Introduction

Endophytic fungi are a unique group of organisms that reside within various tissues and organs of terrestrial and certain aquatic plants. These fungi establish infections that often go unnoticed, as the host tissues remain symptomless, at least temporarily¹. Endophytic fungi are known to engage in mutualistic interactions with their host plants, primarily by enhancing the host’s resistance to herbivores². In most cases, fungi benefit from colonizing a plant host as they gain access to nutrients and protection from various abiotic stresses. Previously, mutualistic symbiosis was primarily associated with mycorrhizal fungi residing inside plant roots. However, it has been recently discovered that many other fungi, especially endophytic fungi, can also engage in mutualistic root symbioses³. Endophytic fungi, throughout most or all of their life cycle, inhabit plant tissues without causing visible symptoms. This diverse group of fungi includes dormant saprophytes and latent pathogens, occupying different habitats and positions in the food chain at various stages of their life cycle. The duration of an endophytic fungus’s latent period can vary over ecological timescales. The transition of an endophytic fungus to a parasitic, pathogenic, or saprophytic lifestyle can be triggered by a single locus mutation in its genome. It is believed that endophytic fungi have evolved from parasites or pathogens by extending their latency periods and reducing their virulence. However, the development of endophytes is likely more complex, involving multiple parallel and reverse trajectories influenced by various factors and selection pressures in different contexts⁴.

Endophyte-associated plants release substances that trigger resistance. It was discovered that, after a pathogen challenge, symbiotic plants activate their defence system more rapidly than non-symbiotic plants⁵. Plant growth-promoting endophytes reside in plant tissues, and their close interaction enhances nutrient exchange, growth promoting hormones and enzyme activity. As a result, the established plant-endophyte relationship promotes plant health through several mechanisms⁶. A greater understanding of native plant endophytes might clarify their capabilities and opportunities in promoting plant growth.

Endophytes also represent a novel source of bioactive secondary metabolites. Several reports have shown that endophytes enhance the fitness of their host plants by direct production of bioactive secondary metabolites. It is evident that the secondary metabolites produced by the endophytes are medicinally important as like the phytochemicals, originally believed to be produced only by their host plants⁷.

The Lamiaceae (Labiatae) family is renowned for its diversity and well-established reputation.. Many plants within this family possess a pleasant fragrance due to external glandular structures that produce volatile oils. Coleus aromaticus, also known as Plectranthus amboinicus, is a herbaceous plant belonging to this family. It is characterized by its large size, fragrant nature, and perennial growth habit. Its leaves are used in treatment of common cold, cough, headache and some common digestive disorders⁸.

Coleus aromaticus has been worked out very well in terms of its phytochemistry and isolation of several chemical constituents has been done. Many endophytic fungi has been isolated from different parts of this plant, especially from the leaves and petiole which has the medicinal properties.  Present study thus focus in the isolation, identification, extracellular enzyme activity, plant growth promoting traits of endophytic fungi from the roots of Coleus aromatics and molecular identification of the most promising endophyte from among isolated.

Materials and Methods

Collection and Surface sterilization of the plant material

Fresh and healthy absorption roots of C. aromaticus were collected from its natural habitat from different parts of Palakkad, Kerala. Plants were carefully selected to be free from pathogens, diseases, and fertilizer application. The collected roots were then brought to the research laboratory of Govt. Victoria College, Palakkad to maintain sterile conditions throughout the experiments and subjected to a series of sterilization steps ⁹.

Inoculation of the plant material

Ampicillin was added to Potato Dextrose Agar (PDA), which was then transferred to petri plates in the sterilised inoculation chamber and allowed to solidify in the laminar air flow. Each Petri dish was inoculated with three root segments (1 cm each), for a total of nine segments (3×3=9). To confirm surface sterilization, sterilized spots were randomly imprinted on another PDA plate. The inoculated plates were incubated at 25°C for 14 days and observed regularly during this period. After two weeks, any fungal mycelium emerging from the cut ends of the root segments was sub-cultured, and pure cultures were stored at 4°C on PDA slants

