The microecological mechanism of Cordyceps chanhua promoting soil nitrogen cycling

The microecological mechanism of Cordyceps chanhua promoting soil nitrogen cycling | 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 The microecological mechanism of Cordyceps chanhua promoting soil nitrogen cycling Gongping Hu, Youcui Yang, Tao Wang, Jiaojiao Qu, Zhongshun Xu, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6682575/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background The nitrogen cycle is crucial to the function of the Earth's biosphere. Entomogenous fungihave been proven to promote nitrogen metabolism and cycling in host insects, and transfer nitrogen from insects to soil. However, little is known about the microecological mechanism of entomogenous fungusparticipating in nitrogen cycling and the microecological impact of exonitrogen from entomogenous fungus on soil. Results Here, we report that the entomogenous fungus Cordyceps chanhua secretes nitrate nitrogen and organic nitrogen from its mycelia into the soil environment and absorbs ammonium nitrogen, nitrite nitrogen and hydroxylamine nitrogen from the soil environment into the C. chanhua . Along with the nitrogen exchange process, the bacterial communities related to nitrogen metabolism in the sclerotium of C. chanhua emerge in the soil environment, promoting the soil organic nitrogen cycle process. Redundancy analysis strongly demonstrated that the endogenous/symbiotic bacterial communities within C. chanhua have the greatest impact on ammonium nitrogen and organic nitrogen at the genus level. During the growth process of C. chanhua , the diversity of the bacterial community in its microenvironment significantly decreased. Consistent with this, this study also verified that the exonitrogen of C. chanhua can reduce the diversity of bacterial communities in the soil environment and enrich the bacterial group of Sporosarcina spp., which has a positive promoting effect on nitrogen metabolism. Furthermore, we isolated three highly active nitrogen-transforming dominant strains from the sclerotia of C. chanhua , which further indicates that the nitrogen transport of C. chanhua is closely related to the bacterial community in its mycelia. Conclusions The results of this study demonstrate that the associated/endophytic bacteria of C. chanhua facilitates the participation of C. chanhua in soil nitrogen cycling in its microenvironment. Cordyceps chanhua Soil nitrogen cycle Exonitrogen Bacterial community Growth and development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background The nitrogen cycle is a crucial process in the Earth's biosphere, which ensures the recycling of nitrogen and promotes the balance and stability of ecosystems [ 1 , 2 ]. Soil nitrogen cycling directly affects the stability of ecosystem services and functions, and is closely related to the dynamic balance of soil ecosystem [ 3 ]. The soil nitrogen cycle mainly includes key processes such as absorption of biological residues, biological nitrogen fixation, ammonification, nitrification and denitrification. These processes form an interconnected network system predominantly driven by soil microorganisms [ 4 ]. In nature, microorganisms harmoniously interact and participate in every reaction of the nitrogen cycle [ 5 ], continuously converting nitrogen into various biologically available forms of nitrogen [ 6 ]. For example, nitrogen in the atmosphere is mainly fixed by various nitrogen conversion reactions carried out by complex microbial networks with diverse metabolic functions and converted into inorganic nitrogen or small molecular organic nitrogen, which can be used for the growth of other organisms [ 7 – 9 ], and these microorganisms play ecological functions related to the nitrogen cycle [ 10 ]. In addition to nitrogen-fixing microorganisms, there are also many microorganisms in nature that can transform nitrogen form [ 5 ], such as Proteus , Bacteroides , and other bacteria can reduce nitrite to NO [ 11 ]. Soil fungi are important biological groups in terrestrial ecosystems [ 12 ], which are widely distributed in various soil environments and maintain ecosystem functions by decomposing organic matter and participating in nutrient cycling [ 13 ]. Some fungi in soil will form fungal mycelia during their growth and development, and the interconnection of fungal mycelia will form a three-dimensional network structure [ 14 ], which can effectively improve the absorption and utilization of inorganic nitrogen by organisms [ 15 , 16 ]. Fungi in soil influence the soil nitrogen cycle by regulating the temporal and spatial flow of nitrogen in the ecosystem [ 17 – 19 ]. Entomogenous fungi, as a special group of microorganisms, play a unique role in the nitrogen cycle [ 20 ]. Cordyceps fungi are a special class of entomogenous fungi that live in insects and produce spores on the adult worms of host insects [ 21 ]. During their growth and development, cordyceps fungi can obtain nutrients from the host insects, including nitrogen compounds [ 22 ]. When the fruiting bodies of cordyceps fungi grow out of insect carcasses and release spores, some of the nitrogen in their bodies may be returned to the surrounding environment through catabolism, thereby participating in the nitrogen cycle [ 23 ]. Some cordyceps fungi can break down large nitrogen compounds such as proteins into small substances like amino acids and ammonia when decomposing insect carcasses. These substances can then be utilized by other organisms or further converted into other nitrogen forms to enter the nitrogen cycle [ 24 ]. Other studies have shown that after entomogenous fungi infects insects, it may change the decomposition rate of insect carcasses, thereby affecting the speed and amount of nitrogen released into the environment [ 25 ]. From the perspective of the global nitrogen cycling, there is a close relationship between entomogenous fungi and plants [ 26 ]. It has been found that the transfer of nitrogen source from insects to plants through the mycelium of entomogenous fungi is widespread in nature [ 27 , 28 ]. For example, the entomogenous fungi Metarhizium anisopliae can establish a symbiotic relationship with many plant species [ 29 , 30 ], in which M. anisopliae can transfer nitrogen to plants, and the plants can also transfer carbon-containing compounds synthesized by photosynthesis to M. anisopliae , from which both plants and M. anisopliae benefit and are able to grow better [ 31 , 32 ]. Behie et al. [ 32 ] assessed that insects supply at least 0.4 to 4.0 g of nitrogen per square meter of soil, and Metarhizium sp. plays an efficient role in nitrogen transport and increases plant biomass. In addition, up to 31% of plant nitrogen in ecosystems is transferred to insects by phytophagous insects [ 33 ]. When insects are infected by entomogenous fungi, they break down and release nitrogen-rich compounds into the soil [ 34 ], which can be taken up and utilized by plants and other soil microorganisms. More than 60% of insects that die in nature are killed by fungi [ 35 ], so entomogenous fungi can promote nitrogen return and thus play an important role in nitrogen cycling in the ecosystem. C. chanhua is a fungus-insect complex formed by fungus of Cordyceps sp. parasitizing on cicadae [ 36 ], which mainly consists of the stroma, coremium, spore powder, body wall of the insect, mycelium, and sclerotia (Fig. S1 ). Wild C. chanhua grow in natural habitats with rich and complex vegetation, and can now be cultured artificially (Fig. S2). From the perspective of microbial ecology, the microbial communities in vivo and in vitro play a large role in the growth and development of C. chanhua [ 37 ]. The interaction between fungi and bacteria in the fungal network system is also an important part of the network [ 36 ]. In natural ecosystems, fungi often form a complex microbial network with other microorganisms [ 14 , 38 , 39 ]. The microenvironment surrounding the hyphae of fungi is full of living organisms such as bacteria living together with fungi [ 40 ], which in turn can affect the metabolism of fungi. It has been found that fungi provide nutrients and habitats for some bacteria, and conversely, these bacteria also provide some nutrients to the fungal host [ 41 ]. Some of which are nitrogen nutrients, and this process may involve bacterial communities related to nitrogen transformation [ 42 ]. Nelson et al. [ 43 ] showed that bacterial communities in soil are closely related to soil nitrogen cycling. It has been found that bacterial uptake and utilization of various nitrogen sources are often tightly regulated in response to available nitrogen sources [ 44 ]. Previous studies have shown that different nitrogen forms can have different effects on the resistance of pathogenic microorganisms [ 45 ]. For example, inorganic nitrogen will affect the resistance of Nicotiana tabacum L. to pathogenic microorganisms [ 45 ], and nitrate nitrogen in soil can regulate the symbiotic relationship between legumes and rhizobia [ 46 ], etc. Zeng et al. [ 37 ] discovered that the total content of amino acids and essential amino acids in the fruiting body of sterile-cultivated C. chanhua was lower than that in wild C. chanhua , suggesting that bacteria may play a role in nitrogen transport and metabolism in C. chanhua . In our previous study, we found that C. chanhua can transfer nitrogen from the insect body to the soil environment and also transfer nitrogen from the soil environment to the insect body [ 47 ]. However, little is known about the specific form of nitrogen transfer and the amount of nitrogen transfer. Therefore, it is necessary to explore the microecological mechanism by which C. chanhua promotes the soil nitrogen cycle. The solution to this problem is conducive to demonstrating the composition and structural change patterns of exogenic nitrogen elements during the life history of entomogenous fungi. It not only helps to develop new types of fertilizer products and restore soil ecology, but also provides a reference basis for precise fertilization in agriculture and offers new ideas for improving soil fertility. We collected samples of inner sclerotium, bacteriospheric soil, and control soil of C. chanhua at different growth stages. The objectives were (i) to determine the nitrogen form and content in the growth and development of C. chanhua ; (ii) To determine the relationship between the nitrogen content of C. chanhua and soil bacterial communities; (iii) To investigate the regulation of nitrogen secretion on the structure and function of bacterial community in soil during the growth of C. chanhua ; (iv) To verify the existence of bacterial strains related to nitrogen conversion in the sclerotium of C. chanhua . The results of this study will reveal the ecological functions of entomogenous fungi in natural ecosystems and the interactions between these fungi and microenvironment ecological networks from the perspective of nitrogen cycle. This will considerably enhance our understanding of the mechanism by which entomogenous fungi participate in soil nitrogen cycling from the perspective of endophytic bacteria. Materials and methods C. chanhua cultivation and sample acquisition The strains of C. chanhua were revitalized and activated, and the activated colonies were selected for fermentation in liquid PDA medium, injected into the disinfected tussah silkworm ( Antheraea pernyi ) pupae, cultivated in a sterile environment until the rigor stage, and then buried in the soil environment, as shown in Fig. S3. According to our previous method, inner sclerotium (IS), bacteriospheric soil (BS: soil tightly wrapped about 0.2 cm of the insect body) and control soil (CS: soil without C. chanhua in the same conditions) were taken from C. chanhua at the rigor stage (P1), membrane formation stage (P2) and mature stage (P3) respectively [ 47 ]. Part of the sample was used for routine nitrogen analysis, and the other part of the sample was ground evenly with liquid nitrogen and stored in an ultra-low temperature refrigerator at -80 ℃ until sent to Guangdong MeGG Gene Sequencing Company for high-throughput sequencing analysis. Routine nitrogen testing and calculation of nitrogen conversion rate The total nitrogen content of different samples was determined by using the Kjeldahl nitrogen determination method (LY/T1228-2005), the determination of nitrate nitrogen by phenol disulfonic acid colorimetric method (LY/T1228-2005), and the determination of ammonium nitrogen by indigo phenol blue colorimetric method (LY/T1228-2005). The content of hydroxylamine was determined according to GB/T 6685 − 2007. Spectrophotometric colorimetric determination of nitrite nitrogen content in samples (LY/T1228-2005). According to the method of Datta et al. [ 48 ], the formula for calculating the organic nitrogen content in the sample is ω 0 = ω t - ω i (ω a + ω N ). In the formula, ω 0 represents organic nitrogen, ω t represents total nitrogen, ω i represents inorganic nitrogen, ω a represents ammonium nitrogen, and ω N represents nitrate nitrogen. The nitrogen content secreted or absorbed by C. chanhua was determined using the following formula: ω n = (JC - S n ) - (Sm n - Sck n ), the detailed derivation process of the formula is provided in Supplementary Text S1. The nitrogen conversion rate in the microenvironment of C. chanhua was calculated using the following formula [ 49 ]: (1) Net nitrogen mineralization rate = [(M t +N t ) - (M 0 + N 0 )]/t; (2) Net nitrification rate = (N t -N 0 )/t; (3) Net nitrogen ammonification rate = (M t -M 0 )/t. In the formula, M 0 and M t are the NH 4 + -N content in the soil of C. chanhua just cultivated with soil cover and when C. chanhua mature, mg·kg − 1 ; N 0 and N t were NO 3 − -N contents (mg·kg − 1 ) in the freshly covered soil and at mature stage of C. chanhua ; And t is the corresponding soil sampling days, d, which is 30 d in this study; The unit of nitrogen conversion rate in the equation is mg·kg − 1 ·d − 1 . Bacterial community analysis A total of 21 samples (3 replicates) were collected from IS, BS, and CS of C. chanhua at the P1, P2, and P3 stages, and were sent to Guangdong Mager Gene Technology Co., LTD for 16S rRNA amplicon sequencing analysis. Detailed information about DNA extraction, PCR amplification, OTU annotation, bacterial community diversity analysis, bacterial community structure analysis, bacterial community function prediction analysis in different samples, and bacterial community co-occurrence network analysis can be found in the Supporting Information (Text S2). The effect of exocrine nitrogen aqueous solution on bacterial community structure Fresh soil was collected from the ground of Pinus massoniana forest of Guizhou University (106°39'26"E, 26°27'10"N) and shipped back to the laboratory. After sifting and fully mixing, it was divided into six pots with sterilized surfaces. The soil weight of each pot was 10 kg and the humidity was balanced for 7 d. Two groups were set up in the experiment (Fig. S4), in the experimental group: Aqueous solutions of C. chanhua exonitrogen were added to the soil in 3 pots according to the experimental data of C. chanhua exonitrogen form content (The results of this study showed that in P3 period, the contents of organic nitrogen and nitrate nitrogen in C. chanhua bodies were 0.4469 g/kg and 0.1179 g/kg, respectively. Therefore, in this study, organic nitrogen and nitrate nitrogen were replaced by arginine and potassium nitrate to prepare exogenous nitrogen aqueous solution [ 50 , 51 ], and the ratio was aseptic ultra-pure water: arginine: potassium nitrate = 1000:0.4469:0.1179, that is, 0.4469 g arginine and 0.1179 g potassium nitrate were added to 1 L aseptic ultra-pure water). Soil samples were collected for 30 days (the time from the P1 to P3 of C. chanhua ) and recorded as SNN, during which droplets of C. chanhua exonitrogen aqueous solution were added to keep soil moist. Control group: Sterile ultra-pure water was added to 3 pots of soil, and soil samples were collected 30 days later and recorded as SNck, during which sterile ultra-pure water was added to keep the soil moist. High-throughput sequencing technology was used to detect the bacterial community composition, diversity, and function prediction of the experimental group and the control group, respectively, to compare the differences in soil bacterial community composition with or without C. chanhua exonitrogen aqueous solution dripping, and to analyze the effects of exonitrogen compounds on bacterial community structure. Isolation, culture, and identification of bacteria related to nitrogen conversion in IS of C. chanhua . Collect C. chanhua cultivated under ecological soil cover in P3 stage, remove the soil and mycoderm of C. chanhua , grind 10 g IS samples, and add them to 90 mL normal saline with glass beads. Place it on a shaking table at 28 ℃ for 30 min, fully break up the bacterial micelles, obtain 10 − 1 bacterial solution, and add normal saline to dilute the bacterial solution to 10 − 4 . They were coated on LB medium and beef extract-peptone medium with a coating stick on a sterile operating table and cultured in an incubator at 28 ℃ (Fig. S5), observed and separated every 12 hours, and cultured for about 2 days. The number of bacteria strains isolated from different media was counted according to different colony morphology, and plate scribing was performed for isolation (Fig. S6). The plate medium was cultured in an incubator at 28 ℃ for 1 day, and representative single colonies were selected according to the different morphology characteristics of bacterial colonies, and added into sterilized LB liquid medium or beef extract-peptone liquid medium, and enriched and cultured for 24 h on a shaking table at 28 ℃ and 220 r/min. Ammonium nitrogen and nitrite nitrogen were determined in the cultured bacterial solution. The determination methods were shown in Text S3 and Text S4, and the standard curves were shown in Fig. S7 and Fig. S8 respectively. Bacterial DNA was extracted by chelex 100 method (Fig. S9) from strains with higher ammonia nitrogen and nitrite nitrogen content (see Text S5 for specific methods). The PCR amplification products obtained in the experiment were immediately sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are spliced through DNA MAN software, the spliced gene sequences were analyzed by Blast homologous sequences through NCBI ( http://www.ncbi.nlm.nih.gov ), a preliminary determine its genus name, and get the same in the Genebank strains of the genus 16S rRNA gene sequences, MEGA_11.0.13 software was used to perform multiple sequence comparison between the sequenced sequences and the r DNA-ITS sequences downloaded from GenBank, and the phylogenetic tree was constructed using the Neighbor-joining. Data analysis and availability Data analysis methods are described in Text S6. Results The mutual transfer of nitrogen between inner sclerotium of C. chanhua and the soil environment. As can be seen from Fig. 1 , there was no significant difference in the nitrogen content of CS samples in P2 stage and P3 stage, indicating that the influence of external environmental conditions on the experimental results could be negligible. During the growth of C. chanhua , the total nitrogen content in IS in C. chanhua decreased significantly (Fig. 1 a). Compared with CS, the total nitrogen content in Sm2 (Bacteriospheric soil samples of C. chanhua at P2 stage) was significantly higher than that in Sm3 (Bacteriospheric soil samples of C. chanhua at P3 stage). Therefore, it was speculated that during the growth of C. chanhua , the nitrogen in the insect body was excreted into the soil environment. Our study found that with the growth of C. chanhua , the contents of nitrate nitrogen and organic nitrogen in the body of C. chanhua gradually decreased (Fig. 1 b, Fig. 1 c). The content of nitrate nitrogen and organic nitrogen in soil at P3 stage was higher than that at P2 stage. Therefore, we speculated that nitrate and organic nitrogen are secreted into the soil environment during the growth of C. chanhua . In contrast, the contents of nitrite nitrogen, hydroxylamine nitrogen, and ammonium nitrogen in IS of C. chanhua gradually increased with the growth of C. chanhua (Fig. 1 d to f). The contents of nitrite nitrogen, hydroxylamine nitrogen, and ammonium nitrogen in soil at P3 stage were lower than those at P2 stage. It can be inferred that nitrite nitrogen, hydroxylamine nitrogen, and ammonium nitrogen in the soil environment are absorbed into IS of C. chanhua during the ontogenesis of C. chanhua . To verify the above conjectures, this study deduced the formula ω n = (JC - S n ) - (Sm n - Sck n ) and calculated the nitrogen content exchanged between IS in C. chanhua and the soil environment (Fig. 2 ). Both ω 2 and ω 3 were greater than 0 at P2 and P3 stages (Fig. 2 a), indicating that nitrate and organic nitrogen were secreted into the soil environment from IS of the C. chanhua in P2 and P3 stages, and the contents of nitrate and organic nitrogen in the P2 stage were 66.53 mg/kg and 103.98 mg/kg, respectively, and those in the P3 stage were 117.92 mg/kg and 446.88 mg/kg, respectively. However, the ω 2 and ω 3 of the contents of nitrite, ammonium, and hydroxylamine transported nitrogen in P2 and P3 stages were all less than 0, indicating that C. chanhua absorbed nitrite, ammonium, and hydroxylamine nitrogen from the soil at P2 and P3 stages. According to Fig. 2 b ~ c, the contents of nitrite nitrogen, ammonium nitrogen, and hydroxylamine nitrogen absorbed from the soil environment in the P2 stage were 1151.45 mg/kg, 0.21 mg/kg and 1.17 mg/kg, respectively, and those in the P3 stage were 568.02 mg/kg and 1.17 mg/kg, 2.32 mg/kg, respectively. At P2 and P3 stages, the total nitrogen content was greater than 0, indicating that the content of nitrogen secreted by the resoil-cultivated C. chanhua was greater than that absorbed from the soil environment. The cultivation of C. chanhua by overlaying soil could increase the rate of nitrogen transformation in soil (Fig. 3 ). Compared with CS (0.56 mg·kg − 1 ·d − 1 ), the net nitrogen mineralization rate of BS (3.30 mg·kg − 1 ·d − 1 ) was increased by 473.09% ~ 525.78%. The net nitrogen nitrification rate in BS was 4.80 mg·kg − 1 ·d − 1 , which was 8.46 times of that in the ambient soil (0.57 mg·kg − 1 ·d − 1 ) (Fig. 3 a). Figure 3 b shows that compared with CS (-0.04 mg·kg − 1 ·d − 1 ), the net nitrogen ammonification rate of BS decreased by 180.54% ~ 382.86%. In conclusion, C. chanhua could increase the rates of soil net nitrogen mineralization and nitrification, and reduce the rates of soil net nitrogen ammonification. Bacterial community composition According to the results of high-throughput sequencing, a total of 2,794,234 valid sequences were obtained from the 21 samples sequenced in this experiment, the total number of bases was 898,572,190, and the number of sequences in each sample was 74,622 − 336,952. OTUs belonging to 35 phyla, 93 classes, 191 orders, 272 families, 559 genera, and 9,692 species were detected in 21 groups of samples in P1, P2, and P3 after optimized sequence clustering. In the dilution curve, it can be found that with the increase of the number of sample sequences, the dilution curve of this sequencing tended to be flat and did not rise (Fig. S10), indicating that the amount of sequencing data was reasonable and large enough to reflect most of the bacterial community diversity information in IS, BS and CS samples. Figure 4 a and 3 b clearly show the species composition information of the bacterial communities at the phylum level and the genus level for different samples at different growth stages of C. chanhua . At the phylum level (Fig. 4 a), Proteobacteria, Firmicutes, and Actinobacteria were the top three dominant phyla in the relative abundance of bacterial community in IS of C. chanhua . Proteobacteria (98.39%) was the dominant phylum of JC (Inner sclerotium of C. chanhua at P1 stage), followed by Firmicutes (1.13%). When the cultivation continued to P2 stage, the number of Proteobacteria (68.34%) in S2 (Inner sclerotium of C. chanhua at P2 stage) significantly decreased, while that of Firmicutes (26.95%) and Bacteroidetes (4.6%) increased. When the culture continued to P3 stage, the number of Proteobacteria (60.09%) in S3 (Inner sclerotium of C. chanhua at P3 stage) continued to decrease, but it was still the dominant phylum. The abundance of Firmicutes (19.89%) and Bacteroidetes (0.54%) also decreased, while that of Actinobacteria (19.30%) increased. With the growth and development of C. chanhua , the number of Proteobacteria in IS of C. chanhua gradually decreased, but the dominant phylum in the growth and development of C. chanhua . The abundance of Firmicutes and Bacteroidetes first increased and then decreased, with the highest relative abundance at P2 stage. The number of Proteobacteria showed a relatively increasing trend, with the following order: P3 > P2 > P1. The results of network topology analysis (Fig. S11) showed that at the phylum level, the bacterial symbiotic network structure of IS samples of C. chanhua was relatively simple compared with soil samples. Compared with the P3 stage, the bacterial symbiotic network structure in the P2 stage soil samples was relatively simple. As shown in Fig. 4 a, the bacterial community composition of BS samples and CS samples was similar at the P2 and P3 stages of C. chanhua . In P2 stage, Proteobacteria was the predominant phylum in Sm2 and Sck2 samples, accounting for 48.63% and 49.62%, respectively. However, at P3 stage, the number of Proteobacteria in Sm3 and Sck3 samples decreased, accounting for 33.80% and 34.16%, respectively, but still occupied the main advantage. At P2 stage, Acidobacteria (16.47%) and Bacteroidetes (21.59%) were the predominant phyla. Acidobacteria (23.53%) was the dominant phylum in CS, while Bacteroidetes (5.64%) was relatively less than that in BS. Other bacterial communities, such as Actinobacteria, Chloroflexi, and Verrucomicrobiae, accounted for 5.21%, 2.24%, and 1.81% of the total abundances in BS, and 7.10%, 3.64%, and 3.34% in CS, respectively. At P3 stage, Actinobacteria (25.34%), Bacteroidetes (15.25%), and Firmicutes (13.86%) were dominant in BS samples. In CS samples, Actinobacteria (21.90%), Acidobacteria (14.49%), and Bacteroidetes (8.44%) were the dominant phyla. In addition, the abundance of Proteobacteria (13.86%) and Bacteroidetes (15.25%) in BS samples was relatively lower than that of Proteobacteria (1.52%) and Bacteroidetes (8.24%) in CS, while the number of Acidobacteria (Sm3: 4.87%; Sck3: 14.49%) was relatively increased. Therefore, during the growth of C. chanhua from P2 to P3 stage, the abundance of Proteobacteria, Acidobacteria, and Bacteroidetes decreased, the abundance of Firmicutes and Actinobacteria increased, and the relative abundance of other bacteria did not change much. At different stages of C. chanhua growth, the dominant bacterial groups at the genus level of the samples of IS, BS, and CS in C. chanhua had their characteristics (Fig. 4 b). The bacterial community composition of IS in C. chanhua was relatively simple compared with that in soil. The dominant genera of JC were Stenotrophomonas (20.96%) and Achromobacter (11.89%). Bacillus (23.50%) was the dominant group of S2, and Cedecia (10.30%), Sphingobacter (4.54%), Roamella (3.35%), and Serratia (1.78%) were the secondary dominant groups. In S3 samples, Achromobacter (40.64%), Staphylococcus (19.11%), and Pseudomonas (14.31%) were the main dominant bacteria. Therefore, at different growth stages of C. chanhua , the bacterial groups in IS have different characteristics at the genus level, and the main dominant bacterial groups are also different. In the soil samples, Sphingobacter (14.75%) was the main dominant bacterial group in Sm2 samples. RB41 (3.70%), Bradyrhizobium (3.13%), Pseudomonas (2.52%), Oligotrophomonas (2.04%), Sphingomonas (1.82%), Candidatus_Solibacter (1.81%) is the secondary dominant bacterial group. In addition, it also contains Chryseobacterium (1.49%), Burkholderia-Caballeronia-Paraburkholderia (1.38%), Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium (1.37%), Bryobacte r (1.35%), Reyranella (1.32%), etc. In Sm3 samples, Staphylococcus (12.04%) was the main dominant genus, Sphingobacter (8.94%), Achromobacter (8.38%), and Brevibacterium (7.22%) were also dominant. It is not difficult to see from Fig. 4 b that the bacterial community composition in CS samples at both P2 and P3 stages was more complex than that in BS samples. At the same stage, the composition of bacterial communities in the samples of BS and CS had their characteristics, but the relative abundance of the most dominant bacterial groups in the samples of BS was higher than that in CS. For example, the relative abundance of the dominant group Sphingobacter in Sm2 (14.75%) was higher than that of the dominant group RB41 in Sck2 (3.87%). The relative abundance of the dominant group Staphylococcus in Sm3 (12.04%) was higher than that of the dominant group Paenarthrobacter in Sck3 (3.56%). Venn analysis of the OTU levels of IS samples within the three growth stages of C. chanhua revealed JC and S2 had 15 OTUs, S3 had 103 OTUs, and S2 and S3 had 34 OTUs (Fig. 4 c). A total of 18 OTUs were detected in IS of the three stages of C. chanhua growth, indicating that part of the bacterial community persisted during the growth of C. chanhua . As can be seen from Fig. 4 c, JC has 1,386 OTUs, S2 has 451 OTUs, and S3 has 1,915 OTUs. In addition, a total of 1,496 OTUs were detected in the JC sample, 492 OTUs were detected in S2 sample, and 2,034 OTUs were detected in S3 sample. With the growth of C. chanhua , the bacteria in IS samples of C. chanhua showed a trend of decreasing first and then increasing. Venn analysis was performed on BS of C. chanhua at different growth stages and CS (Fig. 4 d). In P2 stage, Sm2 had a total of 14,192 OTUs, and 10,969 OTUs were unique. Sck2 had a total of 16,084 OTUs and a characteristic of 12,861 OTUs. In P3 stage, there were 12,437 OTUs in Sm3 samples, and 10,478 OTUs were unique. The Sck3 sample had a total of 17,893 OTUs, and 15,384 OTUs were unique. There were 940 OTUs between the soil samples at P2 and P3 stage, and 1,357 OTUs between CS samples. There were 2,832 OTUs between BS and CS at P2 stage. In P3 stage, there were 1,639 OTUs between BS and CS. A total of 583 OTUs were identified between BS and CS at different growth stages. It is not difficult to find from Fig. 4 d that with the growth and development of C. chanhua , the number of OTUs in BS is