阅读说明：本技术 一株解毒抑菌生防菌及其应用 (Detoxifying and bacteriostatic biocontrol bacterium and application thereof ) 是由 龚双军 杨立军 李佳静 向礼波 薛敏峰 史文琦 曾凡松 刘美玲 陈婷婷 袁斌 阙 于 2021-08-17 设计创作，主要内容包括：本发明公开了一株解毒抑菌生防菌及其应用,本发明所述的生防菌Glarealozoyensis P1已保藏在中国典型培养物保藏中心,保藏编号为CCTCC NO:M2021019。本发明所述Glarea lozoyensis P1发酵液的50倍稀释液对小麦赤霉病菌的孢子发芽抑制率为95.23％,无菌滤液的50倍稀释液对菌丝生长的抑制率为53.30％,并且该菌与小麦赤霉菌共培养后,未检测到真菌毒素,完全抑制毒素的产生,说明该菌具有抑制毒素产生的效果,在小麦赤霉菌防治方面有很大的应用前景,同时该菌还对小麦白粉病具有较好的室内活性。本发明中的技术路线合理,可为大规模工业化生产提供可靠的中试数据和设计依据,为开发相关其生物制剂及大田推广奠定基础。(The invention discloses a detoxification and bacteriostasis biocontrol bacterium and application thereof, wherein the biocontrol bacterium Glaarlozoyensis P1 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2021019. The 50-time diluent of the Glarea lozoyensis P1 fermentation liquid has a spore germination inhibition rate of 95.23% for wheat scab, the 50-time diluent of the sterile filtrate has an inhibition rate of 53.30% for hypha growth, and after the bacterium is co-cultured with wheat scab, mycotoxin is not detected, and the generation of toxin is completely inhibited, so that the bacterium has an effect of inhibiting the generation of toxin, has a great application prospect in the prevention and treatment of wheat scab, and has good indoor activity for wheat powdery mildew. The method has reasonable technical route, can provide reliable pilot plant test data and design basis for large-scale industrial production, and lays a foundation for developing related biological agents and field popularization.)

1. A strain of detoxication bacteriostasis biocontrol bacteria is characterized in that: the biocontrol bacterium is Glarea lozoyensis P1, which has been preserved in China center for type culture Collection at 1 month and 6 days 2021, and the preservation number is CCTCC NO: M2021019.

2. The use of Glarea lozoyensis P1 according to claim 1, wherein: the Glarea lozoyensis P1 can be used for preventing and treating wheat scab and wheat powdery mildew.

3. The method of claim 1, wherein the culture medium comprises the following components: 40g/L of mannitol, 33g/L of enzyme hydrolyzed casein, 10g/L of yeast extract, 5g/L of ammonium sulfate, 9g/L of monopotassium phosphate and 1L of supplementary distilled water.

Technical Field

The invention belongs to the technical field of microorganisms, and relates to application of fungi in the field of biocontrol, in particular to a Glarea lozoyensis P1 strain and application thereof in preventing and treating wheat scab and wheat powdery mildew.

Background

Wheat scab is the first disease of wheat caused by fusarium fungi, and the occurrence of wheat scab not only causes the yield reduction of wheat, but also threatens the safety of people and animals due to toxins (such as DON, ZEN and the like) generated by fusarium. Because the rainfall days of the wheat in the flowering period are more, the wheat scab is greatly influenced by climatic factors, and the rainfall and airflow provide favorable conditions for the occurrence and prevalence of the wheat scab and bring a lot of difficulties for chemical prevention and control; in addition, the conventional breeding has certain difficulty in breeding disease-resistant varieties, and breakthrough progress is not obtained all the time. For the prevention and treatment of wheat scab, carbendazim or a compound preparation thereof is used in actual production, although the effect is good, the problem of drug resistance is caused after continuous use. The weakening of the control effect leads to the increasing difficulty in controlling the gibberellic disease, and how to effectively control the gibberellic disease on the premise of protecting the environment is the first problem of current research.

Antagonistic bacteria and their metabolites play an important role in biological control. Active compounds of novel biocontrol bacteria resources are important sources for developing biopesticides. The biocontrol bacterium Glarea lozoyensis P1 obtained in the research is not reported as a biocontrol resource, the fermentation liquor has high activity on mycelial growth and spore germination of the wheat scab germ, and the biocontrol bacterium can inhibit the generation of wheat scab vomitoxin. Meanwhile, the strain also has higher activity on erysiphe graminis.

