Identification, molecular characterization, and plant growth promoting activities of endophytic fungi of Jasminum sambac, Camellia sinensis, and Ocimum basilicum

2.1 Chemicals and materials

Potato dextrose broth (PDB; M403), agar-agar (GRM666), chloramphenicol (TC204), and tryptophan (M1339) were procured from Hi-Media Laboratories Pvt. Ltd., Mumbai, India. Agarose (137049), α-ketobutyrate (K401), and ethidium bromide (E7637) were purchased from Sigma-Aldrich, USA. All other chemicals such as ethanol and sodium hypochlorite were of analytical grade and purchased from Thermo Fisher Scientific, USA.

2.2 Collection of samples

Sampling was done from different locations of Riyadh regions namely Botanical Nurseries of Al-Muzahmiya and Al-Kharj Governorates in December (2018). There were samples of various parts including roots, leaves, and stems of aromatic and medicinal plants. Samples were brought to the laboratory with utmost care that were later used for the selective isolation and purification of fungal endophytes. Samples were thoroughly washed first with running tap water followed by sterile distilled water to remove soil and dust particles, and tightly wrapped in ziplock bags under humid conditions and stored at 8 °C. The plant samples were taxonomically identified and validated by the plant herbarium unit of the Department of Botany and Microbiology, King Saud University, Riyadh, Saudi Arabia as shown in Table 1.

2.3 Isolation of fungal endophytes

Microbes and epiphytes were removed by immersion of J. sambac, C. sinensis, O. basilicum parts sequentially for one minute in each of the following: 75% ethanol, 1–13% NaOCl, and again in 75% ethanol. Small segments measuring sizes of approximately 0.5–1.0 cm were cut and and three times rinsed with autoclaved water. These small sections were dried using towels of blotting papers. Four pieces were kept in potato dextrose agar (PDA) medium with 50 mg/L chloramphenicol to allow fungal growth at 28 ± 2 °C for two weeks. From these master plates, fungi were relocated to new PDA plates not amended with antibiotics (Suryanarayanan and Kumaresan, 2001).

2.4 Phenotypic determination of isolates

Colony morphology, secretion of pigments, spore structures, and a few characteristics of hyphae including spores were examined using standard mycological manuals (Domsch et al., 1980). Staining of mycelium was done with cotton blue dye for microscopic observation. Furthermore, extra-taxonomic papers on fungal endophytes pertaining to specific genus and species were referred (Barnett and Hunter, 1998). For the color distinction of cultures, the ‘Methuen Handbook of Color' was used as a reference (Wanscher, 1978).

2.5 Polyphasic identification of fungal endophytes

Genus-level determination of fungal endophytes was conducted based on phenotypic and microscopic investigations. For species-level characterization, a widely accepted molecular method was employed. Genomic DNAs of fungal isolates were sequenced by using the service of Macrogen Inc., Seoul, South Korea. The ITS region of the rRNA gene was analyzed using primers ITS1: 5′ (TCC GTA GGT GAA CCT GCG G) 3′ and ITS4: 5′ (TCC TCC GCT TAT TGA TAT GC) 3′.

Sequences amplified by PCR were also analyzed by the Basic Local Alignment Search Tool (BLAST) of NCBI (https://www.ncbi.nlm.nih.gov). Maximum score, total score, query cover, and percentage identity from the NCBI website were considered. Identified sequences were submitted to NCBI and accession numbers were obtained. Maximum likelihood method was followed for phylogentic analysis and tree preparation using MEGA 7.0 to establish relationships among isolates while the Maximum Composite Likelihood method to calculateevolutionary distances. DNA samples were amplified using the following PCR (reaction volume 25 µl) conditions: 95 °C for 2 min, 40 cycles at 95 °C for 30 s (denaturation), 55 °C for 30 s (annealing), and extension at 72 °C for 1 min (elongation), and final elongation at 72 °C for 10 min. PCR amplification was confirmed by running 1.2% agarose gel electrophoresis and analyzed by a gel documentation system.

2.6 Determination of IAA production by fungal isolates

Fungi were grown in PDB with added tryptophan (100 µg/ml). The cultures were incubated for four days at 26 °C for 4 days and spun for 10 min at 10,000 rpm. Supernatant served as a source of IAA using the method of Bric et al. (Bric et al., 1991). Supernatant was added to Salkowski’s reagent (1:2 ratio) and a few drops of H₃PO₄ were added to it. Mixtures were kept under a dark environment for at least 30 min. The absorbance of pink color solution was recorded at 530 nm. Amount of IAA in the supernatant was calculated using data from the standard curve of IAA.

