A strain Cd2 and its application

Abstract

The present invention belongs to the technical field of microbiology, and specifically relates to a strain Cd2 and an application thereof. A strain Cd2, the taxonomic name of the strain Cd2 is Pseudomonas aeruginosa, and the deposit number is GDMCC No.64110. The present invention identifies the screened strains by morphological observation, Gram staining and 16S rRNA sequence sequencing, and determines the inhibitory effect of the strains on the crucifer black rot fungus and the fruit cane mildew fungus Pseudomonas spores, thereby obtaining a strain Cd2, which has a strong inhibitory effect on the crucifer black rot fungus and the fruit cane mildew fungus Pseudomonas spores, and brings broad application prospects to the field of biological control of plant diseases caused by the crucifer black rot fungus and the fruit cane mildew disease.

Description

Technical Field

[0001] The invention belongs to the technical field of microbiology, and specifically relates to a strain Cd2 and an application thereof. Background Art

[0002] Crucifer black rot (Xanthomonas campestris pv. Campestris, Xcc) is a pathogen that causes black rot in cruciferous plants (including many important economic crops, such as cabbage, cauliflower, kale, mustard, radish and turnip, etc.). Cruciferous plants are an important type of food crop, with a large planting area and market share. They are the main component of the human table and have huge economic value. Cruciferous black rot invades plants through water holes or wounds, proliferates in the apoplast space of plant cells and extends along the vascular bundles. When the lesions become larger and larger, the leaves will turn black, and the shape of the lesions will become V-shaped, so it is called black rot. Black rot has a strong infectious ability and has a very serious impact on the growth and development of cruciferous plants growing in tropical and subtropical areas.

[0003] In the past, the strategy for the prevention and control of black rot should be based on agricultural prevention and control, using disease-free seeds, supplemented by comprehensive measures of chemical prevention and control. For example, timely spraying of 72% agricultural streptomycin sulfate, new phytocin, 50% DT, 60% chlorothiazide, 10% high-efficiency fungicide, 14% copper chloramine water solution, etc. at the early stage of the disease. However, the long-term use of chemical agents will induce pathogens to develop drug resistance, and the drug residues of chemical synthetic fungicides have potential great harm to human and environmental safety. Therefore, biological control has gradually replaced the use of chemical fungicides and has become the main way to control plant diseases. Among them, the application of antagonistic microorganisms in biological control has become a research hotspot.

[0004] Sugarcane (also known as fresh sugarcane) is rich in nutrition, rich in easily absorbed sugars such as glucose, sucrose and fructose, as well as various vitamins, amino acids and trace elements necessary for the human body, especially the iron content is as high as 9mg / kg, so it is also called blood-tonifying fruit. Sugarcane is deeply loved by consumers because of its sweet taste, rich nutrition and the effect of nourishing the body. At present, the sugarcane planting area in my country has expanded from the eight provinces (regions) in the south of the Yangtze River to 22 provinces (cities and regions). The total area of ​​sugarcane fields in the country is about 270,000 hectares, the annual total output exceeds 20 million tons, and the output value exceeds 20 billion yuan. my country's sugarcane planting is mainly concentrated in southern regions such as Guangxi, Guangdong, Fujian, Yunnan, Hainan, Sichuan, Jiangxi, etc. The sugarcane planting area in Guangxi alone exceeds 27,000 hectares, accounting for about 10% of the national planting area, ranking first in the country. Every year, the concentrated harvesting season of sugarcane is from around New Year's Day to around the Spring Festival. A large amount of sugarcane is transported to the consumer market in northern my country. However, sugarcane is very susceptible to infection by pathogens during transportation and storage, deteriorates, and produces microbial toxins. Since the first case of poisoning caused by spoiled sugarcane occurred in my country in 1972, there have been cases of poisoning caused by eating spoiled sugarcane almost every year. Fungi can be isolated from moldy sugarcane and identified as Arthrinium arundinis. The metabolites of Arthrinium arundinis contain 3-nitropropionic acid, which is a neurotoxin that mainly damages the central nervous system. Studies have confirmed that ingestion of spoiled sugarcane containing high concentrations of 3-NPA can lead to severe food poisoning, which is generally manifested clinically as poisoning symptoms 2-5 hours after eating spoiled sugarcane. Mild cases are accompanied by nausea, vomiting, abdominal pain, diarrhea and other digestive tract symptoms. Some patients experience syncope, blurred vision, and inability to stand. Severe cases have trembling hands and feet, paroxysmal tonic convulsions, and even coma. There is currently no effective treatment. Therefore, how to prevent sugarcane from mildewing during storage or transportation is a scientific problem that needs to be solved urgently.

