Trichoderma longibrachiatum strain tv26, microbial inoculum comprising same and use thereof

Abstract

The present application relates to a strain of Trichoderma longibrachiatum TV26, a microbial agent comprising the same and an application thereof. The strain of Trichoderma longibrachiatum TV26 has a high and persistent antagonistic effect on Pseudomonas lachrymans, and can play a key role in the biological control of cucumber spot disease. Further, through the study of its endogenous metabolites, it is found that the volatile metabolite of turanose as a synergist can significantly improve the colonization ability of the strain on cucumber leaves, so as to be used for developing a more efficient compound microbial agent. Whether the strain of the present application or the compound microbial agent thereof has the characteristics of high efficiency, persistence, safety and environmental protection when used for antagonizing Pseudomonas lachrymans and preventing cucumber spot disease caused by Pseudomonas lachrymans, and therefore has high industrial value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial and biological pesticide technology, specifically relating to a Trichoderma longifolia strain TV26, a fungal agent containing it, and its application. Background Technology

[0002] Mycorrhizal fungus (Phyllosticta cucurbitacearum Sacc. Cucumber spot disease (C. spp.) is a typical plant pathogenic fungus that primarily infects cucurbitaceous crops, causing a disease characterized by "spot disease," with the most significant damage occurring in cucumbers. Cucumber spot disease caused by this pathogen is a devastating foliar disease in cucumber cultivation: in the early stages, small pale yellow spots appear on the leaves; as the disease progresses, these spots gradually enlarge into yellowish-brown circular lesions with a sunken center and clear edges; in severe cases, multiple lesions merge, causing leaves to wither and fall off, resulting in a sharp drop in crop photosynthetic efficiency and ultimately severely restricting cucumber yield and quality.

[0003] Currently, the control of cucumber spot disease still heavily relies on chemical pesticides, such as 75% chlorothalonil wettable powder, 80% mancozeb wettable powder, and 25% pyraclostrobin emulsifiable concentrate. Although these chemical pesticides achieve disease control effects in the short term, long-term use can easily lead to multiple problems: First, they can cause resistance in cucumber gray sclerotium, significantly reducing the effectiveness of subsequent control; second, they can cause pesticide residues in agricultural products to exceed standards, threatening food safety; and third, they can disrupt the soil microecological balance, causing environmental risks.

[0004] In contrast, research on efficient and environmentally friendly biological control technologies for cucumber leaf spot disease is still insufficient, and related reports are extremely scarce. Furthermore, among existing fungal biological agents, especially foliar sprays, the stability of their field application effects is often unsatisfactory. The fundamental reason for this lies primarily in their weak colonization ability in the foliar environment—after spraying, the fungal agent struggles to effectively establish a population and survive sustainably in the complex environment of the leaf surface, resulting in a lack of long-lasting control or growth-promoting effects. Therefore, exploring safe, efficient, and environmentally friendly biological control methods for cucumber leaf spot disease is urgently needed.

[0005] Trichoderma longifolia ( Trichoderma longibrachiatum It is one of the most promising bacteria for the biological control of plant diseases. It has been reported to play a role in a variety of plant diseases, but there are no reports of its use in the control of the above-mentioned pathogens and the plant diseases caused by them. Summary of the Invention

[0006] Purpose of the invention

[0007] To address the problems of existing chemical pesticide control methods for cucumber spot disease caused by *A. cucumeroides*, such as easy development of pesticide resistance, excessive pesticide residues in agricultural products, significant environmental impact, and poor colonization ability of biological agents in crop foliage, this invention provides a *Trichoderma longicornis* strain TV26, an inoculum containing it, and its applications. The *Trichoderma longicornis* strain TV26 of this invention exhibits highly effective antagonism against *A. cucumeroides* and control of cucumber spot disease caused by *A. cucumeroides*, playing a crucial role in the biological control of crops.

[0008] Furthermore, this invention, through research on the endogenous metabolites and activities of the *Trichoderma longicornis* strain, discovered that menobiose, one of its metabolites, can act as a synergist to significantly enhance the field application efficacy of *Trichoderma longicornis* strain TV26. Specifically, menobiose can promote the colonization of *Trichoderma longicornis* strain TV26 on the surface of cucumber leaves, thereby enhancing the strain's antagonistic effect against cucumber leaf spot pathogen *Agrostis spp.*, effectively solving the problem of poor field control efficacy of biological agents due to weak colonization ability.

[0009] Solution

[0010] To achieve the objectives of this invention, the following technical solution is provided.

[0011] In a first aspect, the present invention provides a Trichoderma longifolia strain TV26, the taxonomic name of which is... Trichoderma longibrachiatum It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 21, 2025, with accession number CGMCC NO.42133.

[0012] The *Trichoderma longifolia* of this invention was isolated from the Forest Protection Institute Laboratory of the Shandong Academy of Forestry Sciences. The isolation source was wild *Auricularia auricula-judae* (collected near a reservoir in Rizhao City). Through comparative experiments, the inventors discovered that the isolated *Trichoderma longifolia* (… Trichoderma longibrachiatum The strain TV26 has an antagonistic effect against Azolla mellea, and has a strong control effect on cucumber spot disease caused by Azolla mellea, and has the function of effectively inhibiting the pathogen of cucumber spot disease.

