Molecular Identification of Endophytic Bacteria from Silybum marianum and Their Effect on Brassica napus Growth under Heavy Metal Stress

Abstract

Silybum marianum has been used for a variety of purposes all over the world. This plant is used to treat cancer, liver disease, and inflammation. Eleven endophytic bacteria were isolated from S. marianum collected from heavy metal polluted sites and identified using 16s rRNA sequencing in the current investigation. Three isolates stood out for having many features, including heavy metal resistance, plant growth stimulation, plant hormones, heavy metal toxicity remediation, and antibacterial action. SJLC (6.06 µg/L) and SJRB (5.98 µg/L) were the greatest producers of IAA among the isolates and were identified as Bacillus sp. and Lysinibacillus sp., respectively. Root and shoot length have improved as a result of IAA production. The SJLC was found to be effective against four of the pathogens tested. The strain SJLC showed the most activity against Bacillus cereus with a 20 mm zone of inhibition, followed by the isolate SJRB, which showed a 16 mm zone of inhibition against B. cereus. The same isolates also show inhibition against X. campestris. Almost majority of the Brassica napus plants inoculated with bacterial isolates were able to translocate and degrade heavy metals like Cr. Therefore, it was concluded that these isolates are capable to grow in highly polluted environments.

1. Introduction

Endophytes are a group of microorganisms that have the capability of entering inside the plant hosts, inhabiting the intercellular spaces as well as the xylem vessels. They exist in a variety of tissue types within a wide range of plants, inhabiting the plant systemically with fungal or bacterial colonies and biofilms. They are ubiquitous and inhabit the majority of the plants. They have been isolated from almost all plants studied until now. Endophytes live symbiotically with plants. Although they grow inside the plants, they show no visible symptoms of disease or infection [1,2]. The close association between endophytes and internal tissues of the host plant has gradually gained commercial and scientific interest due to the potential to enhance plant growth and quality [3]. Endophytic bacteria are well-known to closely interact with the host plants and hence could be used as potential bio-control agents in crop production. Endophytes that live in association with the plants benefit both themselves and the plants [4,5].

A variety of chemicals such as chlorinated solvents, several heavy metals, pesticides, and many others have been identified in various natural resources such as water, soil, and air [6,7]. Due to their carcinogenicity, cytotoxicity, and mutagenicity, heavy metals are of great concern to the health of humans [8,9]. Being an eco-friendly and cost-effective technology, phytoremediation uses plants to clean contaminated soil and is currently receiving significant worldwide attention [10,11]. A large number of plant species have the ability to store heavy metals in their tissues. However, phytoremediation deals with a different variety of contaminants and has several restrictions on the level of sites [12,13]. Moreover, phytoremediation success is based upon the ability of plants to tolerate high levels of metal concentrations, while producing a large plant biomass. Being significant in practical applications, microbe and metal tolerant plant associations have been gaining enormous attention for their role in accumulation, uptake, mobilization, or immobilization of heavy metals from the polluted environment and, consequently, enhancement of plant growth. The interaction between plants and microbes have been useful for cleaning up polluted soils [10,14,15].

Beneficial interactions between plants and microbes that improve the health and development of plants have been the subject of utmost interest. Their potential role in the improved biodegradation of soil pollutants has been examined in a recent work. The main focus of these studies is mostly based upon bacteria that arises from the rhizosphere of plants [16,17]. The majority of endophytes with novel chemical production have shown xenobiotic degradation capability naturally or they may introduce degradative traits acting as vectors. Some endophytes have the ability to show resistance to heavy metals, antimicrobials, and organic compounds degradation. This could possibly arise when exposed to certain chemicals in the soil or plant niche. With the enhanced phytoremediation, the natural ability to degrade xenobiotics is inspected [18,19]. Endophytic bacteria produce certain chemicals which promote the quality and growth of the plant.

Phytohormones play a significant role in signaling and regulation of plant growth, development, and improving their quality. Based on their role in biochemical, physiological, and genetic aspects, the most studied phytohormones are auxin and IAA (indole acetic acid) in particular. Different plant hormones such as gibberellins, cytokines, and IAA are produced by both endophytic and non-endophytic bacteria [20]. Different bacteria e.g., Bacillus, Methylophaga, Paenibacillus, Agromyces, and Microbacterium bacteria have been reported to produce, cytokinins, gibberellins, and indole acetic acid (IAA), which increase plant growth and biomass of the plant including root and shoot [21]. The plant kingdom’s most important trait is the ability to produce phytohormones. However, among plants and soil-associated prokaryotes, phytohormone production also varies [22]. Plant hormones synthesized by endophytic bacteria play a vital role in pathogenesis, growth stimulation, and in symbiotic interactions between plants and microbes [23]. Some plant-associated bacteria release high amounts of indole-3-acetic acid, which may elicit responses and alter the internal plant hormone balance of the host, the nature of which depends on the concentration generated by phytohormones, type of host, and its hormone sensitivity [10].

