Nocardia nematophila for biological control of root-knot nematodes and its application

Abstract

The present application discloses a Nocardia nematophila for biological control of root-knot nematodes and its application, which is the Nocardia nematophila strain PE06 with CCTCC NO: M 20221105. This strain and its metabolites can effectively control the root-knot nematode disease of cigar tobacco leaves, and have the characteristics of low use cost, high control efficacy and no residue. It can enrich the existing microbial species for controlling root-knot nematodes and has no pollution during application.

Description

Technical Field

[0001] The present application relates to the technical field of microbial biocontrol, in particular to Nocardia nematophila for biological control of root-knot nematodes and its application. Background Art

[0002] Root-knot nematode disease caused by the infection of Meloidogyne spp. is an important soil-borne disease of various crops. More than 90 species of root-knot nematodes have been reported globally, and more than 50 species are distributed in China. Among them, the four most harmful species are Meloidogyne incognita, Meloidogyne hapla, Meloidogyne arenaria, and Meloidogyne javanica (Liu Weizhi, 2000). The annual loss of tobacco production caused by root-knot nematodes worldwide is estimated to be 15%

[0003] (Shew, Lucas, 1991).

[0004] Cigar is a special tobacco product made from raw tobacco leaves after drying, fermentation, and aging processes. It has the characteristics of cigar flavor type, rich aroma, strong taste, strong strength, alkaline flue gas, and a small ratio of tar to nicotine. Cigar tobacco leaves are mainly widely planted in countries such as Cuba, Indonesia, the United States, the Dominican Republic, Honduras, Brazil, Mexico, Jamaica, and Nicaragua. According to statistics, in the first half of 2015, the production and sales volume of cigarettes in China decreased by 3.11% and 2.23% respectively, but Chinese cigars bucked the trend. The output increased by 37.2%, the sales volume increased by 82%, and the sales amount increased by 55.4%. Cigars have become a new economic growth point in the Chinese tobacco industry (Wang Wenhua, 2015).

[0005] Currently, there is a serious shortage of high-quality domestic cigar tobacco leaves, and both the cigar wrapper and binder are completely dependent on imports. In recent years, China has introduced a variety of cigar tobacco leaf varieties and carried out large-scale trial plantings in provinces such as Zhejiang, Hainan, Sichuan, Hubei, and Yunnan. The cigar industry is gradually taking shape (Wang Yanyan, et al., 2020).

[0006] Multiple tobacco-growing areas in Yunnan Province have excellent geographical and climatic conditions for growing cigar tobacco leaves. The soil is mainly composed of sand and clay, and it is one of the most potentially advantageous production areas for developing high-quality cigar raw materials in China. In recent years, more than 11,000 mu of cigar tobacco leaves have been planted in Yunnan Province, and root-knot nematode disease has occurred to varying degrees. The main root-knot nematode species infecting cigar tobacco leaves are Meloidogyne javanica, Meloidogyne incognita, and Meloidogyne arenaria (Ma Guimei, et al., 2022). Since growing cigar wrapper varieties requires building shade net sheds and the input cost is relatively high, continuous cropping often occurs on the same piece of land for many years, resulting in the gradual aggravation of root-knot nematode disease. Root-knot nematode disease has become one of the important diseases hindering the sustainable development of the cigar tobacco leaf industry in the tobacco-growing areas.

[0007] For a long time, the control of root-knot nematodes has mainly relied on spraying chemical pesticides. However, due to the high toxicity of chemical nematicides, the problems of agricultural product and ecological pollution are very prominent. Currently, chemical nematicides such as ethoprophos, methyl bromide, and aldicarb are almost banned or restricted in use in China. This has led to even fewer available chemical nematicide varieties, exacerbating the difficulty of nematode control.

[0008] Nematode biocontrol microorganisms can kill nematodes through predation, parasitism, toxin production, etc., and are important microbial groups that control nematode populations in nature (Zhang & Hyde, 2014). The virulence factors of nematode-killing bacteria that cause nematode death include: parasporal crystal proteins (Cry), invasive enzymes, toxic small molecule metabolites, volatile active substances, etc. (Colagiero et al., 2017). Nematicides developed using biocontrol bacteria Bacillus firmus (trade names: BioNem, VoTiVo), Pasteuria penetrans (Econem), Burkholderia cepacia (Deny, Blue Circle), Paenobacillus macerans & B. amyloliquefaciens (BioYield), Bacillus spp. mixture (Biostart) have been registered and applied in different countries (Priyank et al., 2019). There are currently few types of biocontrol bacteria for root-knot nematodes, and the varieties are single.

