The Influence of Plant Growth-Promoting Bacteria and Humic Substances on the Rooting of Black Poplar (Populus nigra L.) Cuttings

TL;DR

This study shows that combining bacteria and humic substances improves the rooting of black poplar cuttings by affecting plant hormones and gene activity.

Contribution

The study reveals a novel synergistic effect of PGPR and HSs on black poplar rooting through hormonal and genetic mechanisms.

Findings

DA1.2 bacteria increase auxin levels and PnRGF9 gene expression, promoting root formation.

DA1.2 bacteria and HSs reduce ABA content, enhancing rooting by inhibiting ABA synthesis and breakdown.

Combined use of DA1.2 bacteria and HSs was most effective for black poplar cutting rooting.

Abstract

Plants of the genus Populus are among the most economically important woody plants and are an experimental model system used to study woody species. We studied the ability of plant growth-promoting bacteria (PGPR) and humic substances (HSs) to influence the rooting of cuttings, a process that plays an important role in vegetative propagation. We used strains of bacteria from the collection of microorganisms of the Ufa Institute of Biology: Pseudomonas protegens DA1.2 and Enterobacter ludwigii BLK. For the experiment, cuttings of black poplar (Pópulus nigra L.) were used. They were placed in aqueous solution with either the addition of bacteria, HSs, or their combination. After 15 days, the number and length of adventitious roots were measured, and they were sampled for hormone immunoassay analysis and the determination of gene expression. The contents of the auxin indole-3-acetic acid (IAA) and abscisic acid (ABA) were measured, since both substances have an antagonistic effect on rooting. It was shown that the activation of root formation was due, on the one hand, to increased auxin levels under the influence of IAA-producing bacteria, resulting in increased expression of the PnRGF9 gene that encodes the root meristem growth factor. On the other hand, the activation of rooting was due to a decrease in ABA content resulting from its breakdown by bacteria of the DA1.2 strain, which catabolize this hormone. HSs, which can inhibit ABA synthesis, also played a role. As a result, the preparation for the rooting of black poplar cuttings with the greatest effectiveness was observed in the combined use of DA1.2 bacteria and HSs.

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1. Introduction

Plants of the genus Populus, found primarily in the Northern Hemisphere, are among the most economically important woody plants [1]. They are characterized by rapid growth and successful adaptation to poor-quality soils. The rapid growth of poplars is considered and used as a source of bioenergy, leveraging the rapid production of plant (wood) biomass for further use in small-scale energy production (for example, the production of pellets or biofuels) [2,3,4]. Such plantations are short-term (usually no more than 12 years old), and when studying the characteristics of growth and development, the formation of above-ground phytomass is primarily assessed [5,6,7,8,9]. Therefore, it is important to constantly renew such plantations, which requires high-quality planting material. Due to their small genome size and the potential for genetic transformation, poplars have become an experimental model system used to study tree species [10,11]. The root system of poplars (including black poplar) is studied primarily in terms of the characteristics of rooting ability in cuttings [12,13,14,15,16,17]. The formation of adventitious roots on cuttings from lignified shoots of poplars facilitates their vegetative propagation and ensures their widespread use on plantations [18]. Cuttings are widely used for propagation, as they allow for the rapid production of elite clones [19]. A strong root system is essential for providing reliable support, which is especially important when creating windbreaks [20]. Recently, the ability of cuttings to root has become an important trait used in plant breeding, and efforts have been directed toward identifying the mechanisms that control this trait [21]. However, plant breeding is a lengthy and expensive process. At the same time, rooting ability can be enhanced by manipulating cuttings [22]. Plant growth-promoting bacteria (PGPR) have been shown to both stimulate root growth in woody plants [23] and accelerate the rooting process of their cuttings [24,25]. Positive effects of humic substances (HSs) on the root system have also been found [26,27,28], including effects on the plant’s hormonal balance [29]. The combined use of PGPR and HSs is increasingly used to grow various crops due to their multiple effects on plant development [30,31]. Thus, treatment of plants with a complex of bacteria and HSs revealed their additive effect: the growth-stimulating effect of the combination of bacteria and HSs on plant roots turned out to be more effective compared to when used separately [17]. However, the effect of the combination of HSs and bacteria on adventitious root formation in poplar cuttings has yet to be studied and is the subject of this study. Two strains of bacteria that produce the auxin indole-3-acetic acid (IAA) were selected for this purpose, as this hormone is known to influence plant rooting [32]. One of the strains used in our study (Pseudomonas protegens DA1.2) also demonstrated the ability to catabolize the hormone abscisic acid (ABA). Its involvement in this study was of interest because ABA is an auxin antagonist in the regulation of root growth [33,34]. Our work was aimed at comparing the formation of adventitious roots in black poplar cuttings with the hormonal response during treatment with bacteria and HSs in order to identify the mechanisms that influence the rooting of cuttings. The project’s objectives also included studying the expression of the poplar RGF9 (root meristem growth factor) gene, which encodes a peptide that interacts with auxins in regulating the formation of adventitious roots [35,36].

