CN112725228A - Virginia streptomycete capable of preventing and treating peach brown rot and application thereof - Google Patents

Abstract

The invention relates to a Virginia streptomycete capable of preventing and treating peach brown rot, which is preserved in China general microbiological culture Collection center in 12-month-14-month-2020 with the preservation number of CGMCC No.21376, and also relates to an application of the Virginia streptomycete in preventing and treating peach brown rot. The streptomyces virginiae has strong inhibiting effect on various plant pathogenic bacteria such as peach brown rot and the like, and the metabolite of the streptomyces virginiae has good prevention and control effects on the growth of the peach brown rot pathogenic bacteria and the spread of fruit scabs. The strain can be used for producing environment-friendly biological pesticides by a fermentation thallus or secondary metabolite extraction technology, and has important commercial development and application values.

Description

Virginia streptomycete capable of preventing and treating peach brown rot and application thereof

Technical Field

The invention relates to the field of disease control of fruit trees, in particular to streptomyces virginiae 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 the streptomyces virginiae capable of preventing and treating the peach brown rot, which is preserved in the common microorganism center of China general microbiological culture Collection center (CGMCC) in 12, 14 and 2020 with the preservation number of CGMCC No. 21376.

The invention also provides application of the streptomyces virginiae in preventing and treating brown rot of peach.

The invention also provides a method for preventing and treating peach brown rot, which comprises the step of applying the culture product of the streptomyces virginiae to fruit.

In a specific embodiment, the method comprises the steps of:

s1: culturing the streptomyces virginiae to prepare a streptomyces virginiae sterile fermentation broth;

s2: applying the sterile fermentation broth of Streptomyces virginiae to the fruit.

In a specific embodiment, in S1, the streptomyces virginiae is cultured in medium No. gao.

In a specific embodiment, S1 includes the steps of:

s11: inoculating the streptomyces virginiae into a Gao's No. one liquid culture medium for culture to obtain a streptomyces virginiae culture;

s12: and removing hyphae in the culture of the streptomyces virginiae to obtain the sterile fermentation liquor of the streptomyces virginiae.

In a specific embodiment, S11, the Streptomyces virginiae strain was inoculated at a concentration of 10% and cultured at 28 ℃ for 5 days in the dark at 180 rpm.

In one embodiment, in S2, the fruit is stored at an ambient condition of 30 ℃ and 60% humidity.

The present invention also provides a method of inhibiting the growth of pathogens of brown rot of peach in an environment comprising the step of applying to said environment a culture product of the aforementioned streptomyces virginiae.

In a particular embodiment, administering the culture product of Streptomyces virginiae to the environment is accomplished by adding a sterile fermentation broth prepared by the Streptomyces virginiae to the environment.

The streptomyces virginiae has strong inhibiting effect on various plant pathogenic bacteria such as peach brown rot, and the strain fermentation liquid has good prevention and control effects on the growth of the peach brown rot pathogenic bacteria and the spread of fruit scabs. 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.

Biological material preservation

The purified Streptomyces virginiae XDS1-5 is preserved in China general microbiological culture Collection center (CGMCC) at 12 months and 14 days in 2020 with the preservation number of CGMCC No. 21376.

Drawings

FIG. 1 is a photograph of a plate morphology of Streptomyces virginiae XDS 1-5;

FIG. 2 is a photograph showing the morphology of a single colony of Streptomyces virginiae XDS 1-5;

FIG. 3 is a photomicrograph of spore chains and aerial hyphae of Streptomyces virginiae XDS 1-5;

FIG. 4 shows the effect of Streptomyces virginiae XDS1-5 on the biofilm of Monilinia fructicola, where A is the conductivity change curve in the extracellular fluid of Monilinia fructicola under different treatments and B is the extracellular fluid DNA content under different treatments;

FIG. 5 shows the inhibitory effect of a Streptomyces virginiae XDS1-5 sterile fermentation broth on Monilinia persica pathogens;

FIG. 6 shows the control of isolated fruit brown rot by a sterile fermentation broth of Streptomyces virginiae XDS 1-5.

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 Streptomyces virginiae XDS1-5

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 XDS1-5 with an inhibiting effect on peach brown rot related fungi is separated in the research process.

The strain XDS1-5 was cultured on the Gao's first culture plate at 28 ℃ for 5-7d to obtain a plate morphology photograph as shown in FIG. 1, a single colony photograph as shown in FIG. 2, and a spore chain and aerial morphology as shown in FIG. 3.

The strain XDS1-5 was inoculated into the test medium and observed for its biochemical reaction characteristics. The results showed that this strain can utilize D-aspartic acid, adenosine, L-malic acid, g-cyclodextrin, glycogen, glycine and citric acid. The strain has the characteristics of gelatin liquefaction, no coagulation in the early stage of milk and peptonization in the later stage; producing hydrogen sulfide and melanin.

The bacterial strain XDS1-5 was preliminarily identified as Streptomyces virginiae by combining the colony morphology and hyphal microscopic morphology photographs, and the 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 shown in SEQ ID NO. 1 is obtained by taking the genome DNA of the strain XDS1-5 as a template, carrying out electrophoresis detection after PCR amplification and sequencing. 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 virginiae (Streptomyces virginiae).

