Diversity and biological Potential of endophytes of Pterocarpus erinaceus used in Benin (West Africa): A Step Towards Conservation and New Pharmacological Resources

doi:10.21203/rs.3.rs-4698194/v1

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Diversity and biological Potential of endophytes of Pterocarpus erinaceus used in Benin (West Africa): A Step Towards Conservation and New Pharmacological Resources

https://doi.org/10.21203/rs.3.rs-4698194/v1

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Background

Pterocarpus erinaceus is a plant widely used in traditional medicine in Benin, treating various diseases such as diabetes, ulcers, parasitic infections, hypertension, and infertility. This plant is under strong anthropogenic pressure due to its overexploitation, putting it in danger of extinction. The study aims to explore the diversity and biological potential of endophytes of Pterocarpus erinaceus, with the aim of discovering new pharmacological resources while contributing to the conservation of this valuable species.

Methods

Fresh leaves, stems and roots of Pterocarpus erinaceus, collected in two phytogeographical zones of Benin, Savalou and Abomey-Calavi of Benin, were sterilized to eliminate surface microorganisms prior to cultivation and isolation of endophytes. Isolated endophytes were tested for various enzymatic activities (catalase, cellulase, protease, lipase and amylase) and their antibacterial activities were evaluated by measuring inhibition diameters. The content of total polyphenols and flavonoids was determined by spectrophotometry.

Results

The results showed that bacterial endophytes have a higher colonization (80.55%) and isolation (69.44%) rates than fungal endophytes (44.44% and 50% respectively). Bacterial endophytes expressed more enzymatic activities such as lecithinase and amylase, whereas fungal endophytes showed little. Only two fungal isolates (HT1a and HF2b) showed antibacterial activity, and one isolate (HF2b) showed antifungal action against Candida albicans, while no bacterial isolate demonstrated significant antimicrobial activity. Fungal isolates show significant variation in polyphenol content, while bacterial isolates show similar levels. As far as flavonoids are concerned, bacterial strains such as F3b and SR2C have high levels, unlike fungal endophytes which contain very few.

Conclusion

These results underscore the importance of conserving Pterocarpus erinaceus while exploring its endophytes for potential therapeutic applications.

Pterocarpus erinaceus

Endophytes

Enzymes activities

Polyphenol content

Pterocarpus erinaceus is a very popular plant in traditional medicine in Benin. It is used in the treatment of many pathologies, including diabetes, ulcers, intestinal worms, hypertension, and female and male infertility (Dougnon et al., 2020; Fanou et al., 2020; Klotoé et al., 2013b; Ouinsavi et al., 2021; Vissoh et al., 2024). This popularity is part of a broader context where the importance of traditional medicine in primary health care has been recognized since the declaration of Alma Ata in the guidelines of the Regional Health for All Policy in the Twenty-first Century (WHO, 1978). Indeed, the World Health Organization (WHO) recommends the integration of traditional medicine into national health systems and policies in order to improve access to care and promote health (WHO, 2013).

In Benin, more than 70% of the Beninese population continues to use this natural medicine for their primary health care needs (WHO, 2010). The country's cultural diversity and its ancestral heritage in traditional medicine are a subject of growing interest for scientific research. The flora of Benin is rich and diverse, including about 2807 plant species (Akoegninou et al., 2006). It presents more than 2800 taxa, with information on the type of environment in which they live (dense semi-deciduous humid forest, grassy savannah, wooded savannah, mangrove, etc.) and the names of some localities where they have been observed or collected. The book is highly recommended for traditional healers in Benin and also provides plant names in 21 national languages in addition to French (Akoegninou et al., 2006). Ethnobotanical surveys conducted from 2010 to the present day provide information on the use of a multitude of medicinal plants by the population for various pathologies (Klotoé et al., 2013a; Vissoh et al., 2024). This use results in strong anthropogenic pressure that threatens plant biodiversity and sometimes leads to the disappearance of species. Pterocarpus erinaceus is one of these plants that is under this anthropogenic pressure. So coveted and exploited, with a significant increase in the trade in its timber, it is now on the International Union for Conservation of Nature's red list as a species in danger of extinction (IUCN, 2024). In addition, several scientific studies have proven multiple pharmacological properties such as analgesic, antibacterial, anti-diabetic, anti-inflammatory, antioxidant properties of P. erinaceus (Atchou et al., 2021; Houmènou et al., 2018; Noufou et al., 2016; Tittikpina et al., 2018). In view of these multiple properties and traditional medicinal uses, it is imperative to find alternatives to preserve its population. Research on endophytes is promising in this direction, as endophytes, which live in symbiosis with plants, have the ability to produce bioactive compounds similar to those of their hosts. These microorganisms could offer a sustainable source of therapeutic substances, thus avoiding the overexploitation of Pterocarpus erinaceus. Fungal endophytes, microorganisms that live inside plant tissues, are increasingly being studied for their beneficial properties. Indeed, some enzymes produced by these endophytes, such as proteases, have interesting benefits for human health (Khan et al., 2017b). They produce complex and unique classes of secondary metabolites that can constitute new avenues for pharmaceutical discoveries (Tousif et al., 2023). Studies have shown that these endophytes are undeniable sources of extracellular enzymes that could constitute new products for medical exploitation in the fight against pathogen resistance to existing conventional antibiotics (Firáková et al., 2007; Komeil and Saad, 2021). The present study therefore aims to explore the diversity and biological potential of these endophytes, with the aim of discovering new pharmacological resources while contributing to the conservation of this valuable species. It will answer the following questions:

