CN108085259B - Biocontrol endophytic fungi and application thereof in preventing and controlling gray mold of economic crops - Google Patents
Biocontrol endophytic fungi and application thereof in preventing and controlling gray mold of economic crops Download PDFInfo
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Abstract
The invention belongs to the technical field of crop disease control, and particularly relates to biocontrol endophytic fungi DA-1 and application thereof in gray mold control of cash crops. Specifically, the invention discloses a biocontrol plant endophytic fungus DA-1 which is Arcopius aureus and has a preservation number of CGMCC NO. 14156. The invention also discloses the application of the biocontrol endophytic fungi DA-1: inhibiting the growth of botrytis cinerea hyphae; is used for preventing and treating gray mold of cash crops.
Description
Technical Field
The invention belongs to the technical field of crop disease control, and particularly relates to biocontrol endophytic fungi DA-1 and application thereof in gray mold control of cash crops.
Background
Botrytis cinerea (Botrytis cinerea) caused by Botrytis cinerea is the second most important plant fungal disease in the world, and the annual cost for the disease control exceeds 1 billion of euros. The pathogenic bacteria can infect plants through various infection modes of hypha, conidium and sclerotium, and can quickly initiate soft rot of the plants, so that the organ of the plants is damaged and the function of the plants is lost. In the later period of the pathogen infection, a plurality of conidia which are clearly visible to the naked eyes can be generated on the surface of the plant, and can be transmitted to the adjacent surface of the plant for infection through wind power and water flow.
The method for preventing and treating the gray mold is still mainly chemical prevention and treatment, and has the advantages of rapidness, low cost and high efficiency. The main classes of the medicaments currently used are: seven major bactericides such as benzimidazoles, dicarboximides, anilinopyrimidines, pyrroles, amides, succinic dehydrogenase bactericides and the like. However, due to the frequent use of chemical pesticides and the characteristics of quick propagation genetic variation and high suitability of the botrytis cinerea, the botrytis cinerea quickly generates resistance, and the control effect of the bactericide is influenced. The biological control method can effectively solve and avoid the resistance problem of the bactericide, has high efficiency, no toxicity, no harm, no pollution and no drug resistance, meets the requirements of people on green food, and provides guarantee for the sustainable development of agriculture, so the research on the biological control of plant diseases is more and more focused.
The endophytic Fungi (endophytic Fungi) refers to a large class of Fungi living in plant living tissues without causing any direct and obvious plant diseases, and is closely related to host plants, so that the plants are used as habitats, and the endophytic Fungi have various biological effects on the host plants, such as growth promotion, disease prevention, endogenous nitrogen fixation and the like.
The endophyte of the plant has rich diversity, mainly comprises species diversity, host diversity, habitat diversity, form diversity and genetic diversity, and is an important treasury of a biocontrol strain; secondly, the endophytic fungi have stronger competitive advantage in the plant body when the disease outbreak occurs; finally, the endophytic fungi are from the plant body and are easier to plant compared with biocontrol strains separated from soil.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a biocontrol plant endophytic fungus DA-1 and an application thereof, wherein the biocontrol plant endophytic fungus DA-1 has a good inhibition effect on the hypha growth of botrytis cinerea and has a good control effect on gray mold of field economic crops.
In order to solve the technical problem, the invention provides a biocontrol plant endophytic fungus DA-1 which is Arcopius aureus and has a preservation number of CGMCC NO. 14156.
The invention also provides the application of the biocontrol endophytic fungi DA-1: inhibiting the growth of botrytis cinerea hyphae.
As an improvement of the use of the biocontrol endophytic fungi DA-1 of the invention: is used for preventing and treating gray mold of cash crops.
As a further improvement of the use of the biocontrol endophytic fungi DA-1 of the invention: the cash crop is strawberry.
