CN111187724B - Endophytic fungus with dark blueberry root and application thereof - Google Patents
Endophytic fungus with dark blueberry root and application thereof Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- 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
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Abstract
The invention provides a blueberry root dark color endophytic fungus with septa and application thereof, belonging to the field of microbial technology application. The blueberry dark-root endophytic fungus (DSE) strain is Pezicula sp.FLR13, and the collection number is GDMCC No. 60777. The strain has potential application values of promoting growth of blueberries and enhancing disease resistance of plants in blueberry cultivation, and promotes preservation of blueberry fruits. In addition, the strain can antagonize sclerotinia sclerotiorum and has good effect of promoting tomato seed germination and root growth.
Description
Technical Field
The invention belongs to the field of microbial technology application, and particularly relates to a blueberry root deep color septate endophytic fungus and application thereof.
Background
Blueberry, also known as Vaccinium, belongs to the genus Vaccinium (Vaccinium) of Ericaceae, and is a new small berry tree species with high economic value and wide development prospect worldwide. Blueberries belong to plants with shallow roots and have no root hairs, are typical host plants of ericoid mycorrhiza fungi (ERM), and can also be infected and colonized by Dark-color endophytic fungi (DSE) and arbuscule mycorrhiza fungi (AM).
DSEs are small soil fungi that colonize plants and are generally found in the epidermis, cortex, etc. of healthy plant roots, either within cells or in the intercellular spaces. Colonies of DSE cultures are diverse in color, from dark brown to light yellow, and even white. Two typical characteristics of DSE formed in plant root system are dark color hypha with septum and microsclerotia, which can promote the absorption of mineral nutrition and organic nutrient of host, and improve the drought resistance, heavy metal resistance and disease resistance of host. The blueberry industry in China develops rapidly, and the cultivation area and the yield jump the top of the world. However, the blueberry root system is fine and underdeveloped, has no root hair, is easily damaged by adversity stress, and is lack of corresponding flora in most blueberry cultivation areas. Therefore, beneficial microbial resources at the roots of the blueberries are fully excavated, and high-efficiency DSE strains with disease resistance and growth promotion effects on the blueberries are screened, so that the method has very important significance for enhancing the disease resistance of the blueberries and promoting the healthy and stable development of the blueberry industry.
Disclosure of Invention
The invention aims to provide a novel blueberry root dark color separated endophytic fungus strain, which can promote the growth of blueberries, antagonize main pathogenic bacteria of the blueberries and effectively preserve the fruits of the blueberries, and in addition, the strain can antagonize sclerotinia sclerotiorum and has good effects of promoting the germination of tomato seeds and the growth of root systems.
The invention provides a blueberry root dark color septate endophytic fungus, and the blueberry root dark color septate endophytic fungus strain is Pezicula sp.FLR13, and the preservation number is GDMCC No. 60777.
The invention also provides application of the blueberry root dark-color septate endophytic fungi in promoting growth of blueberries.
The invention also provides application of the blueberry root dark-color septate endophytic fungi in antagonism of pathogenic bacteria of blueberries.
Furthermore, the blueberry pathogenic bacteria comprise blueberry pathogenic bacteria including blueberry botrytis cinerea, blueberry intercalary stem canker and blueberry phomopsis stem canker.
The invention also provides application of the dark-color endophytic fungi at the root of the blueberry in promoting preservation of blueberry fruits.
The invention also provides application of the blueberry root dark color septate endophytic fungi in antagonism of sclerotinia sclerotiorum.
The invention also provides application of the blueberry root dark-color septate endophytic fungi in promoting tomato seed germination.
The invention also provides application of the blueberry root dark-color septate endophytic fungi in promoting growth of tomato roots.
The invention has the following advantages:
the blueberry DSE strain Pezicula sp.FLR13 provided by the invention can promote the growth of potted blueberry seedlings, can antagonize main pathogenic bacteria of blueberries, is effective in keeping the blueberry fruits fresh, and indicates that the strain has potential application values in the aspects of promoting the growth of the blueberries, enhancing the disease resistance of plants and the like in blueberry cultivation. In addition, the strain can antagonize sclerotinia sclerotiorum and has good effects of promoting tomato seed germination and root growth.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention.
