Complex microbial inoculant for preventing and treating rice bakanae disease as well as preparation method and application of complex microbial inoculant

Abstract

The invention discloses a composite microbial inoculant for preventing and treating rice bakanae disease as well as a preparation method and application of the composite microbial inoculant, the composite microbial inoculant is composed of a fungal hypha suspension of plant endophytic fungi phomopsis sp. And a bacterial suspension formed by mixing pseudomonas otitis media BF72 and bacillus subtilis BF86 according to an equal proportion; the amount (dry weight) of fungal hyphae in the composite bacterial agent is 0.01 g / 5ml, and the effective viable count of the composite bacterial suspension is 1 * 10 < 8 >-10 < 9 > CFU. The composite microbial agent screened, combined and constructed by the invention has an obvious inhibition effect on the occurrence of the rice bakanae disease, improves the growth state of diseased rice, reduces the abundance of pathogenic bacteria in the rice, and improves the POD, SOD and PAL activities of the overground part and the underground part of the rice, so that the resistance of the rice to the pathogenic bacteria is enhanced, and the control effect of the composite microbial agent is superior to that of a chemical agent. The method is low in operation cost, has universality and is easy for large-scale production.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and biofertilizers, and in particular relates to a microbial composite agent for preventing and controlling rice seedling disease, and a preparation method and application thereof. Background Art

[0002] Rice (Oryza sativa L.) is the most important food crop in the world and an important food source for more than half of the world's population. China is the world's largest rice producer and consumer, accounting for nearly 30% of the world's annual rice production and consumption, and more than 60% of the population relies on rice as their staple food. Therefore, rice occupies an important position in my country's entire agricultural production system.

[0003] During the planting process, rice is often subjected to various biological and abiotic stresses in the natural environment. Among them, pests and diseases are the most important type of biological stress affecting rice yield, including infections by insects, fungi, bacteria and nematodes. At present, rice seedling blight is widely prevalent in Asia. Under field conditions, the disease threatens the quality and yield of rice, and the loss can reach 95% under severe infection. At present, the prevention and control methods for rice seedling blight mainly include cultivating resistant rice varieties and chemical control. However, rice varieties with high resistance to seedling blight are very rare; although chemical control is economical and efficient, it is easy for pathogens to gradually develop drug resistance, and it is not friendly to humans and animals, and it is harmful to the environment.

[0004] In recent years, more people have turned their attention to biological control. Beneficial microorganisms growing inside and outside plants control plant diseases by secreting antimicrobial substances, competing with pathogens for nutrients and ecological niches, inducing plant resistance, and promoting plant growth. However, due to changes in the planting environment, differences in water and fertilizer management, and farming measures, the long-term use of a single strain of bacteria has problems such as single functional effects and poor adaptability. Therefore, the research trend of microbial agents has gradually tended to multi-strain, multifunctional composite microbial agents. How to efficiently and stably exert antibacterial effects and be safe and effective is still a key issue that needs to be solved in the development of composite agents. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a microbial composite agent for preventing and controlling rice seedling disease, and a preparation method and application thereof. By screening bacteria that have good antagonistic effects on Fusarium fujikura, the main pathogen of rice seedling disease, and combining these bacteria with a plant endophytic fungus Phomopsis sp., a multifunctional and efficient cross-kingdom synthetic bacterial community with both disease resistance and growth promotion is obtained. Finally, a potted plant experiment is used to verify that the composite agent can effectively alleviate the occurrence of rice seedling disease. Compared with chemical control, it has the advantages of low cost, economy and environmental protection, and has good application prospects.

[0006] In order to solve the problems of the prior art, the technical solution adopted by the present invention is:

[0007] A microbial composite agent for preventing and treating rice seedling bakanae disease comprises a fungal mycelium suspension and a bacterial suspension. The strain of the fungal mycelium suspension is Phomopsis 80439, and the strain of the bacterial suspension comprises Pseudomonas otitidis BF72 and Bacillus subtilis BF86.

