A method for alleviating continuous cropping obstacles in melons using Trichoderma longifolia

Abstract

This invention belongs to the field of soil improvement and crop cultivation technology in facility agriculture, specifically relating to a method for alleviating continuous cropping obstacles in melons using *Trichoderma longicornis*. The method involves applying *Trichoderma longicornis* to the soil where melons are continuously grown to inhibit or alleviate continuous cropping obstacles. The method provided by this invention, using *Trichoderma longicornis* to alleviate continuous cropping obstacles in melons, achieves multiple effects—antibacterial activity, growth promotion, soil improvement, microbial community regulation, and enhanced stress resistance—through a scientific application of the microbial agent. This method can significantly inhibit the core pathogen of continuous cropping melons, *Fusarium oxysporum* melon-specific strain, promote the growth of continuously cropped melon plants, improve the physicochemical properties and biological activity of the continuously cropped soil, regulate the soil microbial community structure, restore the microecological balance, and simultaneously enhance the antioxidant capacity of melon plants, thus fundamentally solving the problem of continuous cropping obstacles in melons.

Description

Technical Field

[0001] This invention belongs to the field of soil improvement and crop cultivation technology in facility agriculture, specifically relating to a method for alleviating continuous cropping obstacles of melons using Trichoderma longifolia. Background Technology

[0002] Muskmelon is an important economic fruit and vegetable crop in my country, and continuous cropping is a common practice in greenhouse cultivation. However, continuous cropping of muskmelons can easily lead to serious continuous cropping obstacles, mainly manifested as deterioration of soil physical and chemical properties, imbalance of soil microbial community, and large accumulation of soil-borne pathogens (such as Fusarium oxysporum melon-specific strain), which in turn inhibits the growth of muskmelon plants, reduces their antioxidant capacity, and ultimately reduces the yield and quality of muskmelons.

[0003] Currently, methods to alleviate crop rotation obstacles mainly include soil disinfection, crop rotation, and the application of chemical fertilizers or pesticides. Soil disinfection is costly and easily disrupts the soil microecology; crop rotation is limited by planting structure and land resources, making it difficult to promote in greenhouse cultivation; excessive application of chemical fertilizers can further aggravate soil compaction and salinization, while chemical pesticides easily cause pesticide residues and kill beneficial soil microorganisms, disrupting the soil ecological balance. Therefore, developing a simple, environmentally friendly, and stable biological method to alleviate muskmelon rotation obstacles has become an urgent need for the sustainable development of the muskmelon greenhouse cultivation industry. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide a method for alleviating the obstacles of continuous cropping of melons using Trichoderma longifolia, addressing problems such as deterioration of soil physical and chemical properties, imbalance of microbial communities, accumulation of pathogens, and inhibited plant growth after continuous cropping of melons. This method achieves soil improvement and promotes plant growth in continuously cropped melon fields, and is simple to operate, environmentally friendly, and has stable application effects. The technical problem to be solved is not limited to the described technical subject matter; those skilled in the art will clearly understand other technical subjects not mentioned herein through the following description.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a method for inhibiting or alleviating continuous cropping obstacles of melons, including applying Trichoderma longifolia to the soil in which melons are continuously grown to inhibit or alleviate continuous cropping obstacles of melons.

[0007] The continuous cropping soil mentioned in the above method refers to soil that has been continuously planted with melons for 4 years.

[0008] In some embodiments, the continuous cropping obstacle is caused by *Fusarium oxysporum* melon-specific variant.

[0009] The method also includes applying Trichoderma longifolia to melon plants grown in the continuous cropping soil.

[0010] In the above method, the inhibition or alleviation of continuous cropping obstacles in melons includes at least one of the following: A1) Inhibits Fusarium oxysporum; A2) Increase plant height; A3) Increase leaf area; A4) Increase the number of leaves; A5) Increase the activity of urease, sucrase, and neutral phosphatase in the soil; A6) Increase the content of available potassium, total phosphorus, alkaline nitrogen, and available phosphorus in the soil; A7) Increases the activity of root antioxidant enzymes; A8) Regulate the microbial community in the root soil.

[0011] Available potassium is defined as potassium in the soil that can be directly absorbed and utilized by plants.

[0012] Total phosphorus is defined as the total amount of all forms of phosphorus in the soil.

[0013] Alkaline available nitrogen is defined as nitrogen in the soil that can be absorbed and utilized by the current crop, including inorganic nitrogen and easily decomposed organic nitrogen.

[0014] Available phosphorus is defined as the phosphorus component in the soil that can be absorbed and utilized by plants, also known as readily available phosphorus.

