Arthrobacter strain hp3 and its use in degrading atrazine and relieving tobacco phytotoxicity

Abstract

This invention discloses a strain of *Arthrobacter*, HP3, and its application in degrading atrazine and alleviating herbicide damage in tobacco. The preservation number of strain HP3 is CGMCC No. 37363. The *Arthrobacter* strain HP3 and its microbial agent provided by this invention can efficiently degrade atrazine technical in the soil and remove its toxic intermediate metabolites, thereby achieving complete detoxification of pesticide residues. Simultaneously, this agent can significantly optimize the rhizosphere microecological environment, enrich beneficial microorganisms, and directly act on tobacco plants, effectively reversing atrazine damage and promoting their growth recovery. This provides an efficient, green, and comprehensive biological solution for addressing the safety production issues of subsequent tobacco crops in grain-tobacco rotation areas.

Description

Technical Field

[0001] This invention relates to a strain of *Arthrobacter* HP3 and its application in degrading atrazine and alleviating tobacco pesticide damage, belonging to the field of environmental microbiology technology and agricultural bioremediation. Background Technology

[0002] Atrazine, a widely used herbicide, causes serious phytotoxicity problems to subsequent sensitive crops such as tobacco due to its residues in the soil. These problems manifest as growth inhibition, chlorophyll destruction, and reduced yield and quality, and have become one of the key bottlenecks restricting safe agricultural production.

[0003] Currently, atrazine treatment employs two main methods: physicochemical remediation and microbial remediation. Physicochemical remediation, such as soil washing or chemical oxidation, can rapidly reduce pollutant concentrations, but it is costly, prone to secondary pollution, and difficult to apply to large-scale farmland. In contrast, microbial remediation, with its environmental friendliness and low cost, is considered a highly promising green alternative technology and has received increasing attention both domestically and internationally in recent years. Currently, numerous atrazine-degrading microbial resources have been discovered and applied. For example, literature (CN109234187A) discloses an atrazine-degrading bacterium and its application; literature (CN110358696A) discloses a microbial agent for degrading atrazine pesticide residues in soil; and literature (CN115927076A) discloses a strain of *Pseudomonas aeruginosa* Atrazine16, its agent, and its application in atrazine degradation.

[0004] However, existing microbial remediation technologies still have significant limitations. First, current research and development largely focus on the degradation efficiency of atrazine parent compounds by strains under laboratory conditions, while generally neglecting their actual mitigation effects on crop phytotoxicity in real soil-plant systems. Many degrading strains, while highly efficient at removing atrazine, may not simultaneously reverse its damage to crop physiological functions, leading to a disconnect between remediation effects and crop health recovery. Second, existing technical solutions (especially single strains) often have limited functions, lacking beneficial regulation of the rhizosphere micro-ecosystem and failing to systematically activate the crop's inherent resilience, thus exhibiting insufficient stability and effectiveness in complex field environments. Finally, there is a lack of in-depth mechanistic analysis, including multi-omics evidence such as the rhizosphere microbiome and plant transcriptome, regarding how strains systematically reduce phytotoxicity through the synergistic effects of "degradation-microecological regulation-plant response." Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an Arthrobacter spp. strain HP3 with high degradation efficiency and the ability to systematically alleviate the damage caused by atrazine in tobacco.

[0006] Another object of the present invention is to provide a microbial agent containing the strain HP3 that is easy to produce and apply.

[0007] Another object of the present invention is to provide the application of the strain HP3 or the inoculant in the degradation of atrazine, remediation of contaminated soil and mitigation of phytotoxicity.

[0008] Another objective of this invention is to provide a method for remediating atrazine-contaminated soil and ensuring the safe production of subsequent tobacco crops.

[0009] The technical solution of this invention is:

[0010] In a first aspect, the present invention provides an Arthrobacter genus strain HP3, the preservation number of which is CGMCC No. 37363.

[0011] In a second aspect, the present invention provides a microbial inoculant comprising at least one of the following: live cells, dormant cells, metabolites or fermentation broth of the strain HP3, and an agriculturally acceptable carrier.

[0012] Preferably, the viable count of strain HP3 in the bacterial agent is not less than 1×10⁻⁶. 7 CFU / g or CFU / mL; the carrier includes one or more of peat moss, vermiculite, kaolin, bentonite or trehalose.

[0013] Thirdly, the present invention provides the application of the strain HP3 or the microbial agent in the degradation of atrazine.