Disclosure of Invention

On the one hand, the invention develops a novel biocontrol bacterium with higher control effect on wheat scab. The biocontrol bacteria provided by the invention have been preserved in China center for type culture Collection (address: Wuhan university in Wuhan, China) in 2021, month 1 and day 6, and the preservation number is as follows: CCTCC NO: M2021019, name: glarea lozoyensis P1.

The other aspect of the invention is to use the developed novel biocontrol bacterium for preventing and treating wheat scab and wheat powdery mildew.

The invention also provides a fermentation medium of the Glarea lozoyensis P1, which comprises the following components: 40g/L of mannitol, 33g/L of enzyme hydrolyzed casein, 10g/L of yeast extract, 5g/L of ammonium sulfate, 9g/L of monopotassium phosphate and 1L of supplementary distilled water.

Compared with the prior art, the invention has the beneficial effects that: compared with a control, the fermentation liquor of the Glarea lozoyensis P1 has a remarkable inhibition rate on hypha growth of 100% by 10 times of dilution liquor, the fermentation liquor of 50 times of dilution liquor has an inhibition rate on hypha growth of 53.3%, and in addition, the fermentation liquor of 50 times of dilution liquor of the Glarea lozoyensis P1 has an inhibition effect on spore germination of more than 95%. More particularly, the Glarea lozoyensis P1 inhibits the production of the wheat scab germ toxin, and the Glarea lozoyensis P1 and wheat scab are co-cultured, so that vomitoxin is not detected, and the inhibition rate of the toxin reaches 100%. The Glarea lozoyensis P1 has obvious inhibition effect on growth of wheat gibberella and gibberella vomitus toxin.

The technical scheme shows that: the Glarea lozoyensis P1 has reliable source, wide application range to natural conditions such as temperature, pH and the like, strong tolerance to adverse environment, ecological balance maintenance, adaptability to disease ecosystem, easy culture and preservation and good effect of inhibiting wheat scab, and the Glarea lozoyensis P1 also has higher inhibiting effect on wheat powdery mildew, so the Glarea lozoyensis P1 has great application prospect in agriculture.

The method has reasonable technical route, can provide reliable pilot plant data and design basis for large-scale industrial production, realizes the industrial production of the Glarea lozoyensis P1 by liquid fermentation, and lays a foundation for developing related biological agents and field popularization.

Drawings

FIG. 1 is a colony morphology of Glarea lozoyensis P1 according to the present invention;

FIG. 2 is an agarose gel electrophoresis image of ITS amplified fragment of Glarea lozoyensis P1 strain according to the present invention. M represents the DNA molecular weight; 1-2 represent Glarea lozoyensis P1 samples;

FIG. 3 shows that the fermentation broth of Glarea lozoyensis P1 of the present invention after culturing for 25 days and 50 days on different media has 10-fold dilution effect on the growth of gibberellic hyphae of wheat, wherein 1 is # 1 medium, 2 is # 2 medium, 3 is # 3 medium, 4 is # 4 medium, 5 is # 5 medium, 6 is # 6 medium, and 7 is # 7 medium;

FIG. 4 is a graph showing the effect of fermentation broth of Glarea lozoyensis P1 on the growth of Fusarium graminearum filaments in # 6 medium for 50 days, where CK is a blank control;

FIG. 5 shows the effect of 5-fold, 10-fold and 50-fold dilution treatment of the fermentation broth of Glarea lozoyensis P1 on the germination of Gibberella cerevisiae spores;

FIG. 6 shows the inhibitory effect of the fermentation broth of Glarea lozoyensis P1 on Erysiphe graminis.

Detailed Description

The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the scope of the present invention is not limited to these examples. All changes, modifications and equivalents that do not depart from the spirit of the invention are intended to be included within the scope thereof.

Example 1 isolation and screening of antagonistic strains

The biocontrol bacterium Glarea lozoyensis P1 is obtained by screening deposited soil samples from forest trees planted in Badong county of Enshi city by the first inventor in 8 months of 2020. The strain grows on a PDA culture medium, and after the strain is cultured for 20 days at 25 ℃, the colony grows irregularly and is pink, and the colony morphology is shown in figure 1.

