CN113564057B - A detoxifying and antibacterial biocontrol bacterium and its application - Google Patents

Abstract

The invention discloses a detoxification, antibacterial, and biocontrol bacterium and its application. The biocontrol bacterium Glarealozoyensis P1 described in the invention has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M2021019. The 50-fold dilution of the Glarea lozoyensis P1 fermented liquid of the present invention has an inhibitory rate of 95.23% to the spore germination of wheat head blight, and the 50-fold dilution of the sterile filtrate has a 53.30% inhibitory rate to mycelial growth, and the bacteria After co-cultured with Gibberella wheat, no mycotoxins were detected, and the production of toxins was completely inhibited, indicating that the fungus has the effect of inhibiting the production of toxins, and has great application prospects in the control of Gibberella wheat. The disease has good indoor activity. The technical route in the invention is reasonable, can provide reliable pilot test data and design basis for large-scale industrial production, and lay the foundation for the development of related biological preparations and field promotion.

Description

A detoxifying and antibacterial biocontrol bacterium and its application

technical field

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

Background technique

Wheat head blight is the No. 1 wheat disease caused by Fusarium fungi. Its occurrence will not only cause wheat yield reduction, but also threaten human and animal safety due to the toxins (such as DON, ZEN, etc.) produced by Fusarium. Due to the large number of rainy days during the wheat flowering period, wheat scab is greatly affected by climatic factors. Rainfall and airflow provide favorable conditions for the occurrence and prevalence of wheat scab, but also bring many difficulties to chemical control; There are certain difficulties in conventional breeding of disease-resistant varieties, and no breakthrough has been made. For the control of wheat head blight, carbendazim or its compound preparation is used in actual production. Although the effect is better, there is a problem of drug resistance after continuous use. The weakening of the control effect makes the control of scab more and more difficult. How to effectively control scab under the premise of protecting the environment is the primary problem of current research.

In biological control, antagonistic bacteria and their metabolites have played an important role. Active compounds of novel biocontrol bacteria resources are important sources for the development of biopesticides. The biocontrol fungus Glarea lozoyensis P1 obtained in this research has not been reported as a biocontrol resource. Its fermentation liquid has high activity on the mycelium growth and spore germination of Sclerotia spp. Can inhibit the production of vomitoxin by Gibberella wheat. At the same time, the fungus also has high activity against wheat powdery mildew.

Contents of the invention

One aspect of the invention is to develop a novel biocontrol fungus with high control effect on wheat scab. The biocontrol bacterium provided by the present invention has been preserved in the China Type Culture Collection Center (Address: Wuhan University, Wuhan, China) on January 6, 2021, with the preservation number: CCTCC NO: M 2021019, and the name: Glarea lozoyensis P1.

Another aspect of the present invention is to use the developed novel biocontrol bacteria for the prevention and treatment of wheat scab and wheat powdery mildew.

The present invention also provides the fermentation medium of the Glarea lozoyensis P1, including the following components: mannitol 40g/L, enzyme hydrolyzed casein 33g/L, yeast extract 10g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 9g/L, add distilled water to 1L.

Compared with the prior art, the beneficial effect of the present invention is: the fermentation liquid of Glarea lozoyensis P1 of the present invention has significant inhibitory effect on mycelia growth and spore germination of Gibberella wheat, compared with the control, the fermentation of Glarealozoyensis P1 The inhibitory rate of the 10-fold dilution of the fermentation broth to mycelial growth is 100%, and the inhibition rate of the 50-fold dilution of the fermented liquid is 53.3%. In addition, the 50-fold dilution of the fermentation broth of Glarea lozoyensis P1 has the same effect on the spore germination The inhibition effect is above 95%. Even more outstandingly, Glarea lozoyensis P1 inhibits the production of toxin from Gibberella tritici. When Glarea lozoyensis P1 was co-cultured with Gibberella tritici, no vomitoxin was detected, and the inhibition rate of the toxin reached 100%. It shows that the Glarea lozoyensis P1 of the present invention has a significant inhibitory effect on the growth of Gibberella wheat and Gibberella vomitoxin.

