Methylobacterium mays, and application thereof

Abstract

The present application belongs to the technical field of microorganisms, and particularly relates to a strain of Stenotrophomonas maltophilia capable of improving the reproduction of tobacco aphids and application thereof. Stenotrophomonas maltophilia ) FZECTP.H3, which was preserved in the Guangdong Provincial Microbial Culture Collection Center on September 5, 2023, has a preservation number of GDMCC No: 63770 and a preservation address of 100, Martyrs Road, Yuexiu District, Guangzhou, Guangdong. The Stenotrophomonas maltophilia FZECTP.H3 of the present application has a significant promoting effect on the tobacco aphid production, which has application value or practical significance for indoor long-term aphid preservation and improvement of the scale reproduction of tobacco aphid parasitoid, is a beneficial ecological regulation technology in green ecological prevention and control, and is worthy of demonstration and gradual application in tobacco production.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Stenotrophomonas maltophilia that can enhance the reproductive capacity of tobacco aphids and its applications. Background Technology

[0002] The tobacco aphid, *Myzuspersicae* (Sulzer), is widely distributed in tobacco-growing areas of both northern and southern my country. It is characterized by large populations and a long period of damage, occurring at all stages of tobacco growth. The aphid not only directly punctures the tobacco leaves, causing malformation and secreting honeydew that induces mold and mildew, but it also indirectly transmits various pathogens, leading to the spread of other insect-borne diseases and various viral diseases. This results in a decline in the quality and yield of tobacco leaves, causing significant economic losses to tobacco production (Laie et al., 2011; Li et al., 2013; Lai et al., 2017; Zhang et al., 2019). Biological control of the tobacco aphid involves using natural enemies to reduce its population. These natural enemies are generally predatory or parasitic insects (Gu et al., 2015). *Aphidius gifuensis* Ashmead is a parasitic natural enemy insect with a strong parasitic ability against tobacco aphids. It controls and reduces the tobacco aphid population by laying eggs on the aphids, which then hatch and feed on the aphids' internal tissues and organs, causing the aphids to die from malnutrition (An et al., 2017; Yang et al., 2017; Bai et al., 2020; Chen et al., 2020; Lai et al., 2021). Huang et al. (2008), through investigations in fields with *Aphidius gifuensis* release, conventional fields, and fields treated with chemical pesticides, found that *Aphidius gifuensis* has a control effect comparable to chemical pesticides and can effectively reduce damage to the ecological environment. However, in the large-scale propagation of the tobacco aphid parasitic wasp, it was found that as the number of propagation generations increased, the population of the tobacco aphid parasitic wasp showed varying degrees of decline. This was mainly manifested in the smaller size of the adult wasps, the shorter lifespan, the decreased parasitic ability, and the decreased rate of aphid emergence. In some cases, the population even became smaller and smaller, which seriously restricted the scale of propagation in the later stages.

[0003] Currently, the rejuvenation of tobacco aphids and aphid wasps mainly involves domestication in the wild and the introduction of wild populations. However, the process is very complex and cumbersome, and the results are not satisfactory, failing to achieve the goal of treating both the symptoms and the root cause. Insect endophytic bacteria can be horizontally transmitted through eggs or between individuals, playing an extremely important role in the reproduction and dispersal capabilities of insects. For example, Wolbachia can induce parthenogenesis in parasitic wasps, enhance female fertility and male fertility, accelerate population growth, and make it easier for parasitic wasps to enter new habitats and establish populations (Aeschilimann, 1990; Stouthamer et al., 1993).

[0004] Symbiotic bacteria are widely found in insects of the orders Hemiptera and Hymenoptera, and they have formed a mutually beneficial symbiotic relationship with their hosts through long-term evolution (Douglas, 2015). Symbiotic bacteria can be divided into two main categories: primary symbiotic bacteria and secondary symbiotic bacteria. Primary symbiotic bacteria have a close relationship with their host insects, providing them with essential nutrients and spreading vertically via eggs. These symbiotic bacteria are essential for the normal survival and development of the host insects (Moranetal., 2008; Kogaetal., 2012). Secondary symbiotic bacteria are not necessarily essential for the host's survival. Some of these symbiotic bacteria can spread vertically via eggs, while others can spread horizontally between different individuals (Brumin et al., 2012; Caspi-Fluger et al., 2012). Secondary symbiotic bacteria are diverse in type and function, and different symbiotic bacteria have different effects on their hosts (Yang et al., 2014). Known functions of secondary symbiotic bacteria include participating in the host insect's defense against natural enemies, resisting pathogen infection, providing nutrients to the host, enhancing the host's heat resistance, assisting the host in virus transmission, regulating the host's reproduction, and improving the host's adaptability. Rickettsia can influence the host's reproductive behavior by inducing parthenogenesis and male killing (Giorgini et al., 2010; Hagimori et al., 2006; Giorgini et al., 2010). Yan et al. (2017) found that after treating peach aphids with rifampicin, the expression level of the DNAK gene in *Buchnera aphidicola* was significantly reduced, and the number of offspring produced by peach aphids was significantly lower than that of healthy peach aphids. The secondary symbiotic *Serratia symbiotica* has a beneficial effect on peach aphid reproduction, increasing the daily reproductive rate, but also has an adverse effect on peach aphid development, delaying nymphal development (Liu Yanhong, 2016). Wheat aphids infected with Regiella insecticola showed lower net proliferation rate and intrinsic growth rate at medium and low densities and suitable temperatures compared to naturally uninfected and desymbiotic strains. However, at 31°C and high densities, the net proliferation rate and intrinsic growth rate of infected and desymbiotic strains no longer differed significantly, and in some aphid genotypes, the infection rate was significantly higher than that of desymbiotic strains (Lei Haixia, 2017). When wheat aphids were cultured alone at 25°C, Regiella insecticola infection reduced their litter size; at 31°C with 4 hours of heat shock per day, Regiella insecticola infection significantly reduced the net proliferation rate and intrinsic growth rate of wheat aphids at high densities (Ma Tingting, 2014).