Measurement of endophytic fungal diversity and occurrence

Measures of species richness and evenness are used to calculate species diversity. The Shannon diversity index (H’) and Pielou’s evenness index (J’) was calculated as follows:

𝑆ℎ𝑎𝑛𝑛𝑜𝑛 𝑑𝑖𝑣𝑒𝑟𝑠𝑖𝑡𝑦 𝑖𝑛𝑑𝑒𝑥 (𝐻’) = Σ𝑖𝑝𝑖 * 𝑙(𝑝𝑖)

𝑃𝑙𝑖𝑒𝑜𝑢’𝑠 𝑒𝑣𝑒𝑛𝑛𝑒𝑠𝑠 𝑖𝑛𝑑𝑒𝑥(𝐽’) = 𝐻’/𝑙𝑛(𝑆)

Measurement of fungal occurrence Inoculated plates were observed regularly for two weeks and the colonization rate, colonization frequency and isolation rate was calculated using standard formulas ^{10, 11}.

Microscopic and macroscopic examination of endophytic fungi

Microscopic examination was done by slide culture technique. The fungi was cultured on PDA media on the top of sterile cavity slide. Stained with lactophenol cotton blue, and examined under a low- and high-power in stereo microscope ¹².

Macroscopic features of endophytic fungi were observed for its growth in Potato Dextrose Agar (PDA) after incubating for two weeks and both the sides of the petri dishes were observed regularly for nature, colour and texture of hyphae.

Detection of extracellular enzyme production

Endophytic fungus isolated from the roots of Coleus aromaticus were subjected to five different extracellular assays such as cellulase, amylase, laccase, tyrosinase and protease.

 Amylolytic activity

The fungus was grown in Glucose Yeast Extract Peptone Agar (GYP) medium with 0.2% soluble starch. After incubation the plates were flooded with 1% iodine in 2% Potassium iodide and observed ¹³.

Cellulase activity

The medium used was Glucose Yeast Extract Peptone Agar supplemented with 0.5% Carboxy-methylcellulose. After 3–5 days of fungal growth, the plates were soaked in a 0.2% aqueous Congo red solution and destained with 1M NaCl for 15 minutes. Although the medium appeared red, the presence of yellow patches around the fungal colony indicated cellulose degradation ¹³.

Protease activity

The fungi were grown on GYP agar medium, adjusted to pH 6 with 0.4% gelatin, and protease assay was conducted. After 5 days of incubation, the culture plates were flooded with saturated aqueous ammonium sulphate and formation of a clear zone around the fungal colony indicated gelatin digestion, while undigested gelatin precipitated upon contact with the ammonium sulphate ¹⁴.

Tyrosinase activity

Tyrosinase activity of the fungal isolates was assessed by allowing them to grow on GYP agar medium. A mixture of 0.11% p-cresol and 0.05% glycine was applied to the fungal colony’s surface after it had grown for five days. After 24 hours, culture plates were checked for the development of a reddish brown colour surrounding the colony, which denoted the presence of the tyrosinase enzyme ¹⁴.

Laccase activity

The medium containing 0.05g of 1-napthol per litre was used for assessing laccase activity. Due to laccase’s oxidation of 1-napthol, the colourless medium turns blue as the fungus develops ¹³.

Plant growth promoting activity

Mung bean seeds (Vigna radiata L.) were surface-sterilized with sodium hypochlorite (2 minutes) and then rinsed with sterile distilled water for 2-3 times. A fungal culture suspension filtrate was then applied to the sterilized seeds, with 50 seedlings in each sample treated with 50 ml of the suspension. The seeds were left for 4 hours to facilitate inducer penetration, then incubated and air-dried aseptically in a laboratory environment at 25°C before further research. Seeds kept in sterilized distilled water served as the control ¹⁵. Experiments were performed in triplicate for each sample. After germination, root length and shoot length of the seedlings were measured using ruler following standard protocols ¹⁶.

