Aliou et al.

Bulletin of the National

Bulletin of the National Research Centre (2024) 48:128
https://doi.org/10.1186/s42269-024-01284-1 Research Centre

RESEARCH Open Access

Antimicrobial and enzymatic potential

of Pterocarpus erinaceus Poir. endophytes used
in Benin (West Africa)
Sophia Aliou1,2, Brice Fanou1, Jean Robert Klotoé1,2* , Eric Agbodjento1, Victorien Dougnon1, Norbert Gbesso1,
Toussaint Yangambele1, Curiace Hinnilo1, Toussaint Sovègnon1, Remi Akotègnon1, Alda Yemadje2,
Guevara Nonviho2, Louis Fagbohoun3 and Lamine Baba‑Moussa4

Abstract
Background Pterocarpus erinaceus Poir. 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 anthropo‑
genic pressure due to its overexploitation, putting it in danger of extinction. The study aims to explore the antimicro‑
bial and enzymatic activities of endophytes produced by Pterocarpus erinaceus.
Methods Fresh leaves, stems, and roots of P. 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 antimicrobial 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 P. erinaceus while exploring its endophytes
for potential therapeutic applications.
Keywords Pterocarpus erinaceus, Endophytes, Enzymes activities, Polyphenol content

*Correspondence:
Jean Robert Klotoé
jrklotoe@yahoo.fr
Full list of author information is available at the end of the article

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Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 2 of 15

Background microorganisms could offer a sustainable source of thera-

Pterocarpus erinaceus Poir. is a very popular plant in tra- peutic substances, thus avoiding the overexploitation of
ditional medicine in Benin. It is used in the treatment P. erinaceus. Fungal endophytes, microorganisms that
of many pathologies, including diabetes, ulcers, intesti- live inside plant tissues, are increasingly studied for their
nal worms, hypertension, and female and male infertil- beneficial properties. Indeed, some enzymes produced
ity (Dougnon et al. 2020; Fanou et al. 2020; Klotoé et al. by these endophytes, such as proteases, have interesting
2013b; Ouinsavi et al. 2021; Vissoh et al. 2024). This benefits for human health (Khan et al. 2017b). They pro-
popularity is part of a broader context where the impor- duce complex and unique classes of secondary metabo-
tance of traditional medicine in primary health care has lites that can constitute new avenues for pharmaceutical
been recognized since the declaration of Alma Ata in the discoveries (Tousif et al. 2023). Studies have shown that
guidelines of the Regional Health for All Policy in the these endophytes are undeniable sources of extracellular
Twenty-first Century (WHO 1978). Indeed, the World enzymes that could constitute new products for medi-
Health Organization (WHO) recommends the integra- cal exploitation in the fight against pathogen resistance
tion of traditional medicine into national health systems to existing conventional antibiotics (Firáková et al. 2007;
and policies in order to improve access to care and pro- Komeil and Saad 2021). The present study therefore aims
mote health (WHO 2013). to explore the antimicrobial and enzymatic activities of
In Benin, more than 70% of the Beninese population endophytes produced by P. erinaceus. It will answer the
continues to use this natural medicine for their primary following questions:
health care needs (WHO 2010). The country’s cultural
diversity and its ancestral heritage in traditional medicine • What types of endophytes can be detected in P. eri-
are a subject of growing interest for scientific research. naceus based on the methods employed in this study?
The flora of Benin is rich and diverse, including about • What are the antimicrobial and enzymatic activities
2807 plant species (Akoegninou et al. 2006). It presents of endophytes produced by P. erinaceus?
more than 2800 taxa, with information on the type of
environment in which they live (dense semi-deciduous
humid forest, grassy savannah, wooded savannah, man- Methods
grove, etc.) and the names of some localities where they Plant material
have been observed or collected. The book is highly The plant material consists of the leaves, stem, and fresh
recommended for traditional healers in Benin and also roots of P. erinaceus collected in the town of Savalou and
provides plant names in 21 national languages in addi- in Abomey-Calavi in the botanical garden of the Univer-
tion to French (Akoegninou et al. 2006). Ethnobotanical sity of Abomey-Calavi. Its healthy organs with no visible
surveys conducted from 2010 to the present day pro- symptoms of disease have been carefully selected. They
vide information on the use of a multitude of medicinal have been certified at the National Herbarium of Benin
plants by the population for various pathologies (Klotoé under the number YH 952/HN. The samples collected
et al. 2013a; Vissoh et al. 2024). This use results in strong in this way were immediately transported under aseptic
anthropogenic pressure that threatens plant biodiver- conditions to the laboratory (Xiaoxiang et al. 2019).
sity and sometimes leads to the disappearance of spe-
cies. Pterocarpus erinaceus is one of these plants that Bacterial strains
are under this anthropogenic pressure. So coveted and The microbial material consisted of four pathogenic bac-
exploited, with a significant increase in the trade in its teria, 02 Gram-negative bacilli, 01 Gram-positive cocci
timber, it is now on the International Union for Conser- strain, and 01 fungal strain, provided by the Research
vation of Nature’s red list as a species in danger of extinc- Unit in Applied Microbiology and Pharmacology of nat-
tion (IUCN 2024). In addition, several scientific studies ural substances (URMAPha) of Polytechnic School of
have proven multiple pharmacological properties such Abomey-Calavi (EPAC). They are Escherichia coli ATCC
as analgesic, antibacterial, anti-diabetic, anti-inflamma- 25922; Pseudomonas aeruginosa ATCC 9027; Staphylo-
tory, antioxidant properties of P. erinaceus (Atchou et al. coccus aureus ATCC 6528 and C. albicans ATCC 90028.
2021; Houmènou et al. 2018; Noufou et al. 2016; Tittik-
pina et al. 2018). In view of these multiple properties and Preparation of the organs
traditional medicinal uses, it is imperative to find alterna- After the samples had been collected and sent to the
tives to preserve its population. Research on endophytes laboratory, the organs were rinsed with tap water to
is promising in this direction, as endophytes, which live remove dust and other waste that might end up on their
in symbiosis with plants, have the ability to produce bio- surface (Marchut-Mikołajczyk et al. 2023). After rinsing,
active compounds similar to those of their hosts. These the organs were cut into small fragments that underwent
Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 3 of 15

sterilization according to the method described by


Isolation rate (IR) = Number of segments yielding a number of
Sharma and Mallubhotla (2022) to remove all microor-
  
