The cultivation of edible mushrooms is considered to be an economically viable biotechnological application as their fruiting body represent an effective food source. In addition, they also use various wastes from industries, agriculture, forestry as well as food processing as growth substrate, thereby reducing environmental pollution (Sanchez, 2010). When growing edible mushrooms, the initial quality of strains directly affects the cultivation yield. However, strain degradation is also a common problem in many artificially-cultivated edible mushrooms as the degenerated strains have some peculiar characteristics such as less primordium, an extended production cycle and an abnormal fruiting body after cultivation, which altogether result in huge economic losses to the growers (Sun et al., 2017).

Volvariella volvacea is a mushroom that is industrially grown in many tropical and subtropical regions, ranking it as the fifth most cultivated mushroom in terms of annual global production (Payapanon et al., 2011). In addition to its unique aroma and texture during cooking, this fungus has also been reported to produce an antitumor polysaccharide as well as an immunosuppressive protein while having both immunomodulatory and medicinal effects (Ahlawat et al., 2008b; Wang et al., 2008). Moreover, compared with other edible mushrooms, V. volvacea possesses a shorter growth cycle of only 10–12 days from sowing to harvest (Hou et al., 2017). However, despite these unique properties, strain degradation remains an important factor restricting the industrial application of V. volvacea.

Volvariella volvacea is a tropical fungus, requiring temperatures of 28–34°C for mycelial growth and the development of its fruiting body. At the same time, low temperatures below 15°C can negatively impact its growth while causing chilling damage to its fruiting body, and routine storage temperatures (4°C) will cause V. volvacea cryogenic autolysis (Bao et al., 2013). Therefore, V. volvacea cannot be stored at low temperatures like other edible mushrooms, which makes it strain degradation particularly serious (Zhao et al., 2019). Regular subculturing is a commonly applied approach to preserve and rejuvenate various fungal species (Ayala-Zermeno et al., 2017), but excessive subculturing may also induce the biosynthesis of toxins, DNA methylation and chromosome remodeling as shown in Cordyceps militaris (Yin et al., 2017; Xin et al., 2019), thereby leading to strain degradation that hinders industrial applications.

Although the cultivation techniques (Ahlawat et al., 2011; Bao et al., 2013), breeding techniques (Zhao et al., 2010), and physiology (Diamantopoulou et al., 2016; Li et al., 2017) of V. volvacea have been thoroughly investigated, yet little is known about the characteristics of degenerated strains, especially regarding the mechanism that leads to degeneration. In this study, continuous subculturing of mycelia was used to obtain degenerated strains of V. volvacea. The characteristics of the mycelia and fruiting bodies, the reactive oxygen species (ROS) content as well as enzymatic activities were then measured to explore the physiological changes in subcultured V. volvacea strains. The results indicated that successive subculturing of this fungus negatively impacted mycelial growth, the formation of fruiting bodies, lignocellulose degradation, ROS accumulation and the activities of antioxidant enzymes. This work could form the basis of subsequent studies to gain a better understanding of the degeneration mechanism of edible fungi.

Volvariella volvacea is a typical grass rot fungus which uses straws, wheat straws, waste cottons or cotton seed shells as growth substrate. To grow on these media, V. volvacea secretes extracellular enzymes to decompose lignocellulose into small molecules which then provide the required nutrients to the fungus (Park et al., 2014). Given that lignocellulose consists of cellulose, hemicellulose and small amounts of lignin, the decomposition process involves the breakdown of long chain cellulose into short oligosaccharides by EG. The latter is then further degraded into cellulobiose by CBH, before being eventually decomposed into D-glucose by BGL (Wang et al., 2020). As far as hemicellulose is concerned, with xylan being the main component, it is hydrolyzed into short chain xylo-oligosaccharides by Xyl prior to degradation into xylose (Mamimin et al., 2021). In this study, the enzymatic activities of EG, CBH, BGL, and Xyl measured in the medium broth after culture on a lignocellulosic substrate decreased with increasing numbers of subculture (Figure 3), and this reduced V. volvacea’s ability to degrade cellulose and hemicellulose in the medium. Consequently, there was a decrease in the nutrients and energy available to the mycelia, resulting in a decrease in the overall mycelial growth rate of the subcultured strains. In the experiment, it was also noted that both the mycelial biomass procuded in PDB (Figure 1) and lignocellulases accumulated in lignocellolsic substrates (Figure 3) decreased with increasing subculture. However, extracellular lignocellulase activity was linked with lesser biomass production remains to be further studied.

