Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. niveum in soil

Authors

  • Jun Peng,

    1. Key Laboratory of the Ministry of Agriculture for Integrated Pest Management on Tropical Crops, Institute of Environmental and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, China
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  • Yuanfeng Zhan,

    1. Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan, China
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  • Fanyun Zeng,

    1. Key Laboratory of the Ministry of Agriculture for Integrated Pest Management on Tropical Crops, Institute of Environmental and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, China
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  • Haibo Long,

    1. Key Laboratory of the Ministry of Agriculture for Integrated Pest Management on Tropical Crops, Institute of Environmental and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, China
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  • Yuelin Pei,

    1. Key Laboratory of the Ministry of Agriculture for Integrated Pest Management on Tropical Crops, Institute of Environmental and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, China
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  • Jianrong Guo

    Corresponding author
    1. Key Laboratory of the Ministry of Agriculture for Integrated Pest Management on Tropical Crops, Institute of Environmental and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, China
    • Correspondence: Jianrong Guo, Key Laboratory of the Ministry of Agriculture for Integrated Pest Management on Tropical Crops, Institute of Environmental and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, Hainan, China. Tel.: +86 898 66969305; fax: +86 898 66969211; e-mail: guojianrong@hotmail.com

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Abstract

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is one of the major limiting factors for watermelon production worldwide. Rapid and accurate detection of the causal pathogen is the cornerstone of integrated disease management. In this paper, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay was developed for the rapid and quantitative detection of Fon in soil. Positive products were amplified only from Fon isolates and not from any other species or formae speciales of F. oxysporum tested, showing a high specificity of the primer sets. The detection limit of the RealAmp assay was 1.2 pg μL−1 genomic DNA or 103 spores g−1 of artificially inoculated soil, whereas real-time PCR could detect as low as 12 fg μL−1 or 102 spores g−1. The RealAmp assay was further applied to detect eight artificially inoculated and 85 field soil samples. No significant differences were found between the results tested by the RealAmp and real-time PCR assays. The RealAmp assay is a simple, rapid and effective technique for the quantitative detection and monitoring of Fon in soil under natural conditions.

Introduction

Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum (Fon), is one of the most severe diseases in watermelon and a major limiting factor for watermelon production in the world (Martyn & McLanghlin, 1983). Currently, no effective fungicides or chemical disinfectants are available because Fon can generate thick-walled chlamydospores that are highly resistant to soil fumigation (Besri, 2008). Rapid and accurate detection and monitoring of the causal pathogen is the cornerstone of integrated disease management practice. The identification and detection of the pathogen is traditionally based on either symptoms on the host or culture-dependent isolation of the pathogen from infected host tissues. However, these classical approaches are laborious, time-consuming and sometimes inaccurate. Predictive soil sampling needs to rely on semi-selective media, which only allow for crude estimates of fungal propagules in soil (Nash & Snyder, 1962). Morphological identification requires a great knowledge of Fusarium taxonomy, and it is not possible to identify the pathogen formae speciales using the microscope. Therefore, highly sensitive and rapid detection assays are need for direct identification of Fon in soil.

In recent years, real-time PCR has been widely used as a classic quantitative detection method in the diagnosis of soil-borne pathogens (Brierley et al., 2009) such as Plasmodiophora brassicae (Wallenhammar et al., 2012), Phytophthora infestans (Lees et al., 2012) and Fon (Zhang et al., 2005). A DNA-based soil testing service using real-time PCR is now operating in Australia to assist growers in predicting the likely extent of losses from various soil-borne diseases before a crop is planted (Ophel-Keller et al., 2008). The rapidity and specificity of PCR-based molecular methods have been reported for the detection and differentiation of Fon from other F. oxysporum (Fo) isolates in Taiwan (Lin et al., 2010). Moreover, a real-time PCR assay has been developed for quantitative detection of Fon for field surveys and epidemiological investigations (Zhang et al., 2005). However, quantification of plant pathogens in soil DNA extracts using real-time PCR needs an expensive thermal cycler, specialized reaction reagents and 2–3 h running time.

