New strain for preventing and treating asparagus root rot and application thereof

Abstract

The present application belongs to the field of biological control, and particularly relates to a new strain for preventing and treating asparagus root rot and application thereof. The Pseudarthrobacter oxydans NJ01 is suitable for a slightly acidic environment, and can effectively inhibit the common pathogenic bacteria of asparagus root rot, i.e. Fusarium solani, Fusarium oxysporum and Fusarium proliferatum. It has been verified through experiments that the Pseudarthrobacter oxydans NJ01 has high disease prevention efficiency in preventing and treating Neijiang asparagus root rot, and the plant protection agent prepared from the Pseudarthrobacter oxydans NJ01 has a very wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological control, specifically relating to a new strain for controlling asparagus root rot and its application. Background Technology

[0002] Root rot is a soil-borne disease and one of the most significant diseases affecting crop production. With the development of greenhouse cultivation, continuous cropping has become increasingly prevalent, leading to the accumulation of diseased plant debris in the soil, providing ample inoculum and nutrients for pathogens. Furthermore, excessive planting density, poor ventilation and light penetration, improper fertilizer and water management, and frequent periods of overcast and rainy weather can all contribute to the spread of root rot. Root rot can occur throughout the entire growth period of a crop, but it is most susceptible from transplanting to before flowering. In the early stages, the above-ground leaves turn yellow, wilt, and wither in the morning and evening. In later stages, the stems turn brown and rot. Once the disease develops, it is difficult to control and its severity increases year by year, making prevention and control challenging.

[0003] Currently, the main methods for controlling root rot in production include chemical control, agricultural control, and biological control. Biological control is one of the important means of controlling root rot, especially in situations where the use of chemical agents is restricted or environmental requirements are strict. Biological control is receiving increasing attention due to its environmental friendliness and sustainability.

[0004] Biological control primarily involves introducing beneficial microorganisms to inhibit the growth of pathogens, thereby reducing the occurrence of root rot. These beneficial microorganisms include bacteria and fungi. For example, Bacillus subtilis, Paenibacillus polymyxa, and Serratia plymuthica have been shown to effectively inhibit root rot caused by Fusarium oxysporum. However, there are currently no reports on the control of root rot by Pseudomonas oxysporum, especially asparagus root rot. Summary of the Invention

[0005] To address the aforementioned problems, the present invention aims to provide a novel strain that can be used to prevent and control asparagus root rot, particularly asparagus root rot in Neijiang.

[0006] This invention provides a *Pseudarthrobacter oxydans* NJ01, which was deposited on November 29, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with accession number GDMCC No. 65567 and classified as *Pseudarthrobacter oxydans*. The deposit address is No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0007] The present invention also provides the use of the aforementioned *Pseudomonas oxysporum* in the preparation of a plant protectant for the prevention and / or treatment of asparagus root rot.

[0008] Furthermore, the plant protectant is a plant protectant for the prevention and / or treatment of asparagus root rot.

[0009] Furthermore, the aforementioned asparagus root rot includes Neijiang asparagus root rot.

[0010] Furthermore, the plant protectant has the effect of inhibiting the pathogenic fungus of Fusarium asparagus root rot.

[0011] Furthermore, the pathogenic bacteria include Fusarium solanum, Fusarium oxysporum, and Fusarium moniliforme.

[0012] Furthermore, the plant protectant is a formulation made with *Pseudomonas oxysporum* as the active ingredient and acceptable excipients.

[0013] Furthermore, the formulation includes solutions, powders, ointments, and sprays.

[0014] Furthermore, the viable count of *Pseudomonas oxysporum* in the formulation is ≥1×10⁻⁶. 8 CFU / mL.

[0015] The present invention also provides a plant protectant for preventing root rot, which includes the aforementioned *Pseudomonas oxysporum*.

[0016] The "plant protectant" described in this invention refers to a substance or preparation used to protect plants or plant products from harmful organisms, affect plant life processes, preserve plant products, eliminate undesirable plants, or control the growth of undesirable plants, including microbial pesticides.