[0013] The long-branched Trichoderma of the present invention ( Trichoderma longibrachiatum Strain TV26 was identified as *Trichoderma longichair* through morphological identification, physiological and biochemical characterization, and 16S rRNA sequencing. Trichoderma longibrachiatum The 16S rRNA gene sequence is shown in SEQ ID NO: 1.

[0014] The colony color of the *Trichoderma longicornis* strain TV26 is grayish-green, with a light brown reverse side, and it contains no water-soluble pigments. Furthermore, the *Trichoderma longicornis* (… Trichoderma longibrachiatum TV26 has conidiophores that are not clearly specialized and are slender, while its conidia are oblong or short columnar, and more preferably, the surface of the conidia is smooth.

[0015] In a second aspect, the present invention provides a fungicide comprising at least one selected from the group consisting of *Trichoderma longicornis* as described in the first aspect above. Trichoderma longibrachiatum Live, freeze-dried, inactivated cells, and cultures of strain TV26.

[0016] Optionally, the strain culture is the fermentation broth of the strain, the fermentation broth supernatant, and / or the aforementioned concentrate, dried product, or volatile product.

[0017] The formulation of the microbial agent is a conventional formulation in the art, which may be a liquid or solid preparation. The choice of formulation can be adjusted according to the application requirements, and is preferably a liquid or powder preparation.

[0018] In a preferred embodiment, the microbial agent further comprises synergists and / or excipients.

[0019] In a preferred embodiment, the synergist promotes the colonization of Trichoderma longifolia strain TV26 on the surface of the crop to be applied.

[0020] Optionally, the synergist is the Trichoderma longifolia ( Trichoderma longibrachiatum The volatiles of the fermentation broth of strain TV26, preferably, the synergist is menobiose.

[0021] In some preferred embodiments, the long-branched Trichoderma ( Trichoderma longibrachiatum The effective viable count of strain TV26 in the inoculum is ≥2.5 billion / g.

[0022] In some preferred embodiments, the synergist is menobiose, which has a mass percentage content of 0.5% to 1.5% in the bacterial agent.

[0023] Optionally, the crop surface to be applied is the surface of a cucumber leaf.

[0024] Optionally, the excipients are selected from one or more of high-mesh diatomaceous earth, glucose, starch, amino acid powder, sodium dodecyl sulfonate, and sodium dodecyl sulfate.

[0025] In some preferred embodiments, the glucose content is 1-1.5% by mass.

[0026] In some preferred embodiments, the starch content is 3-4.5% by mass.

[0027] In some preferred embodiments, the amino acid powder has a mass percentage content of 0.1-0.4%.

[0028] In some preferred embodiments, the sodium dodecyl sulfonate or sodium dodecyl sulfate has a mass percentage of 5-7%.

[0029] Thirdly, the present invention provides the application of the Trichoderma longifolia strain TV26 as described in the first aspect above or the fungal agent as described in the second aspect above in the preparation of biological pesticides.

[0030] In a feasible implementation, the biopesticide is used for any one or more of the following purposes:

[0031] (1) Used to antagonize *Gnaphalium affine*;

[0032] (2) Used to prevent and control cucumber spot disease caused by sclerotium tumefaciens.

[0033] Fourthly, the present invention provides the use of live, freeze-dried, inactivated strains and / or strain cultures of the *Trichoderma longicornis* strain TV26 as described in the first aspect above (optionally, the strain culture is the fermentation broth, fermentation supernatant and / or the aforementioned concentrate, dried product or volatile product), and / or menobiose in the preparation of pesticides for antagonizing *Trichoderma graminearum*.

[0034] Fifthly, the present invention provides the use of live, freeze-dried, inactivated cells and / or strain cultures of the *Trichoderma longicornis* strain TV26 as described in the first aspect above (optionally, the strain culture is the fermentation broth, fermentation supernatant and / or the aforementioned concentrate, dried product or volatile product of the strain), and / or menobiose in the preparation of pesticides for controlling cucumber spot disease caused by *Acer calamus*.

[0035] In a sixth aspect, the present invention provides a method for antagonizing *Trichoderma longicornis* and / or controlling cucumber spot disease caused by *Trichoderma longicornis*, the method comprising: applying to the plant (particularly its leaves) or its growing medium (e.g., soil) an effective amount of live, freeze-dried, inactivated bacterial cells and / or strain culture of *Trichoderma longicornis* strain TV26 as described in the first aspect above (optionally, the strain culture is the fermentation broth, fermentation supernatant and / or the aforementioned concentrate, dried product or volatile product of the strain), and / or menobiose.

[0036] Beneficial effects

[0037] (1) The Trichoderma longifolia strain TV26 of the present invention has excellent and efficient antagonistic effect against Acer flavomarginata, and its inhibition rate against Acer flavomarginata reaches 82.38%; its sterile fermentation broth has an inhibition rate against Acer flavomarginata of 63.07%, and its volatile products have an inhibition rate against Acer flavomarginata of 80.74%, playing a key role in the biological control of crops.