Lignin molecules not only wrap cellulose but are also closely linked to it through covalent and hydrogen bonds. Thus, it is only when the lignin is broken down, along with the chemical bonds, that the cellulose can be exposed for enzymatic hydrolysis (Veluchamy and Kalamdhad, 2017). This hydrolysis process can be carried out by Lac and MnP which degrade lignin into smaller compounds such as phenolic monomers (Tellez-Tellez et al., 2008). Lac is widely distributed among fungi for which it carries out lignin degradation (Simonic et al., 2010), and as such, it plays an important role in the morphogenesis of V. volvacea’s fruiting body (Chen et al., 2003). Similarly, for Hypsizygus marmoreus, the Lac-1 gene is involved in primordium initiation by increasing laccase activity (Zhang et al., 2015), while it has been reported that the expression of the Lac-4 gene increases rapidly during the development from primordium to pinhead stage (Chen et al., 2004). In this study, Lac, including lac-1 and lac-4 gene expression, decreased the most rapidly among the lignocellulase. As a result, even though all of the subcultured strains formed primordia, V. volvacea stopped producing fruiting bodies after S12 (Figure 6; Table 1), probably due to the continuous decrease in Lac activity and Lac gene expression. Given the key role of laccase in the formation of fruiting bodies in V. volvacea, laccase could be used as a biomarker to assess the degree of degradation of V. volvacea.

Frequent strain degradation has become a major issue in the production of fungi, especially the degradation of economically-important ones such as edible mushrooms. This has resulted in great losses to production while making strain preservation a challenging process (Chen et al., 2017; Sun et al., 2017). For cultivation purposes, tip mycelium subculture is often used to maintain the vitality of edible mushrooms, but in the long run, successive subculturing can lead to strain degradation. In this context Shah et al. (2007), found that repeated subculturing could rapidly change the properties of spore surfaces and the virulence of Metarhizium anisopliae. Similarly, Sun et al. (2017) reported low growth rates and cellulase activities for C. militaris strains which had degenerated as a result of subculture as well as their insufficient capacity to form fruiting bodies and produce pigment. In the case of Yin et al. (2017), it was shown that strains began to degenerate at the third generation, with their fruiting bodies displaying incomplete growth on the fourth one. Finally Kim et al. (2014), reported slow vegetative growth, tight mycelial pads, and few or no fruiting bodies as some of the symptoms of degraded F. velutipes strains. In line with the above, and particularly the findings of (Sun et al., 2017), this study showed that the density of aerial mycelia, along with their growth rate and biomass, gradually decreased as the degeneration level increased (Figure 1). The cultivation experiment further showed that, compared with S0, the biological efficiency of subcultured strains S1–S12 was significantly reduced, while the production cycle was significantly prolonged (p < 0.05). Eventually, after 12 months of continuous subculture, the degenerated strains S12–S20 no longer produced fruiting bodies (Figure 2).

Although the process of fungal degeneration in nature had long been recognized as a common phenomenon, yet little was known about its causes. In this context, Wang et al. (2005) first proposed that the degeneration of filamentous fungi was a characteristic of an aging fungi, with fungal colony degeneration being an aging phenomenon caused by damage to mycelium cells under oxidative stress (Li et al., 2008). The study of filamentous fungal aging began in the 1950s with Podosporium anthracis (Osiewacz, 2011). Fungi in the aging process first show the loss of sporulation ability before a gradual decrease in the growth rate of mycelia. Eventually, they stop growing, and the tip mycelia begin to die (Bertrand, 2000). These observations were, in fact, consistent with the results of this study. Indeed, after subculturing several strains on PDA plates for over 2 years, severe degradation/aging was observed during the late stages of the experiment, thus leading to difficulties in establishing mycelial growth.

ROS are compounds such as O₂⁻, H₂O₂, and OH which are produced during the metabolic processes of aerobic organisms (Kim and Holmes, 2012). They are not only important for the growth of edible mushrooms but also participate in various cell differentiation and development processes, including the apex polar growth of mycelia (Lara-Ortiz et al., 2003), cell signal transmission (Veal et al., 2007), and the synthesis of secondary metabolites (Reverberi et al., 2008). ROS levels also influence primordium initiation and fruiting body development in edible fungi. For instance, the antioxidant kojic acid (KA) could regulate primordium initiation in H. marmoreus by influencing ROS levels (Zhang et al., 2017). Similarly, Xiong et al. (2013) found that overexpression of GPX could increase fungal abilities to scavenge cellular ROS and protect fungi against oxidative stress. Finally, a degraded C. militaris strain had its fruiting ability restored by genetic transformation with the GPX gene.