decreasing, while the number of OTUs in CS is gradually increasing. However, with the growth of C. chanhua , the number of OTU in IS increased, indicating that with the growth of C. chanhua , some bacteria in the soil environment migrated to the inner sclerotium in the C. chanhua through the mycoderm. In addition, the amount of bacterial OTU in soil environment was much higher than that in IS samples of C. chanhua . A total of 41 genera of bacteria were found in different samples at different growth stages of C. chanhua (Fig. 4 e), the proportions of each of the 41 genera are shown in Fig. 4 f. They were Achromobacter (27.72%), Alcaligenes (11.60%), Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium (11.13%) and Aquisphaera (10.37%), Bacillus (4.82%), Bradyrhizobium (4.69%), Lysobacter (4.69), Ensifer (3.86%) and Burkholderia-Caballeronia-Paraburkholderia (2.42%), Chitinophaga (1.9%), Bosea (1.89%) and 30 other bacteria genera with relatively small proportions. These 41 genera were detected in IS, BS, and CS of C. chanhua at different growth stages, indicating that they not only participate in the growth and development of C. chanhua , but also exist widely in the soil environment. It can be seen from Fig. 4 e that JC samples have 3 endemic genera, namely Alcanivorax , Prevotella , and Pigmentiphaga . The S2 sample had two endemic genera, 28-YEA-48 and Lactococcus respectively. The endemic genera of S3 samples were 11 genera, including Verticia , Mumia , Modestobacte r, Limnohabitans , and so on. The endemic genera in Sm2 samples included 20 genera, such as Polyangium , Rhodanobacter , Rhodoblastus , Herbaspirillum , Candidatus_Jidaibacter and Biernaprussia , etc. In Sck2 samples, 11 genera were unique, including Acidocella , Salinispora , Parachlamydia , Variibacter , Acidiphilium , etc. The unique genera in Sm3 are Actinopolymorpha , Sphaerisporangium , Planomicrobium , and 33 other genera. The specific genera in Sck3 samples included 44 genera, including Cnuella, Bacteriovorax , Azoarcus , Anaerocolumna , Erythrobacter , and Microbulbifer . There were 41 bacterial genera involved in the growth and development of C. chanhua , the bacterial communities in C. chanhua at different growth stages were different and had their characteristics. In addition, the bacteria genera in the sclerotium first decreased and then increased, S2 < JC 0.5). There were significant differences in the abundance of bacterial communities of the other 18 genera (Fig. 4 g), including Achromobacter , Bacillus , Sphingobacter , Stenotrophomonas , Morganella , Staphylococcus , Pseudomonas , Cedecea and so on. Among them, Achromobacter had a large abundance in S3, which was significantly different from JC. The abundance of Oligotrophomonas and Serratia were higher at P1 stage, but decreased significantly with the growth of C. chanhua . Cedecea and Ramococcus only appeared in IS at P2 stage, so it was assumed that these two bacteria were related to the formation of the C. chanhua mycoderm. Staphylococcus , Brevibacterium , Saccharopolyspora , Candidatus_Solibacter , Bryobacter , and Candidatus_udaeter almost only appear in IS of C. chanhua at P1 and P3 stage, while their abundance is 0 during P2 stage. Therefore, we assume that these bacteria may be unfavorable to the formation of membranes. The bacterial communities of Sck2 and Sck3 had similar genus levels, and the abundance of Arthrobactoides , Streptomyces , and Pseudomonas in Sck3 was significantly higher than that of Sck2 at the genus level. Except for these bacteria, the abundance of other bacterial communities was basically the same. The abundance of Staphylococcus , Achromobacter , and Brevibacterium in Sm3 was significantly higher than that in Sm2, while the abundances of other bacterial groups had little or no difference. In addition, Arthrobacter and Saccharopolyspora existed only in P3 stage, not in P2 stage. Bacterial community diversity The Coverage index of each copy reached 100% (Table 1 ), indicating that the data measured by this sequencing adequately reflected the bacterial community diversity in each sample. The Ace and Chao indices of IS samples in different cultivation periods were 229.333-721.333, and those of the soil samples were 456-6940.667, respectively. Among them, there was no significant difference in the Ace and Chao indices among the JC, S2, and S3 samples ( p > 0.05), indicating that cultivation time had little effect on the richness of bacterial community in IS of C. chanhua cultivated under soil cover. The Ace index and Chao index in the soil of C. chanhua in P2 stage were significantly higher than those in P3 stage ( p < 0.05), indicating that the richness of soil bacterial community could be reduced by planting C. chanhua in soil. In addition, with the growth of C. chanhua , the bacterial richness in BS tended to decrease, while the richness in CS basically did not change. The possible reason was that the exosomes of C. chanhua produced natural selection for BS bacteria, which enriched some groups and led to a decline in the overall community diversity. The results of Beta diversity analysis showed that the distance between S2, S1, and S3 samples was relatively long (Fig. 4 h), indicating that the bacterial community β diversity in S2 samples was significantly different from that between S1 and S3 samples. According to the PCoA diagram, other samples except Sm3 were aggregated separately, indicating that there was no significant difference in Beta diversity of bacterial community between the sclerotium samples in C. chanhua and the control soil samples. In other words, the bacterial community composition in IS and CS of C. chanhua in different growth and development stages was similar, while the bacterial community composition in BS changed significantly. In addition, the distance between IS of C. chanhua and the soil samples was relatively far, indicating that the diversity of Beta of bacterial communities in the inner sclerotium of C. chanhua was significantly different from that in BS and CS. At the same stage, the distance between the soil samples (such as Sm2 and Sck2, Sm3 and Sck3) was relatively close, indicating that there was no significant difference in Beta diversity of bacterial community between BS and CS at the same stage. Table 1 Bacterial Alpha diversity index between all samples Sample Ace index Chao index Coverage (%) Shannon index Simpson index JC 539.000 ± 97.766c 539.000 ± 97.767c 100 1.019 ± 0.082a 0.424 ± 0.117a S2 229.333 ± 9.838c 229.333 ± 9.838c 100 0.832 ± 0.494a 0.136 ± 0.005b S3 721.333 ± 143.497c 721.333 ± 143.497c 100 0.523 ± 0.526a 0.305 ± 0.106a Sm2 5956.667 ± 288.506a 5956.667 ± 288.506a 100 1.015 ± 0.336a 0.006 ± 0.001b Sck2 6940.667 ± 724.827a 6940.667 ± 724.827a 100 1.068 ± 1.354a 0.001 ± 0.001b Sm3 4506 ± 968.052b 4506 ± 968.052b 100 1.131 ± 0.321a 0.017 ± 0.005b Sck3 6683 ± 577.01a 6683 ± 577.01a 100 1.590 ± 1.012a 0.003 ± 0.0003b Note: Data in the table are Mean ± SD; Different letters in the same column indicate a significant difference (Duncan’s test, p < 0.05). Functional prediction analysis of bacterial communities associated with nitrogen cycle More detailed descriptions of the KEGG and COG functions of the bacterial communities in each sample can be found in the Supplementary Information (Text S7). Figure 5 a shows that the relative abundance of KEGG functions of amino acid metabolism related to nitrogen cycling in bacterial communities in each sample ranged from 11.89–14.30%. As shown in Fig. 5 b, the amino acid metabolism function of the bacterial communities in IS samples of C. chanhua was higher than that of the soil bacterial communities, indicating that the relative abundance of bacterial communities related to nitrogen cycle in C. chanhua was higher than that in the soil samples. There was no significant difference in the relative abundance of amino acid metabolism in KEGG function of the bacterial community in BS samples, but the amino acid metabolism function of bacterial communities in Sm3 samples was significantly higher than that in Sm2 samples. Therefore, it can be inferred that part of the bacterial community with amino acid metabolism in IS of C. chanhua was excreted into the soil environment along with nitrogen during the growth and development of C. chanhua . As can be seen from Fig. 5 c, the relative abundance of amino acid transport and metabolism, the COG function related to the nitrogen cycle, in the bacterial communities in each sample were 9.83–11.11%, which was larger than that of other COG gene functional families. As shown in Fig. 5 d, the amino acid transport and metabolism function of the bacterial community in IS of C. chanhua was higher than that of the soil bacterial community, indicating that the relative abundance of the bacterial community related to the nitrogen cycle in the C. chanhua was higher than that in the soil samples. The relative abundance of amino acid transport metabolism in IS samples of C. chanhua was S2 < JC < S3, indicating that the bacterial community related to nitrogen cycle in C. chanhua showed a tendency to increase with the growth of C. chanhua , but the overall trend was upward. From the soil samples, the relative abundance of amino acid transport metabolism in COG function of bacterial community in CS samples was not significantly different, while the amino acid transport metabolism of bacterial community in Sm3 samples was significantly higher than that in Sm2 samples. Therefore, during the growth and development of C. chanhua , some amino acid metabolizing bacterial communities in IS of C. chanhua were secreted into the soil along with nitrogen, that is, the related bacterial communities in C. chanhua can promote the soil nitrogen cycle. Correlation analysis between soil bacterial community diversity and soil nitrogen Redundancy analysis between soil nitrogen and bacterial community was performed at phylum level (Fig. 6 a). The cumulative interpretation rates of the first and second ranking axes were 66.6% and 23.5%, respectively, and the cumulative interpretation rates reached 90.1%, indicating that the first and second ranking axes could better reflect the correlation between soil nitrogen form and bacterial community at phylum level. The contents of Firmicutes, Proteobacteria and Actinobacteria in the sclerotium samples and soil samples were positively correlated with organic nitrogen (Org-N) and nitrite nitrogen (NO 2 − -N), and it is negatively correlated with ammonium nitrogen (NH 4 + -N) and nitrate nitrogen (NO 3 − -N) and has the greatest influence on organic nitrogen content. Chloroflexi and Acidobacteria are positively correlated with ammonium nitrogen and nitrate nitrogen, and negatively correlated with organic nitrogen and nitrite nitrogen. Among them, each bacterial group has the greatest influence on organic nitrogen and ammonium nitrogen. At the genus level (Fig. 6 b), in each sample, Sphingobacterium , Staphylococcus , Serratia , Achromobacter , Bacillus , Saccharopolyspora and Saccharopolyspora were found to be oligotrophic Stenotrophomonas , Brevibacterium and Pseudomonas were positively correlated with organic nitrogen and nitrite nitrogen, but negatively correlated with ammonium nitrogen and nitrite nitrogen. RB41 was positively correlated with ammonium nitrogen and nitrate nitrogen, and negatively correlated with organic nitrogen and nitrite nitrogen, in which each bacterial community had the greatest influence on ammonium nitrogen and organic nitrogen. Effects of aqueous solution of nitrogen secreted by C. chanhua on soil bacterial communities The results of OTUs cluster analysis showed that 25,213 OTUs were detected in 27 phyla, 74 classes, 223 orders, 433 families, 766 genera, and 1,414 species in SNN and SNck in six samples (there replicates). As can be seen from Fig. S11, Shannon-Winner exponential curve becomes flatter and no longer increases with the increase in the number of sample sequences, indicating that the depth of sequencing data can more comprehensively reflect the bacterial community information in sequenced SNN and SNck samples. At the phylum level, the bacterial community composition of SNN was simpler than that of SNck (Fig. 7 a). In SNN samples, Firmicutes (85.94%) was the dominant phylum, and Proteobacteria (8.08%) was the minor dominant phylum. In SNck samples, Proteobacteria (52.51%) is the main dominant group, while Acidobacteria (13.40%) and Actinobacteria (11.16%) are the secondary dominant groups. Therefore, the bacterial community in the soil samples can be reduced at the phylum level by adding C. chanhua exonitrogen aqueous solution to the soil. At the genus level, the dominant bacterial groups in the soil samples of SNN and SNck are not the same, but the number of dominant bacterial communities is similar (Fig. 7 b). The bacterial community of SNN samples was dominated by Sporosarcina (58.72%). The bacterial groups such as Pseudomonas (3.91%), Bacillus (2.83%), Psychrobacillus (2.42%) and Staphylococcus (1.47%) also accounted for a certain proportion. Compared with SNN, the bacterial community composition of SNck was more complex, it was mainly composed of Massilia (5.19%), Sphingomonas (4.56%), Candidatus_Solibacter (3.59%), Bradyrhizobium (3.52%) and Streptomyces (2.61%), RB41 (2.55%), Burkholderia-Caballeronia-Paraburkholderia (1.37%), Aquisphaera (1.30%), Reyranella (1.06%) and other bacterial groups were composed. Therefore, at the geneus level, the bacterial community composition in SNN and SNck samples was different, indicating that the aqueous nitrogen solution secreted by C. chanhua is able to change the bacterial community in the soil environment. The analysis of the significance of differences between groups showed (Fig. 7 c) that the relative abundance of SNN and SNck in soil samples accounted for no significant difference in the average relative abundance of the top 15 bacterial genera ( p > 0.05), indicating that the bacterial communities in SNN and SNck samples were similar, that is, the aqueous solution of C. chanhua exonitrogen had no significant effect on soil bacterial genera. Venn analysis of SNN and SNck showed that there were 416 OTUs in SNN and SNck, 3,381 OTUs were unique to SNN and 18,287 OTUs were unique to SNck (Fig. 7 d). The amount of bacterial OTUs in SNN was much lower than that in SNck samples, suggesting that aqueous solution of nitrogen secreted by C. chanhua can reduce the diversity of bacterial community in soil environment. The α diversity index of bacterial communities in SNN samples of the experimental group and SNck samples of the control group was compared and analyzed (Fig. 7 e to h). The Ace and Chao indices in SNN and SNck samples were between 1,232-7,196, respectively, and SNN was significantly lower than SNck. In addition, Shannon index showed that there was no significant difference in bacterial diversity between SNN and SNck samples, while Simpson index showed that SNN was significantly higher than SNck. Therefore, aqueous solution of nitrogen secreted by C. chanhua can affect the diversity of bacterial community in soil environment. Ace, Chao and Shannon indices of soil bacterial communities were decreased, indicating reduced richness and diversity. Conversely, the increase in the Simpson index reflects a decrease in community evenness, signifying greater dominance by fewer bacterial taxa [ 52 ]. Functional prediction analysis of bacterial communities in SNN and SNck samples Figure 8 a shows that 35 shared functions of bacterial communities in SNN and SNck samples at the the secondary pathway level include amino acid metabolism, biosynthesis of other secondary metabolites, and cancer: overview, carbohydrate metabolism, cardiovascular disease, cell growth and death, etc.; The bacterial community in SNck samples had two unique structures, including cellular community-eukaryotes and sensory system. There were no endemic structures in the SNN soil samples. Therefore, the C. chanhua exonitrogen aqueous solution can reduce the function of bacterial community in soil environment. More detailed descriptions of the KEGG and COG functions of the bacterial communities can be found in the Supplementary Information (Text S8). As can be seen from Fig. 8 b, the relative abundance of amino acid metabolism, the KEGG function closely related to the nitrogen cycle, in SNN and SNck samples ranged from 12.78–13.86%, was second only to carbohydrate metabolism (12.97%-13.14%). As can be seen from Fig. 8 d, the relative abundance of amino acid metabolism, a KEGG function closely related to nitrogen cycle, in SNN and SNck samples was 12.78%-13.86%, second only to carbohydrate metabolism (12.97%-13.14%). In SNN and SNck samples, the COG functions closely related to the nitrogen cycle, amino acid transport metabolism, accounted for a relative abundance of 12.78–13.86%, higher than other COG functions (Fig. 8 c). As can be seen from Fig. 8 d, in SNN and SNck samples, the relative abundances of KEGG function -- amino acid metabolism, which is closely related to the nitrogen cycle, are 13.86% and 12.78%, respectively; and the relative abundances of COG function -- amino acid transport metabolism are 9.25% and 10.52%, respectively. SNN is higher than SNck in both cases. It can be further demonstrated that C. chanhua exocrine nitrogen aqueous solution can improve the function related to nitrogen cycling in soil environment, that is, C. chanhua exocrine nitrogen aqueous solution can promote soil nitrogen cycling. Isolation and identification of bacterial strains related to nitrogen conversion in C. chanhua . The bacteria isolated and purified by dilution coated plate method and plate scribing method were cultured in LB medium or beef paste peptone medium for 48 h, and the ammonium nitrogen and nitrite nitrogen contents in the bacterial broth were measured. Among them, 15 bacterial strains had significantly higher ability to produce ammonium nitrogen or nitrite nitrogen than other strains, including eight bacteria on LB medium and seven bacteria on beef extract peptone medium, designated as L1 ~ L8 and B1 ~ B7 respectively. As can be seen from Fig. 9 a, the content of ammonium nitrogen in bacterial fluid of strains L4 and B5 was significantly higher than that of other strains. And the content of nitrous nitrogen in bacterial fluid of strains L4 and B1 was significantly higher than that of other strains (Fig. 9 b). Therefore, the B1 strain isolated from IS in C. chanhua can produce NO 2 − -N, the B5 strain can produce NH 4 + -N, and the L4 strain can produce both NH 4 + -N and NO 2 − -N. The colony morphology of L4, B1 and B5 on plate medium is shown in Fig. S14, and the colony color and morphology of L4, B1 and B5 are shown in Table S2. DNA samples were detected by 2% agarose gel electrophoresis, and the PCR characteristic amplification bands of bacterial strains L4, B1 and B5 were clear and bright at about 1,500 bp, with appropriate concentration and no drag (Fig. S15). Gene sequencing results showed that strains B1, B5 and L4 were associated with Delftia sp. (GenBankaccession: MK414965.1) and Delftia acidovorans (GenBankaccession: MK414884.1), the 16S rRNA nucleotide sequence identity of Pseudomonas protegens (MK235212.1) was 99.92%, 99.85% and 100.00%, respectively (Table S3). The taxonomic status of the three bacteria is shown in Table S4. The three bacteria belong to one kingdom, one phylum, one classes, two orders, two families, two genera. By constructing phylogenetic tree with sequences with high similarity (as shown in Fig. 9 c ~ e), it was further revealed that B1 and B5 belonged to Delftia sp., and L4 belonged to Pseudomonas sp. As can be seen from Fig. 4 b, Delftia spp. belonging to B1 and B5 do not belong to the dominant bacteria genera in IS, BS and CS samples of C. chanhua at different growth stages. Pseudomonas sp. belonging to L4 strain which can produce both ammonium nitrogen and nitrite nitrogen was the main dominant bacterial group in S3 samples, accounting for 14.31% relative abundance. In addition, Fig. 6 b shows that the Pseudomonas sp. is positively correlated with the two factors of organic nitrogen and nitrite nitrogen, and negatively correlated with ammonium nitrogen and nitrate nitrogen. Therefore, the Pseudomonas sp. in C. chanhua can promote nitrogen cycling. Figure 10 a shows that the relative abundance of Pseudomonas in C. chanhua decreases first and then increases. In particular, the abundance of Pseudomonas in P3 stage of C. chanhua is much higher than that in P1 stage and P2 stage. Therefore, with the growth of C. chanhua , the content of Pseudomonas related to nitrogen cycle in the C. chanhua is on the rise. The relative abundance of Pseudomonas in the soil samples at P2 stage was greater than that at P3 stage, indicating that the proportion of Pseudomonas in the soil increased with the extension of cultivation time. The content of Pseudomonas in BS was also increasing, so it could be speculated that some Pseudomonas were secreted into the soil with nitrogen during the growth of C. chanhua . Figure 10 b shows that the relative abundance of Pseudomonas in SNN samples was 3.91%, and that in SNck samples was only 0.03%. Therefore, the aqueous nitrogen solution secreted by C. chanhua promoted the enrichment of Pseudomonas in the soil environment, which further verified that endophyte Pseudomonas promoted the participation of C. chanhua in soil nitrogen cycling to a certain extent. Discussion In the process of soil nitrogen cycling, different forms of nitrogen have their unique functions and effects. Soil total nitrogen can be divided into organic nitrogen and inorganic nitrogen, which is an index to measure the nitrogen fertilizer power of soil [ 53 ]. This study found that nitrogen in the C. chanhua cultivated with soil covered would be secreted into the soil environment, and the nitrogen content in BS gradually increased with the extension of cultivation time, suggesting that the cultivation of C. chanhua by covering soil could improve soil fertility. Soil organic nitrogen in C. chanhua is considered to be an important factor in maintaining soil quality and fertility [ 54 ], accounting for more than 80% of soil nitrogen [ 55 ]. Inorganic nitrogen includes nitrate nitrogen, ammonium nitrogen, nitrite nitrogen, hydroxylamine nitrogen, etc. Nitrite nitrogen is one of the nitrogen compounds occurring in nature, and its content is important for understanding soil nitrogen utilization and loss, evaluating soil fertility, and rational nitrogen application [ 56 ]. Nitrate and organic nitrogen are one of the available nitrogen sources that can be directly absorbed by plants [ 57 ]. As an important soil fertility indicator, an increase in soil nitrate content can promote vegetable growth within a certain numerical range [ 58 ]. Ammonium is the main source of nitrogen nutrients absorbed by plants and an important product or reactant in soil nitrogen transformation [ 59 ]. Hydroxylamine (NH 2 OH) and nitrite nitrogen (NO 2 − -N) can chemically react with iron, manganese, and organic matter to produce nitrous oxide and high N 2 O content contributes to global warming [ 60 , 61 ]. The intermediate products of soil nitrogen cycling, ammonium nitrogen, hydroxylamine nitrogen, and nitrite nitrogen, are inhaled by C. chanhua from the soil environment and used for the growth and development of C. chanhua , thereby reducing the contents of ammonium, hydroxylamine, and nitrite nitrogen in the soil environment and increasing the contents of organic nitrogen and nitrite nitrogen. Therefore, the growth of C. chanhua promoted the process of soil nitrogen cycling. In terms of the stability and diversity of bacterial communities, hydroxylamine nitrogen in the soil environment decreases bacterial diversity, whereas ammonium nitrogen can maintain it when increased to a certain extent [ 62 ]. Therefore, cultivation of C. chanhua in soil can maintain the diversity of bacterial communities to some extent. All processes of the soil nitrogen cycle are microbiologically driven [ 63 ], with ammonification, the decomposition of organic nitrides by microorganisms to produce ammonia [ 64 ], the rate-limiting step in the soil nitrogen cycle and a central link in the global nitrogen cycle [ 65 , 66 ]. During the growth of C. chanhua cultivated under soil cover, IS of C. chanhua secreted organic nitrogen into the soil environment, which not only ensures the normal growth of secreted, but also increases the ammonification rate, thereby accelerating the soil nitrogen cycling process [ 67 ]. Denitrification is mainly driven by facultative anaerobic nitrate reducing bacteria [ 68 ]. During denitrification, autotrophic denitrifying microorganisms using carbohydrate metabolism and heterotrophic denitrifying microorganisms using organic carbon sources as electron donors gradually reduce nitrate (NO 3 − ) to nitrite (NO 2 − ) [ 69 ]. In this study, we found that nitrate in C. chanhua cultivated in the soil would be discharged into the soil environment, which would increase denitrification and promote soil nitrogen cycling. Soil net nitrogen mineralization rate is an important indicator of soil nitrogen cycle and supply capacity, reflecting the dynamic process of soil nitrogen transformation from organic to inorganic forms, which is of great significance for agricultural production and ecosystem management [ 70 ]. By increasing the net nitrogen mineralization rate of soil, it can increase the nitrogen supply capacity of soil, improve plant growth and yield [ 71 ], while reducing the risk of nitrogen loss and protecting the environment [ 72 ]. This study found that the soil net nitrogen mineralization rate of BS was significantly higher than that of CS, indicating that C. chanhua can provide more directly absorbable and utilizable nitrogen for the plants in its microenvironment, thereby promoting the growth and development of the plants [ 73 ]. Bei et al. [ 74 ] research found that the activity and quantity of microorganisms in the soil increase with the increase of the net nitrogen mineralization rate, because microorganisms can utilize newly added inorganic nitrogen for growth and metabolism. In addition, the increase in the net nitrogen mineralization rate of the soil will also make the nitrogen cycle process in the soil more active, which is conducive to maintaining soil fertility and the health of the ecosystem [ 75 ]. Therefore, C. chanhua can increase the net nitrogen mineralization rate in the soil environment, thereby promoting the process of soil nitrogen cycling. The nitrification rate of soil net nitrogen is an important indicator for measuring nitrification in the soil nitrogen cycle process, reflecting the dynamic process of nitrogen transformation from ammonium nitrogen to nitrate nitrogen in the soil [ 76 ]. This study found that the nitrification rate of net nitrogen in BS of C. chanhua was significantly higher than that in CS. The increase in the net nitrogen nitrification rate means that more ammonium nitrogen in the soil is converted into nitrate nitrogen, and nitrate nitrogen is more easily absorbed and utilized by plants, thereby enhancing the soil's nitrogen supply capacity and promoting plant growth [ 77 ]. Li et al. [ 78 ] found that higher net nitrogen nitrification rates contribute to the maintenance of ecosystem health and stability. Therefore, C. chanhua promoted soil nitrogen cycling by increasing net nitrogen nitrification efficiency in the soil environment. Soil net ammonification rate was defined as the rate at which soil organic nitrogen was decomposed into ammonium by microorganisms in a certain period of time [ 79 ]. The net ammonification rates of both BS and CS were negative, indicating that the reduction rate of ammonium in soil exceeded the conversion rate of organic nitrogen to ammonium during the growth stage of C. chanhua [ 80 ]. The possible reason was that the cultivation of C. chanhua increased organic matter and microbial activity in the soil environment, which might rapidly absorb ammonium nitrogen in the soil for growth and metabolism [ 79 ]. In this study, we reported that the net nitrogen ammonification rate in BS was significantly lower than that in CS. The possible reason was that the cultivation of C. chanhua reduced the microbial activity in the soil environment, which reduced the decomposition and transformation of organic nitrogen, leading to a decrease in the net nitrogen ammonification rate [ 81 ]. In conclusion, C. chanhua increased soil nitrogen transformation rate, thereby enhancing soil nitrogen cycling. Cordyceps chanhua and its habitat contain not only a large number of fungal community structures [ 69 ], but also abundant bacterial communities [ 82 ]. In this study, it was found that the bacterial community diversity in IS of C. chanhua decreased first and then increased, which was contrary to the results of other researchers [ 47 ], possibly due to the different cultivation environments [ 69 ]. In this study, soil cover was cultivated in the laboratory, sterile water was added for moisturizing, and the amount of water for soil-covered C. chanhua was different from the rainfall in the natural environment. Previous studies have shown that water factor is a major factor affecting microbial activity, gene expression, and community composition [ 82 ]. Therefore, this also leads to the change of bacterial community diversity during the growth of C. chanhua . In this study, the bacterial community composition analysis showed that Stenotrophomonas , Achromobacter , and Serratia were dominant in JC sample. Bacillus and Cedecea were the dominant groups in S2 sample. Achromobacter , Staphylococcus , and Pseudomonas were the dominant bacterial groups in S3 sample. The results of this study were not exactly the same as those of other studies. Qu et al. [ 69 ] studied the bacterial communities of wild C. chanhua , and the results showed that the most dominant bacterial genus was Cedecea , followed by Rickettsia and Burkholderia-Paraburkholderia . Huang et al. [ 83 ] analyzed the microflora of wild C. chanhua in Anji, Zhejiang and found that Achromobacter, f__Enterobacteriaceae_Unclassified, Stenotrophomonas , Burkholderia-Caballeronia-Paraburkholderia Allorhizobium , Neorhizobium, Pararhizobium , Rhizobium are the main dominant bacterial groups in the C. chanhua community. Therefore, the bacterial communities of C. chanhua have different characteristics in different growth environments and different growth stages. Even if there are the same bacterial groups, the abundance is different. As one of the most abundant and abundant microbial populations in the soil microbial community, bacteria