CN112481160A - Paenibacillus polymyxa and application thereof in prevention and control of pear diseases - Google Patents
Paenibacillus polymyxa and application thereof in prevention and control of pear diseases Download PDFInfo
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Abstract
The invention discloses a paenibacillus polymyxaPaenibacillus polymyxa) FX1 and application thereof, wherein the strain is preserved in China general microbiological culture collection center in 9 months and 18 days in 2020, and the preservation number is CGMCC NO. 20680; the paenibacillus polymyxa belongs to the first-level strain without safety identification in China; the bacterial strain FX1 and the biocontrol microbial inoculum thereof have strong inhibitory activity on main pathogenic fungi on pear trees, can inhibit the expansion of anthracnose spots indoors, can also inhibit the occurrence of anthracnose, ring spot, black spot and rot in fields, and also have the effect of promoting healing of the rot on branches and trunks; the strain and the preparation thereof can reduce the use of chemical pesticides in the field and ensure the ecological environmentThe environment is safe, the fruit quality is safe, the social and economic benefits are good, and the application prospect is wide.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to paenibacillus polymyxa and application thereof in prevention and control of pear diseases.
Background
Pear is one of the major fruits in the world. In recent years, the cultivation area of the pear trees in China is more than 90 million hectares, the yield is stabilized at about 1600 million tons and accounts for more than two thirds of the world, and the healthy development of the pear industry is very important for the development of the fruit industry in China and even the world. The disease is one of important factors for limiting the healthy development of the pear industry, and the pear trees in China mainly have fungal diseases such as anthracnose, ring spot, rot, scab and the like. Anthracnose is harmful to leaves and fruits, the fruit disease rate is up to more than 70 percent and fruit farmers are disastrous in the large outbreak of Dangshan county, Anhui province, which is an important place for pear fruit production in 2008. In recent years, anthracnose causes early leaf drop in southern pear producing areas, seriously influences flowering and fruiting of pear trees in the next year, and has become a key control object in the pear producing areas in China. The ring rot is the damage to the branches, leaves and fruits, the incidence rate of the branches reaches 100 percent when the branches are serious, the disease rate of the fruits can reach 30 to 50 percent, and the branches are almost completely rotten after being stored for 1 month. The rot is a harmful branch, has the characteristics of high incidence rate, wide incidence area and difficult control, and can cause a great amount of dead trees or garden damage due to serious pear gardens, tree scabs and incomplete branches.
The chemical pesticide is mainly sprayed for preventing and treating crop diseases in China for a long time, pathogenic bacteria generate resistance after long-term use of the chemical pesticide, the dosage is continuously increased, the prevention effect is reduced year by year, and serious negative effects are brought to the ecological environment and the quality safety of agricultural products. Biological control utilizes beneficial microorganisms to control pathogenic microorganisms, is safe and pollution-free to the environment, and plays an important role in green control. At present, except scab, few preventive agents are registered on pear trees in China, and especially biocontrol microorganisms are not registered. The continuous development and excavation of high-efficiency biocontrol microbial resources have great significance for protecting the environment and producing the agriculture safely and healthily.
Paenibacillus polymyxa (B) ((B))Paenibacillus polymyxa) Belong toThe strain which is exempted from safety identification at the first stage in China is a strain which is newly classified from the original bacillus in 1994 by Ash et al and is also an important plant rhizosphere growth-promoting strain. At present, 13 parent drugs and bactericide products of paenibacillus polymyxa are registered in China, are widely used on field crops and fruits and vegetables, can prevent and treat wheat scab, rice sheath blight, cucumber angular leaf spot, watermelon anthracnose, tomato bacterial wilt and the like, and are not registered on pears for use.
The invention takes main fungal diseases of pear trees as targets, and selects paenibacillus polymyxa with stronger antagonistic activity from biocontrol microorganisms, preliminarily prepares a biocontrol microbial inoculum for application, and provides new resources for biological control of fruit tree diseases.
Disclosure of Invention
The invention aims to provide a paenibacillus polymyxa with broad-spectrum antibacterial effect, the strain has obvious antagonistic effect on various pear pathogenic fungi, can make up the deficiency of biological control on pear diseases in the currently disclosed patent, and has wide application prospect in the technical field of biological pesticides.
