Salt-tolerant pink spiral promyces and microbial fertilizer thereof

Abstract

The application provides a salt-tolerant and alkali-tolerant promoting pink spiral polyangium strain, and the preservation number is CGMCC No.42331.The application also provides application of the pink spiral polyangium strain and a microbial fertilizer containing the pink spiral polyangium strain.The pink spiral polyangium XRLZ-13-1 strain provided by the application is derived from soil in Tibet, has excellent environmental adaptability, and can stably grow under high-salt (NaCl concentration 5%) and high-alkali (pH 9.0) double stress.Application of the strain fermentation liquor in a saline-alkali environment can significantly improve seed germination rate, accelerate seed germination process, promote tomato seedling growth and root development, increase biomass, induce plant resistance, and improve soil nutrition indexes.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Polyspora pinkis*, microbial fertilizer containing it, and its applications. Background Technology

[0002] Soil salinization and alkalization are becoming increasingly prominent problems, seriously threatening global agricultural production and the ecological environment, and even affecting human survival. Statistics show that over 1.3 billion hectares of land worldwide (approximately 10% of the global soil area) are affected by salinization, leading to soil structure damage, nutrient imbalance, impaired root function, osmotic pressure imbalance in plant cells, and toxicity. Soil degradation and reduced crop yields and quality due to salinization have become major constraints on sustainable agricultural development.

[0003] To mitigate the impact of salt-alkali stress on crop growth, various physical and agronomic measures have been tried and promoted. For example, improving irrigation methods to reduce salt accumulation, using mulch to reduce surface water evaporation, planting salt-tolerant crops, and adopting crop rotation patterns all contribute to enhancing the ecological stability of saline-alkali land. Simultaneously, various chemical remediation methods are widely used, such as applying gypsum and acid conditioners to lower soil pH and improve soil structure, thereby aiding crop growth. However, these methods are often costly, cumbersome to implement, have poor long-term effects, and may cause secondary environmental problems. In recent years, the use of salt-tolerant microorganisms, especially rhizosphere bacteria, to alleviate salt-alkali stress has attracted much attention. These beneficial microorganisms can reduce the physiological damage to plants caused by salt and alkali through multiple mechanisms and improve soil properties. They can secrete plant hormones, produce extracellular polysaccharides, increase nutrients, promote root growth, regulate ion balance within plants, activate defense systems, and scavenge harmful reactive oxygen species. Furthermore, rhizosphere microorganisms can regulate soil pH balance, increase microbial diversity, and restore soil function.

[0004] Pink Spiral Polyporus ( Clonostachys rosea Originally named *Ascomycota pulcherrima*, it belongs to the phylum Ascomycota, class Coccidioides, order Hypocreales, and family Rhizocarpaceae. It is a facultative saprophytic fungus widely distributed in nature. *Ascomycota pulcherrima* is also a biocontrol fungus, capable of parasitizing various plant diseases caused by fungi and nematodes. Furthermore, this type of fungus can promote plant growth, induce plant resistance, and improve the soil microenvironment. It is a beneficial soil microorganism that promotes plant growth and is environmentally friendly. Utilizing *Ascomycota pulcherrima* to alleviate soil salinization and alkalization is a highly attractive research topic. Summary of the Invention

[0005] To address the problems existing in the prior art, the first objective of this invention is to provide a salt- and alkali-tolerant Polyspora pinkis var. spp. strain.

[0006] A second objective of this invention is to propose the application of the aforementioned *Polyspora pinkis* strain;

[0007] A third objective of this invention is to provide a microbial fertilizer containing the aforementioned *Polyspora pinkis* strain.

[0008] The technical solution for achieving the above-mentioned objective of this invention is as follows:

[0009] A salt- and alkali-tolerant *Alternaria pinki* strain was isolated from farmland soil in Resa Township, Lazi County, Shigatse, Tibet. The strain number is XRLZ-13-1, and the preservation number is CGMCC No. 42331.

[0010] Our survey of fungal resources in the Tibet Autonomous Region revealed that *Aspergillus pinki* could be isolated from nearly 50% of the sampling sites. Tibet's high altitude, strong ultraviolet radiation, poor soil, and severe salinization make it an ideal environment to extract important microbial resources like *Aspergillus pinki* from these extreme habitats. Further utilization of these microorganisms could improve agricultural production efficiency, ensure food safety, protect the ecological environment, and meet the needs of sustainable and healthy agricultural development. Furthermore, this fungus exhibits rapid growth, strong sporulation capacity, and is easy to cultivate on a large scale, demonstrating significant application potential.

[0011] The application of the aforementioned *Alternaria pinki* strain in the cultivation of crops in saline-alkali land and the improvement of saline-alkali land.

[0012] Microbial fertilizer containing the aforementioned *Polyporus pulveratus* strain, wherein the microbial fertilizer is a fermentation broth obtained by liquid fermentation of *Polyporus pulveratus* strain XRLZ-13-1, or a mixture of the fermentation broth with excipients and / or soil conditioners and then formulated; the dosage form of the microbial fertilizer is a liquid, granules, or powder.

