Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Development of loop-mediated isothermal amplification assay for the detection of Pythium myriotylum S. Fukuta1, R. Takahashi1, S. Kuroyanagi1, Y. Ishiguro2, N. Miyake1, H. Nagai1, H. Suzuki3, T. Tsuji3, F. Hashizume4, H. Watanabe5 and K. Kageyama2 1 2 3 4 5
Agri-environmental Division, Aichi Agricultural Research Center, Nagakute, Aichi, Japan River Basin Research Center, Gifu University, Gifu, Japan Division of Regenerative Technology, Mie Prefecture Agricultural Research Center, Matsusaka, Mie, Japan Division of Biotechnology, Mie Prefecture Agricultural Research Center, Matsusaka, Mie, Japan Agri-environmental Division, Gifu Prefectural Agricultural Technology Center, Gifu, Japan
Significance and Impact of the Study: This study shows the first LAMP assay for the detection of Pythium myriotylum. The primer set designed from ITS region of P. myriotylum can detect the pathogen in field sample with a fast and convenient method. Analysis of the annealing curve of the LAMP reaction products increases the reliability of the LAMP diagnosis. This study shows that the diagnostic method using the LAMP assay is useful for monitoring P. myriotylum in the field.
Keywords annealing curve, diagnosis, loop-mediated isothermal amplification, Pythium myriotylum. Correspondence Shiro Fukuta, Agri-environmental Division, Aichi Agricultural Research Center, 1-1 Sagamine, Yazako, Nagakute, Aichi 480-1193, Japan. E-mail: [email protected] 2014/0056: received 10 January 2014, revised 28 February 2014 and accepted 3 March 2014 doi:10.1111/lam.12244
Abstract This study reports the development of a loop-mediated isothermal amplification (LAMP) reaction for the detection of Pythium myriotylum. The primer set targeting the ITS sequence of P. myriotylum worked most efficiently at 60°C and allowed the detection of P. myriotylum DNA within 30 min by fluorescence monitoring using a real-time PCR instrument. The peak denaturing temperature of amplified DNA was about 870°C. In specificity tests using eight Pythium myriotylum strains, 59 strains from 39 species of Pythium, 11 Phytophthora strains and eight other soil-borne pathogens, LAMP gave no cross-reactions. The detection limit was 100 fg of genomic DNA, which was as sensitive as PCR. LAMP could detect P. myriotylum in hydroponic solution samples, and the results coincided with those of the conventional plating method in almost all cases. The LAMP method established in this study is a simple and sensitive tool for the detection of P. myriotylum.
Introduction Pythium species contain economically important plant pathogens and have been frequently found to cause diseases in soilless greenhouse systems. Pythium can contaminate irrigation water supplies and spread rapidly in hydroponically grown crops. Early infection by Pythium species causes yield losses in many vegetables and ornamental crops. Pythium myriotylum Drechsler is a causal agent of root rot in economically important crops including peanuts, tomato, rye, wheat, oats, cucumber, soya bean, sorghum, tobacco, cabbage and maize (Wang et al. 2003a). Rapid and reliable detection is essential for an effective control of these diseases. Traditionally, detection
and identification of Pythium species have been achieved by plating methods, bait methods or a combination of both (Pettitt et al. 2002; Watanabe et al. 2007, 2008). However, it is difficult, laborious and time-consuming to detect Pythium species based on morphological characteristics, partly because these characteristics vary under different culture conditions and involve intraspecific phenotypic variation (Pettitt et al. 2002). Within the past two decades, molecular diagnosis has become an alternative strategy for the identification of Pythium species. Especially, the oospores of P. myriotylum are deeply dormant, so the detection of P. myriotylum is difficult at times (Kageyama and Ui 1982; Wang and Chang 2003; Wang et al. 2003b). In fact, P. myriotylum
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Sensitivity of LAMP assay The sensitivity of the LAMP reaction performed with genomic DNA of P. myriotylum (strain C41) in the range of 1 ng to 1 fg was investigated at 60 and 625° C. The sensitivity was 100 fg of genomic DNA at 60°C (Fig. 2a) and 1 pg at 625°C (Fig. 2b). Consequently,
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The dependence of the LAMP reaction on thermal conditions was investigated. The LAMP reaction was performed at 60, 625, 65 or 675°C for 60 min. One hundred pg of genomic DNAs of Pythium species of phylogenetic clade B1 (Levesque and de Cock 2004; P. myriotylum, P. arrhenomanes, P. graminicola, P. plurisporium, P. sulcatum and P. torulosum) were used as templates (Fig. 1). Nonspecific reactions to Pythium clade B1 species were not observed at any reaction temperature. Fluorescence of the reaction with P. myriotylum DNA began to increase after 15 min at 60 and 625°C (Fig. 1a,b) and after 20 min at 65°C (Fig. 1c) and 675°C (Fig. 1d). The annealing curves of the positive LAMP reactions had the same peak temperature at 867–872°C (data not shown). 50
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was not detected at all using the soil dilution method (Ishiguro et al. 2013). Useful detection methods based on polymerase chain reaction (PCR) have been developed that involve species-specific SCAR markers (Ahonsi et al. 2010), PCR-restriction fragment length polymorphisms (PCR-RFLP) (Wang et al. 2003a; Kageyama et al. 2005; G omez-Alpızar et al. 2011) and real-time PCR (Schroeder et al. 2006; Le Floch et al. 2007). Diagnosis by PCR is more sensitive and faster than traditional culture methods. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that relies on autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase (Notomi et al. 2000). The LAMP assay was developed for the detection of plant pathogenic viruses (Fukuta et al. 2003, 2004, 2012), viroids (Boubourakas et al. 2009), fungi (Tomlinson et al. 2010), bacteria (Rigano et al. 2010) and oomycetes pathogen (Tomlinson et al. 2007; Dai et al. 2012; Fukuta et al. 2013a,b). LAMP requires four different primers designed specifically to recognize six distinct regions on the target gene. The LAMP method has advantages over other DNA amplification methods: (i) LAMP can amplify nucleic acids under isothermal conditions at temperatures between 60 and 68°C (Notomi et al. 2000); (ii) LAMP is highly specific as it relies on a set of four specifically designed primers; (iii) in the LAMP assay, DNA amplification can be monitored by fluorescence of the LAMP reaction solution using intercalating dye (Notomi et al. 2000). The aim of this study was to develop a LAMP assay for P. myriotylum ITS sequence amplification and to evaluate the potential of this method for diagnostic purposes.
