A Phytophthora conserved transposon-like DNA element as a potential target for soyabean root rot disease diagnosis

Authors


E-mail: wangyc@njau.edu.cn

Abstract

A transposon-like element, A3aPro, with multiple copies in the Phytophthora sojae genome, was identified as a suitable detection target for this devastating soyabean root rot pathogen. The PCR primers TrapF1/TrapR1 were designed based on unique sequences derived from the transposon-like sequence. A 267-bp DNA fragment was amplified using this primer pair, the specificity of which was evaluated against 118 isolates of P. sojae, 72 isolates of 25 other Phytophthora spp., isolates of Pythium spp. and isolates of true fungi. In tests with P. sojae genomic DNA, detection sensitivities of 10 pg and 10 fg DNA were achieved in standard PCR (TrapF1/TrapR1) and nested PCR (TrapF1/TrapR1 and TrapF2/TrapR2), respectively. Meanwhile, PCR with TrapF1/TrapR1 primers detected the pathogen at the level of a single oospore, and even one zoospore. These primers also proved to be efficient in detecting pathogens from diseased soyabean tissues, residues and soils. In addition, real-time quantitative PCR (qPCR) assays coupled with the TrapF1/TrapR1 primers were developed to detect and quantify the pathogen. The results demonstrated that the TrapF1/TrapR1 and TrapF2/TrapR2 primer-based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in plants and in production fields.

Introduction

Phytophthora sojae is currently one of the most devastating pathogens of soyabean (Glycine max), causing damping off to seedlings and root rot to older plants, with an annual worldwide loss of $1–2 billion (Wrather & Koenning, 2006). Since first being reported around 1950 in Indiana and Ohio (Kaufmann & Gerdemann, 1958), P. sojae has become widespread in many soyabean-producing countries (Schmitthenner, 1985; Erwin et al., 1996). In the past few years, the disease also has caused serious soyabean losses in Heilongjiang province of China (Zhu et al., 2000). Root rot caused by Phytophthora may be found at any stage of soyabean development. Areas that receive heavy rain may suffer plant mortality and yield losses of up to 100% in parts of affected fields. Phytophthora sojae is a homothallic species that produces a lot of oospores in infected soyabean tissues (Lee et al., 1993). One oospore develops after an antheridium fertilizes an oogonium. Once the oospore germinates, the inner wall is absorbed and the germ tube produces either a sporangium or mycelium (Schmitthenner, 1999). Phytophthora sojae produces heterokont, biflagellate zoospores that are released from sporangia into water. The zoospores rapidly develop into adhesive cysts upon reaching the potential host plants. Infection occurs when cysts germinate to form appressoria or produce hyphae. The hyphae directly penetrate the plant epidermis, causing serious root rot (Enkerli et al., 1997; Tyler, 2007). Growing soyabeans in continuous monoculture over several years results in increased losses to disease (Schmitthenner, 1985). Considering that increasing numbers of soyabean shipments are being traded to and from different countries and areas, root rot caused by P. sojae poses a serious threat to worldwide soyabean production. Therefore, rapid, accurate and sensitive detection is important for managing this disease and preventing its spread. The current protocols to regulate the spread of P. sojae are based on the Chinese quarantine industry standards SN/T1131-2002 and SN/T2474-2010. The detection methods of SN/T1131-2002 involve baiting from soils or isolation through selective media from infected plant tissues, followed by identification based on morphological characteristics (Canaday & Schmitthenner, 1982; Oudemans, 1999). This traditional detection method plays an important role, but the isolation and accurate identification of P. sojae is very complex and time-consuming. DNA-based techniques provide an effective means for identification of plant pathogens. The polymerase chain reaction (PCR) offers advantages over more traditional methods of pathogen detection because the organisms need not be cultured and the technique is rapid, with high sensitivity and specificity (Ersek et al., 1994; Bonants et al., 1997; Lacourt & Duncan, 1997; Cullen et al., 1999). The specificity of PCR-based detection technology is determined by the target gene sequences used, and many primers have been designed based on the internal transcribed spacer (ITS) region (Cooke et al., 1995; Bonants et al., 1997; Tooley et al., 1997; Trout et al., 1997; Liew et al., 1998; Schubert et al., 1999; Judelson & Tooley, 2000; Winton & Hansen, 2001; Grote et al., 2002; Ippolito et al., 2002). The methods of SN/T2474-2010 are based on the ITS region with primers PS1 and PS2 for molecular detection of P. sojae, drafted in the laboratory of the present study (Wang et al., 2006). Apart from the ITS region, other genes, such as β-tubulin (Fraaije et al., 1999), actin-coding sequences (Weiland & Sundsbak, 2000), translation elongation factor 1-alpha (EF-1α; O’Donnell et al., 1998), cytochrome oxidase (Martin et al., 2004), elicitin (Lacourt & Duncan, 1997), etc. are easy to detect. However, in other cases, sequences of these conserved genes are not sufficiently variable to distinguish between closely related species (Wang et al., 2006). As a result, a target with high specificity and efficiency is needed for rapid detection of P. sojae in soyabean production fields and in soil and residues carried with transported soyabeans. This will be very important for controlling the dispersal of P. sojae and maintaining ‘Phytophthora-free’ soyabean production.

