Microbial complex microbial inoculant for promoting straw degradation and wheat germination and application of microbial complex microbial inoculant

Abstract

The invention relates to the technical field of agricultural microorganisms, in particular to a composite microbial inoculant for promoting straw degradation and wheat germination and application of the composite microbial inoculant. The invention provides a compound microbial inoculant for promoting straw degradation and wheat germination. The effective components of the compound microbial inoculant are trichoderma reesei and aspergillus niger. According to the method, the functions of promoting straw degradation and promoting wheat germination and growth can be achieved at the same time only through scientific combination and inoculation strategies of lignocellulose degradation fungi, and the antagonism possibly generated by mixed use of multiple fungicides is avoided.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbial technology, specifically to a microbial compound agent that promotes straw degradation and wheat germination and its application. Background Technology

[0002] Crop straw is an abundant renewable biomass resource, mainly composed of cellulose, hemicellulose, and lignin. Returning straw to the field is currently the mainstream treatment method, which can improve soil structure and increase organic matter. However, under natural conditions, straw degrades slowly, leading to untimely nutrient release. Undegraded straw can also inhibit the germination and seedling growth of subsequent crops, and even provide a breeding ground for pests and diseases, severely limiting its application effectiveness.

[0003] Utilizing microorganisms to accelerate straw degradation is an environmentally friendly and efficient solution. In existing technologies, filamentous fungi such as *Trichoderma reesei*, *Aspergillus niger*, and *Neurospora crassa* are recognized as highly efficient lignocellulose-degrading bacteria. For example, *Trichoderma reesei* produces abundant endo- and exo-glucanases, but its β-glucosidase activity is insufficient; *Aspergillus niger* is an excellent producer of β-glucosidase and hemicellulase; and *Neurospora crassa*, as a model strain, possesses a comprehensive lignocellulose-degrading enzyme system. However, current research largely focuses on the enzymatic characteristics of single strains or enzyme production through industrial fermentation, or simply mixes them for straw degradation. It has not explored the comprehensive impact of these microbial agents on crop (especially wheat) germination and growth while degrading straw, and there is a lack of optimized inoculation processes to coordinate the synergistic effects between strains. Furthermore, in actual field applications, most microbial agents are prone to unstable effects due to strain competition and improper inoculation timing, and may even exhibit negative effects such as inhibiting seed germination.

[0004] Therefore, there is an urgent market demand and significant application value in developing a specialized compound microbial agent that can simultaneously achieve rapid degradation of straw and effectively promote wheat germination and growth. Summary of the Invention

[0005] Previous studies have largely focused on the effects of three filamentous fungi—Trichoderma reesei (T), Aspergillus niger (A), and Neurospora crassa (N)—on straw degradation. This invention utilizes a microbial compound inoculant composed of these three fungi to investigate their impact on wheat germination and growth while simultaneously degrading straw. This invention provides a microbial inoculation strategy and theoretical basis for optimizing straw return technology, and has reference value for promoting the resource utilization of agricultural waste and sustainable agricultural development.

[0006] To achieve the above objectives, the present invention provides the following technical solution: On the one hand, the present invention provides a microbial compound inoculant that promotes straw degradation and wheat germination, wherein the effective components of the microbial compound inoculant are Trichoderma reesei and Aspergillus niger.

[0007] Preferably, the effective components of the microbial compound agent are Trichoderma reesei, Aspergillus niger, and Neurospora crassa.

[0008] Preferably, the inoculation volume ratio of Trichoderma reesei to Aspergillus niger is 4-6:1.

[0009] Preferably, the inoculation volume ratio of Trichoderma reesei, Aspergillus niger, and Neurospora crassa is 4-6:1:1.

[0010] Preferably, the microbial compound inoculant is inoculated in a timed manner, first inoculating Trichoderma reesei, and then inoculating Aspergillus niger within 48-72 hours (starting from the time of Trichoderma reesei inoculation).

[0011] Preferably, the microbial compound inoculant is inoculated in a timed manner, first inoculating Trichoderma reesei, and then inoculating Aspergillus niger and Neurospora crassa within 48-72 hours (starting from the time of Trichoderma reesei inoculation).

[0012] On the other hand, the present invention provides the application of the microbial compound inoculant in promoting straw degradation, wheat germination, and wheat growth.

[0013] Preferably, the indicators of straw degradation include straw degradation rate and cellulase activity, the indicators of wheat germination include wheat germination ratio, and the indicators of wheat growth include seedling height, fresh weight, and above-ground dry weight.

[0014] On the other hand, a method for promoting straw degradation and wheat germination is provided, the method comprising the following steps: The microbial compound inoculant of the present invention is inoculated onto straw; Wheat should be sown within 72-168 hours after inoculation.