[0013] Furthermore, the fermentation broth is filtered or concentrated by centrifugation and then mixed with the excipients and / or soil conditioner to prepare a formulation;

[0014] The soil conditioner is one or more of the following: straw powder, biochar, humic acid, fly ash, organic fertilizer, gypsum, desulfurized gypsum, silicon fertilizer, wood ash, and potassium humate.

[0015] The auxiliary materials are one or more of the following: diatomaceous earth, light calcium carbonate, talc, attapulgite, bentonite, kaolin, starch, dextrin, peat moss, and rice husk powder.

[0016] A preferred embodiment of the present invention is that the fermentation broth of the *Polyspora pinkis* strain is prepared by the following steps:

[0017] 1) Inoculum preparation: The *Polyspora pinkis* strain was inoculated onto potato dextrose agar (PDA) medium and cultured at 26-28°C for 6-10 days. The spores were washed off to prepare a spore suspension; or mycelial blocks were cut off as inoculum.

[0018] 2) Seed culture: The spore suspension or mycelial blocks were inoculated into potato dextrose broth (PDB) medium and cultured in the dark with shaking at 25-28°C for 48-84 h to obtain seed culture;

[0019] 3) Liquid fermentation culture: Prepare fermentation culture medium, sterilize and cool it, then inoculate it with the seed liquid and culture at 25~30℃ for 48~120 h to obtain fermentation broth.

[0020] More preferably, in step 3), the fermentation medium comprises per liter: 10-40 g sucrose, 5-15 g yeast extract, 5-15 g soybean meal, 0.5-2 g K2HPO4, 0.5-1.0 g MgSO4•7H2O, and 0.05-0.1 g FeSO4•7H2O; with a pH value of 5.5-6.5.

[0021] pH can be adjusted using a 1-20% NaOH solution, a 1-10% HCl solution, or other pH adjusters known in the art.

[0022] The liquid fermentation culture conditions in step 3) are as follows: seed culture inoculum volume 0.5-5%, rotation speed 150-250 r / min, culture temperature 25-28℃, and culture time 3-4 days.

[0023] The content of the obtained fermentation broth was (0.5~5)×10. 8 Spores / mL.

[0024] This microbial fertilizer can be used directly as the fermentation liquid, or it can be formulated with additives, soil conditioners, or both. The following are the preferred proportions for the additives and soil conditioners.

[0025] The fermentation liquid is obtained through fermentation, and then filtered through a plate and frame filter or centrifuged and concentrated to obtain a bacterial sludge with a water content of 50-70%. Auxiliary materials are added to the bacterial sludge or directly to the fermentation liquid. After mixing and drying, the mixture is crushed or granulated. The amount of the auxiliary materials added is 50-120% of the bacterial sludge or 80-150% of the fermentation liquid by mass percentage.

[0026] The microbial fertilizer contains a soil conditioner in a mass ratio of 1:1 to 10 (fermentation liquid to soil conditioner), or the fermentation liquid is filtered or centrifuged and concentrated to obtain a microbial mud with a water content of 50 to 70%, in which the microbial mud to soil conditioner ratio is 1:0.5 to 5.

[0027] Another preferred embodiment of the present invention is that the soil conditioner is one of the following: fly ash, humic acid, biochar, organic fertilizer, gypsum, desulfurized gypsum, silicon fertilizer, wood ash, potassium humate, and straw powder, with a bacterial content of 0.5~5×10⁻⁶. 8 The mass ratio of spores / mL fermentation broth to soil conditioner is 1:1~5.

[0028] The beneficial effects of this invention are as follows:

[0029] The *Alternaria pinki* strain XRLZ-13-1 provided by this invention originates from soil in Tibet and exhibits excellent environmental adaptability, capable of stable growth under high salinity (5% NaCl concentration) and high alkalinity (pH 9.0) conditions. Applying the fermentation broth of this strain to a saline-alkali environment significantly improves seed germination rate, accelerates seed germination, promotes tomato seedling growth and root development, and increases plant height and biomass.

[0030] In pot experiments, tomato plants under salt-alkali stress showed severely inhibited growth, with stunted growth, yellowing, curling, tip burn, and premature senescence of leaves, and a significant decrease in root length and fresh weight. However, after application of XRLZ-13-1 bacterial solution, tomato plant height and root length increased by 21.6% and 113.6%, respectively, and root dry weight increased twofold. Measurements of stress-related enzymes and peroxidation metabolites showed significantly increased activities of superoxide dismutase, peroxidase, and catalase, while malondialdehyde and hydrogen peroxide levels were significantly reduced, effectively alleviating oxidative damage caused by salt-alkali stress. Furthermore, it was found that *Alternaria pinki* could increase root soil microbial diversity and improve the abundance of beneficial microorganisms, thereby enhancing soil health.

[0031] The study also found that after applying XRLZ-13-1 bacterial solution, the available nitrogen and organic matter content in the soil increased by 14.1% and 37.3%, respectively, showing a significant difference compared with the saline-alkali soil control. P < 0.05). The application of *Polyporus pilosa* compound soil conditioner significantly increased the content of available nitrogen, phosphorus, potassium, and organic matter in the soil, and a certain degree of decrease was also observed in soil pH testing. P < 0.05). This indicates that Pink Spiral Polyporus umbellatus fertilizer can effectively improve soil nutrient indicators and alleviate saline-alkali stress.

