Application of bacillus thuringiensis TG5 in promoting tomato to resist fusarium oxysporum

Abstract

The invention discloses application of bacillus thuringiensis TG5 in promoting tomatoes to resist fusarium oxysporum, and belongs to the technical field of tomato disease resistance. The bacillus thuringiensis TG5 fermentation liquor and phoxim are combined and applied to tomato plants, the tomato disease incidence rate caused by fusarium oxysporum can be reduced, stress faced by the tomato plants can be relieved, and the tomato fruit quality can be improved. Besides, the bacillus thuringiensis TG5 can promote the degradation of phoxim and reduce the residual quantity of phoxim in tomato fruits to 1.18 ug / kg, which is far lower than the maximum residual limit standard of pesticides in food, so that the bacillus thuringiensis TG5 shows a higher safety level. The bacillus thuringiensis TG5 is matched with a traditional chemical pesticide to realize reduction and synergism in use of the chemical pesticide, pesticide residues are also reduced, theoretical and data support is provided for future production, application and popularization, and the bacillus thuringiensis TG5 has important significance on green and sustainable agricultural development.

Description

Technical Field

[0001] The present invention relates to the technical field of tomato disease resistance, and particularly relates to the application of Bacillus thuringiensis TG5 in promoting tomatoes to resist Fusarium oxysporum. Background Art

[0002] Tomato (Solanum lycopersicum) has a long cultivation history and is widely planted. According to the data of the Food and Agriculture Organization of the United Nations (FAO) in 2023, its global planting area has reached 5.4×10 6 hectares. However, tomatoes are frequently infected by various pathogenic microorganisms including Fusarium oxysporum, which induces various plant diseases and causes serious economic losses.

[0003] For a long time, chemical pesticides have been widely used in agricultural production: the organophosphorus pesticide phoxim is widely used for pest control, fludioxonil (FX) and the sterol enzyme inhibitor (DMI) pyrisoxazole are used to control gray mold in cherry tomatoes. However, the control spectrum and scope of chemical pesticides are specific. For example, abamectin has stomach toxicity and contact toxicity to mites and insects, and pyraclostrobin can be used to control various diseases caused by fungi on crops, but neither of them is effective against plant diseases caused by Fusarium oxysporum, indicating the limitations of the use of chemical pesticides. At the same time, the amount of chemical pesticides used is large, and after long-term use, it will cause residues and enrichment of pesticide substances, metabolic intermediates or derivatives in the soil and plant tissues, which is harmful to animals and humans. Therefore, biological control is of great significance for the development of green and sustainable agriculture.

[0004] Bacillus thuringiensis is a biological pesticide that was promoted and applied earlier. It can effectively control pests such as tomato leafminer, cotton bollworm, Spodoptera litura, fall armyworm, and pod borer, and has significant control effects on tomato bacterial wilt, gray mold, root-knot nematode disease, and bacterial leaf spot caused by Pseudomonas syringae, becoming a powerful competitor to traditional insecticides. In addition, compared with other biological pesticides such as Bacillus cereus, Bacillus thuringiensis shows a significantly easy-to-elute characteristic in the attachment strength to plant tissues, which is suitable for timely picking and marketing after pesticide application on fruits and vegetables. Although the functions of phoxim and Bacillus thuringiensis are somewhat understood currently, the efficacy after their combined use remains unknown. Summary of the Invention

[0005] In view of the above-mentioned prior art, the object of the present invention is to provide the application of Bacillus thuringiensis TG5 in promoting tomato resistance to Fusarium oxysporum.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a composition for promoting tomato resistance to Fusarium oxysporum is provided, and the composition comprises Bacillus thuringiensis TG5 and phoxim.

