CN112899181A - Actinomycetes with inhibition effect on peanut soil-borne pathogenic fungi and screening method thereof - Google Patents

Actinomycetes with inhibition effect on peanut soil-borne pathogenic fungi and screening method thereof Download PDF

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CN112899181A
CN112899181A CN202011351022.4A CN202011351022A CN112899181A CN 112899181 A CN112899181 A CN 112899181A CN 202011351022 A CN202011351022 A CN 202011351022A CN 112899181 A CN112899181 A CN 112899181A
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易图永
黎杨凯
洪艳云
李林
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract

The invention discloses streptomyces luteorusu (streptomyces luteus) with an inhibiting effect on peanut soil-borne pathogenic fungiStreptomyces luteoverticillatus) The strain has a preservation number of CCTCC No: m2020262. The fermentation liquor of the strain has an inhibiting effect on southern blight, peanut root rot and peanut crown rot, and the inhibiting rates of the fermentation liquor diluted by 5 times on the three pathogenic bacteria are 77.71%, 42.62% and 63.18% respectively. At present, no report that the same biocontrol bacterium can treat three peanut soil-borne fungal diseases exists in China. The strain has no pathogenicity to human and livestock, and can be safely used in peanut soilThe biological control of the fungal diseases can reduce the usage amount of chemical pesticides, reduce environmental pollution, promote the development of green agriculture, and have good application prospect.

Description

Actinomycetes with inhibition effect on peanut soil-borne pathogenic fungi and screening method thereof
Technical Field
The invention relates to a new strain with an inhibiting effect on peanut soil-borne pathogenic fungi and a screening method thereof.
Background
The soil-borne fungal diseases of the peanuts comprise southern blight, root rot and crown rot of the peanuts and the like of the peanuts, and are one of the main diseases in peanut production. Wherein the pathogenic bacteria of southern blight of peanut is Sclerotium rolfsii (Sclerotium rolfsii), the colony is circular and white on PDA culture medium, the hypha develops and radially grows, and the hypha aggregates to form Sclerotium in later stage. The pathogenic bacteria of the peanut root rot disease are Fusarium spp, wherein the dominant pathogenic bacteria are Fusarium solani (F.solani), the microconidium is colorless, oval or kidney-shaped, the size of the microconidium is 5-12 multiplied by 2-4 mu m, and the main pathogenic bacteria are single cells; the large conidium is sickle-shaped or crescent-shaped and has 3 to 5 diaphragms; the chlamydospores are spherical, unigenic or syngenic and have a diameter of 7-11 mu m. The pathogenic bacteria of the peanut crown rot disease are Aspergillus niger (Aspergillus niger), conidia of the Aspergillus niger are round and single-cell, colorless or pale yellow and light brown, cross-grown and 2.5-5 mu m in size. The top of the conidiophores expands to be nearly spherical with the diameter of 700-800 mu m, and single-layer or double-layer small peduncles are radially arranged on the conidiophores, wherein the top-layer small peduncles are spore-forming cells and have the capability of producing spores for multiple times.
The Sclerotium rolfsii, the rhizoctonia solani and the crown rot can survive in plant disease residues, seeds and soil, and can be accumulated year by year and occur year after year. The germs are transmitted by soil, wind and rain, agricultural operation and the like, and can be infected in each growth period of the peanuts. In planting areas where stubble rotation is difficult due to water shortage and soil impoverishment, diseases occur particularly, and the quality and yield of peanuts are seriously affected.
At present, chemical control is the main method for controlling soil-borne fungal diseases of peanuts. However, if a certain chemical agent is used for a long time, the drug resistance is easy to generate, and the prevention and treatment effect is influenced. Due to the characteristics of soil-borne fungal diseases of peanuts, the peanuts need to be controlled by applying the pesticide for multiple times in the growth period of the peanuts, and a large amount of chemical agents are used to cause environmental pollution, generate a phytotoxicity phenomenon and have potential harm to human health. Therefore, screening out the microorganism with the inhibiting effect on the peanut soil-borne pathogenic fungi is the best way for preventing and treating the peanut soil-borne fungal diseases.
