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Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment
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Soil Biology & Biochemistry xxx (2014) 1e5
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Short communication
Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment Q4
Wei Wei a, Kazuo Isobe a, *, Yutaka Shiratori b, Tomoyasu Nishizawa a, c, Nobuhito Ohte d, Yuta Ise a, Shigeto Otsuka a, Keishi Senoo a a
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan Niigata Agricultural Research Institute, Niigata 940-0826, Japan Department of Bioresource Science, College of Agriculture, Ibaraki University, Ibaraki 300-0393, Japan d Department of Forest Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan b c
Q1
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 June 2014 Received in revised form 21 November 2014 Accepted 26 November 2014 Available online xxx
Fungal denitrification in soils is receiving considerable attention as one of the dominant N2O production processes. However, because of the lack of a methodology to detect fungal denitrification-related genes, the diversity and ecological behavior of denitrifying fungi in soil remains unknown. Thus, we designed a primer set to detect the fungal nitrite reductase gene (nirK) and validated its sensitivity and specificity. Through clone library analyses, we identified congruence between phylogenies of the 18S rRNA gene and nirK of denitrifying fungal isolates obtained from the surface-fertilized cropland soil and showed that fungi belonging to Eurotiales, Hypocreales, and Sordariales were primarily responsible for N2O emissions in the soil. © 2014 Published by Elsevier Ltd.
Keywords: Fungal denitrification Fungal nitrite reductase N2O emission
Nitrous oxide (N2O) is a potent greenhouse gas (IPCC, 2007) and is involved in stratospheric ozone depletion (Ravishankara et al., 2009). It is produced through microbial denitrification, in which nitrate and nitrite are reduced to gaseous N2O (Isobe and Ohte, 2014). Fungal denitrification in soil has recently received considerable attention as an N2O production process. We previously performed an antibiotic assay and isolated denitrifying fungi to demonstrate the dominance of fungal denitrification in a surfacefertilized cropland soil (Wei et al., 2014). This has also been reported in grassland and forest soils using the same methodology (Laughlin and Stevens, 2002; Blagodatskaya et al., 2010). Many fungal species are known to produce N2O (Shoun et al., 1992; Wei et al., 2014); however, the diversity and ecological behavior of denitrifying fungi in soil, unlike denitrifying bacteria, remains unknown, probably because of the lack of a methodology to detect fungal denitrification-related genes. In addition, the ability to denitrify varies at the species level (Wei et al., 2014), making it
* Corresponding author. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: þ81 3 5841 5140; fax: þ81 3 5841 8042. E-mail address: [email protected] (K. Isobe).
difficult to identify denitrifying fungi based on their taxonomic position. Previous studies revealed that Fusarium oxysporum and Cylindrocarpon tonkinese, the most thoroughly characterized denitrifying fungi (Nakanishi et al., 2010), use copper-containing nitrite reductase (nirK) to reduce nitrite to nitric oxide, bearing a close resemblance to its bacterial counterpart (Kobayashi and Shoun, 1995; Kim et al., 2010). Additionally, fungal cytochrome cd1-type nitrite reductase remains undiscovered. Thus, developing a methodology to detect fungal nirK should lead to the precise identification of denitrifying fungi and elucidation of their ecological behavior. Consequently, the objectives of this study were to design suitable PCR primers to detect fungal nirK and use these primers to investigate the diversity of fungal nirK and identify the denitrifying fungi in surface-fertilized cropland soil where fungal denitrification is dominant (Wei et al., 2014). We searched full-length nirK fungal sequences from the public databases, NCBI Microbial Genomes (http://www.ncbi.nlm.nih. gov/genomes) and Functional Gene Repository (http://fungene. cme.msu.edu/index.spr) and obtained 15 sequences belonging to Ascomycota. We also obtained the representative sequences of nirK from diverse bacterial phyla and Euryarchaeota from database. Then, we generated the phylogenetic tree of nirK
http://dx.doi.org/10.1016/j.soilbio.2014.11.026 0038-0717/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Wei, W., et al., Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.026
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Fusarium oxysporum 5507 99 Fusarium oxysporum Fo5176 Fusarium fujikuroi IMI 58289 100 Fusarium lichenicola NBRC:30561 80 Nectria haematococca mpVI 77-13-4 Neosartorya fischeri NRRL 181 Aspergillus fumigatus Af293 100 100 100 Trichophyton verrucosum HKI 0517 100 Arthroderma benhamiae CBS 112371 Arthroderma otae CBS 113480 Myceliophthora thermophila ATCC 42464 72 Chaetomium globosum CBS 148.51 Aspergillus terreus NIH2624 Ajellomyces dermatitidis SLH14081 100 Ajellomyces capsulatus NAm1 Burkholderia pseudomallei 1106a 97 Ralstonia solanacearum GMI1000 Pseudoxanthomonas suwonensis 11-1 93 Bdellovibrio bacteriovorus HD100 Leptospira biflexa Patoc 1 75 Solitalea canadensis DSM 3403 Flavobacterium johnsoniae: Fjoh 2418 Kangiella koreensis: Kkor 2024 Pseudoalteromonas haloplanktis TAC125 Azospirillum brasilense Sp245 Caulobacter segnis ATCC 21756 74 Pseudomonas stutzeri CCUG 29243 Haloarcula hispanica ATCC 33960 Haloferax mediterranei ATCC 33500 100 Actinoplanes missouriensis 431 Actinosynnema mirum DSM 43827 100 Agrobacterium tumefaciens C58: Atu4382 98 Sinorhizobium fredii NGR234: NGR c09950 85 Brucella suis ATCC 23445: BSUIS B0265 Ochrobactrum anthropi ATCC 49188 82 100 Pseudomonas entomophila: PSEEN5226 Shewanella denitrificans: Sden 3482 Alcaligenes faecalis NCIB 8687 Pseudomonas fluorescens Pf-5: PFL 5501 69 Achromobacter xylosoxidans: AXYL 02390
Fungi
Prokaryote
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0.1 Fig. 1. Tree of maximum likelihood phylogeny of database-retrieved full-length NirK amino acid sequences of fungi, bacteria, and archaea. Bootstrap values (500 replicates) greater than 70% are denoted by dots above the branches and branch lengths correspond to sequence differences, which are indicated by a scale bar. Sequences from fungi are within the gray box.
