A nematicidal seaweed fermentation product and its application

Abstract

The present invention discloses a nematicidal seaweed fermentation product and its application. It belongs to the technical field of seaweed application. The nematicidal seaweed fermentation product includes a seaweed fermentation product; the seaweed fermentation product is obtained by inoculating a strain with high alginate lyase production into a seaweed fermentation medium for culture, and then inoculating a biocontrol strain for fermentation and culture. The nematicidal seaweed fermentation product of the present invention effectively combines the growth-promoting function and the biocontrol function of seaweed active substances. In addition to effectively alleviating diseases such as soil nematodes, it has a certain promoting effect on crop growth, and provides new insights into overcoming the problems of single function of biocontrol strains or resource utilization of algae. The nematicidal seaweed fermentation product of the present invention can effectively inhibit root-knot nematodes and pine wood nematodes, and a variety of substances not found in single-bacteria fermentation products are found in the fermentation product, which can be used to improve the control effect of bananas, tomatoes and other crops on root-knot nematodes in field production, and can also promote the growth of cucumber seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of seaweed application, and particularly relates to a nematicidal seaweed fermentation product and application thereof. Background Art

[0002] Seaweed is widely used in food, medicine, agriculture and other fields because of its natural, green, environmentally friendly and abundant resources. In agricultural production, the functional substances in seaweed are considered to have important research significance and application prospects in promoting plant resistance and growth promotion, inducing crop disease prevention and control, etc. For example, Liu Ruizhi (2009) found that a mixture of brown algae fermentation products containing oligosaccharides, oligotrisaccharides and a small amount of oligotetrasaccharides and oligopentasaccharides can significantly increase hormone content and amylase activity and promote pea growth. Jing Hua and Wang Kangjian (2018) found that in terms of preventing rice blast, the best control effect was achieved when the concentration of seaweed fermentation products was 25-100 mg / kg; and 50 mg / kg of seaweed fermentation products had better control effects on rice blast and sheath blight than 50 mg / kg of shell fermentation products. Although seaweed fermentation products have shown good potential in enhancing crop disease resistance and stress resistance, there are few reports on the use of seaweed fermentation products in pest control.

[0003] For the prevention and control of nematodes and other pests, the commonly used methods are mainly chemical control and biological control, such as the use of avermectin, the discovery of microorganisms with biocontrol functions and the application of their fermentation products. Compared with the latter, chemical agents have faster and higher control efficiency, but poor stability and strong photolysis. The residues after excessive application are very likely to cause serious environmental pollution and drug resistance. Using biocontrol strains and their fermentation products to control crop diseases and insect pests is a green and effective control method. Common biocontrol strains include Trichoderma harzianum, Paecilomyces lilacinus, Bacillus subtilis, etc. Tao Shuxing et al. (2013) found that Bacillus subtilis 2-3-2 has a significant control effect on root-knot nematodes, and its control effect is positively correlated with the activity of the chitinase produced. However, the disadvantages of using a single biocontrol strain are gradually emerging during use, including poor stress resistance, low control efficiency, single functionality, and weak induction to plants. Chinese patent application 201911229372.0 reported a method for co-cultivation of Trichoderma and Bacillus and the use of their co-metabolites. It was found that after optimizing the carbon source, nitrogen source type and dosage of the co-cultivation of Trichoderma atroviride SG3403 and Bacillus subtilis 22, their metabolites had significantly better antagonistic effects on wheat fusarium and growth promotion of wheat seedlings than the individual metabolites of Trichoderma atroviride or Bacillus subtilis. At present, the research on the synergistic fermentation products of biocontrol strains in the areas of pests and diseases and growth promotion has become a hot topic. However, there are few reports on the research and application of co-fermentation of seaweed and biocontrol strains in mitigating soil nematodes. Seaweed is rich in active substances, which can play a good synergistic role in the production of fertilizers and pesticides. It is worth noting that the abundant small-molecule organic matter in seaweed can also serve as a nutrient source, providing conditions for the growth of strains. However, due to the complexity of the seaweed cell wall components, ordinary biocontrol strains are difficult to use directly, which greatly limits the development and application of algae resources. Summary of the invention

[0004] The primary purpose of the present invention is to provide a nematicidal seaweed fermentation product.

[0005] Another object of the present invention is to provide the application of the nematicidal seaweed fermentation product.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A nematicidal seaweed fermentation product comprises a seaweed fermentation product; the seaweed fermentation product is obtained by inoculating a strain with high alginate lyase production into a seaweed fermentation medium for culture, and then inoculating a biocontrol strain for fermentation culture.

[0008] After the strain with high alginate lyase production is inoculated into the seaweed fermentation medium for cultivation, the process further comprises the step of adjusting the pH of the medium to 7.0.

[0009] The strain with high alginate lyase production is preferably Microbulbifer sp. SH-1.

[0010] The microbubifer sp. SH-1 of the present invention has been deposited at the General Microbiological Center of the Chinese Academy of Sciences Institute of Microbiology, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on December 7, 2018, with a deposit number of CGMCC No. 16906. The above strain has been disclosed in Chinese patent ZL201910406996.9, a strain SH-1 producing alginate lyase and its application.

[0011] The inoculation amount of the strain with high alginate lyase production is preferably 0.5% to 1.5%; more preferably 1%.

[0012] The seaweed in the seaweed fermentation medium is preferably at least one of Sargassum fusiformis, Laminaria japonica, Ascophyllum nodosum, Sargassum truncatum, N. fasciata and N. fasciata; more preferably at least one of Laminaria japonica and Sargassum fusiformis.

[0013] The concentration of seaweed in the seaweed fermentation medium is preferably 10.0 to 30.0 g / L.

[0014] The components of the seaweed fermentation medium are more preferably: 30.0 g / L kelp powder, 5.0 g / L ammonium sulfate, 1.0 g / L magnesium sulfate, 1.0 g / L dipotassium hydrogen phosphate, 0.02 g / L ferrous sulfate, 15.0 g / L sodium chloride, pH 7.0-7.5.

[0015] The biocontrol strain is preferably Trichoderma harzianum.

[0016] The conditions for fermenting and culturing the biocontrol strain are preferably: pH 6.5-8.0, fermentation temperature 28-34° C., and fermentation time 24-72 h; more preferably: pH 7.0, fermentation temperature 32° C., and fermentation time 36 h.

[0017] The inoculation amount of the biocontrol strain is preferably 0.5% to 1.5%; more preferably 1%.

[0018] The nematicidal seaweed fermentation product also includes a stabilizer.

[0019] The stabilizer includes an inhibitor and a suspending agent.

[0020] Since seaweed fermentation products contain a variety of active organic small molecules, these substances are easily affected by changes in environmental temperature, microorganisms, storage time, etc., their composition and activity vary greatly, and they are prone to odor and bloating, etc., so inhibitors need to be added; in addition, in order to prevent the seaweed fermentation products from producing precipitated substances, suspending agents are added.

[0021] The inhibitor and the suspending agent are preferably calculated at a mass ratio of 0.5-0.7:2-5; more preferably calculated at a mass ratio of 0.6:3.

[0022] The inhibitor preferably comprises at least two of potassium sorbate, benzoic acid and kasperone; more preferably comprises kasperone and benzoic acid.

[0023] The preparation method of the inhibitor is preferably as follows: dissolving kasone and benzoic acid in water and stirring to obtain the inhibitor.

[0024] The kasone and benzoic acid are preferably calculated at a mass ratio of 0.1-0.5:0.5-0.1; more preferably calculated at a mass ratio of 0.3:0.3.

[0025] The kasonide and water are preferably calculated at a mass volume ratio (g:L) of 0.1 to 0.5:0.1; more preferably calculated at a mass volume ratio (g:L) of 0.3:0.1.

[0026] The stirring conditions are preferably: 30-50° C., 100-140 rpm, stirring for 0.5-1 h; more preferably: 40° C., 120 rpm, stirring for 0.5-1 h.

