Characterisation of ganoderma species using morphological, molecular and biochemical markers and evaluation of substrate enhancement influence on their development and biochemical profile.

Abstract

Ganoderma, also known as Reishi mushroom, is used for its potential health benefits in several countries. The current study characterised Ganoderma species using molecular and biochemical markers and evaluated the substrate enhancement influence on its development and biochemical profile. The overall research study consisted of four objectives. The first objective focused on the isolation and characterisation of fifteen fungal specimens collected from the three provinces of South Africa, namely, Mpumalanga, KwaZulu-Natal, and North- West. Five fungal specimens were collected in each province and growth media potato dextrose agar (PDA), malt extract agar (MEA), and sabouraud dextrose agar (SDA) were used to grow sample isolates. After 8 days of incubation, MEA recorded the highest mycelial diameter followed by PDA and SDA. Samples were identified using comparative morphology traits and supported by internal transcribed spacer region (ITS) and phylogenetic analyses. Based on the ITS of ribosomal DNA, fungal samples KG3SY219 and MG1SY119 were found to be closely related to Ganoderma resinaceum and Ganoderma austroafricanum, respectively. The species were further characterised by biochemical compounds, including antioxidants, proteins, essential elements, and heavy metals. The antioxidant capacity exhibited a higher radical scavenging activity in G. austroafricanum compared to G. resinaceum. The concentrations of total phenolics, flavonoids, proteins, essential elements and heavy metals were more abundant in G. austroafricanum compared to G. resinaceum. This study also evaluated the effect of different growth conditions on mycelial growth and development of Ganoderma austroafricanum and Ganoderma resinaceum. The experimental treatments included three levels of pH (4, 6 & 8), temperature (20, 25 & 30 °C), and different types of plant residues namely; beech sawdust (BS), sugarcane bagasse (SB), and buffalo grass (BG). Three independent in vitro experiments were conducted, PDA and MEA were used as standard growth media to grow each fungal species. Mycelial growth and development were measured over 9 days where they reached maximum growth. The culture media pH results demonstrated that the maximum growth for mycelia was reached on day 9 for both species. Typically, G. resinaceum showed the highest mycelial growth for both cultures except for days 6 and 9 where the mycelial growth of the species was decreased by low levels (pH 4) and high levels (pH 8), respectively. The addition of BG to the growth media delayed the mycelial growth of G. resinaceum for both growth media (PD+MEA).

Experiment three investigated the effect of different substrates on the development, total biomass, and biochemical profile of Ganoderma species. The experiment involved growing G. austroafricanum (GA) and G. resinaceum (GR) on different substrates; beech sawdust (BS), sugarcane bagasse (SB), and buffalo grass (BG), and suspension of all substrates (BSSBBG). The growth parameters such as pileus size, weight, total biomass, and biological efficiency were measured. Additionally, each substrate was analysed for biochemical composition. The biochemical composition of the harvested samples was also analysed to determine the levels of biochemical compounds such as minerals, antioxidants, and protein. The substrate pH levels demonstrated that all substrates were within the optimal growth pH range (5-6). SB exhibited greater levels in the majority of essential elements such as Zn and K, also, heavy metals Pb and Hg. The results on the development and total biomass production of Ganoderma species revealed significant variations across different substrates. In terms of development, GRBS was faster to reach the 100% rate of all production parameters in 40-52 days after inoculation. However, GASB exhibited higher quantities in total yield and biological efficiency. In addition, pileus from GASB demonstrated higher concentrations of all evaluated biochemical compounds. GASB also yielded higher levels of DPPH, phenolic compounds, flavonoids, and protein. Experiment four examined the impact of substrate fortified with essential elements on the development, total biomass, and biochemical compounds of Ganoderma species. The experiment involved growing G. austroafricanum (GA) and G. resinaceum (GR) on beech enhanced with elements; no element (Control), Zn(NO3)2.6H2O (Zn), Fe2SO4 7H2O (Fe), Na2SeO3 (Se), and suspension of all essential elements (ZnFeSe). The growth parameters such as the pileus size, weight, total biomass, and biological efficiency were measured. In addition, the biochemical profile of Ganoderma spp. was analysed to evaluate the concentration of compounds. The development and total biomass production findings for the substrate fortified with essential elements exhibited significant differences. GRZn developed expeditious, reaching 100% of all production parameters in 52 days after inoculation. In comparison to all treatments, GAFe showed larger quantities in total yield and biological efficiency. The substrate enhancement with Zn had a significant increase in the majority of minerals. GAZn exhibited higher concentrations of essential elements such as Zn, K, and Mg. Higher levels of heavy metals such as Cd, Pb, and As were recorded from GAControl. GASe produced higher

levels of DPPH, phenolic compounds, flavonoids, and protein reading. These findings demonstrate the variability of morphological characteristics, biochemical compounds, and growth conditions requirements between Ganoderma species. These findings provide valuable insights into the diversity, taxonomy, and potential therapeutic applications of Ganoderma species in South Africa. Further investigation is required to identify Ganoderma species and its pharmaceutical properties.