INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
Apr. 1986, p. 241-245
0020-7713/86/020241-05$02.00/0
Copyright 0 1986, International Union of Microbiological Societies
Vol. 36, No. 2
Deoxyribonucleic Acid Homologies of Hyphomicrobium spp.
Hyphomonas spp. and Other Hyphal, Budding Bacteria
M I N E R GEBERS, BEATE MARTENS, UTA WEHMEYER, AND PETER HIRSCH*
Institut fur Allgemeine Mikrobiologie, Universitat Kiel, 0-2300 Kiel, Federal Republic of Germany
The levels of genetic relatedness of 19 Hyphomicrobium strains which utilize one-carbon compounds were
determined by deoxyribonucleic acid (DNA)-DNA hybridization in solution under optimal conditions (S1
nuclease technique). Most of these hyphomicrobia fell into four groups with high levels of relatedness (level of
homology within each group, 86 to 110%). These groups were only distantly related to each other (levels of
homology between groups, 1 to 9%). Three additional groups of C1-utilizing hyphomicrobia were represented
by only one strain each. In addition, the levels of DNA-DNA homology of four Hyphomonas species and 11
Hyphomicrobium-Hyphomonas-likeisolates were determined. Seven of these isolates formed three groups
containing two or three strains each; the level of homology within each group was 94 to 120%. These groups
of peptide-utilizing strains were related to Hyphomonas spp. at a DNA-DNA homology level of 13 to 43 % ;thus,
they represented new species of Hyphomonas. Four of the isolates had less than 10% DNA homology with either
Hyphomicrobium reference strains or Hyphomonas spp. Strain B-1408 was distinguished by its DNA base
composition of 46.19 mol% guanine plus cytosine, which is 14 mol% below the average base composition of
Hyphomicrobium spp. or Hyphomonas spp. The levels of genetic relatedness of other hyphal, budding bacteria,
such as Pedomicrobium spp., genus F, Rhodomicrobium vannielii, and genus T, to Hyphomicrobium sp. strain
MC-750 were too low to be evaluated by DNA-DNA hybridization techniques.
The genus Hyphomicrobium was described in 1898 as the
first of a group of hyphal, budding bacteria (35). Later,
morphologically similar organisms belonging to the genera
Rhodomicrobium ( 3 , Hyphomonas (29), and Pedomicrobium (1, 2) were discovered. While Rhodomicrobium
spp. were clearly distinguished by their ability to produce
photosynthetic pigments ( 5 , 23, 28), the remaining three
genera could not be differentiated with certainty by morphological and physiological properties (6, 28). However,
Pedomicrobium spp. could be distinguished from other
budding and hyphal bacteria by means of deoxyribonucleic
acid (DNA) base composition and nucleotide distribution (7,
lo), genome size (19), and DNA-DNA homology (8, 9). The
differentiation of Hyphomicrobium spp. from Hyphomonas
spp. by means of morphology, DNA base composition (9,
10,21), nucleotide distribution (lo), and genome size (19,27)
was still afflicted with uncertainty. However, DNA-DNA
hybridization experiments (9, 26) performed with 20
Hyphomicrobium strains and 3 strains of Hyphomonas spp.
revealed 2% homology, at most. Ribosomal ribonucleic
acid-DNA hybridization between Hyphomicrobium strain
B-522 and three Hyphomonas strains detected high thermal
denaturation (T,) values, which indicated distant relatedness between these representatives of the two genera (25).
Unfortunately, the first description of Hyphomicrobium
vulgare (35)did not provide information about the utilization
of one-carbon compounds by this organism. On the other
hand, most of the isolates presently named Hyphomicrobium
specialize on these compounds (11, 14, 16). Therefore, in
practice the term Hyphomicrobium has been applied to
Cl-utilizing strains, while Hyphomonas is used for amino
acid utilizers (12, 28, 29). Since this way of distinguishing
these genera has not been officially confirmed, we chose to
rely on DNA-DNA homology to determine the taxonomic
positions of 11 Hyphomicrobium-Hyphomonas-likeisolates
and two new Hyphomonas species (36). Seven of these
strains had levels of DNA homology of between 13 and 43%
* Corresponding author.
when they were compared with Hyphomonas polymorpha
PS-728T (T = type strain), Hyphomonas neptunium
LE-670T, or Hyphomonas jannaschiana VP-1382, which
were previously shown to be genetically related to other
Hyphomonas species (9).