[0015] The present invention also provides the use of *Trichoderma longicornis* in inhibiting or alleviating continuous cropping obstacles in plants or in the preparation of products that inhibit or alleviate continuous cropping obstacles in plants, wherein the inhibition or alleviation of continuous cropping obstacles in plants is any one of the following: B1) Inhibits Fusarium oxysporum; B2) Increase plant height; B3) Increase the area of ​​plant leaves; B4) Increase the number of plant leaves; B5) Increases the activity of antioxidant enzymes in plant roots.

[0016] In the above application, the continuous cropping obstacle is caused by planting melons continuously for 4 years.

[0017] In the above application, the plant is melon, and the Fusarium oxysporum is the melon-specific Fusarium oxysporum.

[0018] The present invention also provides the application of *Trichoderma longicornis* in improving continuously cropped soil or in preparing products for improving continuously cropped soil, wherein the improved continuously cropped soil is at least one of the following: C1) Increases the activity of urease, sucrase, and neutral phosphatase in the soil; C2) Increase the content of available potassium and alkaline nitrogen in the soil; C3) Regulates the microbial community in the soil around plant roots; In the above applications, the plant is any of the following: G1) Dicotyledons; G2) Cucurbitales plants; G3) Cucurbitaceae plants; G4) Cucumber genus; G5) Melon.

[0019] Continuous cropping soil refers to soil in which melons have been planted for two or more consecutive crops.

[0020] In one embodiment, the continuous cropping soil is soil that has been continuously planted with melons for 4 years.

[0021] In some embodiments, the continuous cropping obstacle is caused by *Fusarium oxysporum* melon-specific variant.

[0022] The *Fusarium oxysporum* cultivar described in this article is the *Fusarium oxysporum* cultivar (melon-specific). Fusarium oxysporum f. sp. melonis ), number TG1605240301.

[0023] The Trichoderma longifolia mentioned in this article is Trichoderma longifolia ( Trichoderma longibrachiatum ).

[0024] This invention provides a method for alleviating continuous cropping obstacles in melons using *Trichoderma longicornis*. Through a scientific application method of the inoculant, it achieves multiple effects including antibacterial activity, growth promotion, soil improvement, microbial community regulation, and enhanced stress resistance. This method can significantly inhibit the core pathogen of continuous cropping melons, *Fusarium oxysporum* melon-specific strain, promote the growth of continuously cropped melon plants, improve the physical and chemical properties and biological activity of continuously cropped soil, regulate the soil microbial community structure, restore microecological balance, and simultaneously enhance the antioxidant capacity of melon plants, thus fundamentally solving the problem of continuous cropping obstacles in melons. It is suitable for soil improvement, plant growth promotion, and yield and quality enhancement in long-term continuous cropping of melons. Attached Figure Description

[0025] Figure 1 This study demonstrates the inhibitory effect of *Trichoderma longicornis* on *Fusarium wilt*, the pathogen of melon. The left side represents the control group (pathogen + sterile water), while the right side represents the treatment group (pathogen + *Trichoderma longicornis*).

[0026] Figure 2 The effect of *Trichoderma longifolia* treatment on the growth of continuously cropped melons. Note: From left to right in the figure, the treatments are CK, NC, and... T. longibrachiatum. (TL), the three basins are repeated three times in sequence.

[0027] Figure 3 The effects of Trichoderma longifolia treatment on the aboveground growth of continuously cropped melons.

[0028] Figure 4The effects of Trichoderma longifolia on soil enzyme activity in continuously cropped melons.

[0029] Figure 5 The effects of Trichoderma longifolia on the antioxidant system of melon roots after continuous cropping.

[0030] Figure 6 This is a Venn diagram showing the distribution of OTUs in soil fungal samples. Note: In Figure A, different colors represent different samples; overlapping numbers represent the number of species shared by each group, and non-overlapping numbers represent the number of species unique to each group; Figure B is a bar chart where the vertical axis represents the total number of OTUs in each sample.

[0031] Figure 7 This is a Venn diagram showing the distribution of OTUs in soil bacterial samples. Note: In Figure A, different colors represent different samples; overlapping numbers represent the number of species shared by each group, and non-overlapping numbers represent the number of species unique to each group; Figure B is a bar chart where the vertical axis represents the total number of OTUs in each sample.

[0032] Figure 8 The composition of the fungal community in the soil of continuous melon cropping at the phylum level.

[0033] Figure 9 The composition of the fungal community in the soil of continuous melon cropping at the genus level.