[0009] Zhang et al. (2022) isolated a bacterium R-N-C8 from the rhizosphere soil of Arabidopsis thaliana T and reported a new species of Nocardioides nematodiphilus using this as a type strain, but there is no report on the biocontrol characteristics of this strain against plant parasitic nematodes.

[0010] The relevant literatures in the above text are as follows:

[0011] 1: Priyank HM, Chinnannan K, Kadirvelu K, et al., Plant growth promoting rhizobacteria (PGPR): A potential alternative tool for nematodes bio-control. Biocatalysis and Agricultural Biotechnology, 2019, 17: 119 - 128.

[0012] 2: Shew HD, Lucas GB. Compendium of tobacco diseases. United States: American Phytopathological Society, 1991.

[0013] 3: Zhang KQ, Hyde KD. 2014. Nematode-trapping fungi. Dordrecht, Neth.: Springer, pp. 1 - 392.

[0014] 4: Zhang XM, Mo WT, Wei YQ, Li X, Ma GM, Yang YS, Wang XJ, Liu JJ, Liu ZY, Zhou XK. Nocardioides nematodiphilus sp. nov., isolated from rhizosphere of Arabidopsis thaliana. Int. J. Syst. Evol. Microbiol.

[0015] 2022; 72:005271. DOI 10.1099 / ijsem.0.005271.

[0016] 5: Ma Guimei, Tan Tao, Yang Dong, Liu Tong, Mo Minghe, Wang Yang, Jiang Fangrong, Zhang Yinghe, Zhao Hua, Liu Jianjin. 2022. Pathogen identification of root-knot nematode disease of cigar tobacco in Jiangcheng, Yunnan and control effect of biogenic ammonia fumigation. Journal of Yunnan University (Natural Sciences Edition), 44:1 - 7. DOI: 10.7540 / j.ynu.20220171.

[0017] 6: Liu Weizhi. Plant pathogenic nematology. Beijing: China Agricultural Press, 2000.

[0018] 7: Wang Wenhua. The economic operation of the tobacco industry has been steadily improved in the first half of 2015 [EB / OL]. Oriental Tobacco Network, 2015 - 7 - 15.

[0019] 8: Wang Yanyan, Liu Guoxiang, Xiang Xiaohua, et al. Overview of main production areas and variety resources of cigar tobacco at home and abroad [J]. Chinese Tobacco Science, 2020, 41(3):93 - 98. Summary of the Invention

[0020] In view of the problems of difficult control of root-knot nematodes and relatively few biocontrol bacterial strains in the prior art, the present application provides a Nocardia nematophila for biocontrol of root-knot nematodes and its application.

[0021] The present application provides a Nocardioides nematodiphilus for biological control of root-knot nematodes and its application, which is the Nocardioides nematodiphilus strain PE06 with CCTCC NO: M20221105.

[0022] In the present application, the Nocardioides nematodiphilus strain PE06 was isolated from the tobacco stalks of healthy tobacco plants of the Yunyan 87 tobacco variety in Ning'er County, Pu'er City, Yunnan Province. This strain has good killing effects on various root-knot nematodes and can be used for the control of root-knot nematodes in various plants.

[0023] Preferably, the root-knot nematode is any one of Meloidogyne incognita, Meloidogyne javanica, and Meloidogyne arenaria. Specifically, it can control the above three root-knot nematode diseases.

[0024] Preferably, the plant for biological control by Nocardioides nematodiphilus is a cigar tobacco plant; the root-knot nematode is a second-instar larva; preferably, the mortality rate of strain PE06 against Meloidogyne javanica J2 is more than 99%. It is particularly suitable for the control of root-knot nematode diseases in cigar tobacco plants.

[0025] Another aspect of the present application also provides a fermentation broth for biological control of root-knot nematodes, including: the Nocardioides nematodiphilus for biological control of root-knot nematodes as described above.

