Phytomicrobiome of Helianthus annuus: in vitro assessment of plant growth-promoting traits and heavy metal tolerance

Abstract

Endophytic bacteria are usually found within plant tissues. They enhance plant growth, with potential agricultural and environmental applications. They might enhance the plant tolerance to abiotic stresses and inhibit the metal toxicity within metal hyperaccumulator plants. The majority of endophytic bacteria possess plant growth-promoting features. They secrete secondary metabolites that improve plant growth and physiological functions. Twelve endophytic bacteria strains were isolated from the root, stem and leaves of Helianthus annuus plant grown in contaminated soil with the heavy metals lead, zinc and chromium. The isolates were identified and characterized by colony morphology, biochemical tests including gram, spore staining, catalase, starch hydrolysis, hydrogen cyanide and auxin production, phosphate solubilization and nitrogen fixation, along with 16S rRNA gene-based molecular identification of bacteria. Five bacterial isolates showed positive results in all Plant growth-promoting traits. The molecular identification through amplification of bacteria’s 16S rRNA using PCR has shown eight Bacillus spp., with two Acinetobacter spp., Providencia vermicola and Enterobacter cloacae. The findings highlight the diversity of endophytic bacteria associated with the COH3 variety of Helianthus annuus plant grown in HM-contaminated soil and evaluate their plant growth-promoting and heavy metal tolerance under in vitro conditions. Further studies are required to evaluate their efficiency under field conditions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08905-z.

Key message

This study highlights the diversity of the endophytic phytomicrobiome in Helianthus annuus. Their plant growth-promoting features and heavy metal tolerance indicate strong potential for use as ameliorating agents to enhance heavy metal tolerance in further field studies.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08905-z.

Introduction

For decades, researchers focused on finding eco-friendly solutions to the environmental problems facing the Earth, particularly those resulting from the industrial revolution and the unplanned disposal of industrial waste, in addition to the excessive use of agricultural chemicals [1].

Heavy metal pollution is one of the most serious contemporary environmental problems [2]. Heavy metals are known for their high atomic weight and high density [3]. Some heavy metals are naturally present in the biosphere, while others are released as a result of human activities [4]. The biological importance of some heavy metals such as iron, zinc, copper, chromium and manganese cannot be neglected due to their roles in enzyme activities, hormone function and receptor sites, but only at low concentrations [5]. Negative effects appear when heavy metal concentrations increase and accumulate in the food chain [6, 7]. The real-world contamination levels of Zn, Pb and Cr range from 150—5000, 20—1000 and 0.5—3950 mg/kg, respectively. While the permitted limits of HMs in drinking water are 3, 0.01and 0.003 mg/L of Zn, Pb and Cr, respectively [8, 9]. The risks of such heavy metals outweigh their benefits [4]. Environmental pollution with lead, thallium, cadmium, and antimony is evident as a result of industrial emissions [6]. The risk of cancer increases with repeated and high exposure to chromium and antimony [10]. Mental deformities appear in children poisoned with lead. Itai-itai disease results from cadmium poisoning, and Minamata disease is caused by mercury poisoning [6]. Heavy metals also have neurotoxic, nephrotoxic, hepatotoxic, and genotoxic effects on the human body [10]. Therefore, it is recommended to avoid industrial areas with high emissions of such metals [11–13]. Eco-friendly solutions such as bioremediation and phytoremediation have contributed to the remediation of heavy metals contaminated soil and water [5, 14–16].

