Functional seedling culture substrate for preventing and treating root-knot nematode and application of functional seedling culture substrate

Abstract

The invention belongs to the technical field of agricultural biology, and particularly discloses a functional seedling culture substrate for preventing and treating root-knot nematode and application of the functional seedling culture substrate. The functional seedling raising substrate is composed of a solid seedling raising substrate and a compound microbial agent uniformly dispersed in the solid seedling raising substrate. The compound microbial agent is prepared by carrying out mixed fermentation on bacillus velezensis RKN1111, bacillus laterosporus CDD-01 and paenibacillus mucilaginosus HX-02. The invention further discloses a preparation method of the compound microbial agent. When the functional seedling culture substrate is used for seedling culture, the colonization rate of the compound microbial agent in seedling rhizosphere is greatly improved, the inside and the outside of root lumps of cultured germchit are rich in active biocontrol bacteria during transplanting, the germchit can continuously play a high-efficiency and stable prevention and control role after transplanting, and the growth of the germchit is remarkably promoted. According to the method, the prevention and control opportunity of the root-knot nematode is greatly advanced from a field period to a controllable seedling raising period, active immunization is achieved, and the technical problems that colonization is difficult and the effect is unstable when a biocontrol microbial inoculum is applied to traditional root irrigation or fields are effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and plant protection, specifically relating to a functional seedling substrate for controlling root-knot nematodes and its application. Background Technology

[0002] Root-knot nematodes are a devastating soil-borne disease that harms greenhouse and high-efficiency agriculture. Chemical pesticide control presents problems such as residues, resistance, and environmental damage. Biological control, represented by microbial inoculants, represents a green alternative.

[0003] However, existing microbial agents used to control root-knot nematodes have significant functional limitations. For example, Bacillus laterosporus with accession number CGMCC No. 28825 has an inhibitory effect on southern root-knot nematode eggs in its fermentation broth and bacterial suspension, but lacks the function of promoting plant growth. Bacillus laterosporus with accession number CGMCC No. 24137 can reduce the number of root knots and has a growth-promoting effect, but it does not show an inhibitory effect on the hatching of root-knot nematode eggs. The single function limits its comprehensive control efficacy.

[0004] More importantly, the application of publicly available microbial agents, whether containing a single strain or a compound strain, is mostly concentrated on root dipping during transplanting or direct application to the field soil. This application has many inherent defects: (1) The protection is short-lived. Root dipping is an immediate treatment before transplanting. The inoculant only adheres to the surface of the root system. After transplanting, the effective number of viable bacteria decreases rapidly due to soil dilution, competition from other bacteria, and environmental stress. It is difficult to form a long-term stable rhizosphere protective barrier and cannot cover the entire susceptible stage of the seedling from planting to maturity. (2) The protection is not uniform. During the root dipping process, the inoculant is easily insufficiently applied to the root system, resulting in some roots lacking effective protection. (3) The operation is not adaptable enough. In large-scale seedling and transplanting scenarios, root dipping requires an additional step of treating each plant individually, which is not only inefficient but also prone to problems such as missed treatment or inconsistent treatment standards. (4) It is impossible to build a protective system in advance. The risk of root-knot nematode infection of seedlings exists from the later stage of seedling cultivation. Root dipping can only provide temporary protection at the time of transplanting and fails to block nematode infection from the source of the seedling stage, thus weakening the control effect.

[0005] As the core environment for seedling growth, the seedling substrate can potentially overcome the aforementioned shortcomings and reduce the risk of root-knot nematode infection by incorporating biocontrol agents during the seedling stage. While adding microbial agents to the seedling substrate is a mature technology, existing single biocontrol agents have significant limitations in seedling substrates: firstly, intense competition among bacteria and large temperature and humidity fluctuations within the substrate lead to a rapid decline in the effective viable count of the agent, typically dropping to 1×10⁻⁶ within 30 days. 6Below CFU / g, long-term efficacy cannot be maintained; on the other hand, single strains lack the ability to improve the substrate microenvironment, making them vulnerable to contamination by other microorganisms and environmental stress, further exacerbating activity decay. Consequently, the control effect of microbial agents against root-knot nematodes decreases significantly with the extension of the seedling cycle. Conventional compound microbial agents mostly focus on functional synergy, without screening for synergistic strains specific to the unique environment of the seedling substrate, making it difficult to achieve the dual goals of long-term viable bacterial survival and stable control effects. Summary of the Invention

