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TitleStudies on distribution and colonization of facultativemethylotrophic bacteria Methylobacterium spp. on the perillaplant( Dissertation_全文 )
Author(s) Mizuno, Masayuki
Citation 京都大学
Issue Date 2013-05-23
URL https://doi.org/10.14989/doctor.k17792
Right 許諾条件により要旨は2014-05-01に公開
Type Thesis or Dissertation
Textversion ETD
Kyoto University
Studies on distribution and colonization of facultative methylotrophic bacteria
Methylobacterium spp. on the perilla plant
Masayuki Mizuno
2013
CONTENTS
Introduction 1
Chapter I
Distribution of pink-pigmented facultative methylotrophs on vegetable leaves. 4
Chapter II
Methylobacterium spp. on red perilla leaves and seeds planted at different sites in Japan.
16
Chapter III
Dominant colonization and inheritance of Methylobacterium sp. strain OR01 on perilla
plants. 25
Conclusion 38
References 40
Acknowledgements 46
Publications 48
Award 50
1
Introduction
In 1969, Ogata et al. firstly reported on a yeast capable of utilizing
methanol, Candida boidinii.1) At that time, supplying source of methanol in
nature was not clarified yet. Nemecek-Marshall et al. revealed that most
plants emit methanol, especially during early stages of leaf expansion – it is
probably produced as a by-product of pectin metabolism during cell wall
synthesis, 3) and Fall and Benson reviewed as “Leaf methanol - the simplest
natural product from plants”.4) In 2006, methane emission from terrestrial
plants under aerobic conditions was reported by Keppler et al.5)
Accordingly, C1 compounds assimilators, methanotrophs and
methylotrophs, have been noticed as hopeful candidate to enhance
agricultural production and to prevent global warming.
In Japanese agricultural science and business, microorganisms,
principally phytopathogenic bacteria and fungi in rhizosphere, were main
targets from the viewpoint of the infectious disease prevention for crop
production. Ruinen firstly introduced concept of the phyllosphere,6) which
comprises the aerial parts of terrestrial plants, and it has been known to
provide an extensive habitat for microorganisms. Especially, leaf surface is
vast, covering surface area of approximately 109 km2 and comprising the
main interface between terrestrial biomass and the atmosphere.7)
Methylobacterium spp. has been found ubiquitously in water, soil,
and air, which are the dominant member of pink-pigmented facultative
methylotrophs (PPFMs), and utilize methanol as the sole carbon and energy
source. Methylobacterium spp. is known to synthesize plant hormones such
2
as auxins8,9,10) and cytokinins11,12), to promote plant growth,25) and to form
strawberry aroma,13) and these phenomena are recently expressed as
mutualism between plants and microorganisms.
Methylobacterium is one of the most abundant bacterial genera in the
phyllosphere, between 104 ~ 107 colony forming units (CFU) per gram
fresh weight of plant material.14) The community composition study of
phyllosphere demonstrated that the predominant species belong to α- and
γ-proteobacteria, and are dependent on plant species.15) Up to 42.8% of the
microbial community in the phyllosphere of soybean are -proteobacteria,
including Methylobacterium species as one of the main components.16)
Knief et al. reported plant species and, more strongly, location influenced
the phyllospheric Methylobacterium community composition.17) In the
review entitled “Microbial life in the phyllosphere”, Vorholt discussed that
insights into the underlying structural principles of indigenous microbial
phyllosphere populations will help us to develop a deeper understanding of
the phyllosphere microbiota and will have applications in the promotion of
plant growth and plant protection.2)
Perilla is a herb of the mint family (Labiatae) native to eastern
Asia,18) and is widely used as food in Japan and other Asian countries, and
also has medicinal value.19) There are green and red varieties, and the
former is a popular protherb, and the latter is mainly used as a coloring and
flavoring ingredient in pickles made in Japan. In the Ohara area in the
northeastern part of Kyoto city, Kyoto, Japan, the characteristic red perilla
is used extensively as one of the most important ingredients for Shiba-Zuke
(local pickles) production.
3
In the present study, I studied the distribution of PPFMs on the leaves
and seeds of various commercially important vegetables. Accordingly, I
studied special relationship between the perilla plant and PPFMs, and I
tried to grasp key factor to approach the origin of Methylobacterium spp. in
the perilla plant.
4
Chapter I
Distribution of pink-pigmented facultative methylotrophs on vegetable
leaves.
Summary
I investigated on the distribution of pink-pigmented facultative
methylotrophs (PPFMs) on the leaves of various vegetables. All kinds of
vegetable leaves tested gave pink-pigmented colonies on agar plates
containing methanol as sole carbon source. The numbers of PPFMs on the
leaves, colony-forming units (CFU) per g of fresh leaves, differed among
the plants, although they were planted and grown at the same farm.
Commercial green perilla, Perilla frutescens viridis (Makino) Makino,
gave the highest counts of PPFMs (2.4 - 4.1 x 107 CFU/g) of all the
commercial vegetable leaves tested, amounting to 15% of total microbes on
the leaves. The PPFMs isolated from seeds of two varieties of perilla, the
red and green varieties, exhibited high sequence similarity as to the 16S
rRNA gene to two different Methylobacterium species, M. fujisawaense
DSM5686T and M. radiotolerans JCM2831T respectively, suggesting that
there is specific interaction between perilla and PPFMs.
5
Introduction
The plant phyllosphere supports a large and complex microbial
community, and bacteria are considered to be the dominant microbial
inhabitants of the phyllosphere. Especially, leaves constitute a very large
microbial habitat. The terrestrial leaf surface area that might be colonized
by microbes is over 6.4 x 108 km2, supporting bacterial populations of
about 1026 cells. As an ecological niche, the plant phyllosphere supports
highly abundant Methylobacterium spp. of 104 ~ 107 colony forming units
(CFU) per leaflet.25) The bacterial genus Methylobacterium is a well-studied
example of pink-pigmented facultative methylotrophs (PPFMs) that belong
to α-proteobacteria class, and use methanol as sole carbon and energy
source. These bacteria are not considered to be passive passengers on plant
leaves, but are known to stimulate seed germination and plant
development,11,25) and to contribute towards the aroma of strawberry.13) In
this chapter, the distribution of these PPFMs on the leaves and seeds of
various commercially important vegetables was studied. In addition, I
investigated to determine whether these bacteria exhibit any specific
interaction with plants.