[0005] Endosymbiotic bacteria and insects have formed a very close symbiotic relationship over a long period of evolution, playing a vital role in insect growth, development, and evolution. Wolbachia was detected in most of the parasitic wasps *Aphidius spp.* in tobacco-growing areas of Fujian Province, and *Rickettsia* was found in almost all of them (Ju Yufang, 2019). These two endophytic bacteria have significant effects on the reproduction, parasitism, sex ratio, and individual size of parasitic wasps such as *Trichogramma spp.*, *Trichogramma spp.*, and *Trichogramma spp.* (Silva et al., 2000; Hagimori et al., 2006; Xiang Yu et al., 2006; Giorgini et al., 2010; Liu Shuping, 2011; Li Peiguang et al., 2015). However, the effects of Wolbachia and *Rickettsia* on the reproduction, parasitism, sex ratio, and individual size of *Aphidius spp.* have not been reported. Furthermore, symbiotic bacteria can induce parthenogenesis in their hosts (Xiang et al., 2006), and parthenogenesis has significant implications for the biocontrol efficacy and industrialized production of parasitic wasps: (1) Parthenogenetic individuals will have higher population growth rates and higher puncture-laying rates. (2) Since they do not need to mate with male individuals, parthenogenetic individuals are more likely to enter new habitats and establish populations at low population densities. (3) Since male individuals do not need to be reared, parthenogenesis can reduce the cost of large-scale cultivation. The influence of symbiotic bacteria on the host can be used to control the growth, development, and reproduction of tobacco aphids and improve the reproductive efficiency and parasitism rate of tobacco aphid parasitoid wasps.

[0006] Therefore, addressing the problems encountered in the artificial breeding and practical application of the tobacco aphid wasp, this invention, based on research into the genetic degradation and rejuvenation techniques of the tobacco aphid wasp, uses insect endophytic bacteria as a starting point. It employs an immersion method to inoculate tobacco aphids with endophytic bacteria, screening out beneficial insect endophytic bacteria that influence the reproductive capacity of tobacco aphids, and applying them to the artificial rearing process to maintain the genetic integrity of tobacco aphids for a long time. This has application value or practical significance for long-term indoor aphid control and improving the large-scale breeding of tobacco aphid wasps. It is a beneficial ecological regulation technology in green ecological control and is worthy of demonstration and gradual promotion in tobacco production. Summary of the Invention

[0007] The purpose of this invention is to provide a strain of Stenotrophomonas maltophilia that can enhance the reproductive capacity of tobacco aphids and its applications.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A strain of Stenotrophomonas maltophilia FZECTP.H3, which can enhance the reproductive capacity of tobacco aphids, was deposited on September 5, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No: 63770. The deposit address is No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0009] The application of the aforementioned Stenotrophomonas maltophilia FZECTP.H3 in improving the reproductive capacity of tobacco aphids is specifically manifested in increasing the aphid production of tobacco aphids.

[0010] A bacterial agent containing the aforementioned Stenotrophomonas maltophilia FZECTP.H3.

[0011] Furthermore, the above-mentioned bacterial agent is a bacterial suspension, and the bacterial content in the bacterial suspension is 0.97 × 10⁻⁶. 6 ~0.97×10 10 cfu / ml.

[0012] Furthermore, the above bacterial suspension was obtained by the following method: Stenotrophomonas maltophilia FZECTP.H3 was inoculated into NA liquid medium and cultured at 37°C with shaking at 230 r / min until OD... 600 The bacterial concentration was set to 1.0, and then centrifuged at 8000g for 5 minutes at 4°C. The bacterial precipitate was collected, washed three times with 0.85% sterile sodium chloride solution, and then resuspended in 0.85% sterile sodium chloride solution until the bacterial count reached 0.97 × 10⁻⁶. 6 ~0.97×10 10 CFU / ml was used to obtain a bacterial suspension.

