Mulberry endophytic enterobacter H62S42 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses mulberry endophytic enterobacter H62S42 and application, and the preservation number of the mulberry endophytic enterobacter H62S42 is CCTCC (China Center For Type Culture Collection) M 20252730. The mulberry endophytic enterobacter H62S42 has various characteristics of promoting plant growth and enhancing stress resistance, and can help mulberry to relieve waterlogging stress.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a mulberry endophytic Enterobacter H62S42 and its applications. Background Technology

[0002] Since its construction, the Three Gorges Reservoir area has formed a drawdown zone with a vertical drop of 30 meters and an area of ​​over 300 square kilometers. This area experiences a cyclical alternation of "summer water and winter land" each year. In summer, the water level reaches as high as 175 meters, with submersion lasting 4 to 6 months, while in winter the water level drops to 145 meters, exposing the land. This unique water-land alternation environment has led to serious ecological and environmental problems, including soil degradation, vegetation destruction, and microbial community imbalance, as detailed below: 1. Deterioration of soil environment: Long-term flooding leads to soil hypoxia, death of aerobic microorganisms, deterioration of soil physical and chemical properties, serious loss of nutrients, and production of a large number of reducing toxic substances; 2. Destruction of vegetation system: The original vegetation has died in large numbers under long-term flooding stress, forming "ecological desertification", which poses a serious threat to the traditional economic forest of mulberry trees in the Three Gorges Reservoir area; 3. Imbalance in the microbial community: Periodic flooding also leads to drastic changes in the structure of the soil microbial community, a sharp decline in the number of beneficial microorganisms, and degradation of the soil ecosystem function.

[0003] To address the aforementioned ecological and environmental problems in the drawdown zone of the Three Gorges Reservoir area, existing vegetation restoration techniques mainly include physical drainage, the use of chemical amendments, the introduction of flood-tolerant plants, and the application of common microbial agents. While these existing techniques have some effectiveness, they generally suffer from high costs and difficulties in implementation, short-lived effects and potential secondary pollution, or failure to effectively adapt to the unique periodic flooding environment of the Three Gorges Reservoir area.

[0004] Therefore, developing a microbial remediation technology specifically adapted to the unique habitat of the drawdown zone in the Three Gorges Reservoir area is of great significance for alleviating waterlogging stress on mulberry trees, promoting mulberry tree growth, and ecological restoration. Summary of the Invention

[0005] This invention relates to a bacterial strain isolated from mulberry trees along the Pengxi River (a tributary of the Yangtze River) in Kaizhou District, Chongqing, China, which, after identification, belongs to Enterobacteriaceae (…). Enterobacter kobei It has a variety of properties that promote plant growth and enhance stress resistance, which can help mulberry trees alleviate waterlogging stress.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions: This invention provides, in one aspect, a strain of endophytic enterobacterium from mulberry trees (… Enterobacter kobei H62S42, with accession number CCTCC M 20252730.

[0007] Another aspect of the present invention provides a bacterial suspension or powder containing the mulberry endophytic enterobacter H62S42 of the present invention.

[0008] In another aspect, the present invention provides a microbial agent comprising the above-mentioned bacterial suspension or bacterial powder of the present invention.

[0009] In another aspect, the present invention provides a fertilizer containing the mulberry endophytic enterobacter H62S42 of the present invention.

[0010] In another aspect, the present invention provides the application of the mulberry endophytic enterobacter H62S42 of the present invention in the preparation of products that promote plant growth and enhance plant stress resistance.

[0011] Preferably, the application of Enterobacter H62S42 in the preparation of microbial agents that help mulberry trees alleviate waterlogging stress.

[0012] Preferably, the above applications include one or more of the following combinations: (a) Application of Enterobacter H62S42 in the preparation of products that produce ACC deaminase; (b) Application of Enterobacter H62S42 in the preparation of products that produce indole-3-acetic acid; (c) Application of Enterobacter H62S42 in the preparation of products with phosphorus-solubilizing and potassium-solubilizing capabilities; (d) Application of Enterobacter H62S42 in the preparation of products with nitrogen-fixing capabilities.

[0013] In another aspect, the present invention provides a method for improving the ability of mulberry trees to alleviate waterlogging stress by applying the above-mentioned mulberry endophytic Enterobacter H62S42 strain, bacterial suspension or powder, microbial agent or fertilizer to the roots of mulberry trees.

[0014] The preservation information of *Enterobacter mulberry* H62S42 in this invention is as follows: Preservation institution: China Center for Type Culture Collection (CCTCC); Address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China; Deposit date: December 1, 2025; Accession number: CCTCC M 20252730; Classification and nomenclature: Mulberry endophytic enterobacter ( Enterobacter kobei ).

