Antagonistic strain Penicillium crustosum LZ31 for efficiently preventing and controlling sugarcane smut and application of antagonistic strain Penicillium crustosum LZ31

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an antagonistic strain Penicillium crustosum LZ31 capable of efficiently preventing and controlling sugarcane smut and application of the antagonistic strain Penicillium crustosum LZ31. The invention relates to an antagonistic strain LZ31, which is characterized in that the taxonomic name of the antagonistic strain LZ31 is Penicillium cutaneum, the antagonistic strain LZ31 is preserved in Guangdong Microbial Culture Collection Center on August 23, 2024, and the preservation number of the antagonistic strain LZ31 is GDMCC No: 65052. According to the invention, strains for inhibiting the growth of the sugarcane smut are screened to obtain a fungus strain LZ31 with relatively strong resistance, and after molecular biological identification, the strain LZ31 is Penicillium crustosum. A pot experiment shows that the strain LZ31 can efficiently prevent and control the sugarcane smut, can promote growth of sugarcane plants and accumulation of biomass, can provide support for development of microbial preparations and bacterial fertilizers for preventing and controlling the sugarcane smut, and has important practical value for green and high-quality development of the sugarcane industry.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to an antagonistic strain Penicillium crustosum LZ31 for effectively preventing and controlling sugarcane smut and an application thereof. Background Art

[0002] Sugarcane smut is widely found in the main sugarcane producing areas of South my country, Central China and Southwest China. In severe years, it can cause a yield loss of up to 20%-50%. In extreme cases, it can even lead to a complete crop failure. Therefore, it is called the "cancer" of sugarcane. Sugarcane smut is a fungal disease caused by the genus Sporisorium of the class Basidiomycetes. It is mainly spread by air. The main prevention and control measures include disease-resistant breeding, soaking seed stems with chemicals, scalding seedlings with hot water, and using biological pesticides and biological organic fertilizers. At present, the most widely used biological control method for sugarcane smut is to cultivate and promote the planting of new disease-resistant sugarcane varieties, such as Guitang No. 28 and Guitang No. 29 (Guitang 02-761), etc. However, there are problems such as long sugarcane breeding cycle and poor resistance of high-quality varieties. The pathogenicity of the pathogen differentiates quickly, and the new subspecies may be the dominant subspecies, resulting in the new bred sugarcane smut-resistant varieties gradually losing their resistance and becoming susceptible varieties. Chemical components such as benzoprochloraz and triadimefon can effectively inhibit the germination of thick-walled spores of sugarcane whip smut fungus (Sporisorium scitamineum), but due to the thick bark of sugarcane stems, ordinary agents are difficult to penetrate, and the economic cost is high, which has certain harm to the ecological environment and is difficult to apply on a large scale.

[0003] Microbial control is the effect of functional microorganisms inhibiting pathogens through various pathways. It has the characteristics of high environmental compatibility and low cost. It is an important way to prevent and control plant diseases and is also considered to be one of the most promising control methods. The use of functional microorganisms to control sugarcane smut mainly includes two aspects: biological pesticides and biological organic fertilizers. Biological pesticides for the control of sugarcane smut are mainly divided into two types: the application of living microorganisms and microbial metabolites. They are biological preparations for the targeted control of sugarcane smut. Compared with chemical pesticides, they are safer and more lasting, and plants are not likely to develop drug resistance after long-term use, so they have good application prospects. Therefore, it is of great significance to screen microbial strains for the control of sugarcane smut.

[0004] The inventors studied the microbial strains in the sugarcane planting soil environment and screened out an antagonistic strain LZ31 that is antagonistic to sugarcane smut fungus and has a preventive and control effect on sugarcane smut, which is of great significance for the development of microbial preparations and bacterial fertilizers for preventing and controlling sugarcane smut.

[0005] 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

[0006] The object of the present invention is to provide an antagonistic strain Penicillium crustosum LZ31 for effectively preventing and controlling sugarcane smut and an application thereof.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] An antagonistic strain LZ31, the taxonomic name of the antagonistic strain LZ31 is Penicillium crustosum, which was deposited in the Guangdong Microbiological Culture Collection Center on August 23, 2024, with the preservation number GDMCC No: 65052.

[0009] The present invention also provides the use of the antagonistic strain LZ31 in efficiently preventing and controlling agricultural fungal diseases.

[0010] The present invention also provides that the agricultural fungal diseases include sugarcane smut.

[0011] The present invention also provides that the pathogen of the agricultural fungal disease is Sporisorium scitamineum.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention screens strains that inhibit the growth of sugarcane smut fungi, and obtains a fungal strain LZ31 with strong resistance. After molecular biological identification, strain LZ31 is Penicillium crustosum. Through potted plant experiments, it was found that strain LZ31 can not only effectively prevent and control sugarcane smut, but also promote the growth of sugarcane plants and the accumulation of biomass. It can provide support for the development of microbial preparations and bacterial fertilizers for preventing and controlling sugarcane smut, and has important practical value for the green and high-quality development of the sugarcane industry.