[0038] (2) Research on the active metabolites of the *Trichoderma longicornis* strain TV26 of this invention revealed that menobiose can act as a synergist. Specifically, menobiose can promote the colonization of *Trichoderma longicornis* strain TV26 on cucumber leaves, enabling it to better control cucumber spot disease caused by *Acer maculatum*, thus exhibiting excellent synergistic effects. The application of menobiose as a synergist improved the colonization effect of *Trichoderma longicornis* strain TV26 on cucumber leaf surface. The synergist of this application is directly extracted from the metabolites of *Trichoderma longicornis* strain TV26 without adding exogenous substances, avoiding secondary pollution sources and providing a new approach for the efficient field application of microbial preparations.

[0039] (3) The biological agent of the present invention containing Trichoderma longifolia strain TV26 and mesobiose has excellent effect in preventing and controlling cucumber spot disease. Compared with chemical agents, the inhibition rate is increased by 10% or more. Attached Figure Description

[0040] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the implementation. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0041] Figure 1 This describes the colony morphology of strain TV26.

[0042] Figure 2 These are the microscopic characteristics of strain TV26.

[0043] Figure 3 This is the phylogenetic tree of strain TV26. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprising of," etc., will be understood to include the stated elements or components, and does not exclude other elements or other components.

[0045] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0046] Example 1: Rapid isolation and purification of Trichoderma longifolia strain TV26

[0047] The *Trichoderma longicornis* strain TV26 involved in this invention was isolated in June 2017 at the Forest Protection Institute of the Shandong Academy of Forestry Sciences. The isolation source was wild *Auricularia auricula-judae* (collected near Rizhao Reservoir in Rizhao City). The isolation method employed was the dilution coating method. The specific isolation steps are as follows:

[0048] (1) Sample pretreatment: The collected wood ear fungus was rinsed three times with 75% alcohol for 2-3 minutes each time, then rinsed three times with sterile water, and dried in a clean bench.

[0049] (2) Preparation of sample suspension: Accurately weigh 3-5g of processed wood ear fungus, grind it thoroughly in a sterile mortar, and add it to an Erlenmeyer flask containing 100mL of sterile water (containing glass beads). Add 1-2 drops of Tween 80. Place the Erlenmeyer flask at 30℃ and 150r / min and shake it for 20-30min to make the fungi uniformly free in the sterile water, thus preparing the sample suspension.

[0050] (3) Preparation of toxin-containing plates: *Staricella mellea* (the pathogen was isolated from diseased leaves using the dilution plating method and verified by Koch's postulate) was inoculated onto PDA medium plates and activated by incubation at 28°C for 3-5 days. After activation, the *Staricella mellea* strain was inoculated into PDB medium and incubated at 28°C and 150 r / min with constant temperature shaking for 3-5 days. The mycelium was filtered using sterile gauze to obtain the *Staricella mellea* spore fermentation broth. The spore fermentation broth was inoculated at 1% in a modified PDA medium at 40-45°C, shaken well, and quickly poured onto plates to prepare toxin-containing plates.

[0051] Both PDA and PDB media were commercial products purchased from Beijing Aoboxin; the modified PDA media was made by adjusting the pH to around 6.0 based on the commercial PDA media.

[0052] (4) Dilution and coating: Use a pipette to add 1 mL of sample suspension to a test tube containing 9 mL of sterile water to prepare a dilution of 10. -1 The bacterial suspension; and so on, diluted to 10... -3 Dilution: In a clean bench, 100 μL of bacterial suspension at each dilution was spread onto a toxic plate, with three plates for each dilution.

[0053] (5) Culture: Place the coated plates in an incubator at 28°C and culture for 3-5 days. Observe the colony morphology of the newly grown strain regularly to screen for target strains. If a clear zone appears, it can be preliminarily determined that the strain can inhibit *Gnaphalium affine*, and thus it is the target strain. At the same time, the target strain can also be determined based on its unique colony morphology and strain growth rate.

[0054] (6) Purification: The target strain is purified by single-cell culture, and cultured at 28°C inverted position until a single colony grows on the plate; the purification medium is PDA medium.

[0055] (7) Preservation: Inoculate the purified single colony into a PDA medium test tube, incubate at 28℃ for 3-5 days, and then store at 4℃ for later use.

[0056] The results showed that, after rapid isolation, based on different colony morphologies, clear zones, and growth rates, 30 strains were initially isolated and numbered TV01 to TV30.

[0057] Example 2: Screening of TV26, an antagonistic bacterium with highly efficient antagonism against *Gnaphalium affine*

[0058] 1. Detection of antagonistic effects of different strains against *Gnaphalium affine*

[0059] The inhibitory effect of strains TV01~TV30 (hereinafter referred to as test strains) obtained in Example 1 on the growth of *Stellaria media* was detected by plate confrontation culture method.