To this end, this study used an identifiable target, named A3aPro, which is a 300-bp deletion element upstream (1·5 kb in the promoter region) of the avirulence gene Avr3a in P. sojae race 7, when compared to races 2 and 12 (Fig. S1), to develop a PCR assay for P. sojae. The objectives of the study were to: (i) develop species-specific primers for detecting P. sojae based on the identifiable target A3aPro; (ii) evaluate the sensitivity of the primers using PCR and real-time SYBR® Green I quantitative PCR (qPCR) assays; (iii) use the assay to detect the pathogen in diseased soyabean tissues; and (iv) determine whether the primers can be used to detect the pathogens in soil samples from different regions.

Materials and methods

Source of isolates

Phytophthora sojae isolates were obtained from diseased soyabean stems collected in different areas of Heilongjiang province in China from 2002 to 2011. All tested P. sojae isolates were isolated using a leaf disc baiting method from diseased soyabean plots (Jinhuo & Anderson, 1998). Using the same method, additional P. sojae isolates were baited from soyabean residues and soil carried by soyabeans imported from the USA, Brazil, Argentina and Canada. The P. sojae isolates, as well as isolates of Phytophthora spp., Pythium spp., Fusarium spp. and various other pathogens used in this study, are maintained in a collection in the Department of Plant Pathology, Nanjing Agricultural University, and are listed in Table 1.

Table 1. Isolates of fungi and oomycetes used to screen Phytophthora sojae (TrapF1/TrapR1) and universal (ITS1/ITS4) primers
SpeciesIsolation/originNo. isolatesPCR product
Host/substrateSourceaITS1/ITS4TrapF1/TrapR1
  1. aATCC, American Type Culture Collection; CBS, Centraalbureau voor Schimmelcultures; CGMCC, China General Microbiological Culture Collection; NJAU, Nanjing Agricultural University.