isolates ≥ 1 / Total number of segments × 100
ganisms from the surface (epiphytes) and to ensure that Morphological and cultural identification of pure colonies
the isolates would come from within them. Briefly, they Macroscopic observation considered color, general col-
were immersed successively in 70% ethanol for 6 min, ony appearance, pigments, colony size and shape, and
sodium hypochlorite or 6% bleach for 4 min, and then in growth diameter (Khan et al. 2017a). Microscopic obser-
70% ethanol for 4 min. The fragments were then rinsed vation was carried out using the scotch technique (Nacef
three times with sterile distilled water and dried on sterile et al. 2022). This method consists of performing a sim-
tea towel paper to remove epiphytes (Khan et al. 2017a, b; ple staining with methylene blue by depositing a drop of
Ratnaweera et al. 2015). For bacterial endophytes, after methylene blue on a clean, dry microscope slide. Indeed,
this step, the organs were cut into pieces of one cm/1 cm young structures of the fungal colonies were taken with
each, then isolated on NA medium, and incubated in the tape and placed on the slide previously bearing a drop of
oven at 35 °C for 24 h. methylene blue so that the structures of the fungus were
colored and visible to observation. Any excess methylene
Sterility check blue around the blade has been gently cleaned with ster-
The method described by Li et al. (2020) with some mod- ile tissue. The slide was examined under a microscope at
ifications was used for this sterility check. To ensure that 400 and 1000 magnifications. Identification was essen-
the surfaces are sterile and that the colonies to be cul- tially based on the study of the mycelium and the nature
tured actually come from the internal tissues, the third of the differentiated organs.
rinse water was inoculated with Potato Dextrose Agar
(PDA) medium supplemented with Chloramphenicol at
150 mg/l for endophytic fungi (Salo and Novero 2020). Enzymatic activity of isolated endophytes
For bacterial endophytes, the third rinse water was inoc- The catalase activity of endophytes was investigated fol-
ulated on NA medium and incubated in the oven at 35 °C lowing the methodology applied by Attia et al. (2020).
for 24 h. Thus, the absence of colonies on the media indi- Isolated endophytes were tested for hydrogen peroxide
cates that surface sterilization has been done well and ­(H2O2) hydrolysis by the slide method. Indeed, fungal
that all epiphytic microorganisms are eliminated (Mar- strains grown on PDA agar and then incubated at 30 °C
sola et al. 2022). for 7 days were used. A drop of hydrogen peroxide was
placed on the test slides, and then, a 1­ mm2 fragment of
Culture of decontaminated fragments, isolation, young preculture of fungal endophytes was taken using
and purification of fungal endophytes a platinum loop and deposited in the drop of hydrogen
After sterilization, the samples were cut into ­ 1mm2 peroxide. Peroxidase production was visually detected
fragments and placed in petri dishes containing Potato by observing the immediate bubbling (Attia et al. 2020).
Dextrose Agar (PDA) agar supplemented with 0.5 g/l of The cellulase activity of endophytes was tested by
chloramphenicol (PDA) and previously autoclaved at using PDA medium supplemented with 0.5% carboxy-
121 °C for 15 min, and aseptically supplemented with methylcellulose was used. After 3–5 days of growth
10 g/l of chloramphenicol to inhibit bacterial growth of the fungal strains, the plates were flooded with a
(3 segments per dish). Different dishes were incubated 0.2% Congo red aqueous solution and discolored with
at 37 °C for 5 to 7 days. For bacterial endophytes, the 1 M NaCl for 15 min. The appearance of yellow areas
organs were cut into pieces of one cm/1 cm each, then around the fungal colony testifies to the production of
isolated on NA medium, and incubated in the oven cellulase by the fungal colony (Marsola et al. 2022).
at 35 °C for 24 h. After incubation, all the fungal endo- Protease activity was assessed using the method of
phytes that form colonies were then isolated and placed Usman et al. (2023). A PDA medium containing 0.4%
in new petri dishes containing PDA, until pure colonies casein (pH 6.0) was used for this purpose. After an
were obtained. Each isolated and purified mushroom was incubation period of 3 to 5 days, casein degradation
stored on PDA at 4 °C. The same process was followed for appears as a clear area around the colonies indicat-
bacterial endophytes using NA medium. After the incu- ing the production of the protease by the fungal strain
bation period, endophyte colonization and isolation rates (Usman et al. 2023).
were calculated using the following formulas (Jayatilake To test the lipase activity of endophytic fungi,
et al. 2020): peptone agar medium (peptone 10 g, NaCl 5 g,

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

Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 4 of 15

­CaCl2HO—0.1 g, agar—16 g, distilled water—1L; pH The experiment was carried out in triplicate for each fun-
6.0) supplemented with 1% Tween 20 was used. After gal endophyte. Finally, plates containing MHA medium
the incubation period of 3 to 5 days, the appearance of and endophyte fungal were incubated at 37 °C for 24 h
a visible precipitate around the colonies indicates posi- before observation of the zones of inhibition. The diam-
tive lipase activity promoted by the formation of cal- eters of these zones were measured and averaged in mm
cium salts of lauric acid released by the enzyme (Hawar (Jayatilake and Munasinghe 2020).
2022).
Amylase activity was investigated using the method
Screening fungal endophytes for antifungal activity
used by Taneja et al. (2023). A PDA medium contain-
Antifungal activity was assessed using the agar cylinder
ing 0.2% starch (pH 6.0) was used. After an incubation
technique as described by Jayatilake and Munasinghe
period of 3 to 5 days, starch degradation appears in the
(2020) with slight modifications. This method involved
form of a clear area around the colonies after a revelation
inoculating young cultures of the pathogenic fungus
with Lugol concentrated at 2% testifying to the produc-
(Candida albicans) onto nutrient agar and then placing
tion of amylase by the fungal strain (Taneja et al. 2023).
agar plates containing fungic endophytic of around 6 mm
diameter (from a 7-day culture on a PDA plate) onto
Antimicrobial activity of isolated endophytes the same agar. The inoculated plates were incubated at
of Pterocarpus erinaceus 28 °C. After incubation for 3–5 days, the diameters of the
Screening for antibacterial activity of bacterial endophytes zone of inhibition around the disk were measured and
The antibacterial activity of bacterial endophytes was recorded.
performed using the agar diffusion technique follow-
ing the methodology described by Marchut-Mikołajczyk
et al. (2023). To this end, all bacterial endophyte strains Screening for antifungal activity of bacterial endophytes
were cultured in nutrient broth (NB) for 24 h at 37 °C. The antifungal activity of bacterial endophytes was deter-
After incubation, 10 ml of each endophytic bacterial mined by the previously used agar cylinder method
culture was centrifuged at 1000 rpm for 10 min and the applied by Photolo et al. (2020). A fungal strain of Can-
supernatant recovered. 24-h colonies of the test strains dida albicans was grown overnight at 37 °C in Mueller
were plated on MH agar using the depletion technique. Hinton (MH) broth. Then 10 µl of the broth was inocu-
6-mm-diameter wells were then drilled into the agar lated onto MH agar, and disks (approx. 6 mm) of each
using a sterile cork borer. Next, 100 µl of the supernatants endophytic bacterium from an overnight culture on
previously obtained from each endophytic bacterium was nutrient agar (NA) medium were plated onto the same
deposited in each well. The dish was incubated for 24 h at MH agar. The plates were incubated at 37 °C for 24 to
37 °C after a 15-min pre-diffusion on the bench. Inhibi- 72 h. The experiment was performed three times. After
tion diameters around the wells were read and recorded. 72 h, the inhibition diameters were read off in mm.
The test was performed three times. Gentamicin was
used as the positive control and sterile distilled water as Determination of total polyphenols of endophytes
the negative control (Marchut-Mikołajczyk et al. 2023). of Pterocarpus erinaceus
The determination of total polyphenols was performed
Screening fungal endophytes for antibacterial activity by spectrophotometry, according to the colorimetric
For the evaluation of the antibacterial activity of isolated method using the Folin-Ciocalteux reagent of Singleton
endophytes, the Agar cylinder method was used (Pelo et al. (1999). A volume of 50 μL of solution (1/100 dis-
et al. 2020). Various pathogenic bacteria were first inocu- tilled water) of a broth in which the endophytes have
lated onto nutrient agar (NA) to obtain young cultures. been previously cultured was added to 250 μL of the 10%
It is composed of peptone, NaCl, meat extract, yeast Folin–Ciocalteu reagent (diluted 10 in distilled water)
extract, and agar. It was used in this study to determine and 750 μL of an aqueous solution of sodium carbonate
the effectiveness of isolated endophytes in inhibiting ­(Na2CO3) at 75 g/L.
the growth of pathogenic bacteria and fungi. Suspen- After 8 min of incubation, 950 μL of distilled water was
sions (turbidity 0.5 McFarland) were then prepared from added and mixed with the vortex and then incubated
these young cultures (Jayatilake and Munasinghe 2020). for 2 h in the dark at room temperature. After incuba-
1 ml of suspension was inoculated onto Mueller Hinton tion, optical densities (ODs) were read at 760 nm using
agar (MHA) using the depletion method. Agar plates the CECIL CE 2041 spectrophotometer. The reading was
containing endophytic champions about 6 mm in diam- taken against a blank consisting of a mixture of 250 μL
eter from young cultures (on chlorophenicol-free PDA of FCR (Folin–Ciocalteu reagent) and 750 μL of N ­ a2CO3
medium) were picked and plated onto MHA medium. and 1 mL of distilled water.
Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 5 of 15