Recently, the Eiken Chemical Company Ltd (Tokyo, Japan) developed a loop-mediated isothermal amplification (LAMP) method, which is also available for quantification of DNA (Notomi et al., 2000). The LAMP assay is performed under isothermal conditions, employing a DNA polymerase with strand-displacing activity and a set of four specially designed primers which recognize a total of six distinct sequences on the target DNA to be amplified. The amplified products contain single-stranded loops, allowing primers to bind without the need for repeated cycles of thermal denaturation (Notomi et al., 2000; Nagamine et al., 2001). Positive LAMP reaction can be visualized with the naked eye by adding DNA-intercalating dyes such as ethidium bromide, SYBR Green I, propidium iodide and Quant-iT PicoGreen, or metal-ion indicators such as hydroxynaphthol blue (HNB; Goto et al., 2009), CuSO4 (Zoheir & Allam, 2011) and calcein (Tomita et al., 2008). The reaction can also be monitored in real-time, allowing quantitative detection of the target (Tomlinson et al., 2010; Bekele et al., 2011). The ESE-Quant tube scanner using fluorescent dye is a simple and cost-effective system for a real-time detection of parasite DNA (Lucchi et al., 2010; Njiru et al., 2012).

However, few examples of real-time fluorescence loop-mediated isothermal amplification assays are available for direct quantitative detection of soil-borne pathogens in soil. Therefore, the objectives of this study were (1) to develop a RealAmp assay for rapid and quantitative detection of the Fon in soil, and (2) to verify the feasibility of using the LAMP-based quantitative detection assay by comparing both artificially and naturally infested soil samples with the classic real-time PCR assay.

Materials and methods

Preparation of mycelia and microconidia

Fifteen isolates of F. oxysporum and two other species were used in this study (Table 1). The isolates were maintained in a collection at our institute. For genomic DNA extraction, the fungi were cultivated in potato dextrose broth (PDB, 20 g glucose and 4 g potato extract in 1 L H2O) medium on a shaker for 6 days at 25 °C, and collected on filter paper and stored at −70 °C until use. Microconidia of Fon were prepared by growing the fungus on PDA (20 g glucose, 4 g potato extract and 17 g agar in 1 L H2O) at 25 °C for 10 days in an incubator (Haixiang MJX-150, Shanghai, China). Microconidia were harvested from the plates by rubbing the surface mycelium gently with a rubber swab and collected it in distilled water. Hyphal debris was removed from the spores by centrifuging the crude spore preparation through a 40% sucrose pad. Spores were adjusted to 108 spores mL−1 for soil inoculation by counting them in a hemocytometer.

Table 1. Fungal species and isolates used to test the specificity of the RealAmp assay
Species/isolatesOriginal hostsOriginRealAmp assayConventional PCR
  1. ATCC, American Type Culture Collection (Manassas, VA, USA); CGMCC, China General Microbiological Culture Collection Center; +, with target product; –, without target product.

Fusarium oxysporum f. sp. niveum (race 0)WatermelonBeijing++
F. oxysporum f. sp. niveum (race 1)WatermelonBeijing++
F. oxysporum f. sp. niveum ATCC62940 (race 2)WatermelonATCC++
F. oxysporum f. sp. niveum WatermelonTaiwan++
F. oxysporum f. sp. niveum WatermelonCGMCC++
F. oxysporum f. sp. niveum WatermelonCGMCC++
F. oxysporum f. sp. niveum WatermelonCGMCC++
F. oxysporum f. sp. niveum WatermelonHainan++
F. oxysporum f. sp. niveum WatermelonHainan++
F. oxysporum f. sp. niveum WatermelonJiangsu++
F. oxysporum f. sp. cubense race 1WatermelonJiangsu
F. oxysporum f. sp. cubense race 4Banana (Musa spp.)Hainan
Foxysporum f. sp. cucumerium CucumberJiangsu
Foxysporum f. sp. lactucae LettuceJiangsu
Foxysporum f. sp. luffae LoofahJiangsu
Ascochyta fabae Vicia faba Fujian
Mycosphaerella melonis WatermelonFujian

Preparation of soil samples

The artificially inoculated soil samples were prepared by adding 1 mL of the Fon spore suspension into 10 g of autoclaved soil substrate in 15-mL conical tubes. The tubes were vortexed at maximum speed for 1 min, air-dried and stored at −70 °C prior to DNA extraction.

Field soil samples were collected from areas with two different types of the watermelon production on the Hainan Island in 2010–2011, respectively. In one area, watermelon had been planted previously. In this area, 15–20 soil samples were randomly collected from the chosen fields. The other area was a watermelon-growing area with Fusarium wilt appearance. The samples in that area were taken directly from under the infected watermelon root. All soil samples were taken within a 10-cm depth as described by Heap & McKay (2004). Samples were air-dried and stored at 10 °C. In all, 85 soil samples were collected and used for genomic DNA extraction.

