Degradation characteristics of lignocellulose by endophytic fungi of Taxillus chinensis

Degradation characteristics of lignocellulose by endophytic fungi of Taxillus chinensis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Degradation characteristics of lignocellulose by endophytic fungi of Taxillus chinensis Lisha Song, Ru Chen, Lingjian Gui, Wenjing Liang, Cuihong Yang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9401766/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background There are several endophytic fungal strains from Taxillus chinensis that can degrade lignocellulose from different substrates. We systematically explored their enzyme-producing characteristics and degradation efficiency on these substrates with the aim of providing theoretical support for the screening and application of high-efficiency degrading strains. Results Among the 4 tested strains, the highest total organic matter mass loss rate (55.50%) was observed when wheat bran was used as the substrate for 44 days of fermentation with the average degradation rate reaching a peak at 9 d (4.28%/d). All 4 strains showed consistent temporal dynamic characteristics with the 4 substrates: the lignin peroxidase activity peaked at 9 d of fermentation, with the highest enzyme activity of 23.52 U/mL. For strain P6, when wheat bran was used as the substrate, the laccase activity peaked at 16 days of fermentation (108.39 U/mL), which was significantly higher than those in other substrates and at different fermentation times. For strains 4 and N6, with mulberry leaves and wheat bran as substrates, the manganese peroxidase activity reached the highest at 16 and 30 days of fermentation (118.32 and 111.69 U/mL), respectively. For strain N6, when mulberry xylem was used as the substrate, the cellulase activity peaked at 2 days (0.67 U/mL), which was higher than that in other substrates and at different fermentation times. Conclusions The degradation efficiency of the 4 screened endophytic fungal strains from T. chinensis on different lignocellulosic substrates, as well as the activities of key degrading enzymes such as lignin peroxidase and laccase, all showed significant strain specificity, substrate specificity, and temporal dynamic characteristics. The results of this study can provide a theoretical basis and practical reference for the excavation of high-efficiency lignin-degrading strain resources and the optimization of their fermentation conditions. Taxillus chinensis endophytic fungi lignocellulose degrading enzymes fungus solid-state fermentation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Lignocellulose, the most abundant natural polymer in nature, is composed mainly of cellulose, hemicellulose, and lignin, which interweave to form a naturally compact structural complex [ 1 ]. The annual global production of lignocellulose is approximately 7.5 billion tons, which is equivalent to 10 times the annual total energy consumption. However, its high structural stability and resistance to degradation results in a low utilization efficiency for this process. At present, traditional disposal methods of agricultural and forestry wastes, (such as in-situ returns to fields, industrial raw material processing, feed preparations, and direct combustion, have several limitations. Among them, incineration tends to cause regional haze and increases the difficulty of air pollution control [ 2 – 3 ]. Therefore, the potential development of efficient and environment-friendly lignocellulose degradation technologies is of great practical significance. During microbial degradation of lignin, various changes occur in its chemical structure, including side-chain oxidation and demethylation. Lignin-degrading enzymes depolymerize the linkages within this macromolecule to ultimately degrade it. The key enzymes involved in degradation by white-rot fungi mainly include lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac) [ 4 – 6 ]. The synergistic action of these enzymes can effectively decompose the refractory lignin components [ 7 – 8 ]. In addition to these three major enzymes, other auxiliary ones also participate in the process. LiP (EC 1.11.1.14), also known as diarylpropane oxygenase, contains Fe³⁺ and was first discovered in the extracellular medium of fungi under nitrogen-limiting conditions [ 9 ]. It is a globular helical glycoprotein with 343 amino acid residues and a molecular weight of 38–46 kDa. It is initially oxidized by H₂O₂ to form compound I, which then reacts with the substrate to generate compound II. In comparison to conventional peroxidases, LiP can oxidize aromatic hydrocarbons with only 2–3 ether substituents, thereby reducing the stability of the lignin molecular bonds by promoting radical formation, and leads to lignin depolymerization [ 10 ]. MnP (EC 1.11.1.13) is a heme-containing, glycosylated peroxidase with a molecular weight of about 45 kDa. Its amino acid sequence shares high similarity with LiP, and its catalytic process is similar to those of LiP, horseradish peroxidase (HRP), and other peroxidases [ 8 , 11 ]. The unique feature of this enzyme is that it uses Mn as an electron donor. Due to the lack of a corresponding tryptophan residue, it cannot directly oxidize non-phenolic lignin but generates highly oxidative Mn³⁺ through its manganese-binding site [ 12 ], which exerts a secondary oxidative effect via diffusion. In addition, it cooperates with other lignin-degrading enzymes to break macromolecular bonds, threby playing an essential role in lignin degradation [ 11 ]. Lac (EC 1.10.3.2), also known as hydroquinone: oxidoreductase) belongs to the multicopper oxidase family. As a copper-containing polyphenol oxidase, it plays a central role in lignocellulose degradation due to its broad substrate specificity and high catalytic efficiency [ 13 – 14 ]. Lac was first found in lacquer trees and later reported in many plants, microorganisms, and animals. Laccases from different sources differ significantly in content, structure, and properties [ 15 – 18 ]. Among them, microbial laccases, including those from fungus and bacteria, which can possess high redox potentials, are dominant enzyme sources for lignocellulose degradation [ 2 ]. Lac can catalyze the conversion of various aromatic compounds such as monophenols, diphenols, and polyphenols, while reducing molecular oxygen to water. It is widely used in industrial production, environmental protection, food processing, as well as other fields [ 19 – 22 ]. However, insufficient catalytic efficiency under harsh conditions limits its practical applications, so the screening of novel strains with extreme environmental tolerances and high enzyme production is crucial [ 23 – 24 ]. White-rot fungi account for approximately 80% of all wood-rotting fungi and they are widely distributed in forest ecosystems and are natural high-yield lac producers and efficient lignocellulose degraders [ 25 – 29 ]. Their unique degradation system consists of Lac, MnP, and LiP, which can efficiently degrade lignin by catalyzing the cleavage of lignin chemical bonds while maintaining the structural integrity of cellulose, without producing pigments during the process [ 30 – 31 ]. For example, Xu et al. [ 32 ] screened Auricularia polytricha 5.584 from 9 white-rot fungi strains, and the lignin degradation rate reached 36.24% after 15 days of submerged fermentation. Li [ 33 ] found that culture and fermentation conditions directly affected lac production and the synergistic effect of lignocellulolytic enzymes in Trametes versicolor , highlighting the importance of optimizing fungal culture conditions. Fermentation protocols are the key factors that can affect lac yield. Submerged fermentation has long been the mainstream method for enzyme production due to uniform nutrient distribution and sufficient microbial absorption. In contrast, solid-state fermentation (SSF) has gradually become a research hotspot because it is closer to the natural living environment of microorganisms, with simplified technology, low energy consumption, less pollution, and high product recovery rates [ 34 – 35 ]. Lignocellulosic materials, including corn stover, wheat bran, and wood sawdust, as SSF substrates not only provide carbon and nitrogen but also mineral elements, such as K and Mg for microbial growth and enzyme synthesis. In addition, they contain natural inducers such as flavonoids and phenols, which can significantly improve fungal lac yield, making them low-cost and high-quality nutrient sources [ 36 – 38 ]. Taxillus chinensis (DC.) Danser, a traditional Chinese medicinal herb, was first recorded in Shennong ' s Classic of Materia Medica as a high-grade medicine. Its dried leafy stems and branches have the effects of expelling wind-dampness, tonifying the liver and kidneys, strengthening tendons and bones, and the capacity to prevent miscarriages. It is officially listed in the Pharmacopoeia of the People ' s Republic of China (2025 Edition) [ 39 ]. Due to low natural parasitism rates and heavy reliance on wild resources, the contradiction between increasing market demand and insufficient supply of T. chinensis has become a prominent concern. It is urgent to alleviate this problem by improving the parasitism rates, analyzing reproductive mechanisms, and promoting artificial cultivations [ 40 – 42 ]. With respect to its parasitic mechanism, T. chinensi s penetrates the epidermal cell wall, which is composed mainly of lignin and cellulose, of host plants through specialized haustoria to establish a “physiological bridge” for obtaining water and mineral nutrients while maintaining its own photosynthesis [ 40 – 42 ]. Studies have shown that white-rot fungi can invade host plants by degrading their cell walls [ 43 ], and the role of endophytic fungi in lignocellulose decomposition has been preliminarily explored. However, most of the relevant studies have tended to focus on the enzymatic characteristics of single strains [ 44 – 45 ]. Previous studies in our laboratory have screened 4 endophytic fungal strains with high lignocellulolytic enzyme production from T. chinensis parasitizing different hosts [ 45 – 46 ]. Based on this, the present study selected 4 different substrates of mulberry bark, xylem, and leaves, as well as wheat bran. We investigated their effects on the kinetic characteristics of their ligninolytic enzymes (lac, MnP, and LiP) and cellulase production in a systematic way. In addition, we assessed the substrate degradation rates of the 4 endophytic fungi. The optimal substrate suitable for the growth of T. chinensis was determined at the enzymatic level, with the aim of providing high-quality microbial resources and theoretical support for efficient lignin degradation as well as utilization of different substrate resources. Materials and methods The test strains Four endophytic fungal strains isolated from Taxillus chinensis (DC.) Danser were used. The plant material identified as T. chinensis was collected from Nanning city, Gugnaxi. Taxonomic identification was performed by Yude Peng based on morphological characteristics. A voucher specimen (accession no.SJS-NN-101) was deposited at the Guangxi Key Laboratory of High-Quality Formation and Utilization of Dao-di Herbs, which is publicly accessible. These were strains 4 ( Colletotrichum sp.), 15 ( Nigrospora sphaerica ), N6 ( Diaporthe phaseolorum ), and P6 ( Pestalotiopsis sp.) [ 45 ]. The NCBI accession numbers (ITS and beta-tubulin sequences) of each strain were MZ823601/MZ964759, MZ823600/MZ934421, MZ823599/MZ934420, and MZ823598/MZ934419, respectively. All strains were preserved in a 4 ℃ refrigerator in the plant pathology laboratory of Guangxi Botanical Garden of Medicinal Plants for subsequent use. Solid-state fermentation of the strains Wheat bran used in the experiment was purchased from Zhonghe Modern Agricultural Development Group Co., Ltd., and its pretreatment was by the method of Tao et al [ 47 ] with slight modifications. Mulberry leaves and branch bark epidermis and xylem were obtained from the Research Base of Guangxi Medicinal Botanical Garden. The samples rinsed with clean water and then distilled water, and dried in a 50 ℃ oven for 24 hours. They were then crushed, passed through a 100-mesh sieve to prepare the culture substrates, which were sealed and stored for later use. 2.0 g of each prepared substrate was added to 250 mL Erlenmeyer flasks with 15 mL of 0.5% malt extract and the mixture was autoclaved at 121 ℃ for 30 minutes. After cooling, the test strains were activated and cultured at a constant temperature of 28℃ for 5 ~ 7 days, and 6 mm diameter mycelial discs were taken with a sterile puncher and inoculated into the sterilized flasks. The humidity was maintained at 90%, and the flasks were cultured in the dark at 28℃ in an incubator. During the fermentation and culture period, sampling was carried out on days 2, 9, 16, 23, 30 and 44, respectively. Each treatment carried out in triplicate. Enzyme solution preparation and calculation of the average degradation rate Total organic matter (TOM) mass loss and average (MLR) degradation rate were used as evaluation indicators to assess the degradation efficiency of the 4 strains on 4 substrates (wheat bran as well as mulberry leaves, bark and xylem) and these were determined based on Hao et al. [ 48 ] with slight modifications. After each sampling, 10 mL of sodium acetate buffer solution (pH 6.0, 50 mmol/L) was pre-cooled to 4℃ in an Erlenmeyer flask, and extracted at 28℃ and 120 r·min⁻¹ for 20 minutes. Dried Whatman No.1 filter paper was weighed, and the mixture was filtered with this filter paper. The filtrate was collected and centrifuged at 4℃ and 10 000 × g for 10 minutes. The supernatant was used as the crude enzyme extract for the subsequent determination of various enzyme activities. The precipitate from the centrifuge tube was combined with the filter paper filtrate, placed on the original filter paper, and dried at 72 ℃ and weighed to calculate MLR. Sample MLR (%) = (Initial dry mass of sample - Dry mass of sample after decomposition) / Initial dry mass of sample × 100%. Average degradation rate (%) = TOM MLR / Culture days. Determination of enzyme activities The activity of lac was determined by the method of Buswell et al. [ 49 ]. 