[0006] The purpose of this invention is to provide a functional seedling substrate for controlling root-knot nematodes and its application. Seedling cultivation using this functional substrate significantly improves the colonization rate of compound microbial agents in the seedling rhizosphere. The resulting infected seedlings are rich in active biocontrol bacteria both inside and outside the root ball at transplanting time, providing continuous, highly efficient, and stable control after transplanting, and significantly promoting crop growth. This invention advances the timing of root-knot nematode control from the field stage to the controllable seedling stage, achieving "active immunization" and effectively solving the technical problems of difficult colonization and unstable effects associated with traditional root irrigation or field application of biocontrol agents.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A functional seedling substrate for preventing and controlling root-knot nematodes, the functional seedling substrate is composed of a solid seedling substrate and a compound microbial agent uniformly dispersed therein; The solid seedling substrate includes a basic nutrient substrate, pore-regulating components, prebiotics, organic nutrients, and a pH buffer. The compound microbial agent is composed of Bacillus vesiculosus (B. vesiculosus). Bacillus velezensis RKN1111, Bacillus laterosporus ( Bacillus laterosporus CDD-01 and gelatinous Bacillus ( Paenibacillus mucilaginous It consists of HX-02.

[0008] Furthermore, the Bacillus belye ( Bacillus velezensis RKN1111 is currently deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number: CGMCC No. 29300, deposit date: December 12, 2023. This strain was published in Chinese Patent CN118344995A on July 16, 2024.

[0009] Lateral spores (Bacillus retroflexus) Bacillus laterosporusCDD-01 is now deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. Accession number: CGMCC No. 34632. Deposit date: May 22, 2025.

[0010] The gelatinous spore-forming bacteria ( Paenibacillus mucilaginosus HX-02 is currently deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number: CGMCC No. 30949, deposit date: June 14, 2024; this strain was published in Chinese Patent CN 119307420 A on January 14, 2025.

[0011] Furthermore, the basic nutrient matrix is ​​at least one of peat moss, plant straw powder, mushroom residue, cow dung, wheat bran, and earthworm castings; the pore-regulating component is perlite; the prebiotic is tryptophan; the organic nutrient is humic acid; and the pH buffer is light calcium carbonate and phosphate.

[0012] Furthermore, the amount of the basic nutrient substrate added is 50% to 75% of the dry weight of the solid seedling substrate, the amount of the pore conditioning component added is 20% to 45% of the dry weight of the solid seedling substrate, the amount of the prebiotic added is 0.01% to 0.5% of the dry weight of the solid seedling substrate, the amount of the organic nutrient agent added is 0.1% to 3% of the dry weight of the solid seedling substrate, and the amount of the pH buffer added is 0.5% to 5% of the dry weight of the solid seedling substrate.

[0013] The present invention also provides a method for preparing the above-mentioned functional seedling substrate, comprising the following steps: (1) Take the refrigerated Bacillus belyss RKN1111, Bacillus laterosporus CDD-01 and Bacillus spp. HX-02 and inoculate them into sterilized LB medium. After seed culture and large-scale fermentation, the fermentation broths of Bacillus belyss RKN1111, Bacillus laterosporus CDD-01 and Bacillus spp. HX-02 were obtained respectively. (2) The fermentation broths of Bacillus belyssus RKN1111, Bacillus laterosporus CDD-01, and Bacillus hygroscopicus HX-02 obtained in step (1) were mixed at a volume ratio of 1:1:1, and then inoculated at a volume ratio of 3×10⁻⁶ per gram. 9 The CFU / g ratio is used to inoculate the mixed fermentation broth into the sterilized solid seedling substrate, and then directly stir it evenly to obtain the functional seedling substrate.

[0014] Furthermore, the seed culture and fermentation conditions are 37°C and 180 r / min.

