A strain of Trichoderma and its microcapsule preparation method and application

Abstract

The invention discloses a strain of Trichoderma and a preparation method and application of its microcapsules, belonging to the field of plant disease biocontrol bacteria. Trichoderma afroharzianum )YZ‑1 has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241084. The African harzianum YZ‑1 provided by the present invention has excellent control effect on pepper white rot, with a control effect of up to 97.57%; in addition, it also has antagonistic activity against five other plant pathogenic fungi on pepper and tobacco, with an inhibition rate between 51.12±1.20% and 81.09±1.28%. At the same time, the present invention uses sodium alginate and montmorillonite K‑10 as wall materials to prepare microcapsules of African harzianum YZ‑1 by an extrusion method, and the potted plant control effect is 74.13%. The African harzianum and its microcapsules provided by the present invention have great potential to be developed into commercial live bacterial biocontrol preparations.

Description

Technical Field

[0001] The invention relates to the field of plant disease biocontrol bacteria, in particular to a strain of Trichoderma and a preparation method and application of its microcapsules. Background Art

[0002] Biocontrol agents refer to biological products made from microorganisms (such as bacteria, fungi, viruses, etc.) and their metabolites, which are used to prevent and control plant diseases. The research and application of biocontrol agents can effectively alleviate the problems of environmental pollution, enhanced disease resistance and threats to human health caused by the use of chemical pesticides, and are very necessary for the sustainable development of agriculture.

[0003] However, the instability of biological agents in soil due to temperature, humidity, ultraviolet radiation and other reasons is the main challenge in their use in agriculture. Microencapsulation is an innovative and effective technology, which refers to the process of encapsulating active ingredients in a protective coating or matrix. It can act as a barrier to protect microorganisms from biotic or abiotic stresses and increase their stability. It can also overcome the limitations of other formulations, such as solid or liquid formulations, in storage and application, and extend the life of microorganisms by controlling the release of microorganisms and improve the efficiency of their use.

[0004] Microcapsule performance is determined by the characteristics of the carrier or encapsulating material, which plays a vital role in the success and efficiency of microencapsulation of biocontrol agents. Sodium alginate is a material commonly used as a carrier. It is a polysaccharide composed of α-l-glucuronic acid and β-mannuronic acid units. Its non-toxicity, biodegradability, and easy gelation with various crosslinking agents such as calcium ions make it a preferred carrier material for encapsulating cells, drugs and other bioactive molecules for delivery and release. However, alginate has limitations such as high mechanical stiffness, uneven size, and poor physical properties, making it unsuitable for providing long-term stability of encapsulated microorganisms. Therefore, in order to overcome these limitations, suitable fillers are usually added to the formulation, and montmorillonite is one of them. It has a large surface area, excellent swelling ability, and is naturally present in soil, making it a suitable filler encapsulation.

[0005] At present, there are relatively few biocontrol agents found in the prevention and treatment of pepper white rot. For example, CN 104988079B discloses a Trichoderma harzianum JSNL1404, which has a strong antibacterial ability against Dendrobium officinale white rot; another example is CN 117327593A discloses a Trichoderma harzianum ZM1, which has a preventive effect on peanut white rot. There are relatively few biocontrol agents for the prevention and treatment of pepper white rot, and there are even fewer reports on Trichoderma microcapsules. Therefore, there is an urgent need to screen a Trichoderma strain with a high biocontrol effect on pepper white rot, and realize its application through microencapsulation. Summary of the invention

[0006] The purpose of the present invention is to provide a strain of Trichoderma and a method for preparing and applying its microcapsules to solve the problems existing in the above-mentioned prior art. The African Trichoderma harzianum YZ-1 and its microcapsules provided by the present invention have excellent control effect on pepper white rot and have great potential to be developed into a commercial live bacterial biocontrol preparation.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The invention provides a strain of Trichoderma afroharzianum YZ-1. The preservation number of the strain of Trichoderma afroharzianum YZ-1 is CCTCC NO: M 20241084.

[0009] The present invention also provides a Trichoderma harzianum YZ-1 microcapsule, comprising the Trichoderma harzianum YZ-1.

[0010] The present invention also provides a method for preparing the Trichoderma harzianum YZ-1 microcapsules, comprising the following steps:

[0011] Take 1-4 g of montmorillonite K-10 and dissolve it in 100-500 mL of deionized water, add 6-9 g of sodium alginate, and then add glycerol and mix thoroughly to obtain a montmorillonite-sodium alginate solution;

[0012] Take the spore liquid of Trichoderma harzianum YZ-1, centrifuge to obtain spores, add the montmorillonite-sodium alginate solution to suspend the spores, and obtain a spore concentration of 1×10 7 ~1×10 9 spores / mL of montmorillonite-alginate spore suspension;

[0013] The montmorillonite-sodium alginate spore suspension was squeezed into a 2% CaC12 solution and washed with sterile water to obtain Trichoderma harzianum YZ-1 microcapsules.