(Fig. 1), and found that fungal nirK formed a monophyletic cluster distinct from the prokaryotic nirK with 100% bootstrap support. nirK is a two-domain enzyme including two copper centers, types 1 and 2 (Sakurai and Kataoka, 2007). We designed the primer sets based on homologs of the copper center type 1 domain (Fig. S1). Because widely used primers for bacterial nirK (such as primer set F1aCu/R3Cu and nirK2F/nirK5R, Braker et al., 1998; Hallin and Lindgren, 1999) also target this region, we can compare fungal nirK sequences with the large quantity of bacterial nirK sequences. We designed the forward primer to anneal with four conserved amino acids (tyrosine, valine, glutamine, and proline) and reverse primer to anneal with four conserved amino acids (aspartic acid, lysine, glycine, and alanine; Fig S1). Most of these amino acids were not conserved in prokaryotic nirK (Fig S1). The primer set specific to fungal nirK sequences, nirKfF (50 -TACGGGCTCATGtaygtnsarcc-30 ) and nirKfR (50 AGGAATCCCACAscnccyttntc-30 ), were designed based on the ConsensuseDegenerate Hybrid Oligonucleotide Primer (CODEHOP) algorithm (Rose et al., 1998). Both primers consist of the
degenerate core region (lowercase letters) corresponding to all possible codons specifying the conserved amino acids and consensus clamp region (uppercase letters) containing a single most common (consensus) nucleotide sequence derived from the rest of amino acids in the primer-designed region (Fig S1). We validated the specificity and sensitivity of the designed primer set, nirKfF/nirKfR, using fungal and prokaryotic strains. We used seventeen denitrifying and three nondenitrifying fungal strains isolated from the collected organic fertilizer (COF) and residual soil (RS) from a surface-fertilized cropland soil where fungal denitrification predominates (Wei et al., 2014). We also used ten prokaryotic strains (nine bacterial and one archaeal) obtained from culture collections (Japan Collection of Microorganisms, Koyadai, Japan or the Biological Resource Center (NBRC), Kazusakamatari, Japan; Table 1 and Fig. 1). Their abilities to produce N2O were confirmed as described previously (Isobe et al., 2011). Genomic DNA was extracted as described previously (Wei et al., 2014), and PCR was performed with the designed primers. The PCR mixture and conditions are described in
Please cite this article in press as: Wei, W., et al., Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.026
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Table 1 Primer validation using denitrifying fungal isolates and prokaryotic strains. Strain number
Fungi COF-2 COF-3 COF-5 COF-6 COF-7 COF-8 COF-10 COF-11 COF-12 COF-13 COF-16 COF-17 COF-19 COF-20 RS-1 RS-3 RS-5 RS-6 RS-8 RS-9 Bacteria ATCC-21756 ATCC-49188 NCIB-8687 NBRC-13243 DSM-43827 CCUG-29243 NBRC-100993 NBRC-106385 Archaea ATCC-33500 a b c d
Q2
Taxonomic assignment
N2O productiona
PCR products using primersb nirKfF/nirKfR
F1aCu/R3Cu
Fusarium oxysporum Actinomucor elegans Fusarium equiseti Fusarium solani Rhizomucor sp. Fusarium equiseti Fusarium oxysporum Fusarium oxysporum Fusarium equiseti Fusarium oxysporum Bionectria ochroleuca Fusarium oxysporum Fusarium solani Fusarium solani Aspergillus niger Bionectria ochroleuca Fusarium oxysporum Penicillium purpurogenum Fusarium avenaceum Fusarium oxysporum
þþþ þþ þþ e þþþ þþ þþþ þþþ þþ þþ e e þþþ þþ þ þþþ þþþ þþ þ þþ
þþþ e e e e e þþþ þþþ e þþ e e þþ þþ þ þþþ þþþ þþ þ þþþ
ec e e e 300, 700d e e e e e e e 400 e e e e e e e
Caulobacter segnis Ochrobactrum anthropi Alcaligenes faecalis Actinoplanes missouriensis Actinosynnema mirum Pseudomonas stutzeri Pseudoalteromonas haloplanktis Pseudoxanthomonas suwonensis
þ þ þ þþ þ þ þ þþþ
e e e e e e e e
þþþ þþ 700, 1200 e 300, 900 e 900
Haloarcula hispanica
þþ
e
e
N2O production: þ, 0e10 mg/day/g-biomass; þþ, 10e100 mg/day/g-biomass; þþþ, >100 mg/day/g-biomass. The concentration of PCR products: þ, 0e20 ng/ml; þþ, 20e50 ng/ml, þþþ, >50 ng/ml. Not amplified. Numbers indicate approximate sizes of nonspecific amplification products.
