Paenibacillus sp., as a promising candidate for development of a novel technology of inoculant production
A bacterial strain IMBG156 producing exopolysaccharide (EPS) was isolated from siliceous rock and identified as a Paenibacillus species by partial sequencing its 16S rDNA. Paenibacillus sp. IMBG156 was used in a novel technology for inoculant production based on co-cultivating this bacterium with an...
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Kozyrovska, N.O. Negrutska, V.V. Kovalchuk, M.V. Voznyuk, T.N. 2019-06-17T11:04:42Z 2019-06-17T11:04:42Z 2005 Paenibacillus sp., as a promising candidate for development of a novel technology of inoculant production / N.O. Kozyrovska, V.V. Negrutska, M.V. Kovalchuk, T.N. Voznyuk // Біополімери і клітина. — 2005. — Т. 21, № 4. — С. 312-318. — Бібліогр.: 27 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0006F7 https://nasplib.isofts.kiev.ua/handle/123456789/155709 574:539 A bacterial strain IMBG156 producing exopolysaccharide (EPS) was isolated from siliceous rock and identified as a Paenibacillus species by partial sequencing its 16S rDNA. Paenibacillus sp. IMBG156 was used in a novel technology for inoculant production based on co-cultivating this bacterium with any bacterium of choice. Paenibacillus sp. provides in situ the bacterial cells of the inoculant with EPS, a carrier, and most likely with a source of carbon and energy. The partner bacterium designates a type of inoculant (biopesticide or biofertiliser). The strain IMBG156 does not destroy the signaling system of Gram-negative partners, based on acylated homoserine lactones, stimulates plant growth, and is rather competitive in the plant rhizosphere and soil. A prototype of the inoculant based on dual-culture Paenibacillus sp. IMBG156 - Pseudomonas sp. IMBG163 exhibits a noticeably longer shelf life than monoculture of Pseudomonas sp. IMBG163. (ЕПС), виділено з силікатної породи та ідентифіковано як Paenibacillus sp. на основі визначення первинної нуклеотидної послідовності 16S рДНК Paenibacillus sp. IMBG156 використа но у технологи виробництва мікробних препаратів, яка базуться на спільному культивуванні цієї бактерії та бактерії за вибором. Paenibacillus sp. постачає in situ бактерійні клітини носієм (ЕПС) та, найвірогідніше, є джерелом вуглецю і енергії. Бактерія-партнер визначає тип препарату (біодобриво, біопестицид). Штам IMBG156 не пошкоджує сигнальної системи грамнегативних бактерій-партнерів, яка грунтується на ацильованих гомосеринлактонах, стимулює ріст рослини і є доволі конкурентною у ризосфері. У демонстраційному препараті, створеному на основі бактерій-партнерів Paenibacillus sp. IMBGI56—Pseudomonas sp. IMBG163, довше зберігається необхідний титр бактерій, ніж у монокультурі Pseudomonas sp. IMBG163. A bacterial strain IMBG156 producing exopolysaccharide (EPS) was isolated from siliceous rock and identified as a Paenibacillus species by partial sequencing its 16S rDNA. Paenibacillus sp. IMBG156 was used in a novel technology for inoculant production based on co-cultivating this bacterium with any bacterium of choice. Paenibacillus sp. provides in situ the bacterial cells of the inoculant with EPS, a carrier, and most likely with a source of carbon and energy. The partner bacterium designates a type of inoculant (biopesticide or biofertiliser). The strain IMBG156 does not destroy the signaling system of Gram-negative partners, based on acylated homoserine lactones, stimulates plant growth, and is rather competitive in the plant rhizosphere and soil. A prototype of the inoculant based on dual-culture Paenibacillus sp. IMBG156 - Pseudomonas sp. IMBG163 exhibits a noticeably longer shelf life than monoculture of Pseudomonas sp. IMBG163. Acknowledgements. We are grateful to Prof. B. Lugtenberg (Leiden University) for providing sequencing data of the rrn gene of Paenibacillus sp. IMBG156 and to Prof. Diethelm Kleiner for critical reading of the manuscript. This work partially supported by Ukrainian Branch of World Wide Laboratory, subcontract N 901 -02-5281-5700-WLU. en Інститут молекулярної біології і генетики НАН України Біополімери і клітина Клітинна біологія Paenibacillus sp., as a promising candidate for development of a novel technology of inoculant production Paenibacillus sp. — перспективна бактерія для створення технології виробництва бакпрепаратів для рослин Paenibacillus sp. — перспективная бактерия для создания технологии производства бакпрепаратов для растений Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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| title |
