Materials
The wild macrofungi grew in decaying leaves and wood in evergreen deciduous broadleaved forest dominated by Quercus linna were collected. The leaves used for fermentation from E. ulmoides ‘Huazhong 6’, which were collected from the ‘Eucommia Resource Nursery of Institute’ of Agro-Bioengineering of Guizhou University. Cellulase Test Kit (Code No. A138-1-1) was purchased from Nanjing Jiancheng Bioengineering Institute (China), and α-L-arabinofuran glycosidase Test Kit (Code No. BC4765) were purchased from Beijing Solarbio Science & Technology Co., Ltd (China). The 2-hydroxy-3, 5-dinitrobenzoic acid (DNS, Cas No. 609-99-4), D-(+)-Xylose (Cas No. 58-86-6), xylan (poly-β-D-xylopyranose (1→4) Cas No. 9014-63-5), D-(+)-glucose (Cas No. 50-99-7), dimethyl sulfoxide (DMSO, Cas No. 67-68-5) and p-nitrophenol acetate (Cas No. 830-03-5) were purchased from Sigma-Aldrich (Merck, USA). The petroleum ether (AR) and absolute ethanol (AR) were purchased from Tianjin Kemiou Chemical Reagent Co., Ltd (China).
Methods
Field Survey and Macrofungi Collection
According to the method of Senanayake et al. (2020), field investigation of macrofungi were carried out in Pingba Town, Jinsha County, Bijie City, Guizhou Province of China (105°47’—106°44’ E, 27°07’—27°46’ N) from May to September, 2021. The altitude sampling site is 739-1460 m and the average annual temperature is about 15℃, and the forest coverage rate is 56.2% and the average annual rainfall is 1030-1100 mm. According to difference of vegetation types and terrain, the wild macrofungi grew in decaying branches and leaves on the ground and in live tree trunks on different slopes were investigated. The collected wild macrofungi were photographed, recorded and stored, and the fruiting bodies were brought back to the laboratory for subsequent research.
Fungal Identification and Pure Culture of Mycelium
Based on the method reported by Zhou (2009), the morphological characteristics (e.g. pileus, stipitipellis, lamella and teleblem, etc) of the collected wild macrofungi (fruiting body) were observed and identified. According to the method of Passari et al. (2016), the base of the fruiting body stalk was removed and placed in the inoculating chamber. After surface sterilization with 75% alcohol, a flame-sterilized scalpel was used to make a longitudinal cut in the middle of fruiting body stalk and then the fruiting body were broken off by hand and five small squares were cut at the junction of the pileus and stipitipellis of macrofungi with an inoculating shovel. Then, a small piece of tissue was selected and quickly transferred to the beveled PDA medium (20 g·L-1 glucose, 2 g·L-1 peptone, 2 g·L-1 yeast extract, 0.5 g·L-1 MgSO4·7H2O,0.46 g·L-1 KH2PO4,20 g·L-1 agar powder, natural pH) for culture at 28 ± 2℃, and the mycelium formed from unpolluted tissue block. After 3 to 5 secondary cultures, the pure mycelium were acquired for subsequent studies. Total genomic DNA of pure mycelium was extracted according to the method of Huang et al. (2018) and Wu (2015) (with minor modifications), and the primer ITS1/ITS4 and LROR/LR5 were used to amplify conserved regions including internal transcribed spacer (ITS) and ribosome large subunit DNA (LSU) according to the method reported by Febriansyah et al. (2018) respectively. The primer sequence for ITS and LUS amplification were shown in Table 1. Finally, the amplified products were sequenced by Shengong Bioengineering (Shanghai) Co., LTD. Blast alignment was performed according to the method of Roy et al. (2018) and the phylogenetic tree was constructed by MEGA 7.
Table 1
Effects of Different Carbon Sources on Mycelium Growth
According to the method of Liu et al. (2021), the purified mycelium was inoculated on the medium (2 g·L-1 peptone, 2 g·L-1 yeast extract, 0.5 g·L-1 MgSO4·7H2O, 0.46 g·L-1 KH2PO4, 20 g·L-1 agar powder, natural pH) supplemented with glucose or xylan as sole carbon source respectively, with the medium without carbon source as control. Each group was independently repeated for 3 times, and the morphology of hypha and mycelium of macrofungi was observed and photographed. Meanwhile, mycelium growth amount was calculated according to the following Formula 1.
