Application of streptomyces fargesii and fermentation liquor thereof in inhibition of colletotrichum truncatum resisting picolinamide

Abstract

The invention relates to application of streptomyces mnorhizosphere and fermentation liquor thereof in inhibition of colletotrichum truncatum resisting picolinamide, and belongs to the technical field of biological prevention and control of crop diseases. The biocontrol strain 14-3 is named as streptomyces farnesii, is preserved in the Guangdong Microbial Culture Collection Center, and has the preservation number of GDMCC No. 63693. The strain shows remarkable antagonistic activity on colletotrichum truncatum with picolinamide resistance, and fermentation liquor of the strain can effectively inhibit mycelial growth of pathogenic bacteria, germination of conidia and formation of attachment spores and can cause spore splitting decomposition. In addition, fermentation liquor of the strain has a good prevention and treatment effect on soybean anthracnose caused by infection of colletotrichum truncatum resisting picolinamide, and shows good application potential in the aspect of crop drug resistance treatment.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for crop diseases, specifically the application of rhizosphere Streptomyces in controlling the drug resistance of Anthracnose. Background Technology

[0002] Soybean anthracnose is a major disease affecting soybeans worldwide, and it is prevalent in soybean-producing areas of Northeast, North, East, Northwest, and South my country, generally more severe in the south than in the north, less severe in cooler regions, and more severe in tropical and subtropical regions. The pathogen primarily damages pods, stems, and seedlings, causing seedling death, stem dieback, and pod drying without grain formation. The most common pathogen causing soybean anthracnose is *Anthracnose spp.* Chemical control is currently the main approach to controlling soybean anthracnose. Pyridabenamide, a pyridinamide fungicide developed by Corteva, acts on mitochondrial electron transport chain complex III, affecting ATP production by blocking electron transfer between cytochrome b and c1, thereby interfering with the energy cycle within the pathogen. Studies have shown that excessive reliance on fungicides can lead to pesticide residues exceeding limits and pathogen resistance. Therefore, there is an urgent need to explore safe and effective control measures.

[0003] Biological control, with its advantages of safety, environmental friendliness, strong targeting, and low likelihood of developing resistance, has become an important development direction for green control of plant diseases in recent years. Actinomycetes are an important class of biocontrol bacteria; currently, about 52% of the antibiotics discovered are produced by actinomycetes. Castaño et al. found that applying Streptomyces A19 significantly reduced the incidence of tomato wilt and increased the total yield of tomatoes. Studies have shown that a 10% fermentation broth of Streptomyces nojiri 9-13 inhibited the mycelial growth of three anthracnose fungi in soybeans by 56.67%–87.04%, and the inhibitory activity was positively correlated with the concentration of the fermentation broth. Furthermore, a 10% fermentation broth inhibited the germination of conidia of all three anthracnose fungi by 100%. Bercovich et al. found that Streptomyces eurocidicus not only inhibits plant pathogenic fungi but also promotes soybean growth and protects it from fungal infection. Studies by Wang Xuandong et al. have shown that *S. zaomyceticus*, a fungus containing saarmycin, is active against a variety of plant pathogens and can promote the growth and development of rice seedlings.

[0004] Actinomycetes effectively regulate soil microbiota and improve the soil microenvironment by producing active substances such as antibiotics, growth regulators, hydrolytic enzymes, and alkaloids, thereby inducing plant resistance and controlling plant diseases while promoting growth. Therefore, utilizing actinomycetes for anthracnose control is of great significance. Summary of the Invention

[0005] The primary objective of this invention is to address the problems of *Anthracnose spp.* causing anthracnose in crops and resulting in serious economic losses, as well as the ease with which the use of chemical fungicides leads to drug resistance in pathogens. This invention provides the application of rhizosphere *Streptomyces* strains in inhibiting *Anthracnose spp.* resistant to pyridabenamide.

[0006] Another object of the present invention is to provide the application of the fermentation broth of Streptomyces rhizosphere strains in inhibiting pyridine-resistant Anthracnose.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] Application of *Streptomyces rhizosphaerihabitans* strain 14-3 in controlling drug resistance in *Streptomyces rhizosphaerihabitans*, deposited at Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No. 63693, deposit date: August 3, 2023; *Streptomyces rhizosphaerihabitans* strain 14-3 exhibits antagonistic activity against pyridine amide fungicides in *Streptomyces rhizosphaerihabitans*.

[0009] The aforementioned Streptomyces rhizosphaerihabitans strain 14-3 exhibits antagonistic activity against pyridine-lactamazole-resistant Anthracnose.

