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Development of LAMP Assays Using a Novel Target Gene for Specific Detection of Pythium terrestris, Pythium spinosum, and ‘Candidatus Pythium huanghuaiense’

    Affiliations
    Authors and Affiliations
    • Hui Feng1 2
    • Wenwu Ye1 2
    • Zhuoyuan Liu1 2
    • Yang Wang1 2
    • Jiajia Chen1 3
    • Yuanchao Wang1 2
    • Xiaobo Zheng1 2
    1. 1Department of Plant Pathology and The Key Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
    2. 2The Key Laboratory of Integrated Management of Crop Diseases and Pests (Ministry of Education), Nanjing, Jiangsu 210095, China
    3. 3College of Landscape Architecture, Jiangsu Vocational College of Agriculture and Forestry, Zhenjiang 212400, China

    Abstract

    Pythium terrestris, Pythium spinosum, and ‘Candidatus Pythium huanghuaiense’ are closely related species and important pathogens of soybean that cause root rot. However, the sequences of commonly used molecular markers, such as rDNA internal transcribed spacer 2 and cytochrome oxidase 1 gene, are similar among these species, making it difficult to design species-specific primers for loop-mediated isothermal amplification (LAMP) assays. The genome sequences of these species are also currently unavailable. Based on a comparative genomic analysis and de novo RNA-sequencing transcript assemblies, we identified and cloned the sequences of the M90 gene, a conserved but highly polymorphic single-copy gene encoding a Puf family RNA-binding protein among oomycetes. After primer design and screening, three LAMP assays were developed that specifically amplified the targeted DNA sequences in P. terrestris and P. spinosum at 62°C for 70 min and in ‘Ca. Pythium huanghuaiense’ at 62°C for 60 min. After adding SYBR Green I, a positive yellow-green color (under natural light) or intense green fluorescence (under ultraviolet light) was observed by the naked eye only in the presence of the target species. The minimum concentration of target DNA detected in all three LAMP assays was 100 pg·μl−1. The assays also successfully detected the target Pythium spp. with high accuracy and sensitivity from inoculated soybean seedlings and soils collected from soybean fields. This study provides a method for identification and cloning of candidate detection targets without a reference genome sequence and identified M90 as a novel specific target for molecular detection of three Pythium species causing soybean root rot.

    The genus Pythium includes many important plant pathogens that frequently cause seed, seedling, and root rot in economically important crops such as soybean, wheat, corn, and cotton (Rojas et al. 2017; Zitnick-Anderson and Nelson 2015). Pythium terrestris, Pythium spinosum, and ‘Candidatus Pythium huanghuaiense’ are three major species that are pathogenic to soybean, an important grain and oil crop worldwide. The soilborne P. terrestris was originally isolated from soil samples, hence its name; it can cause preemergence damping-off and root rot in soybean (Paul 2002; Zitnick-Anderson and Nelson 2015). P. spinosum infects the seeds and young roots of soybean and many other plant species, resulting in wilt, dieback, damping-off, and root rot (Chellemi et al. 2000; Zhang et al. 2018). ‘Ca. Pythium huanghuaiense’ was identified from soybean plants with seed and root rot (Feng et al. 2020). In a recent investigation in China, a total of 32 Pythium and Phytopythium species were identified from soybean roots and field soil, and P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense’ were the first (17%), second (15%), and fourth (8%) most frequently isolated species, respectively. The identified isolates of these species were pathogenic not only to soybean, but also to wheat within the crop rotation (Feng et al. 2020).

    Specific to the soybean root rot caused by several Pythium species, the aggressiveness and fungicide sensitivity of Pythium species and their adaptability to environmental factors such as temperature are often different (Broders et al. 2007; Rizvi and Yang 1996); thus, accurate identification of the pathogenic species is always a fundamental aspect of disease diagnosis and control strategy designing. Thus far, the majority of molecular assays developed for the detection of Pythium species are based on DNA technologies (Schroeder et al. 2013) such as polymerase chain reaction (PCR) (Wang et al. 2003), PCR-restriction fragment length polymorphism (Gomez-Alpizar et al. 2011), real-time PCR (Van der Heyden et al. 2019), oligonucleotide arrays (Tambong et al. 2006), and loop-mediated isothermal amplification (LAMP) (Feng et al. 2019a).

