CN109022311B - Alcaligenes faecalis BC13 and application thereof in prevention and treatment of crop bacterial diseases - Google Patents
Alcaligenes faecalis BC13 and application thereof in prevention and treatment of crop bacterial diseases Download PDFInfo
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Abstract
The invention discloses an alcaligenes faecalis (A)Alcaligenes faecalis) BC13 and application thereof in preventing and controlling crop bacterial diseases. The strain of the alcaligenes faecalis BC13 is preserved in Guangdong province microorganism culture collection center in 2018, 7 months and 10 days, and the preservation number is GDMCC No. 60412. The strain BC13 has good inhibition effect on crop bacterial soft rot, bacterial wilt, rice bacterial leaf blight and Pantoea, can obviously reduce the occurrence of the crop bacterial soft rot, has good effect in pot experiment, and shows that the strain BC13 can be used for developing biological pesticides for the crop bacterial diseases. Meanwhile, the strain has good bacteriostatic action on part of plant pathogenic fungi, such as rhizoctonia solani and peronophythora litchii.
Description
Technical Field
The invention relates to the technical field of biological control, and particularly relates to an alcaligenes faecalis BC13 and application thereof in control of crop bacterial diseases.
Background
Bacterial diseases are plant diseases caused by pathogenic bacterial infection, such as soft rot, bacterial wilt and the like, and can cause serious economic loss.
Such as Dieckea zeae (D.), (Dickeya zeae) Besides monocotyledons, dicotyledons can be infected simultaneously, and the host range of the dicotyledons comprises rice, corn,Sugarcane, banana, brachiaria, iris, blackberry lily, hemerocallis fulva, pennisetum, pineapple, potato, tobacco, Chinese cabbage, carrot, peanut, impatiens balsamina, chrysanthemum and other plants. The reported serious plant diseases caused by the Dieckea zeae comprise rice bacterial basal rot and banana bacterial soft rot, and the diseases caused by the Dieckia zeae have latent infection phenomena, so that a large amount of serious yield loss of crops is caused.
The existing prevention and control methods mainly comprise the following steps: (1) planting disease-resistant varieties: the method is the most economical and effective method for preventing and treating the bacterial soft rot of rice and bananas. Researches show that plantain, Brazil banana and emperor banana have certain resistance to banana bacterial soft rot. However, with the increase of the planting time of the disease-resistant variety, the pathogenic bacteria may generate new physiological races, thereby turning the disease-resistant variety into a disease-susceptible variety. (2) Using chemical agents: at present, the medicines for preventing and treating bacterial diseases including soft rot are few in varieties, and mainly comprise copper preparations, antibiotic bactericides and the like. However, chemical agents have certain toxicity to human and livestock, and can cause problems of drug resistance of germs, water resource pollution and the like.
Therefore, the search for suitable biological control methods is a development direction and an effective way to control bacterial diseases.
Disclosure of Invention
Aiming at the defects of the existing crop bacterial disease control technology, the invention provides an alcaligenes faecalis BC13, which has better inhibition effect on various crop pathogenic bacteria, provides a new way for using microorganisms to replace chemical synthesis bactericides, and can be used as a biological pesticide for development and utilization.
The invention also aims to provide application of the Alcaligenes faecalis BC13 in preventing and treating bacterial diseases and/or fungal diseases of crops.
Another object of the present invention is to provide a biological agent for controlling crop bacterial diseases.
Another object of the present invention is to provide a method for controlling bacterial diseases of crops.
The above object of the present invention is achieved by the following technical solutions:
alcaligenes faecalis (A)Alcaligenes faecalis) BC13, wherein the strain is preserved in Guangdong province microorganism culture collection center in 2018, 7, 10, the strain preservation number is GDMCC No. 60412, and the preservation address is No. 59, 5, of the Michelia Tokyo 100, Guangzhou city.
Strain BC13 is a gram-negative bacterium; BC13 has round colony on LB culture medium, milk white, smooth and regular edge, concave shape, opacity, catalase production, amylase production, gelatin hydrolysis, galactose, mannitol, fructose, trehalose, glucose and sucrose, and can utilize different organic acids and amino acids as carbon sources.
According to researches, the strain BC13 can form an obvious inhibition zone on a flat plate containing bacterial liquids of rice bacterial basic rot EC1, banana bacterial soft rot MS2 and MS3, does not influence the growth of rice and banana seedlings in a greenhouse potting test, and can effectively inhibit the infection of the MS2 on the banana seedlings. Besides, BC13 is found to have good bacteriostatic effect on ralstonia solanacearum, rice bacterial leaf blight and Pantoea, so the BC13 strain can be used for developing biopesticides aiming at crop bacterial diseases. The discovery of the strain is beneficial to reducing the abuse problem of chemical agents, and provides resources for preventing and treating plant bacterial diseases by using a biological control method. Meanwhile, the strain has good bacteriostatic action on part of plant pathogenic fungi, such as rhizoctonia solani and peronophythora litchii.
