Phytochemical Analysis and Antimicrobial Activity of Ganoderma lucidum Against Selected Bacteria and Fungi of Medical Importance

Phytochemical Analysis and Antimicrobial Activity of Ganoderma lucidum Against Selected Bacteria and Fungi of Medical Importance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Phytochemical Analysis and Antimicrobial Activity of Ganoderma lucidum Against Selected Bacteria and Fungi of Medical Importance Sarah Solomon, Oyekale Adesola, Adeola Ayanyinka, Adeniyi Ifejesu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6261550/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction : Ganoderma lucidum , a medicinal mushroom widely studied for its bioactive properties, holds promise for antimicrobial applications. This study aimed to identify G. lucidum samples collected from Osun States, Nigeria, and evaluate their antimicrobial potential, bioactive compound profile and the molecular characterization. Mushroom samples collected during the rainy season of June 2024, were morphological and molecular identification using DNA extraction, PCR amplification, and genome sequencing. Extracts were prepared using hot water and methanol, and bioactive compounds were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). Antimicrobial activity was assessed using the agar well diffusion method against bacterial and fungal strains, and statistical analyses were performed using SPSS. BLAST analysis of showed strong alignments with multiple Ganoderma sp.with Ganoderma lucidum showing the highest similarity with 99.55%. Phylogenetic analysis confirmed its close evolutionary relationship with G. lucidum and the ITS sequence was identified as AS_ITS1_C06_09. GC-MS profiling identified bioactive compounds with antimicrobial and therapeutic potential. Antimicrobial assays demonstrated significant inhibitory effects, particularly from methanol extracts. These findings support the pharmaceutical relevance of G. lucidum and its potential applications in antimicrobial formulations. This study highlights its potential as a source of bioactive compounds with antimicrobial properties, for pharmaceutical and nutraceutical applications. Ganoderma lucidum phytochemical analysis antimicrobial activity medical importance GC-MS PCR authentication medicinal mushroom infectious diseases Figures Figure 1 Figure 2 Introduction Ganoderma species, particularly Ganoderma lucidum , are well known for their medicinal and bioactive properties, including antimicrobial, antioxidant, and immunomodulatory effects. These fungi contain diverse secondary metabolites such as polysaccharides, triterpenoids, and phenolic compounds, which contribute to their therapeutic potential( 1 ). With the rising global concern over antimicrobial resistance, there is an increasing need to explore natural sources for novel bioactive compounds, and medicinal mushrooms have emerged as promising candidates. While the pharmacological properties of G. lucidum are well documented, limited research exists on its antimicrobial efficacy against multidrug-resistant bacteria and pathogenic fungi in Nigeria ( 2 ) Despite several studies highlighting the antimicrobial and bioactive properties of G. lucidum , data on its effectiveness against specific bacterial and fungal strains endemic to Nigeria remain scarce. Additionally, there has been minimal exploration of the phytochemical composition of G. lucidum from different ecological zones in Nigeria, and limited comparisons between the efficacy of aqueous and methanolic extracts ( 3 ). Furthermore, the molecular characterization of indigenous Ganoderma species remains underexplored, restricting a comprehensive understanding of their genetic diversity and pharmaceutical potential. These gaps should be addresses for enhancing local fungal biodiversity in antimicrobial drug discovery. The increasing prevalence of antimicrobial-resistant pathogens necessitates the search for alternative treatment strategies. G. lucidum , a widely recognized medicinal mushroom, represents a valuable natural resource for bioactive compound discovery ( 4 ). Molecular identification of Nigerian G. lucidum , profiling its phytochemical composition using GC-MS isolates ensures accurate species characterization, evaluating its antimicrobial activity against clinically relevant bacterial and fungal strains in Nigeria provides insights into its bioactive compounds, forming a potential as a natural antimicrobial agent and a foundation for pharmaceutical applications. This study aims to collect and identify Ganoderma species from selected ecological zones in Nigeria using morphological and molecular techniques. It seeks to analyze the phytochemical composition of G. lucidum extracts through GC-MS profiling and assess their antimicrobial activity against selected bacterial and fungal pathogens. Furthermore, the study will evaluate the genetic diversity of G. lucidum isolates through phylogenetic analysis and compare the efficacy of aqueous and methanolic extracts in inhibiting microbial growth. MATERIALS AND METHODS 2.1. Sample Collection. Mushroom samples were collected during the rainy season in June 2024 from Ofatedo Osogbo (Osun State) and Owode Ogbomosho (Oyo State), Nigeria. Two specimens, exhibiting morphological characteristics consistent with Ganoderma , were confirmed through molecular identification. The samples were harvested using sterile tools and preserved by drying and refrigeration for subsequent analysis. These locations were selected due to their favorable ecological conditions for fungal growth, with the samples intended for antimicrobial and bioactive compound studies. 2.2. Sample Preparation. Fresh mushrooms were sliced and air-dried in an oven (Selecta, Madrid, Spain) at 50°C until a constant weight was achieved. The dried material (60 g) was ground into a fine powder using a hammer stirrer (IKA, Staufen, Germany). A 30 g portion was extracted in 500 mL of hot water and methanol and stirred for 96 h to dissolve phytochemicals. The extract was filtered using Whatman No. 1 filter paper (5). 2.3. Molecular Identification 2.3.1. DNA Extraction DNA was extracted using the ZYMO DNA extraction kit. A 50 mg wet sample was resuspended in 200 μL of water in a ZR BashingBead™ Lysis Tube, with 750 μL BashingBead™ Buffer added. The mixture was processed in a bead beater for 5 min, centrifuged at 10,000 × g for 1 min, and the 400 μL supernatant was transferred to a Zymo-Spin™ III-F Filter. Following sequential filtrations and centrifugations at 10,000 × g, 100 μL of DNA Elution Buffer was added, yielding 2.80 ng/μL DNA. 2.3.2. Primer Design and PCR Amplification The ITS1 and ITS4 primers (6) were used to amplify the ITS ribosomal region following(7). PCR was performed with 12.5 μL PCR master mix, 1.25 μL of each 10 mM primer, and 10 μL of diluted DNA template, adjusted to 25 μL with sterile deionized water. Thermal cycling conditions included initial denaturation at 94°C for 5 min, followed by 40 cycles of 94°C for 30 s, 50°C for 30 s, and a final extension at 72°C for 1 min. PCR products were purified using Montage™ PCR Centrifugal Filter Devices (Millipore Corp., USA)(8). 2.3.3. Genome Sequencing and Analysis Genome sequencing was conducted using PacBio Sequel (long-read) and Illumina NovaSeq (short-read) platforms. De novo assembly was performed with SMARTdenovo1 and polished using Pilon(9). Assembly integrity was assessed with BUSCO (10). The ITS1-5.8S-ITS2 region (588 bp) was analyzed using BLAST with 99.55% identity, 99.55% query coverage, and an E-value ≤1.00E-135. Sequences were matched to Ganoderma lucidum (taxid:5315). Phylogenetic relationships were inferred using Maximum Likelihood (ML) analysis in MEGA 7(11). 2.4. Extract Concentration Aqueous extracts of G. lucidum were concentrated using a freeze dryer (Model LGJ-10) at 50°C. Methanolic extracts were concentrated via evaporation in a water bath at 50°C. 2.5. Test Organisms Bacterial isolates ( E. coli, Staphylococcus aureus, Enterobacter aerogenes, Pseudomonas aeruginosa ) were obtained from the Microbiology Laboratory, Ladoke Akintola University, Ogbomosho, Nigeria. Fungal strains ( Aspergillus niger, Rhizopus stolonifer, Penicillium sp., A. flavus ) were sourced from Bowen University, Iwo, Nigeria. Cultures were maintained on agar slants at 4°C to prevent contamination(5) 2.6. Phytochemical Analysis (GC-MS) GC-MS analysis was performed using an Agilent Technologies Model 7000 GC-MS Triple Quad (TQQQ) system with Hunter workstation software (B.04.00). The electron ionization potential was 70 eV. Compounds were separated using an OPTIMA-5 column (30 m × 250 μm × 0.25 μm) at 360°C. Helium was the carrier gas (flow rate: 1.129 mL/min; split ratio: 5:1). A 2.5 μL sample was injected via an automatic liquid sampler. Compounds were identified by comparing mass spectra with the NIST database, using retention indices calculated via Kovat’s formula with n -alkanes (C9–C33) as standards(12) 2.7. Inoculum Preparation Test organisms were sub-cultured on nutrient agar and potato dextrose agar, then incubated at 37°C for bacteria and 24°C for fungi for 24–36 h. Bacterial suspensions were adjusted to a 0.5 McFarland standard (~1.5 × 10⁸ CFU/mL). 