[0027] The suspending agent preferably includes at least two of dimethyl sulfoxide, methyl pyrrolidone, dioctyl sodium sulfosuccinate, sodium alkylbenzene sulfonate, succinic acid diester sulfonate, sodium carboxymethyl cellulose, polyglutamic acid and hydroxyethyl cellulose; more preferably includes dimethyl sulfoxide and methyl pyrrolidone.

[0028] The preferred method for preparing the suspension is: dissolving dimethyl sulfoxide and methyl pyrrolidone in water and stirring to obtain the inhibitor.

[0029] The dimethyl sulfoxide and methyl pyrrolidone are preferably calculated at a mass ratio of 1.3-1.7:1.7-1.3; more preferably calculated at a mass ratio of 1.5:1.5.

[0030] The dimethyl sulfoxide and water are preferably calculated at a mass volume ratio (g:L) of 1 to 5:0.1; more preferably at a mass volume ratio (g:L) of 1.5:0.1.

[0031] The stirring conditions are preferably: 30-50° C., 100-140 rpm, stirring for 0.5-1 h; more preferably: 40° C., 120 rpm, stirring for 0.5-1 h.

[0032] The stabilizer is obtained by mixing an inhibitor with a suspending agent and stirring.

[0033] The stirring conditions are preferably: 30-50° C., 100-140 rpm, stirring for 0.5-1 h; more preferably 40° C., 120 rpm, stirring for 0.5-1 h.

[0034] The molecular weight of the nematicidal seaweed fermentation product is preferably at least one of greater than 10 kDa, 5 to 10 kDa, 3 to 5 kDa and less than 3 kDa.

[0035] The nematicidal seaweed fermentation product is used in the preparation of nematicidal drugs and / or drugs for promoting crop growth.

[0036] The nematodes include but are not limited to at least one of root-knot nematodes and pine wood nematodes.

[0037] The crops include bananas, cucumbers and tomatoes.

[0038] Compared with the prior art, the present invention has the following advantages and effects:

[0039] (1) The present invention provides a nematicidal seaweed fermentation product, which has good nematicidal effect and has good application prospects in the field of plant protection.

[0040] (2) The nematicidal seaweed fermentation product of the present invention has algae as its main fermentation substrate, which is a pure natural green resource, thus realizing the resource utilization and development of algae.

[0041] (3) The nematicidal seaweed fermentation product preparation process of the present invention produces antibacterial and anti-insect substances that are not found in the nematicidal seaweed fermentation product of a single strain, and increases the content of growth-promoting substances in the product. The nematicidal seaweed fermentation product of the present invention can effectively inhibit root-knot nematodes and pine wood nematodes, and a variety of substances that are not found in single-bacteria fermentation products are found in the fermentation product, which can be used to improve the control effect of bananas, tomatoes and other crops on root-knot nematodes in field production, and can also promote the growth of cucumber seedlings.

[0042] (4) The nematicidal seaweed fermentation product of the present invention has a good effect on the control of nematodes in crops such as bananas and tomatoes, and has a certain growth-promoting effect on cucumber seedlings. It can be used to reduce or partially replace the amount of pesticides used, thereby alleviating environmental problems caused by excessive application of pesticides. The nematicidal seaweed fermentation product of the present invention effectively combines the growth-promoting function and the biocontrol function of seaweed active substances. In addition to effectively alleviating diseases such as soil nematodes, it has a certain promoting effect on crop growth and provides new insights into overcoming the problems of single function of biocontrol strains or resource utilization of algae. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The figures are colony images of Microbulbifer sp. SH-1 and Trichoderma harzianum strains; wherein: A is a colony image of Microbulbifer sp. SH-1 on a sodium alginate solid culture medium; B is a colony image of Trichoderma harzianum on a PDA solid culture medium; C is a scanning electron microscope image of Microbulbifer sp. SH-1; and D is a scanning electron microscope image of Trichoderma harzianum.

[0044] Figure 2 The electron microscope scanning images of the co-growth of Microbulbifer sp. SH-1 and Trichoderma harzianum strains under different fermentation processes; A is a physical picture of the co-growth of Microbulbifer sp. SH-1 seed solution and Trichoderma harzianum strain seed solution inoculated simultaneously in process ①; B is a physical picture of the co-growth of Microbulbifer sp. SH-1 seed solution in process ② first, and then directly inoculated with Trichoderma harzianum strain seed solution 12 hours later; C is a physical picture of the co-growth of Microbulbifer sp. SH-1 seed solution in process ③ first, and then adjust the pH of the culture medium to 7.0 after 12 hours, and then inoculate Trichoderma harzianum strain seed solution; D is a physical picture of the co-growth of Microbulbifer sp. SH-1 seed solution in process ④ first, and then directly inoculate Microbulbifer sp. SH-1 seed solution after 12 hours; E is a physical picture of the co-growth of Trichoderma harzianum strain seed solution in process ⑤ first, and then adjust the pH of the culture medium to 7.0 after 12 hours, and then inoculate Microbulbifer sp. sp.) Actual picture of SH-1 seed liquid co-growth.

[0045] Figure 3 This is a graph showing the effects of seaweed fermentation products under different fermentation processes on the corrected mortality rate of nematodes.

[0046] Figure 4 The figures are the results of the effects of seaweed fermentation products obtained under different fermentation conditions on the corrected mortality rate of nematodes; wherein: A is the results of the effects of seaweed fermentation products under different pH conditions on the corrected mortality rate of nematodes; B is the results of the effects of seaweed fermentation products under different fermentation temperatures on the corrected mortality rate of nematodes; C is the results of the effects of seaweed fermentation products under different fermentation times on the corrected mortality rate of nematodes; D is the results of the effects of seaweed fermentation products under different seaweed species in seaweed fermentation medium on the corrected mortality rate of nematodes; E is the results of the effects of seaweed fermentation products under different seaweed concentrations on the corrected mortality rate of nematodes.

[0047] Figure 5This is a graph showing the effects of different nematicidal seaweed fermentation products and 5% avermectin microemulsion on the corrected mortality of root-knot nematodes.

[0048] Figure 6 Map of the experimental base for using nematicidal seaweed fermentation products to control root-knot nematodes in field bananas.

[0049] Figure 7 The graphs show the control effects of different nematicide products on root-knot nematodes in field tomatoes; A shows the experimental effects of different nematicide products on root-knot nematodes in field tomatoes; B shows the control effects of different nematicide products on tomato root-knot nematodes; C shows the effects of different nematicide seaweed fermentation products on the theoretical yield of tomatoes.

[0050] Figure 8 The figures are graphs showing the toxic effects of different nematicidal seaweed fermentation products on pine wood nematodes; wherein A is a graph showing the toxicity results of sterile seaweed fermentation medium on pine wood nematodes; B is a graph showing the toxicity results of nematicidal seaweed fermentation products of Microbulbifer sp. SH-1 on pine wood nematodes; C is a graph showing the toxicity results of nematicidal seaweed fermentation products of Trichoderma harzianum on pine wood nematodes; and D is a graph showing the toxicity results of nematicidal seaweed fermentation products prepared in Example 3 on pine wood nematodes.

[0051] Fig. 9 The figures are as follows: A is a figure showing the effect of different pH conditions on the corrected nematode mortality of the nematode fermented product prepared in Example 3; B is a figure showing the effect of different temperatures on the corrected nematode mortality of the nematode fermented product prepared in Example 3; and C is a figure showing the effect of different placement times on the corrected nematode mortality of the nematode fermented product prepared in Example 3.

[0052] Fig.10 This is a qualitative analysis result diagram of the nematicidal algae fermentation product of Microbulbifer sp. SH-1, the nematicidal algae fermentation product of Trichoderma harzianum, and the nematicidal algae fermentation product prepared in Example 3.