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was the reaction substrate and it was carried out at 30 ℃. The amount of enzyme required to catalyze the oxidation of 1µmol ABTS per minute was defined as 1 enzyme activity unit (U) and this was measured by the absorbance change at 420 nm. The molar extinction coefficient of ABTS at 420 nm was 36000 L/(mol·cm). LiP activity was determined by Tien et al. [ 50 ] method with veratryl alcohol as the substrate and this was performed at 30℃. The amount of enzyme required to change the absorbance at 310nm change in 1mL of the reaction mixture to reach 0.1 per minute was defined as 1 enzyme activity unit (U), resulting in the production of veratraldehyde. The activity of MnP was determined by the method of Orth et al. [ 51 ] and used the changes in absorbance of the phenol red at 610 nm. The amount of enzyme required for the absorbance of 1mL of the reaction solution to increase by 0.1 unit per minute was defined as 1 enzyme activity unit (U·mL⁻¹). The activity of cellulase was determined by the 3,5-dinitrosalicylic acid method. The enzyme activity was calculated by measuring the content of reducing sugars produced after cellulase degraded cellulose. A standard curve was constructed with 10 mg of anhydrous glucose standard (loss on drying < 0.2%) from the kit. This was dissolved in 1 mL of ultrapure water to prepare a glucose stock solution, which was then diluted to 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, and 0 mg·mL⁻¹, respectively. 1 mL of fermentation broth was ultrasonicated ice bath conditions to lyse the cells and centrifuged at 4 ℃ and 8000×g for 10 minutes. The supernatants were stored on ice and 50µL samples were used for determinations for measurements by addition of 150µL of the 3,5-dinitrosalicylic acid reagent. The absorbance at 540 nm [ 52 – 53 ] was measured and the cellulase activity was calculated by using the standard curve. Statistical analysis All experimental data were analyzed by one-way analysis of variance (ANOVA) using SPSS 19.0 software, and the results were expressed as means±standard deviations. The homogeneity of variance test was first performed on each group of data and Origin 2018 software was used for plotting the data. Results The mass loss of different substrates of four endophytic fungal strains The mass loss rates (MLRs) of the 4 strains varied significantly across the 4 substrates, mulberry bark, xylem, and leaves, as well as wheat bran, used (Fig. 1 ). The MLR of each strain on mulberry bark displayed an overall trend of first increasing and then decreasing with prolonged incubation times (Fig. 1 A). Strain 15 showed the best degradation performance, with its MLR reaching 50.50% at 30 d of fermentation, which was notably higher than those of the control and the other three strains. The MLRs of the 4 strains on mulberry xylem increased gradually with increased incubation times. At 44 d, strains P6 and 4 achieved significantly higher MLR rates than Strain N6 and the control, at 34.50 and 32.50%, respectively (Fig. 1 B). The MLRs of the strains on mulberry leaves showed a fluctuating pattern of increase to decrease over time (Fig. 1 C). At 44 days, Strains P6 and 15 both showed the highest degradation efficiencies, with a MLR of 62.50%, exceeding the other two strains and the control. The MLRs of the strains using wheat bran as substrate increased continuously with incubation times (Fig. 1 D). Except for the control and Strain N6, Strains 15, P6, and 4 exhibited higher MLRs at 44 d, and reached 55.50, 48.50, and 43.00%, respectively. Overall, Strain 15 showed the highest MLR on mulberry bark and leaves, as well as wheat bran. Strain P6 presented the optimal degradation performance on mulberry xylem and leaves, and wheat bran. Strain 4 achieved the highest MLRs on mulberry xylem and wheat bran. These results suggested that Strains 15, P6, and 4 possess high degradation capacity on diverse substrates. Daily average degradation rates of the four strains with different substrates The average daily degradation rates of the 4 strains on the 4 substrates used all showed a decreasing trend with increased incubation times (Fig. 2 ). However, there were significant differences in the peak times and rates of degradation of the different strains on each substrate used (Fig. 2 , P < 0.05). Using mulberry bark substrate, the average daily degradation rates of strains P6 and 4 decreased gradually with time (Fig. 2 A) but were at relatively high levels from 9 ~ 16 days, ranging from 2.03 to 2.67%, and these were significantly different from other time periods (P < 0.05). The average daily degradation rate of strain N6 reached its peak of 2.56% at 16 days, which was significantly higher than that in other time periods (P < 0.05). That of Strain 15 was 23 d, with the maximum of 1.91%. With mulberry xylem as substrate, except for Strain N6, the average daily degradation rates of the other three strains all decreased with the extension of time and Strain 4 reached the highest average daily degradation rate (3.17%) at 9 days. Strain P6 maintained a relatively high degradation level during 9 ~ 16 days, with rates ranging from 1.56 to 1.61%, which was significantly different from other time periods (Fig. 2 B; P < 0.05). The peak average daily degradation rate of Strain 15 was also at 9 days, with a maximum of 2.00%. The average daily degradation rates of the 4 strains on mulberry leaf substrate all showed a decreasing trend with time, but there were differences observed in the peaks (Fig. 2 C). Strain 4 reached the highest average daily degradation rate (4.39%) at 9 days and Strains 15 and P6 maintained relatively high degradation levels during 9 ~ 16 days, with rate ranging from 3.28 ~ 3.38% and 2.28 ~ 2.39%, respectively (P < 0.05). Strain N6 reached a relatively high degradation level during 16 ~ 23 days, with rates of 2.07–2.19% (P < 0.05). With wheat bran substrate, the average daily degradation rates of the 4 strains all decreased continuously with time, and the highest average daily rate of all the strains was at 9 days. These were 3.56, 3.78, 4.28 and 3.00%, respectively (P < 0.05). Therefore, all the strains reached relatively high average daily degradation rates on wheat bran substrate at 9 d, which were significantly different from other time periods (P < 0.05). The average daily degradation rates of Strain 4 on mulberry xylem and leaves as substrates both reached their peaks at day 9, and these were 3.17 and 4.39%, respectively, (P < 0.05), reflecting their strong short-term degradation advantage. Changes in LiP production by the four strains When mulberry bark was used as the substrate, the LiP activities of the P6, 4, 15, and N6 strains all showed a trend of first increasing and then decreasing as the time of fermentation increased (Fig. 3 A). The activities reached their peaks on the 9th day, and were 6.77, 2.00, 6.94, and 4.17 U/mL, respectively, all of which were higher than that of the control group. When mulberry xylem was used as the substrate, the trend of LiP activity of the 4 strains was similar to that of when mulberry bark was the substrate, and the activities also reached the peak at the 9 days, and were 6.49, 5.14, 5.96, and 3.89 U/mL, respectively (Fig. 3 B). Except for Strain N6, the enzyme activity peaks of the other three strains were all higher than the control group, and none was detected on the second day of culture. When mulberry leaves were used as the substrate, the LiP activities of the 4 strains still showed a trend of first increasing and then decreasing, reaching the peak on day 9, and were 3.33, 21.15, 2.45, and 1.51 U/mL, respectively (Fig. 3 C). Among them, Strain 4 had the highest enzyme activity peak, and except for strain N6, the peaks of the other strains were all higher than that of the control group. With wheat bran as the substrate, the LiP activities of the 4 strains was also consistent and reached the highest on day 9, and were 7.01, 23.52, 4.5, and 2.19 U/mL, respectively (Fig. 3 D). With this substrate, Strain 4 had the highest enzyme activity peak, and the peaks of all 4 strains were also higher than that of the control group. To summarize, under the 4 different substrates and culture times used the LiP activities of the 4 endophytic fungal strains all showed a consistent trend of "first increasing and then decreasing", and peaked on day 9 of culture. Among them, when cultured with mulberry leaves and wheat bran the enzyme activity peaks of Strain 4 were significantly higher than those of the other two substrates and other strains while using the same substrate. Changes in laccase production by the four strains used When mulberry bark was used as the substrate, the lac activities of the P6, 4, 15, and N6 strains all showed a trend of first increasing and then decreasing (Fig. 4 A). Except for strain 15 and the control group, the other strains all reached their peak enzyme activities on the 9th day of culture, with the peak values being 21.88, 3.19, 1.10, and 11.73 U/mL respectively. The peak laccase activity of Strain 15 appeared on the 23rd day, with a value of 62.80 U/mL. When mulberry xylem was used as the substrate, the lac activities of the 4 strains used also showed a trend of first increasing and then decreasing (Fig. 4 B). Except for strains P6 and 4, the other ones all reached their peak activities on the 9th day, with values of 7.35, 9.09, 31.30, and 30.86 U/mL respectively. With mulberry leaves as the substrate, there were differences in the peak lac activities among the 4 strains (Fig. 4 C). Strain P6 reached its peak on day 9, with an activity of 28.94 U/mL. The peak activities of Sstrains 15 and N6 both appeared on the 16th day, and were 5.08 and 3.91 U/mL respectively. and that of Strain 4 was on the 23rd day, at 6.45 U/mL. In addition, the lac activities of all 4 strains were higher than the control group. When wheat bran was used as the substrate, the lac activities of the 4 strains all showed a trend of first increasing and then decreasing, and the peaks occurred on day 16, with peak values of 108.39, 73.20, 11.99, and 68.64 U/mL, respectively (Fig. 4 D). In summary, there were differences in the lac activities of the 4 strains when using different substrates and culture times, and the peak enzyme activities were different. Changes in MnP production by the four strains With mulberry bark, xylem, and leaves and wheat bran as substrates, the MnP activities of P6, 4, 15, and N6 all showed trends of first increasing and then decreasing (Figs. 5 A- 5 D). For mulberry bark, the 4 strains reached their peak activities on day 30, with the peak values being 11.76, 45.64, 45.21, and 78.45 U/mL, respectively, and Strain N6 had the highest activity (Fig. 5 A). For mulberry xylem, except for Strains N6 and 15, the others (P6 and 4) both reached their peak enzyme activities on the 30th day, with peak values of 51.75 and 38.08 U/mL, respectively (Fig. 5 B). When mulberry leaves were used, the peak enzyme activity of strain P6 appeared on day 30, while that of Strain 4 reached the peak on the 16th day, with values being 36.26 and 118.32 U/mL, respectively (Fig. 5 C). With wheat bran as the substrate, the MnP activities reached their peak activities on day 30, with values of 62.28, 55.05, 84.33, and 111.69 U/mL respectively, and strain N6 had the highest activity (Fig. 5 D). In summary, when using different substrates and culture times, the MnP activities of the 4 strains all showed an increasing and then a decreasing trend. Among them, only when Strain 4 used mulberry leaves as the substrate, the peak MnP activity appeared on the 16th day, and that occurred on day 30 for the other strains. Changes in cellulase production by the four strains When mulberry bark, xylem, and leaves were used as substrates, the cellulase activities of Strains P6, 4, 15, and N6 all showed trends of first increasing and then decreasing (Figs. 6 A- 6 C). For mulberry bark, with the exceptions of P6 and the control group, the other three strains all reached their peak cellulase activities on the 2nd day of culture, with peak values being 0.19, 0.37, and 0.21 U/mL respectively (Fig. 6 A). The peak of Strain P6 appeared on day 23, with a value of 0.39 U/mL. With mulberry xylem and the exception of N6, the other three strains all reached their peak enzyme activities on the 2nd day, with the peak values being 0.49, 0.35, and 0.67 U/mL respectively (Fig. 6 B). The peak activity of strain N6 was 0.16 U/mL and appeared on day 9. When mulberry leaves were used as the substrate, the cellulase activities of the 4 strains reached their peaks on the 2nd day, with values of 0.14, 0.15, 0.23, and 0.20 U/mL respectively (Fig. 6 C). With wheat bran as the substrate, except for Strain 15, the cellulase activities of P6, 4, and N6 all showed a trend of first increasing and then decreasing, and all reached their peaks on the 2nd day, with values of 0.27, 0.20, and 0.26 U/mL respectively. That of Strain 15 was on day 9, with a value of 0.28 U/mL (Fig. 6 D). In summary, there were significant differences in the changes of cellulase-producing activities of the 4 endophytic fungi when using different substrates and culture times. Discussion Previous studies have shown that the composition of the fermentation medium is one of the key factors affecting fermentation efficiency [ 53 ], and the differences in enzyme types and their production times also led to differences in the lignin degradation ability of strains on different substrates [ 54 ]. In this study, four endophytic fungal strains isolated from T. chinensis . These were Strains 4 ( Colletotrichum sp.), 15 ( N. sphaerica ), N6 ( D. phaseolorum) , and P6 ( Pestalotiopsis sp.) and were inoculated separately onto the 4 substrates of mulberry, leaves, and xylem, as well as wheat bran to undergo the process of solid-state fermentation. The changes in TOM mass loss, average degradation rates, and lignocellulose-degrading enzyme activities caused by the strains during the degradation of different culture substrates were determined. Our studies showed that all 4 fungi exhibited high lignin degradation levels when wheat bran was used as the substrate, and the highest rate reaching 55.50% under optimal culture conditions. Guo et al. [ 55 ] found that a strain of Pseudomonas guanguanensis isolated from lignin-degrading bacteria the could secrete LiP, MnP, and lac, with a lignin degradation rate of about 38% after 3 days of fermentation. Zhou et al. [ 56 ] found that Alternaria eichhhorniae Strain G6 and Trichoderma shangrilaense Strain G59 could both produce LiP and lac, and the enzyme-producing capacity of the composite strain was significantly improved, with a degradation rate of 54.53% for corn stover. Current studies have shown that the degradation efficiency of composite microbial communities was significantly higher than that of individual strains. In the natural ecosystems, lignin degradation does not primarily result from the independent action of specific strains, but rather it stems from the inhibition and synergism of communities of microorganisms in the same habitat. Therefore, future studies should attempt to construct composite microbial communities, explore their degradation effects on lignocellulose of