Acinetobacter lwoffii GT04 with effect of inhibiting fusarium oxysporum and application of acinetobacter lwoffii GT04

Abstract

The invention relates to acinetobacter lwoffii with an effect of inhibiting fusarium oxysporum and application of the acinetobacter lwoffii, the taxonomic name of the acinetobacter lwoffii is Acinetobacter lwoffii, the preservation number of the acinetobacter lwoffii is GDMCC No: 67188, and the preservation date of the acinetobacter lwoffii is October 30, 2025. The strain can prevent and control root rot by inhibiting tobacco root rot related pathogenic bacteria such as fusarium oxysporum. Therefore, the acinetobacter lwoffii GT04 strain is suitable for preparing related products for preventing and treating tobacco root rot.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial culture and relates to Acinetobacter lwoffii GT04 with the efficacy of inhibiting Fusarium oxysporum and application thereof. BACKGROUND

[0002] Tobacco is one of important economic crops, but the occurrence of diseases and pests seriously affects the yield and quality of tobacco. In recent years, with the change of climate and cultivation mode, the occurrence of tobacco diseases has been increasing year by year. For example, the change of tobacco planting area, cultivation management measures and global climate and other factors, and the occurrence of soil-borne diseases such as tobacco fusarium root rot has been increasing year by year, which further seriously restricts and affects the yield of tobacco.

[0003] Tobacco fusarium root rot is a fungal disease mainly caused by the infection of Fusarium oxysporum, which can occur in the seedling bed stage and the growth stage in the field. The root system of the diseased tobacco plant is rotted and destroys the vascular system, hinders the water absorption of the aboveground part and the nutrient absorption of the underground part, and finally leads to the death of the plant.

[0004] At present, the common prevention and control methods for tobacco fusarium root rot include chemical agents, agricultural measures and biological control. Although the chemical control method has good control effect, it is limited in application due to the problems of pesticide residues and easy drug resistance, which affects the safety of tobacco leaves. The agricultural measures mainly ensure the smooth drainage of the field through high ridge and ditch cultivation, which has weak disease control effect. Biological control has the advantages of high control effect, strong specificity, environmental friendliness and difficulty in producing drug resistance. Therefore, it has important application value to provide a microbial inoculant that can effectively control tobacco root rot. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide Acinetobacter lwoffii GT04 with the efficacy of inhibiting Fusarium oxysporum and application thereof

[0006] To achieve the purpose of the application, the following technical solutions are adopted:

[0007] In the first aspect, the application provides Acinetobacter lwoffii GT04 with the efficacy of inhibiting Fusarium oxysporum, which is taxonomically named as Acinetobacter lwoffii, the preservation number is GDMCC No: 67188, and the preservation date is October 30, 2025.

[0008] The application isolates and preserves a new Acinetobacter lwoffii with the function of inhibiting Fusarium oxysporum from the tobacco leaf tissue of a healthy tobacco plant, and names it as Acinetobacter lwoffii GT04 strain. The strain can inhibit tobacco root rot related pathogens such as tobacco Fusarium oxysporum to achieve the prevention and control of root rot. Therefore, the Acinetobacter lwoffii GT04 strain is suitable for preparing related products for preventing and controlling tobacco root rot.

[0009] In the second aspect, the application provides a culture of the Acinetobacter lwoffii GT04 according to the first aspect, which is prepared by inoculating the Acinetobacter lwoffii GT04 into a culture medium and culturing at 26-30℃ for 24-60h.

[0010] For example, the "26-30℃" can be 26℃, 26.5℃, 27℃, 27.5℃, 28℃, 29℃, 30℃, and other specific point values in the numerical range can be selected, which will not be repeated here.

[0011] For example, the "24-30h" can be 24h, 25h, 26h, 27h, 28h, 29h, 30h, and other specific point values in the numerical range can be selected, which will not be repeated here.

[0012] In the third aspect, the application provides a microbial inoculant with the function of inhibiting Fusarium oxysporum, and the strain in the microbial inoculant includes the Acinetobacter lwoffii GT04 strain according to the first aspect.

[0013] Preferably, in the microbial inoculant, the viable bacterial count of the Acinetobacter lwoffii GT04 is not less than 1×10 10 CFU / mL or 1×10 10 CFU / g, for example, 1×10 10 CFU / mL (CFU / g), 2×10 10 CFU / mL (CFU / g), 5×10 10 CFU / mL (CFU / g), 8×10 10 CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), 5×10 11 CFU / mL (CFU / g), 1×10 12 CFU / mL (CFU / g), etc., and other specific point values in the numerical range can be selected, which will not be repeated here.

[0014] Preferably, the dosage form of the microbial inoculant includes powder, solution, tablet or granule.

[0015] Preferably, the microbial inoculant further comprises a protective agent.

[0016] Preferably, the protective agent comprises any one or a combination of at least two of skimmed milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

[0017] In a fourth aspect, the present application provides use of the Acinetobacter lwoffii GT04 according to the first aspect, the culture according to the second aspect or the microbial inoculant according to the third aspect in the preparation of a product for inhibiting Fusarium oxysporum.