Disclosure of Invention
Aiming at the problems, the inventor collects a peanut rhizosphere soil sample from a peanut field of Hunan agriculture university in the hibiscus area of Changsha, Hunan, acquires a single bacterial colony of bacteria and actinomycetes by a dilution plate method, screens by adopting a plate opposition method to obtain an actinomycetes with a good inhibition effect on peanut soil-borne pathogenic fungi, names the actinomycetes as YY-38, and identifies the actinomycetes as Streptomyces luteovitericus. The inventor finds that the bacterium has no pathogenicity to human and livestock through research, and can be safely used for biological control of soil-borne fungal diseases of peanuts.
The technical scheme provided by the invention is as follows: an actinomycete with an inhibiting effect on peanut soil-borne pathogenic fungi, which has a preservation number of CCTCC No: m2020262 Streptomyces luteovitericus (Streptomyces luteovitericus) was deposited at the China center for type culture Collection, university of Wuhan.
The actinomycete YY-38 (Streptomyces luteovirticus, Latin name: Streptomyces luteovirticus) is preserved in China center for type culture Collection of Wuhan university at 7 months and 1 day 2020 (preservation address: preservation center of Wuchang Lojia mountain Wuhan university, Wuhan, Hubei province), and the preservation number is CCTCC No: m2020262, survival was detected.
Meanwhile, the invention also provides application of the actinomycete in preventing and treating soil-borne fungal diseases of peanuts.
The peanut soil-borne pathogenic fungi are peanut Sclerotium rolfsii, peanut root rot (Fusarium solani) and/or peanut crown rot (Aspergillus niger).
The application utilizes the fermentation liquor or the fermentation liquor extract of the actinomycetes.
The invention also provides an agricultural bactericide, which comprises the actinomycete, preferably fermentation liquor containing the strain, or fermentation liquor extract thereof.
Meanwhile, the invention also provides a screening method of the actinomycetes having the inhibition effect on the peanut soil-borne pathogenic fungi, which comprises the following steps:
(1) taking soil around the peanut rhizosphere, removing surface soil when collecting a soil sample, and taking the soil sample at the depth of 5-20 cm;
(2) separating by dilution method, placing the soil sample into a bottle filled with glass ball and sterile water, oscillating, standing, diluting, and making into 10-4、10-5、10-61mL of the soil diluent is absorbed and dropped on an NA culture medium flat plate, the soil diluent is uniformly coated, and the culture dish is placed in a constant temperature incubator for inverted culture at 28 ℃;
(3) screening biocontrol bacteria with an inhibiting effect on peanut soil-borne pathogenic fungi.
Screening biocontrol bacteria with inhibiting effect on peanut soil-borne pathogenic fungi by adopting a plate confronting method: culturing the purified biocontrol strain with an NA culture medium, culturing peanut sclerotium rolfsii and peanut rhizoctonia solani on a PDA culture medium for 7 days, beating the strain cake with a 7mm puncher, inoculating the strain cake into the center of a culture dish containing the PDA culture medium, uniformly and symmetrically inoculating biocontrol bacteria on the periphery, observing and measuring the diameter of a bacteriostatic circle after culturing for 7-14 days, selecting biocontrol bacteria with obvious antagonistic action, and storing.
The invention has the following beneficial effects:
the actinomycete YY-38 is obtained by separating a peanut rhizosphere soil sample collected from a peanut field in a hibiscus region of Changsha, Hunan province and is identified as Streptomyces luteovirticus, the inhibition rate of fermentation liquor of the strain on southern Sclerotium rolfsii (Sclerotium rolfsii) is 77.71 percent after the strain is diluted by 5 times, the inhibition rate on peanut root rot (Fusarium solani) is 42.62 percent, and the inhibition rate on peanut crown rot (Aspergillus niger) is 63.18 percent. The strain has no pathogenicity to human and livestock, can be safely used for biological control of soil-borne fungal diseases of peanuts, can reduce the use amount of chemical pesticides, prevent the generation of key factors, reduce environmental pollution and promote the development of green agriculture. At present, no report that actinomycetes are used for treating three peanut soil-borne fungal diseases at the same time exists in China. Therefore, the antagonistic strain has good application prospect.
