Trichoderma harzianum and its compound preparation and application

Abstract

The present application relates to the technical field of agricultural biological agents, and specifically provides a Trichoderma harzianum, a compound preparation thereof and application.The Trichoderma harzianum has a significant inhibitory effect on six common plant pathogenic fungi such as Sclerotinia sclerotiorum, Botrytis cinerea and Fusarium oxysporum.The fermentation liquor thereof has a significant growth-promoting effect on a variety of plants such as Chinese cabbage, spinach and flowering Chinese kale in a dilution range of 10-100 times, and can improve the fresh weight, dry weight and chlorophyll content of the plants.The compound liquid is compounded by the Trichoderma harzianum fermentation liquor and 50 ppm concentration of the extract of angelica polymorpha alkaloids, wherein the effective dilution range of the Trichoderma harzianum fermentation liquor in the compound liquid is 10-100 times, the compound liquid has an inhibitory effect on Fusarium oxysporum, and simultaneously promotes the growth of cowpea and Nicotiana benthamiana, realizes the integration of disease prevention and growth promotion, and has a broad agricultural application prospect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biological agent technology, and relates to a Trichoderma harzianum, its compound preparations, and its applications. Background Technology

[0002] Plant diseases are a major factor restricting agricultural production and causing crop yield reduction and quality decline. Currently, chemical pesticides remain the primary means of controlling plant diseases, but long-term use can easily lead to pathogen resistance, excessive pesticide residues, and environmental pollution. Therefore, developing efficient, safe, and environmentally friendly green control technologies has become an urgent need for modern agricultural development.

[0003] Biological control utilizes beneficial microorganisms and their metabolites to inhibit pathogens, and is one of the effective ways to replace chemical pesticides. Trichoderma ( Trichoderma As an important biocontrol fungus, it possesses multiple mechanisms of action, including hyperparasitism, competition, and induction of plant resistance, and is widely used in the control of soil-borne diseases. However, single biological control agents often suffer from problems such as unstable efficacy and slow onset of action.

[0004] Plant-derived pesticides are derived from nature, are easily degradable, and are environmentally friendly. Anise alkaloids are known plant secondary metabolites with antibacterial activity, but their use alone may pose risks of a narrow activity spectrum or toxicity to plants.

[0005] Therefore, the scientific combination of biological control factors (Trichoderma) and plant-derived chemical factors (alkaloids) is expected to leverage their respective advantages and produce synergistic effects, leading to the development of novel green pesticides that integrate growth promotion and antibacterial properties. This is an important research direction in this field. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Trichoderma harzianum strain from a special habitat. Trichoderma harzianum The preparation, which is a compound of the fermentation broth of this strain and anise alkaloids, has significant effects in promoting plant growth and broad-spectrum prevention and control of plant diseases.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, this invention provides a *Trichoderma harzianum* strain isolated from diseased branches of peach trees in the Shannan region of Lhasa, Tibet, named *Trichoderma harzianum*-peach. The strain has the accession number CGMCC No. 42199, the accession date is September 17, 2025, and the accession classification is named... Trichoderma harzianum China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing, China, with a postal code of 100101 and a telephone number of 010-64807355.

[0009] In a second aspect, the present application provides the use of the strain and the fermentation broth containing the strain, and the use is any one of the following:

[0010] A1) use in promoting plant growth;

[0011] A2) use in preparing a product for promoting plant growth;

[0012] A3) use in inhibiting pathogenic fungi;

[0013] A4) use in preparing a product for inhibiting pathogenic fungi;

[0014] The plants include Chinese cabbage, spinach, Chinese flowering cabbage, cowpea, or tobacco;

[0015] The pathogenic fungi include Fusarium oxysporum (Fusarium oxysporum) Fusarium oxysporum ), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) Sclerotinia sclerotiorum ), Rhizoctonia solani (Rhizoctonia solani) Rhizoctonia solani ), Fusarium verticillioides (Fusarium verticillioides) Fusarium verticillioides ), Botrytis cinerea (Botrytis cinerea) Botrytis cinerea ), and Colletotrichum gloeosporioides (Colletotrichum gloeosporioides) Colletotrichum gloeosporioides .

[0016] In a third aspect, the present application provides a compound preparation containing the strain, and the compound preparation further comprises angelica alkaloids, and the preparation method of the compound preparation comprises the following steps:

[0017] Step 1, preparing a strain fermentation stock solution: the strain is activated on a PDA plate medium and cultured in the dark at 25°C; a PSB liquid medium is prepared, and a 0.5 cm diameter Trichoderma harzianum block is punched from the activated plate with an inoculation amount of 1-2 blocks per 100 mL of medium, and is inoculated into the liquid medium; the inoculated medium is placed in a shaker, and is subjected to vibration fermentation at 28°C and 120 rpm for 7 days; after the fermentation is completed, the fermentation broth is filtered using a needle filter to obtain a sterile Trichoderma harzianum fermentation stock solution for standby;

[0018] Step 2, preparing an angelica alkaloid solution: dry angelica plant material is taken, and a reflux extraction method is used to obtain an extract, and an acid-base extraction method is used to extract alkaloid components from the extract; the extracted angelica alkaloids are dissolved in a 10% methanol solution to prepare an angelica alkaloid solution with a concentration of 50 mg / L;

[0019] reflux extraction:

[0020] The reflux extraction method is to use ethanol solvent as the extraction medium, mix the raw material with the solvent, and then heat and distill. The solvent vapor is liquefied by a condensing device, and continuously refluxed to the leaching container to circulate and leach the raw material. Through repeated reflux of the solvent, the effective components are fully dissolved, and the extraction process reaches the end point.

