A pepper seedling raising substrate with Trichoderma asperellum fungicide and biochar as materials has strong seedlings and yield increase

Abstract

This invention belongs to the field of seedling substrate technology, specifically relating to a chili seedling substrate that promotes robust seedling growth and increased yield using Trichoderma as a fungicide and biochar. The Trichoderma asperellum fungicide is composed of equal masses of fermentation broth from strain LZ35 and strain X17; wherein strain LZ35 is taxonomically named Trichoderma asperellum, with accession number GDMCC No: 65053; and strain X17 is taxonomically named Bacillus subtilis, with accession number GDMCC No: 64986. This invention screened strains LZ35 and X17, which showed no antagonistic interaction, from the rhizosphere soil of healthy chili plants. Fermentation broths of strains LZ35 and X17, wheat straw biochar, and substrate were mixed to prepare a seedling substrate. When applied to pepper plug seedling cultivation, it was found that adding the Trichoderma echinosporum agent of this invention can improve the germination potential, germination rate, and survival rate of pepper seedlings, promote pepper seedling growth, and provide support for pepper seedling cultivation and planting.

Description

Technical Field

[0001] This invention belongs to the field of seedling substrate technology, specifically relating to a chili seedling substrate that uses Trichoderma hygroscopica agent and biochar as materials to promote strong seedlings and increased yield. Background Technology

[0002] With the continuous improvement of people's consumption levels, my country's greenhouse vegetable industry has developed rapidly and has become the main direction of vegetable industry development. Currently, the annual output of greenhouse vegetables exceeds 265 million tons, with a value of nearly 700 billion yuan. The greenhouse vegetable industry has become an important way for Chinese farmers to increase their income and create wealth. Chili peppers are one of my country's main vegetables, with a planting area of ​​827,000 hectares in 2021. 2 China's production accounts for about 46% of the world's total. With the increasing area under chili cultivation, it has become an important part of the vegetable industry.

[0003] Seedling raising is the primary and crucial technical link in the vegetable industry. However, with the development of large-scale and modern agricultural production, traditional agricultural breeding methods can no longer meet the needs of current agricultural planting because they cannot guarantee seedling quality. Intensive seedling raising, represented by plug tray seedling raising, has been widely adopted. Intensive seedling raising is simple to operate, saves time and labor, conserves seeds and pesticides, effectively reduces the damage from pests and diseases, overcomes continuous cropping obstacles, facilitates management, and produces robust seedlings. The seedling substrate is the foundation for seedling root growth and is a key element in intensive seedling raising technology. Its quality has a significant impact on the quality of seedlings, and the quality of pepper seedlings has a crucial influence on their yield and fruit quality. Cultivating robust pepper seedlings is a fundamental prerequisite for increasing pepper yield and profits.

[0004] The commonly used vegetable seedling substrates are made of a mixture of peat, perlite and vermiculite, or a mixture of peat and vermiculite. In many cases, they only provide a growing environment for vegetable seedlings and have a single function.

[0005] Soil contains a large number of beneficial microorganisms. Among them, rhizosphere microorganisms in the rhizosphere can promote crop growth and yield through direct (activation of soil nutrients, production of plant hormones, induction of beneficial microbial enrichment, etc.) and indirect (production of antimicrobial substances, induction of plant systemic resistance and stress, nutrient space competition, etc.) mechanisms, while effectively mitigating the damage to soil health. Therefore, research on seedling substrates containing microbial agents is of great significance.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a chili seedling substrate that uses Trichoderma hygroscopica and biochar as materials to promote strong seedlings and increased yield. This substrate can improve the germination potential and germination rate of chili seeds and the survival rate of chili seedlings, thereby promoting the growth of chili seedlings.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A Trichoderma acicularis agent, wherein the Trichoderma acicularis agent is prepared by mixing equal masses of fermentation broth from strain LZ35 and fermentation broth from strain X17; wherein,

[0010] The strain LZ35, taxonomically named *Trichoderma asperellum*, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 23, 2024, with accession number GDMCC No: 65053.

[0011] The strain X17, taxonomically named Bacillus subtilis, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 7, 2024, with accession number GDMCC No: 64986.

[0012] Preferably, the preparation method of the fermentation broth of strain LZ35 is as follows: strain LZ35 is inoculated on PDA medium and cultured at a constant temperature of 28°C until sporulation occurs. The sporulation is washed away with sterile water, the bacterial cells are filtered through four layers of gauze, and the spore concentration is diluted to 1.0 × 10⁻⁶ with sterile water. 7 cfu / g.