[0026] After fermentation with the above-mentioned endophyte, a fermentation broth containing the strain and metabolites can be obtained. The specific fermentation operation can be carried out with reference to the existing method for preparing fermentation broth.

[0027] Preferably, the preparation method includes the following steps: slant culture, liquid seed culture, and fermentation culture to obtain a fermentation broth containing the cells of the strain and its metabolites.

[0028] Preferably, the slant culture conditions: inoculate the Nocardioides nematodiphilus for biological control of root-knot nematodes on the slant of R2A medium and culture at 30°C - 37°C for 1 - 2 days;

[0029] The seed liquid culture conditions: use R2A liquid medium and culture on a shaker at 30°C - 37°C, 100 - 150 rpm for 2 - 3 days to obtain the seed liquid;

[0030] The fermentation broth culture conditions: inoculate the seed liquid into R2A liquid medium at a volume ratio of 1% - 5%, and control the fermentation culture conditions at: temperature 30°C - 35°C, stirring speed 150 - 180 rpm, fermentation time 48 - 72 hours to obtain the fermentation broth;

[0031] Preferably, the volume of the fermentation tank used for fermentation broth culture is 500 - 1000 L.

[0032] The fermentation broth prepared by fermentation under the above conditions can be used for the control of root-knot nematodes. For example, the fermentation broth and its diluent can be sprayed on the plants to achieve control.

[0033] On the other hand, the present application also provides a biocontrol agent for root-knot nematodes. After mixing the above fermentation broth with an adsorption carrier, it is dried at a low temperature until the water content is less than 5%, and then pulverized to obtain the agent. The viable count of strain PE06 in the obtained agent is above 1×10 10 CFU / g.

[0034] Preferably, the adsorption carrier used is soluble corn starch, and the drying temperature is 50°C - 65°C; the adsorption carrier and the fermentation broth are mixed at a volume ratio of 3 - 2:1.

[0035] The agent prepared by this method can effectively maintain the activity of strain PE06, which is convenient for subsequent transportation, sales and use.

[0036] Preferably, using this method: the obtained agent is mixed with the plant seeds at a mass ratio of 1 - 3:1 and evenly mixed, then seedlings are raised, and after obtaining the inoculated seedlings, they are transplanted; by using this method of use, the strain can be colonized at the seed part, so that it is convenient for the subsequent strain to be evenly distributed in each part of the plant as the plant grows and develops, and the control effect is improved. More preferably, the plant seeds are tobacco seeds. This agent can be used for the control of root-knot nematodes of various plants, such as tomato seeds, peanut plants, and cucumber plants.

[0037] Preferably, using this method: the obtained agent is mixed with the plant seeds at a mass ratio of 2 - 3:1 and evenly mixed, then seedlings are raised, and after obtaining the inoculated seedlings, they are transplanted. By using this agent according to this method, the control effect can reach more than 70%, solving the problem of the control effect of pesticides.

[0038] Preferably, using this method: before transplantation, the obtained agent is dissolved and diluted with water, and then the roots of the plants are immersed in the medicine solution and then transplanted;

[0039] Preferably, the dilution multiple of the agent in the method of use is 50 - 100 times; the immersion time of the plant roots is 10 - 20 minutes. Since the main infection part of root-knot nematodes is the root, by using this method of use, the number of this strain in the roots of plants can be effectively increased, achieving effective control.

[0040] The beneficial effects that the present application can produce include:

[0041] 1) The Nocardioides nematodiphilus for biological control of root-knot nematodes provided by this application and its application. The Nocardioides nematodiphilus strain PE06 was isolated from the healthy tobacco stalks of the tobacco variety Yunyan 87 in Ning'er County, Puer City, Yunnan Province. This strain and its metabolites can effectively control the root-knot nematode disease of cigar tobacco leaves, with the characteristics of low usage cost, high control efficiency, and no residue. It can enrich the existing microbial species for root-knot nematode control and has no pollution during application.

[0042] 2) The Nocardioides nematodiphilus for biological control of root-knot nematodes provided by this application and its application. The fermentation broth of this strain has strong lethal activity against the second-stage larvae (J2) of three root-knot nematodes (Meloidogyne incognita, Meloidogyne javanica, Meloidogyne arenaria) that infect cigar tobacco leaves and other plants. It has important application value in the control of root-knot nematode disease of cigar tobacco leaves. The control efficiency of root-knot nematodes on cigar tobacco plants can reach over 70%, which is basically close to the control efficiency of pesticides. It can partially replace pesticides and reduce the usage amount of pesticides.