are closely related to the complexity of soil [ 84 ]. There were 18 common OTUs in JC, S2, and S3 samples, indicating that the growth and development of C. chanhua cannot be separated from some specific bacterial groups, which is consistent with our previous conclusion [ 47 ]. Liang et al. [ 85 ] found that the bacterial communities in C. sinensis were closely related to the external environmental conditions in which they grew, and the bacterial communities in C. chanhua growing in different places were also different [ 84 ]. Mou et al. [ 86 ] analyzed the diversity of bacterial community in the inner sclerotium and membrane of C. chanhua growing in Guiyang, Guizhou Province, and the soil of its habitat, and the results showed that the diversity of bacterial community in the soil environment was significantly higher than that in the inner sclerotium samples, which was similar to the results of this study. In other words, the diversity of bacterial community in soil samples was significantly higher than that in inner sclerotium samples. And the results of previous studies using modern molecular biological methods were similar [ 87 , 88 ]. The microbial composition in IS of C. chanhua was significantly different from that in the soil environment, possibly because the colonization of cordyceps fungi may reshape the internal environment of the infected insects [ 83 ]. Changes in soil physical and chemical properties can easily affect the diversity and composition of bacterial communities; conversely, environmental factors have a greater impact on soil bacterial community structure [ 89 ]. As the main undertakers of biochemical processes in soil, microorganisms have important contributions to soil nutrient conversion, soil fertility and soil health [ 90 ]. Previous studies have found that different nitrogen forms can affect the bacterial groups in soil environment, such as ammonium nitrogen and nitrate nitrogen can affect the total bacterial and nitrogen-fixing bacteria communities in acidic red soil [ 69 ]. In addition, nitrogen morphology can significantly affect the number of bacterial operational taxa and Shannon index [ 91 ]. Luo et al. [ 22 ] found that C. militaris of the same genus as C. chanhua can utilize organic nitrogen and some inorganic nitrogen, among which the utilization level of ammonium nitrogen is higher than that of other inorganic nitrogen, which is similar to the results of this study, that is, each bacterial community has the greatest influence on ammonium nitrogen and organic nitrogen. Organic nitrogen can effectively improve soil physical and chemical properties, enhance soil fertility, and provide a good growth environment for soil bacteria [ 92 ]. Moreover, organic nitrogen can significantly increase the richness and diversity of bacterial communities in soil [ 93 ]. Ammonium nitrogen is the main form directly absorbed by plant roots from soil, and it is also an important index to measure soil fertility [ 94 ]. The content and distribution of soil ammonium nitrogen absorbed and utilized by plant roots not only have an important impact on the soil nitrogen cycling process [ 95 ], but also the level of plant productivity is closely related to the level of soil ammonium nitrogen content [ 96 ]. The plants can directly absorb and utilize nitrate nitrogen and ammonium nitrogen, and changes in the content of these two kinds of nitrogen can affect plant productivity [ 61 ]. Ding et al. [ 97 ] showed that nitrate nitrogen is the main environmental factor affecting soil microbial community, and Yuan et al. [ 98 ] found that soil ammonium nitrogen plays an important role in soil bacterial community, which may be due to the selective use of different forms of nitrogen in soil. The cultivation of C. chanhua in the soil will release excess nitrate nitrogen in the insect and absorb ammonium nitrogen in the soil environment. Therefore, C. chanhua can be associated with it in the later stage, and the mechanism of C. chanhua improving crop quality and yield can be explored from the perspective of nitrogen fertilizer. Soil bacterial community diversity is very sensitive to changes in the external environment, and is often regarded as an early warning indicator of changes in soil ecosystem, an important basis for evaluating soil quality and fertility [ 99 ], and is also commonly used to evaluate the health of soil ecosystem [ 100 ]. One of the important reasons for the changes of soil bacterial community is the changes of soil physical and chemical properties. Soil bacteria are an extremely important part of soil microecosystem, participating in various activities such as soil nutrient transformation [ 101 ]. This study found that exonitrogen aqueous solution of C. chanhua could reduce the richness, diversity and functions of bacterial communities in soil, and increase the relative abundance of functions related to nitrogen cycling. Soil physical and chemical properties may be an important driving factor for the change of soil microbial community [ 102 ]. Since the exocrine nitrogen aqueous solution of cultivated C. chanhua changes soil nitrogen status, soil microorganisms also change correspondingly. The main bacteria regulating the nitrogen conversion process are ammoniating bacteria and denitrifying bacteria, while the number of nitrifying bacteria is relatively small [ 103 ]. Therefore, this study mainly focused on isolation, cultivation and identification of bacterial strains producing high levels of ammonium and nitrite nitrogen. The results showed that B1 and B5 belong to the genus Delftia sp., and B5 is the type species D. acidovorans of the genus. Delftia not only has good biocontrol potential [ 104 , 105 ]. Moreover, it also affects the transformation and mineralization of organic pollutants in the environment [ 106 ], and the environmental bioreactor potential of degradable polyethylene terephthalate to synthetic polyester materials [ 107 ]. It has been reported that most Delftia bacteria are capable of degrading harmful organic matter [ 108 , 109 ], residual pesticides in soil, etc. [ 106 ]. It was identified that strain L4, which can produce both NH 4 + -N and NO 2 − -N, belongs to Pseudomonas protegens , which is closely related to agriculture and can infect and kill pests. It can be used as a biological control agent [ 110 ]. In this study, three strains of bacteria with high ammonium nitrogen or nitrite nitrogen production were isolated from IS of C. chanhua . As an entomogenous fungus, C. chanhua can be applied to biological control [ 111 ]. Whether its biocontrol ability is related to the bacteria in C. chanhua is worthy of further study. Conclusions In this study, it was found that during the growth of C. chanhua absorb ammonium nitrogen, nitrite nitrogen and hydroxylamine nitrogen in the soil, and nitrate nitrogen and organic nitrogen were secreted. C. chanhua increased the rate of soil nitrogen conversion, thus promoting the soil nitrogen cycle in the microenvironment. The growth of C. chanhua decreased the diversity of soil bacterial community, but increased the relative abundance of bacterial community related to nitrogen cycle. The bacterial community inside and outside the C. chanhua mycelia had the greatest influence on ammonium nitrogen and organic nitrogen. Nitrogen excreted by C. chanhua further decreased the diversity of soil bacterial community, but increased the abundance of bacteria associated with nitrogen cycle. The bacteria related to the conversion were isolated from C. chanhua , and it was verified that the endophytic bacteria promoted the participation of C. chanhua in soil nitrogen cycle. Abbreviations P1 the rigor stage P2 membrane formation stage P3 mature stage IS inner sclerotium BS bacteriospheric soil (soil tightly wrapped about 0.2 cm of the insect body) CS control soil (soil without C. chanhua in the same conditions) SNN Soil samples with aqueous solution of exonitrogen from C. chanhua added SNck Soil samples without the addition of exonitrogen aqueous solution of C. chanhua were compared with SNck JC Inner sclerotium of C. chanhua at P1 stage S2 Inner sclerotium of C. chanhua at P2 stage S3 Inner sclerotium of C. chanhua at P3 stage Sm2 Bacteriospheric soil samples of C. chanhua at P2 stage Sck2 Soil samples without C. chanhua at P2 stage, which contrasted with Sm2 Sm3 Bacteriospheric soil samples of C. chanhua at P3 stage Sck3 Soil samples without C. chanhua at P3 stage, which contrasted with Sm3 Declarations Acknowledgements Not applicable. Authors’ contributions G.H. and X.Z. conceived the project; Y.Y. and Z.X. designed experiments; J.Q., T.W., Y.R., Y.Z., Y.H., and Y.D. performed research; G.F., J.Z., and C.D. analyzed data; and G.H., T.H. and X.Z. wrote the paper. Funding We gratefully acknowledge financial support from the National Natural Science Foundation of China (32060038) and Science and Technology Project of Guizhou Province (Qian ke he Foundation [2020]1Z009). Availability of data and materials The data supporting the findings of this study are available within the paper and its supplementary information. The raw amplicon sequence data (16S rRNA gene) generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1141932. Source data are provided in this work. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Mang Q, Gao J, Li Q, Sun Y, Xu G, Xu P. Metagenomic insight into the effect of probiotics on nitrogen cycle in the Coilia nasus aquaculture pond water. Microorganisms. 2024; 12(3): 627. https://doi.org/10.3390/microorganisms12030627. Gold AC, Thompson SP, Piehler MF. Nitrogen cycling processes within stormwater control measures: A review and call for research. Water Res. 2019; 149: 578-587. https://doi.org/10.1016/j.watres.2018.10.036. Isobe K, Ohte N. Ecological perspectives on microbes involved in N-cycling. Microbes Environ. 2014; 29(1): 4-16. https://doi.org/10.1264/jsme2.me13159. Gunina A, Kuzyakov Y. From energy to (soil organic) matter. Glob Chang Biol. 2022; 28(7): 2169-2182. https://doi.org/10.1111/gcb.16071. Kuypers MMM, Marchant HK. Kartal B. The microbial nitrogen-cycling network . Nat Rev Microbiol. 2018; 16(5): 263-276. https://doi.org/10.1038/nrmicro. Mattoo R, Suman BM. Microbial roles in the terrestrial and aquatic nitrogen cycle-implications in climate change. FEMS Microbiol Lett. 2023; 370: fnad061. https://doi.org/10.1093/femsle/fnad061. Nardi JB, Mackie RI, Dawson JO. Could microbial symbionts of arthropod guts contribute significantly to nitrogen fixation in terrestrial ecosystems? Insect Physiol. 2002; 48(8): 751-763. https://doi.org/10.1016/s0022-1910(02)00105-1. Jones DL, Hodge A, Kuzyakov Y. Plant and mycorrhizal regulation of rhizodeposition. New Phytol. 2004; 163(3): 459-480. https://doi.org/10.1111/j.1469-8137.2004.01130.x. Maia LB, Moura JJ. How biology handles nitrite. Chem Rev. 2014; 114(10): 5273-357. https://doi.org/10.1021/cr400518y. Merloti LF, Mendes LW, Pedrinho A, de Souze LF, Ferrari BM, Tsai SM. forest-to-agriculture conversion in amazon drives soil microbial communities and N-cycle. Soil Biology and Biochemistry. 2019; 137: 107567. https://doi.org/10.1016/j.soilbio.2019.107567. Erisman JW, Sutton MA, Galloway J, Klimont Z, Winiwarter W. How a century of ammonia synthesis changed the world. Nature geoscience. 2008; 1(10): 636-639. https://doi.org/10.1038/ngeo325. Tedersoo L, Bahram M, Põlme S, Kõljalg U, Yorou NS, Wijesundera R, Ruiz LV, Vasco-Palacios AM, Thu PQ, Suija A, Smith ME. Global diversity and geography of soil fungi. Science. 2014; 346(6213): 1256688. https://doi.org/10.1126/science.1256688. Fang L, Lakshmanan P, Su X, Shi Y, Chen Z, Zhang Y, Sun W, Wu J, Xiao R, Chen X. Impact of residual antibiotics on microbial decomposition of livestock manures in eutric regosol: implications for sustainable nutrient recycling and soil carbon sequestration. Journal of Environmental Sciences. 2025; 147: 498-511. https://doi.org/10.1016/j.jes.2023.10.021. Boddy L, Wood J, Redman E, Hynes J, Fricker MD. Fungal network responses to grazing. Fungal Genet Biol. 2010; 47(6): 522-30. https://doi.org/10.1016/j.fgb.2010.01.006. Tobar RM, Azcón R, Barea JM. The improvement of plant N acquisition from an ammonium-treated drought-stressed soil by the fungal symbiont in arbuscular mycorrhizae. Mycorrhiza. 1994; 4: 105-108. https://doi.org/10.1007/BF00203769. Jackson LE, Burger M, Cavagnaro TR. Roots nitrogen transformations and ecosystem services. Annu Rev Plant Biol. 2008; 59: 341-63. https://doi.org/10.1146/annurev.arplant.59.032607.092932. Smith ML, Bruhn JN, Anderson JB. The fungus Armillaria bulbosa is among the largest and oldest living organisms. Nature. 1992; 356 (6368): 428-431. https://doi.org/10.1038/356428a0. Pascale FK, Garbaye J, Tarkka M. The mycorrhiza helper bacteria revisited. New Phytol. 2007; 176(1): 22-36. https://doi.org/10.1111/j.1469-8137.2007.02191.x. Murindangabo YT, Kopecký M, Perná K, Konvalina P, Bohatá A, Kavková M, Nguyen TG, Hoang TN. Relevance of entomopathogenic fungi in soil–plant systems. Plant and Soil. 2024 Feb;495(1):287-310. https://doi.org/10.1007/s11104-023-06325-8. Qu J, Zhou Y, Yu J, Zhang J, Han Y, Zou X. Estimated divergence times of Hirsutella (asexual morphs) in Ophiocordyceps provides insight into evolution of phialide structure. BMC Evol Biol. 2018; 18(1): 111. https://doi.org/10.1186/s12862-018-1223-0. Luo L, Dai F, Xu Z. S, Guan J. Q, Fei G. X; Qu JJ; Yao M, Xue Y, Zhou YM, Zou X. Core microbes in Cordyceps militaris sclerotia and their nitrogen metabolism-related ecological functions. Microbiol Spectr. 2024; 12(10): e0105324. https://doi.org/10.1128/spectrum.01053-24. Sharma H, Sharma N, An SSA. Unique bioactives from zombie fungus ( Cordyceps ) as promising multitargeted neuroprotective agents. Nutrients. 2023; 16(1): 102. https://doi.org/10.3390/nu16010102. Wang X, Li Y, Li X, Sun L, Feng Y, Sa F, Ge Y, Yang S, Liu Y, Li W, Cheng X. Transcriptome and metabolome profiling unveils the mechanisms of naphthalene acetic acid in promoting cordycepin synthesis in Cordyceps militaris . Frontiers in Nutrition. 2023; 10: 1104446. https://doi.org/10.3389/fnut.2023.1104446. Kato T, Inagaki S, Shibata C, Takayanagi K, Uehara H, Nishimura K, Park EY. Topical infection of Cordyceps militaris in silkworm larvae through the cuticle has lower infectivity compared to Beauveria bassiana and Metarhizium anisopliae . Current Microbiology. 2025; 82(1): 1-0. https://doi.org/10.1007/s00284-024-03989-y. Jaber LR, Ownley BH. Can we use entomopathogenic fungi as endophytes for dual biological control of insect pests and plant pathogens? Biological Control. 2018; 1(116): 36-45. https://doi.org/10.1016/j.biocontrol.2017.01.018. Klironomos JN, Hart MM. Animal nitrogen swap for plant carbon. Nature. 2001; 410(6829): 651-2. https://doi.org/10.1038/35070643. Dean SL, Farrer EC, Taylor DL, Porras-Alfaro A, Suding KN, Sinsabaugh RL. Nitrogen deposition alters plant-fungal relationships: Linking below ground dynamics to aboveground vegetation change. Molecular Ecology. 2014; 23(6): 1364-1378. https://doi.org/10.1111/mec.12541. Wyrebek M, Huber C, Sasan RK, Bidochka MJ. Three sympatrically occurring species of Metarhizium show plant rhizosphere specificity. Microbiology. 2011; 157(10): 2904-2911. https://doi.org/10.1099/mic.0.051102-0. Sasan RK, Bidochka MJ. The insect-pathogenic fungus Metarhizium robertsii (Clavicipitaceae) is also an endophyte that stimulates plant root development. Am J Bot. 2012; 9(1): 101-7. https://doi.org/10.3732/ajb.1100136. Behie SW, Moreira CC, Sementchoukova I, Barelli L, Zelisko PM, Bidochka MJ. Carbon translocation from a plant to an insect-pathogenic endophytic fungus. Nat Commun. 2017; 8: 14245. https://doi.org/10.1038/ncomms14245. Saikkonen K, Faeth SH, Helander M, Sullivan TJ. Fungal endophytes: A continuum of interactions with host plants. Annual review of Ecology and Systematics. 1998; 29(1): 319-343. https://doi.org/10.1146/annurev.ecolsys.29.1.319. Behie SW, Zelisko PM, Bidochka MJ. Endophytic insect-parasitic fungi translocate nitrogen directly from insects to plants. Science. 2012: 336(6088): 1576-7. https://doi.org/10.1126/science.1222289. Lovett GM, Christenson LM, Groffman PM, Jones CG, Hart JE, Mitchell MJ. Insect defoliation and nitrogen cycling in forests: laboratory, plot, and watershed studies indicate that most of the nitrogen released from forest foliage as a result of defoliation by insects is redistributed within the ecosystem, whereas only a small fraction of nitrogen is lost by leaching. BioScience. 2002; 52(4): 335-41. https://doi.org/10.1641/0006-3568. Behie SW, Bidochka MJ. Ubiquity of insect-derived nitrogen transfer to plants by endophytic insect-pathogenic fungi: An additional branch of the soil nitrogen cycle. Applied and environmental microbiology. 2014: 80(5): 1553-60. https://doi.org/10.1128/AEM.03338-13. Ma M, Luo J, Li C, Eleftherianos I, Zhang W, Xu L. A Life-And-Death Struggle: Interaction of insects with entomopathogenic fungi across various infection stages. Frontiers in immunology. 2024; 14: 1329843. https://doi.org/10.3389/fimmu.2023.1329843. Nxumalo W, Elateeq AA, Sun Y. Can Cordyceps cicada e be used as an alternative to Cordyceps militaris and Cordyceps sinensis ? - A review. J Ethnopharmacol. 2020; 257: 112879. https://doi.org/doi:10.1016/j.jep.2020.112879. Zeng ZY, Mou D, Luo L, Zhong WL, Duan L, Zou X. Different cultivation environments affect the yield bacterial community and metabolites of Cordyceps cicadae . Frontiers in microbiology. 2021; 12: 669785. https://doi.org/10.3389/fmicb.2021.669785. Garbaye J, Bowen GD. Stimulation of ectomycorrhizal infection of Pinus radiata by some microorganisms associated with the mantle of ectomycorrhizas. New Phytologist. 1989: 112: 383-388. https://doi.org/10.1111/j.1469-8137.1989.tb00327.x. Ballhausen MB, de Boer W. The sapro-rhizosphere: carbon flow from saprotrophic fungi into fungus-feeding bacteria. Soil Biology and Biochemistry. 2016; 102: 14-17. https://doi.org/10.1016/j.soilbio.2016.06.014. Haq IU, Zhang M, Yang P, van Elsas JD. The Interactions of bacteria with fungi in soil: emerging concepts. Adv Appl Microbiol. 2014; 89: 185-215. https://doi.org/10.1016/B978-0-12-800259-9.00005-6. Qu QS, Yang F, Zhao CY, Shi XY. Analysis of the bacteria community in wild Cordyceps cicadae and its influence on the production of HEA and nucleosides in Cordyceps cicadae . Journal of Applied Microbiology. 2019; 127(6): 1759-67. https://doi.org/10.1111/jam.14432. Vannini C, Carpentieri A, Salvioli A, Novero M, Marsoni M, Testa L, de Pinto M. C, Amoresano A, Ortolani F, Bracale M, Bonfante P. An interdomain network: the endobacterium of a mycorrhizal fungus promotes antioxidative responses in both fungal and plant hosts. New Phytol. 2016; 211(1): 265-75. https://doi.org/10.1111/nph.13895. Nelson MB, Martiny AC, Martiny JB. Global biogeography of microbial nitrogen-cycling traits in soil. Proc Natl Acad Sci U S A. 2016; 113(29): 8033-40. https://doi.org/10.1073/pnas.1601070113. Prasse D, Schmitz RA. Small RNAs involved in regulation of nitrogen metabolism. Microbiol Spectr. 2018; 6(4): 10-208. https://doi.org/10.1128/microbiolspec.rwr-0018-2018. Gupta KJ, Brotman Y, Segu S, Zeier T, Zeier J, Persijn ST, Cristescu SM, Harren FJ, Bauwe H, Fernie AR, Kaiser WM. The form of nitrogen nutrition affects resistance against Pseudomonas syringae pv. phaseolicola in tobacco. Journal of Experimental Botany. 2013; 64(2): 553-68. https://doi.org/10.1093/jxb/ers348. Wang Q, Huang Y, Ren Z, Zhang X, Ren J, Su J, Zhang C, Tian J, Yu Y, Gao GF, Li L. Transfer cells mediate nitrate uptake to control root nodule symbiosis. Nature Plants. 2020; 6(7): 800-8. https://doi.org/10.1038/s41477-020-0683-6. Hu G, Zhou Y, Mou D, Qu J, Luo L, Duan L, Xu Z, Zou X. Filtration effect of Cordyceps chanhua mycoderm on bacteria and its transport function on nitrogen. Microbiology Spectrum. 2024; 12(1): e01179-23. https://doi.org/10.1128/spectrum.01179-23. Datta D, Paul M, Murshed M, Teng SW, Schmidtke L. Soil moisture organic carbon and nitrogen content prediction with hyperspectral data using regression models. Sensors (Basel). 2022; 22(20): 7998. https://doi.org/doi:10.3390/s22207998. Motamedi E, Safari M, Salimi M. Improvement of tomato yield and quality using slow release NPK fertilizers prepared by Carnauba wax emulsion starch-based latex and hydrogel nanocomposite combination. Sci Rep. 2023; 13(1): 11118. https://doi.org/10.1038/s41598-023-38445-7. Yan S. Yan H. Wang C. Jiang C. Li W. Chen A. Zhang D. Effects of urea combined with nitrification inhibitor (DMPP) on soil nitrogen transformation and N 2 O emission in vegetable field. Journal of Agro-Environment Science (Chinese). 2025; 45: 1-19. http://kns.cnki.net/kcms/detail/12.1347.S.20250124.1021.002.html. Zhou H, Zhang DG, Jiang ZH, Sun P, Xiao HL, Wang YX, Chen JG. Changes in the soil microbial communities of alpine steppe at Qinghai-Tibetan Plateau under different degradation levels. Sci Total Environ. 2019; 651: 2281-2291. https://doi.org/10.1016/j.scitotenv.2018.09.336. Hill TCJ, Walsh KA, Harris JA, Moffett BF. Using ecological diversity measures with bacterial communities. FEMS Microbiology Ecology. 2003; 43(1): 1-11. https://doi.org/10.1111/fem.2003.43.issue-1. Li C, Aluko OO, Yuan G, Li J, Liu H. The responses of soil organic carbon and total nitrogen to chemical nitrogen fertilizers reduction base on a meta-analysis. Sci Rep. 2022; 12(1): 16326. https://doi.org/10.1038/s41598-022-18684-w. Li J, Lu X, Zhu Q, Zhuang Y, Yang W, Qi D. Nitrate δ 15 N and δ 18 O values reveal mariculture impacts on nitrogen cycling in Sansha Bay, SE China. Journal of Marine Science and Engineering. 2025; 13(2): 343. https://doi.org/10.3390/jmse13020343. Liu XJ, Hu B, Chu CC. Nitrogen assimilation in plants: current status and future prospects. J Genet Genomics. 2022; 49(5): 394-404. https://doi.org/10.1016/j.jgg.2021.12.006. Wang C, Zheng MM, Hu AY, Zhu CQ, Shen RF. diazotroph abundance and community composition in an acidic soil in response to aluminum-tolerant and aluminum-sensitive maize ( Zea mays L.) cultivars under two nitrogen fertilizer forms. Plant and Soil. 2018; 424: 463-478. https://doi.org/10.1007/s11104-017-3550-0. Wang TT, Sun SL, Xu YX, Waigi MG, Odinga ES, Vasilyeva GK, Gao YZ, Hu XJ. Nitrogen regulates the distribution of antibiotic resistance genes in the soil-vegetable system. Front Microbiol. 2022; 13: 848750. https://doi.org/10.3389/fmicb.2022.848750. Zayed O, Hewedy OA, Abdelmoteleb A, Ali M, Youssef MS, Roumia AF, Seymour D, Yuan ZC. Nitrogen journey in plants: from uptake to metabolism stress response and microbe interaction. Biomolecules. 2023: 13(10): 1443. https://doi.org/10.3390/biom13101443. Zhu-Barker X, Cavazos AR, Ostrom NE, Horwath WR, Glass JB. The importance of abiotic reactions for nitrous oxide productio. Biogeochemistry. 2015; 126(3): 251-267. https://doi.org/10.1007/s10533-015-0166-4. Liu SR, Han P, Hink L, Prosser JI, Wagner M, Brüggemann N. Abiotic conversion of extracellular NH 2 OH contributes to N 2 O emission during ammonia oxidation. Environ Sci Technol. 2017; 51(22): 13122-13132. https://doi.org/10.1021/acs.est.7b02360. Li YY, Chen XH, Xie ZX, Li DX, Wu PF, Kong LF, Lin L, Kao SJ, Wang DZ. Bacterial diversity and nitrogen utilization strategies in the upper layer of the northwestern Pacific Ocean. Frontiers in microbiology. 2018; 9: 797. https://doi.org/10.3389/fmicb.2018.00797. Francis CA, Beman JM, Kuypers MM. New processes and players in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation. The ISME journal. 2007; 1(1): 19-27. https://doi.org/10.1038/ismej.2007.8. Guan Y, Wu M, Che S, Yuan S, Yang X, Li S, Tian P, Wu L, Yang M, Wu Z. Effects of continuous straw returning on soil functional microorganisms and microbial communities. Journal of Microbiology. 2023; 61(1): 49-62. https://doi.org/10.1007/s12275-022-00004-6. Prosser JI. Autotrophic nitrification in bacteria. Adv Microb Physiol. 1989; 30: 125-81. https://doi.org/10.1016/s0065-2911(08)60112-5. De Boer W, Gunnewiek PK, Veenhuis M, Bock E, Laanbroek HJ. Nitrification at low pH by aggregated chemolithotrophic bacteria. Applied and Environmental Microbiology. 1991; 57(12): 3600-4. https://doi.org/10.1128/aem.57.12.3600-3604.1991. Luo F, Tang G, Hong S, Gong T, Xin XF, Wang C. Promotion of Arabidopsis immune responses by a rhizosphere fungus via supply of pipecolic acid to plants and selective augment of phytoalexins. Science China Life Sciences. 2023; 66(5): 1119-33. https://doi.org/10.1007/s11427-022-2238-8. Ma Y, Zheng X, Fang Y, Xu K, He S, Zhao M. Autotrophic denitrification in constructed wetlands: achievements and challenges. Bioresour Technol. 2020; 318: 123778. https://doi.org/10.1016/j.biortech.2020.123778. Li YJ, Liu X, Wu DF, Chen BH, Ren XJ, Tang J. Effects of continuous croping of greenhouse cucumber on soil fungal abundance and community structure Province. Acta Agriculturae Boreali-occidentalis Sinica. 2020; 35(1): 194-204. https://doi.org/10.1007/s00284-021-02485-x. Rebello S, Nathan VK, Sindhu R, Binod P, Awasthi MK, Pandey A. Bioengineered microbes for soil health restoration: present status and future. Bioengineered. 2021; 12(2): 12839-53. https://doi.org/10.1080/21655979.2021.2004645. Reimer M, Kopp C, Hartmann T, Zimmermann H, Ruser R, Schulz R, Müller T, Möller K. Assessing long term effects of compost fertilization on soil fertility and nitrogen mineralization rate. Journal of Plant Nutrition and Soil Science. 2023; 186(2): 217-33. https://doi.org/10.1002/jpln.202200270. Yadav MR, Kumar R, Parihar CM, Yadav RK, Jat SL, Ram H, Meena RK, Singh M, Verma AP, Kumar UJ, Ghosh A. Strategies for improving nitrogen use efficiency: A review. Agricultural Reviews. 2017; 38(1): 29-40. https://doi.org/10.18805/ag.v0iOF.7306. Anas M, Liao F, Verma KK, Sarwar MA, Mahmood A, Chen ZL, Li Q, Zeng XP, Liu Y, Li YR. Fate of nitrogen in agriculture and environment: agronomic eco-physiological and molecular approaches to improve nitrogen use efficiency. Biological research. 2020; 53: 1-20. https://doi.org/10.1186/s40659-020-00312-4. Wu C, Wei F, Yan B, Liu G, Wang G. Long-term nitrogen and phosphorus fertilization improved crop yield by influencing rhizosphere nitrogen transformation processes. Applied Soil Ecology. 2025; 208: 105968. https://doi.org/10.1016/j.apsoil.2025.105968. Bei S, Li X, Kuyper TW, Chadwick DR, Zhang J. Nitrogen availability mediates the priming effect of soil organic matter by preferentially altering the straw carbon-assimilating microbial community. Science of the Total Environment. 2022; 815: 152882. https://doi.org/10.1016/j.scitotenv.2021.152882. Du L, Zhong H, Guo X, Li H, Xia J, Chen Q. Nitrogen fertilization and soil nitrogen cycling: Unraveling the links among multiple environmental factors functional genes and transformation rates. Science of The Total Environment. 2024; 951: 175561. https://doi.org/10.1016/j.scitotenv.2024.175561. Zou W, Lang M, Zhang L, Liu B, Chen X. Ammonia-oxidizing bacteria rather than ammonia-oxidizing archaea dominate nitrification in a nitrogen-fertilized calcareous soil. Science of the Total Environment. 2022; 811: 151402. https://doi.org/10.1016/j.scitotenv.2021.151402. Henneron L, Kardol P, Wardle DA, Cros C, Fontaine S. Rhizosphere control of soil nitrogen cycling: a key component of plant economic strategies. New Phytologist. 2020; 228(4): 1269-82. https://doi.org/10.1111/nph.16760. Li Z, Zeng Z, Tian D, Wang J, Fu Z, Zhang F, Zhang R, Chen W, Luo Y, Niu S. Global patterns and controlling factors of soil nitrification rate. Global Change Biology. 2020; 26(7): 4147-57. https://doi.org/10.1111/gcb.15119. Cao Q, Zhou Y, Bai Y, Tang F, Wang Y. Net transformation rates of nitrogen in the rhizosphere soil increase with stand age: The roles of nutrient availability and microbial functional guilds. Journal of Soil Science and Plant Nutrition. 2025; 17: 1-5. https://doi.org/10.1007/s42729-025-02234-0. Yuan X, She W, Guo Y, Qiao Y, Liu L, Song C, Qin S, Zhang Y. Linkage between plant nitrogen preference and rhizosphere effects on soil nitrogen transformation reveals a plant resource adaptive strategies in nitrogen-limited soils. Plant and Soil. 2025; 1-8. https://doi.org/10.1007/s11104-025-07220-0. Burger M Jackson LE. Microbial immobilization of ammonium and nitrate in relation to ammonification and nitrification rates in organic and conventional cropping systems. Soil Biology and Biochemistry. 2003; 35(1): 29-36. https://doi.org/10.1146/annurev.arplant.59.032607.092932. Shao JL, Lai B, Jiang W, Wang JT, Hong YH, Chen FB, Tan SQ, Guo LX. Diversity and co-occurrence patterns of soil bacterial and fungal communities of Chinese Cordyceps habitats at Shergyla Mountain, Tibet: Implications for the occurrence. Microorganisms. 2019; 7(9): 284. https://doi.org/10.3390/microorganisms7090284. Huang A, Wu T, Wu X, Zhang B, Shen Y, Wang S, Song W, Ruan H. Analysis of internal and external microorganism community of wild cicada flowers and identification of the predominant Cordyceps cicadae fungus. Frontiers in Microbiology. 2021; 12: 752791. https://doi.org/10.3389/fmicb.2021.752791. Tahat MM, Alananbeh KM, Othman YA, Leskovar D. Soil health and sustainable agriculture. Sustainability. 2020; 12(12): 4859. https://doi.org/10.3390/su12124859. Liang Y, Hong Y, Mai Z, Zhu Q, Guo L. Internal and external microbial community of the Thitarodes moth, the host of Ophiocordyceps sinensi s. Microorganisms. 2019; 7(11): 517. https://doi.org/10.3390/microorganisms7110517. Mou D, Zeng ZY, Zhong WL, Zhou J, Qu JJ, Zou X. Bacterial community structure and function of Cordyceps cicadae microecosystem in Guiyang. Acta Microbiologica Sinica (China). 2019; 61(02): 469-481. https://doi.org/10.13343/j.cnki.wsxb.20200540. Brodie E, Edwards S, Clipson N. Soil fungal community structure in a temperate upland grassland soil. FEMS Microbiol Ecol. 2003; 45(2): 105-14. https://doi.org/10.1016/S0168-6496(03)00126-0. Meyer A, Focks A, Radl V, Welzl G, Schöning I, Schloter M. Influence of land use intensity on the diversity of ammonia oxidizing bacteria and archaea in soils from grassland ecosystems. Microbial ecology. 2014; 67: 161-166. https://doi.org/10.1007/s00248-013-0310-4. Zhang HQ, Zhao XQ, Shi Y, Liang Y, Shen RF. Changes in soil bacterial communities with increasing distance from maize roots affected by ammonium and nitrate additions. Geoderma. 2021; 398: 115102. https://doi.org/10.1016/j.geoderma.2021.115102. Berendsen RL, Pieterse CM, Bakker PA. The rhizosphere microbiome and plant health. Trends Plant Sci. 2012; 17(8): 478-86. https://doi.org/10.1016/j.tplants.2012.04.001. Sun R, Zhang P, Riggins CW, Zabaloy MC, Rodríguez-Zas S, Villamil MB. Long-term N fertilization decreased diversity and altered the composition of soil bacterial and archaeal communities. Agronomy. 