[0014] Fourthly, the present invention provides the application of the strain HP3 or the microbial agent described herein in the preparation of products for remediating atrazine-contaminated soil.

[0015] Fifthly, the present invention provides the application of the strain HP3 or the microbial agent described herein in alleviating atrazine damage to plants and / or promoting the growth of plants under atrazine stress.

[0016] Preferably, the plant is tobacco.

[0017] In a sixth aspect, the present invention provides a method for alleviating atrazine damage to plants, comprising applying the strain HP3 or the microbial agent described above to plants under atrazine stress or their growing environment.

[0018] Preferably, the mitigation of atrazine toxicity is achieved through at least one of the following mechanisms:

[0019] a) Reduce the concentration of atrazine and its toxic intermediate metabolites, deisopropyl atrazine and deethyl deisopropyl atrazine, in the soil;

[0020] b) Increase the relative abundance of Proteobacteria and Sphingosomalids in the rhizosphere soil of plants;

[0021] c) Upregulate the expression levels of photosynthetic system II-related protein genes, glutathione-S-transferase genes, and ABC transporter genes in plant leaves;

[0022] d) Activate the MAPK signaling pathway, phenylpropane biosynthesis pathway, and α-linolenic acid metabolism pathway in plants.

[0023] In a seventh aspect, the present invention provides a method for remediating atrazine-contaminated soil and ensuring the safe production of subsequent crops, comprising applying the strain HP3 or the microbial agent to the soil before or during crop planting. The preferred application amount is to achieve an initial concentration of strain HP3 of 1 × 10⁻⁶ in the soil. 5 ~ 1×10 7 CFU / g.

[0024] This invention, through rigorous pot experiments and multi-omics analysis, demonstrates that the HP3 strain not only efficiently degrades atrazine, but more importantly, it systematically alleviates atrazine damage to sensitive crops (such as tobacco) and promotes their growth. Its mechanism of action is not simply degradation, but rather a synergistic effect achieved through a three-pronged approach: "rhizosphere microecological regulation - soil metabolite remodeling - plant resistance system activation."

[0025] 1. Phenotypic recovery: as shown in the attached document. Figure 4 As shown in Table 1, compared with atrazine alone (AT), HP3 co-treatment with atrazine (AH) significantly increased the maximum leaf length, leaf width and aboveground fresh weight of tobacco by 22.1%, 23.6% and 94.5%, respectively, which directly demonstrates its mitigating effect on herbicide damage.

[0026] 2. Rhizosphere microecological reshaping: as shown in the appendix Figure 5 and 6 As shown, HP3 treatment significantly increased the relative abundance of Sphingomonas, a species with pollutant degradation potential, in rhizosphere soil, indicating that HP3 can recruit and enrich beneficial microorganisms to jointly construct a healthy rhizosphere microenvironment and optimize the microbial community structure.

[0027] 3. Soil toxin removal and metabolic regulation: As shown in Table 2, the contents of key toxic intermediates of atrazine (desopropyl atrazine and deethyldesopropyl atrazine) in the soil of the AH treatment group were significantly lower than those in the AT group. Metabolomics analysis further revealed that HP3 reversed the glycerophospholipid metabolic disorder induced by atrazine.

[0028] 4. Activation of intrinsic plant resistance: For the repair of the photosynthetic system, HP3 treatment significantly upregulated photosynthesis-related genes suppressed by atrazine (see attached). Figure 9 (As shown in A); to enhance detoxification ability, HP3 treatment activated the expression of detoxification and efflux genes such as glutathione-S-transferase (GST) and ABC transporter; to stimulate systemic resistance, HP3 treatment significantly enriched core plant stress resistance pathways such as the MAPK signaling pathway and phenylpropane biosynthesis.

[0029] The beneficial effects of this invention are:

[0030] 1. This invention discovers a degrading bacterium with "plant probiotic function". Through a three-in-one synergistic mechanism of "degradation-regulation-activation", it effectively solves the problem of phytotoxicity of atrazine residues to tobacco. The effect is significant, the mechanism is clear, and it has good prospects for industrial application.

[0031] 2. This invention can significantly alleviate crop phytotoxicity and promote growth. The HP3 strain of this invention overcomes the limitations of existing degrading bacteria with only a single function, possessing both "highly efficient degradation" and "systematic pest control" capabilities. In tobacco pot experiments, compared to atrazine stress alone, application of HP3 significantly reversed atrazine damage, increasing the maximum leaf length, leaf width, and above-ground fresh weight of tobacco by 22.1%, 23.6%, and 94.5%, respectively, thus solving the problem of unstable effects from single-strain degradation applications.

