CN115725448B - Non-toxic acidophilic bacteria capable of inducing crop to resist bacterial diseases and application thereof - Google Patents

Abstract

The invention discloses a non-toxic acidophilic bacteria capable of inducing crop bacterial disease resistance and application thereof, and relates to the technical field of biological control of plant diseases. The non-toxic acidophilic bacteria M292 is obtained by screening from a watermelon acidophilic bacteria Tn5 mutant library constructed in the laboratory. The method of spray inoculation can induce the generation of watermelon bacterial fruit blotch resistance. The nontoxic acidophilic bacteria related by the invention can be used for green biological control of watermelon bacterial fruit blotch.

Description

Non-toxic acidophilic bacteria capable of inducing crop to resist bacterial diseases and application thereof

Technical Field

The invention relates to the technical field of biological control of plant diseases, in particular to a non-toxic acidophilic bacteria capable of inducing crop bacterial disease resistance and application thereof.

Background

The bacterial fruit spot disease of the watermelon is prepared from acidophilic bacteria of the watermelonAcidovorax citrulli) The damage range is wide, and the destructive quarantine disease is strong. The hosts of the disease hazard are expanded from watermelons to various cucurbit crops such as melons, cantaloupes, pumpkins, cucumbers and the like. The leaves and fruits of the plants can be infected by the bacteria, and the leaves are infected and usually have water stain-like lesions which are dark brown, yellow halos are arranged at the edges and are expanded along veins; after the fruits are infected, the surfaces of the fruits are firstly provided with small water-stain-shaped spots, then the color of the disease spots is gradually deepened, the disease spots are enlarged into irregular olive water-shaped spots, the whole surfaces of the fruits which are in contact with the ground are covered for 7-10 days, the surfaces of the fruits are cracked in the later stage of disease, and bacterial pus overflows, so that pulp is rotten, the eating value is lost, and huge losses are caused for melon farmers. The disease is listed in the national agricultural plant quarantine pest list in 11 months 2020. At present, the disease is mainlyThe control method includes planting disease-resistant variety, chemical control, agricultural control, biological control and the like. Although chemical control has a certain effect, due to the occurrence of multi-drug resistant bacteria and the problems of ecological safety, pesticide residue and the like caused by chemical agents, an effective alternative method for chemical control is urgently needed to be searched, and biological control becomes a research hot spot in recent years.

Studies have shown that non-pathogenic strains can control plant diseases through antagonism, competition and redirection, growth promotion, induction of plant to obtain systemic resistance, enhancement of root system and plant development, etc. The non-pathogenic strain has no toxic effect on plants and no environmental pollution, and becomes a new technology and a new way for biological control of plant diseases. However, in the prior art, these methods have limited control effects (mainly for seed treatment) and have not been concerned with inducing plant resistance.

Disclosure of Invention

The invention aims to provide a non-toxic acidophilic bacteria capable of inducing crop bacterial diseases and application thereof.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the nontoxic acidophilic bacteria is watermelon acidophilic bacteriaAcidovorax citrulli) M292, which was preserved in China Center for Type Culture Collection (CCTCC) No. M20221188 at 7.27 of 2022. The preservation address is university of martial arts.

The application of the nontoxic acidophilic bacteria in preventing and controlling watermelon bacterial fruit blotch is provided.

The invention has the advantages that:

(1) the nontoxic acidophilic bacteria M292 has no inhibition effect on the watermelon fruit blotch on a flat plate, but has a certain control effect in pot control experiments, has no pollution, no residue and no toxicity compared with chemical pesticides, has higher safety on the environment, is a green control measure, and provides a new strategy for controlling the watermelon bacterial fruit blotch.

(2) The nontoxic acidophilic bacteria M292 has a resistance induction effect, and when the induction interval is 2d, after the pathogenic fruit spot bacteria 9 d are inoculated, the resistance induction effect of the strain M292 on the watermelon fruit blotch is 66.67%; after inoculation with pathogenic fruit spot bacteria 11 d, the induced resistance effect of strain M292 on watermelon seedling fruit spot is 56.94%.