[0008] As an improvement, the dry weight of the mycelium of Phomopsis 80439 was 0.01 g / 5 ml, and the effective viable count of the composite bacterial suspension was 1×10 8 ~10 9 CFU.

[0009] As an improvement, the Pseudomonas otitidis BF72 was isolated from rice rhizosphere soil and deposited in the China Center for Type Culture Collection on November 4, 2024, with the deposit address being Wuhan University, Wuhan, China, Postal Code 430072, with the deposit number CCTCC NO.M 20242426, and the taxonomic name being Pseudomonas otitidis BF72.

[0010] A further improvement is that the Pseudomonas otitidis BF72 is cultured on LB medium at 37°C for 24 hours, and the colonies are round, yellow-green and transparent, with a smooth and moist surface, regular edges, no halos, a central bulge, a diameter of 1-2 mm, and Gram staining is negative; it can utilize carbon sources such as L-asparagine, L-arginine, γ-hydroxybutyric acid, and has the ability to fix nitrogen, solubilize potassium, and produce protease.

[0011] As an improvement, the Bacillus subtilis BF86 was isolated from rice rhizosphere soil and deposited in the China Center for Type Culture Collection on November 4, 2024, with the deposit address being Wuhan University, Wuhan, China, Postal Code 430072, with the deposit number being CCTCC NO.M 20242427, and the classification name being Bacillus subtilis BF86.

[0012] Further improvement is that the Bacillus subtilis BF86 is cultured on LB medium at 37°C for 24 hours, the colonies are round, dirty white and opaque, with wrinkles on the surface, irregular edges, 2-3mm in diameter, Gram staining is positive, and can produce spores. It has the ability to produce cellulase and protease.

[0013] The preparation method of the above-mentioned microbial composite agent for preventing and controlling rice seedling disease is as follows: the plant endophytic fungus Phomopsis sp. is inoculated into PDB medium at an inoculum rate of 1:10 (v:v), and the culture is shaken for 48 hours, the culture solution is filtered to obtain fungal mycelium, and the mycelium is fully rinsed with sterile water, and then the mycelium is resuspended with sterile water to prepare a fungal mycelium suspension with a dry weight of 0.01 g / 5 ml; Pseudomonas otitis media BF72 and Bacillus subtilis BF86 are inoculated into LB medium respectively, and the culture is shaken until OD 600 The absorbance at nm was 1.0, and the bacteria were washed and resuspended with PBS to make the OD of each bacterium 600 After the value was maintained at 0.01, the mixture was mixed in equal volumes to make the number of viable bacteria reach 1×10 8 ~10 9 CFU, that's it.

[0014] Application of the above-mentioned microbial composite agent for preventing and controlling rice seedling disease in preventing and controlling rice seedling disease.

[0015] As an improvement, the steps of the above application are as follows: transplant the germinated rice seeds into the seedling medium, water the roots with the fungal mycelium suspension of the above-mentioned disease-resistant microbial composite agent for preventing and controlling rice seedling disease, at a watering amount of 5 ml / plant, and water every 7 days with the bacterial suspension of the above-mentioned disease-resistant microbial composite agent for preventing and controlling rice seedling disease, at a watering amount of 5 ml / plant.

[0016] Beneficial effects:

[0017] Compared with the prior art, the microbial composite agent for preventing and controlling rice seedling disease and its preparation method and application of the present invention have the following advantages:

[0018] 1. The microbial composite agent of the present invention is composed of endophytic fungi and bacteria. The composite agent can significantly improve the phenotype of diseased rice, reduce the disease severity and abundance of pathogens of rice seedling blight, increase the POD, SOD and PAL activities of the aboveground and underground parts of rice, thereby enhancing the resistance of rice to pathogens, and can achieve or even exceed the effect of chemical control, and is highly efficient, economical and environmentally friendly.

[0019] 2. The present invention compares the control effects of single bacteria and combined bacteria of the above three strains through greenhouse potted experiments. The composite bacterial agent has the best control effect on rice seedling disease and can increase the root length and biomass of rice under disease conditions, providing an effective composite bacterial agent for controlling rice seedling disease.

