International Journal of

ELSEVIER Food Microbiology 30 (1996) 243-253

Biochemical characteristics of Enterobacter

agglomerans and related strains found in
buckwheat seeds

Kazuo Iimuraa, Akiyoshi Hosonob,*

“MotojiyaCo., Ltd., Matsuntoto390, Japan
bFaculty of Agriculture, Shinshu University,Nagano-Minamiminowa399-45, Japan

Received 9 March 1995; revised 19 June 1995; accepted 25 September 1995

Abstract

Thirty strains of bacteria were randomly isolated and identified from buckwheat seeds.
The phenotypic characteristics of these strains agree well with those of the Enterobacter
agglomerans-Erwinia herbicola complex. On the basis of the difference in indole production
and gas production from D-glucose, the isolates were divided into 3 phenotypic groups, viz.
I, II and III. Twenty two strains were in phenotypic group I, which is negative for indole
production and gas production from D-glucose, and resembles Pantoea agglomerans. All six
strains in phenotypic group II, which is positive for indole production and negative for gas
production from D-glucose, were identified as Erwinia ananas. Two strains in phenotypic
group III, which is negative for indole production and positive for gas production from
D-glucose, were identified as Rahnella aquatilis.

Keywords: Enterobacter agglomerans; Erwinia ananas; Pantoea agglomerans; Rahnella

aquatilis; Buckwheat seed

* Corresponding author.

0168-1605/96/$15.000 1996 Elsevier Science B.V. All rights reserved

PII SO168- 1605(96)00949-X
244 K. Iimura, A. Hosono / ht. J. Food Microbiology 30 (1996) 243-253

1. Introduction

Buckwheat (Fagopyrum esculentum), a native of central Asia, is a minor grain

crols, and has traditionally been used in Japan to produce flour mainly for making
buckwheat noodles. Substantial research on the bacterial flora on buckwheat seeds
was performed by several investigators (Obinata et al., 1987; Miyoshi et al., 1988;
Naito and Shimizu, 1986). We have reported that bacteria on buckwheat seeds
harvested in Nagano and Hokkaido, Japan, were in the range of 105-10*/g, and
have identified Enterobacter agglomerans as one of the dominant species in these
samples (Iimura and Hosono, 1393; Iimura and Hosono, 1994).
E. agglomerans (i.e., synonymous with Erwinia herbicola, Erwinia uredo-vora and
Erwinia stewartii (Dye, 1969a; Sakazaki et al., 1976; Beji et al., 1988)) is a
heterogeneous species, and is considered to be associated with plant materials such
as saprophytes or pathogens, and with animal as well as human reservoirs (Dye,
1969b; Muraschi et al., ,1965). Different taxonomic views have resulted in an
ambiguous situation: phytopathologists prefer to use the nomenclature of Dye,
1969a, i.e., Er. herbicola, whereas clinical bacteriologists use the nomenclature of
Ewing and Fife, 1972, i.e., E. agglomerans. Although many rearrangements
(Sakazaki et al., 1976; Dye, 1981) have been suggested for the ‘Erwinia herbicola-
Enterobacter agglomerans complex’, a consensus has not been reached (Beji et al.,
1988). Ewing and Fife, 1972 investigated the biochemical characteristics of E.
agglomerans and classified it into 11 biogroups. Recently, based on total DNA
homology and electrophoretic protein pattern similarities, some strains of E.
agglomerans and E. herbicola, including the two type strains, were proposed to form
a new genus called Pantoea (Gavini et al., 1989). Referring to Ewing and Fife, 1972
and Gavini et al., 1989, we investigated the biochemical characteristics of E.
agglomerans and related strains isolated from buckwheat seeds.

2. Materials and methods

2.1. Buckwheat seeds

Buckwheat seeds harvested in Nagano (4 samples) and Hokkaido (2 samples),

Japan, were used for the isolation of E. agglomerans strains. The seed samples were
kept in a refrigerator (4°C) and used for the isolation within one month after
harvesting.

