Introduction

The oomycete Phytophthora melonis Katsura, which was first described by Katsura (1976), is conspecific with P. sinesis and has been reported in China, Japan, Egypt, Turkey, Korea, and India (Ho et al. 2007). P. melonis can cause severe damage to cucumbers (Cucumis sativus) in affected fields, and areas receiving heavy rain may suffer from plant mortality and yield losses of up to 80 % (Wang et al. 2007). P. melonis also infects zucchini (Cucurbita pepo L.), hami melon (Cucumis melo L.), wax gourd [Benincasa hispida (Thunb.) Cogn.], pointed gourd (Trichosanthes dioica Roxb.), and pistachio (Pistacia vera L.) (Chen et al. 2012), causing blight, dieback, and root, foot, and crown rots (Mirabolfathy et al. 2001). A multi-locus phylogeny for Phytophthora utilizing markers derived from complete genome sequences shows that P. melonis is closely associated with P. sinensis and they are placed in Clade 7b containing a variety of species, including P. cajani, P. vignae, P. pistaciae, P. sojae, among others (Blair et al. 2008). To control the spread of P. melonis, early detection and diagnosis of the pathogen in the field is essential.

Traditional detection methods, such as baiting and culturing from plant tissue, are difficult, time consuming, and require extensive experience. With the development of DNA-based techniques, polymerase chain reaction (PCR) has become a primary method of plant pathogen identification and detection (Henson and French. 1993; Trout et al. 1997). P. melonis–specific primers based on the internal transcribed spacer (ITS) have been used for rapid identification of the pathogen in infected plant tissues, soil, and water (Wang et al. 2007). Previous studies have suggested that the ITS regions are highly conserved in different fungal species, evolve more quickly than other regions, and may vary among species within a genus (Pavón et al. 2008; Silvar et al. 2005). However, ITS regions are not always sufficiently diverse to allow the separation of closely related taxa (Kroon et al. 2004; Schena and Cooke. 2006; Schena et al. 2006). Therefore, more recent studies have also examined the Ras-related protein gene Ypt1, β-tubulin, elicitin, and the spacer region between the mitochondrially encoded cox1 and cox2 genes in Phytophthora species (Martin et al. 2012; Meng and Wang. 2010; Schena et al. 2008). The Ypt1 gene appears to be a more promising target because its coding (exons) and non-coding (UTRs and introns) regions have polymorphisms which can be used as molecular markers (Haubruck et al. 1987). Based on the Ypt1 gene, specific primers were designed for the detection of Phytophthora species, including Phytophthora inundata, Phytophthora kernoviae, and Phytophthora lateralis (Martin et al. 2012).

The PCR technique offers great promise for plant pathogen detection, most significantly for its advantages in speed, sensitivity, specificity, and flexibility (Trout et al. 1997). However, the associated diagnostic procedures still require long periods of time and expensive laboratory instrumentation and reagents. These drawbacks limit its routine use, especially in poorly resourced laboratories and in rural areas of developing countries. Thus, a rapid, sensitive, simple, and economical detection method for practical applications is preferred. Recently, the novel loop-mediated isothermal amplification (LAMP) technique has been developed (Nagamine et al. 2001; Notomi et al. 2000; Rigano et al. 2010). This assay uses the Bst DNA polymerase, which has displacement activity and a set of four specially designed primers that recognise a total of six distinct sequences of the target DNA (Notomi et al. 2000). This technique, which employs four primers, has been used to perform highly specific and sensitive amplifications of DNA to detect pathogens, including viruses, bacteria, and fungi (Nagamine et al. 2002; Notomi et al. 2000). The LAMP assay can be performed under isothermal conditions ranging between 60 °C and 65 °C, and it produces large amounts of replicate DNA (Notomi et al. 2000). The reaction shows a high tolerance to biological contaminants (Kaneko et al. 2007), which can help to avoid false-negative results due to inhibition of the enzyme (a common problem with PCR). Although LAMP amplification products can be detected by gel electrophoresis, they also should be detected using simple visual inspection by SYBR Green I and calcein intercalating DNA dyes (Niu et al. 2012).

Recently, the LAMP assay has proven to be effective in the diagnosis of oomycetes such as Phytophthora spp. (Martin et al. 2012). Faster, simpler, and more-specific molecular detection of P. ramorum by LAMP in the field was first described by Tomlinson et al. (2007). However, no study has reported the detection of P. melonis using a LAMP assay. In this study, we developed specific PCR and LAMP assays for P. melonis detection based on the Ras-related protein gene Ypt1, and demonstrated that this method is specific and efficient.

