БІОЛОГІЧНІ ВЛАСТИВОСТІ МІКРОСИМБІОНТІВ АРАХІСУ, ПОШИРЕНИХ У ҐРУНТАХ УКРАЇНИ
Objective. Study the presence of nodule bacteria in the soils of Ukraine, capable of nodulatinggroundnut, isolate new strains of rhizobia from peanut nodules, study their morphological, culturaland serological properties and the ability to form symbiosis with different legumes. Methods. Microbiologi...
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| Дата: | 2021 |
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| Формат: | Стаття |
| Мова: | Англійська |
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Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine
2021
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Agriciltural microbiology| _version_ | 1871466246586761216 |
|---|---|
| author | Д. В. , Крутило |
| author_facet | Д. В. , Крутило |
| author_institution_txt_mv | [
{
"author": "Крутило Д. В. ",
"institution": "Інститут сільськогосподарської мікробіології та агропромислового виробництва НААН"
}
] |
| author_sort | Д. В. , Крутило |
| baseUrl_str | https://smic.in.ua/index.php/journal/oai |
| collection | OJS |
| datestamp_date | 2026-07-22T10:10:50Z |
| description | Objective. Study the presence of nodule bacteria in the soils of Ukraine, capable of nodulatinggroundnut, isolate new strains of rhizobia from peanut nodules, study their morphological, culturaland serological properties and the ability to form symbiosis with different legumes. Methods. Microbiological (isolation of nodule bacteria from nodules and cultivation of microorganisms, studying biological properties of strains), serological (producing antisera to Bradyrhizobium lupini367a, studying rhizobia diversity in groundnut nodule populations, studying serological identity ofnew strains), vegetation experiment (studying formation and functioning of groundnut symbioticsystems with nodule bacteria, studying host specificity of new strains of groundnut rhizobia), gaschromatography (determining nitrogen-fixing activity of rhizobia in symbiosis with groundnut),mathematical and statistical. Results. Representatives of two species, B. lupini and B. japonicum,were found in nodule populations of rhizobia during the cultivation of groundnut plants on sodpodzolic soil and leached chernozem. The dominant microsymbionts of groundnut were lupine nodule bacteria of serogroup 367a (54.2 % and 45.8 % according to soils). Fewer nodules wereformed by intensive growing soybean rhizobia of serogroup КВ11 (16.7 % and 12.5 %). The shareof nodule bacteria not classified in the studied serogroups was 21.9 % and 41.7 %. Fifteen newstrains of nodule bacteria were isolated from groundnut nodules, which were morphologically, culturally and serologically identified as B. lupini serogroup 367a (7 of them), B. japonicumserogroup KB11 (4 of them) and Bradyrhizobium sp. (4 of them). New strains of B. lupini fromgroundnut nodules are able to infect white and yellow lupine but do not nodulate soybeans. Strainsidentified as B. japonicum form nodules on soybean roots but do not infect lupine. Serologically unidentified strain Bradyrhizobium sp. AR3, which is able to form a symbiosis with both lupine andsoybean (phenotypes Nod+Fix+) was obtained. Conclusion. For the first time it was established thatgroups of nodule bacteria capable of nodulating groundnut are present in the agrocenoses ofUkraine. Fifteen new strains of groundnut rhizobia were obtained, which were identified as B. lupini, B. japonicum and Bradyrhizobium sp. |
| doi_str_mv | 10.35868/1997-3004.34.3-14 |
| first_indexed | 2025-07-17T12:26:25Z |
| format | Article |
| fulltext |
3
ҐРУНТОВА МІКРОБІОЛОГІЯ
Сільськогосподарська мікробіологія. 2021. Вип. 34. С. 3–14.
ISSN 1997-3004
https://doi.org/10.35868/1997-3004.34.3-14
UDC 631.847.211:633.852.52:631.4
BIOLOGICAL FEATURES OF GROUNDNUT
MICROSYMBIONTS WIDESPREAD IN THE SOILS OF UKRAINE
D. V. Krutylo
Institute of Agricultural Microbiology and Agroindustrial Manufacture, NAAS
97 Shevchenka str., Chernihiv, 14035; Ukraine; e-mail: krutylodv@gmail.com
Objective. Study the presence of nodule bacteria in the soils of Ukraine, capable of nodulating
groundnut, isolate new strains of rhizobia from peanut nodules, study their morphological, cultural
and serological properties and the ability to form symbiosis with different legumes. Methods. Mi-
crobiological (isolation of nodule bacteria from nodules and cultivation of microorganisms, study-
ing biological properties of strains), serological (producing antisera to Bradyrhizobium lupini
367a, studying rhizobia diversity in groundnut nodule populations, studying serological identity of
new strains), vegetation experiment (studying formation and functioning of groundnut symbiotic
systems with nodule bacteria, studying host specificity of new strains of groundnut rhizobia), gas
chromatography (determining nitrogen-fixing activity of rhizobia in symbiosis with groundnut),
mathematical and statistical. Results. Representatives of two species, B. lupini and B. japonicum,
were found in nodule populations of rhizobia during the cultivation of groundnut plants on sod-
podzolic soil and leached chernozem. The dominant microsymbionts of groundnut were lupine no-
dule bacteria of serogroup 367a (54.2 % and 45.8 % according to soils). Fewer nodules were
formed by intensive growing soybean rhizobia of serogroup КВ11 (16.7 % and 12.5 %). The share
of nodule bacteria not classified in the studied serogroups was 21.9 % and 41.7 %. Fifteen new
strains of nodule bacteria were isolated from groundnut nodules, which were morphologically, cul-
turally and serologically identified as B. lupini serogroup 367a (7 of them), B. japonicum
serogroup KB11 (4 of them) and Bradyrhizobium sp. (4 of them). New strains of B. lupini from
groundnut nodules are able to infect white and yellow lupine but do not nodulate soybeans. Strains
identified as B. japonicum form nodules on soybean roots but do not infect lupine. Serologically un-
identified strain Bradyrhizobium sp. AR3, which is able to form a symbiosis with both lupine and
soybean (phenotypes Nod+Fix+) was obtained. Conclusion. For the first time it was established that
groups of nodule bacteria capable of nodulating groundnut are present in the agrocenoses of
Ukraine. Fifteen new strains of groundnut rhizobia were obtained, which were identified as B. lupi-
ni, B. japonicum and Bradyrhizobium sp.