[0032] 3. This invention possesses highly efficient and thorough detoxification capabilities. The HP3 strain not only rapidly degrades atrazine technical grade but also effectively removes its toxic intermediate metabolites (such as deisopropyl atrazine and deethyl deisopropyl atrazine), significantly reducing its residue in the soil and achieving a more thorough detoxification effect than simply degrading the parent compound.

[0033] In summary, the *Arthrobacter* strain HP3 and its microbial agent provided by this invention can efficiently degrade atrazine technical in the soil and remove its toxic intermediate metabolites, thereby achieving complete detoxification of pesticide residues. Simultaneously, this agent can significantly optimize the rhizosphere microecological environment, enrich beneficial microorganisms, and directly act on tobacco plants, effectively reversing atrazine damage and promoting their growth recovery. This provides an efficient, green, and comprehensive biological solution for addressing the safety production issues of subsequent tobacco crops in grain-tobacco rotation areas. Attached Figure Description

[0034] Figure 1 The colony morphology of strain HP3 (A) and its degradation clear zone on atrazine plate (B).

[0035] Figure 2 A phylogenetic tree of strain HP3 constructed based on the 16S rRNA gene sequence;

[0036] Figure 3The degradation kinetics of strain HP3 at different inoculum amounts for 50 mg / L atrazine are shown.

[0037] Figure 4 Phenotypic comparison photos of tobacco plants after 40 days of different treatments;

[0038] Figure 5 Relative abundance of species composition of tobacco rhizosphere soil bacteria (A: phylum level, B: genus level) under different treatments;

[0039] Figure 6 Venn diagrams, numerical statistics, and compositional analysis of ASVs with significant differences among different control groups;

[0040] Figure 7 This section presents bar charts and enrichment bubble charts for KEGG differential metabolites among different comparison groups. In Figure AD, the horizontal axis represents the percentage of metabolites annotated under a specific KEGG pathway out of all annotated metabolites. The vertical axis represents the primary classification of KEGG pathways on the right and the secondary classification on the left. In Figure EH, a larger Ratio (x / y) value and a more rightward-pointing dot indicate a higher enrichment of differential metabolites in that pathway. The color of the dot represents the -log10 (P-value); a redder color indicates a larger -log10 (P-value), and a smaller P-value indicates a higher reliability and statistical significance. The size of the dot reflects the number of differential metabolites in that pathway; a larger dot indicates more differential metabolites in that pathway.

[0041] Figure 8 This is a bubble plot of KEGG pathway enrichment for differentially expressed genes in tobacco leaves in the AH vs AT comparison group. The horizontal axis represents the ratio of the number of differentially expressed genes annotated to the KEGG pathway to the total number of differentially expressed genes, and the vertical axis represents the KEGG pathway. The size of the dot represents the number of genes annotated to the KEGG pathway, and the color from red to purple represents the significance of the enrichment. The redder the color, the more significant the enrichment.

[0042] Figure 9 A heatmap showing the expression patterns of key genes (photosynthesis, ABC transporter, GST, etc.) in tobacco leaves;

[0043] Figure 10 Atrazine residue levels in soil and tobacco leaves under different treatments. Detailed Implementation

[0044] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0045] Example 1: Isolation, Identification and Preservation of Strains HP3

[0046] 1. Enrichment culture: Soil samples collected from Liupanshui, Guizhou Province were inoculated into a basic salt culture medium with atrazine (100 mg / L) as the sole carbon and nitrogen source and cultured at 30℃ and 180 rpm with shaking.

[0047] 2. Isolation and Purification: After three generations of subculturing and enrichment, the enriched solution was plated on a beef extract peptone plate containing 300 mg / L atrazine. Single colonies with a clear zone were picked and repeatedly streaked for purification to obtain a pure strain, named HP3. Figure 1 As shown.

[0048] 3. Molecular identification: Genomic DNA was extracted from HP3, and its 16S rRNA gene was amplified using universal primers 27F / 1492R and sequenced. The sequences were aligned in the EzBioCloud database, and a phylogenetic tree was constructed. The results are as follows: Figure 2 It was determined to be a member of the genus Paenarthrobacter sp.