As a further improvement of the use of the biocontrol endophytic fungi DA-1 of the invention: the method comprises the following steps:
1) preparing biocontrol plant endophytic fungi DA-1 fermentation liquor (the fermentation liquor contains antibacterial substances);
2) separating and purifying the fermentation liquor (namely, separating and purifying the antibacterial substances in the fermentation liquor) to obtain purified fermentation liquor;
3) and diluting the purified fermentation liquor and applying the diluted fermentation liquor to the cash crops.
As a further improvement of the use of the biocontrol endophytic fungi DA-1 of the invention, the step 1) is:
activating biocontrol plant endophytic fungi DA-1 on a PDA culture medium, then beating a fungus cake with the diameter of 0.5cm, cutting the fungus cake in a shape like a Chinese character 'jing', and taking the fungus cake as a strain;
secondly, inoculating the strain into a PD liquid culture medium, and culturing for 4-12 days in a constant-temperature shaking table with the rotating speed of 150rpm and the temperature of 28 ℃ to obtain fermentation liquor.
As a further improvement of the use of the biocontrol endophytic fungi DA-1 of the invention: in the second step, 2 strains are inoculated in 100mL PD liquid culture medium.
As a further improvement of the use of the biocontrol endophytic fungi DA-1 of the invention, the step 2) is: extracting and separating the fermentation liquor from ethyl acetate, concentrating, and performing chromatography by using a silica gel column to obtain purified fermentation liquor.
The purified fermentation liquor contains more purified antibacterial substances, and can be prepared into a biocontrol microbial inoculum.
As a further improvement of the use of the biocontrol endophytic fungi DA-1 of the invention: the chromatography was carried out with methanol as the mobile phase.
The biocontrol plant endophytic fungi DA-1 provided by the invention is separated from the interior of a 'crystal product No. 1' dendrobium officinale plant and is obtained by screening 122 endophytic fungi.
The biocontrol endophytic fungus DA-1 is preserved as Arcopius aureus, and the preservation unit is as follows: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.14156, preservation time 2017, 9 months and 7 days.
The strain is cultured on PDA to observe dark yellow hypha and have a large amount of red pigment precipitate, then a large quantum cyst appears on the surface of a bacterial colony, and a large quantum cyst filament is wound outside the cyst and is long, the tail end of the cyst filament is bent, and a large amount of brown fusiform ascospores are formed.
The bacteriostatic active substance is a lipophilic substance which is more soluble in organic phase ethyl acetate, and the polarity of the substance is a strong polar substance similar to methanol. The biocontrol microbial inoculum of the invention contains more purified bacteriostatic substances.
When the invention is actually sprayed: diluting the purified fermentation liquor to the concentration of 2.4%, selecting the period of the crops which is easy to have gray mold for spraying, and spraying the liquid to the surface of the plants until water drops flow.
The invention has the following technical advantages:
(1) the biocontrol plant endophytic fungus DA-1 is an endophytic fungus obtained by separating in a plant body, has higher planting performance and stronger compatibility with plants compared with other biocontrol strains.
(2) The biocontrol plant endophytic fungus DA-1 is Arcopius aureus, and the thallus of the biocontrol plant endophytic fungus DA-1 shows that the biocontrol plant endophytic fungus DA-1 has a good inhibition effect on the growth of botrytis cinerea hyphae in a flat plate antagonism experiment, and can be applied to biological control of crop botrytis cinerea.
(3) After the effective components of the biocontrol plant endophytic fungus DA-1 fermentation liquor are purified, the control efficiency on the gray mold in the strawberry field is as high as 64.80 percent, the control effect is good, and the biocontrol plant endophytic fungus DA-1 fermentation liquor can be applied to biological control of the gray mold of crops.
(4) The biocontrol endophytic fungi DA-1 is applied to the control of gray mold of crops, can overcome a series of problems caused by the use of chemical pesticides, is beneficial to the pollution-free production of agricultural products, and farmers can not use or reduce the use amount of other chemical pesticides, thereby reducing the expenditure for the farmers and being beneficial to improving the product quality.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a model diagram of antagonistic botrytis cinerea of endophytic fungi DA-1 plate.
