Pythium
species (Pythiales, Oomycetes) are well known as the algal pathogen that causes red rot disease in
Pyropia / 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, only
Pythium porphyrae
has been reported from
Pyropia
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 identify
Pythium
species. In this study, we applied an analysis of both the ITS and
cox
1 regions to clarify the taxonomic relationships of Korean
Pythium
species. From the results, the two closely related
Pythium
species (
P. chondricola
and
P. porphyrae
) showed the same ITS sequence, while the
cox
1 marker successfully discriminated
P. chondricola
from
P. porphyrae
. This is the first report of the presence of
P. chondricola
from the infected blade of
Pyropia yezoensis
in Asia. This finding of the algal pathogen provides important information for identifying and determining the distribution of
Pythium species
. Further studies are also needed to confirm whether
P. chondricola
and
P. porphyrae
are coexisting as algal pathogens of
Pyropia
species in Korea.
INTRODUCTION
Fungal pathogens have plagued the
Pyropia
species of red algae (Rhodophyta) and seriously reduced the output of the
Pyropia
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 in
Porphyra 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 infected
Pyropia
species (
Takahashi et al. 1977
).
Among species of the genus
Pythium, P. chondricola, P. porphyrae
, and
P. 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 in
Pyropia
species (
Takahashi et al. 1977
,
Kim et al. 2014
), while
P. adhaerens
has been isolated from soil and
P. chondricola
was discovered in
Chondrus 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 30
Pythium
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 of
Pythium
. 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 and
cox
1 region of
Pythium, P. adhaerens, P. chondricola
, and
P. porphyrae
formed a single clade (
Levesque and De Cock 2004
,
Robideau et al. 2011
). In another approach,
Park et al. (2001
,
2006)
developed a
P. porphyrae
specific ITS marker and applied it to forecasting red rot disease in
Pyropia 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 related
Pythium
species (
Robideau et al. 2011
). This study has shown the taxonomic usefulness of the
cox
1 region at the species level and suggested it as another candidate molecular marker for DNA barcoding. In this study, we applied the ITS and
cox
1 markers to identify Korean
Pythium
species from an infected blade of
Pyropia yezoensis
, from a
Pythium
culture strain, and from environmental seawater. Using
Pythium
specific ITS primers and newly designed
cox
1 primers, we accurately identified the species of
Pythium
and revealed its taxonomic relationship among other
Pythium
species.
MATERIALS AND METHODS
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. Another
Pythium
strain was also successfully isolated from an infected
Pyropia 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 the
Pyropia
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 used
Pythium porphyrae
specific ITS primers (PP-1/PP-2) (
Park et al. 2001
). To target the
cox
1 region of
Pythium
species, we designed
cox
1 primers having putative specificity for the genus
Pythium
. For the design of these
cox
1 primers, we downloaded the complete
cox
1 sequence of
P. ultimum
(NC_014280) and compared it with other oomycete
cox
1 sequences through the BLAST searching option in GenBank (National Center for Biotechnology Information, NCBI). From the
cox
1 sequence alignment, we found a conserved
cox
1 region among
Pythium
species. The new
cox
1 primers for the genus
Pythium
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 using
amfiXpand
(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 and
cox
1 reference sequences of related
Pythium
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.
RESULTS
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 of
Pythium
species, since the diverse fungus can be present in decaying seaweed (e.g., the infected
Pyropia
blade) and environmental seawater samples. The forward primer (
cox
1-pyth-F1; 5′-ATTAGAATGGAATTAGCACAAC-3′) is positioned at 36405-36426 of the mtDNA of
P. 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
, the
Pythium
culture strain, and the environmental seawater samples. PCR products 707 bp and 428 bp in size were amplified by the ITS and
cox
1 primers (
Fig. 1
). The ITS region was successfully amplified from all three sample sources (the blade of
Pyropia, 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 the
cox
1 region only from the blade of
Pyropia
and the culture strain.
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.
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 in
P. porphyrae
(Korea, AB043506; Japan, AY598673, AB185111, AB043506; USA, JQ898472) and
P. chondricola
(Netherlands, AY598620, HQ643496, HQ643497, HQ643498; USA, HQ643499). The Korean
cox
1 sequences in this study showed 100% similarity with
P. chondricola
(Netherlands, HQ708542, HQ708543, HQ708544; USA, HQ708545), 99% (382/386; matched sequences / total sequences excluding the primer binding sites) with
P. porphyrae
(Japan-HQ708794) and 99% (381/386) with
P. adhaerens
(HQ708462).
In the ITS phylogenetic tree, the Korean
Pythium
ITS sequences clustered in a single clade with
P. porphyrae
(Korea, Japan, and USA) and
P. chondricola
(Netherlands), and also with a more distantly related
P. chondricola
(EF016916) from Thailand (
Fig. 2A
). In contrast, the
cox
1 phylogenetic tree showed that Korean
cox
1 sequences formed a single clade only with
P. 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 Korean
Pythium
species and
P. chondricola
(Netherlands and USA).
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).
DISCUSSION
- Taxonomic resolution of ITS andcox1 regions
Korean
Pythium
isolates (in this study) had the same ITS sequence as
P. porphyrae
and
P. chondricola
sequences that were previously reported. Therefore, the ITS sequence did not seem to provide sufficient information to resolve the taxonomic relationship between
P. porphyrae
and
P. chondricola
. The only difference in the ITS sequences of
P. 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, the
cox
1 sequence showed sufficient variation to discriminate between
P. porphyrae
(HQ708794) and its close relatives,
P. chondricola
(HQ708545) and
P. 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 related
Pythium
species (
Robideau et al. 2011
). From our results, the molecular analysis based on the ITS region could not provide specific information for the identification of
P. porphyrae
. Therefore, the ITS sequence was not a suitable barcoding marker to discriminate
P. chondricola
from
P. porphyrae
.
- Presence ofPythium chondricola / P. porphyraein the aquatic ecosystem
From the PCR analysis of the environmental seawater samples using the
Pythium
specific ITS primer set, we detected
P. chondricola / P. porphyrae
ITS sequences from the coastal seawater of Korea. We did not obtain a
cox
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 than
cox
1 region in detecting
Pythium
.
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 of
P. 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 infect
Pyropia
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 detect
Pythium
species from environmental seawater samples because relatively little biomass of the zoospore exists in the aquatic ecosystem.
- Taxonomic entity of KoreanPythiumspecies
Pythium chondricola
was first isolated from decaying
Chondrus 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 of
P. chondricola
, and discovered diverse sources of
P. chondricola
, including aquatic plants such as
Zostera marina
(flowering plants),
Ulva lactuca
(green algae), and an unidentified red alga. This implies that
P. chondricola
can infect a variety of host plants and that
Pyropia
species is one of them.
Robideau et al. (2011)
reported the presence of
P. porphyrae
(HQ708794) from Japan as a separate taxonomic entity from
P. chondricola. Pythium chondricola
has not been reported from
Pyropia
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 of
P. 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
from
Pyropia
blades during this study. Therefore, further studies targeting the
cox
1 region are strongly requested and should include the wide survey for red rot disease in
Pyropia
cultivation sites and in the natural aquatic ecosystem to confirm whether
P. chondricola
and
P. porphyrae
are coexisting as algal pathogens of
Pyropia
species in Korea.
Acknowledgements
This study was supported by the National Fisheries Research and Development Institute, Korea (RP-2015-AQ-054).
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