In the drawings:
FIG. 1 is an optical microscope photograph of FLR13 after root staining after culturing under sterile conditions. A: the FLR 13-inoculated blueberry root cell characteristics are not obtained; b: and after the FLR13 is infected, the DSE micro-sclerotium structure at the root of the blueberry is formed.
Figure 2 is a morphological feature of FLR 13. A: culturing colony characteristics on a PDA plate for two weeks; b: fusing mycelia; c, rope-shaped hyphae.
FIG. 3 is a phylogenetic tree of Pezicula fungi constructed based on ITS-LSU sequence gene combination.
FIG. 4 shows the growth promoting effect of FLR13 on blueberry. A: FLR13 promotes the growth of blueberry seedlings; b: FLR13 invades blueberry root system and deepens color.
Fig. 5 shows the biological effect of FLR13 on blueberries.
FIG. 6 shows the inhibitory effect of FLR13 fermentation filtrate on Botrytis cinerea (A, B), Phomopsis longissima (C, D) and Phomopsis longissima (E, F). A: culturing bacterial colony characteristics of blueberry botrytis cinerea in PDA for 5 d; b: culturing the botrytis cinerea on a medicine plate (FLR13 fermentation filtrate: PDA 1:9) for 5d for colony characteristics; c: culturing bacterial colony characteristics of the phomopsis longipes in the PDA for 5 d; d: culturing the bacterial colony characteristics of the phomopsis longissima on a medicine plate (FLR13 fermentation filtrate: PDA 1:9) for 5 d; e: culturing bacterial colony characteristics of blueberry interstocet stem canker in PDA for 5 d; d: the bacterial colony characteristics of the blueberry intercropping phimosis are cultured for 5d on a medicine plate (FLR13 fermentation filtrate: PDA ═ 1: 9).
FIG. 7 is a graph showing the effect of FLR13 fermentation filtrate on the rate of blueberry decay during storage.
FIG. 8 is the effect of FLR13 fermentation filtrate on tomato seed germination.
FIG. 9 is the effect of FLR13 fermentation filtrate on tomato root growth.
FIG. 10 is a graph showing the effect of different dilution times of FLR13 fermentation filtrate on tomato root growth.
FIG. 11 shows the antagonistic effect of FLR13 strain on Sclerotinia sclerotiorum.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
On the one hand, the invention provides a blueberry endophytic fungus with dark root (hereinafter referred to as blueberry DSE strain), specifically Pezicula sp. FLR13 with the collection number of GDMCC No. 60777.
The blueberry DSE strain FLR13 is a DSE strain obtained by separating, purifying and obtaining from root systems of a rabbit-eye blueberry variety 'Pink blue', belongs to Ascomycota, Anemomycetales Helotiles, Pezicula, and has the following deposited strain names: FLR13, the strain is preserved in Guangdong province microorganism strain preservation center, and the addresses are as follows: the preservation date of No. 59 building 5 of No. 100 college of Pieli Zhonglu, Guangzhou city is 2019, 9 and 16 days, and the preservation number is GDMCC No. 60777.
The classification status of the mycorrhizal fungi strain FLR13 is determined by joint identification of three methods of morphology, molecular biology and observation of the inoculation morphology of the root system of the blueberry.
In the embodiment of the invention, the morphological characteristics of the strain are as follows: on a PDA culture medium plate, hyphae grow slowly, the diameter of a colony cultured for two weeks is 22.4 +/-2.2 cm, the colony is round in shape, dense and white, and no spore is produced. When the hyphae grow to a certain stage, protrusions are generated, and invade into adjacent hyphae to generate hypha fusion, increase the cell content, and thicken the hyphae. Meanwhile, hyphae are intertwined to form hypha knots. In the embodiment of the invention, the ITS and nrDNA (LSU) gene sequences of the blueberry DSE strain show that the FLR13 strain and the P.rhizophila model strains CBS110602, CBS110604, CBS109839 and CBS110609 are gathered on one branch, but form obvious independent subgroups through the analysis result of a combined phylogenetic tree. The blueberry DSE strain disclosed by the embodiment of the invention can infect blueberry roots and form typical DSE microsclerotia structures in root epidermis and cortex cells.
