A new species of Corynebacterium isolated from field corn is described. The isolates form a homogeneous group that is recognized as a new species, Corynebacterium nebraskense. The organism is characterized by orange-pig-mented colonies, inability to grow on 0.005% triphenyltetrazolium chloride agar, specific bacteriophage sensitivity, and a guanine plus cytosine content of 73.5 mol%. These and other characters differentiate this organism from other described phytopathogenic corynebacteria. The type strain of C. nebraskense is Fur-1 (= ATCC 27822 = NCPPB 2578)

Mandel, Manley

Vidaver, Anne K.

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Papers in Plant Pathology Plant Pathology Department 1974 Corynebacterium nebraskense, a New, Orange-Pigmented Phytopathogenic Species Anne K. Vidaver University of Nebraska–Lincoln, avidaver1@unl.edu Manley Mandel University of Texas System Cancer Center Follow this and additional works at: https://digitalcommons.unl.edu/plantpathpapers  Part of the Plant Pathology Commons Vidaver, Anne K. and Mandel, Manley, "Corynebacterium nebraskense, a New, Orange-Pigmented Phytopathogenic Species" (1974). Papers in Plant Pathology. 253. https://digitalcommons.unl.edu/plantpathpapers/253 This Article is brought to you for free and open access by the Plant Pathology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Plant Pathology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Int J Syst Bacteriol October 1974 24:482-485; doi:10.1099/00207713-24-4-482  Copyright 1974 International Association of Microbiological Societies  Corynebacterium nebraskense, a New, Orange-Pigmented Phytopathogenic Species  Anne K. Vidaver  Department of Plant Pathology, University of Nebraska–Lincoln, Lincoln, Nebraska, 68503,  Manley Mandel The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77025   Abstract A new species of Corynebacterium isolated from field corn is described. The isolates form a homogeneous group that is recognized as a new species, Corynebacterium nebraskense. The organism is characterized by orange-pig-mented colonies, inability to grow on 0.005% triphenyltetrazolium chloride agar, specific bacteriophage sensitivity, and a guanine plus cytosine content of 73.5 mol%. These and other characters differentiate this organism from other described phytopathogenic corynebacteria. The type strain of C. nebraskense is Fur-1 (= ATCC 27822 = NCPPB 2578).    [Includes Errata from Int J Syst Bacteriol January 1975 25:98; doi:10.1099/00207713-25-1-98 ]  INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY Oct. 1974, p. 482435 Copyright 0 1974 International Association of Microbiological Societies Vol. 24 ,  No. 4 Printed in U.S.A. Corynebacterium nebraskense, a New, Orange-Pigmented Phytopathogenic Species’ ANNE K. VIDAVER and MANLEY MANDEL Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska, 68503, and The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77025 A new species of Corynebacterium isolated from field corn is described. The isolates form a homogeneous group that is recognized as a new species, Corynebacterium nebraskense. The organism is characterized by orange-pig- mented colonies, inability to grow on 0.005% triphenyltetrazolium chloride agar, specific bacteriophage sensitivity, and a guanine plus cytosine content of 73.5 mol%. These and other characters differentiate this organism from other described phytopathogenic corynebacteria. The type strain of C. nebraskense is Fur-1 (= ATCC 27822 = NCPPB 2578). A bacterial disease of field corn characterized by a distinctive foliar blight was first observed in south central Nebraska in 1969. In subse- quent years, the disease, called Goss’s bacterial wilt and blight (synonymous with leaf freckles and wilt), was found in other areas in Nebraska as well as in neighboring Iowa and Kansas (1 2). Preliminary investigation showed that a specific corynebacterium was associated with the dis- ease. The bases for differentiating this bac- terium from other phytopathogenic corynebac- teria are presented in this paper. A preliminary report, including host range data, has been published (8). MATERIALS AND METHODS Bacterial isolates. Fifteen isolates were obtained from different field-corn hybrids collected throughout Nebraska over a 3-year period. Isolations were made principally from leaf material, but also from stalks, using