Extraction of genomic DNA

DNA extraction from cultures

Approximately 100 mg of freeze-dried mycelium or conidia were ground in liquid nitrogen, and the total genomic DNA was isolated according to the manufacturer's instructions of E-Z 96® Fungal DNA Kit (Omega). All DNA samples were eluted in 100 μL Tris-EDTA (TE) buffer and stored at −70 °C until required. The DNA concentration of extracts was determined by monitoring absorbance at 260 and 280 nm using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE).

DNA extraction from soil samples

DNA was extracted from soil samples following Li & Hartman (2003), Zhang et al. (2005) and Brierley et al. (2009) with several modifications. Air-dried soil samples (each 10 g) were first lyophilized with liquid nitrogen and then suspended in 20 mL SPCB extraction buffer (Brierley et al., 2009). After vortexing, samples were incubated at 65 °C for 30–60 min and then centrifuged at 12 000 g for 10 min to remove soil and debris. The samples were incubated at 65 °C for 30 min after the addition of 1 mL of 20% (w/v) sodium dodecyl sulfate (SDS). After centrifugation, the supernatant was extracted with an equal volume of chloroform, mixed and re-centrifuged. DNAs were precipitated with 0.3 M sodium acetate (pH 5.2) and an equal volume of isopropanol at −20 °C for 2 h or overnight, and pelleted by centrifugation, washed in 70% ethanol, and dissolved in 100 μL TE buffer. The extracted DNA was further purified by centrifuging through a combined spin column with both PVPP and Sephadex G-75 as described by Cullen et al. (2001). Purified DNA was collected, quantified on a NanoDrop 2000 spectrometer, and stored at −20 °C until use.

LAMP primer design

LAMP primers were designed according to a randomly amplified fragment sequence (accession number EU603504) using primerexplorer V4 software (Eiken Chemical Co. Ltd, Tokyo, Japan). A forward inner primer FIP (5′-CTAGGTGCGGCAGTAAATCCATACTTCACGTCTGTATCCCA-3′) consisted of F1c (the complementary sequence of F1, 5′-CTAGGTGCGGCAGTAAATCCAT-3′, nt 230–209) and F2 (5′-ACTTCACGTCTGTATCCCA-3′, nt 160–178), and a backward inner primer BIP (5′- GGGAGGTAAACCCTGAGACCTGACGGTGCCGATATCCTC-3′) of B1c (the complementary sequence of B1, 5′- GGGAGGTAAACCCTGAGACCT-3′, nt 231–251) and B2 (5′-GACGGTGCCGATATCCTC-3′, nt 294–277). The outer primers F3 (5′-GATTAGCGAAGACATTCACAAG-3′, nt 102–123) and B3 (5′- GAGCGAGACGGTAAAGTC-3′, nt 361–344) were used for the initiation of the LAMP reaction. The primer sequences and their respective binding sites are indicated in Fig. 1. The primer specificity was checked using the basic local alignment search tool (blast) against human DNA and other fungi sequences in public GenBank databases.

Figure 1.

Design of inner and outer primers from a randomly amplified specific sequence (EU603504) from Fon genome and their relative positions inside the sequence. The F1c is complementary to the F1c region of the template sequence, as are B2 and B3. The B1c is identical to the B1c region of the template sequence, as are F3 and F2. The forward primer sequence is indicated above the template sequence, and the reverse primer sequence below the template sequence.

Real-time fluorescence loop-mediated isothermal amplification

The RealAmp reaction was conducted as described previously (Peng et al., 2012) with minor modifications and optimization. The LAMP reaction contained 1.6 μM each of FIP and BIP, 0.2 μM each of F3 and B3, 1.6 mM dNTPs, 1 M betaine, 8 mM MgSO4, 1× ThermoPol reaction buffer (20 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4, 0.1% Triton® X-100, pH 8.8), 8 U of Bst DNA polymerase (New England Biolabs, Ipswich, MA), 1.0 μL of 1 : 1000 dilution SYBR Green I (Invitrogen, Carlsbad, CA), 1 μL of template DNA, and double-distilled water to a final volume up to 25 μL. An equal volume of paraffin oil was then added to the tube to prevent evaporation, followed by the addition of 1 μL of SYBR Green I (Invitrogen) diluted 1 : 10, to the inside of the lid prior to amplification with an improved close-tube visual detection system as described by Peng et al. (2012). The real-time fluorescence loop-mediated isothermal amplification was carried out at 63 °C for 90 min using an ESE-Quant Tube Scanner (ESE GmbH, Stockach, Germany), which was set to collect fluorescence signals at 30-s intervals.