[0014] Furthermore, the preparation method comprises the following steps:

[0015] Take 2.5 g of montmorillonite and dissolve it in 300 mL of deionized water, add 7.5 g of sodium alginate, and then add glycerol and mix thoroughly to obtain a montmorillonite-sodium alginate solution;

[0016] Take the spore liquid of Trichoderma harzianum YZ-1, centrifuge to obtain spores, add the montmorillonite-sodium alginate solution to suspend the spores, and obtain a spore concentration of 1×10 8 spores / mL of montmorillonite-alginate spore suspension;

[0017] The montmorillonite-sodium alginate spore suspension was squeezed into a 2% CaC12 solution and washed with sterile water to obtain Trichoderma harzianum YZ-1 microcapsules.

[0018] The invention also provides a microbial agent containing the African harzianum fungus YZ-1.

[0019] Furthermore, the live bacteria concentration of the microbial agent is 1×10 8 Spores / mL.

[0020] The present invention also provides use of the African harzianum YZ-1, the African harzianum YZ-1 microcapsule or the microbial agent in inhibiting the growth of pathogens, wherein the pathogens include pepper white rot fungus, pepper gray mold fungus, pepper blight fungus, tobacco target spot fungus, pepper damping-off fungus and pepper anthracnose fungus.

[0021] The present invention also provides an application of the African harzianum Trichoderma YZ-1, the African harzianum Trichoderma YZ-1 microcapsule or the microbial agent in preventing and controlling plant diseases, wherein the plant disease is pepper bacterial rot caused by Sclerotium rolfsii.

[0022] The present invention also provides a method for preventing and treating pepper white rot caused by pepper white rot fungus, comprising the following steps:

[0023] Treating pepper plants with the Trichoderma harzianum YZ-1 or its fermentation liquid or bacterial suspension or the Trichoderma harzianum YZ-1 microcapsules;

[0024] The live bacteria concentration in the fermentation liquid or bacterial suspension is 1×10 8 Spores / mL.

[0025] Furthermore, the treatment is: adding a concentration of 1×10 8 The pepper plants are irrigated with the fermentation liquid or bacterial suspension of African harzianum YZ-1 or the African harzianum YZ-1 microcapsules with spores / mL.

[0026] The present invention discloses the following technical effects:

[0027] The present invention isolates and screens a strain of Trichoderma afroharzianum YZ-1. Experimental verification shows that the Trichoderma afroharzianum YZ-1 provided by the present invention has excellent control effect on pepper white rot caused by pepper white rot fungus, with a control effect of up to 97.57%; in addition, it also has antagonistic activity against five other plant pathogenic fungi on pepper and tobacco, with an inhibition rate of 51.12±1.20% to 81.09±1.28%. At the same time, the present invention uses sodium alginate and montmorillonite K-10 as wall materials, and prepares microcapsules of Trichoderma afroharzianum YZ-1 by extrusion, and its potted plant control effect is 74.13%. The Trichoderma afroharzianum and its microcapsules provided by the present invention have great potential to be developed into commercial live bacterial biocontrol preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a graph showing the inhibitory effect of African Trichoderma harzianum YZ-1 on pepper white rot fungus, where the left side is the control; the right side is the confrontation culture of YZ-1 and pepper white rot fungus;

[0030] Figure 2 The colony morphology of Trichoderma harzianum YZ-1 on different culture media; ab: colony morphology on the front and back of PDA; cd: colony morphology on the front and back of CMD. ef: colony morphology on the front and back of SNA;

[0031] Figure 3 This is a multi-gene phylogenetic tree constructed based on ITS and tef-1 gene sequences;

[0032] Figure 4The antagonistic activity of Trichoderma harzianum YZ-1 against different pathogens; the first row is tobacco target spot pathogen (Rhizoctonia solani), pepper gray mold (Botrytis cinerea), pepper anthracnose pathogen (Colletotrichum scovillei), pepper blight pathogen (Phytophthora capsica), pepper damping-off pathogen (Rhizoctonia solani); the second row is tobacco target spot pathogen (Rhizoctonia solani), pepper gray mold (Botrytis cinerea), pepper anthracnose pathogen (Colletotrichum scovillei), pepper blight pathogen (Phytophthora capsica), pepper damping-off pathogen (Rhizoctonia solani) added with Trichoderma harzianum YZ-1;

[0033] Figure 5 The control effects of different treatment groups on pepper white rot; A: Ⅰ is the pepper plant in the pot, Ⅱ is the pepper plant after the soil is removed; B: the relative control effect bar chart of different treatments; Control: blank control; 0.4% Tiofuramide: positive control;