[0053] Fig.11SH-1 nematicidal algae fermentation product, Trichoderma harzianum nematicidal algae fermentation product and the nematicidal algae fermentation product prepared in Example 3, alginate, total algae phenols, mannitol and betaine content result graph; wherein, A is a graph showing the alginate content determination result of the nematicidal algae fermentation product of Microbulbifer sp. SH-1, Trichoderma harzianum nematicidal algae fermentation product and the nematicidal algae fermentation product prepared in Example 3; B is a graph showing the total algae phenol content determination result of the nematicidal algae fermentation product of Microbulbifer sp. SH-1, Trichoderma harzianum nematicidal algae fermentation product and the nematicidal algae fermentation product prepared in Example 3; C is a graph showing the total algae phenol content determination result of the nematicidal algae fermentation product of Microbulbifer sp. SH-1 A is a graph showing the results of determining the betaine content of the nematicidal seaweed fermentation product, the nematicidal seaweed fermentation product of Trichoderma harzianum and the nematicidal seaweed fermentation product prepared in Example 3; D is a graph showing the results of determining the mannitol content of the nematicidal seaweed fermentation product of Microbulbifer sp. SH-1, the nematicidal seaweed fermentation product of Trichoderma harzianum and the nematicidal seaweed fermentation product prepared in Example 3.

[0054] Fig.12 Figure 1 is a graph showing the effects of different treatment groups on the growth promotion of cucumber seedlings; Figure 2 is a graph showing the effects of different treatment groups on cucumber growth; Figure 3 is a graph showing the effects of different treatment groups on cucumber plant height; and Figure 4 is a graph showing the effects of different treatment groups on aboveground biomass of cucumber. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents described in the present invention can be obtained commercially.

[0056] The strain with high alginate lyase production, named Microbulbifer sp. SH-1, was deposited at the General Microbiological Center of the Chinese Academy of Sciences Institute of Microbiology, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, on December 7, 2018, with the deposit number CGMCC No. 16906. The above strain has been disclosed in Chinese patent ZL201910406996.9, a strain producing alginate lyase SH-1 and its application.

[0057] Trichoderma harzianum was purchased from Guangzhou Shenjingya Agricultural Technology Co., Ltd.

[0058] Kelp, Sargassum, Sargassum, Nardella, Ascophyllum, and N. algae were purchased from Guangzhou Shenjingya Agricultural Technology Co., Ltd.

[0059] Example 1: Study on the fermentation process of seaweed fermentation products

[0060] 1. Preparation of culture medium

[0061] (1) Sodium alginate liquid medium: Weigh 10.0 g of sodium alginate, 5.0 g of peptone, 5.0 g of yeast extract, and 10.0 g of sodium chloride, dissolve in water and adjust the volume to 1 L, adjust the pH to 7.0 with 2.0 mol / L sodium hydroxide or acetic acid solution, and sterilize at 121°C for 20 minutes to obtain a sodium alginate liquid medium. A sodium alginate solid medium is obtained by adding 20.0 g of agar powder to the above 1 L of sodium alginate liquid medium.

[0062] (2) PDA medium: Weigh 6.0 g of potato extract powder, 20.0 g of glucose, and 20.0 g of agar powder, dissolve them in deionized water and make up to 1 L, set the pH to natural, and sterilize at 121°C for 20 min to obtain PDA medium.

[0063] (3) Seaweed fermentation medium: Weigh 10.0 g of seaweed dry powder, 5.0 g of ammonium sulfate, 1.0 g of magnesium sulfate, 1.0 g of dipotassium hydrogen phosphate, 0.02 g of ferrous sulfate, and 15.0 g of sodium chloride, dissolve in water and make up to 1.0 L, and sterilize at 121° C. for 20 min to obtain a seaweed fermentation medium.

[0064] 2. Preparation of Nematode Suspension

[0065] Banana fields with many years of planting were collected from banana planting bases in Leiping Town, Daxin County, Chongzuo City, Guangxi. Diseased banana roots and soil were collected. Egg masses of root-knot nematodes were picked from the diseased roots and soil, washed with sterile water, and inoculated into the roots of water spinach grown in the substrate for 20 days. After 15 days, the water spinach roots were dug out and rinsed with clean water. The roots were disinfected with 1.5% sodium hypochlorite for 3 minutes, rinsed with sterile water 3 times, and the egg capsules on the root knots were picked with tweezers and placed in a Behman funnel for incubation. After 7 days of incubation, a large number of highly active second-instar larvae were obtained. The second-instar larvae were separated into water, and the nematode concentration was kept at 200 / ml, which was the second-instar root-knot larvae suspension. Store at 4°C for later use.

[0066] 3. Research on seaweed fermentation technology

[0067] (1) Preparation of seed solution of Microbulbifer sp. SH-1, a strain with high alginate lyase production: 100 μL of Microbulbifer sp. SH-1 bacterial solution (this inoculated bacterial solution is stored at -20°C) was inoculated into a triangular flask containing 100 mL of sodium alginate liquid culture medium, and cultured in a constant temperature shaker at 32°C for 16 h to reach 10 6CFU / mL is the seed solution of Microbulbifer sp.SH-1. Store at 4℃. Colony image and scanning electron micrograph of Microbulbifer sp.SH-1 on sodium alginate solid medium, such as Figure 1 A and 1C.

[0068] (2) Preparation of seed solution of Trichoderma harzianum strain: Inoculate Trichoderma harzianum solution (this inoculated solution is stored at -20°C) in a PDA culture medium plate, incubate at 25-28°C for 72 h, scrape off the Trichoderma spores on the plate with sterile water, and prepare a 1×10 8 The suspension of Trichoderma spores with a concentration of 100 / mL is the seed solution of Trichoderma harzianum strain and is stored at 4°C for future use. The colony image of Trichoderma harzianum on PDA medium and the scanning electron micrograph of the bacteria are shown in Figure 1. Figure 1 B and 1D.

[0069] (3) The above-mentioned Microbulbifer sp. SH-1 seed solution and Trichoderma harzianum strain seed solution were inoculated into the seaweed fermentation medium in sequence, and the process settings were as follows:

[0070] Process ① Inoculating the seed liquid of Microbulbifer sp. SH-1 and the seed liquid of Trichoderma harzianum strain at the same time;

[0071] Process ②: First inoculate the seed solution of Microbulbifer sp. SH-1, and then directly inoculate the seed solution of Trichoderma harzianum strain 12 hours later;

[0072] Process ③: first inoculate the seed solution of Microbulbifer sp. SH-1, then adjust the pH of the culture medium to 7.0 after 12 hours, and then inoculate the seed solution of Trichoderma harzianum strain;

[0073] Process ④: first inoculate the seed solution of Trichoderma harzianum strain, and then directly inoculate the seed solution of Microbulbifer sp. SH-1 12 hours later;

[0074] Process ⑤: first inoculate the seed solution of Trichoderma harzianum strain, adjust the pH of the culture medium to 7.0 after 12 hours, and then inoculate the seed solution of Microbulbifer sp. SH-1;

[0075] Using sterile seaweed fermentation medium as a control, after the two bacterial liquids in all processes were inoculated, they were cultured at a constant temperature of 180 r / min and 32°C for 36 h, then centrifuged at 6000 r / min for 10 min, filtered through four layers of gauze, and the supernatant was collected, which was the seaweed fermentation product. The fermentation products of each process were scanned by electron microscopy to observe the growth status of the two bacterial co-fermentation under each process.

[0076] like Figure 2 As shown, the symbiotic state of the two strains can be observed in processes ②, ③, ④, and ⑤, and the symbiotic state of the strains in processes ② and ③ is better. This indicates that fermenting Microbulbifer sp. SH-1 first and then inoculating Trichoderma harzianum is conducive to the subsequent symbiotic growth of the two strains.

[0077] (4) Toxic effect of seaweed fermentation products on nematodes The seaweed fermentation products prepared by the above processes ① to ⑤ were respectively mixed with the above-prepared second-instar root-knot larvae suspension (200 larvae / mL) at a volume ratio of 9:1 in a 96-well culture plate. Sterile water was used as a blank control. In addition, a commercially available Trichoderma harzianum agent purchased from Jinan Tumuqi Biotechnology Co., Ltd. was used as a comparison. After 24 hours, the number of nematodes that survived and died was observed, and the corrected mortality of nematodes in each treatment was compared. The judgment criteria are as follows: All nematodes that are constantly swinging or in an "S" shape, curled shape, wavy shape, or spiral shape are judged as live worms; all nematodes that are not moving and have a "J" or "C" shape, or have a rigid body and a non-refractive body wall are judged as dead worms.

