Indian Journal of Geo Marine Sciences Vol. 47 (03), March 2018, pp. 558-566

Molecular analysis of microbiota composition and alterations in Pyropia yezoensis infected with red rot disease

Sohrab Khan1,2, *Yunxiang Mao1,2, Dandan Zou1,2,Sadaf Riaz1,2,Liping Qiu1,2& Na Li1,2

1Key Laboratory of Marine Genetics and Breeding (MOE), Ocean University of China, China 2College of Marine Life Sciences Ocean University of China, China

*[E-mail: [email protected]]

Received 14 July 2017; revised 14 August 2017

A variety of microbes resides on the surface of Pyropia yezoensis and the majority of them are unculturable. To identify and explore the different types of microflora present on its surface, we conducted a comparative molecular analysis of Pyropia collected from the three different locations and their subsequent infection with Pythium porphyrae. The samples includes, i.e. healthy Pyropia leaf blades collected from the farm, Pyropia from farm cultured in laboratory for seven days and Pyropia grown in laboratory following artificially infection with P. porphyrae. To investigate the difference in microbial community composition of P. yezoensis, Illumina HiSeq 2000 platform sequencing of the V4-V5 hypervariable region of the 16S ribosomal RNA gene was performed. After performing quality-filtering steps, the range of sequenced reads obtained per microbial sample was between 29, 155 to 65, 615 reads, while the total number of OTU’s found were 7,324, which varied between all the samples. The two most abundant phyla found in all the samples were Proteobacteria and Bacteroidetes. Our study results suggest that there is a change in microbiota composition and abundance due to artificially infecting of P. yezoensis with Pythium spores.

[Keywords: Microflora, Pyropia yezoensis, Pythium porphyrae, Hypervariable region V4-V5, Microbiota composition]

