Fusarium graminearum antagonistic krill BLG-8 and application thereof
By using the aseptic fermentation broth and volatile metabolites of *Fusarium graminearum* BLG-8, the biological control problem of wheat scab in existing technologies has been solved, and effective inhibition of *Fusarium graminearum* and other pathogenic fungi has been achieved, providing a new method for biological control.
Patent Information
- Application Number
- CN202411836839.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-13
- Publication Date
- 2025-11-14
AI Technical Summary
Current technologies lack effective biological control methods to control wheat scab, while chemical control leads to an increase in drug-resistant strains, affecting environmental safety and reducing control effectiveness.
A strain of Kitasatospora purpeofusca BLG-8 was used, which has the characteristic of antagonizing Fusarium graminearum. A sterile fermentation broth and volatile metabolites were prepared by fermentation culture, which inhibited the growth of Fusarium graminearum and had antagonistic ability against a variety of plant pathogenic fungi.
The aseptic fermentation broth and volatile metabolites of strain BLG-8 significantly inhibited the growth of Fusarium graminearum, with an inhibition rate of 43.7-18.0%. It also showed antagonistic effects against a variety of plant pathogenic fungi, providing a theoretical basis and resources for biological control.
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Figure CN120944738A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology and relates to a strain of *Fusarium graminearum* BLG-8 that antagonizes *Fusarium graminearum* and its applications. Background Technology
[0002] Wheat is an important food crop in my country and plays a vital role in the national economy. Fusarium head blight, caused primarily by the fungus *Fusarium graminearum*, is a significant disease affecting wheat production in my country. It leads to reduced grain quantity and the production of shriveled grains. Besides yield loss, *Fusarium graminearum* infection can also secrete various fungal toxins, such as deoxyfucoidanol and zearalenone, which can negatively impact human and animal health. Therefore, *Fusarium graminearum* poses a serious threat to safe wheat production under the current circumstances.
[0003] Due to the lack of effective resistant varieties, chemical control remains the primary method for combating wheat scab. However, the long-term, excessive, and irrational use of chemical agents not only affects environmental safety but also leads to the proliferation of drug-resistant strains in the field, ultimately reducing the effectiveness of chemical control. Using biological control methods to manage wheat scab plays a crucial role in ensuring the safe and sustainable production of wheat. Summary of the Invention
[0004] The purpose of this invention is to provide a strain with biocontrol activity against Fusarium graminearum.
[0005] To achieve the above objectives, the present invention adopts the following technical solution:
[0006] A strain of Kitasatospora purpeofusca BLG-8 was deposited on September 11, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 31942.
[0007] The aerial hyphae of strain BLG-8 on Gao's No. 1 medium are white, while the intramural hyphae are dark brown, and no soluble pigments are produced.
[0008] The physiological and biochemical characteristics of strain BLG-8 are as follows: it can utilize mannose, maltose, sucrose, and glucose; it can tolerate 1% NaCl concentration; it can produce protease; it can liquefy gelatin; it cannot produce cellulase; it cannot produce β-1,3-glucanase; and it does not produce H2S.
[0009] The 16S rRNA sequence of strain BLG-8 is shown in SEQ ID NO.1.
[0010] The aseptic fermentation broth of strain BLG-8 inhibited the colony growth of Fusarium graminearum.
[0011] The volatile metabolites of strain BLG-8 have an inhibitory effect on the colony growth of Fusarium graminearum.
[0012] Strain BLG-8 exhibits antagonistic activity against Alternaria alternata, Botryosphaeria dothidea, Exserohilum turcicum, Colletorichum gloeosporioides, Diaporthe eres, Fusarium oxysporum, F. proliferatum, and F. pseudoograminearum.