[0032] The *Alternaria pinki* strain and its microbial fertilizer provided by this invention not only improve the tolerance of tomatoes to salt and alkali stress but also enhance crop nutrient absorption efficiency and promote plant growth. It also has potential growth-promoting and stress-resistance effects on other crops (such as cucumbers and lettuce). Simultaneously, this strain and technology can be used for the improvement and management of saline-alkali land, helping to restore soil health, improve the ecological environment, and alleviate the problem of insufficient arable land. The strain and its application method provided by this invention are safe, environmentally friendly, and pollution-free, offering a novel and sustainable solution for the ecological restoration of saline-alkali land and healthy crop cultivation, with significant economic and ecological benefits. Attached Figure Description

[0033] Figure 1 The colony morphology of *Polyspora pinkis* XRLZ-13-1 strain on PDA medium. A shows the front of the colony; B shows the back of the colony.

[0034] Figure 2 Homologous phylogenetic tree of ITS rDNA sequence for strain Polyspora pinkis XRLZ-13-1.

[0035] Figure 3 The growth diameter of different *Polyspora pinkis* strains on PDA medium supplemented with salt and alkali.

[0036] Figure 4 The effects of soaking different strains of *Polyspora pinkis* on tomato seed germination under saline-alkali stress.

[0037] Figure 5 The effect of fermentation broth from *Alternaria pinki* strains on the growth of tomato test-tube seedlings in hydroponic saline-alkali solution. From left to right in the figure are: water treatment (no saline-alkali stress), saline-alkali solution control, and *Alternaria pinki* strains XRLZ-13-1, XRDJ-1-2, and XRNM-1-1 treatments.

[0038] Figure 6 The effects of *Alternaria pinki* strains on tomato plant growth in saline-alkali soil are shown. A represents plant growth status; B represents root development. From left to right in the figure, the treatments are: ordinary cultivated soil (no saline-alkali stress), saline-alkali soil control, and *Alternaria pinki* strains XRLZ-13-1, XRDJ-1-2, and XRNM-1-1.

[0039] Figure 7 The effects of fermentation broth of *Alternaria pinki* XRLZ-13-1 on nutritional indicators in tomato leaves were investigated. A represents chlorophyll content; B represents nitrogen content. From left to right in the figure, the soil types are: cultivated soil (no stress), saline-alkali soil control, and saline-alkali soil treated with *Alternaria pinki* XRLZ-13-1 strain.

[0040] Figure 8The effects of *Polyporus pulcherrimus* XRLZ-13-1 fermentation broth on the activity of antioxidant enzymes and peroxidation products in tomato roots were investigated. A represents superoxide dismutase (SOD) activity; B represents catalase (CAT) activity; C represents malondialdehyde (MDA) content; and D represents hydrogen peroxide (H₂O₂) content. From left to right, the figures represent cultivated soil (no stress), saline-alkali soil control, and saline-alkali soil treated with *Polyporus pulcherrimus* XRLZ-13-1 strain.

[0041] Figure 9 The effects of fermentation broth of *Polyspora pulvinata* XRLZ-13-1 on the rhizosphere microbiota of tomato under salt-alkali stress. A represents bacterial composition (phylum level); B represents bacterial composition (genus level); C represents fungal composition (phylum level); D represents fungal composition (genus level).

[0042] Figure 10 This study illustrates the effects of different *Alternaria pinkis* compound microbial fertilizers on tomato growth under saline-alkali stress. From left to right in the figure, the fertilizers are: saline-alkali soil control, *Alternaria pinkis* XRLZ-13-1 fermentation broth, XRLZ-13-1 fermentation broth + fly ash (X+FA), XRLZ-13-1 fermentation broth + humic acid (X+HA), and XRLZ-13-1 fermentation broth + biochar (X+BC). Detailed Implementation

[0043] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0044] Unless otherwise specified, all test materials and instruments used in this instruction manual are commercially available.

[0045] Unless otherwise specified, all parts in the examples are by weight, and all percentages are by weight percentages.

[0046] The *Polyporus pulcherrimus* strain used in this invention was isolated from farmland soil in Resa Township, Lazi County, Shigatse Prefecture, Tibet Autonomous Region by our research team and is labeled as strain XRLZ-13-1. The strain has the CGMCC No. 42331 accession number, was deposited on December 1, 2025, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It is classified and named *Polyporus pulcherrimus*. Clonostachys rosea .

[0047] Example 1: Cultivation of Sclerotium sclerotia

[0048] The preserved Sclerotium sclerotiorum (accession number ACCC 39161) was inoculated onto a PDA plate and incubated in a 28°C incubator for 7-10 days. The mycelial cakes were then punched out and inoculated into carrot agar, with 5-10 cakes per bottle. The plates were incubated at room temperature under light for 20 days. Once the agar was covered with a large number of sclerotia, the carrot agar was rinsed off the surface of the sclerotia with sterile water. The plates were then air-dried and stored in a cool place for later use.

