Trichoderma harzianum agent for promoting growth of allium plants and application thereof

Abstract

The application provides a trichoderma harzianum agent for promoting growth of allium plants and application thereof, the trichoderma harzianum agent is trichoderma harzianum strain MCBC3, the trichoderma harzianum agent is applied in combination with diammonium phosphate, the application amount of the diammonium phosphate is 15 kg per mu, and the application amount of the trichoderma harzianum agent is 1 L per mu; existing trichoderma resource development is concentrated on normal soil environment, and the trichoderma harzianum MCBC3 is screened from saline-alkali soil for the first time, the salt tolerance and alkali tolerance of the trichoderma harzianum MCBC3 directly aim at the double stress of high salt and high alkali in saline-alkali soil, and the technical bottleneck of poor adaptability and low survival rate of traditional strains in extreme environment is solved.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Trichoderma inoculant for promoting the growth of Allium crops in saline-alkali areas and its application. Background Technology

[0002] Allium L. is an important global resource of vegetables, medicinal plants, and ornamental plants. Wild leeks and scallions are two typical examples of Allium species, mainly distributed in the northern grasslands of my country. Wild leeks (Allium senescens L.) are perennial herbaceous plants belonging to the genus Allium in the Liliaceae family, while scallions (Allium schoenoprasum) are bulbous plants belonging to the genus Allium in the Liliaceae family. Both crops have value as feed, food, and medicine, and contain high levels of protein at different growth stages, making them a preferred forage for fattening livestock.

[0003] With the increasing demand for wild chives and scallions, there is a need to continuously expand the cultivation area and growth range of wild chives. However, the available planting areas are mostly arid and semi-arid regions with very limited water resources and serious soil salinization. Therefore, it is necessary to find an effective way to improve soil quality and promote crop growth.

[0004] Applying microbial agents is an effective measure for improving saline-alkali land. Trichoderma, a fungus belonging to the Ascomycota phylum and the Hordeocarpaceae family, is characterized by strong saprophytic properties and rapid growth and reproduction. It can quickly utilize nutrients in the soil to expand, strongly colonize, and survive for extended periods in the plant rhizosphere, making it a beneficial soil microorganism. However, currently, the exploration and application of Trichoderma resources in my country mainly focuses on normal growth environments, while Trichoderma strains under stress environments have not been effectively developed. Therefore, identifying and preserving dominant Trichoderma strains in saline-alkali stress environments can lead to the development of growth-promoting microbial fertilizer products for Allium crops in saline-alkali land, showing promising application prospects. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a Trichoderma inoculant for promoting the growth of Allium plants and its application, as detailed below:

[0006] A Trichoderma inoculant for promoting the growth of Allium plants, wherein the Trichoderma inoculant is Trichoderma harzianum strain MCBC3, which has accession number CGMCCNo.41745 and ITS sequence as shown in SEQ ID NO:1.

[0007] Furthermore, the growth conditions for strain MCBC3 are as follows: culture temperature 28℃, pH=5.0-6.0, carbon source is glucose or sucrose, and it is cultured on PDA medium.

[0008] Moreover, the strain MCBC3 is salt-tolerant and can grow under conditions of 800 mmol NaCl.

[0009] Furthermore, the strain MCBC3 is alkali-resistant and can grow under conditions of 0.5% sodium bicarbonate.

[0010] On the other hand, the present invention provides the application of Trichoderma inoculants for promoting the growth of Allium plants or improving the soil quality in saline-alkali areas.

[0011] Furthermore, the Allium species mentioned include wild leeks and northern leeks.

[0012] Furthermore, the application method includes spraying Trichoderma fungicide onto the roots of Allium crops while simultaneously applying diammonium phosphate.

[0013] Moreover, the application rate is 1L / acre, sprayed once a week for three consecutive weeks.