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Reaction time (min) Figure 1 The fluorescence of the loop-mediated isothermal amplification reaction at 60°C (a), 625°C (b), 65°C (c) or 675°C (d) using Pythium myriotylum primer set. (■) Pythium arrhenomanes; (□) Pythium graminicola; (▲) Pythium myriotylum; ( ) Pythium plurisporium; (♦) Pythium sulcatum; (♢) Pythium torulosum; (●) no template.
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Letters in Applied Microbiology 59, 49--57 © 2014 The Society for Applied Microbiology
LAMP assay for Pythium myriotylum
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60°C was selected as standard reaction temperature for further evaluations of the LAMP detection of P. myriotylum. PCR was performed using the same DNA samples. The amplification product of 149 bp was observed in samples from 1 ng to 100 fg (Fig. 2c), indicating similar sensitivities of LAMP and PCR.
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Specificity of the LAMP assay The specificity of the LAMP assay was tested with DNA from eight P. myriotylum strains, 59 strains belonging to 39 other Pythium species, 11 Phytophthora strains and eight other soil-borne pathogens (Table 1). One hundred pg of genomic DNA extracted from each strain was used. The fluorescence of the LAMP reaction solution of all eight P. myriotylum strains increased detectably after 15 min (Fig. 3a and Table 1). Other Pythium species, Phytophthora species and other soil-borne pathogens gave no amplification (Table 1). The peak annealing temperature of all P. myriotylum strains was almost the same, about 870°C (Table 1), and LAMP amplification products of all P. myriotylum strains showed the same band patterns in agarose electrophoresis (Fig. 3b). Several methods for Pythium species differentiation targeting the rDNA ITS sequence have been developed (Kageyama et al. 2003; Ahonsi et al. 2010), because ITS regions of Pythium species vary in length and sequence. In this study, a specific primer set was designed based on the 30 sequence of ITS2. The specificity of the designed LAMP primer set was confirmed, as it enabled reliable discrimination of closely related B1 clade Pythium species.
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Figure 2 Relative sensitivity of the loop-mediated isothermal amplification (LAMP) reaction at 60°C (a) and 625 °C (b), and PCR (c) for the detection of Pythium myriotylum DNA. The fluorescence of LAMP reaction with P. myriotylum DNA in the range of 1 ng to 1 fg. Agarose gel electrophoretic analysis of the PCR with 1 ng (lane 1), 100 pg (lane 2), 10 pg (lane 3), 1 pg (lane 4), 100 fg (lane 5), 10 fg (lane 6) and 1 fg (lane 7) of P. myriotylum DNA. Arrow shows the band amplified from P. myriotylum DNA. (■) 1 ng; (□) 100 pg; (▲) 10 pg; ( ) 1 pg; (♦) 100 fg; (♢) 10 fg; (●) 1 fg; (○) no template.
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Culture method and LAMP reaction for the detection of P. myriotylum were compared using 22 hydroponic solution samples from tomato cultures (Table 2). Ten samples were positive and 11 samples tested negative by both culture method and LAMP reaction. In one sample collected on 19th December, P. myriotylum was detected by the culture method but not by the LAMP assay. Only one colony was observed growing in culture, indicating that the concentration of P. myriotylum was extremely low in this sample. The peak of denaturing temperature for all positive samples was about 870°C (Table 2), coinciding with that of the LAMP products amplified from P. myriotylum mycelial DNA (Fig. 3a,b). This result showed that the LAMP reaction performed with DNA extracted from hydroponic solution samples can detect P. myriotylum accurately. We have developed a LAMP method for the detection of P. myriotylum. The primer set designed for the ITS region of P. myriotylum amplified the target genes at 60° C most efficiently. The LAMP assay provided reliable results in the range of 1 ng to 100 fg genomic DNA within 30 min. The LAMP method developed in this study, which uses fluorescence to monitor the reaction, shortens the time
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Table 1 List of Pythium, Phytophthora and other soil-borne pathogens used in this study
Species
Clade*
Isolate
Pythium adhaerens P. aphanidermatum
A A A A A A A A A A A B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B2 B2 B2 D D
CBS520.74 TA114 Tju132 UOP390 GA1 GF1089 SSB01-184 05Hay sand1 F01 YnHW-1 N02E2 3-4 NBRC 100102 MAFF425415 NBRC100113 C41 F02 FuOri1 GF46 O03 S06 SP-OP03-1 CBS100117 NBRC100117 TJu143 NBRC107450 MAFF305576 NBRC107365 MAFF241099 NBRC100114 GFSt2-1 CBS234.94 NBRC100115 CBS528.74 1B4162 NBRC104223 CBS266.38 NBRC100108 CBS263.30 NBRC100116 NBRC100119 CO132 IFO31993 CBS285.31 NBRC104222 NBRC107363 1D2S021 C101 NBRC100122 CBS808.96 CBS811.70 CBS259.30
P. P. P. P.
P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.
