Endophythic fungi in bamboo (Dendrocalamus giganteus): Analysis for phytochemicals and antimicrobial property

Introduction

The term "endophyte" originates from the Greek words "endon" (meaning within) and "phyton" (meaning plant), signifying an organism that lives within a host plant. This term is

Fungi

Fungi are an incredibly diverse group that are essential to ecosystem functioning. They play a key role in recycling energy and nutrients and influence plant community composition through symbiotic relationships (Dighton et. al., 2003). Fungal fruiting bodies (sporocarps) are consumed by a variety of invertebrate and vertebrate animals, including humans (Arora, 2001; Johnson, 1996; Lilleskov & Bruns, 2005). Despite their recognized importance, our knowledge about fungal diversity and ecology remains limited. For example, although an estimated 1.5 million species of fungi are believed to exist, only 72,000 have been described (Hawksworth et. al., 2005). Recently, there has been concern about the decline of many fungal species worldwide due to human activity (Arnolds et. al., 1991; Watling, 2005). Therefore, more research is needed to understand fungal diversity, their ecological roles, and the impact of human activities on fungal diversity and function. Fungal diversity can be categorized either taxonomically or by their functional roles. The latest classification schemes identify six phyla and four unplaced subphyla (Hibbett et al., 2007; James et al., 2006a; James et al., 2006b), comprising five major groups: Chytridiomycota, Zygomycota, Ascomycota, Basidiomycota, and Glomeromycotan. Notably, the functional roles of fungi frequently span across these taxonomic boundaries.

Bamboo: Bamboos, belonging to the grass family Poaceae, are renowned for their rapid growth, structural versatility, and ecological significance. These perennial plants are widely distributed across diverse habitats, ranging from tropical forests to temperate regions, and play essential roles in ecosystem functioning and human livelihoods (Li et al., 2016) [10] . Bamboos are characterized by their woody stems, known as culms, which exhibit exceptional strength, flexibility, and resilience. Endophytic fungi have been documented in various species of bamboo, where they inhabit different plant tissues, including roots, stems, and leaves. Studies have revealed a high diversity of fungal endophytes associated with bamboo species, with representatives from both Ascomycota and Basidiomycota phyla (Huang et al., 2019) [11] . These endophytes contribute to the health and resilience of bamboo forests by enhancing nutrient uptake, improving stress tolerance, and protecting against pathogens and herbivores. Furthermore, endophytic fungi in bamboos have been found to produce bioactive compounds with potential applications in medicine, agriculture, and industry. For instance, extracts from endophytic fungi isolated from bamboo have shown antimicrobial and antioxidant activities, suggesting their potential for pharmaceutical and nutraceutical purposes (Wang et al., 2019) [12] . The exploration of endophytic fungi in bamboos represents an emerging area of research with implications for both ecological understanding and biotechnological innovation.

Dendrocalamus giganteus

Dendrocalamus giganteus belongs to the family Poaceae, is one of the largest bamboo species. D. giganteus is one of the twelve high yielding bamboos worth raising as a large scale bamboo plantation, as it is very good for construction, paper production and young shoots are good for 2 vegetable products. Culms are also used for scaffolding, boat masts, rural housing, water pipes, vases, buckets, water pitchers, matting, boards and parquet, furniture, water pots. Culm sheaths are used to make hats. The stems are especially suitable for the production of bamboo boards, which is an ideal material for room decoration and other interior applications such as walls, ceilings, floors, doors, shelves, etc. D. giganteus can be planted to protect soil against erosion. As one of the largest bamboo species, it has a high ornamental value. This bamboo species also produces large amount of biomass. It can give an annual yield of 20 to 30 t/ha, which is 2.7 times higher than that of D. latiflorus (Schröder at al., 2010).

