Application of Ace9 factor in enhancing the toxicity of various Bacillus strains against plant parasitic nematodes

Abstract

This invention discloses the use of an Ace9 factor in enhancing the toxicity of various Bacillus strains against plant parasitic nematodes, belonging to the field of microbial pesticide technology. The invention first discovered that culturing the Ace9 factor can increase the nematicidal activity of the supernatant of Bacillus fermentation broth by more than 50%. Therefore, the Ace9 factor has the potential to be developed as a synergist for microbial pesticides, significantly improving the control efficacy of microbial pesticides against parasitic nematodes.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial pesticides, and specifically relates to a microbial pesticide synergist, and in particular to the use of an Ace9 factor in enhancing the activity of various Bacillus sp. in killing plant parasitic nematodes such as root-knot nematodes and cyst nematodes. Background Art

[0002] Root-knot nematodes are sedentary plant parasitic nematodes of the genus Meloidogyne, family Heteroderidae, order Tylenchida, class Secernentea. They are primarily found in the 3-10 cm topsoil layer and are named for the beaded, nodular root knots or galls they form on plant roots after infection. Root-knot nematodes have a short life cycle, rapid reproduction, and exceptional adaptability. They are widely distributed between tropical and temperate zones and can be spread by agricultural activities. Furthermore, they have a wide host range, encompassing over 3,000 plant species across over 100 families, including vegetables (such as tomatoes, cucumbers, and radishes), grain crops (such as soybeans, wheat, and rice), cash crops (such as watermelon, tea, and various Chinese herbal medicines), fruit trees, and even weeds. Nematodes infect plants by mechanically damaging their roots, leading to complex infections with other pathogens and secondary damage. Consequently, root-knot nematodes are considered the most damaging plant parasitic nematodes to global agriculture. Take China as an example. Since most of the country is located in the subtropical and temperate zones with a suitable climate, and the area of ​​greenhouses has continued to expand in recent years, root-knot nematodes have become seriously ill in my country, covering 27 provinces and regions. Large-scale occurrences have been reported in major agricultural provinces (autonomous regions) such as Heilongjiang, Henan, Hebei, Shandong, Inner Mongolia, Yunnan, Sichuan, Anhui, Jilin, and Jiangsu.

[0003] Currently, in addition to conventional agricultural management and supplementary measures such as film mulching and solarization, the primary control measure is the application of chemical pesticides. However, the long-term application of chemical pesticides can easily lead to problems such as pesticide resistance, and many pesticides are also restricted due to their high toxicity and high residue levels. For reasons of biosafety and environmental protection, green and safe biopesticides are gradually becoming a more promising option. However, biopesticides are currently inferior to chemical pesticides in terms of efficacy, their effectiveness fluctuates greatly due to environmental influences, and their high control costs make them difficult to truly gain mainstream market share. We urgently need to find ways to improve the control effectiveness of biopesticides and develop a new generation of highly effective and stable biopesticides to truly ensure that biopesticides can play a vital role in the green and sustainable development of agriculture.

[0004] 3-Hydroxybutanone, also known as acetoin, is a small molecule alcohol compound. 3-Hydroxybutanone is also an important pharmaceutical raw material [Xiao Xuhui. Synthesis of the antibacterial drug ampicillin hydrochloride [J]. Fine Chemical Intermediates, 34(6): 35-36.]. For example, 3-Hydroxybutanone is used as a raw material to synthesize the intermediate 4-bromomethyl-5-methyl-1,3-dioxolane-2-one, which is then reacted with ampicillin to synthesize ampicillin hydrochloride. At the same time, 3-Hydroxybutanone is a food flavoring that is widely found in crops and fruits. Due to its unique fragrance, it can be used as a flavor enhancer for food, beverages, etc. It is allowed to be consumed in my country's national standards and is also a flavor recommended by the American Food and Extract Association [Ling Guanting. Food Additives Handbook (Second) [M]. Beijing: Chemical Industry Press, 1997].

[0005] A search of the prior art revealed no literature reports on the use of 3-hydroxybutanone to enhance the activity of Bacillus in killing root-knot nematodes. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the object of the present invention is to provide a synergistic substance Ace9 factor that can enhance the toxicity of Bacillus to plant parasitic nematodes when added to the culture medium to cultivate Bacillus.

