A type of leaf-shaped microbacterium, a microbial agent, and the application of leaf-shaped microbacterium.

Abstract

This invention belongs to the field of biotechnology, and particularly relates to a phyllosphaerae microbacterium, a microbial agent, and the application of phyllosphaerae. The phyllosphaerae microbacterium is classified and named *Microbacterium phyllosphaerae*, with the depositary institution being CGMCC, accession number CGMCC No. 24868, and deposit date May 9, 2022. This phyllosphaerae microbacterium can very effectively promote more active root development in early strawberries, forming an early developmental advantage, greatly improving the survival rate of strawberries. Simultaneously, it guides the entire growth cycle of strawberries, improving fruit set rate and actual fruit quality, resulting in a significant increase in the soluble sugar and vitamin C content of strawberries.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to a leaf-shaped microbacterium, a microbial agent, and the application of leaf-shaped microbacterium. Background Technology

[0002] Strawberry is one of the most common hybrids in the genus *Fragaria* of the Rosaceae family. It is a perennial herbaceous plant, 10-40 cm tall, with stems shorter than or nearly equal to the leaves, densely covered with spreading yellow hairs. The leaves are trifoliate; the leaflets have short petioles, are relatively thick, obovate or rhomboid, dark green and nearly hairless on the upper surface, pale green and sparsely hairy on the lower surface, denser along the veins; the petioles are densely covered with spreading yellow hairs. The inflorescence is a cyme, with a short-stalked leaflet below the inflorescence; the flowers are bisexual; the sepals are ovate, slightly longer than the epicalyx; the petals are white, nearly round or obovate-elliptic. It is a very popular economic crop.

[0003] However, strawberry cultivation currently faces certain difficulties. Typically, a single strawberry plant only produces about ten fruits, resulting in a relatively low yield. Furthermore, cultivating strawberry seedlings is quite challenging, as they exhibit poor resistance to diseases and pests, require controlled soil temperature, and are susceptible to rotting and death if transplanted too deeply, or withering if planted too shallowly. Their nutrient requirements are also unique. Consequently, the actual cost of strawberry cultivation remains relatively high.

[0004] In recent years, research has found that microbial fertilizers used in strawberry cultivation can effectively increase strawberry yield and have a significant effect on strawberry cultivation. For example, the use of compound inoculants such as Bacillus amyloliquefaciens, Bacillus subtilis, and Streptomyces cylindrica can effectively achieve disease prevention and yield increase in strawberries. However, research has found that the aforementioned inoculants, such as Bacillus amyloliquefaciens and Bacillus subtilis, mostly play a targeted role in the control of pests and diseases, and only to a certain extent can they decompose some nutrients in fertilizer into smaller molecules that are more conducive to strawberry decomposition, thus improving fertilizer utilization. However, they do not have a significant and direct impact on the actual growth of strawberries. Furthermore, they do not significantly improve the survival rate of strawberry seedlings after transplanting, requiring long-term fertilization. Summary of the Invention

[0005] To address the problems of high cost and difficulty in strawberry cultivation, and the fact that existing microbial fertilizers and compound fertilizers can only provide targeted pest and disease control, this invention provides a leaf-shaped microbacterium, a leaf-shaped microbacterium inoculant, and the application of leaf-shaped microbacterium in strawberry cultivation.

[0006] The purpose of this invention is:

[0007] I. Obtain and screen strains and / or inoculants that are effective for strawberry cultivation;

[0008] II. The survival rate of strawberries after transplanting can be greatly improved by using appropriate strains and / or inoculants;

[0009] III. Achieving effective regulation of strawberry growth;

[0010] IV. To increase strawberry production and improve its quality.

[0011] To achieve the above objectives, the present invention adopts the following technical solution.

[0012] A foliate microbacterium, classified and named Microbacterium phyllosphaerae, is deposited at CGMCC in Beijing, China, with accession number CGMCC No. 24868, on May 9, 2022.

