Burkholderia gladioli H02, fungicide, bacterial fertilizer and application of burkholderia gladioli H02

Abstract

The invention discloses burkholderia gladioli H02, a bacterial agent, a bacterial fertilizer and application of the burkholderia gladioli H02. The strain has a remarkable antagonistic effect on phytopathogens such as sclerotium rolfsii and fusarium oxysporum, has an inhibiting effect on growth of barnyard grass, and has a preservation number of CGMCC (China General Microbiological Culture Collection Center) No.35003. The invention further provides a method for preventing and treating southern blight through combination of H02 and a chemical pesticide thifluzamide for peanut crops. The burkholderia gladioli H02 as well as the microbial agent and the bacterial fertilizer thereof have important significance on control of weed damage, prevention and treatment of soil-borne diseases and pesticide reduction and synergism of agricultural production.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbial control technology. It discloses a strain of Burkholderia gladioli H02, its inoculant, its inoculant fertilizer, and their applications. Background Technology

[0002] Soil-borne diseases and noxious weeds have always been key factors restricting high and stable crop yields and quality improvement. For example, white rot is a soil-borne fungal disease caused by *Sclerotium rolfsii*. Watermelon wilt is another typical soil-borne vascular disease caused by *Fusarium oxysporum* f. sp. *niveum*. Barnyard grass (*Echinochloa crus-galli*) is one of the most noxious and common farmland weeds worldwide. Currently, the control of these diseases and weeds still heavily relies on chemical pesticides. Chemical control is fast-acting, but long-term use alone can easily lead to drug resistance in pathogens. Broad-spectrum fungicides, while killing pathogens, can also harm beneficial microorganisms in the soil, damaging soil health and posing environmental pollution risks. In contrast, biocontrol microorganisms, as the core component of biological pesticides, have significant advantages such as environmental friendliness, strong target specificity, and low resistance induction. However, their colonization efficiency is often affected by environmental factors, and their field application effects are unstable. The combined application of chemical pesticides and biocontrol microorganisms can achieve reduced pesticide use and increased yield while ensuring efficacy, thereby protecting the ecological environment. This is an innovative strategy for plant disease control. Burkholderia is a rich microbial resource, with diverse metabolites that produce various antimicrobial substances, demonstrating great potential in biological control. Several studies have reported on Burkholderia's antagonistic effects on pathogenic fungi and its ability to promote crop growth. For example, patent document CN120025930A discloses a strain of Burkholderia para. D15, which exhibits strong inhibitory effects against Xanthomonas oryzae Xoo, the pathogen causing bacterial blight in rice. It also has a strong inhibitory effect on other pathogens that cause crop diseases, such as rice sheath blight, rice blast, Fusarium graminearum, Sclerotinia sclerotiorum, and bacterial wilt. Patent document CN115975879A discloses a strain of Burkholderia multocida MLR-H8 that can effectively inhibit the mycelial growth of Fusarium graminearum, inhibit the occurrence of corn stem rot, and promote corn plant growth. At the same time, it has a strong inhibitory effect on the mycelial growth of pathogens such as Sclerotinia sclerotiorum, Sclerotinia sclerotiorum, and Fusarium verticillata. However, there are no reports in the prior art that Burkholderia multocida also has the biological function of controlling barnyard grass. Summary of the Invention

[0003] The purpose of this invention is to provide a novel Burkholderia gladioli H02 strain and its prepared inoculants, fertilizers, and applications. The unique value of this strain lies in its functional diversity; a single strain simultaneously possesses multiple biocontrol functions, including controlling barnyard grass, antagonizing Sclerotinia sclerotiorum and Fusarium oxysporum, achieving "multiple effects from one strain," with broad application potential. This provides a new microbial resource and technical solution to address existing production challenges. Furthermore, the invention explores the combined use of biocontrol strain H02 with low-dose chemical fungicide thifluzamide, aiming to achieve enhanced control of peanut white mold through synergistic effects. This not only helps reduce the amount of chemical agents used and delay the development of resistance, but also provides important technical support for developing more scientific and green integrated pest management strategies.

[0004] The objective of this invention is achieved through the following means:

[0005] A strain of Burkholderia gladioli H02, with accession number CGMCCNO: 35003, is deposited at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on June 25, 2025.

[0006] The Gladiolus Burkholderia H02 strain exhibits antagonistic effects against Sclerotium sclerotiorum and Fusarium oxysporum, and also inhibits the growth of barnyard grass.

[0007] The present invention also provides a bacterial agent prepared from the aforementioned Burkholderia gladioli H02.