Silybum marianum belongs to the Asteraceae family and is an annual or biennial medicinal plant. This plant usually inhabits sunny, hot, and dry areas and has the ability to grow in most challenging areas, including heavy metal-stressed areas such as lead, cadmium, and zinc [24,25]. Heavy metal toxicity symptoms can be attributed to a variety of cellular and molecular interactions. Metals may bind to sulfhydryl groups in proteins, inhibiting activity or disrupting structure. Additionally, heavy metals may displace essential elements, causing deficiency effects [26]. Plant growth in metal-polluted areas could be due to the characteristic heavy metal hyper accumulating property of the plant [27]. Previously, fungal endophytes of S. marianum had been isolated and their function as bio-accumulators of heavy metals and their role in the growth of plants have been confirmed [27,28,29]. However, bacterial endophytes of S. marianum have yet to be completely investigated. In this study, a total of eleven bacterial endophytes were isolated and their role in phytoremediation and sustainability in heavy metal-stressed areas was investigated. We suggested that the growth of the plants in such heavy metal affected areas was due to the production of phytohormones. To address this hypothesis, we performed different experiments and found that these endophytes were able to produce IAA, and other metabolites that has the ability to kill the pathogenic bacteria

2. Materials and Methods

2.1. Isolation of Bacterial Endophytes from S. marianum

The S. marianum plant is also known as Saint Mary’s thistle, blessed milk thistle, or Marian thistle. It belongs to the Asteraceae family of thistles species. A typical thistle has red or purple flowers and pale green leaves with white veins, and is an annual or biennial plant. It was originally native to southern Europe and Asia, but is now found worldwide. The plant samples were collected from the premises of The University of Agriculture Peshawar (34°01′ N, 71°28′ E). Healthy plants were washed with tap water in order to remove soil, and then their stems, leaves, and roots were cut into 2–3 cm pieces. These pieces were surface sterilized as described by Costa et al., 2012: three minutes wash in 75% ethanol, then five minutes wash in 5% sodium hypochlorite, and at last five times rinse in a sterilized distilled water. After surface sterilization, the plant materials were cultured on Luria-Bertani Agar media, placed in 9 cm Petri dishes, and incubated at 28 °C for 48 h [30,31]. During the period of incubation, the endophytic bacteria residing inside the root, stem, and leaves form colonies on the media. Individual colonies were selected and for the purpose of purification and preservation, they were sub-cultured by streaking on fresh Luria-Bertani agar plates. After sub-culturing, pure isolates were obtained which was then used for morphological and biochemical characterization [32].

2.2. Molecular Identification

The genomic DNA was isolated following the standard protocol of [33]. The 16S rDNA gene was amplified and sequenced using the 27F (5′-AGA GTT TGA TC(C/A) TGG CTC AG-3′) and 1492R (5′-CGG (T/C) TA CCT TGT TAC GAC TT-3′) primers, as reported in [34]. The BLAST search program of NCBI GenBank database/EzTaxon was used to determine the nucleotide sequence homology of the targeted bacterial isolate. For phylogenetic analysis, the neighbor joining (NJ) method was adopted using MEGA v. 6.1.

2.3. Assessment of IAA Produced by Isolates

To find out the amount of IAA produced by single isolate, a colorimetric technique was accomplished with V.U. Salkowski reagent using the method of Salkowski’s [35]. Isolates were grown in Luria-Bertani broth and incubated for 4 days at 28 °C. After incubation, the broth was centrifuged. The supernatant was preserved and 1 ml of the supernatant was mixed with 2 mL of Salkowski’s reagent and was kept in the dark. The OD (optical density) was recorded at 535 nm using a spectrophotometer [36,37,38].

