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Article

The Identification of Filamentous Cyanobacteria Isolated from Neopyropia Germplasm Bank Illustrates the Pattern of Contamination

by
Yinyin Deng
1,
Cuicui Tian
2,
Chuanming Hu
1,
Guangping Xu
1,
Lien Yang
1,
Qinqin Lu
1 and
Wei Zhou
1,2,*
1
Jiangsu Marine Fisheries Research Institute, Nantong 226007, China
2
Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing 210014, China
*
Author to whom correspondence should be addressed.
J. Mar. Sci. Eng. 2022, 10(6), 838; https://doi.org/10.3390/jmse10060838
Submission received: 31 May 2022 / Revised: 16 June 2022 / Accepted: 19 June 2022 / Published: 20 June 2022
(This article belongs to the Special Issue Algal Cultivation and Breeding)
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Abstract

:
The germplasm bank of economic algae provides biological insurance against environmental changes and pressures for the cultivation industry. However, the red algal free-living conchocelis germplasm of Neopyropia was easily contaminated with filamentous cyanobacteria, which severely affected the growth of Neopyropia germplasm. To date, what and how the filamentous cyanobacteria contaminated Neopyropia germplasm remained unknown. Here, we combined cytological observations with light and electron microscopes and molecular analysis of the 16S rRNA gene to elucidate the pattern of cyanobacteria contamination. Nine filamentous cyanobacteria samples isolated from the Neopyropia germplasm bank were selected. Integrating microscopy observations and phylogenetic analyses of 16S rRNA gene sequences, nine cyanobacteria samples were divided into three groups, including two Leptolyngbya with red pigments (YCR1 and YCR2) and one Nodosilinea with green pigments (YCG3). They had the same asexual reproduction mode, releasing hormogonia to grow new filaments. Due to the high reproductive ability, Leptolyngbya and Nodosilinea were easy to spread in the Neopyropia germplasm. Based on 16S rRNA gene high-throughput sequencing analyses, we found the thallus of Neopyropia (NP1, NP2, and NP3) and surrounding seawater (SW1, SW2, and SW3) were enriched with cyanobacteria, especially with Leptolyngbya and Nodosilinea, indicating the filamentous cyanobacteria contaminated Neopyropia germplasm came from the thallus of Neopyropia or seawater. The results provided a better understanding of the prevention and control of cyanobacteria contamination in the Neopyropia germplasm bank.

