CN114774293A - Stachybotrys botrytis HN17496 strain, biocontrol microbial inoculum and preparation method and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biocontrol bacteria, in particular to a Stachybotrys botrytis HN17496 strain, a biocontrol bacterium agent, and a preparation method and application thereof. The preservation number of the HN17496 strain is CCTCC NO: m2022489. The stachybotrys vinii HN17496 strain provided by the invention has a good biocontrol effect on Helminthosporium putrescens and has no potential safety hazards to people and livestock; the embodiment shows that the Stachybotrys botrytis HN17496 strain and the metabolite thereof provided by the invention have the bacteriostasis rate of 65-72% on Helminthosporium moniliforme, and the biocontrol effect is obvious.
Description
Technical Field
The invention relates to the technical field of biocontrol bacteria, in particular to a Stachybotrys botrytis HN17496 strain, a biocontrol bacterium agent, and a preparation method and application thereof.
Background
Bipolaris sokiniana (bipolaris) is a pathogenic fungus widely distributed in the world and is the main pathogen of wheat root rot. Pathogenic bacteria can infect wheat root systems, stem bases, leaves and ears, and the yield and quality of wheat are seriously influenced. At the present stage, the prevention and control method of the bipolaris merrilat is mainly a method for soaking seeds by using a medicament or spraying a chemical medicament, and both the methods are harmful to the environment; the research on the biocontrol bacterium of the bipolaris cornutus, and 2010 discovers a bacterial strain Stenotrophoromonas maltophilia C3 which can produce a chitin degrading enzyme and has a good control effect on the bipolaris cornutus, but the bacterial strain is a conditional pathogen and is not suitable for serving as the biocontrol bacterium of the bipolaris cornutus. The biocontrol fungus aiming at the creeping liriopsis tricuspidata of the root rot of wheat is only reported.
Disclosure of Invention
In order to solve the problems, the invention provides a stachybotrys uvarum HN17496 strain, a biocontrol microbial inoculum and a preparation method and application thereof. The stachybotrys vinii HN17496 strain provided by the invention has a good biocontrol effect on Helminthosporium umbiliciforme.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a Stachybotrys sp (Stachybotrys sp) HN17496 strain, wherein the preservation number of the HN17496 strain is CCTCC NO: m2022489.
The invention also provides a biocontrol microbial inoculum which comprises the HN17496 strain and/or a metabolite of the HN17496 strain.
Preferably, when the anti-biological inoculant comprises the HN17496 strain, the spore number of the HN17496 strain in the anti-biological inoculant is more than or equal to 10 in each g or each mL of the anti-biological inoculant5CFU。
The invention also provides a preparation method of the biocontrol microbial inoculum, which comprises the following steps:
and (3) inoculating the HN17496 strain into a PDA culture medium, and culturing for 5-7 d to obtain the biocontrol microbial inoculum.
Preferably, the temperature of the culture is 25-28 ℃.
Preferably, when the biocontrol microbial inoculum does not contain the HN17496 strain, the biocontrol microbial inoculum also comprises sterilization treatment after being cultured for 5-7 days.
Preferably, the method of sterilization treatment includes: sterilizing at 121 deg.C for 30 min.
The invention also provides application of the HN17496 strain or the biocontrol microbial inoculum prepared by the preparation method in controlling plant diseases, wherein pathogenic bacteria of the plant diseases comprise Helminthosporium tritici.
Preferably, the plant disease comprises root rot.
Preferably, the plant comprises wheat.
Has the advantages that:
the invention provides a Stachybotrys sp HN17496 strain, wherein the preservation number of the HN17496 strain is CCTCC NO: m2022489. The stachybotrys vinii HN17496 strain provided by the invention has a good biocontrol effect on Helminthosporium putrescens and has no potential safety hazards to people and livestock; the embodiment shows that the Stachybotrys botrytis HN17496 strain and the metabolite thereof provided by the invention have the bacteriostasis rate of 65-72% on Helminthosporium moniliforme, and the biocontrol effect is obvious.
