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Quality of mother spawn is a key factor for high yield of exotic mushrooms; but too much subculturing can lead to reduced yields
Exotic mushrooms spawn quality is one of the most important parts of mushroom production. Spawn is made by colonizing the target mushroom mycelia in select substrates. Common substrates used to make spawn include maize, wheat and millet grains, wood sawdust, with supplements such as rice bran, wheat bran and other nutritional supplementation. Mother spawn is what we have after removing the inoculated mycelial mat from a test tube or Petri-dish to form stock culture and colonizing on spawn substrates. Regular subculturing of mother spawn is a tactic used not only for reducing costs but also a commonly applied approach to preserve and rejuvenate various fungal species. However, excessive subculturing may also induce the biosynthesis of toxins, as well as reduce fruiting body yield. So mushroom spawn production has not been adopted by many farmers due to insufficient knowledge of scientific techniques needed for spawn production and mushroom cultivation. However, in this article I will try to explain the influence of mother spawn and media preparation, preservation, and times of subculturing for commercial mushroom cultivation.

Key words: Exotic mushrooms mother spawn, spawn preservation, spawn substrates, subculturing spawn, Agar media.
Mushroom cultivation is an environmentally friendly industry as it utilizes agrowaste streams. Nutritionally, mushrooms do not contain starchy carbohydrates and are rich in dietary ?ber, proteins, and offer a diverse array of amino acids, minerals, and vitamins. Selecting high vitality mother spawn is the most important factor of commercial mushroom production. This leads not only to high density mycelial growth but also to high yields from the substrate. Thus spawn quality is crucial to mushroom production and the production of spawn by spore print culture, where genetic segregation is the leading method in terms of quantity and quality of mushroom harvest. On the other hand, the mycelium culture method is very suitable from the viewpoint of quantity and quality for preparation and propagation of stock culture for future use. Although this method is not characterized as segregation, the mushrooms produced will be similar to the mother spawn. Originally, farmers used the soil from which mushrooms had grown naturally to produce spawn. They distributed this soil over the surface of substrates which they had prepared previously and, after mycelium covered the surface of the substrate completely, it was used to inoculate other substrates.
When growing exotic mushrooms, the initial quality of spawn directly affects the cultivation yield, but spawn culture degradation is also a common problem in many artificially-cultivated edible mushrooms as the degenerated strains develop peculiar characteristics such as less primordium, an extended production cycle and abnormal fruiting body shape after cultivation, which altogether result in huge economic losses to the growers. Regular subculturing is a commonly applied approach to preserve and rejuvenate various fungal species, but excessive subculturing may also induce the biosynthesis of toxins, thereby leading to strain degradation that hinders industrial applications. Therefore, in this article I will explain spawn culture media, substrate preparation, and subculturing, which can resolve many problems for commercial production when done properly.

Spawn culture media

Most exotic mushrooms are usually inoculated onto MYA (malt yeast agar); PDA (potato dextro agar); or, sometimes, a synthetic liquid medium dependent upon commercial choice, for submerged culture element such as glucose, KH2PO4, KCl, FeSO4-7H2O, trace elements,
Nowadays, grains are a very suitable delivery system for spawn, because they have a hardy shell with an aleurone layer rich in protein and starch. Although some research reports suggesting that mother culture spawn substrate prepared with different sawdust substrates such as Ficus carica, Albizia saman, Swietenia mahagoni, Leucaena leucocephala, Eucalyptus globulus and supplemented with 30% wheat bran and 1% lime as basal substrates are sufficient to cultivate Pleorutus ostreatus (oyster mushrooms). Another recent research article suggests that faster mycelial colonization was observed in spawn substrate containing rice hull, rice bran, and starch in P. ostreatus and P. citrinopileatus mushroom species compared with spawn substrate containing sawdust and rice bran; and the results suggest that an excellent spawn culture can be created using rice hull with rice bran and starch as the main substrate. This spawn culture showed high productivity, using agricultural waste and food waste to optimize the growing media for mushroom production and minimize incubation time.