[0020] 3. The present invention has low operating cost, universal applicability and is easy for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1The two bacteria isolated from the rhizosphere soil of rice in Example 1 have good antagonistic effects on the pathogen of Bakanae disease;

[0022] Figure 2 The phylogenetic tree of the two bacterial strains in Example 1 constructed based on 16s rRNA gene sequencing sequences;

[0023] Figure 3 The diagram is a diagram of the mutual antagonism between the strains that need to be combined in Example 2;

[0024] Figure 4 The control effect of different bacterial strain combinations on rice seedling disease is shown in Figure 1. (a) is the disease index of rice seedling disease, and (b) is the control effect.

[0025] Figure 5 The agronomic index diagram of rice after the application of different strain combinations, (a) is the height of rice plants, (b) is the length of rice root system, (c) is the fresh weight of rice plants, and (d) is the dry weight of rice plants;

[0026] Figure 6 This is a diagram of rice growth in each treatment group of the present invention;

[0027] Figure 7 The figures are the disease degree diagrams of rice bakanae disease in each treatment group of the present invention, (a) is the incidence rate of rice bakanae disease, and (b) is the disease index of rice bakanae disease;

[0028] Figure 8 The abundance diagram of the pathogen of Bakanae disease in rice plants of each treatment group of the present invention;

[0029] Fig. 9 Figures 2 and 3 show the POD, SOD and PAL activities of the aboveground and underground parts of the rice in each treatment group of the present invention on different days, (a) is the POD activity in the rice root, (b) is the POD activity in the rice leaf, (c) is the SOD activity in the rice root, (d) is the SOD activity in the rice leaf, (e) is the PAL activity in the rice root, and (f) is the PAL activity in the rice leaf. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0031] Example 1 Isolation, screening and identification of antagonistic bacteria

[0032] 1. Isolation and purification of test strains

[0033] The tested bacterial strains were isolated from the rhizosphere soil of rice plants in the tillering stage planted in the Botanical Garden of Xianlin Campus of Nanjing Normal University, Nanjing, Jiangsu Province. 1 g of the sample was added to 100 ml of sterile water and cultured in a 37°C incubator for 24 h. The soil sample solution was heated for 10 min. 5 -10 6 The dilution was spread on a plate containing LB solid medium with 2% agar and cultured at 37°C overnight. The strain was isolated and purified from the plate with colonies. The purified strain was inoculated into LB liquid medium and incubated in a shaking incubator at 37°C and 180 rpm for 24 hours. The strain culture solution was mixed with 50% glycerol in a ratio of 1:1 and stored in a -40°C refrigerator.

[0034] The LB medium formula is: 10g peptone, 5g yeast powder, 10g sodium chloride. If preparing solid culture medium, add 15-20g agar, distilled water to 1000mL, pH = 7.0.

[0035] 2. Screening of bacteria with in vitro antagonistic activity

[0036] (1) Prepare potato dextrose medium (PDA / PDB) according to the following formula:

[0037] 200g potatoes, cut into pieces and boil until soft, leaving the soaking juice, 20g glucose, if preparing solid culture medium (PDA), add 15-20g agar, distilled water to make up to 1000mL, pH = 7.0.

[0038] (2) Screening of antagonistic strains by spore germination method

[0039] The isolated bacteria were screened by spore germination method. The specific operation was as follows: single bacterial colonies were selected and placed in liquid LB medium and incubated in a shaking incubator at 37°C and 180 rpm for 24 h. Fusarium hyphae were selected and placed in PDB and incubated in a shaking incubator at 28°C and 180 rpm for 72 h. The Fusarium liquid culture was filtered and the hyphae were centrifuged to obtain the spore precipitate, which was resuspended in sterile water and adjusted to OD 600 The value is 0.1. Take 100 μL of spore suspension and spread it on the PDA plate. After it is naturally air-dried, take 5 μL of bacterial liquid and drop it on the culture medium. Place it in a 28°C incubator and culture it for 2-3 days to observe whether there is a transparent circle around the bacteria that inhibits the germination of Fusarium spores.

