A kind of Trichoderma aspergillus HN01 and its bacterial agent and application

Abstract

The present invention relates to the technical field of microorganisms, and in particular to a strain of Trichoderma aspergillus HN01 and a bacterial agent and application thereof. The strain is preserved in the China Center for Type Culture Collection and is named Trichoderma aspergillus HN01. Trichoderma asperellum HN01, with a deposit number of CCTCC: M 2024589 and a deposit date of March 29, 2024, was isolated and identified based on the soil microbiome. It has a preventive effect on cassava bacterial wilt, 1×10 6 The lesion area of ​​cassava material treated with Trichoderma spinosum spore suspension with spore count / mL is about 59.12% of that of untreated cassava material. In a short period of time, the resistance of cassava to cassava bacterial wilt can be improved, which is of great significance for maintaining the sustainable and healthy development of the cassava industry.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Trichoderma aspergillus HN01 and a bacterial agent and application thereof. Background Art

[0002] Cassava ( Manihot esculenta Crantz, a plant of the genus Cassava in the Euphorbiaceae family, is one of the world's three major tuber crops (potato, sweet potato, cassava), a tropical and subtropical high-starch crop, and the main food source for more than 700 million people in the world. It has a wide range of uses in food, feed processing, industrial raw materials, etc. Cassava has many advantages such as drought and barrenness tolerance, easy growth, low land requirements, relatively low production costs, and high photosynthetic efficiency. It has been widely planted around the world. The cassava planting area in China is second only to rice, sweet potato, sugarcane and corn. Cassava is suitable for planting areas with excellent light, heat and water conditions and rich biodiversity, which is very conducive to the occurrence and harm of various diseases, insects and weeds. It is reported that cassava diseases outside China include bacterial wilt, mosaic virus disease, brown spot disease, anthracnose, vermifuge leaf spot disease, coryneformis leaf spot disease, white spot disease, Phytophthora root rot disease, powdery mildew, brown streak disease, frog skin disease and other diseases. Among them, the main diseases are mosaic virus disease, brown streak disease, powdery mildew and frog skin disease. The main cassava diseases in China are cassava bacterial wilt, root rot and brown spot.

[0003] Cassava bacterial wilt is caused by Xanthomonas cassavaii pv. Xanthomonas axonopodis pv. manihotis ). With the continuous development of China's cassava industry, the exchange of germplasm and products with other countries has become increasingly frequent. Global diseases such as bacterial wilt have occurred in major cassava-growing areas in China, and the damage situation is very serious. In production, the disease is mainly controlled by seedling quarantine, biological control, and drug control, and there is still a lack of effective prevention and control methods. Therefore, analyzing the correlation between the disease resistance of cassava germplasm and the microbiome, thereby exploring typical beneficial microbial germplasm resources, exogenously treating cassava, and improving its disease resistance in the short term, so as to reduce the impact of diseases on the cassava industry and steadily improve the cassava industry, has become a realistic problem that needs to be solved for the sustainable and healthy development of the cassava industry.

[0004] Trichoderma belongs to the class of Hypomycetes, the order of Hypomycetes, the class of Hypomycetes, the class of Myxomycetes, and is widely distributed. It is a component of humus, organic matter, and plant root systems. Studies have also shown that it is a non-toxic plant symbiont. Trichoderma mainly inhibits the growth and reproduction of target pathogens through competition, induction of host resistance, hyperparasitism, and antibiotics. Trichoderma asperellum) is one of the most developed and applied bacteria. How to further improve the disease resistance of cassava so that cassava can be better colonized under relevant environmental stress conditions is a hot topic in current research. At present, there is no method to quickly improve the disease resistance of cassava. Therefore, it is of great significance to use biocontrol bacteria to improve the disease resistance of cassava and maintain the sustainable and healthy development of the cassava industry. Summary of the invention

[0005] In view of this, the present invention aims to provide a Trichoderma aspergillus HN01 and an application thereof for improving the disease resistance of cassava in a short period of time.

[0006] In a first aspect, the present invention provides a kind of Trichoderma aspergillus, named:

[0007] Trichoderma aspergillus HN01

[0008] Trichoderma asperellum HN01

[0009] Deposited in China Center for Type Culture Collection,

[0010] The deposit number is CCTCC: M 2024589,

[0011] The preservation date is March 29, 2024.

