Acta agriculturae Slovenica, 121/3, 1–8, Ljubljana 2025 doi:10.14720/aas.2025.121.3.22058 Original research article / izvirni znanstveni članek Cultivation of some medicinal mushrooms species from Ganoderma on sawdust supplemented with water hyacinth Lauretta OFODILE 1, 2, Viola NICHOLAS-OKPARA 3, 4, Utom-Obong AKPAN 5, Leonard ADAMU-GOV- ERNOR 1, Uchenna KANIFE 1, Emmanuel IKEGWU 6, Emmanuel ANI 1, Adekunle AYODEJI 6, Ndubuisi NWAKANMA 7, Godwin OVIOMA 1, Doyinsola AKINJAYEJU 1, Anjorin SAANU 8 Received March 12, 2025; accepted August 05, 2025 Delo je prispelo 12. marec 2025, sprejeto 5. avgust 2025 1 Department of Biological Science, Yaba College of Technology, Yaba Lagos, P.M.B 2011, Yaba, Lagos, Nigeria 2 corresponding author: nwannemka.ofodile@yabatech.edu.ng 3 Nutrition and Toxicology Division, Federal Institute of Industrial Research Oshodi, P.M.B. 21023 Ikeja, Lagos, Nigeria 4 Center for Excellence in Post-Harvest Technologies, North Carolina A&T State University, 500 Laureate Way, Kannapolis, NC 28081, United States of America 5 Anatomy Programme, Bowen University Iwo, Osun State, Nigeria 6 Department of Statistics, Yaba College of Technology, Yaba, Lagos, P.M.B 2011, Yaba, Lagos, Nigeria 7 Department of Science Laboratory Technology, Yaba College of Technology, Yaba, Lagos, P.M.B 2011, Yaba, Lagos, Nigeria 8 College Central Research Laboratory, Yaba College of Technology, Yaba, Lagos, P.M.B 2011, Yaba, Lagos, Nigeria Cultivation of some medicinal mushrooms species from Ganoderma on sawdust supplemented with water hyacinth Abstract: This study investigated the cultivation of Gano- derma mbrekobenum E.C. Otto, Blanchette, Held, C.W. Barnes & Obodai, G. sessile Murrill, and G. oregonense Murrill us- ing sawdust supplemented with water hyacinth (Eichhornia crassipes Mart.). Ganoderma species have medicinal applica- tions, including immune modulation, antioxidants, and antimi- crobial properties. Substrates with water hyacinth (SWH) con- sisted of 900 g sawdust, 100 g water hyacinth, 100 g rice bran, and 30 g calcium carbonate, adjusted to 65 % moisture while (SNWH) was not supplemented with water hyacinth. The num- ber of primordia was the highest in G. mbrekobenum (8.2), fol- lowed by G. sessile (4.0) and G. oregonense (2.8), but G. sessile and G. oregonense fruited faster (21 days) than G. mbrekobenum (30 days). Fruiting body yield analysis showed that G. mbreko- benum had the highest mass (61.10 g) and biological efficiency (15.42  %). G. sessile had the fastest fruiting time (47.4 days), while G. mbrekobenum took 86.8 days. The biological efficiency of G. oregonense was the lowest at 9.99 % (supplemented) and 5.86  % (unsupplemented). Water hyacinth supplementation improved mushroom yield and G. mbrekobenum demonstrat- ed superior yield, whereas G. sessile had a faster fruiting cycle, making both species suitable for large-scale medicinal mush- room production. Key words: Ganoderma, cultivation, mushrooms, supple- mentation, efficiency, yield, water hyacinth Gojenje nekaterih vrst zdravilnih gob iz rodu Ganoderma na žagovini, dopolnjeni z vodno hijacinto Izvleček: Ta študija je preučevala gojenje gliv Ganoder- ma mbrekobenum E.C. Otto, Blanchette, Held, C.W. Barnes & Obodai, G. sesile Murrill in G. oregonense Murrill na žagovini, dopolnjeni z vodno hijacinto (Eichhornia crassipes Mart.). Vrste iz rodu Ganoderma imajo medicinske lastnosti, kot so imunska modulacija, antioksidativno in protimikrobno de- lovanje. Substrati (SWH) so vsebovali 900 g žagovine, 100 g vodne hijacinte, 100 g riževih otrobov in 30 g kalcijevega kar- bonata s 65 % vlage, medtem, ko (SNWH) ni imel dodatka hijacinte. Vrsti G. mbrekobenum in G. sessile sta kolonizirali petrijevke v 7 dneh, vrsta G. oregonense pa v 8 dneh. Število primordijev je bilo največje pri vrsti G. mbrekobenum (8,2), sledili sta mu G. sessile (4,0) in G. oregonense (2,8). Vrsti G. sessile in G. oregonense sta razvili trosnjake v 21 dneh, vrsta G. mbrekobenum pa v 30 dneh. Vrsta G. mbrekobenum je imela največji pridelek (61,10 g) in največjo biološko učinkovitost (15,42  %), vendar je za razvoj trosnjakov potrebovala več časa (86,8 dni). Vrsta G. sesile je razvila trosnjake najprej (47,4 dni). Biološka učinkovitost vrste G. oregonense je bila najmanjša (9,99  % in 5,86  %). Dodatek vodne hijacinte je izboljšal pridelek, vrsta G. mbrekobenum pa se je izkazala kot najdonosnejša, medtem ko je bila vrsta G. sessile primernejša za hitrejšo pridelavo. Ključne besede: Ganoderma, gojenje, gobe, dodatki sub- stratu, učinkovitost, donos, vodna hijacinta Acta agriculturae Slovenica, 121/3 – 20252 L. OFODILE et al. 1 INTRODUCTION Mushrooms, particularly those from the Ganoder- ma genus, have garnered significant attention due to their medicinal and therapeutic properties (Ekiz et al., 2023). Ganoderma species are widely recognized for their dis- tinctive morphological features, including a hard, woody fruiting body, a white margin, and reddish-brown, dou- ble-walled basidiospores with interwall pillars and a thin hyaline exosporium (Konara et al., 2017). Commonly re- ferred to as “lingzhi” in China and “reishi” in Japan, these mushrooms have been utilized in traditional medicine for centuries (Jin et al., 2012). The bioactive compounds found in Ganoderma species contribute to a variety of pharmacological benefits, including immune modula- tion, antioxidant activity, and antimicrobial properties (Hapuarachchi et al., 2018). The inclusion of Ganoderma in dietary and pharmaceutical products has led to its widespread application in traditional and modern medi- cine. However, despite their medicinal significance, some Ganoderma species also act as pathogens, affecting im- portant crops such as cacao, rubber, tea, and coffee, with G. zonatum Murrill being particularly problematic in the Asian oil palm industry (Yang et al., 2018; Castillo et al., 2022). The medicinal properties of Ganoderma species are attributed to a wide range of bioactive compounds, including polysaccharides, triterpenoids, proteins, and essential vitamins and minerals. These compounds have been shown to exhibit antitumor, anti-inflammatory, im- munomodulatory, and neuroprotective effects (Klupp et al., 2015; Adebesin et al., 2019). Additionally, essential minerals such as calcium, potassium, magnesium, iron, and zinc are found in Ganoderma mbrekobenum, along with trace elements like cobalt, nickel, and manganese (Rana et al., 2021). Over time, environmental factors have significantly impacted the genomic structure of Ganoderma, leading to the emergence of new species. These newly develo- ped species are known to contain unique and beneficial bioactive compounds (Hapuarachchi, 2018). A notable example is Ganoderma mbrekobenum, which has been identified for its valuable bioactive properties. This spe- cies was first identified from Nigeria in 2014 (Ofodile et al., 2022). G. mbrekobenum is characterized by its distinct morphological features, though detailed descriptions are limited. Ganoderma mbrekobenum closely resembles Ga- noderma lucidum (Curtis) Karst in appearance. Howe- ver, molecular analyses, particularly of the ITS and LSU gene regions, have distinguished G. mbrekobenum as a separate species (Otto et al., 2016). Ofodile et al. (2025) reported that G. mbrekobenum could prevent and con- trol uterine tumour in Wistar rats. Isolates have been described as having brownish, red coloured, size-20 cm- 25 cm of fruiting body, red brown stem colour and spo- re print. Strong bioactive substances such as ganoderic acid A, ganoderic acid C1,ganoderic acid S, ganoderiol F, ganodermanondiol have been characterised from G. mbrekobenum (Parihar et al., 2021). Olmesartan medo- xomil, benazeprilat, isopropamide, ramipri glucuronide, desmethyldoxepine, oleamide, phorbol myristate acetate and cepharanthine were first reported from G. mbrekobe- num by Parihar et al. (2021). Desmethyldoxepine is used in the treatment of mild depression, ganoderic acid S has immune resporative effect and anticancer while olmesar- tan medoxomil is used in medicine for high blood pres- sure, heart failuere, diabetics and kidney related diseases (Parihar et al., 2021). G. sessile mature fruiting bodies are laccate (shiny) with a reddish-brown hue and often exhibit a wrinkled margin when dry. G. oregonense, G. sessile have been described as from all over the world, are mainly charac- terized