During the real-time amplification, the fluorescence data were obtained on the 6-carboxyfluorescein (FAM) channel (excitation at 487 nm and detection at 525 nm). The threshold time (Tt) was calculated as the time at which the fluorescence equaled the threshold value. In the plot, the Y-axis denotes the fluorescence units in milli-volts (mV) and the X-axis shows the time in minutes. After the reaction, the LAMP products were detected directly by visual observation of the solution colour by mixing the pre-added SYBR Green I with the reaction solution through gentle centrifugation. The LAMP products (5 μL) were analyzed by electrophoresis on a 2% (w/v) agarose gel and subsequently stained with ethidium bromide.

Specificity and sensitivity test of RealAmp assay

To confirm the specificity of the RealAmp assay, the DNA of 15 isolates of F. oxysporum and two other species were used in the analyses (Table 1). In addition, the small fragment from RealAmp amplification products was cloned and sequenced. The specificity was also validated by conventional PCR using the specific primer set Fon-1/Fon-2 (Fon-1, 5′-CGATTAGCGAAGACATTCACAAGACT-3′; Fon-2, 5′-ACGGTCAAGAAGATGCAGGGTAAAGGT-3′) reported previously by Lin et al. (2010).

To determine the sensitivity of the RealAmp assay, the extracted Fon genomic DNA was adjusted to 120 ng μL−1 and diluted in a 10-fold series (1 × 100 to 1 × 107 dilutions) to assess the detection limit of the RealAmp assay in comparison with real-time PCR. The standard curve was constructed according to the serial dilutions of genomic DNA. The RealAmp products were also detected directly by visual observation of the solution colour. The PCR and RealAmp products (5 μL) were also visualized by electrophoresis.

Real-time PCR

The real-time PCR assay was conducted using the SYBR® Premix Ex Taq™ kit (Takara, Dalian, China) and Fon-1/Fon-2 primer pair in a total volume of 25 μL on a PRISM® 7500 Fast real-Time PCR machine (Applied Biosystems) following the manufacturer's instructions. A standard curve was constructed with eight 10-fold serial dilutions of Fon genomic DNA in triplicate, as described above. Soil DNA extracts were diluted 10-fold before analysis and analyzed in triplicate. The thermal cycling conditions consisted of an initial denaturation for 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 s, annealing at 52 °C for 10 s, and extension at 72 °C for 15 s.

Feasibility test for soil samples

The availability of RealAmp assay for the detection of Fon in artificially inoculated and natural soil samples was investigated. The samples were prepared as described above and then quantitatively detected with both RealAmp and real-time PCR assays, respectively. The results were statistically analyzed by one-way anova and paired Student's t-test using spss program ver. 17.0 (Chicago, IL).

Results

Development of the RealAmp assay for the detection of Fon

Target products were amplified only from DNAs of Fon isolates and not from DNAs of any other formae speciales of F. oxysporum and other species tested, showing a high specificity of the primer sets (Table 1, Fig. 2a). The results were identical to those tested by conventional PCR using the previously reported specific primer pair Fon-1/Fon-2 (Table 1). A single 174-bp PCR product was amplified only from Fon isolates; no amplified bands were observed from other pathogens (Fig. 2b). The colour of LAMP products of Fon isolates changed from orange to green when detected with SYBR Green I, whereas the colour of the other samples remained the original orange (Fig. 2c). The amplification curves obtained using the RealAmp assay indicated that the primer sets were able to specifically amplify the target DNA sequence (Fig. 2d). The sequence of LAMP product amplified by the RealAmp assay perfectly matched the Fon genome sequence (data not shown).

Figure 2.