Paenibacillus sp., as a promising candidate for development of a novel technology of inoculant production |
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Paenibacillus sp., as a promising candidate for development of a novel technology of inoculant production Kozyrovska, N.O. Negrutska, V.V. Kovalchuk, M.V. Voznyuk, T.N. Клітинна біологія |
| title_short |
Paenibacillus sp., as a promising candidate for development of a novel technology of inoculant production |
| title_full |
Paenibacillus sp., as a promising candidate for development of a novel technology of inoculant production |
| title_fullStr |
Paenibacillus sp., as a promising candidate for development of a novel technology of inoculant production |
| title_full_unstemmed |
Paenibacillus sp., as a promising candidate for development of a novel technology of inoculant production |
| title_sort |
paenibacillus sp., as a promising candidate for development of a novel technology of inoculant production |
| author |
Kozyrovska, N.O. Negrutska, V.V. Kovalchuk, M.V. Voznyuk, T.N. |
| author_facet |
Kozyrovska, N.O. Negrutska, V.V. Kovalchuk, M.V. Voznyuk, T.N. |
| topic |
Клітинна біологія |
| topic_facet |
Клітинна біологія |
| publishDate |
2005 |
| language |
English |
| container_title |
Біополімери і клітина |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Paenibacillus sp. — перспективна бактерія для створення технології виробництва бакпрепаратів для рослин Paenibacillus sp. — перспективная бактерия для создания технологии производства бакпрепаратов для растений |
| description |
A bacterial strain IMBG156 producing exopolysaccharide (EPS) was isolated from siliceous rock and identified as a Paenibacillus species by partial sequencing its 16S rDNA. Paenibacillus sp. IMBG156 was used in a novel technology for inoculant production based on co-cultivating this bacterium with any bacterium of choice. Paenibacillus sp. provides in situ the bacterial cells of the inoculant with EPS, a carrier, and most likely with a source of carbon and energy. The partner bacterium designates a type of inoculant (biopesticide or biofertiliser). The strain IMBG156 does not destroy the signaling system of Gram-negative partners, based on acylated homoserine lactones, stimulates plant growth, and is rather competitive in the plant rhizosphere and soil. A prototype of the inoculant based on dual-culture Paenibacillus sp. IMBG156 - Pseudomonas sp. IMBG163 exhibits a noticeably longer shelf life than monoculture of Pseudomonas sp. IMBG163.
(ЕПС), виділено з силікатної породи та ідентифіковано як Paenibacillus sp. на основі визначення первинної нуклеотидної послідовності 16S рДНК Paenibacillus sp. IMBG156 використа но у технологи виробництва мікробних препаратів, яка базуться на спільному культивуванні цієї бактерії та бактерії за вибором. Paenibacillus sp. постачає in situ бактерійні клітини носієм (ЕПС) та, найвірогідніше, є джерелом вуглецю і енергії. Бактерія-партнер визначає тип препарату (біодобриво, біопестицид). Штам IMBG156 не пошкоджує сигнальної системи грамнегативних бактерій-партнерів, яка грунтується на ацильованих гомосеринлактонах, стимулює ріст рослини і є доволі конкурентною у ризосфері. У демонстраційному препараті, створеному на основі бактерій-партнерів Paenibacillus sp. IMBGI56—Pseudomonas sp. IMBG163, довше зберігається необхідний титр бактерій, ніж у монокультурі Pseudomonas sp. IMBG163.