2019; 9(10): 574. https://doi.org/10.3390/agronomy9100574. Wei W, Guan D, Ma M, Jiang X, Fan F, Meng F, Li L, Zhao B, Zhao Y, Cao F, Chen H. Long-term fertilization coupled with rhizobium inoculation promotes soybean yield and alters soil bacterial community composition. Frontiers in Microbiology. 2023; 14: 1161983. https://doi.org/10.3389/fmicb.2023.1161983. Wang X, Feng J, Ao G, Qin W, Han M, Shen Y, Liu M, Chen Y, Zhu B. Globally nitrogen addition alters soil microbial community structure, but has minor effects on soil microbial diversity and richness. Soil Biology and Biochemistry. 2023; 179: 108982. https://doi.org/10.1016/j.soilbio.2023.108982. Christodoulou E, Agapiou A, Anastopoulos I, Omirou M, Ioannides IM. The effects of different soil nutrient management schemes in nitrogen cycling. J Environ Manage. 2019; 243: 168-176. https://doi.org/10.1016/j.jenvman.2019.04.115. Zhang Y, Zhang J, Zhu T, Müller C, Cai Z. Effect of orchard age on soil nitrogen transformation in subtropical China and implications. J Environ Sci (China). 2015; 34: 10-9. https://doi.org/10.1016/j.jes.2015.03.005. Li S, Tian Y, Wu K, Ye Y, Yu J, Zhang J, Liu Q, Hu M, Li H, Tong Y, Harberd NP, Fu X. Modulating plant growth-metabolism coordination for sustainable agriculture. Nature. 2018; 560(7720): 595-600. https://doi.org/10.1038/s41586-018-0415-5. Ding J, Jiang X, Guan D, Zhao B, Ma M, Zhou B, Cao F, Yang X, Li L, Li J. Influence of inorganic fertilizer and organic manure application on fungal communities in a long-term field experiment of Chinese Mollisols. Applied Soil Ecology. 2017; 111: 114-22. https://doi.org/10.1016/j.apsoil.2016.12.003. Yuan Y, Si G, Wang J, Luo T, Zhang G. Bacterial community in alpine grasslands along an altitudinal gradient on the Tibetan Plateau. FEMS microbiology ecology. 2014; 87(1): 121-32. https://doi.org/10.1111/1574-6941.12197. Wen Y, Zhang G, Zhang W, Liu G. Distribution patterns and functional characteristics of soil bacterial communities in desert ecosystems of northern China. Science of The Total Environment. 2023; 905: 167081. https://doi.org/10.1016/j.scitotenv.2023.167081. Mombrikotb SB, Van Agtmaal M, Johnstone E, Crawley MJ, Gweon HS, Griffiths RI, Bell T. The interactions and hierarchical effects of long‐term agricultural stressors on soil bacterial communities. Environmental Microbiology Reports. 2022; 14(5): 711-8. https://doi.org/10.1111/1758-2229.13106. Fu J, Xie X, Zhang S, Kang N, Zong G, Zhang P, Cao G. Rich organic nitrogen impacts clavulanic acid biosynthesis through the arginine metabolic pathway in Streptomyces clavuligerus F613-1. Microbiol Spectr. 2023; 11 (1): e0201722. https://doi.org/10.1128/spectrum.02017-22. Nguyen TM, Ha PT, Le TT, Phan KS, Le TN, Mai TT, Hoang PH. Modification of expanded clay carrier for enhancing the immobilization and nitrogen removal capacity of nitrifying and denitrifying bacteria in the aquaculture system. Journal of bioscience and bioengineering. 2022; 134(1): 41-7. https://doi.org/10.1016/j.jbiosc.2022.04.006. Sørheim R, Torsvik VL, Goksøyr J. Phenotypical divergences between populations of soil bacteria isolated on different media. Microb Ecol . 1989; 17 (2): 181-92. https://doi.org/10.1007/BF02011852. Wen A, Fegan M, Hayward C, Chakraborty S, Sly LI. Phylogenetic relationships among members of the Comamonadaceae and description of Delftia acidovorans (den Dooren de Jong 1926 and Tamaoka et al. 1987) gen. nov. comb. nov. Int J Syst Bacteriol. 1999; 49(2): 567-76. https://doi.org/10.1099/00207713-49-2-567. Han J, Sun L, Dong X, Cai Z, Sun X, Yang H, Wang Y, Song W. Characterization of a novel plant growth-promoting bacteria strain Delftia tsuruhatensis HR4 both as a diazotroph and a potential biocontrol agent against various plant pathogens. Syst Appl Microbiol. 2005; 28 (1): 66-76. https://doi.org/10.1016/j.syapm.2004.09.003. Vásquez-Piñeros MA, Martínez-Lavanchy PM, Jehmlich N, Pieper DH, Rincón CA, Harms H, Junca H, Heipieper HJ. Delftia sp. LCW, a strain isolated from a constructed wetland shows novel properties for dimethylphenol isomers degradation. BMC microbiology. 2018; 18: 1-2. https://doi.org/10.1186/s12866-018-1255-z. Liu J, Xu G, Dong W, Xu N, Xin F, Ma J, Fang Y, Zhou J, Jiang M. Biodegradation of diethyl terephthalate and polyethylene terephthalate by a novel identified degrader Delftia sp. WL-3 and its proposed metabolic pathway. Lett Appl Microbiol. 2018; 67(3): 254-261. https://doi.org/10.1111/lam.13014. Yan H, Yang XJ, Chen J, Yin CH, Xiao CB, Chen H. Synergistic removal of aniline by carbon nanotubes and the enzymes of Delftia sp. XYJ6. J Environ Sci (Chinese). 2018; 23(7): 1165-70. https://doi.org/10.1016/s1001-0742(10)60531-1. Jimenez B, Reboleiro Rivas P, Gonzalez Lopez J, Pesciaroli C, Barghini P, Fenice M. Immobilization of Delftia tsuruhatensis in macro-porous cellulose and biodegradation of phenolic compounds in repeated batch process. J Biotechnol. 2012; 157(1): 148-53. https://doi.org/10.1016/j.jbiotec.2011.09.026. Garrido-Sanz D, Vesga P, Heiman CM, Altenried A, Keel C, Vacheron J. Relation of pest insect-killing and soilborne pathogen-inhibition abilities to species diversification in environmental Pseudomonas protegens . The ISME Journal. 2023; 17(9): 1369-81. https://doi.org/10.1038/s41396-023-01451-8. Horng CT, Yang YL, Chen CC, Huang YS, Chen C, Chen FA. Intraocular pressure-lowering effect of Cordyceps cicadae mycelia extract in a glaucoma rat model. International Journal of Biological Sciences. 2021; 18(4): 1007- 1014. https://doi.org/10.7150/ijms.47912. Additional Declarations No competing interests reported. Supplementary Files SUPPLEMENTALMATERIALwithchangesmarked.docx Supporting Information Additional file 1: Text S1. Derivation of the formula of exocrine nitrogen content of C. chanhua . Text S2. High throughput sequencing and data analysis methods. Text S3. Methods for determination of ammonium nitrogen (NH 4 + -N). Text S4. Methods for determination of nitrite nitrogen (NO 2 - -N). Text S5. Detailed method of extracting bacterial DNA by the Chelex-100 method. Text S6. Data analysis. Text S7. Prediction of KEGG function and COG functions of bacteria in each sample. Text S8. Prediction of KEGG function and COG functions of bacteria in SNN and SNck. Additional file 2: Table S1. Sample names derived from formulas. Table S2. The colour and shape of various strain’s colony. Table S3. Test results of strain 16S rRNA. Table S4. Taxonomic status of strains L4, B1, and B5. Additional file 3: Fig. S1. Structure diagram of C. chanhua . (a) Comic diagram of C. chanhua structure. (b) Cross section of C. chanhua cultivated under soil cover. Fig. S2. Artificial cultivation of C. chanhua . Fig. S3. The experimental method and sample sampling diagram. The red font in the figure represents the sample taken, JC is expressed as the sclerotium in the rigor stage, S2 is expressed as the sclerotium at cortices formation stage, and S3 is expressed as the sclerotium in the mature stage. Sm2 is expressed as the bacteriospheric soil at the stage of membrane formation and Sm3 is expressed as the bacteriospheric soil at the stage of Cordyceps chanhua maturation. Sck2 is expressed as the control soil of membrane formation stage. Sck3 is expressed as control soil at maturity, the same below. Fig. S4. Schematic diagram of preparation of C. chanhua exocrine nitrogen aqueous solution and its effect on soil bacterial community. Fig. S5. The process of isolating bacteria in soils. Fig. S6. The schematic diagram of plate streak. Fig. S7. The standard of ammonia nitrogen concentration line. Fig. S8. The standard of nitrite nitrogen concentration line. Fig. S9. Schematic diagram of extracting bacterial DNA using the chelex 100 method. Fig. S10. Rarefaction curve. Fig. S11. Change of bacterial community co-occurrence network of different samples at the OTU level. (a) IS from three growth stages of C. chanhua . (b) BS and CS at P2 and P3 stages. (c) BS and CS at P2 stage of C. chanhua . (d) BS and CS at P3 stages. (e) Soil samples at P2 and P3 stages. (f) CS samples at P2 and P3 stages of the C. chanhua . Fig. S12. SNN and SNck rarefaction curve. Fig. S13. Analysis of significant differences in KEGG and COG functions of bacterial communities in SNN and SNck samples. (a) Analysis of significant differences in KEGG function of bacterial communities in SNN and SNck soil samples. (b) Analysis of significant difference in prediction of COG function of bacterial communities in SNN and SNck soil samples. Fig. S14. Colony morphology of B1, B5 and L4 on plate medium. Fig. S15. Identification gel map of PCR amplification results. Graphicalabstract.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 25 Oct, 2025 Reviews received at journal 22 Oct, 2025 Reviews received at journal 27 Sep, 2025 Reviewers agreed at journal 20 Sep, 2025 Reviewers agreed at journal 18 Sep, 2025 Reviewers invited by journal 21 Aug, 2025 Submission checks completed at journal 24 Jul, 2025 First submitted to journal 20 Jul, 2025 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. 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1","display":"","copyAsset":false,"role":"figure","size":132421,"visible":true,"origin":"","legend":"\u003cp\u003eThe content of nitrogen form in different samples. The contents of total nitrogen (a), nitrate nitrogen (b), organic nitrogen (c), nitrite nitrogen (d), hydroxylamine nitrogen (e), and ammonium nitrogen (f) in different samples. Data are presented as mean ± SD; ns, \u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05; *, \u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, the figures below is the same.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/940050de42f807bef2af8482.png"},{"id":90298559,"identity":"0ce5e023-a66b-4e58-a16a-7664452a7aa3","added_by":"auto","created_at":"2025-09-01 08:46:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":341083,"visible":true,"origin":"","legend":"\u003cp\u003eNitrogen content in excretion or absorption of \u003cem\u003eC. chanhua\u003c/em\u003e. (a) Analysis of nitrogen content secreted or absorbed by \u003cem\u003eC. chanhua\u003c/em\u003e.(b and c) Local magnification of the amount of nitrogen absorbed by \u003cem\u003eC. chanhua\u003c/em\u003e from the soil environment during the P2 and P3 periods. The arrows indicate magnification of the portion of transferred nitrogen that is less than 0.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/34603620c5dcc97716a7ecdf.png"},{"id":90298552,"identity":"6a8872ff-855b-4e00-923d-37c5247c0d09","added_by":"auto","created_at":"2025-09-01 08:46:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81542,"visible":true,"origin":"","legend":"\u003cp\u003eSoil nitrogen transformation rate. (a) Net nitrogen mineralization rate, net nitrogen nitrification rate, and net nitrogen ammonification rate of BS and CS; (b) Net nitrogen ammonification rate of BS and CS, with arrows showing magnification of the rate of net nitrogen ammonification.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/0a1a3d2f912461d14f3d89cb.png"},{"id":90298574,"identity":"327321f5-0e19-4c5c-9787-5b024e8bfccc","added_by":"auto","created_at":"2025-09-01 08:46:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":248066,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of bacterial community composition and diversity in IS, BS, and CS in different growth stages of \u003cem\u003eC. chanhua.\u003c/em\u003e (a) Relative abundance histograms of bacterial community composition in different samples at Phylum level. (b) Relative abundance histograms of bacterial community composition in different samples at genus level. (c) Statistical analysis of OTUs in IS samples in different growth stages of\u003cem\u003e C. chanhua\u003c/em\u003e. (d) Statistical analysis of OTUs in soil samples in different growth stages of \u003cem\u003eC. chanhua.\u003c/em\u003e(e) Distribution of bacterial communities at the genus level for all samples. (f) The proportion of each of the 41 common genera. (g) Analysis of the significant difference between groups in sclerotium and soil in different growth and development stages of \u003cem\u003eC. chanhu\u003c/em\u003ea. (h) PCoA analysis of β diversity of bacterial communities among different samples.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/40dea49a04ab339d9fa69d42.png"},{"id":90298582,"identity":"5be9504c-e231-4e25-9042-04c9809e957f","added_by":"auto","created_at":"2025-09-01 08:46:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":187459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eC. chanhua\u003c/em\u003e increased the relative abundance of functions related to the nitrogen cycle. (a) Heat map of KEGG functional abundance of bacteria in each sample during the growth and development of \u003cem\u003eC. chanhua\u003c/em\u003e. (b) KEGG function - the relative abundance of amino acid metabolism in each sample. (c) COG functional classification of bacteria in each sample during the growth and development of \u003cem\u003eC.chanhua.\u003c/em\u003e (d) COG function - the relative abundance of amino acid transport and metabolism in each sample.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/7e24b56e49fb39c394f0f08a.png"},{"id":90298571,"identity":"52bbf0ff-c137-4381-8cab-6f101d8e4292","added_by":"auto","created_at":"2025-09-01 08:46:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":64558,"visible":true,"origin":"","legend":"\u003cp\u003eRedundancy analysis between soil nitrogen content and bacterial community. (a) The influence of bacterial community on soil nitrogen form in each sample at the phylum level. (b) Regulation of soil nitrogen form content by bacterial communities in each sample at genus level. In the Figure, red arrows represent nitrogen forms in soil environment, blue arrows represent bacterial taxa in different samples, and the length of arrows represents the degree of correlation between nitrogen forms in soil environment and the data of bacterial taxa in different samples. The blue line indicates the guide line.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/67cd9181837d76f56d3e08af.png"},{"id":90298573,"identity":"043a62cf-3b3f-48fd-9557-1c47315a8c25","added_by":"auto","created_at":"2025-09-01 08:46:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":128986,"visible":true,"origin":"","legend":"\u003cp\u003eThe aqueous solution of exocrine nitrogen of \u003cem\u003eC. chanhua\u003c/em\u003e can reduce the number of soil bacterial communities. (a) Bacterial community relationship diagram of SNN and SNck at phylum level. (b) Relative abundance map of bacterial community composition at the genus level in SNN and SNck samples. (c) The bacterial communities between SNN and SNck samples were significantly different at genus level. (d) Analysis of the amount of common and endemic OTUs in SNN and SNck soil samples. (e to h) α diversity index of bacterial communities in SNN and SNck samples: (e) Ace index; (f) Chao 1 index; (g) Simpson index; (h) Shannon index.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/f63c74fdfdcfb168ba348368.png"},{"id":90298539,"identity":"7ea2eb22-20c3-4963-a25b-c353be0faa56","added_by":"auto","created_at":"2025-09-01 08:46:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":194527,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional prediction analysis of bacterial communities in SNN and SNck. (a) Common and specific functions of bacterial groups in SNN and SNck soil samples. (b) KEGG functional heat maps of bacterial communities in SNN and SNck samples at the secondary taxonomic level. (c) Functional classification of COG of bacterial communities in SNN and SNck samples. (d) Relative abundance maps of functions related to soil nitrogen cycling in SNN and SNck soil samples.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/130eb648ee65ef69d8516612.png"},{"id":90300456,"identity":"0e5e15f1-2863-447e-890a-50c6d243cae3","added_by":"auto","created_at":"2025-09-01 08:54:16","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":226467,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of bacterial strains associated with nitrogen conversion in \u003cem\u003eC. chanhua\u003c/em\u003e. (a) Ammonium nitrogen content of different bacterial strains. (b) Nitrite nitrogen content of different bacterial strains. (c) Phylogenetic tree of 16S rRNA gene sequence of strain B1. (d) Phylogenetic tree of 16S rRNA gene sequence of strain B5. (e) Phylogenetic tree of 16S rRNA gene sequence of strain L4; Lower case letters indicate the significance of α diversity in SNN and SNck bacterial communities at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/99891c70fddd17a8d33e576a.png"},{"id":90298561,"identity":"866926e1-25fb-4ecb-a0d3-1fab2ab3bd69","added_by":"auto","created_at":"2025-09-01 08:46:16","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":191877,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative abundance of \u003cem\u003ePseudomonas\u003c/em\u003e in different samples. (a) The relative abundance of \u003cem\u003ePseudomonas\u003c/em\u003e in each sample. (b) The relative abundance of \u003cem\u003ePseudomonas\u003c/em\u003ein SNN and SNck sample.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/cc2955031bcb586a516c1746.png"},{"id":90304035,"identity":"e689d719-cc4e-4fa1-a889-a7fc2e84b17b","added_by":"auto","created_at":"2025-09-01 09:10:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2895310,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/11f4266c-96b7-4b55-bcdb-4c1bf0f4c4e2.pdf"},{"id":90298542,"identity":"87039384-72b5-4515-b1d3-6211eac00107","added_by":"auto","created_at":"2025-09-01 08:46:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6620119,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting Information\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 1: Text S1\u003c/strong\u003e. Derivation of the formula of exocrine nitrogen content of \u003cem\u003eC. chanhua\u003c/em\u003e. \u003cstrong\u003eText S2.\u003c/strong\u003e High throughput sequencing and data analysis methods.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003cstrong\u003eText S3.\u003c/strong\u003e Methods for determination of ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N).\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003cstrong\u003eText S4. \u003c/strong\u003eMethods for determination of nitrite nitrogen (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N). \u003cstrong\u003eText S5. \u003c/strong\u003eDetailed method of extracting bacterial DNA by the Chelex-100 method. \u003cstrong\u003eText S6.\u003c/strong\u003e Data analysis. \u003cstrong\u003eText S7.\u003c/strong\u003e Prediction of KEGG function and COG functions of bacteria in each sample. \u003cstrong\u003eText S8.\u003c/strong\u003e Prediction of KEGG function and COG functions of bacteria in SNN and SNck.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2: Table S1. \u003c/strong\u003eSample names derived from formulas.\u003cstrong\u003e Table S2. \u003c/strong\u003eThe colour and shape of various strain’s colony. \u003cstrong\u003eTable S3.\u003c/strong\u003e Test results of strain 16S rRNA. \u003cstrong\u003eTable S4.\u003c/strong\u003e Taxonomic status of strains L4, B1, and B5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 3: Fig. S1.\u003c/strong\u003e Structure diagram of \u003cem\u003eC. chanhua\u003c/em\u003e. (a) Comic diagram of \u003cem\u003eC. chanhua\u003c/em\u003e structure. (b) Cross section of \u003cem\u003eC. chanhua\u003c/em\u003e cultivated under soil cover.\u003cstrong\u003e Fig. S2.\u003c/strong\u003e Artificial cultivation of \u003cem\u003eC. chanhua\u003c/em\u003e. \u003cstrong\u003eFig. S3.\u003c/strong\u003e The experimental method and sample sampling diagram. The red font in the figure represents the sample taken, JC is expressed as the sclerotium in the rigor stage, S2 is expressed as the sclerotium at cortices formation stage, and S3 is expressed as the sclerotium in the mature stage. Sm2 is expressed as the bacteriospheric soil at the stage of membrane formation and Sm3 is expressed as the bacteriospheric soil at the stage of \u003cem\u003eCordyceps chanhua\u003c/em\u003e maturation. Sck2 is expressed as the control soil of membrane formation stage. Sck3 is expressed as control soil at maturity, the same below. \u003cstrong\u003eFig. S4. \u003c/strong\u003eSchematic diagram of preparation of \u003cem\u003eC. chanhua \u003c/em\u003eexocrine nitrogen aqueous solution and its effect on soil bacterial community. \u003cstrong\u003eFig. S5.\u003c/strong\u003e The process of isolating bacteria in soils. \u003cstrong\u003eFig. S6.\u003c/strong\u003e The schematic diagram of plate streak. \u003cstrong\u003eFig. S7.\u003c/strong\u003e The standard of ammonia nitrogen concentration line. \u003cstrong\u003eFig. S8.\u003c/strong\u003e The standard of nitrite nitrogen concentration line. \u003cstrong\u003eFig. S9.\u003c/strong\u003e Schematic diagram of extracting bacterial DNA using the chelex 100 method. \u003cstrong\u003eFig. S10.\u003c/strong\u003e Rarefaction curve. \u003cstrong\u003eFig. S11.\u003c/strong\u003e Change of bacterial community co-occurrence network of different samples at the OTU level. (a) IS from three growth stages of \u003cem\u003eC. chanhua\u003c/em\u003e. (b) BS and CS at P2 and P3 stages. (c) BS and CS at P2 stage of \u003cem\u003eC. chanhua\u003c/em\u003e. (d) BS and CS at P3 stages. (e) Soil samples at P2 and P3 stages. (f) CS samples at P2 and P3 stages of the \u003cem\u003eC. chanhua\u003c/em\u003e. \u003cstrong\u003eFig. S12.\u003c/strong\u003e SNN and SNck rarefaction curve. \u003cstrong\u003eFig. S13. \u003c/strong\u003eAnalysis of significant differences in KEGG and COG functions of bacterial communities in SNN and SNck samples. (a) Analysis of significant differences in KEGG function of bacterial communities in SNN and SNck soil samples. (b) Analysis of significant difference in prediction of COG function of bacterial communities in SNN and SNck soil samples. \u003cstrong\u003eFig. S14.\u003c/strong\u003e Colony morphology of B1, B5 and L4 on plate medium. \u003cstrong\u003eFig. S15. \u003c/strong\u003eIdentification gel map of PCR amplification results.\u003c/p\u003e","description":"","filename":"SUPPLEMENTALMATERIALwithchangesmarked.docx","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/bb8d3b736ff50dda052a44fe.docx"},{"id":90298538,"identity":"799e3549-412e-4e03-8718-5495960f4712","added_by":"auto","created_at":"2025-09-01 08:46:16","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":327184,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-6682575/v1/16cc88782557ff7cd5435c63.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The microecological mechanism of Cordyceps chanhua promoting soil nitrogen cycling","fulltext":[{"header":"Background","content":"\u003cp\u003eThe nitrogen cycle is a crucial process in the Earth's biosphere, which ensures the recycling of nitrogen and promotes the balance and stability of ecosystems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Soil nitrogen cycling directly affects the stability of ecosystem services and functions, and is closely related to the dynamic balance of soil ecosystem [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The soil nitrogen cycle mainly includes key processes such as absorption of biological residues, biological nitrogen fixation, ammonification, nitrification and denitrification. These processes form an interconnected network system predominantly driven by soil microorganisms [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In nature, microorganisms harmoniously interact and participate in every reaction of the nitrogen cycle [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], continuously converting nitrogen into various biologically available forms of nitrogen [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For example, nitrogen in the atmosphere is mainly fixed by various nitrogen conversion reactions carried out by complex microbial networks with diverse metabolic functions and converted into inorganic nitrogen or small molecular organic nitrogen, which can be used for the growth of other organisms [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and these microorganisms play ecological functions related to the nitrogen cycle [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In addition to nitrogen-fixing microorganisms, there are also many microorganisms in nature that can transform nitrogen form [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], such as \u003cem\u003eProteus\u003c/em\u003e, \u003cem\u003eBacteroides\u003c/em\u003e, and other bacteria can reduce nitrite to NO [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Soil fungi are important biological groups in terrestrial ecosystems [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], which are widely distributed in various soil environments and maintain ecosystem functions by decomposing organic matter and participating in nutrient cycling [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Some fungi in soil will form fungal mycelia during their growth and development, and the interconnection of fungal mycelia will form a three-dimensional network structure [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which can effectively improve the absorption and utilization of inorganic nitrogen by organisms [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Fungi in soil influence the soil nitrogen cycle by regulating the temporal and spatial flow of nitrogen in the ecosystem [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEntomogenous fungi, as a special group of microorganisms, play a unique role in the nitrogen cycle [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Cordyceps fungi are a special class of entomogenous fungi that live in insects and produce spores on the adult worms of host insects [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. During their growth and development, cordyceps fungi can obtain nutrients from the host insects, including nitrogen compounds [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. When the fruiting bodies of cordyceps fungi grow out of insect carcasses and release spores, some of the nitrogen in their bodies may be returned to the surrounding environment through catabolism, thereby participating in the nitrogen cycle [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Some cordyceps fungi can break down large nitrogen compounds such as proteins into small substances like amino acids and ammonia when decomposing insect carcasses. These substances can then be utilized by other organisms or further converted into other nitrogen forms to enter the nitrogen cycle [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Other studies have shown that after entomogenous fungi infects insects, it may change the decomposition rate of insect carcasses, thereby affecting the speed and amount of nitrogen released into the environment [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. From the perspective of the global nitrogen cycling, there is a close relationship between entomogenous fungi and plants [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. It has been found that the transfer of nitrogen source from insects to plants through the mycelium of entomogenous fungi is widespread in nature [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. For example, the entomogenous fungi \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e can establish a symbiotic relationship with many plant species [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], in which \u003cem\u003eM. anisopliae\u003c/em\u003e can transfer nitrogen to plants, and the plants can also transfer carbon-containing compounds synthesized by photosynthesis to \u003cem\u003eM. anisopliae\u003c/em\u003e, from which both plants and \u003cem\u003eM. anisopliae\u003c/em\u003e benefit and are able to grow better [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Behie et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] assessed that insects supply at least 0.4 to 4.0 g of nitrogen per square meter of soil, and \u003cem\u003eMetarhizium\u003c/em\u003e sp. plays an efficient role in nitrogen transport and increases plant biomass. In addition, up to 31% of plant nitrogen in ecosystems is transferred to insects by phytophagous insects [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. When insects are infected by entomogenous fungi, they break down and release nitrogen-rich compounds into the soil [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], which can be taken up and utilized by plants and other soil microorganisms. More than 60% of insects that die in nature are killed by fungi [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], so entomogenous fungi can promote nitrogen return and thus play an important role in nitrogen cycling in the ecosystem.