Application of yellow microspora fungi in promotion of growth of solanaceae plants

Abstract

The invention relates to application of yellow microspora fungi in promotion of growth of solanaceae plants, and belongs to the technical field of microorganisms. In order to solve the technical problems of narrow host range, dependence on living spores and the like in the prior art related to growth promotion application of the yellow microspora fungi, the method comprises the following steps: respectively treating tomato seeds and seedlings of Bensi tobacco by using yellow microspora fungi spores and fermentation supernate; it is found that tomato growth can be promoted when tomato seeds are coated with a seed coating solution containing yellow microspora fungus spores or tomato seedlings are irrigated with yellow microspora fungus fermentation supernate. The growth of the nicotiana benthamiana can be promoted by soaking the seeds of the nicotiana benthamiana by utilizing the suspension of the spores of the yellow microspora fungi, coating the seeds of the nicotiana benthamiana by utilizing a seed coating solution containing the spores of the yellow microspora fungi, or irrigating the seedlings of the nicotiana benthamiana by utilizing the fermentation supernatant of the yellow microspora fungi. The invention lays a theoretical foundation for the subsequent deep development and utilization of the yellow microspora fungi as the plant probiotics.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of *Xanthomonas* fungi in promoting the growth of Solanaceae plants. Background Technology

[0002] genus *Xanthomonas* Parametarhizium (This is a family of clover established in 2021.) Clavicipitaceae The genus *Xanthomonas* belongs to the fungi genus. *Xanthomonas* fungi are a class of microorganisms with potential plant growth-promoting functions. Studies have reported that their spores can establish a symbiotic relationship with the roots of legumes (such as mung beans and kidney beans) and grasses (such as rice and corn), effectively promoting the growth and development of the host plants, showing good potential as bio-fertilizers or plant growth-promoting agents.

[0003] However, existing research still has significant limitations and gaps: First, the host range is narrow, and the growth-promoting effects on important plant families and genera are unknown. Current research focuses on legumes and grasses, while systematic studies are lacking on whether globally widely cultivated and economically valuable solanaceous plants (such as tomatoes and Nicotiana benthamiana) can achieve symbiosis with Xanthomonas fungi and experience growth promotion. Tomatoes are one of the world's most important vegetable crops, and Nicotiana benthamiana is a key model plant in botanical research. Clarifying the growth-promoting effects of Xanthomonas fungi on them has significant theoretical and applied value.

[0004] Second, the optimal inoculation concentration for small seeds remains unclear. The size and weight of plant seeds significantly influence their interaction with microorganisms. Compared to studied legumes and grasses, tomato and tobacco seeds are smaller and lighter, and their optimal fungal spore inoculation concentrations may differ drastically. Current techniques have not been used for dosage optimization tailored to the characteristics of small seeds, which limits the effective application of this technology in Solanaceae crops.

[0005] Third, existing technologies rely on live spores, creating bottlenecks in productization. Currently, the recognized active ingredient is pure, washed fungal spores. However, live microbial preparations generally suffer from inherent defects such as unstable biological activity, susceptibility to environmental factors (temperature, humidity, pH, etc.), short shelf life, and high costs for large-scale production, which severely limit the commercial development and field promotion of Xanthomonas fungi products.

[0006] Fourth, the growth-promoting mechanism is unclear, and the activity of the metabolites needs further exploration. The specific mechanism by which *Xanthomonas* fungi promote plant growth has not yet been elucidated. A key scientific question arises: does its growth-promoting effect depend entirely on the direct symbiotic interaction between the live fungus and the plant, or do the metabolites produced during its fermentation also possess biological activity? If its fermentation broth, which does not contain live cells (spores), also possesses growth-promoting function, it is hoped that novel stable formulations based on metabolites can be developed, thereby fundamentally overcoming the instability, short shelf life, and high cost of live formulations and achieving technological innovation.

[0007] Therefore, addressing the aforementioned technological gaps, this invention aims to systematically investigate: 1) the growth-promoting effects of *Xanthomonas* fungi on two important Solanaceae plants, tomato and *Nicotiana benthamiana*; 2) the optimal spore inoculation concentration for their small seed characteristics; and 3) more importantly, to evaluate for the first time whether the sterile fermentation filtrate of *Xanthomonas* fungi (i.e., free of any spores or living cells) possesses independent plant growth-promoting properties. This research will provide important theoretical and technological foundations for developing novel, stable, and efficient plant growth-promoting products based on microbial metabolites. Summary of the Invention