A bacterial strain IMBG156 producing exopolysaccharide (EPS) was isolated from siliceous rock and identified as a Paenibacillus species by partial sequencing its 16S rDNA. Paenibacillus sp. IMBG156 was used in a novel technology for inoculant production based on co-cultivating this bacterium with any bacterium of choice. Paenibacillus sp. provides in situ the bacterial cells of the inoculant with EPS, a carrier, and most likely with a source of carbon and energy. The partner bacterium designates a type of inoculant (biopesticide or biofertiliser). The strain IMBG156 does not destroy the signaling system of Gram-negative partners, based on acylated homoserine lactones, stimulates plant growth, and is rather competitive in the plant rhizosphere and soil. A prototype of the inoculant based on dual-culture Paenibacillus sp. IMBG156 - Pseudomonas sp. IMBG163 exhibits a noticeably longer shelf life than monoculture of Pseudomonas sp. IMBG163.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/155709 |
| citation_txt |
Paenibacillus sp., as a promising candidate for development of a novel technology of inoculant production / N.O. Kozyrovska, V.V. Negrutska, M.V. Kovalchuk, T.N. Voznyuk // Біополімери і клітина. — 2005. — Т. 21, № 4. — С. 312-318. — Бібліогр.: 27 назв. — англ. |
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ISSN 0233-7657. Біополімери і клітина. 2005. T. 21. № 4
КЛІТИННА БІОЛОГІЯ
Paenibacillus sp., as a promising candidate
for development of a novel technology of
inoculant production
N . O. Kozyrovska, V . V . Negrutska, M . V. Kovalchuk, T. N . Voznyuk
Institute of Molecular Biology and Genetics of the National Academy of Sciences
150 Zabolotnoho str., 03143 Kyiv, Ukraine
E. mail: kozyr@imbg.org.ua
A bacterial strain IMBG1S6 producing exopolysaccharide (EPS) was isolated from siliceous rock and
identified as a Paenibacillus species by partial sequencing its 16S rDNA. Paenibacillus sp. IMBG156 was
used in a novel technology for inoculant production based on co-cultivating this bacterium with any
bacterium of choice. Paenibacillus sp. provides in situ the bacterial cells of the inoculant with EPS, a
carrier, and most likely with a source of carbon and energy. The partner bacterium designates a type of
inoculant (biopesticide or biofertiliser). The strain IMBG156 does not destroy the signaling system of
Gram-negative partners, based on acylated homoserine lactones, stimulates plant growth, and is rather
competitive in the plant rhizosphere and soil. A prototype of the inoculant based on dual-culture
Paenibacillus sp. IMBG156 — Pseudomonas sp. IMBG163 exhibits a noticeably longer shelf life than
monoculture of Pseudomonas sp. IMBGI63.
Key words: exopolysaccharide, inoculant, partner bacteria
Introduction. The increasing concern on agroche-
micals hazard to health and economical problems have
promoted fundamental research in the area of alter
native agriculture and in search for new agrobiotech-
nologies. Recently, tremendous efforts have been
invested in studies of molecular mechanisms of plant
defense [1—3].
Defense-signaling components have been dis
covered, and new, effective and sustainable alter
natives to pesticides proposed. However, at the pre
sent time a sustainable strategy of crop defense still
relies on usage of microbial biological agents able to
induce system resistance in crop plants. The microbial
inoculants based on the competitive, beneficial for the
plant bacteria are considered as a reasonable alter
native to agrochemicals [4, 5] . It is critical for the
© N. O. K O Z Y R O V S K A , V. V. N E G R U T S K A , M. V. K O V A I jC H U K ,
T. N. V O Z N Y U K , 2 0 0 5
inoculant development that the inoculant product was
in a formulation not only to deliver an adequate
bacterial population but also to have enough product
shelf life. Nowadays, the practical formulations are in
use to prolong survival of Gram-negative bacteria
[6—11], however, the usage of both mineral and
organic carriers for bacteria makes inoculants more
expensive. In our previous research we used the
exopolysaccharide (EPS) mucilan produced by B.
mucilaginosus B-4901 as an inexpensive carrier in the
series of inoculants KLEPS (KLityna (a cell, Ukr.)
and EPS) to prolong Gram-negative bacteria survival
[12]. The inoculants enhanced crop production on
poor soils and exhibited enough shelf life. In spite of
advantages, the technology of KLEPS development
needed a separate stage of EPS manufacture, which
complicated the procedure and raised the price of the
inoculant. The objectives of this study were to isolate
slime-producing bacteria, following the idea to use
mailto:kozyr@imbg.org.ua
PAENIBACILLUS SP., AS A CANDIDATE FOR A NOVEL TECHNOLOGY
EPS as a carrier for inoculant development, and to
simplify the two-stage technology of inoculant ma
nufacture.
Materials and Methods. Bacterial strains. The
microorganisms used in this work were either from
our Institute collection (Paenibacillus sp. IMBG156,
Klebsiella oxytoca IMBG26, Rif, Pseudomonas sp.