[0021] In the experiment, 20 kg of dried H. erectum medicinal products were cut and stored. 1 kg of medicinal materials was taken out every day and added to a round bottom with 10, heated for 2.5 h, and repeated three times. After the solution cooled, the crude extract was poured out. The crude extract was rotary evaporated to obtain the extract. All the medicinal materials were extracted according to the above steps to obtain 160 g of extract.

[0022] acid-base extraction method:

[0023] The obtained extract was dissolved in a 3% tartaric acid solution, the pH was adjusted to 2-3, and the insoluble matter was removed by filtration. The filtrate was extracted with ethyl acetate three times, and concentrated under reduced pressure to obtain the ethyl acetate extract. The extract was combined with the acid water insoluble matter, and sequentially extracted with petroleum ether, ethyl acetate and n-butanol three times, respectively. After concentration under reduced pressure, 90 g of petroleum ether extract, 60 g of ethyl acetate extract and 40 g of n-butanol extract were obtained. The acid water layer after ethyl acetate extraction was adjusted to pH 9-10 with ammonia solution, and extracted with dichloromethane three times. Concentration obtained 39 g of dichloromethane extract; the remaining water layer was adjusted to pH 12-13 with sodium hydroxide solution, and then extracted with n-butanol three times. Concentration obtained 20 g of n-butanol 2 extract. Each part was detected by TLC spotting, and developed with bismuth potassium iodide color developing agent. It was found that the ethyl acetate part developed the most significant color reaction, indicating that the alkaloid content in this part was relatively high. Therefore, the subsequent analysis was carried out for this part.

[0024] Step three, preparation of the complex liquid: the Trichoderma harzianum fermentation stock solution prepared in step one is diluted with sterile water to 10 to 100 times; the Trichoderma harzianum fermentation dilution liquid diluted to 10 to 100 times is added with the H. erectum alkaloid solution prepared in step two, and mixed to obtain the complex liquid.

[0025] In a fourth aspect, the application provides the use of the complex preparation, which is any one of the following:

[0026] A1) for promoting plant growth;

[0027] A2) for preparing a product for promoting plant growth;

[0028] A3) for inhibiting pathogenic fungi;

[0029] A4) use in the preparation of a product for inhibiting a pathogenic fungus;

[0030] The plant includes cowpea or Nicotiana benthamiana;

[0031] The pathogenic fungus includes Fusarium oxysporum f. sp. Vasinfectum.

[0032] In a fifth aspect, the present application provides a method for preventing and treating cowpea wilt and Nicotiana benthamiana wilt caused by Fusarium oxysporum f. sp. Vasinfectum, which comprises soil drenching of cowpea plants or foliar spraying of Nicotiana benthamiana plants with the compound preparation.

[0033] In a sixth aspect, the present application provides a microbial inoculant comprising the strain.

[0034] In a seventh aspect, the present application provides a bio-organic fertilizer, wherein the active ingredient of the bio-organic fertilizer comprises the strain or the microbial inoculant.

[0035] Advantages of the present application:

[0036] (1) Novel strain resource, broad-spectrum biocontrol potential: The present application first isolates a Trichoderma harzianum strain from the Tibetan Plateau, which shows strong inhibitory activity against multiple important plant pathogenic fungi, providing valuable strain resources for the development of new biological pesticides.

[0037] (2) Synergistic effect, functional integration: The fermentation broth of Trichoderma harzianum is innovatively compounded with the aristolochic acid, achieving "biochemical synergy". The compound preparation has the biological functions of Trichoderma harzianum such as growth promotion, hyperparasitism, and induced resistance, and the direct antibacterial chemical function of aristolochic acid, achieving the integration effect of "disease prevention + growth promotion", and realizing the synergistic effect of 1+1≥2.

[0038] (3) Green and safe, attenuated and synergistic: Through compounding, the use concentration of aristolochic acid can be reduced (from 100 ppm effective concentration to 50 ppm), reducing the risk of plant toxicity or environmental residue that may be caused by the single use of plant-derived chemicals, and meeting the development requirements of green agriculture.

[0039] (4) Flexible application method: The compound preparation can be applied through foliar spraying, soil drenching, seed treatment, etc., and can be widely used to effectively prevent and control leaf diseases and soil-borne diseases. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 The phylogenetic tree of the strain of the present application is constructed based on ITS gene sequence.

[0041] Figure 2The colony morphology of the strain of the present invention on different culture media is shown below: A and a are the front and back sides of the colony of the strain on PDA medium; B and b are the front and back sides of the colony of the strain on CMD medium; C and c are the front and back sides of the colony of the strain on SNA medium.

[0042] Figure 3 The microscopic morphological characteristics of the strain of the present invention on PDA medium are shown.

[0043] Figure 4 The hyphal diameter of the strain of the present invention on different culture media

[0044] Figure 5 The effect of different pH values ​​on the mycelial growth of the strain of this invention.