[0049] Example 2: Isolation of *Alternaria pinki* strains from Tibet

[0050] Rhizosphere soil samples were collected from six regions in Tibet (Ali Prefecture, Chamdo City, Lhasa City, Nyingchi City, Nagqu City, and Shigatse Prefecture). The samples were air-dried, passed through a 2 mm sieve, and 100 g of soil was weighed and placed in sterile glass culture bottles. Twelve fresh sclerotia prepared in Example 1 were embedded in each bottle (approximately 2 cm deep) to attract fungi that parasitize other fungi. The culture bottles were placed in a constant temperature incubator at 28°C with 60%–70% humidity. After 30 days, the sclerotia were removed from the soil samples, rinsed thoroughly with tap water, sterilized with 1% NaClO for 30 seconds, and then washed three times with sterile water to remove excess moisture. The sclerotia were then placed in sterile petri dishes lined with three layers of filter paper and incubated at 25°C with humidification. After 7 days, the presence of hyphae on the sclerotia surface was observed. The grown hyphae and spores were streaked onto PDA medium, and single colonies were picked for purification. The isolated parasitic fungi were stored at 4°C.

[0051] Fifty strains of *Alternaria pinki* were isolated from 90 soil samples collected, including 2 strains from Ngari Prefecture, 6 strains from Chamdo City, 3 strains from Nyingchi City, 4 strains from Nagqu City, and 35 strains from Shigatse Prefecture. Figure 1 The image shows the colony morphology of strain XRLZ-13-1 on PDA medium. A is the front of the colony, and B is the back of the colony.

[0052] Example 3 Identification of parasitic bacterium XRLZ-13-1

[0053] The isolated parasitic fungi were identified through colony morphology, microstructure, and ITS sequencing analysis. The identification process for strain XRLZ-13-1 is as follows:

[0054] Morphological identification: The isolated XRLZ-13-1 strain was inoculated into PDA medium and cultured at 26℃ for 7 days. Its colony morphology is as follows: Figure 1 As shown. Hyphae and conidia were collected and observed under a microscope for the characteristics of the conidiophores and conidia of the fungus. The hyphae were colorless and septate, the conidiophores were broom-shaped and branched, and a large number of conidia were produced at the tips of the phialides. The conidia were oval or cylindrical, with a diameter of 3-5 μm. Based on its morphological characteristics, strain XRLZ-13-1 was preliminarily identified as *Aspergillus pinkis*.

[0055] Molecular identification: Hyphae and conidia of strain XRLZ-13-1 were collected, and genomic DNA was extracted using the Ezup column-based fungal genomic DNA extraction kit. PCR amplification was performed using universal fungal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The amplification system consisted of 2 μL DNA template, 1 μL each of the 10 μM ITS primers, and 21 μL of 1.1×T3 Super PCR Mix enzyme. The amplification program was: 98℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 55℃ annealing for 10 s, 72℃ extension for 10 s, for 35 cycles; followed by a 10 min extension at 72℃. After electrophoresis, the PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The obtained sequences were compared for homology using BLAST in the NCBI database, and a phylogenetic tree was constructed using the neighbor-joining method. The results showed that strain XRLZ-13-1 had the highest homology with *Polyspora pinkis*, and the homology comparison phylogenetic tree was as follows: Figure 2 As shown.

[0056] Example 4: Determination of the salt and alkali tolerance of *Polyspora pinkis* strains

[0057] A salt- and alkali-stressed medium was prepared by adding 5% NaCl to PDA medium and adjusting the pH to 9.0 with 10% NaOH solution. The isolated *Polyspora pinkis* strain was inoculated onto PDA plates and incubated at 28°C for 5 days. Then, using a sterile punch, 5 mm diameter mycelial discs were taken from the edge of the colony and inoculated into the center of both a fresh, ordinary PDA plate (control) and a salt- and alkali-stressed PDA plate. The plates were incubated at 28°C for 7 days, and the colony diameter was measured. The relative growth rate (RGR) of each strain was calculated.

[0058] Relative growth rate = Colony diameter on saline-alkali medium / Colony diameter on PDA medium × 100%.

[0059] The results showed that the relative growth rates of different *Alternaria pinki* strains under salt-alkali stress ranged from 7.4% to 83.8%, exhibiting significant differences. P < 0.05 indicates rich genetic diversity within the *Alternaria pinki* species. Some strains exhibit high tolerance, with growth capacity under stress conditions exceeding or equaling half that under normal culture conditions (RGR > 50%). For example, the RGR value of strain XRLZ-13-1 is 83.8%, significantly higher than other tested strains ( P< 0.05), and the RGR value of strain XCBS-2-1 was 55.5%, also showing good salt and alkali tolerance. The vast majority of strains (82%) showed moderate tolerance, with RGR values ​​ranging from 20% to 50%. Some strains, such as XRRB-4-1 and XRSJ-2-3, showed high sensitivity to salt and alkali conditions, and their growth was severely inhibited by salt and alkali stress.

