Fusarium oxysporum and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses fusarium oxysporum, the strain of the fusarium oxysporum is HuN8, the taxonomic name is Fusarium oxysporum, the preservation unit is China General Microbiological Culture Collection Center (CGMCC), the preservation date is December 22, 2023, and the preservation number is CGMCC No.41016. The invention further discloses a preparation method of the fusarium oxysporum. The strain has a remarkable effect on prevention and treatment of wheat basal stem rot, can promote wheat growth, improve fresh weight and plant height, and also can enhance resistance of wheat to pathogenic bacteria such as fusarium graminearum.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to Fusarium oxysporum and application thereof. Background Art

[0002] Wheat stem base rot is a disease of wheat caused by Pseudomonas graminearum and Fusarium graminearum. It mainly harms the base of wheat stems. After the leaf sheaths at the base of the stems are infected, the color gradually changes to dark brown, without cloud-like lesions; as the disease progresses, the internodes at the base of the wheat stems are infected and turn light brown to dark brown. When the field humidity is high, pink or white mold grows at the nodes and internodes, and the stems are easy to break. Wheat stem base rot is a global disease that occurs in wheat-producing areas around the world, including Asia, Africa, North America, South America, and Oceania. It mainly infects the leaf sheaths and stems at the base of wheat at 1-2 nodes, causing wheat to fall over and die prematurely, generally reducing yield by 5-10%, and in severe cases, it can reach more than 50%, or even a total loss.

[0003] At present, the most effective measure for the prevention and control of wheat stem rot is to plant excellent disease-resistant varieties, but there are few wheat varieties that are immune or highly resistant to wheat stem rot. Biological control has been a hot topic in the research of crop diseases in recent years. Therefore, finding fungi that can prevent and control wheat stem rot is an urgent task at present. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a Fusarium oxysporum strain named HuN8, with a taxonomic name of Fusarium oxysporum. The strain has a significant effect on preventing and treating wheat stem base rot.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a Fusarium oxysporum strain, wherein the Fusarium oxysporum strain is HuN8, the taxonomic name is: Fusarium oxysporum, the deposit unit is: General Microbiological Center of China Microbiological Culture Collection Administration, the deposit date is: December 22, 2023, and the deposit number is: CGMCC No. 41016. The ITS gene sequence of the Fusarium oxysporum strain HuN8 is shown in SEQ ID NO.1.

[0007] In a second aspect, the present invention provides a biological bacterial agent comprising the bacterial liquid of the above-mentioned Fusarium oxysporum.

[0008] In a third aspect, the present invention provides the use of the above-mentioned Fusarium oxysporum in preventing and controlling wheat base stem rot.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] The invention separates a Fusarium oxysporum strain HuN8 from bitter melon stems. The strain has a significant effect on preventing and treating wheat stem base rot, can promote wheat growth, increase fresh weight and plant height, and can also enhance wheat resistance to pathogens such as Fusarium graminearum. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a colony morphology diagram of the HuN8 strain of the present invention on PDA culture medium.

[0012] Figure 2 This is a picture of the hyphae morphology of the HuN8 strain of the present invention on PDA culture medium.

[0013] Figure 3 This is a spore morphology diagram of the HuN8 strain of the present invention.

[0014] Figure 4 The colony diameters of the HuN8 strain of the present invention at different growth days.

[0015] Figure 5 The present invention constructs a phylogenetic tree of strain HuN8 based on the rDNA-ITS gene sequence.

[0016] Figure 6 The HuN8 strain of the present invention is pathogenic to wheat coleoptile.

[0017] Figure 7 This is the pathogenicity of the HuN8 strain of the present invention to wheat.

[0018] Figure 8 This is a potted plant diagram showing the control effect of the HuN8 strain of the present invention on wheat stem base rot.

[0019] Fig. 9 This is the control effect of the HuN8 strain of the present invention on wheat stem base rot.

[0020] Fig.10 This is the effect of the HuN8 strain of the present invention on the fresh weight of wheat.

[0021] Fig.11 This is the effect of the HuN8 strain of the present invention on wheat plant height. DETAILED DESCRIPTION

[0022] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified.

[0023] Example 1

[0024] 1 Materials and methods

[0025] 1.1 Test materials

[0026] 1.1.1 Test plants and strains

[0027] Plant pathogens: Fusarium graminearum PH-1 was provided by the Plant Disease Biological Control Laboratory of Henan Agricultural University.