In a second aspect, an embodiment of the invention provides an application of a blueberry DSE strain in promoting blueberry growth. Particularly, the growth of blueberry seedlings is mainly promoted.
Specifically, the embodiment of the invention provides application of fermentation filtrate of a blueberry DSE strain in promoting growth of blueberries.
In a third aspect, the embodiment of the invention provides application of a blueberry DSE strain in antagonism of blueberry pathogenic bacteria.
Specifically, the embodiment of the invention provides application of fermentation liquor of a blueberry DSE strain in antagonism of pathogenic bacteria of blueberries.
Specifically, the pathogenic bacteria of the blueberries comprise Botrytis cinerea (Botrytis cinerea), phoma verticillata (Diaporthe sp.) and Phomopsis sp.
In a fourth aspect, the embodiment of the invention provides an application of a blueberry DSE strain in promoting preservation of blueberry fruits.
In a fifth aspect, an embodiment of the invention provides an application of a blueberry DSE strain in antagonism of Sclerotinia sclerotiorum.
In a sixth aspect, an embodiment of the invention provides an application of a blueberry DSE strain in promoting tomato seed germination and root growth.
Specifically, the embodiment of the invention provides the effect of fermentation filtrate of blueberry DSE strain on promoting tomato seed germination and root growth.
Further, the preparation method of the fermentation filtrate of the blueberry DSE strain comprises the following steps: culturing the blueberry DSE strain on a PDA culture medium for two weeks, taking two fungus cakes (7mm), adding 70ml of liquid PDA culture medium, shaking the fungus at 25 ℃ for 2 weeks to prepare fermentation liquor.
Furthermore, the blueberry DSE strain adopts a plate confrontation method to antagonize blueberry pathogenic bacteria or rape sclerotinia sclerotiorum. For example, a perforator with the inner diameter of 6mm is used for punching fungus cakes on the edges of blueberry pathogenic bacteria and blueberry DSE strains, the fungus cakes are placed into a culture dish with the diameter of 9cm, the distance between the two fungus cakes and the edge of the culture dish is 3cm, and the two fungus cakes are cultured for 5-7 days at a constant temperature of 25 ℃.
Furthermore, the 836bp 28s nrDNA sequence of the Pezicula sp.FLR13 is shown in a sequence table SEQ ID No. 1.
Further, the 499bp ITS sequence of the Pezicula sp.FLR13 is shown in a sequence table SEQ ID No. 2.
The present invention will be described in detail with reference to examples.
Example 1Isolation and characterization of FLR13
1. Separation and purification of blueberry endophytic fungi
The test material was 15-year-old blueberry variety "pink blue". Randomly selecting 3 trees, sampling in east and west directions, removing 5cm thick surface soil, collecting 5-20 cm deep fibrous roots connected with main roots, then placing the collected fibrous roots and soil into a self-sealing bag, storing in a refrigerator at 4 ℃, and processing within 24 hours. Randomly selecting 2cm root samples from each plant sample, washing the root samples with tap water, rinsing the root samples with 75% ethanol for 1min, washing the root samples with sterile water for 3 times, sterilizing the root samples with 0.1% mercuric chloride for 5-10 min, washing the root samples with sterile water for 5 times, placing the root samples on sterile filter paper, removing two ends of the root samples, cutting the root samples into tissue blocks with the size of 5mm, placing the tissue blocks into culture dishes for culture, randomly selecting 100-150 tissue blocks from 4-5 tissue blocks in each dish, and culturing at the constant temperature of 27 ℃. And (5) finding that bacterial colonies grow out around the tissues, separating and purifying in time, and numbering and recording.
In order to verify the disinfection effect of the seed surface, two methods, namely a rinsing liquid inspection method and a tissue imprinting method, are adopted for detection. In the former, the sterile water for rinsing the disinfection material for the last time is coated on a PDA culture medium flat plate, and whether bacterial colonies appear or not is observed after the culture. The tissue block after surface disinfection treatment is pressed into a PDA culture medium flat plate, the surface disinfection material is contacted with the culture medium for 10min, then the disinfection material is removed, and whether bacterial colony appears or not is observed after culture. If the culture medium plates of the two detection methods are aseptically dropped, the surface of the plant material is thoroughly disinfected.