Materials and methods

Sources of isolates

The origin, host, and number of isolates used in this study are listed in Table 1. We tested 36 P. melonis isolates sampled from a wide geographic distribution in China, as well as 34 isolates of 13 different oomycetes (e.g. P. sojae, P. vignae, P. capsici, P. parasitica and P. infestans) and 22 isolates of other fungi isolated (e.g. Fusarium oxysporum, Colletotrichum capsici, Alternaria alternate and Sclerotinia sclerotiorum) from cucumbers and other hosts.

Table 1 Oomycetes and fungi isolates used in the PCR assay
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Culture conditions and DNA extraction

Isolates of Phytophthora spp. were cultured in a tomato juice medium (20 % v/v tomato juice, 0.1 g l−1 CaCO3, and 15 g l−1agar). Mycelia of each Phytophthora and Pythium isolate were obtained by growing the isolates in tomato juice broth at 18–25 °C (temperature dependent on isolate) for at least 5 days. Mycelia of the fungi were grown in potato dextrose broth. The mycelia were harvested by filtration and freeze dried for 36 h. DNA was extracted using the cetyltrimethylammonium bromide (CTAB) procedure, as described previously. Purified DNA was quantified using a spectrophotometer, and aliquots were diluted to 100 ng μl−1 in distilled water as stocks at −20 °C.

Primer design for P. melonis

Alignment of the Ypt1 gene sequences present in National Center for Biotechnology Information databases and belonging to Phytophthora species closely related to P. melonis was used to identify conserved and differing regions, which include P.cinnamomi (DQ270317); P. melonis (EF649778); P. capsici (FJ535571); P. sojae (DQ162958); P. parasitica (DQ864604); P. infestans (JN678988); P. cactorum (HQ850001) (Blair et al. 2008). On the basis of P. melonis (EF649778) regions of divergence, the specific primer pair PmY1F/PmY1R was designed to amplify the P. melonis–specific regions of the Ypt1 gene, and the nested PCR primer pair PmY1F/PmY2R was also developed to increase the sensitivity (Table 2, Fig. 1). Based on the P. melonis Ypt1 gene sequence, a set of LAMP primers comprising two outer (F3 and B3) and two inner (FIP and BIP) primers was designed using the LAMP primer software PrimerExplorer V4 (http://primerexplorer.jp/elamp4.0.0/index.html). The designed primer sequences for P. melonis and their relative position in the sequence are shown in Table 1 and Fig. 1.

Table 2 PCR and LAMP primers used in this study
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Fig. 1
figure 1

Partial sequence of Phytophthora melonis Ypt1 gene (EF649778) and the location and sequences of three PCR primers (PmY1F, PmY1R, and PmY2R) and four LAMP primers (F3, B3, F2, F1c, B2, and B1c) targeting the P. melonis isolate. FIP is a hybrid primer consisting of the F1c sequence and the F2 sequence, BIP is a hybrid primer consisting of the B1c sequence and the B2 sequence. Arrows indicate the direction of extension

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Extraction of P. melonis DNA from cucumber and soil

DNA samples from P. melonis–infected cucumber were performed as previously described (Tooley et al. 1997). A sample (10 mg) of tissue (stem or leaf) was cut from each plant, placed into 10 μl freshly prepared 0.5 M NaOH, and macerated with a plastic pestle. The tubes were then centrifuged at 12,000 × g for 5 min, and 5 μl supernatant was removed and immediately diluted with 195 μl 100 mM Tris (pH 8). DNA from soil and water were isolated as previously described (Wang et al. 2007). Soil DNA samples collected from infected and uninfected cucumbers in the field were extracted using a FastDNA® SPIN Kit for Soil from BIO 101® Systems (Q-Biogene Ltd, Irvine, CA). The samples were then used immediately for PCR or frozen at −20 °C for later use.

Detection of P. melonis by PCR

The Reaction mixtures (final volume, 25 μl) contained 1.4 mM MgCl2, 50 mM KCl, 10 mM Tris–HCl (pH 8.3), 200 μM each dNTPs, 10 pmol of each primer, 1U Taq DNA polymerase, and 20 ng template. All reactions were performed in a PTC200 Thermo Cycler (MJ Research, Watertown, MA, USA) and programmed for an initial denaturation step at 95 °C (5 min) followed by 35 cycles of denaturation for 1 min at 95 °C, annealing for 1 min at 58 °C, extension for 1 min at 72 °C, and a final extension for 5 min at 72 °C. Nested PCR included two rounds of amplification using the universal primers Yph1F/Yph2R for the first round and the P. melonis–specific primers PmY1F/PmY2R for the second round (Table 2), and 1 μl amplified product from the first round as the template and a 203-bp product was amplified. The sensitivity of the primer pair PmY1F/PmY2R was tested by using different concentrations of the template, ranging from 1,000 ng to 1 fg of isolate purified DNA as a template. Negative controls lacking template DNA were performed in each experiment to test for contaminated reagents. All reagents used for PCR amplification were purchased from TaKaRa (Dalian, China). All of the experiments were repeated at least three times.