Introduction been designed. Specific information on the China is the largest producer of Pyropia and phylogeny and functional capabilities of the the most important product of Pyropia is its microbiota of Pyropia is very important to mature blades1. Pyropia yezoensis contains a wide improve our understanding regarding the range of microorganisms on its surface are beneficial and deleterious effects of bacteria on its collectively called as its microbiome. The most growth. Due to recent advances in technology, the important among these microorganisms are field of marine microbial ecology has provided a bacteria, some type of fungi and protists that detailed narrative of bacterial community and its inhabit their surfaces2, 3, 4. Pythium porphyrae (Py. function. As an effect, it is obvious now that porphyrae) is a pathogenic marine oomycete that marine atmosphere has an incredible amount of causes the red rot disease in P. yezoensis, which is bacteria12, 13. Many aquatic eukaryotes possess one of the most serious diseases in Porphyrae steady organizations with microbial associates industry and cultivation farms in Japan5. The and depend on them for growth, nutritional process of beginning of the disease is very similar availability and defense against colonization and to that of plants infected with oomycetes by the predation14, 15, 16, 17. The most incredible work in production of zoospores6. The oomycetes, such as the field of microbial ecology in the past decade is the Pythium genus, recently been separated from the introduction of metagenomics18. other fungi and relocated in the Kingdom Metagenomics is the fastest emerging field in Straminipila or Chromista7, 8. Experiments on research based on studying the uncultivable discovering the red rot illness at preliminary level, organisms for better understanding about the including PCR strategy9, polyclonal antibodies diversity of microbes and their functions19. (Pabs)10 and monoclonal antibodies (MAbs)11 has Environmental samples can be served directly KHAN et al.: MICROBIOTA COMPOSITION AND ALTERATIONS IN P. YEZOENSIS 559 without culturing to know about the different for seven days and the lab grown Pyropia microbial diversity. The molecular microbial yezoensis. Pyropia samples to be infected ecology from environment was initiated in 1990, collected from different sites with different with the direct amplification and sequencing of cultural conditions. The P. porphyrae 16SrRNA genes20. This revolutionized the ways (NBRC23353) was bought from the Biological of studying prokaryotes in the environment. Resource Center of Japan, and maintained on Among the earliest reports, 16S rRNA gene cornmeal seawater agar (CMSA)37. Agar discs was being amplified through PCR after total DNA were transferred to the liquid culture medium extraction by using universal primers i.e. 27f and under axenic conditions for 7 days at 24℃ to 21 1492r , which amplifies almost all the 16S rRNA expand mycelia and 10mM CaCl2 added to the genes, that aid in the study of bacteria in a broad seawater to reduce zoospores as discussed in the range of habitat. Moreover, a variety of preceding report38. pathological and opportunistic communications between macroalgae and viruses are also reported Infection of healthy P. yezoensis via oomycetes in the preceding studies22. Culture-independent zoospores techniques have the advantage of detecting the P. yezoensis leaves were infected with diversity of the entire microbial system, including zoospores by mixing spore solution and healthy that of the yet to be cultured microorganisms23, 24, Pyropia leaves, cultured bottles were kept in the 25, 26. shaking incubator at 15oC, under light 12L: 12D Sequence variations in the 16S ribosomal photocycle using florescent light with intensity of, RNA (rRNA) gene is widely used to characterize 80μmol·s-1·m-2. Provasoli’s enriched seawater the taxonomic diversity present in microbial (PES) medium was added 1ml/L according to the communities27, 28, 29. Metagenomics studies are not experimental procedure39. Samples were observed just limited to bacterial sequences, but also shows for the appearance of infection under the that precise, strain level uniformity can be microscope after every hour of incubation. achieved as in the metagenomics sequencing of E. Healthy algal cells of P. yezoensis were also coli30, V. cholera31, methicillin-resistant maintained as a control under the above mentioned Staphylococcus aureus (MRSA)32 and conditions for each type of algal sample. The tuberculosis33. The sequence of 16SrRNA is healthy and infected Pyropia cells are depicted in composed of nine hypervariable regions mixed figure.1 and 2 respectively. with conserved regions. The sequence of the 16S rRNA gene and its hypervariable regions has been determined for a large number of organisms, and is available from multiple databases such as Greengenes34 and the Ribosomal Database Project35, 36. For taxonomic classification, it is sufficient to sequence individual hypervariable regions instead of the entire gene length. Present study intended to test the diversity of bacterial microflora communities in response to artificial infection of P. yezoenesis with oomycete zoospores that can also bring changes to its Fig.1-Healthy P. yezoensis cells and Fig.2-infected P. yezoensis cells after artificially infecting it with P. porphyrae microbiome. Furthermore, it was aimed to provide under light microscope using 100X lens. in-depth taxonomic characterization of the microbiome of Pyropia managed from different Sample collection and DNA isolation environments. It was also hypothesized that the In total, 31 samples were collected on 0, 1st, Lab microbial community of Pyropia would vary 3rd, 7th and 15th day of infection i.e. 0.2g of each between the two farming places due to the natural sample after infection of all the three types of P. variations in each system’s atmosphere. yezoensis. The control samples (uninfected algae) were also collected at above mentioned days for Materials and Methods each type of Pyropia. In addition, the large red Culturing of P. yezoensis, P. porphyrae and spot samples were collected separately from oomycetes zoospores production infected leaves and all the samples were kept at - Three types of Pyropia was used in this study 80oC until DNA extraction. Total genomic DNA for experimental purpose that includes Pyropia was extracted and purified by phenol/chloroform from the field, Pyropia from field cultured in lab method as previously described40. 560 INDIAN J. MAR. SCI., VOL. 47, NO. 03, MARCH 2018