P. sojae (race 2) Glycine max NJAU5++
P. sojae (race 3) Glycine max NJAU4++
P. sojae (race 6) Glycine max NJAU3++
P. sojae (race 7) Glycine max NJAU5++
P. sojae (race 8) Glycine max NJAU2++
P. sojae (race 12) Glycine max NJAU2++
P. sojae (race 13) Glycine max NJAU3++
P. sojae (race 14) Glycine max NJAU5++
P. sojae (race 17) Glycine max NJAU2++
P. sojae (race 19) Glycine max NJAU2++
P. sojae (race 20) Glycine max NJAU2++
P. sojae (race 28) Glycine max NJAU2++
P. sojae (race 31) Glycine max NJAU2++
P. sojae Glycine max NJAU79++
P. boehmeriae Gossypium sp.NJAU4+
P. ramorum Oak speciesNJAU1+
P. drechsleri Beta vulgaris var. altissimaCBS 292.351+
P. vignae Vigna sinensis Michael D. Coffey1+
P. cambivora Castanea sativa CBS 248.601+
P. rubi RaspberryCBS 967.952+
P. cinnnamomi Cedrus deodara NJAU1+
P. melonis Cucumis sativus NJAU8+
P. brassicae Brassica sp.CBS 178.871+
P. cactorum Malus pumila NJAU19+
Rosa chinensis NJAU1+
P. capsici Capsicum annuum NJAU5+
Lycopersicon esculentum NJAU1+
P. colocasiae UnknownCBS 192.911+
P. cryptogea Gerbera jamesonii NJAU5+
P. drechsleri Beta vulgaris var. altissimaCBS 292.351+
P. erythroseptica Solanum tuberosum CBS 129.231+
P. fragariae var. rubiRaspberryCBS 967.951+
P. hibernalis Cirrus sinensis CBS 270.311+
P. idaei RaspberryCBS 968.951+
P. infestans Solanum tuberosum NJAU2+
P. medicaginis Medicago sativa ATCC 443901+
P. melonis Cucumis sativus NJAU8+
Cucumis sativus NJAU1+
Cucumis sativus NJAU1+
Benincasa hispida NJAU1+
Lagenaria siceraria NJAU1+
Pythium ultimum Irrigation waterNJAU10 
Botrytis cinerea Cucumis sativus NJAU1+
Colletotrichum gossypii Gossypium sp.NJAU1+
C. truncatum Glycine max NJAU8+
Fusarium avenaceum UnknownCGMCC1+
F. culmorum UnknownCGMCC1+
F. equiseti UnknownCGMCC1+
F. graminearum Triticum aestivum NJAU1+
F. oxysporium f. sp. vasinfectum Gossypium sp.NJAU2+
F. oxysporium f. sp. cucumerinum Cucumis sativus NJAU3+
F. oxysporium f. sp. cubense Musa sapientum NJAU1+
F. sambucinum UnknownCGMCC1+
F. solani Gossypium sp.NJAU5+
Glycine max NJAU15+
Macrophoma kawatsukai Malus pumila NJAU1+
Magnaporthe grisea Oryza sativa NJAU3+
Rhizoctonia solani Gossypium sp.NJAU2+
Glycine max NJAU3+
Verticillium dahliae Gossypium sp.NJAU3+
V. albo-atrum Medicago sativa NJAU1+

Culture conditions and preparation of mycelia

Phytophthora isolates were cultured on 10% V8 media at 25°C (Erwin et al., 1996). Mycelia of each Phytophthora and Pythium isolate were obtained by growing the isolates on 10% V8 media at 18–25°C (temperature-dependent isolates) for at least 3 days (Zheng, 1995). Mycelia of the other fungi were grown in potato dextrose broth (Erwin et al., 1996). The mycelia were harvested by filtration and frozen at −20°C.

Source of plant samples

Plant samples exhibiting disease symptoms, collected from the field in Heilongjiang province, China in 2010, were processed at the Department of Plant Pathology, Nanjing Agricultural University. The pathogen was isolated from the diseased plant tissues into pure culture to verify the presence of P. sojae.

Source of soil samples

A total of 360 soil samples were collected from the soyabean production fields in several provinces (Heilongjiang, Jiangsu, Anhui, Shanghai, Xinjiang, Sichuan, Henan and Guizhou) of China from 2002 to 2010. In addition, 33 soil samples were collected from the soil carried with soyabeans imported from the USA, Brazil, Argentina and Canada and described previously (Kalinina et al., 1997; Wang et al., 2006). In commercial processing systems, soyabeans are typically screened to remove contaminated soil and soyabean residues. These soils and residues were collected for PCR assays and the samples evaluated are listed in Table 2.

Table 2. Soil samples used in this study, and numbers that were positive in the A3aPro PCR assay, in the ITS PCR assay and in the baiting assay
SourceNo. of soil samplesNo. A3apro-PCR positiveNo. ITS-PCR positiveNo. leaf-disc baiting positive
Heilongjiang25191719
Jiangsu29202017
Anhui53323031
Shanghai61201817
Xinjiang68181717
Sichuan601096
Henan461098
Guizhou18222
USA24191812
Brazil2111
Argentina1111
Canada6423

Baiting assay

Soyabean field soil (10 g) was moistened in a Petri dish (9 cm diameter), preincubated in the light at 25°C for a week, then flooded with 25 mL sterile distilled water (SDW). Twenty leaf discs (10-mm diameter), freshly cut with a paper punch from the unifoliolate leaves of 10-day-old soyabean cv. Williams seedlings were floated immediately on the surface of the water. After 12 h, the floating leaf discs were removed, washed and placed in another Petri dish with 25 mL SDW. Sporangia emerging from the edge of the infected leaf discs, observed under stereomicroscopy after 48 h of incubation in the water, were recorded as positive results.