[0034] Figure 6 The effects of different treatment groups on the growth of pepper plants; A: pepper plants in pots; B: pepper plants after the soil is removed;

[0035] Figure 7 The hyphae morphology of Trichoderma harzianum YZ-1 after heavy parasitism of pepper Sclerotium rolfsii was observed by microscope (A) and scanning electron microscope (B); a: attachment; b: entanglement; c: dissolution; b: overgrowth;

[0036] Figure 8 Verification of transformants of YZ-1-GFP strain; A: molecular verification; B: fluorescence verification;

[0037] Fig. 9 Observation of YZ-1-GFP1 colonization in pepper plants; 1 day after inoculation, spores were observed attached to the lateral root tips (c); on the 2nd day, they aggregated in the epidermal cells on the root surface (d); 3 days after inoculation, they colonized in the main root (a); on the 5th day, fluorescence was detected in the epidermal cells at the junction of the root and stem (g); 7 days after inoculation, hyphae had penetrated into the epidermal cells of the stem (f); no fluorescence was observed in the leaves (b); e is the cross-section observation of the stem base on the 6th day; h is the observation of the root tip on the 4th day;

[0038] Fig.10 The release of YZ-1 microcapsules in normal saline;

[0039] Fig.11 The particle size statistics of YZ-1 microcapsules; A and B are the particle sizes of empty microcapsules and YZ-1 microcapsules in wet state, respectively; C and D are the particle sizes of empty microcapsules and YZ-1 microcapsules in dry state, respectively;

[0040] Fig.12 The morphology of YZ-1 microcapsules was observed by scanning electron microscope; A and B are the morphologies of empty microcapsules at 200× and 50000×, respectively; C and D are the morphologies of YZ-1 microcapsules at 200× and 50000×, respectively;

[0041] Fig.13 FTIR spectrum of YZ-1 microcapsule; the left side is empty microcapsule; the right side is YZ-1 microcapsule;

[0042] Fig.14 Results of UV stability test of YZ-1 microcapsules; A: dry YZ-1 microcapsules; B: wet YZ-1 microcapsules; C: free YZ-1 spores;

[0043] Fig.15 The temperature stability test results of YZ-1 microcapsules; a, b, c are the survival rates of dry YZ-1 microcapsules at 7°C, 27°C, and 37°C, respectively; d, e, f are the survival rates of wet YZ-1 microcapsules at 7°C, 27°C, and 37°C, respectively;

[0044] Fig.16 The plate inhibition results of YZ-1 microcapsules; YZ-1-GFP1: YZ-1-GFP1 strain; YZ-1-GFP1+SA+K-10: YZ-1-GFP1 microcapsules; the same time: YZ-1-GFP1 microcapsules and pathogen XTBJ-1 were inoculated at the same time; after 1 day: YZ-1-GFP1 microcapsules were inoculated one day before pathogen XTBJ-1 was inoculated; SA+K-10: empty microcapsules; Control: blank control;

[0045] Fig.17 The results of the potted plant test of YZ-1 microcapsules. A is the disease situation of each treatment group after removing the soil, where ae is the blank control, empty microcapsule treatment, YZ-1-GFP1 spore solution treatment, YZ-1-GFP1 microcapsule treatment, and positive control; BG is the determination of various indicators after the disease of each treatment, which are relative protection effect, plant height, stem length, root length, fresh weight, and dry weight. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] In the following embodiments, the pathogenic fungi involved, Botrytis cinerea, Phytophthora capsica, Rhizoctonia solani, Rhizoctonia solani, Colletotrichum scovillei and Sclerotium rolfsii, have been disclosed in the literature (Qiu ZL, Liu SD, Li XG, et al. Identification and mechanism characterization of Streptomyces griseoaurantiacus XQ-29 with biocontrol ability against pepper southern blight caused by Sclerotium rolfsii [J]. Pesticide Biochemistry and Physiology, 2024, 202. DOI: 10.1016 / j.pestbp.2024.105956.).

[0052] The culture medium used in the following examples is:

[0053] Red Bengal medium (RBA): 10 g glucose, 5 g peptone, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 20 g agar, 0.033 g red Bengal, 0.1 g chloramphenicol and 1 L distilled water, pH 7.0-7.2;

[0054] Corn flour and glucose medium (CMD): 40 g corn flour, 20 g agar, 20 g glucose and 1 L distilled water;

[0055] Synthetic nutrient-free agar medium (SNA): KH2PO4 1 g, KNO3 1 g, MgSO4·7 H2O 0.5 g, KC10.5 g, agar 20 g, glucose 0.2 g, sucrose 0.2 g and distilled water 1 L.