Determination of total flavonoids • RFLP

The quantification of flavonoids was carried out by a
method adapted from Zhishen et al. (1999) and Kim For Chrysosporium identification, the ITS2 region
et al. (2003) using aluminum trichloride ­(AlCl3) as the was amplified using ITS86 and ITS4 primers. The ther-
reagent. Thus, 500 μL of an A ­ lCl3 solution (2%) was mal cycling conditions were as follows: initial denatura-
taken and added to 500 μL of diluted endophyte broth. tion at 94 °C for 5 min; 30 cycles of denaturation at 94 °C
To this mixture was added 3 mL of ethanol. The white for 1 min, annealing at 55 °C for 1 min, and extension
consists of 500 μL of A­ lCl3 and 3.5 mL of ethanol. The at 72 °C for 1 min; followed by a final extension at 72 °C
absorbance reading was done with a spectrophotom- for 7 min and cooling at 4 °C. BstUI restriction diges-
eter at 415 nm after a 10-min incubation. Samples were tion was performed on the amplified ITS2 region. Two
prepared in triplicate for each assay. The total flavo- microliters of the amplification product were transferred
noid content was determined in mg rutin equivalent/g to a new Eppendorf tube, where 1 μl BstUI (10,000 U/ml,
extract (μg RuE/g) by the formula used by Ahmed et al. CG/CG; New England BioLabs), 2 μl of BstUI restriction
(2019). buffer, and 15 μl of distilled water were added. The mix-
ture was incubated for 2 h at 60 °C. Following digestion,
Molecular identification of the two most active endophytes the 20 μl DNA restriction mixture was purified by pre-
The two isolates (HT1a and HF2b) which showed anti- cipitation with 2 μl of 3 M sodium acetate, 50 μl of 95%
microbial activity and, respectively, belong to genus ethanol, and 0.2 μl of 20 mg/ml dextran blue, followed by
Chrysosporium spp and Alternaria spp were selected centrifugation at 10,000 g for 30 min. The resulting pel-
for molecular identification. let was washed with 250 μl of 70% ethanol and air-dried.
For fragment length determination, 11.5 μl of forma-
• DNA Extraction mide, 1 μl of the nondigested ITS2 amplicon, 0.3 μl of the
HD400 marker (Applied Biosystems), and 0.1 μl of the
DNA was extracted from presumptively isolated fun- ROX500 marker (Applied Biosystems) were added to the
gal colonies using the Zymo kit (Quick-DNA Fungal/ ethanol-precipitated digest. DNA fragments are analyzed
Bacterial Miniprep Kit) and following the manufactur- by electrophoresis on a 1.5% agarose gel. Migration was
er’s recommendations. carried out for 30 min at 100 V, and images were taken
using Biorad’s Gel Doc EQ imaging system, followed by
interpretation of the results based on comparison of frag-
Nested PCR
ment migration with marker sizes. The specific product
For identification of Alternaria species, the first round of length was found to be 128 bp. The primer sequences
nested PCR was conducted using the universal primer used are summarized in Table 1.
pair ITS1 and ITS4. The reaction mixture, prepared
according to the manufacturer’s recommendations (One-
Statistical analyses
Taq PCR, New England Biolabs, Massachusetts, USA),
The data obtained were subjected to statistical analy-
consisted of 12 µl of OneTaq, 0.5 µl of each primer, 3 µl of
sis using SPSS 26.0 software and Microsoft Excel 2016
DNA, and nuclease-free H₂O to reach a final volume of
spreadsheets. Qualitative variables were expressed as
25 µl. The PCR cycle consisted of 35 cycles with an initial
percentages. Quantitative variables are presented as a
denaturation at 94 °C for 5 min, secondary denaturation
mean and standard deviation. The analysis of variance
at 94 °C for 30 s, annealing at 56 °C for 40 s, extension at
(ANOVA) has a single factor and was used to assess the
72 °C for 45 s, and a final extension at 72 °C for 45 s.
variation in the content of polyphenols, flavonoids, and
For species-specific detection, 1 µl of the first PCR
total tannins of the endophytes studied. The significance
product was used in a subsequent PCR with primers
threshold has been set at 5%.
specific for A. alternata (Al) and A. carthami (AcSP).
The conditions for this PCR were as follows: an initial
denaturation at 94 °C for 2 min, followed by 20 cycles of Results
denaturation at 94 °C for 45 s, annealing at 55 °C for 30 s, Determination of isolation and colonization rates
extension at 72 °C for 45 s, and a final extension at 72 °C The results obtained indicated a colonization rate (CT)
for 10 min. The resulting DNA fragments were analyzed of 44.44%, which means that 44.44% of the segments
by electrophoresis on a 1.5% agarose gel, run for 30 min evaluated were colonized by fungal isolates showing
at 100 V. Images were captured using the Gel Doc EQ the presence of fungi in the analyzed samples (Fig. 1).
imaging system (Bio-Rad), and results were interpreted For bacterial endophytes, 80.55% was obtained as a per-
by comparing the migration of fragments to size markers. centage of colonization. As for the isolation rate (IR),
it is 50%, indicating that half of the segments evaluated
Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 6 of 15

Table 1 Primers sequences

Genes Primers sequences Species Size (pb) References

ITS1-ITS4 F-TCC​GTA​GGT​GAA​CCT​GCG​G Fungi 500–750 Rahman et al. (2020)

R-TCC​TCC​GCT​TAT​TGA​TAT​GC
AL F-GTC​T TT​TGC​GTA​C TT​C TT​GTTT​ Alternaria alternata 249 Zhenyan et al. (2021)
R-GAA​CAG​GCA​TGC​CCT​T TG​GA
AcSP F-GTG​GCC​AGC​C TT​GCT​GAA​T T Alternaria carthami 340 Zhenyan et al. (2021)
R-CCT​C TC​TGT​T TT​TCT​GCG​GG
ITS2-ITS4 F-GTG​AAT​CAT​CGA​ATC​T TT​GAAC​ Chrysosporium group 306 De Baere et al. (2010)
R-TCC​TCC​GCT​TAT​TGA​TAT​GC

Fig. 1 Colonization and isolation rates of fungal and bacterial

endophytes

Fig. 2 Variation in the number of fungal endophytes according

to the collection sites of Pterocarpus erinaceus

yielded at least one fungal isolate. For bacterial endo-

phytes, 69.44% of the segments yielded at least one bacte-
rial isolate (Fig. 1). Thus, there is more colonization for collected at the National Herbarium (38.23%). In terms
bacterial endophytes compared to fungal endophytes. of plant parts, more fungal and bacterial isolates were
The same trend is observed for the isolation rate. These isolated from leaves than other parts for both collection
data suggest that bacterial endophytes are more widely sites.
present and isolated in the samples studied compared to Figure 3 presents the number of bacterial endo-
fungal endophytes. phytes identified in different plant organs (leaf, stem,
root) according to two collection sites: Herbarium of
Diversity of endophyte according to the collection location the University of Abomey-Calavi and in the commune
Analysis of Fig. 2 reveals a marked variability in the of Savalou. The analysis of this figure reveals that the
distribution of fungal endophytes between the two col- roots of the plants collected at Savalou contain the
lection sites and different parts of the plant. A total of highest number of bacterial endophytes (8), while the
thirty-four endophytes were isolated from the plant at leaves of the plants in the Herbarium contain the most
the two collection sites. Samples collected in the com- (5). In general, Savalou plants appear to have a greater
mune of Savalou were richer in endophytes (61.76%) diversity of bacterial endophytes in the stems and roots
than those from the commune of Abomey-Calavi compared to herbarium plants.
Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 7 of 15

Morphological and microscopic characteristics of isolated

fungal endophytes
The results of the study of the cultural and microscopic
characteristics of fungal endophytes of P. erinaceus are
presented in Table 2. 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.
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 out-
line, 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
Fig. 3 Number of bacterial endophytes identified in different plant belong to the genus Chrysosporium spp. HT1c has a
organs (leaf, stem, root) according to two collection sites
fluffy texture with a dark brown center, a whitish outline,
a white and irregular border, a rigid appearance, and a