Additionally, we determined the genetic relatedness of 19
C1-utilizing Hyphomicrobium strains, which split into seven
groups, corresponding to previous results (10, 21, 26).
The DNAs of representative strains of Pedomicrobium
spp., Rhodomicrobium vannielii, and two new genera, preliminarily designated genera F and T (lo), were hybridized
with Hyphomicrobium sp. strain MC-750 DNA to confirm
that they are distinct from the C1-utilizing hyphomicrobia.
MATERIALS AND METHODS
Strains and cultivation. All of the bacterial strains which we
used were supplied by the culture collection of the Institut fur
Allgemeine Mikrobiologie, Kiel, Federal Republic of Germany (Tables 1 and 2). Strains SCH-1427, SCH-1495, and
SCH-1497 were isolated from brackish water by H.
Schlesner, Kiel. Strains SCH-1427 and SCH-1495 were
grown in medium 387+1/4 ASW (10). Strains SCH-1497 and
B-1408 (strain B-1408 was isolated from brackish water by W.
Bockelmann, Kiel) were grown in PYGV medium (33)
supplemented with 250 ml of artificial seawater per liter (20)
and 50 ml of Tris(hydroxymethy1)aminomethane (10) per
liter. Strain P-1258 was isolated from freshwater by J. S.
Poindexter, New York, N.Y., and was cultivated in PSM
medium (6). All of these strains were grown aerobically in the
dark at 30°C. The origins and growth conditions of all of the
other strains studied have been published previously (6, 10).
DNA preparation. The methods used for cell wall disintegration, DNA extraction, and purification have been described previously for most strains (8, 10). Strains
SCH-1427, SCH-1495, SCH-1497, and B-1408 were lysed by
enzyme treatment E (lo), while strain P-1258 was disintegrated mechanically by the cell mill A procedure (10). All
DNAs were sheared to an average double-stranded fragment
M , of 420,000 to 450,000 (8).
24 1
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242
INT.J. SYST.BACTERIOL.
GEBERS ET AL.
TABLE 1. Levels of DNA-DNA homology of Hyphomicrobium spp. and other hyphal, budding bacteria
Source of unlabeled DNA'
Hyphomicrobium spp.
New isolate
New isolate
New isolate
Pedomicrobium ferrugineum
Pedomicrobium manganicum
Pedomicrobium-like
Genus F
Rhodomicrobium vannilii
DSM 162T
Genus T
DNAbase
MC-750
MEV-533gr
EA-617
WH-563
NQ-521gr
ZV -622
ZV -620
ZV -580
KB -677
MC-651
CO-582
CO-559
CO-558
1-551
F-550
H-526
B-522
T-854
Wi -926
SW-815
SW-814
SW-808
P-1196
P-1225
S- 1290T
E- 1129T
ST-1306
WD-1355
869
868
SCH-1315
117gT
1300
ST-1307
27500
27488
27483
27498
27497
27492
27491
27489
27485
27484
33116
33122
33119
33121T
17100T
61.38'
64.69'
63.46'
63.09'
64.11'
64.8d
64.3d
61.77'
62.41'
62.91'
60.54'
60.02'
59.78'
59.40"
59.91'
59.53'
59.34'
57.88'
59.29'
60.23'
59.11'
55.15'
65.7
649
65.v
65.6
65.00'
62.80'
64.41'
64.72'
61.15'
62.8g
62.82'
61.91'
% Homology with labeled DNA from strain?
100
24
20
19
18
14
12
11
11
10
7
5
5
5
4
7
3
5
5
3
5
5
4
5
5
4
103
110
92
100
8
3
3
9
100
100
91
4
1
2
1
1
0
1
2
1
1
1
0
2
4
2
4
5
5
0
0
0
4
0
87
98
91
86
106
88
100
0
4
1
1
0
0
0
0
101
100
0
A
B
B
B
B
C
C
C
D
D
E
E
E
E
E
E
E
F
G
5
4
3
3
2
1
1
~
IFAM, Institut fur Allgemeine Mikrobiologie, Kiel, Federal Republic of Germany; DSM, Deutsche Sammlung von Mikroorganisrnen, Gottingen, Federal
Republic of Germany; ATCC, American Type Culture Collection, Rockville, Md.