6
Materials and Methods
Perilla seeds
Ohara red perilla seeds that harvested in 2009 were generously given
by Doi Shibazuke Honpo, Yase Hanajiricho 41, Sakyo, Kyoto, Japan
601-1251. Green perilla seed (Product no. ATY132L15, Takii & Co., Kyoto,
Japan) was purchased at Takii Shijo shop.
Counting and isolation of PPFMs from fresh vegetable leaves and perilla
seeds
One gram of fresh leaves were homogenized with 100 ml of
ice-cooled sterilized water with Ace Homogenizer (Nihonseiki, Tokyo) at
15,000 rpm for 1 min, and the homogenates were serially diluted and plated
onto AMS (buffered ammonium salts solution20))-methanol agar medium
supplemented with 0.5% (v/v) methanol and 10 μg/ml of cycloheximide.
After 7-10 days of incubation at 28°C, pink-pigmented colonies appeared
on the plates, and the colonies were counted. The numbers of PPFMs were
expressed as CFU (colony forming units)/g of fresh weight. Single colonies
were isolated on plates without cycloheximide for subsequent use.
In the case of seeds, twenty perilla seeds were suspended in 5 ml of
10 mM PBS (phosphate bufferd saline, pH 7.4) in a test tube, and were
shaken for 2 h at 28°C. The supernatant thus obtained was streaked on an
AMS-methanol agar plate supplemented with cycloheximide. After
incubation for 5-7 days, pink colonies were selected and streaked on an
AMS-methanol agar plate for single colony isolation.
7
16S rRNA gene analysis and deposition of nucleotide sequences to DDBJ
The 16S rRNA gene from single-colony isolates were amplified with
the universal eubacterial 16S rRNA gene primers 27f and 1492r.
Sequencing was performed using an automated DNA Sequencer (model
3130; Applied Biosystems, CA) and ca. 1.5-kb sequences were determined.
The nucleotide sequences of the 16S rRNA gene were deposited in DDBJ
under accession numbers AB673234-AB673253.
Agar impression method 21)
One cm2 of disks of leaves was impressed onto AMS- methanol agar
containing cycloheximide (10 ㎍/ml) for 1 min. After removal of the disks,
the plates were incubated for 7-10 days at 28°C. The number of
pink-pigmented colonies appeared on the plates was counted and expressed
as colonies/cm2 of fresh leaves.
Vegetable samples
Most of vegetable leaves were planted and picked off at a farm
(100m2) in the suburbs of Kusatsu, Shiga, Japan. Rest of the vegetable
samples was purchased at neighboring vegetable shops and supermarkets in
Kyoto.
Counting of total microbes on the vegetable leaves
The total microbial count of the leaves was measured by the DAPI
8
(4’,6’-diamidino-2-phenylindole)-staining method. Fresh leaves (1 g) were
mixed with 25 ml of PBSE buffer (130 mM NaCl, 10 mM phosphate buffer,
1 mM EDTA, pH 7.0) in a 50ml-plastic tube, and were processed using an
ultrasonic cleaning device (UT205S, Sharp, Osaka, Japan) for 15 min. The
aqueous phase was treated with 1% (v/v) formaldehyde for 30 min. An
aliquot of the aqueous phase was filtered with a membrane filter (IsoporeTM
0.2 μm GTBP, Millipore, Billerica, MA), the microbes trapped on the filter
were stained with 20 μl of DAPI solution (1 μg/ml), and the total number
of microbes was counted under a fluorescence-inverted microscope (IX70;
Olympus, Tokyo).
9
Results and Discussion
Distribution of PPFMs on vegetable leaves
To study the distribution of PPFMs on vegetable leaves, freshly
sampled leaves of vegetables (listed in Table 1-1) planted at a farm (100
m2) in the suburbs of Kusatsu, Shiga, Japan, were used. The result is
summarized in Table 1-1.
All kinds of vegetable leaves tested exhibited pink-pigmented
colonies on agar plates containing methanol as sole carbon source, but the
CFU values and Methylobacterium species identified differed among the
host plants.
Table 1-1. Distribution of PPFMs on vegetable leaves of various kinds
planted at the same farm
Number of PPFMs
Vegetables (CFU/g fresh weight) Species
Green perilla (Perilla frutescens viridis (1.3±0.47) x 107 M. fujisawaense
(Makino) Makino) M. populi
Small green pepper (Capsicum annuum) (1.3±0.65) x 106 M. aquaticum
Pumpkin (Cucurbita moschata) (1.3±0.32) x 106 M. persicinum
M. extorquens
Bitter melon (Momordica charantia) (8.2±0.22) x 105 M. adhaesivum
Okra (Abelmoschus esculentus) (6.3±0.21) x 105 M. extorquens
Tomato (Solanum lycopersicum) (3.5±0.21) x 105 M. fujisawaense
10
Survey of PPFM profiles on vegetable leaves
Next, the agar impression method21) was employed to obtain total
profiles of the PPFMs on various vegetable leaves. Potherb mustard,
broccoli, crown daisy, rucola, turnip, quing geng cai, Italian parsley and
spinach were Kusatsu farm products, and Japanese radish, Chinese cabbage,
basil, leaf lettuce, komatsu-na and green perilla were purchased from
vegetable shops in Kyoto. The results are shown in Figure 1-1. All kinds of
leaves tested, potherb mustard (Brassica rapa L. var. nipposinica), broccoli
(Brassica oleracea var. italica), crown daisy (Glebionis coronaria), rucola
(Eruca vesicaria), turnip (Brassica rapa L. var. glabra), qing geng cai
(Brassica rapa var. chinensis), Italian parsley (Petroselinum neapolitanum),
spinach (Spnacia oleracea), Japanese radish (Raphanus sativus), Chinese
cabbage (Brassica rapa L. var. glabra Regel), basil (Ocimum basilicum),
leaf lettuce (Lactuca sativa L. var. crispa), komatsu-na (Brassica rapa var.
pervirides), and green perilla (Perilla frutescens viridis (Makino) Makino),
exhibited pink-pigmented colonies on the methanol plates. Corpe and
Rheem reported that epiphytic bacteria are most abundant near the margins
of the abaxial surface of leaves.22) However, in the present study, there was
little difference in PPFM count values between the adaxial and abaxial
sides of the leaves tested.
11
Figure 1-1. PPFM counts for vegetable leaves using the agar impression
method.
