CN116555041A - Strawberry biocontrol endophytic fungus JSNL-B124 and application thereof - Google Patents
Strawberry biocontrol endophytic fungus JSNL-B124 and application thereof Download PDFInfo
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- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G13/00—Protecting plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- 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
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses a biological control bacterium for gray mold and anthracnose of strawberry and application thereof, wherein the biological control bacterium is named as Talaromyces wuschesis JSDL-B124, and the preservation unit is: china general microbiological culture Collection center (CGMCC) with a collection number of CGMCC NO.40436. The invention also discloses application of the composition in preventing and controlling the gray mold of the strawberry caused by B.cinerea and the anthracnose of the strawberry caused by C.siamense. Compared with a control, the biological control bacterium Talaromyces wusches JSNL-B124 provided by the invention has the advantages that the bacterial colony of the experimental group B.cinerea strain is obviously smaller, the inhibition rate reaches 74.15%, the bacterial colony of the experimental group C.siamese strain is obviously smaller, the inhibition rate reaches 50.42%, and the hypha growth of the B.cinerea strain and the C.siamese strain is inhibited.
Description
Technical Field
The invention belongs to the technical field of strawberry biocontrol, and relates to a strawberry biocontrol endophytic fungus JSNL-B124 and application thereof.
Background
The strawberries are delicious and juicy, are rich in nutrition and have important economic value. In recent years, the strawberry industry in China develops rapidly, and the planting area and the yield are the first in the world. However, as the area of strawberry cultivation increases, especially in the application of facility strawberries, fungal diseases are increasingly occurring. Wherein, the gray mold of the strawberries is one of important diseases affecting the yield and quality of the strawberries. The disease is caused by Botrytis cinerea, and mainly infects various plants such as strawberries, grapes, cucumbers, tomatoes, eggplants, peppers and the like, and has a wide host range. The gray mold has wide distribution, high expansion speed and serious hazard, brings great loss to the yield and quality of the fruits and vegetables worldwide, and ranks the second in ten plant diseases worldwide. Gray mold is a fungal disease which is most serious in damage after strawberry flowering, and the disease mainly infects fruits, also infects leaves, fruit stalks, calyx, petals and petioles, causes rot of flowers and fruits, and the disease fruit rate of infected varieties is generally 30% -60%, even in serious cases, the disease fruit rate is dead, and the yield and quality of strawberries are greatly influenced.
Strawberry anthracnose is an important disease in strawberry producing areas worldwide, can be harmful in the whole growth period of strawberries, and mainly occurs in the seedling raising period and the initial planting period of strawberries. Strawberry anthracnose is a major disease on strawberries in recent years. The disease is caused by Colletotrichum spp, which can infect not only strawberries but also grapes, apples, peppers, mangoes, cocoa, bananas, papaya, citrus, peaches, pears, dendrobium officinale and other plants. When strawberry anthracnose occurs, strawberry leaves, petioles, supporting leaves, stolons, petals and fruits can be damaged. The strawberry has high disease risk and high destructive power, generally causes 25% -30% yield reduction of strawberries, and has 80% serious loss even destructive loss. Strawberry anthracnose is the third major disease restricting the strawberry industry in China.
At present, except for planting some high-resistance varieties, the chemical control is mainly carried out on the gray mold and anthracnose of the strawberries, and although the chemical agents can control the gray mold and the anthracnose of the strawberries to a certain extent, a great deal of chemical agents are used to bring great threat to ecological environment and food safety on one hand, on the other hand, the drug resistance of pathogenic bacteria is continuously enhanced, the phenomenon of ineffective pesticide control occurs, and the sustainable development of green agriculture in China is not facilitated. Therefore, finding an environmentally friendly and effective gray mold and anthracnose control measure is a problem to be solved urgently at present. The endophytic fungi (endophyte fungi) are different from pathogenic bacteria, the pathogenic bacteria can cause diseases and reduce the adaptability of host plants, and the endophytic fungi inhabit in asymptomatic host plant tissues, are one of key factors affecting plant growth, nutrition and health, can help plants obtain nutrients, inhibit plant pathogenic bacteria and resist biotic and abiotic stress. Since endophytic fungi are derived from plants, the endophytic fungi are safe to the plants, are environment-friendly and are not easy to generate drug resistance, and the use of the microorganisms for controlling plant diseases is beneficial to maintaining ecological balance. Therefore, endophytic fungi are a source of novel compounds that promote organic agriculture.
