CN114774293B - Stachybotrys HN17496 strain, biocontrol microbial inoculum, preparation method and application thereof - Google Patents
Stachybotrys HN17496 strain, biocontrol microbial inoculum, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biocontrol bacteria, in particular to a strain of Stachybotrys HN17496, a biocontrol bacteria agent, a preparation method and application thereof. The preservation number of the HN17496 strain is CCTCCNO: m2022489. The grape spike mould HN17496 strain provided by the invention has a good biocontrol effect on the wheat root rot vermicular spore, and has no potential safety hazard to people and animals; the embodiment shows that the antibacterial rate of the grape spike mould HN17496 strain and the metabolite thereof on the ophiopogon japonicus is 65-72%, and the biocontrol effect is obvious.
Description
Technical Field
The invention relates to the technical field of biocontrol bacteria, in particular to a strain of Stachybotrys HN17496, a biocontrol bacteria agent, a preparation method and application thereof.
Background
The vermicular spore (bipolaris sorokiniana) of the root rot of wheat is a pathogenic fungus widely distributed in the world and is the main pathogen of the root rot of wheat. Pathogenic bacteria can infect wheat root systems, stalk bases, leaves and ears, and seriously affect the yield and quality of wheat. The control method of the wheat root rot navel vermicular spore at the present stage mainly comprises the steps of seed soaking by medicaments or chemical medicament spraying, and is a control method which is harmful to the environment; few researches on the biocontrol bacteria of the moellendorffium graminearum are carried out, and in 2010, a bacterial strain, namely a stenotrophomonas maltophilia (Stenotrophomonas maltophilia) C3 strain, is discovered, can produce degrading enzyme of chitin, has a good control effect on the moellendorffium graminearum, is a conditional pathogenic bacterium, and is not suitable for being used as the biocontrol bacteria of the moellendorffium graminearum. But the biocontrol fungi for the vermicular spore of the umbilicaria of the wheat root rot are freshly reported.
Disclosure of Invention
In order to solve the problems, the invention provides a Stachybotrys strain HN17496, a biocontrol agent, and a preparation method and application thereof. The grape spike mould HN17496 strain provided by the invention has a good biocontrol effect on the wheat root rot vermicular spore.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a Stachybotrys sp HN17496 strain, wherein the preservation number of the HN17496 strain is CCTCC NO: m2022489.
The invention also provides a biocontrol microbial agent, which comprises the metabolites of the HN17496 strain and/or the HN17496 strain.
Preferably, when the biocontrol microbial agent comprises HN17496 strain, the spore number of HN17496 strain in the biocontrol microbial agent is more than or equal to 10 per g or per mL of biocontrol microbial agent 5 CFU。
The invention also provides a preparation method of the biocontrol microbial inoculum, which comprises the following steps:
inoculating the HN17496 strain into a PDA culture medium, and culturing for 5-7 d to obtain the biocontrol microbial agent.
Preferably, the temperature of the culture is 25-28 ℃.
Preferably, when the biocontrol microbial agent does not contain the HN17496 strain, the biocontrol microbial agent further comprises a sterilization treatment after 5-7 d of culture.
Preferably, the sterilization treatment method comprises the following steps: sterilizing at 121deg.C for 30min.
The invention also provides application of the HN17496 strain or the biocontrol microbial agent prepared by the preparation method in preventing and treating plant diseases, wherein pathogenic bacteria of the plant diseases comprise the ophiopogon root vermiculosus of the wheat root rot.
Preferably, the plant disease includes root rot.
Preferably, the plant comprises wheat.
The beneficial effects are that:
the invention provides a Stachybotrys sp HN17496 strain, wherein the preservation number of the HN17496 strain is CCTCC NO: m2022489. The grape spike mould HN17496 strain provided by the invention has a good biocontrol effect on the wheat root rot vermicular spore, and has no potential safety hazard to people and animals; the embodiment shows that the antibacterial rate of the grape spike mould HN17496 strain and the metabolite thereof on the ophiopogon japonicus is 65-72%, and the biocontrol effect is obvious.