    LAMP is a popular gene-amplification procedure that can rapidly amplify nucleic acids with high specificity, sensitivity, and efficiency under isothermal conditions (Notomi et al. 2000). This method employs a DNA polymerase and a set of specially designed forward and backward inner primers (FIPs and BIPs, respectively) and forward and backward outer primers (F3 and B3, respectively) that recognize a total of six distinct sequences on the target DNA (Mori et al. 2001). Moreover, the method was further improved by forward and backward loop primers (LFs and LBs, respectively) that accelerated the LAMP reaction (Nagamine et al. 2002). LAMP detection assays have been developed for Pythium aphanidermatum (Fukuta et al. 2013), Pythium helicoides (Takahashi et al. 2014), Pythium myriotylum (Fukuta et al. 2014), Pythium irregulare (Feng et al. 2015), Pythium inflatum (Cao et al. 2016), Pythium ultimum (Shen et al. 2017), Pythium uncinulatum (Feng et al. 2019b), and P. spinosum (Feng et al. 2019a), but have not yet been developed for P. terrestris or ‘Ca. Pythium huanghuaiense.’

    The internal transcribed spacer (ITS) regions of the rDNA are broadly used to recognize and detect oomycetes, and most molecular detection assays for Pythium species are based on ITS sequences (Schroeder et al. 2013). However, this region is not adequately flexible to separate some species that are closely related in phylogeny (Kroon et al. 2004; Schena and Cooke 2006), e.g., Phytophthora nemorosa, Phytophthora ilicis, Phytophthora psychrophila, and Phytophthora pseudosyringae in clade 3a of the Phytophthora genus (Martin and Tooley 2003). As a result of the close phylogenetic relationship among P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense,’ not only their ITS2 sequences but also their cytochrome oxidase 1 and elongation factor 1-α (EF1A) gene sequences are similar, making it difficult to design specific LAMP primers that are able to distinguish among the species. For example, the ITS2 sequences differ by only 25 nucleotides between P. terrestris and ‘Ca. Pythium huanghuaiense,’ and our LAMP primers could not distinguish the two species. In previous reports, novel unique targets for the detection of Phytophthora cinnamomi (Dai et al. 2019), Phytophthora sojae (Dai et al. 2013), and P. ultimum were identified by comparative genomic analyses (Shen et al. 2017). However, the reference genome sequences of P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense’ are still lacking.

    The objectives of this study were to identify the conserved, single-copy, and highly polymorphic genes among Pythium species as candidate novel detection targets and develop LAMP assays for the rapid and accurate detection of P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense’ in plant tissues and environment such as field soils.

    Materials and Methods

    Strain sources.

    The P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense’ strains were isolated from diseased soybean roots and field soil collected from various regions in China between 2016 and 2018. The isolates were identified using morphological characteristics and by sequencing of the ITS2 (White et al. 1990) region and cytochrome oxidase I gene (Robideau et al. 2011). PCR to amplify the ITS2 and COI genes was carried out as previously described (Chen et al. 2017). PCR products were sent to GenScript (Nanjing, China) for purification and sequencing. Amplicon sequences were compared with annotated sequences in the GenBank database hosted by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) using BLASTN. The species of each isolate was determined according to its best match in the database, with matches of ≥99% for both the ITS2 and COI regions. All information on isolates used in this study, including the Pythium spp., Phytopythium spp., Phytophthora spp., and fungal pathogens, has been published (Feng et al. 2020; Zeng et al. 2017) and is maintained at the Department of Plant Pathology, Nanjing Agricultural University, China (Table 1).

    Table 1. Isolates used and the results of LAMP assays

    Culture conditions and DNA extraction.

    Mycelia of the Pythium, Phytopythium, and Phytophthora oomycete strains were cultured in V8 juice broth at 25°C for 3 days. Mycelia of the fungal strains were cultured in potato dextrose broth at 28°C for 5 days. The cultured mycelia were harvested by filtration and then stored at –70°C. Mycelial DNA was extracted using the DNAsecure Plant Kit (Tiangen, Beijing, China). DNA concentrations were estimated using a spectrophotometer, and all samples were stored at –20°C.

    Target gene identification and cloning.