different substrates, and analyze the optimal conditions required by different lignocellulose-degrading bacteria, including temperature, pH, light, and types and concentrations of carbon and nitrogen sources. Lignin degradation relies on the synergistic effects of lac, MnP, and LiP. These enzymes can break the chemical bonds in the lignin structure to achieve degradation, and lac plays a leading role in the decomposition process. Its activity can be used as an important indicator reflecting the overall activity of the lignin-degrading enzyme system. During the 44-day full cultivation period, when Strains 4 ( Colletotrichum sp.), 15 ( N. sphaerica ), P6 ( Pestalotiopsis sp.), and N6 ( D. phaseolorum ) were fermented with wheat bran as the lignocellulosic biomass substrate, the activities of lac and cellulase were significantly higher than those of the control group. Zhao et al. [ 57 ] showed that increased lac concentrations were needed for mycelial degradation of lignin. They showed that the laccase activity of the white-rot fungus, Pleurotus eryngii , was the highest in the culture substrate with the highest lignin content, which is consistent with our results. When wheat bran was used as the substrate, cellulase activity increased significantly on the second day of cultivation, while lac activity reached a high level on day 9. Previous studies [ 58 – 60 ] have reported that this phenomenon is closely related to the non-selective lignin degradation mode of white-rot fungi, which consumes large amounts of cellulose. In addition, the reducing sugars and other substances released by cellulase degradation of cellulose can provide the necessary substrates and a suitable reaction environment for lac to act, thereby indirectly regulating its activity. Several others have also reported [ 61 – 63 ] significantly improved the lignin degradation rates by adding wheat bran to the medium, confirming that this substrate can induce the enhancement of enzyme-producing capacity by a strain, which is consistent our findings. When using lac activity as the evaluation index, strain P6 had the highest activity (108.39 U/mL) on the wheat bran substrate. Existing literature reports show that Pestalotiopsis sp. of fungi possess high efficiencies for the production of lac and cellulase, which can effectively degrade forest litter [ 48 ]. This further confirms the excellent enzyme-producing and degradation characteristics of strain P6 in this study. During the early stages of fermentation, the enzyme activities of Strains N6, 4, 15, and P6 on different substrates all showed a gradual upward trend, but as the cultivation times increase, they tend to decrease. This is mainly because the nutrients and oxygen in the substrates were gradually consumed, leading to restricted microbial growth and even death, thereby reducing enzyme production efficiency. The buildup of inhibitor metabolites is another possible explanation for this phenomenon [ 64 ]. In summary, the 4 endophytic fungal strains isolated from T. chinensis screened in this study are all able to degrade lignin efficiently, providing new high-quality strain resources for biological lignin degradation. This can have important theoretical and practical significance for the development of bioenergy and the sustainable utilization technology of lignocellulosic resources. Conclusion The degradation efficiency of the 4 screened endophytic fungal strains from T. chinensis on different lignocellulosic substrates, as well as the activities of key degrading enzymes such as lignin peroxidase and laccase, all showed significant strain specificity, substrate specificity, and temporal dynamic characteristics. The results of this study can provide a theoretical basis and practical reference for the excavation of high-efficiency lignin-degrading strain resources and the optimization of their fermentation conditions. Abbreviations LiP lignin peroxidase MnP manganese peroxidase Lac laccase MLRs the mass loss rates TOM total organic matter ABTS − 2,2' azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) kDa kilodalton HRP horseradish peroxidase SSF solid-state fermentation Declarations Ethics approval and consent to participate Strains 4 ( Colletotrichum sp.), 15 ( Nigrospora sphaerica ), N6 ( Diaporthe phaseolorum ), and P6 ( Pestalotiopsis sp.) were kept in the Plant Pathology Department of the Guangxi Botanical Garden of Medicinal Plants and we obtained written consent from them to use these fungal strains in our studies. The experimental methods conducted in this study complied with all the current Chinese laws and regulations. Consent for publication Not applicable. Conflict of interest The authors declare no competing interest. Authors’ information 1 Guangxi Botanical Garden of Medicinal Plants, Nanning, China, 2 Guangxi Key Laboratory for High-Quality Formation and Utilization of Dao-di Herbs, Nanning, China and 3 Guangxi University of Chinese Medicine, Nanning 530000, China. Funding This work was supported by the Guangxi Natural Science Foundation of China (No. 2024GXNSFAA010371 and 2026GXNSFAA00641220) and the Multidisciplinary Innovative Team for Traditional Chinese Medicine of Guangxi (No. GZKJ2305). Author Contribution Lisha Song implemented the experiments, analyzed and interpreted the data and completed the article writing. Lingjian Gui and Wenjing Liang participated in the experiments and collected the samples. Ni Jiang, Ru Chen and Cuihong Yang integrated information into the tables. Shugen Wei and Jine Fu reviewed and finalized manuscript. Yuqiong Li and Ya Qin generated the figures. Lingyun Wang and Huijie Zhang edited the manuscript. The final manuscript was approved by all authors who agreed to be accountable for the content of this work. Acknowledgement The authors are grateful to Dr. Dev Sooranna of Imperial College London, for English editing of the manuscript. Availability of data and materials ITS and beta-tubulin sequences of 4 strains, 4,15, N6, and P6, were submitted to the GenBank database and the NCBI accession numbers were MZ823601/MZ964759, MZ823600/MZ934421, MZ823599/MZ934420, and MZ823598/MZ934419, respectively. Other data generated and analyzed during this study are included in the article and its supplementary information files: ( https://www.ncbi.nlm.nih.gov/search/all/?term=MZ823600;https://www.ncbi.nlm.nih.gov/pmc/?term=MZ823598;https://www.ncbi.nlm.nih.gov/search/all/?term=MZ823601;https://www.ncbi.nlm.nih.gov/search/all/?term=MZ823599 ) References Liu M, Xu LX, Yin ZX, He DM, Zhang YJ, Liu CQ. Harnessing the potential of exogenous microbial agents: a comprehensive review on enhancing lignocellulose degradation in agricultural waste composting. Arch Microbiol. 2025;207:51. Surwase SV, Patil SA, Srinivas S, Jadhav JP. Interaction of small molecules with fungal laccase: a surface plasmon resonance-based study. Enzym Microb Technol. 2016;82:110–4. Han ML, Zhao YL, Liu TY, Bian LS, Li ZH, Li MX, Li J, Yang XQ, An Q. Effect of alkali lignin on laccase activity and mycelial biomass of Flammulina velutipes : Alkali lignin on laccase activity and mycelial biomass. BioResources. 2025;20:2243–58. Tien M, Kirk TK. Lignin-degrading enzyme from Phanerochaete chrysosporium : purification, characterization, and catalytic properties of a unique H 2 O 2 -requiring oxygenase. P Natl Acad Sci USA. 1984;81:2280–4. Hussaan M, Amna, Javed MT, Akram MS, Ali S. Physiological and molecular basis of bioremediation of micropollutants. Handbook of Bioremediation. Academic Press, 2021; 447–464. Rencoret J, Pereira A, Del Río JC, Martínez AT, Gutiérrez A. Laccase-mediator pretreatment of wheat straw degrades lignin and improves saccharification. Bioenerg Res. 2016;9:917–30. Wong DWS. Structure and action mechanism of ligninolytic enzymes. App Biochem Biotech. 2009;157:174–209. Hofrichter M. Review: lignin conversion by manganese peroxidase (MnP). Enzyme Microb Tech. 2002;30:454–66. Orth AB, Denny M, Tien M. Overproduction of lignin-degrading enzymes by an isolate of Phanerochaete chrysosporium . Appl Environ Microb. 1991;57:2591–6. Kersten PJ, Kalyanaraman B, Hammel KE, Reinhammar B, Kirk TK. Comparison of lignin peroxidase,horseradish peroxidase and laccase in the oxidation of methoxybenzenes. Biochem J. 1990;268:475–80. Sundaramoorthy M, Kishi K, Gold MH, Poulos TL. The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06-A resolution. J Biol Chem. 1994;269:32759–67. Sundaramoorthy M, Kishi K, Gold MH, Poulos TL. Crystal structures of substrate binding site mutants of manganese peroxidase. J Biol Chem. 1997;272:17574–80. Ozer A, Ay Sal F, Belduz AO, Kirci H, Canakci S. Use of feruloyl esterase as laccase-mediator system in paper bleaching. Appl Biochem Biotech. 2020;190:721–31. Leynaud Kieffer Curran LMC, Pham LTM, Sale KL, Simmons BA. Review of advances in the development of laccases for the valorization of lignin to enable the production of lignocellulosic biofuels and bioproducts. Biotechnol Adv. 2022;54:107809. Yoshida HLXIII. Chemistry of lacquer (Urushi). Part I. Communication from the Chemical Society of Tokio. J Chem Soc 1883; 43: 472–86. https://10.1039/ct8834300472 Baldrian P. Fungal laccases - occurrence and properties. Fems Microbiol Rev. 2006;30:215–42. Oztat K, Yavuz AA, Isçen CF. Optimization studies on laccase activity of Proteus mirabilis isolated from treatment sludge of textile industry factories. Braz J Microbiol. 2024;55:1231–41. Scheibel DM, Gitsov IPI, Gitsov I. Enzymes in Green synthetic chemistry: laccase and lipase. Molecules. 2024;29:989. Gupta A, Jana AK. Production of laccase by repeated batch semi-solid fermentation using wheat straw as substrate and support for fungal growth. Bioproc Biosyst Eng. 2019;42:499–512. Wu Y, Ma HF, Cao YJ, Si J, Cui BK. Advances on properties production, purification and immobilization of fungal laccase. Biotechnol Bull. 2019;35:1–10. Al-Rajhi AMH, Bazaid AS, Abdulfattah AM, Abdelghany TM, Shater ARM, Selim S. Evaluation of lignocellulatic activity of enzymes from microwave-irradiated Pleurotus sajor-caju cultivated with wheat straw. BioResources. 2024;19(4):8882–93. Ramirez LS, Dragotakes Q, Casadevall A. A food color-based colorimetric assay for Cryptococcus neoformans laccase activity. Microbiol Spectr. 2024;12:1–16. Pang S, Wu Y, Zhang X, Li B, Ouyang J, Ding M. Immobilization of laccase via adsorption onto bimodal mesoporous Zr-MOF. Process Biochem. 2016;51:229–39. Agrawal K, Chaturvedi V, Verma P. Fungal laccase discovered but yet undiscovered. Bioresour Bioprocess. 2018;5:4. Dai YC. Hymenochaetaceae (Basidiomycota) in China. Fungal Divers. 2010;45:131–343. Dai YC, Yang ZL, Cui BK, Wu G, Yuan HS, Zhou LW, He SH, Ge ZW, Wu F, Wei YL, Yuan Y, Si J. Diversity and systematics of the important macrofungi in Chinese forests. Mycosystema. 2021;40:770–805. Wu F, Man XW, Tohtirjap A, Dai YC. A comparison of polypore funga and species composition in forest ecosystems of China, North America, and Europe. Ecosyst. 2022;9:100051. Zhao H, Wu YD, Yang ZR, Liu HG, Wu F, Dai YC, Yuan Y. Polypore funga and species diversity in tropical forest ecosystems of Africa, America and Asia, and a comparison with temperate and boreal regions of the Northern Hemisphere. Ecosyst. 2024;11:100200. Cui BK, Li HJ, Ji X, Zhou JL, Song J, Si J, Dai YC. Species diversity, taxonomy and phylogeny of Polyporaceae (Basidiomycota) in China. Fungal Diver. 2019;97:137–302. Teigiserova DA, Bourgine J, Thomsen M. Closing the loop of cereal waste and residues with sustainable technologies: an overview of enzyme production via fungal solid-state fermentation. Sustain Prod Consump. 2021;27:845–57. Wang CG, Dai YC, Kout J, Gates GM, Liu HG, Yuan Y, Vlasák J. Multi-gene phylogeny and taxonomy of Physisporinus (Polyporales, Basidiomycota). Mycosphere. 2024;15:1455–521. Xu AM, Li L, Ma S. Screening of lignin degrading enzyme high-producing strain from white rot fungi. J Henan Univ Technol (Natural Sci Edition). 2020;41:78–8289. Li Y, Gao Y, Zhao Y, Lou HY, Wang XW, Niu YW, Yang CM, Zhao RY. Optimization of the culture medium for laccase production by Trametes versicolor and activity detection of fermentation broth in degrading zearalenone. J Henan Univ Technol (natural Sci edition). 2024;45(5):87–96. Rodríguez-Couto S. A promising inert support for laccase production and decolouration of textile wastewater by the white-rot fungus Trametes pubescesns . J Hazard Mater. 2012;233:158–62. Postemsky PD, Bidegain MA, González-Matute R, Figlas ND, Cubitto MA. Pilot-scale bioconversion of rice and sunflower agro-residues into medicinal mushrooms and laccase enzymes through solid-state fermentation with Ganoderma lucidum. Bioresource Technol. 2017;231:85–93. Jasinska A, Góralczyk-Binkowska A, Sobon A, Dlugonski J. Lignocellulose resources for the Myrothecium roridum laccase production and their integrated application for dyes removal. Int J Environ Sci Te. 2019;16(8):4811–22. Xiong X, Li P, Xiang Z, Huang J, Wang J. Influence of different lignocellulosic materials on laccase activity in liquid fermentation of Chinese low- temperature straw mushroom ( Volvariella brumalis ). BioResources. 2024;19:8608–20. Cabrera R, López-Peña D, Asaff A, Esqueda M, Valenzuela-Soto EM. Bioavailability of compounds susceptible to enzymatic oxidation enhances growth of shiitake medicinal mushroom (Lentinus edodes) in solid-state fermentation with vineyard prunings. Int J Med Mushrooms. 2018;20:291–303. National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China 2025. Volume I. Beijing: China Medical Science and Technology; 2025. Xu J. Study on anatomical structure and histochemistry of Taxillus sutchuenensis (Loranthaceae). Hubei: Yangtze University; 2024. Lian T, Guo YX, Tian LY, Liu J, Xu JZ, Yang H, Qiu HL, Qin CS. Histoanatomical observation on the process of Taxillus chinensis infecting Camellia semiserrata . Plant Prot. 2024;50:64–72. Li L. Study on the quality of Taxilli herba resources from Guangxi based on metabolomics. Chengdu: Chengdu University of TCM; 2024. Liu JL, Cheng YH. Screening and identification of lignin-degrading fungi. Agricultural Sci Eng China. 2024;36:16–9. Song LS, Wan LY, Wei SG, Pan LM, Huo J, Fu JE, Jiang N. Screening and identification of high-yield lignocellulose-degrading endophytic fungi from Taxillus chinensis (DC.) seeds. Southwest China J Agricultural Sci. 2023;36:1924–33. Song LS, Pan LM, Jiang N, Fu JE, Wan LY, Wei SG. Effects of endophytic fungi on parasitic process of Taxillus chinensis . Sci Rep. 2022;12:7744. Song L, Huo J, Wan L, Pan L, Jiang N, Fu J, Wei S. Lili He. Differences and biocontrol potential of haustorial endophytic fungi from Taxillus Chinensis on different host plants. BMC Microbiol. 