Table 2 Morphological and microscopic characteristics of fungal endophytes

Codes of isolates Morphology Genus

SR2c Hyaline mushroom, glabrous, brown back, pleated and white underside, raised, rigid, waist 3/2.6 cm, with‑ Penicillium spp.
out diffusible pigment. Presence of simple arthroconidia
HR1c Hyaline mushroom, glabrous, brown back, pleated and green underside, raised, rigid, waist 3/2.6 cm, with‑ Penicillium spp.
out diffusible pigment. Hyphae unseptate, conidiophore branched biseriate in a brush, conidia round in clus‑
ters and chains
HT1c texture, dark brown, white center; whitish outline, white and irregular border, rigid appearance and size Chrysosporium spp.
3/2.6 cm, pigment-free
HF2b Pleated black center and dark green outline, ash backing, irregular border, powdery appearance, 3 cm diam‑ Alternaria spp.
eter and without pigment
HF1b Pleated black center and dark green outline, ash underside, irregular border, cottony look, 3 cm diameter Alternaria spp.
and pigment-free
ST2c Brown black, white reverse, creamy appearance, irregular border, 2 cm diameter, pigment-free and presence Acremonium spp.
of arthrospore
ST2a Pleated black center and dark green outline, ash underside, irregular border, cottony look, 3 cm diameter Not identified
and pigment-free
HF2c Pleated black center and dark green outline, ash underside, irregular border, cottony look, 3 cm diameter Alternaria spp.
and pigment-free
HF1a Pleated black center and dark green outline, ash underside, irregular border, cottony look, 3 cm diameter Not identified
and pigment-free
HF1c Pleated green center and white outline, white underside, regular border, cottony appearance, 3 cm diameter Chrysosporium spp.
and without pigment
HT1c Pleated black center and dark green outline, ash underside, irregular border, cottony appearance, 3 cm diam‑ Penicillium spp.
eter and without pigment
HF3c White colony Unidentified
Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 8 of 15

dimension of 3/2.6 cm, without pigment. HF1c is char- observed, 56% of the strains have endospores while
acterized by a pleated morphology with a green center, 44% do not. Based on this characteristic, the presence
a white outline, a white underside, a regular border, and or absence of endospores 56% of the strains with these
a cottony texture, 3 cm in diameter, without pigment. endospores belongs to the genus Bacillus.
HT1a also has a pleated texture, with a black back and
back, irregular border, stiff appearance, and a 3/2 cm size,
without pigment. Enzymatic activity of isolated endophytes
Isolates HF1b, HF2c, and HF1a belong to the genus Figure 4 shows the percentages of bacterial and fungal
Alternaria spp. All have a pleated morphology with a endophyte isolates expressing various enzymatic activi-
black center and a dark green outline, an ashy underside, ties. For lecithinase, 85.18% of bacterial endophyte iso-
an irregular border, a cottony appearance, and a diameter lates express this activity, while no fungal endophyte
of 3 cm, without pigment. isolates do. Amylase activity was noted in both endo-
Isolates HF3a, SR1c, HF3b, and HF3c (second occur- phyte groups, but higher in bacterial (74%) compared to
rence) belong to the genus Aspergillus spp. HF3a has a fungal (21.05%). The expression of the protease reveals
pleated morphology with a black back and back, an irreg- another marked distinction: 44.45% of bacterial isolates
ular border, and a rigid appearance, measuring 3/2 cm show this activity compared to 0% for fungal isolates.
without pigment. SR1c has a powdery texture, with a Catalase activity is universally expressed in both endo-
beige back color and a whitish underside, an irregular phyte types, with 100% of bacterial and fungal isolates
border, and a cottony texture, 3/2.6 cm in size and with- showing this activity. However, no lipase activity was
out pigment. HF3b shows a powdery texture with alter- detected in either group, and cellulase activity was very
nating beige and brown color, an irregular border, and low in bacteria (3.7%) and absent in fungi.
a cottony appearance, with a diameter of 5 cm without
pigment. Finally, HF3c is a white colony. Isolates ST1a
Screening of antimicrobial activity of fungal endophytes
and HF3c (first occurrence) belong to the genus Fusar-
Antibacterial activity
ium spp. ST1a has a soft morphology, with a beige back
Of the 34 endophytic tested, only two isolates (HT1a
and back, an irregular border, and a creamy appearance,
and HF2b) showed antibacterial activity against the
measuring 2.6/2 cm, without pigment. HF3c is described
three bacterial strains used, namely Pseudomonas aer-
as a black, powdery colony with shades of black and
uginosa, Escherichia coli and Staphylococcus aureus.
white, an irregular border, a cottony texture, and a size of
The mean areas of inhibition measured ranged from 0 to
2.5/2 cm.
35 ± 0.9 mm. HF2b and HT1a isolates were more active
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 cot-
tony texture, and a diameter of 3 cm, without pigment.
Isolates ST2a and HF1a could not be identified. HF2b
belongs to Alternaria spp. It has a pleated morphology
with a black center and a dark green outline, an ashy
underside, an irregular border, a powdery texture, meas-
uring 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 irregu-
lar 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 tex-
ture, others with a mucoid texture, and still others with
a dry texture. Microscopic observation, after gram stain- Fig. 4 Expression of enzymatic activity of bacterial and fungal
endophytes
ing, showed gram-positive bacilli. Of all the samples
Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 9 of 15

on E. coli and Staphylococcus aureus. However, none of Molecular identification of most actives’ endophytes
the bacterial isolates were active against its strains. HF2b and HT1a were confirmed, respectively, as Alter-
naria alternata and Chrysosporium group (Figs. 8 and
Antifungal activity 9).
Of the 34 isolates tested, only HF2b showed fungistatic
action on C. albicans with an inhibition diameter of
23 ± 0.9 mm. However, none of the bacterial isolates were Discussion
active on C. albicans. This study aimed to explore the antimicrobial and enzy-
matic activities of endophytes produced by P. erinaceus.
Determination of the total polyphenol content The results of the determination of the colonization rate
of endophytes and the isolation rate of fungal endophytes indicate that
Figure 5 shows the polyphenol content of fungal iso- there is more colonization for bacterial endophytes com-
lates. Isolates HT1a, HF1b, HF2b, showed high polyphe- pared to fungal endophytes. The same trend is observed
nol contents. Conversely, isolates with lower levels were for the isolation rate. These data suggest that bacterial
HF1c, HR1c. endophytes are more widely present and isolated in the
Figure 6 shows the polyphenol content of bacterial iso- samples studied compared to fungal endophytes. In the
lates. From the analysis of this figure, it appears that there literature, it is reported that the community of bacte-
is no significant difference between the total polyphenol rial endophytes is influenced by biotic and abiotic fac-
content of different isolates. tors, which shape their species composition, community
structure, diversity, and functions (Walitang et al. 2018).
Determination of total flavonoid content of endophytes Environmental factors affect not only the distribution
Figure 7 shows the flavonoid content of different bacte- of a medicinal plant, but also the determination of the
rial endophytic strains. From this figure, it appears that bacterial endophyte that can colonize the host plant
the F3b and SR2C, HT1C and T2b1 strains have high fla- throughout its life cycle (Deng et al. 2011; Wu et al. 2021).
vonoid contents compared to the HT1a, R1b and T1C1 Endophyte diversity is also influenced by host plant char-
strains. As for fungal endophytes, they have a very low acteristics, including genotype (Walitang et al. 2018; Wu
content of flavonoids that can be assimilated to traces. et al. 2021), tissue (Dai et al. 2014), growth stage (age),
and health status (Bogas et al. 2015).

