Characterization, enzymatic and biochemical properties of endophytic bacterial strains of the medicinal plant Ajuga turkestanica (Rgl.) Brig (Lamiaceae)

2.1 Sample collection

Ajuga turkestanica (Rgl.) Brig (Lamiaceae) grows on stony slopes of gypsum and red sandstone, soil and rock mixed areas at an altitude of 600–1000 m on the south-western slopes of the Pamir-Alay mountains of Boysun district of Surkhandarya region (Fig. 1).

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Fig. 1

Map of the area where the medicinal plant A. turkestanica is collected. Boysun district of Surkhandarya region Uzbekistan.

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2.2 Surface sterilization of plant samples

Surface sterilization was performed using Miral and Verma methods to isolate endophytic microorganisms from the medicinal plant A. turkestanica (Verma et al., 2014). Plant samples prepared for surface sterilization were cut into small pieces using sterile tweezers. Plant parts prepared for surface sterilization were washed in running water for fifteen minutes. The washed plant parts were then washed with copious amounts of distilled water. In the next step, plant samples were transferred to a laminar box (Thermo scientific Maxisafe 2020 Germany) provided with aseptic conditions. Plant parts (roots, stems, leaves) were cut into 3–5 cm pieces to facilitate surface sterilization and sterilized for one minute in 70% absolute ethyl alcohol. At the stage of surface sterilization of plant samples are stored in a solution of hypochorite sodium (NaClO) five percent. Also, plant parts divided into small volumes are thoroughly washed in sterile deionized water. To determine how successful the sterilization process was, a pre-prepared Chapek doks agar medium was dripped from the last washed water using a sterile glass rod. If no microorganisms grow from the water droplet medium it confirms that the surface sterilization process was positive.

2.3 Isolation and identification of endophytic bacterial strains

In order to isolate endophytic bacteria from plant specimens the pieces which were initially cut into small pieces, were transferred to Petri dishes containing sterile tweezers Tryptic Soy Agar medium (NaCl 5.0 g;Dextrose 2.5 g; K₂HPO₄ 2.5 g; Agar 15.0 g). Plant specimens were incubated at 28° C for ten to fifteen days after transfer to medium. After incubation, morphologically diverse bacterial colonies were selected and their morphological microscopic and biochemical properties were examined. The isolated bacterial isolates were grown overnight in the Tryptic Soy Broth medium. Cultured endophytic bacterial cultures are centrifuged at a rate of 15,000 revolutions per minute. The supernatant portion of the culture fluid was drained and a precipitate was removed for processing in subsequent stages. The separated precipitate was first washed using 250 µl of sterile deionized water and then 800 µl of ethyl alcohol. The resulting clutoral fluid was again centrifuged at a rate of 15,000 beats per minute to remove the supernatant portion. The precipitate obtained was then dried in a laminar cabinet under aseptic conditions for 5–7 min. The precipitate was then mixed by adding 25 μL of formic acid and acetonitrile. The resulting mixture was then centrifuged again and a total of 1 μL of supernatant was placed on a 96-point tablet and dried. One microliter of MALDI MS solution was then poured into the samples and allowed to dry under aseptic conditions. MALDI TOF MS mass spectra were analyzed using bacterial identification system MALDI Biotyper®. Each sample was analyzed three times to confirm the reliability of the results. The control was carried out on the relevant spectra of existing strains in the collection of sanitary and epidemiological control of the Main Department of Medicine under the Presidential Administration of the Republic of Uzbekistan. According to the results obtained, the bacterial identification scores are as follows: ≥2000 species-level identification means 1700 1999 species-level identification. If the score is <1700, it means that the isolate is not identified.