[0034] Figure 10 The composition of the soil bacterial community at the phylum level in muskmelon continuous cropping soil.

[0035] Figure 11 The composition of the soil bacterial community at the genus level for continuous cropping of melons.

[0036] Figure 12 The effect of microbial inoculants on soil fungal alpha diversity in melon-growing soil. Note: Analysis is performed at the p<0.05 level; no significant differences were found between groups without asterisks.

[0037] Figure 13 The effect of microbial inoculants on soil fungal beta diversity in melon-growing soil.

[0038] Figure 14 The effect of microbial inoculants on soil bacterial alpha diversity in melon-growing continuous cropping. Note: Analysis in the figure is performed at the p<0.05 level; there were no significant differences between groups without asterisks.

[0039] Figure 15 The effect of Trichoderma longifolia inoculant on soil bacterial beta diversity in melon-growing soil. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0043] Fusarium oxysporum melon-specific type ( Fusarium oxysporum f. sp. melonis Thin-skinned melon (item number TG611) (the melon material used in the following examples): This material was donated to the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, and is described in the non-patent literature "Study on the Targeted Control of Melon Wilt Disease by Bacillus vesicularis ZF2 and Its Mechanism of Action [D]. Chinese Academy of Agricultural Sciences, 2023-06-01". It is available to the public from Beijing University of Agriculture. This biological material is only used to replicate the relevant experiments of this invention and cannot be used for other purposes. Fusarium oxysporum is numbered TG1605240301 in the aforementioned literature.

[0044] Potato Dextrose Agar (PDA) medium, a commercially available product, was purchased from Cooler Master.

[0045] The microbial agent used in the experiment was *Trichoderma longifolia* ( Trichoderma longibrachiatum The bacterial agent was produced by Beijing Leili Marine Biological New Industry Co., Ltd., with the product number 207106.

[0046] Test soil: The continuous cropping soil used in the experiment had been planted with melons for 4 consecutive years. The conventional soil was taken from non-continuous cropping soil 50 meters away from the continuous cropping soil.

[0047] The experimental results of the following examples are expressed as mean ± standard deviation. A one-way ANOVA test was used, and P < 0.05 was considered satisfactory. () indicates a significant difference.

[0048] Example 1 I. Experimental Methods 1. Validation of the antifungal properties of Trichoderma longifolia To verify the effectiveness of the method of this invention, a confrontation experiment was first conducted in the laboratory: 0.5g of *Trichoderma longicornis* inoculum was diluted with 50ml of sterile water, and 0.1ml of the diluted solution was evenly spread on PDA medium. After the mycelium grew, the mycelial cakes (obtaining *Trichoderma longicornis* mycelial cakes) were collected using a microbial perforator and transferred to a new PDA medium for further cultivation. Then, the confrontation culture method was used to detect the inhibitory effect of *Trichoderma longicornis* on *Fusarium wilt*, the pathogen of melon. The pathogen mycelial cakes were placed in the center of the PDA plate, and *Trichoderma longicornis* mycelial cakes were inoculated at the four diagonal corners of the plate. The PDA plate inoculated only with pathogen mycelial cakes served as a control, and each treatment was repeated three times. The plates were incubated upside down at 28℃. After 10 days, the diameter of the pathogen colonies in the control and treatment groups was measured, and the inhibition rate was calculated.

[0049] Method for measuring the diameter of pathogenic bacteria: The diameter of pathogenic bacteria colonies is measured using the cross-cross method, and the average value is taken after three repetitions.

[0050]

[0051] 2. Melon seedling cultivation Soak melon seeds in 55℃ water for 30 minutes, let them cool naturally, and continue soaking for 6 hours. Remove the seeds and place them on moist filter paper, then keep them moist at 28℃ to promote germination. Once the seeds show white sprouts, sow them in 72-cell seedling trays and cultivate them until the seedlings have one leaf and one bud.

[0052] 3. Potted plant treatment and application of fungicides Seedlings of uniform growth were transplanted into pots containing continuously cropped soil, one seedling per pot. Each treatment was replicated three times, with three seedlings per replicate. The pot experiment included three treatments: CK (continuous cropped soil) with 50 ml of water (hereinafter referred to as the continuous cropped soil and water group); NC (conventional soil) with 50 ml of water (hereinafter referred to as the conventional soil and water group); and the inoculant treatment group (…). T. longibrachiatum The treatment group (hereinafter referred to as the *Trichoderma longifolia* treatment group, abbreviated as TL) was planted in continuously cropped soil. 50 ml of diluted *Trichoderma longifolia* inoculum was applied at 7, 15, 30, and 50 days post-treatment by soil drenching. The *Trichoderma longifolia* inoculum was prepared by diluting the inoculum (product number 207106) produced by Beijing Leili Marine Biological New Industry Co., Ltd., 800 times with sterile water.

