Strain yc2472 of pantoea agglomerans and application thereof

Abstract

The application provides a widely distributed bacteria strain YC2472 and an application thereof, and belongs to the technical field of biological control. Pantoea vagans The taxonomic name of the YC2472 strain is Pantoea vagans, the preservation unit is the Guangdong Microbial Culture Collection Center, the address is the 5th floor of the No. 59 building of the Guangzhou Martyrs' Road 100 Courtyard, the Institute of Microbiology of the Guangdong Academy of Sciences, the preservation date is July 4, 2024, and the preservation number is GDMCC No: 64827. The application screens a bacteria YC2472 with stable and significant antagonistic activity to the pathogenic bacteria from tobacco rhizosphere soil, and the bacteria YC2472 is identified as Pantoea vagans. Pantoea vagans Through retrieval of domestic and foreign literatures, Pan.vagans the biological control activity of the YC2472 strain to the tobacco Phytophthora has not been reported, so that the application highlights the innovation and potential application value.

Description

Technical Field

[0001] This invention relates to the field of biological control technology, specifically to a widely distributed pan-bacterial strain YC2472 and its applications. Background Technology

[0002] Tobacco black shank, tobacco root rot, and tobacco root black rot, caused by *Phytophthora parasitica* var. *nicotianae*, *Fusarium solani*, and *Thielaviopsis basicola*, are devastating soil-borne diseases in tobacco production. These pathogens infect the tobacco root system and stem base, causing root damage and plant wilting through the secretion of metabolites and toxins, as well as reproduction, ultimately leading to severe yield reduction. In recent years, influenced by monoculture practices and climate change, the prevalence of tobacco black shank has become increasingly severe in core tobacco-producing areas worldwide, with pathogen density accumulating in the field, making control increasingly difficult. Biological control, due to its high efficiency, safety, and environmental friendliness, has become an important strategy for integrated plant disease management. Screening for highly effective antagonistic strains is fundamental to the development of biological control agents. Current research indicates that microorganisms such as *Bacillus*, *Streptomyces*, and *Trichoderma* exhibit antagonistic activity against the aforementioned pathogens. In recent years, rhizosphere microbiome research has revealed that specific groups such as Actinobacteria and Acinetobacter play a key role in suppressing plant diseases. However, such research has mostly remained at the community level, failing to clearly identify key functional strains and systematically elucidate their mechanisms of action. Furthermore, the development of existing biocontrol agents has largely focused on a few "star" strains, while microbial resources such as Pantoea sp., with their enormous biocontrol potential and wide ecological adaptability, have received insufficient attention. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of existing biocontrol strains, such as limited functionality and insufficient environmental adaptability. For the first time, this invention provides a Pan-vagans strain YC2472 that exhibits significant antagonistic activity against the pathogen of tobacco black shank (P. nicotianae) and possesses multiple growth-promoting functions. Currently, research on the biocontrol functions of Pan-vagans, both domestically and internationally, mainly focuses on promoting growth and phosphate solubilization or controlling other diseases; no reports have yet been found regarding the biocontrol potential of this strain against tobacco black shank. This invention not only fills the gap in the application of Pan-vagans in the control of tobacco black shank but also reveals the intrinsic mechanism of its combined antagonistic and growth-promoting functions at the genetic level through whole-genome sequencing, providing valuable strain resources and theoretical basis for the development of efficient and stable novel microbial agents.

[0004] The technical solution of this invention is implemented as follows:

[0005] On one hand, this invention provides a strain YC2472, whose taxonomic name is Pantoea vagans, and its accession number is GDMCC No: 64827. More specifically, the YC2472 strain, with the accession name Pantoea vagans YC2472, is deposited at the Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; accession date: July 4, 2024; accession number: GDMCC No: 64827. The viability of this biological material was tested by the collection center on July 4, 2024, and the result showed that it was viable.

[0006] On the other hand, the present invention provides the antagonistic effect of the YC2472 strain against Phytophthora indica.

[0007] On the other hand, the present invention provides the test results of the growth-promoting and physiological and biochemical characteristics of the YC2472 strain.

[0008] On the other hand, the present invention provides whole genome sequencing results and analysis of the YC2472 strain.

[0009] On the other hand, the present invention provides pan-genome analysis results of the YC2472 strain.

[0010] The present invention further provides the application of the above-mentioned pan-bacterial strain YC2472 in antagonizing tobacco black shank disease.

[0011] As a further improvement of the present invention, the pathogens of tobacco black shank include Phytophthora parasitica var. nicotianae, Fusarium solani, and Thieviopsis basicola.