[0013] The leaf-shaped microbacterium used in this invention is a unique strain isolated from the root system of a bayberry orchard. Studies have shown that the unique strain isolated from the bayberry orchard has a very significant and unique promoting effect on the development of strawberry roots.

[0014] The aforementioned *Microbacterium phyllosphaerae* (LX2-4) not only regulates the soil environment and prevents common strawberry diseases when used on transplanted strawberries or strawberry seedlings after a period of cultivation, but also significantly promotes root development in strawberry seedlings. Especially for vertical root development, early vertical root development greatly improves the early survival rate of strawberry seedlings. Although it may affect the development of the above-ground parts of the strawberry plant to some extent and delay flowering and fruiting, it allows the roots of strawberry seedlings to penetrate deeper into the soil more quickly and rapidly, forming a clear early developmental advantage. The development of vertical roots more effectively improves the actual disease resistance of strawberry seedlings, and the faster formation of deep roots makes them more adaptable to cold environments.

[0015] A leaf-shaped microbacterium inoculant,

[0016] The leaf-shaped microbacterium inoculant contains leaf-shaped microbacterium;

[0017] The bacterial concentration OD of the Microbial agent of the leaf-shaped microbes 600 ≥1.0.

[0018] Controlling the bacterial concentration in the inoculant can ensure that the inoculant contains sufficient effective Micrococcus faecalis in order to produce the corresponding promoting effect.

[0019] As a preferred option

[0020] The bacterial concentration (OD) of the *Microbacterium foliata* inoculum 600 It ranges from 1.0 to 2.0.

[0021] The upper limit of bacterial concentration in the *Microbacterium foliata* inoculant is controlled because the bacterial concentration reaches the OD value. 600 When the value is 2.0, it is almost saturated. Further cultivation is not effective, as it has passed the vigorous logarithmic growth phase and reached the growth saturation period.

[0022] Application of a type of leaf-shaped microbacterium,

[0023] The leaf-shaped microbacterium is used for strawberry cultivation.

[0024] The leaf-shaped microbacterium of this invention has a very significant growth-promoting effect on strawberry cultivation, with a focus on improving the survival rate of strawberry seedlings after transplanting and ultimately increasing strawberry yield.

[0025] As a preferred option

[0026] The specific process for using the leaf-shaped microbes in strawberry cultivation is as follows:

[0027] A microbial agent for *Microbacterium foliatum* was prepared using *Microbacterium foliatum*.

[0028] The gluttonous bacteria inoculant was diluted, and the diluted inoculant solution was applied to strawberry seedlings 0-7 days after transplanting.

[0029] Apply 5-7 days apart, for a total of 2-3 applications.

[0030] When using microbial agents to cultivate strawberry seedlings, they need to be diluted to a certain extent first to avoid the excessive concentration of the agent competing with the strawberry seedlings for nutrients. Otherwise, it will not only fail to produce an effective growth-promoting effect, but will also inhibit the growth and development of the strawberry seedlings.

[0031] As a preferred option

[0032] The bacterial solution was obtained by diluting the gluttonous bacteria agent by 85 to 105 times.

[0033] The bacterial solution is applied in doses of 40–60 mL each time.

[0034] Because the bacterial concentration and activity in the original bacterial agent vary, the dilution ratio of the bacterial agent needs to be controlled according to the actual effect of use.