by laccate pileus. G. oregonense produces large, perennial, woody brackets, commonly known as conks. These fruiting bodies are leathery, with a fan-like or hoof-like appearance, and grow on the trunks of living or dead trees (Ginns, 2017). For centuries, laccate Gano- derma species have been integral to traditional medicine in various Asian cultures, utilized to prevent and treat a multitude of ailments (Galappaththi at al., 2022). Gano- deric acid, endopolysaccharides and polysaccharides from the mycelia of G. oregonense (Boromenskyi et al., 2021; Boromenskyi and Bisko, 2020; Fátima et al., 2021) and polysaccharides from the mycelia G. sessile (Fátima et al., 2021) produced in submerged and solid state culti- vation have been reported. Cultivating Ganoderma mushrooms have become an essential aspect of medicinal mushroom production, as demand continues to grow for their bioactive com- pounds (Wachtel-Galor et al., 2011; Salichanh et al., 2025). To support large-scale production, researchers and commercial growers have explored diverse lignocellulo- sic substrates for optimal cultivation. Various substrates have been explored for optimal growth conditions, in- cluding sawdust, rice bran, wheat straw, sugarcane ba- gasse and corn husks, as they provide essential nutrients for fungal development (Rana et al., 2021; Royse et al., 2017). They provide nutrients such as cellulose, hemi- cellulose, and lignin and influence yield, bioactive com- pound synthesis, and cultivation cost-efficiency (Oke et al., 2022). Proper management of the environmental fac- tors, pH, temperature and humidity are crucial for the best productivity and quality of mushrooms. Most mush- rooms species have their best growth at pH of 5.5-7; ex- treme values usually restrict the development of myce- lium (Atila, 2022; Nguyen et al., 2023) and Luangharn Acta agriculturae Slovenica, 121/3 – 2025 3 Cultivation of some medicinal mushrooms species from Ganoderma on sawdust supplemented with water hyacinth et al. (2020) reported that temperatures 24–28 °C were ideal for fruiting body growth of Ganoderma species. Improvements in cultivation techniques, including tweak in the composition of the substrate and substitution of the substrate with various other agrowastes, are important to increase yield and sustainability (Rashad et al., 2019, Luangharn et al., 2020). Supplementing sawdust with additional organic materials, such as water hyacinth (Eichhornia crassipes), may enhance substrate composition by increasing nitro- gen content and promoting mycelial colonization (Gai- tán-Hernández et al., 2011). Water hyacinth which is an invasive aquatic plant, has been successfully integrated into mushroom cultivation due to its rapid growth and rich organic matter content (Nguyen et al., 2018). Accor- ding to Su et al. (2018) water hyacinth is rich in cellulose, protein, and fat. The dry matter is rich in nutrients, with crude protein of 10–20 % protein, the crude fiber con- tent was 13.74 % and 11.04 %, minerals up to phosphorus 0.32  %, calcium 3.08  % and potassium 4.53  %. By uti- lizing water hyacinth as a supplement, the dual benefits of enhancing mushroom yield and managing an invasive species can be achieved. Given the increasing interest in sustainable mushroom cultivation, exploring the use of sawdust su- pplemented with water hyacinth presents a viable appro- ach for enhancing Ganoderma growth and productivity. This research aims at investigating the effectiveness of this substrate combination in improving the yield of the Ganoderma species and to compare the performance of the three species to utilize the substrate. 2 MATERIALS AND METHODS 2.1 SAMPLE COLLECTION Fresh samples of Ganoderma sessile (Accession Number PQ578284), G. oregonense (Accession Number PQ578286), and G. mbrekobenum (Accession Number PQ578285) collected from the Mushroom Research and Training Laboratory, Yaba College Technology. Sawdust from obeche (Triplochiton scleroxylon K. Schum.) was ob- tained from a sawmill in the Shomolu area of Lagos, sie- ved, and stored for future use. Water hyacinth (Eichhor- nia crassipes Mart.) was gathered from the Lagos Lagoon in Ikorodu and Oworonshoki, Lagos, then dried using an industrial dryer at 50 °C before being shredded into particle sizes ranging from 40 to 100 mm. Additionally, Sorghum bicolor L. grains, rice bran, and calcium carbo- nate (CaCO₃) were sourced from a rice mill in Abeokuta and a chemical market in Ojota, Lagos, respectively. 