[0008] Furthermore, the Bacillus thuringiensis TG5 is a fermentation broth of Bacillus thuringiensis TG5, and the preparation method comprises the following steps:

[0009] Inoculate the Bacillus thuringiensis TG5 strain into a solid medium, culture at 30 - 40 °C for 12 - 36 h, cut the solid medium containing the TG5 strain, inoculate the cut slices into a fermentation medium, stir at 30 - 40 °C at 100 - 400 rpm for 12 - 36 h, and obtain the fermentation broth of Bacillus thuringiensis TG5 after filtration.

[0010] In the second aspect of the present invention, the application of the composition in promoting tomato resistance to Fusarium oxysporum is provided.

[0011] In the third aspect of the present invention, a method for promoting tomato resistance to Fusarium oxysporum is provided, and the composition for promoting tomato resistance to Fusarium oxysporum is applied to tomato plants.

[0012] Furthermore, the viable bacteria count in the fermentation broth of Bacillus thuringiensis TG5 is not less than 1×10 10 CFU / ml.

[0013] Furthermore, the concentration of phoxim is 200 - 800 mg / L.

[0014] Furthermore, it is applied during the growth and fruiting period, the number of applications is 2 - 6 times, and the application amount is 20 - 70 ml per plant per time.

[0015] Furthermore, the application method is one or more of root irrigation, foliar spraying or broadcasting.

[0016] Furthermore, the application method is root irrigation.

[0017] In the fourth aspect of the present invention, the application of Bacillus thuringiensis TG5 in promoting tomato resistance to Fusarium oxysporum is provided, and the application includes reducing the incidence of tomato diseases caused by Fusarium oxysporum, alleviating the stress of tomato plants, improving the quality of tomato fruits, and promoting the degradation of the pesticide phoxim.

[0018] The beneficial effects of the present invention:

[0019] The present invention combines the fermentation broth of Bacillus thuringiensis TG5 and phoxim, and applies it to tomato plants, which can reduce the incidence of tomato diseases caused by Fusarium oxysporum and relieve the stress faced by tomato plants; in terms of improving the quality of tomato fruits, it can increase the average single fruit weight and average yield per plant of tomatoes, and increase the contents of soluble sugar, titratable acid, vitamin C and soluble solids in tomato fruits. In addition, Bacillus thuringiensis TG5 can also promote the degradation of phoxim, reducing the phoxim residue in tomato fruits to 1.18 μg / kg, far lower than the phoxim limit of ≤50 μg / kg for tomato fruits in the "Maximum Residue Limits of Pesticides in Foods GB2763-2021" standard, showing a higher safety level. The combination of Bacillus thuringiensis TG5 and traditional chemical pesticides in the present invention realizes the reduction of chemical pesticide use and the increase of efficiency, and also reduces pesticide residues, providing theoretical and data support for future production application and promotion, and is of great significance for the development of green and sustainable agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a phenotypic diagram of a tomato plant inoculated with Fusarium oxysporum.

[0021] Figure 2 It is a disease diagram of a tomato plant 30 days after inoculation with Fusarium oxysporum.

[0022] Figure 3 It is a diagram of tomato quality; among them, A is a diagram of the average weight of single plants of tomatoes in each treatment; B is a diagram of the single fruit weight of tomatoes in each treatment; C is a diagram of the soluble sugar content of tomatoes in each treatment; D is a diagram of the titratable acid content of tomatoes in each treatment; E is a diagram of the vitamin C content of tomatoes in each treatment; F is a diagram of the soluble solids of tomatoes in each treatment.