One cm2 of disks of leaves was impressed onto AMS-methanol agar
containing cycloheximide. After 5-7 days of incubation at 28℃, pink-pigmented
colonies were counted. Hollow bars, PPFM counts on the adaxial side of the
leaves; solid bars, PPFM counts on the abaxial ones. Error bars show standard
deviations for five replicated measurements.Japanese radish, Chinese cabbage,
basil, leaf lettuce, komatsu-na and green perilla were purchased from local
supermarkets. Other vegetables were planted at the farm described in Table 1.
Among the tested vegetables, Japanese radish, komatsu-na, and green
perilla gave large numbers of PPFMs (> 50 colonies/cm2) on the plates.
The total microbial counts and PPFMs of these three vegetable leaves were
counted using the homogenization method, as described above. The total
microbial cell numbers of the leaves was measured by the DAPI method
>50
12
(Table 1-2).
The highest count of PPFMs was obtained for green perilla leaves
(5.0 x 107 CFU/g of fresh leaves), and the ratio of PPFMs to the total
microbial count (3.3 x 108 cells/g of fresh leaves) was 15%. The counts of
PPFMs in Japanese radish and komatsu-na, however, were negligible
compared to the total microbial counts. Trials to reproduce the high PPFM
values of the leaves of Japanese radish and komatsu-na were unsuccessful.
The reason for this discrepancy is unclear, but might come from several
factors, e.g., planting location and conditions, growth stage, soil, and
surrounding atmosphere.
To determine whether the distribution of PPFMs was dependent on
the geographic location of the perilla plants, the PPFMs from commercial
green perilla leaves planted at different places were analyzed (Table 1-3).
Five samples planted in three prefectures in Japan were purchased from
neighborhood greengrocers and supermarkets, and the PPFMs on their
leaves were evaluated.
Table 1-2. Total microbial counts and PPFMs of vegetable leaves
PPFM Total microbial cells PPFM
(Homogenization) (DAPI stain) (%)
Green perilla 5.0 x 107 3.3 x 108 15.0
Komatsu-na 1.0 x 106 9.7 x 107 1.0
Japanese radish 1.0 x 105 1.1 x 108 0.091
unit:CFU or cells/g fresh leaves
13
Table 3. PPFMs of green perilla leaves planted in different prefectures
Samples PPFMs
(Prefecture of planted place) (CFU/g fresh leaves)
Sample A (Aichi) 4.1 x 107
Sample B (Oita) 2.0 x 107
Sample C (Kochi) 2.0 x 107
Sample D (Oita) 2.1 x 107
Sample E (Aichi) 3.9 x 107
Regardless of geographic location, all of the tested leaves exhibited
high PPFM counts (2.0 - 4.1 x 107 CFU/g of fresh leaves), and the number
of PPFMs was independent of planting site. PPFMs were also detected in
red perilla (Perilla frutescens crispa (Thunb.) Makino) leaves (105-7 CFU/g
fresh leaves).
PPFMs of perilla seeds
Although wide distribution and isolation of PPFMs in the
phyllosphere are widely known, PPFMs of perilla have not been reported
previously. The PPFM counts of perilla were higher than those of other
taxonomically closely related species such as Plectranthus, belonging to
the same family Lamiaceae (Labiatae). The PPFM count of 3.6-7.5
CFU/cm2 was detected for two Plectranthus species.23) In comparison, the
PPFM level in the leaves of perilla was rather high (> 50 CFU/cm2).
I found that the red and green perilla leaves harbored high
populations of PPFMs, and investigated to determine whether a specific
14
interaction exists between PPFMs and the two varieties of perilla. I looked
into the relationship between the two in terms of PPFM occurrence by 16S
rRNA gene sequencing of PPFMs isolated from red perilla seeds and green
perilla seeds. Twelve strains (OR01 to OR12) were isolated from the red
perilla seeds and eight strains (TG01 to TGo8) were isolated from green
perilla seeds, and strains OR01 and TG01 were selected as representatives
of the isolates. Among the 12 PPFM isolates (OR01 to OR12) from red
perilla seeds, the 16S rRNA gene sequence of strain OR09 differed from
that of the other 11 strains by one base at position 93. These 11 strains had
entirely identical 16S rRNA gene sequences and were found to exhibit
highest homology to closest relative Methylobacterium fujisawaense
DSM5686T, with one base difference at position 1176. On the other hand,
all eight PPFM isolates (TG01 to TG08) from the green perilla seeds had
entirely identical 16S rRNA gene sequences, and showed highest sequence
homology to Methylobacterium species, M. radiotolerans JCM2831T, with
one base difference at position 662. Thus the PPFMs from seeds of the red
and green perilla gave different profiles of closest relatives. The 16S rRNA
gene sequence similarity between M. fujisawaense DSM5686T and M.
radiotolerans JCM2831T was 99.3%.
Many studies have investigated the origins of phyllospheric
PPFMs,but still it is under debate. Corpe argues that the paucity of PPFMs
in the air makes it unlikely that the atmosphere is a major contributor of the
methylotrophs encountered on the leaves.21) Holland and Polacco suggested
that leaf-inhabiting PPFMs are probably descendants of seed-borne bacteria
rather than bacteria from the air, soil, or other plants.24) Madahaiyan et al.
15
have reported that PPFMs are transmitted mostly through seeds.25) On the
other hand, Romanovskaya et al. reported that leaves were not colonized
after seed bacteriazation or soil application of a PPFM strain, and were
colonized only after direct application to the phyllosphere, suggesting that
natural leaf colonization occurred via transfer of soil particles.26) There are
various reports on the colonization of PPFMs as well. Omer et al. have
reported that PPFMs isolated from red clover readily colonized in winter
wheat leaves and vice versa in greenhouse experiments, and that the tested
isolates had good potential to colonize the rhizosphere, especially after seed
inoculation.27) According to Knief et al., factors specific to the sites from
which the plant species were collected, more than the plant species
themselves, have a strong influence on the composition of the
phyllospheric Methylobacterium community.17) I found that PPFMs were
highly abundant on green perilla leaves, regardless of the geographic site
from which they were collected.28)
In this chapter, I found that red and green perilla harbored a dominant
population of PPFMs on their leaves and seeds, and that the closest
relatives of PPFMs isolated from red and green perilla seeds differed from
each other in terms of 16S rRNA gene sequence showing similarities to two
different Methylobacterium species. This strongly indicates specific
interaction between perilla and PPFMs. Further investigation focusing on
the origin and inheritance of the PPFMs on perilla seeds will be described
in Chapter II and Chapter III.