Mean of at least two reactions corrected for the background values obtained with the self-reassociation controls. The self-reassociation values obtained by
hybridization with unrelated E. coli K-12 DNA were as follows: strain MC-750,5.9%; strain NQ-521gr, 1.1%; strain ZV-622,0.7%; strain B-522, 1.2%; strain SW814, 1.3%.
Data obtained by T, determinations (10).
Data derived from buoyant density determinations (21).
Data obtained by T, determinations (9).
Data obtained by T, determinations (7).
Data derived from buoyant density determinations (23).
a
DNA base composition. At least six T , profiles of each
DNA species investigated were recorded at 260 nm with a
Gilford model 250 spectrophotometer as described previously (7). From the T, values of these curves the molar
fractions of the DNA bases (guanine-plus-cytosine [G + C]
contents) were calculated by using the following equation
(22): G+C content = (T, in 0.1X SSC/50.2) - 0.990. (IX
SSC is 0.15 M NaCl plus 0.015 M sodium citrate.)
DNA labeling. In vivo labeling of Hyphomicrobium sp.
strain MC-750 DNA was achieved by adding (per milliliter of
exponentially growing culture) 118 kBq each of [methyl3H]thymidine and [8-3H]adenine (specific activities, 684.5
GBq/mmol and 925 GBq/mmol, respectively; Amersham
Buchler, Braunschweig, Federal Republic of Germany).
Extracted, sheared D N A was further purified by
hydroxyapatite column chromatography (3). In vitro iodination of the DNAs of strains NQ-521gr, B-522, ZV-622,
LE-670T, PS-728T, SW-814, SW-815, SCH-1325T, VP-1382,
SCH-1416, and SCH-1417 was done by using the method of
Selin et al. (32) and Na1251(specific activity, 603 MBq/pg of
iodine; Amersham Buchler).
DNA-DNA reassociation experiments. DNA-DN A reassociation experiments were carried out under optimal conditions in solution as described previously (8, 9). Unreacted
DNA fragments were digested with S1 nuclease (4). Reassociated DNA strands were precipitated with 5.5% (wt/vol)
trichloroacetic acid. The hybridization conditions used are
shown in Table 3. Nonspecific binding levels and amounts of
available DNA were determined by using 150 pg of Escherichia coli K-12 DNA and the labeled DNAs. Radioactivity
was expressed as the percentage of available input (Tables 1
and 2). All reactions were performed at least three times.
Tritium-labeled hybrids were counted with a Packard TriCarb model 3390/544 liquid scintillation counter; iodine
decay values were determined with a Berthold model
MAG315 gamma counter.
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DNA HOMOLOGIES OF HYPHAL, BUDDING BACTERIA
VOL. 36, 1986
243
TABLE 2. Levels of DNA-DNA homology of Hyphomonas spp. and other hyphal, budding bacteria
Source of unlabeled DNA"
% Homologv with labeled DNA from strainf
strain
Hyphomonas
polymorpha
Hyphomonas
neptunium
New isolate
New isolate
Hyphomonas
jannaschiana
New isolate
New isolate
New isolate
New isolate
New isolate
Hyphomonas
oceanitis
New isolate
New isolate
New isolate
New isolate
-V..'rV"...V..
(mol% G+C)b PS-728T LE-670'
PS-72gT
33881T
60.07"
PR-727
LE-670T
33880
15444"
60.05"
60.40"
33882
58.w
58.94
6o.B"
SCH-1416
SCH-1497
VP-1382
SW-815
SW-814
SX-821
SCH-1417
SCH-1495
SCH-1325T 33879T
60.29
59.11f
59.66
60.05"
58.77
58.98"
SCH-1427
B-1408
P-1258
SW-808
60.30
46.19
58.53
55.15"
100
10
(28,21)'
(Ill)" (40,20)e
6
100
(23)"
15
26
13
27
(29)"
(15)"
SCH-1416 VP-1382 SW-815 SW-814 SCH-1417 SCH-1325T MC-750
10
3
1
0
i
1
27
2
4
0
4
2
1
100
94
1
6
1
100
3
3
25
2
0
3
2
3
2
10
5
6
100
120
5
7
3
4
100
7
0
0
0
1.2
15
0
1
0
0.7
2
6
3
43
100
2
0
0
(6)"
7
9
9
(3)"
7
2
3
1
38
20
13
9
96
13
9
3
3
2
1
0
0.8
6
0
3
0
1.0
3
0
2
0
0.9
0
0
0
0
2.1
5
3
5
2
1.5
101
100
98
0
1.3
5
5
2
3
5.9
IFAM, Institut fur Allgemeine Mikrobiologie, Kiel, Federal Republic of Germany; ATCC, American Type Culture Collection, Rockville, Md.
