An Economical and Sustainable Non-sterilization Cultivation Method of Oyster Mushroom (Pleurotus spp.) and its Key Influencing Factor

An Economical and Sustainable Non-sterilization Cultivation Method of Oyster Mushroom (Pleurotus spp.) and its Key Influencing Factor | 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 An Economical and Sustainable Non-sterilization Cultivation Method of Oyster Mushroom (Pleurotus spp.) and its Key Influencing Factor Yulong Zhang, Zhijun Li, Dongmei Lin, Jingsi Chen, Linsong Shen, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2158074/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Natural processes inspire human innovation. For example, leafcutter ants inhabiting rainforests of South America cut plant debris, transport them to underground nests, dissociate them into small pieces, and use them to cultivate a specific fungus, which serve as the main food source for the whole colony. Inspired by this, Giant Juncao grass (Cenchrus fungigraminus, name used before: Pennisetum giganteum z.x.lin) was used as the raw material. It is a C4 tall grass with rapid growth, high yield, wide-spread adaptability, nitrogen fixation, low-quality soil requirements, biosafety, and can be cultivated on a large scale in many low value and idle lands such as saline and alkali soils, desert and barren mountains. On this basis, we screened out a non-sterilization-based cultivation method of oyster mushroom with fresh Giant Juncao grass. Using our novel method, the bottleneck technologies in the popularization and application of various oyster mushroom cultivation methods in underdeveloped areas were improved. Took Wei W.'s report as reference that the highest annual output of Giant Juncao grass was 450 tons per hectare in southern China. And according to the conversion rate of the oyster mushroom in this research which was 95% in average (Ratio of total weight of fresh mushroom to dry weight of culture material), and the water content which was 91.5%, about 119.7 tons of fresh oyster mushroom could be produced in 2-3 months. Took the nutrient contents of Pleurotus sapidus cultivated by sterilization cultivation method with fresh Giant Juncao grass reported by Zhixiang X. as reference, it could provide 2.37 tons of high-quality protein. In addition, this study found that, the key to the success of this method was the reduction of the soluble substances in culture materials, which significantly reduced the contamination by Mucor circinelloides and provided an enabling environment for the growth of oyster mushroom. These findings provided an index for quality control and a theoretical basis for further study. If this technology is applied appropriately, it will be able to greatly alleviate malnutrition and food shortages in an economical, organic, and sustainable manner in underdeveloped areas. Giant Juncao grass Non-sterilization Oyster mushroom Sustainable Malnutrition and food shortages Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Malnutrition and food shortages continue to be major socio-economic issues in underdeveloped areas of the earth. Therefore, there is a pressing need to develop sustainable and economically viable food resources that are enriched with nutrition and active compounds. Leafcutter ants from the tropical rain forests of South America dissociate plant debris to use as part of a culture system to mass cultivate a fungus that serve as their main food source [ 1 , 2 ]. This natural paradigm can be mimicked for mass production of edible fungi for human populations. Accordingly, some methods already can cultivate edible fungi, such as oyster mushroom with some herbs and woody plants [ 3 ]. And the edible fungi can grow fast to high fecundity with many nutrients [ 4 , 5 ] and several active compounds that can improve immunity [ 6 , 7 ], anti-tumor [ 8 ] and confer anti-oxidative properties, etc [ 9 , 10 ]. However, several factors hamper large-scale cultivation of edible fungi. Some of these limitations include insufficient supply of wild forage and wood, unsustainable large-scale deforestation [ 3 ], high cost of technology and equipment, high energy consumption, difficulty in drying fresh grass in humid environment, untimely drying, leading to fermentation deterioration and reduction in cultivation efficiency [ 11 – 13 ]. Prolonged raw material treatment cycle and low utilization rate of traditional fermentation cultivation methods [ 14 – 16 ], as well as the need for a large number of toxic antibacterial agents for conventional non-sterilization cultivation approaches [ 17 , 18 ]. Consequently, the conventional cultivation methods employed for oyster mushroom production have not been popularized or applied on a large scale in underdeveloped areas. Therefore, in this study, we aimed to design and select a non-sterilization cultivation method of oyster mushroom with fresh Giant Juncao grass, in view of the shortcomings of various conventional existing methods. Firstly, Giant Juncao grass was used as the raw material. Giant Juncao grass ( Cenchrus fungigraminus , name used before: Pennisetum giganteum z.x.lin ), a C4 tall grass with rapid growth (It could grow to about 3m in three months) [ 19 – 21 ], high yield (The highest annual output of fresh grass in South China was 450 tons per hectare) [ 22 ], wide-spread adaptability (It can grow at an altitude of 200–1500 m, temperature of -2 ~ 45 ℃) [ 23 – 25 ], nitrogen fixation [ 26 ], low-quality soil requirements, and can be cultivated on a large scale in many low value and idle lands such as saline and alkali soils [ 27 – 29 ], desert and barren mountains [ 30 – 32 ]. Therefore, provided an economical, environmentally friendly, sustainable and stable supply of raw materials (It could be harvested continuously for 15–20 years in southern China) with biosafety (which is reproduced by stem, not seeds) and can absorb large amounts of carbon dioxide (In southern China, the annual absorption of carbon dioxide was up to 90 tons per hectare) [ 23 , 33 ]. Therefore, it had been widely promoted in many parts of the world. [ 32 , 34 ]. Further, compared with sterilization cultivation method [ 11 , 35 , 36 ], our present approach did not require high-temperature and high-pressure sterilization, thus, eliminating the need for high technical requirements, the use of large equipment and high energy consumption associated with sterilization. After directly preparing and loading, the samples can be inoculated directly in the open air. For our approach excluded the need for aseptic inoculation, which removed the need for high inoculation technical requirements and large investment in inoculation equipment. Compared with the fermentation cultivation method [ 14 – 16 ], the technique described herein solved the problems of long fermentation time, strict temperature and size requirements, large loss of weight in the fermentation process, and the uncertainty in determining whether the fermentation process was adequate/complete. Compared with the conventional non-sterilization cultivation method [ 17 , 18 ], this approach did not require the use of multiple antibacterial agents with certain toxicity and had a wider tolerance to temperature range, which was the same as that of the sterilization cultivation method. In addition, this method involves crushing the fresh Giant Juncao grass and using it directly, which solves the problems associated with laborious drying procedures of raw materials in humid environment and the relatively slow drying phase, which was easy to cause fermentation deterioration and reduced cultivation efficiency [ 11 , 12 ]. Therefore, we have obtained a short-cycle, recyclable, green, organic and environmentally friendly oyster mushroom cultivation method with high efficiency, fast speed, low technical threshold and low investment, which closely mimicked the leafcutter ants’ cultivation of fungus using plant debris. Additionally, we observed that this method was highly related to the volume of saturated lime water treatment and the infestation degree of a white mold. In the first three days, the mycelia of oyster mushroom in each group recovered normally and grew vigorously. The mycelia recovery and extent of growth in either the non-treatment or low saturated lime water treatment groups were slightly higher than that of the high saturated lime water treatment groups. However, following the third day, the white mold grew rapidly near the oyster mushroom mycelia and some other areas of the mushroom bag. The oyster mushroom mycelia in the no saturated lime water treatment group were covered with this type of white mold in a few days, which led to its complete disappearance. Treatment with lime water was associated with sparser white mold levels in a dose dependent manner, and a concordant increase in oyster mushroom mycelia growth rate. In the equal weight saturated lime water treatment group, we observed small populations of oyster mushroom mycelia that did not disappear. In the four times weight saturated lime water treatment group, this white mold had been very sparsely distributed, which did not affect the growth of oyster mushroom, as the oyster mushroom mycelia could grow to full range/extent normally and produce mushrooms normally. In each treatment group, the addition of corn flour was associated with a slight increase in white mold preponderance and the oyster mushroom mycelia were slightly stronger, but it had no significant effect on the results. Therefore, it was speculated that saturated lime water treatment would dissolve, dilute and take away some soluble substances in the fresh Giant Juncao grass powder that otherwise promoted the growth of this white mold. In further experiments, the white mold was isolated, purified, and identified through the methods of morphology and molecular