Application of fluorescent pseudomonas in prevention and treatment of southern root-knot nematode disease

Abstract

This invention relates to the field of bioengineering technology, and more particularly to the application of *Pseudomonas fluorescens* in the control of southern root-knot nematode disease. The application includes: the use of *Pseudomonas fluorescens*, or a bacterial agent containing *Pseudomonas fluorescens*, or a siderophore secreted by *Pseudomonas fluorescens* in the control of southern root-knot nematode disease; the *Pseudomonas fluorescens* has the preservation number CGMCC No. 16925. This invention has found that *Pseudomonas fluorescens* with preservation number CGMCC No. 16925 can secrete a siderophore that can effectively inhibit the infection and reproduction of southern root-knot nematode disease, reducing the number of root-parasitic nematodes by approximately 46% and the root-knot index by approximately 37.5%, thereby effectively controlling southern root-knot nematode disease and improving crop quality. The *Pseudomonas fluorescens* and its secreted siderophore provided by this invention have significant application value in the integrated control of southern root-knot nematodes throughout their entire growth cycle.

Description

Application of a fluorescent Pseudomonas aeruginosa in the control of southern root-knot nematode disease Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to the application of a fluorescent Pseudomonas bacterium in the prevention and control of southern root-knot nematode disease. Background Technology

[0002] Southern root-knot nematodes are a significant pathogenic organism, causing severe losses to agricultural production. They have a wide host range, parasitizing over 3,000 plant species, including monocots, dicots, herbaceous plants, and woody plants. They are particularly harmful to high-value vegetables such as those in the Solanaceae and Cucurbitaceae families; in tomatoes and cucumbers, infestation can typically result in a 30%–40% reduction in yield, or even total crop failure. Furthermore, the damage caused by southern root-knot nematodes exacerbates soil-borne diseases such as wilt, root rot, and damping-off. Therefore, southern root-knot nematode disease is a major obstacle to current greenhouse agriculture production.

[0003] Traditional methods of controlling root-knot nematodes primarily rely on chemical control. However, chemical agents disrupt the soil's microecological environment, and various nematicides are highly toxic, posing significant safety risks. Once applied to the soil, they can pollute groundwater, and some agents, when absorbed by plants, may indirectly harm humans. Furthermore, using a single nematicide may induce resistance in nematodes, while developing new agents is time-consuming and costly. Therefore, an increasing number of people are exploring integrated approaches that promote environmental sustainability to control nematode diseases.

[0004] Existing studies have found that certain microbial siderophores can antagonize soil-borne pathogens and have certain application potential in the control of soil-borne diseases. However, there are still relatively few microorganisms and siderophores used to control root-knot nematode disease in southern China. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an application of *Pseudomonas fluorescens* in the prevention and control of root-knot nematode disease in southern China.

[0006] In a first aspect, the present invention provides a fluorescent Pseudomonas aeruginosa, or a bacterial agent containing the fluorescent Pseudomonas aeruginosa, or the application of a siderophore secreted by the fluorescent Pseudomonas aeruginosa in the prevention and control of southern root-knot nematode disease.

[0007] The preservation number of the fluorescent Pseudomonas is CGMCC No. 16925.

[0008] The present invention further provides a Pseudomonas fluorescens, or an inoculum containing the Pseudomonas fluorescens, or the application of siderophores secreted by Pseudomonas fluorescens in improving the quality of bananas;

[0009] The preservation number of the fluorescent Pseudomonas is CGMCC No. 16925.

[0010] This invention has discovered that *Pseudomonas fluorescens* strain with accession number CGMCC No. 16925 can secrete a siderophore that can effectively inhibit the reproduction and infection of *Sinus chinensis*, thereby effectively preventing and controlling *Sinus chinensis* disease.

[0011] The fluorescent Pseudomonas species with accession number CGMCC No.16925 has been disclosed in Chinese Patent CN201910030984.0.

[0012] Furthermore, by inhibiting the infection and reproduction of southern root-knot nematodes, the disease can be prevented and / or the quality of bananas can be improved.

[0013] Furthermore, the quality of the banana is defined as one or more of the following: plant height, pseudostem circumference, above-ground fresh weight, root fresh weight, or percentage of fresh leaves.