Some plants, such as Helianthus annuus [17] and Brassica juncea [18] were considered as hyperaccumulators. Some bacterial and fungi species like Aerococcus sp. and Rhodopseudomonas palustris [19] Penicillium citrinum [20] Trametes versicolor [21] and Aspergillus sydowii [22] are also able to rid the water and soil of organic and inorganic pollutants because of their ability to metabolize the pollutants by secreting their extracellular and intracellular enzymes with the bioremediation technique [23, 24]. Microorganisms can convert the pollutants into their simple components which are carbon dioxide, water and less or nontoxic compounds [25]. Mustard is recognized by its ability to accumulate heavy metals through phytoextraction and phytostabilization mechanisms [26, 27]. However, Helianthus annuus accumulates heavy metals through a phytoextraction mechanism and can uptake a wider range of heavy metals in addition to removing organic contaminants [28]. The Helianthus annuus plant was selected for this study due to the widespread availability of its seeds, which are rich in high-quality oil. Helianthus annuus seed oil is nutritionally valued for its notable quantities of unsaturated fatty acids, including linolenic and linoleic acids, which assist in lowering cholesterol levels and preventing artery fat clots. Additionally, it contains significant vitamins such as A, D, E, and K. It is widely recognized as the fourth most important oil seed crop globally, following soybean, oil palm, and canola. Helianthus annuus seeds are a nutrient-dense food, containing elevated amounts of protein, fiber, minerals, and phenolic compounds [29, 30]. COH3 (CSFH 12205) variety of Helianthus annuus was chosen because of the limited research availability on it. This variety has a unique genetic makeup that provides it with notable resistance to leaf spots, necrosis, and powdery mildew caused by fungi. Additionally, Helianthus annuus is a high yielding crop with high oil content suitable for cultivation in Tamil Nadu state. A better understanding of this variety could support the development of innovative breeding techniques used to create more resilient, high-yielding verities with the capacity to hyperaccumulate heavy metals and soil pollutants [31].

The endophytic bacteria present in the tissues of hyperaccumulator plants may also possess the ability to resist heavy metals due to the growth capacity within the tissue containing heavy metals [32, 33], as well as possessing the plant growth-promoting features [34, 35], which contributes to improving nutrient uptake [36]. Additionally, it helps the plant in stress tolerance by maintaining plant growth regulators balance, nutrient level, increase number of leaves, photosynthetic yield and pigments such as chlorophyll and carotenoid content, and improving the antioxidant defence system [15, 37, 38].

IAA production, Nitrogen fixation and solubilization of phosphate are plant growth-promoting traits that can protect the plant from pathogens and increase root distribution and crop yield. they also enhance abiotic stress tolerance [39]. Several genera of endophytic bacteria have been documented as plant growth-promoting such as Enterobacter, Rhizobium, Bacillus, Pantoea, and Klebsiella [40, 41].

Several species of endophytic bacteria have been isolated from some Helianthus annuus varieties and the ability of those endophytic bacteria to enhance plant growth has been verified by testing plant growth-promoting features [1, 42, 43]. COH3 variety of Sunflower was released recently by the Tamil Nadu Agriculture University in 2019. It has high oil and yield content, with a resistance to several diseases and fungi [31].

This research was conducted to find out the endophytic bacterial species isolated from the internal tissues of Helianthus annuus COH3 variety that have been grown in heavy metal-contaminated soil and to screen their plant growth-promoting features and heavy metal tolerance.

Materials and methods

Sample collection and surface sterilization

The seeds of Helianthus annuus COH3 variety were purchased from Tamil Nadu Agricultural University, Coimbatore. The plant was grown in soil that had previously been contaminated with the heavy metals: lead, chromium and zinc (10, 10, 350) mg/kg, respectively, within the limits of the heavy metals pollution allowed according to WHO [8]. The applied heavy metal concentrations were selected to represent controlled exposure conditions while avoiding excessive artificial contamination. These conditions were intended to facilitate the isolation of tolerant endophytic bacteria rather than simulating highly polluted soils. The plant was thoroughly washed with tap water to get rid of soil residues followed by washing with distilled water. Surface sterilization of the isolated plant parts was performed using the method of [44]. Samples were washed with distilled water three times, followed by treatment in 0.1% Tween 20 in distilled water, sterilization with 70% ethanol for 5 min, 10% sodium hypochlorite for 10 min, and 10% sodium hydrogen carbonate for 15 min to confirm that the fungal growth in the samples is prevented. Finally, the samples were washed with autoclaved distilled water to remove the sterilizing agents. The effectiveness of surface sterilization was confirmed by plating aliquots of the final rinse water onto control LB agar plates in triplicate. This method effectively eliminated epiphytic bacteria and fungi from the samples’ surfaces, as demonstrated by the absence of all microbial growth on control plates.