[0043] Biological preservation information:

[0044] The Nocardioides nematodiphilus strain PE06 was preserved in the China Center for Type Culture Collection on July 20, 2022; the address of the preservation unit: Wuhan University, Bayi Road, Hongshan District, Wuhan City, Hubei Province; the preservation number: CCTCC NO: M 20221105. Detailed implementation manners

[0045] To make the purpose, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments.

[0046] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the present invention claimed, but merely represents the selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0047] Examples

[0048] For the materials, seeds, and instruments used in the following examples, unless otherwise specified, they are all obtained through commercial channels.

[0049] Example 1 Obtaining of strain PE06

[0050] The Nocardioides nematodiphilus strain PE06 was isolated from the tobacco stalks of healthy tobacco plants of the Yunyan 87 tobacco variety in Ning'er County, Pu'er City, Yunnan Province. The isolation method of this strain refers to the isolation method disclosed in Feng Yunli, et al. 2011. Screening and population composition analysis of biocontrol bacteria against tobacco black shank in endophytic bacteria of flue-cured tobacco variety NC297. Journal of Yunnan University, 33(4): 488-496.

[0051] The target nematodes used for isolation were: second-stage juveniles (J2) of Meloidogyne javanica. Method for preparing the target nematode suspension: Collect root nodules infected with nematodes from the Pu'er cigar tobacco area, pick egg masses from the root nodules into a petri dish containing sterile water, incubate at 28°C for 5-7 days, and centrifuge and concentrate at 6000 rpm to obtain a J2 suspension. For the isolated strain PE06, after repeating the treatment 3 times, the lethality rate (microscopic examination result) against the second-stage juveniles of Meloidogyne javanica was more than 99%.

[0052] Example 2 Cultivation of Strain PE06 and Preparation of Bacterial Agent

[0053] Test tube slant seed culture of strain PE06: Inoculate this strain onto a conventional R2A medium slant and culture at 30°C for 3 days to obtain slant seeds. The formula of the R2A medium is (g / L): yeast extract 0.5, peptone 0.5, casein hydrolysate 0.5, glucose 0.5, soluble starch 0.5, dipotassium hydrogen phosphate 0.3, anhydrous magnesium sulfate 0.024, sodium pyruvate 0.3, agar 15, pH 7.2.

[0054] Liquid seed culture of strain PE06: Inoculate the slant seeds into a triangular flask containing R2A liquid medium and culture on a shaker at 30°C and 150 rpm for 3 days to obtain liquid seeds.

[0055] Large-scale culture of strain PE06 in a fermenter: Inoculate the liquid seeds into the R2A medium in the fermenter at a ratio of 3% (V / V). The culture conditions in a 500L fermenter are controlled as follows: temperature 30°C - 35°C, stirring speed 150 - 180 rpm, fermentation time 48 - 72 hours.

[0056] Preparation of the bacterial agent of strain PE06: Obtain a fermentation broth containing bacteria and their metabolites through fermenter culture. Mix the fermentation broth with the adsorbent carrier soluble corn starch at a volume ratio of 1:3, dry or air-dry at 55°C until the moisture content is less than 5%, and crush to a particle size less than 100 mesh to prepare the bacterial agent. The viable bacteria count of strain PE06 in the bacterial agent is more than 1×10 10 CFU / g.

[0057] Example 3 Field Test of Controlling Root-Knot Nematode Disease by Treating Seeds with PE06 Bacterial Agent

[0058] Test site: Cigar tobacco leaf planting base, Zhengdong Village, Zhengdong Town, Jiangcheng County, Pu'er City, Yunnan Province.

[0059] Test crop: Cigar tobacco leaves (variety: Yunxue No. 1).

[0060] Test agents: Bacterium agent PE06, with viable bacteria count of 10×10 10 CFU / g, prepared by the method in the above-mentioned embodiment; 10% fosthiazate granules, produced by Shandong United Pesticide Industry Co., Ltd.

[0061] Test method: A total of three treatments were set as follows, with 3 replicates for each treatment and 120 tobacco plants for each replicate.