LAMP reaction

The LAMP reaction was performed using a LoopAmp DNA amplification kit (Eiken Chemicals Co. Ltd, Tokyo, Japan) in a 25-μl volume. The reaction mixture contained 40 pmol each of FIP and BIP, 5 pmol each of F3 and B3 primers, 2.0 μl template DNA (~10 ng), 1 μl Bst DNA polymerase (8 units), and 12.5 μl 2× reaction mix prepared in the kit. Negative controls containing nuclease-free water instead of DNA were included in each assay. To identify the optimal temperature and time for visual LAMP amplification, the reactions were performed in 60 °C, 62 °C, 63 °C, and 65 °C water baths for 15, 30, 45, 60, and 75 min. Finally, the reaction was terminated by heating the reaction mixture at 80 °C for 10 min. Amplification was monitored with SYBR Green I on UV lamp or the products (2 μl) were electrophoresed on 2.0 % agarose gels to determine the optimal conditions (Li et al. 2013), and the results of flourescence correlated well with gel electrophoresis. All of the experiments were repeated at least three times.

LAMP testing of diseased cucumbers

To develop field application of LAMP for P. melonis surveys and management, 15 cucumber wilt samples (stem and leaves) induced by P. melonis and 13 soil samples collected from a greenhouse and inoculated artificially with the pathogen were surveyed using the LAMP method. Diseased cucumbers were grown in a growth chamber. Zoospores for inoculation were performed as previously described (Ristaino 1990). P. melonis was grown on V8 agar petri plates for 1 week. The agar with fungal hyphae was then cut into 0.5-cm squares and placed in a sterile petri dish with sterile distilled water. The solution was replaced with fresh water every 3 days. After 1 week, the sporangia were given a cold shock (1 h at 4 to 6 °C), followed by a 1-h equilibration at room temperature. The zoospores were separated from the solution by filtering through 2 layers of cheesecloth. Zoospores (106 spores/ml) were applied to the cucumbers within 30 min of filtering. Non-infested cucumbers were used as controls. We also collected cucumbers and soil samples from different natural fields in the Fujian province for LAMP detection. DNA extraction was performed and the LAMP react ions were performed at 63 °C for 60 min. Amplification was monitored with gel electrophoresis and SYBR Green I.

Results

Detection of P. melonis using a PCR assay

The specificity of PCR primers was assessed using a large collection of 36 P. melonis isolates, and 56 isolates of other oomycetes and fungi (Table 1). The primer pair PmY1F/PmY1R was able to amplify a unique DNA fragment approximately 410 bp in length (Fig. S1) in all P. melonis isolates from different regions and host plants in China. This primer pair did not yield amplification products when using isolates of any other oomycete or fungus tested. Phytophthora spp. Ypt1 universal primers Yph1F/Yph2R (Table 2), designed to amplify a DNA fragment in Phytophthora spp. (Schena et al. 2008), produced positive PCR reactions in all Phytophthora isolates tested, although the amplification of fungal DNA was not achieved using this primer pair (data not shown).

Optimisation of LAMP assay

LAMP was performed using P. melonis DNA as a template to determine the optimal temperature and reaction time. Optimisation of LAMP reaction temperature and time revealed that the ideal conditions for the primer were 63 °C for 60 min. Therefore, the LAMP assays were performed under these conditions. As expected, the typical ladder-like pattern was observed on 2.0 % agarose gel electrophoresis in all positive samples, but not in the negative controls. Under visual fluorescence detection with SYBR Green I, positive and negative results were easily determined; positive reactions appeared green, whereas the negative control remained orange (Fig. 2).