[0014] Furthermore, the concentration of the siderophores secreted by the fluorescent Pseudomonas is 0.9–900 μM.

[0015] When the siderophore solution concentration is 900 μM, the hatching inhibition rate of Southern Root-knot Nematode eggs reaches 20%, the mortality rate of second-instar larvae of Southern Root-knot Nematode reaches 60%, and at this concentration, it can repel Southern Root-knot Nematode, with a tacticity index of -0.4 for Southern Root-knot Nematode.

[0016] When the siderophore solution concentration was 0.9 μM, the tropism index for *Strombus heterophylla* was -0.2.

[0017] Furthermore, the concentration of the siderophores secreted by the fluorescent Pseudomonas is 9–900 μM.

[0018] When the siderophore concentration is 9 μM, the mortality rate of second-instar larvae of *Strombus heterophylla* reaches over 15%.

[0019] Furthermore, the concentration of the siderophore secreted by the fluorescent Pseudomonas is 70–900 μM, preferably 90–900 μM.

[0020] Furthermore, the application includes applying the fluorescent Pseudomonas aeruginosa, or an agent containing the fluorescent Pseudomonas aeruginosa, or a siderophore secreted by the fluorescent Pseudomonas aeruginosa to the soil before planting crops.

[0021] Furthermore, the application to the soil includes: preparing an aqueous solution of the siderophore secreted by the fluorescent Pseudomonas bacteria and applying it to the soil.

[0022] Furthermore, the siderophore secreted by the fluorescent Pseudomonas was applied to the soil at a rate of 1.5 μmol / kg dry soil.

[0023] Furthermore, the siderophores secreted by the fluorescent Pseudomonas aeruginosa are prepared by the following method:

[0024] The fluorescent Pseudomonas was cultured in an iron-deficient medium, and the bacterial culture was then collected and subjected to adsorption, elution, evaporation, extraction, chloroform extraction, and iron carrier separation.

[0025] Furthermore, the iron-deficient culture medium is iron-deficient SSM culture medium.

[0026] Further, the adsorption includes: adsorption at 8–12°C and 160–200 rpm; and / or,

[0027] The elution includes:

[0028] In the chromatography column, elution was performed sequentially with deionized water and methanol; and / or,

[0029] The evaporation includes:

[0030] Evaporate the eluent obtained from the elution to 15-30 mL; and / or,

[0031] The extraction includes:

[0032] The liquid obtained from the evaporation was saturated with (NH4)2SO4, and then extracted at least once with RNA extraction buffer at 0–8°C and 9000–12000 rpm; and / or,

[0033] The chloroform extraction includes:

[0034] Add deionized water and chloroform to the solution obtained from the extraction, and repeat the extraction at least once at 0–8°C and 6000–10000 rpm, then evaporate to less than 20 mL; and / or,

[0035] The separation of the ferrocarrier includes:

[0036] The solution obtained from the chloroform extraction was separated using an LH20 gel column to obtain the iron carrier.

[0037] Secondly, the present invention provides a product for preventing and controlling southern root-knot nematode disease, comprising *Pseudomonas fluorescens*, or a fermentation product of said *Pseudomonas fluorescens*, or a siderophore secreted by said *Pseudomonas fluorescens*.

[0038] Furthermore, the concentration of the siderophore secreted by the fluorescent Pseudomonas in the product is 700–900 μM.

[0039] Furthermore, the product is an inhibitor.

[0040] The present invention has the following beneficial effects:

[0041] This invention has discovered that *Pseudomonas fluorescens* with accession number CGMCC No. 16925 can secrete a siderophore, which has a certain inhibitory effect on *Strombus heterophylla*. High concentrations of siderophore can kill second-instar larvae of *Strombus heterophylla* and inhibit the hatching of *Strombus heterophylla* eggs, while low concentrations of siderophore can interfere with the directional migration of second-instar larvae of *Strombus heterophylla*.

[0042] The fluorescent Pseudomonas and its secreted siderophores provided by this invention are safe and non-toxic, and will not have a negative impact on crops after application. While inhibiting southern root-knot nematodes, they can also promote crop growth to a certain extent. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0044] Figure 1 is a schematic diagram of the effect of a siderophore with a content of 1.5 μmol / kg·drysoil on banana growth under the condition of infection by southern root-knot nematodes, according to Example 2 of the present invention; wherein, the left figure is the control group inoculated only with southern root-knot nematodes, and the right figure is the treatment group that is simultaneously treated with siderophores.