biology. By measuring the concentration of soluble substances in each group of culture materials with a refractometer, it was found that the key to the success of this method was the reduction of the soluble substances in culture materials, which significantly reduced the contamination caused by Mucor circinelloides and provided an enabling environment for the growth of oyster mushroom. These findings provided an index for quality control and a theoretical basis for further study. Upon optimization and application, the technology described herein can be applied towards alleviating the burden of malnutrition and food shortages in underdeveloped areas. Materials And Methods Materials and instruments Materials The mature and fresh Giant Juncao grass with average height of 3-6 m and growth period of more than 6 months, corn flour, quicklime (calcium oxide) powder, deionized water, 12 cm×24 cm polypropylene mushroom cultivation bags, breathable covers and the spawn of Pleurotus sapidus P969 strain, fresh potatoes, centrifuge tubes were provided by China national engineering research center of Juncao technology; Disposable petri dish (90 mm×20 mm) were purchased from Beijing Labgic; HP Fungal DNA Kit D3195, Gel Extraction Kit D2500 was purchased from Omega Bio-Tek; DNA Marker Ⅲ were purchased from Tiangen Biotech; 2×EasyTaq PCR SuperMix (+dye), GelStain nucleic acid dye, Trans 2K plus DNA Marker, Trans 2K DNA Marker, pEASY-T5 Zero Cloning Kit, Trans1-T1 Phage Resistant Chemically Competent Cell were purchased from Transgen Biotech. Instruments The instruments used in this article are shown in the Table 1: Table 1 Instruments Name Model Brand Clean bench SW-CJ-1F Suzhou Antai Vertical pressure steam sterilizer YXQ-LS-100S II Shanghai Boxun Scientific research-grade positron fluorescence microscope and imaging system Ni-U Nikon Corporation High-speed freezing centrifuge 5415R Sigma Laborzentrifugen PCR instrument S1000 Bio-Rad Gel imaging system GBoxF3 Gene Company Electrophoresis apparatus PowerPacTM Basic Bio-Rad Biochemical incubator LRH-250F Shanghai Bluepard Green gel cutter/Blue light transmission meter OSE-470 Tiangen Biotech Vortex mixer MX-S Scilogex Microwave oven MZC-2070M Qingdao Haier Pipettes Research Plus Eppendorf Thermostatic water bath HH·S21-6-S Shanghai Xinmiao Ice maker IMS-20 Changshu Xueke Ultra-pure water machine WP-RO-30B Sichuan Vortel Analytical balance BSA124A Sartorius Constant temperature culture oscillator ZWY-2102 Shanghai Zhicheng Fridge BCD-216SDN Haier Zhijia Pulverizer DFY-300 Wenling Linda pH meter PB-10 Sartorius Ultra-low temperature freezer Forma 900 series Thermo Fisher Scientific Refractometer LH-Y12 Lohand Biological Methods Screening out the non-sterilization cultivation method of oyster mushroom with fresh Giant Juncao grass Depending on the applied mechanism and previous studies [3, 11, 13, 35-38], the following raw materials were used: Fresh Giant Juncao grass, corn flour and quicklime (CaO), which can also be easily obtained in many underdeveloped areas. This formula provided a highly alkaline environment that was well tolerated by oyster mushroom ( Pleurotus sapidus P969 strain). Nonetheless, few pathogenic microbes can survive in CaO. Gradient experiments were conducted to compare the proportion of corn flour and the volume of saturated lime water used to treat fresh Giant Juncao grass as shown in Table 2, after which we selected the best performing groups. The steps were as follows: Cut down the fresh Giant Juncao grass and shred it into filament fibers or small pieces within 2 cm with a pulverizer. Added saturated lime water of different volumes in each lime water-treated group and soaked for 30 min. Squeezed the excess water out of each lime water-treated group and adjusted the water content of each group to 65%. Added every component to each group as shown in Table 1 and mixed thoroughly. Put the substrate into mushroom cultivation bags, 250 g for each bag, and nine bags for each group. Inoculated the spawn of the oyster mushroom on the surface of the substrate in the open air. Screwed down the breathable covers that came with the mushroom cultivation bags and placing the bags at 25 ℃ for mycelia culture. Selected the best-performing group, conducted mushroom cultivation experiment, and collected relevant data. Table 2 Formula of each experimental group Sample Weight multiples of saturated lime water for raw material treatment (LW) Fresh Giant Juncao grass powder after treatment % Corn flour % CaO % LW×0 0 98 0 2 97.75 0.25 97.5 0.5 97.25 0.75 97 1 LW×1 1 98 0 2 97.75 0.25 97.5 0.5 97.25 0.75 97 1 LW×2 2 98 0 2 97.75 0.25 97.5 0.5 97.25 0.75 97 1 LW×4 4 98 0 2 97.75 0.25 97.5 0.5 97.25 0.75 97 1 LW×8 8 98 0 2 97.75 0.25 97.5 0.5 97.25 0.75 97 1 Isolation, purification and identification of white mold We prepared PDA medium and picked out the culture material with mold from the position where the white mold grew in each treatment group. Next, placed them on PDA medium and made three repetitions for each group. After growing them at 25 ℃ for 5 days, we cut the end parts of the growing mycelia into small squares of 5 mm 2 and placed them on new PDA medium petri dishes to purify the mycelia and observed their growth. After growing to cover more than 2/3 of the petri dish, we observed the morphology of mycelia and sporangia through naked eye and microscopy. Next, we extracted mycelial gDNA from the white mold by using HP Fungal DNA Kit D3195, used the gDNA, 2×EasyTaq PCR SuperMix (+dye) and ITS primers (ITS-5: 5'-GGAAGTAAAAGTCGTAACAAGG-3', ITS-4: 5'-TCCTCCGCTTATTGATATGC-3') to perform PCR amplification of ITS bar code sequence, ran the PCR products through agarose gel electrophoresis, used UV gel imaging, performed gel extraction of PCR products by using Gel Extraction Kit D2500, ligated the gel extraction products into the cloning vector by using pEASY-T5 Zero Cloning Kit, carried out transformation of E. coli competent cells with ligation products by using Trans1-T1 Phage Resistant Chemically Competent Cell and identification of positive clones by performing PCR amplification to complete the TA cloning. Next, we sent the positive clones to Fuzhou Sunya Biotechnology for ITS sequencing. The sequencing results were compared to the nucleotide data base in GenBank via NCBI BLAST. Detailed operation steps are shown in the Supporting Information of Supplementary Information. Measuring the concentration of soluble substances in each group of culture materials with a refractometer We squeezed out the liquid in each group of culture materials, and measured the concentration of soluble substances with a refractometer. After that, compared the result with the contamination caused by M. circinelloides and the growth of oyster mushroom mycelia in each group. The performance of reducing the concentration of soluble substances in culture materials to 2%Brix with Giant Juncao grass in other growth periods We crushed the tender green Giant Juncao grass growing for three months, the Giant Juncao grass growing for one year with high lignification degree, and the dead branches and leaves of Giant Juncao grass separately. Treated them with saturated lime water, used them as the raw materials for non-sterilization culture materials, mixed them with 2% quicklime powder and 0.5% corn flour, and controlled the concentration of soluble substances to 2%Brix measured by refractometer, made mushroom bags as experimental groups. Used the powder of Giant Juncao grass of these three growing periods without treatment as the raw materials for non-sterilization culture materials, mixed them with 2% quicklime powder and 0.5% corn flour, made mushroom bags as control groups, and measured the concentration of their soluble substances by refractometer. Other operations and the spawn were the same as in screening out the non-sterilization cultivation method of oyster mushroom with fresh Giant Juncao grass. Results And Discussion Screening out the non-sterilization cultivation method of oyster mushroom with fresh Giant Juncao grass The Giant Juncao grass that we chose are shown in Figure 1, and the grass powder are shown in Figure 2. The results showed that the mycelia of oyster mushroom ( Pleurotus sapidus P969 strain) in each group recovered normally in the first three days. The mycelial growth speed of the oyster mushroom in the no saturated lime water treatment group (LW×0) and the low-weight lime water treatment groups were even slightly faster than that in the high-weight lime water treatment groups. However, as shown in Figure 3, from the third day onwards, in the LW×0 group, the mycelia of a white mold began to spread near the oyster mushroom mycelia and across some places of the mushroom cultivation bags. At subsequent stages, the mycelia of this white mold became more preponderant, the growth of the oyster mushroom mycelia was hampered and the oyster mushroom mycelia were covered by the white mold mycelia. After 21 days, the oyster mushroom mycelia were almost entirely disappeared, as shown in Table 3. In the same-weight saturated lime water treatment group (LW×1), the mycelia growth of white mold was less than that of the LW×0 group, and the inhibition on the growth of the oyster mushroom mycelia was also alleviated. In the inoculated part, some oyster mushroom mycelia would not be covered by the white mold mycelia, and a small part of the oyster mushroom mycelia remained even after 21 days, as shown in Table 3. The mycelial growth of the white mold was further diminished in the treatment group subjected to twice the weight of lime water (LW×2), which was associated with enhanced growth of the mycelial growth of the oyster mushroom. After 21 days, about 1/3 of the oyster mushroom mycelia did not subside, as shown in Table 3. Starting with four times the weight of