Key words: groundnut microsymbionts, rhizobia nodule populations, Bradyrhizobium lupini,
B. japonicum, serogroups, soybean, lupine.
Introduction. Nodule bacteria (rhizobia) is
a group of nitrogen-fixing microorganisms pre-
vailing in almost all soil and climatic zones of
the Earth and capable of forming specific sym-
biotic organs — nodules — on the roots of leg-
umes [1; 2]. Due to the ability of rhizobia to fix
nitrogen from the atmosphere, synthesize bio-
logically active substances, improve plant nutri-
tion, have an impact on plant resistance to path-
ogens and abiotic stresses, they are widely used
as a basis for microbial preparations [1; 3; 4].
Analysis of the novel studies and publica-
tions. Most species of nodule bacteria that no-
dulate legumes belong to alpha-Proteobacteria
of Rhizobiales (Hyphomicrobiales) and are gro-
uped into four families: Bradyrhizobiaceae (ge-
nus Bradyrhizobium), Rhizobiaceae (genera
Rhizobium, Allorhizobium, Ensifer, Neorhizobi-
© D. V. Krutylo, 2021
4
um, Pararhizobium, Sinorhizobium), Phylobac-
teriaceae (genus Mesorhizobium) and Xantho-
bacteriaceae (genus Azorhizobium) [5–7]. There
have been numerous reports about isolation of
nitrogen-fixing bacteria from legume nodules
belonging to phylogenetically remote rhizobia
genera: Methylobacterium, Devosia, Ochrobac-
trum and Phyllobacterium (alpha-Proteobac-
teria class), as well as Burkholderia, Ralstonia
and Cupriavidus (beta-Proteobacteria class) [8].
Recently, much attention has been paid to
the study of the diversity of nodule bacteria —
microsymbionts of both traditional and rare leg-
umes [2; 9]. The research of representatives of
local groups of specific nodule bacteria may be
important for understanding the processes of
microevolution of rhizobia, as well as in the se-
lection of potential bioagents of microbial pre-
parations.
One of the economically valuable legumes
is a groundnut (Arachis hypogaea L.). Its seeds
contain 45–60 % fat, 25–37 % protein, 15–20 %
carbohydrates. In addition, the product contain
many minerals (sodium, potassium, calcium,
phosphorus, iron) and vitamins (B1, B2, PP, D)
[10; 11].
South America is considered as a homeland
of groundnuts. Currently, this crop is grown in
America, Africa, Australia, Asia and Europe on
an area of about 20–25 million hectares. The
main crops of groundnut are concentrated in
Asia and Africa, and in Europe it is a rare crop
[10; 11].
Groundnut was started to grown in Ukraine
in the second half of the 19th century. The cul-
ture is successfully cultivated in the southern
regions of the country – in the Regions of My-
kolaiv, Odesa, Kherson, Zaporizhzhia and Dnip-
ropetrovsk. The areas of groundnut under culti-
vation in Ukraine are small and concentrated
mainly in the private sector and on farms. How-
ever, recently this culture has been intensively
introduced into production, especially on irri-
gated lands [12].
In addition to the fact that groundnut is a
valuable crop, it is widely used for scientific
purposes as a trap-host in the study of the diver-
sity of local groups of nodule bacteria [13].
The features of groundnut is that it can en-
ter into a symbiotic relationship with both slow-
growing and fast-growing nodule bacteria [14–
16]. More than 30 species of Bradyrhizobium
are currently known to be confirmed as ground-
nut microsymbionts [16–21]. Species such as
Rhizobium tropici, R. (Pararhizobium) giardi-
nii, Neorhizobium galegae and N. huautlense
have been identified in different countries
among fast-growing rhizobia in groundnut nod-
ules [22; 23]. Slow-growing strains in general
dominate in nodule populations of groundnut
rhizobia [14]. It has been noted that in some re-
gions of China, fast-growing nodule bacteria do
not infect groundnut at all [24; 25].
The national literature contains no infor-
mation on soil populations of groundnut nodule
bacteria in agrocenoses of Ukraine. Due to the
low prevalence of the crop, the species of local
rhizobia that may form nodules on groundnut
roots remain unidentified. Therefore, the objec-
tive of our work was to study the presence of
nodule bacteria in Ukrainian soils capable of
nodulating groundnut, to isolate new rhizobia
strains from groundnut nodules, to study their
morphological, cultural and serological proper-
ties and the ability to form symbiosis with dif-
ferent legumes.
Materials and methods. The objects of
study were rhizobia isolated from groundnut
nodules, as well as plants of groundnut (Arachis
hypogaea L., Klynskyi variety), soybean (Gly-
cine max (L.) Merr., Ustia variety), white lupine
(Lupinus albus L., Lybid variety) and yellow
lupine (Lupinus luteus L., Chernihivets variety).