    To identify the conserved, single-copy, and highly polymorphic genes as candidate novel detection targets, the predicted proteins of six genome-sequenced Pythium species (P. aphanidermatum, Pythium arrhenomanes, P. irregulare, Pythium iwayamai, P. ultimum var. sporangiiferum, and Pythium vexans; Panda et al. 2018) were assigned into ortholog groups using the OrthoMCL database (https://orthomcl.org), and the ortholog groups with single member in each species were selected. Using BLASTp (e-value <1e-40), the proteins in the selected ortholog groups were compared with the predicted proteins in P. ultimum var. ultimum genome (with a relatively higher quality; Panda et al. 2018), and the groups with every member having only one hit were finally selected (Fig. 1; Supplementary Table S1).

    Fig. 1.

    Fig. 1. Flow chart of candidate target gene identification and cloning.

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    To obtain the M90 sequences in P. terrestris, P. spinosum, or ‘Ca. Pythium huanghuaiense,’ RNA sequencing (RNA-seq) was performed using mycelial RNA. Total RNAs was extracted using a Total RNA Kit II (Omega, Norcross, GA). RNA libraries were constructed using the VAHTS mRNA-seq V2 Library Prep Kit for Illumina (Vazyme Biotech, Nanjing, China). RNA-seq was conducted on the Illumina HiSeq X Ten platform with a 150-bp paired-end configuration for sequence reads. Raw reads were filtered by removing those containing adapter, poly-N, and low-quality sequences before subsequent analyses. Transcript assembly without use of a reference genome was performed using the Trinity software package (v2.8). Based on the known M90 sequences of P. ultimum (Jiang et al. 2017), the M90 sequences in P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense’ were identified by tBLASTn search (e-value <1e-40) against the transcript assemblies.

    Subsequently, the genomic DNAs of M90 were amplified by PCR with primers M90-PyTer-F (5′-CGTCAGCACACATCTACGGTT-3′) and M90-PyTer-R (5′-GACTGATGACATGCTGGACGA-3′) for P. terrestris, M90-PySpi-F (5′-GCACCAGCACACATCTACGAT-3′) and M90-PySpi-R (5′-GACTGATGACATGCTGGACGA-3′) for P. spinosum, and M90-PyHua-F (5′-CGCACCAGCACACATCTATGA-3′) and M90-PyHua-R (5′-GACTGATGACATGCTGGACGA-3′) for ‘Ca. Pythium huanghuaiense.’ The PCR products were sent to GenScript (Nanjing, China) for purification and sequencing. Sequences were aligned using Muscle (v3.6). The BLAST and Muscle tools are both integrated in the bioinformatics software SeqHunter (v2) (Ye et al. 2010). The RNA-seq data have been deposited in NCBI (BioProject ID: PRJNA625579). The genomic DNA sequences of M90 cloned for LAMP primer design have been deposited in GenBank (accession IDs: MW725028 to MW725030).

    LAMP primer design.

    Based on the alignments of the identified M90 sequences from P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense,’ several sets of candidate LAMP primers were designed for each species using PrimerExplorer V4 (http://primerexplorer.jp/e). In each set of primers, the FIP consisted of F1c and F2 and the BIP comprised B1c and B2, the F3 and B3 outer primers were required to initiate the LAMP reaction, and one or two loop primers (LF or LF and LB) were used to facilitate the LAMP reaction. The designed primers were synthesized by GenScript (Nanjing, China).

    LAMP reaction and detection of the LAMP product.

    Each LAMP assay was performed in a 25-μl reaction mixture containing 0.8 μM each of the FIP and BIP primers, 0.1 μl of each of the F3 and B3 primers, 0.1 μM each of the LF and LB primers, 0.8 M betaine, 1.4 mM dNTPs, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 6 mM MgSO4, 0.1% (vol/vol) Triton X-100, 8 U of Bst DNA polymerase (New England BioLabs, Ipswich, MA, U.S.A.), and 4 μl of the target DNA sample. The reaction mixture was incubated in a PCR Thermal Cycler Dice (TP600; Takara Bio, Shiga, Japan) at 62°C for 70 min for P. terrestris and P. spinosum or 60 min for ‘Ca. Pythium huanghuaiense.’ Each reaction included a positive and negative control, and each sample was assayed at least three times.