2023;23:128. Tao YJ. Study on the Modification and Application of Wheat Bran Dietary Fibre. Jiangnan: Jiangnan University; 2009. Hao JJ, Song FQ, Tian XJ, Huang F, Zhang P, Zhang ZJ. Decomposition of Pinus massoniana Needle Driven by Deuteromycetes—Dynamics of Lignocellulolytic Enzymes. Scientia Silvae Sinicae. 2006;42:69–75. Buswell JA, Cai YJ, Chang S. Effect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Lentinula edodes. Fems Microbiol Lett. 1995;128:81–7. Tien M, Kirk TK. Lignin peroxidase of Phanerochaete chrysosporium . Method Enzymol. 1988;161:238–49. Orth AB, Royse DJ, Tien M. Ubiquity of lignin-degrading peroxidases among various wood-degrading fungi. Appl Environ Microb. 1993;59:4017–23. Ke LX, Wang MY, Wu Q. An exploratory study on elevation of the lignin degradation enzyme activity of Pleurotus ostreatus in submerged cultivation and its decolorization rate to direct sky blue 5B. Acta Sci Circum. 2015;35:1449–56. Su Y, Liu C, Long Z, Ren H, Guo XH. Improved production of spores and bioactive metabolites from Bacillus amyloliquefaciens in solid-state fermentation by a rapid optimization process. Probiotics Antimicro. 2019;11:921–30. Adejoye OD, Fasidi I. Biodegradation of agro-wastes by some Nigerian white-rot fungi. BioResources. 2009;4:816–24. Guo CS, Ye YJ, Chen C, Yang HF, Liu HL, Ning XP, Liu GP, Cai Y. Screening and identification of a lignin-degrading strain and optimization of its degradation conditions. Feed Ind. 2024;45:113–9. Zhou DX, Xu R, Guo HY, Dai HY. Screening and degradation efficiency of low-temperature-tolerant fungal strains for lignocelluose in crop straw. J Northeast Agricultural Univ. 2026;57:74–82. Zhao CM, Du F, Zou YJ, Hu QX, Zheng SY. Study on the relationship between mycelial growth and lignin degrading enzymes of Pleurotus eryngii based on different Substrates. Edible Fungi China. 2020;39:109–14. Shrestha P, Rasmussen M, Khanal SK, Pometto AL, van Leeuwen JH. Solid-substrate fermentation of corn fiber by Phanerochaete chrysosporium and subsequent fermentation of hydrolysate into ethanol. J Agr Food Chem. 2008;56:3918–24. Lai GD, Qin CS, Zhao DY, Qiu HL, Yang H, Tian LY, Lian T, Xu JZ. Isolation and screening of cellulose degrading strains. Forestry Environ Sci. 2021;37:24–32. Wang XX, Qi LH, Song JF, Zhang QG, Liu CC, Zhang Q, Song FF, Xu IL. Screening of cellulose and lignin degrading bacteria and their effect on composting of mushroom residue. J Henan Agricultural Sci. 2024;53:66–79. LI Y. Remediation of polycyclic aromatic hydrocarbons-petroleum hydrocarboncompound contaminated soil by white rot fungi agents / immobilized enzymes. Beijing: Beijing University of Civil Engineering and Architecture, 2023. Miao ZY, Liu YXY, Yao YY, Li HJ. Utilization and modification status of wheat bran nutrient. Cereals Oils. 2020;33:18–20. Xu AM, Li L, Ma Sen, Wang XX. Study on the optimization of laccase fermentation conditions and the degradation of wheat bran by Auricularia polytricha 5.584. J Henan Univ Technol (Natural Sci Edition). 2020;41:65–71. Xu XK. The preparation and functional properties of soluble dietary fiber (SDF) from wheat bran fermented by Neurospora sitophila . Harbin: Harbin University of Commerce; 2021. Tables Tables 1 to 6 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files tables1figure1rawdata224.xlsx tables2figure2rawdata224.xlsx tables4figure4rawdata224.xlsx tables5figure5rawdata224.xlsx tables6figure6rawdata224.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 08 May, 2026 Reviewers invited by journal 05 May, 2026 Editor invited by journal 29 Apr, 2026 Editor assigned by journal 24 Apr, 2026 Submission checks completed at journal 23 Apr, 2026 First submitted to journal 23 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9401766","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":638482661,"identity":"651d0f75-f1c1-4e6a-aab7-09b579900b4b","order_by":0,"name":"Lisha Song","email":"","orcid":"","institution":"Guangxi Botanical Garden of Medicinal Plants","correspondingAuthor":false,"prefix":"","firstName":"Lisha","middleName":"","lastName":"Song","suffix":""},{"id":638482662,"identity":"69bbf103-7f90-466f-a62f-39e287641fdc","order_by":1,"name":"Ru Chen","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Ru","middleName":"","lastName":"Chen","suffix":""},{"id":638482663,"identity":"8f31625a-beb5-486f-a74a-cb5ece7c3a05","order_by":2,"name":"Lingjian Gui","email":"","orcid":"","institution":"Guangxi Botanical Garden of Medicinal Plants","correspondingAuthor":false,"prefix":"","firstName":"Lingjian","middleName":"","lastName":"Gui","suffix":""},{"id":638482664,"identity":"87efd7d7-e4ce-437c-b816-b0f1e2485c63","order_by":3,"name":"Wenjing Liang","email":"","orcid":"","institution":"Guangxi Botanical Garden of Medicinal Plants","correspondingAuthor":false,"prefix":"","firstName":"Wenjing","middleName":"","lastName":"Liang","suffix":""},{"id":638482665,"identity":"740973dc-defd-4b30-b048-aac385449654","order_by":4,"name":"Cuihong Yang","email":"","orcid":"","institution":"Guangxi Botanical Garden of Medicinal Plants","correspondingAuthor":false,"prefix":"","firstName":"Cuihong","middleName":"","lastName":"Yang","suffix":""},{"id":638482666,"identity":"019b1063-df65-4245-9793-5574189c38e1","order_by":5,"name":"Yuqiong Li","email":"","orcid":"","institution":"Guangxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuqiong","middleName":"","lastName":"Li","suffix":""},{"id":638482667,"identity":"3164cf41-30df-4108-9c9b-d79425765636","order_by":6,"name":"Ya Qin","email":"","orcid":"","institution":"Guangxi Botanical Garden of Medicinal Plants","correspondingAuthor":false,"prefix":"","firstName":"Ya","middleName":"","lastName":"Qin","suffix":""},{"id":638482668,"identity":"13a0dcd5-c6c4-44a9-8595-00de8cdbf3eb","order_by":7,"name":"Jine Fu","email":"","orcid":"","institution":"Guangxi Botanical Garden of Medicinal Plants","correspondingAuthor":false,"prefix":"","firstName":"Jine","middleName":"","lastName":"Fu","suffix":""},{"id":638482669,"identity":"89633f6d-9cc2-4eb5-b0ee-c99309355939","order_by":8,"name":"Ni Jiang","email":"","orcid":"","institution":"Guangxi Botanical Garden of Medicinal Plants","correspondingAuthor":false,"prefix":"","firstName":"Ni","middleName":"","lastName":"Jiang","suffix":""},{"id":638482670,"identity":"ccc23875-a4ed-4860-bc96-70e95b811dbf","order_by":9,"name":"Huijie Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIie2QvUoDQRCAd1lYm0XbWTZ6r7BH4BQs9FHWxm2SVs4uEphrBNuA4mMEywkHqe4BBJuzsbcwnCDqnlrY3CWl4H4wUwzzMT+MRSJ/EQjBJ0zxKWME+cee3CpoY4XXB5UYbqvKbaS0CHuO4uQWjm2vkVxPnx5f7sqBKMQS7qX0CMyxJp93KvxmuT/crcqwmDzVM6XGaC6IX1YPnYoAlxmNraIyAwBjHJATHLsVCX71o+yszJu1XoKzvYqCUaafv6cIC865tQrA6Mxw9O0taQ1EKYYnL/puSWZ+rl/x8Ci9KmvS75QkRbGom7xb+XqBCimd/KpQb3+AN+24dV2RSCTyf/kE6WdTit9yuRUAAAAASUVORK5CYII=","orcid":"","institution":"Guangxi Botanical Garden of Medicinal Plants","correspondingAuthor":true,"prefix":"","firstName":"Huijie","middleName":"","lastName":"Zhang","suffix":""},{"id":638482671,"identity":"30f6ebc2-5a9d-4255-b9d7-5efe055af1a6","order_by":10,"name":"Shugen Wei","email":"","orcid":"","institution":"Guangxi Botanical Garden of Medicinal Plants","correspondingAuthor":false,"prefix":"","firstName":"Shugen","middleName":"","lastName":"Wei","suffix":""},{"id":638482672,"identity":"c890a41e-7a71-4993-a8ae-d56d59e2a967","order_by":11,"name":"Lingyun Wan","email":"","orcid":"","institution":"Guangxi Botanical Garden of Medicinal Plants","correspondingAuthor":false,"prefix":"","firstName":"Lingyun","middleName":"","lastName":"Wan","suffix":""}],"badges":[],"createdAt":"2026-04-13 09:27:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9401766/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9401766/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109279624,"identity":"dab85d25-8a74-4370-9c4a-b932efda4676","added_by":"auto","created_at":"2026-05-14 16:35:54","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3155777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe changes observed in mass loss of four endophytic fungalstrains during the degradation of different substrates.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote：A: mulberry bark B: mulberry xylem C: mulberry leaves D: wheat bran.\u003c/p\u003e","description":"","filename":"FIGURE1224.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9401766/v1/1b87a3cee878317bd3f66328.jpg"},{"id":109279625,"identity":"d5289a7f-e8f8-4b59-8104-4d8fa181d98c","added_by":"auto","created_at":"2026-05-14 16:35:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2897285,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe changes observed in the daily average degradation rates of four endophytic fungal strains during the degradation of different substrates.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote：A: mulberry bark B: mulberry xylem C: mulberry leaves D: wheat bran.\u003c/p\u003e","description":"","filename":"FIGURE2224.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9401766/v1/38881d06e561e7ea7d2b99ce.jpg"},{"id":109279626,"identity":"7e21daf5-3f30-4d6b-8046-ff72e63e0832","added_by":"auto","created_at":"2026-05-14 16:35:54","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3115616,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe changes observed in lignin peroxidase of four endophytic fungal strains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote：A: mulberry bark B: mulberry xylem C: mulberry leaves D: wheat bran.\u003c/p\u003e","description":"","filename":"FIGURE3224.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9401766/v1/905115df4be3ba00def5527e.jpg"},{"id":109279627,"identity":"fc65c8d2-e9b9-4f35-b1d6-ee9706a27df7","added_by":"auto","created_at":"2026-05-14 16:35:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2931113,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe changes observed in laccase activities of four endophytic fungal strains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote：A: mulberry bark B: mulberry xylem C: mulberry leaves D: wheat bran.\u003c/p\u003e","description":"","filename":"FIGURE4224.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9401766/v1/7c625bc09d96e883f0a911e5.jpg"},{"id":109296419,"identity":"cd5d9528-7ebc-4a31-8692-0a98c3c96305","added_by":"auto","created_at":"2026-05-15 08:46:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3190133,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe changes observed in manganese peroxidase activities of four endophytic fungalstrains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote：A: mulberry bark B: mulberry xylem C: mulberry leaves D: wheat bran.\u003c/p\u003e","description":"","filename":"FIGURE5224.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9401766/v1/f7c779e578614352750b173f.jpg"},{"id":109296363,"identity":"bc3f3427-d9ea-4cdd-866b-b38ad0777a39","added_by":"auto","created_at":"2026-05-15 08:46:39","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3186595,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe changes observed in cellulase activities of four endophytic fungalstrains.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNote：A: mulberry bark B: mulberry xylem C: mulberry leaves D: wheat bran.\u003c/p\u003e","description":"","filename":"FIGURE6224.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9401766/v1/88d70677b4759d84024f2322.jpg"},{"id":109405691,"identity":"219009de-26b7-4865-a1c1-58a4e75f1796","added_by":"auto","created_at":"2026-05-17 13:19:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18734310,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9401766/v1/7f37589f-e1fc-4fbf-a42f-6f79a3f6ba84.pdf"},{"id":109297856,"identity":"19985ba4-d7f1-4658-b359-dd2ffb8951aa","added_by":"auto","created_at":"2026-05-15 09:06:54","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":15287,"visible":true,"origin":"","legend":"","description":"","filename":"tables1figure1rawdata224.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9401766/v1/6fb55ab135dcde4902b516e0.xlsx"},{"id":109279629,"identity":"ae43f191-8806-47ed-819b-51b7f5e83c29","added_by":"auto","created_at":"2026-05-14 16:35:54","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":13829,"visible":true,"origin":"","legend":"","description":"","filename":"tables2figure2rawdata224.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9401766/v1/f04b0a8e223b4ab551f52b9c.xlsx"},{"id":109279632,"identity":"1c0b932e-bd40-4df3-bb0d-80841361dd57","added_by":"auto","created_at":"2026-05-14 16:35:54","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":13896,"visible":true,"origin":"","legend":"","description":"","filename":"tables4figure4rawdata224.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9401766/v1/d3d51a6b491ca709a36d476c.xlsx"},{"id":109279633,"identity":"765b3c45-4b9a-478b-9429-76cb4a6370b1","added_by":"auto","created_at":"2026-05-14 16:35:54","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":14040,"visible":true,"origin":"","legend":"","description":"","filename":"tables5figure5rawdata224.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9401766/v1/b35336e1a12347a72bfd7647.xlsx"},{"id":109296047,"identity":"f5036920-0513-4bab-94b2-e3d28384c5d0","added_by":"auto","created_at":"2026-05-15 08:44:51","extension":"xlsx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":13521,"visible":true,"origin":"","legend":"","description":"","filename":"tables6figure6rawdata224.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9401766/v1/65dd848388c3f8c8013709b1.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDegradation characteristics of lignocellulose by endophytic fungi of \u003cem\u003eTaxillus chinensis\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eLignocellulose, the most abundant natural polymer in nature, is composed mainly of cellulose, hemicellulose, and lignin, which interweave to form a naturally compact structural complex [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The annual global production of lignocellulose is approximately 7.5\u0026nbsp;billion tons, which is equivalent to 10 times the annual total energy consumption. However, its high structural stability and resistance to degradation results in a low utilization efficiency for this process. At present, traditional disposal methods of agricultural and forestry wastes, (such as \u003cem\u003ein-situ\u003c/em\u003e returns to fields, industrial raw material processing, feed preparations, and direct combustion, have several limitations. Among them, incineration tends to cause regional haze and increases the difficulty of air pollution control [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, the potential development of efficient and environment-friendly lignocellulose degradation technologies is of great practical significance.