1. Introduction

Neopyropia is an economic red alga with important nutritional and functional values, which can be consumed as raw or processed food and as a source of substances beneficial to health [1]. Neopyropia is one of the most important artificially cultivated seaweeds in the world and currently has a global industry of more than 2000 billion US dollars [1]. China, Japan, and South Korea are the main countries for Neopyropia cultivation. According to the data from the Food and Agriculture Organization of the United Nations, Neopyropia production in China has increased year by year rapidly. The annual output reached 1.7 × 106 tons, and the annual output value reached 1000 billion US dollars in 2017. In China, Jiangsu Province has been the main cultivation area of N. yezoensis [2]. However, global warming in recent years has led to a continual increase in seawater temperature. Affected by this, migration to the north coasts of Shandong and Liaoning Provinces has gradually become a tread in N. yezoensis cultivation [3]. With the north migration of cultivation, Neopyropia cultivation is facing new challenges, such as suitable germplasm resources.
The seaweed aquaculture industry relies on the serial subculture of restricted germplasm banks [4]. In China, the National Porphyra Germplasm Bank (NPGB) is the first germplasm bank for Neopyropia, established in 1999. In the past ten years, the NPGB has extensively collected wild populations of bladed and filamentous Bangiales in the intertidal zone from south to north, and make the redefining the genus of Pyropia [5,6,7]. The wild populations of Neopyropia were prepared into free-living conchocelis germplasm for long-term preservation in the laboratory to maintain the species diversity. In addition, the NPGB also created several excellent new varieties with high yield and high light resistance, such as Sutong No. 1 and Sutong No. 2, which were applied to the Neopyropia aquaculture at a scale [8]. The germplasm preservation could quickly and effectively fix excellent economic traits from the cultivars, which provides an important basis for the breeding and application of the Neopyropia industry [9].
The technology of preparing Neopyropia germplasm is quite mature, from haploid thallus to diploid conchocelis through apogamy, and haploid spontaneous diploidization during apogamy was found [10,11,12]. While during the long-term preservation, the germplasm of Neopyropia is susceptible to contamination by other algae, especially cyanobacteria. Cyanobacteria have fast reproduction speed and strongly adaptive ability, mainly relying on the formation of algal colonies and the direct division of cells [13]. Due to the high reproductive ability, cyanobacteria were easy to spread during improper operations of Neopyropia germplasm preservation. Moreover, due to their high biomass accumulation capacity, cyanobacteria severely inhibited the growth and development of Neopyropia germplasm, and they were hardly separated and purified by intertwining with the free-living conchocelis germplasm. Therefore, the contamination of cyanobacteria to Neopyropia germplasm is very unfavorable to the development of the Neopyropia industry.
Cyanobacteria, a widely distributed ancient lineage of oxygenic photosynthetic prokaryotes, constitute a major microbial community in aquatic and terrestrial ecosystems [14]. Cyanobacteria were indigenous members of the epiphytic microbial community on the thallus of Neopyropia [15]. In the ultrastructure of Neopyropia, attachment of filamentous cyanobacteria was found, and they were easier to colonize the holdfast of Neopyropia [13]. In addition, the 16S rRNA gene high-throughput sequencing further indicated that the thallus of Neopyropia and surrounding seawater were predominated by cyanobacteria [16,17]. At present, a polyphasic approach combining morphological, ultrastructural, and molecular studies was the most progressive method in the modern taxonomic study of cyanobacteria [18,19]. The analysis of the 16S rRNA gene sequence was widely used for distinguishing and phylogenetically classifying cyanobacteria, particularly at the genus level [20,21]. So far, there have been no relevant studies on the genus or biological characteristics of cyanobacteria contamination while preserving Neopyropia germplasm. Lacking the basic knowledge of the filamentous cyanobacteria, effective methods for controlling cyanobacteria contamination are still not available. Therefore, in this study, the cytological observations and molecular identification of cyanobacteria isolated from the Neopyropia germplasm bank were analyzed, the reproductive process and the source of contamination were discussed. This work is important for constructing a clean Neopyropia germplasm bank without cyanobacteria contamination and offering healthy and sustainable germplasm for the Neopyropia cultivation industry.

2. Materials and Methods

2.1. The Isolation and Culture of Cyanobacteria

The cyanobacteria samples were collected from the free-living conchocelis germplasm of Neopyropia, which were preserved in NPGB of Jiangsu Marine Fisheries Research Institute (Nantong, China). The cyanobacteria were isolated under a Ni-E light microscope (Nikon, Tokyo, Japan) and cultured in sterilized seawater. The growth conditions were 20 °C with a 12 h/12 h light/dark photoperiod under a light intensity of 80 μmol m−2 s−1.

2.2. The Morphology Observation of Cyanobacteria

Light microscopic investigations of cyanobacteria were performed using Nikon Ni-E equipped with differential interference contrast. Images were taken with a Nikon DS-Ri2 digital camera. The filament diameters and cell length and width were measured with at least 30 repeats. Three groups were also observed by transmission electron microscope (TEM). Samples were fixed in 2.5% glutaraldehyde and subsequently fixed in 1% osmic acid, dehydrated in ethanol series, and embedded with a resin kit. Ultrathin sections were stained with 3% uranyl acetate and 2.7% lead citrate and observed with an HT7800 transmission electron microscope (HITACHI, Tokyo, Japan).