monospermum arrhenomanes graminicola myriotylum
plurisporium sulcatum torulosum aquatile dissotocum pyrilobum acanthicum periplocum oligandrum hypogynum rostratum middletonii parvum takayamanum intermedium irregulare I irregulare II spinosum sylvaticum nagaii paddicum anandrum senticosum undulatum heterothallicum splendes ultimum nunn polymastum chamaehyphon
E1 E1 E2 E2 E2 F F F F F G G H H H I I I J J K
Origin
Carot Soil Spinach Bent grass Bent grass Gomphrena spp. Spinach
Carnation Nemesia spp. Soy bean Kalanchoe Dianthus spp. Curcuma spp. Peanut
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+ + + + + + + +
872 871 870 869 869 870 869 871 (continued)
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Table 1 (Continued)
Species
Clade*
Isolate
P. helicoides
K K K K K K K K K K K K K K K K 1 2 2 3 4 5 6 7 8 9 10
NBRC100107 B5 ori OM6-1 sh Tsu-C1 RoTo ori SP-KS04-A2 ERR-1 Nastr1 GiBeg1 TGhel5 sh1 GUPo1 sh RO-Ph1 sz1 Fuk 3-1 YP1 CBS768.73 2D111 GF101 C94 IFO30696 C71 P0113 P1102 P3826 P6358 CH92-15 P6195 GF749 GFHT6 MAFF72510 HY Type1 S02 IFO32708 Vaal 130308 AiTog
P. oedochium P. ostracodes P. vexans Phytophthora nicotiane Ph. citricola Ph. capsici Ph. nemorosa Ph. palmivora Ph. heveae Ph. humicola Ph. cambivora Ph. cryptogea Ph. insolita Ph. chrysanthemi Aphanomyces sp. Fusarium oxysporum Plasmodiophora brassicae Pyrenochaeta lycopercisi Rhizoctonia solani Saprolegnia sp. Verticillium albo-atrum Sclerotinia sclerotiorum
Origin
Rose Rose Chrysanthemum Rose Strawberry Erica spp. Strawberry Begonia Strawberry Poinsettia Rose Soy bean
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LAMP, loop-mediated isothermal amplification. *Phylogenetic clades in Pythium and Phytophthora species provided by L evesque and de Cock (2004) and Blair et al. (2008), respectively.
required to detect P. myriotylum: the pathogen was detected in 30 min, compared with 60 min required when the reaction is followed by turbidity monitoring (data not shown). As fluorescent dyes bind to double-stranded DNAs, which are amplified by Bst DNA polymerase used in the LAMP reaction, the fluorescence of the LAMP reaction solution increases with DNA accumulation. In contrast, the turbidity of the LAMP reaction solution is caused by an increasing quantity of magnesium pyrophosphate, which is a by-product of the LAMP reaction (Mori et al. 2001). Detection of magnesium pyrophosphate is delayed following DNA amplification. The LAMP reaction with intercalating dyes such as EvaGreen has another advantage. As the amplification
efficiency of the LAMP reaction is extremely high, any contamination of reagents and instruments can easily result in a false-positive result (Peng et al. 2011). Enzyme reactions and electrophoresis using LAMP products raise the danger of contamination, so that LAMP products should not be handled directly if possible. Peak denaturing temperatures can be distinguished clearly by annealing analysis. LAMP products amplified by different LAMP primer sets differ in peak temperature (Fukuta et al. 2013a,b). Therefore, monitoring denaturing peak temperatures helps distinguishing LAMP products without electrophoresis. The denaturing peak temperature of LAMP products amplified by the P. myriotylum primer set was about 87°C. There were no differences of peak tempera-
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and were maintained on cornmeal agar (CMA) at 25°C. Total genomic DNA from mycelia was extracted according to the procedure of Kageyama et al. (2003).
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Figure 3 The fluorescence (a) and agarose gel electrophoretic analysis (b) of the loop-mediated isothermal amplification reaction Pythium myriotylum eight strains, NBRC100113 (lane 1), C41 (lane 2), F02 (lane 3), FuOri1 (lane 4), GF46 (lane 5), O03 (lane 6), S06 (lane 7) and SP-OP03-1 (lane 8). (■) NBRC100113; (□) C41; (▲) F02; ( ) FuOri1; (♦) GF46; (♢) O03; (●) S06; (○) SP-OP03-1.
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ture between P. myriotylum strains. The detection of the pathogen in field samples showed that the LAMP method monitoring the denaturing peak temperature worked successfully. The results of LAMP were conformed to those of the culture method in all samples tested except for one that had very low zoospore concentrations. Pythium diseases have economical impact worldwide, and the development of simple, cost-effective, sensitive and rapid diagnostic techniques is important. The LAMP detection described in this study represents a sensitive, specific and reliable diagnostic protocol. Materials and methods
The LAMP reaction uses four primers (F3, B3, FIP and BIP), which anneal to six regions of the target DNA (Notomi et al. 2000). ITS sequences were chosen as the targets for LAMP primer design. The ITS sequences of P. myriotylum (GenBank Acc. No: GQ121316) and related species of the molecular phylogenetic clade B1 including P. arrhenomanes (EU162759), P. graminicola (AY598625), P. plurispoium (HQ643750), P. sulcatum (DQ528744) and P. torulosum (AY228542) were downloaded from the GenBank database. These sequences were compared by multiple sequence alignment using DNSIS (Hitachi Solutions, Tokyo, Japan). A set of primers for LAMP amplification was designed from the sequences, which differ from those of other Pythium species, using PRIMEREXPLORER V.4 software (http://primerexplorer.jp), and the primers were further modified manually. Consequently, five primers, F3 (50 -GGCGGTATGTTAGGCTTCG-30 ), B3 (50 -TGATATGC TTAAGTCAGCGG-30 ), FIP (50 -GCCCCACACAAGACAG GTACAC-TTGCAGCTGACGGGGTG-30 ), BIP (50 -GTGA GAGTTTCGGCTCCTGCAGG-TCTTATCTTGATATCC AGGTCC-30 ) and B-Loop(50 -CAGGCGGCTTTTTCAAT TG-30 ) were used for the detection of P. myriotylum. LAMP reaction monitoring by fluorescence The LAMP reaction was performed in a total volume of 25 ll. The reaction contained 15 ll of Isothermal Master Mix (Nippon Gene, Osaka, Japan), which contains dsDNA binding dye, 02 lmol l1 each of F3 and B3 primer, 16 lmol l1 each of FIP and BIP primer, 08 lmol l1 of B-Loop primer and 1 ll DNA template solution. The mixtures were incubated at 60–675°C for 30 min while monitoring the fluorescence of the reaction solution using FAM channel by a Step One (ABI, Tokyo, Japan) real-time PCR device, followed by a postamplification anneal analysis step (95 to 80°C, 02°C/s) that was performed to confirm the annealing curve.