[0028] Strain HuN8 was isolated from the bitter melon stems in the bitter melon shed in Hengyang City, Hunan Province, and was deposited in the General Microbiology Center of China Microbiological Culture Collection on January 9, 2024, with the deposit number CGMCC No.41016.

[0029] Wheat seeds dwarf 58 were provided by Xinyang Academy of Agricultural Sciences.

[0030] 1.1.2 Test instruments

[0031] Microwave oven, sterile clean bench, high pressure steam sterilizer, electronic scale, oven, refrigerator, constant temperature and humidity incubator, water bath, shaker, etc.

[0032] 1.1.2 Culture medium used in the experiment

[0033] Potato dextrose agar medium (PDA): cut 200g peeled potatoes into pieces, cook until soft, filter through 4 layers of gauze, add 20g glucose and 20g agar powder, make up to volume in 1L distilled water, and sterilize by high pressure wet heat at 121℃ for 30min.

[0034] Millet culture medium: Weigh a certain amount of commercially available millet, boil it in boiling water for 1 min 40 s, and filter the water with gauze. Place the millet on absorbent paper to air dry for 3-6 h, divide it into 250 mL Erlenmeyer flasks, and sterilize it by high pressure at 121°C for 30 min. Store it at room temperature for later use.

[0035] 1.2 Test methods

[0036] 1.2.1 Isolation and morphological identification of strain HuN8

[0037] Cut a tissue block of 0.5cm×0.5cm×0.2cm from the diseased and healthy junction at the base of the root of the diseased bitter melon, disinfect it with 75% ethanol for 30-40s, and rinse it with sterile water three times. Use sterile filter paper to absorb the moisture on the surface of the tissue. Put the tissue block with the surface moisture absorbed on the PDA plate. Culture it in a constant temperature incubator at 28℃ for 2-3 days. Take out the culture and pick the new mycelium at the edge of the colony for purification culture.

[0038] A 5mm punch was used to punch out a HuN8 strain bacterial cake and inoculated in the center of a culture dish (90 mm) containing a quantitative PDA (15 mL). The dish was cultured in a 28°C incubator. The colony morphology was observed and the colony diameter was measured every day. At the same time, the hyphae of the colony were picked and observed under an optical microscope. The experiment was repeated 3 times.

[0039] 1.2.2 Molecular biological identification of strain HuN8

[0040] First, pour a PDA plate in a clean bench, spread sterilized cellophane on the plate, inoculate a number of bacterial strain blocks on the PDA plate covered with cellophane using an inoculation needle, and culture in a 28°C incubator for 2-3 days for DNA extraction. The DNA extraction method uses the CTAB method. The specific steps are as follows:

[0041] (1) Use a sterile medicine spoon to gently scrape 0.1 g of mycelium cultured on the cellophane, put it into a mortar, add an appropriate amount of liquid nitrogen, grind it into powder, put it into a 2 mL EP tube, add 800 μL of CTAB preheated to 65 °C, put it into a water bath for 30 min, and mix it by turning it upside down every five minutes.

[0042] (2) After the water bath, add 400 μL of DNA extraction phenol and 400 μL of chloroform to the EP tube, shake vigorously to mix, centrifuge at 12,000 r / min for 15 min, take 600 μL of the supernatant and transfer it to a new 1.5 mL EP tube, add an equal volume of chloroform, shake vigorously to mix, and centrifuge at 12,000 r / min for 15 min.

[0043] (3) Take 500 μL of the supernatant and transfer it to a new 1.5 mL EP tube. Add an equal volume of isopropanol, shake gently, and place in a -20°C refrigerator to precipitate for 30 min. Centrifuge at 12,000 rpm for 15 min.

[0044] (4) Discard the supernatant, then wash the precipitate with 75% ethanol 2-3 times, dry the precipitate in a 37°C oven, add 30-50 μL of ddH2O to dissolve it, and place it in a -20°C refrigerator for later use.

[0045] The extracted DNA was amplified by PCR using rDNA-ITS universal primers.

[0046] The universal primers for the fungal ribosomal ITS region used in the experiment are as follows:

[0047] ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′;

[0048] ITS4: 5′-TCCTCCGCTTATTGATATGC-3′.

[0049] Table 1 PCR system

[0050]

[0051]

[0052] PCR amplification conditions:

[0053]

[0054] 1.2.3 Determination of wheat pathogenicity in vitro

[0055] Disinfection of wheat seeds: Use 0.01% mercuric chloride to disinfect the surface of wheat for 5 minutes, and then rinse with sterile distilled water 3-5 times.