\u003c/p\u003e \u003cp\u003eDuring microbial degradation of lignin, various changes occur in its chemical structure, including side-chain oxidation and demethylation. Lignin-degrading enzymes depolymerize the linkages within this macromolecule to ultimately degrade it. The key enzymes involved in degradation by white-rot fungi mainly include lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac) [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The synergistic action of these enzymes can effectively decompose the refractory lignin components [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition to these three major enzymes, other auxiliary ones also participate in the process. LiP (EC 1.11.1.14), also known as diarylpropane oxygenase, contains Fe\u0026sup3;⁺ and was first discovered in the extracellular medium of fungi under nitrogen-limiting conditions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It is a globular helical glycoprotein with 343 amino acid residues and a molecular weight of 38\u0026ndash;46 kDa. It is initially oxidized by H₂O₂ to form compound I, which then reacts with the substrate to generate compound II. In comparison to conventional peroxidases, LiP can oxidize aromatic hydrocarbons with only 2\u0026ndash;3 ether substituents, thereby reducing the stability of the lignin molecular bonds by promoting radical formation, and leads to lignin depolymerization [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMnP (EC 1.11.1.13) is a heme-containing, glycosylated peroxidase with a molecular weight of about 45 kDa. Its amino acid sequence shares high similarity with LiP, and its catalytic process is similar to those of LiP, horseradish peroxidase (HRP), and other peroxidases [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The unique feature of this enzyme is that it uses Mn as an electron donor. Due to the lack of a corresponding tryptophan residue, it cannot directly oxidize non-phenolic lignin but generates highly oxidative Mn\u0026sup3;⁺ through its manganese-binding site [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], which exerts a secondary oxidative effect via diffusion. In addition, it cooperates with other lignin-degrading enzymes to break macromolecular bonds, threby playing an essential role in lignin degradation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLac (EC 1.10.3.2), also known as hydroquinone: oxidoreductase) belongs to the multicopper oxidase family. As a copper-containing polyphenol oxidase, it plays a central role in lignocellulose degradation due to its broad substrate specificity and high catalytic efficiency [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Lac was first found in lacquer trees and later reported in many plants, microorganisms, and animals. Laccases from different sources differ significantly in content, structure, and properties [\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Among them, microbial laccases, including those from fungus and bacteria, which can possess high redox potentials, are dominant enzyme sources for lignocellulose degradation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Lac can catalyze the conversion of various aromatic compounds such as monophenols, diphenols, and polyphenols, while reducing molecular oxygen to water. It is widely used in industrial production, environmental protection, food processing, as well as other fields [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, insufficient catalytic efficiency under harsh conditions limits its practical applications, so the screening of novel strains with extreme environmental tolerances and high enzyme production is crucial [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWhite-rot fungi account for approximately 80% of all wood-rotting fungi and they are widely distributed in forest ecosystems and are natural high-yield lac producers and efficient lignocellulose degraders [\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Their unique degradation system consists of Lac, MnP, and LiP, which can efficiently degrade lignin by catalyzing the cleavage of lignin chemical bonds while maintaining the structural integrity of cellulose, without producing pigments during the process [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. For example, Xu et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] screened \u003cem\u003eAuricularia polytricha\u003c/em\u003e 5.584 from 9 white-rot fungi strains, and the lignin degradation rate reached 36.24% after 15 days of submerged fermentation. Li [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] found that culture and fermentation conditions directly affected lac production and the synergistic effect of lignocellulolytic enzymes in \u003cem\u003eTrametes versicolor\u003c/em\u003e, highlighting the importance of optimizing fungal culture conditions.\u003c/p\u003e \u003cp\u003eFermentation protocols are the key factors that can affect lac yield. Submerged fermentation has long been the mainstream method for enzyme production due to uniform nutrient distribution and sufficient microbial absorption. In contrast, solid-state fermentation (SSF) has gradually become a research hotspot because it is closer to the natural living environment of microorganisms, with simplified technology, low energy consumption, less pollution, and high product recovery rates [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Lignocellulosic materials, including corn stover, wheat bran, and wood sawdust, as SSF substrates not only provide carbon and nitrogen but also mineral elements, such as K and Mg for microbial growth and enzyme synthesis. In addition, they contain natural inducers such as flavonoids and phenols, which can significantly improve fungal lac yield, making them low-cost and high-quality nutrient sources [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eTaxillus chinensis\u003c/em\u003e (DC.) Danser, a traditional Chinese medicinal herb, was first recorded in Shennong\u003csup\u003e'\u003c/sup\u003es Classic of Materia Medica as a high-grade medicine. Its dried leafy stems and branches have the effects of expelling wind-dampness, tonifying the liver and kidneys, strengthening tendons and bones, and the capacity to prevent miscarriages. It is officially listed in the Pharmacopoeia of the People\u003csup\u003e'\u003c/sup\u003es Republic of China (2025 Edition) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Due to low natural parasitism rates and heavy reliance on wild resources, the contradiction between increasing market demand and insufficient supply of \u003cem\u003eT. chinensis\u003c/em\u003e has become a prominent concern. It is urgent to alleviate this problem by improving the parasitism rates, analyzing reproductive mechanisms, and promoting artificial cultivations [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. With respect to its parasitic mechanism, \u003cem\u003eT. chinensi\u003c/em\u003es penetrates the epidermal cell wall, which is composed mainly of lignin and cellulose, of host plants through specialized haustoria to establish a \u0026ldquo;physiological bridge\u0026rdquo; for obtaining water and mineral nutrients while maintaining its own photosynthesis [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Studies have shown that white-rot fungi can invade host plants by degrading their cell walls [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and the role of endophytic fungi in lignocellulose decomposition has been preliminarily explored. However, most of the relevant studies have tended to focus on the enzymatic characteristics of single strains [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies in our laboratory have screened 4 endophytic fungal strains with high lignocellulolytic enzyme production from \u003cem\u003eT. chinensis\u003c/em\u003e parasitizing different hosts [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Based on this, the present study selected 4 different substrates of mulberry bark, xylem, and leaves, as well as wheat bran. We investigated their effects on the kinetic characteristics of their ligninolytic enzymes (lac, MnP, and LiP) and cellulase production in a systematic way. In addition, we assessed the substrate degradation rates of the 4 endophytic fungi. The optimal substrate suitable for the growth of \u003cem\u003eT. chinensis\u003c/em\u003e was determined at the enzymatic level, with the aim of providing high-quality microbial resources and theoretical support for efficient lignin degradation as well as utilization of different substrate resources.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eThe test strains\u003c/h2\u003e \u003cp\u003eFour endophytic fungal strains isolated from \u003cem\u003eTaxillus chinensis\u003c/em\u003e (DC.) Danser were used. The plant material identified as \u003cem\u003eT. chinensis\u003c/em\u003e was collected from Nanning city, Gugnaxi. Taxonomic identification was performed by Yude Peng based on morphological characteristics. A voucher specimen (accession no.SJS-NN-101) was deposited at the Guangxi Key Laboratory of High-Quality Formation and Utilization of Dao-di Herbs, which is publicly accessible. These were strains 4 (\u003cem\u003eColletotrichum\u003c/em\u003e sp.), 15 (\u003cem\u003eNigrospora sphaerica\u003c/em\u003e), N6 (\u003cem\u003eDiaporthe phaseolorum\u003c/em\u003e), and P6 (\u003cem\u003ePestalotiopsis\u003c/em\u003e sp.) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The NCBI accession numbers (ITS and beta-tubulin sequences) of each strain were MZ823601/MZ964759, MZ823600/MZ934421, MZ823599/MZ934420, and MZ823598/MZ934419, respectively. All strains were preserved in a 4 ℃ refrigerator in the plant pathology laboratory of Guangxi Botanical Garden of Medicinal Plants for subsequent use.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSolid-state fermentation of the strains\u003c/h3\u003e\n\u003cp\u003eWheat bran used in the experiment was purchased from Zhonghe Modern Agricultural Development Group Co., Ltd., and its pretreatment was by the method of Tao et al [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] with slight modifications. Mulberry leaves and branch bark epidermis and xylem were obtained from the Research Base of Guangxi Medicinal Botanical Garden. The samples rinsed with clean water and then distilled water, and dried in a 50 ℃ oven for 24 hours. They were then crushed, passed through a 100-mesh sieve to prepare the culture substrates, which were sealed and stored for later use. 2.0 g of each prepared substrate was added to 250 mL Erlenmeyer flasks with 15 mL of 0.5% malt extract and the mixture was autoclaved at 121 ℃ for 30 minutes. After cooling, the test strains were activated and cultured at a constant temperature of 28℃ for 5\u0026thinsp;~\u0026thinsp;7 days, and 6 mm diameter mycelial discs were taken with a sterile puncher and inoculated into the sterilized flasks. The humidity was maintained at 90%, and the flasks were cultured in the dark at 28℃ in an incubator. During the fermentation and culture period, sampling was carried out on days 2, 9, 16, 23, 30 and 44, respectively. Each treatment carried out in triplicate.\u003c/p\u003e\n\u003ch3\u003eEnzyme solution preparation and calculation of the average degradation rate\u003c/h3\u003e\n\u003cp\u003eTotal organic matter (TOM) mass loss and average (MLR) degradation rate were used as evaluation indicators to assess the degradation efficiency of the 4 strains on 4 substrates (wheat bran as well as mulberry leaves, bark and xylem) and these were determined based on Hao et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] with slight modifications. After each sampling, 10 mL of sodium acetate buffer solution (pH 6.0, 50 mmol/L) was pre-cooled to 4℃ in an Erlenmeyer flask, and extracted at 28℃ and 120 r\u0026middot;min⁻\u0026sup1; for 20 minutes. Dried Whatman No.1 filter paper was weighed, and the mixture was filtered with this filter paper. The filtrate was collected and centrifuged at 4℃ and 10 000 \u0026times; g for 10 minutes. The supernatant was used as the crude enzyme extract for the subsequent determination of various enzyme activities. The precipitate from the centrifuge tube was combined with the filter paper filtrate, placed on the original filter paper, and dried at 72 ℃ and weighed to calculate MLR.\u003c/p\u003e \u003cp\u003eSample MLR (%) = (Initial dry mass of sample - Dry mass of sample after decomposition) / Initial dry mass of sample \u0026times; 100%.\u003c/p\u003e \u003cp\u003eAverage degradation rate (%)\u0026thinsp;=\u0026thinsp;TOM MLR / Culture days.\u003c/p\u003e\n\u003ch3\u003eDetermination of enzyme activities\u003c/h3\u003e\n\u003cp\u003eThe activity of lac was determined by the method of Buswell et al. [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was the reaction substrate and it was carried out at 30 ℃. The amount of enzyme required to catalyze the oxidation of 1\u0026micro;mol ABTS per minute was defined as 1 enzyme activity unit (U) and this was measured by the absorbance change at 420 nm. The molar extinction coefficient of ABTS at 420 nm was 36000 L/(mol\u0026middot;cm). LiP activity was determined by Tien et al. [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] method with veratryl alcohol as the substrate and this was performed at 30℃. The amount of enzyme required to change the absorbance at 310nm change in 1mL of the reaction mixture to reach 0.1 per minute was defined as 1 enzyme activity unit (U), resulting in the production of veratraldehyde. The activity of MnP was determined by the method of Orth et al. [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and used the changes in absorbance of the phenol red at 610 nm. The amount of enzyme required for the absorbance of 1mL of the reaction solution to increase by 0.1 unit per minute was defined as 1 enzyme activity unit (U\u0026middot;mL⁻\u0026sup1;).\u003c/p\u003e \u003cp\u003eThe activity of cellulase was determined by the 3,5-dinitrosalicylic acid method. The enzyme activity was calculated by measuring the content of reducing sugars produced after cellulase degraded cellulose. A standard curve was constructed with 10 mg of anhydrous glucose standard (loss on drying\u0026thinsp;\u0026lt;\u0026thinsp;0.2%) from the kit. This was dissolved in 1 mL of ultrapure water to prepare a glucose stock solution, which was then diluted to 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, and 0 mg\u0026middot;mL⁻\u0026sup1;, respectively. 1 mL of fermentation broth was ultrasonicated ice bath conditions to lyse the cells and centrifuged at 4 ℃ and 8000\u0026times;g for 10 minutes. The supernatants were stored on ice and 50\u0026micro;L samples were used for determinations for measurements by addition of 150\u0026micro;L of the 3,5-dinitrosalicylic acid reagent. The absorbance at 540 nm [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] was measured and the cellulase activity was calculated by using the standard curve.