In summary, the prior art lacks a green and efficient probiotic for preventing gray mold and anthracnose of strawberries.
Disclosure of Invention
Aiming at the problems, the invention provides a strawberry biocontrol endophytic fungus Talaromyces wushanicus JSNL-B124 and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides a biocontrol bacterium for controlling strawberry gray mold caused by Botrytis cinerea and strawberry anthracnose caused by anthracnose (Colletotrichum siamense), wherein the biocontrol bacterium is named Talaromyces wushanicus JSNL-B124 and is separated from healthy strawberry stems of white rabbits in period of Zhenjiang city.
Talaromyces wushanicus JSNL-B124, classified and named Talaromyces wushanicus, has been registered and preserved in China general microbiological culture Collection center (CGMCC) at 12/8 of 2022, with the preservation number of CGMCC NO.40436 and the preservation address of North Chen Xiyu No. 1/3 in the Chaoyang area of Beijing city.
In a second aspect, the invention provides an engineering bacterium comprising the nucleic acid fragment of Talaromyces wushanicus JSNL-B124 of the first aspect of the invention.
Further, the base sequence of the nucleic acid fragment includes SEQ ID NO.1 or SEQ ID NO.2.SEQ ID NO.1:
GGCTTTCGAGTGCGGGCCTCGTGGCCCACCTCCCACCCTTGTCTCTAT
ACACCCGTTGCTTTGGCGGGCCCACCGGGGCCACCTGGTCGCCGGGG
GACGTTCGTCCCCGGGCCCGCGCCCGCCGAAGCGCTCTGTGAACCCT
GATGAAGATGGGCTGTCTGAGTACTATGAAAATTGTCAAAACTTTCAG
CAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCG
ATAAGTAATGTGAATTGCAGAATTCCGTGAATCATCGAATCTTTGAAC
GCACATTGCGCCCCCTGGCATTCCGGGGGGCATGCCTGTCCGAGCGTC
ATTTCTGCCCTCAAGCACGGCTTGTGTGTTGGGTGTGGTCCCCCCGGG
GACCTGCCCGAAAGGCAGCGGCGACGTCCGTCTGGTCCTCGAGCGTA
TGGGGCTCTGTCACTCGCTCGGGAAGGACCTGCGGGGGTTGGTCACC
ACCATATTTTACCACGGATGACCTCGGATCAGGTAGGAGTTACCCGCT
GAACTTAAGCATATCAAAAAGCGGGAGGAATTTTTTATTCTSEQ ID NO.2:
ACAGTACGCCGGACTGACCGAAACAAAGTTGTCGGGACGGAAGAGC
TGACCAAAGGGACCAGCGCGGACGGCGTCCATGGTACCGGGCTCCAA
GTCGACGAGGACAGCACGGGGGACGTATTTGTTGCCGGAGGCCTATA
CCATTGTGAGTTTGTTTCGTCATATGGTGGATTGGTTCGACGCACCTCG
TTAAAGTAGACGTTCATACGCTCCAACTGGAGGTCGGAGGAGCCATT
GTAGCTGTTGACGATCAGATATTGTCGGATTGTAGCTGGATTCGAGGT
CGTGAATACTTACACACCAGAGCCATCGAGACCGTGCTCAGCAGAGA
TGATTTGCCTGAAAAAGTCAGCGTGTTGTCGCGACAATTGATAGAAG
GGTTGGGTCGAGTCAAACTCACCAGAAAGCAGCACCAATG
in a third aspect, the invention provides a biocontrol microbial inoculum capable of preventing and controlling strawberry gray mold caused by strawberry gray mold bacteria (b.cinerea) and strawberry anthracnose caused by strawberry anthracnose bacteria (c.siamese), wherein the biocontrol microbial inoculum comprises one or more of the following active ingredients:
a1 Talaromyces wushanicus JSNL-B124 of the first aspect of the invention;
a2 One or more of mycelium, spores or secondary metabolites of Talaromyces wushanicus JSNL-B124 of the first aspect of the invention;
a3 Any one of the engineering bacteria of the second aspect of the invention.
