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.