2.4 Morphological characterization of endophytes

After incubation, the endophytic bacteria isolated from plant parts were purified according to their phenotypic properties. The morphology of endophytic bacterial isolates (color of bacterial colonies, gram staining, shape of colony margins, growth dynamics and nature of colonies) was studied according to Berge’s (Bergey and Holt, 2000, Sayed et al., 2019). The gram method was used for morphological analysis of endophytic bacteria. Reagents used in staining of bacterial isolates by Gram reaction: (crystal violet (basic dye), iodine solution (purple color stabilizer in cell wall, color decolorization of samples (ethanol 70%) and distilled water).

2.5 Biochemical properties of endophytic bacterial isolates

Endophytic bacteria were grown in Simmons citrate agar medium and incubated for 24 h at 28 ± 2 °C. If the bacterial isolates have the property of assimilating citrate the medium color will change from green to blue. Such a change by endophytic bacteria is a positive reaction.

The carbohydrate fermentation property of the isolated bacterial isolates was determined by cultivation in Triple Sugar Iron agar medium (3.0 g beef extract; 20.0 g peptone; 3 g yeast extract; 10.0 g sugar; 10.0 g lactose; 1.0 g dextrose; 0.2 g ferrous sulfate; 5.0 g sodium chloride, 0.3 g sodium thiosulfate; 0.024 g phenol red and 12.0 g agar). Bacterial strains were incubated at 30 ± 2 °C for one day in TSI (Triple Sugar Iron agar) medium. The medium in which endophytic bacteria are grown exhibits an alkaline / acidic state. As a result, if the slope of the medium inside the test tube is red and the bottom is yellow, it means that the bacteria have only carried out dextrose fermentation. When endophytic bacteria grow in a medium acidic state, the slope and bottom of the medium turn yellow. This indicates that the bacteria carried out the fermentation of dextrose, lactose and sucrose.

When endophytic bacteria form a medium alkaline medium in which they grow, the slope and bottom of the medium inside the test tube remain unchanged. This result indicates that carbohydrate fermentation has not taken place. The blackening of the medium inside the solution indicates that hydrogen sulfide was produced by the bacterial isolates. The division of the medium and its rise in the solution indicates that the endophytic bacteria have formed a gas (Patel et al., 2016, Singh and Dubey, 2020).

Bacterial strains were incubated in Glucose Phosphate Broth (Buffered peptone 7.0 g; dextrose glucose 5.0 g; dipotassium phosphate 5.0 g; final pH 6.9 ± 0.2) for two days at 28 °C. At the end of the incubation period, 1 mL of bacterial fluid was transferred to a fresh sterile solution. Then 1–2 drops of methyl red solution were added to the solution. The rapid formation of a red color in the bacterial fluid when a methyl red solution was added was taken as a positive reaction.

2.6 Screening of plant growth promoting

Endophytic bacteria have been isolated from various parts of the A. turkestanica plant which is listed as an endemic species of flora of Uzbekistan. These endophytic bacteria have been found to have plant-enhancing properties used in sustainable agriculture.

The ability of endophytic bacterial strains to break down phosphate salts was studied in the Pikovskaya agar medium (C₆H₁₂O₆ 10.0 g; Ca₃(PO₄)₂ 5.0 g; yeast extract 0.5 g; (NH₄)₂SO₄ 0.5 g; KCl 0.2 g; NaCl 0.2 g; MgSO₄ 0.1 g; MnSO₄·H₂O, 0.002 g; FeSO₄·7H₂O, 0.002 g agar 15 g; distilled water 1 L). Bacterial strains were stored for ten days at a temperature of 28 ± 2 °C after planting in agar medium. Bacterial colonies were monitored during incubation. Bacteria break down phosphate compounds by producing organic acids into the medium. The yellow color formed around the bacterial colony indicates that the phosphate compounds are broken down (Ogale et al., 2018; Jain et al., 2017).