Biological preservation information
The strain Stachybotrys HN17496, latin named Stachybotrys sp, was preserved in China Center for Type Culture Collection (CCTCC) for 4 months and 25 days in 2022, the preservation address is eight-channel 299-No. Wuhan university China center for type culture collection in Wuhan, inc. of Hubei province, the preservation number is CCTCC NO: m2022489. The grape spike mould HN17496 strain provided by the invention has a good biocontrol effect on the wheat root rot vermicular spore.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a partial picture of the primary screening effect of a vermiculella radicata biocontrol strain;
FIG. 2 is a morphological characterization of the rescreened HN17496 strain; wherein A is the form of HN17496 colony on the front surface of the PDA culture medium, B is the form of HN17496 colony on the back surface of the culture dish, C-G are conidiophores, H-I are conidiophores, and the scale in C-I is 10 mu m;
FIG. 3 is a phylogenetic tree of the HN17496 strain;
FIG. 4 is a graph showing the biocontrol effect of the HN17496 strain; wherein A and B are normally grown wheat root rot vermicularia umbilicifolia (no metabolic liquid is added), and C is the bacteriostatic effect of the biocontrol fungus metabolic liquid which is not subjected to autoclaving; d is the bacteriostatic effect of the biocontrol fungus metabolic solution after autoclaving.
Detailed Description
The invention provides a Stachybotrys sp HN17496 strain, wherein the preservation number of the HN17496 strain is CCTCC NO: m2022489.
The HN17496 strain of the present invention preferably has the following characteristics:
1) The colony is black, the spore-forming peduncles grow on the culture medium, and the spore groups are covered by sticky, gray or black;
2) The mycelium can produce dark brown secretion substances, is similar to impurities and is often in a beam shape to form a mycelium bridge;
3) Conidiophores grow in a giant line shape, are single or aggregate, have thin walls, branch or not branch, stand or bend slightly, have 1-2 diaphragms and are smooth, 18.2-64.0x2.8-4.9 mu m, and the top ends of the conidiophores are attached to 1-6 spore-producing cells;
4) The spore-producing cell is in a bottle stem shape, is in a stick shape to be similar to a stick shape, is transparent, semitransparent and dark black, has a smooth surface, and has a diameter of 6.6-11.5X3.4-5.5 mu m and an obvious neck ring;
5) Conidiophores are apoplastic, no diaphragm, nearly circular, oval to nearly cylindrical, transparent, semitransparent to dark olive, thick wall, dark olive to dark brown, smooth surface or warty protrusions, 7.0-11.6x3.3-7.5 μm.
The grape spike mould HN17496 strain provided by the invention has a good biocontrol effect on the wheat root rot vermicular spore, and has no potential safety hazard to people and animals; the embodiment shows that the antibacterial rate of the grape spike mould HN17496 strain and the metabolite thereof on the ophiopogon japonicus is 65-72%, and the biocontrol effect is obvious.
The invention also provides a biocontrol microbial agent, which comprises metabolites of the HN17496 strain and/or the HN17496 strain. The biocontrol microbial agent of the invention preferably achieves a biocontrol effect by inhibiting and/or killing pathogenic microorganisms.
In the present invention, when the biocontrol agent comprises HN17496 strain, the spore number of HN17496 strain in the biocontrol agent is preferably not less than 10 per g or per mL of biocontrol agent 5 CFU。
The invention also provides a preparation method of the biocontrol microbial inoculum, which comprises the following steps:
inoculating the HN17496 strain into a PDA culture medium, and culturing for 5-7 d to obtain the biocontrol microbial agent.
In the present invention, the temperature of the culture is preferably 25 to 28 ℃.
In the present invention, when the biocontrol agent does not contain the HN17496 strain, it is preferable that the culture is further carried out for 5 to 7 days and then sterilization treatment is carried out; the method of sterilization treatment preferably comprises: sterilizing at 121deg.C for 30min.
The invention also provides application of the HN17496 strain or the biocontrol microbial agent prepared by the preparation method in preventing and treating plant diseases, wherein pathogenic bacteria of the plant diseases comprise the ophiopogon root vermiculosus of the wheat root rot. In the present invention, the plant disease preferably includes root rot and the plant preferably includes wheat.