Further, the mycelium of Talaromyces wushanicus JSNL-B124 is prepared by inoculating Talaromyces wushanicus JSNL-B124 into PDA culture medium for culturing.
Further, the biocontrol microbial inoculum comprises biocontrol chemical fertilizers or biocontrol pesticides.
In a fourth aspect, the invention also provides a preparation method of the biocontrol microbial agent in the third aspect, which is characterized by comprising the following steps:
s1, preparing a sterile matrix;
s2, adding mycelium, spores or secondary metabolites of Talaromyces wushanicus JSNL-B124 into the substrate of the S1;
and S3, culturing the substrate obtained in the step S2 for a plurality of days in a sealing way to obtain the biocontrol microbial agent.
Further, the method comprises the following steps:
a1: the substrate was placed in sterile triangular flasks (250 mL) with vent holes, each flask was filled with 200mL of substrate, and sterilized at 121℃for 20min.
A2: talaromyces wushanicus JSNL-B124 was inoculated into a 9cm diameter PDA medium containing 15mL and incubated at 25℃for 3-5d in the absence of light.
A3: cutting mycelia with length of 3mm×3mm from colony edge of Talaromyces wushanicus JSNL-B124, transferring into liquid matrix under aseptic condition, sealing, and culturing at 25deg.C and 160rpm for 7d to obtain Talaromyces wushanicus JSNL-B124 liquid fermentation inoculum.
In a fifth aspect, the invention also provides the application of the biocontrol bacterium, the engineering bacterium or the biocontrol microbial inoculum in preventing and controlling strawberry gray mold caused by strawberry gray mold bacteria (B.cinerea) and strawberry anthracnose caused by strawberry anthracnose bacteria (C.siamense).
In a sixth aspect, the invention also provides a method for controlling gray mold and anthracnose of strawberry, which comprises applying the biocontrol bacteria, engineering bacteria or biocontrol microbial agents provided by the invention to a plant growth environment.
Further, the plant growing environment is plant seedling root or leaf.
Compared with the prior art, the biocontrol bacterium Talaromyces wushanicus JSNL-B124 provided by the invention can obviously reduce the disease severity of the gray mold and the anthracnose of the strawberries, the average biocontrol effect reaches 63.82%, and the growth indexes such as plant height, root length and the like are obviously improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.
FIG. 1 is a graph showing the effect of Talaromyces wushanicus JSNL-B124 on inhibiting the growth of B.cinerea (left) and C.siamese (right) hyphae on a plate in example 1;
FIG. 2 is the effect of Talaromyces wushanicus JSNL-B124 on strawberry plants in example 2;
FIG. 3 is a graph showing the effect of Talaromyces wushanicus JSNL-B124 on B.cinerea-induced gray mold in strawberry in example 3;
FIG. 4 is the effect of Talaromyces wushanicus JSNL-B124 on C.siamese-induced strawberry anthracnose in example 3.
Detailed Description
Embodiments of the technical scheme of the present invention will be described in detail below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and thus are merely examples, which should not be construed as limiting the scope of the present invention. It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.
As used herein, the term "biocontrol bacteria" refers to beneficial microorganisms, primarily bacteria, fungi and actinomycetes, which control plant diseases.
As described in the present invention, the term "engineering bacteria" refers to novel microorganisms processed by modern biological engineering technology, and has the characteristics of multifunction, high efficiency, strong adaptability, etc.