2. Results

The bacterial strains used in the work were initially examined for their ability to produce IAA and catabolize ABA (Table 1). Table 1 shows that both strains had the ability to synthesize IAA, but this property was more pronounced in bacteria of the BLK strain, with its culture fluid demonstrating a concentration approximately 2 times higher compared to the DA1.2 strain.

The addition of bacteria-free HSs to the hydroponic solution used for rooting of black poplar cuttings did not affect either the number of adventitious roots or their length: the tendency for these indicators to increase by one and a half times compared to the control under the influence of HSs was not significant (Figure 1a,b). Images of cuttings with developing adventitious roots are presented in Supplementary Figure S1.

By themselves (without HSs), the BLK strain bacteria significantly increased the length of adventitious roots (Figure 1b); however, the tendency for the number of adventitious roots to increase under their influence was not significant (Figure 1a). Bacteria of the DA1.2 strain turned out to be more effective compared to the BLK strain: their addition to the medium led to a statistically significant increase in both the number of adventitious roots and their length compared to the control. However, the bacteria-induced increase in the number and length of adventitious roots did not exceed twofold compared to the control when applied without HSs. When bacteria were combined with HSs, their impact on rooting increased dramatically: the number of adventitious roots was 3.5 and 5 times higher, and their length was 7 and 10 times greater than in the control, respectively. The influence of DA1.2 was significantly greater than that of BLK when combined with HSs.

The assessment of the transcript level of the PnRGF9 gene revealed an increase in its expression following treatment with bacteria of the DA1.2 strain, both in combination with HSs and without them, as well as in the case of a combination of HSs and bacteria of the BLK strain (Figure 2). Bacteria of the latter strain alone (without HSs) did not cause an increase in the PnRGF9 transcript level. This gene is known to interact with auxins in the process of implementing its regulatory action [37]; while the role of auxins in rooting is well known [38], it was of interest to evaluate the auxin content in plant roots during the formation of adventitious roots.

All bacterial treatments resulted in a significant increase in the IAA content in the roots compared to the control (Figure 3), which is not surprising given the ability of bacteria of these strains to synthesize auxins (see Table 1). The DA1.2 strain stimulated auxin accumulation in roots to a lesser extent than the BLK strain, which corresponds to the lower ability of the DA1.2 strain to accumulate IAA in the medium during in vitro cultivation. Adding HSs to the DA1.2 strain preparation resulted in an even greater increase in IAA content in the roots.

The discrepancy between the ability of the strains to stimulate root formation and auxin accumulation in plants is noteworthy: strain DA1.2 was more effective in stimulating the formation and growth of adventitious roots, but had less of an effect on IAA content in the absence of HSs than strain BLK. Since adventitious roots are influenced not only by auxins but also by ABA [39], it was of interest to evaluate the content of this hormone in plants during the formation of adventitious roots (Figure 4).

All treatments, except for the combination of DA1.2 and HSs, increased ABA content in the roots (in the latter case, ABA content was similar to that of the control). The maximum accumulation of this hormone was recorded under the treatment with the BLK strain without HSs, when it was six times higher than in the control. The addition of HSs to both bacterial strains reduced ABA accumulation by half.