[0015] The present invention also provides the application of the above-mentioned functional seedling substrate in the control of root-knot nematodes.

[0016] The present invention also provides a method for controlling crop root-knot nematodes, which includes using the above-mentioned functional seedling substrate for crop sowing or seedling transplanting to obtain seedlings that meet the standards for field transplanting.

[0017] The present invention also provides the application of the above-mentioned functional seedling substrate in the seedling cultivation of melon, fruit and vegetable crops.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention combines Bacillus belyss RKN1111, Bacillus laterosporus CDD-01 and Bacillus spp. HX-02. The synergistic effect of the three significantly improves the biocontrol agent's tolerance to competition from miscellaneous bacteria in the seedling substrate and fluctuations in temperature and humidity. This ensures that the agent maintains high efficiency throughout the entire seedling cycle and continuously "inoculates" the surrounding soil as the roots grow and the substrate slowly decomposes, forming a long-term, slow-release "biocontrol reservoir" that provides continuous protection for seedlings. The duration of the protective effect far exceeds that of any single field application.

[0019] (2) The present invention first uses a seedling substrate inoculated with compound microbial agents for seedling cultivation, and then transplants the seedlings. This method not only avoids the problems of short protection time and poor uniformity in the root dipping operation, but also eliminates the need for additional individual treatment procedures, greatly improving the efficiency of seedling cultivation and transplanting. Furthermore, it enables the seedlings to establish a strong "self-microbial protection system" before transplanting, and immediately possess the ability to resist nematodes after transplanting. This achieves a fundamental shift from "treatment after disease" to "immunization before transplanting", and the control effect is significantly improved compared with the traditional root dipping method.

[0020] (3) The compound microbial agent described in this invention has the dual core functions of inhibiting the hatching of root-knot nematode eggs and reducing the formation of root knots. In addition to effectively controlling nematodes, the compound microbial agent works synergistically with prebiotics and organic nutrients in the seedling substrate to significantly promote seedling root development and plant growth, improve stress resistance, and reduce the blind use of subsequent chemical pesticides through precise prevention, thereby saving costs and protecting the ecological environment. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to embodiments. These embodiments are used to explain the present invention, but are not intended to limit the scope of the invention.

[0022] Example 1: Isolation, screening, and identification of Bacillus laterosporus CDD-01 1. Test sample The Bacillus laterosporus CDD-01 used in this embodiment was screened and isolated by the Environmental and Microbial Ecology Laboratory of the Innovation Center for Biotechnology of Hebei University and is preserved at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34632.

[0023] The test culture medium (LB) consisted of 1% peptone, 0.5% yeast extract, and 1% NaCl.

[0024] 2. Isolation and screening of Bacillus laterosporus strain CDD-01 Weigh 10.0 g of soil sample (collected in 2023 from a greenhouse in Baoding, Hebei Province), add 90.0 mL of sterile water containing glass beads, let stand for 20 min, shake thoroughly on a rotary shaker for 30 min, then heat in an 80℃ water bath for 20 min, and then cool rapidly. Use a sterile pipette to take 1.0 mL of the above soil suspension and dilute it 1 to 10 times to obtain 1×10⁻⁶ ppm. 1 1×10 2 1×10 3 1×10 4 Soil suspension diluted 10 times... Using a sterile pipette, aspirate 10... times... -2 10 -3 and 10 -4 0.1 mL of the gradient soil suspension was evenly spread on the isolation culture medium plate and incubated at 30℃ for 36-72 h.

[0025] 3. Purification Single colonies with a clear zone were picked and transferred to NA medium slant, incubated at 30°C for 24 h, and stored at 4°C for later use. The colony characteristics of the isolated strains were recorded.

[0026] 4. Identification 4.1 Morphological identification Colony morphology observation: The purified strain was inoculated onto LB plates and incubated at 37°C for 24 hours. The colony shape, color, edge, surface texture, elevation, and whether pigment was produced were recorded.