2.8. Antimicrobial Assay The agar well diffusion method was used. A 24-h broth culture was swabbed onto sterile Mueller-Hinton agar in Petri dishes. Three equidistant wells (6 mm) were created using a sterile cork borer and filled with 0.1 mL of extract. A positive control (chloramphenicol, 50 μg/mL) and a negative control (solvent) were included. Plates were incubated at 37°C for 24 h, and inhibition zones were measured in millimetres (5) 2.9. Statistical Analysis Data were analyzed using SPSS (Version 25.0) and expressed as mean ± standard deviation. Statistical significance was determined using one-way ANOVA, Student’s t -test, and chi-square analysis ( p < 0.05). Bar charts with error bars were generated using Microsoft Excel with Pearson correlation analysis. RESULTS The BLAST analysis of the ITS sequence AS_ITS1_C06_09 yielded significant alignments with sequences belonging to the genus Ganoderm a lucidum . High-confidence matches were observed with multiple species, including Ganoderma oregonense strain , Ganoderma sp. , Ganoderma resinaceum , and Ganoderma lobatum . These alignments were characterized by high Max Scores, consistent Query Coverages, high Percent Identities, and exceptionally low E-values (Table 1). The top alignments for Ganoderma lucidum isolate included sequences such as OQ701698.1 ( Ganoderma lucidum isolate GJN-2 ), MF755277.1 ( Ganoderma lucidum strainNM20 TENN ) and KX055549.1 ( Ganoderma resinaceum strain G62 ), which exhibited Max Scores of 645, Percent Identities of 99.32%, Query Coverages of 75%, and E-values of 0.0. Alignment metrics, indicating consistent taxonomic associations with the query sequence. For Ganoderma sp. , notable alignments included MH 708512 .1 ( Ganoderma isolate 16 ITS-1 . ) and. These sequences exhibited Max Scores of 645, Percent Identities of 99.32%, Query Coverages of 75%, and E-values of 0.0. These results suggest that the query sequence shares substantial similarity with other species in the genus, highlighting its broader taxonomic relevance. Alignments with Ganoderma lucidum sequences were equally significant. High-confidence matches included OQ701698.1 ( Ganoderma lucidum isolate GJN-2 ), which exhibited a Max Score of 620, Percent Identity of 99.55%, Query Coverage of 75%, and an E-value of 0.0. Additional sequences, such as ON394695.1 ( Ganoderma lucidum strain LAU22 ) and MF755277.1 ( Ganoderma lucidum strain NM20 ), displayed Max Scores of 663, Percent Identities of 99.32%, Query Coverages of 75%, and E-values range is0.0. These findings confirm the close taxonomic relationship of the query sequence with Ganoderma lucidum . Table 1: Best hit for AS_ITS1_C06_09 sequence using BLAST Description Scientific Name Max Score Total Score Query Coverage (%) E Value Percent Identity (%) Accession Length Accession Ganodermalucidum isolate GJN-2 ITS-2 Ganoderma lucidum 806 806 75 0.0 99.55 620 OQ701698.1 Ganoderma lucidum strain NM20 (TENN) Ganoderma lucidum 802 802 75 0.0 99.32 663 MF755277.1 Ganoderma strain. LAU22 Ganoderma sp. 802 802 75 0.0 99.32 611 ON394695.1 Ganoderma oregonense strain ASI 7049 Ganoderma oregonense 800 800 75 0.0 99.32 645 JQ520194.1 Ganoderma lucidum ITS1 Ganoderma lucidum 800 800 75 0.0 99.32 557 KT997431.1 Ganoderma lucidum isolate SMCC170.01.61 Ganoderma lucidum 800 800 75 0.0 99.32 627 FJ501561.1 Ganoderma lucidum isolate BNB01 Ganoderma lucidum 800 800 75 0.0 99.32 565 OR995187.1 Ganoderma lucidum isolate GLSM4 Ganoderma lucidum 800 800 75 0.0 99.32 586 KF648564.1 Ganoderma resinaceum strain G62 Ganoderma resinaceum 800 800 75 0.0 99.32 647 KX055549.1 Ganoderma lobatum strain ASI 7061 Ganoderma lobatum 800 800 75 0.0 99.32 645 JQ520166.1 Ganoderma lucidum strain OE-234 Ganoderma lucidum 800 800 75 0.0 99.32 634 AY636059.1 Ganoderma lucidum isolate 16 ITS-1 Ganoderma lucidum 800 800 75 0.0 99.32 612 MH708512.1 Ganoderma oregonense strain ASI 7067 Ganoderma lucidum 795 795 75 0.0 99.10 645 JQ520197.1 Ganoderma sp. ASI 7033 Ganoderma lucidum 795 795 75 0.0 99.10 645 JQ520208.1 Ganoderma lucidum isolate BNB01 Ganoderma lucidum 496 496 74 0.0 86.65 650 OR995187.1 The phylogenetic analysis of the sample AS_ITS1_C06_09 (Figure 2) provided key insights into its evolutionary relationships within the Ganoderma genus. Using the Maximum Likelihood method with 100 bootstrap replicates, the analysis showed a strong clustering of AS_ITS1_C06_09 with Ganoderma sessile (KF605634.1), indicating significant genetic similarity and evolutionary relatedness. High bootstrap values further validated this placement. The phylogenetic tree also revealed distinct clades for other Ganoderma species, illustrating the genus's genetic diversity. Notably, Ganoderma lucidum (ON394695.1), isolated from Ogbomoso, Nigeria, formed a separate, well-supported clade, underscoring its evolutionary divergence and unique genetic identity. Gas Chromatography – Mass Spectrophotometry The Gas Chromatography-Mass Spectrometry (GC-MS) analysis of Ganoderma lucidum Methanol Extract (GLME) and Aqueous Extract (GLAE) revealed a diverse range of bioactive compounds (Figure 3), each identified by its retention time (RT). G. lucidum , a highly versatile medicinal mushroom, is rich in polysaccharides and triterpenoids, which have been traditionally used and are now widely recognized as nutraceuticals and functional foods. Notably, hexadecenoic acid, detected at RTs of 13.782 min (GLME) and 13.456 min (GLAE), plays a vital role in cardiometabolic protection, immune modulation, and liver health. Additionally, the presence of antioxidant and anti-inflammatory compounds underscores the potential therapeutic benefits of these extracts. These findings confirm that the phytochemicals identified contribute to the antimicrobial and bioactive properties of G. lucidum , warranting further investigation into their functional applications. GC-MS Profiling of Bioactive Compounds in Ganoderma lucidum The GC-MS analysis of Ganoderma lucidum extracts identified various bioactive compounds with significant therapeutic potential. The most abundant was octadecanoic acid (RT: 22.737 min, 33.24%), known for its antimicrobial, anti-inflammatory, and lipid metabolism-regulating effects. Another major compound, deca-6,9-diene-2,8-dione (RT: 13.324 min, 22.11%), is also bioactive. Various saturated and unsaturated fatty acids, including hexadecanoic acid, pentadecanoic acid methyl ester, and oleic acid, exhibited cardioprotective, antimicrobial, and antioxidant properties. Beyond fatty acids, D-arabinitol (RT: 10.772 min, 5.32%), a sugar alcohol, displayed antifungal activity. Organic acids like nonane acid and sulfurous acid derivatives suggested preservative-like and antimicrobial functions. Minor bioactive components, such as carbamic acid derivatives (neuroprotective potential) and 4H-1,2,4-triazol-3-ol derivatives (antifungal properties), further enriched the extract's medicinal potential. These findings reinforce the pharmaceutical and nutraceutical significance of G. lucidum , supporting its traditional medicinal use and potential application in antimicrobial and therapeutic formulations. Antibiotic susceptibility test The evaluation of aqueous and methanol extracts of Ganoderma lucidum against bacterial and fungal test organisms. The presence of clear inhibition zones around some of the test organisms confirmed the antimicrobial potency of the extracts. Given that methanol is a more effective solvent for extracting bioactive compounds, the methanol extract likely exhibited stronger activity than the aqueous extract. These results suggest that G. lucidum extracts possess natural antimicrobial properties, making them promising candidates for the development of plant-based antimicrobial agents. The identification of clear inhibition zones against certain bacteria and fungi indicates their potential application in controlling microbial infections. Table 2: GC-MS Profiling of Bioactive Compounds in Ganoderma lucidum : Identification and Functional Significance Compounds peak Retention time Area (%) Molecular weight Decane,2,3,7-trimethyl3-Ethyl-3-methylheptaneUndecane,3-ethyl- 6.944 0.82 116.16 Hexadecane, tridecane, sulfurous acid. 7.499 2.01 158.24 Nonane acid 10.497 3.74 242.40 D-Arabinitol 10.772 5.32 652.9 pentadecanoic acid, 14-methyl-este 19.46 12.87 270.45 Hexadecanoic acid, ethyl ester 19.907 7.45 284.55 deca-6,9-diene-2,8-dione 13..324 22.11 294.47 Heptadecane, 2, 6, 10, 15 Tetramethy 20.95 1.66 296.12 (E)-9-Octadecanoic acid, methyl ester 21.631 1.29 296.48 Octadecanoic acid 22.737 33.24 284.47 Carbamic acid, 2-(dimethylamino)ethyl ester 27.391 1.21 132.16 Oleic acid. 15.367 1.88 281.48 Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester 19.325 0.21 330.50 6,9-Octadecadienoic acid, methyl ester 30.876 0.41 294.50 4H-1,2,4-triazol-3-ol, 5- (phenylmethyl) 18.199 1.55 56.50 Tables 4 and 5 present the zones of inhibition for different concentrations of Ganoderma lucidum methanolic and aqueous extracts against selected test organisms. The methanolic extract demonstrated moderate antibacterial activity against Escherichia coli , Staphylococcus aureus , Candida albicans , Pseudomonas aeruginosa , and Rhizopus stolonifer . The inhibition zones suggest that E. coli was the most sensitive organism, with concentration values at 2, 5, and 10 mg/mL, confirming its greater susceptibility compared to the other test strains. The aqueous extract also exhibited moderate antimicrobial activity, particularly