The invention realizes the technical steps that:
a dilution coating separation method is adopted to separate and screen a biocontrol strain FX1 from rhizosphere soil of a fruit tree, and the strain has strong antagonistic effect on colletotrichum pyricularis, ring spot pathogen, rot pathogen and black spot pathogen on a solid plate. FX1 is identified as Paenibacillus polymyxa (b) by methods such as colony thallus morphology observation, physiological and biochemical indexes, 16S and gyrB gene sequence determination and the likePaenibacillus polymyxa) FX 1. The strain is preserved in China general microbiological culture collection management center (CGMCC for short, the address: No. 3 of West Lu No. 1 of Suzhou province, the national institute of microbiology, postal code 100101 in the morning area of the Chaoyang, Beijing) in 9 and 18 days of 2020, the preservation number is CGMCC number 20680, and the sequence is shown as SEQ ID NO. 1.
The bacterial colony characteristics of the strain on the NA medium: smooth surface without wrinkles, milk white, opaque, rod-shaped thallus, negative gram stain and spore production.
The application of the paenibacillus polymyxa is to prepare the biocontrol microbial inoculum based on the strain, and is used for controlling fungal diseases such as pear anthracnose, pear ring rot, pear black spot, pear rot and the like.
The method for obtaining the microbial inoculum comprises the following steps: picking Paenibacillus polymyxa on solid culture substratePaenibacillus polymyxa) FX1 was inoculated in liquid NB medium and cultured at 25-28 ℃ at 120-600And =1.0, inoculating the mixture into NB culture medium according to the volume ratio of 1-2%, performing amplification culture for 48-72 h under the same condition, and adding sodium benzoate with the mass concentration of 0.2% to obtain the biocontrol microbial inoculum.
Further, the composition of NB medium: 5g/L of peptone, 10g/L of sucrose, 1g/L of yeast powder and 3g/L of beef extract, adding water to a constant volume, adjusting the pH value to 7.0, and sterilizing for 20-25 min by high-pressure steam at 121 ℃.
Further, the paenibacillus polymyxa fungicide is used for preventing and treating anthracnose, ring spot and black spot of pear trees, is sprayed 7-10 days after the pear trees fall flowers, is sprayed once according to the weather and 10-15 days in the growing period, and is timely supplemented after raining until the spraying is finished before bagging. Preferably, the concentration of the thallus in the spray microbial inoculum is 1 multiplied by 108-1×109 cfu/mL。
Furthermore, the paenibacillus polymyxa microbial inoculum is used for preventing and treating pear tree rot, ingredients such as an auxiliary agent, a film-forming agent and the like are added into the microbial inoculum to prepare a gel, and the gel is smeared on scabs of the pear tree rot in spring and autumn to promote the healing of scab wounds.
Compared with the prior art, the invention has the beneficial effects that:
the Paenibacillus polymyxa FX1 provided by the invention is a biocontrol microorganism resource with broad-spectrum antagonistic activity, has a good control effect on pear diseases, can improve the microenvironment of an orchard garden, inhibit the growth and prevalence of germs, reduce the dosage of chemical pesticides, protect the safety of ecological environment, improve the fruit quality and have good social and economic benefits.
Drawings
FIG. 1 is a colony morphology of Paenibacillus polymyxa FX1 on NA plates;
FIG. 2 is a graph showing the effect of Paenibacillus polymyxa FX1 on antagonism of pear pathogenic fungi on PDA plates;
wherein, the graph A is an inoculation schematic diagram, a pathogenic fungus block with the diameter of 5 mm is inoculated in the center of a PDA plate culture dish with the diameter of 9 cm, 3 microliter of cultured FX1 bacterial liquid is inoculated on the upper side, the lower side and the left side of the dish, and the bacterial liquid is not inoculated on the right side of the dish and is reserved as a blank control; FIG. B, C, D, E shows inoculation of Colletotrichum pyricola (B)Colletotrichum fructicola) Perilla, Perilla frutescens (A)Botryosphaeria dothidea) Pear scab bacteria (A), (B)Alternaria alternata) Pear rot pathogen: (Valsa pyri) And antagonistic effect pattern after 5 days of culture.
