Application of protozoan Nephridia nephridia NJAU-Z2 in promoting banana growth and preventing and controlling Fusarium wilt disease

Abstract

The present invention provides the use of the protozoan reniformis NJAU‑Z2 in promoting banana growth and preventing and controlling wilt disease. The use of the protozoan reniformis NJAU‑Z2 with a preservation number of CCTCC NO: C2024242 in increasing the content of ammonium nitrogen in the soil, promoting banana growth and preventing and controlling banana wilt disease. Through pot experiments, it was found that the reniformis NJAU‑Z2 involved in the present invention can increase the content of ammonium nitrogen in the soil, and significantly promote the growth of banana plants, and increase the biomass of banana plants. Indoor experiments found that the reniformis NJAU‑Z2 involved in the present invention can directly inhibit the growth of the pathogen Fusarium oxysporum of banana wilt disease, and pot experiments confirmed that the reniformis NJAU‑Z2 has a good prevention and control effect on banana wilt disease. The development and application of the beneficial protozoa of the present invention is an important agricultural measure to achieve efficient prevention and control of soil-borne diseases and green and sustainable development of agriculture.

Description

Technical Field

[0001] The invention belongs to the technical field of protozoology and plant disease biocontrol, and particularly relates to application of protozoan reniformis NJAU-Z2 in promoting banana growth and preventing and controlling wilt disease. Background Art

[0002] Banana (Musa nana Lour.) is the second largest fruit after citrus, accounting for 16% of the world's total fruit production. In recent years, in addition to the impact of growing climate, market regulation and natural disasters, banana wilt (Fusarium WiltDisease of Banana) is the main factor limiting the further growth of banana production.

[0003] Banana wilt is a vascular wilt disease caused by fungi. The disease is caused by the fungus Fusarium oxysporum f. sp. cubense (Foc). The pathogen usually invades plants through the roots, grows in the roots and vascular tissues of plants, blocks the supply of water and nutrients, and then releases enzymes and plant toxins. Fusaric acid (FA) is a plant toxin produced by Fusarium oxysporum that can cause plant chloroplast damage, reduce the photochemical efficiency of photosystem II, and lead to reduced photosynthesis. After the plant is attacked by Fusarium oxysporum, the most obvious external symptom is that the old leaves turn yellow, wilt, die and gradually separate and split. These leaves bend on the pseudostem and cause the plant to collapse; due to the obstruction of the internal vascular system of the plant, the xylem of the rhizome gradually changes from brown to reddish brown and finally to black, and the root system is seriously affected, eventually leading to root rot and plant death. Fusarium oxysporum is mainly transmitted through the soil. The pathogen survives in the soil for a long time in the form of dormant spores and invades plants through the roots under suitable conditions. Transmission through surface water is the cause of localized large-scale outbreaks of banana wilt. Irrigation causes the pathogen to spread rapidly along river basins, accelerating the spread of the disease.

[0004] So far, there are many methods for the prevention and control of banana wilt, but few effective measures. The current mainstream prevention and control measure is the breeding of disease-resistant varieties, but the cultivation cycle of disease-resistant varieties is long, and Fusarium oxysporum is highly variable, and mutants make the plant's disease resistance ineffective. Chemical disinfection and soilless cultivation are too expensive to maintain, and the economic burden is large, especially chemical disinfection has potential risks to the environment and human health. Crop rotation can reduce the density of pathogens in the soil to a certain extent, but the long-term survival ability greatly increases the chance of Fusarium oxysporum re-infecting banana plants after crop rotation. The tenacious survival ability, variability and diverse transmission ability of Fusarium oxysporum increase the challenges of preventing and controlling banana wilt.

[0005] Protozoa are an important class of single-cell eukaryotic microorganisms that are widely found in soil, water and other environments. They prey on bacteria, fungi and other microorganisms in the ecosystem, promote nutrient cycling and play a role in regulating microbial communities, thereby better maintaining soil health and promoting plant growth. For the prevention and control of banana wilt, protozoa can prey on fungal spores in the soil, such as the spores of Fusarium oxysporum, thereby reducing the number of pathogens; at the same time, the predation of protozoa helps beneficial bacteria release metabolites to inhibit the reproduction of Fusarium oxysporum in order to compete for living space. As a type of biological control, protozoan control is environmentally friendly and does not cause pollution. It can establish a long-term and stable ecological community in the soil, regulate the balance of soil microorganisms, reduce the risk of pathogen resistance, and reduce the cost of long-term wilt control.