[0078] The adjusted mortality rate was calculated as follows:

[0079] Corrected mortality rate (%) = (treatment mortality rate - control mortality rate) / (1 - control group mortality rate) × 100%

[0080] The results are as follows Figure 3 As shown, the corrected mortality rate of the seaweed fermentation product prepared by process ③ against root-knot nematodes is higher than that of other processes, and the corrected mortality rate of the seaweed fermentation product prepared by process ② against root-knot nematodes is higher than that of processes ①, ④, ⑤ and the commercial Trichoderma harzianum agent, while the corrected mortality rate of the seaweed fermentation product prepared by process ① and the commercial Trichoderma harzianum agent against root-knot nematodes is higher than that of processes ④ and ⑤, but the difference in the corrected mortality rate of the seaweed fermentation products prepared by processes ④ and ⑤ against root-knot nematodes is not obvious. This result shows that the seaweed fermentation process of first inoculating (Microbulbifer sp.) SH-1 seed solution and then inoculating Trichoderma harzianum seed solution is better than the seaweed fermentation process of first inoculating Trichoderma harzianum and then inoculating Microbulbifer sp. SH-1 seed solution. Moreover, after the Microbulbifer sp. SH-1 seed solution was fermented for 12 hours, the pH of the culture medium was first adjusted to 7.0, and then the seed solution of the Trichoderma harzianum strain was inoculated. The seaweed fermentation product obtained had a stronger inhibitory effect on the activity of root-knot nematodes, and the effect was better than process ② and commercially available Trichoderma harzianum agents.

[0081] (5) The contents of total phenols and total organic acids in the fermentation products of each process were determined, and the scavenging rates of hydroxyl, superoxide anion and DPPH free radicals of the seaweed fermentation products of each process were determined to compare the antioxidant activities of the seaweed fermentation products.

[0082] The total phenol determination method refers to the Folin phenol colorimetric method (Sun Luchuanyang, Ren Dandan, Wang Shuai, et al. Optimization of the process of extracting brown algae polyphenols from copper algae by bioenzymatic method [J]. Journal of Food Safety and Quality, 2019, 10(08): 2201-2206);

[0083] The total organic acid was determined by NaOH titration method.

[0084] The scavenging rates of hydroxyl, superoxide anion and DPPH free radicals were measured by hydroxyl free radical scavenging ability test kit, superoxide anion scavenging ability test kit and DPPH free radical scavenging ability test kit, respectively. The kits were purchased from Guangzhou Solebow Biotechnology Co., Ltd.

[0085] The results are shown in Table 1. The total phenol and total organic acid contents of the seaweed fermentation products prepared by processes ② and ③ are higher than those of processes ①, ④ and ⑤, while the total phenol and total organic acid contents of the products of process ① are higher than those of processes ④ and ⑤, indicating that the fermentation process of first inoculating Microbulbifer sp. SH-1 and then inoculating Trichoderma harzianum is helpful to increase the content of phenolic and organic acid components in seaweed fermentation products. In addition, the scavenging rates of hydroxyl, superoxide anion and DPPH free radicals of the seaweed fermentation products prepared by processes ② and ③ are also higher than those of processes ①, ④ and ⑤, while the scavenging rates of hydroxyl, superoxide anion and DPPH free radicals of process ① are higher than those of processes ④ and ⑤, indicating that the fermentation process of first inoculating Microbulbifer sp. SH-1 and then inoculating Trichoderma harzianum also improves the antioxidant activity of seaweed fermentation products, and has a good promoting effect on inducing crops to enhance their ability to resist nematode diseases.

[0086] Table 1: Effects of different processes on the total phenolic content, organic acid content and antioxidant capacity of seaweed fermentation products

[0087]

[0088] Example 2: Optimization of fermentation conditions for seaweed fermentation products

[0089] In Example 1, a seaweed fermentation process with a good inhibitory effect on nematode activity was determined, and on this basis, other conditions of the fermentation process (fermentation temperature, culture medium pH, seaweed species, seaweed concentration) were optimized.

[0090] 1. Preparation of culture medium

[0091] (1) Sodium alginate liquid culture medium: Weigh 10.0 g of sodium alginate, 5.0 g of peptone, 5.0 g of yeast extract, and 10.0 g of sodium chloride, dissolve them in water and make the volume to 1 L, adjust the pH to 7.0 with 2.0 mol / L sodium hydroxide or acetic acid solution, and sterilize at 121°C for 20 min to obtain a sodium alginate liquid culture medium.

[0092] (2) PDA medium: Weigh 6.0 g of potato extract powder, 20.0 g of glucose, and 20.0 g of agar powder, dissolve them in deionized water and make up to 1 L, set the pH to natural, and sterilize at 121°C for 20 min to obtain PDA medium.

[0093] (3) Seaweed fermentation medium: Weigh 10.0 g of seaweed dry powder, 5.0 g of ammonium sulfate, 1.0 g of magnesium sulfate, 1.0 g of dipotassium hydrogen phosphate, 0.02 g of ferrous sulfate, and 15.0 g of sodium chloride, dissolve in water and make up to 1.0 L, and sterilize at 121° C. for 20 min to obtain a seaweed fermentation medium.

[0094] 2. Preparation of Nematode Suspension

[0095] Banana fields with long-term planting of bananas were collected from banana planting bases in Leiping Town, Daxin County, Chongzuo City, Guangxi Province. Diseased banana roots and soil were collected. Egg masses of root-knot nematodes were picked from the diseased roots and soil, washed with sterile water, and inoculated into the roots of water spinach grown in the substrate for about 20 days. After 15 days, the water spinach roots were dug out and rinsed with clean water. The roots were disinfected with 1.5% sodium hypochlorite for 3 minutes, rinsed with sterile water 3 times, and the egg capsules on the root knots were picked with tweezers and placed in a Bayman funnel for incubation. After 7 days of incubation, a large number of highly active second-instar larvae were obtained. The second-instar larvae were separated into water, and the nematode concentration was kept at 200 / ml, which was the second-instar root-knot larvae suspension, which was stored at 4°C for later use.

[0096] 3. Experiment on optimization of fermentation conditions of seaweed fermentation products

[0097] (1) Preparation of seed solution of Microbulbifer sp. SH-1, a strain with high alginate lyase production: 100 μL of Microbulbifer sp. SH-1 bacterial solution (this inoculated bacterial solution is stored at -20°C) was inoculated into a triangular flask containing 100 mL of sodium alginate liquid culture medium, and cultured in a constant temperature shaker at 32°C for 16 h to reach 10 6 CFU / mL, which is the seed solution of Microbulbifer sp. SH-1. Store at 4℃.

[0098] (2) Preparation of seed solution of Trichoderma harzianum strain: Inoculate Trichoderma harzianum solution (this inoculated solution is stored at -20°C) in a PDA culture medium plate, incubate at 25-28°C for 72 h, scrape off the Trichoderma spores on the plate with sterile water, and prepare a 1×10 8 The suspension of Trichoderma spores at a concentration of spores / mL is the seed solution of Trichoderma harzianum strain and is stored at 4°C for future use.

[0099] (3) The above-mentioned Microbulbifer sp. SH-1 seed solution and Trichoderma harzianum seed solution were inoculated into the seaweed fermentation medium in sequence according to process ③ in Example 1. Before the inoculation of Microbulbifer sp. SH-1, the seaweed species (Hippophae rhamnoides, Laminaria japonica, Ascophyllum nodosum, Sargassum, N. fasciata, and N. fasciata) and seaweed concentration (based on the results of the seaweed species in the seaweed fermentation medium, the seaweed species with the best nematode inhibition effect was selected for the next step, which were 5.0 g / L, 10.0 g / L, 15.0 g / L, 20.0 g / L, 25.0 g / L, and 30.0 g / L) in the seaweed fermentation medium were tested for their effects on the nematode inhibition effect of the seaweed fermentation product. When inoculating the seed solution of Trichoderma harzianum strain, the pH of the seaweed fermentation medium (5.5, 6.0, 6.5, 7.0, 7.5, 8.0), fermentation temperature (26°C, 28°C, 30°C, 32°C, 34°C, 36°C), fermentation time (calculated after all the strains were inoculated, 0h, 12h, 24h, 36h, 48h, 72h respectively) and other conditions were tested on the effect of seaweed fermentation products on the inhibition of nematodes.