IMBG163, Pseudomonas sp. IMBG168, P. aureo-
faciens IMBG288, Agrobacterium sp. IMBG260, P.
syringae pv. syringae IMBT295, Xanthomonas axono-
podis pv. phaseoli IMBF293) or kindly provided from
other collections: P. fluorescens 13525 from ATCC;
Pseudomonas sp. AP33 from A. Perebityuk (Institute
of Cytology and Genetics of Belarus Academy of
Sciences, Minsk); Pantoea agglomerans IMV56 and
Erwinia carotovora subsp. atroceptica IMB9027 from
R. Gvozdyak (Institute of Microbiology and Virology
of NASU, Kyiv); Agrobacterium tumefaciens A136, A
tumefaciens NT1 (pTiC58AaccR) and A. tumefaciens
NT1 from L. Halda-Alija (Mississippi University).
Culture conditions. The media for bacterial gro
wth were: KB [13] and LB [14] used for all strains
of bacteria, except for Paenibacillus sp. being cultured
in the medium MZ [12]. Zeolite (10 g/1) was added
to LB when needed.
Determination of EPS content. Cells of a two-day
culture were centrifuged at 10,000 g for 30 min, EPS
was extracted from supernatant by 2 v of ethanol and
dried at 37 °C until stable weight was obtained.
Bacteria isolation procedures were performed us
ing the samples of zeolite collected from Sokyrnytzya
(Transkarpatian region) and fragments of silica rocks
originated from Khmelnitsky region. In separate ex
periments 1 g of zeolite or silica rocks (fraction of
5 mm) was incubated in MZ for 48 h at 30 "C. The
accumulating cultures were diluted serially and spre
ad on selective MZ medium. Slime colonies were
collected, bacteria were purified, and identified accor
ding to N. Krasilnikov [15].
Total DNA isolation was performed as recom
mended in [16].
Amplification, purification, and analysis of 16S
rDNA. PCR primers pair of pA (8—27) and pH
(1542—1523) described in [17] was used for identi
fication of the rrn (16S rRNA) gene, and specific
sequences of two selected isolates were detected at the
annealing temperature of 52 °C and the standard
concentration of MgCl2 (1.5 mM). The reaction was
performed with 25 pmol of each primer («Sigma»,
USA), 50 f*M dNTP-mix, 2 U of Taq-DNA-poly-
merase (both reagents from «Fermentas», Lithuania).
Amplification was carried out in ALF thermal cycler
(«Pharmacia», Sweden). Initial denaturation was per
formed at 98 °C for 3 min, then Taq-DNA-poly-
merase was added. The thermal profile involved 28
cycles of penetration temperature of 93 °C for 30 s,
primer annealing at 52 °C for 30 s and 72 "C for
1 min. Amplicons were checked on a 2.0 % agarose
gel. Purification of amplicons was performed with
UltraClean™ PCR Clean-up™ Kit (MoBio Labo
ratories Inc., USA). Analysis of PCR products per
formed by Blast N search program (NCBI, htt-
p://www.ncbi.nlm.nih.gov). A phylogenetic tree was
constructed by the program ClustalW 1.83 (http://w-
ww.genebee.msu.su/genebee.html).
Co-cultivating of partners was performed in MZ.
After 30 h of co-cultivating, the serial dilutions were
made for evaluation of population size. The partner
cultures were detected on LB or KB agar, Paeni
bacillus sp. — on MZ plates.
Assay for production of signaling molecules. Bac
terial strains (separately and in pairs with Paenibacil
lus sp.) were tested in cross-feeding assays for
acylated homoserine lactones (AHLs), using the indi
cator strain A tumefaciens A136 as recommended in
[18].
Antibacterial activity was tested in vitro on Petri
dishes by the standard agar-diffusion assay, using
two layer agar with the upper layer of an indicator
culture soft agar (0.4 % ) .
Detection of the acetylene reductase (nitrogenase)
activity (ARA) was performed according to [19]. The
ARA of K. oxytoca IMBG26 was detected with the gas
chromatograph Tzvet (Cheh Republic) in 14 ml flasks
where the bacterial culture was grown in an N-free
medium supplemented with sucrose or EPS (final
content 1.5 and 1.0 %, respectively) in the presence
of 10 % acetylene within 16 h at 28 °C.
Plant growth conditions, bacteria inoculation and
re-isolation. Ten wheat germinated seeds were inocu
lated with a washed overnight monoculture, a dual
culture or a suspension of bacterial strains mixed in
equal proportions (10* colony forming units, CFU/ml)
and placed in zeolite (Sokyrnytzya deposit). Control
plants were left non-inoculated. The plants were
maintained under natural light at 20 °C in a growth
chamber. The plants were watered once per two days.
