Trichoderma guasei strain NH-1 for preventing and treating cucumber fusarium wilt, promoting growth and improving quality, microbial agent and application of trichoderma guasei strain NH-1 and microbial agent

Abstract

The invention relates to the field of microorganisms, in particular to a Trichoderma guasei strain NH-1 for preventing and treating cucumber fusarium wilt, promoting growth and improving quality, a fungicide and application of the Trichoderma guasei strain NH-1 and the fungicide. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.40021. The Trichoderma guasei NH-1 can effectively promote the growth of cucumbers, effectively prevent and treat fusarium wilt of the cucumbers and effectively improve the quality of the cucumbers, for example, the sugar content and the vitamin C content are improved, and the tannin content is reduced.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to the Trichoderma genus NH-1 strain for controlling cucumber wilt, promoting growth and improving quality, its inoculant, and its application. Background Technology

[0002] cucumber( Cucumis sativus Cucumber (L.) is an annual climbing herbaceous plant belonging to the Cucurbitaceae family and the Cucurbita genus. Utilizing microbial inoculants to improve the quality and taste of cucumbers aligns with the overall requirements of my country's food philosophy and is also an important research area for improving people's quality of life.

[0003] Achieving stable and high yields in cucumber cultivation is crucial for cucumber production. This involves improving cucumber growth capacity to ultimately ensure productivity. In the early stages of cucumber growth, focus should be placed on root development to enhance its resilience. During the growing season, a balanced approach to nutrition, flowering, and fruit setting is necessary to prevent excessive vegetative growth and improve fruit set.

[0004] Cucumber cultivation is highly susceptible to various pathogens, among which cucumber wilt (also known as wilt disease or vine rot) is the most common and devastating disease. It is caused by *Fusarium oxysporum* f.sp. cucumerinum, affecting both seedling and mature cucumber plants. Once it occurs, control is extremely difficult, often resulting in the death of numerous plants and severe yield reduction.

[0005] Therefore, it is essential to develop microbial agents that can improve the disease resistance, promote growth, and enhance the quality of cucumbers.

[0006] Trichoderma galbana ( Trichoderma gamsii Trichoderma is a type of fungus widely distributed in moist habitats such as soil and decaying wood. Its antagonistic ability against various plant pathogenic fungi, such as wheat stem rot and corn stem rot, makes it a popular target for developing biological control agents for use in green agriculture practices. Summary of the Invention

[0007] The purpose of this application is to provide a Trichoderma geysers strain for the prevention and control of cucumber wilt, promoting growth and improving quality.

[0008] Another object of this application is to provide a microbial inoculant comprising the aforementioned Trichoderma galbense strain.

[0009] Another object of this application is to provide the application of the aforementioned Trichoderma galbsiella strain.

[0010] Another objective of this application is to provide a method for preventing and controlling cucumber wilt, promoting growth, and improving quality.

[0011] According to the Trichoderma galbana of this application ( Trichoderma gamsii The strain NH-1 has the accession number CGMCCNo. 40021.

[0012] This invention provides the above-mentioned Trichoderma galbana ( Trichoderma gamsii The following uses of strain NH-1: Control of cucumber wilt disease Promote cucumber growth; and Improve cucumber quality.

[0013] The present invention also provides a microbial inoculant, the inoculant comprising the Trichoderma galbana ( Trichoderma gamsii ) The cells or spores of strain NH-1.

[0014] According to the microbial agent of the present invention, the microbial agent is a powder or a solution.

[0015] The *Trichoderma geysersii* NH-1, with accession number CGMCC No. 40021 in this application, can effectively promote cucumber growth, effectively prevent cucumber wilt, and effectively improve cucumber quality, such as increasing sugar content, vitamin C content, and reducing tannin content.

[0016] The Trichoderma geysinensis of this application ( Trichoderma gamsii The strain NH-1 was classified as *Trichoderma gems*. Trichoderma gamsii It has been deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) on December 28, 2021, with accession number CGMCC No. 40021. Attached Figure Description

[0017] Figure 1 This is a colony morphology diagram of Trichoderma NH-1. Detailed Implementation

[0018] The following provides a detailed description of specific embodiments of this disclosure. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit this disclosure.

[0019] This invention isolated and cultured a new fungal species from soil samples of the Hulunbuir Grassland in Inner Mongolia Autonomous Region. The fungus was identified as belonging to the genus *Trichoderma*. Trichoderma It was named Trichoderma galbana ( ). Trichoderma gamsiiThe specimen has been deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) on December 28, 2021, with accession number CGMCC No. 40021.

