Evaluation of genetic markers for identifying ... - Wiley Online Library

Short Communication Received: 17 January 2011

Revised: 14 April 2011

Accepted: 15 May 2011

Published online in Wiley Online Library: 1 July 2011

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4507

Evaluation of genetic markers for identifying isolates of the species of the genus Fusarium Maiko Watanabe,a∗ Takahiro Yonezawa,b Ken-ichi Lee,c Susumu Kumagai,c Yoshiko Sugita-Konishi,a Keiichi Gotod and Yukiko Hara-Kudoa Abstract BACKGROUND: Members of the genus Fusarium are well known as one of the most important plant pathogens causing food spoilage and loss worldwide. Moreover, they are associated with human and animal diseases through contaminated foods because they produce mycotoxins. To control fungal hazards of plants, animals and humans, there is a need for a rapid, easy and accurate identification system of Fusarium isolates with molecular methods. RESULTS: To specify genes appropriate for identifying isolates of various Fusarium species, we sequenced the 18S rRNA gene (rDNA), internal transcribed spacer region 1, 5.8S rDNA, 28S rDNA, β-tubulin gene (β-tub), and aminoadipate reductase gene (lys2), and subsequently calculated the nucleotide sequence homology with pair-wise comparison of all tested strains and inferred the ratio of the nucleotide substitution rates of each gene. Inter-species nucleotide sequence homology of β-tub and lys2 ranged from 83.5 to 99.4% and 56.5 to 99.0%, respectively. The result indicated that sequence homologies of these genes against reference sequences in a database have a high possibility of identifying unknown Fusarium isolates when it is more than 99.0%, because these genes had no inter-species pair-wise combinations that had 100% homologies. Other markers often showed 100% homology in inter-species pair-wise combinations. The nucleotide substitution rate of lys2 was the highest among the six genes. CONCLUSION: The lys2 is the most appropriate genetic marker with high resolution for identifying isolates of the genus Fusarium among the six genes we examined in this study. c 2011 Society of Chemical Industry Keywords: Fusarium; aminoadipate reductase gene; phylogenetic species concept; molecular phylogenetic analysis

INTRODUCTION

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In recent years, fungal species contaminating foods continue to grow in both number and variety due to the increase in variety and quantity of imported foods, and also due to the development of transport and packaging techniques for foods. Members of the genus Fusarium are well-known as one of the most important plant pathogens causing food spoilage and loss worldwide1 . Moreover, because this genus consists of many important mycotoxin producers, including Fusarium graminearum, F. verticillioides, F. sporotrichioides, and others, it is associated with human and animal diseases through contaminated foods.1,2 To control fungal hazards of plants, animals and humans, and rapidly evaluate a risk for foods contaminated by Fusarium, there is a need for a rapid, easy and accurate identification system. Fungal species are traditionally identified by morphological techniques. However, identification based on morphological characteristics is often complicated, difficult and time-consuming because of many conditions to be tested and the application of skilled techniques in observing and recognising the diagnostic characteristics of the isolates. As a result, molecular tools are required to facilitate identification of fungal isolates. To date, researchers have developed many strategies for rapid and easy molecular identification of fungal isolates, such as a search by the nucleotide sequences homology with reference sequences in database,3 construction of phylogenetic tree with reference

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sequences4 and PCR-based assays with species-specific regions of genes.5 However, there are also some problems in the field of molecular identification of Fusarium species. The genetic markers commonly used for identification have few speciesspecific diagnoses in sequences among the closely related species, such as species complexes.6,7 Although many researchers studied about taxonomy and identification of Fusarium species with various genetic markers, we still cannot find a suitable marker for identification of all Fusarium species. Therefore, to overcome this issue, it is necessary to identify a gene that has a high evolutionary rate and have the species-specific diagnosis.