the saturated lime water treatment group (LW×4), the white mold only appeared sporadically in the form of small mycelial plaque or weak gauze, which did not affect the growth of the oyster mushroom mycelia. In this group, after approximately 21 days, the oyster mushroom mycelia could grow to full extent in the mushroom cultivation bags and did not subside, as shown in Table 3. There were no significant differences observed between the eight times the weight of saturated lime water treatment group (LW×8) and LW×4 group, and each repetition grew normally, with high stability, as shown in Table 3. Therefore, these groups can facilitate normal growth of the oyster mushroom mycelia. As shown in Table 3, In each treatment group, addition of corn flour was associated with a slight increase in the white mold abundance and moderately stronger oyster mushroom mycelia, which was not significant. Based on these observations and previous studies, we deduced that addition of appropriate amount of corn flour is beneficial to improve the mycelial growth of oyster mushroom in this non-sterilization cultivation method. As such, the formula consisting of 0.5% corn flour in the middle of LW×4 group was selected for mushroom production experiments. As shown in Figure 4, the mushroom production experiment revealed that after the oyster mushroom mycelia grew to full extent by the average growth speed of 0.43 cm/d in bags, and was incubated for approximately 14 days, the mushroom bags would turn predominantly white and began to exhibit mushroom primordia. Mushrooms grew three or four times in total, the conversion rate was 95% in average (Ratio of total weight of fresh mushroom to dry weight of culture material), and the water content was 91.5%. Isolation, purification and identification of white mold Morphological identification of the white mold Sequencing and comparison of ITS sequence of the white mold As shown in Figure 5, when the white mold grew to approximately 2/3 of the plate on PDA medium, black sporangia became preponderant in most of the area, with the aerial mycelia exhibiting a curly morphology and the peripheral mycelia appearing white. Microscopy was used to discern the morphology of sporangium, spore and mycelia as well as the interval between mycelia, as shown in Figure 6. The genomic DNA was extracted from the white mold as shown in Figure 7, the ITS fragment was obtained through PCR as shown in Figure 8, and the positive clones of TA cloning were identified through PCR as shown in Figure 9. Compared the ITS sequence to the nucleotide data base in GenBank via NCBI BLAST, the comparison results corresponded to Mucor circinelloides . The ITS sequence is available in the Genbank, MN744376.1, and the length is 662 bp. This sequence-based data corroborated the results obtained via morphological identification. Full-length gels are presented in Supplementary Fig. 7, Fig. 8 and Fig. 9 of the Supplementary Information. Measuring the concentration of soluble substances in each group of culture materials with a refractometer Compared the concentration of soluble substances in each group of culture materials with a refractometer (The result is presented in Supplementary Chart 1 of the Supplementary Information) with the result of screening out the non-sterilization cultivation method of oyster mushroom with fresh Giant Juncao grass, we found that, with the increase of saturated lime water in treatments, the concentration of soluble substances and the contamination caused by M. circinelloides decreased, and the growth of oyster mushroom mycelia increased accordingly. For all groups with the concentration of soluble substances measured by refractometer equal to or less than 2%Brix, the M. circinelloides grew very week, which did not affect the growth of the oyster mushroom mycelia, and after approximately 21 days, the oyster mushroom mycelia could grow to full extent and did not subside. The performance of reducing the concentration of soluble substances in culture materials to 2%Brix with Giant Juncao grass in other growth periods According to the results as shown in Table 4, in the control groups, the group with Giant Juncao grass growing for 3 months and the group with Giant Juncao grass growing for 1 year still had high soluble substances, reached 11%Brix, the M. circinelloides grew vigorously, and the oyster mushroom mycelia could not grow effectively. The group with branches and leaves of dead Giant Juncao grass, had relatively low soluble substances, only 5%Brix, the M. circinelloides grew like gauze, the oyster mushroom mycelia could grow to full but week and had big black patches. In all the experimental groups with the treatment of saturated lime water, the M. circinelloides only appeared sporadically and the oyster mushroom mycelia could grow vigorously to full extent and did not subside in each group. Conclusions This study provided a non-sterilization cultivation method for oyster mushroom with fresh Giant Juncao grass. It saved time, labor, equipment and technical costs, and was green and organic. And the raw material Giant Juncao grass had the advantages of tall plant, high yield, wide adaptability, nitrogen fixation, bio-safety and strong sustainability. Further study showed that the soluble substances of the formula promoted the growth of M. circinelloides , and this substantial growth resulted in the inhibition of oyster mushroom mycelial growth. These findings provided an index for quality control and a theoretical basis for further study. With proper optimization and application, this method can be an efficacious in alleviating the burden of malnutrition and food shortages. Declarations Acknowledgements This work was supported by the Major Special Project of Fujian Province “Research and application of key technologies for innovation and industrialized utilization of Juncao” (2021NZ029009) and Interdisciplinary integration to promote the high-quality development of Juncao science and Industry (XKJC-712021030). Authors ’ Contributions Yulong Zhang wrote the main manuscript text, designed of the study and collection, analysis, and interpretation of data, and performed experiments, wrote original draft and edited the final manuscript. Zhijun Li conducted the experiments, and assisted with manuscript writing, and checked the final edition. Dongmei Lin contributed to the conception and design of the study and supervised the manuscript. Jingsi Chen performed the early experiments and collected some data. Linsong Shen, Fengmei Wen and Mingjun Xie prepared the figures and tables. Hui Lin, Xingsheng Lin and Hailing Luo contributed to funding acquisition and supervised the manuscript. Fan Yan edited the language. Eyalira Jacob Okal analyzed data and performed some experiments. Yingxing Lin did some experiments. Zhanxi Lin was the supervisor, who administrated this project, and contributed to the conception and design of this study, edited the manuscript. All authors read and approved the final manuscript. Funding Fujian Province “Research and application of key technologies for innovation and industrialized utilization of Juncao” (2021NZ029009) and Interdisciplinary integration to promote the high-quality development of Juncao science and Industry (XKJC-71202103) Availability of data and materials All data and materials are available upon request to corresponding author. The datasets generated and/or analyzed during the current study are available in the GenBank repository. Gene data: ITS sequence data: GenBank accession number MN744376.1. Ethics approval and consent to participate The authors declare all experimental research and field studies on plants (either cultivated or wild), including the cultivation and collection of plant material, comply with relevant institutional, national, and international guide-lines and legislation. Consent for publication Not applicable. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Mueller UG, Mikheyev AS, Hong EY, Sen R, Warren DL, Solomon SE, Ishak HD, Cooper M, Miller JL, Shaffer KA, Juenger TE. "Evolution of cold-tolerant fungal symbionts permits winter fungiculture by leafcutter ants at the northern frontier of a tropical ant-fungus symbiosis." Proceedings of the National Academy of Sciences of the United States of America. 2011;108(10):4053-4056. Lechner BE, Josens R. 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Guizhou Agricultural Sciences. 2017;45(07):111-113. Liwei S, Rongqing L, Xiao’an W, Xingde Z. Technical regulations of cultivation for Pennisetum giganteum in semi-arid area of Gansu Province. Forest Science and Technology. 2021;(10):78-80. Ma YX, Li GT, Wang GH, Liang TY, Yan JQZ, Li JJ. Effects of saline-alkali mixed stress on the growth and physiological characteristics of giant juncao (Pennisetum giganteum z.x. lin). applied ecology and environmental research. 2021;19(1):75-94. Qiang W, Jinrong L, Jianying G, Li X, Zhijie Z, Naqi Z. Sand fixation effect of Pennisetum giganteum z.x. Lin in Ulanbuh Desert along the Yellow River. Journal of Inner Mongolia Forestry Science and Technology. 2018;44(02):50-56. Xingsheng L, Zhanxi L, Dongmei L, Hui L, Hailing L, Yingping H, Chunmei L, Chaozhi Z. Effects of planting Pennisetum sp. (Giant Juncao) on soil microbial functional diversity and fertility in the barren hillside. Acta Ecologica Sinica. 2014;34(15):4304-4312. Chen H, Chu XG, Jia Q. Windbreak and sand fixation of sand plants based on intelligent image processing and plant landscape design. Arabian Journal of Geosciences. 2021;14:1-12. Haizhong C. Discussion on the prospect of JUNCAO industry development in high cold areas. Nong Min Zhi Fu Zhi You. 2017;(16):7. Xiaohui Y. China’s Agricultural Technical Cooperation: A Case Study on juncao Aid Projects in Papua New Guinea and Fiji. South-south Cooperation and Chinese Foreign Aid. 2019;143-159. Zhenghui L. Screening of culture formulation of pleurotus pulmonarius using Pennisetum spp. as substitute. Fujian Agricultural Science and Technology. 