[0014] Storage Information

[0015] The strain Penicillium crustosum LZ31 was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on August 23, 2024. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the deposit number is GDMCC No: 65052. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the antagonistic effect of strain LZ31 on the growth of pathogenic bacteria;

[0017] Figure 2 This is a graph showing the inhibitory effect of the fermentation liquid of strain LZ31 on the mycelial growth of pathogenic bacteria;

[0018] Figure 3 The colony morphology and phylogenetic tree of strain Penicillium crustosum LZ31;

[0019] Figure 4 The effect of strain LZ31 on the growth and development of sugarcane plants;

[0020] Figure 5 The effect of strain LZ31 on the number of pathogen spores;

[0021] Figure 6 The effect of strain LZ31 on the incidence of sugarcane smut;

[0022] Figure 7 This is the effect of strain LZ31 on sugarcane biomass accumulation.

[0023] Description of main reference numerals:

[0024] Figure 1 The white round colonies on the plate are pathogens. 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] 1. Strain Isolation and Identification

[0027] 1.1 Strain Isolation

[0028] Soil samples were collected from the macadamia planting base of Guangxi South Asian Tropical Agricultural Science Research Institute in Longzhou City, Guangxi Zhuang Autonomous Region. In 2020, sugarcane was planted in soil samples with high incidence and soil samples with high incidence. The results showed that the incidence of sugarcane smut in the two soils was 38.5% and 5.5%, respectively. After testing, it was found that the number of pathogens in the two soils was 2.36×10 6 cfu / g and 2.05×10 2 cfu / g.

[0029] Add 45 mL of sterile water to a sterilized flask (250 mL), take 5.0 g of soil sample and put it into the flask, place it in a constant temperature shaker at 30°C and 180 rpm and culture it for 1 hour. Pipette 1 mL of soil suspension and add it to 9 mL of sterile water, mix well, and dilute it in a gradient of 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 50 μL of the solution with different gradients was respectively added to beef extract peptone agar (NA) and potato dextrose agar (PDA) plates, and spread evenly with a spreading rod. Three replicates were set for each treatment group.

[0030] The coated NA plates and PDA plates were placed in constant temperature incubators at 37°C and 28°C, respectively, and single colonies with different colony characteristics were picked out and cultured and purified until single colonies appeared on the plates.

[0031] 1.2 Activation of target pathogens

[0032] Collect the newly formed sugarcane smut whips, disinfect the surface, peel off the leaf sheaths, pick up a few black winter spores with sterilized tweezers, and dilute them in sterile water. Dilute the spore solution of sugarcane whip smut fungus (Sporisorium scitamineum) and spread it on the PDA plate. After culturing in a constant temperature incubator at 28℃ for 2 days, use an inoculation loop in the clean bench to inoculate the newly germinated sugarcane whip smut fungus on a new PDA plate and continue culturing.

[0033] 1.3 Preliminary screening of antagonistic strains

[0034] The plate confrontation method was used. When the colony of sugarcane whip smut fungus grew to about 2mm, 4 isolated and purified single strains were inoculated at a distance of 20mm in the center of each plate. The bacteria were inoculated with an inoculation loop and the fungus was inoculated with a puncher with a diameter of 5mm. Three replicates were set for each treatment and culture was continued for 10-15 days. The antagonism degree was determined by the distance from the bacterial / fungal inoculation point to the outer edge of the sugarcane smut colony. The colonies with obvious inhibition of pathogen growth were selected and numbered and recorded. 12 bacterial strains and 24 fungal strains with antagonistic effects on smut pathogens were obtained in the initial screening.

[0035] 1.4 Rescreening of antagonistic strains

[0036] The specific steps are as follows:

[0037] (1) Inoculate the newly germinated cakes of sugarcane whip smut fungus (5 mm in diameter) on a PDA plate. When the diameter of the sugarcane whip smut fungus colony is about 1 cm, inoculate the antagonistic bacteria obtained in the initial screening at a position 1 cm away from the edge of the plate. Only one strain is inoculated on each plate, and 6 replicates are set for each colony. Continue to culture for 10-15 days.

[0038] As a result, a fungus with strong antagonistic ability was obtained, which was numbered LZ31. The antagonistic culture results of the antagonistic strain LZ31 against sugarcane whip smut fungus are shown in Figure 1 .