Fig. 5 Polyphenol content of fungal isolates

Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 10 of 15

Fig. 6 Polyphenol content of bacterial isolates

Fig. 7 Flavonoid content of bacterial isolates

Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 11 of 15

bacterial endophyte in the stems and roots. The differ-

ences observed between the two collection sites suggest
that local environmental and ecological conditions may
play a crucial role in the diversity and distribution of fun-
gal endophytes. Indeed, Deng et al. (2011) reported that
variations in microclimate, soil composition, and sur-
rounding vegetation influence the colonization of plants
by endophytes. Different parts of the plant (leaves, stems,
roots) provide varied habitats for endophytes, with spe-
cific micro-environmental conditions (LIU et al. 2020;
Fig. 8 Electrophoretic profiles of ITS1-ITS4 regions and the AL gene Zhang et al. 2019). In Benin, the commune of Abomey-
for Alternaria alternate. a electrophoretic profile of the ITS1-ITS4
Calavi (first sample collection site), and the commune of
regions (500-750 bp); b electrophoretic profile of the of AL gene
(249 bp); (M): gene ruler 100 bp; HF2b: Alternaria alternata Savalou (second collection site) belong, respectively, to
the Guinean-Congolese zone and the Sudano-Guinean
zone, two distinct phytogeographical zones of the coun-
try. The Guinean-Congolese zone is located in southern
Benin and is characterized by tropical rainforest veg-
etation. 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 mat-
ter 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 cul-
Fig. 9 Electrophoretic and restriction fragment profiles tivation of cash crops such as oil palm, cocoa, and other
of the ITS2-ITS4 region for the Chrysosporium group. a tropical crops (Imorou et al. 2015). The Sudanian zone of
Electrophoretic profile of the ITS2-ITS4 regions (306 bp); b BstUI
northern Benin is influenced by the characteristics of its
restriction fragment profiles of the ITS2-ITS4 region/(M): gene ruler
100 bp; HT1a: Chrysosporium group (128 bp) tropical ferruginous soils, which are often shallower and
less fertile than those in the south. The low organic mat-
ter content and the texture varying from sandy to sandy-
The results reported that the roots of the plants col- clayey, with a moderate to low water holding capacity,
lected at Savalou contain the highest number of bacterial limit microbial diversity. Well-drained, but sometimes
endophytes (8), while the leaves of the plants in the Her- drought-prone soils, combined with sparse savannah veg-
barium contain the most (5). In general, Savalou plants etation, further reduce endophyte diversity. In contrast,
appear to have a greater diversity of bacterial endophytes the central Sudano-Guinean zone, with its intermediate
in the stems and roots compared to herbarium plants. tropical and ferralitic ferruginous soils, has moderate fer-
This richness of the roots and stem in bacterial endo- tility and organic matter content, providing more favora-
phytes is comparable to the results reported by Liu et al. ble conditions for a richer endophytic diversity of plants
(2020) compared to the Paris polyphylla var. yunnanen- (Salako et al. 2018).
sis, a famous and endangered traditional Chinese herb In addition, the endophyte richness in the leaves of the
that has significant medicinal value. This endophyte rich- National Herbarium of the commune of Abomey-Calavi
ness can also be observed in plants growing in unfavora- could be due to factors such as relative humidity, nutrient
ble extreme environments such as Distichlis and Pluchea availability, and interactions with other microorganisms
absinthioides, Gaultheria mucronata and Hieracium (Wu et al. 2021). These results may have applications in
pilosella which grow in extreme environments in Chile. Biotechnology and Agriculture. Indeed, knowledge of
In addition, the results highlight a diversity of fun- the distribution and diversity of endophytes can have
gal endophyte depending on the place of collection. important applications in biotechnology and agricul-
Samples collected at the National Herbarium showed ture, particularly in the development of biopesticides and
a greater richness in endophytes, particularly in the biofertilizers. The endophyte richness of the leaves of the
leaves, unlike Savalou. In contrast, Savalou has a rich
Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 12 of 15

National Herbarium and those of the roots of Savalou HF1c isolates showed lower concentrations. However,
could be explored for beneficial properties. these polyphenol and flavonoid contents are very low
In addition, the results obtained have shown, from the compared to those reported (Toukam et al. 2016) for
analysis of their cultural and microscopic characteristics, the acetate and butanol extracts of the plant. The pres-
that these endophytes belong to six genera, the most rep- ence of polyphenols is often correlated with the anti-
resented of which are Aspergillus spp., Penicillium spp., microbial properties of medicinal plants, which could
Alternaria spp., Fusarium spp. This diversity indicates a partly explain the activities observed. In particular,
rich population of endophytes within P. erinaceus. In the the high polyphenol content of isolate HF2b could be
literature, a number of previous studies have reported the related to its antimicrobial efficacy against both bacte-
presence of these fungal genera as a dominant group of ria and C. albicans.
endophytes residing in association with different medici- These data suggest that some fungal endophytes, such
nal plants (Ferreira et al. 2020; Li et al. 2022; Shah et al. as HF2b and HT1a, possess significant antimicrobial
2018; Talukdar et al. 2021). The variations observed in capabilities, potentially due to their polyphenol content.
cultural and microscopic characteristics, even within the HF2b is particularly distinguished by its dual role, show-
same genus, suggest a specific adaptation of endophytes ing activity against both pathogenic bacteria and C. albi-
to their environment or to the part of the plant colonized. cans. These findings hold promise for the development of
The presence of a diversity of endohytes can have impli- novel antimicrobial agents based on fungal endophytes.
cations for the health and resilience of host plants. The results of this study open up promising prospects
The identified endophytes genus, such as Penicillium for the development of new antibiotics. The fungal iso-
spp and Aspergillus spp, are known for their abilities to lates HT1a and HF2b, which have demonstrated sig-
produce bioactive enzymes and metabolites. nificant antibacterial activity, could serve as a basis for
Regarding bacterial endophytes, most isolates belong creating antibiotics that are effective against bacteria
to the genus Bacillus. Bacillus are Gram-positive bacteria, that are resistant to current drugs. This breakthrough is
which are able to withstand adverse environmental con- crucial in the face of increasing bacterial resistance that
ditions through the production of endospores. This genus threatens global public health. In addition, HF2b fungal
is one of the most common for bacterial endophytes of isolate, with its dual antibacterial and antifungal activity,
medicinal plants (Bolivar-Anillo et al. 2021). is of particular interest for medical applications. It could
The study of the screening of the antimicrobial activ- be explored to treat mixed bacterial and fungal infec-
ity of fungal endophytes has revealed, unlike bacterial tions, which are often complex to manage with current
endophytes, interesting and promising results regarding treatments. This could improve treatment options for
the potential of these microorganisms in the fight against patients suffering from such infections and reduce asso-
bacterial and fungal pathogens. Among the fungal endo- ciated complications.
phytes tested, two isolates in particular, HT1a and HF2b, In addition, research on the total polyphenol content of
demonstrated significant antibacterial activity against endophytes is also highlighted by this study. The observed
strains of Pseudomonas aeruginosa, Escherichia coli, and correlation between polyphenol content and antimicro-
Staphylococcus aureus. These isolates showed notable bial activity suggests that manipulating polyphenol levels
areas of inhibition, with diameters up to 35 ± 0.9 mm, in fungal endophytes may increase their antimicrobial
indicating strong antimicrobial activity. Comparable efficacy. However, bacterial endophytes did not show
results have been obtained for plant extracts from other antibacterial activity against the tested strains. This could
authors in the literature (Tittikpina et al. 2018). Antifun- be explained by the low polyphenol content in these bac-
gal activity was also explored, revealing that only isolate terial endophytes compared to fungal endophytes.
HF2b demonstrated fungistatic action against C. albi- Finally, the implications for agriculture and food pres-
cans, with an inhibition zone of 23 ± 0.9 mm. This sug- ervation are notable. Fungal endophytes could be used
gests that although fewer isolates showed antifungal to develop natural biopesticides or food preservatives,
activity compared to antibacterial activity, some fungal providing an alternative to synthetic chemicals. This
endophytes possess properties that can be exploited to approach could not only reduce reliance on these chemi-
combat fungal infections. Tittikpina et al. (2019) reported cals, but also contribute to more sustainable and environ-
that root extract showed significant antifungal activity mentally friendly agricultural and conservation practices.
against A. fumigatus with a MIC value of 16 μg/mL. In summary, this study highlights the potential of
The polyphenol content of fungal isolates was also endophytic fungi of P. erinaceus as a source of natural
determined, revealing significant variation between dif- antimicrobial agents, offering interesting and diverse
ferent isolates. HR1c, HF2b, and HF1c isolates showed perspectives for medicine, agriculture, and scientific
the highest concentrations of polyphenols, while H and research.
Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 13 of 15