[0013] The beneficial effects of this invention compared to existing technologies are as follows: Strain BLG-8 exhibits good antagonistic activity against *Fusarium graminearum*. After fermentation culture of strain BLG-8 on Gao's No. 1 medium, its sterile fermentation broth significantly inhibited the colony growth of *Fusarium graminearum*, with an inhibition rate of 43.7±1.0%. After cultivation on PDA medium and Gao's No. 1 medium, the volatile metabolites of BLG-8 inhibited the colony growth of *Fusarium graminearum*, with inhibition rates of 18.0±1.4% and 14.0±0.6%, respectively. Regarding the antibacterial spectrum, strain BLG-8 also exhibits antagonistic activity against *Alternaria alternata*, *Staphylococcus aureus*, *Cordyceps militaris*, *Colletotrichum gloeosporioides*, *Metacarpa sacchariformis*, *Fusarium oxysporum*, *Fusarium solani*, and *Fusarium pseudograhamii*. The results of this invention demonstrate that strain BLG-8 can provide strain resources and a theoretical basis for developing highly efficient biocontrol microbial agents against *Fusarium graminearum*. Attached Figure Description
[0014] Figure 1 This illustrates the antagonistic effect of strain BLG-8 on Fusarium graminearum. A represents normally cultured Fusarium graminearum, B represents the confrontation culture of strain BLG-8 and Fusarium graminearum, C represents the mycelium at the colony edge of normally cultured Fusarium graminearum, and D represents the mycelium at the colony edge of Fusarium graminearum after confrontation culture.
[0015] Figure 2 The images show the colony morphology of strain BLG-8 on Gao's No. 1 medium. A shows the front of the colony, and B shows the back of the colony.
[0016] Figure 3 A neighbor-joining phylogenetic tree for strain BLG-8 constructed based on the 16S rRNA gene sequence.
[0017] Figure 4The effect of sterile fermentation broth of strain BLG-8 cultured on Fusarium graminearum colony growth was investigated. A represents the Fusarium graminearum control; B represents the effect of sterile fermentation broth of strain BLG-8 on Fusarium graminearum.
[0018] Figure 5 This study investigated the effects of volatile metabolites produced by strain BLG-8 after solid culture on PDA and Gao's No. 1 medium on the colony growth of *Fusarium graminearum*. A represents the *Fusarium graminearum* control; B represents the effect of volatile substances produced by strain BLG-8 after culture on PDA medium on *Fusarium graminearum*; C represents the effect of volatile substances produced by strain BLG-8 after culture on Gao's No. 1 medium on *Fusarium graminearum*.
[0019] Figure 6 The antagonistic effects of strain BLG-8 on Alternaria alternata, Staphylococcus aureus, Cornus zeylans, Colletotrichum gloeosporioides, Alternaria sacchariformis, Fusarium oxysporum, Fusarium moniliformis, and Fusarium pseudograss. A (left): *Alternaria alternata* control; A (right): Confrontation culture of strain BLG-8 and *Alternaria alternata*; B (left): *Botrytis cinerea* control; B (right): Confrontation culture of strain BLG-8 and *Botrytis cinerea*; C (left): *Cordyceps militaris* control; C (right): Confrontation culture of strain BLG-8 and *Cordyceps militaris*; D (left): *Colletotrichum gloeosporioides* control; D (right): Confrontation culture of strain BLG-8 and *Colletotrichum gloeosporioides*; E (left): *Ceratophyllum demersum* control; E (right): Confrontation culture of strain BLG-8 and *Ceratophyllum demersum*; F (left): *Fusarium oxysporum* control; F (right): Confrontation culture of strain BLG-8 and *Fusarium oxysporum*; G (left): *Fusarium moniliforme* control; G (right): Confrontation culture of BLG-8 and *Fusarium moniliforme*; H (left): *Fusarium pseudograss* control; H (right): Confrontation culture of BLG-8 and *Fusarium pseudograss*. Detailed Implementation
[0020] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and do not constitute a limitation thereof.
[0021] Example 1: Isolation and screening of strain BLG-8.
[0022] Soil samples were collected in Baligou, Huixian County, Xinxiang City, Henan Province in June 2023. 10g of soil sample was placed in 90mL of sterile water and shaken for 30min at 28℃ and 180r / min. The suspension was then serially diluted with sterile water to 10⁻⁶. -1 10 -2 and 10 -3 For each concentration, 100 μL was plated onto Gao's No. 1 agar plates containing 50 μg / mL potassium dichromate and incubated at 28°C for 5-7 days. Single bacteria were then picked for purification.