16
Chapter II
Methylobacterium spp. on red perilla leaves and seeds planted at
different sites in Japan
Summary
Red perilla seeds harvested at Ohara, Kyoto, in 2009, were planted at
geographically 4 different sites (Yamagata, Shizuoka, Mie, and Kyoto
Prefectures) in 2010 and 2011. All of the 16S rRNA sequences of PPFMs
isolated from leaves and seeds planted at Yamagata, Shizuoka, and Kyoto,
and seeds of Mie coincided with that of Methylobacterium sp. OR01.
Exceptionally, the 16S rRNA sequences of PPFMs isolated from leaves
planted in Mie showed the highest sequence similarity to that of M.
radiotolerans JCM2831T. To test the reproducibility of the results, the
seeds harvested at Mie in 2010 were planted again at Mie in 2011. The
closest relatives of PPFMs isolated from leaves and seeds harvested at Mie
in 2011 were coincided with that of strain OR01. Although the reason for
this discrepancy in the closest relatives of isolates from leaves harvested at
Mie between 2010 and 2011 was not clarified yet, PPFM which had the
same 16S rRNA gene sequence as that of Methylobacterium sp. OR01 was
dominant in every PPFM communities of the red perilla plant tested.
PPFMs isolated from red perilla seeds harvested at Ohara in 2010 and 2011
showed the highest sequence similarity to that of Methylobacterium sp.
17
OR01. From these results, I strongly suggest special relationship between
perilla plant and Methylobacterium spp., and its independency on
geographic factor.
18
Introduction
Methylobacterium is one of the most abundant bacterial genera in
phyllosphere, exhibiting 104~107 colony forming units (CFU) per gram
fresh weight of plant material,14) and the bacterial genus Methylobacterium
is a well-studied example of pink-pigmented facultative methylotrophs
(PPFMs) that belong to the α-proteobacteria class and use methanol as the
sole carbon and energy source. Many strains of Methylobacterium genus
are known to promote plant growth by synthesizing plant hormones such as
auxins8-10) and cytokinins,11-12) and through the activity of
1-aminocyclopropane-1-carboxylate deaminase, which lowers ethylene
levels in plants.29,30) Their additional activities are considered to be
involved in nutrient acquisition for plants, and thus the genus is believed to
be one of the major bacteria affecting plant growth.25,29,31,32) The bacteria
also contributes towards the aroma formation of strawberry.13)
In chapter I, I studied on distribution of PPFMs on the leaves of
various vegetables and perilla seeds. I found that leaves of commercial
green perilla (Perilla frutescens viridis (Makino) Makino) gave the highest
number of PPFMs (2.0-4.1 x 107 CFU/g) among the commercial vegetable
leaves tested. PPFMs were also detected in red perilla (Perilla frutescens
crispa (Thunb.) Makino) leaves (105-7 CFU/g fresh leaves). The PPFMs
isolated from seeds of two varieties of perilla, the red and green varieties,
exhibited high sequence similarity of 16S rRNA gene to two different
Methylobacterium species, M. fujisawaense DSM5686T and M.
radiotolerans JCM2831T, respectively. Perilla is a herb of the mint family
19
(Labiatae) native to eastern Asia, and red perilla is widely used as a
coloring and flavoring ingredient in pickles production in Japan. In Ohara
area, the north east part of Kyoto City, Japan, the red perilla leaves are used
extensively as one of the most important ingredients for Shibazuke
(Japanese traditional pickles) production. The origin of PPFMs on
individual plants is still debatable. 17,21,24,25,26,27,33,34,35,36)
In this study, I planted the red perilla seeds, which had been
harvested at Ohara in 2009, at geographically 4 different sites (Yamagata,
Shizuoka, Mie, and Kyoto Prefectures) in 2010 and 2011, and identified
PPFMs isolated from fresh leaves and seeds in order to elucidate the origin
of PPFMs on red perilla seeds.
20
Materials and Methods
Perilla seeds
Red perilla seeds harvested from the Ohara area, Kyoto City, Japan
in 2009, 2010, and 2011 were generous gift from Mr. Hitoshi Yoshimura of
Doi Shibazuke Honpo Co., Ltd., Kyoto, Japan.
Planting of red perilla plant and sample collection
The red perilla seeds (harvested at Ohara in 2009) were planted at
Yamagata Prefecture (Sagae City), Shizuoka Prefecture (Iwata City), Mie
Prefecture (Yokkaichi City), and Kyoto Prefecture (Kyoto City, Kyoto
University, different from the Ohara district) in 2010. Planting sites and a
series of procedures are shown in Figure 1. In all cases, commercial soil
and plastic planters were used for sowing perilla seeds, and no fertilizer and
no insecticide were used during the course of the investigation. The seeds
were sowed in April to May of 2010 at Yamagata, Shizuoka, Mie, and
Kyoto, and leaf samples of plant were removed in the summer. Sampling
timing was different at the different planting sites. The seeds harvested at
Mie in 2010 were sowed and grown at Mie in 2011. Leaf samples were
packed in polyethylene bag and were mailed to Kyoto University in ice,
within 24 h after collection. After the mature plants withered, seed samples
were collected, and mailed to Kyoto University.
21
Figure 1. Experimental strategy used to evaluate the geographic
effect on PPFMs colonized on red perilla plants.
Red perilla seeds harvested from the Ohara area of Kyoto, Japan in 2009
were planted in 2010 at four sites: Yamagata, Shizuoka, Mie, and Kyoto,
Japan. Samples of leaves and seeds collected at planting sites were mailed to
Kyoto University for analysis. Isolation and phylogenetic analysis of PPFMs
were carried out at Kyoto University.
PPFMs on the surface of red perilla seeds
Twenty red perilla seeds were suspended in 5 ml of 10 mM PBS
(phosphate buffered saline, pH 7.4) in a test tube, and shaken for 2 h at
28°C. The supernatant was serially diluted and plated onto AMS (buffered
ammonium salts solution)-methanol agar medium {(NH4)2HPO4 0.03 g,
KCl 0.01 g, Yeast Extract 0.005 g, MgSO4・7H2O 0.01 g, vitamin solution 1
ml, mineral solution 1 ml, methanol (after autoclave) 0.5 ml per 100 ml}.
