CN-121991973-A - DsRNA of targeted BdSpt gene, biocontrol agent and application of biocontrol agent

CN121991973ACN 121991973 ACN121991973 ACN 121991973ACN-121991973-A

Abstract

The invention discloses a dsRNA targeting BdSpt gene, a biocontrol agent and application thereof, wherein the sequence of the dsRNA targeting BdSpt gene is shown as SEQ ID No. 2. The active ingredient of the biocontrol agent is the dsRNA targeting BdSpt gene. The invention also provides application of the ring spot germ BdSpt gene in preventing and treating apple ring spot, and the nucleotide sequence of the ring spot germ BdSpt gene is shown as SEQ ID No. 1. The invention also provides an African trichoderma harzianum engineering strain for expressing dsRNA, which is the African trichoderma harzianum integrated with a dsRNA expression cassette for interfering the expression of a target gene BdSpt gene, wherein the dsRNA expression cassette is shown as SEQ ID No.1, and the African trichoderma harzianum adopts the African trichoderma harzianum with the preservation number of CGMCC No. 41709. The dsRNA of the targeted BdSpt gene can obviously reduce pathogenicity of the ring spot bacteria through interference, the trichoderma harzianum engineering strain can colonize on the surface of plants and continuously express the dsRNA, thereby exerting disease control effect of the ring spot, and the method is environment-friendly.

Inventors

WANG CAIXIA
CUI XINYUE
YUE YANWEN
ZHANG SHAN
Chi Guibing
REN WEICHAO
LIAN SEN
LI BAOHUA

Assignees

青岛农业大学

Dates

Publication Date: 20260508
Application Date: 20260129

Claims (10)

1. The application of the ring spot germ BdSpt gene in preventing and treating apple ring spot is characterized in that the nucleotide sequence of the ring spot germ BdSpt gene is shown as SEQ ID No. 5.
2. The application of the ring spot germ BdSpt gene in preventing and treating apple ring spot according to claim 1, wherein the application method is that BdSpt gene in the apple ring spot germ is knocked out or BdSpt gene in the apple ring spot germ is subjected to RNA interference.
3. A dsRNA targeting BdSpt genes, characterized in that the sequence of the dsRNA is shown in SEQ ID No. 2.
4. A biocontrol formulation comprising the dsRNA targeting BdSpt gene of claim 3 as an active ingredient.
5. Use of the dsRNA targeting BdSpt gene according to claim 3 or the biocontrol formulation according to claim 4 for the preparation of a biologic for controlling apple ring rot.
6. The use according to claim 5, wherein the dsRNA is used at a concentration of 80-400 ng/μl by spraying the leaves of the plant.
7. The trichoderma harzianum engineering strain for expressing dsRNA is characterized by being trichoderma harzianum (Trichoderma afroharzianum) integrated with a dsRNA expression cassette interfering with target gene BdSpt gene expression, wherein the dsRNA expression cassette is shown as SEQ ID No.1, and trichoderma harzianum (Trichoderma afroharzianum) T10 with the preservation number of CGMCC No.41709 is adopted as the trichoderma harzianum.
8. A method of preparing an engineered strain of trichoderma harzianum as in claim 7, comprising the steps of: (1) Constructing a dsRNA expression cassette, namely constructing the dsRNA expression cassette containing a target gene BdSpt by using arm sequences with the same two end sequences and opposite directions and a central sequence with a connecting function in the middle, wherein the nucleotide sequence of the dsRNA expression cassette is shown as SEQ ID No. 1; (2) Constructing a recombinant expression vector, namely connecting a dsRNA expression cassette into a trichoderma expression vector, and constructing a recombinant expression vector containing the dsRNA expression cassette; (3) And (3) converting protoplasts, namely introducing a recombinant expression vector which is successfully constructed and contains the dsRNA expression cassette into Trichoderma African trichoderma harzianum T10 by a PEG-mediated protoplast conversion method, and screening positive transformants to obtain the Trichoderma African trichoderma harzianum engineering strain.
9. The use of the trichoderma harzianum engineering strain as claimed in claim 7 for controlling ring spot.
10. A method of controlling apple ring rot, wherein one or both of the dsRNA of claim 3 or the trichoderma harzianum engineering strain of claim 7 is applied to fruit trees.

