J Gen Plant Pathol (2010) 76:116–121 DOI 10.1007/s10327-010-0224-7 FUNGAL DISEASES Real-time PCR for differential determination of the tomato wilt fungus, Fusarium oxysporum f. sp. lycopersici, and its races Keigo Inami • Chizu Yoshioka • Yasushi Hirano • Masato Kawabe • Seiya Tsushima • Tohru Teraoka Tsutomu Arie • Received: 21 August 2009 / Accepted: 5 January 2010 / Published online: 13 March 2010 Ó The Phytopathological Society of Japan and Springer 2010 Abstract Five primer/probe sets to identify the tomato wilt pathogen, Fusarium oxysporum f. sp. lycopersici (FOL), and its three races selectively were designed based on the rDNA-intergenic spacer and avirulence genes. Realtime PCR using genomic DNA from mycelia and soil DNA with the primer/probe sets allowed the successful identification of FOL and its races. Keywords Avirulence gene
Detection
rDNA-intergenic spacer (IGS) region
Soil DNA Fusarium oxysporum is a mitosporic ascomycete found globally in almost every natural habitat. Although the fungus usually is saprophytic (nonpathogenic), some strains of this soilborne species cause agriculturally and K. Inami and C. Yoshioka have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s10327-010-0224-7) contains supplementary material, which is available to authorized users. K. Inami
C. Yoshioka
T. Teraoka
T. Arie (&) Laboratory of Plant Pathology, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo 183-8509, Japan e-mail: arie@cc.tuat.ac.jp Y. Hirano Saitama Prefectural Agriculture and Forestry Research Center, Kuki, Saitama, Japan M. Kawabe National Agricultural Research Center (NARC), Tsukuba, Ibaraki, Japan S. Tsushima National Institute for Agro-Environmental Sciences (NIAS), Tsukuba, Ibaraki, Japan 123 economically damaging plant diseases. Of the phytopathogenic strains of the fungus, more than 120 formae speciales (ff. sp.) have been reported, each of which causes disease on a well-defined host range of plant species (Agrios 2005). For example, f. sp. lycopersici (FOL) causes wilt disease only on tomato (Solanum lycopersicum L.), while other ff. sp. except radicis-lycopersici (FORL) do not cause diseases on tomato. Within a forma specialis, races are frequently distinguished by their specific pathogenicity to different cultivars. Three races (races 1, 2, and 3) have been reported for FOL to date (Alexander and Tucker 1945; Clayton 1923; Grattidge and O’Brien 1982). The detection and determination of these pathogenic types (ff. sp. and races) of F. oxysporum in the field, seeds, and seedlings are the most important steps in managing diseases caused by the fungus. Although a correlation between phylogeny and physiological characters including pathogenicity is generally expected for a fungal species, almost no correlation between phylogeny and pathogenic types is found in F. oxysporum (Kistler 1997), eliminating the possibility of determining the pathogenic types based on the phylogeny in the species. However, Kawabe et al. (2005) found a correlation between phylogeny and races among the Japanese isolates of FOL, possibly from the independent introduction of each race into Japan. Hirano and Arie (2006) established a PCR-based method to differentiate the pathogenic types of Japanese isolates of F. oxysporum in tomato. This method is useful to identify the pathogenic types of F. oxysporum in culture and of F. oxysporum in tomato tissue because usually a single pathogenic type inhabits tomato tissue (Balogun et al. 2008). However, the method is not feasible for determining the pathogenic type of F. oxysporum in soil cohabitated by other pathogenic types. For example, soil co-infested with races 1 and 2 J Gen Plant Pathol (2010) 76:116–121 117 Japanese FOL isolates (see alignment of the representatives from each race in Fig. S1). Primer/probe sets P1, P2, and P3 were designed to recognize race-specific insertions– deletions and single nucleotide polymorphisms (SNPs) in the rDNA-IGS region in races 1, 2, and 3, respectively (Table 1; Fig. S1). SIX1, determining AVR3, is carried by all the present races of FOL but not by other ff. sp. and nonpathogenic isolates in F. oxysporum and other fungal species. Primer/ probe set L1 (Table 1) designed to amplify and detect a fragment of 43 bp in SIX1 thus determines FOL specifically. SIX4, determining AVR1, is carried specifically by race 1. None of the isolates of the other races of FOL, the other ff. sp. in