Sources of DNA

DNA of mycelia was extracted from pure microbial cultures as described by Schubert et al. (1999). These cultures included isolates of P. sojae, other Phytophthora spp., isolates of Pythium spp. and isolates of true fungi, as listed in Table 1. DNA from mycelia was obtained according to a modified cetyltrimethyl ammonium bromide (CTAB) procedure (Sambrook et al., 1989). DNA from oospores existing in contaminated soils was extracted by a screening method: 200-mesh (76 μm), 300-mesh (54 μm) and 400-mesh (38 μm) screens (all 20-cm diameter) were used to eliminate most of the soil, and a 600-mesh (25 μm) screen (20-cm diameter) was used to collect the oospores. A single oospore was selected under a microscope, rinsed in 100 μL SDW in a 1·5-mL Eppendorf tube and treated with a FastDNA® SPIN Kit for Soil (Q-Biogene) to extract DNA. To extract crude DNA from zoospores, hyphal tip plugs of P. sojae isolates were used to inoculate 30 mL sterile clarified 10% V8 broth in 90-mm Petri dishes to obtain axenically prepared mycelium. Mycelial cultures were incubated at 25°C in the dark for 3 days. Sporulating hyphae were prepared by repeatedly washing 2-day-old hyphae incubated in 10% V8 broth with SDW and incubating the washed hyphae in the dark at 25°C for 4–8 h until sporangia developed on most of them. Zoospores were filtered with Miracloth (Calbiochem) and collected by centrifugation at 2000 g for 2 min. One hundred zoospores were suspended in 100 μL double-distilled water, 0·5 g silica added, and the mixtures vortexed for 1 min, after which 1–10 μL suspension was added to the PCR reaction. DNA of P. sojae from plant samples was prepared using the NaOH method (Wang et al., 1993), which can extract sufficient DNA in an appropriate buffer and use it directly for PCR. DNA from soil samples was extracted directly using a FastDNA® SPIN Kit for Soil (Q-Biogene) according to the manufacturer’s recommendations. DNA concentrations were determined spectrophotometrically or by quantitation on 1% agarose gels stained with ethidium bromide in comparison with commercially obtained standards. DNA was stored at −20°C.

PCR

Each reaction for PCR consisted of 2·5 μL 10 × PCR buffer, 1 μL 2·5 mm dNTP, 2·5 μL 25 mm MgCl2, 0·25 μL each of 20 μm primers, and 0·25 μL Taq DNA polymerase (5 U μL−1), to which were added 1 μL template DNA and then SDW to a final volume of 25 μL. All reactions were performed in a PTC-2000 PCR instrument (MJ Research). The thermal cycling settings used were an initial denaturation at 94°C for 5 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 30 s, then a final extension at 72°C for 10 min. Negative controls lacking template DNA were performed in each experiment to test for contamination. The products of PCR were electrophoresed on 1% agarose gels and stained with ethidium bromide. All of the reagents used for PCR amplification were purchased from TakaRa. PCR was repeated on independent DNA extractions. The amplification was estimated based on at least six replicates.

Real-time PCR

Amplification was monitored in real-time using the 7500 Real-Time PCR System (Applied Biosystems). Reaction volumes (20 μL) contained 2 × SYBR® Premix ExTaqTM (Takara), primers at 10 μm each, ROX reference dye II (50 × ), 2 μL PCR control template, and SDW to the final volume. The reaction mixture was initially incubated at 50°C for 2 min, followed by 30 s denaturation at 95°C, and 40 cycles of 5 s at 95°C and 34 s at 60°C. The standard curve was constructed by plotting the log of a known concentration (10-fold dilution series from 100 ng to 10 fg) of DNA from P. sojae against the threshold cycle (Ct) values and melting curve. The amplification was estimated based on at least six replicates.