2.4. Antimicrobial Activity of the Endophytic Isolates

For the screening of antimicrobial activity of selected isolates, agar disc diffusion method was used according to the method described by Tendencia, [39]. In this method, first the agar plates were inoculated with test microbes (Bacillus cereus, Clavibacter sp., Citrobacter freundii, E. coli and Xanthomonas campestris). Then filter paper disc having the supernatant of selected isolates were placed on nutrient agar plates and was kept for incubation overnight at 37 °C. After overnight incubation, inhibition zones were measured. The zone of inhibition was considered the point at which no growth was visible to the naked eye. The zone was measured using normal mm scale. Streptomycin was used as the standard antibiotic. The pathogenic strains used for this study were provided by King Fahad Center at King Abdulaziz University.

2.5. Pot-Bioassay of the Plants

Seeds of Brassica napus were taken. The seeds were surface sterilized using 70% ethanol for 3 min and then treated with sodium hypochlorite (2% solution) for 3 min, followed by three times washing with sterilized distilled water [34]. The seeds were then germinated by the method of [40].

After germination of seeds, ten days old seedlings were transferred to screen house for further growth as described by Khan [34]. The medium used for plant growth was thoroughly washed and oven dried (at 80 °C for 48 h). The plants were then transferred to the pots filled with 200 g of sand and were treated with MS (Murashige and Skoog) media to provide nutrients. In order to compensate for water loss, distilled water was added to the pots accordingly. After 15–20 days the all the plants were treated with 2 mL dose of different concentrations (0 mM, 7 mM, 12 mM) of Cr stress in the form of chromium nitrate solution and after an hour, 2 mL of bacterial culture (isolates grown in a Luria-Bertani broth at 28 °C incubation for 4 days) was added to the pots while control was treated with Cr stress but not inoculated with bacterial culture.

2.6. Analysis of Plant Growth Attributes

After 2 weeks of treatments, plants were uprooted carefully and its fresh weight, lengths of root, shoot, and leaves were measured. Plant materials were taken in paper bags and oven dried at 100 °C for 10 min, followed by drying at 100 °C to a constant weight. Plant materials were ground into fine powder and 0.1 g of each powdered sample was digested in a mixture of nitric-perchloric acid (HNO₃-HCLO₄). Total Cr concentration was measured by atomic absorption spectrophotometry (AAS).

3. Results

The current research work was carried out to isolate and characterize endophytic bacteria from S. marianum. The endophytes were assessed for phytoremediation of heavy metals (Chromium Cr) and promoting plants growth under Cr stress. Following are the findings of the research work.

3.1. Identification of Endophytic Bacteria

Based on different morphological and biochemical characteristics, eleven different bacterial isolates were obtained and maintained on Luria-Bertani media. All the isolates were circular in shape and opaque in nature with smooth surface. Out of seven isolates, three were isolated from shoot and five from leaves and three from roots. Potent three isolates were selected for molecular identification. Based on 16s rRNA, SJSB was identified as Lysinibacillus sp. SJLC was identified as Bacillus sp. strain YA, and SJRB was identified as Lysinibacillus sp. Phylogenetic tree for these strains is shown in Figure 1. The sequences of the selected strains were submitted to NCBI for accession number.

3.2. Antimicrobial Activity

Bacterial isolates have been checked for antimicrobial activity against five test bacterial strains. The test microbes used were Bacillus cereus, Clavibacter sp., Citrobacter freundii, E. coli, and Xanthomonas compestris. Streptomycin was used as the standard antibiotic. Some isolates showed good antimicrobial activity as compared to others (

Table 1. Endophytic isolates showing activity against different bacterial strains.

S.No	Isolates	Bacillus cereus	Clavibacter sp.	Citrobacter freundii	E. coli	Xanthomonas campestris
1	Streptomycin	28 mm	30 mm	27 mm	30 mm	32 mm
2	SJLC	20	15	13	0	16
3	SJRB	16	9	8	8	11
4	SJSB	11	14	10	7	13

). Out of eleven isolates, SJSB and SJLC were found effective against all the tested pathogens. The isolate SJRB showed the most activity against Bacillus cereus with a zone of inhibition of 20 mm, followed by the strain SJLC with a zone of inhibition of 16 mm. The same isolates show a promising inhibition zone against Xanthomonas campestris.

