appliedsciences
ArticleDistribution of Growth-Inhibiting Bacteria against the Toxic
Dinoflagellate Alexandrium catenella (Group I) in Akkeshi-Ko
Estuary and Akkeshi Bay, Hokkaido, Japan
Yuka Onishi 1, *, Akihiro Tuji 2 , Atsushi Yamaguchi 1
1

2

3

*




and Ichiro Imai 1,3

Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611,
Japan; a-yama@fish.hokudai.ac.jp (A.Y.); imai1ro@fish.hokudai.ac.jp (I.I.)
Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005,
Japan; tuji@kahaku.go.jp
Lake Biwa Museum, 1091 Oroshimo-cho, Kusatsu, Shiga 525-0001, Japan
Correspondence: y.onishi@frontier.hokudai.ac.jp

Abstract: The distribution of growth-inhibiting bacteria (GIB) against the toxic dinoflagellate Alexandrium catenella (Group I) was investigated targeting seagrass leaves and surface waters at the seagrass
bed of Akkeshi-ko Estuary and surface waters of nearshore and offshore points of Akkeshi Bay, Japan.
Weekly samplings were conducted from April to June in 2011. GIBs were detected from surface ofleaves of the seagrass Zostera marina in Akkeshi-ko Estuary (7.5 × 105 –4.7 × 106 colony-forming
units: CFU g−1 wet leaf) and seawater at the stations in Akkeshi Bay (6.7 × 100 –1.1 × 103 CFU mL−1 ).
Sequence analyses revealed that the same bacterial strains with the same 16S rRNA sequences wereisolated from the surface biofilm of Z. marina and the seawater in the Akkeshi Bay. We therefore
strongly suggested that seagrass beds are the source of algicidal and growth-inhibiting bacteria incoastal ecosystems. Cells of A. catenella were not detected from seawaters in Akkeshi-ko Estuary andthe coastal point of Akkeshi Bay, but frequently detected at the offshore point of Akkeshi Bay. It issuggested that A. catenella populations were suppressed by abundant GIBs derived from the seagrassbed, leading to the less toxin contamination of bivalves in Akkeshi-ko Estuary.

Citation: Onishi, Y.; Tuji, A.; Yamaguchi,
A.; Imai, I. Distribution of GrowthInhibiting Bacteria against the Toxic
Dinoflagellate Alexandrium catenella

Keywords: Alexandrium catenella; growth-inhibiting bacteria; Zostera marina; Akkeshi-ko Estuary;
distribution; phylogenetic analysis(Group I) in Akkeshi-Ko Estuary andAkkeshi Bay, Hokkaido, Japan. Appl.
Sci. 2021, 11, 172. https://dx.doi.org/
10.3390/app11010172
Received: 22 October 2020
Accepted: 21 December 2020
Published: 27 December 2020
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claimsin published maps and institutional
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Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This
article is an open access article distributed
under the terms and conditions of theCreative Commons Attribution (CC BY)
license (https://creativecommons.org/
licenses/by/4.0/).

1. Introduction
The toxic dinoflagellate Alexandrium catenella (Miozoa, Dinophyceae) (Group I, formerly A. tamarense and toxic [1]) is known to produce strong neurotoxins similar to pufferfish (Takifugu rubripes, etc.) and cause paralytic shellfish poisoning (PSP) of edible bivalves(Patinopecten yessoensis, Crassostrea gigas, Ruditapes philippinarum, etc.) through the foodchain in coastal seas. The danger of PSP to human health makes it a serious threat inbivalve aquaculture industries worldwide [2,3]. Occurrences of shellfish poisoning and theresultant harvesting closures have been reported in the farming of bivalves such as oysters
and scallops. Hokkaido Island provides the largest production of scallops in Japan, andthe incidents of paralytic shellfish poisonings in Hokkaido are frequent and have a long
history [4].
Akkeshi-ko Estuary (area, 32.31 km2 ) is located in the eastern part of Hokkaido,
and paralytic shellfish toxins in bivalves have only rarely been confirmed in the estuary
during the monitoring by the local government of Hokkaido Prefecture. The most notableproperty of Akkeshi-ko Estuary is the existence of a large-scale seagrass bed that covers
approximately 11–25 km2 of the area (Nakaoka, personal communication).
Bacteria that have the ability to kill microalgae have been isolated from the leaves ofseagrass (Zostera marina), and those bacteria were observed to be present in huge densities [5–10]. These prior investigations were conducted mainly targeting red tide algae, forAppl. Sci. 2021, 11, 172. https://dx.doi.org/10.3390/app11010172

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Appl. Sci. 2021, 11, 172

ample, the raphidophytes Chattonella antiqua, C. marina, C. ovata, Heterosigma akashiwo, andFibrocapsa japonica, and the dinoflagellates Karenia mikimotoi, Margalefidinium (= Cochlodinium) polykrikoides, and Heterocapsa circularisquama. These effective bacteria usually belong to α-proteobacteria, β-proteobacteria, and the phylum Bacteroides [9]. Although theexistence of growth-inhibiting bacteria against the toxic dinoflagellate Alexandrium spp. 2 of 22
has been reported [11–15], the reported bacteria in these studies [11–15] do not have strong
activities. There are only a few studies on the isolation of bacteria with strong growthinhibiting activities or on their existing densities in nature [8,10,16]. In addition, there haveexample, the raphidophytes Chattonella antiqua, C. marina, C. ovata, Heterosigma akashiwo,
been no studies on the dynamics of growth-inhibiting bacteria together with the distribuand Fibrocapsa japonica, and the dinoflagellates Karenia mikimotoi, Margalefidinium (=Cochlotion and temporal fluctuations of A. catenella. We conducted the present study to underdinium) polykrikoides, and Heterocapsa circularisquama. These effective bacteria usually belong
stand the distribution and the composition of growth-inhibiting bacteria against the toxic
to α-proteobacteria, β-proteobacteria, and the phylum Bacteroides [9]. Although the exisdinoflagellate A. catenella in Akkeshi-ko Estuary and Akkeshi Bay, eastern Hokkaido. We
tence of growth-inhibiting bacteria against the toxic dinoflagellate Alexandrium spp. has
also investigated the reasons why shellfish poisoning has rarely occurred in the Akkeshibeen reported [11–15], the reported bacteria in these studies [11–15] do not have strongko Estuary.
activities. There are only a few studies on the isolation of bacteria with strong growthinhibiting activities or on their existing densities in nature [8,10,16]. In addition, there
2. Materials and Methods
have been no studies on the dynamics of growth-inhibiting bacteria together with the
distribution and temporal fluctuations of A. catenella. We conducted the present study to
2.1. Samplingunderstand the distribution and the composition of growth-inhibiting bacteria against the
Samples
were collected
every Wednesday
morning
fromApril
21 toBay,
Juneeastern29, 2011
at
toxic dinoflagellate
A. catenellain Akkeshi-ko
Estuary
andAkkeshi
Hokkaido.
Akkeshi-ko
Estuary
andAkkeshiBayin
Hokkaido,
Japan(Figure1).
Sampling
stations
We also investigated the reasons why shellfish poisoning has rarely occurred in the Akkeshiwere set
the central part of Akkeshi-ko Estuary (Stn. 1, mean depth of 1 m, at the center
koat
Estuary.
of the seagrass field), at the inner part of the Akkeshi Bay (Stn. 2, mean depth of 5.8 m),
Methods
and at 2.
theMaterials
southernand
mouthpart of Akkeshi Bay (Stn. 3, mean depth of 23.4 m). The survey
vessels2.1.
usedwere “Etopirika” (Stn. 1) and “Misago” (Stns. 2, 3), which belong to Akkeshi
Sampling
Marine Station,
Fieldwere
Science
Centerevery
for theNorthern Biosphere,
Hokkaido
University.
Samplescollected
Wednesdaymorning from
21 Aprilto 29 June 2011 at
Water
temperatureandsalinityweremeasuredfrom thesurfaceto nearthe bottom
Akkeshi-ko
Estuary
andAkkeshiBayin Hokkaido,
Japan(Figure1). Samplingstations
using awere
CTDset(conductivity,
depth) Estuarywater quality
meter(AAQ1186s-Pro,at the centraltemperature,
part of Akkeshi-ko(Stn. 1,mean
depth of 1 m, atAlec
the center
Electronics
Tokyo,
Japan),
andinnersamplesseawaterat all staof theCo.,
seagrass
field),
at thepartofofsurface
the Akkeshi
Baywere(Stn. collected
2, mean depthof 5.8 m),
tions. The
seagrass
sampleswerecollected
at
Stn.
1.
The
collected
samplesofseawaterandand at the southern mouth part of Akkeshi Bay (Stn. 3, mean depth 23.4 m). The survey
seagrass
wereusedstoredpolypropylene
bottlessterilized(Stns.
with 2,autoclaving
and werevessels
werein“Etopirika”(Stn. 1)and “Misago”
3), which belong
to Akkeshi
broughtback tothe laboratory.
MarineStation,Field Science Center for the Northern Biosphere, Hokkaido University.

1. The locationsof thethree samplingin Akkeshi-ko
andAkkeshi Bay,
Figure Figure
1. The locationsof the threesampling
stations instations
Akkeshi-ko
Estuary andEstuary
AkkeshiBay,
Hokkaido,
Japan.
Hokkaido, Japan.

