Saxaul forests perform the following functions in the ecosystem: biomass production, stabilization of sand movement, maintenance of soil layer and specific microclimate, ensuring the growth and development of associated plants (e.g., Carex physodes M. Bieb.), establishment of habitat conditions for animals, and support of permanent microbial communities in the root system and endophytic microorganisms living in plant tissues. Saxaul forests are a source of food for farm animals and a valuable fuel for the local population [4–7].
The tissues of various plants have their own unique communities of bacterial endophytes. There are many reports showing that endophytes play an important role in plant growth stimulation and their protection from phytopathogenic microorganisms [8–13]. Bacterial endophytes have advantages over the bacteria inhabiting the rhizosphere. Once inside the tissue, they have direct contact with the plant, and hence easy communication between cells can take place. Thus, they can have a direct beneficial effect on the host. In this process, compounds produced by bacteria directly affect the physiological activity of a host plant and can increase biomass production . Bacteriogenic substances include indole-3-acetic acid (IAA) , siderophores , 1-aminocyclopropane-1-carboxylate (ACC) deaminase , and lytic enzymes, leading to plants beneficial processes such as phosphate solubilization , atmospheric nitrogen fixation , chelation of metal ions in an absorbable form , alleviation of stresses [14,20], and suppression of pathogenic microbiota .
Despite the fact that endophytic communities of various plants have been studied, there is still no information about bacterial endophytes living in black saxaul and their effect on it. So, this is the first report about bacterial endophytes of black saxaul.
The aim of this study was to isolate and identify bacterial endophytes from roots and stem of black saxaul, as well as check their plant beneficial traits.
2. MATERIALS AND METHODS
2.1. Black Saxaul Plants Collection
Five plants of black saxaul (H. aphyllum Minkw.) were carefully isolated from the soil of Kyzylkum Desert in Uzbekistan in springtime. The soil particles were eliminated from roots and shoots by washing them in sterile water.
2.2. Bacteria Isolation
The shoot and root were cut from each other and the pieces (15 g) were sterilized by putting them into glasses with 99.9% ethanol for 2 minutes and 10% sodium hypochlorite for 1 minute. After that, they were put into glasses with sterile water for 2 minutes . The pieces of shoots and roots were longitudinally cut into thin slices. 5 g of each sample was transferred into tubes with 9 ml of sterile tap water for serial dilution (101–105). 100 μl of suspension from each dilution was transferred and spread on Tryptic Soy Agar. The plates were incubated in a thermostat at 30°C. In 4 days, the colonies changing in color and shape were transferred and streaked on plates with Tryptic Soy Agar for purification. The pure cultures were used for DNA isolation. We also checked the outer surface of the root and shoot pieces for sterility by putting them onto Tryptic Soy Agar (TSA) media and incubating for 4 days at 30°C. There were no colonies after incubation.
2.3. Bacteria Identification
The method of Dashti et al.  was used for bacterial DNA isolation. The bacterial colonies were transferred into Eppendorf tubes with 1 ml of Milli-Q water. The colonies were mixed with water by shaking in hand for 1 minute and incubated at 90°C for 20 minutes in a dry block heater. After that, they were centrifuged at 12,000 rpm for 5 minutes. The isolated DNA was visualized using gel electrophoresis.
The extracted DNA was exposed to 16S rRNA gene analysis by means of Polymerase chain reaction (PCR) using the following primers: 27F 5′-GAGTTTGATCCTGGCTCAG-3′ (Sigma-Aldrich, St. Louis, MO) and 1492R 5′-GAAAGGAGGTGATCCAGCC-3′ (Sigma-Aldrich, St. Louis, MO) . The PCR program was as follows: a primary heating step for 30 seconds at 94°?, followed by 30 cycles of denaturation for 15 seconds at 94°C, annealing for 30 seconds at 55°C, and extension for 1.5 minutes at 68°C, then followed by the final step for 20 minutes at 68°C. The PCR products were checked by electrophoresis using GelRed.
The ABI PRISM BigDye 3.1 Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, USA) was used for the sequencing. The obtained sequences were compared with the sequences of the closest relatives from GenBank of the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/).
The evolutionary history was inferred using the neighbor-joining method . The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown above the branches . The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method  and are in the units of the number of base substitutions per site. This analysis involved 41 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1,648 positions in the final dataset. Evolutionary analyses were conducted in MEGA X .