[0014] Furthermore, the Trichoderma inoculant is applied in combination with diammonium phosphate, with the diammonium phosphate application rate being 15 kg / mu and the Trichoderma inoculant application rate being 1 L / mu.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] 1. Wild leeks and northern onions are typical Allium species in the northern grasslands, with value in feed, food and medicine. However, their cultivation is limited by drought and salinization. This invention directly solves the problems of poor soil quality and low nutrient availability in saline-alkali land by applying MCBC3 inoculant in combination with diammonium phosphate, while promoting crop growth and meeting the needs of agricultural production in the northern grasslands.

[0017] 2. Existing Trichoderma resource development focuses on normal soil environments, while this invention is the first to screen Trichoderma harzianum MCBC3 from saline-alkali land. Its salt and alkali tolerance directly targets the dual stress of high salt and high alkali in saline-alkali land, solving the technical bottleneck of poor adaptability and low survival rate of traditional strains in extreme environments.

[0018] 3. The strain MCBC3 provided by this invention not only has salt and alkali tolerance, but also grows rapidly under optimal conditions (28℃, pH 5.0-6.0) (covering the plate in 60 hours), indicating that it has both high-efficiency amplification ability and stress tolerance.

[0019] 4. When the microbial agent of the present invention was applied, the plant height of wild leeks (M4) with microbial agent treatment reached 36.03 cm, which was 18.52% higher than the blank control (M1), 3.71% higher than conventional fertilization (M2), and 4.13% higher than chemical fertilizer + organic fertilizer (M3); the plant height of scallions (M4) reached 39.69 cm, which was 9.52% higher than M1, 5.03% higher than M2, and 6.41% higher than M3. This shows that the synergistic effect of the microbial agent of the present invention with chemical fertilizer is significantly better than that of chemical fertilizer or organic fertilizer alone, and it continues to perform well throughout the crop growth period, verifying its long-term growth-promoting ability.

[0020] 5. The MCBC3 strain provided by this invention colonizes crop roots or soil pores, propagates and grows, and produces metabolic activities. During the metabolic process, it secretes organic acid substances and growth hormones that are beneficial to plant growth. It regulates rhizosphere pH and promotes the dissolution of nutrients (such as phosphorus and potassium), thereby directly improving the physical and chemical properties of saline-alkali soil and creating a suitable microenvironment for crop roots. Attached Figure Description

[0021] Figure 1 The morphological characteristics of Trichoderma strains. Detailed Implementation

[0022] Example 1

[0023] 1. New strains of Trichoderma

[0024] The inoculum was Trichoderma harzianum MCBC3, strain accession number CGMCC: No.41745, accession date January 6, 2025, accession address: Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0025] Its optimal growth conditions are: culture temperature 28℃, pH 5.0-6.0, carbon source glucose or sucrose, and cultured on PDA medium. Trichoderma strain BC3 grows rapidly; after 60 hours of cultivation under optimal conditions, colonies completely cover a 9cm plate. Initially, the colonies are cottony and grayish-white; as the conidia mature, the colonies gradually turn green from the center to the edge. It is salt-tolerant (can grow in 800 mmol NaCl) and sodium bicarbonate-tolerant (can grow in 0.5% sodium bicarbonate). The molecular marker sequence is shown in SEQ ID NO:1.

[0026] 2. Screening and isolation of strains

[0027] The strain was screened in Huwei Grassland, Baicheng City, Jilin Province, using saline soil. The specific method for strain isolation is as follows:

[0028] (1) Preparation and dilution of soil stock solution

[0029] Soil stock solution was prepared by dissolving soil in sterile water. The prepared soil stock solution was then divided into 10... -1 10 -2 10 -3 Gradient dilution;

[0030] (2) Soil stock solution inoculation culture

[0031] The diluted soil stock solution was inoculated onto potato dextrose agar (PDA) medium. Figure 1 );

[0032] (3) Strain purification

[0033] Single colonies of Trichoderma were selected from the culture medium of the initial inoculation and streaked onto a new culture medium to purify the strain.

