Detection of Methylobacterium radiotolerans IMBG290 in potato plants by in situ hybridization
A new bacterial strain of pink-pigmented facultative methylotroph (M. radiotolerans IMBG290) which was previously isolated from in vitro grown potato plantlets after their inoculation with Pseudomonas fluorescens IMBG163 was detected in tissues by in situ hybridization method (ISH/FISH). The presenc...
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| Language: | English |
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Інститут молекулярної біології і генетики НАН України
2009
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| Journal Title: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Cite this: | Detection of Methylobacterium radiotolerans IMBG290 in potato plants by in situ hybridization / O.V. Podolich, N.O. Kozyrovska, A.M. Pirttilа // Біополімери і клітина. — 2009. — Т. 25, № 2. — С. 115-119. — Бібліогр.: 34 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859997696663748608 |
|---|---|
| author | Podolich, O.V. Kozyrovska, N.O. Pirttila, A.M. |
| author_facet | Podolich, O.V. Kozyrovska, N.O. Pirttila, A.M. |
| citation_txt | Detection of Methylobacterium radiotolerans IMBG290 in potato plants by in situ hybridization / O.V. Podolich, N.O. Kozyrovska, A.M. Pirttilа // Біополімери і клітина. — 2009. — Т. 25, № 2. — С. 115-119. — Бібліогр.: 34 назв. — англ. |
| collection | DSpace DC |
| description | A new bacterial strain of pink-pigmented facultative methylotroph (M. radiotolerans IMBG290) which was previously isolated from in vitro grown potato plantlets after their inoculation with Pseudomonas fluorescens IMBG163 was detected in tissues by in situ hybridization method (ISH/FISH). The presence of Methylobacterium rRNA was observed in leaves and stems of potato plantlets, whereas no signal was detected in potato roots. The signal was less abundant in the untreated plants than in the plantlets infected with M. radiotolerans IMBG290.
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| first_indexed | 2025-12-07T16:34:33Z |
| format | Article |
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ÌÎËÅÊÓËßÐÍÀ ² Ê˲ÒÈÍÍÀ Á²ÎÒÅÕÍÎËÎò¯
Detection of Methylobacterium radiotolerans IMBG290 in
potato plants by in situ hybridization
O. V. Podolich, L. P. Ovcharenko1, N. O. Kozyrovska1, A. M. Pirttilà2
Institute of Agroecology of UAAS
12 Metrologichna str., Kyiv, Ukraine, 03680
1Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine
150 Acad. Zabolotnoho str., Kyiv, Ukraine, 03680
2Department of Biology University of Oulu
Linnanmaa, PO Box 3000, FIN-90014 Oulu, Finland
kozyr@imbg.org.ua
A new bacterial strain of pink-pigmented facultative methylotroph (M. radiotolerans IMBG290) which was
previously isolated from in vitro grown potato plantlets after their inoculation with Pseudomonas
fluorescens IMBG163 was detected in tissues by in situ hybridization method (ISH/FISH). The presence of
Methylobacterium rRNA was observed in leaves and stems of potato plantlets, whereas no signal was
detected in potato roots. The signal was less abundant in the untreated plants than in the plantlets infected
with M. radiotolerans IMBG290.
Keywords: in situ hybridization, Methylobacterium radiotolerans IMBG290, potato plantlets.
Introduction. The pink-pigmented facultative
methylotrophic bacteria (PPFMB) are indispensable
inhabitants of the phyllosphere where methanol is
produced [1]. Under natural conditions, colonization of
plant leaves by PPFMB seems to occur via soil particle
transfer to the leaves by air [2]. Bacteria in the genus
Methylobacterium are often isolated from buds, roots
and in vitro plant cultures, and they are also known as
endophytic bacteria, found inside plant tissue [3–8].