Specificity test of the real-time fluorescence loop-mediated isothermal amplification assay (RealAmp assay) for the detection of Fon in comparison with conventional PCR. Lanes 1–4, genomic DNAs of Fusarium oxysporum f. sp. niveum (Fon) race 0, race 1, race 2, artificially inoculated soil samples, respectively; Lanes 5–8, the DNA of Mycosphaerella melonis, F. oxysporum f. sp. cucumerium, Foxysporum f. sp. luffae, and F. oxysporum f. sp. cubense race 4, respectively; Lane M, Trans2K plus II DNA marker. (a) Agarose gel electrophoresis analysis of RealAmp assay amplicon. (b) Conventional PCR using the specific Fon-1/Fon-2 primer set. (c) Visual detection of the RealAmp amplification products. The original orange colour of SYBR green turned green in the positive reaction mixture. (d) The fluorescence units vs. time graph were plotted automatically by the ESE-Quant Tube Scanner.

Sensitivity and standard curve analysis

The electrophoresis results showed that the RealAmp assay could detect as low as about 1.2 pg μL−1 of Fon genomic DNA (Fig. 3a), whereas the detection sensitivity of conventional PCR was about 120 pg μL−1 (Fig. 3b). The colour change of RealAmp products (from orange to green) was clearly observed, ranging from 120 ng μL−1 to 1.2 pg μL−1, and identical to the electrophoresis results (Fig. 3c). This assay represented a standard fluorescence amplification of LAMP products with exponential growth using the ESE-Quant tube scanner (Fig. 3d). The results indicated that the sensitivity of the RealAmp assay was 100 times higher than that of the conventional PCR method.

Figure 3.

Sensitivity test of RealAmp assay using serial dilutions of genomic DNA in comparison with PCR assay. Lanes 1–8 are serial 10-fold dilutions of Fon genomic DNA ranging from 120 to 1.2 × 10−5 ng μL−1, and lane M is Trans2K Plus II DNA marker. (a) Sensitivity test of RealAmp assay. (b) Conventional PCR using the Fon-1/Fon-2 primer set. (c) Visual detection of the RealAmp amplification products. (d) The fluorescence units vs. time amplification curves were plotted automatically using an ESE-Quant Tube Scanner.

To evaluate the performance of the RealAmp system for quantification of pathogens, serial dilutions of Fon genomic DNA were analyzed. As illustrated in Fig. 4, there was a good linearity between the threshold time (Tt) and logarithm of the concentration of genomic DNA, confirming that amplification was reliable and the RealAmp assay could be used for Fon quantification (Fig. 4a). The standard curve was established for further quantification of Fon in soil (Fig. 4b).

Figure 4.

Determination of the detection limit and establishment of standard curve of the RealAmp and real-time PCR assays. Lanes 1–8 correspond to serial 10-fold dilutions of Fon genomic DNA ranging from 120 to 1.2 × 10−5 ng μL−1 in triplicate. (a) The real-time fluorescence unit measured by the RealAmp assay. (b) Standard curve generated by RealAmp assay. (c) Detection limit of real-time PCR. (d) Standard curve obtained with real-time PCR by plotting the log of Fon genomic DNA concentration against the cycle threshold (Ct) values.

Meanwhile, the lowest detection limit of the real-time PCR was 12 fg μL−1 (signal was obtained at CT value of 34.0; Fig. 4c). The real-time PCR represented standard fluorescence amplification of PCR products with an exponential growth, and regression analysis showed a standard curve was linear over at least eight orders of magnitude (R2 > 0.99; Fig. 4d). The standard curve produced by both RealAmp assay and real-time method revealed a good linearity within the detection limit and high correlations between Ct and DNA quantities (R2 > 0.98). The detection limit of the real-time PCR was about 100 times lower than that of the RealAmp assay. All the experiments were performed independently three times, and nearly identical results were obtained.

In artificially inoculated samples, the detection limit of RealAmp was about 103 spores g−1 soil (Fig. 5a) and the real-time PCR assay could detect as low as 102 spores g−1 soil (Fig. 5b). No Fon DNA was detected in the un-inoculated soil samples.

Figure 5.

Quantitative detection of the RealAmp assay in artificially inoculated and partial field soil samples in comparison with real-time PCR. All the samples were tested in triplicate. (a and b) Lanes 1–7 correspond to serial 10-fold dilutions of spores ranging from 106 to 100 spores g−1 artificially inoculated soil. Lane 8 is the negative control. (c) Quantitative detection of partial field soil samples by the RealAmp assay. (d) Statistical comparison of the quantitative results of partial field soil samples between the both assays. (c and d) Lanes 1–2 are the samples collected from the area where watermelon was previously planted. Lanes 3–8 are the samples collected from the watermelon-growing area with Fusarium wilt appearance.