What is the diversity of endophytes present in Pterocarpus erinaceus?
What are the biological potentials of endophytes produced by Pterocarpus erinaceus?

Plant material

The plant material consists of the leaves, stem and fresh roots of Pterocarpus erinaceus collected in the town of Savalou and in Abomey-Calavi in the botanical garden of the University of Abomey-Calavi. Its healthy organs with no visible symptoms of disease have been carefully selected. They have been certified at the National Herbarium of Benin under the number YH 952/HN. The samples collected in this way were immediately transported under aseptic conditions to the laboratory (Xiaoxiang et al., 2019).

Bacterial strains

The microbial material consisted of four pathogenic bacteria, 02 Gram-negative bacilli, 01 Gram-positive cocci strain and 01 fungal strain, provided by the Research Unit in Applied Microbiology and Pharmacology of natural substances (URMAPha) and the Polytechnic School of Abomey Calavi (EPAC). They are Escherichia coli ATCC 25922; Pseudomonas aeruginosa ATCC 9027; Staphylococcus aureus ATCC 6528 and Candida albicans ATCC 90028.

Preparation of the organs

After the samples had been collected and sent to the laboratory, the organs were rinsed with tap water to remove dust and other waste that might end up on their surface (Marchut-Mikołajczyk et al., 2023). After rinsing, the organs were cut into small fragments that underwent sterilization according to the method described by Sharma and Mallubhotla (2022) to remove all microorganisms from the surface (epiphytes) and to ensure that the isolates would come from within them. Briefly, they were immersed successively in 70% ethanol for 6 minutes, sodium hypochlorite or 6% bleach for 4 minutes and then in 70% ethanol for 4 minutes. The fragments were then rinsed three times with sterile distilled water and dried on sterile tea towel paper to remove epiphytes (Khan et al., 2017; Ratnaweera et al., 2015). For bacterial endophyes, after this step, the organs were cut into pieces of one cm/1 cm each, then isolated on NA medium and incubated in the oven at 35°C for 24 hours.

Sterility check

The method described by Li et al. (2020) with some modifications was used for this sterility check. To ensure that the surfaces are sterile and that the colonies to be cultured actually come from the internal tissues, the third rinse water was inoculated with Potato Dextrose Agar (PDA) medium supplemented with Chloramphenicol at 150 mg/l for endophytic fungi (Salo and Novero, 2020). For bacterial endophytes, the third rinse water was inoculated on NA medium and incubated in the oven at 35°C for 24 hours. Thus, the absence of colonies on the media indicates that surface sterilization has been done well and that all epiphytic microorganisms are eliminated (Marsola et al., 2022).

Culture of decontaminated fragments, isolation and purification of fungal endophytes

After sterilization, the samples were cut into 1mm2 fragments and placed in petri dishes containing Potato Dextrose Agar (PDA) agar supplemented with 0.5g/l of chloramphenicol (PDA) and previously autoclaved at 121°C for 15 minutes, and aseptically supplemented with 10g/l of chloramphenicol to inhibit bacterial growth (3 segments per dish). The different dishes were incubated at 37°C for 5 to 7 days. For bacterial endophytes, the organs were cut into pieces of one cm/1 cm each, then isolated on NA medium and incubated in the oven at 35°C for 24 hours. After incubation, all the fungal endophytes that form colonies were then isolated and placed in new petri dishes containing PDA, until pure colonies were obtained. Each isolated and purified mushroom was stored on PDA at 4°C. The same process was followed for bacterial endophytes using NA medium. After the incubation period, endophyte colonization and isolation rates were calculated using the following formulas (Jayatilake et al., 2020) :

Colonization rate CT(%) = Number of Colonized Fragments / Total Number of Fragments×100

Isolation rate (IR)=(Number of segments yielding a number of isolates ≥ 1)/(Total number of segments)×100

Morphological and cultural identification of pure colonies

Macroscopic observation considered color, general colony appearance, pigments, colony size and shape, and growth diameter (Khan et al. 2017a). Microscopic observation was carried out using the scotch technique (Nacef et al., 2022). This method consists of performing a simple staining with methylene blue by depositing a drop of methylene blue on a clean, dry microscope slide. Indeed, young structures of the fungal colonies were taken with tape and placed on the slide previously bearing a drop of methylene blue so that the structures of the fungus were colored and visible to observation. Any excess methylene blue around the blade has been gently cleaned with sterile tissue. The slide was examined under a microscope at 400 and 1000 magnifications. Identification was essentially based on the study of the mycelium and the nature of the differentiated organs.