[0045] Figure 6 The present invention describes the inhibitory effect of the strain on six pathogenic bacteria.

[0046] Figure 7 The plate volatiles of the strain of this invention exhibit inhibitory effects against six pathogens.

[0047] Figure 8 This invention relates to the reparasitic effect of the mycelium of the strain on the mycelium of six pathogenic fungi.

[0048] Figure 9 The results of spraying different concentrations of the fermentation broth of the strain of this invention onto Chinese cabbage.

[0049] Figure 10 The results of spraying spinach with different concentrations of the fermentation broth of the strain of this invention.

[0050] Figure 11 The results after spraying different concentrations of the fermentation broth of the strain of this invention onto flowering cabbage.

[0051] Figure 12 The effect of fermentation broth of the strain of the present invention at different dilution concentrations on plant dry weight: Figure 12 A and a in the figure: above-ground dry weight and underground dry weight of Chinese cabbage after being sprayed with different concentrations of the fermentation broth of the strain of this invention; Figure 12 B and b in the figure: aboveground dry weight and underground dry weight of flowering cabbage after spraying with different concentrations of the fermentation broth of the strain of this invention; Figure 12 C and c in the figure: aboveground dry weight and underground dry weight of spinach after spraying with different concentrations of the fermentation broth of the strain of this invention.

[0052] Figure 13 The effect of fermentation broth of the strain of the present invention at different dilution concentrations on the chlorophyll content of plants.

[0053] Figure 14 The front of the colonies of various plant pathogens on the culture medium treated with different concentrations of anise extract.

[0054] Figure 15 The inhibitory effects of different concentrations of anethole aqueous solutions on *Fusarium wilt* pathogen of cowpea: Figure 15 In the table, A represents PDA plates containing different concentrations of anethole. Figure 15 B in the text represents PDA plates with different concentrations.

[0055] Figure 16 This study compares the growth-promoting effects of soil drenching treatment with the fermentation broth and compound solution of the strain of this invention on cowpea plant growth.

[0056] Figure 17 Phenotypic characteristics of cowpea seedlings infected with Fusarium wilt (yellowing and shedding of leaves and discoloration of roots).

[0057] Figure 18 The effect of inoculation with Fusarium oxysporum after root irrigation treatment with the fermentation broth and compound solution of the strain of this invention on the rhizosphere of cowpea: Figure 18 In this context, A represents the control root drenching treatment. Figure 18 In this context, B represents the root irrigation treatment of the fermentation broth of the strain of this invention. Figure 18 Figure CE in the figure represents the root irrigation treatment with the compound solution, and Figure F represents the relative diseased area.

[0058] Figure 19 The results after spraying different concentrations of the fermentation broth of the strain of this invention onto *Flavus benthamiana* 5 days later.

[0059] Figure 20 The effects of different concentrations of the fermentation broth of the strain of this invention on the fresh weight and leaf area of ​​Nicotiana benthamiana.

[0060] Figure 21 The results after spraying different concentrations of compound solution on the smoke plant 5 days later.

[0061] Figure 22 The effects of different concentrations of the compound solution of the strain of this invention on the fresh weight and leaf area of ​​Nicotiana benthamiana.

[0062] Figure 23 The results of inoculating cowpea with Fusarium wilt pathogen 5 days after spraying different concentrations of fermentation broth onto *Flavus benthamiana*: Figure 23 A in the text stands for Bright. Figure 23 B in the text: UV; Figure 23 C in the text: Dyeing.

[0063] Figure 24 The results of inoculating cowpea with Fusarium wilt pathogen 5 days after spraying different concentrations of compound solution on *Flavus benthamiana*: Figure 24 A in the text stands for Bright. Figure 24 B in the text: UV; Figure 24 C in the text: Dyeing.

[0064] Figure 25Comparison of the effects of different treatments of the fermentation liquor of the strain of the application and the compound liquid on the leaf area and fresh weight of Nicotiana benthamiana.

[0065] Figure 26 Comparison of the inhibitory effects of different treatments of the fermentation liquor of the strain of the application and the compound liquid on the pathogen of Fusarium wilt of cowpea. DETAILED DESCRIPTION

[0066] The specific embodiments of the application are described below to facilitate the understanding of the application for those skilled in the art, but it should be clear that the application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the application as defined and determined by the appended claims, and all applications utilizing the concept of the application are within the scope of protection.

[0067] Example 1 Isolation and functional identification of Trichoderma strains

[0068] 1. Materials and methods

[0069] 1.1 Test materials

[0070] The test Trichoderma strain was isolated from a diseased branch sample of a peach tree in the Shannan area of Lhasa, Tibet.