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were analyzed by one-way analysis of variance (ANOVA) using SPSS 19.0 software, and the results were expressed as means\u0026plusmn;standard deviations. The homogeneity of variance test was first performed on each group of data and Origin 2018 software was used for plotting the data.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eThe mass loss of different substrates of four endophytic fungal strains\u003c/h2\u003e\n \u003cp\u003eThe mass loss rates (MLRs) of the 4 strains varied significantly across the 4 substrates, mulberry bark, xylem, and leaves, as well as wheat bran, used (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The MLR of each strain on mulberry bark displayed an overall trend of first increasing and then decreasing with prolonged incubation times (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Strain 15 showed the best degradation performance, with its MLR reaching 50.50% at 30 d of fermentation, which was notably higher than those of the control and the other three strains. The MLRs of the 4 strains on mulberry xylem increased gradually with increased incubation times. At 44 d, strains P6 and 4 achieved significantly higher MLR rates than Strain N6 and the control, at 34.50 and 32.50%, respectively (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The MLRs of the strains on mulberry leaves showed a fluctuating pattern of increase to decrease over time (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). At 44 days, Strains P6 and 15 both showed the highest degradation efficiencies, with a MLR of 62.50%, exceeding the other two strains and the control. The MLRs of the strains using wheat bran as substrate increased continuously with incubation times (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Except for the control and Strain N6, Strains 15, P6, and 4 exhibited higher MLRs at 44 d, and reached 55.50, 48.50, and 43.00%, respectively. Overall, Strain 15 showed the highest MLR on mulberry bark and leaves, as well as wheat bran. Strain P6 presented the optimal degradation performance on mulberry xylem and leaves, and wheat bran. Strain 4 achieved the highest MLRs on mulberry xylem and wheat bran. These results suggested that Strains 15, P6, and 4 possess high degradation capacity on diverse substrates.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eDaily average degradation rates of the four strains with different substrates\u003c/h3\u003e\n\u003cp\u003eThe average daily degradation rates of the 4 strains on the 4 substrates used all showed a decreasing trend with increased incubation times (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). However, there were significant differences in the peak times and rates of degradation of the different strains on each substrate used (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Using mulberry bark substrate, the average daily degradation rates of strains P6 and 4 decreased gradually with time (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) but were at relatively high levels from 9\u0026thinsp;~\u0026thinsp;16 days, ranging from 2.03 to 2.67%, and these were significantly different from other time periods (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The average daily degradation rate of strain N6 reached its peak of 2.56% at 16 days, which was significantly higher than that in other time periods (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). That of Strain 15 was 23 d, with the maximum of 1.91%. With mulberry xylem as substrate, except for Strain N6, the average daily degradation rates of the other three strains all decreased with the extension of time and Strain 4 reached the highest average daily degradation rate (3.17%) at 9 days. Strain P6 maintained a relatively high degradation level during 9\u0026thinsp;~\u0026thinsp;16 days, with rates ranging from 1.56 to 1.61%, which was significantly different from other time periods (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The peak average daily degradation rate of Strain 15 was also at 9 days, with a maximum of 2.00%. The average daily degradation rates of the 4 strains on mulberry leaf substrate all showed a decreasing trend with time, but there were differences observed in the peaks (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Strain 4 reached the highest average daily degradation rate (4.39%) at 9 days and Strains 15 and P6 maintained relatively high degradation levels during 9\u0026thinsp;~\u0026thinsp;16 days, with rate ranging from 3.28\u0026thinsp;~\u0026thinsp;3.38% and 2.28\u0026thinsp;~\u0026thinsp;2.39%, respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Strain N6 reached a relatively high degradation level during 16\u0026thinsp;~\u0026thinsp;23 days, with rates of 2.07\u0026ndash;2.19% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). With wheat bran substrate, the average daily degradation rates of the 4 strains all decreased continuously with time, and the highest average daily rate of all the strains was at 9 days. These were 3.56, 3.78, 4.28 and 3.00%, respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eTherefore, all the strains reached relatively high average daily degradation rates on wheat bran substrate at 9 d, which were significantly different from other time periods (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The average daily degradation rates of Strain 4 on mulberry xylem and leaves as substrates both reached their peaks at day 9, and these were 3.17 and 4.39%, respectively, (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), reflecting their strong short-term degradation advantage.\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eChanges in LiP production by the four strains\u003c/h2\u003e\n \u003cp\u003eWhen mulberry bark was used as the substrate, the LiP activities of the P6, 4, 15, and N6 strains all showed a trend of first increasing and then decreasing as the time of fermentation increased (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The activities reached their peaks on the 9th day, and were 6.77, 2.00, 6.94, and 4.17 U/mL, respectively, all of which were higher than that of the control group. When mulberry xylem was used as the substrate, the trend of LiP activity of the 4 strains was similar to that of when mulberry bark was the substrate, and the activities also reached the peak at the 9 days, and were 6.49, 5.14, 5.96, and 3.89 U/mL, respectively (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Except for Strain N6, the enzyme activity peaks of the other three strains were all higher than the control group, and none was detected on the second day of culture. When mulberry leaves were used as the substrate, the LiP activities of the 4 strains still showed a trend of first increasing and then decreasing, reaching the peak on day 9, and were 3.33, 21.15, 2.45, and 1.51 U/mL, respectively (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Among them, Strain 4 had the highest enzyme activity peak, and except for strain N6, the peaks of the other strains were all higher than that of the control group. With wheat bran as the substrate, the LiP activities of the 4 strains was also consistent and reached the highest on day 9, and were 7.01, 23.52, 4.5, and 2.19 U/mL, respectively (Fig. \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). With this substrate, Strain 4 had the highest enzyme activity peak, and the peaks of all 4 strains were also higher than that of the control group.\u003c/p\u003e\n \u003cp\u003eTo summarize, under the 4 different substrates and culture times used the LiP activities of the 4 endophytic fungal strains all showed a consistent trend of \u0026quot;first increasing and then decreasing\u0026quot;, and peaked on day 9 of culture. Among them, when cultured with mulberry leaves and wheat bran the enzyme activity peaks of Strain 4 were significantly higher than those of the other two substrates and other strains while using the same substrate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eChanges in laccase production by the four strains used\u003c/h2\u003e\n \u003cp\u003eWhen mulberry bark was used as the substrate, the lac activities of the P6, 4, 15, and N6 strains all showed a trend of first increasing and then decreasing (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Except for strain 15 and the control group, the other strains all reached their peak enzyme activities on the 9th day of culture, with the peak values being 21.88, 3.19, 1.10, and 11.73 U/mL respectively. The peak laccase activity of Strain 15 appeared on the 23rd day, with a value of 62.80 U/mL. When mulberry xylem was used as the substrate, the lac activities of the 4 strains used also showed a trend of first increasing and then decreasing (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Except for strains P6 and 4, the other ones all reached their peak activities on the 9th day, with values of 7.35, 9.09, 31.30, and 30.86 U/mL respectively. With mulberry leaves as the substrate, there were differences in the peak lac activities among the 4 strains (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Strain P6 reached its peak on day 9, with an activity of 28.94 U/mL. The peak activities of Sstrains 15 and N6 both appeared on the 16th day, and were 5.08 and 3.91 U/mL respectively. and that of Strain 4 was on the 23rd day, at 6.45 U/mL. In addition, the lac activities of all 4 strains were higher than the control group. When wheat bran was used as the substrate, the lac activities of the 4 strains all showed a trend of first increasing and then decreasing, and the peaks occurred on day 16, with peak values of 108.39, 73.20, 11.99, and 68.64 U/mL, respectively (Fig. \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In summary, there were differences in the lac activities of the 4 strains when using different substrates and culture times, and the peak enzyme activities were different.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eChanges in MnP production by the four strains\u003c/h2\u003e\n \u003cp\u003eWith mulberry bark, xylem, and leaves and wheat bran as substrates, the MnP activities of P6, 4, 15, and N6 all showed trends of first increasing and then decreasing (Figs. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). For mulberry bark, the 4 strains reached their peak activities on day 30, with the peak values being 11.76, 45.64, 45.21, and 78.45 U/mL, respectively, and Strain N6 had the highest activity (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). For mulberry xylem, except for Strains N6 and 15, the others (P6 and 4) both reached their peak enzyme activities on the 30th day, with peak values of 51.75 and 38.08 U/mL, respectively (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). When mulberry leaves were used, the peak enzyme activity of strain P6 appeared on day 30, while that of Strain 4 reached the peak on the 16th day, with values being 36.26 and 118.32 U/mL, respectively (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). With wheat bran as the substrate, the MnP activities reached their peak activities on day 30, with values of 62.28, 55.05, 84.33, and 111.69 U/mL respectively, and strain N6 had the highest activity (Fig. \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In summary, when using different substrates and culture times, the MnP activities of the 4 strains all showed an increasing and then a decreasing trend. Among them, only when Strain 4 used mulberry leaves as the substrate, the peak MnP activity appeared on the 16th day, and that occurred on day 30 for the other strains.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eChanges in cellulase production by the four strains\u003c/h2\u003e\n \u003cp\u003eWhen mulberry bark, xylem, and leaves were used as substrates, the cellulase activities of Strains P6, 4, 15, and N6 all showed trends of first increasing and then decreasing (Figs. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). For mulberry bark, with the exceptions of P6 and the control group, the other three strains all reached their peak cellulase activities on the 2nd day of culture, with peak values being 0.19, 0.37, and 0.21 U/mL respectively (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The peak of Strain P6 appeared on day 23, with a value of 0.39 U/mL. With mulberry xylem and the exception of N6, the other three strains all reached their peak enzyme activities on the 2nd day, with the peak values being 0.49, 0.35, and 0.67 U/mL respectively (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The peak activity of strain N6 was 0.16 U/mL and appeared on day 9. When mulberry leaves were used as the substrate, the cellulase activities of the 4 strains reached their peaks on the 2nd day, with values of 0.14, 0.15, 0.23, and 0.20 U/mL respectively (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). With wheat bran as the substrate, except for Strain 15, the cellulase activities of P6, 4, and N6 all showed a trend of first increasing and then decreasing, and all reached their peaks on the 2nd day, with values of 0.27, 0.20, and 0.26 U/mL respectively. That of Strain 15 was on day 9, with a value of 0.28 U/mL (Fig. \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In summary, there were significant differences in the changes of cellulase-producing activities of the 4 endophytic fungi when using different substrates and culture times.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have shown that the composition of the fermentation medium is one of the key factors affecting fermentation efficiency [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], and the differences in enzyme types and their production times also led to differences in the lignin degradation ability of strains on different substrates [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In this study, four endophytic fungal strains isolated from \u003cem\u003eT. chinensis\u003c/em\u003e. These were Strains 4 (\u003cem\u003eColletotrichum\u003c/em\u003e sp.), 15 (\u003cem\u003eN. sphaerica\u003c/em\u003e), N6 (\u003cem\u003eD. phaseolorum)\u003c/em\u003e, and P6 (\u003cem\u003ePestalotiopsis\u003c/em\u003e sp.) and were inoculated separately onto the 4 substrates of mulberry, leaves, and xylem, as well as wheat bran to undergo the process of solid-state fermentation. The changes in TOM mass loss, average degradation rates, and lignocellulose-degrading enzyme activities caused by the strains during the degradation of different culture substrates were determined.