[0023] The test strains were symmetrically inoculated 2.5 cm from the center of PDA plates and cultured at 28°C for 2 days. Then, Fusarium graminearum mycelium discs (0.5 cm) were inoculated into the center of the plates. PDA plates inoculated only with Fusarium graminearum served as a control. After culturing for another 3 days, the antagonistic effect was observed. The final antagonistic strain of Fusarium graminearum was obtained, designated BLG-8, with an inhibition rate of 47.3 ± 1.6%. Simultaneously, microscopic observation revealed that the mycelia at the edge of the control colonies exhibited normal branching growth, while the presence of strain BLG-8 affected the mycelial growth of Fusarium graminearum, manifested as thinner hyphae and increased branching. Figure 1 ).
[0024] Example 2: Classification and identification of strain BLG-8.
[0025] (1) Morphological characteristics of strain BLG-8
[0026] Strawberry strain BLG-8 was streaked onto Gao's No. 1 medium and incubated at 28°C for 7 days. The shape, color, and texture of the colonies were then observed. Figure 2 As shown, the aerial hyphae of strain BLG-8 are white, while the intracellular hyphae are dark brown, and no soluble pigments are produced.
[0027] (2) Physiological and biochemical characteristics of strain BLG-8
[0028] Physiological and biochemical tests were performed on strain BLG-8 according to "Rapid Identification and Systematic Classification of Actinomycetes". The specific results are shown in Table 1.
[0029] Table 1 Physiological and biochemical characteristics of strain BLG-8
[0030] (3) 16S rRNA gene sequence analysis of strain BLG-8
[0031] Genomic DNA was extracted from strain BLG-8, and the 16S rRNA gene sequence was amplified using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The amplified product was purified and sequenced by Sangon Biotech (Shanghai) Co., Ltd. (gene sequence shown in SEQ ID NO.1). The sequencing results were compared for homology in the GenBank database, and a phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.0 software. Figure 3As shown, strain BLG-8 clustered with three other strains of K. purepeofusca: NBRC 12905, LMG 20283, and ICMP 1594. Therefore, based on morphological and physiological-biochemical characteristics, strain BLG-8 was ultimately identified as *K. purepeofusca*.
[0032] Example 3: Inhibitory effect of sterile fermentation broth of strain BLG-8 on Fusarium graminearum.
[0033] (1) Preparation of sterile fermentation broth
[0034] Freshly cultured bacterial blocks (2cm) of strain BLG-8 were placed in 100mL of Gao's No. 1 medium and cultured at 28℃ and 180r / min for 5 days. The bacterial solution was collected and centrifuged at 12000r / min for 15min. The supernatant was then filtered through a 0.22μm microporous membrane to remove the bacterial cells, thus obtaining the sterile fermentation broth of the strain.
[0035] The formula for Gao's No. 1 culture medium is as follows: soluble starch 20.0g, NaCl 0.5g, KNO3 1g, K2HPO4·3H2O 0.5g, MgSO4·7H2O 0.5g, FeSO4·7H2O 0.01g, agar 20g, distilled water 1L, pH 7.4-7.6.
[0036] (2) Effect of sterile fermentation broth on the growth of Fusarium graminearum colonies
[0037] The aseptic fermentation broth of strain BLG-8 was added to the melted PDA medium at a final concentration of 10%. After the medium solidified, Fusarium graminearum mycelium (0.5 cm) was inoculated into the center of the plate. A PDA plate with an equal volume of sterile water was used as a control. After incubation at 28°C for 3 days, the colony diameter was measured and the inhibition rate was calculated. Figure 4 As shown, the aseptic fermentation broth of strain BLG-8 can inhibit the colony growth of Fusarium graminearum, with an inhibition rate of 43.7±1.0%.
[0038] Example 4: Inhibitory effect of volatile metabolites of strain BLG-8 on Fusarium graminearum.
