Volume 21
Issue 2
Agronomy
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
DOI:10.30825/5.ejpau.18.2018.21.2, EJPAU 21(2), #02.
Available Online: http://www.ejpau.media.pl/volume21/issue2/art-02.html
ROLE OF ALKYLRESORCINOLS IN COLONIZATION OF WHEAT BY PSEUDOMONAS SP. STRAIN 150
DOI:10.30825/5.EJPAU.18.2018.21.2
Elżbieta G. Magnucka1, Stanisław J. Pietr2
1 Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland
2 Laboratory of Agricultural Microbiology, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
Cereals contain 5-n-alkylresorcinols that are potent antimicrobials.
Despite of this fact various bacteria effectively colonize cereals. The aim of
this study was to determine whether the tolerance of strain PO150, isolated from
wheat rhizosphere, to orcinol could play a role in wheat colonization. The formation
of the petite-sized and non-fluorescent colonies of this strain was observed
already at a dose of 1.0 mg cm-3 orcinol. Its proliferation, in turn, was completely
inhibited by 1.5 mg cm-3 of this phenolic compound. A Tn5 mutant with reduced
tolerance to orcinol more weakly colonized both wheat kernels and the 24-h-old
germinating seeds than the parent strain. Moreover, both the mutant and wild-type
strain adhered more effectively to grains of the cultivar which contained less
5-n-alkylresorcinols.
Thus, our results suggest that resistance to wheat seed alkylresorcinols can
be important for the early stages of its colonization by this rhizobacteria of
the genus Pseudomonas.
Key words: colonization, 5-n-alkylresorcinols, Pseudomonas sp., wheat.
INTRODUCTION
Colonization of the plant seed and, then, its root system by soil-borne microbes are the important steps in nearly all interactions between plants and microorganisms. Plant can actively select microflora that colonize its tissues or organs mainly through the composition of exudates, among which the products of its defense responses are also present [1, 24]. Therefore, not only the ability of microorganisms to acquire plant exudates but also their capability to cope with antimicrobial compounds are essential for effective colonization of the root system [11]. This process is very important because the establishment of large numbers of beneficial rhizobacteria in the immediate vicinity of roots is critical for the success of biological control of roots- and soil-borne diseases [4].
One of the most abundant microorganisms in the rhizosphere of many plants, including wheat, are bacteria of the genus Pseudomonas [2]. These bacteria have excellent nutritional versatility and consequently present a high growth rate in soil. These features enhance their competitiveness in this environment and cause that they are the most effective root-colonizing group of bacteria [2]. Furthermore, the ability of these bacteria to colonize root system, despite the presence of various toxic substances, suggests that they have evolved to cope with the selective pressure exerted by the defense compounds produced especially by young plants. It is common knowledge that seedlings, which are more susceptible to soil-born pathogens than mature plants, abundantly produce antimicrobial compounds to protect themselves against potential damage. In plants, a well-known example of toxicants are phenolic compounds [5]. These compounds represent a large group of molecules widely distributed in the plant kingdom, where they have a variety of functions in growth, development and defense [17]. Among phenols, an important but relatively poorly known group are resorcinolic lipids. These substances, also called 5-n-alkylresorcinols (ARs), are the homologues of 1,3-dihydroxy-5-methylbenzene. They occur mainly in cereals belonging to the Poaceae family [20]. Furthermore, the cereal 5-n-alkylresorcinols are mixtures mainly of saturated derivatives with 13-29 carbon chains [18, 31]. However, both quantitative and qualitative patterns of this class of phenolic lipids in cereal plants can be modulated by various biotic and abiotic factors [7, 27, 38]. Moreover, ARs are very important components of the outer parts of cereal kernels. They are chiefly located in an intermediate layer of the caryopsis, including the hyaline layer, testa and inner pericarp [22]. Resorcinolic lipids were also isolated from cereal seedlings, especially from rye and rice coleoptiles, shoots and leaves [7, 34]. In addition, several alkylresorcinol derivatives were detected in the rice root exudates [3]. The sorghum exudates, for example, contain the resorcinol analogue sorgoleone [33]. Furthermore Dayan and coworkers [6] showed that the sorghum root hairs possess the entire metabolic machinery necessary for the biosynthesis of these compounds.