Species, strains and DNA extraction
PCR
A total of 86 strains were used, including 8 P. myriotylum strains, 59 non-P. myriotylum Pythium strains, 11 Phytophthora strains, which belong to 10 clades, and eight other soil-borne pathogens (Aphanomyces, Fusarium, Plasmodiophora, Pyrenochaeta, Rhizoctonia, Saprolegnia, Verticillium and Sclerotinia spp.), all listed in Table 1. Isolates were collected from various hosts and geographic origins
PCR mixture contained 10 mmol l1 Tris-HCl pH 83, 50 mmol l1 KCl and 15 mmol l1 MgCl2, 02 mmol l1 dNTP mixture, 1 lmol l1 each of primers PyH (50 -CT GTTCTTTCCTTGAGGTG-30 ) (Asano et al. 2010) and kk_MYRR (50 -GAGCCGAAACTCTCACAAGACC-30 ) (Kageyama et al. 2012), 10 ng bovine serum albumin (SigmaAldrich, Tokyo, Japan), 1 unit Fast Start Taq DNA
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Table 2 Detection of Pythium myriotylum using the culture method and the LAMP reaction from hydroponic solution samples from tomato fields
Sample no.
Sampling date
Conventional plating method Concentration (CFU/1000 ml)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
12 September, 2011 12 September, 2011 27 September, 2011 27 September, 2011 7 November, 2011 7 November, 2011 21 November, 2011 21 November, 2011 6 December, 2011 6 December, 2011 19 December, 2011 19 December, 2011 10 January, 2012 10 January, 2012 31 January, 2012 31 January, 2012 13 February, 2012 13 February, 2012 12 March, 2012 12 March, 2012 26 March, 2012 26 March, 2012
1500 0 110 0 200 0 9 0 100 0 1 0 20 0 2 0 100 – 20 – 100 0
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Annealing temperature
77 – 107 – 86 – 155 – 93 – – – 88 – 215 – 95 – 107 – 103 –
871 – 869 – 870 – 871 – 869 – – – 868 – 870 – 870 – 869 – 869 –
LAMP, loop-mediated isothermal amplification.
polymerase (Roche Applied Science, Tokyo, Japan) and 1 ll DNA solution in a total volume of 25 ll. PCR was conducted in a DNA thermal cycler (Gene Amp PCR system 2700; ABI) at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 1 min, with a final extension step at 72°C for 10 min. Amplification products were confirmed by electrophoresis in 2% agarose gels. Gels were stained with ethidium bromide and photographed under ultraviolet light.
Acknowledgements
Detection of P. myriotylum in hydroponic solution samples
References
Hydroponic solution samples were collected from September 2011 to March 2012 from tomato greenhouses with hydroponic culture systems in Mie, Japan. Two 1000-ml hydroponic solution samples were filtered through 5 lm DURAPOREâ (Millipore, Billerica, MA) gridded membrane filters. DNA was extracted from one membrane filter following Kageyama et al. (2003). Extracted DNA (1 ll) was used in the LAMP assay, and the second membrane filter was put on NARM medium (Morita and Tojo 2007) and incubated at 40°C for 24 h. Isolation and detection of P. myriotylum by the culture method were performed according to Kageyama et al. (2003).
This work was supported by the Science and Technology Research promotion program for agriculture, forestry, fisheries and food industry, Ministry of Agriculture, Forestry and Fisheries, Japan. Conflict of Interest There is no conflict of interest to declare.
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by the reverse transcription loop-mediated isothermal amplification. J Virol Methods 160, 63–68. Dai, T.-T., Lu, C.-C., Lu, J., Dong, S., Ye, W., Wang, Y. and Zheng, X. (2012) Development of a loop-mediated isothermal amplification assay for detection of Phytophthora sojae. FEMS Microbiol Lett 334, 27–34. Fukuta, S., Kato, S., Yoshida, K., Mizukami, Y., Ishida, A., Ueda, J., Kanbe, M. and Ishimoto, Y. (2003) Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction. J Virol Methods 112, 35–40. Fukuta, S., Ohishi, K., Yoshida, K., Mizukami, Y., Ishida, A. and Kanbe, M. (2004) Development of immunocapture reverse transcription loop-mediated isothermal amplification for the detection of tomato spotted wilt virus from chrysanthemum. J Virol Methods 121, 49–55. Fukuta, S., Takeyama, K., Suzuki, M., Shichi, M., Ichikawa, K. and Nakanishi, H. (2012) Detection of kyuri green mottle mosaic virus from soil by the immunocapture reverse transcription loop-mediated isothermal amplification reaction. Plant Pathol J 11, 51–59. Fukuta, S., Takahashi, R., Kuroyanagi, S., Miyake, N., Nagai, H., Suzuki, H., Hashizume, F., Tsuji, T. et al. (2013a) Detection of Pythium aphanidermatum in tomato using loop-mediated isothermal amplification (LAMP) with species-specific primers. Eur J Plant Pathol 136, 689–701. Fukuta, S., Tamura, M., Maejima, H., Takahashi, R., Kuwayama, S., Tsuji, T., Yoshida, T., Itoh, K. et al. (2013b) Differential detection of Wheat yellow mosaic virus, Japanese soil-borne wheat mosaic virus and Chinese wheat mosaic virus by reverse transcription loop-mediated isothermal amplification reaction. J Virol Methods 189, 348–354. G omez-Alpızar, L., Saalau, E., Picado, I., Tambong, J.T. and Saborıo, F. (2011) A PCR-RFLP assay for identification and detection of Pythium myriotylum, causal agent of the cocoyam root rot disease. Lett Appl Microbiol 52, 185–192. Ishiguro, Y., Asano, T., Otsubo, K., Suga, H. and Kageyama, K. (2013) Simultaneous detection by multiplex PCR of the high-temperature-growing Pythium species: P. aphanidermatum, P. helicoides and P. myriotylum. J Gen Plant Pathol 79, 350–358. Kageyama, K. and Ui, T. (1982) Survival structure of Pythium spp. in the soils of bean fields. Ann Phytopath Soc Japan 48, 308–313. Kageyama, K., Suzuki, M., Priyatmojo, A., Oto, Y., Ishiguro, K., Suga, H., Aoyagi, T. and Fukui, H. (2003) Characterization and identification of asexual strains of Pythium associated with root rot of rose in Japan. J Phytopathol 151, 485–491. Kageyama, K., Nakashima, A., Kajihara, Y., Suga, H. and Nelson, E.B. (2005) Phylogenetic and morphological analyses of Pythium graminicola and related species. J Gen Plant Pathol 71, 174–182.