Table S1. PCR using the primer set, nirKfF/nirKfR, amplified the nirK fragment (ca. 480 bp) from twelve denitrifying fungal strains, belonging to Ascomycota (Table 1), but did not amplify the fragment from nondenitrifying fungal strains, all prokaryotic strains tested, or denitrifying Fusarium equiseti of Ascomycota, Actinomucor elegans and Rhizomucor sp. of Zygomycota. PCR using the widely-used bacterial nirK primers, F1aCu/R3Cu, did not amplify the fragment with the expected size. Thus, the primer set, nirKfF/ nirKfR, could successfully amplify the diverse fungal nirK of Ascomycota, the most dominant fungal group in soil (Wei et al., 2014), except F. equiseti, with sufficient selectivity and specificity. We analyzed nirK and 18S rRNA phylogenies of the fungal species isolated from COF and RS described above. We constructed the phylogenetic tree of the amplified nirK and the corresponding 18S rRNA gene obtained in our previous study (Wei et al., 2014; Fig. S2). The tree also included the database-retrieved fungal nirK and 18S rRNA genes (Fig. 1). Fungal nirK and 18S rRNA gene-based phylogenies was congruent at the order level of Ascomycota, whereas bacterial nirK and 16S rRNA gene-based phylogenies are known to be incongruent (Jones et al., 2008). This suggested that we could estimate the taxonomic position of denitrifying fungi based on their nirK phylogeny. We also investigated the diversity and phylogeny of fungal nirK in the surface-fertilized cropland soil using the extracted DNAs from COF and RS (Wei et al., 2014). We performed PCR using the primer set, nirKfF/nirKfR, and clone library analyses in the same
way as described previously (Wei et al., 2014). In total, 44 and 26 sequences of fungal nirK were obtained from COF and RS and classified into six COF and three RS OTUs, respectively, with 3% differences using the Mothur program (Schloss et al., 2009). From the phylogenetic tree of nirK and 18S rRNA gene (Fig. 2), the nirK clones were expected to be classified into Hypocreales, Sordariales, and Eurotiales of Ascomycota based on the congruence between the two phylogenies. Based on culture-dependent and DGGE analyses, we previously showed that denitrifying fungi closely related to Fusarium and Bionecter sp. in Hypocreales and to Chaetomium sp. in Sordariales are dominant in soils (Wei et al., 2014). The results of the clone library analysis using fungal nirK in this study strongly indicate that they are actually responsible for the N2O production in the surface-fertilized soil. We previously did not detect the presence of members of Eurotiales based on their 18S rRNA and ITS genes (Wei et al., 2014); however, we obtained the nirK clones and denitrifying isolates (Penicillium purpurogenum and Aspergillus niger) of this order. This shows that the designed primer set can sensitively detect denitrifying fungi regardless of their lower abundance in soils. In conclusion, we designed a novel fungal nirK primer set which can successfully detect the nirK of the most dominant denitrifying fungal group in soil (Ascomycota). The methodology developed here allows to study diversity and ecological behavior of denitrifying fungi and the importance of fungal denitrification in environments.
Please cite this article in press as: Wei, W., et al., Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.026
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Fusarium oxysporum isolate RS-5 Fusarium oxysporum isolate RS-9 Fusarium oxysporum isolate COF-2 Fusarium oxysporum isolate COF-13 Fusarium oxysporum isolate COF-11 Fusarium oxysporum isolate COF-10 92 Fusarium oxysporum Fo5176 Fusarium fujikuroi IMI 58289 Fusarium avenaceum isolate RS-8 Fusarium equiseti isolate COF-12 Fusarium equiseti isolate COF-8 Fusarium oxysporum isolate COF-17 Fusarium equiseti isolate COF-5 Fusarium solani isolate COF-6 99 Fusarium solani isolate COF-19 83 Fusarium solani isolate COF-20 Bionectria ochroleuca isolate COF-16 99 Bionectria ochroleuca isolate RS-3 Nectria haematococca mpVI 77-13-4 62 Chaetomium globosum CBS 148.51 Myceliophthora thermophila ATCC 42464 96 Arthroderma otae CBS 113480 Trichophyton verrucosum HKI 0517 Ajellomyces capsulatus NAm1 Ajellomyces dermatitidis SLH14081 Penicillium purpurogenum isolate RS-6 Aspergillus terreus NIH2624 Aspergillus fumigatus Af293 Aspergillus niger isolate RS-1 65 Neosartorya fischeri NRRL 181 Rhizomucor sp. isolate COF-7 Actinomucor elegans isolate COF-3 99 Actinomucor elegans isolate P.tri.IsoA
(B) 18S rRNA gene
Hypocreales
Sordariales Onygenales
Eurotiales
Zygomycota
0.05
Ascomycota
Fusarium oxysporum isolate RS-5 [AB938230] 61 COF clone OTU-2 (3) [AB938218] Fusarium oxysporum isolate COF-13 [AB938237] Fusarium fujikuroi IMI 58289 Fusarium oxysporum Fo5176 Fusarium avenaceum isolate RS-8 [AB938232] 64 68 RS clone OTU-1 (6) [AB938223] Fusarium oxysporum isolate RS-9 [AB938233] COF clone OTU-1 (21) [AB938217] 92 Fusarium oxysporum isolate COF-10 [AB938235] Hypocreales Fusarium oxysporum isolate COF-11 [AB938236] 69 Fusarium oxysporum isolate COF-2 [AB938234] Bionectria ochroleuca isolate RS-3 [AB938229] 96 COF clone OTU-3 (9) [AB938219] Fusarium solani isolate COF-19 [AB938238] 91 Fusarium solani isolate COF-20 [AB938239] 72 Nectria haematococca mpVI 77-13-4 RS clone OTU-4 (12) [AB938226] RS clone OTU-5 (4) [AB938227] 99 COF clone OTU-6 (1) [AB938222] Sordariales COF clone OTU-4 (2) [AB938220] COF clone