[0039] The inhibitory effect of volatile metabolites of strain BLG-8 on *Fusarium graminearum* was determined using the plate-on-plate method. BLG-8 strain was inoculated onto PDA and Gao's No. 1 agar plates using an inoculation loop and incubated at 28°C for 7 days. A 0.5 cm *Fusarium graminearum* mycelial cake was inoculated in the center of another PDA plate. The two plates containing the 7-day-old BLG-8 culture and the freshly inoculated *Fusarium graminearum* were then inverted face-to-face, and the outer seams of both plates were sealed with sealing film. The plates were then incubated at 28°C for 3 days. PDA plates inoculated only with *Fusarium graminearum* and untreated PDA and Gao's No. 1 plates served as controls. Each treatment was repeated three times.
[0040] Three days later, the antibacterial activity of volatile metabolites of BLG-8 after cultivation on PDA plates and Gao's No. 1 plates was measured. The volatile metabolites on PDA plates showed better antibacterial activity, with an inhibition rate of 18.0±1.4%, while the inhibition rate on Gao's No. 1 plates was 14.0±0.6%.
[0041] Example 5: Antibacterial spectrum analysis of strain BLG-8.
[0042] Using an in-plate confrontation method, strain BLG-8 was first symmetrically inoculated 2.5 cm from the center of a PDA plate and incubated at 28°C for 2 days. Then, eight plant pathogenic fungi, including Alternaria, were inoculated into the center of the plate, and the antagonistic effect was observed after further incubation for 3-5 days. Figure 6 As shown, strain BLG-8 exhibited the strongest antagonistic activity against *Staphylococcus aureus*, with an inhibition rate of 47.4±1.7%; followed by *Fusarium oxysporum* and *Colletotrichum gloeosporioides*, with inhibition rates of 43.7±2% and 43.6±0.7%, respectively; the inhibition rates against *Gnaphalium angularis*, *Fusarium moniliforme*, and *Fusarium pseudograss* were all around 35%; the inhibition rate against *Alternaria alternata* was 31.3±1.3%; and the inhibition rate against *Metaphyllum spp.* was relatively low, at 28.0±0.4%. Therefore, strain BLG-8 possesses a good antibacterial spectrum.
[0043] The 16S rRNA sequence of strain BLG-8 is shown in SEQ ID NO.1.
[0044] SEQ ID NO.1
[0045]
Claims
1. A strain of Kitasatospora purpeofusca BLG-8, characterized in that, It was deposited on September 11, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 31942.
2. The *Tetracentron sinense* BLG-8 according to claim 1, characterized in that, The strain BLG-8 has an antagonistic effect on Fusarium graminearum, which causes wheat scab.
3. The application of aseptic fermentation broth of strain BLG-8 as described in claim 1 in inhibiting Fusarium graminearum.
4. The application of the aseptic fermentation broth of strain BLG-8 as described in claim 3, characterized in that, The aseptic fermentation broth of strain BLG-8 was used to inhibit the mycelial growth of Fusarium graminearum.
5. A method for preparing the aseptic fermentation broth of strain BLG-8 according to claim 4, characterized in that, BLG-8 strain was inoculated into 100 mL of Gao's No. 1 liquid culture medium, and cultured with shaking at 28℃ and 180 r / min for 5 days. The bacterial solution was collected and filtered through a 0.22 μm microporous membrane to obtain sterile fermentation broth.
6. The use of a volatile metabolite of the strain BLG-8 according to claim 1 in inhibiting Fusarium graminearum.
7. The application of a volatile substance from the strain BLG-8 according to claim 6, characterized in that, The volatile metabolites of strain BLG-8 inhibit the mycelial growth of Fusarium graminearum.
8. The use of the strain BLG-8 according to claim 1 in inhibiting Alternaria alternata, Staphylococcus aureus, Cornus zeylans, Colletotrichum gloeosporioides, Alternaria sacchariformis, Fusarium oxysporum, Fusarium moniliformis, and Fusarium pseudograss.