[0039] (2) After culturing strain LZ31 on a PDA plate, a bacterial cake was punched from the edge of the colony with a hole punch and inoculated into the center of the PDA plate. After culturing in a 28°C constant temperature incubator for 7 days, 10 mL of sterile water was added to the PDA plate. The spores and sterile water were mixed evenly with a spreader rod. The culture medium and mycelium were filtered through 4 layers of gauze to obtain a fresh spore suspension. The spore concentration in the spore suspension was determined using a hemocytometer and diluted to adjust the spore concentration to 1.0 × 10 7 cfu / mL.

[0040] (3) 1 mL of strain LZ31 spore suspension (spore concentration 1.0 × 10 7 cfu / mL) was inoculated into a Erlenmeyer flask containing 100 mL of PDB medium and cultured in a constant temperature shaker at 28°C and 180 rpm for 5 days. After the culture was completed, the fermentation product was centrifuged at 20°C and 1000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm sterile water filter to obtain the fermentation filtrate.

[0041] (4) 10 mL of fermentation filtrate and 90 mL of PDA medium cooled to about 50°C were mixed evenly and poured into a culture dish with a diameter of 9 cm. After cooling, an antagonistic plate was formed, and a treatment with an equal amount of sterile water was set as a blank control.

[0042] Use an inoculation loop to inoculate the newly germinated sugarcane whip smut fungus onto a new PDA plate and place it in a 28°C constant temperature incubator for 2 days. Use a hole puncher to punch a 5mm diameter bacterial cake at the edge of the colony and inoculate it into the center of the antagonistic plate and the blank control plate respectively. The colony growth diameter of the sugarcane whip smut fungus was measured regularly for 7 consecutive days, and the inhibitory effect of strain LZ31 on the sugarcane whip smut fungus was calculated. The results are shown in Figure 2 .

[0043]

[0044] Depend on Figure 2 It can be seen that the inhibition rate of the antagonistic strain LZ31 on the mycelial growth of Sugarcane Whip Ustilago canefolia was 97.8% at 15 days.

[0045] 1.5 Identification of antagonistic strains

[0046] 1.5.1 Colony morphology observation

[0047] The strain LZ31 was inoculated on PDA solid medium and cultured at 28°C for 5 days. Figure 3 .

[0048] Depend on Figure 3 It can be seen that the colony of strain LZ31 has a velvety and granular texture, is rough, opaque, has irregular edges, and the conidia are bean green.

[0049] 1.5.2 Molecular Biological Identification

[0050] 2 μL of strain LZ31 spore suspension (spore concentration 1.0×10 7 cfu / mL) were inoculated in a PDA plate and incubated at 28°C until the plate was full. The genomic DNA of strain LZ31 was extracted using a genomic kit. The genomic DNA was used as a template and the universal primers for fungal ITS were the ITS1 region for PCR amplification. The primer sequences are shown in Table 1; the PCR reaction system is shown in Table 2.

[0051] Table 1 Universal primer sequences

[0052]

[0053] Table 2 PCR reaction system

[0054]

[0055] The PCR program was: 95°C, 5 min; 95°C for 30 s, 57°C for 30 s, 72°C for 15 min, 30 cycles; 72°C for 5 min.

[0056] The amplified product was tested by 1% agarose gel electrophoresis to obtain the target band, which was then purified and recovered for sequencing. The sequence was determined as shown in SEQ ID No. 3. The sequence was compared with the NCBI database by BLAST, and the phylogenetic analysis of the isolated antagonistic bacterial sequence was performed by MEGA7.0 software. The developmental tree was constructed as shown in Figure 3 shown.

[0057] According to the comparison results, the phylogenetic tree was constructed by combining the model strain with the highest similarity. The antagonistic strain LZ31 was determined to be Penicillium crustosum through comparison and phylogenetic tree, and was named Penicillium crustosum LZ31.

[0058] 2. Control effect of antagonistic strain LZ31 on sugarcane smut

[0059] 2.1 Test pathogen spore suspension

[0060] 2 μL of spore liquid of Ustilago officinalis stored in -80°C glycerol was taken and inoculated on a PDA solid plate. After culturing in a 28°C constant temperature incubator for 15 days, 10 mL of sterile water was added to the plate. The germinated black spores on the plate were scraped with a sterile coating stick. The obtained spore suspension was passed through four layers of sterile gauze and stored for later use. The spore concentration in the spore liquid was determined with a hemocytometer, and the concentration was diluted with sterile water to adjust the spore concentration to 1.0×10 7 cfu / mL.

[0061] 2.2 Antagonistic bacteria spore suspension

[0062] The spore suspension of strain LZ31 (spore concentration 1.0×10 7 cfu / mL) were inoculated into PDA plates and cultured in a constant temperature incubator at 28°C for 7 days. Then, 10 mL of sterile water was added to the PDA plates, and the spores and sterile water were mixed evenly with a coating stick. The culture medium and mycelium were filtered through 4 layers of gauze to obtain a fresh spore suspension, and the spore concentration in the spore suspension was determined using a hemocytometer.