MgSO4-7H2O; CaCl2?-H2O, thymine-1, vitamin ver. nitrogen source variable etc., in a 500~5000 ml spawn bio flask (Kitamoto et al. 1972), and adjust pH 5.5 ~ 6.0 before sterilization (Fig 3c). Mycelia or liquid spawn produced by submerged fermentation is an alternative method for generating spawn as it produces higher yields and more uniform mycelial biomass in a shorter period, favours mycelium dispersion and adaptation and enables inoculation to be carried out under a relatively more stringent aseptic condition. However, the sterilized agar medium of 10-20 ml (which is the easiest way) each was poured into sterilized Petri dishes and allowed to solidify and then transferred to a 5 ml disc of the mycelial mat on agar. The colony morphology of the succeeding strains was observed and photographed at 3-day intervals until full colonization on 90 mm plates incubated at 25°C. Observations on colony diameter should be taken when the maximum growth has been attained on the selected high density medium and higher mycelial colonization once transferred to the mother spawn substrates (Fig 2b).
Spawn culture preservation
Maintaining and preserving mushroom mother cultures are essential for producing high quality mushrooms spawn. There are several methods of cultivation and preservation required to ensure the viability and morphological, physiological, and genetic integrity of the spawn cultures over time. The primary methods of culture preservation are continuous growth, drying, and freezing. Most spawn cultures can be maintained for a short period by serial transfer and the method is simple, inexpensive, and widely used: inoculation is done by transferring a 5 ml disc of the mycelial mat of mushroom culture in test tubes (screw cap or plugged with cotton or foam) or Petri dishes (wrapped with Para-film to reduce drying) containing an agar medium of choice as previously described. After a culture is established, it is kept at a temperature of between 2°~4°C and must be checked periodically for contamination and desiccation when used further.
Another popular culture-preserving method involves freezing the culture at -20° to -80°C in mechanical freezers (Fig 2a) and spawn cultures grown on agar slants, or micro-tubes that can be placed directly in the freezer; although sterile cryotubes containing 10% glycerol in water are also applicable. The reported successful cryopreservation of Agaricus bisporus using liquid nitrogen (-196°C) also suggested that storage of the strain of A. brunnescens (Peck) without any significant change in mycelial growth is possible using liquid nitrogen. In addition, many reports on longer period storage methods of various exotic mushroom culture such as Lentinula edodes (shiitake), Pleurotus spp. (king oyster, see also: eryngii), Pholiota nameko (nameko), Grifola frondosa (maitake) etc. may be found in the literature.

Preparation of spawn substrates

Spawn substrates were produced using rice hull with rice bran and starch, for which the material percentage was calculated on a dry-wet basis. 74% Rice hull, 20% rice bran, 3% starch, and 1% calcium carbonate were mixed thoroughly, the pH adjusted to between 5.5-6.0 and the moisture maintained at 59_endash_61% (Barua BS et al 2024). Sawdust from various trees, grains, woods, and other agricultural by-products may also be used for preparing spawn media for wood rooting fungi such as shiitake and oyster mushrooms. There are several research results showing a variety of substrate materials to be suitable for shiitake spawn, including hammer-milled corncobs; cottonseed hulls; peanut hulls; sawdust with rice bran as additive; and sawdust mixed with rice bran, to which micronutrients can be added to improve quality of the substrate. Then, 400_endash_750g of substrates (depending on which spawn substrates used) were placed into 1000cc polypropylene bottles (Fig2 A, B). The bottles were sterilized at a peak temperature of 121°C for 30-40 minutes in the autoclave. After cooling at the clean bench, the bottles were inoculated with a piece of mycelia mat from the agar plate medium culture over the substrate’s surface and then incubated for 20_endash_30 days at 25°C in the incubation room. The spawn mycelia then colonized the inside and surfaces of the bottles before being stored at 5ºC ±1ºC before being used to inoculation substrate-filled mushroom cultivation bags.

Measurement of mycelial growth rate & selecting strains

Inoculation was conducted by transferring a 5 ml disc of the mycelial mat from the sterilized PDA or nutrient medium, and the periphery of the culture incubated for 10 days. The plates were then incubated at 25±1C. Observations on colony diameter were taken when the maximum growth was attained on the medium selected to transfer to the mother spawn substrates. The colony diameter was marked on the plate by the cross-marking method and the mycelial growth rate was calculated as mycelial growth rate mm/hr of 72hr (Fig 1 a-d). The mushroom mycelium on agar medium there will appear different forms of mycelium such as rhizomorph mycelium (like the roots of plants) and ‘fluffy’ mycelium (like cotton). Only the rhizomorph mycelium is suitable for further cultivation and fruiting. The primordia, which later become fruiting bodies, are built from this mycelia. Therefore, when producing mother spawn cultures from Petri dishes, it is better to select the rhizomorph mycelium rather than the fluffy and recover well and then only the rhizomorph-growing mycelium is used for further mother culture spawn.
Effect of spawn substrates:

The faster mycelial colonization was observed in spawn substrate containing rice hull, rice bran, and starch in oyster mushroom species compared to spawn substrate containing sawdust and rice bran. It is clear that by adding starch to rice hull, it is possible to create a spawn culture that is equivalent to or better than the conventional culture medium made from sawdust (Fig 2A, B). The best results were on an optimal substrate composed of agrowaste m with a high yield of 120-140g per bag in among substrates from P. ostreatus and P. citrinopileatus obtained. The lowest yield of 55_endash_75g was obtained from a substrate consisting only of wood sawdust supplemented with rice bran (Fig 3 A, B).

Effect of Liquid spawn:

Liquid spawn which could be more uniformly distributed in the substrate was then developed and the minimum incubation period needed for harvesting sufficient amounts of high-quality mushrooms is shortened. Another advantage of liquid spawn is that it allows the automation of inoculation for industrial scale production. Liquid cultures contain live mycelium that has already started to grow and
Figure 1. (a-d) Mycelia growth rate of P. ostreatus on different strains. The red arrow indicates rhizomorphic mycelia (d). Mycelial growth rate was measured by three replicates.
colonize the nutrient solution. This head start allows for faster colonization of your chosen substrate compared to starting from spores, significantly reducing the time required to see results.
Spawn quality and subculturing

Mushroom growers should ensure they source spawn directly from a reliable company laboratory where it is produced. Laboratories produce a wide variety of mushroom strains for commercial use under strict sterile and controlled conditions (Fig 4a).