[0020] The microbial inoculant according to the present invention comprises bacterial cells and a culture medium, wherein the bacterial cells contain *Trichoderma* species with accession number CGMCC No. 40021. The amount of bacterial cells in the microbial inoculant can vary considerably; for example, the viable count of *Trichoderma geysii* species with accession number CGMCC No. 40021 per gram of microbial inoculant can be 10. 7 -10 11 CFU; preferably, the viable count of *Trichoderma geysei* with preservation number CGMCC No. 40021 in each gram of microbial inoculant can be 10. 8 -10 10 CFU; more preferably, the viable count of *Trichoderma geysei* with accession number CGMCC No. 40021 may be 10 per gram of microbial inoculant. 9 -10 10 CFU. During cultivation, the concentration of viable bacteria can be determined using conventional methods, such as hemocytocyte count or OD value observation. The culture medium is potato dextrose agar solid medium (PDA) and / or potato dextrose liquid medium (PD).

[0021] The aforementioned culture media can be sterilized using conventional sterilization methods, such as sterilizing at 115-125℃ and 1.5-2 standard atmospheres for 10-30 minutes.

[0022] The preparation method of the microbial inoculant may include: inoculating *Trichoderma galbana* with preservation number CGMCC No. 40021 into potato dextrose agar (PDA) solid medium and incubating at 25°C for 7 days; then taking 3-4 mycelial blocks and adding them to a seed bottle; and incubating at 25°C and 180 rpm for 48 hours to prepare a seed culture. The seed culture is then added to a seed fermentation tank at a volume ratio of 1:150 for further cultivation, and then added to a fermentation tank containing chlamydospore fermentation medium (containing 2.5% starch, 1.5% yeast powder, 5.0% corn steep liquor, 0.4% CaCO3, 0.01% ZnSO4, and 0.03% MgSO4 by weight) at a volume ratio of 1:20. The medium is incubated at 28°C for 132 hours until at least 90% of the mycelia form chlamydospores, yielding a chlamydospore fermentation broth. The fermentation broth is then adsorbed using diatomaceous earth, centrifuged or filtered through a plate and frame filter, and dried to form a wettable powder.

[0023] The cultured bacterial solution can be used directly as a microbial inoculant. Preferably, the bacterial solution is further processed into a more convenient storage formulation of the microbial inoculant through steps including aseptic filtration and freeze-drying. There are no particular restrictions on the culture conditions for the strain; commonly used conditions in the cultivation of *Trichoderma galbsiella* can be applied, such as shaking culture at a temperature of 25-30°C for 1-2 days.

[0024] Preferably, the preparation method of the microbial agent may further include: mixing the material obtained after cultivation in a fermentation medium with an inert solid carrier and a binder; the inert solid carrier includes at least one selected from clay, silica, diatomaceous earth, kaolin, attapulgite, montmorillonite, bentonite, talc, white carbon black, and calcium carbonate; the binder includes maltose and / or dextrin. Preferably, in the microbial agent, the content of the inert solid carrier is 30-90% by weight, and the content of the binder is 5-20% by weight. The mixed material can be further dried and granulated into granules.

[0025] Optionally, the microbial agent is a powder and / or solution.

[0026] The present invention will be further described in detail below with reference to the embodiments, but the scope of the present invention is not limited to the following embodiments.

[0027] In the following examples, the potato dextrose agar solid medium (PDA) used was prepared as follows: 20g of peeled potatoes were boiled for 20 minutes to extract the juice. 2g of glucose and 1.5g of agar were added to the juice, and water was added to 100mL. The pH was 7.0. The mixture was then poured into two Erlenmeyer flasks, each containing 50mL. After sterilization, slant or plate culture was prepared.

[0028] The fermenter formula for the thick-walled spore fermentation medium is as follows: by weight, it contains 2.5% starch, 1.5% yeast powder, 5.0% corn steep liquor, 0.4% CaCO3, 0.01% ZnSO4, and 0.03% MgSO4. Example 1: Isolation and identification of Trichoderma gems NH-1

[0029] 1.1 Sampling Soil samples were collected near Hailar, Hulunbuir District, Inner Mongolia Autonomous Region. The collection method was as follows: at each sampling point, 10 m of vegetation was observed. 2 Within the quadrats, the five-point diagonal sampling method was used for soil collection. During sampling, after removing 1-2 cm of topsoil, soil around the plant roots was collected, and soil from the rhizosphere of the host plant at a depth of 2-30 cm was taken. After mixing, 200 g of the soil was placed in a sterilized resealable bag, brought back to the laboratory, and marked (collection location, latitude and longitude, altitude, and vegetation cover), and stored at 4°C.