against E. coli , S. aureus , and C. albicans , with concentration values recorded at 10 and 5 mg/mL. Discussion Phylogenetic and Biochemical Characterization of Ganoderma Species The phylogenetic analysis of Ganoderma species revealed strong genetic relationships, with G. lucidum , G anoderma sp. , G. oregonense and G. lucidum forming a well-supported clade. The high bootstrap values reinforce the genetic integrity of this classification. Notably, G. resinaceum exhibited clear genetic distinctiveness, corroborating its status as a unique species within the G. lucidum complex(13). The placement of AS_ITS1_C06_09 alongside G. resinaceum suggests close evolutionary ties, further supported by robust bootstrap values. This study emphasizes the diversity within Ganoderma , where G. lucidum and G. resinaceum demonstrate distinct evolutionary paths. The ITS-based taxonomic approach proved reliable in delineating these relationships. Bioactive Compounds and Functional Potential GC-MS analysis identified several bioactive compounds with significant pharmacological properties. Oxaspiro (C₁₇H₂₄O), exhibits antioxidant and metabolic regulatory functions (14), affirming its potential in mitigating oxidative stress-related diseases. Hexadecenoic acid, found in both methanolic ( G. lucidum methanol extract, GLME) and aqueous extracts ( G. lucidum aqueous extract, GLAE), has cardioprotective and immunomodulatory effects (15). Oleic acid was abundant in G. lucidum , suggesting its potential cardiovascular benefits, aligning with previous reports (16)). The presence of tetradecanoic acid, known for its anti-virulence activity against Pseudomonas aeruginosa (17), highlights the antimicrobial potential of Ganoderma . The compound phenylmethyl exhibited strong antimicrobial activity, inhibiting carbonic anhydrase enzymes and showing broad-spectrum efficacy against bacterial and fungal pathogens (18). Additionally, 1,2-propanediol, 3-benzyloxy-1,2-diacetyl, identified in the aqueous extract, demonstrated antibacterial and antiviral properties, suggesting its potential as a biopharmaceutical agent. The presence of hexadecanoic acid ethyl ester further supports its antimicrobial and anti-inflammatory applications, with potential cosmetic and therapeutic benefits. Notably, GLME contained a higher concentration of bioactive compounds and exhibited a stronger antimicrobial effect compared to GLAE. This suggests solvent-specific extraction efficiency, emphasizing the role of polarity in optimizing bioactive compound recovery. Future studies should explore alternative extraction techniques, such as supercritical fluid extraction, to enhance yield and efficacy. Antimicrobial Efficacy and Clinical Relevance The antimicrobial assays demonstrated that G. lucidum extracts exhibit concentration-dependent activity against clinical pathogens. While the methanol extract showed inhibition against P. aeruginosa , the aqueous extract displayed complete resistance. This highlights the solvent-dependent nature of antimicrobial efficacy, aligning with previous findings(19). The broad susceptibility of Staphylococcus aureus to methanol extracts across all concentrations supports its potent antibacterial properties (20). However, aqueous extracts exhibited inconsistent activity, reinforcing the importance of solvent selection in antimicrobial studies. Both extracts demonstrated activity against Escherichia coli , with resistance observed only at 2 mg/mL in aqueous extracts. This supports previous reports on G. lucidum ’s efficacy against E. coli (21). Interestingly, Rhizopus and Aspergillus spp. displayed resistance to both extracts, consistent with prior studies (22). However, Candida albicans exhibited dose-dependent susceptibility. Differences in susceptibility between Gram-negative and Gram-positive bacteria can be attributed to their cell wall structures. Gram-negative bacteria’s outer membrane and lipopolysaccharide (LPS) layer restrict compound diffusion, whereas the porous structure of Gram-positive bacteria facilitates greater permeability (23). Understanding these mechanisms could aid in optimizing Ganoderma -derived antimicrobials for targeted applications. Implications for Food and Pharmaceutical Industries The findings highlight the vast potential of G. lucidum as a functional food and pharmaceutical resource. With an annual global market exceeding $1.5 billion, G. lucidum ’s bioactive compounds, including triterpenoids and polysaccharides, offer diverse health benefits. Their roles in immune modulation, anticancer therapy, and metabolic regulation position Ganoderma as a promising natural alternative to synthetic pharmaceuticals. The structural diversity of bioactive compounds, such as oxaspiro and methyl esters, underscores their potential applications in nutraceuticals and functional foods. The ability of these compounds to regulate hormonal balance, relieve pain, and inhibit cancer cell proliferation further enhances their therapeutic relevance. Moreover, the antimicrobial properties of G. lucidum suggest its application in food preservation and alternative medicine. The strain isolated from Oshogbo exhibits promising potential for commercial exploitation, particularly in developing countries where affordable, natural antimicrobial agents are needed. Conclusion The identification of bioactive compounds with antimicrobial, antioxidant, and anti-inflammatory properties highlights G. lucidum ’s potential as a natural therapeutic agent. Its solvent-dependent antimicrobial efficacy further underscores the need for optimized extraction techniques. Future research should explore synergistic effects of Ganoderma bioactives with conventional antibiotics to combat drug-resistant pathogens. Additionally, large-scale fermentation and biotechnological approaches could enhance metabolite production, paving the way for commercial applications in pharmaceuticals and functional foods. Declarations Funding This research work received no funding. Author Contribution S.S and A.O conceived and designed the research work. S.S., A.O.O,I.O, A.A and F.D.O. prepared the manuscript text. O.B.A and A.I performed the molecular analysis. I.O. and A.A. provided the isolates. O.O performed the Gas Chromatography Mass Spectrometry analysis. 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Available from: https://www.researchgate.net/scientific-contributions/Kei-Kumakura-2125777496 Mustafin C, Vesnin S, Turnbull A, Dixon M, Goltsov A, Goryanin I. Diagnostics of Ovarian Tumors in Postmenopausal Patients. Diagn Basel Switz. 2022 Oct 28;12(11):2619. Tables Tables 3 to 5 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table345.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6261550","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":431876452,"identity":"102b7513-2649-4ce0-a7cc-05c8c7b5f347","order_by":0,"name":"Sarah Solomon","email":"","orcid":"","institution":"Department of Medical Microbiology and Parasitology, College of Health Sciences, Ladoke Akintola University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Solomon","suffix":""},{"id":431876455,"identity":"6479dfc7-00d2-430c-8a2e-0f94f9304953","order_by":1,"name":"Oyekale Adesola","email":"","orcid":"","institution":"Department of chemical pathology, college of health science, Ladoke Akintola University of technology","correspondingAuthor":false,"prefix":"","firstName":"Oyekale","middleName":"","lastName":"Adesola","suffix":""},{"id":431876456,"identity":"7b8661b3-c708-4c2d-afcf-4363efa8a9d7","order_by":2,"name":"Adeola Ayanyinka","email":"","orcid":"","institution":"Department of Medical Microbiology and Parasitology, College of Health Sciences, Ladoke Akintola University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Adeola","middleName":"","lastName":"Ayanyinka","suffix":""},{"id":431876457,"identity":"43bfdac8-f395-48e5-8875-52191df6829c","order_by":3,"name":"Adeniyi Ifejesu","email":"","orcid":"","institution":"Department of pharmacology and therapeutics, University of Ibadan O","correspondingAuthor":false,"prefix":"","firstName":"Adeniyi","middleName":"","lastName":"Ifejesu","suffix":""},{"id":431876458,"identity":"82fe1403-27a2-4e07-9cd0-8fb1f94480e8","order_by":4,"name":"Olorunfemi Adedolapo","email":"","orcid":"","institution":"Department of Medical Microbiology and Parasitology, College of Health Sciences, Ladoke Akintola University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Olorunfemi","middleName":"","lastName":"Adedolapo","suffix":""},{"id":431876459,"identity":"9a34ecf8-080c-411d-8444-23cb05a51477","order_by":5,"name":"Fiyinfoluwa Ojeniyi","email":"","orcid":"","institution":"Department of Biochemistry, Ladoke Akintola University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Fiyinfoluwa","middleName":"","lastName":"Ojeniyi","suffix":""},{"id":431876460,"identity":"2449e52c-a613-4547-9f9d-b18edcc19804","order_by":6,"name":"Itunuoluwa Oyelayo","email":"","orcid":"","institution":"Department of Medical Microbiology and Parasitology, College of Health Sciences, Ladoke Akintola University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Itunuoluwa","middleName":"","lastName":"Oyelayo","suffix":""},{"id":431876461,"identity":"4a057410-6360-4449-8ec7-f416cb97d93a","order_by":7,"name":"Oluranti Olayinka","email":"","orcid":"","institution":"Departments