Alkylresorcinols are potent antimicrobials and therefore considered an effective defensive chemical barrier against a wide variety of both bacterial and fungal pathogens [21]. For this reason they are often called the natural biofungicides [37]. It was also found that soil bacteria from the genus of Azotobacter, Pseudomonas and Streptomyces produce alkylresorcinols [19, 36]. They are present, for example, in a cyst membrane of Azotobacter vinelandii [12]. Moreover, resorcinolic lipids operate as microbial anabiosis autoinducers in bacteria, also among pseudomonad species [9]. Interestingly, numerous researches showed that extracellular alkylresorcinols can directly induce the transition of bacteria to their stationary stage and the formation of cystlike anabiotic pseudomonad cells [9]. Their action on bacterial cells can be also indirect. It was proved that the presence of these compounds in the medium stimulates the endogenous synthesis of alkylresorcinols by bacteria [29]. This effect suggests that these lipids act as small hormone-like molecules and play a significant role in chemical communication (quorum-sensing) between bacterial cells. Noteworthy is also fact that the rice seedling exudates containing alkylresorcinols induced stress-responses, especially protein damage and oxidative stress in Escherichia coli biosensor [28]. Therefore, this result markedly suggests that resorcinolic lipids may exert a selective pressure on the root colonizing rhizobacteria. However, so far little has been known about their role in cereal rhizosphere colonization by pseudomonads. The present study was conducted to test whether orcinol, the simplest homologue of 5-n-alkylresorcinols, influences growth of the rhizosphere originating strain Pseudomonas sp. PO150. Additionally, we want to test whether the degree of orcinol tolerance can determine the ability of this rhizobacteria to wheat colonize.
Bacterial strains
A fluorescent bacteria of the genus Pseudomonas strain
PO150 was isolated on Gould’s agar medium [14] from the rhizosphere of
winter wheat (Triticum
aestivum L. cultivar (cv.) Kobra) according method described by Oksinska
and coresearchers [30]. This strain was selected from a collection of over 50
pseudomonads because it did not utilize methanol as a sole source of carbon and
energy. Moreover, it turned out to be naturally resistant to chloramphenicol
(Cm; 50 mg L-1), trimethoprim (Tp; 20 mg L-1) and, simultaneously, was sensitive
to kanamycin (Km; 50 mg L-1) (data not shown).
Escherichia coli strain DH5 alpha [15] which harbors a plasmid pRL765 [10] and the helper strain E. coli DH5 alpha (pRK2013) [35] were used to generate random Tn5 transposon insertions in the genome of the chosen isolate. These strains were received from Plant Pathology and Biocontrol Unit, at the Swedish University of Agricultural Sciences (Uppsala, Sweden).
Identification of strain tested
The strain PO150 was identified by determination
of 16S rRNA gene sequence. Total DNA was extracted using GenElute Bacterial Genomic
DNA kit (Sigma-Aldrich, USA) according to the manufacturer’s recommended protocol. PCR amplification
of 16S rRNA gene fragment was performed by using bacterial primer 27f (5′-AGA
GTT TGA TCM TGG CTC AG-3′) as the forward primer and primer 1541r (5′-AAG
GAG GTG ATC CAG CC-3′) [23] as the reverse primer. This amplification was
conducted in a final volume of 0.02 cm3, using approximately 400 ng of DNA, 500
nM of each primer (DNA Gdansk, Gdansk, Poland) and 0.004 cm3 of 5 x hot FIREPol® Blend
Master Mix (Soils BioDyne, Tartu, Estonia). Thirty-five PCR cycles were carried
out in a Mastercycler gradient (Eppendorf AG, German) according to the following
procedure: initial denaturation of DNA and activation of polymerase for 15 min
at 95ºC, denaturation for 1 min at 93ºC, annealing for 45 s at 55ºC,
elongation for 1.5 min at 72ºC and final extension for 10 min at 72ºC.
The amplified DNA was verified by electrophoresis of PCR mixture in 1.5% (w/v)
agarose (Sigma-Aldrich, USA) containing ethidium bromide (0.5 mg cm-3) in 1 × TBE
buffer (0.1 M Tris, 0.09 M boric acid, 1 mM EDTA). The DirectLoad Step Ladder
50 pb (Sigma-Aldrich, USA) was used as a molecular weight marker.
The purified 16S rRNA gene fragment was sequenced using two sets of universal bacterial primers (Genomed S.A., Warsaw, Poland). The first primer set 27f/1541r flanked this gene, in turn, primers 704f (5'-TGT GTA GCG GTG AAA TGC GTA GA-3') and 765r (5'-CTG TTT GCT CCC CAC GCT TTC-3') annealed to its internal fragments [30].