Drawings
FIG. 1 shows the culture characteristics of actinomycetes YY-38 of the present invention, wherein (a) shows a colony morphology and (b) shows a hyphal morphology.
FIG. 2 is a diagram showing the results of PCR amplification gel electrophoresis of the 16SrDNA fragment of actinomycete YY-38 of the present invention.
FIG. 3 is a sequence diagram showing 16SrDNA of actinomycetes YY-38 of the present invention.
FIG. 4 shows a phylogenetic tree constructed based on the 16SrDNA sequence of actinomycetes YY-38 of the present invention.
Detailed Description
Example 1: screening and identification of strains
The screening method of the actinomycetes which has the inhibiting effect on the peanut soil-borne pathogenic fungi comprises the following steps:
1. soil sample collection
Peanut rhizosphere soil is collected from peanut fields of Hunan agriculture university in Hibiscus region of Changsha, Hunan, and is brought back to a laboratory.
2. Isolation of biocontrol bacteria (Actinomycetes)
Separation was performed by dilution. Placing the soil sample into a bottle filled with glass balls and sterile water, oscillating, standing, diluting with sterile water, and making 10-4、10-5、10-6The diluted solution was added to NA medium (3 g of beef extract, 20g of agar, 5g of sodium chloride, 10g of peptone, ddH) by pipetting 100. mu.L of the diluted solution, and adding dropwise2O1000 mL, pH adjusted to 7.2), spread evenly on the plates, and place the dishes in a constant temperature incubator at 28 ℃ for inverted culture.
3. Screening of antagonistic biocontrol bacteria (actinomycetes)
The biocontrol bacteria which have the inhibiting effect on peanut soil-borne pathogenic fungi are preliminarily screened by adopting a plate confronting method. And selecting a single colony of the purified actinomycete strain to culture on an NA culture medium. Inoculating southern blight and crown rot of peanut into the center of PDA culture medium with a puncher with diameter of 7mm, and inoculating biocontrol bacteria symmetrically around. Using a control which is not inoculated with biocontrol bacteria as a control, observing and measuring the diameter of a bacteriostatic zone after CK controls grow over a culture dish, selecting strains with obvious antagonism, purifying and storing by using a glycerol preservation method, and screening 4 strains respectively numbered as YY-1, YY-38, YY-60 and YY-83 which have certain antagonism effect on two pathogenic bacteria.
4. Bacterial antagonistic rescreening
4 strains which are screened to have good antagonistic effect are inoculated in NB culture medium (beef extract 3)g. 5g of sodium chloride, 10g of peptone and ddH2O1000 mL, pH adjusted to 7.2), shake-cultured at 28 ℃ for 2d, centrifuged at 8000r/min at 4 ℃ for 10min, and filtered with a bacterial filter. The filtered fermentation liquor is diluted by 5 times (4mL fermentation liquor and 16mL PDA are mixed and poured into a flat plate) and 10 times (2mL fermentation liquor and 18mL PDA are mixed and poured into a flat plate) to carry out bacteriostatic activity detection, and a culture medium added with the same amount of sterile water is used as a CK control. The southern blight and peanut root rot are detected by a hypha growth rate method, and the peanut crown rot is detected by a spore germination method. The results are shown in tables 1, 2 and 3.
TABLE 1 biocontrol bacteria fermentation broth inhibiting southern blight
Figure BDA0002798829840000041
TABLE 2 inhibitory Effect of biocontrol bacteria fermentation broth on peanut root rot
Figure BDA0002798829840000042
TABLE 3 inhibitory Effect of biocontrol bacteria fermentation broth on crown rot of peanut
Figure BDA0002798829840000043
Figure BDA0002798829840000051
As seen from the above 3 tables, 4 strains all have certain inhibitory action on southern blight, peanut root rot and peanut crown rot, and YY-38 has the best overall inhibitory action on the 3 peanut soil-borne pathogens, so that the strain YY-38 is selected as the optimal biocontrol microbial strain for peanut soil-borne diseases to be preserved.
