Multifunctional pseudomonas Y2-2 and application thereof

Abstract

The invention relates to a multifunctional pseudomonas Y2-2 and application thereof, the strain is a multifunctional microbial strain, can effectively utilize microorganisms to solve the problem of heavy metal environmental pollution remediation, can also restrain various plant diseases and insect pests, and is classified and named as Pseudomonas sp. The strain is preserved in China General Microbiological Culture Collection Center (CGMCC) on April 21, 2025, the address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.34287. The method can effectively utilize microorganisms to repair heavy metal environmental pollution, can inhibit various plant diseases and insect pests, is good in effect and high in efficiency, and has practical popularization and application values.

Description

Technical Field

[0001] This invention relates to the field of microorganisms, and in particular to a multifunctional genus of Pseudomonas (…). Pseudomonas sp.) Y2-2 and its applications. Background Technology

[0002] Plant diseases caused by pathogenic bacteria and fungi pose a serious threat to the safety of plants for human consumption. Globally, losses due to these diseases can reach up to 30.0% in major crops, severely jeopardizing food security and the quality of agricultural products. Currently, developing resistant crop varieties is one strategy to combat plant diseases; however, breeding resistant varieties is a time-consuming process that can take decades. Furthermore, pathogens can develop resistance through mutation and recombination, reducing the effectiveness of this strategy for controlling plant diseases. Therefore, chemical pesticides play a crucial role in preventing damage and yield losses during disease outbreaks. The ever-increasing amount of chemicals used to protect plants each year leads to environmental pollution, pathogen resistance, and damage to agro-ecosystems. In particular, pesticide deposits used to control plant diseases on crops or their products pose a serious environmental threat to human health and food security. Fungicide resistance is well-known worldwide, manifesting as a heritable decrease in the sensitivity of fungi to specific chemicals, leading to crop yield losses and mycotoxin contamination. Therefore, the control of plant pathogens, especially fungal diseases, in agricultural production should receive greater attention and emphasis.

[0003] The situation of heavy metal pollution in farmland soil in my country is very serious. The long-term unreasonable use of pesticides and fertilizers in agricultural production has also aggravated the heavy metal pollution in the soil. Therefore, the impact of heavy metals on biocontrol agents should also be considered in the process of biological control of crops. According to existing reports, there are few studies on the tolerance and accumulation of heavy metals in existing biocontrol agents. Therefore, it is of great significance and application value to screen out microbial agents that can tolerate and accumulate heavy metals and have good control effects on a variety of plant diseases.

[0004] Among various control methods, biological control is generally considered the most effective approach to disease management due to its environmental friendliness, cost-effectiveness, low toxicity, and promotion of sustainable agricultural practices. Microbial agents are a safer alternative and sustainable management method for controlling plant diseases, and they are known for producing antibiotics, siderophores, and volatile fungicides. In plant disease management, microbial agents, as biopesticides, reduce dependence on chemical pesticides while promoting sustainable agricultural development. Currently, most existing research focuses on screening microbial biocontrol agents for a single disease; research on screening microbial biocontrol agents capable of simultaneously inhibiting multiple diseases and pests is limited. Therefore, this invention addresses these shortcomings by screening microbial strains that exhibit highly effective biocontrol against multiple plant diseases, but such studies have not yet been publicly reported. Summary of the Invention

[0005] In view of the above situation and to overcome the shortcomings of the prior art, the purpose of this invention is to provide a multifunctional *Pseudomonas* genus (…). Pseudomonas sp.) Y2-2 and its applications: This strain is a multifunctional microbial strain that can effectively utilize microorganisms to solve the problem of heavy metal environmental pollution remediation, while also inhibiting a variety of plant diseases and pests.

[0006] The technical solution provided by this invention is a multifunctional Pseudomonas genus Y2-2, classified and named Pseudomonas. Pseudomonas sp. was deposited on April 21, 2025 at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34287.

[0007] The application of the aforementioned Pseudomonas Y2-2 in the remediation of heavy metal pollution and the control of plant diseases.