Conclusions Availability of data and materials

All data generated or analyzed during this study are included in this pub‑
This study revealed significant diversity and biological lished. The raw data supporting the conclusion of this article will be made
potential of P. erinaceus endophytes, demonstrating available by the authors without undue reservation.
notable fungal colonization with high colonization and
isolation rates. The isolation of a multitude of endo- Declarations
phytes from leaves, stems, and roots, from two distinct
Ethics approval and consent to participate
sites, has highlighted a diversity of endophyte influ- The research protocol received approval from the Ethics Committee of the
enced by local environmental conditions. The genera Research Unit in Applied Microbiology and Pharmacology of natural sub‑
Aspergillus, Penicillium, Alternaria, and Fusarium were stances at the University of Abomey-Calavi in Benin (Approval No. 0044/2023/
CE/URMAPha/UAC).
the most represented for fungal endophytes and the
genus Bacillus for bacterial endophytes. Consent for publications
The results of the antimicrobial activity screening Not applicable.
showed that the HT1a and HF2b isolates possess sig- Competing interests
nificant antibacterial capabilities, while HF2b also dem- The author declares no competing interests.
onstrated antifungal action. These isolates can serve
Author details
as a basis for the development of new antibiotics, par- 1
Research Unit in Applied Microbiology and Pharmacology of natural
ticularly against bacteria resistant to current drugs. substances, University of Abomey-Calavi (UAC), Abomey, Benin. 2 Multidis‑
The correlation between polyphenols and antimicro- ciplinary Research Laboratory for Technical Education (LARPET), National
University of Science, Technology, Engineering and Mathematics (UNSTIM),
bial activity opens up avenues of research to increase Abomey, Benin. 3 Kaba Laboratory for Research in Chemistry and Applications
the effectiveness of these endophytes. This study high- (LaKReCA), National University of Science, Technology, Engineering and Math‑
lights the potential of endophytic P. erinaceus fungi as ematics (UNSTIM), Abomey, Benin. 4 Laboratory of Biology and Molecular
Typing in Microbiology, University of Abomey-Calavi (UAC), Abomey, Benin.
a source of natural antimicrobial agents, offering inter-
esting prospects for medicine, agriculture, and scien- Received: 10 July 2024 Accepted: 1 December 2024
tific research.
Abbreviations
AlCl3 Aluminum trichloride
CaCl2HO Calcium chloride hydrate References
CT Colonization rate Ahmed R, Tariq M, Hussain M, Andleeb A, Masoud MS, Ali I, Mraiche F, Hasan
H2O2 Hydrogen peroxide A (2019) Phenolic contents-based assessment of therapeutic potential
IR Isolation rate of Syzygium cumini leaves extract. PLoS ONE 14(8):e0221318. https://​doi.​
Na2CO3 Sodium carbonate org/​10.​1371/​journ​al.​pone.​02213​18
NaCl Sodium chloride Akoegninou A, Van Der burg W, Van der Maesen L (2006) Flore Analytique du
NA Nutrient agar Bénin [Analytical Flora of Benin], Backhuys. Backhuys. https://​edepot.​wur.​
PDA Potato dextrose agar nl/​281595
pH Potential of hydrogen Atchou K, Lawson-Evi P, Eklu-Gadegbeku K (2021) In vitro study of protective
ITS Internal transcribed spacer effect of Pterocarpus erinaceus Poir. stem bark and Amaranthus spinosus
bp Base pair L. root extracts on cataractogenesis and glomerulopathy. Bull Natl Res
BstUI Thermo Scientific Bsh1236I (BstUI) restriction enzyme Centre. https://​doi.​org/​10.​1186/​s42269-​021-​00552-8
RFLP Restriction fragment length polymorphism Attia EZ, Farouk HM, Abdelmohsen UR, Mo’men H (2020) Antimicrobial and
PCR Polymerase chain reaction extracellular oxidative enzyme activities of endophytic fungi isolated
DNA Deoxyribonucleic acid from alfalfa (Medicago sativa) assisted by metabolic profiling. S Afr J Bot
134:156–162. https://​doi.​org/​10.​1016/j.​sajb.​2019.​12.​003
Acknowledgements Bogas AC, Ferreira AJ, Araújo WL, Astolfi-Filho S, Kitajima EW, Lacava PT,
The authors would like to sincerely thank Paola Cynthia Emoh Demeni for her Azevedo JL (2015) Endophytic bacterial diversity in the phyllosphere of
invaluable contribution. Amazon Paullinia cupana associated with asymptomatic and symp‑
tomatic anthracnose. Springerplus 4:258. https://​doi.​org/​10.​1186/​
Author contributions s40064-​015-​1037-0
SA, BF, EA, AY, GN, LF, and JRK conceptualized and collected data for this Bolivar-Anillo HJ, González-Rodríguez VE, Cantoral JM, García-Sánchez D,
work, methodology, data analysis, writing and editing. EA, NG, TYB, FB, CH, Collado IG, Garrido C (2021) Endophytic bacteria Bacillus subtilis, Isolated
TS, RA participated in the implementation of the activities of this study. VD from Zea mays, as Potential biocontrol agent against Botrytis cinerea. Biol‑
and LB provided scientific direction. All authors have read and approved the ogy (Basel) 10:492. https://​doi.​org/​10.​3390/​biolo​gy100​60492
published version of the manuscript. Dai JX, Liu XM, Wang YJ (2014) Diversity of endophytic bacteria in Caragana
microphylla grown in the desert grassland of the Ningxia Hui autono‑
Funding mous region of China. Genet Mol Res 13:2349–2358. https://​doi.​org/​10.​
This study was funded by the “Programme de Fonds Compétitifs de Recherche 4238/​2014.​April.3.7
de l’Ecole Doctorale des Sciences, Technologies, Ingénierie et Mathématiques De Baere T, Summerbell R, Theelen B, Boekhout T, Vaneechoutte M (2010)
(PFCR I/ED-STIM)” of the National University of Science, Technology, Engineer‑ Evaluation of internal transcribed spacer 2-RFLP analysis for the identifica‑
ing and Mathematics (UNSTIM) through the “Endo_med” Project. tion of dermatophytes. J Med Microbiol 59:48–54. https://​doi.​org/​10.​
1099/​jmm.0.​013870-0
Deng ZS, Zhao LF, Kong ZY, Yang WQ, Lindström K, Wang ET, Wei GH (2011)
Diversity of endophytic bacteria within nodules of the Sphaerophysa
Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 14 of 15