This gene sequence was submitted to NCBI website (www.ncbi.nlm.nih.gov) to search for similar sequences among those available online by using the BLAST sequence analysis service (the megablast program). The sequence obtained in the study and those retrieved from GenBank database were aligned by Clustal W method, and then a phylogenetic tree was constructed with the neighbor-joining method using DNAStar software package (Lasergene Madison, WI, USA). The 16S rRNA sequence of this strain was deposited in the above mentioned database with the accession number KM058081.
Preliminary test – a determination of Minimal Inhibitory Concentration
for orcinol
The ability of tested strain to multiply in the presence of alkylresorcinol
was tested on 1/10 Tryptic Soy Agar (Difco Laboratories, Inc., USA) supplemented
with different concentrations of orcinol (0.0–2.0 mg cm-3).
Solutions of orcinol in methanol were added to the medium to final concentration
of solvent below 1% (v/v). Inoculation of aforementioned medium was done with
the 48-h-old bacterial cells grown also on 1/10 TSA. Twenty five microliters
of bacterial suspension (6.5 × 108 cfu cm-3) in 0.1 M MgSO4 x 7H2O was
spotted on the solid medium containing various doses of orcinol. After 48 h of
incubation at 28ºC the bacterial growth was compared to its uninhibited
growth on control plate (1/10 TSA with methanol only). The lowest concentration
of orcinol that inhibited the bacterial growth taken as the MIC value. This experiment
was performed in three replicates.
Construction of Tn5 transposon mutant with reduced tolerance
to orcinol
The strain tested is naturally sensitive to kanamycin at a dose of 50 mg L-1.
Transfer of the plasmid pRL765 with a kanamycin resistance
transposon Tn5from E. coli DH5 alpha to strain PO150 conferred upon
it this antibiotic resistance. The recipient, helper and donor strains were grown
till mid log phase at 28ºC in liquid Luria medium (LM) containing (per liter)
bacto tryptone 10 g, yeast extract 6 g, K2HPO4 1.5 g, NaCl 0.5 g, and MgSO4 × 7H2O
0.4 g. This medium was supplemented with appropriate antibiotics: kanamycin (50
and 25 mg L-1 for donor and helper strain respectively) and chloramphenicol (50
mg L-1 for recipient strain). Half a milliliter of both donor and helper strains
and 1 cm3 of recipient strain were centrifuged (3,000 g, 7 min), washed twice
in LM broth, and finally resuspended in 0.1 cm3 of this medium. Then the suspensions
of donor, helper and recipient cells were mixed in 1:1:1 ratio. Fifty microliters
of this mixture was spot – inoculated on solid LM and incubated overnight
at 28ºC to allow conjugal mating. After mating, the cells were transferred
to a eppendorf tube with 0.5 cm3 of LM broth. Serial dilutions of this suspension
were plated on LM agar supplemented with both antibiotics. The kanamycin and
chloramphenicol resistant transconjugants were tested for their ability to grow
on 1/10 TSA and Gould’s medium [14], and for their inability to grow on
1/10 TSA containing orcinol at concentrations ranging from 0.5 to 1.4 mg cm-3.
These orcinol levels were determined on the basis of the preliminary test results.
Plates were incubated at 28ºC for 4 days and then the right transconjugants
were selected.
Utilization of 5-n-alkylresorcinols as a sole source of carbon and
energy
Test was carried out on solid Stanier's mineral medium [32] supplemented with
one of the following compounds: orcinol, 5-n-pentadecylresorcinol and
mixture of 5-n-alkylresorcinol homologues isolated from wheat grains.
These compounds were dissolved in methanol, filtered (pore size 220 nm; Milipore,
Ireland) and, then were introduced into medium at the concentrations of 1 mg
cm-3. Forty-eight-hour-old bacterial cells of the parent strain and its mutant
were used to prepare their suspensions according the above mentioned method.
The bacterial density of each suspension was determined using a calibration curve
assessed by turbidity (optical density at 600 nm; OD600), and adjusted to 6.5 × 108
colony forming units per cm3 (cfu cm-3). Then, the bacterial suspensions were
point – inoculated onto surface of medium. The growth of parent strain and its
mutant was compared to their multiplication on respective controls, i.e. 1/10
TSA with methanol, Stanier’s mineral medium with both glucose and methanol
(positive controls) and mineral medium with this solvent (negative controls)
after 72 h of incubation at 28ºC. Media used for estimation of a transconjugant
growth were supplemented with kanamycin (50 mg L-1). The whole experiment was
performed in three replicates.
Growth of the parent strain and its mutant in the presence of orcinol
The ability of strain Pseudomonas sp. PO150 and its mutant to multiply
in the presence of orcinol was also tested on King’s B medium [16] (pH
7.0) containing different doses of this phenolic compound (0.0–2.0 mg cm-3).