As used herein, the term "gray mold of strawberry (b.cinerea)" is classified as: fungi kingdom (Fungi), ascomycota (Ascomycota), glossomycetes (leotomycete), molluscles (Helotiales), sclerotiniaceae (Sclerotiniaceae), sporotrichum (botryotonia). Is mainly distributed in the united states, sweden, germany, france, china, brazil, japan, korea, argentina, india, chile, etc.
As used herein, the term "strawberry anthracnose" (c.siamese) is classified as: mycota (eumycin), phylum half (deuteromyctina), class of cavitys (Coelomycetes), order of Colletotrichum (melanocardiales), family of collectaceae (melanoceae), genus anthrax (Colletotrichum). Is mainly distributed in the united states, canada, australia, japan, uk, china, belgium, germany, india, vietnam, korea, brazil, panama, mexico, russia, israel, etc.
Example 1 screening of bacteria Talaromyces wushanicus JSNL-B124
1. Isolation of potential biocontrol bacteria
1.1, obtaining a test material: healthy strawberry plants with good growth vigor are selected from the field (Jiangsu nongbo garden), and stem tissues are taken.
1.2, separation and culture of endophytic fungi in strawberry stems: cutting stem tissues of strawberries, uniformly mixing, weighing 2g, and carrying out surface disinfection. To ensure removal of all the periphyton, 100mL of sterile water and 2 drops of tween 20 were added, and the mixture was shaken at 220rpm for 20min at 25 ℃ for 20s,70% (v/v) ethanol for 30s,2.5% (v/v) sodium hypochlorite solution for 2min, and finally rinsed 3-4 times with sterile water, and the surface water was blotted with sterile absorbent paper. The stems were further cut into smaller pieces (0.25 cm) under sterile conditions, and 5 pieces of each sample were randomly selected and placed on PDA plate medium containing ampicillin (50 mg/L) and rifampicin (50 mg/L). The dishes were sealed and incubated at 25℃for 1-2d in the dark. When mycelium emerges from the stem tissue, small pieces of medium grown at the edge of the medium are carefully transferred to a new PDA plate along with the mycelium.
2. Screening for antagonistic bacteria by plate counter method
Plates containing 15mL PDA medium with a diameter of 9cm were symmetrically inoculated with 3-5d of B.cinerea and C.siamese bacterial cakes (diameter 5 mm) and endophytic fungi cakes to be tested, respectively, at a distance of 2cm from the edge, using the PDA plate counter method, and plates inoculated with only B.cinerea and C.siamese bacterial cakes were used as controls. The plates were incubated at 25℃in the dark. After 5d, B.cinerea and C.siamese colony radii were measured and inhibition rates calculated. The inhibition rate is calculated according to the formula: inhibition ratio = (control colony radius-treatment colony radius)/control colony radius x 100%.
Finally, 1 strain with good antagonistic activity to both B.cinerea and C.siamense on a flat plate is obtained through screening, and the strain is particularly characterized in that the growth of pathogenic bacteria hypha is strongly inhibited, and the number is JSNL-B124.
As shown in FIG. 1, the left graph shows that JSNL-B124 is opposite to the B.cinerea strain, the colony of the B.cinerea strain of the experimental group is obviously smaller than that of the control, the inhibition rate reaches 74.15%, the right graph shows that JSNL-B124 is opposite to the C.siamese strain, the colony of the C.siamese strain of the experimental group is obviously smaller than that of the control, the inhibition rate reaches 50.42%, and the hypha growth of the B.cinerea strain and the C.siamese strain are inhibited.
3. Molecular biological identification of the resulting strains
3.1 genomic DNA of strain JSNL-B124 was extracted using TIANGEN kit (DP 320-03).