This ensures only the purest strains are utilized to produce spawn for use by mushroom growers and this is important because it is unlikely that spawn generated in a laboratory will be contaminated by other undesirable fungal or bacterial species that could negatively impact the quality and yield of the mushroom crop (Fig 4n, o-t). Subculturing spawn for commercial mushroom production requires the laboratory to select mother spawn cultures of mushroom strains developed in various climatic and growing conditions (Fig 4d, e). Many farmers have been doing regular subculturing of mother spawn not only to reduce spawn costs but also as a common approach to preserve and rejuvenate various fungal species. However, excessive subculturing may also induce the biosynthesis of toxins, DNA methylation and chromosome remodeling as well as reduce mushroom yields.

These mother strains are cryogenically stored at -8o°C to preserve their potential viability until they are needed for spawn production. For spawn production, a selected mother culture will be removed from cryogenic storage, and, using hi-tech equipment under controlled conditions, it will be multiplied to suitable quantities in a Petri dish before being transferred into a special nutrient mix for further growth (Fig 4b). If the spore or tissue culture comes from wild mushrooms via applied breeding technology (and the culture to mother spawn process differs for each mushroom strain), it can take several months to a year for the spawn of some strains to become commercially ready spawn (Fig 4d,e).
Figure 2. Effects of rice hulls spawn of P. ostreatus, A: 12 days incubation using a substrate of sawdust rice bran and with rice hull rice bran starch. B: 21 days incubation using a substrate with rice hull rice bran starch; and sawdust rice bran. Barua BS et al., published at Journal of Recycling 2024, 9(4), 58.
Figure 3: Substrate yield differs by supplementation
Figure 4 (above): Diagram of stock culture to mother culture and disadvantage of subculturing of mother spawn too much. A: stock culture; B: mushroom culture on agar nutrients plate; C: liquid medium; D & E: mother spawn; F-H: subculturing spawn; I-L: mushroom incubation bags; M & N: mushroom fruiting bodies; O-T: Contaminated bags during incubation and flashing time.

Figure 4 indicates that 1st generation spawn can produce a good mushroom harvest and even 2nd generation subculturing will not decrease it much with the use of sawdust or liquid mother culture. However, when subculturing 3rd or 4th generations the spawn culture will start to produce abnormal or defective fruiting bodies as has been observed in research. In such research observations many of the inoculated substrate blocks are infected by mold during incubation and when the cultivation bags are opened in the fruiting room for the cultivation the substrate blocks are soon damaged by bacteria. Thus, we can speculate that spawn might lose its vitality due to a lack of nutrients in spawn substrates. Many spawn laboratories control their own nutrient mixes for mother spawn production, but when it is subcultured too many times, there will be a decrease in mushroom yields. (Figure 4). However, spawn type has a big influence on mycelial growth and properly prepared spawn will colonize a substrate with healthy and high density mycelium in a shorter period of time. In fact, if the substrate meets the mycelia requirements and all conditions are suitable, bad spawn will produce abnormal and faint mycelial growth, even though it may looks like faster colonization it will sometimes make cauliflower or spongy types of mycelial, which can be seen around spawn bottles mouths and finally get contamination mold or bacteria (Fig 5a,b).
Figure 5 (above). Bad sawdust produces cauliflower-like mycelia around the bottle mouths during incubation and when refrigerated for prolonged times, it becomes spongy (a,b); contaminated liquid spawn attacked by mold can be found in the 1st to 3rd weeks of incubation (d).

In conclusion, when growing exotic mushrooms, the initial quality of the mother spawn has a direct impact on the cultivated yield. However, strain/spawn deterioration is also a common problem in many artificially grown edible mushrooms. Deteriorated strains have specific characteristics such as fewer primary cells, longer production cycles, and abnormal fruiting bodies after cultivation, all of which cause enormous economic losses to the grower. Too much mycelial subculturing may result in inconsistent yields and lower profits in the commercial production of exotic mushrooms. Therefore, single subculturing from mother spawn is recommended to maintain mushroom yields comparable to the original mother spawn and produced with the proper medium and technique.

Acknowledgments: Author would like to thank many researchers for their research article review use as references in this article to complete and the authors declare no conflicts of interest. The author also thanks Chiba University where the author’s PhD degree was completed, and the field experiences gained from Misawa Kinoko in Gunma, Fujishukin co.ltd in Yamanashi, and academic experiences as a Post doctoral researcher at the University of Yamanashi.
Present affiliation: Researcher & foreign division chief at Fujishukin, Yamanashi.

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Quality of mother spawn is a key factor for high yield of exotic mushrooms; but too much subculturing can lead to reduced yields