[0015] Preferably, the method includes the following steps: The microbial compound inoculant of the present invention is inoculated onto straw; Wheat was sown within 120 hours after inoculation.

[0016] Preferably, the straw application rate is 4500-7500 kg / hm². 2 The application rate of the microbial compound inoculant is 452 mL of Trichoderma reesei spore suspension per kilogram of straw (spore concentration of 5 × 10⁻⁶). 4 452 mL of other spore suspensions (spore concentration of 1×10⁻⁶ / mL) and 452 mL of other spore suspensions (spore concentration of 1×10⁻⁶ / mL). 4 (units / mL).

[0017] More preferably, the straw application rate is 4500 kg / hm². 2 The application rate of the microbial compound inoculant is 452 mL of Trichoderma reesei spore suspension per kilogram of straw (spore concentration of 5 × 10⁻⁶). 4 452 mL of other spore suspensions (spore concentration of 1×10⁻⁶ / mL) and 452 mL of other spore suspensions (spore concentration of 1×10⁻⁶ / mL). 4 (units / mL).

[0018] It should be noted that in the pot experiment, 2.21 g of straw was first mixed with 1 mL of Trichoderma reesei spore suspension (spore concentration of 5 × 10⁻⁶). 4 (spores / mL), and after 48 h, add other spore suspensions (spore concentration of 1×10⁻⁶). 4 Therefore, 1 kg of straw corresponds to 452 mL of Trichoderma reesei spore suspension (spore concentration of 5 × 10⁻⁶ spores / mL). 4 452 mL of other spore suspensions (spore concentration of 1×10⁻⁶ / mL) and 452 mL of other spore suspensions (spore concentration of 1×10⁻⁶ / mL). 4 (units / mL).

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention studies the effects of lignocellulose-degrading bacteria such as Trichoderma reesei, Aspergillus niger, and Neurospora crassa on straw degradation and wheat germination promotion. The results show that adding Aspergillus niger has the best straw degradation effect, reaching 43.2%. In hydroponic experiments, the combination of Trichoderma reesei + Aspergillus niger (TA) and Trichoderma reesei + Aspergillus niger + Neurospora crassa (TAN) can improve cellulase activity and wheat germination ratio. The germination ratio of TA compared with the control is 1.5±0.06, and that of TAN is 1.4±0.17. Pot experiments further verify that the combination of TA and TAN can improve the germination ratio of wheat. The germination ratio of TA is 1.77±0.39, and that of TAN is 1.83±0.34. The addition of the combination of TA and TAN can improve wheat seedling height, fresh weight, and aboveground dry weight. The two filamentous fungal combinations verified by this invention have good effects on straw degradation and wheat germination promotion, effectively alleviating the inhibitory effect of straw return to the field on wheat growth, realizing the transformation from growth inhibition to growth promotion. This can not only realize the resource utilization of agricultural waste, but also increase wheat yield, which meets the needs of green agricultural development and is expected to be applied to straw return to the field.

[0020] (2) The innovative time-segmented inoculation strategy of this invention is in line with the growth characteristics of each strain, maximizes the degradation potential of the complex microbial community, and provides a reliable operation plan for practical application.

[0021] (3) This invention does not require the addition of growth-promoting bacteria. It can simultaneously promote straw degradation and wheat germination and growth by using only the scientific combination and inoculation strategy of lignocellulose-degrading fungi, thus avoiding the antagonistic effects that may occur when multiple fungal agents are used together. Attached Figure Description

[0022] Figure 1 For the colonization of microorganisms on straw; A: Uninoculated straw; B: Trichoderma reesei; C: Aspergillus niger; D: Neurospora crassa.

[0023] Figure 2 The degradation rate (%) of straw inoculated with different microorganisms was measured. Control: sterile water; T: Trichoderma reesei; A: Aspergillus niger; N: Neurospora crassa; TA: Trichoderma reesei + Aspergillus niger; TAQ: Trichoderma reesei + Aspergillus niger + QL-Z3; TN: Trichoderma reesei + Neurospora crassa; TNQ: Trichoderma reesei + Neurospora crassa + QL-Z3; TAN: Trichoderma reesei + Aspergillus niger + Neurospora crassa; TANQ: Trichoderma reesei + Aspergillus niger + Neurospora crassa + QL-Z3.

[0024] Figure 3 Germination ratio of wheat after different delayed inoculation times for single-strain degradation of straw; ordinate represents strain and observation time (days / d); abscissa represents delayed inoculation time (hours / h); T: Trichoderma reesei; A: Aspergillus niger; N: Neurospora crassa; Q: QL-Z3.

