Penicillium oxalicum strain, biocontrol agent and application thereof

Abstract

The application discloses a Penicillium oxalicum strain, a biocontrol agent and application. A fungus is isolated and screened from soil, and the fungus is identified as Penicillium oxalicum by combining morphological characteristics and molecular identification, and is named as Penicillium oxalicum PO14 with a preservation number of CGMCC No.22461. The Penicillium oxalicum PO14 has a strong inhibitory effect on Fusarium fujikuroi spore germination and mycelium growth, and can be used for prevention and treatment of rice bakanae disease. The Penicillium oxalicum PO14 strain can be used for development of a biocontrol agent or a biological pesticide, and has a good application prospect in biological prevention of rice bakanae disease.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for plant diseases, and in particular to Penicillium oxalicum strains, biocontrol agents, and their applications. Background Technology

[0002] Rice is one of the world's three major food crops, with a wide planting area and abundant yield. Statistics show that half of the world's population consumes rice, mainly in Asia, Southern Europe, and parts of tropical America and Africa. Therefore, ensuring the yield and quality of rice is of great significance to my country's food production and national economy.

[0003] Rice is susceptible to many fungal diseases during production, especially rice bakanae disease. Since its first report in Japan in 1898, this disease has occurred in almost all rice-growing regions worldwide, causing severe yield losses. Furthermore, the pathogen of rice bakanae disease, *Fusarium fujikuroi*, produces water-soluble metabolites such as fumonisin. These toxins can contaminate grains and their products, and can cause poisoning in humans and animals, seriously endangering food security and the health of humans and livestock.

[0004] Because *Fusarium oxysporum* can produce various gibberellins, it is difficult to cultivate highly resistant varieties. Therefore, the control of rice bakanae disease still relies mainly on chemical seed treatment. In China, 152 pesticide products (including single-agent and compound formulations) are currently registered for the control of rice bakanae disease. Commonly used pesticides contain active ingredients such as carbendazim, prochloraz, cyazofamid, and tebuconazole. However, due to the long-term use of chemical fungicides, the rice bakanae disease pathogen has developed significant resistance to carbendazim and prochloraz, and resistance to cyazofamid is rapidly expanding, making the resistance problem increasingly serious. This makes rice bakanae disease difficult to control and also causes environmental pollution. Therefore, the focus of plant disease control should gradually shift from chemical control to green and safe biological control.

[0005] Biological control offers advantages such as long-lasting effectiveness, low pollution, low residue, low risk of developing resistance, and benefits for human and animal safety and environmental protection. Utilizing biocontrol strains and their metabolites to control rice bakanae disease helps ensure rice safety, reduce ecological pollution, and provides both economic and ecological benefits. Currently, research on the biocontrol of rice bakanae disease mainly focuses on microorganisms such as Actinomycetes spp., Bacillus spp., Pseudomonas spp., and Trichoderma spp. Summary of the Invention

[0006] This invention aims to solve the problem of difficult control of rice bakanae disease, and provides a strain of *Penicillium oxalicum*, a biocontrol agent prepared from this strain, and its applications. The *Penicillium oxalicum* PO14 strain exhibits strong inhibitory effects on both spore germination and mycelial growth of *Fusarium fujikuroi*, and can be used for the control of rice bakanae disease. *Penicillium oxalicum* PO14 provides an excellent strain for the development of biocontrol agents or biological pesticides, and has good application prospects in the biological control of rice bakanae disease.

[0007] The Penicillium oxalicum strain provided by this invention was isolated from field soil in Wupu Farm, Huaixi County, Anhui Province, and classified as Penicillium oxalicum PO14. It was deposited on June 21, 2021, at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 22461.

[0008] The described Penicillium oxalate strain has a strong inhibitory effect on the mycelial growth and spore germination of Fusarium fujikuroi, and has good application in antagonizing Fusarium fujikuroi.

[0009] In addition, the Penicillium oxalate strain can inhibit the mycelial growth of Sclerotinia sclerotiorum, Botrytis cinerea, Rhizoctonia graminearum, Fusarium oxysporum, Cannosporium, Fusarium pseudograss, and Phyllostachys gracilis, exhibiting a good antagonistic effect.