\u003c/p\u003e\u003cp\u003e\u003cem\u003eC. chanhua\u003c/em\u003e is a fungus-insect complex formed by fungus of \u003cem\u003eCordyceps\u003c/em\u003e sp. parasitizing on cicadae [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], which mainly consists of the stroma, coremium, spore powder, body wall of the insect, mycelium, and sclerotia (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Wild \u003cem\u003eC. chanhua\u003c/em\u003e grow in natural habitats with rich and complex vegetation, and can now be cultured artificially (Fig. S2). From the perspective of microbial ecology, the microbial communities in vivo and in vitro play a large role in the growth and development of \u003cem\u003eC. chanhua\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The interaction between fungi and bacteria in the fungal network system is also an important part of the network [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In natural ecosystems, fungi often form a complex microbial network with other microorganisms [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The microenvironment surrounding the hyphae of fungi is full of living organisms such as bacteria living together with fungi [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], which in turn can affect the metabolism of fungi. It has been found that fungi provide nutrients and habitats for some bacteria, and conversely, these bacteria also provide some nutrients to the fungal host [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Some of which are nitrogen nutrients, and this process may involve bacterial communities related to nitrogen transformation [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Nelson et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] showed that bacterial communities in soil are closely related to soil nitrogen cycling. It has been found that bacterial uptake and utilization of various nitrogen sources are often tightly regulated in response to available nitrogen sources [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Previous studies have shown that different nitrogen forms can have different effects on the resistance of pathogenic microorganisms [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. For example, inorganic nitrogen will affect the resistance of \u003cem\u003eNicotiana tabacum\u003c/em\u003e L. to pathogenic microorganisms [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], and nitrate nitrogen in soil can regulate the symbiotic relationship between legumes and rhizobia [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], etc. Zeng et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] discovered that the total content of amino acids and essential amino acids in the fruiting body of sterile-cultivated \u003cem\u003eC. chanhua\u003c/em\u003e was lower than that in wild \u003cem\u003eC. chanhua\u003c/em\u003e, suggesting that bacteria may play a role in nitrogen transport and metabolism in \u003cem\u003eC. chanhua\u003c/em\u003e. In our previous study, we found that \u003cem\u003eC. chanhua\u003c/em\u003e can transfer nitrogen from the insect body to the soil environment and also transfer nitrogen from the soil environment to the insect body [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. However, little is known about the specific form of nitrogen transfer and the amount of nitrogen transfer. Therefore, it is necessary to explore the microecological mechanism by which \u003cem\u003eC. chanhua\u003c/em\u003e promotes the soil nitrogen cycle. The solution to this problem is conducive to demonstrating the composition and structural change patterns of exogenic nitrogen elements during the life history of entomogenous fungi. It not only helps to develop new types of fertilizer products and restore soil ecology, but also provides a reference basis for precise fertilization in agriculture and offers new ideas for improving soil fertility.\u003c/p\u003e\u003cp\u003eWe collected samples of inner sclerotium, bacteriospheric soil, and control soil of \u003cem\u003eC. chanhua\u003c/em\u003e at different growth stages. The objectives were (i) to determine the nitrogen form and content in the growth and development of \u003cem\u003eC. chanhua\u003c/em\u003e; (ii) To determine the relationship between the nitrogen content of \u003cem\u003eC. chanhua\u003c/em\u003e and soil bacterial communities; (iii) To investigate the regulation of nitrogen secretion on the structure and function of bacterial community in soil during the growth of \u003cem\u003eC. chanhua\u003c/em\u003e; (iv) To verify the existence of bacterial strains related to nitrogen conversion in the sclerotium of \u003cem\u003eC. chanhua\u003c/em\u003e. The results of this study will reveal the ecological functions of entomogenous fungi in natural ecosystems and the interactions between these fungi and microenvironment ecological networks from the perspective of nitrogen cycle. This will considerably enhance our understanding of the mechanism by which entomogenous fungi participate in soil nitrogen cycling from the perspective of endophytic bacteria.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eC. chanhua\u003c/b\u003e \u003cb\u003ecultivation and sample acquisition\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe strains of \u003cem\u003eC. chanhua\u003c/em\u003e were revitalized and activated, and the activated colonies were selected for fermentation in liquid PDA medium, injected into the disinfected tussah silkworm (\u003cem\u003eAntheraea pernyi\u003c/em\u003e) pupae, cultivated in a sterile environment until the rigor stage, and then buried in the soil environment, as shown in Fig. S3. According to our previous method, inner sclerotium (IS), bacteriospheric soil (BS: soil tightly wrapped about 0.2 cm of the insect body) and control soil (CS: soil without \u003cem\u003eC. chanhua\u003c/em\u003e in the same conditions) were taken from \u003cem\u003eC. chanhua\u003c/em\u003e at the rigor stage (P1), membrane formation stage (P2) and mature stage (P3) respectively [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Part of the sample was used for routine nitrogen analysis, and the other part of the sample was ground evenly with liquid nitrogen and stored in an ultra-low temperature refrigerator at -80 ℃ until sent to Guangdong MeGG Gene Sequencing Company for high-throughput sequencing analysis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRoutine nitrogen testing and calculation of nitrogen conversion rate\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe total nitrogen content of different samples was determined by using the Kjeldahl nitrogen determination method (LY/T1228-2005), the determination of nitrate nitrogen by phenol disulfonic acid colorimetric method (LY/T1228-2005), and the determination of ammonium nitrogen by indigo phenol blue colorimetric method (LY/T1228-2005). The content of hydroxylamine was determined according to GB/T 6685\u0026thinsp;\u0026minus;\u0026thinsp;2007. Spectrophotometric colorimetric determination of nitrite nitrogen content in samples (LY/T1228-2005). According to the method of Datta et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], the formula for calculating the organic nitrogen content in the sample is ω\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;ω\u003csub\u003et\u003c/sub\u003e - ω\u003csub\u003ei\u003c/sub\u003e (ω\u003csub\u003ea\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ω\u003csub\u003eN\u003c/sub\u003e). In the formula, ω\u003csub\u003e0\u003c/sub\u003e represents organic nitrogen, ω\u003csub\u003et\u003c/sub\u003e represents total nitrogen, ω\u003csub\u003ei\u003c/sub\u003e represents inorganic nitrogen, ω\u003csub\u003ea\u003c/sub\u003e represents ammonium nitrogen, and ω\u003csub\u003eN\u003c/sub\u003e represents nitrate nitrogen. The nitrogen content secreted or absorbed by \u003cem\u003eC. chanhua\u003c/em\u003e was determined using the following formula: ω\u003csub\u003en\u003c/sub\u003e = (JC - S\u003csub\u003en\u003c/sub\u003e) - (Sm\u003csub\u003en\u003c/sub\u003e - Sck\u003csub\u003en\u003c/sub\u003e), the detailed derivation process of the formula is provided in Supplementary Text S1. The nitrogen conversion rate in the microenvironment of \u003cem\u003eC. chanhua\u003c/em\u003e was calculated using the following formula [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]: (1) Net nitrogen mineralization rate = [(M\u003csub\u003et\u003c/sub\u003e+N\u003csub\u003et\u003c/sub\u003e) - (M\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;N\u003csub\u003e0\u003c/sub\u003e)]/t; (2) Net nitrification rate = (N\u003csub\u003et\u003c/sub\u003e-N\u003csub\u003e0\u003c/sub\u003e)/t; (3) Net nitrogen ammonification rate = (M\u003csub\u003et\u003c/sub\u003e-M\u003csub\u003e0\u003c/sub\u003e)/t. In the formula, M\u003csub\u003e0\u003c/sub\u003e and M\u003csub\u003et\u003c/sub\u003e are the NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N content in the soil of \u003cem\u003eC. chanhua\u003c/em\u003e just cultivated with soil cover and when \u003cem\u003eC. chanhua\u003c/em\u003e mature, mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; N\u003csub\u003e0\u003c/sub\u003e and N\u003csub\u003et\u003c/sub\u003e were NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N contents (mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in the freshly covered soil and at mature stage of \u003cem\u003eC. chanhua\u003c/em\u003e; And t is the corresponding soil sampling days, d, which is 30 d in this study; The unit of nitrogen conversion rate in the equation is mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacterial community analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 21 samples (3 replicates) were collected from IS, BS, and CS of \u003cem\u003eC. chanhua\u003c/em\u003e at the P1, P2, and P3 stages, and were sent to Guangdong Mager Gene Technology Co., LTD for 16S rRNA amplicon sequencing analysis. Detailed information about DNA extraction, PCR amplification, OTU annotation, bacterial community diversity analysis, bacterial community structure analysis, bacterial community function prediction analysis in different samples, and bacterial community co-occurrence network analysis can be found in the Supporting Information (Text S2).\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe effect of exocrine nitrogen aqueous solution on bacterial community structure\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFresh soil was collected from the ground of \u003cem\u003ePinus massoniana\u003c/em\u003e forest of Guizhou University (106\u0026deg;39'26\"E, 26\u0026deg;27'10\"N) and shipped back to the laboratory. After sifting and fully mixing, it was divided into six pots with sterilized surfaces. The soil weight of each pot was 10 kg and the humidity was balanced for 7 d. Two groups were set up in the experiment (Fig. S4), in the experimental group: Aqueous solutions of \u003cem\u003eC. chanhua\u003c/em\u003e exonitrogen were added to the soil in 3 pots according to the experimental data of \u003cem\u003eC. chanhua\u003c/em\u003e exonitrogen form content (The results of this study showed that in P3 period, the contents of organic nitrogen and nitrate nitrogen in \u003cem\u003eC. chanhua\u003c/em\u003e bodies were 0.4469 g/kg and 0.1179 g/kg, respectively. Therefore, in this study, organic nitrogen and nitrate nitrogen were replaced by arginine and potassium nitrate to prepare exogenous nitrogen aqueous solution [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], and the ratio was aseptic ultra-pure water: arginine: potassium nitrate\u0026thinsp;=\u0026thinsp;1000:0.4469:0.1179, that is, 0.4469 g arginine and 0.1179 g potassium nitrate were added to 1 L aseptic ultra-pure water). Soil samples were collected for 30 days (the time from the P1 to P3 of \u003cem\u003eC. chanhua\u003c/em\u003e) and recorded as SNN, during which droplets of \u003cem\u003eC. chanhua\u003c/em\u003e exonitrogen aqueous solution were added to keep soil moist. Control group: Sterile ultra-pure water was added to 3 pots of soil, and soil samples were collected 30 days later and recorded as SNck, during which sterile ultra-pure water was added to keep the soil moist. High-throughput sequencing technology was used to detect the bacterial community composition, diversity, and function prediction of the experimental group and the control group, respectively, to compare the differences in soil bacterial community composition with or without \u003cem\u003eC. chanhua\u003c/em\u003e exonitrogen aqueous solution dripping, and to analyze the effects of exonitrogen compounds on bacterial community structure.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsolation, culture, and identification of bacteria related to nitrogen conversion in IS of\u003c/b\u003e \u003cb\u003eC. chanhua\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eCollect \u003cem\u003eC. chanhua\u003c/em\u003e cultivated under ecological soil cover in P3 stage, remove the soil and mycoderm of \u003cem\u003eC. chanhua\u003c/em\u003e, grind 10 g IS samples, and add them to 90 mL normal saline with glass beads. Place it on a shaking table at 28 ℃ for 30 min, fully break up the bacterial micelles, obtain 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e bacterial solution, and add normal saline to dilute the bacterial solution to 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e. They were coated on LB medium and beef extract-peptone medium with a coating stick on a sterile operating table and cultured in an incubator at 28 ℃ (Fig. S5), observed and separated every 12 hours, and cultured for about 2 days. The number of bacteria strains isolated from different media was counted according to different colony morphology, and plate scribing was performed for isolation (Fig. S6). The plate medium was cultured in an incubator at 28 ℃ for 1 day, and representative single colonies were selected according to the different morphology characteristics of bacterial colonies, and added into sterilized LB liquid medium or beef extract-peptone liquid medium, and enriched and cultured for 24 h on a shaking table at 28 ℃ and 220 r/min. Ammonium nitrogen and nitrite nitrogen were determined in the cultured bacterial solution. The determination methods were shown in Text S3 and Text S4, and the standard curves were shown in Fig. S7 and Fig. S8 respectively. Bacterial DNA was extracted by chelex 100 method (Fig. S9) from strains with higher ammonia nitrogen and nitrite nitrogen content (see Text S5 for specific methods). The PCR amplification products obtained in the experiment were immediately sent to Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results are spliced through DNA MAN software, the spliced gene sequences were analyzed by Blast homologous sequences through NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), a preliminary determine its genus name, and get the same in the Genebank strains of the genus 16S rRNA gene sequences, MEGA_11.0.13 software was used to perform multiple sequence comparison between the sequenced sequences and the r DNA-ITS sequences downloaded from GenBank, and the phylogenetic tree was constructed using the Neighbor-joining.\u003c/p\u003e\u003cp\u003e\u003cb\u003eData analysis and availability\u003c/b\u003e\u003c/p\u003e\u003cp\u003eData analysis methods are described in Text S6.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eThe mutual transfer of nitrogen between inner sclerotium of\u003c/b\u003e \u003cb\u003eC. chanhua\u003c/b\u003e \u003cb\u003eand the soil environment.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, there was no significant difference in the nitrogen content of CS samples in P2 stage and P3 stage, indicating that the influence of external environmental conditions on the experimental results could be negligible. During the growth of \u003cem\u003eC. chanhua\u003c/em\u003e, the total nitrogen content in IS in \u003cem\u003eC. chanhua\u003c/em\u003e decreased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Compared with CS, the total nitrogen content in Sm2 (Bacteriospheric soil samples of \u003cem\u003eC. chanhua\u003c/em\u003e at P2 stage) was significantly higher than that in Sm3 (Bacteriospheric soil samples of \u003cem\u003eC. chanhua\u003c/em\u003e at P3 stage). Therefore, it was speculated that during the growth of \u003cem\u003eC. chanhua\u003c/em\u003e, the nitrogen in the insect body was excreted into the soil environment. Our study found that with the growth of \u003cem\u003eC. chanhua\u003c/em\u003e, the contents of nitrate nitrogen and organic nitrogen in the body of \u003cem\u003eC. chanhua\u003c/em\u003e gradually decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). The content of nitrate nitrogen and organic nitrogen in soil at P3 stage was higher than that at P2 stage. Therefore, we speculated that nitrate and organic nitrogen are secreted into the soil environment during the growth of \u003cem\u003eC. chanhua\u003c/em\u003e. In contrast, the contents of nitrite nitrogen, hydroxylamine nitrogen, and ammonium nitrogen in IS of \u003cem\u003eC. chanhua\u003c/em\u003e gradually increased with the growth of \u003cem\u003eC. chanhua\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed to f). The contents of nitrite nitrogen, hydroxylamine nitrogen, and ammonium nitrogen in soil at P3 stage were lower than those at P2 stage. It can be inferred that nitrite nitrogen, hydroxylamine nitrogen, and ammonium nitrogen in the soil environment are absorbed into IS of \u003cem\u003eC. chanhua\u003c/em\u003e during the ontogenesis of \u003cem\u003eC. chanhua\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo verify the above conjectures, this study deduced the formula ω\u003csub\u003en\u003c/sub\u003e= (JC - S\u003csub\u003en\u003c/sub\u003e) - (Sm\u003csub\u003en\u003c/sub\u003e - Sck\u003csub\u003en\u003c/sub\u003e) and calculated the nitrogen content exchanged between IS in \u003cem\u003eC. chanhua\u003c/em\u003e and the soil environment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Both ω\u003csub\u003e2\u003c/sub\u003e and ω\u003csub\u003e3\u003c/sub\u003e were greater than 0 at P2 and P3 stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), indicating that nitrate and organic nitrogen were secreted into the soil environment from IS of the \u003cem\u003eC. chanhua\u003c/em\u003e in P2 and P3 stages, and the contents of nitrate and organic nitrogen in the P2 stage were 66.53 mg/kg and 103.98 mg/kg, respectively, and those in the P3 stage were 117.92 mg/kg and 446.88 mg/kg, respectively. However, the ω\u003csub\u003e2\u003c/sub\u003e and ω\u003csub\u003e3\u003c/sub\u003e of the contents of nitrite, ammonium, and hydroxylamine transported nitrogen in P2 and P3 stages were all less than 0, indicating that \u003cem\u003eC. chanhua\u003c/em\u003e absorbed nitrite, ammonium, and hydroxylamine nitrogen from the soil at P2 and P3 stages. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u0026thinsp;~\u0026thinsp;c, the contents of nitrite nitrogen, ammonium nitrogen, and hydroxylamine nitrogen absorbed from the soil environment in the P2 stage were 1151.45 mg/kg, 0.21 mg/kg and 1.17 mg/kg, respectively, and those in the P3 stage were 568.02 mg/kg and 1.17 mg/kg, 2.32 mg/kg, respectively. At P2 and P3 stages, the total nitrogen content was greater than 0, indicating that the content of nitrogen secreted by the resoil-cultivated \u003cem\u003eC. chanhua\u003c/em\u003e was greater than that absorbed from the soil environment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe cultivation of \u003cem\u003eC. chanhua\u003c/em\u003e by overlaying soil could increase the rate of nitrogen transformation in soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Compared with CS (0.56 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the net nitrogen mineralization rate of BS (3.30 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was increased by 473.09% ~ 525.78%. The net nitrogen nitrification rate in BS was 4.80 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, which was 8.46 times of that in the ambient soil (0.57 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb shows that compared with CS (-0.04 mg\u0026middot;kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u0026middot;d\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), the net nitrogen ammonification rate of BS decreased by 180.54% ~ 382.86%. In conclusion, \u003cem\u003eC. chanhua\u003c/em\u003e could increase the rates of soil net nitrogen mineralization and nitrification, and reduce the rates of soil net nitrogen ammonification.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacterial community composition\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAccording to the results of high-throughput sequencing, a total of 2,794,234 valid sequences were obtained from the 21 samples sequenced in this experiment, the total number of bases was 898,572,190, and the number of sequences in each sample was 74,622\u0026thinsp;\u0026minus;\u0026thinsp;336,952. OTUs belonging to 35 phyla, 93 classes, 191 orders, 272 families, 559 genera, and 9,692 species were detected in 21 groups of samples in P1, P2, and P3 after optimized sequence clustering. In the dilution curve, it can be found that with the increase of the number of sample sequences, the dilution curve of this sequencing tended to be flat and did not rise (Fig. S10), indicating that the amount of sequencing data was reasonable and large enough to reflect most of the bacterial community diversity information in IS, BS and CS samples. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb clearly show the species composition information of the bacterial communities at the phylum level and the genus level for different samples at different growth stages of \u003cem\u003eC. chanhua\u003c/em\u003e. At the phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), Proteobacteria, Firmicutes, and Actinobacteria were the top three dominant phyla in the relative abundance of bacterial community in IS of \u003cem\u003eC. chanhua\u003c/em\u003e. Proteobacteria (98.39%) was the dominant phylum of JC (Inner sclerotium of \u003cem\u003eC. chanhua\u003c/em\u003e at P1 stage), followed by Firmicutes (1.13%). When the cultivation continued to P2 stage, the number of Proteobacteria (68.34%) in S2 (Inner sclerotium of \u003cem\u003eC. chanhua\u003c/em\u003e at P2 stage) significantly decreased, while that of Firmicutes (26.95%) and Bacteroidetes (4.6%) increased. When the culture continued to P3 stage, the number of Proteobacteria (60.09%) in S3 (Inner sclerotium of \u003cem\u003eC. chanhua\u003c/em\u003e at P3 stage) continued to decrease, but it was still the dominant phylum. The abundance of Firmicutes (19.89%) and Bacteroidetes (0.54%) also decreased, while that of Actinobacteria (19.30%) increased. With the growth and development of \u003cem\u003eC. chanhua\u003c/em\u003e, the number of Proteobacteria in IS of \u003cem\u003eC. chanhua\u003c/em\u003e gradually decreased, but the dominant phylum in the growth and development of \u003cem\u003eC. chanhua\u003c/em\u003e. The abundance of Firmicutes and Bacteroidetes first increased and then decreased, with the highest relative abundance at P2 stage. The number of Proteobacteria showed a relatively increasing trend, with the following order: P3\u0026thinsp;\u0026gt;\u0026thinsp;P2\u0026thinsp;\u0026gt;\u0026thinsp;P1. The results of network topology analysis (Fig. S11) showed that at the phylum level, the bacterial symbiotic network structure of IS samples of \u003cem\u003eC. chanhua\u003c/em\u003e was relatively simple compared with soil samples. Compared with the P3 stage, the bacterial symbiotic network structure in the P2 stage soil samples was relatively simple.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, the bacterial community composition of BS samples and CS samples was similar at the P2 and P3 stages of \u003cem\u003eC. chanhua\u003c/em\u003e. In P2 stage, Proteobacteria was the predominant phylum in Sm2 and Sck2 samples, accounting for 48.63% and 49.62%, respectively. However, at P3 stage, the number of Proteobacteria in Sm3 and Sck3 samples decreased, accounting for 33.80% and 34.16%, respectively, but still occupied the main advantage. At P2 stage, Acidobacteria (16.47%) and Bacteroidetes (21.59%) were the predominant phyla. Acidobacteria (23.53%) was the dominant phylum in CS, while Bacteroidetes (5.64%) was relatively less than that in BS. Other bacterial communities, such as Actinobacteria, Chloroflexi, and Verrucomicrobiae, accounted for 5.21%, 2.24%, and 1.81% of the total abundances in BS, and 7.10%, 3.64%, and 3.34% in CS, respectively. At P3 stage, Actinobacteria (25.34%), Bacteroidetes (15.25%), and Firmicutes (13.86%) were dominant in BS samples. In CS samples, Actinobacteria (21.90%), Acidobacteria (14.49%), and Bacteroidetes (8.44%) were the dominant phyla. In addition, the abundance of Proteobacteria (13.86%) and Bacteroidetes (15.25%) in BS samples was relatively lower than that of Proteobacteria (1.52%) and Bacteroidetes (8.24%) in CS, while the number of Acidobacteria (Sm3: 4.87%; Sck3: 14.49%) was relatively increased. Therefore, during the growth of \u003cem\u003eC. chanhua\u003c/em\u003e from P2 to P3 stage, the abundance of Proteobacteria, Acidobacteria, and Bacteroidetes decreased, the abundance of Firmicutes and Actinobacteria increased, and the relative abundance of other bacteria did not change much.\u003c/p\u003e\u003cp\u003eAt different stages of \u003cem\u003eC. chanhua\u003c/em\u003e growth, the dominant bacterial groups at the genus level of the samples of IS, BS, and CS in \u003cem\u003eC. chanhua\u003c/em\u003e had their characteristics (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The bacterial community composition of IS in \u003cem\u003eC. chanhua\u003c/em\u003e was relatively simple compared with that in soil. The dominant genera of JC were \u003cem\u003eStenotrophomonas\u003c/em\u003e (20.96%) and \u003cem\u003eAchromobacter\u003c/em\u003e (11.89%). \u003cem\u003eBacillus\u003c/em\u003e (23.50%) was the dominant group of S2, and \u003cem\u003eCedecia\u003c/em\u003e (10.30%), \u003cem\u003eSphingobacter\u003c/em\u003e (4.54%), \u003cem\u003eRoamella\u003c/em\u003e (3.35%), and \u003cem\u003eSerratia\u003c/em\u003e (1.78%) were the secondary dominant groups. In S3 samples, \u003cem\u003eAchromobacter\u003c/em\u003e (40.64%), \u003cem\u003eStaphylococcus\u003c/em\u003e (19.11%), and \u003cem\u003ePseudomonas\u003c/em\u003e (14.31%) were the main dominant bacteria. Therefore, at different growth stages of \u003cem\u003eC. chanhua\u003c/em\u003e, the bacterial groups in IS have different characteristics at the genus level, and the main dominant bacterial groups are also different.\u003c/p\u003e\u003cp\u003eIn the soil samples, \u003cem\u003eSphingobacter\u003c/em\u003e (14.75%) was the main dominant bacterial group in Sm2 samples. \u003cem\u003eRB41\u003c/em\u003e (3.70%), \u003cem\u003eBradyrhizobium\u003c/em\u003e (3.13%), \u003cem\u003ePseudomonas\u003c/em\u003e (2.52%), \u003cem\u003eOligotrophomonas\u003c/em\u003e (2.04%), \u003cem\u003eSphingomonas\u003c/em\u003e (1.82%), \u003cem\u003eCandidatus_Solibacter\u003c/em\u003e (1.81%) is the secondary dominant bacterial group. In addition, it also contains \u003cem\u003eChryseobacterium\u003c/em\u003e (1.49%), \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e (1.38%), \u003cem\u003eAllorhizobium-Neorhizobium-Pararhizobium-Rhizobium\u003c/em\u003e (1.37%), \u003cem\u003eBryobacte\u003c/em\u003er (1.35%), \u003cem\u003eReyranella\u003c/em\u003e (1.32%), etc. In Sm3 samples, \u003cem\u003eStaphylococcus\u003c/em\u003e (12.04%) was the main dominant genus, \u003cem\u003eSphingobacter\u003c/em\u003e (8.94%), \u003cem\u003eAchromobacter\u003c/em\u003e (8.38%), and \u003cem\u003eBrevibacterium\u003c/em\u003e (7.22%) were also dominant. It is not difficult to see from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb that the bacterial community composition in CS samples at both P2 and P3 stages was more complex than that in BS samples. At the same stage, the composition of bacterial communities in the samples of BS and CS had their characteristics, but the relative abundance of the most dominant bacterial groups in the samples of BS was higher than that in CS. For example, the relative abundance of the dominant group \u003cem\u003eSphingobacter\u003c/em\u003e in Sm2 (14.75%) was higher than that of the dominant group \u003cem\u003eRB41\u003c/em\u003e in Sck2 (3.87%). The relative abundance of the dominant group \u003cem\u003eStaphylococcus\u003c/em\u003e in Sm3 (12.04%) was higher than that of the dominant group \u003cem\u003ePaenarthrobacter\u003c/em\u003e in Sck3 (3.56%).\u003c/p\u003e\u003cp\u003eVenn analysis of the OTU levels of IS samples within the three growth stages of \u003cem\u003eC. chanhua\u003c/em\u003e revealed JC and S2 had 15 OTUs, S3 had 103 OTUs, and S2 and S3 had 34 OTUs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). A total of 18 OTUs were detected in IS of the three stages of \u003cem\u003eC. chanhua\u003c/em\u003e growth, indicating that part of the bacterial community persisted during the growth of \u003cem\u003eC. chanhua\u003c/em\u003e. As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, JC has 1,386 OTUs, S2 has 451 OTUs, and S3 has 1,915 OTUs. In addition, a total of 1,496 OTUs were detected in the JC sample, 492 OTUs were detected in S2 sample, and 2,034 OTUs were detected in S3 sample. With the growth of \u003cem\u003eC. chanhua\u003c/em\u003e, the bacteria in IS samples of \u003cem\u003eC. chanhua\u003c/em\u003e showed a trend of decreasing first and then increasing.\u003c/p\u003e\u003cp\u003eVenn analysis was performed on BS of \u003cem\u003eC. chanhua\u003c/em\u003e at different growth stages and CS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). In P2 stage, Sm2 had a total of 14,192 OTUs, and 10,969 OTUs were unique. Sck2 had a total of 16,084 OTUs and a characteristic of 12,861 OTUs. In P3 stage, there were 12,437 OTUs in Sm3 samples, and 10,478 OTUs were unique. The Sck3 sample had a total of 17,893 OTUs, and 15,384 OTUs were unique. There were 940 OTUs between the soil samples at P2 and P3 stage, and 1,357 OTUs between CS samples. There were 2,832 OTUs between BS and CS at P2 stage. In P3 stage, there were 1,639 OTUs between BS and CS. A total of 583 OTUs were identified between BS and CS at different growth stages. It is not difficult to find from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed that with the growth and development of \u003cem\u003eC. chanhua\u003c/em\u003e, the number of OTUs in BS is decreasing, while the number of OTUs in CS is gradually increasing. However, with the growth of \u003cem\u003eC. chanhua\u003c/em\u003e, the number of OTU in IS increased, indicating that with the growth of \u003cem\u003eC. chanhua\u003c/em\u003e, some bacteria in the soil environment migrated to the inner sclerotium in the \u003cem\u003eC. chanhua\u003c/em\u003e through the mycoderm. In addition, the amount of bacterial OTU in soil environment was much higher than that in IS samples of \u003cem\u003eC. chanhua\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eA total of 41 genera of bacteria were found in different samples at different growth stages of \u003cem\u003eC. chanhua\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), the proportions of each of the 41 genera are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef. They were \u003cem\u003eAchromobacter\u003c/em\u003e (27.72%), \u003cem\u003eAlcaligenes\u003c/em\u003e (11.60%), \u003cem\u003eAllorhizobium-Neorhizobium-Pararhizobium-Rhizobium\u003c/em\u003e (11.13%) and \u003cem\u003eAquisphaera\u003c/em\u003e (10.37%), \u003cem\u003eBacillus\u003c/em\u003e (4.82%), \u003cem\u003eBradyrhizobium\u003c/em\u003e (4.69%), \u003cem\u003eLysobacter\u003c/em\u003e (4.69), \u003cem\u003eEnsifer\u003c/em\u003e (3.86%) and \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e (2.42%), \u003cem\u003eChitinophaga\u003c/em\u003e (1.9%), \u003cem\u003eBosea\u003c/em\u003e (1.89%) and 30 other bacteria genera with relatively small proportions. These 41 genera were detected in IS, BS, and CS of \u003cem\u003eC. chanhua\u003c/em\u003e at different growth stages, indicating that they not only participate in the growth and development of \u003cem\u003eC. chanhua\u003c/em\u003e, but also exist widely in the soil environment.