[0062] Treatment 1: Bacterium agent PE06: tobacco seeds (W / W) = 1:1. When transplanting after seedling raising, the bacterium agent was evenly mixed with the seeds, and then the bacterium-carrying seedlings were obtained by the conventional seedling raising method and then transplanted.

[0063] Treatment 2: Bacterium agent PE06: tobacco seeds (W / W) = 2:1. When transplanting seedlings, the bacterium agent was evenly mixed with the seeds, and then the bacterium-carrying seedlings were obtained by the conventional seedling raising method and then transplanted.

[0064] Treatment 3: Bacterium agent PE06: tobacco seeds (W / W) = 3:1. When transplanting seedlings, the bacterium agent was evenly mixed with the seeds, and then the bacterium-carrying seedlings were obtained by the conventional seedling raising method and then transplanted.

[0065] Treatment 4: 2 kg / mu of 10% fosthiazate granules. For conventional seedling raising and planting, when preparing the land, the granules were evenly spread on the soil surface and mixed into the soil with a tiller.

[0066] Treatment 5: Seedling raising and planting were carried out according to the conventional operation, with a blank control without applying any nematicide.

[0067] Investigation and statistical method: After the harvest of cigar tobacco leaves, the root-knot disease grade index of the plants was investigated (Grade 0: normal roots without root knots; Grade 1: a small amount of root knots on less than one-fourth of the roots; Grade 3: a small amount of root knots on one-fourth to one-third of the roots; Grade 5: root knots on one-third to one-half of the roots; Grade 7: root knots on more than one-half of the roots, with a small amount of adventitious roots having root knots; Grade 9: all roots (including adventitious roots) covered with root knots), and the disease index and control effect were calculated according to the following formula:

[0068] Disease index = (n1×1 + n3×3 + n5×5 + n7×7 + n9×9) / (S×9)×100, where n1 - n9 represent the total number of plants corresponding to the root-knot disease grade index from 1 to 9, and S represents the total number of plants investigated. Control effect (%) = 100(1 - x / y), where x and y represent the disease indices of the treatment and the blank control respectively. The obtained results are shown in the following table:

[0069] Table 1. Control effect results of each treatment on root-knot nematode disease of cigar tobacco leaves

[0070] Treatment Disease index Control efficacy (%) Treatment 1 (PE06 bacterial agent: tobacco seeds = 1:1) 13.25 56.08c Treatment 2 (PE06 bacterial agent: tobacco seeds = 2:1) 9.14 69.71b Treatment 3 (PE06 bacterial agent: tobacco seeds = 3:1) 8.33 72.39a Treatment 4 (10% fosthiazate granules) 8.69 71.20a Treatment 5 (Blank control) 30.17 -

[0071] Note: The same letter after the numbers in the same column indicates no significant difference, otherwise the difference is significant.

[0072] Test results: Table 1 shows that when tobacco seeds are treated with the PE06 bacterial agent during seedling raising, the control effects on root-knot nematodes of cigar tobacco leaves are 56.08%, 69.71% and 72.39% respectively when the ratio of PE06 bacterial agent to tobacco seeds is 1:1, 2:1, and 3:1; the control effect of applying 2 kg of 10% fosthiazate granules per mu is 71.20%. It shows that treating tobacco seeds with the PE06 bacterial agent during seedling raising has a good control effect on root-knot nematode disease of cigar tobacco leaves. When the dosage ratio of PE06 bacterial agent to tobacco seeds is above 2:1, its control effect is not lower than that of the commonly used chemical nematicide fosthiazate.

[0073] Example 4 Field test on preventing and controlling root-knot nematode disease by dipping roots with PE06 bacterial agent during transplanting period

[0074] Test site: Cigar tobacco leaf planting base in Zhengdong Village, Zhengdong Town, Jiangcheng County, Pu'er City, Yunnan Province.

[0075] Test crop: Cigar tobacco leaves (variety: Yunxue No. 1).

[0076] Test agents: PE06 bacterial agent, with a viable bacteria count of 10×10 10 CFU / g, prepared according to the above method of the present invention; 10% fosthiazate granules, produced by Shandong United Pesticide Industry Co., Ltd.