[0010] Based on the good antagonistic effect of the Penicillium oxalicum strain against Fusarium oxysporum, the Penicillium oxalicum strain has good application prospects in the prevention and control of rice bakanae disease.

[0011] The present invention also provides the application of the Penicillium oxalicum strain in the preparation of biocontrol products for the prevention and control of rice bakanae disease.

[0012] Optionally, the biocontrol product is a biocontrol agent or a biological pesticide.

[0013] For example, the present invention provides a biocontrol agent comprising a spore suspension of the Penicillium oxalate strain.

[0014] Optionally, the method for preparing the spore suspension is as follows:

[0015] The activated Penicillium oxalate strain was inoculated into PDA solid medium and cultured for 5–7 days. The spore suspension was obtained by rinsing with sterile water.

[0016] The biocontrol agent can be used to control rice bakanae disease caused by Fusarium oxysporum.

[0017] For example, the present invention provides a method for preventing and controlling rice bakanae disease, comprising:

[0018] Rice seeds were soaked in the biocontrol agent;

[0019] Alternatively, the biocontrol agent can be sprayed onto the base of the rice seedling stem during the rice seedling stage.

[0020] Optionally, the concentration of the biocontrol agent is 1×10⁻⁶. 6 ~1×10 7 cfu / mL.

[0021] Optionally, the rice seeds are soaked for 3 to 5 hours.

[0022] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0023] (1) The Penicillium oxalate PO14 provided by the present invention has a significant inhibitory effect on Fusarium tumefaciens. In the plate confrontation test, it can effectively inhibit the growth of Fusarium graminearum mycelium; its spore suspension can effectively inhibit the germination of Fusarium tumefaciens spores.

[0024] (2) The Penicillium oxalate PO14 provided by the present invention can significantly inhibit the excessive growth caused by Fusarium tumefaciens under pot test conditions. It can be used to develop biological control products and applied to the biological control of rice seedling disease. Attached Figure Description

[0025] Figure 1 The colony morphology of strain 4C17;

[0026] Figure 2 Phylogenetic tree of Penicillium oxalate PO14;

[0027] Figure 3 The results of the plate confrontation test between Penicillium oxalate PO14 and Fusarium oxysporum are shown. A: Fusarium oxysporum, B: B1 is Fusarium oxysporum, B2 is Penicillium oxalate.

[0028] Figure 4 The results show the effect of Penicillium oxalate PO14 spore suspension on the germination of Fusarium spores. A: Fusarium spores of ...

[0029] Figure 5A The diagram shows the control effect of Penicillium oxalate PO14 on rice bakanae disease. A: Water control group, B: Penicillium oxalate PO14 treatment group, C: Fusarium oxysporum control group, D: Fludioxonil treatment group.

[0030] Figure 5B The diagram shows the effect of Penicillium oxalate PO14 on rice plant height. A: Water control group, B: Penicillium oxalate PO14 treatment group, C: Fusarium oxysporum control group, D: Fludioxonil treatment group.

[0031] Figure 6The results of the plate confrontation test between Penicillium oxalate PO14 and seven other pathogens are shown. A: Six pathogens, including A1 Sclerotinia sclerotiorum, A2 Botrytis cinerea, A3 Rhizoctonia zeae, A4 Fusarium oxysporum, A5 Monosporascus cannonballu, A6 Fusarium pseudograminearum, and A7 Gaeumannomyces graminis; B: The confrontation between Penicillium oxalate PO14 and the seven pathogens. Detailed Implementation

[0032] The technical solution of the present invention will be further described below with reference to specific embodiments, but the present invention is not limited thereto.

[0033] The culture media and reagent components involved in the following examples include:

[0034] Potato glucose agar medium (PDA medium): 200g potato, 20g glucose, 12g agar powder, deionized water to a final volume of 1L, sterilize at 121℃ for 20min, natural pH.

