Streptomyces cellulophilus p9-2 capable of promoting apple growth and application thereof

Abstract

The present application relates to a kind of cellulose streptomyces P9-2 that can promote apple growth and its application.The cellulose streptomyces P9-2 preservation number is CGMCC No.36795.The strain has the ability of potassium and organic phosphorus, can produce cellulase, amylase, protease, catalase, and its indole acetic acid production reaches 25.16±1.91mg / L.Pot experiment results show that, applying the strain can significantly promote apple growth, compared with control group, plant height, stem diameter, aboveground fresh weight, underground fresh weight, total root length, total root surface area, total root volume, total root tip number are all improved.It shows that the strain can effectively promote apple plant growth, and is suitable for apple cultivation.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and more specifically, to a cellulosic streptomyces p9-2 that can promote apple growth and its applications. Background Technology

[0002] Apple (Malus domestica) belongs to the genus Malus (Malus Mill.) of the family Rosaceae. It is a deciduous tree and a widely cultivated fruit tree variety. During the apple tree's growth process, applying basal fertilizer in autumn plays a significant role in enhancing the tree's disease resistance, accounting for over 80% of the total annual fertilizer application. In autumn, apple trees transfer a large amount of nutrients to the fruit, resulting in a severe deficiency of nutrients within the tree itself, leading to decreased disease resistance. Therefore, fruit growers typically apply fertilizer in autumn after deep tilling, based on the actual growth of the trees, to supplement the tree's nutrients and enhance its disease resistance. Deep tilling in autumn improves soil structure, removes weeds, disrupts the latent environment of pathogens, and reduces the initial inoculum population. The fertilizers applied are mainly well-rotted manure, compost, green manure, and other organic fertilizers, appropriately supplemented with nitrogen, phosphorus, and potassium fertilizers. Furthermore, fruit growers also combine orchard fertilization, rainfall, and temperature changes with measures such as pruning and fruit thinning to regulate fruit yield, achieving a balance between vegetative and reproductive growth, and preventing tree weakness and alternate bearing.

[0003] In recent years, during apple cultivation, some farmers have relied excessively on fertilizer input and overemphasized yield, applying large amounts of chemical fertilizers. This behavior disrupts the balance of the soil's microecology, leading to serious resource and environmental problems such as soil compaction and nutrient imbalance. It is known that the interaction between plant roots and microorganisms plays a crucial role in plant nutrient absorption, stress tolerance, and disease resistance. Bio-fertilizers, as a new type of environmentally friendly fertilizer, contain beneficial microorganisms such as rhizosphere-promoting bacteria that not only provide nutrients to plants, reduce the probability of pests and diseases, and increase yield, but also improve soil quality and prevent secondary soil pollution. Therefore, they have significant application value and development prospects in apple cultivation. Summary of the Invention

[0004] The purpose of this invention is to provide a cellulosic streptomyces P9-2 that can promote apple growth and its applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A cellulosus Streptomyces P9-2 that can promote apple growth was deposited on November 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), classified and named as Streptomyces cellulosae, with accession number CGMCC No. 36795, and deposited at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The application of the aforementioned Streptomyces cellulose P9-2 in promoting apple growth;

[0008] Promoting apple growth refers to increasing any one or more of the following: plant height, stem diameter, above-ground fresh weight, underground fresh weight, total root length, total root surface area, total root volume, and total number of root tips.

[0009] A microbial agent for promoting apple growth, said microbial agent comprising the aforementioned Streptomyces cellulose P9-2;

[0010] The bacterial agent is a liquid preparation;

[0011] Promoting apple growth refers to increasing any one or more of the following: plant height, stem diameter, above-ground fresh weight, underground fresh weight, total root length, total root surface area, total root volume, and total number of root tips.

[0012] A method for promoting apple growth includes the step of applying the above-mentioned Streptomyces cellulose P9-2 or a fungal agent to apple plants.

[0013] The application method is root irrigation;

[0014] Promoting apple growth refers to increasing any one or more of the following: plant height, stem diameter, above-ground fresh weight, underground fresh weight, total root length, total root surface area, total root volume, and total number of root tips.

[0015] The significant advantages of this invention are:

[0016] The cellulosic streptomyces P9-2 provided by this invention can effectively promote the growth of apple plants, especially the root development, and is suitable for apple cultivation. Attached Figure Description

[0017] Figure 1 Colony diagram and microscopic observation of Streptomyces cellulose P9-2 plate.

[0018] Figure 2 : Evolutionary tree of Streptomyces p9-2, a cellulosic fungus.

[0019] Figure 3 Image showing the results of potted apple seedlings of Streptomyces p9-2, a cellulose-producing fungus. Detailed Implementation

[0020] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0022] The formula of Gao's No. 1 solid culture medium used in the embodiments of the present invention is as follows: soluble starch 20g / L, potassium nitrate 1g / L, sodium chloride 0.5g / L, dipotassium hydrogen phosphate trihydrate 0.5g / L, magnesium sulfate heptahydrate 0.5g / L, ferrous sulfate heptahydrate 0.01g / L, and agar 20g / L.