Drawings

FIG. 1 fruit pathogenicity assay (post inoculation 7 d).

FIG. 2 specific primer amplification of Uighur bacteria, M: DL2000 marker,1: FJAc01 (ref: yang Bing, hu Fangping, cai Xueqing. Functional analysis of watermelon acid phage flagella gene flgM. Proc. Agrobiotechnical journal, 2019); 2: M292.

FIG. 3 panel antagonism of avirulent mutant strains.

FIG. 4 is a graph showing the effect of strain M292 on the induction resistance of watermelon fruit blotch at different induction intervals.

FIG. 5 shows the effect of strain M292 on the induction resistance of watermelon fruit blotches at different induction intervals (inoculation treatment 7D), A: FJAc01, B: 5d+FJAc 01, C: 4d+FJAc 01, D: 3d+FJAc 01, E: 2d+FJAc 01, F: 1d+FJAc 01, G: 0d+FJAc 01, H: CK.

FIG. 6 is a graph showing the effect of strain M292 on the induction resistance of watermelon seedling fruit blotch at different induction intervals.

Detailed Description

Example 1

The nontoxic acidophilic bacteriaAcidovorax citrulli) A method of screening for M292 comprising the steps of:

by the method of three-parent hybrid transformation (ref: kessler B, lorenzo L, timmisk N.1992. A general system to integrate lacZ fusions into the chromosomes of Gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy [ J)]Molecular and General Genetics, 233:293-301.) Tn5 transposable vector pUT gfp/luxAB (ref: the bacterial wilt biological characteristics of the green fluorescent protein gene transferred by cheilose and the like, and Chinese agricultural science) is introduced into a wild watermelon acidophilic bacterial strain FJAc01 (the bacterial strain is separated from diseased watermelon leaves) to obtain a mutant bacterial strain M292. Specific use of acidophilic bacteria of watermelonSex primer SEQ ID5 CCTCCACCAACCAATACGCT/SEQ ID4 ^m GTCATTACTGAATTTCAACA, PCR amplification is carried out by taking the total DNA of M292 strain as a template, and agarose gel electrophoresis detection is carried out on the amplified product. The results in FIG. 2 show that mutant strain M292 resulted in a single target band consistent with the wild type acidophilus watermelon FJAc01 strain. The mutant strain M292 was described as being watermelon acidophilus.

EXAMPLE 2 determination of pathogenicity of mutant Strain M292

Needling inoculation: the concentration of the bacterial suspension of the mutant strain was adjusted to od600=1.0. The method comprises the steps of firstly wiping the surfaces of watermelon fruits with 75% alcohol for sterilization, then manufacturing wounds on the surfaces of the fruits by using a sterile 10 mu l gun head, then dipping the prepared bacterial suspension with sterile cotton, smearing the wound with the sterile cotton, then wetting the new sterile cotton with sterile water drops, applying the sterile cotton on the wounds, bagging for moisturizing, culturing at a constant temperature in a 28 ℃ incubator, and taking wild watermelon acidophilic bacteria FJAc01 bacterial suspension (OD 600 = 0.8) as a positive control and taking the sterile water treatment as a blank control. Each treatment was repeated 3 times, 10 wounds were made each time, the disease condition was observed after 7. 7d, and the strain M292 was nonpathogenic to watermelon fruits, and the results are shown in FIG. 1.

Spray inoculation: the concentration of the bacterial suspension of the mutant strain was adjusted to od600=1.5. Inoculating watermelon seedlings with 2-3 true leaves, uniformly spraying the prepared bacterial suspension on the front and back sides of the leaves, keeping moisture 24 and h on the premise that liquid drops do not drop, and treating the wild watermelon acidophilic bacterial strain FJAc01 bacterial suspension (OD 600 = 0.8) as a positive control and treating the wild watermelon acidophilic bacterial strain FJAc01 bacterial suspension with sterile water as a blank control. Each treatment was repeated 3 times, 6 watermelon seedlings were repeated each time, and the disease was observed after 10. 10 d. Strain M292 was nonpathogenic to watermelon seedlings.