[0013] The application of the above-mentioned bacterial agent containing Stenotrophomonas maltophilia FZECTP.H3 in improving the reproductive capacity of tobacco aphids is specifically manifested in increasing the aphid production of tobacco aphids.

[0014] The significant advantages of this invention are: This invention addresses the genetic degradation of tobacco aphids during long-term enclosed artificial breeding. It screens out *Stenotrophomonas maltophilia* FZECTP.H3, an insect endophytic bacterium beneficial to tobacco aphid reproduction, and applies it to the artificial rearing process to maintain the genetic integrity of tobacco aphids. Experimental results show that *Stenotrophomonas maltophilia* FZECTP.H3 significantly promotes aphid production in tobacco aphids, and the source of *Stenotrophomonas maltophilia* FZECTP.H3 is wingless tobacco aphids collected from tobacco fields, with a concentration of 0.97 × 10⁻⁶. 9 The cfu / ml Stenotrophomonas maltophilia FZECTP.H3 showed the most significant effect on promoting aphid production in tobacco aphids, increasing the number by 39.445 aphids compared to the control, an increase of 87.01%. Attached Figure Description

[0015] Figure 1 The colony morphology of Stenotrophomonas maltophilia FZECTP.H3 on NA plates. Detailed Implementation

[0016] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0017] Example 1 Wingless tobacco aphids of uniform size [Myzus persicae (Sulzer)] were collected from tobacco fields in Longyan Branch of Fujian Provincial Tobacco Agricultural Research Institute without the use of any pesticides. They were then raised indoors on potted tobacco seedlings under the following conditions: temperature 25℃±1℃, relative humidity RH 70%±5%, and photoperiod 16L:8D.

[0018] Wingless tobacco aphids were starved for 24 hours to allow them to empty their bodies of food residue. They were then immediately placed in a -20°C freezer for several minutes to kill them. Afterward, they were transferred to sterile petri dishes for surface disinfection. First, they were soaked in 75% alcohol for 30 seconds, then rinsed 3-4 times with sterile water for 30 seconds each time. The surface-disinfected insects were placed in a sterile mortar, phosphate buffer was added, and they were ground into a homogenate. The phosphate buffer was then diluted to 10... -1 10 -2 10 -3 Take 10 -1 10 -2 10 -3 Diluted solutions of varying concentrations were spread onto nanoplates and incubated at 30°C for 48 hours. New colonies were observed every 24 hours thereafter. The number of colonies with different colors, morphologies, and sizes was counted on each plate. Colonies were then streaked, purified, and cultured to obtain bacterial strains. The shape, color, and morphology of each strain were observed and described. The obtained single colonies were numbered and used to prepare bacterial suspensions for culturing wingless tobacco aphids. A strain that significantly increased aphid production was obtained, named FZECTP.H3, and preserved in glycerol tubes.

[0019] The colony morphology of strain FZECTP.H3 on NA plates is as follows: Figure 1 As shown, the center of the colony is yellow and raised, while the edges of the colony are smooth, pale yellow, and moist.

[0020] The genome of strain FZECTP.H3 was extracted using a genome extraction kit. Using this genome as a template, the 16S rDNA sequence was amplified by PCR. The PCR product was sent to the Fuzhou branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing result is shown in SEQ ID No. 1 of the sequence listing. Sequence alignment of the sequencing results on NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) revealed a 97.81% sequence similarity between this strain and *Stenotrophomonas maltophilia*. Therefore, this strain was identified as *Stenotrophomonas maltophilia*.

[0021] Stenotrophomonas maltophilia FZECTP.H3 was deposited at Guangdong Provincial Center for Microbial Culture Collection, on September 5, 2023, with accession number GDMCC No. 63770, located at No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0022] Example 2 Stenotrophomonas maltophilia FZECTP.H3 was inoculated into NA liquid medium and cultured at 37°C with shaking at 230 rpm until OD. 600 The concentration was set to 1.0, and then centrifuged at 8000g for 5 min at 4℃. The bacterial precipitate was collected, washed three times with 0.85% sterile sodium chloride solution, and then resuspended in 0.85% sterile sodium chloride solution to a concentration of 0.97×10⁻⁶. 6 ~0.97×10 10 CFU / ml was used to obtain a bacterial suspension.

