A composite bacterial agent for preventing and controlling Hami melon wilt and root knot nematode disease and its application

Abstract

The present invention discloses a composite bacterial agent capable of preventing and controlling Hami melon wilt and root-knot nematode disease, comprising Trichoderma fungus NJAU-HNF11 and Bacillus bacteria NJAU-HNB23. The present invention also discloses the use of the composite bacterial agent in preventing and controlling Hami melon wilt, root-knot nematode disease, and promoting Hami melon growth. The composite bacterial agent of the present invention has a significant prevention and control effect on Hami melon wilt and root-knot nematode disease, and also has a significant Hami melon growth-promoting effect. The composite bacterial agent of the present invention can improve production efficiency and ecological environmental benefits, and promote the green, healthy and sustainable development of agriculture.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control, and in particular to a composite bacterial agent capable of preventing and controlling Hami melon wilt and root knot nematode disease and application thereof. Background Art

[0002] Hami melon has a high multiple cropping index, and the soil-borne diseases (such as wilt and nematode diseases) caused by long-term continuous cropping have intensified, and the imbalance of soil nutrients has gradually increased, which is not conducive to the green and healthy development of the Hami melon industry. Fusarium oxysporum is a fungus of the genus Fusarium, which is widely distributed throughout the world. It is a highly pathogenic soil-borne pathogenic fungus and is listed as one of the world's top ten plant pathogenic fungi. The wilt disease caused by Fusarium oxysporum infects plants invades the plants from the intercellular spaces of the root epidermis and root wounds, gradually causing blockage of the vascular bundles. In severe cases, the whole plant dies, which may occur throughout the entire growth period of the plant, causing great damage to production. Root-knot nematode (Meloidogyne sp.) is a plant parasitic nematode that is widely distributed throughout the world. During the invasion process, it is highly destructive to the host root system, affecting the plant's absorption and transport of water and nutrients. The trauma caused also makes it easier for various pathogens to invade the plant body, which can easily induce the occurrence of complex infection. At the same time, root-knot nematodes have the characteristics of rapid population growth, wide distribution range, hidden activities and multiple hosts, which brings many challenges to prevention and control. At present, the prevention and control measures for the above two soil-borne diseases are mainly chemical control. Although chemical control is effective quickly, various chemical drugs have problems such as high toxicity, residue and drug resistance, which may cause damage to the ecological balance of the soil. If overused, it will cause environmental pollution and even affect human health. Therefore, it is urgent to create new biological control products with the characteristics of green, environmental protection, safety and high efficiency. Based on this, the present invention isolated and screened two functional microorganisms that can effectively prevent and control Fusarium wilt and root-knot nematode disease from the soil of healthy Hami melon planting fields, combined them to form a composite bacterial agent, and measured its disease resistance and growth-promoting effects, in order to create a new biological control product that can synergistically prevent and control Fusarium wilt and root-knot nematode disease. Summary of the invention

[0003] The present invention aims to provide a composite bacterial agent capable of preventing and controlling Hami melon wilt and root knot nematode disease and its application, so as to solve the shortcomings of the prior art.

[0004] The purpose of the present invention can be achieved through the following technical solutions:

[0005] The first aspect of the present invention provides a composite bacterial agent for preventing and controlling Hami melon wilt and root-knot nematode disease, comprising Trichoderma fungus NJAU-HNF11 and Bacillus bacteria NJAU-HNB23; the Trichoderma fungus NJAU-HNF11 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of April 15, 2024 and a deposit number of CGMCC No.41158; the Bacillus bacteria NJAU-HNB23 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit date of April 15, 2024 and a deposit number of CGMCC No.30335.

[0006] Furthermore, the composite bacterial agent is prepared by the following steps:

[0007] (1) subjecting the Trichoderma fungus NJAU-HNF11 with a deposit number of CGMCC No. 41158 to liquid fermentation to obtain a NJAU-HNF11 bacterial agent;

[0008] The Bacillus bacteria NJAU-HNB23 with a deposit number of CGMCC No. 30335 is subjected to liquid fermentation to obtain a NJAU-HNB23 bacterial agent;

[0009] (2) Mixing the NJAU-HNF11 bacterial agent and the NJAU-HNB23 bacterial agent to obtain the composite bacterial agent.

[0010] Furthermore, the composite bacterial agent is prepared by the following steps:

[0011] (1) Inoculating spores of Trichoderma fungus NJAU-HNF11 with a deposit number of CGMCC No. 41158 into a liquid culture medium for liquid fermentation, wherein the liquid culture medium includes a PDA liquid culture medium, and the liquid fermentation conditions are: a fermentation temperature of 25-28° C., a rotation speed of 170-190 r / min, and a fermentation time of 96-120 h. After the fermentation, an NJAU-HNF11 bacterial agent is obtained, and the number of spores in the NJAU-HNF11 bacterial agent is ≥ 1×10 8 Pieces / mL;

[0012] The Bacillus bacteria NJAU-HNB23 with a preservation number of CGMCC No. 30335 was inoculated into a liquid culture medium for liquid fermentation. The liquid culture medium included an LB liquid culture medium. The liquid fermentation conditions were as follows: a fermentation temperature of 35-37° C., a rotation speed of 170-190 r / min, and a fermentation time of 12-24 h. After the fermentation, an NJAU-HNB23 bacterial agent was obtained. The bacterial content in the NJAU-HNB23 bacterial agent was ≥1×10 8 CFU / mL;

[0013] (2) Mixing equal volumes of NJAU-HNF11 bacterial agent and NJAU-HNB23 bacterial agent to obtain the composite bacterial agent.