[0056] Wheat planting: Sprinkle the disinfected wheat seeds in a sterile substrate. After about 5-7 days, select wheat plants of uniform size for in vitro pathogenicity determination.

[0057] On the PDA medium plates of HuN8 strain and Fusarium graminearum strain, use a 5mm puncher to punch out bacterial cakes of the same size at the same distance from the edge of the colony, and evenly place the wheat on the sterilized plate. In the wheat coleoptile inoculation test, one bacterial cake is inoculated for each coleoptile, and 5 replicates are made. After inoculation, wet the absorbent paper to cover the wheat roots, seal it with plastic wrap (to keep it moist) and cover it with black cloth, and culture it in the dark at 25℃ for 24h; after 24h, remove the black cloth, alternate light and darkness at 25℃, and culture it for about 4d (the specific time depends on the lesions produced by the wild-type strain infecting the plants), observe the infection and take pictures.

[0058] 1.2.4 Indoor fire prevention effectiveness determination

[0059] Preparation of HuN8 bacterial solution: Use a 5 mm cork puncher to punch out a bacterial cake of the HuN8 strain, inoculate it into 100 mL of PDB medium, and culture it at 28°C, 180 rpm, and shake for 5-7 days.

[0060] Seed treatment: Wheat seeds were soaked with spores of HuN8 strain cultured in PDB liquid medium for 4-7 days. The bacterial solution should cover the wheat seeds. After soaking for 8 hours, filter out the excess bacterial solution and let the wheat seeds air-dry for later use. The wheat in the control group was treated with sterile water.

[0061] Propagation of pathogens: Pick up pathogen cakes and inoculate them into millet culture medium for propagation. After the surface of millet seeds is covered with bacterial tissue (cultured at 25°C for 7-10 days), air-dry naturally and store at room temperature for later use.

[0062] Inoculation and planting: Take 150 g of sterilized soil (sandy loam) in a flowerpot with a diameter of 9 cm, place 10 wheat seeds evenly on its surface, and then cover it with 50 g of sterile soil and 1 g of pathogen inoculum. Each treatment is repeated 4 times.

[0063] Disease investigation and data analysis: After 5 weeks of cultivation, the wheat seedlings were taken out and the roots of the plants were rinsed with clean water. The disease levels of the plants were counted and their disease index was calculated.

[0064] The seedling grading standards are as follows:

[0065] Level 0, no browning symptoms on the whole plant;

[0066] Level 1: the root or underground stem turns brown, or the first leaf sheath shows slight browning symptoms;

[0067] Level 3, the first leaf sheath turns obviously brown, but the leaf sheath does not turn black;

[0068] Level 5: the first leaf sheath becomes obviously black or the second leaf sheath becomes obviously brown;

[0069] Level 7: browning symptoms appear on the third leaf sheath, or the plant is stunted or close to death due to the disease;

[0070] Level 9: The plant died due to the disease.

[0071] The incidence rate and disease index were calculated according to the following formula:

[0072] Incidence rate (%) = number of diseased plants / total number of investigated plants × 100;

[0073] Disease index = ∑ (each grade × number of diseased plants at that disease grade) / (highest grade × total number of investigated plants) × 100;

[0074] Biological control effect (%) = (control disease index - treatment disease index) / control disease index × 100%.

[0075] 1.2.5 Field control effectiveness determination

[0076] Field seed treatment and data statistics are the same as 1.2.4.

[0077] 2 Results and analysis

[0078] 2.1 Identification of strain HuN8

[0079] 2.1.1 Morphological identification of strain HuN8

[0080] The cells were cultured in PDA medium at 28°C in the dark. Figure 1 to Figure 4As shown in the figure, the colonies of HuN8 strain are off-white or pink-purple, round and flat, with fuzzy hyphae. After 5 days of cultivation, the colony diameter is about 70 mm, the hyphae are non-septate, with few branches, and the conidia are oval.

[0081] 2.1.2 Molecular biological identification of strain HuN8

[0082] The PCR product obtained by PCR amplification was sent to Shanghai Sangon Biotechnology Co., Ltd. and sequenced using ITS1 / ITS4 as primers to obtain the ITS gene sequence of strain HuN8, which was 493 bp in length (see SEQ ID NO.1).