[0060] The activated pathogen (i.e., *Asterix mellea*) and the test bacteria were separately inoculated into 0.5 cm mycelial cakes. Two points (on the same horizontal line) 2.5 cm from the center of the PDA plate were selected for inoculation of the pathogen and the test bacteria, respectively. The plates were incubated at 28℃ and recorded as the experimental group. The control group was treated with PDA medium instead of the test bacteria. Each treatment was repeated 3 times. When the pathogen colonies in the control group almost covered the plate, the growth radius of the test bacteria and the pathogen colonies in the experimental group was recorded and the inhibition rate was calculated. The results are shown in Table 1.

[0061] Inhibition rate = [(Coronavirus colony radius in control group - Coronavirus colony diameter in experimental group)] / Coronavirus colony radius in control group × 100%.

[0062] Table 1. Inhibitory effects of different tested bacteria on *Staricella cuspidatum*

[0063] ;

[0064] Note: "-" indicates no antagonistic effect; "+" indicates 0 < inhibition rate ≤ 60%; "++" indicates 60 < inhibition rate ≤ 80%; "+++" indicates inhibition rate > 80%.

[0065] The results are analyzed as follows: According to the results in Table 1, among the 30 strains tested, 23 strains had an inhibition effect of less than 60% against *Azolla mellea*, 6 strains had an inhibition effect between 60% and 80%, and 1 strain had an inhibition effect of more than 80%, that is, strain TV26 had an inhibition effect of 82.38% against *Azolla mellea*.

[0066] 2. Detection of the antagonistic effect of aseptic fermentation filtrate of different strains on *Stellaria media*

[0067] The plate dilution method was used to detect the inhibitory effect of the sterile fermentation filtrate of strains TV01~TV30 (hereinafter referred to as the test strains) obtained in Example 1 on the growth of *Staricella cuspidatum*.

[0068] The activated test bacteria were inoculated into 0.5cm mycelial cakes in PDB medium and cultured with shaking at 28℃ and 150 rpm for 3 days. After culture, the fermentation broth of the test bacteria strains was filtered through four layers of sterile gauze to remove most of the mycelium. The broth was centrifuged at 4℃ and 8000 rpm for 10 min, and the supernatant was collected and filtered three times through a 0.22μm microporous membrane to prepare sterile fermentation filtrates for each test bacterial strain. These filtrates were stored at 4℃ for later use. When the sterilized PDA medium cooled to 55℃, the sterile fermentation filtrates of the test bacteria were mixed with the medium at a ratio of 1:9 (v / v) and poured onto a plate. After cooling, the activated and inoculated (0.5cm) *Staricella mellea* strains were inoculated in the center of the plate, designated as the experimental group. An equal volume of PDB and PDA mixed medium was used as the control group. All plates were incubated at 28℃. When the pathogen colonies in the control group expanded to the edge of the plate, the diameter of the pathogen colonies in each experimental group was measured, and the inhibition rate was calculated. Each treatment was set to 3 replicates, and the results are shown in Table 2.

[0069] Inhibition rate (%) = (Coronavirus colony diameter in control group - Coronavirus colony diameter in experimental group) / Coronavirus colony diameter in control group × 100.

[0070] Table 2. Inhibitory effect of sterile fermentation filtrate of different test bacteria on *Staricella cuspidatum*

[0071] ;

[0072] Note: "-" indicates no antagonistic effect; "+" indicates 0 < inhibition rate ≤ 40%; "++" indicates 40 < inhibition rate ≤ 60%; "+++" indicates 60 < inhibition rate ≤ 80%; "++++" indicates inhibition rate > 80%.

[0073] The results are analyzed as follows: Among the 30 sterile fermentation filtrates tested, the sterile fermentation filtrates of 3 strains showed no inhibitory effect on *Helicobacter pylori*, the sterile fermentation filtrates of 15 strains showed an inhibitory effect of 0-40% on *Helicobacter pylori*, the sterile fermentation filtrates of 11 strains showed an inhibitory effect of 40-60% on *Helicobacter pylori*, and only the sterile fermentation filtrate of 1 strain showed an inhibitory effect of 60-80% on *Helicobacter pylori*, that is, the sterile fermentation filtrate of strain TV26 showed an inhibitory effect of 63.70% on *Helicobacter pylori*.

[0074] 3. Detection of the antagonistic effect of volatile products of different strains on *Gnaphalium affine*

[0075] The inhibitory effect of the volatile products of strains TV01~TV30 (hereinafter referred to as test strains) obtained in Example 1 on the growth of *Stellaria media* was detected by plate-to-plate method.

[0076] Activated pathogens and test bacteria were cultured in 0.5 cm cakes and inoculated separately in the center of PDA plates. The plate inoculated with pathogens was then inverted onto the plate inoculated with test bacteria, sealing the center gap. This was designated as the experimental group. The control group used PDA culture medium cakes instead of test bacteria and was incubated in the same manner. All treatments were incubated in a 28°C incubator, with three replicates for each treatment. When the pathogen colonies in the control group expanded to the edge of the plate, the diameter of the pathogen colonies in each experimental group was measured, and the inhibition rate was calculated. The results are shown in Table 3.

[0077] Inhibition rate (%) = (Coronavirus colony diameter in control group - Coronavirus colony diameter in experimental group) / Coronavirus colony diameter in control group × 100.