[0027] Individual morphological observation: Single colonies were picked from the plate, and Gram-stained smears and spore-stained smears were prepared. The morphology, arrangement, Gram staining results, and the shape, position, and formation of spore crystals of the cells were observed under an optical microscope (1000x oil immersion). Observation of liquid culture characteristics: The strain was inoculated into LB liquid medium and cultured at 37℃ and 180r / min for 24h with shaking. The turbidity of the culture medium, whether a bacterial film was formed, and the precipitation were observed.

[0028] 4.2 Molecular biological identification Genomic DNA extraction: Genomic DNA of strain CDD-01 was extracted using a bacterial genomic DNA extraction kit according to the instructions. The purity and integrity of the DNA were verified by 1% agarose gel electrophoresis.

[0029] PCR amplification: Universal primers for Bacillus 16S rRNA were used.

[0030] Sequencing and alignment: The PCR product was purified and sent to a sequencing company for sequencing to obtain the 16S rRNA gene sequence; the sequence was submitted to the GenBank database and compared with known sequences using the BLAST tool. If it is similar to Bacillus laterosporus (…), the sequence was then compared with known sequences. Brevibacillus laterosporus The homology of the type strain is ≥99%. Based on the morphological and physiological-biochemical characteristics, the strain is identified as Bacillus laterosporus.

[0031] 5. Evaluation Results 5.1 Morphological identification results Based on colony morphology observation and individual morphological staining identification, the morphological characteristics of this isolated and purified strain are similar to those of Bacillus laterosporus (Blanctomyces lateralis). Bacillus laterosporus The typical characteristics are completely identical, as follows: Colony morphology: After being cultured on LB solid medium at 37℃ for 48 hours, strain CDD-01 formed round colonies with a diameter of 2-3 mm. The colony surface was smooth and moist, milky white, with neat edges, moderate elevation, opaque, and no pigment production. Individual morphology: Under an optical microscope, the bacteria are rod-shaped, measuring (0.6-0.8) μm × (2.0-3.0) μm, arranged singly or in pairs, and Gram-positive; the spores are oval, located at one end of the bacterial cell (proximal spores), and the sporangia are enlarged, forming typical laterospore crystals (this is a characteristic structure of Bacillus laterosporus). Liquid culture characteristics: After shaking culture in LB liquid medium for 24 hours, the culture medium was uniformly turbid, with a small amount of bacterial film adhering to the tube wall and slight sediment at the bottom. The sediment was easily dispersed after shaking.

[0032] 5.2 Molecular biological identification results The sequence was submitted to the GenBank database, and homology analysis was performed between it and known sequences in the NCBI nucleic acid database using the BLAST tool.

[0033] The comparison results showed that the 16S rRNA gene sequence of this strain (as shown in SEQ ID No. 1, gene sequence database accession number NR_044826.1) was similar to that of Bacillus laterosporus (…). Bacillus laterosporus The homology of the type strain was over 99%. Combined with the aforementioned morphological identification results, the isolated and purified strain was determined to be *Bacillus laterosporus*. Bacillus laterosporus ).

[0034] Example 2: Preparation of Functional Seedling Substrate 1. Preparation of compound microbial inoculants The test culture medium (LB) consisted of 1% peptone, 0.5% yeast extract, and 1% NaCl.

[0035] Two to three loops of refrigerated *Bacillus laterosporus* strain CDD-01, *Bacillus belyssus* strain RKN1111, and *Bacillus lentigines* strain HX-02 were respectively placed in 50 ml of sterile LB medium and cultured at 37°C and 180 rpm for 12 h to obtain seed culture. The seed culture was then inoculated into LB medium at a 5% v / v inoculation rate and cultured at 37°C and 200 rpm for 48 h. Finally, fermentation broths of *Bacillus laterosporus* CDD-01, *Bacillus belyssus* RKN1111, and *Bacillus lentigines* HX-02 were obtained.

[0036] The fermentation broths of Bacillus laterosporus CDD-01, Bacillus belyssus RKN1111, and Bacillus lentigines HX-02 were mixed at a volume ratio of 1:1:1 to obtain a compound microbial inoculum. The viable count of the compound microbial inoculum was 3 × 10⁻⁶. 9 CFU / ml.

