Penicillium ochrochloron Ynauu-PO resistant to lead and cadmium and capable of antagonizing panax notoginseng root rot and application of penicillium ochrochloron Ynauu-PO

Abstract

The invention provides Penicillium ochrochloron Ynauu-PO resistant to lead and cadmium and capable of antagonizing panax notoginseng root rot and application of the Penicillium ochrochloron Ynauu-PO, and relates to the field of microorganisms. The preservation number of the penicillium ochrochloron Ynauu-PO is CCTCC NO: M 20252802, the strain can have the capabilities of efficiently resisting lead and cadmium and antagonizing panax notoginseng root rot at the same time, and the technical bottlenecks that the survival rate of microorganisms in high-concentration lead and cadmium polluted soil is low and the remediation effect is poor are solved; an environment-friendly biological control scheme is provided, so that the use of chemical pesticides is reduced, and the environmental pollution and the drug resistance of pathogenic bacteria are avoided.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a strain of Penicillium ochraceus Ynau-PO that is resistant to lead and cadmium and antagonistic to the root rot fungus of Panax notoginseng, and its applications. Background Technology

[0002] Soil heavy metal pollution and soil-borne plant diseases are two major challenges restricting agricultural production and the ecological environment. Among current remediation methods, bioremediation, especially microbial remediation, has attracted much attention due to its environmental friendliness and low cost. Studies have reported on certain fungi (such as Aspergillus)... Aspergillus Penicillium Penicillium Some have resistance to heavy metals, while others (such as Trichoderma) have resistance to heavy metals. Trichoderma It has biocontrol function; that is, existing strains usually only have one of the functions of heavy metal tolerance or biocontrol, and cannot achieve the synergy of "remediation" and "disease prevention". Furthermore, strains specifically designed for biocontrol are difficult to survive in heavy metal contaminated soil and cannot play a role.

[0003] There are still certain limitations on the concentrations of heavy metals that Penicillium species can tolerate, and no Penicillium strains that simultaneously possess high resistance to lead / cadmium and antagonism against Panax notoginseng root rot fungus have been reported. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a strain of Penicillium ochraceus Ynau-PO that is resistant to lead and cadmium and antagonistic to the root rot fungus of Panax notoginseng, and its applications. This strain simultaneously possesses highly efficient resistance to lead / cadmium and the ability to antagonize the root rot fungus of Panax notoginseng.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A *Penicillium ochraceus* strain Ynau-PO that is resistant to lead and cadmium and antagonistic to *Panax notoginseng* root rot, characterized in that the preservation number of *Penicillium ochraceus* Ynau-PO is CCTCC NO:M 20252802.

[0006] The ochre-green penicillin Ynau-PO is used to treat lead and / or cadmium-polluted environments, and the ochre-green penicillin Ynau-PO is tolerant to lead concentrations of up to 2000 mg / L and cadmium concentrations of up to 500 mg / L when treating lead and / or cadmium-polluted environments.

[0007] The ochre-green penicillin Ynau-PO is used for the biological control of root rot in Panax notoginseng, and the pathogenic fungus of root rot in Panax notoginseng is at least one of Fusarium oxysporum, Fusarium solanum, Cyclospora destructosa, and Phytophthora spp.

[0008] This invention provides a *Penicillium ochraceus* strain Ynau-PO that is resistant to lead and cadmium and antagonistic to *Panax notoginseng* root rot fungus, and its applications. Compared with existing technologies, its advantages are: This invention overcomes the problem of single-function strains in existing microbial remediation technologies, providing a dual-function microorganism that can both efficiently tolerate heavy metals lead / cadmium and effectively antagonize Panax notoginseng root rot pathogens; it solves the technical bottleneck of low microbial survival rate and poor remediation effect in high-concentration lead-contaminated soil; and it provides an environmentally friendly biological control solution to reduce the use of chemical pesticides and avoid environmental pollution and the development of pathogen resistance. Attached Figure Description