2.2. Isolation of E. agglomerans and related glucose fermentative Gram-negative

bacteria

Methods for the isolation of E. agglomerans and related strains were essentially
the same as those described in our previous paper (Iimura and Hosono, 1993). In
brief, 90 ml of sterile physiological saline was added to 10 g of ground buckwheat
sample. They were treated in a blender (Stomacher Lab-Blender 400, Seward
 K. Iimura, A. Hosono / ht. J. Food Microbiology 30 (1996) 243-253 245

Medical Co., Ltd., London, UK) for 1 min, followed by filtration with sterilized
gauze. A decimal dilution of the filtrate was plated on ‘Eiken’ trypto-soy agar
(TSA) (Eiken Chemical Co., Ltd., Tokyo, Japan) and incubated at 30°C for 48 h.
About ten colonies per sample were randomly isolated. These cultures were
purified, growing on the same medium at 30°C for 48 h. The isolates were examined
for Gram reaction and glucose fermentation. Gram-negative glucose fermentative
bacteria were identified by use of API 20E (Bio Merieux S.A., Marcy-I’Etoile,
France). Biochemical properties of the strains identified as Enterobacter agglomer-
ante were further examined.

2.3. Biochemical tests for E. agglomerans and related strains isolated

2.3.1. Catalase and oxidase tests

Catalase production was detected by the production of bubbles in a 3% hydrogen
peroxide solution at 30°C. The oxidase test was carried out by use of cytochrome
oxidase test paper (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) encompassing
Pserudomonas aeruginosa IF0 12689 as a positive control and Escherichia coli JCM
1649 as a negative control.

2.3.2. Gas formation

Gas formation from glucose was detected with Durham tubes in Bacto OF basal
medium (Difco Laboratories, Detroit, Mich.) incorporated at 1.O% D-glucose.
Incubation was carried out at 30°C for 4 days.

2.3.3. Motility and indole production

This was determined using ‘Nissui’ lysine indole motility medium (LIM medium).
The organisms were incubated at 30°C in prepared tubes of LIM medium by
stabbing. Motility was checked by observing a clouding of the medium or growth
extension from the inoculation line following incubation for 48 h. The presence of
indole was detected with Kovacs’ P-dimethyl-amino-benzalaldehyde reagent.

2.3.4. Pigmentation
The organisms were grown on ‘Nissui’ nutrient agar (nutrient broth solidified
with 1.5% agar, pH 7.2). Growth and pigment production were observed after
incubation at 30°C for 4 days.

2.3.5. Growth at 4”C, 41°C and 44°C

One drop of a light suspension of organisms in distilled water was added to
‘Nissui’ nutrient broth filled to a depth of about 5 cm in test tubes and incubated
in a water bath at 4”C, 41°C and 44°C for 2 to 7 days.

2.3.6. Acid production of sugars and related compounds

The following compounds were incorporated at 1.0% in ‘Eiken’ brom cresol
purple (BCP) semisolid medium: D-glucose, L-arabinose, D-ribose, D-xylose, D-
fructose, D-mannose, L-rhamnose, D-cellobiose, D-galactose, lactose, maltose,
246 K. Iimura,A. Hosono / ht. J. Food Microbiology 30 (1996) 243-253

melibiose, sucrose, trehalose, melezitose, raffinose, glycerol, meso-erythritol,

adonitol, duJcito1, inositol, D-mannitol, D-sorbitol, or-methyl-D-glucoside, salicin,
inulin and starch. Gas formation at 30°C was detected with Durham tubes over a
period of 30 days.

2.3.7. Reduction of nitrate

This was determined by growing the organism in ‘Nissui’ nutrient broth contain-
ing 0.1% of KNO, at 30°C for 2, 3 or 7 days and testing for the presence of nitrite
with dimethyl a-naphtylamine-sulphanilic acid reagent.

2.3.8. H,S production

Cultures were grown in ‘Nissui’ Kligler iron agar at 30°C for 48 h; H2S was
detected by blackening of the medium.

2.3.9. Methyl red (MR) and Voges-Proskauer (V-P) tests

Cultures were grown at 30°C in Bacto MR-VP medium (Difco) containing
glucose, 0.5%; buffered peptone, 0.7%; K,HPO,, 0.5% for 48 h. Acetylmethyl-
carbinol was detected using the Barritt modified method (Sakazaki et al., 1988).

2.3.10. Liquefaction of gelatin

A medium containing beef extract (Kyokuto Pharmaceutical Co., Ltd., Tokyo,
Japan), 1%; peptone (Nissui), 1%; NaCl, 0.5%; gelatin (Difco), 12%; pH 7.0;
sterilized at 121°C for 15 min, was dispensed to a depth of 5 cm in test tubes and
bacterial strains were inoculated by stabbing. Cultures were incubated at 30°C for
30 days. Liquefaction was observed after chilling tubes at 4°C after incubation.

