At present, salinization of arable soils has become a major problem aggravated by the use of irrigated agriculture and climate aridization. Application of plant growth-promoting rhizobacteria (PGPR) makes it possible to increase plant resistance to salt stress (Emtsev et al., 2010; Numan et al., 2018; Chu et al., 2019). Presumably, the principal mechanism of the protective and growth-promoting action of PGPR under salt stress conditions is induction of increased synthesis of various groups of phytohormones, enzymes of the antioxidant system, pyrroloquinoline quinone (PQQ), and specific protective molecules (Alavi et al., 2013; Egamberdieva et al., 2017; Numan et al., 2018). The protective effect of PGPR can manifest through activation of photosynthesis and maintenance of ionic homeostasis due to increased absorption of K+, Ca2+, Mg2+, Na+, and other ions (Pan et al., 2019). The effectiveness of the growth-promoting and protective influence of plant bacterization under soil salinity conditions can be enhanced when using halotolerant PGPR, which was successfully demonstrated for members of Azospirillum, Bacillus, Burkholderia, Pseudomonas, Rhizobium, and other genera (Numan et al., 2018; Chu et al., 2019). In this aspect, the search for and isolation of new groups of halotolerant bacteria exhibiting PGP properties remains a challenge.

Bacteria of the genus Advenella (family Alcaligenaceae, class Betaproteobacteria), despite the small number of described species, are characterized by significant ecological and physiological diversity and are quite widespread in natural ecosystems. The genus Advenella was originally proposed by Coenye et al. (2005) to describe gram-negative oxidase-positive rod-shaped or coccoid bacteria isolated from a variety of human and animal clinical materials. For example, A. incenata, the type species of this genus, was isolated from human sputum (Coenye et al., 2005). In 2009, based on the data of phenotypic and phylogenetic analysis, the genus Tetrathiobacter proposed by Ghosh et al. (2005) and including the species T. kashmirensis and T. mimigardefordensis was reclassified to the genus Advenella (Gibello et al., 2009). However, the former species apparently remains the only genus member exhibiting chemolithotrophic properties. Subsequently, Advenella species were characterized from soil samples, activated sludge, rotted compost, rhizosphere of cultivated and wild plants, sludge from a biogas plant, and other sources (Ghosh et al., 2005; Wübbeler et al., 2006; Matsuoka et al., 2012; Poroshina et al., 2015; Wang et al., 2016; Dobrovol’skaya et al., 2017). Among the 16S rRNA gene sequences of A. kashmirensis and Advenella strains not identified to the species deposited into the GenBank database, at least 18 genes represent isolates from various underground habitats and 10 sequences belong to endophytic and rhizosphere bacteria. A natural reservoir of underground Advenella spp. isolates are the ecosystems of mud volcanoes, groundwater, mines, and caves, including their aerial environment, mineral deposits, and some representatives of cave fauna.

Despite the fact that many Advenella isolates have been found in the microbial communities of the rhizosphere of various plants, the growth-promoting activity of these bacteria remains poorly explored. Certain PGP properties were revealed in several strains, such as A. incenata VA2S3 and Advenella sp. PB-06, which are capable of solubilizing poorly soluble phosphorous compounds and producing auxins (Shahi et al., 2011; Singh et al., 2014), as well as in the A. kashmirensis subsp. methylica PK1T, which produces indole-derived auxin precursors in tryptophan-supplemented medium (Poroshina et al., 2015). Advenella members with PGPR properties that are resistant to high selenium concentrations were described as a part of endophytic microbial communities of selenium hyperaccumulator plants (Sura-de Jong et al., 2015). The species A. incenata isolated from tobacco rhizosphere together with 33 species of other bacteria exhibited antagonistic activity against the phytopathogen Phytopthora nicotianae (Jin et al., 2011). These data indicate the potential of the genus Advenella as a promising source of new PGPR members for the development of multifunctional microbial preparations in crop production.

The aim of the present work was to identify and characterize a new bacterial isolate of the genus Advenella capable of solubilizing mineral and organic forms of phosphorus that are inaccessible to plants, as well as to assess its effect on the growth of wheat plants under conditions of moderate soil salinity.