[0012] The present invention obtains Trichoderma aspergillus HN01 based on the separation and identification of soil microbial groups.

[0013] In a second aspect, the present invention provides the use of the above-mentioned Trichoderma aspergillus HN01 for improving the resistance of cassava to cassava bacterial wilt in a short period of time.

[0014] In a third aspect, the present invention provides a microbial agent comprising the above-mentioned Trichoderma aspergillus HN01.

[0015] In a fourth aspect, the present invention provides a method for preparing the above-mentioned microbial agent, wherein the Trichoderma spinulosa HN01 is placed in a culture medium to obtain the microbial agent.

[0016] Further, the culture medium is LB or PDA culture medium. Further, it is LB culture medium.

[0017] In a fifth aspect, the present invention also provides the use of the above-mentioned microbial agent in improving the resistance of cassava to cassava bacterial wilt in a short period of time.

[0018] In a sixth aspect, the present invention provides a method for improving disease resistance of cassava, wherein the disease resistance is resistance to cassava bacterial wilt, and the above-mentioned microbial agent is injected into the cassava plants or sprayed on the surface of the cassava.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The Trichoderma spinulosum HN01 of the present invention has a strong control effect on cassava bacterial wilt, and 1×10 6 The disease spot area of ​​cassava materials treated with a spore suspension of Trichoderma aspergillus at a spore count of 1×106 / mL is about 59.12% of the disease spot area of ​​cassava materials not treated with a spore suspension of Trichoderma aspergillus at a spore count of 1×106 / mL. This can improve the disease resistance of cassava in the short term, which is of great significance for maintaining the sustainable and healthy development of the cassava industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation on the present invention. In the drawings:

[0022] Figure 1 A schematic diagram of the correlation between Trichoderma in different tissues of cassava and disease resistance of cassava in Example 2 of the present invention;

[0023] Figure 2 Schematic diagram of the antibacterial test of the confrontation culture of Trichoderma acanthosporum and cassava bacterial blight in Example 3 of the present invention, wherein Figure (a) is the antibacterial circle and Figure (b) is the antibacterial rate;

[0024] Figure 3 This is a schematic diagram of the effect of continuous watering with a suspension of Trichoderma aspergillus spores for 5 days on the resistance of cassava to cassava bacterial wilt pathogen in Example 3 of the present invention, wherein (a) shows the number of bacteria and (b) shows the area of ​​lesions. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the invention more clear, the invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described here are only used to explain the invention and do not constitute a limitation of the invention.

[0026] The present invention obtains Trichoderma aspergillus HN01 based on the separation and identification of soil microorganisms, and uses the Trichoderma aspergillus HN01 for induction treatment to improve the resistance of cassava to cassava bacterial wilt. The separation and identification process is as follows:

[0027] First, prepare the microbial metagenomics samples;

[0028] Then use Shotgun-Metagenome to perform deep sequencing of metagenomic functional genes;

[0029] Then, we analyzed and counted the bioinformatics data to analyze the abundance of the microbiome in different parts of different germplasms and the correlation between the abundance of beneficial microorganisms and disease resistance;

[0030] Finally, the Trichoderma was isolated and purified, and the beneficial microorganisms were isolated and identified to obtain Trichoderma acanthosporum HN01, which was then identified by morphology and meristem.

[0031] The antibacterial assay process is as follows:

[0032] In vitro antibacterial assay: Co-culture Trichoderma spinulosum and cassava bacterial wilt pathogen to observe whether there is an inhibition zone and calculate the inhibition rate.

[0033] In vivo antibacterial assay: Cassava seedlings at any stage of growth were sprayed with a suspension of Trichoderma spinulosa spores on the leaves for 5 consecutive days, twice a day. Xam Then, the number of bacteria and the area of ​​lesions were counted.