~
- 0.99.
Mean of at least six T , determinations calculated by using the following equation (22): G+C content = (T, in 0 . 1 SSCbO.2)
Mean of at least two reactions corrected for the background value obtained with the self-reassociation controls. The self-reassociation values obtained by
hybridization with unrelated E. coli K-12 DNA were as follows: strain PS-728', 2.1%; strain LE-670T, 1.5%; strain SCH-1416, 0.8%; strain VP-1382, 1.0%; strain
SW-815, 0.9%; strain SW-814, 1.3%; strain SCH-1417, 1.2%; strain SCH-1325', 0.7%; strain MC-750, 5.9%.
Data from reference 9. The homology values in parentheses are included for comparison.
Data in parentheses from references 9 and 26.
Data from reference 10.
a
RESULTS AND DISCUSSION
The C1-utilizing Hyphomicrobium strains which we studied showed 0 to 24 or 86 to 110% DNA-DNA homology to
each other (Table 1). The hybridization experiments revealed that there were four groups of highly related strains,
which were interconnected at only low levels of homology.
Group E consisted of soil hyphomicrobia which had been
isolated in the presence or absence of carbon monoxide (13,
15, 21). These strains were 86 to 106% homologous to strain
B-522 and were distantly related (4 to 11%) to strain MC-750.
Both group D strains, strain KB-677, which was isolated
from sewage (18), and strain MC-651, which was isolated
from soil (21), were about as closely related to strain MC-750
as group E hyphomicrobia were, but neither isolate had a
significant level of homology to strain B-522 or strain
ZV-622. Therefore, they represent a separate group.
The strains of group C, which were isolated from swamp
TABLE 3. Conditions used for DNA-DNA hybridization reactions
Source of
labeled
DNA
(strain)
MC-750
NQ-521gr
B-522
ZV-622
SW-814
PS-72gT
LE-670T
SCH-1416
VP-1382
SW-815
SCH-1417
SCH-1325T
(I
R
~
$
Amt Of
labeled DNA
Per
hybridizatibn
Ratio of
labeled DNA
11,100 (13,400) 11,100 (13,400) 0.25 or 0.28
(0.23)
0.10
1.15 X lo6
6.71 X lo6
0.07
1.45 x lo6
1.45 x lo6
0.10
1.33 X lo6
4.08 X lo6
0.10
4.51 x lo6
1.35 X lo6
0.10
5.2 x lo6
lo6
0.10
5.2 X lo6
lo6
0.10
lo6
7.3 x lo6
0.10
5.3 x lo6
lo6
0.10
6.0 X lo6
lo6
0.10
4.2 X lo6
lo6
0.10
lo6
5.4 x lo6
1:600 or
1536 (1:652)
1:1,500
1:2,143
1:1,500
1:1,500
1:1,500
1:1,500
1:1,500
1:1,500
1:1,500
1:1,500
1:1,500
sp act after
~ labeling
~ e (cpm/Fg
of DNA)
Sp act for
hybridization
(CDm,Up of
reaction (ugl
~
3H
12'1
1251
1251
1251
1251
1251
'*'I
1251
1251
1251
1251
to unlabeled
DNA
Amt of
S1
Incubation
Filter
time (h)
nuclease
(U)
temp ("C)
type
69
16
1.250"
51
GFIC~
70
68
69
68
69
69
68
69
69
69
68
20
20
20
20
20
20
20
20
20
20
20
500'
500'
500'
500'
500"
500'
500'
500'
500'
500'
500'
56
54
55
54
55
55
54
55
55
55
54
BA83d
BA83d
BA83"
BA83"
BA83d
BA83d
BA83"
BA83d
BA83d
BA83"
BA83"
Hybridization Incubation
temp ("C)
_
.