a modification of a direct puncture method (2) in which a sterile dissecting needle was used to puncture margins of lesions and to streak directly onto agar plates. The orange-pigmented isolates derived from such material reproduced disease symptoms when inoculated into stems of lO-day-old seedlings of either sweet corn (Golden Cross Bantam) or field corn (Nebraska 501-D). Methods. The methods used for characterization of the isolates were generally as described previously (9), except that for deoxyribonucleic acid extraction the cells were treated with lysozyme (100 pg/ml) in the presence of saline-ethylenediaminetetraacetate for 3 h Research conducted under project no. 21-21. Published as paper no. 3841, Journal Series, Ne- braska Agriculture Experiment Station. at 37 C prior to lysis with sodium dodecyl sulfate. All tests were repeated at least three times. Growth on 0.005% triphenyltetrazolium chloride agar or 0.0005% iodonitrotetrazolium chloride agar was determined by using Kelman medium ( 5 ) ,  except that glucose was added separately and the stock solutions of tetrazolium salts were filter-sterilized prior to use. Bacteriophage sensitivity was determined by spot tests with four purified phage isolates. The phages were isolated from soil or plant debris as previously described (11), using three different isolates of the new corynebacterium for initial phage enrichment. (These bacteriophages, @C8, OC11, @C12, and &2A, will be described in a separate communication.) Phages titering 0.5 x 10’ to 1.0 X lo7 plaque-forming units per ml were spotted on lawns of bacteria with a Cooke Microdiluter (Cooke Engineering Co.). RESULTS Morphology. Cells of the new isolates were gram-positive, pleomorphic rods characteristic of coryneform bacteria. All isolates were nonmotile and without flagella (Fig. 1). Cells from 40-h-old cultures grown at 24 C were 0.25 to 0.5 by 0.75 to 2.7 pm, with average dimensions of 0.5 by 2.0 pm, as determined by light and electron microscopy. Cultural characteristics. Growth on nutrient broth-yeast extract agar (1 0), on potato-dex- trose agar, and on a synthetic medium supple- mented with yeast extract was slow, with visible colonies rarely appearing before 3 to 4 days. Yeast extract (Difco) was necessary for growth. Differentiation of colony character- istics was possible after about 5 days. Three colony types were observed on nutrient broth- 482 VOL. 24,1974 CORYNEBACTERIUM NEBRASKENSE SP.NOV. 483 FIG. 1 .  Electron micrographs of Corynebacterium nebraskense sp.nov., isolate DC-1. The line marker indicates 1 pm. yeast extract agar from primary isolations. All colony types were capucine or apricot-orange pigmented at room temperature (22 to 26 C). However, the intensity of pigmentation varied with the temperature and medium. The most frequent colony type was circular, convex, glistening, and butyrous with entire margins which were delimited from a central, darker zone. The second most frequent type was similar except that it lacked the outer margin. Both of these colony types averaged 3 t o  5 mm in diameter after 5 to  7 days. The rarest colony type (isolated only once) was much smaller, reaching a maximal diameter of 1 mm in 5 to 7 days, and also lacked the outer margin. Physiological and biochemical properties. Table 1 shows the characteristics of 15 isolates of the new bacterium in comparison with other species of phytopathogenic corynebacteria. The new bacterium can be differentiated from these other corynebacteria on the basis of fermenta- tion reactions, pigmentation, motility, growth on TTC and INT agar, bacteriophage sensitivity, and mole percent guanine plus cytosine. All isolates of the new pathogen were susceptible to one or more of the phages. At concentrations 10 to 100 times higher than those used in the routine tests reported here, one C. insidiosum strain was sensitive to  all four phages. The isolates were oxidase negative when tested as previously described (9). Temperature relationships. All isolates grew on nutrient broth-yeast extract agar at 10 C but not at 37 C. The optimal temperature for growth was between 24 and 28 C. Type strain description. Pleomorphic, gram- positive, nonmotile rods, approximately 0.5 by 2.0 pm, occurring singly or in pairs at generally obtuse angles. Orange-pigmented colonies on nutrient broth-yeast extract agar ( 10). Grows