[0056] In the following examples, SPSS 23.0 was used to perform one-way ANOVA analysis on all experimental data, and Tukey's test was used for multiple comparisons and significance analysis.

[0057] Example 1 Identification of African Trichoderma harzianum YZ-1

[0058] The present invention obtains an antagonistic fungus with an inhibitory effect on Sclerotium rolfsii (referred to as XTBJ-1 strain) through screening. Through morphological observation and molecular biological identification, the strain is identified as Trichoderma afroharzianum and named YZ-1.

[0059] 1. Isolation and screening of Trichoderma harzianum

[0060] Using Bengal red medium, the African harzianum strains were isolated and purified from the rhizosphere soil of pepper by the dilution plate method. Using pepper Sclerotium rolfsii XTBJ-1 as the target pathogen, African harzianum strains with antibacterial effects were screened by confrontation culture.

[0061] The results of confrontation culture screening are as follows Figure 1 As shown, the present invention screened out a strain of Trichoderma harzianum which has a significant inhibitory effect on the growth of mycelium of Sclerotium rolfsii of pepper, and its plate inhibition rate was 53.04±0.08%, and was named YZ-1.

[0062] 2. Morphological observation

[0063] Inoculate YZ-1 onto PDA, CMD and SNA medium. Observe the growth rate and colony morphology of the strain. Take the young mycelium at the edge of the YZ-1 plate cultured for 3 days and observe the conidia and conidiophores under an optical microscope.

[0064] from Figure 2 It can be seen that YZ-1 has abundant mycelium on PDA medium, which is cotton-like, yellow-green to green, odorless, and produces yellow pigment ( Figure 2 a, b); On CMD medium, the hyphae are abundant and cotton-like, with a large number of conidia growing in clusters at the top of the hyphae, distributed in dots, with yellow to dark green on the front and white to yellow on the back ( Figure 2 c, d); on SNA medium, the hyphae are sparse, white to light green ( Figure 2 (e, f).

[0065] 3. Molecular identification

[0066] The genomic DNA of YZ-1 was extracted by CTAB method. The ITS and tef-1 sequences of YZ-1 strain were amplified using the primers listed in Table 1. Each 50 µL PCR reaction system contained 25 µL FineTaq PCR Super, 2 µL forward primer (10 µM), 2 µL reverse primer (10 µM), 4 µL DNA template and 17 µL H2O. The PCR reaction program was: 95℃ pre-denaturation for 3 min; 35 amplification cycles (95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min); 72℃ extension for 5 min.

[0067] The amplified products were sequenced (Qingke Biotechnology Co., Ltd., Beijing, China), and the obtained sequences were compared with the NCBI database using BLASTn. The maximum likelihood method in MEGA 7.0 software was used to construct a multi-gene phylogenetic tree ( Figure 3 ), combined with the strain morphological characteristics and molecular biological identification, YZ-1 was finally identified as Trichoderma afroharzianum.

[0068] Table 1 Primer sequences used

[0069]

[0070] The above-mentioned African harzianum Trichoderma YZ-1 has been deposited, its deposit number is CCTCC NO: M 20241084, its classification name is Trichoderma afroharzianum, the deposit date is May 28, 2024, the depository is China Center for Type Culture Collection (CCTCC), and the deposit address is Wuhan University, China.

[0071] Example 2 The protective effect of Trichoderma harzianum YZ-1

[0072] 1. Antibacterial spectrum

[0073] The plate confrontation culture method was used to determine the antibacterial spectrum of YZ-1. Botrytis cinerea, Phytophthora capsica, Rhizoctonia solani, Rhizoctonia solani and Colletotrichum scovillei were used as target bacteria, and the target bacteria cake (d=8 mm) was inoculated on the left side of a PDA plate with a diameter of 7.5 cm. YZ-1 was inoculated 5 cm away from the cake. The plate inoculated with only the target bacteria was used as the blank control. Each treatment was repeated 3 times and cultured in a constant temperature incubator at 28℃. When the colony of the control group was about to fill the plate, the colony diameters of the treatment group and the control group were recorded, and the antibacterial rate of YZ-1 against the tested pathogenic fungi was determined.

[0074] The antagonistic activity of YZ-1 against the above five plant pathogenic fungi was detected by plate confrontation method ( Figure 4 ), and found that the inhibition rate of YZ-1 on these strains ranged from 51.12±1.20% to 81.09±1.28% (Table 2). Among them, the growth of tobacco target spot pathogen and pepper gray mold pathogen was significantly inhibited.

[0075] Table 2 Determination of the antibacterial spectrum of YZ-1 against five pathogens

[0076]

[0077] Note: The data in the table are all mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences at the P < 0.05 level by Tukey's HSD test.