Determination of soil texture and heavy metal content

The experimental soil was artificially supplemented with heavy metals and thoroughly homogenized to ensure uniform distribution before being transferred into pots. Soil physicochemical properties, including pH, EC, texture, and metal concentrations were measured to characterize the baseline properties of the homogenized substrate used in the experiment. Determination of soil texture and calcium carbonate was done by the method of [45]. The soil pH was determined in a 1:2.5 soil: water suspension (ratio) by using an electronic pH meter (Beckman 350 pH/temp meter, USA). To determine the total Pb, Cr and Zn, the soil samples were digested using a mixture of three acids: nitric acid (HNO3), perchloric acid (HClO4) and sulfuric acid (H2SO4) [46]. The Atomic Absorption Spectrophotometer (AAS) (Model No. HVO-AAS01-2100) was used to determine the content of heavy metals, with a detection limit of 0.01 mg/L. The absorption of light by free atoms was measured and then compared to the calibration standard curve of the elements to determine the concentrations of heavy metals in parts per million (ppm). All analyses were performed in triplicate.

Isolation of endophytic bacteria

On the sixtieth day of planting, the plant was in the flowering stage. Samples were collected from the leaves, stem, and roots of the plant to detect the endophytic bacteria species in the plant. Proper surface sterilization is done to ensure the disposal of epiphytic bacteria. Isolation of endophytic bacteria was performed by cutting each part of the plant into small pieces which are then crushed in a sterile mortar and pestle. Each tissue sample was serially diluted in autoclaved distilled water and spread on Luria Bertani media in triplicates. LB agar was mainly used for the streaking of endophytes as it presented improved results compared to nutrient agar in recent studies. A final wash water streaking on the control plates is done to ensure the absence of epiphytic bacteria. The samples were incubated for three days at 37 °C to give the endophytic bacteria enough time to grow. The isolates were then purified by repeated streaking on fresh LB media. The purified isolates were stored at 4 °C for further studies [42].

Morphological characterization

After 24 h incubation of the purified bacterial isolates grown on LB media, the morphological characteristics were recorded by comparing the shape, colour, size, elevation, margins and texture of the colony. In addition to the determination of (CFU/ml), Colony-forming units were calculated using the formula: CFU/ml = (Number of colonies × dilution factor)/plated volume.

Serial dilutions were prepared (10^–1- 10^–5) and the final dilution was plated in triplicate [42].

Biochemical characteristics of isolated endophytic bacteria

Several biochemical tests have been conducted for the identification of bacteria. Such as gram staining based on peptidoglycan content, spore staining to detect the endospores, catalase test by using hydrogen peroxide, starch hydrolysis based on amylase activity by using starch agar media and checking the halo clear zone around the colonies after 24 h incubation and applying the iodine for 30 s [44] and Methyl Red–Voges Proskauer test to check the fermentation pathway bacteria used to utilize glucose [47]. All biochemical tests were performed in triplicate.

Plant growth-promoting traits of isolated endophytic bacteria

Several plant growth-promoting features were checked in the bacterial isolates such as nitrogen fixation by streaking the isolates on nitrogen-free media and incubating them for 5 days at 28 °C [48]. Hydrogen cyanide production by streaking on nutrient agar containing 0.4% glycine, with the existence of filter paper soaked before in 2% sodium carbonate in 0.5% picric acid, followed by incubation of isolates for 4 days at 28 °C [49]. Auxin production was performed by using Tryptophan broth culture kept in a shaking incubator for 2 days at 28 °C, and then applying Salkowski reagent (1:1) to the supernatant of centrifuged bacterial broth culture [50]. Phosphate solubilization was detected by streaking on Pikovskaya's agar media after 5 days of incubation at 28 °C. Clear halo zones were observed in positive samples [51, 34].

Identification of isolated phytomicrobiome by 16S rRNA

The DNA of the purified bacterial cultures were isolated and their quality was evaluated using 1% agarose gel electrophoresis; a single band of each bacterial DNA sample of high-molecular-weight DNA was observed. After the PCR amplicons purification, both forward and reverse DNA sequencing reaction were performed with 16S rRNA-F and 16S rRNA-R primers by using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. The consensus sequence of the 16S rRNA gene was generated from forward and reverse sequence data using aligner software. The 16S rRNA gene sequence was used to carry out a BLAST of the NCBI GenBank database. Based on the maximum identity score, the first ten sequences were selected and aligned using the multiple alignment software program Clustal W. The evolutionary history was concluded using the Maximum Likelihood method and the Tamura-Nei model. The phylogenetic tree with the highest log likelihood (−2048.75) is shown. The Codon positions included were 1 st + 2nd + 3rd + Noncoding. There were a total of 1490 positions in the final dataset. The phylogenetic tree was constructed using MEGA version 12.0.10 (64-bit) employing the Neighbour-Joining method with 1000 bootstrap replicates and computing evolutionary distances.