[0040] The Fusarium is a pathogen that can cause typical rice bakanae symptoms, Fusarium fujikura, and is a commercially available material.

[0041] (3) Screening of antagonistic strains by plate confrontation method

[0042] The antagonistic strains initially screened out by the spore germination method were screened again by the plate confrontation method. The specific operation was as follows: a single bacterial colony was picked up in a liquid LB culture medium and placed in a shaking incubator at 37°C and 180rpm for incubation for 24 hours; a round Fusarium cake was placed on one side of a 90mm PDA culture medium using a 5mm diameter puncher, and a vertical line was drawn on the other side 1.5cm away from the fungal cake using a toothpick to dip in the bacterial liquid, and the mycelium inhibition rate of the bacteria on the fungus was calculated after being cultured in a 28°C incubator for 5-7 days.

[0043] 3. Identification of bacterial species and construction of phylogenetic tree

[0044] (1) Molecular identification of the strain was performed by PCR amplification of the 16s rRNA gene of the strain using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTACGACTT-3'). The PCR products were bidirectionally tested by Beijing Qingke Biotechnology Co., Ltd. (Nanjing). The obtained sequences were compared with homologous sequences in GenBank using NCBI-Blast. Genes of closely related species were downloaded from Genbank for phylogenetic analysis. The downloaded Escherichia coli ATCC 1175 was used as an outgroup.

[0045] (2) After the obtained bacterial gene sequences were sorted and edited, a phylogenetic tree was constructed using the maximum likelihood method using MEGA11 software.

[0046] 4. Results and Analysis

[0047] Figure 1 These two antagonistic bacteria, BF72 and BF86, were screened out through spore germination method and plate confrontation method and have obvious inhibitory effects on Fusarium fujikuraensis.

[0048] Phylogenetic analysis Figure 2 As shown, BF72 was identified as Pseudomonas otitidis and was deposited in the China Center for Type Culture Collection on November 4, 2024. The collection address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University Collection Center, Postal Code 430072, Collection Number CCTCC NO.M 20242426, and the taxonomic name is Pseudomonas otitidis BF72.

[0049] The above-mentioned Pseudomonas otitidis BF72 was cultured on LB medium at 37°C for 24 hours, and the colonies were round, yellow-green and transparent, with a smooth and moist surface, regular edges, no halo, a central bulge, a diameter of 1-2 mm, and Gram staining was negative. It can utilize carbon sources such as L-asparagine, L-arginine, and γ-hydroxybutyric acid, and has the ability to fix nitrogen, dissolve potassium, and produce protease.

[0050] BF86 was identified as Bacillus subtilis and was deposited in the China Center for Type Culture Collection on November 4, 2024. The collection address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University Collection Center, Postal Code 430072, the collection number is CCTCC NO.M 20242427, and the classification name is Bacillus subtilis BF86.

[0051] The above-mentioned Bacillus subtilis BF86 was cultured on LB medium at 37°C for 24 hours, and the colonies were round, dirty white and opaque, with wrinkles on the surface, irregular edges, 2-3 mm in diameter, Gram staining was positive, and it was able to produce spores and had the ability to produce cellulase and protease.

[0052] Example 2 Screening of the optimal combination of bacterial flora for prevention

[0053] The endophytic fungus Phomopsis sp. used in the present invention has a deposit number of CFCC80439 and is purchased from the China Forestry Microbial Culture Collection Management Center.

[0054] 1. Evaluation of strain antagonism

[0055] In order to facilitate the subsequent combination of bacterial colonies and verify whether the bacterial colonies can coexist in a friendly manner, it is necessary to evaluate the antagonistic relationship between the strains. The specific operation is as follows: the bacterial strains are activated with LB medium to the logarithmic growth phase. Before the confrontation experiment, the original bacterial culture was centrifuged to retain the bacteria, the residual culture medium was washed away with sterile saline, and the two bacterial strains were adjusted to OD 600 =0.5.