In order to further illustrate the present invention, the strain of Stachybotrys HN17496, the biocontrol agent, the preparation method and the application thereof provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The preparation method comprises the steps of obtaining the grape spike mould HN17496 strain from soil under weeds in Mitsui mountain county by a dilution plate method, wherein the specific method is as follows:
weighing 10g of soil, adding the soil into a triangular flask containing 90mL of sterilized water, placing the triangular flask on a shaking table at 120rpm, and oscillating for 25min to uniformly disperse soil particles in distilled water to obtain a soil suspension with the dilution factor of 10; sucking 1mL from the soil suspension, and placing the suspension into a test tube filled with 9mL of sterilized water to obtain suspension with dilution factor of 100;
the WA culture medium is sterilized at high temperature and then cooled to 45 ℃, added with streptomycin 30 mug/mL and chloramphenicol 4 drops, poured into a culture dish for solidification and cooling, the obtained soil suspension with dilution factor of 100 is sufficiently and evenly shaken, 1mL is absorbed, 5 drops of the soil suspension are dripped into each culture dish, the culture dish is inverted after being uniformly smeared by a sterilized bent glass rod, and the culture dish is placed in a biochemical incubator at 25 ℃ for culture. The ecological environment of the soil sample collection area is simulated, the soil sample collection area is placed on WA culture medium at 25 ℃, and after 30d, the soil sample collection area is observed under a stereoscopic vision. Single spores or colonies were picked up on PDA medium and purified, and the strain was preserved.
Primary screening of wheat root rot navel-flattening vermicular spore biocontrol strain
The purified strain and pathogenic bacteria wheat root rot vermicular spore are subjected to flat plate opposite culture, and the specific steps are as follows:
the purified bacterial strain and pathogenic bacteria are respectively inoculated on a 9cm culture dish for opposite culture, the pathogenic bacteria are inoculated in the middle of a flat plate, four bacterial strains to be detected are inoculated on the periphery of the flat plate, the inoculation diameter of each bacterial strain is 0.5cm, the distance between each bacterial strain and the edge of the culture dish is 1cm, the bacterial strain distance is 2cm, the pathogenic bacterial strain which is not inoculated with the purified bacterial strain is used as a contrast, 3 repetitions are arranged for each treatment and contrast, the bacterial strain is cultured for 4d at 25 ℃, and according to the growth speed and bacteriostasis condition of bacterial colonies, whether antagonism phenomenon occurs among bacterial colonies or not is judged. And (3) selecting biocontrol strains with obvious antibacterial effect (definition for obviously limiting normal growth of the wheat root rot and the navel vermicular spore is effective, and fusion growth is that no antibacterial effect) as a re-screening object. Part of the primary screening effect is shown in figure 1.
As can be seen from FIG. 1, the partially purified strain has a different inhibitory effect on the growth of Helminthosporium graminearum.
Re-screening of wheat root rot umbilicus vermiculosus biocontrol strain
Preparing the biocontrol strain obtained by primary screening into fungus blocks by using a puncher with the diameter of 0.5cm, carrying out opposite culture on a 9cm flat plate, wherein the distance between the biocontrol strain and the fungus blocks is 1cm away from the edge of a culture dish, 3 repetitions are arranged for each group of treatment, pathogenic bacteria which are not inoculated with the biocontrol strain are used as a contrast, culturing for 5-7 d at the constant temperature of about 25 ℃, periodically measuring the radius of a bacterial colony by using a cross method, and calculating the bacteriostasis rate of the bacterial strain to the pathogenic bacteria by using the following formula.
The strain with the best bacteriostasis rate is screened out and is marked as HN17496 strain.