Heavy metal tolerance of the isolated endophytes

The heavy metal tolerance was determined by streaking the isolated endophytes on sterile LB agar. The heavy metals were prepared in different concentrations (100 µg/ml – 2000 µg/ml). Using the agar diffusion method, the minimum inhibition concentration was recorded for each isolate with lead, chromium and zinc when the endophytic bacteria failed to grow. Wells of 6 mm diameter were filled with 100 µL metal solution and the plates were kept for 1 h to allow initial diffusion before incubation. Then, the plates with uniform agar depth of 4 mm were incubated at 37 °C for 24 h. Growth inhibition was determined by measuring the inhibition zone diameter. Each treatment was performed in triplicate across three independent experiments (n = 9). Results were expressed as mean ± SD and analyzed using one-way ANOVA followed by Tukey test (p < 0.05). Growth inhibition criteria were classified based on zone diameter [52].

Results

The soil physicochemical analyses were conducted on the homogenized experimental soil mixture used for pot experiments. As the soil was artificially prepared and homogenized before use, the reported values represent the baseline characteristics of the experimental substrate rather than spatial variability typically observed under field conditions. The chemical and physical properties of the studied soil are shown in (Table 1). A total of 12 bacterial species were isolated from the leaf, stem and root of Helianthus annuus COH3 variety at the reproductive stage on day sixty of the plant life cycle. Out of a total of 12 isolates, 3 bacterial strains were obtained from leaf tissues, 4 strains from stem tissues and 5 strains from root tissues (Fig. 1). The successful isolation and growth of distinct viable bacterial colonies following sterilization confirmed the preservation of internal endophytic bacteria while eliminating surface-associated microorganisms, as confirmed by control plates.

Table 1.

Analysis of the homogenized experimental soil mixture was performed in triplicate

Parameter	Value
pH (1:2.5)	5.37
Soil texture	Silty clay (57% Silt, 43% Clay)
Cr (mg/kg)	7.9
Pb (mg/kg)	8.6
Zn (mg/kg)	273
Electrical conductivity (dS/m)	3.7
Organic matter (g/kg)	16.9
CaCO3 (g/kg)	13.4

Fig. 1.

The endophytic bacterial isolates from Helianthus annuus COH3 variety. A L: source plate of leaf isolates. L1, L2 and L3 are purified isolates from leaf. S: source plate of stem isolates. S1, S2, S3 and S4 are purified isolates from stem. R: source plate of root isolates. R1, R2, R3, R4 and R5 are purified isolates from root. B Results of biochemical tests and plant growth-promoting features

The bacterial isolates appear to have different morphological characteristics in shape, texture, elevation, margins, colony size and colour (Table 2). The isolated bacteria have shown different shapes of colonies, colours, margins, elevation and texture, i.e., Rhizoid, Lobate, Circular and irregular shape, white to creamy and yellow colours, entire to lobate and filamentous margins. Regarding the biochemical tests, 8 strains were gram-positive, and the remaining were gram-negative. In endospore staining, 7 strains were positive, and the remaining were negative. In the catalase test, 7 strains were positive, whereas 11 strains were positive in the starch hydrolysis test. In the MR test, 8 strains of the isolates were positive, while in the VP test, all the isolates showed negative results (Table 2).

Table 2.

Morphological and biochemical characteristics of the isolated endophytic bacteria

Plant part	Isolate Code	Colony Morphology						Biochemical Characteristics
		Shape	Elevation	Texture	Margins	Colour	CFU/ml	Gram reaction	Spore staining	Catalase Test	Starch hydrolysis	MR Test	VP Test
Leaf	L1	Rhizoid	Flat	Rough	Undulate	White	36 × 106	+ Rods	+	+	+	-	-	Bacillus sp.
	L2	Irregular	Flat	Smooth	Entire	Creamy	31 × 106	+ Rods	+	+	-	-	-	Bacillus amyloliquefaciens
	L3	Irregular	Convex	Smooth	Lobate	Creamy	23 × 106	+ Rods	+	-	+	-	-	Bacillus tropicus
Stem	S1	Circular	Flat	Rough	Filamentous	White	41 × 106	_ Rods	-	+	+	+	-	Providencia vermicola
	S2	Circular	Flat	Smooth	Erose	White	14 × 106	+ Rods	+	-	+	+	-	Bacillus thuringiensis
	S3	Circular	Convex	Smooth	Curled	White	31 × 106	+ Rods	+	+	+	+	-	Bacillus subtilis
	S4	Circular	Flat	Smooth	Entire	White	36 × 106	+ Rods	-	-	+	+	-	Bacillus megaterium
Root	R1	Circular	Flat	Smooth	Lobate	Creamy	11 × 106	_ Rods	-	-	+	-	-	Acinetobacter baumannii
	R2	Irregular	Flat	Smooth	Lobate	white	41 × 106	_ Rod	-	+	+	+	-	Enterobacter cloacae
	R3	Irregular	Convex	Rough	Curled	White	31 × 106	+ Rods	+	+	+	+	-	Bacillus paramycoides
	R4	Circular	Flat	Smooth	Erose	Creamy	23 × 106	_ Rods	-	+	+	+	-	Acinetobacter lwoffii
	R5	Irregular	Flat	Smooth	Lobate	White	15 × 106	+ Rods	+	-	+	+	-	Bacillus cereus