Enzymatic activity of isolated endophytes

The catalase activity of endophytes was investigated following the methodology applied by Attia et al. (2020). Isolated endophytes were tested for hydrogen peroxide (H₂O₂) hydrolysis by the slide method. Indeed, fungal strains grown on PDA agar and then incubated at 30°C for 7 days were used. A drop of hydrogen peroxide was placed on the test slides and then a 1mm2 fragment of young preculture of fungal endophytes was taken using a platinum loop and deposited in the drop of hydrogen peroxide. Peroxidase production was visually detected by observing the immediate bubbling (Attia et al., 2020).

The cellulase activity of endophytes was tested by using PDA medium supplemented with 0.5% carboxymethylcellulose was used. After 3 to 5 days of growth of the fungal strains, the plates were flooded with a 0.2% Congo red aqueous solution and discolored with 1M NaCl for 15 minutes. The appearance of yellow areas around the fungal colony testifies to the production of cellulase by the fungal colony (Marsola et al., 2022).

Protease activity was assessed using the method of Usman et al. (2023). A PDA medium containing 0.4% casein (pH 6.0) was used for this purpose. After an incubation period of 3 to 5 days, casein degradation appears as a clear area around the colonies indicating the production of the protease by the fungal strain (Usman et al., 2023).

To test the Lipase activity of endophytic fungi, Peptone Agar medium (peptone 10 g, NaCl 5 g, CaCl₂HO − 0.1 g, agar − 16 g, distilled water − 1L; pH 6.0) supplemented with 1% Tween 20 was used. After the incubation period of 3 to 5 days, the appearance of a visible precipitate around the colonies indicates positive lipase activity promoted by the formation of calcium salts of lauric acid released by the enzyme (Hawar, 2022).

Amylase activity was investigated using the method used by Taneja et al. (2023). A PDA medium containing 0.2% starch (pH 6.0) was used. After an incubation period of 3 to 5 days, starch degradation appears in the form of a clear area around the colonies after a revelation with Lugol concentrated at 2% testifying to the production of amylase by the fungal strain (Taneja et al., 2023).

Antibacterial activity of isolated endophytes of Pterocarpus erinaceus

For the evaluation of the antibacterial activity of isolated endophytes, the Agar cylinder method was used (Pelo et al., 2020). Indeed, the different strains to be tested were revived in nutritious broth and incubated at 37°C for 24 hours. After these broths showed turbidity, they were inoculated using the three-way streak technique on the nutrient agar and incubated at 37°C for 24 hours. After incubation, a few colonies from each dish, all morphologically identical, were scraped using a platinum loop and then discharged into 5 ml of sterile physiological water. These different suspensions were well homogenized and the turbidity afterwards was adjusted to 0.5 McFarland (Jayatilake et al., 2020). The bacterial suspensions were inoculated using a sterile swab using the Kirby Bauer method on Petri dishes containing nutrient agar (RN). The swab is soaked in the bacterial suspension, then wrung out by rotating it on the inner wall of the tube in order to discharge it as much as possible. The GN medium is rubbed by tight streaks over its entire agar surface from top to bottom, turning the boxes 60° each time (Jayatilake et al., 2020). Once the reference strains were sown, agar cylinders of 6mm diameter of young mushroom culture (7 days) were collected and rigorously deposited aseptically, a cylinder in the center of the different boxes previously seeded with reference strains (An et al., 2020). All the boxes were left at room temperature for a period of 2 hours to allow a pre-diffusion of the active substances secreted by fungal endophytes before being incubated at 37°C for 24 hours. After the incubation period, inhibition diameters around deposited cylinders were measured (Munasinghe et al., 2020).

Determination of total polyphenols of endophytes of Pterocarpus erinaceus

The determination of total polyphenols was performed by spectrophotometry, according to the colorimetric method using the Folin-Ciocalteux reagent of Singleton et al. (1999). Briefly, a volume of 50 µL of the dilute solution (1/100 in distilled water) of concentrated extract at 25 mg/mL was added to 250 µL of the 10% Folin-Ciocalteu reagent (diluted 10 in distilled water) and 750 µL of an aqueous solution of Sodium carbonate (Na₂CO₃) at 75 g/L.