PCR amplification of 16S rRNA (9.64%), whereas in farmed samples that were PCR amplification of the 16srRNA was cultured in the laboratory for a week time period, carried out using universal primers 27F the dominant phyla ratio was Proteobacteria (AGAGTTTGATCMTGGCTCAG) and 1492R (68%) and Bacteroidetes (28.58%), while, in the GGTTACCTTGTTACGACTT41 that targets the laboratory samples the dominant phyla were full-length bacterial 16S rRNA gene sequence to Proteobacteria (92.56%), and confirm the presence of ample microbial Bacteroidetes(5.94%). The farmed Pyropia community DNA and to rule out the presence of samples after infecting with Pythium spores any potential inhibitory compounds. The PCR showed a change in their abundance percentage of amplification program for this confirmatory these two phyla, Proteobacteria (88.12%), and reaction was as follows: denaturation at 94℃ for 5 Bacteroidetes (10.88). Whereas, in farm samples min, followed by 35 cycles of denaturation at 94℃ that were cultured in the laboratory for a week for 30sec, annealing at 55℃ for 40sec and time period, the dominant phyla ratio was elongation at 72℃ for 2 min and then final Proteobacteria (86.96 %) and Bacteroidetes elongation at 72℃ for 10mints. The strong PCR (11.86%), while, the major phyla groups in the product upto ~1500bp considered as indicative for laboratory samples were Proteobacteria (89.86%), the presence of microbial community DNA. and Bacteroidetes (7.4%) (Table. 1). In the majority of the environment, Proteobacterial 16S rRNA Illumina sequencing community dominates the bacterial diversity47 and DNA extracted from all the samples were our results were in accord with their findings. send to Novogene Bioinformatics Technology Proteobacteria dominates the coral associated Co., Ltd, Beijing, China for illumina sequencing bacterial community composition of of V4-V5 region of 16SrRNA. Sequenced data Al.grandiflorum accounting for 69% of the was analyzed and filtered using QIIME relative abundance, while, the bacterial (V1.7.0)42. The data sets supporting the results of community composition of Anthothela contains this article are available in the Sequence Read 48% of Proteobacteria as a dominant phylum. Archive (SRA), accessible through NCBI SRP# Bacteroidetes were also present in accession is: SRP105021. Al.grandiflorum as a minor contributor in relation to Proteobacteria, i.e. 2% of the total bacterial Analysis tools abundance48. To confirm differences in the abundance and taxonomy among all the three groups of Pyropia yezoensis, the R statistical software was used for the graphical representation of the relative abundance of bacterial diversity from phylum to species level43. R packages used for the statistical visualization of data include; reshape44, plyr45, scales46 and ggplot247 respectively.

Results and Discussion Total 16, 53,875 reads were obtained from all the sample libraries after quality filtering steps. The average base length ranged from 370bp to 373bp. The total number of OTU’s assembled Fig.3-Abundance proportions of dominant bacterial taxa in Pyropia yezoensis from different sampling at phylum level from the combined libraries were 7324. Due to before and after infection with Pythium porphyrae spores. rarefaction curves, no significant fluctuation in FC=Farm control, TC= Cultured in lab control, LC =Lab the species composition per sequence sample was samples control, while FS=Farm infected, TS= Cultured in observed in our data. Therefore, 29,038 reads per lab infected, LS =Lab sample infected, SP= Spots sample sample were sufficient for the diversity analysis. (infected).

Organisms belong to 27 phyla were precisely Proteobacteria and Bacteroidetes reported as the found after sequencing of all the 31 samples. dominant bacterial communities of the red alga Proteobacteria and Bacteroidetes phyla were the Delisea pulchara49. More recently, the microbial most abundant phyla in all the samples (Fig 3, Fig community of U. australis was reported to have 4). The two most abundant phyla found in the more than five thousand nearly full-length 16S uninfected (control) farmed Pyropia samples were rDNA, out of which 74% of all the sequences Proteobacteria (86.58%), and Bacteroidetes KHAN et al.: MICROBIOTA COMPOSITION AND ALTERATIONS IN P. YEZOENSIS 561

