Formulations and uses for inhibiting plant and human pathogenic fungi

Abstract

The application provides a high-efficiency, stable and broad-spectrum plant source fungicide, which can specifically inhibit various disease fungi, can effectively prevent and treat plant diseases caused by various fungi and fungal infection diseases caused by fusarium solani, can guarantee the quality of agricultural products and human health, can avoid the disadvantages of chemical agents, can meet the development needs of green agriculture and ecological protection, and has important practical application value; the fungicide comprises a blue flower grass extract, and the fungi in the blue flower grass extract are one or more of fusarium tricinctum, alternaria alternata, fusarium reticulatum, fusarium solani and lasiodiplodia theobromae.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to formulations including indigo extract and its use in inhibiting pathogenic fungi in plants and humans. Background Technology

[0002] Plant-derived pesticides originate from secondary metabolites in nature, possessing natural environmental compatibility and degradability. They do not produce persistent residues in nature and can effectively protect non-target organisms, maintaining ecological balance. Their core advantage lies in their complex composition and diverse targets. Through the synergistic antibacterial action of multiple active molecules such as alkaloids, terpenes, and flavonoids, they can effectively circumvent the drug resistance developed by pathogenic fungi such as Fusarium and Alternaria. Furthermore, many plant-derived agents, while directly inhibiting bacteria, can also stimulate the plant's own systemic induced resistance (ISR), enhancing the plant's own "immunity." This provides a new biological opportunity to overcome the continuous cropping obstacle caused by the robust *Alternaria alternata*.

[0003] *Fusarium tricinctum* is a highly pathogenic fungus that poses a significant threat to agriculture and forestry. Its primary damage is causing gummosis in Sichuan pepper, which infects the base of the stem, leading to necrosis of the cortex and the exudation of an amber-colored, gum-like substance. This severely disrupts nutrient transport, causing yellowing leaves and eventual plant death. Furthermore, this fungus has a wide host range, causing various diseases such as pumpkin fruit rot, potato dry rot, and root rot in soybeans and alfalfa. Not only is it infectious and virulent, but it can also co-infect with other pathogens to accelerate disease progression, posing a continuous and severe threat to agricultural and forestry output and ecological security.

[0004] Alternaria ( Alternaria alternata Alternaria is a highly pathogenic fungus with an extremely wide host range. It can infect economic crops such as potatoes, melons, and apples, forest trees such as tea trees and poplars, and medicinal plants such as ginseng and peony. This fungus mainly causes leaf spot, leaf blight, early blight, and black spot, leading to leaf necrosis and inhibited photosynthesis. Besides its harmful effects during the growing season, it is also a significant cause of post-harvest diseases, causing fruits and vegetables to rot during storage. Physiologically, it can induce oxidative metabolic disorders in plants, alter host transcription levels, and even damage plant health by producing toxins. Due to its high pathogenicity, wide distribution, and ease of developing drug resistance, Alternaria has become a key biological stressor restricting the improvement of yield and quality in modern agriculture.

[0005] Fusarium moniliforme ( Fusarium proliferatum*Pseudomonas aeruginosa* is a widely distributed and highly destructive global plant pathogenic fungus. It has an extremely broad host range, infecting grain crops such as rice and corn, as well as various horticultural crops including bananas, chestnuts, melons, and jujubes. This fungus can cause severe ear rot, sheath rot, and various types of fruit rot. Particularly in chestnut production, the internal rot it causes persists throughout the entire growth and storage process, significantly reducing yield and severely deteriorating fruit quality, resulting in huge economic losses. Due to its extremely strong environmental adaptability and the difficulty in controlling it, it has posed a serious and continuous threat to global agricultural production and food security.

[0006] Fusarium solani is a polyphagous fungus that poses both plant pathogenicity and a threat of zoonotic infection. In agriculture, it is a major pathogen causing various crop diseases such as tobacco wilt and root rot, infecting plant roots and destroying the vascular system, leading to widespread crop death and severe yield reduction. In medicine, fungal keratitis is the most common and harmful form of Fusarium solani infection in humans. It can also cause skin and onychomycosis. In individuals with severely compromised immune systems, the fungus can enter the bloodstream through the respiratory tract or skin wounds (fungemia), subsequently spreading to multiple organs throughout the body (such as the lungs, liver, kidneys, brain, and cardiovascular system), resulting in extremely high mortality. This fungus not only seriously threatens agricultural production safety but also poses a severe challenge to human health.