salsula in different regions of Loess Plateau in China. FEMS Microbiol Ecol variations in the endophytic fungal community. Front Microbiol. https://​
76:463–475. https://​doi.​org/​10.​1111/j.​1574-​6941.​2011.​01063.x doi.​org/​10.​3389/​fmicb.​2022.​981070
Dougnon V, Hounsa E, Koudokpon H, Legba BB, Fabiyi K, Sintondji K, Afaton Liu TH, Zhou Y, Tao WC, Liu Y, Zhang XM, Tian SZ (2020) Bacterial diversity
A, Akouta M, Klotoe JR, Bankole H, Baba-Moussa L, Dougnon J (2020) in roots, stems, and leaves of chinese medicinal Plant Paris polyphylla
A Literature Review—Khaya senegalensis, Anacardium ouest L., Cassia var. yunnanensis. Pol J Microbiol 69:91–97. https://​doi.​org/​10.​33073/​
sieberiana DC., Pterocarpus erinaceus, Diospyros mespiliformis, Ocimum pjm-​2020-​012
gratissimum, Manihot esculenta, Vernonia amygdalina Delile, Pseudocedrela Marchut-Mikołajczyk O, Chlebicz M, Kawecka M, Michalak A, Prucnal F,
kotschyi and Daniellia oliveri possess properties for managing infectious Nielipinski M, Filipek J, Jankowska M, Perek Z, Drożdżyński P, Rutkowska
diarrhea. Adv Biosci Biotechnol 11:457–473. https://​doi.​org/​10.​4236/​abb.​ N, Otlewska A (2023) Endophytic bacteria isolated from Urtica dioica
2020.​11100​31 L.-preliminary screening for enzyme and polyphenols production. Microb
Fanou BA, Klotoe JR, Fah L, Dougnon V, Koudokpon CH, Toko G, Loko F (2020) Cell Factories 22:169. https://​doi.​org/​10.​1186/​s12934-​023-​02167-2
Ethnobotanical survey on plants used in the treatment of candidiasis Marsola SJ, Jorge LF, Meniqueti AB, Bertéli MBD, de Lima TEF, Bezerra JL, Lopes
in traditional markets of southern Benin. BMC Complement Med Ther AD, Gazim ZC, do Valle JS, Colauto NB, Linde GA, (2022) Endophytic fungi
20:288. https://​doi.​org/​10.​1186/​s12906-​020-​03080-6 of Brunfelsia uniflora: isolation, cryopreservation, and determination of
Ferreira MC, de Assis JCS, Rosa LH (2020) Diversity of endophytic fungi associ‑ enzymatic and antioxidant activity. World J Microbiol Biotechnol 38:94.
ated with Carapichea ipecacuanha from a native fragment of the Atlantic https://​doi.​org/​10.​1007/​s11274-​022-​03278-5
Rain forest. South Afr J Bot Curr Future Direct Endophyte Res 134:225– Nacef HS, Ferdjioui S, Chaibi S, Ramla K (2022) Activité antimicrobienne des
229. https://​doi.​org/​10.​1016/j.​sajb.​2019.​12.​031 champignons endophytes et de l’huile essentielle de la plante médici‑
Firáková S, Šturdíková M, Múčková M (2007) Bioactive secondary metabolites nale Lavandula officinalis : Etude comparative. LRBPV 12:3192–3203
produced by microorganisms associated with plants. Biologia 62:251– Noufou O, Anne-Emmanuelle H, Claude WOJ, Richard SW, André T, Marius L,
257. https://​doi.​org/​10.​2478/​s11756-​007-​0044-1 Jean-baptiste N, Jean K, Marie-Genevieve DF, Pierre GI (2016) Bio‑
Hawar SN (2022) Extracellular enzyme of endophytic fungi isolated from logical and phytochemical investigations of extracts from Pterocarpus
Ziziphus spina leaves as medicinal plant. Int J Biomater 2022:2135927. erinaceus Poir. (fabaceae) root barks. Afr J Tradit Complement Altern Med
https://​doi.​org/​10.​1155/​2022/​21359​27 14:187–195. https://​doi.​org/​10.​21010/​ajtcam.​v14i1.​21
Houmènou V, Adjatin A, Assogba FM, Gbenou JD, Akoègninou A (2018) Etude Ouinsavi C, Sourou B, Sourou K, Adigla Appolinaire W, Towanou H, Akin Y, Dos‑
Phytochimique Et De Cytotoxicité De Quelques Plantes Utilisées Dans Le sou J (2021) Traditional uses of African rosewood (Pterocarpus erinaceus
Traitement De La Stérilité Féminine Au Sud-Bénin. Eur Sci J 14:156–172. Poir. Fabaceae) through the sociolinguistic groups and the pathways
https://​doi.​org/​10.​19044/​esj.​2018.​v14n6​p156 of conservation and sustainable management in Benin. Int J Biodivers
Imorou IT, Djogbenou PC, Arouna O, Sogbossi ES, Sinsin B (2015) Effets de la Conserv 13:200–213. https://​doi.​org/​10.​5897/​IJBC2​021.​1509
taille et des regions phytogeographiques sur la diversite floristique et la Pelo S, Mavumengwana V, Green E (2020) Diversity and antimicrobial activity
structure des forets sacrees au Benin. Annales des Sciences Agronom‑ of culturable fungal endophytes in Solanum mauritianum. Int J Environ
iques 19, 77–95. https://​www.​ajol.​info/​index.​php/​asab/​artic​le/​view/​ Res Public Health 17:439. https://​doi.​org/​10.​3390/​ijerp​h1702​0439
145332 Photolo MM, Vuyo M, Lungile S, Tlou MG (2020) Antimicrobial and antioxidant
IUCN (2024) The IUCN red list of threatened species [WWW Document]. IUCN properties of a bacterial endophyte, methylobacterium radiotolerans
Red List of Threatened Species. https://​www.​iucnr​edlist.​org/​en. Accessed MAMP 4754, isolated from Combretum erythrophyllum seeds. Int J Micro‑
6.15.24 biol 94:83670. https://​doi.​org/​10.​1155/​2020/​94836​70
Jayatilake PL, Munasinghe H (2020) Antimicrobial activity of cultivable endo‑ Rahman HU, Yue X, Ren X, Zhang W, Zhang Q, Li P (2020) Multiplex PCR assay
phytic and rhizosphere fungi associated with “mile-a-minute”, Mikania to detect Aspergillus, Penicillium and Fusarium species simultaneously.
cordata (Asteraceae). Biomed Res Int 2020:5292571. https://​doi.​org/​10.​ Food Addit Contam 37:1939–1950. https://​doi.​org/​10.​1080/​19440​049.​
1155/​2020/​52925​71 2020.​18108​60
Khan AL, Gilani SA, Waqas M, Al-Hosni K, Al-Khiziri S, Kim YH, Ali L, Kang SM, Ratnaweera PB, de Silva ED, Williams DE, Andersen RJ (2015) Antimicrobial
Asaf S, Shahzad R, Hussain J, Lee IJ, Al-Harrasi A (2017a) Endophytes from activities of endophytic fungi obtained from the arid zone invasive plant
medicinal plants and their potential for producing indole acetic acid, Opuntia dillenii and the isolation of equisetin, from endophytic Fusarium
improving seed germination and mitigating oxidative stress. J Zhejiang sp. BMC Complement Altern Med 15:220. https://​doi.​org/​10.​1186/​
Univ Sci B 18:125–137. https://​doi.​org/​10.​1631/​jzus.​B1500​271 s12906-​015-​0722-4
Khan AL, Shahzad R, Al-Harrasi A, Lee IJ (2017b) Endophytic microbes: Regional Committee for Africa, 50 (2000). Fiftieth session of the WHO Regional
a resource for producing extracellular enzymes. In: Maheshwari Committee for Africa: Final Report.
DK, Annapurna K (eds) Endophytes: crop productivity and protec‑ Salako KV, Moreira F, Gbedomon RC, Tovissodé F, Assogbadjo AE, Glèlè Kakaï RL
tion, vol 2. Springer, Cham, pp 95–110. https://​doi.​org/​10.​1007/​ (2018) Traditional knowledge and cultural importance of Borassus aethio-
978-3-​319-​66544-3_5 pum Mart. in Benin: interacting effects of socio-demographic attributes
Kim DO, Chun OK, Kim YJ, Moon HY, Lee CY (2003) Quantification of polyphe‑ and multi-scale abundance. J Ethnobiol Ethnomed 14:36. https://​doi.​org/​
nolics and their antioxidant capacity in fresh plums. J Agric Food Chem 10.​1186/​s13002-​018-​0233-8
51:6509–6515. https://​doi.​org/​10.​1021/​jf034​3074 Salo EN, Novero A (2020) Identification and characterisation of endophytic
Klotoé JR, Dougnon TV, Koudouvo K, Atègbo JM, Loko F, Akoègninou A, bacteria from coconut (Cocos nucifera) tissue culture. Trop Life Sci Res
Aklikokou K, Dramane K, Gbeassor M (2013a) Ethnopharmacological 31:57–68. https://​doi.​org/​10.​21315/​tlsr2​020.​31.1.4
survey on antihemorrhagic medicinal plants in South of Benin. Eur J Med Shah S, Shrestha R, Maharjan S, Selosse MA, Pant B (2018) Isolation and charac‑
Plants. https://​doi.​org/​10.​9734/​EJMP/​2013/​2093 terization of plant growth-promoting endophytic fungi from the roots of
Klotoé JR, Dougnon TV, Koudouvo K, Atègbo JM, Loko F, Akoègninou A, Dendrobium moniliforme. Plants (Basel) 8:5. https://​doi.​org/​10.​3390/​plant​
Aklikokou K, Dramane KMG (2013b) Ethnopharmacological survey on s8010​005
antihemorrhagic medicinal plants in South of Benin. Eur J Med Plants. Sharma M, Mallubhotla S (2022) Diversity, antimicrobial activity, and antibiotic
https://​doi.​org/​10.​9734/​EJMP/​2013/​2093 susceptibility pattern of endophytic bacteria sourced from Cordia
Komeil D, Saad M (2021) The antagonistic activity of certain fungal endo‑ dichotoma L. Front Microbiol 13:879386. https://​doi.​org/​10.​3389/​fmicb.​
phytes against some phytopathogenic fungi through their metabolites 2022.​879386
and extracellular enzymes. Phyto 101:21–30. https://​doi.​org/​10.​7202/​ Singleton VL, Orthofer R, Lamuela-Raventós RM (1999) Analysis of total phe‑
10826​02ar nols and other oxidation substrates and antioxidants by means of folin-
Li JL, Sun X, Zheng Y, Lü PP, Wang YL, Guo LD (2020) Diversity and community ciocalteu reagent. In: Methods in enzymology, oxidants and antioxidants
of culturable endophytic fungi from stems and roots of desert halophytes part A. Academic Press. https://​doi.​org/​10.​1016/​S0076-​6879(99)​99017-1
in northwest China. MycoKeys 62:75–95. https://​doi.​org/​10.​3897/​mycok​ Talukdar R, Wary S, Hajowary R, Sarma A, Tayung K (2021) Antimicrobial activ‑
eys.​62.​38923 ity of endophytic fungi isolated from some selected ethnomedicinal
Li L, Liu C, Wen W, Li Q, Pan T, Li Z, Qian G, He Y, Xu D (2022) Dendrobine bio‑ plants of Assam, India. In: Patil RH, Maheshwari VL (eds) Endophytes:
synthesis in Dendrobium nobile in four different habitats is affected by the potential source of compounds of commercial and therapeutic
Aliou et al. Bulletin of the National Research Centre (2024) 48:128 Page 15 of 15