[0005] In the past, chemical synthetic fungicides were usually used as the main way to preserve fruits after harvest. However, long-term use of chemical synthetic fungicides can induce pathogens to develop drug resistance, and the drug residues of chemical synthetic fungicides have a potential great hazard to human and environmental safety. Therefore, biological control has gradually replaced the use of chemical fungicides and has become the main way to control post-harvest diseases of fruits and vegetables. Among them, the application of antagonistic microorganisms in biological control has become a research hotspot. At present, antagonistic microorganisms that can inhibit post-harvest diseases of fruits and vegetables include Bacillus, Pseudomonas, Saccharomyces and fungi.

[0006] Pseudomonas sp., as a type of rhizosphere growth-promoting bacteria, is widely used in biological control of plant diseases. The mechanisms of this type of microorganisms in controlling plant diseases mainly include: producing a variety of antibiotics, effective rhizosphere colonization and competition for rhizosphere nutrients, degrading microbial enzymes and inducing systemic resistance, so that it can be developed into a series of biological control products such as live microbial preparations. In recent years, a series of rhizosphere-derived Pseudomonas aeruginosa with good biological control effects have been isolated and identified. These Pseudomonas aeruginosa can control a variety of fungal or bacterial diseases and are called Pseudomonas aeruginosa biocontrol strains.

[0007] Strain Cd2 is a Pseudomonas aeruginosa isolated from rhizosphere soil. It was cultured on plates opposite to Crucifer black rot and Pseudomonas aeruginosa, and was inoculated simultaneously with Crucifer black rot and strain Cd2 by the leaf cutting method. It was found that strain Cd2 had a strong inhibitory effect on Crucifer black rot and cane mildew pathogen Pseudomonas aeruginosa, which can provide technical support for the development of prevention and control of plant diseases caused by Crucifer black rot and the development of new fruit and vegetable preservatives. No related reports have been seen so far.

[0008] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0009] The purpose of the present invention is to provide a strain Cd2 and application thereof.

[0010] The first object of the present invention is to provide a strain Cd2, which was collected and separated from the mineral soil in Hechi, Guangxi Zhuang Autonomous Region, and was identified as Pseudomonas aeruginosa by morphology and molecular biology. It was preserved in the Guangdong Provincial Microbiological Culture Collection Center on December 5, 2023, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation number is: GDMCC No.64110.

[0011] The second object of the present invention is to provide the use of the strain Cd2 in inhibiting the black rot fungus of the Cruciferae family and inhibiting the hyphae growth of Pseudomonas pyrifos.

[0012] The third object of the present invention is to provide the use of the strain Cd2 in preventing and controlling plant diseases caused by crucifer black rot fungi and preventing cane mildew.

[0013] Compared with the prior art, the present invention has the following beneficial technical effects:

[0014] The present invention identifies the screened strains through morphological observation, Gram staining and 16S rRNA sequence sequencing, and determines the inhibitory effects of the strains on the crucifer black rot fungus and the cane mildew fungus Pseudocerus pyri spore, thereby obtaining a strain Cd2, which has a strong inhibitory effect on the crucifer black rot fungus and the cane mildew fungus Pseudocerus pyri spore, and brings broad application prospects to the field of biological control of plant diseases caused by the crucifer black rot fungus and the cane mildew disease.

[0015] Storage Information

[0016] Strain Cd2, the preservation number is GDMCC No.64110, the preservation date is December 5, 2023, the preservation unit is Guangdong Provincial Microbiological Culture Collection Center, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a confrontation culture diagram of strain Cd2 and crucifer black rot pathogen;

[0018] Figure 2 This is a confrontation culture diagram of strain Cd2 and Pseudomonas aeruginosa;

[0019] Figure 3 This is the colony morphology of strain Cd2 on NA plate;

[0020] Figure 4 This is the bacterial morphology of strain Cd2;

[0021] Figure 5 This is the phylogenetic tree of the 16S rDNA gene sequence of strain Cd2.