[0025] Figure 4 The wheat germination ratio for straw degradation by mixed cellulose-degrading bacteria; TA: Trichoderma reesei + Aspergillus niger; TAQ: Trichoderma reesei + Aspergillus niger + QL-Z3; TN: Trichoderma reesei + Neurospora crassa; TNQ: Trichoderma reesei + Neurospora crassa + QL-Z3; TAN: Trichoderma reesei + Aspergillus niger + Neurospora crassa; TANQ: Trichoderma reesei + Aspergillus niger + Neurospora crassa + QL-Z3.

[0026] Figure 5 To promote cellulase activity during wheat germination by degrading straw with mixed cellulose-degrading bacteria; Control: sterile water; TA: Trichoderma reesei + Aspergillus niger; TAN: Trichoderma reesei + Aspergillus niger + Neurospora crassa.

[0027] Figure 6The wheat germination ratio of mixed cellulose-degrading bacteria with added probiotics in the form of straw degradation agents; TAM1: Trichoderma reesei + Aspergillus niger + commercial straw composting agent; TAM2: Trichoderma reesei + Aspergillus niger + plant growth-promoting bacteria; TAM1M2: Trichoderma reesei + Aspergillus niger + commercial straw composting agent + plant growth-promoting bacteria; TANM1: Trichoderma reesei + Aspergillus niger + Neurospora crassa + commercial straw composting agent; TANM2: Trichoderma reesei + Aspergillus niger + Neurospora crassa + plant growth-promoting bacteria; TANM1M2: Trichoderma reesei + Aspergillus niger + Neurospora crassa + commercial straw composting agent + plant growth-promoting bacteria.

[0028] Figure 7 The effect of adding straw on wheat germination rate under hydroponic and potted conditions; A: hydroponics; B: potted.

[0029] Figure 8 The effect of mixed microbial agents on the growth of potted wheat was investigated. A-TA and A-TAN: straw addition amount was 2.2 g / pot; B-TA and B-TAN: straw addition amount was 3.6 g / pot; C-TA and C-TAN: straw addition amount was 5.8 g / pot; TA: Trichoderma reesei + Aspergillus niger; TAN: Trichoderma reesei + Aspergillus niger + Neurospora crassa.

[0030] Figure 9 The effect of adding 2.2 g / pot of straw on wheat seedling height when inoculated with a mixed microbial agent; control: sterile water; TA: Trichoderma reesei + Aspergillus niger; TAN: Trichoderma reesei + Aspergillus niger + Neurospora crassa.

[0031] Figure 10 The effect of adding 2.2 g / pot of straw on wheat root length when inoculated with a mixed microbial agent; control: sterile water; TA: Trichoderma reesei + Aspergillus niger; TAN: Trichoderma reesei + Aspergillus niger + Neurospora crassa.

[0032] Figure 11 The effect of adding 2.2 g / pot of straw on the fresh weight of wheat aboveground parts by inoculation with mixed microbial agents; control: sterile water; TA: Trichoderma reesei + Aspergillus niger; TAN: Trichoderma reesei + Aspergillus niger + Neurospora crassa.

[0033] Figure 12 The effect of adding 2.2 g / pot of straw on the fresh weight of wheat underground parts by inoculation with mixed inoculum; control: sterile water; TA: Trichoderma reesei + Aspergillus niger; TAN: Trichoderma reesei + Aspergillus niger + Neurospora crassa.

[0034] Figure 13 The effect of adding 2.2 g / pot of straw on the aboveground dry weight of wheat by inoculation with mixed microbial agents; control: sterile water; TA: Trichoderma reesei + Aspergillus niger; TAN: Trichoderma reesei + Aspergillus niger + Neurospora crassa.

[0035] Figure 14 The effect of adding 2.2 g / pot of straw on the dry weight of wheat underground parts by inoculation with mixed microbial agents; control: sterile water; TA: Trichoderma reesei + Aspergillus niger; TAN: Trichoderma reesei + Aspergillus niger + Neurospora crassa. Detailed Implementation

[0036] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0037] Example 1 Experimental Materials The wheat seed was Xinnong 538.

[0038] Trichoderma reesei was identified as *Trichoderma reesei* Rut-C30, and *Aspergillus niger* was identified as *Aspergillus niger* NL02. Both were published in the literature “Fang Hao; Song Xiangyang; Zhao Chen; Chang Zheng; Chu Jie; Yong Qiang. Study on cellulase production by mixed fermentation of *Trichoderma reesei* and *Aspergillus niger*. *Forest Products Chemistry and Industry*, 2009, 29(6), 15-19.” The strain name in the literature is… T. reesei Rut-C30 and A. niger NL02. Available to the public from the applicant.

[0039] Neurospora crassa is strain 2489, which originated from the Fungal Genetics Stock Center (FGSC) in the United States and is a commercially available strain.

