Molecular identification of the algal pathogen Pythium chondricola (Oomycetes) from Pyropia yezoensis (Rhodophyta) using ITS and cox 1 markers
- Author: Lee Soon Jeong, Hwang Mi Sook, Park Myoung Ae, Baek Jae Min, Ha Dong-Soo, Lee Jee Eun, Lee Sang-Rae
- Publish: ALGAE Volume 30, Issue3, p217~222, 15 Sep 2015
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ABSTRACT
Pythium species (Pythiales, Oomycetes) are well known as the algal pathogen that causes red rot disease inPyropia / Porphyra species (Bangiales, Rhodophyta). Accurate species identification of the pathogen is important to finding a scientific solution for the disease and to clarify the host-parasite relationship. In Korea, onlyPythium porphyrae has been reported fromPyropia species, with identifications based on culture and genetic analysis of the nuclear internal transcribed spacer (ITS) region. Recent fungal DNA barcoding studies have shown the low taxonomic resolution of the ITS region and suggested the mitochondrial cytochrome c oxidase subunit 1 (cox 1) gene as an alternative molecular marker to identifyPythium species. In this study, we applied an analysis of both the ITS andcox 1 regions to clarify the taxonomic relationships of KoreanPythium species. From the results, the two closely relatedPythium species (P. chondricola andP. porphyrae ) showed the same ITS sequence, while thecox 1 marker successfully discriminatedP. chondricola fromP. porphyrae . This is the first report of the presence ofP. chondricola from the infected blade ofPyropia yezoensis in Asia. This finding of the algal pathogen provides important information for identifying and determining the distribution ofPythium species . Further studies are also needed to confirm whetherP. chondricola andP. porphyrae are coexisting as algal pathogens ofPyropia species in Korea.
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KEYWORD
cox1 , ITS region , Pyropia yezoensis , Pythium chondricola , Pythium porphyrae , red rot disease
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Fungal pathogens have plagued the
Pyropia species of red algae (Rhodophyta) and seriously reduced the output of thePyropia aquaculture industry in Korea and Japan (Kawamura et al. 2005, Kim et al. 2014). Red rot disease is a major algal disease that was first reported inPorphyra tenera (=Pyropia tenera ) from Japan (Arasaki 1947). After that,Pythium species, the etiological agent of red rot disease, has been isolated and characterized from the red rot of infectedPyropia species (Takahashi et al. 1977).Among species of the genus
Pythium, P. chondricola, P. porphyrae , andP. adhaerens are closely related, based on the characteristics of a filamentous non-inflated sporangia, slow growth, and 1-4 diclinous antheridia (Levesque and De Cock 2004). However, these species show different host / substrate-specific relationships (Matsumoto et al. 1999, Levesque and De Cock 2004).Pythium porphyrae is recognized as the only pathogen of red rot disease inPyropia species (Takahashi et al. 1977, Kim et al. 2014), whileP. adhaerens has been isolated from soil andP. chondricola was discovered inChondrus crispus (Levesque and De Cock 2004).To verify the taxonomy of the genus
Pythium , Matsumoto et al. (1999) analyzed the nuclear internal transcribed spacer (ITS) sequences of 30Pythium species. More recently, Levesque and De Cock (2004) examined the phylogenetic relationship among 102 isolates using sequences of the ITS and D1, D2, and D3 regions in nuclear ribosomal DNA ofPythium . In addition, a DNA barcoding study for Oomycetes was conducted using ITS and cytochrome c oxidase subunit 1 (cox 1) markers, and the taxonomic resolution of these two molecular markers was compared (Robideau et al. 2011).Recently, Schroeder et al. (2013) suggested primers for polymerase chain reaction (PCR) based methods for the diagnosis and quantification of
Pythium species. In a molecular phylogenetic tree from the nuclear ribosomal DNA andcox 1 region ofPythium, P. adhaerens, P. chondricola , andP. porphyrae formed a single clade (Levesque and De Cock 2004, Robideau et al. 2011). In another approach, Park et al. (2001, 2006) developed aP. porphyrae specific ITS marker and applied it to forecasting red rot disease inPyropia yezoensis cultivation farms.Due to the increasing economic significance of
Pyropia species, concern for algal diseases in the scientific community and aquaculture industry has been raised (Gachon et al. 2010). Therefore, accurate species identification of the algal pathogen is needed to clarify the host-parasite relationship and to help find a scientific solution for the disease. Recent fungal DNA barcoding study has revealed that the ITS region cannot provide sufficient taxonomic information to discriminate between the closely relatedPythium species (Robideau et al. 2011). This study has shown the taxonomic usefulness of thecox 1 region at the species level and suggested it as another candidate molecular marker for DNA barcoding. In this study, we applied the ITS andcox 1 markers to identify KoreanPythium species from an infected blade ofPyropia yezoensis , from aPythium culture strain, and from environmental seawater. UsingPythium specific ITS primers and newly designedcox 1 primers, we accurately identified the species ofPythium and revealed its taxonomic relationship among otherPythium species.We sampled a blade of