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Kageyama, K., Asano, T., Otsubo, K. and Suga, H. (2012) Simultaneous detection of Pythium aphanidermatum, P. helicoides and P. myriotylum using multiplex PCR. The 2nd Korea-Japan Joint Symposium and the 2012 Annual Meeting of PSJ, Fukuoka. Le Floch, G., Tambong, J., Vallance, J., Tirilly, Y., Levesque, A. and Rey, P. (2007) Rhizosphere persistence of three Pythium oligandrum strains in tomato soilless culture assessed by DNA microarray and real-time PCR. FEMS Microbiol Ecol 61, 317–326. Levesque, C.A. and de Cock, A.W.A.M. (2004) Molecular phylogeny and taxonomy of the genus Pythium. Mycol Res 108, 1363–1383. Mori, Y., Nagamine, K., Tomita, N. and Notomi, T. (2001) Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 289, 150–154. Morita, Y. and Tojo, M. (2007) Modifications of PARP medium using fluazinam, miconazole, and nystatin for detection of Pythium spp. in soil. Plant Dis 91, 1591–1599. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. and Hase, T. (2000) Loopmediated isothermal amplification of DNA. Nucleic Acids Res 28, e63. Peng, Y., Xie, Z., Liu, J., Pang, Y., Deng, X., Xie, Z., Xie, L., Fan, Q. et al. (2011) Visual detection of H3 subtype avian influenza viruses by reverse transcription loop-meditated isothermal amplification assay. Virol J 8, 337. Pettitt, T.R., Wakeham, A.J., Wainwright, M.F. and White, J.G. (2002) Comparison of serological, culture, and bait methods for detection of Pythium and Phytophthora zoospores in water. Plant Pathol 51, 720–727. Rigano, L.A., Marano, M.R., Castagnaro, A.P., Amaral, A.M.D. and Vojnov, A.A. (2010) Rapid and sensitive detection of citrus bacterial canker by loop-mediated isothermal amplification combined with simple visual evaluation methods. BMC Microbiol 10, 176. Schroeder, K.L., Okubara, P.A., Tambong, J.T., Levesque, C.A. and Paulitz, T.C. (2006) Identification and quantification of pathogenic Pythium spp. from soils in eastern Washington using real-time polymerase chain reaction. Phytopathology 96, 637–647. Tomlinson, J.A., Barker, I. and Boonham, N. (2007) Faster, simpler, more-specific methods for improved molecular detection of Phytophthora ramorum in the field. Appl Environ Microbiol 73, 4040–4047. Tomlinson, J.A., Dickinson, M.J. and Boonham, N. (2010) Detection of Botrytis cinerea by loop-mediated isothermal amplification. Lett Appl Microbiol 51, 650–657. Wang, P.H. and Chang, C.W. (2003) Detection of the low-germination-rate resting oospores of Pythium myriotylum from soil by PCR. Lett Appl Microbiol 36, 157–161.
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Wang, P.H., Chung, C.Y., Lin, Y.S. and Yeh, Y. (2003a) Use of polymerase chain reaction to detect the soft rot pathogen, Pythium myriotylum, in infected ginger rhizomes. Lett Appl Microbiol 36, 116–120. Wang, P.H., Wang, Y.T. and White, J.G. (2003b) Speciesspecific PCR primers for Pythium developed from ribosomal ITS1 region. Lett Appl Microbiol 37, 127–132.
Watanabe, H., Taguchi, Y., Hyakumachi, M. and Kageyama, K. (2007) Pythium and Phytophthora species associated with root and stem rots of kalanchoe. J Gen Plant Pathol 73, 81–88. Watanabe, H., Kageyama, K., Taguchi, Y., Horinouchi, H. and Hyakumachi, M. (2008) Bait method to detect Pythium species that grow at high temperatures in hydroponic solutions. J Gen Plant Pathol 74, 417–424.
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ORIGINAL ARTICLE
Development of loop-mediated isothermal amplification assay for the detection of Pythium myriotylum S. Fukuta1, R. Takahashi1, S. Kuroyanagi1, Y. Ishiguro2, N. Miyake1, H. Nagai1, H. Suzuki3, T. Tsuji3, F. Hashizume4, H. Watanabe5 and K. Kageyama2 1 2 3 4 5
Agri-environmental Division, Aichi Agricultural Research Center, Nagakute, Aichi, Japan River Basin Research Center, Gifu University, Gifu, Japan Division of Regenerative Technology, Mie Prefecture Agricultural Research Center, Matsusaka, Mie, Japan Division of Biotechnology, Mie Prefecture Agricultural Research Center, Matsusaka, Mie, Japan Agri-environmental Division, Gifu Prefectural Agricultural Technology Center, Gifu, Japan
Significance and Impact of the Study: This study shows the first LAMP assay for the detection of Pythium myriotylum. The primer set designed from ITS region of P. myriotylum can detect the pathogen in field sample with a fast and convenient method. Analysis of the annealing curve of the LAMP reaction products increases the reliability of the LAMP diagnosis. This study shows that the diagnostic method using the LAMP assay is useful for monitoring P. myriotylum in the field.
Keywords annealing curve, diagnosis, loop-mediated isothermal amplification, Pythium myriotylum. Correspondence Shiro Fukuta, Agri-environmental Division, Aichi Agricultural Research Center, 1-1 Sagamine, Yazako, Nagakute, Aichi 480-1193, Japan. E-mail: [email protected] 2014/0056: received 10 January 2014, revised 28 February 2014 and accepted 3 March 2014 doi:10.1111/lam.12244
Abstract This study reports the development of a loop-mediated isothermal amplification (LAMP) reaction for the detection of Pythium myriotylum. The primer set targeting the ITS sequence of P. myriotylum worked most efficiently at 60°C and allowed the detection of P. myriotylum DNA within 30 min by fluorescence monitoring using a real-time PCR instrument. The peak denaturing temperature of amplified DNA was about 870°C. In specificity tests using eight Pythium myriotylum strains, 59 strains from 39 species of Pythium, 11 Phytophthora strains and eight other soil-borne pathogens, LAMP gave no cross-reactions. The detection limit was 100 fg of genomic DNA, which was as sensitive as PCR. LAMP could detect P. myriotylum in hydroponic solution samples, and the results coincided with those of the conventional plating method in almost all cases. The LAMP method established in this study is a simple and sensitive tool for the detection of P. myriotylum.