[0014] The second aspect of the present invention provides a method for preparing a composite bacterial agent for preventing and controlling Hami melon wilt and root-knot nematode disease, wherein the composite bacterial agent comprises Trichoderma fungus NJAU-HNF11 and Bacillus bacteria NJAU-HNB23; the Trichoderma fungus NJAU-HNF11 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with a deposit date of April 15, 2024 and a deposit number of CGMCC No. 41158; the Bacillus bacteria NJAU-HNB23 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with a deposit date of April 15, 2024 and a deposit number of CGMCC No. 30335;

[0015] The preparation of the composite bacterial agent comprises the following steps:

[0016] (1) subjecting the Trichoderma fungus NJAU-HNF11 with a deposit number of CGMCC No. 41158 to liquid fermentation to obtain a NJAU-HNF11 bacterial agent;

[0017] The Bacillus bacteria NJAU-HNB23 with a deposit number of CGMCC No. 30335 is subjected to liquid fermentation to obtain a NJAU-HNB23 bacterial agent;

[0018] (2) Mixing the NJAU-HNF11 bacterial agent and the NJAU-HNB23 bacterial agent to obtain the composite bacterial agent.

[0019] Furthermore, the preparation of the composite bacterial agent includes the following steps:

[0020] (1) Inoculating spores of Trichoderma fungus NJAU-HNF11 with a deposit number of CGMCC No. 41158 into a liquid culture medium for liquid fermentation, wherein the liquid culture medium includes a PDA liquid culture medium, and the liquid fermentation conditions are: a fermentation temperature of 25-28° C., a rotation speed of 170-190 r / min, and a fermentation time of 96-120 h. After the fermentation, an NJAU-HNF11 bacterial agent is obtained, and the number of spores in the NJAU-HNF11 bacterial agent is ≥ 1×10 8 Pieces / mL;

[0021] The Bacillus bacteria NJAU-HNB23 with a preservation number of CGMCC No. 30335 was inoculated into a liquid culture medium for liquid fermentation. The liquid culture medium included an LB liquid culture medium. The liquid fermentation conditions were as follows: a fermentation temperature of 35-37° C., a rotation speed of 170-190 r / min, and a fermentation time of 12-24 h. After the fermentation, an NJAU-HNB23 bacterial agent was obtained. The bacterial content in the NJAU-HNB23 bacterial agent was ≥1×10 8 CFU / mL;

[0022] (2) Mixing equal volumes of NJAU-HNF11 bacterial agent and NJAU-HNB23 bacterial agent to obtain the composite bacterial agent.

[0023] The third aspect of the present invention provides the use of the above-mentioned composite bacterial agent in preventing and controlling Hami melon wilt, root knot nematode disease, and promoting Hami melon growth.

[0024] Furthermore, during application, the composite bacterial agent is applied 30-35 days after Hami melon is planted, and is applied 2-4 times, with an interval of 10-15 days between each application, and 10-15 mL of the composite bacterial agent is applied to each plant each time.

[0025] Furthermore, the composite bacterial agent is applied by root irrigation.

[0026] Beneficial effects of the present invention:

[0027] The composite bacterial agent of the present invention comprises Trichoderma fungus NJAU-HNF11 and Bacillus bacteria NJAU-HNB23, which has a significant control effect on Hami melon wilt and root knot nematodes, and has a significant Hami melon growth promotion effect. The composite bacterial agent of the present invention can improve production efficiency and ecological environmental benefits, and promote the green, healthy and sustainable development of agriculture.

[0028] The pot test results of the present invention show that the NJAU-HNB23+NJAU-HNF11 composite bacterial agent can not only better reduce the number of Fusarium oxysporum in the soil and the production of root knots in Hami melon plants, but also better promote the growth of Hami melon plants compared with a single bacterial agent. The field test of the present invention further verified that the NJAU-HNB23+NJAU-HNF11 composite bacterial agent can not only significantly reduce the number of pathogenic Fusarium oxysporum and root-knot nematodes in the soil, but also have a significant growth-promoting effect on Hami melon. The composite bacterial agent of the present invention provides composite bacterial resources and technical support for biological control measures for Hami melon wilt and root-knot nematode disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the result of confrontation between the fungus NJAU-HNF11 and Fusarium oxysporum on a plate.

[0030] Figure 2 This is a phylogenetic tree constructed based on the 16SrRNA gene sequence of strain NJAU-HNF11.

