CN112760248A - Streptomyces lincolnensis capable of preventing and treating peach brown rot and application thereof - Google Patents
Streptomyces lincolnensis capable of preventing and treating peach brown rot and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention relates to streptomyces lincolnensis capable of preventing and treating peach brown rot, which is preserved in China general microbiological culture Collection center in 12-month and 17-month 2020 with the preservation number of CGMCC No.21377, and also relates to application of the streptomyces lincolnensis in preventing and treating peach brown rot. The streptomyces lincolnensis has strong inhibiting effect on various plant pathogenic bacteria such as peach brown rot on a flat plate, and volatile metabolites of the streptomyces lincolnensis have good prevention and control effects on the growth of the peach brown rot pathogenic bacteria. The strain has potential commercial development and application values in biological control practice of diseases. The strain also has broad-spectrum antagonistic activity, has strong bacteriostatic action on pathogenic bacteria such as citrus canker pathogen and tobacco angular leaf spot pathogen, and also has good bacteriostatic effect on pathogenic fungi such as tobacco botrytis cinerea, apple canker pathogen and tobacco root black rot pathogen.
Description
Technical Field
The invention relates to the field of fruit tree disease control, and more particularly relates to streptomyces lincolnensis capable of controlling peach brown rot and application thereof.
Background
The peach tree cultivation area and the peach yield are the top of the world in China, and the peach tree cultivation area and the peach yield are important peach producing big countries. The peaches are one of the main fruit resources in China, are planted in the whole country, are mainly distributed in east China and north China at present, are rich in nutrition and sweet in taste, can be processed into fruit juice, cans and the like, are popular with people, and bring huge economic benefits. According to statistics, the total output and the total consumption of peaches in China in 2015 are 1311 million t and 1320 million t respectively, and the total amount accounts for about 66% of the total peach production in the world. However, the excellent fruit rate of peaches in China is low, and the main reason is that rotten fruits are caused by infection of pathogenic microorganisms, so that the quality of peaches is seriously influenced.
Brown rot of peach, also known as fruit rot and sclerotinia, is a fungal disease caused by sclerotinia spp. The pathogenic bacteria can not only harm peach, but also harm stone fruit trees such as plum, cherry, apricot and the like, and kernel fruit trees such as pear, apple and the like. The Monilinia fructicola is particularly seriously harmful to the flowering phase and the fruit ripening phase of peach trees under the warm and humid condition, so that the burnt-out and fruit drop are caused. Moreover, the harm of the Monilinia fructicola on the picked fruits is more serious in the storage, transportation and consumption processes of the Monilinia fructicola on the fruits, and huge losses are brought to the edible value of the fruits and the economic income of fruit growers.
At present, the control of peach brown rot is mainly carried out by chemical agents, the chemical agents have the advantages of simple and convenient operation and low cost, but the use of chemical bactericides can easily cause the drug resistance of pathogenic bacteria to be enhanced and the environmental pollution, and can possibly harm the ecological safety and the human health.
In order to overcome the drug resistance of plant diseases, reduce the pollution of chemical bactericides to the environment, the ecological damage and the residue of chemical pesticides in agricultural and sideline products, the biological control microorganism has unique advantages, and the biological pesticide prepared by utilizing the secondary metabolites of the biological control microorganism has the advantages of no pollution, no residue, difficulty in causing the pests to generate drug resistance, high compatibility with the environment, safety to people and livestock and the like. Therefore, a biocontrol bacterium capable of controlling the brown rot of peach is needed.
Disclosure of Invention
In order to solve the problems, the invention provides streptomyces lincolnensis capable of preventing and treating peach brown rot, which is preserved in China general microbiological culture Collection center in 12-month and 17-day 2020 with the preservation number of CGMCC No. 21377.
The invention also provides application of the streptomyces lincolnensis in preventing and treating the peach brown rot.
The invention also provides a method for preventing and treating the brown rot of peach, which comprises the step of applying the volatile substances produced by the streptomyces lincolnensis to the fruit of the fruit.
In a specific embodiment, the method comprises the steps of:
s1: culturing the streptomyces lincolnensis to obtain a streptomyces lincolnensis culture;
s2: placing the culture of streptomyces lincolnensis in a storage environment of the fruit, and the culture of streptomyces lincolnensis has gaseous communication with the storage environment of the fruit.
In a specific embodiment, in S1, the streptomyces lincolnensis is cultured on a high-grade No. one plate medium.
In one embodiment, in S2, the fruit is stored at an ambient condition of 30 ℃ and 60% humidity.
The invention also provides a method for inhibiting the spore proliferation of the pathogenic bacteria of brown rot of peach in the environment, which comprises the step of applying the volatile substances produced by the streptomyces lincolnensis to the environment.