MATERIALS AND METHODS

The object of study and its isolation. Microbial isolate IB-K1 was isolated from the soil sampled in the aphotic zone (Borody Hall, 312 m from the entrance) of the Kinderlinskaya Cave (Southern Urals). The soil (0–5 cm) was sampled under sterile conditions, placed in plastic bags, and stored at 4°C. Isolation was carried out by inoculating Endo agar with a soil suspension and incubating it for 72 h at 28°C. The resulting single-type burgundy colonies with a metallic sheen were subcultured by serial dilutions on meat peptone agar (MPA) to assess the purity of the culture. Subsequently, a pure culture of IB-K1 was maintained by cultivation on MPA at 28°C.

Phenotypic and genotypic characteristics of the studied strain. The cultural and morphological properties of the novel isolate were studied when it was grown on MPA and in the G1G liquid nutrient medium of the following composition (g/L): glucose, 10; yeast extract, 5; peptone, 4; corn extract, 1; NaH2-PO4·2H2O, 1; K2HPO4·3H2O, 1; distilled water, 1 L; pH 7.6–7.8.

The physiological and biochemical properties of the novel strain were studied according to the recommendations for the identification of microorganisms (Gerhard and Am. Soc. Microbiol., 1981). Utilization of organic compounds (sugars, amino acids, and organic acid salts) as the only carbon source was studied by growing the strain on the mineral basis of liquid Koser’s medium containing 0.1 wt % of the corresponding carbon source and 0.06 wt % of bromothymol blue. The methylotrophic properties of the strain were assessed by its ability to grow in a liquid medium with methanol (0.5% vol/vol) as the sole carbon source (Doronina et al., 2000).

The presence of acid and alkaline phosphatases in the strain was determined by incubating of cell suspension prepared from a single colony of a 24-h culture in physiological saline with an equal volume of 10 mM disodium p-nitrophenyl phosphate solution (Sigma, United States) in phosphate‒citrate (100 mM; pH 4.8) or glycine (40 mM; pH 10.5) buffer, respectively (Gerhardt and Am. Soc. Microbiol., 1981). The culture was incubated for 6 h at 37°C, centrifuged, and the supernatant absorption was measured at 405 nm. The supernatant dilution rate was adjusted in such a way that the optical density values were in the range of Δ0.1‒0.3 units. The relative level of acid and alkaline phosphatase activity was expressed in terms of the average rate of free p-nitrophenol (p-NP) accumulation (μM/mL per min), taking into account the dilution coefficient. The dye concentration was calculated using the calibration curve constructed with commercial p-nitrophenol (Sigma) in the region of linear dependence in the 10–50 μM/mL range.

The temperature range of IB-K1 growth was studied on NA at 4, 20, 28, and 37°C. To assess the effect of pH on its growth, the strain was cultivated in liquid Koser’s medium at 28°C in the 4–9 pH range. The halotolerance of the test culture was assessed by growth in Hottinger broth containing from 1 to 9 wt % of NaCl.

The phylogenetic position of the strain IB-K1 was determined based on analysis of the 16S rRNA gene. Sequencing of the amplified fragment was performed using the Big Dye Terminator reagent kit on a Genetic Analyzer 3500XL automatic sequencer (Applied Biosystems, United States). Homologous 16S rRNA sequences were searched in the GenBank database using the BLAST software. Multiple alignments of nucleotide sequences and the construction of the phylogenetic tree by the neighbor joining method using the Jukes‒Cantor model were carried out with the Mega X 10.0.4 software. The significance of branching was assessed using bootstrap analysis from the specified 500 alternative phylogenetic trees.

Chemolithotrophic growth of isolate IB-K1. The ability of the strain to utilize reduced sulfur compounds was studied by growing the culture on a medium with thiosulfate (MST medium; Davidson and Summers, 1983) as described by Ghosh et al. (2005). The strain was cultivated in Erlenmeyer flasks (250 mL) on an Innova 40R incubator shaker at 28°C and 250 rpm for 6 days. The biomass of a 12-h culture grown in liquid G1G medium under the same conditions and washed with sterile saline solution was used as the inoculum. In the batch culture, the pH of the medium was maintained at 7.4 by adding sterile 2 M Na2CO3 solution. To support chemolithotrophic growth during cultivation, sterile sodium thiosulfate solution was added to the medium after 48 h of incubation in an amount equivalent to the initial concentration (20 mM). Bacterial growth and pH of the culture supernatant (CS) were monitored every 24 h during the cultivation cycle. The growth rate of bacteria was assessed by the optical density of CS at 600 nm.