Endophytic bacteria were grown in King’s B medium (Proteose peptone 20.0 g; K₂HPO₄ 1.5 g; MgSO₄‧7H₂O 1.5 g; distilled water 1 L) in an incubator at a temperature of 28 °C in a shaker with a rotational speed of 170 times per minute for 72 h The bacterial fluid was then taken in 1.5 mL small-volume solutions and centrifuged at 6000 rpm for ten minutes. Take 1 mL of the liquid from the sepernatant portion and add 2% FeCl₃ from the aqueous solution. The appearance of orange and reddish-brown color in the solutions indicates that endophytic bacteria have synthesized siderophores.

Ammonia-synthesizing properties of bacterial strains were screened in a peptone medium. Bacterial cells were inoculated with peptone water without control. To assess the ammonia production of bacterial strains, 1 mL of Nessler reagent was added to a liquid medium with peptone. A change in color to light yellow indicates that minimal amounts of ammonia have been produced. A change from orange to brown indicates that the maximum amount of ammonia has been produced (ALKahtani et al., 2020; George et al., 2020).

The potential for IAA synthesis of bacterial strains was determined in the tryptophan Nutrient Broth medium. Bacterial strains were grown in a shaker incubator for 7 days at a temperature of 28 ± 2 °C after inoculation in the medium. At the end of the storage period, the bacterial fluid in which the strains were grown was centrifuged for ten to fifteen minutes at a speed of 6000 rpm. In the next step, the supernatant was isolated and 1 drop of Salkowski reagent (35 mL of 35% HCIO₄ and 1 mL of 0.5 M FeCl₃·6H₂O) was added to it. After the reagent was added to the supernatant, it was kept in a dark place for thirty minutes. The gradual development of cherry red in the supernatant indicated that bacterial strains synthesized indole acetic acid (Ogale et al., 2018).

Nitrogen fixation properties of endophytic bacteria were determined in Jensen’s medium (Sucrose 20 g;K₂HPO₄ 1.0 g; MgSO₄‧7H₂O 0.5 g; NaCI 0.5 g; FeSO₄‧7H₂O 0.1 g; Na₂MoO₄ 0.005 g; CaCO₃ 2.0 g; Agar 15.0 g). Bacteria cultured in Jensen’s medium were incubated for 7 days at 28 ± 2 °C. The appearance of endophytic bacterial colonies in Petri dishes during incubation indicates that bacterial cultures have the property of nitrogen fixation.

Endophyte bacterial strains were cultured in Nutrient Broth medium to test the activity of ACC deaminase. The cultured bacterial cultures were centrifuged at 8000 rpm for 8 min. Bacterial sediment was isolated and washed with sterilized 0.1 M Tris-HCl (pH 7.5). Bacterial cells were transplanted into a medium of minimal saline agar. A medium with the addition of 0.2 percent ammonium sulfate was used for positive control. Non-nitrogen source DF medium was used as a negative control. The cultures were incubated for 36 h at 28° ± 2. The occurrence of endophytic bacterial colonies in DF minimal saline medium was evaluated as a positive control (Sánchez-Cruz et al., 2019; Ngoma et al., 2013; Mintoo et al., 2019).

To determine the amylolytic activity of endophytic bacteria, starch agar was incubated for 48 h at 28 ± 2 °C. At the end of the incubation period of bacteria, a solution of potassium iodide was poured into Petri dishes and soon drained. In the presence of amylase activity in endophytic bacterial strains, a colorless transparent zone is formed around the bacterial colonies (Ntabo et al., 2018; Malleswari and Bagyanarayana, 2013).

The protease activity of endophytic bacteria was determined in sterile Skim Milk Agar medium (Casein 5.0 g; glucose 1.0 g; yeast extract 2.5 g; Agar 15.0 g; distilled water 1 L, added 7% skim milk). Bacterial strains were stored for two days at a temperature of 28 ± 2 °C after inoculation on the medium. Degradation of proteolytic activity around bacterial colonies after incubation was detected. A transparent zone around the bacterial colonies indicated that the substrate was broken down by proteases (Ntabo et al., 2018).

