Mycelium running of Volvariella volvacea on palm oil empty fruit bunch fibre following different substrate and mycelium treatment

Leading the palm oil industry, Malaysia strives to become the powerhouse of palm oil producers. However, it is crucial that the industry does not influence the environment negatively in any way. Thus, reusing and repurposing palm oil residues in mushroom farming is one of the potential solutions for agricultural waste management. In this study, the objective of this study is to determine the mycelial performance of V. volvacea in palm oil empty fruit bunch (EFB) fibre as substrate and to evaluate the mycelial growth performance of V. volvacea in two different substrate treatment. The substrate treatment involved the non-treated EFB fibres (N substrate) and EFB fibres treated with 10% Calcium Carbonate (CaCO3) (T substrate), and mycelium treatment involved in gamma-irradiated mycelium (G sample) and non-irradiated mycelium (NG sample). Results shows that mycelium inoculated on treated EFB fibres exhibited a higher growth rate compared to non-treated EFB fibres. The full colonization days of mycelial run-on substrate using treated substrate was found reduced from 15 days to 9 days, as compared to the non-treated substrate. However, gamma irradiated mycelium was found giving no significant changes to the rate of mycelial run. In conclusion, substrate treated with CaCO3, increased the growth of mycelium. In future, it is suggested to measure the effect of irradiation using different gamma irradiation exposure.


Introduction
Palm oil empty fruit bunch (POEFB) is one of the processed residues of palm oil fruits. The lignocellulosic characteristic of POEFB allows it to be used as soil fertilizers or used as substrate for mushroom cultivation [1-2]. In mushroom cultivation, it can be used either as a whole stalk or reduced and compressed into pellets [3][4]. In general, using POEFB as substrate in mushroom cultivation is a good approach toward sustainable agriculture as mushrooms are widely consumed as fresh or dried food [5]. This will ultimately add value to POEFB and help to reduce wastes from palm oil mill. Volvariella volvacea or commonly known as paddy straw mushroom is an edible fungus that is known for its nutritional and medicinal properties [6][7]. However, despite its benefits, it has lower yield compared to other edible mushrooms. The utilization of agricultural waste as substrate for mushroom cultivation is IOP Publishing doi:10.1088/1755-1315/1151/1/012054 2 reported to have a significant impact on the yield and biological efficiency of mushrooms [8]. In previous research, substrate composition, addition of supplementation and irradiation of the spawn are among the methods used to improve V. volvacea yield [9][10][11][12].
In addition, current research to induce a new type of mycelium strain by either ultraviolet (UV) radiation, gamma irradiation or chemical mutagenesis is also being studied. As reported by Liu et al.
[13], mutagenesis was used to breed a cold-tolerant of V. volvacea strain to reduce the susceptibility of V. volvacea to low temperature [13]. Mutagenesis can also be used to generate fast-growing V. volvacea strain to improve yield and biological efficiency [14][15]. In mushroom cultivation, spawn is considered the 'seed' of the mushroom and is used to grow the mycelium of the mushroom [9]. By observing the growth of mycelium during spawning stage, the effect that certain parameters have on mushroom yield improvement can be ascertained. For instance, a common method used is by measuring the degree of mycelium running in which the mycelium is grown in substrate-filled test tubes [10]. Therefore, in this study, growth of V. volvacea mycelium following the effect of CaCO2 addition to the substrate preparation was conducted. Effect of gamma-irradiated mycelium was also performed to investigate the influence of gamma irradiation on the growth rate of mycelium.

Preparation of V. volvacea
Fungal culture of V. volvacea was obtained from the Laboratory of Agriculture and Biotechnology, Malaysian Nuclear Agency, Bangi, Malaysia. The mycelium was maintained on potato dextrose agar plates and sub-cultured regularly. The radial growth development of V. volvacea mycelium on PDA plate was monitored and recorded. V. volvacea mycelium was identified through its white or creamy white filamentous structures called hyphae [9]. Figure 1

Preparation of growth substrate
The empty fruit bunch fibre pellet was obtained from Usaha Strategik Sdn Bhd (USSB) company which supplies biomass products. The substrate was prepared by soaking the EFB pellet with water at a 1:1.5 substrate to water ratio. The substrate was then divided into two groups namely treated (T) and nontreated (N) EFB substrate.