∗

Correspondence to: Maiko Watanabe, Division of Microbiology, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 1588501, Japan. E-mail: [email protected]

a Division of Microbiology, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501, Japan b School of Life Sciences, Fudan University, 220 Handan Road, 200433, Shanghai, China c Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan d Food Research Laboratories, Mitsui Norin Co., Ltd, Miyabara 223-1, Fujieda-shi, Shizuoka 426-0133, Japan

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c 2011 Society of Chemical Industry

Evaluation of genetic markers for identifying Fusarium isolates Previously, the ribosomal RNA gene (rDNA) cluster region and the β-tubulin gene (β-tub) were often used as genetic markers for identification and phylogenetic analysis of fungi including Fusarium.4,6,8 It was also indicated that the aminoadipate reductase gene (lys2) was a good phylogenetic marker for relationships both among genera of fungi9 and among species of the genus Byssochlamys and its related genera with higher evolutionary rate than other frequently used genes.10 In the present study, we analysed sequences of four parts of the rDNA cluster region, βtub, and lys2 to specify the gene(s) appropriate for identifying Fusarium isolates. We focused on the identification by an easy and rapid method with the nucleotide sequence homology against sequences in the database.

MATERIALS AND METHODS Strains The strains used in this study are listed in Table 1. According to a nomenclature system based on morphological characteristics proposed by Nelson et al.,11 we selected 24 species of the genus Fusarium and its related genera. This nomenclature is based on traditional species recognition methods. It is a very simple and systemised taxonomy; therefore, it has widespread application for the identification of Fusarium isolates. To cover a wide range of taxonomic groups, we selected additional species, referring to molecular phylogenetic studies.12,13 Each species includes one to three strains, and we tested a total of 48 strains. We further purified all Fusarium strains by the single-spore method.11

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effectively the genes for many Fusarium and Fusarium-related species. The PCR program consisted of an initial denaturing step at 94 ◦ C for 5 min, 35 amplification cycles, and an additional extending step at 72 ◦ C for 3 min. For the primer pairs FF1/FR1, ITS5/NL4, Fulys2-F03mix/Fulys2-R01 and Fulys2-F04mix/Fulys2-R04mix, the amplification cycles were 94 ◦ C for 30 s, 52 ◦ C for 40 s, and 72 ◦ C for 1 min and 10 s. For the primer pair Btu-F01/Btu0-R01, the amplification cycles were 94 ◦ C for 30 s, 60 ◦ C for 40 s, and 72 ◦ C for 1 min. The PCR products were dye-labelled with the primers for each gene used in the amplification reactions using the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and then were sequenced using an ABI 3130 analyser (Applied Biosystems). For PCR products from ITS5/NL4, we used an additional primer of ITS3.16 The lys2 of five strains, namely CBS 169.30, CBS 638.76, MAFF 236504, MAFF 238538 and MAFF 239038 could not be amplified by PCR. For lys2 of F. solani, we amplified and determined the nucleotide sequence using the third strain (NBRC8505), and tentatively used NBRC8505. The sequences determined in this study were deposited in GenBank (Accession Nos. AB586891-587030, AB587032-587079). Data analysis based on DNA sequences The nucleotide sequence datasets of each gene were automatically aligned by the MUSCULE program.19 Alignments were carefully checked by eye and were manually modified; all ambiguous parts were excluded from analysis. For the protein coding genes of β-tub and lys2, we excluded all intron parts and aligned only the exon parts. We used the following exon sequences of the various Fusarium species: F. graminearum (β-tub, FGSG 09530; lys2, FGSG06041 ), F. oxysporum (β-tub, FOXG 06228; lys2, FOXG01867 ), and F. verticillioides (β-tub, FVEG 04081; lys2, FVEG08245 ) as reference of the alignments, downloaded from the Fusarium Comparative Database (http://www.broadinstitute.org/annotation/genome/fusarium group/MultiHome.html). The final lengths of the sequences are as follows: 18S rDNA (509 bp), ITS1 (100 bp), 5.8S rDNA (159 bp), 28S rDNA (545 bp), β-tub (768 bp), and lys2 (963 bp). After making multiple sequence alignments of each gene, the percentages of pair-wise nucleotide sequence homology among all tested strains within each of six genes were calculated by MEGA4.20 Furthermore, the ratios of the nucleotide substitution rate among the six genes were inferred by the maximum likelihood approach using the proportional model.21 Given the ML tree topology (data not shown), the concatenated six-gene sequence was analysed using the BASEML program of PAML ver. 4.222 program with the GTR+ model. The base frequency, the κ parameter, and the shape parameter (α) of the gamma distribution were separately estimated for each of six genes. The branch lengths were simultaneously estimated. The gap sites were treated as missing data.