7. Identification of antagonistic actinomycetes YY-38
(1) Morphological characteristics
When the strain YY-38 grows for 5 days after being spotted on a Gao's I culture medium by using a sterile toothpick, the growth speed of a bacterial colony is slow, the central white color can be seen, the edge is gray white, the surface of the bacterial colony is dry and has certain wrinkles, the bacterial colony grows closely to the surface of the culture medium, the shape is irregular (figure 1a), and hyphae are bent and sparse (figure 1 b).
(2) Physiological and biochemical experiment
The results of the measurement of the physiological and biochemical characteristics of the strains are shown in Table 4: the YY-38 strain is proved to have no capability of hydrolyzing starch and cellulose, cannot generate hydrogen sulfide and liquefy gelatin, cannot reduce nitrate, but has positive peptonization reaction and melanin generation test.
TABLE 4 physiological and biochemical test results for actinomycetes YY-38
Figure BDA0002798829840000052
Note: "+" indicates a positive result, and "-" indicates a negative result
(3)16S rDNA sequencing and sequence analysis
Bacterial DNA was extracted stepwise using the full-scale gold EasyPure bacteriological DNA kit according to the instruction procedures. The YY-38 strain DNA was subjected to PCR amplification using bacterial universal primers 16SrDNA universal primers 27F and 1492R.
27F：5'-AGTTTGATCMTGGCTCAG-3'
1492R：5'-GGTTACCTTGTTACGACTT-3'
The amplification system was 25. mu.L:
Figure BDA0002798829840000061
the PCR amplification procedure was as follows:
Figure BDA0002798829840000062
the YY-38 strain DNA is subjected to PCR amplification, gel electrophoresis detection and PCR product sequencing to obtain a 16SrDNA sequence fragment (figure 3) with the size of 1434bp (figure 2). The sequences were subjected to BLAST alignment at NCBI and found to be of Burkholderia with similar homology to the sequence, and the sequences with higher sequence similarity were downloaded, and subjected to sequence analysis using Mega6 software, and a phylogenetic tree of 16SrDNA sequence of YY-38 strain was constructed using the Neighbor Joining method (FIG. 4). It can be seen that YY-38 strain and Streptomyces luteovirticus (Streptomyces luteovirticus) are on the same branch, the sequence homology of the strain and the Streptomyces luteovirticus is high, and the strain YY-38 can be identified as Streptomyces luteotxiclus (Streptomyces luteovirticus) by combining morphological feature depiction and physiological and biochemical characteristics of the Streptomyces luteotxiclus.
Example 2: application examples
A field test of the fermentation liquor of Streptomyces luteoviteris YY-38 strain for preventing and treating peanut soil-borne diseases is carried out in a hoeing test base of Hunan agricultural university in 5 months in 2020. The peanut variety used for the test is Hunan flower 2008, and the area of the land is 200m2And 3 ridges are planted, 4 rows are planted in each ridge, the row spacing is 30cm, the hole spacing is 12cm, and 1 seed is sowed in each hole. There were 3 treatments, treatment 1: coating the peanut seeds with 25 percent of thiamethoxam, fludioxonil and metalaxyl-M seed coating agent (New Changshan agriculture development Co., Ltd., Hunan) according to the dosage of 350 ml/100 kg of seeds, and sowing after air drying; and (3) treatment 2: diluting fermentation liquor of Streptomyces luteovirticus YY-38 strain by 10 times, soaking peanut seeds for 30min, airing and sowing; treatment 3 (control zone): controlling with clear water, soaking peanut seeds in clear water for 30min, air drying, and sowing; 1 ridge was sown per treatment. And (5) investigating the emergence rate of each treatment after sowing for 20 days, and calculating the emergence improving effect.
Figure BDA0002798829840000071
TABLE 5 emergence rates of Hunan flowers 2008 with different treatments
Figure BDA0002798829840000072
The results of the field emergence test are shown in Table 5. After being diluted by 10 times, fermentation liquor of Streptomyces luteovitericus YY-38 strain treats Hunan flowers 2008 with the emergence rate of 84.34%, which is improved by 52.98% compared with clear water, and has slightly poor effect compared with 25% of seed coating agent of thiamethoxam, fludioxonil and metalaxyl-M, but is obviously better than that of clear water. The biocontrol microbial inoculum has the characteristics of environmental friendliness, no drug resistance and the like, and the test result shows that the biocontrol microbial inoculum YY-38 has a certain application prospect in preventing and controlling peanut soil-borne diseases and improving the peanut emergence rate.