The vitamin solution consists of panthotenate calcium 0.4 g, inositol 0.2 g,
22
niacin 0.4 g, p-aminobenzoic acid 0.2 g, pyridoxine hydrochloride 0.4 g,
thiamine hydrochloride 0.4 g, biotin 0.2 g, and vitamin B12 0.2 g per liter.
The mineral solution consists of CoCl2・6H2O 1.9 g, MnCl2・6H2O 1.0 g,
ZnCl2 0.7 g, H3BO3 0.06 g, Na2MoO4・2H2O 0.36 g, NiCl2・6H2O 0.24 g,
and CuCl2・2H2O 0.02 g per liter. After incubation for 5-7 days at 28°C, the
pink colonies that appeared were counted, and a part of them were isolated
in single colony for 16S rRNA sequence analysis.
23
Results and Discussion
PPFMs isolated from red perilla leaves and seeds planted at different sites
in Japan
According to Knief et al., site-specific factors had a stronger impact
on the Methylobacterium community composition than plant-specific
factors and the Methylobacterium-plant association is not highly specific to
host plant species.17) To compare the geographic effect on
Methylobacterium community composition of the plant leaves and the host-
plant species specificity, red perilla seeds harvested from the Ohara area in
2009 were grown at four geographically different sites in Japan, Yamagata,
Shizuoka, Mie, and Kyoto Prefectures, in 2010. Closest relatives of the
isolated PPFMs from red perilla leaves and seeds harvested at different
sites of Japan were analyzed using 16S rRNA sequencing analysis.
PPFMs were isolated from leaves and seeds harvested from all
locations. Eight randomly chosen strains from each sample were used for
16S rRNA sequencing analysis. All but one of the 16S rRNA sequences of
PPFMs isolated from leaves and seeds planted at Yamagata, Shizuoka, and
Kyoto, and seeds of Mie were identical with that of Methylobacterium sp.
OR01, which is a representative of PPFMs isolated from red perilla seeds
harvested from the Ohara area in 2009. One isolate from the leaves of a
plant collected in Mie (Methylobacterium sp. ML01 as a representative)
had the highest 16S rRNA sequence identity (99.86%) to that of M.
radiotolerans JCM2831T. To test the reproducibility of this finding, the
seeds harvested at Mie in 2010 were planted again at Mie in 2011, and all
24
isolates from leaves and seeds were identical in 16S rRNA sequence to that
of strain OR01. Although the reason for this discrepancy in the closest
relatives of isolates from leaves harvested at Mie in 2010 and 2011 was not
clarified yet, PPFMs that had the same 16S rRNA gene sequence as that of
Methylobacterium sp. OR01 were dominant in all PPFM communities of
the red perilla plant tested. From these results, I suggested specific
PPFM-perilla plant association and geographic independency of PPFM
communities on red perilla plants.
My findings agree with views that leaf-inhabiting PPFMs are
probably the descendants of seed-borne bacteria rather than bacteria from
the air, soil, or water, or derived from other plants.24) In contrast, my result
is not necessarily in agreement with view that site-specific factors have a
stronger impact on the Methylobacterium community composition than
plant-specific factors and that the Methylobacterium-plant association is
not highly host-plant species specific.17)
25
Chapter III
Dominant colonization and inheritance of Methylobacterium sp. strain
OR01 on perilla plants
Summary
In chapters I and II, I suggested specific association between PPFMs
and the perilla plant, and this was not dependent on geographic factor. In
this chapter, I tried to gather information on dominant colonization of
Methylobacterium sp. strain OR01 on perilla plant. In comparison of
susceptibility of PPFMs against perillaldehyde, the difference was not
found among the PPFMs tested.
On the sterilized red perilla seeds, colonization ability of PPFMs,
including type strains, isolated strains and their antibiotics resistant strains,
was tested in agar-aseptic. For characterization of PPFM species detected
on the plates, I utilized whole-cell MALDI-TOF/MS analysis as a powerful
tool in PPFM phylogenetic study. I confirmed vertical transmission of the
kanamycin resistant strain of Methylobacterium sp. OR01 from seeds to
leaves on red perilla plant directly, and found that the strain had higher
ability to colonize on the red perilla plant than not only M. extorquens AM1
but also than Methylobacterium sp. ML01, in competitiveness test of
PPFMs.
26
Introduction
The phyllosphere, which comprises the aerial parts of terrestrial
plants, provides an extensive habitat for microorganisms. Especially, leaf
surface is vast, covering surface area of approximately 109 km2 and
comprising the main interface between terrestrial biomass and the
atmosphere. This environment harbors a substantial microbial population,
consisting of up to ~1026 bacterial cells as well as eukaryotes and archaea,
and the planetary phyllosphere bacterial population is sufficiently large
enough to many processes of global importance and to the behavior of
individual plants on which they thrive.2,7,43) Current knowledge about
phyllosphere colonization by bacteria, which, in contrast to plant pathogens,
do not cause any obvious harm to plants, is limited, and stems mostly from
cultivation-dependent studies. The culturable fraction of the microbial
phyllosphere community varies in both composition and size as a function
of diverse factors, such as time, space, plant species or leaf age.44,45) Recent
cultivation-independent genomics studies on the community composition
of phyllosphere demonstrate that the predominant species belongs to α- and
γ-proteobacteria and are dependent on plant species.15,43)
As described in chapter II, PPFMs with similar 16S rRNA gene
sequence of Methylobacterium sp. OR01 were isolated from leaves and
seeds at every site planted red perilla plant, and specific interaction
between perilla and PPFMs was suggested. On the origin of
Methylobacterium in plants, three possible routes, seeds, soil and
surrounding atmosphere, are considered,17) but it is still debatable. In this
27
chapter, I tried to obtain key factor for Methylobacterium colonization in
red perilla plant. Perillaldehyde is a principal constituent of perilla essential
oil, and has bactericidal activity for food-borne bacteria.40) Accordingly,
whether perillaldehyde is priority factor of the strain OR01 for specific
colonization and survival on perilla plant or not, susceptibility of PPFMs
was compared. Colonization abilities of strain OR01 were compared with
M. extorquens AM1 utilizing each of antibiotics resistant strains by
competitiveness test on agar plates and soil beds.
