ВПЛИВ СПОСОБІВ ЗБЕРІГАННЯ НА ЖИТТЄЗДАТНІСТЬ ТА СИМБІОТИЧНІ ВЛАСТИВОСТІ БУЛЬБОЧКОВИХ БАКТЕРІЙ СОЇ
The viability and symbiotic properties of active strain Bradyrhizobium japonicum 46 after its long-term storage using different methods were studied. It was established that strain storing in evaporative freezing conditions as well at periodic passages the strain preserves its ability to form active...
Gespeichert in:
| Datum: | 2014 |
|---|---|
| Hauptverfasser: | , , , , |
| Format: | Artikel |
| Sprache: | Englisch Ukrainisch |
| Veröffentlicht: |
Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine
2014
|
| Schlagworte: | |
| Online Zugang: | https://smic.in.ua/index.php/journal/article/view/154 |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Назва журналу: | Agriciltural microbiology |
| Завантажити файл: |  |
Institution
Agriciltural microbiology| _version_ | 1871465612628197376 |
|---|---|
| author | Ковалевська, Т. М. Крутило, Д. В. Горбань, В. П. Романова, І. М. Усманова, Т. О. |
| author_facet | Ковалевська, Т. М. Крутило, Д. В. Горбань, В. П. Романова, І. М. Усманова, Т. О. |
| author_institution_txt_mv | [
{
"author": "Т. М. Ковалевська",
"institution": "Інститут сільськогосподарської мікробіології та агропромислового виробництва НААН"
},
{
"author": "Д. В. Крутило",
"institution": "Інститут сільськогосподарської мікробіології та агропромислового виробництва НААН"
},
{
"author": "В. П. Горбань",
"institution": "Інститут сільськогосподарської мікробіології та агропромислового виробництва НААН"
},
{
"author": "І. М. Романова",
"institution": "Інститут сільськогосподарської мікробіології та агропромислового виробництва НААН"
},
{
"author": "Т. О. Усманова",
"institution": "Інститут сільськогосподарської мікробіології та агропромислового виробництва НААН"
}
] |
| author_sort | Ковалевська, Т. М. |
| baseUrl_str | https://smic.in.ua/index.php/journal/oai |
| collection | OJS |
| datestamp_date | 2026-07-22T10:10:43Z |
| description | The viability and symbiotic properties of active strain Bradyrhizobium japonicum 46 after its long-term storage using different methods were studied. It was established that strain storing in evaporative freezing conditions as well at periodic passages the strain preserves its ability to form active symbiosis with soybean plants. Long-term storage under the layer of mineral oil had negatively influenced on the symbiotic properties of B. japonicum 46 strain resulted in only partial restoration of strains virulence, nitrogen fixing activity and competitiveness. |
| doi_str_mv | 10.35868/1997-3004.20.15-22 |
| first_indexed | 2025-07-17T12:24:09Z |
| format | Article |
| fulltext |
THE EFFECT OF STORAGE METHODS ON VIABILITY AND SYMBIOTIC
PROPERTIES OF SOYBEAN NODULE BACTERIA
T. M. Kovalevska, D. V. Krutylo,
V. P. Horban, I. M. Romanova, T. O. Usmanova
During numerous studies conducted
by scientists it was established that efficiency
of nodule bacteria symbiosis with legumes
can be improved via utilizing active and
competitive bacterial strains for inoculation.
Selection of rhizobia according to the
symbiotic activity and warranted maintaining
of advantageous strains in collections is an
important component of scientific studies
aimed at development of the new microbial
drugs for legumes.
Due to the increased demand for
biological drugs, necessity of the manufacture
for keeping cultures of microorganisms viable
and stable according all parameters is also
increased. Therefore, development of long-
term storage methods for advantageous
nodule bacteria strains that would provide
stability of their productive-valuable
properties takes new practical significance
[1].
Methods of lyophilization,
cryopreservation, periodic reseeding, and
keeping under mineral oil layer are mainly
utilized in collections for keeping nodule
bacteria. Advantage of the two latter strains
includes their simplicity, technical
availability, as well as possibility for long-
term keeping of the microorganisms in
metabolically active condition. Disadvantages
include the necessity for regular recovery of
the cultures that increases the risk for
contamination with foreign microflora and
development of spontaneous mutations that
change collection strains. The most common
and effective method of rhizobia long-term
storage is lyophilization. According to the
data of staff members of the MSU
microbiology department [2], strains of pea,
clover, medic nodule bacteria remain viable
during 33-34 years of storage (number of
viable cells was 1×104 – 1×107 cells per
ampoule). At the same time, bacteria expose
to severe effect of low temperatures and
vacuum during lyophilization that may be
accompanied with genetic variations and lose
of strain properties.
