Endophytic fungi with functions of improving stress resistance and promoting growth of fraxinus mandshurica and application thereof

Abstract

The application discloses an endophytic fungus with functions of improving stress resistance and promoting growth of Fraxinus mandshurica and application thereof, and belongs to the technical field of microorganisms. Alternaria tenuissima The endophytic fungus is named as Alternaria tenuissima (SLR21), is preserved in the China General Microbiological Culture Collection Center, and has a preservation number of CGMCC NO.42057. The application screens out the endophytic fungus with functions of improving stress resistance and promoting growth of Fraxinus mandshurica by taking Fraxinus mandshurica as a material, provides new microbial resources for mining of new strains of biological energy functions, and provides a new path for solving the problem of difficult forestation of Fraxinus mandshurica. The fermentation liquor of the endophytic fungus contains high contents of polyphenols, polysaccharides, flavones, salicylic acid and auxin and the like, has high drought resistance, salt and alkali resistance and functions of promoting growth of seedling height, can be used as a seed soaking agent alone, and can be applied to promotion of seed germination, and the fermentation liquor of the endophytic fungus can also be used for root irrigation or spraying treatment of seedlings.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to an endophytic fungus that enhances the stress resistance and growth-promoting function of Manchurian ash and its applications. Background Technology

[0002] Ash ( Fraxinus mandshurica *Rupr.*, a deciduous tree belonging to the genus *Rupr.* of the Oleaceae family, is a Class II protected wild plant in China. Its wood is tough and valuable, making it a prized timber species in my country. With its beautiful grain, it can be used to make various furniture, musical instruments, sporting goods, vehicles, ships, machinery, and special building materials. The bark has medicinal uses, serving as a traditional treatment for tuberculosis and external injuries, and also as an insect repellent. However, its seeds have a long dormancy period, resulting in slow propagation. Seedlings are susceptible to drought, cold damage, insect infestations, and salinity, making it difficult to establish a mature forest.

[0003] Plant endophytic fungi are a microbial resource with wide-ranging applications. They primarily refer to fungi that live within various tissues and organs of healthy plants at certain or all stages of their life cycle. Endophytic fungi can protect host plants by absorbing and maintaining ion balance, enhancing plant osmotic regulation, maintaining photosynthetic and water use efficiency, strengthening antioxidant defense systems, producing secondary metabolites, or regulating signal transduction. Utilizing endophytic fungi to overcome the harm caused by abiotic stresses to plants is a promising strategy, and researchers are increasingly focusing on developing the potential biotechnological applications of endophytic fungi to improve plant drought resistance.

[0004] However, the resources of stress-resistant and growth-promoting strains for Manchurian ash have not yet been fully developed, and existing technologies lack endophytic fungal strains that simultaneously possess significant drought resistance, growth promotion, and phenolic synthesis-inducing functions. Summary of the Invention

[0005] The purpose of this invention is to provide an endophytic fungus that enhances the stress resistance and growth-promoting function of Manchurian ash, thereby solving the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: an endophytic fungus that enhances the stress resistance and growth-promoting function of *Fraxinus mandshurica*, named *Alternaria alternata* (…). Alternaria tenuissima SLR21 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 42057. The deposit address is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is July 14, 2025.

[0007] Another object of the present invention is to provide an application of the above-mentioned endophytic fungus or its fermentation broth in improving the drought resistance of Manchurian ash.

[0008] Preferably, the endophytic fungus or its fermentation broth is used to induce the synthesis of phenolic substances from ash trees.

[0009] Another object of the present invention is to provide an application of the above-mentioned endophytic fungus or its fermentation broth in promoting the growth of Manchurian ash.

[0010] The fermentation broth of the endophytic fungi provided by this invention contains polyphenols, polysaccharides, flavonoids, salicylic acid and auxins.

[0011] Preferably, the endophytic fungus or its fermentation broth is used to improve the antioxidant enzyme activity, leaf nitrogen content, stress resistance substance content and photosynthetic capacity of Manchurian ash.

