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International Journal of 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-Minamiminowa 399-45, Japan
Received 9 March 1995; revised 19 June 1995; accepted 25 September 1995
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; aquatilis; Buckwheat seed Erwinia ananas; Pantoea agglomerans; Rahnella
* Corresponding author. 0168-1605/96/$15.000 1996 Elsevier Science B.V. All rights reserved PII SO1 1605(96)00949-X 68-
1965). Muraschi et al. Recently. whereas clinical bacteriologists use the nomenclature of Ewing and Fife. i. 2. 1969a. Introduction Buckwheat (Fagopyrum esculentum). 1969b. we investigated the biochemical characteristics of E..e. agglomerans and related strains isolated from buckwheat seeds. In brief.. Buckwheat seeds Buckwheat seeds harvested in Nagano (4 samples) and Hokkaido (2 samples). Iimura and Hosono. They were treated in a blender (Stomacher Lab-Blender 400.e. 1981) have been suggested for the ‘Erwinia herbicolaEnterobacter agglomerans complex’. Ewing and Fife. Hosono / ht.244 K. agglomerans strains.2. and has traditionally been used in Japan to produce flour mainly for making buckwheat noodles. agglomerans and classified it into 11 biogroups. based on total DNA homology and electrophoretic protein pattern similarities. Japan. Substantial research on the bacterial flora on buckwheat seeds was performed by several investigators (Obinata et al. . a native of central Asia. were used for the isolation of E.1. 1987. 1994). agglomerans and related glucose fermentative bacteria Gram-negative Methods for the isolation of E. i. 1989). Different taxonomic views have resulted in an ambiguous situation: phytopathologists prefer to use the nomenclature of Dye... We have reported that bacteria on buckwheat seeds harvested in Nagano and Hokkaido. 1988.. and have identified Enterobacter agglomerans as one of the dominant species in these samples (Iimura and Hosono. Naito and Shimizu. E. 90 ml of sterile physiological saline was added to 10 g of ground buckwheat sample. Dye. 1972 and Gavini et al. 1988).. 1989. Seward .. 1976. were in the range of 105-10*/g. Er. synonymous with Erwinia herbicola.. Food Microbiology 30 (1996) 243-253 1...e. Miyoshi et al. herbicola. 1993). A. Materials and methods 2. J.. a consensus has not been reached (Beji et al. herbicola. Isolation of E. agglomerans and E. Sakazaki et al. 1972 investigated the biochemical characteristics of E. 1986). 1972. 1976. Referring to Ewing and Fife. and with animal as well as human reservoirs (Dye. were proposed to form a new genus called Pantoea (Gavini et al. Japan. is a minor grain crols. Although many rearrangements (Sakazaki et al.. including the two type strains. Beji et al. agglomerans (i. Erwinia uredo-vora and Erwinia stewartii (Dye. agglomerans. agglomerans and related strains were essentially the same as those described in our previous paper (Iimura and Hosono. 1393. 1969a. some strains of E. 1988)) is a heterogeneous species. E. 2. Iimura. and is considered to be associated with plant materials such as saprophytes or pathogens. The seed samples were kept in a refrigerator (4°C) and used for the isolation within one month after harvesting.
3. 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. Ltd. 2.. Iimura. Ltd. Tokyo. maltose.4. 41°C and 44°C for 2 to 7 days. Gas formation Gas formation from glucose was detected with Durham tubes in Bacto OF basal medium (Difco Laboratories. A. 2.3. The organisms were incubated at 30°C in prepared tubes of LIM medium by stabbing. Hosono / ht. Japan) and incubated at 30°C for 48 h. A decimal dilution of the filtrate was plated on ‘Eiken’ trypto-soy agar (TSA) (Eiken Chemical Co..O% D-glucose.3. Ltd. Growth at 4”C. London. Marcy-I’Etoile. Dfructose. Biochemical tests for E.2). Catalase and oxidase tests Catalase production was detected by the production of bubbles in a 3% hydrogen peroxide solution at 30°C.2. pH 7. UK) for 1 min. L-arabinose.. growing on the same medium at 30°C for 48 h. Incubation was carried out at 30°C for 4 days. 2.5% agar.. Japan) encompassing Pserudomonas aeruginosa IF0 12689 as a positive control and Escherichia coli JCM 1649 as a negative control.K. The presence of indole was detected with Kovacs’ P-dimethyl-amino-benzalaldehyde reagent. The isolates were examined for Gram reaction and glucose fermentation. France). Detroit.1.A. J. About ten colonies per sample were randomly isolated.. Pigmentation The organisms were grown on ‘Nissui’ nutrient agar (nutrient broth solidified with 1.6.5. D-cellobiose. The oxidase test was carried out by use of cytochrome oxidase test paper (Nissui Pharmaceutical Co.3. Biochemical properties of the strains identified as Enterobacter agglomerante were further examined. lactose. Tokyo.. 2.3.. D-galactose.3. Motility was checked by observing a clouding of the medium or growth extension from the inoculation line following incubation for 48 h. Food Microbiology 30 (1996) 243-253 245 Medical Co. 2. D-ribose. Growth and pigment production were observed after incubation at 30°C for 4 days. 2. . D-xylose. Acid production of sugars and related compounds The following compounds were incorporated at 1. agglomerans and related strains isolated 2.3.0% in ‘Eiken’ brom cresol purple (BCP) semisolid medium: D-glucose.3. D-mannose. Mich. followed by filtration with sterilized gauze. Gram-negative glucose fermentative bacteria were identified by use of API 20E (Bio Merieux S. L-rhamnose. These cultures were purified. Motility and indole production This was determined using ‘Nissui’ lysine indole motility medium (LIM medium).) incorporated at 1.