[0100] (4) The inhibitory effect of seaweed fermentation products on nematodes was determined by the immersion method. The seaweed fermentation products were mixed with the prepared second-instar root-knot larvae suspension (200 larvae / mL) at a volume ratio of 9:1 in a 96-well culture plate. Unsterilized seaweed fermentation medium and sterile water were used as controls. The number of nematodes that survived and died was observed after 24 hours and 48 hours, and the nematode mortality rate of each treatment was compared. The judgment criteria are as follows: All worms that are constantly swinging or in an "S" shape, curled shape, wavy shape, or spiral shape are judged as living worms; all worms that are not moving and have a "J" shape or a "C" shape, or whose worms are rigid and whose body walls have no refraction, are judged as dead worms. The calculation formula for the corrected mortality rate is as follows:

[0101] Corrected mortality rate (%) = (treatment mortality rate - control mortality rate) / (1 - control group mortality rate) × 100%

[0102] The results are as follows Figure 4As shown in the figure, the optimal fermentation conditions for seaweed fermentation products are pH 6.5-8.0, fermentation temperature 28-34°C, culture time 24-72h, kelp and Sargassum fusiformis are the best seaweed species in the seaweed fermentation medium, and the kelp addition concentration is 10.0-30.0g / L. Within this range of conditions, seaweed fermentation products can have a good inhibitory effect on nematodes.

[0103] Example 3: Preparation of nematicidal seaweed fermentation products

[0104] 1. Preparation of culture medium

[0105] (1) Sodium alginate liquid culture medium: Weigh 10.0 g of sodium alginate, 5.0 g of peptone, 5.0 g of yeast extract, and 10.0 g of sodium chloride, dissolve them in water and make the volume to 1 L, adjust the pH to 7.0 with 2.0 mol / L sodium hydroxide or acetic acid solution, and sterilize at 121°C for 20 min to obtain a sodium alginate liquid culture medium.

[0106] (2) PDA medium: Weigh 6.0 g of potato extract powder, 20.0 g of glucose, and 20.0 g of agar powder, dissolve them in deionized water and make up to 1 L, set the pH to natural, and sterilize at 121°C for 20 min to obtain PDA medium.

[0107] (3) Seaweed fermentation medium: Weigh 30.0 g of kelp dry powder, 5.0 g of ammonium sulfate, 1.0 g of magnesium sulfate, 1.0 g of dipotassium hydrogen phosphate, 0.02 g of ferrous sulfate, and 15.0 g of sodium chloride, dissolve in water and make up to 1.0 L, adjust the pH to 7.0-7.5 with 2.0 mol / L sodium hydroxide or acetic acid solution, and sterilize at 121°C for 20 min to obtain a seaweed fermentation medium.

[0108] 2. Preparation of Stabilizer

[0109] (1) Preparation of inhibitor: 0.3 g of kason and 0.3 g of benzoic acid were dissolved in 100 mL of water, stirred at 40° C. and 120 rpm for 0.5 to 1 hour, and cooled to room temperature.

[0110] (2) Preparation of suspension: 1.5 g of dimethyl sulfoxide and 1.5 g of methyl pyrrolidone were dissolved in 100 mL of water, stirred at 40° C. and 120 rpm for 0.5 to 1 hour, and cooled to room temperature.

[0111] (3) Preparation of stabilizer: The inhibitor (1) and the suspending agent (2) were mixed at a mass ratio of 0.6:3 at 40° C. and a stirring speed of 120 rpm for 0.5 to 1 hour, and then cooled to room temperature to obtain a stabilizer.

[0112] 3. Preparation of Nematicidal Seaweed Fermentation Products

[0113] (1) Preparation of seed solution of Microbulbifer sp. SH-1, a strain with high alginate lyase production: 100 μL of Microbulbifer sp. SH-1 bacterial solution (this inoculated bacterial solution is stored at -20°C) was inoculated into a triangular flask containing 100 mL of sodium alginate liquid culture medium, and cultured in a constant temperature shaker at 32°C for 16 h to reach 10 6 CFU / mL, which is the seed solution of Microbulbifer sp. SH-1, a strain with high alginate lyase production. Store at 4°C.

[0114] (2) Preparation of seed solution of Trichoderma harzianum strain: Inoculate Trichoderma harzianum solution (this inoculated solution is stored at -20°C) in a PDA culture medium plate, incubate at 25-28°C for 72 h, scrape off the Trichoderma spores on the plate with sterile water, and prepare a 1×10 8 The suspension of Trichoderma spores at a concentration of spores / mL is the seed solution of Trichoderma harzianum strain and is stored at 4°C for future use.

[0115] (3) Preparation of seaweed fermentation products: First, inoculate 500 mL of seaweed fermentation medium with the seed liquid of Microbulbifer sp. SH-1, which has a high alginate lyase production, in an amount of 1% of the mass of the seaweed fermentation medium. After culturing in a constant temperature shaker at 32°C for 12 h, adjust the pH of the medium to 7.0, and then inoculate with the seed liquid of Trichoderma harzianum, in an amount of 1% of the mass of the seaweed fermentation medium. Continue to culture in a constant temperature shaker at 32°C for 36 h to obtain the seaweed fermentation product.

[0116] (4) Filtration: The seaweed fermentation product was centrifuged at 6000 r / min and 4°C, filtered through four layers of gauze to obtain the seaweed fermentation product supernatant, and stored at 4°C.

[0117] (5) Add a stabilizer to the supernatant of the seaweed fermentation product at a temperature of 40° C. and a stirring speed of 120 rpm. The amount of the stabilizer added is 2.0% (v / v) of the supernatant of the seaweed fermentation product. Stir well for 2 hours and cool to room temperature to obtain a nematicidal seaweed fermentation product.

[0118] Example 4: Study on the indoor inhibitory effect and field control effect of nematicidal seaweed fermentation products on root-knot nematodes

[0119] In order to explore the inhibitory effect of nematicidal seaweed fermentation products on nematodes, seaweed fermentation products fermented by Microbulbifer sp. SH-1 alone, seaweed fermentation products fermented by Trichoderma harzianum alone, nematicidal seaweed fermentation products prepared in Example 3 and 5% avermectin microemulsion commonly used on the market were used as treatment groups, and clean water was used as the control group. The indoor inhibitory effects of the treatment groups and the control group on root-knot nematodes were compared, and field experiments were carried out to compare the field control effects.

[0120] Seaweed fermentation product fermented by Microbulbifer sp. SH-1 alone: ​​Inoculate the seed liquid of Microbulbifer sp. SH-1 into 500 mL of seaweed fermentation medium, with the inoculation amount being 1% of the mass of the seaweed fermentation medium. Culture the mixture in a constant temperature shaker at 32°C for 48 hours to obtain the seaweed fermentation product fermented by Microbulbifer sp. SH-1 alone.

[0121] The preparation method of the nematicidal seaweed fermentation product of Microbulbifer sp. SH-1 is the same as that in Example 3.

[0122] Seaweed fermentation product fermented by Trichoderma harzianum alone: ​​Trichoderma harzianum seed liquid was inoculated into 500 mL of seaweed fermentation medium, the inoculation amount was 1% of the mass of the seaweed fermentation medium, and cultured in a constant temperature shaker at 32° C. for 36 hours to obtain the seaweed fermentation product.

[0123] The preparation method of the nematicidal seaweed fermentation product of Trichoderma harzianum is the same as that in Example 3.