[0022] Description of reference numerals:

[0023] Figure 1 In the middle, the left side is the control group, and the right side is the treatment group;

[0024] Figure 2 The left side is the treatment group and the right side is the control group. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the technical solution of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by technicians in this field without creative work are within the scope of protection of the present invention.

[0026] In the following examples, the culture medium formulation is as follows:

[0027] NA solid culture medium: 5.0 g peptone, 3.0 g beef extract, 2.5 g glucose, 15 g agar powder, dilute to 1 L with distilled water, pH 7.0±0.2.

[0028] NB liquid culture medium: 5.0 g peptone, 3.0 g beef extract, 2.5 g glucose, dilute to 1 L with distilled water, pH 7.0±0.2.

[0029] NY solid culture medium: Yeast extract 3.0 g, Peptone 5.0 g, Glycerol 20.0 g, Agar powder 15 g, dilute to 1 L with distilled water, pH 7.0±0.2.

[0030] NY liquid culture medium: Yeast extract 3.0 g, Peptone 5.0 g, Glycerol 20.0 g, dilute to 1 L with distilled water, pH 7.0±0.2.

[0031] PDA solid culture medium: 200.0 g potato (peeled), 20.0 g glucose, 15 g agar powder, dilute to 1 L with distilled water, pH 7.0±0.2.

[0032] Example

[0033] 1. Isolation and purification of strains

[0034] Soil was collected from the rhizosphere soil of Aster tataricus planted in Hechi, Guangxi Zhuang Autonomous Region. 10.0 g of soil was weighed and placed in a triangular flask containing glass beads and sterile water. The soil was shaken at 28 °C and 180 r / min for 30 min. The stock solution was used for gradient dilution. 10 -3 , 10 -4 and 10 -5 The gradients were spread on NA solid plates and cultured at 37°C for 24 hours. The growth of the colonies was observed, and a plate with moderate density was selected. A single colony was picked for streak purification, and colonies with different morphologies were picked for repeated streak purification, and the purified strains were preserved.

[0035] 2. Strain Screening

[0036] The black rot of Cruciferae and Pseudocercus pyrifolius were used as indicator strains, and the plate confrontation method was used to screen the activity of the isolated bacteria. Among them, the black rot of Cruciferae was provided by Mr. Jiang Bole from the College of Life Sciences of Guangxi University, and Pseudocercus pyrifolius was provided by the Institute of Agricultural Product Quality Safety and Testing Technology of Guangxi Academy of Agricultural Sciences.

[0037] 2.1 Inhibitory activity of the screened strains against black rot pathogens of Cruciferae

[0038] The isolated strain was cultured in NB liquid at 180 r / min and 30°C overnight. Under sterile conditions, 100 μL of the suspension of Cruciferae black rot fungus (OD 600 After the NY solid medium was thoroughly mixed, it was poured into a plate. After solidification, a sterile punch was used to punch holes around the center at a distance of 2 cm. Then 2 μL of the culture solution of the primary screening strain was inoculated into each hole (OD 600 The control group was set up with a treatment inoculated with Cruciferae black rot fungus but not inoculated with the primary screening strain. Each treatment was repeated 3 times. After culturing at 30°C for 5 days, the diameter of the inhibition zone of each treatment was measured by the cross method. The results are shown in Figure 1 .

[0039] 2.2 Inhibitory activity of the screened strains against Pseudomonas pyri

[0040] The isolated strains were cultured in NB liquid at 180 r / min and 30°C overnight. Under sterile conditions, a 5 mm diameter bacterial cake was punched out on the PDA plate inoculated with Pseudomonas spp. using a sterile puncher, and the Pseudomonas spp. bacterial cake was transferred to the center of the culture dish containing PDA, and 2 μL of the culture solution of the primary screening strain was inoculated around the center at 2 cm (OD 600 The control group was set up with a treatment inoculated with the Pseudomonas sporeans cake but not inoculated with the primary screening strains. Each treatment was repeated 3 times. After culturing at 28°C for 5 days, the diameter of the Pseudomonas sporeans in each treatment was measured by the cross method. The inhibition rate was calculated. The results are shown in Figure 2 .