RESULTS AND DISCUSSION We performed pair-wise calculation of the percentages of nucleotide sequence homology among all tested strains within each of the six genes (Table 2). The percentages of inter-species nucleotide sequence homology of 18S rDNA, 5.8S rDNA, ITS1, and 28S rDNA ranged from 96.1 to 100.0%, 65.0 to 100.0%, 93.7 to 100.0% and 91.1 to 100.0%, respectively. The results indicated a concern that these four genes suggest a number of candidate species when we try to identify unknown Fusarium isolates


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DNA extraction, amplification and sequencing We used two subcultures of each Fusarium and Fusarium-related species obtained by the single-spore method for sequencing. We checked their sequence identity between two subcultures to confirm purity of the strain. Samples were cultured on synthetic low nutrient agar (SNA)14 slant media supplemented with chloramphenicol (100 mg L−1 ) at 25 ◦ C for 14 days. Mycelia and conidia from the slant culture were inoculated into 1 mL potato dextrose broth (Difco Laboratories, Franklin Lake, NJ, USA) in a microtube, and were incubated at 25 ◦ C for 3 days. These fungal bodies were clumped by centrifugation at 18 000 × g for 10 min in a microtube. The genomic DNA was extracted from these pellets using the SDS method with minor modifications, as previously described.15 The rDNA cluster region, including the 3 end of the 18S rDNA, the internal transcribed spacer region 1 (ITS1), the 5.8S rDNA, the 5 end of the 28S rDNA, β-tub, and the fragment of lys2 were selected for analyses in this study. We performed amplification reactions with the primer pair for each gene using TaKaRa ExTaq (TaKaRa Bio Inc., Otsu, Japan), according to the manufacturer’s instructions. The ITS1, 5.8S rDNA, and 5 end of the 28S rDNA were amplified in one fragment using the primer pair, ITS516 and NL4.13 For PCR amplification other than that for ITS1, 5.8S rDNA, and 28S rDNA, we used the following primer pairs: FF1/FR117 for 18S rDNA; Btu-F-F01 (5 -CAGACCGGTCAGTGCGTAA3 )/Btu-F-R01 (5 -TTGGGGTCGAACATCTGCT-3 ) for β-tub; and two pairs of Fulys2-F03mix (5 -CTTTGTTGGTGATGTTCTSA3 )/Fulys2-R01 (5 -TGGTAGGTCCGATATCGGT-3 ) or Fulys2F04mix (5 -GCYATGGGDCARATYCTKGT-3 )/Fulys2-R04mix (5 CGGYTCYTCRTTRCGRTCTCT-3 ) for lys2. The primer pairs for β-tub and lys2 used in this study were designed based on sequences derived from primers used in previous studies10,18 that amplified

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Table 1. Strains of the genus Fusarium and Fusarium-related species used in this study Species registered in resource organisation

Species in traditional taxonomic systema

Fusarium larvarum

Fusarium larvarum

Microdochium nivale

Fusarium nivale

Fusarium incarnatum Fusarium culmorum Fusarium asiaticum Fusarium graminearum Fusarium oxysporum