Pyropia yezoensis infected by red rot disease in the field (Dec 2014 , Biando, Gunsan, Korea). A preliminary determination of infection by an algal pathogen was conducted based on the morphological characteristics in the diseased plant. AnotherPythium strain was also successfully isolated from an infectedPyropia yezoensis specimen collected from the Korean coast (Dec 2014, Aphaedo, Shinan, Korea) and cultured under indoor conditions using cornmeal agar (Park et al. 2001). This culture strain has been deposited in the Seaweed Research Center (National Fisheries Research and Development Institute, Mokpo, Korea). We also analyzed DNA templates from previous metagenomic studies of seawater (Lee et al. 2010, 2012, Yoon et al. 2012) and from environmental seawater sampled from the Nakdong River estuary near thePyropia aquaculture region (Aug 5, 2011, Korea).We applied molecular markers from the ITS and
cox 1 regions for this study. For the amplification of the ITS region, we usedPythium porphyrae specific ITS primers (PP-1/PP-2) (Park et al. 2001). To target thecox 1 region ofPythium species, we designedcox 1 primers having putative specificity for the genusPythium . For the design of thesecox 1 primers, we downloaded the completecox 1 sequence ofP. ultimum (NC_014280) and compared it with other oomycetecox 1 sequences through the BLAST searching option in GenBank (National Center for Biotechnology Information, NCBI). From thecox 1 sequence alignment, we found a conservedcox 1 region amongPythium species. The newcox 1 primers for the genusPythium were designed basing on this conserved region.The DNA extraction, PCR, and sequencing were conducted following methods outlined in Lee et al. (2011). Total genomic DNA was extracted from the blade of infected
Pyropia yezoensis using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. We also extracted total genomic DNA from environmental seawater using protocols suggested by Lee et al. (2010). Amplifications were carried out in a final volume of 20 μL usingamfiXpand (GenDEPOT, Barker, TX, USA) with PCR conditions of 2 min at 95℃, 40 cycles of 30 s at 94℃, 30 s at 48℃, and 1 min at 72℃, with a final 7 min extension step at 72℃.PCR products were subsequently sequenced in both directions by commercial sequencing (Genotech, Daejeon, Korea) and chromatograms were analyzed using Sequencher 5.3 (Gene Codes Corporation, Ann Arbor, MI, USA). The sequence similarity analyses of ITS and
cox 1 sequences were conducted BLAST in the GenBank (NCBI). For the phylogenetic analyses, the ITS andcox 1 reference sequences of relatedPythium species were obtained from GenBank, and all sequences were aligned using Clustal X v.1.8 (Thompson et al. 1997). A phylogenetic tree was constructed by the neighbor-joining method (NJ) (Saitou and Nei 1987) using PAUP 4.0 (Swofford 2001). A bootstrap analysis with 2,000 replicates was conducted to assess the robustness of the NJ tree.In addition to the previously developed ITS primers (Park et al. 2001), new PCR primers were designed for the selective amplification of the
cox 1 region ofPythium species, since the diverse fungus can be present in decaying seaweed (e.g., the infectedPyropia blade) and environmental seawater samples. The forward primer (cox 1-pyth-F1; 5′-ATTAGAATGGAATTAGCACAAC-3′) is positioned at 36405-36426 of the mtDNA ofP. ultimum (NC_014280) and the reverse primer (cox 1-pyth-R1; 5′-CTTAAACCWGGAGCTCTCAT- 3′) is bound at position 36813-36832.We successfully obtained PCR bands from the DNA extractions from the infected blade of
Pyropia yezoensis , thePythium culture strain, and the environmental seawater samples. PCR products 707 bp and 428 bp in size were amplified by the ITS andcox 1 primers (Fig. 1). The ITS region was successfully amplified from all three sample sources (the blade ofPyropia, Pythium culture strain, and environmental seawater). Even though the amplification yielded bands that were very weak from the ITS region of the sample from environmental seawater, the sequence was successfully determined. On the other hand, we could amplify thecox 1 region only from the blade ofPyropia and the culture strain.From the similarity analysis using BLAST, the ITS sequences showed no variation among Korean isolates (the blade of
Pyropia, Pythium culture strain, and seawater). In addition, the Korean ITS sequences had 100% sequence similarity with the reference sequences of ITS inP. porphyrae (Korea, AB043506; Japan, AY598673, AB185111, AB043506; USA, JQ898472) andP. chondricola (Netherlands, AY598620, HQ643496, HQ643497, HQ643498; USA, HQ643499). The Koreancox 1 sequences in this study showed 100% similarity withP. chondricola (Netherlands, HQ708542, HQ708543, HQ708544; USA, HQ708545), 99% (382/386; matched sequences / total sequences excluding the primer binding sites) withP. porphyrae (Japan-HQ708794) and 99% (381/386) withP. adhaerens (HQ708462).In the ITS phylogenetic tree, the Korean
Pythium ITS sequences clustered in a single clade withP. porphyrae (Korea, Japan, and USA) andP. chondricola (Netherlands), and also with a more distantly relatedP. chondricola (EF016916) from Thailand (Fig. 2A). In contrast, thecox 1 phylogenetic tree showed that Koreancox 1 sequences formed a single clade only withP. chondricola cox 1 sequences reported from the Netherlands and USA (Fig. 2B).Pythium porphyrae reported from Japan formed a distant sister group of the clade including KoreanPythium species andP. chondricola (Netherlands and USA).> Taxonomic resolution of ITS and