Their 24-h-old cells were scraped from 1/10 TSA slants and suspended in 0.1 M
MgSO4 x 7H2O. One hundred microliters of this suspension (1.25 × 108 cfu
cm-3) was added to 10 cm3 of liquid King’s B medium supplemented with orcinol.
After 48 h of incubation at 28ºC bacterial cultures were spotted on solid
King’s B medium to estimate colony morphology. Three parallel dilution
series were prepared for each sample and plated on this medium. The number of
viable cells was determined by enumeration of cfu grown on solid medium and calculated
per 1 cm3 of the culture. Mutant was cultured on medium with respective dose
of kanamycin.
Colonization of wheat
Seeds of both winter wheat cv. Mikula and spring
winter cv. Hewilla were used to examine the colonization efficiency of the strain
tested and its mutant with decreased tolerance to orcinol. Wheat grains were
surface disinfected by soaking them in 10% (v/v) Ace bleach for 15 min (Procter & Gamble,
Poland). The parent strain PO150 and its mutant PO150/19 were cultured on 1/10
TSA at 28ºC for 48 h. Their homogenous suspensions (6.5 × 108 cfu
cm-3) were prepared in 0.5% (w/v) carboxymethyl cellulose (CMC; Sigma-Aldrich,
USA) in 0.1 M MgSO4 × 7H2O. A part of the disinfected grains of wheat
were inoculated with bacterial suspensions for 1 h with agitation (90 rev min-1),
while the control kernels were soaked in sterile CMC solution only. Then the
seeds were dried overnight in a laminar flow hood. Sonication (16 kHz, 10 min)
in a 0.1 M solution of MgSO4 × 7H2O was used to detach bacterial cells
from seeds. Diluted sonicates were plated on Gould’s solid medium and incubated
for 48–72 h at 28ºC. The ability to adhere to wheat grains was expressed
as the number of cfu per seed.
Pot experiment with autoclaved sand was applied to estimate the ability of both strains to colonize wheat seedlings. The bacterized and control kernels were transferred aseptically to pots containing 75 g of moistened sand (15 cm3 of sterile water per pot). Then, these pots were incubated for up to 72 h at 28ºC in darkness. The number of cfu per 24-, 48- and 72-h-old wheat seedling was determined according method described above.
Isolation and determination of a alkylresorcinol content in wheat kernels
Alkylresorcinols were extracted from whole grains (10 g) according to
the method described previously [38]. The microcolorimetric method described
by Gajda and coworkers [13] was used for quantitative determination of these
compounds. Both analyses were carried out in triplicate.
Statistical analyses
All data were analyzed using Statistica version 5 (StatSoft, Inc., USA).
RESULTS
Identification of isolate
The sequence of the 16S rRNA gene of Pseudomonas sp. strain PO150 (1425
bp) was the most similar to that of P. veronii strain CIP 104664T
(accession number AF064460) [8]. These two strains had 99.7% of the bases in
common. Their sequences differed at four positions: 1010, 1017, 1135 and 1285
(E. coli J01859 numbering system), where C of CIP 104664T is replaced
by T for PO150, G by A, A by T, and G by C, respectively. Albeit, these two strains
formed cluster with a low bootstrap value (49%). Therefore, a precise identification
of this isolate at the species level was impossible.
Ability of parent strain to grow in the presence of different doses of
orcinol – a preliminary test
Under neutral condition (pH 7.0), strain PO150 tolerated high doses of orcinol.
Its minimal inhibitory concentration (MIC) was equal to 1.5 mg cm-3 of the added
compound.
This test was restricted to only orcinol because this compound is less hydrophobic than the constituents with longer alkyl chain and, at high doses tested, it did not precipitate in media.
Tn5 mutagenesis of Pseudomonas strain PO150
A Tn5 transposon library of Pseudomonas sp. PO150 including more than
730 transconjugants was screened for mutants exhibiting the reduced tolerance
to orcinol. Among them, only one mutant, named PO150/19, had markedly impaired
resistance to this compound in comparison to the wild-type strain. This Tn5 mutant
was unable to grow in the presence of orcinol at concentration 1.25 mg cm-3.
It could markedly multiply on 1/10 TSA (pH 7.0) containing no more than 1.0 mg
cm-3 of this compound.