[0035] 0.7 mol / L NaCl solution: 40.9 g NaCl, diluted to 1 L with deionized water, sterilized at 121 °C for 20 min, natural pH.

[0036] CMC culture medium: CMC-Na 15.0g, yeast extract 1g, NH4NO3 1g, KH2PO4 1g, MgSO4·7H2O 0.5g, deionized water to a final volume of 1L, sterilize at 121℃ for 20min.

[0037] Example 1: Soil sample collection and isolation of biocontrol bacteria

[0038] Soil samples were collected in September 2020 from Wupu Farm, Huaixi County, Huaibei City, Anhui Province. 1 g of field soil was weighed and added to 10 mL of 0.7 mol / L sodium chloride solution. The solution was shaken for 1 min, and after mixing, a soil suspension was prepared. The soil suspension was then diluted to 10 mL with 0.7 mol / L sodium chloride solution. -1 10 -2 10 -3Three gradients were used. 100 μL of soil suspensions at different concentration gradients were plated onto PDA medium containing antibiotics, with three replicates for each gradient. The plates were incubated upside down in a 25°C incubator. The plates were observed daily, and single colonies were picked for isolation and purification. The purified strains were stored aseptically at 4°C for later use, and classified and numbered according to the collection region.

[0039] The purified strain was inoculated onto PDA plates and cultured for 3 days. Then, using a 6mm inner diameter punch, holes were made at the edge of the colonies, and mycelial discs were picked and inoculated onto one side of a 9cm diameter PDA plate. On the other side of the plate, 6mm diameter Fusarium oxysporum mycelial discs were inoculated. The distance between the strain and Fusarium oxysporum was approximately 6cm. The PDA plates were placed in an incubator at 25℃ with a 12h photoperiod. After 7 days, the inhibition of Fusarium oxysporum mycelial growth by the strain was observed, and the inhibition rate of mycelial growth by the strain was calculated. Mycelial growth inhibition rate (%) = (Coronation diameter of control group - Colony diameter of treatment group) / Colony diameter of Fusarium oxysporum in control group × 100%. Among them, strain 4C17 showed a significant inhibitory effect on Fusarium oxysporum mycelial growth in the plate confrontation experiment.

[0040] Example 2: Morphological observation and identification of strain 4C17

[0041] Strain 4C17 was inoculated onto PDA plates and incubated at 25°C. Initially, colonies were white and nearly circular, gradually turning bluish-green after 7 days with neat edges. The colony surface was powdery, producing numerous molecular spores accompanied by mycelial villi. No exudate was observed. Figure 1 .

[0042] DNA was extracted from the bacterial strain and amplified by PCR using universal fungal primers ITS1 (5'-TCCGT AGGTGAACCTG CGG-3') (SEQ ID NO: 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (SEQ ID NO: 2). The PCR reaction volume was 25 μL: 1 μL each of forward and reverse primers (10 μmol / L) ITS1 and ITS4, 2.5 μL DNA template, 12.5 μL Taq Mix, and 8 μL ddH2O. The PCR amplification program was: 94℃ for 5 min; 94℃ for 30 s, 53℃ for 1 min, 72℃ for 1 min, 30 cycles; 72℃ for 10 min. The PCR amplification products were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequence analysis. The sequence results are as follows:

[0043] (SEQ ID NO: 3)

[0044] The sequencing results were subjected to BLAST alignment analysis on NCBI and a phylogenetic tree was constructed. Figure 2 Based on morphological analysis, the strain was identified as Penicillium oxalicum, classified and named as Penicillium oxalicum PO14, and deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No: 22461.

[0045] Example 3: Penicillium oxalate PO14 inhibits Fusarium oxysporum mycelial growth.

[0046] Using a 6mm inner diameter punch, holes were made around the outer ring of the activated *Penicillium oxalate* PO14 culture. The culture was then inoculated onto one side of a 9cm diameter PDA plate, and a 6mm diameter *Fusarium oxysporum* disc was inoculated onto the other side. The interval between the two cultures was approximately 6cm. A negative control was established by inoculating only one side of the PDA plate with a *Fusarium oxysporum* disc. The experiment was repeated three times. The plates were incubated at 25℃ with a 12-hour photoperiod, and the antibacterial effect was observed after 7 days. Mycelial growth inhibition rate = (control group colony diameter - treatment group colony diameter) / control group colony diameter × 100%.