2.4. Test for Plant Growth Promotion by Bacterial Endophytes
Isolated bacterial endophytes were cultivated in a nutrient broth medium for 96 hours at 30°C and cells concentration was adjusted up to 108 CFU/ml. The seeds of a cucumber (genotype Orzu) were inoculated with bacteria by soaking in bacterial suspension and sown into 500 ml plastic pots containing 400 ml of soil by volume. We specially prepared four types of soil salinity with NaCl, 0, 25, 50, and 100 mM, by watering with water containing the appropriate NaCl concentration. All pots were set up randomly in five replications for each bacterial strain. Three seeds were sown into each pot. As a control, we used seeds without any inoculation. Plants were grown at 28°C–30°C during the day and 18°C–20°C at night, and after 14 days, the shoots and roots’ dry weight was measured.
2.5. Tests for Plant-Growth-Promoting Properties
2.5.1. IAA production test
The production of IAA was tested according to the method of Sarwar and Kremer . The bacterial suspension was adjusted to 1 × 108 CFU/ml and added to flasks with 10% TSA  supplemented with 5 mmol/l−1 of L-tryptophan and cultivated at 30°C for 24 hours in the dark. The grown bacteria were centrifuged at 8,000 × g for 15 minutes and the supernatant was poured into fresh tubes. The Salkowski reagent (mixture of FeCl3 0.5 mol/l and H2SO4 7.9 mol/l) was added in a 1:1 ratio (v/v) to the supernatant and left at room temperature for 30 minutes in the dark. The appearance of pink color indicated the production of IAA. For the measurement of IAA, a spectrophotometer at 530 nm was used. Different concentrations of IAA solutions were used to construct a standard curve.
2.5.2. Phosphate solubilization test
The ability of endophytes to solubilize inorganic phosphate was tested according to Mehta and Nautiyal . The bacteria were cultured on solid National Botanical Research Institute’s phosphate growth medium (NBRIP) medium (%): glucose 1, Ca3(PO4)2 0.5, MgCl2 0.5, (NH4)2SO4 0.01, MgSO4.7H2O 0.025, KCl 0.02, and agar 1.5. Plates with bacteria were incubated at 28°C for 96 days. The formation of colonies indicated the ability to use inorganic phosphate in the form of Ca3(PO4)2 as a sole phosphate source.
2.5.3. Nitrogen fixation assay
The colonies of each endophyte were streaked onto solid nitrogen-deficient malate medium (g/l): CaCl2 0.02, NaCl 0.1, FeCl3 0.01, KH2PO4 0.4, K2HPO4 0.5, MgSO4·7H2O 0.2, Na2MoO4·2H2O 0.002, sodium malate 5, agar 15, and pH 7.2–7.4, supplemented with 50 mg/l yeast extract. The plates were incubated at 30°C for 96 hours and the appearance of growth indicated the ability to fix N2. The newly grown single colonies were streaked onto plates with the same medium to confirm the ability of nitrogen fixation .
2.5.4. Siderophores production test
Siderophore production was determined by using chrome azurol S (CAS) agar. Isolates were streaked onto CAS agar and incubated at 30°C for 96 days. The appearance of an orange halo around the bacterial colony indicated the production of siderophores .
2.5.5. ACC deaminase production test
The ACC deaminase production by bacteria was tested based on the utilization of ACC as a sole N-source. The endophytes were cultivated on basal medium supplemented with 3.0 mM of ACC. We used (NH4)2SO4 as a positive control without adding N-source as a negative .
2.6. Statistical Analysis
The statistical significance of data was tested by the analysis of variance of the Microsoft Excel 2010 package. Mean comparisons were conducted using the least significant difference test (p = 0.05). The average values of plant growth parameters, IAA production, and the standard deviation were counted based on several replications.
2.7. Accession Numbers
The 16S rRNA gene sequences of the endophytic bacteria of H. aphyllum Minkw. were deposited into GenBank under the following accession numbers MZ443974–MZ443993.
3.1. Bacterial Endophytes’ Identification and Phylogenetic Analysis
A total of 20 bacterial isolates related to different species were isolated from tissues of roots and shoots of black saxaul. The isolates were identified based on a comparison of their 16S rRNA with the closest relatives registered in GenBank. The degrees of their 16S rRNA genes similarities are shown in Table 1.