Biological preservation information
The Stachybotrys botrytis HN17496 strain, the Latin name is Stachybotrys sp, the strain is preserved in China Center for Type Culture Collection (CCTCC) at 25/4/2022, the preservation address is China center for type culture Collection of Wuhan university No. 299 in one eight-way zone of Wuhan City, Hubei province, the preservation number is CCTCC NO: m2022489. The stachybotrys vinii HN17496 strain provided by the invention has a good biocontrol effect on Helminthosporium umbiliciforme.
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 embodiments will be briefly described below.
FIG. 1 is a partial picture of the primary screening effect of a biocontrol strain of Helminthosporium radices;
FIG. 2 is a morphogram map of rescreened HN17496 strain; wherein A is the shape of HN17496 colony on the front side of PDA culture medium, B is the shape of HN17496 colony on the back side of the culture dish, C-G are conidiophores, H-I are conidiophores, and the scale in C-I is 10 μm;
FIG. 3 is a phylogenetic tree of strain HN 17496;
FIG. 4 is a diagram showing the biocontrol effect of HN17496 strain; wherein A and B are normal growing pseudomonads umbilicalis (no metabolic liquid is added), and C is the bacteriostatic effect of the biocontrol fungus metabolic liquid which is not subjected to high-pressure sterilization; d is the bacteriostasis effect of the biocontrol fungus metabolic fluid after high-pressure sterilization.
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 according to the invention preferably has the following characteristics:
1) the colony is black, the spore-forming peduncles grow on the culture medium, and the viscous, gray or black spore group covers the colony;
2) the hyphae can produce dark brown secretion substances, which are similar to impurities and are often bunched to form hypha bridges;
3) conidiophores are giant, grow singly or in an aggregation manner, are thin in wall, branched or not, are vertical or slightly bent, have 1-2 diaphragms, are smooth, have the diameter of 18.2-64.0 multiplied by 2.8-4.9 mu m, and have 1-6 spore-producing cells on the top;
4) the spore-forming cell is of a bottle stalk shape, is transparent, semitransparent, dark black and smooth in surface, is 6.6-11.5 multiplied by 3.4-5.5 mu m in rod shape, and has an obvious neck ring;
5) conidiophores are terminal, have no diaphragm, are nearly round, oval to nearly cylindrical, transparent, semitransparent to dark olive, and have wall thickness, dark olive to black brown, smooth surface or warty protrusion with the diameter of 7.0-11.6 multiplied by 3.3-7.5 mu m.
The stachybotrys vinii HN17496 strain provided by the invention has a good biocontrol effect on Helminthosporium putrescens and has no potential safety hazards to people and livestock; the embodiment shows that the inhibition rate of the Stachybotrys botrytis HN17496 strain and the metabolite thereof on Helminthosporium tritici is 65-72%, and the biocontrol effect is obvious.
The invention also provides a biocontrol microbial inoculum which comprises the metabolites of the HN17496 strain and/or the HN17496 strain. The biocontrol microbial inoculum disclosed by the invention preferably inhibits and/or kills pathogenic microorganisms, so that a biocontrol effect is achieved.
In the invention, when the biocontrol microbial inoculum comprises the HN17496 strain, the number of spores of the HN17496 strain in the biocontrol microbial inoculum is preferably more than or equal to 10 in terms of each g or each mL of biocontrol microbial inoculum5CFU。
The invention also provides a preparation method of the biocontrol microbial inoculum, which comprises the following steps:
and (3) inoculating the HN17496 strain into a PDA culture medium, and culturing for 5-7 days to obtain the biocontrol microbial inoculum.
In the present invention, the temperature of the culture is preferably 25 to 28 ℃.
In the invention, when the biocontrol microbial inoculum does not contain the HN17496 strain, the biocontrol microbial inoculum also preferably comprises sterilization treatment after being cultured for 5-7 days; the method of sterilization treatment preferably includes: sterilizing at 121 deg.C for 30 min.