[0015] The beneficial effects of this invention include at least the following: 1. The mulberry endophytic enterobacter H62S42 provided by this invention can produce ACC deaminase, which helps promote mulberry growth; it is especially suitable for the special ecological environment of the drawdown zone in the Three Gorges Reservoir area, and can alleviate the negative impact of waterlogging on mulberry trees. 2. The mulberry endophytic enterobacter H62S42 provided by this invention has multiple plant growth-promoting properties such as phosphorus solubilization, nitrogen fixation, potassium solubilization, and production of indole-3-acetic acid (IAA). After applying the strain of this invention, the plant height, chlorophyll content, leaf nitrogen content, dry weight, and fresh weight of mulberry seedlings are significantly improved, showing a strong growth-promoting effect. 3. The endophytic enterobacter H62S42 of mulberry trees of the present invention significantly promotes the development of the root system of mulberry seedlings, increases the length of the taproot, the density of lateral roots, the fresh weight of the underground part, the root surface area and the number of root tips, and improves the overall growth vigor and stress resistance of mulberry trees; 4. The use of the mulberry endophytic enterobacter H62S42 of the present invention provides a new resource for the rapid restoration of mulberry forests in the drawdown zone of the Three Gorges Reservoir area, which helps to repair the damaged ecosystem and improve the regional ecological environment. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the present invention will be further described below in conjunction with the accompanying drawings and embodiments. 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: Figure 1 This is a diagram showing the identification results of strain H62S42 of this invention; Figure 2 This is a graph showing the qualitative detection results of the growth-promoting ability of the strain of this invention; Figure 3 A comparative graph showing the effects of the control group and strain H62S42 on seedling growth morphology; Figure 4 A comparison of radicle length results between the control group and seedlings of strain H62S42; Figure 5 A comparison chart showing the lateral root count results of the control group and strain H62S42 seedlings; Figure 6 A graph showing the comparison of fresh weight results between the control group and the H62S42 strain seedlings; Figure 7 A graph showing the comparison of dry weight results between the control group and the H62S42 strain seedlings; Figure 8 This is a figure showing the effect of treatment with strain H62S42 of the present invention on the growth morphology of mulberry seedlings under waterlogging stress; Figure 9 The corresponding figure shows a comparison of the plant height of mulberry seedlings under waterlogging and non-waterlogging stress. Figure 10 The corresponding figure shows a comparison of the fresh weight of the aboveground parts of mulberry seedlings under waterlogging and non-waterlogging stress. Figure 11The corresponding figure shows a comparison of the stem circumference of mulberry seedlings under waterlogging and non-waterlogging stress. Figure 12 The corresponding figure shows a comparison of the relative chlorophyll content of mulberry seedling leaves under waterlogging and non-waterlogging stress. Figure 13 The corresponding figure shows a comparison of the nitrogen content in the leaves of mulberry seedlings under waterlogging and non-waterlogging stress. Figure 14 The corresponding figure shows a comparison of the root surface area of ​​mulberry seedlings under waterlogging and non-waterlogging stress. Figure 15 The corresponding figure shows a comparison of the root tip count results of mulberry seedlings under waterlogging and non-waterlogging stress; Figure 16 The corresponding figure shows a comparison of the fresh weight of the underground parts of mulberry seedlings under waterlogging and non-waterlogging stress. Detailed Implementation

[0017] 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 described clearly and completely below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0018] The *Enterobacter mulberry* H62S42 in the following examples are all *Enterobacter mulberry* (…). Enterobacter kobei H62S42, this strain was deposited at the China Center for Type Culture Collection (CCTCC) on December 1, 2025, with accession number CCTCC M 20252730, and the deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0019] This invention provides a strain of mulberry endophytic enterobacter H62S42, which is derived from mulberry trees along the Pengxi River (a tributary of the Yangtze River) in Kaizhou District, Chongqing, China. The colony is characterized by a viscous texture, opacity, regular edges, a raised center, and Gram-negative staining. Its 16S rDNA sequence includes the sequence shown in SEQ ID NO. 1.

[0020] This invention screened and identified strain H62S42 (hereinafter referred to as strain H62S42), and the specific screening and identification are as follows: First, mulberry trees were sampled after flooding in the Three Gorges Reservoir area, and endophytic bacteria from the sampled stems and roots were isolated in the laboratory using LB medium. Since many studies have reported that bacteria containing ACC deaminase have strong stress resistance and growth-promoting effects, ACC deaminase (1-Aminocyclopropane-1-Carboxylate Deaminase) was used as an indicator to screen the isolated bacteria, selecting those that produced ACC deaminase, ultimately yielding strain H62S42.

[0021] Then, the growth-promoting and stress-resistance potential of strain H62S42 was determined, revealing that it possesses functions such as phosphorus solubilization, nitrogen fixation, potassium solubilization, and IAA production, indicating that it may promote mulberry tree growth. To verify this effect, mulberry seedlings were treated with the fungicide by adding 20 mL of 1x10... 8 A bacterial suspension of CFU / mL was prepared. One month later, the mulberry seedlings treated with H62S42 showed significantly higher plant height, chlorophyll content, leaf nitrogen content, dry weight, and fresh weight than the untreated group, indicating that strain H62S42 does indeed promote mulberry tree growth.

[0022] Secondly, the effect of strain H62S42 on mulberry seed germination was also investigated. It was found that strain H62S42 had no effect on the germination rate of mulberry seeds, but its application significantly promoted post-germination growth, specifically manifested in increased fresh weight, dry weight, radicle length, and number of lateral roots. These results further demonstrate that strain H62S42 promotes mulberry tree growth.

