A weakly toxic strain BLH4 of Sclerotium uniformis and its application

Abstract

The present invention discloses a weak strain BLH4 of Sclerotium uniformis and its application, and belongs to the technical field of plant disease biocontrol bacteria. The deposit number of the weak strain BLH4 is CCTCC M 20241385. The present invention independently separates and screens a weak strain BLH4 of pepper white rot pathogen. According to indoor bioassay, defense enzyme assay, RT-qPCR and other technical means, it is found that the weak strain BLH4 has good disease resistance, stimulates plant immunity-related enzyme activity, and increases the relative expression of defense-related genes. In addition, it has been verified that the strain has an inhibitory effect on a variety of plant pathogens and can be used as a vaccine strain for disease prevention and control.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease biocontrol bacteria, in particular to a weak-virulence strain BLH4 of Sclerotium truncatum and application thereof. Background Art

[0002] White rot of pepper is a common soil-borne disease caused by Sclerotium rolfsii. It occurs in various parts of China and is more serious in warm and humid areas, especially in southern regions such as Guizhou, Fujian, and Hunan. In recent years, the occurrence of white rot of pepper in open field and greenhouse planting areas in Hunan Province has been more serious. The loss of seriously diseased plots infected by white rot of pepper can reach more than 80%, and even cause crop failure. The symptoms of white rot of pepper are rot of the epidermis at the base of the stem, and then the growth of white silky hyphae. After about a week, the plant wilts, the leaves wilt, dry up, and fall off, and the whole plant gradually dies; in the late stage of the disease, many brown or light brown cabbage seed-like small sclerotia are produced on the mycelium of the diseased part. The roots and fruits in contact with the ground can also be infected, and the surface of the infected fruits has white silky hyphae. The sclerotia of white rot have no dormancy period and are highly adaptable to extreme environments. They are extremely difficult to control in agriculture and currently rely mainly on chemical agents.

[0003] At present, there are relatively few biocontrol agents for preventing and controlling pepper white rot, and there is no report on the use of attenuated strains to prevent and control pepper white rot. Therefore, there is an urgent need to screen an attenuated strain with a high biocontrol effect on pepper white rot. Summary of the invention

[0004] The purpose of the present invention is to provide a weak toxic strain BLH4 of Sclerotium uniformis and its application to solve the problems existing in the above-mentioned prior art. The weak toxic strain BLH4 provided by the present invention is environmentally friendly, isolated from Macleaya cordata, and naturally exists in the soil. It has no pathogenicity to pepper, is relatively safe to humans, animals, crops and insects, and is harmless to the environment. It has been verified that the strain has an inhibitory effect on a variety of plant pathogens and can be used as a vaccine strain for disease prevention and control.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a Sclerotium rolfsii attenuated strain BLH4, and the deposit number of the attenuated strain BLH4 is CCTCC M 20241385.

[0007] The present invention also provides the use of the attenuated strain BLH4 and / or its metabolites in inhibiting the growth of plant pathogens, wherein the plant pathogens include pepper Sclerotium rolfsii, pepper anthracnose, tobacco target spot pathogen and pepper gray mold pathogen.

[0008] The present invention also provides the use of the attenuated strain BLH4 and / or its metabolites in the preparation of biocontrol agents.

[0009] The present invention also provides a biocontrol agent, the active ingredients of which include the attenuated strain BLH4 and / or its metabolites.

[0010] The present invention also provides the use of the attenuated strain BLH4 or the biocontrol agent in preventing and controlling plant diseases, wherein the plant diseases include pepper white rot, pepper anthracnose, tobacco target spot and pepper gray mold.

[0011] The present invention also provides a method for preventing and controlling plant diseases, comprising the step of applying a bacterial solution containing the attenuated strain BLH4 to plants; the plant diseases include pepper white rot, pepper anthracnose, tobacco target spot and pepper gray mold.

[0012] Furthermore, the application method includes root irrigation and foliar spraying.