2018;(04):19-22. Huide Y. Formulation screening of oyster mushroom cultivated with Giant Juncao grass meal. Agricultural Technology Service. 2020;37(11):22-23. Suada IK, Sudarma IM, Kim BS, Cha JY, Ohga S. Fungal Contaminant Threaten Oyster Mushroom (Pleurotus ostreatus (Jacq. ex Fr.) Kummer) Cultivation in Bali. Journal- Faculty of Agriculture Kyushu University. 2015;60(2):309-313. Khan MW, Ali M, Khan NA, Khan MA, Rehman A, Javed N. Effect of different levels of lime and pH on mycelial growth and production efficiency of oyster mushroom (Pleurotus SPP.). Pakistan Journal of Botany. 2013;45(1):297-302. Tables Table 3 and 4 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Concentrationofsolublesubstancesineachgroupofculturematerials.xls Table3and4.docx SupplementaryInformation.zip Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-2158074","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":150068432,"identity":"3a8a493a-9ee4-4850-8784-87b95bfae8b9","order_by":0,"name":"Yulong Zhang","email":"","orcid":"","institution":"College of Life Science, Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yulong","middleName":"","lastName":"Zhang","suffix":""},{"id":150068433,"identity":"c18a433e-1ac8-4331-83b5-7175b37a48a6","order_by":1,"name":"Zhijun Li","email":"","orcid":"","institution":"College of Life Science, Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhijun","middleName":"","lastName":"Li","suffix":""},{"id":150068435,"identity":"984caced-0b14-4276-b656-bbf02044871e","order_by":2,"name":"Dongmei Lin","email":"","orcid":"","institution":"College of Life Science, Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dongmei","middleName":"","lastName":"Lin","suffix":""},{"id":150068437,"identity":"5f4da4f7-44ec-41cb-a9a1-d05763fb1808","order_by":3,"name":"Jingsi Chen","email":"","orcid":"","institution":"College of Life Science, Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingsi","middleName":"","lastName":"Chen","suffix":""},{"id":150068441,"identity":"cd8c6a00-62e4-4ffe-a763-d0b276665e92","order_by":4,"name":"Linsong Shen","email":"","orcid":"","institution":"College of Life Science, Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Linsong","middleName":"","lastName":"Shen","suffix":""},{"id":150068442,"identity":"a091f637-3d60-469a-972c-e3366022b08f","order_by":5,"name":"Fengmei Wen","email":"","orcid":"","institution":"College of Life Science, Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fengmei","middleName":"","lastName":"Wen","suffix":""},{"id":150068443,"identity":"e3ac2beb-82ac-4131-b6d1-3ac0660892ea","order_by":6,"name":"Mingjun Xie","email":"","orcid":"","institution":"College of Life Science, Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mingjun","middleName":"","lastName":"Xie","suffix":""},{"id":150068444,"identity":"26b87962-98a7-4a0b-bfa6-04264336b92b","order_by":7,"name":"Hui Lin","email":"","orcid":"","institution":"College of Life Science, Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Lin","suffix":""},{"id":150068445,"identity":"2d091972-d160-4b22-8f6e-3c117c328b2f","order_by":8,"name":"Xingsheng Lin","email":"","orcid":"","institution":"College of Life Science, Fujian Agriculture and Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xingsheng","middleName":"","lastName":"Lin","suffix":""},{"id":150068446,"identity":"7860b048-571a-4657-a72e-527c252116f5","order_by":9,"name":"Hailing Luo","email":"","orcid":"","institution":"College of Life 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University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zhanxi","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2022-10-12 09:59:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2158074/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2158074/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":28969172,"identity":"f2e5d296-fe9b-45a7-9c04-2d7ff7102adf","added_by":"auto","created_at":"2022-11-11 21:01:02","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":689276,"visible":true,"origin":"","legend":"\u003cp\u003eMature Giant Juncao grass\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2158074/v1/004a7594a4426ce554c58310.jpeg"},{"id":28969175,"identity":"8fe5b40c-6848-4620-afcc-f6486e63590a","added_by":"auto","created_at":"2022-11-11 21:01:02","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":951791,"visible":true,"origin":"","legend":"\u003cp\u003eFresh Giant Juncao grass powder\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-2158074/v1/fda2da017a9c44654947a516.jpeg"},{"id":28968527,"identity":"281de9f5-e0c9-4307-b84f-16cb01812359","added_by":"auto","created_at":"2022-11-11 20:53:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":387611,"visible":true,"origin":"","legend":"\u003cp\u003eWhite mold pollution 6 days after inoculation, 0.5% corn flour, LW×0 group\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2158074/v1/f66a4e566d07485a30f80a73.png"},{"id":28968532,"identity":"dbd1151b-ae46-4b51-b226-36b90c9baf87","added_by":"auto","created_at":"2022-11-11 20:53:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":314175,"visible":true,"origin":"","legend":"\u003cp\u003eOyster mushroom growth in LW×4 group with 0.5% corn flour\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2158074/v1/ab95413f3db9ff28547fe3b7.png"},{"id":28969634,"identity":"5ddc9fd2-b4a7-429e-a298-774d5a5a8802","added_by":"auto","created_at":"2022-11-11 21:09:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":450988,"visible":true,"origin":"","legend":"\u003cp\u003eThe white mold grown on PDA medium\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2158074/v1/8373a0549a1e8616424bfc4b.png"},{"id":28968531,"identity":"8dfd2ddf-e5c4-458e-8a01-c9fc46d68790","added_by":"auto","created_at":"2022-11-11 20:53:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":309886,"visible":true,"origin":"","legend":"\u003cp\u003eThe white mold observed under a microscope\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-2158074/v1/5081d5e55c6865c7a4247954.png"},{"id":28969173,"identity":"22c1c36a-9914-41e4-a0cd-e16bb3646248","added_by":"auto","created_at":"2022-11-11 21:01:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":116101,"visible":true,"origin":"","legend":"\u003cp\u003eThe genomic DNA extracted from the white mold\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-2158074/v1/8507d34584969bcfe0611fd0.png"},{"id":28968534,"identity":"cee0ba46-60be-4a2f-9dc2-c67c853ce638","added_by":"auto","created_at":"2022-11-11 20:53:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":174409,"visible":true,"origin":"","legend":"\u003cp\u003eThe ITS fragment of the white mold\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-2158074/v1/9d0f6e2e221fe4faca074f64.png"},{"id":28969635,"identity":"5c610e32-6eae-493f-b8a8-4b746639465d","added_by":"auto","created_at":"2022-11-11 21:09:02","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":115605,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of positive clones\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-2158074/v1/4683afd69a747ba890a0bb75.png"},{"id":29951632,"identity":"69a8c5d6-6733-4fe2-8fe4-a3764c837bfb","added_by":"auto","created_at":"2022-12-06 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20:53:02","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":620761,"visible":true,"origin":"","legend":"","description":"","filename":"Table3and4.docx","url":"https://assets-eu.researchsquare.com/files/rs-2158074/v1/97c17c9ac18951ef180a9610.docx"},{"id":28968537,"identity":"0155f065-0a81-4cb1-b89f-2d9b7c55ac10","added_by":"auto","created_at":"2022-11-11 20:53:12","extension":"zip","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":206407582,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.zip","url":"https://assets-eu.researchsquare.com/files/rs-2158074/v1/92c62fe8cd72a1b972a923ca.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"An Economical and Sustainable Non-sterilization Cultivation Method of Oyster Mushroom (Pleurotus spp.) and its Key Influencing Factor","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMalnutrition and food shortages continue to be major socio-economic issues in underdeveloped areas of the earth. Therefore, there is a pressing need to develop sustainable and economically viable food resources that are enriched with nutrition and active compounds. Leafcutter ants from the tropical rain forests of South America dissociate plant debris to use as part of a culture system to mass cultivate a fungus that serve as their main food source [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This natural paradigm can be mimicked for mass production of edible fungi for human populations. Accordingly, some methods already can cultivate edible fungi, such as oyster mushroom with some herbs and woody plants [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. And the edible fungi can grow fast to high fecundity with many nutrients [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and several active compounds that can improve immunity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], anti-tumor [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and confer anti-oxidative properties, etc [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, several factors hamper large-scale cultivation of edible fungi. Some of these limitations include insufficient supply of wild forage and wood, unsustainable large-scale deforestation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], high cost of technology and equipment, high energy consumption, difficulty in drying fresh grass in humid environment, untimely drying, leading to fermentation deterioration and reduction in cultivation efficiency [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Prolonged raw material treatment cycle and low utilization rate of traditional fermentation cultivation methods [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], as well as the need for a large number of toxic antibacterial agents for conventional non-sterilization cultivation approaches [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Consequently, the conventional cultivation methods employed for oyster mushroom production have not been popularized or applied on a large scale in underdeveloped areas.