3.2 amplification of ITS gene and BenA gene of genomic DNA, respectively. The reaction volume of 30. Mu.L was used for DNA amplification, containing 15. Mu.L of 2X EasyTaq PCR SuperMix (+dyne) primer F/R each 1. Mu.L (10. Mu.M), 2. Mu.L of template DNA and 11. Mu.L of ddH2O, and the PCR amplification procedure was as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 54℃for 45s, elongation at 72℃for 60s for 35 cycles; finally, the extension is carried out for 10min at 72 ℃.
3.3, after the amplified PCR product is subjected to 1% agarose gel electrophoresis, observing under an ultraviolet lamp, and sending the PCR product with a target band to Nanjing qing department biotechnology Co., ltd for sequencing, wherein the sequencing result is as follows: the ITS gene sequence of the strain JSNL-B124 obtained by screening in the step 2 is SEQ ID NO.1, and the BenA gene sequence is SEQ ID NO.2.
3.4, two sequences of SEQ ID NO.1 and SEQ ID NO.2 were aligned on NCBI database website. The comparison results are shown in Table 1. From the results of Table 1, it was estimated that the biocontrol strain JSNL-B124 of the present invention was Talaromyces wushanicus and was named Talaromyces wushanicus JSNL-B124.
Table 1SEQ ID NO.1 and SEQ ID NO.2 sequence alignment
Example 2 effects of Talaromyces wushanicus JSNL-B124 on strawberry self-growth
1. The strain JSNL-B124 was inoculated into 9cm diameter dishes (containing 15ml PDA medium) and incubated at 25℃for 7d in the absence of light.
2. A pot having a diameter of 12cm was selected, and the medium of Talaromyces wushanicus JSNL-B124 for 7d culture was cut into plates of 0.5 cm. Times.0.5 cm. Test set 2, control: 2 PDA flat plates with the length of 0.5cm multiplied by 0.5cm are added into each pot; the experimental group is: 2 flat plates of Talaromyces wushanicus JSNL-B124 of 0.5cm by 0.5cm were added to each pot. Mixing various flat blocks with sterile water and sterilized vermiculite, adding into the pot to 1/3 position, transplanting 3 healthy strawberry seedlings (with the variety of sweet), and covering with a layer of mixture of thin mycelium blocks and vermiculite. 3 replicates were set for each treatment.
3. Each group of strawberries was irrigated with sterile water and after 30d the root length, plant height and fresh weight of the strawberries were counted.
The results are shown in FIG. 2 and Table 2.
TABLE 2 statistical results of strawberry plant height, root length, root weight and fresh weight
Wherein, the numerical expression modes of plant height, root length, root weight and fresh weight in the table are as follows: mean ± standard error.
From the data in fig. 2 and table 2, the plant height, root length, fresh weight and the like of the experimental group are obviously superior to those of the control group, and Talaromyces wushanicus JSNL-B124 can promote the growth of strawberry plants.
Example 3 effects of Talaromyces wushanicus JSNL-B124 on strawberry gray mold caused by B.cinerea and strawberry anthracnose caused by C.siamense
1. Effect of Strain JSNL-B124 on strawberry gray mold caused by B.cinerea
1.1, talaromyces wushanicus JSNL-B124 and B.cinerea were inoculated into 9cm diameter dishes containing 15mLPDA medium, and incubated at 25℃in a dark place for 5d.
1.2, a pot having a diameter of 12cm was selected, and the medium of Talaromyces wushanicus JSNL-B124 cultured for 5 days was cut into flat plates of 0.5 cm. Times.0.5 cm. Test set 2, control: 2 PDA flat plates with the length of 0.5cm multiplied by 0.5cm are added into each pot; the experimental group is: 2 flat plates of JSNL-B124 of 0.5cm by 0.5cm were added to each bowl. Mixing various flat blocks with sterile water and sterilized vermiculite, adding into the pot to 1/3 position, transplanting 3 healthy strawberry seedlings (variety of purple gold 21) with consistent growth vigor into each pot, and covering a layer of mixture of thin mycelium blocks and vermiculite. 3 replicates were set.