[0013] Furthermore, the plant diseases include pepper white rot and pepper gray mold.

[0014] The present invention discloses the following technical effects:

[0015] The present invention independently separated and screened out a weak strain BLH4 of pepper white rot pathogen. According to indoor bioassay, defense enzyme assay, RT-qPCR and other technical means, it was found that the weak strain BLH4 had good disease resistance, stimulated plant immunity-related enzyme activities, and increased the relative expression of defense-related genes.

[0016] The attenuated strain BLH4 provided by the present invention is environmentally friendly, isolated from Macleaya cordata, and naturally exists in the soil. It has no pathogenicity to peppers, is relatively safe to humans, livestock, crops and insects, and is harmless to the environment. It has been verified that the strain has an inhibitory effect on a variety of plant pathogens and can be used as a vaccine strain for disease prevention and control. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] Figure 1 The colony images of BLH4 and XTBJ-1, where A is BLH4; B is XTBJ-1;

[0019] Figure 2The inoculation diagram of BLH4 and XTBJ-1 on pepper, where the left side is BLH4 inoculation and the right side is XTBJ-1 inoculation;

[0020] Figure 3 is the phylogenetic tree of BLH4;

[0021] Figure 4 The figure is a graph showing the inhibitory effect of the volatile substances of the attenuated strain BLH4 on XTBJ-1, wherein the left side is the control; the right side is the cross-culture of BLH4 and Sclerotium rolfsii of pepper;

[0022] Figure 5 The antagonistic activity of the volatile substances of the attenuated strain BLH4 against different pathogens; the first row from left to right are tobacco target spot pathogen (Rhizoctonia solani), pepper anthracnose pathogen (Colletotrichum scovillei), tobacco root rot pathogen (Fusarium commune), pepper gray mold pathogen (Botrytis cinerea), pepper blight pathogen (Phytophthora capsica); the second row from left to right are tobacco target spot pathogen (Rhizoctonia solani), pepper anthracnose pathogen (Colletotrichum scovillei), tobacco root rot pathogen (Fusarium commune), pepper gray mold pathogen (Botrytis cinerea), pepper blight pathogen (Phytophthora capsica) cultured against BLH4;

[0023] Figure 6 The control effects of different treatment groups on pepper white rot; from left to right are pepper plants without inoculation, inoculated with BLH4 only, inoculated with BLH4+XTBJ-1, and inoculated with XTBJ-1 only;

[0024] Figure 7 The effects of BLH4 on the activities of defense-related enzymes in pepper; A is PAL; B is PPO; C is POD; D is CAT;

[0025] Figure 8 The effect of BLH4 on pepper defense-related gene PR1;

[0026] Fig. 9 The effect of BLH4 on pepper defense-related gene ERF;

[0027] Fig.10 The effect of BLH4 on pepper defense-related gene NPR1;

[0028] Fig.11 The effect of BLH4 on pepper defense-related gene Peroxidase;

[0029] Fig.12 This is the colonization of BLH4 at the base of pepper stem;

[0030] Fig.13 The disease resistance of pepper to Botrytis cinerea induced by BLH4 treatment; A is the overall inoculation diagram, divided into three columns, from left to right: control, foliar spraying, and root irrigation; B, C, and D are close-ups of leaves, respectively control, foliar spraying, and root irrigation. DETAILED DESCRIPTION

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] In the embodiments of the present invention, Sclerotium rolfsii, Botrytis cinerea, Phytophthora capsica, Fusarium commune, Rhizoctonia solani and Colletotrichum scovillei are all from the Hunan Key Laboratory of Plant Pest Biology and Control, Hunan Agricultural University.

[0037] The culture medium used in the embodiment of the present invention is:

[0038] Potato dextrose medium (PDA): 200 g potato, 20 g agar, 20 g glucose and 1 L distilled water;

[0039] In the embodiment of the present invention, SPSS23.0 was used to perform one-way ANOVA analysis on all experimental data, and Tukey's test was used for multiple comparisons and significance analysis.