[0037] 3. Preparation of solid seedling substrate In this embodiment, the solid seedling substrate is a common material suitable for plug seedling cultivation. The basic nutrient substrate accounts for 64.1% of the dry weight of the solid seedling substrate and consists of peat moss, plant straw powder, mushroom residue, cow manure, wheat bran, and earthworm castings. The pore conditioning component accounts for 35% of the dry weight of the solid seedling substrate and consists of perlite. The prebiotic accounts for 0.35% of the dry weight of the solid seedling substrate and consists of tryptophan. The organic nutrient agent accounts for 0.2% of the dry weight of the solid seedling substrate and consists of humic acid. The pH buffer accounts for 0.35% of the dry weight of the solid seedling substrate and consists of light calcium carbonate and phosphate.

[0038] 4. Mix the above-mentioned compound microbial agent into the prepared solid seedling substrate, stir for 5 hours, and maintain the moisture content of the seedling substrate at 50%~65% to obtain the functional seedling substrate, which is denoted as functional seedling substrate CDD-01+RKN1111+HX-02.

[0039] For comparison, in this embodiment, the fermentation broths of Bacillus laterosporus CDD-01, Bacillus belyssus RKN1111, and Bacillus spp. HX-02 were respectively mixed with the solidified seedling substrate in step 3 and stirred for 5 hours to obtain functional seedling substrates inoculated with a single microbial agent, which were denoted as functional seedling substrate CDD-01, functional seedling substrate RKN1111, and functional seedling substrate HX-02, respectively. (The volumes of Bacillus laterosporus CDD-01, Bacillus belyssus RKN1111, and Bacillus spp. HX-02 used in preparing the functional seedling substrates inoculated with a single microbial agent were the same as the volumes of the composite microbial agent fermentation broth used in preparing the CDD-01+RKN1111+HX-02 composite microbial agent seedling substrate.)

[0040] Example 3: Inhibitory effect of functional bacterial strains on the hatching of root-knot nematode eggs. 1. Dilute the compound microbial agent prepared in Example 2 by 100 times.

[0041] 2. Obtaining Southern Root-knot Nematode Eggs Collect cucumber roots infected with southern root-knot nematodes and wash away any soil particles adhering to the surface. Cut the roots into 2-4 cm sections with scissors and place them in a beaker. Add 10% sodium hypochlorite and shake thoroughly for three minutes. Pour the contents of the shaken beaker evenly into a sieve (sieve mesh sizes from top to bottom: 80 mesh, 140 mesh, 180 mesh, 200 mesh, 500 mesh). Rinse with sterile water 5-7 times to obtain sufficient nematode eggs and to wash away excess sodium hypochlorite. Rinse the 500 mesh sieve with a wash bottle and collect the rinsing liquid in a beaker to obtain an egg suspension.

[0042] 3. Experimental Methods Approximately 100 fresh eggs were added to the wells of a 48-well cell culture plate. The number of eggs in each well was observed and recorded under a stereomicroscope. Then, 100 μL of a 10-fold diluted compound microbial agent was added to each well. An equal volume of sterile water was used as a blank control. Each experimental group was performed in triplicate. The 48-well cell culture plates containing eggs were placed in a dark environment at 27°C. The number of Southern Root-Knot Nematodes hatching was observed under a stereomicroscope every 12 hours. After 5 days, the hatching inhibition rate of Southern Root-Knot Nematodes was calculated.

[0043] The method for calculating the relative inhibition rate of egg hatching is as follows: Relative inhibition rate % = (Hatching rate of nematodes in control group - Hatching rate of nematodes in treatment group) / Hatching rate of nematodes in control group × 100% 4. Experimental Results The results showed that after 5 days, the hatching rate of Southern Root-knot Nematode eggs in the blank group was 95.63%, the hatching rate of Southern Root-knot Nematode eggs after 100-fold dilution of the compound microbial agent was 10.43%, and the relative inhibition rate of Southern Root-knot Nematode eggs hatching after 100-fold dilution of the compound microbial agent was 89.09%.

[0044] Relative inhibition rate % = (hatching rate of nematodes in control group - hatching rate of nematodes in treatment group) / hatching rate of nematodes in control group × 100%.