FIG. 2 is a growth diagram of antagonistic botrytis cinerea hyphae of a plant endophytic fungus DA-1 plate.
FIG. 3 is a morphological feature diagram of colony, ascocarp and ascospore of endophytic fungi DA-1 for preventing plants.
a-b represents the colony morphology of the endophytic fungi DA-1 of the biocontrol plant growing for 5d on the PDA; c, representing a mature ascosome of endophytic fungi DA-1 of the biocontrol plant on the PDA; d-e represents the DA-1 ascomycetes of the biocontrol plant endophytic fungi; f, representing biocontrol plant endophytic fungi DA-1 ascomycetes; g, representing the end terminal cyst silk of biocontrol plant endophytic fungi DA-1; h represents biocontrol plant endophytic fungus DA-1 ascospore.
FIG. 4 is a diagram showing the growth of botrytis cinerea hyphae in a sterile fermentation liquid for preventing endophytic fungi DA-1.
FIG. 5 is a diagram of the growth of botrytis cinerea hyphae inhibited by biocontrol plant endophytic fungus DA-1 sterile fermentation broth at different times.
FIG. 6 is a diagram for inhibiting the growth of botrytis cinerea hyphae by active crude extract of plant endophytic fungus DA-1 fermentation broth.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1, collection of samples and isolation and screening of biocontrol endophytic fungi.
1. Collection of samples
In 2015, 10 months, dendrobium officinale plants which are good in growth, robust and free of diseases are collected from dendrobium officinale planting bases in Linan city and are used for separating endophytic fungi.
2. Isolation of biocontrol endophytic fungi
Soaking healthy herba Dendrobii tissue in 75% ethanol solution for 1min, sterilizing with 1.5% sodium hypochlorite solution for 2min, rinsing with sterile water for 4 times, sucking water with sterile filter paper, and cutting into pieces of 0.4cm2And (3) placing the small blocks on the surface of PDA, and culturing in a constant temperature incubator at 25 ℃ in the dark. After the mycelium grows out, picking the colony edge agar block to a new plate for purification culture, and storing in a refrigerator at 4 ℃ for later use. And separating to obtain 122 endophytic fungi in total, and screening biocontrol strains.
3. Screening of plate antagonistic activity of endophytic fungi on Botrytis cinerea
(1) Test method
Activating the plant endophytic fungi and the botrytis cinerea stored at 4 ℃ on a PDA plate by adopting a confronting culture method. The cake was punched out with a 0.5cm diameter punch at the edge of the colony of the endophytic fungi and pathogenic bacteria, and the cake of the endophytic fungi was inoculated at a 9cm petri dish a with an inoculating needle, while the cake of the pathogenic bacteria was inoculated at b (see fig. 1). The control was a biocontrol-free endophyte plate. After being placed in a constant temperature incubator at 25 ℃ for dark culture for 7d, the data are measured, and the inhibition rate is calculated.
Calculating the formula: the radial growth inhibition rate is [100 × (longest radius of pathogen-radius of central axis of pathogen)/longest radius of pathogen ].
(2) Analysis of results
As shown in fig. 2, the plate antagonism assay indicates that: the ineffective strain can not inhibit the growth of botrytis cinerea hyphae, and the biocontrol endophytic fungus DA-1 has a strong inhibition effect on the growth of the botrytis cinerea hyphae, so that an obvious inhibition zone is generated. The radial growth inhibition rate reaches 43.32 percent. The control group grew normally.
Example 2 identification of biocontrol endophytic fungi DA-1
(1) Test method
Activating the biocontrol plant endophytic fungi DA-1 on PDA, punching a fungus cake on the edge of the endophytic fungi colony by using a puncher with the diameter of 0.5cm, inoculating the fungus cake to the center of a new PDA culture medium, and placing the new PDA culture medium in a constant-temperature incubator at 25 ℃ for dark culture. Extracting the DNA of the strain, amplifying the Tublin gene sequence and sequencing; the obtained sequence is shown in SEQ ID NO. 1.