[0040] Example 1 Isolation, screening and identification of attenuated strain BLH4

[0041] 1. Isolation and screening of attenuated strain BLH4

[0042] A large area of ​​Bo Le Hui at the traditional Chinese medicine base in Yong'an Town, Liuyang City, Hunan Province suffered from rhizome rot, wilting and yellowing of stems and leaves. Severely infected plants died entirely. The diseased plants were sampled and separated.

[0043] The pathogen was isolated by conventional tissue separation method. Conventional tissue separation method: First, rinse the diseased part of the diseased plant with sterile water, then wipe the surface of the diseased tissue with cotton wool dipped in 75% alcohol, and then cut the plant tissue at the junction of the diseased and healthy plants with scissors and blades, cut into 5cm×5cm tissue blocks, put them in 75% alcohol for 30s, and then put them in 0.1% mercuric chloride for 1min for disinfection, and then rinse them with sterile water for 3 times, and then use sterilized filter paper to absorb excess water, and then place the small piece of plant tissue in the poured PDA plate (the concentration of streptomycin sulfate in the PDA culture medium is 0.05mg / mL) and seal it and place it in a constant temperature incubator at 28℃ for culture. After 2 days, transfer it to a new DDA plate from the edge of the grown colony to obtain a pure culture strain.

[0044] A strain with a different morphology from the normal pepper white fungus was isolated and named BLH4. The normal pepper white fungus (abbreviated as XTBJ-1, the same below) has dispersed hyphae in the form of silk threads and grows faster; while the hyphae of the strain BLH4 are dense, whitish and grow slowly ( Figure 1BLH4 inoculated peppers did not cause disease ( Figure 2 ), so the strain BLH4 was determined to be a weakly toxic strain.

[0045] 2. Identification of the attenuated strain BLH4

[0046] The genomic DNA of BLH4 was extracted by the CTAB method. The ITS sequence of the BLH4 strain was amplified using the primers in Table 1. Each 50 μL PCR reaction system contained 25 μL of FairTaq PCR Super, 2 μL of forward primer (10 μM), 2 μL of reverse primer (10 μM), 4 μL of DNA template and 17 μL of H2O. The PCR reaction program was: 95°C pre-denaturation for 3 min; 35 amplification cycles (95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension for 1 min); 72°C extension for 5 min.

[0047] 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 MEGA7.0 software was used to construct a multi-gene phylogenetic tree ( Figure 3 ).

[0048] Table 1 Primer sequences used

[0049]

[0050] Molecular biological identification showed that the attenuated strain BLH4 was a uniform sclerotium, classified and named Sclerotiumrolfsii. The strain was deposited in the China Center for Type Culture Collection on June 26, 2024, with the collection address at Wuhan University, Wuhan, China, and the collection number is CCTCC M 20241385.

[0051] Example 2 Antibacterial effect of volatile substances from attenuated strain BLH4

[0052] 1. Antibacterial effect on pepper white rot fungus

[0053] The attenuated strain BLH4 was cultured on PDA plates for 5 days, and then cultured with pepper white rot fungus (XTBJ-1). The XTBJ-1 on the PDA plate alone was used as the control. Each treatment was repeated 3 times and the plates were cultured in a constant temperature incubator at 28°C. When the colonies of the control group were about to fill the plate, the colony diameters of the treatment and control groups were recorded, and the inhibition rate of BLH4 against XTBJ-1 was determined.

[0054] By recording the colony diameters of the treatment group and the control group, it was found that the colony diameter of XTBJ-1 in the treatment group was much smaller than that in the control group ( Figure 4), was only 1.78±0.12cm, indicating that BLH4 could inhibit the growth of XTBJ-1 by producing volatile substances, with an inhibition rate of 82.68±0.01%.

[0055] 2. Antibacterial spectrum

[0056] The antibacterial spectrum of BLH4 was determined by the plate-to-plate culture method, and the method was the same as above. The target bacteria tested were Botrytis cinerea, Phytophthora capsica, Fusarium commune, Rhizoctonia solani and Colletotrichum scovillei.