[0053] 4. Indicator Measurement Sixty days after transplanting, the plant height, maximum leaf area, and number of leaves of the two groups of melons were measured. At the same time, the soil physicochemical properties (available potassium, total nitrogen, total phosphorus, total potassium, organic matter, alkaline nitrogen, available phosphorus, pH), soil enzyme activities (urease, sucrase, neutral phosphatase), effects on the antioxidant system of melon roots (malondialdehyde, proline, superoxide dismutase, peroxidase), and soil microbial community diversity were measured.

[0054] pH was determined using the electrode method (NY / T 1377-2007); Organic matter was determined by potassium dichromate titration method (external heating method, NY / T 1121.6-2006); Total nitrogen was determined by the semi-micro Kjeldahl method (NY / T 53-1987); Total phosphorus was determined using the alkali fusion-molybdenum antimony spectrophotometric method (HJ 632-2011); Total potassium shall be determined according to the method shown in NY / T 87-1988; Alkaline hydrolysate nitrogen was determined according to the method shown in LY / T 1228-2015; Available phosphorus was determined according to the method shown in NY / T 1121.7-2014; Available potassium and slow-acting potassium were determined by extraction-flame photometry according to (NT / 889-2004).

[0055] Soil urease determination method: Soil urease activity detection kit (Beijing Solarbio Science & Technology Co., Ltd., product number: BC0120) was used for detection.

[0056] Soil sucrase assay method: Soil sucrase activity assay kit (Beijing Solarbio Science & Technology Co., Ltd., catalog number: BC0240) was used for detection.

[0057] Soil neutral phosphatase assay method: Soil neutral phosphatase activity assay kit (Beijing Solarbio Science & Technology Co., Ltd., catalog number: BC0460) was used for detection.

[0058] Malondialdehyde (MDA) determination method: The MDA content detection kit (Beijing Solarbio Science & Technology Co., Ltd., product number: BC6410, upgraded version) was used for detection.

[0059] Proline determination method: Take 0.2 g of melon root sample in a mortar, add 1.5 ml of 5% sulfosalicylic acid, grind thoroughly into a homogenate, place in a 10 ml centrifuge tube, seal with sealing film, boil in a water bath for 10 min, cool, and centrifuge at 11500×g for 15 min. Mix 1 mL of the supernatant with 2 mL of acidic ninhydrin and 2 mL of glacial acetic acid in a test tube, shake well, boil in a water bath again for 30 min, cool naturally to room temperature, add 5 mL of toluene, shake thoroughly, and allow to separate into layers. Take the upper toluene extract in a cuvette and measure the absorbance at 520 nm. Calculate the proline (Pro) content using a standard curve.

[0060] Superoxide dismutase (SOD) assay method: The SOD activity assay kit (WST-1 method, Beijing Solarbio Science & Technology Co., Ltd., catalog number: BC5160) was used for detection. Refer to the instruction manual for specific steps.

[0061] Peroxidase assay method: The peroxidase activity assay kit (Beijing Solarbio Science & Technology Co., Ltd., catalog number: BC0090) was used for detection.

[0062] Methods for determining soil microbial community diversity: The samples were sent to Meiji Biotechnology Co., Ltd. for testing and analysis.

[0063] II. Experimental Results 1. Inhibitory effect of Trichoderma longifolia on Fusarium wilt pathogens of melon. The results of the confrontation culture experiment showed that the colony diameter of the pathogen in the control group was 7.10 cm (Table 1), while the colony diameter of the pathogen in the *Trichoderma longicornis* treatment group was 2.45 cm. *Trichoderma longicornis* achieved an inhibition rate of 65.49% against *Fusarium oxysporum* cultivar, demonstrating significant biocontrol and antibacterial activity. Figure 1 ).

[0064] Table 1. Inhibitory effect of Trichoderma longifolia on pathogens

[0065] Note: All data in the table are expressed as mean ± standard deviation; different lowercase letters in the same column indicate significant differences between groups at the (P<0.05) level.

