Selatheconidea furcellata intestinal tract extracting solution as well as extracting method and application thereof

Abstract

The invention discloses a cantheconidea furcellata intestinal tract extracting solution as well as an extracting method and application thereof. The extracting method comprises the following steps: S1, performing surface disinfection on cantheconidea furcellata; s2, extracting the intestinal tract by using a PBS (Phosphate Buffer Solution); s3, screening out a microbial strain with bacteriostatic activity from the intestinal tract extracting solution, and transferring the microbial strain to a solid culture medium for culture; s4, selecting bacterial strains with bacteriostatic action, transferring the bacterial strains into a liquid culture medium for further culture, and comparing with a control group, so that the growth condition of the bacterial strains meets the expectation, the growth condition of the bacterial strains around the intestinal extract of the cantheconidea furcellata is poorer, and the diameter of the bacterial strains is obviously smaller than that of the bacterial strains of the control group; the cantheconidea furcellata intestinal tract extracting solution can be used for effectively preventing and treating the root rot; on one hand, the method for preventing and treating the tobacco root rot by utilizing the cantheconidea furcellata intestinal tract extracting solution can be in an experimental biological mode and is environment-friendly; by utilizing the method, target diseases cannot generate drug resistance, non-target organisms such as natural enemies and bees cannot be harmful, and ecological balance cannot be damaged.

Description

Technical Field

[0001] This invention relates to the field of tobacco pest and disease control technology, specifically to an intestinal extract of the forked bug, its extraction method, and its application. Background Technology

[0002] Tobacco root rot is a disease that affects tobacco plants. It can occur from seedling to mature plant stage, primarily damaging the root system and causing black rot. Root rot is a complex disease caused by fungi, leading to root decay and a gradual weakening of the plant's ability to absorb water and nutrients, eventually resulting in the death of the entire plant. Above-ground symptoms include yellowing and wilting of the leaves. In production practice, tobacco is mainly propagated through rhizomes. With the continuous expansion of tobacco planting areas, the incidence of various tobacco diseases is also on the rise, especially root rot, which is extremely serious. Also known as root and stem rot, this disease is a complex disease caused by fungal infection. The pathogen can spread through the soil or rhizomes, gradually harming tobacco growth and has become one of the main factors restricting the healthy development of the tobacco industry.

[0003] Currently, the main method used in production to control root rot is chemical agents, but this is not effective. Furthermore, chemical methods can easily pollute the soil environment and reduce the quality of tobacco products. Summary of the Invention

[0004] The purpose of this invention is to provide an intestinal extract of *Triplophysa fasciata*, its extraction method, and its application, in order to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for extracting intestinal extract of *Triplophysa fasciata*, comprising the following steps:

[0006] S1. Disinfect the surface of the forked-horn bug;

[0007] S2. Use PBS buffer to extract the intestines;

[0008] S3. Screen out microbial strains with antibacterial activity from the intestinal extract and transfer them to a solid culture medium for culture.

[0009] S4. After selecting the strain with antibacterial effect, transfer it to liquid culture medium for further cultivation.

[0010] Preferably, the solid culture medium is TSA or ISP2, and has an inhibition rate of more than 60% against root rot pathogens.

[0011] Preferably, the liquid culture medium formulation includes:

[0012] Yeast extract: 5g / L

[0013] Casein peptone: 10g / L

[0014] The pH value is 7.4±0.2.

[0015] An intestinal extract of *Triplophysa fasciata* obtained by a method for extracting intestinal extract of *Triplophysa fasciata*.

[0016] An application of the intestinal extract of *Triplophysa fasciata*, as described in claim 4, is to use the intestinal extract of *Triplophysa fasciata* as a control agent against tobacco root rot fungi.

[0017] Preferably, the method of using the control agent to control tobacco root rot includes: diluting the intestinal extract of *Triplophysa fasciata* by 20 times and then applying it to the roots of tobacco plants for irrigation or foliar spraying.