\u003c/p\u003e \u003cp\u003eTherefore, in this study, we aimed to design and select a non-sterilization cultivation method of oyster mushroom with fresh Giant Juncao grass, in view of the shortcomings of various conventional existing methods. Firstly, Giant Juncao grass was used as the raw material. Giant Juncao grass (\u003cem\u003eCenchrus fungigraminus\u003c/em\u003e, name used before: \u003cem\u003ePennisetum giganteum z.x.lin\u003c/em\u003e), a C4 tall grass with rapid growth (It could grow to about 3m in three months) [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], high yield (The highest annual output of fresh grass in South China was 450 tons per hectare) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], wide-spread adaptability (It can grow at an altitude of 200\u0026ndash;1500 m, temperature of -2\u0026thinsp;~\u0026thinsp;45 ℃) [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], nitrogen fixation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], low-quality soil requirements, and can be cultivated on a large scale in many low value and idle lands such as saline and alkali soils [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], desert and barren mountains [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, provided an economical, environmentally friendly, sustainable and stable supply of raw materials (It could be harvested continuously for 15\u0026ndash;20 years in southern China) with biosafety (which is reproduced by stem, not seeds) and can absorb large amounts of carbon dioxide (In southern China, the annual absorption of carbon dioxide was up to 90 tons per hectare) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, it had been widely promoted in many parts of the world. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Further, compared with sterilization cultivation method [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], our present approach did not require high-temperature and high-pressure sterilization, thus, eliminating the need for high technical requirements, the use of large equipment and high energy consumption associated with sterilization. After directly preparing and loading, the samples can be inoculated directly in the open air. For our approach excluded the need for aseptic inoculation, which removed the need for high inoculation technical requirements and large investment in inoculation equipment. Compared with the fermentation cultivation method [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], the technique described herein solved the problems of long fermentation time, strict temperature and size requirements, large loss of weight in the fermentation process, and the uncertainty in determining whether the fermentation process was adequate/complete. Compared with the conventional non-sterilization cultivation method [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], this approach did not require the use of multiple antibacterial agents with certain toxicity and had a wider tolerance to temperature range, which was the same as that of the sterilization cultivation method. In addition, this method involves crushing the fresh Giant Juncao grass and using it directly, which solves the problems associated with laborious drying procedures of raw materials in humid environment and the relatively slow drying phase, which was easy to cause fermentation deterioration and reduced cultivation efficiency [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, we have obtained a short-cycle, recyclable, green, organic and environmentally friendly oyster mushroom cultivation method with high efficiency, fast speed, low technical threshold and low investment, which closely mimicked the leafcutter ants\u0026rsquo; cultivation of fungus using plant debris.\u003c/p\u003e \u003cp\u003eAdditionally, we observed that this method was highly related to the volume of saturated lime water treatment and the infestation degree of a white mold. In the first three days, the mycelia of oyster mushroom in each group recovered normally and grew vigorously. The mycelia recovery and extent of growth in either the non-treatment or low saturated lime water treatment groups were slightly higher than that of the high saturated lime water treatment groups. However, following the third day, the white mold grew rapidly near the oyster mushroom mycelia and some other areas of the mushroom bag. The oyster mushroom mycelia in the no saturated lime water treatment group were covered with this type of white mold in a few days, which led to its complete disappearance. Treatment with lime water was associated with sparser white mold levels in a dose dependent manner, and a concordant increase in oyster mushroom mycelia growth rate. In the equal weight saturated lime water treatment group, we observed small populations of oyster mushroom mycelia that did not disappear. In the four times weight saturated lime water treatment group, this white mold had been very sparsely distributed, which did not affect the growth of oyster mushroom, as the oyster mushroom mycelia could grow to full range/extent normally and produce mushrooms normally. In each treatment group, the addition of corn flour was associated with a slight increase in white mold preponderance and the oyster mushroom mycelia were slightly stronger, but it had no significant effect on the results.\u003c/p\u003e \u003cp\u003eTherefore, it was speculated that saturated lime water treatment would dissolve, dilute and take away some soluble substances in the fresh Giant Juncao grass powder that otherwise promoted the growth of this white mold. In further experiments, the white mold was isolated, purified, and identified through the methods of morphology and molecular biology. By measuring the concentration of soluble substances in each group of culture materials with a refractometer, it was found that the key to the success of this method was the reduction of the soluble substances in culture materials, which significantly reduced the contamination caused by \u003cem\u003eMucor circinelloides\u003c/em\u003e and provided an enabling environment for the growth of oyster mushroom. These findings provided an index for quality control and a theoretical basis for further study. Upon optimization and application, the technology described herein can be applied towards alleviating the burden of malnutrition and food shortages in underdeveloped areas.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials and instruments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mature and fresh Giant Juncao grass with average height of 3-6 m and growth period of more than 6 months, corn flour, quicklime (calcium oxide) powder, deionized water, 12 cm\u0026times;24 cm polypropylene mushroom cultivation bags, breathable covers and the spawn of \u003cem\u003ePleurotus sapidus\u003c/em\u003e P969 strain, fresh potatoes, centrifuge tubes were provided by China national engineering research center of Juncao technology; Disposable petri dish (90 mm\u0026times;20 mm) were purchased from Beijing Labgic; HP Fungal DNA Kit D3195, Gel Extraction Kit D2500 was purchased from Omega Bio-Tek; DNA Marker Ⅲ were purchased from Tiangen Biotech; 2\u0026times;EasyTaq PCR SuperMix (+dye), GelStain nucleic acid dye, Trans 2K plus DNA Marker, Trans 2K DNA Marker, pEASY-T5 Zero Cloning Kit, Trans1-T1 Phage Resistant Chemically Competent Cell were purchased from Transgen Biotech.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstruments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe instruments used in this article are shown in the Table 1:\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: center;\"\u003e Instruments\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable align=\"left\" border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"954\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003e\u003cstrong\u003eName\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003e\u003cstrong\u003eModel\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003e\u003cstrong\u003eBrand\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eClean bench\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eSW-CJ-1F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eSuzhou Antai\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eVertical pressure steam sterilizer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eYXQ-LS-100S II\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eShanghai Boxun\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eScientific research-grade positron fluorescence microscope and imaging system\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eNi-U\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eNikon Corporation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eHigh-speed freezing centrifuge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003e5415R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eSigma Laborzentrifugen\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003ePCR instrument\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eS1000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eBio-Rad\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eGel