Estimation of growth in the presence of alkylresorcinolic compounds
as a sole source of carbon and energy
Strain PO150 and its Tn5 transposon
mutant were not able to grow on Stanier’s
mineral medium containing 5-alkylresorcinols tested as well as on medium supplemented
with the mixture of alkylresorcinol homologues isolated from wheat grains. Thereby,
we concluded that this isolate and its insertion mutant did not utilize these
compounds as the sole carbon source. It is worth noting that this test could
be carried out because these strains did not utilize methanol used to dissolve
the above mentioned compounds.
Growth in the presence of orcinol
Supplementation of
King’s B medium with orcinol generally influenced
both pigmentation and number of cfu of both the parent strain and the tested
mutant (Tab. 1). At concentrations in the range from 0.25 to 0.75 mg cm-3, orcinol
had not effect on the cfu number and morphology of the wild-type strain colonies.
However, the addition of 1.0 mg cm-3 of this compound caused a formation of smaller
colonies which did not produce bright yellow fluorescent pigments and hence did
not exhibit fluorescence. At two higher doses (1.0 and 1.25 mg cm-3), orcinol
markedly reduced also the cell number by ca. 0.58 log10 cfu with reference
to control without orcinol. For the parent strain, the negative correlation between
the dose of orcinol in liquid medium and the number of its cfu grown on solid
medium was found (r = -0.95, p < 0.05).
Table 1. Effect of various doses of orcinol on multiplication of Pseudomonas sp. strain PO150 and its Tn5 mutant with decreased resistance to this compound |
mg cm-3 |
mg cm-3 |
mg cm-3 |
mg cm-3 |
mg cm-3 |
mg cm-3 |
mg cm-3 |
mg cm-3 |
mg cm-3 |
|
± 0.13ab * |
± 0.18abc * |
± 0.20abc * |
± 0.16acd * |
± 0.32def |
± 0.08e |
||||
± 0.10ab * |
± 0.13b * |
± 0.24b * |
± 0.11cf |
± 0.04g |
|||||
± Standard
deviation (SD) of means from three separate experiments; n.d. – not detectable (< 2 log10 cfu cm-3) * – fluorescence on King’s B medium Values followed by different letters are significantly different at P = 0.05, according to Duncan's multiple range test. |
The tested mutant PO150/19 was able to grow in the presence of orcinol at two concentrations tested (0.25 and 0.5 mg cm-3). The higher doses of orcinol (0.75 and 1.0 mg cm-3) resulted in the change in pigmentation of its colonies. Moreover, orcinol at concentration of 1.0 mg cm-3 significantly decreased the number of mutant cells; the decline by 1.44 log10 cfu in comparison to control sample without orcinol was observed. In turn, the higher concentrations tested completly inhibited the mutant growth.
Colonization of wheat
The ability of parent strain and
its Tn5 transposon mutant to adhere to seeds and colonize 24-, 48- and 72-h-old
seedlings of two different wheat cultivars was estimated (Tab. 2). It turned
out that the cultivar of wheat tested markedly influenced the colonization pattern
of both PO150 and PO150/19. The number of cells, which adhered to seeds of spring
wheat cv. Hewilla, was approximately 5.59 log10 cfu per seed for the wild type
strain. Its colonization efficacy gradually increased over the 48-h-period; the
cell number of PO150 strain increased by 2.78 log10 units. However, the amount
of its cells recovered at 72 h after sowing was not statistically different from
that observed after 48 h. In the case
of winter wheat cv. Mikula, no significant rise in its cell number was noted
in the interval between 24 and 48 h after adhesion. The next 24 h, however, caused
the marked enhancement of its cell growth. Finally, the number of cfu increased
from 5.75 to 7.80 log10 units. Based on these results, we concluded that strain
PO150 more readily adhered to the seeds and colonized the seedlings of spring
wheat cv. Hewilla than the other wheat tested. The cfu number of parent strain
already after 24 h reached the cell level which, in the case of Mikula cultivar,
was observed after 72 h of its multiplication on this wheat seedlings.
Table 2. Colonization of two wheat cultivars by Pseudomonas sp. strain PO150 and its Tn5 mutant (PO150/19) with decreased tolerance to orcinol |
* Mean values expressing log10 cfu per seed
or seedling ± SD obtained from three independent experiments Values followed by different letters are significantly different at P = 0.05, according to Duncan's multiple range test; |
The Tn5 mutant differed markedly in its ability to colonize wheat cultivars tested. In the case of spring wheat, its colonization ability was widely impaired in comparison to the results demonstrated by the wild-type strain. Albeit, unlike the parent strain, the cell number of this mutant gradually increased over the whole 72-h-period. Its cfu number increased from 4.08 to 7.75 log10 units. The same pattern of colonization by this transposon mutant was observed for spring cultivar. However, the amounts of mutant cells colonizing the 48 and 72-h-old wheat seedlings were not significantly different to cell numbers reached by strain PO150. Finally, the number of mutant cfu increased from 5.29 to 7.77 log10 units. Interestingly, only the numbers of its cells multiplied on Mikula cultivar were positively correlated with time (r = 0.95; p < 0.05).