[0049] Paenarthrobacter ureafaciens strain HP3 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37363, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0050] Example 2: Quantitative analysis of the degradation efficiency of strain HP3

[0051] The activated HP3 bacterial solution was prepared at different final concentrations (10... 6 10 7 and 10 8 (CFU / ml) was inoculated into a basal salt medium containing 50 mg / L atrazine and incubated at 30°C. Samples were taken at different time points, and the atrazine residue was detected by high-performance liquid chromatography. The results are as follows: Figure 3 As shown, in 10 6 10 7 At an inoculum level of CFU / ml, the degradation rate can reach over 99% within 24 hours, while at 10 8At an inoculum level of CFU / ml, the degradation rate reached over 99% after 4 hours.

[0052] Example 3: Pot plant validation and phenotypic analysis of HP3's effect in alleviating atrazine damage in tobacco.

[0053] Yunyan 87 was used as the indicator crop. Control (CK) and treatment levels (AT (0.67 mg / kg atrazine) and HP (10 mg / kg acetic acid) were set up. 7 CFU / gHP3 bacterial agent), AH (10 7 Four treatments were administered: CFU / g HP3 inoculant + 0.67 mg / kg atrazine. The experiment was conducted when the tobacco plants had 6-7 true leaves. The control group (CK) received only soil spraying with water; the AT group received atrazine solution spraying to achieve an effective atrazine concentration of 0.67 mg / kg soil; and the HP group received 10 mg / kg atrazine solution spraying. 8 The concentration of HP3 bacterial solution in soil is 10 CFU / ml. 7 CFU / g, AH group was first sprayed with 10 CFU / g soil. 8 HP3 bacterial culture was diluted to a concentration of 10 CFU / ml. 7 The concentration of CFU / g was then increased, followed by soil spraying with atrazine until the effective concentration reached 0.67 mg / kg. After 40 days of cultivation, agronomic traits were measured.

[0054]

[0055] The agronomic morphology measurement results are shown in Table 1. It can be seen that the growth of tobacco in the AT group was severely inhibited, while the AH group was significantly better than the AT group in all indicators. Among them, the maximum leaf length, leaf width and aboveground fresh weight recovered to 122.1%, 123.6% and 194.5% of the AT group, respectively. Figure 4 The plant phenotypic photos visually demonstrate the significant mitigating effect of HP3 on pesticide damage.

[0056] Example 4: The regulatory effect of HP3 on rhizosphere microecology and soil metabolites

[0057] Rhizosphere soil samples from each treatment in Example 3 were collected for microbial 16S rRNA sequencing and non-targeted metabolomics analysis, and toxin residues in the soil were detected.

[0058] Microbial communities such as Figure 5 and 6 As shown, HP3 treatment significantly altered the bacterial community structure, especially in the AH group, where the relative abundance of the beneficial bacteria Sphingomonas was significantly higher than in the AT group.

[0059] Metabolic pathways such as Figure 7 As shown, KEGG enrichment analysis indicates that HP3 reversed the disruption of key soil metabolic pathways such as glycerol and phospholipid metabolism caused by atrazine.

[0060] The toxin residues are shown in Table 2. The contents of atrazine degradation intermediates deisopropylatrazine and deethylated deisopropylatrazine in the soil of group AH were only 46.9% and 77.6% of those in group AT, respectively, proving that HP3 effectively accelerated the complete decomposition of atrazine in the soil.

[0061]

[0062] Example 5: Transcriptome Regulation Analysis of HP3 on Stress Resistance and Detoxification Systems in Tobacco Plants

[0063] Tobacco leaves and roots from each treatment in Example 3 were subjected to transcriptome sequencing.

[0064] 1. Leaf response showed that compared with the AT group, 4956 genes were upregulated and 4265 were downregulated in the AH group. KEGG enrichment analysis ( Figure 8 The results showed that pathways such as photosynthesis, plant-pathogen interaction, MAPK signaling pathway, and glutathione metabolism were significantly activated.

[0065] 2. Key gene validation: heatmap analysis ( Figure 9 This further confirms that HP3 treatment specifically upregulated the core genes of photosynthesis that were inhibited by atrazine, and continuously overexpressed detoxification genes such as GST and ABC transporter, revealing the intrinsic mechanism by which HP3 alleviates phytotoxicity and enhances tobacco resistance at the molecular level.