[0010] Application of the fermentation broth of *Streptomyces rhizosphaerihabitans* strain 14-3 in inhibiting *Anthracis chinensis* resistant to pyridine amide. The fermentation broth is of strain 14-3 of *Streptomyces rhizosphaerihabitans*, deposited at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No. 63693, date of deposit: August 3, 2023.

[0011] Fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 was used to control soybean anthracnose caused by *Streptomyces rhizosphaerihabitans*, which is resistant to pyridine amide fungicides.

[0012] Fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 was used to control soybean anthracnose caused by pyridine-resistant Anthracnose.

[0013] The preparation method of the fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 is as follows: Streptomyces rhizosphaerihabitans strain 14-3 is inoculated into Kjeldahl Synthesis No. 1 medium, fermented at 26-28°C for 7-8 days, extracted with ethyl acetate, concentrated under reduced pressure and evaporated to dryness, and then dissolved in DMSO to obtain the fermentation broth.

[0014] The fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 showed an inhibitory effect on the mycelial growth of Anthracnose anthelmintic induced by pyridine-based styrax.

[0015] The fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 has an inhibitory effect on conidial germination and appressorium formation of pyridine-treated Anthracnose.

[0016] Compared with the prior art, the advantages of the present invention are as follows:

[0017] Streptomyces rhizosphaerihabitans strain 14-3 exhibits antagonistic activity against pyridine-resistant Anthracnose fungicide. Its fermentation broth significantly inhibits mycelial growth, conidial germination, and appressorium formation in pyridine-resistant Anthracnose fungicide, and can lead to spore lysis. Furthermore, the fermentation broth demonstrates good control of soybean anthracnose caused by pyridine-resistant Anthracnose fungicide, which is of significant importance in reducing the use of chemical fungicides. Attached Figure Description

[0018] Figure 1 Culture characteristics of Streptomyces rhizosphaerihabitans strain 14-3 on Gao's No. 1 medium.

[0019] Figure 2 This is a confrontation culture between *Streptomyces rhizosphaerihabitans* strain 14-3 and *Anthracis chinensis* resistant to pyridine amide. Note: A represents resistant strain R1, B represents resistant strain R2, and CK represents the control group.

[0020] Figure 3This study investigates the inhibitory effect of fermentation broth from *Streptomyces rhizosphaerihabitans* strain 14-3 on the mycelial growth of *Anthracis chinensis* resistant to pyridine amide. Note: A represents resistant strain R1, B represents resistant strain R2, and CK represents the control group.

[0021] Figure 4 This study investigates the inhibitory effect of fermentation broth of *Streptomyces rhizosphaerihabitans* strain 14-3 on conidial germination and appressorium formation against pyridine-treated *Anthracnose spp.* Note: A represents the germination of conidia from the resistant strain R1 after treatment with fermentation broth of strain 14-3; B represents the germination of conidia from the resistant strain R2 after treatment with fermentation broth of strain 14-3; CK represents the control group. Detailed Implementation

[0022] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:

[0023] Example 1: Isolation and identification of Streptomyces rhizosphaerihabitans strain 14-3.

[0024] 1. Soil sample collection

[0025] Three soil samples were collected from Jinggang Mountain in Jiangxi Province. The surface soil was removed, and soil samples were collected from a depth of 5-20 cm. After being marked, the samples were brought back to the laboratory and air-dried naturally.

[0026] 2. Isolation of Actinomycetes

[0027] Separation was performed using the plate dilution method. The air-dried soil sample was ground in a mortar and pestle, and 1 g of the sample was weighed and suspended in 9 mL of sterile water. The solution was shaken at 40°C and 180 rpm for 30 min, then allowed to stand for 5 min. The solution was then diluted 10-fold to prepare 10 different solutions. -2 10 -3 10 -4 Suspensions of different concentrations were taken and added to modified HVA medium (with potassium dichromate at a final concentration of 100-200 ppm) plates. After spreading evenly, the plates were inverted and incubated at 28°C for observation. After 5-7 days, different single colonies were picked and streaked for purification. The purified strains were preserved in a -80°C freezer using the glycerol method.

[0028] 3. Identification of Streptomyces rhizosphaerihabitans strain 14-3

[0029] (1) Observation of morphological characteristics

[0030] Streptomyces rhizosphaerihabitans strain 14-3 grows well on most culture media and does not produce soluble pigments (see Table 1). Under an optical microscope, the aerial hyphae of strain 14-3 are septate and produce a large number of spores. The spore chains are straight or flexible, and the spores are spherical or oval.