Water
2.2. Observationof temperature
Phytoplanktonand salinity were measured from the surface to near the bottom
using a CTD (conductivity, temperature, depth) water quality meter (AAQ1186s-Pro, AlecElectronics Co., Tokyo, Japan), and samples of surface seawater were collected at all
stations. The seagrass samples were collected at Stn. 1. The collected samples of seawaterand seagrass were stored in polypropylene bottles sterilized with autoclaving and werebrought back to the laboratory.
2.2. Observation of Phytoplankton
Seawater samples (250 mL) were fixed with glutaraldehyde with a final concentrationof 1% and concentrated to 50 mL by gravity precipitation. The concentrated sample (0.1 mL)
was placed on a glass slide, and phytoplankton were identified and counted under aninverted microscope (200× to 600× magnification). For the detection and counting of

Appl. Sci. 2021, 11, 172

3 of 22

thecate dinoflagellates, calcofluor staining was employed [17]. The stained sample (0.5 mL)
was taken on a glass slide, and thecate dinoflagellates were observed and counted underblue light excitation with an inverted epifluorescence microscope.
2.3. Enumeration and Cultivation of the Bacteria in the Seawater Samples
Collected seawater samples were serially diluted 10−1 to 10−3 with filtered andautoclaved seawater, and 1 mL of diluted samples was filtered using sterilized nucleopore
filters (pore size 3.0 µm). The obtained filters were placed on an ST10−1 marine agar plate(0.05 g yeast extract, 0.5 g trypticase peptone, and 15 g agar in 1 L seawater [18]) andincubated in order to culture particle-associated bacteria (PAB), i.e., the >3 µm fraction.
The filtrates of 0.1 mL were spread on ST10−1 agar medium using a sterilized conlarge bar(bacteria spreader), and these were cultured as free-living bacteria (FLB), i.e., the <3 µm
fraction.
Triplicate plates were prepared for each dilution level of the PAB and FLB samples,
and the formed colonies were counted after incubation in the dark at 25 ◦ C for 2 weeks.
The numbers of culturable bacteria were calculated from the obtained colony count values
and the dilution level. Untreated seawater samples and the FLB seawater samples at all
three stations were fixed with 25% glutaraldehyde at a final concentration of 1%, stored in
the dark at 4 ◦ C, and used for the direct count of bacteria (see Section 2.5 below).
2.4. Enumeration and Cultivation of the Bacteria on the Leaves of Seagrasses
The collected seagrass (Zostera marina) leaves were put into a sterile bottle (500 mL
volume) and sterilized filtered seawater (200 mL) was added. The bottle was then vigorously shaken 500 times by hand to detach the bacteria and biofilm from the surface ofthe leaves. The suspension seawater in the bottle was diluted 10−1 to 10−4 with sterilized
filtered seawater, and 0.1 mL of each dilution sample was spread on an ST10−1 agar plate.
Triplicate plates were prepared for each dilution level, and the colonies formed werecounted after incubation at 25 ◦ C in the dark for 2 weeks. The wet weights of the leaves
were measured to calculate the densities of bacteria per gram wet weight of Z. marina
leaves. A portion of the suspension seawater sample was placed in a centrifuge tube, fixedwith 25% glutaraldehyde at a final concentration of 1%, and stored in a dark place at 4 ◦ C.
The portion was later used for the direct counting of bacteria.
2.5. Direct Count of the Bacteria
The total bacteria were counted in the samples of untreated seawater, FLB seawater,and biofilm suspension from seagrass leaves fixed with glutaraldehyde and stored in arefrigerator. The samples were stained with DAPI at a final concentration of 1.0 µg mL−1 for
5 min and then collected on a nucleopore filter (pore size 0.2 µm). Nucleopore filters were
stained overnight in advance in 0.2% Sudan Black B solution in 50% ethanol solvent [19,20].
The filter was sealed with a cover slip using non-fluorescent immersion oil.
At least 10 microscopic fields of the total bacteria were observed and enumerated toobtain their abundance values; a Nikon ECLIPSE 80i upright epifluorescence microscope(Nikon, Tokyo, Japan) with a 100× objective and 10× eyepiece was used for this purpose.
The total number of PAB was calculated by subtracting the number of total FLB from thetotal bacteria in the untreated seawater sample. The total number of bacteria per gramattached to Z. marina leaves was calculated using the wet weight of the seagrass leaves.
2.6. Screening of the Growth-Inhibiting Bacteria against A. catenella (Group I)
An axenic strain of the toxic dinoflagellate A. catenella (Group I) isolated from Osaka
Bay (provided by Dr. Keigo Yamamoto, Research Institute of Environment, Agriculture andFisheries, Osaka Prefecture) was maintained in modified SWM-3 medium [21,22] under thefollowing conditions: temperature of 15 ◦ C, the light intensity of 50 µmol photons m−2 s−1 ,and the photo cycle of 14 h light:10 h dark. Well-grown A. catenella cultures were inoculated
into sterilized disposable 48-well microplates (with the cell density of 103 cells mL−1 ).

Appl. Sci. 2021, 11, 172

4 of 22

Bacterial colonies that formed on agar plates from the PAB and FLB fractions of seawaterand from the biofilm of seagrass leaves were randomly selected; small amounts of thecolonies were picked up using a sterilized toothpick, and they were inoculated to theA. catenella culture in each well of the microplate. The densities of bacteria added to thealgal culture by this procedure were approximately 105 –106 cells mL−1 [10,23]. As a control,
the algal culture was set up without the addition of bacteria.
The co-cultures were incubated for 2 weeks under the culture conditions of A. catenella,
prior to microscopic observations of each well to check for A. catenella mortality or morphological changes. Bacteria that killed all A. catenella cells were determined as algicidal
bacteria. The growth-inhibiting bacteria were considered the bacteria that did not entirely
kill A. catenella but the numbers of dinoflagellate cells in the wells decreased or hardly
proliferated from the beginning of the co-culture experiment. For each sample, 48 randomly
selected bacterial strains were tested, and the percentage of tested bacterial strains showing
growth-inhibiting activity was multiplied by the number of viable bacteria to calculate thedensity of the growth-inhibiting bacteria (colony forming units: CFU) against A. catenella.
2.7. 16S Ribosomal RNA Gene Sequencing of Bacteria
The isolated growth-inhibiting bacteria were grown in ST10−1 liquid medium, and bacterial cells in 200 µL culture were collected by centrifugation (2000× g for 5 min.) followedby twice washings with phosphate-buffered saline (PBS). After removing the supernatantfrom the sample, 100 µL of GITC buffer was added and the obtained suspension was storedat −80 ◦ C overnight. Bacterial DNA was extracted by heating in a 70 ◦ C water bath for
1 h. The 16S ribosomal RNA (rRNA) gene was amplified by polymerase chain reaction(PCR) using GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, USA). The
PCR reaction solution was composed of template 1.0 µL universal primers of bacteria 8F(AGAGTTTGATCCTGGCTCAG) and 0.875 µL 1492R (GGTTACCTTGTTACGACTT) or
1525R (AAAGGAGGTGATCCAGCC) [24,25]. Ampdirect Plus 12.5 µL (Shimadzu), MQ
9.625 µL, and KAPA 3G Plant DNA Polymerase (Nippon Genetics Co. Ltd., Tokyo, Japan)
0.125 µL, respectively. The initial denaturizing at 95 ◦ C for 10 min was followed by 45 cycles
at 95 ◦ C for 20 s, 53 ◦ C for 15 s, and 72 ◦ C for 45 s, and the final extension time at 72 ◦ C
was 2 min. PCR products were checked using 1% agarose gel electrophoresis. To purify
DNA template strands, PCR products were purified with AMpure XP (Beckman Coulter,
Inc., Brea, CA, USA) and sequencing was conducted using an ABI PRISM 3130xl genetic
analyzer (Applied Biosystems).
The obtained sequences were assembled using Chromas PRO (Technelysium Pty Ltd.,
Tewantin, Australia) and searched relative species employing Basic Local Alignment
Search Tool (BLAST) of NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic andmolecular evolutionary analyses for obtained sequences were conducted using MEGA
5 software [26]. Alignments were checked manually. The phylogenetic trees were drawn
by maximum-likelihood (ML) and neighbor joining (NJ), and bootstrapping values foreach tree were generated using 1000 replicates. The nucleotide sequences of 16S rDNA
for 54 isolates were deposited in the DDBJ/EMBL/GenBank databases with accession
numbers LC465489 to LC465542.
3. Results
3.1. Water Environments and Seagrass Bed
Figure 2 illustrates the changes in water temperature and salinity at the surveying
points during the survey period. The surface water temperature at Stn. 1 in Akkeshi-ko
Estuary showed the lowest value of 3.9 ◦ C on 21 April, the first day of this study, and it thengradually rose and reached the maximum of 15.7 ◦ C on 23 June (Figure 2a). The salinity
had a relatively high value 29.8 on 21 April, but the lowest value of 23.0 was recorded thenext week (Figure 2b). After that, the salinity gradually ascended and remained stable in
the range of 28.6 to 30.0 after 25 May.

face salinity showed values between 30.6 (12 May) and 32.2 (8 June, Figure 2f). At the 20
m depth of this station, the water temperature showed the lowest temperature of 1.7 °C
on 27 April and the highest at 5.9 °C on 15 June (Figure 2e). The salinity of the 20 m depthtended to be stable and was within the range from 32.3 (21 April) to 32.7 (29 June, Figure2f). The differences in water temperature between the surface layer (0 m) and the 20 m
Appl. Sci. 2021, 11, 172
depth were 2.4–7.1 °C. The differences in salinity between the surface and the bottom werevery small at Stn. 3 compared to Stn. 2.