FIG. 3 shows the indoor inhibition effect of a Paenibacillus polymyxa FX1 microbial inoculum on pear anthracnose;
wherein, the picture A is a negative clear water control, the picture B is a positive control of pear anthracnose pathogen inoculation without spraying FX1 microbial inoculum, the picture C is a result of FX1 microbial inoculum sprayed after one day of anthracnose pathogen inoculation, and the three pictures are the scab spreading results after 7 days of inoculation.
Detailed Description
The invention is further elucidated with reference to the drawings and the detailed description.
Example 1:
the present invention will be described in detail with reference to specific embodiments, but the present invention is not limited thereto. Unless otherwise specified, all technical methods used in the following examples are conventional methods; unless otherwise specified, the test materials used in the following examples are conventional chemical reagents and biochemical reagents.
The invention provides paenibacillus polymyxa for preventing and treating pear fungus diseases, the strain is separated from soil in apple orchard in Feng county of Jiangsu province, biocontrol bacteria are separated and purified from water solution of a soil sample by adopting a dilution and plate coating method, a high-efficiency antagonistic strain is screened by adopting a flat plate antagonistic experiment method, the FX1 strain is found to have good antagonistic effect, and the strain is named as paenibacillus polymyxa (bacillus polymyxa) (based on morphology and 16S gene sequencing resultsPaenibacillus polymyxa) FX1, wherein the bacterial strain is preserved in China general microbiological culture collection center in 2020, 9 months and 18 days, and the preservation number is CGMCCNo. 20680. And the form, the characteristics and the application effect of the strain are shown by combining with related experimental data.
1. The culture conditions of the strains were as follows: NA solid medium composition: 5g/L of peptone, 10g/L of sucrose, 1g/L of yeast powder, 3g/L of beef extract and 15 g/L of agar powder, adding water to a constant volume, adjusting the pH value to 7.0, sterilizing for 20 min by high-pressure steam at 121 ℃, cooling and then using for strain streak culture.
2. Colony characteristics: the surface of the NA culture medium is smooth, has no folds, is milk white and opaque, the thallus is rod-shaped, gram staining is negative, and spores can be produced.
3. The molecular structure is characterized in that: PCR amplification and sequencing are carried out on 16S and gyrB genes of the strain by adopting a primer pair 27F/1492R and UP1/UP2R, the sequences are placed in NCBI for Blast comparison, and the Bacillus polymyxa (Bacillus polymyxa) with the highest sequence similarity is foundPaenibacillus polymyxa) And the similarity is more than 99 percent, and the bacillus polymyxa is identified.
4. The method for preparing the biocontrol microbial inoculum by using FX1 comprises the following steps: picking Paenibacillus polymyxa on solid culture substratePaenibacillus polymyxa) FX1 was inoculated in liquid NB medium, cultured at 28 ℃ for 24 hours at 180rpm, and OD was adjusted600And =1.0, inoculating the mixture into NB culture medium according to the volume ratio of 1% for amplification culture, culturing for 72 hours under the same condition, and adding sodium benzoate with the mass concentration of 0.2% to obtain the biocontrol microbial inoculum.
Composition of NB medium: 5g/L of peptone, 10g/L of sucrose, 1g/L of yeast powder and 3g/L of beef extract, adding water to a constant volume, adjusting the pH value to 7.0, and sterilizing for 20 min by high-pressure steam at 121 ℃.