[0012] Another object of the present invention is to provide a root irrigation agent and foliar spray for improving the drought resistance and / or promoting the growth of Manchurian ash, including the above-mentioned endophytic fungi or their fermentation liquid.

[0013] Another object of the present invention is to provide the application of the above-mentioned endophytic fungus or its fermentation broth in promoting the germination of Manchurian ash seeds.

[0014] Another object of the present invention is to provide a seed soaking agent for promoting the germination of Manchurian ash seeds, comprising the above-mentioned endophytic fungi or their fermentation broth.

[0015] This invention uses *Fraxinus mandshurica* as material to screen out an endophytic fungus that enhances the stress resistance and growth-promoting function of *Fraxinus mandshurica*, providing new microbial resources for the discovery of new strains with bioenergy functions and offering a new approach to solving the problem of difficulty in establishing *Fraxinus mandshurica* forests. This endophytic fungus exhibits high drought resistance and promotes seedling height growth. It can be used alone as a seed soaking agent to promote seed germination, or its fermented bacterial solution can be used for root irrigation or spraying treatment of seedlings.

[0016] The technical solution provided by this invention has the following advantages compared with the prior art: it is green and environmentally friendly; it is highly operable, low in cost, and can be applied on a large scale; it can significantly improve the drought resistance and salt and alkali resistance of Manchurian ash, expand its planting range in arid areas, improve the survival rate and timber yield, and increase economic benefits. Attached Figure Description

[0017] Figure 1 Morphological diagram of the endophytic fungus SLR21 isolated in an embodiment of the present invention;

[0018] Figure 2 This is a graph showing the drought resistance test results of the endophytic fungus SLR21 provided in an embodiment of the present invention;

[0019] Figure 3 This is a graph showing the detection results of IAA and SA content of endophytic fungus SLR21 provided in an embodiment of the present invention;

[0020] Figure 4 The image shows the detection results of the secondary metabolite content of the endophytic fungus SLR21 provided in the embodiments of the present invention.

[0021] Figure 5 The figure shows the effect of root irrigation with endophytic fungus SLR21 on the photosynthetic index of Manchurian ash seedlings, as provided in the embodiments of the present invention; where "SLR" is the root irrigation treatment group and "T0" is the natural drought control group.

[0022] Figure 6 The figure shows the effect of root irrigation with endophytic fungus SLR21 on the stress-resistance enzyme activity of Ash seedlings, as provided in this embodiment of the invention; where "SLR" represents the root irrigation treatment group and "drought" represents the natural drought control group.

[0023] Figure 7 The figure shows the effect of root irrigation with endophytic fungus SLR21 on the stress resistance substances of Manchurian ash seedlings, as provided in the embodiments of the present invention; where "SLR" is the root irrigation treatment group and "drought" is the natural drought control group.

[0024] Figure 8 The effect of root irrigation with endophytic fungus SLR21 on the flavonoid and polyphenol content of Ash seedlings provided in this embodiment of the invention is shown in the figure; where "SLR" is the root irrigation treatment group and "T0" is the natural drought control group.

[0025] Figure 9 Phenotypic diagrams showing the effects of root irrigation and foliar spraying of endophytic fungus SLR21 on the growth and drought resistance of *Fraxinus mandshurica* seedlings provided in embodiments of the present invention; wherein, Figure A shows the effect of root irrigation of SLR21 on the growth of *Fraxinus mandshurica* seedlings, Figure B shows the effect of root irrigation of SLR21 on the drought resistance of *Fraxinus mandshurica* seedlings under severe drought, Figure C shows the effect of foliar spraying of SLR21 on the drought resistance of *Fraxinus mandshurica* seedlings under severe drought, and Figure D shows the effect of spraying of SLR21 on the drought resistance of detached leaves of one-year-old *Fraxinus mandshurica* under a drought environment simulated by 20% PEG-6000.