[0031] Table 1. Culture characteristics of Streptomyces rhizosphaerihabitans 14-3

[0032]

[0033] (2) Physiological and biochemical characteristics

[0034] The starch hydrolysis and nitrate reduction characteristics of Streptomyces rhizosphaerihabitans 14-3 were determined according to the method described in the "Streptomyces Identification Manual". The results are shown in Table 2.

[0035] Table 2. Physiological and biochemical characteristics of Streptomyces rhizosphaerihabitans 14-3

[0036]

[0037] (3) Sequence analysis

[0038] Genomic DNA of Streptomyces rhizosphaerihabitans 14-3 was extracted using a bacterial genome extraction kit, followed by 16S rRNA amplification. The full-length sequence obtained was 1354 bp (see SEQ ID NO:1). The obtained sequence was submitted to the GenBank database for BLAST alignment. Based on morphological characteristics, physiological and biochemical characteristics, and 16S rRNA sequence analysis, strain 14-3 was identified as Streptomyces rhizosphaerihabitans.

[0039] Example 2: Induction and genetic stability of resistance mutants

[0040] Anthracnose bacterium was cultured at 28°C for 7 days on PDA medium. A 5 mm diameter mycelial cake was then collected from the edge of the colony and inoculated onto a medium containing pyridoxine (EC). 90The mutant was cultured at 28°C on PDA plates until a fan-shaped mutant appeared. It was then transferred to another PDA plate and cultured at 28°C for 7 days. The bacterial culture was then transferred to a higher concentration to continue inducing the mutant. The concentration was increased exponentially until colonies appeared on a PDA plate containing 500 μg / mL of the drug. The EC50 of the mutant was then measured. 50 The resistance fold is calculated using the following formula. Based on the resistance fold, the resistance level of the mutant is classified into sensitive, low-resistant, moderately resistant, and highly resistant. Specifically: strains with a resistance fold ≤ 3 are sensitive (S); strains with a resistance fold ≤ 10 are low-resistant (LR); strains with a resistance fold ≤ 100 are moderately resistant (MR); and strains with a resistance fold ≤ 100 are highly resistant (HR). Resistance fold = EC of the resistant mutant. 50 EC of parental susceptible strains 50 .

[0041] The resistant mutants and susceptible strains were subcultured for 10 generations on PDA plates without the pesticide. The EC50 of the first and tenth generation strains against pyridaben was determined using the bacterial growth rate method. 50 The value is used to calculate the change in the drug resistance coefficient of the mutant according to the following formula, and the genetic stability of the mutant is analyzed.

[0042] Change in resistance coefficient = Resistance fold of the 10th generation of the resistant mutant / Resistance fold of the 1st generation of the resistant mutant

[0043] Table 3. Resistance multiples and resistance stability of *Anthracis chinensis* resistant to pyridine amide.

[0044]

[0045] Note: S2 is the parental strain that is susceptible to infection; R1 and R2 are resistant mutants.

[0046] The results showed that two resistant mutants were obtained through indoor drug domestication using *Anthracnose spp.* as the parent strain, with resistance folds ranging from 451.20 to 479, both being highly resistant strains. After subculture, the resistance folds of the 10th generation ranged from 370.17 to 379.33, and the resistance coefficients changed from 0.79 to 0.82, indicating that the resistance to pyridoxine in the resistant mutants is stably inherited (Table 3).

[0047] Example 3: Antagonistic assay of Streptomyces rhizosphaerihabitans 14-3 against pyridoxine-containing Anthracnose strain

[0048] The antagonistic activity of Streptomyces rhizosphaerihabitans strain 14-3 against antagonistic strains R1 and R2 was determined using the plate confrontation culture method. First, strain 14-3 was streaked onto both sides of the edge of PDA medium. After 3 days, a 5 mm diameter mycelial cake of the tested pathogen was inoculated into the center of the plate. After incubation at 28℃ for 5 days, the inhibition band width of strain 14-3 against the tested pathogen was measured (Table 4). The tested pathogen without antagonistic inoculation served as a control. The results showed that strain 14-3 exhibited very strong antagonistic activity against both strains R1 and R2. Figure 2 ).

[0049] Table 4. Antagonistic effect of strain 14-3 against pyridine-based anthracnose fungus.

[0050]

[0051] Example 4: Inhibitory effect of fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 against the mycelial growth of *Anthracis chinensis* induced by pyridine amide.