Alkylresorcinol content in wheat grains
Grains of two wheat cultivars used in colonization experiment were analyzed for
their alkylresorcinol concentration (Tab. 3). It was found that both cultivars
tested contained these phenolic lipids. Moreover, their level was markedly higher
in kernels of spring wheat cv. Hewilla (531.97 mg kg-1) than in seeds of winter
wheat cv. Mikula (462.35 mg kg-1).
Table 3. Kernel weight and total concentration of 5-n-alkylresorcinols in wheat grains |
[mg kg-1]# |
[g]* |
|
(spring wheat) |
||
(winter wheat) |
||
# Mean values expressing concentration of alkylresorcinols ± SD
obtained from three independent experiments; * Mean values expressing dry biomass of grain ± SD (n=25); Values followed by different letters are significantly different at P = 0.05, according to Duncan's multiple range test; |
DISCUSSION AND CONCLUSIONS
The plant exert a selective pressure on soil microflora by producing a wide variety of organic compounds. Their availability and utilization is one of the crucial factors for a successful establishment of bacteria in the rhizosphere. However, antimicrobial compounds which protect plant against infections caused by various pathogens are also present among these exudates. This fact suggests that rhizobacteria have to overcome a chemical stress to have access to seed or root exudates as a nutrient source and then colonize plant tissues or organs.
The tested strain belonging to the genus Pseudomonas as well as its Tn5 mutant did not utilize 5-n-alkylresorcinols as a sole source of carbon and energy. On the other hand, the simplest representative of this phenolic lipids influenced their growth. The cell proliferation of parent strain was completely inhibited in the presence of 1.5 mg cm-3 orcinol in King’s B medium. In turn, the morphological changes in colonies of this bacteria were already observed at the dose of 1.0 mg cm-3. In the case of its Tn5 transposon mutant with reduced resistance to orcinol, this effect was initiated at lower concentration (0.75 mg cm-3). Moreover, the mutant strain was unable to grow in the presence of 1.25 mg cm-3 orcinol. So far, a similar effect of 4-n-hexylresorcinol on Pseudomonas aurantiaca has been described by Mulyukin et al. [29], who proved that these phenotypic changes in colony morphology of this bacteria were connected with the formation of cystlike refractile cells. In the case of our strain however, this phenotypic dissociation influenced by orcinol was observed at much higher doses. It is likely that rhizospheric pseudomonads, especially those isolated from cereal rhizosphere, have the high level of tolerance to this group of compounds. After all, cereals, especially the epicuticular waxes of their grains are full of resorcinolic lipids [20–22]. Moreover, they are distributed in stems, leaves and also in root exudates of cereal [3, 7, 34]. In the case of wheat, there are no literature reports of alkylresorcinol presence in exudates of this plant. However, they were extracted from root tissue of wheat [25]. Additionally, it is highly probable that a content of these compounds can be significantly modified during germination and seedling growth as it was described in the case of rye [26]. These changes, in turn, may result in a quantitative modification of bacteria inhabiting cereals at early stages of their development. Hence the estimation of capabilities of the strain chosen and its mutant with reduced tolerance to orcinol to inhabit the two cultivars of wheat during their development. The analysis of wheat colonization patterns of parent strain and its mutant showed that they differ mainly in abilities to both adhere to kernels and colonize the 24-h-old germinating seeds. The cfu numbers of mutant obtained in these two early phases of colonization were significantly lower than the cell numbers of wild-type strain PO150. This fact suggested that the kernel ARs probably limited the early stages of wheat colonization by its transposon mutant exhibiting lowered resistance to orcinol. It is likely that an influence of these compounds, which are abundantly present in wheat seeds, on mutant cells is responsible for the decline in their abilities to colonize germinating grain. Interactions of alkylresorcinols with proteins or nucleic acids and also their membrane-disturbing properties probably affect both bacterial transition into a dormant state and an inhibition of microbial growth [20, 21]. The fact that the mutant strain PO150/19 more effectively colonized kernels of Mikula cultivar containing less ARs clearly shows that a tolerance to 5-n-alkylresorcinols of cereal grains decides about efficiency of the early colonization of wheat seedlings by this strain of the genus Pseudomonas. Lastly, it is worth to emphasize the fact that it is the first report that presents the relations between rhizobacteria and resorcinolic lipids of wheat.