[0031] Figure 3 This is a phylogenetic tree constructed based on the 16SrRNA gene sequence of strain NJAU-HNB23.

[0032] Figure 4 This is a potted experiment to test the effects of different treatments on the number of root nodes in Hami melon roots.

[0033] Figure 5 This is the field test effect of the NJAU-HNB23+NJAU-HNF11 composite bacterial agent.

[0034] Biomaterial Deposit Information

[0035] The strain NJAU-HNF11 is classified and named Trichoderma sp. and is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 15, 2024, and the deposit number is CGMCC No.41158.

[0036] The strain NJAU-HNB23, classified and named Bacillus sp., is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit address at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is April 15, 2024, and the deposit number is CGMCC No.30335. DETAILED DESCRIPTION

[0037] The present invention is further explained below in conjunction with the examples and drawings. The following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0038] The diameter of the plates (culture dishes) involved in the following examples is 90 mm.

[0039] Example 1 Isolation and screening of functional strains

[0040] 1. Test materials:

[0041] Test pathogens: The pathogen of Hami melon wilt, Fusarium oxysporum, was isolated and preserved by the Modern Agriculture and Green Production Laboratory of Sanya Research Institute of Nanjing Agricultural University from the soil with high incidence of Hami melon wilt.

[0042] Test nematodes: Caenorhabditis elegans and Meloidogyne sp. were provided by Jiangsu Key Laboratory of High-Tech Research on Solid Organic Waste Resource Utilization.

[0043] Test Escherichia coli: Escherichia coli was provided by Jiangsu Provincial Key Laboratory of High-Tech Research on Solid Organic Waste Resource Utilization.

[0044] Test soil: soil from healthy cantaloupe planting fields.

[0045] Test medium:

[0046] LB liquid medium (g / L): peptone 10g, yeast powder 5g, sodium chloride 5g, sterilized at 121°C for 30min. When preparing solid medium (i.e. LB solid medium), add agar powder 2.5w / v%.

[0047] PDB medium (g / L): 6g potato flour, 20g glucose, sterilized at 121℃ for 30min. When preparing PDA medium, add 2.5w / v% agar powder.

[0048] NGM nematode growth medium (g / L): 2.5 g peptone, 3 g sodium chloride, 0.111 g calcium chloride, 0.12 g magnesium sulfate, 0.005 g cholesterol, 3.4 g potassium dihydrogen phosphate, 17 g agar powder, sterilized at 121°C for 30 min.

[0049] 2. Isolation and purification of strains

[0050] The soil suspension described in the following isolation of bacterial strains and isolation of fungal strains was prepared as follows: 18 mL of sterile water was placed in a 50 mL sterile centrifuge tube, 2 g of soil sample was weighed and added to the above sterile water, the shaker temperature was set to 25°C, and the shaker was shaken at 170 r / min for 2 hours to obtain the soil suspension.

[0051] Isolation and purification of bacterial strains: The bacterial strains were isolated by plate dilution and coating method. The details are as follows: 0.1 mL of soil suspension was added with 0.9 mL of sterile water, mixed well, and then diluted with sterile water to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , absorb 10 -5 , 10 -6 , 10 -7 100 μL of the soil dilution was evenly spread on an LB solid culture medium plate and cultured at 37°C until single colonies appeared. Colonies with different morphologies were picked and purified for later use.

[0052] Isolation and purification of fungal strains: The fungal strains were isolated by plate dilution and coating method. The details are as follows: 0.1 mL of soil suspension was added with 0.9 mL of sterile water, mixed well, and then diluted with sterile water to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , absorb 10 -5 , 10 -6 , 10 -7 100 μL of the soil dilution was evenly spread on a PDA medium plate and cultured at 28°C until single colonies appeared. Colonies with different morphologies were picked and purified for later use.

[0053] 3. Preparation of strain fermentation broth:

[0054] Preparation of bacterial fermentation broth: Pick a single colony into 5 mL LB liquid culture medium, culture at 37°C and 170 r / min with shaking for 24 h, and then filter the fermentation broth with a 0.22 μm microporous filter membrane (the purpose is to remove the bacteria in the fermentation broth for better observation of nematodes).

[0055] Preparation of fungal fermentation broth: Inject 5 mL of sterile water into a fungal purification plate cultured at 28 °C for 96 h, mix the spores and sterile water into spore solution using a sterilized spreading stick, pipette 100 μL of spore solution into 100 mL of PDB medium, then culture at 28 °C and 170 r / min for 96 h, and then filter the fermentation broth with a 0.22 μm microporous filter membrane (the purpose is to remove the bacteria in the fermentation broth for better observation of nematodes).

[0056] 4. Preliminary screening of nematicidal strains:

[0057] Caenorhabditis elegans was used as a model nematode for preliminary screening of nematicidal effects. The NGM nematode plate was obtained in the following manner: activated Escherichia coli was inoculated into NGM nematode growth medium (15-20 mL), and cultured at 37° C. for 24 h. After the Escherichia coli covered the surface of the NGM nematode growth medium, Caenorhabditis elegans was inoculated, and cultured at 25° C. for 96 h to obtain the NGM nematode plate.