[0008] The bacterial agent includes liquid or solid formulations; the liquid bacterial agent is prepared by inoculating Burkholderia gladioli H02 into liquid LB or NA medium and incubating it in a constant temperature shaker at 28-30 ℃ and 140-220 rpm for 1-2 days; the solid bacterial agent is prepared by centrifuging the liquid bacterial agent to remove the supernatant to obtain concentrated bacterial sludge, mixing it with a carrier, and then freeze-drying it.

[0009] Furthermore,

[0010] The concentrated bacterial sludge was resuspended in sterile water to prepare a concentration of 10. 10 CFU / mL ~10 11 The bacterial suspension with CFU / mL is prepared by mixing the bacterial suspension and the carrier at a volume ratio of 1:0.5 to 1:2, and then freeze-drying.

[0011] The carrier includes at least one of 5% glycerol, 5% trehalose, and 10% skim milk.

[0012] This invention also provides a microbial fertilizer prepared from Burkholderia gladioli H02. The microbial fertilizer is prepared by mixing a liquid or solid formulation with at least one organic carrier, such as peat moss, charcoal, or corn cob.

[0013] Furthermore, the mass ratio of liquid or solid formulations to organic carriers ranges from 1:100 to 5:100.

[0014] The present invention also provides the application of the aforementioned Burkholderia gladioli H02, the aforementioned inoculant, or the aforementioned inoculant fertilizer in the prevention or control of plant white mold disease.

[0015] Furthermore, the plant mentioned includes peanuts.

[0016] Furthermore, at least one of the aforementioned Burkholderia gladioli, the aforementioned microbial agent, and the aforementioned microbial fertilizer is mixed with thifluzamide for application.

[0017] Specifically, Burkholderia gladioli bacterial suspension is compounded with thifluzamide, and the compound solution contains a concentration of 10. 7 -10 8 The optimal concentration of 10 cfu / mL Burkholderia gladioli HO2 and 5-100 μg / mL thifluzamide is preferred. 8 Burkholderia gladioli HO2 at cfu / mL and thifluzamide at 50 μg / mL.

[0018] The present invention also provides the application of the aforementioned Burkholderia gladioli, the aforementioned inoculum, or the aforementioned inoculum fertilizer in the prevention or control of Fusarium oxysporum or the watermelon wilt disease caused by it.

[0019] The present invention also provides the application of the aforementioned Burkholderia gladioli, the aforementioned microbial agent, or the aforementioned microbial fertilizer in the prevention or control of barnyard grass.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The Gladiolus Burkholderia H02 provided in this application has multiple biocontrol functions for controlling barnyard grass, peanut white mold and watermelon wilt, achieving "one bacterium with multiple effects" and has broad application potential.

[0022] 2. This invention discovers that Burkholderia gladioli H02 has a synergistic effect with thifluzamide in the synergistic control strategy, and the dosage of thifluzamide is significantly reduced compared to the dosage in other compound microbial agents in the prior art, which can achieve the goal of reducing the environmental pollution caused by chemical fungicides. This indicates that this bacterium and its agent have good development and application prospects in related fields such as biopesticides and microbial preparations.

[0023] 3. The invention provides excellent strain resources for the development of new multifunctional microbial pesticides, which is of great significance for promoting the advancement of green pest control technology in agriculture, ensuring the quality and safety of agricultural products, and protecting the agricultural ecological environment. Attached Figure Description

[0024] Figure 1 The antagonistic effect of Burkholderia gladioli H02 on Sclerotium sclerotiorum on the whole organism. (a) is the control, and (b) is the antagonistic effect.

[0025] Figure 2 The growth of Burkholderia gladioli H02 on PDA plates with no added PDA (CK), 0.15 μg / mL thifluzamide, 6.9 μg / mL allicin, and 0.08 μg / mL fludioxonil; (a) is the control, (b) is thifluzamide, (c) is allicin, and (d) is fludioxonil.

[0026] Figure 3 Effect of sterile fermentation broth of Burkholderia gladioli H02 on the growth of Sclerotium oliguriae; (a) is the control, and (b) is the sterile fermentation broth of H02 diluted 10 times.

[0027] Figure 4 Safety test of Burkholderia gladioli H02 on peanut seedling growth; (a) is a comparison of plant growth, with the left side being the control and the right side being the H02 treatment; (b) is a comparison of plant root morphology, with the left side being the control and the right side being the H02 treatment.

[0028] Figure 5 : Burkholderia gladioli H02 and its combined use with thifluzamide in potted plants to control white rot disease; where (a) is blank control, (b) is inoculation control, (c) is synergistic treatment of H02 + thifluzamide + inoculation, (d) is thifluzamide treatment + inoculation, and (e) is H02 treatment + inoculation.