[0026] Among them, the endophytic fungus SLR21 was named Alternaria spp. ( Alternaria tenuissima SLR21 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 42057 on July 14, 2025. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] This invention uses Manchurian ash as material and combines the induction and regulation effect of plant endophytic fungi to establish a drought resistance and domestication technology system for Manchurian ash during the bud and seedling stages, clarifying the relationship between endophytic fungi and the stress resistance and adaptability of Manchurian ash and their regulatory effect on growth.

[0029] Specifically, in one embodiment of the present invention, an endophytic fungus with the function of improving the stress resistance and growth promotion of Manchurian ash is provided, named Alternaria tenuissima SLR21, or endophytic fungus SLR21 for short, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.42057, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is July 14, 2025.

[0030] In another embodiment of the present invention, the application of the above-mentioned endophytic fungus or its fermentation broth in improving the drought resistance of Manchurian ash is also provided.

[0031] Specifically, the endophytic fungus SLR21 or its fermentation broth can be used to induce the synthesis of phenolic substances from ash trees; among which, phenolic substances include flavonoids and other polyphenols.

[0032] In another embodiment of the present invention, the application of the above-mentioned endophytic fungus or its fermentation broth in promoting the growth of Manchurian ash is also provided.

[0033] Specifically, the endophytic fungus SLR21 or its fermentation broth can be used to improve the antioxidant enzyme activity, stress-resistance substance content, and photosynthetic capacity of *Fraxinus mandshurica*. The antioxidant enzyme activity includes the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Stress-resistance substances include soluble protein, malondialdehyde (MDA) content, proline (PRO), and soluble sugars. Photosynthetic capacity includes indicators such as net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), stomatal conductance (Gs), transpiration rate (Tr), chlorophyll content, and leaf nitrogen content.

[0034] In another embodiment of the present invention, a root drenching agent or foliar spray for improving the drought resistance and / or promoting the growth of Manchurian ash is also provided, comprising the above-mentioned endophytic fungi or their fermentation liquid; it should be noted that the root drenching agent or foliar spray may also include solvents, diluents and other components.

[0035] In another embodiment of the present invention, the application of the above-mentioned endophytic fungus or its fermentation broth in promoting the germination of Manchurian ash seeds is also provided.

[0036] In another embodiment of the present invention, a seed soaking agent for promoting the germination of Manchurian ash seeds is also provided, comprising the aforementioned endophytic fungi or their fermentation broth; it should be noted that the seed soaking agent or foliar spray may also include solvents, diluents and other components.

[0037] The following embodiments are implementation examples of the technical solution of the present invention in practical applications, but are not limited thereto. Unless otherwise specified, all materials and reagents involved are commercially available products; unless otherwise specified, all experimental methods used are conventional methods.

[0038] Example 1: This example provides a method for isolating and identifying the endophytic fungus SLR21, specifically including the following steps:

[0039] Isolation of the endophytic fungus SLR21: Under aseptic conditions, ash roots were cut into 0.5 cm segments and placed in PDA medium. The medium was inverted and incubated at 26-28℃. Observations were made daily, and mycelia were picked and grown on new culture dishes. After colonies formed, a small amount of edge mycelia were picked and the process was repeated twice to purify the fungus, yielding the endophytic fungus SLR21. Its morphology is shown in the image below. Figure 1 As shown.

[0040] ITS sequence identification of endophytic fungus SLR21: Hyphae were scraped from well-grown colony plates and placed in 2 mL centrifuge tubes. Endophytic fungal DNA was extracted using the CTAB method. The obtained DNA product was then dissolved in ddH2O and stored at -20℃ for amplification. The full-length ITS sequence was amplified using the universal fungal ITS primers ITS1 and ITS4 under the following reaction conditions: pre-denaturation 94℃ 2 min; denaturation 94℃ 40 s; annealing 57℃ 50 s; extension 72℃ 1 min; 35 cycles; extension 72℃ 10 min; 4℃ forever. 3.0 μL of the amplification product was mixed with 5× Loading Buffer and spotted onto a 3% agarose gel containing 10 mg / mL Aureobasidium red. Electrophoresis was then performed in 1.0× TAE buffer (120 V). After electrophoresis, the gel was removed and placed in a gel imaging system. Based on the results of PCR electrophoresis, the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for unidirectional sequencing with upstream and downstream primers to obtain sequencing results.