These bacteria evidently have an intimate association
with plants, as they promote plant growth and
development by production of phytohormones [2,
9–13]. The genus Methylobacterium belongs to a-
Proteobacteria, and members of this genus are capable
of growing on monocarbon compounds such as
methanol and methylamine, as well as on a wide range
of multicarbon substrates [3]. Associated with their
carbon utilitization capabilities, methylotrophic
bacteria can be efficient destructors of organic
pollutants in the environment [14, 15].
Endophytic community of potato plants varies, and
it is composed by a broad phylogenetic spectrum of
bacteria: a-, b-, and g-Proteobacteria, Flexibacter-
Cytophaga-Bacteroides, gram-positive microorga-
nisms with high G+C-content, and Planctomycetales
[16–18]. Bacteria of the genus Methylobacterium have
not earlier been detected inside the potato in vitro
tissue. In our experiments PPFMB have been detected
in potato plantlet bacterial communities after activating
them with exogenic bacterium [19].
Here we describe localization of a bacterial isolate
capable of utilizing methanol as a source of carbon and
115
ISSN 0233-7657. Á³îïîë³ìåðè ³ êë³òèíà. 2009. Ò. 25. ¹ 2
Ó ²íñòè òóò ìî ëå êó ëÿð íî¿ á³îëî㳿 ³ ãå íå òè êè ÍÀÍ Óêðà¿ íè, 2009
energy in tissues of both uninoculated and inoculated
in vitro-grown potato plants.
Materials and Methods. Potato plantlets. Potato
(Solanum tuberosum L.) cultivar Chervona Ruta
variety of potato was used in this study. In vitro-grown
potato plantlets were grown on Murashige and Skoog
medium (MS) [20] without phytohormones for one and
a half year, in a conditioned room under a light-dark
period of 16/8 h at 24 °C and 150 mE m–2 s–1.
Bacterial strains and culture conditions. PPFMB
isolate was cultured in M9 medium [21] supplemented
with methanol (1.0 %) at 28 °C for 3–4 days.
Inoculation of potato plants. Nodal cuttings of
six-week-old potato plantlets were used for inoculation
with the PPFMB isolate. The methylobacteria culture
was pelleted by centrifugation (3000 g, 10 min), rinsed,
and resuspended in sterile distilled water (SDW) to a
concentration of 108 CFU/ml–1. The plantlets were cut
in 1-cm pieces and incubated with bacterial suspension
for 5 min. The cuttings were dried on a sterile paper and
placed on the MS medium without hormones.
Untreated plants were incubated in SDW instead of the
bacterial culture.
In situ hybridization. Samples of stems, leaves, and
roots of plantlets were taken for in situ hybridization
experiments. Both PPFMB-infected and untreated
plants were analyzed. All samples were surface-
sterilized for 1 min in 70 % ethanol and for 15 min in
6 % calcium hypochlorite, followed by rinsing three
times in SDW. The leaf tissues were cut in pieces of
1.5 ́1.5 mm, stems were cut in 1 mm2- and roots in
1 mm2-pieces. The cuttings were fixed in 2 %
paraformaldehyde, 2,5 % glutaraldehyde, 0.1 M saline
buffer, pH 7.4, at 4 °C under vacuum overnight. The
fixed samples were dehydrated, cleared through
ethanol/t-butanol series and embedded in paraffin.
Oligonucleotide probes MB and E11 [5] comple-
mentary to the unique regions of 16S rRNA of Me-
thylobacterium and eubacteria, respectively, were used
for in situ hybridization. The oligonucleotides were
end-labeled with fluorescein during synthesis or with
digoxigenin, using the DIG Oligonucleotide 3'-end
Labeling Kit («Roche Applied Science», Finland).
Hybridization was performed as described by
Pirttila et al. [5]. The paraffin-embedded specimens
were sectioned, and the 8 mm sections were baked on
silane-coated slides. The paraffin was removed in
xylene prior to hybridization. All samples of stems,
roots and leaves of infected and untreated potato in
vitro plants were hybridized with the probes MB or E11
as described by Pirttila et al. [5].