on 0.0005% iodonitrotetrazolium chloride agar but not on 0.005% triphenyltetrazolium chlo- ride agar. The optimal growth temperature is approximately 26 C. The oxidase reaction is negative. Acid is produced from glucose, su- crose, mannose, xylose, and galactose in 7 days; no acid is produced from arabinose, raffinose, or trehalose. This strain is sensitive to bacterio- phages GC8, GCl l ,  GCl2, and GC2A. The deoxyribonucleic acid contains 73.5 mol% guanine plus cytosine. The type strain is Fur-1; it has been deposited in the American Type Culture Collec- tion (ATCC), Rockville, Md., under the number 27822, and also in the National Collection of Plant Pathogenic Bacteria (NCPPB), Harpenden, England, under the number 2578. DISCUSSION The genus Corynebacterium is a heterogene- ous group of microorganisms characterized by morphological pleomorphism. On the basis of guanine plus cvtosine content, it is clear that, with the possible exception of C. jascians, the phy t opathogenic species of Cory nebacteeriurn differ from other species of this genus (3 ,  4, 6, 13). The plant pathogenic coryneform bacteria had been divided into two broad groups by Lelliott ( 6 ) ;  recently, on the basis of new data, he has divided them (personal communication) into five groups for the forthcoming 8th edition of Bergey’s Manual. The original grouping (6) included the nonmotile, slow-growing species C. fascians, C. insidiosum, C. michiganense, and C. sepedonicum ip pqe group and the flagellated, moderate- to rapid-growing phytopathogenic corynebacteria in a second group. In the new grouping, C. fascians is in group 1, the corynebacteria causing gumming of inflores- cences in group 2, C. sepedonicum is in group 3, C. insidiosum and C. michiganense are in group 4 ,  and the flagellated corynebacteria are in group 5. The differential characters used in this study are generally in accord with those c U 9 d 3 TABLE 1. Comparative characteristics of Corynebacterium nebraskense sp. nov. phytopathogenic and Covynebacteriurn specief 1 NJ ,- , AYpLdu,ctionb from ~ 1 II I I C. jlaccum faciens C. flaccurn faciens subsp. violaceurn C. jlaccum faciens subsp. auran- tiacum C. poinsettiae C mich2ganense C. insidiosum C. sepedonicum C. fascians 2 2 1 + + + + + + +l+l+ Raf- 1 1 Sal- 1 Tre- 1 Rham- 1 Lac- 1 Motil- hose Xylose 'icin halose nose tose ity + + +f + + I+ I' C. nebraskense I 15 I + 1 + 1 + 1 + 1 6/15 I 2 1 9/15 1 - 1 3/15 1 - 1 - - I I browth on:l pigment cytosine % Symbols: +, positive response; -, negative response; +, variable response at 7 days, positive at 10 to 14 days. Readings at 7 days; 24 * 2 C. Y, Yellow; 0, orange; B, blue granules; W, white on nutrient broth-yeast extract agar; V, violet. C. jlaccumfaciens Nb. 21 was positive; ATCC 6887 was negative. Shuster et al. (9). Positive except for strain Th. M. P. Starr, M. Mandel, and D. Kuhn (personal communication). One isolate produced a brown, diffusible pigment after prolonged incubation. h Yamada and Komagata (13) obtained the higher figures; Masuo and Nakagawa (7) obtained the lower figure. J Buoyant density in CsCl of 1.732 g/cm3 relative to Escherichia coli deoxyribonucleic acid (1.710 g/cm3). I + 171' 63-67; 73.51 53.9 M v VOL. 24,1974 CORYNEBACTERIUM NEBRASKENSE SP.NOV. 485 used by Lelliott for his revised grouping. Our data indicate that the new pathogen is closely related to C. michiganese and C. insidiosum. Such a relationship is also supported by preliminary serologic data (8). The new bacterium is distinct from C. michiganense on the basis of differences in xylose and salicin utilization, pigmentation, inability to  grow on triphenyltetrazolium chlo- ride agar, and bacteriophage sensitivity. Lelliot t ( 6 )  listed C. michiganense as not utilizing salicin, but all of our strains were positive. DaSilva and Holt (personal communication; reference 1) found that salicin utilization depended on the strain of C. michiganense tested. C. nebraskense can also be easily differentiated from C. insidiosum, the alfalfa wilt pathogen, on the basis of pigmentation, lactose and raffinose utilization, bacteriophage sensitivity, and growth on iodonitrotetrazolium chloride and triphenyltetrazolium chloride agars. The differences between the new bacterium and previously described species warrant , in our opinion, the establishment of a separate species for the new bacterium for which the name Corynebacterium nebraskense is proposed (ne.bras.ken’se. M.L. adj. nebraskensis pertain- ing to Nebraska), ACKNOWLEDGMENTS This investigation was supported by the Agricultural Research Service Cooperative Agreement No. 12-14- We thank F. L. Lukezic, B. D. Thyr, G. A. Strobel, M. P. Starr, and C. I .  