Stenotrophomonas maltophilia in tomato leaf miner larva intestinal tract and application thereof

Abstract

The invention discloses stenotrophomonas maltophilia in a tomato leaf miner larva intestinal tract and application of the stenotrophomonas maltophilia in the tomato leaf miner larva intestinal tract, and belongs to the technical field of agricultural microorganisms, the stenotrophomonas maltophilia in the tomato leaf miner larva intestinal tract can degrade and metabolize alpha-tomatine, and the nucleotide sequence of the stenotrophomonas maltophilia in the tomato leaf miner larva intestinal tract is shown as SEQ ID No: 1; the stenotrophomonas maltophilia JLU1 (stenotrophomonas maltophilia JLU1) in the intestinal tract of the tomato leaf miner larva has a degradation function and a detoxification metabolism process on alpha-tomatine, and can be used as a target to be applied to design and screening of prevention and control medicaments for the tomato leaf miner; the invention provides a basis for an intestinal functional bacteria prevention and control target theory, is beneficial to developing pest prevention and control medicaments, guides metabolite structure improvement and tomato molecular breeding, and opens up a new thought for green prevention and control.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microorganisms, and in particular relates to oligotrophic mononas maltophilia in the intestinal tract of tomato leafminer larvae and application thereof. Background Art

[0002] The tomato leafminer (Tuta absoluta Meyrick) is an important invasive pest in the world, belonging to the order Lepidoptera and the family Gelechiidae. The tomato leafminer mainly harms tomatoes, potatoes, eggplants, tobacco and other Solanaceae plants, of which tomatoes are the most serious and often cause devastating damage. The tomato leafminer harms with larvae. After hatching, the larvae burrow into the tissues of the host plants, feed on the mesophyll and form tiny tunnels on the leaves. They can also bore into the fruits to form holes, or feed at the junction of the calyx and the fruit, eventually causing serious yield reduction of the host plants, or even destroying the seeds and replanting. In severe cases, it can lead to a yield reduction of 80% to 100%. Due to the late start of research on the control of tomato leafminers in my country, there is currently a lack of efficient and environmentally friendly strategies for the control of tomato leafminers.

[0003] α-tomatidine, also known as tomatine or tomatine, is a steroidal glycoside alkaloid found in tomatoes and other Solanaceae plants. Among them, tomatine is the most abundant in tomato plants. It is a secondary metabolite produced in tomato rhizomes, leaves, and green fruits. The content of tomatine in its fruit gradually decreases with the maturity of the fruit. Tomatine can protect tomato plants from bacteria, fungi, viruses and certain insects during their growth, and has obvious antibacterial and insect repellent effects. As a steroidal alkaloid, tomatine has the biological activity of binding to cholesterol on biological membranes and then lysing membrane structures, thereby giving plants resistance. Tomato leaf miners can feed on tomatoes and are not affected by the toxicity of tomatine in tomatoes, but the mechanism of their resistance is still unclear.

[0004] After a long period of evolution, insects have developed a close symbiotic relationship with their intestinal microbes. Many studies have shown that intestinal microbes play a vital role in insect host adaptability. For example, insect intestinal bacteria can assist insects in nutrient digestion and plant secondary metabolite metabolism. However, the mechanism by which intestinal symbiotic bacteria can help tomato leafminers metabolize tomatine and thus improve their host adaptability is still unclear.

[0005] Stenotrophomonas maltophilia JLU1 is a bacterium isolated from the intestine of the tomato leafminer (Tuta absoluta Meyrick) larvae. Through the analysis of the α-tomatine metabolic function, its important role in assisting the host adaptation of the tomato leafminer is revealed, providing a theoretical basis for using intestinal functional bacteria as a control target for the tomato leafminer, facilitating the development of pest control agents targeting intestinal functional bacteria, guiding the modification and decoration of the tomatine structure, and its application in tomato molecular breeding, opening up a new idea for the green control of the tomato leafminer. Summary of the Invention

[0006] The aim of the present invention is to provide a strain involved in the degradation of α-tomatine in the intestine of the tomato leafminer.