OTU-5 (8) [AB938221] 98 Chaetomium globosum CBS 148.51 Myceliophthora thermophila ATCC 42464 95 Arthroderma otae CBS 113480 Trichophyton verrucosum HKI 0517 Onygenales 99 Ajellomyces capsulatus NAm1 Ajellomyces dermatitidis SLH14081 Penicillium purpurogenum isolate RS-6 [AB938231] 100 Aspergillus terreus NIH2624 RS clone OTU-2 (1) [AB938224] RS clone OTU-3 (3) [AB938225] Eurotiales Aspergillus niger RS-1 [AB938228] Aspergillus fumigatus Af293 100 Neosartorya fischeri NRRL 181 Burkholderia pseudomallei 1106a 89 Ralstonia solanacearum GMI1000 Pseudoxanthomonas suwonensis 11-1 91 Solitalea canadensis DSM 3403 61 Bdellovibrio bacteriovorus HD100 Leptospira biflexa Patoc 1 Kangiella koreensis: Kkor 2024 69 Flavobacterium johnsoniae: Fjoh 2418 Pseudoalteromonas haloplanktis TAC125 Caulobacter segnis ATCC 21756 Azospirillum brasilense Sp245 66 Pseudomonas stutzeri CCUG 29243 60 Haloarcula hispanica ATCC 33960 Haloferax mediterranei ATCC 33500 98 Actinoplanes missouriensis 431 100 Actinosynnema mirum DSM 43827 71 Agrobacterium tumefaciens C58: Atu4382 89 Sinorhizobium fredii NGR234: NGR c09950 Brucella suis ATCC 23445: BSUIS B0265 79 Ochrobactrum anthropi ATCC 49188 100 Pseudomonas entomophila: PSEEN5226 Shewanella denitrificans: Sden 3482 Pseudomonas fluorescens Pf-5: PFL 5501 0.2 Achromobacter xylosoxidans: AXYL 02390 88 Alcaligenes faecalis NCIB 8687 62
(A) nirK
Fungi Prokaryote
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Fig. 2. Tree of maximum likelihood phylogeny of (A) nirK of clones and fungal isolates obtained from the COF and RS amplified with the designed primer set and (B) the 18S rRNA gene obtained in a previous study (Wei et al., 2014). The nirK phylogenetic tree includes prokaryotic nirK. The 18S rRNA gene and nirK of the fungal isolates are highlighted in gray. The numbers in parentheses represent the numbers of fungal nirK clones in the operational taxonomic units. The numbers in square brackets represent accession numbers of the nucleotide sequences of partial fungal nirK from the environmental samples and isolates deposited in the DDBJ/EMBL/GenBank databases. The bootstrap values (>70%) from 500 replicates are indicated next to the branches.
Acknowledgment
References
This work was supported by the Institute for Fermentation, the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, Grants-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science (Nos. 24780055, 25252026, 26292085, and 26712015) and the GRENE/Ecoinformatics project from the Ministry of Education, Culture, Sports, Science and Technology, Japan. This work was also done as part of project, assessment and extension of technologies for mitigating greenhouse gas emission from agricultural soils, launched by MAFF.
Blagodatskaya, E., Dannenmann, M., Gasche, R., Butterbach-Bahl, K., 2010. Microclimate and forest management alter fungal-to-bacterial ratio and N2O-emission during rewetting in the forest floor and mineral soil of mountainous beech forests. Biogeochemistry 97, 55e70. Braker, G., Fesefeldt, A., Witzel, K.P., 1998. Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Applied and Environmental Microbiology 64, 3769e3775. Hallin, S., Lindgren, P.E., 1999. PCR detection of genes encoding nitrite reductase in denitrifying bacteria. Applied and Environmental Microbiology 65, 1652e1657. IPCC, 2007. Climate change 2007 (Chapter 8). In: Mitigation of Climate Changed Contribution of Working Group III to the Fourth Assessment Report of IPCC. Cambridge University Press, Cambridge, pp. 497e540. Isobe, K., Koba, K., Ueda, S., Senoo, K., Harayama, S., Suwa, Y., 2011. A simple and rapid GC/MS method for the simultaneous determination of gaseous metabolites. Journal of Microbiological Methods 84, 46e51. Isobe, K., Ohte, N., 2014. Ecological perspectives on microbes involved in N-cycling. Microbes and Environments 29, 4e16. Jones, C.M., Stres, B., Rosenquist, M., Hallin, S., 2008. Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification. Molecular Biology and Evolution 25, 1955e1966.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2014.11.026.
Please cite this article in press as: Wei, W., et al., Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.026
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W. Wei et al. / Soil Biology & Biochemistry xxx (2014) 1e5 Kim, S.W., Fushinobu, S., Zhou, S., Wakagi, T., Shoun, H., 2010. The possible involvement of copper-containing nitrite reductase (nirK) and flavohemoglobin in denitrification by the fungus Cylindrocarpon tonkinense. Bioscience, Biotechnology, and Biochemistry 74, 1403e1407. Kobayashi, M., Shoun, H., 1995. The copper-containing dissimilatory nitrite reductase involved in the denitrifying system of the fungus Fusarium oxysporum. The Journal of Biological Chemistry 270, 4146e4151. Laughlin, R.J., Stevens, R.J., 2002. Evidence for fungal dominance of denitrification and codenitrification in a grassland soil. Soil Science Society of America 66, 1540e1548. Nakanishi, Y., Zhou, S., Kim, S.W., Fushinobu, S., Maruyama, J., Kitamoto, K., Wakagi, T., Shoun, H., 2010. A eukaryotic copper-containing nitrite reductase derived from a nirK homolog gene of Aspergillus oryzae. Bioscience, Biotechnology, and Biochemistry 74, 984e991. Ravishankara, A.R., Daniel, J.S., Portmann, R.W., 2009. Nitrous oxide (N2O): the dominant ozone-depleting substance emitted in the 21st century. Science 326, 123e125.
