Rhizosphere streptomyces and fermentation liquor thereof are applied to inhibit colletotrichum gloeosporioides resistant to propiconazole

Abstract

The application relates to application of a rhizosphere streptomyces in inhibition of colletotrichum gloeosporioides resistant to propiconazole, and belongs to the technical field of crop disease biological control. The biocontrol strain 14-3 is named as rhizosphere streptomyces, is preserved in the Guangdong Microbial Culture Collection Center, and has a preservation number of GDMCC No. 63693. The strain shows good antagonism to colletotrichum gloeosporioides resistant to propiconazole, the fermentation liquor can significantly inhibit mycelium growth, conidium germination and appressorium formation of the pathogenic bacteria, and can cause spore lysis. In addition, the fermentation liquor of the strain has excellent prevention and treatment effect on soybean anthracnose caused by colletotrichum gloeosporioides resistant to propiconazole.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for crop diseases, specifically the application of rhizosphere Streptomyces and its fermentation broth in the treatment of Anthracnose spp., which is resistant to propiconazole. Background Technology

[0002] Anthracnose, caused by *Colletotrichum truncatum*, is a major economic disease affecting a wide range of crops globally. Currently, fungicides are the primary means of control. However, excessive reliance on fungicides can lead to pesticide residues, soil pollution, and the development of resistance, necessitating the exploration of safe and effective control measures.

[0003] Microbial inoculants have been widely used in the prevention and control of plant diseases in recent years. Beneficial microorganisms can inhibit the growth of plant pathogens, promote soil nutrient cycling, recruit more beneficial rhizosphere microorganisms, induce or enhance plant disease resistance, and promote plant growth. These mechanisms can alter the interaction between plants and pathogens, thereby inhibiting the occurrence of diseases.

[0004] Actinomycetes are an important class of biocontrol bacteria, and most of the antibiotics discovered to date are produced by actinomycetes. Basavarajappa et al. found that *Streptomyces* SND-2 can enhance the disease resistance of mung beans infected with anthracnose by inhibiting the pathogen and promoting plant growth. Li Juan et al. found that *Streptomyces lavendulae* can produce a variety of antibiotics and has an inhibitory effect on the anthracnose pathogen of strawberries. Zeng et al. found that *Streptomyces diastatochromogenes*, an amylase-producing fungus, can reduce the incidence of anthracnose in bananas and maintain banana quality.

[0005] Therefore, biological control of anthrax has significant economic value and practical significance. Summary of the Invention

[0006] The primary objective of this invention is to address the problems of *Streptomyces chinensis* strains in the rhizosphere, which are responsible for the severe economic losses caused by anthracnose in crops, excessive reliance on fungicides leading to pesticide residues, soil pollution, and resistance.

[0007] Another object of the present invention is to provide the application of the fermentation broth of Streptomyces rhizosphere strains in the treatment of drug resistance in Anthracnose.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] Application of *Streptomyces rhizosphaerihabitans* strain 14-3 in inhibiting propiconazole-resistant *Anthracis chinensis*. This strain is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC No. 63693) on August 3, 2023. *Streptomyces rhizosphaerihabitans* strain 14-3 exhibits antagonistic activity against propiconazole-resistant *Anthracis chinensis*.

[0010] Application of the fermentation broth of *Streptomyces rhizosphaerihabitans* strain 14-3 in inhibiting propiconazole-resistant *Anthracis chinensis*. The fermentation broth is *Streptomyces rhizosphaerihabitans* strain 14-3, 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 propiconazole-resistant Anthracnose.

[0012] 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, and extracted with ethyl acetate. The extract is concentrated under reduced pressure, evaporated to dryness, and then dissolved in DMSO to obtain the fermentation broth.

[0013] The fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 showed an inhibitory effect on the mycelial growth of propiconazole-treated Anthracnose.

[0014] The fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 showed an inhibitory effect on the conidial germination and appressorium formation of propiconazole-treated Anthracnose.