[0066] 2. Effects of Trichoderma longifolia on the growth of continuously cropped melon plants Under greenhouse conditions, *Trichoderma longifolia* inoculant has a good mitigating effect on plant dwarfing caused by continuous cropping obstacles. Figure 2 The plant heights of the control (CK), plants grown in conventional soil, and plants treated with microbial agents were 16.61±1.34 cm, 38.22±4.20 cm, and 35.67±1.89 cm, respectively, with highly significant differences (P<0.001); the maximum leaf area was 31.11±1.74 cm², respectively. 255.14±4.07cm 2 51.61±5.70cm 2 The differences were highly significant (P<0.01); the number of leaves were 5, 9, and 9, respectively. Compared with the control (CK) Figure 3 The left image shows that the height of melon plants grown in conventional soil (middle image) increased by 130.10%, the maximum leaf area increased by 77.24%, and the number of leaves increased by 4. The right image shows that the height of melon plants treated with *Trichoderma longipes* increased by 114.75%, the maximum leaf area increased by 65.90%, and the number of leaves increased by 4. This indicates that the *Trichoderma longipes* agent described in this invention can significantly promote the growth of continuously cropped melons, effectively alleviate continuous cropping obstacles, and significantly improve plant height, leaf area, and number of leaves, demonstrating good application effects and promotional value.

[0067] 3. The effect of Trichoderma longifolia on improving the physical and chemical properties of continuously cropped soils In the *Trichoderma longifolia* treatment group (TL), the contents of available potassium, total phosphorus, alkaline nitrogen, and available phosphorus were 133.00 mg / kg, 3.28 g / kg, 50.71 mg / kg, and 199.65 mg / kg, respectively (Table 2), significantly higher than the control group (CK), increasing by 15.32%, 26.15%, 8.7%, and 19.04%, respectively. Total nitrogen, total potassium, and organic matter were all higher than the control group, while pH was lower, but the differences were not statistically significant. This indicates that the application of *Trichoderma longifolia* can optimize the soil fertility structure, providing a richer nutrient base for melon growth. Although the increases in total nitrogen, total potassium, and organic matter content, as well as the decrease in pH, were not statistically significant, this trend reflects, to some extent, a shift in the overall soil physicochemical environment towards a more favorable environment for melon growth, helping to address the problem of soil fertility decline under continuous cropping conditions.

[0068] Table 2. Effects of Trichoderma longifolia on soil physicochemical properties

[0069] Note: Data in the table are mean ± standard deviation. Different letters in the same column indicate significant differences at the P < 0.05 level.

[0070] 4. Effects of Trichoderma longifolia on enzyme activity in continuously cropped soil Compared with the control group (CK) Figure 4In conventional soil (NC), urease activity was slightly higher, but the difference was not statistically significant; however, neutral phosphatase (p<0.0001) and sucrase activities (p<0.001) were significantly higher than those in the control group. After treatment with *Trichoderma longicornis*, the activities of urease, neutral phosphatase, and sucrase in the soil were all higher than those in the control group, but the differences were not statistically significant. The application of *Trichoderma longicornis* (TL) can enhance the activity of various enzymes in the soil, which overall helps to enhance the soil's biological activity and nutrient transformation capacity, providing a more favorable soil micro-ecological environment for melon growth.

[0071] 5. The regulatory effect of Trichoderma longifolia on the antioxidant system of continuously cropped melon roots Compared with the continuous cropping control group (CK) ( Figure 5 In the conventional soil group (NC), the activities of peroxidase (POD) and superoxide dismutase (SOD) were significantly increased (POD: P < 0.001; SOD: P < 0.01), while the contents of proline (Pro) and malondialdehyde (MDA) were significantly decreased (Pro: P < 0.01; MDA: P < 0.05). In the *Trichoderma longifolia* treatment group, the activities of peroxidase (POD) and superoxide dismutase (SOD) were significantly increased (POD: P < 0.01; SOD: P < 0.001), while the contents of proline (Pro) and malondialdehyde (MDA) were significantly decreased (Pro: P < 0.001; MDA: P < 0.001).

[0072] Continuous cropping stress significantly inhibited the activity of antioxidant enzymes in plants and exacerbated membrane lipid peroxidation and proline accumulation. Treatment with Trichoderma longicornis effectively alleviated continuous cropping stress, significantly enhanced antioxidant capacity, and reduced oxidative damage.

[0073] 6. The regulatory effect of Trichoderma longifolia on the soil microbial community structure of continuously cropped melons 6.1 Effects of Trichoderma longifolia on the number of soil fungal OTUs OTUs (Operational Taxonomic Units) are artificially defined taxonomic markers used in phylogenetic, population genetics, and microbial ecology studies to simplify classification analysis and address the challenge of direct and accurate species identification. A total of 1222 OTUs were annotated across all samples. Specifically, 451 OTUs were annotated for continuously cropped soil (CK), 313 for conventional soil (NC), and 458 for the *Trichoderma longifolia* (TL) treatment group. The three groups combined contained 180 OTUs, with the number of OTUs ranging from 313 to 458 across the samples. Figure 6 ).