[0124] 1. Indoor inhibition test of nematodes by nematode-killing seaweed fermentation products

[0125] The specific experimental settings are as follows:

[0126] ①Sterile water control;

[0127] ② Aseptic seaweed fermentation medium (the components of the seaweed fermentation medium are the same as those of the seaweed fermentation medium in Example 3);

[0128] ③ Microbulbifer sp. SH-1 nematicidal seaweed fermentation product;

[0129] ④ Trichoderma harzianum nematicidal seaweed fermentation products;

[0130] ⑤5% avermectin microemulsion (dilution multiple is 2000, refer to the product instructions for use);

[0131] ⑥ The nematicidal seaweed fermentation product obtained in Example 3.

[0132] Referring to the method in Example 1, a suspension of second-instar root-knot larvae (200 larvae / mL) was prepared, and the corrected mortality of nematodes in each treatment was calculated after 24 h and 48 h using the insect immersion method. The results are as follows: Figure 5 shown.

[0133] Depend on Figure 5 It can be seen that the corrected mortality of nematodes by the nematode-killing seaweed fermentation product prepared in Example 3 was significantly higher than that of ② sterile seaweed fermentation medium, ③ nematode-killing seaweed fermentation product of Microbulbifer sp. SH-1 and ④ nematode-killing seaweed fermentation product of Trichoderma harzianum at 24h and 48h, and the mortality of nematodes reached 100% at 48h. However, it was lower than that of 5% avermectin microemulsion treatment at 24h.

[0134] 2. Experiment on the control of banana root-knot nematodes by nematicidal seaweed fermentation products

[0135] The banana variety tested was Williams. Figure 6 As shown, the test site was the soil with serious root-knot nematode disease in the banana planting base of Leiping Town, Daxin County, Chongzuo City, Guangxi. The treatment groups were set up as follows:

[0136] Treatment ① Clean water;

[0137] Treatment ② sterile seaweed fermentation medium (the seaweed fermentation medium components are the same as those in Example 3);

[0138] Treatment ③ Trichoderma harzianum nematicidal seaweed fermentation products;

[0139] Treatment ④ Microbulbifer sp. SH-1 nematicidal seaweed fermentation product;

[0140] Treatment ⑤ 5% avermectin microemulsion (dilution factor is 2000, refer to the product instructions);

[0141] Processing ⑥ The nematicidal seaweed fermentation product prepared in Example 3.

[0142] In the banana root layer, use ①-⑥ to irrigate the roots respectively, dilute 10 times before application (except avermectin, avermectin should be used according to the instructions), apply 500 ml of each treatment group product to each banana each time, and apply once every 15 days. During the banana ripening period, collect banana root samples to analyze the occurrence of root-knot nematodes. Use the control effect to evaluate the severity of nematode disease. For each treatment, select 10 banana trees with similar growth, collect 100 grams of roots from each tree, clean them, and calculate the control effect, as follows.

[0143] Control effect = (control root knot index - treatment root knot index) / control root knot index × 100%

[0144] Root knot index = ∑ (number of diseased plants at each level × number of corresponding levels) / (total number of plants surveyed × highest level value)

[0145] Disease index grading standard: Level 0: healthy root system, no disease and no root knots; Level 1: less than 10% of the roots have root knots, and the root knots are not connected; Level 2: 11%-30% of the roots have root knots, and only a small number of root knots are connected to each other; Level 3: 31%-50% of the roots have root knots, and less than half of the root knots are connected to each other; Level 4: 51%-75% of the roots have root knots, more than half of the root knots are connected to each other, and some of the main and lateral roots are thickened and deformed; Level 5: more than 75% of the roots have root knots, the root knots are connected to each other, and most of the main and lateral roots are thickened and deformed. The results are shown in Table 2:

[0146] Table 2: Field control effect of nematicidal seaweed fermentation products on banana root-knot nematodes

[0147]

[0148]

[0149] As shown in Table 2, consistent with the results of the indoor inhibition test, the nematode control effect of the nematode-killing seaweed fermentation product prepared in Example 3 in the field test was also higher than that of the nematode-killing seaweed fermentation product of Microbulbifer sp. SH-1 and the nematode-killing seaweed fermentation product of Trichoderma harzianum. It is worth mentioning that the control effect of the nematode-killing seaweed fermentation product prepared in Example 3 was more than 70% of that of the 5% avermectin microemulsion.

[0150] Example 5: Study on the field control effect of nematicidal seaweed fermentation products on tomato root-knot nematodes

[0151] In order to explore the control effect of nematicidal seaweed fermentation products on root-knot nematodes, the nematicidal seaweed fermentation products of Microbulbifer sp. SH-1 and Trichoderma harzianum in Example 4, the nematicidal seaweed fermentation products prepared in Example 3 and the 5% avermectin microemulsion commonly used on the market were used as treatments, and clean water was used as a control. The test crop was tomato, and the test site was in a soil area with severe root-knot nematode disease in Leiping Town, Daxin County, Chongzuo City, Guangxi. The specific settings of the treatment groups are as follows,

[0152] ①Clean water;

[0153] ② Aseptic seaweed fermentation medium (the components of the seaweed fermentation medium are the same as those of the seaweed fermentation medium in Example 3);

[0154] ③ Trichoderma harzianum nematicidal seaweed fermentation products;

[0155] ④ Microbulbifer sp. SH-1 nematicidal seaweed fermentation product;

[0156] ⑤5% avermectin microemulsion (dilution multiple is 2000, refer to the product instructions for use);

[0157] ⑥ The nematicidal seaweed fermentation product obtained in Example 3.

[0158] At the root layer of tomato seedlings, use treatments ①-⑥ respectively, dilute 10 times before application (except avermectin, which should be used according to the instructions), apply 500 ml of each treatment group product to each tomato seedling each time, apply once every 20 days until mature and harvest. Observe the growth status of the aboveground part of the tomato, calculate the control effect and theoretical yield of the tomato, the results are as follows Figure 7 shown.

[0159] The control effect and theoretical yield are calculated as follows:

[0160] When the tomatoes are ripe, the tomato roots are removed, the degree of damage is observed, and the control effect is calculated. For details, refer to the control effect evaluation method for bananas in Example 4. The theoretical yield is calculated based on the single plant yield (the number of tomatoes per plant) and the theoretical planting density (theoretical number of trees planted per hectare).

[0161] Control effect = (control root knot index - treatment root knot index) / control root knot index × 100%

[0162] Theoretical output (t / hm 2 ) = single plant yield (t / plant) × theoretical planting density per hectare (plants / hm 2 )

[0163] Depend on Figure 7 It can be seen that the aboveground growth of tomatoes treated with the nematicidal seaweed fermentation product prepared in Example 3 and 5% avermectin microemulsion in ⑥ are significantly better than those in ① and ②, and the wilting degree and yellowing rate of the leaves are also lower; the growth of tomatoes treated with the nematicidal seaweed fermentation product of Trichoderma harzianum and ④ Microbulbifer sp. SH-1 nematicidal seaweed fermentation product is also better than that of ① and ②, but the effect is not as good as that of the nematicidal seaweed fermentation product prepared in Example 3 and 5% avermectin microemulsion in ⑤. The observation results of each treated leaf also show that the tomato leaves treated with the nematicidal seaweed fermentation product prepared in Example 3 and 5% avermectin microemulsion are green, with fewer brown spots on the surface and less yellowing area. The tomato leaves of the other treatments all show different degrees of yellowing, and the tomato leaves treated in ① and ② are obviously dead and diseased. The results show that the nematicidal seaweed fermentation product has a significant control effect on tomato root-knot nematodes.

[0164] Example 6: Indoor inhibitory effect of nematicidal seaweed fermentation products on pine wood nematodes

[0165] In order to more intuitively compare the direct effects of nematicidal seaweed fermentation products on other root-knot nematodes, the effects of nematicidal seaweed fermentation products on pine wood nematodes were observed using an optical microscope.

[0166] The experimental setting was the same as that in Example 4, 1. Indoor inhibition test of nematodes by nematicidal seaweed fermentation products, except that this example only set up the following treatment groups: sterile seaweed fermentation medium, nematicidal seaweed fermentation product of Microbulbifer sp. SH-1, nematicidal seaweed fermentation product of Trichoderma harzianum, and the nematicidal seaweed fermentation product prepared in Example 3.