    After the reaction, the LAMP product was observed after adding 0.25 μl of SYBR Green I solution. Based on the observation directly by the naked eye, an obvious yellow-green (visualized under natural light) or intense green fluorescence (visualized under ultraviolet [UV] light) indicated a positive reaction (a presence of the target DNA sequence in the extract of the sample), whereas a negative reaction (an absence of the target DNA sequence or presence of target DNA below the limit of the assay) was indicated if the color remained orange (under natural light) or no/weak fluorescence was observed (under UV light).

    Specificity of the LAMP assay.

    The different sets of candidate primers designed for each species were tested separately. First, each set of primers for P. terrestris was used to detect its different isolates, and only the primer set(s) that produced positive reactions for all isolates of the target species were selected; the same was true for P. spinosum and ‘Ca. Pythium huanghuaiense.’ Second, the selected primer sets were used to detect other oomycete and fungal species. In particular, because the three target species belong to the Pythium clade F (LeVésque and de Cock 2004), we also collected the other Pythium species in this clade (Pythium attrantheridium, Pythium intermedium, P. irregulare, Pythium kunmingense, and Pythium paroecandrum) to test the specificity. Only the primer sets that produced negative reactions for all isolates of nontarget species were selected. The specificity of each LAMP assay was assessed at least three times.

    Sensitivity of the LAMP assay.

    To determine the detection sensitivity of the LAMP assay using a specific set of primers, further LAMP assays were performed using 10-fold serial dilutions (from 10 ng to 10 fg) of genomic DNA of the corresponding target species. The sensitivity of each LAMP assay was assessed at least three times. The LAMP assays with high sensitivity (at least picogram-level detection) were selected for further assessment.

    Detection in soybean seedlings and field soils.

    Soybean seedlings were inoculated with mycelia to evaluate the application of LAMP assays as tools for detection of P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense’ in diseased plant tissues. After surface disinfection, soybean seeds were planted in sterilized vermiculite and maintained in a glasshouse at 25°C. When the first true leaf expanded, seedlings were inoculated by transferring culture discs (5 mm in diameter) from 3-day-old cultures of Pythium isolates to the soybean hypocotyls using a sterilized metal needle. As a control, seedlings were inoculated with agar plugs from sterile V8 agar. The plants were incubated at 25°C for 4 days under a 12-h photoperiod in the greenhouse. Tissues 2 cm above and below the inoculation region but excluding the inoculation region were subjected to DNA extraction. After washing the tissues with sterile water, DNA was extracted using the DNAsecure Plant Kit (Tiangen, Beijing, China). The DNA samples were evaluated using the developed LAMP assays, and each experiment was repeated at least three times.

    The LAMP assays were also used to detect P. terrestris, P. spinosum, and ‘Candidatus P. huanghuaiense’ from 104 soybean field soil samples, which were collected from Jiangsu (49), Shandong (30), Anhui (15), and Jilin (10) provinces in China in 2020. In each field, five (or four) 1,000-g soil samples were collected randomly using shovel from a depth of 10 cm below the ground surface adjacent to the soybean plant that showed root rot symptoms. Each soil sample was put in a sterile plastic bag, mixed thoroughly, and then divided into three 100-g subsamples (as three replicates). Each subsample was kept in a sterile polypropylene test tube, and the tubes were then placed in coolers on ice and transferred to the laboratory as soon as possible. DNA was extracted from every soil sample (1 g) using a PowerSoil DNA Isolation Kit (MO BIO, Carlsbad, CA, U.S.A.) according to the manufacturer’s protocol. The DNA samples were stored at –20°C within 3 months and evaluated using the developed LAMP assays.

    Results

    Identification and cloning of the novel target gene.

    To identify candidate novel targets for specific detection of Pythium species including P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense,’ we assigned the predicted proteins of six genome-sequenced Pythium species (P. aphanidermatum, P. arrhenomanes, P. irregulare, P. iwayamai, P. ultimum var. sporangiiferum, and P. vexans) using the OrthoMCL database and obtained 1,819 ortholog groups with a single member in each species. An additional sequence comparison of proteins in these ortholog groups with the predicted proteins in P. ultimum var. ultimum (with a genome of higher quality) resulted in 982 groups with every member having only one hit (Supplementary Table S1). Finally, the ortholog group OG5_126869 with a mean protein sequence identity of 79.7% was selected (Fig. 1). The OG5_126869 group contained Puf family RNA-binding proteins encoded by M90 genes. Consistent with our previous analysis (Jiang et al. 2017), the M90 gene is conserved with single copy but high sequence polymorphism among oomycetes.