Kado for some of the cultures used in this study, M. K.  Brakke for the electron microscopy, and R. Carlson for excellent technical assistance. 100-1 1 198 (34). REPRINT REQUESTS Address reprint requests to: Dr. Anne K. Vidaver, The University of Nebraska-Lincoln, Department of Plant Pathology, Agricultural Experiment Station, Lincoln. Neb. 68503. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. LITERATURE CITED DaSilva, G. A. N., and J. G. Holt. 1965. Numerical taxonomy of certain coryneform bac- teria. J. Bacteriol. 90:921-927. Goth, R. W. 1965. Puncture method for isolating bacterial blights of bean. Phytopathology 55:930- 931. Hill, L. R. 1966. A numerical taxonomy study of some corynebacteria and related organisms. J. Gen. Microbiol. 45:3140. Jones, L. A., and S. G. Bradley. 1964. Phenetic classification of Actinomycetes. Develop. Ind. Microbiol. 5 :  267-2 72. Kelman, A. 1954. The relationship of pathoge- nicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phyto- pathology 44: 693-695. Lelliott, R. A. 1966. The plant pathogenic coryneform bacteria. J.  Appl. Bacteriol. 29: 1 14- 118. Masuo, E., and T. Nakagawa. 1969. Numerical classification of bacteria. 111. Computer analysis of “coryneform bacteria.” (3) Classification based on DNA base compositions. Agr. Biol. Chem. Schuster, M. L., B. Hoff, M. Mandel, and I .  Lazar. 1973. Leaf freckles and wilt, a new corn disease, p. 176-191. Proc. 27th Annu. Corn and Sorghum Res. Congr., 14 to 15 December 1972, Chicago, Ill. In D. Wilkinson (ed.), American Seed Trade Association, Washington, D.C. Schuster, M. L., A. K. Vidaver, and M. Mandel. 1968. A purple-pigment-producing bean wilt bacterium, Corynebacterium jlaccumfaciens var. uiolaceum, n. var. Can. J. Microbiol. 14:423-427. Vidaver, A. K. 1967. Synthetic and complex media for the rapid detection of fluorescence of phy topathogenic pseudomonads: effect of the carbon source. Appl. Microbiol. 15:1523-1524. Vidaver, A. K., and M. L. Schuster. 1969. Characterization of Xanthomonas phaseoli bac- teriophages. J. Virol. 4:300-3 08. Wysong, D. S., A. K. Vidaver, H. Stevens, andD. Stenberg. 1973. Occurence and spread of an undescribed species of Corynebacterium patho- genic on corn in the western corn belt. Plant Dis. Reptr. 57:291-294. Yamada, K., and K. Komagata. 1970. Taxonomic studies on coryneform bacteria. 111. DNA base composition of coryneform bacteria. J. Gen. Appl. Microbiol. 16:215-224. 33: 1570-1576. Corynebacterium nebraskense, a New, Orange-Pigmented Phytopathogenic Species ANNE K. VIDAVER AND MANLEY MANDEL Deptzrtment of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68503, and The University of Texas System Cancer Center, M .  D .  Anderson Hospital and Tumor Institute, Houston, Texas 77025 Volume 24, no. 4, p. 484: Table title should read “Comparative characteristics of Coryne- Page 484, Table 1, column 19: Insert a “-” (minus) on data line 6. bacterium nebraskense sp. nov. and other phytopathogenic Corynebacterium species.” New Genus, Coprococcus, Twelve New Species, and Emended Descriptions of Four Previously Described Species of Bacteria from Human Feces LILLIAN V. HOLDEMAN AND W. E. C. MOORE Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Volume 24, no. 2, p. 264, column 2, lines 3 and 4: The sentence “No hydrogen was produced” should read “Copious hydrogen was produced.” 98 

\u3ci\u3eCorynebacterium nebraskense\u3c/i\u3e, a New,
Orange-Pigmented Phytopathogenic Species

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Papers in Plant Pathology Plant Pathology Department 
1974 
Corynebacterium nebraskense, a New, Orange-Pigmented 
Phytopathogenic Species 
Anne K. Vidaver 
University of Nebraska–Lincoln, avidaver1@unl.edu 
Manley Mandel 
University of Texas System Cancer Center 
Follow this and additional works at: https://digitalcommons.unl.edu/plantpathpapers 
 Part of the Plant Pathology Commons 
Vidaver, Anne K. and Mandel, Manley, "Corynebacterium nebraskense, a New, Orange-Pigmented 
Phytopathogenic Species" (1974). Papers in Plant Pathology. 253. 