[0060] Figure 3 This study visually demonstrates the growth and morphological differences of representative strains of *Alternaria pinki* on PDA and saline-alkali media. Highly tolerant strains continued to grow rapidly under stress, forming dense colonies, while sensitive strains grew slowly with sparse hyphae. Through growth assays, 13 salt-alkali tolerant strains, including XRLZ-13-1, XCBS-2-1, XRXT-7-2, and XRDJ-1-2, were screened from 50 *Alternaria pinki* strains for subsequent research on their effects on seed germination and hydroponic seedling growth under salt-alkali stress.

[0061] Example 5: Fermentation culture of *Polyspora pinkis* XRLZ-13-1

[0062] The *Polyspora pinkis* strain XRLZ-13-1 was inoculated onto a PDA plate and cultured for 7 days. 5 mL of sterile water was added to the plate to wash away the spores and prepare a spore suspension. PDB medium (100 mL / 500 mL Erlenmeyer flask) was prepared, autoclaved, and cooled to room temperature. The spore suspension was then inoculated into the medium and cultured in the dark at 27°C for 72 h to prepare a seed culture.

[0063] Fermentation medium was prepared, containing 25 g sucrose, 4 g yeast extract, 6 g soybean meal, 1.0 g K₂HPO₄, 0.5 g MgSO₄·7H₂O, and 0.05 g FeSO₄·7H₂O per liter, with the pH adjusted to 6.0. The fermentation medium was dispensed into Erlenmeyer flasks and sterilized at 121℃ for 30 min. After cooling to room temperature, the seed culture was inoculated into the fermentation medium at a 3% inoculum. The culture was then incubated at 27℃ with shaking at 180 r / min for 4 days. The concentration of the fermentation broth was determined to be 1.6 × 10⁻⁶ using a hemocytometer. 8 Spores / mL.

[0064] Example 6: Effects of *Alternaria pinki* on tomato seed germination under salt-alkali stress

[0065] The previously screened salt-tolerant *Polyspora pinkis* strain was inoculated into fermentation medium and cultured at 28°C and 180 r / min for 72 h to prepare the fermentation broth. Hemocytometer counting was performed, and the fermentation broth concentration was adjusted to 1×10⁻⁶. 7Spores / mL. Simultaneously, a compound saline-alkali solution was prepared to simulate saline-alkali stress. The saline-alkali solution contained NaCl, NaHCO3, Na2SO4, and Na2CO3 in a mass ratio of 1:9:9:1, with a total concentration of 50 mmol / L. Plump and uniformly sized tomato seeds were selected, surface-sterilized with 1% sodium hypochlorite solution for 5 min, rinsed three times with sterile distilled water, and placed in 9 cm petri dishes lined with double-layered filter paper, 25 seeds per dish. 3 mL of the corresponding saline-alkali solution and 1 mL of *Polyspora pinkis* fermentation broth were added to the petri dishes. Sterile water was used as a substitute for the fermentation broth as a saline-alkali stress control, and sterile distilled water was used as a blank control. The petri dishes were placed in a 25℃ constant temperature incubator for germination in the dark, with a quantitative amount of water added daily to compensate for evaporation. The number of germinated seeds was observed and recorded daily, with the radicle length ≥ 1 / 2 of the seed length as the germination standard. Measurements were taken continuously for 7 days, and the germination rate was calculated to evaluate the ability of each strain to alleviate saline-alkali stress. Each treatment was replicated three times.

[0066] The results are as follows Figure 4 As shown, the addition of a saline-alkali solution strongly inhibited tomato seed germination, while treatment with *Polyporus pulcherrima* effectively alleviated this inhibition. Under no-stress conditions (soaking in water), the seed germination rate reached 100% after 7 days, while the germination rate under saline-alkali stress (salt-alkali control) was only 28.7%. Among the *Polyporus pulcherrima* treatments, strain XRLZ-13-1 showed the most significant effect, achieving a tomato seed germination rate of 78.0%, 2.1 times higher than under stress conditions, and exhibiting a rapid and sustained germination trend from the second day. Most of the tested *Polyporus pulcherrima* strains also showed significant alleviating effects on saline-alkali stress. For example, strains XAPL-13-1, XRDJ-1-2, XRNM-1-1, and XRBL-1-1 all achieved tomato seed germination rates above 60%, significantly higher than the stress control.

[0067] Example 7: Effects of *Polyspora pinkis* fermentation broth on the growth of hydroponic tomato seedlings under salt-alkali stress

[0068] Select plump and uniformly sized tomato seeds, surface disinfect them with 1% sodium hypochlorite solution for 5 minutes, and rinse them three times with sterile distilled water. Place the seeds in tissue culture trays for seedling cultivation. When the tomato seedlings have grown to two leaves and one bud, select seedlings with uniform growth and place them in 15 mL test tubes. Add 9 mL of 1 / 8 MS culture medium (containing 1.9 g potassium nitrate, 1.65 g ammonium nitrate, 0.17 g potassium dihydrogen phosphate, 0.37 g magnesium sulfate, 0.44 g calcium chloride, 0.83 mg potassium iodide, 6.2 mg boric acid, 22.3 mg manganese sulfate, 8.6 mg zinc sulfate, 0.25 mg sodium molybdate, 0.025 mg copper sulfate, 0.5 mg cobalt chloride, 0.5 mg nicotinic acid, 37.3 mg disodium EDTA, 27.8 mg ferrous sulfate, 100 mg inositol, 2 mg glycine, 0.1 mg thiamine hydrochloride, and 0.5 mg pyridoxine hydrochloride) and 1 mL of saline-alkali solution (NaCl, NaHCO3, Na2SO4, and Na2CO3 in a mass ratio of 1:9:9:1, with a total concentration of 500 mmol / L) to each test tube, then add 1 mL of a 10% saline solution prepared as described above. 7 Fermentation broth of *Polyspora pinkis* with spores / mL was cultured under indoor light, with an equal volume of 1 / 8 MS medium added as a control. Sterile distilled water was added daily to maintain a consistent liquid level in the test tubes. After 7 days, root length, fresh weight, dry weight, and chlorophyll content of tomatoes were measured. Six seedlings were used per replicate, and each treatment was repeated three times.