[0052] Strain 14-3 was activated on ISP2 medium and cultured at 28°C for 5 days. The spores of strain 14-3 were inoculated into Kjeldahl Synthesis No. 1 solid medium and fermented at 26-28°C for 7-8 days. The spores were extracted three times with ethyl acetate. The extract was concentrated under reduced pressure, evaporated to dryness, and then dissolved in DMSO to prepare a fermentation broth of 20 mg / mL for later use.

[0053] 100 µL of the fermentation broth of strain 14-3 was added to 100 mL of PDA medium cooled to approximately 50°C (not hot to the touch). After mixing, the mixture was poured into a petri dish. A 5 mm diameter mycelial cake of the tested pathogen was inoculated into the center of the PDA medium plate. After incubation at 28°C for 5 days, the colony diameter was measured. Using the same amount of DMSO as a control, the inhibition rate of the fermentation broth on the mycelial growth of the pathogen was calculated (Table 5). The results showed that the fermentation broth of strain 14-3 had a very strong inhibitory effect on the mycelial growth of both resistant strains R1 and R2. Figure 3 ).

[0054] Table 5. Inhibitory effect of fermentation broth of strain 14-3 on mycelial growth of resistant strains.

[0055]

[0056] Example 5: Inhibitory effect of fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 against pyridoxine-induced conidial germination and appressorium formation in *Streptomyces rhizosphaerihabitans*.

[0057] A 5 mm diameter mycelial disc of the tested pathogen was inoculated into PDB liquid medium and cultured at 28°C with shaking at 180 rpm for 7 days. The spore suspension was then collected by filtration and its concentration was adjusted to 1×10⁻⁶. 5 / mL. 1µL of the fermentation broth of strain 14-3 from Example 4 was added to 1 mL of spore suspension. Using the same amount of DMSO as a control, spore suspensions of resistant strains R1 and R2 were cultured at 28°C for 4 h. The inhibition rate of the fermentation broth of strain 14-3 on spore germination and appressorium formation of resistant strains R1 and R2 was calculated (Table 6). The results showed that the fermentation broth of strain 14-3 completely inhibited the conidial germination and appressorium formation of resistant strains R1 and R2, and caused spore cell lysis ( Figure 4 ).

[0058] Table 6. Inhibitory effect of fermentation broth of strain 14-3 on conidial germination of antagonistic strains.

[0059]

[0060] Example 6: Application of fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 in the control of soybean anthracnose.

[0061] Resistant strains R1 and R2 were cultured at 28°C for 5 days. Mycelial cakes were collected from the edge of the colony using a 1 mm diameter punch for later use.

[0062] Sow soybean seeds of variety 3 in a sterile seedling substrate (sterile seedling substrate thickness of about 2 cm) in a seedling tray with a row spacing and plant spacing of 3 cm. After sowing, cover with about 1 cm of nutrient soil, sprinkle water until the nutrient soil is moist, and place in the dark for 3 days under the conditions of 23-28°C and relative air humidity not exceeding 80%. When the soybean sprouts grow to 3-4 cm, pull out the soybean sprouts, select the soybean sprouts with relatively uniform growth and cut off the roots, wash them with sterile water and set them aside.

[0063] The 14-3 Kreutz fermentation broth from Example 4 was prepared with sterile water to a concentration of 200 μg / mL, and the 95% difenoconazole technical material was prepared with sterile water to a concentration of 100 μg / mL as a positive control. Then, soybean seedlings were soaked in the above-mentioned agents for 1 h, and the treatment of soaking in sterile water was used as a negative control. After drying, the seedlings were inoculated with pathogens.

[0064] Take about 2 grams of defatted cotton, roll it into a ball, and place it in a 15 mL centrifuge tube. Add 2 mL of sterile water to each tube. After the cotton ball absorbs the water, place it at the bottom of the centrifuge tube for later use. Use a sterile needle to prick a small wound on the stem of a soybean seedling (about 1 mm from the cotyledon). Use the sterile needle to inoculate the wound with pathogenic fungal cake. Cover the fungal cake with a moistened defatted cotton strip and wrap it to keep it moist. Each replicate inoculates 20 soybean sprouts. The treatment inoculated with sterile agar blocks serves as a control. Place the inoculated soybean sprouts root-and-stem side down in the centrifuge tube, ensuring the base of the sprouts is in contact with the defatted cotton ball. Gently twist the centrifuge tube cap half a turn. Incubate the inoculated centrifuge tubes in the dark at 25°C for 3 days. The experiment included six treatments: A: inoculated only with resistant strain R1; B: inoculated only with resistant strain R2; C: 200 μg / mL of 14-3 Kjeldahl fermentation broth inoculated with resistant strain R1; D: 200 μg / mL of 14-3 Kjeldahl fermentation broth inoculated with resistant strain R2; E: 100 μg / mL of difenoconazole inoculated with resistant strain R1; and F: 100 μg / mL of difenoconazole inoculated with resistant strain R2.