At the end of the experiment (14 days after ino
culation) all plants were harvested and external root
colonization was examined. Root sections of 100 mg
313
file:///www.ncbi.nlm.nih.gov
http://w-
http://ww.genebee.msu.su/
KOZYROVSKA N. O. ET AL.
N o t e . Error represents standard deviation. Treatment is different from the control at p - 0.05 as determined by Student's f-test. Values
followed by the same letter in a column are not significantly different.
were vortexed in 0.9 % NaCl, and serial dilutions
were plated on selective media LB, KB, MZ supple
mented with rifampicin (50 /ig/ml) when needed to
discriminate between bacteria.
Statistical analysis of results. The data on bio
metrical parameters of wheat and bacteria survival are
means from three replications. Statistical analysis was
performed using SigmaPlot 8.0 software. Standard
deviations were calculated for each data point.
Nucleotide sequence accession number. The se
quence generated in this study has been deposited in
the GenBank database under accession number
AY645946.
Results and Discussion. Phenotypic characteris
tics. Two isolates (from zeolite and silica rock,
designed IMBG156 and IMBG157) were characterized
as aerobic Gram-positive rods of (0.2—0.5) • 2.0 fim
which formed spores of 0.5—0.7 /nm. The spore
position was preferentially central but terminal lo
cation was rarely observed. The optimal temperature
of growth was 28 °C, but they grew well in a range
of 10—45 "C. On the agar MZ medium they created
transparent slime colonies of 10.0—13.0 mm diameter
and produced 10.0—13.0 g EPS per 1 1 of liquid
medium. The isolates consumed carbohydrates, ge
nerated acids, and did not utilize amino acids as C
and N sources. Bacteria hydrolyzed starch and could
not grow anaerobically with nitrate as a respiratory
substrate. We did not manage to determine the
taxonomic position of the isolates with Bergy's deter
minative manual. To clarify their systematic position,
it was practical to analyze the phylogenetic marker
gene, rrn, encoding RNA of small subunit of ribosome
(16S).
Analysis of the 16S rDNA amplicon. The 16S
rDNA amplicons were obtained from IMBG156 of
1500 bp in PCR, and a sequence of 621 bp was
deciphered and aligned with the most closely related
bacterial sequences. The sequence shared 97 %
similarity to the deposited sequence for Paenibacillus
velasolus. In a phylogenetic tree based on sequencing
data this isolate clustered with P. velasolus, however,
more efforts were needed to prove or disprove rela-
7-1
Fig 1. Average size of populations of bacterial strains associated with
wheat roots: two weeks after inoculated seed planting
tedness of the isolate to P. velasolus, and in this
study we did not use a species name.
Effects of the bacterium on growth of wheat and
survival in the plant rhizosphere. Relation to the plant
of the assistant bacterium planned for the inoculant
development should be at minimum neutral, but not
antagonistic. The tests showed that strain IMBG156
•31 A
PAENIBACILLUS S P . , AS A C A N D I D A T E FOR A NOVEL TECHNOLOGY
11
-~10 I
3 9
• a monoculture
• a dual culture
• a monoculture+zeolite
—i 1 i 1 1 1 i - i
0 20 40 Time, days
Fig. 2. Dynamics of survival of Pseudomonas sp. IMBG 163 in
dependence on culture conditions
did not cause a phytotoxic effect and increased wheat
shoot height as shown in Table 1. Being applied
together with Pseudomonas sp. IMBG163 for seed
inoculation, it promoted plant growth more efficiently.
In the rhizosphere of wheat inoculated by a rationally
assembled consortium of plant growth promoting
rhizobacteria (Pseudomonas sp. IMBG163, P. aureo
faciens IMBG288, K. oxytoca IMBG26), the strain
IMBG156 was quite competitive on background of
beneficial bacteria (Fig. 1).
Co-cultivation of Paenibacillus sp. IMBG156 with
bacteria of interest. With the idea to keep bacteria
alive in the gel produced by bacteria within co-
cultivation, Paenibacillus sp. and chosen Gram-ne
gative bacteria were cultured by pairs. The results of
experiments on co-cultivating showed practically the
absence of restrictions in picking up partners for
Paenibacillus sp. (Table 2). The partners gained a
population size of log 9—10 CFU/ml. The critical
factors in the one-step procedure of prototype ino-
culant manufacture were the size of population of a
strain that determined a sort of inoculant, and also
the concentration of EPS produced by Paenibacillus
sp. In the case of co-cultivating IMBG 156 together
with K. oxytoca IMBG26 or Pseudomonas sp.
