Pseudomonas aeruginosa and its application in preventing and treating apple replant disease

Abstract

This invention relates to the field of biological control of apple replant disease, specifically to a strain of *Pseudomonas aeruginosa* C35 and its application in controlling apple replant disease. The *Pseudomonas aeruginosa* C35 (… Pseudomonas aeruginosa The strain was deposited on November 20, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.32713. For use, *Pseudomonas aeruginosa* was activated and fermented to obtain the fermentation broth, which was then diluted to 10⁻⁶ liters with sterile LB broth. 8 CFU / mL was kept on standby. The fermentation broth of *Pseudomonas aeruginosa* was applied to the roots of *Malus arborescens* seedlings via root irrigation. 24 hours later, a suspension of *Fusarium oxysporum* HS2 spores was injected into the roots of the seedlings treated with the fermentation broth. The seedlings were cultured in a greenhouse with regular watering. After 30 days of treatment, the severity of disease on the *Malus arborescens* plants was assessed. The disease index of *Malus arborescens* seedlings treated with *Pseudomonas aeruginosa* C35 was only 25.92, achieving a protective control effect of 67.41%, which was significantly different from the control treatment. *Pseudomonas aeruginosa* can reduce the mortality rate of *Malus arborescens* seedlings in the field to 30%, providing biological control for apple replanting disease.

Description

Technical Field

[0001] This invention relates to the field of biological control of apple replant disease, specifically to Pseudomonas aeruginosa and its application in the control of apple replant disease. Background Technology

[0002] Apples play a vital role in my country's economy. my country ranks first globally in both apple planting area and yield, making it one of the world's largest apple-producing regions. However, continuous apple cultivation in the same area can lead to decreased plant vigor and consequently affect tree growth, a condition known as Apple Replant Disease (ARD). ARD, also called continuous cropping obstacle or replant disease, is a serious and widespread problem currently faced in the renovation and upgrading of old apple orchards in my country. The disease primarily manifests as slow growth, small tree size, damaged root systems, weak tree vitality, severe pest and disease infestations, poor quality, and low yield in replanted saplings. It can even cause the death of the entire plant, resulting in significant economic losses for fruit growers and severely impacting the development of the apple industry.

[0003] Important pathogens causing apple replant disease include those belonging to the genera *Fusarium*, *Cyclophorus*, *Rhizoctonia*, *Phytophthora*, and *Pythium*. *Fusarium* is a significant harmful fungus causing apple replanting problems in the Loess Plateau region of Northwest China, and its abundance is positively or negatively correlated with the severity of the disease. Furthermore, *Fusarium moniliforme*, *Fusarium moniliforme*, *Fusarium solani*, and *Fusarium oxysporum* are the main pathogens causing apple replanting problems in the Bohai Bay region of China. Therefore, it is essential to take safe and effective measures to control apple replant disease.

[0004] Currently, the main agricultural control measures for apple replant disease include reasonable crop rotation and intercropping, deep tilling and topsoil application, and increased application of organic fertilizers. In addition, while chemical fungicides can be sprayed to control apple replant disease, due to environmental pollution, impacts on human health, and the emergence of drug-resistant strains, biological control, which uses beneficial microorganisms to control the disease, offers advantages such as safety and environmental friendliness compared to chemical control methods.

[0005] Pseudomonas aeruginosa C35 is a microorganism belonging to the genus Pseudomonas. Bacteria in this genus have functions such as nitrogen fixation, phosphorus solubilization, iron phosphate, and secretion of extracellular enzymes.

[0006] Currently, there are few reports on the application of Pseudomonas aeruginosa as a biocontrol bacterium for the prevention and control of apple replant disease. Therefore, this patent aims to develop a biocontrol bacterium for the green prevention and control of apple replant disease. Summary of the Invention

[0007] This invention provides the application of a strain of Pseudomonas aeruginosa in the prevention and control of apple replant disease.

[0008] Pseudomonas aeruginosa C35 was deposited on November 20, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCCNo.32713.

[0009] The sterile filtrate of Pseudomonas aeruginosa C35 is also within the scope of protection of this invention.

[0010] The fermentation broth is a fermentation broth of Pseudomonas aeruginosa C35 obtained in a liquid culture medium, containing bacterial cells and their metabolites.

[0011] The fermentation broth can effectively control the occurrence of apple replant disease.