2.1.1 Preparation mycelia culture The mycelial cultures of Ganoderma sessile, G. ore- gonense, and G. mbrekobenum were prepared following the method described by Ofodile et al. (2022). To obtain pure cultures, the internal tissue from each fruiting body was transferred onto sterile potato dextrose agar (PDA 1.5 %) and incubated at 25 °C for 72 hours. The resulting cultures were preserved on PDA at 4 °C for subsequent studies. Agar blocks from seven-day-old cultures of each isolate were inoculated onto PDA (2 %) and incubated at 25 °C for spawn production. 2.1.2 Spawn production A mixture of sorghum grains and calcium carbonate (33:1) with 70 % moisture content was prepared, divided into 500 g portions, and packed into jam bottles. The bot- tles were sterilized at 121 °C for 1 hour at 15 psi. Once cooled, each unit was inoculated with five agar blocks (1 cm in diameter) taken from a 12-day-old plate culture of each selected isolate. After a 14-day incubation period, the bottles were fully colonized and subsequently used to inoculate test substrates for cultivation studies. 2.1.3 Substrates preparation On a dry mass basis, a substrate mixture consisting of 900 g sawdust and 100 g water hyacinth (SD+WH), along with 100 g rice bran and 30 g calcium carbon- ate (CaCO₃), was prepared, with the moisture content adjusted to 65  %. The substrate (1130  g dry mass) was packed into polyethylene bags (30 × 43.2 cm in diam- eter) and pasteurized in a locally fabricated pasteurizer at 65 OC-70 °C for six hours, then left to cool overnight. The bags were partially opened under aseptic conditions and the spawns were evenly distributed on top of the sub- strates, the necks twisted and the sterile PVC pipes were inserted, covered with clean tissue papers and secured with rubber bands. Each bag was inoculated with 50 g of grain spawn per substrate bag. The inoculated substrates were incubated at room temperature, ranging from 20- 25 °C at night and 25-30 °C during the daytime. Upon completion of the incubation period, the fully colonized substrate bags were opened and transferred to fruit- ing rooms maintained at 30±2 °C with 90-95 % relative humidity, where they were monitored for fruiting. The fruiting bodies were harvested at maturity when the col- our of the cap was uniform without the whitish tips. Data collected during the fruiting experiment were the spawn run period, the number of days before primordia for- mation, and the number of primordia per bag. Also re- corded were the number of days from commencement of Acta agriculturae Slovenica, 121/3 – 20254 L. OFODILE et al. fruiting body induction to first flush, mass and number of basidiocarp per flush. The biological efficiency (BE), which was calculated as the mass of fresh mushroom harvested/dry mass of substrate × 100 %.The experiment followed a randomized design and was conducted over two growing cycles, with five replications per cycle. 2.2 STATISTICS ANALYSIS The mushroom cultivation experiment followed a randomized complete block design. Data from all expe- riments are presented as the mean ± SD, and analysis of variance (ANOVA) with Tukey’s post hoc test was con- ducted at a 5 % significance level. 3 RESULTS AND DISCUSSION 3.1 PRIMORDIAL DEVELOPMENT The results of primordial emergence for the three Ganoderma species are presented in Figure 2. Primordia formation and fruiting body yield were assessed in G. mbrekobenum, G. sessile, and G. oregonense cultivated on a sawdust alone and sawdust supplemented with water hyacinth substrates. For substrate with water hyacinth, the number of primordia produced by G. mbrekobenum (8.2) was significantly higher than that of G. sessile (4.0) and G. oregonense (2.8) (p < 0.05). However, the primor- dia of G. sessile (21 days) and G. oregonense (21 days) emerged significantly earlier (p < 0.05) compared to G. mbrekobenum (30 days). The number of primordia pro- duced