[0041] Example 2: This example provides a method for detecting the drought resistance of the endophytic fungus SLR21, as follows: Drought is simulated using PDA solid medium containing 10% polyethylene glycol (PEG). Specifically, after culturing the endophytic fungus SLR21 for one week, mycelial discs of the same size (0.5 cm in diameter) are prepared using a punch and inoculated onto PDA medium containing 10% PEG. Each experiment is repeated three times, with a blank control included. Growth is observed after 7 days of culture, and the results are as follows. Figure 2 As shown, the colony diameter of endophytic fungus SLR21 on PEG medium differed from the average colony diameter on normal medium by only 0.2 cm, and its growth was not significantly affected by drought stress.

[0042] Example 3: The activated endophytic fungus strain SLR21 was inoculated into PDB liquid medium, with sterile water as a control. The medium was placed on a shaker and fermented at 28°C and 120 rpm / min to obtain the fermentation broth. Each treatment was repeated in triplicate. After 7 days, 1 mL of the fermentation broth was transferred to a 1.5 mL centrifuge tube and centrifuged at 4°C and 10000 g (g is a unit of relative centrifugal force, representing the multiple of the acceleration due to gravity) for 20 min. The levels of hormones such as IAA and SA in the fermentation broth were measured. The results are as follows: Figure 3 As shown; among them, the content of auxin (IAA) is 6.35 μg / mL and the content of salicylic acid (SA) is 34 μg / mL.

[0043] Example 4: The activated endophytic fungus strain SLR21 was inoculated into PDB liquid medium, with sterile water as a control. The medium was placed on a shaker and fermented at 28°C and 120 rpm / min to obtain the fermentation broth. Each treatment was repeated in triplicate. After 7 days, 1 mL of the fermentation broth was transferred to a 1.5 mL centrifuge tube and centrifuged at 10000g for 20 min at 4°C. The contents of secondary metabolites such as polyphenols, polysaccharides, and flavonoids in the fermentation broth were determined. The results are as follows: Figure 4 As shown; the contents of polyphenols, polysaccharides, and flavonoids are 0.16 mg / mL, 0.42 mg / mL, and 0.44 mg / mL, respectively.

[0044] Example 5: Fermentation broth of endophytic fungus SLR21 (fermentation broth concentration of 1×10⁻⁶ / mL) was used. 6 The spores were fermented using the same method as in Example 2. A root irrigation experiment was conducted on April 10, 2021, at the potted plant experimental field of the College of Life Sciences, Northeast Forestry University. A potted method was used, employing plastic pots with a diameter of 24cm, a height of 19.5cm, and a bottom diameter of 16cm. Each pot contained 6L of soil (black soil and sand in a 3:1 ratio), with one seedling planted per pot, and the same amount of water was applied. The settings were: SLR: root irrigation treatment group; T0: natural drought control group. Root irrigation with the bacterial solution was performed 2-3 times per week for 2 weeks, with 3 seedlings per treatment, repeated 3 times. Photosynthetic indicators, physiological indicators, and phenolic content were measured at soil moisture contents of 45%-50%, 20%-25% (mild drought), 12%-15% (moderate drought), 3%-8% (severe drought), and 45%-50% (after rehydration). Nine seedlings were sampled for each treatment for each indicator measurement.