[0023] Finally, this invention conducted a potted waterlogging experiment. Mulberry seedlings treated with and without the fungicide were subjected to waterlogging. After one month, it was found that the seedlings treated with strain H62S42 had significantly higher physiological indicators, including plant height, fresh weight, dry weight, chlorophyll content, and relative nitrogen content in leaves. This indicates that strain H62S42 helps alleviate waterlogging stress in mulberry trees.

[0024] This invention also provides a bacterial suspension or powder containing the *Enterobacter mulberryis* H62S42 of this invention. In this embodiment, the preparation method of the bacterial suspension or powder can adopt common preparation methods in the prior art.

[0025] In some specific embodiments, the main steps of the bacterial suspension preparation method are as follows: A suitable amount of bacterial cells were picked from the cultured *Enterobacter mulberryii* H62S42 colonies and inoculated into Erlenmeyer flasks containing appropriate LB medium. The flasks were then cultured with shaking at 28–30 °C and 80–200 rpm until the logarithmic growth phase, at which point the bacterial count reached a high level and the bacteria exhibited strong activity. The cultured bacterial suspension was then aseptically transferred to centrifuge tubes and centrifuged at 4000–6000 rpm for 10–15 min. The supernatant was discarded, and the bacterial cells were collected. The cells were washed with sterile physiological saline or buffer solution, and centrifuged again to collect the cells. This step was repeated 2–3 times to remove impurities from the culture medium. Finally, the bacterial suspension was resuspended in an appropriate amount of sterile physiological saline or buffer solution according to the desired concentration to obtain a homogeneous *Enterobacter mulberryii* H62S42 bacterial suspension.

[0026] In some specific embodiments, the main steps of the preparation method of the bacterial powder are as follows: The prepared bacterial suspension was dried using methods such as spray drying or freeze drying. During spray drying, parameters such as inlet and outlet air temperatures and atomizer rotation speed were controlled to rapidly dry the bacterial solution into powder. Freeze drying involved pre-freezing the bacterial suspension at low temperatures until it solidified, followed by sublimation drying under vacuum to obtain *Enterobacter mulberryis* H62S42 bacterial powder. The dried bacterial powder needed to be sealed in packaging and stored in a low-temperature or dry environment to ensure the activity and stability of the bacteria.

[0027] This invention also provides a microbial agent comprising the above-mentioned bacterial suspension or powder of this invention.

[0028] In practical applications, microbial inoculants need to be formulated according to different application requirements and target plants. In addition to containing the mulberry endophytic enterobacter H62S42 bacterial suspension or powder prepared above, appropriate auxiliary ingredients, such as protectants, nutrients, and stabilizers, can also be added.

[0029] In some specific embodiments, the main steps of the preparation method of microbial inoculants are as follows: The bacterial suspension or powder is mixed with auxiliary ingredients in a certain proportion and thoroughly stirred or homogenized under aseptic conditions to ensure uniform dispersion of all components and form a stable mixture. The mixed agent is then dispensed into sealed packaging using suitable materials; the packaging specifications are determined based on actual usage requirements. The dispensed microbial agent undergoes quality testing, including bacterial count testing, activity testing, and impurity testing, to ensure that the agent meets standard requirements.

[0030] This invention also provides a fertilizer containing the mulberry endophytic enterobacter H62S42 of this invention.

[0031] In practical application, a fertilizer formula suitable for mulberry tree growth should be designed, using mulberry endophytic enterobacter H62S42 as the core functional bacterium and combining it with common organic fertilizers, inorganic fertilizers, and micronutrient fertilizers. The proportions of each component in the fertilizer should be adjusted according to the nutrient requirements of mulberry trees at different growth stages to meet their nutritional needs while fully leveraging the growth-promoting and stress-resistance effects of mulberry endophytic enterobacter H62S42.

[0032] In some specific embodiments, the main steps of the preparation method of microbial inoculants are as follows: For the organic fertilizer portion, composting and fermentation are performed first. Organic materials are mixed with an appropriate amount of fermentation inoculant, and moisture, temperature, and aeration conditions are controlled to ensure thorough fermentation and maturation, transforming the organic materials into stable organic matter. The fermented organic fertilizer is then mixed with inorganic fertilizer, micronutrient fertilizer, and a suspension or powder of *Enterobacterium mulberryii* H62S42 according to the formula ratio. During mixing, an appropriate amount of binder can be added to ensure the fertilizer components are tightly bound, forming a granular or powdered fertilizer product. The prepared fertilizer undergoes quality testing, including nutrient content testing, bacterial count testing, and moisture testing, to ensure that the fertilizer quality meets relevant standards.

[0033] This invention also provides an application of the mulberry endophytic enterobacter H62S42 of this invention in the preparation of products that promote plant growth and enhance plant stress resistance.