[0071] 1.1.2 Test strains

[0072] The test 6 pathogenic fungi were Sclerotinia sclerotiorum (Ss), Botrytis cinerea (Bc), Fusarium oxysporum (Fo), Fusarium solani (Fs), Fusarium verticillioides (Fv) and Colletotrichum gloeosporioides (Cg), all of which were preserved in the laboratory. S.sclerotiorum B. cinerea F.oxvsporum R.solani F.verticillioides C.gloeosporioides

[0073] 1.2 Methods

[0074] 1.2.1 Strain isolation and culture

[0075] The collected plant branch samples were sequentially soaked in 75% ethanol for 1 min, 1% sodium hypochlorite for 5 min, and 75% ethanol for 1 min, and then rinsed with sterile water for 3 times. The rinsed samples were cut into small pieces and air-dried in a sterile environment, and then inoculated into PDA medium and cultured at 25°C in the dark. After colonies formed on the surface of the branches, the edge mycelium was picked with an inoculation needle and cultured on PDA medium at 25°C, and repeatedly purified for 3-5 generations. Finally, the purified strain was stored at 4°C. o

[0076] 1.2.2 Molecular identification of strains

[0077] ITS,​​​​​​​tefl, rpb2 Isogenes are commonly used for the identification of various Trichoderma species. Table 1 shows the primer information for molecular identification of Trichoderma species.

[0078] Table 1 Primer information for molecular identification of Trichoderma species

[0079]

[0080] Phylogenetic analysis based on ITS genes, such as Figure 1 As shown.

[0081] 2.1 Biological characteristics of Trichoderma harzianum strains

[0082] 2.1.1 Morphological characteristics

[0083] This study isolated one strain of *Trichoderma harzianum* from plant branches and trunks, which showed significant inhibitory effects on other fungi on agar plates. After 7 days of culture, the isolated strain exhibited abundant hyphae on PDA medium, appearing fluffy or cottony, with conidiophores distributed in a ring and expanding outwards. Figure 2 (A and a in the text); on CMD medium, the hyphae are abundant and appear cottony ( Figure 2 B and b in the text); the hyphae are sparse on SNA medium ( Figure 2 (C and c in the text). Figure 3 The microscopic morphological characteristics of the strain on PDA medium. Figure 4 The diameter of the mycelium of the strain on different culture media.

[0084] 2.1.2 Effects of different pH values ​​on mycelial growth of the strain

[0085] The strain exhibits the most vigorous mycelial growth at pH 7.0, with colony diameters reaching approximately 65 mm. Growth under this condition is significantly superior to other pH levels. When the pH deviates from 7.0 (e.g., acidic pH 2.0 or 3.0, or alkaline pH 8.0 or 9.0), the colony diameter decreases significantly, and mycelial growth is inhibited. Figure 5 ).

[0086] Therefore, the optimal pH for mycelial growth of this strain is around 7.0 (neutral environment), and acidic or alkaline environments will inhibit its growth.

[0087] 2.2 Inhibitory effect of the strain on the pathogen

[0088] according to Figure 6 Analysis of the results revealed that the antagonistic activity of this strain against different pathogens can be divided into three levels:

[0089] High antagonistic activity: against Sclerotinia sclerotiorum The inhibition rate of (Sclerotinia sclerotiorum) exceeds 80%, indicated by the letter "a," signifying its extremely strong antagonistic ability against this pathogen;Fusarium verticillioides The inhibition rate of Fusarium oxysporum f. sp. cubense was about 80%, marked with the letter "b", and the antagonistic effect was significant.

[0090] Medium antagonistic activity: against Colletotrichum gloeosporioides Gloeosporium psidii and Fusarium oxysporum The inhibition rates of Fusarium oxysporum f. sp. cubense and Fusarium solani were both about 60%, marked with the letter "c", and the antagonistic effect was moderate.

[0091] Low antagonistic activity: against Rhizoctonia solani Rhizoctonia solani, the inhibition rate was only about 40% (marked with the letter "e"), and against Botrytis cinerea Botrytis cinerea, the inhibition rate was about 50% (marked with the letter "d"), and the antagonistic ability was relatively weak.

[0092] According to the analysis of the results of Figure 7 , it was found that the volatile substances of the strain also had antagonistic activity against different pathogens, among which the inhibition rate against Fusarium oxysporum f. sp. cubense was about 60%, followed by Fusarium oxysporum Colletotrichum gloeosporioides The inhibition rate against G. psidii was about 40%, and the inhibition rate against the other four fungi was lower.

[0093] According to the analysis of the results of Figure 8 , it was found that the mycelium of the strain had hyperparasitic effect on the mycelium of the six pathogenic fungi, and the mycelium of the strain was intertwined with the mycelium of the pathogenic fungi, leading to rupture. Thus, the growth of the pathogenic fungi was inhibited.

[0094] 2.3 Effect of fermentation broth of the strain on plant growth

[0095] 2.3.1 Treatment setting of fermentation broth of the strain

[0096] Preparation of fermentation stock solution of the strain: the strain was activated on PDA plate medium and cultured at 25°C in the dark; 5 bottles of PSB liquid medium were prepared, and the strain was inoculated in 3 of them with an inoculum of 1-2 pieces per 100 mL of medium, and the rest were not inoculated with the strain as controls and dilutions; the inoculated medium was placed in a shaker and fermented at 28°C and 120 rpm for 7 days; after fermentation, the fermentation broth was filtered using a needle filter to obtain the fermentation stock solution of the strain, which was ready for use.