2.3. The 16S rRNA Gene Analysis of Cyanobacteria

The genomic DNA of cyanobacteria samples was extracted using the Hi-DNAsecure Plant Kit (Tiangen, Beijing, China) according to the manufacturer’s protocol. The amplification of 16S rRNA gene operons for cyanobacteria was performed with specific primers CYA359F and CYA781R [22,23]. A 25 μL PCR system contained 2.5 μL of 10× PCR buffer, 2 μL of 2 mM dNTP mix, 1 μL of 50 ng μL−1 DNA, 1 μL of each primer at 10 μM, 0.3 μL of PrimeStar high-fidelity DNA polymerase (TaKaRa, Kusatsu, Japan) and 17.2 μL H2O. Amplification was under the following conditions: 3-min denaturation at 95 °C, 35 cycles of 30 s denaturation at 95 °C, 30 s annealing at 45 °C, 30 s extension at 72 °C and a final extension of 10 min at 72 °C. After purification from the agarose gel with TIANquick Mini Purification Kit (Tiangen, Beijing, China), the PCR products were cloned into the pMD-19T vector (TaKaRa, Kusatsu, Japan). All amplicons were sequenced by Sangon Biotech (Shanghai, China). In addition, the sequences were deposited into the NCBI database (GenBank accession numbers: MZ508309-MZ508317).
Thirty-eight 16S rRNA gene sequences of cyanobacteria and that of agrobacterium and bacteria were obtained from GenBank. Agrobacterium and bacteria were incorporated as outgroup taxa for phylogenetic analysis. The similarity values of 16S rRNA gene were calculated using MEGA X [24]. All phylogenetic analyses were carried out using PhyloSuite v1.2.1 [25]. The nucleotide sequences of the 13 PCGs were aligned in batches by multiple alignments using the MAFFT 7 plugin [26]. Gaps and ambiguous sites were removed using the Gblocks [27]. The concatenated nucleotide sequences were tested for the best-fit model using ModelFinder [28]. According to the BIC criterion, the TIM3+F+R3 model was the best-fit substitution model for the maximum likelihood (ML) analyses. The best model was selected automatically, and the ML tree was constructed with the IQ-TREE plugin [29]. Support for nodes in the ML tree was measured with 1000 bootstrapping replicates.

2.4. The 16S rRNA Gene High-Throughput Sequencing Analyses

To study the source of cyanobacteria contamination, the 16S rRNA gene high-throughput sequencing of seawater and Neopyropia were analyzed, and the three cyanobacteria were used as positive controls (YCR1, YCR2 and YCG3). The samples of N. yezoensis and surrounding seawater were separately collected from three cultivation farms in the coastal areas of Nantong (32.5489° N 121.8422° E, 32.5422° N 121.1844° E, and 32.5289° N 121.2133° E) of Jiangsu Province, China. The seawater samples were collected far away from the cultivation rafts of Neopyropia, approximately 1 m. The samples were referred to as NP1, NP2, and NP3 for Neopyropia, and SW1, SW2, and SW3 for seawater. The seawater samples were filtered with 0.22 μm filter membranes (Sangon, China). Each sample had three repeats.
The genomic DNA of each sample was extracted using the EZNA® DNA Kit (Omega, Doraville, GA, USA). The 16S rRNA gene of cyanobacteria were amplified with primers CYA359F and CYA781R. The purified PCR products were quantified and every twenty amplicons were mixed equally. The library was constructed using the pooled DNA product, and then sequenced on an Illumina Novaseq 6000 platform (Nanjing GenePioneer, Nanjing, China). The raw reads were deposited into the NCBI database (BioProject ID: PRJNA848439). Raw reads were demultiplexed and quality-filtered using QIIME (version 1.9.1). Operational Units (OTUs) were clustered with 97% similarity cut off using vsearch v2.15.0 (https://github.com/torognes/vsearch (accessed on 28 May 2021)). A single representative sequence from each clustered OTU was aligned to the SILVA database. Due to the choice of primers targeting the 16S rRNA genes of cyanobacteria, OTUs whose taxonomic classification differed from cyanobacteria were excluded. Taxonomic classification for each OTU was determined using the default classifier UCLUST [30] within QIIME. Raw sequencing counts were used to compute relative abundance per taxonomic level. In addition, the relative abundance of the top 20 genera was performed in the vegan package in R. Alpha diversity was calculated for each sample with QIIME to reveal the Observed, Chao1, and Shannon diversity indices.

2.5. Statistical Analysis

All determinations were repeated at least three times and the data were presented as the average measurements ± SD. Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). To determine statistical significance, we employed Student’s t-test. Differences were considered significant at p < 0.05. Values marked with different letters are significantly different from each other.