[0029] Figure 6 Antagonistic effect of Burkholderia gladioli H02 on Fusarium oxysporum on day 4 after inoculation; (a) is the control and (b) is the antagonistic effect.

[0030] Figure 7 Effects of Burkholderia gladioli H02 fermentation broth on barnyardgrass seed germination and seedling growth; (a) shows seed germination, with the left side being the control and the right side being the H02 treatment; (b) shows seedling growth, with the left side being the control and the right side being the H02 treatment. Detailed Implementation

[0031] The following examples are intended to further illustrate the present invention, but not to limit it.

[0032] Example 1: Isolation and screening of peanut white mold pathogen and Burkholderia gladioli H02

[0033] Typical white rot diseased peanut plants with white silky mycelia were collected from the peanut field. In a clean bench, mycelia were picked from the diseased plants, disinfected with 75% alcohol for 1 minute, washed with sterile water, dried with sterile filter paper, and inoculated into PDA plates. The plates were then cultured at 28°C. After the mycelia had fully grown, DNA was extracted and ITS identification was performed. The strain was identified as *Sclerotium sclerotiorum* and used for subsequent experiments.

[0034] Microorganisms were isolated from various soil types in Hunan Province, including dry land, paddy fields, and forest soil, using a dilution method. These isolated microorganisms were then used in confrontation experiments with *Sclerotium regrinum* on PDA plates. Microorganisms exhibiting inhibitory effects on *Sclerotium regrinum* were screened.

[0035] The results of the confrontation showed that strains B-001, HR2C, G2D, H02, H12, H09, 20-5-1 and 21-2-1 had a certain inhibitory effect on the growth of *Sclerotium truncatum*. B-001, HR2C, G2D, H02, 20-5-1 and 21-2-1 were selected for subsequent drug compatibility tests.

[0036] Example 2: Screening of pesticides for controlling peanut white mold disease

[0037] The inhibitory effects of technical grade thifluzamide (96.2%), prothioconazole (95%), and pyraclostrobin (98%) at concentrations of 100 μg / mL, 25 μg / mL, and 5 μg / mL, and the recommended dosages of common commercial fungicides—80% allicin EC, 25 g / L fludioxonil, 8% ningnanmycin, 30% hymexazol Aqueous Solution, 75% chlorothalonil WP, 50% prochloraz manganese WP, and 50% carbendazim WP—on the growth of *Sclerotinia sclerotiorum* were determined. Stock solutions were prepared using methanol as the solvent. PDA plates containing the drug were then prepared using the stock solutions, with drug-free PDA plates serving as the control group. *Sclerotinia sclerotiorum* mycelial cakes with a diameter of 0.5 cm were inoculated in the center of each plate. Three plates were inoculated for each treatment, and the hyphal diameter was measured every 24 hours to calculate the hyphal inhibition rate.

[0038] The results are shown in Table 1. The results indicate that thifluzamide exhibits a good inhibitory effect on white rot. Among commercial pesticides, allicin and fludioxonil show good inhibitory effects on the growth of *Sclerotium truncatum*.

[0039]

[0040]

[0041] Example 3: Compatibility test of biocontrol agents and fungicides for white rot disease

[0042] The compatibility of B-001, HR2C, G2D, H02, 20-5-1, and 21-2-1 with three fungicides and commonly used commercial fungicides was determined using the dilution plating method. The fungicides were diluted with sterile water to prepare LB agar plates containing the fungicide at EC50 values. The H02 fermentation broth was diluted 10 μL with sterile water. 5 Take 100 μL of the solution and spread it evenly on the surface of LB medium. Use an equal amount of sterile water as a blank control. Record the number of colonies and calculate the inhibition rate after 48 h.

[0043] The results of the biocontrol bacteria and fungicide compatibility test showed that all the candidate biocontrol strains grew well on LB plates containing thifluzamide (0.15 μg / mL), prothioconazole (4.9 μg / mL), and pyraclostrobin (4.8 μg / mL) at EC50 concentrations. Among them, strain H02 grew well on all PDA plates and showed good compatibility with all tested fungicides; therefore, H02 was selected for further application research.