After 8 minutes of incubation, 950 µL of distilled water was added and mixed with the vortex and then incubated for 2 hours in the dark at room temperature. After incubation, optical densities (ODs) were read at 760 nm using the CECIL CE 2041 spectrophotometer. The reading was taken against a blank consisting of a mixture of 250 µL of FCR (Folin Ciocalteu Reagent) and 750 µL of Na₂CO₃ and 1 mL of distilled water.

Determination of total flavonoids

The quantification of flavonoids was carried out by a method adapted from Zhishen et al. (1999) and Kim et al. (2003) using aluminum trichloride (AlCl₃) as the reagent. Thus, 500 µL of an AlCl₃ solution (2%) was taken and added to 500 µL of the sample. To this mixture was added 3 mL of ethanol. The white consists of 500 µL of AlCl₃ and 3.5 mL of ethanol. The absorbance reading was done with a spectrophotometer at 415 nm after a 10-min incubation. Samples were prepared in triplicate for each assay. The total flavonoid content was determined in mg rutin equivalent/g extract (µg RuE/g) by the formula used by Ahmed et al. (2019).

Statistical analyses

The data obtained were subjected to statistical analysis using SPSS 26.0 software and Microsoft Excel 2016 spreadsheets. Qualitative variables were expressed as percentages. Quantitative variables are presented as a mean and standard deviation. The analysis of variance (ANOVA) has a single factor and was used to assess the variation in the content of polyphenols, flavonoids and total tannins of the fractions studied. The significance threshold has been set at 5%.

Determination of isolation and colonization rates

The results obtained indicated a colonization rate (CT) of 44.44%, which means that 44.44% of the segments evaluated were colonized by fungal isolates showing the presence of fungi in the analyzed samples (Fig. 1). For bacterial endophytes, 80.55% was obtained as a percentage of colonization. As for the isolation rate (IR), it is 50%, indicating that half of the segments evaluated yielded at least one fungal isolate. For bacterial endophytes, 69.44% of the segments yielded at least one bacterial isolate (Fig. 1). Thus, there is more colonization for bacterial endophytes compared to bacterial endophytes. The same trend is observed for the isolation rate. These data suggest that bacterial endophytes are more widely present and isolated in the samples studied compared to fungal endophytes.

Diversity of Endophyte species according to the collection location

Analysis of Fig. 2 reveals a marked variability in the distribution of fungal endophytes between the two collection sites and the different parts of the plant. A total of thirty-four endophytes were isolated from the plant at the two collection sites. Samples collected in the commune of Savalou were richer in endophytes (61.76%) than those from the commune of Abomey-Calavi collected at the National Herbarium (38.23%). In terms of plant parts, more fungal and bacterial isolates were isolated from leaves than other parts for both collection sites.

Figure 3 presents the number of bacterial endophytes identified in different plant organs (leaf, stem, root) according to two collection sites: Herbarium of the University of Abomey-Calavi and in the commune of Savalou. The analysis of this figure reveals that the roots of the plants collected at Savalou contain the highest number of bacterial endophytes (8), while the leaves of the plants in the Herbarium contain the most (5). In general, Savalou plants appear to have a greater diversity of bacterial endophytes in the stems and roots compared to herbarium plants.

Morphological and microscopic characteristics of isolated fungal endophytes

The results of the study of the cultural and microscopic characteristics of fungal endophytes of Pterocarpus erinaceus are presented in Table 1. Analysis of this table reveals that each isolate is described based on its morphology, the color of the upper and lower sides of the colony, and the microscopic structures observed and their type of belonging. The isolates belong to six genera: Penicillium spp, Chrysosporium spp, Alternaria spp, Aspergillus spp, Fusarium spp, Acremonium spp.

Table 1

Morphological and microscopic characteristics of fungal endophytes
Codes	Morphology	Genre
SR2c	Hyaline mushroom, glabrous, brown back, pleated and white underside, raised, rigid, waist 3/2.6cm, without diffusible pigment. Presence of simple arthroconidia	Penicillium spp
HR1c	Hyaline mushroom, glabrous, brown back, pleated and green underside, raised, rigid, waist 3/2.6 cm, without diffusible pigment. Hyphae unseptate, conidiophore branched biseriate in a brush, conidia round in clusters and chains.	Penicillium spp
HT1c	texture, Dark brown, White Centre; whitish outline, white and irregular border, Rigid appearance and size 3/2.6cm, pigment-free	Chrysosporium spp
HF2b	Pleated Black centre and dark green outline, ash backing, irregular border, powdery appearance, 3cm diameter and without pigment.	Not identified
HF1b	Pleated Black centre and dark green outline, ash underside, irregular border, cottony look, 3cm diameter and pigment-free.	Alternaria spp
ST2c	Pleated Black centre and dark green outline, ash underside, irregular border, cottony look, 3cm diameter and pigment-free.	Acremonium spp
ST2a	Pleated Black centre and dark green outline, ash underside, irregular border, cottony look, 3cm diameter and pigment-free	Not identified
HF2c	Pleated Black centre and dark green outline, ash underside, irregular border, cottony look, 3cm diameter and pigment-free	Alternaria spp
HF1a	Pleated Black centre and dark green outline, ash underside, irregular border, cottony look, 3cm diameter and pigment-free	Not identified
HF1c	Pleated Green centre and white outline, white underside, regular border, cottony appearance, 3cm diameter and without pigment.	Chrysosporium spp
HT1c	Pleated Black centre and dark green outline, ash underside, irregular border, cottony appearance, 3cm diameter and without pigment.	Penicillium spp
HF3c White Colony		Unidentified