Fig. 2
figure 2

Specificity of Phytophthora melonis DNA detection by LAMP. LAMP assay and visual inspection by adding SYBR Green I dye and observing under visible light. Positive reactions turned green after the addition of SYBR Green I. Lane 1, negative control; lane 2, positive P. melonis control; lanes 3–8, P. melonis isolates; lanes 9–16, other oomycete and fungal isolates (P. capsici, P. sojae, P. parasitica, P. infestans, Pythium aphanidermatum, Fusarium oxysporum f.sp. cucumerimum, Rhizoctonia solani, Colletotrichum lagenarium). The same results were obtained in all three replicates

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Specificity of detection of P. melonis using LAMP

The specificity of the LAMP reaction was confirmed by electrophoresis in 2.0 % agarose gels stained with ethidium bromide and direct visual inspection of the LAMP products by adding SYBR Green I dye. As expected, positive reactions were observed in all P. melonis isolates, but not in other Phytophthora spp. or isolates of true fungi (Fig. 2). The LAMP reaction yielded the same results as the PCR assay (Fig. S1). The results indicated that the LAMP technique developed in this study is highly specific for P. melonis.

Sensitivity detection of P. melonis using nested PCR and LAMP assays

The sensitivity of the primers PmY1F⁄PmY1R was tested by using different concentrations of DNA, ranging from 10 μg to 1 pg P. melonis isolate as a purified DNA template. The sensitivity of the primers was further tested by a nested PCR. All of the experiments were repeated at least three times and the amplicons were detected by agarose gel electrophoresis and visual inspection. As shown in Table 3, the minimum detection concentration required for the LAMP assay was 10 fg genomic DNA, i.e. the sensitivity of the LAMP assay was 1000-fold higher than that of conventional PCR with a detection limit of 10 pg for a 60-min reaction (Table 3). However, a second amplification conducted using 1.0 μl amplified product from the first round PCR as the template and the primers PmY1F/PmY2R increased the sensitivity of the assay by at least 1,000 times to 10 fg per 25.0 μl reaction volume (Table. 3). Our results suggest that the nested PCR assay has the same sensitivity as the LAMP assay (Table 3).

Table 3 The sensitivity of LAMP, Nested PCR and PCR assays in detecting Phytophthora melonis using serial DNA dilutions
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Detection of P. melonis from inoculated samples

To demonstrate the applicability of the LAMP assay to field samples, this method was evaluated using plant tissue and soil infested with P. melonis. The plant tissue and soil from healthy cucumbers grown axenically in a greenhouse were used as negative controls, and purified DNA from P. melonis was used as a positive control. DNA extracted from infected tissue and soil samples was subjected to LAMP. A total of 28 samples of P. melonis–infected plant tissue and soil, and three healthy plant tissues were also analysed. All infected plant tissue and infested soil samples tested positive for P. melonis by LAMP, while no healthy sample showed any electrophoresis band or green stain (Fig. 3).

Fig. 3
figure 3

LAMP detection of Phytophthora melonis from infected tissues and soil. a LAMP assay detected by agarose gel electrophoresis. b LAMP assay and visual inspection by adding SYBR Green I dye and observing under visible light. Three independent replicates gave the same results. Lane 1, purified DNA (positive control); lanes 2 and 5, DNA from healthy cucumbers and soil (negative controls); lanes 3 and 4, DNA from infected tissues and infested soil. Lane M, DL2000 DNA molecular marker. The same results were obtained in all three replicates

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Evaluation of PCR and LAMP assays using diseased cucumbers in the field

To evaluate PCR and LAMP assays for the detection of P. melonis, 56 healthy-looking but pathogen-infected cucumbers and eight healthy cucumbers collected from different areas of Fujian, China, between 2009 and 2012 were tested using nested PCR and LAMP assays. The isolation of P. melonis from these samples was performed using conventional isolation methods. The positive-sample ratios were 46/56 (82.1 %) for nested PCR, 48/56 (85.7 %) for the LAMP assay, and 37/56 (66.1 %) for the traditional isolation method (Table 4). Samples obtained from the control group were inspected using Phytophthora isolation, nested PCR, and LAMP assays, and all samples were negative for P. melonis. The isolation of P. melonis from these samples was performed using conventional methods for detecting P. melonis involving the visual examination and culturing of plant tissue (Wang et al. 2007). The Ypt1 LAMP assay reported here, therefore, be used for the visual detection of P. melonis in plants and production fields.