[0018] In a fourth aspect, the present application provides use of the Acinetobacter lwoffii GT04 according to the first aspect, the culture according to the second aspect or the microbial inoculant according to the third aspect in the preparation of a product for promoting the growth and yield of tobacco.

[0019] In a fourth aspect, the present application provides use of the Acinetobacter lwoffii GT04 according to the first aspect, the culture according to the second aspect or the microbial inoculant according to the third aspect in the preparation of a product for preventing and treating tobacco Fusarium root rot.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] The present application isolates and preserves a new Acinetobacter lwoffii strain with the function of inhibiting Fusarium oxysporum from the leaf tissue of healthy tobacco plants, which is named as Acinetobacter lwoffii GT04 strain. The strain can prevent and treat root rot by inhibiting tobacco root rot related pathogens such as Fusarium oxysporum. Therefore, the Acinetobacter lwoffii GT04 strain is suitable for preparing related products for preventing and treating tobacco root rot.

[0022] The GT04 strain involved in the present application is classified as Acinetobacter lwoffii, and is preserved in the Guangdong Microbial Culture Collection Center with the preservation number GDMCC No: 67188, the preservation date October 30, 2025, and the preservation address 5th Floor, Building 59, Guangzhou Xianlie Middle Road 100 Courtyard. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a diagram of the confrontation culture result of the screening and isolation strain (GT04) and Fusarium oxysporum;

[0024] Figure 2 is a diagram of the colony morphology of the screening and isolation strain (GT04) after streak culture on the culture medium for 48 h. DETAILED DESCRIPTION

[0025] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0026] The GT04 strain mentioned below is classified as Acinetobacter lwoffii, with accession number GDMCCNo: 67188.

[0027] The following are the culture media involved:

[0028] Isolation medium (NA): 10 g peptone, 3 g beef extract, 5 g sodium chloride, 18 g agar, 1000 mL water;

[0029] Screening medium (PDA): 200 g potato, 20 g glucose, 18 g agar, add distilled water to 1000 mL.

[0030] Unless otherwise specified, all reagents and consumables used in the following examples are commercially available, and the relevant experimental steps are common operations in the field and have technical meanings that can be conventionally understood by those skilled in the art.

[0031] Example 1

[0032] In this embodiment, a strain of Acinetobacter rouleauxii with inhibitory effects against Fusarium oxysporum was isolated and screened. The steps are as follows:

[0033] (1) Separation: Take 0.5 g of thoroughly sterilized tobacco leaf tissue and place it together with sterilized steel balls into a 2 mL sterile centrifuge tube. Add 600 µL of sterile water and grind the tissue at 30 rpm for 4 min using a tissue homogenizer. Serially dilute the homogenized tissue solution to 10 mL. -4 For each gradient, 100–300 µL of homogenate was spread onto the isolation medium, and each treatment was repeated 4 times. After incubation in the dark at 25–30 °C for 36–72 h, single colonies were picked according to their morphology, color, etc., and then streaked onto the isolation medium for purification. The single colonies were then transferred to test tubes containing isolation medium for preservation.

[0034] (2) Screening: The plate confrontation method was used. A 0.5 cm diameter hole punch was used to attach a block of Fusarium oxysporum mycelium to the center of the screening medium plate. Single colonies isolated in step (1) were symmetrically streaked on both sides. The treatment with only Fusarium oxysporum mycelium was used as a control. The plates were incubated at 26℃ for 3 days. When the control plate reached 2 / 3 of the plate, the confrontation culture results were checked and the width of the inhibition zone was recorded. The antagonistic effect of the isolated strain on the pathogen was judged based on the colony development speed of the two bacteria, the presence or absence of an inhibition zone between the colonies, and whether the mycelium at the edge of the colony was sparse and atrophied. Each treatment was repeated 3 times. The results showed that when the strain isolated in step (1) was confronted with Fusarium oxysporum, the aerial mycelium of Fusarium oxysporum was sparse, and mycelium only formed around the mycelial cake (diameter 4.8 cm). In contrast, the mycelium in the control plate was uniform (diameter 7.2 cm). cm); the growth of Fusarium oxysporum on plates was also limited, and the isolated strain showed an inhibition rate of 33.3% against Fusarium oxysporum (cm); Figure 1 This indicates that the strain can inhibit not only the growth of aerial mycelia of Fusarium oxysporum, but also the growth of mycelia in the culture medium.

[0035] (3) Purification: The selected strains were streaked on purification medium plates to obtain vigorous single colonies. After purification, liquid NA medium was used for expansion culture. After centrifugation to remove the supernatant, the culture was resuspended in sterile 25% glycerol aqueous solution for preservation.

[0036] Example 2

[0037] This embodiment identifies the strains obtained from screening in Example 1, and the steps are as follows:

[0038] (1) Culture characteristics: This strain is a Gram-negative cocci and is non-motile. It is a strict aerobic bacterium, and both the nitrate reaction and oxidase test are negative.