\u003c/p\u003e\u003cp\u003eIt can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee that JC samples have 3 endemic genera, namely \u003cem\u003eAlcanivorax\u003c/em\u003e, \u003cem\u003ePrevotella\u003c/em\u003e, and \u003cem\u003ePigmentiphaga\u003c/em\u003e. The S2 sample had two endemic genera, \u003cem\u003e28-YEA-48\u003c/em\u003e and \u003cem\u003eLactococcus\u003c/em\u003e respectively. The endemic genera of S3 samples were 11 genera, including \u003cem\u003eVerticia\u003c/em\u003e, \u003cem\u003eMumia\u003c/em\u003e, \u003cem\u003eModestobacte\u003c/em\u003er, \u003cem\u003eLimnohabitans\u003c/em\u003e, and so on. The endemic genera in Sm2 samples included 20 genera, such as \u003cem\u003ePolyangium\u003c/em\u003e, \u003cem\u003eRhodanobacter\u003c/em\u003e, \u003cem\u003eRhodoblastus\u003c/em\u003e, \u003cem\u003eHerbaspirillum\u003c/em\u003e, \u003cem\u003eCandidatus_Jidaibacter\u003c/em\u003e and \u003cem\u003eBiernaprussia\u003c/em\u003e, etc. In Sck2 samples, 11 genera were unique, including \u003cem\u003eAcidocella\u003c/em\u003e, \u003cem\u003eSalinispora\u003c/em\u003e, \u003cem\u003eParachlamydia\u003c/em\u003e, \u003cem\u003eVariibacter\u003c/em\u003e, \u003cem\u003eAcidiphilium\u003c/em\u003e, etc. The unique genera in Sm3 are \u003cem\u003eActinopolymorpha\u003c/em\u003e, \u003cem\u003eSphaerisporangium\u003c/em\u003e, \u003cem\u003ePlanomicrobium\u003c/em\u003e, and 33 other genera. The specific genera in Sck3 samples included 44 genera, including \u003cem\u003eCnuella, Bacteriovorax\u003c/em\u003e, \u003cem\u003eAzoarcus\u003c/em\u003e, \u003cem\u003eAnaerocolumna\u003c/em\u003e, \u003cem\u003eErythrobacter\u003c/em\u003e, and \u003cem\u003eMicrobulbifer\u003c/em\u003e. There were 41 bacterial genera involved in the growth and development of \u003cem\u003eC. chanhua\u003c/em\u003e, the bacterial communities in \u003cem\u003eC. chanhua\u003c/em\u003e at different growth stages were different and had their characteristics. In addition, the bacteria genera in the sclerotium first decreased and then increased, S2\u0026thinsp;\u0026lt;\u0026thinsp;JC\u0026thinsp;\u0026lt;\u0026thinsp;S3.\u003c/p\u003e\u003cp\u003eAmong the top 20 bacteria in each sample, there was no significant difference in the abundance of \u003cem\u003ePseudomonas\u003c/em\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.5). There were significant differences in the abundance of bacterial communities of the other 18 genera (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg), including \u003cem\u003eAchromobacter\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eSphingobacter\u003c/em\u003e, \u003cem\u003eStenotrophomonas\u003c/em\u003e, \u003cem\u003eMorganella\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eCedecea\u003c/em\u003e and so on. Among them, \u003cem\u003eAchromobacter\u003c/em\u003e had a large abundance in S3, which was significantly different from JC. The abundance of \u003cem\u003eOligotrophomonas\u003c/em\u003e and \u003cem\u003eSerratia\u003c/em\u003e were higher at P1 stage, but decreased significantly with the growth of \u003cem\u003eC. chanhua\u003c/em\u003e. \u003cem\u003eCedecea\u003c/em\u003e and \u003cem\u003eRamococcus\u003c/em\u003e only appeared in IS at P2 stage, so it was assumed that these two bacteria were related to the formation of the \u003cem\u003eC. chanhua\u003c/em\u003e mycoderm. \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003eBrevibacterium\u003c/em\u003e, \u003cem\u003eSaccharopolyspora\u003c/em\u003e, \u003cem\u003eCandidatus_Solibacter\u003c/em\u003e, \u003cem\u003eBryobacter\u003c/em\u003e, and \u003cem\u003eCandidatus_udaeter\u003c/em\u003e almost only appear in IS of \u003cem\u003eC. chanhua\u003c/em\u003e at P1 and P3 stage, while their abundance is 0 during P2 stage. Therefore, we assume that these bacteria may be unfavorable to the formation of membranes. The bacterial communities of Sck2 and Sck3 had similar genus levels, and the abundance of \u003cem\u003eArthrobactoides\u003c/em\u003e, \u003cem\u003eStreptomyces\u003c/em\u003e, and \u003cem\u003ePseudomonas\u003c/em\u003e in Sck3 was significantly higher than that of Sck2 at the genus level. Except for these bacteria, the abundance of other bacterial communities was basically the same. The abundance of \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003eAchromobacter\u003c/em\u003e, and \u003cem\u003eBrevibacterium\u003c/em\u003e in Sm3 was significantly higher than that in Sm2, while the abundances of other bacterial groups had little or no difference. In addition, \u003cem\u003eArthrobacter\u003c/em\u003e and \u003cem\u003eSaccharopolyspora\u003c/em\u003e existed only in P3 stage, not in P2 stage.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacterial community diversity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Coverage index of each copy reached 100% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), indicating that the data measured by this sequencing adequately reflected the bacterial community diversity in each sample. The Ace and Chao indices of IS samples in different cultivation periods were 229.333-721.333, and those of the soil samples were 456-6940.667, respectively. Among them, there was no significant difference in the Ace and Chao indices among the JC, S2, and S3 samples (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that cultivation time had little effect on the richness of bacterial community in IS of \u003cem\u003eC. chanhua\u003c/em\u003e cultivated under soil cover. The Ace index and Chao index in the soil of \u003cem\u003eC. chanhua\u003c/em\u003e in P2 stage were significantly higher than those in P3 stage (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the richness of soil bacterial community could be reduced by planting \u003cem\u003eC. chanhua\u003c/em\u003e in soil. In addition, with the growth of \u003cem\u003eC. chanhua\u003c/em\u003e, the bacterial richness in BS tended to decrease, while the richness in CS basically did not change. The possible reason was that the exosomes of \u003cem\u003eC. chanhua\u003c/em\u003e produced natural selection for BS bacteria, which enriched some groups and led to a decline in the overall community diversity. The results of Beta diversity analysis showed that the distance between S2, S1, and S3 samples was relatively long (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh), indicating that the bacterial community β diversity in S2 samples was significantly different from that between S1 and S3 samples. According to the PCoA diagram, other samples except Sm3 were aggregated separately, indicating that there was no significant difference in Beta diversity of bacterial community between the sclerotium samples in \u003cem\u003eC. chanhua\u003c/em\u003e and the control soil samples. In other words, the bacterial community composition in IS and CS of \u003cem\u003eC. chanhua\u003c/em\u003e in different growth and development stages was similar, while the bacterial community composition in BS changed significantly. In addition, the distance between IS of \u003cem\u003eC. chanhua\u003c/em\u003e and the soil samples was relatively far, indicating that the diversity of Beta of bacterial communities in the inner sclerotium of \u003cem\u003eC. chanhua\u003c/em\u003e was significantly different from that in BS and CS. At the same stage, the distance between the soil samples (such as Sm2 and Sck2, Sm3 and Sck3) was relatively close, indicating that there was no significant difference in Beta diversity of bacterial community between BS and CS at the same stage.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBacterial Alpha diversity index between all samples\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAce index\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eChao index\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCoverage (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eShannon index\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSimpson index\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e539.000\u0026thinsp;\u0026plusmn;\u0026thinsp;97.766c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e539.000\u0026thinsp;\u0026plusmn;\u0026thinsp;97.767c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.019\u0026thinsp;\u0026plusmn;\u0026thinsp;0.082a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.424\u0026thinsp;\u0026plusmn;\u0026thinsp;0.117a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e229.333\u0026thinsp;\u0026plusmn;\u0026thinsp;9.838c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e229.333\u0026thinsp;\u0026plusmn;\u0026thinsp;9.838c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.832\u0026thinsp;\u0026plusmn;\u0026thinsp;0.494a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.136\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e721.333\u0026thinsp;\u0026plusmn;\u0026thinsp;143.497c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e721.333\u0026thinsp;\u0026plusmn;\u0026thinsp;143.497c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.523\u0026thinsp;\u0026plusmn;\u0026thinsp;0.526a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.305\u0026thinsp;\u0026plusmn;\u0026thinsp;0.106a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSm2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5956.667\u0026thinsp;\u0026plusmn;\u0026thinsp;288.506a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5956.667\u0026thinsp;\u0026plusmn;\u0026thinsp;288.506a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.015\u0026thinsp;\u0026plusmn;\u0026thinsp;0.336a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.006\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSck2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6940.667\u0026thinsp;\u0026plusmn;\u0026thinsp;724.827a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6940.667\u0026thinsp;\u0026plusmn;\u0026thinsp;724.827a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.068\u0026thinsp;\u0026plusmn;\u0026thinsp;1.354a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.001\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSm3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4506\u0026thinsp;\u0026plusmn;\u0026thinsp;968.052b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4506\u0026thinsp;\u0026plusmn;\u0026thinsp;968.052b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.131\u0026thinsp;\u0026plusmn;\u0026thinsp;0.321a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.017\u0026thinsp;\u0026plusmn;\u0026thinsp;0.005b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSck3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6683\u0026thinsp;\u0026plusmn;\u0026thinsp;577.01a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6683\u0026thinsp;\u0026plusmn;\u0026thinsp;577.01a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.590\u0026thinsp;\u0026plusmn;\u0026thinsp;1.012a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.003\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0003b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eNote: Data in the table are Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; Different letters in the same column indicate a significant difference (Duncan\u0026rsquo;s test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFunctional prediction analysis of bacterial communities associated with nitrogen cycle\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMore detailed descriptions of the KEGG and COG functions of the bacterial communities in each sample can be found in the Supplementary Information (Text S7). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea shows that the relative abundance of KEGG functions of amino acid metabolism related to nitrogen cycling in bacterial communities in each sample ranged from 11.89\u0026ndash;14.30%. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, the amino acid metabolism function of the bacterial communities in IS samples of \u003cem\u003eC. chanhua\u003c/em\u003e was higher than that of the soil bacterial communities, indicating that the relative abundance of bacterial communities related to nitrogen cycle in \u003cem\u003eC. chanhua\u003c/em\u003e was higher than that in the soil samples. There was no significant difference in the relative abundance of amino acid metabolism in KEGG function of the bacterial community in BS samples, but the amino acid metabolism function of bacterial communities in Sm3 samples was significantly higher than that in Sm2 samples. Therefore, it can be inferred that part of the bacterial community with amino acid metabolism in IS of \u003cem\u003eC. chanhua\u003c/em\u003e was excreted into the soil environment along with nitrogen during the growth and development of \u003cem\u003eC. chanhua\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eAs can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, the relative abundance of amino acid transport and metabolism, the COG function related to the nitrogen cycle, in the bacterial communities in each sample were 9.83\u0026ndash;11.11%, which was larger than that of other COG gene functional families. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, the amino acid transport and metabolism function of the bacterial community in IS of \u003cem\u003eC. chanhua\u003c/em\u003e was higher than that of the soil bacterial community, indicating that the relative abundance of the bacterial community related to the nitrogen cycle in the \u003cem\u003eC. chanhua\u003c/em\u003e was higher than that in the soil samples. The relative abundance of amino acid transport metabolism in IS samples of \u003cem\u003eC. chanhua\u003c/em\u003e was S2\u0026thinsp;\u0026lt;\u0026thinsp;JC\u0026thinsp;\u0026lt;\u0026thinsp;S3, indicating that the bacterial community related to nitrogen cycle in \u003cem\u003eC. chanhua\u003c/em\u003e showed a tendency to increase with the growth of \u003cem\u003eC. chanhua\u003c/em\u003e, but the overall trend was upward. From the soil samples, the relative abundance of amino acid transport metabolism in COG function of bacterial community in CS samples was not significantly different, while the amino acid transport metabolism of bacterial community in Sm3 samples was significantly higher than that in Sm2 samples. Therefore, during the growth and development of \u003cem\u003eC. chanhua\u003c/em\u003e, some amino acid metabolizing bacterial communities in IS of \u003cem\u003eC. chanhua\u003c/em\u003e were secreted into the soil along with nitrogen, that is, the related bacterial communities in \u003cem\u003eC. chanhua\u003c/em\u003e can promote the soil nitrogen cycle.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eCorrelation analysis between soil bacterial community diversity and soil nitrogen\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRedundancy analysis between soil nitrogen and bacterial community was performed at phylum level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The cumulative interpretation rates of the first and second ranking axes were 66.6% and 23.5%, respectively, and the cumulative interpretation rates reached 90.1%, indicating that the first and second ranking axes could better reflect the correlation between soil nitrogen form and bacterial community at phylum level. The contents of Firmicutes, Proteobacteria and Actinobacteria in the sclerotium samples and soil samples were positively correlated with organic nitrogen (Org-N) and nitrite nitrogen (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N), and it is negatively correlated with ammonium nitrogen (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N) and nitrate nitrogen (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) and has the greatest influence on organic nitrogen content. Chloroflexi and Acidobacteria are positively correlated with ammonium nitrogen and nitrate nitrogen, and negatively correlated with organic nitrogen and nitrite nitrogen. Among them, each bacterial group has the greatest influence on organic nitrogen and ammonium nitrogen. At the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), in each sample, \u003cem\u003eSphingobacterium\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e, \u003cem\u003eSerratia\u003c/em\u003e, \u003cem\u003eAchromobacter\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eSaccharopolyspora\u003c/em\u003e and \u003cem\u003eSaccharopolyspora\u003c/em\u003e were found to be oligotrophic \u003cem\u003eStenotrophomonas\u003c/em\u003e, \u003cem\u003eBrevibacterium\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e were positively correlated with organic nitrogen and nitrite nitrogen, but negatively correlated with ammonium nitrogen and nitrite nitrogen. \u003cem\u003eRB41\u003c/em\u003e was positively correlated with ammonium nitrogen and nitrate nitrogen, and negatively correlated with organic nitrogen and nitrite nitrogen, in which each bacterial community had the greatest influence on ammonium nitrogen and organic nitrogen.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of aqueous solution of nitrogen secreted by\u003c/b\u003e \u003cb\u003eC. chanhua\u003c/b\u003e \u003cb\u003eon soil bacterial communities\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe results of OTUs cluster analysis showed that 25,213 OTUs were detected in 27 phyla, 74 classes, 223 orders, 433 families, 766 genera, and 1,414 species in SNN and SNck in six samples (there replicates). As can be seen from Fig. S11, Shannon-Winner exponential curve becomes flatter and no longer increases with the increase in the number of sample sequences, indicating that the depth of sequencing data can more comprehensively reflect the bacterial community information in sequenced SNN and SNck samples. At the phylum level, the bacterial community composition of SNN was simpler than that of SNck (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). In SNN samples, Firmicutes (85.94%) was the dominant phylum, and Proteobacteria (8.08%) was the minor dominant phylum. In SNck samples, Proteobacteria (52.51%) is the main dominant group, while Acidobacteria (13.40%) and Actinobacteria (11.16%) are the secondary dominant groups. Therefore, the bacterial community in the soil samples can be reduced at the phylum level by adding \u003cem\u003eC. chanhua\u003c/em\u003e exonitrogen aqueous solution to the soil. At the genus level, the dominant bacterial groups in the soil samples of SNN and SNck are not the same, but the number of dominant bacterial communities is similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). The bacterial community of SNN samples was dominated by \u003cem\u003eSporosarcina\u003c/em\u003e (58.72%). The bacterial groups such as \u003cem\u003ePseudomonas\u003c/em\u003e (3.91%), \u003cem\u003eBacillus\u003c/em\u003e (2.83%), \u003cem\u003ePsychrobacillus\u003c/em\u003e (2.42%) and \u003cem\u003eStaphylococcus\u003c/em\u003e (1.47%) also accounted for a certain proportion. Compared with SNN, the bacterial community composition of SNck was more complex, it was mainly composed of \u003cem\u003eMassilia\u003c/em\u003e (5.19%), \u003cem\u003eSphingomonas\u003c/em\u003e (4.56%), \u003cem\u003eCandidatus_Solibacter\u003c/em\u003e (3.59%), \u003cem\u003eBradyrhizobium\u003c/em\u003e (3.52%) and \u003cem\u003eStreptomyces\u003c/em\u003e (2.61%), \u003cem\u003eRB41\u003c/em\u003e (2.55%), \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia\u003c/em\u003e (1.37%), \u003cem\u003eAquisphaera\u003c/em\u003e (1.30%), \u003cem\u003eReyranella\u003c/em\u003e (1.06%) and other bacterial groups were composed. Therefore, at the geneus level, the bacterial community composition in SNN and SNck samples was different, indicating that the aqueous nitrogen solution secreted by \u003cem\u003eC. chanhua\u003c/em\u003e is able to change the bacterial community in the soil environment. The analysis of the significance of differences between groups showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec) that the relative abundance of SNN and SNck in soil samples accounted for no significant difference in the average relative abundance of the top 15 bacterial genera (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating that the bacterial communities in SNN and SNck samples were similar, that is, the aqueous solution of \u003cem\u003eC. chanhua\u003c/em\u003e exonitrogen had no significant effect on soil bacterial genera. Venn analysis of SNN and SNck showed that there were 416 OTUs in SNN and SNck, 3,381 OTUs were unique to SNN and 18,287 OTUs were unique to SNck (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). The amount of bacterial OTUs in SNN was much lower than that in SNck samples, suggesting that aqueous solution of nitrogen secreted by \u003cem\u003eC. chanhua\u003c/em\u003e can reduce the diversity of bacterial community in soil environment.\u003c/p\u003e\u003cp\u003eThe α diversity index of bacterial communities in SNN samples of the experimental group and SNck samples of the control group was compared and analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee to h). The Ace and Chao indices in SNN and SNck samples were between 1,232-7,196, respectively, and SNN was significantly lower than SNck. In addition, Shannon index showed that there was no significant difference in bacterial diversity between SNN and SNck samples, while Simpson index showed that SNN was significantly higher than SNck. Therefore, aqueous solution of nitrogen secreted by \u003cem\u003eC. chanhua\u003c/em\u003e can affect the diversity of bacterial community in soil environment. Ace, Chao and Shannon indices of soil bacterial communities were decreased, indicating reduced richness and diversity. Conversely, the increase in the Simpson index reflects a decrease in community evenness, signifying greater dominance by fewer bacterial taxa [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFunctional prediction analysis of bacterial communities in SNN and SNck samples\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea shows that 35 shared functions of bacterial communities in SNN and SNck samples at the the secondary pathway level include amino acid metabolism, biosynthesis of other secondary metabolites, and cancer: overview, carbohydrate metabolism, cardiovascular disease, cell growth and death, etc.; The bacterial community in SNck samples had two unique structures, including cellular community-eukaryotes and sensory system. There were no endemic structures in the SNN soil samples. Therefore, the \u003cem\u003eC. chanhua\u003c/em\u003e exonitrogen aqueous solution can reduce the function of bacterial community in soil environment. More detailed descriptions of the KEGG and COG functions of the bacterial communities can be found in the Supplementary Information (Text S8). As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, the relative abundance of amino acid metabolism, the KEGG function closely related to the nitrogen cycle, in SNN and SNck samples ranged from 12.78\u0026ndash;13.86%, was second only to carbohydrate metabolism (12.97%-13.14%). As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed, the relative abundance of amino acid metabolism, a KEGG function closely related to nitrogen cycle, in SNN and SNck samples was 12.78%-13.86%, second only to carbohydrate metabolism (12.97%-13.14%). In SNN and SNck samples, the COG functions closely related to the nitrogen cycle, amino acid transport metabolism, accounted for a relative abundance of 12.78\u0026ndash;13.86%, higher than other COG functions (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed, in SNN and SNck samples, the relative abundances of KEGG function -- amino acid metabolism, which is closely related to the nitrogen cycle, are 13.86% and 12.78%, respectively; and the relative abundances of COG function -- amino acid transport metabolism are 9.25% and 10.52%, respectively. SNN is higher than SNck in both cases. It can be further demonstrated that \u003cem\u003eC. chanhua\u003c/em\u003e exocrine nitrogen aqueous solution can improve the function related to nitrogen cycling in soil environment, that is, \u003cem\u003eC. chanhua\u003c/em\u003e exocrine nitrogen aqueous solution can promote soil nitrogen cycling.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsolation and identification of bacterial strains related to nitrogen conversion in\u003c/b\u003e \u003cb\u003eC. chanhua\u003c/b\u003e.\u003c/p\u003e\u003cp\u003eThe bacteria isolated and purified by dilution coated plate method and plate scribing method were cultured in LB medium or beef paste peptone medium for 48 h, and the ammonium nitrogen and nitrite nitrogen contents in the bacterial broth were measured. Among them, 15 bacterial strains had significantly higher ability to produce ammonium nitrogen or nitrite nitrogen than other strains, including eight bacteria on LB medium and seven bacteria on beef extract peptone medium, designated as L1\u0026thinsp;~\u0026thinsp;L8 and B1\u0026thinsp;~\u0026thinsp;B7 respectively. As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea, the content of ammonium nitrogen in bacterial fluid of strains L4 and B5 was significantly higher than that of other strains. And the content of nitrous nitrogen in bacterial fluid of strains L4 and B1 was significantly higher than that of other strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). Therefore, the B1 strain isolated from IS in \u003cem\u003eC. chanhua\u003c/em\u003e can produce NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, the B5 strain can produce NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N, and the L4 strain can produce both NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N. The colony morphology of L4, B1 and B5 on plate medium is shown in Fig. S14, and the colony color and morphology of L4, B1 and B5 are shown in Table S2. DNA samples were detected by 2% agarose gel electrophoresis, and the PCR characteristic amplification bands of bacterial strains L4, B1 and B5 were clear and bright at about 1,500 bp, with appropriate concentration and no drag (Fig. S15). Gene sequencing results showed that strains B1, B5 and L4 were associated with \u003cem\u003eDelftia\u003c/em\u003e sp. (GenBankaccession: MK414965.1) and \u003cem\u003eDelftia acidovorans\u003c/em\u003e (GenBankaccession: MK414884.1), the 16S rRNA nucleotide sequence identity of \u003cem\u003ePseudomonas protegens\u003c/em\u003e (MK235212.1) was 99.92%, 99.85% and 100.00%, respectively (Table S3). The taxonomic status of the three bacteria is shown in Table S4. The three bacteria belong to one kingdom, one phylum, one classes, two orders, two families, two genera. By constructing phylogenetic tree with sequences with high similarity (as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec\u0026thinsp;~\u0026thinsp;e), it was further revealed that B1 and B5 belonged to \u003cem\u003eDelftia\u003c/em\u003e sp., and L4 belonged to \u003cem\u003ePseudomonas\u003c/em\u003e sp.\u003c/p\u003e\u003cp\u003eAs can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cem\u003eDelftia\u003c/em\u003e spp. belonging to B1 and B5 do not belong to the dominant bacteria genera in IS, BS and CS samples of \u003cem\u003eC. chanhua\u003c/em\u003e at different growth stages. \u003cem\u003ePseudomonas\u003c/em\u003e sp. belonging to L4 strain which can produce both ammonium nitrogen and nitrite nitrogen was the main dominant bacterial group in S3 samples, accounting for 14.31% relative abundance. In addition, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb shows that the \u003cem\u003ePseudomonas\u003c/em\u003e sp. is positively correlated with the two factors of organic nitrogen and nitrite nitrogen, and negatively correlated with ammonium nitrogen and nitrate nitrogen. Therefore, the \u003cem\u003ePseudomonas\u003c/em\u003e sp. in \u003cem\u003eC. chanhua\u003c/em\u003e can promote nitrogen cycling. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea shows that the relative abundance of \u003cem\u003ePseudomonas\u003c/em\u003e in \u003cem\u003eC. chanhua\u003c/em\u003e decreases first and then increases. In particular, the abundance of \u003cem\u003ePseudomonas\u003c/em\u003e in P3 stage of \u003cem\u003eC. chanhua\u003c/em\u003e is much higher than that in P1 stage and P2 stage. Therefore, with the growth of \u003cem\u003eC. chanhua\u003c/em\u003e, the content of \u003cem\u003ePseudomonas\u003c/em\u003e related to nitrogen cycle in the \u003cem\u003eC. chanhua\u003c/em\u003e is on the rise. The relative abundance of \u003cem\u003ePseudomonas\u003c/em\u003e in the soil samples at P2 stage was greater than that at P3 stage, indicating that the proportion of \u003cem\u003ePseudomonas\u003c/em\u003e in the soil increased with the extension of cultivation time. The content of \u003cem\u003ePseudomonas\u003c/em\u003e in BS was also increasing, so it could be speculated that some \u003cem\u003ePseudomonas\u003c/em\u003e were secreted into the soil with nitrogen during the growth of \u003cem\u003eC. chanhua\u003c/em\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb shows that the relative abundance of \u003cem\u003ePseudomonas\u003c/em\u003e in SNN samples was 3.91%, and that in SNck samples was only 0.03%. Therefore, the aqueous nitrogen solution secreted by \u003cem\u003eC. chanhua\u003c/em\u003e promoted the enrichment of \u003cem\u003ePseudomonas\u003c/em\u003e in the soil environment, which further verified that endophyte \u003cem\u003ePseudomonas\u003c/em\u003e promoted the participation of \u003cem\u003eC. chanhua\u003c/em\u003e in soil nitrogen cycling to a certain extent.