[0097] ​The experiment was divided into 6 treatments, i.e. 10 times dilution of the fermentation stock solution (hereinafter referred to as dilution 10), 50 times dilution (hereinafter referred to as dilution 50), 100 times dilution (hereinafter referred to as dilution 100), 500 times dilution (hereinafter referred to as dilution 500), 1000 times dilution (hereinafter referred to as dilution 1000) and control (the filtrate of PSB culture solution without inoculating strain agar block), and the formulations of each treatment were as shown in Table 1. The surface-sterilized seeds of Chinese cabbage, spinach and kai-tai were respectively sowed in sterilized and numbered plastic pots, each pot containing 400 g of sterilized soil, and each pot contained 20 seeds, and each treatment was repeated 3 times. After sowing, the seedlings were sprayed with each treatment after the two cotyledons of the control were fully unfolded. The growth indexes of 5 randomly selected seedlings in each pot were measured 20 days after sowing according to the conventional method.

[0098] Table 2 Formulation table of different treatments of strain fermentation broth

[0099]

[0100]

Note

[0101] 2.3.2 Effect on the growth of Chinese cabbage

[0102] Figure 9 The growth of Chinese cabbage after spraying with different concentrations of strain fermentation dilution solution for 14 days can be seen. After spraying with different concentrations of fermentation solution, the growth of Chinese cabbage in each treatment was significantly different. The growth of Chinese cabbage in dilution 10, dilution 50 and dilution 100 was significantly better than that in the control. The growth of Chinese cabbage in dilution 100 was the best, with large leaves and vigorous growth, followed by dilution 50, and the growth of Chinese cabbage decreased with the increase of dilution multiple after dilution 100. The growth of Chinese cabbage in dilution 1000 was not significantly different from that in the control. This shows that the strain fermentation broth has a promoting effect on the growth of Chinese cabbage.

[0103] 2.3.3 Effect on the growth of spinach

[0104] Figure 10 The situation of spinach after spraying with different concentrations of strain fermentation broth for 14 days can be seen. After applying the fermentation broth, the growth of spinach was better than that of the control. It can also be seen from the figure that from dilution 10 to dilution 100, the growth of spinach was significantly improved with the increase of dilution degree, and the growth of spinach in dilution 100 was the most vigorous, with the longest leaves and stems. Then, with the increase of dilution degree, the growth of spinach decreased with the increase of dilution multiple. The growth of spinach in dilution 1000 was better than that in the control, but it was close to the control. This shows that the strain fermentation broth has a promoting effect on the growth of spinach.

[0105] 2.3.4 Effect on the growth of kai-tai

[0106] Figure 11 From the figure, it can be seen that the growth of kailan after spraying different concentrations of strain fermentation broth is obviously better than the control, the plant is larger than the control, the leaf is large, and the growth is obviously better than the control, but this growth is rapidly reduced with the increase of concentration. The growth of dilution 1000 is not different from the control.

[0107] 2.3.5 Effect of strain fermentation broth on plant fresh weight

[0108] From Table 3, it can be seen that after applying strain fermentation broth to Chinese cabbage, spinach and kailan, the fresh weight of the experimental plants can be obviously increased, and the effective concentration is between 10 times and 500 times dilution of the original solution. Different plants have different concentrations of strain fermentation broth. The most effective concentration of small cabbage is dilution 100 and dilution 50, and the fresh weight is increased by 50.00% and 47.37% respectively compared with the control, with extremely significant difference. The second is dilution 10, and the fresh weight is increased by 36.84% compared with the control. After dilution 100, the fresh weight decreases with the decrease of concentration. Except that there is no difference between the control CK and dilution 1000, the differences between the other treatments are extremely significant.

[0109] Table 3 Effect of strain fermentation broth on plant fresh weight

[0110]

[0111]

Note

[0112] The growth fresh weight of spinach increased the most at dilution 50, which increased by 144.38%, followed by dilution 500, dilution 100 and dilution 10, and the growth fresh weight decreased in turn. There is no obvious difference between dilution 100 and dilution 500, but there is extremely significant difference between them and other treatments. The fresh weight of kailan is the most obvious at dilution 100, which is increased by 64.47% compared with the control, with extremely significant difference. The second is dilution 50, which is increased by 51.32% compared with the control, with extremely significant difference. There is no obvious difference between dilution 10 and dilution 500. There is no significant difference between dilution 1000 and the control.

[0113] 2.3.6 Effect of strain fermentation broth on plant dry weight

[0114] Figure 12The dry weight of the three vegetables after spraying with different concentrations of strain fermentation liquor was observed. As shown in the figure, the dry weight of the three vegetables at dilution 10, dilution 50 and dilution 100 was significantly better than the control, and the weight of the plants above and below ground was better than the control, but the growth decreased rapidly with increasing concentration. This shows that the strain fermentation liquor has a certain promoting effect on the growth of the three plants.

[0115] 2.3.7 Effect of strain fermentation liquor on plant chlorophyll content

[0116] According to Table 4 and Figure 13 It can be concluded that the strain fermentation liquor can significantly increase the chlorophyll content of pak choi, spinach and kailan. The effective concentration is between dilution 10 and dilution 500. However, the optimal effective concentration for each crop is different. Compared with the control, pak choi has the best effect at dilution 100 and dilution 50, with an increase of 64.21% and 57.07%, followed by dilution 10, with an increase of 37.89%, and dilution 500, with an increase of 28.84%. For spinach, dilution 50 is the best, with an increase of 100.62%, dilution 100 and dilution 500 increase by 65.84% and 71.62% respectively, which is significantly different from the control. The optimal concentration of kailan for strain fermentation liquor is dilution 100 times, which increases by 55.88% compared with the control, followed by dilution 10, which increases by 29.22% compared with the control, both reaching a significant level. Dilution 1000 and the control have no difference.