The seeds were provided by the National Scien-
tific Centre “Institute of Agriculture of NAAS”,
the Department of Scientific Support of Agroin-
dustrial Manufacture of the Institute of Agricul-
tural Microbiology and Agroindustrial Manu-
facture of the NAAS (NAAS IAMAM). New
strains of groundnut rhizobia are stored in the
collection of the Laboratory of Plant-Microbial
Interactions of NAAS IAMAM.
The composition of nodule bacteria capable
of entering into symbiotic relationships with
groundnut plants was studied in sod-podzolic
soil and leached chernozem. The study was
conducted under the conditions of vegetation
experiment. Soil has been sampled in the fields
of NAAS IAMAM, where inoculated soybeans
were periodically grown. Over the last 5–6
years, legumes have not been sown in the exper-
imental plots, groundnut have never been
grown.
Vegetation experiment was performed ac-
cording to the generally accepted rules in 2 litre
vessels. Before sowing, sterilized (96 % ethyl
ISSN 1997-3004 Сільськогосподарська мікробіологія. 2021. Вип. 34.
5
alcohol) groundnut seeds were moistened with
tap water, inoculation with nodule bacteria was
not performed. The repetition of the experi-
ment was quadruple. Humidity was maintained
at 60 % of maximum water-holding capacity
[26].
In the flowering phase, nodules were se-
lected from the roots of plants. The share of dif-
ferent strains of rhizobia in nodule populations
was determined by analysis of nodule homoge-
nates (48 units) in the agglutination reaction
(Gruber-Widal technique) [27] using a set of
specific immune antisera obtained to active
strains of soybean nodule bacteria B. japonicum
46, M8, KB11, 634b, OR, HR, NR, microsym-
bionts of cowpea Bradyrhizobium sp. B1 and
common bean Rhizobium phaseoli 700, ФБ1,
ФД3 [28; 29].
We also used polyclonal immune O-anti-
serum to the strain of nodule bacteria of lupine
Bradyrhizobium lupini 367a, which was ob-
tained by the method of All-Russian Scientific
and Research Institute of Agricultural Microbi-
ology [30] in our modification. Rhizobia were
grown on solid legume media at 28 °C. In the
logarithmic growth phase, the bacterial mass
was washed off the agar shoals, precipitated by
centrifugation and washed twice with normal
saline solution. 5 mL of normal saline solution
and 5 mL of 2.5 % glutaraldehyde solution were
added to the nodule bacteria cell precipitate (to
release non-specific flagellar H-antigens) and
left in the refrigerator for 24 hours. One day la-
ter, the bacterial cells (antigen) were washed
three times to remove glutaraldehyde, the pre-
cipitate was resuspended in normal saline solu-
tion and the antigen titre was adjusted to 2 · 109
CFU/mL.
The rabbit immunization schedule included
6 injections (at weekly interval) with increasing
doses of antigen (Table 1). The antigen was ad-
ministered subcutaneously using 0.5 mL of
Complete Freund’s Adjuvant (CFA). Blood
sampling was performed three time one week
after the last immunization from the ear vein.
The titre of 367a antiserum was determined
by agglutination reaction. Its specificity was
tested in a cross-agglutination reaction by the
Gruber-Widal technique [27]. Slow- and fast-
growing nodule bacteria of different species and
genera, stored in the Collection of Beneficial
Soil Microorganisms of NAAS IAMAM, were
used as antigens. The titre of antigens for the
agglutination reaction was 106 cells/mL.
Isolation of nodule bacteria from groundnut
nodules was performed according to guidelines
[31]. Morphological and cultural properties of
the obtained isolates of nodule bacteria were
studied according to generally accepted methods
[30; 31].
The serological features of nodule bacteria
isolated from groundnut nodules were studied
using the Gruber-Widal agglutination test [27].
The ability of new strains of groundnut
nodule bacteria to enter into a symbiotic rela-
tionship with cultivated soybeans, white and
yellow lupine was studied in a vegetation exper-
iment. Plants were grown in 2 L vessels on ni-
trogen-free substrate (sterile vermiculite) mois-
tened with 0.2 % KH2PO4 solution. Before sow-
ing, sterilized seeds were treated with a suspen-
sion of nodule bacteria (titre 2 · 109 CFU/mL).
The inoculation load was 200–300 thousand
cells per 1 seed. The repetition of the experi-
ment was quadruple.
The activity of symbiotic nitrogen fixation
was determined by the acetylene-ethylene
method [32] on a gas chromatograph Chrom-4
with a flame ionization detector (column with β-
β΄oxydipropionitrile).
Table 1. Rabbit immunization schedule with B. lupini 367а (antigen administration at weekly
interval)
No. of
injection
Amount of antigen,
mL
Antigen titre,
cell/mL Method of antigen administration
1 1 106 + CFA subcutaneously
2 1 106 intravenously
3 1 107 intravenously
4 2 107 intravenously
5 4 107 intravenously
6 1 108 intravenously
ISSN 1997-3004 Сільськогосподарська мікробіологія. 2021. Вип. 34.
6
Statistical data processing was performed
by conventional methods [33] using computer
programs Microsoft Office Excel 2016 and Sta-
tistica 8.0.
Results and discussion. At the first stage
of the work, vegetation experiment was used to
found out whether nodule bacteria, which are
able to enter into a symbiotic relationship with
groundnut, are present in the agrocenoses of
Ukraine.
The obtained data indicate that nodule bac-
teria that actively infect groundnut, forming
numerous nodules, are present in both studied
soils (sod-podzolic and leached chernozem)
(Fig. 1).