Lipase and lecithinase activity of bacterial strains was determined by growing in Egg Yolk Agar. Bacterial strains were inoculated and stored at a temperature of 30 ± 2 °C for two to three days.The formation of light zones around bacterial colonies after incubation indicates that lecithinase is synthesized by bacteria. The lipase activity of endophytic bacteria was determined as follows. A saturated solution of copper (II) sulfate was poured into the petri dishes and kept for 10–15 min and the excess reagent was discarded (Malleswari and Bagyanarayana, 2013). A greenish-blue color is formed around bacterial colonies that have lipase activity. This condition is considered a positive result.

A drop of endophytic bacterial culture grown in a nutrient broth medium was instilled into a glass plate and 3% hydrogen peroxide (H₂O₂) was added. Cultures with catalase activity form bubbles.

Whatman №1 filter paper was cut into 10 mm circles and immersed in a freshly prepared 1% solution of tetramethyl paraphenyldiamine dihydroxychloride (C₁₀H₁₈Cl₂N₂) and dried. A small amount of endophytic bacterial colony was then instilled into the filter paper with a sterile Petri loop. The formation of a strong dark purple color in five to ten seconds was accepted as a positive reaction of the oxidase test. Whatman №1 filter paper color change was taken as a negative reaction.

Urea is a product of the decarboxylation of amino acids. Hydrolysis of urea results in the formation of ammonia and carbon dioxide. The formation of ammonia in the process alkalizes the medium and a change in pH (6.8–8.1) turns the light yellow to pink. When bacteria have urease activity, the medium turns light yellow to pink within 24 h. (Patel et al., 2016). Bacteria were cultured in Christensen medium to determine the urease enzyme activity of endophytic bacteria. Urea decomposes at high temperatures that are not resistant to heat. Therefore, a 40% solution of urea under aseptic conditions was prepared. 2 g urea was filtered in 5 mL of distilled water in a 0.22 nm millipore paper filter. After sterilization, the medium was cooled to 45–50° and 5 mL sterile 40% urea solution was added under aseptic conditions and poured into Petri dishes. Bacterial strains were transplanted into medium and stored in a thermostat at 28 ± 2 °C for 24 h. When bacterial strains have urease activity the urea in the medium is broken down. The ammonia formed was determined by changing the color of the medium from light yellow to pinkish red.

The property of gelatin hydrolysis of bacterial isolates was determined in Nutrient gelatin (Nutrient agar 23 g/L, gelatin 8 g/L) agar medium. Bacterial strains were grown for three to seven days at a temperature of 30 ± 2 °C in a gelatin agar medium. After incubation, bacterial isolates were poured into saturated Petri dishes from a saturated solution of ammonium sulfate (NH₄)₂SO₄ and kept for 8–10 min, then discarded. Ammonium sulfate precipitates gelatin that has not been hydrolyzed by bacterial isolates and zones are formed around the bacterial colonies (Gamalero et al., 2020).

3.1 Morphological properties of bacterial strains

Twelve strains of bacteria were isolated from the medicinal plant A. turkestanica (root − 7, stem − 3, leaf − 3). Endophytic bacteria are white, yellow, whitish, yellowish in color and consist of flat and ascending colonies 1–2 mm in size. The morphological and microscopic appearance of endophytic bacteria is shown in Fig. 2, the morphological characteristics in Table 1.

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Fig. 2

Macroscopic and microscopic view of endophytic bacterial strains.

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3.2 Biochemical and plant growth promoting properties of endophytic bacteria

The ability to ferment carbohydrates and enzymatic activities (amylase, lipase, lecithinase, caseinase, protease, gelatinase, urease, catalase, oxidase) and plant growth promoting traits (phosphate solubility property, ACC production, siderophore production, indole acetic acid production) were studied. A citrate test is used to determine the ability of microorganisms to use citrate as a carbon source. Pseudomonas kilonensis, Bacillus amyloliquefaciens ssp. bacterial strains of plantarum, Pseudomonas putida, Pseudomonas graminis, Siccibacter colletis, Pseudomonas chlororaphis showed positive results in citrate use test (Table 2 and Fig. 3).