[0078] Table 3. Inhibitory effects of volatile products from different tested bacteria on *Stellaria media*

[0079] ;

[0080] Note: "-" indicates no antagonistic effect; "+" indicates 0 < inhibition rate ≤ 40%; "++" indicates 40 < inhibition rate ≤ 60%; "+++" indicates 60 < inhibition rate ≤ 80%; "++++" indicates inhibition rate > 80%.

[0081] The results are analyzed as follows: Among the volatile products of the 30 strains tested, the volatile products of 4 strains had no inhibitory effect on *Aegilops mellea*, the volatile products of 12 strains had an inhibitory effect on *Aegilops mellea* between 0% and 40%, the volatile products of 8 strains had an inhibitory effect on *Aegilops mellea* between 40% and 60%, the volatile products of 5 strains had an inhibitory effect on *Aegilops mellea* between 60% and 80%, and only 1 strain had an inhibitory effect on *Aegilops mellea* above 80%, that is, the volatile product of strain TV26 had an inhibitory effect on *Aegilops mellea* of 80.74%.

[0082] Based on the combined inhibitory effects of the 30 tested strains themselves, sterile fermentation filtrate, and volatile products on *Staricella cuspidatum*, strain TV26 showed highly efficient inhibition of *Staricella cuspidatum*.

[0083] Example 3: Identification of strain TV26

[0084] 1. Colony morphology and microscopic characteristics

[0085] like Figure 1 As shown, strain TV26 grows rapidly on malt extract agar (MEA) medium. After 5 days in the dark at 25°C, the colony diameter is 50-55 mm, grayish-green, flocculent in texture, and the sporulation area is evenly distributed. The reverse side of the colony is light brown and has no water-soluble pigment.

[0086] like Figure 2 As shown, the conidiophores of strain TV26 are not clearly specialized, are slender, and have simple branches in the middle and lower parts, with a width of 2.5~4.0μm; the conidiophores are flask-shaped, straight or curved, and grow on the conidiophores or their branches, with a size of 4.5~15.3×1.5~3.0μm; the conidia are oblong or short columnar, with a size of 3.5~5.8×2.0~3.5μm and a smooth surface.

[0087] 2. Gene sequencing results

[0088] Strains TV26 were sent to the Institute of Microbiology, Chinese Academy of Sciences for gene identification, including the gene sequence fragment of transcription elongation factor protein, as shown below:

[0089]

[0090] The sequenced sequences were compared with known sequences stored in the public databases GenBank and NCBI. A phylogenetic tree was constructed using the neighbor-joining method in MEGA version 7.0, as follows: Figure 3 As shown. It is clearly visible that strain TV26 and... Trichoderma longibrachiatum (DQ297067.1) forms an independent multi-branch, with the closest kinship.

[0091] Therefore, based on the colony morphology, microscopic characteristics, and sequence characteristics of strain TV26, strain TV26 was identified as *Trichoderma longicornis*. Trichoderma longibrachiatum This strain was deposited at the China General Microbiological Culture Collection Center on July 21, 2025, with accession number CGMCC NO.42133.

[0092] Example 4: Detection of the colonization-promoting effect of metabolic active products of strain TV26

[0093] Example 2 confirmed that the aseptic fermentation filtrate of strain TV26 had a certain antagonistic effect on *Agropyron cristatum*. To this end, LC-MS was used to analyze the main components of the metabolically active products of strain TV26, revealing 242 components in 9 major categories: 101 organic acids and their derivatives, 2 alkaloids and their derivatives, 3 phenylpropanoids and polyketides, 42 organic heterocyclic compounds, 23 organic oxygen-containing compounds, 31 lipids and lipid molecules, 16 nucleoside nucleotides and analogs, 8 organic nitrogen-containing compounds, and 16 benzene ring compounds. Based on the detection results, 20 pure samples of water-soluble and relatively abundant metabolically active products were selected to test their effect on the colonization ability of strain TV26 on cucumber leaf surfaces. The specific steps are as follows:

[0094] The activated strain TV26 was inoculated into liquid BPY medium and cultured at 28℃ and 150 r / min for 3 days to produce an effective ingredient concentration of 1×10⁻⁶. 8 CFU / mL standard spore mixture. Twenty purified metabolites were dissolved in the standard spore mixture to a final concentration of 0.05%. The solution was sprayed evenly onto both sides of the leaves of cucumber potted seedlings (the seedlings were in the early stages, raised in trays, and transplanted 15 days later). Each seedling was sprayed with 10 mL of the solution, and three seedlings were sprayed per treatment, designated as the experimental group. The control group received the standard spore mixture. Seven days after treatment, cucumber leaves from each treatment were randomly selected and rinsed several times with sterile water. 5 g of the solution was accurately weighed and ground into a paste with 10 mL of sterile water in a mortar. The paste was then transferred to an Erlenmeyer flask containing glass beads and brought to a final volume of 100 mL. The paste was then diluted 10 times using the 10-fold dilution method. -31 mL of the sample was spread onto a selective medium, with each gradient repeated three times. After spreading, the samples were incubated at 28°C for 3 days, and the colonies on the surface of the medium were counted. The bacterial count (CFU) per gram of fresh tissue was calculated, and the results are shown in Table 4.