Known from above technical scheme: Glarea lozoyensis P1 source of the present invention is reliable, wide to the adaptation range of natural conditions such as temperature, pH, strong to the tolerance of adverse environment, can maintain ecological balance, can adapt to disease ecosystem, easy cultivation and Preservation has a good effect on inhibiting wheat scab, and Glarea lozoyensis P1 also has a high inhibitory effect on wheat powdery mildew. Therefore, Glarea lozoyensis P1 has great application prospects in agriculture.

The technical route of this application is reasonable, which can provide reliable pilot test data and design basis for large-scale industrial production, realize the industrial production of Glarea lozoyensis P1 by liquid fermentation, and lay the foundation for the development of related biological agents and field promotion.

Description of drawings

Fig. 1 is the colony morphology of Glarea lozoyensis P1 of the present invention;

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

Fig. 3 is that Glarea lozoyensis P1 of the present invention is cultivated on different substratum for 25 days, 50 days, and the fermented liquid dilution 10 times after treatment affects the growth of Gibberella wheat mycelium, wherein 1 is 1# substratum, and 2 is 2 # medium, 3 is 3# medium, 4 is 4# medium, 5 is 5# medium, 6 is 6# medium, 7 is 7# medium;

Fig. 4 is the graph of the influence of the fermented liquid of Glarea lozoyensis P1 described in the present invention on the growth of wheat scab mycelium when cultured in 6# medium for 50 days, wherein CK is a blank control;

Fig. 5 is the effect of 5 times, 10 times and 50 times dilution treatment of Glarea lozoyensis P1 fermentation broth of the present invention on the germination of Gibberella wheat spores;

Fig. 6 is the inhibitory effect of the Glarea lozoyensis P1 fermentation broth of the present invention on wheat powdery mildew.

Detailed ways

The following are specific examples of the present invention, and further describe the technical solution of the present invention, but the protection scope of the present invention is not limited to these examples. All changes or equivalent substitutions that do not depart from the concept of the present invention are included in the protection scope of the present invention.

Example 1 Isolation and screening of antagonistic strains

The biocontrol fungus Glarea lozoyensis P1 involved in the present invention was obtained by the first inventor of the present application in August 2020 through screening from sedimentary soil samples in forested mountainous areas of Badong County, Enshi City. The bacterium grew on PDA medium, and after 20 days of culture at 25°C, the colony grew irregularly and was pink, and the colony morphology was shown in Figure 1.

Colony morphology and ITS sequence determination of embodiment 2Glarea lozoyensis P1

Colony morphological characteristics: solid medium (plate, slant medium), accurately weighed 20g of malt extract powder, 1g of soybean peptone, 20g of glucose, 20g of agar, put it in a 1000ml measuring bottle, add water to dissolve and make up to volume, and use 6mol/L The sodium hydroxide solution was adjusted to pH 7.0-7.3. Colony growth color on solid medium: pink, very slow growth, wrinkled and fluffy colony; ciliate at the edge of the colony.

Amplification and sequencing identification of ITS:

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

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

The PCR reaction system was 25 μl, and the amplification program was: 94°C for 5 minutes; 94°C for 30s, 56°C for 30s, 72°C for 30s, 35 cycles; 72°C for 10 minutes.

The PCR product was separated and detected with 1.5% agarose gel, and the PCR product was purified and recovered with Takara gel recovery kit, connected to the T vector, and then transformed into Escherichia coli. Two positive clones were selected for each sample, and the forward and reverse bidirectional sequencing was performed on automatic sequencing with M13 universal primers. The obtained sequences were edited with the Edit seq software and DNAtools in the Dnastar Package, and aligned with MegAlign.

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

Combined with colony morphology and molecular identification, this strain was recorded as Glarea lozoyensis P1.