Morphological and Biochemical characteristics of the isolated endophytic bacteria. (L).- endophytes isolated from leaf, (S).- endophytes isolated from stem, (R).- endophytes isolated from root

Abbreviations: mm millimeter, CFU/mL colony forming units per milliliter, MR Methyl Red, VP Voges-Proskauer, + positive result, - negative result

Regarding the plant growth-promoting feature tests, 10 bacterial strains were able to fix nitrogen. Only 5 isolates were able to produce hydrogen cyanide which is regulated by specific conditions, such as pathogen-rich environments, to inhibit pathogenic fungi or other microbes in the rhizosphere. By contrast, all the isolates were able to produce auxin (IAA), and the visible halos around the colonies of 11 bacterial strains proved their ability to solubilize phosphate (Table 3).

Table 3.

Plant growth-promoting features of isolated endophytic bacteria

Plant part	Isolate Code	Plant Growth Promoter tests
		Nitrogen Fixation	Phosphate solubilization	Auxin Production	HCN Production
Leaf	L1	+	+	+	+
	L2	+	+	+	-
	L3	+	+	+	+
Stem	S1	+	+	+	+
	S2	+	+	+	+
	S3	+	+	+	-
	S4	-	+	+	-
Root	R1	-	+	+	-
	R2	+	+	+	-
	R3	+	+	+	-
	R4	+	-	+	-
	R5	+	+	+	+

The table presents differences in the abilities of the isolated bacterial species on nitrogen fixation, phosphate solubilization, Auxin and hydrogen cyanide production

Abbreviations: HCN Hydrogen cyanide, + positive result,—negative result, L endophytes isolated from leaf, S endophytes isolated from stem, R endophytes isolated from root

After the morphological and biochemical studies on the isolates, gene characterization was performed for the endophytes.

Based on the highest similarity in nucleotide homology and phylogenetic tree, the tested samples were identified as follows: L1, Bacillus sp.; L2, Bacillus amyloliquefaciens; L3, Bacillus tropicus; S1, Providencia vermicola; S2, Bacillus thuringiensis; S3, Bacillus subtilis; S4, Bacillus megaterium; R1, Acinetobacter baumannii; R2, Enterobacter cloacae; R3, Bacillus paramycoides; R4, Acinetobacter lwoffii; and R5, Bacillus cereus (Fig. 2). The heavy metal tolerance of the endophytic bacterial isolates was evaluated by determining their minimum inhibitory concentrations for lead, chromium, and zinc under in vitro conditions. All isolates demonstrated the ability to grow in the presence of at least one of the tested metals, although the tolerance levels varied considerably among strains. Overall, the isolates exhibited the highest tolerance to Pb, followed by Zn, while Cr showed the strongest inhibitory effect. The MIC values ranged from 200 to 1600 µg/ml for Pb, 100 to 1000 µg/ml for Zn, and 100 to 900 µg/ml for Cr. Among the tested isolates, Bacillus amyloliquefaciens showed the highest tolerance, sustaining growth at concentrations up to 1600 µg/ml for Pb, 1000 µg/ml for Zn, and 800 µg/ml for Cr. In contrast, isolates such as Bacillus tropicus and Bacillus megaterium exhibited a comparatively lower tolerance, with growth inhibition observed at lower concentrations. Fig. 3 shows the detailed heatmap distribution of MIC values across isolates and metals illustrating the relative tolerance patterns.