[0077] Test method: A total of three treatments were set as follows, with 3 replicates for each treatment and 120 tobacco plants for each replicate.

[0078] Treatment 1: 0.5 kg / mu of the strain PE06 bacterial agent. Before transplanting, dissolve the bacterial agent obtained in Example 2 in about 100 kg of water, mix well, soak the roots of tobacco seedlings in the medicine for about 10 minutes, and then transplant.

[0079] Treatment 2: 1 kg / mu of the strain PE06 bacterial agent. During transplanting, dissolve the bacterial agent in about 100 kg of water, mix well, soak the roots of tobacco seedlings in the medicine for about 10 minutes, and then transplant.

[0080] Treatment 3: 1.5 kg / mu of the strain PE06 bacterial agent. During transplanting, dissolve the bacterial agent in about 100 kg of water, mix well, soak the roots of tobacco seedlings in the medicine for about 10 minutes, and then transplant.

[0081] Treatment 4: 2 kg / mu of 10% fosthiazate granules. When preparing the land, evenly spread the granules on the soil surface and mix them into the soil with a tiller.

[0082] Treatment 5: Blank control without applying any nematicide.

[0083] Investigation and statistical method: After harvesting cigar tobacco leaves, investigate the root-knot nematode disease index of the plants (Grade 0: normal roots; Grade 1: a small number of root knots on less than one-fourth of the roots; Grade 3: a small number of root knots on one-fourth to one-third of the roots; Grade 5: root knots on one-third to one-half of the roots; Grade 7: root knots on more than one-half of the roots, and a small number of adventitious roots with root knots; Grade 9: all roots (including adventitious roots) covered with root knots), and calculate the disease index and control effect according to the following formula:

[0084] Disease index = (n1×1 + n3×3 + n5×5 + n7×7 + n9×9) / (S×9)×100, where n1 - n9 represent the total number of plants corresponding to root-knot nematode disease index levels 1 - 9 respectively, and S represents the total number of plants investigated.

[0085] Control effect (%) = 100(1 - x / y), where x and y represent the disease indices of the treatment and the blank control respectively.

[0086] The obtained results are shown in the following table:

[0087] Table 2. Control effect results of each treatment on root-knot nematode disease of cigar tobacco leaves

[0088] Treatment Disease index Control efficacy (%) Treatment 1 (0.5 kg / acre of PE06 bacterial agent) 12.67 62.31c Treatment 2 (1 kg / acre of PE06 bacterial agent) 9.43 71.95b Treatment 3 (1.5 kg / acre of PE06 bacterial agent) 8.26 75.43a Treatment 4 (10% fosthiazate granules) 10.25 69.51b Treatment 5 (Blank control) 33.62 -

[0089] Note: The same letter after the numbers in the same column indicates no significant difference, otherwise there is a significant difference.

[0090] Experimental results: Table 2 shows that when applying 0.5, 1, and 1.5 kg of PE06 bactericide per mu, the control effects on root-knot nematode of cigar tobacco leaves are 62.31%, 71.95%, and 75.43% respectively; the control effect of applying 2 kg of 10% fosthiazate granules per mu is 69.51%. It shows that applying 1 kg or more of PE06 bactericide per mu has a control effect on root-knot nematode of cigar tobacco leaves above 70%, and its control effect is not lower than that of the commonly used chemical nematicide fosthiazate.

[0091] Preparation of the bactericide in Example 5

[0092] The difference from Example 2 is as follows: Slant culture conditions: Inoculate Nocardia nematophila for biocontrol of root-knot nematodes on the slant of R2A medium and culture at 37°C for 2 days;

[0093] Seed liquid culture conditions: Use R2A liquid medium and culture on a shaker at 37°C and 150 rpm for 3 days to obtain the seed liquid;

[0094] Fermentation broth culture conditions: Inoculate the seed broth into the R2A liquid medium at a volume ratio of 5%, and control the fermentation culture conditions at: temperature 35°C, stirring speed 180 rpm, fermentation time 72 hours to obtain the fermentation broth; mix soluble corn starch and the fermentation broth at a volume ratio of 3:1.

[0095] The volume of the fermenter used for culturing the fermentation broth is 1000 L.

[0096] The drying temperature during the preparation of the microbial agent is 50°C.