[0034] The *Enterobacterium mulberryii* H62S42 of this invention can produce plant growth regulators such as indole-3-acetic acid, which can stimulate plant cell division and elongation, promote root growth and development, increase root absorption area, thereby improving the plant's ability to absorb water and nutrients and promoting overall plant growth. Furthermore, the *Enterobacterium mulberryii* H62S42 of this invention also has nitrogen-fixing ability, converting atmospheric nitrogen into ammoniacal nitrogen that plants can utilize, providing nitrogen nutrition to plants, reducing the amount of chemical nitrogen fertilizer used, and improving the nitrogen supply in the soil, which is beneficial to plant growth. In addition, the phosphorus-solubilizing and potassium-solubilizing abilities of *Enterobacterium mulberryii* H62S42 can activate insoluble phosphorus and potassium in the soil, converting them into effective forms that plants can absorb and utilize, improving the utilization rate of phosphorus and potassium nutrients in the soil, and meeting the plant's demand for phosphorus and potassium elements.

[0035] The *Enterobacterium mulberry* H62S42 of this invention can also produce ACC deaminase, which can degrade the ethylene precursor ACC produced by plants under abiotic stress, reducing ethylene synthesis and thus reducing the inhibitory effect of ethylene on plant growth, enhancing the plant's resistance to abiotic stresses such as drought, waterlogging, and salinity. After colonizing around the plant roots, the *Enterobacterium mulberry* H62S42 of this invention can form a biofilm, improving the microecological environment of the plant roots, inhibiting the growth and reproduction of pathogens, reducing the occurrence of plant diseases, and improving the plant's disease resistance.

[0036] In some specific embodiments, Enterobacter H62S42 is used in the preparation of microbial agents that help mulberry trees alleviate waterlogging stress.

[0037] Waterlogging can lead to an oxygen-deficient environment in mulberry tree roots, affecting normal root respiration, reducing root vitality, and weakening the ability to absorb water and nutrients. This, in turn, affects the growth of the above-ground parts of the mulberry tree, resulting in yellowing and curling of leaves, slow growth, and even death. The *Enterobacterium mulberryii* H62S42 in this invention, when used in a microbial agent, can produce ACC deaminase, reducing the ethylene content in the mulberry tree under waterlogging stress, mitigating the inhibitory effect of ethylene on mulberry growth, and maintaining normal physiological metabolic functions. Furthermore, it can improve the microecological environment of the mulberry root system, promote the growth and reproduction of beneficial root microorganisms, enhance root vitality and absorption capacity, improve the efficiency of water and nutrient utilization, and enhance the mulberry tree's adaptability to waterlogging stress.

[0038] In some specific embodiments, the above applications include one or more combinations of the following: (a) Application of Enterobacter H62S42 in the preparation of products that produce ACC deaminase; (b) Application of Enterobacter H62S42 in the preparation of products that produce indole-3-acetic acid; (c) Application of Enterobacter H62S42 in the preparation of products with phosphorus-solubilizing and potassium-solubilizing capabilities; (d) Application of Enterobacter H62S42 in the preparation of products with nitrogen-fixing capabilities.

[0039] This invention also provides a method for improving the ability of mulberry trees to alleviate waterlogging stress.

[0040] In some specific embodiments, the method of the present invention includes applying a strain of *Enterobacter mulberry* H62S42 to the roots of mulberry trees. Specifically, the cultured *Enterobacter mulberry* H62S42 strain is prepared into a bacterial suspension of a certain concentration using sterile water or buffer solution. This suspension is then applied to the soil surrounding the roots of the mulberry trees via root irrigation. The application amount is determined based on the growth stage of the mulberry tree and soil conditions, generally 10–50 mL of bacterial suspension per mulberry tree, with a bacterial count concentration of 1 × 10⁻⁶. 6 -1×10 8 CFU / mL.

[0041] In some specific embodiments, the method of the present invention includes applying a bacterial suspension or powder containing the mulberry endophytic enterobacter H62S42 strain to the roots of mulberry trees. Specifically, the prepared mulberry endophytic enterobacter H62S42 bacterial suspension or powder can be directly mixed with water in a certain proportion and applied as a root irrigation, or the bacterial powder can be mixed evenly with an appropriate amount of fine soil, sprinkled on the soil surface around the roots of the mulberry tree, and then gently tilled into the soil to ensure full contact between the bacterial powder and the soil. The application rate should be adjusted according to the product instructions and actual conditions to ensure that the bacteria can effectively colonize around the mulberry root system.

[0042] In some specific embodiments, the method of the present invention includes applying a microbial agent containing the mulberry endophytic enterobacter H62S42 strain to the roots of mulberry trees. Specifically, the microbial agent containing mulberry endophytic enterobacter H62S42 is applied according to common usage methods and dosages in the art, and can be applied by hole application, trench application, or root irrigation. Hole application involves digging several small holes around the roots of the mulberry tree, applying the agent into the holes, and then covering it with soil; trench application involves digging trenches between the rows of mulberry trees, evenly spreading the agent into the trenches, and then covering it with soil; root irrigation involves diluting the agent and directly watering it into the soil around the roots of the mulberry tree.