Claims (8)

1. An actinomycete with an inhibiting effect on peanut soil-borne pathogenic fungi, wherein the preservation number is CCTCC No: streptomyces luteus of M2020262 (S. luteus: (S. luteus))Streptomyces luteoverticillatus) And is preserved in the China center for type culture Collection of Wuhan university.
2. Use of the actinomycete for inhibiting a soil-borne peanut fungal pathogen of claim 1 for controlling a soil-borne peanut fungal disease.
3. Use according to claim 2, characterized in that: the peanut soil-borne pathogenic fungus is peanut sclerotium rolfsii (A)Sclerotium rolfsii) Peanut root rot bacteria (A)Fusarium solanic) And/or crown rot of peanut (Aspergillus niger）。
4. Use according to claim 3, characterized in that: it is applied by utilizing the fermentation liquor of the actinomycetes or the extract of the fermentation liquor.
5. An agricultural bactericide, which is characterized in that: comprising the actinomycete of claim 1.
6. An agricultural fungicide according to claim 5 in which: comprising a fermentation broth of the actinomycete of claim 1, or a fermentation broth extract thereof.
7. A method for preparing a fermentation broth for inhibiting peanut soil-borne pathogenic fungi, which is characterized in that actinomycete strain as claimed in claim 1 is inoculated in a culture medium of 3 g/L beef extract, 10 g/L tryptone and 5 g/L sodium chloride and fermented at the temperature of 28 ℃, the temperature of 220 r/min, the pH value of 7.2 and the fermentation time of 48 h.
8. The method for screening actinomycetes having an inhibitory effect on peanut soil-borne pathogenic fungi according to claim 1, characterized by comprising the steps of:
(1) collecting a peanut rhizosphere soil sample, removing surface soil, and taking a soil sample at a depth of 5-20 cm;
(2) diluting the soil sample by dilution separation method in a bottle filled with glass ball and sterile water, shaking, standing, diluting, and making into 10-4、10-5、10-6Diluting the solution, sucking 1mL of the dilution, dripping the dilution on an NA culture medium plate, uniformly coating, and placing the culture dish in a constant-temperature incubator for inverted culture at 28 ℃;
(3) screening biocontrol bacteria with an inhibiting effect on peanut soil-borne pathogenic fungi;
screening of the peanut southern blight bacteria by plate confronting method (Sclerotium rolfsii) And peanut root rot bacteria: (Fusarium solani) Biocontrol bacteria with inhibiting effect: culturing the purified biocontrol strain by using an NA culture medium, culturing peanut southern blight and peanut root rot on a PDA culture medium for 7 days, beating a fungus cake, placing in the center of a culture dish containing the PDA culture medium, symmetrically placing the screened biocontrol bacteria around the culture dish, observing and measuring the diameter of a bacteriostatic circle after culturing for 7-14 days, selecting biocontrol bacteria with obvious antagonistic action, and storing.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105838642A (en) * 2016-04-29 2016-08-10 山东省花生研究所 Actinomycetes for preventing and treating peanut diseases and application of actinomycetes
CN108192829A (en) * 2018-01-13 2018-06-22 鲁东大学 A kind of gamboge streptomyces verticillus and its biological control agent and application
CN111778174A (en) * 2020-05-18 2020-10-16 湖南农业大学 Bacillus subtilis with inhibiting effect on citrus sand skin disease and screening method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105838642A (en) * 2016-04-29 2016-08-10 山东省花生研究所 Actinomycetes for preventing and treating peanut diseases and application of actinomycetes
CN108192829A (en) * 2018-01-13 2018-06-22 鲁东大学 A kind of gamboge streptomyces verticillus and its biological control agent and application
CN111778174A (en) * 2020-05-18 2020-10-16 湖南农业大学 Bacillus subtilis with inhibiting effect on citrus sand skin disease and screening method thereof

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