[0015] Compared with the prior art, the advantages of the present invention are as follows:

[0016] Streptomyces rhizosphaerihabitans strain 14-3 exhibits antagonistic activity against propiconazole-resistant Anthracnose. Its fermentation broth significantly inhibits mycelial growth, conidial germination, and appressorium formation in propiconazole-resistant Anthracnose, and can lead to spore lysis. Furthermore, the fermentation broth shows good control efficacy against soybean anthracnose caused by propiconazole-resistant Anthracnose, and can be used for resistance management, which is of great significance for reducing fungicide use and environmental pollution. Attached Figure Description

[0017] Figure 1 Culture characteristics of Streptomyces rhizosphaerihabitans strain 14-3 on Gao's No. 1 medium.

[0018] Figure 2 This is a confrontation culture between *Streptomyces rhizosphaerihabitans* strain 14-3 and propiconazole-resistant *Anthracis chinensis*. Note: A represents the resistant strain R1, B represents the resistant strain R2, and CK represents the control group.

[0019] Figure 3 This study investigates the inhibitory effect of fermentation broth from *Streptomyces rhizosphaerihabitans* strain 14-3 on the mycelial growth of *Anthracis chinensis* resistant to propiconazole. Note: A represents resistant strain R1, B represents resistant strain R2, and CK represents the control group.

[0020] Figure 4 This study investigates the inhibitory effect of fermentation broth of *Streptomyces rhizosphaerihabitans* strain 14-3 on conidial germination and appressorium formation against propiconazole-treated *Anthracis chinensis*. 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

[0021] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:

[0022] Example 1: Isolation and identification of Streptomyces rhizosphaerihabitans strain 14-3.

[0023] 1. Soil sample collection

[0024] 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.

[0025] 2. Isolation of Actinomycetes

[0026] 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.

[0027] 3. Identification of Streptomyces rhizosphaerihabitans strain 14-3

[0028] (1) Observation of morphological characteristics

[0029] 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.

[0030] Table 1. Culture characteristics of Streptomyces rhizosphaerihabitans 14-3

[0031]

[0032] (2) Physiological and biochemical characteristics

[0033] The starch hydrolysis and nitrate reduction characteristics of Streptomyces rhizosphaerihabitans 14-3 were determined according to the method described in the "Handbook of Streptomyces Identification". The results are shown in Table 2.

[0034] Table 2. Physiological and biochemical characteristics of Streptomyces rhizosphaerihabitans 14-3

[0035]

[0036] (3) Sequence analysis

[0037] 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.

[0038] Example 2: Induction and genetic stability of resistant mutants.

[0039] 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 propiconazole (EC). 90 The mutant was cultured at 28°C on a PDA plate 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, each mutant is classified into four resistance levels: 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 = (Resistant mutant EC) / (Resistant mutant EC) 50 Value / Parental susceptible strain EC 50 value.

[0040] The resistant mutant and the susceptible strain were subcultured for 8 generations on PDA plates without the pesticide. The EC50 of the 1st and 8th generation strains to propiconazole was determined by the mycelial growth rate method. 50 Value. Calculate the coefficient of change in drug resistance of the mutant using the following formula to analyze the genetic stability of the mutant.

[0041] Drug resistance change coefficient = resistance multiple of the 8th generation of the resistant mutant / resistance multiple of the 1st generation of the resistant mutant.

[0042] Table 3. Resistance folds and resistance stability of propiconazole-resistant *Anthracis pinnatifida*.

[0043]

[0044] Note: *S3 is a susceptible strain, while R1 and R2 are resistant mutants.

[0045] 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 3.00 to 3.10, both being low-resistance strains. After subculture, the resistance fold in the 8th generation of the resistant mutants was 3.01, and the resistance coefficient changed from 0.97 to 1.00, indicating that the resistance to propiconazole in the resistant mutants is stably inherited (Table 3).

[0046] Example 3: Antagonistic assay of Streptomyces rhizosphaerihabitans 14-3 against propiconazole-treated Anthracnose.

[0047] 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 ).

[0048] Table 4. Antagonistic effect of strain 14-3 against propiconazole-treated Anthracnose spp.

[0049]

[0050] Example 4: The inhibitory effect of fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 on the mycelial growth of Anthracnose spp. antagonized by propiconazole.

[0051] 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.

[0052] 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 ).