[0023] The formula for the PDA solid culture medium used in this embodiment of the invention is as follows: 200g of peeled potatoes, cut into pieces and boiled in distilled water for 10 minutes, then filtered through gauze, 20g of glucose and 15-20g of agar are added to the filtrate, and distilled water is added to bring the volume to 1000mL.

[0024] The formula for the PDB liquid culture medium used in this embodiment of the invention is as follows: 200g of peeled potatoes, cut into pieces and boiled in distilled water for 10 minutes, then filtered through gauze, 20g of glucose is added to the filtrate, and distilled water is added to make up to 1000mL.

[0025] Example 1: Isolation and identification of Streptomyces cellulose P9-2

[0026] 1.1 Isolation and purification of strains

[0027] The intestinal contents of earthworms were collected using chicken manure as a substrate. Thirty earthworms were randomly selected from a plastic basin containing chicken manure bait and placed on moist filter paper to crawl. After removing surface debris, they were quickly immersed in liquid nitrogen for freezing. Subsequently, they were dissected under a dissecting microscope, and the intestinal contents were collected and placed in sterile centrifuge tubes, then refrigerated at 4°C for later use.

[0028] Actinomycetes in earthworm intestinal contents were isolated using a combination of serial dilution and plate coating. 10g of earthworm intestinal contents were weighed and added to a 250mL Erlenmeyer flask containing 90mL of sterile water (containing glass beads). The flask was placed in a shaker and shaken at 30℃ and 180 r / min for 30min to ensure thorough dispersion of microorganisms. After standing for a short time, 1mL of the supernatant was serially diluted to prepare 1×10⁻⁶ plates.-3 1×10 -4 1×10 -5 and 1×10 -6 Sample dilutions. Take 100 μL of each of the above dilutions and add it to the center of a Gao's No. 1 solid medium plate. Use a sterile spreader to evenly spread the bacterial culture across the entire plate surface. Perform three replicates for each dilution. After multiple streak purifications, pure culture strain P9-2 is obtained and stored in glycerol at low temperature.

[0029] 1.2 Identification of strains

[0030] 1.2.1 Identification of morphological characteristics of strains

[0031] Morphological characteristics of strain P9-2 were identified, and its growth status was observed and recorded. Results showed that strain P9-2 grew well on PDA solid medium. The colony surface was wrinkled and dry, with yellowish-brown aerial hyphae and dark brown hyphae on the substrate, showing visible pigment deposition. The hyphae had a slender, filamentous structure, consistent with the typical culture characteristics of actinomycetes. Photographs of its plate culture and Gram staining are shown below. Figure 1 .

[0032] 1.2.2 Identification of Physiological and Biochemical Characteristics

[0033] The physiological and biochemical characteristics of strain P9-2 were determined according to the methods in the "Manual of Systematic Identification of Common Bacteria" and "Bergey's Manual of Bacterial Identification". The results are shown in the table below.

[0034]

[0035] Note: "+" indicates a positive reaction; "-" indicates a negative reaction.

[0036] Quantitative detection of indoleacetic acid (IAA) production by strain P9-2: Glycerol-preserved strain P9-2 was inoculated onto Gao's No. 1 solid medium plates and activated at 28℃ for 5-7 days; single colonies were picked with an inoculation loop and inoculated into PDB liquid medium, and cultured at 28℃ with shaking at 120 rpm until OD500 was achieved. 600The concentration was 0.6, yielding the seed culture. 2 mL of this seed culture was inoculated into 20 mL of ISP1 liquid medium containing tryptophan (formulation: 0.5 g L-tryptophan, 5 g casein peptone, 3 g yeast extract, 1 L distilled water, pH 7.2), and cultured at 28℃ with shaking at 120 rpm for 72 h. The culture was centrifuged at 5000 g for 10 min, and the supernatant was collected. An equal volume of Salkowski reagent (preparation method: 4.5 g FeCl3 per liter of 10.8 mol / L H2SO4 solution) was added, and the mixture was reacted at room temperature in the dark for 30 min before measuring the absorbance at 530 nm. IAA standard solutions of different concentration gradients were precisely prepared and reacted with an equal volume of Salkowski reagent. The absorbance at 530 nm was measured using the same method. A standard curve was plotted with IAA concentration on the x-axis and absorbance on the y-axis, and a regression equation was fitted. The experiment was repeated three times, and the IAA yield of strain P9-2 was calculated to be 25.16 ± 1.91 mg / L based on the standard curve.

[0037] 1.2.3 Molecular identification

[0038] Genomic DNA was extracted from bacterial strain P9-2 using the Ezup column-based bacterial genomic DNA extraction kit. Using the extracted genomic DNA as a template, the 16S rDNA gene fragment of strain P9-2 was amplified by PCR. After detection by gel electrophoresis, the PCR product was excised and recovered from the gel, and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The 16S rDNA gene sequence of strain P9-2 is shown in SEQ ID NO.1.