5. Indoor control effect test of pear anthracnose and ring spot by using prepared microbial inoculum
The test site is in the plant protection institute laboratory of agricultural science institute of Jiangsu province. The pome is purchased in a supermarket and is the Korla bergamot pear. Selecting healthy pear fruits with uniform size and no scratches or wounds on the surface, washing the surface with clear water, disinfecting the surface with 75% ethanol, and drying at room temperature. Lightly punching a wound on the surface of the pear by using an aseptic puncher (with the aperture of 5 mm), connecting the activated anthracnose and ring spot of the pear to the wound, adding water to the lower part of the pear to keep the pear moist, and culturing for one day at the temperature of 28 ℃. FX1 microbial inoculum is uniformly sprayed on the surface of the inoculated pome one day later, and NB bacterial solution is sprayed on the control group one day later after the pathogenic bacteria are inoculated for treatment. The negative control is not inoculated with germs, and is sprayed with clear water. And (5) observing and measuring the diameter of the disease spot after the culture is maintained for 7-10 days again, and calculating the prevention and treatment effect according to the area of the disease spot. Calculating the formula: control effect = (control group lesion area-treatment group lesion area)/control group lesion area × 100%.
The test results are shown in table 1:
indoor control test of paenibacillus polymyxa FX1 on pear anthracnose and pear ring rot
The results in table 1 show that after treatment by the FX1 bacterial liquid, the anthracnose spots on the surfaces of the pears have small spread area, and compared with a control group, the control effect is over 75%, the spreading of the ring spot is well inhibited, and the effect is similar to that of anthracnose, which indicates that the FX1 bacterial liquid can well inhibit the anthracnose and ring spot of the pears indoors.
6. Field control experiment for pear anthracnose by using microbial inoculum
The test site is located in the Fuyuan fruit Cooperation society of sentence-holding city in Jiangsu province. The variety is rich water and lucky water. A shed frame type cultivation mode and conventional management. 5 pear trees are selected at intervals of one row in the pear orchard as test treatment groups, 3 treatment groups are selected in total and sprayed with FX1 microbial inoculum, and 3 treatment groups are selected at intervals and sprayed with clear water as control treatment. The spraying time of the agent is 10 days after the flower falls, and the agent is sprayed for 4 times before bagging. And after rain, the water is timely sprayed once again. In the mature period, 100 leaves and 30 fruits are randomly selected from each treated pear tree to count the sizes of anthracnose lesions. And (4) counting the number of disease grades and disease indexes according to the grading standards of 0, 1, 3, 5, 7 and 9. Disease index = (number of diseased leaves per grade × number of grades)/(number of investigated total leaves × 9) × 100. The prevention and treatment effect = (disease index of control group-disease index of treatment group)/disease index of control group x 100%.
The test results are shown in table 2:
field control experiment of paenibacillus polymyxa FX1 on pear anthracnose
The results in table 2 show that FX1 bacterial liquid sprayed in the field can effectively inhibit the incidence rate and disease index of pear anthracnose on leaves and fruits, and compared with a control, the control effect reaches more than 85%.
7. Test for field control of pear rot by biocontrol microbial inoculum
The experimental site was located in the orchard of the Dashahe river, Xuzhou city, Feng county, Jiangsu province. The variety is crisp pears. The old trees in the pear orchard are more, the tree vigor is weaker, and the branches and the trunks are rotted and have more scabs. Selecting 5 pear trees as an experimental treatment group in a garden, selecting 5-10 rot scabs with shallow scabs and relatively short occurrence time from each pear tree in the spring rotten disease occurrence period, scraping the scabs by using a special scraper, and smearing FX1 biocontrol microbial inoculum to protect wounds after scraping the scabs clean. And 5 pear trees are selected as blank controls, and the blank controls are smeared with clear water for cleaning after the scabs are scraped. And investigating the healing condition of the scars in late autumn, and counting the healing rate of the scars.
The test results are shown in table 3:
the results in table 3 show that the FX1 biocontrol microbial inoculum can effectively promote the healing of the field pear rot scab, 41 of 45 scabs of a curettage treatment disease coated with FX1 microbial inoculum can heal and recover growth, only 2 control groups heal, the healing rate is greatly improved, and the FX1 microbial inoculum has a good healing effect.