[0008] To address the deficiencies and gaps in the application of *Microsporum* fungi, this invention utilizes *Microsporum* spores and fermentation supernatant (containing only fungal metabolites and no fungal cells) to treat tomato and *Nicotiana benthamiana* seeds and seedlings, respectively. It was found that coating tomato seeds with a seed coating solution containing *Microsporum* spores, or irrigating tomato seedlings with the fermentation supernatant of *Microsporum* spores, both promoted tomato growth. Similarly, soaking *Nicotiana benthamiana* seeds in a *Microsporum* spore suspension, coating *Nicotiana benthamiana* seeds with a seed coating solution containing *Microsporum* spores, or irrigating *Nicotiana benthamiana* seedlings with the fermentation supernatant of *Microsporum* spores, all promoted *Nicotiana benthamiana* growth.

[0009] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide the application of a spore suspension of *Microsporidium* fungi in promoting the growth of Solanaceae plants, including tomato and *Nicotiana benthamiana*; wherein the *Microsporidium* fungi are *Microsporidium longiflorum* (… Parametarhizium changbaiense ) or Xingan microsporidium ( Parametarhizium hingganense ).

[0010] In one embodiment of the present invention, the application involves using a seed coating solution made from a suspension of spores of the genus *Microsporidium* to coat tomato seeds or *Tobacco Benzoinus* seeds.

[0011] In one embodiment of the present invention, the concentration of the spore suspension is 1×10⁻⁶. 6 Cells / mL - 1×10 7per mL.

[0012] Preferably, the concentration of the spore suspension is 1×10⁻⁶. 6 per mL.

[0013] In one embodiment of the present invention, the seed coating solution further contains a film-forming agent.

[0014] In one embodiment of the present invention, the film-forming agent is gum arabic powder.

[0015] In one embodiment of the present invention, the application involves soaking tobacco seeds in a suspension of spores of the genus *Microsporidium*.

[0016] In one embodiment of the present invention, the concentration of the spore suspension is 1×10⁻⁶. 5 Cells / mL - 1×10 8 per mL.

[0017] A second objective of this invention is to provide the application of fermentation supernatant of *Microsporidium* fungi in promoting the growth of Solanaceae plants, including tomato and *Nicotiana benthamiana*; wherein the *Microsporidium* fungi are *Microsporidium longiflorum* (… Parametarhizium changbaiense ) or Xingan microsporidium ( Parametarhizium hingganense ).

[0018] In one embodiment of the present invention, the application is to irrigate tomato seedlings or tobacco seedlings with the fermentation supernatant of Microsporum fungi.

[0019] In one embodiment of the present invention, when the solanaceous plant is tomato, the fermentation supernatant is a 10% or 20% diluted fermentation supernatant; when the solanaceous plant is tobacco Benzoate, the fermentation supernatant is the original fermentation supernatant of Microsporidium dauricum.

[0020] The beneficial effects of this invention are: This invention is the first to discover the growth-promoting effect of *Xanthomonas* fungi on Solanaceae crops. The growth-promoting effect of *Xanthomonas* fungi on tomatoes was investigated using a seed coating method, with a concentration of 1×10⁻⁶. 6 cells / mL and 1×10 7 Seed coating solution containing 1 × 10⁶ spores / mL of *Microspora* fungi has a growth-promoting effect on tomato plants, and the spore concentration is 1 × 10⁶. 6 The best growth-promoting effect was observed at a spore concentration of 1×10⁶ / mL. Compared to the control group, the effect was significantly better when the spore concentration was 1×10⁶ / mL. 6Treating tomato seeds with a seed coating solution of *Microsporum longiflorum* at a concentration of 1 × 10⁶ spores / mL significantly increased stem diameter by 16.73%, above-ground fresh weight by 12.43%, and below-ground fresh weight by 88.73% at 21 days of growth. The above-ground dry weight increased by 33.46%, and the below-ground dry weight by 65.14%, demonstrating significant growth-promoting effects. Compared to the control group, treatment with a spore concentration of 1 × 10⁶ spores / mL significantly improved growth. 6 Treating tomato seeds with a seed coating solution of *Microsporum xinganense* spores / mL significantly increased stem diameter by 19.12%, above-ground fresh weight by 13.55%, underground fresh weight by 36.24%, and above-ground dry weight by 89.15% after 21 days of growth, demonstrating a significant growth-promoting effect. In a seed soaking experiment on *Tobacco Benzovia*, soaking seeds with a *Microsporum* spore suspension effectively promoted root growth, with the *Microsporum xinganense* spore suspension at 1×10⁻⁶ mL showing particularly strong effects. 8 The concentration of *Microsporidium xinganense* spores at 1×10⁶ / mL showed the best effect on promoting root growth in *Nicotiana benthamiana*, increasing root length by 119.35%. The *Microsporidium xinganense* spore suspension at 1×10⁶ / mL also showed the best effect. 7 The concentration of 1×10⁶ / mL showed the best effect on promoting root growth in *Nicotiana benthamiana*, increasing root length by 161.29%. In the seed coating experiment of *Nicotiana benthamiana*, 1×10⁶ / mL… 6 Seed coating solution of *Microsporidium xinganense* at a concentration of [number] cells / mL has a growth-promoting effect on *Nicotiana benthamiana*, increasing plant height by 64.30%, stem diameter by 12.78%, effective leaf number by 21.44%, total plant weight by 29.01%, aboveground fresh weight by 30.69%, aboveground dry weight by 38.89%, underground fresh weight by 24.00%, and underground dry weight by 50.00%.