[0045] The methods for measuring photosynthetic indicators are as follows: Testing was conducted between 9:00 AM and 11:00 AM. The third leaf from each group was randomly selected, and the net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), stomatal conductance (Gs), and transpiration rate (Tr) were measured using a Li-6400 portable photosynthesis system. Chlorophyll and nitrogen content of the leaves were measured using a plant nutrient analyzer (LD-YD). These indicators were measured at soil moisture contents of 45%-50%, 20-25% (mild drought), 12%-15% (moderate drought), 3%-8% (severe drought), and 45%-50% (after rehydration). The results are as follows: Figure 5 As shown.

[0046] The methods for determining antioxidant enzyme activity are as follows: Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were determined using the nitroblue tetrazolium photoreduction method, the guaiacol colorimetric method, and the ultraviolet spectrophotometric absorption method, respectively. The results are as follows: Figure 6 As shown.

[0047] The methods for determining the content of osmotic substances are as follows: the contents of soluble protein, malondialdehyde (MDA), proline (PRO), and soluble sugar were determined using the Coomassie brilliant blue method, the thiobarbituric acid heating method, and the acidic ninhydrin colorimetric method, respectively. The determination results are as follows: Figure 7 As shown.

[0048] The determination methods for phenolic substances are as follows: Flavonoid content determination: The above-ground and underground parts of *Fraxinus mandshurica* were dried at 65℃ and the flavonoid content was determined. Polyphenol content determination: 0.1 g of sample was accurately weighed into a 10 mL centrifuge tube, 3.5 mL of 60% ethanol was added, and the mixture was extracted in a 70℃ water bath for 1 h, followed by sonication for 1 h. This process was repeated twice. The samples were filtered, and the filtrates were combined and centrifuged (10000 r / min, 5 min). 0.1 mL of the supernatant was accurately pipetted, 0.5 mL of 10% FoLin-phenol reagent was added, followed by 1.4 mL of 7.5% saturated sodium carbonate solution. After mixing again, the mixture was reacted at room temperature (25℃) in the dark for 30 min. The absorbance at 760 nm was measured, and the polyphenol content was calculated based on the absorbance. The determination results are as follows: Figure 8 As shown.

[0049] In addition, the phenotypic effects of the fermentation broth of the endophytic fungus SLR21 provided in this embodiment of the invention on the growth and drought resistance of *Fraxinus mandshurica* seedlings through root irrigation are as follows: Figure 9 As shown in A and B.

[0050] The above test results show that root irrigation with the fermentation broth of the endophytic fungus SLR21 can alleviate the physiological state of *Fraxinus mandshurica* under drought conditions, specifically as follows:

[0051] like Figure 5 As shown, when the soil moisture content was 3-8%, under severe drought, the photosynthetic rate of the SLR group was 6.72 times that of the T0 group, the stomatal conductance was 1.2 times that of the T0 group, the transpiration decreased by 2.5%, the CO2 concentration decreased by 29.2%, and the CO2 utilization rate was improved.

[0052] like Figure 6 As shown, the SOD activity of seedlings generally increased with increasing drought severity, reaching its highest level under severe drought. Under mild drought, the SOD activity of the SLR group was 1.04 times that of the T0 group; the CAT activity of seedlings in all groups generally increased, reaching its highest level under severe drought. Under mild drought, the SLR group was 1.41 times that of the T0 group; with increasing drought severity, the POD activity of seedlings in all groups generally increased, reaching its highest level under severe drought. Under severe drought, the SLR group decreased by 26.3% compared to the T0 group. Root irrigation with bacterial solution increased CAT activity, while SOD activity showed no significant change; POD activity decreased.

[0053] like Figure 7 As shown, under severe drought, the PRO content in the leaves of plants in the SLR group increased by 9.55% compared to the T0 group, the MDA content decreased by 33%, and the soluble sugar content increased by 16%. With increasing drought severity, the nitrogen content of seedlings in all groups generally showed a decreasing trend, reaching its lowest point under severe drought. Under moderate drought, the nitrogen content in the SLR group was 1.95 times that of the T0 group. Under severe drought, the nitrate nitrogen content in the leaves of plants in the SLR group increased by 12% compared to the drought control group.

