An antagonistic strain Penicillium crustosum LZ31 against sugarcane smut and its application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to an antagonistic strain Penicillium crustosum LZ31 for efficiently preventing and controlling sugarcane smut and its application. 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 a deposit number of GDMCC No: 65052. The present invention screens strains that inhibit the growth of sugarcane smut fungi to obtain a fungal strain LZ31 with strong resistance, and then molecular biological identification shows that strain LZ31 is Penicillium crustosum. Through potted plant experiments, it was found that strain LZ31 can not only efficiently prevent and control sugarcane smut, but also promote the growth of sugarcane plants and the accumulation of biomass, which 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.

Description

Technical Field

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

[0002] Sugarcane smut widely exists in the main sugarcane producing areas in South China, Central China and Southwest China. In severe years, it can cause a yield loss of up to 20%-50%, and even lead to complete crop failure in extreme cases. Therefore, it is called the "cancer" of sugarcane. Sugarcane smut is a fungal disease caused by Sporisorium scitamineum of the genus Sporisorium in the class Basidiomycetes, and it is mainly spread by air. The main control measures include disease-resistant breeding, soaking seed stems with chemicals, hot water scalding of seedlings, 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 sugarcane disease-resistant varieties, such as Guitang 28, Guitang 29 (Guitang 02-761), etc. However, there are problems such as a long sugarcane breeding cycle and poor resistance of high-quality varieties. The pathogenicity of this pathogen differentiates quickly, and new races may become the dominant races, resulting in the gradual loss of resistance of the newly selected original sugarcane smut-resistant varieties and being easily reduced to susceptible varieties. Chemical components such as propiconazole and triadimefon can effectively inhibit the germination of chlamydospores of Sporisorium scitamineum, but due to the relatively thick sugarcane stem skin, ordinary chemicals are difficult to penetrate, and the economic cost is relatively high, which has a certain harm to the ecological environment and is difficult to be applied on a large scale.

[0003] Microbial control is that functional microorganisms achieve the effect of inhibiting pathogenic bacteria through various ways, and has the characteristics of high environmental compatibility and low cost. It is an important way for plant disease control and is also considered one of the most potential control methods. Using functional microorganisms to control sugarcane smut mainly includes two aspects: biological pesticides and biological organic fertilizers. The biological control of sugarcane smut by biological pesticides is mainly divided into two types: applying microbial live bodies and microbial metabolites. They are biological agents for targeted control of sugarcane smut, and their efficacy is relatively safer and more persistent than chemical pesticides. And plants are not prone to develop drug resistance after long-term use, and have good application prospects. Therefore, screening microbial strains for controlling sugarcane smut is of great significance.

[0004] The inventor studied the microbial strains in the sugarcane planting soil environment and screened out an antagonistic strain LZ31 that is antagonistic to sugarcane smut bacteria and has a prevention and control effect on sugarcane smut, which is of great significance for the development of microbial agents and bacterial fertilizers for 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 The growth antagonistic effect diagram of strain LZ31 against pathogenic bacteria;

[0017] Figure 2 The inhibitory effect diagram of the fermentation broth 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 influence of strain LZ31 on the growth and development of sugarcane plants;

[0020] Figure 5 The influence of strain LZ31 on the spore number of pathogenic bacteria;

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

[0022] Figure 7 The influence of strain LZ31 on the biomass accumulation of sugarcane.

[0023] Description of main reference numerals:

[0024] Figure 1 In, the flat white circular colony is the pathogenic bacterium. Detailed implementation manners

[0025] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0026] 1. Isolation and identification of strains

[0027] 1.1 Isolation of strains

[0028] Soil samples were collected from the macadamia nut planting base of the Guangxi South Subtropical Agricultural Science Research Institute in Longzhou City, Guangxi Zhuang Autonomous Region. Sugarcane was planted in 2020 in highly diseased soil and soil samples. As a result, the incidences of sugarcane smut in the two soils were 38.5% and 5.5% respectively. After detection, the numbers of pathogenic bacteria in the two soils were 2.36×10 6 cfu / g and 2.05×10 2 cfu / g.

[0029] Add 45 mL of sterile water to a sterilized Erlenmeyer flask (250 mL). Take 5.0 g of soil sample and put it into the above Erlenmeyer flask. Incubate it in a constant temperature shaker at 30 °C and 180 rpm for 1 h. Pipette 1 mL of the soil suspension into 9 mL of sterile water, mix well, and dilute it stepwise to 10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 solutions. Pipette 50 μL of solutions with different gradients into plates of nutrient agar (NA) and potato dextrose agar (PDA) respectively, and spread them evenly with a spreader. Set 3 replicates for each treatment group.

[0030] Place the spread NA plates and PDA plates in a constant temperature incubator at 37 °C and 28 °C respectively for incubation. Pick single colonies with different colony characteristics, culture and purify them until single colonies appear on the plates.

