Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2018
Volume 21
Issue 2
Topic:
Agronomy
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Magnucka E. , Pietr S. 2018. ROLE OF ALKYLRESORCINOLS IN COLONIZATION OF WHEAT BY PSEUDOMONAS SP. STRAIN 150
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. Magnucka, Stanisław J. Pietr
Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland

 

ABSTRACT

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.

MATERIALS AND METHODS

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
Strain
Number of viable cell [log10 cfu cm-3] at various doses of orcinol in the medium
0
mg cm-3
0.25
mg cm-3
0.5
mg cm-3
0.75
mg cm-3
1.00
mg cm-3
1.25
mg cm-3
1.5
mg cm-3
1.75
mg cm-3
2.00
mg cm-3
PO 150
9.00
± 0.13ab *
8.94
± 0.18abc *
8.87
± 0.20abc *
8.79
± 0.16acd *
8.53
± 0.32def
8.31
± 0.08e
n.d.
n.d.
n.d.
PO150/19
9.00
± 0.10ab *
9.09
± 0.13b *
9.12
± 0.24b *
8.67
± 0.11cf
7.56
± 0.04g
n.d.
n.d.
n.d.
n.d.
± 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
Strain
Wheat cultivar
Log10 of cfu per seed or seedling*
0 h
24 h
48 h
72 h
PO150
HEWILLA(spring wheat)
5.59 ± 0.16a
7.78 ± 0.02b
8.37 ± 0.16c
8.34 ± 0.09c
PO150/19
4.08 ± 0.09d
6.88 ± 0.06e
7.42 ± 0.00f
7.75 ± 0.16b
PO150
MIKULA(winter wheat)
5.75 ± 0.07g
7.45 ± 0.05f
7.48 ± 0.11f
7.80 ± 0.06b
PO150/19
5.29 ± 0.03h
6.77 ± 0.09e
7.40 ± 0.23f
7.77 ± 0.20b
* 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
Wheat cultivar
Content of ARs
[mg kg-1]#
Weight of kernel
[g]*
HEWILLA
(spring wheat)
531.97 ± 11.28a
0.044 ± 0.00a
MIKULA
(winter wheat)
462.3 ± 9.65b
0.043 ± 0.00a
# 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).

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Accepted for print: 4.06.2018
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
Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland
phone/fax: +48 71 320 6521
Grunwaldzka 53
50-375 Wrocław
Poland
email: stanislaw.pietr@upwr.edu.pl

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