[0030] 1.2 Isolation and purification of strain NH-1: 1.2.1 Soil sample preparation: Using the dilution plate method, 10 μL of each sample was taken. -3 100 μL of a soil sample dilution was spread onto a PDA medium plate, and then spread evenly using a sterile spreader. This process was repeated three times on the PDA medium. The plate was incubated at 28°C in a mold incubator. Fungal colonies were observed after 5-10 days. Suspected Trichoderma colonies appearing on the plates were counted and further purification was performed.

[0031] 1.2.2 Culture and purification of bacterial strains: Suspected Trichoderma strains were picked from the plates and placed on PDA medium for multiple rounds of purification culture until the strain was completely purified, with a total culture period of more than three weeks. The purified Trichoderma strains were temporarily stored in a PDA slant at 4°C, while three aliquots were inoculated into 30% glycerol tubes and stored in an ultra-low temperature freezer.

[0032] 1.3 Identification of Trichoderma gems NH-1 1.3.1 Morphological identification of the isolated suspected Trichoderma colonies: The morphology of hyphae, the morphology and color of spore hyphae, the manner of spore attachment, the arrangement and morphology of spores, etc., are observed under an optical microscope, and the diameter of hyphae, the size of spore hyphae and spores are measured.

[0033] Mycelial discs of the same diameter were taken from the edge of purified NH-1 colonies and placed in the center of PDA medium. The growth rate, growth status, colony color, and mycelial morphology of the strain were observed. The results showed that after culturing at 27℃ for 5-7 days, the isolated and purified NH-1 strain produced radially round colonies with a green surface, regular edges, and a pale green back. The spores were green, and the sporulation rate was high (see...). Figure 1 ).

[0034] 1.3.2 Molecular biological identification: A suitable amount of mycelium from strain NH-1 was scraped and placed in a sterile centrifuge tube. DNA was extracted and collected according to the procedure of the fungal genome extraction kit. The internal transcribed spacer (ITS) and translation elongation factor 1α (TEF) of ribosomal RNA-encoding genes were analyzed. tef The fourth and fifth introns and most of the last exon of 1α), and rpb2 The gene for the second subunit of RNA polymerase was amplified. After successful amplification, the sample was sent for sequencing to obtain the sequence of the ITS region of strain NH-1. rpb 2 genes and tef1α gene sequence. In 2021, Cai and Druzhinina proposed new criteria for classifying Trichoderma species: all must simultaneously meet the following requirements. rpb 2≥99%, tef Only when three conditions are met—α ≥ 97%, ITS ≥ 76%—can a Trichoderma species be identified. Using this method for Trichoderma species identification, strain NH-1 was... rpb 2 genes and tef Sequences of the 1α gene and the ITS region were compared using NCBI Blast and found that strain NH-1... rpb The similarity of gene 2 to *Trichoderma galbana* is ≥99% (99.05-99.68%), and strain NH-1... tef The 1α gene shows a high degree of similarity to *Trichoderma galbana*. tef The 1α sequence showed a similarity of ≥97%, and the ITS sequence of T114 shared ≥76% similarity with *Trichoderma galbana*. Therefore, we confirmed that the *Trichoderma* species was classified as *Trichoderma galbana*. Example 2: Preparation of Microbial Inoculants

[0035] A slant culture of *Trichoderma galbana* with preservation number CGMCC No. 40021 was inoculated into a PDA and incubated at 25°C for 7 days. Three to four mycelial blocks were then added to a seed bottle. The mixture was then shaken at 25°C and 180 rpm for 48 hours to prepare a seed culture. The seed culture was added to a seed fermentation tank at a volume ratio of 1:150 and then added to a fermentation tank containing chlamydospore fermentation medium at a volume ratio of 1:20. The mixture was incubated at 28°C for 132 hours until 90% of the mycelia formed chlamydospores, yielding a chlamydospore fermentation broth. The broth was then adsorbed with diatomaceous earth, centrifuged or filtered through a plate and frame filter, and dried to form a wettable powder. This wettable powder is the microbial inoculum of this embodiment, containing 4 × 10⁻⁶ viable *Trichoderma galbana* cells. 9 CFU / g.