[0065] Three days after inoculation, a disease index survey was conducted based on the extent of lesion expansion. The disease grading criteria were as follows: Grade 0, asymptomatic; Grade 1, lesion area less than 10% of stem area; Grade 3, lesion area greater than or equal to 10% and less than 25% of stem area; Grade 5, lesion area greater than or equal to 25% and less than 50% of stem area; Grade 7, lesion area greater than or equal to 50% and less than 75% of stem area; Grade 9, lesion area greater than or equal to 75% of stem area. The disease index was calculated using the formula: Disease Index = [∑(Number of diseased plants at each grade × Relative grade value) ÷ (Total number of plants surveyed × 9)] × 100. The control effect was calculated using the formula: Control effect (%) = [(Disease index of blank control area - Disease index of treated area) ÷ Disease index of blank control area] × 100.

[0066] Table 6. Control effect of fermentation broth of strain 14-3 on soybean anthracnose caused by resistant strains.

[0067]

[0068] Experimental results showed that the control efficacy of 200 μg / mL fermentation broth of strain 14-3 and 100 μg / mL difenoconazole against soybean anthracnose induced by resistant strains R1 and R2 was 67.54%, 67.19%, 68.09%, and 68.60%, respectively, all of which were safe for soybeans and showed no phytotoxicity. At the 5% significance level, the control efficacy of 200 μg / mL fermentation broth of strain 14-3 against soybean anthracnose was not significantly different from that of 100 μg / mL difenoconazole. Therefore, the control efficacy of 200 μg / mL fermentation broth of strain 14-3 against soybean anthracnose induced by *Anthracnose pyridaben* is comparable to that of 100 μg / mL difenoconazole, indicating good application prospects.

Claims

1. The use of a Streptomyces rhizosphaerihabitans strain in inhibiting Colletotrichum gloeosporioides resistant to pyridylamide, characterized in that: the strain is Streptomyces rhizosphaerihabitans strain 14-3, which is preserved in the Guangdong Microbial Culture Collection Center, with the accession number GDMCC No. 63693 and the preservation date of August 3, 2023; the Streptomyces rhizosphaerihabitans strain 14-3 has antagonistic effect on Colletotrichum gloeosporioides resistant to pyridylamide.

2. The use of a Streptomyces rhizosphaerihabitans strain fermentation broth in inhibiting Colletotrichum gloeosporioides resistant to pyridylamide, characterized in that: the fermentation broth is Streptomyces rhizosphaerihabitans strain 14-3 fermentation broth, the strain is Streptomyces rhizosphaerihabitans strain 14-3, which is preserved in the Guangdong Microbial Culture Collection Center, with the accession number GDMCC No. 63693 and the preservation date of August 3, 2023; the Streptomyces rhizosphaerihabitans strain 14-3 fermentation broth is applied to prevent and control soybean anthracnose caused by Colletotrichum gloeosporioides resistant to pyridylamide; the preparation method of the Streptomyces rhizosphaerihabitans strain 14-3 fermentation broth is as follows: inoculate Streptomyces rhizosphaerihabitans strain 14-3 into Keshi synthetic No. 1 medium, ferment at a temperature of 26-28°C for 7-8 days, extract with ethyl acetate, concentrate and evaporate the extract under reduced pressure, then dissolve it with DMSO to obtain the fermentation broth.

3. The use of the Streptomyces rhizosphaerihabitans strain fermentation broth according to claim 2 in inhibiting Colletotrichum gloeosporioides resistant to pyridylamide, characterized in that: the Streptomyces rhizosphaerihabitans strain 14-3 fermentation broth has inhibitory effect on the mycelial growth of Colletotrichum gloeosporioides resistant to pyridylamide.

4. The use of the Streptomyces rhizosphaerihabitans strain fermentation broth according to claim 2 in inhibiting Colletotrichum gloeosporioides resistant to pyridylamide, characterized in that: the Streptomyces rhizosphaerihabitans strain 14-3 fermentation broth has inhibitory effect on the conidial germination and appressorium formation of Colletotrichum gloeosporioides resistant to pyridylamide. ​ ​ ​ ​ ​ ​

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