    Because the genome sequences of P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense’ have not been reported, no reference sequences were available for M90 gene cloning. We performed RNA-seq to obtain de novo transcript assemblies and identify the M90 sequences in each species. Based on 2.9- to 3.8-Gb clean reads generated from RNA-seq of mycelial RNA, 32,496, 32,670, and 26,931 transcripts were assembled for P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense,’ respectively. One transcript per species was identified as homologous to the reported P. ultimum M90 sequences (Jiang et al. 2017). According to the alignment of the three identified M90 transcripts (Supplementary Data File S1), a 1.4-kb region (without introns) with abundant polymorphisms was selected as the candidate target region for LAMP primer design. Before LAMP primer design, the genomic DNA of this region was amplified by PCR and confirmed by sequencing in each species. After a screening of the different sets of candidate primers for each species, one set of primers was finally obtained for the LAMP assays of P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense,’ respectively (Fig. 2; Table 2), and the assays were termed M90-PyTer-LAMP, M90-PySpi-LAMP, and M90-PyHua-LAMP, respectively.

    Fig. 2.

    Fig. 2. Alignments of partial genomic sequences of M90 gene used to design the loop-mediated isothermal amplification primers for Pythium terrestris, Pythium spinosum, and ‘Candidatus Pythium huanghuaiense.’ The primers were designed using PrimerExplorer V4 (http://primerexplorer.jp/e). The primers B3, F1c, B2, and LF are the reverse complement sequences of the M90 gene.

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    Table 2. The primers used for the loop-mediated isothermal amplification assays

    Optimization of LAMP reaction conditions.

    Based on SYBR Green I, positive or negative reaction were easily determined by the naked eye. The color of the reaction mixture changed to yellow-green (under natural light) or intense green fluorescence (under UV light) for positive reactions, whereas the original orange color (under natural light) or no/weak fluorescence (under UV light) was a negative reaction. With the reaction reagents optimized as previously indicated (Duan et al. 2015; Lu et al. 2015), the LAMP assays were performed using pure P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense’ genomic DNA as template to determine the optimal reaction temperature and time. For example, with the reaction temperature of 62°C and reaction times >70 min, the M90-PyTer-LAMP assay cannot distinguish P. terrestris from ‘Ca. Pythium huanghuaiense’ and the M90-PySpi-LAMP assay cannot distinguish P. spinosum from P. kunmingense; with a reaction temperature of 62°C and reaction times >60 min, the M90-PyHua-LAMP assay cannot distinguish ‘Ca. Pythium huanghuaiense’ from P. terrestris. In addition, the LAMP assays possessed relatively low sensitivity (>1 ng·μl−1) when the M90-PyTer-LAMP and M90-PySpi-LAMP assays were conducted for <70 min and the M90-PyHua-LAMP assay was conducted for <60 min. Finally, the optimal LAMP assays specifically amplified the target DNAs in P. terrestris and P. spinosum at 62°C for 70 min and in ‘Ca. Pythium huanghuaiense’ at 62°C for 60 min.

    Specificity of the LAMP assays.

    The specificity of the LAMP assays was further assessed using isolates of P. terrestris, P. spinosum, ‘Ca. Pythium huanghuaiense’; other Pythium, Phytopythium, and Phytophthora species; and some fungal pathogens (Table 1). The 36 P. terrestris isolates, isolated from soybean roots and field soil, all exhibited positive reactions in the M90-PyTer-LAMP assay, whereas the other 53 non–P. terrestris strains (including P. attrantheridium, P. intermedium, P. irregulare, P. kunmingense, and P. paroecandrum belonging to Pythium clade F) all showed no color change or fluorescence, similar to the negative control (Table 1; Figs. 3 and 4). The same specificity result was observed in the test for P. spinosum and ‘Ca. Pythium huanghuaiense,’ that is, 32 P. spinosum isolates showed positive reactions, whereas non–P. spinosum isolates showed negative reactions using M90-PySpi-LAMP (Table 1; Fig. 3; Supplementary Fig. S1). Twenty ‘Ca. Pythium huanghuaiense’ isolates showed positive reactions, whereas non–‘Ca. Pythium huanghuaiense’ isolates showed negative reactions, using M90-PyHua-LAMP (Fig. 3; Table 1; Supplementary Fig. S2).