of Microbiology, Bowen University Iwo","correspondingAuthor":false,"prefix":"","firstName":"Oluranti","middleName":"","lastName":"Olayinka","suffix":""},{"id":431876462,"identity":"8875e11a-f239-45f0-8fdb-670d54c6298b","order_by":8,"name":"Oluyinka Oladele Opaleye","email":"","orcid":"","institution":"Department of Medical Microbiology and Parasitology, College of Health Sciences, Ladoke Akintola University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Oluyinka","middleName":"Oladele","lastName":"Opaleye","suffix":""},{"id":431876463,"identity":"65be0fe9-b749-492a-98a3-11f0b42855c0","order_by":9,"name":"Olusola Ojurongbe","email":"","orcid":"","institution":"Department of Medical Microbiology and Parasitology, College of Health Sciences, Ladoke Akintola University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Olusola","middleName":"","lastName":"Ojurongbe","suffix":""},{"id":431876464,"identity":"12cae089-8468-44f5-85c7-d14c01643491","order_by":10,"name":"Olowe Adekunle","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYHACxgNAQo4PwgGxEwjrASkzZgPqbSBJS2Ib0Vr42Q8fOPCxzSa9jf2M+YMPNXeAIjkGzAW/cGuR7ElLODizLS23jSfHsHHGsWdAkTcGzDP7cGsxOJBjcJh32+HcNoYcw2behsMMBjeAtvD24NFy/g1Iy/90Nv43hs1/gVrsCWq5AbblQAKbBNAWRpAtEkAtPD/w+GXGM6Bf/iUbtkk8K5zZc+wwj8SZZwWHeRtwa+HnTz744MMZO3kgY8OHHzWH5fjbkzc+5vmDWwsG4AERBxjbSNACBaTYMgpGwSgYBcMdAABlMldi1ZaD3wAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Medical Microbiology and Parasitology, College of Health Sciences, Ladoke Akintola University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Olowe","middleName":"","lastName":"Adekunle","suffix":""}],"badges":[],"createdAt":"2025-03-19 12:23:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6261550/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6261550/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79158083,"identity":"57714eb9-3b3e-4e50-ac07-c89ba4eabb15","added_by":"auto","created_at":"2025-03-25 06:49:53","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":508688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 2: Phylogenetic Tree of Sample AS_ITS1_C06_09 and Related \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGanoderma\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eSequences\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6261550/v1/2925a59a1e5475129dabe95e.jpeg"},{"id":79157050,"identity":"08542692-e67a-454c-97ef-29eb251c7746","added_by":"auto","created_at":"2025-03-25 06:33:53","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":682919,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 3: GC-MS result identifying the bioactive compounds present in GLME\u0026amp;GLAE with their retention time.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6261550/v1/314013eb1dda690275e7c2e4.jpeg"},{"id":79163244,"identity":"c8387449-9ee5-43cb-9ecc-b07209b5befe","added_by":"auto","created_at":"2025-03-25 07:53:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2328466,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6261550/v1/2bb02191-92d2-4756-9e11-01a3da245230.pdf"},{"id":79157054,"identity":"df51ae2f-3baa-4268-82d9-5cbff956dc98","added_by":"auto","created_at":"2025-03-25 06:33:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":457936,"visible":true,"origin":"","legend":"","description":"","filename":"Table345.docx","url":"https://assets-eu.researchsquare.com/files/rs-6261550/v1/32c59d67306bec4cad9f6f8c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phytochemical Analysis and Antimicrobial Activity of Ganoderma lucidum Against Selected Bacteria and Fungi of Medical Importance","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eGanoderma\u003c/em\u003e species, particularly \u003cem\u003eGanoderma lucidum\u003c/em\u003e, are well known for their medicinal and bioactive properties, including antimicrobial, antioxidant, and immunomodulatory effects. These fungi contain diverse secondary metabolites such as polysaccharides, triterpenoids, and phenolic compounds, which contribute to their therapeutic potential(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). With the rising global concern over antimicrobial resistance, there is an increasing need to explore natural sources for novel bioactive compounds, and medicinal mushrooms have emerged as promising candidates. While the pharmacological properties of \u003cem\u003eG. lucidum\u003c/em\u003e are well documented, limited research exists on its antimicrobial efficacy against multidrug-resistant bacteria and pathogenic fungi in Nigeria (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eDespite several studies highlighting the antimicrobial and bioactive properties of \u003cem\u003eG. lucidum\u003c/em\u003e, data on its effectiveness against specific bacterial and fungal strains endemic to Nigeria remain scarce. Additionally, there has been minimal exploration of the phytochemical composition of \u003cem\u003eG. lucidum\u003c/em\u003e from different ecological zones in Nigeria, and limited comparisons between the efficacy of aqueous and methanolic extracts (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Furthermore, the molecular characterization of indigenous \u003cem\u003eGanoderma\u003c/em\u003e species remains underexplored, restricting a comprehensive understanding of their genetic diversity and pharmaceutical potential. These gaps should be addresses for enhancing local fungal biodiversity in antimicrobial drug discovery.\u003c/p\u003e \u003cp\u003eThe increasing prevalence of antimicrobial-resistant pathogens necessitates the search for alternative treatment strategies. \u003cem\u003eG. lucidum\u003c/em\u003e, a widely recognized medicinal mushroom, represents a valuable natural resource for bioactive compound discovery (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMolecular identification of Nigerian \u003cem\u003eG. lucidum\u003c/em\u003e, profiling its phytochemical composition using GC-MS isolates ensures accurate species characterization, evaluating its antimicrobial activity against clinically relevant bacterial and fungal strains in Nigeria provides insights into its bioactive compounds, forming a potential as a natural antimicrobial agent and a foundation for pharmaceutical applications.\u003c/p\u003e \u003cp\u003eThis study aims to collect and identify \u003cem\u003eGanoderma\u003c/em\u003e species from selected ecological zones in Nigeria using morphological and molecular techniques. It seeks to analyze the phytochemical composition of \u003cem\u003eG. lucidum\u003c/em\u003e extracts through GC-MS profiling and assess their antimicrobial activity against selected bacterial and fungal pathogens. Furthermore, the study will evaluate the genetic diversity of \u003cem\u003eG. lucidum\u003c/em\u003e isolates through phylogenetic analysis and compare the efficacy of aqueous and methanolic extracts in inhibiting microbial growth.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e2.1. Sample Collection.\u003c/p\u003e\n\u003cp\u003eMushroom samples were collected during the rainy season in June 2024 from Ofatedo Osogbo (Osun State) and Owode Ogbomosho (Oyo State), Nigeria. Two specimens, exhibiting morphological characteristics consistent with \u003cem\u003eGanoderma\u003c/em\u003e, were confirmed through molecular identification. The samples were harvested using sterile tools and preserved by drying and refrigeration for subsequent analysis. These locations were selected due to their favorable ecological conditions for fungal growth, with the samples intended for antimicrobial and bioactive compound studies.\u003c/p\u003e\n\u003cp\u003e2.2. Sample Preparation.\u003c/p\u003e\n\u003cp\u003eFresh mushrooms were sliced and air-dried in an oven (Selecta, Madrid, Spain) at 50\u0026deg;C until a constant weight was achieved. The dried material (60 g) was ground into a fine powder using a hammer stirrer (IKA, Staufen, Germany). A 30 g portion was extracted in 500 mL of hot water and methanol and stirred for 96 h to dissolve phytochemicals. The extract was filtered using Whatman No. 1 filter paper (5).\u003c/p\u003e\n\u003cp\u003e2.3. Molecular Identification\u003c/p\u003e\n\u003cp\u003e2.3.1. DNA Extraction\u003c/p\u003e\n\u003cp\u003eDNA was extracted using the ZYMO DNA extraction kit. A 50 mg wet sample was resuspended in 200 \u0026mu;L of water in a ZR BashingBead\u0026trade; Lysis Tube, with 750 \u0026mu;L BashingBead\u0026trade; Buffer added. The mixture was processed in a bead beater for 5 min, centrifuged at 10,000 \u0026times; g for 1 min, and the 400 \u0026mu;L supernatant was transferred to a Zymo-Spin\u0026trade; III-F Filter. Following sequential filtrations and centrifugations at 10,000 \u0026times; g, 100 \u0026mu;L of DNA Elution Buffer was added, yielding 2.80 ng/\u0026mu;L DNA.\u003c/p\u003e\n\u003cp\u003e2.3.2. Primer Design and PCR Amplification\u003c/p\u003e\n\u003cp\u003eThe ITS1 and ITS4 primers (6) were used to amplify the ITS ribosomal region following(7). PCR was performed with 12.5 \u0026mu;L PCR master mix, 1.25 \u0026mu;L of each 10 mM primer, and 10 \u0026mu;L of diluted DNA template, adjusted to 25 \u0026mu;L with sterile deionized water. Thermal cycling conditions included initial denaturation at 94\u0026deg;C for 5 min, followed by 40 cycles of 94\u0026deg;C for 30 s, 50\u0026deg;C for 30 s, and a final extension at 72\u0026deg;C for 1 min. PCR products were purified using Montage\u0026trade; PCR Centrifugal Filter Devices (Millipore Corp., USA)(8).