Fusarium semitectum Fusarium culmorum Fusarium graminearum

Fusarium dimerum

Fusarium dimerum

Fusarium merismoides

Fusarium merismoides

Fusarium equiseti

Fusarium equiseti

Fusarium acuminatum

Fusarium acuminatum

Fusarium lateritium

Fusarium lateritium

Fusarium proliferatum

Fusarium proliferatum

Fusarium subglutinans

Fusarium subglutinans

Fusarium verticillioides

Fusarium moniliforme

Fusarium solani

Fusarium solani

Fusarium avenaceum

Fusarium avenaceum

Fusarium decemcellulare

Fusarium decemcellulare

Fusarium oxysporum

Fusarium kyushuense

–

Fusarium langsethiae

–

Fusarium poae

Fusarium poae

Fusarium sporotrichioides

Fusarium sporotrichioides

Gibberella tricincta Fusarium tricinctum

Fusarium tricinctum Fusarium tricinctum

Strain no. b

CBS 169.30 CBS 638.76 (Isotype strain) CBS 116 205 (Isotype strain) MAFFc 236 681 MAFF 236 521 MAFF 241 212 MAFF 240 264 MAFF 240 270 MAFF 240 304 MAFF 240321 CBS 632.76 (Neotype strain) MAFF 237465 CBS 634.76 (Type strain) MAFF 236504 MAFF 236434 MAFF 236 723 CBS 485.94 MAFF 236 716 ATCCd 34 203 (Type strain) MAFF 235 344 MAFF 840 045 CBS 216.76 (Type strain) MAFF 237 651 ATCC 38 016 MAFF 235 376 CBS 576.78 (Epitype strain) CBS 100 312 MAFF 240 085 MAFF 238 538 MAFF 239 038 NBRCe 8505 ATCC 200255 (Type strain) MAFF 239 206 MAFF 238 421 MAFF 238 422 MAFF 237 645 (Ex Holotype strain) NRRLf 6490 (Type strain) CBS 113 234 (Holotype strain) FRCg T-0992 FRC T-1000 FRC T-0796 MAFF 305 947 ATCC 34 914 CBS 119 839 MAFF 236 639 ATCC 38 183 (Type strain) CBS 393.93 (Epitype strain) MAFF 235 551

a

Nelson et al.11 Centraalbureau voor Schimmelcultures. c Ministry of Agriculture, Forestry and Fisheries. d American Type Culture Collection. e National Institute of Technology and Evaluation, Biological Resource Center. f Agricultural Research Service Culture Collection in United stats Department of Agriculture. g Fusarium Research Center in Penn State University. b

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Evaluation of genetic markers for identifying Fusarium isolates

Table 2. Comparison of nucleotide sequence homologies of nucleotide among six genes Nucleotide sequence homology (%) Gene

Intra-species

18S rDNA ITS 1 5.8S rDNA 28S rDNA β-tuba lys 2b

99.2–100.0 84.0–100.0 98.1–100.0 98.9–100.0 93.0–100.0 87.0–100.0

a b

Inter-species 96.1–100.0 65.0–100.0 93.7–100.0 91.1–100.0 83.5–99.4 56.5–99.0

β-tubulin gene. Aminoadipate reductase gene.

www.soci.org species such as species complex, and must evaluate this gene for identifying isolates to these species. The current classification schemes of the fungi, including Fusarium species, are mainly based on the morphological species concept.23,24 An inconsistency between morphological species concept and phylogenetic species concept with molecular phylogenetic analysis13 is thought to be one of the major difficulties in identification of Fusarium isolates. We must reconstruct a reliable species tree based on not only molecular but also morphological and other biological characteristics, and believe that the lys2 sequences will be useful for this purpose. The reliable species tree will shed light on the new insight of the whole picture of the taxonomic system of the genus Fusarium, and such a study is now in progress.

CONCLUSION Table 3. Comparison of ratios of nucleotide substitution rate of sequences among six genes Gene 18S rDNA ITS 1 5.8S rDNA 28S rDNA β-tuba lys 2b

Ratio of substitution rate 1.0 13.9 1.5 4.0 10.1 26.3

a

β-tubulin gene. b Aminoadipate reductase gene.

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ACKNOWLEDGEMENTS We wish to express our special thanks to Professor Masami Hasegawa and Dr Hidenori Nishihara for their helpful comments and technical support. This study was supported by a Health and Labour Sciences Research grant from the Ministry of Health, Labour and Welfare of Japan, and the Cooperative Research Program of Medical Mycology Research Center, Chiba University (09-14).