[0031] 1.2 Activation of target pathogenic bacteria

[0032] Collect the newly formed sugarcane smut whips. After surface disinfection, peel off the leaf sheaths, pick a little black teliospores with sterilized forceps, and stir and dilute them in sterile water. Dilute the spore suspension of Sporisorium scitamineum and spread it on a PDA plate. Incubate it in a constant temperature incubator at 28 °C for 2 d. Then, in a laminar flow hood, inoculate the newly germinated Sporisorium scitamineum onto a new PDA plate with an inoculation loop and continue to culture.

[0033] 1.3 Primary screening of antagonistic strains

[0034] Adopt the plate confrontation method. When the colony of Sporisorium scitamineum grows to about 2 mm, inoculate 4 isolated and purified single strains at a distance of 20 mm from the center of each plate. For bacteria, inoculate with an inoculation loop; for fungi, inoculate with a punch with a diameter of 5 mm. Set 3 replicates for each treatment and continue to culture for 10 - 15 d. Determine the antagonistic degree by the distance from the bacteria / fungi inoculation point to the outer edge of the sugarcane smut pathogen colony, and select colonies with obvious inhibition of pathogen growth, number and record them. 12 bacteria and 24 fungi with antagonistic effects on the smut pathogen are obtained through primary screening.

[0035] 1.4 Re-screening of antagonistic strains

[0036] The specific steps are as follows:

[0037] (1) Inoculate the freshly germinated Ustilago scitaminea H. Syd. cake (5 mm in diameter) on a PDA plate. When the diameter of the Ustilago scitaminea H. Syd. colony is about 1 cm, inoculate the initially screened antagonistic bacteria 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. Then continue the incubation for 10 - 15 days.

[0038] As a result, a fungus with strong antagonistic ability was obtained, numbered LZ31. The antagonistic culture results of the antagonistic strain LZ31 against Ustilago scitaminea H. Syd. are shown in Figure 1 .

[0039] (2) After culturing the strain LZ31 on a PDA plate, use a borer to punch out a cake at the edge of the colony and inoculate it in the center of the PDA plate. After culturing in a constant temperature incubator at 28 °C for 7 days, add 10 mL of sterile water to the PDA plate. Use a spreader to mix the spores and sterile water evenly, and filter the culture medium and mycelium with four layers of gauze to obtain a fresh spore suspension. Use a hemocytometer to measure the spore concentration in the spore suspension, and dilute and adjust the spore concentration to 1.0×10 7 cfu / mL.

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

[0041] (4) Mix 10 mL of the fermentation filtrate and 90 mL of PDA medium cooled to about 50 °C evenly, pour it into a petri dish with a diameter of 9 cm, and form an antagonistic plate after cooling. Set the treatment with an equal amount of sterile water as the blank control.

[0042] Use an inoculation loop to inoculate the freshly germinated Ustilago scitaminea H. Syd. on a new PDA plate, and place it in a constant temperature incubator at 28 °C for 2 days. Use a borer to punch out a cake with a diameter of 5 mm at the edge of the colony, and inoculate it in the center of the antagonistic plate and the blank control plate respectively. Continuously measure the growth diameter of the Ustilago scitaminea H. Syd. colony at regular intervals for 7 days, and calculate the inhibitory effect of the strain LZ31 on Ustilago scitaminea H. Syd. The results are shown in Figure 2 .

[0043]

[0044] It can be seen from Figure 2 that the inhibition rate of the antagonistic strain LZ31 on the mycelial growth of Ustilago scitaminea H. Syd. is 97.8% at 15 days.

[0045] 1.5 Identification of the antagonistic strain

[0046] 1.5.1 Colony Morphology Observation

[0047] Inoculate strain LZ31 onto PDA solid medium and culture it at 28°C for 5 days. The results are as follows Figure 3 .

[0048] It can be seen from Figure 3 that the colony texture of strain LZ31 is velvety and granular, the surface is rough, opaque, the edge is irregular, and the conidia are pea green.

[0049] 1.5.2 Molecular Biology Identification

[0050] Inoculate 2 μL of the spore suspension of strain LZ31 (spore concentration 1.0×10 7 cfu / mL) into a PDA plate and culture it in a constant temperature incubator at 28°C until the plate is covered. Use a genomic kit to extract the genomic DNA of strain LZ31. Using the genomic DNA as a template and the fungal ITS universal primer for the ITS1 region, perform PCR amplification. Among them, 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 is as follows: 95°C, 5 min; 95°C for 30 s, 57°C for 30 s, 72°C for 15 min, cycle 30 times; 72°C, 5 min.

[0056] The amplified product is examined by 1% agarose gel electrophoresis to obtain the target band. Cut the gel to purify and recover the target band, and perform sequence determination. The obtained sequence is shown as SEQ ID No.3. The sequence is compared through the NCBI database, and the phylogenetic analysis of the isolated antagonistic bacteria sequence is performed using the MEGA7.0 software. The constructed phylogenetic tree is as Figure 3 shown.

[0057] According to the comparison results, a phylogenetic tree is constructed in combination with the type strain with the highest similarity. Through the comparison and the phylogenetic tree, it is determined that the antagonistic strain LZ31 is Penicillium crustosum, and it is named Penicillium crustosum LZ31.