[0057] The antibacterial activity of BLH4 against the five plant pathogenic fungi was tested by the plate matching method. Figure 5 ), and found that the inhibition rate of BLH4 on these strains ranged from 8.03±0.04% to 60.90±0.04% (Table 1). Among them, the growth of tobacco target spot pathogen and pepper anthracnose pathogen was significantly inhibited.

[0058] Table 2 Determination of the antibacterial spectrum of BLH4 against 5 pathogens

[0059]

[0060] 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.

[0061] Example 3 Control effect of attenuated strain BLH4 on pepper white rot

[0062] The BLH4 cake was placed in PDB and cultured for 5 days to obtain mycelial tissue. The pepper plants were irrigated with 10 mL of water per plant 21 days after transplantation. The root irrigation was carried out with clean water as the control. Three days later, three fresh XTBJ-1 mycelial blocks (d = 8 mm) were inoculated at the base of the plant stem and covered with moist soil. The mycelial tissue was cultured at 28 ° C. The disease situation of each treatment was observed after 5 days. In order to observe the disease situation more intuitively, the soil at the root of the inoculated pepper was removed, and the excess impurities were washed with clean water, and the pepper was dried naturally. The length of the lesions at the base of the pepper stem and the length of the pepper stem were measured, and the disease index was calculated.

[0063] 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 grading standard is 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 the lesion at the base of the stem) / (stem length of the plant)] × 100.

[0064] The disease index calculation formula is: disease index = [Σ(number of diseased plants at this level × disease level) / (total number of plants surveyed × highest disease level)] × 100.

[0065] The relative protective effect calculation formula is: Relative protective effect (%) = [(disease index of the control group - disease index of the prevention and treatment group) / disease index of the prevention and treatment group] × 100.

[0066] 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 6 ). In contrast, plants treated with BLH4 fermentation liquid did not show these symptoms. The disease index of plants treated with BLH4 fermentation liquid was 14.29, while that of control plants was 88.57, with a relative control effect of 83.87%.

[0067] Example 4 Effect of the attenuated strain BLH4 on pepper plants

[0068] 1. Effects on pepper defense-related enzyme activities

[0069] The treatment method in Example 3 was adopted and divided into 4 treatments: inoculation with XTBJ-1 only, inoculation with BLH4 only, inoculation with BLH4+XTBJ-1, and no inoculation. The leaves of the pepper plants were randomly sampled on 0d, 1d, 3d, and 5d after inoculation with XTBJ-1, and then the activities of phenylalanine ammonia lyase (PAL), catalase (CAT), polyphenol oxidase (PPO), and peroxidase (POD) were detected on the pepper leaves.

[0070] The results showed that the attenuated strain BLH4 could enhance the activity of these disease resistance-related enzymes ( Figure 7 ).

[0071] 2. Effects on pepper defense-related genes

[0072] The treatment method in Example 3 was adopted and divided into 4 treatments: inoculation with XTBJ-1 only, inoculation with BLH4 only, inoculation with BLH4+XTBJ-1, and no inoculation. The leaves of the pepper plants were randomly sampled at 0d, 1d, 3d, and 5d after inoculation with XTBJ-1, and then the pepper leaves were quantitatively detected for salicylic acid response factor (PR1), ethylene response factor (ERF), salicylic acid response factor (NPR1), and peroxidase gene (Peroxidase).

[0073] The results showed that the attenuated strain BLH4 could stimulate the immune pathway faster and stronger after the plants were inoculated with XTBJ-1, thereby inducing plant resistance ( Figure 8-11 ).

[0074] 3. Colonization of pepper

[0075] The BLH4 liquid cultured for 5 days was broken up and used to irrigate the pepper roots, with 10 mL per plant. Samples were taken from the base of the pepper stems at 3, 6, 9, and 12 days after root irrigation, and the samples were decolorized in 95% alcohol boiling water for 15 minutes, then washed in 50% alcohol for 15 minutes, and then transparentized in 50mmol / L NaOH solution twice, each time for 15 minutes, and finally stained in direct yellow dye (final concentration of 0.002%, W / V) dissolved in 0.1mol / LTris-HCl buffer (pH8.5) for 5 minutes, and then directly prepared and observed.