[0078] 2. Live control effect on pepper white rot

[0079] The YZ-1 bacterial cake was cultured in PD for 7 days, and the mycelia were obtained by gauze filtration. The filtered liquid was centrifuged at 6000 rpm for 10 min, and the supernatant was collected as the YZ-1 fermentation filtrate. The precipitate was collected and dissolved with sterile water, and the mycelia were added and mixed to obtain the YZ-1 bacterial suspension with a concentration of 10 8 Spores / mL.

[0080] The YZ-1 bacterial cake was cultured in PD for 7 days. After 7 days, all the cultured bacterial liquid was broken to obtain the YZ-1 fermentation liquid with a concentration of 10 8 Spores / mL.

[0081] YZ-1 fermentation filtrate, YZ-1 bacterial suspension and YZ-1 fermentation liquid were used to irrigate the pepper plants 21 days after transplantation, 10 mL per plant. One day later, four fresh XTBJ-1 mycelium blocks (d=8 mm) were inoculated at the base of the plant stem and covered with moist soil, and cultured at 28°C. 0.4% thiothiocarb was used as a positive control. The disease conditions of each treatment were observed after 5 days. In order to observe the disease conditions more intuitively, the matrix of the inoculated pepper roots was removed, the excess impurities were washed with water, and the peppers were dried naturally. The length of the lesions at the base of the pepper stems was measured, and the disease index was calculated.

[0082] The severity of the disease (disease grade) is defined by the ratio of the length of the lesion to the length of the pepper stem, and the standards are as follows: Grade 0: X: 0, no symptoms; Grade 1: yellowing and falling of leaves, X≤1%; Grade 3: X≤10%; Grade 5: X≤20%; Grade 7: X≤30%; Grade 9: X>30%, plant death. X(%)=[(length of lesion at stem base) / (stem length of plant)]×100.

[0083] Calculation method of disease index: disease index = [Σ(number of diseased plants of this level × disease level) / (total number of plants surveyed × highest disease level)] × 100. Calculation formula for relative protection effect: relative protection effect (%) = [(disease index of control group - disease index of prevention and treatment group) / disease index of prevention and treatment group] × 100.

[0084] The control plants showed symptoms of yellowing and wilting leaves 5 days after the inoculation of XTBJ-1 mycelium at the stem base. After removing the soil from the roots, obvious brown spots were visible on the stems ( Figure 5 In contrast, plants treated with YZ-1 fermentation broth and bacterial suspension did not show these symptoms. The disease index of plants treated with YZ-1 fermentation broth and bacterial suspension was 1.23, while that of control plants was 50.62, with a relative control effect of 97.57% ( Figure 5 (B). It is worth noting that some diseases still existed in the plants treated with YZ-1 fermentation filtrate, resulting in a relative control efficiency of only 60.98%.

[0085] YZ-1 fermentation filtrate, YZ-1 bacterial suspension and YZ-1 fermentation liquid were used for root irrigation of pepper plants at the stage of four leaves and one heart, 10 mL per plant, once every 7 days, for three times, and water treatment was used as a control. After 5 days, the growth status was observed ( Figure 6 ), and the various indicators of pepper plants under different treatments were measured (Table 3). The results showed that there was no significant difference in plant height and stem length between plants treated with YZ-1 fermentation liquid, but their leaves were larger and their roots were denser, while those treated with fermentation filtrate were thinner.

[0086] Table 3 Determination of various indicators of pepper plants under different treatments

[0087]

[0088] Example 3 Study on the biocontrol mechanism of Trichoderma harzianum YZ-1

[0089] 1. Heavy parasitism on the hyphae of pepper white rot fungus

[0090] After the confrontation between the African Trichoderma harzianum strains YZ-1 and XTBJ-1, an obvious overlapping area appeared at the junction. In order to further explore the plate antibacterial mechanism of Trichoderma, the hyphae at the junction were observed. YZ-1 and XTBJ-1 were respectively connected to the two ends of the PDA plate, and a sterilized slide was placed in the middle. When the hyphae of the two were in contact on the slide, the slide was removed, clean water was added to the surface, and the cover glass was covered and observed under a microscope.

[0091] After co-culturing YZ-1 and XTBJ-1 for 7 days, the mycelial block at the junction of the two was cut off with a blade, fixed in 2.5% glutaraldehyde solution, and observed by SEM.

[0092] By observing the overlapping hyphae of strain YZ-1 and XTBJ-1 in dual culture under a microscope, it was found that YZ-1 could attach to, entangle in, and decompose the host hyphae ( Figure 7 Scanning electron microscopy showed that YZ-1 could wrap around and attach to the hyphae of XTBJ-1, depriving the host hyphae of nutrients and growing rapidly, while causing the host hyphae to disintegrate ( Figure 7 (B) This indicates that YZ-1 can parasitize the hyphae of S. rolfsii.