[0040] QL-Z3 is a lignin-degrading bacterium, disclosed in the literature “Hu Xiaofeng, Zhang Duoduo, Zhou Yunheng, Wei Yahong, Chen Shaolin. Identification and degradation characteristics of a lignin-degrading bacterium. Biotechnology Bulletin. 2019, 35 (09): 172-177.” It is available to the public from the applicant.

[0041] The commercial straw composting agent M1 is sourced from Henan Yifuyuan Biotechnology Development Co., Ltd.

[0042] The plant growth-promoting bacterium M2 is *Lactobacillus bruneri* HAC1b23.7, see patent (Qin Baofu, Wen Jianzhong, Li Quanshun, Cao Rang, et al. *A type of *Lactobacillus bruneri* and its application in promoting plant growth. Northwest A&F University. Publication No. CN118421512A). The straw comes from corn stalks harvested in the autumn of 2024 in Huyi District, Xi'an City. It is washed, dried, and chopped or crushed to 20 mesh.

[0043] Example 2 Experimental Method 2.1 Microbial culture Neurospora crassa: The strain was cultured in 50 mL of VM solid medium in a 30°C dark incubator for 3 days, then transferred to a 25°C light incubator for 6 days. The spores were rinsed with 10 mL of sterile water, filtered through double gauze, and the spore suspension was collected into a 10 mL centrifuge tube.

[0044] The formulation of 50 mL VM solid culture medium is as follows: 20 mL of 50×Vogel's, 10 g / L of agar, and 15 g / L of sucrose. The 50×Vogel's medium contains: 126.8 g / L of trisodium citrate dihydrate (Na3Citrate·2H2O), 5 g / L of CaCl2·2H2O (dissolved separately in water before addition), 250 g / L of KH2PO4, 10 g / L of MgSO4·7H2O, 100 g / L of NH4NO3, 10 mL / L of trace elements, and 10 mL / L of biotin. Finally, 5 mL / L of chloroform is added to prevent microbial contamination of the salt solution.

[0045] Trichoderma reesei and Aspergillus niger: The spores were cultured in 50 mL PDA medium (potato extract 3 g / L, glucose 20 g / L, agar 14 g / L) at 30°C in the dark for 9 days. The spores were then washed with 10 mL of sterile water, filtered through double-layered gauze, and collected in 10 mL centrifuge tubes. The spores were then diluted to a concentration of 1×10⁻⁶. 4 per mL.

[0046] QL-Z3: Incubate in 5 mL LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride) at 30°C and 200 rpm for 12 hours.

[0047] Commercial straw composting agent M1 and plant growth-promoting bacteria M2: dilute 10,000 times for later use.

[0048] 2.2 Determination of straw degradation rate Accurately weigh 4.5 g of straw, 2-3 cm in length, and place it in a 250 mL Erlenmeyer flask. Submerge the straw in pure water for 3 hours, then discard the water. For group T, add 500 μL of diluted *Trichoderma reesei* spore solution, and after 48 hours, add 100 μL of sterile water. For groups TA, TAQ, TN, TNQ, TAN, and TANQ, add 500 μL of diluted *Trichoderma reesei* spore solution, and after 48 hours, add 100 μL of each of the other bacterial solutions. For group A, add 500 μL of sterile water, and after 48 hours, add 100 μL of diluted *Aspergillus niger* spore solution. For group N, add 500 μL of sterile water, and after 48 hours, add 100 μL of diluted *Neurospora crassa* spore solution. The control group receives an equal volume of sterile water. Incubate at 28℃ for 7 days, then dry at 65℃ and weigh.

[0049] 2.3 Hydroponic experiment to determine the optimal straw-degrading strain Accurately weigh 0.25 g of straw powder and spread it evenly in a petri dish (9.0 cm in diameter). Add 5 mL of sterile water and 100 μL of spore / bacterial suspension. Place two sheets of sterile filter paper on top, cover and seal. Incubate in an incubator for 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 192 h. Then, place 40 wheat seeds on the filter paper. The incubation conditions are 22℃, 12000 lex light for 16 h, and darkness for 8 h. After 24 h of incubation, transfer the top filter paper and wheat seeds to another clean, sterile petri dish, add 10.0 mL of sterile water, and continue incubation. The control group received 100 μL of sterile water instead of the bacterial suspension. Observe wheat germination every 24 h. Repeat three times.

[0050] 2.4 Hydroponic experiment to determine the effect of combined bacterial strains on wheat germination ratio Accurately weigh 0.25 g of corn stalk powder and spread it evenly in a petri dish (9.0 cm in diameter). Add sterile water and spore / fungus suspension, place two sterile filter papers inside, and seal the dish. Place the dish in an incubator and incubate for 48 h. Then add delayed inoculation spore / fungus suspension and incubate for 120 h. Inoculate with wheat seeds and continue incubating for 24 h. Transfer the top filter paper and wheat seeds to another clean, sterile petri dish, add 10.0 mL of sterile water to each dish, and continue incubating. Observe the germination of wheat every 24 h. Repeat 3 times.