[0095] Bacterial count (CFU / g) = number of colonies on plate × dilution factor × 100mL / 5g.

[0096] Liquid BPY medium: 5 g / L beef extract, 10 g / L peptone, 5 g / L yeast extract, 5 g / L glucose, 5 g / L sodium chloride, pH 6.8~7.0.

[0097] Selective culture medium: 5 g / L beef extract, 10 g / L peptone, 5 g / L yeast extract, 5 g / L glucose, 5 g / L sodium chloride, 300 μg / mL spectinomycin, 20 g / L agar, pH 6.8–7.0. This culture medium was determined to be suitable for screening strain TV26 through antibiotic resistance testing.

[0098] Table 4. Effects of different metabolites on the colonization effect of strain TV26

[0099] ;

[0100] The results were analyzed as follows: Among the 20 pure metabolites tested, only when a final concentration of 0.05% melinotose was added to the TV26 standard spore mixture did the colonization effect of strain TV26 on cucumber leaf surface significantly improve, from 2.8 × 10⁻⁶. 3 CFU / g increased to 3.5×10 6 CFU / g, and other metabolites did not significantly enhance the colonization effect of strain TV26. This indicates that an appropriate concentration of menobiose can significantly improve the colonization ability of TV26 on plant leaf surfaces. It is further speculated that menobiose can act as a functional adjuvant, playing an important role in promoting the better disease resistance function of strain TV26.

[0101] Example 5: Preparation of biological agent from strain TV26

[0102] 1. Deep liquid fermentation of strain TV26

[0103] (1) Activation of strain

[0104] Take a small piece of the purified TV26 culture from the test tube in Example 1 out of the refrigerator and inoculate it onto a PDA medium plate in a clean bench. Incubate at 28-30°C for 36-48 hours. Observe for contamination. If contamination is found, purification needs to be continued until a pure culture single colony is obtained.

[0105] PDA: 200g potatoes (boiled), 20g glucose, 15-20g agar, 1000mL water, natural pH.

[0106] (2) Shake flask seed preparation

[0107] After activation of strain TV26, take the plate with the fastest growth as the seed plate; use an inoculation hook to take a small piece from the edge of the colony and inoculate it again on the PDA medium plate, and incubate at 28~30℃ for 36~48h; after the plate has grown well, use an inoculation spatula to take a large piece from the edge of the colony and inoculate it in PDB medium, and incubate at 28~30℃ and 120~150r / min for 20~26h to prepare the shake flask seed.

[0108] PDB: 200g potatoes (cooked into juice), 20g glucose, 1000mL water, natural pH.

[0109] (3) Liquid fermentation

[0110] Seed tank fermentation: Following the seed tank formula, the ingredients are added and sterilized using normal industrial sterilization methods. Seeds are then introduced into a shake flask and fermented for 20-24 hours at a temperature of 28-30℃, a rotation speed of 100-120 r / min, and an aeration ratio of 1:0.8. The fermentation process is complete. The gas used is sterile air that has undergone three stages of filtration.

[0111] The fermentation medium formula for the seed tank is as follows (by mass percentage): 1-2.5% soybean meal powder, 2-3% corn flour, 0.1-0.5% yeast extract, 0.01-0.5% potassium dihydrogen phosphate, 0.1-0.3% dipotassium hydrogen phosphate, 0.01-0.1% sodium carbonate, 0.01-0.07% manganese sulfate, and the remainder is water.

[0112] Expanded fermentation: Add materials according to the expanded culture formula, sterilize according to normal industrial sterilization method, and then add the fermentation liquid to the seed tank. Ferment for 30-36 hours at a temperature of 28-30℃, a rotation speed of 100-120r / min, and an aeration ratio of 1:0.8 to complete the expanded fermentation.

[0113] This completes the deep liquid fermentation process of strain TV26.

[0114] 2. Preparation of TV26 strain mother powder (solid-state fermentation)

[0115] (1) Formula and ingredients

[0116] Formula: Corn flour 13.5-15%, rice husk powder 55.5-58.0%, wood flour 19-21%, wheat bran 2.5-3%, soybean meal powder 2.5-3.5%, calcium carbonate 1.0-1.5%, potassium dihydrogen phosphate 0.3-0.5%. After adding water for half an hour, squeeze the mixture by hand until water seeps out between your fingers but does not drip; it should drip slightly. The final moisture content should be maintained at 45-50%. After sterilization, maintain the pH at 6-6.5, preferably close to 6.5.

[0117] (2) Sterilization fermentation

[0118] The solid culture medium was sterilized in a large-scale fungal fermentation apparatus and then inoculated at an inoculum rate of 10% to 15% after cooling. The seed culture was used as the expansion fermentation broth. The fermentation temperature was maintained at around 28 to 30°C, and the fermentation time was 5 to 7 days. The humidity was maintained at 85% to 90% in the early stage and 80% to 85% in the later stage. Clean air was required to be introduced during the sporulation process.

[0119] (3) Drying

[0120] After full sporulation, the TV26 bacterial blocks were collected and dried using a fluidized bed, with the moisture content controlled at around 11-13%.