[0063] 2.3 Sugarcane stems for testing

[0064] The "Guitang No. 42" sugarcane seed stems come from the Institute of South Subtropical Agricultural Sciences of Guangxi Academy of Agricultural Sciences.

[0065] 2.4 Test location

[0066] Greenhouse of the Institute of South Subtropical Agricultural Sciences, Guangxi Academy of Agricultural Sciences, time: January 28, 2022 to June 20, 2022.

[0067] 2.5 Test methods

[0068] (1) Disinfect the surface of sugarcane with 75% alcohol, cut into 15-20 cm long stem segments, and cultivate them in seedling trays with seedling medium. After the sugarcane stem segments sprout, select seedlings with basically the same growth, soak them in a spore suspension of sugarcane whip smut fungus for 30 minutes, take them out, dry them naturally, and then transplant them.

[0069] (2) The spore suspension of strain LZ31 was prepared at a concentration of 1×10 7 Add the cfu amount to the soil, mix well and put it into pots, 5kg soil per pot. Transplant the sugarcane seedlings treated in step (1) into pots on January 28, 2022, 1 plant per pot, and set the treatment with the same amount of sterile water as the blank control. Set 3 replicates for each treatment, and 30 pots for each replicate. Regularly observe and record the number of diseased plants in each treatment, and calculate the incidence of each treatment when the disease situation is stable (no new diseased plants for 2 consecutive weeks).

[0070] The incidence rates of different treatments were calculated according to the following formula:

[0071] Incidence rate = number of diseased sugarcane plants / total number of treated sugarcane plants × 100%.

[0072] (3) At the end of the pot experiment, five sugarcane plants were randomly selected from each treatment to collect soil, rhizosphere and root samples. The samples were stored in a -80°C refrigerator and DNA was subsequently extracted for fluorescence quantitative PCR experiments. The number of pathogens was determined using specific primers using Quantitative Real-Time PCR technology. The number of copies of pathogens per gram of dry soil was calculated based on the sample threshold (Ct), and the results were expressed in logarithms (Log 10 copies·g -1 DW); at the same time, the biomass of individual sugarcane plants was measured. The biomass of sugarcane plants was accurately measured by collecting fresh sugarcane samples and using an analytical balance to determine the biomass of sugarcane plants. The average value was taken as the biomass of individual sugarcane plants. Figure 4-7 .

[0073] Table 3 Pathogen fluorescence quantitative primer sequences

[0074]

[0075] Depend on Figure 4 It can be seen that compared with the blank control, the antagonistic strain LZ31 can effectively prevent and control sugarcane smut. In addition, after the antagonistic strain LZ31 was treated, the height and stem diameter of the sugarcane plants were significantly higher than those of the blank control group, indicating that the antagonistic strain can also promote the growth of sugarcane plants.

[0076] Depend on Figure 5 It can be seen that compared with the blank control, the number of sugarcane whip smut fungus spores in the bulk soil, rhizosphere soil and root system of sugarcane plants was significantly reduced after treatment with the antagonistic strain LZ31.

[0077] Depend on Figure 6 It can be seen that the incidence of sugarcane smut in the blank control group was 35.6%, and the incidence of sugarcane smut after treatment with the antagonistic strain LZ31 was 4.4%. It can be seen that the antagonistic strain of the present invention can significantly reduce the incidence of sugarcane smut, thereby effectively preventing and controlling sugarcane smut.

[0078] Depend on Figure 7 It can be seen that compared with the blank control, the biomass of sugarcane was significantly increased after treatment with the antagonistic strain LZ31, indicating that the antagonistic strain LZ31 can promote the accumulation of biomass of sugarcane plants.

[0079] In summary, the antagonistic strain LZ31 of the present invention can not only effectively prevent and control sugarcane smut, but also promote the growth of sugarcane plants and the accumulation of biomass, and can provide support for the development of microbial preparations and bacterial fertilizers for preventing and controlling sugarcane smut.

[0080] 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. An antagonistic strain LZ31, characterized in that: The taxonomic name of the antagonistic strain LZ31 is Penicillium crustosum, which was deposited in the Guangdong Microbiological Culture Collection Center on August 23, 2024, with the collection number GDMCC No: 65052.

2. Use of the antagonistic strain LZ31 according to claim 1 in the efficient prevention and control of agricultural fungal diseases.

3. The use according to claim 2, characterized in that: The agricultural fungal diseases include sugarcane smut.

4. The use according to claim 2, characterized in that: The pathogen of the agricultural fungal disease is Sporisorium scitamineum.

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