Introduction Pythium species contain economically important plant pathogens and have been frequently found to cause diseases in soilless greenhouse systems. Pythium can contaminate irrigation water supplies and spread rapidly in hydroponically grown crops. Early infection by Pythium species causes yield losses in many vegetables and ornamental crops. Pythium myriotylum Drechsler is a causal agent of root rot in economically important crops including peanuts, tomato, rye, wheat, oats, cucumber, soya bean, sorghum, tobacco, cabbage and maize (Wang et al. 2003a). Rapid and reliable detection is essential for an effective control of these diseases. Traditionally, detection
and identification of Pythium species have been achieved by plating methods, bait methods or a combination of both (Pettitt et al. 2002; Watanabe et al. 2007, 2008). However, it is difficult, laborious and time-consuming to detect Pythium species based on morphological characteristics, partly because these characteristics vary under different culture conditions and involve intraspecific phenotypic variation (Pettitt et al. 2002). Within the past two decades, molecular diagnosis has become an alternative strategy for the identification of Pythium species. Especially, the oospores of P. myriotylum are deeply dormant, so the detection of P. myriotylum is difficult at times (Kageyama and Ui 1982; Wang and Chang 2003; Wang et al. 2003b). In fact, P. myriotylum
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Sensitivity of LAMP assay The sensitivity of the LAMP reaction performed with genomic DNA of P. myriotylum (strain C41) in the range of 1 ng to 1 fg was investigated at 60 and 625° C. The sensitivity was 100 fg of genomic DNA at 60°C (Fig. 2a) and 1 pg at 625°C (Fig. 2b). Consequently,
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The dependence of the LAMP reaction on thermal conditions was investigated. The LAMP reaction was performed at 60, 625, 65 or 675°C for 60 min. One hundred pg of genomic DNAs of Pythium species of phylogenetic clade B1 (Levesque and de Cock 2004; P. myriotylum, P. arrhenomanes, P. graminicola, P. plurisporium, P. sulcatum and P. torulosum) were used as templates (Fig. 1). Nonspecific reactions to Pythium clade B1 species were not observed at any reaction temperature. Fluorescence of the reaction with P. myriotylum DNA began to increase after 15 min at 60 and 625°C (Fig. 1a,b) and after 20 min at 65°C (Fig. 1c) and 675°C (Fig. 1d). The annealing curves of the positive LAMP reactions had the same peak temperature at 867–872°C (data not shown). 50
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was not detected at all using the soil dilution method (Ishiguro et al. 2013). Useful detection methods based on polymerase chain reaction (PCR) have been developed that involve species-specific SCAR markers (Ahonsi et al. 2010), PCR-restriction fragment length polymorphisms (PCR-RFLP) (Wang et al. 2003a; Kageyama et al. 2005; G omez-Alpızar et al. 2011) and real-time PCR (Schroeder et al. 2006; Le Floch et al. 2007). Diagnosis by PCR is more sensitive and faster than traditional culture methods. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that relies on autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase (Notomi et al. 2000). The LAMP assay was developed for the detection of plant pathogenic viruses (Fukuta et al. 2003, 2004, 2012), viroids (Boubourakas et al. 2009), fungi (Tomlinson et al. 2010), bacteria (Rigano et al. 2010) and oomycetes pathogen (Tomlinson et al. 2007; Dai et al. 2012; Fukuta et al. 2013a,b). LAMP requires four different primers designed specifically to recognize six distinct regions on the target gene. The LAMP method has advantages over other DNA amplification methods: (i) LAMP can amplify nucleic acids under isothermal conditions at temperatures between 60 and 68°C (Notomi et al. 2000); (ii) LAMP is highly specific as it relies on a set of four specifically designed primers; (iii) in the LAMP assay, DNA amplification can be monitored by fluorescence of the LAMP reaction solution using intercalating dye (Notomi et al. 2000). The aim of this study was to develop a LAMP assay for P. myriotylum ITS sequence amplification and to evaluate the potential of this method for diagnostic purposes.
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Letters in Applied Microbiology 59, 49--57 © 2014 The Society for Applied Microbiology
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60°C was selected as standard reaction temperature for further evaluations of the LAMP detection of P. myriotylum. PCR was performed using the same DNA samples. The amplification product of 149 bp was observed in samples from 1 ng to 100 fg (Fig. 2c), indicating similar sensitivities of LAMP and PCR.
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Specificity of the LAMP assay The specificity of the LAMP assay was tested with DNA from eight P. myriotylum strains, 59 strains belonging to 39 other Pythium species, 11 Phytophthora strains and eight other soil-borne pathogens (Table 1). One hundred pg of genomic DNA extracted from each strain was used. The fluorescence of the LAMP reaction solution of all eight P. myriotylum strains increased detectably after 15 min (Fig. 3a and Table 1). Other Pythium species, Phytophthora species and other soil-borne pathogens gave no amplification (Table 1). The peak annealing temperature of all P. myriotylum strains was almost the same, about 870°C (Table 1), and LAMP amplification products of all P. myriotylum strains showed the same band patterns in agarose electrophoresis (Fig. 3b). Several methods for Pythium species differentiation targeting the rDNA ITS sequence have been developed (Kageyama et al. 2003; Ahonsi et al. 2010), because ITS regions of Pythium species vary in length and sequence. In this study, a specific primer set was designed based on the 30 sequence of ITS2. The specificity of the designed LAMP primer set was confirmed, as it enabled reliable discrimination of closely related B1 clade Pythium species.
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Figure 2 Relative sensitivity of the loop-mediated isothermal amplification (LAMP) reaction at 60°C (a) and 625 °C (b), and PCR (c) for the detection of Pythium myriotylum DNA. The fluorescence of LAMP reaction with P. myriotylum DNA in the range of 1 ng to 1 fg. Agarose gel electrophoretic analysis of the PCR with 1 ng (lane 1), 100 pg (lane 2), 10 pg (lane 3), 1 pg (lane 4), 100 fg (lane 5), 10 fg (lane 6) and 1 fg (lane 7) of P. myriotylum DNA. Arrow shows the band amplified from P. myriotylum DNA. (■) 1 ng; (□) 100 pg; (▲) 10 pg; ( ) 1 pg; (♦) 100 fg; (♢) 10 fg; (●) 1 fg; (○) no template.