[0041] The results showed that a strain of antagonistic bacteria with strong inhibitory effect on black rot pathogens of Cruciferae and Pseudomonas aeruginosa was obtained, and the strain was numbered Cd2.

[0042] 3. Identification of strain morphology

[0043] 3.1 The Cd2 strain was inoculated on NA medium and cultured at 30°C for 24 hours. The colony morphology of the strain was observed. The results are shown in Figure 3 .

[0044] from Figure 3 It can be seen that strain Cd2 appears as a single colony on the NA plate, which is smooth, slightly raised, has neat and wavy edges, and is yellow-green in color.

[0045] 3.2 Take the above cultured strain Cd2 and perform Gram staining and microscopic examination. The observed morphological results are photographed microscopically. The results are shown in Figure 4 .

[0046] Gram staining revealed that strain Cd2 was Gram-negative. Figure 4 It can be seen that the morphology of the bacteria Cd2 is: no spores, the bacteria are slender and of varying lengths.

[0047] 4. Strain Sequencing and Phylogenetic Tree Construction

[0048] 4.1 Preparation of DNA template

[0049] (a) Place 1.5 mL of overnight bacterial culture solution in a sterilized EP tube, centrifuge at 12,000 rpm for 1 min, discard the supernatant, and collect the bacteria;

[0050] (b) adding 400 μL of lysis buffer to the precipitate and mixing it by repeatedly pipetting; wherein the lysis buffer comprises: 40 mM Tris-Ac (pH 7.8), 20 mM NaAc, 1 mM EDTA, and 1% SDS (w / v);

[0051] (c) Add 200 μl of 5 M NaCl, mix thoroughly, and centrifuge at 12,000 rpm for 10 min;

[0052] (d) Take 600 μL of the supernatant from step (c), add an equal volume of phenol / chloroform (1:1), gently invert several times to mix, centrifuge at 12000 rpm for 10 min, and transfer the supernatant to another clean EP tube;

[0053] (e) Add an equal volume of chloroform to the supernatant, mix well, centrifuge at 12000 rpm for 10 min, and transfer the supernatant to another clean EP tube;

[0054] (f) adding an equal volume of isopropanol to the supernatant, mixing, placing at room temperature for 10 min, centrifuging at 12000 rpm for 15 min, discarding the supernatant, and retaining the precipitate;

[0055] (g) washing the resulting precipitate with 70% ethanol by volume and drying it to obtain DNA;

[0056] (h) Dissolve the dried DNA in 30 μL double distilled water (ddH2O) and store at -20°C.

[0057] 4.2 PCR amplification

[0058] The 16S rDNA sequence PCR reaction system shown in Table 1 below was used for amplification. The sequence of 16S rDNA is shown in Table 2 below.

[0059] Table 1 16S rDNA sequence PCR reaction system

[0060]

[0061] Table 2 16S rDNA sequences

[0062]

[0063] PCR reaction program: denaturation at 95°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, for a total of 35 cycles; extension at 72°C for 5 min.

[0064] 4.3 The PCR amplification product was sequenced by Guangzhou Qingke Biotechnology Co., Ltd., and its nucleotide sequence is shown in SEQ ID No.3.

[0065] 4.4 The measured 16S rDNA sequences were compared with the 16S rDNA sequences of bacteria in the GenBank gene library using MAFFT (v7.490). The phylogenetic tree was constructed using the maximum likelihood method using IQ-TREE (v2.0.7) and displayed using ggtree (v3.6.2). The phylogenetic tree is shown in Figure 5 .

[0066] Through Genebank database comparison and IQ-TREE (v2.0.7) analysis, strain Cd2 was determined to be Pseudomonas aeruginosa, the strain code was Cd2, and it was named Pseudomonas aeruginosa Cd2.

[0067] 5. Inhibitory effect of strain Cd2 fermentation broth on crucifer black rot infection

[0068] 5.1 Preparation of strain Cd2 fermentation broth

[0069] The strain Cd2 suspension (OD 600 The value was 1.0) was inoculated into a conical flask containing 100 mL of NB medium at a 1% inoculation amount and cultured at 28°C for 72 h to obtain the fermentation broth of strain Cd2.