[0051] Seven strain combinations and their addition ratios were set. TA group: Trichoderma reesei + Aspergillus niger, with Aspergillus niger inoculation delayed by 48 h, inoculation volume ratio 5:1 (500 μL:100 μL); TACQ group: Trichoderma reesei + Aspergillus niger + QL-Z3, with Aspergillus niger inoculation delayed by 48 h, inoculation volume ratio 5:1:1; TN group: Trichoderma reesei + Neurospora crassa, with Neurospora crassa inoculation delayed by 48 h, inoculation volume ratio 5:1; TNQ group: Trichoderma reesei + Neurospora crassa + QL-Z3, with Neurospora crassa inoculation delayed by 48 h, inoculation volume ratio 5:1:1; TAN group: Trichoderma reesei + Aspergillus niger + Neurospora crassa, with Aspergillus niger and Neurospora crassa inoculation delayed by 48 h, inoculation volume ratio 5:1:1; TANQ group: Trichoderma reesei + Aspergillus niger + Neurospora crassa + QL-Z3, with Aspergillus niger and Neurospora crassa inoculation delayed by 48 h, inoculation volume ratio 5:1:1:1. The control group was treated with 100 μL of sterile water instead of the bacterial culture. The experiment was repeated three times.

[0052] 2.5 Verification of optimal inoculation conditions using potted plants Nine treatments were set up: ① 2.21 g straw (4500 kg / hm² of straw) 2); ② 3.68 g of straw (7500 kg / hm² of straw) 2 ); ③ 5.88 g of straw (12000 kg / hm of straw) 2 ); ④ 2.21 g straw + TA group mixed bacteria; ⑤ 3.68 g straw + TA group mixed bacteria; ⑥ 5.88 g straw + TA group mixed bacteria; ⑦ 2.21 g straw + TAN group mixed bacteria; ⑧ 3.68 g straw + TAN group mixed bacteria; ⑨ 5.88 g straw + TAN group mixed bacteria.

[0053] Plastic pots (7 cm × 5 cm × 7.8 cm) were used, each containing approximately 21 g of soil, 1.0 mL of spore suspension, and 5 wheat seeds. Eight pots were used per group, totaling 40 plants, with 3 parallel groups. The potting mix contained 68% total nitrogen, 27% phosphorus anhydride, and 36% potassium oxide. After thoroughly mixing the straw with the top 3 cm of soil in the pots, 1.0 mL of Trichoderma reesei spore suspension was sprayed onto the surface (specifically, the spore concentration here is 5 × 10⁻⁶). 4 (spores / mL), and after 48 h, spray with 1.0 mL (1×10⁻⁶) of other spore suspension. 4 The control group was sprayed with an equal volume of sterile water and inoculated with wheat seeds at a depth of approximately 1 cm. Culture conditions were 22℃, 12000 lex light for 16 h, followed by 8 h darkness. Plant growth was observed every 24 h, and observed for 7 days after wheat germination. The experiment was repeated three times.

[0054] 2.6 Determination of cellulase activity The culture medium from the above hydroponic experiment was collected for the determination of filter paper enzyme activity. The standard method recommended by the International Union of Pure and Applied Chemistry (IUPAC) (Determination of Cellulase Activity in Feed Additives: NY / T 912-2020 [S]. Beijing: China Standards Press, 2020) was adopted. A brief description is as follows: 50 mg of 1×1 cm filter paper was added to each test tube, along with 0.5 mL of the hydroponic supernatant and 1 mL of 0.05 M citrate buffer. The test tubes were stoppered and placed in a constant temperature water bath at 80 rpm and 50℃ for 16 h. Immediately after removal, 3 mL of DNS reagent was added, and the mixture was reacted in boiling water for 10 min. After cooling, the absorbance of the supernatant was measured at 550 nm.

[0055] 2.7 Measurement Indicators and Analytical Methods Straw degradation rate and wheat germination rate were determined using conventional statistical methods.

[0056] Straw degradation rate: The initial weight of straw minus the mass of straw dried on day n is used as the base. The ratio of the mass difference before and after straw degradation to the initial straw mass is calculated for each group.

[0057] Germination rate: Number of germinated seeds / Number of seeds tested × 100%.

[0058] Germination ratio: The ratio of the germination rate of each treatment to the baseline was calculated using the total seed germination rate of the control group as the baseline (Germination Ratio, GR = Germination rate of experimental group / Germination rate of control group).

