A mycoparasite, a mycoparasite agent and application of the mycoparasite

Abstract

This invention belongs to the field of biotechnology, and specifically relates to a variovorax, a variovorax inoculant, and the application of the variovorax. The variovorax is classified and named *Variovorax soil*, its depositary institution is CGMCC, its accession number is CGMCCNo.24867, and its deposit date is May 9, 2022. This variovorax can very effectively promote the development of horizontal roots in strawberry seedlings and control nutrient levels in the soil, as well as promote the expansion and development of the strawberry plant crown, reducing field shading. This significantly improves the quality and weight of strawberry fruits, and also helps increase the early survival rate of strawberry seedlings.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a glutathione, a glutathione agent, and the application of glutathione. Background Technology

[0002] Strawberries, also known as red berries, belong to the Rosaceae family. Originally from Europe, they were later introduced to my country. Strawberries are a fruit known for their excellent taste, vibrant color, and high nutritional value. The fruit is brightly colored, with exceptionally soft and juicy flesh, boasting an edible component of up to 98%. They are rich in various essential vitamins, amino acids, minerals, fiber, anthocyanins, and other nutrients. China's strawberry industry has developed for 30 years, making China the world's largest strawberry producer. With the continuous improvement of Chinese people's consumption levels and living standards, the demand for strawberries is constantly rising, and their positioning is gradually shifting from a common fruit to a premium one. However, with the continuous expansion of strawberry planting areas, the use of pesticides and fertilizers has increased exponentially, leading to a decline in fruit quality and a series of problems such as soil and environmental pollution. With the shift in the positioning of strawberries and the increasing demand for healthy living, the issue of reducing pesticide and fertilizer use while developing new fertilizer sources and improving strawberry quality has received widespread attention.

[0003] With increasing health awareness and pollution control, organic agriculture is receiving growing attention from researchers and the public. Microbial inoculants, also known as microbial fertilizers, are bioactive products or inoculants that include beneficial bacteria from one or more fungal strains. They are used to promote growth and yield by increasing root utilization of nutrients in the soil, such as fixing nitrogen, dissolving phosphorus, or breaking down cellulose.

[0004] However, research has found that while microbial fertilizers can be used in strawberry cultivation and effectively increase strawberry yield, playing a beneficial role in strawberry cultivation—for example, the combination of agents such as Bacillus amyloliquefaciens, Bacillus subtilis, and Streptomyces fulvicina can effectively prevent disease and increase yield—they almost completely fail to improve strawberry quality. This is because strawberries have a low survival rate and are difficult to cultivate; simply increasing the amount of fertilizer not only fails to effectively improve strawberry quality but can also easily lead to mass mortality. Therefore, how to use microbial fertilizers to improve strawberry fruit quality in a gentler way is an important research direction. Summary of the Invention

[0005] To address the challenges of high cultivation difficulty and the lack of effective methods to improve strawberry survival rates while enhancing fruit quality, this invention provides a leech bacterium, a fungal agent prepared from the leech bacterium, and the application of the leech bacterium 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. Significantly improves the quality of strawberry fruit.

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

[0012] A glutathione bacterium, classified and named Variovorax soil, is deposited at CGMCC with accession number CGMCCNo.24867 on May 9, 2022.

[0013] The *Variovorax soil* strain LXC3-3 of this invention is a unique strain isolated from the root system of a bayberry orchard. Research shows that this unique strain has a unique guiding effect on the development of strawberry plant roots, not by directly promoting it, but by altering the root development trend. Since strawberry root development in its early stages is primarily vertical, especially immediately after transplanting, vertical root development effectively enhances the disease resistance and environmental adaptability of strawberry seedlings. The *Variovorax soil* strain of this invention can, to a certain extent, maintain a constant soil temperature, improve soil aeration and looseness, and protect the strawberry seedling roots, thereby enhancing the disease resistance of the strawberry seedlings. On the other hand, the phytophagium bacterium of this invention differs from conventional phytophagium bacteriums. Conventional phytophagium bacteriums have good nitrogen-fixing ability and can convert insoluble phosphates into soluble phosphates that are more easily absorbed by plants. However, the phytophagium bacterium of this invention can form a unique nitrogen fertilizer cycle, which can control the nitrogen content in the soil, reduce nitrogen loss, and control the absorption of nitrogen fertilizer by strawberry plants. This characteristic is usually unfavorable for the cultivation of cash crops, but it is indeed particularly beneficial for the cultivation of strawberries. This is mainly because in the early growth and development of strawberries, nitrogen will greatly increase the leaf area of ​​strawberries, causing shading in the field, which will lead to a reduction in the number of flowers, smaller fruits, and induce strawberry plant diseases. However, research has found that the phytophagium bacterium of this invention can dynamically control the content of absorbable nitrogen in the soil to a certain extent, keeping it within the range suitable for strawberry growth, and has a very good positive impact on the development of strawberries and flowering and fruiting during the ripening period. On the other hand, the root-promoting effect of the phytophagous bacteria of this invention differs from that of conventional phytophagous bacteria. Conventional phytophagous bacteria mainly promote the absorption of nutrients by the roots, making the roots thicker and more developed. However, the phytophagous bacteria of this invention stimulates the natural branching ability of the vertical roots through biological action, resulting in more new roots that are absorbing roots, thus promoting the development of the horizontal root system and the expansion of the strawberry plant crown. Combined with the control of nitrogen absorption, this makes it less likely for the above-ground parts of the strawberry plant to form a shading effect. The development of the horizontal root system also promotes the earlier and smoother transformation of the above-ground parts into reproductive organs, thereby improving the development effect of reproductive organs.