https://digitalcommons.unl.edu/plantpathpapers/253 
This Article is brought to you for free and open access by the Plant Pathology Department at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Papers in Plant Pathology by 
an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
Int J Syst Bacteriol October 1974 24:482-485; doi:10.1099/00207713-24-4-482  
Copyright 1974 International Association of Microbiological Societies 
 
Corynebacterium nebraskense, a New, 
Orange-Pigmented Phytopathogenic Species 
 
Anne K. Vidaver  
Department of Plant Pathology, University of Nebraska–Lincoln, Lincoln, Nebraska, 68503, 
 
Manley Mandel 
The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 
77025  
 
Abstract 
A new species of Corynebacterium isolated from field corn is described. The isolates form a 
homogeneous group that is recognized as a new species, Corynebacterium nebraskense. The organism is 
characterized by orange-pig-mented colonies, inability to grow on 0.005% triphenyltetrazolium chloride 
agar, specific bacteriophage sensitivity, and a guanine plus cytosine content of 73.5 mol%. These and 
other characters differentiate this organism from other described phytopathogenic corynebacteria. The 
type strain of C. nebraskense is Fur-1 (= ATCC 27822 = NCPPB 2578).  
 
 
[Includes Errata from Int J Syst Bacteriol January 1975 25:98; doi:10.1099/00207713-25-1-98 ]  
INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY 
Oct. 1974, p. 482435 
Copyright 0 1974 International Association of Microbiological Societies 
Vol. 24 ,  No. 4 
Printed in U.S.A. 
Corynebacterium nebraskense, a New, Orange-Pigmented 
Phytopathogenic Species’ 
ANNE K. VIDAVER and MANLEY MANDEL 
Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska, 68503, and The University of 
Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77025 
A new species of Corynebacterium isolated from field corn is described. The 
isolates form a homogeneous group that is recognized as a new species, 
Corynebacterium nebraskense. The organism is characterized by orange-pig- 
mented colonies, inability to grow on 0.005% triphenyltetrazolium chloride 
agar, specific bacteriophage sensitivity, and a guanine plus cytosine content of 
73.5 mol%. These and other characters differentiate this organism from other 
described phytopathogenic corynebacteria. The type strain of C. nebraskense is 
Fur-1 (= ATCC 27822 = NCPPB 2578). 
A bacterial disease of field corn characterized 
by a distinctive foliar blight was first observed 
in south central Nebraska in 1969. In subse- 
quent years, the disease, called Goss’s bacterial 
wilt and blight (synonymous with leaf freckles 
and wilt), was found in other areas in Nebraska 
as well as in neighboring Iowa and Kansas (1 2). 
Preliminary investigation showed that a specific 
corynebacterium was associated with the dis- 
ease. The bases for differentiating this bac- 
terium from other phytopathogenic corynebac- 
teria are presented in this paper. A preliminary 
report, including host range data, has been 
published (8). 
MATERIALS AND METHODS 
Bacterial isolates. Fifteen isolates were obtained 
from different field-corn hybrids collected throughout 
Nebraska over a 3-year period. Isolations were made 
principally from leaf material, but also from stalks, 
using a modification of a direct puncture method (2) 
in which a sterile dissecting needle was used to 
puncture margins of lesions and to streak directly onto 
agar plates. The orange-pigmented isolates derived 
from such material reproduced disease symptoms 
when inoculated into stems of lO-day-old seedlings of 
either sweet corn (Golden Cross Bantam) or field corn 
(Nebraska 501-D). 