[0039] (2) Genomic DNA was extracted from the strain screened and preserved in Example 1 according to the instructions of the bacterial genomic DNA extraction kit. PCR amplification was performed using primers gyrB-F / R under standard conditions. After gel recovery, the amplified product was ligated into the PMD-19T vector, transformed into competent E. coli DH5α cells by heat shock, and colonies were picked for colony PCR identification using primers M13F / M13R. Positive clones were sent to a sequencing company for sequencing identification. The sequence of this strain is shown in SEQ ID No:1. The sequenced sequence was compared with the nucleic acid sequence in GeneBank, and the results showed that the strain was Acinetobacter rouxii.

[0040] SEQ ID No:1:

[0041]

[0042] Based on the identification results of Example 2, the strain was confirmed to belong to Acinetobacter ruthenicus and named Acinetobacter ruthenicus lwoffii GT04 strain.

[0043] Example 3

[0044] Test on the control effect of Fusarium root rot in tobacco

[0045] (1) Indoor experiment in a light-incubation chamber (temperature 25℃, relative humidity 60%, 12 h / 12 ​​h light / dark culture): 50-day-old Yunyan 87 tobacco seedlings were transplanted into flowerpots inoculated with Fusarium oxysporum pathogen, covered with sterile substrate, and fermented with GT04 strain (1×10⁻⁶) fermentation broth. 10 Dilute 5 times (CFU / mL) and apply 5 mL to the rootstock of each tobacco plant, with 3 replicates. After 7 days, the disease severity of each treatment group was investigated, and the disease index and relative control efficacy were calculated. The results showed that strain GT04 had a good control effect on Fusarium root rot in tobacco. The disease index of the treatment group treated with GT04 fermentation liquid was 7.4, while the disease index of the control group without fermentation liquid was as high as 28.9. After root irrigation with GT04 fermentation liquid, the occurrence of Fusarium root rot in tobacco can be effectively controlled, with a relative control efficacy of 74.4%. It can be seen that the intervention of strain GT04 can effectively inhibit the occurrence of Fusarium root rot.

[0046] (2) Field efficacy test (Dali Yunlong, Dali Wanqiao, Qujing Jiuxian): GT04 fermentation broth (1×10 10 The inoculant (CFU / mL) was diluted 10 times and applied at a rate of 200 mL per plant to the rootstock. Applications were made on the day of transplanting, 14 days, and 28 days after transplanting, with three replicates for each treatment. A water-based treatment served as a control. Disease incidence was assessed 14 days after application. Disease severity was categorized by disease grade as shown in Table 1. Disease index and relative control efficacy were calculated using the following formulas, and the results are shown in Table 2.

[0047] ;

[0048] .

[0049] Table 1

[0050]

[0051] Table 2

[0052]

[0053] The results in the table show that, in field tests, strain GT04 achieved an average control rate of 59.0% against Fusarium root rot in tobacco. Furthermore, Acinetobacter rudenifolia strain GT04 demonstrated significant efficacy in controlling Fusarium root rot in tobacco. Therefore, this Acinetobacter rudenifolia strain GT04 is suitable for preparing products related to the control of tobacco root rot.

[0054] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0055] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0056] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. An Acinetobacter rudenifolia GT04 strain with inhibitory effects against Fusarium oxysporum, characterized in that, The taxonomic name of GT04 is Acinetobacter lwoffii, with accession number GDMCC No: 67188 and accession date of October 30, 2025.

2. A culture of Acinetobacter GT04 according to claim 1, characterized in that, The culture was prepared by inoculating Acinetobacter lutea GT04 into a culture medium and culturing it at 26-30°C for 24-60 h.

3. A microbial agent with the ability to inhibit Fusarium oxysporum, characterized in that, The strain in the microbial agent includes the Acinetobacter GT04 strain as described in claim 1.

4. The microbial agent as described in claim 3, characterized in that, In the microbial inoculant, the viable count of Acinetobacter ruthenium GT04 is not less than 1 × 10⁻⁶. 10 CFU / mL or 1×10 10 CFU / g.

5. The microbial agent as described in claim 3, characterized in that, The dosage forms of the microbial agents include powders, solutions, tablets, or granules.

6. The microbial agent as described in claim 3, characterized in that, The microbial agent also includes a protectant.

7. The microbial agent as described in claim 6, characterized in that, The protective agent includes any one or a combination of at least two of the following: skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol, or xylitol.

8. The use of Acinetobacter GT04 as described in claim 1, or the culture as described in claim 2, or the microbial agent as described in any one of claims 3-7, in the preparation of a product inhibiting Fusarium oxysporum.

9. The use of Acinetobacter GT04 as described in claim 1, or the culture as described in claim 2, or the microbial agent as described in any one of claims 3-7, in the preparation of tobacco growth-promoting and yield-increasing products.

10. The use of Acinetobacter GT04 as described in claim 1, or the culture as described in claim 2, or the microbial agent as described in any one of claims 3-7, in the preparation of a product for controlling Fusarium root rot in tobacco.

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