[0117] Table 4 Effect of strain fermentation liquor on plant chlorophyll content

[0118]

[0119]

Note

[0120] Example 2 Preparation and functional identification of strain compound preparation

[0121] 1. Cumin alkaloid extract inhibition experiment

[0122] The specific extraction method is as follows: take dry cumin 30 kg, extract 1 kg of extract by reflux method, and then extract 160 g of alkaloids from it by acid-base extraction method.

[0123] Reflux extraction:

[0124] Reflux extraction is to use ethanol solvent as extraction medium, mix the raw material with the solvent and heat distillation, the solvent vapor is liquefied by condensing device, and the raw material is continuously extracted by the circulating extraction of the solvent through repeated reflux, until the extraction process reaches the end point.

[0125] The experiment takes H. erectum 20 kg of dry medicinal products, cuts and stores. 1 kg of medicinal materials is taken every day and added to 10 in a round bottom, heated for 2.5 h, and repeated three times. After the solution is cooled, the crude extract is poured out, and the crude extract is rotary evaporated to obtain the extract. All the medicinal materials are extracted according to the above steps to obtain 160 g of extract.

[0126] Acid-base extraction method:

[0127] The obtained extract is dissolved in 3% tartaric acid solution, the pH is adjusted to 2-3, and the insoluble matter is removed by filtration, and the filtrate is extracted with ethyl acetate for 3 times, and concentrated under reduced pressure to obtain the ethyl acetate extract. The extract and acid water insoluble matter are combined, and are extracted with petroleum ether, ethyl acetate and n-butanol respectively for 3 times, and concentrated under reduced pressure to obtain 90 g of petroleum ether extract, 60 g of ethyl acetate extract and 40 g of n-butanol extract. The acid water liquid after ethyl acetate extraction is adjusted to pH 9-10 with ammonia solution, and extracted with dichloromethane for 3 times, and concentrated to obtain 39 g of dichloromethane extract; the remaining water layer is adjusted to pH 12-13 with sodium hydroxide solution, and extracted with n-butanol for 3 times, and concentrated to obtain 20 g of n-butanol 2 extract. Each part is detected by TLC point plate, and after coloring with bismuth potassium iodide color developing agent, it is found that the coloring reaction of the ethyl acetate part is the most significant, which indicates that the alkaloid content of the part is relatively high, so the follow-up experiment is carried out for this part.

[0128] The previous research of the laboratory has proved that the alkaloid extract of H. erectum has inhibitory effect on a variety of plant pathogens (Botrytis cinerea (Bc), Fusarium oxysporum (Fo), Sclerotinia sclerotiorum (Ss), Rhizoctonia solani (Rs)) Figure 14 ), including Botrytis cinerea (Bc), Fusarium oxysporum (Fo), Sclerotinia sclerotiorum (Ss), Rhizoctonia solani (Rs) Botrytis cinerea ), Sclerotinia sclerotiorum (Ss) Fusarium oxysporum ), Sclerotinia sclerotiorum (Ss) Sclerotinia sclerotiorum ), Rhizoctonia solani (Rs) Rhizoctonia solani ), Rhizoctonia solani (Rs)

[0129] 2. Determine the optimal working concentration of H. erectum alkaloids compounded with the fermentation broth of the strain of the present application

[0130] Preparation of the fermentation stock solution of the strain of the present application: the strain XZT was activated on PDA plate medium and cultured in the dark at 25°C; the PSB liquid medium was prepared, and 1-2 pieces of the strain block with a diameter of 0.5 cm were inoculated into 100 mL of the medium at an inoculation amount; the inoculated medium was placed in a shaker and subjected to vibration fermentation at 28°C and 120 rpm for 7 days; after the fermentation, the fermentation liquid was filtered using a needle filter to obtain the fermentation stock solution of the strain, which was ready for use.

[0131] First, the angelica alkaloids were dissolved in 10% methanol to prepare a mother liquor (1 g of angelica alkaloids was dissolved in 100 mL of MeOH), which was then added to the PDA medium to prepare plates containing different final concentrations (such as 10, 50, and 100 ppm) of angelica alkaloids. Some of the plates were also inoculated with the fermentation stock solution of the strain. In the center of all the plates, the cowpea wilt pathogen was inoculated. After 5 days of culture, it was found that: (1) all the compound treatments could inhibit the growth of the pathogen; (2) at concentrations of 10 ppm and 50 ppm, the compound had the best antibacterial effect; (3) at a concentration of 100 ppm, although the growth of the pathogen was also strongly inhibited, this was due to the methanol introduced during the preparation of the high-concentration mother liquor, which inhibited the active substances in the strain fermentation liquor. This interference effect caused by the solvent increased with increasing concentration. To avoid the interference of the methanol solvent and ensure the best synergistic antibacterial effect of angelica alkaloids and strain fermentation liquor, 50 ppm (50 mg / L) was finally selected as the optimal concentration for subsequent compound experiments. Figure 15

[0132] 3. Preparation scheme of the compound solution

[0133] Different dilution multiples of the strain fermentation liquor (dilution 10, dilution 50, dilution 100, dilution 500, dilution 1000) and the control were mixed with 50 ppm of the angelica alkaloid mother liquor to obtain a series of compound solutions.