[0056] (1) Confrontation between bacteria: 100 μL of each bacterial suspension was spread on an LB solid plate. After air-drying, 5 μL of the non-spread bacterial suspension was taken and dropped on three different locations on the air-dried culture medium. The plate was cultured at 28°C for 24 h, and the formation of a transparent ring around the bacteria was observed.

[0057] (2) Confrontation between fungi and bacteria: Use a 5 mm diameter puncher to place a circular fungal cake on one side of a 90 mm PDA culture medium. On the other side, use a toothpick to dip into bacterial liquid and draw a vertical line 1.5 cm away from the fungal cake. Culture at 28°C for 7 days to observe whether the bacteria have an inhibitory effect on fungal hyphae.

[0058] 2. Preparation of single bacterial inoculum

[0059] The plant endophytic fungus Phomopsis sp. was inoculated into PDB medium at an inoculum ratio of 1:10 (v:v), and cultured in a shake flask for 48 hours. The obtained culture solution was filtered to obtain fungal mycelium, which was fully rinsed with sterile water and then resuspended with sterile water to prepare a fungal mycelium suspension with a dry weight of 0.01 g / 5 ml.

[0060] The two antagonistic bacteria were inoculated into LB medium and cultured overnight. The bacterial solution that reached the logarithmic growth phase was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, the bacteria were washed 2-3 times with sterile PBS, and the bacterial solution was resuspended to OD 600 =0.01, to obtain a single bacterial suspension.

[0061] 3. Preparation of combined bacterial suspension and biological composite bacterial agent

[0062] The single bacterial suspensions of the above two bacterial strains are mixed in equal proportions to prepare a composite bacterial suspension.

[0063] A microbial composite agent for preventing and controlling rice seedling disease comprises a fungal mycelium suspension and a bacterial suspension, wherein the strain of the fungal mycelium suspension is Phomopsis 80439, and the strain of the bacterial suspension comprises Pseudomonas otitidis BF72 and Bacillus subtilis BF86; wherein the dry weight of the fungal mycelium is 0.01 g / 5 ml, and the effective viable count of the composite bacterial suspension is 1×10 8 ~10 9 CFU.

[0064] 4. Comparison of the control effects of single bacteria and combined bacterial flora on rice seedling disease

[0065] Single bacteria Phomopsis (E+F), single bacteria Pseudomonas otitidis BF72 (BF72+F), single bacteria Bacillus subtilis BF86 (BF86+F), combination of Phomopsis and Pseudomonas otitidis BF72 (E+BF72+F), combination of Phomopsis and Bacillus subtilis BF86 (E+BF86+F), combination of Phomopsis, Pseudomonas otitidis BF72 and Bacillus subtilis BF86 (E+BF72+BF86+F) (volume ratio of composite bacterial suspension was 1:1) were set to control rice seedling blight caused by Fusarium fujikura, and the group inoculated with Fusarium fujikura alone (F) was used as the negative control.

[0066] After disinfecting the rice seeds, soak them in sterile water for 1-2 days to allow the seeds to absorb water and swell. Then spread the seeds evenly on a culture dish covered with sterilized filter paper, soak the filter paper with sterile water to keep the moisture, wait for the seeds to germinate, select the germinated rice seeds with the same bud length and plant them in sterile vermiculite, and inoculate the roots with sterile water or 5 ml / plant of the fungal mycelium suspension of the endophytic fungus Phomopsis after 7 days. Then inoculate with sterile water, single bacterial suspension or composite bacterial suspension 5 ml / plant again after 7 days.

[0067] After 48 hours, the spore suspension of Fusarium fusarius was inoculated and the rice was irrigated with nutrient solution regularly. After 14 days, the disease degree of rice in each treatment group was counted, the agronomic traits of rice plants in each treatment group were measured, and the control effects of single bacteria and combined bacteria were compared.