Isolates SR2c, HR1c, and HT1c (first occurrence) belong to the genus Penicillium spp and have similar characteristics. SR2c is a hyaline and hairless fungus with a brown, pleated back and a white underside, raised, rigid, 3/2.6 cm in size, without diffusible pigment, and with simple arthroconidia. HR1c shares similar features with a brown back and green underside, but also has unseptate hyphae and brush-branched conidiophores with cluster-shaped and chain-shaped round conidia. HT1c is characterized by a black center, a dark green outline, an ashy underside, an irregular border, and a cottony texture 3 cm in diameter, with a white colony.

Isolates HT1c (second occurrence), HF1c, and HT1a belong to the genus Chrysosporium spp. HT1c has a fluffy texture with a dark brown center, a whitish outline, a white and irregular border, a rigid appearance, and a dimension of 3/2.6 cm, without pigment. HF1c is characterized by a pleated morphology with a green center, a white outline, a white underside, a regular border, and a cottony texture, 3 cm in diameter, without pigment. HT1a also has a pleated texture, with a black back and back, irregular border, stiff appearance, and a 3/2 cm size, without pigment.

Isolates HF1b, HF2c, and HF1a belong to the genus Alternaria spp. All have a pleated morphology with a black center and a dark green outline, an ashy underside, an irregular border, a cottony appearance, and a diameter of 3 cm, without pigment

Isolates HF3a, SR1c, HF3b, and HF3c (second occurrence) belong to the genus Aspergillus spp. HF3a has a pleated morphology with a black back and back, an irregular border, and a rigid appearance, measuring 3/2 cm without pigment. SR1c has a powdery texture, with a beige back color and a whitish underside, an irregular border, and a cottony texture, 3/2.6 cm in size and without pigment. HF3b shows a powdery texture with alternating beige and brown color, an irregular border, and a cottony appearance, with a diameter of 5 cm without pigment. Finally, HF3c is a white colony.

Isolates ST1a and HF3c (first occurrence) belong to the genus Fusarium spp. ST1a has a soft morphology, with a beige back and back, an irregular border, and a creamy appearance, measuring 2.6/2 cm, without pigment. HF3c is described as a black, powdery colony with shades of black and white, an irregular border, a cottony texture, and a size of 2.5/2 cm.

Isolate ST2c belongs to the genus Acremonium spp. It has a pleated morphology with a black center and a dark green outline, an ashy backing, an irregular border, a cottony texture, and a diameter of 3 cm, without pigment.

Isolates HF2b, ST2a, and HF1a could not be identified. HF2b has a pleated morphology with a black center and a dark green outline, an ashy underside, an irregular border, a powdery texture, measuring 3 cm in diameter, without pigment. Similarly, ST2a and HF1a have a pleated morphology with a black center and a dark green outline, an ashy underside, an irregular border, and a cottony texture, with a diameter of 3 cm and no pigment.

Morphological and microscopic characteristics of isolated bacterial endophytes

Morphological analysis of isolated bacterial colonies of P. erinaceus showed that they were mainly brown in color, with regular margins of different sizes (small, medium and large). There are a few colonies with a smooth texture, others with a mucoid texture, and still others with a dry texture. Microscopic observation, after gram staining, showed gram-positive bacilli. Of all the samples observed, 56% of the strains have endospores while 44% do not. Based on this characteristic, the presence or absence of endospores 56% of the strains with these endospores belong to the genus Bacillus.

Enzymatic activity of isolated endophytes

Figure 4 shows the percentages of bacterial and fungal endophyte isolates expressing various enzymatic activities. For lecithinase, 85.18% of bacterial endophyte isolates express this activity, while no fungal endophyte isolates do. Amylase activity was noted in both endophyte groups, but higher in bacterial (74%) compared to fungal (21.05%). The expression of the protease reveals another marked distinction: 44.45% of bacterial isolates show this activity compared to 0% for fungal isolates. Catalase activity is universally expressed in both endophyte types, with 100% of bacterial and fungal isolates showing this activity. However, no lipase activity was detected in either group, and cellulase activity was very low in bacteria (3.7%) and absent in fungi.