[0012] The present invention has the following beneficial effects:

[0013] (1) This invention is the first to discover and verify the significant antagonistic effect of Pan. vagans YC2472 on the pathogen of tobacco black shank (P. nicotianae), with a plate inhibition rate of 51.33%, filling the gap in the biocontrol research of this strain on tobacco black shank. At the same time, this strain has both nitrogen fixation and potassium solubilization functions, and can achieve a dual effect of "disease prevention-promoting", effectively overcoming the problem of single function of existing biocontrol strains.

[0014] (2) Whole genome analysis showed that the strain has abundant carbohydrate and amino acid metabolism genes and contains a cluster of 7 secondary metabolite synthesis genes, including nonribosomal peptide synthase (NRPS), revealing the material basis of its biocontrol potential from a genetic perspective.

[0015] (3) Further pan-genome comparison revealed that the NRPS and Terpene gene clusters are widely present in multiple Pan. vagans strains, suggesting that this species has the potential for widespread application in biocontrol; at the same time, the 246 genes unique to YC2472 provide genetic resources for its functional expansion. This study not only provides excellent strains for the green control of tobacco black shank, but also lays the species and genomic foundation for the development of broad-spectrum, stable microbial pesticides based on widely distributed pan-dense bacteria. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This diagram illustrates the morphological characteristics and phylogenetic analysis of strain YC2472 of this invention. Figure 1 A: Colony morphology (front view) of YC2472 cultured on TSA solid medium at 30°C for 48 h. Figure 1 B: Gram staining microscopic results of YC2472 (100×); Figure 1 C: A phylogenetic tree constructed based on the atpD and infB multi-gene sequences shows that YC2472 belongs to the Pantoea vagans.

[0018] Figure 2 The diagram shows the antagonistic effect and life-promoting activity of strain YC2472 of this invention against Phytophthora nicotineae. Figure 2 A: The antagonistic effect of YC2472 against Phytophthora indicum; Figure 2 B: Results showing that YC2472 has nitrogen-fixing ability on Assumption solid medium; Figure 2 C: This indicates that the YC2472 potassium-solubilizing medium has potassium-solubilizing ability.

[0019] Figure 3 A circular diagram of the YC2472 genome visually illustrates its genomic composition;

[0020] Figure 4 Orthologous base group (COG) analysis for YC2472 to demonstrate the overall functional classification of the gene;

[0021] Figure 5 Gene ontology (GO) analysis of YC2472 provides detailed functional annotations of the genes.

[0022] Figure 6 KEGG analysis of YC2472 was conducted to explore the role of genes in the metabolic network.

[0023] Figure 7 This represents a potential secondary metabolite gene cluster in YC2472;

[0024] Figure 8 This diagram shows a comparative analysis of the types of secondary metabolite gene clusters of YC2472 and four other antagonistic bacteria of Phytophthora indicum.

[0025] Figure 9 Figure A shows the pan-genome composition of YC2472 and 12 other Pan.vagans strains; Figure B shows the count of conserved and specific genes of YC2472 and 12 other Pan.vagans strains.

[0026] Figure 10 COG classification diagram of the core genes of YC2472 and 12 other Pan.vagans strains;

[0027] Figure 11 COG classification diagram of genes unique to YC2472; Figure 12 This refers to the types of secondary metabolite gene clusters of YC2472 and 12 other Pan.vagans strains. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0029] This invention provides a strain of Pantoea vagans (hereinafter referred to as Pan.vagans) YC2472, taxonomically named Pantoea vagans, deposited at the Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on July 4, 2024, with accession number GDMCC No: 64827. The viability of this biological material was tested by the collection center on July 4, 2024, and the result showed that it was viable.

[0030] Example 1: The bacteria of the present invention is Pan. vagans strain YC2472.

[0031] Strain YC2472 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 4, 2024; the address of the depository is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the accession number is GDMCC No: 64827.

[0032] When strain YC2472 was cultured on TSA solid medium (15 g / L tryptone, 5 g / L soybean peptone, 5 g / L sodium chloride, 15 g / L agar) at 30ºC for 3 days, the colonies were milky white, round, smooth and moist with regular edges. Figure 1 A); Gram staining showed a negative reaction ( Figure 1 B); Phylogenetic analysis based on atpD and infB gene sequences ( Figure 1 C) indicates that the strain is Pan.vagans.

[0033] Example 2: Strain YC2472 can antagonize Phytophthora indica and possesses growth-promoting and physiological / biochemical properties.

[0034] 1. Strain YC2472 can antagonize Phytophthora nicotineae.