Example 2
Identification of the rescreened Strain
The re-screened HN17496 strain of example 1 was inoculated onto PDA medium and cultured at 25℃for 7d to obtain a colony having a diameter of 2.2cm, and the morphological characteristics of the colony were as follows:
the colony is black, the spore-forming peduncles grow on the culture medium, the spore groups from gray to black are covered, and the back of the culture dish is dark brown. There is no ascar shell. Hyphae produce dark brown secreted substances, which are as impurities, often in bundles, forming hyphae bridges. The conidiophore is giant, single or aggregate growing, thin wall, branch or no branch, vertical or slightly bending, 1-2 diaphragms, smooth, 18.2-64.0 μm long, 2.8-4.9 μm wide, and 1-6 spore-producing cells attached and growing on the top. The spore-producing cell is in the shape of bottle stalk, stick or stick-like, transparent, semitransparent or dark black, smooth in surface, 6.6-11.5X3.4-5.5 mu m, and has obvious neck ring. The conidiophore grows on the top, has no diaphragm, is nearly round, oval or nearly cylindrical, is transparent, semitransparent to deep olive, has a wall thickness, is deep olive or black brown, has smooth surface or warty protrusions, has the conidiophore growth of 7.0-11.6 mu m, and has the conidiophore width of 3.3-7.5 mu m. Preliminary identification of HN17496 strain was a Stachybotrys (Stachybotrys) fungus.
HN17496 and similar species thereof are combined to construct a phylogenetic tree based on cmdA, ITS, LSU, rpb, tub2 and tef-1a by using a Bayesian method, and the numerical value PP on a node is more than or equal to 0.50, and the method specifically comprises the following steps:
the evolution tree comprises 100 strains of Stachybotrys, a phylogenetic tree is constructed by combining six genes of cmdA, ITS, LSU, rpb, tub2 and tef-1a, which are 4276 bases in total, 3772 brief information sites passimon-informative character (692 bases in cmdA, 376 brief information sites, 549 bases in ITS, 189 brief information sites in LSU, 830 bases in 70 brief information sites, 777 bases in rpb, 2826 brief information sites in tub2, 287 bases in tub2, 113 brief information sites in tub 1, 1141 bases in tef-1a, 450 brief information sites in tub), peethambara sundara (CBS 646.77) in outer group, and obvious distinction between the inner and the intermediate species and the inner species of the Stachybotrys and the approximated species thereof, and high node support rate.
The HN17496 strain is independently branched on a phylogenetic tree (figure 3), the mycelium of the strain is often in a bundle shape to form a mycelium bridge, and the conidium surface is smooth or has wart-shaped protrusions and is distinguished from other species; HN17496 was identified as a new species by a combination of morphology and molecular system: stachybotrys sp.1.
The screened strain is named as Stachybotrys sp HN17496 and is sent to China Center for Type Culture Collection (CCTCC) with the collection number of CCTCC NO: m2022489.
Example 3
Biocontrol effect of HN17496 Strain
The HN17496 strain selected in example 2 was inoculated onto PDA medium and cultured for 5 days at 25℃and then a cake (cake diameter: 5 mm) was made on the colony edge of HN17496 strain by a puncher in a sterile super clean bench, and then the cake was transferred into a triangular flask containing 150ml of PDB liquid medium and cultured for 7 days in a constant temperature shaking incubator at 25℃at 100 r/min. After 7d incubation, filtration was performed with sterile gauze (eight layers) and then with sterile filter paper. Filtering 30mL of filtrate with a bacterial filter (0.22 μm) in a sterile ultra-clean workbench to obtain biocontrol strain culture solution (CL), and reserving at 4 ℃. And (3) taking 30mL of filtrate, and autoclaving in an autoclave at 121 ℃ for 30min to obtain crude toxin extract (CT) of the biocontrol strain bacteria, wherein the crude toxin extract is reserved at 4 ℃.
The method comprises the steps of detecting the biological activity of a metabolic solution by adopting a mixed plate method, uniformly mixing the metabolic solution with a PDA culture medium according to a ratio of 1:4 (V/V), pouring the mixture into a plate, inoculating a pathogenic bacterium (wheat root rot navel vermicular spore) bacterial cake (d=5 mm) at the center of the plate, culturing for 5 days at 25 ℃, observing the morphological change of a bacterial colony, measuring the bacterial colony diameter by using a crisscross method, calculating the bacterial growth inhibition rate of hyphae (the formula is as follows), taking the plate without the metabolic solution as a negative control, and repeating the test for 3 times.
The bacteriostasis test effect is shown in figure 4, wherein A, B is a C, D culture dish with no metabolic liquid added and a diameter of 5 days reaching 5cm, and the growth of the Pythium gracile is slow and the diameter of 5 days reaching less than 1cm.