2.3.1 I. Urease test

Christensen’s urea agar (Christensen, 1946) was used with incubation for 48 h.
The same medium without urea was used as a control for avoiding misjudgment.

2.3.12. Phenylalanine deaminase test

Phenylalanine agar (Ewing et al., 1957) was used with incubation at 30°C for 48
h. The production of phenylpyruvic acid (by deaminase) was indicated by the
formation of the characteristic green color upon the addition of an acidified ferric
chloride solution to the culture.

2.3.13. Decarboxylase test

Moeller’s medium (Moeller, 1955) containing 0.3% agar was employed, with
bromothymol blue replacing bromocresol purple indicator. Results were taken after
4 days of incubation at 30°C.

2.3.14. Deoxyribonuclease (DNase) test

Plates .of ‘Eiken’ DNA agar were heavily spotted with loopfuls of organisms from
nutrient agar and incubated at 30°C for 48 h. DNase production was detected by
flooding plates with n-HCl and observing zones of clearing around growth.
 K. Iimura, A. Hosono / Int. J. Food Microbiology 30 (1996) 243-253 241

2.3.15. Malonate utilization test

Malonate utilization was determined using ‘Eiken’ malonate broth. Observations
were made at 30°C over 48 h.

2.3.16. Citrate utilization test

Citrate utilization was determined using Bacto Simmons citrate agar (Difco).
Observations were made at 30°C over 4 days.

2.3.17. Growth in KCN

Test tubes (16-150 mm), filled to a depth of 5 cm with ‘Eiken’ KCN broth base
containing the same volume of 0.015% KCN solution were each inoculated with a
loopful of a 24 h nutrient broth culture, tightly closed with rubber stoppers and
observed for growth at 30°C over 48 h. Growth was indicated by a clouding of the
medium.

2.3.18. Digestion of es&in

This was examined by the methods described by Sakazaki (Sakazaki et al., 1988).

2.3.19. Kauffmann-Petersen’s organic acid utilization test

Kauffmann-Petersen’s test was examined by the methods described by Sakazaki
(Sakazaki et al., 1988). p-Galactosidase was determined using an ONPG (o-nitro-
phenyl-l)-galactosidase) Disc (Nissui).

3. Results and discussion

We used the API 20E system to clarify the taxonomic position of the strains
isolated from buckwheat seed samples. Table 1 shows the API 20E system codes of
30 strains isolated. The API 20E system codes 1005132 (strain T-33), 1005133
(strains T-3, T-6, T-8, T-12, T-17, T-18, T-21, T-24 and T-34) and 1205132 (strains
T-11 and T-35) were interpreted with the API data base (API system 3rd edition)
as E. agglomerans. Codes 1005332 (strain T-22), 1005333 (T-l, T-2, T-15 and T-19)
and 1045573 (strain T-9) were assigned to E. agglomerans or to Erwinia sp. Codes
1045773 (strain T-14) and 1245773 (strain T-4, T-10, T-23 and T-32) were assigned
to E. agglomerans, Erwinia sp., Klebsiella oxytoca or Klebsiella planticola, whereas
code 1205573 (strains T-5, T-36) was assigned to Rahnella aquatilis. However, the
API 20E system code 1205133 (strains T-7, T-13, T-16, T-20 and T-31) was not in
this data base.
Table 2 shows phenotypic characteristics of the 30 strains isolated. These strains
showed positive reactions for the following productions: catalase, anaerobic
growth, motility at 30°C gelatin liquefaction, acetoin from D-glucose and a-galac-
tosidase, and acids from D-glucose, L-arabinose, D-ribose, D-xylose, D-fructose,
D-mannose, L-rhamnose, D-galactose, maltose, sucrose, trehalose and D-mannitol.
The negative results were for cytochrome oxidase, urease, arginine dihydrolase,
lysine and ornithine decarboxylase, and H,S. These results agree well with those of
the E. agglomerans-E. herbicola complex. (Brenner et al., 1984).
248 K. Iimura, A. Hosono / ht. J. Food Microbiology 30 (1996) 243-253