The capacity of isolate IB-K1 for chemolithotrophic growth was also assessed on MST agar supplemented with 0.01% phenol red to control acidification of the medium during accumulation of sulfur oxidation products (Ghosh et al., 2005).

Determining the phosphate-mobilizing activity of isolate IB-K1. The growth rate of the strain and its ability to solubilize poorly soluble calcium, aluminum, and iron orthophosphates, as well as the calcium‒magnesium salt of inositol phosphoric (phytic) acid, were studied during its cultivation on Muromtsev’s and Menkina’s agar media according to the methodological recommendations (Szegi, 1979). The culture was stabbed on the surface of the nutrient medium and incubated for 14 days at 28°C; next, the diameters of bacterial colonies and the surrounding clear zones indicating phosphate solubilization were measured. The phosphate solubilization index (SI) was calculated using the formula SI = (Dcol-ony + Dzone)/Dcolony (Moawad and Vleck, 1996). The experiment was carried out in four replicates.

The ability of strain IB-K1 to solubilize calcium orthophosphate was assessed by submerged cultivation in Muromtsev’s liquid medium for 10 days at 28°C and 250 rpm. The phosphate mobilization rate was assessed by the change in the pH of the medium, by the level of accumulation of soluble phosphate anions in the culture medium, as well as by the degree of conversion (%) of the added insoluble phosphates. The concentration of phosphate anions was determined using the Fiske‒Subbarow method (Lurie, 1971).

Analysis of auxin (IAA) production in a submerged IB-K1 culture. To assess the level of IAA synthesis, the strain was grown for 7 days in liquid G1G medium at 28°C and 250 rpm. The titer of viable cells in the culture was determined by serial dilutions after two and six days of cultivation. To assess IAA production, the culture was sampled on days 1, 3, and 7. The samples were centrifuged for 30 min at 3260 g to remove bacterial biomass; the clarified CS was acidified with 1 N HCl solution to pH 2–3 and then subjected to double extraction with diethyl ether in a volume ratio of organic and aqueous phases of 1 : 3. IAA was re-extracted from the combined organic phase with 1% NaHCO3 solution in a ratio of 1 : 3 (aqueous phase : organic phase). After the organic phase was removed and discarded, the aqueous phase was acidified to pH 2–3, and phytohormones were extracted twice with diethyl ether (1 : 3) and methylated with diazomethane (Veselov et al., 1992). The IAA content was determined using ELISA with an appropriate test system (Veselov et al., 1992).

Evaluation of the effect of seed bacterization with strain IB-K1 on wheat productivity under conditions of moderate soil salinity. The effect of seed bacterization on wheat growth and productivity in the presence of NaCl in soil was studied in hard spring wheat Triticum durum Desf. ‘Bashkirskaya 27’. The seeds were treated with bacterial biomass obtained after 48 h of submerged cultivation of strain IB-K1 in G1G medium under the described conditions. The cells were collected by centrifugation at 2486 g for 20 min and suspended in sterile tap water to obtain an inoculum density of 104–106 CFU/seed for plant treatment. A 0.4% Na-CMC solution of moderate viscosity (Sigma) was used as a sticking agent. The field experiment was carried out on the territory of the Ufa district of the Republic of Bashkortostan in 2018 according to the generally accepted method (Dospekhov, 1985). The soil of the experiment was clay-illuvial agrochernozem with a moderate humus content (6.29%), slightly acidic pH, a high content of absorbed bases with the predominance of calcium and magnesium (Ca2+, 350 cmol/(equiv)/kg; Mg2+, 120 cmol/(equiv)/kg), and a moderate supply of mobile phosphorus and alkaline hydrolysable nitrogen (175 mg/kg). Experimental plots with an area of 1 m2 were arranged in one lane in four replicates. Before sowing, 1000 g of NaCl in the form of an aqueous solution were added to each plot by sprinkling. The soil content of water-soluble salts was determined according to the standard method (Agrokhimicheskie methody…, 1975). The assessment was made on the basis of an integral sample taken after harvesting from different sites of the plot, at a soil layer depth of up to 10 cm. The yield structure was analyzed according to the standard scheme (Dospekhov, 1985).