    Fig. 3.

    Fig. 3. Universal applicability of the A, M90-PyTer loop-mediated isothermal amplification (LAMP) assay for detection of Pythium terrestris, B, the M90-PySpi-LAMP assay for detection of P. spinosum, and C, the M90-PyHua-LAMP assay for detection of ‘Candidatus Pythium huanghuaiense’. A positive reaction was indicated by a color change from orange to yellow-green under natural light and an intense green fluorescence under UV light.

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    Fig. 4.

    Fig. 4. Specificity of the M90-PyTer loop-mediated isothermal amplification assay for detection of Pythium terrestris. A positive reaction was indicated by a color change from orange to yellow-green under natural light and an intense green fluorescence under UV light.

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    Sensitivity of the LAMP assays.

    Serial 10-fold dilutions (from 10 ng to 10 fg) of pure P. terrestris, P. spinosum, or ‘Ca. Pythium huanghuaiense’ genomic DNA were used to evaluate the sensitivity of each LAMP assay. The minimum concentration detected in the LAMP assays of P. spinosum, P. terrestris, and ‘Ca. Pythium huanghuaiense’ was 100 pg·μl–1 (Fig. 5). The sensitivity was evaluated at least three times, and all results were consistent.

    Fig. 5.

    Fig. 5. Sensitivity of the loop-mediated isothermal amplification assays for detection of the indicated species. A positive reaction was indicated by a color change from orange to yellow-green under natural light and an intense green fluorescence under UV light. Plus and minus signs indicate the positive and negative controls, respectively.

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    Detection in inoculated soybean tissues and collected field soil samples.

    To evaluate the LAMP assays for detection of the pathogens in infected plants, DNA was extracted from soybean seedlings inoculated with P. spinosum, P. terrestris, or ‘Ca. Pythium huanghuaiense’ mycelia. Three Pythium species were respectively detected in the inoculated soybean seedlings and showed positive reactions consistent with the positive control, whereas samples from noninoculated healthy soybean seedlings showed no color change, consistent with the negative control (Fig. 6A). Based on the 104 soil samples collected from fields in 2020, P. terrestris was detected in eight samples from all four provinces (Anhui, five; Shandong, Jiangsu, and Jilin, one each); P. spinosum was detected in seven samples from three provinces (Anhui and Jiangsu, three each; Jilin, one); ‘Ca. Pythium huanghuaiense’ was detected in only one sample from Jiangsu province (Fig. 6B; Table 3). In addition, P. spinosum, P. terrestris, and/or ‘Ca. Pythium huanghuaiense’ could be isolated from a portion of the LAMP-positive soil samples, rather than from the LAMP-negative soil samples (Table 3). These results indicated that the LAMP assays were useful for the detection of P. spinosum, P. terrestris, and ‘Ca. Pythium huanghuaiense’ in infected tissues or soil samples collected in field.

    Fig. 6.

    Fig. 6. Application of the loop-mediated isothermal amplification assays for detection of the three Pythium species in inoculated A, soybean seedlings and B, soybean field soils. A positive reaction was indicated by a color change from orange to yellow-green under natural light and an intense green fluorescence under UV light. The plus and minus signs indicate the positive and negative controls, respectively. A, samples 1 to 4 and 5 to 8 were collected from four different inoculated soybean seedlings and four different healthy soybean seedlings, respectively. B, results of 12 samples shown as examples. Samples 1, 6, and 8 to 12 are soil samples from Anhui province; sample 2 is a soil sample from Shandong province; samples 3 to 5 are soil samples from Jiangsu province; and sample 7 is a soil sample from Jilin province.

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    Table 3. Summary of the LAMP detection results for the collected soybean field soils

    Discussion

    The process of disease control requires highly specific and sensitive detection of pathogen infection. P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense’ are the three major Pythium pathogens causing soybean root rot, one of the most economically important diseases in soybean production. In this study, we developed three LAMP assays for specific detection of the three closely related species using the M90 gene, a new target identified from comparative genomic analysis.