[0093] 2. Colonization analysis in pepper plants

[0094] Green fluorescent protein (GFP) was used as a reporter gene to observe the colonization of strain YZ-1 in pepper roots. The plasmid pCT74 carrying the hygromycin B phosphotransferase gene (hygr) was inserted into strain YZ-1 using the method described by Hua et al. in Oral vaccine against IPNV based on antibiotic-free resistance recombinant Lactobacillus casei expressing CK6-VP2fusion protein (2021) to construct the YZ-1-GFP strain. Positive transformants were identified and screened on PDA plates containing 150 µg / mL hygromycin B.

[0095] The YZ-1-GFP strain was cultured on a PDA plate for 7 days, scraped with a spreader, washed with sterile water, and filtered through gauze to obtain a concentration of 1×10 8 Conidia solution of spores / mL. The obtained conidia solution was irrigated to the roots of pepper plants at the four-leaf and one-heart stage, 10mL per plant. Pepper plants were sampled at different time points after root irrigation, the soil was carefully removed, the roots were rinsed with sterile water, and then sliced ​​with a lancet to prepare slides. Various parts of the pepper plants were observed using a fluorescence microscope.

[0096] Thirty-three transformants were obtained by hygromycin screening, of which 20 were used to observe mycelial luminescence under a fluorescence microscope. Among them, YZ-1-GFP-1, YZ-1-GFP-2, and YZ-1-GFP-3 had the brightest and most uniform fluorescence, and the colony morphology did not change. YZ-1-GFP-1 still had obvious fluorescence after 10 passages ( Figure 8 ), so YZ-1-GFP-1 was selected for subsequent experiments. YZ-1-GFP-1 spores were collected and inoculated into the roots of pepper plants. At the same time, sterile water was set as a control treatment group. On the first day after inoculation, YZ-1-GFP-1 spores adhered to the lateral root tips, gathered in the root epidermal cells on the second day, colonized the main root on the third day, and fluorescence was visible in the epidermal cells at the root-stem junction on the fifth day. On the seventh day, the hyphae had grown into the stem epidermal cells ( Fig. 9 ). No fluorescence was detected in the roots of pepper plants in the control treatment group.

[0097] Example 4 Preparation of Trichoderma harzianum YZ-1 microcapsules and determination of various indicators

[0098] 1. Microcapsule preparation method

[0099] Take 2.5 g of montmorillonite K-10 and dissolve it in 300 mL of deionized water, stir at room temperature, add 7.5 g of sodium alginate and continue to stir until it is completely dissolved, then add glycerol and mix thoroughly to obtain a montmorillonite-sodium alginate solution.

[0100] Take the YZ-1 spore solution, centrifuge at 5000 rpm for 10 min, remove the supernatant and get the spores. Add the montmorillonite-sodium alginate solution to suspend the spores and stir well to make the concentration of 1×10 8 Spores / mL, montmorillonite K-10-sodium alginate spore suspension was obtained. A magnetic stirrer was added to the 2% CaC12 solution, and the above montmorillonite K-10-sodium alginate spore suspension was squeezed into it with a syringe to obtain calcium alginate-montmorillonite microcapsules. The microcapsules were collected by gauze filtration, eluted with sterile water, and YZ-1 microcapsules were obtained, which were transferred to a conical flask and stored at 4°C.

[0101] 2. Determination of various indicators

[0102] 2.1 Encapsulation efficiency

[0103] 1 mL of a concentration of 1×10 8 Spores / mL montmorillonite K-10-sodium alginate spore suspension is dripped into a sterilized 2% CaC12 solution. 1 mL of the used CaC12 solution is taken and diluted to apply on the plate, and the free spore concentration is calculated under a microscope. Encapsulation efficiency (%) = (N0-Ne) / N0×100%, where N0 and Ne represent the spore concentration used to make microcapsules and the free spore concentration, respectively. The encapsulation efficiency was measured to be 99.98%. It can be seen that the preparation method provided by the present invention can well encapsulate Trichoderma harzianum in microcapsules.

[0104] 2.2 Sustained release rate

[0105] Weigh 0.3 g of YZ-1 microcapsules, add them to 7.5 mL of 0.9% saline, and incubate at 27°C. Dilutions of 100 μL were taken out every 5 days and plated, and the spore concentration was calculated under a microscope until the 65th day.

[0106] The CaCl2 solution used to prepare YZ-1 microcapsules was taken and plated, and the number of free colonies was calculated. The sustained release rate (EN%) was 99.98%. Fig.10 As shown, it reaches a peak at 25 days and then gradually decreases over time.

[0107] 2.3 Particle size

[0108] The particle sizes of empty microcapsules (montmorillonite K-10-calcium alginate) and YZ-1 microcapsules (YZ-1 spores + montmorillonite K-10-calcium alginate) were calculated using a stereo microscope, with 500 microcapsules for each treatment. The particle sizes of the two microcapsules were calculated again after drying at 27°C for 12 h.