[0023] Figure 4 It is a diagram of pesticide residues in the soil and tomato fruits of each treatment; among them, A is a diagram of the phoxim content in the rhizosphere soil of tomatoes in each treatment; B is a diagram of the phoxim content in tomato fruits in each treatment; C is a diagram of the fludioxonil content in the rhizosphere soil of tomatoes in each treatment; D is a diagram of the fludioxonil content in tomato fruits in each treatment. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] It should be noted that the following detailed description is illustrative and is intended to provide further illustration of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0025] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

[0026] Crop high yields rely heavily on pesticides. However, the abuse of pesticides poses considerable challenges to soil health and food safety. Excessive pesticide use can lead to a series of systematic problems such as soil quality degradation and groundwater pollution, further threatening ecosystem diversity. Pesticides can be absorbed through the leaves or roots of plants, resulting in harmful effects such as extensive damage to plant tissues, inhibited growth, and reduced yield and quality. In addition, the average leaching rate of pesticides in the soil is 5% of the total mass of pesticides applied, and the leached pesticides may harm soil organisms and pollute water bodies through runoff and leaching. Moreover, excessive pesticide stress can also alter crop gene expression, disrupt tissue cell signal transduction, thereby interfering with the normal cell metabolism process of plant tissues and even inducing toxic stress; some pesticides can even interact at the molecular level with proteins, nucleic acids, etc. in plant tissues, causing significant changes in protein structure and physiological functions, ultimately affecting crop quality.

[0027] As basic components of human diet, the pesticide residues in vegetables and fruits are related to people's food safety. The compounding and synergistic effects between chemical pesticides and biological pesticides can, to the greatest extent, reduce pesticide residues while ensuring the control effect, which is a strategy representing the development direction of the green circular industry.

[0028] The test materials not specifically described used in the embodiments of the present invention are all conventional test materials in the art and can be obtained through commercial channels. In the present invention, the Bacillus thuringiensis used is Bacillus thuringiensis TG-5. The whole genome of Bacillus thuringiensis TG-5 (B.thuringiensis TG5) has been publicly disclosed, and its accession number is GWHBOWN00000000. It is also publicly used in the inventor's previous patent "CN 115868506B Application of Bacillus thuringiensis in Dissolving Phosphorus in Soil, Promoting Plant Growth and Regulating Soil Enzyme System Metabolism". (The 16S rRNA gene sequence of the TG-5 strain is saved in the NCBI database with the accession number RID-8MG9P79F013QUERY-39355, and this bacterium is identified as Bacillus thuringiensis). The applicant undertakes to publicly distribute the biological material of Bacillus thuringiensis TG-5 to the public for free for at least twenty years. The substrate required for the pot experiments of the present invention was purchased from Shandong Lukang Zhonghe Environmental Protection Technology Co., Ltd., and the phoxim and fludioxonil used were purchased from Shandong Huayang Pesticide Group Co., Ltd. The tomato seed variety used is Micro Tom tomato, which was donated by the Meng Qingwei team of the College of Life Sciences, Shandong Agricultural University.

[0029] Example 1

[0030] 1.1 Preparation of the fermentation broth of Bacillus thuringiensis TG-5

[0031] The Bacillus thuringiensis strain TG5 was inoculated into an LB medium petri dish and cultured at 37°C for 24 h. The LB plate containing the TG5 strain was divided into slices of 1 cm 2 each. Each 1 cm 2 slice was inoculated into 100 mL of fermentation medium and stirred at 220 rpm on a shaker at 37°C for 24 h. Subsequently, the fermentation broth was filtered and collected in a sterile environment for later use.

[0032] 1.2 Determination of viable bacteria count in the fermentation broth

[0033] After diluting the prepared fermentation broth 100-fold, 100 μL was taken and evenly spread on an LB plate. There were three replicates for each treatment. After 24 h, the number of visible colonies on each LB plate was determined. Then, the viable bacteria count N per milliliter was calculated using the following formula:

[0034] N (CFU / mL) = average number of colonies per plate / volume × dilution factor

[0035] The quantitatively detected fermentation broth of Bacillus thuringiensis TG5 was reserved for later use.