imaging system\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eGBoxF3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eGene Company\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eElectrophoresis apparatus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003ePowerPacTM Basic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eBio-Rad\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eBiochemical incubator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eLRH-250F\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eShanghai Bluepard\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eGreen gel cutter/Blue light transmission meter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eOSE-470\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eTiangen Biotech\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.bing.com/images/search?q=vortex+mixer\u0026id=0F613760E0D7559E6A57032E68ED882C73EA1130\u0026FORM=IQFRBA\u0026tsc=ImageHoverTitle\" target=\"https://english.sogou.com/_blank\"\u003eVortex mixer\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eMX-S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003e\u003ca href=\"http://www.hbzhan.com/st73343/list_369031.html\" target=\"E:已分类文件课题相关资料自己课题1毕业课题_blank\"\u003eScilogex\u0026nbsp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eMicrowave oven\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eMZC-2070M\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eQingdao Haier\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003ePipettes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eResearch Plus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eEppendorf\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003e\u003ca href=\"http://www.sogou.com/link?url=hedJjaC291OOtQUMV1fnm_6E9Am8AaUh9-pmlp36ByTTDSjx_SvgCZNwmXi7aECZ91F-2uYGIlhFhNG1zqiUdA..\u0026query=Thermostatic+Thermostat+water+bath++English\" target=\"https://english.sogou.com/_blank\"\u003eThermostatic water bath\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eHH\u0026middot;S21-6-S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eShanghai Xinmiao\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eIce maker\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eIMS-20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eChangshu Xueke\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eUltra-pure water machine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eWP-RO-30B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eSichuan Vortel\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eAnalytical balance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eBSA124A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eSartorius\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eConstant temperature culture oscillator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eZWY-2102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eShanghai Zhicheng\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eFridge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eBCD-216SDN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eHaier Zhijia\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003ePulverizer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eDFY-300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eWenling Linda\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003epH meter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003ePB-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003e\u003ca href=\"https://www.baidu.com/link?url=1_jo_G-J9C_9lKCiXI_sS1kLSWNfih2FEXpeTHD4B40CGX6vWpEhTEcPP0ZF9ULz\u0026wd=\u0026eqid=e1148282000d73aa00000006625acd7f\" target=\"https://www.baidu.com/_blank\"\u003eSartorius\u0026ensp;\u003c/a\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eUltra-low temperature freezer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eForma 900 series\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eThermo Fisher Scientific\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"55.136268343815516%\"\u003e\n \u003cp\u003eRefractometer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.9958071278826%\"\u003e\n \u003cp\u003eLH-Y12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.867924528301888%\"\u003e\n \u003cp\u003eLohand Biological\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening out the non-sterilization cultivation method of oyster mushroom with fresh Giant Juncao grass\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepending on the applied mechanism and previous studies [3, 11, 13, 35-38], the following raw materials were used: Fresh Giant Juncao grass, corn flour and quicklime (CaO), which can also be easily obtained in many underdeveloped areas. This formula provided a highly alkaline environment that was well tolerated by oyster mushroom (\u003cem\u003ePleurotus sapidus\u003c/em\u003e P969 strain). Nonetheless, few pathogenic microbes can survive in CaO. Gradient experiments were conducted to compare the proportion of corn flour and the volume of saturated lime water used to treat fresh Giant Juncao grass as shown in Table 2, after which we selected the best performing groups. The steps were as follows:\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eCut down the fresh Giant Juncao grass and shred it into filament fibers or small pieces within 2 cm with a pulverizer.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAdded saturated lime water of different volumes in each lime water-treated group and soaked for 30 min.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSqueezed the excess water out of each lime water-treated group and adjusted the water content of each group to 65%.\u003c/li\u003e\n \u003cli\u003eAdded every component to each group as shown in Table 1 and mixed thoroughly.\u003c/li\u003e\n \u003cli\u003ePut the substrate into mushroom cultivation bags, 250 g for each bag, and nine bags for each group.\u003c/li\u003e\n \u003cli\u003eInoculated the spawn of the oyster mushroom on the surface of the substrate in the open air.\u003c/li\u003e\n \u003cli\u003eScrewed down the breathable covers that came with the mushroom cultivation bags and placing the bags at 25\u0026nbsp;℃\u0026nbsp;for mycelia culture.\u003c/li\u003e\n \u003cli\u003eSelected the best-performing group, conducted mushroom cultivation experiment, and collected relevant data.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp style=\"text-align: center;\"\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp style=\"text-align: center;\"\u003eFormula of each experimental group\u003c/p\u003e\n\u003cdiv align=\"center\"\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"633\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.480252764612954%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSample\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e\u003cstrong\u003eWeight multiples of saturated lime water for raw material treatment (LW)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFresh Giant Juncao grass powder after treatment %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCorn flour %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.638230647709321%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCaO %\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"12.480252764612954%\"\u003e\n \u003cp\u003eLW\u0026times;0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"24.96050552922591%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"12.638230647709321%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"12.480252764612954%\"\u003e\n \u003cp\u003eLW\u0026times;1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"24.96050552922591%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"12.638230647709321%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"12.480252764612954%\"\u003e\n \u003cp\u003eLW\u0026times;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"24.96050552922591%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"12.638230647709321%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"12.480252764612954%\"\u003e\n \u003cp\u003eLW\u0026times;4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"24.96050552922591%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"12.638230647709321%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"12.480252764612954%\"\u003e\n \u003cp\u003eLW\u0026times;8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"24.96050552922591%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.96050552922591%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"5\" valign=\"top\" width=\"12.638230647709321%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation, purification and identification of white mold\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe prepared PDA medium and picked out the culture material with mold from the position where the white mold grew in each treatment group. Next, placed them on PDA medium and made three repetitions for each group. After growing them at 25\u0026nbsp;℃\u0026nbsp;for 5 days, we cut the end parts of the growing mycelia into small squares of 5 mm\u003csup\u003e2\u003c/sup\u003e and placed them on new PDA medium petri dishes to purify the mycelia and observed their growth. After growing to cover more than 2/3 of the petri dish, we observed the morphology of mycelia and sporangia through naked eye and microscopy.