[0069] from Figure 5 As can be seen, tomato seedling growth was severely hampered under salt-alkali stress, with plants exhibiting wilting. However, inoculation with *Alternaria pinki* XRLZ-13-1, XRNM-1-1, and XRDJ-1-2 significantly improved seedling growth. Specifically, treatment with XRLZ-13-1 fermentation broth increased the dry weight of the aboveground parts and roots of tomato seedlings by 1.5 times and 6.5 times, respectively, compared to the stress control. Inoculation with XRLZ-13-1 and XRNM-1-1 increased leaf chlorophyll content by 3.7 times and 3.5 times, respectively, compared to the control, and even by 19.4% and 15.7% compared to the non-stress condition.

[0070] Example 8: Effects of *Alternaria pinki* on tomato growth, antioxidant enzymes, and peroxidation products in saline-alkali soil.

[0071] Preparation of saline-alkali soil: Topsoil (0-20 cm depth) was collected from farmland at the Langfang Experimental Base of the Chinese Academy of Agricultural Sciences, air-dried, and passed through a 2 mm sieve. Analytical grade NaCl, Na₂SO₄, Na₂CO₃, and NaHCO₃ were added to each kilogram of soil at a ratio of 0.4%, and the mixture was thoroughly mixed to prepare saline-alkali soil. Soil samples were prepared according to national standards, and their electrical conductivity (EC) and pH value were tested. The initial values ​​were EC = 22.5 dS / m and pH = 8.51.

[0072] Greenhouse potted tomatoes were used to determine the response of tomato plants to salt-alkali stress after treatment with *Aspergillus pilosa* XRLZ-13-1. Ordinary topsoil, saline-alkali soil, and saline-alkali soil were prepared with *Aspergillus pilosa* solution (2 mL / pot, fermentation broth prepared in Example 5, concentration 1.6 × 10⁻⁶). 8 Treatments were administered with spores / mL (300g soil per pot). Ten days later, the growth of tomatoes under different treatments was as follows: Figure 6 As shown: After treatment with *Alternaria pinkis* fermentation broth, tomato seedlings did not wilt, and the plants had robust stems and dark green leaves. Especially after adding XRLZ-13-1 fermentation broth, the tomato growth was no different from that in ordinary cultivated soil. The chlorophyll and nitrogen content of the leaves were measured using a plant nutrient analyzer, and the results are as follows: Figure 7 As shown, compared with the saline-alkali soil control, the chlorophyll and nitrogen content increased by 64.1% and 63.2% respectively after bacterial treatment.

[0073] Tomato roots grown for 10 days were collected, soil clumps were washed off, and the roots were flash-frozen in liquid nitrogen and ground for later use. The activities of superoxide dismutase (SOD) and catalase (CAT), as well as the contents of the peroxidation products malondialdehyde (MDA) and hydrogen peroxide (H2O2), were determined using a kit according to the manufacturer's instructions. Figure 8 As shown, saline-alkali stress (saline-alkali soil in the figure) led to increased activity of antioxidant enzymes and accumulation of peroxidation products in the roots. Because saline-alkali stress activated the plant's antioxidant system, compared with normal soil, the activities of SOD and CAT in tomatoes in saline-alkali soil increased by 1 time and 3.9 times, respectively. Peroxidation products MDA and H2O2 also accumulated significantly, with contents increasing by 0.8 times and 1.1 times, respectively. After application of XRLZ-13-1 inoculant, the activities of antioxidant enzymes increased to varying degrees, with CAT activity showing a significant difference, increasing by 1.2 times. Meanwhile, the contents of MDA and H2O2 decreased by 31.6% and 39.4%, respectively, compared to the saline-alkali control, approaching those of normal soil.