Obtained from Miles Laboratories, Inc., Elkhart, Ind.
Obtained from Whatman, Maidstone, Kent, United Kingdom.
Obatined from Sigma Chemie, Taufkirchen, Federal Republic of Germany.
Obtained from Schleicher & Schuell, Dassel, Federal Republic of Germany.
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244
INT. J. SYST.BACTERIOL.
GEBERS ET AL.
soil by G. A. Zavarzin (personal communication), were
almost identical to each other. They were only distantly
related to strain MC-750 (strain ZV-580, 14%) and to the
other reference strains. The exposed surface antigens were
serologically identical (30). These three strains may be
descendants of one original isolate (37).
The group B strains were isolated from brackish water (13,
17). They were homologous to representative strain NQ521gr. This was not surprising, since strains NQ-521gr and
EA-617 were subcultures of the original strain B of Mevius
(24) and were maintained over a period of 16 years by
different investigators.
Obviously, these cultures did not suffer dramatic changes
in their DNA base sequences during subculturing, but the
serological relationship of strains NQ-521gr and MEV-533gr
was completely lost (30). However, previously published
data on the levels of DNA-DNA homology between strain
EA-617 and strain NQ-521gr (101%) and between strain
EA-617 and strain MEV-533s (70%) differed by 30%; these
values were calculated from filter hybridization data by
Moore and Hirsch (26).
Strains MC-750, Wi-926, and T-854 were not sufficiently
homologous to any other hyphomicrobia to be included in
one of the four groups.
Supported by the results of previous studies on DNA base
composition (21) and nucleotide distribution (lo), on DNADNA homology (26), and on ribosomal RNA-DNA relatedness (25), our results suggest that the C1 utilizing
Hyphomicrobium strains should be separated according to
the group designations (groups A through G) (Table 1).
Numerical taxonomy studies (P. Hirsch and R. R. Colwell,
unpublished data) revealed that strain MC-750 is the
hyphothetical median organism of 84 Hyphomicrobium
strains and is the strain that is most similar to the original
description of Hyphomicrobium vulgare (35). If levels of
DNA-DNA homology of 20 or 25% were used as the minimal
values for relatedness on the generic level (31, 34), none of
our groups B through G could be classified as
Hyphomicrobium spp. Furthermore, most of the presently
known C1-utilizing hyphomicrobia would have to be described as members of new genera. However, with our
still-limited knowledge of the relatedness of hyphal and
budding bacteria and with more than 80 intensely investigated strains still awaiting proper classification, we suggest
some caution in applying generic limits to hyphal, budding
bacteria by using definitions employed for different bacterial
groups. From a practical point of view, we recommend
designating groups A through E (Table 1) a s
Hyphomicrobium species, to recognize their distant relatedness to this genus. Eventually further changes in their
taxonomic rank (species designations) should await collection of more comprehensive data. The proper classification
of groups F and G will require further investigation.
The levels of DNA-DNA homology of Hyphomonas spp.
and some hew isolates (Table 2) which grew on media
containing peptides confronted us with the same questions
concerning taxonomic ranking as discussed above. Recently, Hyphomonas neptunium LE-670T was transferred
from the genus Hyphomicrobium to Hyphomonas (28) because of its morphological and physiological characteristics
(9, 12, 28, 29) and levels of DNA-DNA homology (9, 26).
Data from these previous homology studies were included
for comparison in Table 2. The present hybridization experiments revealed 6 and 10% levels of homology between
DNAs of Hyphomonas polymorpha and Hyphomonas
neptunium; the previous results were between 20 and 40%.
Although technical differences between the experiments
(e.g., membrane filter technique versus S1 nuclease technique; DNA fragment M , of 600,000 versus DNA fragment
M , of 420,000 to 450,000) reduced the comparability of the
results, a fixed limit for generic relatedness of 20 or 25%
DNA-DNA homology does not seem to be applicable to
these bacteria. Until stronger evidence calls for a change, we
recommend keeping strain LE-670T as Hyphomonas
neptunium. This was confirmed by levels of homology of 13
to 27% between new isolates SCH-1416 and SCH-1497 and
both Hyphomonas type strains (strains PS-728 and LE-670).