The percent of identity of 16S rRNA gene from black saxaul isolates and the closest relatives from GenBank ranged from 99.38 to 99.79. The isolates represent three phyla: Firmicutes, Actinobacteria, and Proteobacteria. The most numerous is Firmicutes with 13 representatives: HAPH2, HAPH3, HAPH4, HAPH5, HAPH6, HAPH7, HAPH8, HAPH9, HAPH10, HAPH14, HAPH15, HAPH19, and HAPH20. Among 20 isolates, only 4 were related to Actinobacteria (HAPH1, HAPH12, HAPH13, and HAPH17) and 3 to Proteobacteria (HAPH11, HAPH16, and HAPH18). The isolates were divided into three classes: Bacilli (13 isolates), Actinobacteria (4 isolates), and Gammaproteobacteria (3 isolates). There were representatives of five orders: Bacillales (12), Micrococcales (4), Lactobacillales (1), Oceanospirillales (2), and Pseudomonadales (1). All isolates were related to 13 genera: Bacillus (HAPH9, HAPH4, HAPH6, HAPH7, HAPH8, and HAPH2), Arthrobacter (HAPH1), Priestia (HAPH3 and HAPH5), Enterococcus (HAPH10), Halomonas (HAPH11), Isoptericola (HAPH12), Kocuria (HAPH13), Planococcus (HAPH14), Planomicrobium (HAPH15), Pseudomonas (HAPH16), Rothia (HAPH17), Salinicola (HAPH18), and Staphylococcus (HAPH19 and HAPH20).
|Table 1: Endophytes isolated from black saxaul (H. aphyllum Minkw.) and their closest relatives from GenBank.|
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|Figure 1: Phylogenetic tree of endophytic bacteria (HAPH1–HAPH20) of black saxaul with its closest relatives registered in GenBank of NCBI.|
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Based on 16S rRNA gene similarities using the neighbor-joining method, the phylogenetic tree was constructed (Fig. 1).
3.2. Plant Growth-Promotion Activity of Endophytic Bacteria
The strains of endophytic bacteria were tested for the ability to stimulate plant growth on the example of cucumber. The seeds of cucumber were inoculated with bacterial suspension and cultivated in pots. Although black saxaul is a halophyte, we decided to check whether its endophytes can benefit plants in salinity conditions. That is why for cucumber growing we used four types of soil salinization with NaCl: 0, 25, 50, and 100 mM. We measured shoot and root dry weight as the main growth parameter because the speed of a plant’s dry biomass accumulation depends on the speed of cell division and its growth. The results are shown in Table 2.
It can be seen from Table 2 that the majority of endophytes stimulated plant growth of a cucumber less or more actively at different degrees of soil salinity. The strain Bacillus amyloliquefaciens HAPH2 appeared to be the most active plant growth-promoting bacteria at 0 and 25 mM NaCl in soil. At 0 mM NaCl, the cucumber seeds’ inoculation with this strain resulted in 17.9% and 18.8% increase in shoot and root dry weight as compared to the control, respectively. At 25 mM NaCl, the effect of this strain was even more and it increased shoot dry weight up to 22.3% and root dry weight up to 27.2% in comparison with the control. At 50 mM NaCl, the strain B. amyloliquefaciens HAPH2 increased cucumber plants shoot dry weight up to 38.1% and root dry weight up to 37.2% as compared to the control. At 100 mM NaCl, this strain raised the shoot dry weight up to 65.2% and root dry weight up to 216.7%. However, at 50 and 100 mM NaCl, the strain B. amyloliquefaciens HAPH2 was less effective than Pseudomonas kilonensis HAPH16. The seeds’ inoculation with strain P. kilonensis HAPH16 resulted in 39.9% and 34.9% increase in shoot and root dry weight at 50 mM NaCl as compared to control, respectively. At 100 mM NaCl, P. kilonensis HAPH16 raised the shoot dry weight up to 68.3% and root dry weight up to 225%. It should be noted that strains Arthrobacter agilis HAPH1, Bacillus cereus HAPH4, Bacillus pumilus HAPH6, Brevibacterium frigoritolerans HAPH9, Kocuria polaris HAPH13, Rothia terrae HAPH17, Staphylococcus epidermidis HAPH19, and Staphylococcus warneri HAPH20 did not show or showed very low insignificant changes in shoot and root dry weight after seeds’ inoculation.
3.3. Plant Growth-Promoting Traits of Endophytic Bacteria
The endophytic bacteria were analyzed for their plant growth-promoting characteristics: nitrogen fixation, phosphates solubilization, and production of IAA, ACC deaminase, and siderophores (Table 3).