We initiated study of the long-term
storage conditions of advanced nodule
bacteria strains in 2006. Considering that the
main requirement to rhizobia collections is
the guarantee for symbiotic properties
preservation, significant attention was paid to
the investigation of functional activity of the
strains put for the long-term storage.
The objective of this work was to
study effect of different storage methods on
viability and symbiotic properties of
Bradyrhizobium japonicum 46 master seed
strain.
Materials and methods. Master seed
strain B. japonicum 46 (В-23), which is stored
in the collection of useful soil
microorganisms of the Institute of
Agricultural Microbiology and Agro-
Industrial Manufacture of NAAS was used in
the work [3, 4].
B. japonicum 46 strain was isolated in
1995, and it is maintained in the collection by
periodic reseeding method. In 2006 this strain
was put for long-term storage under the
mineral oil layer and in 2007 in lyophilized
dry condition.
For lyophilization B. japonicum 46
culture was cultivated on leguminous (lupine)
agar before initiation of the stationary growth
phase. Protective sucrose-gelatine medium
(10:1) was used. Lyophilization of bacteria
was conducted on the base of D. K.
Zabolotnyi Institute of Microbiology and
Virusology.
Viable cells count in the samples
following lyophilization and 5 and 6 years
following storage was measured via critical
dilution method by calculation of number of
colonies on the surface of leguminous
agarized medium.
Culture for maintaining under mineral
oil was cultivated on solid and semifluid
(0.3% agar) leguminous medium to stationary
growth phase, primed with sterile mineral oil
with layer of 1.5 cm, and stored at room
temperature. Bacterial viability was studied
by exhaustive seeding method on 4 sectors of
the surface of agarized culture medium. Count
of sectors colonized with bacteria was
measured, and culture activity was evaluated
by 4-point scale. Following long-term storage,
nodule bacteria cultures were renewed by
three-time reseeding on the optimal culture
medium.
Symbiotic properties and efficiency of
B. japonicum 46 strain stored by different
methods were studied under conditions of
field studies on meadow-chernozemic leached
slightly clay-loam soil with the following
agrochemical parameters: content of humus in
the plow layer (according to Tiurin) – 3.5 %,
easily hydrolysed nitrogen (according to
Kornfield) – 95.0 mg, extractable phosphorus
(Р2О5) (according to Kirsanov) – 251 mg,
exchangeable calcium (K2О) (according to
Kirsanov) – 108 mg per 1 kg of solid
(experimental field of the Institute of
Agricultural Microbiology and Agro-
Industrial Manufacture of NAAS). Ustia
variety of soybean was used in the
experiments. Before planting, soybean seeds
were treated with suspension of
microorganisms assuming 200-300 cells per 1
seed. Area of the accounting site is 5.0 m2,
fourfold repeatability.
Initiation of the experiments,
sampling, yield accounting were conducted
according to the current rules. At the phases
of budding, flowering and legumes formation
amount of nodules was counted, their weight
and nitrogenase activity were determined [5].
Competitiveness (ratio of the number of the
formed by strain nodules to total number of
the studied nodules from each variant,
expressed as percentage) of the B. japonicum
46 strain following long-term storage was
evaluated by agglutination reaction.
During the work, antiserums to
B. japonicum 46 strain and to three typical
representatives of soybean nodule bacteria:
B. japonicum М-8, B. japonicum КВ11,
B. japonicum 634б were used. Antiserums
were obtained by the method described earlier
(method of All-Russian Research Institute for
Agricultural Microbiology (ARRIAM) in our
version) [6].
Activity of the symbiotic nitrogen
fixation was determined by acetylenic method
[7] on the gas chromatograph “Chrom-4” with
flame-ionization detector (column with β-
β΄oxydipropionitrile). Thermostat temperature
is 500С. Consumption of gases: hydrogen –
30 mL/min, nitrogen – 100 mL/min, air – 500
mL/min.