[0043] In some specific embodiments, the method of the present invention includes applying fertilizer containing the mulberry endophytic enterobacter H62S42 strain to the roots of mulberry trees. Specifically, the fertilizer containing mulberry endophytic enterobacter H62S42 is applied to the soil as base fertilizer before planting the mulberry trees. The amount of fertilizer is determined according to soil fertility and the growth needs of the mulberry trees, generally 50-200 kg of fertilizer per acre. During the growth of the mulberry trees, topdressing can also be applied according to the actual situation, using trenching or hole application to apply the fertilizer into the soil, followed by covering with soil and watering to promote the dissolution and absorption of the fertilizer and improve the mulberry trees' ability to alleviate waterlogging stress.

[0044] I. Isolation and Screening of Mulberry Endophytic Enterobacter H62S42 1. Sample Collection In May 2024, mulberry tree samples were collected from the water level fluctuation zone along the Pengxi River (a tributary of the Yangtze River) in Kaizhou District, Chongqing, China. The geographical coordinates of the collection site were 31°08'N, 108°30'E. The collected mulberry trees included two varieties, Gui Sang You 62 and Gui Sang You 6, and the growth conditions of these mulberry trees varied.

[0045] 2. Isolation of endophytic bacteria Endophytic bacteria were isolated using a segmented process. Surface-sterilized root and stem segments (approximately 5 cm) were aseptically cut using a sterile scalpel. The fragments were transferred to NA, R-2A, GA, and LB culture dishes. The dishes were incubated at 28°C, with daily checks for microbial growth at the tissue edges. Colonies exhibiting distinct morphological characteristics were selected from each culture medium type and streaked onto LB medium for purification.

[0046] 3. Strain screening and nomenclature After purifying single colonies, strains were screened by testing for ACC deaminase production. Finally, an Enterobacter strain capable of producing ACC deaminase was obtained and named H62S42.

[0047] 4. ACC deaminase detection Following the method of Penrose et al., the activity of ACC deaminase was quantitatively determined using a modified Dworkin and Foster (DF) medium. The specific procedures are as follows: First, the isolates were cultured in basal DF medium, then transferred to ADF medium supplemented with 3 mmol ACC, and cultured with shaking at 25 °C and 180 r / min for 10 days. Isolates that grew vigorously in ADF medium were confirmed to utilize ACC as a nitrogen source. Next, following the method of Penrose et al., the production of ACC deaminase in these cultures was determined by the α-ketobutyrate colorimetric method.

[0048] II. Enterobacteriaceae endophytic in mulberry trees ( Enterobacter kobei Identification of H62S42 1. Morphological and physiological-biochemical identification of strain H62S42 Referring to Bergey's Manual of Bacterial Identification, the strain was inoculated onto LB medium and incubated at 37 °C for 1-2 days. Single colony morphology was observed and Gram staining was performed. Specific results are shown below. Figure 1 As shown. In Figure 1 In the diagrams, A, B, and C represent the morphological observation, Gram staining, and phylogenetic tree results of strain H62S42, respectively.

[0049] according to Figure 1The results showed that strain H62S42 was pale yellow, viscous, opaque, with neat edges and a raised center, and was Gram-negative.

[0050] Physiological and biochemical indicators such as glucose, lactose, galactose, and maltose were measured using bacterial micro-biochemical identification tubes. The specific results are shown in Table 1 below.

[0051] Table 1. Physiological and biochemical test results of strain H62S42

[0052] 2. Molecular biological identification (1) Strains culture and DNA extraction Strain H62S42 was inoculated into LB liquid medium and cultured at 37 °C and 180 r / min for 24 h. After culture, the bacterial cells were collected by centrifugation, and bacterial DNA was extracted using a bacterial genome extraction kit.

[0053] (2) PCR amplification Primer selection: Universal primers 27F (SEQ ID NO. 2) (AGAGTTTGATCMTGGCTCAG) and 1492R (SEQ ID NO. 3) (GGTTACCTTGTTACGACTT) were used for amplification.

[0054] Reaction system: The PCR reaction system was set at 25 μL, which included 12.5 μL of 2× rapid Taq enzyme mixture, 1 μL each of upstream and downstream primers (concentration of 10 μM), and 10 ng of template DNA.

[0055] Thermal cycling parameters: initial denaturation was performed at 95 °C for 4 minutes; followed by 30 cycles, each cycle consisting of denaturation at 94 °C for 30 seconds, annealing at 55 °C for 45 seconds, extension at 72 °C for 1 minute; and finally extension at 72 °C for 8 minutes.

[0056] (2) Product detection and sequencing After the PCR products were detected by 1% agarose gel electrophoresis, they were sent to Sangon Biotech for sequencing.

[0057] (3) Sequence analysis and phylogenetic tree construction Sequence assembly and alignment: After the 16S rRNA gene sequence of the strain was assembled by Seqman, the assembled sequence was subjected to BLAST sequence alignment analysis on NCBI.

[0058] Sequence screening: Select 16S rRNA gene sequences with more than 97% sequence similarity to the strain.

[0059] Phylogenetic tree construction: The MEGA11 software was used to construct a phylogenetic tree using the Neighbor-Joining method to determine the taxonomic position of the strains.