[0036] Example 2

[0037] 2.1 Experimental design and grouping

[0038] This experiment was conducted in the climate chamber of Shandong Agricultural University in Tai'an, Shandong Province. The temperature in the climate chamber was kept constant at 23°C, the relative humidity was 70%, the illumination was 6000 Lux for 16 h, and the dark period was 8 h for cultivation. Tomato seeds were soaked in warm water at 55°C for 30 minutes, naturally cooled to room temperature, sown in the substrate of a flat tray, and watered thoroughly. After 30 days of growth, tomato seedlings with the same growth specifications were transplanted into flower pots with a diameter of 10 cm and a height of 8 cm for planting.

[0039] Tomato plants grown in protected cultivation are extremely vulnerable to infection by Fusarium oxysporum, which can cause root (stem) basal rot. Fludioxonil (FX) has a significant control effect on crop root rot and is not easily residual. It is the mainstream control pesticide in the current market. In order to study the control effects of traditional chemical pesticides and biological pesticides on tomato root (stem) basal rot caused by Fusarium oxysporum infection. The specific grouping is as follows:

[0040] The well-grown and consistent tomato seedlings after planting were divided into five groups, with 10 replicates in each group. Since the pesticide dose may affect the microbial community, three different pesticides, phoxim, fludioxonil, and Bacillus thuringiensis TG5, were dissolved in sterile water respectively, and each pesticide was directly applied to the soil by irrigation at the recommended dose: PX group, 50 mL / plant, concentration of phoxim 400 mg / L; FX group: 50 mL / plant, concentration of fludioxonil 500 mg / L; TG5 group: 50 mL / plant, concentration of 2×10 10Bacillus thuringiensis TG5 fermentation broth at CFU / ml; TG5+PX group: 50 ml / plant, phoxim with a final concentration of 400 mg / L was dissolved in 2×10 10 CFU / ml Bacillus thuringiensis TG5 fermentation broth; the control group was only irrigated with the same amount of sterile water (CK).

[0041] There were a total of four pesticide treatments after the tomato was planted, all of which were carried out during the growth and fruiting period of the tomato. The first treatment should be carried out 0 days after planting the tomato seedlings, and then on the 30th, 45th, and 60th days after planting respectively. The pesticide treatment method was carried out by root irrigation according to the dose of 50 ml per plant each time as described above, and the watering was the same and appropriate at other times.

[0042] On the 21st day after planting, the pathogen of Fusarium oxysporum with an inoculation concentration of 1×10 8 spores / ml was used, and the dose was 25 ml / plant. The incidence of neck rot and root rot of tomato plants in each group was counted on the 36th and 51st days after planting respectively.

[0043] 2.2 Statistics of tomato diseases

[0044] The incidence of neck rot and root rot of tomato plants in each group on the 36th and 51st days after transplanting and planting was counted, and the incidence rate and disease index of the plants were calculated. The calculation methods are as follows:

[0045] (1) Incidence rate (%) = number of diseased seedlings / total number of seedlings × 100

[0046] (2) Disease index = (∑ number of diseased plants at each level × representative value of disease grading) / highest disease grade × total number of investigations × 100 for the disease condition. The specific grading is shown in Table 1.

[0047] Table 1 Disease grading

[0048]

[0049] 2.3 Rhizosphere sampling and treatment of tomatoes

[0050] When the tomato plants were planted for 100 days, the fruits were picked, and the rhizosphere soil of each group was collected. The soil of each group was divided into two parts. One part of the rhizosphere soil was stored at 4°C and sent to Shandong Pengbo Biotechnology Co., Ltd. together with the tomato fruits to determine the average single fruit yield, average plant yield, total soluble sugar, total titratable acid, vitamin C content, and total soluble solid content, and the pesticide residues in the tomato fruits and rhizosphere soil were detected by quantitative PCR method; the other part of the rhizosphere soil sample was centrifuged at 4°C and 8000 g for 5 minutes, and after removing the supernatant, the precipitate was stored at -80°C and sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for metagenomic determination. The tomato leaves were collected for the detection of the activities of superoxide dismutase, phenylalanine ammonia-lyase, polyphenol oxidase, and malondialdehyde.