\u003c/p\u003e\n\u003cp\u003e2.3.3. Genome Sequencing and Analysis\u003c/p\u003e\n\u003cp\u003eGenome sequencing was conducted using PacBio Sequel (long-read) and Illumina NovaSeq (short-read) platforms. De novo assembly was performed with SMARTdenovo1 and polished using Pilon(9). Assembly integrity was assessed with BUSCO (10). The ITS1-5.8S-ITS2 region (588 bp) was analyzed using BLAST with 99.55% identity, 99.55% query coverage, and an E-value \u0026le;1.00E-135. Sequences were matched to \u003cem\u003eGanoderma lucidum\u003c/em\u003e (taxid:5315). Phylogenetic relationships were inferred using Maximum Likelihood (ML) analysis in MEGA 7(11).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.4. Extract Concentration\u003c/p\u003e\n\u003cp\u003eAqueous extracts of \u003cem\u003eG. lucidum\u003c/em\u003e were concentrated using a freeze dryer (Model LGJ-10) at 50\u0026deg;C. Methanolic extracts were concentrated via evaporation in a water bath at 50\u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.5. Test Organisms\u003c/p\u003e\n\u003cp\u003eBacterial isolates (\u003cem\u003eE. coli, Staphylococcus aureus, Enterobacter aerogenes, Pseudomonas aeruginosa\u003c/em\u003e) were obtained from the Microbiology Laboratory, Ladoke Akintola University, Ogbomosho, Nigeria. Fungal strains (\u003cem\u003eAspergillus niger, Rhizopus stolonifer, Penicillium sp., A. flavus\u003c/em\u003e) were sourced from Bowen University, Iwo, Nigeria. Cultures were maintained on agar slants at 4\u0026deg;C to prevent contamination(5)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.6. Phytochemical Analysis (GC-MS)\u003c/p\u003e\n\u003cp\u003eGC-MS analysis was performed using an Agilent Technologies Model 7000 GC-MS Triple Quad (TQQQ) system with Hunter workstation software (B.04.00). The electron ionization potential was 70 eV. Compounds were separated using an OPTIMA-5 column (30 m \u0026times; 250 \u0026mu;m \u0026times; 0.25 \u0026mu;m) at 360\u0026deg;C. Helium was the carrier gas (flow rate: 1.129 mL/min; split ratio: 5:1). A 2.5 \u0026mu;L sample was injected via an automatic liquid sampler. Compounds were identified by comparing mass spectra with the NIST database, using retention indices calculated via Kovat\u0026rsquo;s formula with \u003cem\u003en\u003c/em\u003e-alkanes (C9\u0026ndash;C33) as standards(12)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.7. Inoculum Preparation\u003c/p\u003e\n\u003cp\u003eTest organisms were sub-cultured on nutrient agar and potato dextrose agar, then incubated at 37\u0026deg;C for bacteria and 24\u0026deg;C for fungi for 24\u0026ndash;36 h. Bacterial suspensions were adjusted to a 0.5 McFarland standard (~1.5 \u0026times; 10⁸ CFU/mL).\u003c/p\u003e\n\u003cp\u003e2.8. Antimicrobial Assay\u003c/p\u003e\n\u003cp\u003eThe agar well diffusion method was used. A 24-h broth culture was swabbed onto sterile Mueller-Hinton agar in Petri dishes. Three equidistant wells (6 mm) were created using a sterile cork borer and filled with 0.1 mL of extract. A positive control (chloramphenicol, 50 \u0026mu;g/mL) and a negative control (solvent) were included. Plates were incubated at 37\u0026deg;C for 24 h, and inhibition zones were measured in millimetres (5)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.9. Statistical Analysis\u003c/p\u003e\n\u003cp\u003eData were analyzed using SPSS (Version 25.0) and expressed as mean \u0026plusmn; standard deviation. Statistical significance was determined using one-way ANOVA, Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test, and chi-square analysis (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05). Bar charts with error bars were generated using Microsoft Excel with Pearson correlation analysis.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eThe BLAST analysis of the ITS sequence \u003cstrong\u003eAS_ITS1_C06_09\u003c/strong\u003e yielded significant alignments with sequences belonging to the genus \u003cem\u003eGanoderm\u003c/em\u003e\u003cem\u003ea lucidum\u003c/em\u003e. High-confidence matches were observed with multiple species, including \u003cem\u003eGanoderma\u0026nbsp;\u003c/em\u003e\u003cem\u003eoregonense strain\u003c/em\u003e, \u003cem\u003eGanoderma sp.\u003c/em\u003e, \u003cem\u003eGanoderma resinaceum\u003c/em\u003e, and \u003cem\u003eGanoderma\u0026nbsp;\u003c/em\u003e\u003cem\u003elobatum\u003c/em\u003e. These alignments were characterized by high Max Scores, consistent Query Coverages, high Percent Identities, and exceptionally low E-values (Table 1).\u003c/p\u003e\n\u003cp\u003eThe top alignments for \u003cem\u003eGanoderma\u0026nbsp;\u003c/em\u003e\u003cem\u003elucidum\u003c/em\u003eisolate included sequences such as \u003cem\u003eOQ701698.1\u003c/em\u003e(\u003cem\u003eGanoderma lucidum isolate GJN-2\u003c/em\u003e\u003cem\u003e),\u0026nbsp;\u003c/em\u003eMF755277.1\u0026nbsp;(\u003cem\u003eGanoderma\u0026nbsp;\u003c/em\u003e\u003cem\u003elucidum strainNM20 TENN\u003c/em\u003e) and \u003cem\u003eKX055549.1\u003c/em\u003e (\u003cem\u003eGanoderma\u0026nbsp;\u003c/em\u003e\u003cem\u003eresinaceum strain G62\u003c/em\u003e), which exhibited Max Scores of 645, Percent Identities of 99.32%, Query Coverages of 75%, and E-values of 0.0. \u0026nbsp; Alignment metrics, indicating consistent taxonomic associations with the query sequence.\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eGanoderma sp.\u003c/em\u003e, notable alignments included \u003cem\u003eMH\u003c/em\u003e\u003cem\u003e708512\u003c/em\u003e\u003cem\u003e.1\u003c/em\u003e (\u003cem\u003eGanoderma\u0026nbsp;\u003c/em\u003e\u003cem\u003eisolate 16 ITS-1\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e) and. These sequences exhibited Max Scores of 645, Percent Identities of 99.32%, Query Coverages of 75%, and E-values of 0.0. These results suggest that the query sequence shares substantial similarity with other species in the genus, highlighting its broader taxonomic relevance.\u003c/p\u003e\n\u003cp\u003eAlignments with \u003cem\u003eGanoderma lucidum\u003c/em\u003e sequences were equally significant. High-confidence matches included \u003cem\u003eOQ701698.1\u003c/em\u003e (\u003cem\u003eGanoderma lucidum isolate GJN-2\u003c/em\u003e), which exhibited a Max Score of 620, Percent Identity of 99.55%, Query Coverage of 75%, and an E-value of 0.0. Additional sequences, such as \u003cem\u003eON394695.1\u003c/em\u003e (\u003cem\u003eGanoderma lucidum strain LAU22\u003c/em\u003e) and \u003cem\u003eMF755277.1\u003c/em\u003e (\u003cem\u003eGanoderma lucidum strain NM20\u003c/em\u003e), displayed Max Scores of 663, Percent Identities of 99.32%, Query Coverages of 75%, and E-values range is0.0. These findings confirm the close taxonomic relationship of the query sequence with \u003cem\u003eGanoderma lucidum\u003c/em\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: Best hit for AS_ITS1_C06_09 sequence using BLAST\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"955\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003eScientific Name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eMax Score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003eTotal Score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003eQuery Coverage (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003eE Value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003ePercent Identity (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003eAccession Length\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003eAccession\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanodermalucidum isolate GJN-2 ITS-2\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma lucidum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e806\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e806\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e620\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eOQ701698.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma lucidum strain \u0026nbsp;NM20 (TENN)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma lucidum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e802\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e802\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e663\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eMF755277.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma strain. \u0026nbsp;LAU22\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma sp.\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e802\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e802\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e611\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eON394695.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma oregonense strain ASI 7049\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma oregonense\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e645\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eJQ520194.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma lucidum ITS1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma lucidum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e557\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eKT997431.