[0034] (4) Secondary purification of strains

[0035] Use a punch to collect mycelial cakes from the colonies, and use an inoculation needle to transfer the mycelial cakes into the center of a new PDA medium, with the mycelial side facing down, to improve the purity of the strain.

[0036] (5) Identification and preservation of strains

[0037] The cultured Trichoderma strain was used for strain sequencing, and Trichoderma harzianum MCBC3 was selected for strain preservation based on the sequencing results.

[0038] (6) Preservation of microbial strains

[0039] The main methods for preserving laboratory microbial strains include glycerol preservation, test tube slant preservation, and freeze-drying preservation.

[0040] Example 2

[0041] The experimental area was located in Wuyuan County, Bayannur City, Inner Mongolia Autonomous Region (location information: longitude 108.2041, latitude: 41.0512). The basic physicochemical properties of the tested soil were as follows: organic matter 21.60 g / kg, total phosphorus 3.11 g / kg, total potassium 12.45 g / kg, available nitrogen 51.45 mg / kg, available phosphorus 8.91 mg / kg, available potassium 64.53 mg / kg, and pH value 8.76.

[0042] Using scallions and wild leeks as the test crops, the experiment was designed with four treatments:

[0043] Treatment ①: No fertilizer applied (blank), recorded as M1;

[0044] Treatment ②: Apply 25 kg / mu of fertilizer, recorded as M2;

[0045] Treatment ③: 25 kg / mu of chemical fertilizer + 200 kg / mu of organic fertilizer, recorded as M3;

[0046] Treatment ④: 15 kg / mu of chemical fertilizer + 1 L / mu of MCBC3 Trichoderma solution, designated as M4;

[0047] Treatment ⑤: Trichoderma MCBC3 solution (1L / acre), designated as M5.

[0048] Each treatment was replicated 6 times. Diammonium phosphate was used as the chemical fertilizer, and the organic fertilizer was purchased from Inner Mongolia Mengcao Plant Nutrition Technology Co., Ltd. (product name: Mengcao Organic Fertilizer, N+P2O5+K2O≥4%, organic matter≥30%), with an application rate of 200 kg / mu.

[0049] Chemical and organic fertilizers were applied to the soil as base fertilizer. The test crops were planted by transplanting. After seedling establishment, Trichoderma inoculant was sprayed on the crop roots at a rate of 1 L / mu, applied to the soil around the seedlings, once a week for three weeks, followed by normal watering. Plant height was observed monthly during the crop growth period, for a total of three times. Soil samples were collected during the last crop height observation to observe the effects of the Trichoderma inoculant solution on soil physicochemical properties.

[0050] (I) The impact of Trichoderma mycelium solution on soil

[0051] Table 1. Effects of different treatments on soil pH and available nutrients

[0052]

[0053] The effects of different treatments on soil pH and available nutrients are shown in Table 1. From the perspective of soil physicochemical properties after the crop growth period, the soil pH of treatment M4 was 8.68, which was 0.1, 0.11, 0.12, and 0.08 units lower than the blank control (M1), conventional fertilization (M2), chemical fertilizer + organic fertilizer (M3), and single application of Trichoderma solution (M5), respectively. This indicates that applying Trichoderma solution MCBC3 to saline-alkali land has a promoting effect on reducing soil pH and improving soil salinity. The soil organic matter content of treatment M4 was significantly lower than that of treatment M5. The organic matter content was 37.20 g / kg, which was higher than that of the blank control (M1), conventional fertilization (M2), chemical fertilizer + organic fertilizer (M3) treatment and single application of Trichoderma solution (M5), indicating that the application of Trichoderma solution MCBC3 on saline-alkali land has a promoting effect on increasing soil organic matter content. As for soil available nitrogen and available phosphorus, the M4 treatment increased the content of soil available nitrogen and available phosphorus compared with other treatments, especially the activation effect of soil phosphorus, which increased the content of available phosphorus in the soil.