[0045] Figure 2 is a schematic diagram of how the iron carrier provided in Embodiment 2 of the present invention improves the height of banana seedlings in soil affected by root-knot nematode disease in southern China.

[0046] Figure 3 is a schematic diagram of how the iron carrier provided in Embodiment 2 of the present invention improves the pseudostem circumference of banana seedlings in soil affected by southern root-knot nematode disease.

[0047] Figure 4 is a schematic diagram of how the iron carrier provided in Embodiment 2 of the present invention improves the aboveground fresh weight of banana seedlings in soil affected by southern root-knot nematode disease.

[0048] Figure 5 is a schematic diagram of how the iron carrier provided in Embodiment 2 of the present invention improves the root fresh weight of banana seedlings in soil affected by southern root-knot nematode disease.

[0049] Figure 6 is a schematic diagram of how the iron carrier provided in Embodiment 2 of the present invention increases the proportion of fresh leaves in banana seedlings in soil infested with southern root-knot nematode disease.

[0050] Figure 7 is a schematic diagram of how the iron carrier provided in Embodiment 2 of the present invention reduces the number of root-parasitic nematodes in banana seedlings.

[0051] Figure 8 is a schematic diagram of how the iron carrier provided in Embodiment 2 of the present invention reduces the root knot index of banana seedlings.

[0052] Figure 9 is a schematic diagram showing the mortality rate of different concentrations of siderophores on second-instar larvae of *Strombus heterophylla* provided in Example 3 of this invention.

[0053] Figure 10 is a schematic diagram of the interference of different concentrations of siderophores on the directional migration of second-instar larvae of *Symplocos spp.* provided in Embodiment 3 of the present invention.

[0054] Figure 11 is a schematic diagram of the ferrocarrier inhibiting the hatching of southern root-knot nematode eggs provided in Embodiment 3 of the present invention. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0056] The reagents and preparation methods involved in the embodiments of the present invention are as follows:

[0057] (1) SSM medium

[0058] Dissolve the following in 1000 mL of deionized water: 1 g (NH4)2SO4; 4 g C4H6O4; 0.2 g MgSO4. Dissolve the following in 20 mL of deionized water: 6 g K2HPO4; 3 g KH2PO4. Adjust the pH to 7.0 with potassium hydroxide solution, sterilize at 121℃ for 20 min, and then add the mixture.

[0059] (2) CAS testing solution

[0060] CAS Detection Solutions: Prepare 2 mM CAS stock solution and 1 mM FeCl3 stock solution separately. Weigh 0.0219 g CTAB and dissolve it in 25 mL of ultrapure water. Weigh 4.3079 g anhydrous piperazine and dissolve it in 30 mL of ultrapure water. Adjust the pH to 5.6 with 12 M concentrated hydrochloric acid to obtain piperazine buffer. Mix 7.5 mL of 2 mM CAS solution with 1.5 mL of 1 mM FeCl3, and slowly add it to 25 mL of CTAB solution while stirring to prevent excessive foaming. Then add 30 mL of piperazine buffer and mix well. Before use, weigh 0.0873 g 5-sulfosalicylic acid and add it to the test solution. Transfer the solution to a 100 mL volumetric flask, make up to volume, and gently shake to mix.

[0061] Unless otherwise specifically mentioned, the reagents and methods used in the examples can all be tested using conventional formulations and methods in the art.

[0062] Example 1

[0063] This embodiment provides a method for isolating and purifying fluorescent Pseudomonas siderophores, as detailed below:

[0064] 1. The strain used in this embodiment is *Pseudomonas* sp. 1502 IPR-01 (abbreviated as IP1), which is deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode 100101), classified as *Pseudomonas fluorescens*, with accession number CGMCC NO. 16925. This strain is disclosed in Chinese Patent CN201910030984.0.

[0065] 2. This invention has discovered that this strain can produce microbial siderophores. The specific steps for isolating and purifying siderophores from bacterial culture IP1 are as follows:

[0066] (1) The test strain IP2 was activated to maintain a consistent growth state;

[0067] (2) The test strains with the same state were inoculated into iron-deficient SSM medium for inducing iron production. After inoculation, the strains were cultured in a shaker at 28°C and 180 rpm.