[0014] A gluttonous bacteria agent,

[0015] The gluttonous bacteria agent contains gluttonous bacteria;

[0016] The bacterial concentration OD of the gluttonous bacteria inoculant 600 ≥1.0.

[0017] Controlling the bacterial concentration in the microbial agent can ensure that the agent has enough active gluttons to produce the corresponding promoting effect.

[0018] As a preferred option

[0019] The bacterial concentration OD of the gluttonous bacteria agent 600 It ranges from 1.0 to 2.0.

[0020] The upper limit of bacterial concentration in the *Vampire Digestives* 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.

[0021] An application of a gluttonous bacterium,

[0022] The gluttonous bacteria are used for strawberry cultivation.

[0023] The glutathione bacterium of this invention has a very significant growth-promoting effect when used in strawberry cultivation, with a focus on improving the survival rate of strawberry seedlings after transplanting and ultimately enhancing the quality of strawberry fruits.

[0024] As a preferred option

[0025] The specific process of using the gluttonous bacteria in strawberry cultivation is as follows:

[0026] The glutathione was used to prepare the glutathione inoculum;

[0027] The glutathione was used to prepare the glutathione inoculum;

[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 glutathione bacteria in strawberry seedling cultivation, it is necessary to dilute it to a certain extent first to avoid the excessive concentration of the inoculant competing with the strawberry seedlings for nutrients. This would not only fail to produce an effective growth-promoting effect, but would 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 glutathione bacteria of this invention can very effectively promote the development of horizontal roots in strawberry seedlings and control the nutrient levels in the soil, as well as promote the expansion and development of the strawberry plant crown, reduce the shading effect in the field, and significantly improve the quality and weight of strawberry fruits. At the same time, the glutathione bacteria are beneficial to improving the early survival rate of strawberry seedlings. Attached Figure Description

[0037] Figure 1 SEM characterization of the glutathione LXC3-3 of this invention Figure 1 ;

[0038] Figure 2 SEM characterization of the glutathione LXC3-3 of this invention Figure 2 ;

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

[0040] Figure 4 Macroscopic photographs of strawberry plants in group LXC3-3-2. 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 gluttonous bacteria of the present invention.

[0045] The strain used in all embodiments of this invention is Variophora spp. LXC3-3, which is classified and named Variophora spp., deposited at CGMCC with accession number CGMCC No. 24867 on 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 spindle-shaped, and the surface of the strain is not smooth, resembling an earthworm.

[0048] Example 2

[0049] Preparation of *Gnaphalium affine* inoculum:

[0050] Using R2A medium as the propagation system for the strain, single colonies of the glutathione 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 to 2.5, which yields the gluttonous bacteria 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 gluttonous bacteria:

[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 2 months 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 same day after transplanting; in the experimental groups JX 14-13 to JX 14-15 in the table, the same type of strain was used for the same substitution test. The strain used to replace LXC3-3 was *Gnaphalium affine* from Nanjing Agriculture and Forestry University, and its accession number is CGMCC NO.5623.

[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 the LXC3-3-2 experimental group, Figure 4 As shown. From Figure 3 and Figure 4 The comparison clearly shows that the strawberry plants treated with the LXC3-3 fungus of this invention have a looser root system, and the development of horizontal roots is significantly higher than that of the CK group. Furthermore, the crown expansion of the strawberry plants in the LXC3-3-2 experimental group is significantly greater than that of the CK group. Figure 3 The CK group shown has a relatively larger number of leaves but a slightly smaller leaf area than the CK group. This means that while ensuring nutrient synthesis, more gaps are formed to avoid the shading effect in the field, which could hinder reproductive development in the later stages.