Screening of antimicrobial activity of fungal endophytes

Antibacterial Activity

Of the 34 endophytic tested, only two isolates (HT1a and HF2b) showed antibacterial activity against the three bacterial strains used, namely Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus. The mean areas of inhibition measured ranged from 0 to 35 ± 0.9 mm. HF2b and HT1a isolates were more active on E. coli and Staphylococcus aureus. However, none of the bacterial isolates were active against its strains.

Antifungal activity

Of the 34 isolates tested, only HF2b showed bacteriostatic action on Candida albicans with an inhibition diameter of 23 ± 0.9 mm. However, none of the bacterial isolates were active on Candida albicans.

Determination of the total polyphenol content of endophytes

Figure 5 shows the polyphenol content of fungal isolates. Isolates HT1a, HF1b, HF2b, showed high polyphenol contents. Conversely, isolates with lower levels were HF1c, HR1c.

Figure 6 shows the polyphenol content of bacterial isolates. From the analysis of this figure, it appears that there is no significant difference between the total polyphenol content of the different isolates.

Determination of total flavonoid content of endophytes

Figure 7 shows the flavonoid content of different bacterial endophytic strains. From this figure, it appears that the F3b and SR2C, HT1C and T2b1 strains have significantly high flavonoid contents compared to the HT1a, R1b and T1C1 strains. As for fungal endophytes, they have a very low content of flavonoids that can be assimilated to traces

This study aimed to explore the diversity and biological potential of endophytes of Pterocarpus erinaceus, with the aim of discovering new pharmacological resources while contributing to the conservation of this valuable species. The results of the determination of the colonization rate and the isolation rate of fungal endophytes indicate that there is more colonization for bacterial endophytes compared to bacterial endophytes. The same trend is observed for the isolation rate. These data suggest that bacterial endophytes are more widely present and isolated in the samples studied compared to fungal endophytes. In the literature, it is reported that the community of bacterial endophytes is influenced by biotic and abiotic factors, which shape their species composition, community structure, diversity, and functions (Walitang et al., 2018). Environmental factors affect not only the distribution of a medicinal plant, but also the determination of the bacterial endophyte that can colonize the host plant throughout its life cycle (Deng et al., 2011; Wu et al., 2021). Endophyte diversity is also influenced by host plant characteristics, including genotype (Walitang et al. 2018; Wu et al., 2021), tissue (Dai et al., 2014), growth stage (age), and health status (Bogas et al., 2015).

The results reported that the roots of the plants collected at Savalou contain the highest number of bacterial endophytes (8), while the leaves of the plants in the Herbarium contain the most (5). In general, Savalou plants appear to have a greater diversity of bacterial endophytes in the stems and roots compared to herbarium plants. This richness of the roots and stem in bacterial endophytes is comparable to the results reported by LIU et al. (2020) compared to the Paris polyphylla var. yunnanensis, a famous and endangered traditional Chinese herb that has significant medicinal value. This endophyte richness can also be observed in plants growing in unfavorable extreme environments such as Distichlis and Pluchea absinthioides, Gaultheria mucronata and Hieracium pilosella which grow in extreme environments in Chile.

In addition, the results highlight a diversity of fungal endophyte species depending on the place of collection and. Samples collected at the National Herbarium showed a greater richness in endophytes, particularly in the leaves, unlike Savalou. In contrast, Savalou has a rich bacterial endophyte in the stems and roots. The differences observed between the two collection sites suggest that local environmental and ecological conditions may play a crucial role in the diversity and distribution of fungal endophytes. Indeed, Deng et al. (2011) reported that variations in microclimate, soil composition, and surrounding vegetation influence the colonization of plants by endophytes. The different parts of the plant (leaves, stems, roots) provide varied habitats for endophytes, with specific micro-environmental conditions (LIU et al., 2020; Zhang et al., 2019). In Benin, the first sample collection site c the National Herbarium, the commune of Abomey-Calavi (first sample collection site) and the commune of Savalou (second collection site) belong respectively to the Guinean-Congolese zone and the Sudano-Guinean zone, two distinct phytogeographical zones of the country. The Guinean-Congolese zone is located in southern Benin and is characterized by tropical rainforest vegetation. The soils of the Guinea-Congo zone, located in southern Benin, are mainly ferralitic, rich in iron and aluminum, and well-drained. Although often acidic and low in nutrients like calcium, magnesium, and potassium, they can become very fertile with proper management and fertilization. The texture of these soils varies from sandy to clayey, with a strong presence of organic matter on the surface. However, soils are prone to erosion due to heavy rainfall, requiring conservation practices to maintain their fertility. They are suitable for the cultivation of cash crops such as oil palm, cocoa, and other tropical crops (Imorou et al., 2015). The Sudanian zone of northern Benin is influenced by the characteristics of its tropical ferruginous soils, which are often shallower and less fertile than those in the south. The low organic matter content and the texture varying from sandy to sandy-clayey, with a moderate to low water holding capacity, limit microbial diversity. Well-drained, but sometimes drought-prone soils, combined with sparse savannah vegetation, further reduce endophyte diversity. In contrast, the central Sudano-Guinean zone, with its intermediate tropical and ferralitic ferruginous soils, has moderate fertility and organic matter content, providing more favorable conditions for a richer endophytic diversity of plants (Salako et al., 2018).