[0058] First, scrape 1 / 5 of the culture medium from the NGM nematode plate into a sterile plate, add 3 mL of sterile water, and mix manually for 3 minutes to allow the nematodes to fully enter the sterile water. The pipette draws 100 μL, 50 μL, and 20 μL of three gradient nematode suspensions, and observes the number of nematodes in different gradient nematode suspensions with an inverted microscope and calculates the average number of nematodes per mL of nematode suspension. Then, add 30 μL of strain fermentation liquid (bacterial fermentation liquid or fungal fermentation liquid) and 30 μL of nematode suspension to a 96-well cell culture plate (ensuring that the number of live nematodes is ≥30, calculated based on the average number of nematodes per mL of nematode suspension), set up 3 replicates for each strain fermentation liquid, place it in a 25°C incubator for static culture, and record the nematode mortality rate after 24 hours. Four strains with nematode mortality of more than 30% and no antagonism between each other were selected, of which three were bacteria, named NJAU-HNB2, NJAU-HNB3, and NJAU-HNB23; and one was a fungus, named NJAU-HNF11.

[0059] 5. Screening of antagonistic bacteria:

[0060] Four functional bacteria with good nematicidal effects were used in plate confrontation tests with Fusarium oxysporum.

[0061] Screening of bacteria against Fusarium oxysporum: Add 5mL of sterile water to the plate of Fusarium oxysporum cultured at 28℃ for 96h, mix the spores and sterile water with a sterilized coating stick to form a spore solution, then draw 2.5μL of the spore solution, place it in the center of the PDA plate, and culture it at 28℃ for 48h; pick a single bacterial colony in 100mL LB liquid culture medium, culture it at 37℃ and 170r / min for 24h to obtain a bacterial solution; 2.5μL of bacterial solution was symmetrically inoculated on the Fusarium oxysporum plate cultured for 48h, that is, 2.5μL of bacterial solution was inoculated in the four directions of front, back, left and right, respectively, and placed in a 37℃ incubator for culture, observed every 8h, and transferred to a 28℃ incubator for culture after the bacterial colony grew out (24h-48h), observed every 8h, and confirmed the antagonistic effect after continuous observation for 2d. The results showed that none of the three strains of bacteria had an inhibitory effect on Fusarium oxysporum.

[0062] Screening of anti-Fusarium oxysporum fungi: Add 5 mL of sterile water to the PDA plate of Fusarium oxysporum cultured at 28°C for 96 hours and the PDA plate of the fungus to be screened, respectively. Use a sterilized coating stick to mix the spores and sterile water into spore liquid, then take 2.5 μL of spore liquid respectively, and symmetrically inoculate the spore liquid of Fusarium oxysporum and the spore liquid of the fungus to be screened on the PDA plate, put it in a 28°C incubator for culture, observe it every 8 hours, and confirm the confrontation effect after continuous observation for 5 days. The results showed that the fungus NJAU-HNF11 has a significant inhibitory effect on Fusarium oxysporum ( Figure 1 ).

[0063] Example 2 Toxicity of the fermented liquid of the strain to root-knot nematodes

[0064] Preparation of root-knot nematode suspension: Take the tomato roots enriched with root-knot nematodes, wash the soil on the root surface with clean water and disinfect with 1% sodium hypochlorite for 2 minutes (disinfect the root surface, the root-knot nematodes are inside the root system and have no effect), then chop the tomato roots with root knots into about 1 mm, use 100 mesh, 500 mesh, and 1000 mesh sieves to collect the root-knot nematodes, and use sterile water to collect the root-knot nematodes in a sterilized centrifuge tube. Observe and count the root-knot nematodes with an inverted microscope, and select the live root-knot nematodes for later use.

[0065] In a 96-well cell culture plate, a 60 μL confrontation system was prepared, including: 30 μL root-knot nematode suspension (number of live root-knot nematodes ≥ 30), 30 μL strain fermentation broth (bacterial fermentation broth or fungal fermentation broth, prepared as the strain fermentation broth in Part 3 of Example 1), and sterile water treatment was set as a control (CK) (i.e., 30 μL sterile water replaced 30 μL strain fermentation broth), and 3 replicates were set for each treatment. The cells were statically cultured in a 25°C incubator, observed under an inverted microscope after 24 h and 48 h, and the root-knot nematode corrected mortality was calculated according to the following formula.

[0066] Root-knot nematode mortality = number of root-knot nematodes that died in a treatment / number of root-knot nematodes observed in a treatment × 100%.