[0023] The effect of Stenotrophomonas maltophiliae FZECTP.H3 on the reproductive capacity of tobacco aphids was determined in an artificial climate chamber at a temperature of (25±1)℃, a relative humidity of (70±5)%, and a photoperiod of 16L:8D. Inoculation was performed using the immersion method. Before inoculation, fourth-instar nymphs of the tested wingless tobacco aphids were gently transferred to a nylon mesh using a clean brush. The nylon mesh was then immersed in 50 mL of the test bacterial suspension or 0.85% sterile sodium chloride solution for 5 seconds (control). After inoculation, excess suspension remaining on the nylon mesh was blotted dry with clean filter paper. Leaflets with a diameter of 2 cm were made from the leaves of Yunyan 87 tobacco plants. Inoculated aphids were transferred to these leaflets, one aphid per leaflet. The leaflets were then placed in a 3 cm diameter plastic box lined with filter paper and damp cotton. The plants were reared at a temperature of (25±1)℃, a relative humidity of (70±5)%, and a photoperiod of 16L:8D. The aphids' molting, reproduction, and mortality were observed and recorded every 12 hours, and newly born nymphs were removed. The cotton at the bottom of the plastic box was kept moist as needed, and the leaflets were replaced every 2 days.

[0024] The effects of Stenotrophomonas maltophilia FZECTP.H3 on the reproductive capacity of the wingless tobacco aphid are shown in Table 1. Stenotrophomonas maltophilia FZECTP.H3 significantly promoted aphid production in the wingless tobacco aphid (F... 5,12 =40.938, P<0.01); when the concentration of Stenotrophomonas maltophilia FZECTP.H3 was 0.97×10 9 At a concentration of cfu / ml, the effect on promoting aphid production in wingless tobacco aphids was greatest, increasing by 39.445 aphids / aphid compared to the control, an increase of 87.01%; when the concentration of Stenotrophomonas maltophilia FZECTP.H3 was 0.97×10⁻⁶, the effect was even greater. 6 At a concentration of cfu / ml, the effect on promoting aphid production in wingless tobacco aphids was minimal, increasing production by 12 aphids / aphid compared to the control, a 26.47% increase; the concentration of Stenotrophomonas maltophilia on aphid production was 0.97 × 10⁻⁶. 8 0.97×10 7 At cfu / ml, the effect on promoting the aphid production of wingless tobacco aphids was moderate, increasing by 15.333 aphids / aphid and 12.667 aphids / aphid compared to the control, representing increases of 33.82% and 27.94%, respectively.

[0025] Table 1. Effects of Stenotrophomonas maltophilia on the reproductive capacity of tobacco aphids. Note: The data in the table are mean ± standard error. Different lowercase letters in the same column indicate significant differences (one-way ANOVA, LSD test, P < 0.05).

[0026] The above results indicate that *Stenotrophomonas maltophilia* FZECTP.H3, collected from tobacco fields, significantly promotes aphid production in tobacco aphids, especially at a concentration of 0.97 × 10⁻⁶. 9 The CFU / ml ratio has the most significant promoting effect, which has application value or practical significance for long-term aphid control indoors. It is a beneficial ecological regulation technology in green ecological control and is worth demonstrating and gradually promoting in tobacco production.

[0027] 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 strain of Stenotrophomonas maltophilia FZECTP.H3 that can enhance the reproductive capacity of tobacco aphids, characterized by: Classified as Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia FZECTP.H3 was deposited on September 5, 2023, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 63770), located at No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

2. The application of Stenotrophomonas maltophiliae FZECTP.H3 as described in claim 1 in improving the reproductive capacity of tobacco aphids.

3. The application according to claim 2, characterized in that: Application of Stenotrophomonas maltophilia FZECTP.H3 in increasing aphid production in tobacco aphids.

4. A bacterial agent containing Stenotrophomonas maltophilia FZECTP.H3 as described in claim 1.

5. The microbial agent according to claim 4, characterized in that: The bacterial agent is a bacterial suspension, and the bacterial content in the bacterial suspension is 0.97 × 10⁻⁶. 6 ~0.97×10 10 cfu / ml.

6. The microbial agent according to claim 5, characterized in that: The bacterial suspension was obtained by the following method: Stenotrophomonas maltophilia FZECTP.H3 was inoculated into NA liquid medium and cultured at 37°C with shaking at 230 r / min until OD. 600 The bacterial concentration was set to 1.0, and then centrifuged at 8000g for 5 minutes at 4°C. The bacterial precipitate was collected, washed three times with 0.85% sterile sodium chloride solution, and then resuspended in 0.85% sterile sodium chloride solution until the bacterial count reached 0.97 × 10⁻⁶. 6 ~0.97×10 10 CFU / ml was used to obtain a bacterial suspension.

7. The application of the bacterial agent containing Stenotrophomonas maltophilia FZECTP.H3 as described in claim 4 in improving the reproductive capacity of tobacco aphids.

8. The application according to claim 7, characterized in that: Application of bacterial agents containing Stenotrophomonas maltophilia FZECTP.H3 in increasing aphid production in tobacco aphids.

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