[0068] (3) After culturing for 48 h, the supernatant was collected by centrifugation at 4℃ and 10000 rpm for 25 min.

[0069] (4) Adsorption of macroporous resin XAD4: After adding the supernatant to macroporous resin XAD4, place it on a shaker and shake for 3 hours at 10°C and 180 rpm.

[0070] (5) Elution: Pour macroporous resin XAD4 into the chromatography column, add deionized water to elute, and after the deionized water has been eluted, add 50% methanol to elute. Collect the eluted liquid in a centrifuge tube, and use CAS to detect the iron carrier yield. Collect the liquid in the centrifuge tube that detected the iron carrier.

[0071] (6) Rotary evaporation: The liquid collected in (5) is rotary evaporated to 20 mL;

[0072] (7) Liquid-liquid extraction: Add small amounts of saturated (NH4)2SO4 to the rotary evaporated liquid multiple times until it is just saturated. Add RNA extraction solution and centrifuge at 4℃ and 10000rpm for 5min. Take the lower layer solution and place it in a new centrifuge tube. Add RNA extraction solution to the upper layer solution and mix the newly precipitated lower layer solution with the previously collected lower layer solution. Repeat 2-3 times.

[0073] (8) Add deionized water and chloroform to the lower layer solution, centrifuge at 4℃ and 8000 rpm for 5 min, collect the upper layer and place it in a new centrifuge tube, discarding the lower layer solution. Add 50 mL of chloroform to the upper layer solution, centrifuge at 4℃ and 8000 rpm for 5 min, collect the upper layer solution and place it in a new centrifuge tube, discarding the lower layer solution. Repeat this step 2-3 times. Add twice the volume of diethyl ether to the upper layer solution, centrifuge at 4℃ and 8000 rpm for 5 min, collect the lower layer solution and place it in a new centrifuge tube. Repeat this step 2-3 times. Rotary evaporate the lower layer solution to less than 20 mL.

[0074] (9) Pass the liquid from (8) through an LH20 gel column, one drop every 30 seconds, in 15 mL tubes. Collect the five tubes with the highest iron carrier concentration using CAS detection solution, and freeze-dry them for later use. The freeze-dried yellow-brown powder is then reconstituted with deionized water to obtain the iron carrier solution.

[0075] Example 2

[0076] This embodiment demonstrates the effect of fluorescent Pseudomonas siderophores on banana growth, specifically using potted banana plants. The experimental procedure is as follows:

[0077] 1. Test materials:

[0078] The tested crop, Banana Williams B6 (Musa AAA Cavendish CV. Williams), was obtained from tissue culture seedlings from Guangxi Xiangfeng Seed Industry Co., Ltd.

[0079] The southern root-knot nematode was obtained from diseased water spinach grown in the greenhouse of the College of Resources and Environment, China Agricultural University.

[0080] Ferric carrier: purified by column chromatography, with the specific preparation method being the same as in Example 1.

[0081] 2. Test methods:

[0082] (1) Banana cultivation: Transplant banana tissue culture seedlings to seedling trays and cultivate them on coconut coir for 60 days, watering them with Garden's culture solution once a week, and watering them appropriately during the period;

[0083] (2) When the banana seedlings have grown to 4 unfolded leaves, transplant them. Each pot contains 3 kg of soil. After the seedlings have recovered for 1 week, apply 5 ml of 900 μM iron carrier solution to each pot. Use an equal amount of sterile water as a control. Set up 5 replicates for each treatment. Apply once a week.

[0084] (3) Nematode inoculation: Two weeks after transplanting banana seedlings, make four small holes about 2 cm deep within 1 cm-2 cm from the base of the banana stem and water them appropriately. Inoculate each pot with about 3,000 second-instar larvae of southern root-knot nematodes and collect samples two months later.