[0018] Compared with the prior art, the beneficial effects of the present invention are: the use of the intestinal extract of *Triplophysa fasciata* to prevent and control root rot achieves the following effect: compared with the control group, the growth of the strains is as expected, the growth of the strains around the intestinal extract of *Triplophysa fasciata* is poor, and the diameter of the strains is significantly smaller than that of the control group, indicating that the intestinal extract of *Triplophysa fasciata* can effectively prevent and control root rot.

[0019] Using the intestinal extract of the forked bug to control tobacco root rot is an experimental biological approach that is environmentally friendly. Furthermore, this method does not lead to drug resistance in the target disease or harm non-target organisms such as natural enemies and bees, thus avoiding disruption of the ecological balance. This method saves labor and management, conserves land resources, and reduces crop pests and diseases to some extent. Attached Figure Description

[0020] Figure 1 Photographs of the intestines of *Triplophysa fasciata* A and B, and the intestinal extract culture dish of *Triplophysa fasciata* provided in Example 2 of this application;

[0021] Figure 2 A comparative diagram showing the growth of the A and B root rot pathogens inoculated on culture dishes (control group) and the experimental treatment group in Example 2 of this application. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise specified, all materials and instruments used in the following examples were obtained commercially; and all detection methods and strain culture methods used are existing methods unless otherwise specified.

[0024] Please see Figures 1-2 This invention provides a technical solution: a method for extracting intestinal extract of *Triplophysa fasciata*, comprising the following steps:

[0025] S1. Material Preparation:

[0026] Forked-horn bug individuals: Select healthy forked-horn bug individuals;

[0027] PBS buffer: used for intestinal extraction;

[0028] Culture medium: such as TSA or ISP2 medium, used to culture the extracted microorganisms;

[0029] S2. Intestinal extract of *Trigonella asiatica*:

[0030] First, disinfect the surface of the forked-horn bug; usually, 70% ethanol or other suitable disinfectants can be used for disinfection.

[0031] Next, dissect the forked-horned bug and remove its intestinal portion; handle the operation carefully to avoid contaminating other parts;

[0032] The gut is extracted using PBS buffer; PBS buffer helps to dissolve microorganisms or other active substances in the gut.

[0033] S3. Bacterial screening and culture:

[0034] Microbial strains with antibacterial activity were screened from intestinal extracts; these strains grew on culture media and showed the ability to inhibit root rot pathogens.

[0035] Select strains with antibacterial effects and transfer them to solid culture medium (such as TSA or ISP2) for culture;

[0036] The selected strain is cultured, usually at 28°C, and colony growth is observed after 24 hours.

[0037] S4. Fermentation and preparation of the extract:

[0038] After selecting strains with antibacterial activity, they are transferred to liquid culture medium for further cultivation; the culture medium formulation includes:

[0039] Yeast extract: 5g / L

[0040] Casein peptone: 10g / L

[0041] pH value: 7

[0042] Other ingredients: to be adjusted according to requirements;

[0043] The strain was cultured in a constant temperature shaker (28°C, 200 rpm) for 24 hours to promote its growth and fermentation.

[0044] Application of intestinal extract of *Fusarium oxysporum* in the control of plant root rot, where the fungus causing root rot is *Fusarium oxysporum*. *Fusarium oxysporum* is a fungal pathogen that causes root rot in tobacco, widely distributed globally and posing a threat to tobacco growth. This strain belongs to the genus *Fusarium* and is a common soil-borne pathogen in tobacco and other plants.

[0045] The intestinal extract of *Stachys formosanus* showed inhibitory effects on *Fusarium oxysporum* root rot pathogens with diameters of 8 mm and 84 mm, respectively; the intestinal extract of *Stachys formosanus* also showed inhibitory effects on root rot pathogens.

[0046] A control agent for the intestinal extract of the forked bug, wherein the active ingredient of the control agent is the intestinal extract of the forked bug and its fermentation products or the fermentation liquid of the intestinal extract of the forked bug.

[0047] The method of using the control agent is as follows: After transplanting tobacco seedlings or rhizomes, the fermented bacterial solution of the intestinal extract of the forked bug is diluted 20 times and then applied to the plants for irrigation or foliar spraying.