[0039] The obtained 16S rDNA gene sequence was subjected to BLAST homology comparison in the NCBI database, similar sequences were downloaded, and a phylogenetic tree of strain P9-2 was constructed using MEGA software and the Neighbor-Joining method. Figure 2 As shown, strain P9-2 naturally aggregates with Streptomyces cellulosae.

[0040] Based on the morphological characteristics, physiological and biochemical features, and 16S rDNA phylogenetic tree of strain P9-2, it was identified as Streptomyces cellulosae.

[0041] Streptomyces cellulosae P9-2 was deposited on November 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), classified as Streptomyces cellulosae, with accession number CGMCC No. 36795, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0042] Example 2: Growth-promoting effect of Streptomyces cellulose P9-2 on apples

[0043] 2.1 Preparation of Streptomyces cellulose P9-2 culture medium

[0044] Glyceryl cultured Streptomyces p9-2 was inoculated onto Gao's No. 1 solid medium plates and activated at 28°C for 5–7 days. Single colonies were picked using an inoculation loop and inoculated into PDB liquid medium, and cultured at 28°C with shaking at 120 rpm until the culture volume reached 2 × 10⁻⁶. 7 The CFU / mL concentration yielded the Streptomyces cellulose P9-2 culture medium, which was used for subsequent apple growth promotion experiments.

[0045] 2.2 Apple seedling pot experiment and growth index determination

[0046] Healthy G935 rootstock apple seedlings with a height of 12-15cm and uniform growth were selected and transplanted into plastic pots with a diameter of 15cm and a height of 17cm. The cultivation substrate was a commercial organic seedling substrate. Two treatments were included in the experiment: (1) a Streptomyces cellulose P9-2 culture medium treatment group; and (2) a PDB liquid culture medium control group. Inoculation was performed using the root irrigation method, with 50mL of the corresponding treatment solution applied to each seedling approximately 2cm from the root. Each treatment group was replicated 10 times. The seedlings were then cultured routinely for 6 weeks after inoculation. After cultivation, plant height, stem diameter, above-ground fresh weight, and underground fresh weight were measured, and comparison images of plant and root growth were taken. At the same time, the apple roots were carefully removed from the substrate and gently rinsed with purified water to remove residual substrate and avoid root damage. The rinsed roots were placed on a transparent root plate or scanning tray containing a small amount of water and scanned using a ScanMaker i800 Plus GXY-A root scanner (Zhejiang, China). The obtained images were analyzed using Scan Wizard EZ software to obtain structural parameters such as total root length, total root surface area, total root volume, and total number of root tips.

[0047] Depend on Figure 3 As shown in the table below, Streptomyces cellulose P9-2 can promote apple plant growth and optimize the root morphology of apple plants.

[0048] Compared with the control group, the *Streptomyces cellulose* P9-2 treatment group showed an increase of 18.6% in plant height, 5.23% in stem diameter, 42.51% in aboveground fresh weight, and 108.03% in underground fresh weight, with the most significant increase in underground fresh weight. Compared with the control group, the *Streptomyces cellulose* P9-2 treatment group also showed an increase of 31.07% in total root length, 66.83% in total root surface area, 136.33% in total root volume, and 24.1% in total root tip number, with the largest increase in total root volume.

[0049]

[0050] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A cellulosic streptomyces P9-2 that promotes apple growth, characterized in that: The Streptomyces cellulosae P9-2 was deposited on November 27, 2025, at the China General Microbiological Culture Collection Center (CGMCC), classified and named Streptomyces cellulosae, with accession number CGMCC No. 36795, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. The application of Streptomyces cellulose P9-2 as described in claim 1 in promoting apple growth.

3. The application according to claim 2, characterized in that: Promoting apple growth refers to increasing any one or more of the following: plant height, stem diameter, above-ground fresh weight, underground fresh weight, total root length, total root surface area, total root volume, and total number of root tips.

4. A fungicide for promoting apple growth, characterized in that: The microbial agent comprises Streptomyces cellulose P9-2 as described in claim 1.

5. The microbial agent according to claim 4, characterized in that: The bacterial agent is a liquid preparation.

6. The microbial agent according to claim 4, characterized in that: Promoting apple growth refers to increasing any one or more of the following: plant height, stem diameter, above-ground fresh weight, underground fresh weight, total root length, total root surface area, total root volume, and total number of root tips.

7. A method for promoting apple growth, characterized in that: The step includes applying the Streptomyces cellulose P9-2 as described in claim 1 or the inoculum as described in any one of claims 4 to 6 to apple plants.

8. The method according to claim 7, characterized in that: The method of application is root irrigation.

9. The method according to claim 7, characterized in that: Promoting apple growth refers to increasing any one or more of the following: plant height, stem diameter, above-ground fresh weight, underground fresh weight, total root length, total root surface area, total root volume, and total number of root tips.

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