Fig. 2.

Phylogenetic tree based on 16S rRNA gene sequences showing the genetic relative relationship between the endophytic bacterial isolates from Helianthus annuus and closely related reference strains retrieved from the NCBI GenBank database. The tree was constructed using the neighbor-joining method, and bootstrap values of 1000 replicates are shown at branch points to indicate the confidence level of each node

Fig. 3.

Heatmap showing the minimum inhibitory concentration of lead, chromium, and zinc for the endophytic bacterial isolates obtained from Helianthus annuus. Each cell represents the MIC value in µg/ml of a given heavy metal for a specific isolate, defined as the lowest concentration at which visible bacterial growth was inhibited under the tested conditions. Color intensity represents MIC values with orange to brown colours indicating higher tolerance and blue to green colours indicating lower tolerance as shown in the scale bar. All measurements were performed in triplicates under identical experimental conditions

Discussion

Many studies incorporated the Helianthus annuus plant as it is a hyperaccumulator for heavy metals and can tolerate the heavy metal presence in plant tissues, in addition to its importance as an oilseed crop [17]. Helianthus annuus has a high biomass, giving it a broad tolerance to metals, including lead, cadmium, and zinc [53]. Helianthus annuus was selected for this study due to its high tolerance to heavy metals present in the soil used [54, 55].

Plant growth-promoting features may provide the plant tissues with some secondary metabolites that can contribute to plant adaptation under abiotic stress conditions [40, 39, 34, 56]. Indole acetic acid is an auxin that plays a crucial role in enhancing plant tolerance to abiotic stresses, such as stress responses. It primarily regulates root growth and structure for increased water and nutrient uptake by influencing gene expression through auxin response factors (ARFs). ARFs are DNA-binding transcription factors that regulate the expression of genes involved in stress tolerance, such as osmoregulation, antioxidant defense, nutrient uptake, and improved plant resistance to heavy metals [57, 58].

This study was performed to investigate the endophytic bacterial species isolated from the internal tissues of Helianthus annuus plants grown in soil artificially contaminated with heavy metals. 12 strains of endophytic bacteria were isolated from the internal tissues of the leaf, stem and root of the plant after surface sterilization to exclude the epiphytic bacteria. The successful isolation and growth of distinct viable bacterial colonies following sterilization confirmed the preservation of endophytic bacteria while eliminating surface-associated microorganisms, as confirmed by control plates. Since the 16S rRNA gene sequence for each bacterial species is specific, it can help in exact bacterial identification [59]. Three strains obtained from leaf tissues were identified as Bacillus sp., Bacillus amyloliquefaciens and Bacillus tropicus. Four strains from stem tissues were identified as Providencia vermicola, Bacillus thuringiensis, Bacillus subtilis and Bacillus megaterium. The five strains from the root tissues were Acinetobacter baumannii, Enterobacter cloacae, Bacillus paramycoides, Acinetobacter lwoffii and Bacillus cereus. Based on 16S rRNA gene sequence analysis, isolate L1 showed 99.93–100% similarity to the Bacillus cereus group, including Bacillus cereus, Bacillus thuringiensis, and Bacillus sp. Due to the high sequence similarity within this group, species-level identification could not be conclusively determined. Therefore, the L1 isolate was designated as Bacillus sp. (Supplementary Figure S1).

Geographical distribution, seasonal variation, and soil type lead to the presence or absence of some endophytes in plant tissues and might change their distribution among leaf, stem, and root tissues. Phytomicrobiome distribution is influenced by several factors, such as nutrient availability, soil pH, and temperature. Some microbes prefer acidic conditions, while others thrive in alkaline conditions. Bacterial species have varying ranges of optimal temperatures for growth. Soil texture and structure affect the soil’s ability to retain water, aeration, and nutrient availability. The amount and type of organic matter, along with essential elements such as nitrogen, phosphorus, and potassium, directly influence microbial growth and activity [60, 61]. Additionally, variations in heavy metal concentrations in plant tissues can affect the abundance and diversity of endophytic bacteria. Bacterial populations may decline in number as heavy metal concentrations increase unless they can tolerate heavy metals. Bacterial membranes and genetic material can be damaged by heavy metals, thus impairing their survival and reproduction [62].

The results of isolating endophytic bacterial species may differ depending on the type of isolation medium. In our study, isolation was tested using nutrient agar and Luria–Bertani agar. The results were more efficient using LB agar, which is consistent with previous studies [42, 44].