In a particular embodiment, the administration of the volatile substances produced by the streptomyces lincolnensis into the environment is achieved by placing the streptomyces lincolnensis culture in the environment.
The streptomyces lincolnensis has strong inhibiting effect on various plant pathogenic bacteria such as peach brown rot on a flat plate, and volatile metabolites of the streptomyces lincolnensis have good prevention and control effects on spore production of the peach brown rot. Because the strain is separated from soil, has natural harmony and compatibility with natural ecology, has no toxic or side effect and no residue on soil ecology compared with chemical pesticides, and has potential commercial development and application value in biological control practice of diseases. The strain also has broad-spectrum antagonistic activity, has strong bacteriostatic action on pathogenic bacteria such as citrus canker pathogen and tobacco angular leaf spot pathogen, and also has good bacteriostatic effect on pathogenic fungi such as tobacco botrytis cinerea, apple canker pathogen and tobacco root black rot pathogen.
Biological material preservation
The purified streptomyces lincolnensis JCP1-7 is preserved in China general microbiological culture Collection center (CGMCC) at 12.17.2020, and the preservation number is CGMCC No. 21377.
Drawings
FIG. 1 is a photograph of a plate morphology of Streptomyces lincolnensis JCP 1-7;
FIG. 2 is a photograph of a single colony morphology of Streptomyces lincolnensis JCP 1-7;
FIG. 3 is a photomicrograph of spore chains and aerial hyphae of Streptomyces lincolnensis JCP 1-7;
FIG. 4 is a chromatogram analysis of a Streptomyces lincolnensis JCP1-7 volatile metabolite;
FIG. 5 shows the inhibitory effect of volatile substances of Streptomyces lincolnensis JCP1-7 on Monilinia fructicola pathogens;
FIG. 6 shows the control effect of the volatile substances of Streptomyces lincolnensis JCP1-7 on peach brown rot of isolated fruits.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
1. Identification and preservation of Streptococcus lincolensis JCP1-7
The group collects rhizosphere soil of non-farmland ecosystems such as lawns, bamboo groves, street trees and the like from 4 regions of Beibei, Shaping dam, south and south banks in Chongqing city, and a plurality of strains obtained by separation are primarily screened and re-screened, and a strain JCP1-7 with an inhibiting effect on peach brown rot related fungi is separated in the research process.
The strain JCP1-7 was cultured on the Gao's I plate at 28 ℃ for 5-7d, and the morphology of the plate was shown in FIG. 1, the single colony was shown in FIG. 2, and the morphology of the spore chain and aerial growth was shown in FIG. 3.
The strain JCP1-7 was inoculated into the test medium and observed for its biochemical reaction characteristics. The results show that the strain has the characteristics of gelatin liquefaction and milk coagulation peptonization, and has slight brown pigment; production of hydrogen sulfide and melanoid; there was slight growth on the cellulose.
The strain JCP1-7 was primarily identified as Streptomyces lincolnensis by combining the colony morphology and hyphal microscopic morphology photographs, and biochemical characteristics.
Genomic DNA of the strain was extracted with bacterial universal primers 27F (5' -AGAGAGTTTGATCCTGGCTCAG-3) and 1492R (5-TACGGCTACCTTGACGACTT-3). The 16S rDNA sequence obtained by taking the genome DNA of the strain JCP1-7 as a template and carrying out electrophoresis detection and sequencing after PCR amplification is shown as SEQ ID NO. 1. And performing Blast comparison on the obtained sequence result in NCBI (national center of Biotechnology information) to construct an evolutionary tree. The obtained sequence alignment result combines morphological and biochemical characteristics, and shows that the strain belongs to Streptomyces lincolnensis (Streptomyces lincolnensis).
The strain is preserved in China general microbiological culture Collection center (CGMCC) of the microbiological research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, 12 months and 17 days in 2020, with the preservation number: CGMCC No. 21377.
2. Detection and analysis of volatile substances released by streptomyces lincolnensis JCP1-7
Streaking Streptomyces lincolnensis JCP1-7 on Gao's synthetic No. I culture medium, culturing at 28 deg.C for 7d, washing spores with sterile water, filtering the mixture with three-layer mirror wiping paper to obtain sporesThe suspension was finally diluted with sterile water to a spore concentration of 1X 108spores/mL. The spore suspension was inoculated at a ratio of 2% into Gao's No. I liquid medium and cultured with shaking at 180rpm at 28 ℃ for 4d to obtain a seed solution. 100mL of Gao's synthetic No. one medium was poured into a 250mL triangular flask and sterilized, and left to stand at room temperature for 3 days to remove water on the surface of the medium. Sucking 1mL of seed solution into a triangular flask and coating the seed solution uniformly. The cells were incubated at 28 ℃ for 14 days.