[0021] This study is the first to discover that the fermentation supernatant of *Microsporum* fungi (i.e., containing only fungal metabolites and no fungal cells) has a growth-promoting effect on tomatoes and *Nicotiana benthamiana*. In the experiment on the promotion of tomato seedlings by *Microsporum* fungi fermentation supernatant, water and YM liquid medium were used as control groups, and there was no significant difference between the two groups. Compared with the YM liquid medium group, in terms of plant height, 10% fermentation supernatant of *Microsporum longiflorum* increased by 19.26%, and 20% fermentation supernatant of *Microsporum longiflorum* increased by 19.85%; 10% fermentation supernatant of *Microsporum xinganense* increased by 25.74%, and 20% fermentation supernatant of *Microsporum xinganense* increased by 15.78%. Compared with the YM liquid culture medium group, in terms of root length, the 10% fermentation supernatant of *Microsporidium longiflorum* increased by 19.14%, and the 20% fermentation supernatant of *Microsporidium longiflorum* increased by 19.20%; the 10% fermentation supernatant of *Microsporidium xinganense* increased by 22.27%, and the 20% fermentation supernatant of *Microsporidium xinganense* increased by 10.46%.

[0022] The fermentation supernatant of *Xanthomonas xinganensis* increased the plant height of *Nicotiana benthamiana* by 27.45% and the root length by 13.96%, showing a significant difference compared to the control. This invention lays a theoretical foundation for further in-depth development and utilization of *Xanthomonas* fungi as plant probiotics. Attached Figure Description