Statistical data processing was
conducted according to B. O. Dospekhov [8]
and using software application Statistica 7.0.
Therefore, summarizing obtained
results it can be concluded that for long-term
storage of B. japonicum 46 master seed strain
all three studied methods can be used. The
best is periodic reseeding method and
lyophilization which provide high strain
viability and stability of its symbiotic
properties during long term. Maintaining
under mineral oil can be recommended as the
additional method providing reseeding of
nodule bacteria cultures following every 2-3
years. The methods require further
investigation for longer terms of preservation
and higher number of nodule bacteria strains.
At the same time, established fact of
maintaining of B. japonicum 46 strain
symbiotic properties in case of long-term in
vitro storage suggests usefulness of the above
mentioned methods for storage of the
advantageous and master seed strains of
soybean nodule bacteria.
|
| id | oai:ojs2.smic.in.ua:article-154 |
| institution | Agriciltural microbiology |
| keywords_txt_mv | keywords |
| language | English Ukrainian |
| last_indexed | 2026-07-23T01:04:48Z |
| publishDate | 2014 |
| publisher | Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine |
| record_format | ojs |
| resource_txt_mv | smicinua/6b/01f817fd176c0c83189e46c5fb41e66b.pdf |
| spelling | oai:ojs2.smic.in.ua:article-1542026-07-22T10:10:43Z THE EFFECT OF STORAGE METHODS ON VIABILITY AND SYMBIOTIC PROPERTIES OF SOYBEAN NODULE BACTERIA ВПЛИВ СПОСОБІВ ЗБЕРІГАННЯ НА ЖИТТЄЗДАТНІСТЬ ТА СИМБІОТИЧНІ ВЛАСТИВОСТІ БУЛЬБОЧКОВИХ БАКТЕРІЙ СОЇ Ковалевська, Т. М. Крутило, Д. В. Горбань, В. П. Романова, І. М. Усманова, Т. О. Bradyrhizobium japonicum, long-term storing, symbiotic system, soybean Bradyrhizobium japonicum, симбіотичні властивості, соя, тривале зберігання The viability and symbiotic properties of active strain Bradyrhizobium japonicum 46 after its long-term storage using different methods were studied. It was established that strain storing in evaporative freezing conditions as well at periodic passages the strain preserves its ability to form active symbiosis with soybean plants. Long-term storage under the layer of mineral oil had negatively influenced on the symbiotic properties of B. japonicum 46 strain resulted in only partial restoration of strains virulence, nitrogen fixing activity and competitiveness. Вивчено життєздатність та симбіотичні властивості виробничого штаму Bradyrhizobium japonicum 46 після тривалого зберігання різними способами. Встановлено, що досліджуваний штам у ході зберігання в сублімовано-висушеному стані та за періодичних пересівів не втрачає здатності до активного симбіозу з соєю. За тривалого підтримання під шаром мінерального масла симбіотичні властивості (вірулентність, азотфіксувальна активність, конкурентоздатність) штаму повністю не відновлюються. Institute of Agrocultural Microbiology and Agro-industrial Manufacture of NAAS of Ukraine 2014-12-22 Article Article Рецензована Стаття application/pdf application/pdf https://smic.in.ua/index.php/journal/article/view/154 10.35868/1997-3004.20.15-22 Agricultural microbiology; Vol. 20 (2014): Agriciltural microbiology; 15-22 Сільськогосподарська мікробіологія; Том 20 (2014): Сільськогосподарська мікробіологія; 15-22 1997-3004 10.35868/1997-3004.20 en uk https://smic.in.ua/index.php/journal/article/view/154/193 https://smic.in.ua/index.php/journal/article/view/154/194 Авторське право (c) 2014 T. M. Kovalevska, D. V. Krutylo, V. P. Horban, I. M. Romanova, T. O. Usmanova https://creativecommons.org/licenses/by/4.0 |