28
Materials and Methods
Susceptibility test of PPFMs against perillaldehyde
Two type strains, M. fujisawaense DSM5686T and M. radiotolerans
JCM2831T, and isolated strains, Methylobacterium sp. OR01 and ML01,
and M. extorquens AM1 were applied for the test. Perillaldehyde was
dissolved in 10% (v/v) Tween 20. After autoclaving the media, the filtered
perillaldehyde solution was added into the media aseptically. Pre-cultured
PPFM on AMS-methanol plate for 7 days at 28°C were streaked on the
plates supplemented with different concentrations (0, 500, 1000, 2000 ppm)
of perillaldehyde, and grown at 28°C for 7-10 days.
PPFMs on red perilla seeds
To reveal relationship between sterilization processes and PPFM
numbers on red perilla seeds, I studied effect of individual sterilization
procedures on PPFMs. The seeds were treated separately with individual
ways.
Generation of antibiotics resistant strains
Strains carrying antibiotics resistant DNA genes in the chromosome
were generated by electroporating the vector.37) Methylobacterium sp.
OR01 was transformed with pUT mini-Tn5 km (Biomedal Co., Sevilla,
Spain) to yield the kanamycin resistant strain and was designated as strain
OR01 KMR. M. extorquens AM1 was transformed with pCM16838) to yield
the tetracycline resistant strain AM1 TCR. Transformants were selected on
29
agar plates containing appropriate antibiotics: kanamycin (KM) 20 μg/ml
or tetracycline (TC) 10 μg/ml. There was no difference in growth between
the transformants and the parental strains in liquid culture either on
succinate or methanol.
Colonization test of PPFMs on red perilla plants
Methylobacterium sp. OR01, Methylobacterium sp. ML01, M.
extorquens AM1, and antibiotics resistant strains, OR01 KMR and AM1
TCR, were used solely or in combination to compare their colonization
abilities on red perilla plants. The strains were grown on AMS- succinate
(0.2%) at 28°C for 40 h, and the cells were collected, washed with
sterilized water, and suspended in sterilized water to obtain a suspension
with an OD600nm of 0.5. In the case of mixed inoculation of multiple strains,
0.5 ml of each cell suspension was put in one tube and centrifuged cells
were resuspended in 0.5 ml of water. Perilla seeds were treated with 40°C
water for 5 min, with 70% ethanol for 1 min, and with 1% antiformin
(containing 0.3% (v/v) Tween 20) for 5 min, and washed with sterilized
water for 5 times. The seeds were soaked in 0.5 ml of single or mixed cell
suspension for 4 h with gentle shaking at 2 rpm using a ROTATOR RT-5
(Taitec, Saitama, Japan) at room temperature. The seeds incubated with
PPFMs were sowed onto Hoagland agar (16 oz/500 ml bottle, Nalgene),
and grown in the NK Biotron LH-220 (Nihon Ikakikai Seisakusho, Osaka,
Japan) for 2 weeks. The system was operated at 25°C under 14 h light and
10 h dark cycle.
30
Sampling of fresh leaves, PPFM counting and isolation of PPFM strains
Fresh leaves were removed from the the perilla seedlings aseptically,
weighed, and put in 2.5 ml of sterilized water in a 5 ml tube. The mixture
was processed using an ultrasonic cleaning device (UT205S, Sharp, Osaka,
Japan) for 15 min, and the aqueous phase was serially diluted and plated
onto AMS-methanol agar supplemented with 10 μg/ml of cycloheximide.
For selective counting of antibiotics resistant strains, the appropriate
antibiotics were included in the media. After 7-10 days of incubation at
28°C, pink-pigmented colonies that appeared on the plates were counted.
Some of the colonies were isolated in pure state for additional analysis.
Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF/MS) analysis
Whole-cell MALDI-TOF/MS analysis was carried out by the method
of Tani et al.39) using a Bruker UltrafleXtreme MALDI-TOF/MS (Bruker
Daltonics K.K., Yokahama, Japan). Five to ten mg of PPFM cells grown on
AMS-methanol agar for 5 days were placed into a tube, and then 300 μl of
water and 900 μl of ethanol were added, and mixed. The mixture was
centrifuged to remove the supernatant, and the residue was dried. The dried
cells were extracted with 70% formic acid and acetonitrile (1:1, v/v), the
extract was applied to a steel target plate and overlayed with matrix
solution containing saturated sinapic acid in 50% (v/v) acetonitrile and
2.5% (v/v) tri-fluoro-acetic acid. The protein molecular weight profiles
were obtained by MALDI-TOF/MS (m/z range, 2,000-20,000). Cluster
analysis of the whole-cell MALDI-TOF/MS data was conducted using
31
SpecAlign software and XLSTAT software.
32
Results and Discussion
Susceptibility of PPFMs to perillaldehyde
Perillaldehyde is the major essential oil of perilla and is known as a
bacteriostatic and bacteriocidal substance towards a variety of
microorganisms. For example, perillaldehyde at 500 ppm killed completely
Escherichia coli.40) As a part of the investigation to determine why PPFMs
with the same 16S rRNA gene sequence as that of Methylobacterium sp.
OR01 were predominantly isolated from perilla leaves and seeds at almost
all planting sites, the susceptibility of PPFMs to perillaldehyde was tested.
Two type strains, M. fujisawaense DSM5686T and M. radiotolerans
JCM2831T, two isolated strains, Methylobacterium spp. OR01 and ML01,
and M. extorquens AM1 as an example of well-studied Methylobacterium
were grown on AMS-methanol plates supplemented with 500 to 2000 ppm
perillaldehyde, and then growth of the strains were compared. At 1000 ppm
of perillaldehyde or less, all PPFM tested could grow, however, none of the
strains grew on media containing 2000 ppm perillaldehyde (data not
shown). That is, different susceptibilities among the strains applied was not
observed in this test. Thus, the dominant recovery of specific PPFMs is not
related to perillaldehyde susceptibility of the strains isolated from red
perilla plants.
Competitiveness of diverse Methylobacterium strains on red perilla plants
Knief et al.41) reported that the colonization ability of PPFMs on
Arabidopsis thaliana was dependent on bacterial species, and strains of M.
33
extorquens 157, M. hispanicum GP34T, M. radiotolerans JCM2831T and M.
fujisawaense DSM5686T were defined as competitive strains. In contrast,
M. populi BJ001T was very weak or not detectable, and M. extorquens
SM14 and M. extorquens AM1 were also less competitive or very weak,
even being present of competitiveness variation in M. extorquens species.
In order to compare the colonization ability of Methylobacterium sp.