In addition, the endophyte richness in the leaves of the National Herbarium of the commune of Abomey-Calavi could be due to factors such as relative humidity, nutrient availability, and interactions with other microorganisms (Wu et al., 2021). These results may have applications in Biotechnology and Agriculture. Indeed, knowledge of the distribution and diversity of endophytes can have important applications in biotechnology and agriculture, particularly in the development of biopesticides and biofertilizers. The endophyte richness of the leaves of the National Herbarium and those of the roots of Savalou could be explored for beneficial properties.

In addition, the results obtained have shown, from the analysis of their cultural and microscopic characteristics, that these endophytes belong to six genera, the most represented of which are Aspergillus spp, Penicillium spp, Alternaria spp, Fusarium spp. This diversity indicates a rich population of endophytes within Pterocarpus erinaceus. In the literature, a number of previous studies have reported the presence of these fungal genera as a dominant group of endophytes residing in association with different medicinal plants (Ferreira et al., 2020; Li et al., 2022; Shah et al., 2018; Talukdar et al., 2021). The variations observed in cultural and microscopic characteristics, even within the same genus, suggest a specific adaptation of endophytes to their environment or to the part of the plant colonized. The presence of a diversity of endohytes can have implications for the health and resilience of host plants.

The identified endophytes, such as Penicillium and Aspergillus, are known for their abilities to produce bioactive enzymes and metabolites.

Regarding bacterial endophytes, most isolates belong to the genus Bacillus. Bacillus are Gram-positive bacteria, which are able to withstand adverse environmental conditions through the production of endospores. This genus is one of the most common for bacterial endophytes of medicinal plants (Bolivar-Anillo et al., 2021).

The study of the screening of the antimicrobial activity of fungal endophytes has revealed, unlike bacterial endophytes, interesting and promising results regarding the potential of these microorganisms in the fight against bacterial and fungal pathogens. Among the fungal endophytes tested, two isolates in particular, HT1a and HF2b, demonstrated significant antibacterial activity against strains of Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus. These isolates showed notable areas of inhibition, with diameters up to 35 ± 0.9 mm, indicating strong antimicrobial activity. Comparable results have been obtained for plant extracts from other authors in the literature (Tittikpina et al., 2018). Antifungal activity was also explored, revealing that only isolate HF2b demonstrated bacteriostatic action against Candida albicans, with an inhibition zone of 23 ± 0.9 mm. This suggests that, although fewer isolates showed antifungal activity compared to antibacterial activity, some fungal endophytes possess properties that can be exploited to combat fungal infections. Tittikpina et al. (2019) reported that root extract showed significant antifungal activity against A. fumigatus with a MIC value of 16 µg/mL.

The polyphenol content of fungal isolates was also determined, revealing significant variation between different isolates. HR1c, HF2b and HF1c isolates showed the highest concentrations of polyphenols, while H and HF1c isolates showed lower concentrations. However, these polyphenol and flavonoid contents are very low compared to those reported (Toukam Djouonzo et al., 2016) for the acetate and butanol extracts of the plant. The presence of polyphenols is often correlated with the antimicrobial properties of medicinal plants, which could partly explain the activities observed. In particular, the high polyphenol content of isolate HF2b could be related to its antimicrobial efficacy against both bacteria and Candida albicans.

These data suggest that some fungal endophytes, such as HF2b and HT1a, possess significant antimicrobial capabilities, potentially due to their polyphenol content. HF2b is particularly distinguished by its dual role, showing activity against both pathogenic bacteria and Candida albicans. These findings hold promise for the development of novel antimicrobial agents based on fungal endophytes

The results of this study open up promising prospects for the development of new antibiotics. The fungal isolates HT1a and HF2b, which have demonstrated significant antibacterial activity, could serve as a basis for creating antibiotics that are effective against bacteria that are resistant to current drugs. This breakthrough is crucial in the face of increasing bacterial resistance that threatens global public health. In addition, HF2b fungal isolate, with its dual antibacterial and antifungal activity, is of particular interest for medical applications. It could be explored to treat mixed bacterial and fungal infections, which are often complex to manage with current treatments. This could improve treatment options for patients suffering from such infections and reduce associated complications.

