Application of Ace9 factor in enhancing activity of multiple bacilli in killing plant parasitic nematodes

Abstract

The invention discloses an application of an Ace9 factor in enhancing the activity of killing plant parasitic nematodes by multiple bacilli, and belongs to the technical field of microbial pesticides. It is found for the first time that the nematicidal activity of bacillus fermentation liquid supernatant can be improved by 50% or above through Ace9 factor culture, and therefore the Ace9 factor has the potential of being developed into a microbial pesticide synergist, and the control effect of microbial pesticide on parasitic nematodes can be greatly improved.

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 application of an Ace9 factor in enhancing the activity of multiple Bacillus in killing plant parasitic nematodes such as root-knot nematodes and cyst nematodes. Background Art

[0002] Root-knot nematodes are sedentary plant parasitic nematodes belonging to the genus Heteroderidae, order Tylenchida, class Secernentea, and class Tylenchida. They are mainly distributed in the topsoil of 3 to 10 cm. They are named because they form beaded or nodular root knots or galls on the plant roots after infection. Root-knot nematodes have a short life cycle, reproduce quickly, and have a strong ability to adapt to the environment. They are widely distributed between tropical and temperate zones and can spread with agricultural activities. In addition, root-knot nematodes have a very wide host range, covering more than 3,000 plant species from more than 100 families, including vegetables (such as tomatoes, cucumbers, and radishes), food crops (such as soybeans, wheat, and rice), cash crops (such as watermelons, tea, and various Chinese herbal medicines), fruit trees, and even weeds. After infection, nematodes will cause mechanical damage to the plant roots, thereby causing complex infections with other pathogens and forming secondary damage. Therefore, root-knot nematodes are regarded as the most harmful 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 are seriously ill in my country, covering 27 provinces and regions. There have been reports of large-scale occurrences in major agricultural provinces (autonomous regions) such as Heilongjiang, Henan, Hebei, Shandong, Inner Mongolia, Yunnan, Sichuan, Anhui, Jilin, and Jiangsu.

[0003] At present, in addition to conventional agricultural management and auxiliary means such as film covering and sun exposure, the main prevention and control measures are the application of chemical pesticides. However, the long-term application of chemical pesticides is prone to cause problems such as drug resistance, and many pesticides are also restricted due to high toxicity, high residues and other problems. For the sake of biosafety and environmental ecological protection, green and safe biological pesticides are gradually becoming a more promising choice. However, the current prevention effect of biological pesticides is often inferior to that of chemical pesticides, and the effect fluctuates greatly due to environmental influences, and the prevention and control cost is high, making it difficult to truly occupy the mainstream market. We urgently need to find ways to improve the prevention and control effect of biological pesticides, develop a new generation of efficient and stable biological pesticides, and truly let biological pesticides escort 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, and then reacts 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 eaten 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 3-hydroxybutanone enhancing the activity of Bacillus in killing root-knot nematodes. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a synergistic substance Ace9 factor which 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 originates from the screening of a strain of Bacillus cereus BMB2600 (strain collection number CCTCC NO: M 2022893, preserved in the China Type Culture Collection Center of Wuhan University, Hongshan District, Wuhan City, Hubei Province, China on June 15, 2022, and classified and named Bacillus cereus BMB2600) with obvious toxic effect on root-knot nematodes in the preliminary work of the laboratory. In view of the obvious changes in the nematicidal activity of the bacteria over time, the inventors analyzed the components of the fermentation broth of the strain at different periods, 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] In further experiments, it was found that 3-hydroxybutanone and 2,3-butanediol only appeared during the period when the BMB2600 strain had significant nematicidal activity, but the inventors treated root-knot nematodes with 3-hydroxybutanone or 2,3-butanediol up to 10 mg / mL, and found that they had no significant effect on nematodes, indicating that 3-hydroxybutanone and 2,3-butanediol themselves did not have toxic activity against root-knot nematodes. Further, the inventors added Ace9 factor or 2,3-butanediol to the culture medium at a concentration of 3 mg / ml to culture the BMB2600 strain, and found that the nematicidal ability of the fermentation supernatant of the BMB2600 strain was significantly improved. Subsequently, the inventors used the same concentration of the BMB2600 strain fermentation supernatant to carry out biological activity determination, and adding 3-hydroxybutanone or 2,3-butanediol to culture can increase its in vitro nematicidal activity by more than 50%, which indicates that 3-hydroxybutanone and its reduction products have the potential to be developed as microbial pesticide synergists. In addition, it is known from relevant literature that 3-hydroxybutanone and its reduction product 2,3-butanediol can be converted into each other during microbial fermentation, which explains why the reduction product 2,3-butanediol of 3-hydroxybutanone 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 scheme: the use of Ace9 factor in enhancing the activity of various Bacillus to kill plant parasitic nematodes, or the use of Ace9 factor in the preparation of Bacillus biological pesticide synergist, 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 the polymers of 3-hydroxybutanone can be converted into monomeric compounds during microbial fermentation, the polymers of 3-hydroxybutanone can be added to the Bacillus culture medium as a synergist, which can also play a role in improving 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 velez, Bacillus cereus, Bacillus toyota, Bacillus tropicalis, Bacillus stratosphericus, Bacillus proteolyticus, Bacillus mycoides, Bacillus mobilis, Bacillus licheniformis and the like.