Strains SCH-1416 and SCH-1497 were 94% homologous to
each other and thus may represent a new Hyphomonas
species.
Strains SW-815 and SX-821 were 43 and 38% homologous,
respectively, to Hyphomonas jannaschiana VP-1382 and
were completely homologous to each other and to strain
SW-814. These three strains, which were isolated from
brackish water (13), were only 5% homologous to
Hyphomicrobium sp. strain MC-750 (Table 1); they may
constitute an additional new species of Hyphomonas.
Homologous strains SCH-1417 and SCH-1495 shared 20
and 13% DNA homology, respectively, with Hyphomonas
jannaschiana VP-1382, as well as 13 and 9% DNA homology, respectively, with strain SW-815 and 9% DNA homology with Hyphomonas neptunium LE-670T. Although these
homology values were below the arbitrary limit of 20 or 25%
for generic relatedness (31,34), we suggest that both of these
strains should be added to the genus Hyphomonas.
The levels of DNA homology between Hyphomonas
oceanitis SCH-1325T and other Hyphomonas strains were
less than 10% (Table 2). Nevertheless, we suggest keeping
this strain as a Hyphomonas species, because its morphology, physiology, and lysing behavior of the cell envelope
suggest some relatedness to Hyphomonas (36).
Strain SCH-1427 was 15% homologous to Hyphomonas
oceanitis SCH-1325T and less than 8% homologous to all
other reference DNAs; thus, its taxonomic position remains
unclear.
Red-pigmented strain B-1408 was clearly different from all
other strains studied on the basis of its DNA base composition (46.19 mol% G+C) (Table 2). Such a low G+C content
is quite unusual for the hyphal, budding bacteria which we
studied; on@ orange-red-pigmented strain SCH-1415, which
was investigated previously (lo), had a similar value (46.34
mol% G+C). Most likely, both of these strains belong to a
new genus of pigmented, hyphal, budding bacteria.
Unlike all Hyphomonas strains which we studied, the cell
envelopes of strains P-1258 and SW-808 resisted enzymatic
and detergent treatments, so the cells had to be disrupted by
grinding in a cell mill (10). Strain SW-808 was not homologous to either the C1-utilizinghyphomicrobia (Table 1)or the
Hyphomonas reference strains (Table 2).
Reciprocal hybridization reactions between the reference
strains of the C1-utilizing hyphomicrobia and Hyphomonas
spp. revealed different levels of DNA-DNA homology for
each pair of strains depending on which genome carried the
radioactive label (Table 1). These deviations correlated to
some extent with the different sizes of the genomes (19), so
that, if the smaller genome was labeled, it yielded high
homology values and vice versa. The reciprocal reactions
between strain NQ-52lgr and strain ZV-622 gave almost
identical results (8 and 9% homology); since the genome size
of strain ZV-580 was similar to that of strain NQ-521gr (19),
we assume that the genome size of strain ZV-622 also falls
into this range.
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VOL. 36, 1986
DNA HOMOLOGIES OF HYPHAL, BUDDING BACTERIA
All of the other genera of hyphal, budding bacteria tested
were only 1 to 5% homologous to Hyphomicrobium sp.
strain MC-750. The genetic distances between these genera
will be determined by ribosomal ribonucleic acid-DNA hybridization and by 16s ribosomal ribonucleic acid oligonucleotide sequencing.
ACKNOWLEDGMENTS
We thank J. L. Johnson, Blacksburg, Va., and R. L. Moore,
Calgary, Alberta, Canada, for helpful discussions on DNA labeling
and hybridization procedures.
Part of this work was supported by a grant from the Deutsche
Forschungsgemeinschaft, Bonn-Bad Godesberg, Federal Republic
of Germany, to P.H.
LITERATURE CITED
1. Aristovskaya, T. V. 1961. Accumulation of iron in breakdown of
organomineral humus complexes by microorganisms. Dokl.
Akad. Nauk SSSR 136:954-957. (In Russian.)
2. Aristovskaya, T. V. 1963. On the decomposition of organic
mineral compounds in podzolic soils. Pochvoved. Akad. Nauk
SSSR 1:3042. (In Russian.)
3. Bernardi, G. 1969. Chromatography of nucleic acids on
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