Methods. The methods used for characterization of 
the isolates were generally as described previously (9), 
except that for deoxyribonucleic acid extraction the 
cells were treated with lysozyme (100 pg/ml) in the 
presence of saline-ethylenediaminetetraacetate for 3 h 
Research conducted under project no. 21-21. 
Published as paper no. 3841, Journal Series, Ne- 
braska Agriculture Experiment Station. 
at 37 C prior to lysis with sodium dodecyl sulfate. All 
tests were repeated at least three times. 
Growth on 0.005% triphenyltetrazolium chloride 
agar or 0.0005% iodonitrotetrazolium chloride agar 
was determined by using Kelman medium ( 5 ) ,  except 
that glucose was added separately and the stock 
solutions of tetrazolium salts were filter-sterilized 
prior to use. 
Bacteriophage sensitivity was determined by spot 
tests with four purified phage isolates. The phages 
were isolated from soil or plant debris as previously 
described (11), using three different isolates of the 
new corynebacterium for initial phage enrichment. 
(These bacteriophages, @C8, OC11, @C12, and &2A, 
will be described in a separate communication.) Phages 
titering 0.5 x 10’ to 1.0 X lo7 plaque-forming units 
per ml were spotted on lawns of bacteria with a Cooke 
Microdiluter (Cooke Engineering Co.). 
RESULTS 
Morphology. Cells of the new isolates were 
gram-positive, pleomorphic rods characteristic 
of coryneform bacteria. All isolates were 
nonmotile and without flagella (Fig. 1). Cells 
from 40-h-old cultures grown at 24 C were 0.25 
to 0.5 by 0.75 to 2.7 pm, with average 
dimensions of 0.5 by 2.0 pm, as determined by 
light and electron microscopy. 
Cultural characteristics. Growth on nutrient 
broth-yeast extract agar (1 0), on potato-dex- 
trose agar, and on a synthetic medium supple- 
mented with yeast extract was slow, with 
visible colonies rarely appearing before 3 to 4 
days. Yeast extract (Difco) was necessary for 
growth. Differentiation of colony character- 
istics was possible after about 5 days. Three 
colony types were observed on nutrient broth- 
482 
VOL. 24,1974 CORYNEBACTERIUM NEBRASKENSE SP.NOV. 483 
FIG. 1 .  Electron micrographs of Corynebacterium nebraskense sp.nov., isolate DC-1. The line marker indicates 
1 pm. 
yeast extract agar from primary isolations. All 
colony types were capucine or apricot-orange 
pigmented at room temperature (22 to 26 C). 
However, the intensity of pigmentation varied 
with the temperature and medium. The most 
frequent colony type was circular, convex, 
glistening, and butyrous with entire margins 
which were delimited from a central, darker 
zone. The second most frequent type was 
similar except that it lacked the outer margin. 
Both of these colony types averaged 3 t o  5 mm 
in diameter after 5 to  7 days. The rarest colony 
type (isolated only once) was much smaller, 
reaching a maximal diameter of 1 mm in 5 to 7 
days, and also lacked the outer margin. 
Physiological and biochemical properties. 
Table 1 shows the characteristics of 15 isolates 
of the new bacterium in comparison with other 
species of phytopathogenic corynebacteria. The 
new bacterium can be differentiated from these 
other corynebacteria on the basis of fermenta- 
tion reactions, pigmentation, motility, growth 
on TTC and INT agar, bacteriophage sensitivity, 
and mole percent guanine plus cytosine. 
All isolates of the new pathogen were 
susceptible to one or more of the phages. At 
concentrations 10 to 100 times higher than 
those used in the routine tests reported here, 
one C. insidiosum strain was sensitive to  all four 
phages. 
The isolates were oxidase negative when 
tested as previously described (9). 
Temperature relationships. All isolates grew 
on nutrient broth-yeast extract agar at 10 C but 
not at 37 C. The optimal temperature for 
growth was between 24 and 28 C. 