5 of 22

Figure2. Temporalchangesof watertemperature(a,c,e)andmonitored
salinity (b,d,f)
at the three
Figure 2. Temporalchangesof watertemperature(a,c,e)and salinity(b,d,f)at themonitoredthreestationsEstuary
in Akkeshi-ko(Stn. 1)and
Akkeshistations in Akkeshi-ko(Stn. 1) Estuary
and Akkeshi
Bay
(Stn.
2 and Bay
Stn. (Stn.
3). 2 and Stn. 3).

The surface water temperature at Stn. 2 fluctuated in the range of 3.1 ◦ C (21 April) to
14.8 ◦ C (23 June, Figure 2c). The salinity varied from 27.0 on 27 April to 30.9 on 23 June
(Figure 2d). Regarding the water temperature at the 5 m depth, the lowest value (3.0 ◦ C)
was recorded on May 6 and the highest value (8.7 ◦ C) was on June 8 (Figure 2c). The
salinity at the 5 m depth fluctuated between 29.8 on 23 June and 32.4 on 30 June (Figure 2d).
The differences in water temperature between the surface layer (0 m) and the 5 m depth
were 2.7–8.6 ◦ C, and the differences in salinity were more pronounced with a maximum
difference of 8.4 (27 April, Figure 2d).
The water temperature of the surface layer (0 m) at Stn. 3 (at the mouth of Akkeshi
Bay) was in the range between 2.4 ◦ C (21 April) and 12.1 ◦ C (29 June, Figure 2e). The
surface salinity showed values between 30.6 (12 May) and 32.2 (8 June, Figure 2f). At the
20 m depth of this station, the water temperature showed the lowest temperature of 1.7 ◦ C
on 27 April and the highest at 5.9 ◦ C on 15 June (Figure 2e). The salinity of the 20 m depthtended to be stable and was within the range from 32.3 (21 April) to 32.7 (29 June, Figure 2f).
The differences in water temperature between the surface layer (0 m) and the 20 m depth
were 2.4–7.1 ◦ C. The differences in salinity between the surface and the bottom were verysmall at Stn. 3 compared to Stn. 2.
Concerning the seagrass Z. marina at Stn. 1 in Akkeshi-ko Estuary, the lengths ofthe leaves were <1 m on 21 April, and the growth tips of the leaves did not reach the sea
surface. The leaves had become longer and reached the sea surface on the last observation
day (29 June). Biofilms developed especially on the surface of the leaves that reached thesea surface, and large amounts of microorganisms including diatoms were confirmed in
the biofilms.

Concerning the seagrass Z. marina at Stn. 1 in Akkeshi-ko Estuary, the lengths of theleaves were <1 m on April 21, and the growth tips of the leaves did not reach the sea
surface. The leaves had become longer and reached the sea surface on the last observation
day (29 June). Biofilms developed especially on the surface of the leaves that reached theAppl. Sci. 2021,
11,surface,172
sea
and large amounts of microorganisms including diatoms were confirmed in
the biofilms.

6 of 223.2. Fluctuations of Phytoplankton
3.2. Fluctuations of Phytoplankton
Figure 3 illustrates the changes in the total cell counts and the taxon composition of
Figure 3 illustrates the changes in the total cell counts and the taxon composition
phytoplankton in thewater samples
Stations
1–3.samples
The totalatnumbers
phytoof surface
phytoplankton
in the atsurfacewater
Stationsof
1–3.
The total numbers of
4–2.0 × 106 cells L−1 (Figure 3a). The centric diatoms
plankton cells at Stn.
1
were
4.7
×
10
4
6
−
phytoplankton cells at Stn. 1 were 4.7 × 10 –2.0 × 10 cells L 1 (Figure 3a). The centric
Chaetoceros spp. anddiatoms
Thalassiosira
spp. (Bacillariophyta)
appeared
at high densities appearedat Stn. at high densities
Chaetoceros
spp. and Thalassiosira
spp. (Bacillariophyta)
1 during April before
thegrowthofZ.
marina,but
thesegroupsbecameminor
inMay
and became minor in
at Stn. 1 during April before the growth of Z. marina, but these groups
thereafter. The occurrencesof
Euglenoids
were
more
frequentat
Stn.
1(Figure3).
PennateMay and thereafter. The occurrences of Euglenoids were more frequent at Stn. 1 (Figure 3).
diatoms became predominantfrom thesecondweek of May,accounting
for weeka maximum
Pennate diatoms
becamepredominantfromthe secondof May, accounting for aof 99% of the phytoplankton
community
(3rd
week
of
June).
The
increaseinpennatemaximum of 99% of the phytoplankton community (3rd week ofdiaJune). The increase intoms appeared to bepennate
coincident
with appeared
the growthmarina. The
appearance
ofof
dinoflagdiatoms
toof
beZ.
coincident
withthe growth
Z. marina. The appearance
ellates was confirmed
at this station, andof themat
wereof dinoflagellates
wasmostconfirmed
thisheterotrophic
station, and Protoperidinium
most of them were heterotrophic
spp. (Miozoa, Dinophyceae).
The
toxic
dinoflagellateA.
catenella(GroupI) was not conProtoperidinium spp. (Miozoa, Dinophyceae). The
toxic dinoflagellate
A. catenella (Group I)
firmed.
was not confirmed.

Figure of
3. cellTemporalchangesof celldensities (#)
and phytoplankton
communities in composition
Figure 3. Temporal changes
densities(○) andphytoplankton
communities
in composition(histograms)
in the surface
Stn.Stn.
1 (a),
Stn. 2 (b), and Stn. 3 (c).
(histograms) in the surface
water collected
at Stn.water
1 (a),collected
Stn. 2 (b),atand
3 (c).

At Stn.
2, theoftotal
cell numbersfluctuatedof phytoplankton
within a relatively lowAt Stn. 2, the total cellnumbers
phytoplankton
within a fluctuatedrelatively low4 –5.0 × 105 cells L−1 (Figure 3b). The taxonomic composition
density
range
at
2.7
×
10
4
5
−1
density range at 2.7 × 10 –5.0 × 10 cells L (Figure 3b). The taxonomic composition varied
greatly.
The centric(13–50%)
diatoms in
predominated(13–50%)in April,and then the ratios ofgreatly. The centricvaried
diatoms
predominatedApril, and thenthe ratiosof EuEuglenoidandpennatediatomsincreased
inMay(42–97%).
The
appearanceof the genus
glenoid and pennate diatoms increased in May (42–97%). The appearance of the genus
Alexandrium
wasnotconfirmed
at
Stn.
2.
Alexandrium was not confirmed at Stn. 2.
The total numbers of phytoplankton cells at Stn. 3 fluctuated over a wide range
at 7.8 × 104 –1.6 × 106 cells L−1 (Figure 3c). The proportion of pennate diatoms and Euglenoids increased slightly from April to May and decreased thereafter. Centric diatoms
were overall predominant throughout the survey period, and most of them were Chaetoceros
spp. At this station, the most important point was the appearance of A. catenella with aconfirmed presence 7 times out of 11 samplings, and the detected maximum cell density
reached 1.0 × 103 cells L−1 (Figure 4).

The total numbers of phytoplankton cells at Stn. 3 fluctuated over a wide range at 7.8
× 104–1.6 × 106 cells L−1 (Figure 3c). The proportion of pennate diatoms and Euglenoids
increased slightly from April to May and decreased thereafter. Centric diatoms were overall predominant throughout the survey period, and most of them were Chaetoceros spp.
At this station, the most important point was the appearance of A. catenella with a conAppl. Sci. 2021, 11, 172
firmed presence 7 times out of 11 samplings, and the detected maximum cell density
reached 1.0 × 103 cells L−1 (Figure 4).

7 of 22

FigureTemporal changes
inthe
thetoxic
abundanceof cells of
the toxic dinoflagellate
Alexandrium catenella
Figure 4. Temporal changes
in the4.abundanceof cells of
dinoflagellate
Alexandrium
caten(Group
I)
in
the
surfaceseawater
at
Stn.
3.
No
cells
of
A.
catenella
weredetected
at Stns. 1 and 2.
ella (Group I) in the surface seawater at Stn. 3. No cells of A. catenella were detected at Stns. 1 and
2.

3.3. Bacteria on the Seagrass Leaves

Figure 5 presents the total numbers of bacteria and the culturable bacteria per unit wet
3.3. Bacteria on the Seagrass Leaves
weight of the seagrass leaves collected at Stn. 1 and their proportions of the total bacteria
Figure 5 presents the total numbers of bacteria and the culturable bacteria 7per unit(%). The direct counts of bacteria ranged from 4.1 × 10 to 6.0 × 108 cells g−1 wet leaf
wet weight of the seagrass leaves collected at Stn. 1 and their proportions of the total bac(Figure 5a). The lowest value was detected on 27 April, and the highest value was observed
teria (%). The direct counts of bacteria ranged from 4.1 × 107 to 6.0 × 108 cells g−1 wet leaf
on 29 June. The culturable bacteria were in the range of 9.1 × 106 to 7.5 × 107 CFU g−1 wet(Figure 5a). The lowest value was detected on 27 April, and the highest value was obleaf (Figure 5b). The ratios of the number of culturable bacteria to the total bacteria wereservedon 29 June. The culturable bacteria were in the range of 9.1 × 106 to 7.5 × 107 CFU 8 of 23
2021, 11, x FOR PEER
REVIEW
2.26–28.8% (Figure 5c), which is higher than the values obtained for the seawater samples
g−1 wet leaf (Figure 5b). The ratios of the number of culturable bacteria to the total bacteria
at Stn. 1 (described later; see Figure 6).
were 2.26–28.8% (Figure 5c), which is higher than the values obtained for the seawater
samples at Stn. 1 (described later; see Figure 6).