[0035] The beneficial effects of this invention are:

[0036] The leaf-shaped microbacterium of this invention can very effectively promote more active root development in strawberries in the early stages, forming an early developmental advantage, greatly improving the survival rate of strawberries, and at the same time guiding the entire growth cycle of strawberries, improving the fruit set rate and actual fruit quality, and significantly increasing the content of soluble sugars and vitamin C in strawberries. Attached Figure Description

[0037] Figure 1 SEM characterization of the leaf-shaped microbacteria of the present invention Figure 1 ;

[0038] Figure 2 SEM characterization of the leaf-shaped microbacteria of the present invention Figure 2 ;

[0039] Figure 3 Macroscopic photographs of strawberry plants in group CK;

[0040] Figure 4 Macroscopic photographs of strawberry plants in group LX2-4-2. Detailed Implementation

[0041] The present invention will be further described clearly and in detail below with reference to specific embodiments and the accompanying drawings. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0042] Unless otherwise specified, all raw materials used in the embodiments of the present invention are commercially available or obtainable by those skilled in the art; unless otherwise specified, all methods used in the embodiments of the present invention are methods mastered by those skilled in the art.

[0043] Example 1

[0044] This embodiment is used to illustrate the biological characteristics of the leaf-shaped microbacterium described in this invention.

[0045] The strain used in all embodiments of this invention is Microbacterium phyllosphaerae LX2-4. Specifically, the Microbacterium phyllosphaerae is classified and named Microbacterium phyllosphaerae. The depositary institution is CGMCC, the accession number is CGMCCNo.24868, and the deposit date is May 9, 2022.

[0046] Characterization was performed using cell slide culture and scanning electron microscopy, as shown in the following figures. Figure 1 and Figure 2 The SEM image shown.

[0047] from Figure 1 and Figure 2 As can be seen, the overall morphology of the strain is more spindle-shaped or spherical, with a smooth surface and regular edges.

[0048] Example 2

[0049] Preparation of Microbacterium leafii inoculum:

[0050] Using R2A medium as the propagation system for the strain, single colonies of the leaf-shaped microbacterium described in Example 1 were inoculated onto R2A medium and cultured at 30±1℃ until the bacterial concentration reached OD500. 600 The concentration is 1.0–2.5, which yields the leaf-shaped microbacterium inoculum.

[0051] The R2A culture medium formulation includes: 0.25 g / L tryptone, 0.5 g / L acid-hydrolyzed casein, 0.5 g / L yeast extract, 0.5 g / L soluble starch, 0.3 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate, 0.3 g / L sodium pyruvate, 12.0 g / L agar, 0.25 g / L peptone, 0.5 g / L glucose, and a pH of 7.2 ± 0.2.

[0052] Example 3

[0053] Application trials of Microbes folliculorum:

[0054] The experiment was conducted in the strawberry experimental field of Zhejiang Academy of Agricultural Sciences;

[0055] First, strawberry seedlings were selected. All selected seedlings were 15 days old and of the same growth. The root length difference of all seedlings was ≤0.5cm, which was suitable for transplanting. They were transplanted to the experimental field. During transplanting, controllable factors such as seedling spacing, transplanting depth, light environment, and soil temperature were kept basically the same. The cultivation and application operations after transplanting were also exactly the same. Only the inoculant was used as a single-factor variable in the experiment.

[0056] The experiment selected the following 19 sets of data for comparison, with 17 strawberry seedlings in each set.

[0057]

[0058]

[0059] In the table: x = 0 means application on the day of transplanting; in the Bio-13 to Bio-15 test groups in the table, the same type of strain was used for the same substitution test. The strain used to replace LX2-4 was Microbacterium spheroidum purchased from ATCC (preservation number Bio-100287).

[0060] Among them, the strawberry plants were characterized after 35 days of cultivation. Macroscopic photographs of the strawberry plants in the CK group are shown below. Figure 3 As shown in the macroscopic photographs of strawberry plants in group LX2-4-2, Figure 4 As shown. From Figure 3 and Figure 4The comparison shows that the strawberry roots treated with the leaf-shaped microbacterium LX2-4 of this invention are significantly more developed. The average root length of the CK group is about 14cm, while the average root length of the LX2-4-2 group is about 21cm, and the root length of the original strawberry seedlings is about 8cm.

[0061] All root lengths are vertical root lengths.