F. oxysporum, nonpathogenic F. oxysporum, nor other fungal species have SIX4 (Houterman et al. 2008). Thus, primer/probe set R1 (Table 1) designed to amplify and detect a 57-bp fragment in SIX4 selectively determines FOL race 1. Briefly, five sets of primers and probes were designed to distinguish FOL and its races using real-time PCR (Table 1). Primers and TaqMan probes carrying a reporter (FAM) and a quencher with a minor groove binder (MGB) Tm gives positive reactions with both primer sets sp13 and sp23 (Hirano and Arie 2006), a reaction identical to that with soil infested with race 3. According to the gene for gene hypothesis (Flor 1956), the relation between FOL races and tomato cultivars can be explained by interactions between the three avirulence genes (AVRs) in FOL and the three resistance genes in tomato. Resistance genes I, I-2, and I-3 in tomato theoretically confer resistance to the isolates possessing AVR1, AVR2, and AVR3, respectively. In other words, the genotype of races 1, 2, and 3 are AVR1 AVR2 AVR3, avr1 AVR2 AVR3, and avr1 avr2 AVR3, respectively. Recently, van der Does et al. (2008) and Houterman et al. (2008) found genes SIX4 and SIX1 each of which corresponds to AVR1 and AVR3 in FOL, suggesting that these avirulence genes can be used for race determination. In this study, we present primer/probe sets, designed based on the rDNA intergenic spacer (IGS), SIX4, and SIX1 to establish molecular-based practical techniques to distinguish FOL races. Alignment of the rDNA-IGS nucleotide sequences (583–598 bp) amplified with primers FIGS11 ? FIGS12 (Kawabe et al. 2005) from genomic DNA presents nucleotide sequence polymorphisms among races in the Table 1 Primer/probe sets developed in this study for selectively identifying Fusarium oxysporum f. sp. lycopersici (FOL) and races of FOL Primer/probe set Sequence Nucleotide position in database FOL-specific Set L1, based on SIX1 (AJ608703) Primers Probe SIX1f 50 -GGGAGCCCCAGATATTTTTCA-30 SIX1r 50 -GGATGCTGCCACCTTATCCA-30 10156–10137 SIX1pr 50 -TTGACCTACACGGAATAT-30 10118–10135 10095–10115 Race 1-specific Set P1, based on rDNA-IGS (AB106019) Primers Probe sp1-2f 50 -GCTGGCGGATCTGACACTGT-30 367–386 sp1-2r 50 -CCTAAACCACATATCTCGTCCAAA-30 466–443 sp1-2pr 50 -TTTCGTACTTGCCAGGTTG-30 421–439 Set R1, based on SIX4 (AM234064) Primers SIX4f 50 -TGTCACGCGTGCAGGAAA-30 Probe 1519–1536 SIX4r 50 -TGGGCCTTGAGTCGAATGA-30 1557–1575 SIX4pr 50 -CCAGGAATAGGACGAAAG-30 1538–1555 Race 2-specific Set P2, based on rDNA-IGS (AB106027) Primers Probe sp2-2f 50 -CGGTGCAGGGTAGTAGTCGAGTTA-30 sp2-2r 50 -CAACACACAGCCGACCAGACT-30 139–119 sp2-2pr 50 -ACTTGGTGGAGTTCCGTC-30 100–117 78–98 Race 3-specific Set P3, based on rDNA-IGS (AB106044) Primers Codes in the parentheses are GenBank accession numbers Probe sp3f 50 -GTCGGTTCGAGGATCGATTC-30 184–203 sp3r 50 -AAGACAAACCAGCCTAGGGTAGAC-30 262–239 sp3pr 50 -CCGTCGATGATATGTGATGTA-30 214–234 123 118 enhancer were designed using the Primer Express program (Applied Biosystems, Foster City, CA, USA) and synthesized by Applied Biosystems Japan (Tokyo, Japan). Realtime PCR was performed using an Applied Biosystems 7300 Real-time PCR System (Applied Biosystems). The reaction mixture (50 ll) contained 25 ll of TaqMan Universal Master Mix (Applied Biosystems), 20 ng of template DNA, 900 nM primers (forward and reverse each), and 250 nM TaqMan probe. Conditions for the reaction followed the manufacturer’s protocol: 50°C for 2 min, 95°C for 10 min, 40 cycles 9 (95°C for 15 s, at annealing temperature for 1 min). Annealing temperature for P1, P3, and L1 was set to 63°C and for P2 and R1 to 61°C. Fungal isolates used in this study (Table 2) were maintained on potato sucrose (PS) agar. Genomic DNA (ca. 300–900 ng/ll) was isolated from mycelia statically cultured in PS broth at 25°C for 2 weeks as described previously (Arie et al. 1997). When genomic DNA isolated from the fungal mycelia was used as a template, all primer/ probe sets successfully detected the respective target isolates and races of FOL (Fig. 1a). None of the other ff. sp. or nonpathogenic isolates were detected with any of the primer/probe sets except for MAFF 103044 (FORL) and NRRL 26406 (f. sp. melonis) with P1 set (data not shown) and Fo304 with P2 set. These findings were congruent with Kawabe et al. (2005) who reported that MAFF 103044 and NRRL 26406 are included in FOL race 1 clade (A2) in the rDNA-IGS based phylogeny and with