1.3, repeatedly washing the surface of the B.cinerea PDA plate growing for 5 days by using sterile water, so that the conidium is uniformly scattered in the water. Filtering the obtained conidium suspension with double-layer gauze, counting with a hemocytometer under an optical microscope, calculating the concentration of B.cinerea spore suspension, and adjusting to 1.0X10 6 The sample was kept at one/mL.
1.4, irrigating each group of strawberries every other day with sterile water, spraying and inoculating 2 drops of tween 20 into the B.cinerea spore suspension obtained in 1.3 to each treated plant by using a small sprayer after 7d, enabling the leaves and petioles to be full of water drops but not drop, and covering a plastic film for airtight moisture preservation after inoculation. The incidence was recorded 1 week after inoculation, and the incidence and disease index of the leaves were calculated.
Wherein, leaf disease progression grading criteria: 0: no disease spots; 1: the area of the disease spots accounts for less than 5% of the area of the whole leaf (fruit); 2: the area of the disease spots accounts for 6% -15% of the area of the whole leaf (fruit); 3: the area of the disease spots accounts for 16% -30% of the area of the whole leaf (fruit); 4: the area of the disease spots accounts for 31% -50% of the area of the whole leaf (fruit); 5: the area of the disease spots accounts for more than 50% of the area of the whole leaf (fruit).
Incidence (%) = number of diseased leaves (petiole, stolons, flowers, fruits) per total leaves (petiole, stolons, flowers, fruits) x 100; disease index= [ Σ (number of onset at each stage×number of disease stages)/(number of highest onset stages×total number of investigation) ]×100; control effect (%) = (control group disease index-experimental group disease index) ×100.
The results are shown in FIG. 3 and Table 3.
TABLE 3 statistical results of strawberry gray mold incidence, disease index and control effect
As can be seen from the graph 3 and the table 3, the biocontrol bacterium Talaromyces wushanicus JSNL-B124 can obviously reduce the disease severity of plant gray mold, and the biocontrol effect reaches 75.12%, which shows that Talaromyces wushanicus JSNL-B124 has the effect of preventing or controlling the strawberry gray mold caused by B.cinerea.
2. Effect of strain JSNL-B124 on strawberry anthracnose caused by c.siamense
2.1, talaromyces wushanicus JSNL-B124 and C.siamense were inoculated into 9cm diameter dishes containing 15mL of DA medium, and incubated at 25℃in the absence of light for 5d.
2.2, setting two groups of treatment transplanted strawberries by adopting the method of the step 1.2. The strawberry variety is mauve 21.
2.3 preparing a C.siamese spore suspension by the method of step 1.3, adjusting to 1.0X10 5 The sample was kept at one/mL.
2.4, irrigating each group of strawberries every other day with sterile water, spraying and inoculating 2 drops of tween 20 into each treated plant with the C.siamese spore suspension obtained in 2.3 by using a small sprayer after 14d, enabling the leaves and petioles to be full of water drops but not drop, and covering a plastic film after inoculation for airtight moisture preservation. After 1 week of inoculation, the onset of disease was recorded, and the incidence and index of disease of leaves, petioles and stolons were calculated.
Wherein, leaf disease progression grading criteria: 0: no disease spots; 1: brown spots with the size of the needle point are arranged on the edge of the leaf blade, or brown disease spots appear on the leaf blade, and the area of the disease spots accounts for less than 5 percent of the area of the leaf blade; 3: nearly circular or spindle-shaped gray brown lesions appear, and the area of the lesions accounts for 6% -10% of the area of the leaves; 5: black necrotic spots appear in the center of the disease spots, or local disease spots of the leaf are connected, and the area of the disease spots accounts for 11% -25% of the area of the leaf; 7: typical disease spots, enlarged black necrotic spots or dead leaf edges, and the disease spot area accounts for 26% -50% of the leaf area; 9: the area of the disease spots accounts for more than 50% of the area of the leaf, or the leaf is dead.