3. Results

3.1. The Morphology of Cyanobacteria

The cyanobacteria samples were mainly embedded in the free-living conchocelis germplasm of Neopyropia. In the present study, nine samples of cyanobacteria were isolated. Based on the pigmentation and cell size, the samples could be divided into three phenotypes: the filaments of YCR101, YCR102, and YCR103 were purple-red with small cells (Figure 1a); the filaments of YCR201, YCR202, and YCR203 were purple-red with larger cells (Figure 1e), while the filaments of YCG301, YCG302 and YCG303 were green (Figure 1i). The color of red cyanobacterial strains was quite similar to the conchocelis of Neopyropia, especially N. yezoensis, which would hardly distinguish them with naked eyes.
The filaments of YCR101, YCR102, and YCR103 were all uniseriate, without false branching (Figure 1a). Their sheaths were quite clear and thin. The filaments were slightly motile, particularly at the 10–20 cells of the tips. In addition, the dome-shaped apical cells were easy to release hormogonia to grow new filaments (Figure 1b). They were segmented with evident constriction at the cross-walls in the filaments. Cells were barrel-shaped (2.34–2.37 μm long × 1.18–1.28 μm wide) or isodiametric (2.36–2.57 μm in diameter), and the cell size of YCR101, YCR102, and YCR103 had no significant differences (Table 1). The filaments of YCR201, YCR202, and YCR203 were quite similar to YCR101, YCR102, and YCR103, except for cell size (Figure 1e). They had much larger cell width (1.33–1.48 μm) and diameter (3.23–3.27 μm) with significant differences. The apical hormogonia were released by gliding through an opening in the sheath at the end of filaments. In addition, they reproduced with motile hormogonia (Figure 1f). The filaments of YCG301, YCG302, and YCG303 were quite different. They had green pigments with round or conical apical cells (Figure 1i). Compared to YCR101, YCR102, and YCR103, the cells of YCG301, YCG302, and YCG303 were significantly narrow, with smaller cell width (1.04–1.08 μm) and diameter (2.08–2.25 μm). Moreover, their asexual reproduction pattern was also by hormogonia (Figure 1j). According to the morphology, the nine cyanobacteria samples could divide into three different groups.
The ultrastructural features of three groups (YCR101, YCR201, and YCG301) were observed. The results showed similar features in longitudinal and cross-sections, which were all uniseriate filaments composed of long cylindrical cells with constrictions at the cross-walls. The number of parietal thylakoids varied from 3 to 4 with parallel arrangements at the inner periphery of the cells in YCR101 (Figure 1d) and YCG301 (Figure 1l) and from 7 to 8 in YCR201 (Figure 1h). The differences between the strains also included the structure of the sheath. The YCR101 and YCR201 possessed thin, compact sheaths with fibrils parallel to the filaments. While YCG301 had a bi-layered sheath, in which the internal layer was made up of fibrils running parallel to the long axis of the filament, and the external layer was formed by disordered fibrils that were sometimes grouped into bundles (Figure 1k,l). Polyphosphate bodies were present in the cytoplasm of most cells or observed occasionally.

3.2. The 16S rRNA Gene Identification of Cyanobacteria

To further identify the cyanobacteria, the 16S rRNA gene of nine samples were amplified. The amplification products of 16S rRNA gene were 424 bp in length. The sequences were deposited in GenBank, and their accession numbers are presented in Figure 2. The p-distance values calculated by MEGA X were shown in Table 2. The 16S rRNA gene sequences of YCR101, YCR102, and YCR103 were the same, with 100% similarity. The YCR201 and YCR203 were the same, and YCR202 had only two base pair differences. The YCG301, YCG302, and YCG303 also had identical 16S rRNA gene sequences. Compared to the sequences of YCR101, YCR102, and YCR103, the YCR201, YCR202 and YCR203 had 98.1–98.6% similarity (p-distance = 0.014–0.019). However, the YCG301, YCG302, and YCG303 were less compatible, with a 0.093 p-distance value. Based on the similarity values of the 16S rRNA gene, we divide nine samples into three groups, namely YCR1 (YCR101, YCR102, and YCR103), YCR2 (YCR201, YCR202, and YCR203), and YCG3 (YCG301, YCG302, and YCG303).
To accurately resolve the phylogenetic relationship of these filamentous cyanobacteria, the 16S rRNA gene sequences from different cyanobacteria were downloaded, including Aphanizomenon, Pseudanabaena, Microcystis, Prochlorothrix, Toxifilum, Leptolyngbya, and Nodosilinea. The bacterium and agrobacterium were used as outgroup taxa. An ML phylogenetic tree was constructed using the PhyloSuite package (Figure 2). In the phylogenetic tree, the YCR1 group, along with the YCR2 group, formed two different clusters, but all resolved within a fully-supported clade of Leptolyngbya with 100% bootstrap. This clade of Leptolyngbya were all marine species from different countries, including two L. ectocarpi and two similar species. There was another clade of Leptolyngbya formed by L. frigida from the Antarctic, L. crispate from the desert, and L. foveolarum and L. boryanum from freshwater. While the YCG3 group stayed a distinct clade with Nodosilinea with 100% bootstrap, among which YCG3 clustered into a well-supported subclade that was sister to an unidentified species of Nodosilinea from the rock surface of intertidal zones. The other subclade included different species from the soil, rocks, marine, hot springs, or freshwater. Therefore, the results indicated the YCR1 and YCR2 groups belong to the genus of Leptolyngbya. In addition, the YCG3 group was a member of Nodosilinea.