8. Test for field control of pear black spot and ring spot by biocontrol microbial inoculum
The test site is located in the Fuyuan fruit Cooperation society of sentence-holding city in Jiangsu province. The variety is rich water and lucky water. A shed frame type cultivation mode and conventional management. 5 pear trees are selected at intervals of one row in the pear orchard as test treatment groups, 3 treatment groups are selected in total and sprayed with FX1 microbial inoculum, and 3 treatment groups are selected at intervals and sprayed with clear water as control treatment. The spraying time of the agent is 15 days after the flower falls, and the agent is sprayed for 4 times before bagging. And after rain, the water is timely sprayed once again. And randomly selecting 100 leaves for counting the sizes of black spot and ring spot of each pear tree two weeks after the fruits are harvested. And (4) counting the number of disease grades and disease indexes according to the grading standards of 0, 1, 3, 5, 7 and 9. Disease index = (number of diseased leaves per grade × number of grades)/(number of investigated total leaves × 9) × 100. The prevention and treatment effect = (disease index of control group-disease index of treatment group)/disease index of control group x 100%.
The test results are shown in table 4:
the results in table 4 show that the FX1 biocontrol microbial inoculum can effectively prevent and treat black spot and ring spot on pear leaves, the prevention and treatment effects are close to 90%, and the field performance of the microbial inoculum is good.
The tests show that the Paenibacillus polymyxa FX1 and the biocontrol microbial inoculum thereof provided by the invention can effectively control main fungal diseases of pear trees, have good control effects on leaf and fruit diseases such as anthracnose, ring spot and the like and branch diseases such as rot and the like, and have good application prospects in biological control of fruit tree diseases.
The above-mentioned experiments are only preferred examples of the present invention, and should not be construed as limiting the technical scope of the present invention, and all modifications, substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
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tcgcactgga aactggggag cttgagtgca gaagaggaga gtggaattcc acgtgtagcg 660
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tgacgctgag gcgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc 780
cgtaaacgat gaatgctagg tgttaggggt ttcgataccc ttggtgccga agttaacaca 840
ttaagcattc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacgggga 900
cccgcacaag cagtggagta tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960
cttgacatcc ctctgatcgg tctagagata gacctttcct tcgggacaga ggagacaggt 1020
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Claims (7)
1. Paenibacillus polymyxa (A)Paenibacillus polymyxa) FX1, which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC number 20680 and the preservation date of 2020, 9 months and 18 days.
2. A biocontrol bacterial agent prepared from the Paenibacillus polymyxa FX1 of claim 1.
3. The biocontrol microbial inoculum of claim 2, wherein the biocontrol microbial inoculum is prepared by the following method: inoculating Paenibacillus polymyxa FX1 into liquid NB medium for culturing for 24-36 h, and adjusting OD600And =1.0, inoculating the liquid NB culture medium according to the volume ratio of 1-2% for amplification culture for 48-72 h, and adding sodium benzoate with the mass concentration of 0.2% to obtain the biocontrol microbial inoculum.
4. The biocontrol microbial inoculum of claim 3 wherein: the temperature of the culture is 25-28 ℃, and the rotation speed is 120-180 rpm.
5. The biocontrol microbial inoculum of claim 3, wherein the preparation process of the NB culture medium is as follows: adding 5g of peptone, 10g of sucrose, 1g of yeast powder, 3g of beef extract and 1000mL of distilled water, adjusting the pH value to 7.0, and performing high-pressure steam sterilization to obtain the NB culture medium.
6. The biocontrol microbial inoculum of claim 5 wherein the temperature of autoclaving is 121 ℃ and the sterilization time is 20-25 min.
7. The use of the biocontrol bacterial agent of any one of claims 2-6 for controlling anthracnose, ring spot, black spot, rot of pear trees.
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|---|---|---|---|---|
| CN113817640A (en) * | 2021-09-26 | 2021-12-21 | 中国科学院微生物研究所 | Paenibacillus polymyxa HZ-9 and application thereof |
| CN113817640B (en) * | 2021-09-26 | 2023-01-17 | 中国科学院微生物研究所 | Paenibacillus polymyxa HZ-9 and application thereof |
| CN116622589A (en) * | 2023-06-30 | 2023-08-22 | 江苏省农业科学院 | Bacillus bailii and application thereof in preventing and controlling fruit tree diseases |
| CN116622589B (en) * | 2023-06-30 | 2024-05-24 | 江苏省农业科学院 | Bacillus bailii and application thereof in preventing and controlling fruit tree diseases |
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| CN112481160B (en) | 2021-09-28 |
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