A3aPro-based primer design

The A3aPro sequence was acquired from the Joint Genome Institute (JGI) P. sojae genome database (http://www.jgi.doe.gov/) (position: scaffold 80: 317485–317760) and the entire A3aPro sequence from P. sojae was submitted to GenBank (accession no. JX118829). Using the A3aPro sequence of P. sojae as a bait for a blastn search did not show any similarity with other sequenced isolates of P. infestans, P. ramorum or Hyaloperonospora parasitica. Similar A3aPro sequences were then obtained from the genome databases for P. infestans, P. ramorum and H. parasitica. Phytophthora infestans T30-4 DNA sequence was available from the Broad Institute (http://www.broad.mit.edu/) (position: supercontig 1849: 1900–2350); P. ramorum DNA sequence was available from the Joint Genome Institute (JGI; http://www.jgi.doe.gov/) (position: scaffold 1220: 1–342); H. parasitica genome sequence was available from http://vmd.vbi.vt.edu/ (position: contig 159: 13356–13809). The P. sojae primers TrapF1 (5′-CGGTGGCTCTCGGCATTCGTG-3′) and TrapR1 (5′-CACCCTACTGTTATAGACACG-3′) were designed based on the A3aPro sequence (Fig. 1). In order to increase the sensitivity of the standard PCR, TrapF2 (5′-ATCTGACGGTGGCTCTCGGC-3′) and TrapR2 (5′-TTACACCCTACTGTTATAGAC-3′) were similarly designed for nested PCR (Fig. 1).

Figure 1.

 Nucleotide sequence alignment of the target region A3aPro of Phytophthora sojae and similar sequences of other species used for designing PCR primers. The nucleotide sequence of the sense strand of A3aPro DNA is shown. The schematic diagram shows the positions of the primers TrapF1/TrapR1 and TrapF2/TrapR2 for amplification.

Results

Specificity of primers TrapF1 and TrapR1

The specificity of the P. sojae primers TrapF1 and TrapR1 was evaluated using 118 isolates of P. sojae and 136 other isolates of fungi and oomycetes (Table 1). The 118 isolates representing P. sojae were able to amplify a unique DNA sequence of 267 bp (Fig. S2c) with primers TrapF1 and TrapR1. However, isolates representing all of the other Phytophthora species and the other species tested yielded no amplification products (Fig. S2a,b) with primers TrapF1 and TrapR1. All of the DNA preparations from fungi and oomycetes amplified a product with the ITS universal primers ITS1/ITS4 (Table 1), proving that the DNA preparations were suitable for PCR amplification.

Sensitivity of TrapF1 and TrapR1

To determine the detection sensitivity of the standard PCR, DNA from P. sojae P6497 was serially diluted from 1 ng to 100 ag, and 1 μL of each dilution was used as a template for PCR using the conditions previously described. A nested PCR protocol using the primers (TrapF1/TrapR1 and TrapF2/TrapR2) was also assessed for sensitivity: the serially diluted template DNA was initially amplified using the outer primers TrapF2/TrapR2, as previously described; then 1 μL of the products from PCR with primers TrapF2/TrapR2 was used as a template for secondary amplification with the TrapF1/TrapR1 primers. The detection limit of the standard PCR was 10 pg μL−1 (Fig. 2a). The nested PCR could detect as little as 10 fg DNA μL−1 (Fig. 2b) (9·3 copies in genome equivalents) (http://www.uri.edu/research/gsc/resources/cndna.html). Other P. sojae isolates (R3, R20, R31) were also tested; the results showed the same sensitivity (data not shown). At least six replicates of each dilution were evaluated to assess the sensitivity of the A3aPro PCR reaction.

Figure 2.

 Sensitivity of the A3aPro PCR method using serially diluted genomic DNA (1 ng–100 ag) with Phytophthora sojae isolate P6497 as template. M, DL100 bp DNA ladder marker. –, no template DNA. (a) Standard PCR using the primers TrapF1/TrapR1; detection limit 10 pg μL−1. (b) Nested PCR using the product of primers TrapF2/TrapR2 as template and TrapF1/TrapR1 as the primers; detection limit 10 fg μL−1.

Crude DNA from zoospores and oospores was used as template for testing the sensitivity of primers TrapF1 and TrapR1. Ten concentrations of zoospores and oospores were tested, ranging from 1000 to 1 μL−1. With a 1 μL−1 concentration, DNA could be extracted from one to 10 zoospores or oospores as template in a 25-μL reaction volume, all resulting in amplification of the 267-bp band (Fig. S3). Therefore, DNA extracted from a single oospore or even one zoospore was also sufficient for PCR detection. At least six replicates of each dilution were evaluated to assess the sensitivity of the A3aPro PCR reaction.