5. in
Bacterial
numbers
in theeelgrass
ZosteramarinasamplesfromStn. 1: the direct counts (a),Figure 5. Bacterial Figure
numbers
the eelgrass
ZosteramarinasamplesfromStn. 1:the direct
countsthe(CFU)
viable(b),counts(b), and theofto
viabledirect counts (c).
(a), the viable counts
and (CFU)
the percentagesof percentages
viable counts
directcounts
countsto(c).

3.4. Bacteria in the Seawater
Figure 6 describes the fluctuations of bacteria at Stn. 1, presenting the total number(direct count (DC)), free-living bacteria (DC of FLB, the <3 µm fraction), particle-associated bacteria (DC of PAB, the >3 µm fraction), the culturable bacteria (viable count (VC)),
and their proportions (%) to the total number. The total counts (DC) of bacteria ranged
from 5.4 × 105 to 3.0 × 106 cells mL−1 (Figure 6a). The lowest value was recorded on June 2,
and the highest value was recorded on June 8.

Appl.
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9 of 23

8 of 22

Figure 6.inBacterial
numbers
in theseawater
1 (0(a),m):thethe direct counts (a), theFigure 6. Bacterial numbers
the seawater
samplesfromStn. 1 (0samplesm): the fromdirectStn.
countscounts(CFU) (b), andthe percentagesof viablecountsviable counts (CFU)viable(b), andthe percentagesof viablecounts to direct
counts(c). to direct counts (c).

3.4. Bacteria in the Seawater
The densities of FLB were between 3.9 × 105 and 1.5 × 106 cells mL−1. The numbers of
6 describes
fluctuations
bacteria at
Stn.of1,PAB
presenting
PAB were 6.1 × 104–1.6Figure
× 106 cells
mL−1. Thethe
densitiesof FLBofexceeded
those
in nine the total number(directcount(DC)),
free-living
bacteria
(DC
of
FLB,
the
<3
µm
fraction),of the 11 surveys (the exceptions were the May 18 and June 8 surveys). Concerning theparticle-associatedbacteria(DC ofµmand
fraction),
thedensities
culturable(viablenumbers of culturable
bacteria
at PAB,
Stn. 1thefor>3
FLB
PAB, theof bacteria
FLB were
8.8 × count (VC)), andtheirproportions
(%)
to
the
total
number.
The
total
counts(DC)ofbacteria ranged from
2
4
−1
1
3
−1
10 –2.0 × 10 CFU mL and those of PAB were 4.0 × 10 –3.7 × 10 CFU mL , showing wide
5 to 3.0 × 106 cells mL−1 (Figure 6a). The lowest value was recorded on 2 June,
5.4
×10
fluctuations (Figure 6b). FLB exceeded PAB in 10 surveys (the exception was June 2). The
and thebacteria
highesttovalue
was bacteria
recordedshowedon Junerelatively
8.
proportion of culturable
the total
low values of 0.11–
Thedensities
of
FLB
were
between
3.9
×105 and 1.5 × 106 cells mL−1 . The numbers
1.93% for FLB and 0.01–1.37% for PAB4(Figure 6c).
6
−1 . The densities of FLB exceeded those of PAB inof PAB
were 6.1of×bacteria
10 –1.6 ×
mLwaters
Figure 7 shows
the densities
in 10
the cellssurfaceat Stn. 2. The total counts
nine offrom
the 11
were
the May
andJunecounts
8 surveys). Concerning−1 (Figure(DC) of bacteria ranged
3.9surveys
× 105 to(the1.9 ×exceptions
106 cells mL7a).18The
directthe numbers of culturable
bacteria
at
Stn.
1
for
FLB
and
PAB,
the
densities of FLB wereof FLB were between 3.8–8.0
× 105 cells mL−1, and those of PAB were 5.1 × 103–1.1 × 106
2 –2.0 × 104 CFU mL−1 and those of PAB were 4.0 × 101 –3.7 × 103 CFU mL−1 ,8.8
×10cells mL−1. In particular, the PAB densities were characterized by low values after June.
showing wide fluctuations (Figure 6b). FLB exceeded PAB in 10 surveys (the exception was
The density of PAB exceeded the density of FLB only once, on May 18. Concerning the2 June). The proportion of culturable bacteria to the total bacteria showed relatively low
numbers of culturable bacteria (VC), the FLB densities were 1.0 × 102–1.3 × 104 CFU mL−1
values of 0.11–1.93% for FLB and 0.01–1.37% for PAB (Figure 6c).
and the PAB densities were 2.1 × 102–6.5 × 103 CFU mL−1 (Figure 7b). The FLB fraction of
Figure 7 shows the densities of bacteria in the surface waters at Stn. 2. The total
the culturable bacteria tended to be higher in density than PAB.5The proportionof viablecounts (DC) of bacteria ranged from 3.9 × 10 to 1.9 × 106 cells mL−1 (Figure 7a). The
bacteria to the total number of bacteria was 0.03–2.18% in FLB and5 0.19–22.91%
in PAB
direct counts of FLB were between 3.8–8.0 × 10 cells mL−1 , and those of PAB were
(Figure 7c).
3
6−1
5.1 × 10 –1.1 × 10 cells mL . In particular, the PAB densities were characterized by
low values after June. The density of PAB exceeded the density of FLB only once, on
18 May. Concerning the numbers of culturable bacteria (VC), the FLB densities were
1.0 × 102 –1.3 × 104 CFU mL−1 and the PAB densities were 2.1 × 102 –6.5 × 103 CFU mL−1
(Figure 7b). The FLB fraction of the culturable bacteria tended to be higher in density than
PAB. The proportion of viable bacteria to the total number of bacteria was 0.03–2.18% in
FLB and 0.19–22.91% in PAB (Figure 7c).

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10 of 23

Figure 7. Bacterial numbers
surface seawater
samplesfromStn. 2: seawater
the direct counts(a), theFigurein7.theBacterial
numbers
in thesurfacesamplesfrom Stn. 2: the direct counts (a), theviable counts (CFU) (b), and the percentages of viable counts to direct counts (c).

viable counts (CFU) (b), and the percentages of viable counts to direct counts (c).

Figure 8 presents the surface seawater results from the offshore point of Stn. 3. The
Figure 8 presents the surface seawater results from the offshore point of Stn. 3. The total
total counts of bacteria were very stable, showing values in the narrow range of 2.3–4.1 ×countsof direct
bacteria
stable,× 10
showingvalues5 cells mL−1
105 cells mL−1 (Figure8a). The
countswereof FLBvery
were 1.8–2.9
and thatin the narrow range of 2.3–
−1 (Figure
5 cells
4.12.4×× 10
1045–2.2
cells8a). The
directcountsFLB were 1.8–2.9 × 105 cells mL−1
counts of PAB were× 10mLmL−1, respectively.
FLB
showedhigherofdensi45−ties except on Mayand
6. Regarding
the numbersof culturable
bacteria,
those
FLBmL
were 12.8
that countsof PAB were
2.4 × 10–2.2 ×10 ofcells, respectively. FLB showed higher
1–3.8 × 103 CFU mL−1. The values
× 102–5.1 × 103 CFUdensities
mL−1, andexcept
those ofon
PAB
were
8.0
×
10
6 May. Regarding the numbers of culturable bacteria, those of FLB wereat Stn. 3 were generally lower
than those at the other two stations (Figure 8b). The per2.8 × 102 –5.1 × 103 CFU mL−1 , and those of PAB were 8.0 × 101 –3.8 × 103 CFU mL−1 . The
centages of culturable bacteria to the direct counts of bacteria were 0.12–2.17% for FLB
atPAB
Stn.fractions
3 werewere
generallylower
those at(Figure
the otherand 0.13–8.35% forvalues
PAB. The
higher thanthethan
FLB fractions
8c). two stations (Figure 8b). The

l. Sci. 2021, 11, x FOR PEER REVIEW

percentages of culturable bacteria to the direct counts11ofbacteria were 0.12–2.17% for FLB
of 23
and 0.13–8.35% for PAB. The PAB fractions were higher than the FLB fractions (Figure 8c).

Figure 8. Bacterial numbers
in 8.
theBacterial
surface seawater
samplesfromStn. 3: thedirect counts(a), thefrom Stn. 3: the direct counts (a), theFigurenumbers
in thesurfaceseawatersamples
viable counts (CFU) (b), and the percentages of viable counts to direct counts (c).

viable counts (CFU) (b), and the percentages of viable counts to direct counts (c).

3.5. Bacteria that Inhibit the Growth of A. catenella (Group I)
To examine the growth-inhibiting activities against A. catenella (Group I), we carriedout co-culture experiments with an axenic A. catenella strain using the bacteria isolated
from the seawater and the surface of the seagrass leaves. No bacteria possessing algicidal

Appl. Sci. 2021, 11, 172

10 of 223.5. Bacteria that Inhibit the Growth of A. catenella (Group I)
To examine the growth-inhibiting activities against A. catenella (Group I), we carriedout co-culture experiments with an axenic A. catenella strain using the bacteria isolated
from the seawater and the surface of the seagrass leaves. No bacteria possessing algicidal
activity were detected, but growth-inhibiting bacteria were detected. A. catenella increased
the cell densities in the control wells with no addition of bacteria during the 2-week culture
period (Figure 9a,b). On the other hand, swimming cells of A. catenella were not found in
the wells showing growth-inhibiting activity by bacteria, and all cells sank to the bottom
Appl. Sci. 2021, 11, x FOR PEER REVIEW
12 ofof23
the wells (Figure 9c). In addition, most of the A. catenella cells had escaped from the theca(Figure 9d).