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Rose, T.M., Schultz, E.R., Henikoff, J.G., Pietrokovski, S., McCallum, C.M., Henikoff, S., 1998. Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Research 26, 1628e1635. Sakurai, T., Kataoka, K., 2007. Structure and function of type I copper in multicopper oxidases. Cellular and Molecular Life Sciences 64, 2642e2656. Schloss, P.D., Westcott, S.L., Ryabin, T., Hall, J.R., Hartmann, M., Hollister, E.B., Lesniewski, R.A., Oakley, B.B., Parks, D.H., Robinson, C.J., Sahl, J.W., Stres, B., Thallinger, G.G., Van Horn, D.J., Weber, C.F., 2009. Introducing mothur: opensource platform-independent, community-supported software for describing and comparing microbial communities. Applied and Environmental Microbiology 75, 7537e7541. Shoun, H., Kim, D.H., Uchiyama, H., 1992. Denitrification by fungi. FEMS Microbiology Letters 94, 277e282. Wei, W., Isobe, K., Shiratori, Y., Nishizawa, T., Ohte, N., Otsuka, S., Senoo, K., 2014. N2O emission from cropland field soil through fungal denitrification after surface applications of organic fertilizer. Soil Biology and Biochemistry 69, 157e167.
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Soil Biology & Biochemistry xxx (2014) 1e5
Contents lists available at ScienceDirect
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Short communication
Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment Q4
Wei Wei a, Kazuo Isobe a, *, Yutaka Shiratori b, Tomoyasu Nishizawa a, c, Nobuhito Ohte d, Yuta Ise a, Shigeto Otsuka a, Keishi Senoo a a
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan Niigata Agricultural Research Institute, Niigata 940-0826, Japan Department of Bioresource Science, College of Agriculture, Ibaraki University, Ibaraki 300-0393, Japan d Department of Forest Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan b c
Q1
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 June 2014 Received in revised form 21 November 2014 Accepted 26 November 2014 Available online xxx
Fungal denitrification in soils is receiving considerable attention as one of the dominant N2O production processes. However, because of the lack of a methodology to detect fungal denitrification-related genes, the diversity and ecological behavior of denitrifying fungi in soil remains unknown. Thus, we designed a primer set to detect the fungal nitrite reductase gene (nirK) and validated its sensitivity and specificity. Through clone library analyses, we identified congruence between phylogenies of the 18S rRNA gene and nirK of denitrifying fungal isolates obtained from the surface-fertilized cropland soil and showed that fungi belonging to Eurotiales, Hypocreales, and Sordariales were primarily responsible for N2O emissions in the soil. © 2014 Published by Elsevier Ltd.
Keywords: Fungal denitrification Fungal nitrite reductase N2O emission
Nitrous oxide (N2O) is a potent greenhouse gas (IPCC, 2007) and is involved in stratospheric ozone depletion (Ravishankara et al., 2009). It is produced through microbial denitrification, in which nitrate and nitrite are reduced to gaseous N2O (Isobe and Ohte, 2014). Fungal denitrification in soil has recently received considerable attention as an N2O production process. We previously performed an antibiotic assay and isolated denitrifying fungi to demonstrate the dominance of fungal denitrification in a surfacefertilized cropland soil (Wei et al., 2014). This has also been reported in grassland and forest soils using the same methodology (Laughlin and Stevens, 2002; Blagodatskaya et al., 2010). Many fungal species are known to produce N2O (Shoun et al., 1992; Wei et al., 2014); however, the diversity and ecological behavior of denitrifying fungi in soil, unlike denitrifying bacteria, remains unknown, probably because of the lack of a methodology to detect fungal denitrification-related genes. In addition, the ability to denitrify varies at the species level (Wei et al., 2014), making it
* Corresponding author. Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: þ81 3 5841 5140; fax: þ81 3 5841 8042. E-mail address: [email protected] (K. Isobe).
difficult to identify denitrifying fungi based on their taxonomic position. Previous studies revealed that Fusarium oxysporum and Cylindrocarpon tonkinese, the most thoroughly characterized denitrifying fungi (Nakanishi et al., 2010), use copper-containing nitrite reductase (nirK) to reduce nitrite to nitric oxide, bearing a close resemblance to its bacterial counterpart (Kobayashi and Shoun, 1995; Kim et al., 2010). Additionally, fungal cytochrome cd1-type nitrite reductase remains undiscovered. Thus, developing a methodology to detect fungal nirK should lead to the precise identification of denitrifying fungi and elucidation of their ecological behavior. Consequently, the objectives of this study were to design suitable PCR primers to detect fungal nirK and use these primers to investigate the diversity of fungal nirK and identify the denitrifying fungi in surface-fertilized cropland soil where fungal denitrification is dominant (Wei et al., 2014). We searched full-length nirK fungal sequences from the public databases, NCBI Microbial Genomes (http://www.ncbi.nlm.nih. gov/genomes) and Functional Gene Repository (http://fungene. cme.msu.edu/index.spr) and obtained 15 sequences belonging to Ascomycota. We also obtained the representative sequences of nirK from diverse bacterial phyla and Euryarchaeota from database. Then, we generated the phylogenetic tree of nirK
http://dx.doi.org/10.1016/j.soilbio.2014.11.026 0038-0717/© 2014 Published by Elsevier Ltd.