[0008] This invention relates to a multifunctional Pseudomonas genus Y2-2, classified and named Pseudomonas. Pseudomonas sp. can effectively utilize microorganisms to remediate heavy metal environmental pollution, while also inhibiting various plant diseases and pests. It has good effects, high efficiency, and practical application value. Attached Figure Description

[0009] Figure 1 This is a diagram illustrating the inhibitory effect of the Pseudomonas spp. Y2-2 of this invention on various plant diseases; Among them, 1. *Pythium cornii*; 2. *Pythium cucurbita*; 3. *Fusarium oxysporum* watermelon-specific type; 4. *Fusarium graminearum*; 5. *Pseudomonas syringae* kiwifruit pathogenic species; 6. *Sclerotium sclerotium*. Detailed Implementation

[0010] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances.

[0011] The present invention is illustrated by the following embodiments.

[0012] This invention relates to the acquisition of a multifunctional Pseudomonas spp. Y2-2: On March 13, 2024, soil samples were collected from Yangguo Village, Puyang County, Henan Province (115.2005°E, 35.5371°N). A bacterium, Y2-2, capable of tolerating and accumulating heavy metals and exhibiting control effects against multiple plant diseases, was isolated and purified from the soil. Strain Y2-2 was identified through colony morphology and molecular biology 16S DNA analysis and classified as *Pseudomonas*. PseudomonasThe accession was deposited on April 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 34287. This strain has the function of remediating heavy metal pollution and preventing plant diseases, enabling its application in remediating heavy metal pollution and preventing plant diseases.

[0013] The above-mentioned strains were first screened and preserved by the applicant. They exhibit strong inhibitory effects against heavy metals and various plant diseases, and have broad application value in treating soil heavy metals and various plant diseases. Experiments have shown very good beneficial accumulation and control effects. Relevant information is as follows: 1. Screening and identification of strains On March 13, 2024, soil samples were collected from Yangguo Village, Puyang County, Henan Province (115.2005°E, 35.5371°N). A bacterium Y2-2 that can tolerate and accumulate heavy metals and has a control effect on a variety of plant diseases was isolated and purified from the soil.

[0014] Strain Y2-2 was identified by colony morphology and molecular biology 16S DNA analysis. Single colonies were picked and inoculated into LB liquid medium and cultured at 37°C and 180 rpm for 24 h. PCR amplification was then performed directly using the bacterial culture. The primer sequences used for PCR amplification were: front primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), and back primer 1492R (5'-TACGGTTACCTTGTTACGACTT-3').

[0015] PCR amplification program: 95℃ pre-denaturation for 10 min, 94℃ denaturation for 1 min, 55℃ annealing for 1 min, 72℃ extension for 1 min, 35 cycles. PCR products were detected by 1% agarose gel electrophoresis and then sent to BGI Genomics for sequencing analysis. The sequencing results were compared for homology using BLAST in GenBank.

[0016] The strain isolated by comparing the 16S rDNA sequence of strain Y2-2 with known sequences in Genbank was identified as Pseudomonas and deposited (see deposit certificate).

[0017] 2. Activity test 2.1 Disease resistance experiment of strain Y2-2 The tested plant pathogens were: *Pythium spp.* (corn mold), *Pythium spp.* (melon mold), *Fusarium oxysporum* (watermelon-specific strain), *Fusarium graminearum* (grass mold), *Sclerotium spp.* (sclerotium spp.) and *Pseudomonas syringae* (a pathogenic strain of *Actinidia chinensis*). The first five were fungal pathogens, while *Pseudomonas syringae* was a bacterial pathogen. *Pythium spp.* is a pathogenic fungus causing corn stem rot; *Pythium spp.* mainly infects seedlings and fruits of cucurbits, beans, solanaceae, tobacco, corn, cotton, and sugar beets, causing damping-off, root and stem rot, and fruit rot; *Fusarium oxysporum* var. *hypocreum* primarily infects watermelon and melons, leading to watermelon wilt; *Fusarium graminearum* mainly infects the ears, stems, stem bases, and roots of cereal crops such as wheat, barley, rice, and oats, causing ear rot, stem rot, stem base rot, and root rot; *Sclerotium spp.* can cause soil-borne diseases, mainly affecting the roots and stems of seedlings and young trees, such as peanut white mold; *Pseudomonas syringae* var. *hypocreum* is the pathogen of bacterial canker in kiwifruit, mainly infecting branches, leaves, and fruits of kiwifruit trees, harming plant growth, fruit quality, and yield.