[0074] Example 9: Effects on the rhizosphere microbiome of tomato

[0075] Tomatoes were grown in a greenhouse (planting method as in Example 8). Soil samples were collected from both untreated (CK) and treated (XRLZ-13-1) soils. High-throughput sequencing was used to analyze the microbial community composition of the soil bacteria and fungi, and Mothurv 1.30.1 was used to calculate various α-diversity indices. The results showed that the application of XRLZ-13-1 had no significant effect on the α-diversity of the soil bacterial community, but significantly increased the abundance of the fungal community. The Sobs, Ace, and Chao indices increased from 455.7, 467.4, and 485.4 to 541.3, 562.4, and 558.9, respectively, with the increases in Ace and Chao indices reaching highly significant levels. P < 0.001). Microbial classification, sequencing, and gene function annotation cluster analysis of soils under different treatments showed that the composition of the microbial community in saline-alkali soils changed after the application of XRLZ-13-1 bacterial solution. The pink spiral polycystic spp. agent increased the relative abundance of beneficial microbial groups, such as *Sphingomonas* and *Microbranchium* (e.g., ...). Figure 9 (As shown). Sphingomonas can produce catalase under saline-alkali conditions, and can play a role in nitrogen fixation and promote carbon cycling under high salinity conditions. Microbranchs help improve the bioavailability of soil phosphorus.

[0076] Example 10 Preparation of Pink Spiral Polyporus Microbial Fertilizer

[0077] Biochar was selected as a soil conditioner and compounded with *Polyspora pinkis* to prepare microbial fertilizer. The fermentation broth of *Polyspora pinkis* XRLZ-13-1 obtained in Example 5 was placed in a low-speed centrifuge and centrifuged at 4000 r / min for 10 min. The supernatant was discarded, and the bacterial sludge was collected. The moisture content of the bacterial sludge was determined to be 58%. Diatomaceous earth and peat moss (diatomaceous earth to peat moss mass ratio 1:1) were added to the bacterial sludge at a 1:1 mass ratio and mixed thoroughly until the material was homogeneous. The resulting mixture was placed in a clean enamel dish and allowed to dry naturally in a cool, ventilated place until constant weight. It was then pulverized to prepare the microbial inoculant.

[0078] The microbial agent and biochar were then mixed at a mass ratio of 1:2 and stirred thoroughly to prepare a compound microbial fertilizer. The prepared sample was a dark brown powder with a loose texture and good adsorption properties, suitable for field trials.

[0079] Weigh 1.0 g of the prepared microbial fertilizer, add 9 mL of sterile water and shake. Perform serial dilutions, and spread 100 μL onto a PDA plate containing 50 mg / L penicillin and 50 mg / L streptomycin sulfate. Incubate at 28℃ for 3 days and count the components. Perform three replicates. The content of the active ingredient in the microbial fertilizer was determined to be 4.2 × 10⁻⁶. 7 CFU / g.

[0080] Example 11: Effects of Pink Spiral Polyporus Microbial Fertilizer on Tomato Growth

[0081] Fermentation broth of *Polyspora pinkis* XRLZ-13-1 was prepared according to the method described in Example 5, and the concentration was determined to be 1.4 × 10⁻⁶ using a hemocytometer. 8 Spores / mL. Meanwhile, saline-alkali soil was prepared according to the method in Example 8, and 300 g of soil was added to each pot. The application effect of *Polyspora pinkis* fertilizer on tomatoes was comprehensively evaluated through pot experiment. A total of 5 treatments were set up: (1) planting in saline-alkali soil (CK); (2) adding 2 mL of *Polyspora pinkis* XRLZ-13-1 fermentation broth (XRLZ); (3) XRLZ-13-1 strain fermentation broth + fly ash (X+FA, mass ratio 1:3); (4) XRLZ-13-1 strain fermentation broth + humic acid (X+HA, mass ratio 1:3); (5) XRLZ-13-1 strain fermentation broth + biochar (X+BC, mass ratio 1:3).

[0082] Seedlings were raised in seedling trays and transferred to pots of different treatments when they reached two leaves and a central bud, one seedling per pot. An individual tray was placed under each pot, and water seeping from the trays was re-poured into the pot to ensure consistent soil composition. Ten days after transplanting, plant height, root length, fresh weight, and dry weight were measured. Each treatment consisted of 15 plants, with three replicates.

[0083] Tomato growth status as follows Figure 10 As shown, different treatments under salt-alkali stress had significant differences in their effects on tomato growth. P < 0.05). Compared with the control (CK), the application of *Aspergillus pilaris* XRLZ-13-1 fermentation broth (XRLZ) significantly alleviated the inhibitory effect of salt-alkali stress on tomato growth, increasing root length and biomass by more than 40%, especially the aboveground dry weight, which increased by 3.2 times. The application of compound microbial fertilizer, especially the treatment with *Aspergillus pilaris* + biochar (X+BC), further enhanced the growth-promoting effect, increasing aboveground and root biomass by more than 50%, demonstrating a significant synergistic effect. This indicates that the application of *Aspergillus pilaris* and biochar compound microbial fertilizer can effectively improve plant growth vitality and stress resistance under salt-alkali stress, while also improving the rhizosphere environment.

[0084] Example 12: Effects of Pink Spiral Polyporus Microbial Fertilizer on Soil Physicochemical Indicators

[0085] Saline-alkali soil was prepared, and a greenhouse pot experiment was conducted using the pink spiral polyspora microbial fertilizer prepared in Example 11. Three treatments were set up: (1) planting in saline-alkali soil (CK); (2) treatment with pink spiral polyspora XRLZ-13-1 fermentation broth (XRLZ); (3) treatment with XRLZ-13-1 fermentation broth + biochar (X+BC).