[0053] Table 5. Inhibitory effect of fermentation broth of strain 14-3 on mycelial growth of resistant strains.

[0054]

[0055] Example 5: The inhibitory effect of fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 on the antagonistic effect of propiconazole on the germination of conidia and appressorium of Anthracnose.

[0056] 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 3 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 ).

[0057] Table 6. Inhibitory effect of fermentation broth of strain 14-3 on conidial germination of antagonistic strains.

[0058]

[0059] Example 6: Application of fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 in the control of soybean anthracnose.

[0060] 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.

[0061] 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.

[0062] The fermentation broth of strain 14-3 from Example 3 was prepared with sterile water to a concentration of 200 μg / mL. The 98% pyraclostrobin technical grade was prepared with sterile water to a concentration of 100 μg / mL as a positive control. Soybean seedlings were then soaked in the above-mentioned agents for 1 hour. The treatment with sterile water soaking was used as a negative control. After drying, the seedlings were inoculated with soybean anthracnose.

[0063] 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). Inoculate the wound with soybean anthracnose fungal cake using a sterile needle. Cover the fungal cake with a moistened defatted cotton strip and wrap it to retain moisture. Inoculate 20 soybean sprouts per replicate. Use the treatment with sterile agar blocks as a control. Place the inoculated soybean sprouts root-and-stem side down in the centrifuge tube, so that the base of the soybean 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: inoculation with resistant strain R1 only; B: inoculation with resistant strain R2 only; C: 200 μg / mL of Kjeldahl fermentation broth of strain 14-3 + inoculation with resistant strain R1; D: 200 μg / mL of Kjeldahl fermentation broth of strain 14-3 + inoculation with resistant strain R2; E: 100 μg / mL of pyraclostrobin + inoculation with resistant strain R1; and F: 100 μg / mL of pyraclostrobin + inoculation with resistant strain R2.

[0064] 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.

[0065] Table 7. Control efficacy of fermentation broth of strain 14-3 against soybean anthracnose caused by resistant strains.

[0066]

[0067] Experimental results showed that the control efficacy of 200 μg / mL fermentation broth of strain 14-3 and 100 μg / mL pyraclostrobin against soybean anthracnose induced by resistant strains R1 and R2 was 70.17%, 70.12%, 70.76%, and 71.00%, respectively (Table 7), 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 pyraclostrobin. Therefore, the control efficacy of 200 μg / mL fermentation broth of strain 14-3 against soybean anthracnose induced by propiconazole-treated *Anthracnose spp.* is comparable to that of 100 μg / mL pyraclostrobin, indicating good application prospects.

Claims

1. The application of rhizosphere Streptomyces strains in inhibiting propiconazole-resistant Anthracnose, characterized by: The strain described is Streptomyces rhizosphaerihabitans strain 14-3, deposited at Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No. 63693, deposit date: August 3, 2023; The aforementioned Streptomyces rhizosphaerihabitans strain 14-3 exhibits antagonistic activity against propiconazole-treated Anthracnose.

2. The application of fermentation broth from rhizosphere Streptomyces strains in inhibiting propiconazole-resistant Anthracnose, characterized by: The fermentation broth is the fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3, which is deposited at the Guangdong Provincial Microbial Culture Collection Center with accession number GDMCC No. 63693 and deposit date of August 3, 2023. Fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 was used to control soybean anthracnose caused by propiconazole-resistant Anthracnose. 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, and extracted with ethyl acetate. The extract is concentrated under reduced pressure, evaporated to dryness, and then dissolved in DMSO to obtain the fermentation broth.

3. The application of the fermentation broth of the rhizosphere Streptomyces strain according to claim 2 in inhibiting propiconazole-resistant Anthracnose bacillus, characterized in that: The fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 showed an inhibitory effect on the mycelial growth of propiconazole-treated Anthracnose.

4. The application of the fermentation broth of the rhizosphere Streptomyces strain according to claim 2 in inhibiting propiconazole-resistant Anthracnose bacillus, characterized in that: The fermentation broth of Streptomyces rhizosphaerihabitans strain 14-3 showed an inhibitory effect on the conidial germination and appressorium formation of propiconazole-treated Anthracnose.

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