[0059] At the end of the eight-day germination period, 10 plants with consistent growth were taken from each replicate to measure seedling height, root length, fresh weight of above-ground parts, fresh weight of underground parts, dry weight of above-ground parts, and dry weight of underground parts.

[0060] The surface of the straw before and after inoculation was observed using a stereomicroscope (Qitian B4550-N080).

[0061] Data analysis was performed using OriginPro 2024.

[0062] Example 3 Experimental Results 3.1 Degradation of straw by different strains The colonization of three main filamentous fungi on straw was observed. Figure 1 The results showed that, compared with uninoculated straw (control), straw inoculated with *Trichoderma reesei*, *Aspergillus niger*, and *Neurospora crassa* exhibited significant mycelial growth after 96 h of cultivation, with spores and mycelia visible under a 4x microscope. This indicates that *Trichoderma reesei*, *Aspergillus niger*, and *Neurospora crassa* can colonize and grow on straw.

[0063] Different microorganisms and mixtures thereof were inoculated onto straw, and the degradation rate was calculated. The results are as follows: Figure 2 (Significance analysis was performed using Tukey's method: different letters represent...) P As shown in the figure (<0.05, the same letter indicates no significant difference), compared with the natural degradation of the control, the addition of selected bacteria and combinations, except for the TANQ group, significantly improved the degradation rate of straw. In the single-strain inoculation treatment, the degradation rate of straw after seven days after inoculation with Aspergillus niger (A) reached 43.2%, which was 1.3 times that of the control, showing the best degradation effect. When two or three bacteria were mixed for degradation, the simultaneous inoculation with TNQ showed the best effect, with a degradation rate of 42.0%, and there was no significant difference in degradation rate among other groups. However, the degradation effect of four bacteria did not reach the expected level.

[0064] The above results indicate that inoculation with *Aspergillus niger* alone is the most effective method for straw degradation. *Aspergillus niger* possesses a highly efficient ability to degrade hemicellulose, an amorphous, branched polymer with a low degree of polymerization, making it more easily hydrolyzed than cellulose and lignin. The degradation rate of *Aspergillus niger* in combination with other strains was lower than that of treatment A alone, suggesting that complex interactions, such as competitive inhibition and nutrient competition, may exist among different microbial communities, which to some extent inhibit the full realization of their degradation efficiency. Since the above strains all have significant effects on straw degradation, their effects on wheat will be further tested in future studies.

[0065] 3.2 Hydroponic experiments were conducted to determine the optimal inoculation conditions for promoting straw degradation and wheat germination. Since microbial growth may inhibit wheat seed germination, wheat inoculation was delayed to reduce its impact on wheat growth. Inoculation was designed with delays of 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, and 192 h, and wheat germination was observed.

[0066] Figure 3 (The germination ratio calculation control is based on the case with only straw added.) This section shows the germination of wheat seeds inoculated at different delay times after straw degradation by different microorganisms. The results showed significant differences in wheat germination ratios among different treatments and at different delay times under single-strain treatments. This difference was clearly presented by a color gradient from dark green (low value, inhibiting germination) to yellow (high value, promoting germination). *Trichoderma reesei* and *Aspergillus niger* had a positive effect on wheat germination, especially when wheat was inoculated at a delay of 120 h. *Neurospora crassa* only had a positive effect on germination when wheat was inoculated at a delay of 0 h. QL-Z3 showed inhibitory effects at most delay times.

[0067] By observing the growth of the three filamentous fungi in pairs, it was found that the growth rate of *Aspergillus niger* ≥ *Neurospora crassa* > *Trichoderma reesei*. This indicates that the slower growth rate of *Trichoderma reesei* may be the main reason for the poor degradation effect of mixed cultures. Previous studies have reported that in a co-culture system of *Aspergillus niger* and *Trichoderma reesei*, if *Aspergillus niger* is inoculated 48 hours later, and the inoculation volume ratio of *Trichoderma reesei* to *Aspergillus niger* is 5:1, the highest filter paper enzyme activity can be achieved on day 5 of fermentation. This condition is the optimal inoculation mode for achieving synergistic effects between the two. Based on the differences in the growth rates of the three filamentous fungi, this embodiment adopts the following time-segmented inoculation strategy: first, *Trichoderma reesei* spore suspension is inoculated; 48 hours later, *Neurospora crassa* and / or *Aspergillus niger* are inoculated; finally, wheat seeds are inoculated 120 hours later. The changes in seed germination ratio with observation time (1-5 days) under different microbial combinations are shown below. Figure 4 (The germination ratio calculation control was based on the case with only straw added; the significance analysis used the Tukey method:*) P <0.05,**P <0.01, *** P As shown in the figure (<0.001), the TA and TAN treatment groups showed significant differences compared to the other groups. The germination ratios of TA and TAN on the second day were significantly higher than the control, after which TA remained stable at 1.5±0.06, and TAN remained stable at 1.4±0.17; while the germination ratios of the other treatment groups (TAQ, TN, TNQ, TANQ) did not show significant differences compared to the control. In conclusion, the TA and TAN combination significantly promoted seed germination; while other microbial combinations did not show a germination-promoting effect.