Figure 9. Photomicrographs showing the growth-inhibiting activities of bacteria against the toxic
Figure 9. Photomicrographs showing the growth-inhibiting activities of bacteria against the toxic
dinoflagellate Alexandrium catenella (Group I). (a) The control cells on day 0, (b) the control cells on
dinoflagellate Alexandrium catenella (Group I). (a) The control cells on day 0, (b) the control cells
day 14, (c) the cells on day 14 affected by growth-inhibiting bacteria, and (d) the cells killed byon
day 14,Scale(c) thecells
onµm,
dayexcept
14 affectedby growth-inhibiting
bacteria, and (d) the cells killed bybacteria.
bars:
100
for panel(d).
bacteria. Scale bars: 100 µm, except for panel (d).

The results in the surface water at Akkeshi Bay (Stn. 2) were 1.9 × 101–1.1 × 103 CFU
Growth-inhibiting bacteria on the leaves of Z. marina collected in Akkeshi-ko EstuarymL−1 for FLB (6 detections) and 1.7 × 101–4.3 × 102 CFU mL−1 for PAB5 (5 detections,Figurewere detected in 7 of the 11 surveys, and the densities were 7.5 × 10 –4.7 × 106 CFU g−1
10c). Offshore at Stn. 3, growth-inhibiting bacteria were detected only once on June 8 fromwet weight (Figure 10a). In seawater
samples at the same station, growth-inhibiting
the FLB with the value of 1.5 × 102 CFU mL−1 (Figure 10d). On the other hand, PAB werebacteria were detected three times for FLB and fourtimes for PAB during the entire
confirmed five times, and the densities were 1.1 × 101–6.4 × 102 CFU mL−1. Growth-inhibsurvey period (Figure 10b). The densities of FLB with growth-inhibiting activity wereitingbacteria
moremLfrequently
detectedin thefractionof0PABthan
Stn.
2 –7.9 ×were−1 , and those−1 .3 in
1.9
× 10
102 CFU
of PAB
were
6.7 × 10–6.0 ×102 FLB
CFUatmL
offshore.
The results in the surface water at Akkeshi Bay (Stn. 2) were 1.9 × 101 –1.1 × 103 CFU mL−1 for FLB (6 detections) and 1.7 × 101 –4.3 × 102 CFU mL−1 for PAB(5 detections, Figure 10c). Offshore at Stn. 3, growth-inhibiting bacteria were detected only
once on 8 June from the FLB with the value of 1.5 × 102 CFU mL−1 (Figure 10d). On the other
hand, PAB were confirmed five times, and the densities were 1.1 × 101 –6.4 × 102 CFU mL−1 .
Growth-inhibiting bacteria were more frequently detected in the fraction of PAB than FLB
at Stn. 3 in offshore.

Appl.
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11 of 22

Figure 10. Temporal changes in the numbers of growth-inhibiting bacteria against Alexandrium
Figure 10. Temporalchanges in the numbers of growth-inhibiting bacteria against Alexandrium
catenella(GroupI) detectedfrom Zostera
marina(a) seawaters
and surface
seawaters
collected
catenella (GroupI) detected
from
Zostera marina(a) andsurfacecollected
at Stn.
1 (b),at Stn. 1 (b), Stn. 2
(c),
and
Stn.
3
(d).
PAB:
particle-associatedbacteria(the>3µm
fraction:).
FLB:
free-living bacteria
Stn. 2 (c), and Stn. 3 (d). PAB: particle-associated bacteria (the >3 µm fraction: ■). FLB: free-living(theµm fraction:
bacteria (the <3µm<3fraction:□). ).

3.6. The
16SSequencerRNA GeneSequence
Analyses
3.6. The 16S rRNA
Gene
Analysesof theBacteriaof the Bacteria
The phylogenetic trees constructed by 16S rRNA gene analyses (~1300 bp) of 54 strains
The phylogenetic trees constructed by 16S rRNA gene analyses (~1300 bp) of 54
of growth-inhibiting bacteria against A catenella (Group I) are provided in Figures 11–14.
strains of growth-inhibiting bacteria against A catenella (Group I) are provided in Figures
These strains include the class α-proteobacteria (10 strains, 18.5%, Figure 14), β-proteobacteria
11–14. These strains include the class α-proteobacteria (10 strains, 18.5%, Figure 11), β(one strain, 1.9%, Figure 14), γ-proteobacteria (18 strains, 33.3%, Figure 11), the Cytophagaproteobacteria (one strain, 1.9%, Figure 11), γ-proteobacteria (18 strains, 33.3%, Figure 12),
Flavobacterium-Bacteroidetes (CFB) group (23 strains, 42.6%, Figure 12), Actinobacteria
the Cytophaga-Flavobacterium-Bacteroidetes (CFB) group (23 strains, 42.6%, Figure 13),(one strain, 1.9%), and Firmicutes (Bacillus sp.) (one strain, 1.9%, Figure 13). As the major
Actinobacteria (one strain, 1.9%), and Firmicutes (Bacillus sp.) (one strain, 1.9%, Figure 14).
groups among them, α-proteobacteria, γ-proteobacteria, and the CFB group accounted for
As the major groups among them, α-proteobacteria, γ-proteobacteria, and the CFB grouphigh proportions and were the main taxa.
accounted for high proportions and were the main taxa.
Table 1 summarizes the bacterial strains showing homology of ≥98.7% with previously
reported bacterial strains. Multiple strains of growth-inhibiting bacteria isolated from

Appl. Sci. 2021, 11, 172. Sci. 2021, 11, x FOR PEER REVIEW

12 of 22

different sources were related to the same closest bacterial relative. For example, the strains
AK1, AK2, and AK 21 were related to Zobellia laminariae with the homologies of 100%,100%, and 99.92%, respectively. Strains AK1 and AK2 were isolated from seawater at Stn.
16 of 23
3, and AK21 was obtained from the surface of a leaf of the seagrass Zostera marina collected
at Stn. 1.

Figure 11. tree
Phylogenetic
tree for γ-proteobacteria
on16S
the rRNAbasis ofgene
16S sequencesrRNA gene(~1300
sequences (~1300 bp).
Figure 12. Phylogenetic
for γ-proteobacteriaon the basis of
See thefor the explanationof the tree.
bp). See the legend
oflegend
Figure of
11Figure
for the14
explanationof the tree.

4.2. Total Bacteria and Culturable Bacteria
Total counts of approximately 106−107 cells mL−1 of bacteria exist in coastal seawater[33], and 105−106 cells mL−1 exist in open sea [34]. In the present study, the total counts ofbacteria were 5.4 × 105–3.0 × 106 cells mL−1 at Stn. 1, 3.9 × 105–1.9 × 106 cells mL−1 at Stn. 2,
and 2.3 × 105–4.1 × 105 cells mL−1 at Stn. 3 (Figures 6a, 7a, and 8a). These results are notsignificant deviations from the reported values. Comparing the direct counts of FLB andPAB at each station, the FLB tended to exceed the PAB on the whole at all three stations(Figures 6a, 7a, and 8a). It is speculated that PAB are attached to particles and utilize therelatively rich organic matter of detritus, whereas FLB uptake dissolved oligotrophic organic matter in seawater [35].
In the previous studies conducted in Akkeshi Bay, the reported densities of culturable bacteria were 102–104 CFU mL−1 [30]. In the present study, the values of culturable
bacteria were detected within that range (Figures 6b, 7b, and 8b). In Akkeshi-ko Estuary(Stn. 1), FLB tended to be more abundant than PAB. However, the densities and/or proportions (%) of PAB tended to exceed those of FLB at Stns. 2 and 3. It is considered thatheterotrophic bacteria can exist as FLB at areas with relatively higher concentrations of
dissolved organic matter such as Akkeshi-ko Estuary, and that the proportion of PAB increased as the stations went offshore with less dissolved organic matter.
A review noted that the proportions of culturable bacteria to the total counts of bacteria were 0.001−0.1% in seawater of the open ocean [36]. These proportions were reported

[39]. It[39].
is reasonable
to haveratio of22.91%of culturable
bacteria
to total
It is reasonable
to thehavehighest
the highest
ratio
of 22.91%
of culturable
bacteria
to totalcountscounts
in thein
Akkeshi
Bay
area
with
high
productivity(Figure
7c,
PAB).
The
proportions
the Akkeshi Bay area with high productivity (Figure 7c, PAB). The proportionsof culturable
bacteria
to the todirectcountscountsof bacteria
attached
to the tosurfaceof the ofseagrassof culturable
bacteria
the directof bacteria
attached
the surface
the seagrassleavesleaves
were >20%
in
six
of
the
11
surveys(Figure5c).
Similarresults(1–20%)
werereported
were >20% in six of the 11 surveys (Figure 5c). Similar results (1–20%) were reported
for11,
bacteria
in the inbiofilmon theonsurfaceof reedin lakes
OhnumaQuasi-National
for bacteria
the biofilm
the surface
ofstems
reed stems
in in
lakes
in Ohnuma
Quasi-National
Appl. Sci. 2021,
172
Park, Park,
Hokkaido,
JapanJapan[40]. It[40].
thusItseems
that many
bacteria
possess
high viability
due todue to
Hokkaido,
thus seems
that many
bacteria
possess
high viability
the organic
matterprovidedbytheleavesofseagrass.
the organic matter provided by the leaves of seagrass.