[0058] 2. Control Effect of Antagonistic Strain LZ31 on Sugarcane Smut

[0059] 2.1 Tested Pathogen Spore Suspension

[0060] Absorb 2 μL of the Sporisorium scitamineum spore suspension stored in glycerol at -80 °C, inoculate it on a PDA solid plate, and culture it in a constant temperature incubator at 28 °C for 15 d. After black conidia are produced, add 10 mL of sterile water to the plate, and scrape the germinated black spores on the plate with a sterile spreader. After passing the obtained spore suspension through four layers of sterile gauze, store it for later use. Use a hemocytometer to measure the spore concentration in the spore suspension, and dilute the concentration with sterile water to adjust the spore concentration to 1.0×10 7 cfu / mL.

[0061] 2.2 Test antagonistic bacteria spore suspension

[0062] Inoculate the spore suspension of strain LZ31 (spore concentration 1.0×10 7 cfu / mL) into a PDA plate, and culture it in a constant temperature incubator at 28 °C for 7 d. Then add 10 mL of sterile water to the PDA plate, mix the spores and sterile water evenly with a spreader, filter the culture medium and mycelium with four layers of gauze to obtain a fresh spore suspension, and use a hemocytometer to measure the spore concentration in the spore suspension.

[0063] 2.3 Test sugarcane seed stems

[0064] The sugarcane seed stems of "Guitang 42" are from the South Subtropical Agricultural Science Research Institute of Guangxi Academy of Agricultural Sciences.

[0065] 2.4 Test site

[0066] The greenhouse of the South Subtropical Agricultural Science Research Institute of Guangxi Academy of Agricultural Sciences, time: from January 28, 2022 to June 20, 2022.

[0067] 2.5 Test method

[0068] (1) Disinfect the surface of sugarcane with 75% alcohol, cut it into stem segments 15 - 20 cm long, and cultivate it in a seedling tray with a seedling substrate. After the sugarcane stem segments germinate, select the seedlings with basically the same growth vigor, soak them in the Sporisorium scitamineum spore suspension for 30 min, take them out, and transplant them after natural drying.

[0069] (2) Add the spore suspension of strain LZ31 to the soil at an addition amount of 1×10 7 cfu per gram of soil, mix it evenly and then load it into pots, 5 kg of soil per pot. Transplant the sugarcane seedlings treated in step (1) into the pots on January 28, 2022, 1 seedling per pot, and set the treatment with an equal amount of sterile water added as a blank control. Each treatment has 3 replicates, and each replicate has 30 potted plants. Regularly observe and record the number of diseased plants in each treatment, and calculate the incidence rate of each treatment when the disease condition is stable (no new diseased plants for 2 consecutive weeks).

[0070] Calculate the incidence of different treatments according to the following formula:

[0071] Incidence = Number of diseased sugarcane plants / Total number of sugarcane plants in the treatment × 100%.

[0072] (3) At the end of the pot experiment, randomly select 5 pots of sugarcane plants from each treatment to collect their soil, rhizosphere, and root samples. Store the samples in a -80°C refrigerator, and then extract DNA for fluorescence quantitative PCR experiments. Using Quantitative Real-Time PCR technology and specific primers, determine the number of pathogenic bacteria. Calculate the copy number of pathogenic bacteria per gram of dry soil based on the sample threshold (Ct), and express the results in logarithm (Log 10 copies·g -1 DW); at the same time, measure the biomass of single sugarcane plants. The biomass of sugarcane is accurately measured by collecting fresh sugarcane samples using an analytical balance to obtain the sugarcane biomass weight, and its average value is used as the biomass of single sugarcane plants. The results are shown in Figures 4 - 7 .

[0073] Table 3 Fluorescent quantitative primer sequences of pathogenic bacteria

[0074]

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

[0076] It can be seen from Figure 5 that compared with the blank control, after treatment with the antagonistic strain LZ31, the number of Sporisorium scitamineum spores in the soil, rhizosphere soil, and roots of sugarcane plants is significantly reduced.

[0077] It can be seen from Figure 6 that the incidence of sugarcane smut in the blank control group is 35.6%, and the incidence of sugarcane smut after treatment with the antagonistic strain LZ31 is 4.4%. It can be seen that the antagonistic strain of the present invention can significantly reduce the incidence of sugarcane smut, and thus effectively prevent and control sugarcane smut.

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

[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 agents and bacterial fertilizers for preventing and controlling sugarcane smut.

[0080] The foregoing description of specific exemplary embodiments of the invention has been presented for purposes of illustration and example. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that, according to the above teaching, many changes and variations are possible. The purpose of selecting and describing exemplary embodiments is to explain the specific principles of the invention and its practical application, so that those skilled in the art can implement and utilize various different exemplary embodiments of the invention, as well as various different selections and changes. The scope of the invention is intended to be defined 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 ( Penicillium crustosum ), which was deposited in the Guangdong Microbial Culture Collection Center on August 23, 2024, with the deposit number GDMCC No: 65052.

2. Use of the antagonistic strain LZ31 as claimed in claim 1 in highly effectively preventing and controlling sugarcane smut.

3. The application according to claim 2, characterized in that, The pathogen of the sugarcane smut is Sporisorium scitamineum Ustilago scitaminea Syd. .

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