[0023] Figure 1 Phenotypic observations of tomato plants corresponding to different concentrations of *Microspora leucosus* seed coating solution; from left to right in the figure are the control group, 10... 5 Processing group, 10 6 Processing group, 10 7 Processing group, 10 8 Treatment group, scale bar in the figure is 4 cm; Figure 2 Phenotypic observations of tomato plants corresponding to different concentrations of *Microsporidium xinganense* seed coating solution; from left to right in the figure are the control group, 10... 5 Processing group, 10 6 Processing group, 10 7 Processing group, 10 8 Treatment group, scale bar in the figure is 4 cm; Figure 3 Figure 1 shows the effect of different concentrations of seed coating solutions from two species of *Xanthomonas* fungi on the phenotype of tomato plants; among them... Figure 3 Figure A in the figure shows the effect of different concentrations of seed coating solutions from two different fungi, *Xanthomonas*, on the plant height of tomato plants. Figure 3 Figure B in the figure shows the effect of different concentrations of seed coating solutions from two different fungi, *Xanthomonas*, on the stem diameter of tomato plants. Figure 3 Figure C in the figure shows the effect of different concentrations of seed coating solutions from two different fungi on the aboveground fresh weight of tomato plants. Figure 3 Figure D in the figure shows the effect of different concentrations of seed coating solutions from two different fungi of the genus *Xanthomonas* on the underground fresh weight of tomato plants. Figure 3 E in the figure represents the effect of different concentrations of seed coating solutions from two different fungi on the aboveground dry weight of tomato plants. Figure 3 F in the figure represents the effect of different concentrations of seed coating solutions from two types of Xanthomonas fungi on the underground dry weight of tomato plants. Multiple comparisons were performed using Duncan's method or nonparametric tests to test for significant differences (P<0.05). Different letters indicate that there are significant differences in the data among the concentration treatments. Figure 4 The figure shows the effects of seed coating solutions from two species of *Xanthomonas* fungi on biochemical indicators of tomato plants; among them... Figure 4 In the figure, A represents the result of the detection of total nitrogen content in tomato leaves; Figure 4 In the figure, B represents the result of the chlorophyll content detection in tomato leaves; Figure 4 In the figure, C represents the detection results of total phenolic content in tomato roots; Figure 4 D in the figure represents the results of the detection of total flavonoid content in tomato roots; Duncan's method was used for multiple comparisons to test for significant differences (P<0.05), and different letters indicate that there are significant differences in the data among the concentration treatments; Figure 5 The figure shows the effect of different concentrations of *Xanthomonas* fungal fermentation supernatant (i.e., containing only fungal metabolites and no fungal cells) on tomato plant growth; among them, Figure 5 Figure A in the figure shows the effect of different concentrations of fermentation supernatant of *Xanthomonas* fungi on the plant height of tomato plants. Figure 5 Figure B in the figure shows the effect of different concentrations of *Xanthomonas* fungal fermentation supernatant on the root length of tomato plants. Multiple comparisons were performed using Duncan's method or nonparametric tests to test for significant differences (P<0.05). Different letters indicate that there are significant differences in the data among the different concentration treatments. Figure 6 The effect of different concentrations of *Microspora longiflora* spore suspensions on the root growth of *Nicotiana benthamiana* is shown in the figure; from left to right in the figure are the control group, 10... 5 Group, 10 6 Group, 10 7 Group, 10 8 Group; the scale bar in the figure is 1 cm; Figure 7 The effect of different concentrations of *Microspora xinganensis* spore suspension on the root growth of *Nicotiana benthamiana* is shown in the figure; from left to right in the figure are the control group, 10... 5 Group, 10 6 Group, 10 7 Group, 10 8 Group; the scale bar in the figure is 1 cm; Figure 8 The figure shows the effect of different concentrations of spore suspensions of two fungi, *Xanthomonas*, on the root length of *Nicotiana benthamiana*. Multiple comparisons were performed using Duncan's method, with P < 0.05. Different letters indicate significant differences between different treatments. Figure 9 Figure 1 shows the effect of *Microsporidium xinganense* seed coating solution on the phenotype of *Nicotiana benthamiana* plants; among them... Figure 9 A and Figure 9 C in the figure represents the control group; Figure 9 B and Figure 9 D in the figure represents the treatment group with *Xanthomonas xinganensis*; the scale bar in the figure is 4 cm. Figure 10 Figure 1 shows the effect of *Microsporidium xinganense* seed coating solution on the growth of *Nicotiana benthamiana* plants; among them... Figure 10 In the figure, A represents the measurement results of tobacco plant height; Figure 10 In the figure, B represents the measurement results of the tobacco plant stem diameter; Figure 10 C in the figure represents the statistical results of the number of effective leaves of tobacco plants; Figure 10 In the figure, D represents the result of the measurement of the whole plant weight of tobacco; Figure 10 E in the figure represents the results of measuring the fresh weight of the aboveground parts, dry weight of the aboveground parts, fresh weight of the underground parts, and dry weight of the underground parts of tobacco plants; in A, B, and D, the asterisks indicate significant differences evaluated using the t-test: **P<0.01, ***P<0.001; in C and E, the asterisks indicate significant differences evaluated using the non-parametric test: ***P<0.001. Figure 11 The effect of watering *Nicotiana benthamiana* seedlings with the fermentation supernatant of *Microsporum* fungi on *Nicotiana benthamiana* growth is shown in the figure. Figure 11 Figure A in the diagram shows the results of the measurement of the height of Tobacco Benedictine plant; Figure 11 Figure B in the figure shows the results of root length measurement in Nicotiana benthamiana; A was compared using a nonparametric test (P < 0.05), and B was compared using Duncan's method for multiple comparisons (P < 0.05). Different letters indicate significant differences between different treatments. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents, culture media and instruments used are all conventional materials, reagents, culture media and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0026] The test plants used in this invention are from the following sources: tomato ( Solanum lycopersicum cv. AilsaCraig), Benedict's tobacco ( Nicotiana benthamiana (This information is kept by our laboratory.)

[0027] The microorganisms used in this invention are as follows: Microsporidium longiflorum ( Parametarhizium changbaiense) and Xingan microsporidium ( Parametarhizium hingganense This is disclosed in Chinese Patent Application No. 202210962001.9, entitled "Application of a Microsporum fungus in promoting plant growth and improving plant stress resistance." (Gao S, Meng W, Zhang L, Yue Q, Zheng X and Xu L (2021)) Parametarhizium ( Clavicipitaceae Before the publication of *Microsporum changbaiense* gen. nov. With Two New Species as a Potential Biocontrol Agent Isolated From Forest Litters in Northeast China. Front. Microbiol. 12:627744. doi: 10.3389 / fmicb.2021.627744), *Microsporum changbaiense* was known as *Metarhizium anisopliae*. Metarhizium changbaiensis The fungus *Xanthomonas xinganensis* is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 19143 and deposit date of December 5, 2019. *Xanthomonas xinganensis* was formerly known as *Metarhizium xinganensis*. Metarhizium hingganensis (This entry is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 19144 and deposit date of December 5, 2019).