Molecular identification and sequence analysis

Molecular identification of the fungi showing most colonization frequency was done. The DNA was isolated by using the NucleoSpin® Plant II Kit (MachereyNagel). The sequencing reaction was done in a PCR thermal cycler (GeneAmp PCR System 9700, Applied Biosystems) using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) following the manufacturer’s protocol. The large subunit (LSU) ribosomal DNA primers were used for sequencing. The primers used were forward LROR(F) 5’- ACCCGCTGAACTTAAG C 3’- and reverse LR7 (R) 3’- TACTACCACCAAGATCT- 5’. The sequences so received were analysed using the BLAST to find the closest match. The sequence quality was checked using Sequence Scanner Software v1 (Applied Biosystems). Sequence alignment and required editing of the obtained sequences were carried out using Geneious Pro v5.1 ¹⁷.

Results and Discussion

The elucidation of fungal endophytes’ abilities is a burgeoning field in research, offering ample scope for exploration and discovery. This study thus primarily aims to elucidate the fungal endophytes and their biological activities isolated from the roots of Coleus aromaticus.

Endophytic colonization in roots of C. aromaticus

A total of five endophytic fungi were obtained from the healthy root tissue of C. aromaticus, namely, Trichoderma sp. (TC1), Fusarium spp. (FC2, FC3, FC5) and an unidentified fungal endophyte (C4). The absence of any microbial growth in the imprinted petri dish confirmed the effectiveness of root segment sterilization. Trichoderma is a widely studied genus of fungi, well-known for its vast range of biological functions, potential as biocontrol agents, and capacity to stimulate plant development ^{18, 19}.

The genus Fusarium is recognized as one of the most significant genera of fungi, as it is responsible for causing numerous plant diseases. However, it is one of the most prevalent endophytic fungal genera with about 70 species. It is a cosmopolitan genus of filamentous ascomycete fungi and is in the third level of endophytes after Aspergillus and Penicillium species ^{20, 21}. Studies have shown that Fusarium can form symbiotic relationships with host plants, and that it may help plants grow and develop their defence mechanisms highlighting its importance as a key endophyte in plant-microbe interactions and highlight its potential uses in biotechnology, agriculture, and ecological research ¹⁰.

Distribution of endophytic fungi in root cells of C. aromaticus

Thin sections were observed under a microscope. The endophytic fungal colonies was observed as tiny dots and lines in the cortical cells. The cortical cells infected with endophytic fungi is depicted in Fig. 1 (a-d).

The results demonstrate the lactophenol cotton blue stain’s effectiveness in detecting widespread colonisation along the cortex’s cells, which indicates tight contact between endophytes in various structural and trophic sub niches in the host. Microscopy combined with lactophenol cotton blue staining provides valuable insights into the presence, distribution, and interactions of fungal endophytes within plant hosts ²².

Figure 1: (a – d): Cross section of roots of Coleus aromaticus.Click here to view Figure

Endophytic fungal diversity and occurrence

After a period of two to three days, fungal growth was observed in nine root segments that were inoculated into three petri plates. The fungal colonies exhibited distinct morphologies, a total of nine fungal isolates and five different types of endophytic fungi were identified. To obtain pure cultures, five morphologically different fungal isolates were sub cultured, as shown in Fig. 2. The endophytic fungal diversity observed is recorded in Fig.3.

Shannon diversity index and Pielou’s evenness index of endophytic fungi from the roots of

C. aromaticus were:

Shannon diversity index (H’) =1.52

Pielou’s evenness index (J’) =1.6696

This study showed that the fungal endophytic colonization rate in the roots was 1, while the isolation rate was 0.55. The data is presented in Table 1.

According to many studies fungal diversity is higher in roots but endophytic diversity can change with samples, seasons and location ²³. In the present study, FC3 showed the highest colonization and isolation frequencies, while TC1 and FC2 had the lowest. The isolation frequency indicated the abundance of endophytes from tissues among the total isolates. The results demonstrated that FC3 is the dominant endophyte in C. aromaticus. However, this study focused only on plants from Palakkad district and expanding to other regions could reveal greater endophytic diversity.