3.3. The Source of Cyanobacteria Contaminations

As we had known these cyanobacteria and their reproduction from Neopyropia germplasm, where did these cyanobacteria come from? During the germplasm preparing process, the cyanobacteria may come from seawater or the thallus of Neopyropia. Therefore, we checked the 16S rRNA gene high-throughput sequencing of seawater (SW1, SW2, and SW3) and the thallus of N. yezoensis (NP1, NP2, and NP3), and three cyanobacteria groups (YCR1, YCR2, and YCG3) were sequenced as positive controls.
A total of 2,315,898 effective sequences of DNA were identified after the quality control process, and 2974 OTUs were obtained (Table S1). According to the SILVA database, all OTUs classified at the order level of cyanobacteria could be assigned into eight orders. To test the community richness and diversity of seawater and Neopyropia, the randomly selected sequences were used to evaluate the alpha diversity (Observed, Chao1, and Shannon) of each dataset. The Observed and Chao1 show species richness, and Shannon reflect relative abundance. Although the means of the three indices were quite different, a much greater cyanobacteria diversity was shown in seawater (SW1, SW2, and SW3) than in N. yezoensis (NP1, NP2, and NP3) (Figure 3), and three cyanobacteria groups (YCR1, YCR2, and YCG3) had the lowest diversity. The seawater samples were less evenly distributed, with a wide distribution in all diversity indices. Therefore, the results indicated seawater had much higher cyanobacteria diversity than N. yezoensis.
The differences in cyanobacteria relative abundance between seawater and Neopyropia were further demonstrated at the genus level (Figure 4). In the top 20 genera of all samples, the compositions of cyanobacteria attached to the thallus of Neopyropia were quite different. Leptolyngbya was the dominant genus among NY1 (38.49%) and also present at a small scale in NY2 and NY3 samples (0.01 and 1.45%). The thallus of Neopyropia also had a small percentage of Nodosilinea in NY1 and NY2 samples (0.01 and 0.06%). For seawater, SW1 and SW2 samples had Nodosilinea as the dominant genus with a little Leptolyngbya (0.23 and 0.46%). While SW3 mainly had Leptolyngbya (15.67%). As the positive controls, YCR1 and YCR2 had Leptolyngbya as the dominant genus, and YCG3 had Nodosilinea as the dominant genus. In total, the 16S rRNA gene high-throughput sequencing showed that Neopyropia and seawater were enriched with different kinds of cyanobacteria, especially with Leptolyngbya or Nodosilinea as the dominant genus.

4. Discussion

The germplasm bank for economical macroalgae is always necessary, which could provide biological insurance against environmental change both locally and globally and pressures on the cultivation industry [4]. However, the macroalgae were known to contain diverse microbiomes on their surfaces, including cyanobacteria [31,32,33]. Cyanobacteria were part of the epiphytic microbiome on the thallus of Neopyropia, which had the potential to modify inorganic nutrient uptake of Neopyropia and cause disease [13,15].