[0060] III. Verification of the growth-promoting and stress-resistance potential of endophytic Enterobacter h62S42 in mulberry trees 1. IAA testing IAA detection was performed on freshly cultured isolates. The isolates were placed in LB liquid medium supplemented with tryptophan (0.2% w / v) and incubated at 30 °C for 72 h. After incubation, the isolates were centrifuged (3000 rpm, 30 min), and 1 mL of the supernatant was reacted with 2 mL of Salkowsky reagent (a mixture of 50 mL of 35% perchloric acid and 1 mL of 0.5 M ferric chloride). After incubating the reaction system in the dark for 20 min, the formation of the pink chromogen was measured at 530 nm, and the synthesis of IAA was confirmed spectrophotometrically.

[0061] 2. Evaluation of other plant growth-promoting (PGP) characteristics The remaining plant growth-promoting (PGP) characteristics were evaluated using selective media, as follows: Phosphorus dissolution assessment: Phosphorus dissolution was assessed on Pikovskaya agar supplemented with 0.5% tricalcium phosphate (Ca3(PO4)2) according to the method of Kumar and Ram (2014).

[0062] Nitrogen fixation activity assay: Nitrogen fixation activity was determined in nitrogen-free Ashby medium according to the methods of Shi et al. (2023) and Alexander (1965).

[0063] Potassium solubility activity determination: The potassium solubility activity was determined in Aleksandrov agar containing potassium aluminum silicate according to the method of Hu et al. (2006).

[0064] After culturing at 30 °C for 5 days, the presence of positive activity was confirmed by visually observing the hydrolysis zones (including those related to phosphate, nitrogen fixation, and potassium release). Specific results are as follows: Figure 2 As shown. In Figure 2 In the diagrams, A through E represent the phosphorus-solubilizing, nitrogen-fixing, potassium-solubilizing, ACC deaminase-producing, and IAA-producing capabilities of strain H62S42, respectively.

[0065] A bacterial strain is considered to have the corresponding ability if it produces a clear zone on the corresponding phosphorus-solubilizing, nitrogen-fixing, and potassium-solubilizing media. If the bacteria can grow in ADF (the medium becomes turbid), it indicates that the strain has the ability to produce ACC deaminase; if the bacterial culture medium reacts with Salkowski's reagent to turn pink, the strain has the ability to produce IAA.

[0066] according to Figure 2 The results show that, through qualitative experiments, strain H62S42 exhibits significant plant growth-promoting properties. Strain H62S42 can produce hydrolysis zones on the corresponding phosphorus-solubilizing, potassium-releasing, and nitrogen-fixing media, indicating that the strain has the ability to solubilize phosphorus, release potassium, and fix nitrogen. Strain H62S42 can grow in ADF medium, indicating that the strain has the ability to produce ACC deaminase. The medium of strain H62S42 reacts with Salkowski's reagent to turn pink, indicating that the strain has the ability to produce indoleacetic acid (IAA).

[0067] IV. Verification of the effect of endophytic enterobacter H62S42 on promoting mulberry tree growth 1. Preparation of experimental materials and grouping From a population of healthy mulberry seedlings, mulberry seedlings of uniform growth were carefully selected. The selected seedlings were similar in height, number of leaves, and size to ensure consistent initial experimental conditions and minimize the impact of individual differences on the results. The selected mulberry seedlings were then divided into two groups: a control group and an H62S42 treatment group.

[0068] 2. Experimental treatment settings Control group (CK): Sterile water was applied to the mulberry seedlings in this group. In practice, an appropriate amount of sterile water was accurately drawn using a pipette and slowly and evenly poured around the roots of the mulberry seedlings to ensure that the sterile water could fully penetrate into the soil around the roots, providing the seedlings with basic water needs, without introducing other microorganisms or substances that may affect growth.

[0069] H62S42 treatment group: The roots of mulberry seedlings in this group were irrigated with H62S42 bacterial suspension at a concentration of 1×10⁻⁶. 8 CFU / mL. Before irrigation, thoroughly shake the prepared H62S42 bacterial suspension to ensure uniform bacterial distribution. Then, use a pipette to draw an appropriate amount of the bacterial suspension and slowly irrigate it around the roots of the mulberry seedlings using the same irrigation method as the control group, so that the bacterial suspension can fully contact the roots and provide specific microbial activity for mulberry growth.

[0070] 3. Observation and Recording of Experimental Results After completing the above treatments, the two groups of mulberry seedlings were placed in the same suitable growing environment, including the same light, temperature, and humidity conditions, for a 30-day cultivation and observation period. During the cultivation period, the growth status of the mulberry seedlings was checked regularly, and relevant data were recorded.

[0071] Thirty days later, the growth indicators of the two groups of mulberry seedlings were measured and compared in detail. The specific results are as follows: Figures 3 to 7 As shown in Table 2, the specific data is as follows.