[0066] Example 6: Field efficacy verification of HP3 in alleviating herbicide damage from tobacco atrazine.

[0067] The field trial was conducted in tobacco fields with severe atrazine herbicide residues, where herbicide damage had occurred the previous year. The flue-cured tobacco variety was Yunyan 87. Two control groups (CK, conventional production practices) and a field trial (HT, application of HP3 inoculant at a concentration of 10) were established. 7 The bacterial solution (CFU / mL) was applied to the rhizosphere of the tobacco plants at a rate of 50 mL per plant. All treatments followed standardized cultivation management practices, and other agronomic measures were implemented according to local standardized production techniques to ensure the comparability of experimental results. Agronomic traits were measured at 40 and 80 days after transplanting. At harvest, the tobacco leaves were harvested in strata to calculate the post-cured tobacco leaf yield, and soil herbicide residues were also measured.

[0068] The results of agronomic trait testing are shown in Table 3. Atrazine herbicide had a certain inhibitory effect on the agronomic traits of flue-cured tobacco. After application of atrazine-degrading bacteria HP3, the plant height, stem circumference, and maximum leaf area of ​​flue-cured tobacco plants recovered by 16.79%, 7.76%, and 30.13% respectively from the inhibitory effects of atrazine on plant height, stem circumference, and maximum leaf area 80 days after transplanting.

[0069]

[0070] Table 4 shows the economic characteristics of flue-cured tobacco in the field. After applying the atrazine-degrading strain, the yield, value, percentage of high-grade tobacco, and average price of flue-cured tobacco were significantly higher than those of the herbicide control. Specifically, the yield per mu (667 square meters) and the value per mu increased by 19.17% and 15.62%, respectively, and the average price increased by 0.68 yuan per kilogram. These results indicate that the herbicide-degrading bacteria HP3 can significantly alleviate the toxic effects on the soil caused by atrazine application.

[0071]

[0072] Soil herbicide residues such as Figure 10 As shown, after using HP3 degrading bacteria, the atrazine residue in soil and tobacco leaves was significantly reduced compared with the control, by 73.28% and 89.63%, respectively.

Claims

1. A strain of *Arthrobacter*, HP3, characterized in that, The preservation number of the *Arthrobacter* strain HP3 is CGMCC No. 37363.

2. A microbial inoculant, characterized in that, It comprises at least one of the live cells, dormant cells, metabolites or fermentation broth of strain HP3 as described in claim 1, and an agriculturally acceptable carrier.

3. The microbial agent as described in claim 2, characterized in that, The viable count of strain HP3 in the bacterial agent is not less than 1×10⁻⁶. 7 CFU / g or CFU / mL; the carrier includes one or more of peat moss, vermiculite, kaolin, bentonite or trehalose.

4. The use of strain HP3 as described in claim 1 or the microbial agent as described in any one of claims 2-3 in the degradation of atrazine.

5. The use of strain HP3 according to claim 1 or the microbial agent according to any one of claims 2-3 in the preparation of products for remediating atrazine-contaminated soil.

6. The application of strain HP3 as described in claim 1 or the microbial agent as described in any one of claims 2-3 in alleviating atrazine damage to plants and / or promoting the growth of plants under atrazine stress.

7. The application as described in claim 6, characterized in that, The plant in question is tobacco.

8. A method for alleviating phytotoxicity caused by atrazine in plants, characterized in that, This includes applying the strain HP3 of claim 1 or the microbial agent of any one of claims 2-3 to plants or their growing environment under atrazine stress.

9. The method according to claim 8, characterized in that, The mitigation of atrazine phytotoxicity is achieved through at least one of the following mechanisms: a) Reduce the concentration of atrazine and its toxic intermediate metabolites, deisopropyl atrazine and deethyl deisopropyl atrazine, in the soil; b) Increase the relative abundance of Proteobacteria and Sphingosomalids in the rhizosphere soil of plants; c) Upregulate the expression levels of photosynthetic system II-related protein genes, glutathione-S-transferase genes, and ABC transporter genes in plant leaves; d) Activate the MAPK signaling pathway, phenylpropane biosynthesis pathway, and α-linolenic acid metabolism pathway in plants.

10. A method for remediating atrazine-contaminated soil and ensuring the safe production of subsequent crops, characterized in that, This includes applying the strain HP3 of claim 1 or the microbial agent of any one of claims 2-3 into the soil before or during crop planting.

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