[0048] The fermentation medium for the intestinal extract of *Triplophysa fasciata*, per 1L, contains approximately 5g of yeast extract, approximately 10g of tryptone, and has a pH of approximately 7.4.

[0049] The fermentation culture temperature of the intestinal extract of *Triplophysa fasciata* was 37℃, the shaking speed was 190 rpm, and the culture time was 24 h.

[0050] Example 1

[0051] Isolation, purification and screening of microorganisms from the gut reagent of *Trichoderma harzianum*

[0052] I. Experimental Methods

[0053] Microbial isolation and purification of intestinal extract from *Triplophysa fasciata*:

[0054] Sampling was conducted in tobacco fields in Chuxiong, Yunnan. Single-egg masses of female insects were collected and subjected to 20 generations of sister mating to produce offspring. A portion of the offspring were randomly selected and cultured in the laboratory.

[0055] 2. Feeding

[0056] Forked-horned bugs were kept in a 40cm×40cm×40cm cubic ventilated plastic box. They were fed mealworms once a day and kept in a suitable humidity and temperature (25–28℃, relative humidity 60–70%).

[0057] 3. Obtaining intestinal extracts from *Tripterygium wilfordii*.

[0058] (1) Select insects: Take 5 nymphs of the 4th to 5th instar of the forked horn bug.

[0059] (2) Ice bath treatment: Take 500g of ice at -5℃, spread it evenly in an ice box, place the forked bug on the ice surface for 3–5 minutes to make it unconscious, so as to facilitate subsequent operations.

[0060] 4. Isolation and purification of bacterial strains

[0061] In a sterile environment such as a laminar flow hood, wipe the surface of the forked horn bug with a 75% ethanol swab for 1 minute; then rinse and soak the surface of the bug with sterile water 3 times, 1 minute each time, to remove any alcohol residue.

[0062] The prepared forked bug was placed in a sterile petri dish and dissected using sterile dissecting tools (such as scissors and forceps) to obtain intact intestinal tissue. The intestinal tissue was then gently rinsed in sterile water to remove any attached tissue or impurities.

[0063] Transfer the cleaned intestinal tissue into a 50mL sterile centrifuge tube, add PBS buffer to 50mL, place on a 28℃ constant temperature shaker, and shake at 150rpm for 20 minutes to allow the intestinal microorganisms to be fully released into the solution.

[0064] Then, take 100 μL of the original shake solution and perform serial dilutions of 10-2 times using sterile water. Take 100 μL of the diluted liquid and inoculate it onto a TSA (tryptone soybean agar) solid medium plate. Spread it evenly with a sterile spreader, seal it, and incubate it in a 28°C incubator for 12–24 hours.

[0065] After the colonies have grown, single colonies of different morphologies are selected and inoculated into LB liquid medium for propagation and culture, which will be used for subsequent identification and functional analysis.

[0066] 2. Microbial screening of intestinal extracts from *Triplophysa fasciata*

[0067] 1. Initial screening of the biocontrol function of the strain

[0068] The initial screening of the biocontrol function of the strains was conducted using a plate confrontation experiment. The specific steps were as follows: Root rot pathogens were pre-inoculated into PDA (potato dextrose agar) medium (a fungal block of approximately 5 mm was punched in the center of the medium for inoculation). Different isolated and purified bacteria were then spotted around the mycelium at a distance of 10 mm, forming a cross shape with four strains. A plate inoculated only with root rot pathogen strains served as a control. The plates were incubated at 25°C for 6 days. When the blank plate reached full coverage, the plates inoculated with root rot pathogen strains were observed, recorded, and selected bacteria exhibiting inhibition zones for further use.

[0069] 2. Rescreening of strains for biocontrol function

[0070] The screening method was the same as above, but after each plate was inoculated with fungi, the same strain was inoculated in the four corners. The control was inoculated with root rot pathogens and the plate was fully covered before the inhibition zone of the strain was recorded.