13 of 22

Figure12.
Phylogenetic
tree
forof
the
16S(~1300
rRNA
gene
sequences
FigureFigure
13. Phylogenetic
tree fortheCFBof 16SrRNA
genegroup
sequencesbp).
See
theSee the(~1300 bp). See the13. Phylogenetic
tree
forthegroup
CFB group
16SCFBrRNA
geneofsequences(~1300bp).
FigureFigure
11 legendfortheexplanation
of
the
tree.
The
outgroup
isEscherichia
coli.
Figure14
legendfortheexplanation
of
the
tree.
The
outgroup
isEscherichia
coli.
11 legend for the explanation of the tree. The outgroup is Escherichia coli.

Figure 13. Phylogenetic tree for Actinobacteria and Bacilli (Firmicutes) of 16S rRNA gene sequences
FigureFigure
14. Phylogenetic
tree fortree
Actinobacteria
and Bacilli(Firmicutes)
of 16S ofrRNAgene sequences
14. Phylogenetic
fortheActinobacteria
and Bacilli(Firmicutes)
16S
rRNA(~1300bp). See
Figure14 legend
for theexplanationofthetree. gene sequences(~1300(~1300
bp). Seethe
Figure11legendfortheexplanation
of
the
tree.
bp). See the Figure 11 legend for the explanation of the tree.

Appl. Sci. 2021, 11, 172

14 of 22

Table 1. Closest bacterial relatives of 16S rRNA gene sequences with growth inhibiting bacteria against Alexandrium catenella (Group I). Bacterial strains with homology more than 98.7%
were described. FLB: Free-living bacteria (<3 µm fractions), PAB: Particle-associated bacteria (>3 µm fractions).

StrainName

DDBJ
Accession
Number

AK1
AK2
AK8
AK9
AK10
AK11
AK12
AK13
AK16
AK18
AK19
AK20
AK21
AK24
AK25
AK27
AK28
AK31
AK32
AK34
AK36
AK37
AK41
AK42
AK45
AK47
AK48
AK50
AK51
AK52
AK53
AK54

LC465489
LC465490
LC465496
LC465497
LC465498
LC465499
LC465500
LC465501
LC465504
LC465506
LC465507
LC465508
LC465509
LC465512
LC465513
LC465515
LC465516
LC465519
LC465520
LC465522
LC465524
LC465525
LC465529
LC465530
LC465533
LC465535
LC465536
LC465538
LC465539
LC465540
LC465541
LC465542

Origin
Date
April
April
April
April
April
April
April
April
May
May
May
May
May
May
May
May
May
May
May
June
June
June
June
June
June
June
June
June
June
June
June
June

21
21
27
27
27
27
27
27
6
6
12
12
12
18
18
25
25
25
25
2
2
2
8
8
8
8
8
15
15
15
15
15

Area
2011
Akkeshi Bay
2011
Akkeshi Bay
2011 Akkeshi-ko Estuary2011 Akkeshi-ko Estuary2011 Akkeshi-ko Estuary
2011
Akkeshi Bay
2011
Akkeshi Bay
2011
Akkeshi Bay
2011 Akkeshi-ko Estuary
2011
Akkeshi Bay
2011
Akkeshi Bay
2011
Akkeshi Bay
2011 Akkeshi-ko Estuary
2011
Akkeshi Bay
2011
Akkeshi Bay
2011 Akkeshi-ko Estuary2011 Akkeshi-ko Estuary2011 Akkeshi-ko Estuary
2011
Akkeshi Bay
2011 Akkeshi-ko Estuary2011 Akkeshi-ko Estuary
2011
Akkeshi Bay
2011 Akkeshi-ko Estuary2011 Akkeshi-ko Estuary2011 Akkeshi-ko Estuary
2011
Akkeshi Bay
2011
Akkeshi Bay
2011 Akkeshi-ko Estuary2011 Akkeshi-ko Estuary2011 Akkeshi-ko Estuary2011 Akkeshi-ko Estuary2011 Akkeshi-ko Estuary

Closely Related Strain
Station FLB/PAB/Leaf
Stn. 3Stn. 3Stn. 1
Stn. 1
Stn. 1
Stn. 2
Stn. 2
Stn. 2
Stn. 1
Stn. 2
Stn. 3Stn. 2
Stn. 1
Stn. 3Stn. 3Stn. 1
Stn. 1
Stn. 1
Stn. 2
Stn. 1
Stn. 1
Stn. 3Stn. 1
Stn. 1
Stn. 1
Stn. 3Stn. 3Stn. 1
Stn. 1
Stn. 1
Stn. 1
Stn. 1

PAB
PAB
Leaf
PAB
PAB
PAB
FLB
FLB
Leaf
PAB
PAB
PAB
Leaf
PAB
PAB
Leaf
Leaf
PAB
PAB
Leaf
PAB
PAB
Leaf
Leaf
FLB
PAB
FLB
PAB
PAB
PAB
FLB
FLB

Name

DDBJ
Accession
NumberClassification

Homology

Zobellia laminariae
Zobellia laminariae
Bacteroidetes bacterium KMM 3949
Labrenzia sp. AB434f
Leifsonia sp. SS12.6
Neptuniibacter sp. CAR-SF
Kordia antarctica strain IMCC3317
Kordia sp. LNM-4
Kordia antarctica strain IMCC3317
Bacillus baekryungensis strain YD13
Cellulophaga sp. AB405d
Roseobacter sp. r22
Zobellia laminariae
Vibrio sp. D4058
Vibrio sp. D4058
Krokinobacter sp. NBRC 100818
Sulfitobacter sp. MBEF04
Marinobacter sp. 132Z-4
Sphingomonas sp. NF4-4
Alteromonas sp. KT1101
Vibrio sp. HNS024
Aquimarina sp. XH-W2
Sulfitobacter sp. MBEF05
Alteromonas sp. TO01
Sulfitobacter sp. MBEF04
Tenacibaculum sp. 35
Tenacibaculum sp. 35
Alteromonas sp. USC168
Sphingomonas sp. NF4-4
Alteromonas sp. KT1101
Alteromonas sp. W-6
Vibrio sp. HNS024

AB121975
AB121975
AY271625
FR821171
KC160899
AB086227
JX456458
AB758592
JX456458
JN210568
FR821154
JQ661116
AB121975
DQ480136
DQ480136
AB681258
AB733554
JX310148
FJ196036
AF235128
JN128258
KF017576
AB733554
AB049730
AB733554
JX454522
JX454522
HQ441215
FJ196036
AF235128
DQ923440
JN128258

Flabobacteria
Flabobacteria
Flabobacteria
α-Proteobacteria
Actinobacteria
γ-Proteobacteria
Flabobacteria
Flabobacteria
Flabobacteria
Bacilli
Flabobacteria
α-Proteobacteria
Flabobacteria
γ-Proteobacteria
γ-Proteobacteria
Flabobacteria
α-Proteobacteria
γ-Proteobacteria
α-Proteobacteria
γ-Proteobacteria
γ-Proteobacteria
Flabobacteria
α-Proteobacteria
γ-Proteobacteria
α-Proteobacteria
Flabobacteria
Flabobacteria
γ-Proteobacteria
α-Proteobacteria
γ-Proteobacteria
γ-Proteobacteria
γ-Proteobacteria

100
100
98.92
100
99.42
99.86
99.64
99.47
99.25
99.79
99.93
99.93
99.92
99.66
99.51
99.71
100
99.58
99.49
99.79
99.86
99.57
99.56
99.86
100
99.85
99.85
99.78
98.75
99.79
99.59
99.93

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15 of 22

4. Discussion
4.1. Hydrological Parameters
Akkeshi-ko Estuary is a lagoon with a maximum depth of 9 m [27]. Several rivers flow
into the estuary. The Bekanbeushi River accounts for 98.8% of the total water flow into
Akkeshi-ko Estuary [28]. The basin area of Bekanbeushi River is 447 km2 . The common
flow rate is estimated to be ~10 billion kg week−1 , and it doubles to ~20 billion kg week−1during the snow-melting season [28]. The hydrological environment of Stn. 1 located in thecentral part of Akkeshi-ko Estuary thus showed a tendency of low-salinity conditions dueto the influence of the increased water from the Bekanbeushi River during the snow-melting
season (Figure 2b).
Station 2 was located at the inner part of Akkeshi Bay, and it tended to show low
salinity (Figure 2d). The phytoplankton taxa were dominated by Euglenoids and pennate
diatoms from April to early June (Figure 3b). Pennate diatoms were reported to be more
dominant than centric diatoms in Akkeshi-ko Estuary [29]. Station 2 is located in coast of
Akkeshi Bay close to the mouth of Akkeshi-ko Estuary where the estuarine waters flow out
Appl. Sci. 2021, 11, x FOR PEER REVIEW
15 of 23and have significant impacts.
A thermocline was observed in Akkeshi Bay at a depth of ~5 m [30,31], and a similar
phenomenon was observed at Stn. 3. The seawater from the open ocean and the estuarine
are mixed
andthe influenceof oceanopen ocean
was thought
to be rather
waterwater
are mixed
at Stn.at3,Stn.
and3,theinfluenceof openwaterwater
was thought
to be rather(Figurestrongstrong(Figure2e,f). 2e,f).