(2) Analysis of results
Biocontrol plant endophytic fungus DA-1 is separated from the stem of 'Jingpin No. 1' dendrobium officinale, dark yellow hyphae and a large amount of red pigment precipitates can be observed after the biocontrol plant endophytic fungus DA-1 is cultured on PDA for 4 days, then a large quantum capsule body appears on the surface of a bacterial colony, the large quantum capsule body is wound outside the ascocarp body, the ascocarp filament is long, the tail end is bent, and a large amount of brown fusiform ascocarp spores are arranged (see figure 3). The alignment of the Tublin gene sequence of this strain in NCBI showed the highest homology to Arcopius aureus.
The biocontrol endophytic fungus DA-1 is preserved as Arcopius aureus, and the preservation unit is as follows: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, on Beijing, with a deposit number: CGMCC NO.14156, preservation time 2017, 9 months and 7 days.
Example 3 determination of antagonistic Activity of biocontrol endophytic fungus DA-1 fermentation broth against Botrytis cinerea
1. Preparation of biocontrol plant endophytic fungi fermentation liquor
The biocontrol plant endophytic fungi DA-1 is cultured on a PDA culture medium, 2 fungus cakes with the diameter of 0.5cm are punched at the edge of a bacterial colony by a puncher, the bacterial colony is cut into small blocks on sterile filter paper in a shape like a Chinese character 'jing' by a sterile scalpel, the small blocks are placed into a triangular flask filled with 100mL of PD liquid culture medium, and the shaking culture (28 ℃ C.; 150rpm) is carried out for 8 days in a constant temperature shaking table. And filtering the cultured fermentation liquor by using two layers of mirror paper to obtain biocontrol plant endophytic fungi DA-1 fermentation liquor.
2. Activity determination of biocontrol plant endophytic fungus DA-1 fermentation liquor on botrytis cinerea hyphae
(1) Test method
Adding the fermentation liquor into melted PDA with the concentration of 1% (v/v), inoculating Botrytis cinerea to the center of the plate, placing the plate in a constant temperature incubator at 25 ℃ for culture, taking sterile distilled water as a blank control, and measuring the colony diameter after 3 d. The formula is as follows: inhibition was 100% × (control colony diameter-treated colony diameter)/control colony diameter.
(2) Analysis of results
As shown in FIG. 4, the biocontrol plant endophytic fungus DA-1 fermentation liquid can inhibit the growth of botrytis cinerea hyphae, and the inhibition rate is 53.33%.
Example 4 biocontrol of the Activity of the production of the fermentation broth of endophytic fungi DA-1 of plants
(1) Test method
Culturing biocontrol endophytic fungi DA-1 on PDA culture medium, punching 2 bacterial cakes with diameter of 0.5cm from the edge of the bacterial colony by using a puncher, cutting the bacterial colony into small blocks on sterile filter paper in a shape like a Chinese character 'jing' by using a sterile scalpel, putting the small blocks into a triangular flask filled with 100mL PD liquid culture medium, repeating the operation for 3 times, and performing shaking culture (28 ℃ C.; 150rpm) in a constant temperature shaking table for 4, 8 and 12 days respectively. Adding fermentation liquor fermented at different times into melted PDA, inoculating Botrytis cinerea bacterial cake to the center of the plate, culturing in a constant temperature incubator at 25 deg.C for 3d, measuring colony diameter, and using sterile distilled water as blank control. Each treatment was repeated 3 times. The formula is as follows: inhibition was 100% × (control colony diameter-treated colony diameter)/control colony diameter.
(2) Analysis of results
And (3) measuring the inhibition rate of the botrytis cinerea on fermentation liquor for controlling endophytic fungi DA-1 of the plants at different times by using a hypha growth method. The results show that: with the prolonging of the culture time, the inhibition effect of the fermentation liquid on the botrytis cinerea hyphae is improved, and the inhibition rate is highest on the 12 th day of culture, as shown in figure 5.