Therefore, the application of the alcaligenes faecalis BC13 in preventing and treating bacterial diseases and/or fungal diseases of crops is also within the protection scope of the invention.
Preferably, the crop bacterial disease comprisesD.zeaeBacterial diseases caused by ralstonia solanacearum, rice bacterial blight or Pantoea oryzae; the fungal diseases comprise fungal diseases caused by rhizoctonia solani or peronophythora litchi.
More preferably, the bacterial diseases include, but are not limited to, bacterial basal rot of rice, bacterial soft rot of banana, bacterial soft rot of clivia, bacterial wilt of solanaceae crops, bacterial leaf blight of rice, soft rot of rice or peach rot; the fungal diseases include but are not limited to rice sheath blight disease and downy mildew of litchi.
The invention also claims a biological preparation for controlling crop bacterial diseases, which comprises the Alcaligenes faecalis BC 13.
Preferably, the concentration of the Alcaligenes faecalis BC13 is OD600 =1.0~1.5。
More preferably, the concentration of the Alcaligenes faecalis BC13 is OD600 =1.3。
Preferably, the biological agent is prepared from a bacterial liquid and/or a fermentation liquid of alcaligenes faecalis BC 13.
The invention also provides a method for preventing and controlling crop bacterial diseases, which comprises the step of treating soil or plant materials by using the Alcaligenes faecalis BC13 or the biological agent.
Preferably, the treatment is a seeding treatment.
More preferably, the vaccination treatment is an injection vaccination or a puncture vaccination.
The invention has the following beneficial effects:
the alcaligenes faecalis BC13 provided by the invention has a remarkable inhibition effect on various pathogenic bacteria, especially on crop bacterial soft rot, ralstonia solanacearum, rice bacterial leaf blight and Pantoea, can obviously reduce the occurrence of the crop bacterial soft rot, and has a good control effect in a potting test. Moreover, the strain BC13 has no pathogenicity, can be used for developing biological pesticides aiming at crop bacterial diseases, and is used for biologically preventing and treating the crop bacterial diseases. Meanwhile, the strain has good bacteriostatic action on part of plant pathogenic fungi, such as rhizoctonia solani and peronophythora litchii.
Drawings
FIG. 1 is a clustering diagram of strain BC13 based on the 16S rDNA gene sequence.
FIG. 2 shows the determination of the bacteriostasis spectrum of the biocontrol bacterium BC13 on crop pathogenic bacteria.
FIG. 3 shows the determination of the bacteriostasis spectrum of the biocontrol bacterium BC13 on crop pathogenic fungi.
FIG. 4 shows that strain BC13 significantly reduces the occurrence of bacterial soft rot symptoms on Chinese cabbage, potato and carrot.
FIG. 5 shows that the strain BC13 remarkably reduces the occurrence of banana bacterial soft rot disease symptoms.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, but the examples are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Example 1 isolation and screening of Pseudomonas BC13
1. Collecting samples: soil samples with soil surface of 5 cm below are dug in the cabbage rhizosphere soil in village under the great mountains of Committee in the country of Kyoho of Kwangsi Hepu Qu-zhang, and the cabbage rhizosphere soil is bagged and stored and taken back to a laboratory for strain separation.
2. Strain separation: the soil sample is subjected to strain separation by adopting a dilution coating plate method. Specifically, the collected soil sample is fully and uniformly mixed by hand, 4.0 g is weighed, soaked and uniformly shaken in 20 mL sterile water, and then shake-cultured for 20 min at 28 ℃ and 180 rpm for gradient dilution. Taking the dilution factor as 105～109The diluted solution of 100 mu L is coated on an LB flat plate, and is sealed after being dried at room temperature, and is inverted and cultured in an incubator at 28 ℃ for 24 hours. And picking single colonies growing on the LB flat plate, labeling, placing in a 2 mL centrifuge tube containing 500 muL liquid LB, and shaking and culturing on a shaker overnight at 28 ℃ and 180 rpm for later use.