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the process of soil nitrogen cycling, different forms of nitrogen have their unique functions and effects. Soil total nitrogen can be divided into organic nitrogen and inorganic nitrogen, which is an index to measure the nitrogen fertilizer power of soil [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This study found that nitrogen in the \u003cem\u003eC. chanhua\u003c/em\u003e cultivated with soil covered would be secreted into the soil environment, and the nitrogen content in BS gradually increased with the extension of cultivation time, suggesting that the cultivation of \u003cem\u003eC. chanhua\u003c/em\u003e by covering soil could improve soil fertility. Soil organic nitrogen in \u003cem\u003eC. chanhua\u003c/em\u003e is considered to be an important factor in maintaining soil quality and fertility [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], accounting for more than 80% of soil nitrogen [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Inorganic nitrogen includes nitrate nitrogen, ammonium nitrogen, nitrite nitrogen, hydroxylamine nitrogen, etc. Nitrite nitrogen is one of the nitrogen compounds occurring in nature, and its content is important for understanding soil nitrogen utilization and loss, evaluating soil fertility, and rational nitrogen application [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Nitrate and organic nitrogen are one of the available nitrogen sources that can be directly absorbed by plants [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. As an important soil fertility indicator, an increase in soil nitrate content can promote vegetable growth within a certain numerical range [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Ammonium is the main source of nitrogen nutrients absorbed by plants and an important product or reactant in soil nitrogen transformation [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Hydroxylamine (NH\u003csub\u003e2\u003c/sub\u003eOH) and nitrite nitrogen (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N) can chemically react with iron, manganese, and organic matter to produce nitrous oxide and high N\u003csub\u003e2\u003c/sub\u003eO content contributes to global warming [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. The intermediate products of soil nitrogen cycling, ammonium nitrogen, hydroxylamine nitrogen, and nitrite nitrogen, are inhaled by \u003cem\u003eC. chanhua\u003c/em\u003e from the soil environment and used for the growth and development of \u003cem\u003eC. chanhua\u003c/em\u003e, thereby reducing the contents of ammonium, hydroxylamine, and nitrite nitrogen in the soil environment and increasing the contents of organic nitrogen and nitrite nitrogen. Therefore, the growth of \u003cem\u003eC. chanhua\u003c/em\u003e promoted the process of soil nitrogen cycling. In terms of the stability and diversity of bacterial communities, hydroxylamine nitrogen in the soil environment decreases bacterial diversity, whereas ammonium nitrogen can maintain it when increased to a certain extent [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Therefore, cultivation of \u003cem\u003eC. chanhua\u003c/em\u003e in soil can maintain the diversity of bacterial communities to some extent.\u003c/p\u003e\u003cp\u003eAll processes of the soil nitrogen cycle are microbiologically driven [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], with ammonification, the decomposition of organic nitrides by microorganisms to produce ammonia [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], the rate-limiting step in the soil nitrogen cycle and a central link in the global nitrogen cycle [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. During the growth of \u003cem\u003eC. chanhua\u003c/em\u003e cultivated under soil cover, IS of \u003cem\u003eC. chanhua\u003c/em\u003e secreted organic nitrogen into the soil environment, which not only ensures the normal growth of secreted, but also increases the ammonification rate, thereby accelerating the soil nitrogen cycling process [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Denitrification is mainly driven by facultative anaerobic nitrate reducing bacteria [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. During denitrification, autotrophic denitrifying microorganisms using carbohydrate metabolism and heterotrophic denitrifying microorganisms using organic carbon sources as electron donors gradually reduce nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) to nitrite (NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. In this study, we found that nitrate in \u003cem\u003eC. chanhua\u003c/em\u003e cultivated in the soil would be discharged into the soil environment, which would increase denitrification and promote soil nitrogen cycling.\u003c/p\u003e\u003cp\u003eSoil net nitrogen mineralization rate is an important indicator of soil nitrogen cycle and supply capacity, reflecting the dynamic process of soil nitrogen transformation from organic to inorganic forms, which is of great significance for agricultural production and ecosystem management [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. By increasing the net nitrogen mineralization rate of soil, it can increase the nitrogen supply capacity of soil, improve plant growth and yield [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], while reducing the risk of nitrogen loss and protecting the environment [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. This study found that the soil net nitrogen mineralization rate of BS was significantly higher than that of CS, indicating that \u003cem\u003eC. chanhua\u003c/em\u003e can provide more directly absorbable and utilizable nitrogen for the plants in its microenvironment, thereby promoting the growth and development of the plants [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Bei et al. [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] research found that the activity and quantity of microorganisms in the soil increase with the increase of the net nitrogen mineralization rate, because microorganisms can utilize newly added inorganic nitrogen for growth and metabolism. In addition, the increase in the net nitrogen mineralization rate of the soil will also make the nitrogen cycle process in the soil more active, which is conducive to maintaining soil fertility and the health of the ecosystem [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Therefore, \u003cem\u003eC. chanhua\u003c/em\u003e can increase the net nitrogen mineralization rate in the soil environment, thereby promoting the process of soil nitrogen cycling. The nitrification rate of soil net nitrogen is an important indicator for measuring nitrification in the soil nitrogen cycle process, reflecting the dynamic process of nitrogen transformation from ammonium nitrogen to nitrate nitrogen in the soil [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. This study found that the nitrification rate of net nitrogen in BS of \u003cem\u003eC. chanhua\u003c/em\u003e was significantly higher than that in CS. The increase in the net nitrogen nitrification rate means that more ammonium nitrogen in the soil is converted into nitrate nitrogen, and nitrate nitrogen is more easily absorbed and utilized by plants, thereby enhancing the soil's nitrogen supply capacity and promoting plant growth [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. Li et al. [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] found that higher net nitrogen nitrification rates contribute to the maintenance of ecosystem health and stability. Therefore, \u003cem\u003eC. chanhua\u003c/em\u003e promoted soil nitrogen cycling by increasing net nitrogen nitrification efficiency in the soil environment. Soil net ammonification rate was defined as the rate at which soil organic nitrogen was decomposed into ammonium by microorganisms in a certain period of time [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. The net ammonification rates of both BS and CS were negative, indicating that the reduction rate of ammonium in soil exceeded the conversion rate of organic nitrogen to ammonium during the growth stage of \u003cem\u003eC. chanhua\u003c/em\u003e [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. The possible reason was that the cultivation of \u003cem\u003eC. chanhua\u003c/em\u003e increased organic matter and microbial activity in the soil environment, which might rapidly absorb ammonium nitrogen in the soil for growth and metabolism [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. In this study, we reported that the net nitrogen ammonification rate in BS was significantly lower than that in CS. The possible reason was that the cultivation of \u003cem\u003eC. chanhua\u003c/em\u003e reduced the microbial activity in the soil environment, which reduced the decomposition and transformation of organic nitrogen, leading to a decrease in the net nitrogen ammonification rate [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. In conclusion, \u003cem\u003eC. chanhua\u003c/em\u003e increased soil nitrogen transformation rate, thereby enhancing soil nitrogen cycling.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCordyceps chanhua\u003c/em\u003e and its habitat contain not only a large number of fungal community structures [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e], but also abundant bacterial communities [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. In this study, it was found that the bacterial community diversity in IS of \u003cem\u003eC. chanhua\u003c/em\u003e decreased first and then increased, which was contrary to the results of other researchers [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], possibly due to the different cultivation environments [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. In this study, soil cover was cultivated in the laboratory, sterile water was added for moisturizing, and the amount of water for soil-covered \u003cem\u003eC. chanhua\u003c/em\u003e was different from the rainfall in the natural environment. Previous studies have shown that water factor is a major factor affecting microbial activity, gene expression, and community composition [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Therefore, this also leads to the change of bacterial community diversity during the growth of \u003cem\u003eC. chanhua\u003c/em\u003e. In this study, the bacterial community composition analysis showed that \u003cem\u003eStenotrophomonas\u003c/em\u003e, \u003cem\u003eAchromobacter\u003c/em\u003e, and \u003cem\u003eSerratia\u003c/em\u003e were dominant in JC sample. \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eCedecea\u003c/em\u003e were the dominant groups in S2 sample. \u003cem\u003eAchromobacter\u003c/em\u003e, \u003cem\u003eStaphylococcus\u003c/em\u003e, and \u003cem\u003ePseudomonas\u003c/em\u003e were the dominant bacterial groups in S3 sample. The results of this study were not exactly the same as those of other studies. Qu et al. [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e] studied the bacterial communities of wild \u003cem\u003eC. chanhua\u003c/em\u003e, and the results showed that the most dominant bacterial genus was \u003cem\u003eCedecea\u003c/em\u003e, followed by \u003cem\u003eRickettsia\u003c/em\u003e and \u003cem\u003eBurkholderia-Paraburkholderia\u003c/em\u003e. Huang et al. [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e] analyzed the microflora of wild \u003cem\u003eC. chanhua\u003c/em\u003e in Anji, Zhejiang and found that Achromobacter, f__Enterobacteriaceae_Unclassified, \u003cem\u003eStenotrophomonas\u003c/em\u003e, \u003cem\u003eBurkholderia-Caballeronia-Paraburkholderia Allorhizobium\u003c/em\u003e, \u003cem\u003eNeorhizobium, Pararhizobium\u003c/em\u003e, \u003cem\u003eRhizobium\u003c/em\u003e are the main dominant bacterial groups in the \u003cem\u003eC. chanhua\u003c/em\u003e community. Therefore, the bacterial communities of \u003cem\u003eC. chanhua\u003c/em\u003e have different characteristics in different growth environments and different growth stages. Even if there are the same bacterial groups, the abundance is different. As one of the most abundant and abundant microbial populations in the soil microbial community, bacteria are closely related to the complexity of soil [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. There were 18 common OTUs in JC, S2, and S3 samples, indicating that the growth and development of \u003cem\u003eC. chanhua\u003c/em\u003e cannot be separated from some specific bacterial groups, which is consistent with our previous conclusion [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLiang et al. [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e] found that the bacterial communities in \u003cem\u003eC. sinensis\u003c/em\u003e were closely related to the external environmental conditions in which they grew, and the bacterial communities in \u003cem\u003eC. chanhua\u003c/em\u003e growing in different places were also different [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. Mou et al. [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e] analyzed the diversity of bacterial community in the inner sclerotium and membrane of \u003cem\u003eC. chanhua\u003c/em\u003e growing in Guiyang, Guizhou Province, and the soil of its habitat, and the results showed that the diversity of bacterial community in the soil environment was significantly higher than that in the inner sclerotium samples, which was similar to the results of this study. In other words, the diversity of bacterial community in soil samples was significantly higher than that in inner sclerotium samples. And the results of previous studies using modern molecular biological methods were similar [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. The microbial composition in IS of \u003cem\u003eC. chanhua\u003c/em\u003e was significantly different from that in the soil environment, possibly because the colonization of cordyceps fungi may reshape the internal environment of the infected insects [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eChanges in soil physical and chemical properties can easily affect the diversity and composition of bacterial communities; conversely, environmental factors have a greater impact on soil bacterial community structure [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. As the main undertakers of biochemical processes in soil, microorganisms have important contributions to soil nutrient conversion, soil fertility and soil health [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. Previous studies have found that different nitrogen forms can affect the bacterial groups in soil environment, such as ammonium nitrogen and nitrate nitrogen can affect the total bacterial and nitrogen-fixing bacteria communities in acidic red soil [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. In addition, nitrogen morphology can significantly affect the number of bacterial operational taxa and Shannon index [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. Luo et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] found that \u003cem\u003eC. militaris\u003c/em\u003e of the same genus as \u003cem\u003eC. chanhua\u003c/em\u003e can utilize organic nitrogen and some inorganic nitrogen, among which the utilization level of ammonium nitrogen is higher than that of other inorganic nitrogen, which is similar to the results of this study, that is, each bacterial community has the greatest influence on ammonium nitrogen and organic nitrogen. Organic nitrogen can effectively improve soil physical and chemical properties, enhance soil fertility, and provide a good growth environment for soil bacteria [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. Moreover, organic nitrogen can significantly increase the richness and diversity of bacterial communities in soil [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. Ammonium nitrogen is the main form directly absorbed by plant roots from soil, and it is also an important index to measure soil fertility [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. The content and distribution of soil ammonium nitrogen absorbed and utilized by plant roots not only have an important impact on the soil nitrogen cycling process [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e], but also the level of plant productivity is closely related to the level of soil ammonium nitrogen content [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]. The plants can directly absorb and utilize nitrate nitrogen and ammonium nitrogen, and changes in the content of these two kinds of nitrogen can affect plant productivity [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Ding et al. [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e] showed that nitrate nitrogen is the main environmental factor affecting soil microbial community, and Yuan et al. [\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e] found that soil ammonium nitrogen plays an important role in soil bacterial community, which may be due to the selective use of different forms of nitrogen in soil. The cultivation of \u003cem\u003eC. chanhua\u003c/em\u003e in the soil will release excess nitrate nitrogen in the insect and absorb ammonium nitrogen in the soil environment. Therefore, \u003cem\u003eC. chanhua\u003c/em\u003e can be associated with it in the later stage, and the mechanism of \u003cem\u003eC. chanhua\u003c/em\u003e improving crop quality and yield can be explored from the perspective of nitrogen fertilizer.\u003c/p\u003e\u003cp\u003eSoil bacterial community diversity is very sensitive to changes in the external environment, and is often regarded as an early warning indicator of changes in soil ecosystem, an important basis for evaluating soil quality and fertility [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e], and is also commonly used to evaluate the health of soil ecosystem [\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e]. One of the important reasons for the changes of soil bacterial community is the changes of soil physical and chemical properties. Soil bacteria are an extremely important part of soil microecosystem, participating in various activities such as soil nutrient transformation [\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e]. This study found that exonitrogen aqueous solution of \u003cem\u003eC. chanhua\u003c/em\u003e could reduce the richness, diversity and functions of bacterial communities in soil, and increase the relative abundance of functions related to nitrogen cycling. Soil physical and chemical properties may be an important driving factor for the change of soil microbial community [\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e]. Since the exocrine nitrogen aqueous solution of cultivated \u003cem\u003eC. chanhua\u003c/em\u003e changes soil nitrogen status, soil microorganisms also change correspondingly.\u003c/p\u003e\u003cp\u003eThe main bacteria regulating the nitrogen conversion process are ammoniating bacteria and denitrifying bacteria, while the number of nitrifying bacteria is relatively small [\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e]. Therefore, this study mainly focused on isolation, cultivation and identification of bacterial strains producing high levels of ammonium and nitrite nitrogen. The results showed that B1 and B5 belong to the genus \u003cem\u003eDelftia\u003c/em\u003e sp., and B5 is the type species \u003cem\u003eD. acidovorans\u003c/em\u003e of the genus. \u003cem\u003eDelftia\u003c/em\u003e not only has good biocontrol potential [\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e, \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e]. Moreover, it also affects the transformation and mineralization of organic pollutants in the environment [\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e], and the environmental bioreactor potential of degradable polyethylene terephthalate to synthetic polyester materials [\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e]. It has been reported that most \u003cem\u003eDelftia\u003c/em\u003e bacteria are capable of degrading harmful organic matter [\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e, \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e], residual pesticides in soil, etc. [\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e]. It was identified that strain L4, which can produce both NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N and NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e-N, belongs to \u003cem\u003ePseudomonas protegens\u003c/em\u003e, which is closely related to agriculture and can infect and kill pests. It can be used as a biological control agent [\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e]. In this study, three strains of bacteria with high ammonium nitrogen or nitrite nitrogen production were isolated from IS of \u003cem\u003eC. chanhua\u003c/em\u003e. As an entomogenous fungus, \u003cem\u003eC. chanhua\u003c/em\u003e can be applied to biological control [\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e]. Whether its biocontrol ability is related to the bacteria in \u003cem\u003eC. chanhua\u003c/em\u003e is worthy of further study.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, it was found that during the growth of \u003cem\u003eC. chanhua\u003c/em\u003e absorb ammonium nitrogen, nitrite nitrogen and hydroxylamine nitrogen in the soil, and nitrate nitrogen and organic nitrogen were secreted. \u003cem\u003eC. chanhua\u003c/em\u003e increased the rate of soil nitrogen conversion, thus promoting the soil nitrogen cycle in the microenvironment. The growth of \u003cem\u003eC. chanhua\u003c/em\u003e decreased the diversity of soil bacterial community, but increased the relative abundance of bacterial community related to nitrogen cycle. The bacterial community inside and outside the \u003cem\u003eC. chanhua\u003c/em\u003e mycelia had the greatest influence on ammonium nitrogen and organic nitrogen. Nitrogen excreted by \u003cem\u003eC. chanhua\u003c/em\u003e further decreased the diversity of soil bacterial community, but increased the abundance of bacteria associated with nitrogen cycle. The bacteria related to the conversion were isolated from \u003cem\u003eC. chanhua\u003c/em\u003e, and it was verified that the endophytic bacteria promoted the participation of \u003cem\u003eC. chanhua\u003c/em\u003e in soil nitrogen cycle.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eP1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ethe rigor stage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eP2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emembrane formation stage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eP3\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emature stage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003einner sclerotium\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ebacteriospheric soil (soil tightly wrapped about 0.2 cm of the insect body)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003econtrol soil (soil without C. chanhua in the same conditions)\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSNN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSoil samples with aqueous solution of exonitrogen from C. chanhua added\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSNck\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSoil samples without the addition of exonitrogen aqueous solution of C. chanhua were compared with SNck\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eJC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInner sclerotium of C. chanhua at P1 stage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eS2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInner sclerotium of C. chanhua at P2 stage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eS3\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInner sclerotium of C. chanhua at P3 stage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSm2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBacteriospheric soil samples of C. chanhua at P2 stage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSck2\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSoil samples without C. chanhua at P2 stage, which contrasted with Sm2\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSm3\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBacteriospheric soil samples of C. chanhua at P3 stage\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSck3\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSoil samples without C. chanhua at P3 stage, which contrasted with Sm3\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.H. and X.Z. conceived the project; Y.Y. and Z.X. designed experiments; J.Q., T.W., Y.R., Y.Z., Y.H., and Y.D. performed research; G.F., J.Z., and C.D. analyzed data; and G.H., T.H. and X.Z. wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe gratefully acknowledge financial support from the National Natural Science Foundation of China (32060038) and Science and Technology Project of Guizhou Province (Qian ke he Foundation [2020]1Z009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available within the paper and its supplementary information. The raw amplicon sequence data (16S rRNA gene) generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1141932. Source data are provided in this work.\u003c/p\u003e\n\u003ch3\u003eEthics approval and consent to participate\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eConsent for publication\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMang Q, Gao J, Li Q, Sun Y, Xu G, Xu P. Metagenomic insight into the effect of probiotics on nitrogen cycle in the \u003cem\u003eCoilia nasus\u003c/em\u003e aquaculture pond water. Microorganisms. 2024; 12(3): 627. https://doi.org/10.3390/microorganisms12030627.\u003c/li\u003e\n\u003cli\u003eGold AC, Thompson SP, Piehler MF. Nitrogen cycling processes within stormwater control measures: A review and call for research. Water Res. 2019; 149: 578-587. https://doi.org/10.1016/j.watres.2018.10.036.\u003c/li\u003e\n\u003cli\u003eIsobe K, Ohte N. Ecological perspectives on microbes involved in N-cycling.\u003cem\u003e \u003c/em\u003eMicrobes Environ. 2014; 29(1): 4-16. https://doi.org/10.1264/jsme2.me13159.\u003c/li\u003e\n\u003cli\u003eGunina A, Kuzyakov Y. From energy to (soil organic) matter. Glob Chang Biol. 2022; 28(7): 2169-2182. https://doi.org/10.1111/gcb.16071.\u003c/li\u003e\n\u003cli\u003eKuypers MMM, Marchant HK. Kartal B. The microbial nitrogen-cycling network\u003cem\u003e. \u003c/em\u003eNat Rev Microbiol. 2018; 16(5): 263-276. https://doi.org/10.1038/nrmicro.\u003c/li\u003e\n\u003cli\u003eMattoo R, Suman BM. Microbial roles in the terrestrial and aquatic nitrogen cycle-implications in climate change. FEMS Microbiol Lett. 2023; 370: fnad061. https://doi.org/10.1093/femsle/fnad061.\u003c/li\u003e\n\u003cli\u003eNardi JB, Mackie RI, Dawson JO. Could microbial symbionts of arthropod guts contribute significantly to nitrogen fixation in terrestrial ecosystems?\u003cem\u003e \u003c/em\u003eInsect Physiol. 2002; 48(8): 751-763. https://doi.org/10.1016/s0022-1910(02)00105-1.\u003c/li\u003e\n\u003cli\u003eJones DL, Hodge A, Kuzyakov Y. Plant and mycorrhizal regulation of rhizodeposition. New Phytol. 2004; 163(3): 459-480. https://doi.org/10.1111/j.1469-8137.2004.01130.x.\u003c/li\u003e\n\u003cli\u003eMaia LB, Moura JJ. How biology handles nitrite. Chem Rev. 2014; 114(10): 5273-357. https://doi.org/10.1021/cr400518y.\u003c/li\u003e\n\u003cli\u003eMerloti LF, Mendes LW, Pedrinho A, de Souze LF, Ferrari BM, Tsai SM. forest-to-agriculture conversion in amazon drives soil microbial communities and N-cycle. Soil Biology and Biochemistry. 2019; 137: 107567. https://doi.org/10.1016/j.soilbio.2019.107567.\u003c/li\u003e\n\u003cli\u003eErisman JW, Sutton MA, Galloway J, Klimont Z, Winiwarter W. How a century of ammonia synthesis changed the world. Nature geoscience. 2008; 1(10): 636-639. https://doi.org/10.1038/ngeo325.\u003c/li\u003e\n\u003cli\u003eTedersoo L, Bahram M, P\u0026otilde;lme S, K\u0026otilde;ljalg U, Yorou NS, Wijesundera R, Ruiz LV, Vasco-Palacios AM, Thu PQ, Suija A, Smith ME. Global diversity and geography of soil fungi. Science. 2014; 346(6213): 1256688. https://doi.org/10.1126/science.1256688.\u003c/li\u003e\n\u003cli\u003eFang L, Lakshmanan P, Su X, Shi Y, Chen Z, Zhang Y, Sun W, Wu J, Xiao R, Chen X. Impact of residual antibiotics on microbial decomposition of livestock manures in eutric regosol: implications for sustainable nutrient recycling and soil carbon sequestration. Journal of Environmental Sciences. 2025; 147: 498-511. https://doi.org/10.1016/j.jes.2023.10.021.\u003c/li\u003e\n\u003cli\u003eBoddy L, Wood J, Redman E, Hynes J, Fricker MD. Fungal network responses to grazing. Fungal Genet Biol. 2010; 47(6): 522-30. https://doi.org/10.1016/j.fgb.2010.01.006. \u003c/li\u003e\n\u003cli\u003eTobar RM, Azc\u0026oacute;n R, Barea JM. The improvement of plant N acquisition from an ammonium-treated drought-stressed soil by the fungal symbiont in arbuscular mycorrhizae. Mycorrhiza. 1994; 4: 105-108. https://doi.org/10.1007/BF00203769.\u003c/li\u003e\n\u003cli\u003eJackson LE, Burger M, Cavagnaro TR. Roots nitrogen transformations and ecosystem services. Annu Rev Plant Biol. 2008; 59: 341-63. https://doi.org/10.1146/annurev.arplant.59.032607.092932.\u003c/li\u003e\n\u003cli\u003eSmith ML, Bruhn JN, Anderson JB. The fungus \u003cem\u003eArmillaria bulbosa\u003c/em\u003e is among the largest and oldest living organisms. Nature. 1992; 356 (6368): 428-431. https://doi.org/10.1038/356428a0.\u003c/li\u003e\n\u003cli\u003ePascale FK, Garbaye J, Tarkka M. The mycorrhiza helper bacteria revisited. New Phytol. 2007; 176(1): 22-36. https://doi.org/10.1111/j.1469-8137.2007.02191.x.\u003c/li\u003e\n\u003cli\u003eMurindangabo YT, Kopeck\u0026yacute; M, Pern\u0026aacute; K, Konvalina P, Bohat\u0026aacute; A, Kavkov\u0026aacute; M, Nguyen TG, Hoang TN. Relevance of entomopathogenic fungi in soil\u0026ndash;plant systems. Plant and Soil. 2024 Feb;495(1):287-310. https://doi.org/10.1007/s11104-023-06325-8.\u003c/li\u003e\n\u003cli\u003eQu J, Zhou Y, Yu J, Zhang J, Han Y, Zou X. Estimated divergence times of \u003cem\u003eHirsutella\u003c/em\u003e (asexual morphs) in \u003cem\u003eOphiocordyceps\u003c/em\u003e provides insight into evolution of phialide structure. BMC Evol Biol. 2018; 18(1): 111. https://doi.org/10.1186/s12862-018-1223-0.\u003c/li\u003e\n\u003cli\u003eLuo L, Dai F, Xu Z. S, Guan J. Q, Fei G. X; Qu JJ; Yao M, Xue Y, Zhou YM, Zou X. Core microbes in \u003cem\u003eCordyceps militaris \u003c/em\u003esclerotia and their nitrogen metabolism-related ecological functions. Microbiol Spectr. 2024; 12(10): e0105324. https://doi.org/10.1128/spectrum.01053-24.\u003c/li\u003e\n\u003cli\u003eSharma H, Sharma N, An SSA. Unique bioactives from zombie fungus (\u003cem\u003eCordyceps\u003c/em\u003e) as promising multitargeted neuroprotective agents. Nutrients. 2023; 16(1): 102. https://doi.org/10.3390/nu16010102.\u003c/li\u003e\n\u003cli\u003eWang X, Li Y, Li X, Sun L, Feng Y, Sa F, Ge Y, Yang S, Liu Y, Li W, Cheng X. Transcriptome and metabolome profiling unveils the mechanisms of naphthalene acetic acid in promoting cordycepin synthesis in \u003cem\u003eCordyceps militaris\u003c/em\u003e. Frontiers in Nutrition. 2023; 10: 1104446. https://doi.org/10.3389/fnut.2023.1104446.