4.1. Leptolyngbya and Nodosilinea Contaminated Neopyropia Germplasm

The classification of cyanobacteria based on morphological traits is difficult because many specimens are morphologically poorly distinguishable and phenotypically plastic. Therefore, the approach combining morphological, ultrastructural, and molecular studies is especially suitable for the taxonomy of filamentous cyanobacteria [18]. Together with the morphological, ultrastructural features and 16S rRNA gene analyses, our results fully supported that the filamentous cyanobacteria-contaminated Neopyropia germplasm were two Leptolyngbya with red pigments (YCR1 and YCR2) and one Nodosilinea with green pigments (YCG3). The filaments of YCR2 were quite similar to YCR1, except for larger cell width, which was likely caused by the double numbles of parietal thylakoids. The morphological features of YCR2 revealed its similarity to a previously described strain of Leptolyngbya (VRUC184), which had 6–10 parietal thylakoids with larger cell width [18]. The sequence similarity of the 16S rRNA gene between YCR1 and YCR2 was quite close (98.1–98.6%) and formed two different subclades but with low bootstrap value in the phylogenetic tree. We cannot identify if YCR1 and YCR2 are the same species of Leptolyngbya because the analyses of the 16S rRNA gene sequence were insufficient for the establishment of closely related species of cyanobacteria [20,21].
The YCG3 was quite different from YCR1 and YCR2, which had green pigments, round or conical apical cells, and a bi-layered sheath. The cytomorphology of YCG3 was quite similar to three red strains of Leptolyngbya (VRUC192, VRUC198, and VRUC135), which also had conical apical cells and a bi-layered sheath [18]. Compared to the sequences of YCR1 and YCR2, YCG3 had only 90.7–91.0% similarity. In addition, YCG3 stayed a distinct clade with Nodosilinea with well-supported bootstrap, indicating YCG3 was a member of Nodosilinea. As described previously, the genus Leptolyngbya has been reported to be polyphyletic based on genetic diversity, with exceptional morphological diversity [34]. The genus Nodosilinea has separated and diverged from the genus recently and possess the facultative ability to form nodules in the filaments under low-light condition [21].

4.2. Leptolyngbya and Nodosilinea Were from Seawater and Neopyropia

The germplasm preparing process of Neopyropia was easy by cutting thallus into tissues and placing seawater for static culture. Recent research showed that Neopyropia and the surrounding seawater in China were predominately attached by cyanobacteria [16,17]. Our 16S rRNA gene high-throughput sequencing showed that the thallus of Neopyropia (NP1, NP2, NP3) and seawater samples (SW1, SW2, SW3) were enriched with different kinds of cyanobacteria, and seawater had a much higher diversity than N. yezoensis. The relative abundance of cyanobacteria also showed that the thallus of Neopyropia and seawater were mainly attached by Leptolyngbya or Nodosilinea, which was in accord with cyanobacteria contaminated Neopyropia germplasm. The results indicated Leptolyngbya and Nodosilinea contaminated Neopyropia germplasm came from seawater or the thallus of Neopyropia.
Ecological habitat is an important criterion used to define genera of cyanobacteria. Leptolyngbya was distributed in a diverse range of ecological habitats, including freshwater, hypersaline, and marine. In the phylogenetic tree, YCR1 and YCR2 were most close to the species of L. ectocarpi. L. ectocarpi is an epiphyte of various seaweeds, epizoic on animals, mud, and walls of glass vessels with seawater widely distributed in littoral and sublittoral zones of coasts [35]. Our two groups of Leptolyngbya (YCR1 and YCR2) lived in marine or on the surface of Neopyropia, which is similar to the habitat of L. ectocarpi. The genus of Nodosilinea was distributed in marine, freshwater, and terrestrial [36]. In addition, YCG3 was clustered with an unidentified species of Nodosilinea from the rock surface of the intertidal zone, different from other identified species, including N. nodulosa from marine, N. bijugata from freshwater, and N. epilithica, N. conica, and N. chupicuarensis from subaerial.