The detection of the digoxigenin label was
performed with the DIG Nucleic Acid Detection Kit
(«Roche Applied Science»), and the slides were
viewed with optical microscope (Optiphot-2
Photomicroscope, «Nikon», Japan) under bright field
illumination. For the fluorescein label, the samples
were viewed under fluorescent light with fluor
objectives, episcopic-fluorescence attachment EF-D,
and the filter set UV-1A («Nikon»).
DNA staining. The RNase-treated sections were
stained with ethidium bromide (10 mg/ml) for 45 min in
the dark. The slides were rinsed with SDW, air dried in
the dark and viewed immediately under the fluorescent
light.
Bacterial DNA isolation, PCR and sequencing.
Bacterial DNA isolation was performed with
UltraCleanTM Microbial DNA isolation kit (MoBio
Laboratories Inc., USA). To determine the nucleotide
sequence of the rrs gene (16S ribosomal RNA) a PCR
product was amplified with primers pA and pH
described by Edwards et al. [22], cleaned with
UltraCleanTM PCR Clean-up DNA purification kit
(MoBio Laboratories Inc., USA) and cloned into vector
pTZ57R/T using InsTAcloneTM PCR Cloning Kit
(«Fermentas», Lithuania). The PCR product was then
sequenced with primers M13/pUC (forward and
reverse) («Fermentas») by the Sanger method [23]
using «Amersham» (USA) sequencing kit Cy5
AutoCycle and apparatus ALF express («Pharmacia
Biotech.», Sweden). The nucleotide sequence was
analyzed with the basic local alignment search tool
(BLAST) and Vector NTI 8.0 program (Infomax Inc.,
USA).
Nucleotide sequence accession number. The
sequence generated in this study has been deposited in
the GenBank database under accession number
EF583689.
Results. The endophytic isolate M1, originated
from in vitro-grown potato [19], was characterized by
sequencing the 16S rDNA, and a comparison of
specific sequence of the rrs gene with sequences
116
PODOLICH O. V. ET AL.
Ðè ñóí êè äî ñòàòò³ O. V. Podolich òà ñï³âàâò.
ISSN 0233-7657. Á³îïîë³ìåðè ³ êë³òèíà. 2009. Ò. 25. ¹ 2
Fig. 1. In situ hybridization of leaf tissues of in vitro potato plant with both
digoxigenin and fluorescence-labeled oligonucleotide probe MB: A, B, C – leaf
control, digoxigenin detection; D – leaf control, fluorescein detection; E – leaf
infected with M. radiotolerans, fluorescein detection; F, G – leaf infected with M.
radiotolerans, digoxigenin detection (arrows – highest signal intensity; x/v – xy-
lem vessel; p – parenchyma; v – vascular tissues). Scale bar = 50 mm
PODOLICH O. V. ET AL.
Fig. 3. In situ hybridization of root
tissues of in vitro potato plant with
digoxigenin-labeled oligonucleoti-
de probe MB: A – root of infected
plant with M. radiotolerans; B –
root control (arrows – highest
signal intensity; x – xylem; c/c –
center cylinder; d/v – differential
vessel). Scale bar = 50 mm
Fig. 2. In situ hybridization of stem tissues of in vitro potato plant with both digoxigenin (A) and fluorescence-labeled (C, D) oligonucleotide
probe MB and DNA staining (B): A, B, C – stem tissues of plantlets infected with M. radiotolerans; D – stem control (arrows – highest signal
intensity; b – biofilm; p – parenchyma; n – nuclear; x – xylem). Scale bar = 50 mm
deposited to GenBank suggested that the M1 isolate
belongs to methylotrophic bacteria, having the highest
homology to M. radiotolerans (96–100 %) and
M. organophilum (90 %). A detailed analysis of the
sequence demonstrated that the region conserved
between methylobacteria was present and a variable
region of 50 bp (nucleotides 901–951) was identical to
M. radiotolerans. The sequence was 99 % identical and
had a seqmatch score of 0.933 with type strains of
M. radiotolerans (JCM 2831, DSM 1819) in RDP
(Ribosomal DNA Project).