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Culture method and LAMP reaction for the detection of P. myriotylum were compared using 22 hydroponic solution samples from tomato cultures (Table 2). Ten samples were positive and 11 samples tested negative by both culture method and LAMP reaction. In one sample collected on 19th December, P. myriotylum was detected by the culture method but not by the LAMP assay. Only one colony was observed growing in culture, indicating that the concentration of P. myriotylum was extremely low in this sample. The peak of denaturing temperature for all positive samples was about 870°C (Table 2), coinciding with that of the LAMP products amplified from P. myriotylum mycelial DNA (Fig. 3a,b). This result showed that the LAMP reaction performed with DNA extracted from hydroponic solution samples can detect P. myriotylum accurately. We have developed a LAMP method for the detection of P. myriotylum. The primer set designed for the ITS region of P. myriotylum amplified the target genes at 60° C most efficiently. The LAMP assay provided reliable results in the range of 1 ng to 100 fg genomic DNA within 30 min. The LAMP method developed in this study, which uses fluorescence to monitor the reaction, shortens the time
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Table 1 List of Pythium, Phytophthora and other soil-borne pathogens used in this study
Species
Clade*
Isolate
Pythium adhaerens P. aphanidermatum
A A A A A A A A A A A B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B2 B2 B2 D D
CBS520.74 TA114 Tju132 UOP390 GA1 GF1089 SSB01-184 05Hay sand1 F01 YnHW-1 N02E2 3-4 NBRC 100102 MAFF425415 NBRC100113 C41 F02 FuOri1 GF46 O03 S06 SP-OP03-1 CBS100117 NBRC100117 TJu143 NBRC107450 MAFF305576 NBRC107365 MAFF241099 NBRC100114 GFSt2-1 CBS234.94 NBRC100115 CBS528.74 1B4162 NBRC104223 CBS266.38 NBRC100108 CBS263.30 NBRC100116 NBRC100119 CO132 IFO31993 CBS285.31 NBRC104222 NBRC107363 1D2S021 C101 NBRC100122 CBS808.96 CBS811.70 CBS259.30
P. P. P. P.
P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P. P.
monospermum arrhenomanes graminicola myriotylum
plurisporium sulcatum torulosum aquatile dissotocum pyrilobum acanthicum periplocum oligandrum hypogynum rostratum middletonii parvum takayamanum intermedium irregulare I irregulare II spinosum sylvaticum nagaii paddicum anandrum senticosum undulatum heterothallicum splendes ultimum nunn polymastum chamaehyphon
E1 E1 E2 E2 E2 F F F F F G G H H H I I I J J K
Origin
Carot Soil Spinach Bent grass Bent grass Gomphrena spp. Spinach
Carnation Nemesia spp. Soy bean Kalanchoe Dianthus spp. Curcuma spp. Peanut
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Peak denaturing temperature
+ + + + + + + +
872 871 870 869 869 870 869 871 (continued)
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Table 1 (Continued)
Species
Clade*
Isolate
P. helicoides
K K K K K K K K K K K K K K K K 1 2 2 3 4 5 6 7 8 9 10
NBRC100107 B5 ori OM6-1 sh Tsu-C1 RoTo ori SP-KS04-A2 ERR-1 Nastr1 GiBeg1 TGhel5 sh1 GUPo1 sh RO-Ph1 sz1 Fuk 3-1 YP1 CBS768.73 2D111 GF101 C94 IFO30696 C71 P0113 P1102 P3826 P6358 CH92-15 P6195 GF749 GFHT6 MAFF72510 HY Type1 S02 IFO32708 Vaal 130308 AiTog
P. oedochium P. ostracodes P. vexans Phytophthora nicotiane Ph. citricola Ph. capsici Ph. nemorosa Ph. palmivora Ph. heveae Ph. humicola Ph. cambivora Ph. cryptogea Ph. insolita Ph. chrysanthemi Aphanomyces sp. Fusarium oxysporum Plasmodiophora brassicae Pyrenochaeta lycopercisi Rhizoctonia solani Saprolegnia sp. Verticillium albo-atrum Sclerotinia sclerotiorum
Origin
Rose Rose Chrysanthemum Rose Strawberry Erica spp. Strawberry Begonia Strawberry Poinsettia Rose Soy bean
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LAMP, loop-mediated isothermal amplification. *Phylogenetic clades in Pythium and Phytophthora species provided by L evesque and de Cock (2004) and Blair et al. (2008), respectively.
required to detect P. myriotylum: the pathogen was detected in 30 min, compared with 60 min required when the reaction is followed by turbidity monitoring (data not shown). As fluorescent dyes bind to double-stranded DNAs, which are amplified by Bst DNA polymerase used in the LAMP reaction, the fluorescence of the LAMP reaction solution increases with DNA accumulation. In contrast, the turbidity of the LAMP reaction solution is caused by an increasing quantity of magnesium pyrophosphate, which is a by-product of the LAMP reaction (Mori et al. 2001). Detection of magnesium pyrophosphate is delayed following DNA amplification. The LAMP reaction with intercalating dyes such as EvaGreen has another advantage. As the amplification
efficiency of the LAMP reaction is extremely high, any contamination of reagents and instruments can easily result in a false-positive result (Peng et al. 2011). Enzyme reactions and electrophoresis using LAMP products raise the danger of contamination, so that LAMP products should not be handled directly if possible. Peak denaturing temperatures can be distinguished clearly by annealing analysis. LAMP products amplified by different LAMP primer sets differ in peak temperature (Fukuta et al. 2013a,b). Therefore, monitoring denaturing peak temperatures helps distinguishing LAMP products without electrophoresis. The denaturing peak temperature of LAMP products amplified by the P. myriotylum primer set was about 87°C. There were no differences of peak tempera-
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and were maintained on cornmeal agar (CMA) at 25°C. Total genomic DNA from mycelia was extracted according to the procedure of Kageyama et al. (2003).
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Figure 3 The fluorescence (a) and agarose gel electrophoretic analysis (b) of the loop-mediated isothermal amplification reaction Pythium myriotylum eight strains, NBRC100113 (lane 1), C41 (lane 2), F02 (lane 3), FuOri1 (lane 4), GF46 (lane 5), O03 (lane 6), S06 (lane 7) and SP-OP03-1 (lane 8). (■) NBRC100113; (□) C41; (▲) F02; ( ) FuOri1; (♦) GF46; (♢) O03; (●) S06; (○) SP-OP03-1.