by G. mbrekobenum was also significantly higher than that of the other species for the substrate without water hyacinth but the primordia of G. mbrekobenum (21.2 days). and G. sessile (30 days) emerged significantly (p < 0.05) faster than that of G. sessile (68.4 days) Similar patterns have been reported in other Gano- derma cultivation studies. Under artificial cultural condi- tions, G. lucidum forms primordia after inoculation for 43–48 days (Ngo et al., 2019). Ganoderma sinense J.D. Zhao, L.W. Hsu & X.Q. Zhang, demonstrated that pri- mordia formation occurred between 38 to 41 days after inoculation, with variations attributed to different sub- strate compositions. The study found that substrates en- riched with wheat bran accelerated primordia initiation and enhanced biological efficiency (Nguyen et al., 2023). This suggests that substrate composition plays a crucial role in the timing of primordia emergence. 3.2 FRUITING BODY MASS AND BIOLOGICAL EFFICIENCY The primordia were monitored until full fruit body development which is the matured basidiocarp, with the resulting fruiting bodies displayed in Figure 3. Fruiting body yield and biological efficiency are documented in Table 2. The mass of fruiting bodies (g) produced by G. mbrekobenum (61.15 ± 1.01 g) was significantly higher (p = 0.05) than that of G. sessile and G. oregonense on the substrate with water hyacinth (SWH). Additionally, G. sessile produced significantly heavier fruiting bodies than G. oregonense (p < 0.05). Similar results were produced on substrates without water hyacinth (SNWH), although the mass of fruiting bodies was significantly higher on SWH than without water hyacinth SNWH, for G. mbre- kobenum and G. oregonense. The biological efficiency (BE) of G. mbrekobenum, G. sessile, and G. oregonense in utilizing the substrate SWH were 15.42 %, 9.99 %, and 10.44 %, respectively while in SNWH, BE were 12.62 %, 10.20 % and 5.86 %. During cultivation, G. sessile deve- loped four fruiting bodies, while G. mbrekobenum and G. oregonense each produced approximately two fruiting bodies on the substrate supplemented with water hya- cinth. On the other hand, these species had longer time to fruitbodies production than on substrate supplemen- ted with water hyacinth but were only significant for G. oregonense and G. sessile. In this study, G. sessile produced fruiting bodies approximately 47.4 days post-inoculation, whereas G. mbrekobenum required about 86.8 days, indicating a sig- nificantly slower development. G. oregonense also exhib- ited a slower fruiting process compared to the other two species. Similar disparities in fruiting times have been documented in other Ganoderma species. Ganoderma resinaceum Boud. was reported to produce fruiting bod- Figure 2: Time to primordia development and number of pri- mordia during cultivation of Ganoderma species Key: SWH = substrate with water hyacinth; SNWH = substrate without wa- ter hyacinth, Days to primordial = days taken before primordia was formed, show significant difference at 5 % (p < 0.005) be- tween species. Acta agriculturae Slovenica, 121/3 – 2025 5 Cultivation of some medicinal mushrooms species from Ganoderma on sawdust supplemented with water hyacinth ies approximately two months of post-inoculation, while another unidentified Ganoderma species required three months under similar conditions (El-Fallal et al., 2015). These differences highlight the influence of species-spe- cific genetic factors and environmental conditions on the developmental timelines of Ganoderma fruiting bodies (Makarenkova et al., 2021; De Oliveira Campos et al., 2024). Additionally, research on MYB transcription fac- tors in Ganoderma species indicates that these genes play crucial roles in various developmental and physiological activities, suggesting that genetic differences among spe- cies can impact their development (Wang et al., 2020). A study investigated the effects of various supple- ments, including rice bran and oilseed cakes, on the cul- tivation of Calocybe indica Purkay. & A. Chandra. The findings revealed that supplementing rice straw with these additives increased both the yield and quality of the mushrooms (Alam et