Table 4 Comparison of isolation, nested-PCR, LAMP for the detection of Phytophthora melonis in collected plant samples
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Discussion

Traditional methods for the identification of P. melonis are time consuming and require considerable expertise to differentiate between species of Phytophthora based on morphologic characteristics. The LAMP reactions may be completed within 60 min by incubating at 63 °C and terminated by heating the reaction mixture at 80 °C for 10 min. Results can be visually inspected using SYBR Green I and confirmed by gel electrophoresis. The LAMP method has an extremely high amplification efficiency, due in part to its isothermal nature (Notomi et al. 2000); there is no time lost arising from changes in temperature for enzyme function, and the inhibition that occurs at later stages of amplification, a typical problem with PCR, is less likely to take place (Osawa et al. 2007). In addition, LAMP amplifies DNA at higher concentrations than PCR, making it convenient for visualisation of amplification products without gel electrophoresis in resource-poor settings. Moreover, the sensitivity of the Nested PCR assay was 1000-fold higher than that of conventional PCR with a detection limit of 10 fg, the detection limit of the Nested PCR assay is equivalent to the LAMP assay (Table 3) and Real-time PCR (Poppert et al. 2005; Skottman et al. 2007). Our results suggest that nested PCR assay has the same sensitivity as the LAMP assay in the P. melonis detection.

Historically, the ITS region has been used because it is possible to design general primers and the regions shows relatively high polymorphism. More recently, several alternative nuclear loci such as 60S ribosomal protein L10, β-tubulin, enolase, HS protein 90, large subunit rRNA, TigA gene fusion, and translation elongation factor 1α, and mitochondrial loci such as cox1, cox2, nad1, nad9, rps10, and secY, have been sequenced for phylogenetic resolution within Phytophthora, and these loci are also useful for molecular identification purposes (Martin et al. 2012). Molecular tools based on the restriction fragment length polymorphism (RFLP) of ITS region and ITS sequencing in Indian P. melonis isolate has suggested that they are 100 % similar with the five isolates of GenBank including a P. sinensis (Guharoy et al. 2006). Meanwhile, the Ypt1 gene has been applied for the rapid detection of P. nicotianae (Meng and Wang. 2010). To the best of our knowledge, this is the first report of the application of the Ypt1 for the detection of P. melonis. The greatest advantage of this gene is that its alternating conserved and variable regions allow the development of nested PCR with a first round using universal primers Yph1F/Yph2R, and a second round using species-specific primers. This allows the use of a common amplified product from the first amplification as a template for all specific primers, significantly reducing time and cost (Schena et al. 2006). LAMP is a novel, sensitive and rapid detection technique and can be applied for disease diagnosis in the field. The LAMP primers were selected from the Ypt1 gene, which is highly specific to P. melonis. The four primers (F3, B3, forward inner [FIP], and backward inner [BIP]) targeted six regions of P. melonis (Fig. 1) and provided additional levels of specificity when compared to PCR primers (i.e. by targeting two regions). Using this detection system, target DNA was amplified within 60 min under isothermal condition at 63 °C plus 10 min at 80 °C and visualised on agarose gel. Also, LAMP was observed directly in the reaction tube by addition of SYBR Green I for a naked-eye inspection. The sensitivity assay showed that this method also had a detection limit of 10 fg DNA template. Of the 36 P. melonis and other non–P. melonis strains tested, the LAMP assay had 100 % inclusivity and 100 % exclusivity. This level of specificity was the same as that of nested PCR–based assays evaluated simultaneously in this study. Overall, these data revealed that the LAMP method was equivalent to the nested PCR method in specificity and sensitivity for P. melonis detection. Considering that the LAMP method has an advantage in its performance and low cost, this technique is more suitable for the detection of P. melonis in the field.

Several studies have reported the use of the LAMP method to detect various pathogens using an expensive real-time turbidimeter for reaction confirmation (Mori and Notomi. 2009; Nakao et al. 2010; Yamazaki et al. 2010). The use of expensive equipment decreases the versatility of LAMP and limits the use of this procedure, especially in developing countries. Thus, rapid and unambiguous visual inspection of LAMP results is essential for diagnostics, and several fluorescent intercalating dyes (such as SYBR Green I or calcein) have been developed to enable visual discrimination of positive samples. Here, the addition of SYBR Green I after incubation resulted in a colour change from orange to green in positive amplifications, which could be judged under natural or ultraviolet light, and agreed with the results of gel electrophoresis. However, all fluorescent intercalating dyes should be systematically compared and analysed for their rapidity, simplicity, sensitivity, and cost effectiveness in the LAMP assay (Dai et al. 2012; Mao et al. 2012). Then, the most effective and practical indicator should be employed for LAMP detection.

The LAMP protocol described in this study represents a very sensitive, specific, and rapid diagnostic protocol for P. melonis detection. To the best of our knowledge, this is the first study to use the LAMP technique to detect P. melonis. This protocol is useful for detecting low levels of P. melonis in plant tissues and it can be used to confirm the early stages of P. melonis infection at a relatively low level. Thus, P. melonis–carrying cucumbers can be identified during the early stages of infection, and management can be devised before the infection becomes epidemic.