applications. Springer, Singapore, pp 91–102. https://​doi.​org/​10.​1007/​

978-​981-​15-​9371-0_6
Taneja T, Garg H, Sharma I, Singh R (2023) Isolation and Identification of
endophytic fungi and evaluation of their interactions with plants from
Syzygium cumini (Jamun) and Ocimum tenuiflorum (Tulsi). J Indian Bot
Soc 234:103. https://​doi.​org/​10.​61289/​jibs2​023.​12.​23.​1145
Tittikpina NK, Nana F, Fontanay S, Philippot S, Batawila K, Akpagana K, Kirsch
G, Chaimbault P, Jacob C, Duval RE (2018) Antibacterial activity and
cytotoxicity of Pterocarpus erinaceus Poir extracts, fractions and isolated
compounds. J Ethnopharmacol 212:200–207. https://​doi.​org/​10.​1016/j.​
jep.​2017.​10.​020
Tittikpina NK, Sandjo LP, Nana F, Vaillant V, Fontanay S, Philippot S, Diop YM,
Batawila K, Akpagana K, Kirsch G, Duval RE, Jacob C, Chaimbault P (2019)
Investigation of the antifungal activity of Pterocarpus erinaceus led to the
identification of two new diarylpropanoids from its roots. Phytochem Lett
32:110–114. https://​doi.​org/​10.​1016/j.​phytol.​2019.​05.​013
Toukam DP, Ladoh Yemeda CF, Alembert T, Mbafor J (2016) Antiradical activity,
total phenolic and flavonoid content of extracts from the stem bark of
Pterocarpus erinaceus. Sch Acad J Biosci (SAJB) 4:473–477. https://​doi.​org/​
10.​21276/​sajb.​2016.4.​6.4
Tousif MI, Touseef S, Sadeer N, Jugreet S, Zengin G, Legoabe L, Imran M,
Mahomoodally F (2023) Phenolics from endophytic fungi as natural
α-glucosidase inhibitors: a comprehensive review. J Mol Struct. https://​
doi.​org/​10.​1016/j.​molst​ruc.​2023.​135852
Usman MM, Idi A, Aisami A, Maigari FU (2023) A review on endophytic fungi: a
natural source of industrial enzymes. Asian J Plant Biol 5:7–11. https://​doi.​
org/​10.​54987/​ajpb.​v5i1.​820
Vissoh ACS, Klotoé JR, Fah L, Agbodjento E, Koudokpon H, Togbe E, Saïdou S,
Dougnon V (2024) Knowledge and practices of traditional management
of child malnutrition and associated pathologies in Benin. J Ethnobiol
Ethnomed 20:47. https://​doi.​org/​10.​1186/​s13002-​024-​00684-x
Walitang DI, Kim CG, Kim K, Kang Y, Kim YK, Sa T (2018) The influence of host
genotype and salt stress on the seed endophytic community of salt-
sensitive and salt-tolerant rice cultivars. BMC Plant Biol 18:51. https://​doi.​
org/​10.​1186/​s12870-​018-​1261-1
WHO (1978) Déclaration d’Alma-Ata. Conférence internationale sur les soins de
santé primaires, Alma-Ata (URSS), 6–12 Sept 1978. Organisation mondiale
de la santé, Genève
WHO (2010) Guidelines for registration of traditional medicines in the who
African region
WHO (2013) WHO traditional medicine strategy. WHO library cataloguing-in-
publication data
Wu W, Chen W, Liu S, Wu J, Zhu Y, Qin L, Zhu B (2021) Beneficial relationships
between endophytic bacteria and medicinal plants. Front Plant Sci.
https://​doi.​org/​10.​3389/​fpls.​2021.​646146
Xiaoxiang D, Xu F, Dan D, Gao T (2019) Diversity and bioactivities of fungal
endophytes from Distylium chinense, a rare waterlogging tolerant plant
endemic to the Three Gorges Reservoir [WWW Document]. https://
microbialcellfactories.biomedcentral.com/articles/https://​doi.​org/​10.​
1186/​s12934-​023-​02167-2. Accessed 4.11.24
Zhang Q, Acuña JJ, Inostroza NG, Mora ML, Radic S, Sadowsky MJ, Jorquera
MA (2019) Endophytic bacterial communities associated with roots
and leaves of plants growing in Chilean extreme environments. Sci Rep
9:4950. https://​doi.​org/​10.​1038/​s41598-​019-​41160-x
Zhenyan C, Yihua Y, Yang S, Xuping S, Xiaoping Y (2021) Simultaneous detec‑
tion of four pathogens in Dendrobium officinale by nested multiplex PCR
assay. Crop Prot 140:105445. https://​doi.​org/​10.​1016/j.​cropro.​2020.​105445
Zhishen J, Mengcheng T, Jianming W (1999) The determination of flavonoid
contents in mulberry and their scavenging effects on superoxide radicals.
Food Chem 64:555–559. https://​doi.​org/​10.​1016/​S0308-​8146(98)​00102-2

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