[0007] The realization of the present invention originated from the screening of a strain of Bacillus cereus BMB2600 (culture collection number CCTCC NO: M 2022893, deposited in the China Type Culture Collection of Wuhan University, Hongshan District, Wuhan City, Hubei Province, China on June 15, 2022, and classified as Bacillus cereus BMB2600) with obvious toxic effect on root-knot nematodes in the early laboratory work. In view of the fact that the nematicidal activity of this bacterium changes significantly over time, the inventors analyzed the components of the fermentation broth of this strain at different stages and identified a differential substance Ace9 factor, namely 3-hydroxybutanone and its reduction product 2,3-butanediol, from the fermentation broth with significant nematicidal activity.

[0008] Further experiments revealed that 3-hydroxybutanone and 2,3-butanediol only appeared during the period when the BMB2600 strain had significant nematicidal activity. However, the inventors treated root-knot nematodes with 3-hydroxybutanone or 2,3-butanediol at concentrations up to 10 mg / mL and found that neither had a significant effect on the nematodes. This suggests that 3-hydroxybutanone and 2,3-butanediol themselves do not have toxic activity against root-knot nematodes. Furthermore, the inventors added Ace9 factor or 2,3-butanediol to the culture medium at a concentration of 3 mg / ml and cultured the BMB2600 strain. They found that the nematicidal ability of the fermentation supernatant of the BMB2600 strain was significantly improved. Subsequently, the inventors conducted bioactivity assays using the same concentrations of BMB2600 fermentation supernatant. Adding 3-hydroxybutanone or 2,3-butanediol to the culture increased its in vitro nematicidal activity by more than 50%. This phenomenon suggests that 3-hydroxybutanone and its reduction products have the potential to be developed as microbial pesticide synergists. In addition, according to relevant literature, 3-hydroxybutanone and its reduction product 2,3-butanediol can be converted into each other during microbial fermentation, which explains why the reduction product of 3-hydroxybutanone, 2,3-butanediol, can also enhance the insecticidal activity of microbial fermentation liquid when used as an additive.

[0009] Based on the above research results, the purpose of the present invention can be achieved by the following technical solutions: the use of Ace9 factor in enhancing the activity of various Bacillus species in killing plant parasitic nematodes, or the use of Ace9 factor in preparing Bacillus species biological pesticide synergists, wherein the Ace9 factor is selected from at least one of the following: 3-hydroxybutanone, 2,3-butanediol, and a polymer of 3-hydroxybutanone.

[0010] It should be noted that, since polymers of 3-hydroxybutanone can be converted into monomeric compounds during microbial fermentation, polymers of 3-hydroxybutanone can be added to Bacillus culture medium as a synergist to also enhance the nematicidal activity of the fermentation product.

[0011] Further preferably, the 3-hydroxybutanone includes various chiral isomers, and the chiral isomers are (R)-3-hydroxybutanone and (S)-3-hydroxybutanone.

[0012] More preferably, the 2,3-butanediol includes various chiral isomers, and the chiral isomers are (R,R)-2,3-butanediol, (S,S)-2,3-butanediol, (R,S)-2,3-butanediol, and (S,R)-2,3-butanediol.

[0013] Further preferably, the polymer of 3-hydroxybutanone is a dimer of 3-hydroxybutanone.

[0014] Further preferably, the Bacillus includes Bacillus thuringiensis, Bacillus subtilis, Bacillus megaterium, Bacillus death valley, Bacillus agglomerans, Bacillus velezensis, Bacillus cereus, Bacillus toyota, Bacillus tropicalis, Bacillus stratosphericus, Bacillus proteolyticus, Bacillus mycoides, Bacillus mobilis, Bacillus licheniformis, etc.

[0015] Further preferably, the Bacillus biopesticide is a biopesticide that uses Bacillus and its metabolites as active ingredients and kills plant parasitic nematodes.

[0016] Further preferably, the plant parasitic nematodes are root-knot nematodes and cyst nematodes.