[0167] Preparation of second-instar pine wood nematode suspension:

[0168] The pine wood nematodes were inoculated on a PDA plate covered with Botrytis cinerea, and after culturing at 25°C for 5-6 days, the nematode suspension was separated and collected using the Baermann funnel method. The collected nematode suspension was centrifuged at 3500r / min for 3min, the supernatant was discarded, and it was washed with sterile water for more than 3 times to obtain a nematode water suspension. Subsequently, the collected nematode water suspension was mixed with 3% hydrogen peroxide in a volume ratio of 1:1, allowed to stand for 10min, disinfected, centrifuged at 3500r / min for 3min, the supernatant was discarded, and it was washed with sterile water for 3 times to obtain a second-instar pine wood nematode suspension.

[0169] The toxic effects of the above-mentioned sterile seaweed fermentation medium, the nematicidal seaweed fermentation product of Microbulbifer sp. SH-1, the nematicidal seaweed fermentation product of Trichoderma harzianum and the nematicidal seaweed fermentation product prepared in Example 3 on pine wood nematodes were tested using the immersion method. The above-mentioned different treatment groups were respectively mixed with the prepared second-instar pine wood nematode suspension (200 nematodes / mL) at a volume ratio of 9:1 and placed in a 96-well culture plate. The number of surviving and dead nematodes was observed after 24 hours. The judgment criteria are as follows: All worms that are constantly swinging or in an "S" shape, curled shape, wavy shape, or spiral shape are judged as living worms; all worms that are not moving and have a "J" shape or a "C" shape, or whose worm bodies are rigid and whose body walls have no refractive index are judged as dead worms. The results are as follows: Figure 8 shown.

[0170] from Figure 8 It can be seen that the nematodes are more active under the treatment of sterile seaweed fermentation medium and Trichoderma harzianum nematicidal seaweed fermentation products. Under the microscope, most nematodes are in S shape and are active (see Figure 8 A, 8C). After treating root-knot nematodes with nematicidal seaweed fermentation products of Microbulbifer sp. SH-1 for 24 hours, some dead nematodes became stiff, elongated, and hook-shaped, while most live nematodes were in a "C" shape (see Figure 8B). After 24 hours of treatment of pine wood nematodes with the nematocidal seaweed fermentation product prepared in Example 3, the nematode mortality rate reached more than 80%. From the appearance, the nematodes treated with the nematocidal seaweed fermentation product prepared in Example 3 had stiff bodies and a hook shape ("C" shape), and most of them were brown (see Figure 8 D).

[0171] Example 7: Study on the stability of nematicidal seaweed fermentation products

[0172] In order to study the stability of the nematicidal ability of the nematicidal seaweed fermentation product, the pH, temperature, storage time and other conditions of the nematicidal seaweed fermentation product prepared in Example 3 were changed, and its insecticidal effect on root-knot nematodes was tested. The details are as follows:

[0173] (1) The nematicidal seaweed fermentation product prepared in Example 3 was adjusted to pH 3, 4, 5, 6, 7, 8, 9, and 10 with 1 mol / L NaOH and HCl, and cultured at 25° C. and 180 r / min for 2 h. The pH was then adjusted back to the original pH, and the nematicidal effects of the nematicidal seaweed fermentation products treated with different pH values ​​were detected 24 h later using the insect immersion method.

[0174] (2) The nematicidal seaweed fermentation product prepared in Example 3 was kept at -20°C, 4°C, 10°C, 25°C, 30°C, 40°C, 50°C, and 60°C for 2 hours, and after being brought to room temperature, the nematicidal effect of the nematicidal seaweed fermentation product treated at different temperatures after 24 hours was detected by the insect immersion method;

[0175] (3) The nematicidal seaweed fermentation product prepared in Example 3 was placed at room temperature for 30 days, 50 days, 70 days, 90 days, 110 days, 130 days, 150 days, and 170 days, respectively. The nematicidal effect of the nematicidal seaweed fermentation product at different storage times was detected after 24 hours using the insect immersion method.

[0176] The results are as follows Fig. 9 As shown, the pH tolerance range of the nematode-killing seaweed fermentation product obtained in Example 3 is between 4 and 9. When the pH is lower than 4 or higher than 9, the inhibitory effect on nematodes will be relatively weakened ( Fig. 9 A); the temperature tolerance range is -20 ℃ ~ 60 ℃, and the inhibitory effect of the nematode-killing seaweed fermentation product prepared in Example 3 on nematodes can still maintain more than 70% ( Fig. 9 B); The tolerance range of storage time is that after 90 days of storage at room temperature, the nematode corrected mortality rate begins to gradually weaken, but the inhibitory effect on nematodes can still be maintained at more than 60% ( Fig. 9 C). The above results show that the nematicidal seaweed fermentation product obtained in Example 3 has good stability and good application value in practical production.

[0177] Example 8: Indoor inhibitory effect of different molecular weights of nematicidal algae fermentation products on root-knot nematodes

[0178] Comparison of nematicidal seaweed fermentation products with different molecular weights is helpful to explore the intrinsic mechanism of nematode inhibition and further process optimization. The nematicidal seaweed fermentation product prepared in Example 3 was centrifuged at 4°C and 6000r / min using cellulose acetate ultrafiltration membranes with different pore sizes to obtain 4 nematicidal seaweed fermentation product components with molecular weights of >10kDa, 5-10kDa, 3-5kDa, and <3kDa, and their insecticidal effects on root-knot nematodes were tested.

[0179] The results are shown in Table 3. The corrected mortality of the nematicidal seaweed fermentation products with a molecular weight less than 3 kDa against root-knot nematodes was significantly higher than that of the nematicidal seaweed fermentation products with a molecular weight of >10 kDa, 5-10 kDa and 3-5 kDa at 24 h and 48 h, while the nematicidal seaweed fermentation products with a molecular weight of 5-10 kDa and 3-5 kDa were significantly higher than that of the nematicidal seaweed fermentation products with a molecular weight of >10 kDa. The above results show that the main active components of the nematicidal fermentation products are mainly in the part with a molecular weight less than 3 kDa, and purifying this part of the components in production is helpful for the application of the product.

[0180] Table 3: Corrected mortality of nematicidal algae fermentation products of different molecular weights against root-knot nematodes

[0181]

[0182]

[0183] Example 9: Metabolite Analysis of Nematicidal Seaweed Fermentation Products

[0184] In order to further explore the intrinsic mechanism of nematode inhibition by nematode-killing seaweed fermentation products, their components were analyzed. The nematode-killing seaweed fermentation product of Microbulbifer sp. SH-1, the nematode-killing seaweed fermentation product of Trichoderma harzianum, and the nematode-killing seaweed fermentation product prepared in Example 3 were used as samples. Each sample was mixed with methanol at a volume ratio of 1:4, allowed to stand at -20°C for 1 hour, centrifuged at 12000 rpm for 10 minutes at 4°C, the supernatant was freeze-dried, and the dried product was re-dissolved with methanol at a ratio of 0.02 g / mL to obtain a test solution. The test solution was sent to the Analysis and Testing Center of South China Agricultural University for qualitative analysis and identification of the product using ultra-high pressure liquid chromatography-quadrupole tandem time-of-flight mass spectrometry. The results are as follows Fig.10 And as shown in Table 4.

[0185] Table 4: Analysis of different substances in fermentation broth components

[0186]

[0187]

[0188] Depend on Fig.10 As shown in Table 4, 151, 124, and 188 substances were detected in the nematicidal algae fermentation product of Microbulbifer sp. SH-1, the nematicidal algae fermentation product of Trichoderma harzianum, and the nematicidal algae fermentation product prepared in Example 3, respectively. Through the material analysis of the database, a total of 21 fermentation products were detected in the nematicidal algae fermentation product prepared in Example 3 that were not present in the nematicidal algae fermentation product of Microbulbifer sp. SH-1 and the nematicidal algae fermentation product of Trichoderma harzianum. By consulting relevant materials, it was found that substances such as Fosfomycin, Xanthohumol D, Nummularine A, and Mulberrofuran Q in the products were confirmed in existing reports to have good effects in antibacterial, pest and disease resistance, and inducing crop defense mechanisms.