[0061] Specifically, the vertical root length, relative growth rate, root fresh weight, and aboveground fresh weight of each experimental group were characterized, recorded, and their mean values ​​were calculated. The results are shown in the table below.

[0062] Experimental group number Vertical root length relative growth rate Fresh root weight Fresh weight of aboveground parts CK 14.11cm / 9.95g 17.02g LXC3-3-1 16.07cm 34.09％ 8.84g 20.62g LXC3-3-2 17.16cm 53.04％ 8.54g 22.25g LXC3-3-3 16.83cm 47.30％ 8.71g 21.01g LXC3-3-4 15.73cm 28.17％ 8.93g 19.96g LXC3-3-5 16.99cm 50.09％ 8.52g 22.39g LXC3-3-6 16.02cm 33.22％ 8.61g 21.65g LXC3-3-7 14.91cm 13.91％ 8.97g 19.36g LXC3-3-8 16.67cm 44.52％ 8.81g 21.34g LXC3-3-9 15.09cm 17.04％ 9.03g 18.91g LXC3-3-10 14.97cm 14.96％ 8.84g 19.21g LXC3-3-11 15.03cm 16.00％ 8.99g 18.97g LXC3-3-12 14.89cm 13.57％ 9.04g 19.04g JX14-13 17.96cm 66.96％ 10.11g 20.69g JX14-14 18.36cm 73.91％ 10.97g 21.03g JX14-15 17.81cm 64.35％ 10.76g 20.91g LXC3-3-16 17.33cm 56.00％ 9.02g 21.65g LXC3-3-17 13.65cm -8.00％ 8.26g 17.35g LXC3-3-18 18.05cm 68.52％ 9.31g 18.91g

[0063] In the table: the relative growth rate is the relative elongation rate of the vertical roots compared to the CK group.

[0064] Specifically, the formula for calculating the relative growth rate is as follows.

[0065] In the formula: the original strawberry seedling's vertical root length is 8.36cm.

[0066] As can be seen from the table above, the *L.XC3-3* fungus of this invention has a very significant guiding effect on the root development of strawberry plants, altering the root development trend. In particular, the comparison of root fresh weight shows that the strawberry plants treated with *L.XC3-3* have generally lower root fresh weights than the control group (CK group), but higher vertical root lengths. Figure 3 , Figure 4 The comparison shows that the horizontal root system of strawberry plants treated with the LXC3-3 bacterium of the present invention is significantly more developed. Therefore, it can be understood that although the root mass density of strawberry plants decreased after treatment with LXC3-3, the strawberry plants were able to obtain and acquire nutrients thanks to the effective decomposition of fertilizer nutrients by the bacterium. Furthermore, the soil became looser under the action of the bacterium, resulting in a greater vertical root development depth than the control group. Most notably, the aboveground fresh weight of each experimental group treated with LXC3-3 was significantly higher than that of the control group, indicating that LXC3-3 has a very significant promoting effect on the aboveground development of strawberry plants.

[0067] By comparing test groups with equal bacterial concentrations, it can be seen that different bacterial concentrations should be applied to 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 the glutathione LXC3-3. 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... 600 When the concentration is 2.5, it can be found that even if the dilution factor is further reduced, it 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 glutathione bacterium of the present invention.

[0068] Compared with the phytophagous bacteria used in the JX14-13 to JX14-15 experimental groups, the effects of the phytophagous bacteria LXC3-3 of this invention on the root system are completely different. Phytophagous bacteria JX14 effectively promoted the development of the vertical root system of strawberry plants, making the strawberry plants more resistant to diseases, and the fresh weight of the roots was also much higher than that of the experimental groups treated with phytophagous bacteria LXC3-3 of this invention. However, in terms of the development of the aboveground parts, it was not as good as that of the experimental groups treated with phytophagous bacteria LXC3-3 of this invention. This indicates that phytophagous bacteria JX14, which has a typical root-promoting effect, promotes development by directly promoting root growth and absorption, while phytophagous bacteria LXC3-3 of this invention focuses on changing the actual development trend of the strawberry plant's root system, thereby forming a developmental advantage in the aboveground parts. The analysis of soil available nitrogen content revealed that the nitrogen content in the control group (CK) decreased rapidly due to the natural loss of nitrogen fertilizer. In contrast, the nitrogen content in the JX14-14 experimental group remained at a relatively high level for an extended period, exhibiting a gradually accelerating downward trend. This indicates that the JX14 bacterium has a good nitrogen-fixing effect, and that nitrogen demand and consumption increase with the development of strawberry plants. The LXC3-3-2 experimental group showed a rapid decrease in nitrogen content in the early stages after fertilization, but subsequently exhibited a relatively stable fluctuation trend that lasted for a considerable period. This suggests that the LXC3-3 bacterium also possesses some nitrogen-fixing ability, but more importantly, it has the ability to regulate available nitrogen through alkaline solutions. This is beneficial for dynamically regulating and controlling the available nitrogen content in the soil, offering unique advantages for strawberry cultivation.