[0068] 5. Results and Analysis

[0069] Figure 3 The figure shows the mutual antagonism between the two antagonistic bacteria, Pseudomonas otitidis BF72 and Bacillus subtilis BF86, on the LB plate and with the endophytic fungus Phomopsis on the PDA plate. It can be seen from the figure that there is no antagonism between the two bacteria, Pseudomonas otitidis BF72 and Bacillus subtilis BF86. Therefore, the two bacteria were made into a composite bacterial suspension for subsequent experimental inoculation; but the two bacteria inhibited the hyphae growth of the endophytic fungus Phomopsis. Therefore, the subsequent bacterial combination inoculation adopted the method of inoculating the hyphae suspension of the endophytic fungus Phomopsis on rice 7 days before the bacterial suspension or the composite bacterial suspension.

[0070] Figure 4 The figure shows a comparison of the effects of single bacteria and combined bacterial communities on rice seedling disease control. As can be seen from the figure, the combined bacterial community of plant endophytic fungi Phomopsis spp., Pseudomonas otitidis BF72 and Bacillus subtilis BF86 has the mildest disease compared with single bacteria and other combined bacterial communities, and has the best control effect on rice seedling disease.

[0071] Figure 5 Figure 1 is a graph of agronomic indicators of single bacteria and combined bacterial communities 14 days after infection with Fusarium fujikura. It can be seen from the figure that the plants in the F treatment group as the negative control are the tallest, but the fresh weight and dry weight of the plants are significantly lower than those of other treatment groups, showing significant symptoms of rice seedling blight: leggy and slender; while both the single bacteria and combined bacterial community treatment groups can improve the condition of rice seedling blight caused by Fusarium fujikura, and the three-strain combination of plant endophytic fungi Phomopsis, Pseudomonas aeruginosa BF72 and Bacillus subtilis BF86 (E+BF72+BF86+F) has the best growth condition, which can significantly increase the root length of rice and the biomass accumulation of rice.

[0072] Example 3 Comparison and application of the control effect of composite bacterial agent and chemical agent

[0073] 1. Potted experiment design

[0074] The potted plant experiment selected 6 treatment groups, including sterile water (CK), chemical agent fludioxonil (CH), combined bacterial community E+BF72+BF86 (S) with the best control effect, pathogen spore suspension (F), chemical agent treatment followed by pathogen spore suspension (CF), and combined bacterial community treatment followed by pathogen spore suspension (SF). Details are shown in Table 1 below.

[0075] Table 1 Treatment group

[0076]

[0077] After disinfection, rice seeds were soaked in sterile water or chemical agent mefluoxetine (CH and CF treatment groups) for 24 hours, then the seeds were evenly spread in a petri dish covered with sterile filter paper, the filter paper was soaked with sterile water to keep moisture, and the seeds were waited for whitening and germination. The germinated rice seeds with the same bud length were selected and planted in sterile vermiculite. After 7 days, the roots were inoculated with sterile water or fungal mycelium suspension of plant endophytic fungi Phomopsis (S and SF treatment groups) 5ml / plant, and then inoculated with sterile water or composite bacterial suspension (S and SF treatment groups) 5ml / plant, and 48 hours later, the spore suspension of Fusarium fusariosus was inoculated (F, CF and SF treatment groups), and the rice nutrient solution was irrigated every 5 days. After 14 days, the disease degree of rice seedling disease in each treatment group, the abundance of pathogens in the aboveground and underground parts of rice, and the agronomic traits of the plants were counted.

[0078] 2. Determination of agronomic traits of rice

[0079] Five rice plants were randomly selected from each treatment group, and their plant height, root length, and above- and underground dry weight were measured.

[0080] 3. Investigation on the severity of rice seedling disease

[0081] The grading standards for rice bakanae disease are as follows (using a 0-5 rating scale):

[0082] Disease level 0: The plant is healthy and has no disease symptoms;

[0083] Disease level 1: Plants are dwarfed; leaves are narrow and yellow;

[0084] Disease level 2: The plants are thin and weak; the leaves are narrow and light yellow;

[0085] Grade 3: Rice leaves are narrow and long; the leaves are light yellow; the internodes are thin and long; the root system is short;

[0086] Disease level 4: Rice is seriously affected by seedling blight and the top is necrotic;

[0087] Disease level 5: Plant death caused by seedling blight.