On the basis of the differences in indole production and gas production from
D-glucose, the isolates were divided into 3 phenotypic groups, viz., I, II and III.
Out of the 30 strains isolated, 22 strains (73.3% of the total isolates) were negative
for indole production and gas production from D-glucose, which were grouped in
phenotype I. The API 20E system codes of these strains were 1005132, 1005133,
1005332, 1005333, 1205132 and 1205133. These codes were characterized by the
modes of citrate utilization, and of acid production from amygdalin and inositol as
illustrated in Fig. 1. However, all the strains involved in this group were negative
for acid production on the BCP semisolid medium and positive for citrate utiliza-
tion on the Bacto Simmons citrate agar (Table 2). Many taxonomical data have
been published on the E. agglomerans-E. herbicola complex (Gavini et al., 1983;
Brenner et al., 1984; Verdonck et al., 1987; Beji et al., 1988). Recently, Gavini et al.,

Table 1
Enterobucter ugglomerans isolated from buckwheat seeds and their API 20E profile numbers

Strain number Buckwheat harvested API 20E profile

Place Year

T-33 Hata, Nagano 1991 1005132

T-3 Azumi, Nagano 1991 1005133
T-6 Kawahigasi, Hokkaido 1991 1005133
T-8 Kawahigasi, Hokkaido 1991 1005133
T-12 Uryu, Hokkaido 1991 1005133
T-17 Uryu, Hokkaido 1991 1005133
T-18 Uryu, Hokkaido 1991 1005133
T-21 Matsumoto, Nagano 1991 1005133
T-24 Matsumoto, Nagano 1991 1005133
T-34 Hata, Nagano 1991 1005133
T-22 Matsumoto, Nagano 1991 1005332
T-l Azumi, Nagano 1991 1005333
T-2 Azumi, Nagano 1991 1005333
T-15 Uryu, Hokkaido 1991 1005333
T-19 Uryu, Hokkaido 1991 1005333
T-11 Kawahigasi, Hokkaido 1991 1205132
T-35 Hata, Nagano 1990 1205132
T-7 Kawahigasi, Hokkaido 1991 1205133
T-13 Uryu, Hokkaido 1991 1205133
T-16 Uryu, Hokkaido 1991 1205133
T-20 Uryu, Hokkaido 1991 1205133
T-31 Saku, Nagano 1990 1205133
T-9 Kawahigasi, Hokkaido 1991 1045573
T-14 Uryu, Hokkaido 1991 1045773
T-4 Azumi, Nagano 1991 1245773
T-10 Kawahigasi, Hokkaido 1991 1245773
T-23 Matsumoto, Nagano 1991 1245773
T-32 Saku, Nagano 1990 1245773
T-5 Azumi, Nagano 1991 1205573
T-36 Hata, Nagano 1991 1205573
Table 2
Penotypic characteristics of Enterohacter ugglomeruns and related strains isolated from buckwheat seeds

Characteristics Phenotype I Pontoea agglomerans (22 Phenotype II Erwinia ananas (6 Phenotype III Rahnella
strains) Reaction strains) Reaction aquatilis(2 strains) Reac-
tion
h
Growth at 4°C (+) (33)
Growth at 41°C _ _
Growth at 44°C _ _
Production of yellow pigment + +
Motility + +
Catalase + +
Cytochroomoxidase _ -
KCN (growth) 95 83
Gelatin liquefaction + +
Indole production _ +
Voges-Proskauer reaction + +
Nitrate reduced to nitrite 95 _
H,S production _ -
Hydrolysis of esculin 91 61
Gas production from D-glucose - _
Arginine dihydrolase (Moeller) _ _
Lysine decarboxylase _ _
Omithine ,decarboxylase _ _
Phenylalanine deaminase + -
Deoxyribonuclease - 17
p-Galactosidase + +
Urease _ _
Utilization of:
Citrate (Simmons) + +
Malonate 91 _
D-Tartrate (K-P) 5 _
Mutate (K-P) _ _
Acid produced from:
L-Arabinose + +
Table 2 (continued)

Characteristics Phenotype I Puntoea agglomerans (22 Phenotype II Erwinia ananus Phenotype III Rahnella
strains) Reaction (6 strains) Reaction aquatilis (2 strains) Reac-
tion
3
k?
D-Ribose + + +
2
D-Xylose + + +
“3
D-Fructose + + +
.k
D-Glucose + + +
D-tiannose + + + 9
L-Rhamnose + + + P
0
- + .
D-Cellobiose +
D-Galactose + f + $
Lactose t + 4
Maltose + + +
- 3
Melibiose 83 + B
Sucrose + + +
+ 3
Trehalose + + 3
Melezitose - - - &
Rafkose 5 67 a-..
ij-
Glyserol 18 f s’o P
meso-Erytheritol - - -
ti
Adonitol - - T=
Dulcitol - -t
- - 8
Inositole 83
D-Mannitol + + + k
D-sd’rbitol - + + ‘;-
a-Methyl-D-glucoside - - !z
Salicin 86 83 +
Inulin - - -
Starch - - -