In situ hybridization was performed on tissues of
leaves, stems and roots of in vitro-grown potato plants.
The specific for bacteria in the Methylobacterium
genus probe MB was used for hybridization. Both
untreated and M. radiotolerans-inoculated plants were
surface-disinfected prior a processing for hybridization
in order to avoid epiphyte contamination of the
samples.
The hybridization signals of Methylobacterium
rRNA were detected in leaves of untreated plants
mainly at the tip of the leaf in sponge and palisade
parenchyma and xylem vessels (Fig. 1, A–D, see inset).
In the infected leaf tissues, the signals were present in
the xylem vessels (Fig. 1, E–G) and in parenchyma
tissues (Fig. 1, E). In the stems of infected and
untreated plants the Methylobacterium rRNA was
mainly detected in the parenchyma and the vascular
tissues (Fig. 2, A–C, see inset). In the untreated plants
the signal was weaker than in infected plants (Fig. 2,
D). DNA staining of infected stem samples was
observed in areas indicated by in situ hybridization,
supporting the presence of bacteria in the genus
Methylobacterium inside cells of parenchyma tissues
(Fig. 2, B). Specifically, in the stem parenchyma both
types of hybridization signals and the DNA staining
revealed biofilms (Fig. 2, A–D). Based on the signals
bacteria were found at highly localized areas in the
biofilm (Fig. 2, A–C). In the root of the infected plants,
Methylobacterium rRNA was detected in vascular (Fig.
3, A, see inset) and parenchyma tissues. In the untreated
plants Methylobacterium rRNA was not detected in the
root tissues (Fig. 3, B).
Discussion. Methylobacteria have earlier been
found as endophytes in plant tissues [5, 6, 24] but the
locations, infection processes and roles of these
bacteria have rarely been studied. The methylotrophs
are suspected to supply the plant with growth
stimulating compounds or to help the plant in
activation of defense system for a better adaptation to
the environment. The results of the in situ hybridization
experiment showed the presence of Methylobacterium
rRNA in all organs (leaves, stems and roots) of the
inoculated potato plants. Our results provide the first
direct evidence that methylotrophs exist inside in vitro
potato tissues. These bacteria may exist as regular
inhabitants of potato plants since we found bacteria in
plantlets after in vitro cultivation of one and half year.
In the untreated plants, the Methylobacterium rRNA
was detected only in the leaves and stems. It means that
methylobacteria preferably reside in the aerial parts of
these plants. This is also supported by the fact that the
hybridization signal was the most abundant in the
stems, slightly less in the leaves and the lowest in the
root tissues of Methylobacterium-inoculated potato
plantlets.
Since these bacteria were detected by molecular
method and could not be isolated on selective media,
they may normally exist in an unculturable state inside
the potato plants. Methylobacteria can persist in
natural environments by forming biofilms [25].
Biofilms are specifically often found on plant surfaces
and provide a mechanism for the bacteria to survive in
harsh environments [26]. Biofilms are also an
unculturable state of the bacteria. In the in situ
hybridization experiments, a distinct biofilm structure
was detected in the stem parenchyma of the in
vitro-grown potato plants. Therefore, a regular
existence as biofilms inside the potato tissues may
explain the lack of culturability of these bacteria.
Intercellular spaces and xylem vessels are the most
commonly reported locations of endophytic bacteria
[27–31]. In the present study the Methylobacterium-
specific in situ hybridization signals were mainly
located in the xylem vessels of leaves and stems, but
not in the root vessels of untreated potato plants. This is
in agreement with the data of absence of Serratia
marcescens rice root interior [30], as well as the
endophyte Enterobacter asburiae in cotton roots [32].