[0097] Preparation of the microbial agent in Example 6

[0098] The difference from Example 2 lies in: Slant culture conditions: Inoculate Nocardia nematophila for biological control of root-knot nematodes on the R2A medium slant and culture at 37°C for 1 day;

[0099] Seed broth culture conditions: Use the R2A liquid medium to culture on a shaker at 30°C and 100 rpm for 2 days to obtain the seed broth;

[0100] Fermentation broth culture conditions: Inoculate the seed broth into the R2A liquid medium at a volume ratio of 5%, and control the fermentation culture conditions at: temperature 30°C, stirring speed 150 rpm, fermentation time 48 hours to obtain the fermentation broth; mix soluble corn starch and the fermentation broth at a volume ratio of 2:1; ^

[0101] The drying temperature during the preparation of the microbial agent is 65°C.

[0102] The control efficacy results of the microbial agents obtained in Examples 5 - 6 are similar to those of the microbial agent obtained in Example 2, and will not be elaborated here.

[0103] Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Nocardia nematophila strain PE06 for biological control of root-knot nematodes ( Nocardioides nematodiphilus ), characterized in that The preservation number of the Nocardioides nematodiphilus strain PE06 for biocontrol of root-knot nematodes is CCTCC NO: M20221105.

2. A fermentation broth for biological control of root-knot nematodes, characterized in that, Including: The Nocardioides nematodiphilus strain PE06 for biocontrol of root-knot nematodes as described in claim 1.

3. The fermentation broth for biological control of root-knot nematodes according to claim 2, wherein The preparation method includes the following steps: slant culture, liquid seed culture, and fermentation culture are carried out in sequence to obtain a fermentation broth containing the bacterial cells of this strain and its metabolites.

4. The fermentation broth for biological control of root-knot nematodes according to claim 3, characterized in that, Slant culture conditions: Inoculate the Nocardioides nematodiphilus strain PE06 for biocontrol of root-knot nematodes on an R2A medium slant and culture at 30°C - 37°C for 1 - 2 days; Seed liquid culture conditions: Use an R2A liquid medium and culture on a shaker at 30°C - 37°C and 100 - 150 rpm for 2 - 3 days to obtain a seed liquid; Fermentation broth culture conditions: Inoculate the seed liquid into an R2A liquid medium at a volume ratio of 1 - 5%, and control the fermentation culture conditions at: temperature 30°C - 35°C, stirring speed 150 - 180 rpm, fermentation time 48 - 72 hours to obtain a fermentation broth.

5. The fermentation broth for biological control of root-knot nematodes according to claim 3, characterized in that, The volume of the fermenter used for fermentation broth culture is 500 - 1000 L.

6. A biocontrol agent against root-knot nematodes, characterized in that, Mix the fermentation broth for biological control of root-knot nematodes according to any one of claims 2 to 5 with an adsorption carrier, then dry it at a low temperature until the water content is less than 5%, and then crush it to obtain a biological control agent for root-knot nematodes; the viable count of strain PE06 in the biological control agent for root-knot nematodes is above 1×10 10 CFU / g.

7. The biocontrol agent against Meloidogyne spp. according to claim 6, wherein The adsorption carrier used is soluble corn starch, and the drying temperature is 50°C - 65°C; the adsorption carrier and the fermentation broth are mixed at a volume ratio of (2 - 3):

1.

8. A method for using the biocontrol agent against root-knot nematodes as described in claim 6 or 7, characterized in that, Mix the biocontrol agent for root-knot nematodes and the plant seeds evenly at a mass ratio of (1 - 3):1, then carry out seedling raising, and transplant after obtaining the bacteria-carrying seedlings.

9. The usage method according to claim 8, wherein, Mix the biocontrol agent for root-knot nematodes and the plant seeds evenly at a mass ratio of (2 - 3):1, then carry out seedling raising, and transplant after obtaining the bacteria-carrying seedlings.

10. The method of use according to claim 9, wherein Before transplanting, dissolve and dilute the biocontrol agent for root-knot nematodes in water, then immerse the planting roots in the medicinal solution and then transplant.

11. The usage method according to claim 10, wherein, The dilution multiple of the biocontrol agent for root-knot nematodes is 50 - 100 times; the immersion time of the plant roots is 10 - 20 minutes.