[0012] The beneficial effects of this invention are as follows: This invention provides the application of Pseudomonas aeruginosa C35 with accession number CGMCC No.32713 in the prevention and control of apple replant disease. This not only enriches the Pseudomonas species resource bank and broadens its field and application scope for the prevention and control of plant diseases, but also provides a new solution for the prevention and control of apple replant disease. Attached Figure Description

[0013] To clearly illustrate the implementation examples in the specific implementation methods, the following figures are added for supplementary explanation.

[0014] Figure 1 Results of functional activity assays for Pseudomonas aeruginosa C35. From left to right: C35 phosphorus solubility index (organic phosphorus), phosphorus solubility index (inorganic phosphorus), nitrogen fixation activity, ironophilic activity, and indoleacetic acid production.

[0015] Figure 2 The protective and curative effects of Pseudomonas aeruginosa C35 against replant disease caused by Fusarium oxysporum. From top to bottom, the images show the protective and curative effects of C35 against replant disease caused by Fusarium oxysporum.

[0016] Figure 3 The effect of Pseudomonas aeruginosa C35 on the growth of Malus baccata seedlings in the field. From top to bottom, the growth status of Malus baccata seedlings under the CK and C35 treatments on days 30, 60, and 90 are shown.

[0017] Figure 4 A single colony image of Pseudomonas aeruginosa C35.

[0018] Figure 5 Gram staining image of Pseudomonas aeruginosa C35.

[0019] Figure 6 Molecular identification diagram of Pseudomonas aeruginosa C35.

[0020] Biological Preservation Information

[0021] Pseudomonas aeruginosa C35 was isolated from rhizosphere soil collected from apple orchards of different varieties at Hebei Agricultural University. It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 20, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain number is C35, and the accession number is CGMCC No. 32713. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described below with reference to the accompanying drawings. The following embodiments are intended to facilitate a better understanding of the present invention, but do not limit the present invention.

[0023] Example 1

[0024] Screening of apple rhizosphere bacteria and determination of the functional activity of C35.

[0025] Rhizosphere soil samples of apple varieties such as 'Jinxiu Hong' and 'Zhongqiu Wang' were collected from the apple experimental orchard of Hebei Agricultural University and brought back to the laboratory. 0.5g of soil was prepared into a suspension with sterile water, diluted, and then inoculated into solid culture media such as LB and PDA. Colonies were picked and purified to obtain single colonies. These single colonies were then shaken to prepare a fermentation broth, mixed with glycerol solution, and stored at -80℃ for later use. Strains that produced a clear zone diameter of grade III on the phosphorus-solubilizing, nitrogen-fixing, iron-loving, protease, and cellulase detection media from the initial screening were further screened, and their ability to secrete IAA was determined to identify strains with strong growth-promoting effects.

[0026] Depend on Figure 1 It can be seen that strain C35 produces a large clear zone, has a high activity level, and has the ability to secrete IAA. Its phosphorus solubility index (organic phosphorus, inorganic phosphorus) is 4.07a and 3.47a, respectively, nitrogen fixation activity is 1.32b, and iron affinity activity is 3.32a.

[0027] Example 2

[0028] The control effect of Pseudomonas aeruginosa C35 on apple replant disease caused by Fusarium oxysporum.

[0029] After a concentration of 7.5×10 6 Most Malus halliana seedlings treated with a Fusarium oxysporum spore suspension at a concentration of 1 / mL for 30 days showed stunting symptoms, accompanied by wilting, death, and yellowing of leaves. The disease index of Malus halliana seedlings treated with Pseudomonas aeruginosa C35 was 25.92, with a protective control effect of 67.41%, while the disease index of the blank control group was 80.55. Figure 2(Above). The therapeutic effect of this strain on Malus halliana seedlings was determined through a greenhouse pot experiment. The disease index of Malus halliana seedlings treated with C35 was 38.89, with a therapeutic effect of 53.33%, while the disease index of the blank control group was 83.33. Figure 2 Down).

[0030] Depend on Figure 2 Table 1 shows that this strain has a certain protective and therapeutic effect against apple replanting disease, and can effectively reduce the damage of Malus baccata seedlings to pathogens, but the therapeutic effect is lower than the protective effect.

[0031] Table 1. Protective and curative effects of C35 against replanting diseases caused by Fusarium oxysporum.

[0032]

[0033] Example 3

[0034] The effect of Pseudomonas aeruginosa C35 on the growth of Malus baccata seedlings in the field.