Example 3 results of antagonistic measurement of bacterial strain M292 on West Nile-associated disease

Wild watermelon acidophilus FJAc01 bacterial suspension (OD 600 = 1.0) (the ratio of the wild watermelon acidophil FJAc01 bacterial suspension to the culture medium is 1:100) is added into KB culture medium cooled to 45 ℃, shaking is carried out uniformly, plates are prepared, 2.5 mu L of mutant strain M292 (OD 600 = 1.5) is evenly inoculated in each plate, and the plates are placed in an incubator 48 h at 28 ℃ to observe whether a bacteriostasis zone appears.

The results showed that no zone of inhibition was present on the plates, indicating that the non-pathogenic mutant M292 had no antagonism against wild watermelon acidophilus FJAc01 (FIG. 3).

Example 4 determination of the resistance-inducing effect of Strain M292 on fruit blotches of watermelon fruit at different induction intervals

The surface of watermelon fruits is firstly sterilized by using 75% alcohol, then a wound is manufactured on the surface of the fruits by using a sterile 10 mu l gun head, then sterile cotton is dipped in strain M292 bacterial suspension (the concentration is regulated to be OD 600=1.0) and smeared on the wound, new sterile cotton is wetted by sterile water drops and smeared on the wound, and 0d, 1d, 2d, 3d, 4d and 5d of inoculated wild watermelon fruit blotch bacteria FJAc01 bacterial suspension (OD 600=0.8) are inoculated, bagging and moisturizing are carried out, and the culture box is incubated at constant temperature at 28 ℃ so as to only inoculate the wild watermelon fruit blotch bacteria bacterial suspension as a control. Each treatment was repeated 3 times, 10 wounds were inoculated each time, the onset was observed, and statistics were performed according to the disease grading criteria of table 1.

TABLE 1 grading Standard for bacterial fruit blotch disease of watermelon (fruit)

As a result, when the induction interval was 2d, the control effect of the bacterial suspension of the inoculated strain M292 was 66.67% after inoculation of the pathogenic bacteria 9 d.

EXAMPLE 5 determination of the resistance-inducing effect of Strain M292 on fruit blotch of watermelon seedlings at various induction intervals

The watermelon seeds are planted into a plug after germination accelerating, inoculated when the watermelon grows to 2-3 true leaves, the strain M292 bacterial suspension (OD 600 = 1.5) is uniformly sprayed on the front and back sides of the leaves, and after inoculation, 0d, 1d, 2d, 4d, 6 d, 8 d and 10d are inoculated with the wild watermelon fruit spot bacteria FJAc01 bacterial suspension (OD 600 = 0.8) based on the condition that liquid drops do not drop, and the treatment of only inoculating the wild watermelon fruit spot bacteria bacterial suspension is used as a control, wherein each treatment is repeated 3 times, and 6 watermelon seedlings are repeated each time. The onset was observed and counted according to the criteria for disease classification in table 2.

TABLE 2 disease classification criteria for bacterial fruit blotch of watermelon (seedlings)

When the induction interval period is 2d, after the pathogenic bacteria 11 and d are inoculated, the control effect of the inoculated strain M292 bacterial suspension is 56.94 percent.

The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (2)

1. A non-toxic acidophilic bacteria capable of inducing crop to resist bacterial diseases is characterized by that said bacteria are watermelon acidophilic bacteriaAcidovorax citrulli) M292, which was preserved in China Center for Type Culture Collection (CCTCC) No. M20221188 at 7.27 of 2022.

2. Use of the non-toxic acidophilic bacteria according to claim 1 for controlling bacterial fruit blotch of watermelons.