3. Discussion

HSs alone did not significantly affect root formation in poplar cuttings in our experiments. Therefore, HSs per se received little attention in this study. We previously recorded an increase in root mass in linden and pine trees when HSs were added to their rhizosphere [23]. The effect of HSs on root growth was attributed to the presence of auxin-like substances in the roots. The presence of IAA in the HSs structure was established using gas chromatography combined with mass spectrometry [26], allowing the authors to explain the effect of HSs on the growth and development of the root system of maize. However, the effect of HSs depends on the species, age, and the specific process being assessed. Although in our experiments, humic substances themselves did not have a significant effect on the formation of adventitious roots in poplar cuttings, their addition to bacteria increased rooting. This additive effect may be due to the ability of HSs to stimulate bacterial growth rate [40] and increase root colonization with bacteria [41]. Other aspects of HSs/bacterial interaction will be discussed below.

The ability of the treatments used in the present experiments to influence the rooting of poplar cuttings was studied in connection with their influence on plant hormones. Experiments with tea softwood cuttings demonstrated that adventitious root formation depends on local hormone gradients, measured with an immunoassay at the base of semi-lignified shoots [42]. The present experiments were conducted on heavily lignified poplar stem cuttings. Since the accumulation of phenolic compounds in highly lignified stem tissues could interfere with the immunoassay [43], hormones were extracted only from young adventitious root tissues. However, the study of local auxin gradients at the base of the poplar cuttings could be a target for further research after adapting the hormonal immunoassay to work with heavily lignified tissue. This can be performed, for example, using polyvinylpolypyrrolidone, which reduces nonspecific cross-reactions between antibodies and phenolic compounds [43].

Stimulation of adventitious root formation and growth was observed with both bacterial strains studied (more noticeable with strain DA1.2). The influence of rhizosphere bacteria on root growth and development is easily explained by the fact that many of them are capable of producing auxins [44]. Since plants are capable of auxin absorption from the soil solution [45], increased concentration of auxins in plants treated with auxin-producing bacteria may be attributed to the uptake of microbial hormone by plants. Some bacteria are also capable of influencing auxin metabolism in plants, thereby affecting the concentration of this hormone. For example, bacterial volatile organic compounds influenced host genes associated with auxin synthesis [46]. However, the latter mechanism of bacterial action on host auxins is discussed much less frequently. Of the two strains we studied, greater accumulation of IAA was detected in the medium on which the BLK strain bacteria grew, indicating their ability to produce more auxins compared to bacteria of the DA1.2 strain. At first glance, it seems puzzling that the BLK strain’s ability to stimulate root formation was less pronounced than that of the DA1.2 strain. This discrepancy between the ability to produce auxins and stimulate adventitious root growth can be explained by analyzing the ABA levels in the roots, which were higher in plants treated with the bacteria of the BLK strain. Interactions between auxin and ABA regulate numerous processes in plants, including root development [47]. It has been shown that the formation of adventitious roots depends on the ratio of auxins and ABA [42], and while auxins stimulate rooting, ABA inhibits it [48].

Bacteria are able to increase the root ABA levels by stimulating ABA synthesis by plants, and an increase in the expression level of the NCED gene, responsible for ABA synthesis, was detected in barley plants treated with Bacillus subtilis IB22 [49]. The increase in the ABA level in plants under the influence of the BLK strain may be associated with the ability of bacteria of this strain to produce a lot of auxins, while auxins have been shown to up-regulate the expression of the NCED gene and thereby promote the accumulation of this hormone [50]. Further experiments are needed to confirm the hypothesis that the increase in ABA levels was caused by BLK strain-induced expression of some genes responsible for ABA synthesis in poplar cuttings.

Plants treated with bacteria of the DA1.2 strain had significantly lower ABA levels than those treated with the BLK strain. This was apparently due to the ability of this strain of bacteria to catabolize ABA (see Table 1). It was previously shown that bacteria with the ability to catabolize ABA reduced ABA’s level in soil and lettuce plants, the accumulation of which was detected at increased planting density [51]. As mentioned above, the formation of adventitious roots on cuttings depends on the IAA/ABA ratio [47]. Lower ABA levels might facilitate adventitious roots formation and development [35]. Therefore, decreased concentration of ABA recorded in the roots of poplar cuttings treated with ABA-degrading bacteria explains the more noticeable stimulation of root formation with this treatment.