[0085] 3. Test Results:

[0086] The experimental results are shown in Figures 1-8: The application of iron carriers to soils infected with nematodes significantly improved banana growth indicators (Figures 1-5) and significantly reduced banana disease indicators (Figures 6-8). When the iron carrier concentration was 900 μM and the application rate was 1.5 μmol / kg dry soil, it not only ensured the normal growth and development of banana plants, but also inhibited the infection and reproduction of southern root-knot nematodes. The number of root-parasitic nematodes decreased by about 46% (Figure 7), and the root-knot index decreased by about 37.5% (Figure 8).

[0087] Example 3

[0088] This embodiment further verifies the inhibitory effect of the siderophore solution on southern root-knot nematodes using an indoor simulation experiment, as follows:

[0089] 1. Test materials:

[0090] The methods for obtaining the tested Southern root-knot nematodes and siderophores were the same as in Example 2.

[0091] 2. Test methods:

[0092] (1) Using sterile water as a control, four concentration gradients of 900 μM iron carrier aqueous solution were set up: undiluted, diluted 10 times, 100 times, and 1000 times. Each concentration gradient treatment was set up in 6 replicates.

[0093] (2) Mix 1 ml of the above-mentioned siderophore aqueous solution of different concentrations with 1 ml of suspension of second instar nematode larvae (about 100 nematodes) and determine the effect of different concentrations of siderophore aqueous solution on the mortality rate of second instar larvae of Southern Root-knot Nematode.

[0094] (3) Mix 1 ml of the above-mentioned iron carrier aqueous solution of different concentrations with 3 Southern root-knot nematode egg masses of similar size. Every 24 hours, count the number of second-instar larvae of Southern root-knot nematode hatched in the centrifuge tube under a microscope, and calculate the relative inhibition rate of different concentrations of functional substances on the hatching of Southern root-knot nematode eggs at different times.

[0095] (4) Inject 20 μl of second-instar larval suspension of Southern root-knot nematode into the center of the agar plate. Inject different concentrations of siderophore aqueous solution and sterile water into the left and right sides of the agar plate 1 cm away from the center. Calculate the tactic index of Southern root-knot nematode based on the migration ratio of nematodes to the left and right sides of the agar plate.

[0096] 3. Test Results:

[0097] The experimental results are shown in Figures 9-11. When the concentration of the Pseudomonas siderophore solution was 900 μM, the hatching inhibition rate of Southern Root-Knot Nematode eggs reached 20% (Figure 11), and the mortality rate of second-instar larvae of Southern Root-Knot Nematode reached 60% (Figure 9). Moreover, at this concentration, it could repel Southern Root-Knot Nematode, and the tacticity index for Southern Root-Knot Nematode was -0.4 (Figure 10). When the concentration of the siderophore solution was 0.9 μM, the tacticity index for Southern Root-Knot Nematode was -0.2 (Figure 10).

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a siderophore secreted by *Pseudomonas fluorescens* in the control of southern root-knot nematode disease; the preservation number of *Pseudomonas fluorescens* is CGMCC No. 16925; the concentration of the siderophore secreted by *Pseudomonas fluorescens* is 0.9~900 μM.

2. The application according to claim 1, characterized in that, The application includes applying siderophores secreted by the fluorescent Pseudomonas bacteria to the soil before planting crops.

3. The application according to claim 1 or 2, characterized in that, The siderophores secreted by *Pseudomonas fluorescens* are prepared by the following method: *Pseudomonas fluorescens* is cultured in an iron-deficient medium, and then the bacterial solution is collected and subjected to adsorption, elution, evaporation, extraction, chloroform extraction, and siderophore separation; the extraction includes: adding (NH4)2SO4 to the evaporated liquid until saturated, and then extracting at least once with RNA extraction buffer at 0-8°C and 9000-12000 rpm; and / or, the chloroform extraction includes: adding deionized water and chloroform to the extracted solution, centrifuging at 0-8°C and 6000-10000 rpm, taking the upper layer solution and placing it in a new centrifuge tube, discarding the lower layer solution, repeating the extraction at least once, and then evaporating to less than 20 mL.

4. The application according to claim 3, characterized in that, The adsorption includes adsorption at 8-12°C and 160-200 rpm; and / or, the elution includes eluting with deionized water and methanol sequentially in a chromatography column; and / or, the evaporation includes evaporating the eluent obtained from the elution to 15-30 mL; and / or, the iron support separation includes separating the iron support from the solution obtained from the chloroform extraction using an LH20 gel column.

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