Gamma irradiation of V. volvacea mycelium
Irradiated (G) mycelium was done by exposing V. volvacea mycelium grown on PDA plate at 100Gy. Irradiation of mycelium was done at an acute gamma irradiation facility in Malaysian Nuclear Agency, Bangi, Malaysia using Biobeam GM 8000 (GmbH, Germany). The control sample was brought along during the transport of mycelium from the main laboratory to the facility. This is done according to standard protocol to ensure that both the irradiated and non-radiated mycelium experience the same environmental and physical changes that might occur during transportation such as temperature, humidity, or external pressure. Two samples were irradiated at a time to serve as duplicates. An example of fully grown mycelium of normal and irradiated mycelium of V. volvacea on PDA plate can be seen from Figure 2(a) and Figure 2

Degree of mycelium running and morphological analysis
A small cube of about 6mm was cut from PDA-grown mycelium and transferred onto EFB substrates in test tubes under laminar flow. Each T and N substrates were inoculated with normal and irradiated mycelium of V. volvacea. Each parameter tested were carried out in duplicate. The data was taken every 3 days after the inoculation of mycelium into test tubes until the substrate was fully colonized. The degree of mycelium running was marked and measured on the straight lines drawn on the test tube [16]. The measurement was expressed as mm/d.
Growth rate = degree of mycelium running total days of mycelium running (1) The mycelial growth characteristics including the colour and density of the hyphae was also observed and recorded. The measurement for the mycelial growth characteristics were adapted from Ahlawat & Kaur [17].

Effect of irradiation and substrate treatment on degree of mycelium running
Results showed the V. volvacea mycelium relatively have a higher growth rate on treated EFB substrate compared to non-treated EFB substrate. Mycelium inoculated on treated substrate has mean ratio of above 3.0 for both irradiated and non-radiated mycelium. There were no data taken of samples inoculated on treated substrate after day 9 because the mycelium has fully colonized the substrate after 9 days. On the other hand, samples inoculated on non-treated EFB substrate took more than 9 days to fully colonize the whole substrate. Gamma-irradiated mycelium inoculated on non-treated EFB substrate have the slowest growth rate at 0.99.

Effect of irradiation and substrate composition on mycelial growth characteristics
As seen in Table 2, mycelium grown on treated EFB substrates appear to have more dense and compact hyphae. Two samples with the highest mycelial density were GN1 and GT2. Both samples contain different substrate compositions but were inoculated using the irradiated mycelium of V. volvacea. All mycelium samples appear visibly white or creamy white. An example of fully colonized treated and non-treated substrate can be seen in Figure 3(a) and Figure 3(b).

Discussion and Conclussion
The low yield of V. volvacea leads to various introductions of new strategies and techniques for yield improvement. Preliminary investigation using mycelium running method can facilitate researchers in studying the effects of certain variables on mushroom yield. EFB substrate treated with 10% CaCO3 yielded better results both in terms of growth rate and morphological characteristics of mycelium. This result is in correlation with several studies involving substrate supplementation in V. volvacea cultivation where substrate supplementation using organic substances and micronutrients leads to an increase in mushroom yield and biological efficiency [11,18]. The supplementation of substrate with CaCO3 can be deduced due to its role as pH stabilizer. Bhadana et al. [19] uses the combination of calcium carbonate and calcium phosphate in the spawn mushroom of oyster mushroom. The combination treatment of substrate exhibits more spawn growth than the non-treated substrate. Similarly, Khan et al. [20] emphasizes that 2% lime supplementation in substrate provided the best condition for oyster mushroom to grow. Both studies agreed that calcium carbonate plays an important role as pH stabilizer in substrate. Furthermore, Thuc et al. [21] list the optimal pH for V. volvacea cultivation during both mycelium formation and fruiting body induction is basic pH between 6 to 7.
In this study, we attempt to investigate the effect of gamma irradiation on mycelial growth of V. volvacea. Irradiation of mushroom spawn is an uncommon method in mushroom production. Although there had been little studies done on this topic, there are evidences of mushroom strain improvement induced by irradiation. A study done by Jyothi and Thara [22] for instance, found that gamma-irradiated mushroom spawn enhances the biological efficiency of Pleurotus species and reduces the number of days required for primordial initiation. Moreover, a study using UV-irradiated mycelium instead of gamma irradiation also resulted in a 30% improvement in biological efficiency of the mutant strain [14]. Harfi et al. [23] achieve similar results with white button mushrooms in which gamma-irradiated mushroom spawn generate variants that exhibit higher yield than the control strain. Thus, based on the result from this study, the growth rate and morphological characteristics of gamma-irradiated mycelium are similar to that of a normal mycelium. Apart from that, it also has denser hyphae compared to the non-irradiated mycelium. This has raised the possibility of using gamma-irradiated spawn for V. volvacea cultivation. In the future, a trial cultivation in an in-vivo setting using growth substrate can be done to affirm the production efficiency of gamma-irradiated V. volvacea spawn. In conclusion, treatment of EFB fibre with CaCO3 is better for V. volvacea than non-treated EFB fibres and gamma-irradiated mycelium can survive similar growth conditions as non-irradiated mycelium. However, further studies using higher number of replicates need to be carried out to improve the credibility of the results. Another experiment with a higher dosage of gamma irradiation can also be conducted in the future.