[0007] The strain involved in the degradation of α-tomatine provided by the present invention is from the intestine of the tomato leafminer (Tuta absoluta Meyrick) larvae, and its species name is Stenotrophomonas maltophilia JLU1. Its 16S rDNA sequence is as shown in SEQ ID No: 1. This DNA sequence is the full length of the 16S rDNA of Stenotrophomonas maltophilia, consisting of 2655 nucleotides.

[0008] Stenotrophomonas maltophilia from the intestine of the tomato leafminer larvae can be applied in the process of α-tomatine degradation and can also be used in the biological technology field for controlling the tomato leafminer.

[0009] Inhibiting the intestinal strain Stenotrophomonas maltophilia from the tomato leafminer larvae, affecting the degradation function of Stenotrophomonas maltophilia on α-tomatine, and interfering with the detoxification and metabolism process of α-tomatine in the tomato leafminer larvae can be used as a target in the design and screening of pest control agents for the tomato leafminer.

[0010] Through strain screening, isolation, identification, selective culture, and growth curve determination, the present invention proves that Stenotrophomonas maltophilia has an in vitro degradation effect on tomatine, indicating that Stenotrophomonas maltophilia (JLU1) is a tomatine-degrading intestinal functional bacterium of Tuta absoluta Meyrick. Currently, there is no report on the tomatine degradation function of Stenotrophomonas maltophilia (JLU1). The present invention designs, synthesizes, and screens compounds with inhibitory strain activity using this strain as a target or α-tomatine as a lead, and further develops new pest insecticides or provides a theoretical basis for tomato molecular breeding, thereby achieving efficient and green control of Tuta absoluta Meyrick. That is, an important use of Stenotrophomonas maltophilia (JLU1) provided by the present invention is to disrupt the detoxification metabolism process of Tuta absoluta Meyrick larvae by affecting the action of this strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a photograph of the intestinal structure of Tuta absoluta Meyrick larvae;

[0012] Figure 2 It is a photograph of the isolation and culture of intestinal bacteria of Tuta absoluta Meyrick;

[0013] Among them: a - f are the growth conditions of colonies after intestinal homogenate dilutions diluted to 1.0×10-1, 1.0×10-2, 1.0×10-3, 1.0×10-4, 1.0×10-5, and 1.0×10-6 times are spread on LB plates;

[0014] Figure 3 It is a schematic diagram of the metagenomic determination and analysis of intestinal bacteria of Tuta absoluta Meyrick enriched by a restrictive medium;

[0015] Among them: α-tomatine in the restrictive medium is the sole carbon source;

[0016] Figure 4 It is the growth curve of Stenotrophomonas maltophilia in a restrictive medium with α-tomatine as the sole carbon source. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention will be described below in conjunction with the drawings and through specific examples. The methods in the examples are all conventional methods unless otherwise specified.

[0018] Example 1

[0019] Intestinal Dissection of Tuta absoluta Meyrick and Isolation and Culture of Intestinal Bacteria

[0020] Randomly select 10 third-instar larvae of Tuta absoluta with similar size and state. After 24 hours of starvation treatment, soak them in 75% alcohol for disinfection for 30 seconds, then rinse them 3 times with sterile water. Then place the larvae in a disposable petri dish filled with sterile PBS buffer and perform aseptic dissection using dissecting forceps under a stereomicroscope (as Figure 1 shown). Completely peel off the intestinal tissue, place the collected intestinal tissue in a 1.5 mL centrifuge tube containing 100 μL of PBS buffer, homogenize it, then add 900 μL of PBS buffer, and mix well with a vortex oscillator. The obtained intestinal homogenate is spread on an LB plate, and the situation of the isolated and cultured intestinal bacteria is as Figure 2 shown.