[0069] Further comparison between the LXC3-3-2 and LXC3-3-16 experimental groups reveals significant differences in various indicators, particularly in vertical root length. The vertical root length of the LXC3-3-16 group is significantly greater than that of the LXC3-3-2 group, indicating that the early-stage *L.C. 3* fungus effectively inhibits vertical root development to some extent. Regarding survival rate, the CK group had a survival rate of 82.4%, while the LXC3-3-2 group had a survival rate of 100%, and the LXC3-3-16 group had a survival rate of 94.1%, demonstrating that the early-stage *L.C. 3* fungus also has a significant protective effect on the plant's root system. In actual strawberry cultivation, to improve the survival rate of strawberry seedlings, the fungal solution should be applied appropriately on the day of transplanting or the following day to provide protection for the strawberry seedling root system.

[0070] Comparing the LXC3-3-2 and LXC3-3-17 experimental groups, it was found that frequent application of the LXC3-3 bacterial solution of *Bacillus thuringiensis* creates strong nutrient competition for strawberry plants, which is detrimental to their development. This is because the OD concentration of the original bacterial inoculum is... 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 LXC3-3-2 and LXC3-3-18 experimental groups also shows that excessively long intervals between bacterial suspension applications do not produce the corresponding beneficial effects.

[0071] After the first strawberry harvest, the average number of strawberries harvested and the weight of a single fruit in each experimental group were recorded and the mean of each group was calculated. Part of the data obtained is shown in the table below.

[0072] experimental group Number of fruits / plant Single fruit weight experimental group Number of fruits / plant Single fruit weight CK 7.2 18.2g JX14-14 10.4 16.3g LXC3-3-2 6.9 pieces 26.1g LXC3-3-16 6.4 20.6g

[0073] As can be seen from the table above, the number of fruits harvested per strawberry plant after treatment with the phytophagous bacterium LXC3-3 of this invention did not increase significantly compared with the CK group, and even decreased somewhat. However, the increase in single fruit weight was very significant, which can greatly improve the rate of large strawberry fruits. It is gratifying that the phytophagous bacterium LXC3-3 of this invention, in addition to changing the root development trend, also significantly promotes the development of the above-ground reproductive organs of strawberry plants, making the development of strawberry fruits more excellent.

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

[0075] The following table shows some of the data records and their mean values.

[0076] experimental group Vitamin C content Soluble sugars experimental group Vitamin C content Soluble sugars CK 0.96mg / g 1.83％ JX14-14 0.99mg / g 1.91％ LXC3-3-2 1.16 mg / g 2.31％ LXC3-3-16 1.03 mg / g 2.16％

[0077] As can be seen from the table above, the glutathione LXC3-3 of this invention significantly improves the quality of strawberry fruit, with soluble sugar content and vitamin C content being much higher than those of the CK group and the glutathione control group (JX14-14).

[0078] In summary, it can be seen that the phytophagous bacterium LXC3-3 of this invention has a significant beneficial effect on the propagation of strawberry plants, and has a very significant effect on improving the survival rate, fruit size, and fruit quality of strawberry seedlings.

Claims

1. A glutathione bacterium LXC3-3, characterized in that, The glutathione LXC3-3 is classified and named Variovora x soil. The depositary institution is CGMCC, the accession number is CGMCC No. 24867, and the deposit date is May 9, 2022.

2. A gluttonous bacteria agent, characterized in that, The gluttonous bacteria agent contains the gluttonous bacteria LXC3-3 according to claim 1; The bacterial concentration (OD600) of the *Gnaphalium affine* inoculant is ≥1.

0.

3. The gluttonous bacteria agent according to claim 2, characterized in that, The bacterial concentration (OD600) of the *Gnaphalium affine* inoculant is 1.0~2.

0.

4. The application of the glutathione LXC3-3 according to claim 1, characterized in that, The gluttonous bacterium LXC3-3 was used for strawberry cultivation.

5. The application of the glutathione LXC3-3 according to claim 4, characterized in that, The specific process of using the gluttonous bacterium LXC3-3 for strawberry cultivation is as follows: The glutathione LXC3-3 was used to prepare the glutathione inoculum; The gluttonous bacteria inoculant was diluted, and the diluted inoculant 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 glutathione LXC3-3 according to claim 5, characterized in that, The bacterial solution was obtained by diluting the gluttonous bacteria agent by 85 to 105 times.

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