IMBG163, the culture gel contained 1.4±0.05E+9 or
2.5±0.09E+9 CFU/ml of a bacterium-partner, res
pectively, and not less than 20.0 g/1 of EPS.
A term of the inoculant shelf life is important
when the inoculant is based on Gram-negative bac
teria. We showed that survival of Pseudomonas sp.
IMBG 163 was prolonged in the dual culture with
Paenibacillus sp. in comparison with the monoculture
(Fig. 2). A better survival of IMBG 163 was also
observed in a minimal medium supplemented with
zeolite. The results showed that the strain IMBG 163
preserved the population size at the level of log
9 CFU/ml in both dual culture and monoculture
grown in the presence of zeolite within 2 months. On
the contrary, the population size of IMBG 163 de
clined from log 10 to log 7 CFU/ml without co-
inoculation by Paenibacillus sp. IMBG156 or addition
of the mineral to the medium. This clearly de
monstrated that both Paenibacillus and zeolite sup
ported survival of Pseudomonas sp. IMBG163. K.
oxytoca IMBG26 was able to grow and reduce ace
tylene (518 nM C 2 H 4 / h flask) in the nitrogen-free
medium where 1.0 % EPS (derived from Paeni
bacillus sp. IMBG156) was used as a carbon source,
and this suggested that EPS produced by Paeni
bacillus had the potential to support growth and
activity of the partner.
315
•Indicator phytopathogenic bacteria: I — P. syringae pv. syringae IMBG295; II — Erwinia carotovora subsp. atroceptica IMB9027; III —
Xanthomonas axonopodis pv. phaseoli IMBG293; **diameter of zone of suppression of indicator bacteria (mm): 2 — 10.0—20.0; 3 —
21.0—30.0; 4 — 31.0.
Detection of antibacterial activity in dual cultures.
Signaling systems play a role in bacteria-bacteria and
plant-bacteria communications [20]. Antibacterial ac
tivity of some pseudomonads is controlled with AHLs
[21, 22]. It is well known that Gram-positive bacteria
are able to destroy AHLs of Gram-negative neighbors
[23 ]. To compose dual bacterial pairs, it is important
to know that quorum signaling is not impaired by
co-cultivation. Bacteria used in experiments on co-
cultivation were tested in cross-feeding assays for
AHL4_14 detection earlier [24 ]. Few of them produced
AHLs (Pseudomonas sp. IMBG163, Pseudomonas sp.
IMBG168 and P. auerofaciens IMBG288), and no
difference was observed between mono- and dual
Pseudomonas-Paenibacillus cultures with respect to
AHL production. The results represented in Table 3
showed inhibition of pathogenic bacteria by both
monocultures of pseudomonads and appropriate dual
Pseudomonas-Paenibacillus cultures. This may mean
that strain Paenibacillus sp. IMBG156 did not impair
AHLs produced by a partner and indirectly demon
strated integrity of AHLs in dual cultures.
Paenibacillus sp. IMBG156 has been selected as
a bacterium-nurse for the dual-culture technology of
inoculant development, first of all, due to production
of large amounts of EPS. The strain IMBG156
provided the living cells of a bacterium-partner, the
second species of two-component consortium, with a
carbon source and apparently caused better survival
of the latter. In this study IMBG156 displayed
commensal interactions in the pairs with other bac
terial strains and synergistic positive impact on the
plant. These results are consistent with those ob
tained for other bacteria acting synergistically on the
plant development [25, 26]. IMBG156 was quite
competitive in the plant rhizosphere bacterial com
munity and in the soil, in contrast to the known data
on the gradual replacement of Paenibacillus by Pseu
domonas [27]. These additional beneficial features
make the strain rather promising for application for
seed inoculation in programs of plant health care and
soil remediation in company with biocontrol bacteria.
The inoculants, containing both Paenibacillus sp.
IMBG156 and a partner bacterium, can be stored for
a relatively long period of time in the presence of
large amounts of EPS produced in situ without
preservatives and conventional carriers. This finding
is based on a concept of keeping bacteria alive under
storage of the dual culture in a gel and may be
explained, first of all, by the fact that growing any
Gram-negative bacterium with Paenibacillus sp.