The invention also provides application of the HN17496 strain or the biocontrol microbial inoculum prepared by the preparation method in controlling plant diseases, wherein pathogenic bacteria of the plant diseases comprise Helminthosporium tritici. In the present invention, the plant disease preferably includes root rot, and the plant preferably includes wheat.
In order to further illustrate the invention, the strain stachybotrys HN17496, the biocontrol microbial inoculum, and the preparation method and application thereof provided by the invention are described in detail in the following with reference to the examples, but the strain and the biocontrol microbial inoculum are not to be construed as limiting the scope of the invention.
Example 1
The method comprises the following steps of obtaining a Stachybotrys botrytis HN17496 strain from the soil under weeds of the Saxatilis praecox in Shandong mountain county by a dilution plate method, wherein the method comprises the following specific steps:
weighing 10g of soil, adding the soil into a triangular flask containing 90mL of sterile 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 of the suspension from the soil, and putting the suspension into a test tube filled with 9mL of sterilized water to obtain a suspension with a dilution multiple of 100;
sterilizing WA culture medium at high temperature, cooling to 45 ℃, adding streptomycin 30 mu g/mL and 4 drops of chloramphenicol, pouring the mixture into a culture dish, solidifying and cooling, fully shaking the obtained soil suspension with the dilution multiple of 100, sucking 1mL, dripping 5 drops of the soil suspension into each culture dish, uniformly smearing the soil suspension by using a sterilized bent glass rod, inverting the culture dish, and culturing in a biochemical incubator at 25 ℃. Simulating the ecological environment of the soil sample collection place, setting the temperature to be 25 ℃ on WA culture medium, and observing under a stereoscope after 30 days. Selecting single spore or colony on PDA culture medium, purifying, and preserving strain.
Preliminary screening of rhizopus niveus biocontrol strain
Carrying out plate confronting culture on the purified strain and pathogenic bacteria Erwinia umbilicalis of the wheat root rot, which comprises the following steps:
respectively inoculating the purified strains and pathogenic bacteria to a 9cm culture dish for opposite culture, inoculating the pathogenic bacteria in the middle of a flat plate, connecting four strains to be detected on the periphery of the flat plate, wherein the inoculation diameter of each strain is 0.5cm, the distance between the strains is 1cm from the edge of the culture dish, the distance between the strains is 2cm, 3 times of treatment and comparison are performed by taking pathogenic strains which are not connected with the purified strains as comparison, culturing for 4d at 25 ℃, and whether antagonism occurs between colonies or not according to the growth speed and the bacteriostasis condition of bacterial colonies. Selecting biocontrol strains with obvious bacteriostatic effect (the definition of obviously limiting the normal growth of the Helminthosporium tritici is effective, and the fusion growth is without bacteriostatic effect) as objects for re-screening. The partial primary screening effect diagram is shown in figure 1.
As can be seen from FIG. 1, the partially purified strain has an inhibitory effect on the growth of Helminthosporium tritici, and the inhibitory effects are different.
Double screening of Meretrix meretrix et Schlegeli biological control strain
Using a puncher with the diameter of 0.5cm to prepare the bio-control strains obtained by primary screening into fungus blocks, carrying out opposite culture on a 9cm flat plate, wherein the distance between the bio-control strains and the culture dish edge is 1cm, 3 treatment settings are set for each group, using pathogenic bacteria without bio-control strains as a reference, carrying out constant temperature culture at about 25 ℃ for 5-7 d, periodically measuring the radius of the bacterial colonies by using a cross method, and calculating the bacteriostasis rate of the strains on the pathogenic bacteria by using the following formula.
And screening out the strain with the best antibacterial rate, and marking as the strain HN 17496.