Hirano and Arie (2006) that Fo304 was detectable by sp23. However, these isolates are distinguishable from FOL by FOL-specific detection set L1 that was developed in this study. Thus, combining phylogeny-based and AVR-gene-based primer/ probe sets achieved accurate diagnosis in this study. In addition, combined mixtures of DNAs from FOL races (race 1, MAFF 305121; race 2, JCM 12575; race 3, Chz1-A) and non-FOL F. oxysporum (Rif-1, NBRC 31984, T-2A, T-2C, 9901, 101-2, and F-4) were subjected to realtime PCR as templates under the same conditions, and we successfully detected each FOL race in the template (data not shown). The correlation between the amount of template DNA and threshold cycle value (Ct) for each set is illustrated in Fig. S2. The minimum detectable amount of DNA was 2.0 9 10-4 ng for P1, P2, and P3, 2.0 9 10-3 ng for R1, and 2.0 9 10-2 ng for L1. DNA isolated from soil infested with FOL was subjected to real-time PCR. Soil (Fuchu, Tokyo; Type, andosol; pH 7.2; EC 0.275 mS/cm) artificially infested with FOL was prepared. Each FOL isolate, MAFF 305121 (race 1), JCM 12575 (race 2), or Chz1-A (race 3), was cultured on PS broth for 5 days at 25°C with shaking (120 strokes/min), and the bud cells were collected by centrifugation (90509g). The washed bud cells (107 cells) in distilled water were mixed 123 J Gen Plant Pathol (2010) 76:116–121 Table 2 Fungal isolates used in this study Fungal species and isolate Source Phylogenetic Avirulence geneb cladea SIX4 SIX1 (AVR1) (AVR3) Fusarium oxysporum f. sp. lycopersici Race 1 103036 MAFF A2 ? ? 305121 MAFF A2 ? ? Race 2 103043 MAFF A1 - ? 12575c JCM A1 - ? Race 3 H-1-4 Y. Hosobuchi A3 - ? Chz1-A This laboratory A3 - ? This laboratory - - - NBRC - - - SUF - - - MAFF - - - This laboratory - - - MAFF - - - This laboratory - - - This laboratory - - - This laboratory - - - F. Kodama - - - ATCC - - - MAFF - - - T-2A K. Watanabe - - - T-2C K. Watanabe - - - 9901 K. Watanabe - - - 101-2 K. Watanabe - - - F-4 S. Suwa - - - Fo304 Y. Amemiya A2 - - This laboratory - - - H. Tateishi & Y. Miyake - - - This laboratory - - - f. sp. cucumerinum Rif-1 f. sp. fragariae 31984 f. sp. apii 1017 f. sp. melongenae 103051 f. sp. matthioli 880116a f. sp. niveum 305608 f. sp. lilii 851209k f. sp. glycines 851209m f. sp. spinaciae 880803e-2 f. sp. asparagi FokF233 f. sp. nicotianae 15645 f. sp. batatas 103070 Non-pathogenic isolates F. roseum 0016-2 F. moniliforme N-68 Verticillium dahliae 060714-2 J Gen Plant Pathol (2010) 76:116–121 119 Table 2 continued Fungal species and isolate Source Phylogenetic Avirulence geneb cladea SIX4 SIX1 (AVR1) (AVR3) Sclerotinia sclerotiorum 060328a-1 This laboratory - - - This laboratory - - - This laboratory - - - Mucor sp. 0016 2-2 Rhizopus sp. 0046 2-1 MAFF GeneBank of the Ministry of Agriculture, Forestry and Fisheries of Japanese government (Tsukuba, Japan), JCM Japan Collection of Microorganisms (RIKEN, Wako, Japan); NBRC NITE (National Institute of Technology and Evaluation Biological Resource Center, Kazusa, Japan), SUF Culture Collection of Fusarium in Shinshu University (Ueda, Nagano, Japan), ATCC American Type Culture Collection (Manassas, USA) a Corresponding to Kawabe et al. (2005); -, included in none of the three FOL clades b ?, posesses SIX4 or SIX1; -, null c = 880621a-1 in Hirano and Arie (2006) into 60 g of sterilized (autoclaved at 121°C for 3 h) soil by drenching and incubated for 7 days at 25°C. DNA in the soil sample (0.3 g) was isolated using a FastDNA SPIN Kit for Soil (Q-BioGene, Montreal, Canada) following the standard protocols established in the eDNA Project by NIAS (Tsukuba, Japan; Morimoto and Hoshino 2008). Normally, about 120 ng/ll soil DNA were obtained by this method. Under the same conditions, all primer/probe sets detected each corresponding race in the soil successfully (Fig. 1b). In the preliminary experiment, we also used DNA from field soil naturally infested with FOL as a template and FOL and its races were detectable (data not shown). As Kawabe et al. (2005) described, each of the FOL races found in Japan seems to be clonal today. Thus, primer/probe sets P1, P2, and P3 can distinguish phylogenetic clade A2, A1, and A3, respectively, and will be useful to determine races presently in Japan. Nevertheless, emergence of new races in each clade and/or invasion of foreign FOL may invalidate this phylogenybased method using primer/probe sets P1, P2, and P3 in