Materials and Methods

Phytochemicals screening: Phytochemical screening refers to the extraction, screening, and identification of the medicinally active substances found in plants (Harborne et al., 1995) [13] . Phytochemicals are naturally occurring bioactive compounds found in the leaves, stems, bark, roots, fruits, and seeds of plants that serve as a defense mechanism and protect against various diseases (Dewick et al., 2009) [14] . Phytochemicals are categorized into primary and secondary compounds. Primary constituents include chlorophyll, proteins, and common sugars, while secondary constituents comprise terpenoids, alkaloids, Carbohydrates, flavonoids, and saponins. The secretion of these compounds varies from plant to plant, with some plants producing them in larger quantities and others in minimal amounts (Wagner et al., 2009) [15] . Phytochemical screening was performed at School of engineering and technology Nagaland University. All the materials and laboratory equipment's were present here at the collage. The sample used for phytochemical screening were different type of Endophytes in the D. giganteus bamboos from different parts of Nagaland (Wokha, Kiphire and Longlang) [14] . Terpenoids can induce apoptosis, inhibit angiogenesis, and modulate immune responses to prevent cancer cell growth (Thoppil, Bishayee 2011) [15] . They inhibit viral replication and interfere with the viral lifecycle, reducing infection severity. Additionally, terpenoids disrupt bacterial cell walls and membranes, inhibiting bacterial growth and serving as potential antibiotic alternatives. (Burt at el., 2004) [17] . It begins with the addition of 100 µL of the plant extract to a clean test tube, followed by the careful incorporation of 2 mL of chloroform and subsequent introduction of 3 mL of concentrated sulfuric acid in a slow, controlled manner to prevent excessive heat generation, ensuring thorough mixing by gentle swirling to facilitate the reaction between the components. The test tube is then held vertically to allow for careful observation at the interface of the chloroform and sulfuric acid layers, where the formation of a distinct reddish-brown ring indicates the presence of terpenes within the extract.

Reagents used

Test for flavonoids

Flavonoids are especially known for their potent antioxidant activities, which play a significant role in promoting cardiovascular health and preventing cancer (Harbone et al., 1995). These compounds help neutralize free radicals, thereby reducing oxidative stress and inflammation. In addition to their antioxidant properties, flavonoids exhibit a range of other health benefits, including antihistamine effects, antimicrobial activity, memory enhancement, antidiabetic properties, and mood-boosting capabilities. Their diverse pharmacological actions make flavonoids valuable in both nutritional and therapeutic contexts To confirm the presence of flavonoids in a sample, 100 µL of the sample suspected to contain flavonoids is carefully transferred into a clean test tube, followed by the addition of 2 mL of ammonia solution, and then 1 mL of concentrated sulfuric acid is slowly and cautiously added down the side of the tube to prevent excessive heat generation, ensuring thorough mixing by gentle swirling; the appearance of a distinct yellow coloration or precipitate at the interface of the solution confirms the presence of flavonoids.

Test for alkaloids

Alkaloids contain one or more nitrogen atoms usually in a heterocyclic ring and are crystalline and non-volatile solids (Dewick et al., 2009) [14] . In plants, they act as reservoirs for protein synthesis and may function as protective substances against animal or insect attacks. Alkaloids are used as anesthetic agents and for reducing headaches and fever. Wagner's test: It begins with the addition of 100 µL of Wagner's reagent (iodine in potassium iodide) to a test tube containing 100 µL of the sample suspected to contain alkaloids, followed by careful swirling to ensure thorough mixing and allowing the reaction to proceed; the appearance of a distinct brown or reddish precipitate at the interface of the reagent and sample confirms the presence of alkaloids.

Test for carbohydrates

Carbohydrates are essential for life as they form a major portion of our diet, serving as vital body fuels and the primary source of energy for metabolic processes. They are crucial for the proper functioning of our bodies, providing the necessary energy to sustain physical activities and maintain physiological functions (Dewick et al., 2009) [14] . Additionally, carbohydrates are integral to the structural materials of all living cells, playing a key role in cell structure and function, as well as in the storage and transport of energy within biological systems.

Benedict's test: Begins with the addition of 100 µL of Benedict's reagent (containing copper sulfate, sodium citrate, and sodium carbonate) to 1 mL of the sample suspected to contain carbohydrates in a clean test tube, followed by gently heating the mixture in a water bath or using a heating mantle at approximately 80-100°C for 3-5 minutes, during which the reducing sugars present in the sample react with the copper ions in the reagent, resulting in the formation of a varying intensity of orange-red to reddish-brown precipitate, the intensity of which correlates with the concentration of reducing sugars, thereby providing a qualitative and semi-quantitative indication of the presence of carbohydrates.