2.7 Determination of ACC deaminase activity

Activity of the ACC deaminase enzyme was quantified using the method of Yedidia et al. (Yedidia et al., 1999). Briefly, 20 ml synthetic medium was inoculated with the spore suspensions (20 µl) of endophytic fungal isolates. The medium was allowed to incubate for two days at 28 °C. Fungal mycelia were washed and inoculated to a synthetic medium (5 ml) amended with 3 mM ACC but without adding ammonium. After the induction period, samples were processed as described earlier (Yedidia et al., 1999). The absorbance at λ_max = 540 nm was recorded and ACC deaminase activity was measured due to the generation of α-ketobutyrate and expressed as mmol of α-ketobutyrate mg⁻¹ protein h⁻¹. A standard curve of α-ketobutyrate was used and values were calculated.

2.8 Siderophore production assay

A disc inoculation method was used for the detection of the siderophore activity of endophytic fungal isolates. Seven to eight days old cultures of fungi were used. Mycelial discs measuring 4 mm in size were placed on Chrome Azurol S (CAS) agar media. CAS plates with isolated strains were allowed to incubate at 30 °C for a week. The orange-colored halo zone around the disc was monitored and isolates were designated positive or negative based on the halo zone surrounding fungal inoculation on a blue medium.

2.9 Data analysis

The colonization frequency (CF) of isolated strains was calculated by using the following formula: $$\mathit{CF}\left(\%\right)=\frac{\mathit{Number}ofsegmentscolonizedbyendophytespecies}{\mathit{Total}numberofsegmentsexamined}\times 100$$

Relative frequency percentage for every fungus was presented as: $$\mathit{RF}\left(\%\right)=\frac{\mathit{Number}ofisolatesofonespecies}{\mathit{Total}numberofisolates}\times 100$$

The isolation rate was calculated by the total number of isolated fungi divided by the total number of segments. $$\mathit{RF}\left(\%\right)=\frac{\mathit{Total}numberofisolates}{\mathit{Total}numberofsegments}\times 100$$

3.1 Isolation and morphological identification

Eighty-four isolates were collected from three aromatic and medicinal plants J. sambac, C. sinensis, and O. basilicum. Fungal isolates recovered from plants were separated into twenty morpho-types based on the similarity of their cultural characteristics that include both macro- and microscopic features including colony morphology, plate color (above and reverse), and mycelium (Fróhlich et al., 2000) (Table 4).

Based on macro and microscopic assays done for culture characteristics and morphology, and following molecular identification, the isolates were identified as- one species of Neopestalotiopsis from four isolates, one species of Trichoderma from three isolates, four species of Alternaria from thirty-five isolates, three species of Fusarium from eight isolates, one species of Curvularia from eight isolates, one species of Colletotrichum from two isolates, one species of Phoma from three isolates, one species of Aspergillus from three isolates, two species of Cladosporium from four isolates, one species of Chaetomium from two isolates, one species of Myrothecium from three isolates, one species of Neodidymelliopsis from two isolates (Fig. 1A). Among the various isolates, the genus Alternaria was most dominant with an overall colonizing frequency (CF) of 41.5%, and this genus was also the dominant endophyte group in previous studies (Mohammad Golam Dastogeer et al., 2020) (Fig. 1 A-B).

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Fig. 1

Bar diagram showing counts of fungal isolates (A) and colonization frequency (%) (B).