associated microbial communities58, 59, 60, 61. Most of the differential OTUs belonged to the Phyla Proteobacteria and Bacteroidetes62. One of the factors that influence the change in the composition and bacterial richness observed in our study is may be due to the associated community of microorganisms, as it showed clear difference in microbial composition of farmed samples cultured in laboratory for seven days subsequent to infection. Our results also showed some resemblance with the findings of a research study on macro algae Delisea pulchra and its 63 Fig.4-Heat map showing of Pyropia yezoensis from different related microbial communities . sampling and their abundance proportions before and after Peudomonas xanthomarina and Halomonas infection with Pythium porphyrae spores. FC=Farm control, cupida were the two most dominant species found TC= Cultured in lab control, LC =Lab samples control, while in all the samples (Fig.5, Fig.6). At species level, FS=Farm infected, TS= Cultured in lab infected, LS =Lab sample infected, SP= Spots sample (infected). Peudomonas xanthomarina was highly found in the laboratory samples before and after infection were classified as Alphaproteobacteria (5.36%, 4.9%), and in the infected spots sample (Proteobacteria) and 13% were grouped into (4.3%), while, the lowest amount was found in the Bacteroidetes50. On the other hand, Bacteroidetes farm samples cultured in laboratory (0.24% & was the abundant phylum found in all the samples 0.24%). Halomonas cupida was dominant in the in another report, which is a diverse and largely farm samples cultured in laboratory (4.4% & distributed phylum, as well as members present in 3.04%), while lowest in the laboratory samples both fresh and salt-water ecosystems. Forty-one (0.04% & 0.1%) and in infected spot sample clones were analyzed from the cow dung (0.2%). microbiota that also represents Bacteroidetes and The bacterial species diversity present in the Proteobacteria as their abundant phyla51. A farm samples varied between the highest and general feature connected with environmental lowest amount of level (Table. 2). This study Bacteroidetes is their ability to degrade composite showed Pseudomonas xanthomarina and glycans, such as cellulose, hemicelluloses, chitin, Halomonasas as their abundant microbial agarose and alginate52. Bacteroidetes also play a community found on the surface of P. yezoensis. role in the natural genetic transmission of Pseudomonas xanthomarina is a bacterium, found antimicrobial genes53. in marine ascidians, unlike many other members 64 A research study carried out on microbiome of the Pseudomonas genus, it is not fluorescent . of rainbow trout from aquarium and farm settings, The genus Pseudomonas has been reported to be a the four dominant phyla includes; Proteobacteria, genetically and metabolically diverse bacterial 65, 66, 67 while, in contrast to the rainbow trout intestinal group . samples, the tank biofilm sample was dominated Pseudomonas xanthomarina, a bacterium, by members of Proteobacteria and responsible for the fast biodegradation of Bacteroidetes54. Commonly detected organisms in Phenanthrene, not reported previously in the another study were bacteria associated with the degradation of PAHs. Although, this ability is phyla Proteobacteria, Bacteroidetes and common in the phylogenetically closely related P. Cyanobacteria55. In additional, a pyrosequencing- stutzeri, as previously reported in a study based study on the diversity of different types of conducted on the isolation of bacterial community 68 algae taken from a coral reef ecosystem, 22 from contaminated soil . The majority of the different types of phyla were found, which shows isolates retrieved from algal surfaces contains the the high diversity present on macroalgae56. genera Pseudomonas. Halomonas is a genus of Taxonomic profiling of bacterial communities in a halophilic Proteobacteria, also belongs to phylum gilthead seabream (Sparus aurata) hatchery57, Proteobacteria. A study carried out on reported Proteobacteria, Bacteroidetes and heterotropical bacterial flora of shrimps and crab Firmicutes as the three most dominant phyla ponds showed Holomonas as their second most 69 accounted for more than 98.5% of all the retrieved bundant bacterial community . quality filtered sequence reads. The transfer of marine organisms from their natural environment to an aquarium can have an impact on their 562 INDIAN J. MAR. SCI., VOL. 47, NO. 03, MARCH 2018

Table.1.Different phylum proportions of all the samples, representing Proteobacteria and Bacteroidetes as their most abundant phyla

Samples Proteobacteria Bacteroidetes Gracilibacteria Planctomycetes Others FC_1_0 0.993 0.006 0.000 0.000 0.000 FC_1_1 0.986 0.013 0.000 0.000 0.000

FC_1_3 0.859 0.105 0.006 0.003 0.002

FC_1_7 0.681 0.193 0.123 0.000 0.002 FC_1_15 0.810 0.165 0.001 0.011 0.012 FS_2_0 0.998 0.001 0.000 0.000 0.000 FS_2_1 0.989 0.010 0.000 0.000 0.000

FS_2_3 0.971 0.025 0.001 0.000 0.002

FS_2_7 0.826 0.146 0.026 0.000 0.000 FS_2_15 0.622 0.362 0.010 0.001 0.002 TC_3_0 0.409 0.578 0.001 0.000 0.002 TC_3_1 0.926 0.068 0.001 0.000 0.005 TC_3_3 0.856 0.135 0.001 0.001 0.006

TC_3_7 0.573 0.402 0.000 0.019 0.004

TC_3_15 0.636 0.246 0.001 0.028 0.039 TS_4_0 0.957 0.038 0.003 0.001 0.001 TS_4_1 0.903 0.087 0.001 0.001 0.006 TS_4_3 0.851 0.138 0.003 0.001 0.005

TS_4_7 0.782 0.199 0.001 0.012 0.004

TS_4_15 0.855 0.131 0.000 0.006 0.003 LC_5_0 0.963 0.029 0.002 0.003 0.000 LC_5_1 0.971 0.023 0.001 0.001 0.000 LC_5_3 0.948 0.035 0.000 0.006 0.000 LC_5_7 0.898 0.080 0.001 0.007 0.001