2. Screening of antagonistic strains
Screening antagonistic strains of 3 blueberry plant pathogenic bacteria of blueberry botrytis cinerea, blueberry diaporthe bijuga canker and blueberry phomopsis canker by adopting a plate confronting method.
Using a puncher with the inner diameter of 6mm to punch fungus cakes on the edges of pathogenic bacteria of test plants and endophytic fungi colonies, putting the cakes into a culture dish with the diameter of 9cm, keeping the distance between the two cakes and the edge of the culture dish at 3cm, using a plate inoculated with the pathogenic bacteria as a reference, setting 3 times of repetition for each treatment, and after placing the plate at the temperature of 25 ℃ for constant-temperature culture for 7 days, recording the width of an antagonistic zone of the pathogenic bacteria and the endophytic bacteria to be detected.
3. Detection of bacterial strain infection on blueberry root system
And (3) observing the colonization condition of the strain in the blueberry root system by using a microscope by adopting a trypan blue staining method.
The specific process is as follows:
1) fixing: root segments with the diameter of 0.5-1 mm are selected and cut into small segments with the length of 2-5 cm, and the small segments are fixed in FAA solution (5 ml of formalin, 5ml of glacial acetic acid and 90ml of 70% ethanol).
2) Purifying: the roots fixed in the FAA fixative solution were taken out, washed clean with tap water, and cut into root segments of about 3 cm. The root sample was completely immersed in the solution by adding 10% (w/v) KOH solution. The container is put into a water bath kettle and heated for 70min at the temperature of 90 ℃.
3) Cleaning: and pouring the KOH solution after heating, and rinsing the root sample in a beaker with clear water for 3-5 times until the root sample does not appear yellow any more.
4) Softening: adding alkaline hydrogen peroxide (10% H) into the cleaned root sample202) And (4) standing at room temperature for 20-30 min or until the root sample is softened.
5) And (3) cleaning again: removing alkaline H 202And rinsing with clear water for several times.
6) Dyeing: adding 1% HCl solution, soaking for 5min, and removing acid solution. Trypan blue dye solution (10mL 85% lactic acid 10mL phenol, 10mL glycerol, 10mL pure water, 15mg Trpanblne) is added to stain the mixture in a water bath at 90 ℃ for 20-30 min or overnight at room temperature.
7) And (3) decoloring: and after dyeing, soaking the root sample in lactic acid glycerol or pure glycerol for decoloring for 12-24 h.
8) Tabletting: the 50% glycerol is used as floating agent for flaking, and microscopic examination is carried out.
4. Results
The separated strains are primarily screened by a plate confronting method, and the strain FLR13 separated from the root of the pink blue has stronger bacteriostatic activity on 3 blueberry germs. The strain FLR13 is inoculated to blueberry seedlings under aseptic condition for co-culture for 3 months, root systems are collected, and after dyeing, the typical microscler nucleus structure of DSE is observed in cells of blueberry root epidermis and cortex infected by FLR13 (figure 1B), and the trace of infection is not found in the root system cells of the missed seedlings (figure 1A).
Example 2Morphological and molecular biological characterization of FLR13
1. Morphological identification FLR13 bacterial cakes with the diameter of 5mm are placed on a PDA plate and cultured at the constant temperature of 26 ℃ for two weeks. The strain grows slowly, is cultured for two weeks, has the colony diameter of 22.4 +/-2.2 cm, is white and does not produce spores (figure 2A). Morphological characteristics of the mycelia were observed under a microscope, and FLR13 mycelia were septate mycelia, and when the mycelia grew to a certain stage, they produced protrusions, invaded adjacent mycelia, and produced hypha fusion, increased cell contents, and thickened mycelia (FIG. 2B). Meanwhile, hyphae intertwine into rope-like hyphae, and cell fusion occurs around the rope-like hyphae, so that hyphae knot increases and thickens (fig. 2C).