When growing groundnuts on sod-podzolic
soil, an average of 118 nodules were formed on
the roots of one plant. The soil population of
groundnut microsymbionts in leached cherno-
zem turned out to be more numerous, which is
indirectly evidenced by the higher number of
formed nodules — 163 units/plant (Table 2).
The mass of root nodules per plant was at the
level of 0.21 and 0.23 g, respectively. The nod-
ules were red upon cutting, which indicates the
active fixation of molecular nitrogen. Despite
the lower number of root nodules in plants on
sod-podzolic soil, their activity was higher
(3.08 μg N/plant per hour), compared to plants
grown on leached chernozem — 2.46 μg
N/plant per hour. The dry aboveground mass of
plants grown on different soil types correlated
with the nitrogenase activity of the nodules:
1.55 g/plant and 1.49 g/plant, respectively.
A
B
Fig. 1. Nodules on groundnut roots upon cultivation on sod-podzolic soil (А) and leached
chernozem (B).
Table 2. Symbiotic parameters of groundnut plants upon cultivation on different soils (vege-
tation experiment, flowering phase)
Type of soil Dry aboveground
mass, g/plant
Number of
nodules,
units/plant
Mass of nodules,
g/plant
Activity of symbiotic
nitrogen fixation,
µg N/plant per hour
Sod-podzolic 1.55 ± 0.07 118.17 ± 7.73 0.21 ± 0.01 3.08 ± 0.28
Leached chernozem 1.49 ± 0.05 162.58 ± 5.45 0.23 ± 0.01 2.46 ± 0.23
ISSN 1997-3004 Сільськогосподарська мікробіологія. 2021. Вип. 34.
7
The next step was to determine which spe-
cies of nodule bacteria infected groundnut roots.
To solve this task, we used the serological
method. Typing of rhizobia in groundnut nodule
populations was performed using specific anti-
sera obtained to different strains of soybean rhi-
zobia B. japonicum 46, M8, KB11, 634b, OR,
HR, NR, microsymbionts of cowpea Bradyrhi-
zobium sp. B1 and common beans Rhizobium
phaseoli 700, ФБ1, ФД3.
Given that, according to the literature,
groundnuts can be nodulated by representatives
of different species of the genus Bradyrhizobi-
um [14], we have developed a rabbit immuniza-
tion schedule and obtained antiserum to stand-
ard strain B. lupini 367а to identify potential
microsymbionts in the nodules — lupine rhizo-
bia (Bradyrhizobium lupini). The titre of 367a
antiserum in the agglutination reaction was
1:5,120, working dilution 1:500.
The specificity of the obtained antiserum
was tested in the agglutination reaction with
homo- and heterologous strains of nodule bacte-
ria (Table 3).
As Table 3 shows, 367a antiserum did not
react with any of the 20 strains of nodule bacte-
ria belonging to the phylogenetically distant
genera Rhizobium, Sinorhizobium, Neorhizobi-
um and Mesorhizobium. Representatives of the
genus Bradyrhizobium — 13 strains of soybean
rhizobia, 4 strains of microsymbionts of cowpea
and 3 strains isolated from tick trefoil nodules,
also had no common antigenic determinants
with strain B. lupini 367a.
It should be noted that 10 strains of rhizobia
isolated from nodules of different species of lu-
pine were serologically similar to B. lupini 367a.
That is, they all belong to one serogroup —
367a. Only strain B. lupini 3 did not react with
the studied antiserum and can be assigned to
another serogroup.
Thus, we obtained a group-specific antise-
rum to strain B. lupini 367a, which was also
used to identify rhizobia of this species in
groundnut nodule populations.
As a result of serological analysis of no-
dules, it was first established that rhizobia be-
longing to two species, B. lupini and B. japoni-
cum, infected plant roots when cultivating gro-
undnuts both on sod-podzolic soil and leached
chernozem (Table 4). The dominant microsym-
bionts of groundnuts were lupine nodule bac-
teria of serogroup 367a (54.2 % and 45.8 %,
respectively). The share of intensive growing
Table 3. Results of agglutination reaction of different species of rhizobia with 367а antise-
rum
Strains of microorganisms (antigens) /
(host plant) 367а antiserum
Rhizobium simplex 820 –
R. phaseoli 700, ФБ1, Н6, ФБ2, ФБ3, ФБ4, ФА1, ФД1, ФД2, ФД3 –
R. leguminosarum bv. viceae 250a (pea), 0419 (bean), Ч14 (everlasting pea) –
R. trifolii 1326 –
Neorhizobium galegae 0703 –
Sinorhizobium meliloti 425a –
Mesorhizobium ciceri 522, 065, Н12 –
B. japonicum 1967T, 46, М8, КВ11, 634b, OR, HR, NR, КС22, КС23, СК1,
СК5, КН10 –
B. lupini 367а +++
B. lupini 30л +++
B. lupini 40422, 5500/4, 58543, 3а, 5л, 8л +++
B. lupini 1, 6 (ornamental lupine) +++
B. lupini 3 (mutable lupine) –
B. lupini 6 (mutable lupine) +++
Bradyrhizobium sp. В1, В2, В3, В4 (cowpea) –
Bradyrhizobium sp. 1, 2, 3 (tick trefoil) –
ISSN 1997-3004 Сільськогосподарська мікробіологія. 2021. Вип. 34.