\u003c/p\u003e \u003cp\u003eOur studies showed that all 4 fungi exhibited high lignin degradation levels when wheat bran was used as the substrate, and the highest rate reaching 55.50% under optimal culture conditions. Guo et al. [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] found that a strain of \u003cem\u003ePseudomonas guanguanensis\u003c/em\u003e isolated from lignin-degrading bacteria the could secrete LiP, MnP, and lac, with a lignin degradation rate of about 38% after 3 days of fermentation. Zhou et al. [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] found that \u003cem\u003eAlternaria eichhhorniae\u003c/em\u003e Strain G6 and \u003cem\u003eTrichoderma shangrilaense\u003c/em\u003e Strain G59 could both produce LiP and lac, and the enzyme-producing capacity of the composite strain was significantly improved, with a degradation rate of 54.53% for corn stover. Current studies have shown that the degradation efficiency of composite microbial communities was significantly higher than that of individual strains. In the natural ecosystems, lignin degradation does not primarily result from the independent action of specific strains, but rather it stems from the inhibition and synergism of communities of microorganisms in the same habitat. Therefore, future studies should attempt to construct composite microbial communities, explore their degradation effects on lignocellulose of different substrates, and analyze the optimal conditions required by different lignocellulose-degrading bacteria, including temperature, pH, light, and types and concentrations of carbon and nitrogen sources.\u003c/p\u003e \u003cp\u003eLignin degradation relies on the synergistic effects of lac, MnP, and LiP. These enzymes can break the chemical bonds in the lignin structure to achieve degradation, and lac plays a leading role in the decomposition process. Its activity can be used as an important indicator reflecting the overall activity of the lignin-degrading enzyme system. During the 44-day full cultivation period, when Strains 4 (\u003cem\u003eColletotrichum\u003c/em\u003e sp.), 15 (\u003cem\u003eN. sphaerica\u003c/em\u003e), P6 (\u003cem\u003ePestalotiopsis\u003c/em\u003e sp.), and N6 (\u003cem\u003eD. phaseolorum\u003c/em\u003e) were fermented with wheat bran as the lignocellulosic biomass substrate, the activities of lac and cellulase were significantly higher than those of the control group. Zhao et al. [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] showed that increased lac concentrations were needed for mycelial degradation of lignin. They showed that the laccase activity of the white-rot fungus, \u003cem\u003ePleurotus eryngii\u003c/em\u003e, was the highest in the culture substrate with the highest lignin content, which is consistent with our results. When wheat bran was used as the substrate, cellulase activity increased significantly on the second day of cultivation, while lac activity reached a high level on day 9. Previous studies [\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] have reported that this phenomenon is closely related to the non-selective lignin degradation mode of white-rot fungi, which consumes large amounts of cellulose. In addition, the reducing sugars and other substances released by cellulase degradation of cellulose can provide the necessary substrates and a suitable reaction environment for lac to act, thereby indirectly regulating its activity. Several others have also reported [\u003cspan additionalcitationids=\"CR62\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] significantly improved the lignin degradation rates by adding wheat bran to the medium, confirming that this substrate can induce the enhancement of enzyme-producing capacity by a strain, which is consistent our findings.\u003c/p\u003e \u003cp\u003eWhen using lac activity as the evaluation index, strain P6 had the highest activity (108.39 U/mL) on the wheat bran substrate. Existing literature reports show that \u003cem\u003ePestalotiopsis\u003c/em\u003e sp. of fungi possess high efficiencies for the production of lac and cellulase, which can effectively degrade forest litter [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. This further confirms the excellent enzyme-producing and degradation characteristics of strain P6 in this study. During the early stages of fermentation, the enzyme activities of Strains N6, 4, 15, and P6 on different substrates all showed a gradual upward trend, but as the cultivation times increase, they tend to decrease. This is mainly because the nutrients and oxygen in the substrates were gradually consumed, leading to restricted microbial growth and even death, thereby reducing enzyme production efficiency. The buildup of inhibitor metabolites is another possible explanation for this phenomenon [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, the 4 endophytic fungal strains isolated from \u003cem\u003eT. chinensis\u003c/em\u003e screened in this study are all able to degrade lignin efficiently, providing new high-quality strain resources for biological lignin degradation. This can have important theoretical and practical significance for the development of bioenergy and the sustainable utilization technology of lignocellulosic resources.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe degradation efficiency of the 4 screened endophytic fungal strains from T. chinensis on different lignocellulosic substrates, as well as the activities of key degrading enzymes such as lignin peroxidase and laccase, all showed significant strain specificity, substrate specificity, and temporal dynamic characteristics. The results of this study can provide a theoretical basis and practical reference for the excavation of high-efficiency lignin-degrading strain resources and the optimization of their fermentation conditions.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLiP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elignin peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMnP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emanganese peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLac\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elaccase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMLRs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ethe mass loss rates\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTOM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etotal organic matter\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eABTS\u0026thinsp;\u0026minus;\u0026thinsp;2,2'\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eazino-bis(3-ethylbenzothiazoline-6-sulfonic acid)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ekDa\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ekilodalton\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehorseradish peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSSF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esolid-state fermentation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eStrains 4 (\u003cem\u003eColletotrichum\u003c/em\u003e sp.), 15 (\u003cem\u003eNigrospora sphaerica\u003c/em\u003e), N6 (\u003cem\u003eDiaporthe phaseolorum\u003c/em\u003e), and P6 (\u003cem\u003ePestalotiopsis\u003c/em\u003e sp.) were kept in the Plant Pathology Department of the Guangxi Botanical Garden of Medicinal Plants and we obtained written consent from them to use these fungal strains in our studies. The experimental methods conducted in this study complied with all the current Chinese laws and regulations.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eAuthors\u0026rsquo; information\u003c/h2\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eGuangxi Botanical Garden of Medicinal Plants, Nanning, China, \u003csup\u003e2\u003c/sup\u003eGuangxi Key Laboratory for High-Quality Formation and Utilization of Dao-di Herbs, Nanning, China and \u003csup\u003e3\u003c/sup\u003eGuangxi University of Chinese Medicine, Nanning 530000, China.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Guangxi Natural Science Foundation of China (No. 2024GXNSFAA010371 and 2026GXNSFAA00641220) and the Multidisciplinary Innovative Team for Traditional Chinese Medicine of Guangxi (No. GZKJ2305).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLisha Song implemented the experiments, analyzed and interpreted the data and completed the article writing. Lingjian Gui and Wenjing Liang participated in the experiments and collected the samples. Ni Jiang, Ru Chen and Cuihong Yang integrated information into the tables. Shugen Wei and Jine Fu reviewed and finalized manuscript. Yuqiong Li and Ya Qin generated the figures. Lingyun Wang and Huijie Zhang edited the manuscript. The final manuscript was approved by all authors who agreed to be accountable for the content of this work.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful to Dr. Dev Sooranna of Imperial College London, for English editing of the manuscript.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eITS and beta-tubulin sequences of 4 strains, 4,15, N6, and P6, were submitted to the GenBank database and the NCBI accession numbers were MZ823601/MZ964759, MZ823600/MZ934421, MZ823599/MZ934420, and MZ823598/MZ934419, respectively. Other data generated and analyzed during this study are included in the article and its supplementary information files: (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/search/all/?term=MZ823600;https://www.ncbi.nlm.nih.gov/pmc/?term=MZ823598;https://www.ncbi.nlm.nih.gov/search/all/?term=MZ823601;https://www.ncbi.nlm.nih.gov/search/all/?term=MZ823599\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/search/all/?term=MZ823600;https://www.ncbi.nlm.nih.gov/pmc/?term=MZ823598;https://www.ncbi.nlm.nih.gov/search/all/?term=MZ823601;https://www.ncbi.nlm.nih.gov/search/all/?term=MZ823599\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLiu M, Xu LX, Yin ZX, He DM, Zhang YJ, Liu CQ. Harnessing the potential of exogenous microbial agents: a comprehensive review on enhancing lignocellulose degradation in agricultural waste composting. Arch Microbiol. 2025;207:51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSurwase SV, Patil SA, Srinivas S, Jadhav JP. Interaction of small molecules with fungal laccase: a surface plasmon resonance-based study. Enzym Microb Technol. 2016;82:110\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan ML, Zhao YL, Liu TY, Bian LS, Li ZH, Li MX, Li J, Yang XQ, An Q. Effect of alkali lignin on laccase activity and mycelial biomass of \u003cem\u003eFlammulina velutipes\u003c/em\u003e: Alkali lignin on laccase activity and mycelial biomass. BioResources. 2025;20:2243\u0026ndash;58.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTien M, Kirk TK. Lignin-degrading enzyme from \u003cem\u003ePhanerochaete chrysosporium\u003c/em\u003e: purification, characterization, and catalytic properties of a unique H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-requiring oxygenase. P Natl Acad Sci USA. 1984;81:2280\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHussaan M, Amna, Javed MT, Akram MS, Ali S. Physiological and molecular basis of bioremediation of micropollutants. Handbook of Bioremediation. Academic Press, 2021; 447\u0026ndash;464.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRencoret J, Pereira A, Del R\u0026iacute;o JC, Mart\u0026iacute;nez AT, Guti\u0026eacute;rrez A. Laccase-mediator pretreatment of wheat straw degrades lignin and improves saccharification. Bioenerg Res. 2016;9:917\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong DWS. Structure and action mechanism of ligninolytic enzymes. App Biochem Biotech. 2009;157:174\u0026ndash;209.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHofrichter M. Review: lignin conversion by manganese peroxidase (MnP). Enzyme Microb Tech. 2002;30:454\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrth AB, Denny M, Tien M. Overproduction of lignin-degrading enzymes by an isolate of \u003cem\u003ePhanerochaete chrysosporium\u003c/em\u003e. Appl Environ Microb. 1991;57:2591\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKersten PJ, Kalyanaraman B, Hammel KE, Reinhammar B, Kirk TK. Comparison of lignin peroxidase,horseradish peroxidase and laccase in the oxidation of methoxybenzenes. Biochem J. 1990;268:475\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSundaramoorthy M, Kishi K, Gold MH, Poulos TL. The crystal structure of manganese peroxidase from \u003cem\u003ePhanerochaete chrysosporium\u003c/em\u003e at 2.06-A resolution. J Biol Chem. 1994;269:32759\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSundaramoorthy M, Kishi K, Gold MH, Poulos TL. Crystal structures of substrate binding site mutants of manganese peroxidase. J Biol Chem. 1997;272:17574\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzer A, Ay Sal F, Belduz AO, Kirci H, Canakci S. Use of feruloyl esterase as laccase-mediator system in paper bleaching. Appl Biochem Biotech. 2020;190:721\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeynaud Kieffer Curran LMC, Pham LTM, Sale KL, Simmons BA. Review of advances in the development of laccases for the valorization of lignin to enable the production of lignocellulosic biofuels and bioproducts. Biotechnol Adv. 2022;54:107809.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshida HLXIII. Chemistry of lacquer (Urushi). Part I. Communication from the Chemical Society of Tokio. J Chem Soc 1883; 43: 472\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://10.1039/ct8834300472\u003c/span\u003e\u003cspan address=\"https://10.1039/ct8834300472\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaldrian P. Fungal laccases - occurrence and properties. Fems Microbiol Rev. 2006;30:215\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOztat K, Yavuz AA, Is\u0026ccedil;en CF. Optimization studies on laccase activity of Proteus mirabilis isolated from treatment sludge of textile industry factories. Braz J Microbiol. 2024;55:1231\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScheibel DM, Gitsov IPI, Gitsov I. Enzymes in Green synthetic chemistry: laccase and lipase. Molecules. 2024;29:989.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta A, Jana AK. Production of laccase by repeated batch semi-solid fermentation using wheat straw as substrate and support for fungal growth. Bioproc Biosyst Eng. 2019;42:499\u0026ndash;512.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Y, Ma HF, Cao YJ, Si J, Cui BK. Advances on properties production, purification and immobilization of fungal laccase. Biotechnol Bull. 2019;35:1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Rajhi AMH, Bazaid AS, Abdulfattah AM, Abdelghany TM, Shater ARM, Selim S. Evaluation of lignocellulatic activity of enzymes from microwave-irradiated Pleurotus sajor-caju cultivated with wheat straw. BioResources. 