[0076] The results showed that a large number of hyphae were found at the base of the stems of the treatment group, but not in the control group. Moreover, the number of hyphae increased gradually with the increase of time, indicating that BLH4 can colonize at the base of pepper stems ( Fig.12 ).

[0077] 4. Inducement of resistance to other pathogens

[0078] The fermented liquid of BLH4 cultured for 5 days was sprayed on the leaves and irrigated on the roots of peppers. Each treatment was repeated 9 times, and 10 mL was sprayed or irrigated on each plant. The pepper plants sprayed or irrigated with clean water were used as the control. After 3 days of treatment, pepper gray mold (Botrytis cinerea) was inoculated, the disease of peppers was observed, the number and area of ​​lesions were recorded, and the disease index and relative control effect were calculated.

[0079] The severity of pepper gray mold disease is as follows (in leaves): 0: no lesions; 1: 3 lesions on a single leaf, lesion area <1%; 3: 4-6 lesions on a single leaf, lesion area 1%-10%; 5: 7-10 lesions on a single leaf, lesion area 10%-25%; 7: 11-20 lesions on a single leaf, some densely packed into pieces, lesion area 25%-50%; 9: 1 / 4 or more lesions on a single leaf, lesion area >50%. The disease index calculation method and relative control efficiency calculation formula are detailed in Example 3.

[0080] The leaves of the control plants turned yellow, with large areas of lesions, and a large number of leaves fell off. However, only a small number of lesions appeared on the leaves of the plants treated with BLH4 fermentation liquid spraying and root irrigation, and a few leaves turned yellow, but no leaves fell off ( Fig.13 ). In the foliar spraying treatment group, the disease index of the plants treated with BLH4 fermentation liquid was 25.93, and that of the control plants was 85.19, with a relative control effect of 69.56%. In the root irrigation treatment group, the disease index of the plants treated with BLH4 fermentation liquid was 40.74, and that of the control plants was 87.63, with a relative control effect of 52.17%. This indicates that the attenuated strain BLH4 also has a certain control effect on other pathogens, such as pepper gray mold (Botrytis cinerea).

[0081] 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 Sclerotium uniformis ( Sclerotium rolfsii ) is a attenuated strain BLH4, characterized in that The deposit number of the attenuated strain BLH4 is CCTCC M 20241385.

2. The use of the attenuated strain BLH4 according to claim 1 in inhibiting the growth of plant pathogens, characterized in that: The plant pathogens are pepper white rot fungus, pepper anthracnose fungus, tobacco target spot fungus and pepper gray mold fungus.

3. The use of the attenuated strain BLH4 according to claim 1 in the preparation of a biocontrol agent, characterized in that: The biocontrol agent is used for inhibiting the growth of pepper white rot fungus, pepper anthracnose fungus, tobacco target spot fungus and pepper gray mold fungus.

4. A biocontrol agent, characterized in that: The active ingredient comprises the attenuated strain BLH4 described in claim 1.

5. Use of the attenuated strain BLH4 according to claim 1 or the biocontrol agent according to claim 4 in preventing and controlling plant diseases, characterized in that: The plant diseases are pepper white rot, pepper anthracnose, tobacco target spot and pepper gray mold.

6. A method for preventing and controlling plant diseases, characterized in that: The method comprises the step of applying a bacterial solution containing the attenuated strain BLH4 according to claim 1 to plants; the plant diseases are pepper white rot, pepper anthracnose, tobacco target spot and pepper gray mold.

7. The method according to claim 6, characterized in that The application methods include root irrigation and foliar spraying.

8. The method according to claim 6, characterized in that The plant diseases are pepper white rot and pepper gray mold.

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