It was also important to understand how the increased bacterial efficiency was achieved when HSs were added to the bacterial preparation. The combined treatment with HSs and both bacterial strains reduced ABA levels compared to plants treated with either bacteria alone. How could the effect of HSs on ABA accumulation be explained? HSs are known to influence the metabolism of hormones in plants [52]. It has been shown that treatment with HSs significantly reduces the activity of the oxidase that catalyzes the final step in ABA synthesis from its precursor (aldehyde) [53], which explains the decrease in this hormone level under HS treatment. The most noticeable decrease in ABA accumulation under the influence of humic substances was observed when they were used in combination with the BLK strain, which led to a twofold decrease in ABA content compared to plants treated only with the BLK strain. The decrease in the ABA level in plants treated with a combination of HSs and the BLK strain was accompanied by a significant increase in the number and length of adventitious roots on poplar cuttings and was probably the cause of this. Although the effect on rooting was weaker than that of the combination of humic acids with DA1.2 bacteria, the combination of humic acids with BLK bacteria deserves further study as a way to enhance plant rooting.

The higher IAA levels in the roots of plants treated with a complex of HSs and DA1.2 bacteria, compared to the effect of this strain alone, also deserve explanation. ABA is known to increase the expression of the GH3 gene, which encodes an enzyme that catalyzes the inactivation of auxins by their conjugation [54]. The ABA level was extremely low in the roots of plants treated with the complex of HSs and DA strain bacteria, which apparently prevented the inactivation of IAA via conjugation and thereby contributed to the maintenance of high levels of this hormone.

The specific PnRGF9 gene encodes meristem root growth factor (RGF) and plays a critical role in regulating cell division and differentiation in the root meristem. RGFs are involved in maintaining stem cell activity and stimulating the formation of new roots, including adventitious roots [35]. This is especially important for plants propagated vegetatively (for example, by cuttings), since the formation of adventitious roots directly affects the success of rooting and the adaptation of plants to stressful conditions. It has previously been shown that the expression of the poplar-specific PnRGF9 gene increases under the influence of auxin, indicating its involvement in auxin-dependent root formation processes [11]. In our work, we also discovered this relationship: treatment with DA1.2 bacteria and/or humic substances increased the level of IAA and simultaneously increased the expression of PnRGF9, which confirms the hypothesis that auxins are involved in the regulation of root formation in poplar plant cuttings after their bacterial treatment. The IAA-overproducing BLK strain did not increase the activity of this gene, and despite elevated IAA levels, BLK did not enhance root number and exhibited a weaker stimulatory effect compared to DA1.2. This may indicate a blockage of the auxin signaling pathway due to high levels of ABA, an auxin antagonist. ABA is known to influence auxin signaling [55]. It has been proposed that the gene network enhancing RGF transcription under auxin action involves lateral organ boundary domain (LBD) transcription factors [35], which are repressed by ABA [56]. Thus, the mere presence of high amounts of IAA does not necessarily stimulate the activation of this gene expression and the increase in root apical meristem activity [36] and adventitious root growth observed in other treatments.

4. Materials and Methods

4.1. Plant Growth Conditions and Treatments

For experiments, we used cuttings of black poplar (Populus nigra L.) (Supplementary Figure S1). Cuttings 20 cm long were prepared from the South Ural Botanical Garden-Institute in November, after the leaves had fallen and the buds had formed. They were stored until spring at a low positive temperature (about 4 °C). In February, the cuttings were placed in natural 11 h day-time light conditions at 20 °C room temperature with constant aeration. A suspension of bacteria (1 mL·L^−1^, CFU 10^8^), HS concentrate (32 g·L^−1^), and their combination were added to a solution of distilled water. Thus, the working solution contained 1·10^5^ mL^−1^ colony-forming units (CFUs) of microorganisms and 1.6 mg·L^−1^ humic substances. Twice a week, the water in which the cuttings grew was changed, and the same amount of bacteria and humates was added. After 15 days, the number and length of adventitious roots were measured, and they were sampled for hormone immunoassay analysis and the determination of gene expression. Root length was measured with an electronic caliper with an accuracy of 0.1 mm. Roots longer than 1 mm of first-order branching were selected and counted.