[0045] Example 4: Verification of the colonization ability of functional seedling substrate The functional seedling substrates prepared in Example 2 were filled into 72-well trays and randomly arranged. Each treatment was replicated three times. Samples were collected at five points on days 0, 7, 14, 21, and 30 to determine the viable bacterial count and take the average value. The results are shown in Table 1.

[0046] Table 1. Dynamic changes in viable bacterial counts in different functional seedling substrates.

[0047] Table 1 shows that the colonization effect of the combined strains of *Bacillus laterosporus* CDD-01, *Bacillus belyssus* RKN1111, and *Bacillus mucilaginosa* HX-02 in the seedling substrate was significantly better than that of a single strain. Using the dilution plating method, the three-strain combination stably colonized in the substrate, with a viable count reaching 6 × 10⁶ on day 30. 7 CFU / g.

[0048] The combined bacterial strains exhibited a synergistic colonization effect: in the initial state (day 0), the viable bacterial count in each substrate reached 3 × 10⁻⁶. 9 CFU / g; With prolonged culture time, the viable cell count in all substrates showed a decreasing trend, but the viable cell count in the composite strain substrate was consistently significantly higher than that in the single strain substrate. After 30 days of culture, the viable cell count in the composite strain substrate remained at 6.00 × 10⁻⁶. 7 The CFU / g count was only 2.52 × 10⁻⁶ CFU / g, while the viable count of a single strain in the substrate was only 2.52 × 10⁻⁶. 7 ~3.12×10 7 The CFU / g ratio indicates a synergistic effect among the compound strains, which can significantly improve the colonization stability of the strains in the seedling substrate, laying the foundation for long-term biocontrol efficacy.

[0049] The strain exhibited good colonization stability: regardless of whether it was a single strain or a combination of strains, although the viable count decreased slightly during the 30-day detection period, it eventually stabilized and remained at 10. 7 The CFU / g level indicates that all tested strains can achieve long-term stable colonization in the seedling substrate, with the compound strain exhibiting superior colonization persistence.

[0050] Example 5: Verification of the potted plant control effect and growth-promoting effect of functional seedling substrate 1. Test crop: cucumber, transplanted at the four-leaf stage.

[0051] 2. Hatching of Southern Root-Knot Nematodes Taken from the southern root-knot nematode ( Meloidogyne incongnita Infected cucumber roots were cleaned to remove soil particles adhering to the surface. Mature egg sacs were picked from the roots using sterile forceps under a stereomicroscope. The picked egg sacs were thoroughly washed with a 2% sodium hypochlorite solution for 3 minutes, and then rinsed with sterile water to remove any remaining sodium hypochlorite solution. The cleaned egg sacs were placed in a 500-mesh sieve in a petri dish containing sterile water and incubated in a dark incubator at 27°C for 3-4 days. The hatched Southern Root-Knot Nematodes J2s were collected for experimental use.

[0052] 3. Experimental methods: They were divided into five treatment groups and two control groups, as follows: CK1 (blank seedling substrate): Seedlings were raised in a seedling substrate that was not inoculated with any active microorganisms. The seedlings were transplanted after 30 days without any biocontrol treatment. CK2 (positive control group): Same as CK1, but conventional chemical nematicide (10% abamectin) was applied to the seedlings at the time of transplanting. T1 (Invention Group): The functional seedling substrate CDD-01+RKN1111+HX-02 prepared in Example 2 was used for seedling cultivation, and the seedlings were transplanted directly with the clumps attached. T2 (Conventional biological control group): Same as CK1, but when transplanting seedlings, the same volume of RKN1111, CDD-01 and HX-02 mixed spore suspension as T1 group was used for root irrigation. T3 (single strain substrate group): seedlings were raised using the functional seedling substrate RKN1111 prepared in Example 2, and transplanted directly with the seed clumps. T4 (single strain substrate group): seedlings were raised using the functional seedling substrate CDD-01 prepared in Example 2, and transplanted directly with the seedling clumps. T5 (single strain substrate group): seedlings were raised using the functional seedling substrate HX-02 prepared in Example 2, and transplanted directly with the seedling clump.