[0088] Incidence rate = number of diseased plants / total number of plants × 100%.

[0089] Disease index = 100% × ∑ (number of diseased plants at each level × representative value of each level) / (total number of plants surveyed × highest representative value).

[0090] 4. Determination of pathogen abundance in rice plants

[0091] AxyPrep Plant Genomic DNA Kit was used to extract DNA from the aboveground and underground parts of rice. The extracted DNA was identified by 1% (w / v) agarose gel electrophoresis and stored at -20°C. The content of Fusarium fujikuraensis, the pathogen of rice seedling disease, in the aboveground and underground parts of rice was determined by qRT-PCR reaction using Fusarium fujikuraensis-specific primers 45I-f1 (5′-CTGTTCGAGCGTCATTTCAA-3′) and 45I-r1 (5′-TGATCCGAGGTCAACATTCA-3′).

[0092] 5. Detection of antioxidant enzyme and disease resistance enzyme activity in rice

[0093] 0.1 g of fresh rice leaf and root tissues were collected and immediately ground in liquid nitrogen. The activities of peroxidase (POD), superoxide dismutase (SOD) and phenylalanine ammonia lyase (PAL) were determined using a test kit (Jiangsu Addison Biotechnology Co., Ltd., Yancheng, China) according to the instructions. One POD activity unit is expressed as 0.01 change in absorbance per gram of tissue per milliliter of reaction system per minute; one SOD activity unit is expressed as the amount of SOD inhibition percentage of 50% per gram of tissue per milliliter of reaction system; one PAL activity unit is expressed as 0.01 change in absorbance per gram of tissue per milliliter of reaction system per minute.

[0094] 6. Results and Analysis

[0095] Figure 6 The graphs show the growth of rice in each treatment group. As can be seen from the figure, in the treatment without inoculation of Fusarium fujikura, the composite bacterial agent has a significant effect on promoting the growth of the aboveground part of rice compared with the control group and the chemical agent treatment group; in the treatment with inoculation of Fusarium fujikura, the composite bacterial agent can significantly alleviate the occurrence of leggy and spindly symptoms caused by rice seedling baka, and is even better than the chemical agent treatment group.

[0096] Figure 7The figure shows the disease severity of rice seedling bakanae in each treatment group. It can be seen from the figure that the composite bacterial agent can significantly reduce the incidence and disease index of rice seedling bakanae caused by Fusarium fusarifolia, which is consistent with the effect of the chemical agent treatment group.

[0097] Figure 8 is the abundance diagram of the pathogen of Bakanae disease in rice plants of each treatment group; Figure 8 As shown, the synthetic bacterial community can significantly reduce the content of Fusarium fujikura in the aboveground and underground parts of rice, especially the content of Fusarium fujikura in the aboveground part, which is even lower than that of the chemical treatment group.

[0098] Fig. 9 The following are the activities of POD, SOD and PAL in the aboveground and underground parts of rice in each treatment group on different days. It can be seen from the figure that one day after the inoculation of Fusarium fujikura, the composite bacterial agent can significantly increase the activity of antioxidant enzyme POD in the underground part of rice and the activity of disease resistance-related enzyme PAL, as well as the activity of antioxidant enzyme POD and SOD in the aboveground part; 14 days after the inoculation of Fusarium fujikura, the composite bacterial agent can still significantly increase the activities of two antioxidant enzymes POD and SOD in the underground and aboveground parts of rice.

[0099] The results showed that the composite bacterial agent can enhance rice's resistance to Fusarium fujikori by continuously improving the activity of rice's antioxidant enzymes and disease resistance-related enzymes.

[0100] In summary, by screening bacteria that have good antagonistic effects on Fusarium fujikura, the main pathogen of rice seedling disease, and combining these bacteria with a plant endophytic fungus Phomopsis sp., a multifunctional and efficient cross-kingdom synthetic bacterial community with both disease resistance and growth promotion was obtained. Finally, potted experiments were used to verify that the composite bacterial agent can effectively alleviate the occurrence of rice seedling disease. Compared with chemical control, it has the advantages of low cost, economy and environmental protection, and has good application prospects.