[0006] The applicant previously screened a protozoan reniformis NJAU-Z2 (202411328795.9) that can prevent and control strawberry wilt. However, the physiological species of Fusarium oxysporum that causes banana wilt is Fusarium oxysporumf. sp. cubense, while the physiological species of Fusarium oxysporum that causes strawberry wilt is Fusarium oxysporum f.sp. fragariae. Different physiological species have different drug resistance. Therefore, without research, it is impossible to predict whether the protozoan reniformis NJAU-Z2 can also be used to prevent and control banana wilt, and it is even more impossible to predict whether it has a growth-promoting effect on bananas. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the present invention provides the use of protozoan Nephridia NJAU-Z2 in promoting banana growth and preventing and controlling wilt disease.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] Application of protozoan Colpoda steinii NJAU-Z2 in increasing soil ammonium nitrogen content, promoting banana growth and / or controlling banana wilt. Protozoan Colpoda steinii NJAU-Z2 has been disclosed in 202411328795.9.

[0010] Application of a culture of protozoan Colpoda steinii NJAU-Z2 in increasing soil ammonium nitrogen content, promoting banana growth and / or controlling banana wilt disease.

[0011] As a preferred embodiment of the present invention, the culture of the protozoan Renilla NJAU-Z2 is prepared by the following preparation method: inoculating the protozoan Renilla NJAU-Z2 into PAS liquid culture medium, adding inactivated Escherichia coli DH5α, placing in an incubator at 20° C., and standing for 48 hours.

[0012] The culture of the present invention is used in preparing products for increasing the ammonium nitrogen content in soil, promoting banana growth and / or preventing and treating banana wilt.

[0013] As a preferred embodiment of the present invention, the product is a biological pesticide or a biological fertilizer.

[0014] As a further preferred embodiment of the present invention, the culture is used in the preparation of a biological fertilizer for increasing the ammonium nitrogen content in the soil and promoting the growth of bananas.

[0015] As a further preferred embodiment of the present invention, the culture is used in the preparation of biological pesticides for preventing and treating banana wilt.

[0016] The protozoan reniform insect Colpoda steinii NJAU-Z2 involved in the present invention is a reniform insect isolated and screened from the rhizosphere soil of healthy crop plants in the experimental field of the Sanya Research Institute of Nanjing Agricultural University, which has a significant predatory effect on Fusarium oxysporum and can increase the ammonium nitrogen content to promote banana growth. According to the cyst and living body morphological characteristics of the protozoan reniform insect NJAU-Z2 and the 18S rDNA gene sequence analysis, it was identified as Colpodasteinii, and it was preserved in the China Type Culture Collection Center in 2024 (address: Wuhan University Collection Center, Wuchang District, Wuhan City, Hubei Province, with a preservation number of CCTCC NO: C2024242). The present invention studies the growth-promoting effect of the reniform insect on bananas and the control effect on banana wilt, providing a scientific basis for the biological control of banana wilt and the development and utilization of biocontrol agents.

[0017] Beneficial effects: The present invention isolates and screens the protozoan Colpoda steinii from the rhizosphere of healthy crops, which can increase the ammonium nitrogen content in the soil, promote the growth of bananas, and has a significant inhibitory ability on the pathogenic bacteria of banana wilt. Potted plant experiments show that the Colpoda steinii NJAU-Z2 has a growth-promoting effect on bananas and can increase the ammonium nitrogen content in the soil. Fungal pathogen co-cultivation experiments show that the Colpoda steinii NJAU-Z2 has a significant preventive and therapeutic effect on banana wilt. Therefore, the Colpoda steinii of the present invention has a growth-promoting effect on bananas, and is of great significance for the restoration of continuously cropped soil and the prevention and control of soil-borne diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0019] Figure 1 In Example 1 of the present invention, the control group CK ( Figure 1 Left) and the treatment group inoculated with NJAU-Z2 Colpoda_steinii ( Figure 1 (right) The growth conditions of banana plants.

[0020] Figure 2 The ammonium nitrogen content in the soil of the control group CK and the treatment group inoculated with the Nephrolepis spp. NJAU-Z2 ( Figure 2 Left) and banana biomass ( Figure 2 Right) Difference histogram.