[0086] Tomato plants were transplanted, and rhizosphere soil samples were collected from different treatment groups 10 days later and stored at -80℃. Soil physicochemical properties were determined according to national standards. A certain amount of soil sample was weighed, diluted 5 times and 2.5 times with water respectively, and soil electrical conductivity and pH were measured. Total nitrogen was determined using an elemental analyzer, available nitrogen was determined using the alkaline hydrolysis-diffusion method, total phosphorus and available phosphorus were determined using the molybdenum-antimony colorimetric method, total potassium and available potassium were determined using flame photometry, and soil organic matter was determined using the potassium dichromate oxidation-titration method.

[0087] The results of soil physicochemical index determination are shown in Table 1. After applying XRLZ-13-1 bacterial solution, the available nitrogen and organic matter content in the soil increased by 14.1% and 37.3% respectively compared with the saline-alkali soil control, showing significant differences. P < 0.05). The microbial fertilizer prepared by combining XRLZ-13-1 strain with biochar also increased the content of total phosphorus, available phosphorus and available potassium, thus playing a synergistic role, and significantly reduced the soil pH, indicating that the application of *Alternaria pinki* microbial fertilizer can effectively improve soil nutrient indicators and alleviate salt and alkali stress.

[0088] Table 1. Effects of Pink Spiral Polyporus microbial fertilizer on soil physicochemical properties

[0089]

[0090] Note: TN: Total Nitrogen; TP: Total Phosphorus; TK: Total Potassium; AN: Available Nitrogen; AP: Available Phosphorus; AK: Available Potassium; SOM: Soil Organic Matter.

[0091] Although the present invention has been described above through embodiments, those skilled in the art should understand that any improvements and modifications made to the present invention without departing from its spirit and essence should fall within the protection scope of the present invention.

Claims

1. A salt- and alkali-tolerant *Alternaria pinki* strain, characterized in that, The accession number is CGMCC No.42331.

2. The application of the *Alternaria pinki* strain according to claim 1 in the cultivation of crops in saline-alkali land and the improvement of saline-alkali land; wherein the crop is tomato.

3. A microbial fertilizer containing the *Alternaria pinkis* strain according to claim 1, characterized in that, The microbial fertilizer is a fermentation broth obtained by liquid fermentation of the *Polyspora pinkis* strain, or a preparation obtained by mixing the fermentation broth with excipients and soil conditioner; the microbial fertilizer is in the form of liquid, granules, or powder; the fermentation broth is filtered or centrifuged and concentrated, then excipients and soil conditioner are added to prepare the preparation; after fermentation, the fermentation broth is filtered or centrifuged and concentrated to obtain a bacterial mud with a water content of 50-70%; excipients are added to the bacterial mud, or directly to the fermentation broth; after mixing and drying, it is pulverized or granulated; the amount of excipients added is 50-120% of the bacterial mud by mass percentage, or 80%-150% of the fermentation broth; the microbial fertilizer contains soil conditioner, and the mass ratio of added soil conditioner is 1:1-10 (fermentation broth: soil conditioner), or the fermentation broth is filtered or centrifuged and concentrated to obtain a bacterial mud with a water content of 50-70%, and the bacterial mud: soil conditioner ratio is 1:0.5-5; The soil conditioner is one of the following: fly ash, humic acid, biochar, organic fertilizer, gypsum, desulfurized gypsum, silicon fertilizer, wood ash, potassium humate, and straw powder, with a bacterial content of 0.5~5×10⁻⁶. 8 The mass ratio of spores / mL fermentation broth to soil conditioner is 1:1~5; the auxiliary materials are one or more of diatomaceous earth, light calcium carbonate, talc, attapulgite, bentonite, kaolin, starch, dextrin, peat moss, and rice husk powder.

4. The microbial fertilizer according to claim 3, characterized in that, The fermentation broth of the *Polyspora pinkis* strain was prepared by the following steps: 1) Inoculum preparation: The *Polyspora pinkis* strain was inoculated onto potato dextrose agar medium and cultured at 26-28°C for 6-10 days. The spores were washed off to prepare a spore suspension; or mycelial blocks were cut off as inoculum. 2) Seed culture: The spore suspension or mycelial blocks are inoculated into PDB medium and cultured in the dark with shaking at 25-28℃ for 48-84 h to obtain seed culture; 3) Liquid fermentation culture: Prepare fermentation culture medium, sterilize and cool it, then inoculate it with the seed liquid and culture at 25~30℃ for 48~120h to obtain fermentation broth.

5. The microbial fertilizer according to claim 4, characterized in that, In step 3), the fermentation medium comprises the following components per liter: sucrose 10-40 g, yeast extract 5-15 g, soybean meal powder 5-15 g, K2HPO4 0.5-2 g, MgSO4•7H2O 0.5-1.0 g, FeSO4•7H2O 0.05-0.1 g; and a pH value of 5.5-6.

5.

6. The microbial fertilizer according to claim 4, characterized in that, In step 3), the liquid fermentation culture conditions are as follows: seed liquid inoculation amount 0.5~5%, rotation speed 150~250 r / min, culture temperature 25~28℃, culture time 3~4 days.

Images