2. Molecular biological identification
The genomic DNA of the strain was extracted using the instructions of the UNIQ-10 column type fungal genome extraction kit (Biotechnology engineering, Shanghai, Ltd.). Primers were performed using ITS 1:
5'-ACACAAGCTTTCCGTAGGTGAACCTGCGG-3'/ITS4：
5'-ACACGGATCCTCCTCCGCTTATTGATATGC-3' and 5.8S-R: 5'-TCGATGAAGAACGCAGC-3'/LR-5: 5'-ATCCTGAGGGAAACTTC3' to amplify ITS and 28s nrDNA gene sequences, respectively.
The PCR reaction system is as follows: phusion Master Mix (2X) 15. mu.L, forward and reverse primers (5. mu.M) 2. mu. L, gDNA (10 ng/. mu.L) 2. mu.L (10-20 ng) each, and water was added to a total volume of 30. mu.L. PCR reaction procedure: pre-denaturation at 94 ℃ for 1 min; denaturation at 94 ℃ for 30sec, annealing at 50 ℃ for 45sec, extension at 72 ℃ for 30sec, 30 cycles; preserving at 72 deg.C for 10min and 4 deg.C. The PCR product was subjected to electrophoresis on a 1.5% agarose gel to determine the sample quality. Purification and sequencing of the PCR products were performed by Shanghai bioengineering.
Obtaining sequences, comparing the sequences in NCBI/Genbank, downloading model strains, performing multi-sequence splicing by sequence matrix software, using MEGA7.0 software for the associated genes by adopting a Maximum Likelihood Method (ML), setting a Bootstrap value as 1000, constructing a phylogenetic tree, using Rhizodermea Veluwensis CBS 110605 as an outer group, and analyzing and comparing the genetic relationship among strains.
BLASTN alignment shows that the 28s nrDNA sequence has 99.52% homology to P.rhizophila (KR859033) and the ITS sequence has 99.2% homology to P.rhizophila (MN 131303). It was subjected to joint phylogenetic tree analysis with the sequence of the model strain downloaded from Genbank. FLR13 strain and p. rhizophila model strains CBS110602, CBS110604, CBS109839 and CBS110609 clustered in one branch, but formed distinct independent subgroups (fig. 3), indicating that FLR13 strain belongs to the sessile fungus, Pezicula, a new species. From morphological characteristics and phylogenetic analyses, this strain was identified and named Pezicula sp.
Example 3Biological effects of FLR13 inoculation on potted blueberry seedlings
Preparation of FLR13 fermentation broth
The FLR13 was plated on PDA plates for 2 weeks and inoculated into 250ml of Erlenmeyer flasks, and 70ml of liquid PDA medium was added to 2 cakes (7mm in diameter) per flask at 25 ℃ at 150rpm/min for 2 weeks.
2. Inoculation experiment
Domesticated and cultured 3-month blueberry pot culture tissue culture seedlings are ground into uniform slurry by a sterilized mortar through FLR13 zymogen suspension, root dipping treatment is carried out, sterile water is used as a control, and then the blueberry pot culture tissue culture seedlings are planted in a sterilized culture medium (humus soil: moss: pine needle: 1: 1), 10 plants are treated every time, and 3 times of treatment are repeated. Culturing at 23 deg.C in artificial climatic chamber, and periodically pouring sterile water. Culturing for 3 months, and measuring various physiological indexes of blueberry seedling such as plant height, stem thickness, root length, leaf width and the like. The experimental data were analyzed using SPSS16.0 statistical software.
3. Results
The strain FLR13 is inoculated to blueberry seedlings under aseptic condition for co-culture for 3 months, and the growth vigor of the blueberry seedlings inoculated with the FLR13 is found to be obviously better than that of a control, mainly manifested by increased plant height, obviously increased leaf area and leaf thickness, thick and strong stems, increased total weight and no obvious change in root length (figure 4A); the roots of the blueberries inoculated with FLR13 were darkened and dark brown (FIG. 4B). The data were analyzed using SPSS statistical software to find: the plant height, stem thickness, leaf width and total fresh weight of the control group and the treatment group are obviously different (P is less than 0.05).
Wherein, the overground part growth is improved by 24.13%, the stem thickness is improved by 43.32%, the leaf area is increased by 88.10%, and the total fresh weight is improved by 54.19% (fig. 5).