[0017] Still more preferably, the root-knot nematodes are southern root-knot nematodes, northern root-knot nematodes, peanut root-knot nematodes and javanic root-knot nematodes.

[0018] Still more preferably, the cyst nematodes are soybean cyst nematodes, potato golden wireworms and potato white wireworms.

[0019] Compared with the prior art, the present invention has the following advantages and improvements:

[0020] (1) Industrial products such as acetoin (3-hydroxybutanone) and 2,3-butanediol are inexpensive and can significantly enhance the nematicidal activity of microbial fermentation products by adding them at a low concentration of 3 g / L.

[0021] (2) The addition of Ace9 factor not only enhances the nematicidal activity of existing microbial pesticides, but also reduces the amount of microbial pesticides used, thereby reducing the cost of use;

[0022] (3) Adding the Ace9 factor can make the fermentation broth of some strains that originally had no nematicidal activity produce high nematicidal activity, significantly expanding the range of microbial choices and thus reducing the potential possibility of nematodes developing resistance to microbial agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Changes of nematicidal activity of Bacillus cereus BMB2600 with fermentation time.

[0024] Figure 2 :Bacillus cereus BMB2600 has a differential substance Ace9 factor when it has nematicidal activity.

[0025] Figure 3 : Non-toxic activity of Ace9 factor against root-knot nematodes.

[0026] Figure 4 :The effect of adding Ace9 factor on the nematicidal activity of the fermentation supernatant of the strain.

[0027] Figure 5 : Toxic activity of BMB2600 fermentation broth supplemented with 3-hydroxybutanone against various nematodes; Mi: root-knot nematode, Hg: cyst nematode, Dd: sweet potato stem nematode, Ce: Caenorhabditis elegans.

[0028] Figure 6 :Toxicidal activity of ICPM culture medium fermentation supernatants of various Bacillus strains against root-knot nematodes.

[0029] Figure 7 :Ace9 factor has an enhancing effect on the toxicity of most Bacillus with nematicidal activity.

[0030] Figure 8 : Adding Ace9 factor improves the potted control effect of Bacillus against root-knot nematodes.

[0031] Figure 9 : Actual photos of tomato roots in each treatment group compared with CK. DETAILED DESCRIPTION

[0032] The following are specific embodiments of the present invention, which describe the technical solutions of the present invention in detail. If no specific technical operations or conditions are specified in the examples, the operations are performed according to the techniques or conditions described in the literature in the field. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased normally.

[0033] Example 1: Detection of Different Substances in the Supernatant of BMB2600 Fermentation at Different Time Periods

[0034] The growth curve of the strain was determined by sampling the Bacillus cereus BMB2600 strain at various time points while incubating in ICPM medium at 37°C and 220 rpm. The fermentation supernatant at each growth point was then assayed for insecticidal activity. The specific steps for the in vitro bioactivity assay against root-knot nematodes using a 96-well plate are as follows:

[0035] (1) Pipette nematode J2s that had been hatched for 3 days into a 1.5 ml centrifuge tube, centrifuge at 5000 rpm for 1 min, and discard the supernatant;

[0036] (2) Resuspend the nematodes in an appropriate amount of sterile water and determine the nematode density under a microscope. The final nematode density is approximately 30 nematodes / μL.

[0037] (3) In a clean bench, the fermentation supernatant and nematodes were pipetted into the corresponding wells of a 96-well bioassay plate (20-40 J2s per well). Sterile water and ICPM medium were used as blank controls. The total volume per well was 100 μL. Each treatment was repeated three times.

[0038] (4) Seal the 96-well bioassay plate with sealing film and place it in a 20°C incubator. After 4 hours, count the total number of nematodes and the number of deaths in each well.

[0039] The nematicidal activity of the fermentation supernatant of Bacillus cereus BMB2600 first increased and then decreased with the extension of fermentation time ( Figure 1 ). The 8-hour fermentation supernatant with significant toxicity to Bacillus cereus BMB2600 and the 33-hour fermentation supernatant with no significant toxicity were selected for GC-MS analysis, and a differential substance was finally identified, which was 3-hydroxybutanone ( Figure 2 ).