[0109] The wet empty microcapsules are spherical and white in color. The wet YZ-1 microcapsules are green and spherical. The dried empty microcapsules are slightly yellow, while the dried YZ-1 microcapsules are dark green. The diameters of the empty microcapsules and the African Trichoderma YZ-1 microcapsules are mainly concentrated at 3200μm. Due to water loss, the diameters of the dried microcapsules are concentrated at 1200 ~ 1400μm ( Fig.11 ).

[0110] 2.4 Microcapsule morphology

[0111] The YZ-1 microcapsules and empty microcapsules were dried at 27 °C for 24 h. The external morphology of both microcapsules was observed using scanning electron microscopy (SEM). The microcapsules were dehydrated at 50 °C for 24 h before being coated with colloidal gold. Images were captured using a SEM (Hitachi SU8010) operating at 20 kV.

[0112] Scanning electron microscopy showed that the empty microcapsules were approximately spherical with a smooth surface. In contrast, the YZ-1 microcapsules showed slight protrusions and wrinkles, and the pore structure on the surface was more obvious ( Fig.12 ), which may promote the release of spores after water absorption and swelling.

[0113] 2.5 Fourier Transform Infrared Spectroscopy (FTIR)

[0114] At 400~4000 cm -1 The infrared spectra of the microcapsules were recorded on FTIR (Thermo Nicolet IS5, BRUKERTENSOR II) using KBr particle technology within the range of 200 nm. There were two treatments: YZ-1 microcapsules and empty microcapsules.

[0115] FTIR spectrum of microcapsules Fig.13 3379.72, 2941.62, 1614.42 and 1036.46 cm -1 The bands are OH, CH, OC=O and COC groups. -1 There is a strong and broad absorption band at 2151.88cm, which belongs to OH, which is related to adsorption of water and expansion rate. -1 The single peak at indicated that the microcapsules interacted with Trichoderma YZ-1.

[0116] 2.6 Stability

[0117] UV stability: Wet and dry YZ-1 microcapsules (1 g) were placed on plates, with free YZ-1 spores as controls. They were exposed to a 253.7 wavelength UV lamp, emitting UV light from a distance of 100 cm for 0, 0.5, 1, 2, 4, 9, and 24 h. 0.1 g was taken each time, suspended in 5 mL of 0.2 M NaHCO3 and 0.06 M sodium citrate solution, stirred for 15 hours until completely dissolved, and diluted to plate. The survival rate was expressed as the percentage of surviving conidia in the sample after UV irradiation relative to the conidia before irradiation. All treatments were repeated 3 times.

[0118] After 24 hours of ultraviolet irradiation, free YZ-1 spores died completely, with a mortality rate of 100%, while the mortality rates of dry and wet YZ-1 microcapsules were 87% and 91%, respectively ( Fig.14 ). Encapsulated YZ-1 was more resistant to UV light than unencapsulated YZ-1. This result emphasizes the beneficial role of microcapsules in enhancing the UV resistance of biocontrol agents.

[0119] Temperature stability: Wet and dry YZ-1 microcapsules (1 g) were placed in 7°C, 27°C, and 37°C environments for culture. Every 15 days, 0.1 g was taken and suspended in 5 mL of 0.2 M NaHCO3 and 0.06 M sodium citrate solution, stirred for 15 hours until completely dissolved and diluted and plated until 60 days later. The survival rate was expressed as the percentage of the number of conidia in the samples at different time points relative to the number of conidia on day 0. All treatments were repeated 3 times.

[0120] Temperature stability results are as follows Fig.15 As shown, the results show that both dry and wet microcapsules have the highest stability at 7°C and the worst stability at 37°C. At the same time, the dry microcapsules have higher storage stability.

[0121] 2.7 Antibacterial effect of plate

[0122] The antibacterial effect of YZ-1 microcapsules was observed by confrontation culture. There were four treatments: empty microcapsules, YZ-1-GFP1 microcapsules, YZ-1-GFP1 and blank control. The YZ-1-GFP1 microcapsule treatment was divided into two treatments: simultaneous inoculation with XTBJ-1 and inoculation of YZ-1-GFP1 microcapsules one day before inoculation of XTBJ-1.

[0123] When YZ-1-GFP1 microcapsules and XTBJ-1 were inoculated on PDA at the same time, the inhibition rate was 37.24%. The inhibition rate was 71.51% when XTBJ-1 was inoculated 1 day after YZ-1-GFP1 microcapsules were inoculated. The inhibition rate of YZ-1-GFP1 cake was 59.03% ( Fig.16 ).