[0074] 6.2 Effects of Trichoderma longifolia on the number of soil bacterial OTUs A total of 13,889 OTUs were annotated across all samples, with 4,758 OTUs annotated in the continuous cropping soil (CK), 4,432 OTUs in the conventional soil (NC), and 4,699 OTUs in the Trichoderma longifolia (TL) treatment group. The four groups contained a total of 3,060 OTUs, with the number of OTUs ranging from 4,432 to 4,699 across the samples. Figure 7 ).

[0075] 6.3 Effects of microbial agents on soil fungal community composition High-throughput sequencing data from the samples were annotated and summarized at different taxonomic levels, totaling annotations for species in 14 phyla, 39 classes, 64 orders, 120 families, and 201 genera. At the phylum level ( Figure 8 The control group (CK) mainly consisted of Ascomycota, Basidiomycota, Glomeromycota, Chytridiomycota, Blastodiomycota, Mortierellomycota, and Olpidiomycota, with a total relative abundance of 87.63%. The blank control group (NC) mainly consisted of Ascomycota, Basidiomycota, and Mortierellomycota. The *Trichoderma* group consisted mainly of Ascomycota, Mortierellomycota, and Aphelidiomycota, with a total relative abundance of 86.82%. The *Trichoderma longifolia* (TL) treatment group was primarily composed of Ascomycota, Basidiomycota, Olpidiomycota, Chytridiomycota, and Blastodiomycota, with a total relative abundance of 88.82%. The relative abundance of each of these phyla was above 1%. The highest relative abundance was found in Ascomycota, accounting for 76.29%, 74.29%, and 74.12% in the CK, NC, TL, and TH treatment groups, respectively.

[0076] At the genus level, different treatments significantly regulated the composition of soil fungal communities in melon-growing continuous cropping systems. Figure 9 The dominant bacterial genera in the control group (CK) were *Basilella* (…). Talaromyces Aspergillus ( ) Aspergillus ), Gibellulopsis Genus, Cladosporium ( Cladosporium The dominant bacterial genera in the blank control group (NC) included *Pseudo-red-shell* (…). Emericellopsis ), Gibellulopsis Genus, genus *Ctenophora* ( Acremonium ), genus *Pyrophyllus* Lophotrichus ), Cladosporium ( Cladosporium The dominant genus in the *Trichoderma* (TL) treatment group was *Trichoderma*. The fungal community in continuously cropped soil differed significantly from that in conventional soil; continuous cropping led to the development of *Basilaria* genus. Talaromyces The soil is enriched with fungi of the genera *Pseudocarpus*, exhibiting high community complexity. *Pseudocarpus* species are also found in conventional soil. Emericellopsis ), Gibellulopsis The high proportion of genus indicates a more stable community structure. Trichoderma was the dominant genus in the TL treatment composition, with a relative abundance of 40.28%, indicating that Trichoderma successfully colonized and dominated the community, and may improve the soil microecology through antagonistic and growth-promoting effects.

[0077] 6.4 Effects of microbial agents on soil bacterial community composition High-throughput sequencing data from the samples were annotated and summarized at different taxonomic levels. For continuously cropped soils, species from 49 phyla, 140 classes, 313 orders, 527 families, and 1011 genera were annotated. For conventional soils, species from 45 phyla, 131 classes, 308 orders, 517 families, and 973 genera were annotated. For the *Trichoderma longicornis* treatment group, species from 49 phyla, 139 classes, 316 orders, 531 families, and 1022 genera were annotated.

[0078] At the level of the door ( Figure 10 The soil was mainly composed of Pseudomonas, Bacillota, Acidobacteriota, Chloroflexota, Actinomycetota, Bacteroidota, and Cyanobacteriota. These phyla accounted for 83.68%, 84.46%, and 82.01% of the total abundance in conventional soil, continuously cropped soil, and the *Trichoderma longicornis* treatment group, respectively. Among these, Bacillota had the highest abundance in conventional soil, accounting for 27.45%; while Pseudomonas had the highest abundance in continuously cropped soil, conventional soil, and the *Trichoderma longicornis* treatment group, accounting for 22.64% and 21.55%, respectively, representing increases of 51.74% and 44.44% compared to conventional soil (14.92%).