[0036] Following the above method, five Trichoderma species isolated together with the Trichoderma galvanum with accession number CGMCC No. 40021 of this application were used to prepare microbial inoculants, namely Trichoderma galvanum 1, Trichoderma galvanum 2, Trichoderma galvanum 3, Trichoderma galvanum 4 and Trichoderma galvanum 5. Example 3: Trichoderma galbsiella strain promotes cucumber growth and improves cucumber quality.

[0037] The various microbial agents prepared in Example 2 above were used for testing, and a control group was set up without the application of wettable powder.

[0038] The experimental setup was a paper cup experiment. Microbial wettable powders of *Trichoderma galbsiella* (accession number CGMCC No. 40021), *Trichoderma galbsiella* 1, *Trichoderma galbsiella* 2, *Trichoderma galbsiella* 3, *Trichoderma galbsiella* 4, and *Trichoderma galbsiella* 5, were mixed with soil at a ratio of 10 μL / cup. 7 The total number of spores was diluted with water at a 1:1 ratio with soil. Then, cucumber seeds of consistent quality were selected and planted in paper cups, with five plants replicated for each treatment. After one month, cucumber growth was statistically analyzed, primarily focusing on the number of true leaves and plant height. For changes in cucumber quality, the treated cucumber plants were transplanted to greenhouses, and the sugar and tannin content of the final fruits was calculated after ripening. For control of cucumber wilt disease, the disease index and incidence rate were statistically analyzed. The results are listed in Table 1.

[0039] Table 1 Number of true leaves of cucumber (pieces) Standard deviation of true leaf number Cucumber plant height (cm) Standard deviation of plant height Cucumber contains reducing sugar (calculated as glucose) g / 100g Cucumber Vitamin C (mg / 100g) Tannin content in cucumbers (mg / 100g) control group 6.5 0.577 49.58 5.910 2.7 6.35 1.24 x 10 2 <!-- 4 -->]]> Trichoderma ghim's NH-1 8.67 1.528 58.1 3.348 3.0 6.87 <![CDATA[1.13×10 2 ]]> Trichoderma galbana 1 6.5 1.517 47.25 4.742 2.6 6.23 <![CDATA[1.25×10 2 ]]> Trichoderma galbana 2 6.75 1.643 42.75 4.663 2.45 6.19 <![CDATA[1.23×10 2 ]]> Trichoderma 3 6 3.209 38.05 13.666 2.57 6.32 <![CDATA[1.21×10 2 ]]> Trichoderma 4 4.5 2.081 30.55 5.950 1.98 6.12 <![CDATA[1.25×10 2 ]]> Trichoderma 5 5.5 1.923 37.15 11.232 2.63 6.51 <![CDATA[1.27×10 2 ]]> .

[0040] As shown in Table 1, compared with the control group, the *Trichoderma gems* NH-1 of this application increased the number of true leaves and plant height of cucumber by 33.4% and 17.2%, respectively; compared with *Trichoderma gems* 1 to *Trichoderma gems* 5, it increased the number of true leaves and plant height of cucumber by 28%-93% and 23%-90%, respectively. These data further indicate that *Trichoderma gems* NH-1 with accession number CGMCC No. 40021 can effectively promote the growth of cucumber.

[0041] As shown in Table 1, the Trichoderma geysii NH-1 of this application has significant advantages over the control group in increasing the sugar content and vitamin C content of cucumbers and reducing the tannin content.

[0042] In the statistical analysis of cucumber sugar content, the Trichoderma geysersii NH-1 of this application increased the sugar content of cucumber by 11.11% compared with the control group; compared with the control group, the sugar content of cucumbers in Comparative Examples 1-5 decreased by 2.59%-26.66%.

[0043] In the statistical analysis of vitamin C content data in cucumbers, compared with the control group, Example 2 showed an 8.19% increase in vitamin C content in cucumbers; Trichoderma 1 to Trichoderma 5 showed a 2.51% increase in vitamin C content in cucumbers compared with the control group; and Trichoderma 1 to Trichoderma 4 showed a decrease in vitamin C content in cucumbers ranging from 0.47% to 3.62% compared with the control group.