Type strain description. Pleomorphic, gram- 
positive, nonmotile rods, approximately 0.5 by 
2.0 pm, occurring singly or in pairs at generally 
obtuse angles. Orange-pigmented colonies on 
nutrient broth-yeast extract agar ( 10). Grows 
on 0.0005% iodonitrotetrazolium chloride agar 
but not on 0.005% triphenyltetrazolium chlo- 
ride agar. The optimal growth temperature is 
approximately 26 C. The oxidase reaction is 
negative. Acid is produced from glucose, su- 
crose, mannose, xylose, and galactose in 7 days; 
no acid is produced from arabinose, raffinose, 
or trehalose. This strain is sensitive to bacterio- 
phages GC8, GCl l ,  GCl2, and GC2A. The 
deoxyribonucleic acid contains 73.5 mol% 
guanine plus cytosine. 
The type strain is Fur-1; it has been 
deposited in the American Type Culture Collec- 
tion (ATCC), Rockville, Md., under the number 
27822, and also in the National Collection of 
Plant Pathogenic Bacteria (NCPPB), Harpenden, 
England, under the number 2578. 
DISCUSSION 
The genus Corynebacterium is a heterogene- 
ous group of microorganisms characterized by 
morphological pleomorphism. On the basis of 
guanine plus cvtosine content, it is clear that, 
with the possible exception of C. jascians, the 
phy t opathogenic species of Cory nebacteeriurn 
differ from other species of this genus (3 ,  4, 6, 
13). 
The plant pathogenic coryneform bacteria 
had been divided into two broad groups by 
Lelliott ( 6 ) ;  recently, on the basis of new data, 
he has divided them (personal communication) 
into five groups for the forthcoming 8th edition 
of Bergey’s Manual. The original grouping (6) 
included the nonmotile, slow-growing species C. 
fascians, C. insidiosum, C. michiganense, and C. 
sepedonicum ip pqe group and the flagellated, 
moderate- to rapid-growing phytopathogenic 
corynebacteria in a second group. In the new 
grouping, C. fascians is in group 1, the 
corynebacteria causing gumming of inflores- 
cences in group 2, C. sepedonicum is in group 
3, C. insidiosum and C. michiganense are in 
group 4 ,  and the flagellated corynebacteria are 
in group 5. The differential characters used in 
this study are generally in accord with those 
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v 
VOL. 24,1974 CORYNEBACTERIUM NEBRASKENSE SP.NOV. 485 
used by Lelliott for his revised grouping. Our 
data indicate that the new pathogen is closely 
related to C. michiganese and C. insidiosum. 
Such a relationship is also supported by 
preliminary serologic data (8). 
The new bacterium is distinct from C. 
michiganense on the basis of differences in 
xylose and salicin utilization, pigmentation, 
inability to  grow on triphenyltetrazolium chlo- 
ride agar, and bacteriophage sensitivity. Lelliot t 
( 6 )  listed C. michiganense as not utilizing 
salicin, but all of our strains were positive. 
DaSilva and Holt (personal communication; 
reference 1) found that salicin utilization 
depended on the strain of C. michiganense 
tested. C. nebraskense can also be easily 
differentiated from C. insidiosum, the alfalfa 
wilt pathogen, on the basis of pigmentation, 
lactose and raffinose utilization, bacteriophage 
sensitivity, and growth on iodonitrotetrazolium 
chloride and triphenyltetrazolium chloride 
agars. 
The differences between the new bacterium 
and previously described species warrant , in our 
opinion, the establishment of a separate species 
for the new bacterium for which the name 
Corynebacterium nebraskense is proposed 
(ne.bras.ken’se. M.L. adj. nebraskensis pertain- 
ing to Nebraska), 
ACKNOWLEDGMENTS 
This investigation was supported by the Agricultural 
Research Service Cooperative Agreement No. 12-14- 
We thank F. L. Lukezic, B. D. Thyr, G. A. Strobel, 
M. P. Starr, and C. I .  Kado for some of the cultures 
used in this study, M. K.  Brakke for the electron 
microscopy, and R. Carlson for excellent technical 
assistance. 
100-1 1 198 (34). 