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we extracted mycelial gDNA from the white mold by using HP Fungal DNA Kit D3195, used the gDNA, 2\u0026times;EasyTaq PCR SuperMix (+dye) and ITS primers (ITS-5: 5\u0026apos;-GGAAGTAAAAGTCGTAACAAGG-3\u0026apos;, ITS-4: 5\u0026apos;-TCCTCCGCTTATTGATATGC-3\u0026apos;) to perform PCR amplification of ITS bar code sequence, ran the PCR products through agarose gel electrophoresis, used UV gel imaging, performed gel extraction of PCR products by using Gel Extraction Kit D2500, ligated the gel extraction products into the cloning vector by using pEASY-T5 Zero Cloning Kit, carried out transformation of \u003cem\u003eE. coli\u003c/em\u003e competent cells with ligation products by using Trans1-T1 Phage Resistant Chemically Competent Cell and identification of positive clones by performing PCR amplification to complete the TA cloning. Next, we sent the positive clones to Fuzhou Sunya Biotechnology for ITS sequencing. The sequencing results were compared to the nucleotide data base in GenBank via NCBI BLAST. Detailed operation steps are shown in the Supporting Information of Supplementary Information.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasuring the concentration of soluble substances in each group of culture materials with a refractometer\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe squeezed out the liquid in each group of culture materials, and measured the concentration of soluble substances with a refractometer. After that, compared the result with the contamination caused by \u003cem\u003eM. circinelloides\u003c/em\u003e and the growth of oyster mushroom mycelia in each group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe performance of reducing the concentration of soluble substances in culture materials to 2%Brix with Giant Juncao grass in other growth periods\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe crushed the tender green Giant Juncao grass growing for three months, the Giant Juncao grass growing for one year with high lignification degree, and the dead branches and leaves of Giant Juncao grass separately. Treated them with saturated lime water, used them as the raw materials for non-sterilization culture materials, mixed them with 2% quicklime powder and 0.5% corn flour, and controlled the concentration of soluble substances to 2%Brix measured by refractometer, made mushroom bags as experimental groups. Used the powder of Giant Juncao grass of these three growing periods without treatment as the raw materials for non-sterilization culture materials, mixed them with 2% quicklime powder and 0.5% corn flour, made mushroom bags as control groups, and measured the concentration of their soluble substances by refractometer. Other operations and the spawn were the same as in screening out the non-sterilization cultivation method of oyster mushroom with fresh Giant Juncao grass.\u003c/p\u003e"},{"header":"Results And Discussion","content":"\u003cp\u003e\u003cstrong\u003eScreening out the non-sterilization cultivation method of oyster mushroom with fresh Giant Juncao grass\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Giant Juncao grass that we chose are shown in Figure 1, and the grass powder are shown in Figure 2. The results showed that the mycelia of oyster mushroom (\u003cem\u003ePleurotus sapidus\u003c/em\u003e P969 strain) in each group recovered normally in the first three days. The mycelial growth speed of the oyster mushroom in the no saturated lime water treatment group (LW\u0026times;0) and the low-weight lime water treatment groups were even slightly faster than that in the high-weight lime water treatment groups. However, as shown in Figure 3, from the third day onwards, in the LW\u0026times;0 group, the mycelia of a white mold began to spread near the oyster mushroom mycelia and across some places of the mushroom cultivation bags. At subsequent stages, the mycelia of this white mold became more preponderant, the growth of the oyster mushroom mycelia was hampered and the oyster mushroom mycelia were covered by the white mold mycelia. After 21 days, the oyster mushroom mycelia were almost entirely disappeared, as shown in Table 3. In the same-weight saturated lime water treatment group (LW\u0026times;1), the mycelia growth of white mold was less than that of the LW\u0026times;0 group, and the inhibition on the growth of the oyster mushroom mycelia was also alleviated. In the inoculated part, some oyster mushroom mycelia would not be covered by the white mold mycelia, and a small part of the oyster mushroom mycelia remained even after 21 days, as shown in Table 3. The mycelial growth of the white mold was further diminished in the treatment group subjected to twice the weight of lime water (LW\u0026times;2), which was associated with enhanced growth of the mycelial growth of the oyster mushroom. After 21 days, about 1/3 of the oyster mushroom mycelia did not subside, as shown in Table 3. Starting with four times the weight of the saturated lime water treatment group (LW\u0026times;4), the white mold only appeared sporadically in the form of small mycelial plaque or weak gauze, which did not affect the growth of the oyster mushroom mycelia. In this group, after approximately 21 days, the oyster mushroom mycelia could grow to full extent in the mushroom cultivation bags and did not subside, as shown in Table 3. There were no significant differences observed between the eight times the weight of saturated lime water treatment group (LW\u0026times;8) and LW\u0026times;4 group, and each repetition grew normally, with high stability, as shown in Table 3. Therefore, these groups can facilitate normal growth of the oyster mushroom mycelia.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown in Table 3, In each treatment group, addition of corn flour was associated with a slight increase in the white mold abundance and moderately stronger oyster mushroom mycelia, which was not significant. Based on these observations and previous studies, we deduced that addition of appropriate amount of corn flour is beneficial to improve the mycelial growth of oyster mushroom in this non-sterilization cultivation method. As such, the formula consisting of 0.5% corn flour in the middle of LW\u0026times;4 group was selected for mushroom production experiments. As shown in Figure 4, the mushroom production experiment revealed that after the oyster mushroom mycelia grew to full extent by the average growth speed of 0.43 cm/d in bags, and was incubated for approximately 14 days, the mushroom bags would turn predominantly white and began to exhibit mushroom primordia. Mushrooms grew three or four times in total, the conversion rate was 95% in average (Ratio of total weight of fresh mushroom to dry weight of culture material), and the water content was 91.5%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation, purification and identification of white mold\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological identification of the white mold\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequencing and comparison of ITS sequence of the white mold\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 5, when the white mold grew to approximately 2/3 of the plate on PDA medium, black sporangia became preponderant in most of the area, with the aerial mycelia exhibiting a curly morphology and the peripheral mycelia appearing white. Microscopy was used to discern the morphology of sporangium, spore and mycelia as well as the interval between mycelia, as shown in Figure 6. The genomic DNA was extracted from the white mold as shown in Figure 7, the ITS fragment was obtained through PCR as shown in Figure 8, and the positive clones of TA cloning were identified through PCR as shown in Figure 9. Compared the ITS sequence to the nucleotide data base in GenBank via NCBI BLAST, the comparison results corresponded to \u003cem\u003eMucor circinelloides\u003c/em\u003e. The ITS sequence is available in the Genbank, MN744376.1, and the length is 662 bp. This sequence-based data corroborated the results obtained via morphological identification. Full-length gels are presented in Supplementary Fig. 7, Fig. 8 and Fig. 9 of the Supplementary Information.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasuring the concentration of soluble substances in each group of culture materials with a refractometer\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompared the concentration of soluble substances in each group of culture materials with a refractometer (The result is presented in Supplementary Chart 1 of the Supplementary Information) with the result of screening out the non-sterilization cultivation method of oyster mushroom with fresh Giant Juncao grass, we found that, with the increase of saturated lime water in treatments, the concentration of soluble substances and the contamination caused by \u003cem\u003eM. circinelloides\u0026nbsp;\u003c/em\u003edecreased, and the growth of oyster mushroom mycelia increased accordingly. For all groups with the concentration of soluble substances measured by refractometer equal to or less than 2%Brix, the \u003cem\u003eM. circinelloides\u003c/em\u003e grew very week, which did not affect the growth of the oyster mushroom mycelia, and after approximately 21 days, the oyster mushroom mycelia could grow to full extent and did not subside.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe performance of reducing the concentration of soluble substances in culture materials to 2%Brix with Giant Juncao grass in other growth periods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e According to the results as shown in Table 4, in the control groups, the group with Giant Juncao grass growing for 3 months and the group with Giant Juncao grass growing for 1 year still had high soluble substances, reached 11%Brix, the \u003cem\u003eM. circinelloides\u003c/em\u003e grew vigorously, and the oyster mushroom mycelia could not grow effectively. The group with branches and leaves of dead Giant Juncao grass, had relatively low soluble substances, only 5%Brix, the \u003cem\u003eM. circinelloides\u003c/em\u003e grew like gauze, the oyster mushroom mycelia could grow to full but week and had big black patches. In all the experimental groups with the treatment of saturated lime water, the \u003cem\u003eM. circinelloides\u003c/em\u003e only appeared sporadically and the oyster mushroom mycelia could grow vigorously to full extent and did not subside in each group.