[0124] 2.8 Potted plant protection effect

[0125] In order to determine the control effect of YZ-1 microcapsules on pepper white rot, pepper seedlings were divided into five treatments: empty microcapsules, YZ-1-GFP1 microcapsules, YZ-1-GFP1, blank control and positive control. Empty microcapsules (3 g), YZ-1-GFP1 microcapsules (3 g), YZ-1-GFP1 spore solution (10 mL), clean water (10 mL) and 0.4% thiothiocarb (10 mL) were inoculated in peppers at the four-leaf and one-heart stage. All microcapsules were buried in the roots of pepper plants through soil, and all liquids were used for root irrigation. Each treatment was repeated 12 times. After 7 days, the treatment was repeated. After 14 days, four fresh XTBJ-1 mycelium blocks (d=8 mm) were inoculated at the base of the plant stem and covered with moist soil, and cultured at 28°C to keep the moisture. After 5 days, the disease index was calculated, and the fresh weight, dry weight and other indicators were measured.

[0126] The control effect of YZ-1-GFP1 spore liquid was 42.19%. In contrast, the relative control efficiency of YZ-1-GFP1 microcapsules reached 74.13%. Pepper plants treated with microcapsules showed better growth conditions ( Fig.17 ).

[0127] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Trichoderma harzianum (Trichoderma afroharzianum )YZ-1, characterized in that The deposit number of the African harzianum Trichoderma YZ-1 is CCTCC NO: M 20241084.

2. An African Trichoderma harzianum YZ-1 microcapsule, characterized in that: It comprises the African harzianum Trichoderma YZ-1 described in claim 1.

3. The method for preparing the African Trichoderma harzianum YZ-1 microcapsules according to claim 2, characterized in that: The following steps are involved: Take 1-4 g of montmorillonite and dissolve it in 100-500 mL of deionized water, add 6-9 g of sodium alginate, and then add glycerol and mix thoroughly to obtain a montmorillonite-sodium alginate solution; Take the spore liquid of Trichoderma harzianum YZ-1, centrifuge to obtain spores, add the montmorillonite-sodium alginate solution to suspend the spores, and obtain a spore concentration of 1×10 7 ~1×10 9 spores / mL of montmorillonite-alginate spore suspension; The montmorillonite-sodium alginate spore suspension was squeezed into a 2% CaC12 solution and washed with sterile water to obtain Trichoderma harzianum YZ-1 microcapsules.

4. The preparation method according to claim 3, characterized in that: The following steps are involved: Take 2.5 g of montmorillonite and dissolve it in 300 mL of deionized water, add 7.5 g of sodium alginate, and then add glycerol and mix thoroughly to obtain a montmorillonite-sodium alginate solution; Take the spore liquid of Trichoderma harzianum YZ-1, centrifuge to obtain spores, add the montmorillonite-sodium alginate solution to suspend the spores, and obtain a spore concentration of 1×10 8 spores / mL of montmorillonite-alginate spore suspension; The montmorillonite-sodium alginate spore suspension was squeezed into a 2% CaC12 solution and washed with sterile water to obtain Trichoderma harzianum YZ-1 microcapsules.

5. A microbial agent, characterized in that: Contains the African harzianum Trichoderma YZ-1 described in claim 1.

6. The microbial agent according to claim 5, characterized in that The live bacteria concentration of the microbial agent is 1×10 8 Spores / mL.

7. Use of the African harzianum YZ-1 according to claim 1, the African harzianum YZ-1 microcapsule according to claim 2, or the microbial agent according to any one of claims 5 to 6 in inhibiting the growth of pathogenic bacteria, characterized in that: The pathogens include pepper white rot fungus, pepper gray mold fungus, pepper blight fungus, tobacco target spot fungus, pepper damping-off fungus, pepper anthracnose fungus.

8. Use of the African Trichoderma harzianum YZ-1 according to claim 1, the African Trichoderma harzianum YZ-1 microcapsule according to claim 2, or the microbial agent according to any one of claims 5 to 6 in preventing and controlling plant diseases, characterized in that: The plant disease is pepper white rot caused by Sclerotium rolfsii.

9. A method for preventing and treating pepper white rot caused by Sclerotium rolfsii, characterized in that: The following steps are involved: Treating pepper plants with the African harzianum YZ-1 or its fermentation liquid or bacterial suspension described in claim 1 or the African harzianum YZ-1 microcapsules described in claim 2; The live bacteria concentration in the fermentation liquid or bacterial suspension is 1×10 8 Spores / mL.

10. The method according to claim 9, characterized in that The treatment is: the concentration is 1×10 8 The pepper plants are irrigated with the fermentation liquid or bacterial suspension of Trichoderma harzianum YZ-1 with spores / mL or the Trichoderma harzianum YZ-1 microcapsules described in claim 2.

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