[0071] II. Results Analysis:

[0072] Table 1 shows the inhibitory effect of the intestinal extract of *Triplophysa fasciata* on tobacco root rot pathogens.

[0073]

[0074] From Table 1 and Figure 1 It was observed that in the experimental group inoculated with the intestinal extract of *Triplophysa fasciata*, the colony diameter of the root rot pathogen was significantly reduced, with an average diameter of 29.4 mm, which was significantly smaller than that of the control group (84.0 mm), and the inhibition rate reached 65%. This indicates that the intestinal extract of *Triplophysa fasciata* has a strong inhibitory effect on this pathogen.

[0075] In the control group, only root rot pathogens were inoculated without any other interference treatments. The pathogens grew well in the culture medium, with intact colony morphology, rapid expansion, and the largest colony diameter.

[0076] The results in summary indicate that the intestinal extract of *Triplophysa fasciata* can significantly inhibit the growth of the pathogen causing tobacco root rot, demonstrating a clear antibacterial effect with a stable inhibition rate of 60%–70%, providing a theoretical basis for the development of subsequent biocontrol products.

[0077] Example 2

[0078] Preparation of biocontrol agent from intestinal extract of *Triplophysa fasciata*: Intestinal extract of *Triplophysa fasciata* was streaked onto solid ISP1 medium (tryptone: 5.0 g / L, yeast extract: 3.0 g / L, agar: 15 g / L, pH 7.0 ± 0.2, sterilized at 121℃ for 10 minutes) until single colonies were obtained. The culture conditions were: 28℃ for 14 h in a constant temperature incubator. After single colonies grew, they were picked and transferred to test tubes containing 6 mL of ISP1 liquid medium and placed in a constant temperature shaker at 28℃ and 200 rpm for 24 h to obtain the biocontrol agent.

[0079] Example 3

[0080] Preparation of biocontrol agent from intestinal extract of *Triplophysa fasciata*: Intestinal extract of *Triplophysa fasciata* was streaked onto solid culture medium ISP2 (ISP2 solid culture medium formula: yeast extract: 3.0 g / L, malt extract: 9.0 g / L, agar: 15 g / L, glucose: 5.0 g / L, pH 7.5, sterilized at 121℃ for 15 minutes) until single colonies were obtained. The culture conditions were: 14 h in a constant temperature incubator. After single colonies grew, they were picked and transferred to test tubes containing 10 mL of ISP2 liquid culture medium (without agar), and then incubated in a constant temperature shaker at 28℃ and 200 rpm for 24 h to obtain the biocontrol agent.

[0081] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for extracting intestinal extract of *Triplophysa fasciata*, characterized in that: Includes the following steps: S1. Disinfect the surface of the forked-horn bug; S2. Use PBS buffer to extract the intestines; S3. Screen out microbial strains with antibacterial activity from the intestinal extract and transfer them to a solid culture medium for culture. S4. After selecting the strain with antibacterial effect, transfer it to liquid culture medium for further cultivation.

2. The method for extracting the intestinal extract of *Triplophysa fasciata* according to claim 1, characterized in that: The solid culture medium is TSA or ISP2, and has an inhibition rate of more than 60% against root rot pathogens.

3. The intestinal extract of *Triplophysa fasciata* according to claim 1, its extraction method, and its application, characterized in that: The liquid culture medium formulation includes: Yeast extract: 5g / L Casein peptone: 10g / L The pH value is 7.4±0.

2.

4. The intestinal extract of *Triplophysa fasciata* prepared by the extraction method according to any one of claims 1-3.

5. An application of an intestinal extract of *Triplophysa fasciata*, characterized in that: The intestinal extract of *Trichoderma harzianum* as described in claim 4 is used as a control agent against tobacco root rot fungi.

6. The application of the intestinal extract of *Triplophysa fasciata* according to claim 5, characterized in that: The method of using the control agent to control tobacco root rot includes: diluting the intestinal extract of *Triplophysa fasciata* 20 times and applying it to the roots of tobacco plants for irrigation or foliar spraying.

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