The different days of embodiment 3 different substratum fermentations affect the mycelia growth of Gibberella wheat

The mycelial growth rate method was used to determine the effect of the fermentation broth of Glarea lozoyensis P1 cultured in 7 different fermentation media and different fermentation days on the mycelial growth of Gibberella tritici. Using the solid medium in Example 1, culture Glarea lozoyensis at 25°C for 15 days, dig out pieces from the well-grown plate medium, transfer to 7 different mediums, and fill 300ml of 1L Crush them in a triangular flask and culture them on a shaker at 25°C and 200r/min for 10d, 25d and 50d.

The formulations of the seven fermentation media are as follows:

1# medium: Mannitol 40g/L, peptone 20g/L, yeast extract 10g/L, add distilled water to 1L.

2# medium: Mannitol 40g/L, yeast nitrogen base (Yeast Nitrogen Base, YNB without ammonium sulfate) 6.7g/L, add distilled water to 1L.

3# medium: mannitol 40g/L, peptone 20g/L, yeast extract 10g/L, yeast nitrogen base (YeastNitrogen Base, YNB does not contain ammonium sulfate) 6.7g/L, add distilled water to 1L.

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

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

6# Medium: Mannitol 40g/L, Casein enzymatic hydrolysate (N-Z-Amine) 33g/L, yeast extract 10g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 9g/L, supplement Distilled water to 1L.

7# medium: mannitol 100g/L, casein enzymatic hydrolysate (N-Z-Amine) 33g/L, yeast extract 10g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 9g/L, supplement Distilled water to 1L.

Fermentation liquid was taken at 10 days, 25 days and 50 days of fermentation respectively, centrifuged at 5000 g for 15 min, supernatant was taken, and filtered with a 0.22 μm aqueous bacterial filter to obtain a sterile fermentation filtrate. According to the volume 1:9 (that is, 1ml fermentation broth + 9 ml), add the fermentation broth and culture medium into the petri dish and mix well. Each culture medium was replicated three times. After the culture was solidified, it was inoculated with the cultured Gibberella tritici, and cultured in the dark at 25°C for 3 days. Colony diameters were measured by the cross method. The results are shown in Table 1.

As can be seen from Table 1, there is a large difference in the inhibition of the growth of Gibberella wheat by different fermentation media (Fig. 3). After 10 days of fermentation broth treatment, the colony growth of Gibberella wheat was larger than the colony diameter of 25 days and 50 days of fermentation; after 25 days of fermentation broth treatment, except for the No. 6 medium formula, the colony diameters of the other 6 formula fermentation broth treatments It should be less than 50 days for fermentation broth treatment. It may be that the nutrients in the different media affect the composition of the active substances. After 50 days of fermented liquid treatment, the fermented liquid treated with the No. 6 medium formula had the smallest colony diameter of Gibberella tritici, only 0.8 cm (Fig. 4).

Table 1 Effects of different fermentation media and different days on the mycelial growth of wheat head blight

The inhibitory effect of embodiment 4 optimal culture medium fermented liquid different dilution multiples to wheat head blight

The 6# medium in Example 3 was selected to ferment the biocontrol bacterium Glarea lozoyensis P1, and it was shaken at 25° C. and 180 rpm for 50 days. Set three gradients of 10-fold, 20-fold and 50-fold dilution of the fermentation broth, according to the 10-fold dilution (ie 1mL fermentation broth + 9ml medium), the 20-fold dilution (ie 0.5mL fermentation broth + 9.5mL medium), and the dilution of 50 times (ie 200 μL of fermentation broth + 9.8 mL of culture medium), the fermentation broth and culture medium were added to the petri dish and mixed. Each dilution factor was repeated three times. After the culture was solidified, it was inoculated with the cultured Gibberella tritici and cultured in the dark at 25°C for 3 days. Colony diameters were measured by the cross method. The formula for calculating the growth inhibition rate of colony expansion:

The test results are shown in Table 2. As can be seen from Table 2, different dilutions of the Glarea lozoyensis P1 fermentation broth have inhibitory effects on wheat head blight, and the inhibition rates of 10-fold, 20-fold and 50-fold dilutions are 100%, 64.72% and 53.3% respectively.