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In the current study, J. sambac showed the highest number of fungi as 39 isolates with a colonization rate of 27 % and isolation rate (IR) of 0.81, followed by 27 isolates from C. sinensis, CF of 18.7 % and IR 0.56, and O. basilicum has recorded the lowest CF, 12.5 %, by 18 isolates. In a previous study, (Sharmila et al., 2017) reported the isolation of endophytic fungi and the colonization rate was recorded in leaf tissues as well as few reports conducted on J. sambac, therefore, our study confirmed this endophytic biodiversity (Table 4). Xie et al. recently reported the isolation of endophytic fungi from C. sinensis (L.), the fungal diversity, and biological functions (Xie et al., 2020). They have also reported the plant growth-promoting activities of those fungi such as siderophore production, secretion of phytohormones, ACC deaminase enzyme, etc. Moreover, the variations in the composition of fungal isolates were assigned to seasonal or cultivar changes were shown (Xie et al., 2020). Endophytic fungal isolates have also been obtained from various habitats, environments, and climates. Similar to these conditions, Riyadh's vicinity conditions are characterized by extreme temperatures, the prevalence of drought, and floral diversity in natural habitats. Fungi generally show the ability to survive in such extreme conditions and to adapt to most habitats. Moreover, heterogeneous conditions may encourage a high diversity of fungal communities in desert ecosystems (Verma et al., 2012).

Among the isolated endophytic fungi, 13 species Trichoderma virens, Alternaria eichhorniae, Alternaria alstroemeriae, Alternaria alternata, Alternaria tenuissima, Fusarium sp., Fusarium equiseti, Fusarium anthophilum, Colletotrichum trifolii, Aspergillus niger, Cladosporium tenuissimum, Penicillium glabrum, and Chaetomium sp. were distributed through the four aromatic and medical plants, whereas other species have shown a specific tendency for their hosts were: Neopestalotiopsis sp., Curvularia subpapendorfii, Phoma multirostrata, Cladosporium perangustum, Myrothecium inundatum, and Neodidymelliopsis sp. (Figs. 2 and 3). In this investigation, endophytic fungi have displayed host or organ specificity, and there was little intersection between the leaf, stem, and root assemblages, which have been consistent with previous studies (Rajulu et al., 2016).

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Fig. 2

Macroscopic and microscopic features of five genera of fungal endophytes: A- Neopestalotiopsis clavispor, B- Trichoderma virens, C- Fusarium sp., D- Alternaria eichhorniae, E- Alternaria alternata, F- Alternaria alstroemeriae, G- Curvularia subpapendorfii, H- Penicillium glabrum, I- Alternaria tenuissima, and J- Fusarium equiseti. Images of Petri dishes in each panel represent pictures from the upper and lower side. The length of the scale bar is 20 µm.

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Fig. 3

Macroscopic and microscopic characteristics of nine genera endophytes fungi isolated from aromatic and medical plants: A- Fusarium anthophilum, B- Colletotrichum trifolii, C- Curvularia subpapendorfii, D- Cladosporium tenuissimum, E- Cladosporium perangustum, F- Aspergillus niger, G- Chaetomium sp., H- Myrothecium inundatum, I- Curvularia subpapendorfii, and J- Neodidymelliopsis sp. Images of Petri dishes in each panel represent pictures from the upper and lower side. The length of the scale bar is 20 µm.

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In this study, some genera have been isolated for the first time confirming that Saudi flora is rich in endophytic fungi. One hundred forty-four segments (leaves-steams-roots) of J. sambac, C. sinensis, and O. basilicum have produced 39, 27, and 18 endophytic strains with colonization rates at 27 % 18.7 % and 12.5 %, respectively (Table 2). Environmental conditions significantly affect the structure of fungal communities and populations (Gashgari et al., 2016). Further, plants that belong to the same geoenvironmental locations may show a higher degree of smilalrity between the taxa and species (D’Amico et al., 2008).

Interestingly, the section of leaves recorded the highest relative frequency (27.7 %), while the frequencies for stems (19.4 %) and roots (9.02 %) were notably less (Table 3). Therefore, it seems that endophytes favorably colonize the leaves due to greater surface area, rich nutrition, and thin walls (Figs. 2 and 3).

Eighty-four endophyte fungi have been classified into 12 genera sequentially subdivided under three classes: dothideomycetes, sordariomycetes, and eurotiomycetes. Dothideomycetes have included six genera as Alternaria, Curvularia, Phoma sp., Cladosporium, Neodidymelliopsis, six genera of isolates were classified in sordariomycetes class as Neopestalotiopsis, Trichoderma, Fusarium, Colletotrichum, Chaetomium, Myrothecium, while eurotiomycetes has contributed only two genera of endophytic fungi that were Aspergillus and Penicillium. The majority of endophytes were filamentous ascomycetes supporting the former studies belonging to the Ascomycota phylum (Clay, 1990). Similar results were also obtained with seven medicinal plants collected from the Al-Gouf governorate's salt marshes in North Saudi Arabia (Gashgari et al., 2016).