It can be seen from Table 3 that, from 20 strains, only 5 possessed all checked plant growth-promoting characteristics: B. amyloliquefaciens HAPH2, Priestia endophytica HAPH5, Bacillus subtilis HAPH7, Bacillus toyonensis HAPH8, and P. kilonensis HAPH16. These strains showed N2-fixation, phosphate solubilization, IAA, ACC, deaminase, and siderophores production. The highest amounts of produced IAA were observed in seven strains: B. amyloliquefaciens HAPH2 (172.12 μg/ml), P. endophytica HAPH5 (134.65 μg/ml), B. subtilis HAPH7 (163.86 μg/ml), B. toyonensis HAPH8 (119.03 μg/ml), Isoptericola halotolerans HAPH12 (123.35 μg/ml), Planomicrobium soli HAPH15 (141.32 μg/ml), and P. kilonensis HAPH16 (159.47 μg/ml). The following eight strains did not have any of the checked traits and the IAA level was too low: S. epidermidis HAPH19, B. cereus HAPH4, B. frigoritolerans HAPH9, Enterococcus faecalis HAPH10, K. polaris HAPH13, R. terrae HAPH17, Salinicola tamaricis HAPH18, and A. agilis HAPH1. Four strains possessed three important characteristics: P. soli HAPH15, I. halotolerans HAPH12, Planococcus salinarum HAPH14, and Halomonas sulfidaeris HAPH11. The strains Priestia aryabhattai HAPH3 and S. warneri HAPH20 could solubilize phosphates and produce siderophores, but their IAA production was insignificant. The strain B. pumilus HAPH6 could only solubilize phosphate.
|Table 2: Effect of endophytic bacteria from black saxaul on the dry weight of cucumber (Cucumis sativus) (genotype Orzu) plants grown at different concentrations of NaCl after 14 days of pot experiment.|
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|Table 3: Plant growth-promoting properties of the isolated endophytes.|
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As a result of the conducted research, we isolated 20 different species of endophytic bacteria from black saxaul. These bacteria are related to 3 phyla, 3 classes, 5 orders, 8 families, and 13 genera. Some of them demonstrated high plant growth-promoting activity in the stimulation of a cucumber plant’s growth. To explain the activity, we conducted tests for some important plant growth-promoting traits. There were many reports about plant growth-promoting properties of endophytic bacteria isolated from different plants [8,13,35].
In our research, the strains B. amyloliquefaciens HAPH2, P. endophytica HAPH5, B. subtilis HAPH7, B. toyonensis HAPH8, H. sulfidaeris HAPH11, I. halotolerans HAPH12, P. soli HAPH15, and P. kilonensis HAPH16 showed the highest plant growth-promoting activity. Gamez et al.  reported on B. amyloliquefaciens Bs006 which was selected after screening as one of the best plant growth-promoting rhizobacteria. The strain promoted banana growth on the same level as chemical fertilization and was proposed to be used as a formulation of a new biofertilizer. Park et al.  reported on Bacillus aryabhattai SRB02 (now P. endophytica) isolated from the rhizosphere of soybean which significantly promoted the growth of rice and soybean by producing phytohormones. There were especially many reports about different strains of endophytic B. subtilis isolated from Camellia oleifera , Theobroma cacao L. , and Zea mays  and their antimicrobial and plant growth-promoting properties. The endophytic bacteria B. toyonensis COPE52 stimulated shoot and root length and increased biomass and chlorophyll content of blueberry plants (Vaccinium spp. var. Biloxi) . The bacteria H. sulfidaeris HAPH11, I. halotolerans HAPH12, and P. soli HAPH15 are considered halotolerant. Zhou et al.  studied the effect of halotolerant rhizobacteria isolated from halophytes on the growth of sugar beet under salt stress and observed growth stimulation activity of some bacterial strains. However, they did not find plant growth-promoting activity in strain I. halotolerans TGT-T12. Shurigin et al.  also isolated endophytic strains H. sulfidaeris JST7 and P. soli JST11 from halophyte Seidlitzia rosmarinus Ehrenb. ex Boiss. The strains did not show any plant growth-promoting (PGP) activity. Egamberdieva et al.  reported on antibacterial and antifungal efficacy of endophytic P. kilonensis isolated from horseradish (Armoracia rusticana G. Gaertn., B. Mey. & Scherb.). Due to its antimicrobial properties, P. kilonensis could protect plants against microbial diseases.