[0068] The changes in enzyme activity during straw degradation under TA and TAN treatments were assessed by measuring cellulase activity in petri dishes. The time of introduction of the first microorganism was designated as day 0, and cellulase activity was measured on days 3, 5, and 7 (after wheat seed inoculation). The results are as follows: Figure 5 (OD) 550 The value is positively correlated with cellulase activity and represents cellulase activity; significance analysis was performed using the Tukey method:** P <0.01, *** P As shown in the figure (<0.001). On the 3rd day after inoculation, the cellulase activities of the control group, TA treatment group, and TAN treatment group were all at low levels, with the TA group showing a significant increase compared to the control group. At this time, the exogenously added microorganisms had not yet fully multiplied, and their enzyme secretion capacity was similar to the straw self-degradation rate. As the culture time was extended to the 5th day, the enzyme activity of the TA treatment group showed an upward trend; the enzyme activity of the TAN treatment group was more significantly enhanced. This means that more straw was degraded at this time. Then, on the 7th day, the cellulase activity remained at the same level. The cellulase activity detection verified that TA and TAN treatments can directly and effectively increase the cellulase activity in the petri dishes, thereby accelerating the degradation of straw. On the 7th day, the straw in the petri dishes had been degraded for some time. At this time, wheat seeds were inoculated, and an increase in the germination rate was observed on the 2nd day after sowing. Therefore, it is speculated that the increase in the wheat germination rate may be directly related to the degradation of straw. The enzyme activity of the control group remained low, confirming that the natural degradation rate of straw is slow, and its weak enzyme activity mainly comes from a small number of inherent microorganisms or endogenous enzymes attached to the straw itself.

[0069] To further test the synergistic effect of other microbial agents and the above agents on seed germination, the effects of a commercial straw composting agent M1 and a plant growth-promoting bacterium M2 on the above experiments were compared. M1 is composed of various beneficial microbial communities, including lactic acid bacteria, yeast, Bacillus, photosynthetic bacteria, and actinomycetes. Based on the above TA and TAN agents, M1 and M2 agents were added separately to form new treatment groups: TAM1, TAM2, TAM1M2, TANM1, TANM2, and TANM1M2. The results are as follows: Figure 6(The germination ratio calculation control is based on the case with only straw added; the significance analysis uses the Tukey method:*) P <0.05,** P <0.01, *** P As shown in the figure (<0.001), the germination ratios of TAM2 and TANM1M2 were significantly higher than those of the control in the early stages of wheat germination. Specifically, the germination ratio of the TANM1M2 treatment group increased sharply to 1.9±0.05 on the second day, significantly higher than other groups, indicating that this compound microbial agent has a very strong germination-promoting ability in the early stages of wheat germination. Apart from this, no significant difference was observed in the germination ratios of other treatments compared to the control; TANM1 even showed an inhibitory effect. Although the multi-strain compound inoculation (TANM1M2) can significantly increase the seed germination rate in the short term, its effect is time-sensitive. These results indicate that TA and TAN are superior to other strain combinations in promoting wheat germination.

[0070] 3.3 Optimal inoculation conditions were verified using potted plants. Undegraded or excessive amounts of straw can negatively impact wheat growth. In hydroponics, adding 0.25 g of straw powder to a petri dish reduces the germination rate by 31.4%. Figure 7 (A, 0.25g / plate), reduced by 23.1% when potted ( Figure 7 (For example, B, 2.2g / pot). Therefore, different straw addition amounts were selected when validating the above inoculant in pots. Based on a straw return rate of 7000-7500 kg / ha for the whole season, three gradients were designed: A - 2.205 g straw powder / pot (straw 4500 kg / ha). 2 B-3.675 g straw powder / pot (7500 kg / hm of straw) 2 C-5.88 g straw powder / pot (straw 12000 kg / hm) 2 ).