The bacterial endophytes spread systemically but not in
root inner tissue after seed inoculation. In our study the
absence of the methylobacteria in roots of untreated
117
DETECTION OF M. RADIOTOLERANS IMBG290 IN POTATO PLANTS BY IN SITU HYBRIDIZATION
potato plants may be explained by the putative role of
these bacteria in scavenging monocarbon wastes
within the leaf parenchyma tissues where these com-
pounds are produced in a larger scale.
In general, it is considered that some bacterial
endophytes do not inhabit living vegetative cells [29,
30, 33]. However, immunogold labeling allowed the
precise location of endophyte E. asburiae within
epidermal cells [32] and intact root cortical cells of
cotton [34]. Using the in situ hybridization method,
Pirttila and co-authors detected species in the genus
Methylobacterium in the meristem cells of Scots pine
buds [5]. In this study we observed bacterial rRNA
hybridization signals inside cells of stem parenchyma
of in vitro-grown potato plants inoculated with
M. radiotolerans IMBG290. However, the cells
appeared to be senescing, and this may explain the
bacterial presence in these cells.
Î. Â. Ïî äîë³÷, Ë. Ï. Îâ÷à ðåí êî, Í. Î. Êî çè ðî âñüêà,
À. Ì. ϳðòò³ëà
Âèç íà ÷åí íÿ ëî êàë³çàö³¿ Methylobacterium radiotolerans
IMBG290 ó òêà íè íàõ êàð òîïë³ ìå òî äîì ã³áðè äè çàö³¿ in situ
Ðå çþ ìå
Íî âèé øòàì ðî æå âî-ï³ãìåí òî âà íî¿ ôà êóëü òà òèâ íî¿ ìå òè -
ëîò ðîô íî¿ áàê òå𳿠M. radiotolerans IMBG290, âèä³ëå íèé
ðàí³øå ç êóëü òó ðè òêà íèí êàð òîïë³ ï³ñëÿ îá ðîá êè áàê òåð³ºþ
Pseudomonas fluorescens IMBG163, âèç íà ÷å íî â òêà íè íàõ êàð -
òîïë³ ìå òî äîì ã³áðè äè çàö³¿ in situ (ISH/FISH). Íà ÿâí³ñòü ðè áî -
ñîì íî¿ 16S ÐÍÊ ñïîñ òåð³ãàëè â ëèñò³ òà ñòåá ëàõ ïðîá³ðêî âèõ
ðîñ ëèí, ïðî òå ¿¿ íå çíàé äå íî â êîð³íí³ êàð òîïë³. Ó ðîñ ëèí, îá -
ðîá ëå íèõ M. radiotolerans IMBG290, ã³áðè äè çàö³éíèé ñèã íàë âè -
ÿ âèâ ñÿ ñèëüí³øèì, í³æ ó êîí òðîëü íèõ (íå îá ðîá ëå íèõ) ðîñ ëèí.
Êëþ ÷îâ³ ñëî âà: ã³áðè äè çàö³ÿ in situ, Methylobacterium
radiotolerans IMBG290, æèâö³ êàð òîïë³.
Î. Â. Ïî äî ëè÷, Ë. Ï. Îâ÷à ðåí êî, Í. À. Êî çû ðîâ ñêàÿ,
À. Ì. Ïèðò òè ëà
Îïðå äå ëå íèå ëî êà ëè çà öèè Methylobacterium radiotolerans
IMBG290 â ðàñ òå íè ÿõ êàð òî ôå ëÿ ìå òî äîì ãèá ðè äè çà öèè in situ
Ðå çþ ìå
Íî âûé øòàìì ðî çî âî-ïèã ìåí òè ðî âàí íîé ôà êóëü òà òèâ íîé ìå -
òè ëîò ðîô íîé áàê òå ðèè M. radiotolerans IMBG290, âû äå ëåí -
íûé ðà íåå èç êóëü òó ðû òêà íåé êàð òî ôå ëÿ ïî ñëå îá ðà áîò êè
áàê òå ðè åé Pseudomonas fluorescens IMBG163,îïðå äå ëåí â òêà -
íÿõ êàð òî ôå ëÿ ìå òî äîì ãèá ðè äè çà öèè in situ (ISH/FISH). Ïðè -
ñó òñòâèå 16S ðÐÍÊ M. radiotolerans íà áëþ äà ëè â ëèñ òüÿõ è
ñòåá ëÿõ, íî íå â êîð íÿõ êàð òî ôå ëÿ. Ó ðàñ òå íèé, îá ðà áî òàí -
íûõ M. radiotolerans IMBG290, ãèá ðè çà öè îí íûé ñèã íàë áûë
ñèëü íåå, ÷åì ó êîí òðîëü íûõ (íå îá ðà áî òàí íûõ) ðàñ òå íèé.