[0189] In addition, the present invention detected the changes in the contents of alginic acid, total seaweed phenols, mannitol, betaine and other substances in the nematicidal seaweed fermentation product of Microbulbifer sp. SH-1, the nematicidal seaweed fermentation product of Trichoderma harzianum, and the nematicidal seaweed fermentation product prepared in Example 3. The specific operation is as follows:

[0190] Main active substance content determination method:

[0191] (1) Determination of alginic acid: refer to the “m-hydroxybiphenyl spectrophotometric method” in “NY / T 3174-2017 Determination of alginic acid content in water-soluble fertilizers”. The results are as follows: Fig.11 As shown in A.

[0192] (2) Determination of total phenols in seaweed was carried out according to the Folin phenol colorimetric method (Sun Luchuanyang, Ren Dandan, Wang Shuai, et al. Optimization of the process of extracting brown algae polyphenols from copper algae by bioenzymatic method [J]. Journal of Food Safety and Quality, 2019, 10(08): 2201-2206). The results are as follows: Fig.11 As shown in B.

[0193] (3) The determination of mannitol and betaine was carried out by ultra-high performance liquid chromatography-tandem mass spectrometry (Zhang Wenxi, Tian Qiong, Cui Weiguo. Simultaneous determination of mannitol and betaine in seaweed fertilizer by ultra-high performance liquid chromatography-tandem mass spectrometry [J]. Chinese Agricultural Science Bulletin, 2015, 31(11): 177-181.). The results are as follows: Fig.11 C, as shown in 11D.

[0194] Depend on Fig.11It can be seen that the contents of alginate, total seaweed phenols and betaine in the nematicidal seaweed fermentation product prepared in Example 3 are significantly higher than those in other single bacteria fermentation treatments, which indicates that the nematicidal seaweed fermentation product prepared in Example 3 may have a potential growth-promoting effect on plant growth.

[0195] Example 10: Study on the growth-promoting effect of nematicidal seaweed fermentation products on cucumber seedlings

[0196] In order to verify the growth-promoting effect of nematicidal fermentation products, different nematicidal seaweed fermentation products were applied to cucumber seedlings to observe the effects on the biomass and plant height of cucumber seedlings. The treatment groups were set as follows:

[0197] Treatment ①: clean water;

[0198] Treatment ②: sterile seaweed fermentation medium (the components are the same as those of the seaweed fermentation medium in Example 3);

[0199] Treatment ③: nematicidal algae fermentation product of Microbulbifer sp. SH-1 (prepared in Example 4);

[0200] Treatment ④: Trichoderma harzianum nematicidal seaweed fermentation product (prepared in Example 4);

[0201] Treatment ⑤: containing 50% of the nematicidal seaweed fermentation product prepared in Example 3 (diluted with water);

[0202] Treatment ⑥: containing 70% of the nematicidal seaweed fermentation product prepared in Example 3 (diluted with water);

[0203] Treatment ⑦: 100% of the nematicidal seaweed fermentation product prepared in Example 3.

[0204] The specific tests are as follows:

[0205] Pot experiment: The cucumber seeds were sterilized with 75% alcohol for 15 minutes, and rinsed three times with deionized water for later use. 1.5 kg of soil was weighed for each pot, and 2 seeds were sown in the middle of the pot. Starting from the sowing day, the seeds were irrigated every 10 days according to the above treatment group settings. Each treatment was irrigated with 500 mL. Each treatment group was diluted 100 times before irrigating. After 40 days, the cucumber seedlings were collected and the plant height and aboveground biomass were measured. The results are as follows: Fig.12 shown.

[0206] Depend on Fig.12 It can be seen that, from the overall growth perspective, the nematicidal seaweed fermentation product prepared in Example 3 containing 50%, 70% and 100% of the nematicidal seaweed fermentation product had the best growth-promoting effect on cucumber seedlings ( Fig.12 A), the plant height and biomass were significantly higher than those treated with Microbulbifer sp. SH-1 nematicidal algae fermentation product and Trichoderma harzianum nematicidal algae fermentation product ( Fig.12 B, 12C), indicating that the nematicidal seaweed fermentation product prepared in Example 3 of the present invention has a certain growth-promoting effect on crops.

[0207] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A nematicidal seaweed fermentation product, characterized in that: The invention comprises seaweed fermentation products; the seaweed fermentation products are obtained by inoculating a strain with high alginate lyase production into a seaweed fermentation medium, adjusting the pH of the medium to 7.0 after fermentation for 12 hours, and then inoculating the biocontrol strain for fermentation and culture; The high-yield alginate lyase strain is Microvesicles ( Microbulbifer sp. )SH-1; The biocontrol strain is Trichoderma harzianum.

2. The nematicidal seaweed fermentation product according to claim 1, characterized in that: The seaweed in the seaweed fermentation medium is at least one of Sargassum fusiformis, Laminaria japonica, Ascophyllum nodosum, Sargassum truncatum, N. fasciata and N. fasciata; The concentration of seaweed in the seaweed fermentation medium is 10.0-30.0 g / L.

3. The nematicidal seaweed fermentation product according to claim 2, characterized in that: The components of the seaweed fermentation medium are: 30.0 g / L of kelp dry powder, 5.0 g / L of ammonium sulfate, 1.0 g / L of magnesium sulfate, 1.0 g / L of dipotassium hydrogen phosphate, 0.02 g / L of ferrous sulfate, and 15.0 g / L of sodium chloride; the pH of the seaweed fermentation medium is 7.0-7.

5.

4. The nematicidal seaweed fermentation product according to claim 1, characterized in that: The conditions for fermentation culture of biocontrol strains are: pH 6.5-8.0, fermentation temperature 28-34°C and fermentation time 24-72h; The inoculation amount of the high-yield alginate lyase strain is 0.5% to 1.5%; The inoculation amount of the biocontrol strain is 0.5% to 1.5%.

5. The nematicidal seaweed fermentation product according to claim 1, characterized in that: The nematicidal seaweed fermentation product also includes a stabilizer; The stabilizer includes an inhibitor and a suspending agent; The inhibitor and the suspending agent are calculated in a mass ratio of 0.5-0.7:2-5; The stabilizer is obtained by mixing an inhibitor with a suspending agent and stirring; The inhibitor comprises at least two of potassium sorbate, benzoic acid and kasone; The suspending agent comprises at least two of dimethyl sulfoxide, methyl pyrrolidone, dioctyl sodium sulfosuccinate, sodium alkylbenzene sulfonate, succinic acid diester sulfonate, sodium carboxymethyl cellulose, polyglutamic acid and hydroxyethyl cellulose.

6. The nematicidal seaweed fermentation product according to claim 5, characterized in that: The preparation method of the inhibitor is as follows: dissolving kason and benzoic acid in water and stirring to obtain the inhibitor; Wherein, the kason and benzoic acid are calculated in a mass ratio of 0.1-0.5:0.5-0.1; The mass volume ratio of the cassonne to water is 0.1-0.5 g:0.1 L; The preparation method of the suspension is as follows: dissolving dimethyl sulfoxide and methyl pyrrolidone in water and stirring to obtain the suspension; The dimethyl sulfoxide and methyl pyrrolidone are calculated in a mass ratio of 1.3-1.7:1.7-1.3; The dimethyl sulfoxide and water are calculated according to a mass volume ratio of 1-5 g:0.1L.

7. The nematicidal seaweed fermentation product according to claim 1, characterized in that: The nematicidal seaweed fermentation product includes a portion with a molecular weight less than 3 kDa.

8. Use of the nematicidal seaweed fermentation product according to any one of claims 1 to 7 in preparing nematicidal drugs and / or promoting cucumber growth.

9. The use according to claim 8, characterized in that: The nematodes include at least one of root-knot nematodes and pine wood nematodes.

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