[0062] Other shape characteristics include root fresh weight and aboveground fresh weight, and the specific characteristics are shown in the table below.

[0063]

[0064]

[0065] In the table: the relative growth rate is the relative elongation rate of the vertical roots compared to the CK group.

[0066] Specifically, the formula for calculating the relative growth rate is as follows.

[0067] In the formula: the original strawberry seedling's vertical root length is 8.36cm.

[0068] As can be seen from the table above, the strawberry seedlings treated with *Microbacterium foliatum* LX2-4 of this invention showed a very significant promotion effect on root development. Compared with the control group, the relative elongation rate reached approximately 123%, meaning that the vertical root growth length was more than twice that of the control group. This clearly demonstrates an excellent effect in promoting the deep development of the strawberry vertical root system. Furthermore, due to the significantly improved root development, the above-ground fresh weight of the strawberry plants also increased significantly.

[0069] Furthermore, comparisons between test groups with equal bacterial concentrations show that different bacterial concentrations require different dilution ratios. For example, the bacterial concentration OD... 600 When the concentration is 1.0, the optimal dilution factor should be around 10 times, which represents the relationship between the concentration of common beneficial bacteria and the dilution factor, while the bacterial concentration OD... 600 When the concentration is 1.5, the dilution factor increases accordingly, and the optimal factor should be around 11 times. However, further increasing the bacterial concentration to the OD value will not yield optimal results. 600 At a concentration of 2.0, the dilution factor actually decreased, which may be related to the physiological activity of *Microbacterium leafii* LX2-4, and the bacterial concentration OD in the inoculum. 600 At a concentration of 2.0, physiological and proliferative activities are reduced, thus allowing for smaller dilutions; a dilution of approximately 8 times yields relatively better results. Further increases in bacterial concentration to OD... 600When the concentration is 2.5, it can be found that even further reduction of the dilution factor is difficult to produce a very obvious optimization effect, which also indicates that the bacterial concentration has a significant impact on the actual use effect of the leaf-shaped microbacterium of the present invention.

[0070] Compared with the *Microbacterium foliatum* (preservation number Bio-100287) used in the Bio-13 to Bio-15 experimental groups, the *Microbacterium foliatum* LX2-4 of this invention showed significantly better growth-promoting effects. Although the fresh weight of roots and aboveground parts in the Bio-13 to Bio-15 experimental groups was higher than that in the CK group, indicating that they also produced certain growth-promoting effects, they did not have a significant effect on the vertical development of the root system. It can also be seen that the *Microbacterium foliatum* isolated by this invention is different from the conventional *Microbacterium foliatum*, and this difference makes it have a significant effect on the vertical development of strawberry roots.

[0071] Further comparison of the LX2-4-2 and LX2-4-16 experimental groups reveals that their growth-promoting effects are relatively similar. However, there are differences in the actual survival rates of the strawberry seedlings. The survival rate of the LX2-4-2 experimental group was 100%, meaning all 17 experimental seedlings survived. In contrast, the survival rates of the CK group and the LX2-4-16 experimental group were 82.4% and 88.2%, respectively. This indicates that applying the LX2-4 bacterial solution of *Microbacterium foliatum* on the day of transplanting has a significant positive impact on the survival rate of strawberry seedlings. The study found that applying the bacterial solution on the day of transplanting protects the root system of the strawberry seedlings. In actual strawberry cultivation, if the goal is to improve the survival rate of strawberry seedlings, the bacterial solution should be applied appropriately on the day of transplanting or the following day to provide protection for the strawberry seedling root system. Comparing the LX2-4-2 and LX2-4-17 experimental groups, it was found that frequent application of the bacterial solution resulted in root and aboveground fresh weight indices of strawberries being similar to the CK group, but root development was slightly better than the CK group. This may be because the bacterial strain competes with the strawberry seedlings for nutrients, thus promoting root development to some extent while affecting the overall development trend. This is also due to the OD concentration of the original bacterial agent. 600 When the bacterial concentration is 1.0, the bacteria exhibit high propagation activity and consume a large amount of nutrients, while the bacterial concentration OD in the original inoculum is low. 600 When the concentration is high, the interval between bacterial suspension applications can be appropriately shortened. A comparison between the LX2-4-2 and LX2-4-18 experimental groups also shows that excessively long intervals between bacterial suspension applications do not produce the corresponding beneficial effects.