[0067] Corrected root-knot nematode mortality = (treatment root-knot nematode mortality - control root-knot nematode mortality) / (1 - control root-knot nematode mortality) × 100%

[0068] Table 1 Corrected mortality of single mycorrhizal root-knot nematodes

[0069]

[0070] Note: Different letters in the table indicate significant differences (P<0.05)

[0071] By analyzing the test results in Table 1, it can be seen that the treatment with the fermentation liquid of strain NJAU-HNB23 reached a corrected mortality rate of 92.47% after 48 hours, which was significantly higher than that of other treatments. The results show that the fermentation liquid of strain NJAU-HNB23 can effectively kill root-knot nematodes, and the killing effect of root-knot nematodes is significantly higher than that of other strains.

[0072] Example 3 Identification of strains NJAU-HNF11 and NJAU-HNB23

[0073] The strain NJAU-HNF11 was cultured on PDA medium at 28°C for 96 hours. The mycelium was white, the colonies were round with regular edges, the spores were green or yellow-green, the spores were easy to pick, and the mycelium was tightly connected to the culture medium. The 16srRNA gene sequence of the strain was compared with similar sequences, and a phylogenetic tree was constructed. The results showed that the strain NJAU-HNF11 had the highest homology with Trichoderma aureoviride. Combining the results of the phylogenetic tree comparison analysis of the colony morphology and the 16S rRNA gene sequence, the strain NJAU-HNF11 was identified as a Trichoderma sp. ( Figure 2 ). The phylogenetic tree results showed that the genus was harmless to crops and non-pathogenic to humans and animals. The strain NJAU-HNF11 has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.41158.

[0074] The strain NJAU-HNB23 was cultured on LB solid medium at 37°C for 24 hours. The colonies were white, round, moist and smooth, with neat edges and easy to pick. The 16S rRNA gene sequence of the strain was compared with similar sequences and a phylogenetic tree was constructed. The results showed that the strain NJAU-HNB23 had the highest homology with Bacillus altitudini. Combining the results of the phylogenetic tree comparison analysis of the colony morphology and the 16S rRNA gene sequence, the strain NJAU-HNB23 was identified as a Bacillus sp. ( Figure 3 ). The phylogenetic tree results showed that the genus was harmless to crops and non-pathogenic to humans and animals. The strain NJAU-HNB23 has been deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC No.30335.

[0075] Example 4: Potted plant experiment to study the effects of functional bacteria NJAU-HNF11 and NJAU-HNB23 on the aboveground growth of Hami melon plants, the number of soil pathogenic bacteria Fusarium oxysporum and the root growth

[0076] The potted plant test site is located in the culture room of Sanya Research Institute of Nanjing Agricultural University, Sanya City, Hainan Province. The test period is from November 2023 to January 2024. The test cantaloupe variety is "Dongfang 2029". The test agent is NJAU-HNB23 agent (take the activated NJAU-HNB23, pick a single colony and add it to 200mL LB liquid culture medium and shake it in a shaker. The culture temperature is 37°C, the speed is 170r / min, and the fermentation time is 12h to obtain NJAU-HNB23 agent. The bacterial content in the NJAU-HNB23 agent is ≥1×10 8 CFU / mL) and NJAU-HNF11 agent (the activated NJAU-HNF11 was inoculated on a PDA medium plate, wrapped with a sealing film, and placed in a 28°C incubator for 96 h to obtain a purified fungal strain plate; about 5 mL of sterile water was injected into the purified fungal strain plate, and the spores and sterile water were mixed with a sterile coating rod to form a spore liquid, and 200 μL of the spore liquid was pipetted with a pipette and added to 200 mL of PDB medium for shaking culture in a shaker. The culture temperature was 28°C, the rotation speed was 170 r / min, and the fermentation time was 120 h to obtain the NJAU-HNF11 agent, and the number of spores in the NJAU-HNF11 agent was ≥1×10 8 The test soil was a high-incidence soil inoculated with Fusarium oxysporum and root-knot nematodes.

[0077] The potted plant experiment was set up with 4 treatments, namely 1) CK, application of clean water; 2) NJAU-HNB23, application of NJAU-HNB23 microbial agent; 3) NJAU-HNF11, application of NJAU-HNF11 microbial agent; 4) NJAU-HNB23+NJAU-HNF11, application of NJAU-HNB23+NJAU-HNF11 composite microbial agent (composite microbial agent obtained by mixing NJAU-HNB23 microbial agent and NJAU-HNF11 microbial agent in a volume ratio of 1:1). Each treatment was repeated 5 times.

[0078] The pot experiment was conducted in a greenhouse, with the temperature set at 30℃ for 16h with light on, 26℃ for 8h with light off, and the air humidity at 60%. Hami melon seedlings were self-cultivated, and after germination for 7d, the seedlings with consistent growth were selected and transplanted into flower pots (the flower pots were 8.5cm high, 7.5cm in diameter, 6.8cm in diameter at the bottom, and about 120mL of soil and 10mL of vermiculite were used for each pot, and the soil and vermiculite were mixed evenly for use). One Hami melon seedling was transplanted in each pot, and water was poured until water seeped out from the bottom of the pot after transplanting. Subsequently, water was poured twice a week, with 50mL of water poured into each pot each time. 10d after transplanting, 1mL of bacterial agent was applied to each pot by root irrigation. After transplanting for 40d, 5 pots of potted plants were taken from each treatment for index determination (fresh weight of aboveground part, dry weight of aboveground part, plant height, and number of Fusarium oxysporum).