14. Phylogenetictree
forβ-proteobacteria
α- and β-proteobacteria
isolates
based
thegene16S rRNA
gene
FigureFigure
11. Phylogenetic
tree for αandisolates basedon the16S onrRNAsesequences(~1300
bp).
The
tree
was
constructed
using
the
maximum
likelihood
method(ML).
quences (~1300 bp). The tree was constructed using the maximum likelihood method (ML). Numbers atNumbers
nodes indicate
bootstrap(neighbormethod(NJ)/ML).
the after theat nodesindicatevalues
bootstrap
valuesjoining(neighborjoining
method Numbers(NJ)/ML).after
Numbers
strain nametheareaccession
numbers.
PAB: particle-associatedbacteria,
FLB: free-living
bacteria,
strain are
namethe accession
numbers.
PAB: particle-associatedbacteria,
FLB: free-living
bacteria,
Leaf: isolated
from afrom
leaf ofthe seagrass
ZosteraZostera
marina.marina.
The bacterialname AKis AK
the strain
iso- isolated
Leaf: isolated
a leafof the seagrass
The bacterial
nameis the strain
lated ininthis
thisstudy.
study.The
Theoutgroupoutgroupalgicidal
algicidalbacteria
bacteriaare
arereported
reported byby previous
previous study
study [32].
[32].

Appl. Sci. 2021, 11, 172

16 of 224.2. Total Bacteria and Culturable Bacteria
Total counts of approximately 106 −107 cells mL−1 of bacteria exist in coastal seawater [33], and 105 −106 cells mL−1 exist in open sea [34]. In the present study, the total countsof bacteria were 5.4 × 105 –3.0 × 106 cells mL−1 at Stn. 1, 3.9 × 105 –1.9 × 106 cells mL−1 atStn. 2, and 2.3 × 105 –4.1 × 105 cells mL−1 at Stn. 3 (Figures 6a, 7a and 8a). These results
are not significant deviations from the reported values. Comparing the direct counts of
FLB and PAB at each station, the FLB tended to exceed the PAB on the whole at all threestations (Figures 6a, 7a and 8a). It is speculated that PAB are attached to particles and utilize
the relatively rich organic matter of detritus, whereas FLB uptake dissolved oligotrophic
organic matter in seawater [35].
In the previous studies conducted in Akkeshi Bay, the reported densities of culturable
bacteria were 102 –104 CFU mL−1 [30]. In the present study, the values of culturable bacteria
were detected within that range (Figures 6b, 7b and 8b). In Akkeshi-ko Estuary (Stn. 1),
FLB tended to be more abundant than PAB. However, the densities and/or proportions (%)
of PAB tended to exceed those of FLB at Stns. 2 and 3. It is considered that heterotrophic
bacteria can exist as FLB at areas with relatively higher concentrations of dissolved organic
matter such as Akkeshi-ko Estuary, and that the proportion of PAB increased as the stations
went offshore with less dissolved organic matter.
A review noted that the proportions of culturable bacteria to the total counts ofbacteria were 0.001−0.1% in seawater of the open ocean [36]. These proportions werereported to be higher in coastal waters [37,38]. For example, in seawater of Maizuru Bay,
Japan, the total counts of bacteria were 105 –106 cells mL−1 and culturable bacteria were
104 –105 CFU mL−1 [37]. In the surface waters along the Adriatic coast, the proportions ofculturable bacteria to the total counts of bacteria were 0.1–22% [38].
In other environments such as Lake Biwa, the culturable bacteria were 1.7 × 103 –1.0 × 105 CFU mL−1 when the total bacteria were 1.3 × 106 –1.4 × 107 cells mL−1 . In
the Yodo River, the only river flowing out from Lake Biwa, the total culturable bacteria
were 7.1 × 104 –1.4 × 106 CFU mL−1 , and the total direct counts of bacteria were 2.5 × 106 –4.0 × 107 cells mL−1 [39]. It is reasonable to have the highest ratio of 22.91% of culturable
bacteria to total counts in the Akkeshi Bay area with high productivity (Figure 7c, PAB).
The proportions of culturable bacteria to the direct counts of bacteria attached to the surfaceof the seagrass leaves were >20% in six of the 11 surveys (Figure 5c). Similar results (1–20%)
were reported for bacteria in the biofilm on the surface of reed stems in lakes in Ohnuma
Quasi-National Park, Hokkaido, Japan [40]. It thus seems that many bacteria possess highviability due to the organic matter provided by the leaves of seagrass.
In general, most of the heterotrophic bacteria in natural environments are unculturable on agar media, and this is one of the major issues that has long been unsolved in
microbiology [41]. In the present study, growth-inhibiting bacteria were cultured and isolatedusing ST10−1 agar medium, and the bacteria were limited to those that can be cultured using
this medium.
4.3. Distribution of Growth-Inhibiting Bacteria against A. catenella (Group I)
Bacteria that inhibit the growth of A. catenella (Group I) were detected from thesurface of the seagrass (Z. marina) leaves of Akkeshi-ko Estuary (Stn. 1) with a maximum density of 4.7 × 106 CFU g−1 wet leaf (Figure 10a). It is reported that growthinhibiting bacteria against A. catenella attached to seagrasses, and the maximum density
was 7.5 × 107 CFU g−1 wet leaf collected from the biofilm of Z. marina at the center ofPadilla Bay in Puget Sound, Washington, U.S.A. [10]. The density of the growth-inhibiting
bacteria was 1.8 × 108 CFU g−1 wet leaf from the surface of Z. japonica leaves at the same
location, and 6.9 × 105 CFU g−1 wet thallus from the biofilm on the surface of Ulva lactuca
collected at Cattle Point in Puget Sound. Growth-inhibiting bacteria against A. catenella
were also detected at densities of 4 × 104 –1 × 106 g−1 wet weight from the seaweeds Laminaria japonica, Corallina pilulifera, and Ulva pertusa at Shinori Coast, Hakodate, Japan [16].

Appl. Sci. 2021, 11, 172

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The existence of growth-inhibiting bacteria on seagrasses and seaweeds thus appears to becommon in coastal seas.
Herein, PAB isolated from seawater in seagrass beds (Stn. 1) were detected at the
maximum density of 6.0 × 102 CFU mL−1 and FLB were detected at the maximum densityof 7.9 × 102 CFU mL−1 as growth-inhibiting bacteria (Figure 10b). There have beenvery few investigations [10,16] of the distribution and abundance of growth-inhibiting
bacteria against A. catenella in nature. The density of 4.1 × 103 CFU mL−1 of growthinhibiting bacteria against A. catenella was obtained in the seawater of Holmes Harbor,
Puget Sound [10]. Approximately 1 × 102 –2 × 103 cells mL−1 of growth-inhibiting bacteria
against A. catenella were detected in the seawater of the seaweed bed of Shinori Coastin Hakodate, Hokkaido, Japan [16]. The densities of growth-inhibiting bacteria obtained
from Akkeshi-ko Estuary and Akkeshi Bay are slightly lower than those studied in Puget
Sound [10], but they are similar to the values reported from Shinori Coast, Japan [16]. It
should be noted that the existence of growth-inhibiting bacteria with high frequency wasconfirmed in the present study.
Our comparison of FLB and PAB as growth-inhibiting bacteria in seawater revealed
that the densities of PAB tended to exceed those of FLB at Stn. 3, located offshore(Figure 10d). PAB were defined as bacteria that attach to particles and use the particles as
a nutrient source, and they defined FLB as bacteria that use dissolved organic matter as
a nutrient source [35]. The activity of PAB in terms of thymidine incorporation rate wasrevealed to be 10–100 times higher than that of FLB [35]. It is reported that there are larger
numbers of algicidal bacteria against red tide flagellates in the fraction of PAB than that of
the FLB category in seawater samples collected at approximately 20 km off Himeji in the
Seto Inland Sea of Japan [42]. Considering the previous reports and this study’s findings,
we speculate that the greater part of growth-inhibiting bacteria are in the PAB fraction. We
also speculate that when PAB are detached from the particles they had attached themselves
to, they would become FLB and presumably reach an inactivated state within a short time.
Gene sequence analyses of 16S rRNA were carried out herein for the obtained strainsof growth-inhibiting bacteria. Some of these bacteria from seawater were found to be thesame types of bacteria isolated from the surface of the seagrass Z. marina. For example, thestrains AK28 (Stn. 1, leaf), AK41 (Stn.1, leaf), and AK45 (Stn. 1, FLB) were closely related
to Sulfitobacter sp. strain MBEF04 of the class α-proteobacteria, with homologies of 100%,
99.56%, and 100%, respectively (Figure 14). As these three strains were isolated from Stn. 1
and showed very high homologies, they are highly likely to be the same species. A similar
presumption is possible for the strains AK34 (Stn. 1, leaf, homology 99.79%), AK52 (Stn. 1,
PAB, homology 99.79%), and AK53 (Stn. 1, FLB, homology 99.59%), which all have basesequences close to that of Alteromonas sp. KT1101 belonging to the class γ-proteobacteria(Figure 11). In the CFB group, the strains AK1 (Stn. 1, leaf), AK2 (Stn. 3, PAB), and AK21
(Stn. 3, PAB) are related to Zobellia laminariae (the homologies were 100%, 100%, and 99.92%,
respectively), and the strains AK15 (Stn. 2, FLB), AK29 (Stn. 1, leaf), and AK49 (Stn. 1, PAB)
are closely related to Lewinella persicus with 100% homology for each strain (Figure 12).
As described above, several strains of growth-inhibiting bacteria showed the same
16S rRNA gene sequences, and these strains were isolated from seagrass leaves and theseawater outside Akkeshi-ko Estuary. These results strongly suggest that the seagrassarea is one of the significant habitats of growth-inhibiting bacteria against A. catenella in
the coastal area. The same situation was reported about the algicidal bacteria against
the fish-killing raphidophyte Chattonella antiqua (Ochrophyta, Raphidophyceae) from theseagrass (Z. marina) leaves and surrounding seawater in the Seto Inland Sea, Japan [43]. The
distribution of three strains of algicidal bacteria Alteromonas sp., i.e., strains S, K, and D [44],against the above mentioned raphidophyte C. antiqua was investigated in the seagrass bed
and surrounding sea area in Japan’s Seto Inland Sea, and the study revealed the abundant
existence of these algicidal bacteria in seawater from the seagrass bed compared to theseawater of the surrounding area [45]. This report suggested that the seagrass bed was amajor source of algicidal Alteromonas strains to the surrounding sea area.