REPRINT REQUESTS 
Address reprint requests to: Dr. Anne K. Vidaver, 
The University of Nebraska-Lincoln, Department of 
Plant Pathology, Agricultural Experiment Station, 
Lincoln. Neb. 68503. 
1. 
2. 
3. 
4. 
5. 
6. 
7. 
8. 
9. 
10. 
11. 
12. 
13. 
LITERATURE CITED 
DaSilva, G. A. N., and J. G. Holt. 1965. 
Numerical taxonomy of certain coryneform bac- 
teria. J. Bacteriol. 90:921-927. 
Goth, R. W. 1965. Puncture method for isolating 
bacterial blights of bean. Phytopathology 55:930- 
931. 
Hill, L. R. 1966. A numerical taxonomy study of 
some corynebacteria and related organisms. J. 
Gen. Microbiol. 45:3140. 
Jones, L. A., and S. G. Bradley. 1964. Phenetic 
classification of Actinomycetes. Develop. Ind. 
Microbiol. 5 :  267-2 72. 
Kelman, A. 1954. The relationship of pathoge- 
nicity in Pseudomonas solanacearum to colony 
appearance on a tetrazolium medium. Phyto- 
pathology 44: 693-695. 
Lelliott, R. A. 1966. The plant pathogenic 
coryneform bacteria. J.  Appl. Bacteriol. 29: 1 14- 
118. 
Masuo, E., and T. Nakagawa. 1969. Numerical 
classification of bacteria. 111. Computer analysis 
of “coryneform bacteria.” (3) Classification based 
on DNA base compositions. Agr. Biol. Chem. 
Schuster, M. L., B. Hoff, M. Mandel, and I .  Lazar. 
1973. Leaf freckles and wilt, a new corn disease, 
p. 176-191. Proc. 27th Annu. Corn and Sorghum 
Res. Congr., 14 to 15 December 1972, Chicago, 
Ill. In D. Wilkinson (ed.), American Seed Trade 
Association, Washington, D.C. 
Schuster, M. L., A. K. Vidaver, and M. Mandel. 
1968. A purple-pigment-producing bean wilt 
bacterium, Corynebacterium jlaccumfaciens var. 
uiolaceum, n. var. Can. J. Microbiol. 14:423-427. 
Vidaver, A. K. 1967. Synthetic and complex 
media for the rapid detection of fluorescence of 
phy topathogenic pseudomonads: effect of the 
carbon source. Appl. Microbiol. 15:1523-1524. 
Vidaver, A. K., and M. L. Schuster. 1969. 
Characterization of Xanthomonas phaseoli bac- 
teriophages. J. Virol. 4:300-3 08. 
Wysong, D. S., A. K. Vidaver, H. Stevens, andD. 
Stenberg. 1973. Occurence and spread of an 
undescribed species of Corynebacterium patho- 
genic on corn in the western corn belt. Plant Dis. 
Reptr. 57:291-294. 
Yamada, K., and K. Komagata. 1970. Taxonomic 
studies on coryneform bacteria. 111. DNA base 
composition of coryneform bacteria. J. Gen. 
Appl. Microbiol. 16:215-224. 
33: 1570-1576. 
Corynebacterium nebraskense, a New, Orange-Pigmented 
Phytopathogenic Species 
ANNE K. VIDAVER AND MANLEY MANDEL 
Deptzrtment of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68503, and The University of 
Texas System Cancer Center, M .  D .  Anderson Hospital and Tumor Institute, Houston, Texas 77025 
Volume 24, no. 4, p. 484: Table title should read “Comparative characteristics of Coryne- 
Page 484, Table 1, column 19: Insert a “-” (minus) on data line 6. 
bacterium nebraskense sp. nov. and other phytopathogenic Corynebacterium species.” 
New Genus, Coprococcus, Twelve New Species, and Emended 
Descriptions of Four Previously Described Species of Bacteria 
from Human Feces 
LILLIAN V. HOLDEMAN AND W. E. C. MOORE 
Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 
Volume 24, no. 2, p. 264, column 2, lines 3 and 4: The sentence “No hydrogen was produced” 
should read “Copious hydrogen was produced.” 
98 


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\u3ci\u3eCorynebacterium nebraskense\u3c/i\u3e, a New, Orange-Pigmented Phytopathogenic Species

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