In addition, research on the total polyphenol content of endophytes is also highlighted by this study. The observed correlation between polyphenol content and antimicrobial activity suggests that manipulating polyphenol levels in fungal endophytes may increase their antimicrobial efficacy. However, bacterial endophytes did not show antibacterial activity against the tested strains. This could be explained by the low polyphenol content in these bacterial endophytes compared to fungal endophytes.

Finally, the implications for agriculture and food preservation are notable. Fungal endophytes could be used to develop natural biopesticides or food preservatives, providing an alternative to synthetic chemicals. This approach could not only reduce reliance on these chemicals, but also contribute to more sustainable and environmentally friendly agricultural and conservation practices.

In summary, this study highlights the potential of endophytic fungi of Pterocarpus erinaceus as a source of natural antimicrobial agents, offering interesting and diverse perspectives for medicine, agriculture and scientific research.

This study revealed significant diversity and biological potential of Pterocarpus erinaceus endophytes, demonstrating notable fungal colonization with high colonization and isolation rates. The isolation of a multitude of endophytes from leaves, stems and roots, from two distinct sites, has highlighted a diversity of endophyte species influenced by local environmental conditions. The genera Aspergillus, Penicillium, Alternaria and Fusarium were the most represented for fungal endophytes and the genus Bacillus for bacterial endophytes.

The results of the antimicrobial activity screening showed that the HT1a and HF2b isolates possess significant antibacterial capabilities, while HF2b also demonstrated antifungal action. These isolates can serve as a basis for the development of new antibiotics, particularly against bacteria resistant to current drugs. The correlation between polyphenols and antimicrobial activity opens up avenues of research to increase the effectiveness of these endophytes. This study highlights the potential of endophytic P. erinaceus fungi as a source of natural antimicrobial agents, offering interesting prospects for medicine, agriculture and scientific research.

AlCl₃ aluminum trichloride

CaCl₂HO Calcium chloride hydrate

CT colonization rate

H₂O₂ hydrogen peroxide

IR isolation rate

Na₂CO₃ Sodium carbonate

NaCl Sodium chloride

PDA Potato Dextrose Agar

pH Potential of Hydrogen

Ethics approval and consent to participate

The research protocol received approval from the Ethics Committee of the Research Unit in Applied Microbiology and Pharmacology of Natural Substances at the University of Abomey-Calavi in Benin (Approval No. 0044/2023/CE/URMAPha/UAC).

Consent for publications

Not applicable.

Competing interests

The author declares no competing interests.

Funding

This study was funded by the "Programme de Fonds Compétitifs de Recherche de l'Ecole Doctorale des Sciences, Technologies, Ingénierie et Mathématiques (PFCR I/ED-STIM)" of the National University of Science, Technology, Engineering and Mathematics (UNSTIM) through the "Endo_med" Project.

Author contributions

SA, BF, EA, AY, GN, LF and JRK conceptualized and collected data for this work, methodology, data analysis, writing and editing. EA, NG, TYB, FB, CH, TS, RA, participated in the implementation of the activities of this study. VD and LB provided scientific direction. All authors have read and approved the published version of the manuscript.

Acknowledgements

The authors would like to sincerely thank Paola Cynthia Emoh Demeni for her invaluable contribution.

Availability of data and materials

All data generated or analyzed during this study are included in this published. The raw data supporting the conclusion of this article will be made available by the authors without undue reservation.

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Diversity and biological Potential of endophytes of Pterocarpus erinaceus used in Benin (West Africa): A Step Towards Conservation and New Pharmacological Resources

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Abstract

Figures

Background

Methods

Plant material

Bacterial strains

Preparation of the organs

Sterility check

Culture of decontaminated fragments, isolation and purification of fungal endophytes

Morphological and cultural identification of pure colonies

Enzymatic activity of isolated endophytes

Determination of total flavonoids

Statistical analyses

Results

Determination of isolation and colonization rates

Diversity of Endophyte species according to the collection location

Morphological and microscopic characteristics of isolated fungal endophytes

Morphological and microscopic characteristics of isolated bacterial endophytes

Enzymatic activity of isolated endophytes

Screening of antimicrobial activity of fungal endophytes

Antibacterial Activity

Antifungal activity

Determination of the total polyphenol content of endophytes

Determination of total flavonoid content of endophytes

Discussion

Conclusions

Abbreviations

Declarations

Competing interests

Funding

Author contributions

Acknowledgements

Availability of data and materials

References

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