[0007] Strong Earth Red Shell Fungus ( Ilyonectria robusta *Gnaphalium affine* is the core pathogen causing root rot in ginseng and American ginseng from Northeast China. This bacterium possesses extremely strong broad-spectrum pathogenicity and cross-host infectivity, with even greater invasiveness towards ginseng, making it the leading cause of "continuous cropping obstacles" and significant economic losses in ginseng fields. Research reveals that its population exhibits extremely high genetic diversity and significant differentiation with varying geographical environments, with marked differences between strains from the Changbai Mountains and other producing areas. This complex biological characteristic makes it difficult to apply single control methods universally, becoming a key technological bottleneck restricting the green and sustainable development of my country's ginseng industry.

[0008] Faced with the severe challenges posed by the aforementioned fungal diseases, traditional chemical control methods are increasingly revealing their insurmountable limitations and potential ecological hazards. For a long time, humans have over-reliant on synthetic chemical pesticides. While this has suppressed disease outbreaks in the short term, it has led to the serious "3R" problem (resistance, toxicity, and resurgence). Pathogenic fungi such as *Alternaria alternata* and *Alternaria solani* have rapidly evolved resistance mechanisms under continuous chemical pressure, resulting in ever-increasing control costs and significantly reduced efficacy. Simultaneously, the overuse of chemical pesticides has damaged soil microbial diversity, weakened the self-regulating function of agroforestry ecosystems, and created harmful residues in soil and plant tissues. This is particularly problematic in high-quality medicinal herbs and agricultural products such as ginseng and Sichuan pepper, where pesticide residues directly threaten consumer safety. This one-way governance model, sacrificing the environment and quality, is no longer suitable for the strategic needs of modern agriculture for sustainable development and food safety. Developing more efficient and safer alternative control technologies is now imperative. Against this backdrop, developing plant-derived pesticides is not only an upgrade to the control model but also a core strategic choice for implementing the concept of green agriculture. Therefore, the development of plant-derived pesticides has irreplaceable strategic value for ensuring food security, the quality of Chinese medicinal materials, and the construction of ecological civilization, and is a key path to a new pattern of high-quality agricultural development.

[0009] Therefore, there is an urgent need to develop efficient, stable, and broad-spectrum plant-derived fungicides that can effectively control various plant diseases caused by fungi. Summary of the Invention

[0010] This invention provides a highly efficient, stable, and broad-spectrum plant-derived fungicide that can specifically inhibit various pathogenic fungi. It can effectively prevent and control plant diseases caused by various fungi, as well as fungal infections caused by Fusarium solanum, ensuring the quality of agricultural products and protecting human health. It also avoids the drawbacks of chemical agents, meets the needs of green agriculture and ecological protection development, and has important practical application value.

[0011] The present invention provides an antifungal preparation comprising bluewort extract.

[0012] Furthermore, the aforementioned bluewort extract is a water extract or an alcohol extract.

[0013] The water extracts in this invention can be extracted using hot water extraction, cold water soaking, distillation, etc.; the alcohol extracts in this invention can be extracted using hot alcohol extraction, cold alcohol soaking, reflux extraction, etc.

[0014] Furthermore, the aforementioned fungi are one or more of the following: Fusarium trifidum, Alternaria alternata, Fusarium moniliforme, Fusarium solanaceum, and Rhizoctonia solani.

[0015] Furthermore, the above-mentioned formulations also include pharmaceutically acceptable excipients.

[0016] Furthermore, the concentration of the above-mentioned bluewort extract is 2-6 mg / mL.

[0017] Furthermore, the concentration of the above-mentioned bluewort extract is 2.5 or 5 mg / mL.

[0018] The present invention also provides the use of any of the above-mentioned formulations for inhibiting plant pathogenic fungi.

[0019] Furthermore, the aforementioned fungi are one or more of the following: Fusarium trifidum, Alternaria alternata, Fusarium moniliforme, Fusarium solanaceum, and Rhizoctonia solani.