The 12 isolated endophytic bacteria exhibited varying biochemical traits that distinguish each bacterial species. Several strains demonstrated particular plant growth-promoting features, such as phosphate solubilization or nitrogen fixation, while others were characterized by their ability to produce hydrogen cyanide or auxin. Hydrogen cyanide production is a strain-specific trait. Unlike more common PGPR traits such as IAA production or phosphate solubilization, the availability of the hcnABC operon is required for HCN biosynthesis [63, 64].

The bacterial genera identified in this study, particularly Bacillus spp. and Enterobacter spp., have been widely reported as plant-associated endophytes with potential roles in metal tolerance and plant–microbe interactions. Previous studies have shown that some species within these genera are capable of tolerating or interacting with heavy metals through mechanisms such as biosorption, accumulation, or transformation [65–68]. However, it is important to note that such functional capabilities were not directly evaluated in the present study. Therefore, the current findings are limited to the in vitro characterization of metal tolerance, and further studies are required to investigate the functional roles of these isolates under in planta and environmental conditions.

The endophytic bacterial species isolated from the tissues of hyperaccumulator plants may have a role in stimulating plant growth and enhancing plant tolerance by secreting bioactive compounds that confer plant growth-promoting features. Therefore, the findings of this study contribute to understanding the heavy metal tolerance of these endophytes. Besides their beneficial effects, some endophytes are pathogenic, and caution must be exercised while using them in agricultural and environmental applications. For example, the use of Bacillus cereus in agriculture poses environmental risks, such as the potential for disease, toxin production that can cause food poisoning manifesting as diarrhea and vomiting, and environmental disturbance. It requires careful screening and risk assessment before use. Furthermore, the spread and distribution of the microbes cannot be limited once introduced into the environment, particularly for those capable of forming biofilms or producing environmentally resistant spores, which may pose long-term risks. This study showed that the endophytic bacteria enhance plant growth through various mechanisms, including nitrogen fixation, phosphate solubilization, and the production of auxins and hydrogen cyanide. This aligns with the results of previous studies [69–71]. Heavy metals were applied at moderate concentrations under controlled conditions to avoid excessive environmental contamination. These conditions were intended to allow the isolation of tolerant endophytes while maintaining environmentally relevant exposure levels. Furthermore, the use of moderate concentrations minimizes the risk of inducing acute toxicity that could inhibit microbial survival or distort natural plant–microbe interactions. This approach enables the isolation of relevant endophytes adapted to realistic exposure conditions. It is important to note that the aim of this study was not to simulate highly polluted environments, but rather to establish a controlled framework for the preliminary characterization of endophytic bacteria with metal tolerance. Future studies should evaluate the performance of these isolates under higher levels of contamination and in planta systems to further evaluate their functional potential.

Conclusion

This study showed the diversity of endophytic bacteria isolated from Helianthus annuus tissues. The findings suggest that the isolated endophytic bacteria possess traits that may contribute to plant growth promotion and heavy metal tolerance under controlled conditions. Further in planta studies are required to evaluate their ecological roles, their ability to enhance plant growth, and their potential applications under natural and stress conditions. Future research prospects are to investigate the application of isolated endophytes to other hyperaccumulator plants, verify the released secondary metabolites, and the deep mechanism of the phytomicrobiome in heavy metal tolerance.

Abbreviations

AAS

Atomic absorption spectroscopy

CFU

Colony-forming unit

HCN

Hydrogen cyanide

HM

Heavy metals

IAA

Indole-3-acetic acid

LB agar

Luria Bertani Agar

PCR

Polymerase chain reaction

PGPR

Plant growth-promoting rhizobacteria

Authors’ contributions

AK designed the study, acquired and analyzed the data and drafted the manuscript with figures and tables. IS gave valuable inputs and revised the manuscript. SB, GPKJ, MN and RG revised and edited the whole manuscript.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. The authors have no relevant financial or non-financial interests to disclose.

Data availability

The datasets supporting the conclusions of this article are included within the article and its additional files.

Declarations

Ethics approval and consent to participate

This article does not contain any studies performed by the authors on human participants or animals. The methods implemented were carried out in accordance with relevant guidelines and regulations. The authors confirm that SRM Institute of Science and Technology approved all experimental protocols.

Consent of publication

Not applicable.

Competing interests

The authors declare no competing interests.