[0013] Preferably, the fermentation broth of strain X17 is prepared as follows: strain X17 is inoculated onto LB liquid medium and cultured on a shaker at 37°C and 180 rpm / min until the logarithmic growth phase; the bacterial concentration is then diluted to 1.0 × 10⁻⁶ using LB liquid medium. 8 cfu / g.

[0014] The present invention also provides a seedling substrate comprising the aforementioned Trichoderma acicularis agent.

[0015] Preferably, the seedling substrate is composed of a mixture of substrate, biochar, and Trichoderma hygroscopic agent in a mass ratio of 1:0.1:0.4-0.5.

[0016] As a preferred embodiment, the preparation method of the biochar is as follows: take wheat straw, wash it, dry it to constant weight, crush it into small pieces of 3-5 cm, place it in a carbonization furnace, pyrolyze it at 450-600℃ for 2-3 hours, cool it naturally to room temperature, rinse it with water, dry it to constant weight, crush it and pass it through a 150-200 mesh sieve to obtain the biochar.

[0017] Preferably, the matrix is ​​a mixture of peat moss and vermiculite in a mass ratio of 7:3.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention isolates and screens bacterial strains from the rhizosphere soil of healthy chili pepper plants. A plate confrontation experiment was conducted on well-growing strains to screen for strains LZ35 and X17, which showed no antagonistic effect. Molecular biological identification confirmed that strain LZ35 is *Trichoderma echinococcus* and strain X17 is *Bacillus subtilis*. A seedling substrate was prepared by mixing the fermentation broth of strains LZ35 and X17, wheat straw biochar, and a substrate. When applied to chili pepper plug seedling cultivation, the addition of the *Trichoderma echinococcus* agent of this invention improved the germination potential, germination rate, and survival rate of chili pepper seedlings, promoting seedling growth and providing support for chili pepper seedling cultivation and planting.

[0020] Preservation Information

[0021] Strain LZ35, taxonomically named *Trichoderma asperellum* LZ35, was deposited on August 23, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 65053.

[0022] Strain X17, taxonomically named Bacillus subtilis X17, was deposited on August 7, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 64986. Attached Figure Description

[0023] Figure 1 The colony and phylogenetic tree of strain LZ35;

[0024] Figure 2 This is a colony and phylogenetic tree of strain X17. Detailed Implementation

[0025] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0026] In the following examples, the PDA culture medium formula is: 200g potato, 20g glucose, 15g agar, 1000mL sterile water, pH 7.0.

[0027] The LB liquid culture medium formula is: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1000mL sterile water, pH 7.0.

[0028] 1. Isolation and purification of bacterial strains

[0029] Rhizosphere soil from healthy chili pepper plants was collected and prepared into a soil suspension. A gradient dilution plating method was used, with 0.1 mL of each suspension applied at different rates. -3 10 -4 10 -5 Fungi were screened on PDA medium using dilutions, with three replicates per gradient, and incubated at 28°C for 3-10 days. Simultaneously, 0.1 mL of 10... -5 10 -6 10 -7 Bacteria were screened on LB medium using dilutions, with three replicates per gradient, and incubated at 37°C for 1-2 days. Then, the well-growing fungi and bacteria were isolated, purified, and numbered for differentiation.

[0030] 2. Screening and identification of fungi and bacteria

[0031] The isolated and purified strains with good growth were subjected to plate confrontation experiments to screen out strains that did not have antagonistic effects on each other. DNA was extracted using the PowerSoil DNA kit (Mobio Laboratories, Carlsbad, CA, USA), and the taxonomic position of the strains was determined by 16S / ITS rDNA amplicon sequencing.

[0032] After screening, one fungal strain LZ35 and one bacterial strain X17 were obtained. Molecular biological identification confirmed that strain LZ35 is *Trichoderma asperellum*, and strain X17 is *Bacillus subtilis*. The morphological characteristics and phylogenetic trees of the two strains are shown below. Figure 1 and 2 The base sequences are shown in SEQ ID No. 1 and SEQ ID No. 2 of the sequence listing.

[0033] 3. Preparation of LZ35 and X17 fermentation broths

[0034] Strain LZ35 was inoculated onto PDA medium and cultured at 28°C until sporulation occurred. The sporulation was eluted with sterile water, and the bacterial cells were filtered through four layers of gauze before the spore concentration was diluted to 1.0 × 10⁻⁶ with sterile water.7 cfu / g.