Hypha growth amount (HGA) = (mycelium diameter - inoculum diameter) /2 Formula 1
Effects of Xylan Carbon Source on Cellulase and Xylanase Activities
To investigated the effects of xylan carbon source on CLE and XLE activities, the depurative mycelium block (d=1 cm) was taken out from solid medium with aseptic puncher and transferred the mycelium to liquid medium (2 g·L-1 peptone, 2 g·L-1 yeast extract, 0.5 g·L-1 MgSO4·7H2O,0.46 g·L-1 KH2PO4, natural pH) contained xylan, with no-carbon source medium as control. At different days after shaking culture with 180 r·min-1 under 28℃, an equivalent amount of pure culture was taken out to measure XLE activity according to the method reported by Meng et al. (2021) and CLE activity was measured according to the instruction of Cellulase Test Kit (Nanjing Jiancheng Bioengineering Institute). Each treatment was repeated 3 times.
Effects of Xylan Carbon Sources on Activities of Tissue Degradation-correlated Enzymes during Fermentation
To further survey the decomposability of C. disseminatus mycelium to leaf tissue, the purified mycelium was inoculated into a triangular flask containing sterilized Eucommia leaves for solid fermentation according to the method of Qian et al. (2020). The freshly picked E. ulmoides leaves were dried at 60℃ to a constant weight, then were gently rubbed and crushed for fermentation. Meanwhile, an equivalent amount of liquid medium was added to the leaves solid matrix as the nutrient for mycelial fermentation at initial stage. The supplemental liquid medium included carbon-free medium (CFM) 2 g·L-1 peptone, 2 g·L-1 yeast extract, 0.5 g·L-1 MgSO4·7H2O, 0.46 g·L-1 KH2PO4, natural pH), xylan-carbon medium (XCM) (20 g·L-1 xylan, 2 g·L-1 peptone, 2 g·L-1 yeast extract, 0.5 g·L-1 MgSO4·7H2O, 0.46 g·L-1 KH2PO420 g·L-1 agar powder, natural pH), with supplemental sterile water as control. Weighed exactly 5.0 g crushed E. ulmoides leaves to clean culture flask and mixed with supplemental liquid medium according to a ratio of 1 leaves : 1 liquid medium, and an equivalent amount (~1 cm2 mycelium block) was inoculated into leaves matrix above for continuous fermentation at 28℃ under dark conditions. At different days (3, 5 and 7 d) after fermentation, the activities of XLE and CLE were determined according to description above, and the activities of AXE and α-L-AF were determined according to the method of Krastanova et al. (2005) and the operation of α-L-arabinofuran glycosidase Test Kit (Beijing Solarbio Science & Technology Co., Ltd) respectively.
Effects of Different Conditions on Extraction Rate of Gutta-percha
At 7 d and 14 d after fermentation, the fermented E. ulmoides leaves were dried at 60℃ to constant weight, and gutta-percha extraction was conducted according to the method reported by Wei et al. (2021) and Yuan et al. (2021) (with minor modifications). The dried E. ulmoides leaves and petroleum ether were mixed according to a solid-liquid ratio of 1 : 20, and the gutta-percha was extracted on a Soxhlet extractor at 90℃ for 24 h. After that, the extract was placed at a low temperature of -20℃ for 3 h to precipitate the gutta-percha. After natural drying of precipitation, the dried precipitates was refined E. ulmoides gum, and the yield of refined gum was calculated according to Formula 2.
Refined gum yield (RGY) = (Dry mass of refined gutta-percha from extraction/total dry mass of sample) × 100% Formula 2
Determination of Gutta-percha Based on Fourier Transform Infrared Spectroscopy
Accurately weighed 10 mg of gutta-percha dried to constant weight for weighed for tablet pressing according to the method of Cui et al. (2022). The processed tablet samples were placed into a Fourier Transform Infrared Spectroscopy (FTIR) (spotlight 200) for scanning identification, comparison and analysis. The spectral region ranged from 4000-400 cm-1, and the maximum resolution was 2.0 cm-1. Addtionally, the detector was a DTGS detector and the light source is air cooled ceramic light source.
Data Analysis and Data Processing
IBM SPSS Statistics 18.0 were used for data analysis and GraphPad Prism 9.0 were used for mapping respectively.