2024;19(4):8882\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRamirez LS, Dragotakes Q, Casadevall A. A food color-based colorimetric assay for \u003cem\u003eCryptococcus neoformans\u003c/em\u003e laccase activity. Microbiol Spectr. 2024;12:1\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePang S, Wu Y, Zhang X, Li B, Ouyang J, Ding M. Immobilization of laccase via adsorption onto bimodal mesoporous Zr-MOF. Process Biochem. 2016;51:229\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgrawal K, Chaturvedi V, Verma P. Fungal laccase discovered but yet undiscovered. Bioresour Bioprocess. 2018;5:4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai YC. Hymenochaetaceae (Basidiomycota) in China. Fungal Divers. 2010;45:131\u0026ndash;343.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDai YC, Yang ZL, Cui BK, Wu G, Yuan HS, Zhou LW, He SH, Ge ZW, Wu F, Wei YL, Yuan Y, Si J. Diversity and systematics of the important macrofungi in Chinese forests. Mycosystema. 2021;40:770\u0026ndash;805.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu F, Man XW, Tohtirjap A, Dai YC. A comparison of polypore funga and species composition in forest ecosystems of China, North America, and Europe. Ecosyst. 2022;9:100051.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao H, Wu YD, Yang ZR, Liu HG, Wu F, Dai YC, Yuan Y. Polypore funga and species diversity in tropical forest ecosystems of Africa, America and Asia, and a comparison with temperate and boreal regions of the Northern Hemisphere. Ecosyst. 2024;11:100200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCui BK, Li HJ, Ji X, Zhou JL, Song J, Si J, Dai YC. Species diversity, taxonomy and phylogeny of Polyporaceae (Basidiomycota) in China. Fungal Diver. 2019;97:137\u0026ndash;302.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeigiserova DA, Bourgine J, Thomsen M. Closing the loop of cereal waste and residues with sustainable technologies: an overview of enzyme production via fungal solid-state fermentation. Sustain Prod Consump. 2021;27:845\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang CG, Dai YC, Kout J, Gates GM, Liu HG, Yuan Y, Vlas\u0026aacute;k J. Multi-gene phylogeny and taxonomy of Physisporinus (Polyporales, Basidiomycota). Mycosphere. 2024;15:1455\u0026ndash;521.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu AM, Li L, Ma S. Screening of lignin degrading enzyme high-producing strain from white rot fungi. J Henan Univ Technol (Natural Sci Edition). 2020;41:78\u0026ndash;8289.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Gao Y, Zhao Y, Lou HY, Wang XW, Niu YW, Yang CM, Zhao RY. Optimization of the culture medium for laccase production by Trametes versicolor and activity detection of fermentation broth in degrading zearalenone. J Henan Univ Technol (natural Sci edition). 2024;45(5):87\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-Couto S. A promising inert support for laccase production and decolouration of textile wastewater by the white-rot fungus \u003cem\u003eTrametes pubescesns\u003c/em\u003e. J Hazard Mater. 2012;233:158\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePostemsky PD, Bidegain MA, Gonz\u0026aacute;lez-Matute R, Figlas ND, Cubitto MA. Pilot-scale bioconversion of rice and sunflower agro-residues into medicinal mushrooms and laccase enzymes through solid-state fermentation with Ganoderma lucidum. Bioresource Technol. 2017;231:85\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJasinska A, G\u0026oacute;ralczyk-Binkowska A, Sobon A, Dlugonski J. Lignocellulose resources for the Myrothecium roridum laccase production and their integrated application for dyes removal. Int J Environ Sci Te. 2019;16(8):4811\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiong X, Li P, Xiang Z, Huang J, Wang J. Influence of different lignocellulosic materials on laccase activity in liquid fermentation of Chinese low- temperature straw mushroom (\u003cem\u003eVolvariella brumalis\u003c/em\u003e). BioResources. 2024;19:8608\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCabrera R, L\u0026oacute;pez-Pe\u0026ntilde;a D, Asaff A, Esqueda M, Valenzuela-Soto EM. Bioavailability of compounds susceptible to enzymatic oxidation enhances growth of shiitake medicinal mushroom (Lentinus edodes) in solid-state fermentation with vineyard prunings. Int J Med Mushrooms. 2018;20:291\u0026ndash;303.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNational Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China 2025. Volume I. Beijing: China Medical Science and Technology; 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu J. Study on anatomical structure and histochemistry of \u003cem\u003eTaxillus sutchuenensis\u003c/em\u003e (Loranthaceae). Hubei: Yangtze University; 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLian T, Guo YX, Tian LY, Liu J, Xu JZ, Yang H, Qiu HL, Qin CS. Histoanatomical observation on the process of \u003cem\u003eTaxillus chinensis\u003c/em\u003e infecting \u003cem\u003eCamellia semiserrata\u003c/em\u003e. Plant Prot. 2024;50:64\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi L. Study on the quality of \u003cem\u003eTaxilli herba\u003c/em\u003e resources from Guangxi based on metabolomics. Chengdu: Chengdu University of TCM; 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu JL, Cheng YH. Screening and identification of lignin-degrading fungi. Agricultural Sci Eng China. 2024;36:16\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong LS, Wan LY, Wei SG, Pan LM, Huo J, Fu JE, Jiang N. Screening and identification of high-yield lignocellulose-degrading endophytic fungi from \u003cem\u003eTaxillus chinensis\u003c/em\u003e(DC.) seeds. Southwest China J Agricultural Sci. 2023;36:1924\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong LS, Pan LM, Jiang N, Fu JE, Wan LY, Wei SG. Effects of endophytic fungi on parasitic process of \u003cem\u003eTaxillus chinensis\u003c/em\u003e. Sci Rep. 2022;12:7744.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong L, Huo J, Wan L, Pan L, Jiang N, Fu J, Wei S. Lili He. Differences and biocontrol potential of haustorial endophytic fungi from \u003cem\u003eTaxillus Chinensis\u003c/em\u003e on different host plants. BMC Microbiol. 2023;23:128.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTao YJ. Study on the Modification and Application of Wheat Bran Dietary Fibre. Jiangnan: Jiangnan University; 2009.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHao JJ, Song FQ, Tian XJ, Huang F, Zhang P, Zhang ZJ. Decomposition of Pinus massoniana Needle Driven by Deuteromycetes\u0026mdash;Dynamics of Lignocellulolytic Enzymes. Scientia Silvae Sinicae. 2006;42:69\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuswell JA, Cai YJ, Chang S. Effect of nutrient nitrogen and manganese on manganese peroxidase and laccase production by Lentinula edodes. Fems Microbiol Lett. 1995;128:81\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTien M, Kirk TK. Lignin peroxidase of \u003cem\u003ePhanerochaete chrysosporium\u003c/em\u003e. Method Enzymol. 1988;161:238\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrth AB, Royse DJ, Tien M. Ubiquity of lignin-degrading peroxidases among various wood-degrading fungi. Appl Environ Microb. 1993;59:4017\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKe LX, Wang MY, Wu Q. An exploratory study on elevation of the lignin degradation enzyme activity of Pleurotus ostreatus in submerged cultivation and its decolorization rate to direct sky blue 5B. Acta Sci Circum. 2015;35:1449\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSu Y, Liu C, Long Z, Ren H, Guo XH. Improved production of spores and bioactive metabolites from \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e in solid-state fermentation by a rapid optimization process. Probiotics Antimicro. 2019;11:921\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAdejoye OD, Fasidi I. Biodegradation of agro-wastes by some Nigerian white-rot fungi. BioResources. 2009;4:816\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo CS, Ye YJ, Chen C, Yang HF, Liu HL, Ning XP, Liu GP, Cai Y. Screening and identification of a lignin-degrading strain and optimization of its degradation conditions. Feed Ind. 2024;45:113\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou DX, Xu R, Guo HY, Dai HY. Screening and degradation efficiency of low-temperature-tolerant fungal strains for lignocelluose in crop straw. J Northeast Agricultural Univ. 2026;57:74\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao CM, Du F, Zou YJ, Hu QX, Zheng SY. Study on the relationship between mycelial growth and lignin degrading enzymes of Pleurotus eryngii based on different Substrates. Edible Fungi China. 2020;39:109\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShrestha P, Rasmussen M, Khanal SK, Pometto AL, van Leeuwen JH. Solid-substrate fermentation of corn fiber by \u003cem\u003ePhanerochaete chrysosporium\u003c/em\u003e and subsequent fermentation of hydrolysate into ethanol. J Agr Food Chem. 2008;56:3918\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLai GD, Qin CS, Zhao DY, Qiu HL, Yang H, Tian LY, Lian T, Xu JZ. Isolation and screening of cellulose degrading strains. Forestry Environ Sci. 2021;37:24\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang XX, Qi LH, Song JF, Zhang QG, Liu CC, Zhang Q, Song FF, Xu IL. Screening of cellulose and lignin degrading bacteria and their effect on composting of mushroom residue. J Henan Agricultural Sci. 2024;53:66\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLI Y. Remediation of polycyclic aromatic hydrocarbons-petroleum hydrocarboncompound contaminated soil by white rot fungi agents / immobilized enzymes. Beijing: Beijing University of Civil Engineering and Architecture, 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao ZY, Liu YXY, Yao YY, Li HJ. Utilization and modification status of wheat bran nutrient. Cereals Oils. 2020;33:18\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu AM, Li L, Ma Sen, Wang XX. Study on the optimization of laccase fermentation conditions and the degradation of wheat bran by Auricularia polytricha 5.584. J Henan Univ Technol (Natural Sci Edition). 2020;41:65\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu XK. The preparation and functional properties of soluble dietary fiber (SDF) from wheat bran fermented by \u003cem\u003eNeurospora sitophila\u003c/em\u003e. Harbin: Harbin University of Commerce; 2021.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 6 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbit","sideBox":"Learn more about [BMC Biotechnology](http://bmcbiotechnol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bbit/default.aspx","title":"BMC Biotechnology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Taxillus chinensis, endophytic fungi, lignocellulose degrading enzymes fungus, solid-state fermentation","lastPublishedDoi":"10.21203/rs.3.rs-9401766/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9401766/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThere are several endophytic fungal strains from \u003cem\u003eTaxillus chinensis\u003c/em\u003e that can degrade lignocellulose from different substrates. We systematically explored their enzyme-producing characteristics and degradation efficiency on these substrates with the aim of providing theoretical support for the screening and application of high-efficiency degrading strains.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAmong the 4 tested strains, the highest total organic matter mass loss rate (55.50%) was observed when wheat bran was used as the substrate for 44 days of fermentation with the average degradation rate reaching a peak at 9 d (4.28%/d). All 4 strains showed consistent temporal dynamic characteristics with the 4 substrates: the lignin peroxidase activity peaked at 9 d of fermentation, with the highest enzyme activity of 23.52 U/mL. For strain P6, when wheat bran was used as the substrate, the laccase activity peaked at 16 days of fermentation (108.39 U/mL), which was significantly higher than those in other substrates and at different fermentation times. For strains 4 and N6, with mulberry leaves and wheat bran as substrates, the manganese peroxidase activity reached the highest at 16 and 30 days of fermentation (118.32 and 111.69 U/mL), respectively. For strain N6, when mulberry xylem was used as the substrate, the cellulase activity peaked at 2 days (0.67 U/mL), which was higher than that in other substrates and at different fermentation times.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe degradation efficiency of the 4 screened endophytic fungal strains from \u003cem\u003eT. chinensis\u003c/em\u003e on different lignocellulosic substrates, as well as the activities of key degrading enzymes such as lignin peroxidase and laccase, all showed significant strain specificity, substrate specificity, and temporal dynamic characteristics. The results of this study can provide a theoretical basis and practical reference for the excavation of high-efficiency lignin-degrading strain resources and the optimization of their fermentation conditions.\u003c/p\u003e","manuscriptTitle":"Degradation characteristics of lignocellulose by endophytic fungi of Taxillus chinensis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-14 16:35:48","doi":"10.21203/rs.3.rs-9401766/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"189266636627649350636219313772498559786","date":"2026-05-09T03:42:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-06T02:09:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-30T03:20:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-24T06:27:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-24T02:19:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Biotechnology","date":"2026-04-24T02:14:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bbit","sideBox":"Learn more about [BMC Biotechnology](http://bmcbiotechnol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bbit/default.aspx","title":"BMC Biotechnology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"94bc2f6e-7fab-44e8-abd8-603ff9928f9d","owner":[],"postedDate":"May 14th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"189266636627649350636219313772498559786","date":"2026-05-09T03:42:39+00:00","index":31,"fulltext":""},{"type":"reviewersInvited","content":"9","date":"2026-05-06T02:09:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-30T03:20:55+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T16:35:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-14 16:35:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9401766","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9401766","identity":"rs-9401766","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}