Please cite this article in press as: Wei, W., et al., Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.026
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Fusarium oxysporum 5507 99 Fusarium oxysporum Fo5176 Fusarium fujikuroi IMI 58289 100 Fusarium lichenicola NBRC:30561 80 Nectria haematococca mpVI 77-13-4 Neosartorya fischeri NRRL 181 Aspergillus fumigatus Af293 100 100 100 Trichophyton verrucosum HKI 0517 100 Arthroderma benhamiae CBS 112371 Arthroderma otae CBS 113480 Myceliophthora thermophila ATCC 42464 72 Chaetomium globosum CBS 148.51 Aspergillus terreus NIH2624 Ajellomyces dermatitidis SLH14081 100 Ajellomyces capsulatus NAm1 Burkholderia pseudomallei 1106a 97 Ralstonia solanacearum GMI1000 Pseudoxanthomonas suwonensis 11-1 93 Bdellovibrio bacteriovorus HD100 Leptospira biflexa Patoc 1 75 Solitalea canadensis DSM 3403 Flavobacterium johnsoniae: Fjoh 2418 Kangiella koreensis: Kkor 2024 Pseudoalteromonas haloplanktis TAC125 Azospirillum brasilense Sp245 Caulobacter segnis ATCC 21756 74 Pseudomonas stutzeri CCUG 29243 Haloarcula hispanica ATCC 33960 Haloferax mediterranei ATCC 33500 100 Actinoplanes missouriensis 431 Actinosynnema mirum DSM 43827 100 Agrobacterium tumefaciens C58: Atu4382 98 Sinorhizobium fredii NGR234: NGR c09950 85 Brucella suis ATCC 23445: BSUIS B0265 Ochrobactrum anthropi ATCC 49188 82 100 Pseudomonas entomophila: PSEEN5226 Shewanella denitrificans: Sden 3482 Alcaligenes faecalis NCIB 8687 Pseudomonas fluorescens Pf-5: PFL 5501 69 Achromobacter xylosoxidans: AXYL 02390
Fungi
Prokaryote
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0.1 Fig. 1. Tree of maximum likelihood phylogeny of database-retrieved full-length NirK amino acid sequences of fungi, bacteria, and archaea. Bootstrap values (500 replicates) greater than 70% are denoted by dots above the branches and branch lengths correspond to sequence differences, which are indicated by a scale bar. Sequences from fungi are within the gray box.
(Fig. 1), and found that fungal nirK formed a monophyletic cluster distinct from the prokaryotic nirK with 100% bootstrap support. nirK is a two-domain enzyme including two copper centers, types 1 and 2 (Sakurai and Kataoka, 2007). We designed the primer sets based on homologs of the copper center type 1 domain (Fig. S1). Because widely used primers for bacterial nirK (such as primer set F1aCu/R3Cu and nirK2F/nirK5R, Braker et al., 1998; Hallin and Lindgren, 1999) also target this region, we can compare fungal nirK sequences with the large quantity of bacterial nirK sequences. We designed the forward primer to anneal with four conserved amino acids (tyrosine, valine, glutamine, and proline) and reverse primer to anneal with four conserved amino acids (aspartic acid, lysine, glycine, and alanine; Fig S1). Most of these amino acids were not conserved in prokaryotic nirK (Fig S1). The primer set specific to fungal nirK sequences, nirKfF (50 -TACGGGCTCATGtaygtnsarcc-30 ) and nirKfR (50 AGGAATCCCACAscnccyttntc-30 ), were designed based on the ConsensuseDegenerate Hybrid Oligonucleotide Primer (CODEHOP) algorithm (Rose et al., 1998). Both primers consist of the
degenerate core region (lowercase letters) corresponding to all possible codons specifying the conserved amino acids and consensus clamp region (uppercase letters) containing a single most common (consensus) nucleotide sequence derived from the rest of amino acids in the primer-designed region (Fig S1). We validated the specificity and sensitivity of the designed primer set, nirKfF/nirKfR, using fungal and prokaryotic strains. We used seventeen denitrifying and three nondenitrifying fungal strains isolated from the collected organic fertilizer (COF) and residual soil (RS) from a surface-fertilized cropland soil where fungal denitrification predominates (Wei et al., 2014). We also used ten prokaryotic strains (nine bacterial and one archaeal) obtained from culture collections (Japan Collection of Microorganisms, Koyadai, Japan or the Biological Resource Center (NBRC), Kazusakamatari, Japan; Table 1 and Fig. 1). Their abilities to produce N2O were confirmed as described previously (Isobe et al., 2011). Genomic DNA was extracted as described previously (Wei et al., 2014), and PCR was performed with the designed primers. The PCR mixture and conditions are described in
Please cite this article in press as: Wei, W., et al., Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.026
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Table 1 Primer validation using denitrifying fungal isolates and prokaryotic strains. Strain number
Fungi COF-2 COF-3 COF-5 COF-6 COF-7 COF-8 COF-10 COF-11 COF-12 COF-13 COF-16 COF-17 COF-19 COF-20 RS-1 RS-3 RS-5 RS-6 RS-8 RS-9 Bacteria ATCC-21756 ATCC-49188 NCIB-8687 NBRC-13243 DSM-43827 CCUG-29243 NBRC-100993 NBRC-106385 Archaea ATCC-33500 a b c d
Q2
Taxonomic assignment
N2O productiona
PCR products using primersb nirKfF/nirKfR
F1aCu/R3Cu
Fusarium oxysporum Actinomucor elegans Fusarium equiseti Fusarium solani Rhizomucor sp. Fusarium equiseti Fusarium oxysporum Fusarium oxysporum Fusarium equiseti Fusarium oxysporum Bionectria ochroleuca Fusarium oxysporum Fusarium solani Fusarium solani Aspergillus niger Bionectria ochroleuca Fusarium oxysporum Penicillium purpurogenum Fusarium avenaceum Fusarium oxysporum
þþþ þþ þþ e þþþ þþ þþþ þþþ þþ þþ e e þþþ þþ þ þþþ þþþ þþ þ þþ
þþþ e e e e e þþþ þþþ e þþ e e þþ þþ þ þþþ þþþ þþ þ þþþ
ec e e e 300, 700d e e e e e e e 400 e e e e e e e
Caulobacter segnis Ochrobactrum anthropi Alcaligenes faecalis Actinoplanes missouriensis Actinosynnema mirum Pseudomonas stutzeri Pseudoalteromonas haloplanktis Pseudoxanthomonas suwonensis
þ þ þ þþ þ þ þ þþþ
e e e e e e e e
þþþ þþ 700, 1200 e 300, 900 e 900
Haloarcula hispanica
þþ
e
e
N2O production: þ, 0e10 mg/day/g-biomass; þþ, 10e100 mg/day/g-biomass; þþþ, >100 mg/day/g-biomass. The concentration of PCR products: þ, 0e20 ng/ml; þþ, 20e50 ng/ml, þþþ, >50 ng/ml. Not amplified. Numbers indicate approximate sizes of nonspecific amplification products.