IMBG156 results in stimulation of EPS production
which can serve both as a carbon and energy source
for the bacterium-partner. In case when EPS serves
as a carrier, the organisms appear to establish struc
tured populations where cells are not aggregated.
Under this condition the bacteria are positioned in a
heterogeneous environment with gradients of nutri
ents and waste products as a consequence of diffusion
and mass transport processes, and it is therefore to
be expected that this heterogeneity is reflected in the
physiology of the individual cells and better survival.
EPS keeps up water and nutritional regime, and
therefore bacterial cells are physiologically active
under storage, in contrast to a dry KLEPS formu
lation where bacteria were dormant. The dual-culture
technology based on co-cultivating the bacterium
Paenibacillus sp. IMBG156 and any bacterium of
choice is simpler and less expensive compared to the
previous technology [12].
PAENIBACIIXUS St., AS A CANDIDATE FOR A NOVEL TECHNOLOGY
Acknowledgements. We are grateful to Prof.
B. Lugtenberg (Leiden University) for providing se
quencing data of the rrn gene of Paenibacillus sp.
IMBG156 and to Prof. Diethelm Kleiner for critical
reading of the manuscript. This work partially sup
ported by Ukrainian Branch of World Wide Labo
ratory, subcontract N 901 -02-5281-5700-WLU.
H. О. Козировська, В. В. Негруцька, М. В. Ковальчук,
Т. М. Вознюк
Paenibacillus sp. — перспективна бактерія для створення
технології виробництва бакпрепаратів для рослин
Резюме
Штам бактерій IMBG156, який продукує екзополісахарид
(ЕПС), виділено з силікатної породи та ідентифіковано як
Paenibacillus sp. на основі визначення первинної нуклеотидної
послідовності 16S рДНК Paenibacillus sp. IMBG156 використа
но у технологи виробництва мікробних препаратів, яка ба-
зуться на спільному культивуванні цієї бактерії та бактерії
за вибором. Paenibacillus sp. постачає in situ бактерійні кліти
ни носієм (ЕПС) та, найвірогідніше, є джерелом вуглецю і
енергії. Бактерія-партнер визначає тип препарату (біодоб-
риво, біопестицид). Штам IMBG156 не пошкоджує сигнальної
системи грамнегативних бактерій-партнерів, яка грунтує
ться на ацильованих гомосеринлактонах, стимулює ріст рос
лини і є доволі конкурентною у ризосфері. У демонстрацій
ному препараті, створеному на основі бактерій-партнерів
Paenibacillus sp. IMBGI56—Pseudomonas sp. IMBG163, довше
зберігається необхідний титр бактерій, ніж у монокультурі
Pseudomonas sp. IMBG163.
Ключові слова: екзополісахариди, бакпрепарат, бактерії-
партнери.
Н. А. Козыровская, В. В. Негруцкая, М. В. Ковальчук,
Т. Н. Вознюк
Paenibacillus sp. — перспективная бактерия для создания
технологии производства бакпрепаратов для растений
Резюме
Штамм бактерии IMBG156, продуцирующий экзополисахарид
(ЭПС), выделен из силикатной породы. Он идентифицирован
как Paenibacillus sp. на основе определения первичной нуклео-
тидной последовательности 16S рДНК. Paenibacillus sp.
IMBG156 использован в технологии производства микробиоло
гических препаратов, основанной на совместном культивиро
вании этой и выбранной бактерий. Paenibacillus sp. снабжает
in situ клетки бактерий носителем (ЭПС) и является, наибо
лее вероятно, источником углерода и энергии. Бактерия-пар
тнер определяет тип препарата (биоудобрение, биопестицид).
Штам IMBG156 не повреждает сигнальной системы грам-
мотрицательных бактерий-партнеров, которая основывается
на аицлированных гомосеринлактонах, стимулирует рост
растений и является достаточно конкурентоспособной в ри
зосфере. В демонстрационном препарате, созданном на базе
бактерий-партнеров Paenibacillus sp. IMBG156—Pseudomonas
sp. IMBG163, дольше сохраняется необходимый титр бакте
рий, чем в монокультуре Pseudomonas sp. IMBG163.
Ключевые слова: экзополисахариды, бакпрепарат, бактерии-
партнеры
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