[0079] like Figure 11 As shown, at the genus level, there were significant differences in the dominant genera and taxa of the soil bacterial communities among the different treatment groups: in continuously cropped soils norank_o_Vicinamibacterales Sphingosomalidosis (Sphingosomalidosis) Sphingomonas ), norank_f_ Vicinamibacteraceae The relative abundance is relatively high, while the abundance of Bacillus spp. in conventional soil is relatively high. SporosarcinaThe abundance increased significantly. norank_o_Vicinamibacterales , norank_f_Vicinamibacteraceae The abundance was similar to that of continuously cropped soils, and at the same time, [the following occurred]. Paenibacillus, Mesobacillus Beneficial functional genera. Compared with continuously cropped soils, the community structure of the *Trichoderma longicornis* group changed significantly: the *Nitrifying Spirulina* genus (…) was present in the *Trichoderma longicornis* treatment group. Nitrospira ), Bryobacteria ( Bryobacter The abundance of beneficial microorganisms was significantly increased compared to soils with continuous cropping. In summary, continuous cropping stress leads to an imbalance in the soil bacterial community structure and a decrease in the abundance of beneficial functional genera. However, the application of Trichoderma longifolia can effectively alleviate the obstacles of continuous cropping of melons by regulating the relative abundance of core genera, optimizing the composition of the soil bacterial community, promoting the enrichment of beneficial microorganisms, and regulating the relative abundance of core genera.

[0080] 6.5 Alpha diversity analysis of soil fungal communities Alpha diversity reflects the diversity or richness of soil microbial communities. After applying microbial inoculants to soil continuously cropped with melons, the fungal community diversity showed corresponding changes. Figure 12 The *Trichoderma longicornis* treatment group showed higher Chao and Sobs indices, reflecting community richness, than the control group, indicating an increase in species abundance, but the differences were not statistically significant. The Shannon index, reflecting community diversity, decreased while the Simpson index increased, indicating a decrease in overall fungal community diversity and an increased dominance of a few dominant groups, but these differences also did not reach a statistically significant level. These changes in diversity indices suggest that *Trichoderma longicornis* treatment has a certain regulatory effect on the richness and diversity of fungal communities in continuously cropped soils, but it did not significantly alter the community structure.

[0081] 6.6 Beta diversity analysis of soil fungal communities β-diversity is used to measure the degree of difference in species composition among different samples. Principal coordinate analysis (PCoA) based on Bray-Curtis distance shows that ( Figure 13 The PC1 axis explained 37.36% of the community variation, and the PC2 axis explained 25.63%, with a cumulative explained rate of 62.99%, effectively reflecting the differences in fungal community structure among samples. The conventional soil control (NC) and continuous cropping control (CK) samples were completely separated on the PC1 axis, with no overlap of 95% confidence ellipses, indicating that continuous cropping significantly altered the rhizosphere fungal community structure of melon (P<0.05). Both the continuously cropped soil water group (CK) and the *Trichoderma longifolia* treatment group (TL) were completely separated from the CK group, forming independent clusters on the positive and negative PC2 axes, respectively, with no ellipse overlap between the two groups. This indicates that *Trichoderma longifolia* can significantly reshape the fungal community structure of continuously cropped soil, forming two entirely new community structures different from those of continuously cropped soil and healthy soil.

[0082] 6.7 Alpha diversity analysis of soil bacterial communities After applying microbial inoculants to soil continuously cropped with melons, soil bacterial community diversity showed a positive change. Figure 14 The Chao and Sobs indices of the *Trichoderma longifolia* treatment group were higher than those of the control group, reflecting an increase in the number of soil microbial species, but the differences were not statistically significant. The Shannon index was not significantly different from the control group, while the Simpson index was significantly lower (P<0.05), indicating that the application of microbial agents can effectively restore bacterial diversity in continuously cropped soils and further optimize community evenness, making the community structure more balanced. Overall, the changes in various diversity indices show that the richness and diversity of the bacterial community in continuously cropped soils treated with *Trichoderma longifolia* are shifting towards a healthier direction, significantly optimizing the evenness of the bacterial community, and playing a positive regulatory role in improving the bacterial community structure under continuous cropping obstacles.

[0083] 6.8 Analysis of Beta Diversity of Soil Bacterial Communities Principal coordinate analysis (PCoA) based on Bray-Curtis distance shows that ( Figure 15 The PC1 axis explained 50.62% of the community variation, and the PC2 axis explained 13.06%, for a cumulative explained rate of 63.68%, effectively reflecting the differences in bacterial community structure among samples. The continuous cropping control (CK) and the conventional soil control (NC) samples were completely separated on the PC1 axis, with no overlap of 95% confidence ellipses, indicating that continuous cropping significantly altered the rhizosphere bacterial community structure of melon (P<0.05). The *Trichoderma longicornis* treatment group (TL) was completely separated from the CK group, forming independent clusters, indicating that *Trichoderma longicornis* can significantly reshape the bacterial community structure of continuously cropped soils, and this community structure differs significantly from the healthy community in conventional soil.