[0017] The "Neijiang Asparagus" mentioned in this invention refers to Asparagus cochinchinensis (Lour.) Merr., a plant of the Liliaceae family, which is a traditional Chinese medicine from Dongxing District, Neijiang City, Sichuan Province.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The *Pseudarthrobacter oxydans* NJ01 of this invention thrives in slightly acidic, high-temperature environments and can effectively inhibit common pathogens of asparagus root rot, such as *Fusarium solanum*, *Fusarium oxysporum*, and *Fusarium solani*. Experimental verification shows that *Pseudarthrobacter oxydans* NJ01 can effectively control asparagus root rot in Neijiang, with a disease control efficiency of 51.03%. The formulation of *Pseudarthrobacter oxydans* NJ01 into a microbial pesticide for the control of asparagus root rot has a very broad market prospect.

[0020] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0021] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0022] Figure 1 Streak plate plot of strain NJ01;

[0023] Figure 2 pH affects the OD of strain NJ01 bacterial culture 600 Effects of absorbance (A) and temperature on the OD of strain NJ01 bacterial culture 600 Effect of absorbance (B);

[0024] Figure 3 The effects of different concentrations of strain NJ01 bacterial solution on the incidence of root rot of Asparagus cochinchinensis in Neijiang (A), the inhibition rate of strain NJ01 bacterial solution on Fusarium solani, Fusarium oxysporum, and Fusarium moniliforme (B), the effects of different concentrations of strain NJ01 bacterial solution on the average disease index (C), and the effects of different concentrations of strain NJ01 bacterial solution on the average disease control efficiency (D). Detailed Implementation

[0025] The raw materials, reagents, and equipment used in the specific embodiments of this invention were all obtained through commercial purchase. Example 1: Screening and efficacy experiment of *Pseudomonas oxysporum*.

[0026] I. Methods

[0027] 1. Strain screening and bacterial culture preparation

[0028] Multiple types of arthrobacteria were screened from soil samples obtained from the sorghum-asparagus intercropping base in the Guobei Town Asparagus Industrial Park, Dongxing District, Neijiang City, Sichuan Province, using extreme dilution and streak plating methods. After physicochemical analysis, category identification, and testing of some safety indicators, *Pseudarthrobacter oxydans* NJ01 was selected as the strain that met the basic requirements in the initial tests. A bacterial suspension was prepared using *Pseudarthrobacter oxydans* NJ01, with a viable count ≥1×10⁻⁶. 8 CFU / mL.

[0029] 2. Identification and Preservation

[0030] The NJ01 strain was identified as *Pseudarthrobacter oxydans*, and was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 29, 2024. The deposit address is No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province; the accession number is GDMCC No. 65567.

[0031] Its identification characteristics are as follows:

[0032] (1) Morphological characteristics and biochemical properties

[0033] The morphology of Gram-positive bacteria on tryptic soy broth (TSB) solid medium: colonies are basically round, with a moist and smooth surface, white, and opaque. Figure 1 Through oxidative fermentation testing, strain NJ01 was found to be an aerobic bacterium with an optimal growth temperature of 32-36℃ and an optimal pH of 5-7.

[0034] (2) Sequence analysis of 16S rDNA

[0035] The sequence is as follows (SEQ ID NO.1):

[0036]

[0037] The BLAST tool was used on the NCBI website to compare the similarity of the sequence in the gene bank. The results showed that the 16S rDNA of strain NJ01 had a 99.27% ​​similarity to the 16S rDNA of Arthrobacter sp. X38. Based on the morphological and physiological and biochemical characteristics, strain NJ01 was identified as a new strain of Pseudarthrobacter oxydans.

[0038] This strain was deposited on November 29, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No. 65567, classified as Pseudarthrobacter oxydans, and the deposit address is No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0039] 3. Effects of growth conditions on strain NJ01

[0040] (1) pH value

[0041] The initial pH of TSB liquid medium was adjusted to 3, 4, 5, 6, 7, 8, 9, 10, and 11 using 1M NaOH and 1M HCl, respectively, and then filtered through a sterile membrane for sterilization. 200 μL of TSB liquid medium at different pH values ​​was added to a 96-well plate. 4 μL of activated strain NJ01 bacterial suspension was inoculated into each well, with three replicates for each pH gradient. One well served as a negative control without bacterial suspension. The 96-well plates were incubated at 32°C for 3 days, and the OD values ​​were read using a microplate reader. 600 The values ​​and results are shown in Table 1 and Figure 2 A.