[0009] Figure 1 This is a schematic diagram showing the growth of Penicillium ochraceus Ynau-PO under different concentrations of lead stress according to the present invention; Figure 2 This is a schematic diagram illustrating the inhibition rate of Penicillium ochraceus Ynau-PO under different concentrations of lead stress according to the present invention; Figure 3 The image shows the growth of *Penicillium ochraceus* Ynau-PO under different concentrations of cadmium stress according to this invention. Figure 4 This is a schematic diagram illustrating the inhibition rate of *Penicillium ochraceus* Ynau-PO under different concentrations of cadmium stress according to the present invention. Figure 5 This is a schematic diagram showing the antagonistic effect of the present invention, *Penicillium ochraceum* Ynau-PO, against different root rot pathogens. The upper part shows the growth of root rot pathogens, and the lower part shows the growth of root rot pathogens after inoculation with *Penicillium ochraceum* Ynau-PO. Figure 6 The inhibition rate of the ochre-green Penicillium Ynau-PO of this invention against different root rot pathogens; Figure 7 This diagram illustrates the incidence rates of Panax notoginseng in different treatment groups according to the present invention. Detailed Implementation

[0010] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0011] The *Penicillium ochraceus* Ynau-PO in the following examples has the accession number CCTCC NO: M 20252802, and the proposed classification name is *Penicillium ochraceus* Ynau-PO. Penicillium ochrochloron Ynau-PO; deposited at the China Center for Type Culture Collection, dated December 8, 2025.

[0012] Example 1: 1. Isolation and identification of strains: Soil samples were collected from the tailings area in Lancang Lahu Autonomous County, Pu'er City, Yunnan Province (22°55′34″N, 99°56′4″E) using a five-point sampling method. After being brought back to the laboratory, 10g of soil was placed in a 250mL sterile Erlenmeyer flask, 90mL of sterile water was added, and the flask was incubated at 30℃ and 180rpm for 1 hour with shaking. This yielded a 10⁻⁶ dilution. -1 Soil suspension, diluted to 10... -1 1.0 mL of soil suspension was added to 9.0 mL of sterile water and mixed thoroughly, resulting in 10 mL of the solution. -2 The soil dilution solution was then serially diluted in this manner to prepare solutions with concentration gradients of 10. -3 -10 -7 The soil dilution solution was taken in batches of 100 μL each. -3 -10 -7 The soil dilution was spread onto Pb using a sterile spreader. 2+ On PDA plates with concentrations of 0, 20, 100, 300, 600, and 2000 mg / L, Cd 2+ A Penicillium strain was purified from PDA plates at concentrations of 0, 5, 10, 50, 100, 300, and 500 mg / L. The ITS rDNA sequence of this strain was sequenced and identified as Penicillium, and named Penicillium ochre-green Ynau-PO.

[0013] 2. Cultivation characteristics: When the obtained Penicillium ochreae-PO strain was placed on a PDA plate, the colonies were bluish-green, fluffy, and grew rapidly, with the optimal growth temperature being 25-28℃.

[0014] 3. Lead and cadmium resistance test: The strain was inoculated into a Pb-containing environment. 2+ On PDA plates with concentrations of 0, 20, 100, 300, 600, and 2000 mg / L, Cd 2+ The samples were incubated on PDA plates at concentrations of 0, 5, 10, 50, 100, 300, and 500 mg / L for 7 days at 28°C. Results are as follows: Figure 1 , Figure 2 , Figure 3 , Figure 4 As shown in Tables 1 and 2 below: Table 1. Pb at different concentrations 2+ Inhibition rate on the growth of Penicillium ochraceus Ynau-PO strain Table 2. Cd at different concentrations 2+ Inhibition rate on the growth of Penicillium ochraceus Ynau-PO strain As shown in the table above, strain *Penicillium ochreaceae* Ynau-PO could grow at all tested concentrations. With increasing Pb²⁺ concentration, the colony diameter increased slightly. The inhibition rates at concentrations of 20-2000 mg / L were (in ascending order of concentration): -2.74%, -3.21%, -9.57%, -9.16%, and 5.14%. At 2000 mg / L (extremely high lead concentration), the inhibition rate was only 5.14%, indicating that the strain maintained good activity. With increasing Cd²⁺ concentration, the colony diameter showed a trend of "promotion at low concentrations and inhibition at high concentrations" (5 mg / L and 10 mg / L were low-concentration promotions, while 50 mg / L, 100 mg / L, 300 mg / L, and 500 mg / L were high-concentration inhibitions). The inhibition rates corresponding to each concentration from 5 to 500 mg / L (in ascending order of concentration) were: -7.54%, -0.98%, 6.07%, 14.75%, 48.69%, and 49.34%, respectively. This strain maintained its activity even under extremely high concentrations of 2000 mg / L Pb²⁺ and 500 mg / L Cd²⁺, demonstrating excellent heavy metal tolerance.