4.2. Bacterial Strain and Cultural Media

We used two strains of Gram-negative bacteria from the collection of microorganisms of the Ufa Institute of Biology isolated from natural sources: Pseudomonas protegens DA1.2 deposited in the All-Russian Collection of Microorganisms (VCM B-3542D) and deposited in the collection of microorganisms of the Ufa Institute of biology (UIB-56), and Enterobacter ludwigii BLK deposited in the collection of microorganisms of the Ufa Institute of biology (UIB-51). Bacteria were cultivated in Erlenmeyer flasks with King’s B medium (2% peptone, 1% glycerol, 0.15% K_2_HPO_4_, 0.15% MgSO_4_·7H_2_O) on an Innova 40 R shaker (Eppendorf, New Brunswick, NJ, USA) (160 rpm) for 48 h at 28 °C. The number of cells in cultures was measured by applying serial dilutions to King B medium with agar-agar (15 g·L^−1^) and then counting the number of CFUs. The bacterial culture was diluted with sterile water to give a working solution containing (1.0 ± 0.5)·10^5^ CFU·mL^−1^.

4.3. Measurement of the Content of Plant Hormones in the Culture Liquid Using High-Performance Liquid Chromatography–Mass Spectrometry (HPLC-MS)

To study the degradation of ABA by bacteria, they were grown for 10 days in Raymond’s liquid medium [57], to which ABA was added to a final concentration of 100 mg·L^−1^. To study the IAA synthesis by bacteria, ABA was not added. The culture liquid was subjected to centrifugation at 8000× g followed by ultrafiltration through cassettes with a pore diameter of 1 kDa (Sartocon slice cassette, Mainz, Germany). Samples were analyzed on an LC-20 Prominence HPLC system with an SPD-M20A diode array detector (Shimadzu, Tokyo, Japan). A PerfectSil Target ODS-3 HD 5 µm (150 × 4.6 mm^2^) column (MZ-Analysentechnik, Mainz, Germany) was used. A 50% solution of acetonitrile in 0.1% acetic acid was used as the mobile phase at an elution rate of 0.4 mL·min^−1^. The volume of the injected sample was 5 µL. HPLC–MS analysis was performed on an LCMS-IT-TOF tandem liquid chromatography–mass spectrometer (Shimadzu, Tokyo, Japan) using electrospray ionization (ESI) in the negative ion mode. The trifluoroacetic acid solution was used as a standard to adjust the sensitivity and resolution, and to perform the mass number calibration. ABA and IAA concentration was calculated from a calibration curve constructed using a standard (Sigma-Aldrich, St. Louis, MO, USA). Chromatographic profiles of these measurements are presented in Supplementary Figures S2 and S3.

4.4. Extraction of Humic Substances

The source of humic substances was the brown coal from the Tyulganskoe deposit in the Orenburg region of the Russian Federation. Coal was mixed with 0.1 M KOH in a ratio of 1:10, and HSs were extracted for two hours with stirring at 1500 rpm. The precipitate was removed by centrifugation at 12,000 rpm for 10 min. A solution was obtained—a concentrate containing the sum of humic substances (humates and potassium fulvates with a total concentration of 32 g·L^−1^).

4.5. Extraction of IAA and ABA and Their Immunoassay

Adventitious roots were detached from poplar cuttings, homogenized in 80% ethanol, and incubated overnight at 4 °C. Aqueous supernatant, obtained after ethanol evaporation, was used to purify the samples of ABA and IAA, as described previously [51,58]. After adjusting the pH to 2.5 with HCl, the extract was partitioned with diethyl ether. Subsequently, free IAA and ABA were transferred from the organic phase into 1% sodium hydrocarbonate (pH 7–8). Readjusting the pH of the aqueous phase to 2.5, the re-extraction with ether gave the secondary ether extract, which was methylated, and the solvent was evaporated. The procedure released the samples containing free hormones from their conjugated forms, and the concentration of free hormones was immunoassayed. The final residue was dissolved in 100 µL of 80% ethanol and used for enzyme-linked immunosorbent assay with specific anti-ABA or anti-IAA antibodies. The specificity of the enzyme immunoassay was confirmed by low cross-reactivity of antibodies with hormone precursors and derivatives, as well as by the coincidence of chromatographic distribution of immunoreactivity with the position of hormonal standards detected in the purified hormonal samples. Recovery of IAA and ABA was calculated using the partition constants of hormones between aqueous and organic phases and was found to be about 90%. The reliability of the immunoassay of ABA was also confirmed by comparison of the results from the immunoassay with those of a physicochemical assay [50].