8
Table 4. Ability of the representative of nodule bacteria soil populations to colonize ground-
nut roots (vegetation experiment)
Type of soil
Share of nodule bacteria strains in nodules, %
46
М
8
КВ
11
63
4b
O
R
H
R
N
R
36
7а
В
1
70
0
Ф
Б1
Ф
Д
3
O
th
er
*
Sod-podzolic 0 0 16.7 0 0 0 0 54.2 0 0 0 0 29.1
Leached
chernozem 0 0 12.5 0 0 0 0 45.8 0 0 0 0 41.7
Note. * — nodule bacteria are not assigned to studied serogroups.
soybean rhizobia of serogroup КВ11 in nodules
was 16.7 % and 12.5 %. A significant number
of nodules (21.9 % and 41.7 %) were formed by
nodule bacteria, which do not belong to the
studied serogroups. We believe that unidentified
groundnut microsymbionts may be represen-
tatives of new serogroups of B. lupini and
B. japonicum or may belong to other species of
specific nodule bacteria.
The ability of groundnut to interact with
soybean and lupine rhizobia present in soil po-
pulations can be explained by its symbiotic pro-
perties, namely compatibility with a wide range
of microsymbionts, which is noted in the works
of many researchers [14; 15]. Our data suggest
that groundnut infection with different species
of nodule bacteria occurs not only in tropical
and subtropical countries [13; 14; 16], but also
in moderate climates where groundnut has never
been grown. Moreover, groundnut plants are
able to select strains from the soil that belong to
phylogenetically distant species. In our case,
these are B. lupini and B. japonicum. Interes-
tingly, the main host plants of these nodule bac-
teria (lupine and soybean) belong to different
tribes of Leguminosae family: Genisteae and
Phaseoleae, respectively, and groundnut is the
member of Dalbergieae tribe [34]. The for-
mation of nodules on groundnut roots by lupine
and soybean nodule bacteria (B. lupini 367a and
B. japonicum KB11) may indicate that they
have a much wider range of hosts. And such a
migration of microsymbionts between macro-
symbionts may facilitate emergence of new
genotypes of rhizobia capable to infect various
leguminous plants.
To characterize nodule bacteria, which en-
tered into a symbiotic relationship with ground-
nut plants, 15 isolates were isolated from root
nodules.
The obtained cultures grow well at 26–
28 °С on a modified bean medium [31]. Accor-
ding to the growth rate on agar medium, isolates
are divided into two groups: group I — colonies
appear on Day 4 to 7 of growth; they are round,
translucent, mucous, whitish, the diameter of the
colonies is 2–4 mm (Fig. 2). Group II — colo-
nies appear on Day 8 to 10 of growth; they are
round, opaque, whitish, the diameter of the col-
onies is 1.0–1.5 mm.
According to the morphology, at Day 7 of
culture growth, the cells are motile, have the
shape of slightly curved rods, are gram-nega-
tive, do not form spores (Fig. 3). As cultures
aging, cells lose mobility.
All isolates of groundnut nodule bacteria do
not grow on MPA. Litmus milk is not pepto-
nized, changing the reaction of the medium to
alkaline. Most isolates on the surface of milk do
not form a transparent zone. Only three isolates
such as Bradyrhizobium sp. AR3, AR4 and AR5
are characterized by the formation of a small
(1 mm) mucous ring.
According to morphological and cultural
properties (cell shape, colony size and growth
rate on agar bean medium with mannitol,
growth on MPA and milk with litmus), the new
strains belong to the genus Bradyrhizobium.
In the final portion of the experiments, the
identity of new strains of groundnut
microsymbionts with collectible cultures of
nodule bacteria and their ability to enter into
symbiosis with soybean and lupine plants were
studied.
Serological identification of groundnut
rhizobia was performed using the agglutination
reaction with 9 specific antisera: 46, M8, KB11,
364b, OR, HR, NR, 367a and B1. The results
are given in Table 5.
It was found that 7 new strains: Bradyrhi-
zobium sp. AR1, AR2, AR7, AR9, AR12, AR13
and AR15 reacted positively with antiserum to
ISSN 1997-3004 Сільськогосподарська мікробіологія. 2021. Вип. 34.
9
A
B
Fig. 2. Morphology of the colonies isolated from groundnut nodule bacteria on solid bean
medium: A — group I, B — group II.
A
B
Fig. 3. Morphology of cells isolated from groundnut nodule bacteria ( 600): A — group I,
B — group II.
the strain of nodule bacteria of lupine B. lupini
367a. Four cultures, namely Bradyrhizobium sp.
AR6, AR8, AR10 and AR14, were included in
the serogroup KB11, which consists of intensive
growing soybean nodule bacteria. Four strains
of Bradyrhizobium sp. AR3, AR4, AR5 and
AR11 did not interact with used antisera.
According to the main morphological, cul-
tural and serological characteristics, new stra-
ins of groundnut microsymbionts can be classi-
fied as B. lupini and B. japonicum. The taxo-
nomic status of the four cultures needs to be
clarified.
Under the conditions of vegetation experi-
ment, it was found that all strains of groundnut
nodule bacteria belonging to serogroup 367a
(Bradyrhizobium sp. AR1, AR2, AR7, AR9,
AR12, AR13 and AR15) entered into an active
symbiotic relationship with lupine (phenotype
Nod+Fix+) but did not nodulate soybean (pheno-
type Nod–) (Table 5). Strains of Bradyrhizobium
sp. AR6, AR8, AR10 and AR14, serologically
related to B. japonicum KB11, in contrast, in-
fected soybeans (phenotype Nod+Fix+) and did
not form nodules on the roots of white and yel-
low lupine (phenotype Nod–).
ISSN 1997-3004 Сільськогосподарська мікробіологія. 2021. Вип. 34.