[0079] Plant height: Use a tape measure to measure from the soil surface of the cantaloupe plant to the top of the main stem.

[0080] Fresh weight and dry weight of aboveground parts: The aboveground parts of Hami melon plants were cut off and weighed to obtain the fresh weight of aboveground parts. The aboveground parts of Hami melon plants were then placed in an oven and dried at 65°C until constant weight was obtained, and then weighed to obtain the dry weight of aboveground parts.

[0081] Number of Fusarium oxysporum: The number of Fusarium oxysporum in the rhizosphere of Hami melon was determined by the dilution spreading counting method. The specific steps are as follows:

[0082] Cut off the Hami melon plants from the soil surface, gently shake off the loose soil and large particles attached to the root surface, and remove impurities such as stones; take 20mL of sterile water in a 50mL sterile centrifuge tube, put the treated Hami melon roots (about 5g) into the centrifuge tube, oscillate at 25℃ and 170r / min for 1h until there is no obvious soil attached to the root surface, remove the roots with sterile tweezers, and obtain the rhizosphere soil suspension. Add 0.9mL of sterile water and 0.1mL of rhizosphere soil suspension to a 1.5mL sterile centrifuge tube, mix well, and then dilute with sterile water to 10 -2 , 10 -3 , 10 -4 , 10 -5 , absorb 10 -3 , 10 -4 , 10 -5 100 μL of the soil dilution was evenly spread on a K2 selective medium plate using a spreading rod, and cultured in a 28°C incubator until single colonies appeared and then counted.

[0083] Table 2 Growth of aboveground parts of Hami melon plants and soil pathogenic fungus Fusarium oxysporum in potted plant experiment

[0084]

[0085] Note: Different letters in the table indicate significant differences (P<0.05)

[0086] The aboveground growth of Hami melon plants in each treatment group is shown in Table 2. From the perspective of plant height, the Hami melon plants treated with the NJAU-HNB23+NJAU-HNF11 compound microbial agent were significantly higher than those treated with the CK and single microbial agents; from the perspective of aboveground fresh weight, the aboveground fresh weight of the plants treated with the NJAU-HNB23+NJAU-HNF11 compound microbial agent was significantly higher than those treated with the CK and single microbial agents; from the perspective of aboveground dry weight, the aboveground dry weight of the plants treated with the NJAU-HNB23+NJAU-HNF11 compound microbial agent was also the highest, which was significantly better than the CK and NJAU-HNF11 single microbial agent treatments. Combining the aboveground fresh weight, aboveground dry weight and plant height, it can be clearly seen that the growth of Hami melon plants treated with the NJAU-HNB23+NJAU-HNF11 compound microbial agent was significantly better than that of the CK and single microbial agent treatments. The number of Fusarium oxysporum in the CK treatment was significantly higher than that in the bacterial agent treatment, and the number of Fusarium oxysporum in the treatment with the NJAU-HNB23+NJAU-HNF11 composite bacterial agent was the lowest. Figure 4 It can be seen that the Hami melon roots treated with the NJAU-HNB23+NJAU-HNF11 composite microbial agent had no obvious root knots, the CK treatment had the largest number of root knots, and the single microbial agent treatment also had root knots to varying degrees. From Example 1, it can be seen that NJAU-HNF11 can effectively antagonize the pathogenic fungus Fusarium oxysporum of Hami melon wilt, and from Example 2, it can be seen that NJAU-HNB23 can effectively kill root-knot nematodes. From the results of the potted plant test, it can be seen that the NJAU-HNB23+NJAU-HNF11 composite microbial agent can not only better reduce the number of Fusarium oxysporum in the soil and the generation of root knots in Hami melon plants compared to a single microbial agent, but also better promote the growth of Hami melon plants.

[0087] Example 5 Field effect verification of NJAU-HNB23+NJAU-HNF11 composite microbial agent