    Many reported LAMP detection targets were based on housekeeping genes (Parkinson et al. 2009), presence of singleton genes, or sequences identified by suppression subtractive hybridization (Ballard et al. 2011). However, LAMP primers based on the ITS sequence failed to distinguish P. ultimum from its related Pythium spp. because of insufficient polymorphisms (Shen et al. 2017). This issue was also apparent in the distinction among P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense’; although our developed LAMP primers based on the ITS2 sequence could distinguish P. spinosum from P. terrestris (Feng et al. 2019a), they failed to distinguish P. spinosum from ‘Ca. Pythium huanghuaiense.’ In addition, although bioinformatics tools have been increasingly used to identify targets for molecular diagnostics in many plant pathogens (Bühlmann et al. 2012; Gétaz et al. 2017), the genome sequences of three Pythium species are not available for a direct identification of novel specific targets. Thus, finally, we performed a comparative genomic analysis based on the genome-available Pythium spp. to identify candidate detection targets and used a de novo sequencing method based on RNA-seq and transcript assembly to obtain their sequences in P. terrestris, P. spinosum, and ‘Ca. Pythium huanghuaiense’ for primer design. At the same sequencing depth, RNA-seq, in which only transcripts other than the complete genome are sequenced, is a relatively more economical and handy method to obtain assemblies with high coverage and quality compared with genome sequencing; however, the candidate transcript should be confirmed by PCR amplification and genomic DNA sequencing and checked for introns.

    The M90 sequences not only exhibited high polymorphisms among the three closely related Pythium species but also showed consistent identity within species in our further sequencing of PCR fragments from additional species. The intraspecific identity of M90 gene sequences was also found in Phytophthora sojae based on a comparative genomic analysis among its four representative species (Ye et al. 2016). Therefore, the M90 gene could also be a potential target for molecular detection and identification of other Pythium species, and even other oomycete species. In addition to M90, we also provided a complete list of the identified candidate target genes, which would be alternative opinions for the development of other molecular detection assays in the future (Supplementary Table S1).

    Compared with conventional and real-time PCR assays, LAMP assays are more rapid, simpler, and more sensitive. LAMP assays require only a heating block or water bath instead of a thermal cycler to incubate the reaction mixture. These assays can be finished in approximately 1 h under isothermal conditions, whereas a typical 30-cycle PCR takes nearly 2 h. The results of LAMP assays are easier to determine because the amplified products can be detected visually by addition of SYBR Green I. The sensitivity evaluation showed that the detection limit of all three developed LAMP assays was 100 pg·μl–1, which has reached the sensitivity level useful for disease detection. High sensitivity would easy to be subject to aerosol pollution, resulting in false-positive results (Wan et al. 2019; Yuan et al. 2018). Compared with traditional PCR, LAMP assays required relatively low purity of template DNA to detect the target from the mixed DNA of the diseased tissue or soil, indicating that the LAMP assays are reliable and suitable for diagnosis of the three Pythium species in the field.

    DNA-binding dyes such as the fluorescent dye SYBR Green I possess specific molecular structures that allow them to bind selectively to double-stranded DNA and cause a visible color change of the dye. The sensitivity of detection using DNA-binding dyes is considerably higher than that obtained according to turbidity alone (Zhang et al. 2014). Drawbacks of the DNA-binding dyes include inhibition of the LAMP biochemical amplification process, meaning that the reagents must be added at the end point, after LAMP reaction (Mori et al. 2006). The risk of contamination of amplicons is increased because of the need to open the reaction tube to add the dye. DNA aerosol drifting into other reaction tubes may result in a certain degree of green fluorescence under UV light. However, the negative readings under bright light may show a distinct orange color different from green, and, under UV light, the fluorescence intensity of positive reaction and negative reaction vary greatly (positive reaction showed intense green fluorescence, whereas negative reaction showed weak fluorescence).

    In conclusion, this study provides a de novo method for identification of novel target sequences without a reference genome sequence and revealed M90 as a novel specific target for molecular detection and identification of Pythium spp. and even other oomycete species. The three developed assays will be useful for pathogen detection and disease diagnosis of soybean root rot.

    The author(s) declare no conflict of interest.

    Literature Cited

    The author(s) declare no conflict of interest.

    Funding: This work was supported by grants from the National Natural Science Foundation of China (31972250; 31721004), the China Agriculture Research System (CARS-004-PS14), and the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions (PPZY2015B157).