[0015] Further preferably, the Bacillus biological pesticide is a biological pesticide 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 progress:

[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 improves the nematicidal activity of existing microbial pesticides, but also reduces the amount of microbial pesticides used, thereby reducing the cost of use;

[0022] (3) The addition of the Ace9 factor can cause the fermentation broth of some strains that originally had no nematicidal activity to produce high nematicidal activity, significantly expanding the range of microbial options 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 or not 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 nematodes, Hg: cyst nematodes, Dd: sweet potato stem nematodes, Ce: Caenorhabditis elegans.

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

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

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

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

[0032] The following are specific embodiments of the present invention, and the technical scheme of the present invention is described in detail. If no specific technical operation or condition is specified in the embodiments, the operation is performed according to the technology or condition described in the literature in the field. If no manufacturer is specified for the reagents or instruments used, 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 different time periods under the culture conditions of ICPM medium, 37°C, and 220rpm, and the OD600 was detected. The insecticidal activity of the fermentation supernatant at each growth point was determined. The specific steps for the in vitro bioactivity determination of root-knot nematodes using a 96-well plate are as follows:

[0035] (1) Pipette nematode J2s that have 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, use a pipette to add the fermentation supernatant and nematodes to the corresponding wells of a 96-well bioassay plate (20-40 J2s per well), and use sterile water and ICPM medium as blank controls. The total volume of each well is 100 μL; each treatment is 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 8h fermentation supernatant with significant killing activity and the 33h fermentation supernatant with no significant killing activity of Bacillus cereus BMB2600 were selected for GC-MS analysis, and finally a differential substance was 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. The solution was prepared with sterile water to form 10 mg / mL, 5 mg / mL, 1 mg / mL, 500 μg / mL, and 100 μg / mL solutions and the biological activity was determined. 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, the Ace9 factor 3-hydroxybutanone was exogenously added to the ICPM medium for the cultivation of the BMB2600 strain to evaluate its effect on the nematicidal activity of the BMB2600 strain. The results showed that the addition of the Ace9 factor 3-hydroxybutanone could significantly enhance the nematode-killing activity of the fermentation supernatant of the BMB2600 strain ( Figure 4 ), and the fermentation liquid of BMB2600 supplemented with 3-hydroxybutanone showed high toxicity against root-knot nematodes and cyst nematodes ( Figure 5 ).

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

[0043] Using the existing Bacillus resources in the 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: transfer the strains preserved in glycerol tubes to LB plates, culture them at 28°C overnight, pick out single colonies in LB liquid culture medium the next day, culture and activate them at 28°C and 220rpm, then inoculate them in ICPM culture medium at a ratio of 1%, and culture them at 37°C and 220rpm for a specified time. Centrifuge to remove the bacteria, and prepare the fermentation supernatant at a concentration of 50% with sterile water 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 nematicidal ability when cultured with exogenous addition of Ace9 factor 3-hydroxybutanone, using sterile water to dilute the fermentation supernatant to 20%, 10%, and 5% in total. 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 plant experiment to verify that the Ace9 factor enhances the nematode-killing activity of Bacillus