[0044] In the statistical analysis of cucumber tannin content, compared with the control group, the Trichoderma ghimiglia NH-1 of this application reduced the cucumber tannin content by 8.87%; compared with the control group, the tannin content of Trichoderma ghimiglia 1 to Trichoderma ghimiglia 5 did not change significantly. Among them, Trichoderma ghimiglia 2 and Trichoderma ghimiglia 3 decreased by 0.81% and 2.42% respectively, while Trichoderma ghimiglia 4 and Trichoderma ghimiglia 5 increased by 0.81%, 0.81% and 2.42% respectively.

[0045] These data further demonstrate that the Trichoderma hymenopterus NH-1 with accession number CGMCC No. 40021 of this application can effectively improve the quality of cucumbers, such as increasing sugar content, vitamin C content and reducing tannin content. Example 4: Control of cucumber wilt with Trichoderma galbestris

[0046] The various microbial agents prepared in Example 2 above were used for testing, and a control group was set up without the application of wettable powder.

[0047] The experimental setup was a paper cup experiment. The wettable powders of *Trichoderma galbsiella* NH-1, *Trichoderma galbsiella* 1, *Trichoderma galbsiella* 2, *Trichoderma galbsiella* 3, *Trichoderma galbsiella* 4, and *Trichoderma galbsiella* 5 from this application were mixed with soil at a concentration of 10 mg / cup. 7 The total number of spores; pathogens were measured using OD. 600 The pathogen solution was diluted 400 times with a ratio of water:pathogen:soil of 0.5:0.5:1. Then, cucumber seeds of consistent quality were selected and planted in paper cups, with 5 plants per treatment. After one month, the growth of the cucumbers was analyzed, and the disease index and incidence rate of cucumber wilt were calculated based on the severity level of the disease.

[0048] The severity of cucumber wilt disease is typically assessed using a standardized grading system based on the severity of plant symptoms. According to the People's Republic of China Agricultural Industry Standard "Technical Regulations for Identification of Resistance to Major Cucumber Diseases, Part III: Technical Regulations for Identification of Cucumber Resistance to Fusarium Wilt," the disease severity is classified into five levels: - Level 0 (Asymptomatic): The plant shows no signs of disease and grows normally; - Grade 1 (mild symptoms): Characterized by yellowing of cotyledons, but without wilting; - Level 2 (moderate symptoms): The cotyledons begin to wilt, and the plant shows obvious signs of wilting; - Level 3 (more severe symptoms): Both cotyledons and true leaves are wilted, or the plant shows signs of stunting; - Level 4 (Severe Symptoms): The plant dies and cannot recover; This grading system is mainly used for indoor disease resistance identification, quantifying disease severity by observing the reactions of cotyledons and true leaves.

[0049] Disease index = 100 × ∑ (number of diseased leaves at each level × representative value at each level) / (total number of leaves surveyed × highest representative value). Relative prevention and control effect (%) = (Control disease index - Treatment disease index) / Control disease index × 100. The results are shown in Table 2.

[0050] Table 2 .

[0051] As shown in Table 2, compared with the pathogen control group, the average disease index of *Trichoderma gems* NH-1 was 30, and the relative control efficacy was 63.08%. Compared with other *Trichoderma gems* species, *Trichoderma gems* NH-1 had the lowest average disease index, which was 13.67-23.7% lower than that of *Trichoderma gems* 1-5. The relative control efficacy of *Trichoderma gems* NH-1 was the highest, which was 13.67-23.7% higher than that of *Trichoderma gems* 1-5. These data further demonstrate that *Trichoderma gems* NH-1, with accession number CGMCC No. 40021, can effectively control cucumber wilt.

[0052] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0053] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0054] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. Trichoderma galbana ( Trichoderma gamsii strain NH-1, characterized in that, The Trichoderma gems ( Trichoderma gamsii The preservation number of strain NH-1 is CGMCC No. 40021.

2. The Trichoderma galbana as described in claim 1 ( Trichoderma gamsii The following applications of strain NH-1: To prevent and control cucumber wilt; to promote cucumber growth; and to improve cucumber quality.

3. A method for preventing and controlling cucumber wilt, promoting growth, and improving quality, characterized in that, The method includes applying the Trichoderma galbana of claim 1 to a cucumber. Trichoderma gamsii The steps for strain NH-1.

4. A microbial inoculant, characterized in that, The microbial agent includes *Trichoderma gems* as described in claim 1. Trichoderma gamsii ) The cells or spores of strain NH-1.

5. The microbial agent according to claim 4, characterized in that, The microbial agent is a powder or solution.

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