2.12. trehalose.1% of KNO. 0.3. The same medium without urea was used as a control for avoiding misjudgment.10.. 2. 0. Deoxyribonuclease (DNase) test Plates .3.3. gelatin (Difco). 1946) was used with incubation for 48 h. 2. 12%. Gas formation at 30°C was detected with Durham tubes over a period of 30 days. Phenylalanine deaminase test Phenylalanine agar (Ewing et al. K. inositol. 0. 1%. glycerol.5%.0. 1988). Liquefaction of gelatin A medium containing beef extract (Kyokuto Pharmaceutical Co.A. peptone (Nissui). salicin. 2.3. buffered peptone. Iimura. Urease test Christensen’s urea agar (Christensen. 2. inulin and starch. 0.5% for 48 h.1 I.246 K. Tokyo. duJcito1.of ‘Eiken’ DNA agar were heavily spotted with loopfuls of organisms from nutrient agar and incubated at 30°C for 48 h. Ltd. Japan)..3.5%. Food Microbiology 30 (1996) 243-253 melibiose.. Results were taken after 4 days of incubation at 30°C. Hosono / ht.. or-methyl-D-glucoside.8. 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. Reduction of nitrate This was determined by growing the organism in ‘Nissui’ nutrient broth containing 0. sucrose. 2. raffinose. Decarboxylase test Moeller’s medium (Moeller.3. 1957) was used with incubation at 30°C for 48 h. Cultures were incubated at 30°C for 30 days. Acetylmethylcarbinol was detected using the Barritt modified method (Sakazaki et al. 3 or 7 days and testing for the presence of nitrite with dimethyl a-naphtylamine-sulphanilic acid reagent.13.. Methyl red (MR) and Voges-Proskauer (V-P) tests Cultures were grown at 30°C in Bacto MR-VP medium (Difco) containing glucose. 2.3.7. at 30°C for 2. pH 7.HPO.7%.3% agar was employed. DNase production was detected by flooding plates with n-HCl and observing zones of clearing around growth. Liquefaction was observed after chilling tubes at 4°C after incubation. D-mannitol. D-sorbitol. 2. H. .3. melezitose. NaCl. J.S production Cultures were grown in ‘Nissui’ Kligler iron agar at 30°C for 48 h. 1%. was dispensed to a depth of 5 cm in test tubes and bacterial strains were inoculated by stabbing. with bromothymol blue replacing bromocresol purple indicator. sterilized at 121°C for 15 min. 1955) containing 0.9. adonitol. meso-erythritol. H2S was detected by blackening of the medium.14.