[0035] With a concentration of 1×10 8 A single-strain fermentation broth (CFU / mL) was used to drench the roots of Malus halliana seedlings in the experimental garden at a rate of 300 mL per seedling, with LB liquid medium serving as a control. Drenching was performed every 10 days, and plant height, stem diameter, and other parameters were measured on days 30, 60, and 90 after drenching. The results showed that the C35 treatment slightly improved all parameters compared to the control (CK), but the differences were not statistically significant. Figure 3 As shown in the figure, from top to bottom, the growth status of Malus halliana seedlings treated with CK and C35 on days 30, 60, and 90 is as follows. The seedling mortality rate of Malus halliana seedlings treated with single-strain C35 was 30%, which was lower than that of the control group.

[0036] according to Figure 3 This indicates that C35 treatment has a certain protective effect on Malus halliana seedlings, reducing the mortality rate and promoting their growth to some extent.

[0037] Example 4

[0038] Identification of Pseudomonas aeruginosa C35.

[0039] C35 colonies were streaked in LB medium to observe colony morphology. Gram staining was used to determine whether the bacteria were Gram-positive or Gram-negative. Using their DNA as a template, PCR amplification was performed using universal primers for 16S rDNA and the gyrB gene. The PCR products were purified and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing sequences were compared with those obtained by NCBI to construct a phylogenetic tree.

[0040] like Figure 4As shown, strain C35 colonies on LB solid medium are round and translucent green. Figure 4 ).

[0041] like Figure 5 As shown, strain C35 is Gram-negative, indicating it is a Gram-negative bacterium. Figure 5 ).

[0042] like Figure 6 As shown, after the sequence was aligned with NCBI, a phylogenetic tree was constructed, and C35 was identified as Pseudomonas aeruginosa (A is a phylogenetic tree constructed based on the 16S rDNA sequence, and B is a phylogenetic tree constructed based on the gyrB gene sequence).

Claims

1. A strain of Pseudomonas aeruginosa C35 ( Pseudomonas aeruginosa The strain was deposited at the China General Microbiological Culture Collection Center on November 20, 2024, with the accession number CGMCC NO.32713.

2. The use of Pseudomonas aeruginosa C35 according to claim 1, characterized in that... It can control the incidence of apple replant disease and effectively reduce the occurrence of the disease. 3.(1) First, Pseudomonas aeruginosa C35 was inoculated into LB liquid medium and shaken to obtain the fermentation broth of the bacterium; (2) Eight-ridged crabapple seedlings with uniform leaf size and growth were selected for the experiment. The biocontrol bacteria fermentation solution was irrigated into the roots of the seedlings. 24 hours later, a suspension of Fusarium oxysporum HS2 spores was instilled into the roots of the biocontrol-treated seedlings. A control group was treated with a Fusarium oxysporum spore suspension plus blank LB liquid medium. The seedlings were cultivated in a greenhouse and watered regularly. After 30 days of treatment, morphological and physiological indicators of the seedlings were measured, and the severity of disease was assessed. The disease index was calculated to evaluate the control effect and achieve control of apple replanting disease.

4. The method for controlling apple replant disease using Pseudomonas aeruginosa C35 according to claim 2, characterized in that, The LB liquid culture medium mentioned in step (1) is as follows (1L): 10g peptone, 10g sodium chloride, 5g yeast powder, distilled water to a final volume of 1000mL, pH adjusted to 7, and sterilized at 121℃ for 20min.

5. The method for controlling apple replant disease using Pseudomonas aeruginosa C35 according to claim 2, characterized in that, The conditions for the first and second activation cultures in step (1) were: 28℃ for 24h; and the conditions for centrifugation were: 25℃, 5000 rpm, and 10min.

6. The method for controlling apple replant disease using Pseudomonas aeruginosa C35 according to claim 2, characterized in that, In step (2), the biocontrol bacteria fermentation liquid is poured into the roots of the Malus baccata seedlings, with each seedling receiving 50 mL of the liquid.

7. The method for controlling apple replant disease using Pseudomonas aeruginosa C35 according to claim 2, characterized in that, In step (2), the volume of Pseudomonas aeruginosa C35 fermentation broth added to the root of each crabapple seedling was the same as that of the pathogen suspension, both being 50 mL; the concentration of Pseudomonas aeruginosa C35 fermentation broth used was 1×10⁻⁶. 8 The inoculation concentration of the spore suspension of the pathogen *Fusarium oxysporum* HS2 was 7.5 × 10⁻⁶ CFU / mL. 6 per ml.

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