[0040] Example 2: Detection of nematicidal activity of cultured strains after exogenous addition of Ace9 factor

[0041] First, the in vitro toxicity of Ace9 factor (3-hydroxybutanone, 2,3-butanediol) against root-knot nematodes was determined. Ace9 factor was prepared with sterile water into solutions of 10 mg / mL, 5 mg / mL, 1 mg / mL, 500 μg / mL, and 100 μg / mL and subjected to biological activity assays. The results showed that Ace9 factor itself did not have nematicidal activity ( Figure 3 ). We also found that the dimer of 3-hydroxybutanone and the chiral isomer of 2,3-butanediol did not have direct insecticidal activity. Subsequently, Ace9 factor 3-hydroxybutanone was exogenously added to ICPM medium for the cultivation of BMB2600 strain to evaluate its effect on the nematode killing activity of BMB2600 strain. The results showed that the addition of Ace9 factor 3-hydroxybutanone could significantly enhance the nematode killing activity of the fermentation supernatant of BMB2600 strain ( Figure 4 ), and the fermentation broth of BMB2600 supplemented with 3-hydroxybutanone showed high toxicity against root-knot nematodes and cyst nematodes ( Figure 5 ).

[0042] Example 3: Synergistic effect of exogenously added Ace9 factor on the nematicidal activity of various Bacillus fermentation supernatants

[0043] Using the existing Bacillus resources in this laboratory, the inventors tested the in vitro toxicity of 32 species, totaling 56 strains of Bacillus, against southern root-knot nematodes. The specific operation method is: the strains preserved in the glycerol tube are transferred to the LB plate, cultured at 28°C overnight, and a single colony is picked out the next day and placed in LB liquid culture medium, cultured and activated at 28°C and 220rpm, and then inoculated into ICPM culture medium at a ratio of 1%, and cultured at 37°C and 220rpm for a specified time. The bacteria are removed by centrifugation and the fermentation supernatant is prepared with sterile water at a concentration of 50% for the biological activity determination of root-knot nematodes. From them, 13 species, totaling 18 strains of Bacillus with obvious toxicity to root-knot nematodes were identified ( Figure 6), that is, the mortality rate is ≥80%.

[0044] Furthermore, the inventors tested the toxicity of the above 18 strains with certain nematode killing ability when cultured with exogenous addition of Ace9 factor 3-hydroxybutanone, using sterile water to dilute the fermentation supernatant to 20%, 10%, and 5% concentration gradients. The results showed that Ace9 factor 3-hydroxybutanone can produce different degrees of synergistic effects on 14 of the Bacillus strains ( Figure 7 ).

[0045] Example 4: Potted experiment verification of the Ace9 factor enhancing the nematode-killing activity of Bacillus

[0046] Four strains of Bacillus tropicus (BMB2537), BMB2586 (B. safensis), BMB25102 (B. mycoides), BMB25109 (B. megaterium), and BMB2600 (B. cereus), which demonstrated excellent synergistic effects, were selected for further potted experiments. Healthy tomato seedlings of uniform growth and size were selected as test seedlings. 2,000 second-instar larvae were inoculated at the roots of the tomato seedlings, along with 20 mL of bacterial solution. A control group was inoculated with equal volumes of ICPM medium and Lufuda dilution. After treatment, the plants were cultured in a greenhouse. The potted experiments included four treatments: CK (equal volumes of ICPM medium), ICPM culture of the strain, culture of the strain in ICPM supplemented with the Ace9 factor 3-hydroxybutanone, and a positive control (Lufuda dilution). Each treatment was replicated five times. After 45 days, the number of root knots was counted. The tomato plants were gently pulled up and the complete root system was taken out. The roots were then rinsed under running water and the number of root knots was counted visually. The disease status of the plants was expressed as the average number of root knots per gram of root tissue.