Êëþ ÷å âûå ñëî âà: ãèá ðè äè çà öèÿ in situ, Methylobacterium
radiotolerans IMBG290, ÷å ðåí êè êàð òî ôå ëÿ.
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ÓÄÊ 576.5:582.951:577.2
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DETECTION OF M. RADIOTOLERANS IMBG290 IN POTATO PLANTS BY IN SITU HYBRIDIZATION
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| id | nasplib_isofts_kiev_ua-123456789-5648 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-12-07T16:34:33Z |
| publishDate | 2009 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Podolich, O.V. Kozyrovska, N.O. Pirttila, A.M. 2010-02-01T14:17:55Z 2010-02-01T14:17:55Z 2009 Detection of Methylobacterium radiotolerans IMBG290 in potato plants by in situ hybridization / O.V. Podolich, N.O. Kozyrovska, A.M. Pirttilа // Біополімери і клітина. — 2009. — Т. 25, № 2. — С. 115-119. — Бібліогр.: 34 назв. — англ. 0233-7657 https://nasplib.isofts.kiev.ua/handle/123456789/5648 576.5:582.951:577.2 A new bacterial strain of pink-pigmented facultative methylotroph (M. radiotolerans IMBG290) which was previously isolated from in vitro grown potato plantlets after their inoculation with Pseudomonas fluorescens IMBG163 was detected in tissues by in situ hybridization method (ISH/FISH). The presence of Methylobacterium rRNA was observed in leaves and stems of potato plantlets, whereas no signal was detected in potato roots. The signal was less abundant in the untreated plants than in the plantlets infected with M. radiotolerans IMBG290. en Інститут молекулярної біології і генетики НАН України Молекулярна і клітинна біотехнології Detection of Methylobacterium radiotolerans IMBG290 in potato plants by in situ hybridization Визначення локалізації Methylobacterium radiotolerans IMBG290 у тканинах картоплі методом гібридизації in situ Определение локализации Methylobacterium radiotolerans IMBG290 в растениях картофеля методом гибридизации in situ Article published earlier |
| spellingShingle | Detection of Methylobacterium radiotolerans IMBG290 in potato plants by in situ hybridization Podolich, O.V. Kozyrovska, N.O. Pirttila, A.M. Молекулярна і клітинна біотехнології |
| title | Detection of Methylobacterium radiotolerans IMBG290 in potato plants by in situ hybridization |
| title_alt | Визначення локалізації Methylobacterium radiotolerans IMBG290 у тканинах картоплі методом гібридизації in situ Определение локализации Methylobacterium radiotolerans IMBG290 в растениях картофеля методом гибридизации in situ |
| title_full | Detection of Methylobacterium radiotolerans IMBG290 in potato plants by in situ hybridization |
| title_fullStr | Detection of Methylobacterium radiotolerans IMBG290 in potato plants by in situ hybridization |
| title_full_unstemmed | Detection of Methylobacterium radiotolerans IMBG290 in potato plants by in situ hybridization |
| title_short | Detection of Methylobacterium radiotolerans IMBG290 in potato plants by in situ hybridization |
| title_sort | detection of methylobacterium radiotolerans imbg290 in potato plants by in situ hybridization |
| topic | Молекулярна і клітинна біотехнології |
| topic_facet | Молекулярна і клітинна біотехнології |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/5648 |
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