[0047] See Figure 3 Compared with the control group A, Fusarium tumefaciens (B1) in treatment group B was significantly inhibited by Penicillium oxalate PO14 (B2), with a mycelial growth inhibition rate of 48.4%.

[0048] Example 4: Effect of Penicillium oxalate PO14 on Fusarium spore germination in Fujikura

[0049] Three 6mm diameter discs were placed on the edge of a Fusarium oxysporum agar plate and then placed in an Erlenmeyer flask containing 100mL of CMC culture medium. The plates were incubated at 25℃ and 180rpm with shaking for 3 days, followed by filtration through three layers of filter paper. The filtrate was centrifuged at 5000rpm for 10min to obtain Fusarium oxysporum spores. Penicillium oxalate PO14 was activated onto a PDA plate and incubated at 25℃ for 7 days. The Penicillium oxalate PO14 plate was then washed with 10mL of sterile water to obtain a certain concentration of Penicillium oxalate spores. 1mL of 10... 5 CFU / mL Penicillium oxalate PO14 spores and 1 mL 10 5 Mix CFU / mL Fusarium spores and incubate at 180 rpm for 4 hours on a shaker. Add 1 mL of 10... 5 CFU / mL Fusarium spores were used as a negative control. After 4 hours, 7 mm diameter filter paper discs were placed in the center of PDA plates, and 10 μL of the mixed solution and Fusarium spore solution were spotted onto the filter paper discs. The plates were incubated at 25℃, and spore germination was observed after 3 days. The results are as follows: Figure 4 As shown, in the control group, Fusarium spores germinated normally on the plate, and the hyphae grew rapidly to the edge of the plate. Figure 4 A); in the treatment group ( Figure 4 B) Only Penicillium oxalate PO14 spores germinated and grew on the plate, indicating that PO14 spores significantly inhibited the germination of Fusarium spores.

[0050] Example 5: Rice Bakanae Disease Control Experiment

[0051] Rice seeds were disinfected by soaking in a 3% sodium hypochlorite solution for 3 hours, followed by rinsing three times with sterile water. The sterile seeds were then soaked at 25℃ for 24 hours and germinated at 34℃ for 24 hours. When the sprouts reached half the grain length, uniformly sized sprouts were selected and divided into four groups of 30 seeds each. The sprouts were placed in 100ml Erlenmeyer flasks. Group A was the blank control group, Group B was the PO14 + Fusarium oxysporum treatment group, Group C was the Fusarium oxysporum treatment group, and Group D was the Fusarium oxysporum + fludioxonil treatment group. 19mL of distilled water and 1mL of Fusarium oxysporum spore suspension (1×10⁻⁶) were added to each treatment group's Erlenmeyer flask. 6 CFU / mL); 20 mL of distilled water was added to the conical flask of the control group. After culturing at 28℃ and 90 rpm for 12 h with shaking, 1 mL of Penicillium oxalate PO14 spore suspension (1×10⁻⁶ CFU / mL) was added to group B. 6 (CFU / mL) Add 1 mL of fludioxonil to group D, and continue shaking culture for 12 h under the same conditions. Repeat the experiment three times. Select 30 seedlings with uniform growth and sow them in seedling trays. Cultivate at 28℃, 12h light, and 70-80% relative humidity. After about 14 days of seedling cultivation, when etiolation and lateral rooting occur, perform statistical analysis. Disease incidence (%) = Number of diseased seedlings / Total number of seedlings surveyed × 100.