Example 2
Identification of rescreened strains
The HN17496 strain rescreened in example 1 was inoculated into PDA medium and cultured at 25 ℃ for 7 days to obtain a colony with a diameter of 2.2cm, and the morphological characteristics of the colony are as follows:
the colony is black, the spore-forming peduncle grows on the culture medium, the spore group from gray to black is covered, and the back of the culture dish is dark brown. There is no asco-shell. The hyphae produce dark brown secretion substances, like impurities, and are often bunched to form hypha bridges. Conidiophores are giant, grow singly or in an aggregation manner, are thin in wall, branched or not, are vertical or slightly bent, have 1-2 diaphragms, are smooth, have the conidiophores of 18.2-64.0 mu m in length and the conidiophores of 2.8-4.9 mu m in width, and have 1-6 spore-producing cells attached to the top ends. The spore-forming cells are of a bottle stalk shape, a stick shape or a stick-like shape, transparent, semitransparent or dark black, smooth in surface, 6.6-11.5 multiplied by 3.4-5.5 mu m and provided with an obvious neck ring. Conidia are terminal, have no diaphragm, are nearly round, oval or nearly cylindrical, are transparent, are semitransparent to dark olive, have a wall thickness, dark olive or black brown, have smooth surfaces or warty protrusions, have the length of 7.0-11.6 mu m, and have the width of 3.3-7.5 mu m. The primary characterization of HN17496 strain as Stachybotrys (Stachybotrys) fungus.
HN17496 and ITS approximate species are constructed by Bayesian method based on six genes cmdA, ITS, LSU, rpb2, tub2 and tef-1a to construct phylogenetic tree, the value PP on the node is more than or equal to 0.50, specifically as follows:
the evolutionary tree comprises 100 strains of Stachybotrys, a phylogenetic tree is constructed by combining six genes including cmdA, ITS, LSU, rpb2, tub2 and tef-1a, 4276 bases and 3772 simple-information sites parsimony-informative character (cmdA has 692 bases and 376 simple information sites; ITS has 549 bases and 189 simple information sites; LSU has 830 bases and 70 simple information sites; rpb2 has 777 bases and 2826 simple information sites; tub2 has 287 bases and 113 simple information sites; tef-1a has 1141 bases and 450 simple information sites), and an outer population is Peetharara sundara (CBS 646.77) and has higher genetic distance with various species of Stachybotrys and similar genera, and the genetic distance between the species and the species of the Stachybotrys is larger, and the intraspecies and the support node differentiation rate is higher.
HN17496 strain is in phylogenetic tree (FIG. 3), and has independent branch, large difference, and its hypha is often fasciculate, forms hypha bridge, and the surface of conidiophore is smooth or has warty protuberance, and is distinguished from other species; by combining morphology and molecular systematics, HN17496 was identified as a new species: stachybotrys sp.1.
The screened strain is named as Stachybotrys sp HN17496 and is sent to China center for type culture Collection with the collection number of CCTCC NO: m2022489.
Example 3
Biocontrol effect of HN17496 strain
The HN17496 strain selected in example 2 was inoculated on PDA medium and cultured for 5 days at 25 deg.C, then the colony edge of the HN17496 strain was punched with a punch in a sterile ultra-clean bench (the diameter of the cake is 5mm), and then the cake was transferred to a triangular flask containing 150ml of PDB liquid medium and cultured for 7 days in a constant temperature shaking incubator at 25 deg.C (100 r/min). After 7 days of incubation, the cells were filtered through sterile gauze (eight layers) and then through sterile filter paper. Filtering 30mL of filtrate in a sterile ultra-clean workbench by using a bacterial filter (0.22 μm) to obtain a biocontrol strain culture solution (CL), and keeping the biocontrol strain culture solution at 4 ℃ for later use. And sterilizing 30mL of filtrate at 121 deg.C for 30min to obtain crude toxin extract (CT) of biocontrol strain bacteria at 4 deg.C.