[0053] Ten pots were treated, with three replicates. Each pot was inoculated with 1000 root-knot nematode larvae after transplanting. After 45 days of culture, the plant height, fresh weight, dry weight, and chlorophyll content of the cucumber plants were measured. At the same time, the number of root knots was investigated, and the root knot reduction rate was calculated.

[0054] The root knot reduction rate is calculated as follows: Root knot reduction rate % = (Root knot index of control group - Root knot index of treatment group) / Index of control group × 100%.

[0055] The effects of different treatments on the control and growth promotion of root-knot nematodes in potted plants are shown in Table 2.

[0056] Table 2. Effects of different treatments on potted plant control and growth promotion of southern root-knot nematodes.

[0057] Note: Different letters in the table indicate that different treatments for the same test indicator have significant differences.

[0058] As shown in Table 2, the functional seedling substrate (T1 group) of the present invention has the best nematode control effect: the number of root knots in the T1 group is only 56, and the root knot reduction rate is 64.10%, which is slightly better than the chemical nematicide control group and significantly better than the conventional biological control group and the single strain substrate group.

[0059] In addition, the functional seedling substrate described in this invention has a significant growth-promoting effect on cucumbers: the plant height, fresh weight, dry weight and chlorophyll content of the T1 group were the highest among all treatment groups, which were not only significantly higher than the blank control group (CK1) and the single strain substrate group (T3 / T4 / T5), but also slightly better than the chemical agent control group (CK2).

[0060] Intact root balls were collected from seedlings in each treatment group at the time of transplanting. 5g of root samples were weighed, washed and dried, and the viable bacteria count in the root ball matrix and the viable bacteria count inside the root were tested.

[0061] Viable bacteria count in root ball substrate: Place the root ball in 45 mL of sterile physiological saline and vortex for 30 min to prepare a bacterial suspension.

[0062] Viable bacterial count inside roots: After shaking, the root samples were homogenized and transferred to 45 mL of sterile physiological saline to prepare a bacterial suspension. After serial dilution of the two bacterial suspensions, 0.1 mL was spread on LB solid medium and incubated at 37°C for 48 h. The number of colonies was counted and the viable bacterial count (CFU / g) was calculated. Each treatment was repeated in 3 replicates.

[0063] The test results are shown in Table 3.

[0064] Table 3. Viable bacterial counts in the root interior and root ball (matrix) under different treatments

[0065] Comparing the results of T1 with those of T3-T5, it was shown that the combination of Bacillus laterosporus CDD-01, Bacillus belyssus RKN1111, and Bacillus spp. HX-02 improved its colonization ability in seedling roots. Comparing the results of T1 with those of T2, it was shown that inoculating the active microbial composition into the seedling substrate further enhanced its colonization ability.

[0066] Example 6: Determination of Physicochemical Indicators and Verification of Standard Compliance of Functional Seedling Substrate 1. Sources of experimental materials and samples: The functional seedling substrate CDD-01+RKN1111+HX-02 prepared in Example 2 and the blank seedling substrate CK1 described in Example 5 were used.

[0067] 2. Detection method: Referring to the national standard "Seedling Substrate" (GB / T 39469-2020) and the agricultural industry standard "Vegetable Seedling Substrate" (NY / T 2118-2012), the following core physicochemical indicators were selected, and the values ​​were taken from three parallel measurements: pH: Water extraction potentiometric method (NY / T 1121.2-2006); Electrical conductivity (EC): Water immersion potential method (NY / T 1121.1-2006); Organic matter: Potassium dichromate oxidation-external heating method (NY / T 1121.6-2006); Total nitrogen: Kjeldahl method (NY / T 1121.24-2012); Total phosphorus: Molybdenum-antimony colorimetric method (NY / T 1121.23-2012); Total potassium: flame photometry (NY / T 1121.12-2006); Moisture content (fresh sample): drying method (GB 7959-2012); Total porosity: ring cutter method (LY / T 1215-1999); Survival rate of functional strains: dilution plating method (LY / T 1215-1999).

[0068] The test results are shown in Table 4.