REFERENCES 1 Pitt JI and Hocking AD, Fungi and Food Spoilage. Springer, Berlin (2009). 2 Marasas WFO, Nelson PE and Toussoun TA, Toxigenic Fusarium Species. The Pennsylvania State University Press, University Park (1984). 3 Park B, Park J, Cheong KC, Choi J, Jung K, Kim D, et al, Cyber infrastructure for Fusarium: Three integrated platforms supporting strain identification, phylogenetics, comparative genomics and knowledge sharing. Nucleic Acids Res 39:D640–D646 (2010). 4 Azor M, Gene J, Cano J, Manikandan P, Venkatapathy N and Guarro J, Less-frequent Fusarium species of clinical interest: correlation between morphological and molecular identification and antifungal susceptibility. J Clin Microbiol 47:1463–1468 (2009). 5 Jurado M, Vazquez C, Marin S, Sanchis V and Teresa Gonzalez-Jaen M, PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in maize. Syst Appl Microbiol 29:681–689 (2006). 6 O’Donnell K, Cigelnik E and Nirenberg HI, Molecular systematics and phylogeography of the Gibberella fujikuroi species complex of Fusarium. Mycologia 90:465–493 (1998). 7 O’Donnell K, Molecular phylogeny of the Nectria haematococca–Fusarium solani species complex. Mycologia 92:919–938 (2000). 8 Guadet J, Julien J, Lafay JF and Brygoo Y, Phylogeny of some Fusarium species, as determined by large-subunit rRNA sequence comparison. Mol Biol Evol 6:227–242 (1989). 9 An K-D, Nishida H, Miura Y and Yokota A, Aminoadipate reductase gene: A new fungal-specific gene for comparative evolutionary analyses. BMC Evol Biol 2:6 (2002). 10 Watanabe M, Kato Y, Togami K, Yamanaka M, Wakabayashi K, Ogawa H, et al, Evaluation of gene index for identification of Byssochlamys spp. J Food Hyg Soc Jpn 49:82–87 (2008).


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based on nucleotide sequence homologies against reference sequences in database, because these four genes had several species combinations that had 100% sequence homology in this study. In contrast, the percentages of inter-species nucleotide sequence homology of β-tub and lys2 ranged from 83.5 to 99.4% and 56.5 to 99.0%, respectively. The result indicated that these two genes have a high possibility of identifying unknown Fusarium isolates when the sequence homology against reference sequences in database is more than 99.0%. Table 3 shows the ratios of the nucleotide substitution rates of each gene in genus Fusarium and related genera. The higher this rate is, the easier it is to accumulate the species-specific diagnostic substitution in the nucleotide sequence of the genes. Our result indicated that when the value of 18S rDNA was set to 1.0, the value of 5.8S rDNA, ITS1, 28S rDNA, β-tub and lys2 were 13.9, 1.5, 4.0, 10.1 and 26.3. Therefore, the nucleotide substitution rate of lys2 was the highest among the six genes. On the whole, the lys2 is the most appropriate gene as the genetic marker among the six genes used in this study for accurate identification of isolates to various species in the genus Fusarium based on the nucleotide sequence homology. In this study, we evaluated six genetic markers for identifying isolates to the species of the genus Fusarium, and specified that the lys2 is the most appropriate gene. Therefore, when the lys2 is applied to identifying isolates of taxonomic groups which have been a subject of controversy for many years, this gene is expected to resolve difficulties for identification. In future studies, we must analyse lys2 sequences of strains including very closely related

Our analyses of the pair-wise calculation of the percentages of nucleotide sequence homology among all tested strains and the ratios of the nucleotide substitution rates of each gene suggested that lys2 is the most appropriate genetic marker with a high resolution for identifying isolates of the genus Fusarium among the six genes we examined in this study. When lys2 is applied to identifying isolates of taxonomic groups that have been a subject of controversy for many years, this gene is expected to resolve difficulties for identification.

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18 O’Donnell K and Cigelnik E, Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogenet Evol 7:103–116 (1997). 19 Edgar RC, MUSCLE: A multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5:113 (2004). 20 Tamura K, Dudley J, Nei M and Kumar S, MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599 (2007). 21 Yang Z, Maximum-likelihood models for combined analyses of multiple sequence data. J Mol Evol 42:587–596 (1996). 22 Yang Z, PAML4: Phylogenetic analysis by maximum likelihood. Mol Biol Evol 24:1586–1591 (2007). 23 Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, et al, Phylogenetic species recognition and species concepts in fungi. Fungal Genet Biol 31:21–32 (2000). 24 Kirk PM, Cannon PF, Minter DW and Stalpers JA, eds, Dictionary of the Fungi. CAB International, Wallingford, UK (2008).

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