Table S1. PCR using the primer set, nirKfF/nirKfR, amplified the nirK fragment (ca. 480 bp) from twelve denitrifying fungal strains, belonging to Ascomycota (Table 1), but did not amplify the fragment from nondenitrifying fungal strains, all prokaryotic strains tested, or denitrifying Fusarium equiseti of Ascomycota, Actinomucor elegans and Rhizomucor sp. of Zygomycota. PCR using the widely-used bacterial nirK primers, F1aCu/R3Cu, did not amplify the fragment with the expected size. Thus, the primer set, nirKfF/ nirKfR, could successfully amplify the diverse fungal nirK of Ascomycota, the most dominant fungal group in soil (Wei et al., 2014), except F. equiseti, with sufficient selectivity and specificity. We analyzed nirK and 18S rRNA phylogenies of the fungal species isolated from COF and RS described above. We constructed the phylogenetic tree of the amplified nirK and the corresponding 18S rRNA gene obtained in our previous study (Wei et al., 2014; Fig. S2). The tree also included the database-retrieved fungal nirK and 18S rRNA genes (Fig. 1). Fungal nirK and 18S rRNA gene-based phylogenies was congruent at the order level of Ascomycota, whereas bacterial nirK and 16S rRNA gene-based phylogenies are known to be incongruent (Jones et al., 2008). This suggested that we could estimate the taxonomic position of denitrifying fungi based on their nirK phylogeny. We also investigated the diversity and phylogeny of fungal nirK in the surface-fertilized cropland soil using the extracted DNAs from COF and RS (Wei et al., 2014). We performed PCR using the primer set, nirKfF/nirKfR, and clone library analyses in the same
way as described previously (Wei et al., 2014). In total, 44 and 26 sequences of fungal nirK were obtained from COF and RS and classified into six COF and three RS OTUs, respectively, with 3% differences using the Mothur program (Schloss et al., 2009). From the phylogenetic tree of nirK and 18S rRNA gene (Fig. 2), the nirK clones were expected to be classified into Hypocreales, Sordariales, and Eurotiales of Ascomycota based on the congruence between the two phylogenies. Based on culture-dependent and DGGE analyses, we previously showed that denitrifying fungi closely related to Fusarium and Bionecter sp. in Hypocreales and to Chaetomium sp. in Sordariales are dominant in soils (Wei et al., 2014). The results of the clone library analysis using fungal nirK in this study strongly indicate that they are actually responsible for the N2O production in the surface-fertilized soil. We previously did not detect the presence of members of Eurotiales based on their 18S rRNA and ITS genes (Wei et al., 2014); however, we obtained the nirK clones and denitrifying isolates (Penicillium purpurogenum and Aspergillus niger) of this order. This shows that the designed primer set can sensitively detect denitrifying fungi regardless of their lower abundance in soils. In conclusion, we designed a novel fungal nirK primer set which can successfully detect the nirK of the most dominant denitrifying fungal group in soil (Ascomycota). The methodology developed here allows to study diversity and ecological behavior of denitrifying fungi and the importance of fungal denitrification in environments.
Please cite this article in press as: Wei, W., et al., Development of PCR primers targeting fungal nirK to study fungal denitrification in the environment, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.11.026
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Fusarium oxysporum isolate RS-5 Fusarium oxysporum isolate RS-9 Fusarium oxysporum isolate COF-2 Fusarium oxysporum isolate COF-13 Fusarium oxysporum isolate COF-11 Fusarium oxysporum isolate COF-10 92 Fusarium oxysporum Fo5176 Fusarium fujikuroi IMI 58289 Fusarium avenaceum isolate RS-8 Fusarium equiseti isolate COF-12 Fusarium equiseti isolate COF-8 Fusarium oxysporum isolate COF-17 Fusarium equiseti isolate COF-5 Fusarium solani isolate COF-6 99 Fusarium solani isolate COF-19 83 Fusarium solani isolate COF-20 Bionectria ochroleuca isolate COF-16 99 Bionectria ochroleuca isolate RS-3 Nectria haematococca mpVI 77-13-4 62 Chaetomium globosum CBS 148.51 Myceliophthora thermophila ATCC 42464 96 Arthroderma otae CBS 113480 Trichophyton verrucosum HKI 0517 Ajellomyces capsulatus NAm1 Ajellomyces dermatitidis SLH14081 Penicillium purpurogenum isolate RS-6 Aspergillus terreus NIH2624 Aspergillus fumigatus Af293 Aspergillus niger isolate RS-1 65 Neosartorya fischeri NRRL 181 Rhizomucor sp. isolate COF-7 Actinomucor elegans isolate COF-3 99 Actinomucor elegans isolate P.tri.IsoA
(B) 18S rRNA gene
Hypocreales
Sordariales Onygenales
Eurotiales
Zygomycota
0.05
Ascomycota