Table 2 Inhibitory effect of different times of dilution of Glarea lozoyensis P1 fermentation broth on mycelial growth of Gibberella wheat

The influence of embodiment 5 optimal culture medium fermented liquid on the spore germination of wheat scab

The test of spore germination of Gibberella wheat was determined by the spore germination method. First, the conidia of Gibberella wheat were prepared, and the Gibberella wheat PH-1 preserved in our laboratory was activated on fresh PDA medium, and kept in the dark at 25°C. After 3 days of cultivation, 10 pieces (8 mm) of colony edge hyphae were taken and transferred to 150 mL of carboxymethyl cellulose ester liquid medium (CMC), placed on a constant temperature shaker, and shaken at 25 °C and 150 r/min for 5 days. The conidia suspension of the tested strain was filtered with double gauze, and the spore concentration was adjusted to 1×10 ⁴ by counting with a hemocytometer. The No. 6 medium in Example 3 was selected, and the fermented biocontrol bacteria Glarea lozoyensis P1 was shaken at 25° C. and 180 rpm for 50 days. Three gradients of 5-fold, 10-fold and 50-fold dilution of the fermentation broth were set, and the total volume observed for spore germination was 1 mL (spore-fermentation broth mixed sample). Dilute the fermented broth with an aqueous solution of conidia, and add the fermented broth and spore solution to a 1.5mL centrifuge tube as follows: 200 μL: 800 μL (diluted 5 times); 100 μL: 900 μL (diluted 10 times); 20 μL: 980 μL (diluted 10 times); 50 times). After configuration, place it at 25°C and incubate for 4-8 hours, and use the one without fermentation broth as the blank control. Five fields of view were randomly selected under the microscope, and 20 spores were observed in each field of view, and the germination of the spores was recorded. The results are shown in Table 3 and Figure 5.

As can be seen from Table 3, along with the increase of the dilution multiple of Glarea lozoyensis P1 fermented liquid, the inhibitory rate to Gibberella wheat conidia gradually decreases, and when diluted 5-10 times, the inhibitory rate is up to 100%, and when the diluted multiple is 50 times , the inhibition rate was 95.23%. It shows that the fermentation broth of Glarea lozoyensis P1 has a significant inhibitory effect on the spore germination of Gibberella

Table 3 Inhibitory effect of Glarea lozoyensis P1 fermentation broth on spore germination of Gibberella

Example 6 Research on the Degradation Effect of Glarea lozoyensis P1 Fermented Liquid on Mycotoxins

Weigh 200g of rice, add 600g of deionized water and put it in a 1L Erlenmeyer flask for sterilization. After sterilization, stir it evenly with a glass rod. A total of 6 bottles are needed. Then each bottle was inoculated with the same mycelia block of Gibberella wheat, among which 3 bottles were inoculated with biocontrol fungus Glarealozoyensis P1, and the remaining 3 bottles were used as blank control. Shake the bottle every other day during cultivation at 25°C to allow Gibberella and biocontrol bacteria Glarea lozoyensis P1 to grow evenly. After 30 days of growth, the rice toxin medium was collected into tinfoil paper and dried in an oven at 70°C for toxin detection. The detection of mycotoxins was entrusted to the Institute of Agricultural Product Quality Safety and Nutrition, Jiangsu Academy of Agricultural Sciences. The test results are shown in Table 4.

It can be seen from Table 4 that only the three control treatments inoculated with wheat head blight could detect toxins and the content was also high, while the three treatments inoculated with the biocontrol fungus Glarea lozoyensis P1 could not detect any toxins , indicating that the biocontrol bacteria can inhibit the production of gibberellin in wheat.