[0020] The present invention also provides the use of the above-described formulation in the preparation of a drug for preventing and treating human fungal infections.

[0021] Furthermore, the aforementioned human fungal infections are caused by Fusarium solanum, and include fungal keratitis, dermatophyte and onychomycosis, and systemic disseminated fungal infections.

[0022] This invention utilizes extracts from the above-ground parts of *Cynanchum paniculatum* to treat: 1) pumpkin fruit rot, potato dry rot, and soybean and alfalfa root rot caused by *Fusarium trifidum*; 2) leaf spot, leaf blight, early blight, and black spot caused by *Alternaria alternata*; 3) severe ear rot, sheath rot, and various fruit rot diseases in rice, corn, and other grain crops, as well as bananas, chestnuts, melons, jujubes, and other horticultural crops caused by *Fusarium solani*; and 4) various crop diseases and fungal horn rot caused by *Fusarium solanum*, including tobacco wilt and root rot. 5) Antifungal experiments were conducted on pathogens such as root rot of Chinese medicinal herbs such as ginseng and American ginseng caused by *Cyperus rotundus*. It was found that the extract of the aerial parts of *Cyperus rotundus* at a concentration between 2-6 mg / mL had an antifungal rate of 100% against *Fusarium trifidum*, *Alternaria alternata* and *Fusarium solani*. The antifungal rate against *Fusarium solanum* reached 98.12% and the antifungal rate against *Cyperus rotundus* reached 92.73%, thus finding a broad-spectrum antifungal natural drug preparation. Attached Figure Description

[0023] Figure 1 The image shows the inhibitory effect of the extract on Fusarium trifidum. Figure 2 The toxicity curve of the extract against the mycelial growth of Fusarium trifidum is shown. Figure 3 The graph shows the inhibitory effect of the extract on Alternaria alterniflora. Figure 4 The toxicity curve of the extract against the mycelial growth of Alternaria alterniflora is shown. Figure 5 The image shows the inhibitory effect of the extract on Fusarium moniliforme, a specialized strain of bitter melon. Figure 6 The toxicity curve of the extract against the growth inhibition of Fusarium hyphae is shown. Figure 7 The image shows the inhibitory effect of the extract on Fusarium solani. Figure 8 The toxicity curve of the extract against the mycelial growth of Fusarium solani is shown. Figure 9 The image shows the inhibitory effect of the extract on *Streptomyces rubrum*. Figure 10 The toxicity curve of the extract against the mycelial growth of *Streptomyces rubrum* is shown. Figure 11 The graph shows the inhibitory effects of different concentrations of extracts on different pathogens. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to examples. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained in the art without creative effort should fall within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0026] Example 1: Preparation method of extract from the above-ground parts of blue violet grass Step S1: Take some fresh sample material from the blue clover field and dry it quickly in an oven (stacked forced-air drying oven, model DHG-9120L-3) at 28℃. Step S2 involves crushing the material from S1 in a pulverizer (Fang Gongzi FGZ-2500C - knob); In step S3, the powder obtained in S2 is mixed with 75% ethanol at a ratio of 1:3 and then subjected to ultrasonic extraction at 30 °C for 30 min. After filtration, the powder is soaked again. The above steps are repeated three times, and finally the filtrates are combined.

[0027] In step S4, the filtrate from S3 was poured into a vacuum distillation concentrator (IKA, Shanghai Yarong Biochemical Instrument Factory) and concentrated at 45 ℃ to 100 mg / mL, which was then used as the technical grade of Flame Tree Flower Extract.

[0028] Example 2 Antibacterial effect experiment 2.1 Experimental Procedure Step S1: Prepare potato glucose agar (PDA) medium. Weigh 200.0 g peeled potato pieces, 20.0 g glucose, and 15 g agar, and add deionized water to 1000 mL. Sterilize by steaming at 121 ℃ and 0.1 MPa for 20 min.

[0029] Step S2: Activation of the test bacteria (source: provided by the Traditional Chinese Medicine Quality Testing Laboratory of the School of Biological and Brewing Engineering, Taishan University). In a clean bench (Shanghai Boxun Medical Bio-Instrument Co., Ltd.), pour the melted PDA medium into the petri dish near the flame of an alcohol lamp. Repeatedly heat the inoculation needle and punch in the outer flame of the alcohol lamp. After cooling, punch holes along the edge of the colony using the punch, and then use the inoculation needle to place the punched bacterial cake in the center of the solidified PDA medium. Seal the dish and incubate at 25°C for 7 days.