[0052] See Figure 5A In the blank control group (A), rice plants grew normally with an average plant height of 14.3 cm. In the Fusarium oxysporum treatment group (C), rice bakanae disease was severe, with diseased seedlings exhibiting excessive growth, thin leaf sheaths, and yellowing leaves; the disease incidence rate reached 100%, and the average plant height was approximately 23.8 cm. After treatment with Penicillium oxalate PO14 and fludioxonil, rice bakanae disease was significantly inhibited. The disease incidence rate in group B was 25.0%, with an average plant height of only 16.6 cm; in group D, the disease incidence rate was 9.8%, with an average plant height of only 15.0 cm. Analysis shows that Penicillium oxalate PO14 has a significant inhibitory effect on rice bakanae disease and can be used to develop biocontrol products for the biological control of rice bakanae disease.

[0053] Example 6: Inhibitory effect of Penicillium oxalate PO14 on other pathogens

[0054] Using a 6mm inner diameter punch, holes were made around the outer ring of the activated *Penicillium oxalate* PO14 culture medium. The culture was then inoculated onto one side of a 9cm diameter PDA plate. On the other side of the plate, seven pathogenic fungi were inoculated with discs containing 6mm diameter fungi: *Sclerotinia sclerotiorum*, *Botrytis cinerea*, *Rhizoctonia zeae*, *Fusarium oxysporum*, *Monosporascus cannonballu*, *Fusariumpseudograminearum*, and *Gaeumannomyces graminis*. The spacing between the two fungal inoculations was approximately 6cm. A negative control was performed by inoculating pathogenic fungi discs only on one side of the PDA plate; the inoculation was repeated three times. The plates were incubated at 25℃ with a 12-hour photoperiod, and the antibacterial effect was observed after 7 days.

[0055] See Figure 6 Penicillium oxalate PO14 significantly inhibited the mycelial growth of seven pathogenic fungi: *Sclerotinia sclerotiorum* (A1), *Botrytis cinerea* (A2), *Rhizoctonia graminearum* (A3), *Fusarium oxysporum* (A4), *Cannomonosporium* (A5), *Fusarium pseudograss* (A6), and *Tetranychus graminearum* (A7). Among these, *Penicillium oxalate PO14 showed the lowest inhibition rate against *Fusarium pseudograss* and the highest inhibition rate against *Tetranychus graminearum*.

[0056] This invention screened out a Penicillium oxalate PO14 strain that has a good antagonistic effect against Fusarium oxysporum, providing an effective way to solve the problem of difficult control of rice seedling blight.

[0057] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make appropriate changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on the present invention.

Claims

1. A Penicillium oxalicum strain, characterized in that, It is classified and named Penicillium oxalicum PO14, with accession number CGMCC No. 22461.

2. The application of the Penicillium oxalate strain according to claim 1 in antagonizing Fusarium tumefaciens, Sclerotinia sclerotiorum, Botrytis cinerea, Rhizoctonia graminearum, Fusarium oxysporum, Cannosporium, Fusarium pseudograss, and Phyllostachys gracilis.

3. The application of the Penicillium oxalicum strain according to claim 1 in the prevention and control of rice bakanae disease.

4. The application of the Penicillium oxalicum strain according to claim 1 in the preparation of biocontrol products for the prevention and control of rice bakanae disease.

5. A biocontrol agent, characterized in that, A spore suspension containing the Penicillium oxalate strain as described in claim 1.

6. The biocontrol agent according to claim 5, characterized in that, The method for preparing the spore suspension is as follows: The activated Penicillium oxalate strain was inoculated into PDA solid medium and cultured for 5–7 days. The spore suspension was obtained by rinsing with sterile water.

7. The application of the biocontrol agent according to claim 5 in the control of rice bakanae disease caused by Fusarium oxysporum.

8. A method for controlling rice bakanae disease, characterized in that, Rice seeds are soaked in the biocontrol agent as described in claim 5; Alternatively, during the rice seedling stage, the biocontrol agent as described in claim 5 can be sprayed onto the base of the rice seedling stem.

9. The method according to claim 8, characterized in that, The concentration of the biocontrol agent is 1 x 10 6 ~ 1 x 10 7 cfu / mL; The rice seeds were soaked for 3 to 5 hours.

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