(Brenner et al. T-6. 2.K. Observations were made at 30°C over 48 h.015% KCN solution were each inoculated with a loopful of a 24 h nutrient broth culture. T-13.17. Growth in KCN Test tubes (16-150 mm).16. T-23 and T-32) were assigned to E. T-10. L-rhamnose.3. 1984). L-arabinose. trehalose and D-mannitol. D-mannose. Results and discussion We used the API 20E system to clarify the taxonomic position of the strains isolated from buckwheat seed samples. Observations were made at 30°C over 4 days. J. motility at 30°C gelatin liquefaction. T-8. D-fructose. These strains showed positive reactions for the following productions: catalase. Table 2 shows phenotypic characteristics of the 30 strains isolated. tightly closed with rubber stoppers and observed for growth at 30°C over 48 h. filled to a depth of 5 cm with ‘Eiken’ KCN broth base containing the same volume of 0.15. 1988). 1005133 (strains T-3.. Klebsiella oxytoca or Klebsiella planticola. T-15 and T-19) and 1045573 (strain T-9) were assigned to E.3. Table 1 shows the API 20E system codes of 30 strains isolated. Digestion of es&in This was examined by the methods described by Sakazaki (Sakazaki et al. The negative results were for cytochrome oxidase. 1988). However. acetoin from D-glucose and a-galactosidase.S. T-21. the API 20E system code 1205133 (strains T-7. T-2. T-17.19. Codes 1005332 (strain T-22). herbicola complex. and H. 3. Erwinia sp. T-36) was assigned to Rahnella aquatilis. anaerobic growth. Growth was indicated by a clouding of the medium. 2. p-Galactosidase was determined using an ONPG (o-nitrophenyl-l)-galactosidase) Disc (Nissui). agglomerans or to Erwinia sp. lysine and ornithine decarboxylase. maltose.. 1005333 (T-l. and acids from D-glucose. D-xylose. Malonate utilization test Malonate utilization was determined using ‘Eiken’ malonate broth. D-galactose.18. agglomerans.3. T-20 and T-31) was not in this data base.3. 2. T-12. urease.3. 2. Codes 1045773 (strain T-14) and 1245773 (strain T-4. A. agglomerans-E. The API 20E system codes 1005132 (strain T-33). Kauffmann-Petersen’s organic acid utilization test Kauffmann-Petersen’s test was examined by the methods described by Sakazaki (Sakazaki et al.. whereas code 1205573 (strains T-5. sucrose. These results agree well with those of the E. agglomerans. Citrate utilization test Citrate utilization was determined using Bacto Simmons citrate agar (Difco). . arginine dihydrolase. T-18. Iimura. Hosono / Int. 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. Food Microbiology 30 (1996) 243-253 241 2. D-ribose. T-16..
Brenner et al. Verdonck et al.. herbicola complex (Gavini et al. Nagano Kawahigasi. agglomerans-E. Hokkaido Uryu. Nagano Saku. 1984. 1.. Nagano Hata. Hokkaido Matsumoto. 1205132 and 1205133. 1005332. 1983. Hokkaido Uryu. Nagano Kawahigasi. 22 strains (73. Hokkaido Saku.. Nagano Azumi. Many taxonomical data have been published on the E. Hokkaido Uryu. Hokkaido Azumi. Nagano Matsumoto. Hosono / ht. Hokkaido Uryu. Hokkaido Uryu. and of acid production from amygdalin and inositol as illustrated in Fig. viz. Nagano Uryu. Nagano Hata.. Beji et al. Nagano Kawahigasi. Out of the 30 strains isolated.248 K. However.. J.3% of the total isolates) were negative for indole production and gas production from D-glucose. Recently. Hokkaido Uryu. 1005333. Nagano Azumi. 1987. 1988). II and III. Hokkaido Hata.. The API 20E system codes of these strains were 1005132. Nagano Matsumoto. Nagano Azumi. Gavini et al. Food Microbiology 30 (1996) 243-253 On the basis of the differences in indole production and gas production from D-glucose. Iimura. Hokkaido Kawahigasi. which were grouped in phenotype I. These codes were characterized by the modes of citrate utilization. Nagano Azumi. Table 1 Enterobucter ugglomerans isolated from buckwheat seeds and their API 20E profile numbers Strain number Buckwheat harvested Place Year 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1991 1990 1991 1991 1991 1991 1990 1991 1991 1991 1991 1991 1990 1991 1991 API 20E profile T-33 T-3 T-6 T-8 T-12 T-17 T-18 T-21 T-24 T-34 T-22 T-l T-2 T-15 T-19 T-11 T-35 T-7 T-13 T-16 T-20 T-31 T-9 T-14 T-4 T-10 T-23 T-32 T-5 T-36 Hata. A. Nagano Kawahigasi. 1005133. all the strains involved in this group were negative for acid production on the BCP semisolid medium and positive for citrate utilization on the Bacto Simmons citrate agar (Table 2). Hokkaido Uryu. Hokkaido Matsumoto. Hokkaido Uryu. I. Hokkaido Kawahigasi. the isolates were divided into 3 phenotypic groups. Nagano 1005132 1005133 1005133 1005133 1005133 1005133 1005133 1005133 1005133 1005133 1005332 1005333 1005333 1005333 1005333 1205132 1205132 1205133 1205133 1205133 1205133 1205133 1045573 1045773 1245773 1245773 1245773 1245773 1205573 1205573 .