Acknowledgments
This research was supported by grant from The National Science Centre (N N310 729240).
REFERENCES
- Bais H.P., Weir T.L., Perry L.G., Gilroy S., Vivanco J.M., 2006. The role of root exudates in rhizosphere interactions with plants and other organisms. Annu. Rev. Plant Biol., 57, 233–266.
- Botelho G.R., Mendonca-Hagler L.C., 2006. Fluorescent Pseudomonads associated with the rhizosphere of crops-an overview. Braz. J. Microbiol., 37, 401–416.
- Bouillant M.L., Jacoud C., Zanella I., Faure-Bonvin J., Bailly R., 1994. Identification of 5-(12-heptadecenyl)-resorcinol in rice root exudate. Phytochemistry, 35, 769–771.
- Chin-A-Woeng T.F.C., Bloemberg G.V., Mulders I.H.M., Dekkers L.C., Lugtenberg B.J.J., 2000. Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot. Mol. Plant-Microbe Interact., 13, 1340–1345.
- Daniel O., Meier M.S., Schlatter J., Frischknecht P., 1999. Selected phenolic compounds in cultivated plants: ecologic functions, health implication, and modulation by pesticides. Environ. Health Persp., 107, 109–114.
- Dayan F.E., Watson S.B., Nanayakkara N.P.D., 2007. Biosynthesis of lipid resorcinols and benzoquinones in isolated secretory plant root hairs. J. Exp. Bot., 58, 3263–3272.
- Deszcz L., Kozubek A., 2000. Higher cardol homologs (5-alkylresorcinols) in rye seedlings. Biochim. Biophys. Acta., 1483, 241–250.
- Elomari M., Coroler M., Hoste B., Gillis M., Izard D., Leclerc H., 1996. DNA relatedness among Pseudomonas strains isolated from natural mineral waters and proposal of Pseudomonas veronii sp. nov. Int. J. Syst. Evol. Microbiol., 46, 1138–1144.
- El’Registan G.I., Tsyshnatii G.V., Duzha M.V., Pronin S.V., Mityushina L.L., Savel’eva N.D., et al. 1980. Regulation of Pseudomonas carboxydoflava growth and development by specific endogenous factors. Microbiology, 49, 561–565.
- Figurski D.H., Helinski D.R., 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA, 76, 1648–1652.
- Flores H.E., Vivanco J.M., Loyola-Vargas V.M., 1999. 'Radicle' biochemistry: the biology of root-specific metabolism. Trends Plant Sci., 4, 220–226.
- Funa N., Ozawa H., Hirata A., Horinouchi S., 2006. Phenolic lipid synthesis by type III polyketide synthases is essential for cyst formation in Azotobacter vinelandii. Proc. Natl. Acad. Sci. USA, 103, 6356–6361.
- Gajda A., Kulawinek M., Kozubek A., 2008. An improved colorimetric method for the determination of alkylresorcinols in cereals and whole-grain cereal products. J. Food Comp. Anal., 21, 428–434.
- Gould W.D., Hagedorn C., Bardinelli T.R., Zablotowicz R.M., 1985. New selective media for enumeration and recovery of fluorescent pseudomonads from various habitats. Appl. Environ. Microbiol., 49, 28–32.
- Hanahan D., 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol., 166, 557–580.
- King E.O., Ward M.K., Raney D.E., 1954. Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med., 44, 301–307.
- Kobayashi A., Kim M.J., Kawazu K., 1996. Uptake and exudation of phenolic compounds by wheat and antimicrobial components of the root exudate. Z. Naturforsch., 51c, 527–533.
- Kozubek A., 1985. Isolation of 5-n-alkyl-, 5-n-alkenyl- and 5-n-alkadienyl-resorcinol homologs from rye grains. Acta Aliment. Polon., 9, 185–198.
- Kozubek A., Pietr S.J., Czerwonka A., 1996. Alkylresorcinols are abundant lipid components in different strains of Azotobacter chrococcum and Pseudomonas spp. J. Bacteriol., 178, 4027–4030.
- Kozubek A., Tyman J.H.P., 1999. Resorcinolic lipids, the natural non-isoprenoid phenolic amphiphiles and their biological activity. Chem. Rev., 99, 1–26.