[0028] The experimental method involved in this invention is as follows: 1. Preparation method of seed coating solution and fermentation supernatant of *Microsporum* fungi (i.e., containing only fungal metabolites and no fungal cells). 1) Preparation method of seed coating solution for *Microsporum* fungi Two strains of *Microsporum* were inoculated onto YMA (10 g malt extract, 2 g yeast extract, 20 g agar, 1 L water) agar plates (90 mm in diameter) and incubated at 25°C for 14 days to prepare spore suspensions. Sterilized 0.05% Tween 80 solution was added to the two cultured *Microsporum* strains. Spores were evenly scraped from the plates using a triangular spreader, and the liquid was filtered through sterile medical gauze to remove fungal mycelium. The liquid was transferred to sterilized centrifuge tubes and centrifuged at 5000 rpm for 5 min. The supernatant was discarded, and an appropriate amount of 0.05% Tween 80 solution was added to suspend the spores, thus obtaining the spore suspension. The spore concentration in the spore suspension was determined using an optical microscope and a hemocytometer. The suspensions were prepared to the following concentrations: 1 × 10⁻⁶. 8 cells / mL, 1×10 7 cells / mL, 1×10 6 cells / mL, 1×10 5 By adding 3% (w / v) gum arabic powder to spore suspensions of different concentrations at a concentration of spores / mL, a viscous seed coating solution is prepared.

[0029] 2) Preparation method of fermentation supernatant of *Microsporum* fungi The preparation method of fermentation supernatant (i.e., containing only fungal metabolites and no fungal cells) is as follows: Inoculate 3-4 0.6 mm microsporidium fungal cakes into YM liquid medium (10 g malt extract, 2 g yeast extract, 1 L water), incubate at 25℃ and 180 rpm for 5 days (for tomato treatment) or 7 days (for tobacco treatment), obtain fermentation broth, centrifuge at 5000 rpm, collect the supernatant, filter it through a 0.22 μm filter membrane to remove fungal cells (mycelial fragments and spores) to obtain the original fermentation supernatant without living cells.

[0030] 2. Seed treatment methods 1) Tomato seed treatment methods Select whole tomato seeds and place them in a 1.5 mL centrifuge tube. Add 75% alcohol to sterilize for 5 minutes. Rinse three times with sterile water, then spread them evenly on an iron plate and let them air dry naturally for later use.

[0031] 2) Treatment methods for tobacco seeds (Tobacco Benedict) Place tobacco seeds in a 1.5 mL centrifuge tube and rinse with sterile water. Add 75% alcohol for 1 min to disinfect, rinse with sterile water, add 10% sodium hypochlorite solution for 1 min to disinfect, rinse with sterile water 5-8 times, add an appropriate amount of sterile water, seal, and place at 4℃ for vernalization for 2-3 days.

[0032] 3. Phenotypic determination methods for tomato and Nicotiana benthamiana plants Phenotypic determination of tomato plants: When significant differences in plant traits occurred between the treatment group and the control group, i.e., when the plants grew to 21 days, the tomato traits were measured. For Tobacco Benedict, the traits were measured 30 days after bud break. The basic indicators included are as follows: 1) Plant height (cm): Measure the distance from the point where the roots and stem separate to the top of the main stem, i.e. the growing point, using a ruler.

[0033] 2) Stem diameter (mm): Measure the stem diameter 1 cm below the point where the true leaves unfold, using vernier calipers.

[0034] 3) Fresh weight of plants (g): After cleaning and drying the plants, weigh the fresh weight of the above-ground parts and the fresh weight of the underground parts separately using an electronic balance. (Weigh 3 plants together and take the average value.) 4) Plant dry weight (g): After preheating the oven to 105℃, place the plants in the oven for 30 minutes to kill the green color, then remove them and place them in a preheated oven at 75℃ until constant weight is achieved. After cooling to room temperature, weigh the above-ground and underground dry weights separately using an electronic balance. (Weigh 3 plants together and take the average value.) 5) Number of effective leaves: The number of true leaves grown by the plant.