10
Table 5. Characteristics of nodule bacteria isolated from groundnut nodules
Strains of nodule bacteria Serogroup
Symbiotic phenotypes
Glycine max Lupinus albus Lupinus luteus
B. lupini 367а 367а Nod– Nod+Fix+ Nod+Fix+
B. japonicum КВ11 КВ11 Nod+Fix+ Nod– Nod–
Bradyrhizobium sp. AR1 367а Nod– Nod+Fix+ Nod+Fix+
Bradyrhizobium sp. AR2 367а Nod– Nod+Fix+ Nod+Fix+
Bradyrhizobium sp. AR3 Х Nod+Fix+ Nod+Fix+ Nod+Fix+
Bradyrhizobium sp. AR4 Х Nod– Nod+Fix+ Nod+Fix+
Bradyrhizobium sp. AR5 Х Nod+Fix+ Nod– Nod–
Bradyrhizobium sp. AR6 КВ11 Nod+Fix+ Nod– Nod–
Bradyrhizobium sp. AR7 367а Nod– Nod+Fix+ Nod+Fix+
Bradyrhizobium sp. AR8 КВ11 Nod+Fix+ Nod– Nod–
Bradyrhizobium sp. AR9 367а Nod– Nod+Fix+ Nod+Fix+
Bradyrhizobium sp. AR10 КВ11 Nod+Fix+ Nod– Nod–
Bradyrhizobium sp. AR11 Х Nod– Nod+Fix+ Nod+Fix+
Bradyrhizobium sp. AR12 367а Nod– Nod+Fix+ Nod+Fix+
Bradyrhizobium sp. AR13 367а Nod– Nod+Fix+ Nod+Fix+
Bradyrhizobium sp. AR14 КВ11 Nod+Fix+ Nod– Nod–
Bradyrhizobium sp. AR15 367а Nod– Nod+Fix+ Nod+Fix+
Note: Nod+Fix+ — formation of nitrogen-fixing nodules; Nod– — nodules are not formed, Х —
unidentified serogroups.
Among the group of serologically
unidentified strains, rhizobia which formed
nodules on the roots of lupine, but did not infect
soybeans (Bradyrhizobium sp. AR4 and AR11),
and bacteria that infected soybeans, but were
unable to initiate nodule formation on the roots
of different species of lupine (Bradyrhizobi-
um sp. AR5) were also found. It is noteworthy
that the nodule bacteria of groundnut Brady-
rhizobium sp. AR3 formed an active symbiosis
with both soybean plants (Nod+Fix+ pheno-
types) and lupine plants (Nod+Fix+ phenotypes).
A strain of B. japonicum 631 isolated from
soybean nodules is known from the literature,
and it can form nitrogen-fixing nodules on the
roots of soybean and lupine plants [4]. Further
study of a new strain of Bradyrhizobium sp.
AR3 using modern molecular genetic methods
will allow species identification.
Conclusion. It was established for the first
time that communities of nodule bacteria capa-
ble of nodulating groundnut are present in the
agrocenoses of Ukraine. Representatives of two
species, B. lupini and B. japonicum, were found
in nodule populations of rhizobia during the cul-
tivation of groundnut plants on sod-podzolic
soil and leached chernozem. The dominant mi-
crosymbionts of groundnut were lupine nodule
bacteria of serogroup 367a (54.2 % and 45.8 %,
respectively). Fewer nodules were formed by
intensive growing soybean rhizobia of
serogroup КВ11 (16.7 % and 12.5 %). The
share of nodule bacteria not classified in the
studied serogroups was 21.9 % and 41.7 %.
Fifteen new strains were isolated from
groundnut nodules, which were morphological-
ly, culturally and serologically identified as
B. lupini serogroup 367a (7 of them), B. japoni-
cum serogroup KB11 (4 of them) and Bradyrhi-
zobium sp. (4 of them).
New strains of B. lupini from groundnut
nodules are able to infect white and yellow lu-
pine but do not nodulate soybeans. Strains iden-
tified as B. japonicum form nodules on soybean
roots but do not infect lupine. Serologically uni-
dentified strain Bradyrhizobium sp. AR3, which
is able to form a symbiosis with both lupine and
soybean (phenotypes Nod+Fix+) was obtained.
The author would like to express his sincere
gratitude to Izabella Viacheslavivna Volkova,
ISSN 1997-3004 Сільськогосподарська мікробіологія. 2021. Вип. 34.
11
a research fellow of the Laboratory of Virology
at NAAS IAMAM, for her help in obtaining an-
tisera to the strain of lupine nodule bacteria.
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Received 20.08.2021
https://doi.org/10.35868/1997-3004.34.3-14
УДК 631.847.211:633.852.52:631.4
БІОЛОГІЧНІ ВЛАСТИВОСТІ МІКРОСИМБІОНТІВ АРАХІСУ,
ПОШИРЕНИХ У ҐРУНТАХ УКРАЇНИ
Д. В. Крутило
Інститут сільськогосподарської мікробіології та агропромислового виробництва НААН, м. Чернігів
e-mail: krutylodv@gmail.com
Мета. Дослідити наявність у ґрунтах України бульбочкових бактерій, здатних нодулю-
вати арахіс, виділити нові штами ризобій із бульбочок арахісу, вивчити їхні морфолого-
культуральні й серологічні властивості та здатність формувати симбіоз із різними бобо-
вими культурами. Методи. Мікробіологічні (виділення бульбочкових бактерій із бульбочок
та культивування мікроорганізмів, вивчення біологічних властивостей штамів), серологічні
ISSN 1997-3004 Сільськогосподарська мікробіологія. 2021. Вип. 34.