Scanning electron microscopy (SEM). Colonization of wheat plant rhizoplane by the studied strain in a model experiment was assessed using scanning electron microscopy (SEM). Seeds of T. durum Desf. ‘Bashkirskaya 27’ were treated with a bacterial preparation as described above, except that they had been pre-sterilized in a 1% merthiolate solution (1 min) and then washed twice (5 min) with sterile distilled water. Sterilized wheat seedlings that had not undergone bacterization were used as a control. The treated seeds were allowed to germinate for four days at 22°C on wet filter paper in Petri dishes in the dark. The germinated roots were washed with sterile sodium phosphate buffer (100 mM; pH 7.3) and fixed in 5% (vol/vol) glutaraldehyde solution (PanReac AppliChem, Spain) for 5 h. Next, the samples were dehydrated by sequential treatment with aqueous ethanol solutions of increasing concentrations (10–96%), then with a mixture of ethanol and acetone in a ratio (vol/vol) of 3 : 1, 1 : 1, and 1 : 3, and, finally, with pure acetone. After drying, the samples were coated with gold in a vacuum deposition chamber and analyzed by scanning electron microscopy on a TESCAN Vega 3 microscope (Czech Republic) at the Institute for Metals Superplasticity Problems, Russian Academy of Sciences (Ufa, Russia).

Statistical data processing. Statistical data were processed using the standard MS Office Excel 2007 package (12.0.6611.1000). The data were presented as the mean and the standard deviation (SD). The significance of differences was assessed by the Student’s t-test at P = 0.05.

RESULTS AND DISCUSSION

Strain IB-K1 was isolated on Endo agar when assessing the content of coliform bacteria in the soil of the Kinderlinskaya Cave (Southern Urals). While the isolate formed colonies similar to those of coliform bacteria (presence of metallic luster), it did not grow at 37°C as it is typical of this group. The isolate represented the main morphotype of bacteria isolated on Endo medium from a soil sample taken along the wall of the Borody Hall. With a total titer of 5.3 × 106 CFU/g of copiotrophic bacteria isolated on MPA, the titer of colonies corresponding in morphology to isolate IB-K1 was no less than 1.7 × 103 CFU/g. On rich nutrient media, strain IB-K1 formed round, opaque, shiny, pale pinkish-yellowish convex colonies of a homogeneous structure, with a smooth surface and an even edge. On Endo agar, the isolate colonies acquired a flat profile, red pigmentation, and a metallic sheen of the surface, with the other traits remained the same. IB-K1 cells were gram-negative nonmotile short rods (1.75–2.5 × 0.6–0.75 µm) that did not form capsules or spores. The isolated strain was tested for purity by several passages on MPA and then identified based on analysis of the 16S rRNA gene and comparison of the key physiological and biochemical properties.

Analysis of the 16S rRNA gene revealed that strain IB-K1 belongs to β-proteobacteria, namely, to the genus Advenella. In addition to the validated members of A. kashmirensis, the analysis included closely related strains isolated from different ecological niches, including rock surfaces (Sugiyama et al., 2017), ferromanganese cave deposits, and the rhizosphere of higher plants. Phylogenetic relationships of strain IB-K1 within the genus Advenella and its closest environment in the Alcaligenaceae family are shown in the tree (Fig. 1), where Advenella microorganisms form a clade with an identity level of 94–99.9% and high bootstrap support values. The IB-K1 16S rRNA gene sequence was found to have maximum identity (99.9%) with A. kashmirensis (NR 074872) and somewhat lower identity with A. kashmirensis subsp. methylica (99.8%). The phylogenetic affinity of strain IB-K1 to the type species A. kashmirensis WT 001 (Ghosh et al., 2005), as well as to the strains JCM 28735 (LC133766), clone RR1A10 (AY695719), and M66 (KC464861), is confirmed by the high sequence identity of their 16S rRNA genes (99.9‒100%) and manifests itself in clustering with high bootstrap values. The 16S rRNA gene sequence (1446 bp) of strain IB-K1 was deposited into the GenBank NCBI database under accession number OK336511 (https://www. ncbi.nlm.nih.gov/nuccore/OK336511). The strain IB-K1 itself was deposited into the All-Russian Collection of Microorganisms (VKM) under accession number B-3251.