[0035] This invention utilizes strain YC2472 cultured in TSB liquid medium (15 g / L tryptone, 5 g / L soybean peptone, 5 g / L sodium chloride) at 30°C and 180 rpm for 3 days in a constant temperature shaker. The supernatant was collected by centrifugation. A plate confrontation experiment was conducted using the Oxford cup method. A 6 mm hole punch was used to create a disc of *Phytophthora tobaccoeris*, which was placed in the center of an OA plate. Oxford cups were placed at three equilateral points 20 mm away from the disc, and 150 μL of the fermentation supernatant of YC2472 was injected into each. TSB liquid medium was used as a control. The plates were incubated in the dark at 28°C for 7 days. The results showed that strain YC2472 exhibited an inhibition rate of 51.33% against *Phytophthora tobaccoeris*. Figure 2 A).

[0036] 2. Assay for the life-promoting activity of strain YC2472

[0037] The YC2472 strain of this invention was streaked on Assumption medium (mannitol 10.0 g / L, K2HPO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, CaSO4·2H2O 0.1 g / L, CaCO3 5.0 g / L, agar 15.0 g / L) and cultured at 30°C for 3 days. The strain was then picked and cultured again under the same conditions on Assumption medium for the next generation. If the next generation still survived, then the YC2472 strain possesses nitrogen-fixing ability. Figure 2 B); The activated YC2472 strain was spot-caught or streaked onto potassium-solubilizing solid medium (sucrose 5.0 g / L, MgSO4·7H2O 0.5 g / L, Na2HPO4 2.0 g / L, FeCl3 0.005 g / L, CaCO3 0.1 g / L, potassium feldspar powder 1.0 g / L, agar 15.0 g / L) plates with potassium feldspar powder as the sole potassium source. The plates were incubated upside down in a 30℃ incubator for 7 days. If the strain showed good growth, then YC2472 possesses potassium-solubilizing ability. Figure 2 C).

[0038] 3. Physiological and biochemical characteristics of strain YC2472

[0039] The physiological and biochemical characteristics of YC2472 were determined using conventional methods. YC2472 exhibits the abilities of catalase, anaerobic growth, ONPG, acid production from arabinose, nucleic acid, xylose, mannitol, and sucrose, and can utilize six substances: glucose, mannose, sorbitol, ornithine, Tween 40, and Tween 80.

[0040] Example 3: Whole genome sequencing and pangenome analysis of strain YC2472

[0041] 1. Whole genome sequencing and analysis

[0042] Whole-genome sequencing of YC2472 yielded a 5,004,432 bp genome draft (GC content 56.49%) containing 4,738 coding genes. Figure 3 Through multi-database functional annotation, COG annotation successfully categorized 4,150 genes. Figure 4 ), annotated to 10,400 genes in the GO database ( Figure 5 ), KEGG annotation shows ( Figure 6 The number of genes involved in carbohydrate and amino acid metabolism is the largest (339 and 348, respectively).

[0043] 2. Prediction of gene clusters for secondary metabolite synthesis

[0044] Seven secondary metabolite synthesis gene clusters were predicted using antiSMASH. Figure 7 This includes non-ribosomal peptide synthases (NRPS), terpenes, and nitrogen-containing azole ribosomal post-synthesis modified peptides.

[0045] 3. Comparative analysis of potential antagonistic gene clusters

[0046] To investigate the genetic mechanism of YC2472's antagonism against Phytophthora indicum, four previously reported antagonistic bacteria (Table 1) were selected for comparison of gene clusters related to the synthesis of primary metabolites. Figure 8 The results showed that the NRPS and terpene gene clusters were prevalent in all five bacterial strains.

[0047] Table 1. Fungi with antagonistic effects against Phytophthora indicum

[0048]

[0049] 4. Pangenome and BGCs Distribution Analysis

[0050] A pan-genome analysis of 13 Pan. vagans strains (Table 2) identified a total of 6,912 orthologous genomes (OGs), of which the core genome accounted for 40.86% (2,824 OGs), and 246 genes were unique to YC2472. Figure 9 Core gene function annotation (COG) shows its extensive involvement in metabolism and environmental adaptation. Figure 10 , 11 Of these, 87.4% of the unique genes have unknown functions, potentially containing novel functional genes. Further analysis of the distribution of BGCs in 13 bacterial strains predicted 13 gene clusters (…). Figure 12 NRPS and terpene were present in more than 80% of the strains (with a few exceptions), suggesting that the antagonistic potential of Pan. vagans against Phytophthora nicotineis may be species-wide.

[0051] Table 2. Genomes of the 12 selected Pan.vagans strains

[0052]

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pantothecin strain YC2472, characterized in that, The taxonomic name of strain YC2472 is Pantoeavagans, and its depository is Guangdong Provincial Microbial Culture Collection Center, located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is July 4, 2024, and the accession number is GDMCC No: 64827. The strain exhibits antagonistic activity against Phytophthora nicotineae.

2. The application of the pan-bacterial strain YC2472 as described in claim 1 in antagonizing tobacco black shank disease caused by Phytophthora parasitica var. nicotianae.

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