[0044]

[0045] Example 4: Screening of fermentation medium and fermentation time for H02

[0046] The effect of fermentation time on the antibacterial activity of H02 fermentation broth under LB and NA culture conditions was determined: H02 seed culture was inoculated into LB or NA culture medium at a 1% inoculum and cultured with shaking at 28℃ and 160 r / min. Fermentation broth samples were collected on days 1, 2, 3, 4, and 5 of shaking culture. The supernatant was obtained by centrifugation at 10000 g for 5 min at 4℃, and then filtered through a 0.22 μm microporous membrane to obtain sterile fermentation broth. The sterile fermentation broth was added to soluble PDA medium at approximately 50℃ to prepare PDA medium containing a 10-fold dilution of the sterile fermentation broth. After cooling, neat *Sclerotium sclerotium* mycelial discs with a diameter of 5 mm were inoculated into the center of each disc and placed in a 28℃ incubator for dark incubation. The colony diameter was measured daily, and the antibacterial activity of the fermentation broth was calculated. The results showed that the inhibitory effect of H02 fermentation broth on *Sclerotium sclerotium* generally followed a pattern of first decreasing, then increasing, and then decreasing again over time. Taking into account factors such as time, cost and viable cell count, the optimal fermentation time is 24 h. Under this condition, there is no significant difference in antibacterial activity between NA and LB cultures, both being approximately 30%.

[0047] Example 5: Determination of the effect of strain H02 on peanut seedling growth:

[0048] Peanut seeds, disinfected with 1% sodium hypochlorite for 10 minutes, were placed in a sterile germination box for germination treatment. Once the seedlings reached the cotyledon unfolding stage, a slight wound was made at the base of the stem using a sterile scalpel. The experimental materials were divided into two groups, and the seeds were sprayed or drenched with a concentration of 1×10⁻⁶. 7 20 mL of H02 bacterial suspension (CFU / mL) and 20 mL of water were added. After treatment, the mixture was placed in an environment with a temperature of 28℃ and a humidity of over 80%, and cultured under light for 12 hours followed by 12 hours in the dark. The growth of peanut seedlings in each treatment was observed and recorded to assess the safety of the H02 strain for peanut seedling growth.

[0049] Experimental results showed that peanut seedlings inoculated with H02 through wounds showed no disease symptoms and exhibited slightly stronger growth than the water control. The results indicate that H02 poses no disease risk to peanut seedling growth. Figure 4 ).

[0050] Example 6: Indoor pot experiment on the control of peanut white mold by the combined use of H02 and thifluzamide

[0051] Peanuts that have sprouted and shown white tips were planted in square pots containing peat moss and vermiculite and cultured at 26℃ until they reached the two-leaf-one-heart stage. HO2 was inoculated into NA culture medium and cultured with shaking for 24 hours, and then diluted with sterile water to a concentration of 10%. 8 A bacterial suspension of CFU / mL was prepared. Thifluzamide was weighed and added to the HO2 bacterial solution to prepare a 5 μg / mL thifluzamide + 10... 8 CFU / mL H02; 25 μg / mL thifluzamide + 10 8 CFU / mL H02; 50 μg / mL thifluzamide + 10 8 CFU / mL H02; 100 μg / mL thifluzamide + 10 8 A mixture of fungal and fungal agents (cfu / mL HO2) was used. Peanut seedlings were treated by instilling 20 mL of the fungal suspension or a mixture of fungal suspensions containing various concentrations of thifluzamide around the roots. After 24 hours, two white silkworm cakes were inoculated 2 cm deep and 2 cm wide on each side of the rootstock. An inoculation control (inoculated only with *Sclerotium truncatum* fungal cakes) and a blank control (treated only with water, without *Sclerotium truncatum* inoculation) were also set up. After inoculation, the seedlings were placed at 28℃ and above 90% humidity, with 12 hours of daylight and darkness at night. Normal management was maintained, and the disease incidence of peanut seedlings under each treatment condition was observed and recorded, and the control effect was calculated. Potted results showed that 15 days after inoculation, peanut seedlings in the blank control showed no disease symptoms, while almost all seedlings inoculated only with *Sclerotium truncatum* fungal cakes died, with a disease rate of 92%. For peanut seedlings treated with thifluzamide alone, the disease incidence decreased with increasing thifluzamide concentration; peanuts treated with 100 μg / mL thifluzamide showed no disease symptoms. The incidence rate of peanut seedlings treated with pure H02 was 35%, while the incidence rate of 25 μg / mL thifluzamide + 10 μg / mL was lower.8 The incidence rate of peanut seedlings treated with cfu / ml H02 was 13%, and 50 μg / mL thifluzamide + 10 8 CFU / mL H02 and 100 μg / mL thifluzamide + 10 8 Peanuts treated with cfu / ml H02 showed no disease symptoms. Considering efficacy and cost, the optimal concentration of thifluzamide compatible with H02 is 50 μg / mL. This concentration is 50% lower than the recommended field concentration of 100 μg / mL (for example, a 240 g / L suspension: 20 ml per acre diluted in 30-45 kg of water, resulting in a concentration of approximately 106 μg / mL). Therefore, the combined use of thifluzamide and H02 can achieve reduced pesticide use and increased efficacy in the control of peanut white mold disease.