LC_5_15 0.848 0.128 0.001 0.011 0.000 LS_6_0 0.931 0.063 0.000 0.002 0.001 LS_6_1 0.875 0.056 0.001 0.009 0.003 LS_6_3 0.967 0.027 0.002 0.001 0.001 LS_6_7 0.947 0.043 0.000 0.002 0.002

LS_6_15 0.773 0.181 0.000 0.024 0.001

SP_7_15 0.590 0.376 0.007 0.020 0.000

Nutrients supply, physical damage or presence of Some kind of bacterial infection can also disturb pathogens needs to be explored in order to its flora, the macroalgae cultured in the farm completely understand the circumstances that under 15℃ and in laboratory conditions at 10℃, so develop certain changes or progression in the the temperature difference may also be a factor in microflora of P. yezoensis. The alterations in the bringing out the changes in its flora composition. microbial communities of the P. yezoensis before The phyla found in the farm and laboratory and after infection with Pythium porphyrae infected and uninfected (control) samples shows a transferred from farm to laboratory samples could little change, while the samples which were result in several possible ways or outcomes. The cultured in lab for a week before infection get change in microbial communities could be due the more time to use to the laboratory conditions and infection with P. porphyrae and may be due to the nutrients. Therefore, it shows more difference change in environment, nutrients available to it in from the other two samples, it can also be due to farm and laboratory samples. the difference in their structural morphology. KHAN et al.: MICROBIOTA COMPOSITION AND ALTERATIONS IN P. YEZOENSIS 563

Table.2. Different bacterial species proportions of all the samples, representing

Pseudomonas xanthomarina and Halomonas cupida as their abundant species

Samples Others Pseudomonas Halomonas Tenacibaculum Colwellia xanthomarina cupida soleae aestuarii FC_1_0 0.995 0.001 0.001 0.001 0 FC_1_1 0.995 0 0 0.002 0.001

FC_1_3 0.91 0.001 0.004 0.041 0.026 FC_1_7 0.789 0.023 0.004 0.032 0.035

FC_1_15 0.857 0.003 0.093 0.016 0.001

FS_2_0 1 0 0 0 0 FS_2_1 0.986 0.001 0.003 0.002 0.001 FS_2_3 0.987 0.001 0.004 0.002 0 FS_2_7 0.928 0.005 0.001 0.002 0.024 FS_2_15 0.904 0.042 0.006 0.004 0.006

TC_3_0 0.993 0.001 0 0.004 0

TC_3_1 0.982 0.001 0.001 0.005 0.002 TC_3_3 0.915 0.004 0.048 0.007 0.003 TC_3_7 0.886 0.002 0.031 0.043 0.001 TC_3_15 0.763 0.004 0.14 0.017 0.002

TS_4_0 0.974 0.001 0.001 0.003 0.004 TS_4_1 0.949 0.002 0.004 0.004 0.008

TS_4_3 0.92 0.004 0.008 0.009 0.01

TS_4_7 0.857 0.005 0.059 0.017 0.001

TS_4_15 0.835 0.009 0.08 0.014 0.002

LC_5_0 0.958 0.035 0 0 0 LC_5_1 0.986 0.009 0 0 0 LC_5_3 0.862 0.128 0.001 0 0 LC_5_7 0.975 0.022 0 0 0

LC_5_15 0.909 0.074 0.001 0 0 LS_6_0 0.968 0.02 0 0.001 0 LS_6_1 0.96 0.019 0.002 0.001 0 LS_6_3 0.977 0.013 0 0.001 0 LS_6_7 0.85 0.133 0.002 0.001 0 LS_6_15 0.873 0.06 0.001 0.001 0

SP_7_15 0.758 0.043 0.002 0 0

Fig.5-Abundance proportions of dominant bacterial taxa in Pyropia yezoensis from different sampling at species level Fig.6-Heat map showing of Pyropia yezoensis from different before and after infection with Pythium porphyrae spores. sampling and their abundance proportions before and after FC=Farm control, TC= Cultured in lab control, LC =Lab infection with Pythium porphyrae spores at species level. samples control, while FS=Farm infected, TS= Cultured in FC=Farm control, TC= Cultured in lab control, LC =Lab lab infected, LS =Lab sample infected, SP= Spots sample samples control, while FS=Farm infected, TS= Cultured in (infected). lab infected, LS =Lab sample infected, SP= Spots sample (infected). 564 INDIAN J. MAR. SCI., VOL. 47, NO. 03, MARCH 2018

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