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 14 days in 2020, with the preservation number: CGMCC No. 21376.

2. Preparation of Streptomyces virginiae XDS1-5 sterile fermentation broth

Preparing a seed solution: preparing millet immersion liquid culture medium and sterilizing, subpackaging in 100mL triangular flasks on an ultra-clean workbench, subpackaging each flask with 40mL, inoculating antagonistic bacteria cake prepared in advance (activating the strain on the Gao's No. one culture medium for 5d), placing the centrifuge tube in a shaking table, and performing shaking culture at 28 deg.C and 180rpm in the dark for 72h to obtain seed liquid.

Preparing a Gao's first liquid culture medium, sterilizing, subpackaging in 250mL triangular bottles on an ultra-clean workbench, subpackaging 80mL each bottle, transferring the seed liquid into the Gao's first liquid culture medium according to the inoculation amount of 10% by using a liquid transfer gun (the gun head is sterilized and 1mL of the gun head needs to be cut off), placing in a shaking table, and carrying out dark shaking culture at 28 ℃ and 180rpm for 5 days to obtain the fermentation liquid. Centrifuging the fermentation liquid in a centrifuge at 6000rpm for 15min, and suction filtering the supernatant with 0.22 μm microporous membrane for sterilization to obtain sterile fermentation liquid.

3. Damage effect of Streptomyces virginiae XDS1-5 on biofilm of peach brown rot pathogen

The damage effect of the streptomyces virginiae XDS1-5 on the biofilm of the brown rot pathogen of peach is detected by the following method: fermenting and culturing strain XDS1-5, centrifuging, collecting mycelium with sterilized double-layer gauze, and freeze drying for 48 hr with freeze dryer. 200mg of the freeze-dried mycelium was weighed into a test tube, and the sterile fermentation filtrate of the strain XDS1-5 was added to the tube to make a volume of 10 mL. Three replicates of each treatment were run against the addition of only the same volume of sterile fermentation filtrate. And testing the conductivity of each treatment group by using a conductivity meter after 0min, 5min, 10min, 15min, 20min, 25min, 30min and 1h respectively.

If the cell membrane permeability is damaged, the contents inside the cell leak out of the cell membrane, so that the degree of influence of the cell membrane permeability can be judged by measuring the solution outside the cell membrane, the contents of the cell contain DNA and RNA, and the DNA can be detected by an ultraviolet spectrophotometer at 260 nm. Therefore, after the conductivity was measured, the hyphae of each treatment group were removed, the culture broth was collected, and the DNA content of the solution of each treatment group was measured with an ultraviolet spectrophotometer. Each treatment was repeated 3 times.

As shown in FIG. 4, the XDS1-5 strain fermentation liquor treatment of the germs can significantly increase the bacterial liquid conductivity and DNA content, which indicates that the fermentation liquor treatment can damage the brown rot cell membrane, change the permeability, and cause the exosmosis of various water-soluble substances including electrolytes in the cells.

4. Bacteriostatic effect of Streptomyces virginiae XDS1-5 sterile fermentation broth

The XDS1-5 strain sterile fermentation broth obtained in section 2 was mixed with PDA medium cooled to about 45 ℃ at a ratio of 1:9, and the mixture was poured onto a plate, and an equal volume of sterile water was added to the control group. In a clean bench, punching a hole of a puncher with the diameter of 4mm for the previously cultured Monilinia fructicola, inoculating the fungus cake to the center of a PDA flat plate, and inversely culturing in a constant-temperature incubator at 28 ℃. 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 hypha growth inhibition rate was calculated, and the inhibition rate (%) was (control colony diameter-treated colony diameter)/control colony diameter × 100.

The result is shown in figure 5, the bacterial strain XDS1-5 sterile fermentation liquor has stronger bacteriostatic action on the Monilinia fructicola. When the treatment is carried out for 96 hours, the inhibition rate of the Monilinia fructicola after the treatment of the sterile fermentation liquor of the antagonistic bacteria XDS1-5 is 84.36 percent; after the strain is continuously treated for 7d, the inhibition rate of the strain XDS1-5 is 80.29%, from the growth of the plate colonies in FIG. 5, the growth speed of contrast CK hyphae is high, the colonies are gray brown or gray yellow, the colonies are obviously arranged in a concentric circular ring shape, and a large number of conidia are generated; and the hypha growth and spore production of the Monilinia fructicola are obviously inhibited by the XDS1-5 fermentation liquor.

5. Disease control effect of streptomyces virginiae XDS1-5 sterile fermentation broth

The in vitro fruit method is adopted to determine the disease control effect of the streptomyces virginiae XDS1-5 on the peach brown rot.

