification with Phytophthora genus-specific primers (around 470 bp). Gray arrowhead amplicons using P. nicotianae-specific primers (267 bp). White arrowhead: amplicons using plant primers (approximately 140 bp)

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Discussion

Here we tested our previously designed primer pair to detect Phytophthora genus-specific, which were based on the primers of Schena et al. (2008) and Bi et al. (2019), using mycelial DNA from 155 taxa (including subspecies, varieties and hybrids), representing members from all Phytophthora clades (1–10; Abad et al. 2022). All the tested taxa were detected (Table 1). When non-targets of the primer pair were tested, among 97 isolates representing 55 species of closely related genera, DNA was amplified from only three species and from a small portion of the isolates tested for four species. Thus, our improved primers detected all species of Phytophthora tested, although a few closely related species of Pythium, Globisporangium and Phytopythium yielded false positives (Table 1). Many more taxa were tested than in previous reports of genus-specific detection: 101 species (Bilodeau et al. 2014), 45 species (Scibetta et al. 2012), 35 species (Schena et al. 2008), and 136 taxa (Miles et al. 2015). Therefore, our primers will benefit quarantine efforts, where strict border control is required and detection without omission is of utmost importance.

The detection limit of the simplex PCR using mycelial DNA of P. ramorum, P. kernoviae, and P. nicotianae was 100 fg for all three species (Supplementary Fig. S1), the same detection limit as that of the nested PCR of Schena et al. (2008). We were able to achieve the same level of sensitivity as that of Schen et al. (2008) in one round of amplification. In a comparison of the electrophoretic results for the 155 taxa in the specificity test, the intensity of the amplicon band varied among species, even though 100 pg of DNA was used in all cases (data not shown). Amplification failed only for a few PCR replicate tests for P. aquimorbida (2 of 5 tests) and P. macilentosa (1 of 4 tests), both minor pathogens. Thus, the detection results for plant samples that are potentially infected with those two species should be evaluated carefully. However, for other species, positive results were obtained in all repeated tests.

For the duplex and triplex PCR for diseased plants, the composition of the reaction solution, including the concentrations of primers and magnesium, were optimized so that differences in amplification between primers due to competition between polymerase and substrates could be reduced. The detection limits for Phytophthora DNA in the multiplexed PCRs using mixtures containing plant DNA were 10 times higher than those of simplex PCR using only mycelial DNA (Supplementary Figs. S1–S3). Knowing that detection sensitivity is better with higher quality DNA, we modified the extraction method of Kageyama et al. (2003) to obtain higher quality DNA and increased the sensitivity to 100 fg (data not shown). For the present study, considering the huge number of samples tested during quarantine inspections, we selected the Kaneka Easy DNA Extraction Kit version 2 method for its simple, rapid extraction of DNA (Hieno et al. 2019). Thus, users should choose DNA extraction and detection methods (simplex PCR or multiplex PCR) that are best for their situation and objectives.

Our newly designed triplex PCR was able to simultaneously determine the presence of a Phytophthora species and P. nicotianae, the only Phytophthora species not subject to quarantine in Japan, in a one-tube reaction. In addition, Ypt1 regions have accumulated sufficient mutations to be highly discriminative of species so that the genus-specific primer can be used sequencing the amplicons to identify the species. In addition, the simplex PCR amplicons from DNA extracted from diseased plants (naturally infected tobacco sample no. 1 with P. nicotianae and rhododendron inoculated with P. kernoviae or P. lateralis; Supplementary Table S1) were subjected to a sequencing analysis, and these species were identified (data not shown). Although we previously developed a LAMP assay using a QProbe to simultaneously detect Phytophthora spp. and P. nicotianae (Hieno et al. 2020) that is a rapid, accurate highly applicable method for import/export quarantine inspections, it cannot be used to identify the actual species of Phytophthora, except for P. nicotianae. By comparing the advantages of each detection method, it is possible to select the method that best meets the conditions required by the user.

Molecular detection methods can also be used for practical inspection of nonsymptomatic plants. Fichtner et al. (2012) pointed out that symptomless infections by P. kernoviae in North American native plants may thwart pathogen detection and underscore the importance of implementing a proactive and adaptive biosecurity plan. Harris and Webber (2016) also pointed out that symptomless infections of larch by P. ramorum can lead to an underestimation of infection plants. We are confident that our optimized detection method can contribute to more effective quarantine control.