The 14 d-cultured triangular flask is sent to Shanghai micro-spectrum chemical service technology Limited company for GC-MS analysis, and volatile substances are respectively adsorbed by adopting a headspace solid-phase microextraction method for determination. The volatile matter content of strain JCP1-7 was determined.
As shown in Table 1 and FIG. 4, 28 volatile substances, mainly alcohols, organic acid esters, terpenes, and the like, were detected from the volatile substances produced by Streptomyces lincolnensis JCP 1-7. Wherein the relative content of the dimethydecalin is 34.047%, and the relative contents of the octahydronaphthalene compound, the tetramethylvinyl alkane, the methyl 2-methylbutyrate and the 2-methylisothianol are respectively 13.050%, 10.683%, 6.217% and 4.059%.
TABLE 1 compositional analysis of volatile materials produced by JCP1-7
3. Bacteriostatic effect of streptomyces lincolnensis JCP1-7 volatile substance
The influence of volatile substances produced by the strain JCP1-7 on the growth of the peach brown rot hyphae is measured by adopting a plate-to-buckle method.
Dipping the antagonistic bacteria to be detected by using an inoculating loop, inoculating the antagonistic bacteria to a Gao's first culture medium, culturing for 3d, inoculating the activated 7d peach brown rot fungus cake to the center of another PDA plate after the antagonistic bacteria is colonized, buckling and sealing the two plates, and culturing in a constant-temperature incubator at 28 ℃. Each treatment was repeated 3 times, and the PDA plates inoculated with only brown rot and the Gao's number one plates without any treatment were used as controls. After 24h incubation, observations were made every 12h and colony diameters were measured up to 96 h. When the culture was continued to 7d, the colony diameter was measured again and the hyphal growth inhibition rate was calculated.
The result is shown in figure 5, the volatile substance produced by the strain JCP1-7 has stronger bacteriostatic action on the Monilinia fructicola.
4. Disease control effect of streptomyces lincolnensis JCP1-7 volatile substance
The isolated fruit method is adopted to determine the disease control effect of streptomyces lincolnensis JCP1-7 on peach brown rot.
The strain JCP1-7 is spread and inoculated in a disposable sterile culture dish (70mm) into which the Gao's first culture medium is poured, the contrast is that only the Gao's first culture medium is poured, the strain is inversely cultured for 5 days at the constant temperature of 28 ℃, and the culture dish is removed after the strain is colonized and then the strain is placed at the bottom of a plastic incubator. The perforated partition board is quickly placed to ensure that the volatile products of the bacterial strains can act on the peach fruits, 12 fruits with basically consistent appearances, such as no mechanical trauma, no pest and drug damage, mature degree and the like, are placed on the upper layer of the partition board. The method of treatment of each fruit was as follows: cleaning with sterile water, sterilizing in 75% ethanol for 2min, rinsing with sterile water for 3 times, and air drying. Selecting two points on the male surface and the female surface of the waist of each peach fruit, perforating 3mm deep by using a sterile puncher with the diameter of 6mm to form an artificial wound, and sucking the redundant peach juice of the wound by using sterile filter paper. Taking Monilinia fructicola cake with diameter of 4mm, and pasting hypha surface on fruit wound. Sealing the plastic incubator with a transparent cover, placing the sealed plastic incubator in a constant-temperature incubator with the temperature of 30 ℃ and the humidity of 60% for dark culture for 3d, observing the disease occurrence condition of the peach fruits every 24h, measuring the transverse diameter and the longitudinal diameter of the disease spots, calculating the area of the disease spots according to an oval area formula, and calculating the inhibition rate.
In-vitro inoculation experiments show that after inoculation is carried out for 48 hours, the lesion extension inhibition rate after JCP1-7 treatment is 25.47%; after 72h of inoculation, the lesion of the control group completely covered one side of the peach fruit, and the lesion had a large number of spores and appeared grayish brown.
The spore amount of brown rot germs on peach fruit scabs is calculated by using a blood counting chamber: the spores produced on the fruits were gently washed with a sterile cotton swab into 20mL of sterile distilled water at 72h, and after filtering the spore suspension with a double gauze, the sporulation yield of the peach brown rot strain was determined using a hemocytometer and an optical microscope. The control effect of the JCP1-7 strain on isolated fruits with peach brown rot was determined.