Example 2 colony morphology and ITS sequencing of Glarea lozoyensis P1

And (3) colony morphology characteristics: precisely weighing 20g of malt extract powder, 1g of soybean peptone, 20g of glucose and 20g of agar in a solid culture medium (a flat plate and a slant culture medium), adding water to dissolve and fix the volume, and adjusting the pH to 7.0-7.3 by using 6mol/L sodium hydroxide solution. Colony growth color on solid medium: pink, very slow growth speed, and wrinkled and villous colony; the colony is ciliated.

Amplification and sequencing identification of ITS:

forward primer for PCR amplification 62F: ACGCCCACCCTTGTGTAATA (SEQ ID NO: 2);

518R：CCTACCTGATCCGAGGTCAA(SEQ ID NO:3)。

the PCR reaction system is 25 μ l, and the amplification procedure is: 5min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃, 30s at 72 ℃ and 35 cycles; 10min at 72 ℃.

The PCR product was separated and detected with 1.5% agarose gel, purified and recovered with Takara gel recovery kit, ligated to T vector, and transformed into E.coli. 2 positive clones were picked for each sample and sequenced in both forward and reverse directions with M13 universal primers on automated sequencing. The resulting sequences were edited using Edit seq software in Dnastar Package and DNATOLs, and aligned using MegAlign.

The ITS gene of the fungus is amplified and determined (shown in a sequence table SEQ ID NO: 1), and an agarose gel electrophoresis pattern of a PCR product is shown in figure 2. The sequence size obtained by sequencing is 457bp, the sequence size is compared at NCBI website, and the similarity with Glarea lozoyensis reaches 99%. Therefore, the bacterium was identified as Glarea lozoyensis.

This strain was designated as Glarea lozoyensis P1 in combination with colony morphology and molecular characterization.

Example 3 Effect of different media fermentation on the growth of wheat gibberellic hyphae on different days

The influence of fermentation liquor of Glarea lozoyensis P1 on the growth of wheat gibberella hyphae when the fermentation liquor is cultured in 7 different fermentation culture media and different fermentation days is measured by adopting a hypha growth rate method. Culturing Glarea lozoyensis in the solid medium of example 1 at 25 ℃ for 15 days, digging out pieces from the well-grown plate medium, transferring into 7 different media, loading into 300ml of 1L triangular flask, crushing, and culturing on a shaker at 25 ℃ and 200r/min for 10d, 25d, and 50 d.

The formula of 7 kinds of fermentation culture media are respectively as follows:

1# Medium: 40g/L of mannitol, 20g/L of peptone and 10g/L of yeast extract, and distilled water is supplemented to 1L.

2# Medium: 40g/L of mannitol, 6.7g/L of Yeast Nitrogen source Base (Yeast Nitrogen Base, YNB does not contain ammonium sulfate), and distilled water is supplemented to 1L.

3# Medium: 40g/L mannitol, 20g/L peptone, 10g/L Yeast extract, 6.7g/L Yeast Nitrogen source Base (Yeast Nitrogen Base, YNB ammonium sulfate free), and distilled water to 1L.

4# Medium: 40g/L mannitol, 20g/L peptone, 10g/L yeast extract, 5g/L ammonium sulfate, 1g/L potassium dihydrogen phosphate, and distilled water to 1L.

5# Medium: 40g/L mannitol, 33g/L peptone, 10g/L yeast extract, 5g/L ammonium sulfate, 9g/L potassium dihydrogen phosphate, and distilled water to 1L.

6# Medium: 40g/L of mannitol, 33g/L of enzymatically hydrolyzed Casein (N-Z-Amine), 10g/L of yeast extract, 5g/L of ammonium sulfate and 9g/L of monopotassium phosphate, and distilled water is supplemented to 1L.

7# Medium: 100g/L of mannitol, 33g/L of enzymatically hydrolyzed Casein (N-Z-Amine), 10g/L of yeast extract, 5g/L of ammonium sulfate and 9g/L of monopotassium phosphate, and distilled water is supplemented to 1L.