Sensitivity of TrapF1 and TrapR1 using real-time PCR

The suitability of the species-specific primers TrapF1/TrapR1 for qPCR was demonstrated by a pilot study using SYBR Green chemistry and validated using standard P. sojae isolate P6497. A qPCR using DNA extracted from pure cultures of standard P. sojae isolate P6497 produced an amplicon with template concentrations from 100 ng (average Ct = 11·9) to 10 pg (average Ct = 25·3). Electrophoresis on 1% agarose gels showed that the amplicons were the predicted size (267 bp) (data not shown). The standard curve showed a linear correlation between the logarithm of the concentration and the Ct values, with a correlation coefficient (R2) of 0·9991. The relationship between Ct (y) values and the log of DNA concentration (= log DNA concentration) for P. sojae was expressed by the equation = −3·382+ 18·689 (Fig. S4). At least six replicates of each dilution were evaluated to assess the sensitivity of the A3aPro qPCR reaction.

Detection in diseased soyabean tissues

DNA samples of P. sojae from different regions of Heilongjiang province in 2010 were prepared using the NaOH method. A 267-bp DNA fragment was amplified using the specific primer pair TrapF1/TrapR1, while no PCR product was amplified in the negative controls (Fig. 3). The presence of P. sojae was verified by isolating the pathogen from the tissue into pure culture. In addition, to confirm that no PCR inhibitors were present in healthy soyabean tissues, the primers ITS1 and ITS4, which amplify a 700-bp DNA band (data not shown), were used in PCR analyses as a control. The PCR amplifications were repeated at least three times per sample.

Figure 3.

 Amplification of Phytophthora sojae DNA from diseased soyabean tissues (at least six replicates) using TrapF1/TrapR1 primers. M, DL100 bp DNA ladder marker. Lane 1, positive control, 10 ng P. sojae DNA; lanes 2–13, amplified products using DNA of diseased soyabean tissues from different regions of Helongjiang province (2, Haerbin; 3, Jiamusi; 4, Qiqihaer; 5, Mudanjiang; 6, Jixi; 7, Daqing; 8, Yichun; 9, Hegang; 10, Shuangyashan; 11, Heihe; 12, Wuchang; 13, Qitaihe); lane 14, healthy plant tissues; lane 15, no template DNA.

Detection in infested field soils

To evaluate the A3aPro PCR assay for detection of P. sojae, 360 soil samples collected from different regions of China from 2002 to 2010, plus 33 samples from the USA, Brazil, Argentina and Canada, were tested by the A3aPro PCR assay and the ITS PCR assay, which has been previously described (Wang et al., 2006). Using the A3aPro PCR-based method, 156 of the 393 samples (39·7%) produced 267-bp bands (Fig. S5), compared with 144/393 (36·6%) using the ITS PCR assay and 134/393 (34%) using the baiting method (Table 2). Hence, the A3aPro PCR assay reported here may be used for detection of P. sojae in plants and production fields.

Comparison of previously published primers PS1/PS2 and the newly developed primers TrapF1/TrapR1

The new developed primers TrapF1 and TrapR1 produce a product of 267 bp. Isolates representing all of the other species tested yielded no amplification product with primers TrapF1 and TrapR1 (Fig. S2). Wang et al. (2006) reported detection of P. sojae with primers PS1 and PS2 down to 1 fg DNA in an ITS-based PCR assay, but several other Phytophthora species, such as P. melonis and P. drechsleri, cross-reacted in the assay (Fig. S6a); only when the annealing temperature was increased to more than 66°C was the amplification of a specific 330-bp PCR product from P. drechsleri and P. melonis prevented (Fig. S6b).

Discussion

Research on the interaction between soyabean and P. sojae will greatly increase understanding of the disease mechanisms and help to control crop damage. The avirulence gene Avr3a from P. sojae was recently identified (Qutob et al., 2009), and elucidation of its structure has improved understanding of the evolution and characteristics of Avr genes. This will also aid in identification of additional Avr genes in the future. Given this research background, a DNA transposon-like sequence of Avr3a, designated A3aPro, which has a high copy number in the genome of P. sojae and is species-specific, was used to develop the specific and sensitive PCR primers TrapF1/TrapR1 for rapid detection of P. sojae.