Example 5 separation, purification and Activity tracking determination of active substances in biocontrol endophytic fungi DA-1 fermentation broth
(1) Test method
And pouring 500mL of original fermentation liquor after 8 days of culture into a separating funnel, adding 500mL of ethyl acetate solvent, fully oscillating, standing for 24h, allowing the fermentation liquor to flow out from the lower opening of a liquid separating funnel to serve as extracted fermentation liquor, and storing at 4 ℃. The ethyl acetate solvent was poured out through the top, and ethyl acetate was completely evaporated by using a rotary evaporator (70 ℃ C., 40rpm), and the ethyl acetate was dissolved in 40mL of a methanol solution and poured out as a crude extract. 3mL of the crude extract was uniformly loaded on a 150 mesh silica gel column (diameter 1cm, length 20cm, containing about 50g of 150 mesh silica gel), rinsed with methanol, and 5mL of the solution was collected from the lower part of the column using a 10mL centrifuge tube. The 5 th centrifuge tube was taken out, and dissolved in 3mL of methanol after rotary evaporation to obtain a methanol purified solution.
3mL of the original fermentation broth and 3mL of the extracted fermentation broth and 0.24mL of the methanol purified solution are added into a PDA culture medium (100mL) respectively, and the plates are inverted. Punching 1 fungus cake on the colony edge of Botrytis cinerea with a 0.5cm diameter puncher, inoculating to the PDA center, culturing in a constant temperature incubator at 25 deg.C in the dark, and measuring the inhibition rate of hypha growth after 3 d.
(2) Analysis of results
The biocontrol endophytic fungi DA-1 fermentation liquor is subjected to effective substance separation and activity tracking, and the results shown in figure 6 show that: the effective substances of the fermentation liquor (namely, extracted fermentation liquor) of the original fermentation liquor of the strain DA-1 after being extracted by the ethyl acetate solvent are obviously reduced, the inhibition rate of the original fermentation liquor on botrytis cinerea is only 7.49%, the inhibition rate of the original fermentation liquor on botrytis cinerea is 56.57%, and the inhibition rate of the methanol purification liquor on botrytis cinerea is 49.00%. The result shows that the effective substances of the strain DA-1 fermentation liquor are concentrated and retained in the purified fermentation liquor after separation and purification, and the active substances are lipophilic substances which are more soluble in ethyl acetate, and have polarity similar to that of methanol (the effective substances are concentrated by the purpose of purification and separation).
Example 6 evaluation of Effect of biocontrol plant endophytic fungus DA-1 fermentation broth active substance on prevention and treatment of strawberry Botrytis cinerea
(1) Test method
In a natural strawberry greenhouse, 9 cells are randomly divided for a control effect test, 3 treatment cells including DA-1, AT-6 and clear water are set, and each treatment cell is 3.
Group DA-1: the purified bacteriostatic substance bio-control fungicide (namely, the methanol purified solution prepared in example 5) prepared by the method is prepared into a liquid medicine with the concentration of 2.4%, and the liquid medicine is sprayed on the surfaces of strawberry plants, so that water drops flow (the using amount is about 300L/mu), and the greenhouse is managed conventionally.
XS-6 group: XS-6 is plant endophytic fungus, is Xylaria sp fungus, is prepared into XS-6 fermentation liquor according to the method in the embodiment 3, is diluted into liquid medicine with the concentration of 5.0 percent, and the spraying mode and the using amount are the same as the above;
clear water: spraying with clear water directly in the same manner and dosage;
(2) analysis of results
The results of the tests carried out on day 7 after spraying are shown in Table 1 below.
TABLE 1 DA-1 field control of strawberry gray mold effect
As shown in the table 1, under the conventional management condition of a greenhouse, the occurrence of gray mold of strawberry can be obviously inhibited by spraying biocontrol plant endophytic fungi DA-1, the average control effect reaches 64.80 percent, and the control XS-6 is only 14.31 percent.