3. Strain screening: activating banana bacterial soft rot pathogen MS2 on an LB plate, picking a single colony in a 50 mL centrifuge tube containing 15 mL LB culture solution, shaking and culturing at 28 ℃ and 180 rpm by shaking overnight for standby. Pouring 15 mL of melted solid LB culture medium in a square culture dish of 10 x 10 cm, drying, pouring 15 mL of agarose (cooling to 50-60 ℃ and adding 150 muL of MS2 bacterial liquid respectively) with the concentration of 1% on the square culture dish paved with LB, and drying. And punching by using a puncher with the diameter of 5 mm, after the culture medium is picked up by the sterilized toothpick, injecting 20 mu L of overnight-cultured bacterial liquid to be detected into the hole, drying and sealing. And (3) placing the plate in an incubator at 28 ℃, culturing for 16-18 h, and observing whether a transparent bacteriostatic zone exists. Thus screening to obtain a biocontrol strain BC 13.
Example 2 identification of biocontrol bacterium BC13
1. Morphological and physiological biochemical identification
Strain BC13 is a gram-negative bacterium; BC13 has round colony on LB culture medium, milk white, smooth and regular edge, concave shape, opacity, catalase production, amylase production, gelatin hydrolysis, and acid production by using galactose, mannitol, fructose, trehalose, glucose and sucrose as carbon sources, and can use different organic acids and amino acids as carbon sources.
2. Molecular identification
To clarify the classification status of the strain BC13 obtained in example 1, the BC13 strain was identified by a Phylogenetic tree analysis (Phylogenetic analysis) using the 16S rDNA sequence in combination with physiological and biochemical assays.
The 16S rDNA sequence analysis is a molecular biology identification means for bacteria identification, the invention constructs a phylogenetic tree by analyzing the 16S rDNA sequence and applying MEGA 6.0 software and adopting a Neighbor-join method, analyzes the evolutionary relationship of BC13, and the result shows that: 16S rDNA sequence of BC13 andAlcaligenes faecalisthe homology of BDB4, P156 and ZD02 reaches 99.47 percent and is equal toA. faecalisqz-13, DSM 30030, SeY03 homology up to 99.40%, 99.13%, 98.93%, respectively, toA. aquatilisThe homologies of C _11, C _6 and C _15 respectively reach 99.13%, 99.07% and 98.93%, and phylogenetic relationship analysis is carried out on the homologies and the 16S rDNA sequences of 43 closely related strains to find that BC13 and C _15 have homologyA. faecalisIs closest (fig. 1).
By combining the results, the strain BC13 is identified to belong to the alcaligenes faecalisAlcaligenes faecalis。
EXAMPLE 3 determination of the bacteriostatic spectra of the biocontrol bacterium BC13
1. In order to better study the biocontrol potential of the strain BC13 obtained in example 1, the bacterial inhibition spectrum of the strain was determined. The specific operation is as follows:
(1) it was investigated whether BC13 has the ability to inhibit the growth of other pathogenic bacteria. The pathogenic bacteria MS2 is replaced by the same culture conditions and method by using the same method for screening the strain as in example 1Equal amount of rice basal rot fungus EC1, banana bacterial soft rot MS3, ralstonia solanacearum EP1 (the first layer 15 mL LB is changed into 15 mL TTC), rice white leaf blight fungus Xoo, rice soft rot fungusPantoea ananatis SC7, peach rot pathogenP. ananatis PP1, the remaining steps were unchanged.
(2) Whether BC13 has the ability to inhibit the growth of pathogenic fungi was explored. The fungi tested included colletotrichum capsici, colletotrichum gloeosporioides, fusarium oxysporum, rhizoctonia solani, peronophythora litchii and ustilago sugarcane. Wherein the smut pathogen adopts a plate confronting method. 20 mL of PDA was placed on a 13X 13 square petri dish and cut into rectangular strips with a width of 0.5 cm and a length of 6 cm, each block being 0.5 cm apart, after the plates were dried. And (3) putting 1 mu L of BC13 bacterial liquid cultured overnight at one end of the PDA strip, and uniformly putting the smut "+" basidiospore and "-" basidiospore and the mixed liquid of two basidiospores in equal amount in the middle. And (3) after the bacterial liquid is dried, placing the bacterial liquid in an incubator at 28 ℃ for inverted culture for 1-2 days, observing the growth condition of the smut, and recording the test result after white villous hyphae are formed on a control plate.
Punching holes with a 0.5 cm puncher after the rest fungi are recovered, inversely placing the fungus cakes in the center of a PDA (personal digital assistant) plate, dripping 2 muL of overnight cultured BC13 and Escherichia coli DH5 alpha bacterial liquid at the same distance from the fungus cakes, sealing after the bacterial liquid is dried, just placing in an incubator at 28 ℃ for culturing for 3-4 days, observing the growth condition of hyphae, and recording the experimental result.