Description

DsRNA of targeted BdSpt gene, biocontrol agent and application of biocontrol agent Technical Field The invention relates to the technical field of microorganisms, in particular to dsRNA (double-stranded ribonucleic acid) targeting BdSpt gene, a biocontrol agent and application thereof. Background Fruit tree diseases caused by pathogenic fungi (Botryosphaeria dothidea) of apple ring rot are taken as one of important diseases in the existing fruit tree field, and the yield and quality of fruits are seriously affected. Apple ring rot is widely generated in main apple producing areas in China, and can not only infect fruit tree trunks and fruits to cause branch ulcers and fruit rot to seriously obstruct the normal growth of fruit trees, but also greatly reduce the quality of the fruits to cause obvious economic loss. Therefore, the prevention and treatment of apple ring rot is an important problem facing the fruit tree planting industry. At present, the prevention and treatment of apple ring spot still takes chemical bactericides as the main material, but the traditional prevention and treatment method is easy to cause two key problems that pathogenic bacteria generate drug resistance, the prevention and control effect is gradually reduced, and the widely used chemical pesticides pollute the environment. Therefore, with the promotion of green agriculture, a more environment-friendly apple ring rot control method needs to be sought. In recent years, RNA interference (RNAi) technology has shown great application potential in the field of plant disease prevention and control by virtue of the characteristics of high specificity and environmental friendliness. The technology designs double-stranded RNA (dsRNA) by targeting key pathogenic genes of pathogenic bacteria, and can effectively inhibit the growth and pathogenicity of pathogenic bacteria. However, the research on key pathogenic genes of the ring spot bacteria is less at present, and dsRNA products which are effectively applied to preventing and treating apple ring spot based on RNA interference technology are not available. Accordingly, there is a need for further improvements in the art. Disclosure of Invention Aiming at the problems, the invention provides dsRNA targeting BdSpt gene, a biocontrol agent containing the dsRNA and application thereof in apple ring rot control. In order to solve the problems, the application provides the following technical scheme: In a first aspect, the application provides application of a ring spot germ BdSpt gene in preventing and treating apple ring spot, and the nucleotide sequence of the ring spot germ BdSpt gene is shown as SEQ ID No. 5. The application constructs BdSpt gene knockout strain of the colletotrichum gloeosporioides by means of molecular biology, researches the functional shape of the knockout strain, and discovers that BdSpt gene plays a key regulation and control role in the nutritional metabolism, host adaptability and pathogenic process of colletotrichum gloeosporioides. Compared with the wild type, the colony expansion diameter is only 30% -50% of that of the wild type, the growth capacity of the knocked-out bacteria in different culture mediums (PDA, MM) and host simulation environments (fruit homogenate, bark extract) is obviously reduced, and secondly, the pathogenicity of the knocked-out bacteria on apple fruits and branches is reduced by 70% -80%, the disease spot diameter is reduced from 2.7-3.7 cm of the wild type to 0.6-0.7 cm, and the gene is proved to be an important gene necessary for the disease causing of the ring spot. Based on the function of BdSpt gene, the ring spot germ BdSpt gene is applied to preventing and treating ring spot of apples, and the application method is that BdSpt gene in ring spot pathogenic bacteria of apples is knocked out or BdSpt gene in ring spot pathogenic bacteria of apples is subjected to RNA interference, so that BdSpt expression is inhibited. In a second aspect, the application provides a dsRNA targeting BdSpt genes, the sequence of which is shown in SEQ ID No. 2. The dsRNA is designed aiming at BdSpt genes, the pathogenicity of the ring spot bacteria is obviously inhibited in vitro, after the dsRNA with the concentration of 80-120 ng/mu L is treated for 5 days, the ring spot is reduced by 0.5-1.0 cm, and the inhibiting effect is more obvious when the dosage effect is 400 ng/mu L. In a third aspect, the application also provides a biocontrol agent, the active ingredient of which is the dsRNA targeting BdSpt genes. In a fourth aspect, the application also provides an application of the dsRNA of the targeted BdSpt gene or the biocontrol agent in preparing a biological agent for preventing and treating apple ring rot. Optionally, the dsRNA is used at a concentration of 80-400 ng/μl in the application by spraying the leaves of the plant. In a fifth aspect, the application also provides a trichoderma harzianum engineering strain for expressing dsRNA, wherein the tr