Example 4FLR13 experiment for antagonistic effect on blueberry disease germs
Culturing FLR13 strain on PDA culture medium for two weeks, collecting two bacterial cakes (7mm), adding 70ml liquid PDA culture medium, culturing at 25 deg.C for 150r/min, and shaking for two weeks to obtain fermentation broth. Centrifuging the fermentation liquid at 5000rpm for 20min, and filtering with 0.22 μm filter membrane to obtain filtrate supernatant. Preparing a drug-containing flat plate by taking 1mL of the filtered supernatant and 9mL of PDA culture medium cooled to 45-50 ℃, respectively inoculating 3 blueberry pathogenic bacteria cakes (7mm) in the center of the drug plate, culturing for 5 days at 25 ℃, measuring the diameters of bacterial colonies of the pathogenic bacteria, and setting a blank control.
The results show that the bacteriostatic rates of the strain FLR13 on blueberry botrytis cinerea, blueberry phomopsis stem canker and blueberry intercropping stem canker are 82% (fig. 6A, 6B), 91% (fig. 6C, 6D) and 99% (fig. 6E, 6F) respectively through activity determination of fermentation filtrate, and the strain can remarkably inhibit the growth of blueberry botrytis cinerea, blueberry stem canker and blueberry intercropping stem canker after being treated by the FLR 13.
Example 5Protection of blueberry fruit by FLR13Fresh effect
The blueberry fruits are cleaned, and soaked in the FLR13 filtrate for 24 hours to be soaked in clear water to be used as a control group (CK). Naturally drying, placing into a fresh-keeping bag, sealing, culturing at 22 deg.C under constant temperature and humidity, and detecting the index at intervals of 2 d. Each treatment had 30 fruits, 3 replicates.
The results show (fig. 7) that the decay rate of the control group rose significantly and the rise of the fermentation filtrate group was slower throughout the storage period. After 9 days of storage, the decay rate of the fermentation filtrate group was 24.4%, which was lower than 52.2% of the control group. After 16 days of storage, the incidence of disease was 100% in the control group, while the decay rate in the fermentation filtrate group was 73.3%. The FLR13 fermentation filtrate has good fresh-keeping effect. As proved by previous experiments, the fermentation filtrate of the FLR13 has good broad-spectrum antibacterial activity and also has good antibacterial effect on microorganisms of blueberry gray mold causing blueberry rot, so that the occurrence of blueberry rot is delayed.
Example 6Effect of FLR13 on promoting germination and root growth of tomato seeds
Sterilizing the surfaces of tomato seeds (75% alcohol for 1min, 1.5% sodium hypochlorite for 15min, and washing with sterile water for 6-7 times), naturally drying, soaking the seeds in 100-fold and 1000-fold diluted FLR13 original filtrate for 8h, respectively, soaking the seeds in distilled water for comparison, culturing in a 12D/12L artificial climate box at 25 ℃ for 10D, counting the germination rate of the tomatoes, and measuring the length of the main roots of the tomato seedlings. 30 seeds were treated each, 3 replicates.
The results show that FLR13 filtrate has significant effect on both germination and root growth of tomato seeds (fig. 8), (fig. 9).
The germination of tomato seeds is improved by 28.75% by the original FLR13 filtrate compared with that of a Control (CK); after being diluted by 100 times, the FLR13 filtrate has the best production effect on the tomato root length, and the yield is increased by 76.92%.
The root length of the tomato soaked in the FLR13 original filtrate is increased by 40.38%; after 1000-fold dilution of FLR13 filtrate, tomato root length increased 51.92% (fig. 10).