[0054] It is worth emphasizing that, compared with other treatment groups, the M4 treatment still showed the most significant effect in terms of fertilizer application reduction, proving that the combined application of diammonium phosphate and Trichoderma inoculant can play a synergistic role.

[0055] (II) Effects of Trichoderma mycelium solution on the height of wild leek plants

[0056] The effects of different treatments on the plant height of *Allium harzianum* are shown in Table 2. From the last observation of the crop's growth period, the plant height of treatment M4 reached 36.03 cm, which was 5.63 cm, 1.29 cm, 1.43 cm, and 0.95 cm higher than the control (M1), conventional fertilization (M2), chemical fertilizer + organic fertilizer (M3), and single application of *Trichoderma harzianum* solution (M5), respectively. The growth rates were 18.52%, 3.71%, 4.13%, and 2.71%, respectively. Application of *Trichoderma harzianum* fertilizer had a good growth-promoting effect on *Allium harzianum*, and the plant height of treatment M4 was superior to other treatments throughout the entire crop growth period. *Trichoderma harzianum* MCDC3 bacterial solution had a good growth-promoting effect on *Allium harzianum* in saline-alkali soil environments, and also showed synergistic effects when combined with chemical fertilizer application.

[0057] Table 2. Effects of different treatments on the height of wild leeks (unit: cm)

[0058]

[0059] (III) Effects of Trichoderma mycelium solution on the height of scallion plants

[0060] Applying Trichoderma fertilizer also has a good growth-promoting effect on scallions. Throughout the entire crop growth period, the plant height of M4 is still better than that of other treatments.

[0061] Based on the last observation of the growth period of the scallion crop, the plant height of M4 reached 39.69 cm, which was 3.45 cm, 1.90 cm, 1.43 cm and 1.68 cm higher than the blank control (M1), conventional fertilization (M2), chemical fertilizer + organic fertilizer (M3) treatment and single application of Trichoderma solution (M5), respectively. The growth rates were 9.52%, 5.03%, 6.41% and 4.42% (as shown in Table 3).

[0062] The experimental results show that Trichoderma harzianum MCDC3 bacterial solution has a good growth-promoting effect on scallions in saline-alkali soil environment, and can be used in combination with chemical fertilizers to enhance the planting effect.

[0063] Table 3. Effects of different treatments on the height of scallion plants (unit: cm)

[0064]

[0065] Example 3

[0066] Experiments were continued in the same experimental area, using new wheatgrass ( Psathyrostachys juncea (Fisch.) Nevski The variety (Mengnong No. 4) was used as the test crop. Four treatments were designed in the experiment:

[0067] Treatment ①: No fertilizer applied (blank), recorded as N1;

[0068] Treatment ②: Apply 25 kg / mu of fertilizer, recorded as N2;

[0069] Treatment ③: MCBC3 Trichoderma solution (1L / acre) is designated as N3;

[0070] Treatment ④: 15 kg / mu of chemical fertilizer + 1 L / mu of MCBC3 Trichoderma solution, recorded as N4;

[0071] Each treatment was repeated 6 times.

[0072] New wheatgrass is sown in rows at a rate of 1.5 kg / mu with a row spacing of 40 cm.

[0073] Apply diammonium phosphate as base fertilizer in strips. After the crop seedlings are established, spray the roots with Trichoderma fungicide once a week at a rate of 1L / acre. After three applications, water normally. Spray the fungicide solution again during the jointing and heading stages of the crop.