cox 1 regionsKorean
Pythium isolates (in this study) had the same ITS sequence asP. porphyrae andP. chondricola sequences that were previously reported. Therefore, the ITS sequence did not seem to provide sufficient information to resolve the taxonomic relationship betweenP. porphyrae andP. chondricola . The only difference in the ITS sequences ofP. porphyrae /P. chondricola was in the poly A sequence at the 5′ end of the ITS region (6 in HQ643499 vs. 7 in other isolates). On the other hand, thecox 1 sequence showed sufficient variation to discriminate betweenP. porphyrae (HQ708794) and its close relatives,P. chondricola (HQ708545) andP. adhaerens (HQ708462) (Fig. 2).A recent DNA barcoding study also reported the greater discriminative power in the
cox 1 region than in the ITS region, allowing a clarification of the taxonomic relationships among the closely relatedPythium species (Robideau et al. 2011). From our results, the molecular analysis based on the ITS region could not provide specific information for the identification ofP. porphyrae . Therefore, the ITS sequence was not a suitable barcoding marker to discriminateP. chondricola fromP. porphyrae .> Presence of
Pythium chondricola / P. porphyrae in the aquatic ecosystemFrom the PCR analysis of the environmental seawater samples using the
Pythium specific ITS primer set, we detectedP. chondricola / P. porphyrae ITS sequences from the coastal seawater of Korea. We did not obtain acox 1 amplicon from these samples. Because nuclear ribosomal DNA has a high number of tandem repeat sequences (Rogers and Bendich 1987), PCR amplification with ITS primers could be more efficient thancox 1 region in detectingPythium .The ITS sequences from environmental samples could have originated from fungal zoospores of
P. chondricola / P. porphyrae in the seawater. Kawamura et al. (2005) also reported the distribution ofP. porphyrae in the seafloor sediment (Ariake Sea, Japan) basing on an ITS sequence analysis. Therefore,P. chondricola / P. porphyrae appears to be present in the Korean aquatic ecosystem and could infectPyropia species. This study provides important information to monitor the distribution of red rot disease. In addition, more sensitive, high efficiency methods should be developed to detectPythium species from environmental seawater samples because relatively little biomass of the zoospore exists in the aquatic ecosystem.> Taxonomic entity of Korean
Pythium speciesPythium chondricola was first isolated from decayingChondrus crispus collected in and near the salt lake Grevelingen in the Netherlands (De Cock 1986). Since it was as a new species,P. chondricola has only been reported from the Netherlands and USA using molecular markers (Robideau et al. 2011). De Cock (1986) examined different fungal isolates from the same origin and locality as earlier isolates ofP. chondricola , and discovered diverse sources ofP. chondricola , including aquatic plants such asZostera marina (flowering plants),Ulva lactuca (green algae), and an unidentified red alga. This implies thatP. chondricola can infect a variety of host plants and thatPyropia species is one of them.Robideau et al. (2011) reported the presence of
P. porphyrae (HQ708794) from Japan as a separate taxonomic entity fromP. chondricola. Pythium chondricola has not been reported fromPyropia species or aquatic environments of Korea or Japan (Kawamura et al. 2005, Park et al. 2006, Uzuhashi et al. 2015). To our knowledge, the finding ofP. chondricola in this study is the first report of this species in the Asian region (Global Catalogue of Microorganisms [GCM]; The Barcode of Life Data System [BOLD]) (Ratnasingham and Hebert 2007, Wu et al. 2013).We could hardly find
P. porphyrae fromPyropia blades during this study. Therefore, further studies targeting thecox 1 region are strongly requested and should include the wide survey for red rot disease inPyropia cultivation sites and in the natural aquatic ecosystem to confirm whetherP. chondricola andP. porphyrae are coexisting as algal pathogens ofPyropia species in Korea.-
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[Fig. 1.] Amplification of the nuclear ribosomal DNA internal transcribed spacer (ITS) region and mitochondrial DNA of the cox1 region using Pythium porphyrae specific ITS primers (1) (Park et al. 2001) and cox1 primers (2). Total genomic DNA was extracted from a culture strain (Shinan, Korea) and used as template DNA for polymerase chain reaction.
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[Fig. 2.] Neighbor-joining phylogenetic analyses of Pythium species using the internal transcribed spacer (ITS) (A) and cox1 (B) regions. The numbers at the nodes of the tree indicate the bootstrap values (>50). Samples NKC2 (coastal seawater, Busan, Korea) and Nakdong River estuary (Busan) originated from environmental seawater. Sequences marked with asterisks represent isolates that were genetically identified by both the ITS and cox1 regions. ITS (HQ643498) and cox1 (HQ708544) sequences were isolated from a P. chondricola ex-type specimen (CBS 203.85) (Robideau et al. 2011).