| spellingShingle | Bradyrhizobium japonicum симбіотичні властивості соя тривале зберігання Ковалевська, Т. М. Крутило, Д. В. Горбань, В. П. Романова, І. М. Усманова, Т. О. ВПЛИВ СПОСОБІВ ЗБЕРІГАННЯ НА ЖИТТЄЗДАТНІСТЬ ТА СИМБІОТИЧНІ ВЛАСТИВОСТІ БУЛЬБОЧКОВИХ БАКТЕРІЙ СОЇ |
| title | ВПЛИВ СПОСОБІВ ЗБЕРІГАННЯ НА ЖИТТЄЗДАТНІСТЬ ТА СИМБІОТИЧНІ ВЛАСТИВОСТІ БУЛЬБОЧКОВИХ БАКТЕРІЙ СОЇ |
| title_alt | THE EFFECT OF STORAGE METHODS ON VIABILITY AND SYMBIOTIC PROPERTIES OF SOYBEAN NODULE BACTERIA |
| title_full | ВПЛИВ СПОСОБІВ ЗБЕРІГАННЯ НА ЖИТТЄЗДАТНІСТЬ ТА СИМБІОТИЧНІ ВЛАСТИВОСТІ БУЛЬБОЧКОВИХ БАКТЕРІЙ СОЇ |
| title_fullStr | ВПЛИВ СПОСОБІВ ЗБЕРІГАННЯ НА ЖИТТЄЗДАТНІСТЬ ТА СИМБІОТИЧНІ ВЛАСТИВОСТІ БУЛЬБОЧКОВИХ БАКТЕРІЙ СОЇ |
| title_full_unstemmed | ВПЛИВ СПОСОБІВ ЗБЕРІГАННЯ НА ЖИТТЄЗДАТНІСТЬ ТА СИМБІОТИЧНІ ВЛАСТИВОСТІ БУЛЬБОЧКОВИХ БАКТЕРІЙ СОЇ |
| title_short | ВПЛИВ СПОСОБІВ ЗБЕРІГАННЯ НА ЖИТТЄЗДАТНІСТЬ ТА СИМБІОТИЧНІ ВЛАСТИВОСТІ БУЛЬБОЧКОВИХ БАКТЕРІЙ СОЇ |
| title_sort | вплив способів зберігання на життєздатність та симбіотичні властивості бульбочкових бактерій сої |
| topic | Bradyrhizobium japonicum симбіотичні властивості соя тривале зберігання |
| topic_facet | Bradyrhizobium japonicum long-term storing symbiotic system soybean Bradyrhizobium japonicum симбіотичні властивості соя тривале зберігання |
| url | https://smic.in.ua/index.php/journal/article/view/154 |
| work_keys_str_mv | AT kovalevsʹkatm theeffectofstoragemethodsonviabilityandsymbioticpropertiesofsoybeannodulebacteria AT krutilodv theeffectofstoragemethodsonviabilityandsymbioticpropertiesofsoybeannodulebacteria AT gorbanʹvp theeffectofstoragemethodsonviabilityandsymbioticpropertiesofsoybeannodulebacteria AT romanovaím theeffectofstoragemethodsonviabilityandsymbioticpropertiesofsoybeannodulebacteria AT usmanovato theeffectofstoragemethodsonviabilityandsymbioticpropertiesofsoybeannodulebacteria AT kovalevsʹkatm vplivsposobívzberígannânažittêzdatnístʹtasimbíotičnívlastivostíbulʹbočkovihbakteríjsoí AT krutilodv vplivsposobívzberígannânažittêzdatnístʹtasimbíotičnívlastivostíbulʹbočkovihbakteríjsoí AT gorbanʹvp vplivsposobívzberígannânažittêzdatnístʹtasimbíotičnívlastivostíbulʹbočkovihbakteríjsoí AT romanovaím vplivsposobívzberígannânažittêzdatnístʹtasimbíotičnívlastivostíbulʹbočkovihbakteríjsoí AT usmanovato vplivsposobívzberígannânažittêzdatnístʹtasimbíotičnívlastivostíbulʹbočkovihbakteríjsoí AT kovalevsʹkatm effectofstoragemethodsonviabilityandsymbioticpropertiesofsoybeannodulebacteria AT krutilodv effectofstoragemethodsonviabilityandsymbioticpropertiesofsoybeannodulebacteria AT gorbanʹvp effectofstoragemethodsonviabilityandsymbioticpropertiesofsoybeannodulebacteria AT romanovaím effectofstoragemethodsonviabilityandsymbioticpropertiesofsoybeannodulebacteria AT usmanovato effectofstoragemethodsonviabilityandsymbioticpropertiesofsoybeannodulebacteria |