Fig. 1.
figure 1

Dendrogram of the phylogenetic position of strain A. kashmirensis IB-K1 among representatives of the genus Advenella. The tree was constructed by the neighbor-joining method. The numbers show the statistical significance of branching of 500 alternative trees determined using bootstrap analysis (values above 50% are given). The scale shows the evolutionary distance corresponding to 5 nucleotide substitutions per 1000 nucleotides.

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The isolated strain was an oxidase- and catalase-positive facultative anaerobe; in the absence of atmospheric oxygen, it was able to carry out nitrate respiration and ferment glucose. The temperature and pH ranges of its growth were 4–28°C and pH 5–9, respectively. Unlike other representatives of A. kashmirensis, strain IB-K1 also exhibited good growth at NaCl concentrations of at least 7 wt % (Table 1). Moderate halotolerance of A. kashmirensis IB-K1 indirectly characterizes its viability at elevated ionic strengths of the soil solution under conditions of soil salinity. The strain could utilize ammonium salts and urea as the only nitrogen source; another its feature was the ability to grow in the presence of sulfur-containing compounds: thiosulfate and taurine. Some morphological, physiological,-and biochemical properties of isolate IB-K1 that distinguish it from the previously characterized members of A. kashmirensis are summarized in Table 1; in its other properties, it was similar to the most-studied strains of this species (data not shown).

Table 1. Morphological, physiological, and biochemical characteristics of strain IB-K1 in comparison with A. kashmirensis strains described previously
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Isolate IB-K1, like the type strain A. kashmirensis WT 001, was able to grow in the presence of reduced sulfur compounds, in particular, in a liquid medium with sodium thiosulfate (MST). In the first 24 h of submerged cultivation, alkalization of the medium to pH 8.6 was registered; it was followed by acidification to pH 5.5 at 48 h, which coincided with the growth maximum. At 48 h, the growth curve reached a stable plateau (Fig. 2). In the presence of thiosulfate, strain IB-K1 exhibited rapid dynamics and significant growth rates. A culture with an initial titer of 106 CFU/mL reached 109 CFU/mL after 120 h of cultivation in MST medium. Although the decrease in the pH of MST at two days of cultivation (Fig. 2) due to thiosulfate oxidation followed the pattern observed for strain WT 001, additional introduction of thiosulfate to the medium after reaching a plateau (48 h) did not lead to a further increase in culture growth as described previously for A. kashmirensis WT 001 (Ghosh et al., 2005). In a similar experiment performed on MST agar with 0.01% phenol red, bacterial growth observed after 24 h upon inoculation with a prick was accompanied by acidification of the medium after 48 h of cultivation (the indicator color changed in the decreasing pH 8.4–6.8 range; data not shown). These results confirm that strain IB-K1 is capable of mixotrophic type of nutrition and can be assigned to facultative chemolithotrophs utilizing thiosulfate as an energy source.

Fig. 2.
figure 2

Growth dynamics (1) of A. kashmirensis IB-K1 and changes in CS pH (2) in the presence of 20 mM sodium thiosulfate as the sole source of energy (MST medium, 28°C, 250 rpm). The vertical arrow indicates the moment (48 h) where the culture medium was supplemented with sodium thiosulfate solution in an amount equivalent to its initial concentration (20 mM) and 2 M Na2CO3 solution to adjust pH to 7.4.

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Thus, in terms of chemolithotrophic properties, isolate IB-K1 showed similarity to the type strain A. kashmirensis WT 001, whose genome contains a complete set of genes for oxidation of inorganic sulfur (soxCDYZAXWB; Ghosh et al., 2011). This strain oxidizes thiosulfate to sulfate with the formation of tetrathionate as the main intermediate; in its further oxidation a key role belongs to glutathione (Pyne et al., 2017). The source of isolation of the chemolithotrophic strain WT 001 was an integral sample of orchard soil of non-rhizosphere origin in the temperate climatic zone of the union territory of Jammu and Kashmir (India) (Ghosh et al., 2005). In contrast, strain IB-K1 was isolated from the soils of a carbonate cave with gypsum inclusions, the isotopic composition of which gives evidence of their bacterial origin and indicates the presence of conditions for the life of sulfur cycle bacteria (Chervyatsova et al., 2016). At the same time, A. kashmirensis subsp. methylica PK1, which is unable to oxidize sulfur compounds, was isolated from the rhizosphere of sedge growing on calcite travertines, i.e., on a mineral substrate (Poroshina et al., 2015).