The endophytic bacteria from black saxaul were tested for plant growth-promoting properties. The strains B. amyloliquefaciens HAPH2, P. endophytica HAPH5, B. subtilis HAPH7, B. toyonensis HAPH8, H. sulfidaeris HAPH11, I. halotolerans HAPH12, P. soli HAPH15, and P. kilonensis HAPH16, which showed high plant stimulatory activity, were positive in a minimum of three of five tests for plant growth-promoting properties : nitrogen fixation, phosphates solubilization, and production of IAA, ACC deaminase, and siderophores. Nitrogen (N) is one of the most important chemical elements for plants growth and an essential component of all enzymes and proteins, nucleic acids of DNA, and chlorophyll . In the form of N2, it is a very stable and inert gas that does not come into reactions. However, rhizospheric and endophytic bacteria can converse N2 into ammonia which easily dissolves in water and directly feed a plant . Muangthong et al.  reported on the isolation and characterization of endophytic nitrogen-fixing bacteria from sugarcane. Potassium also is one of the most important nutrients for plant growth. Some endophytes produce organic acids which can be excreted into soil and convert phosphate complexes into orthophosphates for plant absorption and usage. Thus, such endophytic phosphates solubilizing bacteria were proposed for usage as biofertilizers . IAA is related to phytohormones, which stimulates seed germination, increases the root system, and regulates biosynthesis of metabolites and resistance to extreme conditions like salt and drought stresses . Endophytes due to the production of IAA can stimulate plant root growth through supplying the plant with more nutrients from soil .
In our research, the cucumber plants grown at different NaCl concentrations and seeds’ inoculation with some endophytes resulted in higher shoot and root dry mass as compared to control plants. The endophytes P. kilonensis HAPH16, P. endophytica HAPH5, B. subtilis HAPH7, B. toyonensis HAPH8, I. halotolerans HAPH12, P. salinarum HAPH14, P. soli HAPH15, and B. amyloliquefaciens HAPH2 produced ACC deaminase. Ethylene is a hormone which is overproducing in plants in stress conditions like high or low-temperature stresses, high salt concentration, drought, flooding, and so on. In such conditions, ethylene production leads to defoliation, fastened vegetation period, and decrease of plant growth and leads to yield lowering. 1-Aminocyclopropane-1-carboxylic acid (ACC) is an ethylene precursor and the enzyme ACC deaminase is involved in plant growth promotion through cleavage of ACC and lowering of ethylene level in the plant. The lower the ethylene level, the lower the stress in the plant in stress conditions. In this way, ACC deaminase-producing bacteria can stimulate plant growth in stress conditions like high salt concentration .
The bacteria S. warneri HAPH20, P. aryabhattai HAPH3, P. endophytica HAPH5, B. subtilis HAPH7, B. toyonensis HAPH8, H. sulfidaeris HAPH11, P. salinarum HAPH14, P. kilonensis HAPH16, and B. amyloliquefaciens HAPH2 produced siderophores. Yadav  reported that endophytes that produce siderophores can make iron available for the plant through iron chelating that is especially important for plants growing in iron-deficient soils. Siderophores production by bacteria is one of the important mechanisms of plant pathogenic fungi biocontrol .
The above-stated data reported by different scientists about various mechanisms of plant growth promotion explain why some of the endophytes from black saxaul can stimulate the growth of cucumber plants in salt stress conditions.
The strains B. amyloliquefaciens HAPH2, P. endophytica HAPH5, B. subtilis HAPH7, B. toyonensis HAPH8, H. sulfidaeris HAPH11, I. halotolerans HAPH12, P. soli HAPH15, and P. kilonensis HAPH16 isolated from tissues of black saxaul demonstrated a high plant growth-promoting activity of cucumber in four soil salinity levels (0, 25, 50, and 100 mM) after seeds’ inoculation. It can be explained that salt-tolerant bacteria can live inside halophytic plants, which do not lose their activity in high salt concentrations. These strains were able to fix nitrogen, solubilize phosphates, and produce IAA, ACC deaminase, and siderophores which are considered as the main plant growth-promoting traits. After field experiments, the best plant growth promoters can be used as bioinoculants for plants growth improvement in soil salinity conditions.
6. AUTHORS’ CONTRIBUTION
VS and BA carried out the experiments. VS analyzed data. BA statistically analyzed results. KD and VS wrote the draft of the manuscript. KD conducted the critical revision of the manuscript. ZI worked out the concept and design and supervised and funded the experiments. All the authors contributed equally to this manuscript and agreed to submit it for publication.
There is no funding to report.
8. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
9. ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
10. PUBLISHER’S NOTE
This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
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