[0046] Four strains of Bacillus BMB2537 (B.tropicus), BMB2586 (B.safensis), BMB25102 (B.mycoides), BMB25109 (B.megaterium) and BMB2600 (B.cereus) with good synergistic performance were selected for further pot experiments. Specifically, healthy tomato seedlings with uniform growth and size were selected as test seedlings. 2000 second-instar larvae were inoculated at the roots of tomato seedlings, and 20 mL of bacterial solution was added. The control group was inoculated with an equal volume of ICPM medium and Lufuda dilution. After the treatment, it was placed in a greenhouse environment for culture. There were 4 treatments in the pot experiment, namely: CK (equal amount of ICPM medium), ICPM bacterial solution of the strain, bacterial solution after the strain was cultured in ICPM with the addition of Ace9 factor 3-hydroxybutanone, and positive control (Lufuda dilution), and 5 replicates were set for each treatment. 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 in the figure, five representative strains of Bacillus, BMB2537, BMB2586, BMB25102, BMB25109 and BMB2600, also showed certain control effects on root-knot nematodes in conventional ICPM medium, with control effects of 48%, 56%, 14%, 30% and 51% respectively. After root irrigation with fermentation liquid with Ace9 factor added, the number of root knots per gram of root tissue was significantly reduced compared with the blank control group, and the control ability of the fermentation liquid of the above strains on root-knot nematodes was significantly improved, with control effects of 85%, 62%, 71%, 68% and 89% respectively. The pot experiment further proved that the Bacillus screened by the above method has the potential of practical application value in the control of root-knot nematode diseases.

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

[0049] The strain used in the field test was Bacillus thuringiensis HAN055 (strain collection number: CCTCC NO: M 2015608, deposited in China Type Culture Collection Center of Wuhan University, Hongshan District, Wuhan City, Hubei Province, China on October 16, 2015, and classified as Bacillus thuringgiensis HAN055). The fermentation time was 32-36h, treatment group 1 was the conventional fermentation broth of HAN055, and treatment group 2 was the fermentation broth of HAN055 after adding 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 farming water as negative control, and Lu Fu Da dilution as positive control, with 10 replicates for each treatment.

[0050] After 45 days of field test, the results were statistically analyzed, and the root knots formed by root-knot nematode infection were graded and evaluated, which were divided into ten levels. The specific grading method is shown in 2.2.6.3 (Desaeger et al. 2011, Lu et al. 2017). After all the evaluations were completed, the root knot index and relative control effect were calculated. The statistical results are shown in Table 1. Compared with the control of cultivated water, the root knot index of the test plants applied with HAN055 fermented bacterial solution and HAN055-Ace9 fermented bacterial solution was reduced, and the control effect on southern root-knot nematode infection was increased. There was no significant difference in the control effect of HAN055 fermented bacterial solution when 150mL / plant and 300mL / plant were applied, while the control effect of HAN055-Ace9 fermented bacterial solution increased with the increase of application amount. At the same time, compared with HAN055 fermented bacteria solution, the root knot index of the plants applied with HAN055-Ace9 fermented bacteria solution was lower. When the application amount was 300mL / plant, its protective effect was 1.65 times that of HAN055 fermented bacteria solution; and the protective effect of the highest application amount was close to that of the chemical control Lu Fu Da. Fig. 9 The following figure compares each treatment group with CK. The roots of the CK group formed larger root knots and showed root rot. The root knots of the plants treated with bacterial solution were significantly reduced. In addition, compared with HAN055-Ace9, the roots of HAN055 were stronger, and no obvious large root knots appeared by naked eye observation, indicating 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. The application of Ace9 factor in enhancing the activity of multiple Bacillus in killing plant parasitic nematodes, or the application of Ace9 factor in preparing Bacillus biological pesticide synergist, wherein the Ace9 factor is selected from at least one of the following: 3-hydroxybutanone, 2,3-butanediol, and polymers of 3-hydroxybutanone.

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 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.

4. The use according to claim 1, characterized in that: The polymer of 3-hydroxybutanone is a dimer of 3-hydroxybutanone.

5. The use according to claim 1, characterized in that: The bacillus includes bacillus thuringiensis, bacillus subtilis, bacillus megaterium, bacillus death valley, bacillus argyi, bacillus velez, bacillus cereus, bacillus toyota, bacillus tropicalis, stratospheric bacillus, proteolytic bacillus, bacillus mycoides, bacillus mobilis, and bacillus licheniformis.

6. The use according to claim 1, characterized in that: The Bacillus biological pesticide is a biological pesticide which uses Bacillus and its metabolites as active ingredients and kills plant parasitic nematodes.

7. The use according to claim 1 or 6, characterized in that: The plant parasitic nematodes are root-knot nematodes and cyst nematodes.

8. The use according to claim 7, characterized in that: The root-knot nematodes are southern root-knot nematodes, northern root-knot nematodes, peanut root-knot nematodes and Javan root-knot nematodes.

9. The use according to claim 7, characterized in that: The cyst nematodes are soybean cyst nematodes, potato golden nematodes and potato white nematodes.

Images