[0047] The results are as follows Figure 8 As shown, five representative Bacillus strains, BMB2537, BMB2586, BMB25102, BMB25109, and BMB2600, also demonstrated moderate efficacy against root-knot nematodes in conventional ICPM medium, with efficacy rates of 48%, 56%, 14%, 30%, and 51%, respectively. Root irrigation with fermentation broth supplemented with the Ace9 factor significantly reduced the number of root knots per gram of root tissue compared to the blank control. The fermentation broths of these strains significantly enhanced their ability to control root-knot nematodes, with efficacy rates of 85%, 62%, 71%, 68%, and 89%, respectively. Pot experiments further demonstrated the potential of the Bacillus strains screened through this method for practical application in root-knot nematode control.

[0048] Example 5: Field Experimental Verification of the Ace9 Factor to Enhance the Nematode Killing Activity of Bacillus

[0049] The field experiment used the strain Bacillus thuringiensis HAN055 (Culture Collection Number: CCTCC No. M 2015608, deposited with the China Center for Type Culture Collection, Wuhan University, Hongshan District, Wuhan, Hubei Province, China, on October 16, 2015, and designated Bacillus thuringgiensis HAN055). The fermentation time was 32-36 hours. Treatment Group 1 consisted of a conventional HAN055 fermentation broth, while Treatment Group 2 consisted of a HAN055 fermentation broth supplemented with 0.5% Ace9. The field test plants were four-leaf stage Jinpeng No. 3 tomato seedlings of similar size and growth. Before transplanting the tomato plants, the soil was treated with pesticides. The treatment groups were HAN055 fermentation liquid (Group A: 150 mL / plant, Group B: 300 mL / plant), HAN055-Ace9 fermentation liquid (Group C: 150 mL / plant, Group D: 300 mL / plant), field tillage water as negative control, and Lu Fuda dilution as positive control. Each treatment was repeated 10 times.

[0050] After 45 days of field testing, the results were compiled and the root knot formation caused by root-knot nematode infection was graded into a ten-level scale. The detailed grading method is described in Section 2.2.6.3 (Desaeger et al. 2011, Lu et al. 2017). After all evaluations were completed, the root knot index and relative control efficacy were calculated. The statistical results are shown in Table 1. Compared with the cultivated water control, the root knot index of the test plants treated with the HAN055 and HAN055-Ace9 fermented solutions decreased, indicating increased control efficacy against southern root-knot nematode infection. There was no significant difference in the control efficacy of the HAN055 fermented solution at 150 mL / plant and 300 mL / plant, while the control efficacy of the HAN055-Ace9 fermented solution increased with increasing application rate. At the same time, compared with the HAN055 fermentation solution, the root knot index of the plants applied with the HAN055-Ace9 fermentation solution was lower. When the application amount was 300 mL / plant, its prevention effect was 1.65 times that of the HAN055 fermentation solution; and the prevention effect of the highest application amount was close to that of the chemical control Lu Fu Da. Figure 9 The following figure compares each treatment group with the CK group. The roots of the CK group formed large root knots and rotted. The root knots of the plants treated with the bacterial solution were significantly reduced. In addition, compared with HAN055 and HAN055-Ace9, the roots of HAN055 were stronger, and no obvious large root knots appeared when observed with the naked eye. This shows that adding Ace9 during the fermentation process can significantly enhance the field nematicidal activity of the original Bt strain HAN055.

[0051] Table 1

[0052]

Claims

1. Application of Ace9 factor in enhancing the toxicity of Bacillus against plant parasitic nematodes; The Ace9 factor is 3-hydroxybutanone; The Bacillus is Bacillus thuringiensis HAN055 CCTCC NO: M2015608, Bacillus cereus BMB2600 CCTCC NO: M2022893 or Bacillus megaterium BMB25109 CCTCC NO: M2022898; The plant parasitic nematodes are root knot nematodes and cyst nematodes.

2. The use according to claim 1, characterized in that The 3-hydroxybutanone includes various chiral isomers, and the chiral isomers are (R)-3-hydroxybutanone and (S)-3-hydroxybutanone.

3. The use according to claim 1, characterized in that The root-knot nematodes are southern root-knot nematodes, northern root-knot nematodes, peanut root-knot nematodes and javanic root-knot nematodes.

4. The use according to claim 1, characterized in that The cyst nematodes are soybean cyst nematodes, potato golden nematodes and potato white nematodes.

Images