the future. This possibility is congruent with the previous apprehension expressed by Hirano and Arie (2006). On the other hand, primer/probe sets L1 and R1 detecting avirulence genes and determining FOL and race 1 in FOL, respectively, seem to be more feasible than the primer/probe sets based on phylogeny because it is based on pathogenicity-determining factors. However, these two primer/probe sets are predicted to be invalidated by any mutations on the genes outside of the probe sequence (ca. 20 bp). Avirulence genes generally evolve more quickly than intergenic regions. Therefore, we propose that phylogeny-based and avirulence gene-based primer/probe sets should be used in combination to compensate for possible shortcomings. So far, several studies have reported the selective detection of the pathogens by real-time PCR, such as silver scurf pathogen of potato (Helminthosporium solani) (Cullen et al. 2001), root rot pathogens of citrus (Phytophthora nicotianae and P. citrophthora) (Ippolito et al. 2004), vector of soilborne cereal mosaic virus (Polymyxa graminis) (Ratti et al. 2004), root and stem rot pathogen of soybean (Phytophthora sojae) (Wang et al. 2006), wilt pathogen of Brassica crops (Verticillium longisporum) (Saito et al. 2007), three nematode parasites, root-knot nematode (Meloidogyne javanica), lesion nematode (Pratylenchus zeae) and dagger nematode (Xiphinema elongatum) (Berry et al. 2008), and bacterial wilt pathogen (Ralstonia solanacearum) (Huang et al. 2009). In particular, Okubara et al. (2005) and Lievens et al. (2006) selectively detected various soilborne pathogens by real-time PCR. Fusarium oxysporum is a complex species composed of nonpathogenic strains and phytopathogenic strains in which many pathogenic types have been reported. This has made selective detection of the pathogenic types in F. oxysporum difficult. However, using the primer/probe sets we designed in this study, we selectively detected the pathogenic types of F. oxysporum on tomato. Now we are designing other selective primer/probe sets to distinguish race 3 from races 1 and 2 based on the SNPs in SIX3 recently reported by Houterman et al. (2009). Lievens et al. (2009) reported the usefulness of several primer sets for PCR based on AVR genes to identify FOL races. The race selective primer/probe sets for realtime PCR developed in this study may enable us to quantify FOL races in soil in the future to estimate the risk of occurrence of wilt disease in the field. In this study, we showed the thresholds of the amount of detectable DNA template but no other quantitative information. The ‘‘quantity of FOL in soil’’ includes several meanings, such as the amount of FOL DNA in the template, the number of inocula, and the estimated disease severity caused by FOL. Estimation of the disease severity is especially important and in demand in the field; however, disease severity must be greatly affected by soil characteristics and other factors. Therefore, with more detailed experiments, we are examining the relation between the quantity of FOL in the soil and the severity of disease using the five primer/probe sets. For more practical determination of the pathogenic types, we are also investigating the use of the specific probes in microarrays. 123 120 J Gen Plant Pathol (2010) 76:116–121 a b Fig. 1 Curves obtained by real-time PCR using specific primer/probe sets designed in this study. Phylogeny-based primer/probe sets, P1 (race 1), P2 (race 2), P3 (race 3); AVR-based primer/probe sets, L1 (Fusarium oxysporum f. sp. lycopersici including races 1–3), R1 (race 1). Vertical and horizontal axes show strength of fluorescence (DRn) and cycle number (Ct), respectively. a Genomic DNA (20 ng) from mycelia was used as a template. The fungal isolates including FOL were used (Table 2). b DNA (20 ng) purified from soil each infested with a FOL isolate was used as a template Acknowledgments We thank Yuji Hosobuchi (Sakata Seed, Kimitsu, Japan), Hideaki Tateishi (Kureha, Iwaki, Japan), Taiji Miyake (Kureha), Ken Watanabe (Ibaraki Agricultural Center, Kasama, Japan), Suminaga Suwa (Gunma prefecture, Gunma, Japan), Yoshimiki Amemiya (Chiba University, Matsudo, Japan), and Fujio Kodama (previously at Hokkaido Central Agricultural Experiment 123 J Gen Plant Pathol (2010) 76:116–121 Station, Naganuma, Japan) for providing fungal isolates. 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