Detection in field soil samples

To evaluate the usefulness of this RealAmp assay for the detection of Fon in field soil samples, 85 samples collected from different watermelon-growing regions were compared between RealAmp and real-time PCR. Among them, 78 samples were positive and seven samples were negative (partial results shown in Fig. 5c and d). In the real-time PCR assay, 80 samples were positive and five negative. Two samples were negative using the RealAmp assay but positive with real-time PCR. The detection rates of real-time PCR and RealAmp were 80/85 (94.1%) and 78/85 (91.8%) for the field samples in this study, respectively. Analysis by a paired Student's t-test showed no significant differences between the results tested by RealAmp and those by real-time PCR (n = 85, P = 0.38).

Discussion

In this study, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay was developed for the rapid and quantitative detection of Fon directly from soil DNA extracts. No expensive reagent and equipment are needed with this assay, only a portable fluorescence reader (ESE-Quant Tube Scanner). The ESE-Quant tube scanner is a major advancement for the quantitative detection of soil-borne pathogens because the device enables single-step amplification and product detection. The device has an eight-tube holder heating block with adjustable temperature settings and spectral devices to detect amplified product using fluorescence spectra (Lucchi et al., 2010; Njiru et al., 2012).

To ensure high specificity, the RealAmp assay uses four primers that recognize six regions on the target DNA. No positive products were amplified from DNAs of other tested formae speciales of Fo, only from different races of Fon, which was identical to results with the PCR method as described by Lin et al. (2012). The sequence of the LAMP fragment showed a 100% identity to the random primer amplified fragment of Fon in GenBank (data not shown). These results demonstrated the high specificity of the RealAmp assay for the detection of Fon.

The RealAmp assay could detect up to 1.2 pg μL−1 Fon genomic DNA. The detection limit was 100 times lower than that of conventional PCR, indicating the LAMP-based assays may have an increased tolerance for inhibitory substances in DNA extracts and lower detection limits in comparison with conventional PCR. This is in accordance with the previous report by Kaneko et al. (2007) that also showed a better tolerance of LAMP to biological substances compared with PCR.

To evaluate the usefulness of the RealAmp assay for the quantitative detection of Fon directly in soil, the real-time PCR was introduced to verify the quantifying results. The relationship between the both assays has been reported previously. Lin et al. (2012) showed that the real-time PCR assay is more sensitive for the diagnosis of toxoplasmosis, but the LAMP assay can be used as an alternative tool for the detection of Fon in field. Additionally, for the diagnosis of tuberculous pleurisy, the LAMP has a much higher specificity with a slightly lower sensitivity compared with real-time PCR (Yang et al., 2011). To verify the suitability of the RealAmp assay for quantitative detection of the pathogen in soil, 85 soil samples collected from different regions were compared with the real-time PCR assay. With the exception of two samples, no significant differences were found between the results obtained in the both assays. Thus the RealAmp assay is an alternative method for quantification of the pathogens, especially in soil which contains contaminants that may inhibit DNA polymerase activity.

The RealAmp assay developed in this study may be more attractive for the detection under natural conditions as it is much faster and has a higher tolerance for inhibitors of DNA preparations from soil samples than conventional PCR. These characteristics increase the versatility of the assay, and its suitability for routine testing laboratories. As a simple and effective approach for the quantitative detection of Fon in soil under natural conditions, it is quite valuable for monitoring of disease epidemiology and for integrated management.

Acknowledgements

The work was supported by the Basic Scientific Research Special Fund for Central Nonprofit Scientific Research Institutes (1630042012001&1630042013021), the National Natural Science Foundation of China (31301628), Hainan Provincial International Cooperation Key Project (2012-GH-007), Special Fund for Agro-scientific Research in the Public Interest (200903049) and Institute Talent Scientific Research Foundation (Hzs1201). We thank Prof. Dr Xu from Beijing Academy of Agriculture and Forestry, Prof. Dr Kong from Hebei Academy of Agricultural Sciences, who kindly provided us with some isolates of F. oxysporum, and Dr Westphal from Department of Agronomy, Julius Kühn-Institut, Germany, for his kind corrections and suggestions during the preparation of the article.

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