Stage number grading criteria for petioles and stolons: 0: no disease spots; 1: pale red and faded halos appear at the affected part, or reddish brown spots, and the length of the spots is less than 3.0mm;2, enlarging the disease spots to form fusiform depressions with blackish brown, wherein the length of the disease spots is 3-10.0mm;3: typical spindle-shaped lesions, with a length of 11-20.0 mm, encircling the petioles or stolons; 4: blackening of petiole or stolon parts, drying shrinkage, and necrosis of the whole petiole, wherein the length of a disease spot is more than 20.0 mm; 5: the rootstock is necrotic and the plant dies.
Incidence (%) = number of diseased leaves (petiole, stolons, flowers, fruits) per total leaves (petiole, stolons, flowers, fruits) x 100; disease index= [ Σ (number of onset at each stage×number of disease stages)/(number of highest onset stages×total number of investigation) ]×100; control effect (%) = (control group disease index-experimental group disease index) ×100.
The results are shown in Table 4.
TABLE 4 statistics of strawberry anthracnose morbidity, disease index and control effect
From fig. 4 and table 4, it can be known that the biocontrol bacterium Talaromyces wushanicus JSNL-B124 of the invention can obviously reduce the disease severity of plant anthracnose, and the average biocontrol effect reaches 52.52%, which indicates that Talaromyces wushanicus JSNL-B124 has the effect of preventing or controlling strawberry anthracnose caused by c.siamense.
The numerical values set forth in these examples do not limit the scope of the present invention unless specifically stated otherwise. In all examples shown and described herein, unless otherwise specified, any particular value is to be construed as exemplary only and not as limiting, and thus, other examples of exemplary embodiments may have different values.
Claims (9)
1. The biocontrol bacteria for the gray mold and the anthracnose of the strawberries are Talaromyces wushanicusJSNL-B124, and are characterized in that the biocontrol bacteria are as follows: china general microbiological culture Collection center (CGMCC) with a collection number of CGMCC NO.40436.
2. An engineered bacterium comprising the nucleic acid fragment of Talaromyces wushanicusJSNL-B124 of claim 1.
3. The engineering bacterium according to claim 2, wherein the sequence of the nucleic acid fragment is SEQ ID NO.1 or SEQ ID NO.2.
4. A biocontrol microbial inoculum capable of preventing and controlling strawberry gray mold caused by B.cinerea and strawberry anthracnose caused by C.siamese is characterized in that the biocontrol microbial inoculum comprises one or more of the following active ingredients:
a1 A Talaromyces wuschechikus JSNL-B124 according to claim 1;
a2 A) one or more of the hyphae, spores or secondary metabolites of Talaromyces wusches jsnl-B124 of claim 1;
a3 An engineered bacterium according to any one of claims 2 to 3.
5. The biocontrol microbial inoculum according to claim 4 wherein the mycelia of Talaromyces wushanicusJSNL-B124 are prepared by inoculating Talaromyces wuschesis JSDL-B124 to PDA culture medium.
6. The method for preparing the biocontrol microbial agent as claimed in any one of claims 4 to 5, which is characterized by comprising the following steps:
s1, preparing a sterile matrix;
s2, adding hyphae, spores or secondary metabolites of Talaromyces wusches JSNL-B124 into the substrate of the S1;
and S3, culturing the substrate obtained in the step S2 for a plurality of days in a sealing way to obtain the biocontrol microbial agent.
7. The use of the biocontrol bacterium of claim 1, the engineering bacterium of any one of claims 2-3 or the biocontrol bacterium agent of any one of claims 4-5 for controlling the gray mold of strawberry caused by b.cinerea and the anthracnose disease of strawberry caused by c.siamense.
8. The use of claim 7, wherein the strawberry disease comprises strawberry gray mold and strawberry anthracnose.
9. A method for controlling gray mold and anthracnose of strawberries, which is characterized in that the method comprises applying the biocontrol bacterium of claim 1, the engineering bacterium of any one of claims 2-3 or the biocontrol microbial agent of any one of claims 4-5 to a plant growth environment.
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