[0051] Example 3: Experimental Related Indicators and Result Statistics

[0052] 3.1 Incidence of Tomato Diseases

[0053] The statistical results of tomato plant diseases on the 36th and 51st days after transplanting and planting are shown in Table 1 and Table 2 below respectively.

[0054] Table 1 Statistical Results of Tomato Plant Diseases on the 36th Day after Transplanting and Planting

[0055]

[0056]

[0057] Table 2 Statistical Results of Tomato Plant Diseases on the 51st Day after Transplanting and Planting

[0058]

[0059] The phenotypic diagram of tomato plants is shown in Figure 1 and Figure 2 . 15 days after inoculation with Fusarium oxysporum, compared with the control group CK, the disease incidence of tomatoes treated with TG5+PX decreased by 60.0%; compared with the PX and FX treatments, the disease incidence of tomatoes treated with TG5+PX decreased by 33.0% and 60.0% respectively( Figure 1 ). 30 days after inoculation with Fusarium oxysporum, compared with the control treatment, the disease incidence of tomatoes treated with TG5+PX decreased by 53.3%. However, there was no difference in the disease incidence of tomatoes treated with PX (phoxim) and FX (fludioxonil); compared with the PX and FX treatments, the disease incidence of tomatoes treated with TG5+PX decreased by 53.3% and 53.3% respectively( Figure 2 ), which also indicates that, compared with traditional chemical pesticides such as phoxim and fludioxonil, the combination of Bacillus thuringiensis TG5 and phoxim has better effects in controlling tomato root rot caused by Fusarium oxysporum.

[0060] Bacillus thuringiensis TG5 alone or in combination with phoxim greatly increased the resistance of tomato plants to Fusarium oxysporum infection.

[0061] 3.2 Physiological Indexes of Tomato Resistance

[0062] Superoxide dismutase, phenylalanine ammonia-lyase, polyphenol oxidase and malondialdehyde are widely present in the cytoplasm, chloroplasts and mitochondria of tomato cells. As physiological indexes of resistance, the numerical values indicate the stress expression levels of tomato plants in aspects such as biosynthesis, tissue metabolism, and defense responses, that is, the degree of growth restriction or virus stress faced by tomato plants.

[0063] Compared with the control group, the activities of superoxide dismutase, phenylalanine ammonia-lyase, polyphenol oxidase and malondialdehyde in the leaves treated with TG5+PX decreased by 49.2%-59.5% at the time of tomato fruit picking; the activities of phenylalanine ammonia-lyase, polyphenol oxidase and malondialdehyde in the leaves treated with PX decreased by 31.4%-43.3%; the activities of phenylalanine ammonia-lyase, polyphenol oxidase and malondialdehyde in the leaves treated with FX decreased by 27.1%-41.3%; the activities of superoxide dismutase in the leaves treated with PX and FX increased by 12.6% and 7.8% respectively.

[0064] Various resistance physiological indexes such as superoxide dismutase, phenylalanine ammonia-lyase, polyphenol oxidase and malondialdehyde in tomato plants are widely present in the cytoplasm, chloroplasts and mitochondria of tomato cells. The numerical values indicate the stress expression levels of tomato plants in aspects such as biosynthesis, tissue metabolism and defense responses, that is, the degree of stress faced by tomato plants.

[0065] 3.3 Tomato fruit quality

[0066] Bacillus thuringiensis TG5 alone or in combination with phoxim has an impact on the single fruit weight and yield of tomato plants, and the impact is significant. The results are as Figure 3 shown.

[0067] Compared with the control, the average single fruit weight and average yield per plant of tomatoes treated with TG5+PX increased by 32.8% and 175.2% respectively; compared with the PX and FX treatments, the average single fruit weight of tomatoes treated with TG5+PX increased by 14.9% and 10.2% respectively, and the average yield per plant of tomatoes treated with TG5+PX increased by 137.7% and 110.2% respectively ( Figure 3 A, B in).