[0034] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0035] Example 1

[0036] Preparation and deep sequencing of microbial metagenomic samples:

[0037] In a clean bench, rhizosphere samples of different germplasms were transferred to enzyme-free sterile centrifuge tubes, and 2 times the extract solution of PBS buffer was added to mix thoroughly. Then, the samples were quickly placed in a high-speed refrigerated centrifuge and centrifuged at 4000g at 4°C to collect microorganisms.

[0038] Extraction and purification of microbial metagenomic DNA in samples: Since the Shotgun-Metagenome deep sequencing technology is to sequence the genomic DNA of all microorganisms in the sample, it has strict requirements on sample DNA extraction, large total amount of DNA obtained, and high integrity requirements, so that more objective and reliable results can be obtained in the subsequent DNA fragmentation and data splicing and annotation process. Therefore, the present invention uses MBIO's feces extraction kit, combined with lysozyme, a gentle cell wall disruption method, to perform multiple large-scale extractions to ensure the acquisition rate and quality of DNA.

[0039] Genomic DNA fragmentation and library construction: Use a physical method based on the ultrasonic principle to randomly break the DNA into fragments of 200-500bp in size, smooth and repair the DNA ends, add an "A" base to the 3' end, and then add a "Y"-shaped adapter to both ends of the DNA. PCR amplification connects the two ends to different adapter sequences, and finally detects the quality of the constructed library.

[0040] Shotgun-Metagenome deep sequencing: The above-constructed library was used for high-throughput sequencing using a second-generation sequencing platform (Illumina Hiseq 2500). The data from the sequencing machine was quality checked and impurities removed to remove low-quality sequences and fragments less than 70 bp in length, ensuring the output of high-quality data for subsequent bioinformatics analysis.

[0041] Example 2

[0042] Bioinformatics data analysis and statistics:

[0043] First, the high-quality sequence was compared with the host cassava genome, and the host genome sequence fragments and other eukaryotic sequences introduced by improper operation were removed, and only the genome sequence of the microorganism was retained. Then, based on the K-mer idea, the IDBA-UD splicing software was used to splice the small fragment sequence. After obtaining the spliced ​​sequence, the N50 value representing the splicing effect was calculated, and the qualified Contig was used for subsequent gene prediction. Since the number of gene predictions for each sample is very large, it is necessary to remove redundancy after merging all predicted genes to form a single gene set. After obtaining the completed gene set, the gene set was annotated for species and function through the Greengenes, Unite, NR database, EggNOG database and KEGG database, so as to obtain the sample's bacterial abundance distribution, functional gene abundance and metabolic pathway enrichment. For metabolomics data, the analysis software provided by NMR was used to analyze and determine each metabolite.

[0044] After completing the above preliminary analysis, multivariate statistical methods and R open source software were used to integrate and mine all bioinformatics data. Figure 1 As shown, the analysis found that the correlation coefficients between the abundance of Trichoderma in different tissue parts and the disease resistance of cassava were different, among which the correlation coefficient between the abundance of Trichoderma in the stem and the disease resistance of cassava reached 0.54.

[0045] Example 3

[0046] Isolation and identification of beneficial microorganisms:

[0047] In a clean workbench, transfer the stem samples of different germplasms to enzyme-free sterile centrifuge tubes, add PBS buffer with 2 times the extract and mix thoroughly. Then spread the mixture evenly on the LB plate, place it at 28°C for 2 days, and then pick a single colony for further separation, purification and identification.

[0048] In vitro antibacterial assay:

[0049] The concentration of the activated cassava bacterial wilt pathogen was 1×10 6Spore count / mL, take 100mL of LB medium and wait for it to cool to 40℃, add 1mL of the above bacterial solution and mix quickly and evenly, pour the plate, add about 10mL of the bacterial culture medium to each culture dish, and wait for the culture medium to completely cool and solidify. Take a vigorously growing Trichoderma acanthosporum plate, punch a hole with a 5mm puncher to obtain a bacterial cake, and inoculate it to the center of the plate with cassava bacterial wilt bacteria, and inoculate an LB plate of the corresponding size as a control. Each test treatment was repeated 5 times, and the test was repeated 3 times. Place it in a constant temperature incubator at 28℃ for cultivation, observe it every 24 hours, and observe whether there is an inhibition zone as the fungal colony grows. After 3 days, the size of the inhibition zone was measured by the cross method, and the inhibition rate was calculated.