2. Experimental results show that the strain BC13 has strong bacteriostatic action on rice basal saprophytic bacteria EC1, banana bacterial soft rot bacteria MS2 and MS3, solanaceae ralstonia solanacearum EP1, rice bacterial blight Xoo, rice soft rot bacteria SC7, peach rot bacteria PP1, rice sheath blight bacteria and phytophthora capsici (shown in figures 2 and 3), and has weak bacteriostatic effect on pepper anthracnose bacteria, colletotrichum gloeosporioides, banana fusarium oxysporum and usticum (shown in figure 3), which indicates that the strain BC13 can be used for developing a biocontrol agent specially aiming at crop bacterial diseases.
Example 4 determination of the Effect of biocontrol bacteria BC13 on bacterial Soft rot
1. Activation of banana bacterial soft rot on LB solid plateSelecting single colony of MS2 strain and BC13 strain, placing in 50 mL centrifuge tube containing 10 mL LB, placing in 28 deg.C, shaking table at 180 rpm overnight, collecting 1 mL OD600The BC13 bacterial liquid (about 1.3) was mixed with an equal amount of MS2 bacterial liquid. The MS2 bacterial liquid is added with 1 mL LB bacterial liquid to be mixed as positive control, and LB culture medium is used as negative control. And respectively inoculating potato, Chinese cabbage and banana seedlings in different treatments. Each treatment was 3 replicates. Selecting 12 pink banana seedlings with similar growth vigor, respectively taking 200 mu L of LB, MS2+ LB, BC13+ LB, BC13+ MS2 mixed liquid by using a 1 mL injector, injecting the mixed liquid into pseudostems of the pink bananas, repeating the treatment for 3 times, observing the growth condition of the pink banana seedlings every day, and recording the growth condition.
2. Experimental results show that the strain BC13 can obviously reduce the incidence rate of bacterial soft rot on Chinese cabbage, potato, carrot (shown in figure 4) and banana (shown in figure 5).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. An Alcaligenes faecalis (Alcaligenes faecalis) BC13, characterized in that the strain is preserved in Guangdong province microorganism strain preservation center 7-10 days 2018, and the strain preservation number is GDMCC No. 60412.
2. The use of alcaligenes faecalis BC13 according to claim 1 for controlling bacterial and/or fungal diseases in crops;
zeae, ralstonia solanacearum, rice bacterial blight or Pantoea oryzae caused bacterial diseases; the fungal diseases comprise fungal diseases caused by rhizoctonia solani and peronophythora litchii;
the bacterial diseases comprise bacterial basal rot of rice, bacterial soft rot of banana, bacterial soft rot of clivia, bacterial wilt of solanaceae crops, bacterial leaf blight of rice, soft rot of rice or peach rot; the fungal diseases comprise rice sheath blight disease and downy mildew of litchi.
3. A biological agent for controlling bacterial diseases in crops, comprising alcaligenes faecalis BC13 according to claim 1.
4. The biological agent according to claim 3, wherein the concentration of Alcaligenes faecalis BC13 is OD600 ═ 1.0-1.5.
5. The biological agent according to claim 3, wherein the biological agent is prepared from a bacterial solution and/or a fermentation broth of Alcaligenes faecalis BC 13.
6. A method for controlling bacterial diseases in crops, characterized in that soil or plant material is treated with the Alcaligenes faecalis BC13 according to claim 1 or the biological agent according to any one of claims 3 to 5.
7. The method for controlling according to claim 6, characterized in that the treatment is a vaccination treatment.
8. The method for controlling according to claim 7, characterized in that the inoculation treatment is an injection inoculation or a stab inoculation.
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| CN110878263A (en) * | 2019-05-06 | 2020-03-13 | 信阳师范学院 | Application of alcaligenes faecalis and metabolite thereof in prevention and treatment of storage-period grain and oil aspergillus flavus and toxin |
| CN112694983B (en) * | 2019-10-23 | 2022-05-06 | 中国科学院南海海洋研究所 | Marine fungus-bacterium symbiont, metabolite thereof and application thereof in preparation of antibacterial drugs |
| CN110872518B (en) * | 2019-11-20 | 2021-10-08 | 广西金穗生态科技集团股份有限公司 | Acid soil conditioner and preparation method and application thereof |
| CN111909871B (en) * | 2020-07-31 | 2021-12-14 | 扬州大学 | Alcaligenes faecalis for antagonizing peach bacterial perforators and application |
| CN116240148B (en) * | 2023-04-04 | 2023-09-08 | 云南省农业科学院农业环境资源研究所 | Alcaligenes faecalis SZ-220101 and application thereof in preventing and controlling plant diseases |
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