[0083] Sequence alignment was performed using the Blastn method on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to obtain the nucleic acid sequences of strains with close genetic relationships. The phylogenetic tree was constructed using the neighbor-joining method using MEGA7.0 software. The HuN8 phylogenetic tree is shown in Figure 1. Figure 5 Combining morphological and phylogenetic tree analysis, the HuN8 strain was identified as Fusarium oxysporum.

[0084] 2.2 Determination of pathogenicity of HuN8 strain to wheat

[0085] 2.2.1 Determination of the pathogenicity of HuN8 strain to wheat in vitro

[0086] In order to determine the ability of strain HuN8 to infect wheat coleoptiles, a wheat coleoptile inoculation experiment was conducted. Figure 6 The results showed that Fusarium graminearum can cause dark brown spots on wheat coleoptiles, while HuN8 inoculation does not cause brown spots on coleoptiles, and inoculation of strain HuN8 does not cause symptoms on wheat coleoptiles.

[0087] 2.2.1 Determination of pathogenicity of HuN8 strain to wheat in vivo

[0088] In order to further verify the ability of strain HuN8 to infect wheat coleoptiles, a live pot inoculation experiment was conducted. Figure 7 The results showed that the wheat coleoptiles inoculated with HuN8 did not produce brown spots and grew well, further proving that the inoculation of strain HuN8 did not cause symptoms in wheat coleoptiles and did not affect the normal growth of wheat.

[0089] 2.3 Biological control effect of HuN8 strain on wheat stem rot

[0090] 2.3.1 Effect of HuN8 strain on wheat stem rot in indoor potted plants

[0091] To explore the indoor control effect of HuN8 strain on wheat stem rot, a pot experiment was conducted to verify the results. Figures 8 to 11 The results showed that the resistance to wheat stem rot increased after the seeds were soaked with HuN8 mycelium solution (see Figure 8-Figure 9 ); Compared with the wheat stem base inoculated with Fusarium graminearum after water treatment, the wheat stem base inoculated with Fusarium graminearum after HuN8 soaking treatment showed a reduced browning symptom and a significantly decreased disease index (Table 2). In addition, the wheat grew well, with a higher fresh weight and plant height (see Figure 10-11 , Table 3), indicating that strain HuN8 can not only promote the growth of wheat, but also enhance the resistance of wheat to Fusarium graminearum.

[0092] Table 2 Statistics on the indoor control effect of strain HuN8 on wheat stem rot

[0093]

[0094] Table 3 Statistics of the effect of HuN8 strain on wheat plant height and fresh weight

[0095] Plant height(mm) Fresh weight(g) HuN8 <![CDATA[319.83±19.35 c ]]> <![CDATA[0.52±0.08 b ]]> PH-1 <![CDATA[304.95±25.75 b ]]> <![CDATA[0.39±0.09 c ]]> HuN8+PH-1 <![CDATA[350.46±16.86 a ]]> <![CDATA[1.07±0.22 a ]]> CK <![CDATA[344.58±14.38 a ]]> <![CDATA[0.53±0.17 b ]]>

[0096] 2.3.2 Field control effect of HuN8 strain on wheat stem rot

[0097] To verify the biological control effect of strain HuN8 on wheat stem base rot, field tests were conducted in two fields from October 23 to May 20, 2023. The results are shown in Table 4. According to the statistical results, the incidence index of wheat stem base rot in the control group of field 1 was 25.73, while the incidence index of the HuN8 treatment group was only 9.07, and the control effect reached 64.77%. The control effect of the SD-V treatment group in field 2 reached 51.5%. It can be seen that strain HuN8 has a good control effect on wheat stem base rot.

[0098] Table 4 Statistics on the field control effect of strain HuN8 on wheat stem rot

[0099]

[0100] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. A Fusarium oxysporum, characterized in that The Fusarium oxysporum strain is HuN8, and its taxonomic name is: Fusarium oxysporum , Depository unit: General Microbiology Center of China Microbiological Culture Collection Administration, Deposit date: December 22, 2023, Deposit number: CGMCC No.41016.

2. The Fusarium oxysporum according to claim 1, characterized in that The ITS gene sequence of the Fusarium oxysporum strain HuN8 is shown in SEQ ID NO.

1.

3. A biological bacterial agent, characterized in that: A bacterial liquid containing the Fusarium oxysporum according to claim 1.

4. Use of the Fusarium oxysporum according to claim 1 or 2 in preventing and treating wheat stem base rot.

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