[0030] Step S3: Preparation of drug-containing plates: The original drug obtained in S4 was added to a sterilized PDA culture medium at 45℃. The drug and culture medium were mixed to prepare drug-containing plates with concentrations of 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, and 0.3125 mg / mL. A culture medium with an equal volume of sterile water was used as a blank control. Each treatment was repeated 3 times.

[0031] In step S4, use a 0.5 cm punch to collect mycelial cakes from the activated target strain culture medium. Use an inoculation needle to pick up the mycelial cakes and inoculate them into the center of the drug-containing plate in step S7. Seal the inoculated drug-containing plate and incubate it upside down in a 25°C incubator for 5-7 days. Measure the colony diameter. Step S5: Data Processing. Colony diameter was measured using the cross-multiplication method, and the effect of the drug-containing plates on fungal colony morphology was recorded by photographing. The inhibition rate was calculated using Excel software, and virulence analysis was performed using IBM SPSS Statistics 26.

[0032] Mycelial growth inhibition rate (100%) ×100%; 2.2 Experimental Results As shown in Table 1 below, the virulence of the aboveground extracts of *Hemiberlesia argyi* against the tested pathogens shows that the inhibitory effects of the aboveground extracts on the tested pathogens, from highest to lowest, are as follows: *Fusarium trifidum*, *Alternaria alternata*, and *Fusarium solani* all showed an inhibition rate of 100%; the inhibition rate against *Fusarium solani* reached 98.12%, and the inhibition rate against *Fusarium solani* reached 92.73%. Table 1 shows that the corresponding EC50 values ​​are 1.67 mg / mL, 1.563 mg / mL, 0.931 mg / mL, 0.699 mg / mL, and 2.154 mg / mL, respectively; Table 2 and... Figures 1-11 (a~f represent CK, 0.3125 mg / mL, 1.25 mg / mL, 2.5 mg / mL, and 5 mg / mL, respectively.) The results show that the extract from the above-ground parts of *Lysimachia christinae* at a concentration of 5 mg / mL exhibited 100% inhibition against *Fusarium trifidum*, *Alternaria alternata*, and *Fusarium solani*; the inhibition rate against *Fusarium solani* reached 98.12%, and the inhibition rate against *Fusarium solani* reached 92.73%. It is a broad-spectrum antibacterial agent with excellent antibacterial effects.

[0033] Table 1. Virulence determination of extracts from the aboveground parts of *Isodon japonicus* against the tested pathogens.

[0034] Table 2. Inhibitory effects of extracts from the aboveground parts of *Isodon japonicus* on five plant pathogens.

Claims

1. A fungicide preparation, characterized in that: The formulation includes bluewort extract.

2. The formulation according to claim 1, characterized in that: The bluewort extract is a water extract or an alcohol extract.

3. The formulation according to claim 1, characterized in that: The fungus is one or more of the following: Fusarium trifidum, Alternaria alternata, Fusarium moniliforme, Fusarium solanum, and Rhizoctonia solani.

4. The formulation according to claim 1, characterized in that: The formulation also includes pharmaceutically acceptable excipients.

5. The formulation as described in claim 1, characterized in that: The concentration of the bluewort extract is 2-6 mg / mL.

6. The formulation as described in claim 5, characterized in that: The concentration of the bluewort extract is 2.5 or 5 mg / mL.

7. Use of the formulation according to any one of claims 1-6 for the preparation of a medicament for inhibiting diseases caused by plant fungi.

8. The use as described in claim 7, characterized in that: The fungus is one or more of the following: Fusarium trifidum, Alternaria alternata, Fusarium moniliforme, Fusarium solanum, and Rhizoctonia solani.

9. Use of the formulation according to any one of claims 1-6 for the preparation of a medicament for the prevention and treatment of human fungal infections.

10. The use as described in claim 9, characterized in that: The human fungal infections are caused by Fusarium solanum, and include fungal keratitis, dermatophyte and onychomycosis, and systemic disseminated fungal infections.

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