OR01 with other strains on red perilla plants, sterilized red perilla seeds
were incubated with mixed cell suspensions of strains of Methylobacterium
sp. OR01, Methylobacterium sp. ML01, and M. extorquens AM1. Two
weeks after aseptic growth on Hoagland agar, PPFMs were collected from
fresh leaves of the seedlings. PPFM populations colonizing on the leaves
were evaluated by colony counting on AMS-methanol agar (data not
shown), and eight randomly chosen strains were analyzed by whole-cell
MALDI-TOF/MS analysis. Different species of bacteria can be
discriminated by individual profile of MALDI-TOF/MS spectra of whole
cell sample because most of observed peaks can be attributed to ribosomal
proteins, which are abundant in cells and easy to be ionized.39)
Phylogenetic analysis of Methylobacterium species has been established
using whole-cell MALDI-TOF/MS. Figure 3-1 shows the mass profiles of
Methylobacterium sp. OR01, Methylobacterium sp. ML01, M. extorquens
AM1, and 8 PPFM strains, no. 1 - 8, isolated from leaves. All PPFMs
isolated from leaves showed almost identical spectral patterns and high
similarity values to strain OR01. These results indicated that
Methylobacterium sp. OR01 had a greater ability to colonize red perilla
plants than M. extorquens AM1 and Methylobacterium sp. ML1.
34
Figure 3-1. Whole-cell MALDI-TOF/MS profiles of strains of
Methylobacterium species and their dendrogram.
The Dendrogram was calculated as previously described.39) The spectra (m/z
3,000- 9,000) of relative intensities are shown as gel-like images using mMass 5.4.1
software (http://www.mmass.org/).
Direct transmission of PPFM from seeds to leaves
Although Methylobacterium sp. OR01 was detected as the major
PPFM colonizing red perilla seeds and leaves in competitveness test with
strain ML01 and M. extorquens AM1, there is no evidence to support direct
transmission and/or survival of the PPFM from seeds to leaves. Seed-borne
transmission of plant-pathogenic bacteria was documented for
Xanthomonas spp. by Darrasse et al.42) Vorholt pointed out that dedicated
analyses were required to elucidate the relative contributions of vertical
transmission via seeds and horizontal transmission via soil, air and/or other
plants.2) In this study, I constructed antibiotics-resistant strains of PPFM
and tested whether the strain inoculated to seeds could grow with the plant
35
and eventually be found on leaves or not.
First, I investigated on the effect of sterilization procedures on
PPFMs and other bacteria on red perilla seeds. Chanprame et al. reported
that PPFMs are present on the surfaces of most plant tissues, surface
disinfestations alone can effectively remove them so that uncontaminated
tissue cultures can be initiated in most cases.23) The PPFM numbers of
perilla seeds after warming and sterilization processes were as follows
(Table 3-1). The PPFM number of the untreated seeds was 1.1 x 107 CFU/g.
Almost all PPFMs of the seeds were removed by the warming process
(40°C water for 5 min), and the residual PPFM count was 1.8 x 104
CFU/mg, corresponding to 1/1000 of that of untreated seeds. After
treatment with 70% ethanol for 1 min or 1%(v/v) antiformin for 5 min,
PPFMs were not detected. Since the weight of one red perilla seed is
approximately 1 mg, ca.104 cells are estimated to colonize or be attached to
each red perilla seed (approximately 1 mm in diameter).
Table 3-1. Change in numbers of PPFMs from red perilla seeds after
warming and sterilization processes
treatments PPFMs
intact seeds 1.1 ± 0.16 x 107
water treatment (40°C x 5 min) 1.8 ± 0.16 x 104
70% ethanol (1 min) 0
1% antiformin (5 min) 0
CFU/g of seeds
36
Table 3-2. A competitive colonization test between Methylobacterium sp.
OR01 and M. extorquens AM1 using antibiotics resistant
strains, OR01 KMR and AM1 TCR
Number of PPFMsa (CFU/g fresh leaves) on
Incubated strains AMS-methanol AMS-methanol + KM AMS-methanol + TC
none (3.9 ± 0.16) x 107 n.d.b n.d.
OR01 KMR (6.4 ± 0.39) x 107 (6.8 ± 0.40) x 107 n.d.
AM1 TCR (5.6 ± 0.41) x 107 n.d. (4.6 ± 0.26) x107
OR01 KMR + AM1 TCR (9.0 ± 0.83) x 107 (8.9 ± 0.26) x 107 n.d.
a Means ± standard deviations of three replicated measurements are shown.
b Not detected.
After sterilization, red perilla seeds were soaked in the cell suspension
of kanamycin-resistant strain of Methylobacterium sp. OR01, strain OR01
KMR, for 4 h, and then sowed onto Hoagland agar. Two weeks after
growing aseptically, PPFMs were collected from the fresh leaves of the
seedlings. PPFM population colonizing the plants was evaluated by colony
counting on AMS-methanol agar or AMS-methanol agar supplemented
with kanamycin (Table 3-2). Even in the case of seeds that were not
incubated with PPFMs, PPFMs were detected on AMS-methanol but not on
AMS-methanol with kanamycin after rinsing the leaves. The 16S rRNA
gene sequences of randomly-chosen PPFMs isolated from the leaves
derived from seeds without prior PPFM incubation were identical with that
of strain OR01, suggesting that some cells survived after seed sterilization
in this experiment. On the contrary, in the case of seeds incubated with
37
strain OR01 KMR, kanamycin-resistant PPFMs were detected on
kanamycin-supplemented plates at almost the same level as those detected
on plates without kanamycin. All colonies appeared on AMS-methanol
plates could grow on AMS-methanol agar supplemented with kanamycin.
Thus, vertical transmission of Methylobacterium sp. OR01 from red perilla
seeds to leaves was confirmed.
In mixed incubations with strains OR01 KMR and AM1 TCR on
perilla seeds, strain OR01 KMR clearly dominated over strain AM1 TCR,
suggesting that Methylobacterium sp. OR01 had a greater ability to
colonize red perilla plant than M. extorquens AM1.
In this study, competitiveness of Methylobacterium sp. OR01, whose
closest relative was M. fujisawaense DSM5686T, defined as a competitive
strain in the phyllosphere of A. thaliana,41) was also confirmed in the
phyllosphere of red perilla plants. In general, competitive strains in the
phyllosphere must have ability to adapt to the phyllosphere environment,
where they are exposed to temperature shifts, desiccation, nutrient
limitation, and UV irradiation. But, my results that the wide and successive
appearance of PPFMs, which had the same 16S rRNA sequence as that of
Methylobacterium sp. OR01, observed in leaves and seeds of the red perilla
planted in different area over 3 years indicated that there must be some
factors regulating the species-level specificity between the red perilla plant
and PPFMs other than general competitiveness or perillaldehyde resistance.