[0054] like Figure 8 As shown, after rehydration, compared with the natural drought control group, the flavonoid content in the stems of the SLR group increased by 35%; the polyphenol content increased by 25%; and the flavonoid content in the leaves increased by 50%.

[0055] Example 6: Fermentation broth of endophytic fungus SLR21 (fermentation broth concentration of 1×10⁻⁶ / mL) was used. 6 Experiment 1: Three seedlings were planted per pot and watered equally. The setup was as follows: SLR21: bacterial solution spraying treatment group; CK: sterile water spraying control group. Bacterial solution spraying was performed every two days for two weeks. Three seedlings constituted one replicate, for a total of six replicates. Experiment 2: Using the second pair of branches from the top of one-year-old *Fraxinus mandshurica* seedlings as material, 20% PEG-6000 was used to simulate drought, and the leaves were sprayed with bacterial solution. The setup was as follows: SLR21: bacterial solution spraying treatment group; CK: sterile water spraying control group. Bacterial solution spraying was performed once daily for three consecutive days. One branch constituted one replicate, for a total of three replicates. The results are as follows: Figure 9 As shown in C and D, the SLR21 bacterial solution spraying treatment significantly enhanced the drought resistance of Manchurian ash seedlings and detached leaves, and the leaves were more upright and drought-resistant than the CK control group.

[0056] The above results indicate that the endophytic fungus strain SLR21 possesses drought resistance and promotes seed production in *Fraxinus mandshurica*. Root irrigation with the fermentation broth of SLR21 resulted in a more developed root system and an increased number of lateral roots in *Fraxinus mandshurica* seedlings. This treatment significantly enhanced the activity of antioxidant enzymes in the seedling stage, increased the content of stress-resistant substances, improved photosynthesis, and increased leaf nitrogen content, thereby enhancing photosynthetic performance and promoting growth. Simultaneously, it induced the synthesis of phenolic substances in *Fraxinus mandshurica*, conferring higher drought resistance to the seedlings. Furthermore, phenotypically, spraying with SLR21 bacterial solution resulted in more robust leaves and significantly improved drought resistance in *Fraxinus mandshurica*.

[0057] In summary, the solutions provided by the embodiments of the present invention are pollution-free, low-cost, highly operable, can be used on a large scale, and are highly efficient. They offer a practical new technology for effectively solving problems such as slow growth and poor drought resistance in the seedling, planting, and forestation processes of Manchurian ash.

[0058] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. An endophytic fungus that enhances the stress resistance and growth-promoting function of Manchurian ash, characterized in that, Named Alternaria spp. ( Alternaria tenuissima SLR21 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 42057.

2. The application of an endophytic fungus or its fermentation broth as described in claim 1 in improving the drought resistance of Manchurian ash.

3. The application according to claim 2, characterized in that, The endophytic fungus or its fermentation broth is used to induce the synthesis of phenolic substances from ash trees.

4. The application according to claim 2, characterized in that, The fermentation broth of the endophytic fungi contains polyphenols, polysaccharides, flavonoids, salicylic acid, and auxins.

5. The application of an endophytic fungus or its fermentation broth as described in claim 1 in promoting the growth of Manchurian ash.

6. The application according to claim 5, characterized in that, The endophytic fungi or their fermentation broth are used to increase the nitrogen content, soluble sugars, and photosynthetic capacity of Ash leaves.

7. A root drenching agent or foliar spray for improving the drought resistance and promoting the growth of Manchurian ash, characterized in that, Includes the endophytic fungus or its fermentation broth as described in claim 1.

8. The application of an endophytic fungus as described in claim 1 or its fermentation broth in promoting the germination of Manchurian ash seeds.

9. A seed soaking agent for promoting the germination of Manchurian ash seeds, characterized in that, Includes the endophytic fungus or its fermentation broth as described in claim 1.

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