3.2 ITS sequence and phylogenetic analysis

Twenty-five isolates were subjected to morphological assays followed by molecular analysis to confirm the identification of fungi based on ITS1 and ITS4 sequences in rDNA genes. Identification of isolates was constructed on the highest similarity from BLAST results. Gene bank accession numbers of twenty endophytic fungi were obtained (Table 4).

Besides morphological, molecular charactrization by ITS region of the rRNA gene was employed to confirm initial observations and was an extremely useful and appropriate tool for estimating species diversity. The ITS region is reliable for the identification fungi (Gherbawy and Elhariry, 2016). The isolates that have been used for the sequencing analysis, their codes and GenBank numbers are provided in Table 4. Phylogenetic tree for twenty isolates of endophytic fungi (Table 5) was constructed by MEGA 7.0 program that confirms all strains belonging to Dothideomycetes, Sordariomycetes, or Eurotiomycetes classes of Ascomycota division (Fig. 4).

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Fig. 4

Maximum composite likelihood phylogenetic tree of endophytic fungi isolated from aromatic and medical plants based on ITS sequences in rRNA genes. The Clustal W was used for sequence alignment in MEGA 7.0. Bootstrap percentage values obtained from 1000 replications of the data set are shown at nodes. The scale bar shows the average number of nucleotide substitutions per site.

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3.3 Plant growth-promoting (PGP) traits of fungal endophytes

Three PGP traits of all identified fungal isolates were explored (Table 6). Biosynthesis of IAA by fungal isolates ranged from 2.1 to 56 µg/ml. Among those, Aspergillus niger and Penicillum glabrum were found as high producers of IAA (56.3 and 48.2 µg/ml, respectively) with 100 µg/ml tryptophan. Some isolates showed low production of IAA that include Myrothecium inundatum, Chaetomium sp., and Curvularia subpapendorfii while some strains like Cladosporium tenuissimum and Neodidymelliopsis sp. did not produce IAA. Similarly, variable results were observed for ACC deaminase activity and siderophore production. Here, Penicillum glabrumi and Aspergillus niger showed a positive reaction for siderophore production and also produced high amounts of α-ketobutyrate (18.2 and 15.2 μmol α-KB mg⁻¹ Protein h⁻¹, respectively) as a result of ACC deaminase activity. Only 12 isolates were positive for siderophore activity. Overall, aromatic, and medical plants are vital stores of endophytes fungi, wherein it is infrequently finding a fungus-free plant. The medicinal and aromatic plants used in the current study are known to be rich for their medicinally active phytochemical compositions such as the production of catechins, caffeine, and other polyphenolic compounds by C. sinensis (Aboulwafa et al., 2019), various bioactive phytochemicals by J. sambac (Wu et al., 2021), and polyphenolic secondary metabolites by O. basilicum (Pirbalouti et al., 2017) that have shown few or many clinically useful properties including radical scavenging, antioxidant, antimicrobial, antiviral, anticancer, and anticonvulsant, hypoglycaemic, and hypolipidemic properties (Rubab et al., 2017). Similar to their host plants, the endophytic microbes also produce some valuable phytochemical with a variable range of activities. For example, endophytic microbes associated with C. sinensis produced secondary metabolites that could produce antimicrobial activities against Staphylococcus aureus and Bacillus subtilis (Shan et al., 2018). Moreover, endophytic microbes produced some plant growth-regulating substances like IAA and ACC deaminase promoting plant (Shan et al., 2018). In corroboration with our results, J. sambac also showed to have various species of the genus Colletotrichum (Gioia et al., 2020). Also, J. sambac contained Phyllosticta capitalensis fungus living in endophytic mode. Similar to the antimicrobial and antioxidant activities of O. basilicum (Pirbalouti et al., 2017), the extracts from its endophytic fungi (total of eight genera) also produced antibacterial and antioxidant effects against nine clinical pathogens of humans (Atiphasaworn et al., 2017). Among them, Nigrospora sp. Exhibited the highest antibacterial and antioxidant potential. This suggests that in some cases the medicinally important host and its endophytic fungi share common bioactive properties. Moreover, the biostimulants produced by the endophytic fungus can also modulate the production of secondary metabolites of the host plant as has been shown in the case of O. basilicum L (Saia et al., 2021).