Claims

1. A microbial composite agent for preventing and controlling rice seedling disease, characterized in that: The invention comprises a fungal mycelium suspension and a bacterial suspension. The strain of the fungal mycelium suspension is Phomopsis 80439, and the strain of the bacterial suspension comprises Pseudomonas otitidis BF72 and Bacillus subtilis BF86.

2. The microbial composite agent for preventing and controlling rice bakanae disease according to claim 1, characterized in that: The dry weight of mycelium of Phomopsis 80439 was 0.01 g / 5 ml, and the effective viable count of the composite bacterial suspension was 1 × 10 8 ~10 9 CFU.

3. The microbial composite agent for preventing and controlling rice bakanae disease according to claim 1, characterized in that: The Pseudomonas otitidis BF72 was isolated from rice rhizosphere soil and deposited in the China Center for Type Culture Collection on November 4, 2024. The deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University Collection Center, Postal Code 430072, deposit number CCTCC NO. M 20242426, and the taxonomic name is Pseudomonas otitidis Pseudomonas otitidis BF72 。 4. The microbial composite agent for preventing and controlling rice bakanae disease according to claim 3, characterized in that: The colonies of the Pseudomonas otitidis BF72 are round, yellow-green and transparent, with a smooth and moist surface, regular edges, no halos, a central bulge, a diameter of 1-2 mm, and Gram-negative staining; they can utilize carbon sources such as L-asparagine, L-arginine, and γ-hydroxybutyric acid, and have the ability to fix nitrogen, dissolve potassium, and produce protease.

5. The microbial composite agent for preventing and controlling rice bakanae disease according to claim 1, characterized in that: The Bacillus subtilis BF86 was isolated from rice rhizosphere soil and deposited in the China Center for Type Culture Collection on November 4, 2024. The deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University Collection Center, Postal Code 430072, with a deposit number of CCTCC NO. M 20242427, and is classified as Bacillus subtilis Bacillus subtilis BF86 。 6. The microbial composite agent for preventing and controlling rice bakanae disease according to claim 5, characterized in that: The Bacillus subtilis BF86 colony is round, dirty white and opaque, has wrinkles on the surface, irregular edges, a diameter of 2-3 mm, is Gram-positive, can produce spores, and has the ability to produce cellulase and protease.

7. A method for preparing a composite microbial agent for preventing and controlling rice bakanae disease according to any one of claims 1 to 6, characterized in that: The plant endophytic fungus Phomopsis was inoculated into PDB medium at a volume ratio of 1:10, and the culture was shaken for 48 hours. The culture solution was filtered to obtain fungal mycelium, which was fully rinsed with sterile water, and then the mycelium was resuspended with sterile water to prepare a fungal mycelium suspension with a dry weight of 0.01 g / 5 ml; Pseudomonas otitidis BF72 and Bacillus subtilis BF86 were inoculated into LB medium respectively, and the culture was shaken until OD 600 The absorbance at nm was 1.0, and the bacteria were washed and resuspended with PBS to make the OD of each bacterium 600 After the value was maintained at 0.01, the mixture was mixed in equal volumes to make the number of viable bacteria reach 1×10 8 ~10 9 CFU, that's it.

8. Use of the composite microbial agent for preventing and controlling rice bakanae disease according to any one of claims 1 to 6 in preventing and controlling rice bakanae disease.

9. The use according to claim 8, characterized in that: The germinated rice seeds are transplanted into a seedling medium, and the roots are watered with a fungal mycelium suspension of the above-mentioned disease-resistant microbial composite agent for preventing and controlling rice seedling disease at a rate of 5 ml / plant. Every 7 days, the roots are watered with a bacterial suspension of the above-mentioned disease-resistant microbial composite agent for preventing and controlling rice seedling disease at a rate of 5 ml / plant.

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