[0042] Table 1 Effects of different pH values ​​on the growth of strain NJ01

[0043] <![CDATA[OD 600 ]]> 0.008 0.210 1.013 1.623 1.015 0.091 0.001 0.001 0.001 <![CDATA[OD 600 ]]> 0.006 0.211 1.179 2.112 1.314 0.171 0.001 0.001 0.001 <![CDATA[OD 600 ]]> 0.008 0.208 0.984 2.414 1.254 0.301 0.001 0.001 0.001

[0044] From Table 1 and Figure 2 As can be seen from Figure A, strain NJ01 grows better in environments with a pH of 5–7, with optimal growth at pH 6, indicating it is a microorganism suited to slightly acidic environments. Studies have shown that the lower the soil pH, the more severe the root rot, and strain NJ01's rapid and abundant reproduction in acidic environments provides the conditions for its control of root rot.

[0045] (2) Temperature

[0046] Add 200 μL of TSB liquid medium to a 96-well plate, and inoculate 4 μL of activated strain NJ01 bacterial suspension into different wells, with three replicates for each temperature. Incubate multiple 96-well plates at 28, 30, 32, 34, 36, 38, and 40 °C for 3 days, and read the OD values ​​using a microplate reader. 600 The values ​​and results are shown in Table 2 and Figure 2 B.

[0047] Table 2 Effects of different temperatures on the growth of strain NJ01

[0048] <![CDATA[OD 600 ]]> 1.095 1.152 2.851 1.952 2.103 1.754 1.608 <![CDATA[OD 600 ]]> 1.135 0.986 2.794 2.034 1.961 1.813 1.587 <![CDATA[OD 600 ]]> 1.071 1.172 2.883 2.229 2.035 1.704 1.725

[0049] From Table 2 and Figure 2 B shows that strain NJ01 grows better in environments with temperatures of 32-36℃, with the optimal growth at 32℃. Root rot is prevalent during the hot and rainy summer months, and asparagus thrives in warm and humid climates. Therefore, asparagus is prone to root rot outbreaks in summer, especially the traditional medicinal herb from Neijiang, Sichuan Province. In Neijiang, Sichuan Province, the high temperatures and excessively moist soil during summer exacerbate the root rot problem. Strain NJ01's rapid reproduction in hot and humid environments provides excellent conditions for controlling root rot.

[0050] 4. Inhibitory effect of strain NJ01 on the growth of root rot pathogens.

[0051] Common pathogens of asparagus root rot, such as *Fusarium solanum*, *Fusarium oxysporum*, and *Fusarium solani*, were cultured for 4 days and placed in 96-well plates containing 200 μL of potato dextrose agar (PDA) liquid medium. 4 μL (1 × 10⁻⁶) of activated strain NJ01 bacterial solution at different concentrations were added to each well. 4 CFU / mL, 1×10 8 CFU / mL and TSB liquid medium (as a negative control) were inoculated into different wells, with three replicates for each gradient. The 96-well plates were incubated at 32°C for 3 days, and the OD values ​​were read using a microplate reader. 600 The antibacterial rate was evaluated and the results are shown in Table 3. Figure 3 B.

[0052] Table 3. Inhibition rate of strain NJ01 against different root rot pathogens.

[0053] Strain NJ01-1 76.74％ 88.66％ 80.66％ Strain NJ01-2 70.39％ 83.15％ 74.39％ Strain NJ01-3 71.44％ 90.28％ 72.27％

[0054] From Table 3 and Figure 3 B shows that strain NJ01 has a significant growth inhibitory effect on all three root rot pathogens, indicating that strain NJ01 is expected to control asparagus root rot.

[0055] 5. Verification of the disease control effect of strain NJ01 against asparagus root rot

[0056] NJ01 bacterial culture preparation: Inoculate the bacterial strain into TSB liquid medium, incubate overnight at 37℃ and 180 rpm, centrifuge to remove the supernatant, wash twice with 1×PBS, and adjust the bacterial concentration to 1×10⁻⁶. 4 CFU / mL and 1×10 8 CFU / mL.

[0057] Preparation of pathogenic fungal spore suspension: Three pathogenic fungi (Fusarium solani, Fusarium oxysporum, and Fusarium solani) were inoculated into PDA liquid medium and cultured at 28℃ and 180 rpm for 3 days. The supernatant was discarded by centrifugation, and the spores were washed twice with 1×PBS to adjust the spore concentration to 1×10⁻⁶. 8 CFU / mL.