The key factors responsible for the latent potential for plant colonization of
specific Methylobacterium species still remain to be solved.
38
Conclusion
This thesis describes microbial interactions between vegetable leaves
and PPFMs, specific association of Methylobacterium and red perilla
plants.
In chapter I, I studied on distribution of PPFMs on vegetable leaves,
and found that all vegetable leaves contained PPFMs, and vegetable leaves,
which planted at same farm (100 m2) simultaneously, gave different PPFM
numbers and different Methylobacterium species. From these findings, I
suggested species specific relationship between vegetable species and
Methylobacterium species. Next, I firstly found the highest PPFM counts
on the leaves of commercial green perilla leaves, and the numbers of
colonized PPFMs were independent on geographic factor. Red perilla
leaves harbored the most abundant PPFMs.
I isolated two Methylobacterium strains from red and green perilla
varieties, which exhibited high sequence similarity to the 16S rRNA gene
sequence to two distinct Methylobacterium species, M. fujisawaense
DSM5686T and M. radiotolerans JCM2831T, respectively. These suggest
that there are specific interactions between perilla and the PPFMs. I
selected as representatives of PPFMs isolated from two varieties of perilla,
Methylobacterium sp. OR01 for red perilla and TG01 for green perilla.
In chapter II, I planted red perilla seeds at 4 different sites to ensure
the origin of Methylobacterium on red perilla plants, and studied
geographic effect on Methylobacterium community composition on red
perilla plants. As a result, wide and deep distribution of the strain OR01 on
39
red perilla plant were confirmed regardless of geographic factors on
Methylobacterium community composition. From these results, I suggest
specific interaction between perilla and PPFMs.
In chapter III, I tried to get informations on the origin of
Methylobacterium on perilla plants. In colonization test of PPFMs on
sterilized perilla seeds, vertical transmission of Methylobacterium sp.
OR01 from seeds to leaves was demonstrated.
I hope this thesis will be a start point to pursue the origin of
Methylobacterium on perilla plants, and to clarify strong, deep, fantastic
and marvelous relationship between red perilla plants and
Methylobacterium sp. OR01.
40
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Acknowledgements
I wish to express many thanks to Professor Yasuyoshi Sakai,
Laboratory of Microbial Biotechnology, Division of Applied Life Sciences,
Graduate School of Agriculture, Kyoto University, for his directions of this
study, helpful advices and valuable discussions during the course of this
study.
I would like to express hearty thanks to Associate Professor Hiroya
Yurimoto, Laboratory of Microbial Biotechnology, Division of Applied
Life Sciences, Graduate School of Agriculture, Kyoto University, for his
helpful advices, valuable discussions, and continuous warm- hearted
encouragement during the course of this study.
I am grateful to Assistant Professor Masahide Oku, Laboratory of
Microbial Biotechnology, Division of Applied Life Sciences, Graduate
School of Agriculture, Kyoto University, and Associate Professor Jun
Hohseki, Research Unit for Physiological Chemistry, the Center for the
Promotion on Interdisciplinary Education and Research, Kyoto University,
for their valuable discussions and warm supports.
I am deeply grateful to Assistant Professor Naoko Yoshida,
Toyohashi University of Technology for her thoughtful guidance and
provision of precious samples of fresh vegetables.
I say very best thank you to Dr. Hiroyuki Iguchi, Laboratory of
Microbial Biotechnology, Division of Applied Life Sciences, Graduate
School of Agriculture, Kyoto University, for his invaluable suggestions,
guidance and heart-full advices. I also say thanks to Mr. Hiroki Taga for his
47
technical assistance.
My thanks are due to Dr. Kousuke Kawaguchi, Dr. Zhenyu Zhai, Dr.
Naoki Tamura, and all members of Laboratory of Microbial Biotechnology,
for their friendliness.
I indebted to Mr. Hitoshi Yoshimura of Doi Shibazuke Honpo Co.,
Ltd. and to the company, for their generous gift of important red perilla
seeds for Shibazuke production.
I also say thank you very much to Mr. Tohru Tuji, Mr. Hiroyuki
Shirai, Mr. Tomoya Mori, Mr. Takao Akamine, Mrs. Aya Ohta, and Mrs.
Fumi Mizuno for their helpful works on perilla planting and related
activities.
Finally, but not the least, I thank my family for their warm
encouragement and affectionate supports.
48
Publications
(a) Masayuki Mizuno, Hiroya Yurimoto, Naoko Yoshida, Hiroyuki Iguchi
and Yasuyoshi Sakai.
Distribution of Pink-Pigmented Facultative Methylotrophs on Leaves
of Vegetables.
Biosci. Biotechnol. Biochem. 76: 578-580 (2012)
(b) Masayuki Mizuno, Hiroya Yurimoto, Hiroyuki Iguchi, Akio Tani and
Yasuyoshi Sakai.
Dominant Colonization and Inheritance of Methylobacterium sp.
Strain OR01 on Perilla Plants.
Biosci. Biotechnol. Biochem. 77: 000-000 (2013)
49
Publications (continued)
Not relating to this thesis
(a) Masayuki Mizuno, Yukiji Shimojima, Takashi Iguchi, Isao Takeda, and
Saburo Senoh.
Fatty acid composition of hydrocarbon assimilating yeast.
Agr. Biol. Chem., 30: 506-510 (1966)
(b) Masayuki Mizuno, Yukiji Shimojima, Toshiaki Sugawara, and Isao
Takeda.
An antibiotic 24010.
J. Antibiotics, 24: 896-899 (1971)
(c) Masayuki Mizuno, Yohei Chiba, Yutaka Kimura, Yoshitaka Nadachi,
Hiroshi Nabetani, and Mitsutoshi Nakajima.
Process development for high quality chicken extract circulatable at
ordinary temperature from carcass of culled chicken.
Nippon Nōgeikagaku Kaishi, 78: 494-499 (2004)
50
Award
(a) 2004 Technical Award:The Japan Society for Food Engineering.
Hiroshi Nabetani, Nobuya Yanai, and Masayuki Mizuno.
Process development for isolation and purification of antioxydative
dipeptides from carcass of culled chicken, and development of
antioxydative activity evaluating system for antioxydative compounds.