[0050] After 3 days of co-culture of Trichoderma aspergillus and pathogenic bacteria, Figure 2 As shown in (a), the number of pathogenic bacteria in the treatment group is significantly less than that in the control group, and the inhibition zone is more obvious. Figure 2 As shown in (b), the inhibition rate of Trichoderma aspergillus reached 33.75%.

[0051] In vivo antibacterial assay:

[0052] For cassava seedlings at any stage of growth, the height is 15-30cm, and 1×10 6 Sporangium suspension of Trichoderma spinulosa with spore count / mL, twice a day; at other times, water according to normal conditions or irrigate with rainwater according to natural conditions; 20-40 days, every 667m 2 Water 300-600L; after 40 days, irrigate with rainwater according to natural conditions.

[0053] Cassava bacterial wilt pathogen ( Xam , isolated from cassava diseased leaves at the teaching base of the College of Tropical Agriculture and Forestry, Hainan University) was activated on LB (containing 10g NaCl, 10g tryptone, and 5g yeast extract per liter) plates, and a single colony was picked and cultured in 10mL LB culture medium (28℃, 180rpm) for 12h, and then the bacterial solution was transferred to a new LB culture medium for continued culture until the bacterial solution concentration reached OD600≈0.6. After centrifugation at 4000rpm and room temperature for 10min, the supernatant was discarded. The bacterial solution was suspended and diluted with 10mM MgCl2 solution (molar mass concentration, i.e., 95.21mg MgCl2 per liter of water) to OD600≈0.2, and the bacterial solution was injected into the mesophyll on both sides of the main vein with a syringe. With the injection point as the center, use a puncher (1 cm in diameter) to take leaf discs of the same size, disinfect with 75% alcohol for 3 min, wash with sterile water 4 times, grind into a homogenate, dilute with sterile water, drop the homogenate on the LB plate, culture at 28°C overnight, and count the number of bacteria. At the same time, count the area of ​​lesions 6 days after inoculation.

[0054] By continuously spraying 1×10 6 The cassava materials treated with a suspension of Trichoderma spinulosum with a spore count / mL were found to be more resistant to cassava bacterial wilt than the untreated cassava materials. Figure 3 As shown in (a), the bacteria surviving on cassava reproduce faster and have more bacteria. At 6 days after infection, the log value of the number of bacteria reached about 5.83, while the number of bacteria treated with the suspension of Trichoderma spinulosa spores reached about 5.22. Figure 3 The leaf lesion area is shown in (b) and the lesion area of ​​the control group is about 0.22 cm 2 , while the treatment group was only 0.13cm 2 , that is, 1×10 6 The lesion area of ​​cassava material treated with Trichoderma aculeatus spore suspension with spore count / mL is about 59.12% of the lesion area of ​​untreated cassava material, indicating that the treatment with Trichoderma aculeatus spore suspension can improve the resistance of cassava to cassava bacterial wilt.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Trichoderma spinulosa, designated as: Trichoderma aspergillus HN01 Trichoderma asperellum HN01, It is deposited in the China Center for Type Culture Collection, with the deposit number CCTCC: M 2024589 and the deposit date of March 29, 2024.

2. Use of Trichoderma aspergillus HN01 according to claim 1 in improving the resistance of cassava to cassava bacterial wilt.

3. A microbial agent, characterized in that: The invention comprises the Trichoderma aspergillus HN01 according to claim 1.

4. The method for preparing the microbial agent according to claim 3, characterized in that: The Trichoderma aspergillus HN01 described in claim 1 is inoculated into a culture medium to obtain the microbial agent.

5. The method for preparing the microbial agent according to claim 4, characterized in that: The culture medium is LB or PDA culture medium.

6. Use of the microbial agent as claimed in claim 3 in the resistance of cassava to cassava bacterial wilt.

7. A method for improving disease resistance of cassava, characterized in that: The disease resistance in the method for improving the disease resistance of cassava is resistance to cassava bacterial wilt, and the method for improving the disease resistance of cassava is injecting the microbial agent as claimed in claim 3 into the cassava plants or spraying the surface of the cassava.

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