Reactive oxygen species production and removal are usually balanced in healthy organisms but certain conditions such as pathological changes or aging alter this balance, resulting in a rapid increase in ROS levels. Excessive ROS levels, if not removed in time, can cause serious damage to DNA, enzymes or lipid metabolism within an organism (Dong et al., 2009; Holley et al., 2011). These damages, often in the form of protein misfolding (Dukan et al., 2000) and lipid peroxidation (Altomare et al., 2021), occur randomly and accumulate in cells, thereby affecting cell functions. In addition, ROS damage to DNA may also be an important factor that accelerates aging, especially with studies reporting increased oxidative damage to nuclear DNA in aging body cells (Beckman and Ames, 1998). On the other hand, mitochondrial DNA is more sensitive to oxidative stress than nuclear DNA. With mitochondria being the primary source of intracellular ROS and oxidative stress, this can result in mitochondrial dysfunctions which in turn lead to the accumulation of additional ROS. In fact, the mitochondrial theory of aging is based on this “vicious cycle” (Genova et al., 2004; Balaban et al., 2005). As far as fungi are concerned, it has been reported that the mycelia of aging Podosporium anthracis displayed mitochondrial fragmentation which resulted in the accumulation of large amounts of ROS in the mycelia (Scheckhuber et al., 2007). Similarly, the loss of mitochondrial functions in aging cells of Saccharomyces serevisiae was shown to induce the release of excessive ROS (Borghouts et al., 2004). This study found that the O₂⁻ and H₂O₂ content significantly increased after S9 (Figures 4A,B), especially after S12, and it is not unlikely that this could be the main reason for severe degradation of strains S12–S20. However, future studies would be required to further clarify the relationship between mitochondria, nuclei, energy metabolism and V. volvacea degeneration.

In order to avoid oxidative damage, organisms possess antioxidant and enzymatic systems which assist in ROS removal, with the latter acting through the activity of enzymes such as SOD, CAT, and GPX amongst others (Blagosklonny, 2008). SOD is mainly responsible for the catalytic conversion of active O₂⁻ into H₂O₂ (Gao et al., 2020), with CAT further decomposing H₂O₂ into O₂ and H₂O (Ramis et al., 2015). Moreover, H₂O₂ can also oxidize GSH to produce GSSG under the action of GPX, while GR is responsible for the catalytic reduction of GSSG to produce GSH (Flohe, 2013). The activities of antioxidant enzymes (SOD, CAT, GR, and GPX) can directly reflect the ability to scavenge ROS (Chen et al., 2018). The mechanism through which fruiting bodies develop in edible fungi has been a popular research topic in recent years, with genes encoding antioxidant enzymes such SOD (Yan et al., 2016), GPX (Xiong et al., 2013), and CAT (Wang et al., 2017) having a specific relationship with primordium and fruiting body development. In this context, antioxidant enzyme activity assays showed that the activities of SOD, CAT, GR, and GPX initially increased before gradually decreasing with successive subculturing (Figure 5). These results indicated that, during the first few rounds of subculture, the accumulation of ROS induced an increase in the activity of antioxidant enzymes, and as a result, the change in ROS content in the subcultured strains was not significant Figures 4A,B. However, with increasing number of subcultures, the activity of antioxidant enzymes began to decline, thus causing the accumulation of ROS as well as the degradation of V. volvacea. Harman’s “free radical theory” state that aging is the result of the continuous accumulation of ROS (Harman, 1956), and this was well supported by the current results. Therefore, increasing the activity of antioxidant enzymes to reduce the ROS content could potentially delay strain aging, thereby serving as a strategy to prevent the degradation of edible mushroom in the future.

Overall, there was a decrease in the expression of genes related to substrate degradation and ROS scavenging after successive subcultures, and this reduced the activities of lignocellulase and antioxidant enzymes. Consequently, the absorption of nutrients was inhibited and the excessive accumulation of ROS led to the aging of the organism. These processes eventually resulted in the strain degradation of V. volvacea, which was visible as sparse aerial mycelia, decreased yield of fruiting body and prolonged production cycles (Figure 5). Strain degradation is a common problem in different types of cultivated edible mushrooms. While it is expected that, in the future, more research will be focused on this aspect of edible mushrooms, this study nevertheless contributed to an understanding of the process by providing a theoretical basis for the mechanism of degradation in V. volvacea strains. In addition, it also provided a basis for further studies of other functional genes involved in strain degradation.

As a tropical fungus with a short production cycle, V. volvacea has great market potential. However, its development is restricted by strain degradation. In this study, it was found that V. volvacea stopped producing fruiting bodies after continuous subculturing for 13 months. By analyzing ROS content, lignocellulase and antioxidant enzyme activities as well as gene expression by RT-PCR, it was observed that strain degradation in this fungal species was accompanied by a decrease in the activity of substrate-degrading enzymes and an excessive accumulation of ROS. The latter further led to the aging of the organism during successive mycelial subculturing and could have been responsible for the strain degradation in V. volvacea. This study provided a theoretical basis behind strain degradation of edible mushrooms.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

FZ and XL designed the research. XL and CC performed the research. ZC, WW, and JY analyzed the data. FZ and XL wrote the manuscript. All authors contributed to the article and approved the submitted version.

CC is employed by Sinograin Chengdu Storage Research Institute Co., Ltd.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This work was supported by the National Natural Science Foundation of China (Grant No. 32060708).