[0021] Figure 3 The 96-well plate liquid system co-culture of Renilla NJAU-Z2 and Fusarium oxysporum f. sp. cubense in Example 2 of the present invention, before adding Renilla NJAU-Z2 ( Figure 3 Left) and 12h after adding Nephridia NJAU-Z2 ( Figure 3 Right) Growth of Fusarium oxysporum f. sp. cubense spores in a 96-well plate.

[0022] Figure 4 This is a box plot characterizing the difference in the number of Colpoda steinii NJAU-Z2 in the experimental group and the experimental group Foc + Colpoda steiniiNJAU-Z2 after co-culture in the 96-well plate liquid system for 12 hours.

[0023] Figure 5 This is a box plot representing the difference in the number of Fusarium oxysporum f. sp. cubense spores in the experimental group Foc and the experimental group Foc + Colpoda steinii NJAU-Z2 after co-cultivation in a 96-well plate liquid system for 12 hours.

[0024] Figure 6 For the control group Foc (in Example 3 of the present invention, inoculated with the pathogenic fungus Fusarium oxysporum f. sp. cubense Figure 6Left) and the treatment group F + P ( Figure 6 (right) The growth conditions of banana plants. DETAILED DESCRIPTION

[0025] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The experiments in the following examples were repeated three times.

[0026] Example 1 Potted experiment on promoting banana growth by Colpoda steinii NJAU-Z2:

[0027] Two treatments were set up in the experiment: (1) the control group CK inoculated with PAS solution without reniformis; (2) the treatment group Colpoda_steinii inoculated with reniformis Colpoda steinii NJAU-Z2. Each treatment had 6 replicates.

[0028] The banana used for the test was Musa brasiliensis of the Cavendish family, and the protozoa used for the test was Nephrolepis NJAU-Z2, with the deposit number: CCTCC NO: C2024242.

[0029] Preparation of NJAU-Z2: Inoculate NJAU-Z2 into liquid PAS medium, add inactivated E. coli (OD = 0.04), place in a constant temperature incubator at 20°C, and culture. After 48 hours, take 100 μL of the culture medium and place it under an inverted microscope for counting.

[0030] The preparation method of the PAS culture medium is as follows: Solution 1 and Solution 2 are prepared, wherein Solution 1 comprises 0.142 g disodium hydrogen phosphate and 0.136 g potassium dihydrogen phosphate, and water is added to make the volume to 500 mL; Solution 2 comprises 0.12 g sodium chloride, 0.4 mg magnesium sulfate heptahydrate, and 0.6 mg calcium chloride hexahydrate, and water is added to make the volume to 500 mL. Solution 1 and Solution 2 are sterilized at 121° C. for 20 min, and after cooling to room temperature, Solution 1 and Solution 2 are mixed in a ratio of 1:1.

[0031] Cultivation of banana plants: The test bananas were from the Cavendish family of the Hainan farm. The soil for the potted experiment was taken from a banana garden in Chengmai County, Hainan Province, China, and was identified as healthy soil that did not contain pathogens. The potted experiment was carried out in a greenhouse at the Sanya Research Institute of Nanjing Agricultural University. The greenhouse had 16 hours of daylight during the day with an average temperature of 30°C, 8 hours of no light at night, an average temperature of 26°C, and an overall average humidity of 75%. Each replicate in the two treatments set up consisted of a sterile banana plant seedling in a polypropylene pot containing 500 grams of dry soil and hydrated with sterile water. The soil in the Colpoda_steinii pot of the treatment group was inoculated with the culture solution of Nephrolepis NJAU-Z2 at a concentration of 10 3 The control group (CK) was inoculated with the same volume of sterilized PAS solution as the treatment group per gram of soil. The growth of banana potted plants was observed after 6 weeks of continuous cultivation in the greenhouse.

[0032] Figure 1 The control group CK without inoculation of Renilla Figure 1 Left) and the treatment group inoculated with NJAU-Z2 Colpoda_steinii ( Figure 1 (right) The growth conditions of banana plants.

[0033] Figure 2 Soil ammonium nitrogen content in the control group CK without inoculation of Renilla and the treatment group inoculated with Renilla NJAU-Z2 ( Figure 2 Left) and banana biomass ( Figure 2 right).