[0015] 4. Antagonism experiment: Select Fusarium oxysporum Z5 ( Fusarium oxysporum ), Fusarium solani F3 ( Fusarium solani ), Destroyer of Columnar Species RS6 ( Cylindrocarpon destructans ), Phytophthora D-1 ( Phytophthora cactorum The pathogens used in the experiment were all strains from the Key Laboratory of Agricultural Biodiversity and Disease Control, Ministry of Education, Yunnan Agricultural University. Among them, Z5 was previously published in Liu Y, Lu R, Tian G, et al. Root-Secreted Saponins Weaken Soil Disease Suppression Ability by Shaping Rhizosphere Microbial Communities in Panax notoginseng[J]. Microbiological Research, 2025: 128263 (Saponins secreted in the rhizosphere of Panax notoginseng weaken the ability to suppress soil diseases by shaping the rhizosphere microbial community structure[J]. *Microbiological Research*. F3 and D-1 were previously published in: Luo Lifen, Jiang Bingbing, Deng Linmei, et al. Effects of several components in Panax notoginseng root exudates on the growth of root rot pathogens [J]. Southern Agricultural Journal, 2020, 51(12):2952-2961. RS6 was previously published in: Yan Xiubo, Li Gaihui, Luo Lifen, et al. Effects of synergistic application of benzothiadiazole and Trichoderma viride on the growth of Panax notoginseng and root rot [J / OL]. Chinese Journal of Biological Control, 1-10 [2026-02-02]. The inhibitory effect of *Penicillium ochraceus* Ynau-PO on *Fusarium oxysporum* Z5, *Fusarium solani* F3, *Corylus demiseus* RS6, and *Phytophthora* D-1 was detected using the plate confrontation method. Activated *Penicillium ochraceus* Ynau-PO strain on PDA medium was used to prepare 5 mm diameter mycelial discs, which were placed in the center of the plates. Pathogen mycelial discs of 5 mm diameter were prepared using the same method and placed around the perimeter of the *Penicillium ochraceus* mycelial discs at a distance of 2.5 cm. Each treatment was repeated in triplicate, with a blank control containing only the pathogenic strain. The plates were incubated at 28℃ for 4 days. Colony diameter was measured using the cross-cross method, and the colony growth inhibition rate was calculated.

[0016] Inhibition rate (%) = (Control colony diameter (mm) - Treatment colony diameter (mm)) / Control colony diameter (mm) × 100%; Specific experimental results are as follows: Figure 5 , Figure 6 And as shown in Table 3 below: Table 3. Inhibition rate of Penicillium ochraceus Ynau-PO against different pathogens. The inhibition rates of the *Penicillium ochreae* strain Ynau-PO against the root rot pathogens of Panax notoginseng: *Fusarium oxysporum* Z5, *Fusarium solanum* F3, *Cyclocarya pallida* RS6, and *Phytophthora infestans* D-1 were 62.04%, 51.18%, 48.39%, and 23.16%, respectively.