decarboxylase Phenylalanine deaminase Deoxyribonuclease p-Galactosidase Urease Utilization of: Citrate (Simmons) Malonate D-Tartrate (K-P) Mutate (K-P) Acid produced from: L-Arabinose (33) _ _ .S production Hydrolysis of esculin Gas production from D-glucose Arginine dihydrolase (Moeller) Lysine decarboxylase Omithine .Table 2 Penotypic characteristics of Enterohacter ugglomeruns (22 aquatilis and related strains isolated from buckwheat seeds Phenotype II Erwinia ananas (6 strains) Reaction Phenotype III Rahnella (2 strains) Reaction h Characteristics Phenotype I Pontoea agglomerans strains) Reaction (+) _ _ + + + _ 95 + _ + 95 _ 91 _ _ _ + + _ + 91 5 _ + + + + 83 + + + _ 61 _ _ _ _ 17 + _ + _ _ _ + Growth at 4°C Growth at 41°C Growth at 44°C Production of yellow pigment Motility Catalase Cytochroomoxidase KCN (growth) Gelatin liquefaction Indole production Voges-Proskauer reaction Nitrate reduced to nitrite H.
-. + + + + 5 18 83 + + 83 67 f - $ 4 3 B + + + + + + + f t + 83 + + + + + + + + s’o -t + + + - 3 3 & -. delayed reaction.k + + + + + + 9 P 0 . number indicated percentage of positive strains. 0.!z D-Ribose D-Xylose D-Fructose D-Glucose D-tiannose L-Rhamnose D-Cellobiose D-Galactose Lactose Maltose Melibiose Sucrose Trehalose Melezitose Rafkose Glyserol meso-Erytheritol Adonitol Dulcitol Inositole D-Mannitol D-sd’rbitol a-Methyl-D-glucoside Salicin Inulin Starch + 86 - +..Table 2 (continued) Phenotype I Puntoea agglomerans (22 strains) Reaction tion + + + + + + + Characteristics Phenotype II Erwinia ananus (6 strains) Reaction Phenotype III Rahnella aquatilis (2 strains) Reac3 k? 2 “3 . a ijP ti T= 8 k ‘. all strains positive. all strains negative. .
1984 reported that a strain having the code 1245773 could be interpreted with the API data base as E. taking into account that the strain was positive for indole production and negative for gas production from D-glucose. raffinose. Beji et al. Referring to the phenotypic characteristics of P. 1989 reported that E. phenwpic group + III CIT + IN0 + 1245773 CIT IN0 + 1045773 T-14 CIT IN0 1045573 T-9 1205133 T-7 T-l 3 T-l 6 T:E 1205132 T-l 1 T-35 1005333 T-l T-2 T-l 5 T-19 1005332 T-22 1005133 T-3 T-6 T-8 T-12 T-1’:. J. A. Hosono / ht. and results were coded by seven-digit profile numbers as described by the manufactuer. acid production from inositol._. ananas or E. sucrose. uredovora. API 20E profile and phenotype of Enterobacter agglomerans isolated from buckwheat seeds. herbicola complex on the basis of phenotypic characteristics and DNA-DNA hybridization data and proposed the transfer of E. Food Microbiology 30 (1996) 243-253 Entervtmcter zggkmem idated from buckvdheat Seeds 251 zkT-5 indole production 756. INO. agglomerans to Pantoea gen. we recognized that most of the phenotypic characteristics of the strains of phenotypic group I were very similar to P. Gavini et al. were identified either as E. nov. agglomerans (Gavini et al. agglomerans. AMY. as Pantoea agglomerans comb. ananas (Beji et al. in production of ornithine decarboxylase and in acid production from lactose and D-cellobiose. T-9. nov. tests in the API 20E system gave the results that the strains (T-4. sorbitol and salicin. T-21 T-24 1005132 T-33 T:?0 T-23 T-32 _----~--~~~_______. 1989). acid production from amygdalin.. T-23 and T-32) in phenotypic group II. 1989). lactose. maltose. API 20E reaction: CIT. and acids from D-cellobiose. which is positive for indole production and negative for gas production from D-glucose.. Furthermore. but not for adonitol. In reference to the phenotypic characteristics of E. Iimura. API 20E was performed at 30°C for 24 h. As shown in Table 1 and Fig. ananas showed positive reactions for the following: yellow pigment and indole. agglomerans-E. we . but with a few exceptions.. dulcitol and a-methyl-D-glucoside. T-14. T-10. Fig. Mergaert et al. 1988 and Gavini et al. 1.. 1988. agglomerans or as Erwinia sp. ____. viz.. 1.. utilization of citrate. melibiose. phenotypic group II phenotypic group I T-34 c_______________________________________-_____.K. 1989 analyzed the E.