3.3. Assessment of Indoleacetic Acid Production by Endophytic Isolates

The results showed that all of the isolates were able to produce IAA. Addition of a few drops of Salkowski’s reagent to the broth, followed by 30 min incubation at room temperature gave a pink color for a positive test. No color change was observed for negative test. Out of eleven isolates, SJLC (6.06 µg/L) showed maximum IAA production, followed by the isolate SJRB (5.98 µg/L), while SJRC (3.78 µg/L) and SJRA (2.24 µg/L) were the least producer of IAA (Figure 2).

3.4. Pot Culture Experiment Results

After isolation and characterization of endophytic bacteria, these bacteria were inoculated to Brassica napus plants in order to assess the growth of these plants under chromium stress. Root, shoot length, fresh and dry weights were measured. Following were the findings of the experiment.

3.5. Plant Morphology and Growth

B. napus plants when treated with different concentrations of Cr and inoculated with bacterial cultures showed satisfactory results as shown in Figure 3. Increasing concentration of Cr treatment i.e., 7 mM and 12 mM had slightly reduced the root and shoot lengths, however maximum damage was not reported in any plant due to the effective action of bacterial isolates. Although the control which was not inoculated with bacterial culture but only treated with Cr, it stress showed retarded growth with lower leaf chlorosis. Inoculation of B. napus plants with endophytic bacteria showed significant amount of increase in root and shoot length as compared to the ones which were not inoculated with the isolates (Control). Out of eleven, three isolates SJLC (13.8 cm), SJRC (13.1 cm), and SJSD (12.76 cm) had significantly enhanced root length at 7 mM as compared to the control (8.33 cm) as shown in Figure 4. At 12 mM, the isolates SJSB (11.66 cm), followed by SJLC (11.33 cm) had increased root length when compared with the control (5.53 cm). In case of shoot length, at 7 mM Cr stress, maximum length was recorded in the presence of the isolate SJRB (5.7 cm), followed by SJRC (5.0 cm), and SJRA (4.8 cm) respectively (Figure 5). At 12 mM Cr stress, maximum shoot length was recorded in the presence of the isolate SJRA (4.7 cm), followed by SJRC (4 cm) respectively when compared with the control (1.96 cm).

3.6. Plant Biomass

Inoculation of endophytic bacteria to the B. napus plants showed an increase in the fresh and dry weight as compared to the control which was not treated with the isolates but treated with Cr stress. Plant biomass was slightly decreased under the increasing concentrations of Cr. At 7 mM, out of eleven endophytes, seven had shown maximum fresh weight i.e., SJSA > SJSD > SJLB > SJSB > SJRC > SJRA > SJRB > SJLA > Control (Figure 6). At 12 mM, the isolates SJSB (0.62 g), SJSC (0.546 g), and SJLC (0.543 g) had shown increased biomass with respect to control (0.24 g). While in case of dry weight, the isolate SJSA (0.136 g) and SJSD (0.133 g) had shown maximum dry weight at 7 mM Cr concentration while at 12 mM, the isolate SJSB (0.103 g), SJLC (0.08 g), and SJRA (0.08 g) had shown the highest dry weight as compared to the control (0.041 g), as shown in Figure 7.

3.7. Cr Accumulation in Plants Treated with Bacterial Isolates

The individual bacterial isolates had helped variably in the uptake of heavy metal chromium. Each endophyte had played a significant role in the degradation of heavy metals when compared with the control which possessed high levels of Cr concentration (Figure 8). Hence the capability of bacterial endophytes to reduce heavy metal had confirmed their role in phytoremediation. Although Cr concentration was higher in high dose-treated plants (12 mM), but still lower than the control. The Cr level in bacterial culture-treated plants was reduced compared to those that remained untreated (control). Out of eleven isolates, SJSD (0.073 mg/L) had the lowest Cr concentration, followed by the isolate SJLB (0.104 mg/L) at 7 mM concentration when compared with the control (0.247 mg/L). At 12 mM concentration, SJLA (0.115 mg/L) and SJLC (0.118 mg/L) had a minimum value of Cr accumulation as compared to control (0.292 mg/L). The 0 mM of Cr was used as a reference which showed negligible traces in the analysis (Figure 8).

4. Discussion

In this research work, a total of eleven endophytes were isolated from the roots, shoots, and leaves of Silybum marianum, a plant of the Asteraceae Family. However, there have been a few previous studies that have screened endophytes from S. marianum and other Asteraceous plants [28,29,30,31,32,33,34,35,36,37,38,39,40,41]. Out of eleven, four endophytes were found in leaves, four were from shoots, and three from roots. These bacterial isolates were identified on the basis of morphological characteristics [42,43]. They characterized different bacterial isolates by colony shape, size, color, and growth rate.