Figure 2: Inoculation and colonization of endophytic fungi on PDA PlatesClick here to view Figure

Figure 3: Observed fungal diversity in C. aromaticusClick here to view Figure

Table 1: Colonization rate, isolation rate, colonization frequency and isolation frequency in root tissues of C. aromaticus

 Microscopic and macroscopic features of endophytic fungi from C. aromaticus

The microscopic characteristics of endophytic fungi are recorded in Table 2 and Fig. 4 The culture TC1 was recognized as Trichoderma sp., FC2 and FC3 has been identified as Fusarium spp. FC5 was identified as Fusarium sp. from molecular sequencing described later in this study. Thus establishing Fusarium spp. as dominant endophyte in the present study.

Many studies focusing on endophytes in Lamiaceae has revealed Fusarium spp. as a dominant and commonly occurring endophyte in different members of this family. Fusarium sp. has been isolated from C. aromaticus, and Trichoderma sp. from Carissa carandas ¹⁸. Additionally, many species belonging to Fusarium, Trichoderma, and Phoma have been isolated as endophytes from other Lamiaceae plants, including Salvia officinalis, Leonurus cardiaca, Satureja hortensis, and Mentha piperita ²⁴.

While observing the microscopic features the existence of sterile mycelium as endophytes is not rare. However, identification of fungi on the basis of morphological and microscopic characters in absence of spores is quite challenging. Many of these mycelia do not produce spores on artificial media and may fail to do so even under nutrient stress or other adverse conditions. Overall, different fungi exhibit varying requirements of internal and external factors, either individually or in combination, to initiate sporulation ²⁵.

Table 2: Microscopic characters of fungal endophytes of C. aromaticus

In summary, these findings highlight the complex and diverse nature of sporulation in fungal endophytes and emphasize the significance of specific conditions for successful reproductive processes. Cultural characteristics of the fungal isolates and their morphological features are recorded in Table 3.

Cultural behaviours such as linear growth, colony color, pigmentation, and growth pattern play a crucial role in the identification of fungi. To distinguish different fungi, the structure, shape, and arrangement of conidiophores, phialides, and conidia are examined ²⁶.

Figure 4: (a-g): Microscopic view of endophytic fungiClick here to view Figure

Table 3: Morphology of isolated endophytic fungi on PDA medium

Detection of extracellular enzyme production

Fungal endophytes isolated from different plant sources have emerged as valuable resources for organic products in agriculture, industry, and biomedical applications. Many endophytes have exhibited the production of extracellular hydrolase enzymes, including pectinases, cellulases, lipases, amylases, laccases, xylanase, and proteases. These enzymes play a crucial role in the endophytes’ mechanisms to resist pathogenic organisms and acquire nutrients from their host plants. Utilizing enzymes as a natural alternative to harmful chemicals has gained significant attention in recent years and hence there has been many promising researches focusing on enzyme exploration using microorganisms ²⁷. The extracellular enzyme activity of endophytic fungi from C. aromaticus roots is consolidated in Table 4.

All the isolates exhibited positive amylase activity, as indicated by the presence of a clear white halo around the colony. Amylase activity has been shown by many endophytes from Lamiaceae ¹⁴. Fungal amylase finds wide applications in the food and pharmaceutical sectors. Only three isolates (FC3, C4, and FC5) exhibited a positive result for the cellulase test, indicated by the presence of a yellow region surrounding the colony. Cellulase activity may not be observed in all endophytes tested ²⁸. The enzymes such as pectinases, cellulases, and lipases produced by endophytes render resistant mechanisms against pathogenic organisms and also for gaining nutrients from the host. The extracellular enzymes secreted by endophytic fungi have remarkable applications in food, textile, leather, confectionery, agriculture, beverage, and human health ²⁹.

Table 4: Enzyme activity of endophytic fungi from C. aromaticus

All fungal isolates demonstrated negative protease, tyrosinase and laccase activity. However, there are studies indicating positive protease activity by various Fusarium and Trichoderma species ¹³. Tyrosinase is an enzyme involved in lignin degradation and melanin production also its activity is directly proportional to substrate concentrations ³⁰. Laccases have gained attention for their potential applications in pollution detoxification and bioremediation of phenolic compounds. It is important to note that laccase production may vary depending on different plant parts³¹.

The enzyme production differs between fungi and often corresponds to the requirements of its habitat and other factors like age of the host, climatic condition and geographical location, etc. Also, in the present study only limited extracellular enzyme activity assays were conducted, leaving scope for broader exploration.