[0074] (a) Effects of Trichoderma mycelium solution on the height of new wheat straw plants

[0075] The effects of different treatments on the plant height of new wheatgrass are shown in Table 4. Applying Trichoderma harzianum solution (N3) to new wheatgrass did not promote plant height at different growth stages. Plant height was measured at the heading stage. The plant height of the fertilizer-only treatment (N2) was 139.83 cm, which was 21.31%, 25.07%, and 7.31% higher than the no-fertilizer treatment (N1), the fungal solution-only treatment (N3), and the mixed treatment (N4), respectively. These results indicate that *Trichoderma harzianum* (T. harzianum) significantly contributes to the aboveground biomass of new wheatgrass. Trichoderma harzianum MCDC3 has no obvious growth-promoting effect, and its synergistic effect when mixed with chemical fertilizers is also not significant.

[0076] Table 4. Effects of different treatments on the height of new wheatgrass plants (unit: cm)

[0077]

[0078] (II) Effects of Trichoderma mycelium solution on relevant indicators of new wheat straw seed yield

[0079] The effects of different treatments on the seed yield of new wheatgrass are shown in Table 5. The table shows that applying Trichoderma harzianum solution (N3) had no significant effect on increasing crop seed yield. Under the conventional fertilization treatment (N2), the average ear length was 13.07 cm, which was 23.3%, 28.13%, and 14.65% higher than the no-fertilization treatment (N1), the single-application of the fungal solution treatment (N3), and the mixed application of the fungal solution treatment (N4), respectively. Under the conventional fertilization treatment (N2), the average ear weight was 0.243 g, which was 8.97%, 13.02%, and 2.1% higher than the no-fertilization treatment (N1), the single-application of the fungal solution treatment (N3), and the mixed application of the fungal solution treatment (N4), respectively. The results indicate that for the seed yield of new wheatgrass, Trichoderma harzianum (N3) has a significant effect on increasing crop seed yield. Trichoderma harzianum MCDC3 has no obvious growth-promoting effect, and its synergistic effect when mixed with chemical fertilizers is also not significant.

[0080] Table 5. Effects of different treatments on the seed yield of new wheatgrass (unit: cm)

[0081]

[0082] In summary, Trichoderma harzianum ( Trichoderma harzianum MCDC3, a strain screened in saline-alkali soils, exhibits good adaptability to saline-alkali soils and can be used as a microbial fertilizer in the cultivation of crops in saline-alkali lands. However, different crops adapt differently to saline-alkali soil environments, and its adaptability varies with *Trichoderma harzianum* (…). Trichoderma harzianum The interaction effects of MCDC3 also differ. When planting Allium chinense and other Allium species in saline-alkali soil, the interaction effects of Trichoderma harzianum (MCDC3) are observed. Trichoderma harzianum MCDC3 has a positive effect on crop growth and can be developed and applied as a special microbial fertilizer product for Allium crops in saline-alkali land.

Claims

1. The application of a Trichoderma inoculant for promoting the growth of Allium plants, characterized in that, This is used to promote the growth of Allium species in saline-alkali areas, including Allium chinense and Allium spp.; The application method is as follows: the Trichoderma inoculant is applied in combination with diammonium phosphate, wherein the application rate of diammonium phosphate is 15 kg / mu and the application rate of Trichoderma inoculant is 1 L / mu; The Trichoderma inoculum is Trichoderma harzianum strain MCBC3, with accession number CGMCCNo.41745 and ITS sequence as shown in SEQ ID NO:1; The strain MCBC3 exhibits salt and alkali tolerance, and can grow under conditions of 800 mmol NaCl and 0.5% sodium bicarbonate.

2. The application according to claim 1, characterized in that, The growth conditions for strain MCBC3 were as follows: culture temperature 28℃, pH=5.0-6.0, carbon source glucose or sucrose, and cultured on PDA medium.

3. The application according to claim 1, characterized in that, The application method involves spraying Trichoderma fungicide onto the roots of Allium crops while simultaneously applying diammonium phosphate.

4. The application according to claim 1, characterized in that, The Trichoderma inoculant was sprayed once a week for three consecutive weeks.

5. The application according to claim 1, characterized in that, Used to improve the quality of saline-alkali soil.

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