13
(отримання антисироватки до штаму Bradyrhizobium lupini 367а, вивчення різноманіття
ризобій у бульбочкових популяціях арахісу, дослідження серологічної належності нових
штамів), вегетаційного досліду (вивчення формування та функціонування симбіотичних си-
стем арахісу з бульбочковими бактеріями, дослідження хазяйської специфічності нових
штамів ризобій арахісу), газохроматографічний (визначення азотфіксувальної активності
ризобій у симбіозі з арахісом), математично-статистичні. Результати. За вирощування
рослин арахісу на дерново-підзолистому ґрунті та чорноземі вилугуваному в бульбочкових
популяціях ризобій виявлено представників двох видів — B. lupini і B. japonicum. Домінуючи-
ми мікросимбіонтами арахісу були бульбочкові бактерії люпину серогрупи 367а (54,2 % і
45,8% відповідно до ґрунтів). Меншу кількість бульбочок формували інтенсивнорослі ризобії
сої серогрупи КВ11 (16,7 % і 12,5%). Частка бульбочкових бактерій, не зарахованих до дос-
ліджуваних серогруп, становила 21,9 % та 41,7%. Із бульбочок арахісу виділено 15 нових
штамів бульбочкових бактерій, які за морфолого-культуральними та серологічними ознака-
ми ідентифіковані як B. lupini серогрупи 367а (7 од.), B. japonicum серогрупи КВ11 (4 од.) та
Bradyrhizobium sp. (4 од.). Нові штами B. lupini з бульбочок арахісу здатні інфікувати люпин
білий та жовтий, проте не нодулюють сою. Штами, ідентифіковані як B. japonicum, утво-
рюють бульбочки на коренях сої, але не заражають люпин. Отримано серологічно не іден-
тифікований штам Bradyrhizobium sp. AR3, спроможний формувати симбіоз як з люпином,
так і з соєю (фенотипи Nod+Fix+). Висновки. Уперше встановлено, що в агроценозах Украї-
ни наявні угруповання бульбочкових бактерій, здатних нодулювати арахіс. Отримано 15 но-
вих штамів ризобій арахісу, ідентифікованих як B. lupini, B. japonicum та Bradyrhizobium sp.
Ключові слова: мікросимбіонти арахісу, бульбочкові популяції ризобій, Bradyrhizobi-
um lupini, B. japonicum, серогрупи, соя, люпин.
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Отримано 20.08.2022
ISSN 1997-3004 Сільськогосподарська мікробіологія. 2021. Вип. 34.
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| id | oai:ojs2.smic.in.ua:article-451 |
| institution | Agriciltural microbiology |
| keywords_txt_mv | keywords |
| language | English |
| last_indexed | 2026-07-23T01:14:53Z |
| publishDate | 2021 |
| publisher | Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine |
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| resource_txt_mv | smicinua/8c/498edff2e05868cc65627181b763e68c.pdf |
| spelling | oai:ojs2.smic.in.ua:article-4512026-07-22T10:10:50Z BIOLOGICAL FEATURES OF GROUNDNUT MICROSYMBIONTS WIDESPREAD IN THE SOILS OF UKRAINE БІОЛОГІЧНІ ВЛАСТИВОСТІ МІКРОСИМБІОНТІВ АРАХІСУ, ПОШИРЕНИХ У ҐРУНТАХ УКРАЇНИ Д. В. , Крутило groundnut microsymbionts, rhizobia nodule populations, Bradyrhizobium lupini, B. japonicum, serogroups, soybean, lupine мікросимбіонти арахісу, бульбочкові популяції ризобій, Bradyrhizobium lupini, B. japonicum, серогрупи, соя, люпин Objective. Study the presence of nodule bacteria in the soils of Ukraine, capable of nodulatinggroundnut, isolate new strains of rhizobia from peanut nodules, study their morphological, culturaland serological properties and the ability to form symbiosis with different legumes. Methods. Microbiological (isolation of nodule bacteria from nodules and cultivation of microorganisms, studying biological properties of strains), serological (producing antisera to Bradyrhizobium lupini367a, studying rhizobia diversity in groundnut nodule populations, studying serological identity ofnew strains), vegetation experiment (studying formation and functioning of groundnut symbioticsystems with nodule bacteria, studying host specificity of new strains of groundnut rhizobia), gaschromatography (determining nitrogen-fixing activity of rhizobia in symbiosis with groundnut),mathematical and statistical. Results. Representatives of two species, B. lupini and B. japonicum,were found in nodule populations of rhizobia during the cultivation of groundnut plants on sodpodzolic soil and leached chernozem. The dominant microsymbionts of groundnut were lupine nodule bacteria of serogroup 367a (54.2 % and 45.8 % according to soils). Fewer nodules wereformed by intensive growing soybean rhizobia of serogroup КВ11 (16.7 % and 12.5 %). The shareof nodule bacteria not classified in the studied serogroups was 21.9 % and 41.7 %. Fifteen newstrains of nodule bacteria were isolated from groundnut nodules, which were morphologically, culturally and serologically identified as B. lupini serogroup 367a (7 of them), B. japonicumserogroup KB11 (4 of them) and Bradyrhizobium sp. (4 of them). New strains of B. lupini fromgroundnut nodules