[0072] experimental group Number of fruits / plant Single fruit weight experimental group Number of fruits / plant Single fruit weight CK 7.2 18.2g LX2-4-16 9.8 pieces 18.1g LX2-4-2 11.4 18.1g LX2-4-17 6.8 pieces 17.3g Bio-14 7.7 18.3g LX2-4-18 7.3 18.3g

[0073] As can be seen from the table above, the number of fruits harvested per strawberry plant after treatment with the *Microbacterium foliatum* LX2-4 of this invention was significantly increased, with an increase of over 58%. However, there was no significant impact on the weight of individual fruits, which remained at a medium-sized level. It is evident that the main effect of *Microbacterium foliatum* LX2-4 on strawberries is to promote the development of their vertical root system and to promote flowering. This is primarily because the vertical root system meets the developmental needs in advance, allowing the strawberry plant to obtain more nutrients in the early stages of flowering and fruiting, thus greatly improving the flowering and fruiting rates.

[0074] Further quality assessment of the harvested strawberries was conducted, with the main indicators being soluble sugar and vitamin C content.

[0075] experimental group Vitamin C content soluble sugars experimental group Vitamin C content soluble sugars CK 0.96mg / g 1.83％ LX2-4-16 1.05mg / g 1.94％ LX2-4-2 1.07mg / g 1.92％ LX2-4-17 0.95mg / g 1.85％ Bio-14 1.03 mg / g 1.96％ LX2-4-18 0.98mg / g 1.84％

[0076] As can be seen from the table above, the effect of the present invention on improving the quality of strawberry fruit is relatively limited, and the soluble sugar content is even slightly lower than that of the Bio-14 experimental group, but it still has the effect of improving the quality of strawberry fruit.

[0077] In summary, it can be seen that the leaf-shaped microbacterium LX2-4 of the present invention has a significant beneficial effect on the propagation of strawberries, and improves the survival rate, yield and fruit quality of strawberry seedlings.

Claims

1. A leaf-shaped microbacterium, characterized in that, The phyllosphaerae microbacterium is classified and named Microbacterium phyllosphaerae. The depositary institution is CGMCC, the accession number is CGMCC No. 24868, and the deposit date is May 9, 2022.

2. A leaf-shaped microbacterium inoculant, characterized in that, The leaf-shaped microbacterium inoculum contains the leaf-shaped microbacterium according to claim 1; The bacterial concentration (OD600) of the *Microbacterium foliatum* inoculant is ≥1.

0.

3. The leaf-shaped microbacterium inoculant according to claim 2, characterized in that, The bacterial concentration (OD600) of the *Microbacterium foliatum* inoculant is 1.0–2.

0.

4. The application of the leaf-shaped microbacterium according to claim 1, characterized in that, The leaf-shaped microbacteria are used to promote strawberry root development and improve strawberry survival rate.

5. The application of the leaf-shaped microbacterium according to claim 4, characterized in that, The specific process for using the leaf-shaped microbes in strawberry cultivation is as follows: A microbial agent for *Microbacterium foliatum* was prepared using *Microbacterium foliatum*. The microbial agent was diluted, and the diluted solution was applied to strawberry seedlings 0-7 days after transplanting. Apply 5-7 days apart, for a total of 2-3 applications.

6. The application of the leaf-shaped microbacterium according to claim 5, characterized in that, The bacterial solution was obtained by diluting the Microbacterium leafii inoculum by 85 to 105 times.

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