[0134] Table 5 Formulation table of different treatments of the compound solution

[0135]

[0136]

Note

[0137] 4. Effect verification of the compound solution

[0138] 4.1 Promoting growth and disease prevention effect on cowpea

[0139] ​Growth-promoting effect: After 7 days of soil drenching treatment with the bacterial fermentation broth and the compound solution on cowpea plants, the figure shows that the compound solution also has a similar growth-promoting effect on cowpea growth as the bacterial fermentation broth alone. Figure 16 ).

[0140] Disease prevention effect: After a group of eight cowpea seedlings reached the two-leaf stage, their roots were drenched with a fermentation solution of Fusarium wilt pathogen. Seven days later, yellowing and shedding of the leaves were observed. Simultaneously, dissection of the plant's rootstock revealed browning of the vascular bundles. The lower leaves wilted and turned yellow, and discoloration of the roots was visible when the plant was pulled up. Figure 17 ).

[0141] Another group (6 seedlings) of cowpea seedlings at the two-leaf stage were treated with fermentation liquid or compound liquid for 7 days. After that, the cowpea roots were pulled out and cleaned. 5 mm of pathogens were inoculated about 1-2 cm away from the roots and cultured in a moist environment. The area of ​​the lesions was recorded 5 days later. Figure 18 In this context, A represents the control root drenching treatment. Figure 18 In this context, B represents the root irrigation treatment with the fermentation broth of the strain. Figure 18 Figures C to E in the diagram show the root irrigation treatment with the compound solution. Figure 18 Figure F in the figure shows the relative diseased area. It can be seen that the compound solution also exhibits the same inhibitory effect on pathogens as when the bacterial fermentation broth is used alone for root irrigation.

[0142] In conclusion, the compound solution maintained its growth-promoting effect without weakening its ability to induce plant disease resistance.

[0143] 4.2 Growth-promoting and disease-preventing effects on Nicotiana benthamiana

[0144] 4.2.1 Effects of the fermentation broth of the strain on the growth of Nicotiana benthamiana

[0145] Impact on the growth of Tobacco Benedict: Figure 19 The figure shows the growth of Nicotiana benthamiana after spraying with fermentation broth of different concentrations of the strain. As can be seen from the figure, Nicotiana benthamiana grew significantly better than the control at concentrations of 10%, 50%, and 100. The plants were larger and had larger leaves, and the growth was significantly better than the control. However, this growth decreased rapidly with increasing concentration. The growth at concentration 1000 showed no difference from the control.

[0146] Effects on leaf area of ​​*Nicotiana benthamiana*: The table below records the fresh weight and leaf area of ​​*Nicotiana benthamiana* after spraying with fermentation broth of different concentrations of the strain. Table 6 shows that dilutions of 10-500 times significantly promoted the growth of *Nicotiana benthamiana*, but there was no significant difference between the 1000-fold dilution and the control (CK) treatment. Leaf area also showed the same trend (…). Figure 20 ).

[0147] Table 6. Effects of different concentrations of fermentation broth from the strain on the fresh weight and leaf area of ​​*Nicotiana benthamiana*.

[0148]

[0149] 4.2.2 Effect of the compound solution on the growth of Flavescent Benedictine burmannii

[0150] Effects on the growth of *Nicotiana benthamiana*: It can be seen that the growth of *Nicotiana benthamiana* varied significantly among different treatments after spraying with different concentrations of the compound solution. The growth of diluted 10%, diluted 50%, and diluted 100% was significantly better than the control. Diluted 100% showed the best growth, with large leaves and vigorous growth, followed by diluted 50% and diluted 10%. Beyond diluted 500%, the growth of *Nicotiana benthamiana* decreased with increasing dilution ratio. The growth of diluted 1000% showed no significant difference from the control. This indicates that the compound solution has a promoting effect on the growth of *Nicotiana benthamiana*. Figure 21 ).

[0151] Effects on leaf area of ​​Bentham tobacco: The table below records the fresh weight and leaf area of ​​Bentham tobacco after spraying with different concentrations of compound solution. It can be seen from the table that the leaf area of ​​Bentham tobacco was significantly increased compared with CK in the dilution range of 10-500, but there was no significant difference between the 1000-fold dilution and CK treatment, and the fresh weight also showed the same trend.

[0152] Table 7. Effects of different concentrations of the compound solution on the fresh weight and leaf area of ​​Tobacco Benedict.

[0153]

[0154] 4.2.3 The fermentation broth of the strain inhibited the infection of *Benzia benthamiana* leaves by the pathogen causing cowpea wilt.

[0155] Leaves of *Nicotiana benthamiana* plants sprayed with fermentation broth at various concentrations as described in section 4.2.1 were selected. Wounds were made on the same side of the underside of each leaf using an inoculation needle. Activated cowpea wilt pathogens were inoculated onto the wounds. After 3 days of moist incubation, the leaves were stained with TB and DAB, and the area of ​​lesions was quantified. Figure 23 ).