[0084] In summary: 1. Significant antibacterial effect and strong targeting: The long-branch Trichoderma agent selected in this invention has an inhibition rate of 65.49% against Fusarium oxysporum melon-specific type, the main soil-borne pathogen of melon continuous cropping. It can effectively reduce the accumulation of soil pathogens and reduce the occurrence of soil-borne diseases such as melon wilt from the root.

[0085] 2. Comprehensive improvement of continuously cropped soil: The method of this invention can significantly optimize the physical and chemical properties of continuously cropped soil, increase the content of available nutrients in the soil, improve soil enzyme activity, optimize the structure of soil microbial community, alleviate the trend of soil fungalization, restore the soil micro-ecological balance, and achieve biological improvement of continuously cropped soil.

[0086] 3. Enhance the stress resistance of melons and promote plant growth: By regulating the antioxidant system of melon roots, the antioxidant capacity of plants is significantly improved, alleviating oxidative damage caused by continuous cropping stress; at the same time, it effectively promotes the growth of vegetative growth indicators such as melon plant height and leaf area, solving the problem of inhibited plant growth after continuous cropping.

[0087] 4. Environmentally friendly and highly safe: This invention uses biological agents as improvers, eliminating the problems of chemical pesticide and fertilizer residues, and will not damage the soil ecological environment, meeting the development requirements of green agriculture and ecological cultivation.

[0088] 5. Simple operation and easy to promote: The method of this invention does not require complicated equipment and processes. Seedling raising, transplanting, and application of microbial agents are all integrated with conventional melon cultivation and management. The production cost is low and it is easy to promote and apply on a large scale in melon facility continuous cropping cultivation.

[0089] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method of suppressing or alleviating a continuous cropping barrier of melon, characterized by, This includes applying Trichoderma longifolia to the soil where melons are continuously grown to inhibit or alleviate the continuous cropping obstacles of melons.

2. The method of claim 1, wherein, The soil in question is soil that has been continuously planted with melons for four years.

3. The method according to claim 1 or 2, characterized in that, The inhibition or relief of continuous cropping obstacles in melons includes at least one of the following: A1) Inhibits Fusarium oxysporum; A2) Increase plant height; A3) Increase the leaf area; A4) Increase the number of leaves; A5) Increase the activity of urease, sucrase, and neutral phosphatase in the soil; A6) Increase the content of available potassium, total phosphorus, alkaline nitrogen, and available phosphorus in the soil; A7) Increases the activity of root antioxidant enzymes; A8) Regulate the microbial community of the root soil.

4. The use of *Trichoderma longicornis* in inhibiting or alleviating continuous cropping obstacles in plants or in the preparation of products that inhibit or alleviate continuous cropping obstacles in plants, wherein the inhibition or alleviation of continuous cropping obstacles in plants is any one of the following: B1) Inhibits Fusarium oxysporum; B2) Increase plant height; B3) Increase the area of ​​plant leaves; B4) Increase the number of plant leaves; B5) Increases the activity of antioxidant enzymes in plant roots.

5. The application according to claim 4, characterized in that, The plant is any one of the following: G1) Dicotyledons; G2) Cucurbitales plants; G3) Cucurbitaceae plants; G4) Cucumber genus; G5) Melon.

6. The application according to claim 5, characterized in that, The continuous cropping obstacle was caused by planting melons continuously for 4 years.

7. The application according to claim 6, characterized in that, The plant is melon, and the Fusarium oxysporum is a melon-specific variant of Fusarium oxysporum.

8. The application of *Trichoderma longifolia* in improving continuously cropped soil or in preparing products for improving continuously cropped soil, wherein the improved continuously cropped soil is at least one of the following: C1) Increases the activity of urease, sucrase, and neutral phosphatase in the soil; C2) Increase the content of available potassium, total phosphorus, alkaline nitrogen, and available phosphorus in the soil; C3) Regulates the microbial community in the soil around plant roots; C4) Increase the number of beneficial bacteria in the soil.

9. The application according to claim 8, characterized in that, The plant is any one of the following: G1) Dicotyledons; G2) Cucurbitales plants; G3) Cucurbitaceae plants; G4) Cucumber genus; G5) Melon.

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