[0058] Healthy Asparagus tubers from Neijiang were washed with clean water, then rinsed with 75% alcohol for 2 minutes, followed by 0.8% sodium hypochlorite solution for 10 minutes, and then rinsed 7-8 times with sterile water. The sterilized tubers were then placed in petri dishes (lined with three layers of sterile filter paper and sprayed with sterile water). The tuber tissue was incised with a sterile blade. Three groups were established. Treatment A: 10 μL of pathogenic fungal spore suspension; Treatment B: 10 μL of pathogenic fungal spore suspension and 10 μL of NJ01 bacterial solution (concentration: 1×10⁻⁶). 4 Treatment group C: 10 μL of pathogenic spore suspension and 10 μL of NJ01 bacterial suspension (concentration: 1×10⁻⁶ CFU / mL); Treatment group C: 10 μL of pathogenic spore suspension and 10 μL of NJ01 bacterial suspension (concentration: 1×10⁻⁶ CFU / mL). 8 Treatment was performed using CFU / mL. Each treatment was replicated 3-5 times, and the plants were incubated at 32℃ for 5 days. Disease incidence was observed, disease index was calculated, and disease control efficiency was determined. The severity criteria for root rot were as follows:

[0059] 0 Healthy tubers 1 The area of ​​root lesions is ≤5% 2 The area of ​​root lesions is >5% and ≤25%. 3 The area of ​​root lesions is >25% and ≤50%. 4 The area of ​​root lesions is >50% and ≤75%. 5 The area of ​​root lesions is >75% and ≤100%.

[0060] Disease index = [∑(number of diseased plants × severity) / (total number of plants × highest severity)] × 100%

[0061] Disease prevention efficiency = [(Disease index of treatment group - Disease index of control group) / (1 - Disease index of control group)] × 100%

[0062] The disease severity index and disease prevention efficiency of each group are shown in Table 4 and . Figure 3 A, Figure 3 C~D.

[0063] Table 4. Statistics on the incidence of root rot in asparagus.

[0064] Process Group A 78.33％ - Processing Group B 61.23％ 33.15％ Processing Group C 38.36％ 51.03％

[0065] The results in Table 4 and the figures show that, compared to untreated asparagus roots, the root rot disease in asparagus roots treated with strain NJ01 was effectively controlled after infection with common root rot pathogens. Furthermore, the disease control efficiency increased with increasing NJ01 bacterial concentration. When the NJ01 bacterial concentration was 1×10⁻⁶... 8 At a concentration of CFU / mL, the disease control efficiency reached 51.03%, indicating that strain NJ01 has a significant effect in controlling asparagus root rot.

[0066] In summary, the *Pseudarthrobacter oxydans* NJ01 of this invention can effectively inhibit common root rot pathogens such as *Fusarium solani*, *Fusarium oxysporum*, and *Fusarium solani*. Experimental verification shows that *Pseudarthrobacter oxydans* NJ01 can effectively control asparagus root rot in Neijiang, with a disease control efficiency of 51.03%. The formulation of *Pseudarthrobacter oxydans* NJ01 into a microbial pesticide for the control of asparagus root rot has a very broad market prospect.

Claims

1. A strain of *Pseudomonas oxidans* for controlling asparagus root rot, characterized in that: It is *Pseudorobacter oxidans*, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No. 65567. Pseudarthrobacter oxydans NJ01; The *Pseudomonas oxidans* has the effect of inhibiting pathogens causing asparagus root rot; The pathogens include Fusarium solanum, Fusarium oxysporum, and Fusarium moniliforme.

2. The use of *Pseudomonas oxysporum* as described in claim 1 in the preparation of a plant protectant for the prevention and / or treatment of asparagus root rot, characterized in that: The plant protectant has the effect of inhibiting the pathogenic fungus of Fusarium asparagus root rot; The pathogenic bacteria include Fusarium solanum, Fusarium oxysporum, and Fusarium moniliforme.

3. The use as described in claim 2, characterized in that: The asparagus root rot mentioned includes the Neijiang asparagus root rot.

4. The use as described in claim 2 or 3, characterized in that: The plant protectant is a formulation made with *Pseudomonas oxidans* as the active ingredient and acceptable excipients.

5. The use according to claim 4, characterized in that: The formulations include solutions, powders, ointments, and sprays.

6. The use as described in claim 5, characterized in that: The viable count of *Pseudomonas oxysporum* in the formulation is ≥1×10⁻⁶. 8 CFU / mL.

7. A plant protectant for preventing root rot, characterized in that: It includes *Pseudomonas oxidans* as described in claim 1.

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