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma lucidum isolate SMCC170.01.61\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma lucidum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e627\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eFJ501561.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma lucidum isolate BNB01\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma lucidum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e565\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eOR995187.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma lucidum \u0026nbsp;isolate GLSM4\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma lucidum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e586\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eKF648564.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma resinaceum strain G62\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma resinaceum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e647\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eKX055549.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma lobatum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003estrain ASI 7061\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma lobatum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e645\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eJQ520166.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma lucidum strain OE-234\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma lucidum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e634\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eAY636059.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma lucidum isolate 16 ITS-1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma lucidum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e612\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eMH708512.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma oregonense strain ASI 7067\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma lucidum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e795\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e795\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e645\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eJQ520197.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma sp. ASI 7033\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cem\u003eGanoderma lucidum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e795\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e795\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e99.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e645\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cem\u003eJQ520208.1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 265px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma lucidum isolate BNB01\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 156px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eGanoderma lucidum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e496\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 58px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e496\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e74\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e86.65\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e650\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 121px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eOR995187.1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe phylogenetic analysis of the sample AS_ITS1_C06_09 (Figure 2) provided key insights into its evolutionary relationships within the \u003cem\u003eGanoderma\u003c/em\u003e genus. Using the Maximum Likelihood method with 100 bootstrap replicates, the analysis showed a strong clustering of AS_ITS1_C06_09 with \u003cem\u003eGanoderma sessile\u003c/em\u003e (KF605634.1), indicating significant genetic similarity and evolutionary relatedness. High bootstrap values further validated this placement. The phylogenetic tree also revealed distinct clades for other \u003cem\u003eGanoderma\u003c/em\u003e species, illustrating the genus\u0026apos;s genetic diversity. Notably, \u003cem\u003eGanoderma lucidum\u003c/em\u003e (ON394695.1), isolated from Ogbomoso, Nigeria, formed a separate, well-supported clade, underscoring its evolutionary divergence and unique genetic identity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGas Chromatography \u0026ndash; Mass Spectrophotometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Gas Chromatography-Mass Spectrometry (GC-MS) analysis of \u003cem\u003eGanoderma lucidum\u003c/em\u003e Methanol Extract (GLME) and Aqueous Extract (GLAE) revealed a diverse range of bioactive compounds (Figure 3), each identified by its retention time (RT). \u003cem\u003eG. lucidum\u003c/em\u003e, a highly versatile medicinal mushroom, is rich in polysaccharides and triterpenoids, which have been traditionally used and are now widely recognized as nutraceuticals and functional foods. Notably, hexadecenoic acid, detected at RTs of 13.782 min (GLME) and 13.456 min (GLAE), plays a vital role in cardiometabolic protection, immune modulation, and liver health. Additionally, the presence of antioxidant and anti-inflammatory compounds underscores the potential therapeutic benefits of these extracts. These findings confirm that the phytochemicals identified contribute to the antimicrobial and bioactive properties of \u003cem\u003eG. lucidum\u003c/em\u003e, warranting further investigation into their functional applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGC-MS Profiling of Bioactive Compounds in \u003cem\u003eGanoderma lucidum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GC-MS analysis of \u003cem\u003eGanoderma lucidum\u003c/em\u003e extracts identified various bioactive compounds with significant therapeutic potential. The most abundant was octadecanoic acid (RT: 22.737 min, 33.24%), known for its antimicrobial, anti-inflammatory, and lipid metabolism-regulating effects. Another major compound, deca-6,9-diene-2,8-dione (RT: 13.324 min, 22.11%), is also bioactive. Various saturated and unsaturated fatty acids, including hexadecanoic acid, pentadecanoic acid methyl ester, and oleic acid, exhibited cardioprotective, antimicrobial, and antioxidant properties.\u003c/p\u003e\n\u003cp\u003eBeyond fatty acids, D-arabinitol (RT: 10.772 min, 5.32%), a sugar alcohol, displayed antifungal activity. Organic acids like nonane acid and sulfurous acid derivatives suggested preservative-like and antimicrobial functions. Minor bioactive components, such as carbamic acid derivatives (neuroprotective potential) and 4H-1,2,4-triazol-3-ol derivatives (antifungal properties), further enriched the extract\u0026apos;s medicinal potential. These findings reinforce the pharmaceutical and nutraceutical significance of \u003cem\u003eG. lucidum\u003c/em\u003e, supporting its traditional medicinal use and potential application in antimicrobial and therapeutic formulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibiotic susceptibility test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe evaluation of aqueous and methanol extracts of \u003cem\u003eGanoderma lucidum\u003c/em\u003e against bacterial and fungal test organisms. The presence of clear inhibition zones around some of the test organisms confirmed the antimicrobial potency of the extracts. Given that methanol is a more effective solvent for extracting bioactive compounds, the methanol extract likely exhibited stronger activity than the aqueous extract.\u003c/p\u003e\n\u003cp\u003eThese results suggest that \u003cem\u003eG. lucidum\u003c/em\u003e extracts possess natural antimicrobial properties, making them promising candidates for the development of plant-based antimicrobial agents. The identification of clear inhibition zones against certain bacteria and fungi indicates their potential application in controlling microbial infections. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: GC-MS Profiling of Bioactive Compounds in \u003cem\u003eGanoderma lucidum\u003c/em\u003e: Identification and Functional Significance\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"551\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003eCompounds peak\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003eRetention time\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003eArea (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eMolecular weight\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003eDecane,2,3,7-trimethyl3-Ethyl-3-methylheptaneUndecane,3-ethyl-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e6.944\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e0.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e116.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003eHexadecane, tridecane, sulfurous acid.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e7.499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e2.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e158.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003eNonane acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e10.497\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e3.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e242.40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003eD-Arabinitol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e10.772\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e5.