The A3aPro-based primers TrapF1 and TrapR1 for P. sojae designed here had higher specificity than the previously reported (Wang et al., 2006) primers PS1 and PS2 based on ITS sequences. There was a 97% similarity in ITS sequences between P. sojae and P. melonis or P. drechsleri. Only an increase in annealing temperature to 66°C or more could distinguish among these Phytophthora species. However, a high annealing temperature may reduce the sensitivity of PCR reactions. Thus, increasing the annealing temperature did not resolve this problem. One approach to improving the discrimination between similar species is to design a new assay based on a different region of the genome. Thus, the primers TrapF1 and TrapR1, based on an identifiable target, A3aPro, were designed specifically for P. sojae in the present paper. A 267-bp DNA fragment from the genome of all 118 analysed isolates of P. sojae, regardless of origin, was produced using this primer pair; isolates of other species, including P. melonis and P. drechsleri, amplified no PCR product (Fig. S2).

The detection sensitivity of the TrapF1/TrapR1 primer pair was demonstrated to be 10 pg μL−1 in a standard PCR and increased 1000-fold to 10 fg μL−1 in a nested PCR (Fig. 2). The higher sensitivity of the A3aPro-based primers developed here compared to ITS-derived primers is probably the result of the higher abundance of the A3aPro target, which is more than 200 copies in the JGI database (data not shown). Meanwhile, in tests to assess the sensitivity, real-time PCR combined with fluorescent SYBR Green I dye was used to quantify P. sojae directly from purified DNA, and could detect 10 pg μL−1 pure DNA per 25-μL reaction volume (Fig. S4). Although this was not an improvement on standard PCR, it had the advantage of allowing pathogen levels to be quantified, which may be important for studies investigating levels of pathogen occurrence or the inoculum levels in the field. The PCR detection limit is a single oospore or even one zoospore for the TrapF1/TrapR1 primers (Fig. S3). For P. sojae, the PCR detection sensitivity of a single oospore or one zoospore is biologically useful and would allow the pathogen to be detected at low levels in the field. However, this theoretical limit assumes that there is no inhibition in the PCR reaction.

Combined with rapid NaOH lysis, the PCR assay could be used to detect P. sojae in less than 3 h in infected soyabean tissues (Fig. 3). This is especially important for the import and export trade of soyabeans, or when the target concentration is low. The absence of amplification from healthy roots removed any doubt about the specificity of this primer pair.

Furthermore, the assay allows the rapid detection of the pathogen using primers TrapF1/TrapR1 in soil samples (Fig. S5). In this study, 393 soil samples from China and other countries were tested by the A3aPro PCR assay, ITS PCR assay, and a leaf-disc baiting method for comparison (Table 2). Comparing the positive-sample ratios of the three methods, the newly developed A3aPro PCR assay improved the detection efficiency and was more specific than the ITS assay. Increasing numbers of soyabean shipments are traded to and from different countries, including more than 56 million tons imported into China (where P. sojae is a quarantine pathogen) annually. Therefore, rapid detection of P. sojae in the soil carried with the transported soyabeans is not only important for the soyabean trade between China and other countries, but also for controlling the spread of P. sojae within China. The newly developed PCR detection technology with TrapF1/TrapR1 primers offers an effective way to do this and, in view of its speed and efficiency, it is especially suitable for quarantine situations. It has, in fact, been used in some Chinese Entry–Exit Inspection and Quarantine Bureaus in the past few years.

In conclusion, the Phytophthora conserved transposon-like DNA element is a potential target for soyabean root rot disease diagnosis and the TrapF1/TrapR1 and TrapF2/TrapR2 primer-based PCR assay provides a rapid and sensitive tool for the detection of P. sojae in plant tissues and in soils from production fields. Moreover, this assay could control the dispersal of P. sojae and increase ‘Phytophthora-free’ soyabean production.

Acknowledgements

This research was supported by the National Department Public Benefit Research Foundation (no. 200903004), the National ‘863’ Program (2012AA101501), the ‘948’ project (2010-C17) and Chinese National Science Foundation Committee project (3-20). We thank Michael D. Coffey from University of California Riverside for providing us with the isolate of Phytophthora vignae.

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