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ture between P. myriotylum strains. The detection of the pathogen in field samples showed that the LAMP method monitoring the denaturing peak temperature worked successfully. The results of LAMP were conformed to those of the culture method in all samples tested except for one that had very low zoospore concentrations. Pythium diseases have economical impact worldwide, and the development of simple, cost-effective, sensitive and rapid diagnostic techniques is important. The LAMP detection described in this study represents a sensitive, specific and reliable diagnostic protocol. Materials and methods
The LAMP reaction uses four primers (F3, B3, FIP and BIP), which anneal to six regions of the target DNA (Notomi et al. 2000). ITS sequences were chosen as the targets for LAMP primer design. The ITS sequences of P. myriotylum (GenBank Acc. No: GQ121316) and related species of the molecular phylogenetic clade B1 including P. arrhenomanes (EU162759), P. graminicola (AY598625), P. plurispoium (HQ643750), P. sulcatum (DQ528744) and P. torulosum (AY228542) were downloaded from the GenBank database. These sequences were compared by multiple sequence alignment using DNSIS (Hitachi Solutions, Tokyo, Japan). A set of primers for LAMP amplification was designed from the sequences, which differ from those of other Pythium species, using PRIMEREXPLORER V.4 software (http://primerexplorer.jp), and the primers were further modified manually. Consequently, five primers, F3 (50 -GGCGGTATGTTAGGCTTCG-30 ), B3 (50 -TGATATGC TTAAGTCAGCGG-30 ), FIP (50 -GCCCCACACAAGACAG GTACAC-TTGCAGCTGACGGGGTG-30 ), BIP (50 -GTGA GAGTTTCGGCTCCTGCAGG-TCTTATCTTGATATCC AGGTCC-30 ) and B-Loop(50 -CAGGCGGCTTTTTCAAT TG-30 ) were used for the detection of P. myriotylum. LAMP reaction monitoring by fluorescence The LAMP reaction was performed in a total volume of 25 ll. The reaction contained 15 ll of Isothermal Master Mix (Nippon Gene, Osaka, Japan), which contains dsDNA binding dye, 02 lmol l1 each of F3 and B3 primer, 16 lmol l1 each of FIP and BIP primer, 08 lmol l1 of B-Loop primer and 1 ll DNA template solution. The mixtures were incubated at 60–675°C for 30 min while monitoring the fluorescence of the reaction solution using FAM channel by a Step One (ABI, Tokyo, Japan) real-time PCR device, followed by a postamplification anneal analysis step (95 to 80°C, 02°C/s) that was performed to confirm the annealing curve.
Species, strains and DNA extraction
PCR
A total of 86 strains were used, including 8 P. myriotylum strains, 59 non-P. myriotylum Pythium strains, 11 Phytophthora strains, which belong to 10 clades, and eight other soil-borne pathogens (Aphanomyces, Fusarium, Plasmodiophora, Pyrenochaeta, Rhizoctonia, Saprolegnia, Verticillium and Sclerotinia spp.), all listed in Table 1. Isolates were collected from various hosts and geographic origins
PCR mixture contained 10 mmol l1 Tris-HCl pH 83, 50 mmol l1 KCl and 15 mmol l1 MgCl2, 02 mmol l1 dNTP mixture, 1 lmol l1 each of primers PyH (50 -CT GTTCTTTCCTTGAGGTG-30 ) (Asano et al. 2010) and kk_MYRR (50 -GAGCCGAAACTCTCACAAGACC-30 ) (Kageyama et al. 2012), 10 ng bovine serum albumin (SigmaAldrich, Tokyo, Japan), 1 unit Fast Start Taq DNA
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Table 2 Detection of Pythium myriotylum using the culture method and the LAMP reaction from hydroponic solution samples from tomato fields
Sample no.
Sampling date
Conventional plating method Concentration (CFU/1000 ml)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
12 September, 2011 12 September, 2011 27 September, 2011 27 September, 2011 7 November, 2011 7 November, 2011 21 November, 2011 21 November, 2011 6 December, 2011 6 December, 2011 19 December, 2011 19 December, 2011 10 January, 2012 10 January, 2012 31 January, 2012 31 January, 2012 13 February, 2012 13 February, 2012 12 March, 2012 12 March, 2012 26 March, 2012 26 March, 2012
1500 0 110 0 200 0 9 0 100 0 1 0 20 0 2 0 100 – 20 – 100 0
LAMP for P. myriotylum Detection time (min)*
Annealing temperature
77 – 107 – 86 – 155 – 93 – – – 88 – 215 – 95 – 107 – 103 –
871 – 869 – 870 – 871 – 869 – – – 868 – 870 – 870 – 869 – 869 –
LAMP, loop-mediated isothermal amplification.
polymerase (Roche Applied Science, Tokyo, Japan) and 1 ll DNA solution in a total volume of 25 ll. PCR was conducted in a DNA thermal cycler (Gene Amp PCR system 2700; ABI) at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 1 min, with a final extension step at 72°C for 10 min. Amplification products were confirmed by electrophoresis in 2% agarose gels. Gels were stained with ethidium bromide and photographed under ultraviolet light.
Acknowledgements
Detection of P. myriotylum in hydroponic solution samples
References
Hydroponic solution samples were collected from September 2011 to March 2012 from tomato greenhouses with hydroponic culture systems in Mie, Japan. Two 1000-ml hydroponic solution samples were filtered through 5 lm DURAPOREâ (Millipore, Billerica, MA) gridded membrane filters. DNA was extracted from one membrane filter following Kageyama et al. (2003). Extracted DNA (1 ll) was used in the LAMP assay, and the second membrane filter was put on NARM medium (Morita and Tojo 2007) and incubated at 40°C for 24 h. Isolation and detection of P. myriotylum by the culture method were performed according to Kageyama et al. (2003).
This work was supported by the Science and Technology Research promotion program for agriculture, forestry, fisheries and food industry, Ministry of Agriculture, Forestry and Fisheries, Japan. Conflict of Interest There is no conflict of interest to declare.
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