[0035] 6) Total plant weight (g): The weight of a single plant after it has been cleaned and dried.

[0036] Example 1: Effects of Seed Coating Solution of *Microsporum* on Tomato Growth Grind humus and vermiculite, sieve, and mix evenly (humus:vermiculite = 1:1, v / v) to prepare planting soil for tomato cultivation. Add 20 tomato seeds to every 10 mL of seed coating solution for coating, vortex for 2 minutes to mix, and then air dry. Bury the coated seeds in pots 1 cm below the surface of the planting soil and place them in a full-spectrum artificial light incubator with a 16-hour light / 8-hour dark circadian rhythm. Maintain an ambient temperature of 25-28℃ and a relative humidity of 55-60% RH.

[0037] 1. Effects of seed coating with different concentrations of *Xanthomonas* spores on tomato plant phenotype. To test the optimal coating concentration of two *Xanthomonas* seed coating solutions for tomato seeds, spore suspensions of the two *Xanthomonas* fungi were prepared at 1×10⁻⁶ ppm. 5 cells / mL, 1×10 6 cells / mL, 1×10 7 cells / mL and 1×10 8After establishing a concentration gradient of 1000 seeds / mL, a seed coating solution was prepared. Tomato seeds were then coated and sown. When the tomato plants reached 21 days of growth, the phenotype of the tomato plants was observed and measured.

[0038] The phenotypic observation results of tomato plants are as follows: Figure 1 and Figure 2 As shown in the figure. The effects of different concentrations of seed coating solutions from two species of *Microsporum* on the phenotype of tomato plants are as follows. Figure 3 As shown, by comparing the coating effects of seed coating solutions with different spore concentrations, it can be seen that the spore concentration in the seed coating solutions of the two *Xanthomonas* fungi is 1×10⁻⁶. 6 cells / mL and 1×10 7 It showed a growth-promoting effect on tomatoes at a spore concentration of 1×10⁶ / mL. 6 The optimal spore concentration was 1×10⁶ / mL for promoting germination. Compared to the control group, the spore concentration of 1×10⁶ / mL was significantly better. 6 Treatment of tomato seeds with a seed coating solution of *Microsporum longiflorum* at a concentration of 1 × 10⁶ spores / mL significantly increased plant height by 13.09%, stem diameter by 16.73%, above-ground fresh weight by 12.43%, and below-ground fresh weight by 88.73% at 21 days of growth. The above-ground dry weight increased by 33.46%, and the below-ground dry weight by 65.14%, demonstrating a significant growth-promoting effect. Compared to the control group, treatment with a spore concentration of 1 × 10⁶ spores / mL significantly improved growth. 6 Treating tomato seeds with a seed coating solution of *Microsporidium xinganense* at a concentration of 1 × 10⁶ spores / mL resulted in significant increases in plant height (6.50%), stem diameter (19.12%), above-ground fresh weight (13.55%), underground fresh weight (36.24%), and above-ground dry weight (89.15%) at 21 days of growth, demonstrating a substantial growth-promoting effect. Therefore, subsequent treatments using a spore concentration of 1 × 10⁶ spores / mL were recommended. 6 Tomato seeds were coated with a seed coating solution of 1 seed / mL to study its effect on the biochemical indicators of tomato plants.

[0039] 2. Effects of seed coating with a seed coating solution containing spores of the genus *Microsporum* on the total nitrogen and chlorophyll content of tomato plants. To determine whether *Xanthomonas* fungi affect nutrient absorption in tomato plants, the total nitrogen content and chlorophyll content of tomato leaves were measured. The results are as follows: Figure 4 As shown in Figure A, both *Xanthomonas* fungi significantly promoted the absorption of total nitrogen by tomato leaves. *Xanthomonas longiflora* treatment increased total nitrogen absorption by tomato leaves by 16.51%, while *Xanthomonas xinganensis* treatment increased it by 57.06%. Chlorophyll content is shown in Figure A. Figure 4As shown in B, the treatments with the two strains of *Xanthomonas* significantly increased the chlorophyll content in tomato leaves. The treatment with *Xanthomonas longiflora* increased the chlorophyll content in tomato leaves by 13.69%, while the treatment with *Xanthomonas xinganensis* increased the chlorophyll content in tomato leaves by 18.48%.