[0121] (4) Detection

[0122] The dried TV26 bacterial blocks were pulverized using a specialized pulverizer and sieved through a sieve with a mesh size of 120-150 mesh. The sieved mother powder was analyzed, and the viable spore content of TV26 was determined by the dilution plate method. Based on the above process, a mother powder with a viable spore content of ≥15 billion / g can be prepared.

[0123] 3. Preparation of TV26 strain inoculum

[0124] (1) Blending of raw powder

[0125] Based on the mother powder, high-mesh diatomaceous earth filler was added to prepare a standard raw powder, in which the number of TV26 spores was stabilized at 5 billion / g.

[0126] (2) Finished product blending

[0127] Four TV26 inoculants were prepared and labeled PF1, PF2, PF3 and PF4 respectively.

[0128] PF1: 1.0~1.5% glucose + 3~4.5% starch + 0.1~0.4% amino acid powder + 5~7% sodium dodecyl sulfate (or sodium dodecyl sulfate) + 50% raw powder, with the remainder being high-mesh diatomaceous earth.

[0129] PF2: 1.0~1.5% glucose + 3~4.5% starch + 0.1~0.4% amino acid powder + 5~7% sodium dodecyl sulfate (or sodium dodecyl sulfate) + 50% raw powder + 0.5% mesobiose, with the remainder being high-mesh diatomaceous earth.

[0130] PF3: 1.0~1.5% glucose + 3~4.5% starch + 0.1~0.4% amino acid powder + 5~7% sodium dodecyl sulfate (or sodium dodecyl sulfate) + 50% raw powder + 1.0% mesobiose, with the remainder being high-mesh diatomaceous earth.

[0131] PF4: 1.0~1.5% glucose + 3~4.5% starch + 0.1~0.4% amino acid powder + 5~7% sodium dodecyl sulfate (or sodium dodecyl sulfate) + 50% raw powder + 1.5% mesobiose, with the remainder being high-mesh diatomaceous earth.

[0132] All percentages of the components in the above-mentioned microbial agents are by mass percentages.

[0133] Example 6: Field efficacy test of TV26 strain biological agent

[0134] To clarify the efficacy of the four biological agents in Example 5 in controlling cucumber leaf spot disease in the field, and to further screen the optimal dosage of menobiose, we conducted a field efficacy trial at a cucumber planting base in Zhangqiu, Jinan. Cucumber leaf spot disease has historically been severe in this region.

[0135] 1. Materials and Methods

[0136] (1) Test reagents

[0137] Four biological agents, PF1, PF2, PF3 and PF4, were prepared by Shandong Binnong Technology Co., Ltd., and were in powder form. Among them, Trichoderma longifolia TV26 had an effective viable count of ≥200 million / g.

[0138] 5% Chlorothalonil wettable powder: provided by the test site, a commonly used chemical fungicide in previous years.

[0139] (2) Experimental design

[0140] Based on the disease incidence period in previous years, spraying should be carried out one week in advance. A second application should be applied 15 days after the first, and the entire trial process should consist of two applications. Specific details are as follows:

[0141] Control 1: Water control;

[0142] Control 2: Spray 5% chlorothalonil wettable powder, with the dosage and usage method consistent with previous years;

[0143] PF1: Spray Trichoderma longifolia TV26 biological agent PF1, 100g / mu, diluted 500 times;

[0144] PF2: Spray Trichoderma longifolia TV26 biological agent PF2, 100g / mu, diluted 500 times;

[0145] PF3: Spray Trichoderma longifolia TV26 biological agent PF3, 100g / mu, diluted 500 times;

[0146] PF4: Spray Trichoderma longifolia TV26 biological agent PF4, 100g / mu, diluted 500 times.

[0147] Each of the above-mentioned experimental sites shall have an area of ​​no less than 0.5 acres. The experiment at each site shall be conducted in a greenhouse, and the rest shall be carried out in accordance with routine management.

[0148] (3) Survey and statistical methods

[0149] A field survey was conducted 30 days after the final spraying. The disease severity of cucumber spot disease in each treatment was statistically analyzed, and the control effect was calculated. Using a 5-point sampling method, 20 plants were randomly selected from each treatment, with three replicates.

[0150] Grading criteria for spotted disease:

[0151] Leaf diseases are classified by leaf: Level 0: No lesions; Level 1: 3 lesions on a single leaf; Level 3: 4-6 lesions on a single leaf; Level 5: 7-10 lesions on a single leaf; Level 7: 11-20 lesions on a single leaf; Level 9: Dense lesions on a single leaf covering more than 1 / 4 of the leaf area.

[0152] The disease index and prevention and control effect are calculated using the following formula.

[0153] Disease index = [∑(number of plants at each disease level × representative value of each disease level) / (total number of plants × highest representative value of disease level)] × 100.

[0154] Prevention efficacy (%) = (disease index in control area - disease index in treatment area) / disease index in control area × 100.

[0155] 2. Results Analysis

[0156] The results are shown in Table 5:

[0157] Table 5. Control effects of various agents on cucumber spot disease

[0158] ;

[0159] Note: In the table above, the same letter after the data in the same column indicates that the difference is not significant, and different letters indicate that the difference is significant.