[0088] The field test was conducted at the Batou Base of Sanya Research Institute of Nanjing Agricultural University from December 2023 to February 2024. Two treatments were set up, namely CK treatment and NJAU-HNB23+NJAU-HNF11 treatment. CK treatment is the application of water-soluble fertilizer, and NJAU-HNB23+NJAU-HNF11 treatment is the application of water-soluble fertilizer and NJAU-HNB23+NJAU-HNF11 composite bacterial agent. Water-soluble fertilizer is obtained by diluting the water-soluble fertilizer stock solution 500 times with clean water. The formula of water-soluble fertilizer stock solution is (1L): 100g urea, 50g potassium dihydrogen phosphate, 100mL amino acid (purchased from: Jiangyin Pengyao Lianye Biotechnology Co., Ltd.), 100mL nicotine solution (purchased from: Hainan Yikang Ecological Agriculture Development Co., Ltd.), and the rest is sterile water. NJAU-HNB23+NJAU-HNF11 composite bacterial agent is prepared in the same way as in Example 4. Twenty-five Hami melon seedlings (the Hami melon seedling variety for the test was "Dongfang 2029", purchased from Cuizhenzheng professional seedling base in Ledong Li Autonomous County, Hainan Province, and Hami melon seedlings with consistent growth were selected for each treatment) were planted in each treatment (with a spacing of about 1 m between plants and about 50 cm between rows). 30 days after planting, NJAU-HNB23+NJAU-HNF11 compound bacterial agent and water-soluble fertilizer were applied by root irrigation. In the NJAU-HNB23+NJAU-HNF11 treatment, 10 mL of NJAU-HNB23+NJAU-HNF11 compound bacterial agent was applied to each Hami melon plant. In all treatments, 500 mL of water-soluble fertilizer was applied to each Hami melon plant, once every 15 days, for a total of 2 applications. Ten days after the second application, the indicators (fresh weight of the aboveground part, dry weight of the aboveground part, number of Fusarium oxysporum, and number of root-knot nematodes) of each treatment were measured. Except for the application of NJAU-HNB23+NJAU-HNF11 compound fungicide, other field management measures of each treatment remained consistent.

[0089] The aboveground fresh weight and aboveground dry weight were determined in the same manner as in Example 4.

[0090] Number of Fusarium oxysporum: The number of Fusarium oxysporum in the rhizosphere of Hami melon was determined by the dilution spreading counting method. The specific steps are as follows:

[0091] Cut off the Hami melon plants from the soil surface, gently shake off the loose soil and large particles attached to the root surface, and remove impurities such as stones; take 200mL of sterile water in a 400mL sterile tissue culture bottle, put the treated Hami melon roots (about 40g) into the bottle, shake at 25℃ and 170r / min for 1h until there is no obvious soil attached to the root surface, remove the roots with sterile tweezers, and obtain the rhizosphere soil suspension. Add 0.9mL of sterile water and 0.1mL of rhizosphere soil suspension to a 1.5mL sterile centrifuge tube, mix well, and then dilute with sterile water to 10 -2 , 10 -3 , 10 -4 , 10-5 , absorb 10 -3 , 10 -4 , 10 -5 100 μL of the soil dilution was evenly spread on a K2 selective medium plate using a spreading rod, and cultured in a 28°C incubator until single colonies appeared and then counted.

[0092] Number of root-knot nematodes: Each Hami melon plant was cut from the soil surface, and loose soil and large particles attached to the root surface were gently shaken off, and impurities such as stones were removed; loose soil attached to the root surface was collected for the determination of the number of root-knot nematodes. 20 g of each soil sample was placed in a sterilized centrifuge tube, 30 mL of sterile water was added, and the soil was placed in a centrifuge after shaking, and centrifuged at 3000 r / min for 3 min. After centrifugation, the supernatant was poured out; then, 30 mL of sucrose solution with a concentration of 484 g / L was added to the centrifuge tube, and the tube was placed in a centrifuge again at 3000 r / min. After centrifugation for 3 min, the supernatant was poured into a 500 mesh and 1000 mesh sieve for filtration, and the material in the sieve was rinsed with sterile water into a 50 ml centrifuge tube for collecting root-knot nematodes; finally, the 50 mL centrifuge tube was placed in a water bath and heated at 65 ° C for 30 min to kill the root-knot nematodes, and the root-knot nematodes were counted using an inverted microscope.

[0093] The results are as follows Figure 5 As shown, the aboveground fresh weight and aboveground dry weight of the treatment with NJAU-HNB23+NJAU-HNF11 composite microbial agent were significantly higher than those of the CK treatment. At the same time, the number of pathogenic Fusarium oxysporum and root-knot nematodes in the treatment with NJAU-HNB23+NJAU-HNF11 composite microbial agent was significantly lower than that of the CK treatment. The application of NJAU-HNB23+NJAU-HNF11 composite microbial agent can not only significantly reduce the number of pathogenic Fusarium oxysporum and root-knot nematodes in the soil, but also has a significant growth-promoting effect on Hami melon. In summary, the NJAU-HNB23+NJAU-HNF11 composite microbial agent has a significant prevention and control effect on Hami melon wilt and root-knot nematode disease, and also has a significant growth-promoting effect on Hami melon.

Claims

1. A composite bacterial agent for preventing and controlling Hami melon wilt and root knot nematode disease, characterized in that: It includes Trichoderma fungus NJAU-HNF11 and Bacillus bacterium NJAU-HNB23; the Trichoderma fungus NJAU-HNF11 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, the deposit date is April 15, 2024, and the deposit number is CGMCC No.41158; the Bacillus bacterium NJAU-HNB23 is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, the deposit date is April 15, 2024, and the deposit number is CGMCC No.30335.