[0040] 3. Effects of seed coating solution containing spores of the genus *Microsporum* on the total phenolic and flavonoid content of tomato plants. The total phenolic and total flavonoid contents of tomato plants coated with seed coating solutions from two species of *Xanthomonas* fungi were determined, and the results are as follows: Figure 4 As shown in C and D: Compared with the control group, seed coating with both types of *Xanthomonas* fungi significantly increased the total phenolic content in tomato roots. *Xanthomonas longiflora* treatment increased the total phenolic content in tomato roots by 51.91%, while *Xanthomonas xinganensis* treatment increased it by 97.26%. Compared with the control group, *Xanthomonas longiflora* treatment significantly increased the total flavonoid content in tomato roots by 67.41%, while *Xanthomonas xinganensis* treatment increased it by 28.01%, but the difference was not statistically significant.

[0041] Example 2: Effects of watering tomato seedlings with fermentation supernatant of *Microsporum* fungi on tomato growth The supernatant of the fermentation broth of Microsporum spp. (100%) was diluted with water to 2 times (50%), 5 times (20%), and 10 times (10%). 10 mL of the diluted solution was then poured into the soil around the roots of tomato seedlings that had been growing for 5 days. The effect on promoting tomato growth was observed and measured after 5 days.

[0042] The effects of different concentrations of fermentation supernatant from two different *Xanthomonas* fungi on tomato plant growth are as follows: Figure 5As shown: In this example, water and YM liquid culture medium were used as control groups, and there was no significant difference between the two groups. Compared with the YM liquid culture medium group, in terms of plant height, the 10% fermentation supernatant of *Microsporum praecox* increased by 19.26%, the 20% fermentation supernatant of *Microsporum praecox* increased by 19.85%, the 50% fermentation supernatant of *Microsporum praecox* increased by 5.12%, and the original fermentation supernatant of *Microsporum praecox* increased by 2.43%. The 10% fermentation supernatant of *Microsporum xinganense* increased by 25.74%, the 20% fermentation supernatant of *Microsporum xinganense* increased by 15.78%, the 50% fermentation supernatant of *Microsporum xinganense* increased by 14.61%, and the original fermentation supernatant of *Microsporum xinganense* increased by 5.83%. Compared with the YM liquid culture medium group, in terms of root length, the 10% fermentation supernatant of *Microsporidium praecoxibaris* increased by 19.14%, the 20% fermentation supernatant of *Microsporidium praecoxibaris* increased by 19.20%, and the 50% fermentation supernatant of *Microsporidium praecoxibaris* increased by 11.01%; the 10% fermentation supernatant of *Microsporidium dauricum* increased by 22.27%, and the 20% fermentation supernatant of *Microsporidium dauricum* increased by 10.46%.

[0043] Example 3: Effects of seed soaking in a suspension of *Microsporum* spores on root growth of *Nicotiana benthamiana*. To test whether *Xanthomonas* fungi have a growth-promoting effect on the root growth of *Nicotiana benthamiana*, two *Xanthomonas* fungi were prepared at concentrations of 1×10⁻⁶. 5 cells / mL, 1×10 6 cells / mL, 1×10 7 cells / mL and 1×10 8 Tobacco seeds were soaked in spore suspensions of different concentrations for 16 hours, and then rinsed three times with sterile water. Petri dishes, filter paper, and absorbent cotton were sterilized and prepared for use. In a clean bench, absorbent cotton and filter paper were placed in the petri dishes, moistened with sterile water, and the seeds were evenly arranged in the dishes. The dishes were then sealed and placed in a light incubator at 25°C, with a 12-hour light and 12-hour dark period and a relative humidity of 70%. After 14 days, root length was measured using ImageJ.

[0044] Observe and measure the root length after 14 days of growth, such as Figure 6 and Figure 7 As shown.

[0045] like Figure 8 As shown, *Microsporum* fungi have a growth-promoting effect on tobacco roots. Compared with the control group, different concentrations of *Microsporum longiflorum* treatment groups (1×10⁻⁶) showed a greater effect. 5 Root length increased by 22.58% at a concentration of [number] cells / mL, and by 1×10 [units]. 6 Root length increased by 62.90% at a concentration of [number] cells / mL, and by 1×10 [units]. 7Root length increased by 101.61% at a concentration of [number] cells / mL, and by 1×10 [units]. 8 Root length increased by 119.35% at concentrations of [number] cells / mL, all showing significant differences, with 1×10 [units] showing the highest [value]. 8 A concentration of 1 × 10⁶ cells / mL showed the strongest root-promoting effect. Compared with the control group, different concentrations of *Microsporidium xinganense* treatment groups showed the greatest improvement in root growth. 5 Root length increased by 114.52% at a concentration of [number] cells / mL, and by 1×10 [units]. 6 Root length increased by 130.65% at a concentration of [number] cells / mL, and by 1×10 [units]. 7 Root length increased by 161.29% at a concentration of [number] cells / mL, and by 1×10 [units]. 8 Root length increased by 111.29% at concentrations of [number] cells / mL, all showing significant differences, with 1×10 [units] showing the highest [value]. 7 A concentration of [number] cells / mL has the strongest effect on promoting root growth.