[0160] According to Table 5:

[0161] (1) The control efficacy of biological agent PF1 against cucumber spot disease was 68.12%, which was not significantly different from that of the chemical agent chlorothalonil (67.25%). This result indicates that biological agent PF1 can completely replace chlorothalonil in the control of cucumber spot disease. The active ingredient of biological agent PF1 is Trichoderma longifolia TV26, which is safe and has no drug resistance, and is in line with the development trend of ecological agriculture and green agriculture.

[0162] (2) The control effects of biological agents PF2, PF3, and PF4 on cucumber spot disease were 78.17%, 86.90%, and 84.72%, respectively, which were significantly improved compared with biological agent PF1. This result indicates that different concentrations of menobiose can improve the control effect of Trichoderma longifolia TV26 on cucumber spot disease. Based on the results of Example 4, it is speculated that menobiose may achieve efficient control of cucumber spot disease by increasing the colonization of Trichoderma longifolia TV26 on the surface of cucumber leaves.

[0163] (3) The biological agents PF3 and PF4 had control effects of 86.90% and 84.72% on cucumber spot disease, respectively, but there was no significant difference between the two. Therefore, the optimal formulation of the biological agent can be determined to be PF4, and the optimal addition amount of mesobiose can be determined to be 1.0%.

[0164] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A long-branched Trichoderma ( Trichoderma longibrachiatum strain TV26, characterized in that, The *Trichoderma longifolia* strain TV26 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on July 21, 2025, with accession number CGMCC NO.42133.

2. A microbial agent comprising at least one selected from the group consisting of: live Trichoderma longicornis strain TV26 as claimed in claim 1, fermentation broth of the strain, fermentation supernatant, and / or a concentrate of the fermentation broth and fermentation supernatant of the strain.

3. A microbial agent comprising live bacteria of the Trichoderma longifolia strain TV26 as described in claim 1 and a synergist, wherein the synergist is menobiose.

4. The microbial agent according to claim 3, characterized in that, The synergist can promote the colonization of the Trichoderma longifolia strain TV26 on the surface of the crop to be applied.

5. The microbial agent according to claim 2 or 3, characterized in that, The live bacteria are freeze-dried bacteria.

6. The microbial agent according to claim 2 or 3, characterized in that, The bacterial agent is a liquid preparation or a powder.

7. The microbial agent according to claim 2 or 3, characterized in that, The microbial agent also includes excipients.

8. The microbial agent according to claim 7, characterized in that, The excipients are selected from one or more of high-mesh diatomaceous earth, glucose, starch, amino acid powder, sodium dodecyl sulfonate, and sodium dodecyl sulfate.

9. The microbial agent according to claim 4, characterized in that, The synergist is present in the microbial agent at a concentration of 0.5-1.5 wt%. And / or, the crop surface to be applied is the surface of a cucumber leaf.

10. The use of the *Trichoderma longicornis* strain TV26 as described in claim 1 or the inoculum as described in any one of claims 2-9 in the preparation of a biopesticide, wherein the biopesticide is used for any one or more of the following purposes: (1) Used to antagonize *Gnaphalium affine*; (2) Used to prevent and control cucumber spot disease caused by sclerotium tumefaciens.

11. Use of the live Trichoderma longifolia strain TV26 as described in claim 1 in the preparation of a pesticide for antagonizing Cladosporium globosum.

12. Use of the freeze-dried Trichoderma longifolia strain TV26 as described in claim 1 in the preparation of a pesticide for antagonizing Cladosporium globosum.

13. Use of the fermentation broth, fermentation supernatant and / or concentrate of the fermentation broth and fermentation supernatant of the *Trichoderma longifolia* strain TV26 as described in claim 1 in the preparation of a pesticide for antagonizing *Trichoderma graminearum*.

14. Use of the live Trichoderma longifolia strain TV26 and menobiose as described in claim 1 in the preparation of a pesticide for antagonizing Cladosporium cucumeris.

15. Use of the freeze-dried Trichoderma longifolia strain TV26 and menobiose as described in claim 1 in the preparation of a pesticide for antagonizing Cladosporium cucumeris.

16. Use of the live Trichoderma longifolia strain TV26 as described in claim 1 in the preparation of a pesticide for controlling cucumber spot disease caused by Acer calamus.

17. Use of the freeze-dried strain TV26 of Trichoderma longifolia as described in claim 1 in the preparation of a pesticide for controlling cucumber spot disease caused by Acer calamus.

18. Use of the fermentation broth, fermentation supernatant and / or concentrate of the fermentation broth and fermentation supernatant of the *Trichoderma longifolia* strain TV26 as described in claim 1 in the preparation of a pesticide for controlling cucumber spot disease caused by *Acer calamus*.

19. The use of the live Trichoderma longifolia strain TV26 and menobiose as described in claim 1 in the preparation of a pesticide for controlling cucumber spot disease caused by Acer calamus.

20. Use of the freeze-dried Trichoderma longifolia strain TV26 and menobiose as described in claim 1 in the preparation of a pesticide for controlling cucumber spot disease caused by Acer calamus.

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