Table 4 Inhibitory effect of Glarea lozoyensis P1 fermentation broth on toxin

Example 7 Determination of indoor biological activity of Glarea lozoyensis P1 fermentation broth on wheat powdery mildew bacteria

Using the isolated leaf method. The wheat cultivated to 1 leaf and 1 heart, the first leaf was cut into 30 mm long leaf segments, and placed on 0.5% water agar fresh-keeping medium containing 50 μg/mL 99% benzimidazole (produced by Shanghai Sinopharm Group) for later use. Using the fermentation broth of different fermentation formulas in Example 2, draw 4mL of Glarea lozoyensis P1 50-day fermentation broth and add surfactant Tween 20 (final concentration is 2.5 × 10 ^-4 ), mix thoroughly and place in Porter spray tower (Potter precision laboratory spray tower, British BURKARD instrument company), the spray volume is 350mL/m ² , spraying NA medium containing 2.5×10 ^-4 Tween 20 as negative control, and clear water spraying as blank control , dry naturally, and inoculate conidia of wheat powdery mildew after 24 hours (the inoculum size is 10-20 conidia/10 times visual field). Then cultured in an air-conditioned room at 17°C, protected from light for 12 hours, and then cultivated under 40W fluorescent lamp for 24 hours. After the blank control was fully infected (7d), the disease severity of each leaf of each treatment was investigated. Each treatment was repeated 3 times. The test results are shown in Table 5 and Figure 6.

Control effect (%) = (CK-T) × 100/CK

CK is the average severity of blank control; T is the average severity of treatment.

It can be seen from Table 5 that the activity of 50-day fermentation broths of different fermentation formulas on wheat powdery mildew also differs. Among them, the control effect of No. 1 fermentation liquid reached 90.23%, and the control effect of No. 6 fermentation liquid was 80.14%. It shows that the fermentation broth of the biocontrol bacteria also has high activity against wheat powdery mildew.

Table 5 Antibacterial activity of Glarea lozoyensis P1 fermentation broth against wheat powdery mildew

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present invention can easily think of changes or Replacement should be covered within the protection scope of the present invention.

sequence listing

<110> Institute of Plant Protection Soil and Fertilizer, Hubei Academy of Agricultural Sciences

<120> A Detoxification and Antibacterial Biocontrol Bacteria and Its Application

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<170> SIPOSequenceListing 1.0

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<213> Glarea lozoyensis P1

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agccctgtag cgaggagtat tactacgctt agagccgggt ggcgccgcca ctgattttaa 120

ggcccgcccg gaggcgagag cccaagaccc agcaagctgg attggtttaa atgacgctcg 180

aacaggcatg ccccgcggaa taccacgggg cgcaatgtgc gttcaaagat tcgatgattc 240

actgaattct gcaattcaca ttacttatcg catttcgctg cgttcttcat cgatgccaga 300

accaagagat ccgttgttga aagttttaac tattatatag tactcagacg acactaacat 360

tcagagtttg gggtcctctg gcaggcgcga cccggccgga gccggtgccc gagggcggcc 420

tgccaaagca acatgatata ttacacaagg gtgggcgt 458

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cctacctgat ccgaggtcaa 20

Claims (3)

1. A detoxifying, antibacterial, and biocontrol bacterium, characterized in that: the biocontrol bacterium is Glarea lozoyensis P1, which was preserved in the China Center for Type Culture Collection on January 6, 2021, and its preservation number is CCTCC NO : M 2021019. 2. The application of Glarea lozoyensis P1 according to claim 1, characterized in that: said Glarea lozoyensis P1 is used for preventing and treating wheat scab and wheat powdery mildew. 3. according to the fermentation method of the described Glarea lozoyensis P1 of claim 1, it is characterized in that, the culture medium in the described fermentation method is made of following composition: Mannitol 40 g/L, enzymatic hydrolysis casein 33g/L, yeast Extract 10 g/L, ammonium sulfate 5 g/L, potassium dihydrogen phosphate 9 g/L, add distilled water to 1 L.

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