\u003c/li\u003e\n\u003cli\u003eKato T, Inagaki S, Shibata C, Takayanagi K, Uehara H, Nishimura K, Park EY. Topical infection of \u003cem\u003eCordyceps militaris\u003c/em\u003e in silkworm larvae through the cuticle has lower infectivity compared to \u003cem\u003eBeauveria bassiana\u003c/em\u003e and \u003cem\u003eMetarhizium anisopliae\u003c/em\u003e. Current Microbiology. 2025; 82(1): 1-0. https://doi.org/10.1007/s00284-024-03989-y.\u003c/li\u003e\n\u003cli\u003eJaber LR, Ownley BH. Can we use entomopathogenic fungi as endophytes for dual biological control of insect pests and plant pathogens? Biological Control. 2018; 1(116): 36-45. https://doi.org/10.1016/j.biocontrol.2017.01.018.\u003c/li\u003e\n\u003cli\u003eKlironomos JN, Hart MM. Animal nitrogen swap for plant carbon. Nature. 2001; 410(6829): 651-2. https://doi.org/10.1038/35070643.\u003c/li\u003e\n\u003cli\u003eDean SL, Farrer EC, Taylor DL, Porras-Alfaro A, Suding KN, Sinsabaugh RL. Nitrogen deposition alters plant-fungal relationships: Linking below ground dynamics to aboveground vegetation change. Molecular Ecology. 2014; 23(6): 1364-1378. https://doi.org/10.1111/mec.12541.\u003c/li\u003e\n\u003cli\u003eWyrebek M, Huber C, Sasan RK, Bidochka MJ. Three sympatrically occurring species of \u003cem\u003eMetarhizium\u003c/em\u003e show plant rhizosphere specificity. Microbiology. 2011; 157(10): 2904-2911. https://doi.org/10.1099/mic.0.051102-0.\u003c/li\u003e\n\u003cli\u003eSasan RK, Bidochka MJ. The insect-pathogenic fungus \u003cem\u003eMetarhizium robertsii\u003c/em\u003e (Clavicipitaceae) is also an endophyte that stimulates plant root development. Am J Bot. 2012; 9(1): 101-7. https://doi.org/10.3732/ajb.1100136.\u003c/li\u003e\n\u003cli\u003eBehie SW, Moreira CC, Sementchoukova I, Barelli L, Zelisko PM, Bidochka MJ. Carbon translocation from a plant to an insect-pathogenic endophytic fungus. Nat Commun. 2017; 8: 14245. https://doi.org/10.1038/ncomms14245.\u003c/li\u003e\n\u003cli\u003eSaikkonen K, Faeth SH, Helander M, Sullivan TJ. Fungal endophytes: A continuum of interactions with host plants. Annual review of Ecology and Systematics. 1998; 29(1): 319-343. https://doi.org/10.1146/annurev.ecolsys.29.1.319.\u003c/li\u003e\n\u003cli\u003eBehie SW, Zelisko PM, Bidochka MJ. Endophytic insect-parasitic fungi translocate nitrogen directly from insects to plants. Science. 2012: 336(6088): 1576-7. https://doi.org/10.1126/science.1222289.\u003c/li\u003e\n\u003cli\u003eLovett GM, Christenson LM, Groffman PM, Jones CG, Hart JE, Mitchell MJ. Insect defoliation and nitrogen cycling in forests: laboratory, plot, and watershed studies indicate that most of the nitrogen released from forest foliage as a result of defoliation by insects is redistributed within the ecosystem, whereas only a small fraction of nitrogen is lost by leaching. BioScience. 2002; 52(4): 335-41. https://doi.org/10.1641/0006-3568.\u003c/li\u003e\n\u003cli\u003eBehie SW, Bidochka MJ. Ubiquity of insect-derived nitrogen transfer to plants by endophytic insect-pathogenic fungi: An additional branch of the soil nitrogen cycle. Applied and environmental microbiology. 2014: 80(5): 1553-60. https://doi.org/10.1128/AEM.03338-13.\u003c/li\u003e\n\u003cli\u003eMa M, Luo J, Li C, Eleftherianos I, Zhang W, Xu L. A Life-And-Death Struggle: Interaction of insects with entomopathogenic fungi across various infection stages. Frontiers in immunology. 2024; 14: 1329843. https://doi.org/10.3389/fimmu.2023.1329843.\u003c/li\u003e\n\u003cli\u003eNxumalo W, Elateeq AA, Sun Y. Can \u003cem\u003eCordyceps cicada\u003c/em\u003ee be used as an alternative to \u003cem\u003eCordyceps militaris \u003c/em\u003eand \u003cem\u003eCordyceps sinensis\u003c/em\u003e? - A review. J Ethnopharmacol. 2020; 257: 112879. https://doi.org/doi:10.1016/j.jep.2020.112879.\u003c/li\u003e\n\u003cli\u003eZeng ZY, Mou D, Luo L, Zhong WL, Duan L, Zou X. Different cultivation environments affect the yield bacterial community and metabolites of \u003cem\u003eCordyceps cicadae\u003c/em\u003e. Frontiers in microbiology. 2021; 12: 669785. https://doi.org/10.3389/fmicb.2021.669785.\u003c/li\u003e\n\u003cli\u003eGarbaye J, Bowen GD. Stimulation of ectomycorrhizal infection of \u003cem\u003ePinus radiata\u003c/em\u003e by some microorganisms associated with the mantle of ectomycorrhizas. New Phytologist. 1989: 112: 383-388. https://doi.org/10.1111/j.1469-8137.1989.tb00327.x.\u003c/li\u003e\n\u003cli\u003eBallhausen MB, de Boer W. The sapro-rhizosphere: carbon flow from saprotrophic fungi into fungus-feeding bacteria. Soil Biology and Biochemistry. 2016; 102: 14-17. https://doi.org/10.1016/j.soilbio.2016.06.014.\u003c/li\u003e\n\u003cli\u003eHaq IU, Zhang M, Yang P, van Elsas JD. The Interactions of bacteria with fungi in soil: emerging concepts. Adv Appl Microbiol. 2014; 89: 185-215. https://doi.org/10.1016/B978-0-12-800259-9.00005-6.\u003c/li\u003e\n\u003cli\u003eQu QS, Yang F, Zhao CY, Shi XY. Analysis of the bacteria community in wild \u003cem\u003eCordyceps cicadae\u003c/em\u003e and its influence on the production of HEA and nucleosides in \u003cem\u003eCordyceps cicadae\u003c/em\u003e. Journal of Applied Microbiology. 2019; 127(6): 1759-67. https://doi.org/10.1111/jam.14432.\u003c/li\u003e\n\u003cli\u003eVannini C, Carpentieri A, Salvioli A, Novero M, Marsoni M, Testa L, de Pinto M. C, Amoresano A, Ortolani F, Bracale M, Bonfante P. An interdomain network: the endobacterium of a mycorrhizal fungus promotes antioxidative responses in both fungal and plant hosts. New Phytol. 2016; 211(1): 265-75. https://doi.org/10.1111/nph.13895.\u003c/li\u003e\n\u003cli\u003eNelson MB, Martiny AC, Martiny JB. Global biogeography of microbial nitrogen-cycling traits in soil. Proc Natl Acad Sci U S A. 2016; 113(29): 8033-40. https://doi.org/10.1073/pnas.1601070113.\u003c/li\u003e\n\u003cli\u003ePrasse D, Schmitz RA. Small RNAs involved in regulation of nitrogen metabolism. Microbiol Spectr. 2018; 6(4): 10-208. https://doi.org/10.1128/microbiolspec.rwr-0018-2018.\u003c/li\u003e\n\u003cli\u003eGupta KJ, Brotman Y, Segu S, Zeier T, Zeier J, Persijn ST, Cristescu SM, Harren FJ, Bauwe H, Fernie AR, Kaiser WM. The form of nitrogen nutrition affects resistance against \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003ephaseolicola \u003c/em\u003ein tobacco. Journal of Experimental Botany. 2013; 64(2): 553-68. https://doi.org/10.1093/jxb/ers348.\u003c/li\u003e\n\u003cli\u003eWang Q, Huang Y, Ren Z, Zhang X, Ren J, Su J, Zhang C, Tian J, Yu Y, Gao GF, Li L. Transfer cells mediate nitrate uptake to control root nodule symbiosis. Nature Plants. 2020; 6(7): 800-8. https://doi.org/10.1038/s41477-020-0683-6.\u003c/li\u003e\n\u003cli\u003eHu G, Zhou Y, Mou D, Qu J, Luo L, Duan L, Xu Z, Zou X. Filtration effect of \u003cem\u003eCordyceps chanhua\u003c/em\u003e mycoderm on bacteria and its transport function on nitrogen. Microbiology Spectrum. 2024; 12(1): e01179-23. https://doi.org/10.1128/spectrum.01179-23.\u003c/li\u003e\n\u003cli\u003eDatta D, Paul M, Murshed M, Teng SW, Schmidtke L. Soil moisture organic carbon and nitrogen content prediction with hyperspectral data using regression models. Sensors (Basel). 2022; 22(20): 7998. https://doi.org/doi:10.3390/s22207998.\u003c/li\u003e\n\u003cli\u003eMotamedi E, Safari M, Salimi M. Improvement of tomato yield and quality using slow release NPK fertilizers prepared by \u003cem\u003eCarnauba wax\u003c/em\u003e emulsion starch-based latex and hydrogel nanocomposite combination. Sci Rep. 2023; 13(1): 11118. https://doi.org/10.1038/s41598-023-38445-7.\u003c/li\u003e\n\u003cli\u003eYan S. Yan H. Wang C. Jiang C. Li W. Chen A. Zhang D. Effects of urea combined with nitrification inhibitor (DMPP) on soil nitrogen transformation and N\u003csub\u003e2\u003c/sub\u003eO emission in vegetable field. Journal of Agro-Environment Science (Chinese). 2025; 45: 1-19. http://kns.cnki.net/kcms/detail/12.1347.S.20250124.1021.002.html.\u003c/li\u003e\n\u003cli\u003eZhou H, Zhang DG, Jiang ZH, Sun P, Xiao HL, Wang YX, Chen JG. Changes in the soil microbial communities of alpine steppe at Qinghai-Tibetan Plateau under different degradation levels. Sci Total Environ. 2019; 651: 2281-2291. https://doi.org/10.1016/j.scitotenv.2018.09.336.\u003c/li\u003e\n\u003cli\u003eHill TCJ, Walsh KA, Harris JA, Moffett BF. Using ecological diversity measures with bacterial communities. FEMS Microbiology Ecology. 2003; 43(1): 1-11. https://doi.org/10.1111/fem.2003.43.issue-1.\u003c/li\u003e\n\u003cli\u003eLi C, Aluko OO, Yuan G, Li J, Liu H. The responses of soil organic carbon and total nitrogen to chemical nitrogen fertilizers reduction base on a meta-analysis. Sci Rep. 2022; 12(1): 16326. https://doi.org/10.1038/s41598-022-18684-w.\u003c/li\u003e\n\u003cli\u003eLi J, Lu X, Zhu Q, Zhuang Y, Yang W, Qi D. Nitrate \u0026delta;\u003csup\u003e15\u003c/sup\u003eN and \u0026delta;\u003csup\u003e18\u003c/sup\u003eO values reveal mariculture impacts on nitrogen cycling in Sansha Bay, SE China. Journal of Marine Science and Engineering. 2025; 13(2): 343. https://doi.org/10.3390/jmse13020343.\u003c/li\u003e\n\u003cli\u003eLiu XJ, Hu B, Chu CC. Nitrogen assimilation in plants: current status and future prospects. J Genet Genomics. 2022; 49(5): 394-404. https://doi.org/10.1016/j.jgg.2021.12.006.\u003c/li\u003e\n\u003cli\u003eWang C, Zheng MM, Hu AY, Zhu CQ, Shen RF. diazotroph abundance and community composition in an acidic soil in response to aluminum-tolerant and aluminum-sensitive maize (\u003cem\u003eZea mays\u003c/em\u003e L.) cultivars under two nitrogen fertilizer forms. Plant and Soil. 2018; 424: 463-478. https://doi.org/10.1007/s11104-017-3550-0.\u003c/li\u003e\n\u003cli\u003eWang TT, Sun SL, Xu YX, Waigi MG, Odinga ES, Vasilyeva GK, Gao YZ, Hu XJ. Nitrogen regulates the distribution of antibiotic resistance genes in the soil-vegetable system. Front Microbiol. 2022; 13: 848750. https://doi.org/10.3389/fmicb.2022.848750.\u003c/li\u003e\n\u003cli\u003eZayed O, Hewedy OA, Abdelmoteleb A, Ali M, Youssef MS, Roumia AF, Seymour D, Yuan ZC. Nitrogen journey in plants: from uptake to metabolism stress response and microbe interaction. Biomolecules. 2023: 13(10): 1443. https://doi.org/10.3390/biom13101443.\u003c/li\u003e\n\u003cli\u003eZhu-Barker X, Cavazos AR, Ostrom NE, Horwath WR, Glass JB. The importance of abiotic reactions for nitrous oxide productio. Biogeochemistry. 2015; 126(3): 251-267. https://doi.org/10.1007/s10533-015-0166-4.\u003c/li\u003e\n\u003cli\u003eLiu SR, Han P, Hink L, Prosser JI, Wagner M, Br\u0026uuml;ggemann N. Abiotic conversion of extracellular NH\u003csub\u003e2\u003c/sub\u003eOH contributes to N\u003csub\u003e2\u003c/sub\u003eO emission during ammonia oxidation. Environ Sci Technol. 2017; 51(22): 13122-13132. https://doi.org/10.1021/acs.est.7b02360.\u003c/li\u003e\n\u003cli\u003eLi YY, Chen XH, Xie ZX, Li DX, Wu PF, Kong LF, Lin L, Kao SJ, Wang DZ. Bacterial diversity and nitrogen utilization strategies in the upper layer of the northwestern Pacific Ocean. Frontiers in microbiology. 2018; 9: 797. https://doi.org/10.3389/fmicb.2018.00797.\u003c/li\u003e\n\u003cli\u003eFrancis CA, Beman JM, Kuypers MM. New processes and players in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation. The ISME journal. 2007; 1(1): 19-27. https://doi.org/10.1038/ismej.2007.8.\u003c/li\u003e\n\u003cli\u003eGuan Y, Wu M, Che S, Yuan S, Yang X, Li S, Tian P, Wu L, Yang M, Wu Z. Effects of continuous straw returning on soil functional microorganisms and microbial communities. Journal of Microbiology. 2023; 61(1): 49-62. https://doi.org/10.1007/s12275-022-00004-6.\u003c/li\u003e\n\u003cli\u003eProsser JI. Autotrophic nitrification in bacteria. Adv Microb Physiol. 1989; 30: 125-81. https://doi.org/10.1016/s0065-2911(08)60112-5.\u003c/li\u003e\n\u003cli\u003eDe Boer W, Gunnewiek PK, Veenhuis M, Bock E, Laanbroek HJ. Nitrification at low pH by aggregated chemolithotrophic bacteria. Applied and Environmental Microbiology. 1991; 57(12): 3600-4. https://doi.org/10.1128/aem.57.12.3600-3604.1991.\u003c/li\u003e\n\u003cli\u003eLuo F, Tang G, Hong S, Gong T, Xin XF, Wang C. Promotion of \u003cem\u003eArabidopsis\u003c/em\u003e immune responses by a rhizosphere fungus via supply of pipecolic acid to plants and selective augment of phytoalexins. Science China Life Sciences. 2023; 66(5): 1119-33. https://doi.org/10.1007/s11427-022-2238-8.\u003c/li\u003e\n\u003cli\u003eMa Y, Zheng X, Fang Y, Xu K, He S, Zhao M. Autotrophic denitrification in constructed wetlands: achievements and challenges. Bioresour Technol. 2020; 318: 123778. https://doi.org/10.1016/j.biortech.2020.123778.\u003c/li\u003e\n\u003cli\u003eLi YJ, Liu X, Wu DF, Chen BH, Ren XJ, Tang J. Effects of continuous croping of greenhouse cucumber on soil fungal abundance and community structure Province. Acta Agriculturae Boreali-occidentalis Sinica. 2020; 35(1): 194-204. https://doi.org/10.1007/s00284-021-02485-x.\u003c/li\u003e\n\u003cli\u003eRebello S, Nathan VK, Sindhu R, Binod P, Awasthi MK, Pandey A. Bioengineered microbes for soil health restoration: present status and future. Bioengineered. 2021; 12(2): 12839-53. https://doi.org/10.1080/21655979.2021.2004645.\u003c/li\u003e\n\u003cli\u003eReimer M, Kopp C, Hartmann T, Zimmermann H, Ruser R, Schulz R, M\u0026uuml;ller T, M\u0026ouml;ller K. Assessing long term effects of compost fertilization on soil fertility and nitrogen mineralization rate. Journal of Plant Nutrition and Soil Science. 2023; 186(2): 217-33. https://doi.org/10.1002/jpln.202200270.\u003c/li\u003e\n\u003cli\u003eYadav MR, Kumar R, Parihar CM, Yadav RK, Jat SL, Ram H, Meena RK, Singh M, Verma AP, Kumar UJ, Ghosh A. Strategies for improving nitrogen use efficiency: A review. Agricultural Reviews. 2017; 38(1): 29-40. https://doi.org/10.18805/ag.v0iOF.7306.\u003c/li\u003e\n\u003cli\u003eAnas M, Liao F, Verma KK, Sarwar MA, Mahmood A, Chen ZL, Li Q, Zeng XP, Liu Y, Li YR. Fate of nitrogen in agriculture and environment: agronomic eco-physiological and molecular approaches to improve nitrogen use efficiency. Biological research. 2020; 53: 1-20. https://doi.org/10.1186/s40659-020-00312-4.\u003c/li\u003e\n\u003cli\u003eWu C, Wei F, Yan B, Liu G, Wang G. Long-term nitrogen and phosphorus fertilization improved crop yield by influencing rhizosphere nitrogen transformation processes. Applied Soil Ecology. 2025; 208: 105968. https://doi.org/10.1016/j.apsoil.2025.105968.\u003c/li\u003e\n\u003cli\u003eBei S, Li X, Kuyper TW, Chadwick DR, Zhang J. Nitrogen availability mediates the priming effect of soil organic matter by preferentially altering the straw carbon-assimilating microbial community. Science of the Total Environment. 2022; 815: 152882. https://doi.org/10.1016/j.scitotenv.2021.152882.\u003c/li\u003e\n\u003cli\u003eDu L, Zhong H, Guo X, Li H, Xia J, Chen Q. Nitrogen fertilization and soil nitrogen cycling: Unraveling the links among multiple environmental factors functional genes and transformation rates. Science of The Total Environment. 2024; 951: 175561. https://doi.org/10.1016/j.scitotenv.2024.175561.\u003c/li\u003e\n\u003cli\u003eZou W, Lang M, Zhang L, Liu B, Chen X. Ammonia-oxidizing bacteria rather than ammonia-oxidizing archaea dominate nitrification in a nitrogen-fertilized calcareous soil. Science of the Total Environment. 2022; 811: 151402. https://doi.org/10.1016/j.scitotenv.2021.151402.\u003c/li\u003e\n\u003cli\u003eHenneron L, Kardol P, Wardle DA, Cros C, Fontaine S. Rhizosphere control of soil nitrogen cycling: a key component of plant economic strategies. New Phytologist. 2020; 228(4): 1269-82. https://doi.org/10.1111/nph.16760.\u003c/li\u003e\n\u003cli\u003eLi Z, Zeng Z, Tian D, Wang J, Fu Z, Zhang F, Zhang R, Chen W, Luo Y, Niu S. Global patterns and controlling factors of soil nitrification rate. Global Change Biology. 2020; 26(7): 4147-57. https://doi.org/10.1111/gcb.15119.\u003c/li\u003e\n\u003cli\u003eCao Q, Zhou Y, Bai Y, Tang F, Wang Y. Net transformation rates of nitrogen in the rhizosphere soil increase with stand age: The roles of nutrient availability and microbial functional guilds. Journal of Soil Science and Plant Nutrition. 2025; 17: 1-5. https://doi.org/10.1007/s42729-025-02234-0.\u003c/li\u003e\n\u003cli\u003eYuan X, She W, Guo Y, Qiao Y, Liu L, Song C, Qin S, Zhang Y. Linkage between plant nitrogen preference and rhizosphere effects on soil nitrogen transformation reveals a plant resource adaptive strategies in nitrogen-limited soils. Plant and Soil. 2025; 1-8. https://doi.org/10.1007/s11104-025-07220-0.\u003c/li\u003e\n\u003cli\u003eBurger M Jackson LE. Microbial immobilization of ammonium and nitrate in relation to ammonification and nitrification rates in organic and conventional cropping systems. Soil Biology and Biochemistry. 2003; 35(1): 29-36. https://doi.org/10.1146/annurev.arplant.59.032607.092932.\u003c/li\u003e\n\u003cli\u003eShao JL, Lai B, Jiang W, Wang JT, Hong YH, Chen FB, Tan SQ, Guo LX. Diversity and co-occurrence patterns of soil bacterial and fungal communities of Chinese Cordyceps habitats at Shergyla Mountain, Tibet: Implications for the occurrence. Microorganisms. 2019; 7(9): 284. https://doi.org/10.3390/microorganisms7090284.\u003c/li\u003e\n\u003cli\u003eHuang A, Wu T, Wu X, Zhang B, Shen Y, Wang S, Song W, Ruan H. Analysis of internal and external microorganism community of wild cicada flowers and identification of the predominant \u003cem\u003eCordyceps cicadae\u003c/em\u003e fungus. Frontiers in Microbiology. 2021; 12: 752791. https://doi.org/10.3389/fmicb.2021.752791.\u003c/li\u003e\n\u003cli\u003eTahat MM, Alananbeh KM, Othman YA, Leskovar D. Soil health and sustainable agriculture. Sustainability. 2020; 12(12): 4859. https://doi.org/10.3390/su12124859.\u003c/li\u003e\n\u003cli\u003eLiang Y, Hong Y, Mai Z, Zhu Q, Guo L. Internal and external microbial community of the Thitarodes moth, the host of \u003cem\u003eOphiocordyceps sinensi\u003c/em\u003es. Microorganisms. 2019; 7(11): 517. https://doi.org/10.3390/microorganisms7110517.\u003c/li\u003e\n\u003cli\u003eMou D, Zeng ZY, Zhong WL, Zhou J, Qu JJ, Zou X. Bacterial community structure and function of \u003cem\u003eCordyceps cicadae\u003c/em\u003e microecosystem in Guiyang. Acta Microbiologica Sinica (China). 2019; 61(02): 469-481. https://doi.org/10.13343/j.cnki.wsxb.20200540.\u003c/li\u003e\n\u003cli\u003eBrodie E, Edwards S, Clipson N. Soil fungal community structure in a temperate upland grassland soil. FEMS Microbiol Ecol. 2003; 45(2): 105-14. https://doi.org/10.1016/S0168-6496(03)00126-0.\u003c/li\u003e\n\u003cli\u003eMeyer A, Focks A, Radl V, Welzl G, Sch\u0026ouml;ning I, Schloter M. Influence of land use intensity on the diversity of ammonia oxidizing bacteria and archaea in soils from grassland ecosystems. Microbial ecology. 2014; 67: 161-166. https://doi.org/10.1007/s00248-013-0310-4.\u003c/li\u003e\n\u003cli\u003eZhang HQ, Zhao XQ, Shi Y, Liang Y, Shen RF. Changes in soil bacterial communities with increasing distance from maize roots affected by ammonium and nitrate additions. Geoderma. 2021; 398: 115102. https://doi.org/10.1016/j.geoderma.2021.115102.\u003c/li\u003e\n\u003cli\u003eBerendsen RL, Pieterse CM, Bakker PA. The rhizosphere microbiome and plant health. Trends Plant Sci. 2012; 17(8): 478-86. https://doi.org/10.1016/j.tplants.2012.04.001.\u003c/li\u003e\n\u003cli\u003eSun R, Zhang P, Riggins CW, Zabaloy MC, Rodr\u0026iacute;guez-Zas S, Villamil MB. Long-term N fertilization decreased diversity and altered the composition of soil bacterial and archaeal communities. Agronomy. 2019; 9(10): 574. https://doi.org/10.3390/agronomy9100574.\u003c/li\u003e\n\u003cli\u003eWei W, Guan D, Ma M, Jiang X, Fan F, Meng F, Li L, Zhao B, Zhao Y, Cao F, Chen H. Long-term fertilization coupled with rhizobium inoculation promotes soybean yield and alters soil bacterial community composition. Frontiers in Microbiology. 2023; 14: 1161983. https://doi.org/10.3389/fmicb.2023.1161983.\u003c/li\u003e\n\u003cli\u003eWang X, Feng J, Ao G, Qin W, Han M, Shen Y, Liu M, Chen Y, Zhu B. Globally nitrogen addition alters soil microbial community structure, but has minor effects on soil microbial diversity and richness. Soil Biology and Biochemistry. 2023; 179: 108982. https://doi.org/10.1016/j.soilbio.2023.108982.\u003c/li\u003e\n\u003cli\u003eChristodoulou E, Agapiou A, Anastopoulos I, Omirou M, Ioannides IM. The effects of different soil nutrient management schemes in nitrogen cycling. J Environ Manage. 2019; 243: 168-176. https://doi.org/10.1016/j.jenvman.2019.04.115.\u003c/li\u003e\n\u003cli\u003eZhang Y, Zhang J, Zhu T, M\u0026uuml;ller C, Cai Z. Effect of orchard age on soil nitrogen transformation in subtropical China and implications. J Environ Sci (China). 2015; 34: 10-9. https://doi.org/10.1016/j.jes.2015.03.005.\u003c/li\u003e\n\u003cli\u003eLi S, Tian Y, Wu K, Ye Y, Yu J, Zhang J, Liu Q, Hu M, Li H, Tong Y, Harberd NP, Fu X. Modulating plant growth-metabolism coordination for sustainable agriculture. Nature. 2018; 560(7720): 595-600. https://doi.org/10.1038/s41586-018-0415-5.\u003c/li\u003e\n\u003cli\u003eDing J, Jiang X, Guan D, Zhao B, Ma M, Zhou B, Cao F, Yang X, Li L, Li J. Influence of inorganic fertilizer and organic manure application on fungal communities in a long-term field experiment of Chinese Mollisols. Applied Soil Ecology. 2017; 111: 114-22. https://doi.org/10.1016/j.apsoil.2016.12.003.\u003c/li\u003e\n\u003cli\u003eYuan Y, Si G, Wang J, Luo T, Zhang G. Bacterial community in alpine grasslands along an altitudinal gradient on the Tibetan Plateau. FEMS microbiology ecology. 2014; 87(1): 121-32. https://doi.org/10.1111/1574-6941.12197.\u003c/li\u003e\n\u003cli\u003eWen Y, Zhang G, Zhang W, Liu G. Distribution patterns and functional characteristics of soil bacterial communities in desert ecosystems of northern China. Science of The Total Environment. 2023; 905: 167081. https://doi.org/10.1016/j.scitotenv.2023.167081.\u003c/li\u003e\n\u003cli\u003eMombrikotb SB, Van Agtmaal M, Johnstone E, Crawley MJ, Gweon HS, Griffiths RI, Bell T. The interactions and hierarchical effects of long‐term agricultural stressors on soil bacterial communities. Environmental Microbiology Reports. 2022; 14(5): 711-8. https://doi.org/10.1111/1758-2229.13106.\u003c/li\u003e\n\u003cli\u003eFu J, Xie X, Zhang S, Kang N, Zong G, Zhang P, Cao G. Rich organic nitrogen impacts clavulanic acid biosynthesis through the arginine metabolic pathway in \u003cem\u003eStreptomyces clavuligerus\u003c/em\u003e F613-1. \u003cem\u003eMicrobiol Spectr. \u003c/em\u003e2023; \u003cem\u003e11\u003c/em\u003e(1): e0201722. https://doi.org/10.1128/spectrum.02017-22.\u003c/li\u003e\n\u003cli\u003eNguyen TM, Ha PT, Le TT, Phan KS, Le TN, Mai TT, Hoang PH. Modification of expanded clay carrier for enhancing the immobilization and nitrogen removal capacity of nitrifying and denitrifying bacteria in the aquaculture system. Journal of bioscience and bioengineering. 2022; 134(1): 41-7. https://doi.org/10.1016/j.jbiosc.2022.04.006.\u003c/li\u003e\n\u003cli\u003eS\u0026oslash;rheim R, Torsvik VL, Goks\u0026oslash;yr J. Phenotypical divergences between populations of soil bacteria isolated on different media. \u003cem\u003eMicrob Ecol\u003c/em\u003e. 1989; \u003cem\u003e17\u003c/em\u003e(2): 181-92. https://doi.org/10.1007/BF02011852.\u003c/li\u003e\n\u003cli\u003eWen A, Fegan M, Hayward C, Chakraborty S, Sly LI. Phylogenetic relationships among members of the \u003cem\u003eComamonadaceae\u003c/em\u003e and description of\u003cem\u003e Delftia acidovorans\u003c/em\u003e (den Dooren de Jong 1926 and Tamaoka \u003cem\u003eet al.\u003c/em\u003e 1987) gen. nov. comb. nov. Int J Syst Bacteriol. 1999; 49(2): 567-76. https://doi.org/10.1099/00207713-49-2-567.\u003c/li\u003e\n\u003cli\u003eHan J, Sun L, Dong X, Cai Z, Sun X, Yang H, Wang Y, Song W. Characterization of a novel plant growth-promoting bacteria strain \u003cem\u003eDelftia tsuruhatensis\u003c/em\u003e HR4 both as a diazotroph and a potential biocontrol agent against various plant pathogens. Syst Appl Microbiol. 2005; \u003cem\u003e28\u003c/em\u003e(1): 66-76. https://doi.org/10.1016/j.syapm.2004.09.003.\u003c/li\u003e\n\u003cli\u003eV\u0026aacute;squez-Pi\u0026ntilde;eros MA, Mart\u0026iacute;nez-Lavanchy PM, Jehmlich N, Pieper DH, Rinc\u0026oacute;n CA, Harms H, Junca H, Heipieper HJ. \u003cem\u003eDelftia \u003c/em\u003esp. LCW, a strain isolated from a constructed wetland shows novel properties for dimethylphenol isomers degradation. BMC microbiology. 2018; 18: 1-2. https://doi.org/10.1186/s12866-018-1255-z.\u003c/li\u003e\n\u003cli\u003eLiu J, Xu G, Dong W, Xu N, Xin F, Ma J, Fang Y, Zhou J, Jiang M. Biodegradation of diethyl terephthalate and polyethylene terephthalate by a novel identified degrader \u003cem\u003eDelftia\u003c/em\u003e sp. WL-3 and its proposed metabolic pathway. Lett Appl Microbiol. 2018; 67(3): 254-261. https://doi.org/10.1111/lam.13014.\u003c/li\u003e\n\u003cli\u003eYan H, Yang XJ, Chen J, Yin CH, Xiao CB, Chen H. Synergistic removal of aniline by carbon nanotubes and the enzymes of \u003cem\u003eDelftia \u003c/em\u003esp. XYJ6. J Environ Sci (Chinese). 2018; 23(7): 1165-70. https://doi.org/10.1016/s1001-0742(10)60531-1.\u003c/li\u003e\n\u003cli\u003eJimenez B, Reboleiro Rivas P, Gonzalez Lopez J, Pesciaroli C, Barghini P, Fenice M. Immobilization of \u003cem\u003eDelftia tsuruhatensis \u003c/em\u003ein macro-porous cellulose and biodegradation of phenolic compounds in repeated batch process. J Biotechnol. 2012; 157(1): 148-53. https://doi.org/10.1016/j.jbiotec.2011.09.026.\u003c/li\u003e\n\u003cli\u003eGarrido-Sanz D, Vesga P, Heiman CM, Altenried A, Keel C, Vacheron J. Relation of pest insect-killing and soilborne pathogen-inhibition abilities to species diversification in environmental \u003cem\u003ePseudomonas protegens\u003c/em\u003e. The ISME Journal. 2023; 17(9): 1369-81. https://doi.org/10.1038/s41396-023-01451-8.\u003c/li\u003e\n\u003cli\u003eHorng CT, Yang YL, Chen CC, Huang YS, Chen C, Chen FA. Intraocular pressure-lowering effect of \u003cem\u003eCordyceps cicadae\u003c/em\u003e mycelia extract in a glaucoma rat model. International Journal of Biological Sciences. 2021; 18(4): 1007- 1014. https://doi.org/10.7150/ijms.47912.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sigs","sideBox":"Learn more about [Environmental Microbiome](https://environmentalmicrobiome.biomedcentral.com)","snPcode":"40793","submissionUrl":"https://submission.nature.com/new-submission/40793/3","title":"Environmental Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cordyceps chanhua, Soil nitrogen cycle, Exonitrogen, Bacterial community, Growth and development","lastPublishedDoi":"10.21203/rs.3.rs-6682575/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6682575/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eThe nitrogen cycle is crucial to the function of the Earth's biosphere. Entomogenous fungihave been proven to promote nitrogen metabolism and cycling in host insects, and transfer nitrogen from insects to soil. However, little is known about the microecological mechanism of entomogenous fungusparticipating in nitrogen cycling and the microecological impact of exonitrogen from entomogenous fungus on soil.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eHere, we report that the entomogenous fungus \u003cem\u003eCordyceps chanhua\u003c/em\u003e secretes nitrate nitrogen and organic nitrogen from its mycelia into the soil environment and absorbs ammonium nitrogen, nitrite nitrogen and hydroxylamine nitrogen from the soil environment into the \u003cem\u003eC. chanhua\u003c/em\u003e. Along with the nitrogen exchange process, the bacterial communities related to nitrogen metabolism in the sclerotium of \u003cem\u003eC. chanhua\u003c/em\u003e emerge in the soil environment, promoting the soil organic nitrogen cycle process. Redundancy analysis strongly demonstrated that the endogenous/symbiotic bacterial communities within \u003cem\u003eC. chanhua\u003c/em\u003e have the greatest impact on ammonium nitrogen and organic nitrogen at the genus level. During the growth process of \u003cem\u003eC. chanhua\u003c/em\u003e, the diversity of the bacterial community in its microenvironment significantly decreased. Consistent with this, this study also verified that the exonitrogen of \u003cem\u003eC. chanhua\u003c/em\u003e can reduce the diversity of bacterial communities in the soil environment and enrich the bacterial group of \u003cem\u003eSporosarcina\u003c/em\u003e spp., which has a positive promoting effect on nitrogen metabolism. Furthermore, we isolated three highly active nitrogen-transforming dominant strains from the sclerotia of \u003cem\u003eC. chanhua\u003c/em\u003e, which further indicates that the nitrogen transport of \u003cem\u003eC. chanhua\u003c/em\u003e is closely related to the bacterial community in its mycelia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions \u003c/strong\u003eThe results of this study demonstrate that the associated/endophytic bacteria of \u003cem\u003eC. chanhua\u003c/em\u003e facilitates the participation of \u003cem\u003eC. chanhua\u003c/em\u003ein soil nitrogen cycling in its microenvironment.\u003c/p\u003e","manuscriptTitle":"The microecological mechanism of Cordyceps chanhua promoting soil nitrogen cycling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-01 08:46:10","doi":"10.21203/rs.3.rs-6682575/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-25T18:21:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-22T09:16:20+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-28T01:42:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"263046713582675824294673697591010610853","date":"2025-09-20T12:28:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"202899395012408676109213084193929231677","date":"2025-09-18T04:52:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-21T15:19:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-24T15:18:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Microbiome","date":"2025-07-21T01:38:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"sigs","sideBox":"Learn more about [Environmental Microbiome](https://environmentalmicrobiome.biomedcentral.com)","snPcode":"40793","submissionUrl":"https://submission.nature.com/new-submission/40793/3","title":"Environmental Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f3c64f81-31f9-42bc-864a-72b0e7ab622a","owner":[],"postedDate":"September 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-11T03:38:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-01 08:46:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6682575","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6682575","identity":"rs-6682575","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}