[0072] exist Figures 3 to 7 middle: Figure 3 A comparative graph showing the effects of the control group and strain H62S42 on seedling growth morphology; Figure 4 A comparison of radicle length results between the control group and seedlings of strain H62S42; Figure 5 A comparison chart showing the lateral root count results of the control group and strain H62S42 seedlings; Figure 6 A graph showing the comparison of fresh weight results between the control group and the H62S42 strain seedlings; Figure 7 This graph shows a comparison of the dry weight results of the control group and the H62S42 strain seedlings.

[0073] Table 2. Detailed measurement results of the average growth indicators of the two groups of mulberry seedlings.

[0074] It should be noted that the values ​​in Table 2 above are the averages of multiple parallel experiments, and some are rounded to two decimal places.

[0075] According to Table 2, Figures 3 to 7 The results showed that strain H62S42 exhibited a strong post-germination growth-promoting effect. Strain H62S42 significantly remodeled the root structure, increasing taproot length by 149.8% (p<0.001) and lateral root density by approximately four times (p<0.001). Furthermore, inoculation with strain H62S42 significantly increased seedling biomass accumulation, resulting in a 132.8% increase in fresh weight (p<0.05) and a 112.2% increase in dry weight (p<0.05). Overall, these results indicate that strain H62S42 has the potential to greatly promote mulberry tree growth.

[0076] V. Verification of the effect of endophytic enterobacter H62S42 in alleviating waterlogging stress in mulberry trees 1. Preparation of experimental materials and grouping From a population of healthy mulberry seedlings, mulberry seedlings of uniform growth were carefully selected. The selected seedlings were similar in height, number of leaves, and size to ensure consistent initial experimental conditions and minimize the impact of individual differences on the results. The selected mulberry seedlings were then divided into two groups: a control group and an H62S42 treatment group.

[0077] 2. Experimental treatment settings Control group (CK): This group of mulberry seedlings underwent continuous flooding treatment. Specifically, the seedlings were placed in a specially designed experimental container, and water was slowly poured into the container, ensuring the water level remained above the roots of the seedlings, guaranteeing complete submersion of the roots. This flooding state was maintained throughout the experiment to simulate waterlogging stress.

[0078] H62S42 Treatment Group: Mulberry seedlings in this group received the same flooding treatment as the control group, while simultaneously being irrigated with H62S42 bacterial suspension at the roots. First, a concentration of 1×10⁻⁶ was precisely prepared. 8 Prepare a CFU / mL suspension of H62S42 bacteria and shake it thoroughly to ensure even distribution of the bacteria. Then, use a pipette to accurately draw up an appropriate amount of the bacterial suspension and slowly and evenly pour it around the roots of the mulberry seedlings, ensuring that the bacterial suspension can fully penetrate into the soil around the roots, allowing the bacterial strain to make close contact with the roots and thus exert its potential effect in alleviating waterlogging stress.

[0079] 3. Observation and Recording of Experimental Results Both groups of mulberry seedlings were placed in the same suitable growing environment, including the same light intensity, light duration, temperature range, and air humidity, to eliminate interference from other environmental factors. During the 30-day experimental period, the growth of the mulberry seedlings was observed and recorded regularly.

[0080] Twenty-five days later, the growth indicators of the two groups of mulberry seedlings were measured and compared in detail. The comparison table of average growth indicators is shown in Table 3; the comparison graph is shown below. Figures 8 to 16 As shown. Among them, Figure 8 This indicates the effect of strain H62S42 treatment on the growth morphology of mulberry seedlings under waterlogging stress. Figure 9-16 The effect of strain H62S42 treatment on the growth indicators of mulberry seedlings under waterlogging stress is indicated.

[0081] exist Figure 8 In the figure, the graphs corresponding to A and C represent the effects of different strain treatment groups on the growth morphology of mulberry seedlings under waterlogging conditions; the graphs corresponding to B and D represent the effects of different strain treatment groups on the growth morphology of mulberry seedlings under non-waterlogging conditions; and the graph corresponding to E represents the effects of different strain treatment groups on the growth morphology and leaf mortality of mulberry seedlings under non-waterlogging conditions.

[0082] Figures 9 to 16 The corresponding graphs represent the following indicators: Figure 9 The corresponding figure shows a comparison of the plant height of mulberry seedlings under waterlogging and non-waterlogging stress. Figure 10 The corresponding figure shows a comparison of the fresh weight of the aboveground parts of mulberry seedlings under waterlogging and non-waterlogging stress. Figure 11The corresponding figure shows a comparison of the stem circumference of mulberry seedlings under waterlogging and non-waterlogging stress. Figure 12 The corresponding figure shows a comparison of the relative chlorophyll content of mulberry seedling leaves under waterlogging and non-waterlogging stress. Figure 13 The corresponding figure shows a comparison of the nitrogen content in the leaves of mulberry seedlings under waterlogging and non-waterlogging stress. Figure 14 The corresponding figure shows a comparison of the root surface area of ​​mulberry seedlings under waterlogging and non-waterlogging stress. Figure 15 The corresponding figure shows a comparison of the root tip count results of mulberry seedlings under waterlogging and non-waterlogging stress; Figure 16 The corresponding figure shows a comparison of the fresh weight of the underground parts of mulberry seedlings under waterlogging and non-waterlogging stress.