[0156] 4.2.4 The compound solution inhibits the infection of *Benzia benthamiana* leaves by the pathogen causing cowpea wilt.

[0157] Leaves of *Nicotiana benthamiana* plants sprayed with various concentrations of the compound solution described in 4.2.2 were selected. Wounds were made on the same side of the underside of each leaf using an inoculation needle. Activated cowpea wilt pathogens were inoculated onto the wounds. After 3 days of moist incubation, the leaves were stained with TB and DAB, and the area of ​​lesions was quantified. Figure 24 ).

[0158] 4.2.5 Analysis of the effects of fermentation broth and compound solution on promoting the growth of Tobacco Benedictine aurantiacus

[0159] from Figure 25It can be seen that the strain fermentation liquid and the compound liquid can obviously improve the leaf area and fresh weight of N. benthamiana. In Figure A, the mutual comparison of the two for leaf area found that the strain fermentation liquid was better than the compound liquid at dilution 1000 times, which may be due to the damage of less methanol solvent to active substances; in Figure B, the compound liquid was significantly different from the strain fermentation liquid between dilution 10 to dilution 1000, and there was no difference between dilution 500 and dilution 1000.

[0160] 4.2.6 Analysis of the effect of fermentation liquid and compound liquid on inhibiting the pathogenic fungus of cowpea blight

[0161] From Figure 26 It can be seen that the strain fermentation liquid and the compound liquid can obviously inhibit the pathogenic fungus. In Figure A, the effective concentration of the inhibition effect of the strain fermentation liquid is between dilution 10 to dilution 100; in Figure B, the effective concentration of the inhibition effect of the compound liquid is between dilution 10 to dilution 1000. Figure C, the transverse comparison of the two found that the inhibition effect of the compound liquid is better than that of the strain fermentation liquid at each concentration.

Claims

1. A type of Trichoderma harzianum ( Trichoderma harzianum ) strain, characterized in that, The strain was classified and named Trichoderma harzianum The accession number is CGMCC No.42199, and it was deposited at the China General Microbiological Culture Collection Center on September 17, 2025.

2. The strain according to claim 1, characterized in that, The ITS sequence of the strain is shown in SEQ ID NO:

1.

3. A microbial inoculant, characterized in that, Contains the strain described in claim 1.

4. A bio-organic fertilizer, characterized in that, The active ingredients of the bio-organic fertilizer include the strain described in claim 1 or the microbial agent described in claim 3.

5. A fermentation broth containing the strain described in claim 1.

6. The application of the strain according to claim 1 or the fermentation broth according to claim 5, characterized in that, The application is any one of the following: A1) Its application in promoting plant growth; A2) Application in the preparation of products that promote plant growth; A3) Application in inhibiting pathogens; A4) Application in the preparation of products that inhibit pathogens; The plants mentioned are bok choy, spinach, flowering cabbage, or tobacco. The pathogen is: Fusarium oxysporum (… Fusarium oxysporum ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum Rhizoctonia solani ( ) Rhizoctonia solani ), Fusarium verticillata ( Fusarium verticillioides ), Botrytis cinerea ( Botrytis cinerea ) and Colletotrichum anthracnose ( Colletotrichum gloeosporioides ).

7. A compound preparation, characterized in that, The preparation method of the compound preparation includes the following steps: Step 1: Preparation of fermentation stock solution of the strain described in claim 1: Activate the strain described in claim 1 on PDA agar plates and culture in the dark at 25°C; prepare PSB liquid medium, take 0.5 cm diameter pieces of Trichoderma harzianum from the activated plates, and inoculate 1-2 pieces per 100 mL of medium into the liquid medium; place the inoculated medium in a shaker and ferment at 28°C and 120 rpm for 7 days; after fermentation, filter the fermentation broth using a needle filter to obtain sterile Trichoderma harzianum fermentation stock solution for later use; Step 2: Preparation of anise alkaloid solution: Take dried anise plant material, obtain extract by reflux extraction, and then extract alkaloid components from the extract by acid-base extraction. Dissolve the extracted anise alkaloids in 10% methanol aqueous solution to prepare an anise alkaloid solution with a concentration of 50 mg / L. Step 3: Preparation of the compound solution: Dilute the Trichoderma harzianum fermentation stock solution prepared in Step 1 with sterile water to a ratio of 10 to 100 times; add the fennel alkaloid solution prepared in Step 2 to the Trichoderma harzianum fermentation diluted solution to a ratio of 10 to 100 times, and mix well to obtain the compound solution.

8. The application of the compound preparation according to claim 7, characterized in that, The application is any one of the following: A1) Its application in promoting plant growth; A2) Application in the preparation of products that promote plant growth; A3) Application in inhibiting pathogens; A4) Application in the preparation of products that inhibit pathogens; The plant in question is cowpea or Nicotiana benthamiana; The pathogen is *Fusarium oxysporum*, the pathogen that causes cowpea wilt.

9. A method for preventing and controlling cowpea wilt and tobacco wilt caused by *Fusarium oxysporum*, the pathogen of cowpea wilt, characterized in that, The method involves applying the compound preparation described in claim 7 to the soil for root irrigation of cowpea plants or to the leaves of tobacco plants for spraying.

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