Example 7FLR13 antagonistic Sclerotinia sclerotiorum effect experiment
Screening antagonistic strains by adopting a plate confronting method, punching bacterial cakes on the edges of sclerotium pathogenic bacteria of the rape to be tested and FLR13 bacterial colonies by using a puncher with the inner diameter of 6mm, putting the bacterial cakes into a culture dish with the diameter of 9cm, keeping the distance between the two bacterial cakes and the edge of the culture dish at 3cm, taking a plate inoculated with the pathogenic bacteria as a reference, placing the plate at 25 ℃ for constant-temperature culture for 10 days, and recording the width of an antagonistic zone of the pathogenic bacteria and the endophyte to be tested. The results show that the antagonistic bandwidth of Sclerotinia sclerotiorum and FLR13 is 19mm (FIG. 11), which indicates that FLR13 has significant inhibitory effect on Sclerotinia sclerotiorum.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> university of teacher's university of Anqing
<120> blueberry root deep color endophytic fungi and application thereof
<130> 2019
<141> 2020-03-02
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 836
<212> DNA
<213> Pezicula sp. FLR13
<400> 1
ctcagtaacg gcgagtgaag cggtaacagc tcaaatttga aatctgggtc tttcaggctc 60
cgagttgtaa tttgtagaag atgctttggg tgcggccccg gtctaagttc cttggaacag 120
gacgtcatag agggtgagaa tcccgtatgt gatcggtggc ctgcgcccgt gtaaagctct 180
ttcgacgagt cgggttgttt gggaatgcag ctctaaatgg gtggtaaatt tcatctaaag 240
ctaaatattg gccagagacc gatagcgcac aagtagagtg atcgaaagat gaaaaggact 300
ttggaaagag agttaaacag tacgtgaaat tgttgaaagg gaagcgcttg caaccagact 360
tgcgcggggt cgatcatccg gggttctctc cggtgcactc ggcctcgctc aggccagcat 420
cggtttcggt ggttggataa aggccttggg aatgtgactc ctctcgggga gtgttatagc 480
cctcggtgca atgcagccta ctgggaccga ggaccgcgct tcggctagga tgctggcgta 540
atggttgtaa gcgacccgtc ttgaaacacg gaccaaggag tctaacatct atgcgagtgt 600
ttgggtgtca aacccatacg cgtaatgaaa gtgaacggag gtgagaaccc ttaagggtgc 660
atcatcgacc gatcctgatg tcttcggatg gatttgagta ggagcatagc tgttgggacc 720
cgaaagatgg tgaactatgc ctgaataggg tgaagccaga ggaaactctg gtggaggctc 780
gcagcggttc tgacgtgcaa atcgatcgtc aaatttgggt ataggggcga aagact 836
<210> 2
<211> 499
<212> DNA
<213> Pezicula sp. FLR13 ITS
<400> 2
tgccccctgg gtagacctcc caccctgtgt cgttgtacct ttgttgcttt ggcgggccgc 60
ggggccccgg ccctgcccct ggctccggct agggcgcgcc cgccagagga cctcaaaacc 120
tgaatgttag tgtcgtctga gtgctatata atagttaaaa ctttcaacaa cggatctctt 180
ggttctggca tcgatgaaga acgcagcgaa atgcgataag taatgtgaat tgcagaattc 240
agtgaatcat cgaatctttg aacgcacatt gcgccccttg gtattccggg gggcatgcct 300
gttcgagcgt cattacaacc ctcaagctct gcttggtctt gggcgtcacc ggtcccggtg 360
tgccttaaaa tcagtggcgg cgccatctgg ctctaagcgt agtacatctt ctcgctacag 420
acgcccggtg gatgctggcc agcaaccccc atcttacaag gttgacctcg gatcaggtag 480
ggatacccgc tgaacttaa 499
Claims (8)
1. The endophytic fungi for the blueberry with the dark root is characterized by specifically comprising a strain with the endophytic fungi for the blueberry with the dark rootAmanita sessilifolia (A. juss.), (B. justicita)Peziculasp.) FLR13 with accession number GDMCC number 60777.
2. The use of the dark root septate endophytic fungus of blueberry of claim 1 for promoting the growth of blueberry.
3. The use of the dark root spaced endophyte of blueberry as claimed in claim 1 in antagonizing pathogenic bacteria of blueberry.
4. The use as claimed in claim 3, wherein the pathogenic bacteria of blueberry is Botrytis cinerea, Bull canker of blueberry Interval husk or Bull phomopsis longissima.
5. The use of the dark root spaced endophytic fungus of blueberry of claim 1 for promoting preservation of blueberry fruit.
6. The use of the dark brown root spaced endophytic fungus of blueberry of claim 1 for antagonism of sclerotinia sclerotiorum.
7. Use of the dark root compartmentalized endophyte of the blueberry of claim 1 to promote germination of tomato seeds.
8. The use of the dark root septate endophyte of claim 1 to promote growth of tomato roots.
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