4.6. Gene Expression Analysis

Total RNA from poplar roots was extracted using Lira^®^ (Biolabmix, Moscow, Russia) according to the manufacturer’s instructions. First-strand cDNA was synthesized using the M−MLV reverse transcriptase (Fermentas, M-MLV, Synthol, Moscow, Russia). Oligo(dT)15 was used as a primer, and the reverse transcription reagents were incubated at 37 °C for 1 h in a total volume of 25 µL. Primers for real-time polymerase chain reaction (RT PCR) were devised using the web tool PrimerQuest™ (https://www.idtdna.com/pages/tools/primerquest, accessed on 10 November 2023) (Integrated DNA Technologies, Inc., Coralville, IA, USA). According to the PnRGF9 gene sequence, root meristem growth factor primers (F: 5′-GCAAGGACTTTGCGAGAGGT-3′; R: 5′-TGGAGGCTTCTTACTTGCTGG-3′) [11] were used, and the annealing temperature was between 46 °C and 60 °C. To normalize the expression results of the studied gene, primers were used for the ubiquitin-NEDD8-like protein PnUBQ7 gene (F: 5′-GCAGACCTTGTTTCGCTTTG-3′; R: 5′-GCATCGTCTTCTTCTTCTTCTCT-3′) (Gene ID XM_062117497.1) [59]. A melting curve analysis was conducted to determine the specificity of the reaction (at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s). The quantification of gene expression was performed using a CFX Connect RT PCR Detection System (BioRad Laboratories, Hercules, CA, USA). The qPCR program was as follows: 95 °C for 5 min; 40 cycles of 95 °C for 15 s, 60 °C for 20 s, and 72 °C for 30 s. The efficiency of each primer pair was determined using a tenfold cDNA dilution series in order to reliably determine the fold changes. For detection, a set of reagents, BioMaster HS-qPCR SYBR Blue (2×) (Biolabmix, Novosibirsk, Russia), was used.

4.7. Statistics

The data were processed using MS Excel software and one-way analysis of variance (ANOVA) and Duncan’s multiple comparison test to identify differences between mean values (p < 0.05) using Statistica version 10 (Statsoft, Moscow, Russia). Data in figures and tables are presented as mean values ± standard error. In the figures, mean values that are statistically different from each other are indicated by different letters. The number of biological replicates (n) is provided in the figure’s legends. This number varied when measuring the number and length of adventitious roots in 20 cuttings (n = 20 for each treatment), whereas when measuring transcript and hormone levels, values were obtained for adventitious roots collected from six individual plants (n = 6 for each treatment).

5. Conclusions

Most of the treatments on poplar cuttings applied in our experiments improved their rooting, and the bacterial effects were greatly increased by the addition of HSs. Although the combination of Pseudomonas protegens DA1.2 with HSs was the most effective in stimulating rooting, the effect of Enterobacter ludwigii BLK in combination with humates was also significant.

Our experiments showed that the activation of rooting is caused by both an increase in the level of auxins under the influence of bacteria that produce IAA, and a decrease in the content of ABA, as a result of its breakdown with the participation of bacteria that catabolize this hormone, as well as under the influence of HSs, which are capable of inhibiting ABA synthesis. The greatest efficiency was observed in the combined use of bacteria strain DA1.2 and the HSs preparation, each of which contributed to the reduction in ABA levels in the roots. Further experiments are needed to check the effects of DA1.2 + HSs on root formation in other crops for which successful rhizogenesis is a limiting stage of vegetative propagation.