Plant growth promoting activities

The assay was conducted in vitro to assess the true impact of the endophytes on plant growth. Mung bean seeds treated with fungal culture broth exhibited a faster germination and higher growth rate compared to untreated seeds (Table 5). Furthermore, the root length of treated seeds was found to be longer. Plant growth-promoting endophytes reside within plant tissues and their interaction promotes nutrient exchange and enzyme activity. These endophytes transmit growth hormones to plant tissues, promoting plant growth ³².

Table 5: Effect of fungal inoculations on the growth properties of Vigna radiata

Endophytes have the ability to enhance plant growth by secreting various phytohormones. Endophyte culture filtrates has revealed the presence of different forms of gibberellins (GAs) and indole-3-acetic acid (IAA), which contribute to the growth-promoting abilities of endophytes ³³. The culture filtrate of Fusarium fujikuroi, also known as Gibberella fujikuroi, was the first identified source of gibberellins (GAs), which are plant hormones known to promote stem elongation. Additionally, Fusarium species are known to produce other hormones such as cytokinins (CKs), auxins, and ethylene ³⁴. Present study demonstrated that endophytic fungi, specifically FC5 isolate, contribute to plant growth promotion. Considering the results of enzymatic activity and the effect on growth promotion studies FC5 fungal isolate was subjected to molecular studies.

Molecular identification and sequence analysis

Molecular sequencing method was used to identify the isolate FC5.The obtained sequence was given as input in the BLAST wed interface to get the closest match. The isolate FC5 was identified as Fusarium solani. The obtained sequence was submitted in NCBI under the accession number OR240869. Fusarium solani is a filamentous fungus ³⁵. This species is a varied complex of around 45 phylogenetic and/or biological species known as the Fusarium solani species complex (FSSC). These morphologically similar species are referred to as F. solani. They are common in soil and decomposing plant matter as well as has also been identified as host-specific pathogens for many crops of agricultural value. It has been isolated from other species of Lamiaceae ³⁶.

Non-pathogenic Fusarium species isolated suggest a symbiotic (endophytic or mutualistic) relationship with their plant hosts. Fusarium members can live as saprophytes or endophytes in asymptomatic tissues of numerous plant species in a non-pathogenic manner and it may contribute to the health of their hosts by aiding in the adaptability to environmental challenges. However, these connections with host plants are most likely transient, and it should be emphasized that Fusarium spp. are capable of switching symbiotic lifestyles, and harmful isolates may emerge, particularly in commercial environments ³⁷.

Both Fusarium and Trichoderma, falls under the categories of endophytes, saprobes, and plant pathogens ³⁸. Most endophytes are plant pathogens which resides inside the host plant. They causes various plant diseases therefore studies was mainly based upon to suppress their pathogenicity or virulence. But now, recent works have revealed about their benefits and their role in plant growth promoting activities. By controlling the gene expression of endophytes, they can be turned on to a most beneficial organism in the near future. Since molecular identification of all fungal strains isolated was not conducted, precise species-level identification is strongly recommended for all strains isolated is recommended for a more robust characterization of the endophytic fungal diversity.

Conclusion

Endophytes are a poorly understood class of organisms that can generate a wide range of bioactive substances. It is crucial to assess and emphasize the previous achievements, ongoing research, and recent advances in research related to endophytic microorganisms in order to bring the research community’s attention to this emerging field and the potential exploitation of the available sources for their therapeutic uses in various fields, such as the medical, pharmaceutical, food, and cosmetic industries.

Acknowledgements

The authors are thankful for the infrastructure provided by the PG & research Department of Botany, Govt. Victoria College, Palakkad and Principal, Govt. Victoria College, Palakkad for the facilities provided.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials.

Author Contributions 

Sajitha Menon Kampurath: Conceptualization, Supervision, Methodology, Writing

Sneha Poongodu Velayudhankutty: Data collection, Analysis

Renju Krishna Valsamma: Editing 

Each author mentioned has significantly and directly contributed intellectually to the project and has given their approval for its publication. 

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