In conclusion, the strain has a remarkable inhibition effect on the hypha growth of botrytis cinerea and can effectively prevent and treat the occurrence of botrytis cinerea.
Comparative example, the strains shown in Table 2 were subjected to a plate antagonism test in the same manner as described in example 1, and the results were compared with those of Arcopius aureus DA-1 of the present invention as described in Table 2.
TABLE 2
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
<110> Zhejiang agriculture and forestry university
<120> biocontrol plant endophytic fungi and application thereof in preventing and controlling gray mold of economic crops
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<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213> biocontrol plant endophytic fungus (Arcopius aureus)
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gcgtaagtcg attcgactcg agctgtgcga ccgaccaggt gatgcggtgt actgacacgc 240
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gcacggcctt gacagcaatg gcgtgtatgt gactgtcgcc agtccaactt cggttaaccc 420
cacgctcacc gccctattag gtacaacggc acctcggagc tccagctcga gcgcatgaac 480
gtctatttca acgaggtgag ttgtattgcg aggacccggg ctcaacagta gagttctgct 540
tgctgacggc atcttttctg caggcctccg gcaacaagta tgtcccccgt gccgtcctgg 600
tcgatctgga gcccggcacc atggacgccg tccgcgccgg ccccttcggc cagctcttcc 660
gccccgacaa ctt 673
Claims (8)
1. Biocontrol plant endophytic fungiArcopilusaureusDA-1, which is characterized in that: the preservation number is CGMCC NO. 14156.
2. Use of the biocontrol endophytic fungi DA-1 according to claim 1, characterized in that: inhibition of Botrytis cinerea (A. cinerea)Botrytis cinerea) Hyphae grow.
3. Use of the biocontrol endophytic fungi DA-1 according to claim 2, characterized in that: can be used for preventing and treating gray mold of strawberry.
4. Use of the biocontrol endophytic fungi DA-1 according to claim 2 or 3, characterized in that it comprises the following steps:
1) preparing biocontrol plant endophytic fungi DA-1 fermentation liquor;
2) separating and purifying the fermentation liquor to obtain purified fermentation liquor;
3) and diluting the purified fermentation liquor and applying the diluted fermentation liquor to the cash crops.
5. Use of the biocontrol endophytic fungi DA-1 according to claim 4, characterized in that:
the step 1) is as follows:
activating biocontrol plant endophytic fungi DA-1 on a PDA culture medium, then beating a fungus cake with the diameter of 0.5cm, cutting the fungus cake in a shape like a Chinese character 'jing', and taking the fungus cake as a strain;
secondly, inoculating the strain into a PD liquid culture medium, and culturing for 4-12 days in a constant-temperature shaking table with the rotating speed of 150rpm and the temperature of 28 ℃ to obtain fermentation liquor.
6. Use of the biocontrol endophytic fungi DA-1 according to claim 5, characterized in that: in the second step, 2 strains are inoculated in 100mL PD liquid culture medium.
7. Use of the biocontrol endophytic fungi DA-1 according to claim 4, characterized in that:
the step 2) is as follows: extracting and separating the fermentation liquor from ethyl acetate, concentrating, and performing chromatography by using a silica gel column to obtain purified fermentation liquor.
8. Use of the biocontrol endophytic fungi DA-1 according to claim 7, characterized in that: the chromatography was carried out with methanol as the mobile phase.
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| US7528232B2 (en) * | 2005-05-20 | 2009-05-05 | The University Of Kentucky Research Foundation | Utility of phylloplanins as antibiotics, selective fungicides and for enhancing microbial resistance in crop plants |
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| CN101113414A (en) * | 2006-05-19 | 2008-01-30 | 建德市大洋化工有限公司 | Yew trichoderma strain and its application in preparation of trichoderma ester prime |
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