are able to infect white and yellow lupine but do not nodulate soybeans. Strainsidentified as B. japonicum form nodules on soybean roots but do not infect lupine. Serologically unidentified strain Bradyrhizobium sp. AR3, which is able to form a symbiosis with both lupine andsoybean (phenotypes Nod+Fix+) was obtained. Conclusion. For the first time it was established thatgroups of nodule bacteria capable of nodulating groundnut are present in the agrocenoses ofUkraine. Fifteen new strains of groundnut rhizobia were obtained, which were identified as B. lupini, B. japonicum and Bradyrhizobium sp. Мета. Дослідити наявність у ґрунтах України бульбочкових бактерій, здатних нодулювати арахіс, виділити нові штами ризобій із бульбочок арахісу, вивчити їхні морфологокультуральні й серологічні властивості та здатність формувати симбіоз із різними бобовими культурами. Методи. Мікробіологічні (виділення бульбочкових бактерій із бульбочок та культивування мікроорганізмів, вивчення біологічних властивостей штамів), серологічні  (отримання антисироватки до штаму Bradyrhizobium lupini 367а, вивчення різноманіття ризобій у бульбочкових популяціях арахісу, дослідження серологічної належності нових штамів), вегетаційного досліду (вивчення формування та функціонування симбіотичних систем арахісу з бульбочковими бактеріями, дослідження хазяйської специфічності нових штамів ризобій арахісу), газохроматографічний (визначення азотфіксувальної активності ризобій у симбіозі з арахісом), математично-статистичні. Результати. За вирощування рослин арахісу на дерново-підзолистому ґрунті та чорноземі вилугуваному в бульбочкових популяціях ризобій виявлено представників двох видів — B. lupini і B. japonicum. Домінуючими мікросимбіонтами арахісу були бульбочкові бактерії люпину серогрупи 367а (54,2 % і 45,8% відповідно до ґрунтів). Меншу кількість бульбочок формували інтенсивнорослі ризобії сої серогрупи КВ11 (16,7 % і 12,5%). Частка бульбочкових бактерій, не зарахованих до досліджуваних серогруп, становила 21,9 % та 41,7%. Із бульбочок арахісу виділено 15 нових штамів бульбочкових бактерій, які за морфолого-культуральними та серологічними ознаками ідентифіковані як B. lupini серогрупи 367а (7 од.), B. japonicum серогрупи КВ11 (4 од.) та Bradyrhizobium sp. (4 од.). Нові штами B. lupini з бульбочок арахісу здатні інфікувати люпин білий та жовтий, проте не нодулюють сою. Штами, ідентифіковані як B. japonicum, утворюють бульбочки на коренях сої, але не заражають люпин. Отримано серологічно не ідентифікований штам Bradyrhizobium sp. AR3, спроможний формувати симбіоз як з люпином, так і з соєю (фенотипи Nod+Fix+). Висновки. Уперше встановлено, що в агроценозах України наявні угруповання бульбочкових бактерій, здатних нодулювати арахіс. Отримано 15 нових штамів ризобій арахісу, ідентифікованих як B. lupini, B. japonicum та Bradyrhizobium sp. Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine 2021-11-11 Article Article Рецензована Стаття application/pdf https://smic.in.ua/index.php/journal/article/view/451 10.35868/1997-3004.34.3-14 Agricultural microbiology; Vol. 34 (2021): Agriciltural microbiology; 3-14 Сільськогосподарська мікробіологія; Том 34 (2021): Сільськогосподарська мікробіологія; 3-14 1997-3004 10.35868/1997-3004.34 en https://smic.in.ua/index.php/journal/article/view/451/540 Авторське право (c) 2022 Krutylo D. V. https://creativecommons.org/licenses/by/4.0 |
| spellingShingle | мікросимбіонти арахісу бульбочкові популяції ризобій Bradyrhizobium lupini B. japonicum серогрупи соя люпин Д. В. , Крутило БІОЛОГІЧНІ ВЛАСТИВОСТІ МІКРОСИМБІОНТІВ АРАХІСУ, ПОШИРЕНИХ У ҐРУНТАХ УКРАЇНИ |
| title | БІОЛОГІЧНІ ВЛАСТИВОСТІ МІКРОСИМБІОНТІВ АРАХІСУ, ПОШИРЕНИХ У ҐРУНТАХ УКРАЇНИ |
| title_alt | BIOLOGICAL FEATURES OF GROUNDNUT MICROSYMBIONTS WIDESPREAD IN THE SOILS OF UKRAINE |
| title_full | БІОЛОГІЧНІ ВЛАСТИВОСТІ МІКРОСИМБІОНТІВ АРАХІСУ, ПОШИРЕНИХ У ҐРУНТАХ УКРАЇНИ |
| title_fullStr | БІОЛОГІЧНІ ВЛАСТИВОСТІ МІКРОСИМБІОНТІВ АРАХІСУ, ПОШИРЕНИХ У ҐРУНТАХ УКРАЇНИ |
| title_full_unstemmed | БІОЛОГІЧНІ ВЛАСТИВОСТІ МІКРОСИМБІОНТІВ АРАХІСУ, ПОШИРЕНИХ У ҐРУНТАХ УКРАЇНИ |
| title_short | БІОЛОГІЧНІ ВЛАСТИВОСТІ МІКРОСИМБІОНТІВ АРАХІСУ, ПОШИРЕНИХ У ҐРУНТАХ УКРАЇНИ |
| title_sort | біологічні властивості мікросимбіонтів арахісу, поширених у ґрунтах україни |
| topic | мікросимбіонти арахісу бульбочкові популяції ризобій Bradyrhizobium lupini B. japonicum серогрупи соя люпин |
| topic_facet | groundnut microsymbionts rhizobia nodule populations Bradyrhizobium lupini B. japonicum serogroups soybean lupine мікросимбіонти арахісу бульбочкові популяції ризобій Bradyrhizobium lupini B. japonicum серогрупи соя люпин |
| url | https://smic.in.ua/index.php/journal/article/view/451 |
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