Like A. kashmirensis subsp. methylica PK1 (Poroshina et al., 2015), isolate IB-K1 was able to use methanol as the only carbon source, increasing its titer by two orders of magnitude (from 106 to 108 CFU/mL) after 6 days of cultivation in a medium with 0.5% (vol/vol) methanol. The methylotrophy of A. kashmirensis IB-K1 indicates its potential for symbiosis with plants, since methanol is a natural product of plant metabolism (Galbally and Kirstine, 2011; Poroshina et al., 2015; Doronina et al., 2015). Many methylotrophic bacteria are known to demonstrate growth-promoting activity in relation to mono- and dicotyledonous plants during their colonization and to suppress the development of phytopathogens, diminishing plant damage by bacterial and fungal diseases, nematodes, and other pathogens (Doronina et al., 2015). The plant growth-promoting effect of methylobacteria is mainly related to the synthesis of phytohormones and vitamin B12, as well as their nitrogen-fixing and phosphate-solubilizing activities (Doronina et al., 2015). In this context, strain A. kashmirensis IB-K1 was studied in terms of its plant growth-promoting parameters, in particular, phosphate-mobilizing activity and IAA synthesis.

A. kashmirensis IB-K1 exhibited a high level of synthesis of acid and especially alkaline phosphatases, which indicates its active participation in the mineralization of organic phosphorus (Table 2). According to the study by Nassal et al. (2018) that assessed the effect of some PGPR strains of the genera Bacillus and Pseudomonas on tomato plants, their growth-stimulating effect was mainly due to the increased production of phosphatases in the rhizosphere of plants. When cultivated on agar media containing poorly soluble mineral and organic forms of phosphorus, the strain IB-K1 solubilized calcium orthophosphate and phytin more actively than iron and aluminum phosphates (Table 2). The high phytin solubilization index value on Menkina’s agar may indicate that strain IB-K1 productivity is at least comparable to the maximum values (0.174 U/mL) noted previously for Advenella members (Singh et al., 2014). The calcium orthophosphate solubilization index was 5 points, which exceeds this parameter (2‒4 points) in the previously described Advenella spp. PGP strains (Singh et al., 2014). In a bacterial broth culture, complete calcium orthophosphate solubilization and partial aluminum phosphate solubilization occurred; under these conditions, the pH of the culture medium was observed to decrease from the initial value of pH 7 to 4.4–4.5; these values were stably maintained throughout the entire cultivation period from day 1 to 10 (Table 2). The formation of organic acids by microorganisms is considered as one of the main mechanisms of their phosphate-solubilizing activity, although alternative metabolic bases of this process associated with the release of protons during nitrogen assimilation from ammonium or with H2CO3 formation during respiration are also proposed (Goldstein and Krishnaraj, 2007; Khan et al., 2009). The low index of solubilization of aluminum and iron phosphates by strain IB-K1 may indicate that the culture medium predominantly contained organic acids characterized by a low constant of stability of complexes with these metal ions, e.g., gluconic acid (Khan et al., 2009). Our data suggest that strain IB-K1 should be more effective as a phosphate mobilizer in alkaline or neutral soils mainly containing calcium-bound forms of phosphorus than in acidic soils containing predominantly iron and aluminum phosphates (Hu et al., 2005).