Selecting 12 peach fruits which are healthy in growth and basically consistent in mature, cleaning the peach fruits by using sterile water, putting the peach fruits in 75% alcohol in an ultra-clean workbench for disinfection for 2min, rinsing the peach fruits by using the sterile water for 3 times, and airing the peach fruits. 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. Soaking the wound of peach fruit in 2 kinds of antagonistic bacteria liquid for 10min, respectively, collecting Monilinia fructicola cake with diameter of 4mm, and placing the hypha face on the wound of fruit. Soaking the wound of the fruit in sterile water for 10min, and adding peach fruit of the pathogenic bacteria of peach brown rot as control group, wherein each treatment is repeated for 3 times. Placing the treated peach fruit 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 fruit every 24h, measuring the transverse diameter and the longitudinal diameter of disease spots, calculating the area of the disease spots according to an elliptical area formula, and calculating the inhibition rate. The area of the lesion ═ pi ab (a, b are the length of the major axis and the minor axis of the ellipse, respectively), the inhibition ratio (%) of the lesion ═ S control-S treatment/S control × 100, and the results were recorded by photographing.

As shown in FIG. 6, the inhibition rate of lesion extension by XDS1-5 treatment was 86.63% at 48 hours after inoculation. The control group produced significantly more lesions and mildew layer spores than the treatment group; after 72h of inoculation, the inhibition rate of the lesion extension of the XDS1-5 strain is 77.87%, while the lesion of the control group completely covers one side of the peach fruit, and a large number of spores are formed on the lesion, so that the lesion is grayish brown. The disease spots of the treatment group soaked by the XDS1-5 strain in the same time are obviously smaller, wherein the XDS1-5 of the treatment group can hardly see a gray mildew layer with naked eyes and only shows water stain-shaped disease spots, and the control effect is more obvious.

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

<110> university of southwest

<120> Streptomyces virginiae capable of preventing and treating peach brown rot and application thereof

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ggcaatctgc ccttcactct gggacaagcc ctggaaacgg ggtctaatac cggataccac 120

tcctgcctgc atgggcgggg gttgaaagct ccggcggtga aggatgagcc cgcggcctat 180

cagcttgttg gtggggtaat ggcctaccaa ggcgacgacg ggtagccggc ctgagagggc 240

gaccggccac actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa 300

tattgcacaa tgggcgaaag cctgatgcag cgacgccgcg tgagggatga cggccttcgg 360

gttgtaaacc tctttcagca gggaagaagc gaaagtgacg gtacctgcag aagaagcgcc 420

ggctaactac gtgccagcag ccgcggtaat acgtagggcg caagcgttgt ccggaattat 480

tgggcgtaaa gagctcgtag gcggcttgtc acgtcggatg tgaaagcccg aggcttaacc 540

tcgggtctgc attcgatacg ggctagctag agtgtggtag gggagatcgg aattcctggt 600

gtagcggtga aatgcgcaga tatcaggagg aacaccggtg gcgaaggcgg atctctgggc 660

cattactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt 720

ccacgccgta aacgttggga actaggtgtt ggcgacattc cacgtcgtcg gtgccgcagc 780

taacgcatta agttccccgc ctggggagta cggccgcaag gctaaaactc aaaggaattg 840

acgggggccc gcacaagcgg cggagcatgt ggcttaattc gacgcaacgc gaagaacctt 900

accaaggctt gacatatacc ggaaagcatt agagatagtg ccccccttgt ggtcggtata 960

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acaccgcccg tcacgtcacg aaagtcggta acacccgaag ccggtggccc aacccttggg 1380

agggagc 1387

Claims (10)

1. The streptomyces virginiae capable of preventing and treating peach brown rot is characterized in that the streptomyces virginiae is preserved in China general microbiological culture collection center of China Committee for culture Collection of microorganisms in 12-month and 14-month 2020, and the preservation number is CGMCC No. 21376.

2. Use of the streptomyces virginiae of claim 1 for the control of brown rot in peach.

3. A method of controlling peach brown rot, comprising the step of applying the culture product of Streptomyces virginiae according to claim 1 to fruit.

4. A method according to claim 3, characterized by the steps of:

s1: culturing the streptomyces virginiae to prepare a streptomyces virginiae sterile fermentation broth;

s2: applying the sterile fermentation broth of Streptomyces virginiae to the fruit.

5. The method of claim 4, wherein in S1, the Streptomyces virginiae is cultured in a high-count medium.

6. The method of claim 5, wherein S1 includes the following steps:

s11: inoculating the streptomyces virginiae into a Gao's No. one liquid culture medium for culture to obtain a streptomyces virginiae culture;

s12: and removing hyphae in the culture of the streptomyces virginiae to obtain the sterile fermentation liquor of the streptomyces virginiae.

7. The method according to claim 6, wherein S11, the Streptomyces virginiae is inoculated at a concentration of 10% and cultured at 28 ℃ in the dark at 180rpm for 5 days.

8. The method according to claim 4, wherein the fruit is stored at S2 at an ambient temperature of 30 ℃ and a humidity of 60%.

9. A method of inhibiting the growth of brown rot pathogen in peach comprising the step of applying a culture product of streptomyces virginiae of claim 1 to an environment.

10. The method of claim 9, wherein administering the culture product of streptomyces virginiae into the environment is accomplished by adding a sterile fermentation broth prepared from the streptomyces virginiae into the environment.

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