Antimicrobial screening

Antimicrobial screening is a crucial process in pharmaceutical and medical research aimed at evaluating the ability of substances to inhibit the growth of microorganisms, including bacteria, fungi, and viruses. This screening involves a series of assays designed to assess the antimicrobial potential of natural or synthetic compounds, with the goal of identifying effective agents for the development of new antimicrobial drugs or natural products-based therapies (Palombo et al., 2011) [19] . The screening methods typically include determining the minimum inhibitory concentration (MIC), minimum bactericidal/fungicidal concentration (MBC/MFC), and evaluating the spectrum of antimicrobial activity against a panel of standard microorganisms (Cushnie et al., 2005) [20] . Antimicrobial screening plays a pivotal role in the discovery and development of novel antimicrobial agents to combat infectious diseases and address antibiotic resistance (Cowan et al., 1999) [21] . The samples were collected from leaves and brush of different type of Endophytes in the D. giganteus bamboos from different parts of Nagaland (Wokha, Khipheri and Longland) and was isolated and cultured for 10 days before starting antimicrobial screening.

Requirements

Kanamycin, DMSO, bacterial strain; Staphylococcus aureus (Newman) and Escherichia coli (TLTE11) Procure from NU Lumami headquarter, Ethyl Acetate, 70%, Ethanol, 3% Sodiumhypochloride, petri plates, micropipette, laminar air flow, incubator, burner and centrifuge tube.

Preparation of Media

Nutrient agar was prepared and sterilized at 121°C for 15 minutes. Sterilized media was poured into petri plates and allowed to solidify.

Inoculation

Overnight cultures of bacterial strains (Staphylococcus aureus and Escherichia coli) were prepared. 100 µL of each bacterial culture was evenly spread onto the nutrient agar plates.

Application of Extracts

Using the agar well diffusion method, wells of 6 mm diameter were aseptically punched into the agar plates. 50 µL of the plant extract was added to each well. Kanamycin (50 µg) served as the positive control, and DMSO served as the negative control. The plates were incubated at 37 °C for 24 hours.

Observation

Zones of inhibition around each well were measured to determine antimicrobial activity. Results were compared against the positive (Kanamycin) and negative (DMSO) controls.

Result Phytochemical Screening

The presence of various phytochemicals in the extracts from D. giganteus was determined through standard qualitative tests. The phytochemical analysis revealed that terpenoids were consistently present in all isolates, while flavonoids and carbohydrates were only detected in specific samples.

Conclusion for Phytochemical Screening

The phytochemical screening of endophytic fungi isolated from Dendrocalamus giganteus revealed the presence of a diverse array of bioactive compounds, including terpenoids, alkaloids, flavonoids, and carbohydrates. This diversity of phytochemicals reflects the complex biochemical capabilities of endophytic fungi and highlights their role in the ecological interactions within the plant. The identification of these compounds demonstrates the potential of endophytic fungi as a rich source of natural substances with significant biological activities.

Isolation of Endophytic Fungi:

The isolation process successfully identified multiple endophytic fungi from Dendrocalamus giganteus. These isolates were found to produce a variety of phytochemicals with known therapeutic potentials and exhibited substantial antimicrobial properties. This study underscores the ecological and pharmaceutical significance of endophytic fungi in bamboo, suggesting that they are promising candidates for the development of new natural products for medicinal and agricultural applications.

The isolation and characterization of these fungi pave the way for future research aimed at harnessing their bioactive compounds for drug discovery and biocontrol solutions.

Conclusion for Antimicrobial Screening

Based on the results of the antimicrobial screening, the extracts from Dendrocalamus giganteus demonstrated moderate antibacterial activity against both S.aureus and E. coli. The observed inhibition zones (8±10 mm for S. aureus and 10±14 mm for E. coli) suggest a noticeable but limited effectiveness compared to the positive control, Kanamycin (20 mm±15 mm). These findings indicate that D. giganteus extracts contain compounds with potential antibacterial properties, albeit with a lesser potency than the standard antibiotic.

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