[0021] Example 2

[0022] Screening of α-tomatine-degrading functional bacteria

[0023] Use a medium with α-tomatine as the sole carbon source to screen bacteria with tomatine-degrading function. The formula of the liquid minimal medium without carbon source is: 0.7 g / L NH 4 Cl, 0.5 g / L KH 2 PO4, 1.0 g / L NaCl, 1.5 g / L Na 2 HPO 4 , 0.2 g / L MgSO 4 ·7H 2 O. Dissolve tomatine with DMSO to prepare a stock solution with a concentration of 50 mg / mL. After the liquid minimal medium is sterilized and cooled, pipette 1 mL of the medium into a 1.5 mL centrifuge tube, and then add 20 μL of the tomatine stock solution to make the tomatine concentration in the medium reach 1.0 g / L, thus preparing a liquid medium with tomatine as the sole carbon source. Add 20 μL of the intestinal homogenate prepared by the above method to the medium. After culturing for 24 hours (28 °C, 200 rpm) under dark conditions, pipette 20 μL of the suspension into a new test tube containing the liquid medium, and repeat this re-inoculation process 7 times within one week under dark conditions at 28 °C. Perform 7-fold dilution / enrichment, and analyze the obtained bacterial liquid by third-generation sequencing technology for metagenomics determination. Stenotrophomonas maltophilia JLU1 is the dominant bacterium in the restrictive medium, and the species abundance is as Figure 3 shown.

[0024] Example 3

[0025] I. Isolation and culture of functional bacteria

[0026] Refer to the bacterial solution obtained after multiple rounds of enrichment culture in Example 2, and dilute it stepwise with PBS buffer to 1.0×10-3, 1.0×10-4, 1.0×10-5, and 1.0×10-6 times. Then, pipette 100 μL of the diluted solution and spread it on the LB solid medium. Each treatment is set with 3 replicates, and sterile water treatment is used as the blank control. Incubate in an incubator at a temperature of 28 °C and a relative humidity of 70±5% for 12 h.

[0027] II. Strain Identification of Functional Bacteria

[0028] Screen single colonies with clear edges and non-adherent to each other from the medium, and expand the culture in LB medium. Take the bacterial solution sample and perform sequencing using the primers (F: AGRGTTTGATYNTGGCTCAG; R: TASGGHTACCTTGTTASGACTT) to obtain the sequence of the 16S rDNA of the functional strain. The strain is identified as Stenotrophomonas maltophilia JLU1 by sequence alignment.

[0029] Example 4

[0030] Verify the tomatine degradation function of Stenotrophomonas maltophilia JLU1 by measuring the growth curve. Add 1 mL of liquid medium with tomatine as the sole carbon source to a 1.5 mL centrifuge tube. Refer to the strain obtained in the experiment of Example 3, pipette 10 μL of the bacterial solution and add it to the centrifuge tube, and then place it in an incubator for culture under dark conditions (28 °C, 200 rpm). Use a microplate reader to measure the absorbance value of the bacterial solution at a wavelength of 600 nm every 2 h. Zero with the culture medium without bacteria as the blank control, and perform three replicates for each measurement. The isolated and identified Stenotrophomonas maltophilia JLU1 can grow normally in the tomatine-restricted medium, showing a typical S-shaped bacterial growth curve, with a maximum OD value of about 2.5. However, other strains cannot grow normally in the restricted medium, and the OD value remains 0 (as Figure 4 shown).

[0031]

Claims

1. A Stenotrophomonas maltophilia JLU1 in the intestinal tract of a tomato leafminer larvae can degrade and metabolize α-tomatine. The nucleotide sequence of the Stenotrophomonas maltophilia JLU1 in the intestinal tract of a tomato leafminer larvae is shown in SEQ ID No:

1.

2. The degradation function and detoxification metabolic process of α-tomatine by Stenotrophomonas maltophilia JLU1 in the intestine of tomato leafminer larvae as claimed in claim 1 can be used as a target in the design and screening of tomato leafminer control agents.

Images