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Fig. 3

Plant Growth promoting properties of the bacterial strains. A – Detection of phosphate solubilisation. B – Detection of ammonia production. C – Detection of IAA production. D – Detection of siderophore production. E – Detection of carbohydrate fermentation. F Detection of methyl red test.

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The following results were obtained when studying the carbohydrate fermentation properties of endophytic bacteria. Bacterial endophytes P. kilonensis, B. subtilis ssp, B. amyloliquefaciens ssp. plantarum performed only glucose fermentation. Bacillus amyloliquefaciens ssp. plantarum, P. putida, P. graminis, S. colletis, B. amyloliquefaciens, B.subtilis ssp, B. mojavensis, P. chlororaphis, B.amyloliquefaciens ssp. plantarum endophytic bacterial isolates have been found to carry out the fermentation of glucose, lactose and sucrose (Table 2).

The acid production potential of microorganisms during glucose fermentation is determined by methyl red test screening. Some endophytic bacteria synthesize the final products of glucose when they use glucose. The results showed a positive result of S. colletis bacterial endophyte. Other bacterial isolates presented a negative result. The following results were obtained when the amylolytic activity of endophytic bacteria was examined. Bacterial endophytes P.putida, B. subtilis ssp, P. graminis, B. amyloliquefaciens ssp. plantarum, S. colletis, B. amyloliquefaciens, B. subtilis ssp., B. mojavensis and P. chlororaphis were found to have high amylolytic activity. The results obtained showed high protease (caseinase) activity of B. subtilis ssp., B. amyloliquefaciens, B. subtilis ssp.and B. mojavensis bacterial endophytes. Enzymatic screening revealed B. subtilis, B.amyloliquefaciens, B. mojavensis strains with lipase and lecithinase activity. (Table 3 and Fig. 4).

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Fig. 4

Enzymatic activity of the bacterial strains. A – Detection of citrate utilization. B – Detection of amylase activity. C – Detection of proteolytic activity. D – Detection of urease activity. E – Detection of lecithinase and lipase activities.

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All bacterial endophytes of the medicinal plant A.turkestanica showed a positive oxidase test result. The results of the scientific research showed that the bacterial endophytes P. kilonensis, B. amyloliquefaciens ssp. plantarum, B. mojavensis and P.chlororaphis exhibit urease enzyme activity. Gelatin hydrolysis is a two-step process. The enzyme gelatinase hydrolyzes gelatin and results in the formation of polypeptides. Polypeptides are converted into amino acids and used by the cell during metabolism. When the properties of gelatin hydrolysis of endophytic bacteria were studied B. amyloliquefaciens ssp. plantarum, B. amyloliquefaciens, B. subtilis ssp., B. mojavensis and B. amyloliquefaciens ssp. plantarum gave a positive result on gelatin hydrolysis (Table 3).

Among these bacterial endophytes yellow zones were formed in medium B. amyloliquefaciens ssp. plantarum, S. colletis, B.amyloliquefaciens, B. subtilis ssp., and B. mojavensis. This result confirmed that the above five bacterial endophytes have phosphate dissolving properties. Bacterial strains in the DF saline medium were found to synthesize the enzyme ACC deaminase. The results showed that the bacterial isolates P. kilonensis, P.putida, S. colletis and P.chlororaphis have significantly higher property of forming the enzyme ACC deaminase (Table 3 and Fig. 4).

The synthesis of IAA in endophytic bacterial strains was studied. P. kilonensis, B. amyloliquefaciens ssp. plantarum, P. putida, P. graminis, S. colletis, B. amyloliquefaciens and P. chlororaphis showed a positive result. When 2–3 drops of Salkovsky reagent were added to the culture fluid in which endophytic bacteria were grown, a cherry red color was formed in the culture fluid. The bacterial strains identified during our scientific study were found to have the property of assimilating free nitrogen in the air (Table 3).