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The action of algicidal bacteria on red tide plankton is roughly divided into two types,
i.e., the direct attack type (mainly the phylum Bacteroides) and the algicidal substanceproducing type [46,47]. Algicidal/growth-inhibiting substances are thought to be involvedin the algicidal/growth-inhibiting processes. As the algicidal substances, serine protease [48], prodigiosin [49], isatin [50], questiomycins [51], etc. were identified. Thesechemicals would be of the focus of interest for the investigations on understanding thebasic mechanisms of controlling HABs.
4.4. 16S rRNA Gene Sequence Analyses of Growth-Inhibiting Bacteria against A. catenella(Group I)
The taxonomic groups of A. catenella (Group I) growth-inhibiting bacteria isolated inthis study were the CFB group (42.6%), class γ-proteobacteria (33.3%), class α-proteobacteria(18.5%), class β-proteobacteria (1.9%), and Actinobacteria and Firmicutes (3.7%). Regarding the results of the 16S rRNA gene analyses of algicidal and growth-inhibiting bacteria
isolated from coastal water, most of those bacteria reportedly belonged to the class αproteobacteria, the class γ-proteobacteria, and the CFB group [9]. Total 718 heterotrophic
bacterial strains were collected from Akkeshi Bay and were identified to be Pseudomonas spp.
in the class γ-proteobacteria, Achromobacter spp. in the class β-proteobacteria, and Flavobacterium spp. in the CFB group, and the results noted that these three genera accounted for
80% of the total bacterial strains identified [30].
Concerning the class β-proteobacteria, one strain of AK23 (Stn. 2, FLB) was isolated,
and it is in a taxonomic group with many reports of its existence in freshwater. The mostclosely related strain was the β-proteobacterium strain TEG17 (97.68% homology), whichis the strain of freshwater origin. The strain AK23 of β-proteobacterium was isolated on
18 May. This date is within the period with much snowmelt water flowing into Akkeshiko Estuary through Bekanbeushi River. The presence of the strain of growth-inhibiting
bacteria of freshwater origin at the station in the north of Akkeshi Bay during the snowmelt
period suggests that there may be a supply of growth-inhibiting bacteria against microalgae
including the toxic dinoflagellate A. catenella not only from the seagrass bed but also fromthe Bekanbeushi River.
4.5. The Distribution and Density of Microalgae
Toxic dinoflagellate A. catenella (Group I) cells were frequently detected from thesurface water at Stn. 3 offshore (Figure 4) but were not confirmed at Stn. 1 or Stn. 2 during
the investigation period. The distribution of phytoplankton was previously investigated in
Akkeshi-ko Estuary [29], and it is consistent with the present findings that pennate diatoms
predominated and few centric diatoms were observed, dinoflagellates were few, and no A.
catenella cells were detected. It was reported that the cysts of A. catenella decreased from theoffshore of Akkeshi Bay toward the inner bay, and very few were at points close to the strait
in Akkeshi-ko Estuary; no cysts were detected at the other three stations in Akkeshi-ko
Estuary [52]. It is thus presumed that the survival and completion of the life cycle of A.
catenella are difficult in Akkeshi-ko Estuary after the invasion from Akkeshi Bay due to theexistence of growth-inhibiting bacteria. The absence of paralytic shellfish poisoning (PSP)in Akkeshi-ko Estuary is presumably attributable to the relatively abundant existence ofthe growth-inhibiting bacteria provided by the large-scale seagrass beds.
Pennate diatoms were dense in the seawater at Stn. 1 with the large-scale seagrassbeds. Abundant pennate diatom cells on the surface of seagrass leaves are thought todetach to float in the surrounding seawater. In the seagrass beds in Dongdae Bay, Korea, theaverage density of phytoplankton is reported to be 2.1 × 104 cells L−1 and the minimum is
6.0 × 102 cells L−1 [53]. These values appear to be very low and showed the same tendency
as the present study’s data. The culture experiments [54] were conducted to investigate theinteraction of Zostera spp. and phytoplankton, and the obtained result was a significant
suppression of the growth of the phytoplankton.
The densities of phytoplankton were reported to be low at seagrass beds and high at ano-seagrass area in southern Australia [55]. It is recently reported that the eelgrass habitat

Appl. Sci. 2021, 11, 172

19 of 22studied is consistently associated with a marked reduction in dinoflagellate abundanceboth within the beds and for ≤15 m contiguously outside the beds, relative to nearby sites
without eelgrass [56]. At an offshore area of Osaka Bay, Seto Inland Sea, the densities of thecentric diatoms in the seawater showed higher values about 10–1000 times than those in theseawater from the seagrass bed [6]. In the Akkeshi-ko Estuary, the chlorophyll a amounts
were found to be low in seawater in the Akkeshi-ko Estuary despite sufficient nutrient
conditions, due to the supply of nutrients through the inflow of Bekanbeushi River, and an
intense spring bloom was confirmed in Akkeshi Bay before the growth of Z. marina [57].
In the present study, the survey was conducted 1×/week, and it was revealed thatthe phytoplankton densities in seawater were clearly lower in the seagrass beds thanin the outer area of Akkeshi Bay. We thus propose the following hypothesis. Growthinhibiting bacteria on the surface of seagrasses in Akkeshi-ko Estuary are released into
the seawater, and these bacteria suppress the growth of phytoplankton, leading to thedecrease of plankton abundance. It is reasonable to speculate that planktonic-centric
diatoms predominated in seawater at Stn. 1 in Akkeshi-ko Estuary at the first observationin April when the seagrass had not started to grow (Figure 3). The fluctuations of bacteriathat affect the growth of diatoms should be examined in future studies.
5. Conclusions
The occurrences of harmful algal blooms spread widely due to the eutrophication incoastal areas [58,59], the introduction of causative organisms by human activities such as
ballast water and juvenile transfer [60,61], and climate change [62]. Focusing on changes in
the number of red tide occurrences and seagrass areas, it is pointed out that the increasein the red tide incidents and the decrease in seagrass fields coincided in the Seto Inland
Sea [5]. It was reported that in a coastal area of the southwestern Mediterranean, thedecline of seagrass beds and the onset of a toxic dinoflagellate A. minutum bloom began
simultaneously [63]. It can thus be presumed that the loss of seagrass beds leads to theincrease of HAB occurrences due to land reclamation in coastal areas and the devastationof the coastal environments.
Seagrass communities are sensitive to environmental changes and are highly vulnerable ecosystems [64]. The scale of seagrass beds could quickly decline due to climate change
and the artificial devastation of coastal environments [65,66]. The present findings revealed
a potential ability of seagrass beds to suppress the occurrences of toxic blooms of thedinoflagellate A. catenella (Group I). The importance of seagrass beds should be included
when promoting sound bivalve aquaculture industries. It will be of great significance torestore and/or create seagrass beds in order to maintain rich coastal environments from theviewpoint of the Satoumi concept [67], because doing so is expected to prevent not only theoccurrence of harmful red tides [9], but also toxic blooms, together with the conservationof biodiversity and productivity.
Author Contributions: Y.O. wrote the manuscript. A.T. supported the phylogenetic analysis. Y.O.,
A.Y., and I.I. conceived and designed the experiments. All authors participated in discussions andreview of the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This investigation was supported by a JSPS Research Fellowship (JP242350). The part ofthis work was supported by JSPS KAKENHI Grant Number JP20K20573.
Data Availability Statement: The molecular sequences of 16S rDNA obtained in this study areavailable in DDBJ with accession numbers LC465489 to LC465542.
Acknowledgments: We express our deep appreciation to Masahiro Nakaoka, Director of Akkeshi
Marine Station, Field Science Center for Northern Biosphere, Hokkaido University, and the engineers
Shoichi Hamano and Hidenori Katsuragawa of Akkeshi Marine Station. We are grateful to Tomonori
Isada of the station, and Natsuki Hasegawa of the Japan Fisheries Research and Education Agency
for their helpful advice on Akkeshi-ko Estuary and Akkeshi Bay. We also thank Yasuhide Nakamuraof Hokkaido University (currently Shimane University) and Yuka Mohri of the Department of Botany,

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National Museum of Nature and Science for their kind cooperation in the molecular analyses of the
16S rRNA gene sequences of the bacteria.
Conflicts of Interest: The authors declare no conflict of interest.

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