[0018] Strain Y2-2 was inoculated into LB liquid medium and incubated at 30°C and 180 rpm for 24 h. A 5 mm diameter specimen of the aforementioned plant pathogen was placed in the center of a PDA plate. A small amount of strain Y2-2 was pipette-inoculated onto one side of the PDA plate, ensuring the distance between the strain and the central mycelial cake was approximately 3 cm. A PDA plate inoculated only with the pathogen served as a control. Each treatment was replicated in triplicate.

[0019] The LB liquid medium consists of 5 g / L yeast extract, 10 g / L peptone, and 5 g / L sodium chloride, with a pH of 7.0-7.4. The solid medium is prepared by adding 1.5-2% agar by weight to LB medium and autoclaving at 121°C for 20 minutes. LB plates are prepared by pouring LB solid medium into sterile Petri dishes.

[0020] PDA solid medium: potato 200 g / L, glucose 20 g / L, agar 15-20 g / L. PDA plates are prepared by pouring PDA solid medium into sterile petri dishes.

[0021] Pseudomonas syringae was inoculated into LB liquid medium and cultured at 30°C and 180 rpm for 24 h. 100 μL of the culture solution was spread onto LB plates, and then wells were punched on the LB plates. Strain Y2-2 was inoculated into the wells.

[0022] The plates were placed in a 30℃ constant temperature incubator for incubation. When the control plate was fully covered, the antagonistic effect of strain Y2-2 on plant pathogens was observed.

[0023] The results showed that strain Y2-2 exhibited strong antagonistic effects against all five plant pathogens, indicating that strain Y2-2 has the ability to control multiple plant diseases, such as... Figure 1 As shown, it exhibits inhibitory effects against *Pythium spp.*, *Pythium spp.*, *Fusarium oxysporum* (watermelon-specific strain), *Fusarium graminearum*, *Sclerotium spp.*, and *Pseudomonas syringae* pathogens affecting kiwifruit, indicating that this strain possesses inhibitory functions against multiple pathogens.

[0024] 2.2 Experiment on the tolerance effect of strain Y2-2 to heavy metals Four heavy metals, Cr, Cd, Pb and Cu, were selected for this experiment. Strains were picked from the Y2-2 plate and inoculated into LB liquid medium. The plates were then incubated in a shaker at 30℃ and 180 rpm for 24 h. The strains were then inoculated into LB liquid medium containing each heavy metal and their growth was observed. The results are shown in the table below. The table shows that strain Y2-2 is tolerant to all four heavy metals, but the degree of tolerance varies among the different heavy metals.

[0025] Analysis of the tolerance of strain Y2-2 to heavy metals

[0026] In summary, the strain of this invention is a *Pseudomonas* species that the applicant has screened for the first time and is capable of controlling a variety of plant diseases. Pseudomonas This strain (sp.) Y2-2 exhibits excellent tolerance and accumulation of heavy metals Cr, Cd, Pb, and Cu. In plant diseases, it shows strong inhibitory effects against *Pseudomonas syringae* pathogenic species in kiwifruit, *Pseudomonas corniculatus*, *Pythium citrinum*, *Fusarium oxysporum* watermelon-specific strain, *Sclerotium sclerotium*, and *Fusarium graminearum*, effectively addressing the control of multiple plant diseases and enhancing plant resistance to pathogens. The *Pseudomonas* genus to which this invention pertains is a Gram-negative bacterium with diverse metabolic functions, capable of growing under various environmental conditions and utilizing various organic substrates, demonstrating strong adaptability to diverse agricultural environments. Furthermore, *Pseudomonas* produces a series of bioactive antimicrobial metabolites. These metabolites not only possess strong antifungal and antibacterial properties but also stimulate plant growth by promoting root development and nutrient absorption, thereby improving overall plant health and productivity and enhancing plant resistance to pathogens. Moreover, these metabolites can fix or convert heavy metals into non-toxic or less toxic forms. Strain Y2-2 has wide application value in the prevention and control of various plant diseases, and has great economic and social benefits.

Claims

1. A multifunctional genus of Pseudomonas, Y2-2, classified and named Pseudomonas. Pseudomonas sp. was deposited on April 21, 2025 at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34287.

2. The application of Pseudomonas Y2-2 as described in claim 1 in the remediation of heavy metal pollution and the prevention and control of plant diseases.

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