Respectively fermenting for 10 days, 25 days and 50 days, centrifuging at 5000g speed for 15min, collecting supernatant, and filtering with 0.22 μm water phase bacterial filter membrane to obtain sterile fermentation filtrate. The fermentation broth and the culture medium were added to the petri dish at a volume of 1:9 (i.e. 1ml of fermentation broth +9 ml) and mixed well. Repeating each culture medium for 3 times, inoculating with cultured Gibberella tritici, and culturing at 25 deg.C in dark for 3 days. The colony diameter was measured by the cross method. The results are shown in Table 1.

As can be seen from Table 1, there was a large difference in the inhibition of the growth of Gibberella tritici by the different fermentation media (FIG. 3). Treating the fermentation liquor for 10 days, wherein the colony growth of the gibberella zeae is larger than the colony diameter of the gibberella zeae after fermentation for 25 days and 50 days; the culture medium is treated in 25 days, and the colony diameter of the culture medium treated by other 6 formulas except the formula 6 is smaller than that of the culture medium treated by 50 days. This may be the composition of the active substances that are influenced by the nutritional composition in the different media. Fermentation broth treatment for 50 days, fermentation broth treatment for medium formulation No. 6, the colony diameter of gibberella tritici was the smallest, only 0.8cm (fig. 4).

TABLE 1 Effect of different fermentation media on growth of wheat scab germ filaments on different days

Example 4 inhibitory Effect of different dilution times of optimal Medium fermentation broth on Gibberella zeae

The biocontrol strain Glarea lozoyensis P1 was selected for fermentation using the # 6 medium of example 3, and was shaken at 180rpm at 25 ℃ for 50 days. Setting three gradients of 10-fold dilution, 20-fold dilution and 50-fold dilution of the fermentation liquor, diluting by 20-fold (namely 0.5mL of fermentation liquor and 9.5mL of culture medium) according to the dilution by 10-fold (namely 1mL of fermentation liquor and 9.5mL of culture medium), and adding the fermentation liquor and the culture medium into a culture dish for uniformly mixing by 50-fold dilution (namely 200 mu L of fermentation liquor and 9.8mL of culture medium). Each dilution multiple is repeated for 3 times, after the culture and the inoculation are solidified, the cultured wheat gibberella is inoculated, and the dark culture is carried out for 3 days at the temperature of 25 ℃. The colony diameter was measured by the cross method. The colony expansion growth inhibition rate calculation formula is as follows:

the test results are shown in Table 2. As shown in Table 2, the fermentation broth of Glarea lozoyensis P1 has inhibitory effects on Gibberella zeae at different dilution times, and the inhibition rates of 10-fold, 20-fold and 50-fold dilution are respectively 100%, 64.72% and 53.3%.

TABLE 2 inhibitory effect of different dilution times of fermentation broth of Glarea lozoyensis P1 on growth of gibberellic hyphae of wheat

Example 5 Effect of optimal Medium fermentation broth on spore germination of Gibberella zeae

The spore germination test of wheat gibberellic disease is determined by spore germination method, firstly, the conidia of wheat gibberellic disease is prepared, the wheat gibberellic disease PH-1 stored in the laboratory is activated on fresh PDA culture medium, cultured for 3d under dark condition of 25 deg.C, 10 (8mm) colony edge hypha blocks are taken out and transferred into 150mL carboxymethyl cellulose ester liquid Culture Medium (CMC), placed on a constant temperature shaking bed, shaken for 5d at 25 deg.C and 150r/min, the conidia suspension of the tested strain is respectively filtered by double gauzes, the count of a blood counting chamber is used for adjusting the spore concentration to be 1 × 10⁴. The medium No. 6 used in example 3 was selected and the biocontrol bacterium Glarea lozoyensis P1 was shaken at 25 ℃ and 180rpm for 50 days. Setting three gradients of 5 times, 10 times and 50 times of dilution of fermentation liquor, and observing spore germinationThe total volume of the fermentation was 1mL (spore-fermentation broth mixture). The fermentation liquor is diluted by conidium aqueous solution, and the volumes of the fermentation liquor and the conidium solution are respectively added into a centrifugal tube with the volume of 1.5mL as follows: 200 μ L: 800 μ L (diluted 5-fold); 100 μ L of: 900 μ L (diluted 10-fold); 20 μ L of: 980 μ L (50-fold dilution). After the preparation, the mixture is placed at 25 ℃ for culturing for 4-8h, and a blank control without adding fermentation liquor is used. Under the microscope, 5 fields were randomly selected, 20 spores were observed in each field, and germination of spores was recorded. The results are shown in Table 3 and FIG. 5.