[0083] Table 3. Detailed measurement results of the average growth indicators of the two groups of mulberry seedlings.

[0084] It should be noted that the values ​​in Table 3 above are the averages of multiple parallel experiments, and some are rounded to two decimal places.

[0085] according to Figure 8 The results showed that after 25 days of controlled waterlogging treatment, strain H62S42 significantly alleviated waterlogging stress in mulberry seedlings, superior to the uninoculated control group. Under ultraviolet irradiation, the leaf mortality rate in the groups treated with strain H62S42 under waterlogging conditions was observed to be lower than that in the control group. Figure 8 (C and E in the text). In contrast, the groups that were not flooded exhibited a strong red discoloration (C and E in the text). Figure 8 In the D and F groups, and consistent with previous growth-promoting experiments, strain H62S42 significantly promoted the growth of mulberry seedlings.

[0086] According to Table 3, Figures 9 to 16 The results show that the growth indicators of each group in this invention are compared in detail below: Average fresh weight of aboveground parts: Under flooded conditions, the average fresh weight of aboveground parts increased from 0.37 g in the control group to 0.90 g in the H62S42 treatment group, an increase of 142.5% (p<0.001); under natural conditions, the average fresh weight of aboveground parts increased from 1.40 g in the control group to 3.60 g in the H62S42 treatment group, an increase of 157.4% (p<0.001).

[0087] Average plant height: Under flooded conditions, the plant height increased from 7.10 cm in the control group to 10.11 cm in the H62S42 treatment group, an increase of 42.4% (p<0.001); under natural conditions, the plant height increased from 13.09 cm in the control group to 20.27 cm in the H62S42 treatment group, an increase of 54.9% (p<0.001).

[0088] Chlorophyll content: Under flood conditions (day 25), the H62S42 treatment group maintained a significantly higher chlorophyll content of 19.61±0.42, while the control group was 17.25±0.45 (p<0.01); under natural conditions (day 25), the chlorophyll content of the H62S42 treatment group was 28.29±0.46, while the control group was 22.75±0.44 (p<0.001).

[0089] Nitrogen content: Under flood conditions (day 25), the nitrogen content of the H62S42 treatment group was 8.73±0.12, while that of the control group was 7.83±0.12 (p<0.001); under natural conditions (day 25), the nitrogen content of the H62S42 treatment group was 11.59±0.13, while that of the control group was 9.73±0.11 (p<0.001).

[0090] Root development under flooded conditions: root fresh weight (i.e., fresh weight of the underground part) increased by about 115.4%; root surface area (root shade area) increased by about 93.1%; and the number of root tips increased by about 96.1%.

[0091] Based on the above data comparison, under natural conditions, the treatment group inoculated with H62S42 showed significantly better performance than the uninoculated control group in almost all root structure parameters. In summary, the results of this invention demonstrate that strain H62S42 can enhance the waterlogging resistance of mulberry trees.

[0092] This invention observed the root systems of each group after flooding. Compared to the control group, the treatment group containing strain H62S42 significantly promoted root development in mulberry seedlings under flooding conditions. Specifically, it significantly increased the fresh weight of mulberry roots, root shadow area, and number of root tips. Under natural conditions, the H62S42-inoculated treatment group was significantly superior to the uninoculated control group in almost all root structure parameters. In summary, the results of this invention indicate that strain H62S42 can enhance the flood resistance of mulberry trees.

[0093] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the scope of the present invention should be included within the protection scope of the present invention.

Claims

1. Endogenous Enterobacteriaceae in a mulberry tree ( Enterobacter kobei H62S42, characterized in that, The strain was deposited at the China Center for Type Culture Collection on December 1, 2025, with accession number CCTCC M 20252730.

2. A bacterial suspension or powder containing the mulberry endophytic enterobacter H62S42 as described in claim 1.

3. A microbial inoculant, characterized in that, Includes the bacterial suspension or bacterial powder as described in claim 2.

4. A fertilizer containing the mulberry endophytic enterobacter H62S42 as described in claim 1.

5. The application of the mulberry endophytic enterobacter H62S42 as described in claim 1 in the preparation of products that promote plant growth and enhance plant stress resistance.

6. The application according to claim 5, characterized in that, Application of Enterobacter H62S42 in the preparation of microbial agents to help mulberry trees alleviate waterlogging stress.

7. The application according to claim 5 or 6, characterized in that, Applications include one or more of the following combinations: (a) Application of Enterobacter H62S42 in the preparation of products that produce ACC deaminase; (b) Application of Enterobacter H62S42 in the preparation of products that produce indole-3-acetic acid; (c) Application of Enterobacter H62S42 in the preparation of products with phosphorus-solubilizing and potassium-solubilizing capabilities; (d) Application of Enterobacter H62S42 in the preparation of products with nitrogen-fixing capabilities.

8. A method for improving the ability of mulberry trees to alleviate waterlogging stress, characterized in that, The mulberry endophytic enterobacter H62S42 of claim 1, the bacterial suspension or powder of claim 2, the microbial agent of claim 3, or the fertilizer of claim 4 are applied to the roots of the mulberry tree.

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