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provided a non-sterilization cultivation method for oyster mushroom with fresh Giant Juncao grass. It saved time, labor, equipment and technical costs, and was green and organic. And the raw material Giant Juncao grass had the advantages of tall plant, high yield, wide adaptability, nitrogen fixation, bio-safety and strong sustainability.\u003c/p\u003e \u003cp\u003eFurther study showed that the soluble substances of the formula promoted the growth of \u003cem\u003eM. circinelloides\u003c/em\u003e, and this substantial growth resulted in the inhibition of oyster mushroom mycelial growth. These findings provided an index for quality control and a theoretical basis for further study. With proper optimization and application, this method can be an efficacious in alleviating the burden of malnutrition and food shortages.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Major Special Project of Fujian Province \u0026ldquo;Research and application of key technologies for innovation and industrialized utilization of Juncao\u0026rdquo; (2021NZ029009) and Interdisciplinary integration to promote the high-quality development of Juncao science and Industry (XKJC-712021030).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eYulong Zhang wrote the main manuscript text, designed of the study and collection, analysis, and interpretation of data, and performed experiments, wrote original draft and edited the final manuscript. Zhijun Li conducted the experiments, and assisted with manuscript writing, and checked the final edition. Dongmei Lin contributed to the conception and design of the study and supervised the manuscript. Jingsi Chen performed the early experiments and collected some data. Linsong Shen, Fengmei Wen and Mingjun Xie prepared the figures and tables. Hui Lin, Xingsheng Lin and Hailing Luo contributed to funding acquisition and supervised the manuscript. Fan Yan edited the language. Eyalira Jacob Okal analyzed data and performed some experiments. Yingxing Lin did some experiments. Zhanxi Lin was the supervisor, who administrated this project, and contributed to the conception and design of this study, edited the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFujian Province \u0026ldquo;Research and application of key technologies for innovation and industrialized utilization of Juncao\u0026rdquo; (2021NZ029009) and Interdisciplinary integration to promote the high-quality development of Juncao science and Industry (XKJC-71202103)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data and materials are available upon request to corresponding author. The datasets generated and/or analyzed during the current study are available in the GenBank repository.\u003c/p\u003e\n\u003cp\u003eGene data:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eITS sequence data: GenBank accession number MN744376.1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare all experimental research and field studies on plants (either cultivated or wild), including the cultivation and collection of plant material, comply with relevant institutional, national, and international guide-lines and legislation.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMueller UG, Mikheyev AS, Hong EY, Sen R, Warren DL, Solomon SE, Ishak HD, Cooper M, Miller JL, Shaffer KA, Juenger TE. \u0026quot;Evolution of cold-tolerant fungal symbionts permits winter fungiculture by leafcutter ants at the northern frontier of a tropical ant-fungus symbiosis.\u0026quot; Proceedings of the National Academy of Sciences of the United States of America. 2011;108(10):4053-4056.\u003c/li\u003e\n \u003cli\u003eLechner BE, Josens R. 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Technical regulations of cultivation for Pennisetum giganteum in semi-arid area of Gansu Province. Forest Science and Technology. 2021;(10):78-80.\u003c/li\u003e\n \u003cli\u003eMa YX, Li GT, Wang GH, Liang TY, Yan JQZ, Li JJ. Effects of saline-alkali mixed stress on the growth and physiological characteristics of giant juncao (Pennisetum giganteum z.x. lin). applied ecology and environmental research. 2021;19(1):75-94.\u003c/li\u003e\n \u003cli\u003eQiang W, Jinrong L, Jianying G, Li X, Zhijie Z, Naqi Z. Sand fixation effect of Pennisetum giganteum z.x. Lin in Ulanbuh Desert along the Yellow River. Journal of Inner Mongolia Forestry Science and Technology. 2018;44(02):50-56.\u003c/li\u003e\n \u003cli\u003eXingsheng L, Zhanxi L, Dongmei L, Hui L, Hailing L, Yingping H, Chunmei L, Chaozhi Z. Effects of planting Pennisetum sp. (Giant Juncao) on soil microbial functional diversity and fertility in the barren hillside. Acta Ecologica Sinica. 2014;34(15):4304-4312.\u003c/li\u003e\n \u003cli\u003eChen H, Chu XG, Jia Q. Windbreak and sand fixation of sand plants based on intelligent image processing and plant landscape design. Arabian Journal of Geosciences. 2021;14:1-12.\u003c/li\u003e\n \u003cli\u003eHaizhong C. Discussion on the prospect of JUNCAO industry development in high cold areas. Nong Min Zhi Fu Zhi You. 2017;(16):7.\u003c/li\u003e\n \u003cli\u003eXiaohui Y. China\u0026rsquo;s Agricultural Technical Cooperation: A Case Study on juncao Aid Projects in Papua New Guinea and Fiji. South-south Cooperation and Chinese Foreign Aid. 2019;143-159.\u003c/li\u003e\n \u003cli\u003eZhenghui L. Screening of culture formulation of pleurotus pulmonarius using Pennisetum spp. as substitute. Fujian Agricultural Science and Technology. 2018;(04):19-22.\u003c/li\u003e\n \u003cli\u003eHuide Y. Formulation screening of oyster mushroom cultivated with Giant Juncao grass meal. Agricultural Technology Service. 2020;37(11):22-23.\u003c/li\u003e\n \u003cli\u003eSuada IK, Sudarma IM, Kim BS, Cha JY, Ohga S. Fungal Contaminant Threaten Oyster Mushroom (Pleurotus ostreatus (Jacq. ex Fr.) Kummer) Cultivation in Bali. Journal- Faculty of Agriculture Kyushu University. 2015;60(2):309-313.\u003c/li\u003e\n \u003cli\u003eKhan MW, Ali M, Khan NA, Khan MA, Rehman A, Javed N. Effect of different levels of lime and pH on mycelial growth and production efficiency of oyster mushroom (Pleurotus SPP.). Pakistan Journal of Botany. 2013;45(1):297-302.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 3 and 4 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Giant Juncao grass, Non-sterilization, Oyster mushroom, Sustainable, Malnutrition and food shortages","lastPublishedDoi":"10.21203/rs.3.rs-2158074/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2158074/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNatural processes inspire human innovation. For example, leafcutter ants inhabiting rainforests of South America cut plant debris, transport them to underground nests, dissociate them into small pieces, and use them to cultivate a specific fungus, which serve as the main food source for the whole colony. Inspired by this, Giant Juncao grass (Cenchrus fungigraminus, name used before: Pennisetum giganteum z.x.lin) was used as the raw material. It is a C4 tall grass with rapid growth, high yield, wide-spread adaptability, nitrogen fixation, low-quality soil requirements, biosafety, and can be cultivated on a large scale in many low value and idle lands such as saline and alkali soils, desert and barren mountains. On this basis, we screened out a non-sterilization-based cultivation method of oyster mushroom with fresh Giant Juncao grass. Using our novel method, the bottleneck technologies in the popularization and application of various oyster mushroom cultivation methods in underdeveloped areas were improved. Took Wei W.'s report as reference that the highest annual output of Giant Juncao grass was 450 tons per hectare in southern China. And according to the conversion rate of the oyster mushroom in this research which was 95% in average (Ratio of total weight of fresh mushroom to dry weight of culture material), and the water content which was 91.5%, about 119.7 tons of fresh oyster mushroom could be produced in 2-3 months. Took the nutrient contents of Pleurotus sapidus cultivated by sterilization cultivation method with fresh Giant Juncao grass reported by Zhixiang X. as reference, it could provide 2.37 tons of high-quality protein. In addition, this study found that, the key to the success of this method was the reduction of the soluble substances in culture materials, which significantly reduced the contamination by Mucor circinelloides and provided an enabling environment for the growth of oyster mushroom. These findings provided an index for quality control and a theoretical basis for further study. If this technology is applied appropriately, it will be able to greatly alleviate malnutrition and food shortages in an economical, organic, and sustainable manner in underdeveloped areas.\u003c/p\u003e","manuscriptTitle":"An Economical and Sustainable Non-sterilization Cultivation Method of Oyster Mushroom (Pleurotus spp.) and its Key Influencing Factor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-11 20:52:57","doi":"10.21203/rs.3.rs-2158074/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1c202ae5-2001-47f0-8a6f-396c4f25e3d1","owner":[],"postedDate":"November 11th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-12-06T09:59:14+00:00","versionOfRecord":[],"versionCreatedAt":"2022-11-11 20:52:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2158074","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2158074","identity":"rs-2158074","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}