The biological activity of the metabolic solution is detected by adopting a mixed plate method, the metabolic solution and a PDA culture medium are uniformly mixed according to the ratio of 1:4(V/V) and are inverted, a pathogenic bacterium (Helminthosporium tritici) cake (d is 5mm) is inoculated in the center of the plate, the plate is placed at the temperature of 25 ℃ for culture for 5d, the change of the colony morphology is observed, the diameter of the colony is measured by using a cross method, the hypha growth inhibition rate (formula is shown below) is calculated, and the test is repeated for 3 times by using the plate without the metabolic solution as a negative control.
The bacteriostasis test effect diagram is shown in fig. 4, wherein A, B is that the diameter of 5 days reaches 5cm without adding the metabolic solution, and the diameter of 5 days is less than 1cm when the wheat root is slow to grow by adding the metabolic solution into a C, D culture dish.
The biocontrol fungus HN17496 strain has an inhibition effect on Helminthosporium putrescenti: the average bacteriostasis rate of the non-autoclaved biocontrol strain culture solution is 65 percent, and the average bacteriostasis rate of the autoclaved biocontrol strain culture solution reaches 72 percent. The inhibition effect of the culture solution subjected to high-pressure sterilization on the bipolaris merrilowii root is more obvious, which shows that chemicals having an inhibition effect on pathogenic bacteria of the bipolaris merrilowii root are high-temperature and high-pressure resistant. The invention obtains the biocontrol strain crude toxin extract with increased relative content of bacteriostatic substances by sterilizing without filtering through a bacterial filter (0.22 mu m), thereby showing the phenomenon of obvious increase of bacteriostatic rate.
In conclusion, the stachybotrys cinerea HN17496 strain provided by the invention has a good biocontrol effect on Helminthosporium tritici, and has no potential safety hazards to people and livestock; the embodiment shows that the Stachybotrys botrytis HN17496 strain and the metabolite thereof provided by the invention have the bacteriostasis rate of 65-72% on Helminthosporium moniliforme, and the biocontrol effect is obvious.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A Stachybotrys sp HN17496 strain is characterized in that the preservation number of the HN17496 strain is CCTCC NO: m2022489.
2. A biocontrol bacterial agent comprising the metabolite of HN17496 and/or HN17496 strain of claim 1.
3. The biocontrol microbial inoculum of claim 2, wherein when the biocontrol microbial inoculum comprises HN17496 strain, the number of spores of HN17496 strain in the biocontrol microbial inoculum is not less than 10 per g or per mL of biocontrol microbial inoculum5CFU。
4. A method for preparing the biocontrol microbial inoculum according to claim 2 or 3, characterized by comprising the steps of:
inoculating the HN17496 strain of claim 1 into a PDA culture medium, and culturing for 5-7 days to obtain the biocontrol microbial inoculum.
5. The method according to claim 4, wherein the temperature of the culture is 25 to 28 ℃.
6. The preparation method according to claim 4 or 5, wherein when the biocontrol microbial inoculum does not contain the HN17496 strain, the biocontrol microbial inoculum further comprises a sterilization treatment after culturing for 5-7 days.
7. The method of manufacturing according to claim 6, wherein the method of sterilization treatment comprises: sterilizing at 121 deg.C for 30 min.
8. The HN17496 strain of claim 1, the biocontrol microbial inoculum of claim 2 or 3 or the biocontrol microbial inoculum prepared by the preparation method of any one of claims 4-7, and the application of the biocontrol microbial inoculum in controlling plant diseases, wherein pathogenic bacteria of the plant diseases comprise Helminthosporium tritici.
9. Use according to claim 8, wherein the plant disease comprises root rot.
10. Use according to claim 8 or 9, wherein the plant comprises wheat.
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| CN110088298A (en) * | 2016-08-12 | 2019-08-02 | 维多利亚农业服务控股公司 | The method for characterizing endophyte of plant |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110088298A (en) * | 2016-08-12 | 2019-08-02 | 维多利亚农业服务控股公司 | The method for characterizing endophyte of plant |
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| NL2033035B1 (en) | 2023-12-12 |
| CN114774293B (en) | 2023-08-11 |
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