[0052] Example 7: Determination of the antagonistic effect of strain H02 against Fusarium wilt of watermelon

[0053] H02 strain was inoculated onto PDA plates and subjected to a confrontation experiment against the watermelon-specific strain of *Fusarium oxysporum*, the pathogen of watermelon wilt. The results showed that strain H02 had an antagonistic effect against *Fusarium oxysporum*. Figure 6 ).

[0054] Example 8: Determination of the effect of strain H02 on barnyard grass seed germination and growth:

[0055] Take barnyard grass seeds and incubate them separately with sterile distilled water and HO2 bacterial suspension (NA medium) for 24 hours, approximately 10 7 Seeds were soaked in a solution of CFU / mL for 2 hours, excess liquid was removed, and the seeds were wrapped in damp gauze and placed at 28℃ for germination. After 3 days, the germination status of each treatment was observed and recorded, and the germination rate was calculated. Barnyard grass seeds were also germinated normally. When the barnyard grass reached the two-leaf-one-heart stage, sterile water and HO2 fermentation broth were sprayed onto the uniform barnyard grass leaves until droplets were applied to the leaf surface. After treatment, the leaves were placed in an environment with a temperature of 28℃ and humidity above 80%, with 12 hours of light followed by 12 hours of darkness. Changes in the barnyard grass leaves were observed.

[0056] The results showed that, compared with the control (CK), the germination rate of barnyard grass seeds treated with H02 fermentation broth was significantly reduced, and some seeds turned black and showed necrosis symptoms. The leaves of barnyard grass seedlings treated with H02 fermentation broth showed wrinkling, wilting, and necrosis symptoms. Figure 7 Experimental results show that H02 has a killing effect on barnyard grass.

[0057] Example 9:

[0058] The 16S rDNA, gyrB gene, and recA gene of strain H02 were amplified by PCR using universal primers 27F / 1492R, gyrB F / gyrB R, and recA F / recA R, respectively. The products were then sequenced, and the sequencing results are shown in SEQ ID NO. 1-3. The analysis results indicate that strain H02 is *Burkholderia gladioli*.

Claims

1. A strain of Burkholderia gladioli H02, characterized in that, The accession number is CGMCC NO: 35003.

2. The *Burkholderia gladioli* H02 according to claim 1, characterized in that, This strain exhibits antagonistic effects against *Sclerotium sclerotiorum* and *Fusarium oxysporum*, and also inhibits the growth of barnyard grass.

3. The bacterial agent prepared from Burkholderia gladioli H02 as described in claim 1 or 2.

4. The microbial agent according to claim 3, characterized in that, The bacterial agent includes liquid or solid formulations; the liquid bacterial agent is prepared by inoculating Burkholderia gladioli H02 into liquid LB or NA medium and incubating it in a constant temperature shaker at 28-30 ℃ and 140-220 rpm for 1-2 days; the solid bacterial agent is prepared by centrifuging the liquid bacterial agent to remove the supernatant, mixing the obtained concentrated bacterial sludge with a carrier, and then freeze-drying it.

5. The microbial fertilizer prepared from Burkholderia gladioli HO2 as described in claim 1 or 2, characterized in that, The liquid or solid formulation described in claim 4 is prepared by mixing it with at least one of peat moss, charcoal, and corn cob.

6. The use of Burkholderia gladioli H02 as described in claim 1 or 2, the microbial agent as described in claim 3 or 4, or the microbial fertilizer as described in claim 5 in the prevention or control of plant white mold disease.

7. The application according to claim 6, characterized in that, The plant mentioned includes peanuts.

8. The application according to claim 6, characterized in that, At least one of the aforementioned Burkholderia gladioli, the aforementioned inoculum agent, and the aforementioned inoculum fertilizer is mixed with thifluzamide for application.

9. The use of Burkholderia gladioli as described in claim 1 or 2, the microbial agent as described in claim 3 or 4, or the microbial fertilizer as described in claim 5 in the prevention or control of Fusarium oxysporum or the watermelon wilt disease caused by it.

10. The use of Burkholderia gladioli as described in claim 1 or 2, the microbial agent as described in claim 3 or 4, or the microbial fertilizer as described in claim 5 in the prevention or control of barnyard grass.

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