The results showed that the amount of fruit spores in the control group was 1.05X 108spore/mL, spore count on treatment group JCP1-7 of 9.76X 106The result shows that the amount of spores on the disease spots of the treated group fumigated by the antagonistic strain JCP1-7 at 72 hours is obviously less than that of the control CK (figure 6), but the inhibition effect on the disease spot expansion of peach fruits is limited, and the inhibition rate is only 29.68% and 30%.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
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<120> streptomyces lincolnensis capable of preventing and treating peach brown rot and application thereof
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ctggtagtcc acgccgtaaa cggtgggaac taggtgttgg cgacattcca cgtcgtcggt 780
gccgcagcta acgcattaag ttccccgcct ggggagtacg gccgcaaggc taaaactcaa 840
aggaattgac gggggcccgc acaagcagcg gagcatgtgg cttaattcga cgcaacgcga 900
agaaccttac caaggcttga catacaccgg aaacggccag agatggtcgc ccccttgtgg 960
tcggtgtaca ggtggtgcat ggctgtcgtc agctcgtgtc gtgagatgtt gggttaagtc 1020
ccgcaacgag cgcaaccctt gtcctgtgtt gccagcacgt ccttcgggat ggtggggact 1080
cacaggagac cgccggggtc aactcggagg aaggtgggga cgacgtcaag tcatcatgcc 1140
ccttatgtct tgggctgcac acgtgctaca atggcaggta caatgagctg cgataccgtg 1200
aggtggagcg aatctcaaaa agcctgtctc agttcggatt ggggtctgca actcgacccc 1260
atgaagtcgg agttgctagt aatcgcagat cagcattgct gcggtgaata cgttcccggg 1320
ccttgtacac accgcccgtc acgtcacgaa agtcggtaac acccgaagcc ggtggcccaa 1380
ccccttgtgg agggag 1396
Claims (8)
1. The streptomyces lincolnensis capable of preventing and treating peach brown rot is characterized in that the streptomyces lincolnensis is preserved in China general microbiological culture collection center of China Committee for culture Collection of microorganisms in 12-17 th of 2020 with the preservation number of CGMCC No. 21377.
2. Use of streptomyces lincolnensis according to claim 1 for the control of peach brown rot.
3. A method of controlling brown rot in peach comprising the step of applying the volatile substance produced by streptomyces lincolnensis of claim 1 to the fruit of the fruit.
4. A method according to claim 3, characterized by the steps of:
s1: culturing the streptomyces lincolnensis to obtain a streptomyces lincolnensis culture;
s2: placing the culture of streptomyces lincolnensis in a storage environment of the fruit, and the culture of streptomyces lincolnensis has gaseous communication with the storage environment of the fruit.
5. The method according to claim 4, wherein in S1, the Streptomyces lincolnensis is cultured on a high-grade plate culture medium.
6. The method according to claim 4, wherein the fruit is stored at S2 at an ambient temperature of 30 ℃ and a humidity of 60%.
7. A method of inhibiting spore proliferation of a pathogen from brown rot of peach in an environment comprising the step of administering to the environment a volatile material produced by streptomyces lincolnensis of claim 1.
8. The method according to claim 7, wherein the administration of the volatile substances produced by the Streptomyces lincolnensis into the environment is achieved by placing the Streptomyces lincolnensis culture in the environment.
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| CN114045230A (en) * | 2021-10-22 | 2022-02-15 | 中国农业科学院农业环境与可持续发展研究所 | Actinomycetes for promoting plant root growth and application thereof |
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| CN116240115A (en) * | 2023-03-12 | 2023-06-09 | 西南大学 | Streptomyces griseus capable of preventing and treating pepper epidemic disease and application thereof |
| CN116555043A (en) * | 2023-03-12 | 2023-08-08 | 西南大学 | Streptomyces albus capable of preventing and treating pepper epidemic disease and application thereof |
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| CN114045230A (en) * | 2021-10-22 | 2022-02-15 | 中国农业科学院农业环境与可持续发展研究所 | Actinomycetes for promoting plant root growth and application thereof |
| CN114045230B (en) * | 2021-10-22 | 2023-06-06 | 中国农业科学院农业环境与可持续发展研究所 | Actinomycetes for promoting plant root growth and application thereof |
| CN115851553A (en) * | 2022-12-30 | 2023-03-28 | 西南大学 | Streptomyces virginiae capable of preventing and treating clubroot and application thereof |
| CN115851553B (en) * | 2022-12-30 | 2024-04-26 | 西南大学 | Streptomyces virginiae capable of preventing and treating clubroot and application thereof |
| CN116240115A (en) * | 2023-03-12 | 2023-06-09 | 西南大学 | Streptomyces griseus capable of preventing and treating pepper epidemic disease and application thereof |
| CN116555043A (en) * | 2023-03-12 | 2023-08-08 | 西南大学 | Streptomyces albus capable of preventing and treating pepper epidemic disease and application thereof |
| CN116240115B (en) * | 2023-03-12 | 2024-05-03 | 西南大学 | Streptomyces griseus capable of preventing and treating pepper epidemic disease and application thereof |
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