Table 2. Phosphatase production and parameters of phosphate-solubilizing activity of the strain A. kashmirensis IB-K1
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Strain A. kashmirensis IB-K1 was characterized by a relatively high level of IAA synthesis; maximum auxin production (220 ± 42 ng/mL) was recorded 72 h after the beginning of submerged cultivation in a medium with 1% glucose (Fig. 3). The ability of Advenella strains to produce significant auxin concentrations (12–35 mg/mL) was shown previously by Singh et al. (2014). It should be noted that the culture medium used for cultivation by these authors contained a high concentration of tryptophan (0.05 wt %), which is the main precursor of IAA synthesis. The hormone concentration was determined in CS using the Salkowski reagent for indolic compounds. Although this method is relatively simple, its specificity is low, which may lead to overestimated results (Glickman and Dessaux, 1995). Unlike other Advenella strains that produce heteroauxins, the strain we studied was not able to utilize exogenous L-tryptophan (Table 1), which is a precursor of IAA biosynthesis occurring in bacteria via five currently known pathways (Bungsangiam et al., 2019). This fact indicates that A. kashmirensis IB-K1 has a non-tryptophan dependent mechanism of IAA synthesis (Prinsen et al., 1993). IAA production by bacteria normally involves several pathways simultaneously, and non-tryptophan-dependent synthesis usually occurs under conditions of exogenous precursor deficiency. The combined IAA synthesis involving several pathways is most often observed in rhizobacteria, which allows them to produce this compound in larger quantities compared to members of microbial communities from other ecosystems (Zhang et al., 2019).

Fig. 3.
figure 3

Dynamics of culture parameters during submerged periodic cultivation of A. kashmirensis IB-K1 (7 days, 28°C, 250 rpm): (1) bacterial growth, logCFU/mL; (2) OD600; (3) accumulation of IAA; (4) pH of culture supernatant.

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The ability of isolate IB-K1 to modify the endogenous content of abscisic acid (ABA) in wheat was shown in our previous field experiments in nonsaline soil. Evaluation of the hormonal status of bacterized plants revealed a twofold increase in the ABA content in shoots at the second leaf stage. The structural analysis of the crop showed an increase in the length of the main spike and in the number of productive shoots. Strain A. kashmirensis IB-K1 successfully colonized wheat rhizosphere; culture abundance at the second leaf stage was up to 7.5 × 106 CFU/g of roots and further decreased at the tillering stage to 2.5 × 105 CFU/g (Galimzyanova et al., 2018).

SEM analysis of the root surface of bacterized wheat at the 4-day seedling stage confirmed active col-onization of the root hair zone by A. kashmirensis IB-K1 cells (Fig. 4). In control variants of wheat seedlings, bacterial cells were not detected (data not shown).

Fig. 4.
figure 4

Scanning electron microscopy (SEM) of the root system of 4-day-old seedlings of Triticum durum Desf. ‘Bashkirskaya 27’ obtained after seed colonization with a 48-h culture of A. kashmirensis IB-K1 at an inoculum density of 106 CFU/seed. Magnification: ×1000 (a), ×10 000 (b), and ×18 100 (c). The region shown in the micrographs (b) and (c) corresponds to the selected fragment in the micrograph (a).

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In a field experiment assessing the influence of A. kashmirensis IB-K1 on the development and yield of spring wheat under salt stress conditions, the content of water-soluble salts in the upper 10-cm layer of the soil of experimental plots supplemented with NaCl (1000 g/m2) was 0.33 ± 0.06%, which corresponds to the borderline value between medium and high salinity (Kiryushin, 1996). In general, under moderate salinity, the parameters of wheat yield structure exhibit 20–35% decrease in comparison with plants grown under normal conditions (Table 3). When the seeds were treated with A. kashmirensis IB-K1, wheat productivity (in terms of grain weight per plant) increased in comparison with the control variant by 19% (Table 3). An increase in the number and weight of grains in the main spike was also observed, while the differences in other parameters were insignificant at the stage of harvesting. The positive effect of bacterization may be due to both to IAA production by A. kashmirensis IB-K1 and to its ability to reduce the effect of oxidative stress on plants, which develops under conditions of high salt content as shown previously (Arkhipova et al., 2018).

Table 3. Influence of seed treatment with strain A. kashmirensis IB-K1 on the productivity of the hard spring wheat Triticum durum Desf. ‘Bashkirskaya 27’ associated with artificial salinization in the field experiment
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Thus, strain IB-K1 can be characterized as the first member of the facultative chemolithotrophic species A. kashmirensis isolated from a cave ecosystem and exhibiting PGP properties. The growth-promoting effect of A. kashmirensis IB-K1 is apparently associated with its methylotrophic properties and high phosphate-mobilizing potential, as well as the abilities to synthesize IAA and to colonize wheat rhizosphere. The results of the field experiment indicate that this strain can be efficiently employed to stimulate the growth of agricultural plants, in particular, under conditions of increased salt content in the soil.