Int.. J.J.. cr-methyl-D-glucoside. Int. 24. melibiose. Lab. which are negative for indole production and positive for gas production from D-glucose. and Kersters. maltose. 153-161. K.W. W.G. Microbial. Mielcarek. W. D. Dye. (1969a) A taxonomic study of the genus Erwiniu. Syst. 15. D-ribose.H. B. and Krichevsky. H. raffinose. 38. J. agglomerans. 4-l 1. M.252 K. 1991 reported that R. Berge. Izard. (1984) Attempts to classify herbicola group Enterobacter agglomerans strains by deoxyribonucleic acid hybridization and phenotypic tests. D-mannitol. ‘A typical Erwinias’. Sci. K. Van Vuuren. 833-839. sucrose... and De Ley. C. 81-97.B. inositol. soil.W. M.. F. Dye. (1981) A numerical taxonomic study of the genus Erwiniu. (1983) Etude taxonomiqu de souches appartenant ou apparentees au genre Erwinia group Herbicola... Syst.. J.L. (1946) Urea decomposition as a means of differentiating Proteus and paracolon cultures from each other. Beji. Beji. A. aquatilis strains described by Izard et al.. Acid production was observed from D-glucose. Sci. (1969b) A taxonomic study of the genus Erwinia. et a I’espece Enterobacter ugglomeruns. J.... 39. H. 12. ananas. O. A... D-cellobiose. 77-88. erythritol. and De Ley. D-fructose. aquatilis has also been isolated mostly from water. Erwinia milletiae and Enterobacter agglomerans and redefinition of the taxon by genotypic and phenotypic data..I. Microbial. J. M. aquatilis with the API 20E system.J.H.. Gavini. D-mannose. 1991 reported that R.R. Hosono / Int. J. 1955203. aquatilis is a rhizosphere-associated bacteria and also a nitrogen fixer. Appl. as Pantoeu agglomerans comb. Colonies were smooth.K. Bacterial. All the strains (T-5 and T-36) in phenotypic group III. Int. nov. (the herbicolalathry bacteria). a potential contaminant in lager beer breweries. Can. Christensen. 63-68. Kersters. (1957) Phenylalanine and malonate media and their use in enteric bacteriology. D. limura... 337-345. Gavini. Steigerwalt. 1979 E. H. 13. J. D. Izard. D-sorbitol and salicin but not from melezitose. 52.W.J. lager beer breweries and in a few cases. Fanning. W. 218-235.. J. dulcitol. New Zeal. Lefebvre. (1972) Enterobacter aggfomerans (Beiierinck) comb. References Beji. Syst. D-xylose. R.. Int. Davis. A. Gavini. Food Microbiology 30 (19%) 243-253 have recognized that most of the phenotypic characteristics of the strains of phenotypic group II closely resembled E. arginine dihydrolase and DNase were negative. 223-229. Berge et al.. 22. Bacterial. Dye.. 461-466.. from human clinical specimens. Syst. Leclerc. 37.. Kersters. D. Res. translucent. New Zeal. inulin and esculin (Table 2). W. J. J. . J. J. II. Bacterial. Mergaert. Bacterial. were identified as R. 12. and convex with an entire margin (data not shown).F. trehalose. (1988) Subjective synonymy of Erwinia herbicola. a nitrogen fixing enteric bacterium associated with the rizosphere of wheat and maize. K.. F. New Zeal. Heuhn. Syst. J. J. T. Bacterial. and Achouak. D. The ‘carotovora’ group. Hamze. nov. L-rhamnose. F. (1991) Rahnella aquatilis. IV. and Fife. and description of Puntoeu dispersu sp. J. J. D. and Reavis. D. Ewing. nov. L-arabinose. adonitol. Int. lactose. Brenner. A. Izard.W. F. A. 1972 to Puntoeu gen. Mergaert. G. Hamze et al. nov. 45-55. D-galactose. Mergaert. Food Microbial. These characteristics fit well with those of R. 34. Acetoin was positive but lysine and ornithine decarboxylase. (1991) RuhneNu aquatilis. 4. Hlth. Publ. Agric. (1989) Transfer of Enterobucter ugglomerans (Beijerinck 1988) Ewing and Fife. B. and Leclerc. Knutson. Ewing.R. Gavini. J.
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