4.3. Suggestions for Reducing Cyanobacteria Contamination in Neopyropia Germplasm

In the present study, the three groups of Leptolyngbya and Nodosilinea shared the same asexual reproduction mode, releasing hormogonia to grow new filaments. Hormogonia in the cyanobacteria are short sections of trichomes, which separate from the original trichome after fragmentation and show gliding motility, and are important for short-range relocation and symbiotic colonization of hosts [37]. The characteristics of hormogonia are quite constant in various genera of cyanobacteria [38]. Hormogonia are involved in the dispersal and survival of the species in its natural habitat [39], which makes the cyanobacteria easy to spread in the Neopyropia germplasm. Moreover, due to their high biomass accumulation capacity, cyanobacteria may compete for growth space and nutrients, leading to the inhibited growth and development of Neopyropia germplasm.
Therefore, when we prepare the germplasm, the thallus of Neopyropia should be as clean as possible. Although the method of the germplasm preparation process by NPGB was old yet traditional, we still have some suggestions for reducing cyanobacteria contamination. First, cut off the holdfast of the thallus, which is densely covered by microbial epiphytes, including filamentous cyanobacteria [13]. Second, brush the tissues gently to remove cyanobacteria from the surface. Third, wash the tissues with citric acid [40]. A regular acid wash of the Neopyropia cultivation nets was the most common treatment for diseases caused by the microbiome [1,15]. However, the acid wash is ineffective for all of them, especially cyanobacteria. In the future, further research needs to develop a comprehensive manual including proper chemical treatments to remove epiphytic cyanobacteria from Neopyropia thallus, which will add depth to cleaning the germplasm of Neopyropia.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/jmse10060838/s1; Table S1: The detailed information on 16S rRNA gene sequences for all samples.

Author Contributions

Conceptualization, Y.D. and W.Z.; methodology, L.Y. and Q.L.; validation, Y.D. and L.Y.; formal analysis, C.T.; data curation, G.X. and C.H.; writing—original draft preparation, Y.D.; writing—review and editing, W.Z.; funding acquisition, C.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by China Agriculture Research System of MOF and MARA (CARS-50), Jiangsu Agricultural Industry Technology System [grant number JATS [2021]416, JATS [2021]418, JATS [2021]419], Jiangsu Seed Industry Revitalization Project [grant number JBGS [2021]140, JBGS [2021]033] and Nantong Science and Technology Project [grant number JC2020115].

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data of 16S rRNA gene sequences presented in this study are available on NCBI (https://www.ncbi.nlm.nih.gov/ (accessed on 9 July 2021)).