Fusarium oxysporum isolate RS-5 [AB938230] 61 COF clone OTU-2 (3) [AB938218] Fusarium oxysporum isolate COF-13 [AB938237] Fusarium fujikuroi IMI 58289 Fusarium oxysporum Fo5176 Fusarium avenaceum isolate RS-8 [AB938232] 64 68 RS clone OTU-1 (6) [AB938223] Fusarium oxysporum isolate RS-9 [AB938233] COF clone OTU-1 (21) [AB938217] 92 Fusarium oxysporum isolate COF-10 [AB938235] Hypocreales Fusarium oxysporum isolate COF-11 [AB938236] 69 Fusarium oxysporum isolate COF-2 [AB938234] Bionectria ochroleuca isolate RS-3 [AB938229] 96 COF clone OTU-3 (9) [AB938219] Fusarium solani isolate COF-19 [AB938238] 91 Fusarium solani isolate COF-20 [AB938239] 72 Nectria haematococca mpVI 77-13-4 RS clone OTU-4 (12) [AB938226] RS clone OTU-5 (4) [AB938227] 99 COF clone OTU-6 (1) [AB938222] Sordariales COF clone OTU-4 (2) [AB938220] COF clone OTU-5 (8) [AB938221] 98 Chaetomium globosum CBS 148.51 Myceliophthora thermophila ATCC 42464 95 Arthroderma otae CBS 113480 Trichophyton verrucosum HKI 0517 Onygenales 99 Ajellomyces capsulatus NAm1 Ajellomyces dermatitidis SLH14081 Penicillium purpurogenum isolate RS-6 [AB938231] 100 Aspergillus terreus NIH2624 RS clone OTU-2 (1) [AB938224] RS clone OTU-3 (3) [AB938225] Eurotiales Aspergillus niger RS-1 [AB938228] Aspergillus fumigatus Af293 100 Neosartorya fischeri NRRL 181 Burkholderia pseudomallei 1106a 89 Ralstonia solanacearum GMI1000 Pseudoxanthomonas suwonensis 11-1 91 Solitalea canadensis DSM 3403 61 Bdellovibrio bacteriovorus HD100 Leptospira biflexa Patoc 1 Kangiella koreensis: Kkor 2024 69 Flavobacterium johnsoniae: Fjoh 2418 Pseudoalteromonas haloplanktis TAC125 Caulobacter segnis ATCC 21756 Azospirillum brasilense Sp245 66 Pseudomonas stutzeri CCUG 29243 60 Haloarcula hispanica ATCC 33960 Haloferax mediterranei ATCC 33500 98 Actinoplanes missouriensis 431 100 Actinosynnema mirum DSM 43827 71 Agrobacterium tumefaciens C58: Atu4382 89 Sinorhizobium fredii NGR234: NGR c09950 Brucella suis ATCC 23445: BSUIS B0265 79 Ochrobactrum anthropi ATCC 49188 100 Pseudomonas entomophila: PSEEN5226 Shewanella denitrificans: Sden 3482 Pseudomonas fluorescens Pf-5: PFL 5501 0.2 Achromobacter xylosoxidans: AXYL 02390 88 Alcaligenes faecalis NCIB 8687 62
(A) nirK
Fungi Prokaryote
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 Q3 56 57 58 59 60 61 62 63 64 65
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Fig. 2. Tree of maximum likelihood phylogeny of (A) nirK of clones and fungal isolates obtained from the COF and RS amplified with the designed primer set and (B) the 18S rRNA gene obtained in a previous study (Wei et al., 2014). The nirK phylogenetic tree includes prokaryotic nirK. The 18S rRNA gene and nirK of the fungal isolates are highlighted in gray. The numbers in parentheses represent the numbers of fungal nirK clones in the operational taxonomic units. The numbers in square brackets represent accession numbers of the nucleotide sequences of partial fungal nirK from the environmental samples and isolates deposited in the DDBJ/EMBL/GenBank databases. The bootstrap values (>70%) from 500 replicates are indicated next to the branches.
Acknowledgment
References
This work was supported by the Institute for Fermentation, the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, Grants-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science (Nos. 24780055, 25252026, 26292085, and 26712015) and the GRENE/Ecoinformatics project from the Ministry of Education, Culture, Sports, Science and Technology, Japan. This work was also done as part of project, assessment and extension of technologies for mitigating greenhouse gas emission from agricultural soils, launched by MAFF.
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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2014.11.026.
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W. Wei et al. / Soil Biology & Biochemistry xxx (2014) 1e5 Kim, S.W., Fushinobu, S., Zhou, S., Wakagi, T., Shoun, H., 2010. The possible involvement of copper-containing nitrite reductase (nirK) and flavohemoglobin in denitrification by the fungus Cylindrocarpon tonkinense. Bioscience, Biotechnology, and Biochemistry 74, 1403e1407. Kobayashi, M., Shoun, H., 1995. The copper-containing dissimilatory nitrite reductase involved in the denitrifying system of the fungus Fusarium oxysporum. The Journal of Biological Chemistry 270, 4146e4151. Laughlin, R.J., Stevens, R.J., 2002. Evidence for fungal dominance of denitrification and codenitrification in a grassland soil. Soil Science Society of America 66, 1540e1548. Nakanishi, Y., Zhou, S., Kim, S.W., Fushinobu, S., Maruyama, J., Kitamoto, K., Wakagi, T., Shoun, H., 2010. A eukaryotic copper-containing nitrite reductase derived from a nirK homolog gene of Aspergillus oryzae. Bioscience, Biotechnology, and Biochemistry 74, 984e991. Ravishankara, A.R., Daniel, J.S., Portmann, R.W., 2009. Nitrous oxide (N2O): the dominant ozone-depleting substance emitted in the 21st century. Science 326, 123e125.
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