[0034] The results of potted plant experiments showed that the Nephridia nematode NJAU-Z2 could significantly promote the growth of banana plants and increase the content of ammonium nitrogen in the soil and the biomass of banana plants.

[0035] Example 2 Liquid co-culture experiment of protozoan NJAU-Z2 inhibiting banana oxysporum fungus:

[0036] The tested banana fusarium oxysporum f. sp. cubense, and the tested protozoa were the protozoa Nephrolepis NJAU-Z2 in Example 2, with the deposit number: CCTCC NO: C2024242.

[0037] Preparation of NJAU-Z2: Inoculate NJAU-Z2 into liquid PAS medium, add inactivated E. coli (OD = 0.04), place in a constant temperature incubator at 20°C, and culture. After 48 hours, take 100 μL of the culture medium and count it under an inverted microscope, and dilute the number of NJAU-Z2 to 7×10 3 Cells / mL.

[0038] Preparation of banana wilt pathogen (Fusarium oxysporum f. sp. cubense) suspension: The preserved banana wilt pathogen Fusarium oxysporum was inoculated into liquid PDB (Potato Dextrose Broth) medium and placed in a constant temperature shaker for shaking culture. After 3 days, the culture medium was repeatedly washed three times with sterile water and filtered through a sterilized four-layer gauze to obtain Fusarium oxysporum spore liquid. 10 μL of Fusarium oxysporum spore liquid was drawn into a hemocytometer and placed under an upright microscope for counting. The number of Fusarium oxysporum spore liquid was diluted to 5×10 5 CFU / mL.

[0039] Set up one experimental group (Foc): 100 μL with a concentration of 5×10 5 CFU / mL of Fusariumoxysporum f. sp. cubense spore solution + sterile 100 μL PAS buffer, add 200 μL of the liquid to a 96-well plate, with a total of 6 replicates.

[0040] Set up one experimental group (Colpoda steinii NJAU-Z2): 100 μL of concentration of 7×10 3 Cells / mL of protozoa + sterile 100 μL PAS buffer, add 200 μL of the liquid to a 96-well plate, with a total of 6 replicates.

[0041] Set up one experimental group (Foc + Colpoda steinii NJAU-Z2): 100 μL of 5 × 10 5 CFU / mL of Fusarium oxysporum f. sp. cubense + 100μL with a concentration of 7×10 3 Cells / mL of Nephridia NJAU-Z2, 200 μL of the liquid was added to a 96-well plate, with a total of 6 replicates.

[0042] The 96-well plates of the above three experimental groups were placed in a protozoan incubator at 20°C to observe the growth of protozoa and fungi.

[0043] Figure 3 For the co-culture of Renilla NJAU-Z2 and Fusarium oxysporum f. sp. cubense in a 96-well plate liquid system, before adding Renilla NJAU-Z2 ( Figure 3 Left) and 12h after adding Nephridia NJAU-Z2 ( Figure 3Right) Growth of Fusarium oxysporum f. sp. cubense spores in a 96-well plate.

[0044] Figure 4 This is a box plot characterizing the difference in the number of Colpoda steinii NJAU-Z2 in the experimental group and the experimental group Foc + Colpoda steiniiNJAU-Z2 after co-culture in the 96-well plate liquid system for 12 hours.

[0045] Figure 5 Box plot representing the difference in the number of Fusarium oxysporum f. sp. cubense spores between the experimental group Foc and the experimental group Foc + Colpoda steinii NJAU-Z2 after 12 h of co-cultivation in a 96-well plate liquid system.

[0046] It can be seen from the above that the protozoan NJAU-Z2 can inhibit the proliferation of Fusarium oxysporum by preying on the spores of the banana wilt pathogen Fusarium oxysporum f. sp. cubense. The reniform insect Colpoda steinii NJAU-Z2 showed an inhibitory effect on the banana wilt pathogen Fusarium oxysporum f. sp. cubense.

[0047] Example 3 Potted plant test of Colpoda steinii NJAU-Z2 to inhibit banana wilt disease:

[0048] Two treatments were set up in the experiment: (1) the control group Foc inoculated with Fusarium oxysporum f. sp. cubense; (2) the treatment group F (Foc) + P (Protist) inoculated with the pathogenic fungus Fusarium oxysporum f. sp. cubense and Colpoda steiniiNJAU-Z2. Each treatment had 4 replicates.