[0035] Strain X17 was inoculated onto LB liquid medium and cultured at 37°C on a shaker at 180 rpm / min until the logarithmic growth phase. The bacterial concentration was then diluted to 1.0 × 10⁻⁶ with LB liquid medium. 8 cfu / g.

[0036] 4. The effects of different seedling substrates on pepper tray seedling cultivation

[0037] Prepare the seedling substrate as shown in Table 1. After mixing the seedling substrate evenly, fill the 72-cell trays. In Table 1, the substrate is composed of peat moss and vermiculite mixed in a mass ratio of 7:3. The preparation method of biochar is as follows: take wheat straw, wash it, dry it to constant weight, crush it into small pieces of 5 cm, place it in a carbonization furnace, pyrolyze it at 00℃ for 2.5 h in an anaerobic environment, cool it naturally to room temperature, rinse it with clean water, dry it to constant weight, crush it and pass it through a 150-mesh sieve to obtain the biochar.

[0038] Table 1 Different seedling substrates

[0039] matrix 2000g 2000g 2000g 2000g Biochar 200g 200g 200g 200g Fermentation broth of strain LZ35 -- 800g -- 400g Fermentation broth of strain X17 -- -- 800g 400g sterile water 800g -- -- --

[0040] Select uniformly sized and plump "Tiandeng Zhitianjiao" chili seeds, disinfect them with 75% ethanol for 2 minutes, rinse them three times with sterile water, and then soak them in sterile water for 24 hours. Sow the treated chili seeds into 72-cell trays, cover with 0.5 cm of seedling substrate, and sow one seed per cell. Each seedling substrate was replicated four times, with 25 chili seeds per replicate. The number of germinating seeds was recorded on day 7 and day 14. On day 50, the number of plants, plant height, stem diameter, fresh weight, and dry weight were measured. Germination potential, seed germination rate, survival rate, leaf area, and seedling vigor index were calculated. The results are shown in Table 2.

[0041] in,

[0042]

[0043]

[0044]

[0045]

[0046] Table 2. Effects of different seedling substrates on pepper seedling tray cultivation

[0047] Number of seeds germinated and breaking through the soil on day 7 6 14 11 27 Number of seeds germinated on day 14 79 91 84 97 Number of seedlings that survived on day 50 (plants) 68 84 79 93 Plant height (cm) 9.34 12.08 10.13 18.99 Stem diameter (mm) 1.93 2.49 2.65 4.07 Fresh weight (g) 2.09 9.84 7.50 21.11 Dry weight (g) 0.14 0.23 0.21 0.46 Germination potential (%) 6 14 11 27 Seed germination rate (%) 79 91 84 97 Survival rate (%) 68 84 79 93 Strong seedling index 0.0289 0.0474 0.0549 0.0986

[0048] As shown in Table 1, compared with seedling substrate 1, seedling substrates 2-4 showed significant improvements in plant height, fresh weight, leaf number, germination vigor, seed germination rate, survival rate, and seedling vigor index. Seedling substrate 4 showed significantly better improvements in all aspects than seedling substrates 2 and 3, with statistically significant differences. Overall, adding the Trichoderma echinosporum agent of this invention to the substrate can significantly promote the germination of chili seeds and the growth of seedlings.

[0049] In summary, adding the Trichoderma hygroscopic agent of the present invention to the substrate can improve the germination potential, germination rate and survival rate of chili seeds and promote the growth of chili seedlings.

[0050] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. The application of a seedling substrate in improving the germination potential, germination rate, and survival rate of chili seedlings, characterized in that... The seedling substrate is composed of a mixture of substrate, biochar, and inoculant in a mass ratio of 1:0.1:0.4-0.5; wherein, The matrix is ​​composed of peat moss and vermiculite mixed in a mass ratio of 7:3; The preparation method of the biochar is as follows: take wheat straw, wash it, dry it to constant weight, crush it into small pieces of 3-5 cm, place it in a carbonization furnace, pyrolyze it at 450-600℃ for 2-3 hours, cool it naturally to room temperature, rinse it with water, dry it to constant weight, crush it and pass it through a 150-200 mesh sieve to obtain the biochar. The inoculum is a mixture of equal masses of fermentation broth from strain LZ35 and strain X17; strain LZ35 is taxonomically named *Trichoderma hyacinthus*. Trichoderma asperellum LZ35 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 23, 2024, with accession number GDMCC No: 65053; the taxonomic name of the strain X17 is Bacillus subtilis (…). Bacillus subtilis X17 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 7, 2024, with accession number GDMCC No: 64986.

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