[0068] Compared with the control, the changes in the contents of soluble sugar, titratable acid, vitamin C and soluble solids in tomato fruits treated with TG5+PX were between 27.5%-156.7%; compared with the PX treatment, the changes in the contents of soluble sugar, titratable acid, vitamin C and soluble solids in tomato fruits treated with TG5+PX were between 9.4%-43.9%; compared with the FX treatment, the changes in the contents of soluble sugar, titratable acid, vitamin C and soluble solids in tomato fruits treated with TG5+PX were between 13.9%-42.5 ( Figure 3 C, D, E, F in).

[0069] 3.4 Bacillus thuringiensis TG5 promotes the degradation of phoxim

[0070] The pesticide residue in fruits is one of the most important indicators of fruit quality. As Figure 4As shown in the figure, after the application of phoxim and fludioxonil, there will be pesticide residues in the rhizosphere soil and fruits of tomato plants, and the pesticide residue in tomato fruits is proportional to the residue in the rhizosphere soil. Compared with the phoxim residue of 18.5 μg / kg in the tomato fruits treated with PX, the phoxim in the tomato fruits treated with TG5+PX degraded, and the residue decreased to 1.18 μg / kg, meeting the requirement of the phoxim limit ≤ 50 μg / kg in tomato fruits in the "GB 2763-2021 Maximum Residue Limits of Pesticides in Foods" in China. The fludioxonil residue in the tomato fruits treated with FX was 6.37 μg / kg, meeting the requirement of the fludioxonil limit ≤ 3 mg / kg in tomato fruits in the "GB 2763-2021 Maximum Residue Limits of Pesticides in Foods" in China.

[0071] In summary, the compound combination of the biopesticide Bacillus thuringiensis TG5 and PX has a synergistic effect, increasing the quality and yield of tomatoes. The biopesticide Bacillus thuringiensis TG5 reduces the PX residue in tomato plants and fruits.

[0072] The above are only the preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A composition for promoting tomato resistance to Fusarium oxysporum, characterized in that, The composition described above comprises Bacillus thuringiensis TG5 and phoxim.

2. The composition according to claim 1, wherein The Bacillus thuringiensis TG5 is a fermentation broth of Bacillus thuringiensis TG5, and the preparation method comprises the following steps: Inoculate the Bacillus thuringiensis TG5 strain into a solid medium, culture at 30 - 40 °C for 12 - 36 h, cut the solid medium containing the TG5 strain, inoculate the cut slices into a fermentation medium, stir at 30 - 40 °C at 100 - 400 rpm for 12 - 36 h, and obtain the Bacillus thuringiensis TG5 fermentation broth after filtration.

3. Use of the composition according to claim 1 or 2 in promoting tomatoes to resist Fusarium oxysporum.

4. A method for promoting tomato resistance to Fusarium oxysporum, characterized in that, Apply the composition according to claim 1 or 2 for promoting tomatoes to resist Fusarium oxysporum to tomato plants.

5. The method according to claim 4, characterized in that, The viable count in the fermentation broth of Bacillus thuringiensis TG5 is not less than 1×10 10 CFU / ml.

6. The method according to claim 4, characterized in that The concentration of phoxim is 200 - 800 mg / L.

7. The method according to claim 4, characterized in that Apply during the growth and fruiting period, the number of application times is 2 - 6 times, and the application amount is 20 - 70 ml per plant per time.

8. The method according to claim 4, wherein The application method is one or more of root irrigation, foliar spraying or broadcasting.

9. The method according to claim 7, wherein The application method described above is root irrigation.

10. Use of Bacillus thuringiensis TG5 in promoting tomato resistance to Fusarium oxysporum, characterized in that, The use described above includes reducing the incidence of tomato diseases caused by Fusarium oxysporum, alleviating the stress of tomato plants, improving the quality of tomato fruits, and promoting the degradation of the pesticide phoxim.

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