[0049] The banana used for the test was the Cavendish banana, and the protozoan used for the test was the Nephridia NJAU-Z2 in Example 2, with a deposit number of CCTCC NO: C2024242.

[0050] Preparation of NJAU-Z2: Inoculate NJAU-Z2 into liquid PAS medium, add inactivated E. coli (OD = 0.04), place in a constant temperature incubator at 20°C, and culture. After 48 hours, take 100 μL of the culture medium and place it under an inverted microscope for counting.

[0051] Preparation of banana wilt pathogen (Fusarium oxysporum f. sp. cubense) bacterial suspension: The preserved banana wilt pathogen Fusarium oxysporum was inoculated into liquid PDB medium and placed in a constant temperature shaker for shaking culture. After 3 days, the culture solution was repeatedly washed three times with sterile water and filtered through a sterilized four-layer gauze to obtain the spore solution of Fusarium oxysporum. 10 μL of the spore solution of Fusarium oxysporum was drawn into a hemocytometer and placed under an upright microscope for counting.

[0052] Cultivation of banana plants: The test bananas were from the Cavendish family of the Hainan Farm. The soil for the pot experiment was taken from a banana orchard in Chengmai County, Hainan Province, China, and was identified as healthy soil without pathogens. The pot experiment was carried out in a greenhouse at the Sanya Research Institute of Nanjing Agricultural University. The greenhouse had 16 hours of light during the day, an average temperature of 30°C, and no light for 8 hours at night, an average temperature of 26°C, and an overall average humidity of 75%. Each replicate in the two treatments set up consisted of a sterile banana plant seedling in a polypropylene pot containing 500 grams of dry soil and hydrated with sterile water. The soil in the control group (Foc) flower pot was inoculated with spores of the pathogenic fungus Fusarium oxysporum f. sp. cubense at a concentration of 10 3 per gram of soil; the soil in the flowerpots of the treatment group (F + P) was inoculated with spores of the pathogenic fungus Fusarium oxysporum f. sp. cubense at a concentration of 10 3 Each gram of soil was inoculated with the culture solution of Renilla NJAU-Z2 at a concentration of 10 3 After 6 weeks of continuous cultivation in the greenhouse, the growth of banana potted plants was observed.

[0053] Figure 6 The control group Foc ( Figure 6 Left) and the treatment group inoculated with pathogenic fungus Fusarium oxysporum f. sp. cubense and Renilla NJAU-Z2 ( Figure 6 (right) The growth conditions of banana plants.

[0054] The results of potted experiments showed that the Nephrolepis fasciatus NJAU-Z2 could significantly inhibit the occurrence of banana wilt in complex soil systems.

Claims

1. Protozoan Nephroderma NJAU-Z2 ( Colpoda steinii NJAU-Z2 ) is used in increasing the biomass of banana plants. The protozoan Nephridia NJAU-Z2 is deposited in the China Center for Type Culture Collection with the deposit number CCTCCNO: C2024242.

2. Protozoan Nephridia NJAU-Z2 ( Colpoda steinii The protozoan reniformis NJAU-Z2 is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024242. The protozoan reniformis NJAU-Z2 is prepared by the following preparation method: the protozoan reniformis NJAU-Z2 is inoculated into a PAS liquid culture medium, and inactivated Escherichia coli is added. Escherichia coli DH5α, placed in an incubator at 20°C and allowed to stand for 40-50h; the preparation method of the PAS liquid culture medium is as follows: prepare solution 1 and solution 2, solution 1 comprises 0.142 g disodium hydrogen phosphate and 0.136 g potassium dihydrogen phosphate, and water is added to make the volume to 500 mL; solution 2 comprises 0.12 g sodium chloride, 0.4 mg magnesium sulfate heptahydrate, and 0.6 mg calcium chloride hexahydrate, and water is added to make the volume to 500 mL, solution 1 and solution 2 are sterilized at 121°C for 20 min, and after cooling to room temperature, solution 1 and solution 2 are mixed in a ratio of 1:

1.

3. Use of the culture described in claim 2 in preparing a product for increasing the biomass of banana plants.

4. The use according to claim 3, characterized in that: The product is a biological pesticide or a biological fertilizer.

5. The use according to claim 3, characterized in that: The culture is used in preparing biological fertilizer for increasing the biomass of banana plants.

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