[0017] 5. Field verification of root rot: This pot experiment used one-year-old Panax notoginseng plants as experimental material, employing plastic pots with a diameter of 14.7 cm, a height of 12.5 cm, and a base diameter of 10.5 cm. Each pot contained 1.0 kg of test soil, which was prepared by chelating and maturing a mixture of cadmium-contaminated farmland soil (pH 6.3, total cadmium content 48.70 mg / kg) collected from the Zhehai tailings area of ​​Huize, Yunnan Province, with clean local forest soil at a 1:1 ratio. All treatments used the same batch of prepared soil. The experiment included a single cadmium stress treatment (soil containing only cadmium) and a dual stress treatment of cadmium and root rot pathogen (soil containing cadmium and inoculated with the pathogen). The pathogen selected was Fusarium oxysporum Z5, which showed the highest inhibition rate. F. oxysporum ) and Fusarium solani F3 ( F. solani After being cultured separately on PDA plates (200g potato, 20g glucose, 15g agar, and distilled water to a final volume of 1000ml), spores were collected using sterile water and prepared into spore suspensions with a concentration of 1×10⁻⁶. 6 CFU / mL. The biocontrol agent *Penicillium ochreae* Ynau-PO (accession number CCTCC NO: M 20252802) was prepared using the same method to a concentration of 1×10⁻⁶ CFU / mL. 6CFU / mL spore suspension. All treatments included two groups: one inoculated with Penicillium ochraceum and the other uninoculated (control). The root drenching volume per pot was 80 mL each time, with 5 pots per treatment, 10 plants per pot, and 4 replicates. A completely randomized block design was used.

[0018] For the single cadmium stress treatment group, root drenching was performed four times, with each drenching occurring 15 days apart: the inoculated group was drenched with a suspension of *Penicillium ochraceus* spores each time, while the control group was drenched with an equal volume of sterile water each time. For the dual stress treatment group, root drenching was performed seven times, with each drenching occurring 15 days apart: the first time, the inoculated group was drenched with a suspension of *Penicillium ochraceus* spores, while the control group was drenched with sterile water to promote biocontrol colonization; the second, third, and fourth times, all treatments were drenched with a suspension of pathogen spores to establish the disease; the fifth, sixth, and seventh times, the treatment method was restored to the first time, i.e., the inoculated group was drenched with a suspension of *Penicillium ochraceus* spores, while the control group was drenched with sterile water. Plant growth and disease incidence were observed regularly during the treatment period. The incidence of root rot was investigated 14 days after the last treatment and statistical analysis was performed.

[0019] The results are as follows Figure 7 As shown, under different treatment conditions, inoculation with *Penicillium ochraceus* Ynau-PO significantly reduced the incidence of root rot in *Panax notoginseng*. Under single Cd stress, the incidence rate in the inoculated group was 19.94%, significantly lower than the 29.33% in the control group. Under dual Cd and pathogen stress, the biocontrol effect was also significant. For the Cd+Z5 (*Fusarium oxysporum*) combination, the incidence rate in the inoculated group was only 15.45%, significantly lower than the 28.28% in the control; for the Cd+F3 (*Fusarium solani*) combination, the incidence rate in the inoculated group was 22.18%, still lower than the corresponding control of 30.31%. The results indicate that *Penicillium ochraceus* Ynau-PO has stable biocontrol potential against root rot in *Panax notoginseng*, especially under dual Cd and *Fusarium oxysporum* stress.

[0020] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Penicillium ochraceus resistant to lead and cadmium and antagonistic to the root rot fungus of Panax notoginseng ( Penicillium ochrochloron Ynau-PO, characterized in that, The preservation number of the *Penicillium ochreae* Ynau-PO is CCTCC NO:M 20252802.

2. The application of Ynau-PO as described in claim 1 in the treatment of lead and / or cadmium contaminated environments.

3. The application according to claim 2, characterized in that: The *Penicillium ochraceus* Ynau-PO exhibits tolerance to lead concentrations up to 2000 mg / L and to cadmium concentrations up to 500 mg / L when treating environments contaminated with lead and / or cadmium.

4. The application of the *Penicillium ochraceus* Ynau-PO as described in claim 1 in the biological control of root rot in Panax notoginseng.

5. The application according to claim 4, characterized in that: The pathogenic fungus causing the root rot of Panax notoginseng is Fusarium oxysporum. Fusarium oxysporum Fusarium solani ( ) Fusarium solani ), Destroyer of Columnar Species ( Cylindrocarpon destructans ), Phytophthora ( Phytophthora cactorum At least one of the following.

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