- Kozubek A., Tyman J.H.P., 2005. Bioactive phenolic lipids [in:] Atta-ur-Rahman, editor. Studies in Natural ProductsChemistry. Amsterdam: Elsevier Science Publishers, 30, 111.
- Landberg R., Kamal-Eldin A., Salmenkallio-Marttila M., Rouau X., Aman P., 2008. Localization of alkylresorcinols in wheat, rye and barley kernels. J. Cereal Sci., 48, 401–406.
- Lane D.J., 1991. 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M, editors. Nucleic acid techniques in bacterial systematics. New York: John Wiley and Sons, 115.
- Lugtenberg B.J.J, Chin-A-Woeng T.F.C., Bloemberg G.V., 2002. Microbe-plant interactions: principles and mechanisms. A. van Leeuw. J. Microb. 81, 373–383.
- Magnucka E.G., Oksinska M.P., Lewicka T., 2014. Resorcinolic lipids in winter wheat grains and roots. Electron. J. Pol. Agric. Univ., 17.
- Magnucka E.G., Pietr S.J., Zarnowski R., 2015. Dynamics of alkylresorcinols during rye caryopsis germination and early seedling growth. Z. Naturforsch., 70c, 71–73.
- Magnucka E.G., Suzuki Y., Pietr S.J., Kozubek A., Zarnowski R., 2009. Cycloate, an inhibitor of fatty acid elongase, modulates metabolism of very-long-side-chain alkylresorcinols in rye seedlings. Pest Manag. Sci., 65, 1065–1070.
- Miche L., Belkin S., Rozen R., Balandreau J., 2003. Rice seedling whole exudates and extracted alkylresorcinols induce stress-response in Escherichia coli biosensors. Environ. Microbiol., 5, 403–411.
- Mulyukin A.L., Vakhrushev M.A., Strazhevskaya N.B., Shmyrina A.S., Zhdanov R.I., Suzina N.E., et al. 2005. Effect of alkylhydroxybenzenes, microbial anabiosis inducers, on the structural organization of Pseudomonas aurantiaca DNA and on the induction of phenotypic dissociation. Microbiology, 74, 128–135.
- Oksinska M.P., Wright S.A.J., Pietr S.J., 2011. Colonization of wheat seedlings (Triticum aestivum L.) by strains of Pseudomonas spp. with respect to their nutrient utilization profiles. Eur. J. Soil Biol., 47, 364–373.
- Ross A.B., Kamal-Eldin A., Jung C., Shepherd M.J., Aman P., 2001. Gas chromatographic analysis of alkylresorcinols in rye (Secale cereale L.) grains. J. Sci. Food Agric., 81, 1405–1411.
- Stanier R.Y., Palleroni N.J., Doudoroff M., 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol., 43, 159–271.
- Suzuki Y., Kono Y., Inoue T., Sakurai A., 1998. A potent antifungal benzoquinone in etiolated sorghum seedlings and its metabolites. Phytochemistry, 47, 997–1001.
- Suzuki Y., Saitoh C., Hyakutake H., Kono Y., Samurai A., 1996. Specific accumulation of antifungal 5-alk(en)ylresorcinol homologs in etiolated rice seedlings. Biosci. Biotech. Bioch., 60, 1786–1789.
- Tombolini R., Unge A., Davey M.E., de Bruijn F.J., Jansson J.K., 1997. Flow cytometric and microscopic analysis of GFP-tagged Pseudomonas fluorescent bacteria. FEMS Microbiol. Ecol., 22, 17–28.
- Tsuge N., Mizokami M., Imai S., Shimazu A., Seto H., 1992. Adipostatins A and B, new inhibitors of glycerol-3-phosphate dehydrogenase. J. Antibiot., 45, 886–891.
- Zarnowski R., Kozubek A., 2002. Resorcinolic lipids as natural biofungicides [in:] Dehne HW, Gisi U, Kuck KH, Russel PE, Lyr H, editors. Modern fungicides and antifungal compounds III. Th. Mann Verlag, Bonn: AgroConcept GmbH, 337.
- Zarnowski R., Suzuki Y., 2004. 5-n-alkylresorcinols from grains of winter barley (Hordeum vulgare L.). Z. Naturforsch., 59c, 315–317.
Elżbieta G. Magnucka
Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland
Grunwaldzka 53
50-375 Wrocław
Poland
email: elzbieta.magnucka@upwr.edu.pl
Stanisław J. Pietr
Laboratory of Agricultural Microbiology, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
phone/fax: +48 71 320 6521
Grunwaldzka 53
50-375 Wrocław
Poland
email: stanislaw.pietr@upwr.edu.pl
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