Acknowledgments

The authors appreciate Weizhou Chen from Shantou University for suggestions to distinguish cyanobacteria. We are grateful to Hongchang Ding from Shanghai Ocean University and Hui Shen from Jiangsu Marine Fisheries Research Institute for their kind suggestions for this manuscript. We are also grateful to the editors of JMSE and anonymous reviewers for their feedback on this submission, which has helped improve the quality of this manuscript.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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Jmse 10 00838 g001 550
Figure 1. The morphological observations of three cyanobacteria groups with LM and TEM. (a,b) The filaments and released hormogonia of YCR101; (c,d) The longitudinal and cross-sectional views of YCR101; (e,f) The filaments and released hormogonia of YCR201; (g,h) The cross-sectional views of YCR201; (i,j) The filaments and released hormogonia of YCG301; (k,l) The longitudinal and cross-sectional views of YCG301. The arrows indicated capitate apical cells of YCG301. Th, parietal thylakoids; S, sheath; C, cross-walls; P, polyphosphate body. The bar scales were 5 μm in LM and 0.5 μm in TEM.
Figure 1. The morphological observations of three cyanobacteria groups with LM and TEM. (a,b) The filaments and released hormogonia of YCR101; (c,d) The longitudinal and cross-sectional views of YCR101; (e,f) The filaments and released hormogonia of YCR201; (g,h) The cross-sectional views of YCR201; (i,j) The filaments and released hormogonia of YCG301; (k,l) The longitudinal and cross-sectional views of YCG301. The arrows indicated capitate apical cells of YCG301. Th, parietal thylakoids; S, sheath; C, cross-walls; P, polyphosphate body. The bar scales were 5 μm in LM and 0.5 μm in TEM.
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Figure 2. The Maximum likelihood (ML) phylogenetic tree of 16S rRNA gene of cyanobacteria. Numbers at nodes represent the bootstrap percentages from 1000 replicates. Values below 75% were not shown. The accession numbers for the 16S rRNA gene obtained from GenBank were shown in nodes. The habitat of Leptolyngbya and Nodosilinea were also shown in nodes. The sequences of cyanobacteria from this study were indicated in bold.
Figure 2. The Maximum likelihood (ML) phylogenetic tree of 16S rRNA gene of cyanobacteria. Numbers at nodes represent the bootstrap percentages from 1000 replicates. Values below 75% were not shown. The accession numbers for the 16S rRNA gene obtained from GenBank were shown in nodes. The habitat of Leptolyngbya and Nodosilinea were also shown in nodes. The sequences of cyanobacteria from this study were indicated in bold.
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Figure 3. Alpha-diversity of cyanobacteria among samples from Neopyropia (NP1, NP2, NP3) and seawater (SW1, SW2, SW3). The alpha-diversity indices included Observed (A), Chao1 (B), and Shannon (C).
Figure 3. Alpha-diversity of cyanobacteria among samples from Neopyropia (NP1, NP2, NP3) and seawater (SW1, SW2, SW3). The alpha-diversity indices included Observed (A), Chao1 (B), and Shannon (C).
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Figure 4. Relative abundance of cyanobacteria with samples from Neopyropia (NP1, NP2, and NP3) and seawater (SW1, SW2, and SW3) at the genus level. The numbers right to the figure were the percentages of relative abundance of Leptolyngbya and Nodosilinea.
Figure 4. Relative abundance of cyanobacteria with samples from Neopyropia (NP1, NP2, and NP3) and seawater (SW1, SW2, and SW3) at the genus level. The numbers right to the figure were the percentages of relative abundance of Leptolyngbya and Nodosilinea.
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Table
Table 1. Morphological characteristics of cyanobacteria isolated from Neopyropia germplasm.
Table 1. Morphological characteristics of cyanobacteria isolated from Neopyropia germplasm.
StrainFilament Diameter (μm)Cell Length (μm)Cell Width (μm)Apical Cell ShapePigmentation
YCR1012.57 ± 0.22 a2.36 ± 0.48 a1.28 ± 0.04 aRoundPurple-red
YCR1022.36 ± 0.27 a2.34 ± 0.58 a1.28 ± 0.19 aRoundPurple-red
YCR1032.41 ± 0.14 a2.37 ± 0.55 a1.18 ± 0.18 aRoundPurple-red
YCR2013.27 ± 0.28 b2.44 ± 0.46 a1.38 ± 0.50 bRoundPurple-red
YCR2023.23 ± 0.39 b2.58 ± 0.46 a1.33 ± 0.18 bRoundPurple-red
YCR2033.26 ± 0.40 b2.31 ± 0.45 a1.48 ± 0.16 bRoundPurple-red
YCG3012.25 ± 0.16 c2.45 ± 0.33 a1.08 ± 0.13 cRound or conicalGreen
YCG3022.08 ± 0.21 c2.41 ± 0.29 a1.04 ± 0.14 cRound or conicalGreen
YCG3032.11 ± 0.26 c2.54 ± 0.32 a1.05 ± 0.10 cRound or conicalGreen
Data are expressed as mean ± SD (n ≥ 9). Values marked with the different letters are significantly different from each other within a column (Student’s t-test, p < 0.05).
Table
Table 2. The p-distance of 16S rRNA gene sequences for all samples.
Table 2. The p-distance of 16S rRNA gene sequences for all samples.
StrainYCR101YCR102YCR103YCR201YCR202YCR203YCR301YCR302
YCR1020
YCR10300
YCR2010.0140.0140.014
YCR2020.0190.0190.0190.005
YCR2030.0140.0140.01400.005
YCG3010.0930.0930.0930.0900.0900.090
YCG3020.0930.0930.0930.0900.0900.0900
YCG3030.0930.0930.0930.0900.0900.09000
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Deng, Y.; Tian, C.; Hu, C.; Xu, G.; Yang, L.; Lu, Q.; Zhou, W. The Identification of Filamentous Cyanobacteria Isolated from Neopyropia Germplasm Bank Illustrates the Pattern of Contamination. J. Mar. Sci. Eng. 2022, 10, 838. https://doi.org/10.3390/jmse10060838

AMA Style

Deng Y, Tian C, Hu C, Xu G, Yang L, Lu Q, Zhou W. The Identification of Filamentous Cyanobacteria Isolated from Neopyropia Germplasm Bank Illustrates the Pattern of Contamination. Journal of Marine Science and Engineering. 2022; 10(6):838. https://doi.org/10.3390/jmse10060838

Chicago/Turabian Style

Deng, Yinyin, Cuicui Tian, Chuanming Hu, Guangping Xu, Lien Yang, Qinqin Lu, and Wei Zhou. 2022. "The Identification of Filamentous Cyanobacteria Isolated from Neopyropia Germplasm Bank Illustrates the Pattern of Contamination" Journal of Marine Science and Engineering 10, no. 6: 838. https://doi.org/10.3390/jmse10060838

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