In phytoremediation of soil-metal, plants should be able to deal with huge amounts of heavy metals and, at the same time, reach a high biomass. Although, at high pollution rates, their growth may be limited, resulting in a slow growth rate and small size, minimizing their capacity for phytoextraction [44]. So, plants and their associated microbe’s beneficial relationship could be exploited to promote plant biomass production and thereby determine plant-metal stabilization [10]. The effect of bacterial inoculation on the growth of plants and metal uptake is gaining potential interest and there is an increasing literature reporting on this subject area. In all these studies, increased plant growth is linked with plant-associated bacteria, but the effect on uptake of metal is based on the specific partnerships of plant-microorganisms, and also on the characteristics of the soil [45]. The bioavailability of metals is mostly plant species and element specific, and is influenced clearly by metabolites excreted by applied bacteria [46]. In conclusion, a single organism not only produces some metabolites, but might be manufactured by a plant–microbe association [47]. Hence, in recent years, all the attention has been diverted to the role of endophytic bacteria in phytoremediation of heavy metal-contaminated soils [48] and several authors have reported their use for the purpose of removing, reducing, and extracting heavy metal pollutants from soil [49]. The interaction between hyper-accumulator plants and endophytes has attracted the attention of several researchers, allowing scientists to study more about bacterial communities inhabiting a naturally contaminated environment and their possible role and applications in bioremediation [45].

Our data analysis revealed that endophytic bacteria isolated from Silybum marianum had significantly enhanced the root and shoot length in heavy metal stress conditions. Chaturvedi et al., 2016 concluded that endophytes play a vital role in the growth of plants under heavy metals. Similarly, it proved vital for the increase in fresh and dry weights of the selected plants. Endophytic bacteria have shown great potential in the recent past to be used as agricultural inoculants to enhance the growth of plants and the productivity of many crops [50,51,52,53]. The production of phytohormones is one of the methods used by endophytic bacteria to improve the growth of plants. A significant part of the consideration has been centered on the phytohormone (IAA) that is known to play a significant role in root initiation, cell division, and cell enlargement. In this study, among all the strains tested, isolate SJRB was the best producer of IAA as assessed using the colorimetric assay. Improved growth of plants might be associated with the capability of bacterial isolates that produce indoleacetic acid. It has been noted that IAA production by bacteria could improve the symbiosis of legume–rhizobium and nutrient uptake, thus enhancing shoot length, root length, and as a result, enhancing the development of the plant [54].

The antimicrobial activity of endophytic microbes has been demonstrated for a variety of pathogens of plants [55,56]. In the current study, among the eleven bacterial endophytic strains isolated, almost all showed antagonistic activity against the test microbes. Out of eleven isolates, SJLC, SJRB, and SJSB were found to be effective against all the tested pathogens. The strain SJLC showed the most activity against Bacillus cereus with a zone of inhibition 20 mm, followed by the isolate SJRB showing 16 mm of zone of inhibition against Bacillus cereus. Same isolates also show a promising zone of inhibition against Xanthomonas campestris. The ability of different isolates to reduce or inhibit the growth of different pathogens is due to the secretion of secondary metabolites by endophytic bacteria [57]. From the present study, it is also evident that some endophytic isolates can be developed as potential biocontrol agents [58]. Therefore, to assess the ability of the bacterial isolates, further studies are necessary to determine their role in growth enhancement, defense against pathogens, and plant yield under heavy metal stress conditions. High concentrations of heavy metals can lead to toxic symptoms due to numerous cellular and molecular interactions. For example, metals may bind to protein sulfhydryl groups and inhibit activity or cause structural changes. In addition, an excess of heavy metals can promote the production of free radicals and reactive oxygen species, which may cause oxidative stress [20].

Moreover, plant growth-promoting endophytic bacteria may improve plant growth by enhancing the accessibility or supply of major nutrients [59]. Auxins and ethylene production and modulation play an important role in stress tolerance and plant development [47]. Endophytic bacteria can also be used as a biofertilizer, a well-studied form of nitrogen fixation, which is the conversion of atmospheric nitrogen into ammonia [60]. Moreover, some endophytic plant growth-promoting bacteria through the process of phosphorus solubilization can increase the availability of phosphorus to the plant [61]. Finally, several mechanisms may be involved in biocontrol, including the production of antibiotics and catalase [62].