2. A composite bacterial agent for preventing and controlling Hami melon wilt and root-knot nematode disease according to claim 1, characterized in that: The composite bacterial agent is prepared by the following steps: (1) subjecting the Trichoderma fungus NJAU-HNF11 with a deposit number of CGMCC No. 41158 to liquid fermentation to obtain a NJAU-HNF11 bacterial agent; The Bacillus bacteria NJAU-HNB23 with a deposit number of CGMCC No. 30335 is subjected to liquid fermentation to obtain a NJAU-HNB23 bacterial agent; (2) Mixing the NJAU-HNF11 bacterial agent and the NJAU-HNB23 bacterial agent to obtain the composite bacterial agent.

3. A composite bacterial agent for preventing and controlling Hami melon wilt and root-knot nematode disease according to claim 2, characterized in that: The composite bacterial agent is prepared by the following steps: (1) Inoculating spores of Trichoderma fungus NJAU-HNF11 with a deposit number of CGMCC No. 41158 into a liquid culture medium for liquid fermentation, wherein the liquid culture medium includes a PDA liquid culture medium, and the liquid fermentation conditions are: a fermentation temperature of 25-28° C., a rotation speed of 170-190 r / min, and a fermentation time of 96-120 h. After the fermentation, an NJAU-HNF11 bacterial agent is obtained, and the number of spores in the NJAU-HNF11 bacterial agent is ≥ 1×10 8 Pieces / mL; The Bacillus bacteria NJAU-HNB23 with a preservation number of CGMCC No. 30335 was inoculated into a liquid culture medium for liquid fermentation. The liquid culture medium included an LB liquid culture medium. The liquid fermentation conditions were as follows: a fermentation temperature of 35-37° C., a rotation speed of 170-190 r / min, and a fermentation time of 12-24 h. After the fermentation, an NJAU-HNB23 bacterial agent was obtained. The bacterial content in the NJAU-HNB23 bacterial agent was ≥1×10 8 CFU / mL; (2) Mixing equal volumes of NJAU-HNF11 bacterial agent and NJAU-HNB23 bacterial agent to obtain the composite bacterial agent.

4. A method for preparing a composite bacterial agent for preventing and controlling Hami melon wilt and root-knot nematode disease, characterized in that: The composite bacterial agent includes Trichoderma fungus NJAU-HNF11 and Bacillus bacteria NJAU-HNB23; the Trichoderma fungus NJAU-HNF11 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with a deposit date of April 15, 2024 and a deposit number of CGMCC No.41158; the Bacillus bacteria NJAU-HNB23 is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with a deposit date of April 15, 2024 and a deposit number of CGMCC No.30335; The preparation of the composite bacterial agent comprises the following steps: (1) subjecting the Trichoderma fungus NJAU-HNF11 with a deposit number of CGMCC No. 41158 to liquid fermentation to obtain a NJAU-HNF11 bacterial agent; The Bacillus bacteria NJAU-HNB23 with a deposit number of CGMCC No. 30335 is subjected to liquid fermentation to obtain a NJAU-HNB23 bacterial agent; (2) Mixing the NJAU-HNF11 bacterial agent and the NJAU-HNB23 bacterial agent to obtain the composite bacterial agent.

5. The method for preparing a composite bacterial agent for preventing and controlling Hami melon wilt and root-knot nematode disease according to claim 4, characterized in that: The preparation of the composite bacterial agent comprises the following steps: (1) Inoculating spores of Trichoderma fungus NJAU-HNF11 with a deposit number of CGMCC No. 41158 into a liquid culture medium for liquid fermentation, wherein the liquid culture medium includes a PDA liquid culture medium, and the liquid fermentation conditions are: a fermentation temperature of 25-28° C., a rotation speed of 170-190 r / min, and a fermentation time of 96-120 h. After the fermentation, an NJAU-HNF11 bacterial agent is obtained, and the number of spores in the NJAU-HNF11 bacterial agent is ≥ 1×10 8 Pieces / mL; The Bacillus bacteria NJAU-HNB23 with a preservation number of CGMCC No. 30335 was inoculated into a liquid culture medium for liquid fermentation. The liquid culture medium included an LB liquid culture medium. The liquid fermentation conditions were as follows: a fermentation temperature of 35-37° C., a rotation speed of 170-190 r / min, and a fermentation time of 12-24 h. After the fermentation, an NJAU-HNB23 bacterial agent was obtained. The bacterial content in the NJAU-HNB23 bacterial agent was ≥1×10 8 CFU / mL; (2) Mixing equal volumes of NJAU-HNF11 bacterial agent and NJAU-HNB23 bacterial agent to obtain the composite bacterial agent.

6. Use of the composite bacterial agent described in any one of claims 1 to 3 in preventing and controlling Hami melon wilt, root knot nematode disease, and promoting Hami melon growth.

7. The use according to claim 6, characterized in that: When applied, the composite bacterial agent is applied 30-35 days after Hami melon is planted, and is applied 2-4 times, with an interval of 10-15 days between each application, and 10-15 mL of the composite bacterial agent is applied to each plant each time.

8. The use according to claim 7, characterized in that: The composite bacterial agent is applied in the form of root irrigation.

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