[0069] Table 4 Physicochemical Indicators of Functional Seedling Substrates

[0070] As shown in Table 4, the functional seedling substrate of the present invention meets the requirements of the national standard "Vegetable Seedling Substrate" (NY / T 2118-2012) in all its physicochemical properties. It has moderate pH and EC, sufficient organic matter and nutrient content, and reasonable pore structure, thus meeting the physicochemical property requirements of cucumber seedling substrate.

[0071] Compared with the blank substrate, the addition of functional strains did not change the core physicochemical properties of the seedling substrate, indicating that the strains and the substrate carrier have good compatibility and the product quality is stable and controllable.

[0072] The present invention has been described in detail above with general descriptions and specific embodiments. However, modifications or improvements can be made to the present invention, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A functional seedling substrate for controlling root-knot nematodes, characterized in that, The functional seedling substrate is composed of a solid seedling substrate and a compound microbial agent uniformly dispersed therein; The solid seedling substrate includes a basic nutrient substrate, pore-regulating components, prebiotics, organic nutrients, and a pH buffer. The compound microbial agent is composed of Bacillus vesiculosus (B. vesiculosus). Bacillus velezensis RKN1111, Bacillus laterosporus ( Bacillus laterosporus CDD-01 and gelatinous Bacillus ( Paenibacillus mucilaginosus It is made by fermentation of HX-02.

2. The functional seedling substrate according to claim 1, characterized in that, The Bacillus belesi ( Bacillus velezensis RKN1111, preservation number CGMCC No. 29300; Bacillus laterosporus ( Bacillus laterosporus CDD-01, with accession number CGMCC No. 34632; gelatinous spore-forming bacteria ( Paenibacillus mucilaginosus The accession number of HX-02 is: CGMCC No.30949.

3. The functional seedling substrate according to claim 1, characterized in that, The basic nutrient matrix is ​​at least one of peat moss, plant straw powder, mushroom residue, cow dung, wheat bran, and earthworm castings; the pore-regulating component is perlite; the prebiotic is tryptophan; the organic nutrient is humic acid; and the pH buffer is light calcium carbonate and phosphate.

4. The functional seedling substrate according to claim 1, characterized in that, The basic nutrient substrate is added at 50% to 75% of the dry weight of the solid seedling substrate; the pore conditioning component is added at 20% to 45% of the dry weight of the solid seedling substrate; the prebiotic is added at 0.01% to 0.5% of the dry weight of the solid seedling substrate; the organic nutrient agent is added at 0.1% to 3% of the dry weight of the solid seedling substrate; and the pH buffer is added at 0.5% to 5% of the dry weight of the solid seedling substrate.

5. The method for preparing the functional seedling substrate according to any one of claims 1-4, characterized in that, Includes the following steps: (1) Take the refrigerated Bacillus belyss RKN1111, Bacillus laterosporus CDD-01 and Bacillus spp. HX-02 and inoculate them into sterilized LB medium. After seed culture and large-scale fermentation, the fermentation broths of Bacillus belyss RKN1111, Bacillus laterosporus CDD-01 and Bacillus spp. HX-02 were obtained respectively. (2) The fermentation broth of Bacillus vesicularis RKN1111, the fermentation broth of Bacillus laterosporus CDD-01, and the fermentation broth of Bacillus lentigines HX-02 obtained in step (1) are mixed in a volume ratio of 1:1:

1. Then the mixed fermentation broth is added to the sterilized seedling substrate to obtain the functional seedling substrate.

6. The preparation method according to claim 5, characterized in that, The seed culture and fermentation conditions were 37℃ and 180 r / min.

7. The preparation method according to claim 5, characterized in that, Inoculate with 3×10 per gram of seedling substrate. 9 The ratio of CFU is determined by mixing the fermentation broth into the seedling substrate.

8. The application of the functional seedling substrate according to claim 1 in the control of root-knot nematodes.

9. A method for controlling root-knot nematodes in crops, characterized in that, This includes using the functional seedling substrate described in claim 1 for crop sowing or seedling transplanting to obtain seedlings that meet the standards for field transplanting.

10. The application of the functional seedling substrate according to claim 1 in the seedling cultivation of melon, fruit and vegetable crops.

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