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e652.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003epentadecanoic acid, 14-methyl-este\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e19.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e12.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e270.45\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003eHexadecanoic acid, ethyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e19.907\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e7.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e284.55\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003edeca-6,9-diene-2,8-dione\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e13..324\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e22.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e294.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003eHeptadecane, 2, 6, 10, 15 Tetramethy\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e20.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e296.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003e(E)-9-Octadecanoic acid, methyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e21.631\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e296.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003eOctadecanoic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e22.737\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e33.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e284.47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003eCarbamic acid, 2-(dimethylamino)ethyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e27.391\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e132.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003eOleic acid.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e15.367\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e281.48\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003eHexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e19.325\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e330.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003e6,9-Octadecadienoic acid, methyl ester\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e30.876\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e294.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 287px;\"\u003e\n \u003cp\u003e4H-1,2,4-triazol-3-ol, 5- (phenylmethyl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e18.199\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 50px;\"\u003e\n \u003cp\u003e1.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e56.50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTables 4 and 5 present the zones of inhibition for different concentrations of \u003cem\u003eGanoderma lucidum\u003c/em\u003e methanolic and aqueous extracts against selected test organisms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe methanolic extract demonstrated moderate antibacterial activity against \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eCandida albicans\u003c/em\u003e, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, and \u003cem\u003eRhizopus stolonifer\u003c/em\u003e. The inhibition zones suggest that \u003cem\u003eE. coli\u003c/em\u003e was the most sensitive organism, with concentration values at 2, 5, and 10 mg/mL, confirming its greater susceptibility compared to the other test strains. \u0026nbsp;The aqueous extract also exhibited moderate antimicrobial activity, particularly against \u003cem\u003eE. coli\u003c/em\u003e, \u003cem\u003eS. aureus\u003c/em\u003e, and \u003cem\u003eC. albicans\u003c/em\u003e, with concentration values recorded at 10 and 5 mg/mL.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003ePhylogenetic and Biochemical Characterization of \u003cem\u003eGanoderma\u003c/em\u003e Species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phylogenetic analysis of \u003cem\u003eGanoderma\u003c/em\u003e species revealed strong genetic relationships, with \u003cem\u003eG.\u0026nbsp;\u003c/em\u003e\u003cem\u003elucidum\u003c/em\u003e, \u003cem\u003eG\u003c/em\u003e\u003cem\u003eanoderma\u003c/em\u003e\u003cem\u003e\u0026nbsp;sp.\u003c/em\u003e, G.\u0026nbsp;\u003cem\u003eoregonense\u003c/em\u003e and \u003cem\u003eG. lucidum\u003c/em\u003e forming a well-supported clade. The high bootstrap values reinforce the genetic integrity of this classification. Notably, \u003cem\u003eG. resinaceum\u003c/em\u003e exhibited clear genetic distinctiveness, corroborating its status as a unique species within the \u003cem\u003eG. lucidum\u003c/em\u003e complex(13).\u0026nbsp;The placement of AS_ITS1_C06_09 alongside \u003cem\u003eG. resinaceum\u003c/em\u003e suggests close evolutionary ties, further supported by robust bootstrap values. This study emphasizes the diversity within \u003cem\u003eGanoderma\u003c/em\u003e, where \u003cem\u003eG. lucidum\u003c/em\u003e and \u003cem\u003eG. resinaceum\u003c/em\u003e demonstrate distinct evolutionary paths. The ITS-based taxonomic approach proved reliable in delineating these relationships.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioactive Compounds and Functional Potential\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGC-MS analysis identified several bioactive compounds with significant pharmacological properties. Oxaspiro (C₁₇H₂₄O), exhibits antioxidant and metabolic regulatory functions (14), affirming its potential in mitigating oxidative stress-related diseases. Hexadecenoic acid, found in both methanolic (\u003cem\u003eG. lucidum\u003c/em\u003e methanol extract, GLME) and aqueous extracts (\u003cem\u003eG. lucidum\u003c/em\u003e aqueous extract, GLAE), has cardioprotective and immunomodulatory effects (15).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOleic acid was abundant in \u003cem\u003eG. lucidum\u003c/em\u003e, suggesting its potential cardiovascular benefits, aligning with previous reports (16)). The presence of tetradecanoic acid, known for its anti-virulence activity against \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e(17), highlights the antimicrobial potential of \u003cem\u003eGanoderma\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe compound phenylmethyl exhibited strong antimicrobial activity, inhibiting carbonic anhydrase enzymes and showing broad-spectrum efficacy against bacterial and fungal pathogens (18). Additionally, 1,2-propanediol, 3-benzyloxy-1,2-diacetyl, identified in the aqueous extract, demonstrated antibacterial and antiviral properties, suggesting its potential as a biopharmaceutical agent. The presence of hexadecanoic acid ethyl ester further supports its antimicrobial and anti-inflammatory applications, with potential cosmetic and therapeutic benefits.\u003c/p\u003e\n\u003cp\u003eNotably, GLME contained a higher concentration of bioactive compounds and exhibited a stronger antimicrobial effect compared to GLAE. This suggests solvent-specific extraction efficiency, emphasizing the role of polarity in optimizing bioactive compound recovery. Future studies should explore alternative extraction techniques, such as supercritical fluid extraction, to enhance yield and efficacy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntimicrobial Efficacy and Clinical Relevance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antimicrobial assays demonstrated that \u003cem\u003eG. lucidum\u003c/em\u003e extracts exhibit concentration-dependent activity against clinical pathogens. While the methanol extract showed inhibition against \u003cem\u003eP. aeruginosa\u003c/em\u003e , the aqueous extract displayed complete resistance. This highlights the solvent-dependent nature of antimicrobial efficacy, aligning with previous findings(19).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe broad susceptibility of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e to methanol extracts across all concentrations supports its potent antibacterial properties (20). However, aqueous extracts exhibited inconsistent activity, reinforcing the importance of solvent selection in antimicrobial studies. Both extracts demonstrated activity against \u003cem\u003eEscherichia coli\u003c/em\u003e, with resistance observed only at 2 mg/mL in aqueous extracts. This supports previous reports on \u003cem\u003eG. lucidum\u003c/em\u003e\u0026rsquo;s efficacy against \u003cem\u003eE. coli\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e(21). Interestingly, \u003cem\u003eRhizopus\u003c/em\u003e and \u003cem\u003eAspergillus\u003c/em\u003e spp. displayed resistance to both extracts, consistent with prior studies\u0026nbsp;(22). However, \u003cem\u003eCandida albicans\u003c/em\u003e exhibited dose-dependent susceptibility.