al., 2010). Ganoderma species Substrates Average weight of fruiting body (g) Number of fruiting body Days to fruiting bodies Biological Efficiency (%) G. mbrekobenum SWH 61.10C    1.08 1.80D ± 0.84 86.80Z ± 1.10 15.42 SNWH 50.30BC ± 0.90 1.20D ± 0.45 81.20YZ ± 4.15 12.62 G. oregonense SWH 39.58B ± 12.40 1.20D ± 0.45 50.80G ± 2.95 9.99 SNWH 23.20A ± 2.05 4.20E ± 0.45 76.60XY ± 6.43 5.86 G. sessile SWH 41.34B ± 12.28 4.00E ± 0.71 47.40G ± 6.47 10.44 SNWH 40.40B ± 0.55 2.20D ± 0.84 68.60X ± 0.55 10.20 Table 3: Fruiting body production of Ganoderma species on sawdust with supplements Key: SWH = substrate with water hyacinth; SNWH = substrate without water hyacinth, show significant difference at 5 % (p < 0.005) between spe- cies. Figure 3: Top Left -Right A, B, C Pin heads of G. oregonense, G. mbrekobenum and G. sessile; pin head formation on substrate sup- plemented with water hyacinth. Lower Left –Right A1, B1, C1 G. oregonense, G. mbrekobenum and G. sessile carpophore formation on the substrate. Acta agriculturae Slovenica, 121/3 – 20256 L. OFODILE et al. Gurung et al. (2012) reported the effects of using supplements of rice bran, wheat bran, corn flour and gram flour on the yield of G. lucidum. The results showed that supplementation played a positive role in the my- celia growth and yield of G. lucidum, demonstrating the significance of substrate composition in mushroom cul- tivation. These findings are in line with previous research indicating that BE and fruiting body mass can vary sig- nificantly among Ganoderma species and are influenced by substrate composition and environmental factors. A previous study reported that the yield of G. lucidum was increased when grown on a cultivation substrate sup- plemented with wheat bran (Mehta et al., 2014). Also, the composition of the substrate has a significant effect on mycelium growth and biological efficiency of Reishi mushroom (Lisiecka et al., 2015). During cultivation, G. sessile developed an aver- age of four fruiting bodies per substrate bag, whereas G. mbrekobenum and G. oregonense each produced approxi- mately two. This variation in fruiting body count may be attributed to inherent species differences and their interaction with the cultivation substrate. Optimizing factors such as substrate composition, moisture content, and environmental conditions can influence the number of fruiting bodies produced, as demonstrated in various studies on Ganoderma cultivation (Nguyen et al., 2023). Evaluating these three species of Ganoderma, G. sessile is the most economically viable species due to its fast growth rate marked by the shortest fruiting time (~47.4 days), decent yield and produced more fruiting bodies (~4 per bag). Also the potential for multiple cultiva- tion cycles per year. G. mbrekobenum offers the highest single-batch yield and BE but has limited annual output due to its long fruiting time, increasing operational costs per unit mass. G. oregonense was the least efficient spe- cies economically, due to low yield and poor biological efficiency. Soriano et al. (2022) reported that supplemen- tation was important in G. lucidum cultivation since it increased the biological efficiency and its subsequent economic value than that of un-supplemented substrate. It has been previously shown that G. sessile is equipped with a superior machinery of degrading enzymes allow- ing colonization of lignocellulosic biomass in solid state (Loyd et al. 2019) 4 CONCLUSIONS This study demonstrates that substrate composition and species specificity significantly influence Ganoderma cultivation. Water hyacinth supplementation enhanced yield, offering sustainable use for this invasive plant. G. mbrekobenum exhibited superior biological efficiency, while G. sessile fruited fastest. These findings provide in- sights into optimizing medicinal mushroom production and environmental management. Acknowledgement: This work was funded by the TETFUND National Research Fund (NRF 2020) Grant Number TETF/ES/DR&D-CE/NRF2020/SETI/35/ VOL.1. Declaration Authors declare no Conflict of Interest 5 REFERENCES Adebesin, O. A., Ofodile, L. N. & Dibiana, N. N. (2019). 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