[0070] 5.2 Inhibitory effect of the fermentation liquid of strain Cd2 on the infection of black rot pathogen of Cruciferae

[0071] Select 5-leaf red radish seedlings of the same age and similar size as the host plants for the test. Use the leaf cutting inoculation method. Keep the red radish seedlings moisturized for 12-24 hours one day in advance, and inoculate before 9 o'clock the next day: Cruciferae black rot suspension (blank control group), Cruciferae black rot suspension and strain Cd2 fermentation liquid mixture (volume ratio 1:1 mixture, treatment group), Cruciferae black rot suspension and 1.00g / L ceftriaxone sodium mixture (volume ratio 1:1 mixture, positive control group), and use OD 600The value was 0.01. The sterilized scissors were dipped in the bacterial solution and cut the leaves 1 cm away from the tip perpendicular to the central axis vein. The leaves were kept for 5 seconds during the cutting process. More than 30 leaves of carrot seedlings were treated for each treatment. At the same time, NY liquid medium was inoculated as a healthy control. The leaves were cultured in a greenhouse at 25-30℃. The length of the lesions was measured with a ruler on the 10th day after inoculation, and the inhibitory effect was calculated. The results are shown in Table 3.

[0072]

[0073] Table 3 Inhibitory effects of different treatments on the pathogenicity of black rot fungi of Cruciferae

[0074]

[0075] As shown in Table 3, the inhibitory effect of the fermentation liquid of strain Cd2 on the pathogenicity of crucifer black rot fungi was 46.33%. Compared with the positive control 1.00 g / L ceftriaxone, its inhibitory effect on the pathogenicity of crucifer black rot fungi was close to that of the positive control. This shows that the strain Cd2 of the present invention provides a new resource for the biological control of crucifer black rot fungi.

[0076] 6. Inhibitory effect of the fermentation broth of strain Cd2 on the mycelial growth of Pseudomonas pyri

[0077] 6.1 Preparation of fermentation broth of strain Cd2

[0078] The strain Cd2 suspension (OD 600 The value was 1.0) was inoculated into a conical flask containing 100 mL of NB medium at a 1% inoculation amount and cultured at 28°C for 72 h to obtain the fermentation broth of strain Cd2.

[0079] 6.2 Inhibitory effect of the fermentation broth of strain Cd2 on the mycelial growth of Pseudomonas pyri

[0080] PDA plates with 1%, 5% and 10% of the fermentation liquid of strain Cd2 were prepared respectively. Under aseptic conditions, a sterile punch was used to punch a 5mm diameter bacterial cake on the PDA plate inoculated with Pseudomonas sporeans, and the Pseudomonas sporeans bacterial cake was inoculated into the center of the PDA plate. At the same time, a PDA plate containing prochloraz (Bayer Shipauk, 400g / L) was set as a positive control, and a PDA plate inoculated with Pseudomonas sporeans and without any agent was set as a negative control, and each treatment was repeated three times.

[0081] All PDA plates were cultured at 28°C. When the colonies of the negative control were almost full of the culture dish, the diameter of the Pseudomonas aeruginosa in each treatment was measured by the cross method, and the inhibition rate was calculated. The results are shown in Table 4.

[0082]

[0083] Table 4 Inhibition rate of different treatments on the mycelial growth of Pseudomonas sporeans

[0084]

[0085] As shown in Table 4, the mycelial growth inhibition rate of the fermentation liquid of strain Cd2 on the fruit cane mildew pathogen Pseudocerus pyri spores is 100%. Compared with the positive control prochloraz aqueous emulsion, when the content of the fermentation liquid of strain Cd2 in PDA is 1-10%, its mycelial growth inhibition rate on Pseudocerus pyri spores is the same as that of the positive control. This shows that the strain Cd2 of the present invention has the prospect of being developed into a biological preservative.

[0086] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A strain of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) Cd2, characterized in that The deposit number is GDMCCNo.64110, deposited in Guangdong Provincial Microbiological Culture Collection Center, the deposit date is December 5, 2023, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of the Pseudomonas aeruginosa according to claim 1 in inhibiting the black rot fungus of Cruciferae and inhibiting the hyphae growth of Arthrosporum.

3. Use of the Pseudomonas aeruginosa according to claim 1 in preventing and controlling black rot caused by crucifer black rot fungi and preventing cane mildew caused by Arthrosporum spp.

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