The biocontrol fungus HN17496 strain has the inhibition effect on the helminth vermicularia graminea: the average antibacterial rate of the culture solution of the biocontrol strain which is not subjected to high-pressure sterilization is 65%, and the average antibacterial rate of the culture solution of the biocontrol strain which is subjected to high-pressure sterilization reaches 72%. The inhibiting effect of the culture solution after autoclaving on the root rot of the wheat is more obvious, so that the chemical with inhibiting effect on the pathogenic bacteria of the root rot of the wheat is high-temperature and high-pressure resistant. The invention obtains the phenomenon that the relative content of antibacterial substances in the crude toxin extracting solution of the biocontrol strain is increased by sterilizing without filtering by a bacterial filter (0.22 mu m), so that the obvious antibacterial rate is increased.
In conclusion, the grape spike mould HN17496 strain provided by the invention has a good biocontrol effect on the wheat root rot vermicular spore, and has no potential safety hazard to people and livestock; the embodiment shows that the antibacterial rate of the grape spike mould HN17496 strain and the metabolite thereof on the ophiopogon japonicus is 65-72%, and the biocontrol effect is obvious.
While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.
Claims (10)
1. Grape spike mouldStachybotryssp.) HN17496 strain, characterized in that said HN17496 strain has a accession number cctccc NO: m2022489.
2. A biocontrol microbial agent comprising the metabolites of the HN17496 strain and/or HN17496 strain of claim 1.
3. The biocontrol microbial agent according to claim 2, wherein when the biocontrol microbial agent comprises the HN17496 strain, the spore count of the HN17496 strain in the biocontrol microbial agent is not less than 10 per g or per mL of the biocontrol microbial agent 5 CFU。
4. A method for preparing the biocontrol microbial agent as claimed in claim 2 or 3, which comprises the steps of:
inoculating the HN17496 strain of claim 1 into a PDA culture medium, and culturing for 5-7 d to obtain the biocontrol microbial agent.
5. The method according to claim 4, wherein the temperature of the culture is 25 to 28 ℃.
6. The method according to claim 4 or 5, wherein when the biocontrol agent does not contain the HN17496 strain, the culture is further carried out for 5 to 7 days and then the sterilization treatment is carried out.
7. The method of claim 6, wherein the method of sterilizing comprises: sterilizing at 121deg.C for 30min.
8. The application of the HN17496 strain as claimed in claim 1 or the biocontrol microbial agent as claimed in claim 2 or 3 or the biocontrol microbial agent prepared by the preparation method as claimed in any one of claims 4 to 7 in controlling plant diseases is characterized in that pathogenic bacteria of the plant diseases comprise the plant root rot of the plant.
9. The use according to claim 8, wherein the plant disease comprises root rot.
10. The use according to claim 8 or 9, wherein the plant comprises wheat.
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| NL2033035A NL2033035B1 (en) | 2022-05-27 | 2022-09-15 | Stachybotrys sp. hn17496 strain, microbial biocontrol agent, and preparation method and use thereof |
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| CN103484380A (en) * | 2013-08-26 | 2014-01-01 | 山东省科学院中日友好生物技术研究中心 | Parasitism effect of trichoderma T11-W on root-knot nematode egg and application of trichoderma T11-W |
| CN111500501A (en) * | 2020-05-07 | 2020-08-07 | 中国地质大学（北京） | Streptomyces misonii strain and application thereof in preventing and treating wheat root rot and stem basal rot |
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| JP2022502404A (en) * | 2018-09-28 | 2022-01-11 | バイエル・アクチエンゲゼルシヤフト | Bacterial biocontrol and fatty acid combination |
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| CN103484380A (en) * | 2013-08-26 | 2014-01-01 | 山东省科学院中日友好生物技术研究中心 | Parasitism effect of trichoderma T11-W on root-knot nematode egg and application of trichoderma T11-W |
| CN111500501A (en) * | 2020-05-07 | 2020-08-07 | 中国地质大学（北京） | Streptomyces misonii strain and application thereof in preventing and treating wheat root rot and stem basal rot |
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| Title |
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| 翟妮平.土壤淡色丝孢真菌稀有物种的挖掘及有生防潜力丝孢菌的筛选.《中国优秀硕士学位论文全文数据库 基础科学辑》.2020,A006-189. * |
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