As can be seen from Table 3, the inhibition rate of the fermentation broth of Glarea lozoyensis P1 on the wheat gibberella conidia is gradually reduced with the increase of the dilution factor, the inhibition rate is as high as 100% when the fermentation broth is diluted by 5-10 times, and the inhibition rate is 95.23% when the dilution factor is 50 times. The fermentation liquor of Glarea lozoyensis P1 has obvious inhibition effect on spore germination of gibberella

TABLE 3 spore germination inhibitory effect of Glarea lozoyensis P1 fermentation broth on Gibberella sp

Example 6 investigation of the Effect of Glarea lozoyensis P1 fermentation broth on the degradation of mycotoxins

200g of rice is weighed, 600g of deionized water is added, the rice is placed in a 1L triangular flask for sterilization, and after sterilization, the rice is uniformly stirred by a glass rod, and 6 bottles are needed. Each vial was then inoculated with the same piece of Gibberella tritici mycelium, 3 of which were inoculated with the biocontrol bacterium Glarea lozoyensis P1, leaving 3 vials as blank controls. The flask was shaken every 1 day during the incubation at 25 ℃ to allow the growth of the gibberellin and biocontrol bacterium Glarea lozoyensis P1 to be uniform. After 30 days of growth, the rice toxin culture medium is collected into tin foil paper, and is placed in an oven at 70 ℃ for drying for toxin detection. The detection of the mycotoxin is finished by the research institute of agricultural product quality safety and nutrition of agricultural science college of Jiangsu province. The results of the tests are shown in Table 4.

As can be seen from Table 4, the toxin was detected and the content was high only by the 3 control treatments inoculated with the fungus Fusarium graminearum, while any toxin was not detected by the 3 treatments inoculated with the biocontrol bacterium Glarea lozoyensis P1, indicating that the biocontrol bacterium can suppress the production of the fungus Fusarium graminearum.

TABLE 4 inhibitory Effect of Glarea lozoyensis P1 fermentation broth on toxins

Example 7 indoor bioactivity assay of Glarea lozoyensis P1 fermentation broth for Blumeria graminis

The in vitro leaf segment method is adopted. The wheat cultured to 1 leaf and 1 heart is cut into 30mm long leaf segments by the 1 st leaf and placed on a 0.5 percent water agar fresh-keeping culture medium containing 50 mu g/mL 99 percent benzimidazole (produced by Shanghai national drug group) for later use. Using the fermentation broths of the different fermentation formulations of example 2, 4mL of Glarea lozoyensis P150-day fermentation broth was aspirated and surfactant Tween 20 (final concentration 2.5X 10)^-4) After being sufficiently mixed, the mixture was applied to a spray tower (Potter precision laboratory spray tower, Burgard instruments, UK) at a spray rate of 350mL/m²Spraying the mixture containing 2.5 x 10^-4Treating the NA culture medium of the Tween 20 as a negative control, spraying clear water as a blank control, naturally airing, and inoculating the conidia of the erysiphe graminis (the inoculum size is 10-20 conidia/10 times of visual field) after 24 hours. Then culturing in an air-conditioned room at 17 ℃, keeping out of the sun for 12h, and then culturing under the irradiation of 24h daylight lamp 40W. After the placebo had fully developed disease (7d), the severity of disease development was investigated for each leaf treated. Each treatment was repeated 3 times. The test results are shown in table 5 and fig. 6.

The preventing and treating effect (%) is (CK-T) multiplied by 100/CK

CK is the placebo mean severity; t is the treatment mean severity.

As can be seen from Table 5, the activity of the 50-day fermentation broths of the different fermentation formulations on wheat powdery mildew also differed. Wherein the control effect of the fermentation liquor No. 1 reaches 90.23 percent, and the control effect of the fermentation liquor No. 6 reaches 80.14 percent. The fermentation liquor of the biocontrol bacteria also has higher activity on Erysiphe graminis.

TABLE 5 bacteriostatic activity of Glarea lozoyensis P1 fermentation broth on Erysiphe graminis

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

<110> institute of soil and fertilizer for plant protection of academy of agricultural sciences of Hubei province

<120> detoxification and bacteriostasis biocontrol bacterium and application thereof

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