[0046] Example 4: Effects of *Microsporum xinganense* seed coating solution on the growth of *Nicotiana benthamiana* plants Mix humus and vermiculite in a 2:1 (v:v) ratio and fill a flowerpot. Let it absorb water and set aside. Place tobacco seeds in a container with a spore concentration of 1×10⁻⁶. 6 After soaking the seeds in a seed coating solution of *Microsporidium xinganense* (number of seeds / mL) for 1 hour, the seeds were removed, dried on filter paper, and evenly sown on the soil surface. A film was then placed in a light incubator at 25°C, with a light exposure of 16 hours, a dark exposure of 8 hours, and a relative humidity of 70%. The tobacco plants were transplanted when they had 4-5 true leaves, and data were measured 30 days after germination.

[0047] Depend on Figure 9 and Figure 10 It can be seen that, compared with the control group, the plant height of tobacco treated with *Xanthomonas pungens* increased by 64.30%, stem diameter by 12.78%, number of effective leaves by 21.44%, total plant weight by 29.01%, above-ground fresh weight by 30.69%, above-ground dry weight by 38.89%, underground fresh weight by 24.00%, and underground dry weight by 50.00%, all of which showed significant increases.

[0048] Example 5: Effects of watering *Nicotiana benthamiana* seedlings with fermentation supernatant of *Microsporum* fungi on *Nicotiana benthamiana* growth. 20 mL of undiluted fermentation supernatant of *Microsporum* fungi was poured into the soil around the roots of tobacco seedlings. The effect on promoting tobacco growth was observed after 7 days. The control group was YM liquid.

[0049] The effects of irrigating *Nicotiana benthamiana* seedlings with undiluted fermentation supernatants of two *Microsporum* species on seedling height and root length are as follows: Figure 11As shown, compared with the control group, the height of *Nicotiana benthamiana* plants treated with *Microsporidium longiflorum* fermentation supernatant increased by 13.73%, and the root length increased by 9.46%. The height of *Nicotiana benthamiana* plants treated with *Microsporidium xinganense* fermentation supernatant increased by 27.45%, and the root length increased by 13.96%, showing significant differences.

[0050] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. The application of a spore suspension of *Xanthomonas* fungi in promoting the growth of Solanaceae plants, characterized in that... The Solanaceae plants include tomato and Nicotiana benthamiana; the fungus of the genus *Microsporidium* is *Microsporidium longiflorum* (…). Parametarhizium changbaiense ) or Xingan microsporidium ( Parametarhizium hingganense ).

2. The application according to claim 1, characterized in that, The application involves using a seed coating solution made from a suspension of spores of the genus *Microsporidium* to coat tomato seeds or *Tobacco Benzoinus* seeds.

3. The application according to claim 2, characterized in that, The concentration of the spore suspension is 1×10⁻⁶. 6 Cells / mL - 1×10 7 per mL.

4. The application according to claim 3, characterized in that, The concentration of the spore suspension is 1×10⁻⁶. 6 per mL.

5. The application according to claim 2, characterized in that, The seed coating solution also contains a film-forming agent.

6. The application according to claim 1, characterized in that, The application involves soaking tobacco seeds in a suspension of spores from the genus *Xanthomonas*.

7. The application according to claim 6, characterized in that, The concentration of the spore suspension is 1×10⁻⁶. 5 Cells / mL - 1×10 8 per mL.

8. The application of fermentation supernatant of *Xanthomonas* fungi in promoting the growth of Solanaceae plants, characterized in that... The Solanaceae plants include tomato and Nicotiana benthamiana; the fungus of the genus *Microsporidium* is *Microsporidium longiflorum* (…). Parametarhizium changbaiense ) or Xingan microsporidium ( Parametarhizium hingganense ).

9. The application according to claim 8, characterized in that, The application involves using the fermentation supernatant of *Microsporum* fungi to irrigate tomato seedlings or *Tobacco Benzoinus* seedlings.

10. The application according to claim 8, characterized in that, When the solanaceous plant is tomato, the fermentation supernatant is a 10% or 20% diluted fermentation supernatant; when the solanaceous plant is tobacco Benzoate, the fermentation supernatant is the original fermentation supernatant of Microsporidium dauricum.

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