[0071] Figure 8 (The germination ratio calculation control was based on the case with only straw added; the significance analysis used the Tukey method:*) P <0.05,** P <0.01, *** P<0.001) indicates the effect of different straw addition amounts on wheat germination. The wheat germination rate remained unchanged after 8 days of observation. At all straw addition levels, the wheat germination rate in the microbial inoculation treatment groups showed a certain improvement compared to the control group without inoculant. When the straw addition amount was 2.2 g (A-TA, A-TAN), the germination rates of the TA and TAN inoculation groups were consistently significantly higher than the control group throughout the 2nd to 8th day of the culture period, with TA at 1.77±0.39 and TAN at 1.83±0.34 on day 8. When the straw addition amount was 3.6 g (B-TA, B-TAN), the improvement in germination rate mainly occurred in the later stages of observation, with the TA group showing better results. When the straw addition amount was 5.8 g (C-TA, C-TAN), the TA group only showed a significantly higher germination rate than the control on day 3, while the germination performance of the TAN group was similar to that of the control group with 3.6 g straw. Therefore, pot experiments verified that the TA and TAN groups showed results largely consistent with those of the hydroponic experiments in terms of straw degradation and wheat germination promotion. Adding a low dose of straw (2.2g / pot) is more suitable for the current inoculation method.

[0072] Figure 9-14 The effects of TA and TAN inoculation treatments on wheat seedling growth under low-dose straw addition (2.2 g) were shown, and morphological and biomass parameters were measured on day 15 post-planting. Regarding morphogenesis, both TA and TAN inoculation treatments significantly promoted seedling height in wheat seedlings. Figure 9 The significance analysis used Tukey's method:* P <0.05). However, there was no significant difference in the effect of each treatment group on wheat root length ( Figure 10 In terms of biomass accumulation, microbial inoculation treatment also showed a significant positive effect. Results indicated that the fresh weight of wheat aboveground parts in both the TA and TAN inoculation treatment groups was significantly higher than that in the control group. Figure 11 The significance analysis used Tukey's method:* P <0.05,** P <0.01). The fresh weight of the underground part of TAN was significantly higher than that of the control group ( Figure 12 The significance analysis used Tukey's method:* P <0.05%. Regarding dry matter accumulation, the aboveground and underground parts of wheat showed different characteristics. Both TA and TAN treatments increased the aboveground dry weight ( Figure 13 The significance analysis used Tukey's method:* P <0.05), while the dry weight of the underground portion of each treatment group ( Figure 14There was no statistically significant difference between the control and the control group. In summary, under the condition of 2.2 g straw addition, the promoting effect of inoculation with the compound microbial agents TA and TAN on wheat seedling growth was mainly reflected in a significant increase in seedling height, fresh weight, and aboveground dry weight, but the effect on root elongation was not significant. This indicates that this microbial treatment strategy can effectively improve the early vegetative growth of wheat seedlings.

[0073] While straw incorporation increases soil organic matter and nutrients, it also has negative effects on wheat plant growth. In anaerobic soil environments, straw incorporation promotes the accumulation of phytotoxic substances, thereby inhibiting plant growth. In this invention, by combining straw return to the field with the application of straw-degrading microbial agents, the germination rate, seedling height, and fresh weight of wheat are effectively increased, significantly alleviating the inhibitory effect of straw return alone on wheat growth and development. The filamentous fungal combination of this invention not only degrades straw but also promotes wheat germination and growth.

[0074] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

Claims

1. A microbial compound inoculant that promotes straw degradation and wheat germination, characterized in that, The effective components of the microbial compound agent are Trichoderma reesei and Aspergillus niger.

2. The microbial compound inoculant according to claim 1, characterized in that, The effective components of the microbial compound agent are Trichoderma reesei, Aspergillus niger, and Neurospora crassa.

3. The microbial compound inoculant according to claim 1, characterized in that, The inoculation volume ratio of Trichoderma reesei to Aspergillus niger is 4-6:

1.

4. The microbial compound inoculant according to claim 2, characterized in that, The inoculation volume ratio of Trichoderma reesei, Aspergillus niger, and Neurospora crassa is 4-6:1:

1.

5. The microbial compound inoculant according to claim 1, characterized in that, The microbial compound inoculant is inoculated in a timed manner, first inoculating Trichoderma reesei, and then inoculating Aspergillus niger within 48-72 hours.

6. The microbial compound inoculant according to claim 2, characterized in that, The microbial compound inoculant is inoculated in a timed manner, first inoculating Trichoderma reesei, and then inoculating Aspergillus niger and Neurospora crassa within 48-72 hours.

7. The application of the microbial compound inoculant according to any one of claims 1-6 in promoting straw degradation, wheat germination and wheat growth.

8. The application according to claim 7, characterized in that, The indicators of straw degradation include straw degradation rate and cellulase activity; The indicators for wheat germination include the wheat germination ratio; The indicators for wheat growth include seedling height, fresh weight, and above-ground dry weight.

9. A method for promoting straw degradation and wheat germination, characterized in that, The method includes the following steps: The microbial compound inoculant according to any one of claims 1-6 is inoculated onto straw; Wheat should be sown within 72-168 hours after inoculation.

10. The method according to claim 9, characterized in that, The application rate of straw is 4500-7500 kg / hm. 2 .

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