\u003c/p\u003e\n\u003cp\u003eDifferences in susceptibility between Gram-negative and Gram-positive bacteria can be attributed to their cell wall structures. Gram-negative bacteria\u0026rsquo;s outer membrane and lipopolysaccharide (LPS) layer restrict compound diffusion, whereas the porous structure of Gram-positive bacteria facilitates greater permeability (23). Understanding these mechanisms could aid in optimizing \u003cem\u003eGanoderma\u003c/em\u003e-derived antimicrobials for targeted applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImplications for Food and Pharmaceutical Industries\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe findings highlight the vast potential of \u003cem\u003eG. lucidum\u003c/em\u003e as a functional food and pharmaceutical resource. With an annual global market exceeding $1.5 billion, \u003cem\u003eG. lucidum\u003c/em\u003e\u0026rsquo;s bioactive compounds, including triterpenoids and polysaccharides, offer diverse health benefits. Their roles in immune modulation, anticancer therapy, and metabolic regulation position \u003cem\u003eGanoderma\u003c/em\u003e as a promising natural alternative to synthetic pharmaceuticals.\u003c/p\u003e\n\u003cp\u003eThe structural diversity of bioactive compounds, such as oxaspiro and methyl esters, underscores their potential applications in nutraceuticals and functional foods. The ability of these compounds to regulate hormonal balance, relieve pain, and inhibit cancer cell proliferation further enhances their therapeutic relevance.\u003c/p\u003e\n\u003cp\u003eMoreover, the antimicrobial properties of \u003cem\u003eG. lucidum\u003c/em\u003e suggest its application in food preservation and alternative medicine. The strain isolated from Oshogbo exhibits promising potential for commercial exploitation, particularly in developing countries where affordable, natural antimicrobial agents are needed.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe identification of bioactive compounds with antimicrobial, antioxidant, and anti-inflammatory properties highlights \u003cem\u003eG. lucidum\u003c/em\u003e\u0026rsquo;s potential as a natural therapeutic agent. Its solvent-dependent antimicrobial efficacy further underscores the need for optimized extraction techniques.\u003c/p\u003e \u003cp\u003eFuture research should explore synergistic effects of \u003cem\u003eGanoderma\u003c/em\u003e bioactives with conventional antibiotics to combat drug-resistant pathogens. Additionally, large-scale fermentation and biotechnological approaches could enhance metabolite production, paving the way for commercial applications in pharmaceuticals and functional foods.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research work received no funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.S and A.O conceived and designed the research work. S.S., A.O.O,I.O, A.A and F.D.O. prepared the manuscript text. O.B.A and A.I performed the molecular analysis. I.O. and A.A. provided the isolates. O.O performed the Gas Chromatography Mass Spectrometry analysis. A.O.O performed the statistical analysis and prepared the figures. O.O.O, O.O., O.A. supervised the work. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSharma C, Kumar N, Pandey R, Meis JF, Chowdhary A. 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Atriplex halimus L.: Its biology and uses. J Arid Environ. 2014 Jan;100\u0026ndash;101:111\u0026ndash;21. \u003c/li\u003e\n\u003cli\u003eWaterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 2018 Jul 2;46(W1):W296\u0026ndash;303. \u003c/li\u003e\n\u003cli\u003eKumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Battistuzzi FU, editor. Mol Biol Evol. 2018 Jun 1;35(6):1547\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003eFarooqi W, Alabdullkarim FA, Abukaram TM, Gubran L, Alsulami DS, Albehairi SA, et al. Comparison of Efficacy and Safety of Device-Based Interventions Versus Pharmacological Therapy in the Management of Patients With Advanced Parkinson\u0026rsquo;s Disease: A Literature Review. Cureus. 2024 Dec;16(12):e76044. \u003c/li\u003e\n\u003cli\u003eKeith Vlasak [Internet]. [cited 2025 Mar 20]. Available from: https://www.goodreads.com/author/show/8451611.Keith_Vlasak\u003c/li\u003e\n\u003cli\u003eNational Center for Biotechnology Information [Internet]. [cited 2025 Mar 20]. Available from: https://www.ncbi.nlm.nih.gov/\u003c/li\u003e\n\u003cli\u003eFilbay SR, Dowsett M, Chaker Jomaa M, Rooney J, Sabharwal R, Lucas P, et al. Healing of acute anterior cruciate ligament rupture on MRI and outcomes following non-surgical management with the Cross Bracing Protocol. Br J Sports Med. 2023 Dec;57(23):1490\u0026ndash;7. \u003c/li\u003e\n\u003cli\u003ePiccinin E, Villani G, Moschetta A. Metabolic aspects in NAFLD, NASH and hepatocellular carcinoma: the role of PGC1 coactivators. Nat Rev Gastroenterol Hepatol. 2019 Mar;16(3):160\u0026ndash;74. \u003c/li\u003e\n\u003cli\u003eToral-Ju\u0026aacute;rez MA, Avila RT, Cardoso AA, Brito FAL, Machado KLG, Almeida WL, et al. Drought-tolerant coffee plants display increased tolerance to waterlogging and post-waterlogging reoxygenation. Environ Exp Bot. 2021 Feb 1;182:104311. \u003c/li\u003e\n\u003cli\u003eErbiai EH, Amina B, Kaoutar A, Saidi R, Lamrani Z, Pinto E, et al. Chemical Characterization and Evaluation of Antimicrobial Properties of the Wild Medicinal Mushroom Ganoderma lucidum Growing in Northern Moroccan Forests. Life. 2023 May;13(5):1217. \u003c/li\u003e\n\u003cli\u003eGenetics and Plant Breeding, College of PG-Studies, Central Agricultural University, Umiam-793103, Meghalaya, India, Naveenkumar KL. Morphological Diversity Analysis of Novel Inbred Lines of Maize(Zea Mays L.) for Development of Single Cross Hybrids. Int J Pure Appl Biosci. 2017 Mar 8;5(1):489\u0026ndash;97. \u003c/li\u003e\n\u003cli\u003eWang G, Chang L, Zhang R, Wang S, Wei X, Rickert E, et al. Supplement to: Wang, G et al. (2019): Can targeted defense elicitation improve seaweed aquaculture? Journal of Applied Phycology, 31(3), 1845-1854, https://doi.org/10.1007/s10811-018-1709-6. PANGAEA; 2018 [cited 2025 Mar 20]. Experiment on the effects of oligo-alginate elicitation on juvenile and adult sporophytes of Saccharina japonica and Saccharina latissima. Available from: https://doi.pangaea.de/10.1594/PANGAEA.896664\u003c/li\u003e\n\u003cli\u003eOSF | Lee et al. (2022) [Internet]. [cited 2025 Mar 20]. Available from: https://osf.io/vjpw4/\u003c/li\u003e\n\u003cli\u003eKei Kumakura\u0026rsquo;s research works | Takasaki University of Health and Welfare and other places [Internet]. [cited 2025 Mar 20]. Available from: https://www.researchgate.net/scientific-contributions/Kei-Kumakura-2125777496\u003c/li\u003e\n\u003cli\u003eMustafin C, Vesnin S, Turnbull A, Dixon M, Goltsov A, Goryanin I. Diagnostics of Ovarian Tumors in Postmenopausal Patients. Diagn Basel Switz. 2022 Oct 28;12(11):2619. \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 3 to 5 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ganoderma lucidum, phytochemical analysis, antimicrobial activity, medical importance, GC-MS, PCR authentication, medicinal mushroom, infectious diseases","lastPublishedDoi":"10.21203/rs.3.rs-6261550/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6261550/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eIntroduction\u003c/b\u003e:\u003cem\u003eGanoderma lucidum\u003c/em\u003e, a medicinal mushroom widely studied for its bioactive properties, holds promise for antimicrobial applications. This study aimed to identify \u003cem\u003eG. lucidum\u003c/em\u003e samples collected from Osun States, Nigeria, and evaluate their antimicrobial potential, bioactive compound profile and the molecular characterization.\u003c/p\u003e \u003cp\u003eMushroom samples collected during the rainy season of June 2024, were morphological and molecular identification using DNA extraction, PCR amplification, and genome sequencing. Extracts were prepared using hot water and methanol, and bioactive compounds were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). Antimicrobial activity was assessed using the agar well diffusion method against bacterial and fungal strains, and statistical analyses were performed using SPSS.\u003c/p\u003e \u003cp\u003eBLAST analysis of showed strong alignments with multiple \u003cem\u003eGanoderma\u003c/em\u003esp.with \u003cem\u003eGanoderma lucidum\u003c/em\u003e showing the highest similarity with 99.55%. Phylogenetic analysis confirmed its close evolutionary relationship with \u003cem\u003eG. lucidum\u003c/em\u003e and the ITS sequence was identified as AS_ITS1_C06_09. GC-MS profiling identified bioactive compounds with antimicrobial and therapeutic potential. Antimicrobial assays demonstrated significant inhibitory effects, particularly from methanol extracts. These findings support the pharmaceutical relevance of G. \u003cem\u003elucidum\u003c/em\u003e and its potential applications in antimicrobial formulations.\u003c/p\u003e \u003cp\u003eThis study highlights its potential as a source of bioactive compounds with antimicrobial properties, for pharmaceutical and nutraceutical applications.\u003c/p\u003e","manuscriptTitle":"Phytochemical Analysis and Antimicrobial Activity of Ganoderma lucidum Against Selected Bacteria and Fungi of Medical Importance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-25 06:33:48","doi":"10.21203/rs.3.rs-6261550/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7652b5de-b578-4388-93f3-7b90ae43a497","owner":[],"postedDate":"March 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-10T09:38:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-25 06:33:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6261550","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6261550","identity":"rs-6261550","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}