+, all strains positive; -, all strains negative; number indicated percentage of positive strains; 0, delayed reaction.
 K. Iimura, A. Hosono / ht. J. Food Microbiology 30 (1996) 243-253 251

Entervtmcter zggkmem idated from buckvdheat Seeds

zk-
indole production
T-5
756, +
____- _,
phenwpic
group III

1205133 1205132 1005333 1005332 1005133 1005132

CIT + CIT - CIT -
IN0 + IN0 + IN0 - T-7 T-l 1 T-l T-22 T-3 T-33
1245773 1045773 1045573 T-l 3 T-35 T-2 T-6
T-l 6 T-l 5 T-8
T-14 T-9
T-19 T-12
T:?0 T:E
T-23
T-1’:,
T-32 T-21
_----~--~~~_______, T-24
phenotypic group II T-34
c_______________________________________-_____,
phenotypic group I

Fig. 1. API 20E profile and phenotype of Enterobacter agglomerans isolated from buckwheat seeds. API
20E reaction: CIT, utilization of citrate; AMY, acid production from amygdalin; INO, acid production
from inositol. API 20E was performed at 30°C for 24 h, and results were coded by seven-digit profile
numbers as described by the manufactuer.

1989 analyzed the E. agglomerans-E. herbicola complex on the basis of phenotypic

characteristics and DNA-DNA hybridization data and proposed the transfer of E.
agglomerans to Pantoea gen. nov. as Pantoea agglomerans comb. nov. Referring to
the phenotypic characteristics of P. agglomerans (Gavini et al., 1989), we recognized
that most of the phenotypic characteristics of the strains of phenotypic group I
were very similar to P. agglomerans, but with a few exceptions, viz. in production
of ornithine decarboxylase and in acid production from lactose and D-cellobiose.
As shown in Table 1 and Fig. 1, tests in the API 20E system gave the results that
the strains (T-4, T-9, T-10, T-14, T-23 and T-32) in phenotypic group II, which is
positive for indole production and negative for gas production from D-glucose,
were identified either as E. agglomerans or as Erwinia sp. Mergaert et al., 1984
reported that a strain having the code 1245773 could be interpreted with the API
data base as E. ananas or E. uredovora, taking into account that the strain was
positive for indole production and negative for gas production from D-glucose.
Furthermore, Beji et al., 1988 and Gavini et al., 1989 reported that E. ananas
showed positive reactions for the following: yellow pigment and indole, and acids
from D-cellobiose, lactose, maltose, melibiose, raffinose, sucrose, sorbitol and
salicin, but not for adonitol, dulcitol and a-methyl-D-glucoside. In reference to the
phenotypic characteristics of E. ananas (Beji et al., 1988; Gavini et al., 1989), we
252 K. limura, A. Hosono / Int. J. Food Microbiology 30 (19%) 243-253

have recognized that most of the phenotypic characteristics of the strains of

phenotypic group II closely resembled E. ananas.
All the strains (T-5 and T-36) in phenotypic group III, which are negative for
indole production and positive for gas production from D-glucose, were identified
as R. aquatilis with the API 20E system. Colonies were smooth, translucent, and
convex with an entire margin (data not shown). Acetoin was positive but lysine and
ornithine decarboxylase, arginine dihydrolase and DNase were negative. Acid
production was observed from D-glucose, L-arabinose, D-ribose, D-xylose, D-fruc-
tose, D-mannose, L-rhamnose, D-cellobiose, D-galactose, lactose, maltose, meli-
biose, sucrose, trehalose, raffinose, dulcitol, D-mannitol, D-sorbitol and salicin but
not from melezitose, erythritol, adonitol, inositol, cr-methyl-D-glucoside, inulin and
esculin (Table 2). These characteristics fit well with those of R. aquatilis strains
described by Izard et al., 1979 E. agglomerans. Berge et al., 1991 reported that R.
aquatilis is a rhizosphere-associated bacteria and also a nitrogen fixer. Hamze et al.,
1991 reported that R. aquatilis has also been isolated mostly from water, soil, lager
beer breweries and in a few cases, from human clinical specimens.

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